quantum cosmology indeterminate nematode...
TRANSCRIPT
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 1
choanoflagellateschoanoflagellates
spongessponges
diploblastsdiploblasts
lophotrochozoalophotrochozoa
ecdysozoaecdysozoa(e.g., nematodes, flies)(e.g., nematodes, flies)
deuterostomesdeuterostomes
germ layers and animal evolutiongerm layers and animal evolution
multicellularitymulticellularity
ectoderm + endodermectoderm + endoderm
mesodermmesoderm
Ectoderm: skin, nervous system
Mesoderm: skeleton, muscle, etc.
Endoderm: gut, lungs, etc.
Credit: W. Smith
germ layers in a vertebrategerm layers in a vertebrate
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 2
Quantum cosmologyQuantum cosmology
Nematode developmentNematode development
indeterminateindeterminate
determinatedeterminate
determinacy of determinacy of C. elegans C. elegans developmentdevelopment
Sir John SulstonSir John Sulston
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 3
Major cell types made by founder cells
Gene regulatory cascade Gene regulatory cascade for mesendodermfor mesendoderm
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 4
formation of the formation of the C. elegans C. elegans endodermendoderm
EMS
mesodermmesoderm mesectodermmesectodermendodermendoderm
EEMSMS CCEMSEMS
formation of the formation of the C. elegans C. elegans endodermendoderm
EMS
endodermendoderm
mesodermmesoderm mesectodermmesectoderm
MSMS CCEE
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 5
formation o f the formation o f the C. elegans C. elegans endod ermendod erm
EMS
endod ermendod erm
MSMS CCEEspecification
gastrulationcell division
differentiation
SKN-1
MED-1,2
MSfate
Efate
MEDMED--1/2 1/2 GATAsGATAs specify mesendod ermspecify mesendod erm
MS E
wild type
Cmaternal
zygotic
“ C”
med(-)
“ C”
C
((mesectodermmesectoderm ))
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 6
EMSEMS
MSMSgenesgenes
SKNSKN--11
MEDsMEDs
ENDsENDs
MSMS EE
CCgenesgenes
EMSEMS
PALPAL--11
EMSEMS
MSMSgenesgenes
SKNSKN--11
MEDsMEDs
ENDsENDs CCgenesgenes
CC
PALPAL--11
GSKGSK--33
Mesendoderm Mesendoderm developmentdevelopment
•
wild type
MS
ENDEND--1/3 1/3 GATAsGATAs specify endodermspecify endoderm
end(-)
wild type
MS E
C
C
“C”
SKN-1
MED-1,2
END-1,3
Efate
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 7
ENDEND--1/3 specify endoderm1/3 specify endoderm
SKN-1
MED-1,2
END-1,3
Efate
wild type ubiquitous
ENDEND--1/3 specify endoderm1/3 specify endoderm
SKN-1
MED-1,2
END-1,3
ELT-2,7
Efate
Shoichet, S.A., Malik, T.H., Rothman, J.H., and Shivdasani, R.A. PNAS 97, 4076 (2000)
END-1::EnR
WT
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 8
ELTELT--2/7 2/7 GATAsGATAs control gut differentiationcontrol gut differentiation
SKN-1
MED-1,2
END-1,3
ELT-2,7
differentiation
wtwt
eltelt--2/7(2/7(--))
gut structuralproteins, enzymes
maternal
zygote
SKN-1
MED-1,2
END-1,3
ELT-2,7
EMSEMS
EMSMS
specification
differentiation
E fate
properties of endoderm properties of endoderm regulatory cascaderegulatory cascade
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 9
zygote
SKN-1
MED-1,2
END-1,3
ELT-2,7
EMSEMS
EMSMS
E fate
AB
MS fate
cell cyclegastrulation
inducer
repression of C
1. Redundancy(GATAs)
Features
2. cell division
3. dedicated functions
properties of endoderm properties of endoderm regulatory cascaderegulatory cascade
-MED-1
AAGTATACCTTTTG
CTGTATACTTCCCGC
end-1
end-3
GATA factor
MED-1
H G A T A R
A G A T A CA T
consensus
MED-1 defines a novel GATA class
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 10
fourteen putative MED targets
A G A T A CA T A G A T A CA TN25-100
gene MED sites
F58E10.2F58E10.5F35H8.7ceh-20/F31E3.2F58G4.4C32E12.5ZK849.2T07D1.2ZK177.10, ZK177.1T11A5.5C17C3.7, C17C3.10B0303.8, 9
product
end-1end-3wee-1.1homeoboxLAG-2-likeSox family (HMG)RCC1unknownT-box/unknownoxygen transportbHLH (2)unknown
correlation with embryonic transcriptome
Baugh et al., 2003
5/12 detected
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 11
sox-1::GFP in E, MS descendants
E2MS4
MED-1,2
end-1,3
wee-1.1
sox-1sox-1
EMS
end-1,3
wee-1.1POP-1
Asymmetric cell divisionAsymmetric cell division
=
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 12
MS fate E fate
PP22 induces E fateinduces E fate
EMSEMS
P2P2
MEDsMEDs
EMSEMS
P2P2
MS fate E fate
ENDsENDsENDs
POP-1repPOP-1repMEDsMEDs
POP-1actPOP-1act
Wnt MAPK
PP22 induces E fateinduces E fate
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (K ITP Bio Networks 2/13/03) 13
end-1,3
MS
end-1,3
Models for POP-1 repression
MED displacement
MED inhibition
in vivo detection of protein-DNA interactions
targetarray
fusion proteintranscriptionfactor
GFP
•
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 14
MED-1 binds end-3 promoter
array
E
MS
2
4
MED-1
merge MS E
end-3 end-3
MEDsMEDs
specification of E and MSspecification of E and MS
EMSEMS
P2P2
MS fate E fate
ENDsENDsENDs
POP-1repPOP-1repMEDsMEDs
POP-1actPOP-1act
Wnt,MAPK
POPPOP--1 HIGH1 HIGH POPPOP--1 LOW1 LOW
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 15
High POP-1
Low POP-1
recursive POP-1 asymmetry
A P
Lin et al. (1998) Cell 92, 229–239
dynamics of POP-1dynamics of POP-1
HIGHHIGH
LOWLOW
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 16
coalescence of POPcoalescence of POP--11
end-1 end-3
LG VRsma-1 lin-25 him-5 unc-61 sdc-3
identification of PLPidentification of PLP--11
ENDEND--11-310-565-1,035 0-118-216
PLPPLP--1 =1 = purpur alphaalpha
LefLef--1 site1 site
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 17
asymmetry of nuclear PLPasymmetry of nuclear PLP--11
MS E
POPPOP--1 and PLP1 and PLP--1 in asymmetry1 in asymmetry
POPPOP--11
PLPPLP--11(initial)(initial)
A P
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 18
MEDsMEDs
EMSEMS
P2P2
MS fate E fate
ENDsENDsENDs
POP-1repPOP-1repMEDsMEDs
POP-1actPOP-1act
Wnt MAPK
PP22 induces E fateinduces E fate
MOM-4MOMMOM--44
LIT-1LITLIT--11
NuclearPLP-1
NuclearNuclearPLPPLP--11
MAP kinase pathway and PLPMAP kinase pathway and PLP--11
mom-4(-);lit-1(-)
lit-1(-)
mom-4(-)
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 19
dsRNA dsRNA � �� �
“instant gene knockout”“instant gene knockout”
feed bacteria expressing dsRNAfeed bacteria expressing dsRNA
genomegenome--wide screen (~19,000 genes)wide screen (~19,000 genes)
RNAi screenRNAi screen
E. coliexpressing
dsRNA
worms
mutant progeny(+2 days)
RNAi library screenRNAi library screen
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 20
EE
20 cells20 cells
wildtypewildtype
normal normal endend--1,31,3
reduced reduced endend expression results expression results in excess gut cellsin excess gut cells
22~32 cells22~32 cells
weak weak endend--1/31/3, , sknskn--1, 1, medmed--1/21/2, , plpplp--11
reduced reduced endend--1,31,3
pattern of gut cellspattern of gut cellsin in C. elegansC. elegans
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 21
0.4%815%37VI abn. pattern
0%05%12V no expression
1.7%370.4%1IV faint express.
0.2%40%0III subnormal
0%03%9II large excess
0.3%75%13I slight excess
%N (2229)%N (248)Phenotype
LethalLethal ViableViable
RNAi screen summaryRNAi screen summary
Research GroupResearch Group
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 22
MEDs, POPMEDs, POP--1, END1, END--3, 3, ELTsELTs
Morris MaduroMorris MaduroGina BroitmanGina Broitman-- MaduroMaduro
Farzaneh MasoudFarzaneh Masoud
Keith StrohmaierKeith Strohmaier
PLPPLP--11Eric WitzeEric Witze
Recursive and Combinatorial Signaling in C. elegans development
Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 23
RNAi screen/gut numberRNAi screen/gut number
Not pictured: Cricket WoodNot pictured: Cricket Wood
Isabella MengarelliIsabella Mengarelli
James VangJames VangLeukena CheamLeukena Cheam