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Fig. S1
Wnt/β-catenin signalling reporter properties. Related to Fig. 1. a
Schematic diagrams of two Wnt/β-catenin signalling-reporter constructs: OTM:d2EGFP and OTM:ELuc-CP. Tcf/Lef BS: consensus sequence of the Tcf/Lef-binding site; EGFP: enhanced green fluorescent protein; ELuc: Emerald Luciferase; PEST: degradation sequence derived from mouse ornithine decarboxylase; CL1: degradation sequence derived from yeast; Poly A: SV40 polyadenylation sequence. b OTM:ELuc-CP drives ELuc-CP expression in mouse neuro-2a cells in response to the activation of Wnt/β-catenin signalling. Neuro-2a cells were transfected using a reporter construct with a titrated amount of an expression plasmid-encoding Lef1 CA. Then, luciferase activity was measured. c The half-life of ELuc-CP is approximately 15 min. Neuro-2a cells were transfected using a reporter construct. After 36 hours, cells were treated with 100 ng/ml cycloheximide (CHX) for the indicated times. Then, luciferase activity was measured. The error bars indicate the standard deviations. d The OTM:ELuc-CP-transgenic zebrafish line carries a single copy of the OTM:ELuc-CP reporter. Southern blot analysis of the transgenes in the OTM:ELuc-CP transgenic zebrafish line. Genomic DNA was prepared from the tail fins of this line and used for Southern blot analysis. e OTM:ELuc-CP and OTM:d2EGFP are activated in same areas of zebrafish embryos. Left panel shows d2EGFP fluorescence (green) merged with bright-field of OTM:d2EGFP-transgenic zebrafish at 30 hpf (left view with the anterior side to the left). Right panel shows whole mount in situ hybridization of eluc-cp (blue) in OTM:ELuc- CP-transgenic zebrafish at 30 hpf (left view with the anterior side to the left). Scale bar, 200 μm. f, g Expression gradient of a Wnt/β-catenin-target gene lef1 is noisy during zebrafish AP axis formation and lef1 expression-noise correlates with OTM:d2EGFP reporter-noise. Confocal images showing wholemount fluorescence in situ hybridization with lef1 and gfp mRNA of wild-type (f) or OTM:d2EGFP (g) embryos. f Panels show fluorescence intensity and the magnified views of the noise areas. Scale bar, 20 μm. Left bottom graph shows lef1 mRNA levels in the area indicated in left top panel dot line. g Naturally generated unfit Wnt/β-catenin cells (unfit cells with abnormally high or low OTM:d2EGFP activity) are enclosed with yellow dotted line circles. h, i Gradient of endogenous nuclear β-catenin protein levels is noisy during zebrafish AP axis formation and nuclear β-catenin-noise correlates with OTM:d2EGFP reporter-noise. Confocal images showing whole-mount immunostaining of β-catenin in wild-type (h) or OTM:d2EGFP (i) embryos. h Panels show fluorescence intensity and the magnified views of the noise areas. Scale bar, 20 μm. Graph shows β-catenin protein levels in the area indicated in left top panel dotted line. i Naturally generated unfit Wnt/β-catenin cells (unfit cells with abnormally high or low OTM:d2EGFP activity) are indicated with arrow heads.