Glycans and Development II: Cell adhesion, signaling and tissue differentiation

1. Glycans and Development II: Cell adhesion, signaling and tissue differentiation 4/5/11

2. Glycan-mediated developmental processes post-gastrulation • Notch Signaling—THE PARADIGM for glycan regulation of cellular differentiation • Notch, Fringe, Rumi, Egghead, Brainiac • Tissue-specific functions of differentiated cells • Cell adhesion • Axon outgrowth

3. What is Notch? the interactive fly

4. This is Notch Morgan, TH (1919) The physical basis of heredity.

5. Held L., Imaginal Discs, Cambridge Univ. Press (2005) Wing morphogenesis requires Notch • Imaginal discs are adult organ precursors set aside in early developmental stages • The fate of a subset of wing cells are driven by the activity of the wingless gene, whose expression is controlled by activation of Notch

6. Wing Imaginal Disc Anatomy Butler, M.J., et al. (2003) Development 130, 659-670

7. The wing grows by telescoping outward from the disc

8. Wingless requires Notch signaling • The fate of a subset of wing cells are driven by the activity of the wingless gene, whose expression is controlled by activation of Notch • In the wing disc, Notch-ligand interactions require that Notch be modified by specific glycans

9. Notch is a modular protein that requires specific proteolytic cleavages in order to be activated S2 cleavage event (Golgi) S3 cleavage event (Cell Surface) www.cell-research.com

10. Notch signaling pathways are well conserved and are initiated by defined ligands (Delta, Serrate/Jagged) Lai, E., 2004

12. Mutations in the Notch gene also result in neural hyperplasia Drosophila Xenopus Lai, E., 2004

13. Loss of Notch activation favors neuronal differentiation at the expense of glial production Loss of Notch signaling neurosphere WT neurosphere Grandbarbe, L. 2003

14. Genes that phenocopy Notch • Notch ligands (Delta, Serrate) • Notch modifying enzymes (OFUT/POFUT, Fringe) • And others….

15. Post-translational modifications of Notch • Addition of O-Fuc to serum proteins identified on EGF repeat modules of many clotting factors • Modification subsequently identified on Notch and Notch ligands (Delta and Serrate) • Extent of O-linked modification affects Notch signaling

16. Sequential modification of Notch EGF repeats Essentials of Glycobiology Second Edition

17. OFut1 loss of function Okajima, T., et al (2003) JBC 278, 42340

18. Mouse POFUT-1 KO produces cardiac, vascular and neural defects Shi, S., 2003

19. Fringe Correia, T. et al. (2003) PNAS 100, 6404

20. Fringe expression in the dorsal wing disc Kim, J., 1995

21. Compartmentalization in the wing disc Kim, J., 1995

22. Fringe enhances Delta signaling, inhibits Serrate Essentials of Glycobiology Second Edition

23. Differential activation defines boundary and drives opposite fate decisions (top of wing vs. bottom of wing) Fortini, M. (2000) Nature 406, 357

24. Loss of O-Fut1 phenocopies Notch alleles Okajima, T., 2002

25. Genes that phenocopy Notch • Notch ligands (Delta, Serrate) • Notch modifying enzymes (OFUT/POFUT, Fringe) • And others….

26. Rumi Acar, M., et al. (2008) Cell 132, 247

27. Rumi is a Glucosyltransferase Takeuchi, H. (2010) Sem. Cell Dev. Biology 21, 638-645

28. Mapping modifications onto EGF repeats across species Takeuchi, H. (2010) Sem. Cell Dev. Biology 21, 638-645

29. Genes that phenocopy Notch • Notch ligands (Delta, Serrate) • Notch modifying enzymes (POFT, Fringe, Rumi) • ADD genes responsible for membrane dynamics, cis-interactions, raft formation

30. Brainiac phenocopies Notch (and egghead) Goode, S., 1992

31. Lai, E., 2004

32. Egghead and Brainiac are glycolipid-directed glycosyltransferases Wandell, H., 2005

33. Notch activation occurs within the lipid bilayer S2 cleavage event (Golgi) S3 cleavage event (Cell Surface) www.cell-research.com

34. Glycan-mediated developmental processes post-gastrulation • Notch Signaling—THE PARADIGM for glycan regulation of cellular differentiation • Notch, Fringe, Rumi, Egghead, Brainiac • Tissue-specific functions of differentiated cells • Cell adhesion • Axon outgrowth

35. Glycan functions in differentiated cells • Most well-characterized in Nervous system • Primarily because neurons undergo some of the most dramatic post-differentation behaviors • Retino-tectal projection has been a useful system

36. Atardi, D., 1963

37. Differential topographic adhesion Dorsal retina on ventral tectum Ventral retina on dorsal tectum Barbera, A., 1973

38. Roth/Marchase model --A Reciprocal Gradient: 1. Trypsin-sensitive component concentrated on ventral retina and tectum that recognizes HexNAc Hexosaminidase-sensitive component concentrated on dorsal retina and tectum

39. These and other studies kicked-off a decade long dash to generate antibodies against relevant adhesion molecules --NCAM, neural cell adhesion molecule, identified by reiterative immunization with adhesion screening

40. Even with NCAM blocked, retinal axons sort and steer to appropriate DV positions Thanos, S., 1984

41. NCAM isolated from developing tissues demonstrates aberrant mobility on SDS-PAGE -heavily derivatized with poly-a2,8-NeuAc on complex N-linked glycans (4 - 40 residues)

42. Endo-neuraminidase enhances axon fasciculation Yin, X., 1995

43. Loss of NCAM or PSA results in smaller olfactory bulb and incomplete migration of RMS Bruses, J., 2001

44. PSA as a clutch or modulator of adhesive interactions—the anionic cloud model Bruses, J., 2001

45. NCAM adhesion • Modulated by glycan addition • Nature of modulation is dependent on context • Axon-axon fasciculation is in competition with axon-substrate interactions • Cell migration requires the appropriate traction but too much traction leads to immobilization

46. General Principles (Adhesion, signaling, and tissue differentiation) • Glycans modulate the activity of signaling molecules that drive cellular differentiation • The exact structure of a glycan modification can impart specific information • Altered glycosphingolipid expression can phenocopy the effect of altered signaling flux • Adhesive interactions between embryonic cells can be mediated positively and negatively by glycan-protein interactions • Appropriate cell-cell interactions in the embryo require a hierarchy of interactions; glycans participate at multiple levels, from determination of cell fate to post-differentiated cellular behaviors