germ layers in a vertebrate
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1 germ layers and animal evolution ecdysozoa (e.g., nematodes, flies) lophotrochozoa deuterostomes diploblasts sponges choanoflagellates mesoderm ectoderm + endoderm multicellularity germ layers in a vertebrate ctoderm: skin, nervous system Mesoderm: skeleton, muscle, etc. ndoderm: gut, lungs, etc. redit: W. Smith Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 1
2 Quantum cosmology indeterminate Nematode development determinate determinacy of. elegans development Sir John Sulston Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 2
3 Major cell types made by founder cells Gene regulatory cascade for mesendoderm Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 3
4 formation of the. elegans endoderm mesoderm endoderm mesectoderm formation of the. elegans endoderm mesoderm mesectoderm endoderm Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 4
5 formation of the. elegans endoderm specification gastrulation cell division differentiation endoderm MD-1/2 GAAs specify mesendoderm SKN-1 MD-1,2 maternal zygotic wild type fate fate med(-) (mesectoderm) Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 5
6 Mesendoderm development PAL-1 SKN-1 MDs GSK-3 SKN-1 MDs PAL-1 genes genes NDs genes NDs genes ND-1/3 GAAs specify endoderm SKN-1 MD-1,2 wild type ND-1,3 fate end(-) Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 6
7 ND-1/3 specify endoderm SKN-1 MD-1,2 ND-1,3 fate wild type ubiquitous ND-1/3 specify endoderm SKN-1 MD-1,2 W ND-1,3 L-2,7 ND-1::nR fate Shoichet, S.A., Malik,.H., Rothman, J.H., and Shivdasani, R.A. PNAS 97, 4076 (2000) Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 7
8 L-2/7 GAAs control gut differentiation SKN-1 maternal wt MD-1,2 ND-1,3 L-2,7 elt-2/7( 2/7(-) differentiation gut structural proteins, enzymes zygote properties of endoderm regulatory cascade SKN-1 MD-1,2 specification ND-1,3 L-2,7 differentiation fate Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 8
9 zygote properties of endoderm regulatory cascade SKN-1 AB inducer MD-1,2 fate ND-1,3 cell cycle L-2,7 gastrulation repression of fate Features 1. Redundancy (GAAs) 2. cell division 3. dedicated functions MD-1 defines a novel GAA class MD-1 - G A A G A A G A A G end-1 end-3 GAA factor MD-1 A H G A A R A G consensus A A Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 9
10 fourteen putative MD targets A A G A A N A A G A A gene F F F35H8.7 ceh-20/f313.2 F58G ZK D1.2 ZK177.10, ZK A , B0303.8, 9 product end-1 end-3 wee-1.1 homeobox LAG-2-like Sox family (HMG) R1 unknown -box/unknown oxygen transport bhlh (2) unknown MD sites correlation with embryonic transcriptome 5/12 detected Baugh et al., 2003 Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 10
11 sox-1::gfp in, descendants 4 2 MD-1,2 POP-1 sox-1 end-1,3 wee-1.1 sox-1 end-1,3 wee-1.1 Asymmetric cell division = Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 11
12 P2 induces fate P 2 fate fate P2 induces fate P 2 Wnt MAPK MDs POP-1 rep MDs POP-1 act fate NDs NDs fate Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 12
13 Models for POP-1 repression end-1,3 MD displacement MD inhibition end-1,3 in vivo detection of protein-dna interactions fusion protein GFP transcription factor target array Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 13
14 MD-1 binds end-3 promoter MD-1 array 4 2 merge end-3 end-3 specification of and P 2 Wnt, MAPK MDs POP-1 rep MDs POP-1 act NDs NDs fate fate POP-1 1 HIGH POP-1 1 LOW Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 14
15 recursive POP-1 asymmetry A P High POP-1 Low POP-1 Lin et al. (1998) ell 92, dynamics of POP-1 HIGH LOW Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 15
16 coalescence of POP-1 LG VR sma-1 lin-25 him-5 unc-61 sdc-3 end-1 end-3 identification of PLP-1 Lef-1 1 site -1, ND-1 PLP-1 1 = pur alpha Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 16
17 asymmetry of nuclear PLP-1 POP-1 1 and PLP-1 1 in asymmetry A P POP-1 PLP-1 (initial) Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 17
18 P2 induces fate P 2 Wnt MAPK MDs POP-1 rep MDs POP-1 act fate NDs NDs fate MAP kinase pathway and PLP-1 mom-4(-) MOM-4 lit-1(-) LI-1 mom-4(-);lit-1(-) Nuclear PLP-1 Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 18
19 RNAi screen dsrna instant gene knockout feed bacteria expressing dsrna genome-wide screen (~19,000 genes) RNAi library screen worms mutant progeny (+2 days). coli expressing dsrna Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 19
20 reduced end expression results in excess gut cells wildtype weak end-1/3 1/3, skn-1, med-1/2 1/2, plp-1 normal end-1,3 reduced end-1,3 20 cells 22~32 cells pattern of gut cells in. elegans Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 20
21 RNAi screen summary Lethal Viable Phenotype 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 % % V no expression 12 5% 0 0% VI abn. pattern 37 15% 8 0.4% Research Group Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 21
22 MDs, POP-1, ND-3, Ls Gina Broitman- Maduro Morris Maduro Keith Strohmaier Farzaneh Masoud ric Witze PLP-1 Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 22
23 RNAi screen/gut number Leukena heam Not pictured: ricket Wood Isabella Mengarelli James Vang Dr. Joel Rothman, USB (KIP Bio Networks 2/13/03) 23
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