Combinatorial control of cell fates

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Combinatorial control of cell fates
Signal 1
Signal 2
Selector A
Selector B
Target Gene Z
Target Gene X
Target Gene Y
Cell fate l
Cell fate y
Cell fate z
A relatively small toolkit of signals and
selector genes can specify a wide range of cell
fates by a combinatorial mechanism
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Control of gene expression by selector genes and
signaling pathways
Different signal/selector combinations define
different cell fates and gene expression domains
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Visceral mesoderm induction
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Visceral mesoderm induction
Enhancers
AP axis
DV axis
Wg
mesoderm
tin Mad/Med Tin eve Mad/Med Tin
dTCF bap Mad/Med Tin Slp
Dpp
twi
tin
eve
slp
cardiac mesoderm
Dpp and Wg act cooperatively on eve, but
antagonistically on bap
bap
visceral mesoderm
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Dissecting the regulatory region of bagpipe
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DNaseI protection ("footprinting") assay
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Comparative analysis of bap enhancer
("phylogenetic footprinting")
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Testing the in vivo functions of TF binding sites
identified in vitro
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Genome-wide profiling of gene expression
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Somatic portion of the sex determination hierarchy
Arbeitman, M. N. et al. Development
20041312007-2021
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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)
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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
Plt0.001 (897 females gt males, 679 males gt
females) Adjust significance threshold for
multiple comparisons
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Sex-specific gene expression?

• Is gene expression sexually dimorphic in the
  soma?
• (tudor males versus tudor females, P lt 0.05)
• Is it regulated by the canonical sex
  determination pathway?
• (wild type females versus XX tra / tra, P lt
  0.05)
• 147 genes out of 1576
• Is the gene expressed mainly in the soma?
• (wild type females versus tudor females, P gt
  0.2
• wild type males versus tudor males, P gt 0.2)
• 73 genes out of 147
• (37 females gt males 36 males gt females)
• 10 cDNAs turned out to be chimaeric

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Re-testing candidate genes by Northern blots
good
good
not so good
good
not good at all
control
Arbeitman, M. N. et al. Development
20041312007-2021
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Re-testing candidate genes by Northern blots
Overall 20 out of 32 re-tested candidate genes
were confirmed
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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
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Is the gene regulated by dsx or fru ?
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Gene expression in male internal genitalia
Accessory glands
Anterior ejaculatory duct
Ejaculatory bulb
Testes
Arbeitman, M. N. et al. Development
2004132007-2021
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Gene expression in female internal genitalia
Spermathecae parovaria
Nurse and follicle cells, oviducts
Male-enriched genes
Arbeitman, M. N. et al. Development
20041312007-2021
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What are the functions of dsxM and dsxF ?
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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?
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Overview of early myogenesis
twist snail
Founder cell
Dpp
Mesoderm fate
FCMs
Wg
myoblast competence
RTKs (EGF FGF)
myotube
(MaddTCFPnt)
eve
equivalence group
muscle
Notch
Founder cells (dumbfounded)
Fusion - competent cells (lame duck)
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Combining genetic analysis with FACS
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FACS works
Identified 335 genes with higher expression in
GFP-positive cells Tested 207 by RNA in situ
hybridization True positive rate 95.3
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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.
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Deriving the statistical metric to detect FC/FCM
specificity
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Clustering of gene expression changes by genotype
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Empirical validation of predicted FC and FCM genes
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Functional assay for myoblast development
Co-injection of dsRNA and myosin-tau.GFP RNAi for
mbc and blow reproduces their mutant phenotypes
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FC gene Loss of fusion competence
FCM gene Myotubes replaced by multi-nucleate
spheres