Role of Notch in artery development

描述（由申请人提供）：在胚胎发育期间和整个成年期，器官形成和体内平衡需要血管。由于控制血管形成的信号传导机制在很大程度上是保守的，并且在胚胎和成体阶段是相似的，因此可以利用模型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，以决定细胞的命运和调节信号输出在血管发育。值得注意的是，Notch对于促进动脉内皮细胞命运是必不可少的，而在血管生成期间，它有助于限制内皮细胞对促血管生成因子的反应。然而，关于Notch激活在血管发育的早期阶段发生的时间和地点以及Notch激活如何影响血管形成的不同步骤中的内皮细胞命运知之甚少。此外，已经鉴定了很少的Notch靶基因，其可以介导内皮细胞中Notch活化的作用。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。使用转录响应Notch信号的转基因指示线，我们将通过在活斑马鱼胚胎中的延时分析来可视化Notch激活在发育血管中的时间和空间动态。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期间Notch阳性和Notch阴性内皮细胞的详细命运作图。我们还将通过利用我们使用锌指核酸酶产生斑马鱼敲除系的能力来表征Notch在内皮细胞中的推定直接靶点的功能。这将使得能够在感兴趣的靶基因中快速产生无效等位基因，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的基因中的内源性Notch响应性顺式元件，以明确地表征Notch在调节下游靶标中的作用。最后，我们将结合联合收割机的全球表达谱和全基因组的Notch结合位点的占用率测定，以表征内皮细胞中的Notch响应转录网络。公共卫生相关性：新血管形成的关键步骤是建立正确的血管身份（例如动脉与静脉）。最近的证据表明，在许多先天性人类疾病中，血管特性的丧失可导致血管畸形。本提案中的研究将解决如何在开发过程中确定血管身份。

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.