Role of the miR 221-222 cluster in vascular development

项目摘要 新血管的形成是胚胎发育以及 许多疾病。控制血管形成和重塑的过程是 保存允许使用模型系统来研究这个过程。斑马鱼出现在 作为一个理想的遗传系统，解剖参与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。 在血管发育期间的体内细胞行为。最近的研究表明， 编码RNA，称为microRNA，在血管系统的正常发育中。 然而，人们对miRNAs在这一过程中控制的信号通路知之甚少。使用 通过深度测序的方法，我已经鉴定了许多内皮表达的miRNAs。 此外，这些miRNAs的初步功能特征表明，它们发挥不同的作用， 在血管发育中的特殊作用。在这个建议中，我将研究两个内皮细胞的作用， miR-221和miR-222，通过产生在miRNA内具有靶向缺失的斑马鱼， 使用锌指核酸酶的种子序列。与此同时，我会生成一个内皮细胞特异性的 表达myc标记的Argonaute 2（Ago 2）的转基因系。这条线将允许我纯化miR-221 和miR-222靶向复合物特异性地从内皮细胞通过免疫沉淀 Ago2. miRNA敲除斑马鱼的详细表型分析，沿着 确定相关的内皮细胞特异性miRNA靶点将使我能够确定哪些途径， 细胞行为miR-221或miR-222可能是控制的。这些发现将使我能够剖析 血管发育过程中内皮细胞miRNAs调控的遗传网络。统称 能够将联合收割机对miRNA的靶向遗传操作沿着详细成像 斑马鱼中可用的方法，将使我能够获得关于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.