Homeodomain Proteins Six3 and Six6 in GnRH Neuron Development in Mice

项目总结 促性腺激素释放激素(GnRH)是脊椎动物生殖的重要调节因子。这个 十肽GnRH由GnRH神经元释放，并分泌到脑垂体，在那里信号传播 通过下丘脑-垂体-性腺轴来调节生殖。促性腺激素释放激素神经元起源于 嗅觉定位，通过嗅球迁移到下丘脑，并将轴突投射到正中 他们释放GnRH的地方。当GnRH神经元受损时，GnRH的表达会减弱， 产生条件特发性促性腺激素减退(IHH)。IHH的特点是不育 和/或推迟或缺席青春期。虽然显然是哺乳动物物种繁衍的关键因素， 对GnRH神经元个体发育的遗传调控知之甚少。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项下提议的工作 应用，将确定新的基因靶点，可能为治疗GnRH提供新的治疗方法 缺乏症。我建议研究密切相关的同源结构域转录因子，Sine Oculis相关 同源异型盒3(Six3)和Six6，以确定它们在GnRH缺乏中的作用。我的初步数据显示， Six3和Six6在促性腺激素释放激素神经元发育、存活和迁移中的重要性。我提出了三个目标， 将辨别这些基因在调节GnRH神经元中所起的作用。第一个目标将解决的作用是 Six3基因在促性腺激素释放激素神经元的发育、存活和分化中的作用，而Aim 2将专注于 GnRH神经元特异性作用的Six6。AIM 3将研究受体、配体和细胞间通讯基因 负责GnRH神经元在从嗅觉平台到嗅核的迁移路线上的存活 下丘脑。为了实现这些目标，我将利用两个GnRH神经元特异性敲除小鼠(Six6Flox/ GnRHCre)和全身基因敲除小鼠(Six3Het)，以研究细胞自主和系统意义 Six3和Six6对GnRH神经元发育、存活和迁移的影响。具体分析将涉及详细的 免疫组织化学检测促性腺激素释放激素神经元的存活 评估和血统追踪。我还将确定Six3和Six6对年生育率的贡献 Six6Flox/GnRHCre和Six3Het小鼠。除了这些活体小鼠模型，我还将探索其贡献 Six3和Six6在几个GnRH神经元细胞系中的表达。在这些体外模型中，我将使用Six3和Six3的siRNA 详细说明这些基因在促性腺激素释放激素神经元细胞存活、迁移和表达中的重要性。 描述促性腺激素释放激素神经元迁移途径的已知生存线索。这些调查将使 我想获得关于这种迷人的荷尔蒙释放的新的调节机制的知识； 这将使我们能够阐明困扰约7%夫妇的不孕不育原因。

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.