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

该项目旨在了解生物体如何应对必要基因的可能损失。动物的基本生物过程之一是繁殖，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，是脊椎动物繁殖的管理者，在包括人类在内的许多物种中，GnRH功能的丧失会导致不孕。令人惊讶的是，GnRH基因被抑制的斑马鱼仍然可以生育。该项目研究了没有GnRH的斑马鱼的繁殖过程，以及缺乏GnRH触发未知机制的可能性，这种机制补偿了GnRH的缺失，从而维持了繁殖，从而维持了物种。另一种可能是，斑马鱼将被证明是已知的第一个不依赖gnrh的脊椎动物物种。因此，本研究将研究1)GnRH是否对斑马鱼的生殖是必不可少的，2)补偿是否以及如何传递，以及3)两种潜在的补偿体在替代GnRH中的作用。这项研究为揭示一个关键基因的新适应提供了一个机会，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症治疗。该项目将支持研究生的培养。该项目与卡内基的巴尔的摩生物之眼（BioEYES Baltimore）合作，为2-12年级的学生提供使用活斑马鱼的动手生物学体验，该项目将为巴尔的摩市中学的K-12教师开发一个以生殖为导向的培训模块，从而惠及2万多名学生。下丘脑GnRH （GnRH3）敲除（KO）斑马鱼的正常生殖可以通过其对生殖的可有可无或通过激活补偿机制来解释。1)下丘脑局灶性GnRH3基因在体内被RNAi敲低，2)GnRH受体被特异性拮抗剂阻断，3)GnRH3神经元激光消融对生殖损伤的影响表明，GnRH3是斑马鱼生殖的积极调节因子。沿着GnRH3 KO斑马鱼生殖轴的多个分子变化，指向遗传的多因子补偿，排除了已知的补偿机制，如同源因子（即GnRH2）或神经递质。该提案的假设是GnRH3是斑马鱼繁殖的主要调节因子，其遗传丢失激活了沿脑-垂体轴的遗传复杂补偿机制。GnRH3在控制生殖中的作用将通过病毒递送RNAi来敲低GnRH3，以及大脑中Gnrh受体的KO，特别是Lh和Fsh促性腺激素。GnRH3神经元（相对于GnRH3肽）在控制生殖中的作用将通过GnRH3 KO细胞系和细胞毒素对GnRH3条件性神经元消融进行研究。将在FACS分离的GnRH3神经元中鉴定可能模仿GnRH3功能的补偿因子，然后进行转录组学和蛋白质组学研究。两种有前途的非GnRH3神经肽可能是GnRH3的替代候选（或协同工作），将通过它们的基因KO和确定GnRH3肽是否通过组蛋白修饰调节它们的表达来进行测试。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.