combinatorial control of cell fates
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Combinatorial control of cell fates. Signal 1. Selector A. Signal 2. Selector B. Target Gene Z. Target Gene X. Target Gene Y. Cell fate l. Cell fate y. Cell fate z. - PowerPoint PPT PresentationTRANSCRIPT
Combinatorial control of cell fates
Signal 1 Signal 2Selector A Selector B
Target Gene X Target Gene Y Target Gene Z
Cell fate Cell fate Cell fate
A relatively small “toolkit” of signals and selector genes can specify a wide range of cell fates by a combinatorial mechanism
Control of gene expression by selector genes and signaling pathways
Different signal/selector combinationsdefine different cell fates and geneexpression domains
Visceral mesoderm induction
Dpp
tin
twi
eve
Wg
bap
slpcardiac
mesoderm
visceral mesoderm
mesoderm DV axisAP axis
Visceral mesoderm induction
Enhancers:
tin: Mad/Med + Tin
eve: Mad/Med + Tin + dTCF
bap: Mad/Med + Tin + Slp
Dpp and Wg act cooperatively on eve, but antagonistically on bap
Dissecting the regulatory region of bagpipe
DNaseI protection ("footprinting")
assay
Comparative analysis of bap enhancer ("phylogenetic footprinting")
Testing the in vivo functions of TF binding sites identified in vitro
Genome-wide profiling of gene expression
Arbeitman, M. N. et al. Development 2004;131:2007-2021
Somatic portion of the sex determination hierarchy
Wild-type males and females (8 timepoints)
Males and females lacking germline (progeny of tudor females)
Sex-transformed females (XX; tra / tra)
Pseudomales (XX; dsxD / dsx)
Female intersexes (XX; dsx / dsx)
Male intersexes (XY; dsx / dsx)
Fruitless mutant males (XY; fru / fru)
Genotypes
Sex-specific gene expression?
Comparison of wild-type males and females, 7 timepoints
ANOVA: Level = Global mean + sex + timepoint + residualH0: sex1 = sex2; P=???
1576 out of 4040 genes differ at P<0.001(897 females > males, 679 males > females)
Adjust significance threshold for multiple comparisons
Sex-specific gene expression? 1. Is gene expression sexually dimorphic in the soma?
(tudor males versus tudor females, P < 0.05)
2. Is it regulated by the canonical sex determination pathway?(wild type females versus XX; tra / tra, P < 0.05)
147 genes out of 1576
3. Is the gene expressed mainly in the soma?(wild type females versus tudor females, P > 0.2; wild type males versus tudor males, P > 0.2)
73 genes out of 147
(37 females > males; 36 males > females)10 cDNAs turned out to be chimaeric
Arbeitman, M. N. et al. Development 2004;131:2007-2021
good good
goodnot so good
not good at all
Re-testing candidate genes by Northern blots
control
Re-testing candidate genes by Northern blots
Overall 20 out of 32 re-tested candidate genes were confirmed
Is the gene regulated by dsx or fru ?
Forced-choice statistical model (strain-specific variation is a problem)
Expression level = Xij, where i = genotype, j = replicate
If controlled by dsx, expression should not differ between wild-type males and fru males
If controlled by fru, expression should not differ between tudor females and dsxD / dsx pseudomales
Is the gene regulated by dsx or fru ?
Arbeitman, M. N. et al. Development 2004;13:2007-2021
Gene expression in male internal genitalia
Accessory glands
Anterior ejaculatory duct
Ejaculatory bulb
Testes
Arbeitman, M. N. et al. Development 2004;131:2007-2021
Gene expression in female internal genitalia
Spermathecae &parovaria
Nurse and folliclecells, oviducts
Male-enriched genes
What are the functions of dsxM and dsxF ?
~ Half of the fly genome is deployed sex-specifically(Arbeitman et al 2002, Parisi et al 2003, Ranz et al 2003, …)
Less than 2% of the genome is expressed sex-specifically in the soma ??
Why are they all in the internal genitalia?
Considerations:
- Size of the tissue? - Transcript abundance? - Extent of sexual dimorphism? - Tissue + sex specificity?
Overview of early myogenesis
twistsnail
Mesoderm fate
Dpp
Wg
myoblast competenceRTKs
(EGF & FGF)
eveequivalence
group
Notch
(Mad+dTCF+Pnt)
Fusion - competent cells (lame duck)
Founder cells (dumbfounded)
Founder cell
FCMs
myotube
muscle
Combining genetic analysis with FACS
FACS works
Identified 335 genes with higher expression in GFP-positive cellsTested 207 by RNA in situ hybridizationTrue positive rate 95.3%
Compare gene expression profiles in wild-type and mutant mesodermal cells
12 mutant genotype
Use the behavior of each gene across genotypes to infer the cell type in which it is expressed
Example:
If a gene is upregulated by Wg, Dpp, and RTK/Ras pathways, upregulated by loss of Dl, downregulated by Notch, downregulated by loss of wg - then it is likely to be expressed in FCs.
Deriving the statistical metric to detect FC/FCM specificity
Clustering of gene expression changes by genotype
Empirical validation of predicted FC and FCM genes
Functional assay for myoblast development
Co-injection of dsRNA and myosin-tau.GFPRNAi for mbc and blow reproduces their mutant phenotypes
FC geneLoss of fusion competence
FCM geneMyotubes replaced by multi-nucleate spheres