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Wnts are secreted glycoproteins implicated in diverse processes during embryonic patterning in metazoans. They signal through seven-transmembrane receptors of the Frizzled (Fz) family [1] to stabilise beta-catenin [2]. Wnts are antagonised by several extracellular inhibitors including the product of the dickkopf1 (dkk1) gene, which was identified in Xenopus embryos and is a member of a multigene family. The dkk1 gene acts upstream of the Wnt pathway component dishevelled but its mechanism of action is unknown [3]. Although the function of Dkk1 as a Wnt inhibitor in vertebrates is well established [3-6], the effect of other Dkks on the Wnt/beta-catenin pathway is unclear. Here, we report that a related family member, Dkk2, activates rather than inhibits the Wnt/beta-catenin signalling pathway in Xenopus embryos. Dkk2 strongly synergised with Wnt receptors of the Fz family to induce Wnt signalling responses. The study identifies Dkk2 as a secreted molecule that is able to activate Wnt/beta-catenin signalling. The results suggest that a coordinated interplay between inhibiting dkk1 and activating dkk2 can modulate Fz signalling.
Fig. 1. Interaction of Dkks with Wnt signalling. (a,b) Four-cell stage embryos were microinjected in each blastomere in the animal region with the indicated combinations of mdkk1, mdkk2 or mdkk3 RNA (200pg), Xwnt8 RNA (3pg), Xwnt5A RNA (40pg) and hfz5 RNA (200pg). Animal caps were explanted at stage 8–9, and analysed at stage 11 by RT–PCR for induction of siamois (sia) expression. Xbra expression was monitored to control for the isolation of mesoderm-free animal caps. Expression of histone H4 was used for normalisation. –RT, control in which reverse transcriptase was omitted; embryo, whole-embryo control; Co, uninjected control cap; (c) Fz2, 5 and 8 interact with Dkk1. Embryos (4–8-cell stage) were microinjected in two opposite blastomeres with the indicated RNAs. RNA encoding Fz receptors was either injected alone (–) or coinjected with dkk1 and Xwnt8 RNAs (+). Doses per blastomere were: Xwnt8, 4pg; dkk1, 2.5pg; fz mRNAs, 250pg except mfz3 (125pg) and hfz5 (100pg). Embryos were scored for the formation of complete secondary axis at stage 31; n, number of embryos scored; Rfz1, rat fz1.
Fig. 2. Axis duplication by dkk2. (a) Expression of Xdkk2 determined by in situ hybridisation. Lateral view of a stage 40 Xenopus embryo showing expression of dkk2 in headmesenchyme (hm), somites (so), and lens (le). (b,c) Injected dkk2 RNA induces (b) incomplete and (c) complete secondary embryonic axes. Mouse dkk2 RNA (50–250pg) was injected into one ventralblastomere at the 8-cell stage. (d,e) Injected dkk2 RNA induces siamois expression. Embryos (4–8-cell stage) were uninjected (Co) or injected in the animal region with 500pg Xdkk1, or Xenopus or mouse dkk2 RNA. Animal caps were explanted at stage 8 and analysed at (d) stage 11 or (e) stage 30 by RT–PCR analysis for the expression of the indicated marker genes. (f,g) Injected dkk2 RNA activates a TCF-responsive promoter; 50pg pTOP-luc and 5pg pTK-renilla were coinjected either alone (Co) or with the indicated RNAs (Xwnt8, 25pg; Xfz8, 200pg; mdkk2 or mdkk3, 100pg; GSK-3, 1.5ng). Luciferase activity was determined and normalised against renilla activity. (h) Injected dkk2 RNA cooperates with fz2, fz5, and fz8. Expression of siamois in animal caps injected with 25 pg Xwnt8, or RNAs encoding the indicated Fz receptors (Dfz2, 100pg; Xfz8, 200pg; all others, 500pg) injected with or without 100pg mdkk2 RNA as indicated.
Fig. 3. Dkk1 and Dkk2 antagonise each other. (a–e) Plasmid DNAs were injected into four animal blastomeres of 8-cell stage embryos. Embryos were allowed to develop until stage 40 and are shown in lateral (left) and frontal (right) view. (a,b) Post-MBT expression of dkk2 by injection of plasmid DNA (100pg pCSmdkk2) posteriorises Xenopus embryos. (c–e) Injected dkk1 rescues posteriorisation by Xwnt8, and dkk2 enhances it; 100pg pCSmdkk2, 25pg pCSKAXwnt8 and pCSXdkk1 were injected as indicated. (f,g) Injected dkk1 inhibits axis duplication by dkk2. Embryos (8-cell stage) were coinjected in one ventral-vegetal blastomere with (f) 50pg mdkk2+100pg Xfz8, or (g) 50pg mdkk2+100pg Xfz8+50pg Xdkk1. (h) Injected dkk1 inhibits siamois induction by dkk2. Embryos (4–8-cell stage) were coinjected in the animal region with the indicated combinations of 100pg mdkk2, 200pg Xfz8 and 100pg Xdkk1 RNA. Animal caps were explanted at stage 8 and analysed at stage 11 by RT–PCR for the expression of siamois.
Fig. 4. Properties of Dkk2 signalling. (a) Both dkk1 and dkk2 inhibited Wnt signalling in HEK293T cells. Top-Flash/pTK-renilla plasmid DNAs were cotransfected in HEK293T cells with mouse wnt1/fz8 and/or dkk plasmid as indicated. Luciferase activity was normalised against renilla activity. RLU, relative luciferase units. (b) Dkk2 signals in the presence of cycloheximide. Induction of siamois in animal caps injected with the indicated mRNAs (Xwnt8, 25pg; mdkk2, 100pg; Xfz8, 200pg) at the 8-cell stage and incubated with or without 5μg/ml cycloheximide (CHX) as indicated from the blastula stage onwards until harvest at early gastrula stage. (c) Dkk2 can function in a paracrine fashion. Mouse dkk2 (50pg) and Xfz8 (100pg) RNAs were injected either alone, coinjected (autocrine), or injected separately into two neighbouring blastomeres (paracrine) at the 16-cell stage (left). The resulting embryos were scored at the tadpole stage for complete (grey bar) or incomplete (white bar) axis duplication; n, number of embryos scored.
