Role of Notch in artery development

描述（由申请人提供）：在胚胎发育和整个成人生活中，血管是器官形成和体内平衡所必需的。由于控制血管形成的信号机制在很大程度上是保守的，并且在胚胎和成人阶段是相似的，因此有可能利用模式生物来深入了解这一过程。血管的形成是通过一系列复杂的细胞行为的协调而发生的。最近的证据表明，Notch信号通路在这一过程中起着至关重要的作用，在血管发育过程中决定细胞命运和调节信号输出。值得注意的是，Notch对于促进动脉内皮细胞的命运至关重要，而在血管生成过程中，它有助于限制内皮细胞对促血管生成信号的反应。然而，在血管发育的早期阶段，Notch何时何地激活，以及Notch激活如何影响血管形成的不同阶段内皮细胞的命运，我们知之甚少。此外，很少有Notch靶基因被发现可能介导内皮细胞中Notch激活的作用。在这里提出的研究中，我们将利用斑马鱼作为模型系统来解决Notch信号在血管发育过程中的动态作用。利用对Notch信号有转录反应的转基因指示线，我们将通过对斑马鱼活体胚胎的延时分析，可视化Notch在发育血管中激活的时空动态。此外，我们将建立在所有内皮细胞或notch阳性内皮细胞中表达光转换荧光蛋白的转基因系。这些细胞系将允许在血管发育的多个阶段绘制notch阳性和notch阴性内皮细胞的详细命运图。我们还将利用锌指核酸酶产生斑马鱼基因敲除系的能力，对内皮细胞中Notch的推定直接靶点的功能进行表征。这将使感兴趣的靶基因中的零等位基因快速产生，以确定它们在Notch下游的作用。重要的是，该技术还将允许我们删除感兴趣基因中的内源性Notch应答顺式元件，以明确表征Notch在调节下游靶标中的作用。最后，我们将结合全球表达谱和Notch结合位点占用的全基因组分析来表征内皮细胞中Notch应答转录网络。公共卫生相关性：新血管形成的一个关键步骤是建立适当的血管身份（例如动脉与静脉）。最近的证据表明，在一些先天性人类疾病中，血管特性的丧失可导致血管畸形。本提案中的研究将解决在发育过程中如何确定血管身份的问题。

DESCRIPTION (provided by applicant): Blood vessels are required for organ formation and homeostasis during embryonic development and throughout adult life. Since the signaling mechanisms that govern blood vessel formation are largely conserved and are similar during embryonic and adult stages, it is possible to utilize model organisms to gain insight into this process. Blood vessel formation occurs through the coordination of a complex array of cellular behaviors. Recent evidence indicates that the Notch signaling pathway plays an essential role during this process to determine cell fates and modulate signaling output during vascular development. Notably, Notch is essential for promoting arterial endothelial cell fate while during angiogenesis it helps to limit an endothelial cell's response to pro-angiogenic cues. However, little is known about when and where Notch activation occurs during the early stages of vascular development or how Notch activation affects endothelial cell fates at different steps of blood vessel formation. Furthermore, few Notch target genes have been identified that may mediate the effects of Notch activation in endothelial cells. In the studies proposed here, we will take advantage of the zebrafish as a model system to address the dynamic role of Notch signaling during blood vessel development. Using a transgenic indicator line that is transcriptionally responsive to Notch signaling, we will visualize the temporal and spatial dynamics of Notch activation in developing blood vessels through time-lapse analysis in live zebrafish embryos. In addition, we will establish transgenic lines expressing a photoconvertible fluorescent protein in all endothelial cells or in Notch-positive endothelial cells. These lines will allow detailed fate mapping of Notch-positive and Notch-negative endothelial cells during multiple stages of vascular development. We will also characterize the function of putative direct targets of Notch in endothelial cells by taking advantage of our ability to generate zebrafish knockout lines using zinc finger nucleases. This will enable the rapid generation of null alleles in target genes of interest to determine their role downstream of Notch. Importantly, this technique will also allow us to delete endogenous Notch responsive cis elements in genes of interest to definitively characterize the role of Notch in regulating downstream targets. Finally, we will combine global expression profiling and genome-wide assay of occupancy at Notch binding sites to characterize the Notch-responsive transcriptional network in endothelial cells. PUBLIC HEALTH RELEVANCE: A crucial step in the formation of new blood vessels is the establishment of proper blood vessel identity (e.g. artery versus vein). Recent evidence suggests that loss of blood vessel identity in a number of congenital human diseases can lead to vascular malformations. The studies in this proposal will address how blood vessel identities are determined during development.