Download Genetic approaches to development: Drosophila as a model organism

Genetic approaches to development:
Drosophila as a model organism
Ruth Lehmann
New York University/Howard Hughes Medical Institute
[email protected]
Model Organisms and Innovative Approaches
in Developmental Biology,
Juquehy, Brazil-2005
Lecture: Genetic Analysis in
Drosophila
Ruth Lehmann
[email protected]
Embryogenesis
Gastrulation
Generation time
Life span
Eggs/female
Fertilization
# of autosomes
Sex chromosomes
LIFE CYCLE STATISTICS
C. elegans
Drosophila
14 hrs
24 hrs
2 hrs
3 hrs
3 days
10 days
20 days
2-3 months
300 (+)
~700
Internal
Internal
5
3
XX, XO
XX, XY
zebrafish
48 hrs
6 hrs
3 month
> 3 years
~15 000
external
25
none
Species
Chromosomes
CM
E. coli
S. cerevisiae
C. elegans
D. melangaster
D. rerio
M.musculus
H. sapiens
1
16
6
4
25
20
23
N/A
4000
300
280
2900
1700
3300
DNA
content/haploid
genome inMB
5
12
100
165
1740
3000
3000
Year
sequence
complete
1997
1997
1998
2000
2004
2003
2001
Genes/haploid
4,000
6,000
19,000
14,000
40,000
30,000
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
Gonadcycle
development
The life
of germ cells
Primordial
germ cells
Embryo
TF
Embryo
soma
stem
cells
germ
line
ovariole
Larva/pupa
Adult
stem
cells
egg chambers
oocyte
Why flies????
Genetics
Different Types of Mutageneses
Mutagenic
Effect
Advantages
Disadvantages
EMS
(and other
chemicals)
Base pair
changes (point
mutations)
Random
Saturation
Different types of mutations
Slow gene identification
Large-scale
P-elements
(and other
transposons)
DNA Insertions
(mostly
hypomorphic)
Fast gene identification
Flexible scale
No saturation
Non-random (hotspots)
Deficiency kit
(and other
aberrations)
Chromosome
rearrangements
(gene deletions)
Small-scale
Fast screening
Defined set
Slow gene identification
No real saturation
Ionizing
radiations (Xrays, g-rays)
Chromosome
rearrangements
(gene deletions)
Random
Gene deletions
Slow gene identification
No real saturation
Inefficient
Forward Genetics in Drosophila
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)
Assays:
You can only find what you are looking for
Primary and secondary screens
•Lethality
•Sterility
•Behavior
•Pattern defects: segmentation, eye
•Gene expression:
RNA, protein, lacZ, GFP
General mutagenesis approach to isolate
zygotic genes
EMS
DTS
Bal
P
X
RT
F1 single
DTS
Bal
*Mutagenized chromosome*
Bal
*Mutagenized chromosome*
Bal
F2
F3
X
*Mutagenized chromosome*
Bal
*Mutagenized chromosome*
*Mutagenized chromosome*
Test phenotype
X
Bal
Bal
Identification Of Genes Required For Germ Cell Migration:
Recessive mutations
mutant A
‘blue’ balancer
‘blue’ balancer
mutant A
mutant A
‘blue’ balancer
‘blue’ balancer
mutant A
“blue”-balancer
Germ cell
marker
Drosophila germ cells from in germ plasm that
assembles at the posterior pole during oogenesis
Germ
plasm
Germ
cells
General mutagenesis approach to isolate
mutations in maternal effect genes
EMS
DTS
Bal
P
X
RT
F1 single
DTS
Bal
X
*Mutagenized chromosome*
Bal
RT
F2
F3
F4
*Mutagenized chromosome*
Bal
*Mutagenized chromosome* *Mutagenized chromosome*
Bal
*Mutagenized chromosome*
progeny
X
Bal
Bal
Test phenotype
The “No Germ Cells” Class
wild-type
“no germ cells”
How to identify all genes in a
process?
I. Same gene plays role during many
stages/in many tissues
Flp/FRT technique
*
*
*
*
FRT
>
>
*
*
>
>
>
>
>
>
>
>
*
>
>
>
>
*
>
>
>
>
FRT/FLP application:
analysis of mosaics of mutant and wildtype tissue
Mosaic analysis with eyespecific twin spots
agoago- FRT
P(w+)
>
>
agoP(w+)
P(w+)
>
>
>
>
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 cancer
cell lines
OvoD technique
Soma
Germ
line
FRT/FLP application:
Lineage analysis
+/wt
+/-
hs-flp ;
hs
FRT
FRT
+/+
-/-
FRT
FRT, nls-GFP
-/-
FRT, nls-GFP
FRT, nls-GFP
+/- or +/+
Enhancer/suppressor screens
Sensitized condition:
@ 22.7oC
sevts
@ 24.3oC
R7 present
R7 absent
a ts mutation in kinase domain
EMS
P
F1
sev- ; + ;
sev+
or
Bal, P(sevts)
D
sevD2 ; + ; +
X Y
+ +
sev- ; * ;
*
sev-/Y
+
Bal, P(sevts)
Screen for absence of R7 at 22.7oC
Different Mapping Methods in Drosophila
Method
tools
principle
result
resolution
pro
con
Classical
meiotic
mapping
Based on
visible
markers
Meiotic
Genetic
recombination
map position
Non-molecular
(not all markers
cloned)
genetic
location
Few visible markers
available, often
spaced far away
from mutant
Deficiency
mapping
Based on
available
deficiencies
Complementation
Chromosomal
interval
Non-molecular
(not all
breakpoints
molecularly
mapped)
Fast
mapping to
a certain
region
Not all regions of
the genome
covered
Interactions with
other genes in Df
P-mediated
male
recombination
mapping
Based on
available P
insertions
Homologous
recombination
Position of
mutation
relative to P
(proximal/distal)
Molecular
(if position of P
known)
Precise
molecular
interval
Stepwise process,
slow
Still requires visible
markers
SNP mapping
Based on
molecular
markers
Meiotic
Molecular map
position
Molecular
Markers
neutral
Precise
molecular
interval
Expensive
dependent on
detection method
Forward Genetics in Drosophila
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
Mis/overexpression screens, the good and the bad
wrong gene --- right pathway
Faf-lacZ; Gal4-driver X
nos
nos5’UTR
Gal4-VP16
nos3’UTR
EP-UAS-insertion lines
UAS
Endogenous gene
Expression
in germ cells
“mes”
Gal4
UAS
Endogenous gene
Expression
in soma
st 14 dorsal
a-Vasa
wild type
over- or mis- expression
These screens can be misleading-gene is not expressed
in germ cells and has no phenotype in germ cells
st 9
tre1
hemocytes
st 13
caudal visceral mesoderm
midgut primordia
midgut
glia
..but
RNA of close homolog is localized to the germ plasm and PGCs
tre1 RNA
Stage 3
and mutations in this gene affect PGCs migration
Stage 13
Prabhat Kunwar
Mis/overexpression screens, the good and the bad
“Redundant” genes
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 can identify “redundant”
genes
wun-/- and wun2-/-
of both genes
mes::Gal4; UAS::wun2
Stage 11
either gene
Vasa
Zhang et al. (1997) Nature 385, 64-67
Starz-Gaiano et al. (2001) Development 128, 983-991
How to identify all genes in a
process?
II. Technologies beyond EMS and Pelements
Reverse Genetics in Drosophila
-Dominant negative (GAL4-UAS based)
-RNAi (injection, GAL4-UAS based)
-Homologous recombination
-Tilling
Keep balanced stock
Venken & Bellen, March 2005
Venken & Bellen, March 2005
Knowing when to Stop Screening:
Efficiency 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
•The ultimate stem cell, able to generate new generation
Egg & Sperm
Zygote
Germ line
stem cells
Primordial Germ Cells
Soma
Early segregation protects germ
cells from somatic differentiation
Death
Germ cells are specificied by maternally synthesized
germ plasm or by cell-to cell induction
Germplasm
Drosophila, Xenopus,
zebrafish, C. elegans
Induction
Mouse, axolotl
Genetically distinct pathways control formation of germ
line and soma in the early in Drosophila emrbyo
Nuclear migration
Budding
Polarized
membrane Growth
Germ cells
Somatic cells
Fly and mouse germ cells:
repression of somatic genes
Transcription is repressed in early germ cells
blue: slam RNA
green: Vasa
red: pSer2-CTD
Stein et al. (2002) Development 129(16), 3925-3934
Seydoux & Dunn (1997)
Development 124(11), 2191-2201
pgc RNA is localized to germ plasm
and PGC protein represses transcription in early germ cells
Early Cleavage
Wild-type
Blastoderm
pgc
pgc RNA
Rui Martinho
pSer2-CTD
Vasa
slam and other “somatic genes”are activated in pgc
mutant germ cells
Wild type
pgc
Wild type
pgc
as-pgc
slam RNA
Rui Martinho
tailless mRNA
Martinho et al. ( 2004) Current Biol. 14(2), 159-165
PGC may repress germ cell transcription by interfering
with transcriptional elongation
CTD phosphorylation recruits Set1 and Set2 histone methylases
How are germ cells set aside from somatic cells
Soma
Germ Cells
Somatic signals
pgc
Somatic differentiation
Target specificity suggests that PGC may
repress transcriptional machinery that normally
acts in posterior soma
pgc-/-
tll mRNA
pgc-/-
slam mRNA
pgc-/-
eve mRNA
Cross-regulation of torso and pgc pathways may
inhibit cell specification of soma vs germ cells
Soma
Germ Cells
Torso
pgc
Receptor tyrosine kinase
Somatic target
genes
Germ cell target
genes
Up-regulation of torso represses germ cell formation
Wild-type
(100%)
8 copies torso+
(35%)
(25%)
Rui Martinho
Up-regulation of pgc leads to somatic cellularisation defects
similar to the ones observed in torso loss of function alleles
Wild-type
green: Vasa
6 copies pgc+
blue: DNA
torLOF
Rui Martinho
Antagonism between pgc and torso sets apart somatic cells
from germ cells
Soma
Germ Cells
torso
pgc
Somatic target
genes
Germ cell target
genes
Posterior
soma
Germ
cells
Repression of somatic differentiation via transcriptional
regulation could be critical for germ cell specification
Germ cell specification
in Drosophila
slam RNA
(somatic gene)
Primordial germ cells
Germ cell specification in mice
(Saitou et al., 2002)
Repression of somatic differentiation via transcriptional
regulation a common theme for germ cell specification?
Germ cell specification
in Drosophila
Germ cell specification in mice
(Saitou et al., 2002)
Hoxb1
(somatic gene)
slam RNA
(somatic gene)
fragilis
(germ line marker)
Primordial germ cells
Primordial germ cells
Fly and mouse germ cells:
pgc = stem cells?
The niche concept for stem cell maintenance
From:Spradling et al. (2001) Nature 414, 98-104
Somatic niche
Somatic
niche
Stem cells
Differentiating
Cystoblast
Stem cell
Cystoblast
Egg
chamber
Differentiated
egg chamber
Somatic cells
Germ line
Dpp, a BMP2/4 homologue, is an instructive
stem cell factor
Wild type
Fusome
Vasa
Loss of function
dpp-/-
Fusome
Vasa
Gain of function
hs-dpp
Fusome
Vasa
Xie and Spradling Cell 94, 251-260 (1998)
Xie and Spradling Science 290, 328-330 (2000)
The Drosophila BMP2/4 homologue DPP
signals to germ line stem cells via the niche
Soma/niche
Dpp ligand
Germ line
Niche
Stem cell
Cystoblast
Tkv (type I receptor)
Punt (type II receptor)
MadP, Medea
Target genes
Bam, dad
Vasa
p-Mad
Differentiated
egg chamber
The number of germ cells increases
dramatically during larval stages
EE
250
LL3
150
Number
of adult
GSCs/Ovary
50
24 h
48 h
72 h
96 h
Hours after egg laying
~108 h
Bar: 20 mm
PGCs away from the niche differentiate at the
end of larval development
Mid 3rd instar larva
bam::GFP
1B1
Bam-GFP
hts
Late 3rd instar larva
/early pupa
Early pupa
Zhu CH, Xie T. (2003) Development,130(12):2579-88.
PGC differentiation is repressed during larval
stages by the Dpp pathway
WT
1B1
pMad
ML3
nos-Gal4 X UAS-dad
Lilach
Gilboa
1B1
Vasa
ML3
LL3
1B1
Orb
Restriction of niche controls initial
stem cell selection
Early Larva
Dpp/BMP
Late Larva
Tkv
Smads
Bam
Pum
& Nos
Primordial germ cell = germ line
stem cell?
Vasa
pSer2-CTD
1B1
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.