Ventricular chambers are essential for the rhythmic contraction and relaxation occurring in every heartbeat throughout life. Congenital abnormalities in ventricular chamber formation cause severe human heart defects. How the early trabecular meshwork of myocardial fibres forms and subsequently develops into mature chambers is poorly understood. We show that Notch signalling first connects chamber endocardium and myocardium to sustain trabeculation, and later coordinates ventricular patterning and compaction with coronary vessel development to generate the mature chamber, through a temporal sequence of ligand signalling determined by the glycosyltransferase manic fringe (MFng). Early endocardial expression of MFng promotes Dll4–Notch1 signalling, which induces trabeculation in the developing ventricle. Ventricular maturation and compaction require MFng and Dll4 downregulation in the endocardium, which allows myocardial Jag1 and Jag2 signalling to Notch1 in this tissue. Perturbation of this signalling equilibrium severely disrupts heart chamber formation. Our results open a new research avenue into the pathogenesis of cardiomyopathies.

Rationale:: The Notch signaling pathway is crucial for primitive cardiac valve formation by epithelial–mesenchymal transition, and NOTCH1 mutations cause bicuspid aortic valve; however, the temporal requirement for the various Notch ligands and receptors during valve ontogeny is poorly understood. Objective:: The aim of this study is to determine the functional specificity of Notch in valve development. Methods and Results:: Using cardiac-specific conditional targeted mutant mice, we find that endothelial/endocardial deletion of Mib1-Dll4-Notch1 signaling, possibly favored by Manic-Fringe, is specifically required for cardiac epithelial–mesenchymal transition. Mice lacking endocardial Jag1 , Notch1 , or RBPJ displayed enlarged valve cusps, bicuspid aortic valve, and septal defects, indicating that endocardial Jag1 to Notch1 signaling is required for post–epithelial–mesenchymal transition valvulogenesis. Valve dysmorphology was associated with increased mesenchyme proliferation, indicating that Jag1-Notch1 signaling restricts mesenchyme cell proliferation non–cell autonomously. Gene profiling revealed upregulated Bmp signaling in Jag1 -mutant valves, providing a molecular basis for the hyperproliferative phenotype. Significantly, the negative regulator of mesenchyme proliferation, Hbegf , was markedly reduced in Jag1 -mutant valves. Hbegf expression in embryonic endocardial cells could be readily activated through a RBPJ-binding site, identifying Hbegf as an endocardial Notch target. Accordingly, addition of soluble heparin-binding EGF-like growth factor to Jag1 -mutant outflow tract explant cultures rescued the hyperproliferative phenotype. Conclusions:: During cardiac valve formation, Dll4-Notch1 signaling leads to epithelial–mesenchymal transition and cushion formation. Jag1-Notch1 signaling subsequently restrains Bmp-mediated valve mesenchyme proliferation by sustaining Hbegf-EGF receptor signaling. Our studies identify a mechanism of signaling cross talk during valve morphogenesis involved in the origin of congenital heart defects associated with reduced NOTCH function.

Introduction: Dysregulated NOTCH receptor activity has been implicated in breast cancer but the mechanisms by which NOTCH contributes to transformation are not yet clear, as it has context-dependent effects on the properties of transformed cells. Methods: We have used various in vitro and in vivo carcinogenic models to analyze the impact of Notch signaling in the onset and progression of breast tumors. Results: We found that ectopic expression of the Notch1 intracellular domain (N1ICD) in MCF-7 breast adenocarcinoma cell line caused reduction and delocalization of E-CADHERIN levels and increased migratory and invasive abilities. Notch inhibition in the invasive breast cancer cell line MDA-MB-231 resulted in increased E-CADHERIN expression and a parallel reduction in their invasive capacity. The growth of subcutaneous xenografts produced with MCF-7 cells was boosted after N1ICD induction, in a cell autonomous manner. In vivo Notch1 activation in the mammary gland using the MMTV-Cre driver caused the formation of papillary tumors that showed increased Hes1 and Hey1 expression and delocalized E-cadherin staining. Conclusions: These results confirm NOTCH1 as a signal triggering epithelial-mesenchymal transition in epithelial cancer cells, which may have implications in tumor dissemination, metastasis and proliferation in vivo. The identification of specific factors interacting with NOTCH signaling could thus be relevant to fully understanding the role of NOTCH in breast neoplasia.

Objective—: Calcific aortic valve disease is similar to atherosclerosis in that both diseases result from chronic inflammation and endothelial dysfunction. Heterozygous NOTCH1 mutations have been associated to calcific aortic disease and a bicuspid aortic valve. We investigated whether mice with genetic inactivation of the Notch signaling pathway are prone to develop valve disease when exposed to a predisposing diet. Methods and Results—: Using Doppler echocardiography, histology, immunohistochemistry, quantitative gene expression analysis, and cell culture assays, we examined the effect of a hypercholesterolemic diet supplemented with vitamin D on mice heterozygous for null mutations in the Notch1 receptor or the effector transcription factor gene RBPJk . After 16 weeks on the hyperlipidemic diet, calcific aortic disease was detected in heterozygous RBPJk mice. Analysis of valve leaflets revealed macrophage infiltration, enhanced collagen deposition, proosteogenic protein expression, and calcification. Heterozygous null Notch1 mice displayed milder histopathologic changes and did not develop any significant hemodynamic disturbance. Valvular disease correlated with reduced expression of the Notch target gene Hey1 in valves of RBPJk heterozygous mice fed the hyperlipidemic diet. Consistent with the in vivo data, Notch signaling inhibition in porcine valve interstitial cells led to downregulation of HEY1 transcription, activation of osteogenic markers, and increased calcified nodule formation. Conclusion—: We show that Notch signaling disruption via RBPJk heterozygous inactivation results in aortic valve disease. Notch1 heterozygous mice do not show functional impairment, suggesting that additional Notch receptors may be involved in aortic valve homeostasis and disease. Our data establish a genetic mouse model of calcific aortic valve disease and may help to identify a patient population with reduced valvular NOTCH signaling at risk for developing this disease.