The RET proto-oncogene encodes a receptor-tyrosine kinase that serves as a component of the receptor for the glial cell-line-derived neurotrophic factor (GDNF) and the related factor neurturin (NTN). The analysis of the mice carrying RET and GDNF null mutations has established that RET signaling is required for the normal development of the enteric and sympathetic nervous systems as well as the excretory system. In humans, RET mutations are associated with congenital intestinal aganglionosis (Hirschsprung's disease) and the inherited cancer syndromes. Multiple Endocrine Neoplasia (MEN) Type 2A and 2B. In the proposed studies, we continue our investigations of the roles of the RET gene in the development of the nervous system and in cancer. While the RET-null mice that we have generated will continue to be an important tool in many of these studies, we will also generate several new types of mutant mice with specific alterations in the RET gene. Some of the mutations will alter individual residues that interact with specific intracellular signaling molecules, allowing us to dissect the roles of specific downstream signaling pathways in the diverse developmental functions of RET. Other mutant mice will carry RET alleles with amino acid substitutions found in human MEN2A or MEN2B, representing animal models that may provide new insight into the etiology of these diseases. We will also investigate the molecular basis the MEN2B by using the yeast-two hybrid system to identify proteins that interact differentially with the wild type and mutant kinase domains. In a continuation o four studies on the development of the enteric nervous system (ENS), we will use an organotypic culture system for the embryonic gut to examine the roles of the GDNF and NTN in cell migration during ENS development. The potentially different roles of the two RET co-receptors GFRalpha-1 or GFRalpha-2 in the development of the ENS, as well as other parts of the nervous system, will be investigated through the production of mutant mice. Finally, we will examine the role of Eph receptor tyrosine kinases and their lands in patterning of the ENS.