Sonic Hedgehog Signaling in Zebrafish Branchial Arch Development

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DESCRIPTION (provided by applicant): The proposed studies seek to determine the cellular and molecular mechanisms of Shh signaling in the craniofacial skeleton. This may provide new explanations for the wide range of phenotypes observed in Holoprosencephaly patients. To examine the function of Shh in branchial arch development we analyzed the zebrafish chameleon (con) mutant, harboring a defective Displ protein, con mutant embryos lack all branchial arch cartilage, a defect that can be partially explained by a failure of the branchial arch precursors to maintain the essential chondrogenic transcription factor Sox9a. The proposed research will specifically seek to delineate the signaling mechanism whereby the Shh pathway promotes chondrogenesis in the branchial arches. We hypothesize that Displ acts within pharyngeal endoderm non cell autonomously to release Shh which directs branchial arch cartilage precursors toward chondrogenic differentiation. Utilizing cell transplantation studies, we will generate chimeric embryos to demonstrate the cell autonomy of Displ and Smo function. This will determine which cells in the craniofacial region must release (Displ) and respond (Smo) to the Shh ligand. Furthermore, our work has delineated multiple temporal requirements for Shh signaling in branchial arch development. Utilizing a well-characterized GN1 activity reporter construct, we will generate a novel transgenic zebrafish to precisely determine which cells recieve the Shh ligand and when this occurs during cartilage development. Lastly, human craniofacial diseases arise from the misregulation of the SHH pathway and SOX9, therefore further investigation of this potential regulatory relationship is of considerable interest. In vivo overexpression studies will be performed to assess whether ectopic SoxQa in the branchial arches can rescue inactivation of the Shh pathway. The Shh signal transduction pathway plays a central role in growth and patterning of the brain and face. Disruptions in this pathway during embryonic development lead to a considerable disease burden, most often classified as the spectral disorder Holoprosencephaly. The overall goal of this proposed research is to determine the cellular and molecular mechanism of the Shh pathway that controls craniofacial skeleton development.