A region of the frontonasal process (FNP) which mimics the function of the zone of polarizing activity (ZPA) in the limb has been identified. Like a ZPA, the FNP ectoderm expresses the polarizing signal sonic hedgehog. When grafted to the anterior margin of a host wing bud, FNP ectoderm induces two downstream genes, BMP-2 and HOXD-11 in host tissues. Furthermore, FNP ectoderm contains signals which, when grafted into a host limb bud, evoke duplications of the digit pattern. Since retinoic acid is required for establishing the limb ZPA, it is proposed that retinoic acid also participates in establishing the FNP polarizing region. This hypothesis will be tested by investigating the role of RA in patterning the neural plate and neural ridge, from which the FNP ectoderm and the mesenchyme is derived. Cells located in the most anterior region of the neural ridge contribute to the ectoderm covering the FNP, while those cells located more rostrally contribute to the mesenchymal cells. In the first Specific Aim, the temporal and spatial expression domains of genes that potentially regulate neural plate patterning will be characterized. These include fibroblast growth factor 8, emx-2, pax-6 and otx-2. The murine expression patterns of these candidate genes, and the phenotypes of some null mutants, suggest they are involved in craniofacial development. The second Specific Aim will determine whether retinoic acid participates in patterning the neural plate and neural ridge. Endogenous retinoid signaling will be locally inhibited through the use of retinoid receptor pan-specific antagonists. These antagonists will be locally applied at the neural plate stage of development. The effects of retinoid signal blockade will be assessed by three methods: the effect on gene expression in the neural ridge, the effect on morphology of the craniofacial complex at stage 26 (when most facial development is complete), and the effect on the function and gene expression in the FNP polarizing region. These studies directly address the endogenous role of retinoids in patterning craniofacial primordia, and will further our understanding of how retinoids exert their teratogenic effects of retinoids during embryogenesis.
