Spatiotemporal expression pattern of slc7a14 in zebrafish. (A) The slc7a14 expression increased remarkably from 1 to 7 dpf. (B) Spatial expression pattern of slc7a14 in 3-month-old adult male zebrafish. (C)In situ hybridization (ISH) of slc7a14 in retinas and eyes. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; RPE, retinal pigment epithelium. Scale bar = 50 μm. (D) ISH of slc7a14 in embryos. Scale bar = 100 μm.

Morphology of slc7a14-deficient zebrafish morphants. (A) Lateral view of zebrafish larvae. Embryos injected with doses of 4.0 and 6.0 ng MOs exhibited apparent microphthalmia. Scale bar = 200 μm. (B) Magnified view of larval eyeballs showing the significant reductions in eye area with high-dose MOs. Scale bar = 100 μm. (C) Vertical view of larval eyeballs showing the reductions in eye axis length with high-dose MOs. Scale bar = 100 μm. (D,E) Quantification of eye axis length and the axis-to-body length ratio. N = 46 in each group. Bar plots are shown as the mean ± s.e.m. Data was analyzed using One-way ANOVA followed by Tukey’s post hoc test, ^∗∗P < 0.01, ^∗∗∗P < 0.001 significantly different from control 6.0 ng group.

Immunostaining of zpr-1, zpr-2, and zpr-3 in slc7a14-deficient morphants. (A) In the peripheral retina, the high-dose slc7a14-MOs led to sharp reductions in zpr-3. Weaker fluorescence signals were detected in the high-MO dose groups (slc7a14 4.0 ng and slc7a14 6.0 ng) than in the control group. (B) Statistical results for zpr-3 (n = 10 for each group). (C) The high-dose slc7a14-MOs led to significant reductions in zpr-2. There were few fluorescence signals in the peripheral retina in the high-MO dose group (slc7a14 6.0 ng). The RPE in the low-MO dose group (slc7a14 2.0 ng) was relatively normal compared to that in the wild-type group and control group. (D) Statistical results for zpr-2 (n = 10 for each group). (E) No significant changes were found in cone photoreceptors. (F) Statistical results for zpr-1 (n = 5 for each group). Scale bar = 50 μm. Bar plots were shown as the mean ± s.e.m. T-test was performed between the two groups. ^∗P < 0.05, ^∗∗P < 0.01.

Knockdown of slc7a14 led to increased apoptosis in the zebrafish retina. TUNEL assay was used to detect apoptosis in larval retinas at 3 and 5 dpf. (A,B)Slc7a14 knockdown resulted in significant increase of TUNEL + cells in the retina including GCL, INL, ONL and RPE at 3 dpf (A) and 5 dpf (B). Scale bars = 50 μm. (C,D) Statistical results for the numbers of TUNEL positive cell at 3 dpf (n = 6 for each group) and at 5 dpf (n = 3 for each group). Bar plots were shown as the mean ± s.e.m. T-test was performed between the two groups. ^∗P < 0.05, ^∗∗∗P < 0.001.

Slc7a14-deficient zebrafish morphants showed defective visual behaviors. (A,B) VMR testing in slc7a14-deficient zebrafish morphants. Larvae injected with slc7a14-MO (6.0 ng) showed a weaker ON response and a significantly attenuated OFF response compared to control larvae. N = 12 in each group. (C,D) Quantification of the VMR. N = 12 in each group. (C) Larvae injected with slc7a14 MO (6.0 ng) showed a reduced ON response compared with control larvae. (D) Compared with control larvae, larvae injected with 4.0 ng MO and 6.0 ng slc7a14-MOs exhibited markedly reduced OFF responses. (E,F) OKR testing demonstrated significant reductions in eye movement in the slc7a14-deficient zebrafish morphants compared to the control zebrafish. N = 10 in each group. VMR testing and OKR testing were repeated three times, respectively. Data was analyzed using One-way ANOVA followed by Games–Howell test, ^∗P ≤ 0.05, ^∗∗∗P < 0.001 significantly different from control 6.0 ng group.

Slc7a14 mRNA compensation rescued the phenotypes in slc7a14-deficient morphants. (A) Magnified vertical and lateral views of larval eyeballs. Larvae injected with slc7a14 MO and full-length slc7a14 mRNA showed normal-sized eyeballs at 3 dpf. Scale bar = 100 μm. (B,C) Statistical results for the axial length and ocular area. N = 30 in each group. (D,E) VMR testing showed dramatic recovery of both the ON and OFF responses in slc7a14-deficient zebrafish compensated with mRNA compared to slc7a14-deficient zebrafish without compensation. N = 12 in each group. Rescue experiments were repeated three times. Bar plots are shown as the mean ± s.e.m. Data was analyzed using One-way ANOVA followed by Tukey’s post hoc test, ^∗∗∗P < 0.001 significantly different from slc7a14 6.0 ng group.