Role of the miR 221-222 cluster in vascular development

项目总结 新血管的形成是胚胎发育和发育所必需的。 无数种疾病的威胁。管理血管形成和重塑的过程是 保守地允许使用模型系统来研究这一过程。斑马鱼已经出现了 作为一个理想的遗传系统来剖析涉及血管发育的分子机制。 此外，斑马鱼胚胎的透明度有助于直接观察内皮细胞。 血管发育过程中细胞在体内的行为。最近的研究表明，小型非政府组织 编码RNA，称为microRNAs，在血管系统的正常发育中。 然而，对miRNAs在这一过程中控制的信号通路知之甚少。vbl.使用 采用深度测序的方法，我已经鉴定了一些内皮细胞表达的miRNAs。 此外，这些miRNAs的初步功能特征表明，它们发挥着独特的 在血管发育中的特殊作用。在这项提案中，我将研究两种内皮细胞的作用 MiRNA，miR-221和miR-222，通过在miRNA中产生带有靶向缺失的斑马鱼 使用锌指核酸酶的种子序列。同时，我将产生一种内皮细胞特异性 表达myc基因的转基因细胞株Ago2。这一行将允许我提纯miR-221 和miR-222靶向复合物，通过免疫沉淀从内皮细胞特异性地 AGO2。MiRNA基因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靶点将使我能够确定哪些途径和 细胞行为可能由miR-221或miR-222控制。这些发现将使我能够剖析 血管发育过程中受内皮miRNAs调控的遗传网络。团结在一起， 能够将miRNAs的靶向遗传操作与详细的成像相结合 斑马鱼中可用的方法，将使我能够对miRNAs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.