Establishing the roles of oestrogen receptor 1 (ESR1) in olfactory development and function using novel CRISPR/Cas9-based knockouts in the zebrafish

使用基于 CRISPR/Cas9 的新型斑马鱼基因敲除技术确定雌激素受体 1 (ESR1) 在嗅觉发育和功能中的作用

• 批准号: BB/Y00003X/1
• 负责人: Charles Tyler
• 金额: $ 72.31万
• 依托单位: UNIVERSITY OF EXETER
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FY00003X%2F1
• 关键词: Establishing; roles; oestrogen; receptor; ESR1

In all animals, the sense of smell (olfaction) is fundamental for sensing the outside world with roles including for feeding, avoiding predators, social interactions, and reproduction. Much of the information for these smell associated behaviours is imprinted during early-life and in humans smell dysfunction is an early indicator of various behavioural disorders, including autism. The ways (mechanisms) through which smell develops in early life to influence subsequent animal behaviours, however, are largely unknown. Recently, we discovered that oestrogens (which are steroid hormones) regulate olfactory development in the embryo brain via a novel cell type which we have named oestrogen responsive olfactory bulb (EROB). In this project we will apply highly novel ways (so called CRISPR-Cas9 methods), developed by our industry partner (AstraZeneca), to remove (knock out) the key oestrogen receptor (called esr 1) in a highly controlled cell-specific and precisely-timed manner. This will help us to identify the role of esr1 in the development of smell and smell-mediated behaviour. The CRISPR-Cas9 methods we will develop will also allow other researchers to study other genes with much greater precision in the zebrafish model. In this work we will first knock out esr1 in zebrafish in specific brain cells (called glia, which include EROB) in a highly controlled and timed manner to provide the required zebrafish study models. We will then use these zebrafish models to establish what happens to the anatomy of the brain and the neural circuits in a key region of the brain involved in smell (the olfactory bulb) when esr-1 is knocked out. We will do this by analysing brain sections and measuring the different brain cell types, their structural arrangements and the neural circuits they form. We will then cross breed our esr1 knock out zebrafish with another genetically modified zebrafish in which brain neural activity can be visualised via imaging. With this new zebrafish model we will assess the effects of the glial-specific knock out of esr-1 during embryo development on brain activity in response to selected smells using imaging, and in subsequent juveniles and adults through studies on sections of the brain. Finally, we will use behavioural assessments to determine the consequences of knocking out esr1 in EROB cells on smell-mediated behaviours in larval stages, and on social-interaction in both larval and adult animals. We provide significant pilot data supporting our approach that includes showing that esr1 specifically affects the number of EROB cells during development. As a major step in creating a brain cell- specific conditional esr 1 knock out we have also already incorporated key genetic elements into a zebrafish line to facilitate this. Furthermore, we have established an imaging system which allows us to image neural activity in the whole brain, in real time.Our research will be of significant interest to a diverse audience including academic and industry researchers, and the medical profession, by providing new models to study smell and the roles of oestrogens in brain development and function. Our project will advance genomic editing tools for the research community relevant to anyone studying genes and their function in the zebrafish model. It will also be of great interest to industry and government regulatory bodies, as the models developed, for example, could be applied for advancing the risk assessment of chemicals with oestrogenic activity, supporting evidence-based decision-making for those chemicals. The wider public will benefit also from this research from improved understanding of basic life processes associated with smell, a sense fundamental to animal (including human) life.

在所有动物中，嗅觉（嗅觉）对于以喂食，避免捕食者，社交互动和繁殖的角色感知外界都是基本的。这些相关行为的许多信息在早期生活中都印在了，并且在人类中，气味功能障碍是包括自闭症在内的各种行为障碍的早期指标。然而，气味在早期生命中发展以影响后续动物行为的方式（机制）在很大程度上是未知的。最近，我们发现雌激素（类固醇激素）通过一种新型细胞类型调节胚胎大脑中的嗅觉发育，我们将其命名为雌激素反应式嗅球（EROB）。在这个项目中，我们将采用高度新颖的方法（所谓的CRISPR-CAS9方法），该方法是由我们的行业伙伴（阿斯特拉赛）开发的，以高度控制的细胞特异性且精确的方式去除（敲除）关键的雌激素受体（称为ESR 1）。这将有助于我们确定ESR1在气味和气味介导的行为发展中的作用。我们将开发的CRISPR-CAS9方法还将允许其他研究人员在斑马鱼模型中研究其他基因。在这项工作中，我们将以高度控制和定时的方式在特定的脑细胞中（称为Glia，包括EROB）中的斑马鱼中淘汰ESR1，以提供所需的斑马鱼研究模型。然后，我们将使用这些斑马鱼模型来确定大脑解剖结构的发生，以及当ESR-1被淘汰时，涉及气味的大脑关键区域中的神经回路会发生什么。我们将通过分析脑部切片并测量不同的脑细胞类型，它们的结构排列以及它们形成的神经回路来做到这一点。然后，我们将与另一个转基因斑马鱼淘汰斑马鱼的ESR1将斑马鱼淘汰，其中可以通过成像可视化脑神经活动。通过这种新的斑马鱼模型，我们将通过对胚胎发育过程中的胶质特异性敲除ESR-1对脑活动的影响，以响应使用成像的选定气味，以及随后的少年和成年人，通过对大脑部分的研究。最后，我们将使用行为评估来确定在幼体阶段敲除EROB细胞中敲出ESR1的后果，以及在幼虫和成年动物中的社交交流中。我们提供了支持我们方法的重要试验数据，其中包括表明ESR1在开发过程中特异性影响EROB细胞的数量。作为创建脑细胞特异性有条件ESR 1的主要步骤，我们还已经将关键的遗传元素纳入斑马鱼线以促进这一点。此外，我们已经建立了一个成像系统，该系统使我们能够实时对整个大脑的神经活动进行想象。我们的研究将通过提供新的模型来研究气味和Oestrogens在大脑发育和功能中的角色，包括学术和行业研究人员和医学界，包括学术和行业研究人员和医学界的重要兴趣。我们的项目将促进与研究基因及其在斑马鱼模型中的功能相关的研究社区的基因组编辑工具。例如，由于开发的模型可以用于推进具有生气活动的化学物质的风险评估，这也将引起行业和政府监管机构的极大兴趣，从而支持这些化学物质的基于证据的决策。更广泛的公众也将从这项研究中受益于对与气味相关的基本生命过程的了解，这是对动物（包括人类）生命基础的感觉。