Optic nerve regeneration: gene networks in retina development

描述（由申请人提供）：我们在拟议实验中的长期目标是获得关于控制视网膜发育的基本机制的新知识，并应用这些知识开发治疗视网膜退行性疾病的新方法。我们的策略是使用我们已经创建或将要创建的基因工程小鼠模型。尽管在控制视网膜发育的机制上已经积累了大量的信息，但仍然存在很大的差距。特别是，控制祖细胞决定是增殖还是分化的机制只有模糊的理解。更好的理解对于寻找新的方法来修复受损的视网膜是非常重要的。视网膜神经节细胞（RGC）是视网膜祖细胞（RPC）在发育过程中分化的第一种细胞类型，并且是连接到大脑的视网膜神经元。我们专注于导致研究资助委员会（RPC）承诺成为研究资助局（RGC）的监管事件。在相关实验中，胚胎RPC将用于在已经耗尽其内源性RGC的成人视网膜中重新填充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，以实现增殖和分化之间的平衡。为了解决这一假设，我们提出了三个具体目标。第一个目标将确定Atoh 7是否足以将非RGC转化为RGC命运。初步研究表明，用Atoh 7替换Neurod 1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神经元的RGC分化。第二个目标将确定Atoh 7是否调节Notch信号传导以控制RPC增殖和RGC承诺之间的平衡。在初步实验中，我们发现Atoh 7结合到Notch 1上游的E-box元件，并且Atoh 7负调节Notch 1表达。我们将确定Atoh 7相对于Notch信号传导出现的时间。我们将确定Atoh 7和Notch 1是否参与负反馈循环，以及当Notch 1上的Atoh 7结合位点被删除时RPC增殖是否受到干扰。在第三个目标中，我们将通过将表达Atoh 7的RPC与神经保护因子一起移植到RGC耗尽的小鼠的视网膜中，来优化我们关于重新填充RGC耗尽的视网膜的实验。我们还将确定表达Atoh 7的RPC是否可以在视神经挤压和其他小鼠模型中再生视神经。我们对控制视网膜发育的因素的了解使我们能够将发育概念应用于成人视网膜。我们已经开发出了人类视神经退行性变的现实遗传模型，该模型最终将用于干细胞替代疗法以修复受损的视神经。 公共卫生相关性：为了了解控制多能神经祖细胞向终末分化神经元进展的基本遗传程序，我们研究了被编程为分化为视网膜神经节细胞的视网膜祖细胞。我们使用基因工程小鼠结合策略来阐明视网膜神经节细胞基因调控网络，此外，我们开发了人类视神经变性的现实遗传模型，最终用于干细胞替代疗法，以恢复视网膜神经节细胞并再生受损的视神经。

DESCRIPTION (provided by applicant): 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 cell (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. PUBLIC HEALTH RELEVANCE: In order to understand the fundamental genetic programs controlling the advancement of a multipotent neural progenitor cell to a terminally differentiated neuron, we study retinal progenitor cells that are programmed to differentiate into retinal ganglion cells. We use genetically engineered mice coupled with strategies to elucidate the retinal ganglion cell gene regulatory network, and in addition, we develop realistic genetic models for human optic nerve degeneration for ultimate use in stem cell replacement therapy to restore retinal ganglion cells and regenerate damaged optic nerves.