Xenbase Image ID: 173145

Fig. 4. March2 antagonizes canonical Wnt signaling in Xenopus. (A) Mar2MO (40 ng) increases TOPFlash activity in Xenopus. Error bars represent s.d. of triplicate experiments. P=0.0022. (B) Mar2MO mediates activation of β-catenin (ABC) and phosphorylation of LRP6. Four-cell stage embryos were dorsally injected with CoMO or Mar2MO (40 ng) and analyzed at stage 10.5. Actin and total LRP6: loading controls. (C) GFP-Mar2 mRNA (1 ng) expression but not GFPMar2CS mRNA (1 ng) decreases Wnt8 mRNA (20 pg)-mediated activation of β-catenin and phosphorylation of LRP6 in Xenopus. (D) Mar2 mRNA (1 ng) expression decreases TOPFlash activity induced by Dsh (2 ng) but not by β-catenin (200 pg). Error bars represent s.d. of triplicate experiments. (E) Mar2MO increases the severity of the Wnt8-induced ectopic axis. Wnt8 mRNA (1 pg) was injected with or without Mar2MO (50 ng) at the ventral side of the four-cell stage embryos and phenotypes were analyzed at the tadpole stage. ‘Partial’ indicates axis without head, including eyes and cement glands. ‘Full’ indicates axis with head. Statistical analysis was carried out on three independent experiments. Statistically significant differences between two sets are indicated. P values were obtained using two-tailed Student’s t-test (see also Table S3). (F-H) RT-PCR analyses show that March2 inhibited Wnt8 or Dsh-induced Xnr3 expression in Xenopus animal caps (F,G) but did not inhibit β-catenin-induced Xnr3 expression (H). The amounts of injected mRNAs: Wnt8, 20 and 50 pg; XDsh, 1 and 2 ng; βcatenin, 50 and 100 pg; Mar2, 1 ng. (I-K) March2 did not inhibit FGF (I) and BMP (J); Nodal signals (K) mediated induction of Xbra expression. ODC serves as a loading control. -RT, control RT-PCR without reverse transcriptase; WE, whole embryos. Amounts of injected mRNAs: FGF, 50 and 150 pg; BMP, 50 and 200 pg; Xnr1, 100 and 200 pg; Mar2, 1 ng. Image published in: Lee H et al. (2018) Copyright © 2018. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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