Molecular Biology of Retinal Development

视网膜发育的分子生物学

• 批准号: 6605724
• 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/6605724
• 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; 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（由申请人提供）：该项目的长期目标是 阐明感光细胞命运的分子机制 脊椎动物视网膜中的规格。我们最近的研究表明 驱动感光细胞命运规范的遗传程序涉及 Neurod和Neurogenin2（NGN2）。当前项目解决了四个特定的 问题：（1）在正确的位置在正确的位置表达Neurod和Ngn2 正确的时候是参与感光神经发生的内在因素吗？ 双重标记将用于确定表达NGN2或 神经障碍是增殖，有丝分裂后或两者兼而有之。 （2）NGN2的作用是什么 在光感受器细胞命运测定中？ NGN2是否指定光感受器的命运 独家，或者这也导致了其他类型的视网膜的起源 细胞？功能丧失分析都将用于研究 NGN2的功能，使用NGN2的逆转录病毒驱动的misexpression和Active 在发育中的小鸡视网膜中的抑制构造。视网膜色素上皮 （RPE）将使用第6天的小鸡胚胎衍生的细胞培养物检查 NGN2是否可以选择性地诱导RPE转分化向A 光感受器命运。 （3）受感受器的产生需要神经棒吗？在 其他词，会抑制神经表达和抑制 神经胶质蛋白功能导致否则会导致光感受器不足 正常的视网膜？反义寡核苷酸和插入介导的活性 抑制作用将用于抑制神经障碍表达和神经障碍蛋白 功能分别。将分析视网膜神经发生以确定 神经障碍表达和功能的衰减是否会产生 感受者缺陷视网膜。 （4）神经轨道足以诱导整个 当在RPE异位表达光感受器分化的程序 将为分化环境提供的细胞 小鸡胚胎的视网膜下空间？将分化的细胞放置 进入视网膜神经发生的雏鸡胚胎下空间 被操纵以产生较少的感光细胞。发展潜力 将在分子和细胞上评估显微注射的细胞的 确定这些细胞是否可以发展成善意的水平 感受器。这四项研究有望对遗传控制的新见解 光感受器命运规范。此外，他们可能有临床 含义：神经与其他因素结合使用，可能能够 将培养的RPE细胞转化为感光细胞，作为细胞来源 感光器替代疗法。