1. Wnt signaling in development and disease Vítězslav Bryja, PhD. Institute of Experimental Biology Faculty of Science, Masaryk University Brno, Czech Republic

2. Wnts can activate diverse pathways - family of ligands - glycosylated and palmitoylated extracellular proteins - short range of action, bind to extracellular matrix - only in multicellular animals canonical (eg. Wnt-1 or Wnt-3a) non-canonical (eg. Wnt-5a)

3. Wnt/  -kateninová dráha (= kanonická dráha) - induce axis duplication in Xenopus - induce transformation of mammary cell line C57mg -signal via nuclear translocation of  -catenin

4. Wnt/  -catenin pathway

5. Wnt secretion

6. Purification of Wnt ligands Wnt-3a no detergent detergent added

7. Wnt-5a purification Wnt-conditioned medium Blue Sepharose column Heparin column Superdex gel filtration Medium Blue SepharoseSuperdex Heparin WB: Wnt-5a silver staining 4 liters of media conditioned by fibroblasts expressing HA tagged Wnt-5a

8. Frizzled – crucial receptor of most (all?) Wnt pathways

9. Lrp5/6 – crucial co-receptor of the canonical Wnt pathway

10. DIX 11-93 PDZ 267-339 DEP 433-507 C-term 697-736 Dishevelled -phosphorylated by numerous kinases, significance often unknown -bound by many proteins - significance often unknown -various cellular localization (membrane, cytoplasm, nucleus) -capable of polymerization (Dvl aggregate vs. monomere) -required for signal transduction of most Wnt signalling pathways

11. 61.2%79.5%57.3% Dishevelled

12. Destruction complex A working model for the destruction complex. (1) Initially, the destruction complex contains Axin, GSK3, CK1 and APC (with the 15 aa and 20 aa repeat regions shown). The complex contains other components such as PP2A, which are not shown here. (2) -Catenin enters the complex by binding Axin and potentially the APC 15 aa repeats. This positions the N- terminus of -catenin near CK1 and GSK3. (3) CK1 phosphorylates - catenin at Ser45. (4) GSK3 phosphorylates -catenin at, successively, Thr41, Ser37 and Ser33. (5) The 20 aa repeats, particularly repeat 3, are phosphorylated by a CK1 (and possibly GSK3) which greatly increases their affinity for -catenin. The binding of a phosphorylated 20 aa repeat to -catenin displaces Axin from -catenin. (6) -TRCP1 binds the phosphorylated N-terminus of - catenin, causing the ubiquitination of -catenin by an E2 ligase. APC is then either desphosphorylated within the complex, allowing the ubiquitinated - catenin to leave the complex, or the ubiquitinated -catenin bound to APC leaves the complex and is separated from APC at the proteasome. The complex then returns to Step 1

13. Tyr142 of  -catenin: phosphorylated – binds Bcl9 dephosphorylated – binds  -catenin

14. Epithelio-mesenchymal transition (EMT)

15. Transactivation of target genes

17. Maternal Wnt/  -catenin pathway determines the dorsal pole of the zygote and embryo in Xenopus

19. axis duplication assay:

20. Wnt/  -catenin signaling: transcription, proliferation and cell fate

21. Uncx4.1/Mesogenin Mouse embryo at E8.5: Wnt/  -catenin target genes are expressed in the posterior part of the embryo.

22. Loss of Wnt/  -catenin pathway during gastrulation = loss of posterior body parts wild typeWnt-3a knockout

23. Loss of Wnt/  -catenin inhibitors = loss of anterior body parts wild type vs.Dkk1 knockout

25. Wnt/  -catenin pathway specifies neural crest

26. Neural crest development: - Source of peripheral nervous system,Melanocytes, facial muscles/bones and heart outflow tract Wnt-3a

27. Wnt1/3a DKO

28. Aberrant activation of the Wnt/  - catenin pathway leads to cancer

29. Mutations in the adenomatosis polyposis coli (APC) Familial adenomatous polyposis (FAP)

30. Wnt pathway activators are known oncogenes and Wnt pathway inhibitors are tumor supressors

31. Reya & Clevers 2005, Nature Stem cell niche Hair folicle Bone marrowIntestinal epithelium

32. The effects of Wnts on stem cells in their niche Wnt

33. Lo Celso, C. L. et al. Development 2004;131:1787-1799  -catenin gain-of-function in the epidermis

34. Wnt pathway induces de novo formation of hair follicles Wnt signaling pathway related polymorphism?