Genetic approaches to development - Society for Developmental ...
Genetic approaches to development - Society for Developmental ...
Genetic approaches to development - Society for Developmental ...
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
<strong>Genetic</strong> <strong>approaches</strong> <strong>to</strong> <strong>development</strong>:<br />
Drosophila as a model organism<br />
Ruth Lehmann<br />
New York University/Howard Hughes Medical Institute<br />
lehmann@saturn.med.nyu.edu
Model Organisms and Innovative Approaches<br />
in <strong>Developmental</strong> Biology,<br />
Juquehy, Brazil-2005<br />
Lecture: <strong>Genetic</strong> Analysis in<br />
Drosophila<br />
Ruth Lehmann<br />
Lehmann@saturn.med.nyu.edu
LIFE CYCLE STATISTICS<br />
C. elegans Drosophila zebrafish<br />
Embryogenesis 14 hrs 24 hrs 48 hrs<br />
Gastrulation 2 hrs 3 hrs 6 hrs<br />
Generation time 3 days 10 days 3 month<br />
Life span 20 days 2-3 months > 3 years<br />
Eggs/female 300 (+) ~700 ~15 000<br />
Fertilization Internal Internal external<br />
# of au<strong>to</strong>somes 5 3 25<br />
Sex chromosomes XX, XO XX, XY none
Species<br />
Chromosomes<br />
CM<br />
DNA<br />
content/haploid<br />
genome inMB<br />
Year<br />
sequence<br />
complete<br />
E. coli 1 N/A 5 1997 4,000<br />
S. cerevisiae 16 4000 12 1997 6,000<br />
C. elegans 6 300 100 1998 19,000<br />
D. melangaster 4 280 165 2000 14,000<br />
D. rerio 25 2900 1740 2004 40,000<br />
M.musculus 20 1700 3000 2003 30,000<br />
H. sapiens 23 3300 3000 2001 30,000<br />
Genes/haploid
A brief his<strong>to</strong>ry of fly genetics<br />
•1910 Morgan identifies white<br />
•1930 Calvin Bridges linkage groups and polytene chromosomes<br />
•1930 Sturtevant clonal analysis<br />
•1948 Balancer Chromosomes<br />
•1968 Lewis and Bacher EMS<br />
•1934 <strong>to</strong> 1965 From 572 s<strong>to</strong>cks in 1934 <strong>to</strong> 15 000 in 1965<br />
•1980 Systematic mutant screens <strong>for</strong> embryonic lethals by Nüsslein-Volhard<br />
and Eric Wieschaus<br />
•1980 P element techniques by Rubin and Spradling<br />
•2000 Genome sequenced<br />
•2005 Mutations in 7000 genes deletions <strong>for</strong> most of the genome
The life Gonad cycle <strong>development</strong> of germ cells<br />
Primordial<br />
germ cells<br />
Embryo<br />
Embryo<br />
soma<br />
germ<br />
line<br />
TF<br />
stem<br />
cells<br />
ovariole<br />
Larva/pupa<br />
stem<br />
cells<br />
egg chambers<br />
Adult<br />
oocyte
Why flies????<br />
<strong>Genetic</strong>s
Different Types of Mutageneses<br />
Mutagenic<br />
Advantages<br />
Disadvantages<br />
Effect<br />
EMS<br />
(and other<br />
chemicals)<br />
Base pair<br />
changes (point<br />
mutations)<br />
Random<br />
Saturation<br />
Different types of mutations<br />
Slow gene identification<br />
Large-scale<br />
P-elements<br />
(and other<br />
transposons)<br />
DNA Insertions<br />
(mostly<br />
hypomorphic)<br />
Fast gene identification<br />
Flexible scale<br />
No saturation<br />
Non-random (hotspots)<br />
Deficiency kit<br />
Chromosome<br />
Small-scale<br />
Slow gene identification<br />
(and other<br />
aberrations)<br />
rearrangements<br />
(gene deletions)<br />
Fast screening<br />
Defined set<br />
No real saturation<br />
Ionizing<br />
radiations (Xrays,<br />
g-rays)<br />
Chromosome<br />
rearrangements<br />
(gene deletions)<br />
Random<br />
Gene deletions<br />
Slow gene identification<br />
No real saturation<br />
Inefficient
Forward <strong>Genetic</strong>s in Drosophila<br />
any mutagen<br />
-Zygotic screen (e.g. Wieschaus & Nüsslein-Volhard)<br />
-Maternal-effect screen (e.g. Schüpbach & Wieschaus)<br />
-Maternal-effect clonal screen (e.g. Perrimon, St Johns<strong>to</strong>n)<br />
-Adult clonal screen (e.g. Dickson)<br />
-Modifier (Enhancer/Suppressor) screen (e.g. Rubin)
Assays:<br />
You can only find what you are looking <strong>for</strong><br />
Primary and secondary screens<br />
•Lethality<br />
•Sterility<br />
•Behavior<br />
•Pattern defects: segmentation, eye<br />
•Gene expression:<br />
RNA, protein, lacZ, GFP
P<br />
General mutagenesis approach <strong>to</strong> isolate<br />
zygotic genes<br />
DTS<br />
Bal<br />
X<br />
EMS<br />
F1<br />
single<br />
DTS<br />
Bal<br />
X<br />
RT<br />
*Mutagenized chromosome*<br />
Bal<br />
F2<br />
*Mutagenized chromosome*<br />
Bal<br />
X<br />
F3<br />
*Mutagenized chromosome*<br />
Bal<br />
*Mutagenized chromosome*<br />
*Mutagenized chromosome*<br />
Bal<br />
Bal<br />
Test phenotype
Identification Of Genes Required For Germ Cell Migration:<br />
Recessive mutations<br />
mutant A<br />
‘blue’ balancer<br />
‘blue’ balancer<br />
‘blue’ balancer<br />
mutant A<br />
‘blue’ balancer<br />
mutant A<br />
mutant A<br />
“blue”-balancer<br />
Germ cell<br />
marker
Drosophila germ cells from in germ plasm that<br />
assembles at the posterior pole during oogenesis<br />
Germ<br />
plasm<br />
Germ<br />
cells
P<br />
General mutagenesis approach <strong>to</strong> isolate<br />
mutations in maternal effect genes<br />
DTS<br />
Bal<br />
X<br />
RT<br />
EMS<br />
F1 single<br />
F2<br />
DTS<br />
Bal<br />
X<br />
*Mutagenized chromosome*<br />
Bal<br />
*Mutagenized chromosome*<br />
Bal<br />
RT<br />
X<br />
F3<br />
*Mutagenized chromosome*<br />
Bal<br />
*Mutagenized chromosome*<br />
*Mutagenized chromosome*<br />
Bal<br />
Bal<br />
F4<br />
progeny<br />
Test phenotype
The “No Germ Cells” Class<br />
wild-type<br />
“no germ cells”
How <strong>to</strong> identify all genes in a<br />
process?<br />
I. Same gene plays role during many<br />
stages/in many tissues
Flp/FRT technique<br />
* FRT<br />
><br />
><br />
*<br />
*<br />
><br />
><br />
*<br />
><br />
><br />
><br />
><br />
*<br />
><br />
><br />
*<br />
*<br />
><br />
><br />
*<br />
><br />
>
FRT/FLP application:<br />
analysis of mosaics of mutant and wildtype tissue<br />
Mosaic analysis with eyespecific<br />
twin spots<br />
ago -<br />
P(w + )<br />
FRT<br />
><br />
><br />
ago -<br />
><br />
><br />
ago -<br />
P(w + )<br />
><br />
P(w + )<br />
><br />
KENNETH H. MOBERG, DAPHNE W. BELL, DOKE C. R. WAHRER, DANIEL A. HABER & ISWAR K. HARIHARAN<br />
Nature 413, 311 - 316 (2001);<br />
Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer<br />
cell lines
OvoD technique<br />
Soma<br />
Germ<br />
line
FRT/FLP application:<br />
Lineage analysis<br />
-/-<br />
+/-<br />
+/+<br />
wt<br />
hs-flp ;<br />
hs<br />
FRT<br />
FRT<br />
FRT<br />
FRT, nls-GFP<br />
+/-<br />
-/-<br />
FRT, nls-GFP<br />
FRT, nls-GFP<br />
+/- or +/+
Enhancer/suppressor screens<br />
Sensitized condition:<br />
sev ts +<br />
a ts mutation in kinase domain<br />
@ 22.7 o C<br />
R7 present<br />
@ 24.3 o C<br />
R7 absent<br />
EMS<br />
P<br />
sev -<br />
sev -<br />
;<br />
+<br />
;<br />
Bal, P(sev ts ) sev D2 +<br />
D<br />
X<br />
; ;<br />
Y +<br />
+<br />
+<br />
F1<br />
or<br />
sev -<br />
; * ;<br />
*<br />
sev - /Y + Bal, P(sev ts )<br />
Screen <strong>for</strong> absence of R7 at 22.7 o C
Different Mapping Methods in Drosophila<br />
Method <strong>to</strong>ols principle result resolution pro con<br />
Classical<br />
meiotic<br />
mapping<br />
Deficiency<br />
mapping<br />
P-mediated<br />
male<br />
recombination<br />
mapping<br />
SNP mapping<br />
Based on<br />
visible<br />
markers<br />
Based on<br />
available<br />
deficiencies<br />
Based on<br />
available P<br />
insertions<br />
Based on<br />
molecular<br />
markers<br />
Meiotic<br />
Homologous<br />
recombination<br />
Complementation<br />
Meiotic<br />
<strong>Genetic</strong><br />
recombination<br />
map position<br />
Chromosomal<br />
interval<br />
Position of<br />
mutation<br />
relative <strong>to</strong> P<br />
(proximal/distal)<br />
Molecular map<br />
position<br />
Non-molecular<br />
(not all markers<br />
cloned)<br />
Non-molecular<br />
(not all<br />
breakpoints<br />
molecularly<br />
mapped)<br />
Molecular<br />
(if position of P<br />
known)<br />
Molecular<br />
genetic<br />
location<br />
Fast<br />
mapping <strong>to</strong><br />
a certain<br />
region<br />
Precise<br />
molecular<br />
interval<br />
Markers<br />
neutral<br />
Precise<br />
molecular<br />
interval<br />
Few visible markers<br />
available, often<br />
spaced far away<br />
from mutant<br />
Not all regions of<br />
the genome<br />
covered<br />
Interactions with<br />
other genes in Df<br />
Stepwise process,<br />
slow<br />
Still requires visible<br />
markers<br />
Expensive<br />
dependent on<br />
detection method
Forward <strong>Genetic</strong>s in Drosophila<br />
any mutagen<br />
-Zygotic screen (e.g. Wieschaus & Nüsslein-Volhard)<br />
-Maternal-effect screen (e.g. Schüpbach & Wieschaus)<br />
-Maternal-effect clonal screen (e.g. Perrimon, St Johns<strong>to</strong>n)<br />
-Adult clonal screen (e.g. Dickson)<br />
-Modifier (Enhancer/Suppressor) screen (e.g. Rubin)<br />
P-element based<br />
-Enhancer-trap screen (e.g. Bellen, Jan)<br />
-Overexpression-trap screen (e.g. Rorth)<br />
-Protein-trap screen (e.g. Chia, Cooley)
Venken & Bellen, March 2005
Mis/overexpression screens, the good and the bad<br />
wrong gene --- right pathway<br />
Faf-lacZ; Gal4-driver<br />
X<br />
EP-UAS-insertion lines<br />
Expression<br />
in germ cells<br />
nos<br />
nos5’UTR<br />
Gal4-VP16<br />
nos3’UTR<br />
UAS<br />
Endogenous gene<br />
“mes”<br />
Gal4<br />
UAS<br />
Endogenous gene<br />
Expression<br />
in soma<br />
st 14 dorsal<br />
wild type<br />
a-Vasa<br />
over- or mis- expression
These screens can be misleading-gene is not expressed<br />
in germ cells and has no phenotype in germ cells<br />
st 9<br />
caudal visceral mesoderm<br />
tre1<br />
hemocytes<br />
midgut primordia<br />
st 13<br />
midgut<br />
glia
..but<br />
RNA of close homolog is localized <strong>to</strong> the germ plasm and PGCs<br />
tre1 RNA<br />
Stage 3<br />
and mutations in this gene affect PGCs migration<br />
Stage 13<br />
Prabhat Kunwar
Mis/overexpression screens, the good and the bad<br />
“Redundant” genes<br />
wunen RNA<br />
wunen 2 RNA<br />
Zhang et al, Nature (1997) 385, 64-67; Starz-Gaiano et al, Development (2001) 128, 983-991
Mis/overexpression screens can identify “redundant”<br />
genes<br />
wun -/- and wun2 -/-<br />
of both genes<br />
mes::Gal4; UAS::wun2<br />
either gene<br />
Stage 11<br />
Vasa<br />
Zhang et al. (1997) Nature 385, 64-67<br />
Starz-Gaiano et al. (2001) Development 128, 983-991
How <strong>to</strong> identify all genes in a<br />
process?<br />
II. Technologies beyond EMS and P-<br />
elements
Reverse <strong>Genetic</strong>s in Drosophila<br />
-Dominant negative (GAL4-UAS based)<br />
-RNAi (injection, GAL4-UAS based)<br />
-Homologous recombination<br />
-Tilling
Venken & Bellen, March 2005<br />
Keep balanced s<strong>to</strong>ck
Venken & Bellen, March 2005
Knowing when <strong>to</strong> S<strong>to</strong>p Screening:<br />
Efficiency and Saturation<br />
Wieschaus and Nüsslein-Volhard
Germ Cells<br />
•Set aside early in <strong>development</strong> from somatic cells<br />
•Highly specialized (migration, cell interaction, meiosis)<br />
•The ultimate stem cell, able <strong>to</strong> generate new generation
•The ultimate stem cell, able <strong>to</strong> generate new generation<br />
Egg & Sperm<br />
Zygote<br />
Germ line<br />
stem cells<br />
Primordial Germ Cells<br />
Soma<br />
Early segregation protects germ<br />
cells from somatic differentiation<br />
Death
Germ cells are specificied by maternally synthesized<br />
germ plasm or by cell-<strong>to</strong> cell induction<br />
Germplasm<br />
Induction<br />
Drosophila, Xenopus,<br />
zebrafish, C. elegans<br />
Mouse, axolotl
<strong>Genetic</strong>ally distinct pathways control <strong>for</strong>mation of germ<br />
line and soma in the early in Drosophila emrbyo<br />
Nuclear migration<br />
Budding<br />
Polarized<br />
membrane Growth<br />
Germ cells<br />
Somatic cells
Fly and mouse germ cells:<br />
repression of somatic genes
Transcription is repressed in early germ cells<br />
blue: slam RNA<br />
green: Vasa<br />
red: pSer2-CTD<br />
Stein et al. (2002) Development 129(16), 3925-3934<br />
Seydoux & Dunn (1997)<br />
Development 124(11), 2191-2201
pgc RNA is localized <strong>to</strong> germ plasm<br />
and PGC protein represses transcription in early germ cells<br />
Early Cleavage<br />
Wild-type<br />
Blas<strong>to</strong>derm<br />
pgc<br />
pgc RNA<br />
Rui Martinho<br />
pSer2-CTD<br />
Vasa
slam and other “somatic genes”are activated in pgc<br />
mutant germ cells<br />
Wild type<br />
pgc<br />
Wild type<br />
pgc<br />
as-pgc<br />
slam RNA<br />
tailless mRNA<br />
Rui Martinho<br />
Martinho et al. ( 2004) Current Biol. 14(2), 159-165
PGC may repress germ cell transcription by interfering<br />
with transcriptional elongation<br />
CTD phosphorylation recruits Set1 and Set2 his<strong>to</strong>ne methylases
How are germ cells set aside from somatic cells<br />
Soma<br />
Somatic signals<br />
Germ Cells<br />
pgc<br />
Somatic differentiation
Target specificity suggests that PGC may<br />
repress transcriptional machinery that normally<br />
acts in posterior soma<br />
pgc -/- pgc -/-<br />
pgc -/-<br />
tll mRNA slam mRNA eve mRNA
Cross-regulation of <strong>to</strong>rso and pgc pathways may<br />
inhibit cell specification of soma vs germ cells<br />
Soma<br />
Torso<br />
Recep<strong>to</strong>r tyrosine kinase<br />
Germ Cells<br />
pgc<br />
Somatic target<br />
genes<br />
Germ cell target<br />
genes
Up-regulation of <strong>to</strong>rso represses germ cell <strong>for</strong>mation<br />
Wild-type 8 copies <strong>to</strong>rso +<br />
(100%) (35%) (25%)<br />
Rui Martinho
Up-regulation of pgc leads <strong>to</strong> somatic cellularisation defects<br />
similar <strong>to</strong> the ones observed in <strong>to</strong>rso loss of function alleles<br />
Wild-type<br />
6 copies pgc + <strong>to</strong>r LOF<br />
green: Vasa<br />
blue: DNA<br />
Rui Martinho
Antagonism between pgc and <strong>to</strong>rso sets apart somatic cells<br />
from germ cells<br />
Soma<br />
Germ Cells<br />
<strong>to</strong>rso<br />
pgc<br />
Posterior<br />
soma<br />
Germ<br />
cells<br />
Somatic target<br />
genes<br />
Germ cell target<br />
genes
Repression of somatic differentiation via transcriptional<br />
regulation could be critical <strong>for</strong> germ cell specification<br />
Germ cell specification<br />
in Drosophila<br />
Germ cell specification in mice<br />
(Sai<strong>to</strong>u et al., 2002)<br />
slam RNA<br />
(somatic gene)<br />
Primordial germ cells
Repression of somatic differentiation via transcriptional<br />
regulation a common theme <strong>for</strong> germ cell specification?<br />
Germ cell specification<br />
in Drosophila<br />
Germ cell specification in mice<br />
(Sai<strong>to</strong>u et al., 2002)<br />
Hoxb1<br />
(somatic gene)<br />
slam RNA<br />
(somatic gene)<br />
fragilis<br />
(germ line marker)<br />
Primordial germ cells<br />
Primordial germ cells
Fly and mouse germ cells:<br />
pgc = stem cells?
The niche concept <strong>for</strong> stem cell maintenance<br />
From:Spradling et al. (2001) Nature 414, 98-104
Somatic niche<br />
Somatic<br />
niche<br />
Stem cell<br />
Stem cells<br />
Differentiating<br />
Cys<strong>to</strong>blast<br />
Cys<strong>to</strong>blast<br />
Differentiated<br />
egg chamber<br />
Egg<br />
chamber<br />
Somatic cells<br />
Germ line
Dpp, a BMP2/4 homologue, is an instructive<br />
stem cell fac<strong>to</strong>r<br />
Wild type<br />
Fusome<br />
Vasa<br />
Loss of function<br />
dpp -/-<br />
Fusome<br />
Vasa<br />
Gain of function<br />
hs-dpp<br />
Fusome<br />
Vasa<br />
Xie and Spradling Cell 94, 251-260 (1998)<br />
Xie and Spradling Science 290, 328-330 (2000)
The Drosophila BMP2/4 homologue DPP<br />
signals <strong>to</strong> germ line stem cells via the niche<br />
Soma/niche<br />
Dpp ligand<br />
Niche<br />
Stem cell<br />
Germ line<br />
Tkv (type I recep<strong>to</strong>r)<br />
Punt (type II recep<strong>to</strong>r)<br />
Cys<strong>to</strong>blast<br />
Mad<br />
P<br />
, Medea<br />
Target genes<br />
Bam, dad<br />
Vasa<br />
p-Mad<br />
Differentiated<br />
egg chamber
The number of germ cells increases<br />
dramatically during larval stages<br />
EE<br />
250<br />
150<br />
50<br />
LL3<br />
Number<br />
of adult<br />
GSCs/Ovary<br />
24 h<br />
48 h 72 h 96 h ~108 h<br />
Hours after egg laying<br />
Bar: 20 mm
PGCs away from the niche differentiate at the<br />
end of larval <strong>development</strong><br />
Mid 3rd instar larva<br />
bam::GFP<br />
1B1<br />
Bam-GFP<br />
hts<br />
Late 3rd instar larva<br />
/early pupa<br />
Early pupa<br />
Zhu CH, Xie T. (2003) Development,130(12):2579-88.
PGC differentiation is repressed during larval<br />
stages by the Dpp pathway<br />
WT<br />
1B1<br />
pMad<br />
ML3<br />
nos-Gal4 X UAS-dad<br />
Lilach<br />
Gilboa<br />
1B1<br />
Vasa<br />
ML3<br />
LL3<br />
1B1<br />
Orb
Restriction of niche controls initial<br />
Early Larva<br />
stem cell selection<br />
Dpp/BMP<br />
Late Larva<br />
Tkv<br />
Smads<br />
Pum<br />
& Nos<br />
Bam
Primordial germ cell = germ line<br />
stem cell?<br />
Vasa<br />
pSer2-CTD<br />
1B1<br />
Vasa<br />
Niki Y, Mahowald AP. (2003) Proc Natl Acad Sci U S A; 100(24):14042-5<br />
Gilboa L, Lehmann R. (2004) Curr Biol;14(11):981-6.<br />
Wang Z, Lin H. (2004) Science; 303(5666):2016-9. Epub 2004 Feb 19.
Change language