Chapter 5:

Drosophila
4 BIO- 8 (Group 5)
BUHAYO, Joan PICHON, Jasmine

CAYETANO, Nickey Angelo POLICINA, Monica

ENRIQUEZ, Jigs Vergel TRIA, Hannah

MESA, Hannah Marie Frances

 Introduction
First organism whose development was understood in
molecular detail.

Suitable for genetic experimentation due to its small size and

short life cycle (2 weeks).

In developmental biology, Drosophila, were used to study

mutagenesis to produce mutants with interesting looking
phenotypes.

Drosophila genes are named with the first letter lowercase if the
first allele to be discovered was recessive.
 Insects
All adult and larval insects are
built up of 3 anteroposterior
segments: head, thorax, and
abdomen.

They all exhibit their maximum

morphological similarity during a
phylotypic (extended germband)
stage.

Insect species differ in number of

abdominal segments (ranging
from 8-11)
 For Drosophila
Does not display prosencephalic
head segments

3 gnathal segments appear only

transiently
(T1-3)

Only mesothorax bears wings, and

are represented with balancing
structures called halteres

Parasegments: repeating units which

have the same period but are out
phase with the later segments

8 abdominal segments (A1-8)

Insects
Holometabolous vs. Hemimatabolous life
cycle
 Drosophila life cycle
Holometabolous: undergoes
complete metamorphosis, in which
the larva is different from the adult
structure.
Larva grows and molts 2 times
before becoming a pupa.
Pupa: resting stage where body is
remolded to form the adult.
Much of the body is formed from
the imaginal discs and the
abdominal histoblasts.
 Normal development: Oogenesis
Eggs of Drosophilas are laid with
most of its pattern is specified.

1 germ cell 16 cells (1 oocyte +

15 nurse cells)

par1 gene: oocyte formation (in

Drosophila); cytoplasmic
asymmetry (in C. elegans).

Egg chamber: structure in the Border cells: important for

female Drosophila consisting of determination of anteroposterior
the oocyte and its nurse cells pattern.
surrounded by ovarian follicle
cells.
 Normal development: Oogenesis
Nurse cells: polyploid; export large amounts of RNA and
protein into the oocyte contributing to its increase in size.
Polarization in dorsoventral and anteroposterior axes
Granular pole plasm forms at posterior end.
The follicle cells secrete a tough coat around the egg:
the vitelline membrane and chorion
Fat body: where there is major production of yolk. It is
transported to the ovary via the hemolymph.
 Embryogenesis
Fertilization in the uterus through the micropyle
Development within 24 hours
Pole cells fate: germ cells

Plasma membrane
separates the nuclei
Embryogenesis
Pole cells moved
to the dorsal
portion

Segmentation

Ventral nerve
cord becomes
segregated

More defined segmentation

 Embryogenesis
Dorsal closure of epidermis: enclosure of the yolk mass by the
ventrally located germ band.
Occurs after 10-11 hours.
Simultaneously, head involutes interiorly.
Then, Malphigian tubules (excretory organs) form at the junction of
midgut and hindgut.
Mesoderm gives rise to: fat body, muscles, and gonads.
Central nervous system is formed by the ganglia of the ventral nerve
cord.
 Larval Stages
Consist of 3 instar stages separated by molt
NO LEGS
Head = tucked in interior
* HORNY CEPHALOPHARYNGEAL SKELETON =
most prominent structure
- secreted by stomodeal part of alimentary tract
3 thoracic & 8 visible abdominal segments
DORSAL = T2 - A8 covered with fine hairs
VENTRAL = each segment has DENTRICLE BELT
 Larval Stages
Seg. A8 has posterior
SPIRACLES

Spiracles: openings of tracheal

system

Telson: posterior portion

Imaginal discs = form main

epidermal structure

Histoblasts = form abdominal

segments
 Fate Map
For cellular blastoderm-stage embryo:
use of localized UV irradiation and injection of
horseradish peroxidase

NO region of indeterminancy

PROSPECTIVE REGIONS:
A. Gnathal, Thoracic & Abdominal = 75%
B. Procephalic = anterior 25%
C. Posterior midgut, Malphigian tubules, proctodeum and
germ cells = posterior
Fate Map
 Pole Plasm
Germ plasm of drosophila
Formed during oogenesis
Has determinants: contains gene products of oskar and
vasa
Oskar: maintains germ line / somatic integrity
Anterior/posterior determinant
Vasa: maternal

NOTE: maternal-effect mutation = sterility of offspring

Drosophila developmental genetics
genome size is small
generation time is short
genetic maps are detailed
 Transgenesis
P-element:
- transposable elements
- creation of transgenic lines

Injection of recombinant If P-element has

P-element with plasmid integrated into some
Gene of interest cloned germ cells, injected
into P-element vector or mRNA encoding for
transposase enzyme embryos will be
with marker gene mosaics, and will
into the posterior pole of
early embryos produce uniformly
transgenic offspring
 P-element transgenesis
introduce enhancer traps
create strains carrying reporter constructs
rescue endogenous mutations
ectopic gene expression
insertional mutagenesis
Identification of developmentally important genes

Balancer Chromosomes
suppress recombination
visible marker gene
lethal in homozygous
condition
 Types of Mutation
Most mutations are loss of function which are recessive

smaller amount of gene product/

reduced effectiveness gene product
Hypomorphs = alleles ; Hypomorphic = phenotype

Null alleles Amorphic phenotype

Thermal sensitive alleles - mutation affects thermal stability of
protein product
Low temp. = active protein Establish
High temp. = inactive protein developmental
stages in which
gene products
are required
 Types of Mutation
Some loss of functions mutations are dominant
Haploinsufficiency - 50% level of product
- homozygous more severe than
heterozygous

Often, gain of function mutations are dominant

production of active gene product

Dominant negative: antimorphic - mutant version of gene

interferes with the function of the wild-type
 Cloning of Genes
P- element mutagenesis

Mutation caused by P-element insertion is used as starting point

for cloning of gene

Clones are tested by in situ hybridization

To find which particular p-element they represent

One that lying to the nearest to the genetic map position is then
used as starting point for chromosomal walk
THREE CRITERIA THAT WILL PROVE THAT THE CLONED
CANDIDATE IS THE REQUIRED GENE

1. Several known mutants have identifiable sequence changes

in the candidate gene
2. Candidate gene is not expressed in null mutants
3. By rescuing the phenotype of null mutant by transgenesis
with the candidate gene
Gene function is studied by finding how the mutation
of one gene affects the expression of the others.

A B

If gene A codes for an inducing factor or receptor then

the effect is indirect.
 Three Methods of establishing directness:
1. May be possible to demonstrate interaction between gene A
product & gene B: regulatory region using, band shift assay

1. Gene A & B can be transfected together into Drosophila tissue

culture cells to see whether B is turned on in the absence of
other developmental machinery

2. Domain swap: new DNA recognition domain put onto A &

DNA sequence is inserted into B.
 Developmental Program
Set of subprograms
Anteroposterior axis
Dorsoventral axis: responsible for 3 Maternal systems:
the formation of:
1. Anterior: concerned with the production
1. Mesoderm of gradient of Bicoid protein
2. Neurogenic region
2. Posterior: pole of plasm is an integral
3. Epidermis ( ventral- dorsal)
part, deposits of mRNA of gene called
nanos

3. Terminal: activation of receptor encoded

by torso at both extremities leading to
activation of ERK signaling pathway
Fig. 11.8 Immunostaining of three components of the
maternal systems.
 Fig. 11.9 Hierarchy of steps in the development of
anteroposterior pattern.

bicoid
nanos

Krppel

even skipped

engrailed
Ubx
Fig. 11.10 Territories formed during dorsoventral
specification of the early Drosophila embryo
 Maternal Control of Dorsoventral
Patterning
Dorsal - prototype mutation of dorsalizing class

gurken Loss-of-function phenotype

torpedo = Egfr (ventralizing)

cactus With denticle bands around the

embryo

Loss-of-function
toll (dorsalizing alleles)
Gain-of-function
(ventralizing effect)
Experiments to understand how these genes work
1. Arranging dorsalizing alleles in allelic series

promote ventral development

2. Genes arranged in temporal series
making double mutants

One is dorsalizing Another is ventralizing

Epistasis: Whichever predominates, WINS!!!

Temperature-sensitive mutants:
shifting between permissive and nonpermissive temperature
Experiments to understand how these genes work
3. Germ line or Soma? 4. Know the genes function
Oocyte and Ovarian Using cytoplasmic transplantation
Nurse cells follicle cells From: Wild-type embryos

Mutant Normal Injecting small amount

offsprings offsprings of cytoplasm

gurken
Egfr To: Dorsal group mutants
Toll
pipe
dorsal To rescue Dorsal grp mutants
towards a normal appearance
Done by: FLP system
Experiments led to the ff. account that:
Mutant: indicates loss-of-function

K10: 1st known gene to act

has a dorsalizing effect

Function of its product:

to sequester the mRNA for gurken in the oocyte
nucleus
 Gurken and Egfr Showdown
gurken Egfr
Encode for growth Code for EGF/TGF
factor related to Ventralizing type receptor
vertebrate TGF mutant Required in follicle
Required in oocyte phenotypes cells
Its mRNA is on the
Its mRNA is all over
nucleus which is in
the follicle cells
the dorsal side of
oocyte
Protein product of gurken is secreted only on dorsal side

Stimulates Egfr product in dorsal follicle cells

Activates the ERK signaling pathway

Repression of pipe
What is pipe?
Pipe: Create an active extracellular ligand on ventral side of
oocyte
Codes for an enzyme that
Extracellular
Adds sulfate to
glycosamino -
groups
glycans
Local sulfation of Sequestrates proteins in
glycosaminoglycans ventral side

Including proteases produced

by genes snake and easter
 Local sulfation of Sequestrates proteins Activate the
glycosaminoglycans in ventral side ventral signal

Including
proteases
Protein product
produced by
of the sptzle
genes snake
gene
and easter

Synthesized as
an inactive
Requires precursor
proteolytic
cleavage to
activate
What is sptzle gene?
Sptzle gene: activates a receptor encoded by the Toll gene

What is Toll gene?

Toll gene: present all over the surface of oocyte

has 2 types of mutant:
1. Recessive dorsalizing mutant
- Receptor function is lost
2. Dominant ventralizing mutant
- Receptor is signaling even without the ligand
Activated after fertilization on the ventral side
What is dorsal gene?

Dorsal gene Final gene in the maternal dorsoventral pathway

Transcription factor homologous to vertebrate factor
NFB

During syncytial
Its distribution is equal blastoderm stage
Enters the nuclei on the
initially ventral side

Forms a ventral-dorsal
gradient
 Causes dissociation
Entry to nuclei Releases dorsal
of dorsal protein
protein & allows it
(depends on proximity of from a complex
to enter nuclei
activated Toll) formed with IB

IB - Encoded by gene cactus

cactus - Ventralizing mutant

- Inhibits the action of Dorsal
protein
Absence of cactus = dorsal protein can enter = ventral all over!
nuclei all over the embryo
 Operation of the Dorsoventral System
Pipe is active and
K10 sequestrates enables activation
the mRNA for of sptzle
gurken

Sptzle activates
Protein product of Toll then causes
gurken stimulates nuclear
torpedo (Egfr) translocation fo
Dorsal
Leads to
repression of Dorsal regulates
pipe the zygotic genes
Zygotic Control of Dorsoventral
Patterning
Mesoderm: Defined by 2 transcription factors
encoded by genes twist and snail
 twist and snail showdown
twist snail

Encodes a Code for Encodes a

bHLH protein transcription zinc-finger
factors transcription
Required for Upregulated by factor
correct dorsal protein at
Needed for
mesodermal high nuclear
invagination of
differentiation concentration
the mesoderm
Rhomboid - Coding for transmembrane protein involved in
EGF signaling
- Upregulated by dorsal protein at lower
concentration

- Repressed by snail
- Expressed as a stripe in the neurogenic
region
Dorsal ectoderm - Defined by a homeoprotein encoded by zerknllt

- Repressed by Dorsal protein

- Expressed all over the dorsalizing mutants
Decapentaplegic (dpp) - Also repressed by Dorsal protein

- Encodes a signaling molecule

homologous to vertebrated BMPs

Injection of dpp mRNA

High doses Low doses

amniosersosa Dorsal hairs

Although this suggest gradient-like behavior, it is NOT!

No gradient production of Dpp protein
 Induction of dorsal genes is conducted by Dpp + Screw

Graded-effect is due to sog gene

Another
BMP
homolog
Short gastrulation (sog) - Repressed by snail
gene - Expressed in the lateral belt
- Inhibits screw
- Leads to dorso-ventral gradient of BMP
activity
- Homolog of vertebrate Chordin
tinman - Encodes a homeodomain transcription factor
necessary for heart formation

Full dorsoventral pattern arises from:

Maternal gradient of Dorsal protein: Ventral half

Zygotic gradient of Dpp: Dorsal half
 homologous
Dorsal Dpp - Ventral Sog Ventral BMP4 - Dorsal Chordin
pattern pattern

During evolution, one of the groups turned upside-down

Similar biological acitivity:

Dpp Sog
BMP4 Chordin
Gurken
Inducing factors
sptzle
- Causes a regional patterning of their
competent tissue
Toll - Example of cytoplasmic determinant due to
localized region of activation

Dorsal - Can form gradient that will result to various

protein dorsoventral tissue types
 The anteroposterior system
Events in the egg chamber
Specification of structures along the anteroposterior axis is
controlled by maternal systems

Anterior Posterior
mRNA for bicoid mRNA for nanos
Anterior Posterior

mRNA for mRNA for

Bicoid nanos

Gradient Its protein

of Bicoid product
protein lifts the
inhibition
Dorsoventral system
Border cells - small group of
terminal follicle cells at
each end of the egg
chamber are
specialized

Oocyte produces Gurken

Gurken signal causes

posterization of border cells
Dorsoventral system
Formation of gradient and
polarization of microtubules

Localization of oskar mRNA (posterior)

and bicoid mRNA (anterior)

Nucleus moves around the

exterior of oocyte

gurken mRNA causes

Gurken protein to be secreted
on adjacent follicle cells
Dorsoventral system
Adjacent follicle cells will be
dorsal in character

Why?

Gurken signal stimulates

Egfr
- Repression of
pipe
Why same signal can be involved in polarization
along two axis?
1. Acts at different times

1. Different competence of responding tissue

1. Intervening growth of the oocyte changes the location of

signal
 Bicoid
a maternal-effect gene coding for a homeodomain transcription factor

transcription of bicoid occurs during oogenesis occurs in both oocytes

and nurse cells

mRNA becomes localized at anterior of oocyte

Activator of anterior-specific genes

Bicoid protein concentration gradient

patterns anterior-posterior axis of Drosophila during embryogenesis
regulates zygotic expression of gap genes such as orthodenticle,
hunchback, and Krppel
 Posterior System
Relies on accumulation of nanos mRNA in the
posterior region and action of its protein product in
revoking inhibitions on various gap gene
transcriptions for the formation of the future
abdomen

nanos mRNA is normally localized in the pole

plasm
pole plasm: organized by the oskar gene; situated at
posterior region
 Posterior System
Site of oskar RNA localization and oskar protein
synthesis determines the bodys posterior
region and place of germ cell formation

The nanos protein allows the transcription of

knirps gene, together with pumilio

Double repression: nanos inhibits translation of

Hunchback which represses knirps
 Posterior System
Hunchback: zygotic gap gene that controls
transcription of abdominal gap genes
upregulated by bicoid at the anterior
active during oogenesis; uniformly fills egg with a
concentration of maternally-derived hunchback
mRNA
translation of hunchback mRNA starts at early
cleavage and is inhibited at the posterior half of
embryo by the nanos protein
 Posterior System
Mutations in maternal-effect genes such as nanos, oskar,
and pumilio can cause loss of abdomen

Mutants may be rescued by injection of cytoplasm from the

pole plasm region of wild-type embryo into abdomen of
mutant
Confirms localization of abdomen-forming substance
nanos product: RNA-binding protein
 Terminal System

Involves a signal from

follicle cells to activate
receptors at both terminal
ends, thereby turning on
zygotic genes
Different molecules are
involved but has a
mechanism remarkably
similar to the dorsoventral
system
 Terminal System
Key gene: torso
located in oocytes plasma membrane

torso ligand is produced by follicle cells present only at the

terminal ends

also has a gain-of-function allele that causes suppression of

thorax and abdominal segmentation

encodes cell surface receptor of tyrosine kinase, which

stimulates ERK signal transduction pathway
 Terminal System
Key gene: torso

ligand is encoded by trunk, which is active in the

oocyte and secreted into the perivitelline fluid

Trunk protein is activated at the termini by the product of

torsolike, which is present in the anterior and posterior
border cells
 Terminal System
Receptor Tyrosine Kinase pathway is activated when torso
receptor binds to ligand

Torso activation: upregulation of zygotic gap genes tailless

and huckebein
Tailless: encodes a transcription factor of the nuclear
receptor family
Huckebein: encodes a zinc-finger transcription factor
 Gap Genes
Code for transcription factors

Can diffuse from one nucleus to another and exert effects

directly, with no need for cellcell signaling because early
embryo is a syncytium

Some important members are orthodenticle, hunchback,

Krppel, knirps, and giant
 Gap Genes
Regulatory relationships have been deduced by two types
of experiments:

a) Examination of the expression pattern of gene of interest in the

absence of another
expansion of domain: absent gene has repressive
function
reduction of domain: absent gene is responsible for
positive regulation in normal development

b) Examination of the expression pattern of gene of interest

following uniform overexpression of another gene
 Gap Genes
Top 3: normal expression of three
genes

Bottom 4:
expression of gene A in embryos
mutant () or with ubiquitous
overexpression (*) of B or C

results show B upregulates A

and C represses A.
 Gap Genes
Caudal gene:
encodes a homeodomain transcription factor
along with bicoid, its product is another important gap
gene expression regulator
expressed maternally to produce uniform distribution
of mRNA in oocyte
lack of caudal genes result to severe posterior defects
in embryos

*bicoid and caudal proteins upregulate some gap genes and form
inverse gradients; their gene regulation is autonomous
 Gap Genes
Orthodenticle gene:
encodes a homeodomain transcription factor
upregulated by high levels of bicoid and torso
homeobox gene involved in head development

*Otx2: important vertebrate homolog required for formation

of forebrain and midbrain
 Gap Genes
Hunchback gene:
codes for zinc-finger transcription factor
transcription during oogenesis leaves uniformly distributed mRNA in
egg
**anterior to posterior gradient during cleavage: due to inhibition of nanos**
anterior zygotic domain is directly upregulated by bicoid;
posterior by torso
null alleles of hunchback: large anterior gap, which removes the
labium and thorax
 Gap Genes
Krppel gene:
codes for zinc-finger transcription factor
upregulated by Bicoid and Hunchback; repressed by Knirps
and Giant
null mutants: large deletion of the central part of the body,
comprising the thorax and abdominal segments 15
 Gap Genes
Knirps gene:
codes for transcription factor belonging to the nuclear
hormone receptor family
constitutive gene activity and position of main band in
normal development is regulated by repression due to
Hunchback and Tailless
null mutants: similar to the maternal posterior group,
having abdominal segments 17 replaced by a single
large abdominal segment of uncertain identity
 Gap Genes
Giants gene:
encodes a leucine-zipper transcription factor
upregulated by Bicoid and Caudal, and repressed in
the anterior by Hunchback
mutants have defects in the anterior thorax and in the
abdomen at A5A7
Gap Genes

Tailless and Huckebein genes:

zygotic genes upregulated by Torso
mutants each have terminal defects, which together
add up to the phenotype produced by mutants of the
maternal terminal genes
Gap Genes
 Pair-rule Genes
layer in the developmental hierarchy between the gap genes
and the segment polarity genes.

Controlling the expression of Hox genes and aligning their

domains with the segmental repeating pattern
 Pair-rule Genes
All the pair-rule genes code for transcription factors and the
expression patterns consist of seven stripes, corresponding
to two segment-wide bands of the syncytial blastoderm

mutant phenotypes typically show deletions with a periodicity

of two segments, although more complex and more severe
phenotypes can be found.

Example: even-skipped
 Pair-rule Genes
Primary pair-rule genes are
hairy,
even-skipped (eve),
runt,
fushi-tarazu (ftz)
odd-paired (odd).

Those pair-rule genes designated primary are regulated

mainly by the maternal and gap genes, while those
designated secondary are regulated mainly by the primary
genes.
 Pair-rule Genes
They have complex regulatory regions containing several
enhancers.

General Rule: one enhancer, one stripe

To form a stripe it is necessary that the gene be turned on at

one anteroposterior level and turned off again at a slightly
different level

Example: even skipped gene

Stripe 2 Element
Positive Regulator: Bicoid and Hunchback
Repressed by Giant and Krppel
 Pair-rule Genes
4+6 and the 3+7
enhancers
both active in the
presence of ubiquitous
components and
repressed by
Hunchback and
Knirps.

Hunchback and Knirps

also repress each other,
ensuring that the knirps
domain lies in between the
two zygotic hunchback
domains
 Pair-rule Genes
pair-rule transcription factors upregulate the 14 stripes

activators predominate over inhibitors

Example:
1. engrailed stripes of the odd-numbered parasegmentals
upregulated by paired
Repression of sloppy-paired by eve

2. engrailed stripes of the even-numbered parasegmentals

upregulated by ftz
Repression of odd-paired by eve
 Segment Polarity Genes
The segment polarity genes function to create the
parasegment boundaries of the early embryo

defined by activity of the transcription factor genes engrailed

and cubitus interruptus (ci)

The engrailed cells emit an inducing factor called

Hedgehog, which maintains activity of ci in the neighboring
cells,

And the ci cells emit a factor called

Wingless which maintains activity of engrailed in the
neighboring cells
 Segment Polarity Genes
Engrailed

codes for a
homeodomain
transcription factor and
comes on at the cellular
blastoderm stage,
forming a 14-stripe
pattern by the extended
germ-band stage
 Segment Polarity Genes
ci
encodes a Gli-type zinc finger transcription factor
and is repressed by Engrailed such that its
expression pattern consists of 14 stripes in
between the engrailed stripes
 Segment Polarity Genes
Maintenance of the pattern by the action of Hedgehog and Wingless
systems.
 Hox Genes
The Hox cluster is on a single chromosome, but is split into two
gene groups:

the Antennapedia complex

corresponds to vertebrate paralog groups 16 and contains
the genes labial, proboscipedia, Deformed, Sex combs
reduced, and Antennapedia.

the Bithorax complex.

corresponds to vertebrate paralog groups 710 and
contains the genes Ultrabithorax (Ubx), abdominal-A, and
Abdominal-B.
 Hox Genes
The approximate expression domains of the Hox genes in the
parasegments of the extended germ-band stage

The function of these genes is to impart different characters to the

different segments.
 Hox Genes
Loss-of-function mutations generally produce anteriorizations
and gain-of-function mutations generally produce
posteriorizations.

Ubx gene
which is the homolog of the Hox7 paralog group in
vertebrates.
shows an initial peak in parasegment 6 andsubsequent
lesser expression from parasegment 513
Null mutants of Ubxshow a transformation of parasegments
5 and 6 to parasegment4.
 Hox Genes
Initial control of Ubx expression is by Hunchback, which acts as a
repressor

There is an upregulation by Fushi tarazu, which gives a transient

pair-rule character to the Ubx pattern.

Once it is established, the posterior boundary of Ubx expression

is maintained by repression from Abd-A protein. Ubx itself
represses Antp, so maintaining its posterior boundary.
 Hox Genes
Polycomb and extra sex combs
encode components of key regulatory complexes
which are ubiquitously present and generally
repressive.
Loss-of-function mutants of these chromatin regulators
lead to ectopic Hox activity.
 Anteroposterior body pattern
2 Essential Components of Specification :
A. Repeating Pattern of parasegments
Juxtaposition of cells where engrailed (anterior) and ci
(posterior) are active
B. Nonrepeating sequence of Hox expression
Initiated locally by different concentrations of products of
genes
Pair-rule genes: control register between segment polarity and
Hox genes
Loss- of-function mutants: anteriorization
End of Presentation

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