Optic nerve regeneration: gene networks in retina development

项目摘要 我们在拟议实验中的长期目标是在基础上获得新的知识 控制视网膜发育的机制，并应用这一知识开发治疗视网膜的新方法 退行性疾病。我们的策略是使用我们已经拥有的转基因小鼠模型 创造的或我们将要创造的。尽管已经积累了大量的信息 在控制视网膜发育的机制方面，仍然存在很大的差距。特别是，控制的机制 对于祖细胞是增殖还是分化的决定，人们还知之甚少。更好的 了解这一点对于找到修复受损视网膜的新方法非常重要。视网膜神经节细胞 视网膜节细胞(RGCs)是发育过程中从视网膜祖细胞(RPC)分化出的第一种细胞类型， 连接到大脑的视网膜神经元。我们专注于导致RPC承诺遵守的监管事件 研资局的命运。在相关实验中，胚胎RPC将被用来在成年视网膜中重新填充RGC 已经耗尽了它们的内源性RGC。我们的基本假设是，RPC要区分为 RGCs，Atoh7必须与其他调控因素相结合，以实现增殖和 差异化。为了解决这一假设，我们提出了三个具体目标。第一个目标将决定 Atoh7是否足以将非RGC转变为RGC的命运。初步工作表明，更换 带有Atoh7基因的Neurod1导致内核层RGC基因的异位表达。我们将决定 Atoh7是否能促进发育中和成年视网膜中非RGC神经元的RGC分化。第二 AIM将确定Atoh7是否调节Notch信号以控制RPC增殖之间的平衡 以及研资局的承诺。在初步实验中，我们发现Atoh7与E-box元件上游结合 NOTCH1和Atoh7负性调节Notch1的表达。我们将确定Atoh7的时间 相对于Notch信令显示。我们将确定Atoh7和Notch1是否参与了否定 以及当Notch1上的Atoh7结合位点被删除时，RPC的增殖是否受到干扰。 在第三个目标中，我们将优化我们的实验，通过移植再生RGC耗尽的视网膜。 Atoh7表达的RPC进入RGC耗竭小鼠的视网膜以及神经保护因子。我们还将 检测表达Atoh7的RPC能否在视神经损伤等小鼠中再生视神经 模特们。 我们对控制视网膜发育的因素的了解使我们能够应用发育 成人视网膜的概念。我们已经开发出了人类视神经退化的现实遗传模型 将在干细胞替代疗法中最终用于修复受损的视神经。

Project Summary Our long-term objective in the proposed experiments is to obtain new knowledge on the basic mechanisms that control retinal development and to apply this knowledge to develop novel ways to treat retinal degenerative diseases. Our strategy is to use genetically engineered mouse models that we have already created or that we will create. Although an impressive amount of information has accumulated on the mechanisms that control retinal development, large gaps still remain. In particular, the mechanisms that control a progenitor cell's decision whether to proliferate or differentiate are only vaguely understood. A better understanding is of great importance for finding new ways to repair damaged retinas. Retinal ganglion cells (RGCs) are the first cell type to differentiate from retinal progenitor cells (RPCs) during development and are the retinal neurons that connect to the brain. We focus on the regulatory events that cause RPCs to commit to a RGC fate. In related experiments, embryonic RPCs will be used to repopulate RGCs in adult retinas that have been depleted of their endogenous RGCs. Our underlying hypothesis is that for RPCs to differentiate into RGCs, Atoh7 must integrate with other regulatory factors to achieve a balance between proliferation and differentiation. To address the hypothesis, we proposed three specific aims. The first aim will determine whether Atoh7 is sufficient to convert non-RGCs to a RGC fate. Preliminary work indicates that replacing Neurod1 with Atoh7 leads to ectopic RGC gene expression in the inner nuclear layer. We will determine whether Atoh7 can drive RGC differentiation in non-RGC neurons in developing and adult retinas. The second aim will determine whether Atoh7 regulates Notch signaling to control the balance between RPC proliferation and RGC commitment. In preliminary experiments, we found that Atoh7 binds to E-box elements upstream of Notch1 and that Atoh7 negatively regulates Notch1 expression. We will identify the time at which Atoh7 appears relative to Notch signaling. We will determine whether Atoh7 and Notch1 participate in a negative feedback loop and whether RPC proliferation is perturbed when the Atoh7 binding sites on Notch1 are deleted. In the third aim, we will optimize our experiments on repopulating RGC-depleted retinas by transplanting Atoh7-expressing RPCs into the retinas of RGC-depleted mice along with neuroprotective factors. We will also determine whether Atoh7-expressing RPCs can regenerate optic nerves in optic nerve crush and other mouse models. Our knowledge of the factors controlling retinal development allows us to apply developmental concepts to adult retinas. We have developed realistic genetic models for human optic nerve degeneration that will have ultimate use in stem cell replacement therapy to repair damaged optic nerves.