The overall goal of this research proposal is to characterize the roles of two signaling molecules, sonic hedgehog (shh) and retinoic acid, in craniofacial development. Mutations in the SHH locus are associated with craniofacial defects such as holoprosencephaly, and embryos with deletions in retinoid receptors exhibit craniofacial malformations. Abundant molecular evidence also demonstrates that SHH and retinoid signaling are required in the early stages of craniofacial development, when the neural plate and neural ridge are patterned. However, the contribution of SHH and retinoid signaling to later stages of development, when the craniofacial primordia are patterned, is unknown. Recently epithelial domains in the embryonic face were identified that express shh and exhibit polarizing activity in a functional assay. Retinoic acid is required for the establishment of the limb organizer, and recent evidence from the applicant suggests a similar role in the face. It is hypothesized that retinoid signaling is required to establish organizing centers in the face, and that organizing activity mediated by SHH participates in craniofacial outgrowth and patterning.
Two specific aims are proposed to test this hypothesis. First, gain-of-function and loss-of-function experiments will be used to characterize the role of organizing tissues and SHH function in the craniofacial primordia. This will be achieved by creating ectopic sites of polarizing activity through interspecific transplantations and retrovirally expressed SHH protein. Organizer function will be inhibited through surgical removal and through the use of antibodies that block the function of SHH. In both cases embryos will be evaluated for variations in gene expression and changes in skeletal and soft tissue morphology. Second, biochemical and genetic approaches will be used to test whether retinoids are required to establish organizing centers and induce shh expression in craniofacial primordia. A transient blockade in retinoid signaling will be created using pan-specific retinoid receptor antagonists, and the effect on gene expression and morphology will be studied. Whether the craniofacial malformation in Small-eye mice, caused by a loss of retinoic acid-producing mesenchymal cells in the frontonasal process, is associated with disruptions in organizing activity and shh expression will be determined. Collectively, these experiments will determine the contributions of two important signaling molecules in normal and abnormal development of the embryonic face.
