Mislocalization of EYS protein in pomgnt1 mutant zebrafish retina. Retinal sections from wild-type and pomgnt1sny7 mutant zebrafish were double immunostained with EYS (green fluorescence) and acetylated α-tubulin (red fluorescence). (A and E) Wild-type retina at 2-mpf. (C and G) Wild-type retina at 6-mpf. Most EYS-positive puncta in the wild-type retina were associated with basal end of the acetylated α-tubulin reactivity at both 2- and 6-mpf (arrows). (B and F) Homozygous pomgnt1sny7 mutant retina at 2-mpf. (D and H) Homozygous pomgnt1sny7 mutant retina at 6-mpf. Most EYS-positive puncta were not associated with acetylated α-tubulin but were localized in the outer nuclear layer at both 2- and 6-mpf (arrowheads). (I and J) Counting of EYS-acetylated α-tubulin double staining from the outer nuclear layer to the retinal pigment epithelium (RPE) for 2-mpf and 6-mpf retina, respectively. Note that acetylated α-tubulin reactivity associated with EYS puncta was decreased in the mutant, and acetylated α-tubulin not associated with EYS puncta was increased in the mutant. EYS puncta not associated with acetylated α-tubulin was increased in the mutant. ANOVA; post-hoc Student’s t-test with Bonferroni correction.(K and L) Ratio of EYS and acetylated α-tubulin immunoreactivity for each acetylated α-tubulin-labeled axoneme in rods and cones at 2-mpf, respectively. Note that EYS immunoreactivity at the connecting cilium was reduced by about 5-fold in pomgnt1 mutant retina. Student’s t-test. (M and N) Ratio of EYS and acetylated α-tubulin immunoreactivity per acetylated α-tubulin-labeled axoneme in rods and cones at 6-mpf, respectively. Note that EYS immunoreactivity at the connecting cilium was also reduced in the pomgnt1mutant retina at 6-mpf. Student’s t-test. (O and Q) Total EYS immunoreactivity at 2-mpf and 6-mpf, respectively. Total EYS immunoreactivity was reduced in pomgnt1 mutant retina at both ages. Student’s t-test. (P and R) Histogram representation of total EYS immunoreactivity between wild-type/heterozygous and mutant fish at 2-mpf and 6-mpf, respectively. Note the shift in the number of EYS puncta to lower fluorescence intensities in the pomgnt1 mutant fish at both ages. Scale bar in H: 5 µm for A-D; 2.5 µm for E-H.

Mislocalized EYS in pomgnt1 mutant zebrafish retina was co-localized with synaptogamin-1. Retinal sections from 2-mpf zebrafish were double stained with antibodies against EYS (red fluorescence) and synaptotagmin-1 (green fluorescence). (A) Wild-type inner/outer segment layers showing connecting cilia (CC). Maximal projection and its orthogonal views of three double stained puncta are shown. EYS-immunoreactivity was co-localized with synaptagmin-1 reactivity. (B) Wild-type outer nuclear layer (ONL). Maximal projection and its orthogonal views of two double stained puncta are shown. EYS-immunoreactivity was co-localized with synaptagmin-1 reactivity. (C) Homozygous pomgnt1sny7 mutant outer nuclear layer. Maximal projection and its orthogonal views of three double stained puncta are shown. Mislocalized EYS immunoreactive puncta was co-localized with synaptotagmin-1 reactivity. Scale bar in C: 2 µm.

Loss of photoreceptors in pomgnt1 mutant retinas at 6-mpf but not 2-mpf. Retinal sections from 2-mpf and 6-mpf zebrafish were immunostained with GNAT2 antibody (green fluorescence, A–D) and 1D4 (green fluorescence, E–H). The sections were counter-stained with DAPI. To quantify photoreceptors, DAPI fluorescence confocal images of dorsal and ventral retinas from the optic nerve head to the ciliary margin were each divided into 5-equal compartments. Nuclei within the outer nuclear layer of each compartment were counted (M and N). GNAT2 and 1D4 immunoreactive intensities were measured (I–L). (A) Wild-type GNAT2 immunostaining at 2-mpf. (B) Homozygous pomgnt1sny7 mutant GNAT2 immunostaining at 2-mpf showing similar immunoreactivity compared to wild-type. (C) Wild-type GNAT2 immunostaining at 6-mpf. (D) Homozygous pomgnt1sny7 mutant GNAT2 immunostaining at 6-mpf showing reduced immunoreactivity compared to wild-type. (E) Wild-type 1D4 immunostaining at 2-mpf. (F) Homozygous pomgnt1sny7 mutant 1D4 immunostaining at 2-mpf showing similar immunoreactivity compared to the wild-type. (G) Wild-type 1D4 immunostaining at 6-mpf. (H) Homozygous pomgnt1sny7 mutant 1D4 immunostaining at 6-mpf showing reduced immunoreactivity compared to the wild-type. (I) GNAT2 immunofluorescence intensity quantification of wild-type and mutant fish at 2-mpf. There was no significant difference between wild-type and mutant retinas. (J) 1D4 immunofluorescence intensity quantification of wild-type and mutant fish at 2-mpf. There was no significant difference between wild-type and mutant retinas. (K and L) GNAT2 and 1D4 immunofluorescence intensity (artificial units, AU) in the mutant retina was reduced at 6-mpf. Student’s t-test. (M) Nuclei counting of outer nuclear layer at 2-mpf. There was no significant difference between wild-type and mutant retinas. (N) Nuclei counting of outer nuclear layer at 6-mpf. Nuclei within the outer nuclear layer in the mutant were reduced in number compared to wild-type. P = 0.00026; repeated measures ANOVA. (Oand P) 2-mpf nuclei count in the ONL-R and ONL-C retinal layers, respectively. There was no significant difference between wild-type and homozygous pomgnt1sny7 mutants. (Q and R) 6-mpf nuclei count in the ONL-R and ONL-C retinal layers, respectively. Note the significant reduction of nuclei in both ONL-R and ONL-C in homozygous pomgnt1sny7 mutants at 6-mpf. Together, these results indicated photoreceptor degeneration in mutant retina. Student’s t-test. Scale bar in H: 5 µm.

Reduction and mislocalization of rhodopsin in pomgnt1 mutant retina. Retinal sections from 2-mpf and 6-mpf zebrafish were immunostained with antibody 4D2 (red) and counter-stained with DAPI (blue). (A) 4D2 immunostaining of wild-type retina at 2-mpf. (B) 4D2 immunostaining of homozygous pomgnt1sny7 mutant retina at 2-mpf. (C) Quantification of 4D2 fluorescence intensity at 2-mpf. There was no significant reduction in 4D2 intensity in the mutant fish at this age. Student’s t-test. (D) 4D2 immunostaining of wild-type retina at 6-mpf. (E) 4D2 immunostaining of homozygous pomgnt1sny7 mutant retina at 6-mpf. Note the reduction in 4D2 fluorescence intensity. Note the mislocalization of 4D2 immunoreactivity to the ONL-R (arrows). (F) Quantification of 4D2 fluorescence intensity at 6-mpf. There was a significant reduction in 4D2 intensity in the mutant fish at this age. Student’s t-test. (G) High mag of wild-type 4D2 staining at 6-mpf. Fluorescence intensity of 4D2 in the ONL-R was low. (H–K) High mag of homozygous pomgnt1sny7 mutant 4D2 staining at 6-mpf. Note the mislocalization of 4D2 immunoreactivity to the ONL-R layer (arrows). (L) Ratio of 4D2 fluorescence intensity of outer nuclear layer/outer segment layer (ONL/OS). 4D2 ONL/OS fluorescence ratio was increased in the mutants. Student’s t-test. Scale bar in E: 5 µm for A, B, D and E; scale bar in K: 5 µm for G-K.