EARLY EMBRYOLOGY, FATE

DETERMINATION, AND
PATTERNING IN DROSOPHILA
LIFE CYCLE DROSOPHILA
 DROSOPHILA LIFE CYCLE
• Life cycle by 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 hours after egg laying
Day 11-12: Eclosion (adults emerge from the pupa case).
Females become sexually mature 8-10 hours after eclosion
 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.
 DROSOPHILA DEVELOPMENT

¤ Embryonic development in Drosophila is an orderly sequence of

change and is controlled by the differential expression of genes.

¤ Drosophila display a holometabolous method of development,

meaning that they have three distinct stages of their post –
embryonic life cycle, each with radically different body plans:
larva, pupa and finally, adult(imago
 DROSOPHILA DEVELOPMENT - OVERVIEW

 Fertilization
 Cleavage
 Gastrulation
 Drosophila body plan
 Oocyte formation
 Genetic control of axis specification
 Anterior-posterior
 Dorsal-ventral
 Segmentation genes
 Homeotic genes
 DROSOPHILA 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!

 EARLY DEVELOPMENT OF DROSOPHILA
• Egg is centrolecithal
• After fertilization, series of superficial cleavages
• Blastoderm is syncytial until 13th cleavage (256
nuclei!)
• Nuclei begin dividing centrally, migrate toward the
edges
• Several nuclei migrate to posterior end, form cell
membranes (pole cells)
• Give rise to the adult gametes
• What cells are like this in mammals?
 Superficial Cleavage
Syncytial blastoderm stage
- 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
 GASTRULATION
• 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
 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

 DROSOPHILA 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

 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
- diffuse through syncytial cytoplasm bicoid gradient
- activate or repress zygotic genes

2. Gap genes: first zygotic genes expressed

Divide embryo into regions
- expressed in broad, partially
overlapping domains (~ 3 segments wide) Hunchback overlap Kruppel

- code for transcription factors; transient

-
-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 Genes
Cell fate commitment:
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
 Drosophila Body Plan - Egg Stage

Body axes are determined in the egg by distribution of

maternal mRNAs and proteins.

Translation leads to formations of patterning protein

(e.g. morphogen) gradient within the embryo.

How are asymmetric distributions of messages and proteins

established in the egg?
Oocyte Formation (A-P, D-V Axes)
Drosophila ovariole
Oogonium divide
into 16 cells
1 oocyte
15 nurse cells
all interconnected

nurse cells contribute

mRNA, proteins
cytoplasm
 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
A-P Axis: bicoid / Oskar / nanos
“posteriorized” follicles
produce organized (+/-)
microtubules
Nurse cells manufacture
bicoid and nanos mRNA
- deliver cytoplasm
into oocyte
bicoid binds to dynein
- moves to non-growing
(-) end of microtubules

oscar mRNA forms

complex with kinesin I
- moves toward growing
(+) end of microtubules

Oskar binds nanos mRNA

- retains nanos in
posterior end
 Posterior Specification - 1
At least 9 maternal genes make up the posterior patterning group;
including staufen, oskar, nanos

nanos trap:
Staufen allows
oskar translation
Oskar binds nanos;
remains in posterior

Nanos prevents
hunchback translation
 Model of Anterior-Posterior Patterning
mRNA in oocytes
(maternal messages)

Early cleavage
embryo proteins
hunchback translation
repressed by Nanos
caudal translation
repressed by Bicoid
Posterior Specification - 2
Bicoid Mutants

Bicoid – homeodomain
transcription factor;
morphogen

Martin Klingler
Manipulating Bicoid
Dorsal - Ventral Axis Formation
Further Gurken Effects
The oocyte nucleus (with associated gurken) moves
anteriorly along the dorsal margin

Gurkin/Torpedo interactions “dorsalize” follicle cells

 DISTRIBUTION OF DORSAL

Dorsal:
- large amount = mesoderm
- lesser amount = glial/ectodermal
Dorsal activates genes that create
Dorsal
mesodermal phenotype
- transcribed only in cells with highest
Dorsal concentrations
- these genes have low affinity enhancers
ventral cells
(lots of Dorsal necessary) form medoderm
Dorsal also inhibits dorsalizing genes
 Zygotic Patterning Genes
decapentaplaegic (dpp),
zerknüllt (zen), tolloid are
dorsal patterning genes
- repressed by Dorsal

Intermediate dorsal activates

rhomboid (no Twist or snail)
- determines neural ectoderm
- rhomboid + twist = glial cells

Intermediate dorsal activates fgf8

- fgf8 repressed by snail
- promotes mesodermal ingression
dorsal
High Dorsal – activates twist and snail (low affinity enhancers)
- mesoderm determinants
Dorsal (TF) – expressed ventrally; establishes diffusion gradient dorsally
~ 30 genes directly affected by Dorsal
 Maternal effect genes
bicoid
nanos
Gap genes

huckebein
hunchback
giant

Pair-rule genes

even-skipped
fushi tarazu

Segment polarity genes

engrailed hedgehog

wingless patched
GAP GENE EXPRESSION
 Krüppel Specification by Hunchback
Threshold activation/repression

Wolpert, 2007
Pair-Rule Gene Regulation

e.g. – even-skipped

- each stripe regulated

by a different set
of enhancers
- expression patterns are
stabilized by interactions
among other gene products
e.g. even-skipped
expression limited
by Giant
 Segments and Parasegments
Expression patterns in early embryos are not delineated
by segmental boundaries, but by
- parasegments:fundamental units of embryonic gene expression
Segments and parasegments
organized from
A/P compartments
out of phase
Cells of adjacent
compartments
do not mix

fushi tarazu – pair-rule gene

HOMEOTIC SELECTOR GENES

Homeotic genes specify

- head segments
- labial palps
- antennae
- thoracic segments
- wings
- halteres
- legs
- abdominal segments
Eight Genes Regulate the Identity of
Within the Adult and Embryo

labial (lab)
proboscipedia (pb)
Deformed (Dfd)
Sex combs reduced (Scr)
Antennapedia (Antp)
Ultrabithorax (Ubx)
abdominal A (abd-A)
Abdominal B (Abd-B)
 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.
e.g. Ubx is switched on between certain concentrations of
hunchback to give a broad band of expression near the
middle of the embryo. Later, fushi tarazu and even skipped
sharpen the limits of Ubx expression which comes into
register with the anterior boundaries of specific
parasegments.
 The BX-C and ANT-C genes have extensive non-coding
sequences (introns) that are critical in regulating their
individual expression.
Homeotic Gene Expression

distal-less – jaws, limbs

Antennapeida – Ultrabithorax –
thoracic abdomen
 Patterns of
Expression
Figure . The patterns of expression
compared to the chromosomal
locations of the genes of the HOM
complex. The sequence of genes in
each of the two subdivisions of the
chromosomal complex corresponds
to the spatial sequence in which the
genes are expressed. Note that
most of the genes are expressed at
a high level throughout one
parasegment (dark color) and at a
lower level in some adjacent
parasegments (medium color)
where the presence of the
transcripts is necessary for a normal
phenotype, light color where it is
not). In regions where the expression
domains overlap, it is usually the
most "posterior" of the locally active
genes that determines the local
phenotype. The drawings in the
lower part of the figure represent
the gene expression patterns in
embryos at the extended germ
band stage, about 5 hours after
fertilization.
 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

Figure :. Contribution of BX-C genes — Ubx,

abdA, and AbdB—to determination of
parasegment identity. The numbers above
each larva indicate the parasegments; those
below, the corresponding segments. The
cuticular pattern of larvae is used to assign an
identity to each parasegment (PS), which is
indicated by color, as depicted in the wild type
at the top. Red PS and segment labels indicate
abnormal patterns that do not correspond
exactly to any found in wild-type larvae.
[Adapted from P. A. Lawrence, 1992, The
Making of a Fly: Genetics of Animal Design,
Blackwell Scientific Publications.]
Expression and lethal embryonic phenotypes of homeotic genes. (Adapted from Akam,
1987.)
This phenotypic analysis has been confirmed by the expression patterns of the various
genes in various mutant backgrounds. The anterior limits of the domain of expression of a
particular homeotic gene are presumably set by a collaboration between gap genes
and pair-rule genes (see above). Removal of an anteriorly-acting homeotic has no effect
on expression or phenotype in the domain of a more posteriorly acting homeotic gene.
•Antp expands posteriorly from PS4 to PS6 in a Ubx mutant
•Antp expands posteriorly from PS4 to PS12 in a Ubx AbdA mutant
•Antp expands posteriorly from PS4 to PS14 in a Ubx AbdA AbdB mutant
•Ubx expands posteriorly from PS6 to PS12 in a AbdA mutant
•Ubx expands posteriorly from PS6 to PS14 in a AbdA AbdB mutant etc.
Homeotic Gene Expression in Mutant Embryos
 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
stimulategeneexpression.
Figure 21-52. Fate map of a Drosophila embryo at the cellular blastoderm stage. The
embryo is shown in side view and in cross-section, displaying the relationship between
the dorsoventral subdivision into future major tissue types and the anteroposterior pattern
of future segments. A heavy line encloses the region that will form segmental structures.
During gastrulation the cells along the ventral midline invaginate to form mesoderm,
while the cells fated to form the gut invaginate near each end of the embryo. Thus, with
respect to their role in gut formation, the opposite ends of the embryo, although far apart
in space, are close in function and in final fate. (After V. Hartenstein, G.M. Technau, and
J.A. Campos-Ortega, WilhelmRoux'Arch.Dev.Biol.194:213-216, 1985.)
Terminal Specification - 1

Torso – transmembrane RTK

Torso uniformly distributed

Torso activated by
Torso-like protein
- located only at
ends of egg
Terminal Specification - 2

Torso kinases inactivate

an inhibitor of tailless
and huckebein

Tailless and Huckebein

specify termini

Distinction between
anterior and posterior
= Bicoid

Bicoid = acron
formation
Thank You