The long range goal of the proposed research is the characterization of the cellular and molecular mechanisms that regulate the normal and abnormal development of neural crest cells. The neural crest, a transient structure of the early vertebrate embryo, gives rise to a wide variety of cell types and tissues, such as the peripheral nervous system, pigment cells, endocrine cells, and head mesenchyme. Since several differentiated phenotypes are also expressed by cultured crest cells, in vitro methods have become powerful tools for the analysis of some aspects of neural crest cell development under controlled conditions. For the work described in this proposal, it will be relied on two clonal assays that have been developed in this laboratory. The research plan consists of six major projects: 1) to analyze the formation of the adrenergic, serotoninergic, and melanocytic lineages and to define the role of environmental factors in the normal development of serotoninergic cells and in Hirschsprung's disease, a congenital disorder that is caused by a lack of serotoninergic ganglia in the colon; 2) to analyze exogenous factors that influence the differentiation of peptidergic neurons and determine whether classical transmitters and peptide transmitters can coexist in cultured neural crest cells (an anti-dopamine-Beta-hydroxylase antibody will be prepared as a specific and sensitive probe for adrenergic neurons); 3) to extend the in vitro clonal analysis to head neural crest cells; 4) to follow-up the recent observation that blockage of noradrenalin uptake by a tricyclic antidepressant inhibits adrenergic differentiation; 5) to determine how a replication defective retrovirus that carries a newly discovered oncogene affects neural crest cell differentiation; 6) to evaluate MC540-mediated photosensitization as a means to purge neuroblastoma cells from bone marrow grafts. The proposed research addresses fundamental questions of neural crest cell development and of developmental biology in general. In addition, the proposed work also has implications for the understanding of a) Hirschsprung's disease, b) the possible teratogenicity of tricyclic antidepressants, c) alteration of the differentiative program by an oncogene, and d) the possible therapeutic use of fluorescent lipophilic dyes for the treatment of neuroblastoma. A major characteristic of this proposal is that it relies on two types of in vitro clonal assays that allow a quantitative and thus more objective evaluation of the results.
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