Coronary arterial development is regulated by a Dll4-Jag1-EphrinB2 signaling cascade

Coronaries are essential for myocardial growth and heart function. Notch is crucial for mouse embryonic angiogenesis, but its role in coronary development remains uncertain. We show Jag1, Dll4 and activated Notch1 receptor expression in sinus venosus (SV) endocardium. Endocardial Jag1 removal blocks SV capillary sprouting, while Dll4 inactivation stimulates excessive capillary growth, suggesting that ligand antagonism regulates coronary primary plexus formation. Later endothelial ligand removal, or forced expression of Dll4 or the glycosyltransferase MFng, blocks coronary plexus remodeling, arterial differentiation, and perivascular cell maturation. Endocardial deletion of Efnb2 phenocopies the coronary arterial defects of Notch mutants. Angiogenic rescue experiments in ventricular explants, or in primary human endothelial cells, indicate that EphrinB2 is a critical effector of antagonistic Dll4 and Jag1 functions in arterial morphogenesis. Thus, coronary arterial precursors are specified in the SV prior to primary coronary plexus formation and subsequent arterial differentiation depends on a Dll4-Jag1-EphrinB2 signaling cascade. RNA was isolated at E12.5, from whole hearts, for WT (4 replicate samples, pooling 4 embryos) and Jag1flox;Nfatc1-Cre embryos(4 replicate samples, pooling 4 embryos), and for WT (4 replicate samples, pooling 4 embryos) and Dll4flox;Nfatc1-Cre (4 replicate samples, pooling 4 embryos). It was isolated at E15.5, from heart ventricles, for WT (4 replicate samples, pooling 3 embryos) and Dll4flox; Cdh5(PAC)-CreERT (3 replicate samples, pooling 3 embryos) and for WT (4 replicate samples, pooling 3 embryos) and Dll4GOF;Tie2-Cre (3 replicate samples, pooling 3 embryos). Finally, it was isolated at E16.5, from heart ventricles, for WT (4 replicate samples, from one embryo) and Efnb2flox;Nfatc1-Cre (4 replicate samples, from one embryo).