Cytoskeletal regulation and Yap/Taz activity in differentiating retinal neurons

项目总结/摘要 人类视网膜在受伤后不会再生。这项建议检查视网膜发育，以更好地 了解可以用来启动再生的分子机制。发育中的神经元必须 精确地调节细胞骨架动力学，以建立其独特的形态， 功能目前还不清楚细胞骨架动力学在发育过程中和损伤后是如何变化的。 vivo.是的，相关蛋白（雅普）和Tafazzin（TAZ）是功能相似的蛋白质，对于 通过转录调节维持祖细胞状态。雅普/TAZ的突变导致视网膜病变。 层压缺陷通过一个未知的机制。此外，与其他视网膜神经元不同，视网膜神经节 细胞在发育后继续表达雅普/TAZ。有趣的是，细胞骨架张力可以抑制雅普/TAZ 活性和雅普/TAZ可调节细胞骨架动力学。这创造了一个不寻常的分子反馈回路 结构蛋白和转录调节因子之间的联系。本提案的总体目标是了解 细胞骨架和雅普/TAZ协调视网膜发育的分子机制， 在视网膜再生中的作用。中心假设是，细胞骨架重排所需的 视网膜分化下调雅普/TAZ活性，从而驱使细胞脱离祖细胞状态。与 从这些研究中获得的知识，最终目标是在成体细胞中反向工程这一过程，所以我们 然后可以驱动细胞达到增强再生潜力的状态。为此，我将使用以下组合 创新的体内成像技术、遗传操作和再生测定，以更好地了解 细胞骨架和雅普/TAZ协调视网膜发育的分子机制， 再生 这项提议的初步实验将集中在开发斑马鱼的工具和技术， 细胞骨架来检验整个假设掌握斑马鱼模型系统是一个关键因素 K99阶段的训练潜力，并将补充我以前的工作，检查视网膜 小鼠的发育。拥有老鼠和斑马鱼的技术专长将使我能够回答 最关键的问题，通过实验进入视网膜发育，以及广泛的 分子和基因工具。总之，这个培训计划将使我能够开发一个独立的生产线 研究，可以显着促进视网膜再生疗法的发展，和 最终防止视力丧失。

PROJECT SUMMARY / ABSTRACT The human retina does not regenerate after injury. This proposal examines retinal development to better understand the molecular mechanisms that could be used to initiate regeneration. Developing neurons must precisely regulate cytoskeletal dynamics to establish their unique morphology that is required for proper function. It is not well understood how cytoskeletal dynamics change across development and after injury in vivo. Yes Associated Protein (YAP) and Tafazzin (TAZ) are functionally similar proteins that are critical for maintaining the progenitor state through transcriptional regulation. Mutations to YAP/TAZ result in retinal lamination defects through an unknown mechanism. Additionally, unlike other retinal neurons, retinal ganglion cells continue to express YAP/TAZ after development. Intriguingly, cytoskeletal tension can inhibit YAP/TAZ activity and YAP/TAZ can regulate cytoskeletal dynamics. This creates an unusual molecular feedback loop between structural proteins and transcriptional regulators. The overall goal of this proposal is to understand the molecular mechanisms by which the cytoskeleton and YAP/TAZ coordinate retinal development, and the role they play in retinal regeneration. The central hypothesis is that cytoskeletal rearrangements required for retinal differentiation downregulate YAP/TAZ activity and thus drive cells out of the progenitor state. With the knowledge gained from these studies, the ultimate goal is to reverse engineer this process in adult cells, so we can then drive cells to a state of enhanced regenerative potential. To this end I will use a combination of innovative in vivo imaging techniques, genetic manipulations, and regeneration assays to better understand the molecular mechanisms by which the cytoskeleton and YAP/TAZ orchestrate retinal development and regeneration. The initial experiments in this proposal will focus on developing tools and techniques in zebrafish to investigate the cytoskeleton in order to test the overall hypothesis. Mastery of the zebrafish model system is a key element of the training potential of the K99 phase, and will complement my previous work examining retinal development in mice. Having technical expertise in both mouse and zebrafish will enable me to answer the most critical questions by having experimental access across retinal development, and a wide range of molecular and genetic tools. Taken together this training plan will empower me to develop an independent line of research that can significantly contribute to the development of regenerative therapies for the retina, and ultimately prevent vision loss.