Quantum cosmology indeterminate Nematode developmentonline.itp.ucsb.edu/online/bionet03/rothman/pdf/Rothman.pdf · Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 1 choanoflagellates

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



Quantum cosmologyQuantum cosmology

Nematode developmentNematode development

indeterminateindeterminate

determinatedeterminate

determinacy of determinacy of C. elegans C. elegans developmentdevelopment

Sir John SulstonSir John Sulston



Major cell types made by founder cells

Gene regulatory cascade Gene regulatory cascade for mesendodermfor mesendoderm



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



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 ))



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



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



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



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



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



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

=



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



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

•



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-1actPOP-1act

Wnt,MAPK

POPPOP--1 HIGH1 HIGH POPPOP--1 LOW1 LOW



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



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



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



MEDsMEDs

EMSEMS

P2P2

MS fate E fate

ENDsENDsENDs


POP-1actPOP-1act

Wnt MAPK


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(-)



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



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



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



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



RNAi screen/gut numberRNAi screen/gut number

Not pictured: Cricket WoodNot pictured: Cricket Wood

Isabella MengarelliIsabella Mengarelli

James VangJames VangLeukena CheamLeukena Cheam

Documents

Quantum cosmology indeterminate Nematode developmentonline.itp.ucsb.edu/online/bionet03/rothman/pdf/Rothman.pdf · Dr. Joel Rothman, UCSB (KITP Bio Networks 2/13/03) 1 choanoflagellates