1. Shiqiu Li Dr. Scott Stewart Stankunas Lab UO SPUR 2012

2. Outline 1. Background 2. Question/Hypothesis 3. Methods 4. Earlier Data 5. Results 6. Conclusion/Finale

3. Regeneration? Many mammals, like humans, cannot regenerate lost limbs Zebrafish can

4. What is the mechanism?

5. What initiates osteoblast differentiation and dedifferentiation?

6. WNT and BMP!

7. A Model ? What pathway is involved here? Transforming growth factor beta/Activin?

8. Activin or TGF-β II I Smad2 PP TGF-β and activin are ligands for pathway Both are part of TGF-β superfamily Both have a Type II and a Type I receptor TGF-β Type I receptor: Alk5 Activin Type I receptor: Alk4

9. Activin or TGF-β II I Smad4 Smad2 PP

10. Activin or TGF-β II I Smad4 Smad2 PP Transcription Regulation

11. Outline 1. Background 2. Question/Hypothesis 3. Methods 4. Earlier Data 5. Results 6. Conclusion/Finale

12. Question What role do TGF-β/activin play in zebrafish fin regeneration? Hypothesis TGF-β/Activin signaling pathway is involved in cell proliferation in zebrafish fin regeneration

13. Early differentiation? Proliferation?

14. Outline 1. Background 2. Question/Hypothesis 3. Methods 4. Earlier Data 5. Results 6. Conclusion/Finale

15. Methods Fixate and Dehydrate Xylene and paraffin Section Mount fin(s) Fluorescent microscopy Antibody staining Collect fish Amputate fish Wait Alk Inhibitor X Amputate fish Harvest fin(s)Short wait

16. Outline 1. Background 2. Question/Hypothesis 3. Methods 4. Earlier Data 5. Results 6. Conclusion/Finale

17. Alk4 inhibitors cause reduced proliferation in osteoblasts Sp7 PCNA Sp7 PCNA Alk4iDMSOSp7 PCNA

18. Alk4 inhibitors cause reduced proliferation in osteoblasts P value = 0.0049

19. So does TGF-β/activin induce cell proliferation of all cells in the blastema?

20. Outline 1. Background 2. Question/Hypothesis 3. Methods 4. Earlier Data 5. Results 6. Conclusion/Finale

21. TGF-β/activin do not induce cell proliferation in the blastema of regenerating fin

23. Alk4 inhibitors cause reduced proliferation in osteoblasts P value = 0.9941

24. Outline 1. Background 2. Question/Hypothesis 3. Methods 4. Earlier Data 5. Results 6. Conclusion/Finale

25. Conclusion The proliferation of cells in the blastema are not dependent on TGF-β/activin signaling. Osteoblast proliferation specifically may or may not be dependent on TGF-β/activin signaling due to differences of Alk4/5 inhibitor doses.

26. Future Directions Co-stain control and Alk4/5 inhibited fins with osteoblast markers such as Sp7 and Runx2 to fully determine TGF-β/activin’s role in osteoblast proliferation and early redifferentiation. Inhibit zebrafish with fibroblast growth factor receptor inhibitor and observe what happens using immunofuoresence.

27. Acknowledgements Peter O’Day Adam Unger UO SPUR NIH Stankunas Lab Dr. Scott Stewart Dr. Kryn Stankunas

28. Thank you for listening!!!