Figure 2. Age-dependent abnormalities in the RPE cells of cryba1 cKO mice. (A) RPE flatmounts from 7-mo-old Cryba1^fl/fl and cKO mice were stained with DAPI for nuclei (red) and FITC-phalloidin to label the actin cytoskeleton (green). In Cryba1^fl/fl RPE, cells are cuboidal and regularly arranged with cell membranes clearly defined by phalloidin staining (arrows). Cellular architecture is disordered in the cKO sample with poorly defined cell margins and weaker phalloidin staining. Bar: 50 μm. (B) Immunostaining of 12 mo old Cryba1^fl/fl and cKO retinal sections with the lipid droplet marker PLIN2. PLIN2-positive lipid droplets (arrows) are present in the RPE layer of the Cryba1^fl/fl retina, owing to age-related accumulation of lipids, but are much more prominent in the cKO sample. Based on an established grading system (see refs. 19 and 20), the Cryba1^fl/fl scored 3 and the cKO scored 5 on a scale where 7 was maximal staining intensity. Bar: 10 µm. (C) Fundus photographs showed dark hyperpigmentation of RPE cells and subretinal lesions at the posterior pole in cKO mice by 4 mo of age. With increasing age, the lesions increased in number and were scattered throughout the fundus in cKO mice. n = 7 WT, 10 cKO mice. (D) Transmission electron microscopy of 2-mo-old cKO RPE shows many vacuole-like structures with degenerated membrane-bound cellular organelles (red arrows, center), undigested photoreceptor outer segments (red arrow, right) and loss and truncation of basal infoldings (blue asterisks), as compared with Cryba1^fl/fl (left). Bar: 500 nm. (E) At 9 mo, RPE from cKO mice shows large vacuoles (blue arrows, middle image) containing partially degraded cellular debris. Interestingly, type 1 lysosomes are present in Cryba1^fl/fl (green asterisks, left image), but rarely seen in cKO RPE. Moreover, loss of Cryba1 impedes lysosomal function because more melanosomes (red arrowheads) are retained in cKO, as compared with Cryba1^fl/fl. The right image shows, at higher magnification, a degradative autophagic vacuole (Avd, red arrow) and a very large vacuole containing partially degraded cellular material (blue arrow) in a cKO sample. Bar: 2 μm.