Figure 3. Effect of lebercilin loss on bulge formation, distal axoneme organization, and CC length.

(A) Widefield (original magnification, 63×) images of expanded photoreceptors stained for POC5 (green) and tubulin (magenta) from P10 to P22 in Lca5^gt/gt (HOM) mice and in P22 Lca5^+/gt (HET) mice. Lines in images illustrate the measurements used in C of tubulin width at 3 locations: +500 nm, 0 nm, −500 nm. The distal end of CC inner scaffold marker POC5 was used to set the 0 location. Scale bars: 500 nm. (B) Distal axoneme (above CC) conformations of HET versus HOM photoreceptors at P18 and P22 indicated in percentages. Photoreceptor distal axoneme conformations: normal (HET: 99.6%; HOM: 50.2%), open/broken (HET: 0.4%; HOM: 40.8%), and bent/curled (HET: 0%; HOM: 9%). n = 247 (HET), n = 217 (HOM). (C) Tubulin width measurements of the photoreceptor at the 3 locations depicted in A from P10 to P22. Average tubulin width at each location is indicated by a gray or red dot for HET and HOM, respectively. P28 not included, since most of the distal axonemes are lost at this time point. Only photoreceptors that were stained for tubulin and POC5 were used for the measurements. Three animals per time point. Data presented as mean ± SD; n = 16–29. *P < 0.05, **P < 0.01, ***P < 0.001 by F test. Significance represents the tubulin width dispersion between HET and HOM. (D–F) Widefield (original magnification, 63×) images of expanded photoreceptors stained for tubulin (magenta) and CEP290 (cyan, D), POC5 (green, E), or RP1 (white/gray, F) from P10 to P28 in HOM mice. Scale bars: 500 nm. (G–I) Impact of lebercilin loss on CEP290 length (G), CC inner scaffold length (POC5, H), or RP1-normalized intensity at the bulge region (I) from P10 to P28. Three animals per time point. Data presented as mean ± SD; n = 39–65 (G), n = 19–60 (H), n = 39–72 (I). **P < 0.01, ****P < 0.0001 by Mann-Whitney test.