Vertebrate photoreceptor development and regeneration

项目总结 遗传性视网膜营养不良，如视网膜色素变性(RP)，是导致失明的主要原因 目前还没有治愈方法。视网膜退行性疾病的一种潜在治疗方法是细胞- 以移植为基础的疗法，将前体细胞移植到患病的眼睛中以 更换丢失的光感受器。虽然这是一种令人兴奋的可能性，但 必须克服移植疗法的实施，包括低效地整合 光感受器前体进入受体视网膜，以及获得足够数量的 临床应用的光感受器前体。为了改进这类细胞的体外培养方案 细胞，我们必须对促进规范的转录网络有更好的了解 光感受器前体。我们实验室的长期目标之一是为实现这些目标做出贡献 通过研究斑马鱼的光感受器发育和再生的努力。斑马鱼是 对研究光感受器生物学特别有用，因为它的视网膜含有大量的视锥 除棒材外，还有其他亚型。此外，与哺乳动物不同，斑马鱼的视网膜能够再生 神经元对实验性损伤的反应。本提案中描述的实验将定义 三种转录因子--SOX、SOX11和Her9--在视网膜神经发生中的作用 基因打靶和分子遗传学方法的应用。我们的具体目标如下： 具体目的I：确定Sox4和Sox11在光感受器分化中的作用。使用新的 产生的功能丧失突变体，在这个目标上，我们将1)确定是否存在剂量效应 SOX4/11活性的杆光感受器编号；2)使用最先进的时间推移成像 确定何时何地需要Sox4的表达才能获得正确的杆状感光细胞 开发；3)准确确定Sox4/11如何调节HH和BMP信号；以及4)确定 Sox4/11的分子靶点--特异靶II：确定光感受器的原因和程度 斑马鱼her9突变体的缺陷。在这一目标中，我们将1)确定 Her9突变体的光感受器缺陷；2)确定her9的缺失是否损害视力；3)确定 调节视网膜中her9表达的上游信号通路(S)；和4)决定 Her9介导的血管内皮生长因子在无血管斑马鱼视网膜中的表达是否调节视网膜 祖细胞的增殖和分化。我们提案的完成将弥合重要的差距 在我们对脊椎动物光感受器分化的分子机制的理解中， 揭示与开发治疗人类疾病的方法有关的基本原则 视网膜退行性疾病。

PROJECT SUMMARY Inherited retinal dystrophies such as retinitis pigmentosa (RP) are a leading cause of blindness for which there is currently no cure. One potential treatment for retinal degenerative diseases is cell- based transplantation therapy, whereby precursor cells are transplanted into the diseased eye to replace the lost photoreceptors. While this is an exciting possibility, several challenges to the implementation of transplantation therapies must be overcome, including the inefficient integration of photoreceptor precursors into the recipient retina, and the difficulty of obtaining sufficient numbers of photoreceptor precursors for clinical application. To improve protocols for the in vitro culture of such cells, we must have a better understanding of the transcriptional networks that promote specification of photoreceptor precursors. One of the long-term goals of our laboratory is to contribute to these efforts by studying photoreceptor development and regeneration in the zebrafish. The zebrafish is especially useful for studying photoreceptor biology, because its retina contains numerous cone subtypes in addition to rods. Furthermore, unlike mammals, the zebrafish retina is able to regenerate neurons in response to experimental damage. The experiments described in this proposal will define the role of three transcription factors -- Sox, Sox11, and Her9 -- during retinal neurogenesis through the application of gene targeting and molecular genetic approaches. Our specific aims are as follows: Specific Aim I: Determine the role of Sox4 and Sox11 in photoreceptor differentiation. Using newly generated loss-of-function mutants, in this aim, we will 1) determine whether there is a dosage effect of Sox4/11 activity on rod photoreceptor number; 2) use state-of-the-art time-lapse imaging to determine when and where Sox4 expression is required to achieve proper rod photoreceptor development; 3) determine precisely how Sox4/11 regulate Hh and Bmp signaling; and 4) identify the molecular targets of Sox4/11. Specific Aim II: Determine the cause and extent of photoreceptor defects in the zebrafish her9 mutant. In this aim, we will 1) determine the mechanism for photoreceptor defects in her9 mutants; 2) determine whether loss of Her9 impairs vision; 3) identify the upstream signaling pathway(s) that regulate her9 expression in the retina; and 4) determine whether Her9-mediated VEGF expression in the avascular zebrafish retina regulates retinal progenitor cell proliferation and differentiation. Completion of our proposal will bridge important gaps in our understanding of the molecular mechanisms of vertebrate photoreceptor differentiation, and reveal underlying principles relevant to the development of approaches for the treatment of human retinal degenerative disease.