EARLY EMBRYOLOGY, FATE

DETERMINATION, AND
PATTERNING IN DROSOPHILA
Dr. Joy C. Chavez
Instructor
 Why are
Drosophila
good for genetic
studies?
 Reasons for use in laboratories
o Its care and culture require little equipment, space, and expense even when
using large cultures.
o It can be safely and readily anesthetized (usually with ether, carbon dioxide
 gas, by cooling, or with products such as FlyNap).
o Its morphology is easy to identify once anesthetized.
o It has a short generation time (about 10 days at room temperature), so
several generations can be studied within a few weeks.
o It has a high fecundity (females lay up to 100 eggs per day, and perhaps
2000 in a lifetime).
o Males and females are readily distinguished, and virgin females are easily
isolated, facilitating genetic crossing.
o The mature larva has giant chromosomes in the salivary glands called 
polytene chromosomes, "puffs", which indicate regions of transcription,
hence gene activity. The under-replication of rDNA occurs resulting in only
20% of DNA compared to the brain. Compare to the 47%, less rDNA in 
Sarcophaga barbata ovaries.
o It has only four pairs of chromosomes – three autosomes, and one
pair of sex chromosomes.
o Males do not show meiotic recombination, facilitating genetic
studies.
o Recessive lethal "balancer chromosomes" carrying visible genetic
markers can be used to keep stocks of lethal alleles in a
heterozygous state without recombination due to multiple inversions
in the balancer.
o The development of this organism—from fertilized egg to mature
adult—is well understood.
o Genetic transformation techniques have been available since 1987.
o Its complete genome was sequenced and first published in 2000.
o Sexual mosaics can be readily produced, providing an additional tool
for studying the development and behavior of these flies.
LIFE CYCLE OF DRASOPHILA
 DROSOPHILALIFE
• Life cycle by CYCLE
days
Day 0: Female lays eggs
Day 1: Eggs hatch
Day 2: First instar (one
day in length)
Day 3: Second instar
(one day in length)
Day 5: Third and final
instar (two days in
length)
Day 7: Larvae begin
roaming stage.
Pupariation (pupa
formation) occurs 120
 The Drosophila life cycle represents the differentiation of
two distinct forms: the larva and the Imago (adult).

Metamorphosis: Embryogenesis:
differentiation of differentiation
the imago (adult) of the larva

Imaginal cells are the cells

of the adult or imago.
 DROSOPHILADEVELOPMENT -
OVERVIEW
 Fertilization
 Cleavage
 Gastrulation
 Drosophila body plan
 Oocyte formation
 Genetic control of axis specification
 Anterior-posterior
 Dorsal-ventral
 Segmentation genes
 Homeotic genes
 DRASOPHILA
FERTILIZATION
Eggs are activated prior to fertilization.
- oocyte nucleus has resumed meiotic division
- stored mRNAs begin translation

Eggs have begun to specify axes by the point of fertilization.

Sperm enter at the micropyle.

- probably prevents polyspermy.

Sperm compete with each other!

 Superficial
Cleavagestage
Syncytial blastoderm
- zygotic nuclei undergo 8
divisions
- nuclei migrate to periphery
- karyokinesis continues
Cellular blastoderm stage
- following division 13,
oocyte plasma membrane
folds inward
- partitions off each nucleus
and
associated cytoplasm
- constricts at basal end
SUPERFICIAL CLEAVAGE
 Although nuclei share the same cytoplasm, the cytoplasm is not
uniform in its makeup

• Maternal molecules are distributed differently

 Eventually cells will form plasma membranes and the embryo

will consist of a cellular blastoderm

 Mid-blastula transition occurs slowly, increasing transcription of

zygotic genes
 GASTRULATIO
N
• At MBT, gastrulation begins, forming mesoderm,
endoderm, ectoderm

• Cells fold inward to form ventral furrow

• Embryo bends to from cephalic furrow

• Pole cells are internalized, endoderm invaginates

• Ectoderm converges and extends along midline to

form Germ Band
 GERM
BAND
• Wraps around the embryo

• As it wraps around the dorsal surface, the A-P axis of

the embryo is laid down

• Body segments begin to form

• At the end of germ band extension

• Organs are beginning to form
• Body segmentation is set-up

• Groups of cells called imaginal discs are set aside, these

cells will form adult structures

Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15

 DROSOPHILA LARVAE
• During metamorphosis
• 3 “instar” larvae

• Pupae

• Adult

• After gastrulation, 1st instar larvae is formed

• Has head and tail end

• Repeating segments along axis

• Generally the same type of body plan as adult

Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15

 DRASOPHILA BODY
PLAN
• 3 thoracic segments
• Each different from each other

• 8 abdominal segments
• Each different from each other

• Able to tell the difference in the larvae based on

cuticle
• Covering of the embryo

• Correspond to the adult segments

 T1- legs

T2 – legs &
wings

T3 – legs &
halteres

Segments form along the anterior-posterior axis, then become specialized.

Specification of tissues depends on their position along the primary axes.

A/P and D/V axes established by interactions between the developing

oocyte and its surrounding follicle cells
 GENETICS OF AXIS SPECIFICATION
IN DROSOPHILA

• Controlled by a variety of genes

• Maternal effect genes

• Gap genes

• Pair-rule genes

• Segment polarity genes

• Homeotic selector genes

Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15

 Anterior-Posterior Body Plan
Drosophila use a hierarchy of gene expression to
establish the anterior-posterior body plan.
1. Maternal effect genes (e.g. bicoid, nanos)
Establish polarity:
- mRNAs differentially placed in eggs
- transcriptional or
translational regulatory
proteins; transient bicoid gradient
- diffuse through
syncytial cytoplasm
- activate or repress
zygotic genes
2. Gap genes: first zygotic genes
expressed Divide embryo into regions
- expressed in broad, partially
overlapping domains (~ 3 segments Hunchback overlap
Kruppel

wide)for transcription factors; transient

- code
-
- activated or repressed by maternal effect genes
 3. Pair-rule genes;
Establish segmental plan
- regulated by combinations of gap genes
- code for transcription factors; transient
- divide the embryo into periodic units
- pattern of seven transverse bands
- delimit parasegments even-skipped (red),
4. Segment polarity genes; fuschi tarazu (black)
Set boundaries of segments
(i.e. establish A-P for each segment)
- activated by pair-rule genes
- code for variety of proteins; stable
- divide embryo into 14 segmental units

engrailed
5.Homeotic selector genes;
Provide segmental identity
- interactions of gap, pair-
rule,
- and segment polarity
proteins
- determines
developmental fat
 Segmentation
Cell fate commitment: Genes
Phase 1 – specification
Phase 2 – determination
- early in development cell fate depends on
interactions among protein gradients
- specification is flexible; it can alter in response to
signals from other cells
- eventually cells undergo transition from loose
commitment to irreversible determination

The transition from specification to determination in Drosophila is

mediated by the segmentation genes.
- these divide the early embryo into a repeating series of segmental
primordia along the anterior-posterior axis
Body axes are determined in the egg by distribution of
maternal mRNAs and proteins.
 Anterior-Posterior Axis
Formation

Nurse cells synthesize gurken (TGF-β family)

- gurken mRNA transported toward oocyte nucleus (in posterior region)
Gurken protein localized between nucleus and cell membrane
- Note – Gurken diffuses only a short distance
Torpedo (RTK Gurken receptor) present on follicular cells
Gurken binding results in “posteriorization” of follicles
- posteriorized follicles re-organize egg microtubules; (-) = anterior
 HOMEOTIC SELECTOR GENES

Homeotic genes specify

- head segments
- labial palps
- antennae
- thoracic segments
- wings
- halteres
- legs
- abdominal segments

Neelam Devpura, GSBTM Presentation, MNVSC 28-4-15

 Homeotic-Selector Genes
 Homeotic genes encode nuclear proteins containing a
DNA- binding motif called a homeodomain.
 The products are transcription factors that specify
segment identity by activating multiple gene expression
events.
 The genes are initially activated imprecisely by the
concentration gradients of gap gene products.
 The BX-C and ANT-C genes have extensive non-coding
sequences (introns) that are critical in regulating their
individual expression.
Homeotic Gene Expression
Patterns of Expression
 Homeotic Mutations
•The direction of homeotic transformations depends on whether the
mutation causes loss of homeotic gene function where the gene normally
acts or gain of function where the gene normally does not act.

•Ultrabithorax (Ubx) acts in the haltere to promote haltere development

and repress wing development. Loss of function mutations in Ubx
transform the haltere into a wing.

•Dominant mutations that cause Ubx to gain function in the wing

transform that structure into a haltere.

•In antenna-to-leg transformations of Antennapedia the mutants

reflect a dominant gain of Antennapedia gene function in the
antennae.
Effects of Mutations
in Bithorax
Complex
 Maintaining Hox Gene Expression
 The transcription-control regions of some Hox genes contain binding
sites for their encoded proteins – autoregulatory loop (e.g. lab and
Dfd).
 A second mechanism requires proteins that modulate chromatin
structure. There are two classes – the trithorax group and the
polycomb group.
 Early patterning requires repression as well as activation of gene
expression. Polycomb proteins have a repressive effect on the
expression of Hox genes.
 Polycomb proteins bind multiple chromosomal locations to form
large macromolecular complexes and this becomes “locked in”.
 Trithorax proteins maintain the expression of many Hox genes.
These also form large multiprotein complexes at multiple
chromosomal sites, but mainain an open chromatin structure and
stimulate gene expression.
Thank
You!