The long-term goal of this project is to understand the principles by which polypeptide growth factors their receptors control complex developmental and physiological processes in mammals. The particular focus will be on the class of receptors with tyrosine kinase or tyrosine phosphatase activity. The molecules and cell types that will be studied are directly relevant to many important diseases, including cancer, deafness, malformations of the face and heart, and blood deficiency disorders. Also, much of the work is geared toward using recent techniques to identify new growth factors, which in general have great potential as therapeutic agents. We will extend our studies of a recently identified growth factor, the ligand (KL) for the receptor tyrosine kinase encoded by the c-kit proto-oncogene. Both KL and its receptor are encoded at mouse genetic loci with known mutant alleles, showing that KL is required for the development of some especially interesting cell lineages: cells derived from the neural crest (including melanocytes and probably neurons), germ cells, and hematopoietic cells (including mast cells, red cells and multipotent progenitor cells). Now that KL polypeptides and cDNAs are identified, we are in a position to determine at the molecular level how KL functions. This will be done by three complementary approaches. (1) Analysis of the effects of two different transmembrane forms and the soluble form of KL on cellular processes such as proliferation, differentiation, and migration. (2) Determination of the expression patterns of these different forms of KL in mice. (3) Investigation of the interactions of transmembrane KL with other molecules within the cells that express KL. Much of this work on KL will focus on its control of neural crest cells. This population of multipotent stem cells gives rise to a remarkable range of cell types and tissues, including most of the peripheral nervous system, melanocytes, much of the mesenchyme of the face, and parts of the thymus and heart. These progenitor cells are little characterized at the molecular level, and in order to understand how c-kit and other receptors cooperate to control this complex system it will initially be necessary to know which receptors are present. We propose to use a PCR-based approach to determine the known and possibly unknown receptor-like tyrosine kinases and tyrosine phosphatases that are expressed in neural crest progenitor cells. As another means to understand the biology of the kit family of receptors and ligands, we will study two closely related receptors, flt and flk-2, with unknown ligands. Our approaches will be: (1) to determine where these receptors are expressed, by in situ hybridization; (2) to identify their ligands, using and extending the soluble receptor affinity approach we developed to identify KL; and (3) to study their function by genetic mutant analysis, initially by testing known candidate mouse mutants.
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