1. Next lecture: Induction/Signaling Requirements of inducer and responder cells Cascades of inductive events are involved in forming organs Examples of the kinds of cell communication Important signaling pathways in development –Notch, TGF , SHH, Wnt, FGF

2. “Official glossary” from Wolpert Induction: The process whereby one group of cells signals to another group of cells in the embryo and so affects their development Competence: The ability of a tissue to respond to an inducing signal. Embryonic tissues remain competent for a limited time (Can depend on prior inductive events)

3. Competence factors Not all tissues are able to respond to the same signals. Competence is actively acquired –Receptors, signaling molecule –Transcription factors (chromatin state) Competence factors are specifically required in the responding tissue, not the inducer –Demonstrated in recombination experiments

4. Recombination experiments: First name the players… (fig 6.1 from Gilbert)

5. Then play… Fig 6.3 Gilbert

6. Cascades of inductive events form the organ: Reciprocal induction Of Gilbert

7. Two kinds of induction (plus one) Instructive interaction: A signal or factor which tells the developing cell what it is Permissive interaction: A signal or factor that allows the cell to become what it is Selective interaction. A factor allows the cell to be “selected” into one lineage or another after a stochastic (random) change

8. For example, CD4 vs. CD8 T cells Selection vs. Instruction TCR-I TCR-II CD8 CD4

9. Juxtacrine interactions Involve cell surface receptors on inducer and responding cells (no soluble factors) Cell death/apoptosis pathways –Fas (CD95)/FasL Notch/delta pathway –Involved in many binary cell fate decisions –Examples in flies, worms and mice

10. Cell death pathways Figure 6.27 of Gilbert

11. Notch is involved in a wide array of binary cell fate decisions C. elegans:Ventral uterine vs. Anchor cell and vulval development Drosophila: Neural vs. Epidermal cells Mouse: Embryonic lethal, Demonstrated affects in the immune system and others Human: Notch deficiencies cause birth defects

12. Notch/delta pathway (fig 6.29 of Gilbert) This model is simplistic little evidence for nuclear localization of Notch in developing organisms

13. Players in the Notch pathway Receptor family: Notch/lin-12, glp-1 Ligands: (DSL) Delta, Serrate, Lag2, Jagged Processing: ADAMs (Kuz, TACE) and Secretases (Sel-12/presenilins) Downstream effectors: CBF1, Su(Hairless), Lag1 (Collectively called CSL)

14. Proteolytic processing of Notch is complicated Figure 1 from Weinmaster (2000)Curr. Opin. Genet. Dev. 10:363-369

15. Presenilins Multipass transmembrane proteins Mutated in inherited, early onset Alzheimer's disease Involved in cleavage of amyloid precursor protein (APP) Evidence that they are proteases or co- factors of a protease

16. Evidence linking presenilins with Notch signaling Homology between PS1 and Sel-12 of C.elegans (sel-12 facilitates Notch signaling) PS and Notch deficient animals have similar phenotypes (Drosophila and mice) PS is required for access of Notch to the nucleus and, thus, Notch signaling Defects can be rescued by providing exogenous PS

17. Current view of Notch signaling Figure 2 from Weinmaster (2000) Curr Opin Genet. Dev. 10:363-369

18. Notch involvement in cell fate Examples of Notch involvement in inductive interactions (signaling between non-equivalent cells) Lateral specification (occurs in a population of equivalent cells) –Involves the amplification of a stochastic small difference between equivalent cells

19. Lateral specification in C. elegans From Greenwald (1998) Genes. Dev. 12:1751-62

20. Evidence for feedback mechanism in lateral specification Constitutive active mutants of lin12 have no AC Mutants eliminating lin12 activity have 2 AC Mixing experiments between two types above –Lin12 - cells always became AC –Lin12 WT cells always became VU Behavior different from WT in non-mosaic situation where these cells have equal chance to become AC or VU

21. One way to generate a bias in Notch activity- C. elegans vulval development From Greenwald (1998) Genes. Dev. 12:1751-62 fate adoption is 2 o -1 o -2 o

22. Gilbert Chapter 6 errors Lag2 is not secreted but transmembrane. This is the ligand for lin12/Notch (p.169) Figure 6.40 on T-lymphocyte signaling: The pathways leading from the receptors are not correct. (Notice, there is no reference) Figure 6.19: Details appear to be in dispute

23. Paracrine factors and interactions Transforming Growth Factor (TGF)-  Sonic Hedgehog Wnt Fibroblast Growth Factor (FGF) Retinoic Acid (RA)

24. TGF-  Over 30 members of the TGF-  family Subfamilies, TGF , Activin, BMP, Vg1 Processed proteolytically with the C-terminal region conatining the mature peptide TGF  peptides can homo- or hetero-dimerize TGF  signal transduction involves multiple receptors which activate cytoplasmic “Smads”

25. Smad pathway (fig 6.20) p.159

26. Division of labor among Smads Courtesy of J.F. Doody (J. Massague lab @MSKCC) Smad 6 and 7 are inhibitory proteins induced by antagonists of the TGF  signaling pathway, such as  -interferon (Smad7)

27. The Smad family From Piek, et. al. (1999) FASEB J. 13:2105-2124

28. A more complete TGF  pathway From Piek, et. al. (1999) FASEB J. 13:2105-2124

29. Evidence for non-redundant TGF  family involvement in development (due to expression patterns) TGF-  1 knockout mice have defects in blood and vasculature (many die prenatally) TGF-  2 knockout mice have multiple organ malformation (perinatal death) TGF-  3 knockout mice die shortly after birth due to defects in pulmonary development Information from Piek, et. al. (1999) FASEB J.

30. Next lecture: Induction/Signaling Requirements of inducer and responder cells Cascades of inductive events are involved in forming organs Examples of the kinds of cell communication Important signaling pathways in development –Notch, TGF , SHH, Wnt, FGF