Genetic Hierarchy Underlying Photoreceptor Regeneration

描述(申请人提供)：在大多数脊椎动物中，成年视网膜不能再生丢失的神经元，这会导致视力丧失。相比之下，斑马鱼的视网膜具有再生任何一类因各种侮辱而丢失的视网膜神经元的能力。例如，持续的强光会导致视杆细胞和视锥细胞死亡。光感受器再生起源于穆勒胶质细胞，它重新进入细胞周期并分裂产生神经前体细胞，神经前体细胞继续增殖，然后分化为再生的神经元。人的视网膜也拥有穆勒胶质细胞，但不能再生任何类型的视网膜细胞。我们感兴趣的是确定在光损伤的斑马鱼视网膜中调节光感受器视网膜再生的过程。在这个方案中，我们将研究在再生过程中产生和维持神经前体细胞群处于未分化和增殖状态的分子机制，这将确定在受损的哺乳动物视网膜中诱导完全再生反应的方法。这可能为患有遗传性失明的人提供一种恢复视力的策略，例如视网膜色素变性或黄斑变性。我们将探讨Pax6a、Pax6b和Orig2蛋白在光损伤斑马鱼视网膜神经前体细胞增殖过程中的作用。我们将使用我们开发的一种技术将反义吗啡肽电穿孔到再生的视网膜中。这项技术给了我们强大的能力，在光损伤的视网膜再生过程中有条件地阻止特定蛋白质的翻译。我们将验证这样的假设，即Pax6b蛋白是启动神经前体细胞增殖所必需的，但不是祖细胞转录因子随后表达所必需的，最终是光感受器细胞Opsins(S.A.1)。然后我们将确定Pax6a是否是神经前体细胞持续增殖和OLIG2基因转录所必需的(S.A.2)。最后，我们将确定是否Orig2也是神经前体细胞持续增殖所必需的，以及在没有Orig2的情况下，神经前体细胞是否以Muller神经胶质细胞为代价过早地分化为光感受器(S.A.3)。与公共健康相关：这项提案探索了再生成人眼睛视网膜神经元所需的关键基因检查点。揭示从Muller胶质细胞产生神经前体细胞的分子过程，并刺激这些前体细胞增殖，直到它们必须分化为缺失的神经元，可能会揭示在受损的人视网膜中诱导Muller胶质细胞再生神经元的方法。最终，这些结果可能会导致治疗不同形式的人类失明。

DESCRIPTION (provided by applicant): In most vertebrates, the adult retina is unable to regenerate lost neurons, which results in loss of vision. In contrast, the zebrafish retina possesses the ability to regenerate any class of retinal neurons that are lost due to a variety of insults. For example, constant intense light causes rod and cone photoreceptor death. Photoreceptor regeneration originates from the Muller glia that reenter the cell cycle and divide to produce neuronal progenitors, which continue to proliferate and then differentiate into the regenerated neurons. The human retina also possesses Muller glia, but fails to regenerate any retinal cell class. We are interested in identifying the processes that regulate retinal regeneration of photoreceptors in the light-damaged zebrafish retina. In this proposal, we will investigate the molecular mechanisms that generate and maintain the neuronal progenitor cell population in an undifferentiated and proliferating state during regeneration, which will identify approaches to induce a full regeneration response in the damaged mammalian retina. This could provide a strategy to restore vision to individuals who suffer from genetic forms of blindness, such as retinitis pigmentosa or macular degeneration. We will explore the roles of the Pax6a, Pax6b, and Olig2 proteins during proliferation of neuronal progenitors in the light-damaged zebrafish retina. We will use a technique that we developed to electroporate antisense morpholinos into the regenerating retina. This technique gives us the powerful ability to conditionally block the translation of specific proteins during regeneration of the light-damaged retina. We will test the hypothesis that the Pax6b protein is required for the initiation of neuronal progenitor cell proliferation, but is not required for the subsequent expression of progenitor cell transcription factors, and ultimately, photoreceptor cell opsins (S. A. 1). We will then determine if Pax6a is required for the continued proliferation of the neuronal progenitors and the transcription of the olig2 gene (S. A. 2). Finally, we will determine if Olig2 is also required for the continued proliferation of the neuronal progenitor cells and, if, in the absence of Olig2, the neuronal progenitors prematurely differentiate into photoreceptors at the expense of Muller glial cells (S. A. 3). PUBLIC HEALTH RELEVANCE: This proposal explores key genetic checkpoints that are required to regenerate retinal neurons in the adult eye. Uncovering the molecular processes that generate neuronal progenitor cells from the Muller glia and stimulating these progenitors to proliferate until they must differentiate into the missing neurons may reveal approaches to induce neuronal regeneration from the Muller glia in the damaged human retina. Ultimately, these results may lead to therapies for different forms of human blindness.