Genetic approaches to development: Drosophila as a model organism

Ruth Lehmann New York University/Howard Hughes Medical Institute

lehmann@saturn.med.nyu.edu

Model Organisms and Innovative Approachesin Developmental Biology,

Juquehy, Brazil-2005

Lecture: Genetic Analysis inDrosophila

Ruth LehmannLehmann@saturn.med.nyu.edu

LIFE CYCLE STATISTICSC. elegans Drosophila zebrafish

Embryogenesis 14 hrs 24 hrs 48 hrsGastrulation 2 hrs 3 hrs 6 hrsGeneration time 3 days 10 days 3 monthLife span 20 days 2-3 months > 3 yearsEggs/female 300 (+) ~700 ~15 000Fertilization Internal Internal external# of autosomes 5 3 25Sex chromosomes XX, XO XX, XY none

Species Chromo-somes

CM DNAcontent/haploidgenome inMB

Yearsequencecomplete

Genes/haploid

E. coli 1 N/A 5 1997 4,000S. cerevisiae 16 4000 12 1997 6,000C. elegans 6 300 100 1998 19,000D. melangaster 4 280 165 2000 14,000D. rerio 25 2900 1740 2004 40,000M.musculus 20 1700 3000 2003 30,000H. sapiens 23 3300 3000 2001 30,000

A brief history of fly genetics•1910 Morgan identifies white•1930 Calvin Bridges linkage groups and polytene chromosomes•1930 Sturtevant clonal analysis•1948 Balancer Chromosomes•1968 Lewis and Bacher EMS•1934 to 1965 From 572 stocks in 1934 to 15 000 in 1965•1980 Systematic mutant screens for embryonic lethals by Nüsslein-Volhard

and Eric Wieschaus•1980 P element techniques by Rubin and Spradling•2000 Genome sequenced•2005 Mutations in 7000 genes deletions for most of the genome

Embryo

Gonad development

stemcells

egg chambers

Adult

oocyte

The life cycle of germ cells

EmbryoPrimordialgerm cells

TF

ovariole

Larva/pupa soma

germline

stemcells

Why flies????

Genetics

Different Types of Different Types of MutagenesesMutageneses

No saturationNon-random (hotspots)

Fast gene identificationFlexible scale

DNA Insertions(mostlyhypomorphic)

P-elements(and othertransposons)

Chromosomerearrangements(gene deletions)

Chromosomerearrangements(gene deletions)

Base pairchanges (pointmutations)

MutagenicEffect

Slow gene identificationNo real saturation

Small-scaleFast screeningDefined set

Deficiency kit(and otheraberrations)

Slow gene identificationNo real saturationInefficient

RandomGene deletions

Ionizingradiations (X-rays, g-rays)

Slow gene identificationLarge-scale

RandomSaturationDifferent types of mutations

EMS(and otherchemicals)

DisadvantagesAdvantages

Forward Genetics in Forward Genetics in DrosophilaDrosophila

any mutagen-Zygotic screen (e.g. Wieschaus & Nüsslein-Volhard)-Maternal-effect screen (e.g. Schüpbach & Wieschaus)-Maternal-effect clonal screen (e.g. Perrimon, St Johnston)-Adult clonal screen (e.g. Dickson)-Modifier (Enhancer/Suppressor) screen (e.g. Rubin)

You can only find what you are looking for

•Lethality

•Pattern defects: segmentation, eye

•Gene expression: RNA, protein, lacZ, GFP

Primary and secondary screens

•Sterility

•Behavior

Assays:

General mutagenesis approach to isolatezygotic genes

Bal*Mutagenized chromosome*Xsingle

P

F1

F2

F3

BalDTS X

EMS

RT

BalDTS

Bal*Mutagenized chromosome* X

Bal*Mutagenized chromosome*Bal*Mutagenized chromosome*

Bal*Mutagenized chromosome*

Test phenotype

Identification Of Genes Required For Germ Cell Migration:Recessive mutations

Germ cell marker“blue”-balancer

mutant A

‘blue’ balancer

mutant A

‘blue’ balancer‘blue’ balancer

mutant A‘blue’ balancer

mutant A

Germplasm

Germcells

Drosophila germ cells from in germ plasm thatassembles at the posterior pole during oogenesis

General mutagenesis approach to isolatemutations in maternal effect genes

Bal*Mutagenized chromosome*X

P

singleF1

F2

F3

BalDTS X

EMS

RT

BalDTS

Bal*Mutagenized chromosome* X

Bal*Mutagenized chromosome*Bal*Mutagenized chromosome*

Bal*Mutagenized chromosome*

Test phenotype

RT

progenyF4

The “No Germ Cells” Class

wild-typewild-type ““no germ cellsno germ cells””

How to identify all genes in aprocess?

I. Same gene plays role during manystages/in many tissues

Flp/FRT technique

* FRT>>

>>

>>

*

>>

>>

*

*

>*>*

>>

>*>

>*>

KENNETH H. MOBERG, DAPHNE W. BELL, DOKE C. R. WAHRER, DANIEL A. HABER & ISWAR K. HARIHARAN Nature 413, 311 - 316 (2001); Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancercell lines

>>

ago-

>>

P(w+)

P(w+)

ago-

FRT>>

P(w+)

ago-

Mosaic analysis with eye-specific twin spots

FRT/FLP application: analysis of mosaics of mutant and wildtype tissue

OvoD technique

Germ

line

Soma

+/-

+/+

+/- or +/+

-/-

-/-

+/-

wt

hs-flp ;FRT, nls-GFP

FRT

FRT, nls-GFPFRT

hsFRT, nls-GFPFRT

FRT/FLP application: Lineage analysis

Enhancer/suppressor screens

sevts

+

@ 22.7oC

+

@ 24.3oC

R7 present R7 absent

Sensitized condition:

sev-

sev- DBal, P(sevts) sevD2

++

++

X

sev-

sev-/Y +* *

Bal, P(sevts)

; ; ; ;

; ;

Y

EMS

or

Screen for absence of R7 at 22.7oC

P

F1

a ts mutation in kinase domain

Different Mapping Methods in Different Mapping Methods in Drosophila Drosophila

Based onmolecularmarkers

Based onavailable Pinsertions

Based onavailabledeficiencies

Based onvisiblemarkers

MarkersneutralPrecisemolecularinterval

Precisemolecularinterval

Fastmapping toa certainregion

geneticlocation

Expensivedependent ondetection method

MolecularMolecular mapposition

MeioticSNP mapping

Stepwise process,slowStill requires visiblemarkers

Molecular(if position of Pknown)

Position ofmutationrelative to P(proximal/distal)

Homolo-gousrecom-bination

P-mediatedmalerecombinationmapping

Not all regions ofthe genomecoveredInteractions withother genes in Df

Non-molecular(not allbreakpointsmolecularlymapped)

Chromosomalinterval

Complem-entation

Deficiencymapping

Few visible markersavailable, oftenspaced far awayfrom mutant

Non-molecular(not all markerscloned)

Geneticrecombinationmap position

MeioticClassicalmeioticmapping

Method tools principle result resolution pro con

Forward Genetics in Forward Genetics in DrosophilaDrosophila

any mutagen-Zygotic screen (e.g. Wieschaus & Nüsslein-Volhard)-Maternal-effect screen (e.g. Schüpbach & Wieschaus)-Maternal-effect clonal screen (e.g. Perrimon, St Johnston)-Adult clonal screen (e.g. Dickson)-Modifier (Enhancer/Suppressor) screen (e.g. Rubin)

P-element based-Enhancer-trap screen (e.g. Bellen, Jan)-Overexpression-trap screen (e.g. Rorth)-Protein-trap screen (e.g. Chia, Cooley)

Venken & Bellen, March 2005

st 14 dorsal

a-Vasa

over- or mis- expressionwild type

Mis/overexpression screens, the good and the badwrong gene --- right pathway

Gal4-VP16nos3’UTRnos5’UTRnos

Expression in germ cells

Expression in soma

Gal4“mes”

Endogenous geneUAS

Faf-lacZ; Gal4-driver EP-UAS-insertion linesX

Endogenous geneUAS

tre1

st 9

hemocytes

caudal visceral mesoderm

midgut primordia

glia

midgutst 13

These screens can be misleading-gene is not expressedin germ cells and has no phenotype in germ cells

RNA of close homolog is localized to the germ plasm and PGCs

tre1 RNA

Prabhat Kunwar

Stage 13

Stage 3

and mutations in this gene affect PGCs migration

..but

wunen RNA wunen 2 RNA

Zhang et al, Nature (1997) 385, 64-67; Starz-Gaiano et al, Development (2001) 128, 983-991

Mis/overexpression screens, the good and the bad“Redundant” genes

Zhang et al. (1997) Nature 385, 64-67 Starz-Gaiano et al. (2001) Development 128, 983-991

Stage 11 Vasa

either gene

Mis/overexpression screens can identify “redundant”genes

wun-/- and wun2-/-

mes::Gal4; UAS::wun2

of both genes

How to identify all genes in aprocess?

II. Technologies beyond EMS and P-elements

Reverse Genetics in Reverse Genetics in DrosophilaDrosophila

-Dominant negative (GAL4-UAS based) -RNAi (injection, GAL4-UAS based) -Homologous recombination-Tilling

Venken & Bellen, March 2005

Keep balanced stock

Venken & Bellen, March 2005

Knowing when to Stop Screening:Knowing when to Stop Screening:Efficiency and SaturationEfficiency and Saturation

Wieschaus and Nüsslein-Volhard

Germ Cells

•Set aside early in development from somatic cells

•Highly specialized (migration, cell interaction, meiosis)

•The ultimate stem cell, able to generate new generation

Primordial Germ Cells

•The ultimate stem cell, able to generate new generation

Egg & Sperm

Soma

Death

Germ line stem cells

Zygote

Early segregation protects germcells from somatic differentiation

Germplasm Induction

Germ cells are specificied by maternally synthesizedgerm plasm or by cell-to cell induction

Drosophila, Xenopus,zebrafish, C. elegans

Mouse, axolotl

Nuclear migration

Budding Polarizedmembrane Growth

Germ cells Germ cells Somatic cellsSomatic cells

Genetically distinct pathways control formation of germline and soma in the early in Drosophila emrbyo

Fly and mouse germ cells:repression of somatic genes

Transcription is repressed in early germ cells

blue: slam RNA

Stein et al. (2002) Development 129(16), 3925-3934

green: Vasared: pSer2-CTD

Seydoux & Dunn (1997)Development 124(11), 2191-2201

pgc RNA is localized to germ plasm and PGC protein represses transcription in early germ cells

Blastoderm

Early Cleavage

pgc RNARui Martinho

Wild-type

Vasa

pgc

pSer2-CTD

slam and other “somatic genes”are activated in pgcmutant germ cells

Martinho et al. ( 2004) Current Biol. 14(2), 159-165

Wild type

tailless mRNAslam RNA

Wild type pgc

as-pgc

pgc

Rui Martinho

PGC may repress germ cell transcription by interferingwith transcriptional elongation

CTD phosphorylation recruits Set1 and Set2 histone methylases

Soma Germ Cells

Somatic differentiation

pgcSomatic signals

How are germ cells set aside from somatic cells

tll mRNA slam mRNA eve mRNA

Target specificity suggests that PGC mayrepress transcriptional machinery that normally

acts in posterior soma

pgc-/- pgc-/-pgc-/-

Cross-regulation of torso and pgc pathways mayinhibit cell specification of soma vs germ cells

Soma Germ Cells

Somatic targetgenes

Germ cell targetgenes

pgcTorsoReceptor tyrosine kinase

Up-regulation of torso represses germ cell formation

Wild-type 8 copies torso+

(100%) (35%) (25%)

Rui Martinho

6 copies pgc+ torLOF

green: Vasa blue: DNA

Up-regulation of pgc leads to somatic cellularisation defectssimilar to the ones observed in torso loss of function alleles

Wild-type

Rui Martinho

Posteriorsoma

Germcells

Antagonism between pgc and torso sets apart somatic cellsfrom germ cells

Soma Germ Cells

Somatic targetgenes

Germ cell targetgenes

pgctorso

Repression of somatic differentiation via transcriptionalregulation could be critical for germ cell specification

Germ cell specification in mice(Saitou et al., 2002)

Germ cell specification in Drosophila

Primordial germ cells

slam RNA(somatic gene)

Repression of somatic differentiation via transcriptionalregulation a common theme for germ cell specification?

Hoxb1(somatic gene)

fragilis(germ line marker)

Primordial germ cells

Germ cell specification in mice(Saitou et al., 2002)

Germ cell specification in Drosophila

Primordial germ cells

slam RNA(somatic gene)

Fly and mouse germ cells:pgc = stem cells?

The niche concept for stem cell maintenance

From:Spradling et al. (2001) Nature 414, 98-104

Stem cells

DifferentiatingCystoblast

Somatic cellsGerm line

Eggchamber

Somatic niche

Stem cell

Cystoblast

Differentiated egg chamber

Somaticniche

Dpp, a BMP2/4 homologue, is an instructivestem cell factor

hs-dpp FusomeVasa

Gain of function

Wild typeFusomeVasa

Xie and Spradling Cell 94, 251-260 (1998)Xie and Spradling Science 290, 328-330 (2000)

dpp-/-

Loss of function

FusomeVasa

The Drosophila BMP2/4 homologue DPP signals to germ line stem cells via the niche

Dpp ligand Tkv (type I receptor)Punt (type II receptor)

Target genesBam, dad

Soma/niche Germ line

Mad , MedeaP

Vasap-Mad

Stem cell

Cystoblast

Differentiated egg chamber

Niche

The number of germ cells increasesdramatically during larval stages

48 h 72 h 96 h ~108 h24 h

50

150

250

Hours after egg laying

Numberof adultGSCs/Ovary

LL3

EE

Bar: 20 mm

PGCs away from the niche differentiate at theend of larval development

Zhu CH, Xie T. (2003) Development,130(12):2579-88.

bam::GFP1B1

Bam-GFPhts

Early pupa

Mid 3rd instar larva

Late 3rd instar larva/early pupa

PGC differentiation is repressed during larvalstages by the Dpp pathway

1B1Vasa

ML3

nos-Gal4 X UAS-dad

1B1Orb

LL3

1B1pMad ML3

WT

Lilach Gilboa

Restriction of niche controls initialstem cell selection

Bam

TkvSmads

Early Larva

Late LarvaPum& Nos

Dpp/BMP

Primordial germ cell = germ linestem cell?

1B1Vasa

Vasa

Niki Y, Mahowald AP. (2003) Proc Natl Acad Sci U S A; 100(24):14042-5 Gilboa L, Lehmann R. (2004) Curr Biol;14(11):981-6. Wang Z, Lin H. (2004) Science; 303(5666):2016-9. Epub 2004 Feb 19.

pSer2-CTD