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

在所有动物中，嗅觉(嗅觉)是感知外部世界的基础，其作用包括取食、躲避捕食者、社会互动和繁殖。这些气味相关行为的大部分信息都是在生命早期印记的，在人类中，嗅觉障碍是包括自闭症在内的各种行为障碍的早期指标。然而，气味在早期生命中产生影响随后动物行为的方式(机制)在很大程度上是未知的。最近，我们发现雌激素(即类固醇激素)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的时间方式移除(敲除)关键的雌激素受体(称为ESR1)。这将有助于我们确定ESR1在嗅觉和嗅觉中介行为发展中的作用。我们将开发的CRISPR-Cas9方法也将允许其他研究人员在斑马鱼模型中以更高的精度研究其他基因。在这项工作中，我们将首先在斑马鱼特定的脑细胞(称为胶质细胞，包括EROB)中以高度受控和定时的方式敲除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，大脑的解剖结构和大脑中与嗅觉有关的关键区域(嗅球)中的神经回路会发生什么。我们将通过分析大脑切片并测量不同的脑细胞类型、它们的结构安排和它们形成的神经回路来做到这一点。然后，我们将把我们的ESR1基因敲除的斑马鱼与另一种转基因斑马鱼杂交，在这种斑马鱼中，大脑神经活动可以通过成像来可视化。利用这个新的斑马鱼模型，我们将使用成像技术评估胚胎发育期间胶质细胞特异性ESR-1敲除对大脑活动的影响，并通过对大脑切片的研究来评估随后的青少年和成年斑马鱼对大脑活动的影响。最后，我们将使用行为评估来确定在EROB细胞中敲除ESR1对幼虫阶段的嗅觉调节行为以及对幼虫和成年动物的社会互动的影响。我们提供了重要的试验性数据来支持我们的方法，包括显示ESR1在发育过程中特别影响EROB细胞的数量。作为创造脑细胞特异性条件性ESR1基因敲除的重要一步，我们还已经将关键的遗传元素整合到斑马鱼品系中，以促进这一点。此外，我们还建立了一个成像系统，使我们能够实时地对整个大脑的神经活动进行成像。我们的研究将通过提供新的模型来研究气味和雌激素在大脑发育和功能中的作用，从而引起包括学术和行业研究人员在内的不同受众的极大兴趣。我们的项目将为研究界提供先进的基因组编辑工具，与任何研究斑马鱼模型中的基因及其功能的人相关。这也将引起行业和政府监管机构的极大兴趣，因为例如，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，因为人们更好地理解了与气味相关的基本生命过程，气味是动物(包括人类)生命的基本感觉。

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.