1. Combinatorial control of cell fates Signal 1Signal 2Selector ASelector B Target Gene XTarget 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

2. Control of gene expression by selector genes and signaling pathways Different signal/selector combinations define different cell fates and gene expression domains

3. Visceral mesoderm induction

4. Dpp tin twi eve Wg bap slp cardiac mesoderm visceral mesoderm mesoderm DV axis AP 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

5. Dissecting the regulatory region of bagpipe

6. DNaseI protection ("footprinting") assay

7. Comparative analysis of bap enhancer ("phylogenetic footprinting")

8. Testing the in vivo functions of TF binding sites identified in vitro

12. Genome-wide profiling of gene expression

13. Arbeitman, M. N. et al. Development 2004;131:2007-2021 Somatic portion of the sex determination hierarchy

14. 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

15. Sex-specific gene expression? Comparison of wild-type males and females, 7 timepoints ANOVA: Level = Global mean + sex + timepoint + residual H 0 : 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

16. 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

17. Arbeitman, M. N. et al. Development 2004;131:2007-2021 good not so good not good at all Re-testing candidate genes by Northern blots control

18. Re-testing candidate genes by Northern blots Overall 20 out of 32 re-tested candidate genes were confirmed

19. Is the gene regulated by dsx or fru ? Forced-choice statistical model (strain-specific variation is a problem) Expression level = X ij, 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

20. Is the gene regulated by dsx or fru ?

22. Arbeitman, M. N. et al. Development 2004;13:2007-2021 Gene expression in male internal genitalia Accessory glands Anterior ejaculatory duct Ejaculatory bulb Testes

23. Arbeitman, M. N. et al. Development 2004;131:2007-2021 Gene expression in female internal genitalia Spermathecae & parovaria Nurse and follicle cells, oviducts Male-enriched genes

24. What are the functions of dsxM and dsxF ?

25. ~ 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?

26. Overview of early myogenesis twist snail Mesoderm fate Dpp Wg myoblast competence RTKs (EGF & FGF) eve equivalence group Notch (Mad+dTCF+Pnt) Fusion - competent cells (lame duck) Founder cells (dumbfounded) Founder cell FCMs myotube muscle

27. Combining genetic analysis with FACS

28. FACS works Identified 335 genes with higher expression in GFP-positive cells Tested 207 by RNA in situ hybridization True positive rate 95.3%

29. 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.

30. Deriving the statistical metric to detect FC/FCM specificity

31. Clustering of gene expression changes by genotype

32. Empirical validation of predicted FC and FCM genes

33. Functional assay for myoblast development Co-injection of dsRNA and myosin-tau.GFP RNAi for mbc and blow reproduces their mutant phenotypes

34. FC gene Loss of fusion competence FCM gene Myotubes replaced by multi-nucleate spheres