Genetic Hierarchy Underlying Photoreceptor Regeneration

描述（由申请人提供）：在大多数脊椎动物中，成年视网膜不能再生失去的神经元，这导致视力丧失。相比之下，斑马鱼的视网膜具有再生任何类型的视网膜神经元的能力，这些神经元是由于各种损伤而丢失的。例如，持续的强光会导致杆状和锥状光感受器死亡。光感受器再生起源于Muller胶质细胞，Muller胶质细胞重新进入细胞周期并分裂产生神经元祖细胞，祖细胞继续增殖并分化为再生的神经元。人类视网膜也有穆勒神经胶质，但不能再生任何视网膜细胞类别。我们感兴趣的是确定在光损伤的斑马鱼视网膜的光感受器的视网膜再生调节过程。在本研究中，我们将研究再生过程中产生和维持未分化和增殖状态的神经祖细胞群的分子机制，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。这可能为遗传性失明（如视网膜色素变性或黄斑变性）患者提供一种恢复视力的策略。我们将探讨Pax6a， Pax6b和Olig2蛋白在光损伤斑马鱼视网膜中神经元祖细胞增殖中的作用。我们将使用我们开发的一种技术将反义morpholinos电穿孔到再生的视网膜中。这项技术使我们有能力在光损伤视网膜再生过程中有条件地阻断特定蛋白质的翻译。我们将验证一种假设，即Pax6b蛋白是神经元祖细胞增殖起始所必需的，但不是祖细胞转录因子随后表达所必需的，最终，光感受器细胞视蛋白（s.a.1）。然后，我们将确定Pax6a是否为神经元祖细胞的持续增殖和olig2基因的转录所必需（s.a.2）。最后，我们将确定Olig2是否也是神经元祖细胞持续增殖所必需的，如果，在缺乏Olig2的情况下，神经元祖细胞过早地分化为光感受器，以牺牲Muller胶质细胞为代价（s.a. 3）。公共卫生相关性：本提案探讨了成人眼睛视网膜神经元再生所需的关键遗传检查点。揭示从Muller胶质细胞中产生神经元祖细胞并刺激这些祖细胞增殖直到它们必须分化为缺失的神经元的分子过程，可能会揭示诱导受损视网膜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.