NSF-BSF: Studying the compensatory mechanisms underlying gene loss-of-function in the nervous system

这个项目旨在了解生物体如何应对可能丢失的必要基因。动物的基本生物学过程之一是繁殖，以确保物种的延续。促性腺激素释放激素(GnRH)是大脑中的一种蛋白质，是脊椎动物生殖的调节器，在包括人类在内的许多物种中，它的功能丧失会导致不育。令人惊讶的是，GnRH基因沉默的斑马鱼仍然具有繁殖力。该项目研究了斑马鱼在没有促性腺激素释放激素的情况下如何进行繁殖，以及缺乏促性腺激素释放激素是否可能触发未知的机制，以弥补其缺失，从而维持繁殖，从而维持物种。或者，斑马鱼有可能被证明是第一个已知的不依赖促性腺激素释放激素的脊椎动物物种。因此，这项研究将研究1)促性腺激素释放激素是否对斑马鱼的繁殖是不可或缺的，2)是否以及如何传递补偿，以及3)两个潜在的补偿者在取代促性腺激素释放激素中的作用。这项研究提供了一个揭示一种关键基因的新适应的机会，这种适应可能存在于广泛的物种中，并可能导致潜在的不孕不育治疗方法。该项目将支持研究生的培训。该项目与卡内基的BioEYES Baltimore合作，为2-12年级的学生提供使用活斑马鱼的亲身生物体验，该项目将为巴尔的摩市中学的K-12教师开发以生殖为导向的培训模块，从而覆盖2万多名学生。下丘脑GnRH(GnRH3)基因敲除(KO)斑马鱼的正常繁殖可以通过其生殖能力的可有可无或激活补偿机制来解释。在体内，1)通过RNAi抑制下丘脑GnRH3，2)特异性拮抗剂阻断GnRH受体，3)激光消融GnRH3神经元所引起的生殖损伤，表明GnRH3是斑马鱼生殖的活性调节因子。GnRH3 KO斑马鱼生殖轴上的多个分子变化指向遗传的多因素补偿，排除了已知的补偿机制，如同源因子(即GnRH2)或神经递质。该提议的假设是，GnRH3是斑马鱼繁殖的主要调节因子，其基因缺失激活了沿着脑-脑垂体轴的遗传复杂补偿机制。GnRH3在生殖控制中的作用将通过病毒传递RNAi和大脑中GnRH受体的KO，特别是在促黄体生成素和促性腺激素中被敲除来确定。GnRH3神经元(相对于GnRH3多肽)在控制生殖中的作用将通过GnRH3 KO细胞系和GnRH3细胞毒素条件神经元消融来研究。在FACS分离的GnRH3神经元中，将确定模拟GnRH3神经元内GnRH3功能的可能的补偿因子，然后是转录组学和蛋白质组学。两种有希望的非GnRH3神经肽可能是GnRH3的替代候选(或联合作用)，将通过它们的基因KO和确定GnRH3肽是否通过组蛋白修饰来调节它们的表达来进行测试。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This project aims at understanding how organisms cope with a possible loss of essential genes. One of the fundamental biological processes in animals is reproduction that ensures the continuation of a species. Gonadotropin releasing-hormone (GnRH), a protein in the brain, is the governor of reproduction in vertebrates, and its loss of function in many species, including humans, leads to infertility. Surprisingly, zebrafish in which the GnRH gene has been silenced remain fertile. This project examines how reproduction proceeds in zebrafish without GnRH, as well as the possibility that the lack of GnRH triggers yet unknown mechanisms that compensate for its absence so that reproduction, and therefore the species, are maintained. Alternatively, it is possible that zebrafish will prove to be the first known GnRH-independent vertebrate species. Therefore, the study will examine 1) whether GnRH is indispensable for reproduction in zebrafish, 2) if and how a compensation is conveyed, and 3) the role of two potential compensators in replacing GnRH. This study offers an opportunity to reveal a novel adaptation for a critical gene, with the likelihood that this adaptation exists in a broad range of species, and may lead to potential therapies for infertility. The project will support the training of graduate students. Partnering with Carnegie’s BioEYES Baltimore, which provides students in grades 2–12 with hands-on biology experiences using live zebrafish, the project will develop a reproduction-oriented training module for K-12 teachers from Baltimore City middle schools, thereby reaching over 20,000 students.Normal reproduction in hypothalamic GnRH (GnRH3) knockout (KO) zebrafish can be explained by its dispensability for reproduction or by the activation of a compensatory mechanism. The effect of reproductive impairment induced by 1) focal hypothalamic GnRH3 knockdown via RNAi, in vivo, 2) GnRH receptor blocking by specific antagonists, and 3) Gnrh3 neuron laser-ablation, have indicated that GnRH3 is an active regulator of reproduction in zebrafish. Multiple molecular changes along the reproductive axis of the GnRH3 KO zebrafish, point towards an inherited multi-factorial compensation, ruling out known compensatory mechanisms such as homologous factors (i.e., GnRH2) or neurotransmitters. The hypothesis of the proposal is that GnRH3 is the principal regulator of reproduction in zebrafish and its genetic loss activates inherited complex compensatory machinery along the brain-pituitary axis. The role of GnRH3 in the control of reproduction will be determined via knockdown of Gnrh3 through viral delivery of RNAi, and KO of Gnrh receptors in the brain as well as specifically in Lh and Fsh gonadotropes. The role of GnRH3 neurons (versus GnRH3 peptide) in controlling reproduction will be studied using the GnRH3 KO line and GnRH3 conditional neuronal ablation by cytotoxins. Possible compensating factors that mimic GnRH3 function within GnRH3 neurons will be identified in FACS isolated GnRH3 neurons, followed by Transcriptomics and Proteomics. Two promising non-GnRH3 neuronal neuropeptides that may be replacement candidates for GnRH3 (or that work in conjunction) will be tested through their gene KO and by determining if GnRH3 peptide modulates their expression through histone modifications.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.