Functional analysis of the Hey bHLH factors in vascular development

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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.