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）是感知外部世界的基础，其作用包括进食、躲避捕食者、社交互动和繁殖。这些与气味相关的行为的大部分信息是在生命早期留下的痕迹，而在人类中，气味功能障碍是各种行为障碍（包括自闭症）的早期指标。然而，嗅觉在生命早期发展并影响随后的动物行为的方式（机制）在很大程度上尚不清楚。最近，我们发现雌激素（类固醇激素）通过一种新的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性嗅球（EROB）。在这个项目中，我们将应用由我们的行业合作伙伴（阿斯利康）开发的高度新颖的方法（所谓的 CRISPR-Cas9 方法），以高度受控的细胞特异性和精确定时的方式。这将帮助我们确定 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 方法也将使其他研究人员能够在斑马鱼模型中更精确地研究其他基因。在这项工作中，我们将首先以高度控制和定时的方式敲除斑马鱼特定脑细胞（称为神经胶质细胞，其中包括 EROB）中的 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当 esr-1 被敲除时，大脑的解剖结构以及大脑中与气味有关的关键区域（嗅球）的神经回路会发生什么变化。我们将通过分析大脑切片并测量不同的脑细胞类型、它们的结构排列和它们形成的神经回路来做到这一点。然后，我们将敲除 esr1 的斑马鱼与另一种转基因斑马鱼进行杂交，其中大脑神经活动可以通过成像可视化。借助这个新的斑马鱼模型，我们将通过成像评估胚胎发育过程中神经胶质特异性敲除 esr-1 对大脑响应选定气味的活动的影响，并通过对大脑切片的研究来评估随后的幼年和成年斑马鱼的影响。最后，我们将使用行为评估来确定敲除 EROB 细胞中的 esr1 对幼虫阶段嗅觉介导的行为以及幼虫和成年动物的社交互动的影响。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 在发育过程中特别影响 EROB 细胞的数量。作为创建脑细胞特异性条件 esr 1 敲除的一个重要步骤，我们也已经将关键遗传元件纳入斑马鱼系中以促进这一过程。此外，我们还建立了一个成像系统，使我们能够实时成像整个大脑的神经活动。通过提供新模型，我们的研究将引起包括学术和行业研究人员以及医学界在内的不同受众的极大兴趣研究气味以及雌激素在大脑发育和功能中的作用。我们的项目将为与研究斑马鱼模型中的基因及其功能的任何人相关的研究社区推进基因组编辑工具。行业和政府监管机构也对此非常感兴趣，因为所开发的模型可用于推进具有雌激素活性的化学品的风险评估，支持对这些化学品的循证决策。更广泛的公众也将从这项研究中受益，因为他们对与嗅觉相关的基本生命过程有了更好的理解，嗅觉是动物（包括人类）生命的基本感觉。