FIGURE SUMMARY
Title

Phenylthiourea specifically reduces zebrafish eye size

Authors
Li, Z., Ptak, D., Zhang, L., Walls, E.K., Zhong, W., and Leung, Y.F.
Source
Full text @ PLoS One

PTU treatment specifically reduces eye size starting at 3 dpf.

(A & A′) After PTU treatment (PTU), the production of black pigment or melanization in a larva was suppressed (A′). In addition, the eye size of the PTU-treated larvae also appeared smaller compared with the untreated siblings (control) (A) at 3 dpf. Scale bar = 100 μm. (B & C) To quantify potential differences in eye size, the anterior-posterior length or area of eye and body were measured in multiple experiments conducted on different days. (B) A heatmap that shows the results from 22 independent experiments at 3 dpf. At least 10 embryos were used in each treatment group per experiment. Each column represents one experiment and the individual box represents the Holm-adjusted p-value of a Wilcoxon rank sum test for the comparison of eye or body measurements, or their ratios (eye/body size ratios) between PTU-treated and control larvae. The color of each box represents the difference in the means of the parameter between the two groups and the intensity represents the corresponding p-value of each test (green: PTU < control, p-value <0.001; dark green: PTU < control, p-value >0.001 & <0.05; black: PTU = control, p-value >0.05). The experiments analyzed by area measurements are highlighted by asterisks; otherwise, they were analyzed by length measurements. The PTU treatment specifically reduced the measurements of the eye size compared with the body size. See text for further supporting statistical analyses. (C) A plot of the means of eye/body length ratios of 15 control and PTU-treated larvae at 2–5 dpf obtained from one of the multi-days experiments. The error bars show the 95% confidence intervals.

Histological analysis of zebrafish eyes before and after PTU treatment.

Histological analysis was conducted on 1-μm-thick plastic sections of PTU-treated and control larvae at 4 dpf. The number of different recognizable cell types and categories was counted and the area of the retinas measured. Five sections were analyzed per condition. There are no differences in the counts for all cell types and categories, but the dorsal retinal and lens areas are smaller while the whole retinal area and cell density are marginally higher in the PTU-treated eyes. MZ: marginal zone, HC: horizontal cells, GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. The RPE is indicated by red arrows. Scale bar = 50 μm.

Immunohistochemical analysis of zebrafish eyes before and after PTU treatment.

Immunostaining was conducted on 10-μm-thick cryosections of PTU-treated and control larvae at 4 dpf with the following first antibodies: (A & A′) anti-zpr1 for cones (red arrowhead), (B & B′) anti-zpr2 for RPE (red arrowhead), (C & C′) anti-zpr3 for rods (red arrowhead), (D & D′) anti-Islet1 for GCs, ACs, BCs and HCs, (E & E′) anti-acetylated α-tubulin for the axons of differentiated neurons, (G & G′) anti-PH3 for mitotic cells (red arrowheads) and (H & H′) anti-active caspase3 for apoptotic cells. (F & F′) MCs were visualized by cryosectioning of a transgenic fish Tg(gfap:GFP)mi2001 treated with PTU in the same manner. The cell bodies were visualized by green fluorescence (red arrowhead). All positive signal areas or cell counts were extracted and normalized by retinal area, which was traced in the channel with DAPI nuclei stain. There are no differences in staining of any markers between the PTU-treated and control groups except for zpr2, in which the positive signal/ retinal area was larger in the PTU-treated group. All images are transverse sections; the lens is on the left (the location is indicated by a dotted yellow circle in (B)) and dorsal is up. The approximate retinal cell layers and cell types are indicated in (D) and (F). Scale bar = 50 μm.

PTU suppresses thyroid hormone production but its specific inhibition on eye growth is not related to a general suppression of thyroid hormone synthesis.

(A) Whole-mount immunostaining against T4 was performed on PTU-treated and control larvae at 4 dpf. In controls, the T4+ thyroid follicles were stained as dark brown spots along the ventral pharyngeal midline. This staining was substantially diminished or absent in the PTU-treated larvae and in the control without the first antibody. Scale bar = 50 μm. (B) A boxplot of eye/body size ratios from an experiment of thyroid hormones supplements along with PTU treatment. Specifically, 10 larvae collected from the same parents were treated with nothing (control), PTU, PTU with 0.1 μM TriAc, 10 nM T3 or 30 nM T4. While the T3 supplement seemed to increase the eye/body size ratio of the PTU-treated larvae, statistical analysis on this and six additional independent experiments indicate that this was not a genuine increase. See text for details.

The suppression of thyroid hormone synthesis by six goitrogens reveals that PTU suppression on eye growth is related to inhibition of TPO.

(A) The six goitrogens used include two TPO inhibitors with thiocarbamide group: 0.025% PTuracil and 4 mM MM; two TPO inhibitors without thiocarbamide group: 1mM DHP and 0.5mM RES; and two NIS inhibitors: 0.2% KClO4 and 10mM KSCN. All these chemicals were used to treat zebrafish embryos in the same way as PTU. Whole-mount immunostaining against T4 was performed at 4 dpf as in Figure 4. In controls, T4+ thyroid follicles were stained as dark brown spots along the ventral pharyngeal midline. This staining was substantially diminished or absent in the inhibitors-treated groups and in the control without the first antibody. Scale bar = 50 μm. (B) A heatmap that shows the results from multiple experiments in which the larvae were independently treated with these six goitrogens. These include two TPO inhibitors with thiocarbamide group: 0.025% PTuracil (N of independent experiments = 4) and 4 mM MM (N = 5); two TPO inhibitors without thiocarbamide group: 1mM DHP (N = 4), 0.5mM RES (N = 5); and two NIS inhibitors: 0.2% KClO4 (N = 4) and 10mM KSCN (N = 4). All these chemicals were used to treat zebrafish embryos the same way as PTU and at least 10 larvae were used in each individual treatment in each replication. The eye and body lengths were measured and the corresponding eye/body size ratios calculated. The eye and body measurements and their ratios between inhibitors-treated and control larvae were compared by Wilcoxon rank sum test, and the results were presented in a heatmap as in >Figure 1. The color of each box represents the difference in the means of the parameter and the intensity represents the corresponding Holm-adjusted p-value of each test (green: inhibitor < control, p-value <0.001; dark green: inhibitor < control, p-value >0.001 & <0.05, black: inhibitor = control, p-value >0.05; dark red: inhibitor > control, p-value >0.001 & <0.05; red: inhibitor > control, p-value <0.001). The experiments analyzed at 4 dpf are highlighted by asterisks; otherwise they were analyzed at 3 dpf. A linear mixed-effects model was fitted to analyze the effect of individual inhibitors on the measured parameters at 3 dpf. In general, three (MM, DHP & RES) out of four TPO inhibitors specifically reduced eye/body size ratio while none of the NIS inhibitors did. These observations suggest that a general suppression of thyroid hormone synthesis is not the cause of the specific eye size reduction, but a specific inhibition of TPO or TPO-like activity can reduce eye size.

Tpo is specifically expressed in developing mouse retinas.

In situ hybridization was conducted with a riboprobe specific for Tpo on 20-μm-thick cryosections obtained from mouse embryos at E13.5 (A; transverse section) and P0 (B; coronal section). (A) At E13.5, there was a general expression of Tpo in the whole embryo. (B) By P0, there was a specific expression of Tpo in the retina (black arrowhead) compared with lens (red arrowhead) and the surrounding periocular tissue (asterisks). Scale bar = 200μm for both (A) and (B).

Morphology of zebrafish after treatment with various tyrosinase inhibitors.

Different tyrosinase inhibitors, when applied at the same stage as PTU singly or in combination, suppress pigmentation to a different extent at 3 dpf. The inhibitors and their combinations include: (A) untreated control, (B) 1X PTU, (C) 0.3X PTU, (D) 10 μM MBT, (E) 0.25 mM MQ, (F) 0.7 mM 4HBA, (G) 0.8 mM HPU, (H) 0.3X PTU +0.7mM 4HBA, (I) 0.3X PTU +0.8 mM HPU, (J) 0.8 mM HPU +0.7 mM 4HBA and (K) 0.3X PTU +0.8 mM HPU +0.7 mM 4HBA. Scale bar = 100 μm.

Performance of immunostaining and in situ hybridization after bleaching.

(A) Immunostaining experiments were conducted with untreated, PTU-treated or bleached embryos at 52 hpf. Two first antibodies, anti-PH3 and zpr2 were used. The typical positive signal is indicated by a red arrowhead in each case. Scale bar = 50 μm. (B) An in situ hybridization experiment with tubb5 using PTU-treated or bleached embryos. These samples were obtained from a double in situ hybridization experiment with another gene stained in red; thus the samples, especially the whole-mount embryos, look reddish in general. Scale bar = 50 & 100 μm for sectioned and whole-mount samples respectively. All images were acquired with the same parameters and were not altered during the figure composition to ensure comparability across conditions.

Acknowledgments
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