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: 7678922
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7678922
• 关键词: Address; Adult; Biochemical; Blindness; Candidate Disease Gene; Cell Cycle; Cell Proliferation; Cells; Development; Disease; 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; Stem cells; Techniques; Testing; Translations; Vertebrate Photoreceptors; Vertebrates; Vision; Zebrafish; Zebrafish Proteins; base; cell injury; eye formation; mRNA Expression; nerve stem cell; neurogenesis; paralogous gene; prevent; progenitor; protein expression; protein function; regenerative; research study; response; retinal neuron; retinal regeneration; retinal rods; transcription factor; zebrafish development

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（由申请人提供）：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类型的视网膜神经元，包括光感受器。例如，成年斑马鱼视网膜的光诱导感光细胞损伤诱导Muller神经胶质重新进入细胞周期并产生神经元祖细胞。这些Muller胶质源性祖细胞迁移到外核层并分化成新的视杆细胞和视锥细胞。与斑马鱼相反，人类视网膜Muller细胞不能再生任何视网膜细胞类别。通过了解光损伤斑马鱼视网膜中基于Muller胶质的神经元再生的遗传和生化基础，我们可以确定刺激人类视网膜中类似再生反应的方法。这可能会产生一种策略，以恢复视力的个人与遗传和环境形式的失明。我们确定Pax 6在从Muller神经胶质细胞产生后不久在新的神经元祖细胞中表达。为了确定Pax 6蛋白在这些祖细胞再生过程中的作用，我们需要破坏Pax 6的表达。不幸的是，Pax 6是视网膜发育所必需的，在斑马鱼早期发育过程中Pax 6表达的破坏将阻止眼睛的形成。因此，我们开发了一种电穿孔技术，将反义morpholinos递送到再生成人视网膜的祖细胞中，并有条件地阻断Pax 6蛋白表达。我们的初步结果表明，我们可以阻断Pax 6在神经元祖细胞中的表达，从而阻止其增殖。我们将展示这种新技术的实用性，并确定Pax 6在祖细胞增殖和感光细胞再生中的作用。我们也将开始确定Pax 6的转录目标，可能在视网膜再生反应中发挥作用。 相关性：虽然几个候选基因被确定可能在斑马鱼视网膜再生中发挥重要作用，但必须开发一种方法来测试这些候选基因/蛋白质的功能。标准的正向遗传学方法将不适用于这种分析，因为这些基因中的大多数也是视网膜发育所必需的。我们描述了一种吗啉电穿孔技术，有条件地敲低所需的蛋白质在成人视网膜的表达。为了证明这种方法的力量，我们将检查Pax 6蛋白对斑马鱼视网膜再生的推测功能。许多视觉疾病导致人类光感受器的损失，不能再生。斑马鱼中强大的感光细胞再生涉及表达Pax 6蛋白，因为Muller神经胶质细胞分裂并产生神经元祖细胞，然后更新丢失的感光细胞。了解Pax 6在斑马鱼视网膜再生中的作用可能会为人类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神经胶质细胞再生人类视网膜中的光感受器的能力。

项目成果

Pax6a and Pax6b are required at different points in neuronal progenitor cell proliferation during zebrafish photoreceptor regeneration.

• DOI: 10.1016/j.exer.2010.02.001
• 发表时间: 2010-05
• 期刊: EXPERIMENTAL EYE RESEARCH
• 影响因子: 3.4
• 作者: Thummel, Ryan;Enright, Jennifer M.;Kassen, Sean C.;Montgomery, Jacob E.;Bailey, Travis J.;Hyde, David R.
• 通讯作者: Hyde, David R.