Fig. 7.

Conditional knockout of CREB in EC attenuates DLL4–NOTCH1 signaling and retinal neovascularization. A. Tamoxifen was given intraperitoneally to CREB^fl/fl:Cdh5-CreERT2, and CREB^fl/−:Cdh5-CreERT2 pups on P10 and P11, when the pups were under hyperoxia and at P13 the retinas were isolated from WT (both from normoxia and hypoxia), CREB^i ∆ EC, and CREB^fl/−:Cdh5-CreERT2^T/− pups, and extracts were prepared and an equal amount of protein from each group was analyzed by Western blotting for CREB using its specific antibody and normalized to β-tubulin. B. All the conditions were the same as in panel A except that the retinal extracts from WT, CREB^i ∆ EC pups were analyzed by Western blotting for DLL4, NOTCH1 and CREB levels using their specific antibodies and normalized to β-tubulin. C & D. WT and CREB^iΔEC mice pups after exposure to 75% oxygen from P7 to P12 were returned to normoxia to develop the relative hypoxia. At P15, the retinas were isolated, cross-sections were made and stained by immunofluorescence for CD31 and Ki67 (C) and cells positive for both CD31 and Ki67 were counted (D). E & F. All the conditions were the same as in panel C except that at P17 the retinas were isolated, stained with isolectin B4 and flat mounts were made and examined either for EC filopodia formation or neovascularization. In panel F, retinal vascularization is shown in the first column. Neovascularization is highlighted in red in the second column. The third column shows the selected rectangular areas of the images shown in the first column under 10X magnification. G & H. Retinal neovascularization (G) and avascular areas (H) were determined as described in “Materials and Methods.” The bar graphs represent quantitative analysis of 6 retinas. The values are presented as Mean ± SD. * p < 0.01 vs WT. Scale bar represents 50 μm and 20 μm in panel C, far left column and far right column, respectively, 20 μm in panel E and 300 μm and 50 μm in panel F, far left column and far right column, respectively.