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The Wnt/beta-catenin signaling pathway is responsible for the establishment of dorsoventral axis of Xenopus embryos. The recent finding of the F-box/WD40-repeat protein slimb in Drosophila, whose loss-of-function mutation causes ectopic activation of wingless signaling (Jiang, J., Struhl, G., 1998. Nature 391, 493-496), led us to examine the role of its vertebrate homolog betaTrCp in the Wnt/beta-catenin signaling and dorsal axis formation in Xenopus embryos. Co-injection of betaTrCp mRNA diminished Xwnt8 mRNA-induced axis formation and expression of Siamois and Xnr3, suggesting that betaTrCP is a negative regulator of the Wnt/beta-catenin signaling pathway. An mRNA for a betaTrCp mutant construct (DeltaF), which lacked the F-box domain, induced an ectopic axis and expression of Siamois and Xnr3. Because this activity of DeltaF was suppressed by co-injection of DeltaF TrCP mRNA, DeltaF likely acts in a dominant negative fashion. The activity of DeltaF was diminished by C-cadherin, glycogen synthase kinase 3 and Axin, but not by a dominant negative dishevelled. These results suggest that betaTrCp can act as a negative regulator of dorsal axis formation in Xenopus embryos.
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9784611
???displayArticle.link???Mech Dev
Fig. 1. β-TrCP diminishes the dorsal axis activity of Xwnt8. (A) Ventralized UV-treated embryos (DAI=0). (B) UV-embryos injected with Xwnt8 (5 pg) and β-gal (2.5 ng) mRNAs, which recovered complete axes (DAI=5). (C) UV-embryos injected with Xwnt8 (5 pg) and β-TrCP (2.5 ng) mRNAs, which recovered axes (top: DAI=4; middle: DAI=3; bottom: DAI=2). (D) Expression of Siamois, Xnr3 and a control EF1α in animal caps as examined by RTCR. Animal poles were injected with either β-gal (2 ng) or β-TrCP (2 ng) mRNA at the 2-cell stage and then injected with Xwnt8 rnRNA (5 pg) at the 4-cell stage. The expression level of both Siamois and Xnr3 in animal caps injected with Xwnt8 and β-TrCP mRNAs is lower than that in animal caps injected with Xwnt8 and β-gal mRNA.
Fig. 2. ΔF, F-box deleted mutant of β-TrCP, has dorsal axis activity. (A) A schematic diagram to show the structures of β-TrCP and ΔF lacks an entire region encoding the F-box domain (aa 12657). (B) Double axis embryo produced by ventral injection of ΔF. ΔF mRNA (2 ng) at the 16-cell stage. The embryos have a complete secondary axis with a fully developed head containing two distinct eyes and a cement gland. (C) Expression of Siamois, Xnr3 and a control EF1α in animal caps as examined by RTCR. Animal poles were injected either with β-gal (2 ng) or β-TrCP (2 ng) mRNA at the 2-cell stage and then injected with ΔF mRNA (2 ng) at the 4-cell stage. Expression levels of both Siamois and Xnr3 in animal caps injected with ΔF and β-TrCP mRNAs are lower than those in animal caps injected with ΔF and β-gal mRNAs. (D) Expression of Siamois, Xnr3 and a control EF1α in animal caps as examined by RTCR. Animal poles were injected either with β-gal (2 ng), Xdd1 (2 ng), XGSK3 (2 ng), Axin (2 ng) or C-cadherin (2 ng) mRNA at the 2-cell stage and then injected with ΔF mRNA (2 ng) at the 4-cell stage. The expression level of both Siamois and Xnr3 in animal caps induced by ΔF was diminished by XGSK3, Axin and C-cadherin but not Xdd1.
