Genetic approaches to development: Drosophila as a model ... · UAS Endogenous gene. tre1 st 9...
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Genetic approaches to development: Drosophila as a model organism
Ruth Lehmann New York University/Howard Hughes Medical Institute
Model Organisms and Innovative Approachesin Developmental Biology,
Juquehy, Brazil-2005
Lecture: Genetic Analysis inDrosophila
Ruth [email protected]
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>>
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>*>*
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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-
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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