The Notch signaling pathway with Hey bHLH repressors as effectors is a central signal transduction mechanism for cardiovascular development in the embryo as well as for subsequent physiological and pathological processes. We could show that these genes are essential for determination of arterial cell fate, suppression of the venous program and angiogenic sprouting and remodeling processes. Furthermore, Hey1/2 modulate the cellular hypoxia response. The mechanisms by which Hey proteins control differentiation and cell fate decisions are barely understood, however; the mode of transcriptional regulation, relevant protein interactions and critical target genes are likewise unknown. To further our understanding of Hey functions in endothelial biology we are pursuing the following approaches:
1. We plan to generate embryonic endothelial cells from embryonic stem cells from 2D and 3D cultures using specific selection markers. Through comparative analysis of normal and Hey-deficient cells we want to uncover the relevance of Hey genes in angiogenetic cell fate decisions. Based on inducible Hey transgenes we will assess Hey functions in proliferation, sprouting and arteriovenous differentiation by qRT-PCR, immunofluorescence and functional tests.
2. Expression profiling and chromatin immunoprecipitation (ChIPseq) of endothelia with normal, absent or inducible Hey expression will be employed to identify Hey effectors that define the cellular repertoire of Hey targets. Integration of microarray and ChIP data from different sources will provide insight into specificity and redundancy of individual Hey factors in these cell types.
3. Hey targets will be validated in vivo as well as in vitro through targeted deregulation. We also plan to characterize the antagonism between Hey1/2 and Coup-TFII in the decision between arterial and venous cell fate.
4. Hey functions in adult blood vessels will be analysed by inducing endothelial specific deletions of Hey genes. This will be studied under baseline physiological conditions as well as in insult models of angiogenesis and arteriogenesis.
These approaches will provide a systems level view of Hey target genes and their interactions. It will be interesting to elucidate how repressor proteins can generate instructive signals for cell fate decisions. These results will deepen our understanding of endothelial differentiation, arteriogenesis and cellular hypoxia response and they should provide new insights into the interaction of tip and stalk cells in vascular sprouting.