Blocking R-cadherin function disrupts vascular targeting of HSCs. (A) Scanning laser confocal microscopy analysis of frozen retinal cross-sections demonstrates that 3 vascular plexuses (red) are formed in the normal retina: (1) the superficial vascular plexus (star) in the GCL; (2) the intermediate vascular plexus (arrowhead) at the inner edge of the INL; and (3) the deep vascular plexus (arrow) at the outer edge of the INL. (B) Lin^- HSCs from eGFP transgenic mice (green) injected intravitreally into postnatal day 6 developing mouse eyes become localized to the 3 retinal vascular plexuses. (C) Blocking R-cadherin-mediated adhesion prevented normal targeting and caused many HSCs (green) to become localized to the outer edge of the photoreceptor layer, while (D) HSCs preincubated with control preimmune IgG target normally. (E) A low-magnification image across an entire retina cross-section demonstrates the extent of HSCs mistargeting caused by blocking R-cadherin adhesion. (F) Confocal images through z-planes of the 3 normal vascular plexuses and the outer edge of the photoreceptor layer. Fewer HSCs localize to the normal vascular plexuses and many localize at the outer edge of the photoreceptors when R-cadherin adhesion is blocked. (G) The extent of mistargeting was quantified by the number of HSCs mistargeted to the outer edge of the photoreceptors relative to the total number of retinally incorporated HSCs. Error bars indicate standard deviation. GCL indicates ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer (photoreceptors); blue = DAPI (nuclei); red = isolectin Griffonia simplicifolia (blood vessels); green = HSCs. Size bars = ∼50 μm. Original magnification × 20 (A, C, D, F); × 40 (B); and × 10 (E, which is a montage of 5 photos).