Retinal Pigmented Epithelium Reprogramming and Retina Regeneration

描述（由申请人提供）：视网膜变性导致视力丧失是年龄相关性黄斑变性（AMD）、糖尿病视网膜病变和青光眼的最终结果。目前的治疗方法为这些疾病的晚期患者提供了很少的选择。为了探索可能的治疗方法，重要的是使用具有再生能力的动物模型，如鸡胚胎。胚胎雏鸡视网膜切除术后，暴露于异位成纤维细胞生长因子2 （FGF2）下，通过反分化过程再生视网膜。这个过程涉及视网膜色素上皮（RPE）的重编程，使其去分化，失去色素，增殖并形成神经上皮，最终分化形成所有主要的视网膜细胞类型。对于这一建议，重点将放在去分化的过程，这是理解转分化如何工作的关键。我们的初步数据指向一个两步去分化过程，其中损伤（视网膜切除术）诱导RPE对FGF2产生反应。我们已经确定了一系列在“仅损伤”（步骤1）中上调的因子，包括一些多能性诱导因子（pif）和视野转录因子。此外，我们发现Lin-28（一种PiF，也是斑马鱼Muller胶质细胞转分化的关键参与者）仅在去除视网膜后的鸡眼中添加FGF2（步骤2）时才上调。基于我们的初步数据，我们将研究Lin-28是否需要和足以诱导视网膜切除的鸡眼RPE转分化。我们将通过电穿孔含有Lin-28的质粒进行功能获得实验，并使用morpholinos对Lin28进行功能丧失实验。我们的假设是Lin-28足以完成由损伤信号启动的RPE重编程过程，从而形成新的视网膜。该提案的另一个重点是使用无偏倚的全基因组方法解剖调控成分，包括信号网络和mRNA-miRNA调控模块，并利用最先进的技术（如下一代测序）来执行mRNA-Seq和miRNA-Seq。我们假设鸡RPE去分化的两步过程在每一步都需要一组独特的调控分子。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类在内的哺乳动物的视网膜修复过程，特别是人类RPE的潜在重编程以产生新的神经元。此外，考虑到这些小分子对人类治疗非常理想，鉴定可以重编程RPE的关键miRNA分子的意义也很高。

DESCRIPTION (provided by applicant): 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 som 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, isonly 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.