RPE degeneration in Rev-erbα−/−eyes. (A) Isolated RPE/choroid complex flat mounts from 12-month-old Rev-erbα+/+ and Rev-erbα−/− mice stained with β-catenin (green) and DAPI (blue). White arrows indicate areas with junctional disruption and nuclear fragmentation. Scale bar: 50 μm. (B) Toluidine blue staining of epoxy semi-thin sections of Rev-erbα+/+ and Rev-erbα−/− eyes from 16-month-old mice. Scale bar: 20 μm. (C) Increased sub-RPE deposition of vitronectin and C3 in Rev-erbα−/− vs. Rev-erbα+/+ eyes at 16 months old. Drusen-like deposit (highlighted by black dash line) are shown in Rev-erbα−/− but not Rev-erbα+/+ eyes at 12 months old. Scale bar: 10 μm. (D) Western blot and quantification show higher C3 (α chain) protein levels in Rev-erbα−/− RPE in 12-month-old mice compared with age-matched Rev-erbα+/+ mice. Protein size: C3 α chain (115 kDa). n = 3 mice/group. (E) Electron microscopy (EM) imaging of 6-month-old Rev-erbα−/− mice show outer collagenous (OCL) deposits (indicated by yellow arrowhead) in Bruch's membrane (BrM) and increased BrM thickness compared with WT as quantified. Scale bar: 1 μm. (F) EM images from 18-month-old Rev-erbα−/− eyes show focal loss of photoreceptor outer segments (upper panel, scale bar, 10 μm), vacuolization in the RPE (bottom left panel, marked by asterisks, scale bar, 1 μm), and disorganized outer segment disc membranous material surrounded by the microvilli of the RPE (bottom right panel scale bar, 2 μm), compared with age-matched WT eyes (left panel, scale bar: 10 μm). Error bars indicate SD. *: P < 0.05, ****: P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)