Retinal Ganglion Cell Subtype Specification in Human Retinal Organoids

项目摘要 视网膜神经节细胞(RGC)是将视觉信息从眼睛传递到 大脑。视网膜节细胞的退化或死亡会导致许多致盲情况，包括青光眼。RGC可以 可分为许多亚型，每个亚型都有不同的形态、功能和基因表达谱。而当 在许多脊椎动物模式生物中发现了RGC亚型，在人类视网膜中发现了RGC亚型 还没有得到很好的分子表征。此外，RGC规范的控制机制 亚型仍然知之甚少，尤其是在人类视网膜中。在这个项目中，我将研究人类视网膜和 用于识别人类RGC亚型并确定其生成的控制机制的有机化合物。我的工作将 获得对人类RGC亚型规范的机械见解，并促进干细胞来源的使用 有机化合物在青光眼等视网膜疾病的再生疗法中的应用。 这个项目的主要目标之一是对成年人的RGC亚型进行分子分类。 视网膜。基于已发表的人类和猕猴视网膜的单细胞RNA测序数据，我 生成了一个可测试的基因表达代码来唯一识别每个RGC亚型。我会用这个基因 指导组合表达分析的表达编码(即免疫组织化学和RNA FISH)和 确定人类RGC亚型。我已经用我的基因区分了三个人类RGC亚型 表达式代码。我还将描述这些RGC亚型的形态特征。我将使用组合IHC 而亲脂染料确实在分子和形态上对人类RGC亚型进行了分类(目标1)。 研究人类RGC生物学的一个主要挑战是有限的获得遗传和 药理上可操控的人体组织。人类视网膜类器官为研究提供了一个强有力的模型 在受控条件下发育人体组织。约翰斯顿实验室推进了人类视网膜类有机物质的使用 目的：研究光感受器命运调控机制。我证明了有机化合物是一种容易驯服的 通过开发RGC轴突生长分析来研究RGC生物学的模型系统，这表明 视网膜节细胞迅速延伸轴突，重现它们在体内的能力。此外，我还确定了两个子类型 有机体内的RGC。为了确定人类器官中RGC亚型的谱系，我将评估表达 基于可测试的基因表达编码，并通过在 类器官发育的时间进程(目标2)。转录因子Eome/TBR2、TBR1和TBX5具有 在RGC亚型规范中有牵连。为了研究指定人类RGC亚型的机制，我 将利用CRISPR突变敲除和病毒转染法异位表达这三种 转录因子，并检测人类视网膜器质中RGC亚型群体的变化(目标3)。

Project Summary Retinal ganglion cells (RGCs) are the interneurons that transmit visual information from the eye to the brain. Degeneration or death of RGCs results in a number of blinding conditions, including glaucoma. RGCs can be classified into many subtypes, each with distinct morphologies, functions, and gene expression profiles. While RGC subtypes have been identified in many vertebrate model organisms, RGC subtypes in the human retina have not been well-characterized molecularly. Moreover, the mechanisms controlling the specification of RGC subtypes remain poorly understood, especially in the human retina. In this project, I will study human retinas and organoids to identify human RGC subtypes and determine mechanisms controlling their generation. My work will yield mechanistic insights into human RGC subtype specification and facilitate the use of stem cell-derived organoids in regenerative therapies for retinopathies like glaucoma. One of the major goals of this project is to molecularly classify the RGC subtypes of the adult human retina. Based on published single cell RNA sequencing (scRNA-seq) data in human and macaque retinas, I generated a testable gene expression code to uniquely identify each RGC subtype. I will use this gene expression code to guide combinatorial expression analysis (i.e. immunohistochemistry and RNA FISH) and identify human RGC subtypes. I have already distinguished three human RGC subtypes using my gene expression code. I will also characterize the morphologies of these RGC subtypes. I will use combinatorial IHC and the lipophilic dye DiD to molecularly and morphologically classify human RGC subtypes (Aim 1). A major challenge to studying human RGC biology is the limited access to genetically and pharmacologically manipulatable human tissue. Human retinal organoids provide a powerful model to study developing human tissue in controlled conditions. The Johnston lab advanced the use of human retinal organoids to study the mechanisms controlling photoreceptor fate specification. I showed that organoids are a tractable model system to study RGC biology by developing an RGC axon outgrowth assay, which demonstrated that RGCs rapidly extend axons, recapitulating their in vivo capabilities. Furthermore, I identified two subtypes of RGCs in organoids. To identify the repertoire of RGC subtypes in human organoids, I will assess expression based on the testable gene expression code and complement this approach by conducting scRNA-seq over a time course of organoid development (Aim 2). The transcription factors EOMES/TBR2, TBR1, and TBX5 have been implicated in RGC subtype specification. To investigate mechanisms that specify human RGC subtypes, I will utilize CRISPR mutagenesis to knock out and viral transfection to ectopically express these three transcription factors and examine changes in RGC subtype populations in human retinal organoids (Aim 3).