Drosophila melanogaster Genetic studies Microsurgical manipulation One of the best understood developmental systems 13,600 genes Axis determination Signaling.

Drosophila melanogaster

Genetic studiesMicrosurgical manipulationOne of the best understood developmental systems

13,600 genes

Axis determinationSignaling pathwayTranscriptional regulation

P48-52

4 stages: embryo, larva, pupa, adult

Rapid division9 mins/division9 divisions

13 divisions

Single cell

Transgenic flies

One single epithelial layer –all tissuesMesoderm—muscle, connective tissuesEndoderm---midgut (foregut and hidgut-Ectoderm)Ectoderm---nervous tissue and epidermis

gastrulation

Larva hatch 24 hrsAcron: associated with headTelson:posterior terminal structure3 thorcic and 8 abdominal segments—specialization in cuticle(denticle belts and cuticular structure)

GenitaliaSex combPigmentationSmall wing

p. 421-431

Sex determining signal--- Sex-lethal (X chromosome)

Transformer-spliced + transformer 2

2X higher numerator Repression by autosome

Dosage compensation

Barr bodyXist-non-coding RNA

Male specific geneRepressed by Sxl

Primordial germ cell -special cytoplasmGerm plasm-polar granules, pole plasm

Oskar—organization and assembly of the pole plasmmRNA-posterior pole—3’ untranslated region

Polarization of the body axes during oogenesis

Cyst formation:

16 cell cyst enter a long S phase

only one (Oocyte) continues meiosis

Oocyte—4 ring canals

15 cells become nurse cells

after germarium nurse cells left meiotic cycle, grow rapidly without division, and form polytene chromosomes

A/P during oogenesisThe oocyte move towards one end in contact with follicle cellsBoth the oocyte and the posterior follicle cells express high levels of the E-cadherin

If E-cadherin is removed, the oocyte is randomly positioned.Then the oocyte induces surrounding follicle cell to adopt posterior fate.

Microtubule cytoskeleton reorganization is essential for localization of bicoid and oskar mRNA

Maternal effect mutations---reguired for pole plasm assemblyLack polar granules: grandchildless mutation (homozygote female— Progeny—sterile)

Central role: Oskar, Vasa, and TudorPole cell number = amount of oskar RNAEctopic pole cells: oskar RNA at the anterior pole

MtlrRNA (mitochondrically encoded large ribosomal RNA)+ gcl RNA for pole cell formation

antisense reduce pole cells mtlrRNA rescue UV-irradation

Vasa: DEAD-box RNA helicase—translational regulator

Germ cell—extragonadal origin, migrate to

reach the somatic gonada. posterior endb. gastrulation c. migrate dorsally through the wall of the

posterior midgutd. associate with the somatic gonadal prec

ursorse. GC align with somatic gonadal mesoder

mf. coalesce to form the embryonic gonad

Germ cell migration

PGC migration----Genes and mechanisms

Genetic screen—somatically expressed genesGuidance (cues):Wunen: repulsive signal (exclude migrating pole cells from wrong places)Misexpression wunen: transform a tissue permissive to PGC to repulsive onePhosphatidic acid phosphatase 2 (transmembrane protein) Columbus: factor (gonadal mesoderm) attracts pole cellsMisexpression Columbus—attract PGCs to tissues other than gonadal mesoderm3-hydroxy-3-methylglutaryl coenzymeA reductase (cholesterol biosynthesis in human, but fly does not make cholesterol)  nanos, pumilio mutants stall at the outer gut surfacedifferentiate prematurely---act as complete migration to the somatic gonadsnanos target: RNA binding protein Sex lethal (Sxl)---splicing and translational regulation

also depend on specific germ plasm components, e.g polar granule component (Pgc)

Patterning of the fly embryo

Localized mRNA and ProteinsTranslated after fertilization—

Positional information to activate zygotic genes

parasegment

Pattern in the segment

Segment identities

Temporal sequence

Appendages:imaginal discs—pattern formationEctoderm invagination-epithelium(20-40 cells—larva 1000X)Specification occurs –segment being patterned-according to it

p.350-358

A/P and D/V compartment

Wing blade

Ventral fold under dorsal-double layers of epithelium

Signal region and the compartment

Maintain compartment boundaries—communication between compartmentsHh—10 cells, induces expression of Dpp through activation of Ci

The hedgehog signaling pathway

Without signal—Ci is processed as a repressor into nucleusWith signal---full length Ci acts as an activator inthe nucleus

Intercellular signaling set up PS boundary

Wg distributed asymmetrically—less in posterior (endocytosis and degradation)

TGFb , Activin: R-Smad 2,3BMPs: R-Smad 1, 5, 8Common Smad4Inhibitory Smads: I-Smad6, 7—recruting Smurf (ubiquitin ligase to receptor) Cell, 95,737,1998

Smad= Sma + MadSma-C. elegansMad-Fly

Dpp-secreted into both compartment

Long range signal—expression of spalt

Patterning the A/P axis of the wing disc

Dpp-morphogenLow level—ombHigh level—spalt1. Clones can’t respond to Dpp— no spalt and omb2. Ectopic hh-Dpp—localized activation of spalt and omb around the hh clones3. Ts mutant of dpp—reduction in the region with expression of lowThreshold genes—omb4. Clones expression low or highDpp—distinquish these two types of genes

Ectopic expression of Hh and Dpp

L4—compartment boundaryL3– HhL2 ---adjacent to cells expressing spalt

Ectopic Hh in posterior—no effectsIn anterior—mirrow-symmetric repeated pattern

Hh--Dpp

Expression of Wingless (green) and vestigial

Homeotic selector gene—apterous (Lmx-1)induces fringe and Serrate, then Notch receptor activation –Leading to Wingless expressionWg—achaete, distal-less, vestigial

Wingless (green)Vestigial (red)D/V boundaryDpp, Wg morphogenGFP-dpp active transportation—EndocytosisRegulate their receptorsDpp inhibits receptor—thick veinsDpp high--receptor low, and dpp lowReceptor high—1, prevent spreading2,cells reach threshold at low Dpp

Notch –transmembrane proteinDSL family—Delta, Serrate, Lag-2

Kuzbanian—cleave Notch ECDPresenilin—cleave Notch ICD

nervous system: selection of a single neuroblast (lateral inhibition)

Leg disc extension

Jointed tubes of epidermis—secrete the hard cuticle (exoskeleton), inside:Muscles, nerves

Fate map of the leg imaginal disc

Proximo-distal segmentCenter—distal end

Signaling centers in A/P compartment

Dpp, wg meets—Dll (distal end) homothorax (proximal)

Regional subdivision

Dpp, and Wg induce Dll and inhibit homothoraxActivates dachshund between Dll and hth

Butterfly wing pattern

Eyespot center—distal-less

Segmental identity of imaginal discHomeotic selector genesSimilar signal into different structures—Different interpretation—controlled by Hox genes

Antennapedia—PS4 and 5– 2 pairs of legsIf in head, antennae into legs (clones) –which part of the leg—depends on their position along the P/D axis (positional values are similar)

Hth (proximal) and Dll (distal)—in antennae and legIn combination as selector to specify antennaNo Hth, antenna into legIn leg: antennapedia prevents Hth and Dll acting togetherDominant antennapedia mutant (gene on)—blocks Hth and Dll in antennae disc, so leg forms

Imaginal discs and adult thoracic appendages

Bithorax mutation—Ubx misexpressed T3 into T2 –anterior haltere into Anterior wing

Postbithorax muation (pbx)—Regulatory region of the Ubx—Posterior of the haltere into wing

If both mutations—effect is additive—Four wings