Paclobutrazol (PBZ) exposure reduces eye size in zebrafish embryos. (A) Representative eye photomicrographs (20× magnification) from 120 hours post-fertilization (hpf) embryos treated with (a) 0.1% DMSO (control) or (b) 0.1 ppm; (c) 1 ppm; (d) 5 ppm; or (e) 10 ppm of PBZ. Scale bar: 10 μm; (B) Eye areas from 15 embryos treated with 0.1% DMSO or with 0.1, 1, 5, or 10 ppm of PBZ were measured using ImageJ software, and all values were normalized to the mean of the control group. Bars sharing a letter are not significantly different from one another at p < 0.05, as assessed by one-way ANOVA, followed by Fisher’s least significant difference test. Error bars indicate standard error.

Paclobutrazol exposure significantly reduces the thickness of the photoreceptor layer in zebrafish embryos. (A) Hematoxylin and eosin (H&E) staining of eye sections from zebrafish treated with (a) 0.1% DMSO (control) or with (b) 0.1 ppm; (c) 1 ppm; or (d) 5 ppm. High-magnification images for the photoreceptor layer of the eyes are shown in a’–d’. Reference lines indicate the photoreceptor layer. gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; onl, outer nuclear layer; opl, outer plexiform layer; pcl, photoreceptor cell layer; (B) Thicknesses of the photoreceptor cell layer were measured from control embryos and embryos treated with PBZ at 0.1, 1, or 5 ppm, with 20 embryos used per condition. Each group contains at least 10 embryos; Scale bar: 10 μm. All values were normalized to the mean of the normal group. Bars sharing a letter are not significantly different from one another as assessed by one-way ANOVA, followed by Fisher’s least significant difference test (p < 0.05). Error bars indicate standard error.

Paclobutrazol interferes the development of embryonic photoreceptor cells in zebrafish. Eyes of 72 hpf embryos treated with (A,E) 0.1% DMSO (control) or with (B,F) 0.1 ppm, (C,G) 1 ppm, or (D,H) 5 ppm of PBZ and labeled with riboprobes for gnat1 (rod cell marker) (A–D) or gnat2 (cone cell marker) (E–H) by in situ hybridization. Each group contains at least 20 embryos; Scale bar: 50 μm.

Expression of aldehyde dehydrogenases (aldh1a2 and aldh1a3), encoding key enzymes for retinoic acid (RA) synthesis, is decreased in PBZ-treated embryos. Whole-mount in situ hybridization was used to examine the expression of aldh1a2 (A–D) and aldh1a3 (E–H) in the eyes of 48 hpf zebrafish embryos treated with (A,E) 0.1% DMSO (control) or with (B,F) 0.1 ppm, (C,G) 1 ppm, or (D,H) 5 ppm of PBZ. (I,J) Quantitative PCR analysis of the aldh1a2 (I) and aldh1a3 (J) mRNA levels in embryos treated with 0.1% DMSO or 0.1, 1, or 5 ppm PBZ at 48 hpf. Each group contains at least 20 embryos; Scale bar: 50 μm. Error bars represent standard deviation. Data were analyzed using Student’s t-test; ** p < 0.01 and *** p < 0.001.

Schematic diagram showing the timeline of PBZ exposure, RA treatment, and collection of embryos for analysis. Embryos were independently exposed to PBZ (0, 1, or 5 ppm) with or without RA (1 or 5 nM) from 2 hpf until 72 hpf. At 72 hpf, embryos were collected for analysis of retinal photoreceptor cells via gnat1 and gnat2 in situ hybridization.

Retinoic acid increases embryos’ tolerance to the toxic effects of PBZ on retinal photoreceptor development. Fertilized embryos were incubated with (A,J) 0.1% DMSO (control); (B,K) 1 ppm PBZ; (C,L) 5 ppm PBZ; (D,M) 1 nM RA; (E,N) 1 ppm PBZ + 1 nM RA; (F,O) 5 ppm PBZ + 1 nM RA; (G,P) 5 nM RA; (H,Q) 1 ppm PBZ + 5 nM RA; or (I,R) 5 ppm PBZ + 5 nM RA from 2 to 72 hpf. The development of retinal photoreceptor cells was analyzed by in situ hybridization with digoxigenin-labeled gnat1 and gnat2 cRNA probes at 72 hpf. Each group contains at least 20 embryos; Scale bar: 50 μm.

Schematic diagram showing the timeline of PBZ exposure, the addition of RA, and the collection of embryos for analysis. Embryos were exposed separately to different PBZ concentrations (0, 1, or 5 ppm) from 2 to 72 hpf. After the removal of PBZ, 1 or 5 nM RA was added to the embryos’ water for an additional 48 h. At 120 hpf, embryos were collected and fixed for further examination and evaluation of retinal photoreceptor development.

Paclobutrazol-damaged retinal photoreceptor cells are restored by treatment with RA. Fertilized embryos were incubated with (A,J) 0.1% DMSO (control); (B,K) 1 ppm PBZ; (C,L) 5 ppm PBZ; (D,M) 1 nM RA; (E,N) 1 ppm PBZ + 1 nM RA; (F,O) 5 ppm PBZ + 1 nM RA; (G,P) 5 nM RA; (H,Q) 1 ppm PBZ + 5 nM RA; or (I,R) 5 ppm PBZ + 5 nM RA. The development of retinal photoreceptor cells was analyzed by in situ hybridization with digoxigenin-labeled gnat1 (A–I) and gnat2 (J–R) cRNA probes at 120 hpf. Note that RA was added to the embryo for 48 h, beginning at 72 hpf. Scale bar: 50 μm.

Paclobutrazol treatment does not induce cell death in the eye. Fertilized embryos were incubated with (A) 0.1% DMSO (control); (B) 1 ppm PBZ; (C) 5 ppm PBZ until 60 hpf (A–C) and 108 hpf (D–E), and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed for apoptotic cell analysis. Arrowheads indicate dead cells. Scale bar: 50 μm.

Retinoic acid treatment rescues reduced mitotic cells and eye size of PBZ-treated embryos. Embryos exposed to PBZ concentrations (0, 1, or 5 ppm) with or without 1 nM or 5 nM RA was performed at 2 hpf in embryos’ water. (A) Immunostaining was performed to analyze the retinal cell proliferation using an anti-phospho-histone H3 (PH3) antibody at the embryonic stage of 60 (a–i) and 108 hpf (j–r). Scale bar: 50 μm. The proliferating cell numbers were counted and recorded at (B) 60 and (C) 108 hpf. * p < 0.05, ** p < 0.01, and *** p < 0.001. (D) Eye areas from 15 embryos were measured using ImageJ software, and all values were normalized to the mean of the control group. Bars sharing a letter are not significantly different from one another at p < 0.05, as assessed by one-way ANOVA, followed by Fisher’s least significant difference test. Error bars indicate standard error.