The long term objective of the proposed work is to elucidate some of the mechanisms that govern commitment of pluripotent cells to a particular cell lineage. Quail and mouse neural crest cells will be used as an experimental system. the neural crest originates in the neural folds and gives rise to a wide variety of cell types in the mature vertebrate organism. They include the peripheral nervous system, most primary sensory neurons, endocrine cells, pigment cells, and the cranial ectomesenchymal derivatives, muscle, bone, cartilage, and dermis. The majority of neural crest cells are pluripotent as they leave the neural tube. Moreover, pluripotent cells are also present at final sites of neural crest cell differentiation, supporting the notion of location- specific cues that originate from the embryonic microenvironment and influence neural crest cell differentiation. since up to 9 different cell types are generated in neural crest cell cultures, this is a valuable experimental system to investigate the mechanisms that govern differentiation of pluripotent cells into a wide spectrum of phenotypes. The proposed work is presented in three specific aims. 1) The influence of the four growth factors, neurotrophin-3 (NT-3), basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta), and stem cell factor (SCF) will be investigated by in vitro clonal analysis of quail neural crest cells. The aim is to perform these studies in a define culture medium. Emphasis is placed on discriminating between trophic actions that affect cell growth/survival and influences that are involved in cell commitment. 2) The newly developed in vitro clonal culture system for pluripotent mouse neural crest cell swill be used to analyze differentiation of neural crest cells derived from normal C57BL/6J mice and the mutant strains White spotting (W), Steel (Sl), and Piebald (s). 3) Putative novel regulatory genes involved in cell type specification will be searched for by PCR-amplification of qual RNA obtained from a critical stage in development. to this end, pools of degenerate primers recognizing conserved regions of sequences encoding known DNA-binding proteins will be used. Primary emphasis is placed on helix-loop-helix (HLH) protein-encoding messages, because HLH proteins are known to be involved in cell type specification. However, primers will also be designed to investigate zinc finger sequences, the homeotic gene Hox-7, and the paired box gene , Pax-3. Homologues of interest will be characterized, sequenced, cloned into Bluescript, and subsequently used as probes to screen three existing libraries. The neural crest is of clinical relevance because there are many human neural crest-derived diseases. They include genetic birth defects and malformations caused by teratogens, such as various craniofacial malformations (e.g. cleft lip/cleft palate), congenital defects of the cardiac outflow tract, and pigmentation disorders (e.g., albini...