Role of hedgehog signaling in branching morphogenesis and patterning of the fin ray during zebrafish fin regeneration

Abstract

Zebrafish have the capability to regenerate their fins after injury. Previous studies from our laboratory showed that fin regeneration triggers the re-expression of genes involved in the hedgehog (hh) signaling pathway. One member of the hh family, sonic hedgehog (shh) was suggested to regulate bone patterning based on its expgression pattern and functional analysis. Another member of the hh family, Indian hedgehog (ihha) is expressed in the differentiating scleroblasts, the bone-matrix releasing cells, of each fin ray, and may have a more direct role on bone formation based on its expression pattern and known function in other vertebrates. The present study is aimed at investigating the role of hh signalling in patterning the fin ray regenerate and more specifically its role in ray branching morphogenesis. In a first approach, we used the zebrafish 2.2shh:gfp:ABC transgenic line, in which GFP expression recapitulates the endogenous expression of shh, to ablate the shh-expressing cells using a laser beam. Results show that such ablation leads to a delay of ray bifurcation suggesting that the shh-expressing cells play an important role in branching morphogenesis. In a second approach, we cloned the zebrafish hedgehog interacting protein (hip), a hedgehog antagonist, to investigate the effects of its overexpression on ray patterning. Analysis of hip expression during fin regeneration suggests its involvement in limiting hh signaling on the lateral sides of the dermal bones and in their medial region during branching morphogenesis. Overexpression of hip via in vivo cell transfection in the regenerating fin causes a branching delay, possibly as a result of the altered expression of patched1, the hh receptor and type X collagen, a component of the fin ray. These results suggest that hh signaling is involved in patterning the ray branching during zebrafish fin regeneration. Based on the distinct role of shh and ihha suggested by studies in other systems, ihha may be more involved in the scleroblast proliferation and differentiation, and shh is likely to be responsible to pattern the bone formation by directing the site of scleroblast differentiation or possibly mediating scleroblast migration.