drosophila developmental biology

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DROSOPHILA DEVELOPMENT BIOLOGY

NEELAM DEVPURA(M.Sc., NET, GATE)Ph.D. Scholar, SPU

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Life cycle by days

Day 0: Female lays eggs

Day 1: Eggs hatch

Day 2: First instar

Day 3: Second instar

Day 5: Third and final instar

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

DROSOPHILA LIFE CYCLE

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The Drosophila life cycle represents the differentiation of two distinct forms: the larva and the Imago (adult).

Embryogenesis: differentiationof the larva

Metamorphosis:differentiation ofthe imago (adult)

Imaginal cells are the cellsof the adult or imago.

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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

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Drosophila Development - Overview

Cleavage

Fertilization

Gastrulation Drosophila body plan Oocyte formation Genetic control of axis specification

Anterior-posterior Dorsal-ventral

Segmentation genes Homeotic genes

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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!

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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?

Early Development of Drosophila

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Superficial CleavageSyncytial 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

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SUPERFICIAL CLEAVAGE

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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

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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

Gastrulation

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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

Germ Band

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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 Larvae

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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

Drosophila body plan

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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 developingoocyte and its surrounding follicle cells

T1- legs

T2 – legs & wings

T3 – legs & halteres

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Genetics of Axis Specification in Drosophila

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Controlled by a variety of genes

Maternal effect genes

Gap genes

Pair-rule genes

Segment polarity genes

Homeotic selector genes

Axis Specification

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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 expressedDivide embryo into regions- expressed in broad, partially

overlapping domains (~ 3 segments wide)- code for transcription factors; transient

-activated or repressed by maternal effect genes-

Hunchback Krüppel

overlap

bicoid gradient

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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

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even-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 units

engrailed

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Segmentation Genes

Cell fate commitment:Phase 1 – specificationPhase 2 – determination

- early in development cell fate depends on interactionsamong protein gradients

- specification is flexible; it can alter in response to signalsfrom other cells

- eventually cells undergo transition from loose commitmentto irreversible determination

The transition from specification to determination in Drosophila ismediated by the segmentation genes.

- these divide the early embryo into a repeating series of segmentalprimordia along the anterior-posterior axis

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of each segment

Homeotic Selector Gene

5. Homeotic selector genes;Provide segmental identity- interactions of gap, pair-rule,

and segment polarity proteins- determines developmental fat

Sean Carroll

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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 ofmaternal mRNAs and proteins.

How are asymmetric distributions of messages and proteinsestablished in the egg?

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

nurse cells contributemRNA, proteinscytoplasm

Drosophila ovarioleOogonium divide into

16 cells 1 oocyte

15 nurse cells

all interconnected

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Anterior-Posterior Axis Formation

Torpedo (RTK Gurken receptor) present on follicular cells

Gurken binding results in “posteriorization” of follicles- posteriorized follicles re-organize egg microtubules; (-) = anterior

Gurken 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)

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A-P Axis: bicoid / Oskar / nanos

Nurse cells manufacturebicoid and nanos mRNA- deliver cytoplasminto oocyte

bicoid binds to dynein- moves to non-growing

(-) end of microtubules

oscar mRNA formscomplex with kinesin I- moves toward growing

(+) end of microtubules

Oskar binds nanos mRNA- retains nanos in

posterior end

“posteriorized” folliclesproduce organized (+/-)microtubules

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Posterior Specification - 1

Nanos preventshunchback translation

Oskar binds nanos;remains in posterior

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

nanos trap:

Staufen allowsoskar translation

staufen, oskar, nanos

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Posterior Specification - 2

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Model of Anterior-Posterior Patterning

mRNA in oocytes(maternal messages)

Early cleavageembryo proteins

hunchback translationrepressed by Nanos

caudal translationrepressed by Bicoid

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Bicoid Mutants

Martin Klingler

Bicoid – homeodomaintranscription factor;morphogen

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Manipulating Bicoid

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Dorsal - Ventral Axis Formation:Further Gurken Effects

The oocyte nucleus (with associated gurken) movesanteriorly along the dorsal margin

Gurkin/Torpedo interactions “dorsalize” follicle cells

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Dorsal activates genes that createmesodermal phenotype

- transcribed only in cells with highestDorsal concentrations

- these genes have low affinity enhancers(lots of Dorsal necessary)

Dorsal also inhibits dorsalizing genes

Distribution of Dorsal

Dorsal:- large amount = mesoderm- lesser amount = glial/ectodermal

Dorsal

ventral cellsform medoderm

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Zygotic Patterning Genes

decapentaplaegic (dpp),zerknüllt (zen), tolloid aredorsal patterning genes- repressed by Dorsal

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

Intermediate dorsal activates fgf8- fgf8 repressed by snail

- promotes mesodermal ingressiondorsal

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

47wingless patched

Maternal effect genes

bicoid

nanos

Gap genes

huckebein

hunchback

giant

Pair-rule genes

even-skipped

fushi tarazu

Segment polarity genes

engrailed hedgehog

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Krüppel Specification by Hunchback

Wolpert, 2007

Threshold activation/repression

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Pair-Rule Gene Regulation

e.g. – even-skipped

- each stripe regulatedby a different setof enhancers

- expression patterns arestabilized by interactionsamong other gene products

e.g. even-skippedexpression limitedby Giant

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Segments and Parasegments

Segments and parasegmentsorganized fromA/P compartmentsout of phase

Cells of adjacentcompartmentsdo not mix

Expression patterns in early embryos are not delineatedby segmental boundaries, but by

- parasegments:fundamental units of embryonic gene expression

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Homeotic-Selector/ HOX Genes

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Homeotic Selector Genes

Homeotic genes specify :- head segments

- labial palps- antennae

- thoracic segments- wings- halteres- legs

- abdominal segments

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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)

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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-Selector Genes

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Homeotic Gene Expression

distal-less – jaws, limbs

Antennapeida –thoracic

Ultrabithorax –abdomen

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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.

Patterns of Expression

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•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.

Homeotic Mutations

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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.]

Effects of Mutations in Bithorax Complex

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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.

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Homeotic Gene Expression in Mutant Embryos

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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.

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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, Wilhelm Roux' Arch. Dev. Biol.194:213-216, 1985.)

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Terminal Specification - 1

Torso – transmembrane RTK

Torso uniformly distributed

Torso activated byTorso-like protein- located only at

ends of egg

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Terminal Specification - 2

Distinction betweenanterior and posterior= Bicoid

Bicoid = acronformation

Torso kinases inactivatean inhibitor of taillessand huckebein

Tailless and Huckebeinspecify termini

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