Role of Pax6 in photoreceptor cell regeneration using conditional knockdowns

Pax6 在条件敲低感光细胞再生中的作用

• 批准号: 7450294
• 负责人: David R Hyde
• 金额: $ 22.5万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7450294
• 关键词: Address; Adult; Biochemical Genetics; Blindness; Candidate Disease Gene; Cell Cycle; Cell Proliferation; Cells; Class; Development; Disease; Disruption; Electroporation; Future; Gene Expression; Gene Proteins; Gene Targeting; Genes; Genetic; Human; Individual; Inner Nuclear Layer; Lead; Light; Methods; Microarray Analysis; Molecular; Muller' s cell; Natural regeneration; Neuroglia; Neurons; Nuclear; Photoreceptors; Play; Proteins; Retina; Retinal; Retinal Cone; Role; Role playing therapy; Standards of Weights and Measures; Stem cells; Techniques; Testing; Translations; Vertebrate Photoreceptors; Vertebrates; Vision; Zebrafish; Zebrafish Proteins; base; cell injury; desire; eye formation; mRNA Expression; nerve stem cell; neurogenesis; paralogous gene; prevent; progenitor; protein expression; protein function; research study; response; retinal neuron; retinal regeneration; retinal rods; transcription factor

DESCRIPTION (provided by applicant): The retinas of most adult vertebrates cannot regenerate neurons. In contrast, adult zebrafish regenerate all classes of retinal neurons including the photoreceptors. For example, light-induced photoreceptor cell damage of the adult zebrafish retina induces the Muller glia to reenter the cell cycle and generate neuronal progenitor cells. These Muller glial-derived progenitors migrate to the outer nuclear layer and differentiate into new rods and cones. In contrast to zebrafish, human retinal Muller cells cannot regenerate any retinal cell class. By understanding the genetic and biochemical basis of Muller glia-based neuronal regeneration in the light-damaged zebrafish retina, we may identify approaches to stimulate a similar regeneration response in the human retina. This could yield a strategy to restore vision to individuals with genetic and environmental forms of blindness. We determined Pax6 is expressed in the new neuronal progenitor cells shortly after being generated from the Muller glial cells. To determine the role of the Pax6 protein in these progenitors during regeneration, we need to disrupt Pax6 expression. Unfortunately, Pax6 is required for retinal development and disruption of Pax6 expression during early zebrafish development will prevent eye formation. Thus, we developed an electroporation technique to deliver antisense morpholinos into the progenitor cells of the regenerating adult retina and conditionally block Pax6 protein expression. Our preliminary results demonstrate we can block Pax6 expression in the neuronal progenitor cells, which prevents their proliferation. We will demonstrate the utility of this new technique and determine the role of Pax6 in progenitor cell proliferation and photoreceptor regeneration. We will also begin to identify Pax6 transcriptional targets that may play roles during the retinal regeneration response. Relevance: While several candidate genes were identified that may play important roles for retinal regeneration in zebrafish, a method must be developed to test the function of these candidate genes/proteins. Standard forward genetic approaches will not be amenable for this analysis because most of these genes are also required for retinal development. We describe a morpholino electroporation technique to conditionally knockdown the expression of the desired protein in the adult retina. To demonstrate the power of this method, we will examine the speculative function of the Pax6 protein for zebrafish retinal regeneration.Many vision diseases cause loss of human photoreceptors that cannot be regenerated. Robust photoreceptor regeneration in zebrafish involves expressing the Pax6 protein as the Muller glial cells divide and produce neuronal progenitor cells, which then renew the lost photoreceptors. Understanding the role of Pax6 in zebrafish retinal regeneration may provide clues as to why the human Muller glial cells cannot regenerate photoreceptors, and ultimately lead to the ability to induce endogenous Muller glial cells to regenerate photoreceptors in the human retina.

描述(申请人提供)：大多数成年脊椎动物的视网膜不能再生神经元。相比之下，成年斑马鱼再生所有种类的视网膜神经元，包括光感受器。例如，成年斑马鱼视网膜的光感受器细胞损伤诱导Muller胶质细胞重新进入细胞周期并产生神经前体细胞。这些穆勒胶质来源的祖细胞迁移到外核层，并分化为新的视杆和视锥细胞。与斑马鱼不同，人类视网膜米勒细胞不能再生任何类型的视网膜细胞。通过了解光损伤斑马鱼视网膜中基于Muller胶质细胞的神经元再生的遗传和生化基础，我们可以找到在人类视网膜中刺激类似再生反应的方法。这可能会产生一种策略，使患有遗传性和环境性失明的人恢复视力。我们确定Pax6在从Muller神经胶质细胞产生后不久就在新的神经前体细胞中表达。为了确定Pax6蛋白在这些祖细胞再生过程中的作用，我们需要干扰Pax6的表达。不幸的是，Pax6是视网膜发育所必需的，在斑马鱼发育的早期，破坏Pax6的表达将阻止眼睛的形成。因此，我们开发了一种电穿孔技术，将反义吗啉转移到再生成人视网膜的祖细胞中，并有条件地阻断Pax6蛋白的表达。我们的初步结果表明，我们可以阻止Pax6在神经前体细胞中的表达，从而阻止它们的增殖。我们将展示这项新技术的实用性，并确定Pax6在祖细胞增殖和光感受器再生中的作用。我们还将开始确定可能在视网膜再生反应中发挥作用的Pax6转录靶点。 相关性：虽然已经确定了几个可能对斑马鱼视网膜再生起重要作用的候选基因，但必须开发一种方法来测试这些候选基因/蛋白的功能。标准的正向遗传方法不适用于这种分析，因为这些基因中的大多数也是视网膜发育所必需的。我们描述了一种吗啡诺电穿孔技术，有条件地敲除成人视网膜中所需蛋白质的表达。为了证明这种方法的威力，我们将研究Pax6蛋白对斑马鱼视网膜再生的推测功能。许多视力疾病会导致无法再生的人类光感受器的丧失。斑马鱼强健的光感受器再生涉及随着Muller胶质细胞分裂并产生神经前体细胞，从而更新丢失的光感受器，从而表达Pax6蛋白。了解Pax6在斑马鱼视网膜再生中的作用可能为人类Muller神经胶质细胞不能再生光感受器提供线索，并最终导致诱导内源性Muller神经胶质细胞在人类视网膜再生光感受器的能力。