Role of Notch in artery development

描述(由申请人提供)：在胚胎发育和整个成年生活中，血管是器官形成和动态平衡所必需的。由于控制血管形成的信号机制在很大程度上是保守的，在胚胎和成体阶段是相似的，因此利用模式生物来深入了解这一过程是可能的。血管的形成是通过一系列复杂的细胞行为协调进行的。最近的证据表明，Notch信号通路在这一过程中发挥了重要作用，决定了细胞的命运，并调节了血管发育过程中的信号输出。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，关于Notch激活在血管发育的早期阶段何时何地发生，或者Notch激活如何在血管形成的不同步骤影响内皮细胞命运，人们知之甚少。此外，很少有Notch靶基因被鉴定为可能在内皮细胞中介导Notch激活效应的基因。在本文提出的研究中，我们将利用斑马鱼作为模型系统来研究Notch信号在血管发育中的动态作用。使用对Notch信号有转录反应的转基因指示物，我们将通过在活斑马鱼胚胎中的时间推移分析，可视化Notch在血管发育过程中激活的时间和空间动态。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可转换荧光蛋白的转基因系。这些线条将使Notch阳性和Notch阴性内皮细胞在血管发育的多个阶段得到详细的命运图谱。我们还将利用我们使用锌指核酸酶产生斑马鱼基因敲除线的能力，来表征内皮细胞中假定的Notch直接靶标的功能。这将使在感兴趣的目标基因中快速产生空等位基因来确定它们在Notch下游的作用。重要的是，这项技术还将使我们能够删除相关基因中内源性的Notch响应顺式元件，以确定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全基因组分析Notch结合位点的占有率来表征内皮细胞中Notch反应的转录网络。公共卫生相关性：新血管形成的关键一步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，血管特性的丧失会导致血管畸形。这项提案中的研究将解决血管身份如何在发育过程中确定的问题。

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.