Fig. 1.
Defects in junctional remodelling upon inactivation of PI3Kα in endothelial cells. a Lateral views of intersomitic vessels (ISV) in vehicle and GDC-0326 (50 μM)-treated transgenic Tg(kdrl:EGFP)s843 (shown in red) embryos stained for ZO-1 (green) at 33 h post fertilization (hpf). Single ZO-1 staining is shown in upper row. DA refers to dorsal aorta and DLAV refers to dorsal longitudinal anastomotic vessels. White lines indicate ZO-1 negative staining; punctuate white lines indicate elongation of junction; yellow arrowheads show ring-shape junctions. b Quantification of the length of the dorsal part of the ISVs without ZO-1 (left graph) and length of the ISVs with continuous ZO-1 staining (right graph) in vehicle and GDC-0326 treated embryos (n ≥ 54 ISVs per treatment). c Representative maximum intensity projections of anti-VE-cadherin (green) and isolectin B4 (IB4, red) immunostained control and Pik3caKD/iΔEC mouse retinas at P7. Single channel is shown in upper row. Yellow islets show higher magnification of selected regions shown to the right. Yellow arrowheads indicate vascular segments without VE-cadherin staining; red asterisks indicate VE-cadherin-positive isolated rings or single-dots within the vascular tubes, indicating cell−cell junctional contacts that have not elongated. d, e Quantitative number of VE-cadherin-negative vessels (junctional gaps) per unit area (n ≥ 5 retinas per genotype) (d) and length of vessel structures without VE-cadherin (length of junctional gaps) (n ≥ 6 retinas per genotype) (e). f Confocal immunofluorescence images of primary mouse endothelial cells isolated from control and Pik3caKD/iΔEC mice stained for β-catenin (green) and F-actin (red) after being treated with 4-OHT for 72 h and re-plated on gelatin-coated slides for 24 h. Yellow arrowheads indicate straight junctional pattern; orange arrowheads indicate serrated junctional pattern. g Graph shows the average of β-catenin-positive area along junctional linescans (n ≥ 109 cells from six independent experiments). h Quantification of percentages of cells with serrated, straight or mixed junctions (n = 5 independent experiments). Scale bars, 30 µm (a), 20 µm (c), 10 µm (c small panel, f). Data in b, d, e, g, h represent mean ± SEM (error bars). *P < 0.05, **P < 0.01, ****P < 0.0001 were considered statistically significant. Statistical analysis was performed by the two-sided Mann–Whitney test