Components of the Notch signaling pathway are essential for the development of numerous cell types during embryogenesis. Furthermore, mutations in Notch signaling components are associated with a variety of human diseases, including those affecting cardiovascular function. Within the vascular system, Notch receptors are expressed specifically in arterial endothelial cells and play a crucial role in defining artery identity. Notch signaling is also important for coordinating tip and stalk cell behaviors during th sprouting of new blood vessels, or angiogenesis. Our previous work using the zebrafish as a model system was instrumental in defining the roles of Notch during both artery differentiation and angiogenesis. Importantly, our past and current studies suggest that these are distinct processes governed through different receptor and ligand combinations. Additionally, Notch activation during these processes is dynamic, occurring at different developmental stages and in different endothelial subtypes. Thus, it is likely that there are distinct cellular outputs and morphogenetic responses controlled by Notch in each of these contexts. However, what the target genes are to mediate Notch function in different contexts is not known. To identify functional Notch targets in the vascular system, we propose studies encompassed in two separate Aims.
In Aim 1, we will investigate the requirements of candidate transcriptional repressors that we have found are dependent on Notch in arterial endothelial cells in zebrafish. These studies will rely on the application of site-specific nucleases to generate knockout zebrafish lines deficient for these genes, followed by detailed phenotypic and epistasis analysis. These studies will leverage our vast expertise using the zebrafish for complex genetic analyses coupled with high content phenotypic analysis.
In Aim 2, we will identify direct targets of the NOTCH1 receptor in human arterial endothelial cells to better understand how Notch activation programs artery identity. Taken together, these studies will allow us to understand how Notch may effect endothelial cell behaviors and differentiation in distinct contexts.
In the circulatory system, Notch receptors are required to define artery identity and to form new functional blood vessels. Mutations in Notch receptors are associated with congenital vascular diseases, including aortic valve calcification and CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), a congenital syndrome associated with early-onset stroke. Our proposed studies will allow us to determine how Notch receptors control blood vessel identity and formation, which will provide new insights into their role in vascular disease.
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