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Fig. 5. XTgfbi was required for the full activation of canonical Wnt signalling in Xenopus. (A, B, C) In situ hybridization of organizer genes in morpholino-injected embryos. XTgfbi depletion suppressed expression Gsc, Chd, and Xnot. (D) RT-PCR showed Wnt target gene expression was repressed by XTgfbi depletion. The indicated reagents were injected at 2-cell stage and analyzed in whole embryos at stage 10.5. (E) A representative TOPFLASH assay in stage-10.5 embryos injected with CoMO, MO1 and MO1 with XTgfbi-MT at two-cell stage. The data were analyzed with unpaired two-tailed Student T test (P=0.030, 0.037 respectively). (F) RT-PCR showed the exogenously induced Wnt target gene expression was repressed by XTgfbi knockdown in the animal cap assay. Embryos were injected with the indicated constructs at two-cell stage. Animal caps were dissected out at stage 8 and harvested at stage 10. (G, H) Exogenously induced target gene expression downstream of Nodal/Activin pathway was not affected by XTgfbi depletion. Embryos were injected at two-cell stage with indicated constructs. Animal caps were (G) isolated at stage 8, treated with activin at 10 ng/ml and harvested at stage 11, or (H) isolated at stage 9 and harvested at stage 11. (I) RT-PCR showed XTgfbi depletion does not affect the BMP pathway target gene expression. Embryos were injected at two-cell stage. Animal caps were isolated at stage 8 and harvested at stage 11. (J) RT-PCR showed Xwnt-8 injection increased XTgfbi expression level in animal caps at stage 10. Xwnt-8 mRNA was injected at two-cell stage. Animal caps were isolated at stage 8 and harvested at stage 10. (K) Real-time PCR showed injection of Xwnt-8 at two-cell stage increased XTgfbi expression in whole embryos at stage-15. |
