Role of retinoic acid signaling in fovea development

项目概要： 中央凹是位于黄斑内的高度专门化的视网膜结构，负责高- 敏锐的视觉，这在人类中使关键的任务，如阅读和识别面孔。 黄斑变性等黄斑中心凹疾病是高度衰弱的，通常会导致失明。 然而，目前还没有针对中央凹特异性疾病的可用治疗或疗法。一 这种局限性的主要原因是缺乏合适的实验系统， 研究，因为只有少数物种具有中央凹样高敏度区域（HAA）。的确， 在哺乳动物中，只有灵长类动物的一个子集具有中央凹视网膜。然而，鸟类 包括鸡在内的所有鸡胚都具有中央凹状的HAA，可以作为研究HAA的一个强有力的模型系统 在体内形成中央凹的分子基础。人干细胞衍生的视网膜类器官 （hRetlymphoma）已经成为研究人类视网膜疾病的有前景的体外系统， 当前的协议不能产生中央凹。我们最近发现，维甲酸（RA）信号 在人类的中心凹形成过程中是高度模式化的，并且是鸡的HAA形成所需的。 我们的假设是，在hRetinopathy中RA信号传导的内源性模式的重演是 足以诱导中央凹形成。为了验证这一假设，我们将利用小鸡系统， 阐明体内RA依赖性小凹诱导的时空特性（目的1）; 纵向表征RA信号传导的动力学和RA酶的表达谱 在hRetinopathy中的视网膜发生期间（Aim 2）;实验性地操纵RA信号传导， 概括了在体外hRetrieval系统中体内发现的RA的时空模式（Aim 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，用于精确的空间和时间调节转基因 并优化了整个鸡视网膜的高速高分辨率成像分析和3D h保留。这一努力不仅将推进我们目前对RA机制作用的理解， 信号作为中央凹调节器，但也产生创新和急需的中央凹 对通过疾病影响中央凹的疾病具有高翻译潜力的hRetrieval系统 建模，高通量药物筛选和作为临床相关的中央凹特异性的来源， 细胞移植。

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.