Mechanisms of photoreceptor specification and morphogenesis

项目总结 尽管我们依赖视锥细胞介导的视力，以及视锥细胞的衰弱影响 在我们的中心视力退化的情况下，我们对基因和 调节光感受器数量和空间模式的机制。这背后的原因是 非对称性是视网膜神经母细胞重新进入有丝分裂或分化的根本选择。 斑马鱼幼体视网膜提供了一个无与伦比的遗传模型来识别基本的 整合感光器规格和空间图案化的机制。斑马鱼幼体 视网膜在解剖和功能上以视锥为主，有4种视锥亚型排列成 高度有序的马赛克。棒的数量要少得多，并且沿 背轴/腹轴。遗传工具的范围、基因组资源的可获得性和获得 斑马鱼胚胎，允许一种系统的方法来揭示基因的影响 对细胞命运及其分布的操纵。我们的前提是基于严格的基因 和分子分析，在我们发表的研究和初步数据表明，tbx2b/or和 Six7/LJR是维持以视锥为主的斑马鱼视网膜所必需的。Tbx2b基因突变 由于SWS1-的细胞命运开关，导致棒的增加和均匀分布的5倍 视锥细胞转化为视锥细胞，一种与RD7小鼠相反的表型和增强型S视锥综合征 在人类身上。Six3/6的同源基因Six7的突变导致类似的增加和一致 杆的分布，然而，几个等位基因的特征表明，Six7独立 抑制晚期祖细胞的有丝分裂，是锥体亚型生存所必需的。我们的 目的是了解tbx2b和six7在细胞命运中的潜在作用机制。 调控光感受器的已知因素背景下的决策和空间模式 发展。AIM 1将利用基于转座子的创新基因组编辑工具 转基因，以及更常规的方法来检验Six7调节选择的假设 晚期光感受器前体细胞之间持续有丝分裂或分化。目标2 结合体内和体外方法来验证我们的新假设，即tbx2b调节 光感受器测定的时间，从而通过分子进行空间构图 与已知的光感受器转录因子相互作用。此外，我们未公布的数据 已发表的报告显示，基因表达在遗传后发生了意想不到的变化 这些变化表明这些因素在维持细胞命运方面的作用尚未被认识到。因此， 具体目标的完成将极大地促进机制领域的知识 调节高度特化的、视锥为主的视网膜的产生。

PROJECT SUMMARY Despite our dependency upon cone-mediated vision, and the debilitating impact of cone degeneration upon our central vision, major gaps exist in our knowledge of genes and mechanisms that regulate the number and spatial patterning of photoreceptors. Underlying this asymmetry is the fundamental choice of retinal neuroblasts to re-enter mitosis or differentiate. The larval zebrafish retina provides an unparalleled genetic model to identify fundamental mechanisms integrating photoreceptor specification and spatial patterning. The larval zebrafish retina is anatomically and functionally cone-dominated with 4 cone subtypes arranged in a highly ordered mosaic. Rods are far less numerous and distributed asymmetrically along the dorsal/ventral axis. The range of genetic tools, availability of genomic resources and access to the zebrafish embryo, permit a systematic approach to uncover the impact of genetic manipulations upon cell fate and their distribution. Our premise is based upon rigorous genetic and molecular analysis in our published studies and preliminary data showing that tbx2b/lor and six7/ljr are essential for maintaining the cone-dominated zebrafish retina. Mutations of tbx2b result in a 5-fold increase and uniform distribution of rods due to a cell fate switch of SWS1- cones into rods, a phenotype opposite to that of the rd7 mouse and enhanced-S cone syndrome in humans. Mutations of six7, an orthologue of Six3/6 result in a similar increase and uniform distribution of rods, however characterization of several alleles revealed that six7 independently suppresses mitosis of late stage progenitors, and is essential for survival of a cone subtype. Our goal is to understand the mechanisms underlying functions of tbx2b and six7 in cell fate decisions and spatial patterning in the context of know factors that regulate photoreceptor development. Aim 1 will take advantage of innovative genome editing tools, transposon-based transgenesis, and more routine methods to test the hypothesis that six7 modulates the choice of late stage photoreceptor progenitors between continued mitosis or differentiation. Aim 2 combined in vivo and in vitro approaches to test our novel hypothesis that tbx2b regulates the timing of photoreceptor determination and thereby spatial patterning through molecular interaction with known photoreceptor transcription factors. Furthermore, our unpublished data and published reports show unexpected alterations in gene expression following genetic alterations suggesting unrecognized roles for these factors in maintaining cell fate. Therefore, completion of the specific aims will significantly advance the fields knowledge of mechanisms regulating the generation of a highly specialized, cone-dominated retina.