Notch1 Is Pan-Endothelial at the Onset of Flow and Regulated by Flow

Abstract

Arteriovenous differentiation is a key event during vascular development and hemodynamic forces play an important role. Arteriovenous gene expression is present before the onset of flow, however it remains plastic and flow can alter arteriovenous identity. Notch signaling is especially important in the genetic determination of arteriovenous identity. Nevertheless, the effect of the onset of circulation on Notch expression and signaling has not been studied. The aim of this study is therefore to investigate the interaction of Notch1 signaling and hemodynamic forces during early vascular development. We find that the onset of Notch1 expression coincides with the onset of flow, and that expression is pan-endothelial at the onset of circulation in mouse embryos and only becomes arterial-specific after remodeling has occurred. When we ablate flow in the early embryo, endothelial cells fail to express Notch1. We show that low and disturbed flow patterns upregulate Notch1 expression in endothelial cells in vitro, but that higher shear stress levels do not (≥10 dynes/cm2). Using siRNA, we knocked down Notch1 to investigate the role of Notch1 in mechanotransduction. When we applied shear stress levels similar to those found in embryonic arteries, we found an upregulation of Klf2, Dll1, Dll4, Jag1, Hey1, Nrp1 and CoupTFII but that only Dll4, Hey1, Nrp1 and EphB4 required Notch1 for flow-induced expression. Our results therefore indicate that Notch1 can modulate mechanotransduction but is not a critical mediator of the process since many genes mechanotransduce normally in the absence of Notch1, including genes involved in arteriovenous differentiation.

SoxF factors induce Notch1 expression via direct transcriptional regulation during early arterial development

“…This indicates that the NOTCH1+16 enhancer represents a novel, arterially restricted enhancer within the NOTCH1 locus. Analysis of a stable mouse line expressing the NOTCH1+16:lacZ transgene clearly demonstrated that this enhancer is strongly active from the very early stages of vascular development, mimicking the expression of endogenous Notch by becoming restricted to the arteries by late E9.5 and then maintaining an arterial endothelial cell-restricted expression pattern throughout embryonic development (Chong et al, 2011;Jahnsen et al, 2015) (Fig. 2).…”

“…Ligand binding to the Notch receptor releases the Notch intracellular domain (NICD), which translocates to the nucleus and forms a transcriptional activation complex with the otherwise repressive DNA-bound Rbpj [CSL, Su(H)] (Bray, 2006). Combined ablation of Notch1 and Notch4, which are both principally expressed in arterial endothelial cells during early vascular remodelling (Chong et al, 2011;Jahnsen et al, 2015), results in severe vascular remodelling defects (Krebs et al, 2000), as does ablation of the Notch downstream effector Rbpj or of Dll4, a Notch ligand specific within the vasculature to arteries (Duarte et al, 2004;Gale et al, 2004;Krebs et al, 2004). However, loss of Notch signalling does not fully recapitulate the arterial defects downstream of Vegfa ablation (Carmeliet et al, 1996;Krebs et al, 2000;Lawson et al, 2002), and the arterially restricted gene expression patterns of components of the Notch pathway do not fully overlap with activated Vegfa (Lawson, 2003), suggesting that additional factors are involved in the regulation of Notch-mediated arterial fate.…”

The force within: endocardial development, mechanotransduction and signalling during cardiac morphogenesis

“…It is plausible that the reduced blood flow in myh6 mutants might also have a negative effect on the myofibril maturation of ventricular myocardial cells (Lin et al, 2012); trabeculation defects could thus be a result of changes in sarcomere patterning that affect myocardial protrusions (Staudt et al, 2014). Some evidence suggests that Notch signalling may play a role in mechanosensitive pathways (Jahnsen et al, 2015). During zebrafish trabeculation, cilia play a role in sensing fluid forces and in inducing notch1b expression within the ventricular endocardium which, in turn, is required for correct trabeculation (Samsa et al, 2015).…”

Blood flow modulation of vascular dynamics

“…OSS also activates angiopoietin-2 (Ang-2) expression critical to angiogenesis via canonical β-catenin/Wnt signaling in human aortic EC [75*]. Low and disturbed flow patterns up-regulate Notch1 expression in EC with translational implication for arteriovenous identity [76]. Steady shear stress induces VEGF-Notch signaling pathway to increase expression of the arterial endothelial marker EphrinB2, but down-regulates the venous endothelial marker EphB4 in murine embryonic stem (ES) cells [77].…”