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Secreted Frizzled related proteins (SFRPs) are extracellular regulators of Wnt signaling. These proteins contain an N-terminal cysteine rich domain (CRD) highly similar to the CRDs of the Frizzled family of seven-transmembrane proteins that act as Wnt receptors. SFRPs can bind to Wnts and prevent their interaction with the Frizzled receptor. Recently it has been reported that a splice variant of human Frizzled-4 (FZD4S) lacking the transmembrane and the cytoplasmic domains of Frizzled-4 can activate rather than inhibit Wnt-8 activity in Xenopus embryos. This indicates that secreted CRD containing proteins such as Frizzled ecto-domains and SFRPs may not always act as Wnt inhibitors. It is not known how FZD4S can activate Wnt/beta-catenin signaling and what biological role this molecule plays in vivo. Here we report that the Xenopus frizzled-4 is alternatively spliced to give rise to a putative secreted protein that lacks the seven-transmembrane and the cytoplasmic domains. We performed functional experiments in Xenopus embryos to investigate how this novel splicing variant, Xfz4S, can modulate the Wnt/beta-catenin pathway. We show that Xfz4S as well as the extracellular domain of Xfz8 (ECD8) can act as both activators and inhibitors of Wnt/beta-catenin signaling dependent on the Wnt ligand presented. The positive regulation of Wnt/beta-catenin signaling by the extracellular domains of Frizzled receptors is mediated by the members of low density lipoprotein receptor-related protein (LRP-5/6) that act as Wnt coreceptors. This work provides evidence that the secreted extracellular domains of Frizzled receptors may act as both inhibitors and activators of Wnt signaling dependent on the Wnt ligand presented.
Figure 1. Molecular structure and expression pattern of Xfz4S. (A) Nucleotide and amino acid sequences of Xfz4S. The nucleotide sequence of Exon1 of Xfz4 is in capital and that of Intron1 is in small letters. Two sets of primers (P1F/P1R and P2F/P2R) were used for detection of Xfz4S by RT-PCR. (B) Schematic diagram showing the structure of Xfz4 gene containing two exons (boxes) and one intron. The splicing variants, Xfz4S retaining the intron and the Xfz4 are shown. The coding regions of these splicing variants are indicated by closed boxes and the UTRs by open boxes. (C) Developmental RT-PCR of Xenopus embryos with indicated Nieuwkoop and Faber (NF) stages. Xfz4S mRNA is first detected after mid blastula transition and the expression persist into tadpole stages. Xfz4 mRNA is maternally supplied and is expressed in all stages of development studied. ODC is a loading control. (D) Spatial expression pattern of Xfz4 and Xfz4S in tailbud stage embryos (stage 34). The embryos were hybridized with digoxigenin labelled RNA probes for antisense Xfz4 (Xfz4-AS), antisense Xfz4-intronI (Xfz4S-AS) or sense probe for Xfz4-intronI (Xfz4S-S).
Figure 2. Xfz4S acts synergistically with canonical Wnt ligands in activating the Wnt/β-catenin target gene Xnr3 and inhibits non-canonical Wnt ligands. (A) Experimental scheme. Embryos were injected at 4 cell stage into the animal blastomeres with synthetic mRNA for Xwnt-3a (0.5 pg/embryo), Xwnt-4 (150 pg/embryo), Xwnt-5a (50 pg/embryo), Xwnt-8 (0.5 pg/embryo), Xwnt-8b (15 pg/embryo) or Xwnt-11 (50 pg/embryo), either alone or in combination with 500 pg Xfz4S. Animal caps were dissected out at stage 8 – 9, grown until stage 10.5 at which expression of Xnr3 was analyzed by RT-PCR. (B) Xwnt-3a, -8, -8b induce Xnr3 expression only in combination with Xfz4S. Xwnt-5a, -4 or -11 do not synergize with Xfz4S in inducing Xnr3. (C) Xfz4S inhibits Xnr3 expression induced by coinjection of Hfz5 and non-canonical Wnt-5a class ligands. Wnt-5a class ligands such as Wnt-4 (150 pg/embryo), -5a (50 pg/embryo) or -11 (50 pg/embryo) when injected in combination with 250 pg Hfz5, Xnr3 expression was induced. Induction of Xnr3 expression by these Wnt ligands in combination with Hfz5 was inhibited by coinjection of 500 pg Xfz4S mRNA. (D) Coimmunoprecipitation of Xfz4S and Wnt-5a. Myc-tagged Xfz4S (500 pg/embryo) and flag-tagged Wnt-5a (500 pg/embryo) were injected into Xenopus embryos at 2–4 cell stage. Myc-tagged Xfz4S coimmunoprecipitates with flag-tagged Wnt-5a indicating that they interact. A part of the embryo extract was incubated with mouse IgG, which serves as a control against non-specific binding of proteins. Total embryo extract (EE) shows the expression of Xfz4S-myc and Wnt5a-flag constructs.
Figure 3. Extracellular domain of Xfz8 (ECD8) can activate Wnt/β-catenin pathway in combination with Xwnt-5a. (A) 500 pg/embryo ECD8 mRNA when injected in conjunction with Xwnt-5a (50 pg/embryo), Xnr3 expression was induced in animal cap tissues. Extracellular domains of Xfz7 (ECD7, 300 pg/embryo) or Hfz5 (ECD5, 500 pg/embryo) did not synergize with Xwnt-5a in inducing Xnr3 expression. (B) ECD8 (200 pg/embryo) and Wnt-5a (50 pg/embryo) when injected into the ventral marginal zone, Xnr3 expression was induced. (C) At later stages these embryos developed partial secondary axis without head structures. No secondary axes were observed when ECD8 or Wnt-5a was injected alone.
Figure 4. Activation of Wnt/β-catenin pathway by Xfz4S and ECD8 is LRP dependent. (A) Xfz4S (500 pg/embryo) and Xwnt-3a (0.5 pg/embryo) when coinjected into the animal caps, expression of Xnr3 was induced. This activation of Xnr3 was blocked by coinjection of 300 pg Xdkk1 or 1 ng ΔC-LRP6 and was greatly reduced by 250 pg Xdsh-DIX. (B) Induction of Xnr3 expression by injection of ECD8 (500 pg/embryo) and Xwnt-5a (50 pg/embryo) was blocked by coinjection of 300 pg Xdkk1, 1 ng ΔC-LRP6 or by 250 pg Dsh-dd2. Coinjection of a dishevelled mutant lacking the DIX domain (Dsh-ΔDIX 250 pg/embryo) had no effect on ECD8 plus Wnt-5a induced activation of Xnr3.
Figure 5. Model for the modulation of Wnt/β-catenin pathway by extracellular domains of Frizzled receptors. We propose that a complex formed between Xfz4S and Wnt-3a/-8/-8b could be recognized by LRP and Wnt signaling could be activated in LRP dependent manner. Complexes between Xfz4S and Wnt-4/-5a/-11 would not be recognized by LRP and Xfz4S and Wnt/β-catenin signaling would not be activated by these Wnts.
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