Exogenous retinoic acid induces a stage-specific, transient and progressive extension of Sonic hedgehog expression across the pectoral fin bud of zebrafish.

We have performed a time course analysis of the expression of Sonichedgehog (shh) and patched1 (ptc1) in response to exogenous retinoic acid (RA) application to get some insight into the mechanism(s) underlying the formation of a mirror-image duplication of shh and ptc1 domains of expression in the pectoral fin buds of zebrafish. We have shown that RA exposure during the early stages of pectoral fin development first results in a rapid decrease or complete loss of shh/ptc1 expression. This is followed by reappearance of transcripts in the normal posterior domain, then by a stage-dependent and progressive expansion of the shh domain from the ZPA towards the anterior margin of the bud. Shh transcripts are induced in mesenchymal cells underlying the ventral ectoderm at the base of the bud. Once shh expression is activated in the most anterior cells, the number of shh-expressing cells increases in this region, possibly through an amplification mechanism involving signals from the apical ectodermal ridge. At this time, shh expression disappears from cells centrally located in the bud, resulting in the formation of the two distinct domains. An anterior extension of shh expression is also obtained in syu mutants with impaired shh function, suggesting that shh induction across the fin bud is independent of shh signaling. This study suggests the existence of complex mechanisms controlling the spatial and temporal expression of shh in the developing fin bud.

Bone patterning is altered in the regenerating zebrafish caudal fin after ectopic expression of sonic hedgehog and bmp2b or exposure to cyclopamine.

Amputation of the zebrafish caudal fin stimulates regeneration of the dermal skeleton and reexpression of sonic hedgehog (shh)-signaling pathway genes. Expression patterns suggest a role for shh signaling in the secretion and patterning of the regenerating dermal bone, but a direct role has not been demonstrated. We established an in vivo method of gene transfection to express ectopically genes in the blastema of regenerating fins. Ectopic expression of shh or bmp2 in the blastema-induced excess bone deposition and altered patterning of the regenerate. The effects of shh ectopic expression could be antagonized by ectopic expression of chordin, an inhibitor of bone morphogenetic protein (bmp) signaling. We disrupted shh signaling in the regenerating fin by exposure to cyclopamine and found a dose-dependent inhibition of fin outgrowth, accumulation of melanocytes in the distal region of each fin ray, loss of actinotrichia, and reduction in cell proliferation in the mesenchyme. Morphological changes were accompanied by an expansion, followed by a reduction, in domains of shh expression and a rapid abolition of ptc1 expression. These results implicate shh and bmp2b signaling in the proliferation and/or differentiation of specialized bone-secreting cells in the blastema and suggest shh expression may be controlled by regulatory feedback mechanisms that define the region of bone secretion in the outgrowing fin.