Using zebrafish as a model system for investigating lens development, aging and cataract.

这项提议的目标是使用斑马鱼作为一个模型系统来确定新的调节器 人工晶状体的发育和检查单个和结合的α-晶状体蛋白的损失，小热 对晶状体功能至关重要的休克蛋白，影响晶状体老化和晶状体结晶蛋白的调节 表情。这些实验将使我们更好地了解镜片的发展和 维持晶状体透明度对于开发防止晶状体混浊的治疗方法很重要， 是世界范围内导致失明的主要原因。我们实验室的工作帮助斑马鱼成为一种 研究晶状体发育、功能和疾病的强大模型系统。本科生 过去资助的学生已经克隆并鉴定了三种斑马鱼α-晶体蛋白 并使用CRISPR/Cas9基因编辑来禁用每个人，以检查他们的损失对早期 镜头显影。在这里，我们建议在Aim I中使用我们已经生产的敲击线来建模 α-晶状体蛋白丢失对晶状体透明度维持及基因调控的影响 控制晶状体蛋白质表达的网络。这一目标将包括整合转录本 蛋白质组学分析，以及由此产生的晶状体表型的解剖学检查。我们 假设所产生的晶状体表型将取决于特定的α-晶体蛋白丢失， 提供了一种新的αB-晶体蛋白功能的检查，这是由于在 斑马鱼为单拷贝哺乳动物基因。在AIM II中，我们将使用转录数据集 由我们的合作者从老鼠和斑马鱼组织中产生，以确定可能的 调节晶状体发育的转录因子(TF)。然后，我们将有效地瞄准这些TF 使用CRISPR/CAS9方法，使我们能够直接在注射过程中筛查晶状体表型 胚胎。破坏晶状体正常发育的突变的TF等位基因将被携带到稳定的品系中 详细说明它们在建立和维护镜头透明度方面所起的作用。我们预计这些 两个目标将有效地识别晶状体发展的新调节因素，并澄清α的作用- 晶状体蛋白在维持晶状体透明度方面的作用。建议的实验将提供极好的 在研究环境中为本科生提供培训机会 成功。

The goal of this proposal is to use zebrafish as a model system to identify novel regulators of ocular lens development and examine how loss of single and combined α-crystallins, small heat shock proteins important for lens function, affect lens aging and the regulation of lens crystallin expression. These experiments will produce a better understanding of lens development and maintenance of lens transparency important for developing treatments to prevent lens opacities, the leading cause of blindness worldwide. Work in our laboratory has helped make the zebrafish a powerful model system for studying lens development, function and disease. Undergraduate students supported by past funding have cloned and characterized three zebrafish α-crystallins and used CRISPR/Cas9 gene editing to disable each to examine the impact of their loss on early lens development. Here, we propose in Aim I to use our already produced knockout lines to model the impact of α-crystallin loss on the maintenance of lens transparency and the gene regulatory networks that control lens protein expression. This aim will include an integration of transcriptomic and proteomic analyses, along with anatomical examination of resulting lens phenotypes. We hypothesize that the resulting lens phenotypes will depend on the specific α-crystallin lost, providing a novel examination of αB-crystallin function due to the presence of two orthologs in zebrafish for the single-copy mammalian gene. In Aim II we will use transcriptomic data sets generated by our collaborators from mouse and zebrafish tissues to identify possible novel transcription factors (TFs) regulating lens development. We will then efficiently target these TFs using CRISPR/Cas9 methods that will allow us to screen for lens phenotypes directly in injected embryos. Mutant TF alleles that disrupt normal lens development will be carried into stable lines to detail the role they play in establishing and maintaining lens transparency. We expect that these two aims will efficiently identify novel regulators of lens development and clarify the role of α- crystallins in maintaining lens transparency. The experiments proposed will provide excellent training opportunities for undergraduates in a research setting with a strong track record of student success.