The temporal and spatial expression of pro-opiomelanocortin (POMC), thyrotropin releasing hormone (TRH), pro-enkephalin and luteinizing hormone releasing hormone (LHRH) in the Xenopus frog brain were studied using in situ hybridization histochemistry. The first embryonic POMC and TRH cells were shown t be highly clustered in the neural tribe before the appearance of brain nuclei, and these cells go on to form the adult- like pattern, suggesting that these neuropeptide phenotypes, widely distributed in adult, in fact arise by local induction or local proliferation followed by neuronal migration. The Xenopus LHRH cDNA was cloned and the expression of LHRH mRNA during development was studied. Unlike in mammals, this gene was suppressed during early development and not expressed until the late larval period. Three approaches were used to determine how these neuropeptide systems arise in the Xenopus brain during development. One was to use retinoic acid (RA) which deletes anterior to posterior forebrain regions in a dose dependent manner, to localize neuropeptide precursors. POMC cells were eliminated at 5x10(-8) m RA, providing evidence that the onset of POMC gene expression requires the presence of or interaction with anterior neural plate cells. At 10-7 m RA, TRH expression was greatly enlarged in a giant hindbrain, suggesting that in early embryogenesis, an RA-like molecule may specify the posterior CNS. Another approach was to use surgical techniques to precisely define the embryonic location of POMC precursor cells. Thr third was to use embryonic cell marking with the fluorescent dye, DiI to identify clonal relationships and neural migration. In order to study the role of the POMC gene products during development, homologous recombination technology was used to delete the POMC gene. Using a targeting vector which has the neo gene inserted into the coding region, the POMC gene has been disrupted in mouse embryonic derived stem cells. Work is in progress to produce a germ line carrying the disrupted POMC gene.
