Neurosecretory Gene Expression in the Hypothalamus

项目摘要 控制哺乳动物繁殖的神经回路从视交叉上传递昼夜节律信息。 核团(SCN)通过神经肽精氨酸加压素(AVP)和血管活性肠肽(VIP)传递给 Kispeptin(Kiss)和GnRH(GnRH)神经元。不孕不育可能导致 在神经内分泌水平由GnRH神经元、Kiss神经元和/或昼夜节律起搏器和 脊髓核内的神经肽神经元。促性腺激素释放激素神经元出现在嗅觉平台，迁移到下丘脑， 并将轴突延伸至正中隆起，以响应亲吻信号将促性腺激素释放激素传递到脑下垂体。 迁移障碍、轴突寻路和/或亲吻或促性腺激素释放激素神经元分泌紊乱会导致失败 青春期、生育力和生殖功能。我们已经确定了同源结构域转录因子， Vax1、Six3和Six6都是繁殖的关键。6个基因敲除(KO)小鼠表现出显著的减少 杂合子Vax1和Six3小鼠是不育的(Vax1和Six3 KO小鼠是围产期致死的)。 尽管GnRH神经元数量在e13.5时是正常的，但所有三个KO胚胎的GnRH神经元数量都减少了约90% 下丘脑GnRH神经元E17.5。在Kiss神经元中发现了Vax1、Six3和Six6，它们调节着表达 Kiss启动子在表达Kiss的细胞系中的表达。此外，亲吻神经元中Six3的缺失会导致 不育症。此外，三个KO E17.5胚胎都缺乏SCN形态，不表达AVP和VIP， 6只KO成虫缺乏昼夜节律。本应用程序的总体目标是阐明 这种基本的生殖神经内分泌回路(SCnGnRH)在分子、发育、 和生理水平。我们提出了三个具体目标：目标1将研究分化和成熟 通过确定缺乏这些因素的神经元的命运及其对其影响来研究GnRH神经元在迁移过程中的变化 迁徙路径。目标2将重点介绍Vax1、Six3和Six6在亲吻的发育和功能中的作用 在体外和体内的神经元，重点是直接调节Kiss基因的表达和对生育的影响。目标 3将讨论SCN发展的基本机制，Vax1、Six3和Six6对 AVP和VIP基因的表达，并探讨SCN昼夜节律功能在生育中的作用。我们最重要的是 假设由Vax1、Six3和Six6缺陷引起的不孕不育是由特定基因的改变引起的 下丘脑内神经内分泌细胞群下丘脑吻促性腺激素释放激素生殖 调节发育、激素合成和昼夜节律的神经回路。我们相信这项研究 这些关键的同源结构域蛋白将揭示控制生殖功能的基本机制 和昼夜节律，并提供了对发育重要的新调控途径的有价值的见解 以及这个神经内分泌回路的功能。这一多方面的方法将产生一个全面的理解 它们在体内和体外的生育和昼夜节律中的作用，并提供了对基本的 不孕症和昼夜节律的遗传控制。

Project Summary The neural circuit that controls mammalian reproduction delivers circadian information from the suprachiasmatic nucleus (SCN) via the neuropeptides arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) to the kisspeptin (Kiss) and gonadotropin-releasing hormone (GnRH) neurons, respectively. Infertility can be caused at the neuroendocrine level by genetic defects in GnRH neurons, Kiss neurons, and/or circadian pacemaker and neuropeptide neurons in the SCN. GnRH neurons arise in the olfactory placode, migrate into the hypothalamus, and extend their axons to the median eminence to deliver GnRH to the pituitary in response to Kiss signals. Disrupted migration, neurite pathfinding, and/or disregulated secretion of Kiss or GnRH neurons result in failures of puberty, fertility, and reproductive function. We have determined that the homeodomain transcription factors, Vax1, Six3, and Six6, are all crucial for reproduction. Six6 Knock-out (KO) mice exhibit strikingly decreased fertility, while heterozygote Vax1 and Six3 mice are subfertile (Vax1 and Six3 KO mice are perinatal lethal). Though GnRH neuron numbers are normal at e13.5, all three of the KO embryos have ~90% reductions in hypothalamic GnRH neurons by e17.5. Vax1, Six3, and Six6 are found in Kiss neurons and regulate expression of the Kiss promoter in Kiss-expressing cell lines. In addition, deletion of Six3 from Kiss neurons causes subfertility. Furthermore, all three KO e17.5 embryos lack SCN morphology and fail to express AVP and VIP, and Six6 KO adults lack circadian rhythms. The overall goal of this application is to elucidate the development of this fundamental reproductive neuroendocrine circuit (SCNKissGnRH) at the molecular, developmental, and physiological levels. We propose three Specific Aims: Aim 1 will investigate differentiation and maturation of GnRH neurons during migration by determining the fate of neurons lacking these factors and effects on their migratory pathway. Aim 2 will focus on the roles of Vax1, Six3, and Six6 in the development and function of Kiss neurons in vitro and in vivo with a focus on direct regulation of Kiss gene expression and effects on fertility. Aim 3 will address the fundamental mechanisms of SCN development, the contribution of Vax1, Six3, and Six6 to AVP and VIP gene expression, and investigate the roles of SCN circadian function in fertility. Our overarching hypothesis is that infertility due to defects in Vax1, Six3, and Six6 is caused by alterations in specific hypothalamic neuroendocrine cell populations within the hypothalamic SCNKissGnRH reproductive neurocircuit by regulation of development, hormone synthesis, and circadian rhythms. We believe that studies of these key homeodomain proteins will reveal fundamental mechanisms in the control of reproductive function and circadian rhythms and provide valuable insight into novel regulatory pathways important for the development and function of this neuroendocrine circuit. This multifaceted approach will yield a comprehensive understanding of their roles in fertility and circadian rhythms in vivo and in vitro, and provide further insight into the fundamental genetic control of infertility and circadian rhythms.