HERNANDEZ, L.P.; George Washington University: Hedgehog signaling affects growth and differentiation of pharyngeal cartilages.

Hedgehog (Hh) is expressed in the pharyngeal endoderm during the time that pharyngeal cartilages are developing. Given that the Hh pathway has been implicated in chondrocyte proliferation in long bones this pathway may play a role in pharyngeal cartilage development. I have tested whether Hh signaling is involved in growth and differentiation of the pharyngeal cartilages in zebrafish by both mutant analysis and treatment with a pharmacological agent. I have examined several zebrafish mutants with known mutations within the Hh pathway including sonic you (syu, encoding sonic hedgehog), you too (yot, encoding Gli2) or slow muscle omitted (smu, encoding Smoothened) genes. There were significant differences in growth of arches between mutants and wild type (WT), with arches in yot and syu developing later than WT. Branchial arches in smu never developed. While pharyngeal pouches and overall pharyngeal structure appeared normal prior to chondrogenesis, branchial cartilages failed to form in this mutant. Moreover, the mandibular arch was oddly misshapen in smu mutants. Cyclopamine, an alkaloid, specifically blocks Hedgehog signaling. To test whether differences in growth of arches in mutants was an indirect result of early disruption in other structures, we allowed WT embryos to develop until 12h and 24h before treating with cyclopamine. Larvae sampled at 3d and 4d after treatment showed no trace of branchial cartilage, essentially phenocopying the smu mutation. Moreover, there was no significant growth of the hyoid or mandibular cartilages after treatment with cyclopamine. These data strongly suggest that Hh signaling plays an important role in both growth of all pharyngeal cartilages, and differentiation of branchial cartilages. These findings have significant implications for the evolution of the visceral skeleton in fishes.