This proposal describes the strategy and procedures for expression cloning and characterization of neural inducers and patterning genes in Xenopus laevis. The long term goals are to identify and characterize as many genes as possible that are involved in neurogenesis. Short term objectives are: (1) Construction of a cDNA expression library. This expression library will be derived from dissected dorsal mesoderm and neurectoderm at appropriate stages since these are the tissues where neural inducers are expressed in vivo. (2) Construction of a dorsal specific, full length cDNA, subtracted library. This subtracted library will be constructed with a novel design to obtain full length cDNAs as inserts. (3) expression cloning of neural inducers employing a novel 'reprogramming assay'. This scheme is unique in that it is sensitized for neural inducers and not for mesodermal inducers, thus is free of complications from secondary inductions by mesodermal inducers. (4) In parallel with expression cloning, low stringency PCR will be used to search for new members of known gene families that are involved in embryonic development. (5) characterization of the identified clones. The characterization of these genes will further our knowledge of neural induction, cell-cell signaling, and developmental processes in general. This study could also contribute to the understanding of diseases of nervous system and perhaps, given the fact that many proto-oncogenes are involved in embryonic development, tumorogenesis.
