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Exp Eye Res
2025 Oct 22;262:110699. doi: 10.1016/j.exer.2025.110699.
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RPE65 knockout Xenopus laevis have a compromised but detectable electroretinogram and altered visual responses, without retinal degeneration or altered melanophore dispersion.
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The isomerohydrolase RPE65 is a critical element of the visual cycle, the series of enzymatic reactions by which the chromophore of the visual pigments is regenerated following light exposure. In humans, mutations in the RPE65 gene cause a severe form of blindness called Leber's congenital amaurosis. Studies of Rpe65-/- mice have shown dramatic depletion of 11-cis-retinal in the retina, resulting in a slow retinal degeneration. However, a number of studies suggest that RPE65 may not be necessary for the regeneration of photopigment in all photoreceptor types. Using CRISPR/Cas9 technology, we previously generated RPE65 knockout Xenopus laevis in order to test the involvement of rhodopsin chromophore in the cell death mechanisms associated with rhodopsin mutations and rhodopsin quality control. Here we further characterize the effects of RPE65 knockout in these animals, and show their rod photoreceptors have shortened outer segments that lack detectable rhodopsin photopigment. However, there is no progressive degeneration of rods or cones. Via electroretinography we found greatly reduced but significant responses to light under scotopic and photopic conditions. We also found reduced behavioral sensitivity to light, while light-induced melanophore dispersion was unaffected. RPE65 knockout X. laevis may be a useful system for examining RPE65-independent photosensation mechanisms in vertebrates.
Fig. 1. RPE65 knockout X. laevis have no retinal degeneration, no RPE65 expression, and altered ROS length. A: Confocal microscopy of retinas from 14 d to 3m old animals labeled with antibodies shown (green) and counterstained with wheat germ agglutinin (WGA) (red) and Hoechst 33342 (blue), animals are wildtype (WT), knockout (KO), or have one WT “S” allele (WT S). Sections are labeled with anti-RPE65, anti-long wavelength sensitive opsin 1 (LWS1), anti-short wavelength-sensitive opsin 1 (SWS1) and anti-short wavelength-sensitive opsin 2 (SWS2). B: Western blot showing lack of RPE65 expression in edited animals, including one sample from an animal with one WT S allele. W – WT, E = edited (WT S is “E” labeled in green). C: quantification of Western blot data shown in B. D: Quantification of OS length shown in A. In C and D, the average value and±S.E.M. are shown in red. E: OCT imaging of an older knockout animal (4 years) compared to WT. The OS layer is still intact.
Fig. 2. Difference spectra of WT vs KO animals. Black trace: sample obtained from a WT animal. Red trace: sample obtained from an RPE65 KO animal. Indicated peaks on the black trace are consistent with rhodopsin (525 nm) and meta-II rhodopsin (390 nm). Indicated peaks on the red trace are consistent with the spectra of hemoglobin.
Fig. 3. Electroretinography of WT and RPE65 KO X. laevis. Traces are shown in A, C, E, and G, and B wave amplitudes are graphed in B, D, F and H. A-D are scotopic ERGs on 6-week-old (6w) (A, B) or 3-month-old (3m) (C, D) animals. E-H are photopic ERGS for 6-week-old (E-F) or 3-month-old (G-H) animals. The black arrows indicate an artifact associated with the flash discharge. Flash intensities are indicated beside the traces, in cd∗s/m2. Black traces or plots = WT, Red traces or plots = KO. Error bars are±S.E.M. Traces represent averages of 5–7 animals; similarly, data points are averages of 5–7 animals.
Fig. 4. Spectral sensitivity of WT and RPE65 KO X. laevis investigated by electroretinography. Traces are shown in A, C, E, and G, and B wave amplitudes are graphed in B, D, F and H. A-D are scotopic ERGs at 4 different wavelengths, performed on 3-month-old (3m) WT (black) or KO (red) animals at either medium (A-B) or high intensity (C-D) flashes. Flash wavelength is shown next to the traces. E-H are photopic ERGs performed on 6-week-old (6w) animals (E-F), or 3-month-old animals (G-H) performed at the same wavelengths as above. Error bars are±S.E.M. Traces represent averages of 4–7 animals; similarly, data points are averages of 5–7 animals. Black arrows indicate an artifact associated with the flash discharge.
Fig. 5. Visual function in tadpoles tested using a behavioral assay. Assay results are shown on the left, and representative images from the assay are shown on the right. In the images, the location of each tadpole is marked using a red dot. WT tadpoles prefer to swim over a light background. KO tadpoles show less preference, with a distribution close to random. P values are derived from a 2-sample T test (WT vs KO) and a 1-sample T-test (KO vs 50 %).
Fig. 6. Pigment granule migration in tadpole melanocytes. WT, RPE65 KO, and enucleated WT and RPE65 KO animals were moved from dark to light condition, and photographed at several timepoints (T). Binary images were generated and the area of pigment was measured and plotted (top, representative images below). En = enucleated animal. See text for statistical analysis.
