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Combinatorial control of cell fates Signal 1 Selector A Target Gene X Cell fate y Signal 2 Selector B Target Gene Y Target Gene Z Cell fate z Cell fate l 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 combinations define different cell fates and gene expression domains Visceral mesoderm induction Visceral mesoderm induction mesoderm twi AP axis DV axis Wg Dpp Enhancers: tin: Mad/Med + Tin eve: Mad/Med + Tin + dTCF tin bap: Mad/Med + Tin + Slp eve cardiac mesoderm bap visceral mesoderm 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 Somatic portion of the sex determination hierarchy Arbeitman, M. N. et al. Development 2004;131:2007-2021 Genotypes 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) Sex-specific gene expression? Comparison of wild-type males and females, 7 timepoints ANOVA: Level = Global mean + sex + timepoint + residual H0: 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 Re-testing candidate genes by Northern blots good good not so good good not good at all control Arbeitman, M. N. et al. Development 2004;131:2007-2021 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 ? Gene expression in male internal genitalia Accessory glands Anterior ejaculatory duct Ejaculatory bulb Testes Arbeitman, M. N. et al. Development 2004;13:2007-2021 Gene expression in female internal genitalia Spermathecae & parovaria Nurse and follicle cells, oviducts Male-enriched genes Arbeitman, M. N. et al. Development 2004;131:2007-2021 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 twist snail Founder cell Dpp Mesoderm fate RTKs (EGF & FGF) FCMs Wg myoblast competence myotube (Mad+dTCF+Pnt) eve equivalence group muscle Notch Fusion - competent cells (lame duck) Founder cells (dumbfounded) Combining genetic analysis with FACS FACS works Identified 335 genes with higher expression in GFP-positive cells Tested 207 by RNA in situ hybridization True 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.GFP RNAi for mbc and blow reproduces their mutant phenotypes FC gene Loss of fusion competence FCM gene Myotubes replaced by multi-nucleate spheres