Axis Specification

in Drosophila

Developmental Biology – Biology 4361

July 7, 2009

Drosophila Development - Overview

Cleavage

Fertilization

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 compete with each other! Romano Dallai

Drosophila bifurca

Sperm enter at the micropyle.

- probably prevents polyspermy

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 in Drosophila

cellular blastoderm

Gastrulation

germ band

movementventral furrow

(primordial

germ cells)

anterior midgut

invagination

posterior midgut

invagination

Late Gastrulation / Segmentation

Establishing the Drosophila Body Plan

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

Head

segment

Thoracic

segments T1 – legs

T2 – legs & wings

T3 – legs & halteres

Drosophila Body Plan - Egg Stage

Translation leads to formations of patterning protein

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

Body axes are determined in the egg by distribution of

maternal mRNAs and proteins.

How are asymmetric distributions of messages and proteins

established in the egg?

Drosophila Development - Overview

Cleavage

Fertilization

Gastrulation

Drosophila body plan

Oocyte formation

Genetic control of axis specification

Anterior-posterior

Dorsal-ventral

Segmentation genes

Homeotic genes

Oocyte Formation (A-P, D-V Axes)

modified from Kalthoff, K. 2001.

Analysis of Biological Development.

Drosophila ovariole- oogonium divides into

16 cells

- 1 oocyte

- 15 nurse cells

- all interconnected

nurse cells contribute

mRNA, proteins

cytoplasm

Anterior-Posterior Axis Formation

Torpedo (RTK Gurken receptor) present on follicular cells

Gurkin binding results in “posteriorization” of follicles

- posteriorized follicles re-organize egg microtubules; (-) = anterior

Gurkin protein localized between nucleus and cell membrane

- Note – Gurken diffuses only a short distance

Nurse cells synthesize gurken (TGF-β family)

- gurken mRNA transported toward oocyte nucleus (in posterior region)

Microtubules

Tubulin

dimer

protofilament

(+)

(-)

microtubule

(+)

(-)

Andreas Merdes

disassemblygrowth

(+)

(-)

(+) (-)

kinesindynein

A-P Axis: bicoid / Oskar / nanos

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

“posteriorized” follicles

produce organized (+/-)

microtubules

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

- results in Dorsalactivation

Oocyte

Pipe (ventral cells) eventually triggersnuclear Tollreceptor activity;

Dorsal determines ventral fates

Syncitial blastoderm

Gurken/Torpedoinhibits Pipesynthesis in dorsal cells

D-VPolarity

Distribution of Dorsal

Dorsal activates genes that create

mesodermal phenotype

- transcribed only in cells with highest

Dorsal concentrations

Dorsal also inhibits dorsalizing genes

- these genes have low affinity enhancers

(lots of Dorsal necessary)

Dorsal:

- large amount = mesoderm

- lesser amount = glial/ectodermal

ventral cells

form medoderm

Dorsal

Zygotic Patterning Genes

Dorsal (TF) – expressed ventrally; establishes diffusion gradient dorsally

decapentaplaegic (dpp),

zerknüllt (zen), tolloid are

dorsal patterning genes

- repressed by Dorsal

High Dorsal – activates twist and snail (low affinity enhancers)

- mesoderm determinants

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 ingressiondorsal

~ 30 genes directly affected by Dorsal

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)

- mRNAs differentially placed in eggs

- transcriptional or translational

regulatory proteins

- diffuse through syncytial cytoplasm

- activate or repress zygotic genes

3. Pair-rule genes

4. Segment polarity genes

2. Gap genes

embryonic segmentation genes

Anterior Specification - 1

Bicoid binds to caudal

3’UTR; prevents

translation

Caudal specifies

posterior domain

Anterior Specification - 2

Bicoid controls gap

gene Hunchback*

- anterior patterning

*hunchback is also

maternally expressed

Bicoid Mutants

Martin Klingler

Bicoid – homeodomain

transcription factor;

morphogen

Manipulating Bicoid

Posterior Specification - 1

Nanos prevents

hunchback translation

Oskar binds nanos;

remains in posterior

At least 9 maternal genes make up the posterior patterning group; including

nanos trap:

Staufen allows

oskar translation

staufen, oskar, nanos

Posterior Specification - 2

Model of Anterior-Posterior Patterning

mRNA in oocytes

(maternal messages)

Early cleavage

embryo proteins

hunchback translation

repressed by Nanos

caudal translation

repressed by Bicoid

Terminal Specification - 1

Torso – transmembrane RTK

Torso uniformly distributed

Torso activated by

Torso-like protein

- located only at

ends of egg

Terminal Specification - 2

Distinction between

anterior and posterior

= Bicoid

Bicoid = acron

formation

Torso kinases inactivate

an inhibitor of tailless

and huckebein

Tailless and Huckebein

specify termini

Drosophila Development - Overview

Cleavage

Fertilization

Gastrulation

Drosophila body plan

Oocyte formation

Genetic control of axis specification

Anterior-posterior

Dorsal-ventral

Segmentation genes

Homeotic genes

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

Anterior-Posterior Body PlanDrosophila 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

- 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)

- code for transcription factors; transient

- activated or repressed by

maternal effect genesHunchback Krüppel

overlap

bicoid gradient

Krüppel Specification by Hunchback

Wolpert, 2007

Threshold activation/repression

Anterior-Posterior Body Plan

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 parasegmentseven-skipped (red),

fuschi tarazu (black)

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

Regulation of even-skipped Expression

Wolpert, 2007

Anterior-Posterior Body Plan

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 parasegmentseven-skipped (red),

fuschi tarazu (black)

4. Segment polarity genes;

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 unitsengrailed

Maternal effect genes

Gap genes

hunchback

huckebein

giant

even-skipped

fushi tarazu

Pair-rule genes

Segment polarity genes

engrailed

wingless

hedgehog

patched

bicoid

nanos

fushi tarazu – pair-rule gene

Segments and Parasegments

Segments and parasegments

organized from

A/P compartments

out of phase

Cells of adjacent

compartments

do not mix

Expression patterns in early embryos are not delineated

by segmental boundaries, but by

- parasegments:fundamental units of embryonic gene expression

P A P A P A P A P A P A P

Segments

Compartments

Parasegments

muscles/hinge

nerves

Segments and Parasegments - Adult

5. Homeotic selector genes;

Provide segmental identity

- interactions of gap, pair-rule,

and segment polarity proteins

- determines developmental fate

of each segment

Homeotic Selector Genes

Sean Carroll

Homeotic Selector Genes

Homeotic genes specify:

- head segments

- labial palps

- antennae

- thoracic segments

- wings

- halteres

- legs

- abdominal segments

Homeotic Gene Expression

distal-less – jaws, limbs

Antennapeida –

thoracic

Ultrabithorax –

abdomen

Ultrabithorax MutantUbx-/- transforms 3rd thoracic segment (halteres)…

into duplicate 2nd thoracic segment (wings).

Antennapedia Mutant