Conditional knockout of <i>Ift20</i> ablates primary cilia in the RPE without affecting other retinal layers and causes subtle alterations in RPE ultrastructure.

(a) Representative fluorescent images of E16.5 RPE flatmounts stained for ARL13B (red) and GT335 (green) to visualize primary cilia. Staining for F-Actin (magenta) was used to visualize the cytoskeleton, DAPI to stain nuclear DNA. Scale bars: 10 μm. (b) Quantification of primary cilia in E16.5 RPE revealed that Ift20null;Tyrp2-Cre RPE showed significantly less ciliated cells (13% n = 4 eyes (1,039 cells)) compared to controls (84.3% n = 4 eyes (816 cells)). Statistical analysis was performed using ROUT test (Q = 0.1%) before using unpaired t test (p Ift20+/+;Tyrp2-Cre 84.75% Ift20null;Tyrp2-Cre 13.21%. (c) Cilia length data binned at intervals of 0.5 μm. Cilia remaining in the mutant where more likely to be longer than in control. Ift20+/+;Tyrp2-Cre (n = 4 eyes 191 cilia, Ift20null;Tyrp2-Cre n = 4 eyes 43 cilia). (d) Representative images of histological eye sections from 1-month-old mice. In both Ift20null;Tyrp2-Cre and control RPE, no differences in retinal layers could be observed. Scale bar: 25 μm. (e) Representative TEM image of a photoreceptor connecting cilium from control and mutant mice. Photoreceptor connecting cilia remained intact. Scale bar: 1 μm. (f) Schematic showing a typical photoreceptor primary cilium. (g) Representative TEM images of eye sections showing subretinal gaps in Ift20null;Tyrp2-Cre mice at 1 month of age, while they appear to be gone by 4 months. Scale bars: 10 μm. (h) Representative TEM images of eye sections from 4-month-old mice showing multilayered RPE cells (top), as well as the absence of microvilli and accumulation of debris (bottom, red outline, arrow highlights the continuous apical membrane) in Ift20null;Tyrp2-Cre mice. Scale bars: 10 μm. (i) Measurements taken from TEM images: Bruch’s membrane, RPE thickness and RPE to POS distance. (j) Quantification of RPE thickness measured in Ift20+/+;Tyrp2-Cre and Ift20null;Tyrp2-Cre revealed significant differences between 1 and 4 months, but no differences between control and knockout. Median: 1 month Ift20+/+;Tyrp2-Cre 6.0 μm, Ift20null;Tyrp2-Cre 5.0 μm. 4 months: Ift20+/+;Tyrp2-Cre 6.8 μm, Ift20null;Tyrp2-Cre 6.5 μm. (k) Quantification of RPE to POS distance measured in Ift20+/+;Tyrp2-Cre and Ift20null;Tyrp2-Cre shows highly significant differences between control and knockout. Median: 1 month Ift20+/+;Tyrp2-Cre 0.77 μm, Ift20null;Tyrp2-Cre 1.38 μm. 4 months: Ift20+/+;Tyrp2-Cre 1.24 μm, Ift20null;Tyrp2-Cre 2.09 μm. (l) Quantification of Bruch’s membrane thickness measured in Ift20+/+;Tyrp2-Cre and Ift20null;Tyrp2-Cre reveals only slight increase in the mutant at 1 month of age. Median: 1 month Ift20+/+;Tyrp2-Cre 0.65 μm, Ift20null;Tyrp2-Cre 0.75 μm. 4 months: Ift20+/+;Tyrp2-Cre 0.46 μm, Ift20null;Tyrp2-Cre 0.48 μm. Statistical analysis was performed using an unpaired two-tailed t test. (For j–l: 1 month Ift20+/+;Tyrp2-Cre n = 6 eyes, >60 measurements, Ift20null;Tyrp2-Cre n = 6 eyes, >60 measurements; 4 months Ift20+/+;Tyrp2-Cre n = 4 eyes, >50 measurements, Ift20null;Tyrp2-Cre n = 4 eyes, >50 measurements). Significance levels: >0.05 not significant (ns), S6 Table.