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Fig. 5 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.