In situ hybridization of dio2 and dio3b on consecutive transverse cryosections of the eye of uninjected control zebrafish at 3 dpf. (A) dio2 AS: dio2 is expressed throughout all layers of the retina except IPL and most strongly in the OPL and ONL. (B) dio2 S: negative control. (C) dio3b AS: dio3b is also expressed in all retinal layers except IPL and most strongly in OPL and INL. (D) dio3b S: negative control. The different layers of the retina are indicated as GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer. AS: antisense probe; S: sense probe.

Effects of deiodinase KD on morphometric parameters. (A) Eye size at 29 hpf, 4 and 7 dpf. (B) Body length at 29 hpf, 4 and 7 dpf. (C) Calculated relative eye size (RES) values (observed eye size divided by expected eye size, see Supplementary Fig. S2) at 4 and 7 dpf. Data represent mean + SEM (n ≥ 47 per group). Statistical significance scores are based on coefficients of a mixed effects model (batch = random effect). *p<0.05, **p<0.01, ***p<0.001. SCMO: control group, D1D2MO: KD of D1+D2; D3bMO: KD of D3b

Effects of deiodinase KD on retinal lamination. Hematoxylin and eosin stain on cross sections of the eye of control and KD animals at 3 and 7 dpf. The different layers of the retina are denoted in the control conditions. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer. SCMO: control group, D1D2MO: KD of D1+D2; D3bMO: KD of D3b.

Effects of deiodinase KD on relative rod and cone photoreceptor number. (A-L) Immunohistochemical stainings with Zpr3, Zpr1, anti-blue opsin and anti-UV opsin antibodies on cryosections of the eye of control (A,D,G,J), D1D2MO (B,E,H,K) and D3bMO (C,F,I,L) larvae at 3 dpf. Zpr1 is a marker for both green and red cone photoreceptors, Zpr3 is a marker for rod photoreceptors and anti-blue and anti-UV opsin label the outer segment of blue and UV cone photoreceptors respectively. Blue: DAPI staining of the nuclei; Green: Zpr1/Zpr3+Alexa488 or anti-blue/anti-UV + FITC. (M-P) Data for relative rod/cone number (expressed per retinal surface area) are shown as mean + SEM (n ≥ 5 per group). Groups with no common letter are significantly different (p < 0.05). Data in (N,O,P) followed Gaussian distribution and were analyzed by one-way ANOVA with Tukey post hoc test. Data in (M) did not assume Gaussian distribution and were analyzed by Kruskal-Wallis and Dunn′s post hoc test. SCMO: control group, D1D2MO: KD of D1+D2; D3bMO: KD of D3b.

Optokinetic response test to reveal visual impairments. Semi-quantitative scoring of the optokinetic response (OKR) test at 100 hpf and 7 dpf for control, KD and rescue larvae. Data show a significant delay for D3bMO when compared to control and D1D2MO larvae at 100 hpf and compared to all groups at 7 dpf (p < 0.001 and p < 0.05 respectively, Chi-square statistics). Co-injection with hD3 mRNA partially rescues the phenotype. SCMO: control group, D1D2MO: KD of D1+D2; D3bMO: KD of D3b, D3bMO + hD3 RNA: rescue group; Full OKR: normal optokinetic response with the eyes following the movement of the stripes and returning to their original position; Partial OKR: the eyes follow the movement of the stripes but do not return to their original position after reaching the border of the visual field; no OKR: fish do not respond to the moving stripes.

Analysis of the light response in control and deiodinase KD larvae. The light response was analyzed at 4 dpf (A,B), 5 dpf (C,D) and 7 dpf (E,F). The left column depicts average activity patterns for the different conditions throughout the test period. The right column specifically compares activity in the last 2 min in the dark with activity during the first 10 s of light. Boxes show median with 25th and 75th percentiles. Whiskers represent 10th and 90th percentiles. Statistical comparisons were made using wilcoxon-matched pairs signed rank test. *: significant difference between activity in dark and light, i.e. significant light response (p<0.05). SCMO: control group, D1D2MO: KD of D1+D2; D3bMO: KD of D3b.

Relative rod and cone photoreceptor number in D1D2MO and D3bMO zebrafish at 4 and 7 dpf. Data represent relative cone/rod numbers per retinal surface area (expressed as number ×10^-3/pixel²) and are shown as mean ± SEM (n ≥ 5 per group). The following antibodies were used: Zpr1 (green and red cones), anti-blue opsin (blue cones), anti-UV opsin (UV cones) and Zpr3 (rods). Rod data were obtained in a separate experiment and only counted at 4 dpf. For a given opsin and stage groups with no common letter are significantly different (p < 0.05). Data followed Gaussian distribution and were analyzed by one-way ANOVA with Tukey post hoc test.