Retinal Pigmented Epithelium Reprogramming and Retina Regeneration

描述(申请人提供)：视网膜变性导致视力丧失是老年性黄斑变性(AMD)、糖尿病视网膜病变和青光眼的最终结果。目前的治疗方法对那些患有这些疾病晚期的人几乎没有选择。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。胚胎雏鸡在视网膜切除后，如果暴露于异位成纤维细胞生长因子2(FGF2)，则通过转分化过程再生视网膜。这个过程涉及视网膜色素上皮(RPE)的重新编程以去分化，失去它的色素，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。对于这项提议，重点将放在去分化的过程上，这是理解转分化如何起作用的关键。我们的初步数据指出了一个两步去分化的过程，在这个过程中，损伤(视网膜切除)诱导RPE对FGF2做出反应。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SOM多能性诱导因子(PIF)和眼场转录因子。此外，我们还发现，在斑马鱼Muller胶质细胞转分化过程中起关键作用的PIF基因LIN-28只有在去除视网膜后加入FGF2(步骤2)才能上调。基于我们的初步数据，我们将调查LIN-28是否是诱导视网膜切除鸡眼RPE转分化所必需的和足够的。我们将通过电穿孔含有LIN-28的质粒和使用吗啉对LIN28的功能丧失来进行功能获得实验。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，以生成新的视网膜。这项提案的另一个重点是使用一种无偏见的全基因组方法和利用最先进的技术(如下一代测序)来执行mRNA-Seq和miRNA-Seq，来剖析调控组件，包括信号网络和mRNA-miRNA调控模块。我们假设，鸡RPE去分化所涉及的两步过程在每一步都需要一组独特的调节分子。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重新编程以产生新神经元的可能性。此外，考虑到这些小分子非常适合人类治疗，识别可以重新编程RPE的关键miRNA分子的意义也很大。

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.