Recursive and Combinatorial Signaling in C. elegans development
germ layers and animal evolution
ecdysozoa
(e.g., nematodes, flies)
deuterostomes
lophotrochozoa
mesoderm
diploblasts
sponges
choanoflagellates
ectoderm + endoderm
multicellularity
germ layers in a vertebrate
Ectoderm: skin, nervous system
Mesoderm: skeleton, muscle, etc.
Endoderm: gut, lungs, etc.
Credit: W. Smith
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
Quantum cosmology
indeterminate
Nematode development
determinate
determinacy of C. elegans development
Sir John Sulston
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
Major cell types made by founder cells
Gene regulatory cascade
for mesendoderm
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
formation of the C. elegans endoderm
EMS
MS
EMS
mesoderm
E
C
endoderm
mesectoderm
formation of the C. elegans endoderm
EMS
MS
E
mesoderm
C
mesectoderm
endoderm
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
formation of the C. elegans endoderm
EMS
specification
MS
E
C
gastrulation
cell division
differentiation
endoderm
MEDMED-1/2 GATAs specify mesendoderm
C
SKN-1
maternal
zygotic
wild type
MED-1,2
MS
fate
E
fate
MS E
C
med(-)
(mesectoderm)
mesectoderm)
“C”
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
“C”
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Recursive and Combinatorial Signaling in C. elegans development
Mesendoderm
development
PALPAL-1
EMS
EMS
C
EMS
SKNSKN-1
SKNSKN-1
PALPAL-1
GSKGSK-3
MEDs
C
MS
genes genes
MEDs
ENDs
MS
MS
genes
ENDs
C
genes
E
•
ENDEND-1/3 GATAs specify endoderm
C
SKN-1
wild
wildtype
type
MED-1,2
END-1,3
MS E
C
E
fate
end(-)
MS
“C”
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
ENDEND-1/3 specify endoderm
SKN-1
MED-1,2
END-1,3
E
fate
wild type
ubiquitous
ENDEND-1/3 specify endoderm
SKN-1
WT
MED-1,2
END-1,3
ELT-2,7
E
fate
END-1::EnR
Shoichet, S.A., Malik, T.H., Rothman, J.H.,
and Shivdasani, R.A. PNAS 97, 4076 (2000)
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
ELTELT-2/7 GATAs control gut differentiation
wt
maternal
SKN-1
MED-1,2
END-1,3
eltelt-2/7(2/7(-)
ELT-2,7
differentiation
gut structural
proteins, enzymes
properties of endoderm
regulatory cascade
zygote
SKN-1
EMS
MS
specification
MED-1,2
E
END-1,3
ELT-2,7
differentiation
E fate
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
properties of endoderm
regulatory cascade
zygote
SKN-1
AB
inducer
EMS
MS
MED-1,2
MS fate
E
Features
1. Redundancy
(GATAs)
2. cell division
END-1,3
cell cycle
gastrulation
repression of C
ELT-2,7
3. dedicated
functions
E fate
MED-1 defines a novel GATA class
MED-1
-
G
T
T
T
T
C
C
A
T
A
T
G
A
A
C
G
C
C
C
T
T
C
A
T
A
T
G
T
C
end-1
end-3
consensus
H G A T A R
GATA factor
MED-1
A A G T A T A C
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
fourteen putative MED targets
A A G T A T A C
N
25-100
A A G T A T A C
gene
product
F58E10.2
F58E10.5
F35H8.7
ceh-20/F31E3.2
F58G4.4
C32E12.5
ZK849.2
T07D1.2
ZK177.10, ZK177.1
T11A5.5
C17C3.7, C17C3.10
B0303.8, 9
end-1
end-3
wee-1.1
homeobox
LAG-2-like
Sox family (HMG)
RCC1
unknown
T-box/unknown
oxygen transport
bHLH (2)
unknown
MED sites
correlation with embryonic
transcriptome
5/12 detected
Baugh et al., 2003
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
sox-1::GFP in E, MS descendants
MS 4
E2
MED-1,2
MS
POP-1
E
sox-1
sox-1
end-1,3
end-1,3
wee-1.1
wee-1.1
Asymmetric cell division
=
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
P2 induces E fate
P2
EMS
MS fate
E fate
P2 induces E fate
P2
EMS
MEDs
POP-1rep
ENDs
MS fate
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
Wnt
MEDs
MAPK
POP-1act
ENDs
E fate
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Recursive and Combinatorial Signaling in C. elegans development
Models for POP-1 repression
MS
MED displacement
end-1,3
MED inhibition
end-1,3
in vivo detection of protein-DNA
interactions
GFP
fusion protein
transcription
factor
target
array
•
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
MED-1 binds end-3 promoter
array
MED-1
MS4
E2
merge
MS
E
end-3
end-3
specification of E and MS
P2
Wnt,
MAPK
EMS
MEDs
MEDs
POP-1rep
ENDs
MS fate
POPPOP-1 HIGH
POP-1act
ENDs
E fate
POPPOP-1 LOW
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
recursive POP-1 asymmetry
A
P
High POP-1
Low POP-1
Lin et al. (1998) Cell 92, 229–239
dynamics of POP-1
HIGH
LOW
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
coalescence of POP-1
him-5
sma-1 lin-25
LG VR
unc-61 sdc-3
end-1
end-3
identification of PLP-1
LefLef-1 site
-1,035
-565
-310 -216 -118 0
ENDEND-1
PLPPLP-1 = pur alpha
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
asymmetry of nuclear PLP-1
MS
E
POP-1 and PLP-1 in asymmetry
A
P
POP-1
PLP-1
(initial)
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
17
Recursive and Combinatorial Signaling in C. elegans development
P2 induces E fate
P2
EMS
MEDs
POP-1rep
ENDs
Wnt
MEDs
MAPK
POP-1act
ENDs
E fate
MS fate
MAP kinase pathway and PLP-1
MOM-4
MOM
MOM-4
mom-4(-)
lit-1(-)
LIT-1
LIT
LIT-1
mom-4(-);lit-1(-)
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
Nuclear
PLP-1
PLP
PLP-1
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Recursive and Combinatorial Signaling in C. elegans development
RNAi screen
dsRNA
“instant gene knockout”
feed bacteria expressing dsRNA
genome-wide screen (~19,000 genes)
RNAi library screen
worms
mutant progeny
(+2 days)
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
E. coli
expressing
dsRNA
19
Recursive and Combinatorial Signaling in C. elegans development
reduced end expression results
in excess gut cells
wildtype
E
weak endend-1/3,
1/3, sknskn-1,
,
med1/2,
plp1
med 1/2 plp
normal endend-1,3
reduced endend-1,3
20 cells
22~32 cells
pattern of gut cells
in C. elegans
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
RNAi screen summary
Lethal
Phenotype
Viable
N (248)
%
N (2229)
%
I slight excess
13
5%
7
0.3%
II large excess
9
3%
0
0%
III subnormal
0
0%
4
0.2%
IV faint express.
1
0.4%
37
1.7%
V no expression
12
5%
0
0%
VI abn. pattern
37
15%
8
0.4%
Research Group
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
MEDs, POP-1, END-3, ELTs
Gina BroitmanBroitman- Maduro
Keith Strohmaier
Morris Maduro
Farzaneh Masoud
PLP-1
Eric Witze
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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Recursive and Combinatorial Signaling in C. elegans development
RNAi screen/gut number
Leukena Cheam
James Vang
Isabella Mengarelli
Not pictured: Cricket Wood
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03)
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