LHRH neurons, critical for reproduction, are derived from the olfactory placode and migrate into the brain, where they become integral members of the hypothalamic-pituitary-gonadal axis. We study mechanism(s) underlying LHRH neuronal differentiation and migration in normal/transgenic animals, and nasal explants. Intrinsic and trans- synaptic regulation of LHRH gene expression, peptide synthesis and secretion in embryonic LHRH neurons (outside the CNS) versus postnatal LHRH neurons (in the CNS) is studied using nasal explants and organotypic cultures, respectively.Previous work in our lab using nasal explants 1) showed that outgrowth of olfactory axons and migration of LHRH neurons occur in isolated olfactory pit tissue, 2) revealed olfactory axon guidance cues localized to midline nasal cartilagenous tissue and 3) identified two developmental genes in LHRH neurons; AP-2 (a developmental transcription factor) and clone 18 (a novel gene). Over the past year, we have addressed the role of AP-2 in LHRH development using AP-2 mutant mice. These studies demonstrate that AP-2 is important for maintenance of LHRH gene expression as these neurons enter the CNS. Since the LHRH promoter does not contain an AP-2 consensus sequence, AP-2 must act via an indirect route. With regard to our novel gene, clone 18, we were able to generate an antibody specific for the encoded protein. Mapping of both the mRNA transcript and protein for clone 18 have been done. Although expression is found in a variety of CNS regions, most pertinent to our work, the encoded protein is expressed in the olfactory system and migrating LHRH neurons. Preliminary work in vitro suggests that the protein is associated with the extracellular portion of the cell membrane, making it a candidate molecule for migration. To understand the regulation of LHRH neuronal activity, we have completed work on the developmental changes in LHRH peptide content in mice. These studies demonstrate that LHRH peptide content/cell increases in LHRH neurons in vivo as well as LHRH neurons maintained in nasal explants (embryonic LHRH neurons in vitro). In vitro, the increase in LHRH peptide content is comparable to that measured in vivo, suggesting important maturational events are intrinsic properties of these cells. We have also demonstrated that LHRH neurons maintained in vitro can release LHRH peptide.Work in progress is focused on: 1) isolation of the midline cues which influence olfactory axon outgrowth; 2) the role of clone 18 in LHRH migration, 3) whether LHRH neurons maintained in nasal explants (devoid of brain influences) show rhythmic activity patterns and if so the mechanisms underlying this rhythm, and 4) genes differentially expressed in LHRH neurons as a function of developmental and reproductive state. - GnRH, neuronal migration, axon guidance, pulsatility, nasal explants, single cell pcr.
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