Up-regulation of Notch signaling and angiogenic suppression in BAZF knockout mouse retina. Littermate mice of representing 2 genotypes (BAZF^+/+ and BAZF^−/−) were assessed for retinal angiogenesis and Notch signaling at P5. The retinas were stained with isolectin B4, and then flat-mounted for confocal laser microscopic analysis. (A) Whole retinal vascular bed (i and ii, scale bar: 200 μm) and front (iii and iv, scale bar: 20 μm) of BAZF^+/+ and BAZF^−/− mice. Quantitative analyses of the diameter of the vascular area (B), isolectin B4⁺ area (C), branching points (D), and the number of filopodia (E) in BAZF^+/+ (n = 9) and BAZF^−/− (n = 3) retinas. Error bars represent ± SD (**P < .01, ***P < .001). (F) BAZF immunostaining in whole-mount retina. Both BAZF^+/+ and BAZF^−/− retinas were stained with isolectin B4 and anti-BAZF. Scale Bar: 500 μm (50 μm in the enlarged image). (G) CBF1 immunostaining in whole-mount retina. Retinas were stained with isolectin B4 and anti-CBF1 or normal rabbit IgG as a negative control. Scale bar: 15 μm. (H) HEY1 immunostaining in whole-mount retinal vasculature. Retinas were stained with isolectin B4 and anti-HEY1 antibody. Scale bar: 200 μm. (I) HEY1 localization in retinal vascular plexus. Nuclear localization (arrowheads) of HEY1 in stalk ECs, whereas faint signals in sprouting tip cells (arrows). Endothelia were also stained with isolectin B4. Scale bar: 20 μm. (J) Formation of hyperfused plexus by Notch signal inhibition in retinas. After administration of DMSO or DAPT (100 mg/kg) at P3 and P4, retinas were collected at P5. Retinas were stained with isolectin B4 and Hoechst33342. Images were obtained by a confocal laser microscopy. Scale bar: 200 μm.