Disruption of the furin cleavage site in mouse Notch1 results in cardiovascular malformations due to hypomorphic Notch1 signaling

The highly conserved Notch signaling pathway has been a focus of developmental biologists for decades because of its indispensable role during embryogenesis. Disruptions of the Notch pathway in humans and animal models often result in lethality due to cardiovascular abnormalities, thus indicating a requirement for proper Notch signaling during development of the heart and blood vessel network. Typical activation of this pathway involved engagement of a cell-surface Notch receptor with a ligand on an adjacent cell, which results in proteolytic processing of the Notch molecule to render a downstream transcriptional effect. Three proteolytic activities are required for proper activation of Notch and various aspects of cleavage events have been extensively studied. The first such processing event of Notch is through the action of furin, a proprotein convertase, that cleaves the Notch molecule at a conserved consensus site to generate a heterodimeric molecule that is primed for subsequent proteolysis, and thus activation. While furin cleavage of Notch1 has been widely studied in vitro and in Drosophila, an absolute role for furin proteolysis in mammals has yet to be described. The main goal of my research was to ascertain the requirement for furin cleavage of vertebrate Notch1 by using a mouse model harboring a mutation in the Notch1 gene which deletes the furin cleavage site in the gene sequence, but does not affect translation or expression of the protein. Through a series of biochemical and in vitro studies, combined with an in vivo phenotypical analysis of mutant mice, I found that disruption of the furin cleavage site does not completely prevent downstream proteolysis and activation of the mutant Notch1 protein, however, these activities were decreased, thus resulting in hypomorphic Notch1 signaling. In accordance with the overwhelming role for Notch signaling during cardiovascular development, I found that decreased Notch1 signaling in mutant embryos results in yolk sac vasculature defects and congenital cardiac malformations. I propose that disruption of the furin cleavage site of Notch1 leads to decreased proteolytic processing of Notch, which results in hypomorphic Notch1 signaling that is not sufficient to sustain normal cardiovascular development.