Mechanisms Controlling RPE Development

项目摘要/摘要 视网膜色素上皮(RPE)在眼的发育和功能中起着重要的调节作用 光感受器外节更新、视色素再生、跨上皮运输、视黄醇 储存和防止氧化损伤。因此，RPE结构或生理的改变 由环境或遗传因素引起的疾病最终会导致失明。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，它 对于确定RPE正常发育的机制至关重要。虽然有些基因对 RPE的发育和功能(如MITF、OTX2)是已知的，但RPE特异性基因的表达尚不清楚 启动和维护。我们在小鼠视泡外植体中的初步数据表明，周围的 眼外间充质产生促进RPE发展的信号(S)。此外，我们有证据表明 Wnt/-catenin通路对胚胎RPE分化的延续至关重要。我们假设 间充质激活激活素受体是诱导RPE的早期信号，而Wnt/- 连环蛋白信号随后起作用，以确保RPE的维护和功能。在这项计划中，我们建议在 小鼠眼外间充质的确切时间需求及其在激活激活素和 使用外植体培养和组织特异性基因破坏的WNT/-连环蛋白途径(目标1)。为了调查 Wnt/-catenin信号在维持围产期和出生后RPE中的作用，我们将进行可诱导的， -连环蛋白的组织特异性失活。我们还将确定Wnt/-catenin是否通过TCF/Lef 利用荧光素酶和芯片分析，转录因子直接激活RPE特异性基因表达(目标2)。 利用AP2基因在小鼠胚胎中的破坏和对视泡外植体的成纤维细胞生长因子的处理，我们建议 检测Wnt/-catenin途径的异位和持续激活是否足以阻断 RPE向视网膜的转分化(目标3)。总而言之，这些实验将增进我们对 在哺乳动物眼睛发育过程中控制RPE分化的信号，并可能为 眼部退行性疾病的治疗。

Project Summary/Abstract 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 AP2 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.