This proposal examines the potential role of the neurofibromatosis type 1 (NF1) gene and its protein product in development and differentiation of neuronal lineages from the neural crest (NC), a cell population that appears transiently during embryonic development of all vertebrates. The long-range objectives are to determine how the NC gives rise to the myriad of differentiated cells which arise from it, and most importantly, how neuronal lineages arise from the NC. Understanding the role of NF1 in normal NC development and differentiation will augment information on how the alteration or disruption of the gene results in the aberrant proliferation of certain crest derivatives that characterizes the disease Neurofibromatosis 1 in humans. The experiments outlined in this proposal are designed to determine if NF1 is an important gene in crest development by following its early expression pattern in the premigratory and migratory avian neural crest and in the derivatives of the crest that are involved in NF1. The avian neurofibromatosis 1 (alphaNF1) cDNA which has been cloned and partially sequenced in this lab is highly (at least 86%) homologous to the human NF1 cDNA at the nucleic acid level. Using an avian system as a model has unique advantages for neural crest studies. Not only is the avian system the best studied of all the NC systems and that which has produced the most information about crest development, it is the only system that is accessible and manipulable at very early developmental time points. These experiments are at he interface of molecular and cellular biology and address important questions of stem cell differentiation both in normal development and pathologies such as neurofibromatosis. The specific questions addressed include: 1. How does differentiation by retinoic acid (RA) upregulate alphaNF1 message levels in a src-transformed immortalized quail NC cell line that become neuronal upon differentiation? 2. Do primary avian NC cells express the gene and is it upregulated upon differentiation of NC into a variety of neurons? 3. What is the expression pattern of the gene and the protein product during differentiation of the NC in vivo? 4. If anti-sense constructs of the alphaNFI cDNA or gene are introduce into the src-transformed quail NC cell line, is differentiation and/or alphaNF1 upregulation blocked? 5. Does transplantation of the src-transformed quail NC cell line, onto chick NC pathways that do not lead to neuronal differentiation, lead to upregulation of the alphaNF1 gene?
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