1. Classification of Enteric Progenitors in Zebrafish During Development William Montagne Eisen Lab August 17, 2012

2. Enteric Nervous System (ENS) Background ENS is the largest part of the Peripheral Nervous System Intrinsic innervation of the intestinal tract Modulates gut function Functions autonomously from Central nervous system Composed of different subtypes of neurons and glial cells Wallace et al. (2005) zebrafish

3. ENS Cell Origin Entirely derived from one population of cells. Enteric progenitors migrate to the foregut. Continue to migrate and proliferate to populate the entire digestive tract.

4. Enteric Progenitor Markers Progenitors express specific marker genes (sox10, phox2b and ret) Key roles in migration, survival and proliferation In mouse Ascl1 is present in enteric progenitors but not analyzed in zebrafish

5. Aims Are there distinct subpopulations of enteric progenitors? Is ascl1a an enteric progenitor marker in zebrafish? Do the subpopulations change spatially and/or temporally?

6. Methods RNA probe synthesis Tissue sectioning Immunohistochemistry Fluorescent in situ Hybridization Fluorescent Microscopy Tg (phox2b:GFP)

7. Aims Are there distinct subpopulations of enteric progenitors? Is ascl1a an enteric progenitor marker in zebrafish? Do subpopulations change spatially and/or temporally?

8. 2. 3. 1.Section phox2b:GFP 2.Stain for marker/GFP 3.Focus on Progenitors around the gut 1.

9. phox2b Colocalization with GFP phox2b GFP n=2

10. Distinct progenitor subpopulations sox10 GFP n=2

11. Distinct Progenitor Subpopulations ret GFP n=2

12. Aims Are there distinct subpopulations of enteric progenitors? Is ascl1a an enteric progenitor marker in zebrafish? Do subpopulations change spatially and/or temporally?

13. ascl1a Colocalization with GFP ascl1a GFP n=2

14. Colocalization Summary n=2

15. Aims Is ascl1a an enteric progenitor marker in zebrafish? Are there distinct subpopulations of enteric progenitors? Do subpopulations change spatially and/or temporally?

16. Spatial differences in marker gene expression? Rostral Mid-region Caudal

17. Spatial differences in marker gene expression? n=2

18. Summary ascl1a expressed in enteric progenitors in zebrafish. Based on marker expression, distinct subpopulations of ENS progenitors exist. Spatial differences for ascl1a + /GFP + and ret + /GFP + subpopulations along the gut

19. Future Directions Double FISH with the different progenitor markers FISH at different time points during ENS development Lineage tracing to investigate functional importance of subpopulations ret + /phox2b + sox10 + /phox2b + Neurons

20. Thank You Questions? Judith Eisen Julia Ganz Eisen Lab SPUR Program Peter O’Day SPUR Interns NIH zebrafish facility Histology