The major theme of this proposal is to improve our understanding of how retinoic acid and its receptors contribute to craniofacial development. The first phase of the study will define the temporal and spatial expressions of the specific retinoic acid receptors and cellular retinoic acid binding protein (CRABP) in embryonic mouse craniofacial mesenchyme. This will be done by in situ and Northern blot hybridization. Receptor gene expression from different facial areas will be quantitatively compared using an image analyzer system. Receptor expression will also be studied in embryos exposed to teratogenic doses of exogenous retinoic acid. The second phase of the investigation will analyze the expression of retinoic acid receptors and CRABP in high density micromass cell cultures of mesenchyme taken from different craniofacial areas, with and without exogenous retinoic acid. This study will provide information concerning the role of specific retinoic acid receptors in controlling mesectodermal chondrogenesis. In the studies of both of these phases, special emphasis will be made on observing any differences between first and second brachial arch mesenchyme, because consistent differences in their responses to exogenous retinoids have been found in mice, nonhuman primates, and humans. The third phase probes retinoic acid's role in craniofacial development by blocking expression of retinoic acid receptor genes. Antisense oligonucleotides or retroviruses containing antisense fragments of receptor cDNAs will be used to block receptor expression. The antisense oligonucleotides will be added to the micromass mesectodermal cell cultures. The retroviruses will be injected into the neural fold of mouse embryos, in order to transfect some cells destined to become cranial neural crest. These embryos will then be allowed to grow in whole embryo and micromass cell culture. This series of experiments should provide insight into the developmental regulation of retinoic acid receptors and CRABP during normal craniofacial chondrogenesis and following exogenous application of retinoic acid that is known to induce craniofacial malformations.
