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Fig. 5 hwa can function through β-catenin in a Wnt ligand/receptor-independent manner in zebrafish embryos. (A) Immunostaining of β-catenin. After injection of 50 pg of hwa mRNA at one-cell stage, embryos were immunostained for β-catenin at 512-cell stage and observed by confocal microscopy. Whole embryos are viewed from the animal pole; two opposite marginal areas are viewed at a higher magnification. The ratio of embryos with the representative pattern is indicated. (B) Recovery of the body axis in Mhwatsu01sm embryos by ca-β-catenin or tcf3 overexpression. Embryos were injected at one-cell stage and observed at 24 hpf. The embryos are categorized into five classes (top panel); ratios are shown below. (C) hwa overexpression induces the expression of the β-catenin target genes boz and chd. Wild-type or Mhwatsu01sm embryos were injected with 50 pg of hwa-HA mRNA at the one-cell stage and collected at 4 hpf for WISH or qRT-PCR analysis. Left: WISH results (animal-pole views). Right: qRT-PCR results showing averages from three independent experiments (±SEM). **P < 0.01, ***P < 0.001. (D) hwa overexpression fails to induce boz and chd in β-cat2 morphants. Wild-type or Mhwatsu01sm embryos were injected with 50 pg of hwa-HA mRNA together with 20 ng of control MO (cMO) or β-cat2-MO at the one-cell stage and subjected to WISH at 4 hpf. Embryos are laterally viewed. (E and F) Inhibition of Wnt ligand/receptor signaling has no effect on hwa’s organizer/axis-inducing activity. (E) One blastomere of Tg(gsc:EGFP);Mhwatsu01sm embryos at about 32-cell stage was injected with 50 pg of hwa plus 200 pg of mCherry (In1) or LRP5ΔC-mCherry mRNA (In2). Left: Embryos are shown at 6 and 10 hpf. Embryo ratios are expressed as GFP+/mCherry+/total. Right: Bar graph showing ratios of classes of 30-hpf embryos growing from GFP+ 6-hpf embryos. Note that the class I mutant phenotype did not show in any injected embryos and served only for comparison. (F) Tg(gsc:EGFP);Mhwatsu01sm embryos injected with hwa and mCherry mRNAs as in (E) were incubated in DMSO or 20 μM Wnt-C59. The organizer and body axis are indicated by arrowheads and arrows, respectively. Scale bars, 10 μm (A), 100 μm (all others).