Functional Analysis of Sonic Hedgehog Signaling during Fin Regeneration in Zebrafish

Amanda Smith, Liz Quint, Fabien Avaron, Lynda Laforest and Marie-Andrée Akimenko

Ottawa Health Research Institute
725 Parkdale Avenue, Ottawa, Ontario, K1Y 4E9, Canada

Teleost fish have the ability to regenerate their fins after injury. Sonic hedgehog (shh), its receptor patched1 (ptc1), and the bone morphogenetic factor bmp2b, are re-expressed during fin regeneration in patterns that implicate shh and bmp2b signaling in the patterning of the dermal bones within the regenerating fin. To better understand the role of shh and bmp2b signaling during regeneration, we have performed a functional analysis. We have shown that ectopic expression of shh or bmp2b in the inter-ray region using an in vivo transfection approach, results in excess bone formation that disrupts the normal patterning of the regenerating fin. The effects of shh ectopic misexpression could be antagonized by chordin, an inhibitor of bmp signaling, suggesting that bmp is a downstream target of shh signaling in the fin regenerate. Furthermore, ectopic expression of shh, but not bmp2b, induced the activation of ptc1 expression indicating an absence of reciprocal interaction between bmp2b and shh signaling. In addition, we demonstrated that exposure of regenerating fins to the alkaloid cyclopamine, which specifically interferes with hedgehog signaling, results in a dose dependent reduction in cell proliferation in the regenerative mesenchyme, leading to an inhibition of fin regeneration. Morphological changes were accompanied by a rapid loss of ptc1 expression, and an expansion, followed by a reduction, in domains of shh expression. Recent findings from our group and others, using specific inhibitors for shh and FGF signaling, suggest that growth of the regenerate is controlled by both FGF and shh signaling through a positive feedback loop. Finally, we found that ectopic expression of shh within the ray blastema leads to a down-regulation of endogenous shh expression that is accompanied by a transient decrease of the growth rate of the regenerate. On the one hand, this result further implicates shh signaling in the regulation of the fin growth, and, on the other hand, it indicates a tight regulation of the amount of shh signaling in the regenerating fin.

Therefore, the ectopic bone matrix deposition following ectopic expression of shh and bmp2b and the effects of cyclopamine treatments on fin outgrowth implicate shh signaling in the proliferation and/or differentiation of the specialized bone-secreting cells in the blastema. In addition, the domains of shh expression in regenerating fins may be controlled by regulatory feedback mechanisms that function to define the region of bone secretion. Supported by a grant from the CIHR.