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科普可能失去的必要基因。动物的基本生物学过程之一是确保物种延续的繁殖。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控者，它在许多物种（包括人类）中的功能丧失导致不育。令人惊讶的是，GnRH基因沉默的斑马鱼仍然具有生育能力。该项目研究了没有GnRH的斑马鱼如何繁殖，以及缺乏GnRH触发未知机制的可能性，这些机制可以补偿其缺乏，从而维持繁殖，从而维持物种。另一方面，斑马鱼有可能被证明是第一个已知的不依赖GnRH的脊椎动物物种。因此，该研究将研究1）GnRH是否是斑马鱼繁殖所必需的，2）是否以及如何传达补偿，以及3）两种潜在补偿剂在替代GnRH中的作用。这项研究提供了一个机会，揭示了一个关键基因的新适应，这种适应可能存在于广泛的物种中，并可能导致不孕症的潜在疗法。该项目将支持研究生的培训。该项目与卡内基的BioEYES巴尔的摩合作，为2-12年级的学生提供使用活斑马鱼的实践生物学经验，该项目将为来自巴尔的摩市中学的K-12教师开发一个以生殖为导向的培训模块，从而达到20多个，下丘脑促性腺激素释放激素（GnRH 3）基因敲除（KO）的正常生殖斑马鱼可以解释为它的生殖或补偿机制的激活。通过1）在体内通过RNAi局部下丘脑GnRH 3敲低，2）通过特异性拮抗剂阻断GnRH受体，和3）GnRH 3神经元激光消融诱导的生殖损害的效果表明，GnRH 3是斑马鱼生殖的主动调节剂。沿着GnRH 3 KO斑马鱼的生殖轴的多个分子变化沿着指向遗传的多因子补偿，排除了已知的补偿机制，例如同源因子（即，GnRH 2）或神经递质。该提案的假设是，GnRH 3是斑马鱼繁殖的主要调节因子，其遗传缺失激活了沿脑垂体轴的遗传复杂补偿机制沿着。GnRH 3在控制生殖中的作用将通过经由RNAi的病毒递送的Gnrh 3的敲低和脑中以及特别是Lh和FSH促性腺激素中Gnrh受体的KO来确定。将使用GnRH 3 KO系和细胞毒素促性腺激素释放激素3条件性神经元消融研究GnRH 3神经元（与GnRH 3肽相比）在控制生殖中的作用。模拟GnRH 3神经元内GnRH 3功能的可能补偿因子将在FACS分离的GnRH 3神经元中鉴定，随后进行转录组学和蛋白质组学。两个有前途的非GnRH 3神经元神经肽，可能是替代候选人的GnRH 3（或协同工作）将通过他们的基因KO和通过确定是否GnRH 3肽调节其表达通过组蛋白modifications.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.