Role of the miR 221-222 cluster in vascular development

描述（由申请人提供）：新血管的形成是胚胎发育和许多疾病进展所必需的。控制血管形成和重塑的过程是保守的，允许使用模型系统来研究这一过程。斑马鱼已经成为一个理想的遗传系统来解剖参与血管发育的分子机制。此外，斑马鱼胚胎的透明性有助于直接观察血管发育过程中内皮细胞在体内的行为。最近的研究表明，小的非编码rna，即微rna，在血管系统的正常发育中起着重要作用。然而，在这一过程中，我们对mirna控制的信号通路知之甚少。使用深度测序方法，我已经确定了一些内皮细胞表达的mirna。此外，这些mirna的初步功能表征表明，它们在血管发育中发挥着独特而特定的作用。在本提案中，我将研究两个内皮miRNA， miR-221和miR-222的作用，通过使用锌指核酸酶在miRNA种子序列中产生具有靶向缺失的斑马鱼。同时，我将生成内皮细胞特异性转基因系，表达myc标记的Argonaute 2 （Ago2）。这条线将允许我通过Ago2的免疫沉淀从内皮细胞特异性地纯化miR-221和miR-222靶复合物。对miRNA敲除的斑马鱼进行详细的表型分析，以及识别相关内皮细胞特异性miRNA靶点的能力，将使我能够确定miR-221或miR-222可能控制的途径和细胞行为。这些发现将使我能够在血管发育过程中剖析由内皮mirna调节的遗传网络。总之，结合mirna的靶向遗传操作以及斑马鱼中可用的详细成像方法的能力，将使我能够获得mirna在血管发育中的作用的新见解。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: We are developing technology to facilitate the in vivo study of microRNAs in the vascular system. The resulting data will allow us to gain new knowledge concerning microRNA function in controlling endothelial cell function and blood vessel formation.