Interactions Between ihha and mef2ca in Bone Development

Bones form through the differentiation of mesenchymal cells into bone-forming osteoblasts, and multiple genes regulate this process in order to ensure proper bone size and shape. Myocyte enhancer factor 2ca (mef2ca) encodes a transcription factor that negatively regulates development of the opercle (Op), a craniofacial bone of the zebrafish. Loss of mef2ca can lead to ectopic bone growth along the antero-ventral edge of the Op, yet the developmental mechanism underlying this process is unclear. We tested whether indian hedgehog a (ihha), a positive regulator of Op development acting in the same region of bone that mef2ca negatively regulates, is required for the complete ectopic bone growth seen in mef2ca mutants. With fluorescent in situ hybridization we show that expression of ihha and ptch1, a downstream target of active Hedgehog signaling, is present in ectopic bone growth of mef2ca mutants, suggesting that mef2ca may regulate Ihha signaling to pattern bone. Furthermore, analysis of mef2ca; ihha double mutant larvae reveals many ventrally reduced bones that resemble ihha single mutants; however, many also display the mef2ca mutant phenotype. Therefore, although Ihha is required for certain mef2ca mutant phenotypes, it is not imperative to induce expansion of bone in mef2ca mutants, and thus mef2ca must im- pose its negative regulation of bone development by acting through distinct gene networks in addition to the Hedgehog pathway.