Acylation of Wnt3 is essential for activation of canonical Wnt signaling. (A) Morphological phenotypes at 24 hpf of embryos injected with capped sense RNA of Wnt3-EGFP (200 pg, 53/56 embryos) or Wnt3(S212A)-EGFP (200 pg, 46/49 embryos). (B) Whole mount in situ hybridization (WMISH) showing the loss of capacity in Wnt3(S212A)-EGFP (200 pg) capped sense RNA to activate canonical Wnt signaling. First and second columns left: mCherry WMISH shows upregulation of signaling in the transgenic Tg(7xTcf-Xla.Siam:nlsm-Cherryia) canonical Wnt/ß-catenin reporter embryos by Wnt3-EGFP (36/36) but not by Wnt3(S212A)-EGFP (32/33). Second column right: Direct Wnt/ß-catenin target gene sp5l is upregulated by Wnt3-EGFP (30/32) but not by Wnt3(S212A)-EGFP (29/32). Rightmost column: Wnt3-EGFP (33/34), but not Wnt3(S212A)-EGFP (36/36), completely abolishes the telencephalon, marked by foxg1a (arrowhead), while the midbrain-hindbrain boundary, marked by her5 (arrow), is preserved. (C) Expression levels of mCherry in transgenic Tg(7xTcf-Xla.Siam:nlsm-Cherryia) and sp5l in wt embryos injected with capped sense RNAs of Wnt3-EGFP (200 pg) or Wnt3(S212A)-EGFP (200 pg) and determined by qPCR. Four independent experiments were performed. (D) Average and SD of the mean (error bars) values of pBAR luciferase reporter activity monitoring Wnt/ß-catenin signaling activity (normalized to renilla luciferase activity) in SH-SY5Y cells transfected with wt Wnt3-GFP or Wnt3S212A-GFP. wt Wnt3-GFP significantly activated the reporter whereas Wnt3S212A-GFP did not. Statistical significance was evaluated using unpaired t-test. ***p < 0.001, **p < 0.01, and n.s., non-significant. Error bars represent SD. Three independent experiments were performed.
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