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Figure 8. The actin cytoskeleton of the calyceal processes and their roots is disrupted in pcdh15 morphant larvae. (A) The axoneme, stained with antibodies against acetylated tubulin (red), is present in the morphant cone photoreceptors, but it is often curved and bent. The typical distribution of the USH2 protein Adgrv1 (green) at the base of the axoneme, in the periciliary ridge complex, is preserved in morphant photoreceptors. (B) At the inner segment (IS)–outer segment (OS) interface, there are two F-actin networks: the transverse F-actin network at the apical surface of the IS, and the longitudinal F-actin bundles of the calyceal processes and their roots (arrows). In 4-dpf morphant retinas, morphants display less phalloidin staining at the IS–OS interface (middle) than do the age-matched controls (left). F-actin staining in morphant photoreceptors is restored by the coinjection into the embryo of pcdh15 cRNAs with the pcdh15 MO e6-i6 (see arrows in the right panel). (C) The transverse F-actin network (green) can still be observed in a morphant photoreceptor. (D) Measurements of F-actin fluorescence intensity in the IS–OS interface (FIS–OS) compared with the subjacent region of the outer plexiform layer (OPL) (FOPL, taken as a reference) showed an FIS–OS/FOPL ratio that is 50% lower in the morphants than in the controls. The F-actin cytoskeleton was partially preserved in the retinas of 4-dpf morphants after the coinjection into the embryo of pcdh15 cRNAs and pcdh15 MO e6-i6 (FIS–OS/FOPL ratio 20% smaller than in controls). The FIS–OS/FOPL ratio at 3 dpf: controls, 1.05 ± 0.10 (n = 5); morphants, 0.54 ± 0.1 (n = 6), mean ± SEM; unpaired t test, **, P = 0.007. At 4 dpf: controls, 0.97 ± 0.03 (n = 10); morphants, 0.48 ± 0.06 (n = 20); morphants + cRNAs, 0.81 ± 0.08 (n = 9); unpaired t tests: controls versus morphants, ***, P < 0.0001; morphants versus morphants + cRNAs, **, P = 0.002; controls versus morphants + cRNAs, P = 0.05. Bars, 5 µm. |
