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The Zebrash as a Tool to Study Hematopoiesis, Human Blood Diseases, and Immune Function
Guest Editors: Jason Berman, Elspeth Payne, and Christopher Hall
The Zebrash as a Tool to Study Hematopoiesis, Human Blood Diseases, and Immune Function
Advances in Hematology
The Zebrash as a Tool to Study Hematopoiesis, Human Blood Diseases, and Immune Function
Guest Editors: Jason Berman, Elspeth Payne, and Christopher Hall
Copyright 2012 Hindawi Publishing Corporation. All rights reserved. This is a special issue published in Advances in Hematology. All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Contents
The Zebrash as a Tool to Study Hematopoiesis, Human Blood Diseases, and Immune Function, Jason Berman, Elspeth Payne, and Christopher Hall Volume 2012, Article ID 425345, 2 pages Development and Characterization of Anti-Nitr9 Antibodies, Radhika N. Shah, Ivan Rodriguez-Nunez, Donna D. Eason, Robert N. Haire, Julien Y. Bertrand, Val erie Wittamer, David Traver, Shila K. Nordone, Gary W. Litman, and Jerey A. Yoder Volume 2012, Article ID 596925, 9 pages Characterization of Zebrash von Willebrand Factor Reveals Conservation of Domain Structure, Multimerization, and Intracellular Storage, Arunima Ghosh, Andy Vo, Beverly K. Twiss, Colin A. Kretz, Mary A. Jozwiak, Robert R. Montgomery, and Jordan A. Shavit Volume 2012, Article ID 214209, 9 pages Drift-Diusion Analysis of Neutrophil Migration during Inammation Resolution in a Zebrash Model, Georey R. Holmes, Giles Dixon, Sean R. Anderson, Constantino Carlos Reyes-Aldasoro, Philip M. Elks, Stephen A. Billings, Moira K. B. Whyte, Visakan Kadirkamanathan, and Stephen A. Renshaw Volume 2012, Article ID 792163, 8 pages Novel Insights into the Genetic Controls of Primitive and Denitive Hematopoiesis from Zebrash Models, Raman Sood and Paul Liu Volume 2012, Article ID 830703, 13 pages Myelopoiesis and Myeloid Leukaemogenesis in the Zebrash, A. Michael Forrester, Jason N. Berman, and Elspeth M. Payne Volume 2012, Article ID 358518, 12 pages Neutrophil Reverse Migration Becomes Transparent with Zebrash, Taylor W. Starnes and Anna Huttenlocher Volume 2012, Article ID 398640, 11 pages Through the Looking Glass: Visualizing Leukemia Growth, Migration, and Engraftment Using Fluorescent Transgenic Zebrash, Finola E. Moore and David M. Langenau Volume 2012, Article ID 478164, 8 pages Pathogen Recognition and Activation of the Innate Immune Response in Zebrash, Michiel van der Vaart, Herman P. Spaink, and Annemarie H. Meijer Volume 2012, Article ID 159807, 19 pages Histocompatibility and Hematopoietic Transplantation in the Zebrash, Jill L. O. de Jong and Leonard I. Zon Volume 2012, Article ID 282318, 8 pages Zebrash Thrombocytes: Functions and Origins, Gauri Khandekar, Seongcheol Kim, and Pudur Jagadeeswaran Volume 2012, Article ID 857058, 9 pages
In Vivo Chemical Screening for Modulators of Hematopoiesis and Hematological Diseases, Yiyun Zhang and J.-R. Joanna Yeh Volume 2012, Article ID 851674, 12 pages Genomic Amplication of an Endogenous Retrovirus in Zebrash T-Cell Malignancies, J. Kimble Frazer, Lance A. Batchelor, Diana F. Bradley, Kim H. Brown, Kimberly P. Dobrinski, Charles Lee, and Nikolaus S. Trede Volume 2012, Article ID 627920, 12 pages Hydrogen Peroxide in Inammation: Messenger, Guide, and Assassin, C. Wittmann, P. Chockley, S. K. Singh, L. Pase, G. J. Lieschke, and C. Grabher Volume 2012, Article ID 541471, 6 pages
Hindawi Publishing Corporation Advances in Hematology Volume 2012, Article ID 425345, 2 pages doi:10.1155/2012/425345
Editorial The Zebrash as a Tool to Study Hematopoiesis, Human Blood Diseases, and Immune Function
Jason Berman,1 Elspeth Payne,2 and Christopher Hall3
1 Departments
of Pediatrics, Microbiology and Immunology and Pathology, Dalhousie University and IWK Health Centre, Halifax, NS, Canada B3K 6R8 2 Department of Haematology, University College London Cancer Centre, London, UK 3 Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, New Zealand Correspondence should be addressed to Jason Berman, [email protected] Received 31 August 2012; Accepted 31 August 2012 Copyright 2012 Jason Berman et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Over the last decade, the zebrash has cemented itself as a unique model system for providing new insights into the regulatory factors required for vertebrate hematopoiesis. In particular, the ease of genetic manipulation together with the transparency of embryos facilitating high resolution imaging has enabled the fate mapping of a host of blood cell lineages. Most notably, this has included the detailed evaluation of the origin and emergence of hematopoietic stem cells. Genetic conservation between zebrash and mammals and the construction of well-annotated detailed genomic databases have permitted the use of a number of forward and reverse genetic approaches to study a variety of benign and malignant human blood disorders in this organism. These studies have revealed new molecular players underlying human phenotypes as well as providing platforms both for genetic screens to identify novel interacting partners as well as chemical modier screens to reveal compounds that may represent new therapeutic strategies. Conserved hematopoietic cell biology extends across the innate and adaptive immune systems, fueling a recent growth of research focused on exploiting the advantages of the zebrash system to examine vertebrate host-pathogen interactions and the contributions of individual cell subtypes to innate and adaptive immune responses. This special issue highlights some of the most recent and profound contributions provided by the zebrash model system to understand hematopoiesis, hematopoietic malignancies, and the vertebrate immune system. As a volume, it highlights the tremendous accomplishments achieved in these diverse areas of hematology using the zebrash model
to date and sets the stage for continued advancement in all spheres of hematology, oncology, and immunology using this highly genetically conserved, easily manipulated, and clearly visualized remarkable organism. In the paper entitled Novel insights into the genetic controls of primitive and denitive hematopoiesis from zebrash models, R. Sood and P. Liu review the anatomic sites and developmental waves of primitive and denitive hematopoiesis and emphasize the conservation of critical transcription factors and other genes that regulate these processes. They highlight some of their own recent work in this eld in which they utilize a zebrash runx1 mutant to identify novel insights into the role of runx1 in denitive hematopoiesis and identify a hypomorphic allele of gata1 that provides the opportunity to more precisely attribute the contribution of this transcription factor to various stages of erythroid development. In the report entitled Myelopoiesis and myeloid leukaemogenesis in the zebrash, A. M. Forrester et al. highlight the conservation of myeloid gene regulation in zebrash and describe the recent advances in this eld. A number of studies and approaches are reviewed that have shed light on vertebrate neutrophil, monocyte, eosinophil, and mast cell development and provide a suite of in vivo tools to examine the perturbations associated with premalignant and malignant myeloid disease. Myeloid cells are key players in the innate immune system, an area of increasing investigation using the zebrash model. A. H. Meijer et al. provide an overview of this area in their paper entitled Pathogen recognition and activation
2 of the innate immune response in zebrash. Conservation of toll-like receptors, nucleotide-binding oligomerization domain (NOD)-like receptors, and other key members of the innate immune response are discussed and examined in the context of a number of bacterial pathogens. Novel immunetype receptors (NITRs) and functional orthologues in zebrash of mammalian NK cell receptors are characterized in the paper by J. Yoders group entitled Development and characterization of anti-Nitr9 antibodies. C. Wittmann et al. outline the critical role of hydrogen peroxide as a mediator of inammatory responses in the zebrash in their paper entitled Hydrogen peroxide in inammation:messenger, guide, and assassin. Neutrophil behaviour in response to wounds is dissected in more detail in two papers entitled Neutrophil reverse migration becomes transparent with zebrash by T. W Starnes and A. Huttenlochers group and Driftdiusion analysis of neutrophil migration during inammation resolution in a zebrash model by S. A. Renshaw et al. Huttenlochers group takes advantage of a neutrophil-specic Lyn oxidation mutant to demonstrate that this Src family kinase is a critical link between hydrogen peroxide produced at the site of a wound and neutrophil chemoattraction. The imaging capabilities of the zebrash and photoconversion techniques are subsequently exploited by both groups to reveal the process of neutrophil reverse migration for the rst time. The purpose of this phenomenon and ultimate fate of these reversely travelling cells remain to be determined. However, the zebrash is likely to serve a key role in further elucidating the factors underlying this process. Platelet development and hemostasis in the zebrash is next addressed in two papers entitled Zebrash thrombocytes: functions and origins by P. Jagadeeswaran et al. and Characterization of zebrash von Willebrand factor reveals conservation of domain structure, multimerization, and intracellular storage by J. A. Shavit et al. These reports set the stage for the zebrash to provide new insights into platelet biology and model human bleeding disorders. This special issue also includes a number of papers highlighting the utility of the zebrash as a tool in dissecting oncogenic pathways in leukemia pathogenesis, identifying novel therapies, and improving stem cell transplantation. F. E. Moore and D. M. Langenau summarize the transgenic models of leukemia that have been developed by their laboratory and others in their paper entitled Through the looking glass: visualizing leukemia growth, migration, and engraftment using uorescent transgenic zebrash. They present the opportunities provided by the transparency of zebrash embryos and uorescent labeling to study leukemia cell engraftment, homing, and frequency of leukemia propagating cells. These transgenic leukemia models provide a platform both for further genetic interrogation and high throughput drug screening. In their paper entitled Genomic amplication of an endogenous retrovirus in zebrash T-cell malignancies, J. K. Frazer et al. utilize array comparative genomic hybridization (aCGH) on the genomes of three zebrash T-cell leukemia transgenic lines to identify a novel oncogenic retrovirus. Y. Zhang and J. R. Joanna Yeh describe the process for conducting chemical screens in zebrash embryos in their paper entitled In vivo chemical screening
Advances in Hematology for modulators of hematopoiesis and hematological disease and highlight the tremendous advantages and opportunities inherent in this approach. In particular, they describe the identication of the prostaglandin pathway and COX proteins in two separate screens: as positive regulators of hematopoietic stem cell development and as targets for inhibition in AML1-ETO driven myeloid disease. Finally, J. L. O. de Jong and L. I. O. Zon have contributed, Histocompatibility and hematopoietic transplantation in the zebrash, whereby they extend the zebrash model to studies of matched allogeneic stem cell transplantation, with potential to quantify engraftment and model graft versus host disease. Jason Berman Elspeth Payne Christopher Hall
Hindawi Publishing Corporation Advances in Hematology Volume 2012, Article ID 596925, 9 pages doi:10.1155/2012/596925
of Molecular Biomedical Sciences and Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, NC 27607, USA 2 Immunology Program, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, NC 27607, USA 3 Childrens Research Institute, Department of Pediatrics, University of South Florida College of Medicine, 140 Seventh Avenue South, St. Petersburg, FL 33701, USA 4 Immunology Program, H. Lee Mott Cancer Center and Research Institute, 12902 Magnolia Avenue, Tampa, FL 33612, USA 5 Department of Pathology and Immunology, University of Geneva School of Medicine, Rue Michel-Servet 1, 1211 Geneva 4, Switzerland 6 Department of Cellular and Molecular Medicine and Section of Cell and Developmental Biology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0380, USA 7 Department of Molecular Genetics, All Childrens Hospital, 501 Sixth Avenue South, St. Petersburg, FL 33701, USA Correspondence should be addressed to Jerey A. Yoder, je [email protected] Received 23 March 2012; Revised 13 June 2012; Accepted 27 June 2012 Academic Editor: Christopher Hall Copyright 2012 Radhika N. Shah et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The novel immune-type receptors (NITRs), which have been described in numerous bony sh species, are encoded by multigene families of inhibitory and activating receptors and are predicted to be functional orthologs to the mammalian natural killer cell receptors (NKRs). Within the zebrash NITR family, nitr9 is the only gene predicted to encode an activating receptor. However, alternative RNA splicing generates three distinct nitr9 transcripts, each of which encodes a dierent isoform. Although nitr9 transcripts have been detected in zebrash lymphocytes, the specic hematopoietic lineage(s) that expresses Nitr9 remains to be determined. In an eort to better understand the role of NITRs in zebrash immunity, anti-Nitr9 monoclonal antibodies were generated and evaluated for the ability to recognize the three Nitr9 isoforms. The application of these antibodies to ow cytometry should prove to be useful for identifying the specic lymphocyte lineages that express Nitr9 and may permit the isolation of Nitr9-expressing cells that can be directly assessed for cytotoxic (e.g., NK) function.
1. Introduction
Mammalian natural killer (NK) cells are large, granular lymphocytes of the innate immune system that express several cell surface receptors to regulate cytotoxic function through a complex network of signaling pathways. NK cell receptors include both activating and inhibitory forms that are procient in distinguishing neoplastic or virally infected cells from normal host cells [1, 2]. The regulation of NK cell cytotoxicity is dependent on the integration of signals from activating and inhibitory receptors [3]. Although it is postulated that NK cell receptors arose early in vertebrate
phylogeny, functional data are based primarily on studies of mammalian NK cell receptors [4]. In order to appreciate the origins and evolution of NK cell receptors and their function, it is critical to dene equivalent receptor forms in nonmammalian species. The bony sh represent one of the earliest vertebrate lineages with a functional innate and adaptive immune response that closely parallels that of humans and other mammals [5]. A large multigene family of recently and rapidly evolving inhibitory and activating novel immune-type receptors (NITRs) that share structural and functional characteristics with mammalian NK cell receptors has been identied in
2 multiple sh species [6, 7]. Complete analyses of the NITR gene clusters at the sequence level only have been performed with the zebrash and medaka genomes [811]. Although transcripts of various catsh NITRs have been detected in NK-like, T, B, and macrophage cell lines [12], transcripts of all zebrash NITRs are detectable in the lymphoid, but not the myeloid, lineage [13]. Of the 39 NITR genes that have been identied within the zebrash genome, nitr9 is the only NITR gene that is predicted to encode an activating receptor [10, 11, 14]. Three alternatively spliced transcripts of nitr9 have been characterized: Nitr9-long (Nitr9L), Nitr9short (Nitr9S), and Nitr9-supershort (Nitr9SS), which dier in their extracellular domains [13, 14]. Nitr9L is the most similar to other NITRs in that it possesses two extracellular Ig domains: one of the variable (V) type and one of the intermediate (I) type [6]. Nitr9S arises through cryptic splice donor and acceptor sites within the exon encoding the V domain. Nitr9SS lacks the entire V domain exon. The transmembrane domain of all Nitr9 isoforms possesses a positively charged residue: this feature permits Nitr9L to associate with and signal through the adaptor protein Dap12 [14]. Based on protein structures, Nitr9S and Nitr9SS also are expected to signal via Dap12; however, this has not been veried experimentally. Although nitr9 transcripts have been detected in zebrash lymphocytes, the identication and recovery of Nitr9-expressing cells has not been possible. Herein we describe the derivation of two anti-Nitr9 monoclonal antibodies, demonstrate their utility to recognize recombinant Nitr9 by indirect immunouorescence, ow cytometry, and Western blot analyses, and subsequently identify all three Nitr9 isoforms in zebrash tissues by Western blot analyses. These antibodies should prove useful for: (1) evaluating Nitr9 protein levels within tissues by Western blot, (2) evaluating the distribution of Nitr9 expressing cells within tissues by indirect immunouorescence, (3) dening the specic hematopoietic lineage(s) that express Nitr9 by ow cytometry, and (4) purifying Nitr9 expressing cells by uorescence-activated cell sorting (FACS) for functional characterization.
Advances in Hematology Lymphoid and myeloid cell populations were puried from the kidney of multiple zebrash and pooled as described [16]. Total RNA from isolated cells (1 g) was reverse transcribed (SuperScript III Reverse Transcriptase). cDNAs from tissues and isolated cells were subjected to quantitative PCR (Q-PCR) with TaqMan primers and probes (Life Technologies, Carlsbad, CA) (Table 1). Q-PCR was performed on a single-color MyiQ real-time PCR detection system (Bio-Rad, Hercules, CA) using the protocol: 50C for 2 min, 95C for 10 min, followed by 55 cycles at 95C for 15 s and at 60C for 1 min. The threshold cycle (CT ) value was calculated by the iQ5 Optical System Software (Bio-Rad). Relative transcript levels of nitr9 were normalized to -actin and calculated using the 2CT method [17]. All reactions were carried out as technical triplicates. 2.3. Antibody Development and Purication. The coding sequence of the Nitr9 I domain (nucleotides 298623 of GenBank NM 001005576.1) was amplied by PCR (Table 1) and cloned into pETBlue-1 (EMD Millipore, Billerica, MA), and E. coli Tuner cells (EMD Millipore) were transformed employing a standard procedure. Cells were induced, and the Nitr9 I domain was recovered from inclusion bodies. Swiss Webster mice were immunized with the Nitr9 I domain expressed in E. coli and splenocytes were fused with P3X63Ag8.653 cells (CRL-1580, ATCC, Manassas, VA). Approximately 3,000 individual hybridoma supernatants were screened by an enzyme-linked immunosorbent assay (ELISA) against the denatured recombinant Nitr9 I domain (Immunology Core Facility, University of North Carolina, Chapel Hill). The most strongly reactive 100 supernatants in turn were screened by parallel Western blot analyses and indirect immunouorescence. Two single clones, 19.1.1 (herein referred to as anti-Nitr919 ) and 90.10.5 (herein referred to as anti-Nitr990 ), were selected for additional characterization based on their ability to recognize recombinant Nitr9. Antibody isotypes were determined (IsoStrips: Roche; Indianapolis, IN) to be IgG2b, light chain (90.10.5), and IgG2a, light chain (19.1.1). Antibodies were puried via protein A agarose columns (Upstate Cell Signaling Solutions; Lake Placid, NY). 2.4. Plasmids and Cell Culture. Nitr9 expression cassettes (without epitope tags) were constructed with pcDNA3 (Life Technologies). Epitope (FLAG)-tagged Nitr9 (FLAG-Nitr9) expression cassettes were constructed with the pLF plasmid which incorporates an amino-terminal leader sequence and FLAG epitope [14]. The coding sequences of zebrash nitr9L, nitr9S, and nitr9SS were amplied by PCR and cloned into pcDNA3 or pLF. Nitr9 and FLAG-Nitr9 cassettes were then shuttled into pIRES2-EGFP (Clontech) generating: pNitr9L/EGFP, pNitr9S/EGFP, pNitr9SS/EGFP, pFLAG-Nitr9L/EGFP, pFLAG-Nitr9S/EGFP, and pFLAGNitr9SS/EGFP plasmids (Figure 1). Primer sequences that were used in cloning steps are included in Table 1. Plasmids were transfected into human HEK293T cells using Fugene 6 (Roche) according to the manufacturers instructions and were harvested 48 hr after transfection.
Advances in Hematology
Table 1: Oligonucleotide primer sequences. Purpose Reverse transcriptasePCR: nitr9 Reverse transcriptasePCR: -actin TaqMan Q-PCR: nitr9 (probe = CAAGGTTTGGAAAAGCAC) TaqMan Q-PCR: -actin (probe = CCCATGCCATCCTGC) Amplify nitr9 I domain for bacterial expression construct Amplify nitr9L for FLAG-tagged expression cassette Amplify nitr9S for FLAG-tagged expression cassette Amplify nitr9SS for FLAG-tagged expression cassette Amplify wild type nitr9L, nitr9S and nitr9SS for expression cassettes
a An
Primer sequence GGATTTTTGGACTTTTCTGTC TCCACATGCGGTAACTGTAC GGTATGGAATCTTGCGGTATCCAC ATGGGCCAGACTCATCGTACTCCT GTCAAAGGGACAAGGCTGATAGTT GTTCAAAACAGTGCATGTAAGACTCA CCATCTATGAGGGTTACGCTCTTC AGGATCTTCATCAGGTAGTCTGTCA ATGGAAAAGCACACTGTAGTAa TTATTTAGAGCCATTCCTGTCCb CACCCAAATGCACCACCTGTGTTTGTTAAACc gactgcggccgcTTACTGCTGGTTAGAAACd CACCCAAATGCACCACCTGTGc gactgcggccgcTTACTGCTGGTTAGAAACd CATGATTTAATTCCATCCCAc gactgcggccgcTTACTGCTGGTTAGAAACd gatcggatccgacATGATCAACTTTTGGATTTe gatcgaattcTTACTGCTGGTTAGAAACCGAGf
articial start codon is underlined. articial stop codon is bold. c These primers are designed for blunt PCR cloning into the EcoRV site of pLF. d Overhang (5 ) sequences are in lower case text and include a Not I site for cloning into pLF. e Overhang (5 ) sequences are in lower case text and include a BamHI site for cloning into pcDNA3. f Overhang (3 ) sequences are in lower case text and include an EcoRI site for cloning into pcDNA3.
b An
2.5. Indirect Immunouorescence. HEK293T cells were transfected in four well chamber slides (Thermo Fisher Scientic, Rochester, NY). Transfected cells were washed in phosphate buered saline (PBS), xed with 3% paraformaldehyde for 20 min and treated with 50 mM NH4 Cl, PBS for 5 minutes. Cells were then permeabilized with 1.0% Triton-X-100 in PBS for 5 min, rinsed and blocked with 1% BSA in PBS for 5 min. Permeabilized cells were incubated with the antiNitr919 , anti-Nitr990 , or anti-FLAG antibody for 1 hr, rinsed with PBS, incubated with a phycoerythrin (PE) anti-mouse IgG antibody and DAPI (1 : 1000) for 1 hr, and washed with PBS. Chambers were removed from the slides, and coverslips were mounted using immunomount (Thermo Shandon, Pittsburgh, PA). Cells were photographed at 40x magnication using a Leica DM5000 microscope. 2.6. Flow Cytometry. Transfected HEK293T cells were incubated with the anti-Nitr919 , anti-Nitr990 , or anti-FLAG monoclonal antibody for 1 hr, washed in PBS, and incubated for 30 min with an allophycocyanin- (APC-) conjugated antimouse IgG secondary antibody. Labeled cells were washed and then xed with 3% paraformaldehyde and subjected to ow cytometric analysis (BD FACSCalibur, BD Biosciences, San Jose, CA).
2.7. Western Analyses. Transfected HEK293T cells were washed with PBS and lysed with mammalian protein extraction reagent (M-PER, Pierce, Rockford, IL). Kidney, spleen, intestine, and gills were removed from sacriced adult zebrash and collected directly into tissue protein extraction reagent (T-PER, Pierce) supplemented with protease inhibitors (Pierce) and homogenized. Lysates were centrifuged to remove nuclei, and cell debris and protein concentrations were determined (BCA Protein Assay, Pierce). Proteins were resolved on 12% SDS-polyacrylamide gels and transferred to polyvinylidene diuoride (PVDF) membranes for Western analyses. Membranes were washed in Trisbuered saline with 0.1% Tween 20 (TBST) and incubated in blocking buer (100 mM boric acid, 25 mM Na-Borate, 75 mM NaCl, 5% goat serum, and 5% dry milk powder) for 1 hr. Membranes were incubated overnight with primary antibodies in blocking buer at 4 C. Primary antibodies include anti-Nitr990 , anti-FLAG (M2) mouse monoclonal antibody (Sigma-Aldrich, St. Louis, MO), anti-GFP mouse monoclonal antibody (Roche), and anti-GAPDH rabbit polyclonal antibody (AnaSpec, Fremont, CA). Membranes were washed in TBST, followed by incubation with blocking buer and either horseradish peroxidase-conjugated antimouse IgG secondary antibody (Roche) or horseradish
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Advances in Hematology
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Figure 1: Transcriptional variation and expression of Nitr9. (a) Exon organization of the nitr9 gene depicting the three transcript variants. Primer positions for PCR are indicated below. (b) The predicted Nitr9 protein isoforms encoded by the three nitr9 transcripts. Transmembrane (TM) and immunoglobulin domains (of the variable (V) and intermediate (I) types) of Nitr9 are indicated. The I domain of Nitr9 was used as the antigen for antibody production. The positive charge within the TM domain of Nitr9 is represented by a plus sign. (c) RT-PCR with primers whose positions are depicted in (a) detects transcripts of all three nitr9 isoforms. (d) Quantitative RT-PCR with nitr9 primers (Table 1), whose positions are depicted in (a), and a TaqMan probe that spans an exon-exon boundary reveal relative levels of nitr9L/S transcripts in dierent tissues. (e) Schematic representation of the recombinant Nitr9 expression constructs used in this paper. Constructs include an internal ribosomal entry sequence (IRES2) permitting the expression of two proteins from a single transcript.
peroxidase-conjugated anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). After washing with TBST, the Lumi-LightPLUS western blotting substrate and detection system (Roche) was used to visualize reactivity. 2.8. Endoglycosidase Treatment. Cleared lysates (20 g) from transfected cells were incubated with N-Glycosidase F (PNGase F, New England Biolabs, Ipswich, MA) for 1 hr at 37 C. Cleared lysates (25 g) from zebrash tissues were
precipitated with OrgoSOL buer (G-Biosciences, St. Louis, MO) and resuspended in PNGase buer for treatment with PNGase F.
Advances in Hematology
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Figure 2: Detection of FLAG-tagged isoforms of Nitr9 from transfected cells by indirect immunouorescence. HEK293T cells were transfected with plasmids encoding FLAG-tagged Nitr9 isoforms and EGFP as indicated on top of the panels. FLAG-tagged Nitr9 proteins were detected with (a) an anti-FLAG antibody, (b) anti-Nitr990 or (c) anti-Nitr919 , and a PE conjugated secondary antibody (red). Transfected cells can be identied by EGFP expression (green). DAPI labels the nuclei of all cells (blue). The pIRES2-EGFP parental plasmid was included as a negative control.
encode type I transmembrane cell surface receptors that possess a positively charged residue within the transmembrane domain. The nitr9S isoform is expressed at higher levels in the zebrash spleen, kidney, and intestine than the nitr9L and nitr9SS isoforms, whereas, nitr9L transcripts are the most abundant isoform expressed in gills. Transcripts of nitr9SS are detected in all four tissues at reduced levels relative to the other isoforms (Figure 1(c)). Q-PCR (Table 1) was employed to determine the combined relative levels of nitr9L and nitr9S transcripts in these same tissues as well as in puried lymphoid and myeloid cells (the TaqMan primer/probe set employed in this paper does not detect nitr9SS transcripts). The combined relative expression level of nitr9L and nitr9S transcripts is consistently higher in intestine than in kidney and gill (Figure 1(d)). However, the relative expression level of nitr9L and nitr9S in spleen varied between biological replicates, ranging from levels matching those in intestine to lower levels as observed in kidney and gill. As reported previously, nitr9 transcripts are present at much higher levels in zebrash lymphocytes as compared to myeloid cells [13]. In order to generate monoclonal antibodies that could detect all three Nitr9 isoforms, mice were immunized with a bacterially expressed Nitr9 I domain (see Figure 1(b)), and hybridomas were screened for the production of antibodies that recognize recombinant Nitr9 by ELISA, Western blot and indirect immunouorescence. Two clones, 19.1.1 (herein referred to as anti-Nitr919 ) and 90.10.5 (herein referred to as anti-Nitr990 ), were selected for further evaluation.
3.2. Detection of Nitr9 Isoforms in Transfected Cells by Indirect Immunouorescence. In order to determine if antiNitr919 and anti-Nitr990 could detect all three isoforms of Nitr9 by indirect immunouorescence, HEK293T cells were transfected with plasmids that coexpress EGFP and either a FLAG-tagged or endogenous isoform of Nitr9; in this way, any cell expressing Nitr9 also expresses EGFP (Figure 1(e)). To ensure that the recombinant Nitr9 proteins could be detected by immunouorescence, an anti-FLAG antibody was used to detect all three FLAG-tagged isoforms of Nitr9 in transfected cells (Figure 2(a)). It was then shown that both anti-Nitr919 and anti-Nitr990 recognize FLAG-Nitr9L and FLAG-Nitr9S by immunouorescence, but either fail to bind (anti-Nitr990 ) or bind less eectively (anti-Nitr919 ) to FLAG-Nitr9SS (Figures 2(b) and 2(c)). In contrast, both anti-Nitr919 and anti-Nitr990 eectively recognize all three isoforms of endogenous Nitr9 when expressed in transfected cells albeit with an apparent higher background labeling of cells with anti-Nitr919 (Figure 3). It is possible that the FLAGtag disrupts folding of Nitr9SS or sterically interferes with antibody recognition of the I domain of FLAG-Nitr9SS; this also was observed with Western analyses (discussed below). 3.3. Detection of Nitr9 Isoforms in Transfected Cells by Flow Cytometry. In order to determine if anti-Nitr919 and antiNitr990 could detect all three isoforms of Nitr9 by ow cytometry, HEK293T cells were transfected with plasmids encoding an endogenous or FLAG-tagged isoform of Nitr9
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Advances in Hematology
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Figure 3: Detection of endogenous isoforms of Nitr9 from transfected cells by indirect immunouorescence. HEK293T cells were transfected with plasmids encoding endogenous isoforms of Nitr9 and EGFP as indicated on top of the panels. Nitr9 proteins were detected with (a) antiNitr990 or (b) anti-Nitr919 and a PE conjugated secondary antibody (red). Transfected cells can be identied by EGFP expression (green). DAPI labels the nuclei of all cells (blue).
and EGFP (Figure 1(e)). Flow cytometry was performed using the anti-FLAG, anti-Nitr919 , and anti-Nitr990 antibodies to detect Nitr9 expressing cells. The percentage of double positive FLAG-Nitr9L expressing cells (i.e., EGFP+ and Nitr9+ ) was similar (5563% of EGFP+ cells) when the anti-FLAG or the anti-Nitr9 antibodies were employed (Figure 4(a)). Both anti-Nitr9 antibodies recognize transfected cells expressing the endogenous isoform of Nitr9L with a similar eciency (6173% of EGFP+ cells) (Figure 4(b)). The anti-FLAG monoclonal antibody failed to bind FLAG-Nitr9S and the anti-Nitr919 antibody failed to detect the Nitr9S- or FLAG-Nitr9S-expressing cells (29% of EGFP+ cells) (Figures 4(c) and 4(d)). Although the antiNitr990 antibody detects FLAG-Nitr9S (31% of EGFP+ cells), it does not recognize endogenous Nitr9S (5% of EGFP+ cells). Although the Nitr9S and FLAG-Nitr9S proteins are produced by transfected cells (see Figures 2 and 3 and Western blot results below) they may not be expressed eectively on the cell surface. To determine if cell surface expression of Nitr9S requires coexpression of the signaling adaptor protein Dap12, cells were cotransfected with plasmids encoding Nitr9S and zebrash Dap12. No increase was observed in cell surface labeling by the anti-Nitr9 antibodies (data not shown). The anti-FLAG and anti-Nitr919 antibodies eectively bound FLAG-Nitr9SS (57% and 31% of EGFP+ cells, resp.). The anti-Nitr990 antibody failed to bind FLAG-Nitr9SS, possibly due to steric hindrance by the FLAG tag (Figure 4(e)) since both anti-Nitr9 antibodies were eective at recognizing Nitr9SS (6575% of EGFP+ cells; Figure 4(f)). 3.4. Anti-Nitr990 Binds All Three Isoforms of Nitr9 in Western Analyses. In order to evaluate the ability of the anti-Nitr990 antibody to detect the three isoforms of Nitr9 in Western
analyses, HEK293T cells were transfected with plasmids encoding endogenous and FLAG-tagged isoforms of Nitr9 (Figure 1(e)). Cell lysates were subjected to Western blot analyses using the anti-Nitr990 antibody. All three isoforms of the endogenous Nitr9 as well as the FLAG-tagged Nitr9L and Nitr9S proteins were detected. A binding pattern equivalent to that seen with the anti-FLAG monoclonal antibody positive control is apparent (Figure 5(a)). However, anti-Nitr990 failed to bind the FLAG-tagged Nitr9SS. As mentioned above, this may be a result of the FLAG-tag blocking access to the specic epitope recognized by this antibody. Two proteins bands were detected by anti-Nitr990 in both endogenous and FLAG-tagged Nitr9L and Nitr9S transfections that were also bound by the anti-FLAG antibody. Both observed Nitr9L proteins migrated at a higher molecular weight than the predicted size of Nitr9L (34 kD), and one of the observed Nitr9S proteins was larger than the predicted size of Nitr9S (30 kD). The dierences are consistent with dierential glycosylation (see below). Based on the chemiluminescence exposure times required for detecting the dierent isoforms of Nitr9, anti-Nitr990 appears to exhibit a higher anity for Nitr9L as compared to Nitr9S and Nitr9SS. In parallel experiments, the anti-Nitr919 antibody did not bind endogenous Nitr9S and Nitr9SS proteins (data not shown) and was not characterized further in the Western blot analyses. 3.5. Nitr9 Glycosylation in Transfected Cells. Nitr9L, Nitr9S and Nitr9SS possess three (NMSC, NDSR, and NGSK), two (NMSC and NGSK), and one (NGSK) candidate Nlinked glycosylation sites, respectively. Treatment of lysates from Nitr9 transfected cells with endoglycosidase (PNGase F) results in the detection of only a single protein of
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Figure 4: Detection of Nitr9 isoforms by ow cytometry. HEK293T cells were transfected with plasmids encoding FLAG-tagged (a, c, and e) or endogenous (b, d, and f) isoforms of Nitr9 and EGFP as indicated above the panels. Cells were labeled with an anti-FLAG antibody (top row), anti-Nitr990 (middle row) or anti-Nitr919 (bottom row), and an APC conjugated secondary antibody. Flow cytometric analyses were employed to detect EGFP positive (X axis) and APC positive (Y axis) cells. Isotype-matched antibodies were evaluated as controls for both anti-Nitr9 antibodies and displayed no labeling of transfected cells (data not shown).
the expected size for both Nitr9L and Nitr9S (Figure 5(b)). Both sets of results are consistent with in vivo glycosylation. The observed size of Nitr9SS in transfected cells does not appear to be altered by endoglycosidase treatment, with the limitations of detection, suggesting that it may not be glycosylated. 3.6. Nitr9 Proteins Are Dierentially Expressed in Dierent Tissues of Zebrash. In order to determine if the anti-Nitr990 antibody can recognize endogenous Nitr9, lysates from adult zebrash tissues were treated with endoglycosidase and subjected to Western blot analyses (Figure 5(c)). Nitr9L and Nitr9S were detected at varying levels in the spleen, kidney, gills, and intestine. Nitr9SS was detected only in the spleen, although faint bands also have been observed in intestine (data not shown). A nonspecic band of approximately 28 kD is detected in zebrash tissues as well as in HEK293T cells when the anti-Nitr990 antibody is used with large total protein loads (e.g., 25 g lysate; Figure 5(c)).
4. Conclusions
Three dierent transcript variants from nitr9, the single putative activating NITR gene in zebrash, and their corresponding protein isoforms have been identied and characterized. The utility of the anti-Nitr919 and anti-Nitr990 monoclonal antibodies for detecting recombinant Nitr9 was demonstrated by indirect immunouorescence, ow
cytometry, and Western blot analyses. The antibodies exhibit profound dierences in recognizing the three dierent Nitr9 isoforms. When employed for indirect immunouorescence, both anti-Nitr9 antibodies bound eciently and specically to cells-expressing all three Nitr9 isoforms. Both anti-Nitr9 antibodies are eective for detecting cell surface expression of Nitr9L and Nitr9SS by ow cytometry. The anti-Nitr990 antibody recognized all three Nitr9 isoforms by Western blot analyses, although a higher anity for Nitr9L is noted. When using anti-Nitr990 in Western blot analyses with high levels of protein, a nonspecic band was identied. Although the identity of this protein remains unknown, it may represent a well-conserved member of the Ig superfamily. Marked dierences in the relative levels of Nitr9 transcripts and protein isoforms are apparent. Although the PCR analyses (Figure 1(c)) suggest that nitr9S may be the predominant mRNA isoform in spleen, kidney, and intestine, Western analyses demonstrate that Nitr9L is the predominant protein isoform expressed in kidney. This discrepancy may reect diering transcript and protein stability in dierent tissues or the preferred reactivity of the antibody with Nitr9L (Figure 5(a)). The monoclonal antibodies described here should be useful for further evaluation of Nitr9 protein levels in zebrash tissues by Western blot analyses and identifying Nitr9 expressing cells in tissue sections by indirect immunouorescence. Eorts are underway to purify Nitr9expressing zebrash cells employing FACS in order to characterize their morphology and cytotoxic properties.
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Figure 5: Detection of Nitr9 protein by Western analyses. (a) Western blot analyses of total protein lysates from HEK293T cells transiently transfected with plasmids expressing a Nitr9 isoform and EGFP. Plasmids encode either an endogenous isoform of Nitr9 or a FLAG-tagged Nitr9 as indicated above each lane. The primary antibodies utilized are shown on the left, and the molecular weights of identied bands are shown on the right. The anti-FLAG antibody serves as a positive control for Nitr9 detection, and the anti-GFP antibody indicates transfection eciency of each plasmid. Note the total protein loaded (bottom) for the Nitr9L isoform is ten times less than that for Nitr9S and Nitr9SS plasmids. Exposure times for chemiluminescence detection are indicated in each panel. (b) Nitr9L and Nitr9S are glycosylated. Western blot analyses of endoglycosidase-treated total protein lysates from HEK293T cells that were transfected with plasmids encoding endogenous Nitr9 isoforms. The anti-Nitr990 antibody recognizes all three Nitr9 isoforms at the predicted size (right). (c) Detection of Nitr9 protein from zebrash tissues. Western blot analyses of 25 g of endoglycosidase-treated total protein from zebrash tissues and HEK293T cells. Note that a nonspecic band (28 kD) is detected in HEK293T cells as well as in zebrash kidney and spleen, with high protein loads. Bottom panel indicates loading control using an anti-GAPDH polyclonal antibody.
These antibodies may also prove to be useful for activating (crosslinking) or blocking Nitr9 function in both cell culture and ex-vivo functional assays as well as in dissecting isoformspecic functions of NITRs.
Barb Pryor for editorial assistance. This paper was supported by grants awarded by the National Science Foundation (MCB-0505585 to J. A. Yoder) and the National Institutes of Health (R01 AI057559 to G. W. Litman and J. A. Yoder).
Acknowledgments
The authors are grateful to Bradley Bone and Karen Marcus for assistance with generating hybridomas and performing ELISAs, Janet Dow for assistance with ow cytometry, and
References
[1] R. Biassoni, Human natural killer receptors, co-receptors, and their ligands, Current Protocols in Immunology, chapter 14, unit 14.10, 2009.
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- 34 (Nitr9L) - 30 (Nitr9S)
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- pIRES2-EGFP
- 34 - 30
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[2] L. L. Lanier, Up on the tightrope: natural killer cell activation and inhibition, Nature Immunology, vol. 9, no. 5, pp. 495502, 2008. [3] L. L. Lanier, NK cell recognition, Annual Review of Immunology, vol. 23, pp. 225274, 2005. [4] J. A. Yoder and G. W. Litman, The phylogenetic origins of natural killer receptors and recognition: relationships, possibilities, and realities, Immunogenetics, vol. 63, no. 3, pp. 123141, 2011. [5] G. W. Litman, J. P. Cannon, and L. J. Dishaw, Reconstructing immune phylogeny: new perspectives, Nature Reviews Immunology, vol. 5, no. 11, pp. 866879, 2005. [6] J. A. Yoder, Form, function and phylogenetics of NITRs in bony sh, Developmental and Comparative Immunology, vol. 33, no. 2, pp. 135144, 2009. [7] S. Ferraresso, H. Kuhl, M. Milan et al., Identication and characterisation of a novel immune-type receptor (NITR) gene cluster in the European sea bass, Dicentrarchus labrax, reveals recurrent gene expansion and diversication by positive selection, Immunogenetics, vol. 61, no. 11-12, pp. 773 788, 2009. [8] S. Desai, A. K. Heelnger, T. M. Orcutt, G. W. Litman, and J. A. Yoder, The medaka novel immune-type receptor (NITR) gene clusters reveal an extraordinary degree of divergence in variable domains, BMC Evolutionary Biology, vol. 8, no. 1, article 177, 2008. [9] J. A. Yoder, M. G. Mueller, S. Wei et al., Immune-type receptor genes in zebrash share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster, Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 12, pp. 67716776, 2001. [10] J. A. Yoder, R. T. Litman, M. G. Mueller et al., Resolution of the novel immune-type receptor gene cluster in zebrash, Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 44, pp. 1570615711, 2004. [11] J. A. Yoder, J. P. Cannon, R. T. Litman, C. Murphy, J. L. Freeman, and G. W. Litman, Evidence for a transposition event in a second NITR gene cluster in zebrash, Immunogenetics, vol. 60, no. 5, pp. 257265, 2008. [12] J. Evenhuis, E. Bengt en, C. Snell, S. M. Quiniou, N. W. Miller, and M. Wilson, Characterization of additional novel immune type receptors in channel catsh, Ictalurus punctatus, Immunogenetics, vol. 59, no. 8, pp. 661671, 2007. [13] J. A. Yoder, P. M. Turner, P. D. Wright et al., Developmental and tissue-specic expression of NITRs, Immunogenetics, vol. 62, no. 2, pp. 117122, 2010. [14] S. Wei, J.-M. Zhou, X. Chen et al., The zebrash activating immune receptor Nitr9 signals via Dap12, Immunogenetics, vol. 59, no. 10, pp. 813821, 2007. [15] D. D. Jima, R. N. Shah, T. M. Orcutt et al., Enhanced transcription of complement and coagulation genes in the absence of adaptive immunity, Molecular Immunology, vol. 46, no. 7, pp. 15051516, 2009. [16] J. A. Yoder, T. M. Orcutt, D. Traver, and G. W. Litman, Structural characteristics of zebrash orthologs of adaptor molecules that associate with transmembrane immune receptors, Gene, vol. 401, no. 1-2, pp. 154164, 2007. [17] K. J. Livak and T. D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2CT method, Methods, vol. 25, no. 4, pp. 402408, 2001.
Hindawi Publishing Corporation Advances in Hematology Volume 2012, Article ID 214209, 9 pages doi:10.1155/2012/214209
Research Article Characterization of Zebrash von Willebrand Factor Reveals Conservation of Domain Structure, Multimerization, and Intracellular Storage
Arunima Ghosh,1 Andy Vo,2 Beverly K. Twiss,2 Colin A. Kretz,1 Mary A. Jozwiak,3 Robert R. Montgomery,3 and Jordan A. Shavit2
1 Life
Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA of Pediatrics, University of Michigan, Room 8301 Medical Science Research Building III, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-5646, USA 3 Blood Research Institute, Medical College of Wisconsin, Milwaukee, WI 53226, USA
2 Department
Correspondence should be addressed to Jordan A. Shavit, [email protected] Received 20 April 2012; Revised 18 June 2012; Accepted 26 July 2012 Academic Editor: Elspeth Payne Copyright 2012 Arunima Ghosh et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. von Willebrand disease (VWD) is the most common inherited human bleeding disorder and is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWF is a secreted glycoprotein that circulates as large multimers. While reduced VWF is associated with bleeding, elevations in overall level or multimer size are implicated in thrombosis. The zebrash is a powerful genetic model in which the hemostatic system is well conserved with mammals. The ability of this organism to generate thousands of ospring and its optical transparency make it unique and complementary to mammalian models of hemostasis. Previously, partial clones of zebrash vwf have been identied, and some functional conservation has been demonstrated. In this paper we clone the complete zebrash vwf cDNA and show that there is conservation of domain structure. Recombinant zebrash Vwf forms large multimers and pseudo-Weibel-Palade bodies (WPBs) in cell culture. Larval expression is in the pharyngeal arches, yolk sac, and intestinal epithelium. These results provide a foundation for continued study of zebrash Vwf that may further our understanding of the mechanisms of VWD.
1. Introduction
Vertebrates possess a complex closed circulatory system that requires balanced coordination of various factors that serve to maintain blood ow as well as prevent exsanguination when the system is breached. This is known as hemostasis and consists of a complex array of cellular elements, as well as a network of proteins known as the coagulation cascade. The latter have been highly conserved at the genomic level throughout vertebrate evolution, including mammals, birds, reptiles, and sh [13]. One of the central components of coagulation is von Willebrand factor (VWF), deciencies of which are the basis for the bleeding disorder von Willebrand disease (VWD). The mammalian VWF gene consists of 52 exons, and the
largest, exon 28, contains several functional domains that are frequently mutated in VWD [4]. VWF is a 260 kDa (kilodalton) secreted glycoprotein that assembles into multimers of over 10,000 kDa [5]. At sites of injury, high molecular weight VWF multimers bind to receptors in the vascular subendothelium and tether platelets to form the primary hemostatic plug [6]. Much of our knowledge of VWF function is derived from characterization of mutations in humans and various mammalian model organisms, including mouse, dog, horse, cat, pig, and rabbit [7, 8]. However, relatively little information is available in other vertebrate models, such as the teleost Danio rerio (zebrash). Teleost sh possess highly conserved orthologs of nearly all blood coagulation factors [1, 3] and have been shown to develop thrombosis in response to a laser-induced injury
2 [9]. Zebrash embryonic development is external, rapid, and transparent, greatly simplifying phenotypic screening. Circulation begins approximately 24 hours after fertilization, and vascular development has been well characterized [10]. Forward genetic screens with chemical mutagenesis have been performed to study cardiogenesis, vasculogenesis, and angiogenesis [1114]. Recently exon 28 was cloned from zebrash, and conservation of several VWF functions was demonstrated [15], and in silico assembly of full length zebrash vwf has also been described [16]. We now report cloning and characterization of the full length zebrash vwf cDNA. Zebrash Vwf demonstrates conservation of primary human VWF domain structure, as well as the ability to form pseudo-WeibelPalade bodies (WPBs) and large multimers in cell culture. Unlike mammalian species, at the stages examined it does not appear to be expressed widely in developing endothelium.
Advances in Hematology pi-zVwf-EGFP was constructed by inserting the vwf cDNA into Tol2-i-EGFP [20] in frame with egfp. 2.4. Immunouorescence Analysis. HEK293T cells were maintained in DMEM (Sigma; St Louis, MO) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin (Sigma). Cells were grown on cover slips until they reached 5080% conuence, followed by transfection using FuGENE (Roche, Penzberg, Germany) as per manufacturers instructions. The transfected cover slips were washed in phosphate buered saline (PBS) and xed in 10% formalin at room temperature for 25 minutes, followed by xation/permeabilization at 4 C for 10 minutes in 100% ice cold methanol. After rehydration in PBS for 5 minutes, the cells were incubated with mouse anti-Myc (Santa Cruz Biotechnology, Santa Cruz, California) and rabbit anti-calnexin (Novus Biologicals, Littleton, Colorado) antibodies at dilutions of 1 : 100 and 1 : 500, respectively, at 4 C overnight. Cells were then washed three times in PBS (5 minutes each) and incubated with goat anti-mouse antibody coupled to Alexa Fluor 488 and goat anti-rabbit antibody coupled to Alexa Fluor 594, both at 1 : 200 dilutions for 60 minutes at room temperature. After an additional three washes in PBS, the cover slips were mounted with Prolong Antifade Gold (Invitrogen) and viewed on an inverted Olympus (Melville, New York) confocal microscope. Processing was completed with Olympus FluoView version 5.0. 2.5. Vwf Multimer Analysis. HEK293T (human embryonic kidney) cells were cultured and transfected with pzVwf/V5HISA or an untagged full length human VWF expressing plasmid (pCineoVWF), as previously described [21]. Conditioned medium from pzVwf/V5-HISA transfected cells was puried over nickel columns per manufacturers instructions (GE Healthcare Life Sciences, Uppsala, Sweden). Supernatants were analyzed by electrophoresis through a 0.8% (w/v) HGT(P) agarose (FMC Bioproducts, Rockland, Maine) stacking gel and a 1.5% (w/v) HGT(P) agarose running gel containing 0.1% sodium dodecyl sulfate for 16 hours at 40 volts using the Laemmli buer system and western blotting as previously described [21]. Primary antibodies were a 1 : 5 mixture of anti-V5 antibody (Invitrogen) and anti-HIS antibody (AbD Serotec, Oxford, United Kingdom) or a mixture of monoclonal anti-human VWF antibodies Avw1, 5, and 15 [22]. 2.6. Maintenance of Zebrash Lines and Production of Embryos. Adult zebrash (AB, TL, EK) were maintained and bred according to standard methods [23]. Embryos collected immediately after fertilization were maintained at 28.5 C and treated with 1-phenyl-2-thiourea (PTU) at 68 hpf (hours post fertilization) until xation in order to prevent pigment formation. At specic time points, embryos were dechorionated or euthanized with tricaine, xed using 4% paraformaldehyde in PBS overnight at 4 C, and stored at 20 C in methanol up to one month [24]. 2.7. RNA Isolation and cDNA Synthesis for RT-PCR of Embryos and Larvae. Total RNA was extracted from at least three
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Table 1: List of primers and sequences. Sequence AGTCGGCAGCACATACACAC ATCCGGACAGGTCAGTTCAC CCTGCAGCTTAAACCCAAAG AAAGCTTCATCGTCCAGCTC CTGTTGACGGCAAGTGCTAA TCTCCTGATGCTGGACACAC GACGGCAGTGTAACGACAGA CCTGCAAGAGAGCCGATAAC TGCGTGCTGAATCAAACTGT AGTCGCCAGGGAATTCATAA TTTGATTGACATTTTTATTTATTGTAGTTTA gatttaggtgacactatagCGACATGCAAGTGCAGAAGT taatacgactcactatagggGCTGGGTTTTGCTGTAGGAG gatttaggtgacactatagGGAGTTATCGGCTCTCTTGC taatacgactcactatagggACACAGACTTGCTGCCACAC Description vwf cloning, assembly of fragment 1 (EcoRI-BstBI) vwf cloning, assembly of fragment 1 (EcoRI-BstBI) vwf cloning, assembly of fragment 2 (BstBI-AvaI) vwf cloning, assembly of fragment 2 (BstBI-AvaI) vwf cloning, assembly of fragment 3 (AvaI-SbfI) vwf cloning, assembly of fragment 3 (AvaI-SbfI) vwf cloning, assembly of fragment 4 (SbfI-ApaLI) vwf cloning, assembly of fragment 4 (SbfI-ApaLI) vwf cloning, 3 RACE (ApaLI-SpeI), SpeI vector derived vwf cloning, 5 RACE (NotI-EcoRI), NotI vector derived vwf cloning, amplication of 3 UTR 424 bp vwf riboprobe (exon 28) with SP6 promoter overhang 424 bp vwf riboprobe (exon 28) with T7 promoter overhang 441 bp vwf riboprobe (exons 4752) with SP6 promoter overhang 441 bp vwf riboprobe (exons 4752) with T7 promoter overhang
4 biological replicates per experimental condition using TRIzol RNA isolation reagent (Invitrogen) according to the manufacturers instructions. RNA (1 g) was reverse-transcribed using random hexamers and SuperScript III reverse transcriptase (Invitrogen). First-strand cDNA aliquots from each sample served as templates in PCR reactions using primers for vwf. 2.8. In Situ Hybridization. In situ hybridization was performed essentially as described with a few modications [24]. Full length vwf cDNA in pCR4-TOPO was linearized with NotI and SpeI (antisense and sense transcripts, respectively) and transcribed in vitro using T3 and T7 (Ambion, Austin, Texas), respectively, with digoxigenin labeled nucleotides followed by alkaline hydrolysis per manufacturers instructions (Roche). Alternatively, 424 and 441 bp fragments were amplied from full length cDNA using primers with SP6 or T7 overhangs (Table 1) and transcribed in vitro with digoxigenin labeled nucleotides. Prior to hybridization, riboprobes were heated to 80 C for 35 minutes and chilled immediately on ice for at least 5 minutes. Stained embryos were photographed using a Leica MXFLIII stereouorescent microscope with an Olympus DP-70 digital camera. Embedding was in JB-4 resin as described [25], followed by sectioning at 46 m using a Leica RM2265 ultramicrotome. Imaging of sections was with an Olympus BX-51 upright light microscope and Olympus DP-70 high-resolution digital camera.
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Table 2: Human/zebrash Vwf domain conservation. Domain D1 D2 D D3 A1 A2 A3 D4 B1 B2 B3 C1 C2 CK Total Identities (%) 51 64 51 56 36 28 42 39 58 52 67 50 48 42 46 Positives (%) 70 79 71 69 57 56 58 54 73 64 83 58 63 64 62 Human length 352 360 90 376 220 193 202 372 35 26 25 116 119 90 2813 zebrash length 351 359 88 370 233 193 207 382 34 30 25 107 117 91 2812
Alignment of human and zebrash amino acid sequences using BLAST (http://blast.ncbi.nlm.nih.gov/). Percentage identity represents exact amino acid matches, while positives indicate conserved substitutions. Domain length is in amino acids.
3. Results
3.1. Cloning and Characterization of Zebrash vwf cDNA. According to genomic sequence, the zebrash vwf locus is located on chromosome 18 just downstream of cd9, maintaining conservation of synteny with mammalian species [15]. The full length vwf cDNA was assembled by RT-PCR of four overlapping fragments from total adult zebrash cDNA, followed by RACE to complete the 5 and 3 UTRs (Section 2). The full length sequence is one amino acid shorter than human VWF with 46% overall identity (Table 2). Alignment of zebrash Vwf to human VWF using BLAST shows clear delineation of all known domains (Figure 1(a)) with varying degrees of conservation (Table 2). The least conserved are the A1 and A2 domains, which encompass the entirety of exon 28 (Table 2). As in mammals, the vwf locus consists of 52 exons, but only spans 81 kb (kilobases), as opposed to 176 kb and 134 kb in the human and murine genomes, respectively. Previous iterations of the zebrash genome (prior to Zv7) predicted that exon 28 was split into two exons [17]. Both sequence data from this report and previous work [15, 16] demonstrate clearly that the intervening sequence is actually exonic. Other key features of human VWF are identiable with varying degrees of conservation. The propeptide cleavage site, Arg-Ser, is highly conserved across all species examined except for medaka, and is a part of the extended RX(R/K)R motif (Figure 1(b)) [26]. The putative ADAMTS13 cleavage site in the A2 domain, Phe-Leu, is discernible due to mammalian orthology of anking residues and is conserved across
all sh species (Figure 1(c)). However, the presumed PheLeu cleavage site is only somewhat similar to the highly conserved mammalian and avian Tyr-Met cleavage sequence (Figure 1(c)). More importantly, there is conservation of a leucine orthologous to human Leu1603 (Figure 1(c)), which has been shown to be critical for ADAMTS13-mediated proteolysis of VWF [27]. A number of disulde bonds are required for dimerization and multimerization of human VWF [6]. These are mediated by cysteines at positions 1099, 1142, and several in the C-terminal cystine knot (CK), at 2771, 2773, and 2811, all of which are conserved in zebrash Vwf. In fact, nearly all cysteine residues are completely conserved, with the exception of Cys1669 and Cys1670 , located at the Cterminus of the A2 domain [16] and absent in all sh species examined. There was one cysteine present solely in medaka, four residues N-terminal to the propeptide cleavage site, but its absence in other species makes its signicance unclear. There is a cysteine in zebrash Vwf at position 4, which is not conserved in mammalian species, although genomic sequence information for the other teleost species is absent in this region. 3.2. Expression of Vwf in Mammalian Cell Culture. In order to determine if zebrash Vwf can multimerize, we expressed V5/HIS tagged vwf cDNA in HEK293T cells. A ladder of high molecular weight multimers was detected using a mixture of anti-V5 and anti-HIS antibodies (Figure 2). This included high molecular weight multimers similar in size to human VWF (Figure 2). The zebrash vwf cDNA was cloned into an expression vector in frame with a Myc-HIS tag using the same linker as a human VWF cDNA construct. The latter, when transfected into HEK293T cells, is known to form pseudo-WPBs
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Figure 1: Domain organization of human VWF and multispecies alignment of the VWF propeptide and ADAMTS13 cleavage sites and anking sequences. Sequence alignment was performed using ClustalW2 followed by output using BOXSHADE (Section 2). (a) Domain organization of human VWF. Upper notations indicate known protein-protein interaction domains (Gp: glycoprotein). The solid triangle indicates the propeptide (PP) cleavage site, and the open triangle indicates the ADAMTS13 cleavage site. B indicates domains B1B3. (b) Alignment of sequences surrounding the Arg-Ser (RS, indicated by the solid triangle) human propeptide cleavage site demonstrates a high degree of conservation. Note the extended RX(R/K)R motif present in all species except for medaka. The open triangle indicates the presence of an unconserved cysteine in medaka Vwf. (c) Alignment at the human ADAMTS13 cleavage site (YM, indicated by the solid triangle) and anking sequences demonstrates conservation of the Tyr-Met residues in mammalian and avian species, but a Phe-Leu putative site in teleost sh. The invariant Leu (human residue 1603) is indicated by a white triangle. z: zebrash; h: human; m: mouse; ca: canine; c: chicken; fu: fugu; st: stickleback; med: medaka.
[28, 29]. These structures are produced after VWF has been processed into high molecular weight multimers in the Golgi apparatus. Using an anti-Myc antibody we were able to identify elongated structures consistent with pseudoWPBs in zebrash vwf transfected cells (Figures 3(d) and 3(g)). These were morphologically similar to those found in human VWF transfected cells (Figure 3(a)). Staining with an anti-calnexin antibody to localize endoplasmic reticulum (ER, Figures 3(b), 3(e), and 3(h)) demonstrated no overlap between the structures (Figures 3(c), 3(f), and 3(i)), as expected for WPBs and pseudo-WPBs [28, 29].
3.3. Developmental Patterns of vwf Expression. RT-PCR of whole embryos up to 96 hpf demonstrated increasing levels of vwf expression, with the most intense expression at 96 hpf (Figure 4(f)). Whole-mount in situ hybridization was used to localize expression from the middle of gastrulation (8 hpf) to 120 hpf. Expression of vwf is weakly detectable throughout the embryo at 8 hpf (Figure 4(a)). Stronger expression is observed in 12-hour embryos as a more diuse pattern throughout the embryo (Figure 4(b)). At 48 hours there is diuse expression cranially, which extends caudally (Figure 4(c)). At 96120 hours, strong expression is present
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hVWF hVWF zVwf NHP
Advances in Hematology marker of human vasculature [37]. However, it has not been examined in the developing vertebrate. We hypothesized that there would be widespread expression of zebrash vwf in developing vasculature, but instead found an early broad and then later restricted pattern. A previous study in zebrash identied Vwf protein expression within the vasculature at the larval stage, although the source was not determined [15]. Therefore one possible explanation for the discrepancy with our results is that larval intravascular Vwf is not produced in endothelial cells but rather comes from the yolk sac or pharyngeal arches. Alternatively, endothelial vwf mRNA expression might not be present until later in development. The expression seen in early embryonic development may possibly reect maternally derived transcripts [38], while later expression is clearly of embryonic/larval origin. There is no prior evidence for a role of VWF in gastrulation, although the expression in the pharyngeal arches is intriguing. These structures develop into gills [39], the organs responsible for oxygen exchange in sh. The highest levels of mammalian Vwf mRNA expression have been identied in the lung [36], suggesting the possibility of an evolutionary conserved role of VWF in these structures. In order to produce functional VWF activity, high molecular weight multimers are assembled in the trans-Golgi, packaged into WPBs, and secreted. This is followed by circulation in the blood and tethering of platelets to sites of vessel injury, forming the primary platelet plug [6]. It has been previously shown that zebrash thrombocytes will aggregate in a Vwf-dependent fashion and that morpholinomediated knockdown results in increased bleeding times and hemorrhage [15]. In this paper we have demonstrated that zebrash Vwf has the ability to multimerize and form pseudo-WPBs in mammalian cell culture. Taken together, these data suggest that the basic mechanisms of zebrash Vwf function appear to be conserved. Previous studies have shown evidence for the presence of the Vwf receptor, GpIb, on thrombocytes in zebrash and chicken [40, 41]. If thrombocytes bind Vwf as platelets do in mammals, one might expect a high degree of conservation of the Vwf A1 domain, which encodes the GpIb-binding site. The A2 domain, which encodes the Adamts13 cleavage site, is required for the production of properly sized Vwf multimers. When cleavage is reduced, vascular occlusion can occur, while when enhanced, bleeding results [42]. However, there are notable dierences between mammalian and nonmammalian vertebrate systems. Despite the overall amino acid similarity and conservation of synteny of Vwf, the A1 and A2 domains display the largest degree of divergence when compared to humans. It is tempting to speculate that the A1 domain has evolved a relatively increased or decreased ability to bind thrombocytes in compensation for the latters lesser or greater role in the initiation of primary hemostasis. Shear forces required to expose the A1 and A2 domains are likely to be dierent in zebrash compared to mammals. Despite their functional similarities, nucleated thrombocytes are clearly dierent from anucleate platelets, suggesting the possibility that the two function quite dierently. Studies of avian thrombocytes, which are also nucleated, have led to the
Anti-V5/ anti-HIS
Figure 2: Multimerization of zebrash Vwf in mammalian cell culture demonstrates high molecular weight multimers similar to human VWF. HEK293T cells were transfected with pzVwf/V5HISA, expressing V5-HIS tagged zebrash Vwf (zVwf), or pCineoVWF, expressing untagged human VWF (hVWF). Normal human plasma (NHP) and zebrash and human supernatants were separated by agarose gel electrophoresis, transferred by western blotting, and detected with either a pool of monoclonal anti-hVWF antibodies (Avw1, 5, 15, left panel) or a mixture of anti-V5 and antiHIS antibodies (for tagged zVwf, right panel). The anti-V5/HIS combination detects zVwf with a multimer pattern, including high molecular weight multimers, indistinguishable from that typically observed for human VWF (brackets indicate high molecular weight multimers for both zebrash and human VWF).
in the pharyngeal arches, intestinal epithelium, and inner layer of the yolk sac (Figures 4(e), 4(g), and 4(h)).
4. Discussion
VWD is due to quantitative or qualitative deciency of VWF and has been described in several mammals, including human, horse, cat, pig, rabbit, and dog [7, 8]. Identication and characterization of the human VWF cDNA [3033] enabled the eventual identication of many of these pathogenic mutations as well as partial or full length sequence information in numerous mammalian species [34]. The zebrash genome project [35] assisted in the identication of much of the vwf cDNA [15, 16], but this did not include the complete 5 and 3 UTRS. We have now completed cloning and characterization of the full length zebrash vwf cDNA. We found that vwf displays widespread expression in early embryonic development and then becomes more restricted at the larval stage. Mammalian VWF is widely expressed in vascular endothelial cell beds of the adult mouse [36], and VWF protein is an established clinical pathologic
Advances in Hematology
Myc Calnexin Merge
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Figure 3: Zebrash Vwf forms pseudo-Weibel-Palade bodies (pseudo-WPBs) in mammalian cell culture. pVWF/Myc-HIS (human VWF, (ac)) or pzVwf/Myc-HIS (zebrash Vwf, (di)) plasmids were transfected into HEK293T cells. Anti-Myc antibody conjugated to Alexa Fluor 488 (green channel, (a, d, g)) was used for detection and anti-calnexin antibody conjugated to Alexa Fluor 594 (red channel, (b, e, h)) labeled endoplasmic reticulum (ER). Both constructs demonstrate formation of elongated Myc positive and ER negative structures (absence of yellow signal in the merged panels, (c, f, i)) characteristic of pseudo-WPBs (examples are indicated in (a, d), and (g) by arrowheads). Scale bars, 2.5 m.
hypothesis that human cardiovascular disease may be related to the existence of platelet rather than thrombocyte-initiated primary hemostasis [41]. Further understanding of the role of thrombocytes and Vwf in zebrash and avian hemostasis may have potential implications for the treatment of bleeding and thrombotic disorders.
Acknowledgments
The authors thank the University of Michigan Sequencing and Genotyping Core, and Dave Siemieniak, Susan Spaulding, Kristen Lessl, and Toby Hurd for technical assistance, and Evan Sadler for helpful suggestions. The authors would
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Figure 4: Developmental expression of vwf mRNA. Wild type zebrash ospring were isolated from 8 to 120 hpf, xed, and in situ hybridization was performed (Section 2). (a) Examination at 8 hpf demonstrates weak expression throughout the entire embryo, and staining was completely absent from a sense control. (b) Diuse expression continues at 12 hpf (staining was completely absent from a sense control), followed by more restricted expression cranially with a stripe that extends caudally at 48 hpf (c). Figure 4(d) is a sense probe as negative control at 48 hpf. (e) 96 hpf shows strong expression in the pharyngeal arches. (f) RT-PCR of cDNA isolated from whole zebrash embryos and larvae from 896 hpf. (g, h) Analysis at 120 hpf shows continued expression in the pharyngeal arches, as well as inner yolk sac layer and intestinal epithelium. Experiments in (ae) used full length vwf riboprobes. Results in (g, h) are representative of hybridization with exon 28 and exon 4752 riboprobes (Section 2, Table 1). Abbreviations: p: pharyngeal arches; y: inner layer of yolk sac; i: intestinal epithelium.
especially like to thank David Ginsburg for support and critical reading of the paper. This work was supported by American Heart Association no. 0675025N, the Bayer Hemophilia Awards Program, and the Diane and Larry Johnson Family Scholar Award (J.A.S.), as well as National Institutes of Health P01-HL081588 and R01-HL033721 (R.R.M.).
References
[1] C. J. Davidson, R. P. Hirt, K. Lal et al., Molecular evolution of the vertebrate blood coagulation network, Thrombosis and Haemostasis, vol. 89, no. 3, pp. 420428, 2003. [2] C. J. Davidson, E. G. Tuddenham, and J. H. McVey, 450 million years of hemostasis, Journal of Thrombosis and Haemostasis, vol. 1, no. 7, pp. 14871494, 2003. [3] Y. Jiang and R. F. Doolittle, The evolution of vertebrate blood coagulation as viewed from a comparison of puer sh and sea squirt genomes, Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 13, pp. 75277532, 2003. [4] W. C. Nichols and D. Ginsburg, Von Willebrand disease, Medicine, vol. 76, no. 1, pp. 120, 1997.
[5] R. Schneppenheim and U. Budde, Von Willebrand factor: the complex molecular genetics of a multidomain and multifunctional protein, Journal of Thrombosis and Haemostasis, vol. 9, no. 1, pp. 209215, 2011. [6] J. E. Sadler, Von Willebrand factor assembly and secretion, Journal of Thrombosis and Haemostasis, vol. 7, supplement s1, pp. 2427, 2009. [7] D. Ginsburg and E. J. W. Bowie, Molecular genetics of von Willebrand disease, Blood, vol. 79, no. 10, pp. 25072519, 1992. [8] G. Levy and D. Ginsburg, Getting at the variable expressivity of von Willebrand disease, Thrombosis and Haemostasis, vol. 86, no. 1, pp. 144148, 2001. [9] P. Jagadeeswaran, R. Paris, and P. Rao, Laser-induced thrombosis in zebrash larvae: a novel genetic screening method for thrombosis, Methods in Molecular Medicine, vol. 129, pp. 187195, 2006. [10] K. R. Kidd and B. M. Weinstein, Fishing for novel angiogenic therapies, British Journal of Pharmacology, vol. 140, no. 4, pp. 585594, 2003. [11] J. N. Chen, P. Hater, J. Odenthal et al., Mutations aecting the cardiovascular system and other internal organs in zebrash, Development, vol. 123, pp. 293302, 1996.
Advances in Hematology
[12] D. Y. Stainier, B. Fouquet, J. N. Chen et al., Mutations aecting the formation and function of the cardiovascular system in the zebrash embryo, Development, vol. 123, pp. 285292, 1996. [13] S. W. Jin, W. Herzog, M. M. Santoro et al., A transgeneassisted genetic screen identies essential regulators of vascular development in vertebrate embryos, Developmental Biology, vol. 307, no. 1, pp. 2942, 2007. [14] E. E. Patton and L. I. Zon, The art and design of genetic screens: zebrash, Nature Reviews Genetics, vol. 2, no. 12, pp. 956966, 2001. [15] M. Carrillo, S. Kim, S. K. Rajpurohit, V. Kulkarni, and P. Jagadeeswaran, Zebrash von Willebrand factor, Blood Cells, Molecules, and Diseases, vol. 45, no. 4, pp. 326333, 2010. [16] L. T. Dang, A. R. Purvis, R. H. Huang, L. A. Westeld, and J. E. Sadler, Phylogenetic and functional analysis of histidine residues essential for pH-dependent multimerization of von willebrand factor, Journal of Biological Chemistry, vol. 286, no. 29, pp. 2576325769, 2011. [17] P. A. Fujita, B. Rhead, A. S. Zweig et al., The UCSC genome browser database: update 2011, Nucleic Acids Research, vol. 39, no. 1, pp. D876D882, 2011. [18] M. Goujon, H. McWilliam, W. Li et al., A new bioinformatics analysis tools framework at EMBL-EBI, Nucleic Acids Research, vol. 38, supplement 2, pp. W695W699, 2010. [19] M. A. Larkin, G. Blackshields, N. P. Brown et al., Clustal W and clustal X version 2.0, Bioinformatics, vol. 23, no. 21, pp. 29472948, 2007. [20] D. A. Buchner, F. Su, J. S. Yamaoka et al., Pak2a mutations cause cerebral hemorrhage in redhead zebrash, Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 35, pp. 1399614001, 2007. [21] S. L. Haberichter, S. A. Fahs, and R. R. Montgomery, Von Willebrand factor storage and multimerization: 2 independent intracellular processes, Blood, vol. 96, no. 5, pp. 18081815, 2000. [22] J. Schullek, J. Jordan, and R. R. Montgomery, Interaction of von Willebrand factor with human platelets in the plasma milieu, Journal of Clinical Investigation, vol. 73, no. 2, pp. 421 428, 1984. [23] M. Westereld, The Zebrash Book. A Guide For the Laboratory Use of Zebrash (Danio Rerio), University of Oregon Press, Eugene, Ore, USA, 4th edition, 2000. [24] C. Thisse and B. Thisse, High-resolution in situ hybridization to whole-mount zebrash embryos, Nature Protocols, vol. 3, no. 1, pp. 5969, 2008. [25] J. Sullivan-Brown, M. E. Bisher, and R. D. Burdine, Embedding, serial sectioning and staining of zebrash embryos using JB-4 resin, Nature Protocols, vol. 6, no. 1, pp. 4655, 2011. [26] A. Rehemtulla and R. J. Kaufman, Preferred sequence requirements for cleavage of pro-von Willebrand factor by propeptide-processing enzymes, Blood, vol. 79, no. 9, pp. 2349 2355, 1992. [27] Y. Xiang, R. de Groot, J. T. B. Crawley, and D. A. Lane, Mechanism of von Willebrand factor scissile bond cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), Proceedings of the National Academy of Sciences of the United States of America, vol. 108, no. 28, pp. 1160211607, 2011. [28] G. Michaux, L. J. Hewlett, S. L. Messenger et al., Analysis of intracellular storage and regulated secretion of 3 von Willebrand disease-causing variants of von Willebrand factor, Blood, vol. 102, no. 7, pp. 24522458, 2003.
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[29] J. W. Wang, K. M. Valentijn, H. C. de Boer et al., Intracellular storage and regulated secretion of von Willebrand factor in quantitative von Willebrand disease, Journal of Biological Chemistry, vol. 286, no. 27, pp. 2418024188, 2011. [30] D. Ginsburg, R. I. Handin, and D. T. Bonthron, Human von Willebrand Factor (vWF): isolation of complementary DNA (cDNA) clones and chromosomal localization, Science, vol. 228, no. 4706, pp. 14011406, 1985. [31] D. C. Lynch, T. S. Zimmerman, and C. J. Collins, Molecular cloning of cDNA for human von Willebrand factor: authentication by a new method, Cell, vol. 41, no. 1, pp. 4956, 1985. [32] J. E. Sadler, B. B. Shelton-Inloes, and J. M. Sorace, Cloning and characterization of two cDNAs coding for human von Willebrand factor, Proceedings of the National Academy of Sciences of the United States of America, vol. 82, no. 19, pp. 63946398, 1985. [33] C. L. Verweij, C. J. M. de Vries, B. Distel et al., Construction of cDNA coding for human von willebrand factor using antibody probes for colony-screening and mapping of the chromosomal gene, Nucleic Acids Research, vol. 13, no. 13, pp. 46994717, 1985. [34] ISTH SSC VWF Database, http://www.vwf.group.shef.ac.uk/ index.html. [35] S. C. Ekker, D. L. Stemple, M. Clark, C. B. Chien, R. S. Rasooly, and L. C. Javois, Zebrash genome project: bringing new biology to the vertebrate genome eld, Zebrash, vol. 4, no. 4, pp. 239251, 2007. [36] K. Yamamoto, V. de Waard, C. Fearns, and D. J. Loskuto, Tissue distribution and regulation of murine von Willebrand factor gene expression in vivo, Blood, vol. 92, no. 8, pp. 2791 2801, 1998. [37] M. R. Wick and J. L. Hornick, Immunohistology of soft tissue and osseous neoplasms, in Diagnostic Immunohistochemistry, D. J. Dabbs, Ed., pp. 83136, Saunders, 3rd edition, 2010. [38] A. F. Schier, The maternal-zygotic transition: death and birth of RNAs, Science, vol. 316, no. 5823, pp. 406407, 2007. [39] C. B. Kimmel, W. W. Ballard, S. R. Kimmel, B. Ullmann, and T. F. Schilling, Stages of embryonic development of the zebrash, Developmental Dynamics, vol. 203, no. 3, pp. 253310, 1995. [40] P. Jagadeeswaran, J. P. Sheehan, F. E. Craig, and D. Troyer, Identication and characterization of zebrash thrombocytes, British Journal of Haematology, vol. 107, no. 4, pp. 731 738, 1999. [41] A. A. Schmaier, T. J. Stalker, J. J. Runge et al. et al., Occlusive thrombi arise in mammals but not birds in response to arterial injury: evolutionary insight into human cardiovascular disease, Blood, vol. 118, no. 13, pp. 36613669, 2011. [42] J. E. Sadler, Von Willebrand factor: two sides of a coin, Journal of Thrombosis and Haemostasis, vol. 3, no. 8, pp. 1702 1709, 2005.
Hindawi Publishing Corporation Advances in Hematology Volume 2012, Article ID 792163, 8 pages doi:10.1155/2012/792163
Research Article Drift-Diffusion Analysis of Neutrophil Migration during Inammation Resolution in a Zebrash Model
Geoffrey R. Holmes,1 Giles Dixon,2 Sean R. Anderson,1 Constantino Carlos Reyes-Aldasoro,3 Philip M. Elks,2, 4 Stephen A. Billings,1 Moira K. B. Whyte,2, 4 Visakan Kadirkamanathan,1 and Stephen A. Renshaw2, 4
of Automatic Control and Systems Engineering, University of Sheeld, Sheeld S1 3JD, UK Centre for Developmental and Biomedical Genetics, University of Sheeld, Firth Court, Western Bank, Sheeld S10 2TN, UK 3 University of Sussex School of Engineering and Design Biomedical Engineering Research Group, Brighton BN1 9QT, UK 4 Department of Infection and Immunity, University of Sheeld, Sheeld S10 2JF, UK
1 Department 2 MRC
Correspondence should be addressed to Stephen A. Renshaw, [email protected] Received 17 February 2012; Accepted 22 April 2012 Academic Editor: Christopher Hall Copyright 2012 Georey R. Holmes et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Neutrophils must be removed from inammatory sites for inammation to resolve. Recent work in zebrash has shown neutrophils can migrate away from inammatory sites, as well as die in situ. The signals regulating the process of reverse migration are of considerable interest, but remain unknown. We wished to study the behaviour of neutrophils during reverse migration, to see whether they moved away from inamed sites in a directed fashion in the same way as they are recruited or whether the inherent random component of their migration was enough to account for this behaviour. Using neutrophil-driven photoconvertible Kaede protein in transgenic zebrash larvae, we were able to specically label neutrophils at an inammatory site generated by tailn transection. The locations of these neutrophils over time were observed and tted using regression methods with two separate models: pure-diusion and drift-diusion equations. While a model hypothesis test (the F -test) suggested that the datapoints could be tted by the drift-diusion model, implying a fugetaxis process, dynamic simulation of the models suggested that migration of neutrophils away from a wound is better described by a zero-drift, diusion process. This has implications for understanding the mechanisms of reverse migration and, by extension, neutrophil retention at inammatory sites.
1. Introduction
The fate of neutrophils following completion of the inammatory programme is of critical importance for the outcome of episodes of acute inammation and can determine whether there is prompt healing of a wound or the development of chronic inammation and tissue injury. Neutrophils recruited to sites of inammation may leave the site or die in situ [1]. The most widely accepted mechanism of neutrophil disposal is the programmed cell death or apoptosis, of the neutrophil followed by macrophage uptake and clearance (reviewed in [2]). Recently, other routes have been proposed; neutrophils may move away from the inamed site into the bloodstream (reverse transmigration [3]), by migration
through other tissues (retrograde chemotaxis or reverse migration [46]), or be lost into the inammatory exudate [7, 8]. Current understanding of the process of reverse migration is reviewed elsewhere [9]. The uncertainty as to the in vivo fates of individual cells relates in part to the diculty in following individual cells during inammation resolution in vivo. The transgenic zebrash model is emerging as a key model for the study of vertebrate immunity [10] and allows direct imaging and tracking of individual cells, and of populations of cells allowing their fate to be determined in vivo. Using a transgenic system, in which neutrophils express the uorescent protein Kaede, notable for its ability to change uorescence characteristics on exposure to light, we have assessed the fates of inammatory neutrophils as
2 inammation resolves. Although others have used a similar system to label immune cell populations responding to much smaller stimuli [6], there has been no detailed study of the migratory patterns of neutrophils during inammation resolution following tail transection. Using dynamic modelling techniques based on the driftdiusion equation, we tested the competing hypotheses that neutrophils were directed away from the wound region by proresolution agents produced locally or that they cease responding to existing chemokine gradients and redistribute as a feature of stochastic migratory behaviours.
Advances in Hematology was tested by simulation using a Monte Carlo procedure and the distribution of simulated cell populations compared to the observed data.
2. Methods
2.1. Reagents, Zebrash Lines and Maintenance. All reagents were from Sigma-Aldrich (Poole, UK) unless otherwise stated. Zebrash were maintained according to standard protocols [11]. The Tg(lyz: Gal4)i252 [12] and Tg(UAS: Kaede)s1999t [13] lines are described elsewhere. 2.2. Microscopy, Photoconversion, and Image Processing. For confocal microscopy, a Perkin Elmer Ultra VIEW VoX ERS 6FR Laser Confocal Imaging System (Perkin Elmer INC, USA) with an inverted Olympus IX81 microscope, equipped with six diode laser lines and a Yokogawa CSU-X1 spinning disk, was used to capture images on a 14-bit Hamamatsu C9100-50 Electron Multiplying-Charged Couple Device (EM-CCD) peltier-cooled camera (Hamamatsu Photonics Inc.), through an appropriate lter. For uorescence microscopy, a Nikon Eclipse TE2000-U Inverted Compound Fluorescence Microscope (Nikon UK Ltd) was used with a Hamamatsu 1394 ORCA-ERA (Hamamatsu Photonics Inc.). Images were captured using Volocity build 5.3.2. A Perkin Elmer Ultra VIEW PhotoKinesis device, attached to the microscope described before, was used to photoconvert the Kaede protein using a 405 nm laser line. The device was calibrated using a glass microscope slide (Menzel-Gl azer) covered with uorescent highlighter ink (Stabilo Boss) as a photobleachable substrate (according to manufacturers instructions). Photoconversion was performed using 40% laser energy for 120 cycles of the 405 nm laser line. The embryos were then released from the agarose gel and transferred to fresh E3. The petri dishes containing the embryos were wrapped in tinfoil to prevent background photoconversion. At the timepoints indicated, embryos were again mounted and wideeld uorescence Z-stacks taken. Neutrophil segmentation was performed in Volocity based on uorescence intensity, size, and separate touching objects feature. The XY position of each uorescent cell at each timepoint was determined. 2.3. Dynamic Modelling of Neutrophil Behaviour. Neutrophil centroid coordinates in time were exported into Matlab (MathWorks, MA), for analysis. To describe quantitatively the population dynamics of neutrophils, drift-diusion and pure-diusion variants of the simple random walk model were used ([14] see Supplementary Material for full details available online at doi: 10.1155/2012/792163). Using parameters identied in these models, the behavior of each model
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Figure 1: Inammatory neutrophils exhibit restricted migration away from the site of tissue injury. 3 dpf embryos from transgenic zebrash expressing Kaede in neutrophils were subjected to tailn transection under anaesthesia using a sterile scalpel. The embryos were recovered for 4 hours. At four hours after injury the embryo was mounted in 0.5% low melting point agarose for imaging on a Laser Confocal System (Perkin Elmer Inc). The PhotoKinesis device was then used to photoconvert all neutrophils present within the tip of the tailn. Photoconversion was carried out according to the methods described (120 cycles of 40% 405 nm laser energy), and time-lapse videomicroscopy was performed using a TE2000 uorescent inverted microscope (Nikon). (a) Composite images of DIC overlaid with the red and green uorescence channels showing a representative zebrash tail before (above) and after (below) photoconversion. (b) A montage of DIC images overlaid with the red uorescence channel at then timepoints indicated after tailn injury. The redistribution of photoconverted cells can be clearly seen over time. (c) For each neutrophil in six individual sh, the distance from the wound was calculated using algorithms within Volocity and plotted against time.
injury falls. Where individual cells can be seen and followed over time, the pattern of accumulation of neutrophils during inammation can be accurately determined. This technique has increased sensitivity for detecting continued inux compared to mammalian labelled-cell techniques, and this may explain the dierences seen from rabbit pneumonia
models where inux is no longer detectable shortly after initiation of the inammatory episode [17]. 3.3. Neutrophils Actively Migrate (Drift) toward a Wound. Random walk models are often used in biology to describe the movement dynamics of individuals and populations
Advances in Hematology
Figure 2: At peak inammation, new neutrophils are recruited to the site of injury. Photomontage generated from the time-lapse data used in Figure 1(b), and Supplemental Movie 1, imaged using the GFP lterset, showing neutrophil recruitment to the site of injury over the same timespan. Green neutrophils can be seen to accumulate at the site of injury between 6 and 14 hours after injury.
[14, 18] and particularly for cell movement patterns [1921]. Over short timescales neutrophils exhibit correlated random walk behaviour. However, these local correlations decay over time. The time between our data observations is greater than typical neutrophil persistence times [22] and thus we are able to ignore these local correlations and apply a simple random walk model [18]. To identify any global directional bias apparent in the movement of neutrophils, the simple random walk model was applied to aggregate data. The contribution of active recruitment (chemotaxis) of neutrophils and its reverse (fugetaxis) were examined by establishing the positions of all neutrophils at 4 hours following tail n transection and modelling their behaviour using a driftdiusion equation. Non-photoconverted neutrophils were examined to determine the behaviour of neutrophils not at the wound site at the time of photoconversion. Fitting the drift-diusion equation to the dataset treats the neutrophils as point objects and asks whether they are behaving like simple particles redistributing stochastically (diusion) or whether there is an element of active movement towards or away from a chemical gradient (chemotaxis or fugetaxis). The equation (full description in supplemental data) generates a value for the drift co-ecient, for which non-zero values reect an active rather than purely random migration. The drift was estimated from the linear relationship between time and mean cell distance from the wound (Figure 3). For 6 independent experiments, the coecient estimates ranged in value from 0.11 to 0.95 m/min (Table 1). As expected, in all cases cell populations demonstrated active drift toward the wound, consistent with migration directed by a chemotactic process. 3.4. Migration of Neutrophils away from a Wound Is Better Described by a Zero-Drift, Diusion Process. The same analysis was performed for photoconverted cells present at the site of the wound at the time of photoconversion, 4 hours following the tailn transection (Table 2). Drift-diusion and pure-diusion model ts are compared in Figure 4.
Table 1: Estimated drift coecients for the model of drift-diusion describing cell migration toward the wound. Dataset (1) (2) (3) (4) (5) (6) All data Drift coecient (std dev.) 0.85 (0.13) 0.95 (0.06) 0.11 (0.02) 0.32 (0.02) 0.48 (0.08) 0.37 (0.06) 0.35 (0.03)
Mathematical testing of the t of the two models suggested that the drift-diusion model tted better with the data, but we were alert to the possibility that drift-diusion models might appear superior due to the better ability of quadratic ts to model real, noisy data than simple linear ts. Using modeled data comparing the predicted distributions of neutrophils over time by applying drift-diusion versus pure diusion models gave a dramatic result: the cell population mode of the drift-diusion model moved away from the wound over time (Figure 5, red line), in contrast to the observed data, where the mode remained close to the wound (Figure 5, yellow bars). The pure-diusion model accurately captured this qualitative behavior, more accurately reecting the observed distribution of neutrophils over time (Figure 5, blue line), suggesting that stochastic redistribution might best describe the pattern of neutrophil behavior during inammation resolution. For the larger wounds used in these studies, our data support a stochastic redistribution of neutrophils during inammation resolution. However, to denitively prove this will require more advanced modelling techniques. For smaller wounds, dierent principles may apply. Previous studies have suggested that neutrophils leaving the wound follow the same dynamics as those arriving, having the same velocity
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Figure 3: Nonphotoconverted neutrophils actively migrate into the wound region. (a) Variation over time of mean cell distance from the wound for the nonphotoconverted (green) neutrophils, observed in each subject 16 (black line). Overlaid on each graph is the prediction of mean distance obtained from the linear model used to characterise the initial drift (red line). The time is measured from the start of observations which commenced 4 hours after injury. The cell count in subject 6 (bottom right) was low and sometimes zero near the end of the dataset, which explains the missing sections. (b) Data and model combined over all subjects.
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Figure 4: Inammatory neutrophil behaviour can be tted by pure-diusion and drift-diusion models. (a) Plots of mean squared cell distance from the wound against time for the photoconverted (red) neutrophils for datasets 16. Also shown on each plot are the ts for the linear model corresponding to pure-diusion with zero drift (blue line) and for the drift-diusion model (red line). (b) Data and models combined over all subjects.
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Time: 5 (mins) 100 Normalised cell count Normalised cell count 80 60 40 20 0 50 450 850 Distance (m) 100 80 60 40 20 0 50 450 850 Distance (m) Normalised cell count Time: 105 (mins) 100 Normalised cell count 80 60 40 20 0 50 450 850 Distance (m) Time: 205 (mins) 100 80 60 40 20 0 50 450 850 Distance (m) Normalised cell count Time: 305 (mins) 100 80 60 40 20 0 50 450 850 Distance (m) Time: 405 (mins)
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Figure 5: Simulation reveals a pure-diusion model to be a better t to the real data. Both the drift-diusion model (red line) corresponding to drift (0.26 m/min) and diusion (8.0 m/min) and the pure-diusion model (blue line) corresponding to diusion (41.8 m/min) were simulated 1000 times. The simulations were used to produce a distribution for the spatially binned data of each model. The mean values of cell distribution over space are shown by the red and blue lines, respectively (in terms of distance from the wound). Overlaid on these is a corresponding histogram representation (yellow) of the real data (combined over all sh). The histogram bins have width 100 m and are centered at 50 m to 950 m from the wound. The pure-diusion model shows a correct qualitative prediction of cell distribution whereas the drift-diusion model predicts that the population mode moves away from the wound over time, in contrast to the observed data.
Table 2: Estimated coecients for the drift-diusion model and pure-diusion model of cell migration away from the wound (standard deviation is given in brackets). An F -test value >5 indicates that the drift-diusion model should be preferred to the pure-diusion model. Dataset (1) (2) (3) (4) (5) (6) All data Drift-diusion model Drift coecient 0.25 (0.05) 0.27 (0.07) 0.19 (0.05) 0.21 (0.05) 0.35 (0.07) 0.27 (0.03) 0.26 (0.02) Diusion coecient 4 (10) 23 (15) 13 (10) 32 (11) 8(14) 7 (6) 8 (3) Pure-diusion model Diusion coecient 27 (2) 56 (4) 32 (3) 54 (3) 55 (4) 31 (2) 41.8 (0.10) F -test 38 28 14 14 82 145 267
and directionality [15]. However, those data rely on preselection of tracks directly leaving the wound, and may give dierent results to studies considering the whole population of cells. This approach uses static point data for each neutrophil; an alternative approach would be to investigate the dynamics using individual track data. Such an approach has been applied to proteins in living cells [23, 24] and to in vivo melanoma cell tracks [25]. Care is needed when considering cell tracks as a naive approach could misrepresent shortterm correlations in track direction as biased migration. In
addition, to identify tracks requires faster sampling of observations which must be balanced against total experiment runtime. Although the pure-diusion model appears to t the data well, it consistently underestimates the number of photoconverted cells remaining adjacent to the wound, suggesting some cells are actively retained at the wound site. To completely address this will require the development of systems incorporating multiple models to reect the dynamic mix of neutrophil behaviours present within a single population.
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[10] S. A. Renshaw and N. S. Trede, A model 450 million years in the making: zebrash and vertebrate immunity, Disease Models & Mechanisms, vol. 5, pp. 3847, 2011. [11] C. N usslein-Volhard and R. Dahm, Zebrash, A Practical Approach, Oxford University Press, Oxford, UK, 2002. [12] P. M. Elks, F. J. Van Eeden, G. Dixon et al., Activation of hypoxia-inducible factor-1 (hif-1) delays inammation resolution by reducing neutrophil apoptosis and reverse migration in a zebrash inammation model, Blood, vol. 118, no. 3, pp. 712722, 2011. [13] J. M. Davison, C. M. Akitake, M. G. Goll et al., Transactivation from Gal4-VP16 transgenic insertions for tissue-specic cell labeling and ablation in zebrash, Developmental Biology, vol. 304, no. 2, pp. 811824, 2007. [14] E. A. Codling, M. J. Plank, and S. Benhamou, Random walk models in biology, Journal of the Royal Society Interface, vol. 5, no. 25, pp. 813834, 2008. [15] J. R. Mathias, B. J. Perrin, T. X. Liu, J. Kanki, A. T. Look, and A. Huttenlocher, Resolution of inammation by retrograde chemotaxis of neutrophils in transgenic zebrash, Journal of Leukocyte Biology, vol. 80, no. 6, pp. 12811288, 2006. [16] H. A. Jones, R. J. Clark, C. G. Rhodes, J. B. Schoeld, T. Krausz, and C. Haslett, In vivo measurement of neutrophil activity in experimental lung inammation, American Journal of Respiratory and Critical Care Medicine, vol. 149, no. 6, pp. 16351639, 1994. [17] H. A. Jones, S. Sriskandan, A. M. Peters et al., Dissociation of neutrophil emigration and metabolic activity in lobar pneumonia and bronchiectasis, European Respiratory Journal, vol. 10, no. 4, pp. 795803, 1997. [18] C. S. Patlak, The eect of the previous generation on the distribution of gene frequencies in populations, Proceedings of the National Academy of Sciences of the United State, vol. 39, pp. 10631068, 1953. [19] W. Alt, Biased random walk models for chemotaxis and related diusion approximations, Journal of Mathematical Biology, vol. 9, no. 2, pp. 147177, 1980. [20] L. Li, S. F. Nrrelkke, and E. C. Cox, Persistent cell motion in the absence of external signals: a search strategy for eukaryotic cells, PLoS ONE, vol. 3, no. 5, Article ID e2093, 2008. [21] A. A. Potdar, J. Jeon, A. M. Weaver, V. Quaranta, and P. T. Cummings, Human mammary epithelial cells exhibit a bimodal correlated random walk pattern, PLoS ONE, vol. 5, no. 3, Article ID e9636, 2010. [22] R. T. Tranquillo, E. S. Fisher, B. E. Farrell, and D. A. Lauenburger, A stochastic model for chemosensory cell movement: application to neutrophil and macrophage persistence and orientation, Mathematical Biosciences, vol. 90, no. 1-2, pp. 287303, 1988. [23] H. Qian, M. P. Sheetz, and E. L. Elson, Single particle tracking. Analysis of diusion and ow in two-dimensional systems, Biophysical Journal, vol. 60, no. 4, pp. 910921, 1991. [24] M. P. Sheetz, S. Turney, H. Qian, and E. L. Elson, Nanometrelevel analysis demonstrates that lipid ow does not drive membrane glycoprotein movements, Nature, vol. 340, no. 6231, pp. 284288, 1989. [25] R. Dickinson, Optimal estimation of cell movement indices from the statistical analysis of cell tracking data, AIChE Journal, vol. 39, pp. 19952010, 1993.
4. Conclusions
From this analysis, we conclude that the two key neutrophil migratory behaviours regulating neutrophil numbers during the inammatory responsemovement of neutrophils in and out of woundsare qualitatively dierent processes. Neutrophils are recruited actively towards the site of injury (drift), but as inammation resolves, their movement away is better modelled by stochastic redistribution (diusion). This has implications for our understanding of how neutrophils might be retained at sites of inammation in disease states.
Acknowledgments
The authors gratefully acknowledge that this work was supported by the Engineering and Physical Sciences Research Council (EPSRC), UK; a European Research Council Advanced Investigator Award (S.A.B.); an MRC Senior Clinical Fellowship (S.A.R.) (Reference no. G0701932); and an MRC Centre Grant (G0700091). Microscopy studies were supported by a Wellcome Trust Grant to the MBB/BMS Light Microscopy Facility (GR077544AIA).
References
[1] C. N. Serhan, S. D. Brain, C. D. Buckley et al., Resolution of inammation: state of the art, denitions and terms, The FASEB Journal, vol. 21, no. 2, pp. 325332, 2007. [2] R. Dun, A. E. Leitch, S. Fox, C. Haslett, and A. G. Rossi, Targeting granulocyte apoptosis: mechanisms, models, and therapies, Immunological Reviews, vol. 236, no. 1, pp. 2840, 2010. [3] C. D. Buckley, E. A. Ross, H. M. McGettrick et al., Identication of a phenotypically and functionally distinct population of long-lived neutrophils in a model of reverse endothelial migration, Journal of Leukocyte Biology, vol. 79, no. 2, pp. 303311, 2006. [4] S. B. Brown, C. S. Tucker, C. Ford, Y. Lee, D. R. Dunbar, and J. J. Mullins, Class III antiarrhythmic methanesulfonanilides inhibit leukocyte recruitment in zebrash, Journal of Leukocyte Biology, vol. 82, no. 1, pp. 7984, 2007. [5] C. Hall, M. V. Flores, A. Chien, A. Davidson, K. Crosier, and P. Crosier, Transgenic zebrash reporter lines reveal conserved Toll-like receptor signaling potential in embryonic myeloid leukocytes and adult immune cell lineages, Journal of Leukocyte Biology, vol. 85, no. 5, pp. 751765, 2009. [6] S. K. Yoo and A. Huttenlocher, Spatiotemporal photolabeling of neutrophil tracking during inammation in live zebrash, Journal of Leukocyte Biology, vol. 89, no. 5, pp. 661 667, 2011. [7] P. Follin, Skin chamber technique for study of in vivo exudated human neutrophils, Journal of Immunological Methods, vol. 232, no. 1-2, pp. 5565, 1999. [8] L. Uller, C. G. A. Persson, and J. S. Erjef alt, Resolution of airway disease: removal of inammatory cells through apoptosis, egression or both? Trends in Pharmacological Sciences, vol. 27, no. 9, pp. 461466, 2006. [9] A. Huttenlocher and M. C. Poznansky, Reverse leukocyte migration can be attractive or repulsive, Trends in Cell Biology, vol. 18, no. 6, pp. 298306, 2008.
Hindawi Publishing Corporation Advances in Hematology Volume 2012, Article ID 830703, 13 pages doi:10.1155/2012/830703
Review Article Novel Insights into the Genetic Controls of Primitive and Denitive Hematopoiesis from Zebrash Models
Raman Sood and Paul Liu
Oncogenesis and Development Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA Correspondence should be addressed to Paul Liu, [email protected] Received 28 March 2012; Revised 20 May 2012; Accepted 8 June 2012 Academic Editor: Elspeth Payne Copyright 2012 R. Sood and P. Liu. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hematopoiesis is a dynamic process where initiation and maintenance of hematopoietic stem cells, as well as their dierentiation into erythroid, myeloid and lymphoid lineages, are tightly regulated by a network of transcription factors. Understanding the genetic controls of hematopoiesis is crucial as perturbations in hematopoiesis lead to diseases such as anemia, thrombocytopenia, or cancers, including leukemias and lymphomas. Animal models, particularly conventional and conditional knockout mice, have played major roles in our understanding of the genetic controls of hematopoiesis. However, knockout mice for most of the hematopoietic transcription factors are embryonic lethal, thus precluding the analysis of their roles during the transition from embryonic to adult hematopoiesis. Zebrash are an ideal model organism to determine the function of a gene during embryonicto-adult transition of hematopoiesis since bloodless zebrash embryos can develop normally into early larval stage by obtaining oxygen through diusion. In this review, we discuss the current status of the ontogeny and regulation of hematopoiesis in zebrash. By providing specic examples of zebrash morphants and mutants, we have highlighted the contributions of the zebrash model to our overall understanding of the roles of transcription factors in regulation of primitive and denitive hematopoiesis.
conserved pathways of regulation [1215]. Initial validation of the use of zebrash for hematopoiesis research came from the forward genetic screens. In 1996, two large-scale chemical mutagenesis screens were performed to identify mutants with a variety of phenotypes [16, 17]. Of these, characterization of 46 mutants with blood phenotypes by allelic complementation suggested roles for at least 26 genes in hematopoiesis [18, 19]. Subsequent eorts by several groups identied the underlying genetic defects in many of these mutants by positional cloning or candidate gene approaches. In addition to identifying the genes previously known to have a role in hematopoiesis (e.g., gata1, sptb, and, alas2), these mutants also uncovered novel genes with roles in hematopoiesis, (e.g., slc25a37, slc40a1, and glrx5) [20 25]. Subsequent forward genetic screens focusing on mutants aecting specic hematopoietic lineages have identied additional conserved pathways of regulation between zebrash and mammals [2628]. This led to a surge of activity in zebrash research laboratories, developing a variety of tools for thorough
2 analysis of hematopoiesis. Lineage-specic transgenic lines were generated using promoters of a variety of hematopoietic genes driving uorescent markers (reviewed in [29, 30] and listed in Table 1), allowing for visual observations of hematopoietic lineages in real-time during development. Advances in imaging combined with the ability to perform lineage tracing made it possible to follow the fate of specifically marked cells during development in a live vertebrate animal model [31, 32]. Sorting of hematopoietic cells by uorescence-activated cell sorting (FACS), in vitro culturing using zebrash-specic cytokines and kidney stromal cells, and the ability to perform transplantation have facilitated characterization of hematopoietic potential of dierent mutants [3337]. While forward screens are biased by the phenotype being screened, mutants in any specic gene can be generated using reverse genetic approaches. This has been made possible in zebrash in the last decade by TILLING (Targeting-Induced Local Lesions IN Genomes) [55, 60], and more recently by targeted mutagenesis using zinc-nger and transcriptionactivator-like-eector nucleases (i.e. ZFNs and TALENs) [6164]. Furthermore, eects of gene dosage can be analyzed by injecting suboptimal doses of antisense morpholinos or studying hypomorphic alleles generated by TILLING. In this review, we discuss how the technical advances and genomic tools discussed above went hand-in-hand with the elucidation of genetic controls of hematopoiesis in zebrash.
Advances in Hematology placenta [65, 7072]. HSCs from these sites migrate through circulation to fetal liver to support hematopoiesis during embryogenesis [65, 70, 73]. Recently, Chen and colleagues [74] demonstrated that EMPs and HSCs are derived from two dierent hemogenic endothelial populations. Unlike HSCs, EMPs lack the potential to give rise to lymphocytes. The site of adult hematopoiesis, where HSCs undergo dierentiation to generate lineage-committed progenitors that give rise to all the mature blood cell types and selfrenewal to maintain a constant supply of HSCs, is bone marrow [75]. The prevailing thinking, based on the current data, is that HSCs emerging from the hemogenic endothelial cells in the AGM region of the developing mouse embryo give rise to most (if not all) bone marrow hematopoietic cells [73, 76]. The shifting sites of hematopoiesis are thought to provide specic microenvironment cues required for the specication, and migration of precursors for lineage commitment [77, 78]. Although the overall process of hematopoiesis is well dened, we have just begun to elucidate the exact nature of the molecular controls and lineage relationships using in vitro colony assays and animal models, particularly mice and zebrash. The key questions revolved around the generation, migration, and dierentiation of HSCs into lineage-committed progenitors and how these processes are regulated to maintain a critical balance required for proper functioning of the hematopoietic system. 2.1. Primitive Hematopoiesis in Zebrash. In zebrash, the rst blood cells can be observed in circulation at around 26 hours post fertilization (hpf). However, based on the expression patterns of the genes involved in primitive hematopoiesis, it is clear that the primitive hematopoiesis starts at 11 hpf in the lateral plate mesoderm (LPM) during somitogenesis. The erythroid precursors are observed as bilateral stripes in the posterior lateral mesoderm (PLM) that fuse along the midline to form the intermediate cell mass (ICM) located in the trunk dorsal to the yolk tube extension by 24 hpf [29, 75, 77, 7981]. Primitive myeloid progenitors initiate at the anterior lateral mesoderm (ALM) and dierentiate into macrophages in the rostral blood island [80, 82]. Thus, primitive hematopoiesis in zebrash occurs in two waves, producing primitive macrophages and primitive erythrocytes, respectively. In addition, neutrophils and thrombocytes have also been detected during primitive hematopoiesis in zebrash. However, the origin of neutrophils during primitive hematopoiesis is not clear, as two recent reports presented contradictory data on their origin from either primitive macrophage lineage [83] or primitive erythrocyte lineage [84] using fate-mapping techniques. Thus, primitive blood cells in zebrash appear to have diverse lineages, similar to the mouse [67]. However, further studies are required to clearly dene the lineage relationships between these cell types during primitive hematopoiesis. 2.2. Denitive Hematopoiesis in Zebrash. The hallmark of denitive hematopoiesis is generation of multipotential HSCs that can undergo self-renewal and dierentiation to
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Table 1: Lineage-specic mutant and transgenic lines for zebrash hematopoiesis research. Lineage Marker tal1/scl lmo2 EMPs runx1 runx1 cmyb cd41 Erythropoiesis Myelopoiesis: GMPs gata1 spi1/pu.1 mpx Myelopoiesis: Neutrophils, Macrophages, Monocytes lyz mpeg1 rag1 lck ikzf1/ikaros Mutant lines Mutant designation and mutation type t21384, K183X None hg1, W84X hg1, W84X t25217, I181N hkz3, truncation in transactivation domain None m651 (vlad tepes), R339X hg2, T301K None None None None t26683, R797X None t24980, Q360X [55] [58] [42, 43] [42, 43] [45] [46] Transgenic lines References [38] Line designation PAC-tal1:GFP 5.0tal1:EGFP lmo2:EGFP lmo2:DsRed runx1P1:EGFP runx1P2:EGFP cmyb:EGFP cd41:GFP gata1:GFP gata1:DsRed spi1:EGFP zpu.1:EGFP mpx:GFP lyz:EGFP lyz:DsRed mpeg1:EGFP mpeg1:mCherry rag1:GFP lck:EGFP ikzf1:GFP References [39, 40] [41] [44] [44] Developed by the Zon lab, used in [47] [33, 34] [37, 49] [50, 51] [52] [53] [54] [56] [57] [59]
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produce cells of erythroid, myeloid, and lymphoid lineages. In zebrash HSCs can be identied by their expression of runx1 and cmyb as early as 26 hpf in the ventral wall of the dorsal aorta and hence this region of the embryo is referred to as the AGM [13, 29]. Two recent studies have unequivocally demonstrated the origin of HSCs from the hemogenic endothelium lining the ventral wall of the dorsal aorta using time lapse imaging and lineage tracing in double transgenic lines marking HSCs and endothelial cells with dierent uorescent markers [47, 85]. A novel process of cell transition, termed endothelial hematopoietic transition (EHT), appeared to be involved in the production of HSCs from hemogenic endothelium [85]. Similar to the mouse, a transient multipotent progenitor population of EMPs supports denitive hematopoiesis during embryogenesis and these EMPs originate in the posterior blood island (PBI) of zebrash [86]. The sites of adult hematopoiesis in zebrash are kidney marrow (analogous to the mammalian bone marrow) and thymus (for T cells) [13, 29, 87]. Up until recently, a site analogous to mammalian fetal liver was not recognized in the zebrash. Therefore, HSCs from AGM were presumed to support embryonic denitive hematopoiesis and migrate to thymus and kidney for adult denitive hematopoiesis. However, two independent studies demonstrated the existence of an intermediate site of hematopoiesis posterior to the yolk tube extension, termed caudal hematopoietic tissue (CHT), using imaging and cell tracing techniques [88, 89]. It was proposed that the function of CHT is
analogous to that of the fetal liver in mammals for supporting denitive hematopoiesis during embryogenesis. By tracing the generation and migration of HSCs using cd41:GFPlow cells, Kissa and colleagues [90] validated the migratory route of HSCs as being AGM to CHT and then to thymus and pronephros. Recently, Hess and Boehm [91] elegantly imaged the process of thymopoiesis in real time in zebrash using triple transgenic lines and their data suggested that AGM is a major source of thymus-settling lymphoid progenitors compared to CHT. Thus, based on the current status of our understanding, denitive hematopoiesis in zebrash occurs in two waves: rst wave produces transient EMPs in the PBI region and second wave produces HSCs in the AGM region that migrate to CHT to support larval denitive hematopoiesis and to thymus and kidney marrow to support adult denitive hematopoiesis. It is not clear if the migration of HSCs from AGM to kidney and thymus is via CHT only or also occurs directly as was previously assumed.
4 precursor for hematopoiesis and vasculogenesis [92, 93]. A complex network of regulatory signals is involved in the specication and lineage commitment of precursors during primitive and denitive hematopoiesis in mammals. These include homeobox, notch, vegf, and wnt signaling pathways as well as specic transcription factors, such as Tal1 (Scl), Lmo2, Gata1, Cmyb, Runx1, Spi1 (Pu.1), and Ikzf1 (Ikaros), which are shown to function in a hierarchical manner [5, 9499]. The importance of proper functioning of these transcription factors is evident from the preponderance of mutations and genomic rearrangements disrupting their activity detected in several blood disorders, particularly leukemias and lymphomas [100106]. Animal models, where level of gene activity can be manipulated, have played a critical role in advancing our understanding of the genetic controls of hematopoiesis. However, knockout mice are embryonic lethal at mid-tolate gestation for Tal1, Lmo2, Gata1, Sfpi1 (Pu.1), Myb, and Runx1, thus precluding the examination of their roles in later stages of hematopoiesis [107112]. Conditional knockout is a useful tool to determine the function of these genes later in life; however, it has been dicult to use this technology to study the initiating events of a lineage, especially for the HSCs, since appropriate promoters to drive Cre recombinase expression may not be available. Zebrash provide an advantage over mouse models due to their ability to survive without blood for several days and are, therefore, a suitable model organism for investigating the eects of loss of function of genes that cause embryonic lethality in mice due to the hematopoietic defects. Here, we discuss the contributions of zebrash mutants, morphants, and transgenic lines to our understanding of the regulatory cascade controlling the hematopoiesis process (Table 1 lists the lineage-specic transgenic lines and genetic mutants in transcription factors involved in regulation of hematopoiesis). The common theme in the studies reviewed below is utilization of the unique features of zebrash embryos and available tools for analysis of the disruptions to the gene activity in an eort to understand the overall process. 3.1. Genes Involved at the Hemangioblast Level: tal1 and lmo2. Based on their expression in both hematopoietic and endothelial cells, and the phenotypes of loss of function animal models, the T-cell acute lymphocytic leukemia 1 (TAL1) and the LIM domain only 2 (LMO2) genes are both believed to function at the hemangioblast level [12, 113]. Both genes were identied from translocations occurring in T-cell acute lymphoblastic leukemia, TAL1 from translocation t(1;14) and LMO2 from translocation t(11;14) [102, 104]. TAL1 is a basic helix-loop-helix (bHLH) transcription factor where the bHLH domain is involved in DNA binding as part of a multiprotein complex that includes LMO2 as a bridging protein. LMO2 belongs to the LMO family of zinc-nger proteins that are characterized by 2 LIM domains, each composed of 2 zinc ngers [104]. Knockout mice for Tal1 and Lmo2 died in utero by embryonic days 9.510.5 (E9.5-10.5) due to lack of embryonic erythropoiesis [108, 112]. Thus, their roles during denitive and adult hematopoiesis were investigated by in vitro colony assays, chimeric mice, and/or conditional
Advances in Hematology knockout mice [114, 115]. Failure to produce any myeloid colonies in vitro from Tal1/ yolk sac cells indicated a block at the EMP level [108]. Using conditional knockout mice, Hall and colleagues [114, 116] demonstrated that adult hematopoiesis can occur independent of Tal1 function with minor defects in erythropoiesis and megakaryopoiesis. On the other hand, Lmo2 was shown to be absolutely necessary for adult hematopoiesis based on the analysis of chimeric mice derived from Lmo2/ embryonic stem cells [115]. In zebrash, tal1 is expressed in the ALM and PLM from 11 hpf and in the posterior ICM at 26 hpf, validating its role in primitive hematopoiesis [39, 117, 118]. First direct proof for the exact site of HSC initiation between the dorsal aorta and the posterior cardinal vein being analogous to AGM in zebrash came from the examination of Tg(tal1-PACGFP) embryos by time lapse imaging [40]. Loss-of-function analyses for tal1 have been performed using morpholinos and a genetic truncation mutation, K183X, which deletes the bHLH domain [38, 119121]. Homozygous mutant embryos (tal1 K 183X/K 183X ) exhibited lack of expression of markers of both primitive and denitive lineages and also lacked visible circulation at 26 hpf [38]. These studies not only conrmed the role of Tal1 during primitive hematopoiesis, but also provided direct evidence for the role of Tal1 in the initiation of denitive hematopoiesis. However, mutant embryos died due to pericardial edema and defects in heart morphogenesis and could not be studied for the role of Tal1 in transition of embryonic to adult stages of denitive hematopoiesis. In zebrash, lmo2 expression in the ALM and PLM is detected about 20 minutes after the tal1 expression and phenotype of lmo2 morphants is very similar to the tal1 morphants, supporting their roles as part of the multiprotein complex during hemangioblast development [41, 122]. To date, no genetic mutants have been reported for lmo2. Overall, zebrash studies have conrmed the strict requirements for Tal1 and Lmo2 in initiation of both primitive and denitive hematopoiesis. 3.2. Genes Involved at the HSC Level: runx1 and cmyb. The onset of denitive hematopoiesis in the AGM is marked by the specication of HSCs, which support hematopoiesis throughout the life of a vertebrate. runx1 and cmyb have been used interchangeably as the earliest markers of denitive hematopoiesis due to their expression in the AGM during HSCs specication [12, 123]. However, we have just begun to elucidate their precise roles in HSCs specication, migration to the sites of larval and adult hematopoiesis, and dierentiation into erythroid, myeloid, and lymphoid lineages. RUNX1 belongs to a family of genes (3 members in mammals and 4 in zebrash) that encode for the alpha subunits of a heterodimeric complex that binds DNA through the highly conserved runt domain. A single gene, CBFB, encodes for the beta subunit, which does not bind to DNA by itself but increases the anity of alpha subunits to bind to DNA after heterodimerization through their runt domains [124]. Promoters of many hematopoietic genes, for example, SPI1 and GATA1, contain RUNX1 DNA binding sites [125127]. RUNX1 was rst identied in the t(8;21) translocation frequently observed in acute myeloid leukemias and its
Advances in Hematology dimerization partner, CBFB, is also frequently involved in genomic rearrangements associated with leukemia [100, 128, 129]. Furthermore, mutations aecting the level of RUNX1 activity leading to loss of function, dominant negative gain of function, and/or overexpression are associated with other blood disorders such as familial platelet disorder with predisposition to acute myeloid leukemia and myelodysplastic syndrome, suggesting that the process of hematopoiesis is very sensitive to the level of RUNX1 activity [130132]. Studies using knockout mouse models demonstrated that Runx1 is essential for the initiation of HSCs generation during denitive hematopoiesis as the mutant mice failed to develop fetal liver hematopoiesis and died in utero at E12.5 [111]. Conditional knockout mice were able to develop all lineages but showed defects in megakaryocyte maturation and dierentiation of B and T cells [133, 134]. Recent elegant fate mapping experiments in mouse embryos by Chen and colleagues demonstrated that Runx1 is required for the emergence of HSCs from the hemogenic endothelium [135]. Taken together, these data suggest a strict requirement of Runx1 in the generation of HSCs to initiate denitive hematopoiesis and in further dierentiation of certain lineages but not for the maintenance of HSCs if they are already produced (reviewed in [73]). Zebrash runx1 was identied based on its high similarity to the human RUNX1 in the runt homology domain [123, 136]. Since then, several studies have validated the critical requirement of Runx1 in the initiation of denitive hematopoiesis by morpholinos and characterization of a variety of hematopoietic mutants [95, 97, 136, 137]. As these studies were performed prior to the recognition of CHT being the site of embryonic denitive hematopoiesis, they did not address Runx1 requirements in specication of EMPs and their transient nature precluded analysis of Runx1 requirements in adult hematopoiesis. None of the hematopoietic mutants from forward genetic screens mapped to the runx1 locus. Therefore, our group performed TILLING to identify a truncation mutation, W84X, in the runt domain of runx1 [42, 43]. Homozygous mutant embryos displayed a complete lack of cells expressing markers of HSCs, denitive erythroid, myeloid, and lymphoid lineages in the CHT and thymus between 35 dpf [42, 43]. However, utilizing Tg(cd41:GFP) transgenic zebrash, we were able to demonstrate that cd41+ cells were formed in the runx1W 84X/W 84X sh in the AGM and CHT regions and migrate to the pronephros, even though they were negative for other HSC markers such as cmyb. Based on the analysis of circulating blood cells, the mutant sh displayed 3 distinct phases: rst phase of normal circulating blood cells until around 68 dpf (presumably from normal primitive hematopoiesis), second phase of bloodless stage until around 20 dpf leading to death in most larvae (defective larval denitive hematopoiesis), and astonishingly, 20% of the mutant larvae resumed blood circulation and grew as phenotypically normal adult sh with multilineage adult hematopoiesis [43]. We do not know exactly how these 20% runx1 mutant larvae were rescued. One possibility is that the cd41+ cells observed in these embryos are hematopoiesis-committed or -primed
5 mesoderm cells, which could restart hematopoiesis in permissive conditions, such as compensation by runx2a, runx2b, and runx3 genes or other genetic and/or epigenetic changes. Another scenario is that two waves of denitive hematopoiesis exist, one for larval and the other adult, while Runx1 is only required for the larval stage. For both scenarios, most larvae died due to lack of circulating blood cells resulting from defective larval hematopoiesis. It is interesting to note that alternate runx1 promoters are used during establishment of EMPs and HSCs (Table 1) as demonstrated recently by Lam and colleagues [44]. Similarly, MYB, a cellular homolog of the V-MYB protooncogene, is a critical transcription factor required for denitive hematopoiesis. A number of mouse models, including conventional and conditional knockouts as well as hypomorphic alleles, have been generated for functional analysis of Myb requirements during hematopoiesis, as discussed in a recent review by Greig and colleagues [109]. These studies have highlighted the key dierence between Runx1 and Myb requirements during denitive hematopoiesis to be the generation of HSCs. Myb knockout mice displayed defects in erythroid and myeloid development and died in utero at E15.5, which is much later than the stage when HSCs are generated [109]. Furthermore, M yb/ ES cells were able to produce T cell progenitors in Rag 1/ chimeric mice [138]. Thus, Myb deciency causes a block in HSCs dierentiation and lineage commitment rather than HSCs specication. Lieu and Reddy [139] demonstrated important contributions of Myb to self-renewal and dierentiation of HSCs during adult hematopoiesis. Recently, two groups reported characterization of loss of function mutants for cmyb in zebrash: (1) allele t25127 with a missense mutation, I181N, aecting DNA binding domain and (2) allele hkz3, a splice site mutation leading to truncation of the transactivation domain. These mutants were identied from forward genetic screens for defects in thymopoiesis and lack of lysozyme C (lyz) expression, respectively [45, 46]. Homozygous embryos for either mutation showed lack of denitive hematopoiesis but behaved dierently with respect to survival. cmyb I 181N/I 181N mutant embryos displayed severe anemia and became bloodless by 20 dpf. Although the mutants survived for 2-3 months with stunted growth, there were no detectable hematopoietic cells by FACS or histology [45]. This is in contrast to our nding with runx1W 84X/W 84X mutants, thus suggesting dierential requirements for runx1 and cmyb activities during larval and adult hematopoiesis. On the other hand, most of the cmyb hkz3 mutants (splice site mutation aecting the transactivation domain) died by 10 dpf. The authors did not explain the reason for this dierence. We speculate that the husbandry dierences between laboratories might be the reason for their dierential survival in the absence of blood cells. Using time-lapse imaging of cmyb hkz3 /Tg(cd41:GFP) embryos and lineage tracing, Zhang and colleagues [46] demonstrated an important role for cmyb in the migration of HSCs from ventral wall of the dorsal aorta (VDA) to CHT, thereby proposing that migratory defects of HSCs maybe the cause of failure of denitive hematopoiesis in cmyb decient embryos. Thus, zebrash models of cmyb deciency have
6 provided novel insights into its role in the migration of HSCs from AGM to CHT during denitive hematopoiesis. 3.3. Genes Involved at the Level of Erythropoiesis, Myelopoiesis, and Lymphopoiesis: gata1, spi1, and ikzf1. Dierentiation of HSCs during denitive hematopoiesis into lineagecommitted progenitors, which further dierentiate into mature blood cells, is mediated by lineage-specic transcription factors [77]. Unlike HSCs, these lineage-committed progenitors lack the potential for self-renewal and thus require a constant supply of HSCs for their production [87, 140]. The rst series of lineage-committed multi-potent progenitors are termed common myeloid and common lymphoid progenitors (CMPs and CLPs). In mammals, CMPs further dierentiate into megakaryocyte-erythroid progenitors (MEPs) that produce mature erythrocytes and platelets (erythropoiesis), and granulocyte/macrophage progenitors (GMPs) for the generation of mature myeloid cells (myelopoiesis). CLPs produce mature lymphoid lineage cells (lymphopoiesis). However, intermediate multilineage progenitors have not been identied in zebrash yet, and all lineage relationships are speculative. Here, we have summarized the genetic controls of erythropoiesis, myelopoiesis, and lymphopoiesis in zebrash. Erythropoiesis involves dierentiation of erythroidmyeloid progenitors into mature erythrocytes and thrombocytes. The master regulator of erythropoiesis is GATA1, a transcription factor belonging to the GATA family (6 members) that contains a conserved DNA binding domain consisting of two zinc ngers [140, 141]. Its consensus DNA binding site, WGATAR, is found in regulatory regions of most erythroid-specic genes [142]. Human mutations in GATA1 are associated with anemia, thrombocytopenia and acute megakaryoblastic leukemia in Down Syndrome patients [143]. Gata1 knockout mouse embryos die by E10.5 due to severe defects in erythropoiesis during primitive hematopoiesis, precluding assessment of its role in denitive hematopoiesis without generating conditional knockout mice [107, 144]. The zebrash gata1 gene was identied by crosshybridization with the zinc-nger region of Xenopus Gata1 [145]. Its expression is consistent with the sites of erythropoiesis during primitive hematopoiesis starting at 5somite stage [49]. Using positional cloning of one of the bloodless mutants, termed vlad tepes or vltm651 , identied in the 1996 large-scale forward screens, our group identied a truncation mutation, R339X, distal to the Cterminal zinc-nger domain in Gata1 [23]. As expected, homozygous mutant embryos displayed defects in primitive erythropoiesis and lacked visible circulating blood cells at the onset of circulation. Evaluation of denitive hematopoiesis by WISH revealed similar defects in erythropoiesis but normal development of myeloid and lymphoid lineages, thus demonstrating the specic role of Gata1 in generation of erythroid progenitor cells not only during primitive but also during denitive hematopoiesis [23, 48]. Myelopoiesis involves dierentiation of erythroid-myeloid progenitors into dierentiated macrophages/monocytes, mast cells, and granulocytes, including neutrophils and
Advances in Hematology eosinophils [9, 80, 82]. The master regulator of myelopoiesis is SPI1 (previously known as PU.1), an oncogene originally identied as the site of genomic rearrangements by spleen focus-forming proviral insertion in erythroblastic tumors [103]. SPI1 belongs to the ETS family of transcription factors that bind DNA through a purine rich sequence, termed the PU box [146]. Sfpi1 knockout mice died around E18 due to multilineage defects, implicating additional roles of Sfpi1 in erythropoiesis and lymphopoiesis [110]. In vitro studies have demonstrated the importance of a negative cross-regulation of Gata1 and Sfpi1 during erythroid and myeloid dierentiation from CMPs [140]. Unlike mammals, the sites of erythropoiesis (PLM) and myelopoiesis (ALM) are separate in zebrash during embryogenesis [50, 51]. However, upregulation of myelopoiesis in gata1 morphants and ectopic expression of gata1 in spi1 morphants proved that similar cross-regulation of these two transcription factors is critical for the proper commitments of erythroid and myeloid lineages in zebrash [147, 148]. Lymphopoiesis involves dierentiation of lymphoid progenitors into mature T and B cells that participate in a functional immune system of the organism [11]. Primary lymphoid organs for T-cell maturation in zebrash are bilateral thymii, which are marked by expression of rag1, ikzf1 and lck starting at 72 hpf [56, 57, 59]. Pancreas has been suggested as an intermediate site for the production of B cells [149] between 4 dpf to 3 weeks, at which point B cells become evident in the kidney. However, this remains to be veried, as no good transgenic markers of B cells currently exist to follow their development in real time. The master regulator of lymphopoiesis is the transcription factor IKZF1 (previously known as IKAROS) [150]. IKZF1 contains six zinc-ngers that are involved in DNA binding and protein-protein interactions [151]. By analysis of knockout mice, Wang and colleagues [152] demonstrated dierential requirements of Ikzf1 for B- and T-cell dierentiation during fetal and adult hematopoiesis. Ikzf1 null mice displayed complete blockage of dierentiation of B cells during both fetal and postnatal stages. On the other hand, they displayed blockage of dierentiation of T cells only during the fetal stage. Postnatal T-cell development recovered, albeit with deregulation of CD4 versus CD8 lineage commitment. Overall, their data suggested that Ikzf1 is essential for lymphopoiesis (both B and T cells) during fetal hematopoiesis, but it is dispensable for adult T cell development. Similar to the knockout mice, zebrash with a truncation mutation, Q360X, in ikzf1 (ikz f 1t24980 ), which removes the C-terminal two zinc ngers essential for protein-protein interactions, are adult viable [58]. Mutant sh displayed complete lack of lymphopoiesis during larval stage, and partial recovery after 14 dpf. Although the mutant sh survived and lived up to at least 17 months in nonsterile conditions, they displayed abnormal and inecient lymphoid development. However, it is interesting to note that similar to our observations of two phases of denitive hematopoiesis in runx1 mutants, zebrash lacking Ikzf1 activity potentially demonstrated two phases of lymphoid development. In both cases, the larval phase is gene activity dependent while the adult phase develops to some extent despite the lack of gene activity.
Advances in Hematology
Erythropoiesis gata1
Myelopoiesis spi1
Lymphopoiesis ikzf1
ICM (PE)
AGM (HSCs)
Kidney marrow (HSCs to myeloid cells, B cells, erythrocytes and thrombocytes) Thymus (T cells)
PBI (EMPs)
Start of circulation 3 11 hpf 24 hpf TD 36 hpf Denitive 3 dpf 5 dpf Adult Primitive
Figure 1: A schematic of overall view of zebrash hematopoiesis with shifting sites, types of cells produced at each site, and genes involved, shown in 3 tiers as described below. Tier 1: lineage-specic transcription factors that control primitive and denitive hematopoiesis in zebrash. Tier 2: the sites of action during each stage of hematopoiesis and the types of cells produced at each of the sites. The site boxes are color matched with waves of hematopoiesis and temporally placed according to the developmental stages in Tier 3. Tier 3: the time scale depicting the stage of development in hpf (hours postfertilization) and dpf (days postfertilization) and dierent waves of hematopoiesis. The abbreviations used are as follows: ALM: anterior lateral mesoderm, PLM: posterior lateral mesoderm, PBI: posterior blood island, AGM: aorta-gonad-mesonephros, CHT: caudal hematopoietic tissue, PM: primitive macrophages, PE: primitive erythrocytes, HSCs: hematopoietic stem cells, TD: transient denitive wave.
4. Different Activity-Levels, Domains, and Isoforms of the Same Transcription Factors Are Required during Different Stages of Hematopoiesis
Recent studies have demonstrated the need to address dosage requirements of transcription factors in the hematopoietic cascade as opposed to a simple on versus o situation [153 156]. In zebrash, it is relatively easy to manipulate gene dosage by careful tuning of morpholino doses and generation of hypomorphic alleles using TILLING. Therefore, dierential requirements for some of the transcription factors either in terms of level of activity or dierent isoforms have been demonstrated recently in zebrash, as discussed below. 4.1. Tal1. As discussed previously, Tal1 plays critical roles during both primitive and denitive hematopoiesis. Using dierent doses of morpholinos to completely or partially abolish Tal1 activity, Juarez and colleagues [120] demonstrated dierential requirements of tal1 expression for erythroid specication and maturation during primitive hematopoiesis. Their work showed that lower activity of Tal1 was sucient for primitive erythroid specication but not their maturation. Furthermore, by complementation experiments with wild-type and DNA-binding mutant forms of Tal1, they demonstrated dierential requirements for the DNA-binding activity of Tal1 during erythroid specication and maturation. Their data suggested dierent mechanisms of target gene regulation during erythrocyte specication
and maturation by Tal1: direct binding to promoters of the target genes involved in erythroid maturation and indirect regulation through other protein complexes for genes involved in erythroid specication. Further complexity to Tal1 requirements during primitive and denitive hematopoiesis became obvious from the analysis of its two isoforms: the full-length form termed Tal1- and a shorter form lacking the rst 146 amino acids, termed Tal1-. Using morpholinos to specically target the and forms, Qian and colleagues [157] demonstrated that both forms act redundantly in initiation of primitive hematopoiesis, while only the Tal1- form is required for the specication of HSCs in the AGM to initiate denitive hematopoiesis. Ren and colleagues [158] examined the requirements of Tal1- and Tal1- during angioblast and HSC specication, also demonstrating the requirement for Tal1- in HSC specication. Thus, zebrash research has contributed signicantly to our understanding of the regulation of dierent stages of hematopoiesis by Tal1. 4.2. Gata1. Similar to Tal1, Gata1 activity is crucial for erythropoiesis during both primitive and denitive hematopoiesis. Recently, we described a hypomorphic allele of Gata1 due to a missense mutation, T301K, in its Cterminal zinc nger [48]. This mutation reduces DNA binding anity and diminishes transactivation of target gene expression by Gata1 [48]. The gat a1T 301K/T 301K sh had defective primitive erythropoiesis but normal denitive hematopoiesis. By combining the T301K allele with the
8 Gata1 null allele of vlad tepes, we were able to generate an allelic series with dierent Gata1 activity levels, listed in the descending order: gat a1+/+ , gat a1+/T 301K , gat a1+/vlt , gat a1T 301K/T 301K , gat a1T 301K/vlt , gat a1vlt/vlt . Analysis of sh with these genotypes demonstrated that erythropoiesis during primitive hematopoiesis requires higher activity level of Gata1 than erythropoiesis and thrombopoiesis during denitive hematopoiesis [48].
Advances in Hematology
[5] C. E. Burns, J. L. Galloway, A. C. H. Smith et al., A genetic screen in zebrash denes a hierarchical network of pathways required for hematopoietic stem cell emergence, Blood, vol. 113, no. 23, pp. 57765782, 2009. [6] C. Thisse and B. Thisse, High-resolution in situ hybridization to whole-mount zebrash embryos, Nature Protocols, vol. 3, no. 1, pp. 5969, 2008. [7] C. E. Willett, A. Cortes, A. Zuasti, and A. G. Zapata, Early hematopoiesis and developing lymphoid organs in the zebrash, Developmental Dynamics, vol. 214, no. 4, pp. 323 336, 1999. [8] G. J. Lieschke, A. C. Oates, M. O. Crowhurst, A. C. Ward, and J. E. Layton, Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrash, Blood, vol. 98, no. 10, pp. 30873096, 2001. [9] J. T. Dobson, J. Seibert, E. M. Teh et al., Carboxypeptidase A5 identies a novel mast cell lineage in the zebrash providing new insight into mast cell fate determination, Blood, vol. 112, no. 7, pp. 29692972, 2008. [10] D. Carradice and G. J. Lieschke, Zebrash in hematology: sushi or science? Blood, vol. 111, no. 7, pp. 33313342, 2008. [11] S. A. Renshaw and N. S. Trede, A model 450 million years in the making: zebrash and vertebrate immunity, Disease Models and Mechanisms, vol. 5, no. 1, pp. 3847, 2012. [12] M. A. Thompson, D. G. Ransom, S. J. Pratt et al., The cloche and spadetail genes dierentially aect hematopoiesis and vasculogenesis, Developmental Biology, vol. 197, no. 2, pp. 248269, 1998. [13] J. L. O. de Jong and L. I. Zon, Use of the zebrash system to study primitive and denitive hematopoiesis, Annual Review of Genetics, vol. 39, pp. 481501, 2005. [14] F. Ellett and G. J. Lieschke, Zebrash as a model for vertebrate hematopoiesis, Current Opinion in Pharmacology, vol. 10, no. 5, pp. 563570, 2010. [15] L. Jingd and L. I. Zon, Zebrash as a model for normal and malignant hematopoiesis, Disease Models and Mechanisms, vol. 4, no. 4, pp. 433438, 2011. [16] W. Driever, L. Solnica-Krezel, A. F. Schier et al., A genetic screen for mutations aecting embryogenesis in zebrash, Development, vol. 123, pp. 3746, 1996. [17] P. Hater, M. Granato, M. Brand et al., The identication of genes with unique and essential functions in the development of the zebrash, Danio rerio, Development, vol. 123, pp. 1 36, 1996. [18] D. G. Ransom, P. Hater, J. Odenthal et al., Characterization of zebrash mutants with defects in embryonic hematopoiesis, Development, vol. 123, pp. 311319, 1996. [19] B. M. Weinstein, A. F. Schier, S. Abdelilah et al., Hematopoietic mutations in the zebrash, Development, vol. 123, pp. 303309, 1996. [20] A. Brownlie, A. Donovan, S. J. Pratt et al., Positional cloning of the zebrash sauternes gene: a model for congenital sideroblastic anaemia, Nature Genetics, vol. 20, no. 3, pp. 244250, 1998. [21] A. Donovan, A. Brownlie, Y. Zhou et al., Positional cloning of zebrash ferroportin1 identies a conserved vertebrate iron exporter, Nature, vol. 403, no. 6771, pp. 776781, 2000. [22] E. C. Liao, B. H. Paw, L. L. Peters et al., Hereditary spherocytosis in zebrash riesling illustrates evolution of erythroid -spectrin structure, and function in red cell morphogenesis and membrane stability, Development, vol. 127, no. 23, pp. 51235132, 2000. [23] S. E. Lyons, N. D. Lawson, L. Lei, P. E. Bennett, B. M. Weinstein, and P. Paul Liu, A nonsense mutation in
5. Concluding Remarks
Depicted in Figure 1 is a schematic of the overall view of zebrash hematopoiesis emerging from these studies. It is clear from the above-mentioned studies that zebrash has played a signicant role in our understanding of the genetic controls of hematopoiesis, particularly the dosagespecic requirements during dierent stages. The viability to adulthood with multi-lineage hematopoiesis in runx1 knockout zebrash clearly demonstrated that Runx1 is dispensable for adult hematopoiesis. Similarly, Ikzf1 was found to be dispensable for adult lymphopoiesis. On the other hand, Cmyb was found to be essential for adult hematopoiesis, while dispensable for larval denitive stage. Genetic mutants need to be generated for spi1 to elucidate its exact role in maintaining proper balance between adult erythropoiesis and myelopoiesis. Proper functioning of the genetic controls regulating hematopoiesis is crucial for normal development of all the blood lineages. Mutations in critical genes at many of the steps lead to leukemogenesis. Thus, adult viable mutant zebrash would allow us to understand the process of leukemogenesis. Furthermore, the recent application of next generation sequencing technologies to a variety of leukemia samples have led to the identication of several new genes mutated in leukemias [159, 160]. We anticipate that understanding their roles in normal hematopoiesis using the many advantages of the zebrash model for hematopoiesis research would aid in therapeutic advances in the coming years.
Acknowledgment
This study was supported by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health.
References
[1] J. Bradbury, Small sh, big science, PLoS Biology, vol. 2, no. 5, article E148, 2004. [2] C. Teh, S. Parinov, and V. Korzh, New ways to admire zebrash: progress in functional genomics research methodology, BioTechniques, vol. 38, no. 6, pp. 897906, 2005. [3] D. Wang, L. E. Jao, N. Zheng et al., Ecient genomewide mutagenesis of zebrash genes by retroviral insertions, Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 30, pp. 1242812433, 2007. [4] B. R. Bill, A. M. Petzold, K. J. Clark, L. A. Schimmenti, and S. C. Ekker, A primer for morpholino use in zebrash, Zebrash, vol. 6, no. 1, pp. 6977, 2009.
Advances in Hematology
zebrash gata1 causes the bloodless phenotype in vlad tepes, Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 8, pp. 54545459, 2002. R. A. Wingert, J. L. Galloway, B. Barut et al., Deciency of glutaredoxin 5 reveals Fe-S clusters are required for vertebrate haem synthesis, Nature, vol. 436, pp. 10351039, 2005. G. C. Shaw, J. J. Cope, L. Li et al., Mitoferrin is essential for erythroid iron assimilation, Nature, vol. 440, no. 7080, pp. 96100, 2006. B. M. Hogan, J. E. Layton, U. J. Pyati et al., Specication of the primitive myeloid precursor pool requires signaling through Alk8 in zebrash, Current Biology, vol. 16, no. 5, pp. 506511, 2006. N. S. Trede, T. Ota, H. Kawasaki et al., Zebrash mutants with disrupted early T-cell and thymus development identied in early pressure screen, Developmental Dynamics, vol. 237, no. 9, pp. 25752584, 2008. M. C. Keightley, J. E. Layton, J. W. Hayman et al., Mediator subunit 12 is required for neutrophil development in zebrash, PloS ONE, vol. 6, no. 8, Article ID e23845, 2011. J. Y. Bertrand and D. Traver, Hematopoietic cell development in the zebrash embryo, Current Opinion in Hematology, vol. 16, no. 4, pp. 243248, 2009. P. Zhang and F. Liu, In vivo imaging of hematopoietic stem cell development in the zebrash, Frontiers of Medicine in China, vol. 5, no. 3, pp. 239247, 2011. C. Pardo-Martin, T. Y. Chang, B. K. Koo, C. L. Gilleland, S. C. Wasserman, and M. F. Yanik, High-throughput in vivo vertebrate screening, Nature Methods, vol. 7, no. 8, pp. 634 636, 2010. O. Renaud, P. Herbomel, and K. Kissa, Studying cell behavior in whole zebrash embryos by confocal live imaging: application to hematopoietic stem cells, Nature Protocols, vol. 6, no. 12, pp. 18971904, 2011. H. F. Lin, D. Traver, H. Zhu et al., Analysis of thrombocyte development in CD41-GFP transgenic zebrash, Blood, vol. 106, no. 12, pp. 38033810, 2005. D. Ma, J. Zhang, H. F. Lin, J. Italiano, and R. I. Handin, The identication and characterization of zebrash hematopoietic stem cells, Blood, vol. 118, no. 2, pp. 289297, 2011. D. L. Stachura, J. R. Reyes, P. Bartunek, B. H. Paw, L. I. Zon, and D. Traver, Zebrash kidney stromal cell lines support multilineage hematopoiesis, Blood, vol. 114, no. 2, pp. 279 289, 2009. D. L. Stachura, O. Svoboda, R. P. Lau et al., Clonal analysis of hematopoietic progenitor cells in the zebrash, Blood, vol. 118, no. 5, pp. 12741282, 2011. D. Traver, B. H. Paw, K. D. Poss, W. T. Penberthy, S. Lin, and L. I. Zon, Transplantation and in vivo imaging of multilineage engraftment in zebrash bloodless mutants, Nature Immunology, vol. 4, no. 12, pp. 12381246, 2003. J. Bussmann, J. Bakkers, and S. Schulte-Merker, Early endocardial morphogenesis requires Scl/Tal1, PLoS Genetics, vol. 3, no. 8, article e140, 2007. H. Jin, J. Xu, F. Qian et al., The 5 zebrash scl promoter targets transcription to the brain, spinal cord, and hematopoietic and endothelial progenitors, Developmental Dynamics, vol. 235, no. 1, pp. 6067, 2006. X. Y. Zhang and A. R. F. Rodaway, SCL-GFP transgenic zebrash: in vivo imaging of blood and endothelial development and identication of the initial site of denitive hematopoiesis, Developmental Biology, vol. 307, no. 2, pp. 179194, 2007.
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[41] H. Zhu, D. Traver, A. J. Davidson et al., Regulation of the lmo2 promoter during hematopoietic and vascular development in zebrash, Developmental Biology, vol. 281, no. 2, pp. 256269, 2005. [42] H. Jin, R. Sood, J. Xu et al., Denitive hematopoietic stem/progenitor cells manifest distinct dierentiation output in the zebrash VDA and PBI, Development, vol. 136, no. 4, pp. 647654, 2009. [43] R. Sood, M. A. English, C. L. Belele et al., Development of multilineage adult hematopoiesis in the zebrash with a runx1 truncation mutation, Blood, vol. 115, no. 14, pp. 28062809, 2010. [44] E. Y. N. Lam, J. Y. M. Chau, M. L. Kalev-Zylinska et al., Zebrash runx1 promoter-EGFP transgenics mark discrete sites of denitive blood progenitors, Blood, vol. 113, no. 6, pp. 12411249, 2009. [45] C. Soza-Ried, I. Hess, N. Netuschil, M. Schorpp, and T. Boehm, Essential role of c-myb in denitive hematopoiesis is evolutionarily conserved, Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 40, pp. 1730417308, 2010. [46] Y. Zhang, H. Jin, L. Li, F. X. F. Qin, and Z. Wen, cMyb regulates hematopoietic stem/progenitor cell mobilization during zebrash hematopoiesis, Blood, vol. 118, no. 15, pp. 40934101, 2011. [47] J. Y. Bertrand, N. C. Chi, B. Santoso, S. Teng, D. Y. R. Stainier, and D. Traver, Haematopoietic stem cells derive directly from aortic endothelium during development, Nature, vol. 464, no. 7285, pp. 108111, 2010. [48] C. L. Belele, M. A. English, J. Chahal et al., Dierential requirement for Gata1 DNA binding and transactivation between primitive and denitive stages of hematopoiesis in zebrash, Blood, vol. 114, no. 25, pp. 51625172, 2009. [49] Q. Long, A. Meng, M. Wang, J. R. Jessen, M. J. Farrell, and S. Lin, GATA-1 expression pattern can be recapitulated in living transgenic zebrash using GFP reporter gene, Development, vol. 124, no. 20, pp. 41054111, 1997. [50] K. Hsu, D. Traver, J. L. Kutok et al., The pu.1 promoter drives myeloid gene expression in zebrash, Blood, vol. 104, no. 5, pp. 12911297, 2004. [51] A. C. Ward, D. O. McPhee, M. M. Condron et al., The zebrash spi1 promoter drives myeloid-specic expression in stable transgenic sh, Blood, vol. 102, no. 9, pp. 32383240, 2003. [52] S. A. Renshaw, C. A. Loynes, D. M. I. Trushell, S. Elworthy, P. W. Ingham, and M. K. B. Whyte, Atransgenic zebrash model of neutrophilic inammation, Blood, vol. 108, no. 13, pp. 39763978, 2006. [53] C. Hall, M. Flores, T. Storm, K. Crosier, and P. Crosier, The zebrash lysozyme C promoter drives myeloid-specic expression in transgenic sh, BMC Developmental Biology, vol. 7, article 42, 2007. [54] F. Ellett, L. Pase, J. W. Hayman, A. Andrianopoulos, and G. J. Lieschke, mpeg1 promoter transgenes direct macrophagelineage expression in zebrash, Blood, vol. 117, no. 4, pp. e49e56, 2011. [55] E. Wienholds, S. Schulte-Merker, B. Walderich, and R. H. A. Plasterk, Target-selected inactivation of the zebrash rag1 gene, Science, vol. 297, no. 5578, pp. 99102, 2002. [56] J. R. Jessen, C. E. Willett, and S. Lin, Articial chromosome transgenesis reveals long-distance negative regulation of rag1 in zebrash, Nature Genetics, vol. 23, no. 1, pp. 1516, 1999. [57] D. M. Langenau, A. A. Ferrando, D. Traver et al., In vivo tracking of T cell development, ablation, and engraftment in
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
10
transgenic zebrash, Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 19, pp. 73697374, 2004. M. Schorpp, M. Bialecki, D. Diekho et al., Conserved functions of Ikaros in vertebrate lymphocyte development: genetic evidence for distinct larval and adult phases of T cell development and two lineages of B cells in zebrash, Journal of Immunology, vol. 177, no. 4, pp. 24632476, 2006. B. Bajoghli, N. Aghaallaei, I. Hess et al., Evolution of genetic networks underlying the emergence of thymopoiesis in vertebrates, Cell, vol. 138, no. 1, pp. 186197, 2009. R. Sood, M. A. English, M. Jones et al., Methods for reverse genetic screening in zebrash by resequencing and TILLING, Methods, vol. 39, no. 3, pp. 220227, 2006. Y. Doyon, J. M. McCammon, J. C. Miller et al., Heritable targeted gene disruption in zebrash using designed zincnger nucleases, Nature Biotechnology, vol. 26, no. 6, pp. 702708, 2008. X. Meng, M. B. Noyes, L. J. Zhu, N. D. Lawson, and S. A. Wolfe, Targeted gene inactivation in zebrash using engineered zinc-nger nucleases, Nature Biotechnology, vol. 26, no. 6, pp. 695701, 2008. P. Huang, A. Xiao, M. Zhou, Z. Zhu, S. Lin, and B. Zhang, Heritable gene targeting in zebrash using customized TALENs, Nature Biotechnology, vol. 29, no. 8, pp. 699700, 2011. J. D. Sander, L. Cade, C. Khayter et al., Targeted gene disruption in somatic zebrash cells using engineered TALENs, Nature Biotechnology, vol. 29, no. 8, pp. 697698, 2011. E. Dzierzak and N. A. Speck, Of lineage and legacy: the development of mammalian hematopoietic stem cells, Nature Immunology, vol. 9, no. 2, pp. 129136, 2008. J. Palis, J. Malik, K. E. McGrath, and P. D. Kingsley, Primitive erythropoiesis in the mammalian embryo, International Journal of Developmental Biology, vol. 54, no. 6-7, pp. 1011 1018, 2010. J. Tober, A. Koniski, K. E. McGrath et al., The megakaryocyte lineage originates from hemangioblast precursors and is an integral component both of primitive and of denitive hematopoiesis, Blood, vol. 109, no. 4, pp. 14331441, 2007. K. E. McGrath, J. M. Frame, G. J. Fromm et al., A transient denitive erythroid lineage with unique regulation of the globin locus in the mammalian embryo, Blood, vol. 117, no. 17, pp. 46004608, 2011. J. Palis, R. J. Chan, A. Koniski, R. Patel, M. Starr, and M. C. Yoder, Spatial and temporal emergence of high proliferative potential hematopoietic precursors during murine embryogenesis, Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 8, pp. 45284533, 2001. H. K. A. Mikkola and S. H. Orkin, The journey of developing hematopoietic stem cells, Development, vol. 133, no. 19, pp. 37333744, 2006. K. E. Rhodes, C. Gekas, Y. Wang et al., The emergence of hematopoietic stem cells is initiated in the placental vasculature in the absence of circulation, Cell Stem Cell, vol. 2, no. 3, pp. 252263, 2008. I. M. Samokhvalov, N. I. Samokhvalova, and S. I. Nishikawa, Cell tracing shows the contribution of the yolk sac to adult haematopoiesis, Nature, vol. 446, no. 7139, pp. 10561061, 2007. G. Swiers, M. de Bruijn, and N. A. Speck, Hematopoietic stem cell emergence in the conceptus and the role of Runx1, International Journal of Developmental Biology, vol. 54, no. 67, pp. 11511163, 2010.
Advances in Hematology
[74] M. J. Chen, Y. Li, M. E. de Obaldia et al., Erythroid/myeloid progenitors and hematopoietic stem cells originate from distinct populations of endothelial cells, Cell Stem Cell, vol. 9, no. 6, pp. 541552, 2011. [75] A. Medvinsky, S. Rybtsov, and S. Taoudi, Embryonic origin of the adult hematopoietic system: advances and questions, Development, vol. 138, no. 6, pp. 10171031, 2011. [76] A. C. Zovein, J. J. Hofmann, M. Lynch et al., Fate tracing reveals the endothelial origin of hematopoietic stem cells, Cell Stem Cell, vol. 3, no. 6, pp. 625636, 2008. [77] S. H. Orkin and L. I. Zon, Hematopoiesis: an evolving paradigm for stem cell biology, Cell, vol. 132, no. 4, pp. 631 644, 2008. [78] L. D. Wang and A. J. Wagers, Dynamic niches in the origination and dierentiation of haematopoietic stem cells, Nature Reviews Molecular Cell Biology, vol. 12, no. 10, pp. 643655, 2011. [79] J. Berman, K. Hsu, and A. T. Look, Zebrash as a model organism for blood diseases, British Journal of Haematology, vol. 123, no. 4, pp. 568576, 2003. [80] J. N. Berman, J. P. Kanki, and A. T. Look, Zebrash as a model for myelopoiesis during embryogenesis, Experimental Hematology, vol. 33, no. 9, pp. 9971006, 2005. [81] A. J. Davidson and L. I. Zon, The denitive (and primitive) guide to zebrash hematopoiesis, Oncogene, vol. 23, no. 43, pp. 72337246, 2004. [82] M. O. Crowhurst, J. E. Layton, and G. J. Lieschke, Developmental biology of zebrash myeloid cells, International Journal of Developmental Biology, vol. 46, no. 4, pp. 483492, 2002. [83] D. Le Guyader, M. J. Redd, E. Colucci-Guyon et al., Origins and unconventional behavior of neutrophils in developing zebrash, Blood, vol. 111, no. 1, pp. 132141, 2008. [84] R. M. Warga, D. A. Kane, and R. K. Ho, Fate mapping embryonic blood in zebrash: multi- and unipotential lineages are segregated at gastrulation, Developmental Cell, vol. 16, no. 5, pp. 744755, 2009. [85] K. Kissa and P. Herbomel, Blood stem cells emerge from aortic endothelium by a novel type of cell transition, Nature, vol. 464, no. 7285, pp. 112115, 2010. [86] J. Y. Bertrand, A. D. Kim, E. P. Violette, D. L. Stachura, J. L. Cisson, and D. Traver, Denitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrash embryo, Development, vol. 134, no. 23, pp. 4147 4156, 2007. [87] A. T. Chen and L. I. Zon, Zebrash blood stem cells, Journal of Cellular Biochemistry, vol. 108, no. 1, pp. 3542, 2009. [88] H. Jin, J. Xu, and Z. Wen, Migratory path of denitive hematopoietic stem/progenitor cells during zebrash development, Blood, vol. 109, no. 12, pp. 52085214, 2007. [89] E. Murayama, K. Kissa, A. Zapata et al., Tracing hematopoietic precursor migration to successive hematopoietic organs during zebrash development, Immunity, vol. 25, no. 6, pp. 963975, 2006. [90] K. Kissa, E. Murayama, A. Zapata et al., Live imaging of emerging hematopoietic stem cells and early thymus colonization, Blood, vol. 111, no. 3, pp. 11471156, 2008. [91] I. Hess and T. Boehm, Intravital imaging of thymopoiesis reveals dynamic lympho-epithelial interactions, Immunity, vol. 36, no. 2, pp. 298309, 2012. [92] J. W. Xiong, Molecular and developmental biology of the hemangioblast, Developmental Dynamics, vol. 237, no. 5, pp. 12181231, 2008.
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72]
[73]
Advances in Hematology
[93] K. M. Vogeli, S. W. Jin, G. R. Martin, and D. Y. R. Stainier, A common progenitor for haematopoietic and endothelial lineages in the zebrash gastrula, Nature, vol. 443, no. 7109, pp. 337339, 2006. [94] B. Argiropoulos and R. K. Humphries, Hox genes in hematopoiesis and leukemogenesis, Oncogene, vol. 26, no. 47, pp. 67666776, 2007. [95] C. E. Burns, D. Traver, E. Mayhall, J. L. Shepard, and L. I. Zon, Hematopoietic stem cell fate is established by the NotchRunx pathway, Genes and Development, vol. 19, no. 19, pp. 23312342, 2005. [96] W. K. Clements, A. D. Kim, K. G. Ong, J. C. Moore, N. D. Lawson, and D. Traver, A somitic Wnt16/Notch pathway species haematopoietic stem cells, Nature, vol. 474, no. 7350, pp. 220224, 2011. [97] M. Gering and R. Patient, Hedgehog signaling is required for adult blood stem cell formation in zebrash embryos, Developmental Cell, vol. 8, no. 3, pp. 389400, 2005. [98] D. Liang, J. R. Chang, A. J. Chin et al., The role of vascular endothelial growth factor (VEGF) in vasculogenesis, angiogenesis, and hematopoiesis in zebrash development, Mechanisms of Development, vol. 108, no. 1-2, pp. 2943, 2001. [99] L. Bugeon, H. B. Taylor, F. Progatzky et al., The NOTCH pathway contributes to cell fate decision in myelopoiesis, Haematologica, vol. 96, no. 12, pp. 17531760, 2011. [100] E. de Braekeleer, N. Douet-Guilbert, F. Morel, M. J. Le Bris, C. F erec, and M. de Braekeleer, RUNX1 translocations and fusion genes in malignant hemopathies, Future Oncology, vol. 7, no. 1, pp. 7791, 2011. [101] N. L. DiFronzo, C. T. Leung, M. K. Mammel, K. Georgopoulos, B. J. Taylor, and Q. N. Pham, Ikaros, a lymphoid-cellspecic transcription factor, contributes to the leukemogenic phenotype of a mink cell focus-inducing murine leukemia virus, Journal of Virology, vol. 76, no. 1, pp. 7887, 2002. [102] E. L ecuyer and T. Hoang, SCL: from the origin of hematopoiesis to stem cells and leukemia, Experimental Hematology, vol. 32, no. 1, pp. 1124, 2004. [103] F. Moreau-Gachelin, A. Tavitian, and P. Tambourin, Spi1 is a putative oncogene in virally induced murine erythroleukaemias, Nature, vol. 331, no. 6153, pp. 277280, 1988. [104] C. H. Nam and T. H. Rabbitts, The role of LMO2 in development and in T Cell leukemia after chromosomal translocation or retroviral insertion, Molecular Therapy, vol. 13, no. 1, pp. 1525, 2006. [105] Y. Shima and I. Kitabayashi, Deregulated transcription factors in leukemia, International Journal of Hematology, vol. 94, no. 2, pp. 134141, 2011. [106] R. Shimizu, J. D. Engel, and M. Yamamoto, GATA1-related leukaemias, Nature Reviews Cancer, vol. 8, no. 4, pp. 279 287, 2008. [107] L. Pevny, M. C. Simon, E. Robertson et al., Erythroid dierentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1, Nature, vol. 349, no. 6306, pp. 257260, 1991. [108] R. A. Shivdasanl, E. L. Mayer, and S. H. Orkin, Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL, Nature, vol. 373, no. 6513, pp. 432 434, 1995. [109] K. T. Greig, S. Carotta, and S. L. Nutt, Critical roles for c-Myb in hematopoietic progenitor cells, Seminars in Immunology, vol. 20, no. 4, pp. 247256, 2008.
11
[110] E. W. Scott, M. C. Simon, J. Anastasi, and H. Singh, Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages, Science, vol. 265, no. 5178, pp. 15731577, 1994. [111] T. Okuda, J. van Deursen, S. W. Hiebert, G. Grosveld, and J. R. Downing, AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis, Cell, vol. 84, no. 2, pp. 321330, 1996. [112] A. J. Warren, W. H. Colledge, M. B. L. Carlton, M. J. Evans, A. J. H. Smith, and T. H. Rabbitts, The oncogenic cysteinerich LIM domain protein rbtn2 is essential for erythroid development, Cell, vol. 78, no. 1, pp. 4557, 1994. [113] S. L. DSouza, A. G. Elefanty, and G. Keller, SCL/Tal-1 is essential for hematopoietic commitment of the hemangioblast but not for its development, Blood, vol. 105, no. 10, pp. 38623870, 2005. [114] M. A. Hall, D. J. Curtis, D. Metcalf et al., The critical regulator of embryonic hematopoiesis, SCL, is vital in the adult for megakaryopoiesis, erythropoiesis, and lineage choice in CFU-S12, Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 3, pp. 992997, 2003. [115] Y. Yamada, A. J. Warren, C. Dobson, A. Forster, R. Pannell, and T. H. Rabbitts, The T cell leukemia LIM protein Lmo2 is necessary for adult mouse hematopoiesis, Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 7, pp. 38903895, 1998. [116] M. A. Hall, N. J. Slater, C. Glenn Begley et al., Functional but abnormal adult erythropoiesis in the absence of the stem cell leukemia gene, Molecular and Cellular Biology, vol. 25, no. 15, pp. 63556362, 2005. [117] M. Gering, A. R. F. Rodaway, B. G ottgens, R. K. Patient, and A. R. Green, The SCL gene species haemangioblast development from early mesoderm, The EMBO Journal, vol. 17, no. 14, pp. 40294045, 1998. [118] E. C. Liao, B. H. Paw, A. C. Oates, S. J. Pratt, J. H. Postlethwait, and L. I. Zon, SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrash, Genes and Development, vol. 12, no. 5, pp. 621626, 1998. [119] K. A. Dooley, A. J. Davidson, and L. I. Zon, Zebrash scl functions independently in hematopoietic and endothelial development, Developmental Biology, vol. 277, no. 2, pp. 522536, 2005. [120] M. A. Juarez, F. Su, S. Chun, M. J. Kiel, and S. E. Lyons, Distinct roles for SCL in erythroid specication and maturation in zebrash, The Journal of Biological Chemistry, vol. 280, no. 50, pp. 4163641644, 2005. [121] L. J. Patterson, M. Gering, and R. Patient, Scl is required for dorsal aorta as well as blood formation in zebrash embryos, Blood, vol. 105, no. 9, pp. 35023511, 2005. [122] L. J. Patterson, M. Gering, C. E. Eckfeldt et al., The transcription factors Scl and Lmo2 act together during development of the hemangioblast in zebrash, Blood, vol. 109, no. 6, pp. 23892398, 2007. [123] H. Kataoka, M. Ochi, K. I. Enomoto, and A. Yamaguchi, Cloning and embryonic expression patterns of the zebrash Runt domain genes, runxa and runxb, Mechanisms of Development, vol. 98, no. 1-2, pp. 139143, 2000. [124] M. Ichikawa, T. Asai, S. Chiba, M. Kurokawa, and S. Ogawa, Runx1/AML-1 ranks as a master regulator of adult hematopoiesis, Cell Cycle, vol. 3, no. 6, pp. 722724, 2004.
12
[125] G. Huang, P. Zhang, H. Hirai et al., PU.1 is a major downstream target of AML1 (RUNX1) in adult mouse hematopoiesis, Nature Genetics, vol. 40, pp. 5160, 2008. [126] J. Michaud, K. M. Simpson, R. Escher et al., Integrative analysis of RUNX1 downstream pathways and target genes, BMC Genomics, vol. 9, article 363, 2008. [127] M. R. Tijssen, A. Cvejic, A. Joshi et al., Genome-wide analysis of simultaneous GATA1/2, RUNX1, FLI1, and SCL binding in megakaryocytes identies hematopoietic regulators, Developmental Cell, vol. 20, no. 5, pp. 597609, 2011. [128] R. K. Hyde and P. P. Liu, RUNX1 repression-independent mechanisms of leukemogenesis by fusion genes CBFBMYH11 and AML1-ETO (RUNX1-RUNX1T1), Journal of Cellular Biochemistry, vol. 110, no. 5, pp. 10391045, 2010. [129] N. A. Sangle and S. L. Perkins, Core-binding factor acute myeloid leukemia, Archives of Pathology & Laboratory Medicine, vol. 135, no. 11, pp. 15041509, 2011. [130] J. K. Mangan and N. A. Speck, RUNX1 mutations in clonal myeloid disorders: from conventional cytogenetics to next generation sequencing, a story 40 years in the making, Critical Reviews in Oncogenesis, vol. 16, no. 1-2, pp. 7791, 2011. [131] M. Osato, N. Asou, E. Abdalla et al., Biallelic and heterozygous point mutations in the runt domain of the AML1/ PEBP2B gene associated with myeloblastic leukemias, Blood, vol. 93, no. 6, pp. 18171824, 1999. [132] S. Schnittger, F. Dicker, W. Kern et al., RUNX1 mutations are frequent in de novo AML with noncomplex karyotype and confer an unfavorable prognosis, Blood, vol. 117, no. 8, pp. 23482357, 2011. [133] J. D. Growney, H. Shigematsu, Z. Li et al., Loss of Runx1 perturbs adult hematopoiesis and is associated with a myeloproliferative phenotype, Blood, vol. 106, no. 2, pp. 494504, 2005. [134] M. Ichikawa, T. Asai, T. Saito et al., AML-1 is required for megakaryocytic maturation and lymphocytic dierentiation, but not for maintenance of hematopoietic stem cells in adult hematopoiesis, Nature Medicine, vol. 10, no. 3, pp. 299304, 2004. [135] M. J. Chen, T. Yokomizo, B. M. Zeigler, E. Dzierzak, and N. A. Speck, Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter, Nature, vol. 457, no. 7231, pp. 887891, 2009. [136] C. E. Burns, T. DeBlasio, Y. Zhou, J. Zhang, L. Zon, and S. D. Nimer, Isolation and characterization of runxa and runxb, zebrash members of the runt family of transcriptional regulators, Experimental Hematology, vol. 30, no. 12, pp. 13811389, 2002. [137] M. L. Kalev-Zylinska, J. A. Horseld, M. V. C. Flores et al., Runx1 is required for zebrash blood and vessel development and expression of a human RUNX-1-CBF2T1 transgene advances a model for studies of leukemogenesis, Development, vol. 129, no. 8, pp. 20152030, 2002. [138] R. D. Allen III, T. P. Bender, and G. Siu, c-Myb is essential for early T cell development, Genes and Development, vol. 13, no. 9, pp. 10731078, 1999. [139] Y. K. Lieu and E. P. Reddy, Conditional c-myb knockout in adult hematopoietic stem cells leads to loss of self-renewal due to impaired proliferation and accelerated dierentiation, Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 51, pp. 2168921694, 2009.
Advances in Hematology
[140] L. C. Dor e and J. D. Crispino, Transcription factor networks in erythroid cell and megakaryocyte development, Blood, vol. 118, no. 2, pp. 231239, 2011. [141] R. Ferreira, K. Ohneda, M. Yamamoto, and S. Philipsen, GATA1 function, a paradigm for transcription factors in hematopoiesis, Molecular and Cellular Biology, vol. 25, no. 4, pp. 12151227, 2005. [142] J. J. Welch, J. A. Watts, C. R. Vakoc et al., Global regulation of erythroid gene expression by transcription factor GATA1, Blood, vol. 104, no. 10, pp. 31363147, 2004. [143] W. A. Ciovacco, W. H. Raskind, and M. A. Kacena, Human phenotypes associated with GATA-1 mutations, Gene, vol. 427, no. 1-2, pp. 16, 2008. [144] Y. Fujiwara, C. P. Browne, K. Cunni, S. C. Go, and S. H. Orkin, Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1, Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 22, pp. 1235512358, 1996. [145] H. W. Detrich III, M. W. Kieran, F. Y. Chan et al., Intraembryonic hematopoietic cell migration during vertebrate development, Proceedings of the National Academy of Sciences of the United States of America, vol. 92, no. 23, pp. 1071310717, 1995. [146] P. Kastner and S. Chan, PU.1: a crucial and versatile player in hematopoiesis and leukemia, International Journal of Biochemistry and Cell Biology, vol. 40, no. 1, pp. 2227, 2008. [147] J. L. Galloway, R. A. Wingert, C. Thisse, B. Thisse, and L. I. Zon, Loss of Gata1 but not Gata2 converts erythropoiesis to myelopoiesis in zebrash embryos, Developmental Cell, vol. 8, no. 1, pp. 109116, 2005. [148] J. Rhodes, A. Hagen, K. Hsu et al., Interplay of pu.1 and Gata1 determines myelo-erythroid progenitor cell fate in zebrash, Developmental Cell, vol. 8, no. 1, pp. 97108, 2005. [149] N. Danilova and L. A. Steiner, B cells develop in the zebrash pancreas, Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 21, pp. 1371113716, 2002. [150] K. Georgopoulos, Haematopoietic cell-fate decisions, chromatin regulation and ikaros, Nature Reviews Immunology, vol. 2, no. 3, pp. 162174, 2002. [151] L. B. John and A. C. Ward, The Ikaros gene family: transcriptional regulators of hematopoiesis and immunity, Molecular Immunology, vol. 48, no. 9-10, pp. 12721278, 2011. [152] J. H. Wang, A. Nichogiannopoulou, L. Wu et al., Selective defects in the development of the fetal and adult lymphoid system in mice with an Ikaros null mutation, Immunity, vol. 5, no. 6, pp. 537549, 1996. [153] N. Novershtern, A. Subramanian, L. N. Lawton et al., Densely interconnected transcriptional circuits control cell states in human hematopoiesis, Cell, vol. 144, no. 2, pp. 296 309, 2011. [154] F. Rosenbauer, K. Wagner, J. L. Kutok et al., Acute myeloid leukemia induced by graded reduction of a lineage-specic transcription factor, PU.1, Nature Genetics, vol. 36, no. 6, pp. 624630, 2004. [155] R. Shimizu, S. Takahashi, K. Ohneda, J. D. Engel, and M. Yamamoto, In vivo requirements for GATA-1 functional domains during primitive and denitive erythropoiesis, The EMBO Journal, vol. 20, no. 18, pp. 52505260, 2001. [156] H. Sakamoto, G. Dai, K. Tsujino et al., Proper levels of cMyb are discretely dened at distinct steps of hematopoietic cell development, Blood, vol. 108, no. 3, pp. 896903, 2006.
Advances in Hematology
[157] F. Qian, F. Zhen, J. Xu, M. Huang, W. Li, and Z. Wen, Distinct functions for dierent scl isoforms in zebrash primitive and denitive hematopoiesis, PLoS Biology, vol. 5, no. 5, article e132, 2007. [158] X. Ren, G. A. Gomez, B. Zhang, and S. Lin, Scl isoforms act downstream of etsrp to specify angioblasts and denitive hematopoietic stem cells, Blood, vol. 115, no. 26, pp. 5338 5346, 2010. [159] T. J. Ley, E. R. Mardis, L. Ding et al., DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome, Nature, vol. 456, no. 7218, pp. 6672, 2008. [160] E. R. Mardis, L. Ding, D. J. Dooling et al., Recurring mutations found by sequencing an acute myeloid leukemia genome, The New England Journal of Medicine, vol. 361, no. 11, pp. 10581066, 2009.
13
Hindawi Publishing Corporation Advances in Hematology Volume 2012, Article ID 358518, 12 pages doi:10.1155/2012/358518
of Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada B3H 3J5 of Pediatrics, Microbiology and Immunology, and Pathology, Dalhousie University, Halifax, NS, Canada B3H 3J5 3 IWK Health Centre, Halifax, NS, Canada B3K 6R8 4 Department of Haematology, UCL Cancer Institute, School of Life and Medical Sciences, University College London, London WC1E 6BT, UK
2 Departments
Correspondence should be addressed to Elspeth M. Payne, [email protected] Received 20 April 2012; Accepted 5 June 2012 Academic Editor: Christopher Hall Copyright 2012 A. Michael Forrester et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Over the past ten years, studies using the zebrash model have contributed to our understanding of vertebrate haematopoiesis, myelopoiesis, and myeloid leukaemogenesis. Novel insights into the conservation of haematopoietic lineages and improvements in our capacity to identify, isolate, and culture such haematopoietic cells continue to enhance our ability to use this simple organism to address disease biology. Coupled with the strengths of the zebrash embryo to dissect developmental myelopoiesis and the continually expanding repertoire of models of myeloid malignancies, this versatile organism has established its niche as a valuable tool to address key questions in the eld of myelopoiesis and myeloid leukaemogenesis. In this paper, we address the recent advances and future directions in the eld of myelopoiesis and leukaemogenesis using the zebrash system.
1. Introduction
The zebrash is emerging as a powerful model system in which to study haematopoiesis and leukaemogenesis. In addition to the benets aorded by scale and simplicity of this versatile genetic model system for studying developmental aspects of haematopoiesis, the last decade has seen an explosion of molecular methods and models to facilitate studies informing on haematopoietic disease biology, particularly leukaemogenesis and cancer. At its inception as a cancer model, proliferation and angiogenesis were proposed as phenotypic attributes as readouts relevant to cancer pathogenesis [1]. However, it was the generation of a transgenic zebrash expressing the C-myc oncogene under the control of the rag2 promoter that went on to develop T-cell acute lymphoblastic leukaemia (ALL), which really revolutionized the view of the scientic world on this small organism as a cancer disease model [2]. In the ensuing 10 years, many models of oncogene induced cancer have been generated in zebrash along with mutagenesis strategies to identify novel tumour suppressor genes or chromosome instability loci [35]. The utility of such models to answer
key biological questions continues to grow. In this paper, we focus on developments in the eld of myelopoiesis in the zebrash, cancer models aecting the myeloid lineages, and how these have instructed our knowledge on the biology of these diseases.
2 blood island (PBI), with the emergence of bipotent erythromyeloid progenitors (EMPs). These cells are marked in their undierentiated state by combined expression of gata1 and lmo2 or by expression of cd41 [6]. These cells have both proliferative and dierentiation potential and increase in number, peaking at 3036 hpf. This wave of haematopoiesis gives rise to further erythrocytes and myeloid cells and recently has been shown to give rise to early mast cells in developing embryos [9]. Multipotent denitive haematopoietic stem cells (HSCs) expressing cd41, c-myb, and runx1 arise directly from kdrl-expressing haemogenic endothelium in the ventral wall of the aorta starting around 2628 hpf [10, 11]. These cells then migrate to the caudal haematopoietic tissue (CHT) where they seed and divide giving rise to all lineages of adult blood cells. These cells go on to populate the adult organs of haematopoiesis in the zebrash, the kidney and the thymus. The precise timing of the move from primitive wave haematopoiesis to denitive wave haematopoiesis has yet to be fully established, but evidence from globin gene expression and mutants with normal primitive wave blood production suggests that the major contribution of haematopoiesis comes from denitive HSC derived cells by around 5 days post fertilization [1214].
Advances in Hematology in all leucocytes [21]. The Tg(lysc:eGFP) expresses GFP from 22 hpf, initially in primitive macrophages arising from the ALPM. Expression of eGFP increases and is notable in the CHT (likely labelling and dierentiating denitive myeloid cells) and the developing brain and retina (more likely to represent the on-going expression in a proportion of macrophages). To clarify precisely which cells express the eGFP from the Tg(lysc:eGFP) transgene, Hall et al. performed anti-GFP staining along with uorescent in situ hybridization for mpx, l-plastin, and fms. Dual staining was observed for eGFP with each of these myeloid transcripts; however, there were some eGFP (lysc) expressing cells that did not express mpx, some fms expressing cells that did not express eGFP (lysc), and some l-plastin expressing cells that did not express eGFP. Thus, the Tg(lysc:eGFP) marks primitive macrophages and a majority of developing granulocytes but does not label all mpx positive granulocytes or all fms expressing macrophages [17]. It is conceivable that these subtleties may in time come to give us more detailed information about subpopulation of myeloid cells, such as their stage of dierentiation. More recently transgenic lines using the mpeg1 or fms (csf1r) promoter [22, 23] have been used to distinguish macrophage populations from granulocytic myeloid cells, further enhancing studies of innate immune system. However, fms reporter animals exhibit expression in neural crest-derived xanthophores as well as macrophages, which may result in some limitations in the use of this system. By contrast, the mpeg1 promoter appears exclusive to macrophages, but expression in adult sh is maintained only in zebrash lines generated using direct transgenic approaches, and not detectable in those lines in which mpeg1 is linked to a GAL4/UAS expression system. To further delineate the expression pattern of macrophages and other mononuclear phagocytes in adult zebrash, a promoter fragment of the MHC class II beta gene, mhc2dab, was isolated. By virtue of the combined transgene expression, the Tg(mhc2dab:eGFP) transgenic line in combination with Tg(CD45:dsRed) (which labels all leukocytes except B cells) has now allowed identication of macrophages and dendritic cells as well as B lymphocytes in adult zebrash tissues [24]. Several recent studies have also delineated additional granulocytic subpopulations. Zebrash mast cells can be identied by expression of the cpa5 transcript, and, like their mammalian counterpart are positive for toluidine blue, express mast cell tryptase and Cd117 at the protein level [25], as well as elements of the Tol-like receptor (TLR) pathway as evidenced by coexpression of cpa5 and GFP in the Tg(myd88:eGFP) transgenic line [26]. These cells have also been isolated after xation by ow cytometry of fast red stained in situ hybridization for cpa5 [27]. The distinction of zebrash mast cells from zebrash eosinophils has also been addressed using a BAC-engineered transgenic line expressing GFP from the gata2 promoter. This study conrmed the presence of and described in detail the characteristics of zebrash eosinophils. In the Tg(gata2:eGFP) line, eosinophils express high levels of eGFP and have high forward and side scatter characteristics by ow cytometry. These cells were also demonstrated to be functionally orthologous to human eosinophils [28]. A summary of transgenic
Advances in Hematology
12 hpf pu.1 PM lysc mpx 24 hpf EMP gata1/lmo2 HSPC cd41 runx1 cmyb CLP CMP Mast cells Macrophage Neutrophils Neutrophils hrocytes y cpa5 cpa5 cp mpeg pu.1 pu.1 Erythrocytes lysc lysc GMP csf1r mpx mpx myd88 myd88 Lymphocytes From 36 hpfadulthood
MEP Erythrocytes
Thrombocytes Thro o
Mast cells Macrophage gata2 mpeg Dendritic cell ell ll d88 myd88 csf1r PHA cpa5 mhc2dab/cd45
Figure 1: Overview of zebrash developmental myelopoiesis, key transgenic lines, and lineage identication tools labelling myeloid cell populations during developmental haematopoiesis. (Transgenic lines are shown in green, other specic lineage identiers are in blue.) PM: primitive myelopoiesis; EMP: erythromyeloid progenitors; HSPCs: haematopoietic stem and progenitor cells; CMP: common myeloid progenitor; CLP: common lymphoid progenitor; MEP: megakaryocyte/erythroid progenitor; GMP: granulocyte/monocyte progenitor; PHA: peanut haemaglutinin. Denotes lineages only demonstrated in adult zebrash. Lineage intermediates are shown for clarity but are yet to be isolated as distinct populations in zebrash.
lines and markers facilitating myeloid populations is shown in Figure 1. As well as facilitating assessment of the ontogeny and spectrum of zebrash haematopoietic and immune systems, the utility of this array of transgenic animals extends to a more functional analysis of zebrash haematopoiesis, which will be particularly useful in zebrash disease models. Once again utilizing cell sorting by ow cytometry, Stachura et al. have established an assay system in which to assess the clonogenic myeloerythroid capability of subpopulations of haematopoietic cells [29]. This recent study utilized traditional clonogenic techniques, commonly used for mammalian haematopoietic cell analysis in methylcellulose, facilitated by recombinant zebrash growth factors, erythropoietin and granulocyte colony stimulating factor and serum derived from carp. Such studies are in their infancy in the zebrash system but should lead the way to further capability to assess clonogenic and lineage potential of individual cells and populations. Critically, this will allow more detailed biological analysis of haematopoietic populations which are currently lacking.
determining haematopoietic lineage fate choice has also been elegantly addressed using this model, using reverse genetics and transplantation techniques. More recently transient heterologous overexpression of mutated human oncogenes has provided some mechanistic insight into the potential pathogenetic eects of such genes on normal developmental haematopoiesis and malignant transformation. In addition functional studies have also addressed aspects of the innate immune system using the zebrash (also reviewed elsewhere in this issue of AIH). What follows is a summary of a selection of studies in zebrash that highlight its diverse and unique capacity to answer a range of biological questions pertaining to myelopoiesis. 4.1. A Myeloid Mutant Identied in a Forward Genetic Screen. Several zebrash studies have identied novel genes involved in myelopoiesis. Bolli et al. identied the grechetto mutant with a mutation within the cpsf1 gene from an early pressure genetic screen for genes involved in denitive myelopoiesis at 5 dpf. On further investigation, grechetto mutants displayed pan-haematopoietic defects, arising from apoptotic cell death of developing haematopoietic stem and progenitor cells (HSPCs). The CPSF1 protein is part of a complex of genes required for processing of the 3 UTR and addition of the poly(A) tail on a subset of pre-mRNAs. CPSF1 specically recognizes a canonical polyadenylation signal within these pre-mRNAs. Bolli et al., showed that in grechetto mutants the transcript encoding the snRNP70 lacked a poly(A) tail [13]. This gene was also identied
4 from a screen for abnormal HSC production [30] and is of particular note because of its role in normal pre-mRNA splicing. Since publication of this report in zebrash, both loss of function and gain of function mutations in several genes required for normal splicing have been identied as contributing to the pathogenesis of human myelodysplastic syndromes (MDS) [31, 32]. 4.2. Lineage Fate Choice Studies. Studies in zebrash embryos have also shed light on the lineage fate decisions during developmental haematopoiesis. Elegant studies of Rhodes and Galloway showed the interplay between the major myeloid and erythroid transcription factors pu.1 and gata-1, respectively, in regulating the fate choice between erythropoiesis and myelopoiesis [33, 34]. Building on these studies Monteiro et al. examined the bloodless moonshine mutant carrying a truncating mutation in the transcription intermediate factor-1 (ti f 1) gene. While previous studies had demonstrated a requirement for ti f 1 in maintenance of primitive erythropoiesis [35], denitive haematopoiesis had not been examined. In this study Monteiro et al. showed that HSPCs are specied and emerge normally from the aorta in moonshine mutants. Subsequently ti f 1 is required for normal erythroid dierentiation in the CHT at 4 dpf, while expression of dierentiated myeloid markers (mpx and l-plastin) were expanded in the same region. Moonshine mutants also showed increased levels of pu.1 and reduced levels of gata-1 at this time in the CHT suggesting that ti f 1 may interplay with these transcription factors in the regulation of myeloid versus erythroid fate in progenitor cells derived from denitive HSCs. To determine whether these ndings may also be relevant to other stages of haematopoietic development, expression of erythroid and myeloid lineage markers were assessed in moonshine mutants along with gata-1 and pu.1 morphants at various time points during developmental haematopoiesis [36]. The authors concluded that ti f 1 modulates the erythromyeloid fate choice by regulating the expression of gata-1 and pu.1, and this regulation showed distinct patterns during specic phases of developmental haematopoiesis. This study demonstrated a novel role for ti f 1 as a regulator of cell fate decisions, and also highlighted the dynamic changes in levels of transcription factors and their interactions that occur during developmental haematopoiesis. A recent study by Li et al., has also addresses lineage fate decisions between the macrophage versus the granulocytic lineages. In this study the interferon regulatory factor 8 (irf8) was identied as a novel regulator of terminal myeloid dierentiation downstream of pu.1, that promoted the development of the macrophage lineage at the expense of neutrophils during primitive and denitive haematopoiesis [37]. Morpholino knockdown of irf8 depleted the number of embryonic macrophages and expanded the neutrophil population with the underlying mechanism determined to be a cellular fate switch. There was no denitive evidence for decreased neutrophil apoptosis or increased proliferation to account for increased neutrophil numbers and doublelabelling of l-plastin and mpx or fms in irf8 morphants
Advances in Hematology revealed a predominance of l-plastin and mpx positive cells. Transgenic overexpression of irf8 achieved through generation of a Tg(hsp70:irf8myc ) transgenic line, promoted macrophage development at the expense of neutrophils [37], but could not rescue macrophage development following pu.1-morpholino injection. Interestingly, Irf8-mutant mice develop a chronic-myelogenous-leukaemia- (CML-) like syndrome with elevated numbers of neutrophils [38, 39]. Taken in this context, this study not only identies a novel role for irf8 in normal myelopoiesis, but also highlights mechanisms that could be possibly hijacked during leukaemogenesis. 4.3. Functional Assessment of Human Leukaemia Mutations Using Developmental Myelopoiesis. Novel insights into the biology of haematopoietic malignancies have also been gained using zebrash models expressing haematopoietic oncogenes as detailed in the subsequent section. However, one recent study has harnessed the developmental myeloid phenotype of a zebrash mutant to functionally interrogate the eects of human nonsynonymous sequence variants (NSVs) found in human acute myeloid leukaemia (AML). In this study ddx18 mutant zebrash were shown to have aberrant myelopoiesis resulting from p53-dependent cell cycle arrest. Sanger sequencing of the DDX18 gene then identied 4 NSVs in samples from patients with AML. Rescue experiments were then performed using the ddx18 mutant zebrash and identied that one of the NSVs appeared to exert a dominant negative eect on developmental myelopoiesis [40]. While this study was based on Sanger sequencing targeting the DDX18 gene, it paves the way to utilize the zebrash for other such strategies to interrogate novel NSVs now being identied in the thousands from whole genome and whole exome sequencing eorts, for functional relevance. Furthermore the value of this strategy will become even more powerful as additional models of existing known leukaemic variants and oncogenes become more prevalent, facilitating combined knockdown/overexpression studies using the existing models to test NSVs. 4.4. Heterologous Overexpression Studies. Overexpression and knockdown studies of myeloid oncogenes and tumour suppressor genes, respectively, have also been informative in studies using the zebrash embryo. The nucleophosmin 1 (NPM1) gene encoding the ubiquitous nucleolar phosphoprotein nucleophosmin is lost in over one-third of patients with AML or MDS associated with loss of chromosome 5q [41]. In addition heterozygous gain-of-function mutations in NPM1 are the most common mutations found in AML accounting for one-third of cases with normal karyotype [42]. Structurally, these mutations result in the generation of a novel nuclear export signal and loss of nucleolar localization signal and thus, in contrast to the normal exclusively nucleolar localization of NPM, mutated NPM is located in the nucleolus, nucleoplasm, and cytoplasm [43]. Furthermore, because NPM contains an oligomerisation domain, NPM mutants relocate at least some of the residual wild-type NPM to the cytoplasm and nucleoplasm. Such NPM mutants
Advances in Hematology have therefore been named NPMc+ to denote their cytoplasmic localization. Heterologous overexpression of the most common NPM1 mutation resulting in NPMc+ (NPM mutant A) was undertaken in a study by Bolli et al. Overexpression of NPMc+ resulted in mislocalization of the zebrash orthologues of NPM1 (npm1a and npm1b) to the cytoplasm indicating that human NPM can oligomerize with the zebrash Npm genes. In addition, primitive myeloid cell numbers were increased, as were c-myb expressing cells in the ventral wall of the aorta and gata1/lmo2 double expressing cells in the CHT. This data suggested that NPMc+ mutant protein led to the expansion of HSPCs as well as developing primitive myeloid and erytho-myeloid progenitor cells [44]. Interestingly, such expansion of myeloid progenitors has also subsequently been demonstrated in a mouse knockin model of NPMc+ mediated leukaemia [45]. 4.5. Innate Immune System. Cells of the myeloid lineage form the principle components of the innate immune system and, as such, production and development of such cells are stimulated upon exposure to pathogens. G-CSF/CSF3 and its receptor, CSF3R, have well-established roles in haematopoiesis, directing myeloid dierentiation of HSCs and proliferation of progenitors [46]. In particular, CSF3 is strongly expressed in response to microbiological toxins in the blood, such as bacterial lipopolysaccharide (LPS), to promote myelopoiesis (especially granulocytes) and cellular migration towards the infection site [47]. Zebrash possess a homologous csf3/csf3r signalling axis that functions similarly to its mammalian counterpart [48]. Overexpression of csf3 mRNA expands embryonic myelopoiesis, but loss of zebrash csf3r blocks myelopoiesis entirely with losses of fms-, lyz-, and mpx-expressing populations. Furthermore, exposing embryos to LPS stimulates csf3 and csf3r expression, and leads to an emergency increase in lyz-expressing granulocytes in a csf3r-dependent manner. Inducible nitric oxide (iNOS/NOS2) signalling also participates in the inammatory response to infection. The zebrash homologue, nos2a, appears to be dispensable for normal formation of HSPCs [49]. However, using morpholinos and L-NAME or L-NMMA (pan-NOS pharmacologic inhibitors), Hall et al. determined that Nos2a protein is required downstream of C/ebp to expand the HSPC population (as evidenced by increased c-myb and runx1 expression) and promote myeloid dierentiation in response to Salmonella infection [50]. In this study, zebrash nos2a appears to primarily favour production of neutrophil granulocytes (evidenced by increased lyz expression). Hall et al. further conrmed the importance of csf3r signalling for emergency myelopoiesis during infection, as csf3r morphants could not mount a myeloid response upon exposure to Salmonella.
5 rate around 11% [3], however the incidence of haematopoietic malignancies is rare. Studies of transgenic zebrash, with tissue specic or ubiquitous promoters driving human or murine oncogenes, have however resulted in faithful models of myeloid leukaemias with features of their human disease counterparts. Below is a summary of the existing models of myeloid leukaemia, the novel ndings such models have contributed to our understanding of human myeloid malignancies and a critique of existing and emerging technologies within this eld. 5.1. K-RAS. Le et al. developed a model of K-RAS-mediated malignant disease by generating a Cre/lox-inducible K -RASG12D allele driven by the -actin promoter. Tg(actin:loxP-eGFP-loxP:K -RASG12D ) zebrash crossed to a zebrash carrying a heat shock promoter (hsp70) driving cre expression resulted in the development of a myeloproliferative neoplasm (MPN) between 34 to 66 days of life, with increased myelomonocytes and myeloid precursors in kidney marrow, and a signicant loss of mature erythrocytes [51]. Notably these malignancies occurred in the absence of any heat shock and were rare in animals that had been exposed to heat shock. Sibling animals exposed to heat shock developed more aggressive, nonhaematopoietic neoplasms such as rhabdomyosarcoma and died as a result of these in early life, suggesting that only low doses of activated K-RAS were necessary to transform haematopoietic cells, or that expression of cre from the hsp70 promoter in the haematopoietic lineage was greater or more leaky than in other tissues. 5.2. MOZ-TIF2. Using the pu.1 promoter to drive transgene expression in myeloid cells, Tg(pu.1:MOZ-TIF2-eGFP) sh were the rst to demonstrate overt AML in zebrash at 14 to 26 months of life, showing an accumulation of immature myelomonocytes in the kidney marrow and a reduction in haematopoietic cells within the spleen [52]. It is notable, however, that both Tg(-actin:K-RAS) and Tg(pu.1:MOZTIF2-EGFP) sh showed a low penetrance of disease, and their underlying molecular mechanisms remain unexplored. 5.3. Tel-jak2a. A handful of studies have provided more mechanistic insight into oncogenic activity in zebrash myelopoiesis. In such a study, Tg(pu.1:FLAG-tel-jak2a) sh utilized a fusion oncogene created from the zebrash orthologues of TEL and JAK2, rather than use of human cDNA [53]. In embryos, tel-jak2a expression leads to an accumulation of large myeloid cells in blood smears, induction of the cell cycle, and a gain in cells expressing the myeloid markers pu.1 and l-plastin at 24 hpf. Interestingly, despite a loss of circulating mature erythrocytes by 48 hpf, Tg(pu.1:FLAG-teljak2a) sh also showed expanded distribution of erythroid markers gata1 and e3-globin at 24 hpf and 48 hpf. This is in keeping with other studies of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling having wide-ranging eects on haematopoiesis in zebrash embryos. For example, mutant chordin zebrash that overexpress jak2a also show upregulation of both erythroid
6 and myeloid genetic markers [54]. This phenotype in chordin mutants could be rescued by injection of jak2a morpholino or pharmacological treatment with the Jak2 inhibitor, AG490, and phenocopied in wild-type embryos by injection of constitutively active jak2a mRNA. This study also suggested that the likely mechanism for the haematopoietic phenotype was hyperphosphorylation of Stat5 because the injection of zebrash stat5 mRNA carrying a hyperactive H298R/N714F mutation led to increases in erythroid, myeloid, and B cell numbers [55]. Similar ndings were observed in a zebrash model of the myeloproliferative disease, polycythemia vera (PCV), where erythroid dysregulation by jak2aV581F mRNA could be rescued by injection of stat5 morpholino [56]. Despite these promising embryonic ndings, however, none of the Tg(pu.1:FLAG-teljak2a) transgenic embryos survived to adulthood [53]. 5.4. NUP98-HOXA9. Recently, our group described a myeloid-specic, Cre/lox-inducible Tg(pu.1:NUP98HOXA9) sh line that exhibits MPN in 23% of sh between 19 and 23 months of life [57]. Despite evidence of myeloid proliferation and delayed cell maturation in kidney marrow, no animals were identied with overt AML. However, mechanistic insights were gained at the embryonic level. Following DNA-damaging irradiation, Tg(pu.1:NUP98HOXA9) embryos showed increased numbers of cells in G2-M transition compared to controls and absence of a normal apoptotic response, which may result from an upregulation of bcl2. Furthermore, embryos showed altered haematopoiesis at 28 hpf, with increased myeloid development marked by pu.1, l-plastin, and lysc, at the expense of erythroid development marked by gata1, suggesting that expression of the NUP98-HOXA9 fusion oncoprotein is capable of altering the cell fate and myeloid cell dierentiation. These early phenotypes in Tg(pu.1:NUP98HOXA9) embryos highlight a potential mechanism whereby HSPCs carrying this oncogene have increased likelihood of acquiring additional mutations due to their impaired DNA damage response and also carry an aberrant population of less dierentiated myeloid cells that may be preferentially targeted and thus may mechanistically account for the predisposition of these sh to develop overt MPNs [57]. 5.5. AML1-ETO. Expression of the AML1-ETO oncogene, driven by the heat shock protein 70 (hsp70) promoter also results in disruption of developmental myelopoiesis in zebrash embryos [58]. In this study, embryos show the appearance of cells with blast-like morphology, as well as upregulation of pu.1 and downregulation of gata1 at 2022 hpf. Interestingly, there was a dierential impact on more mature myeloid lineages, with increased granulocytes marked by mpx, but decreased numbers of cells expressing l-plastin. The transforming mechanism was identied as a downregulation of scl, one of the master transcription factors for embryonic haematopoiesis. All phenotypes were rescued by injecting Tg(hsp70:AML1-ETO) embryos with either scl mRNA or pu.1 morpholino.
Advances in Hematology To date, the Tg(hsp70:AML1-ETO) line represents the most successful use of zebrash to study the molecular biology of myeloid leukaemia. Despite the absence of an overt adult disease phenotype, Tg(hsp70:AML1-ETO) embryos have been an instrumental research tool in the identication of genetic and chemical modiers of myeloid oncogenesis. A subset of human AML cases show deletions on chromosome 9q, which are specically associated with the t(8;21) translocation yielding AML1-ETO. The eects of del(9q) result from the loss of two genes, transducin-like enhancer of split 1 (TLE1) and TLE4, in the Notch signaling pathway. A reverse genetics approach used morpholino knockdown of the zebrash TLE homolog, groucho3, in Tg(hsp70:AML1ETO) embryos to show an acceleration of the haematopoietic phenotype, namely the appearance of blast-like cells, the increase in mpx expression, and a loss of circulating erythrocytes [59]. In human AML, the AML1-ETO oncoprotein disrupts epigenetic programming through recruitment of histone deacetylase complexes (HDAC), which can be pharmacologically targeted by HDAC inhibitors such as trichostatin A (TSA). Taking advantage of this phenotype, Yeh and colleagues used the rescue of gata1 expression by TSA as a proof of principle springboard for a chemical modier screen with a library of known bioactive compounds [60]. Interestingly, they identied COX2 inhibitors, such as NS-398 and indomethacin, as novel therapeutic agents against AML1-ETO, and subsequently demonstrated the critical importance of COX2-prostaglandin E2 signalling through the Wnt/-catenin pathway [61] to the altered haematopoiesis in Tg(hsp70:AML1-ETO) sh. This proved to be an important discoverysoon after, this same pathway and therapeutic strategy was identied in a mouse model of Hoxa9;Meis1-induced AML [62]. 5.6. Technical Challenges and Advances. The reason behind the long latency and low penetrance of overt myeloid leukaemia in zebrash models of this disease may lie in part with the lack of available myeloid-targeted promoters that are active in early blood cells. Even with the success of the pu.1 promoter used in several studies, endogenous zebrash pu.1 expression is downregulated during terminal myeloid dierentiation, and has been found to be active in only 2% of adult haematopoietic kidney marrow cells [16]. This could account for the low incidence of AML in Tg(pu.1:MOZ-TIF2-eGFP) sh and the lack of progression to overt AML in Tg(pu.1:NUP98-HOXA9) sh. Targeted promoters have also proven troublesome in other models of sh leukaemia. Sabaawy et al. showed that expression of the oncogene TEL:AML1 from ubiquitous zebrash -actin and xenopus elongation factor 1 (Xef1) promoters but not early lymphoid targeted sh using the rag2 promoter could produce pre-B (ALL) [63]. Such lessons suggest that the use of promoters that are active earlier in zebrash blood development may prove more robust at driving leukaemic transformation. However, the use of ubiquitous promoters carry the caveat of o-target eects, as seen in Tg(-actin:KRASG12D) sh where MPN was one of a spectrum of disease phenotypes, including rhabdomyosarcoma, intestinal
Advances in Hematology hyperplasia, and malignant peripheral nerve sheath tumours [51]. Potency of the oncogenic signal is another hurdle to successfully modelling leukaemia in sh. For example, Tg(pu.1:FLAG-tel-jak2a) sh as well as the early models of Tg(rag2:eGFP-Myc) sh [2] display such severe abnormalities that animals do not survive to breeding age, and so embryos must be reinjected for every study. Cre/loxinducible strategies can be helpful to establish germline transmission of the oncogene, but historically the most reliable method to control Cre activity was to use the hsp70 promoter, which is known to have leaky expression [51, 57]. This in turn has also suggested that oncogene dosage is likely to have a direct impact on the penetrance and type of malignancies induced as described above for the Tg(-actin:loxPeGFP-loxP:K-RASG12D) [51]. Direct use of the hsp70 promoter to drive oncogene expression has proven fruitful in the study of AML1-ETO, but the absence of an adult phenotype may reect the transience of promoter activity following heat-shock activation. Tamoxifen-inducible Cre recombinase (Cre-ERT2) may allow tighter temporal control of transgene expression [64] and can dramatically improve the leaky expression in Tg(hsp70:Cre) animals [65]. Hans et al. show that, even at temperature ranges of 3742 C, recombination events can be blocked completely in Tg(hsp70:Cre-ERT2) animals if tamoxifen is not applied following heat shock. Other intriguing developments include the generation of zebrash with mosaic expression of oncogenic transgenes [66, 67] allowing more detailed analysis of the eect on oncoprotein expression in individual cells. In mice, the use of lineage-restricted myeloid promoters, for example, CathepsinG [68, 69], Mrp8 [69, 70], has not limited the success of oncogenic transformation and, in fact, committed myeloid progenitor cells have been identied as the leukaemiainitiating cell (LIC) in many karyotypes of AML [6973]. In the zebrash, the use of more lineage-restricted myeloid promoters (i.e., lysc, mpx, mpeg, fms) have ourished in the eld of leukocyte tracking [17, 22, 23, 74] so these may ultimately provide alternative tools for future sh models of myeloid leukaemogenesis. Finally, given that overt AML has been achieved in only one zebrash model to date suggests that the acquisition of mutations within collaborating proto-oncogenes and/or inactivation of tumour suppressor genes may occur less readily in the short life expectancy of the zebrash. Alternatively, the acquisition of disease promoting cooperating mutations may be masked by increased genetic redundancy that has resulted from the additional round of gene duplication undergone in the teleost genome. However, the zebrash is well suited to test specic interactions between collaborating oncogenes due to its high fecundity and thus capacity to generate large number of animals with a range of genotypes, as recently demonstrated in neuroblastoma by Zhu et al. [75]. Transgenic sh harbouring multiple oncogenes have also been a successful strategy for modulating the incidence of zebrash ALL [76]. Thus future strategies to assess the contribution of collaborating mutations could be targeted at overexpression/knockdown strategies of two, three, or four genes.
7 Until recently, stable gene knockout studies of tumour suppressor genes have been dicult to achieve in most zebrash laboratories. While the clinical relevance of such models is apparent from mutant alleles derived from targeting induced local lesions in Genomes (TILLING), such as p53 mutant animals [7779], targeted, heritable gene knockdown in zebrash has been a major challenge for the community over the past decade. The last few years have seen a major sea change with the snowballing of technical advances in this regard. Initial reports of zinc nger nuclease- (ZFN-) induced cleavage and repair resulting in gene knockouts from two groups [80, 81] followed shortly by the publication of the oligomerized pool engineering (OPEN) system for in vitro identication and validation of potential gene targeting zinc ngers by Keith Joungs laboratory [82, 83] have highlighted the potential to harness this technology even in smaller laboratories. Less than 2 years later, the same groups had further rened their in vitro and in silico systems to allow accuracy in identication of target sites using bioinformatics alone [84]. Most recently, evidence has shown that transcriptional activator-like nucleases (TALENs), engineered from DNA binding proteins of the Xanthomonas bacteria function even more faithfully in the zebrash system to target the enzymatic cleavage component of the FOK1 endonuclease to within a few bases of the desired double stranded DNA break [85, 86]. Of course we continue to avidly anticipate the optimization of homologous recombination methodologies to nally permit conditional knockin models of disease.
8
In vivo cell proliferation assay
Advances in Hematology
A B Dissociate embroys to single cell suspension and count uorescent cells B/A = fold increase in cell number
Figure 2: Schematic of in vivo cell proliferation assay in xenotransplanted zebrash embryos. Human leukemia cells are uorescently labelled with a cell tracking dye. Approximately 2550 uorescently labelled cells are microinjected into the yolk sac of 48 hpf casper embryos. Embryos are screened using uorescent microscopy for the presence of a uorescent mass at the site of injection. Positive embryos are divided into two groups; one of which is maintained at 35C for 24 h, and the other group is maintained for until the time point of interest with or without drug exposure. At the end of each time point embryos are enzymatically dissociated to a single cell suspension and the number of uorescent cells in the suspension is counted using a semiautomated macro in Image J (NIH, Bethesda, MD). The number of uorescent cells present at the later time point divided by the number of uorescent cells present at 24 h represents the fold increase in cell number. Adapted from Corkery et al. [90].
A number of anatomic sites in the embryo have been trialled for xenografting, but the yolk sac is generally considered the ideal anatomic location and has been used in the leukaemia xenotransplantation studies to date [90, 91]. Incubation of xenografted embryos at 35 C enables growth of injected human cell lines in a fully constituted, 3D, in vivo microenvironment, without compromising zebrash embryogenesis [89, 90, 92]. Two groups, including ours, have exploited xenotransplantation for the study of myeloid leukaemias [90, 93]. Both groups demonstrated successful engraftment and proliferation of CM-DiI uorescently labelled K562 erythroleukemia and NB4 acute promyelocytic leukaemia (APL) cell lines following yolk sac injection in 48 hpf zebrash embryos. Moreover, response to targeted therapy with imatinib mesylate in K562 cells harbouring the BCR-ABL1 oncoprotein or with all-trans retinoic acid (ATRA), a targeted inhibitor of the PML-RAR oncoprotein found in NB4 cells was observed with the addition of these compounds to the water of xenografted embryos. Pruvot et al. observed a reduction in the number of xenografted K562 cells upon exposure to imatinib and a dose-dependent teratogenic eect and death of NB4 cell xenografted embryos treated with ATRA. Our group have developed a robust ex vivo cell proliferation assay to quantify cell numbers over time following xenotransplantation (Figure 2) and demonstrated that xenografted K562 cells specically responded to imatinib, resulting in decreased cell numbers but no embryonic toxicity. Similar results were obtained with ATRA for xenografted NB4 cells. Importantly, when therapeutic agents
were swapped and applied against the opposite cell type, leukaemia cells continued to proliferate demonstrating that human cancer cells can be specically targeted in a zebrash xenotransplantation model. These studies open the door for using the zebrash xenotransplantation platform to rapidly assess the ecacy of novel compounds on the proliferation of human leukaemia cells in vivo. Xenotransplantation could also enable screens of currently available anticancer agents for o-label, in vivo activity against human leukaemia cells. More recently, as has been demonstrated for some gastrointestinal tumours [94], we have undertaken studies using primary leukaemia patient-derived bone marrow (Tugce Balci, Dale Corkery, Graham Dellaire and Jason Berman, unpublished results). We have seen similar robust engraftment, proliferation, and circulation of primary leukaemia samples and conrmed this process to be an active process, requiring functional living cells, as xed control cells remained in the yolk. Other groups have further demonstrated dierential engraftment of human leukemia subpopulations, with engraftment of CD34+ putative leukaemia stem cells but not from CD34 cells, indicating that zebrash models may reect the biology of disease in a similar way as mouse models and enable studies on tumorigenicity and tumour stem cells [93, 95, 96]. In parallel, with other tools, such as the development of syngeneic sh lines (CG1) [76] and the casper mutant sh line that permanently maintains transparency into adulthood [97], xenotransplantation will enable the zebrash to explore questions of leukemia initiating cell frequency, clonogenicity, and the ability to serially transplant
Advances in Hematology disease. Given the complexity of genetic lesions that can present in AML and the heterogeneity of treatment response inherent in this disease, xenotransplantation models could ultimately be used in real-time analysis of primary patient biopsies as an informative diagnostic tool to predict eective therapeutic regimens and/or inform subsequent preclinical murine studies of promising novel agents, ultimately leading to Phase I clinical trials.
9
[2] D. M. Langenau, D. Traver, A. A. Ferrando et al., Myc-induced T cell leukemia in transgenic zebrash, Science, vol. 299, no. 5608, pp. 887890, 2003. [3] A. Amsterdam, K. C. Sadler, K. Lai et al., Many ribosomal protein genes are cancer genes in zebrash, PLoS Biology, vol. 2, no. 5, article E139, 2004. [4] J. L. Moore, L. M. Rush, C. Breneman, M. A. P. K. Mohideen, and K. C. Cheng, Zebrash genomic instability mutants and cancer susceptibility, Genetics, vol. 174, no. 2, pp. 585600, 2006. [5] J. K. Frazer, N. D. Meeker, L. Rudner et al., Heritable Tcell malignancy models established in a zebrash phenotypic screen, Leukemia, vol. 23, no. 10, pp. 18251835, 2009. [6] J. Y. Bertrand, A. D. Kim, E. P. Violette, D. L. Stachura, J. L. Cisson, and D. Traver, Denitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrash embryo, Development, vol. 134, no. 23, pp. 4147 4156, 2007. [7] P. Herbomel, B. Thisse, and C. Thisse, Ontogeny and behaviour of early macrophages in the zebrash embryo, Development, vol. 126, no. 17, pp. 37353745, 1999. [8] M. O. Crowhurst, J. E. Layton, and G. J. Lieschke, Developmental biology of zebrash myeloid cells, International Journal of Developmental Biology, vol. 46, no. 4, pp. 483492, 2002. [9] S. I. Daas, A. J. Coombs, T. B. Balci, C. A. Grondin, A. A. Ferrando, and J. N. Berman, The zebrash reveals dependence of the mast cell lineage on Notch signaling in vivo, Blood, vol. 119, pp. 35853594, 2012. [10] K. Kissa and P. Herbomel, Blood stem cells emerge from aortic endothelium by a novel type of cell transition, Nature, vol. 464, no. 7285, pp. 112115, 2010. [11] J. Y. Bertrand, N. C. Chi, B. Santoso, S. Teng, D. Y. R. Stainier, and D. Traver, Haematopoietic stem cells derive directly from aortic endothelium during development, Nature, vol. 464, no. 7285, pp. 108111, 2010. [12] F. Y. Chan, J. Robinson, A. Brownlie et al., Characterization of adult - and -globin genes in the zebrash, Blood, vol. 89, no. 2, pp. 688700, 1997. [13] N. Bolli, E. M. Payne, J. Rhodes et al., Cpsf1 is required for denitive HSC survival in zebrash, Blood, vol. 117, no. 15, pp. 39964007, 2011. [14] L. Du, J. Xu, X. Li et al., Rumba and Haus3 are essential factors for the maintenance of hematopoietic stem/progenitor cells during zebrash hematopoiesis, Development, vol. 138, no. 4, pp. 619629, 2011. [15] A. C. Ward, D. O. McPhee, M. M. Condron et al., The zebrash spi1 promoter drives myeloid-specic expression in stable transgenic sh, Blood, vol. 102, no. 9, pp. 32383240, 2003. [16] K. Hsu, D. Traver, J. L. Kutok et al., The pu.1 promoter drives myeloid gene expression in zebrash, Blood, vol. 104, no. 5, pp. 12911297, 2004. [17] C. Hall, M. Flores, T. Storm, K. Crosier, and P. Crosier, The zebrash lysozyme C promoter drives myeloid-specic expression in transgenic sh, BMC Developmental Biology, vol. 7, article 42, 2007. [18] S. A. Renshaw, C. A. Loynes, D. M. I. Trushell, S. Elworthy, P. W. Ingham, and M. K. B. Whyte, Atransgenic zebrash model of neutrophilic inammation, Blood, vol. 108, no. 13, pp. 39763978, 2006. [19] J. R. Mathias, M. E. Dodd, K. B. Walters et al., Live imaging of chronic inammation caused by mutation of zebrash
References
[1] J. F. Amatruda, J. L. Shepard, H. M. Stern, and L. I. Zon, Zebrash as a cancer model system, Cancer Cell, vol. 1, no. 3, pp. 229231, 2002.
10
Hai1, Journal of Cell Science, vol. 120, no. 19, pp. 33723383, 2007. C. Hall, M. V. Flores, A. Chien, A. Davidson, K. Crosier, and P. Crosier, Transgenic zebrash reporter lines reveal conserved Toll-like receptor signaling potential in embryonic myeloid leukocytes and adult immune cell lineages, Journal of Leukocyte Biology, vol. 85, no. 5, pp. 751765, 2009. D. Le Guyader, M. J. Redd, E. Colucci-Guyon et al., Origins and unconventional behavior of neutrophils in developing zebrash, Blood, vol. 111, no. 1, pp. 132141, 2008. F. Ellett, L. Pase, J. W. Hayman, A. Andrianopoulos, and G. J. Lieschke, mpeg1 promoter transgenes direct macrophagelineage expression in zebrash, Blood, vol. 117, no. 4, pp. e49e56, 2011. C. Gray, C. A. Loynes, M. K. B. Whyte, D. C. Crossman, S. A. Renshaw, and T. J. A. Chico, Simultaneous intravital imaging of macrophage and neutrophil behaviour during inammation using a novel transgenic zebrash, Thrombosis and Haemostasis, vol. 105, no. 5, pp. 811819, 2011. V. Wittamer, J. Y. Bertrand, P. W. Gutschow, and D. Traver, Characterization of the mononuclear phagocyte system in zebrash, Blood, vol. 117, no. 26, pp. 71267135, 2011. J. T. Dobson, J. Seibert, E. M. Teh et al., Carboxypeptidase A5 identies a novel mast cell lineage in the zebrash providing new insight into mast cell fate determination, Blood, vol. 112, no. 7, pp. 29692972, 2008. S. Daas, E. M. Teh, J. T. Dobson et al., Zebrash mast cells possess an FceopenRI-like receptor and participate in innate and adaptive immune responses, Developmental and Comparative Immunology, vol. 35, no. 1, pp. 125134, 2011. J. T. Dobson, S. Daas, E. R. McBride, and J. N. Berman, Fluorescence-activated cell sorting (FACS) of whole mount in situ hybridization (WISH) labelled haematopoietic cell populations in the zebrash, British Journal of Haematology, vol. 144, no. 5, pp. 732735, 2009. K. M. Balla, G. Lugo-Villarino, J. M. Spitsbergen et al., Eosinophils in the zebrash: prospective isolation, characterization, and eosinophilia induction by helminth determinants, Blood, vol. 116, no. 19, pp. 39443954, 2010. D. L. Stachura, O. Svoboda, R. P. Lau et al., Clonal analysis of hematopoietic progenitor cells in the zebrash, Blood, vol. 118, pp. 12741282, 2011. C. E. Burns, J. L. Galloway, A. C. H. Smith et al., A genetic screen in zebrash denes a hierarchical network of pathways required for hematopoietic stem cell emergence, Blood, vol. 113, no. 23, pp. 57765782, 2009. T. A. Graubert, D. Shen, L. Ding et al., Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes, Nature Genetics, vol. 44, pp. 5357, 2012. E. Papaemmanuil, M. Cazzola, J. Boultwood et al., Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts, The New England Journal of Medicine, vol. 365, pp. 1384 1395, 2011. J. L. Galloway, R. A. Wingert, C. Thisse, B. Thisse, and L. I. Zon, Loss of Gata1 but not Gata2 converts erythropoiesis to myelopoiesis in zebrash embryos, Developmental Cell, vol. 8, no. 1, pp. 109116, 2005. J. Rhodes, A. Hagen, K. Hsu et al., Interplay of pu.1 and Gata1 determines myelo-erythroid progenitor cell fate in zebrash, Developmental Cell, vol. 8, no. 1, pp. 97108, 2005. D. G. Ransom, N. Bahary, K. Niss et al., The Zebrash moonshine gene encodes transcriptional intermediary factor 1, an essential regulator of hematopoiesis, PLoS Biology, vol. 2, no. 8, article E237, 2004.
Advances in Hematology
[36] R. Monteiro, C. Pouget, and R. Patient, The gata1/pu.1 lineage fate paradigm varies between blood populations and is modulated by tif1, The EMBO Journal, vol. 30, no. 6, pp. 10931103, 2011. [37] L. Li, H. Jin, J. Xu, Y. Shi, and Z. Wen, Irf8 regulates macrophage versus neutrophil fate during zebrash primitive myelopoiesis, Blood, vol. 117, no. 4, pp. 13591369, 2011. [38] T. Holtschke, J. L ohler, Y. Kanno et al., Immunodeciency and chronic myelogenous leukemia-like syndrome in mice with a targeted mutation of the ICSBP gene, Cell, vol. 87, no. 2, pp. 307317, 1996. [39] K. Turcotte, S. Gauthier, A. Tuite, A. Mullick, D. Malo, and P. Gros, A mutation in the Icsbp1 gene causes susceptibility to infection and a chronic myeloid leukemia - Like syndrome in BXH-2 mice, Journal of Experimental Medicine, vol. 201, no. 6, pp. 881890, 2005. [40] E. M. Payne, N. Bolli, J. Rhodes et al., Ddx18 is essential for cell-cycle progression in zebrash hematopoietic cells and is mutated in human AML, Blood, vol. 118, no. 4, pp. 903915, 2011. [41] S. Heinrichs, R. V. Kulkarni, C. E. Bueso-Ramos et al., Accurate detection of uniparental disomy and microdeletions by SNP array analysis in myelodysplastic syndromes with normal cytogenetics, Leukemia, vol. 23, no. 9, pp. 1605 1613, 2009. [42] B. Falini, C. Mecucci, E. Tiacci et al., Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype, The New England Journal of Medicine, vol. 352, no. 3, pp. 254266, 2005. [43] B. Falini, B. Bigerna, A. Pucciarini et al., Aberrant subcellular expression of nucleophosmin and NPM-MLF1 fusion protein in acute myeloid leukaemia carrying t(3;5): a comparison with NPMc+ AML, Leukemia, vol. 20, no. 2, pp. 368371, 2006. [44] N. Bolli, E. M. Payne, C. Grabher et al., Expression of the cytoplasmic NPM1 mutant (NPMc+) causes the expansion of hematopoietic cells in zebrash, Blood, vol. 115, no. 16, pp. 33293340, 2010. [45] G. S. Vassiliou, J. L. Cooper, R. Rad et al., Mutant nucleophosmin and cooperating pathways drive leukemia initiation and progression in mice, Nature Genetics, vol. 43, no. 5, pp. 470476, 2011. [46] R. Beekman and I. P. Touw, G-CSF and its receptor in myeloid malignancy, Blood, vol. 115, no. 25, pp. 51315136, 2010. [47] D. R. Barreda, P. C. Hanington, and M. Belosevic, Regulation of myeloid development and function by colony stimulating factors, Developmental and Comparative Immunology, vol. 28, no. 5, pp. 509554, 2004. [48] C. Liongue, C. J. Hall, B. A. OConnell, P. Crosier, and A. C. Ward, Zebrash granulocyte colony-stimulating factor receptor signaling promotes myelopoiesis and myeloid cell migration, Blood, vol. 113, no. 11, pp. 25352546, 2009. [49] T. E. North, W. Goessling, M. Peeters et al., Hematopoietic stem cell development is dependent on blood ow, Cell, vol. 137, no. 4, pp. 736748, 2009. [50] C. J. Hall, M. V. Flores, S. H. Oehlers et al., Infectionresponsive expansion of the hematopoietic stem and progenitor cell compartment in zebrash is dependent upon inducible nitric oxide, Cell Stem Cell, vol. 10, pp. 198209, 2012. [51] M. Lessard, C. H elias, S. Struski et al., Fluorescence in situ hybridization analysis of 110 hematopoietic disorders
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
Advances in Hematology
with chromosome 5 abnormalities: do de novo and therapyrelated myelodysplastic syndrome-acute myeloid leukemia actually dier? Cancer Genetics and Cytogenetics, vol. 176, no. 1, pp. 121, 2007. J. Zhuravleva, J. Paggetti, L. Martin et al., MOZ/TIF2induced acute myeloid leukaemia in transgenic sh, British Journal of Haematology, vol. 143, no. 3, pp. 378382, 2008. S. M. N. Onnebo, M. M. Condron, D. O. McPhee, G. J. Lieschke, and A. C. Ward, Hematopoietic perturbation in zebrash expressing a tel-jak2a fusion, Experimental Hematology, vol. 33, no. 2, pp. 182188, 2005. A. C. H. Ma, A. C. Ward, R. Liang, and A. Y. H. Leung, The role of jak2a in zebrash hematopoiesis, Blood, vol. 110, no. 6, pp. 18241830, 2007. R. S. Lewis, S. E. M. Stephenson, and A. C. Ward, Constitutive activation of zebrash Stat5 expands hematopoietic cell populations in vivo, Experimental Hematology, vol. 34, no. 2, pp. 179187, 2006. A. C. H. Ma, A. Fan, A. C. Ward et al., A novel zebrash jak2aV581F model shared features of human JAK2V617F polycythemia vera, Experimental Hematology, vol. 37, no. 12, pp. 1379e4, 2009. A. M. Forrester, C. Grabher, E. R. Mcbride et al., NUP98HOXA9-transgenic zebrash develop a myeloproliferative neoplasm and provide new insight into mechanisms of myeloid leukaemogenesis, British Journal of Haematology, vol. 55, no. 2, pp. 167168, 2011. J. R. J. Yeh, K. M. Munson, Y. L. Chao, Q. P. Peterson, C. A. MacRae, and R. T. Peterson, AML1-ETO reprograms hematopoietic cell fate by downregulating scl expression, Development, vol. 135, no. 2, pp. 401410, 2008. F. Dayyani, J. Wang, J. R. J. Yeh et al., Loss of TLE1 and TLE4 from the del(9q) commonly deleted region in AML cooperates with AML1-ETO to aect myeloid cell proliferation and survival, Blood, vol. 111, no. 8, pp. 4338 4347, 2008. J. R. J. Yeh, K. M. Munson, K. E. Elagib, A. N. Goldfarb, D. A. Sweetser, and R. T. Peterson, Discovering chemical modiers of oncogene-regulated hematopoietic dierentiation, Nature Chemical Biology, vol. 5, no. 4, pp. 236243, 2009. T. E. North, W. Goessling, C. R. Walkley et al., Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis, Nature, vol. 447, no. 7147, pp. 10071011, 2007. Y. Wang, A. V. Krivtsov, A. U. Sinha et al., The wnt/-catenin pathway is required for the development of leukemia stem cells in AML, Science, vol. 327, no. 5973, pp. 16501653, 2010. H. E. Sabaawy, M. Azuma, L. J. Embree, H. J. Tsai, M. F. Starost, and D. D. Hickstein, TEL-AML1 transgenic zebrash model of precursor B cell lymphoblastic leukemia, Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 41, pp. 1516615171, 2006. C. Mosimann, C. K. Kaufman, P. Li, E. K. Pugach, O. J. Tamplin, and L. I. Zon, Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrash, Development, vol. 138, no. 1, pp. 169177, 2011. S. Hans, D. Freudenreich, M. Gearth, J. Kaslin, A. Machate, and M. Brand, Generation of a non-leaky heat shockinducible Cre line for conditional Cre/lox strategies in zebrash, Developmental Dynamics, vol. 240, no. 1, pp. 108 115, 2011. E. Y. Chen and D. M. Langenau, Zebrash models of rhabdomyosarcoma, Methods in Cell Biology, vol. 105, pp. 383402, 2011.
11
[67] D. M. Langenau, M. D. Keefe, N. Y. Storer et al., Co-injection strategies to modify radiation sensitivity and tumor initiation in transgenic zebrash, Oncogene, vol. 27, no. 30, pp. 4242 4248, 2008. [68] M. Iwasaki, T. Kuwata, Y. Yamazaki et al., Identication of cooperative genes for NUP98-HOXA9 in myeloid leukemogenesis using a mouse model, Blood, vol. 105, no. 2, pp. 784 793, 2005. [69] S. Wojiski, F. C. Guibal, T. Kindler et al., PML-RAR initiates leukemia by conferring properties of self-renewal to committed promyelocytic progenitors, Leukemia, vol. 23, no. 8, pp. 14621471, 2009. [70] F. C. Guibal, M. Alberich-Jorda, H. Hirai et al., Identication of a myeloid committed progenitor as the cancerinitiating cell in acute promyelocytic leukemia, Blood, vol. 114, no. 27, pp. 54155425, 2009. [71] A. V. Krivtsov, D. Twomey, Z. Feng et al., Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9, Nature, vol. 442, no. 7104, pp. 818822, 2006. [72] A. Cozzio, E. Passegu e, P. M. Ayton, H. Karsunky, M. L. Cleary, and I. L. Weissman, Similar MLL-associated leukemias arising from self-renewing stem cells and shortlived myeloid progenitors, Genes and Development, vol. 17, no. 24, pp. 30293035, 2003. [73] B. J. P. Huntly, H. Shigematsu, K. Deguchi et al., MOZ-TIF2, but not BCR-ABL, confers properties of leukemic stem cells to committed murine hematopoietic progenitors, Cancer Cell, vol. 6, no. 6, pp. 587596, 2004. [74] P. M. Elks, F. J. Van Eeden, G. Dixon et al., Activation of hypoxia-inducible factor-1 (hif-1) delays inammation resolution by reducing neutrophil apoptosis and reverse migration in a zebrash inammation model, Blood, vol. 118, no. 3, pp. 712722, 2011. [75] S. Zhu, J. S. Lee, F. Guo et al., Activated ALK collaborates with MYCN in neuroblastoma pathogenesis, Cancer Cell, vol. 21, pp. 362373, 2012. [76] A. C. H. Smith, A. R. Raimondi, C. D. Salthouse et al., Highthroughput cell transplantation establishes that tumorinitiating cells are abundant in zebrash T-cell acute lymphoblastic leukemia, Blood, vol. 115, no. 16, pp. 32963303, 2010. [77] S. Berghmans, R. D. Murphey, E. Wienholds et al., tp53 mutant zebrash develop malignant peripheral nerve sheath tumors, Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 2, pp. 407412, 2005. [78] J. M. Parant, S. A. George, J. A. Holden, and H. J. Yost, Genetic modeling of Li-Fraumeni syndrome in zebrash, DMM Disease Models and Mechanisms, vol. 3, no. 1-2, pp. 4556, 2010. [79] S. Sidi, T. Sanda, R. D. Kennedy et al., Chk1 suppresses a caspase-2 apoptotic response to DNA damage that bypasses p53, Bcl-2, and caspase-3, Cell, vol. 133, no. 5, pp. 864877, 2008. [80] X. Meng, M. B. Noyes, L. J. Zhu, N. D. Lawson, and S. A. Wolfe, Targeted gene inactivation in zebrash using engineered zinc-nger nucleases, Nature Biotechnology, vol. 26, no. 6, pp. 695701, 2008. [81] Y. Doyon, J. M. McCammon, J. C. Miller et al., Heritable targeted gene disruption in zebrash using designed zincnger nucleases, Nature Biotechnology, vol. 26, no. 6, pp. 702708, 2008. [82] M. L. Maeder, S. Thibodeau-Beganny, A. Osiak et al., Rapid open-source engineering of customized zinc-nger
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
12
nucleases for highly ecient gene modication, Molecular Cell, vol. 31, no. 2, pp. 294301, 2008. M. L. Maeder, S. Thibodeau-Beganny, J. D. Sander, D. F. Voytas, and J. K. Joung, Oligomerized pool engineering (OPEN): an open-source protocol for making customized zinc-nger arrays, Nature Protocols, vol. 4, no. 10, pp. 1471 1501, 2009. J. D. Sander, E. J. Dahlborg, M. J. Goodwin et al., Selectionfree zinc-nger-nuclease engineering by context-dependent assembly (CoDA), Nature Methods, vol. 8, no. 1, pp. 6769, 2011. J. D. Sander, L. Cade, C. Khayter et al., Targeted gene disruption in somatic zebrash cells using engineered TALENs, Nature Biotechnology, vol. 29, no. 8, pp. 697698, 2011. P. Huang, A. Xiao, M. Zhou, Z. Zhu, S. Lin, and B. Zhang, Heritable gene targeting in zebrash using customized TALENs, Nature Biotechnology, vol. 29, no. 8, pp. 699700, 2011. E. Y. N. Lam, C. J. Hall, P. S. Crosier, K. E. Crosier, and M. V. Flores, Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells, Blood, vol. 116, no. 6, pp. 909914, 2010. M. Konantz, T. B. Balci, U. G. Hartwig et al., Zebrash xenografts as a tool for in vivostudies on human cancer, Annals of the New York Academy of Sciences. In press. S. H. Lam, H. L. Chua, Z. Gong, T. J. Lam, and Y. M. Sin, Development and maturation of the immune system in zebrash, Danio rerio: a gene expression proling, in situ hybridization and immunological study, Developmental and Comparative Immunology, vol. 28, no. 1, pp. 928, 2004. D. P. Corkery, G. Dellaire, and J. N. Berman, Leukaemia xenotransplantation in zebrashchemotherapy response assay in vivo, British Journal of Haematology, vol. 153, no. 6, pp. 786789, 2011. M. Haldi, C. Ton, W. L. Seng, and P. McGrath, Human melanoma cells transplanted into zebrash proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrash, Angiogenesis, vol. 9, no. 3, pp. 139151, 2006. L. M. J. Lee, E. A. Seftor, G. Bonde, R. A. Cornell, and M. J. C. Hendrix, The fate of human malignant melanoma cells transplanted into zebrash embryos: assessment of migration and cell division in the absence of tumor formation, Developmental Dynamics, vol. 233, no. 4, pp. 15601570, 2005. B. Pruvot, A. Jacquel, N. Droin et al., Leukemic cell xenograft in zebrash embryo for investigating drug ecacy, Haematologica, vol. 96, no. 4, pp. 612616, 2011. I. J. Marques, F. U. Weiss, D. H. Vlecken et al., Metastatic behaviour of primary human tumours in a zebrash xenotransplantation model, BMC Cancer, vol. 9, article 128, 2009. D. Bonnet and J. E. Dick, Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell, Nature Medicine, vol. 3, no. 7, pp. 730 737, 1997. J. C. Y. Wang, T. Lapidot, J. D. Cashman et al., High level engraftment of NOD/SCID mice by primitive normal and leukemic hematopoietic cells from patients with chronic myeloid leukemia in chronic phase, Blood, vol. 91, no. 7, pp. 24062414, 1998. R. M. White, A. Sessa, C. Burke et al., Transparent adult zebrash as a tool for in vivo transplantation analysis, Cell Stem Cell, vol. 2, no. 2, pp. 183189, 2008.
Advances in Hematology
[98] D. G. Ransom, P. Hater, J. Odenthal et al., Characterization of zebrash mutants with defects in embryonic hematopoiesis, Development, vol. 123, pp. 311319, 1996. [99] B. M. Weinstein, A. F. Schier, S. Abdelilah et al., Hematopoietic mutations in the zebrash, Development, vol. 123, pp. 303309, 1996. [100] A. Donovan, A. Brownlie, Y. Zhou et al., Positional cloning of zebrash ferroportin1 identies a conserved vertebrate iron exporter, Nature, vol. 403, no. 6771, pp. 776781, 2000. [101] R. Peravali, J. Gehrig, S. Giselbrecht et al., Automated feature detection and imaging for high-resolution screening of zebrash embryos, BioTechniques, vol. 50, no. 5, pp. 319 324, 2011.
[83]
[84]
[85]
[86]
[87]
[88]
[89]
[90]
[91]
[92]
[93]
[94]
[95]
[96]
[97]
Hindawi Publishing Corporation Advances in Hematology Volume 2012, Article ID 398640, 11 pages doi:10.1155/2012/398640
Doctoral Training Program and Medical Scientist Training Program, University of Wisconsin-Madison, Madison, WI 53706, USA 2 Department of Pediatrics and Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53706, USA Correspondence should be addressed to Anna Huttenlocher, [email protected] Received 3 February 2012; Accepted 8 May 2012 Academic Editor: Christopher Hall Copyright 2012 T. W. Starnes and A. Huttenlocher. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The precise control of neutrophil-mediated inammation is critical for both host defense and the prevention of immunopathology. In vivo imaging studies in zebrash, and more recently in mice, have made the novel observation that neutrophils leave a site of inammation through a process called neutrophil reverse migration. The application of advanced imaging techniques to the genetically tractable, optically transparent zebrash larvae was critical for these advances. Still, the mechanisms underlying neutrophil reverse migration and its eects on the resolution or priming of immune responses remain unclear. Here, we review the current knowledge of neutrophil reverse migration, its potential roles in host immunity, and the live imaging tools that make zebrash a valuable model for increasing our knowledge of neutrophil behavior in vivo.
1. Introduction
Certain of the lower animals, transparent enough to be observed alive, clearly show in their midst a host of small cells with moving extensions. In these animals the smallest lesion brings an accumulation of these elements at the point of damage. In small transparent larvae, it can easily be shown that the moving cells, reunited at the damage point do often close over foreign bodies [1]. Ilya Mechnikov, one of the fathers of immunology, spoke these words at his Nobel Prize lecture in 1908. More than one hundred years after his seminal studies using transparent starsh larvae to illuminate a role for phagocytosis in immunity, we are again exploiting the power of transparent larvae for research on the immune system. Studies of neutrophils in both humans and mammalian model systems have brought great advances in our knowledge of their functions; however, zebrash, a small tropical sh with transparent larvae, have demonstrated that direct observation of neutrophils in live animals can provide important insights that would have otherwise faced signicant technical challenges using mice.
Neutrophils are the most abundant leukocytes in both humans and zebrash, and they are critical for defending the host against microbial infection [2]. In response to wounding, infection, or other inammatory stimuli, neutrophils are rapidly recruited to perform their well-known eector functions: degranulation, phagocytosis, production of reactive oxygen species (ROS), secretion of proinammatory cytokines, and extrusion of neutrophil extracellular traps (NETs) [3, 4]. These responses are acknowledged to kill and sequester microorganisms at their site of entry and promote the activation of the adaptive immune system [4]. Until recently, it was thought that neutrophils, which responded to a wound, had a single fate: death [5, 6]. There remains clear evidence for neutrophil apoptosis in the abundance of pus that emanates from infected wounds, and the clearance of dead neutrophils from the site of inammation has been demonstrated to occur through phagocytosis by macrophages [47]. However, studies of neutrophil wound responses using live zebrash embryos revealed for the rst time that neutrophils could leave an extravascular site of inammation and persist in the host
2 [8, 9]. This reverse migration process requires two distinct, but related steps: migration of neutrophils away from the inamed area and reverse transendothelial migration to enter the vascular lumen. The observation that neutrophils can reverse migrate away from a wound in zebrash [8] and in mice [10] raises new questions about neutrophil functions. First, the mechanism that neutrophils use to perform reverse migration and the signals that trigger it are entirely unknown. The fate of reverse-migrated neutrophils also remains to be explored. However, recent studies have demonstrated that neutrophils can aect the adaptive immune system and regulate systemic inammation in previously unappreciated ways, raising intriguing possibilities for the roles of reverse-migrated neutrophils. Because of the conservation of functions between human and zebrash neutrophils, as well as the many tools available for live imaging and genetic manipulation, zebrash will certainly continue to be a critical resource for elucidating the mechanisms and functions of neutrophil reverse migration. Here, we review the features of zebrash and some of the tools that make them particularly well suited to this task. Additionally, we will discuss the current state of the neutrophil reverse migration eld and its implications for the regulation immune responses.
Advances in Hematology [29]. This method has the disadvantage of being transient and aecting the entire organism. However, more recent developments indicate that shRNA-mediated knockdowns are possible in zebrash, which should allow the creation of transgenics with tissue-specic knockdowns [30]. While previous knockout technology relied on random mutagenesis and screening, new approaches promise to increase the availability of knockout zebrash to the community [23, 31]. Zinc nger nucleases (ZFN) and transcription activatorlike eector nucleases (TALEN) both rely on modular DNA recognition motifs coupled to nucleases to introduce highly localized DNA lesions [3234]. Tissue specic, inducible gene expression systems, such as those that rely on cremediated recombination, are actively being developed and will be critical in assessing the functions of genes whose longterm expression is detrimental [3537]. The relative ease of transgenesis and the growing complement of tools for genetic manipulation are a very attractive point of the zebrash model and should drive advances in understanding leukocyte behavior. Perhaps the single most signicant advantage of zebrash larvae as a model organism is their optical clarity, which allows for noninvasive, live imaging. Furthermore, live zebrash imaging can be accomplished with commonly available confocal microscopes or uorescence stereomicroscopes, obviating the need for highly specialized equipment and techniques used for in vivo imaging in mice. The ability to perform live imaging in zebrash has been enabled by the discovery of cell lineage-specic promoters that can drive expression of uorescent proteins that label cells or other proteins of interest. Two promoters, myeloperoxidase (mpx) and lysozyme c (lyz), are used to drive neutrophilspecic expression [8, 12, 38, 39]. Recent advances have also allowed tissue-specic expression in macrophages using the macrophage-expressed gene-1 (mpeg1) and colony stimulating factor 1 receptor (csf1r) promoters [14, 15]. These promoters have enabled the creation of stable transgenic lines and the characterization of neutrophil or macrophage responses to wounds, infections, and other inammatory stimuli.
Advances in Hematology several imaging techniques from cell biology [40]. The GFPtagged ratiometric probe, pleckstrin homology domain of Akt (PHAKT-EGFP), allowed live imaging of PI(3,4)P2 PI(3,4,5)P3 inside of neutrophils. Additionally, this study made use of photoactivatable Rac, whose activity could be induced in individual neutrophils with the targeted application of 458 nm laser light [41]. Finally, the F-actin probes, Lifeact and utrophin calponin homology domain (UtrCH), allowed simultaneous in vivo imaging of total F-actin and stable F-actin, respectively [42, 43]. Imaging of PHAKT-GFP demonstrated that PI(3,4)P2 -PI(3,4,5)P3 accumulated at the leading edge of migrating neutrophils, and inhibition PI(3)K prevented leading edge PI(3,4,5)P3 production, leading edge protrusion, and neutrophil motility. While photoactivation of Rac in normal neutrophils could be used to precisely control motility, photoactivation in PI(3)K inhibited cells could induce protrusions but not motility. Additionally, Rac activation could not induce proper F-actin polarization in PI(3)K-inhibited cells. This suggested a two-tiered model of PI(3)K activity in migrating neutrophils, where PI(3)K was needed for Rac-mediated leading edge protrusion but was also necessary for Rac-independent F-actin polarization [40]. The question of how neutrophils are recruited to wounds has also been partially answered by the application of advanced imaging techniques to zebrash research. The uorescent, reversible, genetically encoded, ratiometric probe, HyPer, is able to detect hydrogen peroxide production in vivo [44]. Niethammer et al. used this probe to show that wounding zebrash ns induced a burst of hydrogen peroxide that was necessary for the early recruitment of neutrophils to wounds [45]. Wound-produced hydrogen peroxide was subsequently demonstrated to attract neutrophils through the oxidation-mediated activation of the src-family kinase, Lyn. The ability to perform a neutrophil-specic rescue with wild-type and oxidation-mutant Lyn, a major benet of the zebrash system, was critical to conrming this nding [46]. Overall, these advances have demonstrated the value of coupling uorescent bioprobes and uorescent-tagged proteins to live, in vivo studies of neutrophil behavior. The continued innovation of advanced imaging techniques will be critical to future advances in understanding neutrophil behavior.
3 of zebrash neutrophils was the rst direct demonstration that reverse migration was responsible for clearance of neutrophils from the interstitium of wounded tissues [8]. Indeed, studies of neutrophil reverse migration in zebrash larvae have found that neutrophil apoptosis at a wound site is a rare event, occurring in less than 3% of the responding neutrophils [48]. Mathias et al. were able to demonstrate this reverse migratory behavior by tracking woundresponsive neutrophils in the transgenic (Tg) zebrash line, Tg(mpx : GFP), in which GFP is expressed specically in neutrophils. This study further demonstrated that neutrophils undergoing both forward and reverse migration to a wound had nearly equivalent velocity and directionality, implying that each was a robust, active process [8]. Using zebrash, other groups have also observed neutrophil reverse migration under similar experimental conditions [38, 48, 49]. While illuminating, technical challenges prevented the exploration of some questions about the fate and individual behavior of neutrophils responding to a wound. The application of the photoconvertible protein, Dendra2, which can be switched from green to red uorescence with 405 nm light, allowed these questions to be more denitively explored [50]. Dendra2-labeled neutrophils that had reached a wound were photoconverted, permitting detailed tracking of a small number of neutrophils. Thus, it was determined that individual neutrophils often trac between the wound and the vasculature repeatedly before leaving permanently. The signicance of this oscillatory behavior remains unclear, but it may reect the competition of signals between two endpoints. Transgenic zebrash with GFP-labeled vasculature, Tg(i1 : EGFP), demonstrated that some reverse-migrating neutrophils do enter the vasculature, performing a true reverse transendothelial migration (Figures 1(a)1(c)). While the oscillatory neutrophil migration and reverse transendothelial migration appear to be steps along a common pathway in the movement of neutrophils away from inamed tissue, it is not yet clear what triggers the progression between these steps. Finally, the fate of neutrophils that responded to wounds was determined by tracking photoconverted neutrophils for two days after wounding, demonstrating two important ndings: neutrophils survived for multiple days after wounding, and they were found dispersed throughout the body without obvious tissue preferences (Figure 1(d)) [9]. While these observations of neutrophil reverse migration in zebrash were intriguing, they still faced criticism that this could be a zebrash or larva-specic phenomenon and not applicable to mammals. Around the time of the rst observation of neutrophil reverse migration in zebrash, two groups made observations that suggested the existence of neutrophil reverse migration in mammalian systems. Primary human neutrophils that were cocultured on monolayers of endothelial cells in vitro were observed to transmigrate through the endothelium and subsequently perform reverse transendothelial migration to return to the apical surface [51]. These reverse transmigrated (RT) neutrophils demonstrated decreased adhesion to the endothelial surface
Advances in Hematology
Figure 1: Neutrophil reverse migration in zebrash larvae. This diagram illustrates the behavior of neutrophils undergoing reverse migration over an image of a 3-day postfertilization zebrash larva that was PTU-treated to prevent pigmentation. (a) In unwounded larva, neutrophils (green ovals) are present in the caudal hematopoietic tissue (CHT), which is situated between the caudal artery (red shading) and the caudal vein (blue shading). (b) In response to a wound, neutrophils are mobilized from the CHT, and they migrate through the tissue towards a wound. (c) The green to white color change represents the ability to photoconvert individual neutrophils that reach a wound. Neutrophils often migrate between the wound and the vasculature multiple times before nally departing. Neutrophils have been observed performing reverse migration by entering the vasculature and by migrating through tissues in zebrash larvae. (d) Reverse migrated neutrophils (white) are found dispersed throughout the body without an obvious tissue preference 2 days after leaving wounds.
and decreased tendency to undergo forward transendothelial migration as compared to fresh neutrophils. Another interesting nding from this study was a unique surface phenotype for neutrophils that had undergone the reverse migration process. CXCR1, the receptor for the neutrophil chemoattractant IL-8, CD11b (integrin M chain), and CD54 (intercellular adhesion molecule-1) along with other cell surface markers were used to distinguish the dierent neutrophil populations. RT neutrophils were found to be CD11bhigh CD54high CXCR1low , which dierentiated them from freshly isolated (CD11blow CD54low CXCR1high ) neutrophils [51]. Additionally, a study by Maletto et al. found that neutrophils in mice that had been immunized against ova peptide would transport an FITC-labeled ova
peptide from the site of footpad injection to local lymph nodes. Extensive ow cytometric and histopathologic analysis demonstrated that the cells bearing ova-FITC were indeed neutrophils. One caveat of this study was that ova-FITC containing neutrophils were only found in lymph nodes in the ipsilateral, but not contralateral leg, to the site of ova administration, implying that lymphatic drainage may have been responsible for their dissemination [52]. While not denitive proof of reverse migration, these ndings supported the idea that neutrophils could survive after responding to an inammatory stimulus and could aect the immune response in a manner spatiotemporally separated from the site of this stimulus. Subsequent in vivo studies in mice have also demonstrated that neutrophils can
Advances in Hematology directly interact with B cells and T cells in lymphoid tissue, further strengthening the concept of neutrophils existing outside of their conventional roles [10, 5355]. The most direct support for the observations of reverse migration in zebrash has come from a recent intravital imaging study using mice. Woodn et al. used a system in which intrascrotal inammation, induced by ischemia-reperfusion injury, allowed the monitoring of uorescently-labeled neutrophils. Approximately 10% of the transendothelial migration events observed with this assay were reverse transendothelial migration. Additionally, it was observed that ischemia-reperfusion injury disrupted the localization of junctional adhesion molecule C (JAMC) to endothelial junctions and that mice with JAMC/ endothelial cells demonstrated an increase in reverse transendothelial migration, reaching greater than 50% of total transendothelial migration events [10]. It is important to note that this study did not address the migration of neutrophils in the tissue parenchyma, and this will be an interesting area for future study. These ndings of neutrophil reverse migration in mice and in vitro are paralleled by monocyte studies that demonstrated reverse transendothelial migration with similar kinetics and regulation by JAMC [56, 57]. This suggests a possible conservation in the mechanisms that mediate reverse transendothelial migration of neutrophils and macrophages. Several dierences have been observed between zebrash and mammalian reverse migration that await further investigation. While all of the reverse migration events described thus far in mice involve transendothelial migration, some neutrophils are able to disperse throughout the body of the zebrash larva without entering the vasculature. Most apparent is that nearly all wound responsive neutrophils perform reverse migration in zebrash, whereas approximately 10% do so under the observed conditions in mice [9, 10]. We believe that this discrepancy may be the result of using larva versus adult animals, species-specic dierences, or the type of inammatory stimulus. However, the high percentage of reverse migrating neutrophils may provide a substantial benet in studies of reverse migration. Mammalian studies have supported the utility of using zebrash to study reverse migration; however, neither system has yielded a denitive answer on the mechanisms that mediate reverse migration.
5 this process. During the early response to a wound, velocity and directionality are equivalent during forward and reverse neutrophil migration, suggesting that reverse migration is an active process [8]. Therefore, a passive mechanism, such as the loss of wound-derived chemoattractants and the random dispersal of neutrophils, is not likely to be involved. We speculate that the signals that trigger neutrophils to perform reverse migration could include a competing chemoattractant pulling them away from the wound, a chemorepellent pushing them away from the would, or both (Figures 2(a) and 2(b)). Because neutrophils often migrate back to the vasculature after an inammatory response, chemoattractants emanating from the blood or endothelium are attractive targets for promoting migration away from a wound (Figure 2(a)). Interestingly, high concentrations of chemoattractants, including IL-8 (CXCL8), can repel neutrophils in vitro and in vivo [58]. Other leukocytes can also be repelled by high chemokine concentrations. T cells are repelled by high concentrations of SDF-1 (CXCL12) in vivo and in vitro [59], and monocytes can be repelled by high concentrations of eotaxin-3 (CCL26) [60]. Thus, it is also plausible that the wounded tissue could be a source of both chemoattractants and chemorepellents in competition with each other. Previous studies of leukocyte chemorepulsion suggest that a wound chemoattractant at suciently high concentration could also act as a chemorepellent. As a neutrophil approached the wounded tissue, the concentration of chemorepellent would increase, potentially overwhelming the eect of the chemoattractant and driving the neutrophil away from the wound (Figure 2(b)). Neutrophils respond to chemokines in a hierarchical manner, preferring some over others, when faced with competing gradients [61, 62]. The oscillatory migration of neutrophils between wounds and the vasculature suggests that a mechanism of competing chemoattractant gradients between these locations is likely (Figure 2(c)) [8, 9]. In this scenario, the signals promoting migration away from a wound (Figures 2(a)-2(b)) would compete with the signals attracting neutrophils to wounds. One complicating factor for this model is that fresh neutrophils continue to arrive at wounds after some have already reverse migrated, suggesting that neutrophil intrinsic regulation may also be involved in their oscillatory behavior and eventual departure. Therefore, we believe that the most likely explanation for the oscillatory behavior during the early wound response is a combination of competing chemokine gradients and neutrophilautonomous changes in chemokine receptor sensitivity. It is known that neutrophils will internalize and downregulate G protein coupled receptors, including many chemokine receptors, after stimulation [63]. In this model, as neutrophils approach a high concentration of chemoattractant at the wound or vasculature, receptor desensitization would occur and promote migration towards the competing gradient (Figure 2(d)). Failure to internalize the CXCR4 receptor prevents downregulation of CXCR4-mediated signalling and is responsible for the retention of neutrophils in the bone marrow or caudal hematopoietic tissue of WHIM syndrome patients and a zebrash model of WHIM syndrome, respectively [24, 6467]. This suggests that the dynamic regulation
6
(a) Chemoattractant from blood/endothelium
Advances in Hematology
Figure 2: Proposed mechanisms for reversed migration. This diagram illustrates how chemoattractant gradients from the blood (red), endothelium (purple), and wound (green) or chemorepellent gradients (orange) may inuence reverse migration. The color of a chemoattractant receptor matches the gradient to which it responds. (a)(d) Reverse migration in the early wound response. (a) Demonstration of neutrophil reverse migration towards blood or endothelium-derived chemoattractants. (b) Reverse migration of a neutrophil away from a wound-derived chemorepellent. There could be competition between wound-derived chemoattractants (not shown) and chemorepellents promoting reverse migration, or neutrophils may perform fugetaxis from areas of high chemoattractant concentration. (c) Oscillatory behavior of neutrophils suggests competing gradients of chemoattractants may exist between the wound and vasculature. (d) Receptor desensitization, via internalization or other mechanisms, may allow neutrophils to oscillate between the wound and the vasculature while others are still actively responding to the wound. (e)(f) Mechanisms that promote resolution of neutrophil-mediated inammation at wounds. (e) During the healing phase, wounded tissue may gradually produce less neutrophil chemoattractant, shifting the balance to favor reverse migration. (f) Neutrophils that responded to a wound may initiate transcriptional changes favoring reverse migration from the wound. Potential changes include altered expression or sensitivity of chemoattractant receptors.
of chemokine receptor surface expression or its sensitivity to signaling is critical for allowing neutrophils to follow the appropriate gradient of chemoattractant, which could be necessary for performing reverse migration.
The eventual permanent departure of neutrophils from the wound site indicates the second phase of the reverse migration response. During this later phase, the wounded tissue may gradually produce less chemoattractant or more
Advances in Hematology chemorepellent as it heals, shifting the balance towards reverse migration (Figure 2(e)). The time it takes for neutrophils to leave a wound, which can be several hours, makes it possible that transcriptional changes may also be involved. In a mechanism that may be cooperative with declining chemoattractant gradients, neutrophils could be programmed to activate transcriptional changes that favor reverse migration after responding to an inammatory stimulus. The result could modify chemokine receptor expression or sensitivity, shifting the balance towards reverse migration (Figure 2(f)). Clearly, there are many possible mechanisms internal and external to the neutrophil that may drive reverse migration, making this an area ripe for rapid advancement.
7
Table 1: Comparison of surface phenotypes between studies of neutrophil reverse migration and immunomodulation by neutrophils. CD11b (integrin M ), which is a component of Mac-1 integrin, and CD54 (ICAM-1) were the surface molecules with the most overlap between these studies. Blank spaces indicate that the expression of this molecule was not addressed by a particular study. The () indicates that expression of the indicated molecule was elevated over the appropriate control sample of neutrophils (non reverse migrated, not responsible for neutrophil eects on lymphocytes, etc.). CD11b Reverse migration Buckley et al. 2006 [51] Woodn et al. 2011 [10] Immunomodulation Maletto et al. 2006 [52] Ostanin et al. 2012 [53] Pillay et al. 2012 [54] Puga et al. 2012. [55]
CD54
neutrophil inhibitory function, and imaging revealed that H2 O2 was produced at neutrophil T cell contacts, suggesting a model in which activated neutrophils could form a synapselike structure and deliver proliferation-inhibiting H2 O2 to T cells (Figure 3) [54]. Another interesting population of CD11bhigh CD54high neutrophils was recently found in the marginal zone of the spleen. These neutrophils expressed MHC class II, which is normally found on professional antigen presenting cells and had the ability to promote antibody diversication and production by splenic B cells. These neutrophils populated the splenic lymphoid follicles after acquiring gut-associated-microbial products, suppressed T cell proliferation, and promoted antibody production to T cell-independent antigens (Figure 3) [55]. Taken together, these ndings support the idea that neutrophils are able to acquire material from extravascular tissue, return to and survive in lymphoid tissue, and modulate the adaptive immune response. The ability to retrieve antigen outside of the bloodstream and the surface phenotype consistent with reverse migrated neutrophils suggests the possibility that neutrophils could perform reverse migration during their trip back to lymphoid tissues. While the eects of neutrophils on the adaptive immune system described above could be viewed as antiinammatory or immunomodulatory, a proinammatory role has also been proposed for reverse migrated neutrophils [9, 10, 51]. Several lines of evidence lend support to this hypothesis, which stems from the observations that severe, localized trauma can lead to multiple organ failure and neutrophil reverse migration. Neutrophils are thought to be important in the pathogenesis of multiple organ failure, which is associated with states of injury and heightened inammation [73, 74]. While the reverse migration of neutrophils away from a site of inammation may result in local resolution of inammation, the observation by Yoo and Huttenlocher that reverse migrated neutrophils disperse in tissues throughout the body of zebrash suggests the possibility that neutrophils could be promoting
Advances in Hematology
B cell
Inflammation
Figure 3: Potential functions of reverse migrated neutrophils. On the left side of the illustration, neutrophils (tan) are responding to an extravascular inammatory stimulus. After responding to the stimulus, neutrophils perform reverse migration (yellow) and enter the vasculature. This process has been suggested as a mechanism to resolve inammation at the local level. We are proposing the following as potential functions of reverse migrated neutrophils. Neutrophils may modulate T-cell proliferation and cytokine production in an antigenindependent or-dependent manner. Integrin-mediated neutrophil-T cell contact, hydrogen peroxide, and T cell receptor (TCR) signaling have demonstrated importance in neutrophil-mediated regulation of T cell function. Neutrophils may promote antibody diversication, class switching, and production by splenic B cells through the secretion of cytokines. Reverse migrated neutrophils may travel to distant tissues and induce additional inammation. Reverse-migrated neutrophils were implicated in inducing pulmonary inammation in mice.
inammation or tissue damage in these sites (Figure 3) [9]. Additionally, proinammatory conditions such as ischemiareperfusion injury, rheumatoid arthritis, and chronic colitis generate elevated numbers of neutrophils with the reversemigrated surface phenotype [10, 51, 53]. Mice with chronic colitis were found to have neutrophils that presented antigen to T cells in an MHC II-restricted manner, resulting in increased T cell proliferation and proinammatory cytokine production [53]. Furthermore, after ischemia-reperfusion injury in mice, pulmonary edema was observed, and neutrophils with a reverse migrated surface phenotype could be found in the lung, suggesting that this neutrophil population may promote inammation at sites distant from the actual injury (Figure 3) [10]. Reverse migrated neutrophils also produce elevated amounts of reactive oxygen species, which are thought to play a key role in the pathophysiology of multiple organ failure [10, 51, 73, 74]. Although a proor anti-inammatory role for reverse migrated neutrophils remains uncertain, many lines of evidence support the idea that neutrophils can retrieve antigen from extravascular tissues, move to distant organs, and inuence the adaptive immune response, leading us to believe that neutrophil reverse migration could be playing a role in these neutrophil functions.
reverse migration, which are rapidly changing the view that neutrophils are short-lived cells with narrowly dened eector functions. It seems that in at least some circumstances neutrophils can regulate T cell activity and present antigen in the context of MHC II, functions which were previously ascribed to macrophages and dendritic cells, the professional antigen presenting cells [5255]. While evidence in support of the existence of reverse migration in mammals continues to grow, the mechanisms driving reverse migration and the functions of reverse migrated neutrophils remain to be further dened. Continued progress towards these goals will require additional characterization of neutrophil forward and reverse migration coupled with technical advances. In order to fully understand the signaling that drives reverse migration, we must better characterize the signaling that is recruiting neutrophils to wounds. Currently, we know that a gradient of H2 O2 drives the early recruitment of neutrophils to wounds. However, neutrophils still arrive at wounds with a 3060 minute delay in the absence of wound-produced H2 O2 , indicating that other chemoattractants are likely involved at later time points [46]. Additionally, the chemoattractants or chemorepellents that drive neutrophil reverse migration remain entirely unknown. The intracellular signaling that dierentiates forward from reverse migration is also unexplored. Fully characterizing this signaling hierarchy would be facilitated by chemical or genetic screening strategies with the ability to read out changes in neutrophil-mediated inammation.
Advances in Hematology Advances in our imaging capabilities in zebrash will also be critical to further progress in reverse migration studies. While we can observe the movements of neutrophils responding to wounds in zebrash, we currently know little about their other functions in vivo. Tools that allow live imaging of neutrophil activation and eector functionsreactive oxygen species production, phagocytosis, and degranulationwill be particularly valuable. Biosensors that allow the activity of critical signaling pathways to be monitored will help to integrate the roles of extracellular cues and neutrophil eector functions on their wound responses. A FRET-based Rac activity biosensor, which has been applied to the study of primordial germ cell protrusion and migration, is an example of the type of probe that will be integral in understanding the signaling pathways controlling reverse migration [75, 76]. Determining the function of reverse migrated neutrophils should also be a priority. Recent reports that neutrophils can modulate B cell and T cell functions demonstrate the importance of characterizing these interactions in vivo. Approaching this question with 24-day-old zebrash larvae is not possible, as they have not yet developed an adaptive immune system [16]. As a result, the functions of zebrash B and T cells in response to inammatory stimuli are poorly characterized. Techniques that allow simultaneous in vivo imaging of neutrophils, B cells, T cells, and eector molecules in more developed zebrash would allow a more denitive determination of how these interactions shape immune responses. In order to fully understand the role of neutrophil reverse migration, it will be necessary to determine how it impacts immune homeostasis and disease. Models of immunodeciency and inammatory disease have been developed in zebrash larvae [22, 23, 77, 78]. However, a model of neutrophil autonomous inammatory disease has not yet been developed in zebrash. These and future disease models can be used to determine if neutrophil reverse migration is altered in pathologic states. Additionally, determination of the signaling that drives reverse migration will allow this process to be inhibited, which will be informative in understanding how it may inuence pathology. While rapid progress has been made in the characterization of reverse migration in zebrash and mice, much remains to be learned about the underlying mechanisms and functional consequences of this process. However, the availability of powerful tools for genetic manipulation and in vivo imaging makes it clear that the use of transparent zebrash larvae will allow researchers to continue probing the secrets of neutrophil behavior in vivo.
9 received predoctoral funding support from the University of Wisconsin Hematology Training Grant (NIH Grant HL007899; John Sheehan, principal investigator) and the University of Wisconsin Medical Scientist Training Program training grant (NIH grant GM008692; Deane Mosher, principal investigator). The authors would like to thank Qing Deng for contributions to Figure 2 and Sa Kan Yoo for critically reading this paper.
References
[1] I. Mechnikov, On the Present State of the Question of Immunity in Infectious Diseases, Nobel Lecture, 1908. [2] G. J. Lieschke, A. C. Oates, M. O. Crowhurst, A. C. Ward, and J. E. Layton, Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrash, Blood, vol. 98, no. 10, pp. 30873096, 2001. [3] N. Borregaard, Neutrophils, from marrow to microbes, Immunity, vol. 33, no. 5, pp. 657670, 2010. [4] C. Nathan, Neutrophils and immunity: challenges and opportunities, Nature Reviews Immunology, vol. 6, no. 3, pp. 173182, 2006. [5] J. Savill and C. Haslett, Granulocyte clearance by apoptosis in the resolution onammation, Seminars in Cell Biology, vol. 6, no. 6, pp. 385393, 1995. [6] D. L. Bratton and P. M. Henson, Neutrophil clearance: when the party is over, clean-up begins, Trends in Immunology, vol. 32, no. 8, pp. 350357, 2011. [7] G. Cox, J. Crossley, and Z. Xing, Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inammation in vivo, American Journal of Respiratory Cell and Molecular Biology, vol. 12, no. 2, pp. 232 237, 1995. [8] J. R. Mathias, B. J. Perrin, T. X. Liu, J. Kanki, A. T. Look, and A. Huttenlocher, Resolution of inammation by retrograde chemotaxis of neutrophils in transgenic zebrash, Journal of Leukocyte Biology, vol. 80, no. 6, pp. 12811288, 2006. [9] S. K. Yoo and A. Huttenlocher, Spatiotemporal photolabeling of neutrophil tracking during inammation in live zebrash, Journal of Leukocyte Biology, vol. 21, no. 4, pp. 735 745, 2011. [10] A. Woodn, M. B. Voisin, M. Beyrau et al., The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in vivo, Nature Immunology, vol. 12, pp. 761769, 2011. [11] C. M. Bennett, J. P. Kanki, J. Rhodes et al., Myelopoiesis in the zebrash, Danio rerio, Blood, vol. 98, no. 3, pp. 643651, 2001. [12] S. A. Renshaw, C. A. Loynes, D. M. I. Trushell, S. Elworthy, P. W. Ingham, and M. K. B. Whyte, A transgenic zebrash model of neutrophilic inammation, Blood, vol. 108, no. 13, pp. 39763978, 2006. [13] P. Herbomel, B. Thisse, and C. Thisse, Ontogeny and behaviour of early macrophages in the zebrash embryo, Development, vol. 126, no. 17, pp. 37353745, 1999. [14] C. Gray, C. A. Loynes, M. K. B. Whyte, D. C. Crossman, S. A. Renshaw, and T. J. A. Chico, Simultaneous intravital imaging of macrophage and neutrophil behaviour during inammation using a novel transgenic zebrash, Thrombosis and Haemostasis, vol. 105, no. 5, pp. 811819, 2011.
Authors Contribution
T. W. Starnes and A. Huttenlocher wrote the paper.
Acknowledgments
This work was supported by National Institutes of Health (NIH) Grant GM074827 (A. Huttenlocher). T. W. Starnes
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[15] F. Ellett, L. Pase, J. W. Hayman, A. Andrianopoulos, and G. J. Lieschke, mpeg1 promoter transgenes direct macrophagelineage expression in zebrash, Blood, vol. 117, no. 4, pp. e49 e56, 2011. [16] D. M. Langenau, A. A. Ferrando, D. Traver et al., In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrash, Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 19, pp. 73697374, 2004. [17] N. Danilova and L. A. Steiner, B cells develop in the zebrash pancreas, Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 21, pp. 1371113716, 2002. [18] J. T. Dobson, J. Seibert, E. M. Teh et al., Carboxypeptidase A5 identies a novel mast cell lineage in the zebrash providing new insight into mast cell fate determination, Blood, vol. 112, no. 7, pp. 29692972, 2008. [19] K. M. Balla, G. Lugo-Villarino, J. M. Spitsbergen et al., Eosinophils in the zebrash: prospective isolation, characterization, and eosinophilia induction by helminth determinants, Blood, vol. 116, no. 19, pp. 39443954, 2010. [20] E. Colucci-Guyon, J. Y. Tinevez, S. A. Renshaw, and P. Herbomel, Strategies of professional phagocytes in vivo: unlike macrophages, neutrophils engulf only surface-associated microbes, Journal of Cell Science, vol. 124, no. 18, pp. 3053 3059, 2011. [21] D. Le Guyader, M. J. Redd, E. Colucci-Guyon et al., Origins and unconventional behavior of neutrophils in developing zebrash, Blood, vol. 111, no. 1, pp. 132141, 2008. [22] Q. Deng, S. K. Yoo, P. J. Cavnar, J. M. Green, and A. Huttenlocher, Dual roles for Rac2 in neutrophil motility and active retention in zebrash hematopoietic tissue, Developmental Cell, vol. 21, no. 4, pp. 735745, 2011. [23] A. Cvejic, C. Hall, M. Bak-Maier et al., Analysis of WASp function during the wound inammatory responseliveimaging studies in zebrash larvae, Journal of Cell Science, vol. 121, no. 19, pp. 31963206, 2008. [24] K. B. Walters, J. M. Green, J. C. Surfus, S. K. Yoo, and A. Huttenlocher, Live imaging of neutrophil motility in a zebrash model of WHIM syndrome, Blood, vol. 116, no. 15, pp. 28032811, 2010. [25] D. Carradice and G. J. Lieschke, Zebrash in hematology: sushi or science? Blood, vol. 111, no. 7, pp. 33313342, 2008. [26] N. S. Trede, D. M. Langenau, D. Traver, A. T. Look, and L. I. Zon, The use of zebrash to understand immunity, Immunity, vol. 20, no. 4, pp. 367379, 2004. [27] G. J. Lieschke and N. S. Trede, Fish immunology, Current Biology, vol. 19, no. 16, pp. R678R682, 2009. [28] S. A. Renshaw and N. S. Trede, A model 450 million years in the making: zebrash and vertebrate immunity, Disease Models & Mechanisms, vol. 5, no. 1, pp. 3847, 2012. [29] P. A. Morcos, Achieving targeted and quantiable alteration of mRNA splicing with Morpholino oligos, Biochemical and Biophysical Research Communications, vol. 358, no. 2, pp. 521 527, 2007. [30] M. Dong, Y. F. Fu, T. T. Du et al., Heritable and lineagespecic gene knockdown in Zebrash embryo, PLoS ONE, vol. 4, no. 7, Article ID e6125, 2009. [31] E. Wienholds, F. van Eeden, M. Kosters, J. Mudde, R. H. A. Plasterk, and E. Cuppen, Ecient target-selected mutagenesis in zebrash, Genome Research, vol. 13, no. 12, pp. 27002707, 2003. [32] J. E. Foley, J. R. J. Yeh, M. L. Maeder et al., Rapid mutation of endogenous zebrash genes using zinc nger nucleases made
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by oligomerized pool ENgineering (OPEN), PLoS ONE, vol. 4, no. 2, article e4348, 2009. M. Christian, T. Cermak, E. L. Doyle et al., Targeting DNA double-strand breaks with TAL eector nucleases, Genetics, vol. 186, no. 2, pp. 756761, 2010. T. Li, S. Huang, W. Z. Jiang et al., TAL nucleases (TALNs): hybrid proteins composed of TAL eectors and FokI DNAcleavage domain, Nucleic Acids Research, vol. 39, no. 1, pp. 359372, 2011. S. Hans, D. Freudenreich, M. Gearth, J. Kaslin, A. Machate, and M. Brand, Generation of a non-leaky heat shockinducible Cre line for conditional Cre/lox strategies in zebrash, Developmental Dynamics, vol. 240, no. 1, pp. 108 115, 2011. C. Mosimann, C. K. Kaufman, P. Li, E. K. Pugach, O. J. Tamplin, and L. I. Zon, Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrash, Development, vol. 138, no. 1, pp. 169177, 2011. C. Mosimann and L. I. Zon, Advanced zebrash transgenesis with Tol2 and application for Cre/lox recombination experiments, Methods in Cell Biology, vol. 104, pp. 173194, 2011. C. Hall, M. Flores, T. Storm, K. Crosier, and P. Crosier, The zebrash lysozyme C promoter drives myeloid-specic expression in transgenic sh, BMC Developmental Biology, vol. 7, article 42, 2007. A. H. Meijer, A. M. van der Sar, C. Cunha et al., Identication and real-time imaging of a myc-expressing neutrophil population involved in inammation and mycobacterial granuloma formation in zebrash, Developmental and Comparative Immunology, vol. 32, no. 1, pp. 3649, 2008. S. K. Yoo, Q. Deng, P. J. Cavnar, Y. I. Wu, K. M. Hahn, and A. Huttenlocher, Dierential regulation of protrusion and polarity by PI(3)K during neutrophil motility in live zebrash, Developmental Cell, vol. 18, no. 2, pp. 226236, 2010. Y. I. Wu, D. Frey, O. I. Lungu et al., A genetically encoded photoactivatable Rac controls the motility of living cells, Nature, vol. 461, no. 7260, pp. 104108, 2009. J. Riedl, A. H. Crevenna, K. Kessenbrock et al., Lifeact: a versatile marker to visualize F-actin, Nature Methods, vol. 5, no. 7, pp. 605607, 2008. B. M. Burkel, G. Von Dassow, and W. M. Bement, Versatile uorescent probes for actin laments based on the actinbinding domain of utrophin, Cell Motility and the Cytoskeleton, vol. 64, no. 11, pp. 822832, 2007. V. V. Belousov, A. F. Fradkov, K. A. Lukyanov et al., Genetically encoded uorescent indicator for intracellular hydrogen peroxide, Nature Methods, vol. 3, no. 4, pp. 281286, 2006. P. Niethammer, C. Grabher, A. T. Look, and T. J. Mitchison, A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrash, Nature, vol. 459, no. 7249, pp. 996999, 2009. S. K. Yoo, T. W. Starnes, Q. Deng, and A. Huttenlocher, Lyn is a redox sensor that mediates leukocyte wound attraction in vivo, Nature, vol. 480, no. 7375, pp. 109112, 2011. J. Hughes, R. J. Johnson, A. Mooney, C. Hugo, K. Gordon, and J. Savill, Neutrophil fate in experimental glomerular capillary injury in the rat: emigration exceeds in situ clearance by apoptosis, American Journal of Pathology, vol. 150, no. 1, pp. 223234, 1997. P. M. Elks, F. J. Van Eeden, G. Dixon et al., Activation of hypoxia-inducible factor-1 (hif-1) delays inammation resolution by reducing neutrophil apoptosis and reverse
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
Advances in Hematology
migration in a zebrash inammation model, Blood, vol. 118, no. 3, pp. 712722, 2011. S. B. Brown, C. S. Tucker, C. Ford, Y. Lee, D. R. Dunbar, and J. J. Mullins, Class III antiarrhythmic methanesulfonanilides inhibit leukocyte recruitment in zebrash, Journal of Leukocyte Biology, vol. 82, no. 1, pp. 7984, 2007. N. G. Gurskaya, V. V. Verkhusha, A. S. Shcheglov et al., Engineering of a monomeric green-to-red photoactivatable uorescent protein induced by blue light, Nature Biotechnology, vol. 24, no. 4, pp. 461465, 2006. C. D. Buckley, E. A. Ross, H. M. McGettrick et al., Identication of a phenotypically and functionally distinct population of long-lived neutrophils in a model of reverse endothelial migration, Journal of Leukocyte Biology, vol. 79, no. 2, pp. 303311, 2006. B. A. Maletto, A. S. Ropolo, D. O. Alignani et al., Presence of neutrophil-bearing antigen in lymphoid organs of immune mice, Blood, vol. 108, no. 9, pp. 30943102, 2006. D. V. Ostanin, E. Kurmaeva, K. Furr et al., Acquisition of antigen-presenting functions by neutrophils isolated from mice with chronic colitis, The Journal of Immunology, vol. 188, no. 3, pp. 14911502, 2012. J. Pillay, V. M. Kamp, E. van Hoen et al., A subset of neutrophils in human systemic inammation inhibits T cell responses through Mac-1, The Journal of Clinical Investigation, vol. 122, no. 1, pp. 327336, 2012. I. Puga, M. Cols, C. M. Barra et al., B cell-helper neutrophils stimulate the diversication and production of immunoglobulin in the marginal zone of the spleen, Nature Immunology, vol. 13, no. 2, pp. 170180, 2012. P. F. Bradeld, C. Scheiermann, S. Nourshargh et al., JAM-C regulates unidirectional monocyte transendothelial migration in inammation, Blood, vol. 110, no. 7, pp. 25452555, 2007. G. J. Randolph and M. B. Furie, Mononuclear phagocytes egress from an in vitro model of the vascular wall by migrating across endothelium in the basal to apical direction: role of intercellular adhesion molecule 1 and the CD11/CD18 integrins, Journal of Experimental Medicine, vol. 183, no. 2, pp. 451462, 1996. W. G. Tharp, R. Yadav, D. Irimia et al., Neutrophil chemorepulsion in dened interleukin-8 gradients in vitro and in vivo, Journal of Leukocyte Biology, vol. 79, no. 3, pp. 539554, 2006. M. C. Poznansky, I. T. Olszak, R. Foxall, R. H. Evans, A. D. Luster, and D. T. Scadden, Active movement of T cells away from a chemokine, Nature Medicine, vol. 6, no. 5, pp. 543 548, 2000. P. Ogilvie, S. Paoletti, I. Clark-Lewis, and M. Uguccioni, Eotaxin-3 is a natural antagonist for CCR2 and exerts a repulsive eect on human monocytes, Blood, vol. 102, no. 3, pp. 789794, 2003. A. Huttenlocher and M. C. Poznansky, Reverse leukocyte migration can be attractive or repulsive, Trends in Cell Biology, vol. 18, no. 6, pp. 298306, 2008. B. Heit, S. Tavener, E. Raharjo, and P. Kubes, An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients, Journal of Cell Biology, vol. 159, no. 1, pp. 91102, 2002. S. S. G. Ferguson, Evolving concepts in G protein-coupled receptor endocytosis: Tte role in receptor desensitization and signaling, Pharmacological Reviews, vol. 53, no. 1, pp. 124, 2001. P. A. Hernandez, R. J. Gorlin, J. N. Lukens et al., Mutations in the chemokine receptor gene CXCR4 are associated with
11
WHIM syndrome, a combined immunodeciency disease, Nature Genetics, vol. 34, no. 1, pp. 7074, 2003. K. Balabanian, B. Lagane, J. L. Pablos et al., WHIM syndromes with dierent genetic anomalies are accounted for by impaired CXCR4 desensitization to CXCL12, Blood, vol. 105, no. 6, pp. 24492457, 2005. D. H. McDermott, Q. Liu, J. Ulrick et al., The CXCR4 antagonist plerixafor corrects panleukopenia in patients with WHIM syndrome, Blood, vol. 118, no. 18, pp. 49574962, 2011. D. C. Dale, A. A. Bolyard, M. L. Kelley et al., The CXCR4 antagonist plerixafor is a potential therapy for myelokathexis, WHIM syndrome, Blood, vol. 118, no. 18, pp. 49634966, 2011. J. Pillay, I. Den Braber, N. Vrisekoop et al., In vivo labeling with 2 H2 O reveals a human neutrophil lifespan of 5.4 days, Blood, vol. 116, no. 4, pp. 625627, 2010. K. W. Li, S. M. Turner, C. L. Emson, M. K. Hellerstein, and D. C. Dale, Deuterium and neutrophil kinetics, Blood, vol. 117, no. 22, pp. 60526053, 2011. P. S. Tofts, T. Chevassut, M. Cutajar, N. G. Dowell, and A. M. Peters, Doubts concerning the recently reported human neutrophil lifespan of 5.4 days, Blood, vol. 117, no. 22, pp. 60506052, 2011. J. Pillay, I. den Braber, N. Vrisekoop et al., The in vivo half-life of human neutrophils, Blood, vol. 117, no. 22, pp. 60536054, 2011. G. Parsonage, A. Filer, M. Bik et al., Prolonged, granulocytemacrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial broblast activation by IL-17 and TNFalpha, Arthritis Research and Therapy, vol. 10, no. 2, article R47, 2008. T. Tsukamoto, R. S. Chanthaphavong, and H. C. Pape, Current theories on the pathophysiology of multiple organ failure after trauma, Injury, vol. 41, no. 1, pp. 2126, 2010. D. Dewar, F. A. Moore, E. E. Moore, and Z. Balogh, Postinjury multiple organ failure, Injury, vol. 40, no. 9, pp. 912918, 2009. E. Kardash, J. Bandemer, and E. Raz, Imaging protein activity in live embryos using uorescence resonance energy transfer biosensors, Nature Protocols, vol. 6, no. 12, pp. 18351846, 2011. H. Xu, E. Kardash, S. Chen, E. Raz, and F. Lin, G signaling controls the polarization of zebrash primordial germ cells by regulating Rac activity, Development, vol. 139, no. 1, pp. 57 62, 2012. M. E. Dodd, J. Hatzold, J. R. Mathias et al., The ENTH domain protein Clint1 is required for epidermal homeostasis in zebrash, Development, vol. 136, no. 15, pp. 25912600, 2009. J. R. Mathias, M. E. Dodd, K. B. Walters et al., Live imaging of chronic inammation caused by mutation of zebrash Hai1, Journal of Cell Science, vol. 120, no. 19, pp. 33723383, 2007.
[49]
[65]
[50]
[66]
[51]
[67]
[68]
[52]
[69]
[53]
[70]
[54]
[71]
[55]
[72]
[56]
[73]
[57]
[74]
[75]
[58]
[59]
[76]
[60]
[77]
[61]
[78]
[62]
[63]
[64]
Hindawi Publishing Corporation Advances in Hematology Volume 2012, Article ID 478164, 8 pages doi:10.1155/2012/478164
Review Article Through the Looking Glass: Visualizing Leukemia Growth, Migration, and Engraftment Using Fluorescent Transgenic Zebrash
Finola E. Moore1, 2, 3 and David M. Langenau1, 2, 3
1 Department
of Pathology and Cancer Center, Massachusetts General Hospital, Building 149, Charlestown, MA 02129, USA 2 Harvard Stem Cell Institute, Holyoke Center, Suite 727W, 1350 Massachusetts Avenue, Cambridge, MA 02138, USA 3 Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, NRB 0330, Boston, MA 02115, USA Correspondence should be addressed to David M. Langenau, [email protected] Received 15 March 2012; Accepted 23 May 2012 Academic Editor: Elspeth Payne Copyright 2012 F. E. Moore and D. M. Langenau. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zebrash have emerged as a powerful model of development and cancer. Human, mouse, and zebrash malignancies exhibit striking histopathologic and molecular similarities, underscoring the remarkable conservation of genetic pathways required to induce cancer. Zebrash are uniquely suited for large-scale studies in which hundreds of animals can be used to investigate cancer processes. Moreover, zebrash are small in size, optically clear during development, and amenable to genetic manipulation. Facile transgenic approaches and new technologies in gene inactivation have provided much needed genomic resources to interrogate the function of specic oncogenic and tumor suppressor pathways in cancer. This manuscript focuses on the unique attribute of labeling leukemia cells with uorescent proteins and directly visualizing cancer processes in vivo including tumor growth, dissemination, and intravasation into the vasculature. We will also discuss the use of uorescent transgenic approaches and cell transplantation to assess leukemia-propagating cell frequency and response to chemotherapy.
Although characterized by increased circulating white blood counts, chronic leukemias are often much slower growing and take months or years to progress. Leukemias can be further subdivided based on the blood lineage in which cells have become transformed [8]. To date, zebrash models of Acute Lymphoblastic Leukemias (ALL), Acute Myeloid Leukemia (AML), and Myeloproliferative Neoplasms (MPN) have been described. Zebrash rst emerged as a powerful genetic model of leukemia with the description of transgenic approaches in which cMYC was overexpressed in developing thymocytes [7]. Utilizing the rag2 promoter to drive both MYC and GFP expression, transgenic zebrash T-cell acute lymphoblastic leukemias (T-ALLs) could be easily visualized in live animals. In this model, uorescently labeled T cell precursors resident in the thymus were the T-ALL-initiating cell type and disseminated widely over the course of tumor progression [7].
2 Moreover, GFP+ thymocytes exhibited stereotypical homing to the nasal placode, periocular space, and kidney marrow when assessed by serial uorescent imaging over days [7]. Subsequent studies developed conditional approaches to create uorescent transgenic zebrash models of T-ALL that utilized CRE-Lox or tamoxifen-inducible MYC-ER strategies [5, 9]. Interestingly, withdrawal of tamoxifen and subsequent inactivation of MYC expression led to regression of uorescently labeled T-ALL; however, leukemia regression was not observed in pten mutant sh or those that overexpressed activated Akt [9]. These data indicate that Akt pathway activation is sucient for tumor maintenance in this model. Additional studies have utilized uorescence imaging to assess synergy between MYC and Bcl2 [5, 10] and NOTCH1ICD [1]. Moreover, human NOTCH1-intracellular domainEGFP transgene expression induces uorescently labeled TALL with a long latency of >6 months in mosaic and stable transgenic zebrash [6]. Finally, forward genetics screens that utilize ENU (N-Ethyl-N-nitrosourea-) induced mutagenesis are easily performed in zebrash due to their large clutch size and accessible observation of phenotypes. Utilizing this approach, the Trede group mutagenized Tg(lck:GFP) transgenic sh and visualized animals for uorescently labeled T-ALL onset in F1 and F2 animals, identifying both dominant and recessive mutations that aect T-ALL onset [11]. Mapping of mutations that are found in these mutant lines will likely uncover novel mechanisms that drive T-ALL onset and growth in both zebrash and man. Many exciting new models of hematopoietic malignancy have been created including B-cell acute lymphoblastic leukemia (B-ALL), acute myeloid leukemia (AML), and myeloproliferative neoplasm (MPN). For example, Sabaawy et al. developed a model of B-ALL by overexpressing EGFP-TELAML1 from a ubiquitous transgene promoter. In this model, 16 of 545 transgenic animals developed B-ALL by 812 months of age [2]. Zhuravleva et al. generated transgenic zebrash in which the MYST3/NCOA2 fusion gene was expressed under control of the spi1 promoter [12]. 2 of 180 MYST3/NCOA2-EGFP mosaic transgenic animals developed AML at 14 and 26 months. Two models of MPN have also been developed. Le et al. utilized CRE/Lox techniques to conditionally activate kRASG12D in developing embryos [3]. A subset of these animals went on to develop myeloproliferative neoplasm with a latency of 66.2 23.1 days (n = 10 of 19 sh). Forrester et al. also developed a conditional CRE/Lox transgenic approach to model MPN [13]. Specically, NUP98-HOXA9 was conditionally activated in pu.1 expressing cells, leading to 23% of adult NUP98HOXA9-transgenic sh developing MPN by 1923 months of age. Finally, several investigators have utilized heat-shock transgenic approaches to uncover early developmental eects of fusion oncogenes in blood development, including AML1ETO, RUNX1-CBF2T1, and TEL-JAK2 [4, 14, 15]. These heat-shock approaches drive transgene expression during early development and often result in aberrant arrest of cells in early stages of blood development. However, the development of frank leukemia in heat-shock inducible transgenic lines has yet to be reported. Taken together, zebrash have fast emerged as a novel animal model of leukemia and are
Advances in Hematology poised to contribute to our understanding of the molecular pathogenesis of human disease.
Advances in Hematology multiple, linearized transgenes to incorporate into the genome as concatamers when microinjected into one-cell stage zebrash, ultimately culminating in the coexpression of transgenes in developing disease. We have successfully used this approach to show that kRASG12D collaborates with p53 loss to induce early onset embryonal rhabdomyosarcoma [22] and work from Feng et al., elegantly showed that mosaic transgenesis can be used to modify Myc-induced T-ALL through coinjection of activated Akt [10]. We have used similar approaches to develop T-ALLs that coexpress MYC and various uorescent reporters including AmCyan, GFP, zsYellow, dsREDexpress, and mCherry [23, 24]. In these experiments, embryos are coinjected with Myc and uorescent protein under transcriptional control of the rag2 promoter. A small cohort of animals develop uorescently labeled thymi that eventually progresses into T-ALL. Using this approach, we have been able to create T-ALLs in various genetic backgrounds, permitting the creation of syngeneic strain sh that develop multicolored T-ALL (Figure 1) [23]. Finally, we have recently utilized mosaic transgenesis to coexpress Notch1a-ICD, MYC, and GFP by coinjection of three transgenes simultaneously into one-cell stage animals [1]. In summary, while some uorescent transgenic approaches can be limited by fusion stability, early onset of cancer, and genetic background, other uorescent transgenic approaches have been able to overcome these limitations. Such approaches provide rapid assays to identify collaborating oncogenic/tumor suppressor pathways in leukemia.
3 host immune system and subsequent attack of engrafted cells [23, 28]. To avoid immune rejection, Mizgirev and Revskoy recently developed syngeneic zebrash strains and created robust models of transplantable, chemically induced hepatocellular carcinomas, hepatoblastomas, cholangiocarcinoma, and pancreatic carcinoma [2931]. Specically, syngeneic zebrash were created by fertilizing eggs with UV-inactivated sperm, then subjecting eggs to heat-shock [29]. Female gynogenic diploid animals were raised to adulthood and the process repeated. The resulting progeny were genetically similar and could be maintained by incrossing or mating male sh back to the founding mother. Several lines were created using this method including clonal golden strain 1 and 2 (CG1 and CG2). Adoptive transfer of chemical-induced cancers and Tg(rag2:EGFP-Myc-) induced T-ALLs from CG2-strain sh could engraft disease into syngeneic recipients [31]. Moreover, uorescently labeled rhabdomyosarcoma and TALL cells arising in CG1 strain sh could also engraft into nonirradiated, recipient sh [23, 24]. Taken together, these results illustrate the power of cell transplantation and use of syngeneic zebrash to study leukemia cell engraftment.
4. Cell Transplantation Approaches to Examine Tumor Cell Homing and Intravasation into Vessels
Blood cells and their dynamic cell movements can be easily visualized in live uorescent transgenic zebrash. For example, researchers have tracked the migration of various blood lineages including erythroid and macrophage progenitors [25, 3234]. Importantly, hematopoietic stem cell (HSC) movement can also be followed in Tg(CD41:eGFP), Tg(cmyb:GFP), Tg(runx1:GFP), and Tg(lmo2:GFP) transgenic zebrash larvae [3540]. Moreover, uorescently labeled blood cells can also be tracked in adult sh [27, 41]. Capitalizing on cell transplantation approaches, investigators have also utilized uorescence imaging to visualize normal hematopoietic cell homing in live animals. For example, Bertrand et al. visualized HSC homing to the caudal hematopoietic tissue by transplanting Tg(CD41:eGFP; gata1:dsRed) cells into irradiated recipients [36]. We have also described the homing of Tg(lck:GFP)+ T cells back to the thymus following transplantation of cells into larval wildtype sh [42]. While malignant GFP+ T-ALL lymphoblasts also migrate to the thymus, they exhibit robust and specic homing to the olfactory bulb [6, 7]. These studies demonstrate the ease of visualizing cell migration and homing to specic anatomically dened sites within live animals using uorescently labeled normal hematopoietic and leukemic cells. Intravasation of cancer cells into the vasculature is a critical step in cancer progression, allowing the spread of tumor cells beyond the site of origin [43]. The extent to which lymphoblasts disseminate is the clinically dening characteristic of T-lymphoblastic lymphoma (T-LBL) and acute Tlymphoblastic leukemia (T-ALL) [8]. In T-LBL, transformed lymphoblasts are conned to mediastinal masses, while frank leukemia involves dissemination of cells to the marrow.
Advances in Hematology
10 d
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Figure 1: Fluorescently labeled Myc-induced T-ALLs from CG1-strain zebrash engraft into nonirradiated CG1-strain recipients. (a)(c) GFP-labeled T-ALLs were isolated from primary leukemic sh, and 1 103 FACS sorted GFP-labeled leukemia cells were transplanted into nonirradiated CG1-strain animals and scored for engraftment at 10, 20, and 30 days posttransplantation. (d)(f) T-ALL transplant recipients that express Amcyan (d), dsRED (e), and zsYellow (f) under the rag2 promoter. Panels are merged images of uorescent and brighteld photographs. Images were originally published in [23].
Remarkably, this disease transition was recently visualized in zebrash transplanted with uorescently labeled lymphoblasts [10]. For example, RFP+ lymphoblasts from Mycinduced T-ALL were able to intravasate into Tg(i:GFP)labeled vasculature, while cells that overexpressed the antiapoptotic protein Bcl2 were unable to enter the vasculature and, thus, were arrested in a T-LBL state (Figure 2) [10]. Remarkably, treatment of transgenic zebrash that overexpressed MYC and Bcl2 with an antagonist to Sphingosine-1Phosphate (S1P1), a T-cell adhesion and migration protein, promoted invasion into the vasculature [10]. These elegant studies by Feng et al. were the rst to directly visualize the molecular mechanisms governing the transition of T-LBL to T-ALL and underscore the power of imaging dynamic cellular processes in uorescently labeled animals.
responds to the same drugs that are used to treat human TALL patients [31]. In addition, uorescently labeled cells can be assessed for response to radiation. For example, we have shown that engrafted GFP-labeled T-ALLs that coexpress EGFP-bcl2 and the Myc transgene failed to undergo apoptosis following 20 Gy of gamma-irradiation [44]; however, T-ALLs that express only Myc were ablated by 4 days postirradiation, suggesting that Myc-induced T-ALL have an intact p53 DNA damage pathway.
6. Cell Transplantation Approaches to Quantify Leukemia Propagating Cell Frequency and Aggression
Leukemia-propagating cells (LPCs) have the capacity to produce all the other cell types contained within the leukemia, are responsible for continued tumor growth, and ultimately drive relapse. Investigators have used uorescence-activated cell sorting (FACS) to identify unique cell populations and limiting dilution cell transplantation to assess if molecularly dened leukemia cells retain LPC activity in human disease. For example, in AML a rare CD34+, CD38 cell enriches for leukemia-propagating potential [45, 46]. In T-ALL, it has been suggested that CD34+ CD7+ cell populations are enriched in LPCs [47]. Despite enormous eorts aimed at dening if and what cell surface markers dene LPC activity, relatively little is known about the molecular mechanisms that drive leukemia propagating activity. For example, elegant work from Jean Souliers group has shown xenograft transplantation of primary human T-ALL into
Advances in Hematology
EGFP (blood vessel) (a) (b) dsRED (tumor cells) (c) EGFP/dsRED overlay
Myc; Cre
(d)
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Figure 2: Zebrash T-lymphoblasts overexpressing bcl2 spread locally but fail to intravasate into vasculature. (a)(c) dsRED2-expressing lymphoma cells (b) from the Myc; Cre sh intravasate into EGFP-labeled vasculature (a) of the transplant host Tg(i1:EGFP); Casper by 6 days posttransplantation (see arrowheads in (c)). (d)(f) In contrast, dsRED2-expressing lymphoma cells (e) from the Myc; Cre; bcl2 sh fail to intravasate vasculature (d) of the transplant hosts by 6 days posttransplantation (compare (f) with (c)). Note aggregates of the Myc; Cre; bcl2 lymphoma cells in (e) and (f). Scale bar is 10 m. Reprinted from [10].
Treated
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Figure 3: Syngeneic zebrash transplant models of T-ALL are a powerful tool for drug discovery: T-ALL growth is suppressed by cyclophosphamide treatment. Approximately 200 cells/5 nL were engrafted into 5-day-old syngeneic CG2 larvae. Engrafted animals were treated with cyclophosphamide (400 mg/L dissolved in sh water) beginning 5 days posttransplantation. Images of control (a) and treated animals. (b) Tumor growth was assessed based on the percentage of body taken over by GFP+ T-ALL and compared using t -test calculations. This work was performed in [31] and later published in [61].
immune-compromised mice selected for a small subset of clones found within the diagnosis leukemia [48]. These clones contained specic genomic lesions that likely increase leukemia aggression and increase the frequency of LPCs within the bulk of the leukemia mass [48]. Yet, despite the identication of recurrent genomic changes associated within continued clonal evolution, the mechanisms driving these relapse-associated processes are largely unknown. The process by which leukemic cells acquire mutations to increase aggression and frequency of LPCs has been dicult to study in human and mouse models of disease. However, recent work from the Trede group has utilized serially passaged uorescently labeled zebrash T-ALLs to demonstrate that leukemias become more aggressive and develop with shortened latency [49]. To assess genetic changes acquired between the primary and evolved clones, array
comparative hybridization studies were completed to identify recurrent genomic DNA alterations associated with increased aggression. An average of 34 new copy number aberrations (CNAs) were identied in T-ALLs following serial passaging, a majority of which were also found in human T-ALL [49]. Clonal evolution can also result in increased numbers of LPCs contained within the leukemia mass [48]. To directly assess LPC frequency within the bulk of the tumor mass, we have pioneered high-throughput limiting dilution cell transplantation approaches and showed that 1% of Mycinduced T-ALL cells has the capacity to remake leukemia in syngeneic recipient animals [23, 24]. Following serial passaging, a subset of clones can increase LPC activity with up to 16% of cells now capable of inducing leukemia in transplant recipient animals [23]. Similar array CGH studies as described by Rudner et al. [49] are currently underway
6 to identify recurrent CNAs associated with modulating LPC frequency in zebrash T-ALL. Taken together, we believe that unbiased genetic approaches, when coupled with limiting dilution cell transplantation assays in zebrash, will likely uncover the mechanisms driving relapse-associated changes in aggression and LPC frequency in human disease.
Advances in Hematology
[5] D. M. Langenau, H. Feng, S. Berghmans, J. P. Kanki, J. L. Kutok, and A. T. Look, Cre/lox-regulated transgenic zebrash model with conditional myc-induced T cell acute lymphoblastic leukemia, Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 17, pp. 60686073, 2005. [6] J. Chen, C. Jette, J. P. Kanki, J. C. Aster, A. T. Look, and J. D. Grin, NOTCH1-induced T-cell leukemia in transgenic zebrash, Leukemia, vol. 21, no. 3, pp. 462471, 2007. [7] D. M. Langenau, D. Traver, A. A. Ferrando et al., Mycinduced T cell leukemia in transgenic zebrash, Science, vol. 299, no. 5608, pp. 887890, 2003. [8] S. H. Swerdlow, International Agency for Research on Cancer, World Health Organization. WHO Classication of Tumours of Haematopoietic and Lymphoid Tissues, International Agency for Research on Cancer, Lyon, France, 2008. [9] A. Gutierrez, R. Grebliunaite, H. Feng et al., Pten mediates Myc oncogene dependence in a conditional zebrash model of T cell acute lymphoblastic leukemia, Journal of Experimental Medicine, vol. 208, no. 18, pp. 15951603, 2011. [10] H. Feng, D. L. Stachura, R. M. White et al., T-lymphoblastic lymphoma cells express high levels of BCL2, S1P1, and ICAM1, leading to a blockade of tumor cell intravasation, Cancer Cell, vol. 18, no. 4, pp. 353366, 2010. [11] J. K. Frazer, N. D. Meeker, L. Rudner et al., Heritable Tcell malignancy models established in a zebrash phenotypic screen, Leukemia, vol. 23, no. 10, pp. 18251835, 2009. [12] J. Zhuravleva, J. Paggetti, L. Martin et al., MOZ/TIF2-induced acute myeloid leukaemia in transgenic sh, British Journal of Haematology, vol. 143, no. 3, pp. 378382, 2008. [13] A. M. Forrester, C. Grabher, E. R. Mcbride et al., NUP98HOXA9-transgenic zebrash develop a myeloproliferative neoplasm and provide new insight into mechanisms of myeloid leukaemogenesis, British Journal of Haematology, vol. 155, no. 2, pp. 167181, 2011. [14] M. L. Kalev-Zylinska, J. A. Horseld, M. V. C. Flores et al., Runx1 is required for zebrash blood and vessel development and expression of a human RUNX-1-CBF2T1 transgene advances a model for studies of leukemogenesis, Development, vol. 129, no. 8, pp. 20152030, 2002. [15] S. M. N. Onnebo, M. M. Condron, D. O. McPhee, G. J. Lieschke, and A. C. Ward, Hematopoietic perturbation in zebrash expressing a tel-jak2a fusion, Experimental Hematology, vol. 33, no. 2, pp. 182188, 2005. [16] S. W. Park, J. M. Davison, J. Rhee, R. H. Hruban, A. Maitra, and S. D. Leach, Oncogenic KRAS induces progenitor cell expansion and malignant transformation in zebrash exocrine pancreas, Gastroenterology, vol. 134, no. 7, pp. 20802090, 2008. [17] A. T. Nguyen, A. Emelyanov, C. H. Koh et al., A high level of liver-specic expression of oncogenic Kras(V12) drives robust liver tumorigenesis in transgenic zebrash, Disease Models & Mechanisms, vol. 4, pp. 801813, 2011. [18] E. E. Patton and L. I. Zon, Taking human cancer genes to the sh: a transgenic model of melanoma in zebrash, Zebrash, vol. 1, no. 4, pp. 363368, 2005. [19] C. Santoriello, E. Gennaro, V. Anelli et al., Kita driven expression of oncogenic HRAS leads to early onset and highly penetrant melanoma in zebrash, PloS ONE, vol. 5, no. 12, Article ID e15170, 2010. [20] M. Dovey, R. M. White, and L. I. Zon, Oncogenic NRAS cooperates with p53 loss to generate melanoma in zebrash, Zebrash, vol. 6, no. 4, pp. 397404, 2009.
Conict of Interests
The authors declare no competing nancial interests.
Acknowledgments
D. M. Langenau is supported by NIH Grants K01 AR055619, 1RO1CA154923, and 1R21CA156056, an American Cancer Society Research Scholar Grant, Leukemia Research Foundation, the Alex Lemonade Stand Foundation, and the Harvard Stem Cell Institute.
References
[1] J. S. Blackburn, S. Liu, D. M. Raiser et al., Notch signaling expands a pre-malignant pool of T-cell acute lymphoblastic leukemia clones without aecting leukemia-propagating cell frequency, Leukemia. In press. [2] H. E. Sabaawy, M. Azuma, L. J. Embree, H. J. Tsai, M. F. Starost, and D. D. Hickstein, TEL-AML1 transgenic zebrash model of precursor B cell lymphoblastic leukemia, Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 41, pp. 1516615171, 2006. [3] X. Le, D. M. Langenau, M. D. Keefe, J. L. Kutok, D. S. Neuberg, and L. I. Zon, Heat shock-inducible Cre/Lox approaches to induce diverse types of tumors and hyperplasia in transgenic zebrash, Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 22, pp. 94109415, 2007. [4] J. R. J. Yeh, K. M. Munson, Y. L. Chao, Q. P. Peterson, C. A. MacRae, and R. T. Peterson, AML1-ETO reprograms hematopoietic cell fate by downregulating scl expression, Development, vol. 135, no. 2, pp. 401410, 2008.
Advances in Hematology
[21] D. M. Langenau, M. D. Keefe, N. Y. Storer et al., Eects of RAS on the genesis of embryonal rhabdomyosarcoma, Genes and Development, vol. 21, no. 11, pp. 13821395, 2007. [22] D. M. Langenau, M. D. Keefe, N. Y. Storer et al., Co-injection strategies to modify radiation sensitivity and tumor initiation in transgenic zebrash, Oncogene, vol. 27, no. 30, pp. 4242 4248, 2008. [23] A. C. H. Smith, A. R. Raimondi, C. D. Salthouse et al., High-throughput cell transplantation establishes that tumorinitiating cells are abundant in zebrash T-cell acute lymphoblastic leukemia, Blood, vol. 115, no. 16, pp. 32963303, 2010. [24] J. S. Blackburn, S. Liu, A. R. Raimondi, M. S. Ignatius, C. D. Salthouse, and D. M. Langenau, High-throughput imaging of adult uorescent zebrash with an LED uorescence macroscope, Nature Protocols, vol. 6, no. 2, pp. 229241, 2011. [25] D. Traver, B. H. Paw, K. D. Poss, W. T. Penberthy, S. Lin, and L. I. Zon, Transplantation and in vivo imaging of multilineage engraftment in zebrash bloodless mutants, Nature Immunology, vol. 4, no. 12, pp. 12381246, 2003. [26] R. M. White, A. Sessa, C. Burke et al., Transparent adult zebrash as a tool for in vivo transplantation analysis, Cell Stem Cell, vol. 2, no. 2, pp. 183189, 2008. [27] L. Zhang, C. Alt, P. Li, R. M. White, and L. I. Zon, An optical platform for cell tracking in adult zebrash, Cytometry Part A, vol. 81, pp. 176182. [28] D. Traver, A. Winzeler, H. M. Stern et al., Eects of lethal irradiation in zebrash and rescue by hematopoietic cell transplantation, Blood, vol. 104, no. 5, pp. 12981305, 2004. [29] I. V. Mizgireuv and S. Y. Revskoy, Transplantable tumor lines generated in clonal zebrash, Cancer Research, vol. 66, no. 6, pp. 31203125, 2006. [30] I. Mizgirev and S. Revskoy, Generation of clonal zebrash lines and transplantable hepatic tumors, Nature Protocols, vol. 5, no. 3, pp. 383394, 2010. [31] I. V. Mizgirev and S. Revskoy, A new zebrash model for experimental leukemia therapy, Cancer Biology and Therapy, vol. 9, no. 11, pp. 895903, 2010. [32] C. Hall, M. Flores, T. Storm, K. Crosier, and P. Crosier, The zebrash lysozyme C promoter drives myeloid-specic expression in transgenic sh, BMC Developmental Biology, vol. 7, article 42, 2007. [33] X. Y. Zhang and A. R. F. Rodaway, SCL-GFP transgenic zebrash: in vivo imaging of blood and endothelial development and identication of the initial site of denitive hematopoiesis, Developmental Biology, vol. 307, no. 2, pp. 179194, 2007. [34] M. J. Redd, G. Kelly, G. Dunn, M. Way, and P. Martin, Imaging macrophage chemotaxis in vivo: studies of microtubule function in zebrash wound inammation, Cell Motility and the Cytoskeleton, vol. 63, no. 7, pp. 415422, 2006. [35] E. Murayama, K. Kissa, A. Zapata et al., Tracing hematopoietic precursor migration to successive hematopoietic organs during zebrash development, Immunity, vol. 25, no. 6, pp. 963975, 2006. [36] J. Y. Bertrand, A. D. Kim, S. Teng, and D. Traver, CD41+ cmyb+ precursors colonize the zebrash pronephros by a novel migration route to initiate adult hematopoiesis, Development, vol. 135, no. 10, pp. 18531862, 2008. [37] K. Kissa, E. Murayama, A. Zapata et al., Live imaging of emerging hematopoietic stem cells and early thymus colonization, Blood, vol. 111, no. 3, pp. 11471156, 2008. [38] J. Y. Bertrand, N. C. Chi, B. Santoso, S. Teng, D. Y. R. Stainier, and D. Traver, Haematopoietic stem cells derive directly from
7
aortic endothelium during development, Nature, vol. 464, no. 7285, pp. 108111, 2010. E. Y. N. Lam, C. J. Hall, P. S. Crosier, K. E. Crosier, and M. V. Flores, Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells, Blood, vol. 116, no. 6, pp. 909914, 2010. K. Kissa and P. Herbomel, Blood stem cells emerge from aortic endothelium by a novel type of cell transition, Nature, vol. 464, no. 7285, pp. 112115, 2010. C. Hall, M. V. Flores, K. Crosier, and P. Crosier, Live cell imaging of zebrash leukocytes, Methods in Molecular Biology, vol. 546, pp. 255271, 2009. D. M. Langenau, A. A. Ferrando, D. Traver et al., In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrash, Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 19, pp. 73697374, 2004. P. B. Gupta, S. Mani, J. Yang, K. Hartwell, and R. A. Weinberg, The evolving portrait of cancer metastasis, Cold Spring Harbor Symposia on Quantitative Biology, vol. 70, pp. 291297, 2005. D. M. Langenau, C. Jette, S. Berghmans et al., Suppression of apoptosis by bcl-2 overexpression in lymphoid cells of transgenic zebrash, Blood, vol. 105, no. 8, pp. 32783285, 2005. D. Bonnet and J. E. Dick, Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell, Nature Medicine, vol. 3, no. 7, pp. 730 737, 1997. T. Lapidot, C. Sirard, J. Vormoor et al., A cell initiating human acute myeloid leukaemia after transplantation into SCID mice, Nature, vol. 367, no. 6464, pp. 645648, 1994. B. Gerby, E. Clappier, F. Armstrong et al., Expression of CD34 and CD7 on human T-cell acute lymphoblastic leukemia discriminates functionally heterogeneous cell populations, Leukemia, vol. 25, pp. 12491258, 2011. E. Clappier, B. Gerby, F. Sigaux et al., Clonal selection in xenografted human T cell acute lymphoblastic leukemia recapitulates gain of malignancy at relapse, Journal of Experimental Medicine, vol. 208, no. 4, pp. 653661, 2011. L. A. Rudner, K. H. Brown, K. P. Dobrinski et al., Shared acquired genomic changes in zebrash and human T-ALL, Oncogene, vol. 30, pp. 42894296, 2011. M. S. C. C. Ignatius, N. M. Elpek, A. Fuller et al., in vivo imaging of tumor-propagating cells, regional tumor heterogeneity, and dynamic cell movements in embryonal rhabdomyosarcoma, Cancer Cell, vol. 21, no. 5, pp. 680693, 2012. D. P. Corkery, G. Dellaire, and J. N. Berman, Leukaemia xenotransplantation in zebrashchemotherapy response assay in vivo, British Journal of Haematology, vol. 153, no. 6, pp. 786789, 2011. A. M. Cock-Rada, S. Medjkane, N. Janski, N. Yous, and M. Perichon, SMYD3 promotes cancer invasion by epigenetic upregulation of the metalloproteinase MMP-9, Cancer Research, vol. 72, pp. 810820, 2012. B. E. Lally, G. A. Geiger, S. Kridel et al., Identication and biological evaluation of a novel and potent small molecule radiation sensitizer via an unbiased screen of a chemical library, Cancer Research, vol. 67, no. 18, pp. 87918799, 2007. L. M. J. Lee, E. A. Seftor, G. Bonde, R. A. Cornell, and M. J. C. Hendrix, The fate of human malignant melanoma cells transplanted into zebrash embryos: assessment of migration and cell division in the absence of tumor formation, Developmental Dynamics, vol. 233, no. 4, pp. 15601570, 2005.
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
8
[55] I. J. Marques, F. U. Weiss, D. H. Vlecken et al., Metastatic behaviour of primary human tumours in a zebrash xenotransplantation model, BMC Cancer, vol. 9, article 128, 2009. [56] C. Zhao, X. Wang, Y. Zhao et al., A novel xenograft model in zebrash for high-resolution investigating dynamics of neovascularization in tumors, PLoS ONE, vol. 6, no. 7, Article ID e21768, 2011. [57] S. Zhang, Z. Cao, H. Tian et al., SKLB1002, a novel potent inhibitor of VEGF receptor 2 signaling, inhibits angiogenesis and tumor growth in vivo, Clinical Cancer Research, vol. 17, no. 13, pp. 44394450, 2011. [58] V. P. Ghotra, S. He, H. de Bont et al., Automated whole animal bio-imaging assay for human cancer dissemination, PloS ONE, vol. 7, Article ID e31281, 2012. [59] S. He, G. E. Lamers, J. W. Beenakker, C. Cui, V. P. Ghotra et al., Neutrophil-mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrash xenograft model, The Journal of Pathology. In press. [60] A. Eguiara, O. Holgado, I. Beloqui, L. Abalde, and Y. Sanchez, Xenografts in zebrash embryos as a rapid functional assay for breast cancer stem-like cell identication, Cell Cycle, vol. 10, pp. 37513757, 2011. [61] M. S. Ignatius and D. M. Langenau, Fluorescent imaging of cancer in Zebrash, Methods in Cell Biology, vol. 105, pp. 437 459, 2011.
Advances in Hematology
Hindawi Publishing Corporation Advances in Hematology Volume 2012, Article ID 159807, 19 pages doi:10.1155/2012/159807
Review Article Pathogen Recognition and Activation of the Innate Immune Response in Zebrash
Michiel van der Vaart, Herman P. Spaink, and Annemarie H. Meijer
Institute of Biology, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands Correspondence should be addressed to Annemarie H. Meijer, [email protected] Received 3 February 2012; Accepted 22 April 2012 Academic Editor: Christopher Hall Copyright 2012 Michiel van der Vaart et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The zebrash has proven itself as an excellent model to study vertebrate innate immunity. It presents us with possibilities for in vivo imaging of host-pathogen interactions which are unparalleled in mammalian model systems. In addition, its suitability for genetic approaches is providing new insights on the mechanisms underlying the innate immune response. Here, we review the pattern recognition receptors that identify invading microbes, as well as the innate immune eector mechanisms that they activate in zebrash embryos. We compare the current knowledge about these processes in mammalian models and zebrash and discuss recent studies using zebrash infection models that have advanced our general understanding of the innate immune system. Furthermore, we use transcriptome analysis of zebrash infected with E. tarda, S. typhimurium, and M. marinum to visualize the gene expression proles resulting from these infections. Our data illustrate that the two acute disease-causing pathogens, E. tarda and S. typhimurium, elicit a highly similar proinammatory gene induction prole, while the chronic disease-causing pathogen, M. marinum, induces a weaker and delayed innate immune response.
1. Introduction
The use of adult zebrash (Danio rerio) and their transparent ospring as hosts to model infectious diseases caused by human pathogens, or closely related animal pathogens, has recently provided novel insights into pathogenesis, which in many cases could not have been achieved using mammalian models [16]. The power of the zebrash model lies in its suitability for genetic approaches, high-throughput screening, and live imaging studies. Fluorophore-marked transgenic lines are now available that allow unprecedented visualization of pathogen interactions with macrophages and neutrophils, the major phagocytic innate immune cell types of zebrash larvae [711]. As early as one day after fertilization (dpf), zebrash embryos display phagocytic activity towards microbial infections [12] and are able to mount an innate immune response with a transcriptional signature that resembles responses in mammalian or cell culture systems [13]. Adaptive immunity becomes active after approximately three weeks of development [14]. Therefore, innate immunity can be studied during the earlier zebrash embryonic
and larval stages in the absence of T- and B-cell responses. In this paper we focus on signaling pathways involved in pathogen recognition and activation of the innate immune response in zebrash embryos and larvae. We compare the knowledge of the zebrash innate immune system with that of human and mammalian models and discuss results from transcriptomic analyses that show clear specicity in responses to dierent bacterial pathogens, such as Salmonella and Mycobacteria species.
2 This occurs when infection, inammation, or other cellular stresses cause host factors to be present in aberrant locations, or to form abnormal molecular complexes, so called dangerassociated molecular patterns (DAMPs) [17]. PRRs located on the cell surface are scouting the extracellular environment for the presence of microbes. PRRs located on endosomes identify microbes that have entered the phagolysosomal degradation pathway, and cytoplasmic PRRs recognize intracellular cytosolic pathogens or components of internalized microbes [18]. Upon PAMP recognition, PRRs signal the presence of infection and initiate proinammatory and antimicrobial responses by activating several intracellular signaling pathways [19], ultimately leading to activation of gene expression and synthesis of a broad range of molecules. These include proinammatory and chemotactic cytokines and antimicrobial peptides [20]. The dierent families of PRRs present in both humans and zebrash and their downstream signaling pathways are summarized in Figure 1 and will be discussed below. 2.1. Toll-Like Receptors. The most extensively studied class of PRRs are the Toll-like receptors (TLRs), a family of 10 proteins in human. TLRs are named after the Drosophila Toll protein, which functions in dorsoventral patterning and antifungal responses [23]. TLRs are integral glycoproteins which possess an extracellular or luminal, ligand-binding domain with leucine-rich repeat (LRR) motifs and a cytoplasmic signaling Toll/Interleukin-1 (IL-1) receptor homology (TIR) domain [20, 24]. In mammals, the main cell types expressing TLRs are antigen-presenting cells (APCs), including macrophages and dendritic cells, and B lymphocytes [18]. However, most cell types are capable of expressing TLRs, for instance, in response to a localized infection [25]. In mammals, TLR4 recognizes Gram-negative bacteria via the lipid A portion of lipopolysaccharide (LPS), while TLR2 recognizes Gram-positive bacteria via lipoteichoic acid (LTA), lipoproteins, and peptidoglycan, and TLR5 recognizes the motility apparatus protein agellin, which can be present on both Gram types [18]. Other TLRs are specialized in recognizing nuclear acids in endosomal and phagosomal compartments. TLR3 can detect viral replication by binding to double-stranded RNA (dsRNA), TLR7 and TLR8 specically recognize single-stranded RNA (ssRNA) of RNA viruses, and unmethylated CpG DNA present in the genomes of viruses and bacteria is detected by TLR9 [18]. Ligand binding by a TLR will induce it to form homomeric or heteromeric oligomers, which triggers intracellular signal transduction via their TIR domains [18]. The mammalian TLR signaling pathway uses ve dierent TIR-domain-containing adaptor molecules: MYD88, MAL/TIRAP, TRIF/TICAM1, TRAM/TICAM2, and SARM [19, 24]. Among these, MYD88 is the most universal adaptor, since it is used for downstream signaling by all TLRs, with the exception of TLR3 [26]. Downstream signaling via central intermediate molecules such as TRAF6 will eventually lead to the activation of transcription factors, mostly members of the ATF, NFB, AP-1, IRF, and STAT families, regulating the expression of a battery of antimicrobial and proinammatory genes [26].
Advances in Hematology Putative orthologs of mammalian TLRs have been identied in zebrash, in addition to some sh-specic family members [27, 28]. A genome duplication during the evolution of teleost sh most likely explains why zebrash have two counterparts for some of the mammalian TLRs (e.g., tlr4ba/tlr4bb for TLR4 and tlr5a/tlr5b for TLR5), but it is still unknown whether this increase in the number of receptors is associated with diversication in PAMP recognition [4]. Only some of the zebrash TLR ligands are currently known [29]. The specicity of TLR2, TLR3, and TLR5 is conserved between mammals and sh, recognizing lipopeptides, dsRNA, and agellin, respectively [13, 30, 31]. Additionally, the sh-specic TLR22 has been shown to recognize dsRNA and PolyI:C [31]. However, zebrash TLR4 cannot be stimulated by LPS, illustrating that not all ligand specicities are conserved between mammals and zebrash [32, 33]. Signaling intermediates in the pathway downstream of mammalian TLRs have also been identied in zebrash, including homologs of four of the adaptor proteins, Myd88, Mal/Tirap, Trif/Ticam1, and Sarm, and the central intermediate Traf6 [34]. Among these, Myd88 and Traf6 have been functionally studied by knockdown analysis in zebrash embryos, showing their requirement for a proinammatory innate immune response to microbial presence [13, 3537]. Furthermore, triggering of the innate immune response in zebrash embryos also leads to induction of members of the ATF, NFB, AP-1, IRF, and STAT families of transcription factors [13, 38]. 2.2. NOD-Like Receptors. Pathogens that escape the surveillance of cell surface and endosomal PRRs may end up in the cytosol, where nucleotide-binding-oligomerization-domain(NOD-) like receptors (NLRs) detect their presence by intracellular PAMPs and DAMPs [39]. The NLRs constitute a family of 23 proteins in humans. Their dening features are the presence of a centrally located NOD domain responsible for oligomerization, a C-terminal LRR capable of ligandbinding, and an N-terminal protein-protein interaction domain, such as the caspase recruitment domain (CARD), pyrin (PYD), or baculovirus inhibitor repeat (BIR) domain [40]. Two of the NLRs, NOD1 and NOD2, can sense bacterial presence by directly or indirectly detecting molecules produced during synthesis or breakdown of peptidoglycan [40]. NOD1 recognizes g-D-glutamyl-meso-diaminopimelic acid (iE-DAP), a dipeptide produced mostly by Gram-negative bacteria, whilst NOD2 can recognize both Gram types, since it is activated upon binding to muramyl dipeptide (MDP), a more common component of peptidoglycan [41, 42]. Interestingly, both NOD1 and NOD2 have recently been implicated in detection of parasites lacking peptidoglycan, indicating that these receptors can recognize a broader range of pathogens than was originally assumed [43, 44]. Upon ligand-binding, NOD1 and NOD2 recruit the serine/threonine kinase RIPK2 (also known as RIP2) via CARDCARD interactions, eventually leading to the activation of NFB [45, 46]. In addition, NOD1/2 stimulation also induces MAP kinase signaling [47]. Synergistically, with TLR activation, NOD1/2 signaling cascades induce the expression
Advances in Hematology
Mbl Lgals91l Tlr1 Tlr2 Tlr4a/b Tlr5a/b Tlr18 Myd88 Tirap Trif Sarm NFB AP-1 ATF IRF STAT Pro-IL1 Nod1 Nod2 Nod3 Nalp IL1 Dc-sign
TF
L-arginine iNOS
NO
Azurophilic granules
ROS
(Auto)Phagolysosome
Phox
NADP + O 2
Caspase1 Inflammasome
Viral RNA
TF Nucleus
Cytoplasm
Figure 1: Pattern recognition receptors and eector mechanisms of the innate immune system. The localization of Tlrs on the cell surface or on endosomes is hypothetical and based on the known or proposed functions of their homologs in other sh or mammals. The ability of PRRs (depicted in green) to recognize PAMPs present on various types of microorganisms, like bacteria, viruses, and fungi, has been simplied here by depicting microorganisms as rod-like bacteria (in blue). PAMP recognition by PRRs leads to activation of transcription factors (TFs), which translocate to the nucleus and initiate transcription of cytokine genes, antimicrobial genes, and other immune-related genes. Defense mechanisms such as autophagy, ROS and NO production, and degranulation can be immediately activated upon microbial recognition, without de novo gene transcription.
of cytokines and chemokines, such as TNF, IL6, IL8, IL10, and IL12, as well as the production of antimicrobial peptides [46, 48, 49]. Other NLRs, such as IPAF, NALP1, and NALP3, mainly function to create a multiprotein complex known as the inammasome, in which they associate with an adaptor called ASC (apoptosis-associated speck-like protein containing a CARD) and with procaspase 1 [50]. Oligomerization of the proteins in an inammasome via CARD-CARD interactions ultimately leads to the cleavage of procaspase 1 into its active form, caspase 1, which is then available to catalyze the cleavage of accumulated pro-IL1 and pro-IL18 into their secreted forms, biological active IL1 and IL18 [40]. The NLR family member incorporated into these complexes determines which PAMPs and DAMPs are recognized by the inammasome. A role for NALP3 has been established in the recognition of ATP [51], uric acid crystals [52], viral RNA [53], and bacterial DNA [54]. Both NALP1 and NALP3 share NOD2s ability to respond to MDP [55]. Furthermore, NALP1 can associate with NOD2 (Hsu 2008), showing a role for NOD2 in MDP-triggered IL1 activation, separate from its role as an inducer of proinammatory gene expression. Although the function of NLR family members in zebrash is not widely studied, it is known that the canonical members of the mammalian NLR family, NOD1, NOD2, and
NOD3 (or Nlrc3) are conserved. Additionally, a subfamily of NLRs resembling the mammalian NALPs and a unique teleost NLR subfamily are present [34, 56]. Conrmation of the antibacterial role of NOD1 and NOD2 in zebrash was achieved by gene knockdown, resulting in higher bacterial burdens and decreased survival of embryos following Salmonella enterica infection [57]. Moreover, nod1/2 depletion signicantly decreased expression of dual oxidase (DUOX), required for production of reactive oxygen species (ROS) [57]. These ndings illustrate that the family of Nod-like receptors and their downstream signaling pathways are important for antibacterial innate immunity, both in mammals and in zebrash.
2.3. RIG-I-Like Receptors. Another family of cytosolic PRRs, the RIG-I-like receptors (RLRs), consists of three members: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma dierentiation-associated factor 5), and LGP2 (laboratory of genetics and physiology 2). All three members are DExD/H box RNA helicases that can detect the presence of RNA from a broad range of viruses [58]. While expressed at low levels in most tissues, their expression is greatly increased upon viral infections or interferon (IFN) exposure [59, 60]. The RNA helicase domain of RLRs has the capacity to hydrolyze
4 ATP and bind to RNA [61]. Furthermore, RIG-I and MDA5 contain a tandem of CARDs, which facilitate protein-protein interactions [60]. LGP2 lacks the two CARDs and is thought to function as a negative regulator of RIG-I and MDA5 signaling [62]. Following recognition of viral RNA, the CARDs of RIG-I and MDA5 become available for binding to a common mitochondrial signaling adaptor, IPS-1 or MAVS [63]. The subsequent signaling cascade culminates in the induction of transcription factors like interferon regulatory factor 3 (IRF3), IRF7, and NFB [64]. Activation of these transcription factors leads to the production of type I IFN, which binds to the IFN receptor to initiate expression of interferon-stimulated genes (ISGs) [65]. Amongst these ISGs are antiviral proteins, immune-proteasome components, all three RLRs, members of the TLR family, transcription factors like IRF7, and various proinammatory cytokines and chemokines [65]. As such, the RLR-induced pathway works cooperatively with TLR signaling to prepare the cell for elimination of viral infections [58]. Zebrash homologs of RIG-I, MDA5, and DXH58 were identied in a genome search [66]. However, in silico analysis of the predicted proteins revealed that the domain distribution diers between humans and zebrash [66]. For instance, whilst human RIG-I contains two CARDs, one DExD/H domain and a Helicase C domain, zebrash RIGI consists of a single CARD and a DExD/H domain [66]. Whilst functional studies of the RLR pathway are scarce, it is clear that zebrash and other teleosts possess a strong antiviral IFN system, which shares a common evolutionary origin with mammals [67, 68]. The mitochondrial RLR adaptor, IPS-1/MAVS, was recently cloned from salmon and zebrash, and overexpression in sh cells led to a constitutive induction of ISGs [68]. Furthermore, MITA, another adaptor functioning downstream of IPS-1/MAVS and upstream of Tank-binding kinase 1 (TBK1), was cloned from crucian carp (Carassius auratus) and shown to activate zebrash IFN promoter gene constructs, dependent on IRF3 or IRF7 [69]. 2.4. Scavenger Receptors. Scavenger receptors are a large family of transmembrane cell surface receptors, present on macrophages, dendritic cells, mast cells [70], and some endothelial and epithelial cell types [71]. Although originally dened for their role in uptake of low-density lipoproteins (LDL), they are now known to act as PRRs for a wide variety of PAMPs, like LPS, LTA, CpG DNA, yeast zymosan, and microbial surface proteins [72]. Commonly, PAMP binding to a scavenger receptor will induce the cell to directly phagocytose the pathogen [73]. Upregulation of scavenger receptor expression via TLR signaling can be a mechanism to increase phagocytic activity [74]. Moreover, scavenger receptors can also contribute to cytokine production as coreceptors for TLRs [75, 76]. Some of the C-type lectins, discussed below, also display scavenger receptor activity. Based upon their multidomain structure, scavenger receptors are divided into eight subclasses (A-H) (Murphy 2005). Subclasses A and B are the most extensively studied, but members from other subclasses have also been shown to recognize bacterial PAMPs [72]. SR-A, the founding
Advances in Hematology member of subclass A, functions as a phagocytic receptor for bacterial pathogens like Staphylococcus aureus, Neisseria meningitides, Streptococcus pneumonia, and Escherichia coli [7779]. Macrophage receptor with collagenous structure (MARCO), another subclass A member with established PRR activity [80], functions as a phagocytic receptor for S. pneumonia [81] and N. meningitidis [82]. MARCO was shown to cooperate with TLR2 to trigger macrophage cytokine responses to the mycobacterial cell wall glycolipid trehalose dimycolate (TDM) and Mycobacterium tuberculosis [83]. CD36, the most prominent member of subclass B, is a sensor for LTA and diacylated lipopeptide (MALP-2) and also acts as a coreceptor for TLR2 [75]. CD36-mediated phagocytosis of S. aureus was shown to be required for initiation of TLR2/6 signaling [84]. SR-BI (or CLA-1), also in subclass B, can bind to LPS and was implicated in phagocytosis of both Gram-negative and Gram-positive bacteria [85]. As well as their antibacterial roles, CD36 and SR-BI are also known for increasing the pathogenesis of malaria and hepatitis C virus (HCV). CD36 can function as a receptor for erythrocytes that have been parasitized by Plasmodium falciparum, adhering these cells to the venular endothelium of various organs (Pluddemann 2007). Furthermore, SR-BI is used by Plasmodium sporozoites and HCV as an entry site into hepatocytes [72]. Many homologs of the mammalian scavenger receptor family can be identied in the zebrash genome, but a systematic analysis is still awaited. A zebrash homolog of human MARCO was identied as a specic marker for macrophages and dendritic cells from adult zebrash [86], and this gene is also myeloid specic in zebrash embryos [87]. Expression of the cd36 gene was upregulated after exposing zebrash to haemorrhagic septicemia rhabdovirus [88]. In contrast, cd36 expression was downregulated by Mycobacterium marinum infection in adult zebrash and larvae [22]. 2.5. C-Type Lectins. The C-type lectin receptors (CLRs) are a large family of carbohydrate-binding proteins that are highly conserved amongst mammals [89]. The diversity of the CLR family is illustrated by the fact that up to 17 groups are present in vertebrates, with some consisting of soluble serum proteins, whilst others consist of transmembrane proteins. These are mainly expressed in myeloid cells (macrophages and dendritic cells) but also in natural killer cells [90, 91]. The best known CLR in serum is mannose-binding lectin (MBL), a member of the collectin class, which binds to a variety of sugar moieties present on viruses, bacteria, fungi, and protozoa and activates the complement system [92]. In terms of their function as PRRs, the transmembrane CLRs that are expressed on myeloid cells are the most interesting. Transmembrane CLRs can be divided into two groups: the mannose receptor family and the asialoglycoprotein receptor family [93]. CLRs recognize pathogens mainly via ligand binding to mannose, fucose, and glucan carbohydrate structures, which means that together they are capable of recognizing most classes of human pathogens [93]. Like scavenger receptors, CLRs can act as phagocytic receptors
Advances in Hematology for nonopsonized bacteria, leading to their destruction in acidied phagolysosomes [73]. The best-studied member of the asialoglycoprotein receptor family is Dectin-1, which mediates phagocytosis of yeast and the yeast-derived protein zymosan [94]. Phagocytosis induced by CLRs like Dectin1 is not only important for the lysosomal breakdown of pathogens, but also for antigen presentation [95, 96]. Besides their role in phagocytosis, CLRs can directly induce gene expression upon carbohydrate recognition. PAMP recognition by Dectin-1, Dectin-2, and macrophage-inducible Ctype lectin (Mincle) ultimately leads to activation of NFB [9799]. Where Dectin-1 associates with the kinase Syk to activate NFB [100], Dectin-2 and Mincle are dependent on Fc receptor -chain as an adaptor molecule [98, 99]. Other CLRs, for example, DC-specic ICAM3-grabbing nonintegrin (DC-SIGN), induce specic gene expression proles upon pathogen recognition by modulating TLR signalling [93]. When DC-SIGN recognizes mannose or fucose moieties on pathogens such as Mycobacteria, HIV1, measles virus, and Candida albicans, it activates a Raf-1dependent signaling pathway that modulates TLR-induced NFB activation, increasing the production of IL8 and antiinammatory IL10 production [101]. Only a few homologs of CLRs have been described in zebrash. A homolog of the complement activating mannose-binding lectin (MBL) was associated with resistance against Listonella anguillarum [102]. Expression of another soluble lectin, lgals91l, is enriched in zebrash embryonic myeloid cells and is dependent on the Spi1/Pu.1 transcription factor that plays a crucial role in myeloid cell development in vertebrates [87]. A membrane type collectin, CL-P1 (collectin placenta 1), was shown to be involved in vasculogenesis during zebrash embryogenesis [103]. In humans, CL-P1 is mainly expressed on vascular endothelial cells and has been shown to act as a scavenger receptor mediating the phagocytosis of bacteria and yeast [104]. A putative homolog for DC-SIGN has recently been proposed and is upregulated in immune-related tissues following infection by Aeromonas anguillarum [105]. Finally, putative homologs for the mammalian C-type lectin NK cell receptors have been identied in zebrash and are dierentially expressed on cells from the myeloid and lymphoid lineages [106].
5 proteins, including complement factors and other acutephase proteins, make an important contribution to the innate defenses, and strong induction of their encoding genes has been observed in adult and embryonic zebrash infection models [13, 36, 38, 107109]. 3.1. Secreted Peptides and Lipid Mediators of the Innate Immune Response. Cytokines, including interleukins, chemokines, and interferons, are small secreted proteins that steer the hosts immune system into a cytotoxic, humoral, cell-mediated, or allergic response [110]. Since this paper focuses on innate immunity, we will mainly discuss the cytokines produced by or acting on phagocytic cells. A distinction can be made between cytokines that promote a state of inammation and cytokines that are anti-inammatory. The main proinammatory cytokines produced by phagocytes are TNF, IL1, IL1, IL6, and IL8 [110]. TNF- is processed as a membrane-bound protein and, when required, the active soluble factor is cleaved o by the TNF- converting enzyme (TACE) [111]. Similarly, IL1 and IL1 are synthesized as inactive precursors that are only secreted as active cytokines after inammasomemediated cleavage by caspase 1 [112]. The most potent antiinammatory cytokine in humans is IL10, which deactivates the proinammatory cytokine production by macrophages and T cells [113]. The IL10/IL12 balance, maintained by cells of the innate immune system, determines whether adaptive immunity polarizes towards a Th1 (promoted by IL12) or Th2 response. A Th1 response, which activates the bactericidal activities of macrophages, is the most important for controlling intracellular pathogens. The single type II IFN, IFN, is also required for activating macrophage bactericidal functions, while type I IFNs (IFN and IFN) and type III IFN (IFN) function in mounting antiviral responses. Finally, eicosanoid lipid mediators also promote (e.g., prostaglandins and leukotrienes) or inhibit (e.g., lipoxins) inammation, thus synergizing with or antagonizing cytokine functions. Many of the cytokine subfamilies are conserved between zebrash and mammals [34]. However, there has been extensive expansion and diversication of members of the chemokine gene family in zebrash, and their specic functions are yet to be determined [114]. Several of the main cytokines, like IL1, IL6, and IL10, have been cloned and characterized [115117]. Furthermore, the zebrash homolog of interleukin 10 receptor 1 (IL10R1) has recently been identied and seems to contain all the protein domains that are required for its function in anti-inammatory signaling [118]. The proinammatory chemokine IL8 (CXCL8) and it receptors, CXCR1 and CXCR2, are also conserved between mammals and zebrash [119]. In addition, a second IL8/CXCL8 lineage has been identied in both zebrash and common carp (Cyprinus carpio), and the chemotactic properties of carp IL8/CXCL8 molecules of both lineages were demonstrated by in vitro chemotaxis assays using carp leukocytes [120]. Both pro- and anti-inammatory cytokines are upregulated upon infection of zebrash embryos with pathogens such as S. typhimurium [13], P. aeruginosa [121], and E. tarda [122, 123].
6 The role of TNF during Mycobacterium marinum infection of zebrash embryos was studied by knockdown analysis of the TNF receptor (tnfrsf1a), which revealed that intracellular bacterial burdens, granuloma formation, and necrotic death of macrophages are increased in the absence of TNF signaling [124]. The importance of TNF signaling during M. marinum infection was further illustrated when the same model was used to show that a strict balance between proinammatory TNF and anti-inammatory lipoxins is vital for control of mycobacterial infections, with either too much or too little TNF expression leading to a more severe outcome of the disease [1]. Another study using the zebrash model indicates that TNF- is a potent activator of endothelial cells, leading to the production of chemokines, whilst it has little eect on the activation status of phagocytes [125]. This suggests that sh TNF- mainly functions in the recruitment of leukocytes to the site of infection, rather than activating them. The three IFN groups present in humans are not conserved unambiguously in zebrash and other sh species. The type II group of IFNs in zebrash consists of IFN1 and IFN2 [126]. Expression levels of the corresponding genes did not change upon infection of zebrash embryos with E. coli or Y. ruckeri, but was increased by M. marinum infection [126, 127]. Viral infection induced their expression in adult zebrash but not in embryos [126]. IFN1 and IFN2 were shown to bind to dierent receptor complexes, and Janus kinase 2a (Jak2a), but not Jak2b, was shown to be required for intracellular transmission of the IFN signal. Two groups of antiviral IFNs, named IFN1 and IFN2, exist in zebrash, and structural analysis showed that these are evolutionarily closer to type I than to type III human IFNs [34, 67, 128]. IFN1 and IFN2 signal via distinct receptor complexes [67, 129]. All zebrash IFN genes induce the expression of genes that are predicted to be involved in antiviral activities [67]. 3.2. Phagocytosis, Autophagy, and Lysosomal Destruction. Internalization of microorganisms is triggered when they are recognized by phagocytic receptors, mainly by scavenger receptors discussed above. This type of direct phagocytosis is termed nonopsonic phagocytosis, while opsonic phagocytosis relies on host-derived proteins that coat the surface of the microbe thereby enhancing phagocytosis eciency. Opsonins include complement fragments, most notably C3b, which are recognized by complement receptors [130]. Mannose binding lectin, which can initiate C3b formation, and antibodies that bind to Fc receptors (IgG) or that activate complement (IgM) are also considered opsonins. Regardless of which receptor initiates the process, phagocytosis requires the activation of kinases and Rab GTPases that control alterations in the phospholipid membrane and remodeling of the actin cytoskeleton [131]. In macrophages, fusion of the resulting vesicle with early and late endosomes will decrease the pH of the immature phagosome and alter the proteins present on its membrane. Ultimately, maturing phagosomes turn into phagolysosomes when lysosomes fuse with them, mixing their contents [132]. Lysosomes are
Advances in Hematology highly acidic endocytic vesicles (pH < 5.5), containing active proteases and lipases, and hydrolytic enzymes such as cathepsin D [133]. In addition, phagolysosomes also contain bactericidal peptides (defensins) and have the ability to generate toxic oxidative compounds that help microbial degradation [134]. Most of our knowledge about phagosome maturation comes from studies of phagocytosis in macrophages, and much less is known about phagosome maturation in neutrophils. While macrophage phagosomes fuse with endosomes and lysosomes, neutrophil phagosomes obtain their bactericidal properties by fusing with secretory vesicles and granules [135, 136]. In contrast to phagosome maturation in macrophages, neutrophil phagosomes do not acidify in order to become microbicidal [135, 136]. Many intracellular pathogens, like M. tuberculosis, S. typhimurium, and Legionella pneumophila, have evolved the ability to prevent phagosome maturation in macrophages and survive inside these vesicles [137]. To a certain extent, such pathogens can also withstand the hostile environment of the (phago)lysosome. Other pathogens like Listeria monocytogenes, Francisella tularensis, and many viruses can escape the phagosome and enter the cytosol [138]. Mycobacterium marinum, a pathogen studied extensively in zebrash to model human tuberculosis, can survive inside phagosomes but also escape into the cytosol and spread to neighbouring cells by actin-based motility [139, 140]. Phagosomal escape has also been observed for the human pathogen M. tuberculosis and is dependent on a virulence factor, the ESX-/RD1 secretion system, shared by all pathogenic mycobacteria [141]. Together, these data indicate that host cells face numerous pathogens that have developed multiple strategies to avoid the pathway of phagolysosomal degradation. To counter such threats, cells may use autophagy to clear microbes and microbe-containing vesicles from the cytosol. Autophagy is well known as a metabolic process that recycles nutrients by degrading intracellular organelles and proteins. Only recently, it has been recognized that autophagy also plays an important role in the innate immune response against intracellular pathogens [142]. Autophagy is initiated when an autophagosomal isolation membrane is formed around its target, enclosing it entirely in a double-membrane vesicle. This process relies on class III phosphatidylinositol 3-kinase (PI3-kinase) and autophagyrelated genes (Atgs), such as Atg6 (or Beclin-1) [143]. The hallmark of autophagosomes is the presence of Atg8 (or LC3) in their membranes, which is essential for membrane elongation [144]. Similar to a maturing phagosome, the autophagosome also fuses with lysosomes to achieve its degradative properties [145]. In addition, autolysosomes acquire unique antimicrobial properties due to the function of autophagic adaptor protein p62, which delivers cytosolic components to autolysosomes where they are processed into potent antimicrobial peptides [146]. As reviewed elsewhere [147], pathogen-targeted autophagy can be induced by several TLRs and NLRs, TNF-, NFB, and many other immune-related signalling molecules. The transparency of zebrash embryos and availability of uorescent macrophage and neutrophil reporter lines allow for study of the process of phagocytosis in great detail
Advances in Hematology [7, 148150]. It was recently shown that zebrash embryonic macrophages eciently engulf E. coli bacteria from bloodand uid-lled cavities, while neutrophils are hardly capable of phagocytosing bacteria present in uids [150]. However, neutrophils did prove to be highly phagocytic when moving over bacteria present on tissue surfaces. This shows that the type of immune cell that clears an infection not only depends on the PAMPs present on the invading microbe, but also on the characteristics of the infection site. An in vivo phagocytosis assay was used to show that functions of Wasp1, Wasp2, Abi2, and colin regulator 14-3-3 (Ywab) in bacterial phagocytosis are conserved in zebrash [151]. The recent generation of a transgenic zebrash line with GFP-tagged LC3 has enabled in vivo visualization of the interactions between microbes and this core component of the autophagy machinery [152]. The importance of autophagy in the innate immune response of zebrash remains to be studied, but we have shown that LC3-labeled structures accumulate around M. marinum infection sites in zebrash embryos (Figure 2). Furthermore, autophagy-related genes were induced in adult zebrash infected with Citrobacter freundii and zebrash embryos infected with S. typhimurium [37, 153]. 3.3. Oxidative Defenses in Leukocytes. In several systems, it has been shown that neutrophils are the rst immune cells to arrive at the site of infection or wounding. They facilitate their migration by exocytosing granules that contain metalloproteinases and other enzymes that degrade the extracellular matrix [154]. Upon recognition of pathogens, neutrophils release their antimicrobial granules, called azurophils, into phagosomes or the extracellular environment [155, 156]. Azurophils are packed with acidic hydrolases and antimicrobial proteins, such as lysozyme, cathepsins, and myeloperoxidase (MPO) [157]. The primary function of MPO is to react with hydrogen peroxide(H2 O2 ), which subsequently oxidates chloride, tyrosine, and nitrite to form hypochloric acid (HOCl), tyrosine radicals, and reactive nitrogen intermediates [158]. These highly reactive chemicals attack the surface membranes of microbes. Additionally, microbes can be bound by neutrophil extracellular traps (NETs), which are brous networks of granule proteins and chromatin released by neutrophils [159]. While MPO is mostly produced in neutrophils, all professional phagocytes produce high levels of reactive oxygen species (ROS), including superoxide, H2 O2 , and hydroxyl radicals, produced by the enzymes NADPH oxidase (NOX) and dual oxidase (DUOX) [160]. The NOX of phagocytes (Phox) is only activated upon exposure to microorganisms or other pro-inammatory stimuli [161]. When active, Phox is located in the phagosomal membrane and catalyzes the respiratory burst, which consists of the large-scale production of ROS that helps degrade phagocytosed microbes by nonspecically oxidizing protein, DNA, lipid, and carbohydrate [162]. H2 O2 produced during the respiratory burst can also function as a substrate for MPO activity. The oxidative enzyme DUOX may even combine the two functions, by generating H2 O2 as a substrate for its own peroxidase domain [160].
7 Nitric oxide (NO) is produced from the amino acid L-arginine by nitric oxide synthase (NOS) enzymes and functions as a signaling molecule in numerous biological processes as well as having antimicrobial activity [163]. There are two constitutively expressed NOS enzymes, neuronal NOS (nNOS or NOS1) and endothelial NOS (eNOS or NOS3), and one inducible NOS (iNOS or NOS2) that is important in innate immunity. Regulation of NOS2 plays an important role in the inammatory response, and many cells of the immune system are capable of producing NO [164, 165]. NO has cytostatic and cytotoxic antimicrobial eects when high amounts are excreted by immune cells into mammalian tissues, most likely via reactive nitrogen species (RNS) which are generated when NO interacts with O2 [166]. These RNS subsequently lead to lipid peroxidation, DNA damage, oxidation of thiols, and nitration of tyrosine residues [167]. It has recently been shown that Nos2a, the zebrash homolog of NOS2, is also required for the expansion of hematopoietic stem cells and progenitor cells during infection, leading to increased numbers of the required immune cells [168]. This discovery further adds to the importance of NOS2 in the inammatory response. The oxidative defense mechanisms need to be tightly controlled, since high levels of reactive chemicals like ROS and RNS cause tissue damage at sites of infection. Therefore, the resolution phase of inammation is critical in order to restore the tissue to its normal state and prevent chronic inammation. The molecules produced during oxidative defenses are often self-limiting and help initiate resolution of inammation by inducing neutrophil apoptosis [160, 169]. Furthermore, iNOS-induced NO production can be countered by activation of arginase (ARG), which depletes the substrate for iNOS by converting L-arginine to the harmless compounds urea and L-ornithine, thus creating conditions more favorable for wound healing [163, 170]. The zebrash homolog of MPO, ocially named MPX, is specically expressed in neutrophils during embryonic development. Transgenic reporter lines driven by the mpx promoter have made the zebrash a highly suitable model organism to study neutrophilic inammation [8, 171]. In fact, using one of these lines, it was demonstrated for the rst time that H2 O2 produced in the context of wounding not only functions as an antiseptic compound, but also forms a gradient that is required for rapid attraction of leukocytes [172]. However, this H2 O2 gradient is only generated at wounds and does not occur at infected tissues [173]. The formation of this H2 O2 gradient was shown to be dependent on the oxidase activity of Duox. The Src family kinase Lyn has been identied as the redox sensor that mediates neutrophil migration towards the wound [174]. The innate immune function of Duox and the importance of ROS in zebrash were further established by studies showing that knockdown of Duox impaired the ability of zebrash larvae to control enteric Salmonella infections [175]. It has also been shown that zebrash Phox is important in controlling the in vivo growth of the pathogenic fungus Candida albicans [176]. A 5,5-dimethyl-l-pyrroline N-oxide- (DMPO-) based immunospin trap technique has been adopted for in situ detection of ROS production in zebrash embryos [177].
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M. marinum Mma20 Lc3
Advances in Hematology
M. marinum Mma20 Lc3
(a)
(b)
(c)
Figure 2: In situ detection of autophagy by Lc3 accumulation. CMV::LC3-GFP transgenic [15] zebrash embryos (28 hpf) were injected into the caudal vein with 200 colony-forming units (CFU) of M. marinum Mma20 expressing a pMST3::mCherry vector. Confocal images were taken of a tail region of the developing larva at 3 days after infection (3 dpi), a point at which the M. marinum infection (a) has been established. Low levels of Lc3-GFP signal (b) can be observed throughout the cells, whilst brighter regions (indicated by arrowheads) are only observed upon Lc3 accumulation and formation of autophagic membranes associated with bacteria (c). Scale bar: 10 m.
M. marinum Mma20
Anti-nitrotyrosine
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Figure 3: In situ detection of reactive nitrogen species. Wild-type zebrash embryos (Albino; 28 hpf) were injected into the caudal vein with 200 colony-forming units (CFU) of M. marinum Mma20 expressing a pMST3::mCherry vector. Confocal images were taken of a tail region of the developing larva at 3 days after infection (3 dpi), a point at which the M. marinum infection (a) has been established. Embryos were xed in 4% paraformaldehyde at 3 dpi, and immunohistochemistry was performed, using an antinitrotyrosine antibody that detects tissue nitration (b) [21]. Colocalization (c) between bacteria and extensive tissue nitration can be observed at this time point. Scale bar: 10 m.
DMPO is a chemical substrate that binds to reactive oxygen, which can later be detected with an anti-DMPO antibody. This protocol detects the build-up of the conjugated product, thereby showing a cumulative ROS production. Furthermore, a respiratory burst assay has been developed for zebrash embryos, which was used to demonstrate that macrophages and neutrophils are the ROS-producing cells in zebrash [178]. A similar method is available to image the production of NO in zebrash embryos, using a diaminouorescein probe that only becomes uorescent in the presence of NO [179]. As mentioned before, nitration of tyrosine residues is a hallmark of NO production. Forlenza et al. (2008) used an antinitrotyrosine antibody on common carp tissue to visualize the tissue nitration that occurs at sites of Trypanoplasma borreli infection [21]. We used the same antibody for immunohistochemistry on zebrash embryos to visualize the production of RNS in response to M. marinum infection (Figure 3). This technique also visualizes the nitrosative stress that the host tissue suers upon release of RNS. The resolution of inammation that should prevent tissue damage following such stresses
has also been studied in zebrash. This has led to new insights on the mechanisms underlying resolution, including apoptosis and retrograde chemotaxis of neutrophils, with the oxygen-sensing transcription factor hypoxia-inducible factor-1 (Hif-1) playing a role in the control of these mechanisms [171, 180].
Advances in Hematology
Table 1: Transcriptome proling studies on infection models in adult and embryonic zebrash. Bacterial species Mycobacterium marinum Mycobacterium marinum Mycobacterium marinum Salmonella enterica serovar Typhimurium (Salmonella typhimurium) Streptococcus suis Salmonella enterica serovar Typhimurium (Salmonella typhimurium) Edwardsiella tarda Citrobacter freundii Strain M; E11 M ma20; E11 M; E11 SL1027; LPS derivative SF1592 (Ra), HA9801 SL1027; LPS derivative SF1592 (Ra), FL6-60 Not specied Infection model Adult (IP) 28hpf (CV); Adult (IP) Adult (IP) 28hpf (CV) Adult (IP) 28hpf (CV) 28hpf (CV) Adult (IM) Reference Meijer et al. [182] Van der Sar et al. [22] Hegedus et al. [107]
Stockhammer et al. [13] Wu et al. [183] Ordas et al. [38] Van Soest et al. [123] Lu et al. [153]
and : these studies used the same samples but applied microarray analysis and deep sequencing, respectively. (IP): intraperitoneal; (CV): caudal vein; (IM): immersion.
for microarray or RNA sequencing [181]. Expression proling can be done either at whole organism level or on FACS-sorted immune cells from transgenic lines. The latter approach was used to determine the transcriptional signature of early myeloid cells [87]. Microarray analysis of zebrash adults and embryos infected with various pathogens has provided insights into the transcriptome during infection and has provided leads for further functional studies (Table 1). The transcriptional response of both zebrash embryos and adults showed clear conservation with host responses detected in other vertebrate models and human cells. Genes that were induced upon infection included receptors involved in pathogen recognition, signaling intermediates, their downstream transcription factors (like NFB and AP-1), and inammatory mediators. Furthermore, these studies led to the identication of novel immune responsive genes and infection markers, for example, the DNA-damageregulated autophagy modulator 1 gene (dram1), which was identied in a knockdown study of Traf6, a central intermediate in TLR and TNF receptor signaling [37].
4.2. Comparison of Gene Expression Proles Induced by Different Bacterial Pathogens. To illustrate the similarities and dierences in the innate immune response against dierent bacterial pathogens, Figure 4 shows a comparison of the gene expression proles of zebrash infected with Edwardsiella tarda, S. typhimurium, and M. marinum. E. tarda is a Gram-negative, naturally occurring sh pathogen that belongs to the Enterobacteriaceae family. Inside its host, E. tarda is able to resist complement activity and can survive inside macrophages [184]. It causes a progressive disease when injected into the caudal vein of 28 hours after fertilization (hpf) embryos, leading to mortality within 2 days after infection (dpi) [123]. S. typhimurium (short for S. enterica serovar Typhimurium), also belonging to the Gramnegative Enterobacteriaceae family, causes salmonellosis in a broad range of hosts. S. typhimurium is a facultative intracellular species that can survive within phagocytic and nonphagocytic cells. Following internalization, it survives
and replicates in a modied phagosome, known as the Salmonella-containing vacuole. Like E. tarda, injection of S. typhimurium into the caudal vein at 28 hpf leads to a progressive disease which leads to mortality of the embryo during the rst 30 hours after infection (hpi) [13, 185]. In contrast, M. marinum injection at the same stage leads to a chronic infection that persists during larval development. M. marinum is a natural pathogen of teleost sh and a close relative of M. tuberculosis, the causative agent of tuberculosis in humans. Mycobacteria have a thick, waxy, acid-fast staining cell wall containing characteristic lipids that are important for virulence. Both M. marinum and M. tuberculosis have the ability to replicate inside macrophages, eventually causing them to undergo apoptosis. Dependent on secreted virulence factors that are conserved between M. marinum and M. tuberculosis, other macrophages are attracted to the initial infection site. These become infected by phagocytosing the apoptotic remains, which ultimately leads to the formation of a granuloma [186]. Using the zebrash embryo model, Ramakrishan et al. have provided new insights demonstrating the importance of the innate immune system to control M. marinum infection during early stages of pathogenesis [1, 2, 124, 187, 188]. Complementary to previously reported transcriptome data (Table 1), here we present new data comparing the gene expression proles induced by E. tarda, S. typhimurium, and M. marinum under similar conditions (Figure 4). We injected 200 colony-forming units (CFUs) of each pathogen into the caudal vein of 28 hpf zebrash embryos and analyzed the response at 8 hpi. Since M. marinum develops a chronic infection, we also sampled at 4 dpi, a time point at which granulomas are present. Finally, we compared the transcriptome prole of the embryonic samples with data from a previous study, in which adult zebrash were infected with the same strain of M. marinum [22]. The two progressive Gram-negative pathogens, E. tarda and S. typhimurium, induced a strong early immune response at 8 hpi, while the chronic M. marinum infection hardly induced any response at this time point. At 4 dpi, the transcriptome prole of M. marinum-infected embryos did
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Embryo Et 8 hpi tlr18 tlr2 tlr3 tlr4b tlr5a tlr5b tlr18 tlr21 tlr22 nod2 marco cd36 scrab1 scrab2 mbl lgals1l1 lgals3bp lgals9l1 myd88 tirap traf3 traf2b traf4b traf6 irak4 irak3 nfkbiaa nfkbiab ikbkg ripk2 jak1 isg15 ptpn6 pik3ca atf3 fos irf1 irf3 irf7 rel relb jun junb stat3 stat4 stat5.2 spi1 cmyb myca mycb mych mycn rag1 mhc1uea ighm St 8 hpi Mm 8 hpi Mm 4 dpi
Adult Mm 1 dpi Mm 6 dpi il1b il8 il10 il12a il12b il16 il17a/f2 il34 tgfb2 tnfa tnfb ifn ifng1-2 ccl1 ccl20 ccl24 ccl25b cxc46 ccl-chr2e ccl-chr5a ccl-chr10b ccl-chr11b ccl-chr20c ccl-chr20d cxcl-chr1c lyz mpx ncf1 ncf4 nos2 arg2 mmp9 mmp13 casp6 caspb ctsl1a ctsl1b c3b c3c c4-2l c6 c7 c8b c9 chia.1 chia.2 chia.4 chia.3 chia.5 chia.6 mpeg1 irg1 ic3 gabarap Et 8 hpi
Signaling intermediates
Transcription factors
Adaptive
Figure 4: Comparison of the zebrash innate immune response to dierent bacterial pathogens. Gene expression proles of zebrash embryos and adults infected with E. tarda FL6-60 (Et), S. typhimuriumSL1027(St), and M. marinum Mma20 (Mm) are depicted in a heat map. Embryos were infected with 200 CFU of each pathogen into the caudal vein at 28 hpf and snap frozen individually at 8 hpi for E. tarda and S. typhimurium, and at 8 hpi and 4 dpi for M. marinum. Triplicate samples for each infection condition were compared with samples from control embryos (injected with PBS) using a common reference microarray design. The raw data were deposited in the Gene Expression Omnibus database under accession number GSE35474. The data derived from embryonic infections were compared with data from a study in which adult zebrash were infected intraperitoneally with M. marinum Mma20, after which RNA samples were taken at 1 dpi and 6 dpi [22]. The dose of the Mma20 strain used in the adult infection study was lethal within days after the nal sampling point at 6 dpi. Only genes relevant to this paper were included in the heatmap. All selected genes are represented by a minimum of two probes that showed signicant up or downregulation (signicance cut-os for the ratios of infected versus control groups were set at 2-fold with P < 105 ). Upregulation is indicated by increasingly bright shades of yellow, and downregulation is indicated by increasingly bright shades of blue. It should be noted that the genes listed in this gure are named according to sequence homology with mammalian counterparts and in most cases have not yet been conrmed functionally.
Other
Chitinases
Complement
Proteases
Defenses
Cytokines
Advances in Hematology show an immune response, although it was still weaker than the response to E. tarda or S. typhimurium infection at 8 hpi. In adults, the immune response to M. marinum infection has been shown to develop in a similar manner, with hardly any induction of proinammatory genes at 1 dpi and a stronger response at 6 dpi, when the sh began to show symptoms of disease [22]. Infections with E. tarda and S. typhimurium resulted in a remarkably similar transcriptome. Nevertheless, subtle dierences were observed, like the upregulation of Tlr3 that was specic to E. tarda infection in this data set, and the variation in the panel of cytokines expressed upon these infections. Interestingly, various PRRs, for example, Tlr5a and 5b, showed increased expression upon infection, most likely indicating an elevated state of awareness needed to identify the invading pathogens. In contrast, the sh-specic Tlr18, the scavenger receptors CD36, scarb1, and scarb2, and the C-type lectin Mbl were downregulated in some conditions. In many cases, signaling intermediates downstream of PRRs were upregulated, relaying and possibly amplifying the activating signals they receive from their respective receptors. A wide range of transcription factors with well-established functions in immunity (e.g., Atf3, Jun and Fos, Rel, and the IRF and Stat family members) were signicantly upregulated under all conditions tested, except for the 8 hpi time point of M. marinum infection, whereas we observed upregulation of transcription factors of the oncogenic Myc family mainly in adult sh. The hematopoietic transcription factor Spi1 (Pu.1) was upregulated in M. marinum infection of embryos and adults. Genes for the key pro-inammatory cytokines, like TNF (two genes in zebrash: tnfa and tnfb), IL1, and IL8, and for the anti-inammatory cytokine IL10 were induced by infection with any of the three pathogens. Other cytokines appeared to be more specic for certain pathogens or might not be expressed at the specic time point of infection that we sampled. We also observed increased expression of genes involved in eector mechanisms. However, upregulation of the genes encoding lysozyme, myeloperoxidase, and iNos was detectable only in adult zebrash infected with M. marinum. Infection with any of the three pathogens led to increased gene expression of ncf1, a subunit of the neutrophil NADPH oxidase complex. Proteases are an important part of the innate immune response, functioning in reorganizing the extracellular matrix to allow leukocyte migration, in degradation of microbes, and in processing of cytokines. In adult zebrash infected with M. marinum, we observed upregulation of cathepsin-like 1a and 1b (ctsl1a and ctsl1b), members of lysosomal cathepsin family that aids in the destruction of microbes. Expression levels of casp6 and caspb, members of the cysteine-aspartic acid protease (caspase) family involved in apoptosis, were downregulated at dierent stages of infection in adults and embryos. The matrix metalloproteinase (mmp) genes 9 and mmp13 proved to be excellent markers for infection, since their gene expression was induced by E. tarda, S. typhimurium and M. marinum. Our data further suggest that complement activation plays an important role during the early innate immune response, since a large number of complement factor genes
11 show increased expression upon infection. Upregulated expression of the autophagy marker genes lc3 and gabarap in adults infected with M. marinum hints towards a role for autophagy in the control of this infection. Intriguingly, a macrophage-expressed gene with unknown function in immunity, mpeg1 [87], is downregulated during the embryonic immune response against all three pathogens. The mouse homolog of this gene encodes a perforin-like protein that is expressed in mature macrophages and prion-infected brain cells [189]. We have also observed specic upregulation of genes with as of yet unknown function in immunity, like immunoresponsive gene 1 (irg1). This gene is highly conserved in vertebrates and has high homology to bacterial methylcitrate dehydrogenase [190]. We also included some genes involved in adaptive immunity in our comparison, the lymphocyte marker rag1, the immunoglobulin heavy chain gene ighm, and the antigen-presenting major histocompatibility complex class I UEA gene (mhc1uea). Even though no cells of the adaptive immune system are present yet, embryos infected with E. tarda or S. typhimurium increase the expression of the MHC I gene. Finally, upon infection with S. typhimurium and M. marimum, we observe up and downregulation of chitinases, a family of genes which has been attributed a role during the host-microbial interactions involved in the development of acute and chronic inammatory conditions [191].
5. Discussion
Zebrash infectious disease models have started to make an important contribution to the understanding of hostpathogen interaction mechanisms. A good example is the discovery of the mechanism whereby a mycobacterial virulence factor (ESAT6) induces mmp9 expression in host epithelial cells neighboring infected macrophages, which enhances macrophage recruitment and formation of granuloma-like aggregates that provide a replication niche for mycobacteria [2]. The combination of genetics and in vivo imaging in zebrash embryos is unparalleled in other vertebrate models. Furthermore, zebrash embryos provide an ideal model for high-throughput in vivo screening of antimicrobial drug candidates or novel vaccine candidates [192, 193]. Knowledge of the zebrash immune system is also important in high-throughput screening for cancer in zebrash embryos [194]. However, many aspects of zebrash immunity still require further characterization and validation. Currently available transgenic lines clearly distinguish macrophages (marked by csf1r/fms and mpeg1) from neutrophils (marked by mpx and lyz) in embryos and larvae, but there is insucient knowledge of surface markers to identify dierent macrophage and neutrophil subpopulations. Similar to mammals, there is evidence of the existence of subpopulations of classically activated macrophages (M1: high producers of proinammatory mediators, ROS, and NO) and alternatively activated macrophages (M2: high producers of anti-inammatory mediators) in sh [195]. The polarization of macrophages towards these subtypes plays
12 a critical role in the pathology of both infectious diseases and cancer [196]. Furthermore, dierent subpopulations of mammalian neutrophils (N1 and N2) have been recently described that display pro- and antitumorigenic properties [197] and that probably will also turn out to have distinctive functions during infectious disease pathology. Tumor implants in zebrash embryos were shown to attract a heterogeneous population of leukocytes, including cells that express arginase, a marker of alternatively activated macrophages [177]. In addition, the neutrophil markers mpx, mych, and lyz do not show complete overlap [177, 198], and markers such as cxcr3.2 and ptpn6, which are macrophage specic in one-day-old embryos, also label a subset of neutrophils at later stages [87]. Future development of transgenic lines that can distinguish these multiple myeloid subsets would further strengthen the use of zebrash models for innate immunity and infectious disease studies. As detailed in this paper, counterparts of the major vertebrate PRRs and downstream signaling components have been identied in zebrash, but relatively few have thus far been functionally studied in infectious disease models. Recently, new PRRs have been described in mammals, like the INF-inducible dsRNA-activated protein kinase R (PKR) [199], the cytosolic DNA sensor DNA-dependent activator of IFN-regulatory factors (DAI) [200], and a cytosolic DNA receptor named AIM2 (absent in melanoma 2) [201]. Thus far, only the zebrash homolog for PKR has been identied. Furthermore, autophagic adaptors known as sequestosome 1/p62-like receptors (SLRs), conserved between zebrash and human, have recently been suggested as a new category of PRRs, since they have the ability to recognize and capture targets for immune-related autophagy [202]. Various datasets derived from transcriptome analyses have shown the specicity of immune responses to dierent pathogens. In future studies, the analysis of these responses can be rened by FACS sorting of immune cell populations from infected embryos, using labeled pathogens in combination with transgenic lines for dierent immune cell types. For example, it now comes within reach to aim at dissecting the dierences in gene expression between M. marinum-infected macrophages inside a granuloma and recently attracted uninfected macrophages. In addition, simultaneous proling of pathogen and host genes will be a challenging approach to help unravel the complex mechanisms underlying hostpathogen interactions. Transcriptome analysis only reveals altered RNA levels upon infection, and therefore, the application of proteomic and epigenetic analyses are needed to study the regulation of immune responses on dierent levels. Transcriptome studies have revealed infection responsiveness of many genes that have not yet been well studied (for example, dram1, mpeg1, irg1, and irg1l, mentioned above) and an emerging immune function for several chitinaselike proteins during infection [13, 37, 123]. Many zebrash infection models have been described here and in other recent papers [4, 203, 204] that can be used to investigate the functions of these genes in dierent pathogenic interactions, either using morpholino knockdown in embryos or using stable knockout lines which nowadays can be identied very eciently by high-throughput resequencing
Advances in Hematology of mutant libraries or by targeted knock-down approaches using technologies such as zinc nger nucleases (ZFNs) or transcription activator-like eector nucleases (TALENs) [205].
Acknowledgments
The authors thank Dan Klionsky (University of Michigan) for the GFP-Lc3 zebrash line, Maria Forlenza (Wageningen University) for the antinitrotyrosine antibody, and Phil Elks for critically reading the paper. Infectious disease research in our laboratory is supported by the Smart Mix Program of the Netherlands Ministry of Economic Aairs and the Ministry of Education, Culture and Science, the European Commission 7th framework project ZF-HEALTH (HEALTH-F4-2010-242048), and the European Marie-Curie Initial Training Network FishForPharma (PITN-GA-2011289209).
References
[1] D. M. Tobin, J. C. Vary, J. P. Ray et al., The lta4h locus modulates susceptibility to mycobacterial infection in zebrash and humans, Cell, vol. 140, no. 5, pp. 717730, 2010. [2] H. E. Volkman, T. C. Pozos, J. Zheng, J. M. Davis, J. F. Rawls, and L. Ramakrishnan, Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium, Science, vol. 327, no. 5964, pp. 466469, 2010. [3] M. Ludwig, N. Palha, C. Torhy et al., Whole-body analysis of a viral infection: vascular endothelium is a primary target of Infectious hematopoietic necrosis virus in zebrash larvae, PLoS Pathogens, vol. 7, no. 2, Article ID e1001269, 2011. [4] A. H. Meijer and H. P. Spaink, Host-Pathogen interactions made transparent with the zebrash model, Current Drug Targets, vol. 12, no. 7, pp. 10001017, 2011. [5] C. Sullivan and C. H. Kim, Zebrash as a model for infectious disease and immune function, Fish and Shellsh Immunology, vol. 25, no. 4, pp. 341350, 2008. [6] J. P. Allen and M. N. Neely, Trolling for the ideal model host: zebrash take the bait, Future Microbiology, vol. 5, no. 4, pp. 563569, 2010. [7] J. R. Mathias, K. B. Walters, and A. Huttenlocher, Neutrophil motility in vivo using zebrash., Methods in Molecular Biology, vol. 571, pp. 151166, 2009. [8] S. A. Renshaw, C. A. Loynes, D. M. I. Trushell, S. Elworthy, P. W. Ingham, and M. K. B. Whyte, Atransgenic zebrash model of neutrophilic inammation, Blood, vol. 108, no. 13, pp. 39763978, 2006. [9] C. Hall, M. Flores, T. Storm, K. Crosier, and P. Crosier, The zebrash lysozyme C promoter drives myeloid-specic expression in transgenic sh, BMC Developmental Biology, vol. 7, article no. 42, 2007. [10] C. Gray, C. A. Loynes, M. K. B. Whyte, D. C. Crossman, S. A. Renshaw, and T. J. A. Chico, Simultaneous intravital imaging of macrophage and neutrophil behaviour during inammation using a novel transgenic zebrash, Thrombosis and Haemostasis, vol. 105, no. 5, pp. 811819, 2011. [11] F. Ellett, L. Pase, J. W. Hayman, A. Andrianopoulos, and G. J. Lieschke, mpeg1 promoter transgenes direct macrophagelineage expression in zebrash, Blood, vol. 117, no. 4, pp. e49e56, 2011.
Advances in Hematology
[12] P. Herbomel, B. Thisse, and C. Thisse, Ontogeny and behaviour of early macrophages in the zebrash embryo, Development, vol. 126, no. 17, pp. 37353745, 1999. H. P. [13] O. W. Stockhammer, A. Zakrzewska, Z. Hegedus, Spaink, and A. H. Meijer, Transcriptome proling and functional analyses of the zebrash embryonic innate immune response to Salmonella infection, Journal of Immunology, vol. 182, no. 9, pp. 56415653, 2009. [14] S. H. Lam, H. L. Chua, Z. Gong, T. J. Lam, and Y. M. Sin, Development and maturation of the immune system in zebrash, Danio rerio: a gene expression proling, in situ hybridization and immunological study, Developmental and Comparative Immunology, vol. 28, no. 1, pp. 928, 2004. [15] R. Medzhitov and C. Janeway Jr., Innate immune recognition: mechanisms and pathways, Immunological Reviews, vol. 173, pp. 8997, 2000. [16] A. A. Beg, Endogenous ligands of Toll-like receptors: implications for regulating inammatory and immune responses, Trends in Immunology, vol. 23, no. 11, pp. 509512, 2002. [17] P. Matzinger, An innate sense of danger, Annals of the New York Academy of Sciences, vol. 961, pp. 341342, 2002. [18] T. H. Mogensen, Pathogen recognition and inammatory signaling in innate immune defenses, Clinical Microbiology Reviews, vol. 22, no. 2, pp. 240273, 2009. [19] S. Akira and K. Takeda, Toll-like receptor signalling, Nature Reviews Immunology, vol. 4, no. 7, pp. 499511, 2004. [20] S. Akira, S. Uematsu, and O. Takeuchi, Pathogen recognition and innate immunity, Cell, vol. 124, no. 4, pp. 783801, 2006. [21] M. Forlenza, J. P. Scharsack, N. M. Kachamakova, A. J. Taverne-Thiele, J. H. W. M. Rombout, and G. F. Wiegertjes, Dierential contribution of neutrophilic granulocytes and macrophages to nitrosative stress in a host-parasite animal model, Molecular Immunology, vol. 45, no. 11, pp. 3178 3189, 2008. [22] A. M. van der Sar, H. P. Spaink, A. Zakrzewska, W. Bitter, and A. H. Meijer, Specicity of the zebrash host transcriptome response to acute and chronic mycobacterial infection and the role of innate and adaptive immune components, Molecular Immunology, vol. 46, no. 11-12, pp. 23172332, 2009. [23] B. Lemaitre, E. Nicolas, L. Michaut, J. M. Reichhart, and J. A. Homann, The dorsoventral regulatory gene cassette spatzle/Toll/Cactus controls the potent antifungal response in Drosophila adults, Cell, vol. 86, no. 6, pp. 973983, 1996. [24] L. A. J. ONeill and A. G. Bowie, The family of ve: TIRdomain-containing adaptors in Toll-like receptor signalling, Nature Reviews Immunology, vol. 7, no. 5, pp. 353364, 2007. [25] M. Miettinen, T. Sareneva, I. Julkunen, and S. Matikainen, IFNs activate toll-like receptor gene expression in viral infections, Genes and Immunity, vol. 2, no. 6, pp. 349355, 2001. [26] K. Takeda and S. Akira, Microbial recognition by Toll-like receptors, Journal of Dermatological Science, vol. 34, no. 2, pp. 7382, 2004. [27] C. Jault, L. Pichon, and J. Chluba, Toll-like receptor gene family and TIR-domain adapters in Danio rerio, Molecular Immunology, vol. 40, no. 11, pp. 759771, 2004. [28] A. H. Meijer, S. F. Gabby Krens, I. A. Medina Rodriguez et al., Expression analysis of the Toll-like receptor and TIR domain adaptor families of zebrash, Molecular Immunology, vol. 40, no. 11, pp. 773783, 2004.
13
[29] Y. Palti, Toll-like receptors in bony sh: from genomics to function, Developmental and Comparative Immunology, vol. 35, no. 12, pp. 12631272, 2011. [30] C. M. S. Ribeiro, T. Hermsen, A. J. Taverne-Thiele, H. F. J. Savelkoul, and G. F. Wiegertjes, Evolution of recognition of ligands from gram-positive bacteria: similarities and dierences in the TLR2-mediated response between mammalian vertebrates and teleost sh, Journal of Immunology, vol. 184, no. 5, pp. 23552368, 2010. [31] A. Matsuo, H. Oshiumi, T. Tsujita et al., Teleost TLR22 recognizes RNA duplex to induce IFN and protect cells from birnaviruses, Journal of Immunology, vol. 181, no. 5, pp. 34743485, 2008. [32] M. P. Sepulcre, F. Alcaraz-P erez, A. Lopez-Mu noz et al., Evolution of lipopolysaccharide (LPS) recognition and signaling: sh TLR4 does not recognize LPS and negatively regulates NF-B activation, Journal of Immunology, vol. 182, no. 4, pp. 18361845, 2009. [33] C. Sullivan, J. Charette, J. Catchen et al., The gene history of zebrash tlr4a and tlr4b is predictive of their divergent functions, Journal of Immunology, vol. 183, no. 9, pp. 5896 5908, 2009. [34] C. Stein, M. Caccamo, G. Laird, and M. Leptin, Conservation and divergence of gene families encoding components of innate immune response systems in zebrash, Genome Biology, vol. 8, no. 11, article no. R251, 2007. [35] J. M. Bates, J. Akerlund, E. Mittge, and K. Guillemin, Intestinal alkaline phosphatase detoxies lipopolysaccharide and prevents inammation in zebrash in response to the gut microbiota, Cell Host and Microbe, vol. 2, no. 6, pp. 371382, 2007. [36] A. M. Van Der Sar, O. W. Stockhammer, C. Van Der Laan, H. P. Spaink, W. Bitter, and A. H. Meijer, MyD88 innate immune function in a zebrash embryo infection model, Infection and Immunity, vol. 74, no. 4, pp. 24362441, 2006. [37] O. W. Stockhammer, H. Rauwerda, F. R. Wittink, T. M. Breit, A. H. Meijer, and H. P. Spaink, Transcriptome analysis of Traf6 function in the innate immune response of zebrash embryos, Molecular Immunology, vol. 48, no. 1-3, pp. 179 190, 2010. [38] A. Ordas, Z. Hegedus, C. V. Henkel et al., Deep sequencing of the innate immune transcriptomic response of zebrash embryos to Salmonella infection, Fish and Shellsh Immunology, vol. 31, no. 5, pp. 716724, 2011. [39] K. Kersse, M. J. M. Bertrand, M. Lamkan, and P. Vandenabeele, NOD-like receptors and the innate immune system: coping with danger, damage and death, Cytokine and Growth Factor Reviews, vol. 22, no. 5-6, pp. 257276, 2011. nez, Intracellular [40] T. D. Kanneganti, M. Lamkan, and G. Nu NOD-like receptors in host defense and disease, Immunity, vol. 27, no. 4, pp. 549559, 2007. [41] S. E. Girardin, I. G. Boneca, L. A. M. Carneiro et al., Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan, Science, vol. 300, no. 5625, pp. 15841587, 2003. [42] S. E. Girardin, J. P. Hugot, and P. J. Sansonetti, Lessons from Nod2 studies: towards a link between Crohns disease and bacterial sensing, Trends in Immunology, vol. 24, no. 12, pp. 652658, 2003. [43] G. K. Silva, F. R. S. Gutierrez, P. M. M. Guedes et al., Cutting edge: nucleotide-binding oligomerization domain 1dependent responses account for murine resistance against Trypanosoma cruzi infection, Journal of Immunology, vol. 184, no. 3, pp. 11481152, 2010.
14
[44] M. H. Shaw, T. Reimer, C. S anchez-Valdepe nas et al., T cell-intrinsic role of Nod2 in promoting type 1 immunity to Toxoplasma gondii, Nature Immunology, vol. 10, no. 12, pp. 12671274, 2009. [45] N. Inohara, T. Koseki, J. Lin et al., An induced proximity model for NF-B activation in the Nod1/RICK and RIP signaling pathways, Journal of Biological Chemistry, vol. 275, no. 36, pp. 2782327831, 2000. [46] K. Kobayashi, N. Inohara, L. D. Hernandez et al., RICK/ Rip2/CARDIAK mediates signalling for receptors of the innate and adaptive immune systems, Nature, vol. 416, no. 6877, pp. 194199, 2002. [47] J. H. Park, Y. G. Kim, M. Shaw et al., Nod1/RICK and TLR signaling regulate chemokine and antimicrobial innate immune responses in mesothelial cells, Journal of Immunology, vol. 179, no. 1, pp. 514521, 2007. [48] E. Voss, J. Wehkamp, K. Wehkamp, E. F. Stange, J. M. Schr oder, and J. Harder, NOD2/CARD15 mediates induction of the antimicrobial peptide human beta-defensin-2, Journal of Biological Chemistry, vol. 281, no. 4, pp. 2005 2011, 2006. [49] E. Noguchi, Y. Homma, X. Kang, M. G. Netea, and X. Ma, A Crohns disease-associated NOD2 mutation suppresses transcription of human IL10 by inhibiting activity of the nuclear ribonucleoprotein hnRNP-A1, Nature Immunology, vol. 10, no. 5, pp. 471479, 2009. [50] F. Martinon, K. Burns, and J. Tschopp, The Inammasome: a molecular platform triggering activation of inammatory caspases and processing of proIL-, Molecular Cell, vol. 10, no. 2, pp. 417426, 2002. [51] K. Tsuchiya, H. Hara, I. Kawamura et al., Involvement of absent in melanoma 2 in inammasome activation in macrophages infected with Listeria monocytogenes, Journal of Immunology, vol. 185, no. 2, pp. 11861195, 2010. [52] D. L. Kastner, I. Aksentijevich, and R. Goldbach-Mansky, Autoinammatory disease reloaded: a clinical perspective, Cell, vol. 140, no. 6, pp. 784790, 2010. [53] S. C. Eisenbarth, O. R. Colegio, W. OConnor, F. S. Sutterwala, and R. A. Flavell, Crucial role for the Nalp3 inammasome in the immunostimulatory properties of aluminium adjuvants, Nature, vol. 453, no. 7198, pp. 1122 1126, 2008. [54] M. Chamaillard, S. E. Girardin, J. Viala, and D. J. Philpott, Nods, nalps and naip: intracellular regulators of bacterialinduced inammation, Cellular Microbiology, vol. 5, no. 9, pp. 581592, 2003. [55] T. Henry, A. Brotcke, D. S. Weiss, L. J. Thompson, and D. M. Monack, Type I interferon signaling is required for activation of the inammasome during Francisella infection, Journal of Experimental Medicine, vol. 204, no. 5, pp. 987 994, 2007. [56] K. J. Laing, M. K. Purcell, J. R. Winton, and J. D. Hansen, A genomic view of the NOD-like receptor family in teleost sh: identication of a novel NLR subfamily in zebrash, BMC Evolutionary Biology, vol. 8, no. 1, article no. 42, 2008. [57] S. H. Oehlers, M. V. Flores, C. J. Hall, S. Swift, K. E. Crosier, and P. S. Crosier, The inammatory bowel disease (IBD) susceptibility genes NOD1 and NOD2 have conserved antibacterial roles in zebrash, Disease Models and Mechanisms, vol. 4, no. 6, pp. 832841, 2011. [58] Y. M. Loo and M. Gale, Immune signaling by RIG-I-like receptors, Immunity, vol. 34, no. 5, pp. 680692, 2011. [59] D. C. Kang, R. V. Gopalkrishnan, L. Lin et al., Expression analysis and genomic characterization of human melanoma
Advances in Hematology
dierentiation associated gene-5, mda-5: a novel type I interferon-responsive apoptosis-inducing gene, Oncogene, vol. 23, no. 9, pp. 17891800, 2004. M. Yoneyama, M. Kikuchi, T. Natsukawa et al., The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses, Nature Immunology, vol. 5, no. 7, pp. 730737, 2004. T. Saito, R. Hirai, Y. M. Loo et al., Regulation of innate antiviral defenses through a shared repressor domain in RIG1 and LGP2, Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 2, pp. 582587, 2007. M. Yoneyama, M. Kikuchi, K. Matsumoto et al., Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity, Journal of Immunology, vol. 175, no. 5, pp. 28512858, 2005. I. Scott, The role of mitochondria in the mammalian antiviral defense system, Mitochondrion, vol. 10, no. 4, pp. 316320, 2010. S. Paz, Q. Sun, P. Nakhaei et al., Induction of IRF-3 and IRF-7 phosphorylation following activation of the RIG-I pathway, Cellular and Molecular Biology, vol. 52, no. 1, pp. 1728, 2006. Y. M. Loo, J. Fornek, N. Crochet et al., Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity, Journal of Virology, vol. 82, no. 1, pp. 335345, 2008. J. Zou, M. Chang, P. Nie, and C. J. Secombes, Origin and evolution of the RIG-I like RNA helicase gene family, BMC Evolutionary Biology, vol. 9, no. 1, article no. 85, 2009. D. Aggad, M. Mazel, P. Boudinot et al., The two groups of zebrash virus-induced interferons signal via distinct receptors with specic and shared chains, Journal of Immunology, vol. 183, no. 6, pp. 39243931, 2009. S. Biacchesi, M. LeBerre, A. Lamoureux et al., Mitochondrial antiviral signaling protein plays a major role in induction of the sh innate immune response against RNA and DNA viruses, Journal of Virology, vol. 83, no. 16, pp. 7815 7827, 2009. F. Sun, Y.-B. Zhang, T.-K. Liu, J. Shi, B. Wang, and J.F. Gui, Fish MITA serves as a mediator for distinct sh IFN gene activation dependent on IRF3 or IRF7, Journal of Immunology, vol. 187, no. 5, pp. 25312539, 2011. N. Medic, F. Vita, R. Abbate et al., Mast cell activation by myelin through scavenger receptor, Journal of Neuroimmunology, vol. 200, no. 1-2, pp. 2740, 2008. J. E. Murphy, P. R. Tedbury, S. Homer-Vanniasinkam, J. H. Walker, and S. Ponnambalam, Biochemistry and cell biology of mammalian scavenger receptors, Atherosclerosis, vol. 182, no. 1, pp. 115, 2005. A. Pl uddemann, C. Neyen, and S. Gordon, Macrophage scavenger receptors and host-derived ligands, Methods, vol. 43, no. 3, pp. 207217, 2007. T. Areschoug and S. Gordon, Pattern recognition receptors and their role in innate immunity: focus on microbial protein ligands, Contributions to Microbiology, vol. 15, pp. 4560, 2008. S. E. Doyle, R. M. OConnell, G. A. Miranda et al., Toll-like receptors induce a phagocytic gene program through p38, Journal of Experimental Medicine, vol. 199, no. 1, pp. 8190, 2004. K. Hoebe, P. Georgel, S. Rutschmann et al., CD36 is a sensor of diacylglycerides, Nature, vol. 433, no. 7025, pp. 523527, 2005.
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72]
[73]
[74]
[75]
Advances in Hematology
[76] P. Jeannin, B. Bottazzi, M. Sironi et al., Complexity and complementarity of outer membrane protein A recognition by cellular and humoral innate immunity receptors, Immunity, vol. 22, no. 5, pp. 551560, 2005. [77] L. Peiser, P. J. Gough, T. Kodama, and S. Gordon, Macrophage class A scavenger receptor-mediated phagocytosis of Escherichia coli: role of cell heterogeneity, microbial strain, and culture conditions in vitro, Infection and Immunity, vol. 68, no. 4, pp. 19531963, 2000. [78] L. Peiser, M. P. J. De Winther, K. Makepeace et al., The class A macrophage scavenger receptor is a major pattern recognition receptor for Neisseria meningitidis which is independent of lipopolysaccharide and not required for secretory responses, Infection and Immunity, vol. 70, no. 10, pp. 53465354, 2002. [79] M. S. Arredouani, Z. Yang, A. Imrich, Y. Ning, G. Qin, and L. Kobzik, The macrophage scavenger receptor SR-AI/II and lung defense against pneumococci and particles, American Journal of Respiratory Cell and Molecular Biology, vol. 35, no. 4, pp. 474478, 2006. [80] O. Elomaa, M. Kangas, C. Sahlberg et al., Cloning of a novel bacteria-binding receptor structurally related to scavenger receptors and expressed in a subset of macrophages, Cell, vol. 80, no. 4, pp. 603609, 1995. [81] M. Arredouani, Z. Yang, Y. Y. Ning et al., The scavenger receptor MARCO is required for lung defense against pneumococcal pneumonia and inhaled particles, Journal of Experimental Medicine, vol. 200, no. 2, pp. 267272, 2004. [82] S. Mukhopadhyay, Y. Chen, M. Sankala et al., MARCO, an innate activation marker of macrophages, is a class A scavenger receptor for Neisseria meningitidis, European Journal of Immunology, vol. 36, no. 4, pp. 940949, 2006. [83] D. M. E. Bowdish, K. Sakamoto, M. J. Kim et al., MARCO, TLR2, and CD14 are required for macrophage cytokine responses to mycobacterial trehalose dimycolate and Mycobacterium tuberculosis, PLoS Pathogens, vol. 5, no. 6, Article ID e1000474, 2009. [84] L. M. Stuart, J. Deng, J. M. Silver et al., Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain, Journal of Cell Biology, vol. 170, no. 3, pp. 477485, 2005. [85] T. G. Vishnyakova, R. Kurlander, A. V. Bocharov et al., CLA1 and its splicing variant CLA-2 mediate bacterial adhesion and cytosolic bacterial invasion in mammalian cells, Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 45, pp. 1688816893, 2006. [86] V. Wittamer, J. Y. Bertrand, P. W. Gutschow, and D. Traver, Characterization of the mononuclear phagocyte system in zebrash, Blood, vol. 117, no. 26, pp. 71267135, 2011. [87] A. Zakrzewska, C. Cui, O. W. Stockhammer, E. L. Benard, H. P. Spaink, and A. H. Meijer, Macrophage-specic gene functions in Spi1-directed innate immunity, Blood, vol. 116, no. 3, pp. e1e11, 2010. [88] P. Encinas, M. A. Rodriguez-Milla, B. Novoa, A. Estepa, A. Figueras, and J. Coll, Zebrash n immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach, BMC Genomics, vol. 11, no. 1, article no. 518, 2010. [89] A. N. Zelensky and J. E. Gready, The C-type lectin-like domain superfamily, FEBS Journal, vol. 272, no. 24, pp. 61796217, 2005.
15
[90] M. J. Robinson, D. Sancho, E. C. Slack, S. LeibundGutLandmann, and C. R. Sousa, Myeloid C-type lectins ininnate immunity, Nature Immunology, vol. 7, no. 12, pp. 1258 1265, 2006. [91] L. L. Lanier, NK cell recognition, Annual Review of Immunology, vol. 23, pp. 225274, 2005. [92] M. Takahashi, D. Iwaki, A. Matsushita et al., Cloning and characterization of mannose-binding lectin from lamprey (Agnathans), Journal of Immunology, vol. 176, no. 8, pp. 48614868, 2006. [93] T. B. H. Geijtenbeek and S. I. Gringhuis, Signalling through C-type lectin receptors: shaping immune responses, Nature Reviews Immunology, vol. 9, no. 7, pp. 465479, 2009. [94] J. Herre, A. S. J. Marshall, E. Caron et al., Dectin-1 uses novel mechanisms for yeast phagocytosis in macrophages, Blood, vol. 104, no. 13, pp. 40384045, 2004. [95] D. M. Underhill, E. Rossnagle, C. A. Lowell, and R. M. Simmons, Dectin-1 activates Syk tyrosine kinase in a dynamic subset of macrophages for reactive oxygen production, Blood, vol. 106, no. 7, pp. 25432550, 2005. [96] P. J. Tacken, R. Torensma, and C. G. Figdor, Targeting antigens to dendritic cells in vivo, Immunobiology, vol. 211, no. 6-8, pp. 599608, 2006. [97] N. C. Rogers, E. C. Slack, A. D. Edwards et al., Syk-dependent cytokine induction by dectin-1 reveals a novel pattern recognition pathway for C type lectins, Immunity, vol. 22, no. 4, pp. 507517, 2005. [98] K. Sato, X. L. Yang, T. Yudate et al., Dectin-2 is a pattern recognition receptor for fungi that couples with the Fc receptor chain to induce innate immune responses, Journal of Biological Chemistry, vol. 281, no. 50, pp. 3885438866, 2006. [99] S. Yamasaki, M. Matsumoto, O. Takeuchi et al., C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia, Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 6, pp. 18971902, 2009. [100] O. Gross, A. Gewies, K. Finger et al., Card9 controls a nonTLR signalling pathway for innate anti-fungal immunity, Nature, vol. 442, no. 7103, pp. 651656, 2006. [101] S. I. Gringhuis, J. den Dunnen, M. Litjens, B. van het Hof, Y. van Kooyk, and T. H. Geijtenbeek, C-Type Lectin DC-SIGN modulates toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-B, Immunity, vol. 26, no. 5, pp. 605616, 2007. [102] A. N. Jackson, C. A. McLure, R. L. Dawkins, and P. J. Keating, Mannose binding lectin (MBL) copy number polymorphism in Zebrash (D. rerio) and identication of haplotypes resistant to L. anguillarum, Immunogenetics, vol. 59, no. 11, pp. 861872, 2007. [103] M. Fukuda, K. Ohtani, S.-J. Jang et al., Molecular cloning and functional analysis of scavenger receptor zebrash CLP1, Biochimica et Biophysica Acta, vol. 1810, no. 12, pp. 11501159, 2011. [104] K. Ohtani, Y. Suzuki, S. Eda et al., The membrane-type collectin CL-P1 is a scavenger receptor on vascular endothelial cells, Journal of Biological Chemistry, vol. 276, no. 47, pp. 4422244228, 2001. [105] A. F. Lin, L. X. Xiang, Q. L. Wang, W. R. Dong, Y. F. Gong, and J. Z. Shao, The DC-SIGN of zebrash: insights into the existence of a CD209 homologue in a lower vertebrate and its involvement in adaptive immunity, Journal of Immunology, vol. 183, no. 11, pp. 73987410, 2009.
16
[106] P. G. Panagos, K. P. Dobrinski, X. Chen et al., Immunerelated, lectin-like receptors are dierentially expressed in the myeloid and lymphoid lineages of zebrash, Immunogenetics, vol. 58, no. 1, pp. 3140, 2006. [107] Z. Hegedus, A. Zakrzewska, V. C. Agoston et al., Deep sequencing of the zebrash transcriptome response to mycobacterium infection, Molecular Immunology, vol. 46, no. 15, pp. 29182930, 2009. [108] B. Lin, S. Chen, Z. Cao et al., Acute phase response in zebrash upon Aeromonas salmonicida and Staphylococcus aureus infection: striking similarities and obvious dierences with mammals, Molecular Immunology, vol. 44, no. 4, pp. 295 301, 2007. [109] I. Rojo, O. M. de Il arduya, A. Estonba, and M. A. Pardo, Innate immune gene expression in individual zebrash after Listonella anguillarum inoculation, Fish and Shellsh Immunology, vol. 23, no. 6, pp. 12851293, 2007. [110] S. P. Commins, L. Borish, and J. W. Steinke, Immunologic messenger molecules: cytokines, interferons, and chemokines, Journal of Allergy and Clinical Immunology, vol. 125, no. 2, supplement 2, pp. S53S72, 2010. [111] C. Perez, I. Albert, K. DeFay, N. Zachariades, L. Gooding, and M. Kriegler, A nonsecretable cell surface mutant of tumor necrosis factor (TNF) kills by cell-to-cell contact, Cell, vol. 63, no. 2, pp. 251258, 1990. [112] D. P. Cerretti, C. J. Kozlosky, B. Mosley et al., Molecular cloning of the interleukin-1 converting enzyme, Science, vol. 256, no. 5053, pp. 97100, 1992. [113] I. Lalani, K. Bhol, and A. R. Ahmed, Interleukin-10: biology, role in inammation and autoimmunity, Annals of Allergy, Asthma and Immunology, vol. 79, no. 6, pp. 469484, 1997. [114] H. Nomiyama, K. Hieshima, N. Osada et al., Extensive expansion and diversication of the chemokine gene family in zebrash: identication of a novel chemokine subfamily CX, BMC Genomics, vol. 9, article no. 222, 2008. [115] M. O. Huising, R. J. M. Stet, H. F. J. Savelkoul, and B. M. L. Verburg-Van Kemenade, The molecular evolution of the interleukin-1 family of cytokines; IL-18 in teleost sh, Developmental and Comparative Immunology, vol. 28, no. 5, pp. 395413, 2004. [116] D.-C. Zhang, Y.-Q. Shao, Y.-Q. Huang, and S.-G. Jiang, Cloning, characterization and expression analysis of interleukin-10 from the zebrash (Danio reriori), Journal of Biochemistry and Molecular Biology, vol. 38, no. 5, pp. 571 576, 2005. [117] M. Varela, S. Dios, B. Novoa, and A. Figueras, Characterisation, expression and ontogeny of interleukin-6 and its receptors in zebrash (Danio rerio), Developmental and Comparative Immunology, vol. 37, no. 1, pp. 97106, 2012. [118] L. Grayfer and M. Belosevic, Molecular characterization of novel interferon gamma receptor 1 isoforms in zebrash (Danio rerio) and goldsh (Carassius auratus L.), Developmental & Comparative Immunology, vol. 36, no. 2, pp. 408 417, 2012. [119] S. H. B. Oehlers, M. V. Flores, C. J. Hall et al., Expression of zebrash cxcl8 (interleukin-8) and its receptors during development and in response to immune stimulation, Developmental and Comparative Immunology, vol. 34, no. 3, pp. 352359, 2010. [120] L. M. van der Aa, M. Chadzinska, E. Tijhaar, P. Boudinot, and B. M. Lidy verburg-Van kemenade, CXCL8 chemokines in teleost sh: two lineages with distinct expression proles during early phases of inammation, PLoS One, vol. 5, no. 8, Article ID e12384, 2010.
Advances in Hematology
[121] A. E. Clatworthy, J. S. W. Lee, M. Leibman, Z. Kostun, A. J. Davidson, and D. T. Hung, Pseudomonas aeruginosa infection of zebrash involves both host and pathogen determinants, Infection and Immunity, vol. 77, no. 4, pp. 12931303, 2009. [122] M. E. Pressley, P. E. Phelan, P. Eckhard Witten, M. T. Mellon, and C. H. Kim, Pathogenesis and inammatory response to Edwardsiella tardainfection in the zebrash, Developmental and Comparative Immunology, vol. 29, no. 6, pp. 501513, 2005. [123] J. J. Van Soest, O. W. Stockhammer, A. Ordas, G. V. Bloemberg, H. P. Spaink, and A. H. Meijer, Comparison of static immersion and intravenous injection systems for exposure of zebrash embryos to the natural pathogen Edwardsiella tarda, BMC Immunology, vol. 12, Article ID 58, 2011. [124] H. Clay, H. E. Volkman, and L. Ramakrishnan, Tumor necrosis factor signaling mediates resistance to mycobacteria by inhibiting bacterial growth and macrophage death, Immunity, vol. 29, no. 2, pp. 283294, 2008. [125] F. J. Roca, I. Mulero, A. Lopez-Mu noz et al., Evolution of the inammatory response in vertebrates: sh TNF- is a powerful activator of endothelial cells but hardly activates phagocytes, Journal of Immunology, vol. 181, no. 7, pp. 5071 5081, 2008. [126] D. Aggad, C. Stein, D. Sieger et al., In vivo analysis of Ifn1 and Ifn-2 signaling in zebrash, Journal of Immunology, vol. 185, no. 11, pp. 67746782, 2010. [127] D. Sieger, C. Stein, D. Neifer, A. M. Van Der Sar, and M. Leptin, The role of gamma interferon in innate immunity in the zebrash embryo, Disease Models and Mechanisms, vol. 2, no. 11-12, pp. 571581, 2009. [128] O. J. Hamming, G. Lutfalla, J. P. Levraud, and R. Hartmann, Crystal structure of zebrash interferons I and II reveals conservation of type I interferon structure in vertebrates, Journal of Virology, vol. 85, no. 16, pp. 81818187, 2011. [129] J. P. Levraud, P. Boudinot, I. Colin et al., Identication of the zebrash IFN receptor: implications for the origin of the vertebrate IFN system, Journal of Immunology, vol. 178, no. 7, pp. 43854394, 2007. [130] K. Kwiatkowska and A. Sobota, Signaling pathways in phagocytosis, BioEssays, vol. 21, no. 5, pp. 422431, 1999. [131] P. J. Sansonetti, Phagocytosis, a cell biology view, Journal of Cell Science, vol. 113, part 19, pp. 33553356, 2000. [132] A. Pitt, L. S. Mayorga, P. D. Stahl, and A. L. Schwartz, Alterations in the protein composition of maturing phagosomes, Journal of Clinical Investigation, vol. 90, no. 5, pp. 19781983, 1992. [133] T. E. Tjelle, T. Lvdal, and T. Berg, Phagosome dynamics and function, BioEssays, vol. 22, no. 3, pp. 255263, 2000. [134] H. Tapper, Out of the phagocyte or into its phagosome: signalling to secretion, European Journal of Haematology, vol. 57, no. 3, pp. 191201, 1996. [135] A. W. Segal, How neutrophils kill microbes, Annual Review of Immunology, vol. 23, pp. 197223, 2005. [136] W. L. Lee, R. E. Harrison, and S. Grinstein, Phagocytosis by neutrophils, Microbes and Infection, vol. 5, no. 14, pp. 1299 1306, 2003. [137] S. Duclos and M. Desjardins, Subversion of a young phagosome: the survival strategies of intracellular pathogens, Cellular Microbiology, vol. 2, no. 5, pp. 365377, 2000. [138] J. Gruenberg and F. G. Van Der Goot, Mechanisms of pathogen entry through the endosomal compartments,
Advances in Hematology
Nature Reviews Molecular Cell Biology, vol. 7, no. 7, pp. 495 504, 2006. J. M. Davis, H. Clay, J. L. Lewis, N. Ghori, P. Herbomel, and L. Ramakrishnan, Real-time visualization of Mycobacteriummacrophage interactions leading to initiation of granuloma formation in zebrash embryos, Immunity, vol. 17, no. 6, pp. 693702, 2002. L. M. Stamm, J. H. Morisaki, L. Y. Gao et al., Mycobacterium marinum escapes from phagosomes and is propelled by actin-based motility, Journal of Experimental Medicine, vol. 198, no. 9, pp. 13611368, 2003. N. van der Wel, D. Hava, D. Houben et al., M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells, Cell, vol. 129, no. 7, pp. 12871298, 2007. B. Levine, N. Mizushima, and H. W. Virgin, Autophagy in immunity and inammation, Nature, vol. 469, no. 7330, pp. 323335, 2011. E. Itakura, C. Kishi, K. Inoue, and N. Mizushima, Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG, Molecular Biology of the Cell, vol. 19, no. 12, pp. 53605372, 2008. H. Nakatogawa, J. Ishii, E. Asai, and Y. Ohsumi, Atg4 recycles inappropriately lipidated Atg8 to promote autophagosome biogenesis, Autophagy, vol. 8, no. 2, 2012. M. A. Sanjuan and D. R. Green, Eating for good health: linking autophagy and phagocytosis in host defense, Autophagy, vol. 4, no. 5, pp. 607611, 2008. M. Ponpuak, A. S. Davis, E. A. Roberts et al., Delivery of cytosolic components by autophagic adaptor protein p62 endows autophagosomes with unique antimicrobial properties, Immunity, vol. 32, no. 3, pp. 329341, 2010. R. Sumpter and B. Levine, Autophagy and innate immunity: triggering, targeting and tuning, Seminars in Cell and Developmental Biology, vol. 21, no. 7, pp. 699711, 2010. C. Hall, M. V. Flores, A. Chien, A. Davidson, K. Crosier, and P. Crosier, Transgenic zebrash reporter lines reveal conserved Toll-like receptor signaling potential in embryonic myeloid leukocytes and adult immune cell lineages, Journal of Leukocyte Biology, vol. 85, no. 5, pp. 751765, 2009. D. Le Guyader, M. J. Redd, E. Colucci-Guyon et al., Origins and unconventional behavior of neutrophils in developing zebrash, Blood, vol. 111, no. 1, pp. 132141, 2008. E. Colucci-Guyon, J.-Y. Tinevez, S. A. Renshaw, and P. Herbomel, Strategies of professional phagocytes in vivo: unlike macrophages, neutrophils engulf only surface-associated microbes, Journal of Cell Science, vol. 124, no. 18, pp. 3053 3059, 2011. J. Ulvila, L. M. Vanha-Aho, A. Kleino et al., Colin regulator 14-3-3 is an evolutionarily conserved protein required for phagocytosis and microbial resistance, Journal of Leukocyte Biology, vol. 89, no. 5, pp. 649659, 2011. C. He, C. R. Bartholomew, W. Zhou, and D. J. Klionsky, Assaying autophagic activity in transgenic GFP-Lc3 and GFP-Gabarap zebrash embryos, Autophagy, vol. 5, no. 4, pp. 520526, 2009. A. Lu, X. Hu, J. Xue, J. Zhu, Y. Wang, and G. Zhou, Gene expression proling in the skin of zebrash infected with Citrobacter freundii, Fish and Shellsh Immunology, vol. 32, no. 2, pp. 273283, 2012. C. Delclaux, C. Delacourt, M. P. DOrtho, V. Boyer, C. Lafuma, and A. Harf, Role of gelatinase B and elastase in human polymorphonuclear neutrophil migration across
17
basement membrane, American Journal of Respiratory Cell and Molecular Biology, vol. 14, no. 3, pp. 288295, 1996. H. Sengelov, L. Kjeldsen, and N. Borregaard, Control of exocytosis in early neutrophil activation, Journal of Immunology, vol. 150, no. 4, pp. 15351543, 1993. K. A. Joiner, T. Ganz, J. Albert, and D. Rotrosen, The opsonizing ligand on Salmonella typhimurium inuences incorporation of specic, but not azurophil, granule constituents into neutrophil phagosomes, Journal of Cell Biology, vol. 109, no. 6 I, pp. 27712782, 1989. M. Faurschou and N. Borregaard, Neutrophil granules and secretory vesicles in inammation, Microbes and Infection, vol. 5, no. 14, pp. 13171327, 2003. C. Zhang, J. Yang, J. D. Jacobs, and L. K. Jennings, Interaction of myeloperoxidase with vascular NAD(P)H oxidasederived reactive oxygen species in vasculature: implications for vascular diseases, American Journal of Physiology, Heart and Circulatory Physiology, vol. 285, no. 6, pp. H2563H2572, 2003. V. Brinkmann, U. Reichard, C. Goosmann et al., Neutrophil extracellular traps kill bacteria, Science, vol. 303, no. 5663, pp. 15321535, 2004. J. D. Lambeth, NOX enzymes and the biology of reactive oxygen, Nature Reviews Immunology, vol. 4, no. 3, pp. 181 189, 2004. B. M. Babior, The activity of leukocyte NADPH oxidase: regulation by p47PHOX cysteine and serine residues, Antioxidants and Redox Signaling, vol. 4, no. 1, pp. 3538, 2002. D. Roos and C. C. Winterbourn, Immunology: lethal weapons, Science, vol. 296, no. 5568, pp. 669671, 2002. E. Peranzoni, I. Marigo, L. Dolcetti et al., Role of arginine metabolism in immunity and immunopathology, Immunobiology, vol. 212, no. 9-10, pp. 795812, 2008. C. Bogdan, Nitric oxide and the immune response, Nature Immunology, vol. 2, no. 10, pp. 907916, 2001. S. El-Gayar, H. Th uring-Nahler, J. Pfeilschifter, M. R ollingho, and C. Bogdan, Translational control of inducible nitric oxide synthase by IL-13 and arginine availability in inammatory macrophages, Journal of Immunology, vol. 171, no. 9, pp. 45614568, 2003. G. Wu and S. M. Morris, Arginine metabolism: nitric oxide and beyond, Biochemical Journal, vol. 336, no. 1, pp. 117, 1998. S. Pfeier, A. Lass, K. Schmidt, and B. Mayer, Protein tyrosine nitration in cytokine-activated murine macrophages. Involvement of a peroxidase/nitrite pathway rather than peroxynitrite, Journal of Biological Chemistry, vol. 276, no. 36, pp. 3405134058, 2001. C. J. Hall, M. V. Flores, S. H. Oehlers et al., Infectionresponsive expansion of the hematopoietic stem and progenitor cell compartment in zebrash is dependent upon inducible nitric oxide, Cell Stem Cell, vol. 10, no. 2, pp. 198 209, 2012. J. Arnhold and J. Flemmig, Human myeloperoxidase in innate and acquired immunity, Archives of Biochemistry and Biophysics, vol. 500, no. 1, pp. 92106, 2010. J. D. Shearer, J. R. Richards, C. D. Mills, and M. D. Caldwell, Dierential regulation of macrophage arginine metabolism: a proposed role in wound healing, American Journal of Physiology, Endocrinology and Metabolism, vol. 272, no. 2, pp. E181E190, 1997. J. R. Mathias, B. J. Perrin, T. X. Liu, J. Kanki, A. T. Look, and A. Huttenlocher, Resolution of inammation by retrograde
[139]
[155]
[156]
[140]
[157]
[141]
[158]
[142]
[143]
[159]
[144]
[160]
[145]
[161]
[146]
[162] [163]
[147]
[164] [165]
[148]
[149]
[166]
[150]
[167]
[151]
[168]
[152]
[169]
[153]
[170]
[154]
[171]
18
chemotaxis of neutrophils in transgenic zebrash, Journal of Leukocyte Biology, vol. 80, no. 6, pp. 12811288, 2006. P. Niethammer, C. Grabher, A. T. Look, and T. J. Mitchison, A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrash, Nature, vol. 459, no. 7249, pp. 996999, 2009. Q. Deng, E. A. Harvie, and A. Huttenlocher, Distinct signalling mechanisms mediate neutrophil attraction to bacterial infection and tissue injury, Cellular Microbiology, vol. 14, no. 4, pp. 517528, 2012. S. K. Yoo, T. W. Starnes, Q. Deng, and A. Huttenlocher, Lyn is a redox sensor that mediates leukocyte wound attraction in vivo, Nature, vol. 480, no. 7375, pp. 109112, 2011. M. V. Flores, K. C. Crawford, L. M. Pullin, C. J. Hall, K. E. Crosier, and P. S. Crosier, Dual oxidase in the intestinal epithelium of zebrash larvae has anti-bacterial properties, Biochemical and Biophysical Research Communications, vol. 400, no. 1, pp. 164168, 2010. K. M. Brothers, Z. R. Newman, and R. T. Wheeler, Live imaging of disseminated candidiasis in zebrash reveals role of phagocyte oxidase in limiting lamentous growth, Eukaryotic Cell, vol. 10, no. 7, pp. 932944, 2011. Y. Feng, C. Santoriello, M. Mione, A. Hurlstone, and P. Martin, Live imaging of innate immune cell sensing of transformed cells in zebrash larvae: parallels between tumor initiation and wound inammation, PLoS Biology, vol. 8, no. 12, Article ID e1000562, 2010. A. C. Hermann, P. J. Millard, S. L. Blake, and C. H. Kim, Development of a respiratory burst assay using zebrash kidneys and embryos, Journal of Immunological Methods, vol. 292, no. 1-2, pp. 119129, 2004. S. Lepiller, V. Laurens, A. Bouchot, P. Herbomel, E. Solary, and J. Chluba, Imaging of nitric oxide in a living vertebrate using a diaminouorescein probe, Free Radical Biology and Medicine, vol. 43, no. 4, pp. 619627, 2007. P. M. Elks, F. J. Van Eeden, G. Dixon et al., Activation of hypoxia-inducible factor-1 (hif-1) delays inammation resolution by reducing neutrophil apoptosis and reverse migration in a zebrash inammation model, Blood, vol. 118, no. 3, pp. 712722, 2011. M. De Jong, H. Rauwerda, O. Bruning, J. Verkooijen, H. P. Spaink, and T. M. Breit, RNA isolation method for single embryo transcriptome analysis in zebrash, BMC Research Notes, vol. 3, article no. 73, 2010. A. H. Meijer, F. J. Verbeek, E. Salas-Vidal et al., Transcriptome proling of adult zebrash at the late stage of chronic tuberculosis due to Mycobacterium marinum infection, Molecular Immunology, vol. 42, no. 10, pp. 11851203, 2005. Z. Wu, W. Zhang, Y. Lu, and C. Lu, Transcriptome proling of zebrash infected with Streptococcus suis, Microbial Pathogenesis, vol. 48, no. 5, pp. 178187, 2010. P. S. Srinivasa Rao, T. M. Lim, and K. Y. Leung, Functional genomics approach to the identication of virulence genes involved in Edwardsiella tardapathogenesis, Infection and Immunity, vol. 71, no. 3, pp. 13431351, 2003. A. M. van der Sar, R. J. P. Musters, F. J. M. van Eeden, B. J. Appelmelk, C. M. J. E. Vandenbroucke-Grauls, and W. Bitter, Zebrash embryos as a model host for the real time analysis of Salmonella typhimurium infections, Cellular Microbiology, vol. 5, no. 9, pp. 601611, 2003. D. M. Tobin and L. Ramakrishnan, Comparative pathogenesis of Mycobacterium marinum and Mycobacterium tuberculosis, Cellular Microbiology, vol. 10, no. 5, pp. 1027 1039, 2008.
Advances in Hematology
[187] H. E. Volkman, H. Clay, D. Beery, J. C. W. Chang, D. R. Sherman, and L. Ramakrishnan, Tuberculous granuloma formation is enhanced by a Mycobacterium virulence determinant, PLoS Biology, vol. 2, no. 11, article e367, 2004. [188] H. Clay, J. M. Davis, D. Beery, A. Huttenlocher, S. E. Lyons, and L. Ramakrishnan, Dichotomous Role of the Macrophage in Early Mycobacterium marinum Infection of the Zebrash, Cell Host and Microbe, vol. 2, no. 1, pp. 2939, 2007. [189] K. Spilsbury, M. A. OMara, Wan Man Wu, P. B. Rowe, G. Symonds, and Y. Takayama, Isolation of a novel macrophage-specic gene by dierential cDNA analysis, Blood, vol. 85, no. 6, pp. 16201629, 1995. [190] B. Chen, D. Zhang, and J. W. Pollard, Progesterone regulation of the mammalian ortholog of methylcitrate dehydratase (Immune Response Gene 1) in the uterine epithelium during implantation through the protein kinase C pathway, Molecular Endocrinology, vol. 17, no. 11, pp. 23402354, 2003. [191] H. T. Tran, N. Barnich, and E. Mizoguchi, Potential role of chitinases and chitin-binding proteins in host-microbial interactions during the development of intestinal inammation, Histology and Histopathology, vol. 26, no. 11, pp. 1453 1464, 2011. [192] R. Carvalho, J. de Sonneville, O. W. Stockhammer et al., A high-throughput screen for tuberculosis progression, PLoS One, vol. 6, no. 2, Article ID e16779, 2011. [193] E. J. M. Stoop, T. Schipper, S. K. Rosendahl Huber et al., Zebrash embryo screen for mycobacterial genes involved in the initiation of granuloma formation reveals a newly identied ESX-1 component, Disease Models and Mechanisms, vol. 4, no. 4, pp. 526536, 2011. [194] M. G. Morash, S. E. Douglas, A. Robotham, C. M. Ridley, J. W. Gallant, and K. H. Soanes, The zebrash embryo as a tool for screening and characterizing pleurocidin hostdefense peptides as anti-cancer agents, Disease Models and Mechanisms, vol. 4, no. 5, pp. 622633, 2011. [195] G. F. Wiegertjes and M. Forlenza, Nitrosative stress during infection-induced inammation in sh: lessons from a hostparasite infection model, Current Pharmaceutical Design, vol. 16, no. 38, pp. 41944202, 2010. [196] F. O. Martinez, L. Helming, and S. Gordon, Alternative activation of macrophages: an immunologic functional perspective, Annual Review of Immunology, vol. 27, pp. 451 483, 2009. [197] Z. G. Fridlender, J. Sun, S. Kim et al., Polarization of tumorassociated neutrophil phenotype by TGF-: N1 versus N2 TAN, Cancer Cell, vol. 16, no. 3, pp. 183194, 2009. [198] A. H. Meijer, A. M. van der Sar, C. Cunha et al., Identication and real-time imaging of a myc-expressing neutrophil population involved in inammation and mycobacterial granuloma formation in zebrash, Developmental and Comparative Immunology, vol. 32, no. 1, pp. 3649, 2008. [199] A. J. Sadler, O. Latchoumanin, D. Hawkes, J. Mak, and B. R. G. Williams, An antiviral response directed by PKR phosphorylation of the RNA helicase A, PLoS Pathogens, vol. 5, no. 2, Article ID e1000311, 2009. [200] A. Takaoka, Z. Wang, M. K. Choi et al., DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response, Nature, vol. 448, no. 7152, pp. 501505, 2007. [201] V. Hornung, A. Ablasser, M. Charrel-Dennis et al., AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inammasome with ASC, Nature, vol. 458, no. 7237, pp. 514518, 2009.
[172]
[173]
[174]
[175]
[176]
[177]
[178]
[179]
[180]
[181]
[182]
[183]
[184]
[185]
[186]
Advances in Hematology
[202] V. Deretic, Autophagy as an innate immunity paradigm: expanding the scope and repertoire of pattern recognition receptors, Current Opinion in Immunology, vol. 24, no. 1, pp. 2131, 2012. [203] D. M. Tobin, R. C. May, and R. T. Wheeler, Zebrash: a see-through host and a uorescent toolbox to probe hostpathogen interaction, PLoS Pathogens, vol. 8, no. 1, Article ID e1002349, 2012. [204] S. A. Renshaw and N. S. Trede, A model 450 million years in the making: zebrash and vertebrate immunity, Disease Models and Mechanisms, vol. 5, no. 1, pp. 3847, 2012. [205] K. J. Clark, D. F. Voytas, and S. C. Ekker, A Tale of two nucleases: gene targeting for the masses? Zebrash, vol. 8, no. 3, pp. 147149, 2011.
19
Hindawi Publishing Corporation Advances in Hematology Volume 2012, Article ID 282318, 8 pages doi:10.1155/2012/282318
of Hematology-Oncology and Stem Cell Transplant, Department of Pediatrics, The University of Chicago, KCBD 5120, Chicago, IL 60637, USA 2 Stem Cell Program and Division of Hematology/Oncology, Childrens Hospital Boston-Dana, Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, and Harvard Medical School, Boston, MA 02115, USA Correspondence should be addressed to Jill L. O. de Jong, [email protected] Received 7 March 2012; Accepted 1 May 2012 Academic Editor: Jason Berman Copyright 2012 J. L. O. de Jong and L. I. Zon. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The zebrash has proven to be an excellent model for human disease, particularly hematopoietic diseases, since these sh make similar types of blood cells as humans and other mammals. The genetic program that regulates the development and dierentiation of hematopoietic cells is highly conserved. Hematopoietic stem cells (HSCs) are the source of all the blood cells needed by an organism during its lifetime. Identifying an HSC requires a functional assay, namely, a transplantation assay consisting of multilineage engraftment of a recipient and subsequent serial transplant recipients. In the past decade, several types of hematopoietic transplant assays have been developed in the zebrash. An understanding of the major histocompatibility complex (MHC) genes in the zebrash has lagged behind transplantation experiments, limiting the ability to perform unbiased competitive transplantation assays. This paper summarizes the dierent hematopoietic transplantation experiments performed in the zebrash, both with and without immunologic matching, and discusses future directions for this powerful experimental model of human blood diseases.
1. Introduction
In the past few decades, the zebrash has emerged as an outstanding vertebrate animal model for studying developmental hematopoiesis (reviewed in [1, 2]). In this same time frame, the understanding of the biology of adult hematopoietic stem cells has also blossomed, predominantly due to hematopoietic transplantation experiments performed in mice (reviewed by Orkin and Zon in [3]). To capitalize on the advantages of the zebrash model (small size, high fecundity, rapid maturation, external fertilization, and the ability to perform large-scale genetic and chemical screens), a zebrash hematopoietic transplantation assay was needed. Developing a transplantation assay in the zebrash required a dierent approach than that used in mice. While dierential expression of CD45 isoforms is generally used to distinguish between donor and recipient cells in murine transplant assays, these reagents are not available for
zebrash. Instead, scientists have utilized transgenic technology to make zebrash expressing green uorescent protein (GFP) or other uorochromes under the inuence of an ubiquitous or a tissue-specic promoter. These uorescently labeled donor cells are transplanted into uorochromenegative recipients, and engraftment is monitored at various time points after transplant.
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Erythroid Myeloid
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101 Precursors Lymphoid 100 200 400 600 Forward scatter (FSC)
(a) Myeloid
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Lymphoid 100 Max (%) 80 60 40 20 0 100 101 102 103 FL1-H:: gfp 44% 100 80 60 40 20
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Figure 1: Flow cytometry analysis of zebrash whole kidney marrow from a marrow transplant recipient. Zebrash transplant recipients were irradiated and injected with 5 105 marrow cells from a transgenic -actin:GFP donor. Whole kidney marrow from a representative recipient was dissected 3 months later and analyzed by ow cytometry. (a) The forward scatter (FSC) versus side scatter (SSC) prole of zebrash whole kidney marrow shows four cell populations: erythroid, lymphoid, myeloid, and precursor cells. (b) Histograms for GFP expression of cells within the lymphoid, myeloid and precursor gates show multilineage engraftment with GFP+ donor cells (blue lines). The red lines show GFP expression in a wild-type-negative control sh.
is currently the standard procedure for identifying multilineage engraftment after hematopoietic transplantation in zebrash (Figure 1(a)). In addition, hematopoietic transplantation was used to rescue two dierent mutant embryos. The Vlad tepes (gat a1/ ) mutation is homozygous lethal by 14 days after fertilization, and these embryos have a complete absence of erythroid cells [5]. Approximately 100 1000 whole kidney marrow (WKM) cells from a gata1GFP transgenic donor were injected into the circulation of gat a1/ zebrash embryos 48 hours after fertilization (hpf). While untransplanted control embryos did not survive past 14 dpf, 2060% of the transplant recipients survived long term, up to 8 months after transplant. All surviving recipients had circulating GFP+ red blood cells, indistinguishable from the gata1-GFP donors [4]. Taking these embryonic transplant experiments one step further, donor marrow was isolated from double transgenic -actin-GFP/gata1-dsRED sh, in order to monitor donorderived cells from multiple lineages. The -actin-GFP transgene is expressed by almost all zebrash cell types, including
all leukocytes. Erythrocytes do not express actin, so they are marked by the gata1-dsRED transgene instead. For these experiments, the transplant recipients were bloodless (bls) mutants, a dominant, partially penetrant mutation resulting in absent primitive hematopoiesis, but preserved adult hematopoiesis [6]. Injection of double-positive WKM cells into 48 hpf bls mutants allowed independent tracking of GFP+ leukocytes and dsRED+ erythrocytes in the recipient embryos [4]. Sustained multilineage donor-derived cells were visible in the circulation of transplant recipients at 8 weeks after transplantation, indicating successful engraftment of long-term hematopoietic repopulating cells. 2.2. Adult Marrow Cells into Adult Recipients. Following up on their transplantation experiments into embryos, Traver et al. subsequently performed transplantation of WKM cells into adult recipients [7]. After using ionizing radiation as pretransplant conditioning to ablate the recipients hematopoietic cells, including the immune system, approximately 1 106 -actin-GFP/gata1-dsRED donor marrow
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(a)
(b)
Figure 2: Direct visualization of engrafted GFP+ and mCherry+ marrow donor cells in casper recipients. 40 103 WKM cells from a transgenic ubiquitin:GFP donor were mixed with 80 103 WKM cells from a transgenic ubiquitin:mCherry donor and injected into the circulation of a casper recipient sh. The photos are taken 4 weeks after transplantation and show engraftment of (a) GFP+ and (b) mCherry+ cells in the kidney (white arrows).
cells were delivered into the recipients circulation by direct intracardiac injection. When irradiated with 40 Gy, a lethal dose, all the untransplanted animals died by 14 days after irradiation. However, >70% of the animals receiving WKM cells after irradiation were rescued, and survived at least 30 days after irradiation. As in the experiments with embryonic transplant recipients, GFP+ leukocytes and dsRED+ erythrocytes were visible in the circulation of the engrafted adult recipients using uorescence light microscopy [7]. FACS analysis of recipient WKM showed robust multilineage engraftment with >86% GFP+ cells up to 8 weeks after transplant (Figure 1(b)). 2.3. Embryonic HSCs into Embryos. Similar to murine embryonic HSCs, the rst HSCs in the developing zebrash are located in the aorta-gonad-mesonephros (AGM) [8]. Initial experiments to identify these HSCs in zebrash relied upon anatomic similarities with murine embryonic HSCs. Cells expressing cmyb, runx1, and CD41 are observed in the ventral wall of the dorsal aorta in zebrash embryos 2436 hpf [912], similar to the expression noted in the ventral wall of the aorta in murine embryos [13]. These cmyb+ and runx1+ cells were presumed to be embryonic denitive HSCs, although functional evaluation of these cells was lacking. Using CD41 as another marker of embryonic HSCs, Bertrand et al. sorted CD41+ /gata1 donor cells by ow cytometry from CD41-eGFP/gata1-dsRED double transgenic embryos at 72 hpf [14]. These cells were then injected into the sinus venosus of age-matched wild-type embryos. Within one day after transplant, donor-derived cells were observed in the caudal hematopoietic tissue (CHT) and thymi of recipients. Although the transplanted donor cells had been dsRED negative, subsequent erythroid dierentiation of engrafted cells revealed dsRED+ cells in the circulation of recipients [14]. These experiments helped to prove that CD41+ cells in the AGM are capable of colonizing denitive hematopoietic organs, namely, the thymus and CHT, in developing zebrash, and therefore, this population includes the rst developing HSCs in the embryo. 2.4. A Competitive Transplantation Assay for Chemical Screening. Capitalizing on the relative ease of in vivo chemical screening using the zebrash model, Li et al. have utilized a competitive hematopoietic transplantation assay to search
for chemicals that enhance hematopoietic engraftment (manuscript submitted). Marrow cells from actin-GFP sh were incubated ex vivo in chemicals from a panel of more than 2000 known bioactive compounds. After pretreatment, the actin-GFP WKM was mixed at a standard ratio with WKM from commercially available red Glosh, and transplanted into casper recipient sh [15]. Normally kidney marrow uorescence is not visible in an adult animal due to the presence of pigmentation in the skin. However, casper sh are homozygous for two pigment mutations, roy and nacre, and therefore have transparent skin, allowing visualization of engrafted uorescent marrow cells in vivo. Unlike prior studies examining engraftment at a single time point by FACS analysis of multilineage WKM populations, this screen also followed the level of GFP+ and RFP+ cells in the kidney of anesthetized recipients at several time points after transplant (Figure 2). The ratio of green : red marrow cells by uorescence microscopy in vivo was highly correlated with the green : red ratio measured by ow cytometry of the dissected WKM cell preparation. All chemicals identied in the screen that stimulated enhanced engraftment were also tested in murine transplants to validate the eects in an immune-matched mammalian transplant assay. In total, ten compounds were identied in the screen that resulted in enhanced green : red ratio, and these are currently undergoing further evaluation.
4 to maintain highly inbred sh lines. Despite this disadvantage, signicant progress has still been made developing hematopoietic transplantation methods in the zebrash over the past decade, as described above. As more sophisticated transplantation experiments are designed to ask more complex questions about stem cell biology, the need for immune matching becomes more critical. When transplanting any allogeneic tissue into an adult recipient with a competent immune system, one would expect a lack of immune matching to result in rejection of the transplanted tissue (reviewed in [17]). In the zebrash, immune matching is not required in embryonic recipients younger than 5 days after fertilization, as thymic development is not apparent until then [18]. By 46 weeks after fertilization, the cellular and humoral immune system is fully functional and would be capable of rejecting any transplanted tissue that was not histocompatible [19, 20]. Pretransplant conditioning with radiation is commonly used to suppress the immune system of adult murine and zebrash recipients, and in the case of hematopoietic transplants to give the added advantage of clearing the marrow niche. For zebrash recipients receiving a sublethal dose of radiation, the transplanted tissue is still rejected once the recipients immune cells recover, approximately 4 weeks after irradiation [21]. Another consequence of immune mismatch between transplant donors and recipients occurs uniquely in the setting of hematopoietic transplantation. When engrafted immune cells recognize the recipient as nonself, an immune response is mounted against the recipients tissues resulting in graft-versus-host disease (GVHD), a phenomenon that is also observed clinically in human allogeneic bone marrow transplant [22]. Therefore, the importance of immune matching in hematopoietic transplantation impacts not only initial engraftment, but also the health and survival of the recipient if the engrafted hematopoietic cells attack the host.
Advances in Hematology normal HSC function, the donor chimerism would reveal an equal mix of engrafted hematopoietic cells from both donors. Immune matching of both donors and the recipient is an essential component of any competitive hematopoietic transplantation assay. Otherwise, one cannot rule out biased immune rejection of one donors cells compared to the other, and the engraftment winner may merely reect immunologic dierences and not a dierence in stem cell biology. A variation of the competitive hematopoietic transplantation assay is the limit dilution assay. This method is the gold standard for quantitating HSC content and also requires all donors and recipients to be immunologically matched. This assay involves transplantation of serially diluted marrow cells such that fewer and fewer marrow cells are given to subsequent transplant recipients, while a constant number of wild-type marrow cells are given simultaneously to radioprotect the recipients. Engraftment and donor chimerism are evaluated for each recipient, and then Poisson statistics are used to calculate the number of long-term repopulating cells contained in the original marrow population [24]. The ability to perform these competitive and quantitative experiments using zebrash HSCs will be essential to characterize stem cell mutants and asking questions about HSC biology. Therefore, a better understanding of the histocompatibility genes in the zebrash is needed so that these assays can be performed with proper immune matching.
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Table 1: Mean percentage of GFP+ cells in engrafted recipient zebrash receiving MHC-matched or -unmatched donor marrow. Only Chr 19 matched [35] Myeloid matched Myeloid unmatched Lymphoid matched Lymphoid unmatched
Data are mean S.D.
Chr 1, 8, 19 all matched 52.36 25.43 11.58 7.03 9.51 12.32 3.47 4.601 P = 0.0036 P = 0.047
P = 0.0002 P = 0.05
that they have class I and II genes located on distinct chromosomes [29]. In the zebrash, at least three relevant loci have been identied. Chromosome 19 contains class I genes as well as some antigen-processing genes, making the locus syntenic to the human MHC locus [30, 31]. However, there are no class II genes on chromosome 19. Instead the zebrash class II alpha and beta genes are located on chromosome 8 [26, 32]. Chromosome 1 contains additional class I genes, termed ze genes, which appear most similar to mammalian nonclassical Class I genes [33]. Finally, the L genes, class I genes unique to teleost sh, are located on chromosomes 3 and 8, although they are less polymorphic than other class I genes, and their precise function is not clear [34]. While DNA sequence analyses of the zebrash MHC genes show similarities with MHC genes of many species, virtually no data are available to evaluate the function or even the cell-surface expression of the class I and II genes in zebrash. Prior to the transplantation experiments described below, no functional evaluation of any zebrash MHC genes had been performed.
the same MHC genotype and also into unrelated wildtype recipients, presumed to be mismatched. Survival and donor chimerism were signicantly improved in the matched recipients compared with the unmatched recipients (Table 1), indicating the importance of immune matching at the chromosome 19 MHC locus for hematopoietic engraftment [35]. These experiments were the rst functional evaluation of any zebrash MHC genes in a transplantation assay. These rst experiments did not specically type for class II genes located on chromosome 8, or other class I genes on other chromosomes. It may be that coincidental matching at the class II locus occurred for a signicant number of the related matched recipients in these experiments, thereby contributing to improved donor chimerism. We expected that immune matching at the class II locus would also be important for hematopoietic engraftment. Therefore, we performed additional transplantation experiments matching the donors and recipients at three separate loci: the two class I loci on chromosomes 1 and 19 and the class II locus on chromosome 8. 2.5 105 WKM cells from -actin-GFP+ donor sh were transplanted into both completely matched recipients and unmatched, unrelated recipients. Long-term engraftment at 3 months after transplant showed similar donor chimerism results as the transplant experiments with matching at only the chromosome 19 locus (Table 1). These data suggest that matching of the class I genes at the chromosome 19 locus is the most important for tissue histocompatibility in a transplantation assay, and that the additional MHC loci on chromosomes 1 and 8 play a minimal role. Further experiments are underway to individually test the class I genes on chromosome 1 and the class II genes on chromosome 8 to determine the contribution, if any, of these loci to histocompatibility in tissue transplantation.
6 recipients are approximately 3-4 months of age (J. L. O. de Jong and L. I. Zon, unpublished data). This may be due to colonization of older sh with bacterial or fungal pathogens that overwhelm and kill the immune-compromised host after transplantation. Maintaining excellent water quality is also critically important to recipient survival. We hypothesized that treatment with prophylactic antibiotics for a few days immediately after transplant might improve survival. However, placing transplant recipients o system in sh water containing antibiotics paradoxically caused decreased survival, as sh being treated in this way suered from quickly deteriorating water quality and high ammonia levels (C. Lawrence, personal communication). While it is impractical to keep a therapeutic level of antibiotics in the large volume of water circulating through an entire aquatic system, the ability to maintain water quality at a consistently high standard resulted in improved survival of our transplant recipients, even without antibiotics. Determining the appropriate radiation dose for pretransplant conditioning of recipient sh has also proven more challenging than initially anticipated. Water can greatly attenuate the radiation dose over a short distance. For example, at a depth of 1 cm of water, we have observed that the radiation dose at the bottom of the dish is decreased by about 1015% compared with the radiation dose at the surface of the water (J. L. O. de Jong, unpublished data). Therefore, it is critically important that sh be placed in a minimal volume and depth of water to ensure that all recipients receive an equivalent radiation dose. The minimum lethal dose of radiation for zebrash was rst reported to be 40 Gy [7]. However, subsequent work showed that this dose was not optimal for pretransplant conditioning, as the mortality of sh was 100%, even after receiving a radio-protective dose of WKM cells. A sublethal dose of 25 Gy provided for maximal survival with engraftment, so this was the dose selected for most experiments [35]. This result suggests that while the hematopoietic compartment is the most radiation-sensitive tissue in the zebrash, as in mammals, there is a narrow therapeutic index for lethal radiation damage to other tissues. To minimize the radiation injury to nonhematopoietic tissues, many protocols for murine and human bone marrow transplants utilize fractionated radiation dosing. We have now initiated a standard conditioning protocol of 30 Gy split into two equal fractions of 15 Gy, where the two fractions are given 24 hours apart. The survival of these recipients is comparable to animals receiving 25 Gy as a single dose (J. L. O. de Jong, unpublished data). Finally, we have observed that dierent sh lines have varying sensitivities to radiation. For example, when comparing sh from the AB strain that have been bred to homozygosity at the MHC loci, some were signicantly more sensitive to a given radiation dose than others (Figure 3). This result suggests that a radiation dose-response titration should be performed for each strain of recipients to be transplanted in order to determine the optimal radiation dose. Alternatively, conditioning with chemotherapeutic medications such as cyclophosphamide [36] could be used, although these have not been tested for pretransplant conditioning of zebrash donors.
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0.6
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Figure 3: Survival of dierent zebrash lines in response to radiation. Kaplan-Meier survival curves are shown for four dierent zebrash strains after irradiation with a total dose of 25 Gy, delivered in two equal fractions of 12.5 Gy separated by 24 hours. Twenty one sh were irradiated in each group. CG1 is a clonal homozygous diploid sh line generated by parthenogenesis [21, 38]. UDA, UXA2, and UBA are inbred zebrash lines derived from a single mating pair of AB parents [35]. Each line was named for the homozygous class I MHC gene at its chromosome 19 locus. The results demonstrate 100% mortality for the CG1 sh by day 22, and by day 37 for the UDA sh. In contrast, the UBA and UXA2 sh lines both had approximately 80% survival at 40 days after irradiation.
Advances in Hematology line is generated, it can be used to make transgenic donors with uorochrome-labeled marrow cells. These donors could then be used to perform competitive HSC transplants using immunologically identical donors and recipients. Developing a homozygous diploid sh line from casper sh would be even more useful, as the advantages of analyzing engraftment at many time points could also be realized in the setting of an immune-matched competitive transplant. Eorts are currently underway to generate these sh. 8.2. Minor Histocompatibility Antigens. Further work will also be valuable to identify all the specic class I and II genes important for histocompatibility in the zebrash, both for a basic understanding of zebrash immunology, as well as the implications for optimizing future transplant experiments. When a zebrash mutant has a postulated HSC defect, scientists need to have immune-matched recipients to test whether marrow cells from the mutant zebrash have awed engraftment in a competitive transplantation assay. Without immune matching, such an assay will be dicult to interpret. The ability to immunotype any random zebrash, and thereby select appropriately matched donors and recipients would allow for a much quicker time frame to perform these experiments, compared with generations of inbreeding, which may be unsuccessful given the history of prior attempts to generate such inbred zebrash lines. However, even having a donor with perfect matching at the MHC locus, human bone marrow transplant recipients are still at risk for GVHD, likely due to mismatched minor histocompatibility antigens on other chromosomes. Therefore, identifying both major and minor histocompatibility antigens throughout the genomes that are relevant for transplant rejection and GVHD in the zebrash will be critical to prospectively determine optimally matched donors and recipients. This information will clearly be useful for zebrash experiments, as described above. In addition, identifying signicant minor histocompatibility antigens in the zebrash would suggest minor histocompatibility antigens that may also be relevant for human bone marrow transplantation and GVHD. Such work may impact the selection of human bone marrow transplant donors to minimize this potentially devastating outcome after human BMT. 8.3. Developing a Zebrash Model for GVHD. Finally, in the process of fully characterizing the zebrash histocompatibility genes, we expect to identify recipients with GVHD. To date, we have observed transplant recipients that develop severe edema and ascites resulting in aring of their scales. This condition in the zebrash is generically termed dropsy and likely can result from a myriad of causes. We postulate that in the setting of hematopoietic transplantation, some of these recipient sh may have GVHD, although further work is needed to fully characterize the dropsy phenotype after transplant and conrm the pathophysiology of this diagnosis. By characterizing the GVHD phenotype in zebrash and developing a zebrash model of GVHD, one could exploit the advantages of genetic and small moleculebased screening to further characterize the pathways that
7 regulate GVHD. Such experiments may discern mechanisms to minimize GVHD while maximizing the graft-versusleukemia eect in bone marrow transplant patients.
9. Conclusion
As a model for human disease, the zebrash holds numerous advantages. Gaining knowledge of the functional Class I and II genes in the zebrash will enhance our understanding of basic zebrash biology, as well as the ability to use this versatile animal model to ask questions about tissue transplantation, including hematopoietic stem cells, other normal tissues and cancers cells. This work will likely inform mammalian biology, improving our understanding of human HSCs, and has the potential to impact the treatment of patients undergoing bone marrow transplantation.
Acknowledgment
The authors would like to thank Dr. V. Binder for helpful discussions and for providing the photos in Figure 2. J. L. O. de Jong is supported by grants from the NIH NIDDK (5K08DK074595, 1R03DK091497). L. I. Zon is supported by HHMI, and grants from the NIH NHLBI (5R01HL48801-19) and NIDDK (5RO1DK53298-14).
References
[1] L. Jing and L. I. Zon, Zebrash as a model for normal and malignant hematopoiesis, Disease Models & Mechanisms, vol. 4, no. 4, pp. 433438, 2011. [2] D. Carradice and G. J. Lieschke, Zebrash in hematology: sushi or science? Blood, vol. 111, no. 7, pp. 33313342, 2008. [3] S. H. Orkin and L. I. Zon, Hematopoiesis: an evolving paradigm for stem cell biology, Cell, vol. 132, no. 4, pp. 631644, 2008. [4] D. Traver, B. H. Paw, K. D. Poss, W. T. Penberthy, S. Lin, and L. I. Zon, Transplantation and in vivo imaging of multilineage engraftment in zebrash bloodless mutants, Nature Immunology, vol. 4, no. 12, pp. 12381246, 2003. [5] S. E. Lyons, N. D. Lawson, L. Lei, P. E. Bennett, B. M. Weinstein, and P. P. Liu, A nonsense mutation in zebrash gata1 causes the bloodless phenotype in vlad tepes, Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 8, pp. 54545459, 2002. [6] E. C. Liao, N. S. Trede, D. Ransom, A. Zapata, M. Kieran, and L. I. Zon, Non-cell autonomous requirement for the bloodless gene in primitive hematopoiesis of zebrash, Development, vol. 129, no. 3, pp. 649659, 2002. [7] D. Traver, A. Winzeler, H. M. Stern et al., Eects of lethal irradiation in zebrash and rescue by hematopoietic cell transplantation, Blood, vol. 104, no. 5, pp. 12981305, 2004. [8] A. Medvinsky and E. Dzierzak, Denitive hematopoiesis is autonomously initiated by the AGM region, Cell, vol. 86, no. 6, pp. 897906, 1996. [9] C. E. Burns, T. DeBlasio, Y. Zhou, J. Zhang, L. Zon, and S. D. Nimer, Isolation and characterization of runxa and runxb, zebrash members of the runt family of transcriptional regulators, Experimental Hematology, vol. 30, no. 12, pp. 13811389, 2002.
8
[10] M. L. Kalev-Zylinska, J. A. Horseld, M. V. Flores et al., Runx1 is required for zebrash blood and vessel development and expression of a human RUNX-1-CBF2T1 transgene advances a model for studies of leukemogenesis, Development, vol. 129, no. 8, pp. 20152030, 2002. [11] D. Ma, J. Zhang, H. F. Lin, J. Italiano, and R. I. Handin, The identication and characterization of zebrash hematopoietic stem cells, Blood, vol. 118, no. 2, pp. 289297, 2011. [12] H. F. Lin, D. Traver, H. Zhu et al., Analysis of thrombocyte development in CD41-GFP transgenic zebrash, Blood, vol. 106, no. 12, pp. 38033810, 2005. [13] T. E. North, M. F. de Bruijn, T. Stacy et al., Runx1 expression marks long-term repopulating hematopoietic stem cells in the midgestation mouse embryo, Immunity, vol. 16, no. 5, pp. 661672, 2002. [14] J. Y. Bertrand, A. D. Kim, S. Teng, and D. Traver, CD41+ cmyb+ precursors colonize the zebrash pronephros by a novel migration route to initiate adult hematopoiesis, Development, vol. 135, no. 10, pp. 18531862, 2008. [15] R. M. White, A. Sessa, C. Burke et al., Transparent adult zebrash as a tool for in vivo transplantation analysis, Cell Stem Cell, vol. 2, no. 2, pp. 183189, 2008. [16] M. Shinya and N. Sakai, Generation of highly homogeneous strains of zebrash through full sib-pair mating, Genes Genomes Genetics, vol. 1, no. 5, pp. 377386, 2011. [17] J. Chinen and R. H. Buckley, Transplantation immunology: solid organ and bone marrow, Journal of Allergy and Clinical Immunology, vol. 125, no. 2, supplement 2, pp. S324S335, 2010. [18] C. E. Willett, A. Cortes, A. Zuasti, and A. G. Zapata, Early hematopoiesis and developing lymphoid organs in the zebrash, Developmental Dynamics, vol. 214, no. 4, pp. 323336, 1999. [19] S. H. Lam, H. L. Chua, Z. Gong, T. J. Lam, and Y. M. Sin, Development and maturation of the immune system in zebrash, Danio rerio: a gene expression proling, in situ hybridization and immunological study, Developmental & Comparative Immunology, vol. 28, no. 1, pp. 928, 2004. [20] D. M. Langenau and L. I. Zon, The zebrash: a new model of T-cell and thymic development, Nature Reviews Immunology, vol. 5, no. 4, pp. 307317, 2005. [21] A. C. Smith, A. R. Raimondi, C. D. Salthouse et al., Highthroughput cell transplantation establishes that tumor-initiating cells are abundant in zebrash T-cell acute lymphoblastic leukemia, Blood, vol. 115, no. 16, pp. 32963303, 2010. [22] M. E. Flowers, Y. Inamoto, P. A. Carpenter et al., Comparative analysis of risk factors for acute graft-versus-host disease and for chronic graft-versus-host disease according to National Institutes of Health consensus criteria, Blood, vol. 117, no. 11, pp. 32143219, 2011. [23] L. E. Purton and D. T. Scadden, Limiting factors in murine hematopoietic stem cell assays, Cell Stem Cell, vol. 1, no. 3, pp. 263270, 2007. [24] C. Taswell, Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis, Journal of Immunology, vol. 126, no. 4, pp. 16141619, 1981. [25] S. Beck, D. Geraghty, H. Inoko, and L. Rowen, Complete sequence and gene map of a human major histocompatibility complex. The MHC sequencing consortium, Nature, vol. 401, no. 6756, pp. 921923, 1999. [26] N. Kuroda, F. Figueroa, C. OhUigin, and J. Klein, Evidence that the separation of Mhc class II from class I loci in the zebrash, Danio rerio, occurred by translocation, Immunogenetics, vol. 54, no. 6, pp. 418430, 2002.
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[27] B. W. Murray, H. S ultmann, and J. Klein, Analysis of a 26-kb region linked to the Mhc in zebrash: genomic organization of the proteasome component /transporter associated with antigen processing-2 gene cluster and identication of ve new proteasome subunit genes, Journal of Immunology, vol. 163, no. 5, pp. 26572666, 1999. [28] J. Neefjes, M. L. Jongsma, P. Paul, and O. Bakke, Towards a systems understanding of MHC class I and MHC class II antigen presentation, Nature Reviews Immunology, vol. 11, no. 12, pp. 823836, 2011. [29] J. Bingulac-Popovic, F. Figueroa, A. Sato et al., Mapping of mhc class I and class II regions to dierent linkage groups in the zebrash, Danio rerio, Immunogenetics, vol. 46, no. 2, pp. 129134, 1997. [30] V. Michalov a, B. W. Murray, H. S ultmann, and J. Klein, A contig map of the mhc class I genomic region in the zebrash reveals ancient synteny, Journal of Immunology, vol. 164, no. 10, pp. 52965305, 2000. [31] J. G. Sambrook, F. Figueroa, and S. Beck, A genome-wide survey of major histocompatibility complex (MHC) genes and their paralogues in zebrash, BMC Genomics, vol. 6, article 152, 2005. [32] H. Sultmann, W. E. Mayer, F. Figueroa, C. OhUigin, and J. Klein, Organization of mhc class II B genes in the zebrash (Brachydanio rerio), Genomics, vol. 23, no. 1, pp. 114, 1994. [33] C. P. Kruiswijk, T. T. Hermsen, A. H. Westphal, H. F. J. Savelkoul, and R. J. M. Stet, A novel functional class I lineage in zebrash (Danio rerio), carp (Cyprinus carpio), and large barbus (Barbus intermedius) showing an unusual conservation of the peptide binding domains, Journal of Immunology, vol. 169, no. 4, pp. 19361947, 2002. [34] J. M. Dijkstra, T. Katagiri, K. Hosomichi et al., A third broad lineage of major histocompatibility complex (MHC) class I in teleost sh; MHC class II linkage and processed genes, Immunogenetics, vol. 59, no. 4, pp. 305321, 2007. [35] J. L. de Jong, C. E. Burns, A. T. Chen et al., Characterization of immune-matched hematopoietic transplantation in zebrash, Blood, vol. 117, no. 16, pp. 42344242, 2011. [36] I. V. Mizgirev and S. Revskoy, A new zebrash model for experimental leukemia therapy, Cancer Biology and Therapy, vol. 9, no. 11, pp. 895902, 2010. [37] I. V. Mizgireuv and S. Y. Revskoy, Transplantable tumor lines generated in clonal zebrash, Cancer Research, vol. 66, no. 6, pp. 31203125, 2006. [38] I. Mizgirev and S. Revskoy, Generation of clonal zebrash lines and transplantable hepatic tumors, Nature Protocols, vol. 5, no. 3, pp. 383394, 2010. [39] G. Streisinger, C. Walker, N. Dower, D. Knauber, and F. Singer, Production of clones of homozygous diploid zebra sh (Brachydanio rerio), Nature, vol. 291, no. 5813, pp. 293296, 1981.
Hindawi Publishing Corporation Advances in Hematology Volume 2012, Article ID 857058, 9 pages doi:10.1155/2012/857058
1. Introduction
Hemostasis is a defense mechanism to prevent loss of blood in the event of an injury in an organism that has a vasculature [1]. It consists of the platelet response to injury which results in platelet aggregation and plugging the wound, termed primary hemostasis, followed by the interplay of a complex cascade of coagulation factors on the platelet surface ultimately resulting in a brin clot, termed secondary hemostasis. After their primary hemostatic function platelets, also repair the damaged endothelium [2]. In primary hemostasis platelets adhere to collagen in the subendothelial matrix in response to injury and are subsequently activated by a complex signaling cascade resulting in secretion of their granular contents. These contents also result in the amplication of platelet aggregation at the site of injury and formation of a platelet plug which is stabilized further with help of brin [1]. This hemostatic plug prevents loss of blood from the site of injury. Thus, platelets that play a role in hemostasis and defects in platelet function have been shown to be involved in bleeding disorders as well as many pathophysiological conditions like thrombosis, inammation, and even cancer
[3]. Platelets have a number of receptors on their membrane surface that help regulate signaling pathways in platelets. A substantial amount of research has been done in studying platelet development and function mostly using human platelets [24] murine models [4], and identication of a number of factors and their roles in platelet function [2 4]. Recently, to identify novel factors involved in platelet function, N-ethyl-N-nitrosourea (ENU) mutagenesis and genomic screens of genes aecting platelet development and function have been attempted in mice [5]. However, they are expensive, less ecient, and have lower throughput. In humans, several novel quantitative trait loci associated with platelet-signaling pathways have been identied: however, these studies require additional functional evaluation using either animal models or human subjects [6]. Thus, study of platelet function requires a model system that is ecient, less costly, and amenable to higher-throughput screen, with hemostatic pathways similar to those found in humans [7]. The hemostatic system of invertebrates diers from that of vertebrates and therefore cannot be used as a model organism to study hemostasis [8]. In this regard, we wondered whether Danio rerio (Zebrash) previously used as a genetic
2 model to study developmental biology could be used as a genetic model to study hemostasis especially platelet biology [1]. Its high fecundity, external fertilization, transparency at early stages of development, and availability of large-scale mutagenesis methods are some of the features that make it a useful model system, thus attracting our attention [9, 10]. However, the challenge was to prove whether zebrash thrombocytes and their functional pathways are similar to those found in platelets. For this, characterization of thrombocytes and their functional pathways was required as well as technology suitable for large-scale screens. Therefore, we developed the required technologies ourselves and found them sucient enough to warrant their utility for the study of hemostatic function. Recently, several groups utilized our zebrash model to study hemostasis and discovered several factors regulating hemostasis [11]. This paper provides an overview on the zebrash thrombocyte characterization and development as well as other advances made not only in our laboratory but also from other laboratories which have applied the knowledge and technology that we developed in studying thrombocyte biology.
Advances in Hematology time of laser injury (TTO), time to attachment of rst cell from the time of laser injury (TTA) and also time taken to dissolution of the aggregate (TTD). Several reviews regarding the development of the zebrash model for the study of thrombocyte function using laser-induced thrombosis assays from our laboratory are available [1821].
4. Thrombocyte Microparticles
Platelet microparticles are the microvesicles released by platelets upon activation and have been shown to be involved in thrombin generation [25]. These are 0.11.0 m in diameter and posses most receptors found on platelets
Advances in Hematology
(a)
(b)
Figure 1: Zebrash thrombocyte electron micrographs. (a) Zebrash thrombocyte. Open canalicular like system is shown by arrowhead; N: nucleus; (b) An activated thrombocyte. Thrombocyte in an aggregation reaction; activated thrombocyte is shown by a thick arrow, thrombocyte in the aggregate shows lopodia shown by a thin arrow; E: erythrocyte [12].
pathological conditions including meningococcal sepsis [30], disseminated intravascular coagulation [31], and myocardial infarction [32]. We recently identied thrombocyte microparticles in zebrash and determined that they possess the membrane protein IIb, which is also found in thrombocytes. Positive labeling of zebrash microparticles with FITC annexin V suggests that microparticles could be a result of thrombocyte apoptosis [33]. To elucidate the role of microparticles in hemostasis, Kim et al. used CD41-GFP labeled zebrash and studied microparticle aggregation/agglutination in the presence of dierent agonists. Thrombin, ADP, and collagen did not aggregate thrombocyte microparticles; however, ristocetin induced agglutination in microparticles derived from thrombocytes as well as non-thrombocytes, suggesting that the agglutination is dependent on vWF. During laser injury, we have shown that the thrombocyte microparticles are the rst players to arrive at the site of injury (Figure 3), even before the young thrombocytes [33].
(a) (b) (c)
Figure 2: Young and mature thrombocytes forming independent clusters in an aggregation reaction. Top to bottom, the panels show four dierent thrombocyte clusters. (a) bright eld image; (b) DiIlabeled thrombocytes and mepacrine-labeled thrombocytes as green or orange; (c) DiI-labeled thrombocytes [23].
such as P-selectin, GPIb, and IIb3 [26]. Microparticle formation from platelets is believed to occur when the asymmetry of the membrane phospholipid is lost and phophotidylserine is externalized [27, 28]. Platelet derived microparticles are thought to promote platelet interaction with subendothelial matrix in an IIb3-dependent manner [29]. Elevated levels of microparticles are observed in many
Advances in Hematology
SEM M L Y MP EC
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SEM M L Y MP EC E
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SEM M L Y MP YC
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SEM M L MC Y MP YC
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SEM M L MC Y MP YC
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Figure 3: Schematic representation of sequential steps in growing arterial thrombus. Panels (a) through (e) show the sequence of events in thrombus growth. Arrowhead shows the site of laser injury in (a), (b) shows initiation of thrombus with the formation of microparticle (MP) clusters (MPC) followed by young thrombocyte (Y) clusters (YC) shown in (c) and then followed by a mixture of mature thrombocyte (M) clusters (MC) and YC as shown in (d) and (e) EC indicates endothelial cell; SE, subendothelial matrix; (e) erythrocytes; L, leukocytes. Arrows show the direction of blood ow.
conduct forward genetic screens for hemostatic function. Another mutant which has relevance to thrombocyte function is the fade out mutant which reiterates several aspects of Hermansky-Pudlak syndrome [34]. Furthermore, in the large-scale genome TILLING project spearheaded by Sanger Institute, several mutations in genes related to thrombocyte function were found. However, these will have to be sorted out and their functional evaluation performed in the near future. We have also applied the knockdown technology developed by Ekker and his coworkers to study hemostatic function [35]. We used knockdown of clotting factors to establish the proof of principle and suggested that we could study the thrombocyte functions by knockdowns [1, 17, 36]. Knockdowns of thrombocyte-specic genes selected by microarray RNA analysis has resulted in identifying four genes (acvr1, ift122, poldip2 and ripk5) all of whose deciencies, in addition to other abnormalities, gave either a
hemorrhagic phenotype or prolongation of TTO phenotype [37, 38]. Since then, several knockdowns aecting thrombocyte function have appeared (see Table 1). Schulte-Merker and his group silenced myosin light chain kinase gene mlck1a that is expressed in thrombocytes by knockdown and found this gene is important in thrombus formation [39]. By using knockdowns and our zebrash thrombosis model, OConnor et al. have identied four novel genes (bambi, lrrc32, dcbld2 and esam) involved in platelet function [11]. These genes were selected from comparative transcript analysis of platelets and megakaryocytes together with nucleated blood cells, endothelial cells and erythroblasts. In this work, they used CD41-GFP zebrash to estimate thrombocyte aggregation during arterial thrombosis by measuring thrombocyte surface area (TSA) which essentially provides similar information as the TTO assay. Another group has also used the zebrash model to decipher the role of prkca (PKC) and prkcb (PKC) genes in thrombocyte function; by knockdown
Advances in Hematology
Table 1: Summary of the silencing of genes by knockdown methods aecting thrombocyte formation. Gene acvr1 ift122 poldip2 ripk5 mlck1a bambi lrrc32 dcbld2 esam prkca (PKC) prkcb (PKC) itga2b (CD41) scl c-mpl runx1 miR-126/c-myb fog1 mastl march2 max smox pttg11p emilin1 sufu arhgef3 ak3 rnf45 jmjd1c tpma nbeal2 rgs18 Functional evaluation Laser thrombosis Laser thrombosis Laser thrombosis Laser thrombosis Laser thrombosis Laser thrombosis Laser thrombosis Laser thrombosis Laser thrombosis Laser thrombosis Laser thrombosis Laser thrombosis/thrombocyte aggregation assays Thrombocyte formation Thrombocyte formation Whole mount in situ hybridization/immunostaining Thrombocyte formation Thrombocyte formation Thrombocyte formation Thrombocyte formation Thrombocyte formation Thrombocyte formation Thrombocyte formation Thrombocyte formation Thrombocyte formation Thrombocyte formation Thrombocyte formation Thrombocyte formation Thrombocyte formation Thrombocyte formation Thrombocyte formation Thrombocyte formation Phenotype Hemorrhagic/Prolonged TTO Hemorrhagic/Prolonged TTO Hemorrhagic/Prolonged TTO Hemorrhagic/Prolonged TTO Prolonged TTO Prolonged TTA/reduced thrombus surface area Prolonged TTA/Reduced TSA Increased TSA Increased thrombus size Reduced TSA Reduced TSA Reduced TSA/Prolonged TTO/no aggregation of thrombocytes Reduction in GFP+ cells in CD41-GFP transgenic zebrash line Reduction in GFP+ cells in CD41-GFP transgenic zebrash line Accumulation of hematopoietic progenitors Decrease in CD41 : EGFP+ thrombocytes in a double transgenic reporter line Tg (cd41 : EGFP) : Tg (gata1 : dsRed) Failure to generate eGFP+ cells in CD41-GFP transgenic zebrash line Reduction in GFP+ cells in CD41-GFP transgenic zebrash line Reduction in GFP+ cells in CD41-GFP transgenic zebrash line Reduction in GFP+ cells in CD41-GFP transgenic zebrash line Reduction in GFP+ cells in CD41-GFP transgenic zebrash line Reduction in GFP+ cells in CD41-GFP transgenic zebrash line Reduction in GFP+ cells in CD41-GFP transgenic zebrash line Reduction in GFP+ cells in CD41-GFP transgenic zebrash line Absence of GFP+ cells in CD41-GFP transgenic zebrash line Absence of GFP+ cells in CD41-GFP transgenic zebrash line Absence of GFP+ cells in CD41-GFP transgenic zebrash line Absence of GFP+ cells in CD41-GFP transgenic zebrash line Absence of GFP+ cells in CD41-GFP transgenic zebrash line Abrogation of thrombocyte formation Thrombocytopenia
Reference [36, 37] [36, 37] [36, 37] [36, 37] [39] [11] [11] [11] [11] [40] [40] [11, 41] [48] [48] [52] [55] [56] [58] [59] [59] [59] [59] [59] [59] [60] [60] [60] [60] [60] [61] [62]
expression of these genes, they showed that knockdown with either morpholino leads to attenuated thrombus formation [40]. This group has also used the TSA method but in their recent review they suggested that the manual TSA measurements may be time consuming and may not be accurate when using uorescence measurements in CD41GFP larvae although OConnor et al. have calculated TSA for every minute of the time course and eectively used this method in their work [11, 41]. Although the knockdown methods combined with the laser-induced thrombosis method have the ability to demonstrate the function of the gene that plays a role in thrombosis, biochemical studies on thrombocytes cannot be performed because there is no way to study thrombocyte function by collecting blood samples from the larvae. In order to study the pathways involved in thrombocyte signaling, a
knockdown in adult zebrash was needed. Therefore, we used Vivo morpholino and created an adult Glanzmanns thrombasthenia phenotype by knockdown of the itga2b (CD41) gene [42]. With this advancement, it is now possible to study the biochemistry of thrombocytes after knockdown since we have already developed blood collection methods, thrombocyte assays and thrombocyte separation methods [14, 43, 44].
6 called intermediate cell mass (ICM) where macrophages and erythrocytes are generated, resulting in primitive hematopoiesis. The third and fourth waves are called denitive hematopoiesis and produce erythromyeloid progenitors and hematopoietic stem cells (HSCs), respectively. The third wave may start as early as 24 hpf but peaks at around 30 hpf in caudal hematopoietic tissue (CHT) also called the posterior blood island. The fourth wave starting at 32 36 hpf occurs within endothelial cells of the ventral wall of the dorsal aorta, comparable to mammalian aorta-gonadmesonephros (AGM). The HSCs produced from the fourth wave colonize the CHT and adult hematopoietic organs, the kidney and thymus (Figure 4). Unfortunately, at the time we began our studies with zebrash thrombocytes zebrash thrombopoiesis, received little attention due to the lack of labeling of zebrash thrombocytes and the inability to follow their development. Therefore, we took advantage of labeling of circulating thrombocytes in vivo by intravital microscopy in order to test when thrombocytes appear in the circulation during development. We found by DiI labeling, which specifically labels thrombocytes, that thrombocytes were present in the circulation around 36 hpf, almost coinciding with the fourth wave of hematopoiesis that occurs within the ventral wall of dorsal aorta suggesting precursors for thrombocytes must exist prior to 36 hpf [22]. Subsequently, Handins laboratory developed a transgenic zebrash (CD41-GFP zebrash) where they used the CD41 gene promoter to drive GFP expression. In this line they found green uorescent cells owing in the blood stream around 48 hpf; after this observation they asked us to test whether these cells aggregate using our thrombosis and thrombocyte aggregation assays. When we performed aggregation assays and laser injury thrombosis assays, a green uorescent aggregate formed, establishing that Handins green uorescent cells were in fact thrombocytes [47]. Furthermore, it also provided the possibility of quantifying the intensity of the thrombocyte aggregates. Subsequently, knockdown of transcription factor gene scl and the receptor for a cytokine thrombopoietin cmpl gene resulted in reduction of GFP-labeled thrombocytes, suggesting the presence of C-mpl receptor on zebrash thrombocytes [48]. C-mpl receptor mRNA was shown to be present in the thrombocytes as early as 42 hpf [49]. Using this transgenic line, Lin et al. determined that the GFP+ thrombocytes were not present in the ICM and, therefore, are not part of primitive hematopoiesis [48]. However, they found nonmobile GFP+ thrombocytes between the dorsal aorta and caudal vein at 40 and 48 hpf that they suggested to correspond to the AGM although not having classical AGM features. The circulatory GFP+ thrombocytes appeared rst at 48 hpf [48]. FACS analysis of the GFP+ cells from mesonephros detected two distinct populations: one with bright uorescence (GFPHigh ), considered to be welldierentiated with typical thrombocyte morphology (scant cytoplasm and spindle shape), and the other with weak uorescence (GFPLow ) and larger than GFPHigh thrombocytes with undierentiated morphology (round) and basophilic cytoplasm. Further studies by Kissa and coworkers revealed that the GFPLow cells appeared rst at 33 to 35 hpf as single cells between the dorsal aorta and the postcardinal vein
Advances in Hematology
DA AGM AV YE
CHT
Figure 4: Schematic representation of thrombocyte development in zebrash larva. DA, dorsal aorta; AV, axial vein; AGM, area corresponding to mammalian aorta- gonad- mesonephros; CHT, caudal hematopoietic tissue; K, kidney; Y, yolk; YE, yolk extension; lled small circles and ovals represent GFPLow and GFPHigh thrombocytes, respectively. The yellow and blue lines with arrows correspond to the routes of immigration of the thrombocytes. Thymus is not shown. Black circle and outline show the eye and the zebrash body, respectively.
and they migrate subsequently to CHT and thymus via the axial vein rather than dorsal aorta [50]. Bertrand and his colleagues rened these studies and found that these cells appear as early as 27 hpf in the trunk randomly between axial vessels and conrmed their migration to CHT, thymus and pronephros along with the nding of their migration along the pronephric tubules [49]. These immigrants to kidney supposedly initiate adult hematopoiesis in the developing kidney. They also found migration of the GFPLow cells between axial vessels and pronephric ducts and back to vessels. A recent study from Handins laboratory revealed that the GFPLow cells injected into irradiated adult zebrash showed production of GFP+ cells in kidneys by long term multilineage reconstitution, suggesting that they have the features of HSCs while GFPHigh cells did not reconstitute [51]. 6.2. Identication of Factors Aecting Thrombocyte Development. Several transcription factors such as Fli-1, Fog1, GATA-1 (Zg1), NFE2, and Runx1 which have been found in megakaryocytes have also been identied in zebrash [46]. runx1 morpholino injected zebrash embryos lack a normal circulation and accumulate immature hematopoietic progenitors [52]. The CD41-GFP cells were also found to express Runx1. Using CD41-GFP zebrash, the truncated Runx1 developed normal CD41+ HSCs, indicating there is a Runx1-independent secondary pathway to generate HSCs [53]. Another factor, c-Myb a negative regular of megakaryocytopoiesis, has been identied in zebrash [54]. Functional knockdown of miR-126, a key regulator of c-myb in zebrash, resulted in an increase in erythrocytes and a decrease in thrombocytes, proving that the cell fate decision is regulated by the micro RNA [55]. Yet another factor Fog1, a cofactor that interacts with GATA-1 and GATA-2 has been shown to play a role in erythroid and megakaryocyte dierentiation [56]. fog1 morpholino injected in CD41-GFP zebrash embryos failed to generate GFP+ mature thrombocytes suggesting that Fog1 is necessary for thrombocyte development [57]. Recently, thrombocyte maturation in the circulation has been studied in adult zebrash, revealing that
Advances in Hematology the gata1 promoter becomes weaker and i1 promoter gets stronger in mature thrombocytes and is conversely regulated in young thrombocytes [24]. In addition to these studies, a transient knockdown of mastl in zebrash resulted in deciency of circulating thrombocytes [58]. More recently, knockdowns of genes, march2, max, smox, pttg1lp, emilin1, and sufu resulted in a severe decrease in the number of thrombocytes indicating that these genes are important for thrombocyte development [59]. These genes were selected for knockdowns by genomewide analysis studies (GWAS) for genes adjacent to binding sites for GATA-1, GATA-2, Runx1, Fli-1, and SCL using primary human cells. Another study by Gieger et al. used meta-analyses of GWAS for mean platelet volume and platelet count and identied 68 genomic loci and from these loci four genes (arhgef3, ak3, rnf145, and jmjd1c) were silenced in zebrash which led to the ablation of both primitive erythropoiesis and thrombocyte formation. Silencing of tpma, the orthologue of tpm1 transcribed in megakaryocytes but not in other blood cells, abolished the formation of thrombocytes, but not erythrocytes [60]. In addition to these ndings, silencing of nbeal2 and rgs18 in zebrash resulted in reduction in thrombocyte formation [61, 62]. The silencing of genes by knockdown methods aecting thrombocyte formation is summarized in Table 1.
7 aggregation/adhesion functional assays, and determination of their mechanism of action will all be within reach in the next decade.
References
[1] P. Jagadeeswaran, M. Gregory, K. Day, M. Cykowski, and B. Thattaliyath, Zebrash: a genetic model for hemostasis and thrombosis, Journal of Thrombosis and Haemostasis, vol. 3, no. 1, pp. 4653, 2005. [2] G. Dav` and C. Patrono, Mechanisms of disease: platelet activation and atherothrombosis, The New England Journal of Medicine, vol. 357, no. 24, pp. 24822494, 2007. [3] P. Harrison, Platelet function analysis, Blood Reviews, vol. 19, no. 2, pp. 111123, 2005. [4] R. J. Westrick, M. E. Winn, and D. T. Eitzman, Murine models of vascular thrombosis (Eitzman series), Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 27, no. 10, pp. 2079 2093, 2007. [5] K. D. Mason, M. R. Carpinelli, J. I. Fletcher et al., Programmed anuclear cell death delimits platelet life span, Cell, vol. 128, no. 6, pp. 11731186, 2007. [6] C. I. Jones, S. Bray, S. F. Garner et al., A functional genomics approach reveals novel quantitative trait loci associated with platelet signaling pathways, Blood, vol. 114, no. 7, pp. 1405 1416, 2009. [7] T. Thijs, H. Deckmyn, and K. Broos, Model systems of genetically modied platelets, Blood, vol. 119, no. 7, pp. 1634 1642, 2012. [8] D. Carradice and G. J. Lieschke, Zebrash in hematology: sushi or science? Blood, vol. 111, no. 7, pp. 33313342, 2008. [9] L. Solnica-Krezel, A. F. Schier, and W. Driever, Ecient recovery of ENU-induced mutations from the zebrash germline, Genetics, vol. 136, no. 4, pp. 14011420, 1994. [10] G. Streisinger, C. Walker, and N. Dower, Production of clones of homozygous diploid zebra sh (BrachyDanio rerio), Nature, vol. 291, no. 5813, pp. 293296, 1981. [11] M. N. OConnor, I. I. Salles, A. Cvejic et al., Functional genomics in zebrash permits rapid characterization of novel platelet membrane proteins, Blood, vol. 113, no. 19, pp. 4754 4762, 2009. [12] P. Jagadeeswaran, J. P. Sheehan, F. E. Craig, and D. Troyer, Identication and characterization of zebrash thrombocytes, British Journal of Haematology, vol. 107, no. 4, pp. 731 738, 1999. [13] T. Grosser, S. Yusu, E. Cheskis et al., Developmental expression of functional cyclooxygenases in zebrash, Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 12, pp. 84188423, 2002. [14] S. Kim, M. Carrillo, V. Kulkarni, and P. Jagadeeswaran, Evolution of primary hemostasis in early vertebrates, PLoS ONE, vol. 4, no. 12, article e8403, 2009. [15] M. Carrillo, S. Kim, S. K. Rajpurohit, V. Kulkarni, and P. Jagadeeswaran, Zebrash von Willebrand factor, Blood Cells, Molecules, and Diseases, vol. 45, no. 4, pp. 326333, 2010. [16] M. R. Lang, G. Gihr, M. P. Gawaz, and I. M uller, Hemostasis in Danio rerio: is the zebrash a useful model for platelet research? Journal of Thrombosis and Haemostasis, vol. 8, no. 6, pp. 11591169, 2010. [17] M. Gregory, R. Hanumanthaiah, and P. Jagadeeswaran, Genetic analysis of hemostasis and thrombosis using vascular occlusion, Blood Cells, Molecules & Diseases, vol. 29, no. 3, pp. 286295, 2002.
7. Future Studies
Despite the advances in genetic studies of thrombocyte function and development in zebrash, many novel genes involved in thrombocyte origins and functions remain to be identied. For example, even though embryonic GFPLow thrombocytes have been identied as HSCs and their role in repopulating the kidney for initiating the subsequent generation of thrombocytes from HSCs has not yet been investigated. Thus, we have no information regarding genes involved in the production of thrombocyte precursor cells in adult zebrash. Likewise, studies of genes involved in maturation from young to mature thrombocytes, as well as genes controlling the production of thrombocyte microparticles are in the beginning stages. Since our laser-induced thrombosis assays for studying hemostasis have already found applications, we anticipate more such studies of this kind will be performed to assess the role of novel human genes relevant to hemostasis and thrombocyte development and function [35]. Recently developed technologies such as Genome TILLING [63, 64], zinc nger nuclease, or other nuclease/s (TALEN) based knockout methods [6568] are also anticipated to complement the already available methods for studying functions of genes involved in thrombocyte function and production. However, large-scale silencing of genes to study thrombocyte development and production are still prohibitively expensive. Thus, future development of cost-eective gene silencing methodologies is required to attempt a functional genomics approach to analyze thrombocytes using the zebrash model. Once the genes are identied, utilizing Vivo morpholino technology, we predict that characterization of the phenotypes by thrombocyte
8
[18] P. Jagadeeswaran, M. Carrillo, U. P. Radhakrishnan, S. K. Rajpurohit, and S. Kim, Laser-induced thrombosis in Zebrash, Methods in Cell Biology, vol. 101, pp. 197203, 2011. [19] P. Jagadeeswaran, M. Cykowski, and B. Thattaliyath, Vascular occlusion and thrombosis in zebrash, Methods in Cell Biology, vol. 76, pp. 489500, 2004. [20] P. Jagadeeswaran, Y. C. Liu, and J. P. Sheehan, Analysis of hemostasis in the zebrash, Methods in Cell Biology, no. 59, pp. 337357, 1999. [21] P. Jagadeeswaran, R. Paris, and P. Rao, Laser-induced thrombosis in zebrash larvae: a novel genetic screening method for thrombosis, Methods in Molecular Medicine, vol. 129, pp. 187195, 2006. [22] M. Gregory and P. Jagadeeswaran, Selective labeling of zebrash thrombocytes: quantitation of thrombocyte function and detection during development, Blood Cells, Molecules, and Diseases, vol. 28, no. 3, pp. 418427, 2002. [23] B. Thattaliyath, M. Cykowski, and P. Jagadeeswaran, Young thrombocytes initiate the formation of arterial thrombi in zebrash, Blood, vol. 106, no. 1, pp. 118124, 2005. [24] P. Jagadeeswaran, S. Lin, B. Weinstein, A. Hutson, and S. Kim, Loss of GATA1 and gain of FLI1 expression during thrombocyte maturation, Blood Cells, Molecules, and Diseases, vol. 44, no. 3, pp. 175180, 2010. [25] R. J. Berckmans, R. Nieuwland, A. N. B oing, F. P. H. T. M. Romijn, C. E. Hack, and A. Sturk, Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation, Thrombosis and Haemostasis, vol. 85, no. 4, pp. 639646, 2001. [26] D. E. Connor, T. Exner, D. D. F. Ma, and J. E. Joseph, The majority of circulating platelet-derived microparticles fail to bind annexin V, lack phospholipid-dependent procoagulant activity and demonstrate greater expression of glycoprotein Ib, Thrombosis and Haemostasis, vol. 103, no. 5, pp. 1044 1052, 2010. [27] A. Tesse, M. C. Mart nez, F. Meziani et al., Origin and biological signicance of shed-membrane microparticles, Endocrine, Metabolic and Immune Disorders, vol. 6, no. 3, pp. 287294, 2006. [28] M. C. Mart nez, A. Tesse, F. Zobairi, and R. Andriantsitohaina, Shed membrane microparticles from circulating and vascular cells in regulating vascular function, American Journal of Physiology, vol. 288, no. 3, pp. H1004H1009, 2005. [29] M. Merten, R. Pakala, P. Thiagarajan, and C. R. Benedict, Platelet microparticles promote platelet interaction with subendothelial matrix in a glycoprotein IIb/IIIa-dependent mechanism, Circulation, vol. 99, no. 19, pp. 25772582, 1999. [30] R. Nieuwland, R. J. Berckmans, S. McGregor et al., Cellular origin and procoagulant properties of microparticles in meningococcal sepsis, Blood, vol. 95, no. 3, pp. 930935, 2000. [31] P. A. Holme, N. O. Solum, F. Brosstad, M. Roger, and M. Abdelnoor, Demonstration of platelet-derived microvesicles in blood from patients with activated coagulation and brinolysis using a ltration technique and Western blotting, Thrombosis and Haemostasis, vol. 72, no. 5, pp. 666671, 1994. [32] M. Gawaz, F. J. Neumann, I. Ott, A. Schiessler, and A. Sch omig, Platelet function in acute myocardial infarction treated with direct angioplasty, Circulation, vol. 93, no. 2, pp. 229237, 1996. [33] S. Kim, M. Carrillo, U. P. Radhakrishnan, and P. Jagadeeswaran, Role of thrombocyte and non-thrombocyte microparticles in hemostasis, Blood Cells Molecules and Diseases, vol. 48, no. 3, pp. 188196, 2012.
Advances in Hematology
[34] R. Bahadori, O. Rinner, H. B. Schonthaler et al., The zebrash fade out mutant: a novel genetic model for Hermansky-Pudlak syndrome, Investigative Ophthalmology and Visual Science, vol. 47, no. 10, pp. 45234531, 2006. [35] A. Nasevicius and S. C. Ekker, Eective targeted gene knockdown in zebrash, Nature Genetics, vol. 26, no. 2, pp. 216220, 2000. [36] K. Day, N. Krishnegowda, and P. Jagadeeswaran, Knockdown of prothrombin in zebrash, Blood Cells, Molecules, and Diseases, vol. 32, no. 1, pp. 191198, 2004. [37] A. Srivastava et al., The role of acvr1, ift122, poldip2 and ripk5 in zebrash hemostasis, in Proceedings of the International Conference for Zebrash Development and Genetics, p. 577, Madison, Wis,USA, 2008. [38] P. Jagadeeswaran and P. Rao, Role of KIAA0472, a Novel Ser/Thr Kinase in zebrash thrombosis, in Proceedings of the International Conference on Zebrash Development and Genetics, Madison, Wis, USA, 2006. [39] E. Tournoij, G. J. Weber, J. W. N. Akkerman et al., Mlck1a is expressed in zebrash thrombocytes and is an essential component of thrombus formation, Journal of Thrombosis and Haemostasis, vol. 8, no. 3, pp. 588595, 2010. [40] C. M. Williams et al., Protein kinase C alpha and beta are positive regulators of thrombus formation in vivo in a zebrash (Danio rerio) model of thrombosis, Journal of Thrombosis and Haemostasis, vol. 9, no. 12, pp. 24572465, 2011. [41] C. M. Williams and A. W. Poole, Using zebrash (Danio rerio) to assess gene function in thrombus formation, Methods in Molecular Biology, vol. 788, pp. 305319, 2012. [42] S. Kim, U. P. Radhakrishnan, S. K. Rajpurohit, V. Kulkarni, and P. Jagadeeswaran, Vivo-Morpholino knockdown of IIb: a novel approach to inhibit thrombocyte function in adult zebrash, Blood Cells, Molecules, and Diseases, vol. 44, no. 3, pp. 169174, 2010. [43] P. Jagadeeswaran, M. Gregory, S. Johnson, and B. Thankavel, Haemostatic screening and identication of zebrash mutants with coagulation pathway defects: an approach to identifying novel haemostatic genes in man, British Journal of Haematology, vol. 110, no. 4, pp. 946956, 2000. [44] V. Kulkarni et al., Separation of young and mature thrombocytes by a novel immuno-selection method, Blood Cells Molecules and Diseases, vol. 48, no. 3, pp. 183187, 2012. [45] J. F. Amatruda and L. I. Zon, Dissecting hematopoiesis and disease using the zebrash, Developmental Biology, vol. 216, no. 1, pp. 115, 1999. [46] A. J. Davidson and L. I. Zon, The denitive (and primitive) guide to zebrash hematopoiesis, Oncogene, vol. 23, no. 43, pp. 72337246, 2004. [47] H. Lin et al., Production and characterization of transgenic zebrash (Danio rario) with uorescent thrombocytes and thrombocyte precursors, Blood, vol. 98, p. 2147, 2001. [48] H. F. Lin, D. Traver, H. Zhu et al., Analysis of thrombocyte development in CD41-GFP transgenic zebrash, Blood, vol. 106, no. 12, pp. 38033810, 2005. [49] J. Y. Bertrand, A. D. Kim, S. Teng, and D. Traver, CD41+ cmyb+ precursors colonize the zebrash pronephros by a novel migration route to initiate adult hematopoiesis, Development, vol. 135, no. 10, pp. 18531862, 2008. [50] K. Kissa, E. Murayama, A. Zapata et al., Live imaging of emerging hematopoietic stem cells and early thymus colonization, Blood, vol. 111, no. 3, pp. 11471156, 2008.
Advances in Hematology
[51] D. Ma, J. Zhang, H. F. Lin, J. Italiano, and R. I. Handin, The identication and characterization of zebrash hematopoietic stem cells, Blood, vol. 118, no. 2, pp. 289297, 2011. [52] M. L. Kalev-Zylinska, J. A. Horseld, M. V. C. Flores et al., Runx1 is required for zebrash blood and vessel development and expression of a human RUNX-1-CBF2T1 transgene advances a model for studies of leukemogenesis, Development, vol. 129, no. 8, pp. 20152030, 2002. [53] R. Sood, M. A. English, C. L. Belele et al., Development of multilineage adult hematopoiesis in the zebrash with a runx1 truncation mutation, Blood, vol. 115, no. 14, pp. 28062809, 2010. [54] Y. Zhang et al., cMyb regulates hematopoietic stem/progenitor cell mobilization during zebrash hematopoiesis, Blood, vol. 118, no. 15, pp. 40934101, 2011. [55] C. Grabher, E. M. Payne, A. B. Johnston et al., Zebrash microRNA-126 determines hematopoietic cell fate through cMyb, Leukemia, vol. 25, no. 3, pp. 506514, 2011. [56] A. G. Muntean and J. D. Crispino, Dierential requirements for the activation domain and FOG-interaction surface of GATA-1 in megakaryocyte gene expression and development, Blood, vol. 106, no. 4, pp. 12231231, 2005. [57] J. D. Amigo, G. E. Ackermann, J. J. Cope et al., The role and regulation of friend of GATA-1 (FOG-1) during blood development in the zebrash, Blood, vol. 114, no. 21, pp. 46544663, 2009. [58] H. J. Johnson, M. J. Gandhi, E. Shazadeh et al., In vivo inactivation of MASTL kinase results in thrombocytopenia, Experimental Hematology, vol. 37, no. 8, pp. 901908, 2009. [59] M. R. Tijssen, A. Cvejic, A. Joshi et al., Genome-wide analysis of simultaneous GATA1/2, RUNX1, FLI1, and SCL binding in megakaryocytes identies hematopoietic regulators, Developmental Cell, vol. 20, no. 5, pp. 597609, 2011. [60] C. Gieger et al., New gene functions in megakaryopoiesis and platelet formation, Nature, vol. 480, no. 7376, pp. 201208, 2011. [61] C. A. Albers, A. Cvejic, R. Favier et al., Exome sequencing identies NBEAL2 as the causative gene for gray platelet syndrome, Nature Genetics, vol. 43, no. 8, pp. 735737, 2011. [62] S. Louwette, V. Labarque, C. Wittevrongel et al., Regulator of G-protein signaling 18 controls megakaryopoiesis and the cilia-mediated vertebrate mechanosensory system, The FASEB Journal, vol. 26, no. 5, pp. 21252136, 2012. [63] J. Kulinski, D. Besack, C. A. Oleykowski, A. K. Godwin, and A. T. Yeung, CEL I enzymatic mutation detection assay, BioTechniques, vol. 29, no. 1, pp. 4448, 2000. [64] E. Wienholds, F. van Eeden, M. Kosters, J. Mudde, R. H. A. Plasterk, and E. Cuppen, Ecient target-selected mutagenesis in zebrash, Genome Research, vol. 13, no. 12, pp. 2700 2707, 2003. [65] Y. Doyon, J. M. McCammon, J. C. Miller et al., Heritable targeted gene disruption in zebrash using designed zincnger nucleases, Nature Biotechnology, vol. 26, no. 6, pp. 702 708, 2008. [66] S. C. Ekker, Zinc nger-based knockout punches for zebrash genes, Zebrash, vol. 5, no. 2, pp. 121123, 2008. [67] K. J. Clark, D. F. Voytas, and S. C. Ekker, A TALE of two nucleases: gene targeting for the masses? Zebrash, vol. 8, no. 3, pp. 147149, 2011. [68] D. Caroll and B. Zhang, Primer and interviews: advances in targeted gene modication, Developmental Dynamics, vol. 240, no. 12, pp. 268896, 2011.
Hindawi Publishing Corporation Advances in Hematology Volume 2012, Article ID 851674, 12 pages doi:10.1155/2012/851674
Review Article In Vivo Chemical Screening for Modulators of Hematopoiesis and Hematological Diseases
Yiyun Zhang1, 2 and J.-R. Joanna Yeh1, 2
1 Cardiovascular 2 Department
Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA of Medicine, Harvard Medical School, Boston, MA 02115, USA
Correspondence should be addressed to J.-R. Joanna Yeh, [email protected] Received 22 February 2012; Accepted 26 April 2012 Academic Editor: Jason Berman Copyright 2012 Y. Zhang and J.-R. J. Yeh. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In vivo chemical screening is a broadly applicable approach not only for dissecting genetic pathways governing hematopoiesis and hematological diseases, but also for nding critical components in those pathways that may be pharmacologically modulated. Both high-throughput chemical screening and facile detection of blood-cell-related phenotypes are feasible in embryonic/larval zebrash. Two recent studies utilizing phenotypic chemical screens in zebrash have identied several compounds that promote hematopoietic stem cell formation and reverse the hematopoietic phenotypes of a leukemia oncogene, respectively. These studies illustrate ecient drug discovery processes in zebrash and reveal novel biological roles of prostaglandin E2 in hematopoietic and leukemia stem cells. Furthermore, the compounds discovered in zebrash screens have become promising therapeutic candidates against leukemia and included in a clinical trial for enhancing hematopoietic stem cells during hematopoietic cell transplantation.
1. Introduction
Zebrash has been used eectively as a vertebrate model for studying blood cell development and function (for reviews see [15]). It is an advantageous model because the optical clarity of its embryos, and their ex utero development enables easy and real-time detection of hematopoietic cells during development. A wide variety of tools and reagents have been developed for in vivo labeling and imaging of blood cells and for investigating blood cell function (for reviews of these methods and protocols, see [610]). In addition, transient and stable genetic manipulation can link hematopoietic genes to their functions [1116]. Added to this arsenal of research tools available in zebrash is in vivo chemical screening [1720]. By exposing zebrash embryos to a chemical library, bioactive compounds that aect any complex developmental and physiological processes may be identied. Furthermore, in vivo chemical screening may be used for uncovering chemical agents that modify a disease phenotype in a whole animal. The compounds that induce a unique biological eect may serve as invaluable probes for identifying critical components of biological pathways,
and compounds that can reverse a disease phenotype in vivo may have therapeutic potential or shed light on an eective therapeutic target. This innovative approach has created a unique utility for the zebrash model in chemical biology and contributed to its emerging role in drug discovery (for additional reviews see [2124]).
2. Linking Genes to Their Functions: In Vivo Chemical Screens versus Genetic Screens
Both genetic and in vivo chemical screens may be used to dissect genetic pathways that regulate specic biological processes. However, an in vivo chemical screen oers the advantage of temporal control that a traditional genetic screen does not. In a genetic screen, gene function is aected from conception. Thus, the role of a gene in early embryonic development may preclude characterization of its roles during later stages. On the other hand, in a chemical screen, compounds that aect the function of a gene can be administered at specic time points and for xed durations chosen by the investigator so that its roles at dierent developmental stages may
2 be distinctly determined. In addition, in a genetic screen, the roles of a protein family may sometimes be masked by functional redundancy of its family members. However, chemical modulators may exhibit similar activities against multiple members of a protein family and can, therefore, reveal their in vivo cumulative roles. It should be noted that some compounds may aect multiple cellular proteins and thus their on-target eects should be carefully veried using additional chemical agents as well as genetic manipulations. Taken together, in vivo chemical screens may complement traditional genetic approaches and uncover hematopoietic genes that cannot be identied in genetic screens.
Advances in Hematology important advances in analytical research tools including mass spectrometry, proteomics, genomics, metabolomics, expressional proling, and chemical informatics as well as novel in vivo labeling methods, the eciency and success rate of target identication have improved signicantly in recent years [3134]. In addition, in vivo chemical screens are sometimes performed using chemical libraries consisting of known bioactive compounds, so that the signaling pathways mediating a disease phenotype can be uncovered relatively quickly once chemical suppressors of the phenotype are identied.
Advances in Hematology
Step 1
Step 2
1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 12
Step 3
1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 12
Step 4
Step 5
1 A B C D E F G H 2 3 4 5 6 7 8 9 10 11 12
Figure 1: Chemical screening using zebrash embryos. Step 1Wild-type, reporter, or mutant zebrash are crossed to obtain embryos. Step 2Once reaching an investigator-specied developmental stage (usually between 05 days after fertilization), embryos are arrayed into multi-well plates either manually or by automation. Step 3Compounds from a chemical library are added into the wells containing the embryos using a multichannel pipette or a pin-transfer device. Step 4After reaching the developmental stage for phenotype manifestation, which is usually within hours to a couple of days after the compound treatment, embryos may be subjected to staining procedures, reporter, or functional assays to detect chemical-induced phenotypes or reversal of genetic phenotypes. The images shown here depict dierential hematopoietic gene expression between the compound-treated (red circle) and vehicle-treated (black circle) embryos as detected by RNA in situ hybridization. Step 5In vivo phenotypes can be detected by visual inspection or by automated imaging and recording. Thus, the whole screening procedure, once optimized, may be automated for high-throughput experimentation and nished within a few days. In addition, a wide range of phenotypes may be detected in vivo.
be processed by automated liquid handling machines or may be recorded using automated imaging systems and analyzed using customized software [51, 5356]. Thus, conducting chemical screening in zebrash provides great potential for identifying modulators of hematopoiesis and hematological diseases.
4 For example, if a screen is based on the reduction of the signals in a reporter assay, it may be prone to identifying false positives such as toxic compounds. In this case, a quick visual scan of embryo/larva viability before conducting the reporter assay may help exclude those nonspecic hits. In addition, since proper embryonic development depends on precise execution of multiple sequential processes, compounds added at dierent times will have the opportunity to aect dierent developmental steps. Thus, the timing and duration of chemical treatment are also likely to aect the screening outcomes. If a screen utilizes a transgenic line, additional validation steps should be incorporated to examine whether the hit compounds may aect the promoter used for driving the transgene or the stability of the transgene itself. For example, in one of the screens that we have performed, we have identied several hits that suppress the heat shock promoter used for driving the expression of an oncogene rather than the activity of the oncogene [20]. Whenever possible, positive controls should be used to validate that zebrash models exhibit similar molecular machineries and pharmacological responses as humans do (if the screening purpose is drug discovery) for the biological processes under investigation. This conrmation beforehand will facilitate the likelihood of relevantly translating the ndings from zebrash screens to human conditions. In addition, it is important to conduct a pilot screen using 100300 compounds and one screening plate of untreated embryos/larvae to evaluate the robustness and potential variables of the screening methods, including the degrees of natural variations among dierent clutches of embryos/larvae. A pilot screen may also provide information as regard to the potential hit rates. On one hand, in vivo screening methods may cast a broad net for identifying compounds that elicit the phenotype-of-interest through various mechanisms. On the other hand, if the hit rates are higher than 1-2%, researchers may wish to incorporate secondary screening strategies or consider a dierent screening method in order to limit the hits to the ones that are likely to be of potential interest to the investigators. For example, we previously showed that immediately after the expression of the leukemia oncogene AML1-ETO, gata1 expression is abolished, whereas myeloperoxidase (mpo) expression is increased at a later time point [57]. We conducted a chemical suppressor screen and identied various compounds that can restore gata1 expression in the presence of AML1-ETO [20]. We have also veried the therapeutic potential of some of the hits identied in this screen, and these results will be discussed in more detail later. Conceivably, a chemical suppressor screen can also be performed based on the reversal of mpo upregulation in the same zebrash model. The latter screening strategy may not only identify compounds that directly antagonize AML1-ETOs eects but also additional compounds that suppress the accumulation of mpo+ cells through AML1-ETO-unrelated mechanisms. The choices of screening designs are subject to each investigators discretion. 5.2. Hit Evaluation and Translation to Humans. The potency, eectiveness, and specicity of the conrmed hits obtained
Advances in Hematology from zebrash screens have already been demonstrated in vivo. Thus, these hits have a high probability of being eective in other in vivo systems. Both hematopoietic and other nonhematopoietic eects of these candidate compounds should be evaluated further in embryonic/larval zebrash. The eects of the candidate compounds on cell dierentiation, proliferation, or survival can be evaluated using whole-mount RNA in situ hybridization, whole-mount immunostaining or staining with lineage-specic cytological dyes such as Sudan Black for neutrophils and o-dianisidine for hemoglobin. These in vivo eects may be assessed facilely using embryonic/larval zebrash. For example, we have found that AML1-ETO can reprogram hematopoietic cell fate decisions, converting the erythroid cell fate to the granulocytic cell fate. We have also found that nimesulide, a chemical suppressor of AML1-ETO, can reverse these eects in zebrash. AML1-ETO has been shown to suppress erythroid dierentiation in mammalian cells, and we have conrmed that nimesulide can also reverse AML1-ETOs eects in cultured cells [20]. The eects of candidate compounds on leukocyte or thrombocyte function can also be assessed in embryonic/larval zebrash using an injury model for neutrophil chemotaxis, a bacterial infection model for phagocytosis, or a laser-induced coagulation assay [47, 58, 59]. Moreover, lineage-specic hematopoietic cells can be isolated from control and compound-treated embryos/larvae of various uorescent reporter lines mentioned earlier by ow cytometry for transcriptional proling analysis. Interestingly, the nonhematopoietic eects may sometimes provide instrumental information as to the mechanisms of action of the candidate compounds. For example, a candidate compound may cause a developmental phenotype similar to the phenotype caused by other genetic mutations or other chemicals with known bioactivities, suggesting that the candidate compound acts through a similar pathway as these other modulations do. The eects of the candidate compounds can also be evaluated in adult zebrash using standard hematopoietic assays adapted from mouse models, including irradiation followed by hematopoietic cell transplantation and irradiation recovery assays, as well as leukemic cell xenograft and limiting dilution transplantation [37, 6064]. The zebrash provides the investigator the exibility at which point to verify the eects of these compounds in mammalian systems. While the degree of conservation between zebrash and mammals in hematopoiesis and in the functions of many hematopoietic cell lineages is high, conservation of humoral regulators and the adaptive immune system is presently less clear. However, rapid advancement in those areas is anticipated. For those biological processes already shown to be highly conserved, the translatability of the screening hits from zebrash to humans will likely to be high.
Advances in Hematology between zebrash and mammals [72, 73]. Thus, it is a suitable model for investigating the genetic pathways regulating hematopoiesis and hematological diseases. As in mammals, during embryonic development, zebrash rst exhibit a primitive wave of hematopoiesis and later produce several intermediate cell types that eventually contribute to denitive hematopoiesis (for more detailed reviews see [74, 75]). During primitive hematopoiesis, which begins around 11 hours after fertilization (hpf), zebrash embryos produce myeloid and erythroid cells in two anatomically separate locations. Myeloid cells, which express the transcription factor pu.1, are formed in the anterior lateral plate mesoderm (ALM), while erythroid progenitors expressing the gata1 transcription factor are formed in the posterior lateral plate mesoderm (PLM). It has been shown that hematopoietic cell fate in both blood islands is determined by the expression of these two genes. While knockdown of pu.1 induces erythropoiesis in the ALM, knockdown of gata1 promotes myelopoiesis in the PLM [76, 77]. These results indicate that primitive hematopoiesis in embryonic zebrash produces bi-potent hematopoietic progenitor cells. Thus, these two synchronously specied blood populations may be useful for identifying important genes that regulate myeloid and erythroid cell fate determination. In a later section of this paper, we will discuss a study that utilizes these cells to uncover some of the AML1-ETOs oncogenic eects that lead to acute myeloid leukemia [20, 57]. In zebrash, multipotent hematopoietic stem cells (HSCs) originate in the hemogenic endothelium of the aorta, which is equivalent to the aorta-gonad-mesonephros (AGM) in mammals [78]. Using in vivo lineage-tracing experiments, it has been shown that these newly emerged HSCs will subsequently colonize a transient hematopoietic tissue called the caudal hematopoietic tissue (CHT), which may be comparable to another mammalian embryonic hematopoietic site in the fetal liver [7981]. Finally, HSCs from those regions will migrate to and seed both kidney (equivalent to bone marrow in mammals) and thymus, the nal hematopoietic organs that remain through adult life [7981]. As in mammals, zebrash HSCs express runx1 and cmyb, and runx1 deciency abrogates denitive hematopoiesis in sh [78, 8284]. Several major signaling pathways that regulate HSC formation and homeostasis in mouse models also aect HSC formation in zebrash, such as the Hedgehog (Hh) pathway and the Notch-Runx pathway [78, 85]. Recently, an in vivo chemical screen in zebrash has identied important roles of the prostaglandin-E2-(PGE2-) Wnt signaling pathway in HSC formation [19, 86], which will be discussed in more detail later. These ndings suggest that zebrash and mammals utilize similar genetic circuitry for regulating HSC formation. 6.2. Hematological Disease Models. Due to the easiness of inspecting blood cell phenotypes in zebrash embryos, a large number of blood mutants have been isolated in three large-scale genetic screens [11, 14, 87, 88]. Many of these blood mutants have defects in the maturation or iron transport of erythrocytes, and their related phenotypes and orthologous gene mutations have been dened in humans
5 [8991]. Transgenesis approaches have also been used to create various hematological disease models in zebrash, of which the majority are blood cancer models [20, 38, 57, 92 96]. In these studies, ectopic expression of human oncogenes resulted in zebrash phenotypes reminiscent of human leukemia characteristics. In addition, investigators can now perform ecient targeted gene disruption in zebrash using engineered zinc nger nucleases (ZFNs) and transcription activator-like eector (TALE) nucleases [13, 16, 97100]. In the future, many of these hematological disease models may be used for chemical suppressor screens. The vast array of research tools available in the zebrash model combined with in vivo chemical screening will prove useful in providing novel insights into the molecular mechanisms and potential therapy for hematological diseases.
6 niche. Furthermore, Goessling et al. showed that PGE2 promotes HSC formation by activating the Wnt/-catenin signaling pathway [86]. In their screen, North et al. also found 22 compounds that might regulate HSC formation through their eects on blood ow, such as compounds aecting - and -adrenergic receptors, Ca2+ or Na+ /K+ channels, nitric oxide (NO) synthesis, or the angiotensin pathway [103]. They showed that blood ow had a positive impact on cmyb/runx1 expression, suggesting that the hemodynamic force on the endothelium might be an inducing factor for the emergence of HSCs. In addition, the authors found that NO donors could stimulate HSC formation even in the silent heart mutant, which does not exhibit blood ow. Using mosaic transplantation experiments, they discovered that NO positively regulated HSC through cell-autonomous signaling.
Advances in Hematology vascular niche [110]. Since hCB contains both HSCs and endothelial cells, the authors postulated that dmPGE2 might promote HSC formation from hemogenic endothelial cells, analogous to the scenario in developing zebrash embryos. Alternatively, Butler et al. have shown that endothelial cells can provide signals for retaining HSC multipotency [111]. Finally, Goessling et al. provided evidence demonstrating preclinical safety of their regimen in nonhuman primate autologous transplantation [110]. Thus, from its initial discovery using an in vivo chemical screen in zebrash, PGE2 is now entering a Phase I clinical trial.
Advances in Hematology and the upregulation of pu.1 in multipotent hematopoietic progenitors, suggesting a conversion of erythroid to myeloid cell fate. Moreover, the accumulated hematopoietic cells strongly expressed the myeloperoxidase (mpo) gene, indicative of a granulocytic cell fate. A previous study had shown that AML1-ETO downregulates c/ebp, resulting in a maturation block of the granulocytic cells in human t(8; 21) AML [122]. In the zebrash model, we also observed a dramatic reduction of c/ebp expression, suggesting that only two days after its expression in zebrash embryos, AML1ETO induced an accumulation of granulocytic blast cells resembling the clinical features of human t(8; 21) AML. 9.3. Chemical Screening in the Zebrash Model of AML-ETO. A library of 2,000 bioactive compounds was screened using the Tg(hsp:AML1-ETO) zebrash model [20]. The screening compounds were added to embryos at 1216 hpf, followed by 1 hour of heat treatment to induce AML1-ETO expression. Fifteen hit compounds were identied by restored gata1 expression in the transgenic embryos as measured by RNA in situ hybridization. We found that some of the compounds aected the heat shock response in zebrash, preventing AML1-ETO expression. In addition, we identied a histone deacetylase (HDAC) inhibitor sodium valproate as a chemical suppressor of AML1-ETOs eects. HDAC is a transcription corepressor that is known to interact with the ETO moiety of the AML1-ETO protein [123]. It has been shown that recruitment of HDAC is critical for AEs function, and that an HDAC inhibitor trichostatin A (TSA) induces dierentiation and apoptosis of a t(8; 21) AML cell line [124]. We have shown previously that TSA also reversed the hematopoietic phenotype of Tg(hsp:AML1-ETO) zebrash [57]; therefore, the discovery of sodium valproate validated the biological relevance of the chemical screen performed on the AML1-ETO zebrash model. Interestingly, nimesulide, a selective COX-2 inhibitor, was also identied in this screen [20]. Subsequently, we showed that treatments with indomethacin (a nonselective COX inhibitor), NS-398 (a selective COX-2 inhibitor), and nimesulide not only restored gata1 expression but also inhibited increased expression of mpo in the transgenic embryos. Furthermore, we demonstrated that these drugs eects were on target because they could be reversed by supplementing a downstream metabolite PGE2. Thus, the hematopoietic dierentiation defects induced by AML1-ETO in vivo can be rescued by inhibiting the COX enzymes.
7 colorectal carcinoma and breast cancers [125, 126]. Moreover, PGE2 had been shown to promote colon cancer cell growth via a -catenin-dependent signaling pathway [127, 128]. As in zebrash, we found that AML1-ETO induced ptgs2 but not ptgs1 expression in the K562 human myeloid leukemia cell line [20]. AML1-ETO induced the activity of a -catenin reporter and inhibited erythroid dierentiation in these cells, and both eects could be abrogated by NS398. Subsequently, we found that genetic knockdown of catenin rescued AML1-ETOs eects in zebrash embryos [20]. Thus, AML1-ETO aects hematopoietic dierentiation through the COX-2/-catenin pathway in both zebrash and human leukemia cells. Since the publication of these ndings, we have obtained strong evidence indicating that AML1-ETO also signals through a COX-2/-catenin pathway in mouse bone marrow cells (Zhang et al., unpublished results). We have found that COX inhibitors can eectively suppress in vitro serial replating of hematopoietic stem/progenitor cells expressing AML1-ETO as well as AML1-ETO-mediated tumorigenesis in various in vivo mouse models (Zhang et al., unpublished results). Two recent studies have also explored the roles of the COX enzymes and -catenin in leukemia stem cells expressing other leukemia oncogenes [129, 130]. In one of the studies, Wang et al. showed that either the MLL-AF9 fusion oncoprotein or coexpression of Hoxa9 and Meis1a could induce ptgs1 expression and -catenin activation. In addition, inhibiting COX activities using indomethacin attenuated leukemia development induced by MLL-AF9 or by coexpression of Hoxa9 and Meis1a oncogenes [129]. In the other study, Steinert et al. found that a nonselective COX inhibitor sulindac prevented -catenin from being activated and reduced in vivo growth of HSCs expressing PML/RAR or PLZF/RAR oncogenes [130]. Collectively, these results suggest that inhibiting the COX enzymes using nonsteroidal anti-inammatory drugs (NSAIDs) can suppress oncogenic function and -catenin activation in AML leukemia stem cells. Interestingly, casebased studies have also suggested an inverse relationship between NSAID usage and AML incidence [131, 132]. Although PGE2 can induce -catenin expression and augment some aspects of HSC function as discussed above, it has been shown that loss of -catenin does not aect normal hematopoiesis in adult mice [133136]. At present, a major obstacle for achieving long-term survival of AML patients is relapse. Although chemotherapy can eectively induce remission in the majority of patients, more than 50% of the patients experience relapse within a year after remission [137, 138]. In sum, these results suggest that NSAIDs may impair leukemia stem cell function and thus their clinical ecacy in preventing AML relapse should be explored.
8 screening in discovering potential new therapeutics. In these cases, the use of an in vivo screening platform allowed the identication of compounds that may act in a noncell autonomous fashion such as hemodynamic forces, bypassed the well-known technical diculties involved in culturing hematopoietic or leukemia stem cells, and also circumvented the obstacles conferred by undruggable targets or unknown disease mechanisms. Both of the studies uncovered novel biological mechanisms as well as strong candidates for clinical therapeutic use. It is important to note that most of the advantageous features of the zebrash model occur at its embryonic and larval stages. Thus, a disease phenotype under investigation must manifest during these stages in order to be most eectively exploited for chemical screening. Since multitudinous signaling pathways acting together in zebrash during early development are also likely to play important roles in maintaining homeostasis in adults and may be disrupted or reactivated during disease progression, a surrogate embryonic phenotype can often be very useful for identifying potential disease modulators. For example, compounds that suppress T-cell development in embryonic zebrash may demonstrate potent inhibitory eects against T-cell leukemia [18]. Overall, drug discovery in zebrash benets from the feasibility of high-throughput chemical screening, closer physiological similarities to human than invertebrate screening strategies, and the ability to create complex disease models not achievable in vitro. The possibility of detecting a wider range of hematopoietic phenotypes using innovative assays promises an ever-increasing role for zebrash in future drug discovery processes.
Advances in Hematology
[5] L. Jingd and L. I. Zon, Zebrash as a model for normal and malignant hematopoiesis, Disease Models and Mechanisms, vol. 4, no. 4, pp. 433438, 2011. [6] C. Hall, M. V. Flores, K. Crosier, and P. Crosier, Live cell imaging of zebrash leukocytes, Methods in Molecular Biology, vol. 546, pp. 255271, 2009. [7] P. Jagadeeswaran, M. Carrillo, U. P. Radhakrishnan, S. K. Rajpurohit, and S. Kim, Laser-induced thrombosis in zebrash, Methods in Cell Biology, vol. 101, pp. 197203, 2011. [8] P. Li and L. I. Zon, Stem cell migration: a zebrash model, Methods in Molecular Biology, vol. 750, pp. 157168, 2011. [9] D. L. Stachura and D. Traver, Cellular dissection of zebrash hematopoiesis, Methods in Cell Biology, vol. 101, pp. 75110, 2011. [10] A. M. Taylor and L. I. Zon, Zebrash tumor assays: the state of transplantation, Zebrash, vol. 6, no. 4, pp. 339346, 2009. [11] B. M. Weinstein, A. F. Schier, S. Abdelilah et al., Hematopoietic mutations in the zebrash, Development, vol. 123, pp. 303309, 1996. [12] J. D. Sander, J.-R. J. Yeh, R. T. Peterson, and J. K. Joung, Engineering zinc nger nucleases for targeted mutagenesis of zebrash, Methods in Cell Biology, vol. 104, pp. 5158, 2011. [13] J. D. Sander, L. Cade, C. Khayter et al., Targeted gene disruption in somatic zebrash cells using engineered TALENs, Nature Biotechnology, vol. 29, no. 8, pp. 697698, 2011. [14] D. G. Ransom, P. Hater, J. Odenthal et al., Characterization of zebrash mutants with defects in embryonic hematopoiesis, Development, vol. 123, pp. 311319, 1996. [15] A. Nasevicius and S. C. Ekker, Eective targeted gene knockdown in zebrash, Nature Genetics, vol. 26, no. 2, pp. 216220, 2000. [16] P. Huang, A. Xiao, M. Zhou, Z. Zhu, S. Lin, and B. Zhang, Heritable gene targeting in zebrash using customized TALENs, Nature Biotechnology, vol. 29, no. 8, pp. 699700, 2011. [17] M. F. Cusick, J. E. Libbey, N. S. Trede, D. D. Eckels, and R. S. Fujinami, Human T cell expansion and experimental autoimmune encephalomyelitis inhibited by Lenaldekar, a small molecule discovered in a zebrash screen, Journal of Neuroimmunology, vol. 244, no. 1-2, pp. 3544, 2012. [18] S. Ridges, W.L. Heaton, D. Joshi et al., Zebrash screen identies novel compound with selective toxicity against leukemia, Blood. In press. [19] T. E. North, W. Goessling, C. R. Walkley et al., Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis, Nature, vol. 447, no. 7147, pp. 10071011, 2007. [20] J. R. J. Yeh, K. M. Munson, K. E. Elagib, A. N. Goldfarb, D. A. Sweetser, and R. T. Peterson, Discovering chemical modiers of oncogene-regulated hematopoietic dierentiation, Nature Chemical Biology, vol. 5, no. 4, pp. 236243, 2009. [21] I. T. Helenius and J. R. Yeh, Small zebrash in a big chemical pond, Journal of Cellular Biochemistry, vol. 113, no. 7, pp. 22082216, 2012. [22] D. Kokel and R. T. Peterson, Chemobehavioural phenomics and behaviour-based psychiatric drug discovery in the zebrash, Briengs in Functional Genomics and Proteomics, vol. 7, no. 6, pp. 483490, 2008. [23] P. J. Schlueter and R. T. Peterson, Systematizing serendipity for cardiovascular drug discovery, Circulation, vol. 120, no. 3, pp. 255263, 2009.
Conict of Interests
The authors declare no competing nancial interests.
Acknowledgments
The authors thank Taneli Helenius, Caroline Burns, and Randall Peterson for helpful comments on the paper. Funding for this work was provided by U.S. National Institutes of Health Grants R01 CA140188 (J.-R. J. Yeh), K01 AG031300 (J.-R. J. Yeh) and Massachusetts General Hospital Clain Distinguished Scholar Award (J.-R. J. Yeh).
References
[1] S. A. Renshaw and N. S. Trede, A model 450 million years in the making: zebrash and vertebrate immunity, Disease Models and Mechanisms, vol. 5, no. 1, pp. 3847, 2012. [2] E. Payne and T. Look, Zebrash modelling of leukaemias, British Journal of Haematology, vol. 146, no. 3, pp. 247256, 2009. [3] J. Y. Bertrand and D. Traver, Hematopoietic cell development in the zebrash embryo, Current Opinion in Hematology, vol. 16, no. 4, pp. 243248, 2009. [4] F. Ellett and G. J. Lieschke, Zebrash as a model for vertebrate hematopoiesis, Current Opinion in Pharmacology, vol. 10, no. 5, pp. 563570, 2010.
Advances in Hematology
[24] L. I. Zon and R. T. Peterson, In vivo drug discovery in the zebrash, Nature Reviews Drug Discovery, vol. 4, no. 1, pp. 3544, 2005. [25] M. A. Lindsay, Target discovery, Nature Reviews Drug Discovery, vol. 2, no. 10, pp. 831838, 2003. [26] A. L. Hopkins and C. R. Groom, The druggable genome, Nature Reviews Drug Discovery, vol. 1, no. 9, pp. 727730, 2002. [27] T. H. Keller, A. Pichota, and Z. Yin, A practical view of druggability, Current Opinion in Chemical Biology, vol. 10, no. 4, pp. 357361, 2006. [28] S. M. Paul, D. S. Mytelka, C. T. Dunwiddie et al., How to improve RD productivity: the pharmaceutical industrys grand challenge, Nature Reviews Drug Discovery, vol. 9, no. 3, pp. 203214, 2010. [29] B. Munos, Lessons from 60 years of pharmaceutical innovation, Nature Reviews Drug Discovery, vol. 8, no. 12, pp. 959968, 2009. [30] D. C. Swinney and J. Anthony, How were new medicines discovered? Nature Reviews Drug Discovery, vol. 10, no. 7, pp. 507519, 2011. [31] S. E. Ong, B. Blagoev, I. Kratchmarova et al., Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics, Molecular & Cellular Proteomics, vol. 1, no. 5, pp. 376386, 2002. [32] C. Laggner, D. Kokel, V. Setola et al., Chemical informatics and target identication in a zebrash phenotypic screen, Nature Chemical Biology, vol. 8, no. 2, pp. 144146, 2012. [33] P. L. Ross, Y. N. Huang, J. N. Marchese et al., Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents, Molecular and Cellular Proteomics, vol. 3, no. 12, pp. 11541169, 2004. [34] A. Westman-Brinkmalm, A. Abramsson, J. Pannee et al., SILAC zebrash for quantitative analysis of protein turnover and tissue regeneration, Journal of Proteomics, vol. 75, pp. 425434, 2011. [35] J. Giacomotto and L. S egalat, High-throughput screening and small animal models, where are we? British Journal of Pharmacology, vol. 160, no. 2, pp. 204216, 2010. [36] C. Sachidanandan, J. R. J. Yeh, Q. P. Peterson, and R. T. Peterson, Identication of a novel retinoid by small molecule screening with zebrash embryos, PLoS One, vol. 3, no. 4, Article ID e1947, 2008. [37] D. M. Langenau, A. A. Ferrando, D. Traver et al., In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrash, Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 19, pp. 73697374, 2004. [38] D. M. Langenau, D. Traver, A. A. Ferrando et al., Mycinduced T cell leukemia in transgenic zebrash, Science, vol. 299, no. 5608, pp. 887890, 2003. [39] K. Hsu, D. Traver, J. L. Kutok et al., The pu.1 promoter drives myeloid gene expression in zebrash, Blood, vol. 104, no. 5, pp. 12911297, 2004. [40] H. F. Lin, D. Traver, H. Zhu et al., Analysis of thrombocyte development in CD41-GFP transgenic zebrash, Blood, vol. 106, no. 12, pp. 38033810, 2005. [41] X. Y. Zhang and A. R. F. Rodaway, SCL-GFP transgenic zebrash: in vivo imaging of blood and endothelial development and identication of the initial site of denitive hematopoiesis, Developmental Biology, vol. 307, no. 2, pp. 179194, 2007. [42] Q. Long, A. Meng, M. Wang, J. R. Jessen, M. J. Farrell, and S. Lin, GATA-1 expression pattern can be recapitulated in
9
living transgenic zebrash using GFP reporter gene, Development, vol. 124, no. 20, pp. 41054111, 1997. C. Hall, M. Flores, T. Storm, K. Crosier, and P. Crosier, The zebrash lysozyme C promoter drives myeloid-specic expression in transgenic sh, BMC Developmental Biology, vol. 7, article no. 42, 2007. C. Gray, C. A. Loynes, M. K. B. Whyte, D. C. Crossman, S. A. Renshaw, and T. J. A. Chico, Simultaneous intravital imaging of macrophage and neutrophil behaviour during inammation using a novel transgenic zebrash, Thrombosis and Haemostasis, vol. 105, no. 5, pp. 811819, 2011. J. R. Mathias, B. J. Perrin, T. X. Liu, J. Kanki, A. T. Look, and A. Huttenlocher, Resolution of inammation by retrograde chemotaxis of neutrophils in transgenic zebrash, Journal of Leukocyte Biology, vol. 80, no. 6, pp. 12811288, 2006. S. H. Oehlers, M. V. Flores, K. S. Okuda, C. J. Hall, K. E. Crosier, and P. S. Crosier, A chemical enterocolitis model in zebrash larvae that is dependent on microbiota and responsive to pharmacological agents, Developmental Dynamics, vol. 240, no. 1, pp. 288298, 2011. B. Thattaliyath, M. Cykowski, and P. Jagadeeswaran, Young thrombocytes initiate the formation of arterial thrombi in zebrash, Blood, vol. 106, no. 1, pp. 118124, 2005. M. N. OConnor, I. I. Salles, A. Cvejic et al., Functional genomics in zebrash permits rapid characterization of novel platelet membrane proteins, Blood, vol. 113, no. 19, pp. 47544762, 2009. K. N. Adams, K. Takaki, L. E. Connolly et al., Drug tolerance in replicating mycobacteria mediated by a macrophageinduced eux mechanism, Cell, vol. 145, no. 1, pp. 3953, 2011. E. L. Benard, A. M. van der Sar, F. Ellett, G. J. Lieschke, H. P. Spaink, and A. H. Meijer, Infection of zebrash embryos with intracellular bacterial pathogens, The Journal of Visualized Experiments , no. 61, article e3781, 2012. R. Carvalho, J. de Sonneville, O. W. Stockhammer et al., A high-throughput screen for tuberculosis progression, PLoS One, vol. 6, no. 2, Article ID e16779, 2011. C. A. Loynes, J. S. Martin, A. Robertson et al., Pivotal advance: pharmacological manipulation of inammation resolution during spontaneously resolving tissue neutrophilia in the zebrash, Journal of Leukocyte Biology, vol. 87, no. 2, pp. 203212, 2010. S. L. Walker, J. Ariga, J. R. Mathias et al., Automated reporter quantication in vivo: high-throughput screening method for reporter-based assays in zebrash, PLoS One, vol. 7, no. 1, Article ID e29916, 2012. A. Vogt, H. Codore, B. W. Day, N. A. Hukriede, and M. Tsang, Development of automated imaging and analysis for zebrash chemical screens, Journal of Visualized Experiments, vol. 40, article e1900, 2010. C. A. Lessman, The developing zebrash (Danio rerio): avertebrate model for high-throughput screening of chemical libraries, Birth Defects Research Part C, vol. 93, no. 3, pp. 268280, 2011. J. R. Yeh and K. M. Munson, Zebrash small molecule screen in reprogramming/cell fate modulation, Methods in Molecular Biology, vol. 636, pp. 317327, 2010. J. R. J. Yeh, K. M. Munson, Y. L. Chao, Q. P. Peterson, C. A. MacRae, and R. T. Peterson, AML1-ETO reprograms hematopoietic cell fate by downregulating scl expression, Development, vol. 135, no. 2, pp. 401410, 2008.
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
10
[58] F. Ellett, L. Pase, J. W. Hayman, A. Andrianopoulos, and G. J. Lieschke, mpeg1 promoter transgenes direct macrophagelineage expression in zebrash, Blood, vol. 117, no. 4, pp. e49e56, 2011. [59] S. K. Yoo, Q. Deng, P. J. Cavnar, Y. I. Wu, K. M. Hahn, and A. Huttenlocher, Dierential regulation of protrusion and polarity by PI(3)K during neutrophil motility in live zebrash, Developmental Cell, vol. 18, no. 2, pp. 226236, 2010. [60] B. Pruvot, A. Jacquel, N. Droin et al., Leukemic cell xenograft in zebrash embryo for investigating drug ecacy, Haematologica, vol. 96, no. 4, pp. 612616, 2011. [61] A. C. H. Smith, A. R. Raimondi, C. D. Salthouse et al., Highthroughput cell transplantation establishes that tumor-initiating cells are abundant in zebrash T-cell acute lymphoblastic leukemia, Blood, vol. 115, no. 16, pp. 32963303, 2010. [62] R. M. White, A. Sessa, C. Burke et al., Transparent adult zebrash as a tool for in vivo transplantation analysis, Cell Stem Cell, vol. 2, no. 2, pp. 183189, 2008. [63] D. Traver, B. H. Paw, K. D. Poss, W. T. Penberthy, S. Lin, and L. I. Zon, Transplantation and in vivo imaging of multilineage engraftment in zebrash bloodless mutants, Nature Immunology, vol. 4, no. 12, pp. 12381246, 2003. [64] D. Traver, A. Winzeler, H. M. Stern et al., Eects of lethal irradiation in zebrash and rescue by hematopoietic cell transplantation, Blood, vol. 104, no. 5, pp. 12981305, 2004. [65] D. L. Stachura, O. Svoboda, R. P. Lau et al., Clonal analysis of hematopoietic progenitor cells in the zebrash, Blood, vol. 118, no. 5, pp. 12741282, 2011. [66] V. Wittamer, J. Y. Bertrand, P. W. Gutschow, and D. Traver, Characterization of the mononuclear phagocyte system in zebrash, Blood, vol. 117, no. 26, pp. 71267135, 2011. [67] P. Jagadeeswaran, J. P. Sheehan, F. E. Craig, and D. Troyer, Identication and characterization of zebrash thrombocytes, British Journal of Haematology, vol. 107, no. 4, pp. 731738, 1999. [68] C. M. Bennett, J. P. Kanki, J. Rhodes et al., Myelopoiesis in the zebrash, Danio rerio, Blood, vol. 98, no. 3, pp. 643651, 2001. [69] G. J. Lieschke, A. C. Oates, M. O. Crowhurst, A. C. Ward, and J. E. Layton, Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrash, Blood, vol. 98, no. 10, pp. 30873096, 2001. [70] G. Lugo-Villarino, K. M. Balla, D. L. Stachura, K. Ba nuelos, M. B. F. Werneck, and D. Traver, Identication of dendritic antigen-presenting cells in the zebrash, Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 36, pp. 1585015855, 2010. [71] C. E. Willett, A. G. Zapata, N. Hopkins, and L. A. Steiner, Expression of zebrash rag genes during early development identies the thymus, Developmental Biology, vol. 182, no. 2, pp. 331341, 1997. [72] N. Hsia and L. I. Zon, Transcriptional regulation of hematopoietic stem cell development in zebrash, Experimental Hematology, vol. 33, no. 9, pp. 10071014, 2005. [73] H. D. Song, X. J. Sun, M. Deng et al., Hematopoietic gene expression prole in zebrash kidney marrow, Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 46, pp. 1624016245, 2004. [74] J. L. O. De Jong and L. I. Zon, Use of the zebrash system to study primitive and denitive hematopoiesis, Annual Review of Genetics, vol. 39, pp. 481501, 2005. [75] A. T. Chen and L. I. Zon, Zebrash blood stem cells, Journal of Cellular Biochemistry, vol. 108, no. 1, pp. 3542, 2009.
Advances in Hematology
[76] J. Rhodes, A. Hagen, K. Hsu et al., Interplay of pu.1 and Gata1 determines myelo-erythroid progenitor cell fate in zebrash, Developmental Cell, vol. 8, no. 1, pp. 97108, 2005. [77] J. L. Galloway, R. A. Wingert, C. Thisse, B. Thisse, and L. I. Zon, Loss of Gata1 but not Gata2 converts erythropoiesis to myelopoiesis in zebrash embryos, Developmental Cell, vol. 8, no. 1, pp. 109116, 2005. [78] C. E. Burns, D. Traver, E. Mayhall, J. L. Shepard, and L. I. Zon, Hematopoietic stem cell fate is established by the NotchRunx pathway, Genes and Development, vol. 19, no. 19, pp. 23312342, 2005. [79] J. Y. Bertrand, A. D. Kim, E. P. Violette, D. L. Stachura, J. L. Cisson, and D. Traver, Denitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrash embryo, Development, vol. 134, no. 23, pp. 41474156, 2007. [80] K. Kissa, E. Murayama, A. Zapata et al., Live imaging of emerging hematopoietic stem cells and early thymus colonization, Blood, vol. 111, no. 3, pp. 11471156, 2008. [81] E. Murayama, K. Kissa, A. Zapata et al., Tracing hematopoietic precursor migration to successive hematopoietic organs during zebrash development, Immunity, vol. 25, no. 6, pp. 963975, 2006. [82] H. Jin, R. Sood, J. Xu et al., Denitive hematopoietic stem/progenitor cells manifest distinct dierentiation output in the zebrash VDA and PBI, Development, vol. 136, no. 4, pp. 647654, 2009. [83] T. North, T. L. Gu, T. Stacy et al., Cbfa2 is required for the formation of intra-aortic hematopoietic clusters, Development, vol. 126, no. 11, pp. 25632575, 1999. [84] T. E. North, M. F. T. R. De Bruijn, T. Stacy et al., Runx1 expression marks long-term repopulating hematopoietic stem cells in the midgestation mouse embryo, Immunity, vol. 16, no. 5, pp. 661672, 2002. [85] M. Gering and R. Patient, Hedgehog signaling is required for adult blood stem cell formation in zebrash embryos, Developmental Cell, vol. 8, no. 3, pp. 389400, 2005. [86] W. Goessling, T. E. North, S. Loewer et al., Genetic Interaction of PGE2 and Wnt signaling regulates developmental specication of stem cells and regeneration, Cell, vol. 136, no. 6, pp. 11361147, 2009. [87] C. E. Burns, J. L. Galloway, A. C. H. Smith et al., A genetic screen in zebrash denes a hierarchical network of pathways required for hematopoietic stem cell emergence, Blood, vol. 113, no. 23, pp. 57765782, 2009. [88] A. Amsterdam, R. M. Nissen, Z. Sun, E. C. Swindell, S. Farrington, and N. Hopkins, Identication of 315 genes essential for early zebrash development, Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 35, pp. 1279212797, 2004. [89] B. H. Paw, A. J. Davidson, Y. Zhou et al., Cell-specic mitotic defect and dyserythropoiesis associated with erythroid band 3 deciency, Nature Genetics, vol. 34, no. 1, pp. 5964, 2003. [90] A. Donovan, A. Brownlie, Y. Zhou et al., Positional cloning of zebrash ferroportin1 identies a conserved vertebrate iron exporter, Nature, vol. 403, no. 6771, pp. 776781, 2000. [91] D. G. Ransom, N. Bahary, K. Niss et al., The Zebrash moonshine gene encodes transcriptional intermediary factor 1, an essential regulator of hematopoiesis, PLoS Biology, vol. 2, no. 8, article e237, 2004. [92] J. Chen, C. Jette, J. P. Kanki, J. C. Aster, A. T. Look, and J. D. Grin, NOTCH1-induced T-cell leukemia in transgenic zebrash, Leukemia, vol. 21, no. 3, pp. 462471, 2007.
Advances in Hematology
[93] X. Le, D. M. Langenau, M. D. Keefe, J. L. Kutok, D. S. Neuberg, and L. I. Zon, Heat shock-inducible Cre/Lox approaches to induce diverse types of tumors and hyperplasia in transgenic zebrash, Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 22, pp. 94109415, 2007. [94] H. E. Sabaawy, M. Azuma, L. J. Embree, H. J. Tsai, M. F. Starost, and D. D. Hickstein, TEL-AML1 transgenic zebrash model of precursor B cell lymphoblastic leukemia, Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 41, pp. 1516615171, 2006. [95] H. Feng, D. L. Stachura, R. M. White et al., T-lymphoblastic lymphoma cells express high levels of BCL2, S1P1, and ICAM1, leading to a blockade of tumor cell intravasation, Cancer Cell, vol. 18, no. 4, pp. 353366, 2010. [96] A. M. Forrester, C. Grabher, E. R. Mcbride et al., NUP98HOXA9-transgenic zebrash develop a myeloproliferative neoplasm and provide new insight into mechanisms of myeloid leukaemogenesis, British Journal of Haematology, vol. 155, pp. 167181, 2011. [97] Y. Doyon, J. M. McCammon, J. C. Miller et al., Heritable targeted gene disruption in zebrash using designed zinc-nger nucleases, Nature Biotechnology, vol. 26, no. 6, pp. 702708, 2008. [98] J. E. Foley, J. R. J. Yeh, M. L. Maeder et al., Rapid mutation of endogenous zebrash genes using zinc nger nucleases made by oligomerized pool ENgineering (OPEN), PLoS One, vol. 4, no. 2, Article ID e4348, 2009. [99] X. Meng, M. B. Noyes, L. J. Zhu, N. D. Lawson, and S. A. Wolfe, Targeted gene inactivation in zebrash using engineered zinc-nger nucleases, Nature Biotechnology, vol. 26, no. 6, pp. 695701, 2008. [100] J. D. Sander, E. J. Dahlborg, M. J. Goodwin et al., Selectionfree zinc-nger-nuclease engineering by context-dependent assembly (CoDA), Nature Methods, vol. 8, no. 1, pp. 6769, 2011. [101] C. S. Williams, M. Mann, and R. N. DuBois, The role of cyclooxygenases in inammation, cancer, and development, Oncogene, vol. 18, no. 55, pp. 79087916, 1999. [102] T. Grosser, S. Yusu, E. Cheskis, M. A. Pack, and G. A. FitzGerald, Developmental expression of functional cyclooxygenases in zebrash, Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 12, pp. 84188423, 2002. [103] T. E. North, W. Goessling, M. Peeters et al., Hematopoieticstem cell development is dependent on blood ow, Cell, vol. 137, no. 4, pp. 736748, 2009. [104] C. Delaney, S. Heimfeld, C. Brashem-Stein, H. Voorhies, R. L. Manger, and I. D. Bernstein, Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution, Nature Medicine, vol. 16, no. 2, pp. 232236, 2010. [105] A. E. Boitano, J. Wang, R. Romeo et al., Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells, Science, vol. 329, no. 5997, pp. 1345 1348, 2010. [106] C. C. Zhang, M. Kaba, S. Lizuka, H. Huynh, and H. F. Lodish, Angiopoietin-like 5 and IGFBP2 stimulate ex vivo expansion of human cord blood hematopoietic stem cells as assayed by NOD/sCiD transplantation, Blood, vol. 111, no. 7, pp. 3415 3423, 2008. [107] K. K. Ballen, E. J. Shpall, D. Avigan et al., Phase I trial of parathyroid hormone to facilitate stem cell mobilization,
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Biology of Blood and Marrow Transplantation, vol. 13, no. 7, pp. 838843, 2007. J. Hoggatt, P. Singh, J. Sampath, and L. M. Pelus, Prostaglandin E2 enhances hematopoietic stem cell homing, survival, and proliferation, Blood, vol. 113, no. 22, pp. 5444 5455, 2009. H. E. Broxmeyer, S. Cooper, D. M. Hass, J. K. Hathaway, F. B. Stehman, and G. Hangoc, Experimental basis of cord blood transplantation, Bone Marrow Transplantation, vol. 44, no. 10, pp. 627633, 2009. W. Goessling, R. S. Allen, X. Guan et al., Prostaglandin E2 enhances human cord blood stem cell xenotransplants and shows long-term safety in preclinical nonhuman primate transplant models, Cell Stem Cell, vol. 8, no. 4, pp. 445458, 2011. J. M. Butler, D. J. Nolan, E. L. Vertes et al., Endothelial cells are essential for the self-renewal and repopulation of notchdependent hematopoietic stem cells, Cell Stem Cell, vol. 6, no. 3, pp. 251264, 2010. G. Nucifora, D. J. Birn, P. Erickson et al., Detection of DNA rearrangements in the AML1 and ETO loci and of an AML1/ ETO fusion mRNA in patients with t(8;21) acute myeloid leukemia, Blood, vol. 81, no. 4, pp. 883888, 1993. B. Lutterbach, J. J. Westendorf, B. Linggi, S. Isaac, E. Seto, and S. W. Hiebert, A mechanism of repression by acute myeloid leukemia-1, the target of multiple chromosomal translocations in acute leukemia, Journal of Biological Chemistry, vol. 275, no. 1, pp. 651656, 2000. R. K. Hyde, Y. Kamikubo, S. Anderson et al., Cbfb/Runx1 repression-independent blockage of dierentiation and accumulation of Csf2rb-expressing cells by Cbfb-MYH11, Blood, vol. 115, no. 7, pp. 14331443, 2010. M. Higuchi, D. OBrien, P. Kumaravelu, N. Lenny, E. J. Yeoh, and J. R. Downing, Expression of a conditional AML1ETO oncogene bypasses embryonic lethality and establishes a murine model of human t(8;21) acute myeloid leukemia, Cancer Cell, vol. 1, no. 1, pp. 6374, 2002. J. C. Mulloy, J. Cammenga, F. J. Berguido et al., Maintaining the self-renewal and dierentiation potential of human CD34+ hematopoietic cells using a single genetic element, Blood, vol. 102, no. 13, pp. 43694376, 2003. J. C. Mulloy, J. Cammenga, K. L. MacKenzie, F. J. Berguido, M. A. S. Moore, and S. D. Nimer, The AML1-ETO fusion protein promotes the expansion of human hematopoietic stem cells, Blood, vol. 99, no. 1, pp. 1523, 2002. T. Okuda, Z. Cai, S. Yang et al., Expression of a knockedin AML1-ETO leukemia gene inhibits the establishment of normal denitive hematopoiesis and directly generates dysplastic hematopoietic progenitors, Blood, vol. 91, no. 9, pp. 31343143, 1998. M. Schwieger, J. L ohler, J. Friel, M. Scheller, I. Horak, and C. Stocking, AML1-ETO inhibits maturation of multiple lymphohematopoietic lineages and induces myeloblast transformation in synergy with ICSBP deciency, Journal of Experimental Medicine, vol. 196, no. 9, pp. 12271240, 2002. M. L. Kalev-Zylinska, J. A. Horseld, M. V. C. Flores et al., Runx1 is required for zebrash blood and vessel development and expression of a human RUNX-1-CBF2T1 transgene advances a model for studies of leukemogenesis, Development, vol. 129, no. 8, pp. 20152030, 2002. Y. Choi, K. E. Elagib, L. L. Delehanty, and A. N. Goldfarb, Erythroid inhibition by the leukemic fusion AML1-ETO is associated with impaired acetylation of the major erythroid
[108]
[109]
[110]
[111]
[112]
[113]
[114]
[115]
[116]
[117]
[118]
[119]
[120]
[121]
12
transcription factor GATA-1, Cancer Research, vol. 66, no. 6, pp. 29902996, 2006. T. Pabst, B. U. Mueller, N. Harakawa et al., AML1ETO downregulates the granulocytic dierentiation factor C/EBP in t(8;21) myeloid leukemia, Nature Medicine, vol. 7, no. 4, pp. 444451, 2001. B. A. Hug and M. A. Lazar, ETO interacting proteins, Oncogene, vol. 23, no. 24, pp. 42704274, 2004. J. Wang, Y. Saunthararajah, R. L. Redner, and J. M. Liu, Inhibitors of histone deacetylase relieve ETO-mediated repression and induce dierentiation of AML1-ETO leukemia cells, Cancer Research, vol. 59, no. 12, pp. 27662769, 1999. H. S. Kim, H. G. Moon, W. Han et al., COX2 overexpression is a prognostic marker for stage III breast cancer, Breast Cancer Research and Treatment, pp. 19, 2011. C. E. Eberhart, R. J. Coey, A. Radhika, F. M. Giardiello, S. Ferrenbach, and R. N. Dubois, Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas, Gastroenterology, vol. 107, no. 4, pp. 11831188, 1994. J. Shao, C. Jung, C. Liu, and H. Sheng, Prostaglandin E2 stimulates the -catenin/T cell factor-dependent transcription in colon cancer, Journal of Biological Chemistry, vol. 280, no. 28, pp. 2656526572, 2005. M. D. Castellone, H. Teramoto, B. O. Williams, K. M. Druey, and J. S. Gutkind, Medicine: prostaglandin E2 promotes colon cancer cell growth through a Gs-axin--catenin signaling axis, Science, vol. 310, no. 5753, pp. 15041510, 2005. Y. Wang, A. V. Krivtsov, A. U. Sinha et al., The wnt/-catenin pathway is required for the development of leukemia stem cells in AML, Science, vol. 327, no. 5973, pp. 16501653, 2010. G. Steinert, C. Oancea, J. Roos et al., Sulindac sulde reverses aberrant self-renewal of progenitor cells induced by the AML-associated fusion proteins PML/RAR and PLZF/ rar, PLoS One, vol. 6, no. 7, Article ID e22540, 2011. C. M. Kasum, C. K. Blair, A. R. Folsom, and J. A. Ross, Non-steroidal anti-inammatory drug use and risk of adult leukemia, Cancer Epidemiology Biomarkers and Prevention, vol. 12, no. 6, pp. 534537, 2003. J. M. Pogoda, J. Katz, R. McKean-Cowdin, P. W. Nichols, R. K. Ross, and S. Preston-Martin, Prescription drug use and risk of acute myeloid leukemia by French-American-British subtype: results from a Los Angeles County case-control study, International Journal of Cancer, vol. 114, no. 4, pp. 634638, 2005. G. Jeannet, M. Scheller, L. Scarpellino et al., Long-term, multilineage hematopoiesis occurs in the combined absence of -catenin and -catenin, Blood, vol. 111, no. 1, pp. 142 149, 2008. U. Koch, A. Wilson, M. Cobas, R. Kemler, H. R. MacDonald, and F. Radtke, Simultaneous loss of - and -catenin does not perturb hematopoiesis or lymphopoiesis, Blood, vol. 111, no. 1, pp. 160164, 2008. M. Cobas, A. Wilson, B. Ernst et al., -catenin is dispensable for hematopoiesis and lymphopoiesis, Journal of Experimental Medicine, vol. 199, no. 2, pp. 221229, 2004. C. Zhao, J. Blum, A. Chen et al., Loss of -catenin impairs the renewal of normal and CML stem cells in vivo, Cancer Cell, vol. 12, no. 6, pp. 528541, 2007. A. Redaelli, M. F. Botteman, J. M. Stephens, S. Brandt, and C. L. Pashos, Economic burden of acute myeloid leukemia: a literature review, Cancer Treatment Reviews, vol. 30, no. 3, pp. 237247, 2004.
Advances in Hematology
[138] R. M. Stone, M. R. ODonnell, and M. A. Sekeres, Acute myeloid leukemia, American Society of Hematology. Education Program, pp. 98117, 2004.
[122]
[123] [124]
[125]
[126]
[127]
[128]
[129]
[130]
[131]
[132]
[133]
[134]
[135]
[136]
[137]
Hindawi Publishing Corporation Advances in Hematology Volume 2012, Article ID 627920, 12 pages doi:10.1155/2012/627920
of Pediatrics, University of Utah, Salt Lake City, UT 84112, USA of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA 3 Department of Pathology, Brigham and Womens Hospital, Boston, MA 02115, USA
2 Department
Correspondence should be addressed to J. Kimble Frazer, [email protected] and Nikolaus S. Trede, [email protected] Received 21 February 2012; Accepted 17 April 2012 Academic Editor: Elspeth Payne Copyright 2012 J. Kimble Frazer et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Genomic instability plays a crucial role in oncogenesis. Somatically acquired mutations can disable some genes and inappropriately activate others. In addition, chromosomal rearrangements can amplify, delete, or even fuse genes, altering their functions and contributing to malignant phenotypes. Using array comparative genomic hybridization (aCGH), a technique to detect numeric variations between dierent DNA samples, we examined genomes from zebrash (Danio rerio) T-cell leukemias of three cancerprone lines. In all malignancies tested, we identied recurring amplications of a zebrash endogenous retrovirus. This retrovirus, ZFERV, was rst identied due to high expression of proviral transcripts in thymic tissue from larval and adult sh. We conrmed ZFERV amplications by quantitative PCR analyses of DNA from wild-type sh tissue and normal and malignant D. rerio T cells. We also quantied ZFERV RNA expression and found that normal and neoplastic T cells both produce retrovirally encoded transcripts, but most cancers show dramatically increased transcription. In aggregate, these data imply that ZFERV amplication and transcription may be related to T-cell leukemogenesis. Based on these data and ZFERVs phylogenetic relation to viruses of the murine-leukemia-related virus class of gammaretroviridae, we posit that ZFERV may be oncogenic via an insertional mutagenesis mechanism.
1. Introduction
Zebrash are an emerging animal model for the study of lymphocytic cancers. A landmark 2003 study rst described that transgenic murine Myc (mMyc) misexpression could induce D. rerio T-cell acute lymphoblastic leukemia (T-ALL) [1]. Since that initial report, several other zebrash ALL models have been described, utilizing transgenic mammalian TEL-AML1 (human), NOTCH1 (human), MYC (murine and human), and AKT2 (murine) in similar fashion [25]. In addition, we used a phenotypic mutagenesis screen to create three further zebrash models with heritable T-ALL predisposition [6]. All but one of the eight lines cited above are prone to T-ALL, not B-cell-lineage cancers. Like human T-ALL, D. rerio T-ALL often arises in or spreads to the thymus and forms tumors. Hence, these seven zebrash
lines actually more accurately model two related lymphocyte malignancies, T-ALL and T-cell lymphoblastic lymphoma (T-LBL). In fact, mMyc zebrash have even been used to investigate the molecular changes that accompany the transition between T-LBL and T-ALL [7]. Because the molecular origins of T-ALL and T-LBL are not completely understood, these zebrash models provide opportunities to investigate the genetic underpinnings of these diseases oncogenesis. In addition, they also facilitate inquiries designed to reveal features associated with T-ALL and T-LBL progression. For example, in the aforementioned study, Feng et al. demonstrated that changes in BCL2, S1P1, and ICAM1 expression were linked to autophagy, intercellular adhesion, and intravascular invasion, thereby governing the T-LBL to T-ALL transition [7]. Similarly, Gutierrez et al. used transgenic zebrash T-ALL to study the dependence of
2 MYC-driven cancers upon Pten and Akt for disease persistence and progression [5]. While these two studies utilized D. rerio models to investigate candidate genes of suspected importance to disease progression, zebrash T-cell cancers can also serve as a means for candidate gene discovery. In our own work, we utilized serial allo-transplantation of D. rerio T-ALL as an experimental approach to model clinically aggressive neoplasia [8]. Similar strategies have been employed by other groups, using serially allo-grafted murine T-cell lymphomas or xeno-transplanted human T-ALL into immunodecient mice [9, 10]. In our study, we performed aCGH to seek acquired genomic changes common to serially passaged D. rerio T-ALL and refractory/relapsed human T-ALL. Several candidate genes met this criterion, including C7orf60 (zebrash homologue, zgc: 153606 ), a gene whose amplications were linked to both accelerated T-ALL progression in sh and inferior outcomes in human T-ALL patients [8]. Although our study concentrated on acquired copy number aberrations (CNAs) shared by zebrash and human T-ALL, we also identied other genomic amplications and deletions seen only in D. rerio cancers. Amongst these, two recurring copy number gains were observed in every sample, and with further scrutiny we found that both regions corresponded to the same endogenous retrovirus (ERV). This genomically integrated provirus is predicted to have 24 integration sites in the zebrash genome [11], and its ploidy and genomic positions may vary between individual animals, complicating its inquiry. In this paper, we use two independent methodologies to show that this multicopy ERV undergoes further amplication in both normal and neoplastic zebrash T cells, which could create new and potentially oncogenic integrations. Some cancers showed very high ZFERV copy number, well above that seen in T lymphocytes. We also demonstrate the expression of retrovirally encoded RNAs by both normal and cancerous D. rerio T cells, with most malignancies displaying signicantly elevated proviral transcription relative to normal T cells. Our ndings, and further characterization of this ERV, will be essential to understanding how this biologically active retrovirus impacts normal and malignant zebrash T lymphocyte biology.
Advances in Hematology 2.2. Dissections and Fluorescence-Activated Cell Sorting (FACS). Zebrash thymi and GFP+ tumors were dissected, with preparation of single cell suspensions and FACS performed as described previously [6]. BD FACSVantage and FACSAria II SORP (Becton Dickson) instruments were used for FACS. GFP intensity and side- and forward-scatter were gating parameters for GFP+ lymphocyte collections. 2.3. Nucleic Acid Purications. Genomic DNA for aCGH and qPCR was extracted from FACS-puried GFP+ T cells and matched tailn tissue using the DNeasy Blood and Tissue Kit (Qiagen) as described previously [8]. Total RNA for qRTPCR assays was extracted from FACS-puried T lymphocytes and T cell cancers with Trizol (Invitrogen) or the RNeasy Mini-Kit (Qiagen) according to manufacturer instructions. RNA samples were treated with RNase-Free DNase (Qiagen) according to manufacturer instructions prior to qRT-PCR. 2.4. Array Comparative Genomic Hybridization (aCGH). Genomic DNA was labeled with the BioPrime Labeling Kit (Invitrogen), puried, quantied, and hybridized to Zv6based Zebrash Genomic Arrays (NimbleGen) as reported previously [8]. Arrays were analyzed using the G2565CA Microarray Scanner System with SureScan High Resolution Technology (Agilent) and normalized using Agilent Feature Extraction software. Copy-number analysis was conducted using the Rank Segmentation algorithm with Nexus Copy Number 5.0 software (BioDiscovery). Detailed descriptions of the aCGH methods used and copy number analyses performed are available in the supplemental sections of the report by Rudner et al. [8]. 2.5. Quantitative Polymerase Chain Reactions (qPCR). A LightCycler CFX96 (Bio-Rad) was used for qPCR assays. Briey, IQ SYBR Green Supermix (Bio-Rad) was used to amplify genomic DNA from various tissue types. Pooled thymocyte DNA (our limiting sample) was spectrophotometrically quantied and then diluted 1 : 100 for use in qPCR. DNA from tailn tissue and GFP+ tumor cells were diluted to identical concentrations, with 2 L of each DNA used in reactions with total volumes of 25 L, and other components added according to manufacturer instructions. All reactions were performed in triplicate. SYBR Green signals were used to derive estimates of relative ZFERV copy number. Since true ZFERV copy number is unknown, values were arbitrarily normalized to 1 copy/haploid genome. Thus, a ZFERV relative copy number equal to 3 indicates three times as many ZFERV copies/genome (e.g., if germline copy number = 3 copies/haploid genome, ZFERV relative copy number = 3 indicates 9 copies/haploid genome). All qPCR results with pol and env were normalized to elf2a, present in 1 copy/D. rerio haploid genome. Primers and reaction conditions were as follows: Forward pol primer: CGC-CCC-ACA-CAT-CACATA Reverse pol primer: CAA-CCA-TCA-CAG-AACAGA
Advances in Hematology Forward env primer: ATG-TTT-GGG-GAA-TGGAAG-G Reverse env primer: TTT-GAT-AAG-GAG-GTGGGT-TTT Forward elf2a primer: TGG-AGG-TGG-AGG-TGAGAA-CT Reverse elf2a primer: GAG-TGG-TTG-TGT-AAGCAT-TTC-G Denaturation: 95 C 3 minutes 40 cycles: 95 C 10 seconds 59 C 40 seconds Melt curve analysis55 C95 C. 2.6. Quantitative Reverse Transcription Polymerase Chain Reactions (qRT-PCR). Total RNA (200 ng/sample) from FACS-puried normal and malignant T cells was assayed with the iScript One-step RT-PCR Kit with SYBR Green (Bio-Rad) using the aforementioned equipment. Reactions were run in triplicate. Results with pol and env were normalized to elf2a, assayed in parallel qRT-PCRs. Expression fold changes were calculated by the 2Ct method. Primers and reaction conditions were as follows: Forward pol primer: CAG-CAC-AAA-CGA-AAATGG-TCT Reverse pol primer: TGG-CTC-CTC-AGT-GTCTCC-TT Forward env primer: AGA-GGG-AAA-GGA-TGGGAT-GT Reverse env primer: TGT-TGG-ATG-TGG-TCTGGT-CT Forward elf2a primer: ATG-AGA-CAA-TGG-GGAGAG-CA Reverse elf2a primer: GGA-TGC-GGC-TGG-AGTTTC Denaturation: 95 C 5 minutes 40 cycles: 95 C 10 seconds 52 C 10 seconds 72 C 30 seconds Melt curve analysis55 C95 C. 2.7. Statistical Analyses. The students t -test was used to compare dierences in genomic relative copy number or fold changes in RNA expression. P values < 0.05 were considered signicant.
4
High-copy signal otg T-ALL1 srk T-ALL1 srk T-ALL2 otg T-ALL2 hlk T-ALL1 hlk T-ALL2 srk T-ALL3 hlk T-ALL3 26.4 Mb 26.6 Mb 62.1 Mb Chr.7 Chr.14 62.3 Mb 5/6 probes 6/6 probes 6/6 probes 6/6 probes 6/6 probes 5/6 probes 2/6 probes 6/6 probes
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Figure 1: Recurrent amplications of a small genomic locus in zebrash T-ALL. Ten kb loci on chromosomes 7 and 14 (Zv6 genomic assembly) show high-copy gains with multiple probes in these regions (black arrows) of 8/8 D. rerio T-ALL samples tested. Signal intensities above a high-copy gain threshold (upper green line) indicate a greater than 2-fold increase in copy number. Individual probes and their intensities are depicted as blue dots; areas with 3 adjacent probes above the high-copy gain threshold use green dots to denote those probes. Seven cancers exhibited high-copy signals for 5/6 probes in this region, while srk T-ALL3 had high-copy signals for only 2/6 probes.
this is made even more taxing by its sequence composition. ZFERV harbors several redundant sequence tracks, including 5 and 3 long terminal repeats (LTRs) and a 517 bp repeat region (RR) containing 9 consecutive repeat elements (see Figure 2). When compounded with potential variability resulting from strain-specic ZFERV integrations, it is perhaps predictable that ZFERV has not received denitive chromosomal map position(s). Consequently, the current NCBI zebrash genome actually suppresses ZFERV sequences and curates them so they do not appear on the Zv9 assembly at all. In the original report describing ZFERV, Shen and Steiner conducted studies to address some of these questions concerning copy number and genomic localization: to prove that ZFERV was integrated into the D. rerio germline, they tested sperm DNA from several T ubingen (T u) sh and veried an integration site common to each of their genomes [11]. Additionally, using Southern blots of T u genomic DNA, they detected 24 bands hybridizing to a ZFERV env probe, implying a maximum of four retroviral copies per haploid genome [11]. However, not all T u sh showed identical hybridization patterns. This could be due to restriction site polymorphisms in the T u strain but might also suggest that dierent sh, even from the same strain, can possess dierent ZFERV copy number and integration sites. Moreover, when Southern hybridizations with an LTR-based probe were performed, 810 bands were seen. Mostbut not allof these entities were shared by dierent T u sh [11]. As with prior results, this nding might be attributable to variability in ZFERV copy number and genomic position between dierent sh. Another interpretation that must be considered
is that homologous LTRs from other related retroviruses and/or incomplete ZFERV proviral genomes (having 1 LTR, but no env) would yield a similar experimental outcome. In spite of these uncertainties, our aCGH data remain convincing as evidence of somatically acquired ZFERV amplications in D. rerio T-cell cancers. None of our aCGH probes correspond to LTR sequences, and 5/6 derive from the retroviral gag, pol, or env genes (Figure 2). Furthermore, even if repeat elements had been used in hybridizations, our method of comparing neoplastic to non-malignant DNA from the same animal is designed to normalize for CNV discrepancies between dierent sh. Therefore, we conclude that zebrash T-cell malignancies acquire non-germline ZFERV copies at some point after fertilization, but whether amplications precede and contribute to oncogenesis is unclear. Because ZFERV transcription occurs in normal zebrash T cells [11], we were curious whether normal D. rerio T lymphocytes might also have ZFERV copy gains. To determine if retroviral amplications also occur in nonleukemic T cells, we investigated ZFERV in normal zebrash T lymphocytes. To emulate our aCGH comparisons, we developed quantitative PCR (qPCR) assays for two ZFERV genomic regions. Using DNA from cancers with gains identied by aCGH, we veried these assays ability to detect ZFERV copy number gains (data not shown). Next, we employed these qPCRs of amplicons from the pol and env regions (locations shown in Figure 2) to test genomic DNA from tailn tissue and FACS-puried T cells of wild-type (WT) adult zebrash. Thymocytes were obtained from WT WIK
Advances in Hematology
0 2 kb 4 kb 6 kb 8 kb 10 kb 12 kb
18903962 gag
39638603 pol #
860010531 env #
Figure 2: ZFERV Genomic Organization. The ZFERV retrovirus is comprised of two 695 bp long terminal repeats (LTRs), a 517 bp repeat region (RR) containing 9 direct repeats, and ORFs for 3 proteins: gag, pol, and env [11]. The gag and pol genes share the same reading frame and are predicted to be translated from one transcript by read-through of a stop codon; env uses a dierent reading frame and is probably a distinct transcript [11]. aCGH probe sites are shown beneath the ZFERV genome schematic: probes found on Zv6 chromosomes 7 ( ) and 14 (# ) are dispersed throughout ZFERV. One aCGH probe had sequences corresponding to the RR (## ), and has multiple binding sites in this area. The 5 remaining aCGH probes map to ORFs. Amplicons from qPCR and qRT-PCR assays are also depicted (not shown to scale). The pol and env qPCR products are 168 and 169 bp, respectively; qRT-PCR products are 225 bp for pol and 234 bp for env.
D. rerio carrying an lck::EGFP transgene [12]. Since the zebrash lck promoter is T cell specic, T lymphocytes from this line are GFP+ . However, unlike sh with T-ALL or TLBL, adult (>6 months of age) WT sh have signicantly fewer T cells (approximately 5 104 GFP+ thymocytes/sh; our unpublished observations). Consequently, we pooled thymic tissue from several WT sh for FACS purications. We then analyzed amplicons from both ZFERV regions to independently assay copy number dierences. Tailn DNAs were tested individually or in small groups to ascertain whether there were appreciable germline CNV dierences in WIK strain sh (Figure 3, lanes 16 and 8 10). As seen in these data, qPCR of pol (Figure 3(a)) and env (Figure 3(b)) show little deviation between n DNA from dierent WIK sh, implying that CNV was minimal in these strain-related animals (lanes 7 and 11). Because copy number was so uniform, this further suggests that ZFERV amplication does not occur in n tissue. Thus, we conclude that ZFERV status in n tissue likely represents true germline copy number, and that this level is relatively stable between individual sh. In contrast, normal T cells pooled from these same WT sh showed signicant ZFERV gains relative to tailn DNA (Figure 3, lanes 12, 13). On average, WIK T cells had 2- to 3fold as many ZFERV copies as matched tail DNA (compare lane 11 to 14). Since germline copy number is unknown, we cannot deduce the real number of ZFERV copies in these T cells. Nonetheless, if prior data suggesting 24 copies/haploid genome are accurate [11], these results indicate normal T cells may average up to 12 copies per haploid genome, or 24 copies/diploid T cell. If correct, this would compute to 16 new ZFERV integrations, on average, in each T cell. Because we used T lymphocytes pooled from several WIK sh in these studies, we cannot denitively conclude whether all animals T cells bore evidence of ZFERV amplication. It is possible that only one or a few sh have ZFERV gains, with DNA from those sh skewing the average upward. However, even in one sh, T lymphocytes constitute a nonclonal population. It is possibleperhaps even likelythat ZFERV
copy number varies on a cell-to-cell basis. ZFERV amplications may occur in T cells themselves; alternatively, they might take place earlier along the hematopoietic stem cell/T cell progenitor dierentiation spectrum. We have not tested precursor populations, as these are impossible to obtain in D. rerio owing to the dearth of antibodies to cell surface receptors. Irrespective of its precise timing, we conclude that thymocytes acquire additional genomic ZFERV copies at some point after fertilization, exactly like those detected in our aCGH analyses of zebrash T-cell cancers. Notably, there is precedent proving that ZFERV is active in zebrash T cells. This retrovirus was originally discovered from a thymic cDNA library, after adult D. rerio thymus had been subtracted against 2-day postfertilization (dpf) larval sh, which have not yet developed T lymphocytes [11]. This study identied 43 clones hybridizing to only adult thymic cDNA. Of these, 21 clones also showed thymusspecic staining in 7-dpf in situ hybridizations (ISH). After sequencing, Shen and Steiner recognized that all 21 clones derived from various segments of the ZFERV genome [11]. So, not only was ZFERV transcribed by both 7-dpf and adult thymocytes, its expression in these cells was signicantly higher than in other tissues by these two methodologies. Subsequent ISH experiments in 4-dpf, 5-dpf, and 3-monthold juvenile sh, as well as Northern blotting of RNA from adult sh thymocytes, conrmed these ndings [11]. Together, these prior studies and our own new ndings demonstrate that ZFERV is highly transcribed by larval and adult D. rerio thymocytes and that ZFERV amplications occur in the genomes of normal and malignant zebrash T cells. To further expand our understanding of these phenomena, we next compared ZFERV amplications in cancerprone thymocytes and neoplastic T cells to WT T cells. For these experiments, we used our qPCR assays to compare ZFERV copy number in two other T-cell malignancy predisposed lines, hlk and MYC-ER. Both of these lines are prone to T-LBL and T-ALL, allowing ZFERV quantication of their germlines, their premalignant T lymphocytes,
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WT WIK lck::EGFP pol 4 P = 8.49 106 4 env
Advances in Hematology
P = 4.71 1010
Relative copy #
Relative copy #
0 1 Tail 1 2 Tail 2 3 Tail 3 4 Tail 4 5 Tail 5 6 Tail 6 7 Tails 16 avg. 8 Tails 79 9 Tails 1012 10 11 12 13 14 Tails 115 avg. T cells 115 avg. Tails 1315 T cells 1015 T cells 19
0 1 Tail 1 2 Tail 2 3 Tail 3 4 Tail 4 5 Tail 5 6 Tail 6 7 Tails 16 avg. 8 Tails 79 9 Tails 1012 10 11 12 13 14 Tails 115 avg. T cells 115 avg. Tails 1315 T cells 1015 T cells 19
(a)
(b)
Figure 3: ZFERV amplications in WT WIK D. rerio T cells. Genomic DNA from 15 WIK lck::EGFP sh was analyzed by qPCR of the ZFERV pol (a) and env (b) genes. White bars depict results from tail DNA of individual sh (lanes 16) or groups of 3 sh (lanes 810). Black bars show calculated means of 6 singly tested tails (lane 7) or all 15 tails (lane 11). Tails 79 sample (lane 8) was arbitrarily assigned copy number equal to 1, and this DNA was used as the reference standard for all subsequent qPCRs. T cells pooled from 9 or 6 WT sh (gray bars) had 2- to 3-fold gains in ZFERV. Mean copy number was higher in T cells than tailn DNA for the 15 sh cohort (lane 11 versus 14). Zebrash elf2a (1 copy/haploid genome) qPCR was used to normalize pol and env results (not shown). Water-only template controls lacked detectable product (not shown). Reactions were performed in triplicate, and error bars show standard deviations (env qPCR of tails 2 and 5 had standard deviations too small to be seen).
and their malignant T cells. All MYC transgenic sh have hypertrophic thymi, likely reecting abnormal T-cell proliferation and physiology. In contrast, hlk sh carry an unidentied mutation, display normal-appearing thymi, and the molecular basis for their cancer predisposition is unknown. T-ALL or T-LBL aicts roughly 35% of hlk homozygotes by one year [6], reecting a requirement for additional mutations to promote malignant transformation [8]. By comparison, WT lck::EGFP sh rarely develop Tcell cancers (<0.1%, our unpublished observations) and have normal T-cell development and physiology [12]. Thus, using these samples we could investigate whether normal, abnormal, and neoplastic T cells all exhibited similar degrees of ZFERV amplication. As in earlier experiments, we examined tailns from individual sh to ascertain ZFERV germline variability. Tails from single hlk and MYC-ER sh (Figures 4 and 5, lanes 3 8) demonstrated consistent copy number between animals. Moreover, both hlk and MYC-ER tails had ZFERV CNV similar to the WT WIK line (compare lane 1 to other white bars in Figures 4 and 5). Based on these results, identical for both the pol and env regions, we conclude that all 3 lines have approximately equivalent germline copies of ZFERV. In pooled premalignant T cells (i.e., thymocytes from hlk and MYC-ER sh lacking tumors or other non-thymic GFP), genomic ZFERV was again elevated relative to tailn DNA from the same sh (Figures 4 and 5, compare gray bars in lanes 10-11 to white bars in lanes 38). Overall, mean T-cell
ZFERV copy number was roughly 3-fold above germline in WT, 4-fold higher in hlk, and 5-fold increased in MYC-ER (compare lane 9 to 12 in both gures). Since WT, hlk, and MYC-ER thymocytes all showed approximately equivalent gains, we infer that genomic integration is not appreciably enhanced in T lymphocytes of either cancer-prone genotype. Thus, while retroviral amplication is clearly a common feature of all D. rerio T cells, cancer predisposition probably does not directly originate from increased susceptibility to ZFERV integration, as these events evidently transpire in normal T cells regularly. However, it is plausible that cancer predisposing mutations and ZFERV copy number gains may cooperate to promote malignant transformation of T cells, as amplications were uniformly present in every T-ALL sample examined by aCGH. To investigate how WT and cancer-prone T cells compare to actual neoplasias in the hlk and MYC-ER lines, we also analyzed malignancies from these same genetic backgrounds. We performed pol and env qPCRs on 3 hlk and 5 MYC-ER sh, each of which had large thymic tumors and/or extensive GFP+ disease in extra-thymic areas (Figures 4 and 5). Tail DNA from these 8 sh all had similar germline ZFERV content to previously tested tailn samples (compare lanes 1315 in Figure 4 and lanes 1317 in Figure 5 to other white bars in both gures). Like hlk T lymphocytes, hlk cancers had ZFERV amplication (Figure 4, lanes 1618). However, gains were similar in magnitude to those seen in hlk premalignant T cells (compare lane 12 versus 19). In MYC-ER cancers,
Advances in Hematology
hlk pol P = 1.18 105 P = 0.64 Relative copy # 7 6 5 4 3 2 1 0 env P = 4.53 104 P = 0.13
5 Relative copy # 4 3 2 1 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 WT tails 79 WT T cells 115 avg. hlk tail 1 hlk tail 2 hlk tail 3 hlk tail 4 hlk tail 5 hlk tail 6 hlk tails 16 avg. hlk T cells 13 hlk T cells 46 hlk T cells 1-6 avg. hlk T-ALL 4 tail hlk T-ALL 5 tail hlk T-ALL 6 tail hlk T-ALL 4 cancer hlk T-ALL 5 cancer hlk T-ALL 6 cancer hlk T-ALL cancers avg.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 WT tails 79 WT T cells 115 avg. hlk tail 1 hlk tail 2 hlk tail 3 hlk tail 4 hlk tail 5 hlk tail 6 hlk tails 16 avg. hlk T cells 13 hlk T cells 46 hlk T cells 1-6 avg. hlk T-ALL 4 tail hlk T-ALL 5 tail hlk T-ALL 6 tail hlk T-ALL 4 cancer hlk T-ALL 5 cancer hlk T-ALL 6 cancer hlk T-ALL cancers avg. (b)
(a)
Figure 4: ZFERV amplications in hlk zebrash. DNA from 6 hlk sh with normal phenotype and 3 with GFP+ cancers was tested by qPCR of pol (a) and env (b). White bars show tail DNA of individual normal (lanes 38) or T-ALL+ (lanes 1315) sh. All were statistically similar to each other and to WT Tails 79 (lane 1). Pooled T cells from hlk sh without T-ALL (gray bars) had ZFERV gains comparable to normal WIK T cells (lane 2). Mean copy number was higher in hlk T cells than tails (lane 9 versus 12) in the same animals. Diagonally striped bars show amplications in neoplastic T cells of 3 hlk sh (lanes 1618). Mean copy gains were similar in non-malignant and malignant hlk T cells (lane 12 versus 19). Other details are as described in the legend to Figure 3.
ZFERV gains were also detected (Figure 5, lanes 1822). As in hlk, benign and malignant MYC-ER T cells did not show appreciable copy number dierences (compare lane 12 to 23). We also tested 2 other malignancies by qPCR. In one WT lck::EGFP sh, we noticed a large GFP+ thymic tumor. In our experience, the spontaneous occurrence of T-cell cancer in WT sh is exceedingly rare, so we used this opportunity to investigate whether ZFERV amplication accompanied this event. Tail DNA indicated this animal had normal ZFERV germline content (Figure 6, compare lane 1 versus 9), and cancerous T cells from this sh showed approximately 5.5-fold higher copy number (lane 10). This degree of amplication is roughly twice that seen in normal WIK T cells and more closely resembled typical copies in MYC-ER T cells and cancers (compare lane 10 to lanes 4, 6, and 8). However, since this result reects only one tumor, no general conclusions can be drawn about retroviral amplication in the rare cancers of WT sh. Lastly, in one additional hlk cancer, we found remarkably high ZFERV levels, showing 25- to 30-fold amplication above germline (lanes 11 and 12). This degree of copy number gain is nearly ten times higher than the other 9 T-ALLs we examined by qPCR, or the 8 tested previously by aCGH. Nonetheless, this infrequent scenario clearly demonstrates that ZFERV can parasitize the zebrash genome in striking fashion, as this cancer likely harbors as many as 50100 newly acquired retroviral copies. Taken together, we conclude that virtually all MYCdriven, hlk-, srk-, and otg -induced, or even spontaneous zebrash T-cell cancers have ZFERV amplications. However, since nearly all benign, cancer-prone, and malignant T cells show similar genomic levels, the absolute amount of ZFERV amplication does not appear to be an important
oncogenic determinant. This is not surprising, as it is likely that the site rather than the number of integrations is the crucial factor. To pursue this premise, one could identify new loci where ZFERV has integrated into cancer genomes, with the hypothesis that these might lie near or within protooncogenes or tumor suppressors. We have initiated such studies, and they are currently in progress. As an adjunct, we chose to investigate transcription of ZFERV-derived RNAs. We reasoned that integrations into transcriptionally permissive genomic sites might be accompanied by increased ZFERV RNA expression, perhaps signifying active proviral copies. While these insertions might not denote sites where oncogenes or tumor suppressor reside, it could serve as a proxy for ZFERV promoter potency in the genome overall. If so, this predicts that cancers would have higher ZFERV transcription than normal T cells and perhaps premalignant T lymphocytes as well. To conduct these studies, we developed quantitative Reverse Transcription-Polymerase Chain Reactions (qRTPCR) of the ZFERV pol and env genes (amplicon locations shown in Figure 2). As for qPCR, we used pooled normal T cells from WT WIK sh as our reference. Recall that even normal T lymphocytes highly express ZFERV transcripts [11], so these RNAs are already plentiful in the cells used as our standard. Results for pol and env were highly reproducible between two pooled T-cell samples from dierent groups of WT sh, and this value was arbitrarily assigned an expression level of 1 (Figure 7, lanes 1 and 2). By comparison, pooled pre-cancerous T cells from hlk sh exhibited approximately 6-fold and 7-fold enhanced pol and env transcription, respectively (lane 3, white bar). Likewise, pooled premalignant T cells from MYC-ER sh also had higher ZFERV transcripts (lane 9; pol: 9-fold increase,
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MYC-ER pol 6 5 4 3 2 1 0 P = 7.46 109 P = 0.002 8 7 6 5 4 3 2 1 0 Relative copy no. Relative copy no. env P = 4.33 104
Advances in Hematology
P = 0.11
1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 17 18 19 20 21 22 23 WT tails 79 WT T cells 115 avg. MYC-ER tail 1 MYC-ER tail 2 MYC-ER tail 3 MYC-ER tail 4 MYC-ER tail 5 MYC-ER tail 6 MYC-ER tails 16 avg. MYC-ER T cells 13 MYC-ER T cells 46 MYC-ER T cells 16 avg. MYC-ER T-ALL 1 tail MYC-ER T-ALL 2 tail MYC-ER T-ALL 3 tail MYC-ER T-ALL 4 tail MYC-ER T-ALL 5 tail MYC-ER T-ALL 1 cancer MYC-ER T-ALL 2 cancer MYC-ER T-ALL 3 cancer MYC-ER T-ALL 4 cancer MYC-ER T-ALL 5 cancer MYC-ER T-ALL cancer avg.
1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 17 18 19 20 21 22 23 WT tails 79 WT T cells 115 avg. MYC-ER tail 1 MYC-ER tail 2 MYC-ER tail 3 MYC-ER tail 4 MYC-ER tail 5 MYC-ER tail 6 MYC-ER tails 16 avg. MYC-ER T cells 13 MYC-ER T cells 46 MYC-ER T cells 16 avg. MYC-ER T-ALL 1 tail MYC-ER T-ALL 2 tail MYC-ER T-ALL 3 tail MYC-ER T-ALL 4 tail MYC-ER T-ALL 5 tail MYC-ER T-ALL 1 cancer MYC-ER T-ALL 2 cancer MYC-ER T-ALL 3 cancer MYC-ER T-ALL 4 cancer MYC-ER T-ALL 5 cancer MYC-ER T-ALL cancer avg. (b) env 35 30 25 20 15 10 5 0 1 WT tails avg. 2 hlk tails avg. 3 MYC-ER tails avg. Relative copy no. P = 0.15 P = 0.18 4 WT T cells avg. 5 hlk T cells avg. 6 MYC-ER T cells avg. 7 hlk T-ALL avg. 8 MYC-ER T-ALL avg. 9 WT GFP+ tumor tail 10 WT GFP+ tumor 11 hlk T-ALL 7 tail 12 hlk T-ALL 7 cancer (b)
(a)
Figure 5: ZFERV amplications in MYC-ER zebrash. Phenotypically normal (n = 6) or T-ALL+ (n = 5) MYC-ER sh were tested by qPCR of pol (a) and env (b). White bars display tail DNA from single normal (lanes 38) or diseased (lanes 1317) sh. MYC-ER tails had similar copy number to each other (compare lanes 38 and 1317) and to WT Tails 79 (lane 1). T cells pooled from groups of 3 normal MYC-ER sh (gray bars) showed ZFERV amplication; higher gains were seen in MYC-ER than WT T cells (lane 2 versus 12; P values 5.86 104 for pol, 0.15 for env). T cells showed 4- to 5-fold higher ZFERV copy than matched tails (lane 9 versus 12). Diagonally hatched bars depict amplications in T-ALL cells from 5 MYC-ER sh (lanes 1822). Cancer ZFERV levels were well above paired tails (compare lanes 1317 to 1822). Slightly lower gains were seen in cancerous than non-malignant MYC-ER T cells (lane 12 versus 23); this reached statistical condence for pol, but not env. Other details are as listed in Figure 3s legend.
Additional cancers pol 30 25 20 15 10 5 0 1 WT tails avg. 2 hlk tails avg. 3 MYC-ER tails avg. Relative copy no. P = 5.86 104 P = 0.19
4 WT T cells avg.
10 WT GFP+ tumor
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Figure 6: ZFERV amplications in two other D. rerio T-cell cancers. One WT WIK sh spontaneously developed a GFP+ thymic tumor and was tested by qPCR of pol (a) and env (b). Average copy numbers of other samples tested previously are shown as black bars. Germline ZFERV copy number in this sh (lane 9) was similar to the 15 WIK sh already examined (lane 1). This tumor showed 5.5-fold amplication (lane 10, diagonal bar), similar to non-malignant T cells and cancers from WT, hlk, and MYC-ER sh (lanes 48). One other hlk T-ALL exhibited high-level, 25- to 30-fold gains (lane 12), although its germline copy number (lane 11) was comparable to other sh (lanes 13).
env: 2.5-fold increase). So, while ZFERV genomic amplication did not dier impressively between WT and cancerprone T cells (3- to 5-fold; Figure 6), expression of retroviral transcripts was more pronounced in T cells from both cancer-prone genotypes. We also examined T-cell cancers from both lines (n = 10; 4 hlk, 6 MYC-ER). In hlk malignancies, all 4 cancers (lanes
47, gray bars) showed increased pol and env compared to normal T cells. One cancer (hlk T-ALL 4; lane 4) resembled premalignant hlk T cells in its transcriptional prole. This same tumor had also been tested by qPCR and showed comparable ZFERV amplication to non-malignant hlk T cells (see Figure 4, lanes 12 and 16). So, in this instance, copy number mirrored ZFERV expression. Three other hlk cancers
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ZFERV RNA expression P = 1.67 80 70 60 50 40 30 20 10 0 Relative expression 105
pol expression 60 50 4 P = 2.84 10 40 30 20 10 0 1 2 3 4 5 hlk T cells hlk T-ALL 4 cancer hlk T-ALL 7 cancer WIK T cells 1631 WIK T cells 3249 P = 0.012 Relative expression
env expression
P = 0.011
3 hlk T cells
9 10 11 12 13 14 15 16 MYC-ER T-ALL cancer avg. MYC-ER T cells MYC-ER T-ALL 10 cancer MYC-ER T-ALL 11 cancer MYC-ER T-ALL 6 cancer MYC-ER T-ALL 7 cancer MYC-ER T-ALL 8 cancer MYC-ER T-ALL 9 cancer
9 10 11 12 13 14 15 16 MYC-ER T-ALL cancer avg. MYC-ER T-ALL 10 cancer MYC-ER T-ALL 11 cancer MYC-ER T cells MYC-ER T-ALL 6 cancer MYC-ER T-ALL 7 cancer MYC-ER T-ALL 8 cancer MYC-ER T-ALL 9 cancer
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(b)
Figure 7: ZFERV gene expression by normal and abnormal D. rerio T cells. Total RNA was tested by qRT-PCR of the ZFERV pol (a) and env (b) genes. Normal T cell RNA pooled from WIK sh (n = 16 and 18; lanes 1, 2) were used as control, and the WIK T cells 1631 sample was arbitrarily set to an expression value = 1. Premalignant T lymphocytes from hlk (n = 6) and MYC-ER (n = 3) sh had higher expression than WT sh (white bars; lanes 3, 9), and this higher transcription reached statistical signicance. Individual cancers from hlk (n = 4; lanes 47) and MYC-ER (n = 6; lanes 1015) sh are depicted with gray bars. Cancer cells from these sh invariably showed higher pol and env transcripts than T cells from WT sh, and nearly always had elevated RNA expression relative to normal T cells from these same two lines. Mean expression of pol and env in malignant T cells (black bars; lanes 8, 16) exceeded both WT and premalignant T cell transcript levels. Two cancers highlighted by asterisks (hlk T-ALL 4, hlk T-ALL 7; lanes 4, 5) were also tested for genomic copy number by qPCR. The hlk T-ALL 4 cancer had ZFERV copy number similar to hlk premalignant T cells (see Figure 4), and its pol and env expression also resembled hlk T cells. Cells from the hlk T-ALL 7 sample had high-level genomic ZFERV gains (see Figure 6), and likewise demonstrated dramatically increased ZFERV transcription.
(Figure 7, lanes 57) had greater pol and env transcription than hlk premalignant T cells, with at least 2-fold increases in both transcripts. One of these (hlk T-ALL 7; lane 5) had markedly higher levels, with 13-fold pol upregulation and 7-fold higher env than non-malignant hlk T cells (lane 3). The hlk T-ALL 7 sample was also analyzed by qPCR and exhibited high copy gains (Figure 6, lane 12), providing a second example that correlated genomic copy number to ZFERV transcriptional activity. Overall, mean transcription was 5-fold greater for pol and 3-fold higher for env in hlk cancers than their pre-cancerous T lymphocytes (compare lane 3 versus 8), although the hlk T-ALL 7 cancer skews this result somewhat. That notwithstanding, every hlk cancer showed 4-fold upregulation of both transcripts relative to WT thymocytes, proving that higher ZFERV expression does coincide with malignancy. Similar ndings were also obtained in 6 MYC-ER cancers (lanes 1015, gray bars). Although transcript levels varied in individual cancers, mean pol expression was 2-fold increased and env was 3-fold higher in all six malignancies compared to MYC-ER premalignant T cells (compare lane 9 versus 16). Expression of pol and env by the same tumor usually followed the same trend. However, some cancers did have discordant transcription of these two genes. Despite these disparities, MYC-ER cancers averaged 18- and 7-fold higher pol and env, respectively, than normal T cells from WT sh, further implicating ZFERV in zebrash T-cell oncogenesis.
Though ZFERV copy number and transcriptional activity correlated in the two cancers where we evaluated both genomic and expression data, variation between pol and env in the same tumor requires another explanation. Unlike ZFERV gag -pol, which is thought to be transcribed as a single RNA, pol and env come from distinct transcripts [11]. Thus, these genes could be dierentially regulated. In addition, other factors may impact overall ZFERV transcription. As noted previously, certain integration sites might foster retroviral activity. In addition, cancers with very high copy number might be expected to have commensurate RNA levels, and our limited data support this. Another potential factor regulating transcription pertains to normal patterns of ZFERV expression in T lymphocytes. While it is known that D. rerio T cells normally make ZFERV RNA ([11] and this work), it is not known if all T-lineage cells do, or rather if only some T lymphocyte developmental stages have active ZFERV. Since T-ALL can exhibit dierentiation arrests at multiple maturational stages [13, 14], it is possible that individual cancers with diering arrest points might also demonstrate dierent ZFERV transcription patterns. Unfortunately, the lack of antibody reagents able to recognize zebrash T cell surface markers currently limit testing of this latter hypothesis. Despite these limitations, our ndings indicate that both genomic amplication and transcription of ZFERV may impact normal D. rerio T-cell biology and oncogenesis. In
10 particular, our results bolster the notion that new retroviral integrations could be pathologic on the molecular level. Stably integrated retroviral elements are common in vertebrate genomes, with nearly 10% of the human genome comprised of ERVs or their derivatives [15]. However, most ERVs are inert due to their accrual of point mutations and partial deletions. ZFERV is atypical in that its genes apparently retain unmutated ORFs. Moreover, these genes are robustly transcribed by D. rerio T cells as veried by ISH, Northern blotting, and qRT-PCR ([11] and this paper). The abundance of ZFERV RNA in T lymphocytes is perhaps not surprising, as ZFERVs LTR was the most potent promoter among several transcriptional regulatory sequences assayed in a carp (Cyprinus carpio) epithelial cell line, including the oft-used CMV promoter [16]. Rather, ZFERVs apparent T-cell specicity may be the more intriguing nding. Shen and Steiner identied putative binding sites for the lymphoid transcription factors Ikaros and Tcf3 (E47) in the ZFERV LTR, but also for other factors (FOS/JUN, C/EBP, STAT, NF-B, and others) that are more general activators of transcription [11]. Indeed, the sequencing of ZFERV-derived transcripts by EST projects from several other tissue types suggests that non-T-cells may transcribe ZFERV also [11]. Whether this nding reects low-level ZFERV transcription by other cell types, or lowlevel T-cell contamination in these tissues, is not clear. In either case, the atypical persistence of intact ZFERV ORFs, and their transcriptional activity in zebrash T cells, raises the question of whether ZFERV proteins might serve a functional purpose. Selective pressure would normally favor mutations disabling a potentially genotoxic retrovirus. Instead, we hypothesize that ZFERV may in fact serve some important biologic role, accounting for its paradoxical maintenance as an active retrovirus in the zebrash genome. ZFERVs apparent absence in the genomes of other Danio genera [11] implies that its entry into zebrash is fairly recent in evolutionary terms, but ZFERV sequences have been identied from several dierent strains, suggesting that its integration is pervasive in the species. It is not known whether ZFERV is present in all D. rerio, and to our knowledge, this question has not been investigated. To date, the closest relative to ZFERV is an exogenous piscine retrovirus, SSSV. Curiously, this virus is linked to swim bladder leiomyosarcomas in Atlantic salmon, and like our results with ZFERV, these tumors show high copy number proviral SSSV integration [17]. Besides its close relation to SSSV, ZFERV also shares sequence conservation and similar genomic structure with gammaretroviridae of the murine leukemia virus (MLV) class [11, 17]. MLV-related retroviruses are known to be oncogenic by insertional mutagenesis [18], and the determinants governing their preferred integration sites have been the subject of intense scientic scrutiny [1921]. Although an obvious ZFERV homologue has not been identied in humans, other MLV-related sequences have been detected in human cell lines. However, it appears that these retroviral sequences may have been acquired by human cells during xenografting into murine recipients or result from reagent contamination by murine DNA [22, 23]. In addition, a
Advances in Hematology long ORF on human chromosome 14 bears high homology to ZFERVs env, and upstream sequences contain a short gag -pol element [24]. So, ZFERV-related retroviruses are evidently integrated in the human genome as well. Incorporating all these circumstantial data, it becomes plausible that ZFERV integrationslike SSSV and MLVmay not only be oncogenic in zebrash, but might also have relevance for human biology in general.
4. Conclusions
Nearly a decade ago, Shen and Steiner discovered a zebrash endogenous retrovirus, ZFERV, based on its high transcriptional activity in larval and adult D. rerio thymocytes [11]. Their work suggested that multiple copies of ZFERV existed in the zebrash genome, and since that time, the loci where ZFERV resides still have not been denitively assigned. These diculties are probably attributable to the fact that this multicopy locus may vary in copy number and genomic positioning in dierent sh. Amidst this backdrop, we have found that ZFERV copy number is increased still further in every D. rerio T-cell malignancy we examined from 4 dierent genetic lines. Somewhat surprisingly, our results demonstrate that ZFERV amplication is not unique to cancerous T cells. Rather, copy number gains also occur in T lymphocytes of WT D. rerio, the same cells where ZFERV transcription was rst identied. Moreover, ZFERV copy number appears to be fairly consistent amongst normal, premalignant, and malignant T cells (Figure 6, lanes 48), although individual cancers can occasionally show even higher levels of ZFERV in their genomes. It is possible that individual normal T cells have similar variability in ZFERV copy number, but this has not been experimentally addressed. As seen with genomic amplications, ZFERV transcription occurs within normal, pre-cancerous, and neoplastic T cells. Our results suggest that expression of ZFERV RNAs is higher in cancer samples, but we do not recognize a consistent trend from one cancer to the next. Nonetheless, these commonalties between normal and malignant T lymphocytes imply that ZFERV activation and amplication may be a normal feature of zebrash T cell biology, with no pathologic consequence. Still, ZFERVs abundant transcription, apparently functional ORFs, and ability to undergo genomic amplication all allude to its oncogenic potential. Compounded with mutations like hlk, srk, and otg that confer malignancy predisposition, ZFERV may help promote T-cell transformation. Given that the closest phylogenetic relatives of ZFERV are an exogenous piscine retrovirus linked to sarcomagenesis and MLV-class retroviruses that are leukemogenic via genomic integration, it is tempting to speculate that ZFERV may contribute to cellular immortalization by similar mechanisms. Certainly, ZFERV integrations in crucial genomic sites could have transformative properties. For example, integration within a tumor suppressor gene might render it unable to generate its normal protein product, thereby ablating function. Conversely, integrations into the promoter or
Advances in Hematology enhancer regions of proto-oncogenes might augment their transcription. Since ZFERV appears to be specically and highly expressed by thymocytes, this scenario could be analogous to the translocation of proto-oncogenes into the T-cell receptor loci, which are well described in T-ALL [25, 26]. However, proof of this hypothesis will require identication of somatically acquired ZFERV integrations at these genomic sites. At this point, the possibility that ZFERV amplications may contribute to T-cell oncogenesis remains an open question that will require further investigation to resolve decisively.
11
ZNF198-broblast growth factor receptor-1 chimeric tyrosine kinase, Blood, vol. 114, no. 8, pp. 15761584, 2009. E. Clappier, B. Gerby, F. Sigaux et al., Clonal selection in xenografted human T cell acute lymphoblastic leukemia recapitulates gain of malignancy at relapse, Journal of Experimental Medicine, vol. 208, no. 4, pp. 653661, 2011. C. H. Shen and L. A. Steiner, Genome structure and thymic expression of an endogenous retrovirus in zebrash, Journal of Virology, vol. 78, no. 2, pp. 899911, 2004. D. M. Langenau, A. A. Ferrando, D. Traver et al., In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrash, Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 19, pp. 73697374, 2004. A. A. Ferrando, D. S. Neuberg, J. Staunton et al., Gene expression signatures dene novel oncogenic pathways in T cell acute lymphoblastic leukemia, Cancer Cell, vol. 1, no. 1, pp. 7587, 2002. A. A. Ferrando and A. T. Look, Gene expression proling in T-cell acute lymphoblastic leukemia, Seminars in Hematology, vol. 40, no. 4, pp. 274280, 2003. E. S. Lander, L. M. Linton, B. Birren et al., Initial sequencing and analysis of the human genome, Nature, vol. 409, no. 6822, pp. 860921, 2001. S. Ruiz, C. Tafalla, A. Cuesta, A. Estepa, and J. M. Coll, In vitro search for alternative promoters to the human immediate early cytomegalovirus (IE-CMV) to express the G gene of viral haemorrhagic septicemia virus (VHSV) in sh epithelial cells, Vaccine, vol. 26, no. 51, pp. 66206629, 2008. T. A. Paul, S. L. Quackenbush, C. Sutton, R. N. Casey, P. R. Bowser, and J. W. Casey, Identication and characterization of an exogenous retrovirus from Atlantic salmon swim bladder sarcomas, Journal of Virology, vol. 80, no. 6, pp. 29412948, 2006. H. Mikkers and A. Berns, Retroviral insertional mutagenesis: tagging cancer pathways, Advances in Cancer Research, vol. 88, pp. 5399, 2003. X. Wu, Y. Li, B. Crise, and S. M. Burgess, Transcription start regions in the human genome are favored targets for MLV integration, Science, vol. 300, no. 5626, pp. 17491751, 2003. C. Cattoglio, G. Maruggi, C. Bartholomae et al., High-denition mapping of retroviral integration sites denes the fate of allogeneic T cells after donor lymphocyte infusion, PLoS One, vol. 5, no. 12, Article ID e15688, 2010. L. Biasco, A. Ambrosi, D. Pellin et al., Integration prole of retroviral vector in gene therapy treated patients is cell-specic according to gene expression and chromatin conformation of target cell, EMBO Molecular Medicine, vol. 3, no. 2, pp. 89 101, 2011. O. Cingoz and J. M. Con, Endogenous murine leukemia viruses: relationship to XMRV and related sequences detected in human DNA samples, Advances in Virology, vol. 2011, Article ID 940210, 10 pages, 2011. J. Blomberg, A. Sheikholvaezin, A. Elfaitouri et al., Phylogeny-directed search for murine leukemia virus-like retroviruses in vertebrate genomes and in patients suering from myalgic encephalomyelitis/chronic fatigue syndrome and prostate cancer, Advances in Virology, vol. 2011, Article ID 341294, 20 pages, 2011. P. Villesen, L. Aagaard, C. Wiuf, and F. S. Pedersen, Identication of endogenous retroviral reading frames in the human genome, Retrovirology, vol. 1, p. 32, 2004. B. Johansson, F. Mertens, and F. Mitelman, Clinical and biological importance of cytogenetic abnormalities in childhood
[10]
[11]
[12]
Acknowledgments
The authors appreciate the contributions of Lynnie Rudner, Ph.D., Alexandra Keefe, and Nathan Meeker, M.D., who also participated on this project. They thank Alejandro Gutierrez, M.D., and A. Thomas Look, M.D., for sharing the MYC-ER zebrash line used in some of these studies. J. Kimble Frazer was supported by Eunice Kennedy Shriver NICHD Award K08-HD053350 and the CHRC at the University of Utah. Nikolaus S. Trede was supported by NIAID Award R21AI079784 and the Huntsman Cancer Foundation. Huntsman Cancer Institute core facilities supported by NCI P30CA042014 also contributed to this work.
[13]
[14]
[15]
[16]
References
[1] D. M. Langenau, D. Traver, A. A. Ferrando et al., Mycinduced T cell leukemia in transgenic zebrash, Science, vol. 299, no. 5608, pp. 887890, 2003. [2] H. E. Sabaawy, M. Azuma, L. J. Embree, H. J. Tsai, M. F. Starost, and D. D. Hickstein, TEL-AML1 transgenic zebrash model of precursor B cell lymphoblastic leukemia, Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 41, pp. 1516615171, 2006. [3] H. Feng, D. M. Langenau, J. A. Madge et al., Heat-shock induction of T-cell lymphoma/leukaemia in conditional Cre/ lox-regulated transgenic zebrash, British Journal of Haematology, vol. 138, no. 2, pp. 169175, 2007. [4] J. Chen, C. Jette, J. P. Kanki, J. C. Aster, A. T. Look, and J. D. Grin, NOTCH1-induced T-cell leukemia in transgenic zebrash, Leukemia, vol. 21, no. 3, pp. 462471, 2007. [5] A. Gutierrez, R. Grebliunaite, H. Feng et al., Pten mediates Myc oncogene dependence in a conditional zebrash model of T cell acute lymphoblastic leukemia, Journal of Experimental Medicine, vol. 208, no. 18, pp. 15951603, 2011. [6] J. K. Frazer, N. D. Meeker, L. Rudner et al., Heritable Tcell malignancy models established in a zebrash phenotypic screen, Leukemia, vol. 23, no. 10, pp. 18251835, 2009. [7] H. Feng, D. L. Stachura, R. M. White et al., T-lymphoblastic lymphoma cells express high levels of BCL2, S1P1, and ICAM1, leading to a blockade of tumor cell intravasation, Cancer Cell, vol. 18, no. 4, pp. 353366, 2010. [8] L. A. Rudner, K. H. Brown, K. P. Dobrinski et al., Shared acquired genomic changes in zebrash and human T-ALL, Oncogene, vol. 30, no. 41, pp. 42894296, 2011. [9] M. Ren, X. Li, and J. K. Cowell, Genetic ngerprinting of the development and progression of T-cell lymphoma in a murine model of atypical myeloproliferative disorder initiated by the
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
12
and adult acute lymphoblastic leukemia, Annals of Medicine, vol. 36, no. 7, pp. 492503, 2004. [26] P. Van Vlierberghe, R. Pieters, H. B. Beverloo, and J. P. P. Meijerink, Molecular-genetic insights in paediatric T-cell acute lymphoblastic leukaemia, British Journal of Haematology, vol. 143, no. 2, pp. 153168, 2008.
Advances in Hematology
Hindawi Publishing Corporation Advances in Hematology Volume 2012, Article ID 541471, 6 pages doi:10.1155/2012/541471
of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), 76133 Karlsruhe, Germany Cycle and Cancer Genetics Laboratory, Peter MacCallum Cancer Centre, East Melbourne, VIC 3002, Australia 3 Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia Correspondence should be addressed to C. Grabher, [email protected] Received 9 February 2012; Accepted 29 April 2012 Academic Editor: Christopher Hall Copyright 2012 C. Wittmann et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Starting as a model for developmental genetics, embryology, and organogenesis, the zebrash has become increasingly popular as a model organism for numerous areas of biology and biomedicine over the last decades. Within haematology, this includes studies on blood cell development and function and the intricate regulatory mechanisms within vertebrate immunity. Here, we review recent studies on the immediate mechanisms mounting an inammatory response by in vivo analyses using the zebrash. These recently revealed novel roles of the reactive oxygen species hydrogen peroxide that have changed our view on the initiation of a granulocytic inammatory response.
1. Introduction
The innate immune system comprises the cells and mechanisms that defend the host from infection by other organisms or damage to tissue integrity, in a nonspecic manner. This means that the cells of the innate system recognise and respond to pathogens and trauma in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. The innate immune system provides an immediate defence. A typical vertebrate immune response depends on the orchestrated motility and activity of various haematopoietic compartments and their interactions that ultimately control the magnitude of the response [13]. Inammation is one of the rst responses of the immune system to infection or irritation. Stimulated by factors released from injured cells, it serves to establish a physical barrier against the spread of infection. This further promotes healing of any damaged tissue following the clearance of pathogens or cell debris. Molecules produced during inammation sensitise pain receptors, cause localised vasodilatation of blood vessels, and attract phagocytes, especially neutrophils and macrophages, which then trigger other parts of the immune system. Failure to initiate a response allows uncontrolled proliferation of invading microorganisms and severe tissue
damage that may become fatal. Failure to resolve an immune response can also cause severe tissue damage, due to persistent degranulation, and may lead to chronic inammation, which ceases to be benecial to the host. Overall, inammation is now recognised as a central feature of prevalent pathologies, such as atherosclerosis, cancer, asthma, thyroiditis, inammatory bowel disease, autoimmune disease, as well as Alzheimers and Parkinsons disease [46]. Hence, the regulation of an inammatory response is an active eld of research. New players or novel functions of old players continue to be identied and we are only beginning to understand their specic function at the corresponding level during inammation. Hydrogen peroxide is an example of a molecule with a long known function for pathogen clearance in inammation. Here, we discuss how recent work using the zebrash model has revealed a pivotal role of hydrogen peroxide in mounting an inammatory response.
2 superoxide, hydroxyl, peroxyl, and alkoxyl; or the nonradicals: hypochlorous acid, ozone, singlet oxygen, and the current topic in focus, hydrogen peroxide [7]. ROS generation can either occur as a by-product of cellular metabolism (e.g., in mitochondria through autoxidation of respiratory chain components) or it can be created by enzymes with the primary function of ROS generation [8]. Enzymes capable of rapidly increasing local H2 O2 levels include the family of NADPH oxidases [7] and other oxidases such as xanthine oxidase [9] and 5-lipoxygenase [10]. The mammalian NADPH oxidase family encompasses 7 members, which are NOX1-5 and DUOX1-2. To date, a single isoform of duox and four nox genes (nox1, 2, 4, 5) have been identied in the zebrash genome [11]. Each member is capable of converting NADPH to NADP+ and then transporting the freed electrons across membranes. DUOX enzymes are capable of direct hydrogen peroxide production, while NOXes1-5 produce superoxide, which is rapidly converted to H2 O2 by a separate superoxide dismutase or occurs spontaneously [12]. H2 O2 may subsequently be utilised by peroxidase, such as thyroperoxidase, to produce thyroid hormones or myeloperoxidase and lactoperoxidase to generate more potent ROS. However, if not consumed, high concentrations of H2 O2 may result in DNA damage and modications of proteins, lipids and other molecules [13]. Thus, to avoid H2 O2 -mediated deleterious eects, excess H2 O2 is usually rapidly catalysed or reduced by various antioxidant enzymes: such as glutathione peroxidase and catalase [14].
Advances in Hematology membrane to the extracellular or intra-phagosomal space. Superoxide is further converted into hydrogen peroxide either through spontaneous dismutation, which involves the consumption of two protons, or facilitated by the catalytic activity of superoxide dismutase. Hydrogen peroxide alone and in conjunction with the amplication activity of myeloperoxidase (MPO) is responsible for bacterial killing [23, 24]. MPO, which is abundantly present in phagocyte granules, catalyses the conversion of halides and pseudohalides such as Cl , I , Br , and SCN to form hypohalous acids or pseudohypohalous acids. HOCl, however, is the primary MPO product in neutrophils responsible for bacterial killing. 3.1.2. Hydrogen Peroxide Mounting an Inammatory Response. Recent advances accomplished by utilising the model organism zebrash greatly expanded our view of H2 O2 mediated cellular activities. The optical transparency of zebrash larvae oers the unique advantage of real-time monitoring an immune response in a whole animal context. This is in contrast to in vitro studies and/or end-point analyses of stained tissues. Additionally, a recently developed genetically encoded H2 O2 sensor provided an elegant solution for investigating the role of hydrogen peroxide dynamics during an immune response in vivo [25]. The previous view on the critical mechanisms in immediate inammation focused on the activity of damageassociated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). Tissue damage results in the release of intracellular DAMPs usually hidden from the immune system (i.e., ATP, uric acid, lipids, DNA, nuclear proteins) or extracellular DAMPs released through degradation of extracellular matrix upon tissue injury (i.e., hyaluronan, byglycan, heparan sulfate). The receiving cell senses these signals through 5 dierent types of pattern recognition receptors (PRRs). Activation of these receptors in turn activates downstream NFkB, MAPK, or type I interferon-signalling pathways that are important for inammatory and antimicrobial responses. The signicance of DAMPs, PAMPs, and PRRs is comprehensively reviewed elsewhere [26, 27]. However, the mechanisms for immediate immune cell recruitment were not well dened. Recently, Niethammer et al. described for the rst time that wounded epithelium of zebrash larvae produces a tissue-scale gradient of H2 O2 mediating leukocyte recruitment [28]. This nding was in contrast to the prevalent view that leukocytes undergoing an oxidative burst response were the only source of H2 O2 at a site of trauma or infection [29]. The authors employed the genetically encoded ratiometric HyPer sensor to visualise H2 O2 in vivo and in real time. HyPer consists of the bacterial H2 O2 -sensitive transcription factor, OxyR, fused to a circularly permutated yellow uorescent protein (YFP). Cysteine oxidation of OxyR induces a conformational change in the YFP that increases emission excited at 500 nm and decreases emission excited at 420 nm. This change is rapidly reversible within the reducing cytoplasmic environment, which allows dynamic monitoring of the intracellular hydrogen peroxide concentration [30].
3. Functional Activities of H2 O2
H2 O2 is also involved in many regulatory cellular events including the activation of transcription factors, cell proliferation, and apoptosis [8]. H2 O2 produced from the mitochondrial electron transport chain has been shown to play a role in haematopoietic cell dierentiation and cell division in ies [15, 16]. NADPH oxidase generated H2 O2 can aect cardiac dierentiation [17], vascularisation [18], and angiogenesis [19]. In targeting cysteine and methionine residues of protein kinases and phosphatases, H2 O2 is capable of modulating a number of principal signalling cascades including ERK, JNK, p38, MAPK, and PI3K/Akt [20, 21]. 3.1. Inammation-Related Functions 3.1.1. Respiratory Burst. The classical physiological role attributed to H2 O2 is its capability to induce bacterial killing [12]. NOX2 is the enzyme responsible for phagocyte respiratory burst responses and is expressed in neutrophils, eosinophils, monocytes/macrophages, as well as nonphagocytic cells such as broblasts, cardiomyocytes, haematopoietic stem cells, and endothelial cells [7]. Under resting conditions neutrophil NOX2 resides in secondary granules, which upon activation of neutrophils fuse with phagosomal as well as plasma membranes [22]. The NADPH-oxidase-mediated respiratory burst response of neutrophils generates two superoxide anions by transporting two electrons from one NADPH across the
Advances in Hematology Tailn transection on zebrash larvae induced a rapid increase in H2 O2 levels extending approximately 100200 m from the wound margin as a decreasing concentration gradient. Furthermore, gradient formation preceded leukocyte arrival at the scene and H2 O2 levels started to decrease again with accumulation of immune cells. Generation of the gradient as well as leukocyte recruitment was dependent on the activity of Duox in epithelial cells. Both, genetic knockdown of Duox and chemical inhibition of oxidase activity abolished gradient formation and signicantly decreased absolute numbers of leukocytes at the wound margin, without aecting general cellular motility. These ndings were corroborated by a study in drosophila focusing on prioritising competing signals by migrating macrophages [31] emphasising the crucial role of the tissue scale gradient of H2 O2 for leukocyte attraction. A study, also using zebrash larvae, demonstrated that newly oncogene-transformed cells and their neighbours attracted leukocytes through H2 O2 signalling. Utilising the H2 O2 -indicating dye, acetyl-pentauorobenzene sulphonyl uorescein, and 5,5-dimethyl-l-pyrroline N-oxide (DMPO) that reports a history of ROS exposure, it was shown that H2 O2 was stochastically and momentarily produced around V12RAS expressing cells in the epidermis. Like wounded epithelial cells, transformed cells generated H2 O2 in a Duox dependent manner, highlighting parallels between oncogene-transformed cells and mechanical induced injury initiation of the host inammatory response [32]. 3.1.3. Hydrogen Peroxide as a Signalling Molecule in Inammation. Functional roles of H2 O2 during inammation have been observed previously. Mechanistically, hydrogen peroxide can modulate protein function by reversible chemical modication of protein thiols, which can result in conformational changes aecting DNA binding, enzymatic activity, multimerisation, or protein complex formation. For example, the NFkB/Rel family, key regulatory molecules in the transcription of many genes involved in inammation, is a well-known redox-sensitive transcription factor family [33]. H2 O2 -induced activation of NFkB, which includes tyrosine phosphorylation of IkB and activation of IKK by H2 O2 has been reported [34, 35]. Moreover, H2 O2 can activate the release of high mobility group 1 protein from macrophages resulting in amplication of proinammatory stimuli [36] or modulate leukocyte adhesion molecule expression and leukocyte endothelial adhesion [29]. VCAM1, an endothelial scaold on which leukocytes migrate, can activate signals in endothelial cells required for VCAM1-dependent leukocyte migration. Leukocyte binding to VCAM-1 stimulates NOX2 in endothelial cells, resulting in the generation of H2 O2 , which locally activates matrix metalloproteinases (MMPs). These MMPs in turn degrade matrix and endothelial cell surface receptors in cell junctions facilitating leukocyte transendothelial migration [37, 38]. These examples show how H2 O2 can act as an intracellular or local signalling molecule but long-distance intercellular mechanisms of H2 O2 -mediated leukocyte recruitment were less well dened.
3 The open question of how leukocytes may receive the signal to initiate directional migration was recently addressed in another elegant study using the zebrash model by Yoo et al. [39]. They have identied the SRC family kinase (SFK) Lyn as a redox sensor in neutrophils that detects hydrogen peroxide emanating from wounds and guiding their migration. Yoo and colleagues were able to provide direct evidence for punctate SFK activation at the leading edge of neutrophils in response to wounding. Through the knockdown of Duox, which is responsible for H2 O2 production at the wound margin, they have explored the role of H2 O2 in SFK activation. Duox knockdown prevented SFK phosphorylation indicating that activation of neutrophil SFKs may be dependent on the presence of hydrogen peroxide levels at wounds. Further evidence suggesting that SFKs can act as a redox sensor was provided by utilisation of SFK inhibitors that resulted in impairment of early neutrophil accumulation, while having no eect on epithelial hydrogen peroxide bursts [39]. Proling SFK family members in zebrash myeloid cells identied the Lyn kinase as a promising candidate acting as the redox sensor in neutrophils and macrophages. Morpholino knockdown of Lyn impaired directional migration of neutrophils to a tailn wound in zebrash larvae. Further in vitro investigation revealed that hydrogen peroxide directly activates Lyn through the oxidation of Cys466, leading to downstream signalling, for example, Erk activation. This in vitro evidence was elegantly conrmed in vivo using a combination of genetic knockdown of Lyn and neutrophil-specic transgenic reconstitution of a Cys466 mutant or wild-type Lyn-GFP fusion. In conclusion, these two sophisticated studies demonstrated a novel role of H2 O2 as mediator of immediate inammation and revealed aspects of the mechanisms resulting in leukocyte recruitment to a site of trauma (Figure 1). Evidence is accumulating that H2 O2 signalling to phagocytes is a widely conserved mechanism present not only in zebrash [28, 32, 39] but also ies [31] and mammals [39, 40].
4. Outlook
The discovery of a new biological mechanism opens up a new line of research and poses numerous new questions to address. The most obvious being: How is Duox activated in epithelial cells upon wounding and how is the H2 O2 gradient resolved? One hypothesis would place calcium as the immediate injury signal to the wounded cell in order to produce hydrogen peroxide through Duox. Physical disruption of plasma membranes results in an uncontrolled inux of calcium [41]. Giving credence to this hypothesis, evidence exists showing that DUOX activation by calcium regulates H2 O2 generation [42]. In order to avoid excess tissue damage and persistent granulocyte recruitment/retention, the presence of the hydrogen peroxide gradient must be tightly regulated. Regulation could occur on the enzymatic level in terms of H2 O2 production as well as on the molecular level in terms of H2 O2
Advances in Hematology
H2 O2 establishment
2NADPH 2NADP+ + 2H+ EF-hands Ca2+ ? Ca2+ Duox Ca2+ Peroxide homology domain
H2 O2
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H2 O2 H2 O2 Migrating neutrophil
Lyn pLyn
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H2 O2 modulation by neutrophils
Glutathione Phagosome peroxidase2H O 2O2 4H+ 2 Nox2 2O2 4O2 2H2 O2 H2 O2 2H2 O2 Catalase SOD 4O2 2H+ Mpo 2Cl 2HOCl H2 O2 Mpo 2HOCl Cl 2H2 O
H2 O2
Bacteria
Figure 1: The role of hydrogen peroxide during the inammatory response. (a) Upon tissue injury/trauma, epithelial cells adjacent to damaged cells activate the NADPH oxidase, Duox. Duox generates and establishes a localised tissue scale gradient of hydrogen peroxide, (b) Potential cellular events that result in Duox activation in epithelial cells. Disruption of epithelial cell membranes by mechanical trauma could lead to an increased inux of calcium in adjacent cells. Calcium binding to the EF-hand domain of Duox (residing in plasma membranes of epithelial cells), may initiate generation of hydrogen peroxide. (c) A tissue scale gradient of hydrogen peroxide acts as the rst attraction signal for leukocytes. (d) Neutrophils sense hydrogen peroxide emanating from the wound partly through Lyn, a Src family kinase. Oxidation of Cys466 activates Lyn, resulting in autophosphorylation (pLyn) and punctate appearance of pLyn at the neutrophil leading edge is observed. (e) At the site of injury, neutrophils may alter hydrogen peroxide levels, both by consuming epithelial-derived hydrogen peroxide (A) or by local production of hydrogen peroxide through oxidative bursts (B). (f) Antioxidants, such as glutathione peroxidase and catalase could catalyse the decomposition of hydrogen peroxide into oxygen and water, while myeloperoxidase (Mpo) may consume hydrogen peroxide to produce hypochlorous acid (A). Neutrophils are equipped with multiple mechanisms to kill foreign organisms, one of them being the generation of ROS. Upon activation, phagosomal Nox2 generates superoxide, which is further converted into hydrogen peroxide by superoxide dismutase (SOD). Hydrogen peroxide alone and in conjunction with hypochlorous acid, generated by myeloperoxidase and other ROS exert bactericidal functions (B).
Advances in Hematology stability. Oxidase activity results in membrane depolarisation due to the electrogenic properties of the enzymes to the point of NADPH oxidase inhibition. Prolonged H2 O2 production depletes the NADPH pools, which may automatically result in cessation of H2 O2 generation. Alternatively or in addition, neutrophil MPO could be responsible for the decrease in hydrogen peroxide levels upon arrival at the wound [24]. This mechanism suggests new approaches to therapeutically modulate both the onset of the cellular inammatory response and its resolution, particularly as it involves a small, relatively unstable signalling molecule and is dependent on multiple enzymatic steps amenable to pharmacologic intervention.
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[10] A. T. Demiry urek and R. M. Wadsworth, Superoxide in the pulmonary circulation, Pharmacology and Therapeutics, vol. 84, no. 3, pp. 355365, 1999. [11] B. T. Kawahara, M. T. Quinn, and J. D. Lambeth, Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes, BMC Evolutionary Biology, vol. 7, article 109, 2007. [12] B. Rada and T. Leto, Oxidative innate immune defenses by Nox/Duox Family NADPH oxidases, Contributions to Microbiology, vol. 15, pp. 164187, 2008. [13] N. Driessens, S. Versteyhe, C. Ghaddhab et al., Hydrogen peroxide induces DNA single- and double-strand breaks in thyroid cells and is therefore a potential mutagen for this organ, Endocrine-Related Cancer, vol. 16, no. 3, pp. 845856, 2009. [14] T. L. Leto and M. Geiszt, Role of Nox family NADPH oxidases in host defense, Antioxidants and Redox Signaling, vol. 8, no. 9-10, pp. 15491561, 2006. [15] E. Owusu-Ansah, A. Yavari, S. Mandal, and U. Banerjee, Distinct mitochondrial retrograde signals control the G1-S cell cycle checkpoint, Nature Genetics, vol. 40, no. 3, pp. 356361, 2008. [16] E. Owusu-Ansah and U. Banerjee, Reactive oxygen species prime Drosophila haematopoietic progenitors for dierentiation, Nature, vol. 461, no. 7263, pp. 537541, 2009. [17] J. Li, M. Stous, L. Serrander et al., The NADPH oxidase NOX4 drives cardiac dierentiation: role in regulating cardiac transcription factors and MAP kinase activation, Molecular Biology of the Cell, vol. 17, no. 9, pp. 39783988, 2006. [18] S. Lange, J. Heger, G. Euler, M. Wartenberg, H. M. Piper, and H. Sauer, Platelet-derived growth factor BB stimulates vasculogenesis of embryonic stem cell-derived endothelial cells by calcium-mediated generation of reactive oxygen species, Cardiovascular Research, vol. 81, no. 1, pp. 159168, 2009. [19] R. Colavitti, G. Pani, B. Bedogni et al., Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR, Journal of Biological Chemistry, vol. 277, no. 5, pp. 31013108, 2002. [20] K. Hensley, K. A. Robinson, S. P. Gabbita, S. Salsman, and R. A. Floyd, Reactive oxygen species, cell signaling, and cell injury, Free Radical Biology and Medicine, vol. 28, no. 10, pp. 1456 1462, 2000. [21] S. P. Gabbita, K. A. Robinson, C. A. Stewart, R. A. Floyd, and K. Hensley, Redox regulatory mechanisms of cellular signal transduction, Archives of Biochemistry and Biophysics, vol. 376, no. 1, pp. 113, 2000. [22] W. M. Nauseef, Assembly of the phagocyte NADPH oxidase, Histochemistry and Cell Biology, vol. 122, no. 4, pp. 277291, 2004. [23] W. M. Nauseef, Contributions of myeloperoxidase to proinammatory events: more than an antimicrobial system, International Journal of Hematology, vol. 74, no. 2, pp. 125133, 2001. [24] S. J. Klebano, Myeloperoxidase: friend and foe, Journal of Leukocyte Biology, vol. 77, no. 5, pp. 598625, 2005. [25] L. Pase, C. J. Nowell, and G. J. Lieschke, In vivo real-time visualization of leukocytes and intracellular hydrogen peroxide levels during a zebrash acute inammation assay, Methods in Enzymology, vol. 506, pp. 135156, 2012. [26] G. Y. Chen and G. Nu nez, Sterile inammation: sensing and reacting to damage, Nature Reviews Immunology, vol. 10, no. 12, pp. 826837, 2010. [27] O. Takeuchi and S. Akira, Pattern recognition receptors and inammation, Cell, vol. 140, no. 6, pp. 805820, 2010.
Authors Contribution
C. Wittman and P. Chockley contributed equally to this paper.
Acknowledgments
CW was supported by a PhD fellowship from the Helmholtz program BioInterfaces international graduate school (BIFIGS). Further support was provided by a KIT-RISC grant and by a Marie Curie International Reintegration Grant within the 7th European Community Framework Program (PIRG07-GA-2010-267552) to CG. The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government.
References
[1] J. Banchereau and R. M. Steinman, Dendritic cells and the control of immunity, Nature, vol. 392, no. 6673, pp. 245252, 1998. [2] K. Hoebe, E. Janssen, and B. Beutler, The interface between innate and adaptive immunity, Nature Immunology, vol. 5, no. 10, pp. 971974, 2004. [3] C. N. Serhan and J. Savill, Resolution of inammation: the beginning programs the end, Nature Immunology, vol. 6, no. 12, pp. 11911197, 2005. [4] L. M. Coussens and Z. Werb, Inammation and cancer, Nature, vol. 420, no. 6917, pp. 860867, 2002. [5] P. Libby, Inammation in atherosclerosis, Nature, vol. 420, no. 6917, pp. 868874, 2002. [6] C. L. Van Hove, T. Maes, G. F. Joos, and K. G. Tournoy, Chronic inammation in asthma: a contest of persistence vs resolution, Allergy, vol. 63, no. 9, pp. 10951109, 2008. [7] K. Bedard and K. H. Krause, The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology, Physiological Reviews, vol. 87, no. 1, pp. 245313, 2007. [8] M. Rojkind, J. A. Dom nguez-Rosales, N. Nieto, and P. Greenwel, Role of hydrogen peroxide and oxidative stress in healing responses, Cellular and Molecular Life Sciences, vol. 59, no. 11, pp. 18721891, 2002. [9] C. A. Pritsos, Cellular distribution, metabolism and regulation of the xanthine oxidoreductase enzyme system, ChemicoBiological Interactions, vol. 129, no. 1-2, pp. 195208, 2000.
6
[28] P. Niethammer, C. Grabher, A. T. Look, and T. J. Mitchison, A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrash, Nature, vol. 459, no. 7249, pp. 996999, 2009. [29] C. K. Sen and S. Roy, Redox signals in wound healing, Biochimica et Biophysica Acta, vol. 1780, no. 11, pp. 13481361, 2008. [30] V. V. Belousov, A. F. Fradkov, K. A. Lukyanov et al., Genetically encoded uorescent indicator for intracellular hydrogen peroxide, Nature Methods, vol. 3, no. 4, pp. 281286, 2006. [31] S. Moreira, B. Stramer, I. Evans, W. Wood, and P. Martin, Prioritization of competing damage and developmental signals by migrating macrophages in the Drosophila embryo, Current Biology, vol. 20, no. 5, pp. 464470, 2010. [32] Y. Feng, C. Santoriello, M. Mione, A. Hurlstone, and P. Martin, Live imaging of innate immune cell sensing of transformed cells in zebrash larvae: parallels between tumor initiation and wound inammation, PLoS Biology, vol. 8, no. 12, Article ID e1000562, 2010. [33] H. Jay Forman and M. Torres, Redox signaling in macrophages, Molecular Aspects of Medicine, vol. 22, no. 4-5, pp. 189216, 2001. [34] S. Schoonbroodt, V. Ferreira, M. Best-Belpomme et al., Crucial role of the amino-terminal tyrosine residue 42 and the carboxyl- terminal PEST domain of IB in NF-B activation by an oxidative stress, Journal of Immunology, vol. 164, no. 8, pp. 42924300, 2000. [35] Z. Yin, V. N. Ivanov, H. Habelhah, K. Tew, and Z. Ronai, Glutathione S-Transferase p elicits protection against H2 O2 induced cell death via coordinated regulation of stress kinases, Cancer Research, vol. 60, no. 15, pp. 40534057, 2000. [36] D. Tang, Y. Shi, R. Kang et al., Hydrogen peroxide stimulates macrophages and monocytes to actively release HMGB1, Journal of Leukocyte Biology, vol. 81, no. 3, pp. 741747, 2007. [37] J. S. Alexander and J. W. Elrod, Extracellular matrix, junctional integrity and matrix metalloproteinase interactions in endothelial permeability regulation, Journal of Anatomy, vol. 200, no. 6, pp. 561574, 2002. [38] J. M. Cook-Mills, Hydrogen peroxide activation of endothelial cell-associated MMPS during VCAM-1-dependent leukocyte migration, Cellular and Molecular Biology, vol. 52, no. 4, pp. 816, 2006. [39] S. K. Yoo, T. W. Starnes, Q. Deng, and A. Huttenlocher, Lyn is a redox sensor that mediates leukocyte wound attraction in vivo, Nature, vol. 480, pp. 109112, 2011. [40] I. V. Klyubin, K. M. Kirpichnikova, and I. A. Gamaley, Hydrogen peroxide-induced chemotaxis of mouse peritoneal neutrophils, European Journal of Cell Biology, vol. 70, no. 4, pp. 347351, 1996. [41] A. Draeger, K. Monastyrskaya, and E. B. Babiychuk, Plasma membrane repair and cellular damage control: the annexin survival kit, Biochemical Pharmacology, vol. 81, no. 6, pp. 703712, 2011. [42] S. Rigutto, C. Hoste, H. Grasberger et al., Activation of dual oxidases Duox1 and Duox2: dierential regulation mediated by cAMP-dependent protein kinase and protein kinase Cdependent phosphorylation, Journal of Biological Chemistry, vol. 284, no. 11, pp. 67256734, 2009.
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