Mechanisms Controlling RPE Development

项目总结/摘要 视网膜色素上皮细胞（RPE）是眼睛发育和功能所必需的，因为它介导 光感受器外节更新，视色素再生，跨上皮转运，维甲酸 储存和防止氧化损伤。因此，RPE结构或生理学的改变 由环境或基因干扰引起的疾病最终会导致失明。重要的是，不正确的RPE 发育损害眼睛的生长，并可能导致严重的先天性缺陷，如小眼症。因此 对于确定RPE正常发育的潜在机制至关重要。虽然一些基因对 RPE发育和功能（例如Mitf、Otx 2）是已知的，但不清楚RPE特异性基因表达如何影响RPE的发育和功能。 发起和维护。我们在小鼠视泡外植体中的初步数据表明， 眼外间充质产生促进RPE发育的信号。另外，我们有证据表明 Wnt/β-catenin通路对于胚胎RPE分化的持续是必需的。我们假设 间充质激活素受体的激活作为诱导RPE的早期信号，而Wnt/- 连环蛋白信号传导稍后起作用以确保RPE的维持和功能。在这个项目中，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 使用外植体培养物和组织特异性基因破坏的Wnt/β-连环蛋白途径（Aim 1）。探讨 Wnt/β-连环蛋白信号在维持新生和出生后RPE中的作用，我们将进行诱导， - 连环蛋白的组织特异性失活。我们还将通过TCF/LEF来确定Wnt/β-连环蛋白是否 使用荧光素酶和ChIP测定，转录因子直接激活RPE特异性基因表达（Aim 2）。 使用小鼠胚胎中的AP 2基因破坏和视泡外植体的FGF处理，我们提出， 测试Wnt/-连环蛋白通路的异位和持续激活是否足以阻断 RPE向视网膜的转分化（Aim 3）。总之，这些实验将促进我们对 在哺乳动物眼睛发育过程中控制RPE分化的信号， 治疗性治疗眼部变性疾病。

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.