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Fig. 6 hwa can function through β-catenin in a Wnt ligand/receptor-independent manner in Xenopus embryos. (A) hwa or wnt8 overexpression delays cytosolic β-catenin degradation. Top: Illustration of experiment design (dosage: hwa or wnt8 mRNA, 200 pg; β-catenin MO or standard MO, 20 ng). Bottom: Immunoblotting results and quantification of cytosolic β-catenin levels. For immunoblotting, membrane fraction in embryo lysate was removed using digitonin buffer. Quantitative data are derived from three experiments. (B) hwa fails to induce the body axis in embryos depleted of β-catenin. Embryos were first injected with 20 ng of β-cat-MO or standard MO at one-cell stage and then injected with 200 pg of hwa and 200 pg of β-gal mRNAs into one ventral blastomere at four-cell stage, and were observed at stage 26. (C) dkk1 overexpression inhibits secondary axis induction of wnt8 but not hwa. mRNAs were injected into one ventral blastomere of four-cell stage embryos. Left: Categories of embryos at stage 32. Right: Ratios of embryos in each category. (D) hwa overexpression induces the body axis of embryos derived from oocytes depleted of lrp6. Oocytes were injected with 6 ng of lrp6 antisense oligodeoxynucleotide (lrp6-AS) and one blastomere of the resulting embryos at two-cell stage was injected with 20 pg of hwa mRNA. Embryos were observed at stage 28 (left panel). (E) Embryos from the experiment shown in (D) were also collected at stage 10 for RT-PCR analysis of dorsal markers. qRT-PCR results are based on four technical repeats. *P < 0.05, ***P < 0.001. Scale bars, 1 mm.