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