The objectives of this project are to study the molecular and cellular mechanism regulating the developmental expression of three neuropeptide genes coding for the pro-opiomelanocortin (POMC), thyrotropin releasing hormone TRH and the pro-[met]enkephalin family of peptides, and to investigate the role of these peptides in development, particularly in the central nervous system (CNS). Two model vertebrate systems: the frog (Xenopus laevis) and mouse were used. The temporal and spatia expression of POMC, TRH and [met]enkephalin mRNA during development have been mapped by in situ hybridization histochemistry. The first appearance of POMC mRNA in the mouse CNS was in the region of the presumptive arcuate nucleus, at embryonic day 10-1/2 (E10-1/2), and in the anterior lobe and intermediate lobe of the pituitary at E12-1/2 and E14-1/2 respectively. Immunocytochemical and biochemical analyses showed that the POMC was expressed at E10-1/2 but processing of the prohormone began only a day later, at E11-1/2. The major embryonic POMC-derived products were ACTH 1-17, des acetyl alpha-MSH, and beta-endorphin(1-31) and beta-endorphin(1-27) inbrain and ACTH(1-39) and beta-endorphin(1-31) indicating some differences in processing between embryos and adult. Characterization of opiate receptors in embryonic mouse brain revealed the first expression of the mu receptors which bind specifically to beta-endorphin(1-31), but not the truncated or acetylated forms of the peptide, at E12.5. These studies indicate that the POMC system is functional as early as E12.5 in the mouse CNS. In the frog, migrating pituitary precursor cells were found to express mature POMC mRNA at the same time that they reach the infundibular wall at stage 28. The first POMC expressing cells in the CNS was found in embryonic forebrain (the posterior diencephalon) at stage 31 and they were juxtaposed to the first POMC cells found in the pituitary primordium. The finding of the first brain and pituitary POMC cells juxtaposed at the juncture of the diencephalic and infundibulum walls suggest brain-pituitary interactions in the setting up of this system. The frog POMC and pro-enkephalin genes including the 5' upstream regulatory region have been cloned and sequenced. Analysis of the 5' upstream region of the POMC gene have identified the promoter sequence and a cyclic AMP regulatory element. Work is now in progress to identify the elements responsible for tissue-specificity expression and the trans acting factors involved in activating these genes during development.
