Role of the miR 221-222 cluster in vascular development

项目概要 新血管的形成是胚胎发育和进展所必需的 的多种疾病。控制血管形成和重塑的过程是 保守允许使用模型系统来研究这个过程。斑马鱼出现了 作为剖析血管发育分子机制的理想遗传系统。 此外，斑马鱼胚胎的透明度有利于内皮细胞的直接可视化 血管发育过程中的体内细胞行为。最近的工作涉及小型非 编码 RNA（称为 microRNA）参与血管系统的正常发育。 然而，人们对 miRNA 在此过程中控制的信号通路知之甚少。使用 通过深度测序方法，我鉴定出了许多内皮表达的miRNA。 此外，这些 miRNA 的初步功能表征表明它们发挥着独特的作用 在血管发育中发挥特殊作用。在这个提案中，我将研究两种内皮细胞的作用 miRNA、miR-221 和 miR-222，通过生成 miRNA 内带有靶向删除的斑马鱼 使用锌指核酸酶的种子序列。同时，我将生成内皮细胞特异性 表达 myc 标记的 Argonaute 2 (Ago2) 的转基因系。这条线可以让我纯化 miR-221 miR-222 通过免疫沉淀特异性靶向内皮细胞的复合物 前2. miRNA 敲除斑马鱼的详细表型分析，以及 识别相关的内皮细胞特异性 miRNA 靶标将使我能够确定哪些途径和 miR-221或miR-222可能控制细胞行为。这些发现将使我能够剖析 血管发育过程中内皮 miRNA 调控的遗传网络。在一起， 将 miRNA 的靶向基因操作与详细成像相结合的能力 在斑马鱼中可用的方法将使我能够获得关于 miRNA 在 血管发育。

PROJECT SUMMARY The formation of new blood vessels is required for embryonic development as well as the progression of numerous diseases. The processes that govern blood vessel formation and remodeling are conserved allowing the use model systems to investigate this process. The zebrafish has emerged as an ideal genetic system to dissect the molecular mechanisms involved in vascular development. Furthermore, the transparency of the zebrafish embryos facilitates direct visualization of endothelial cell behaviors in vivo during blood vessel development. Recent work has implicated small non- coding RNAs, referred to as microRNAs, in the proper development of the vascular system. However, little is known about the signaling pathways that miRNAs control during this process. Using deep sequencing methods, I have identified a number of endothelial expressed miRNAs. Furthermore, initial functional characterization of these miRNAs suggests that they play distinct and specific roles in vascular development. In this proposal, I will investigate the role of two endothelial miRNAs, miR-221 and miR-222, by generating zebrafish bearing targeted deletions within miRNA seed sequences using zinc finger nucleases. In parallel, I will generate an endothelial cell-specific transgenic line expressing myc tagged Argonaute 2 (Ago2). This line will allow me to purify miR-221 and miR-222 target complexes specifically from endothelial cells through immunoprecipitation of Ago2. The detailed phenotypic analysis of miRNA-knockout zebrafish, along with the ability to identify relevant endothelial-cell specific miRNA targets will allow me to determine what pathways and cell behaviors miR-221 or miR-222 may be controlling. These findings will then enable me to dissect the genetic networks regulated by endothelial miRNAs during vascular development. Together, the ability to combine targeted genetic manipulation of miRNAs along with the detailed imaging approaches available in the zebrafish, will allow me to gain novel insights onto the role of miRNAs in vascular development.