Role of retinoic acid signaling in fovea development

项目总结： 黄斑中心凹是一种高度专业化的视网膜结构，位于黄斑内，负责高度... 敏锐的视觉，在人类中能够完成关键的任务，如阅读和识别人脸。 黄斑变性等黄斑中心凹疾病使人非常虚弱，并经常导致失明。 然而，目前还没有可用的治疗方法或治疗中心凹特定的疾病。一个 造成这一限制的主要原因是缺乏合适的实验系统。 研究表明，只有少数物种有中央凹状高敏感区(HAA)。的确， 在哺乳动物中，只有一小部分灵长类动物有视网膜凹陷。然而，鸟类物种 包括雏鸡，具有中心凹状的HAA，可以作为一个强大的模型系统来研究 活体中心凹形成的分子基础。人干细胞来源的视网膜有机体 (HRetOrg)已成为研究人类视网膜疾病的一种有前途的体外系统，但 目前的治疗方案不能产生中心凹。我们最近发现维甲酸(RA)信号 在人类的凹形成过程中高度图案化，是雏鸟形成HAA所必需的。 我们的假设是，hRetOrg中RA信号的内源性模式的概括是 足以诱导中心凹的形成。为了验证这一假设，我们将利用CHICK系统 阐明体内RA依赖的中心凹诱导的时空特性(目标1)； RA信号动态和RA酶表达谱的纵向表征 在hRetOrg的视网膜形成期(目标2)；实验性地操纵RA信号来 概述了在体外hRetOrg系统(AIM)体内发现的RA的时空模式 3)。为了实现这一目标，我们开发了一种基于转座子的创新分子策略- 和多西环素诱导系统，用于转基因的精确时空调控 并优化了全鸡视网膜和3D的高速高分辨率成像分析 HRetOrg。这一努力不仅将促进我们目前对RA的机械作用的理解 作为中心凹调节器的信号，也产生了创新的和急需的中心凹 HRetOrg系统对通过疾病影响中心凹的疾病具有高翻译潜力 建模、高通量药物筛选和作为临床相关中心凹特异性的来源 用于移植的细胞。

PROJECT SUMMARY: The fovea is a highly specialized retinal structure located within the macula responsible for high- acuity vision, which in humans enables critical tasks such as reading and recognizing faces. Foveal diseases such as macular degeneration are highly debilitating and often lead to blindness. However, there are no current available treatments or therapies for fovea-specific disorders. A major reason underlying this limitation is the lack of suitable experimental systems for foveal studies, as only a small number of species have fovea-like high acuity areas (HAA). Indeed, among mammals, only a subset of primates has foveated retinas. However, avian species including the chick, possess fovea-like HAA and can be used as a powerful model system to study the molecular underpinnings of fovea formation in vivo. Human stem cell-derived retinal organoids (hRetOrg) have emerged as a promising in vitro system to study human retinal disorders but current protocols fail to generate foveae. We have recently found that retinoic acid (RA) signaling is highly patterned during foveogenesis in humans and is required for HAA formation in the chick. Our hypothesis is that recapitulation of endogenous patterns of RA signaling in hRetOrg is sufficient to induce fovea formation. To test this hypothesis, we will utilize the chick system to elucidate the spatiotemporal properties of RA-dependent fovea induction in vivo (Aim 1); longitudinally characterize the dynamics of RA signaling and expression profile of RA enzymes during retinogenesis period in hRetOrg (Aim 2); experimentally manipulate RA signaling to recapitulate the spatiotemporal patterns of RA found in vivo in the in vitro hRetOrg system (Aim 3). To achieve this we have developed an innovative molecular strategy based on transposon- and doxycycline-inducible systems for precise spatial and temporal regulation of transgenesis and optimized high-speed high-resolution imaging analysis of whole chick retinas and 3D hRetOrg. This effort will not only advance our current understanding of the mechanistic role of RA signaling as a fovea regulator but also generate an innovative and much needed foveated hRetOrg system with high translational potential for diseases affecting the fovea through disease modeling, high-throughput drug screening and as a source of clinically-relevant fovea-specific cells for transplantation.