Development And Regulation Of The Gonadotropin Releasing Hormone System

GnRH-1（也被称为LHRH）神经元，对生殖至关重要，来源于鼻位并迁移到大脑，在那里它们成为下丘脑-垂体-性腺轴的组成部分。我们研究了GnRH-1神经元在正常/转基因动物和鼻腔外植体中的分化、迁移和轴突靶向的机制。使用这些相同的模型，我们的工作还解决了GnRH-1神经元中调节（内在和跨突触）GnRH基因表达、肽合成和分泌的机制。多种方法用于识别和理解在引导GnRH-1神经元到达其在中枢神经系统中的最终位置中发挥作用的众多分子和因素。这些包括从迁移和非迁移细胞中获得的文库的差异筛选，在迁移路线的关键位置检查差异表达的分子，敲除小鼠中GnRH-1系统发育的形态学检查，以及体外分子扰动和随后监测GnRH-1神经元运动。随着GnRH-1神经元的迁移，它们也成熟了，这两个过程实际上可能是相关联的。为了研究GnRH-1神经元的成熟，我们采用钙显像、电生理和生化方法检测了GnRH-1神经元的活性和肽分泌。

GnRH-1 (also known as LHRH) 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-1 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-1 neurons. Multiple approaches are used to identify and understand the multitude of molecules and factors which play a role in directing the GnRH-1 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-1 system in knockout mice, and perturbation of molecules in vitro and subsequent monitoring of GnRH-1 neuronal movement. As GnRH-1 neurons migrate they also mature and the two processes may in fact be linked. To investigate the maturation of GnRH-1 neurons we use calcium imaging, electrophysiology and biochemical measures to examine GnRH-1 neuronal activity and peptide secretion. Over the past year two studies were finished: 1) Gene expression screenings of GnRH-1 neurons identified the Amyloid Precursor Protein (APP) Binding-protein1 (FE65) gene. FE65, in association with APP, can regulate B1-integrin dynamics, actin cytoskeleton and cell motility. In addition, FE65/APP signaling has been described in neuro/glial cell fate determination, with increased neurogenesis reported in cortical neuronal progenitor cell cultures from APP-/- and FE65-/- 97kD isoform specific KO mice. Fe65 is expressed in neurons in two isoforms: 97KD and 60kD. The 97kD FE65 contains a tryptophan, tryptophan (WW) domain that is necessary for FE65 nuclear translocation and mediated gene transcription. The 60kD protein, from an alternative start codon, has a truncated WW domain and cannot activate gene expression. To determine the role of Fe65 in GnRH-1 development (migration or neurogenesis), two mouse lines were analyzed: one deficient for the 97kD isoform but retaining the FE65 60kD protein and the other deficient for both FE65 isoforms. Overlapping phenotypes were found. In both lines, no changes in GnRH-1 neuronal migration were detected. However, a 25% increase in total GnRH-1 cell number during embryonic development was found. BrdU birth tracing and spatiotemporal tracking of GnRH-1 cell precursors demonstrated lack of the N terminal portion of FE65 extended the timing of GnRH-1 neurogenesis in the developing nasal placode without affecting proliferation of GnRH-1 neuronal progenitors or cell death. Thus, neurogenesis of specific progenitor cells in the developing vomeronasal organ increased in the absence of the fully functional nuclear translocation/gene transcription domain of FE65. These data highlight a unique role for the 97kDa isoform of FE65 in controlling GnRH-1 neurogenesis and suggest that GnRH-1 cells originate from multipotent progenitors able to generate distinct cell types as GnRH-1 neurogenesis declines in response to environmental changes. 2) The nasal placodes were thought to be ectodermally derived tissue thickenings that arise from the anterior end of the neural plate. Starting from E10.5 in mouse, the nasal placodes invaginate and give rise to cell types including GnRH-1 neurons that migrate into the brain, olfactory sensory neurons that project to the olfactory bulbs and olfactory ensheathing cells (OECs) that provide guidance for olfactory sensory axons. Although our earlier work had ruled out GnRH-1 cells being derived from the anterior pituitary, the lineage of GnRH-1 cells was still controversial - nasal placode or neural crest. Thus, a project using early identified neuronal/GnRH-1 markers and genetic lineage tracing was performed to investigate ontogenesis of the GnRH-1 system and its relation with the developing olfactory system. A neural crest specific Wnt-1Cre mouse line and an ectodermal specific Crect mouse line were used. These mice were crossed with Rosa reporter mouse lines and the lineage of cells associated with the nasal placode determined. We proved that neural crest cells mingle with ectodermal cells in the developing nasal placode and give rise to subpopulations of GnRH-1 neurons (30%), olfactory and vomeronasal cells (7%) and to the entire population of OECs (100%). These data demonstrate that OECs and peripheral Schwann cells share a common NC origin and that within the GnRH-1 neuronal population, neurons originate from distinct ectodermal and neural crest progenitors within the placode. By redefining the concept of the nasal placode as a structure with ectodermal and neural crest cellular components, we provide a new framework for understanding certain pathologies effecting olfaction and sexual development, such as Kallmann Syndrome, which may be in part neural crest disorders. New investigations using Cre-lox-mice to specifically remove molecules of interest from GnRH-1 cells during development will be continued this year with specific studies on the early development of the GnRH-1 neurons and the location of their progenitor cells in relation to nasal placodal cells and neural crest. In progress are studies examining the role of growth factor receptors (FGFR1, PDGF), NELF (a migrational molecule), and cytokines, in GnRH-1 development as well as in situ characterization of the migration of GnRH-1 neurons (real time microscopy). In addition, we continue to study the role of estrogen on GnRH-1 neuronal activity and are finishing studies monitoring GnRH-1 neuronal activity in nasal explants generated from estrogen receptor knockout mice. Other studies include examining/identifying 1) the maturation of ion transporters associated with GnRH-1 neuronal activity, 2) molecules that modulate GnRH-1 neuronal activity that participate in reproductive functions such as adiponectin, 3) midline cues which influence olfactory axon outgrowth and 4) the dynamics of GnRH-1 neuronal migration.