ERG analysis of rep1 mutant larvae. (A) ERG responses from 5 dpf wild-type (Left) and mutant (Right) to 200-msec flashes of light at the designated wavelengths. Each waveform was based on the average response from 10 stimulus presentations. Stimulus irradiance (15 log quanta s^-1·cm^-2) was the same across all panels. Bars represent the light stimulus. (B) Graph of the average irradiance-response functions from wild-type (filled circles; n = 11) and mutant (open circles; n = 14) subjects based on the b-wave amplitude at three different wavelengths. Error bars represent ±1 SEM. A mixed-design ANOVA found a significant difference (P d 0.05) between the wild-type and mutant responses at the last five irradiances tested.

Histological sections of 4.5 dpf wild-type and rep1 mutant retinas. (A) Wild-type retinas at 4.5 dpf have fully laminated and retinal ganglion cells (GCL), amacrine and bipolar interneurons (INL), and photoreceptors (ONL) differentiated, and the optic nerve (O.N.) is apparent. (B) Lamination and cellular differentiation is not affected in rep1 mutants, but eye size is reduced and the RPE layer appears irregular. (C) High magnification of sections from light-adapted wild-type retinas showing the rod (r) outer segments positioned distally from the cone (c) outer segments. (D) Sections of rep1 mutants showing areas of RPE hypertrophy (arrows) into the photoreceptor layer and other regions where the RPE is almost devoid of pigmentation (arrowhead). Rod and cone outer segments are not normally positioned and are shorter than wild-type.

Transmission electron microscopy of 4.5 dpf wild-type and rep1 mutant retinas. (A) Electron micrographs of wild-type retinas reveal an orderly array of photoreceptor outer segments and the uniform thickness of the RPE. Melanosome maturation within the RPE is normal. (B–D) Electron micrographs from multiple rep1 retinas showing degeneration of the RPE and photoreceptors. Arrows in B and C indicate large vacuoles observed in the RPE of rep1 mutants. Arrowheads in D indicate outer segment material not digested by the RPE. Melanosome size and maturation vary more dramatically and are less dense than what is seen in wild type.

Opsin trafficking is unaffected in rep1 mutants. Retinal cryosections were stained with markers for rod and cone photoreceptors in wild type (A–D) and rep1 mutants (E–H). The 1D1 marker labels rhodopsin (Rho), whereas antibodies are against blue opsin (BOPS) and green opsin (GOPS) in the photoreceptor outer segments of wild-type and rep1 mutants. The zpr-1 antibody labels the red/green double cones and reveals the disheveled morphology of the photoreceptors. All sections were counterstained with DAPI to visualize nuclei. Pyknotic nuclei, a hallmark of cell death, are seen in rep1 mutants and indicated by arrows. (Scale bar, 20 μm.)