Role of Pax6 in photoreceptor cell regeneration using conditional knockdowns

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别的视网膜神经元，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元祖细胞。这些Muller胶质细胞衍生的祖细胞迁移到外核层并分化成新的杆状细胞和锥状细胞。与斑马鱼不同，人类视网膜Muller细胞不能再生任何一类视网膜细胞。通过了解光损伤斑马鱼视网膜中Muller神经胶质细胞再生的遗传和生化基础，我们可能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，使遗传和环境形式的失明患者恢复视力。我们确定Pax6在Muller胶质细胞生成后不久在新的神经祖细胞中表达。为了确定Pax6蛋白在这些祖细胞再生过程中的作用，我们需要破坏Pax6的表达。不幸的是，Pax6是视网膜发育所必需的，在斑马鱼发育早期，Pax6表达的中断将阻止眼睛的形成。因此，我们开发了一种电穿孔技术，将反义morpholinos传递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，我们可以阻断Pax6在神经祖细胞中的表达，从而阻止它们的增殖。我们将展示这种新技术的实用性，并确定Pax6在祖细胞增殖和光感受器再生中的作用。我们也将开始确定Pax6转录靶点，可能在视网膜再生反应中发挥作用。

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.