Mechanisms Controlling RPE Development

描述(申请人提供)：视网膜色素上皮(RPE)对眼睛的发育和功能是必不可少的，因为它介导光感受器外节更新、视觉色素再生、跨上皮运输、视黄醇储存和保护免受氧化损伤。因此，环境或遗传干扰引起的RPE结构或生理变化最终可能导致失明。重要的是，不适当的RPE发育会损害眼睛的生长，并可能导致严重的先天性缺陷，如小眼球。因此，确定RPE正常发育的机制至关重要。虽然一些对RPE发育和功能至关重要的基因(如MITF、OTX2)已知，但RPE特异性基因的表达是如何启动和维持的尚不清楚。我们在小鼠视泡外植体中的初步数据表明，周围的眼外间充质产生一个信号(S)来促进RPE的发展。此外，我们有证据表明，Wnt/β-catenin通路对于胚胎RPE分化的延续是必不可少的。我们假设，间充质激活激活素受体作为早期信号诱导RPE，而Wnt/β-catenin信号则在较晚时起作用，以确保RPE的维持和功能。在这个项目中，我们建议通过外植体培养和组织特异性基因中断，在小鼠身上研究眼外间充质的确切时间需求及其在激活激活素和Wnt/β-连环蛋白通路中的作用(目标1)。为了研究Wnt/β-catenin信号在维持围产期和出生后RPE中的作用，我们将对-catenin进行可诱导的、组织特异性的失活。我们还将使用荧光素酶和芯片分析来确定Wnt/β-catenin是否通过TCF/Lef转录因子直接激活RPE特异性基因的表达(目标2)。利用小鼠胚胎中AP2a基因的破坏和视泡外植体的成纤维细胞生长因子处理，我们建议测试Wnt/β-catenin途径的异位和持续激活是否足以阻止RPE向视网膜的转分化(目标3)。总之，这些实验将促进我们对哺乳动物眼睛发育过程中控制RPE分化的信号的理解，并可能为治疗眼睛退行性疾病提供线索。 公共卫生相关性：视网膜色素上皮(RPE)对于脊椎动物眼睛的发育和功能是必不可少的。RPE发育异常会损害眼睛的生长，并导致先天性缺陷，如小眼球。组织和细胞之间的信号转导是调节发育的基本机制；然而，许多控制RPE发育的调节信号仍有待定义。总之，这里提出的实验目标将促进我们对控制RPE发展的信号的理解，并可能为治疗眼睛退行性疾病的新的假定靶点提供线索。

DESCRIPTION (provided by applicant): The retinal pigment epithelium (RPE) is essential for development and function of the eye as it mediates photoreceptor outer segment renewal, regeneration of visual pigments, trans-epithelial transport, retinoid storage and protection against oxidative damage. Accordingly, alterations in RPE structure or physiology caused by environmental or genetic perturbations can ultimately cause blindness. Importantly, improper RPE development impairs eye growth and can result in severe congenital defects such as microphthalmia. Thus, it is critical to identify the mechanisms underlying normal development of the RPE. While some genes crucial for RPE development and function (e.g. Mitf, Otx2) are known, it is unclear how RPE-specific gene expression is initiated and maintained. Our preliminary data in mouse optic vesicle explants suggests that the surrounding extraocular mesenchyme produces a signal(s) to promote RPE development. Furthermore, we have evidence that the Wnt/¿-catenin pathway is essential for continuation of embryonic RPE differentiation. We hypothesize that activin receptor activation by the mesenchyme acts as an early signal to induce the RPE, while Wnt/¿-catenin signaling acts later to ensure RPE maintenance and function. In this project, we propose to examine in mouse the exact temporal requirement of extraocular mesenchyme and its role in activating the activin and Wnt/¿-catenin pathways using explant cultures and tissue-specific gene disruption (Aim 1). To investigate the role of Wnt/¿-catenin signaling in maintenance of the peri- and postnatal RPE, we will perform inducible, tissue-specific inactivation of -catenin. We will also determine whether Wnt/¿-catenin through TCF/LEF transcription factors directly activates RPE-specific gene expression using luciferase and ChIP assays (Aim 2). Using AP2a gene disruption in the mouse embryo and FGF treatment of optic vesicle explants, we propose to test whether ectopic and sustained activation of the Wnt/¿-catenin pathway is sufficient to block transdifferentiation of RPE into retina (Aim 3). Together, these experiments will advance our understanding of the signals that control RPE differentiation during mammalian eye development and may provide clues for therapeutic treatment of degenerative diseases in the eye. PUBLIC HEALTH RELEVANCE: The retinal pigment epithelium (RPE) is essential for development and function of the vertebrate eye. Abnormal RPE development impairs eye growth and results in congenital defects such as microphthalmia. Signaling between tissues and cells is a fundamental mechanism by which development is regulated; however, many of the regulatory signals controlling RPE development remain to be defined. Together, the goal of experiments proposed here will advance our understanding of the signals that control RPE development and may provide clues for new putative targets for therapeutic treatment of degenerative diseases in the eye.