Expression pattern of lhx4 in zebrafish retinal development. All figures are horizontal sections along the temporal-nasal axis (T-N). (A) The expression of Lhx4 was not detected in the retina at 24 hpf. (B) The expression of Lhx4 in the dorsal-nasal of the retina was weak at 48 hpf. (C) Lhx4 was expressed in the ONL and INL at 72 hpf. (D–F) The expression of Lhx4 in the ONL and INL was gradually weakened, and the signal in the CMZ was relatively strong from 96 hpf to 6 dpf. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; OPL, outer plexiform layer; IPL, inner plexiform layer. Scale bar = 20 μm.

Effects of lhx4 knockdown via vivo-MO in the eyes on the ACs differentiation. All figures are horizontal sections along the temporal-nasal axis (T-N). (A–C’’) Immunofluorescence staining with different ACs markers, GABA, Parvalbumin, and Calretinin in WT, vCT, and 4vMO retinas at 84 hpf. Blue, DAPI staining of the nuclei. Scale bar = 20 μm. (a–c) Statistical analysis of GABA⁺, Parvalbumin⁺, and Calretinin⁺ cells in WT, vCT, and 4vMO retinas at 84 hpf. ns, P > 0.05; ****, P < 0.0001; vCT vs. WT; 4vMO vs. vCT. Results are presented as the mean ± SEM (n ≥ 10).

Effects of lhx4 knockdown via vivo-MO in the eyes on the other retinal neuronal differentiation. All figures are horizontal sections along the temporal-nasal axis (T-N). (A–D”) Immunofluorescence staining with other retinal neural markers, PKCα (at 84 hpf), Zpr1(at 84 hpf), Rhodopsin (at 84 hpf), and Zn8 (at 72 hpf) in WT, vCT, and 4vMO retinas. Blue, DAPI staining of the nuclei. Scale bar = 20 μm. (a–d) Statistical analysis of PKCα⁺, Zpr1⁺, Rhodopsin⁺, and Zn8⁺ cells in WT, vCT, and 4vMO retinas. ns, P > 0.05; ****, P < 0.0001; vCT vs. WT; 4vMO vs. vCT. Results are presented as the mean ± SEM (n ≥ 10).

Effects of lhx4 knockdown via vivo-MO in the eyes on the expression of vsx1, vsx2, and bhlhe23 at 60 hpf. ns, P > 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; vCT vs. WT; 4vMO vs. vCT. Results are presented as mean ± SEM (n = 3 biological replicates).

Effects of lhx4 knockdown via vivo-MO in the eyes on the apoptosis in the retina. All figures are horizontal sections along the temporal-nasal axis (T-N). (A?F) Immunofluorescence staining with Caspase3 at 60 hpf and 72 hpf. Blue, DAPI staining of the nuclei. Scale bar?=?20 ?m. (G,H) Statistical analysis of the number of Caspase3+ cells in WT, vCT, and 4vMO retinas at 60 hpf and 72 hpf. ns, P?>?0.05; vCT vs. WT; 4vMO vs. vCT. Results are presented as the mean?±?SEM (n???10).

The response of zebrafish larvae to the light stimulus at 5 dpf. (A) The average swimming speed of the larvae during the last 2 min dark and 2 min light period. (B) The swimming speed during the last 2 min dark and the first 10 s of the 2 min light period. ns, P > 0.05; **, P < 0.01; ***, P < 0.001; vCT vs. WT; 4vMO vs. vCT. Results are presented as the mean ± SEM (n ≥ 40).