Fig. 5
Early retinal angiogenesis is delayed in Atf6−/− mice. (a) Wholemounts of Atf6+/+ and Atf6−/− mice at P3, P7, P10, and P14 labeled with IB4 to visualize vasculature. Retina area is outlined. (b) Retina area did not differ significantly between Atf6+/+ and Atf6−/− retinas at P3, P7, P10, and P14 (data represent mean ± SEM, p > 0.05, two-way ANOVA, n = 3/group). (c) Vascular fraction was significantly reduced in Atf6−/− retinas compared to Atf6+/+ retinas at P7 (n = 6/group) but showed no significant difference at P3 (n = 6/group) or P10 and P14 (n = 3/group) (data represent mean ± SEM, *p < 0.05, two-way ANOVA). (d) Wholemounts were analyzed for branch number in fields from central (250 μm away from the optic disc), mid-peripheral (750 μm away from the optic disc), and peripheral (1.25 mm away from the optic disc) regions (black boxes, 250 μm x 250 μm) in the superior region (left). Schematic of vascular plexus development (center). ImageJ (FIJI) quantification workflow (right). (e) Confocal microscopy images of IB4-stained wholemounts at P7 (superficial layer), P10 (superficial and deep layers), and P14 (superficial, deep, and intermediate layer) in Atf6+/+ and Atf6−/− retinas. (f) Branch counts in the superficial layer at P7, P10, and P14. At P7, branch number is significantly greater in the central retina of Atf6−/− mice compared to Atf6+/+ mice (data represent mean ± SEM, *p < 0.01, two-way ANOVA, n = 7/group). At P10 and P14, no differences in the central retina (data represent mean ± SEM, p > 0.05, two-way ANOVA, n = 3–4/group). Mid-peripheral and peripheral regions showed no differences at any time points (P7, n = 7/group; P10 and P14, n = 3–4/group). Branch numbers in the deep layer at P10 and P14 and the intermediate layer at P14 showed no differences between groups. (g) Representative images of endothelial tip cells at P7 in Atf6+/+ and Atf6−/− retinas labeled with IB4 (left). Tip cell number is significantly greater in Atf6−/− retinas (right, data represent mean ± SEM, *p < 0.05, Student’s t-test, n = 3–4/group). Sup. superior; temp. temporal; inf. Inferior. Scale bar = 1 mm (A); 1 mm (D); 50 μm (E); 50 μm (G).
