Retinal angiogenesis is impaired in the absence of tissue macrophages. Double-labeling of transverse sections through a wild-type mouse eye at 11.5 dpc (A) and a wild-type mouse retina at P21 (B) with IB4 and F4/80 (A) or an antibody specific for smooth muscle actin (SMA); cell nuclei in panel B were labeled with DAPI. (C-I) Whole mount labeling of the wild-type retinal vasculature at P4 (C), P9 (D-F) and P21 (G-I). (C) Some macrophages (solid arrows) interact with endothelial tip cells (solid arrowheads) at the vascular front; others embrace emerging vascular bridges (wavy arrows). (D-E) Activated caspase 3 (aC3) is present in regressing vessel segments near arteries (yellow); note capillary narrowing at the junction of the regressing vessel segment with a stable vessel (open arrowheads) and a macrophage at one of the narrow ends (solid arrow); the area boxed in panel D is shown at higher magnification in panel E. (F) Collagen IV and IB4 expression are retained by acellular capillaries (feathered arrow). (G-I) IB4-positive vessel regression profiles (red arrows) and stable intersections (red arrowheads) between nonregressing vessel segments at P21 in the deep plexus of the indicated genotypes. Scale bars represent 50 μm (except in A, 100 μm). (J-K) Quantitation of stable intersections (J) and regression profiles (K) in the deep plexus at P21; n = 3. Error bars represent SD of the mean. P values were determined by comparing the measurements obtained for both types of mutants to the control, which contained the measurements obtained for wild-types from both groups. SP indicates superficial plexus; IP, intermediate plexus; DP, deep plexus; ONL, outer nuclear layer; INL, inner nuclear layer; RGC, retinal ganglion cell layer; OPL, outer plexiform layer; PR, photoreceptor layer; a, artery; and v, vein.