1-phenyl 2-thiourea (PTU) activates autophagy in zebrafish embryos

Abstract

1-phenyl 2-thiourea (PTU) is a Tyr (tyrosinase) inhibitor that is extensively used to block pigmentation and improve optical transparency in zebrafish (Danio rerio) embryo. Here, we reported a previously undescribed effect of PTU on macroautophagy/autophagy in zebrafish embryos. Upon 0.003% PTU treatment, aberrant autophagosome and autolysosome formation, accumulation of lysosomes, and elevated autophagic flux were observed in various tissues and organs of zebrafish embryos, such as skin, brain, and muscle. Similar to PTU treatment, autophagic activation and lysosomal accumulation were also observed in the somatic tyr mutant zebrafish embryos, which suggest that Tyr inhibition may contribute to PTU-induced autophagic activation. Furthermore, we demonstrated that autophagy contributes to pigmentation inhibition, but is not essential to the PTU-induced pigmentation inhibition. With the involvement of autophagy in a wide range of physiological and pathological processes and the routine use of PTU in zebrafish research of autophagy-related processes, these observations raise a novel concern in autophagy-related studies using PTU-treated zebrafish embryos.Abbreviations: 3-MA: 3-methyladenine; Atg: autophagy-related; BSA: bovine serum albumin; CHT: caudal hematopoietic tissue; CQ: chloroquine; GFP: green fluorescent protein; hpf: hour-post-fertilization; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NGS: normal goat serum; PtdIns3K: class III phosphatidylinositol 3-kinase; PTU: 1-phenyl 2-thiourea; RFP: red fluorescent protein; Sqstm1: sequestosome 1; tyr: tyrosinase.

Keywords: 1-phenyl 2-thiourea; autophagy; melanogenesis; tyrosinase; zebrafish embryo.

Figure 1.

1-phenyl-2-thiourea (PTU) induces aberrant autophagosome and autolysosome formation in zebrafish embryos. (A) Representative bright-field images showing the pigmentation of 2 days-post-fertilization (dpf) zebrafish embryo treated with E3 (Control, CTRL) and 0.003% (200 μM or 1X) PTU in E3 from around 1 dpf. Scale bar: 0.5 mm; (B) Western blot results showing the dose-dependent accumulation of Lc3-I, Lc3-II, and the degradation of Sqstm1/p62 proteins in various doses of PTU-treated zebrafish embryos compared with CTRL. Mean relative ratio of Lc3-I:Tuba (tubulin, alpha), Lc3-II:Tuba, and Sqstm1:Tuba were presented under the bands. 50 embryos were collected per group for three independent experiments. One-way ANOVA was applied and significant increase (p < 0.05) in Lc3-I:Tuba and Lc3-II: Tuba was detected under PTU treatments compared with control in a dose-dependent manner, while significant decrease (p < 0.05) was found in Sqstm1:Tuba in 2X and 4X-treatment compared with CTRL. (C) Schematic diagram showing the position (periderm or basal epidermal cells above the eye) of imaging. Representative images from nine Tg(GFP-Lc3) zebrafish embryos treated with PTU and stained with LysoTracker from three independent experiments were shown. Three independent areas were selected from individual animals, and the relative number of GFP-Lc3⁺, LysoTracker⁺, and Merged (GFP-Lc3⁺ and LysoTracker⁺) puncta per cell was quantified. Red arrowhead, GFP-Lc3⁺ and LysoTracker⁺ puncta. **, p < 0.01 compared with CTRL. Scale bar: 10 μm (Merged), 5 μm (Enlarged). (D) Schematic diagram showing the position (cells in the midbrain) of imaging. The relative number of GFP-Lc3⁺, LysoTracker⁺, and Merged (GFP-Lc3⁺ and LysoTracker⁺) puncta per cell in neurons of midbrain was counted based on Z-Stack (10 layers out of 100 layers) image. Representative images of nine Tg(GFP-Lc3) zebrafish embryos treated with PTU and/or 3-MA and stained with LysoTracker from three independent experiments were shown. Red arrowhead, GFP-Lc3⁺ and/or LysoTracker⁺ puncta. *, p < 0.05, **, p < 0.01 compared with CTRL; ^##, p < 0.01 compared with PTU. Scale bar: 40 μm (Merged), 6 μm (Enlarged)

Figure 2.

PTU elevates autophagic flux in zebrafish embryos. (A) Schematic diagram showing the position (dorsal view of skin on the head) of imaging. Autophagic flux was detected by using GFP-Lc3-RFP-Lc3ΔG probe. The ratio of GFP-Lc3:RFP-Lc3ΔG was calculated based on the mean fluorescent intensity in the selected area. Representative images of nine zebrafish embryos from three independent experiments were shown. Red arrowhead, GFP-Lc3⁺ and RFP-Lc3ΔG⁺ puncta. **, p < 0.01 compared with control (CTRL). Scale bar: 100 μm. (B) Schematic diagram showing the position (midbrain) of imaging. GFP-Lc3 and RFP-Lc3ΔG co-localized puncta were detected in the midbrain of 24 hpf embryo treated with PTU from 6 hpf. Red arrowhead, GFP-Lc3⁺ and/or RFP-Lc3ΔG⁺ puncta. Scale bar: 100 μm. Representative images of nine zebrafish embryos from three independent experiments were shown. (C) Western blot results showing the level of Lc3-I and Lc3-II proteins in CTRL and PTU groups treated with chloroquine (CQ). Mean relative ratio of Lc3-II:Tuba was presented under the bands. 50 embryos were collected per group for three independent experiments. Two-way ANOVA with Tukey post hoc was applied and significant increase (p < 0.05) in Lc3-II:Tuba was detected between PTU and PTU+CQ but not CTRL and CTRL+CQ. (D) Schematic diagram showing the position (cells in the midbrain) of imaging. The relative number of GFP-Lc3⁺ and LysoTracker⁻, GFP-Lc3⁻ and LysoTracker⁺, and GFP-Lc3⁺ and LysoTracker⁺ puncta per cell in neurons of midbrain was counted based on Z-Stack (10 layers out of 100 layers) images. Representative images of nine Tg(GFP-Lc3) zebrafish embryos stained with LysoTracker treated with PTU and/or CQ from three independent experiments were shown. Red arrowhead, GFP-Lc3⁺ and/or LysoTracker⁺ puncta. **, p < 0.01 compared with CTRL; ^##, p < 0.01 compared with PTU. Scale bar: 40 μm and 3 μm (Enlarged)

Figure 3.

Aberrant autophagosome and autolysosome formation in tyr^Mut zebrafish embryos. (A) Representative bright-field images showing the pigmentation of 2 dpf zebrafish embryo injected with tyr gRNA+Cas9 protein (tyr^Mut) and their sibling control (CTRL). Scale bar: 0.5 mm. (B) Western blot results showing the level of Lc3-I and Lc3-II proteins in CTRL and tyr^Mut groups treated with chloroquine (CQ). Mean relative ratio of Lc3-II:Tuba was presented under the bands. 50 embryos were collected per group for three independent experiments. Two-way ANOVA with Tukey post hoc was applied, and a significant increase (p < 0.05) of Lc3-I and Lc3-II were detected in tyr^Mut compared with CTRL while no significant differences (p > 0.05) in Lc3-II:Tuba were detected between tyr^Mut treated with CQ and tyr^Mut. (C) Schematic diagram showing the position (periderm or basal epidermal cells above the eye) of imaging. Representative images of nine tyr^Mut Tg(GFP-Lc3) zebrafish embryos stained with LysoTracker from three independent experiments were shown. Three independent areas were selected from individual animals, and the relative number of GFP-Lc3⁺, LysoTracker⁺ and Merged (GFP-Lc3⁺ and LysoTracker⁺) puncta per cell were quantified. Red arrowhead, GFP-Lc3⁺ and LysoTracker⁺ puncta. *, p < 0.05 compared with CTRL. Scale bar: 10 μm (Merged), 5 μm (Enlarged). (D) Schematic diagram showing the position (cells in the midbrain) of imaging. The relative number of GFP-Lc3⁺ and LysoTracker⁻, GFP-Lc3⁻ and LysoTracker⁺, and GFP-Lc3⁺ and LysoTracker⁺ puncta per cell in neurons of midbrain were counted based on Z-Stack (10 layers out of 100 layers) images. Representative images of nine tyr^Mut Tg(GFP-Lc3) zebrafish embryos treated CQ and stained with LysoTracker from three independent experiments were shown. Red arrowhead, GFP-Lc3⁺ and/or LysoTracker⁺ puncta. **, p < 0.01 compared with CTRL; ^##, p < 0.01 compared with tyr^Mut. Scale bar: 40 μm (Merged), 3 μm (Enlarged)

Figure 4.

L-tyrosine, but not autophagic modulators, restores melanogenesis in PTU-treated zebrafish embryos. (A) Representative bright-field images showing the pigmentation of 2 dpf zebrafish embryo treated with autophagic modulators, including E3, dimethyl sulfoxide (DMSO), chloroquine (CQ), 3-methyladenine (3-MA), rapamycin and L-tyrosine with or without PTU. Red arrowhead, melanin shown in Enlarged. Scale bar: 0.5 mm. (B) Schematic diagram showing the position (cells in the midbrain) of imaging. The relative number of GFP-Lc3⁺, LysoTracker⁺, and Merged (GFP-Lc3⁺ and LysoTracker⁺) puncta per cell were counted based on Z-Stack (10 layers out of 100 layers) images. Representative images of nine Tg(GFP-Lc3) zebrafish treated with PTU, L-tyrosine, or PTU+L-tyrosine and stained with LysoTracker prior to imaging from three independent experiments were shown. Red arrowhead, GFP-Lc3⁺ and/or LysoTracker⁺ puncta. **, p < 0.01 compared with CTRL; ^##, p < 0.01 compared with L-tyrosine. Scale bar: 40 μm (Merged), 6 μm (Enlarged)