Non-acylated Wnts Can Promote Signaling

Abstract

Wnts are a family of 19 extracellular ligands that regulate cell fate, proliferation, and migration during metazoan embryogenesis and throughout adulthood. Wnts are acylated post-translationally at a conserved serine and bind the extracellular cysteine-rich domain (CRD) of Frizzled (FZD) seven-pass transmembrane receptors. Although crystal structures suggest that acylation is essential for Wnt binding to FZDs, we show here that several Wnts can promote signaling in Xenopus laevis and Danio rerio embryos, as well as in an in vitro cell culture model, without acylation. The non-acylated Wnts are expressed at levels similar to wild-type counterparts and retain CRD binding. By contrast, we find that certain other Wnts do require acylation for biological activity in Xenopus embryos, although not necessarily for FZD binding. Our data argue that acylation dependence of Wnt activity is context specific. They further suggest that acylation may underlie aspects of ligand-receptor selectivity and/or control other aspects of Wnt function.

Keywords: Frizzled; Wnt; Xenopus laevis; acylation; lipid modification.

FIGURE 1. Effect of site1 and site 2 mutations on xWnt8 activity

(A) Crystal structure of the xWnt8/mFZD8 CRD complex (PDB: 4F0A), with ‘thumb’ and ‘index finger’ projections on xWnt8 binding to the CRD at sites 1 and 2 respectively (Janda et al., 2012). Residues mutated in site 1 (green) and site 2 (blue) are marked. The palmitoleoyl chain and S187 are red. (B) Representative dorsalization phenotypes observed upon ectopic xWnt8 expression in ventral cells of Xenopus laevis embryos. The top row shows tailbud-stage embryos with corresponding phenotype scores. Example phenotypes are shown in the bottom two rows. Yellow arrow = partial axis duplication; black = full axis duplication; red = radial dorsalization. (C) Quantitation of dorsalization phenotypes in Xenopus embryos for site 1 and site 2 mutations. Total number of embryos scored (across 3 biological replicates) is listed for each bar. Dorsal Scores for xWnt8^WT and xWnt8^S187A are from the dataset in Figure 2A, represented here for comparison. (D) Initial RT-PCR quantitation of Siamois and Xnr3 induction for each variant, represented as mean ± SEM (n = 3). Significance denoted as ‘ns’ (p ≥ 0.05), * (p ≤ 0.05), ** (p ≤ 0.01), or *** (p ≤ 0.001). (E) Expression of injected xWnt8 variants assessed by Western blotting of mid gastrula stage embryos. Representative of at least three repeats. (F) Dorsalization phenotypes observed in zebrafish embryos upon ectopic expression of xWnt8^WT or xWnt8^S187A mRNA. Pictures (top row) show representative embryos at 1 day post fertilization displaying normal (left), moderately dorsalized (“twisted”, center), or highly dorsalized (“bustled”, right) phenotypes. Quantitation of observed phenotypes is shown below, with number of embryos scored across at least two biological replicates listed for each bar. See also Figure S1.

FIGURE 2. xWnt8 retains biological activity without acylation

(A) xWnt8^S187A causes dorsalization of Xenopus embryos. Data are represented both as mean dorsal score (left – with number of embryos injected above each bar) and phenotype frequency (right), scored across 4 biological replicates. (B) Dose-response curve for Siamois and Xnr3 expression (RT-PCR) induced by xWnt8^WT and xWnt8^S187A (n = 3), as in Figure 1D. Although the difference in maximal Xnr3 expression at 500 pg mRNA for xWnt8^WT and xWnt8^S187A appears statistically significant (p ≤ 0.05), biological significance is unclear. (C) Inhibition of xWnt8^WT and xWnt8^S187A signaling by xFZD8 CRD. xWnt8 and xFZD8 CRD mRNA were co-injected and RT-PCR performed at gastrula stage. Siamois and Xnr3 induction is represented as in Figure 1D (n = 4). (D) xWnt8^WT and xWnt8^S187A proteins co-immunoprecipitate with myc-tagged xFZD8 CRD in embryos co-injected with both mRNAs. The band in lanes 2 and 6 of the α-myc IP blot corresponds to mouse α-myc antibody heavy chain, which co-migrates with the CRD (~50kD). Representative of at least three biological repeats. See also Figure S2.

FIGURE 3. mWnt1 requires acylation for biological activity

(A) mWnt1^S224A does not dorsalize Xenopus embryos. Mean dorsal scores (left) were calculated for tailbud-stage embryos, with injected embryo number listed above each bar (scored across 3 biological replicates). Right: photographs of representative embryos. (B) mWnt1^S224A fails to induce Siamois or Xnr3. Data presented as in Figure 1D (n = 3). (C) mWnt1^WT and mWnt1^S224A proteins are expressed at similar levels as assessed by Western blotting of injected mid gastrula stage embryos. Representative of at least two biological repeats. See also Figure S3.

FIGURE 4. Effects of acylation site mutations in hWnt3a and mWnt5a

(A) hWnt3a^S209A dorsalizes Xenopus embryos. Mean dorsal score data (left) are represented as in Figure 2A, for 3 biological replicates, with representative embryos shown at right (arrows as in Figure 1B). (B) Signaling by hWnt3a^WT and hWnt3a^S209A is inhibited by xFZD8 CRD (n = 3), as described for xWnt8 in Figure 2C. (C) Co-immunoprecipitation, as in Figure 2D, shows that hWnt3a^WT and hWnt3a^S209A both interact with xFZD8 CRD. The faint band in lanes 2 and 6 of the α-myc IP blot is α-myc heavy chain. Representative of at least three biological repeats. (D) mWnt5a^S244A does not affect CE in Xenopus embryos. Data represent the percent of individuals injected with mWnt5a^WT or mWnt5a^S244A mRNA displaying CE defects at neurula stages, with the number of scored individuals (across 3 biological replicates) listed above each bar. Photographs show representative tailbud stage embryos injected with 0 pg and 100 pg mRNA. (E) mWnt5a^WT and mWnt5a^S244A proteins are expressed at similar levels in mid gastrula stage embryos. See also Figure S4.