Phenylthiourea Specifically Reduces Zebrafish Eye Size

Phenylthiourea (PTU) is commonly used for inhibiting melanization of zebrafish embryos. In this study, the standard treatment with 0.2 mM PTU was demonstrated to specifically reduce eye size in larval fish starting at three days post-fertilization. This effect is likely the result of a reduction in retinal and lens size of PTU-treated eyes and is not related to melanization inhibition. This is because the eye size of tyr, a genetic mutant of tyrosinase whose activity is inhibited in PTU treatment, was not reduced. As PTU contains a thiocarbamide group which is presented in many goitrogens, suppressing thyroid hormone production is a possible mechanism by which PTU treatment may reduce eye size. Despite the fact that thyroxine level was found to be reduced in PTU-treated larvae, thyroid hormone supplements did not rescue the eye size reduction. Instead, treating embryos with six goitrogens, including inhibitors of thyroid peroxidase (TPO) and sodium-iodide symporter (NIS), suggested an alternative possibility. Specifically, three TPO inhibitors, including those that do not possess thiocarbamide, specifically reduced eye size; whereas none of the NIS inhibitors could elicit this effect. These observations indicate that TPO inhibition rather than a general suppression of thyroid hormone synthesis is likely the underlying cause of PTU-induced eye size reduction. Furthermore, the tissue-specific effect of PTU treatment might be mediated by an eye-specific TPO expression. Compared with treatment with other tyrosinase inhibitors or bleaching to remove melanization, PTU treatment remains the most effective approach. Thus, one should use caution when interpreting results that are obtained from PTU-treated embryos

PTU treatment specifically reduces eye size starting at 3 dpf.

<p>(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 <i>p</i>-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 <i>p-</i>value of each test (green: PTU < control, <i>p-</i>value <0.001; dark green: PTU < control, <i>p</i>-value >0.001 & <0.05; black: PTU  =  control, <i>p-</i>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.</p

Properties of the solutions used to treat developing zebrafish larvae.

<p>The osmolarity and pH of the chemicals in E3 were determined. For osmolarity, the measurements are combined from at least two different experiments because there was no difference between individual experiments (ANOVA, <i>F</i>(1,56)  = 0.24, <i>p-</i>value  = 0.62). See text for details. While for pH, the measurements of solutions prepared with two different sources of water were different (ANOVA, <i>F</i>(1,46)  = 5.35, <i>p</i>-value  = 0.025); thus each preparation is presented separately.</p