Embryonic regulation of GnRH neuron migration and function

描述(申请人提供)：中枢神经系统(CNS)控制生殖是由分泌促性腺激素释放激素(GnRH)的一群神经内分泌细胞介导的。这些促性腺激素释放激素神经元起源于中枢神经系统外，在胚胎发育过程中迁移到腹侧端脑和下丘脑。促性腺激素释放激素神经元的不正确迁移是某些形式的生殖疾病的基础，即卡尔曼综合征。然而，胚胎调控GnRH神经元迁移和功能的机制却知之甚少。这在一定程度上是因为GnRH神经元数量很少，在大多数脊椎动物胚胎或完整的成年大脑中无法接触到。为了克服这些障碍，我们利用斑马鱼的几个实验特征来识别胚胎迁移过程中的GnRH神经元，并监测完整神经网络中的电生理活动。本研究的目的是阐明促性腺激素释放激素神经元迁移和功能的体内机制。在转基因斑马鱼胚胎中，GnRH神经元在出生后不久就表达绿色荧光蛋白，并增加胚胎迁移过程中自发动作电位的放电率。我们假设GnRH神经元电活动增加是体内调节迁移速度、路线或目的地的机制。我们将：1)确定迁移的GnRH神经元何时何地获得神经生理活动，2)确定正常的GnRH神经生理学是否需要刻板迁移，以及3)确定神经元活动是否调节GnRH神经元的胚胎迁移。这项建议中概述的实验结合了转基因斑马鱼、最先进的显微镜和单细胞电生理学，以监测体内GnRH神经元的迁移和功能。为了确定GnRH神经元迁移和神经元活动之间的功能关系，我们将通过操纵特定Kallmann基因的mRNA表达来阻断GnRH神经元迁移。相反，我们将使用GnRH神经元特异性转基因只在GnRH神经元中过度表达人类内向整流钾通道，并在迁移过程中沉默电活动。总之，这些分析测试了GnRH神经元迁移和发育获得性电活动之间的功能关系。此外，该系统允许使用GnRH神经元迁移的体内模型对人类基因功能(野生型或突变型)进行细胞自主测试。我们开发了一个强大的模型系统，并提出了一种创新的实验方法，以促进我们对GnRH神经元在体内迁移的理解。进一步了解引导GnRH神经元迁移的机制对于确定出生缺陷的病因和预测有关生殖成熟和生育的临床结果至关重要。斑马鱼的这些结果是对正在进行的临床研究的补充，目的是确定(候选)基因的功能，这些基因是人类生殖疾病中GnRH神经元异常迁移的分子病因学基础。 公共卫生相关声明：几种形式的人类生殖疾病是由分泌促性腺激素释放激素(GnRH)的专门神经内分泌细胞的不适当迁移或功能引起的。这项建议的目的是通过利用转基因斑马鱼的实验特征来确定胚胎GnRH神经元迁移的分子机制和神经生理学。进一步了解引导GnRH神经元迁移的机制对于确定出生缺陷的病因和预测人类生殖成熟和生育的临床结果至关重要。

DESCRIPTION (provided by applicant): Central nervous system (CNS) control of reproduction is mediated by a population of neuroendocrine cells that secrete gonadotropin-releasing hormone (GnRH). These GnRH neurons originate outside the CNS and migrate into the ventral telencephalon and hypothalamus during embryogenesis. Incorrect migration of GnRH neurons underlies some forms of reproductive disease, namely Kallmann Syndrome. However, the embryonic mechanisms regulating GnRH neuron migration and function are poorly understood. In part, this is because GnRH neurons are few in number and inaccessible in most vertebrate embryos or the intact adult brain. To overcome these barriers, we exploit several experimental features of zebrafish to identify GnRH neurons during embryonic migration and monitor electrophysiological activity in an intact neural network. The goal of this proposal is to elucidate the in vivo mechanisms of GnRH neuron migration and function. In transgenic zebrafish embryos, GnRH neurons express green fluorescent protein soon after birth and increase the rate of spontaneous action potential firing during embryonic migration. We hypothesize that increased electrical activity in GnRH neurons is an in vivo mechanism to regulate migratory rate, route, or destination. We will: 1) Determine when and where migratory GnRH neurons acquire neurophysiological activity, 2) Determine whether stereotyped migration is required for normal GnRH neurophysiology, and 3) Determine whether neuronal activity regulates embryonic migration of GnRH neurons. Experiments outlined in this proposal combine transgenic zebrafish, state-of-the-art microscopy, and single cell electrophysiology to monitor GnRH neuron migration and function in vivo. To determine the functional relationship between GnRH neuron migration and neuronal activity, we will disrupt GnRH neuron migration by manipulating mRNA expression of specific Kallmann genes. Conversely, we will use a GnRH neuron-specific transgene to overexpress a human inward rectifying potassium channel only in GnRH neurons and silence electrical activity during migration. Together, these analyses test the functional relationship between GnRH neuron migration and developmentally acquired electrical activity. Moreover, this system permits cell autonomous tests of human gene function (wild-type or mutant) using an in vivo model of GnRH neuron migration. We have developed a powerful model system and propose an innovative experimental approach to advance our understanding of GnRH neuron migration in vivo. A further understanding of the mechanisms guiding GnRH neuron migration is essential to determine the etiology of birth defects and predict clinical outcomes regarding reproductive maturation and fertility. These results in zebrafish complement ongoing clinical research efforts aimed toward identifying the function of (candidate) genes that underlie the molecular etiology of abnormal GnRH neuron migration in human reproductive disease. Public Health Relevance Statement: Several forms of human reproductive disease are caused by the inappropriate migration or function of specialized neuroendocrine cells that secrete gonadotropin-releasing hormone (GnRH). The goal of this proposal is to identify the molecular mechanisms and neurophysiology of embryonic GnRH neuron migration by exploiting the experimental features of transgenic zebrafish. A further understanding of the mechanisms guiding GnRH neuron migration is essential to determine the etiology of birth defects and predict clinical outcomes regarding reproductive maturation and fertility in humans.