Cone subtype specification in human retinas and organoids

人类视网膜和类器官中的视锥细胞亚型规范

• 批准号: 10327705
• 负责人: Robert John Johnston
• 金额: $ 51.69万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10327705
• 关键词: Adult; Autoimmune Diseases; Cells; Color; Color Visions; Color blindness; Cone; Cone dystrophy; Data; Development; Dissection; Ensure; Exhibits; Genes; Goals; Graves' Disease; Human; Human Development; Hyperthyroidism; Image; In Situ Hybridization; Light; Macular degeneration; Maintenance; Maps; Methods; Modeling; Morphology; Mus; Neurons; Opsin; Organoids; Patients; Pattern; Pharmacology; Play; Primates; Process; Proteins; Protocols documentation; RNA; Red-Green Color Blindness; Regulation; Resolution; Retina; Retinal Cone; Retinitis Pigmentosa; Role; SW opsin; Science; Seminal; Signal Transduction; Spatial Distribution; Specific qualifier value; System; Testing; Therapeutic; Thyroid Gland; Thyroid Hormones; Tissues; To specify; Tretinoin; Vision; Vision Disorders; Work; Zebrafish; analysis pipeline; cell fate specification; cell type; detector; experimental study; fetal; hormonal signals; hormone regulation; human stem cells; insight; regenerative therapy; repaired

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.

项目摘要：三种视锥细胞亚型的规格使白天和夜晚的高敏锐度成为可能 人类的三色视觉。在终末分化过程中，视锥细胞表达视蛋白，定义其 蓝色（S-视蛋白）、绿色（M-视蛋白）或红色（L-视蛋白）光检测器的功能。三种视锥细胞亚型如何 人类中产生的物质仍然很大程度上未知。阐明这些机制对于 了解和治疗视觉疾病，包括黄斑变性、视锥细胞营养不良、视网膜炎 色素变性、黄斑中心凹发育不全和色盲。该项目的目标是确定机制 控制人类视网膜中 S、M 和 L 视锥细胞的规格。 锥体亚型规范分两步进行。首先，在 S 和 L/M 之间做出决定 圆锥的命运。如果选择了 L/M 命运，则随后在 L 和 M 锥体命运之间做出选择。要研究这些 过程中，我们通过基因和药理学方法操纵人类视网膜类器官来测试其机制 视网膜发育。我们发现甲状腺激素（TH）调节基因的动态表达 视网膜确保早期的低 TH 信号传导以指定 S 视锥细胞，并确保较晚的高 TH 信号传导以产生 L/M 视锥细胞。我们的 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 在治疗和视力修复方面具有广阔的应用前景（Eldred et al., 2018, Science）。 我们发现 S、M 和 L 视锥细胞是在发育过程中按时间顺序生成的。如何 人们对时间和空间输入整合到人类视网膜中以生成视锥细胞模式的了解还知之甚少。 我们开发了解剖、成像和细胞识别方法来评估锥体的空间分布 亚型并生成完整人类视网膜上第一个视锥细胞亚型图（目标 1）。我们发现 TH 信号传导的时间调节对于锥体亚型命运的建立和维持至关重要。到 评估甲状腺失调和视锥细胞命运可塑性，我们将定义 TH 调节的时间窗口 类器官并检查迟发性甲状腺失调患者视网膜中的视锥细胞命运。我们将 确定 TH 调节基因在类器官锥体亚型规范中的作用（目标 2）。此外，我们 发现视黄酸 (RA) 信号传导在决策 1 的早期诱导 S 视锥细胞命运并抑制 L/M 视锥细胞命运， 并在决策 2 后期诱导 L 视锥细胞命运并抑制 M 视锥细胞命运。我们将表征时间窗口 RA 作用并评估 RA 调节基因在锥体亚型规范中的功能和表达 类器官的命运（目标 3）。 这些实验将描述 TH 和 RA 信号传导的调节如何指定视锥细胞亚型 人类视网膜。此外，这些实验将首次提供对 L 和 M 锥体的直接机械见解 对于理解红/绿色盲和发展具有重要意义的规范 黄斑变性的再生疗法。