GnRH neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain where they become integral members of the hypothalamic-pituitary-gonadal axis. We study mechanism(s) underlying GnRH neuronal differentiation, migration and axonal targeting in normal/transgenic animals, and nasal explants. Using these same models, our work also addresses the mechanisms regulating (intrinsic and trans-synaptic) GnRH gene expression, peptide synthesis and secretion in GnRH neurons. Multiple approaches are used to identify and understand the multitude of molecules and factors which play a role in directing the GnRH neurons to their final location in the CNS. These include differential screening of libraries obtained from migrating versus non-migrating cells, examination of molecules differentially expressed at key locations along the migratory route, morphological examination of the development of the GnRH system in knockout mice, and perturbation of molecules in vitro and subsequent monitoring of GnRH neuronal movement. As GnRH neurons migrate they also mature and the two processes may in fact be linked. To investigate the maturation of GnRH neurons we use calcium imaging, electrophysiology and biochemical measures to examine GnRH neuronal activity and peptide secretion. In addition, we collaborate with labs performing human genetic screening of Kallman patients. Once a mutation is identified, we analyze the expression pattern in mice and perform biological assays to determine the outcome of the mutated gene on GnRH development. Over the past year, one article and one book chapter were published, and one mouse line created: Article) One key signaling pathway known to influence neuronal migration involves the extracellular matrix protein Reelin. Typically, signaling of Reelin occurs via apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VLDLR), and the cytoplasmic adapter protein disabled 1 (Dab1). However, non-canonical Reelin signaling has been reported, though no receptors have yet been identified. The literature indicated Dab1-independent Reelin signaling impacts GnRH neuronal migration. Our paper study was initiated to delineate the non-canonical Reelin signaling pathways used by GnRH neurons. Chronic treatment of nasal explants with CR-50, an antibody known to interfere with Reelin homopolymerization and Dab1 phosphorylation, decreased the distance GnRH neurons and OECs migrated. Normal migration of these two cell types was observed when Reelin was co-applied with CR-50. Immuno-cytochemistry was performed to determine if OECs might transduce Reelin signals via the canonical pathway, and subsequently indirectly altering GnRH neuronal migration. We showed that in mouse: (1) both OECs and GnRH cells express ApoER2, VLDLR and Dab1, and (2) GnRH neurons and OECs show a normal distribution in the brain of two mutant reeler lines. These results indicate that the canonical Reelin pathway is present in GnRH neurons and OECs, but that Reelin is not essential for development of these two systems in vivo. Book Chapter) This chapter focuses on the development of the GnRH neuronal system. Topics include: 1) development of the nasal placode (where GnRH neurons come from), 2) the axonal tracks on which GnRH cells migrate, 3) signaling factors that affect GnRH development/migration, 4) genes associated with disruption of GnRH development that have been linked to patients with Kallmans Syndrome (anosmia and delayed or absent puberty), 5) cytoskeletal dynamics that control GnRH neuronal movement, and 6) a nasal explant model for the study of the GnRH system. New Research Strategy) Our lab has shown that the GnRH cell population can be developmentally subdivided into a group expressing placodal ectodermal markers and a smaller group expressing neural crest markers. To test whether these two subpopulations are functionally distinct, we created a GnRH exon two flox/flox mouse line using CRISPR technology. We have established the flox/flox line and have begun mating females to two different reporter lines - one a neural crest cre line and the other a placodal ectoderm cre line. Over the next year, we should generate our first GnRH specific subpopulation mutants and begin to address the function, if any, of these two developmentally distinct group of GnRH cells.
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