Wnt Signaling-Independent Regulation of Dorsoventral Patterning by the Axin/JNK Signaling Cascade (A) Genetic interaction between μ-catenin and axin. Ten nanograms of μ-cat2-MO was injected into one-cell embryos alone or together with 300 pg axin mRNA or 150 pg axinΔMID mRNA. In situ hybridization of marker genes was performed at the shield stage. (B) JNK mRNA, JNK-MO, aida mRNA, aida-MO, or aida antibody was injected into one-cell embryos, and expression of bozozok was analyzed in sphere-stage embryos. (C) JNK mRNA, JNK-MO, and wnt8-MO were injected singly or in combination as indicated. In situ analysis of tbx6 expression was performed at the shield stage. (D) JNK signaling has no effect on Wnt signaling as assessed with LEF-1 luciferase reporter and β-catenin stability assay. HEK293T cells were transfected with 0.5 μg of LEF-1 luciferase reporter plasmid together with 1.5 μg of MKK4, JNK, MKK4-KM, JNK-APF, or MKK4-RNAi alone or in combination for the LEF-1 luciferase activity. None of the plasmids affected basal or wnt-1-induced reporter expression. For the β-catenin stability assay, HEK293T cells were transfected with 4 μg of MKK4, JNK, MKK4-KM, JNK-APF, or MKK4-RNAi alone or in combination together with 0.5 μg of GFP, 1 μg of Myc-β-catenin, and 1.5 μg of wnt-1. Results showed that JNK signaling did not affect the β-catenin protein level. (E) A model for dual activities of Axin in dorsal development of vertebrate embryos. In the canonical Wnt pathway, μ-catenin promotes dorsalization. Axin reduces μ-catenin levels by serving as a scaffold for the assembly of μ-catenin degradation complex, and hence attenuates dorsalization. Axin also exerts an opposing role, i.e., promotion of dorsalization, by means of activation of JNK. Aida, by virtue of repressing Axin-induced JNK activation through disrupting Axin homodimerization, attenuates Axin-enhanced dorsalization.
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