Abstract
1-phenyl 2-thiourea (PTU) is a tyrosinase inhibitor commonly used to block pigmentation and aid visualization of zebrafish development. At the standard concentration of 0.003% (200 µM), PTU inhibits melanogenesis and reportedly has minimal other effects on zebrafish embryogenesis. We found that 0.003% PTU altered retinoic acid and insulin-like growth factor (IGF) regulation of neural crest and mesodermal components of craniofacial development. Reduction of retinoic acid synthesis by the pan-aldehyde dehydrogenase inhibitor diethylbenzaldehyde, only when combined with 0.003% PTU, resulted in extraocular muscle disorganization. PTU also decreased retinoic acid-induced teratogenic effects on pharyngeal arch and jaw cartilage despite morphologically normal appearing PTU-treated controls. Furthermore, 0.003% PTU in combination with inhibition of IGF signaling through either morpholino knockdown or pharmacologic inhibition of tyrosine kinase receptor phosphorylation, disrupted jaw development and extraocular muscle organization. PTU in and of itself inhibited neural crest development at higher concentrations (0.03%) and had the greatest inhibitory effect when added prior to 22 hours post fertilization (hpf). Addition of 0.003% PTU between 4 and 20 hpf decreased thyroxine (T4) in thyroid follicles in the nasopharynx of 96 hpf embryos. Treatment with exogenous triiodothyronine (T3) and T4 improved, but did not completely rescue, PTU-induced neural crest defects. Thus, PTU should be used with caution when studying zebrafish embryogenesis as it alters the threshold of different signaling pathways important during craniofacial development. The effects of PTU on neural crest development are partially caused by thyroid hormone signaling.
Highlights
Zebrafish (Danio rerio) are small fresh-water tropical fish that have gained popularity as a vertebrate model for biological research over the past 20 years [1]
In the presence of 0.003% phenyl 2-thiourea (PTU) which was added to the media at 12 hpf, treatment with 10 mM DEAB at 24 hpf caused thickening and overlapping of extraocular muscles as well as poor formation and occasional absence of the oblique muscles (Figure 1C,D) Importantly addition of only 0.003% PTU at 12 or 22 hpf did not alter craniofacial development (Figure 1K,L compared to Figure 1I,J; Figure S1D compared to Figure S1C; Figure S2C,D compared to Figure 1I,J) at 72 hpf
PTU blocks the synthesis of melanin from tyrosine as well as the intermediates required for catecholamine synthesis
Summary
Zebrafish (Danio rerio) are small fresh-water tropical fish that have gained popularity as a vertebrate model for biological research over the past 20 years [1]. Zebrafish are amenable for studying organismal development because of their small size, extracorporeal development, and accessibility for forward and reverse genetics. In vivo imaging of live zebrafish embryos, combined with transgenic approaches, can yield real-time viewing of development [2]. In situ hybridization and immunostaining) employed at specific time points, zebrafish models have yielded significant insights into embryogenesis, organogenesis, tissue regeneration and pathogenesis. Zebrafish embryos should remain transparent to achieve optical clarity during in vivo imaging and easy detection of whole mount color reagents. Embryos are treated with the tyrosinase inhibitor 1-phenyl 2-thiourea (PTU) to block endogenous melanogenesis [3,5]. Addition of PTU prior to 24 hpf effectively but reversibly inhibits pigmentation until approximately 120 hpf
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