Retinal Pigmented Epithelium Reprogramming and Retina Regeneration

项目摘要 视网膜变性导致视力丧失是老年性黄斑变性的最终结局 (AMD)糖尿病视网膜病变和青光眼。目前的治疗方法为那些患有 这些疾病的晚期。为了探索可能的治疗方法，重要的是使用动物 具有再生能力的模型，如鸡胚。鸡胚再生出 视网膜，视网膜切除术后，如果暴露于异位成纤维细胞，通过转分化过程 生长因子2（FGF 2）。这一过程涉及视网膜色素上皮细胞的重编程 (RPE)去分化，失去色素，增殖并形成神经上皮， 分化形成所有主要的视网膜细胞类型。对于本提案，重点将放在过程上 这是理解转分化如何运作的关键。我们的初步数据 指出一个两步去分化过程，其中损伤（视网膜切除术）诱导RPE变得 能够对FGF 2做出反应。我们已经确定了一系列与“损伤”相关的上调因子， 仅”（步骤1）包括一些多能性诱导因子（PiF）和眼区转录因子。在 此外，我们还发现Lin-28，一种PiF，在Muller胶质细胞转分化中起关键作用， 斑马鱼，仅在视网膜去除后在鸡眼中加入FGF 2（步骤2）时上调。 根据我们的初步数据，我们将研究是否需要Lin-28，并且是否足以诱导 视网膜切除鸡眼的RPE转分化。我们将通过以下方式进行功能增益实验： 电穿孔含有Lin-28的质粒，并使用吗啉代对Lin-28进行功能丧失。我们 假设是Lin-28足以完成RPE重编程过程， 损伤信号使新的视网膜。该提案的另一个重点是剖析监管机构， 包括信号网络和mRNA-miRNA调控模块的组件， 全基因组方法，并利用最先进的技术，如下一代 测序以进行mRNA-Seq和miRNA-Seq。我们假设两步过程 参与鸡RPE去分化需要一套独特的调节分子， 步这项研究将对再生医学领域产生重大影响，因为 所获得的信息可以外推到包括人在内的哺乳动物的视网膜修复过程， 特别是对人类RPE的潜在重编程以产生新的神经元。也 鉴定能够重编程RPE的关键miRNA分子的意义是很高的， 这些是人类治疗非常需要的小分子。

Project Summary Retinal degeneration leading to vision loss is the ultimate outcome of age related macular degeneration (AMD), diabetic retinopathy and glaucoma. Current therapies offer few options to those suffering from late stages of these diseases. In order to explore possible therapies, it is important to use animal models with regenerative capabilities such as the chick embryo. Embryonic chicks regenerate their retina, following retinectomy, via the process of trans-differentiation if exposed to ectopic fibroblast growth factor 2 (FGF2). This process involves the reprogramming of the retinal pigmented epithelium (RPE) to dedifferentiate, losing its pigment, proliferating and forming a neuroepithelium that eventually differentiates to form all major retina cell types. For this proposal, the emphasis will be on the process of dedifferentiation which is key to understanding how transdifferentiation works. Our preliminary data point to a two-step dedifferentiation process where injury (retinectomy) induces the RPE to become competent to respond to FGF2. We have identified a series of factors that are up-regulated with "injury only" (step 1) including some pluripotency inducing factors (PiFs) and eye field transcriptional factors. In addition, we have found that Lin-28, a PiF and a critical player in Muller glia transdifferentiation in zebrafish, is only up-regulated upon addition of FGF2 (step 2) in the chick eye after retina removal. Based on our preliminary data, we will investigate whether Lin-28 is required and sufficient to induce RPE transdifferentiation in retinectomized chick eyes. We will perform gain-of-function experiments by electroporating a plasmid containing Lin-28 and loss-of-function using morpholinos against Lin28. Our hypothesis is that Lin-28 is sufficient to complete the RPE reprogramming process initiated by injury signals to make new retina. The other focus of this proposal is on dissecting regulatory components including signaling networks and mRNA-miRNA regulatory modules using an unbiased, genome wide approach and taking advantage of state of the art technology such as Next Generation Sequencing to perform mRNA-Seq and miRNA-Seq. We hypothesize that the two-step process implicated in chick RPE dedifferentiation requires a unique set of regulatory molecules at each step. This study will have a significant impact on the field of regenerative medicine since the information obtained can be extrapolated to the process of retina repair in mammals including humans, and specifically on the potential reprogramming of human RPE to generate new neurons. Also the significance of identifying key miRNA molecules that could reprogram RPE is high considering these are small molecules highly desirable for human therapeutics.