Cone subtype specification in human retinas and organoids

项目概述：三种视锥细胞亚型的规格使高敏锐度的白天和 人类的三色视觉在终末分化过程中，视锥细胞表达视蛋白，视蛋白定义视锥细胞的分化。 作为蓝光（S-视蛋白）、绿色（M-视蛋白）或红光（L-视蛋白）检测器的功能。三种锥细胞亚型 在人体内产生的能量仍然是未知的。阐明这些机制将是至关重要的 了解和治疗视力障碍，包括黄斑变性、视锥细胞营养不良、视网膜炎 色素沉着症、中央凹发育不全和色盲。本项目的目标是确定 控制人视网膜中S、M和L视锥的规格。 锥细胞亚型规范分两步进行。首先，在S和L/M之间进行决策 命运锥如果选择L/M结局，则在L和M视锥结局之间进行后续选择。研究这些 过程中，我们遗传和人工操纵人类视网膜类器官，以测试机制， 视网膜发育我们发现，甲状腺激素（TH）调节基因的动态表达， 视网膜确保低TH信号早期指定S锥和高TH信号晚期产生L/M锥。我们 一项工作建立了人类视网膜类器官作为确定人类发育机制的模型， 有希望用于治疗和视力修复（Eldred等人，2018年，科学）。 我们发现，S，M，和L锥产生的时间进展过程中的发展。如何 时间和空间输入被整合在人视网膜中以产生视锥图案的方法知之甚少。 我们开发了解剖、成像和细胞鉴定方法来评估锥状细胞的空间分布。 亚型，并生成完整人类视网膜中锥细胞亚型的第一张图（目的1）。我们发现 TH信号传导的时间调节对于视锥细胞亚型命运的建立和维持是关键的。到 评估甲状腺失调和视锥细胞命运可塑性，我们将确定TH调节的时间窗， 类器官和检查晚发性甲状腺功能失调患者视网膜中的视锥细胞命运。我们将 确定TH调节基因在类器官中视锥细胞亚型特化中的作用（目的2）。另外我们 发现视黄酸（RA）信号传导诱导S视锥命运并在决策1早期抑制L/M视锥命运， 并在决策2后期诱导L视锥命运并抑制M视锥命运。我们将描述时间窗 RA的作用，并评估RA调节基因的功能和表达在视锥细胞亚型的规范 在类器官中的命运（目标3）。 这些实验将描述TH和RA信号的调节如何指定视锥细胞中的亚型。 人类视网膜此外，这些实验将提供第一个直接的机制洞察L和M锥 对理解红/绿色色盲和发展具有重要意义的规范 黄斑变性的再生疗法。

Project Summary: The specification of three cone photoreceptor subtypes enables high-acuity daytime and trichromatic color vision in humans. During terminal differentiation, cone cells express opsins that define their functionalities as blue (S-opsin), green (M-opsin), or red (L-opsin) light detectors. How the three cone subtypes are generated in humans remains largely unknown. Elucidating these mechanisms will be critical for understanding and treating visual disorders including macular degeneration, cone dystrophy, retinitis pigmentosa, foveal hypoplasia, and color blindness. The goal of this project is to determine the mechanisms controlling the specification of S, M, and L cones in the human retina. Cone subtype specification occurs in a two-step process. First, a decision occurs between S and L/M cone fates. If the L/M fate is chosen, a subsequent choice is made between L and M cone fates. To study these processes, we genetically and pharmacologically manipulated human retinal organoids to test mechanisms of retinal development. We found that dynamic expression of thyroid hormone (TH)-regulating genes within the retina ensures low TH signaling early to specify S cones and high TH signaling late to produce L/M cones. Our work established human retinal organoids as a model for determining mechanisms of human development with promising utility for therapeutics and vision repair (Eldred et al., 2018, Science). We found that S, M, and L cones are generated in a temporal progression during development. How temporal and spatial inputs are integrated in the human retina to generate patterns of cones is poorly understood. We developed dissection, imaging, and cell identification approaches to assess the spatial distributions of cone subtypes and generate the first maps of cone subtypes across intact human retinas (Aim 1). We found that temporal regulation of TH signaling is critical for establishment and maintenance of cone subtype fates. To evaluate thyroid dysregulation and cone fate plasticity, we will define the temporal windows of TH regulation in organoids and examine cone fates in retinas from patients with late-onset thyroid dysregulation. We will determine the roles of TH-regulating genes in cone subtype specification in organoids (Aim 2). Additionally, we found that retinoic acid (RA) signaling induces S cone fate and suppresses L/M cone fate early in decision 1, and induces L cone fate and suppresses M cone fate late in decision 2. We will characterize temporal windows of RA action and assess the function and expression of RA-regulating genes in the specification of cone subtype fates in organoids (Aim 3). These experiments will delineate how regulation of TH and RA signaling specify cone subtypes in the human retina. Moreover, these experiments will provide the first direct mechanistic insight into L and M cone specification with important implications for understanding red/green color blindness and developing regenerative therapeutics for macular degeneration.