Neurosecretory Gene Expression in the Hypothalamus

项目摘要 控制哺乳动物生殖的神经回路从视交叉上神经传递昼夜节律信息。 通过神经肽精氨酸加压素（AVP）和血管活性肠肽（VIP）， kisspeptin（Kiss）和促性腺激素释放激素（GnRH）神经元。不孕症可能是由 在神经内分泌水平，GnRH神经元、Kiss神经元和/或昼夜节律起搏器的遗传缺陷， SCN中的神经肽神经元。GnRH神经元出现在嗅基板，迁移到下丘脑， 并将它们的轴突延伸到正中隆起，以响应Kiss信号将GnRH输送到垂体。 Kiss或GnRH神经元的迁移、轴突寻路和/或分泌失调导致失败 青春期，生育能力和生殖功能。我们已经确定同源结构域转录因子， Vax 1、Six 3和Six 6对生殖都至关重要。Six 6敲除（KO）小鼠表现出显著降低的 Vax 1和Six 3基因敲除小鼠的生育力低下，而杂合子Vax 1和Six 3基因敲除小鼠的生育力低下（Vax 1和Six 3基因敲除小鼠是围产期致死的）。 虽然GnRH神经元数量在e13.5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%。 e17.5下丘脑GnRH神经元。Vax 1、Six 3和Six 6在Kiss神经元中发现并调节表达 在表达Kiss的细胞系中的Kiss启动子。此外，从Kiss神经元中删除Six 3会导致 生育力低下此外，所有三个KO e17.5胚胎都缺乏SCN形态并且不能表达AVP和VIP， 而六个六KO成年人缺乏昼夜节律。本申请的总体目标是阐明 这种基本的生殖神经内分泌回路（SCN-Kiss-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子的神经元的命运及其对它们的影响， 迁移途径目标2将重点关注Vax 1，Six 3和Six 6在Kiss的发展和功能中的作用 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力的影响。目的 3将讨论SCN发展的基本机制，Vax 1，Six 3和Six 6对 AVP和VIP基因的表达，探讨SCN的昼夜节律功能在生育中的作用。我们的总体 有一种假说认为，由于Vax 1、Six 3和Six 6缺陷导致的不育是由特定基因的改变引起的， 下丘脑SCN内的下丘脑神经内分泌细胞群 通过调节发育、激素合成和昼夜节律来调节神经回路。我们相信研究 这些关键同源结构域蛋白的研究将揭示生殖功能控制的基本机制 和昼夜节律，并提供有价值的见解，新的调控途径，重要的发展 和神经内分泌回路的功能。这种多方面的方法将产生一种全面的理解 它们在体内和体外的生育力和昼夜节律中的作用，并提供了对基本的 不育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.