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- Open Access
A secreted splice variant of the Xenopus frizzled-4 receptor is a biphasic modulator of Wnt signalling
© Gorny et al.; licensee BioMed Central Ltd. 2013
- Received: 27 March 2013
- Accepted: 11 November 2013
- Published: 19 November 2013
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.
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.
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.
- Splice variant frizzled-4 receptor
- Wnt signalling
- Dorsal fin
- Neural crest
Fz4-v1 demonstrates a dose-dependent, biphasic activity in modulating Wnt/β-catenin-dependent signalling
SFRPs consist of two domains, an N-terminal cysteine-rich domain (CRD) and a C-terminal Netrin-like domain [1–3]. Based on similarity with the CRD region of Frizzled receptors, SFRPs were originally described as classical Wnt antagonists , however, recent data also describe agonistic functions of SFRPs [5–9]. These gain-of-function experiments in cultured cells and Xenopus embryos demonstrated that low levels of SFRPs enhance the Wnt/β-catenin pathway, but high levels of SFRPs suppress Wnt signalling [7, 8].
Xenopus Fz4-v1 is an SFRP-type protein which is generated from the Frizzled-4 receptor mRNA by intron retention. In contrast to classical SFRPs Fz4-v1 includes a CRD domain but lacks a Netrin-like domain . Such splice variants have been described for both the human and Xenopus Frizzled-4 receptor and were previously named Fz4S [5, 9]. Xenopus Fz4-v1 is a secreted protein and can modulate Wnt/β-catenin signalling in a non-cell autonomous manner (Additional file 1: Figure S1).
Both human and Xenopus Fz4 splice variants were shown to enhance the activity of Wnt ligands, which activate the Wnt/β-catenin pathway [5, 9]. Xenopus Fz4-v1 also interacts with Wnt ligands of the Wnt5a class, but its effect on β-catenin-independent Wnt signalling has not been assessed . Because previous experiments had only demonstrated an activating function of Fz4-v1, we tested whether Fz4-v1 might also have an inhibitory activity on Wnt/β-catenin signalling.
In addition, we performed Topflash-Luciferase reporter experiments in Xenopus embryos in order to quantitatively measure Wnt/β-catenin signalling strength (Figure 1G, H). Xenopus embryos were injected with synthetic mRNAs for wnt3a, fz4-v1 and the Topflash-Luciferase reporter plasmid. Wnt activity was measured at gastrula stage. Fz4-v1 alone did not activate the Topflash-Luciferase reporter. However, Luciferase activity generated by low doses of wnt3a (0.05 pg and 0.1 pg) was enhanced by co-injection of 5 and 50 pg of fz4-v1, but repressed by 1000 pg. In contrast, strong Wnt/β-catenin signals triggered by high doses of wnt3a (5 pg) were inhibited by both, high (1000 pg) and low (5 and 50 pg) amounts of fz4-v1 (Figure 1H).
This analysis revealed that Fz4-v1 can act as a biphasic modulator of Wnt/β-catenin signalling. Therefore Fz4-v1 behaves like classical SFRPs, which activate Wnt signalling at low and inhibit at high doses [7, 8]. The presence of the CRD domain is sufficient to induce biphasic activity, because Fz4-v1 is lacking the NTR domain. Recent structural analysis of Wnt8 interaction with the CRD domain of Fz8 could explain the finding that low doses of SFRPs activate, but high doses inhibit the activity of Wnt ligands . Low CRD concentration could weaken the attachment of the lipid modified Wnt proteins to the plasma membrane and ECM. Wnts would become more diffusible and association with the receptors would be facilitated. High concentration of CRD could induce the clustering of Wnt/CRD complexes, rendering them inactive. Since SFRPs can bind to Frizzled receptors, it would also be plausible that high concentrations of secreted CRD domains could cause receptor silencing.
In case of strong Wnt/β-catenin signals induced by high concentration of Wnt ligands one can assume that all the endogenous Wnt receptors are occupied. In this case the CRD domains could compete with Wnt ligands for the receptors, which would result in reduced Wnt/β-catenin activity.
Fz4-v1 activates the Wnt/β-catenin-independent JNK pathway
PCP signalling can be measured using an ATF-Luciferase reporter , which we made use to analyze the effects of Fz4-v1 (Figure 2C). ATF reporter, wnt11 and fz4-v1 RNAs were injected animally into the two ventral blastomeres of Xenopus embryos at the 4-cell stage, and Luciferase activity was analyzed at gastrula stage. Low doses of wnt11 were only able to induce ATF reporter activity when combined with high amounts of fz4-v1. ATF reporter activity induced by high concentrations of wnt11 was further enhanced by co-injection of fz4-v1 (Figure 2C). The ATF reporter experiment indicated that Fz4-v1 augments Wnt11-mediated PCP signalling. This was confirmed in Xenopus animal cap experiments (Figure 2D-G). Ectodermal animal cap explants stimulated by BVg1, a TGF-β growth factor , elongate due to convergent-extension (CE) movements that are controlled by the PCP pathway. Explants injected with RNA coding for BVg1 elongated as expected, but co-expression of Fz4-v1 inhibited CE movements. JNK is a downstream effector of the PCP pathway, and co-expression of a dominant negative form of Xenopus JNK1, JNK-APF, antagonized the inhibitory effect of Fz4-v1 in the explants (Figure 2D-G). This demonstrates that Fz4-v1 inhibits CE movements by hyper-activation of PCP signalling and confirms the results of the ATF reporter assay.
In vivo function of Fz4-v1
To analyze the endogenous function of Fz4-v1 during Xenopus embryogenesis we performed antisense morpholino knock-down experiments using a translation-blocking morpholino that targets the 5′-UTR. The efficiency and specificity of the morpholino is shown in the Supplements (Additional file 2: Figure S2). Since Fz4-v1 is generated by intron retention, the antisense morpholino also blocks translation of fz4 mRNA. Injection of the Fz4/Fz4-v1 morpholino at the 2-or 4-cell stage had no adverse effect on early embryogenesis, suggesting that Fz4-v1 and Fz4 do not affect maternal and early zygotic Wnt signalling, since such interference are expected to result in dorsoventral patterning defects.
Furthermore, it was shown that a calcium-sensitive epithelial-mesenchymal-transition (EMT) event essential for dorsal fin induction is controlled by Wnt11-R , which could be modulated by Fz4-v1. Our data suggest that Fz4-v1 controls Wnt signalling in the head and trunk neural crest and somites, and thereby contributes to the development of the dorsal fin.
Xenopus embryo manipulations
Xenopus laevis frogs were obtained from Nasco and all experiments complied with local and international guidelines for the use of experimental animals. Xenopus eggs were obtained from females injected with 500 IU human chorionic gonadotropin (Sigma), and were fertilized in vitro. Embryos were dejellied with 2% cysteine hydrochloride (pH8) and embryos were microinjected in 1x MBSH (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.41 mM CaCl2, 0.33 mM Ca(NO3)2, 10 mM HEPES (pH7.4), 10 μg/ml penicillin). The embryos were cultured in 0.1x MBSH and staged according to Nieuwkoop and Faber .
Animal cap elongation assay
For the animal cap elongation assay, 4-cell-stage embryos were injected animally into two opposing blastomeres with synthetic RNAs. Animal caps were excised at stage 9 and cultivated in 1x MBSH together with 10 ng/μl gentamycine overnight.
Synthesis of CAP-RNA and morpholinos for microinjection
Capped RNAs were synthesized form linearized plasmids using the mMessage mMachine Kit (Ambion).
pCS2-Wnt3a (mouse), pCS2-Wnt8b, pCS2-Wnt11, pCS2-Fz4, pCS2-Fz4-v1, pCS2-Fz4-myc, pCS2-Fz4-v1-myc and pCS2-JNK-APF (all Xenopus) were linearized with Not1 and pSP64T-BVg1 (Xenopus) was linearized with EcoRI. Sense RNA was transcribed by SP6 polymerase.
For knock-down experiments antisense Fz4/Fz4-v1 morpholino oligonucleotide (5′-ATTATTCTTCTTCTGTTGCCGCTGA-3′) or control morpholino (5′-CCTCTTACCTCAGTTACAATTTATA-3′) was injected.
Whole-mount in situ hybridization and Luciferase reporter assay
Embryos were fixed in MEMFA and whole-mount in situ hybridization was performed as described . pBluescriptSK–Sox10  was linearized with EcoRI, and DIG-labelled antisense RNA was transcribed by T3 polymerase. pCR2.1–Twist  was linearized with HindIII, and DIG-labelled antisense RNA was transcribed by T7 polymerase. pCR-Blunt II-TOPO-Fz4-IntronI  was linearized with BamHI, and DIG-labelled antisense RNA was transcribed by T7 polymerase. Whole-mount in situ hybridization for fz4-v1 was performed using a double (5′ and 3′) DIG-labelled LNA probe (5′-AGTATAGAAAGTAAACCCCCTGTG-3′) from Exiqon, according to manufacturer’s instructions.
For reporter assays 4-cell stage embryos were injected animally with 80 pg M50 Super 8x Topflash  or 50 pg ATF-Luciferase reporter plasmid  in combination with 8 pg or 5 pg TK-Renilla-Luciferase reporter plasmid. The reporter plasmids were injected alone or in combination with synthetic RNAs. Triplicates of 5 embryos were lysed according to the manufacturer’s protocol (Promega) and 20 μl of cell lysate was used for Luciferase detection.
We thank Kirsten Linsmeier for technical support and Sarah Cramton for critically reading the manuscript. This work was supported by a research grant of the Deutsche Forschungsgemeinschaft (DFG) (STE 613/8-2).
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