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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