ONTOGENY OF THE LUTINIZING HORMONE RELEASING HORMONE SYSTEM

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LHRH neurons are derived from the olfactory placode and migrate into the brain. The factors dictating cell-specific LHRH gene expression, and the migratory mechanism(s) used by the LHRH neurons during movement into the CNS are currently unknown. We continue to study the prenatal development of the LHRH system both in vivo as well as through the use of in vitro models. We have established that: (1) LHRH neurons are apposed to peripherin positive fibers in nasal and forebrain regions; (2) the olfactory axon/LHRH migratory pathway is N-CAM positive in nasal regions; (3) GABAergic cells are present along this pathway; and (4) olfactory epithelial cells in the olfactory pit express peripherin mRNA. Based on these findings, we hypothesize that peripherin-positive olfactory axons delineate the entire LHRH migratory pathway. We are using embryonic explants, which maintain large numbers of LHRH expressing cells, to test the hypothesis that LHRH neuron migration occurs via interactions with olfactory fibers. Consistent with the in vivo findings, in explants, LHRH neurons are also associated with peripherin-positive fibers, GABAergic cells, and N-CAM-positive pathways. We have shown that LHRH cells in vivo do not posttranslationally modify the cleaved LHRH peptide to the amidated form until reaching the brain. However, in embryonic explants, LHRH cells can produce amidated peptide after 3 days in culture, in the absence of brain tissue. Using transgenic mice containing the LHRH promoter fused to a luciferase reporter, we have targeted luciferase mRNA and product in LHRH neurons. Using a second transgenic line containing the neuron-specific enolase promoter fused to LacZ, neuron-specific enolase positive-nonLHRH cells were detected along the nasal migratory path. In non-mammalian species, two LHRH systems have been described: one located in forebrain (analogous to mammalian LHRH) and the other in midbrain. We cloned a LHRH gene in Xenopus laevis, an organism well-suited for the study of cell-cell interactions. The cDNA showed high homology to mammalian LHRH and encoded a mRNA localized to forebrain cells only. In the frog, LHRH is not expressed until the late larval period, but the location of the LHRH cells indicates that this neuronal population also originates in the olfactory placode, in regions accessible to ablation and transplantation studies. Currently, we are determining: (1) the phenotype(s) of the peripherin-positive cells; (2) the relevance of this peripherin- positive axonal system to the CNS pathway on which LHRH cells migrate; (3) the identity of cells expressing N-CAM and the role of N-CAM in LHRH movement in nasal regions; and (4) whether the GABAergic cells arise from the olfactory placode and share a common lineage wish the LHRH cells during development. Finally, we will use the olfactory placodes of transgenic animals to generate explant cultures and determine if LHRH cells can be identified in situ during migration.