Fig. 2

Tcf/Lef-miniP:dGFP reporter expression reflected Wnt/β-catenin signaling in vivo. (A) Dkk1-GFP or ΔTcf3-GFP expression inhibited Tcf/Lef-miniP:dGFP reporter activation. Homozygous Tcf/Lef-miniP:dGFP transgenic zebrafish Line-2 was crossed with heterozygous hsp70:Dkk1-GFP or hsp70:ΔTcf3-GFP transgenic zebrafish. The collected embryos were exposed to heat shock at 37 °C from 5 to 6, 10 to 11, or 22 to 23 hpf. They were then classified as hsp70:Dkk1-GFP carriers (+/-), hsp70:ΔTcf3-GFP carriers (+/-), or non-carriers (-/-) by determining whether Dkk1-GFP or d2EGFP expression was detected by in situ hybridization at 8, 12, or 25 hpf. To determine d2EGFP mRNA expression, the PEST sequence-coding region of d2EGFP was used as a probe (the schematic diagram shown in the lower panel). The left and middle panels show embryos with the ventral side to the left. The right panels show embryos with the anterior side to the left. Scale bar, 100 μm. (B) Tcf/Lef-miniP:dGFP reporter activity was enhanced by BIO treatment. Left-side views of Line-2 treated with 10 μM BIO from 24 to 30 hpf, with the anterior side to the left. (C) XAV939 or IWR-1 treatment reduced the β-catenin protein level. Zebrafish embryos were treated with DMSO or 10 μM XAV939 or IWR-1 from 5 to 8 hpf. Extracts were harvested from embryos at 8 hpf and immunoblotted with anti-β-catenin and anti-α-tubulin antibodies. (D) XAV939 or IWR-1 treatment reduced Tcf/Lef-miniP:dGFP reporter activity. Panels show lateral views of Line-2 treated with DMSO, 10 μM XAV939, or IWR-1 from 18 to 28 hpf, with the anterior side to the left. d2EGFP-expressing cells were visualized by fluorescence microscopy (lower panels). Bright-field (BF) images are shown in the upper panels. Scale bar, 200 μm.