1 Cell and Molecular Biology Graduate Group (School of Medicine), University of Pennsylvania, Philadelphia, PA 19104, USA
2 Department of Medicine (School of Medicine), University of Pennsylvania, Philadelphia, PA 19104, USA
3 Department of Animal Biology (School of Veterinary Medicine), University of Pennsylvania, Philadelphia, PA 19104, USA
4 Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA 19104, USA
*Author for correspondence (e-mail: pklein{at}hhmi.upenn.edu)
Accepted July 6, 2001
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SUMMARY |
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Key words: Xenopus, Xwnt, Frizzled, Neural crest, Xfz3, Slug, Vertebrate embryo, Melanocyte
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INTRODUCTION |
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Frizzled proteins consist of an extracellular N terminus containing a cysteine-rich domain (CRD) believed to be important for ligand binding, seven hydrophobic domains proposed to be transmembrane segments, and a C-terminal tail believed to be important for cytoplasmic signaling (Vinson et al., 1989). Wg protein has been shown to bind to the surface of Schneider cells that express frizzleds, and a CRD with a GPI anchor also recruits wg to the cell surface (Bhanot et al., 1996). Rat Fz1 expressed in Xenopus can also recruit Myc tagged Xwnt8 to the surface of animal cap cells (Yang-Snyder et al., 1996). Although Wnt proteins have been notoriously difficult to purify in an active form, Xwnt8 in conditioned medium, similar to wg, has been shown to bind to the CRD of Fz2 and vertebrate Fz4, Fz5, Fz7 and Fz8 (Hsieh et al., 1999). Importantly, ectopic expression of frizzleds in cells that do not normally respond to Wnts confers Wnt-dependent signal transduction, as assessed by accumulation of armadillo/ß-catenin protein (Bhanot et al., 1996). These observations have provided compelling evidence that frizzled proteins are functional Wnt receptors, although the transmembrane proteins encoded by arrow/LRP-5 and LRP-6 (LDL receptor-like proteins) may interact with frizzleds and serve as co-receptors for Wnts (Pinson et al., 2000; Tamai et al., 2000; Wehrli et al., 2000).
Because of the large number of Wnts and frizzleds expressed in vertebrate embryos (Wang et al., 1996), it is important to define which Wnts interact with specific frizzleds. Straightforward biochemical analysis, particularly binding assays, have in general been limited by the difficulty in obtaining purified soluble Wnts, except in limited cases. An alternative and informative approach has been to examine synergy between ligand and receptor pairs. Using this approach, He et al. have demonstrated that Xwnt5a, when ectopically expressed with human FZ5 (FZD5 Human Gene Nomenclature Database; originally named hfz5), is able to activate the canonical Wnt pathway and cause duplication of the dorsal axis in Xenopus embryos, despite the inability of Xwnt5a to do so on its own (He et al., 1997), which suggests a functional interaction between Xenopus Xwnt5a and human FZ5. Similarly, Xenopus Xwnt8 and rat Fz1 have been shown to synergize in the activation of dorsal specific gene expression (Yang-Snyder et al., 1996) and Xfz8 has been shown to synergize with Xwnt8 in dorsal axis duplication assays (Deardorff et al., 1998). Recently, two groups have also demonstrated synergy between Xwnt8b and Xfz7 in the dorsal axis duplication assay in Xenopus (Medina et al., 2000; Sumanas et al., 2000).
Using this approach, we have examined eight Wnts and five frizzleds for potential interactions in the dorsal axis induction assay in Xenopus. This screen identified a remarkably strong interaction between Xwnt1 and Xfz3, while none of the other Wnts tested showed significant interaction with Xfz3. As Xwnt1 and Xfz3 are both expressed in the dorsal neural tube in Xenopus, zebrafish and mouse (Fig. 2a) (Borello et al., 1999; Dorsky et al., 1998; Shackleford and Varmus, 1987; Shi et al., 1998; Wilkinson et al., 1987; Wolda et al., 1993) and Xwnt1 has been implicated in the regulation of neural crest development (Dorsky et al., 1998; Ikeya et al., 1997; Saint-Jeannet et al., 1997), we also examined whether Xfz3 may also play a role in neural crest development. We find that Xfz3 is able to induce neural crest in embryos and in explants and that inhibition or loss of Xfz3 blocks endogenous neural crest formation. These observations suggest that endogenous Xwnt1 and Xfz3 act as a ligand-receptor pair in the induction of neural crest.
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MATERIALS AND METHODS |
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In vitro transcription, microinjection and explants
Capped synthetic RNAs were generated using an SP6 mMessage mMachine kit from Ambion (Austin, TX). RNA in a volume of 10 nl was injected into embryos as described (Deardorff et al., 1998). For synergy assays, each RNA preparation was tested independently in a dose-response analysis to identify concentrations that were ineffective alone in dorsal axis duplication or neural crest induction assays. pCS2/nßgal was used to generate lineage tracer RNA. For morpholino oligo injections (Heasman et al., 2000), a Xfz3 antisense oligo with the sequence 5'-CGCAAAGCCACATGCACCTCTTGAA-3' was purchased from Gene Tools (Corvallis, OR) and was dissolved in DEPC-treated water. 5 nl of oligo (0.4 ng/nl) was injected into one dorsolateral animal blastomere at the 32-cell stage together with mRNA encoding ß-galactosidase. Embryos were cultured until stage 18 and then fixed and stained for ß-galactosidase activity and Xslug expression as described below.
Fertilization and embryo culture were performed as described previously (Deardorff et al., 1998). The neural crest induction assay in animal pole explants was performed as described previously (Saint-Jeannet et al., 1997), except that chordin was used instead of noggin; briefly, embryos were injected at the 1-2 cell stage in the animal pole with mRNA for chordin alone or with Xwnt1, frizzleds or frizzled deletion constructs in combinations and at concentrations indicated in the figure legends. At stage 9, animal pole explants were removed using a Gastromaster, were cultured as described previously (Deardorff et al., 1998) and were then harvested at the stages indicated in the figure legends.
RT-PCR
RT-PCR methods and primers for EF-1, muscle actin and NCAM are described elsewhere (Deardorff et al., 1998). Primers for Xslug, Xsnail, Xtwist (LaBonne and Bronner-Fraser, 1998) and ADAM-13 (Alfandari et al., 1997) were as reported. In addition, primers for Xpax3, Xfz3 and Xwnt1 were as follows (in each case 25 cycles of amplification were used and conditions were otherwise as described (Deardorff et al., 1998): Pax3, U-ACCACATTCACTGCAGAGC D-AACCACACTTGAACTCGCG; Xfz3, U-TAACAATCATCCTG-CTCGC D-TTGTACCCAAGTTGTCTCC; Xwnt1, U-TGCTGTTT-CTGCCTTGGGTG D-GCGTCGGTTCCTAAAATGCC
In situ hybridization, histology and lineage tracing
Whole-mount in situ hybridization was performed as described (Deardorff et al., 1998). Probes detected Xfz3, Xslug (Mayor et al., 1995) and Xwnt1 (Wolda et al., 1993). Lineage tracing was performed by co-injecting ß-galactosidase RNA and staining the embryos using Red-Gal (Research Organics) as a substrate.
Immunoblotting
Xenopus embryos were injected at the animal pole at the one cell stage with Xfz3 mRNA and varying doses of Xfz3 morpholino antisense oligonucleotide (as indicated in Fig. 8), developed to stage 10 and lysed in embryo lysis buffer (20 mM Tris, pH 7.5, 140 mM NaCl, 10% glycerol, 1 mM DTT, 2 mM sodium vanadate, 25 mM NaF, 1% Nonidet P-40, and protease inhibitor cocktail for mammalian cells (Sigma)). Protein extracts were analyzed by western blot using -Xfz3 monoclonal antibodies raised against GST-Xfz3 C terminus, amino acids 499-664 (Tan et al., 2001).
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RESULTS |
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Wnts have been shown to cooperate with neuralizing signals to induce robust expression of neural crest markers in Xenopus (Bang et al., 1999; Chang and Hemmati-Brivanlou, 1998; LaBonne and Bronner-Fraser, 1998; Saint-Jeannet et al., 1997). As Xfz3 synergizes with Xwnt1 and is expressed in tissues that develop into neural crest, we tested whether Xfz3 can induce neural crest in ectodermal explants. We find that Xfz3, similar to Xwnt1, induces Xslug in a dose-dependent manner in animal cap explants that have been neuralized by expression of chordin, but not in non-neuralized explants (Fig. 3A). Furthermore, Xfz3, like Xwnt1, also induces the neural crest markers Xsnail (Essex et al., 1993), Xtwist (Hopwood et al., 1989) and ADAM-13 (Alfandari et al., 1997)( Fig. 3B). This induction of neural crest by Xfz3 is not a result of secondary induction by paraxial mesoderm as indicated by the absence of muscle actin in these explants (Fig. 3B). Interestingly, Xfz3 does not reduce the expression of the anterior neural marker Otx2 or induce expression of the posterior neural marker hoxB9 (data not shown), indicating that Xfz3, in contrast to Xwnt1, Xwnt3a, Xwnt8 and Xwnt7b (McGrew, 1995; Fredieu, 1997; Chang, 1998), does not posteriorize the anterior neural tissue induced in these assays.
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In order to determine whether Xfz3 induces neural crest at the same time as endogenous neural crest formation, animal caps expressing chordin and Xfz3 were harvested at several intervals from stage 12 (late gastrula) until stage 25 (tadpole). In parallel, caps expressing chordin and Xwnt1, as well as whole embryos, were also harvested and Xslug expression was measured as above. As shown in Fig. 4, Xslug expression is strongly induced by stage 14, similar to the effect of Xwnt1 and similar to Xslug expression in whole embryos. Thus, the timing of Xslug induction by Xfz3 is similar to both Xwnt1 and endogenous Xslug expression.
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The observation that overexpression of Xfz3 leads to neural crest induction raises the question of whether Xfz3 functions constitutively or is dependent on an endogenous ligand (such as Xwnt1). To address this question, Xfz3 was co-expressed with Nxfz3 in neuralized animal caps. Nfxz3 prevented neural crest induction by full-length Xfz3, suggesting that the activity of Xfz3 in this assay is dependent on an endogenous ligand (Fig. 5C). Furthermore, a Xfz3 construct that lacks the CRD (Xfz3N) has no detectable activity (Fig. 5c), indicating that the CRD is required for Xfz3 function in this assay. These observations are in contrast to Xfz8, which still dorsalizes when co-expressed with a construct encoding only the CRD (Nxfz8) (Deardorff et al., 1998) and shows apparent constitutive activity when the CRD is deleted (data not shown).
Specificity of Xfz3 in neural crest induction
To test whether neural crest induction is a specific activity of Xfz3 or a general attribute of frizzleds, we analyzed Xfz2, Xfz3, Xfz7 and Xfz8 for induction of neural crest in animal caps. Neural crest induction was observed with Xfz3, but not with Xfz2 or Xfz7 and only weakly with Xfz8, as assessed by Xslug induction (Fig. 6A). Thus, Xfz3 is functionally different from the other frizzleds with respect to induction of neural crest. This is supported by the sequence comparison of Xfz3 that places it in a different sequence subfamily from the other three frizzleds tested (see dendrogram in Fig. 6B). Furthermore, the CRD domains of Xfz2 and Xfz7 (Nfz2 and Nfz7) do not inhibit neural crest induction, indicating that Nfz3 is a specific inhibitor of neural crest induction (Fig. 6C), although it remains possible that Nfz3 interacts with other unidentified Wnts in this assay.
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DISCUSSION |
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Biochemical analysis of Wnt/frizzled binding available to date (Hsieh et al., 1999) appears to correlate with functional data we have obtained from dorsal axis synergy assays. Thus, Xwnt8 binds to Xfz8 but not Xfz3 (Hsieh et al., 1999), and similarly synergizes with Xfz8 but not with Xfz3 (this work). Clearly it will be essential to correlate functional analysis with direct binding assays for Xwnt1when sufficient levels of active, soluble protein are attainable.
Several other findings also support a specific functional interaction between Xwnt1 and Xfz3. Xfz3 alone does not induce axis duplication (or expression of dorsal mesodermal markers), even when expressed at high levels (M. A. D. and P. S. K., unpublished)(Shi et al., 1998; Sheldahl, 1999), despite the presence of several maternal Wnt mRNAs (Gradl et al., 1999). However, Xfz3 greatly enhances the response to ectopic Xwnt1, suggesting that Xfz3 is both specific for Xwnt1 and that its activity in these assays is dependent on ligand activation. Indeed, the induction of neural crest by ectopic Xfz3 is blocked by co-expression of Nfz3, suggesting that this activity depends on the endogenous Xwnt1 ligand in early embryos. Other Xwnts that do not appear to synergize with Xfz3, including Xwnt3a, Xwnt7b and Xwnt8, are also able to induce neural crest in neuralized explants; these ligands may function through a promiscuous Xfz, such as Xfz8 (Fig. 1B), which is present in animal caps (data not shown). Alternatively, these ligands may interact with Xfz9, which is expressed in a pattern similar to Xfz3 in anterior neural tissues in the tadpole (Wheeler and Hoppler, 1999).
Previous work has suggested that overexpression of mouse Xfz3, which is closely related to Xenopus Xfz3, activates protein kinase C (PKC) but not expression of the Wnt-dependent gene siamois (Xsia) in Xenopus (another Wnt-dependent dorsal gene, Xnr3, was weakly activated (Sheldahl et al., 1999)). By contrast, a more recent report showed that overexpression of Xenopus Xfz3 can induce the dorsal markers Xsia and Xnr3, consistent with ectopic activation of the canonical Wnt pathway, although that work did not show dorsal axis induction by Xfz3 (Umbhauer et al., 2000). These apparently conflicting observations may reflect differences in the constructs used (for example mouse versus Xenopus) or in the level of overexpression of the respective mRNAs. Consistent with both studies, we found that neither Xfz3 (Fig. 1) nor mouse Fz3 (Fzd3 Mouse Genome Informatics; data not shown) induces secondary dorsal axes in the absence of Xwnt1. However, Xfz3 in combination with Xwnt1 robustly induces secondary axes, implying that the Xwnt1/Xfz3 interaction activates the canonical Wnt/ß-catenin pathway. The ability of frizzleds to function in both canonical and non-canonical pathways has been reported for Drosophila fz and fz7, and raises the interesting possibility that the ligand may determine which pathway is activated by a given fz (Bhanot et al., 1999; Bhat, 1998; Chen and Struhl, 1999; Djiane et al., 2000; Kennerdell and Carthew, 1998; Medina et al., 2000). As the canonical Wnt/ß-catenin pathway has been strongly implicated in neural crest formation (Dorsky et al., 1998; Dorsky et al., 2000; Saint-Jeannet et al., 1997), it seems likely that activation of this pathway by Xwnt1/Xfz3 interaction is also responsible for neural crest induction by this putative ligand-receptor pair observed here. However, the possibility that activation of PKC (or another effector) by Xfz3 plays a role in neural crest induction cannot be ruled out.
Work in the mouse has suggested that Wnts are required for the expansion of neural crest progenitors (Ikeya et al., 1997; Dunn et al., 2000), while work in Xenopus and zebrafish (Bang et al., 1999; Chang and Hemmati-Brivanlou, 1998; Dorsky et al., 1998; LaBonne and Bronner-Fraser, 1998; Saint-Jeannet et al., 1997) supports a more direct role for Wnt signaling in specification of neural crest lineage. The work presented here, together with our related work on the Xfz3-interacting protein Kermit (Tan et al., 2001), suggests that Xfz3 signaling is required for neural crest formation; however, the generation of neural crest is a dynamic process and it is not yet clear whether Xwnt1/Xfz3 signaling is involved in the earliest steps in neural crest induction or plays a later role in maintenance and/or proliferation of neural crest.
Fz3 expression in Xenopus is mostly restricted to the developing CNS, especially the dorsal neural tube, but also including the developing optic and otic vesicles (Shi et al., 1998)(data not shown). Thus, while this work identifies a role for Xfz3 in neural crest induction, possible additional roles are not addressed here. Indeed, Xfz3 appears to play a role in the early development of the eye, as overexpression of Xfz3 can lead to ectopic formation of complete eyes and antagonists of Xfz3 function, such as Nfz3, block endogenous eye formation (Rasmussen et al., 2001).
In this work, we have identified a synergistic interaction between Xwnt1 and Xfz3, which are co-expressed in the dorsal neural tube. Xfz3 is specifically required for Xwnt1-mediated neural crest induction in explants. Furthermore, Xfz3 is required for the formation of neural crest in the developing vertebrate embryo. Finally, this work suggests a functional interaction between endogenous Wnt and frizzled family members in the developing vertebrate embryo.
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ACKNOWLEDGMENTS |
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