The major objective of this project is to isolate and characterize cDNAs that are involved in craniofacial development. The strategy will be to examine both a set of craniofacial-specific homeobox genes that have already been isolated and novel genes that will be identified by two independent approaches. One approach will be to use degenerate PCR primers against homeobox genes with a subtracted cDNA library prepared from day 42-52 human fetal craniofacial tissues. This approach has been used to identify two novel homeobox genes, H6 and LD71, that are expressed in the craniofacial region. The second approach will be to isolate chick cranial crest-specific genes using the newly described differential mRNA display protocol. The rationale for this approach is that only the cranial crest, and not the trunk crest, can give rise to mesectoderm. The hypothesis that cranial crest cells are predetermined by specific gene expression will be tested. The temporal and spatial expression patterns will be determined for both the homeobox genes and the avian crest genes by in situ hybridizations to confirm differential expression. The importance of the cranial crest-specific genes will be assessed by using avian branchial arch explant cultures and micromass mesenchymal cell cultures. Likewise the functional significance of two craniofacial homeobox genes that have been identified, avian H6 (GH6) and Xenopus Msx- 1, will be determined in chick explant cultures and Xenopus whole embryo approaches, respectively. The genes will be overexpressed by infection of explanted tissues and cells with a recombinant infectious avian retrovirus. Conversely, gene expression will be interfered with by treating the cultures with modified sense and antisense oligonucleotides. The effectiveness of the treatments will be examined by RNase protection assays and effects on morphogenesis and differentiation assessed. The role of any genes that exhibit interesting expression patterns or functional activities will then be pursued by isolating the murine homologs for transgenic mice studies in collaboration with Project 4. The chromosomal position of LD71 and any new homeobox genes will be mapped in the human to help establish a database of craniofacial loci for the genetics of Project 2. These studies are expected to help lead to the identification of genes involved in craniofacial development and to their possible roles in craniofacial abnormalities.