Homeodomain Proteins Six3 and Six6 in GnRH Neuron Development in Mice

项目摘要 促性腺激素释放激素（GnRH）是脊椎动物生殖的主要和必需的调节因子。的 十肽GnRH由GnRH神经元释放并分泌到垂体，信号在垂体中传播 通过下丘脑-垂体-性腺轴调节生殖。GnRH神经元起源于 嗅基板，通过嗅球迁移到下丘脑，并将轴突投射到正中神经 释放GnRH的隆起。当GnRH神经元受损时，GnRH表达减弱， 产生特发性低促性腺激素性腺功能减退症（IHH）。IHH的特点是不育 和/或青春期延迟或缺失。尽管这显然是哺乳动物繁殖的关键因素， 对GnRH神经元个体发育的遗传调节因子知之甚少。通过这个研究项目，我计划 阐明控制GnRH神经元迁移和成熟的发育和遗传机制， 从而阐明了IHH的新机制。通过本F31建议的工作， 应用，将确定新的遗传靶点，可以提供治疗GnRH的新疗法 缺陷我建议研究密切相关的同源结构域转录因子，sine oculis相关 同源框3（Six 3）和Six 6，以确定其在GnRH缺乏症中的作用。我的初步数据显示 Six 3和Six 6在GnRH神经元发育、存活和迁移中重要性。我提出三个目标， 将辨别这些基因在调节GnRH神经元中的作用。第一个目标将涉及以下方面的作用： Six 3基因在GnRH神经元的发育、存活和分化中的作用，而Aim 2将专注于 GnRH神经元特异性作用Six 6.目标3将研究受体、配体和细胞间通讯基因， 是负责GnRH神经元存活沿着迁移路线从嗅基板到 下丘脑为了实现这些目标，我将利用GnRH神经元特异性敲除小鼠（Six 6 Flox/ GnRHCre）和全身敲除小鼠（Six 3 Het）研究细胞自主和系统性影响 Six 3和Six 6对GnRH神经元发育、存活和迁移的影响。具体分析将涉及详细 使用GnRH神经元染色、凋亡的免疫组织化学评估GnRH神经元存活 评估和血统追踪。我还将确定Six 3和Six 6对生育率的贡献， Six 6 Flox/GnRHCre和Six 3 Het小鼠。除了这些在体小鼠模型，我将探讨的贡献， Six 3和Six 6对几种GnRH神经元细胞系的作用。在这些体外模型中，我将使用Six 3的siRNA， Six 6来详细说明这些基因在GnRH神经元细胞存活、迁移和表达中的重要性。 描述GnRH神经元迁移途径的已知生存线索。这些调查将使 让我获得有关这种迷人的激素释放的新调节机制的知识; 并且，这将使我们能够阐明占所有夫妇约7%的不孕症的原因。

PROJECT SUMMARY Gonadotropin-releasing hormone (GnRH) is a primary and essential regulator of vertebrate reproduction. The decapeptide GnRH is released by GnRH neurons and secreted to the pituitary where the signal is propagated throughout the hypothalamic-pituitary-gonadal axis to regulate reproduction. GnRH neurons originate in the olfactory placode, migrate through the olfactory bulb to the hypothalamus, and project axons into the median eminence where they release GnRH. When GnRH neurons are compromised, GnRH expression is attenuated, producing the condition Idiopathic Hypogonadotropic Hypogonadism (IHH). IHH is characterized by infertility and/or delayed or absent puberty. Although clearly a crucial factor in the reproduction of mammalian species, little is known about the genetic regulators of GnRH neuronal ontogeny. With this research project, I plan to elucidate developmental and genetic mechanisms that control the migration and maturation of GnRH neurons, and therefore illuminate the novel mechanisms responsible for IHH. Through the work proposed under this F31 application, new genetic targets will be identified that could provide novel therapies for treating GnRH deficiency. I propose to study the closely related homeodomain transcription factors, sine oculis-related homeobox 3 (Six3) and Six6, to determine their role in GnRH deficiency. My preliminary data show a profound importance of Six3 and Six6 in GnRH neuron development, survival, and migration. I am proposing 3 aims that will discern the roles these genes play in the regulation of GnRH neurons. The first aim will address the role of the Six3 gene in the development, survival, and differentiation of GnRH neurons, while Aim 2 will focus on the GnRH neuron-specific role of Six6. Aim 3 will study receptors, ligands, and cell-cell communication genes that are responsible for GnRH neuron survival along the migratory route from the olfactory placode to the hypothalamus. To execute these aims, I will utilize both GnRH neuron-specific knock-out mice (Six6Flox/ GnRHCre), and full-body knock-out mice (Six3Het) to investigate the cell autonomous and systemic implication of Six3 and Six6 on GnRH neuron development, survival, and migration. Specific analysis will involve detailed assessment of GnRH neuron survival using immunohistochemistry for GnRH neuron staining, apoptotic assessment, and lineage tracing. I will also determine the contribution of Six3 and Six6 to fertility in the Six6Flox/GnRHCre and Six3Het mice. In addition to these in vivo mouse models, I will explore the contribution of Six3 and Six6 to in several GnRH neuron cell lines. In these in vitro models I will use siRNA for Six3 and Six6 to detail the importance of these genes in GnRH neuron cell survival, migration, and in the expression of known survival cues that delineate the migratory pathway of GnRH neurons. These investigations will enable me to garner knowledge concerning the novel regulatory mechanisms of this fascinating hormone's release; and, will allow us to shed light on causes of infertility plaguing ~7% of all couples.