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获得新的知识， 控制视网膜发育的机制，并应用这些知识来开发治疗视网膜病变的新方法。 退化性疾病我们的策略是使用我们已经拥有的基因工程小鼠模型 创造或我们将创造。尽管已经积累了大量关于 控制视网膜发育的机制，仍然存在很大的差距。特别是，控制 对于祖细胞是增殖还是分化的决定，人们只有模糊的理解。更好的 理解对于寻找修复受损视网膜的新方法非常重要。视网膜神经节细胞 视网膜前体细胞（RGC）是在发育过程中从视网膜前体细胞（RPC）分化的第一种细胞类型， 连接大脑的视网膜神经元。我们专注于导致RPC承诺的监管事件， RGC的命运。在相关的实验中，胚胎RPC将被用于在成人视网膜中重建RGCs， 已经耗尽了他们的内源性RGC。我们的基本假设是，对于RPC分化为 RGC，Atoh 7必须与其他调节因子整合，以实现增殖和增殖之间的平衡。 分化为了解决这个假设，我们提出了三个具体目标。第一个目标将决定 Atoh 7是否足以将非RGC转化为RGC命运。初步工作表明，更换 Neurod 1与Atoh 7导致RGC基因在内核层中的异位表达。我们将确定 Atoh 7是否可以驱动发育和成年视网膜中非RGC神经元中的RGC分化。第二 目的是确定Atoh 7是否调节Notch信号传导，以控制RPC增殖之间的平衡， 和研资局的承诺。在初步的实验中，我们发现Atoh 7结合到E-box元件上游， Notch 1和Atoh 7负调控Notch 1的表达。我们将确定Atoh 7 这与Notch信号有关。我们将确定Atoh 7和Notch 1是否参与了一个阴性反应， 反馈环以及当Notch 1上的Atoh 7结合位点缺失时RPC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，通过移植重建RGC缺失的视网膜， 表达Atoh 7的RPC与神经保护因子一起沿着进入RGC缺失小鼠的视网膜。我们还将 确定表达Atoh 7的RPC是否可以在视神经损伤和其他小鼠中再生视神经 模型 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。我们已经开发出人类视神经退化的现实遗传模型， 将最终用于干细胞替代疗法，以修复受损的视神经。

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.