Development And Regulation Of The Gonadotropin Releasing Hormone System

GnRH（也称为LHRH）神经元，对生殖至关重要，来源于鼻基板并迁移到大脑，在那里它们成为下丘脑-垂体-性腺轴的组成成员。我们在正常/转基因动物和鼻外植体中研究GnRH神经元分化、迁移和轴突靶向的潜在机制。使用这些相同的模型，我们的工作也解决了机制调节（内在的和跨突触）GnRH基因的表达，肽的合成和分泌在GnRH神经元。多种方法被用于识别和理解在将GnRH神经元引导到其在CNS中的最终位置中发挥作用的众多分子和因子。 这些包括从迁移与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沿着差异表达的分子的检查，在敲除小鼠中GnRH系统的发育的形态学检查，以及体外分子的扰动和随后的GnRH神经元运动的监测。 随着GnRH神经元迁移，它们也成熟，这两个过程实际上可能是联系在一起的。 为了研究GnRH神经元的成熟，我们使用钙成像、电生理和生化措施来检查GnRH神经元的活性和肽分泌。 在过去的一年里，完成了两项研究： 1)基质衍生生长因子（SDF-1）和γ-氨基丁酸（GABA）是两种细胞外因子，在发育过程中调节神经元迁移，并可能协同作用。这种相互作用的分子机制尚不清楚。SDF-1和GABA均已显示分别通过加速和减慢迁移来调节GnRH神经元迁移的速率。因此，该系统用于探索这些分子在发育期间产生协调细胞运动的机制。GABA和SDF-1显示通过激活去极化或超极化信号通路对细胞运动速度产生相反的作用，GABA通过氯的变化，SDF-1通过钾的变化。还发现GABA和SDF-1协同作用以促进线性运动而不是随机运动。因此，这些信号通路的同时激活导致细胞速度的严格控制和GnRH神经元迁移通路方向性的沿着改善。 2)神经回路的正确组装需要新生的神经元从它们的起源地迁移到它们的最终位置。嗜轴性神经元迁移的机制知之甚少，神经元通过沿着轴突通路向其目的地移动。GnRH神经元迁移沿着嗅觉轴突从鼻子到前脑在发展过程中，并被用作一个模型的嗜轴性迁移。我们在小鼠中研究了从钙信号到细胞骨架动力学的运动调节。活体成像显示，在嗜轴细胞迁移过程中，钙离子的活性很强，通过IP 3受体释放的钙离子被发现可以刺激迁移。这通过涉及钙传感器钙/钙调蛋白蛋白激酶激酶（CaMKK）、AMP激活的激酶（AMPK）和RhoA/ROCK的信号通路发生。通过对表达肌动蛋白- GFP或Lifeact-RFP的GnRH神经元成像，发现钙释放刺激主导过程肌动蛋白流离开细胞体。相反，肌动蛋白收缩在细胞后部不受这种钙信号通路。这些研究结果是第一次测试的调节细胞骨架动力学在axophilic迁移，并揭示了运动的机制，具有广泛的影响，其他CNS人群的迁移。 新的研究正在表征骨形态发生蛋白-4（Bmp 4）和成纤维细胞生长因子-8（Fgf 8）在发育中的嗅觉/犁鼻系统中定义上皮与神经源性命运的作用。使用不同的敲入小鼠系和Cre-lox介导的谱系追踪，分析Fgf 8表达和细胞谱系与Bmp 4及其拮抗剂Noggin表达的关系。这些实验将确定Fgf 8是否足以诱导嗅窝的外胚层祖细胞获得神经命运以及诱导GnRH神经元特化。此外，为了在发育过程中从GnRH细胞中特异性去除感兴趣的分子并检查迁移变化，正在使用Cre-lox小鼠和/或药理学/分子操作，并使用真实的时间显微镜对GnRH神经元的迁移进行原位表征。 正在进行的具体研究检查了生长因子受体（VEGR 1和FGFR 1）的作用。最后，我们继续研究GnRH神经元的生理学，重点是GABA和脂联素。我们正在研究为什么GABA仍然兴奋性GnRH神经元在成人通过评估氯转运蛋白和GABA受体亚型的表达在发展。在人类和动物模型中，代谢功能障碍通常与生殖异常有关。脂联素是由白色脂肪组织分泌的一种外周激素，在能量平衡和食欲调节中起重要作用。 我们发现GnRH神经元的亚群表达AdipoR 2。因此，我们将确定脂联素是否可以直接作用于GnRH神经元使用全细胞膜片钳和钙成像。通过脂联素对GnRH神经元活性的调节将证明能量平衡与控制生殖的神经元之间的直接联系。

GnRH (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 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. Over the past year, two studies were finished: 1) Stromal derived growth factor (SDF-1) and gamma-aminobutyric acid (GABA) are two extracellular cues that regulate neuronal migration during development and may act synergistically. The molecular mechanisms of this interaction were unclear. Both SDF-1 and GABA have been shown to regulate the rate of GnRH neuronal migration by accelerating and slowing migration, respectively. As such, this system was used to explore the mechanism by which these molecules act to produce coordinated cell movement during development. GABA and SDF-1 were shown to exert opposite effects on the speed of cell movement by activating depolarizing or hyperpolarizing signaling pathways, GABA via changes in chloride and SDF-1 via changes in potassium. GABA and SDF-1 were also found to act synergistically to promote linear rather than random movement. The simultaneous activation of these signaling pathways, therefore, results in tight control of cellular speed and improved directionality along the migratory pathway of GnRH neurons. 2) Proper assembly of neural circuits requires newly born neurons to migrate from their place of origin to their final location. Little is known about the mechanisms of axophilic neuronal migration, whereby neurons travel along axon pathways to navigate to their destinations. GnRH neurons migrate along olfactory axons from the nose into the forebrain during development, and were used as a model of axophilic migration. We investigated in mice the regulation of movement from calcium signals to cytoskeletal dynamics. Live imaging revealed robust calcium activity during axophilic migration, and calcium release through IP3 receptors was found to stimulate migration. This occurred through a signaling pathway involving the calcium sensor calcium/calmodulin protein kinase kinase (CaMKK), AMP-activated kinase (AMPK) and RhoA/ROCK. By imaging GnRH neurons expressing actin- GFP or Lifeact-RFP, calcium release was found to stimulate leading process actin flow away from the cell body. In contrast, actin contractions at the cell rear were unaffected by this calcium signaling pathway. These findings are the first to test the regulation of cytoskeletal dynamics in axophilic migration, and reveal mechanisms of movement that have broad implications for the migration of other CNS populations. New investigations are characterizing the role of Bone Morphogenic Protein-4 (Bmp4) and Fibroblast growth factor-8 (Fgf8) in defining epithelial versus neurogenic fate in the developing olfactory/vomeronasal system. Using different knock-in mouse lines and Cre-lox mediated lineage tracing, Fgf8 expression and cell lineage is being analyzed in relation to the expression of Bmp4 and its antagonist Noggin. These experiments will determine whether Fgf8 is sufficient to induce ectodermal progenitors of the olfactory pit to acquire neural fate as well as induce GnRH neuronal specification. In addition, to specifically remove molecules of interest from GnRH cells during development and examine migrational changes, Cre-lox-mice and/or pharmacological/molecular manipulations are being employed and in situ characterization of the migration of GnRH neurons done using real time microscopy. Specific studies in progress examine the role of growth factor receptors (VEGR1 and FGFR1). Lastly, we continue to study the physiology of GnRH neurons focusing on GABA and adiponectin. We are examining why GABA remains excitatory to GnRH neurons in the adult by evaluating chloride transporters and expression of GABA receptor subtypes over development. In humans and animal models, metabolic dysfunctions are often linked to reproductive abnormalities. Adiponectin, a peripheral hormone secreted by white adipose tissue, is important in energy homeostasis and appetite regulation. We found that a subpopulation of GnRH neurons express AdipoR2. Thus, we will determine whether adiponectin can directly act on GnRH neurons using whole cell patch clamping and calcium imaging. Modulation of GnRH neuronal activity by adiponectin would demonstrate a direct link between energy balance and neurons controlling reproduction.