CRIP1在胚胎血管发育中的功能和作用机制研究

Formation of a cohesive, seamless and contiguous network of blood vessels carrying blood is essential for proper function of cardiovascular system. However the molecular and cellular mechanisms driving the formation of vascular tubes remain to be fully elucidated. The advantage that the zebrafish embryos can survive for about seven days in the absence of blood flow in combination with the embryo transparency has made zebrafish an excellent model to study blood vessel formation. .Very recently, we have characterized a CRIP1 gene in zebrafish, which belongs to group 2 LIM proteins gene family, and encodes a highly conserved protein. Whole-mount in situ hybridization showed that CRIP1 is specifically expressed in posterior cardinal vein plexus as well as pronephric tubule. We also showed that CRIP1 protein is co-localized with the Flk1, a blood vessel marker, in the posterior blood vessels. Moreover, knockout of CRIP1 by Talens leads to abnormal blood vessel formation, in specific, caudal vein plexus failed to consolidate into a single lumen. All these preliminary results suggest that CRIP1 is required for the formation of vascular tubes. However, its precise role and mode of action remain enigmatic, and demand further detailed study. .The aim of this proposal is thus to explore the function and mechanisms of CRIP1 in blood vessel formation. We will first study subcellular localization of CRIP1. Then we will analyze the implication of CRIP1 in the formation of blood vessels by TALENs-mediated gene knockout; the blood vessels phenotype in mutants will be determined by in vivo time-lapse imaging, and the expression of vessel marker genes will be analysed by in situ hybridization. We will also examine whether the vascular system function properly in CRIP1 mutants. Furthermore, we plan to test whether CRIP1 could affect the migration and angiogenesis in vitro in human cell line. To provide a molecular mechanism of CRIP1 action, we project to identify putative CRIP1 protein partners, and to analyze the biochemical and functional interaction between CRIP1 and its interaction partners. All together, these experiments should help to elucidate the mechanism of CRIP1 in the formation and function of blood vessels. They will also provide novel insights into the gene regulatory network underlying blood vessel formation.

血管系统的精确建成是胚胎发育的重要事件，形成有效管腔是其中的关键环节，探索其形成机制是血管发育的研究热点。我们前期研究发现斑马鱼CRIP1基因在尾静脉血管丛、肾管等管腔结构中表达，其蛋白与Flk1共存于血管上，敲除后血管发育异常，尾静脉血管丛无法形成正常的静脉, 提示其在血管发育中起重要作用，但作用机制尚不明确。我们拟：首先确定CRIP1的亚细胞定位；在突变体中通过活体成像观察血管发生、发育并检测血管标志性基因表达情况；在哺乳动物细胞中通过细胞转染和RNAi确定CRIP1是否调控细胞迁移及血管生成；筛选与CRIP1相互作用的蛋白，重点关注其中影响血管发育或细胞极性的蛋白，确定CRIP1与候选作用蛋白是否共定位、共注射实验明确在血管发育过程中的功能互作。本研究将从细胞、分子、生化多层次揭示CRIP1在血管发育中的功能及作用机制，构建血管发育调控网络，为血管发育异常疾病的分子机制提供重要参考。

血管系统的精确建成是胚胎发育的重要事件，形成有效管腔是其中的关键环节，探索其形成机制是血管发育的研究热点。其中驱动尾静脉重塑的分子和细胞水平机制尚不清楚。我们发现斑马鱼crip1基因，编码含有Lim结构域的蛋白，属于第二类群Lim蛋白。其在尾静脉血管丛等管腔结构中表达，与Flk1共存于血管上。crip1敲除后纯合突变体有不足30%在受精后5天出现了水肿，小头以及身体弯曲等表型。内源性的血管内皮碱性磷酸酶染色以及crip1突变体与转基因Tg(flk:egfp)杂交，筛选带荧光的crip1纯合突变体结果均显示：与野生型斑马鱼的尾部静脉一条很完整而且平直的血管不同，crip1纯合突变体尾静脉血管发育缺陷-尾静脉的宽度加大，为多个不正常、小的管腔结构，而且形态极不规则。血液在尾部静脉处的流速变缓，甚至停滞。检测突变体中一些血管特异的marke基因，发现angpt1h和vegfr3的表达降低，而vegfr1, vegfr2和cdh5在突变体中的表达增加，进一步说明Crip1影响了尾静脉的管腔重塑过程。对作用机制进行探讨发现：Crip1敲除会影响细胞连接，ZO-1在细胞边界的表达明显弱于对照组。转录组分析Crip1敲除时，与血管、心脏发育相关的信号通路有粘着斑信号通路和肌动蛋白细胞骨架调控通路受影响最大。我们还筛选了与Crip1相互结合蛋白，nuclear factor kappa B (NF-κB) 复合体的一个组分-p65是我们感兴趣的蛋白。免疫共沉淀结果显示Crip1能够与p65相互结合，具体是与p64的Rel功能域结合。在细胞系中敲降Crip1能够上调TNFα诱导的p65的磷酸化水平，但TNFα诱导的IκBα的降解并没有任何影响，提示Crip1对于p65磷酸化水平的影响并不依赖IκBα。敲降Crip1会降低切割的caspase8和caspase3的水平，抑制细胞凋亡。以上结果表明在斑马鱼胚胎中Crip1通过与p65相互结合，影响其磷酸化水平，调控细胞凋亡，从而参与尾静脉重塑过程。除此发现crip2在尾静脉也有较高水平的表达，敲除导致背主静脉、节间静脉和尾静脉丛的发育受影响，可能是因内皮细胞的不迁移或相聚集导致尾静脉管腔发育缺陷。本项目的研究揭示了Crip家族蛋白在尾静脉重塑过程中的功能与机制，对于研究相关的发育缺陷具有理论意义。