Molecular Biology of Retinal Development

视网膜发育的分子生物学

• 批准号: 6399508
• 负责人: SHU-ZHEN WANG
• 金额: $ 28.7万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6399508
• 关键词: cell differentiation; cell proliferation; cell type; chick embryo; data collection methodology /evaluation; developmental neurobiology; gene expression; genetic regulatory element; immunocytochemistry; in situ hybridization; molecular biology; neurogenesis; northern blottings; organ culture; polymerase chain reaction; protein structure function; radionuclide double label; retina; retinal pigment epithelium; transcription factor; visual photoreceptor; western blottings

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the molecular mechanisms underlying photoreceptor cell fate specification in the vertebrate retina. Our recent studies suggest that the genetic program that drives photoreceptor cell fate specification involves neuroD and neurogenin2 (ngn2). The current project addresses four specific questions: (1) Are neuroD and ngn2 expressed at the right place during the right time to be intrinsic factors participating in photoreceptor neurogenesis? Double labeling will be used to determine whether cells expressing ngn2, or neuroD, are proliferating, postmitotic, or both. (2) What is the role of ngn2 in photoreceptor cell fate determination? Does ngn2 specify photoreceptor fate exclusively, or does it also lead to the genesis of other types of retinal cells? Both gain- and loss-of-function analyses will be used to study the function of ngn2, using retroviral-driven misexpression of ngn2 and an active repression construct in the developing chick retina. Retinal pigment epithelial (RPE) cell cultures derived from day 6 chick embryos will be used to examine whether ngn2 can selectively induce RPE transdifferentiation towards a photoreceptor fate. (3) Is neuroD required for photoreceptor production? In other words, will the suppression of neuroD expression and the repression of NeuroD protein function result in a photoreceptor deficit in an otherwise normal retina? Antisense oligonucleotides and Engrailed-mediated active repression will be used to suppress neuroD expression and NeuroD protein function, respectively. Retinal neurogenesis will be analyzed to determine whether attenuation of neuroD expression and function will produce photoreceptor-deficient retinas. (4) Is neuroD sufficient to induce the entire program of photoreceptor differentiation when ectopically expressed in RPE cells that will be provided with an environment for differentiation within the subretinal space of a chick embryo? Transdifferentiating cells will be placed into the subretinal space of chick embryos whose retinal neurogenesis has been manipulated to produce fewer photoreceptor cells. The developmental potential of the microinjected cells will be assessed at the molecular and cellular levels to determine whether these cells can develop into bona fide photoreceptors. These four studies promise new insight into the genetic control of photoreceptor fate specification. Furthermore, they may have clinical implications: neuroD, in conjunction with other factors, may be able to transform cultured RPE cells into photoreceptor cells as a source of cells for photoreceptor replacement therapies.

描述(申请人提供)：本项目的长期目标是 阐明光感受器细胞命运的分子机制 脊椎动物视网膜的规格。我们最近的研究表明， 驱动光感受器细胞命运的遗传程序包括 Neurod和Neurogenin2(Ngn2)。目前的项目涉及四个具体的 问题：(1)Neurod和Ngn2在 成为参与光感受器神经发生的内在因素是合适的时机吗？ 将使用双重标记来确定表达Ngn2或 神经细胞是增殖的、有丝分裂后的或两者兼而有之。(2)Ngn2的作用是什么？ 光感受器细胞命运的决定？Ngn2是否决定了光感受器的命运 或者它也导致了其他类型的视网膜的发生 细胞？将使用增益分析和损失函数分析来研究 Ngn2的功能，利用逆转录病毒驱动的Ngn2的错误表达和活性 发育中的雏鸡视网膜中的抑制结构。视网膜色素上皮 从6日龄鸡胚胎中提取的(RPE)细胞培养将被用于检测 Ngn2是否可以选择性地诱导RPE转分化为 光感受器命运。(3)光感受器的产生需要神经递质吗？在……里面 换句话说，对神经性表达的抑制和对 神经蛋白功能导致光感受器缺陷 视网膜正常吗？反义寡核苷酸及其载体介导的活性 抑制将被用来抑制神经D表达和神经D蛋白 函数。将对视网膜神经发生进行分析以确定 神经表达和功能的减弱是否会产生 光感受器缺陷的视网膜。(4)神经质是否足以诱导整个 RPE异位表达时光感受器分化的程序 将为细胞提供分化的环境 小鸡胚胎的视网膜下空间？转分化细胞将被放置 进入视网膜下空间的小鸡胚胎，其视网膜神经发生已经 被操控以产生更少的感光细胞。发展潜力 将在分子和细胞层面进行评估 以确定这些细胞是否能发育成真正的 光感受器。这四项研究为基因控制提供了新的视角。 光感受器命运规范。此外，他们可能有临床上的 暗示：神经症，结合其他因素，可能能够 将培养的RPE细胞转化为光感受器细胞作为细胞来源 光感受器替代疗法。