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BACKGROUND: Activation of the Wnt signalling cascade is primarily based on the interplay between Wnt ligands, their receptors and extracellular modulators. One prominent family of extracellular modulators is represented by the SFRP (secreted Frizzled-related protein) family. These proteins have significant similarity to the extracellular domain of Frizzled receptors, suggesting that they bind Wnt ligands and inhibit signalling. The SFRP-type protein Fz4-v1, a splice variant of the Frizzled-4 receptor found in humans and Xenopus, was shown to augment Wnt/β-catenin signalling, and also interacts with those Wnt ligands that act on β-catenin-independent Wnt pathways.
FINDINGS: Here we show that Xenopus Fz4-v1 can activate and inhibit the β-catenin-dependent Wnt pathway. Gain-of-function experiments revealed that high Wnt/β-catenin activity is inhibited by low and high concentrations of Fz4-v1. In contrast, signals generated by low amounts of Wnt ligands were enhanced by low concentrations of Fz4-v1 but were repressed by high concentrations. This biphasic activity of Fz4-v1 was not observed in non-canonical Wnt signalling. Fz4-v1 enhanced β-catenin-independent Wnt signalling triggered by either low or high doses of Wnt11. Antisense morpholino-mediated knock-down experiments demonstrated that in early Xenopus embryos Fz4-v1 is required for the migration of cranial neural crest cells and for the development of the dorsal fin.
CONCLUSIONS: For the first time, we show that a splice variant of the Frizzled-4 receptor modulates Wnt signalling in a dose-dependent, biphasic manner. These results also demonstrate that the cystein-rich domain (CRD), which is shared by Fz4-v1 and SFRPs, is sufficient for the biphasic activity of these secreted Wnt modulators.
Figure 1. Fz4-v1 has a biphasic, dose-dependent activity in modulating Wnt/β-catenin-dependent signalling. (A-F) 4-cell stage embryos were injected on the ventral marginal side with 0.5 pg wnt8b RNA (lo) or 10 pg (hi) alone, or in combination with 250 pg fz4-v1 RNA (lo) or 1000 pg (hi) or with 250 pg (lo) or 1000 pg (hi) fz4-v1 RNA alone. Formation of secondary body axes was scored at neurula (st. 20, B-F) and tadpole stages (st. 38, B’-F’). Frequency of secondary axis formation: (B, B’) 71.9% (n = 36), (C, C’) 0% (n = 24), (D, D’) 0% (n = 20), (E, E’) 0% (n = 24), (F, F’) 54.2% (n = 24). (G) At the 4-cell stage blastomeres were injected in the animal region with 80 pg Topflash-Luciferase reporter plasmid in combination with wnt3a RNA (0.05 pg, 0.1 pg, 5 pg) and fz4-v1 RNA (5 pg, 50 pg, 1000 pg). (H) Luciferase activity was measured at gastrula stage (st. 11). Error bars represent standard deviation (SD). (*) indicates significant difference (Student’s t test, p < 0.05).
Figure 2. Fz4-v1 perturbs morphogentic movements by activation of the Wnt/β-catenin-independent JNK pathway. (A, B) Embryos were injected radially at the 4-cell stage with 1 ng fz4-v1 RNA. At tailbud stages (st. 25) 22% displayed spina bifida and neural tube closure defects (n = 45). (C) At the 4-cell stage 50 pg of the ATF-luciferase reporter plasmid was injected ventral-animally alone, or in combination with 100 pg (low) or 500 pg (high) wnt11 RNA and fz4-v1 RNA (5, 50 and 1000 pg). Reporter activity was analyzed at late gastrula stages (st. 12). Error bars represent SD. (*) indicate significant difference to control or wnt11 alone, respectively (grey bars) (Student’s t test, *p < 0.05, **p < 0.001). (D-H) 4-cell stage embryos were injected at the animal pole region with bvg1 RNA (100 pg) alone, or in combination with fz4-v1 (600 pg) and jnk-apf (1 ng). At blastula stages (st. 9) the caps were explanted and elongation was scored at stage 25. (D) Non-injected controls (n = 20) showed no elongation but bvg1-injected explants (E) did (n = 27). (F) Co-injection of fz4-v1 partially inhibited explant elongation (n = 29), which was restored by JNK-APF (n = 27) (G). (H) Quantification of the animal cap experiments.
Figure 3. Fz4-v1 function is required for dorsal fin development. (A-D) Embryos were injected at the 2- to 4-cell stage with 50 ng antisense Fz4/Fz4-v1 morpholino oligonucleotide (MO) alone (B, Bâ) (nâ=â10), or in combination with 500 pg fz4-v1 RNA (C, Câ) (nâ=â29) or 500Â pg fz4 RNA (D, Dâ) (nâ=â23). At the tailbud stage (st. 30) formation of the dorsal fin was compared to uninjected control embryos (A, Aâ) (nâ=â40). (Aâ-Dâ) Vibratome cross-sections of the embryos shown in A-D. White arrows indicate the dorsal fin. (E) Quantification of the experiment. (df) dorsal fin. (F)in situ hybridization for sox10 and twist mRNA in tailbud-stage embryos (st. 32) injected unilaterally with Fz4/Fz4-v1 MO or the combination of MO and fz4-v1 RNA. Asterices indicate the injected side of the embryos. (NI) non-injected side of the embryo, (wt) wild-type embryos. (G) Quantification of the CNC phenotypes.
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