Fig. 1

Fig. 1

Generation of new Wnt/β-catenin signaling reporters. (A) Schematic diagrams of Wnt/β-catenin signaling-reporter constructs. Tcf/Lef BS: consensus sequence of the Tcf/Lef-binding site. PolyA: SV40 polyadenylation sequence. (B) The Wnt/β-catenin signaling-reporter constructs drive d2EGFP expression in HEK293 cells in response to the activation of Wnt/β-catenin signaling. HEK293 cells were transfected using a reporter gene with a control empty vector (Vector) or an expression plasmid-encoding mouse Wnt-1 (Wnt-1) or β-catenin (β-catenin), as indicated. Scale bar, 200 μm. (C) Comparison of reporter activities in zebrafish embryos transiently introduced with various Wnt/β-catenin signaling-reporter constructs. Left side views of 30-hpf zebrafish embryos injected with either Tcf/Lef-miniP:dGFP, Tcf/Lef(MT)-miniP:dGFP, or Tcf/Lef-tkP:dGFP with Tol2 transposase mRNA, where the anterior is to the left. Cells expressing d2EGFP were visualized by fluorescence microscopy (right panels). Bright-field (BF) images are shown in the left panels. Scale bar, 200 μm. Fluorescence was observed in embryos injected with Tcf/Lef-miniP:dGFP (55%, n=22) and Tcf/Lef-tkP:dGFP (45%, n=20). In contrast, Tcf/Lef(MT)-miniP:dGFP did not express d2EGFP (n=43). dmb: dorsal midbrain, ov: otic vesicle, mff: median fin fold, pfb: pectoral fin bud. (D) Southern blot analysis of the transgenes in Tcf/Lef-miniP:dGFP transgenic zebrafish lines. Genomic DNA was prepared from the tail fins of Line-1, Line-2, and Line-3, and used for Southern blot analysis. (E) Comparison between Line-1, Line-2, and Line-3 reporter activities. Panels show the left side views of 30-hpf Line-1, Line-2, and Line-3 embryos, with the anterior to the left. Cells expressing d2EGFP were visualized by fluorescence microscopy (right panels). Bright-field (BF) images are shown in the left panels. Scale bar, 200 μm.