Functional analysis of the Hey bHLH factors in vascular development

以HY蛋白为转导分子的Notch信号通路现已成为脊椎动物胚胎心血管发育和成体病理的关键信号系统。我们和其他人已经证明，这些基因对于建立动脉认同感、抑制静脉命运以及发育中的动脉的血管新生发芽和重塑至关重要。Hey1/2也调节细胞的缺氧反应。然而，对HY蛋白影响分化和完成细胞命运改变的机制还知之甚少。我们将利用小鼠ES细胞分化和遗传选择在2D和3D培养条件下产生胚胎内皮细胞。野生型和Heyless突变细胞的表达谱将确定血管生成命运决定的潜在下游中介。与此同时，我们将扩展我们的全基因组染色质IP分析(ChIPseq)，以确定内皮细胞中HY蛋白的完整结合位点。这将为我们提供一个关于嘿靶标的系统生物学观点，我们将使用强制表达/RNAi和我们的基因敲除小鼠进一步验证。其中一个重点将是破译嘿和Coup-TFII在动脉/静脉决策中的相互拮抗作用。我们希望这些数据能极大地提高我们对血管新生萌发中的内皮分化、动脉命运决定以及尖端/柄细胞通讯的理解。

The Notch signalling pathway with Hey proteins as bHLH transducers is now firmly established as a key signalling system for embryonic cardiovascular development and adult pathologies in vertebrates. We and others have shown that these genes are essential for establishing arterial identity, suppression of venous fate and angiogenic sprouting and remodelling of developing arteries. Hey1/2 also modulate the cellular hypoxia response. However, there is little insight into the mechanisms whereby Hey proteins influence differentiation and accomplish cell fate changes. We will utilize mouse ES cell differentiation with genetic selection to generate embryonic endothelia in 2D and 3D culture conditions. Expression profiling of wild type and Heyless mutant cells will identify potential downstream mediators of angiogenic fate decisions. In parallel we will expand our whole genome chromatin IP analyses (ChIPseq) to define the full complement of binding sites for Hey proteins in endothelial cells. This will provide us with a systems biology view of Hey targets that we will further validate using forced expression/RNAi and our knockout mice. One focus will be to decipher the reciprocal antagonistic action of Hey and Coup-TFII in artery/vein decisions. We expect these data to greatly enhance our understanding of endothelial differentiation, arterial fate decision, and tip/stalk cell communication in angiogenic sprouting.