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This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Dishevelled (Dsh) is a key component of multiple signaling pathways that are initiated by Wnt secreted ligands and Frizzled receptors during embryonic development. Although Dsh has been detected in a number of cellular compartments, the importance of its subcellular distribution for signaling remains to be determined.
We report that Dsh protein accumulates in cell nuclei when Xenopus embryonic explants or mammalian cells are incubated with inhibitors of nuclear export or when a specific nuclear-export signal (NES) in Dsh is disrupted by mutagenesis. Dsh protein with a mutated NES, while predominantly nuclear, remains fully active in its ability to stimulate canonical Wnt signaling. Conversely, point mutations in conserved amino-acid residues that are essential for the nuclear localization of Dsh impair the ability of Dsh to activate downstream targets of Wnt signaling. When these conserved residues of Dsh are replaced with an unrelated SV40 nuclear localization signal, full Dsh activity is restored. Consistent with a signaling function for Dsh in the nucleus, treatment of cultured mammalian cells with medium containing Wnt3a results in nuclear accumulation of endogenous Dsh protein.
These findings suggest that nuclear localization of Dsh is required for its function in the canonical Wnt/β-catenin signaling pathway. We discuss the relevance of these findings to existing models of Wnt signal transduction to the nucleus.
The specification of cell fates during embryonic development frequently depends on inductive interactions, which involve transmission of extracellular signals from the cell surface to the nucleus. In the transforming growth factor β (TGFβ) signal transduction pathway, Smad proteins that are initially associated with TGFβ receptors move to the nucleus to regulate target genes . Another example of a direct link between the cell surface and the nucleus during embryonic development is the proteolytic cleavage and nuclear translocation of the cytoplasmic fragment of the Notch receptor . In contrast, multiple steps appear to be required for a Wnt signal to reach the nucleus. In this molecular pathway, signals from Frizzled receptors are transduced to Dishevelled (Dsh), followed by inactivation of the β-catenin degradation complex that includes the adenomatous polyposis coli protein (APC), Axin and glycogen synthase kinase 3 (GSK3) [3,4]. Stabilization of β-catenin is thought to promote its association with members of the T-cell factor (Tcf) transcription factor family in the nucleus, resulting in the activation of target genes [5,6]. As well as the canonical β-catenin-dependent pathway, Frizzled receptors also activate small GTPases of the Rho family, protein kinase C and Jun-N-terminal kinases (JNKs) to regulate planar cell polarity in Drosophila and convergent extension cell movements and tissue separation in Xenopus [7-12]. Thus, the Wnt/Frizzled pathway serves as a model for molecular target selection during signal transduction.
Dsh is a common intracellular mediator of several pathways activated by Frizzled receptors and is composed of three conserved regions that are known as the DIX, PDZ and DEP domains . Different domains of Dsh are engaged in specific interactions with different proteins, thereby leading to distinct signaling outcomes . Daam, a formin-related protein, promotes RhoA activation by Dsh , whereas Frodo, another Dsh-binding protein, is required for Wnt/β-catenin signaling in the nucleus . These interactions may take place in various cellular compartments, linking specific activities of Dsh to its distribution inside the cell. Dsh is found in a complex with microtubules and with the actin cytoskeleton [15-17]. Dsh is also associated with cytoplasmic lipid vesicles, and this localization was shown to require the DIX domain [7,16,18]. Overexpressed Frizzled receptors can recruit Dsh to the cell membrane in Xenopus ectoderm, and this redistribution requires the DEP domain [7,18,19]. The DIX domain is essential for the Wnt/β-catenin pathway, whereas the DEP domain plays a role in the planar cell polarity pathway [7,8,16,18,20,21]. Thus, the specific subcellular localization of Dsh may be crucial for local signaling events.
The current study was based on our initial observation that a Dsh construct lacking the carboxy-terminal DEP domain was found in cell nuclei. We have now identified a nuclear export signal in the deleted region and also discovered that Dsh proteins accumulate in the nuclei of Xenopus ectodermal cells and mammalian cells upon inhibition of nuclear export. Dsh also accumulated in the nuclei after stimulation of mammalian cells with Wnt3a-containing culture medium. By analyzing various mutant Dsh constructs in Xenopus ectoderm, we show that the signals responsible for Dsh nuclear localization reside in a novel domain and that the nuclear translocation of Dsh is essential for its ability to activate Wnt/β-catenin signaling.
We studied the subcellular distribution of fusions of Dsh with green fluorescent protein (GFP) in Xenopus ectodermal cells. In contrast to Dsh-GFP, which is localized in punctate structures within the cytoplasm [7,18], the Ds2 construct, lacking the carboxy-terminal region, accumulates in the nucleus (Figure 1a–c), indicating that the carboxyl terminus contains sequences for nuclear export. Indeed, we found a potential leucine-rich nuclear export signal (NES) in Dsh at positions 510–515, corresponding to the conserved consensus M/LxxLxL (single letter amino-acid code, where x is any amino acid) [22,23]. When leucines 513 and 515 in this putative NES were substituted with alanines, the mutated Dsh fusion construct, DsNESm, was localized predominantly in the nucleus (Figure 1a,d), demonstrating that the sequence is a functional nuclear export signal.
To examine whether inhibition of nuclear export abrogates Dsh activity, we compared the abilities of DsNESm and wild-type Dsh-GFP to induce secondary axes in frog embryos. Although the molecular mechanism operating during axis induction remains to be elucidated, this assay faithfully reflects the biological activity of Dsh in the canonical Wnt/β-catenin pathway [14,16,18,24]. DsNESm, which was expressed at similar levels to the wild-type Dsh-GFP (data not shown), induced secondary axes at least as efficiently as Dsh-GFP (Table (Table1).1). Induced axes contained pronounced head structures with eyes and cement glands (Figure 1e–g). These results suggest that Dsh may function in the nucleus to trigger dorsal axial development.
Accumulation of DsNESm in the nucleus implies that the wild-type Dsh shuttles between the nucleus and the cytoplasm. We therefore studied the subcellular distribution of Dsh in Xenopus embryonic cells under conditions in which nuclear export is blocked. When ectodermal cells expressing Dsh-GFP were incubated with N-ethylmaleimide (NEM), an inhibitor of the nuclear export receptor CRM1/exportin [25,26], Dsh-GFP was detected predominantly in the nucleus, compared to the punctate cytoplasmic pattern of Dsh-GFP in untreated cells (Figure 2a,b). This effect was specific to full-length Dsh-GFP, as Ds3, a Dsh construct that lacks 48 amino acids adjacent to the PDZ domain (Figure (Figure1a),1a), did not accumulate in the nucleus after NEM treatment (Figure 2e,f). The nuclear retention of Dsh-GFP was also observed using leptomycin B (LMB), another inhibitor of CRM1-dependent nuclear export [22,23] (Figure 2c,d). These results indicate that Dsh shuttles between the cytoplasm and the nucleus, and that its abundance in the cytoplasm is due to highly efficient nuclear export.
To ensure that the Dsh-GFP fusion behaves similarly to the endogenous Dsh protein, we examined the localization of endogenous Dvl2, a mammalian homolog of Dsh, in human and rat tissue culture cells. Human embryonic kidney (HEK) 293 cells treated with LMB accumulated Dvl2 in the nucleus, contrasting with the cytoplasmic localization of Dvl2 in untreated cells (Figure 3a–c). We also evaluated the subcellular localization of endogenous Dvl2 in Rat-1 fibroblasts, which are known to respond to Wnt signaling. Fractionation of cells into nuclear and cytoplasmic protein pools revealed only a small amount of endogenous Dvl2 in intact nuclei, whereas after NEM treatment, Dvl2 was localized predominantly in the nuclear fraction (Figure (Figure3d).3d). The efficiency of subcellular fractionation was controlled for by staining with antibodies to glyceraldehyde phosphate dehydrogenase (GAPDH) and nuclear lamins. These proteins remained exclusively cytoplasmic or nuclear, respectively, in both untreated and NEM-treated cells (Figure (Figure3d).3d). Thus, our data show that Dsh translocates into the nucleus and is actively exported into the cytoplasm of both Xenopus ectodermal cells and mammalian fibroblasts.
To identify specific amino-acid sequences that direct the transport of Dsh to the nucleus, we studied the subcellular distribution of mutated Dsh-GFP fusion constructs (Figure (Figure4a).4a). The removal of the DIX and PDZ domains (Ds1) did not eliminate nuclear translocation in response to NEM or LMB (Figure 4a–d), indicating that these two domains are not required for the nuclear import. Similarly, the DEP domain is not required for Dsh nuclear localization (Ds2; Figure 1a,c). Comparison of Ds1 and Ds2 (see Figure Figure4a),4a), both capable of nuclear localization, reveals a short stretch of shared amino acids located between the PDZ and DEP domains. Strikingly, the removal of just this 48 amino-acid region abrogated nuclear import of Dsh in the presence of NEM or LMB (Ds3; Figures 2e,f and and4a).4a). Together these experiments identify amino acids 333–381 as the region required for nuclear localization of Dsh.
Although this short sequence is highly conserved in all Dsh homologs from Hydra to humans (Figure (Figure4j),4j), it does not bear detectable similarity to nuclear localization signals characterized in other proteins . This sequence may interact directly with components of the nuclear import machinery or bind to a protein that itself binds a karyopherin/importin and mediates the nuclear import of Dsh by a piggyback mechanism. Interestingly, this region overlaps a novel proline-rich domain identified by mutational analysis of Dsh in Drosophila . To define further the specific amino acids necessary for nuclear localization, a panel of Dsh constructs with point mutations spanning the conserved region was examined (data not shown). Nuclear import was eliminated with the substitution of three amino acids, converting IVLT into AVGA (DsNLSm; Figure 4a,e–g,j), indicating that these three amino acids are critical.
To determine whether nuclear localization of Dsh is required for its activity, we compared the abilities of DsNLSm and wild-type Dsh to induce secondary axes in frog embryos. We also assessed activation of a luciferase reporter construct for Siamois , an immediate target of Wnt/β-catenin signaling. DsNLSm had impaired ability to induce secondary axes and to activate the Siamois reporter when compared with wild-type Dsh (Figure 5a,b; Table Table1).1). Furthermore, DsNLSm failed to stabilize β-catenin (Figure (Figure5c).5c). This difference was not due to differences in protein expression, as both constructs were present in embryo lysates at similar levels (Figure (Figure5c).5c). Thus, these findings indicate that the nuclear localization of Dsh is critical for its functional activity in the β-catenin pathway.
Not only was the function of DsNLSm in the β-catenin pathway impaired, but we found that this construct behaved as a dominant inhibitor of Wnt signaling and prevented the activation of the Siamois reporter by Xwnt3a and Xwnt8 RNAs (Figure 6a,b). Consistent with these observations, another construct lacking the region responsible for the nuclear localization (Ds3; see Figure Figure4a)4a) also suppressed Wnt signaling (Figure (Figure6b).6b). Despite these inhibitory properties, dorsally injected DsNLSm RNA, like Xdd1, a dominant negative deletion mutant , did not suppress primary axis formation (data not shown).
Impaired activity of the DsNLSm construct may be due to its inability to translocate to the nucleus, or due to a coincidental elimination of a binding site for an essential cofactor that functions together with Dsh in the cytoplasm. To exclude the latter possibility, the IVLT sequence of Dsh NLS was replaced with KKKRK, an unrelated NLS from SV40 T antigen . This construct, DsSNLS, relocated to the nucleus even in the absence of nuclear export inhibitors (Figure 4a,i). Notably, all activities of wild-type Dsh, including induction of complete secondary axes, activation of the Siamois promoter and β-catenin stabilization were significantly restored in DsSNLS (Figure 5a–c; Table Table1).1). In contrast to DsNLSm, DsSNLS did not inhibit the ability of Wnt ligands to activate pSia-Luc (Figure (Figure6b),6b), consistent with its being a positive regulator of the Wnt pathway. We note that the signaling activity of DsSNLS was not enhanced compared to wild-type Dsh, suggesting that the rate of the nuclear translocation of Dsh rather than its steady state levels in the nucleus is critical for target gene activation. It is also possible that other nuclear components, rather than Dsh, become rate-limiting for signaling. Overall, the simplest interpretation of our data is that the nuclear import of Dsh is essential for its activity.
We next examined the ability of DsNLSm to bind critical Wnt signaling components, such as casein kinase 1ε (CK1ε), a positive regulator of the β-catenin pathway [30,31], or Axin, a negative regulator [20,32-36], both of which are known to bind Dsh. Both DsSNLS, enriched in the nucleus, and DsNLSm and Ds3, which do not enter the nucleus, bound CK1ε and XARP, a Xenopus Axin-related protein  (Figure (Figure7).7). Thus, these mutated Dsh constructs retain the ability to associate with critical components of the Wnt/β-catenin pathway, arguing that defective nuclear translocation of DsNLSm is likely to be responsible for its inability to activate β-catenin signaling.
Besides the β-catenin pathway, Dsh also functions in a planar cell polarity (PCP) pathway, which involves Rho GTPase and JNK and controls morphogenetic movements in early embryos [8,9,37-39]. We asked whether mutations in DsNLSm influence the β-catenin pathway exclusively or affect the PCP pathway as well. First, we observed that both Dsh-GFP and DsNLSm-GFP were efficiently recruited to the cell membrane by overexpressed Xfz8, a Frizzled family member  (Figure (Figure8a).8a). As Dsh relocalization to the cell membrane in response to Frizzled is associated with its ability to signal in the PCP pathway [7,8], this observation suggests that DsNLSm can respond to Frizzled signaling independent of β-catenin.
In Xenopus, the PCP pathway involving Dsh is implicated in the control of convergent extension movements [24,41,42]. Overexpression of the Xdd1 deletion mutant leads to the development of short embryos when expressed in dorsal marginal cells (; Figure Figure8b).8b). Severe convergent extension defects (Figure (Figure8b)8b) were observed in 22%, and mild defects were observed in 28% of the embryos injected with Xdd1 RNA (N = 35). In contrast, only mild morphogenetic defects were observed in embryos coinjected with Dsh (15%; N = 40) or DsNLSm RNA (18%; N = 39), indicating that both Dsh and DsNLSm partially rescued the effect of Xdd1. This indicates that DsNLSm is active in noncanonical PCP-like signaling. We also examined whether DsNLSm activates c-Jun N-terminal kinase (JNK), which is thought to function downstream of Dsh in the PCP pathway [8,37-39]. Both DsNLSm and Dsh activated JNK at equivalent levels (Figure (Figure8c),8c), suggesting that nuclear localization of Dsh is not required for its function in noncanonical signaling.
Our findings are consistent with a scenario in which Wnt signaling may cause nuclear translocation of Dsh followed by formation of a stable β-catenin/Tcf3 complex and transcriptional activation of target genes. In support of this hypothesis, Dsh was reported to move to the nucleus in response to Wnt signaling in primary embryonic kidney cells . In Rat-1 cells, we did not detect a significant change in Dsh distribution in response to Wnt signals (data not shown), possibly due to highly efficient nuclear export of Dsh in these cells. But immunofluorescence staining for Dvl2 revealed the nuclear accumulation of the protein in HEK293 and MCF7 cells after 3–6 h stimulation with Wnt3a-containing medium (Figure (Figure9a,9a, and data not shown). The effect was quantified by measuring nuclear to cytoplasmic (N/C) ratios of fluorescence intensity. The N/C ratio averaged 28% after 6 h treatment with the control medium, but increased to 91% after stimulation with Wnt3a-conditioned medium (Figure (Figure9b).9b). These observations are consistent with the view that Dsh regulates Wnt-dependent gene targets in the nucleus.
In the current view, Wnt signaling causes inactivation of the β-catenin degradation complex, leading to stabilization and nuclear translocation of β-catenin . Given that Dsh is genetically upstream of the β-catenin degradation complex [3,4] and that β-catenin degradation is thought to occur in the cytoplasm , Dsh nuclear import is unexpected. Nevertheless, our data demonstrate that Dsh shuttles between the cytoplasm and the nucleus and that its presence in the nucleus is critical for signaling. One explanation of these results is that β-catenin degradation may occur in the nucleus. Consistent with this possibility, APC, Axin and GSK3, components of the β-catenin degradation complex, have also recently been found to shuttle between the cytoplasm and the nucleus [22,23,44-47]. Moreover, Frat/GBP, a positive regulator of β-catenin, has been reported to expel GSK3 from the nucleus . We show that the ability of Dsh constructs to enter the nucleus correlates with their ability to stabilize β-catenin (Figure (Figure5c).5c). These observations indicate that Wnt/β-catenin signaling may depend on the nuclear localization of pathway components.
Alternatively, nuclear localization of Dsh may affect β-catenin stability indirectly, by regulating protein interactions that sequester β-catenin in the nucleus, thereby preventing its cytoplasmic degradation . Although we did not detect a significant change in nuclear import of β-catenin-GFP in Xenopus ectoderm cells overexpressing Dsh (data not shown), this process may be cell-context-dependent. On the other hand, we recently showed that Frodo, a nuclear Dsh-interacting protein, associates with Tcf3 and influences Tcf3-dependent transcription . It is thus possible that Frodo links Tcf3 and Dsh to regulate Wnt target genes. Future studies should examine molecular components critical for the nuclear function of Dsh.
GFP-tagged Dsh constructs were all derived from the DshGFP-RN3 plasmid that encodes the Xdsh protein fused at amino acid 724 to the first amino acid of GFP (Figures (Figures1a,1a, ,4a).4a). Ds1 lacks the first 332 amino-terminal amino acids. Ds2 is the carboxy-terminal deletion of Xdsh, starting with amino acid 383. Ds3 lacks amino acids 334–381. In DsNLSm, the IVLT residues at positions 334–337 were replaced with AVGA, whereas in DsSNLS the same region is replaced with KKKRK, the SV40 T antigen NLS . In DsNESm, L513 and L515 were substituted for alanines.
To generate these constructs, DshGFP-pRN3 was used as a template. The in-frame deletion in Ds3 was made by PCR. Other GFP fusion constructs were synthesized with specific primers and PfuI DNA polymerase followed by DpnI digestion of the template . The following primers were used: 5'-GTCCATAAACCGGGGCCCGCAGTCGGCGCCGTGGCCAAATGCTGG-3' for DsNLSm; 5'-ACACTAGGCCGCAGAATGCCCATTGTCCTGACCGTG-3' for Ds1; 5'-TCCATAAACCGGGGCCAAAGAAGAAGCGAAAGGTGGCCAAATGCTGGGA-3' for DsSNLS; 5'-TTCCCAGTGTACCCCGGGGCCATGGTGAGCAAGGGC-3' for Ds2, and 5'-GAGAACTATGACCAACGCTAGCGCGAATGACAACGATGGAT-3' for DsNESm. All constructs were verified by sequencing. Myc-tagged Dsh mutant constructs were made by replacing mutated regions with corresponding regions of Myc-Dsh . Cloning details are available as an Additional data file with the online version of this article.
In vitro fertilization, culture and microinjections of Xenopus eggs were essentially as described previously . Stages were determined according to Nieuwkoop and Faber . Axis induction was carried out by injecting mRNAs encoding different Dsh constructs (1 ng) into a single vegetal ventral blastomere at the 4–8-cell stage and assessed when the injected embryos reached stage 36–40. To monitor axis extension defects, 0.6 ng of Xdd1 RNA was injected alone or with 2 ng of Dsh or DsNLSm RNA into two dorsovegetal blastomeres of 4-cell embryos and the injected embryos were allowed to develop until sibling embryos reached stage 32.
For subcellular localization of Dsh-GFP constructs, mRNAs were injected into the animal pole region of 2–4-cell embryos. Animal cap explants were dissected at stages 9–10.5, incubated for 60 min in 10 mM N-ethylmaleimide (NEM; Sigma, St Louis USA) in 0.8 × MMR (Marc's Modified Ringer's solution, 1 × MMR: 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM HEPES, pH 7.4), or in control (0.8 × MMR), then fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30–45 min, washed three times in PBS, and mounted in 70% glycerol, 30% PBS containing 25 mg/ml of diazabicyclo(2,2,2)-octane (Sigma). Leptomycin B was used at 50 ng/ml in low-calcium medium (76 mM NaCl, 1.4 mM KCl, 0.2 mM CaCl2, 0.1 mM MgCl2, 0.5 mM Hepes, 1.2 mM sodium phosphate, (pH 7.5), 0.6 mM NaHCO3 and 0.06 mM EDTA) for one hour prior to fixation. In some experiments, nuclei were stained by addition of 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) to the final PBS wash. For membrane localization studies, Xfz8 RNA was coinjected with RNAs encoding the Dsh constructs in the animal-pole region; animal-cap explants were dissected at stage 9–9.5 and mounted for observation. Fluorescence was visualized using a Zeiss Axiophot microscope.
For luciferase assays, pSiaLuc reporter plasmid (20–40 pg) was coinjected with mRNAs encoding Xwnt3a  or Xwnt8  and different Dsh constructs into one or two animal-ventral blastomeres or into one ventral-vegetal blastomere at the 4–8-cell stage. Luciferase activity was measured as described .
Rat-1 fibroblasts, human embryonic kidney (HEK) 293 cells and MCF7 human breast carcinoma cells were cultured in 1 × Dulbecco's Modified Eagle Medium (DMEM; Gibco/Invitro-gen, Carlsbad, USA) supplemented with 10% fetal calf serum and 5 μg/ml gentamicin. Conditioned medium was prepared from L cells stably transfected with Wnt3a as described , with the medium from untransfected L cells serving as a control.
For immunocytochemistry, HEK293 cells were treated with 50 ng/ml LMB for 14 h while MCF7 cells were treated with Wnt3a or control conditioned medium for 1, 3, 6 or 8 h. Cells were fixed with 4% paraformaldehyde, immersed in methanol, and incubated with anti-Dvl2 antibodies and then Cy3-conjugated anti-rabbit IgG. Nuclei were stained by addition of 1 μg/ml DAPI as described for animal-cap cells. Fluorescence was observed under the Zeiss Axiophot microscope; 10–15 cells from each group were randomly picked up for measurement of the nuclear and cytoplasmic staining intensity using Image-Gauge software (Fuji Film, Tokyo, Japan).
For subcellular fractionation, confluent cultures of Rat-1 cells were harvested by scraping plates and resuspended in hypotonic lysis buffer containing 1 mM EGTA, 1 mM EDTA, 2 mM MgCl2, 10 mM KCl, 1 mM DTT, 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 μg/ml pepstatin. Cells were swollen for 30 min, and broken open with 25 strokes in a tight fitting Dounce homogenizer. Lysates were layered into tubes containing 1 M sucrose in hypotonic lysis buffer, and spun at 1600 × g for 10 min. Supernatant remaining above the sucrose cushion was used as the cytoplasmic fraction. The pellet, containing nuclei, was resuspended in an equivalent volume of hypotonic lysis buffer.
Immunoprecipitation and western analysis were carried out with cell and embryo lysates as described . To prepare embryo lysates at stage 10+, four animal blastomeres of 4–8-cell embryos were injected with RNAs encoding different forms of Dsh, ΔRGS-Axin , Flag-β-catenin , CK1ε  and HA-XARP . To generate anti-Xdsh polyclonal antibodies, rabbits were immunized with a carboxy-terminal half of Xdsh (amino acids 301–736) fused to GST. First, GST beads were used for purification of anti-GST antibodies. Subsequently anti-Xdsh antibodies were affinity-purified on GST-Xdsh (301–736) beads. Polyclonal anti-Dvl2 antibody was generated in rabbits and affinity-purified on PVDF membrane blotted with human Dvl2 (79–249) . A small aliquot of anti-human Dvl2 was obtained from M. Snyder (Yale University, New Haven, USA). Anti-GAPDH antibody was a gift from A. Stuart-Tilley and S. Alper (Beth Israel Deaconess Medical Center, Boston, USA), anti-lamin antibody was from F. McKeon (Harvard Medical School, Boston, USA). Anti-β-tubulin antibodies were from BioGenex (San Ramon, USA), anti-Flag M2 antibody was from Sigma and anti-CK1ε antibodies were from BD Biosciences (Palo Alto, USA). Anti-Myc and anti-HA monoclonal antibodies are hybridoma supernatants of 9E10 and 12CA5 cells (Roche Applied Science, Indianapolis, USA).
Four-cell embryos were injected with 4 ng Dsh or DsNLSm RNA into four animal blastomeres. Embryo lysates were prepared at stage 10.5 and in vitro kinase assays were carried out essentially as described , except that phosphorylated c-Jun-GST was detected with anti-phospho-c-Jun-specific antibodies (Cell Signaling Technology, Beverly, USA) by western blotting rather than with autoradiography.
The following is provided as an additional data file with the online version of this article. Additional data file 1, containing cloning details of Dsh mutant constructs.
Cloning details of Dsh mutant constructs
We thank S. Alper, F. McKeon and M. Snyder for antibodies, and X. He, F. Costantini and J. Graff for plasmids, J. Kitajewsky for Rat-1 cells, R. Nusse for L cells transfected with Wnt3a, and J. Martinez, Y. Yoneda and M. Yoshida for leptomycin B. We also thank V. Krupnik and M. Lisovsky for help with the generation of anti-Xdsh and anti-Dvl2 antibodies. We are grateful to J. Green, V. Krupnik, B. Neel, N. Perrimon and members of this laboratory for reading of the manuscript and useful discussions. This work was supported by NIH grants to S.Y.S.