Xenbase Image ID: 171220

Figure 3. Rapgef5 Acts Downstream of β-Catenin Cytoplasmic Stabilization (A) The ability to induce secondary axes in development can be used as a readout of Wnt signaling activity. Uninjected embryos have a single axis (dotted green line), while embryos injected with β-catenin mRNA can have a second embryonic axis (two dotted red lines), which are readily detected in stages 16–19 embryos. Injection of WT, ST, or NLS-tagged β-catenin can induce secondary axes. Rapgef5 depletion significantly decreases the percentage of secondary axes induced by WT and ST β-catenin. Injection of GSK3 mRNA reduces secondary axes induced by WT β-catenin but has no effect on ST β-catenin. The ability of NLS-β-catenin to induce secondary axes is unaffected by reduction of Rapgef5 levels. (B) Rapgef5 knockdown reduces luciferase activity in embryos injected with WT or ST β-catenin mRNA in a TOPFlash assay. Data are represented as mean ± SD. (C) Wnt signaling activity is reduced in ST β-catenin murine embryonic fibroblasts (δexon3) following siRNA depletion of Rapgef5. The schematic depicts the Catnblox(ex3) mouse allele. Exon 3 (E3), containing the GSK3 phosphorylation sites that target β-catenin for cytoplasmic degradation, is flanked by loxP sites allowing for its conditional removal and production of a ST β-catenin allele. WT, WT cells; Fop, ST β-catenin cells transfected with FOPFlash-negative control; Top, ST β-catenin cells transfected with TOPFlash reporter plasmid; Top + R5 siRNA, ST β-catenin cells transfected with Rapgef5 siRNA and TOPFlash reporter plasmid; Top + C siRNA, ST β-catenin cells transfected with control siRNA and TOPFlash reporter plasmid. A Renilla luciferase transfection control was included in each treatment to allow normalization. Data are represented as mean ± SEM. (D) Pharmacological inhibition of GSK3 by the addition of BIO between stages 9 and 11 results in increased β-catenin signaling and loss of anterior development in Xenopus embryos. Depletion of Rapgef5 can counteract this effect and rescue development of the head, demonstrating that Rapgef5 regulates Wnt signaling downstream of GSK3. ∗p < 0.05, ∗∗∗p < 0.005. Image published in: Griffin JN et al. (2018) Copyright © 2018. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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