Figures and data
Endocardium and vascular endothelium of dorsal aorta showed different patterns of Notch1 activation and Dll4 ligand expression.
A, Schematic representation of 3 types of endothelial cells and their corresponding localizations. B, Type I cells are NICD-high and Dll4-high. Type II cells are NICD-high and Dll4-low. Type III cells are NICD-low and Dll4-low. Scale bars, 100 µm. (n = 3 embryos) C, Representative images showing Dll4 protein and NICD in wild-type and endothelial-specific Dll4-deleted (Tie2cre;Dll4flox/flox) E9.5 mouse hearts. Scale bars, 100 µm. (wild-type: n = 3 embryos; Tie2cre;Dll4flox/flox: n = 3 embryos)
Notch activation in cushion endocardium is dependent on blood flow.
A, Echocardiography of control and dofetilide treated embryos. (0 h: n = 12; 1 h: n = 15; 3 h: n = 12; 5 h: n = 9). B, Expression of NICD, Dll4, Jag1, total Notch1, p-SMAD1/5 and Sox9, Twist1 in the E9.5 dorsal aorta endothelium (arrow) and endocardium (arrowhead). C, Sagittal E10.5 hematoxylin-eosin stained sections demonstrated hypocellularity in both superior (arrowhead) and inferior AV cushion (arrow) caused by dofetilide treatment. Quantification of mesenchymal cell density in superior (below left) and inferior (below right) AV cushion (Control: n = 16 embryos; Dofetilide: n = 14 embryos). OFT, outflow tract; A, atrium; V, ventricle. D, Representative heart defects induced by maternal dofetilide treatment. pmVSD, perimembranous ventricular septal defect; DORV, double-outlet right ventricle; mVSD, muscular VSD; OA, overriding aorta; BAV, bicuspid aortic valve; AVSD, atrioventricular septal defect; BPV, bicuspid pulmonary valve; ra, right atrium; la, left atrium; ao, aorta; rv, right ventricle; lv, left ventricle; s, interventricular septum; pa, pulmonary artery. Scale bars, 100 µm (B, C), 500 µm (D). (n > 3 for every group and each condition)
Flow-responsive mTORC2-PKCε activity is required for Notch activation in the cushion endocardium.
A, Phospho-PKCSer660 and phospho-AKTSer473 levels in the atrioventricular (AV) canal endocardium and proximal OFT endocardium (arrowhead) after dofetilide treatment. Each point in the quantification chart represents one embryo. B, NICD, pPKC and pAKT expression in cultured E9.5 heart in response to ex vivo staurosporine treatment (100 nM). Each point in the quantification chart represents one embryo. C, NICD expression in AV canal endocardium in wild-type, Prkce and Prkch single knockout and double knockout embryos. Each point in the quantification chart represents one embryo. D, NICD, p-PKCSer660 and p-AKTSer473 staining in AVC endocardium (arrowhead) after dofetilide treatment and after rescue by PMA. E, Sagittal E10.5 HE staining sections demonstrates acellularized superior (arrowhead) and inferior AV cushion (arrow) caused by dofetilide which were rescued by in PMA treatment (2 mg/kg). OFT, outflow tract; A, atrium; V, ventricle. Mesenchymal cell density was quantitated in superior and inferior AV cushion (Control: n = 14 embryos; Dofetilide: n = 11 embryos; Dofetilide + PMA: n = 9 embryos). F, Heart defect rate caused by maternal dofetilide and PMA treatment (Control: n = 37; Dofetilide: n = 62; Dofetilide + PMA: n = 27). G, NICD, phospho-PKCSer660, and phospho-AKTSer473 in AV canal endocardium (arrowhead) in wild-type and Rictor null mice (Rictor+/+: n = 6; Rictor−/−: n = 8). Scale bars, 100 µm.
Shear stress-induced alteration of membrane lipid microstructure activated mTORC2-PKC-Notch signaling pathway.
A, Caveolin-1 and VE-cadherin expression in mouse E9.5 AV canal, dorsal aorta, atrium and ventricle endocardium, demonstrating luminal (arrowhead) and lateral (arrow) surface localization. Ex vivo dofetilide treatment (0.2 μg/ml) of cultured E9.5 embryos for 3 h caused retraction of Caveolin-1 and VE-cadherin from luminal surface of the AVC endocardial cells to the lateral cell adhesion sites which could be rescued by co-treatment with cholesterol (1 mg/ml). B, Representative images and quantifications of scanning electron microscopy on E 9.5 embryonic heart AV canal endocardial cells at their luminal surfaces, with caveolae structure presentively pointed by arrowhead. Each point in the quantification chart represents one embryo. C, Loss of NICD, phospho-PKCSer660, and phospho-AKTSer473 in AVC endocardium (arrowhead) by ex vivo dofetilide treatment could be rescued by cholesterol. The rescue failed in Rictor null hearts. Each point in the quantification chart represents one embryo.Scale bars, 100 µm (A, C). Scale bars, 10 µm (A, zoom-in), 1 µm (B).
Pharmacogenetic interaction causing heart defects.
A, Heart defect rate significantly increased in Notch1 heterozygous embryos treated with dofetilide (1.8 mg/kg) at E9.5. (Saline Notch1+/+: n = 48, Saline Notch1+/−: n = 37, Dofetilide Notch1+/+: n = 31, Dofetilide Notch1+/−: n = 16). B, Types of heart defects in the dofetilide and Notch1+/− combined group. C, Heart defect rate significantly increased in Notch1 heterozygous embryos treated with Verapamil (15 mg/kg) at E9.5. (Saline Notch1+/+: n = 18, Saline Notch1+/−: n = 9, Verapamil Notch1+/+: n = 8, Verapamil Notch1+/−: n = 12). D, Types of heart defects in the verapamil combined with Notch1+/− group. E, Representative echocardiography and quantifications of heartbeat in control and verapamil treated E 9.5 embryos. Each point in the quantification chart represents one embryo.
Working model of the establishment of Notch activation pattern by mechanical cues.
The establishment of Notch activation pattern by mechanical cues involves a series of events in the developing heart tube. At E8.5, the arterial endothelium and non-EMT endocardium exhibit low shear stress, high VEGF, Dll4, and Notch signaling. One day later, the endocardium undergoes patterning and becomes capable of epithelial-to-mesenchymal transition (EMT) only in the AVC and proximal OFT regions. This patterning is achieved through restricted expression of BMP2 and NICD in these specific areas. EMT requires the downregulation of VEGF signaling by the endocardium, enabling EMT to occur. Additionally, Dll4 is downregulated in endocardium to prevent widespread Notch activation. Simultaneously, the high shear stress present in the AVC and proximal OFT regions leads to increased membrane lipid order, which activates the mTORC2-PKC-Notch pathway and promotes EMT. On the other hand, regions flanking these valve-forming areas experience lower shear stress, resulting in inactive Notch signaling and an inability to undergo EMT.
