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Address correspondence to Randall T. Moon, Dept. of Pharmacology, Campus Box 357750, University of Washington School of Medicine, Seattle, WA 98195. Tel.: (206) 543-1722. Fax: (206) 543-0858. E-mail: rtmoon{at}u.washington.edu
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Abstract |
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Key Words: Dishevelled; PKC; Wnt; calcium; signal transduction
* Abbreviations used in this paper: CamKII, calcium/calmodulin-dependent protein kinase II; Dsh, Dishevelled; Fz, Frizzled; JNK, jun-N-terminal kinase; MO, morpholino; PCP, planar cell polarity; PTX, pertussis toxin; TnIc, cardiac troponin I; wt, wild type.
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Introduction |
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The vertebrate WntCa2+ pathway is stimulated by certain Wnt ligands and Fz receptors and involves the mobilization of intracellular Ca2+ and the activation of the calcium-responsive enzymes PKC and calcium/calmodulin-dependent protein kinase II (CamKII) (for review see Kühl et al., 2000a,b; Huelsken and Behrens, 2002). WntCa2+ activity functions in promoting ventral cell fate and antagonizing dorsal cell fate during early Xenopus development, in regulating gastrulation movements, and in regulating heart development (Kühl et al., 2000a,b; Pandur et al., 2002). The PCP pathway has been studied primarily in Drosophila and involves an array of proteins, two of which are also required for Wntß-catenin signaling: Fz receptors and the cytoplasmic protein Dsh (for reviews see Shulman et al., 1998; Peifer and McEwen, 2002; Wharton, 2003).
The term "noncanonical Wnt signaling" has been used to describe both the WntCa2+ pathway and the PCP pathway, primarily to differentiate either pathway from the Wntß-catenin pathway. In vertebrates, noncanonical Wnt signaling has been shown to be very important for the control of convergent extension movements during gastrulation (for reviews see Smith et al., 2000; Darken et al., 2002; Kühl, 2002; Wallingford et al., 2002; Yamanaka et al., 2002). Regulation of convergent extension by Wnts has been attributed to the activation of the PCP pathway (Smith et al., 2000). However, activation of the WntCa2+ pathway has also been implicated in the regulation of convergent extension movements (Torres et al., 1996; Kühl et al., 2001), and its effectors (Ca2+, PKC, and CamKII) act downstream of Wnts and Fzs that reportedly also activate the PCP pathway (Kühl et al., 2000a; Medina et al., 2000; Winklbauer et al., 2001). Thus, these two vertebrate pathways may overlap in terms of function and, conceivably, mechanisms of signaling.
Dsh plays a dual role in the Wntß-catenin and PCP pathways, and has been termed a molecular switch between the two (for review see Wharton, 2003). Dsh is comprised of different domains, including the DEP, PDZ, and DIX domains (Fig. 1 h), and analysis of Dsh deletion constructs and Drosophila genetics have been used to separate the role of Dsh in the Wntß-catenin pathway versus the PCP pathway (Axelrod et al., 1998; Boutros et al., 1998; Penton et al., 2002). That the PCP pathway is downstream of Wnt signaling in vertebrates was first suggested when Dsh was found to affect Wnt and Fz function during gastrulation in zebrafish (Heisenberg et al., 2000) and Xenopus (Tada and Smith, 2000; Wallingford et al., 2000). One construct, DshDIX, which is not competent to activate the Wntß-catenin pathway but can signal via the PCP pathway, has been found to rescue loss of Wnt-11 function during gastrulation in Xenopus (Tada and Smith, 2000) and zebrafish (Heisenberg et al., 2000). Finally, data from PCP signaling in flies suggest that activation of the small GTPase Rho and jun-N-terminal kinase (JNK) lies downstream of Dsh (for review see Adler and Lee, 2001). In vertebrates, activation of Rho by Wnts and Fz also involves Dsh, and the protein Daam1, which links Dsh to Rho (Habas et al., 2001).
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Results |
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Heterotrimeric G proteins have been shown to be downstream of Wnt- and Fz-mediated activation of the Wntß-catenin and WntCa2+ pathways (Slusarski et al., 1997a,b; Sheldahl et al., 1999; Kühl et al., 2000a; Liu et al., 2001), but the hierarchy between G proteins and Dsh remains unclear. The A protomer subunit of pertussis toxin (PTX) catalyzes the ADP ribosylation of specific G protein subunits of the Gi family and prevents the interaction of the receptor and G protein (Gilman, 1987). Co-expression of the A protomer of PTX partially inhibits the rat Fz-2 (Rfz2)mediated membrane translocation of XPKC (red signal in Fig. 1 f, compared with Rfz2 alone, e; Sheldahl et al., 1999), yet does not block activation of XPKC by XDsh
DIX (red signal in Fig. 1 g, compared with d).
XDshDIX activates intracellular calcium flux in a PTX-insensitive manner
We have previously demonstrated that misexpression of both XWnt-5A and Rfz2 stimulates an increase in intracellular Ca2+ release in zebrafish embryos (Slusarski et al., 1997a,b). We therefore asked whether XDsh and XDshDIX would also stimulate Ca2+ flux in this in vivo assay. Injection of RNA encoding wt XDsh is sufficient to stimulate a mild increase in Ca2+ release (Fig. 2 c), above levels observed in uninjected embryos (Fig. 2 a), in the region of the embryo injected with RNA and Texas red lineage tracer (Fig. 2 d, n = 6). By comparison, misexpression of XDsh
DIX (Fig. 2 e) stimulates Ca2+ release well above endogenous levels, as further demonstrated by the localized distribution of the RNA and Texas red lineage tracer (Fig. 2 f, n = 11) spatially overlapping the localized elevated Ca2+ flux (Fig. 2 e).
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XDshDIX activates CamKII in a PTX-insensitive manner
As a third independent assay for the activation of the WntCa2+ pathway, we monitored activation of CamKII (Kühl et al., 2000a). Injection of XDshDIX or Rfz2 RNA at the two-cell stage resulted in a twofold activation of CamKII activity in Xenopus embryos at stage 7 before the onset of zygotic transcription (Fig. 3). Consistent with the observed increase in Ca2+ fluxes, injection of RNA encoding wt XDsh slightly increases CamKII activity. Whereas activation of CamKII by Rfz2 was sensitive to the A protomer of PTX, the effect of XDsh
DIX was insensitive to this treatment (Fig. 3). In summary, our data reveal that XDsh
DIX is able to activate three effectors of the WntCa2+ pathway, Ca2+, PKC, and CamKII, in a PTX-insensitive manner.
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As an independent method to test whether Dsh function is required for WntCa2+ signaling to activate PKC, we then asked whether decreasing levels of endogenous XDsh would decrease translocation to the membrane of ectopic XPKC. To accomplish this, we injected an antisense morpholino (MO) oligonucleotide, designed to hybridize with the 5' untranslated region of XDsh mRNA and inhibit its translation (for review see Heasman, 2002). We found that this MO (XDshMO), when injected into fertilized Xenopus eggs, was capable of reducing levels of endogenous XDsh protein in animal caps explanted from stage-8 embryos, without affecting the levels of a control protein (Fig. 5 e). Furthermore, translocation of ectopic XPKC to the membrane in response to Xfz7 was largely inhibited by this MO (Fig. 5 b, compared with control, a, shows reduced membrane localization of XPKC and elevated cytoplasmic XPKC). When a control MO was injected, membrane translocation of XPKC induced by Xfz7 was still observed (Fig. 5 c, compared with control, a). Coinjection of XDshMO with XDshHA RNA, which does not contain the 5' untranslated region targeted by XDshMO, rescues the ability of Xfz7 to activate XPKC translocation to the membrane (Fig. 5 d, compared with b and a). Injection of this RNA also restores levels of Dsh to those found in control MO-injected animal caps (Fig. 5 e). Injection of XDshMO did not block membrane translocation of XPKC by the pharmacological PKC activator PMA (unpublished data), suggesting that the MO does not act upon XPKC directly.
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As Wnt-11mediated activation of noncanonical Wnt signaling through PKC and JNK is required for heart development in Xenopus (Pandur et al., 2002), we investigated whether injection of XDshMO into eight-cell embryos leads to heart defects, as monitored by reduced or missing contracting tissue. Indeed, after injecting XDshMO into early Xenopus embryos, nearly 86% of embryos had a reduced or missing heart, compared with 20% of embryos injected with a control MO (Fig. 6, a and b). Interestingly, nearly 30% of XDshMO-injected embryos completely lacked contracting tissue, whereas this phenotype was not observed in embryos injected with the control MO. We confirmed the reduced heart phenotype independently by analyzing the expression of the cardiac marker gene cardiac troponin I (TnIc) by whole mount in situ hybridization. Indeed, XDshMO-injected embryos showed a strong reduction in TnIc staining (Fig. 6 c; 64% of XDshMO-injected embryos, n = 53, vs. 16% of control MO-injected embryos, n = 50). These data indicate that disrupting the function of XDsh interferes with normal heart formation, though it is unknown at present whether XDsh is required within the developing heart or in noncardiac cells. In addition, embryos injected with XDshMO display a penetrant moderate to weak shortening of the axis, suggestive of an effect on convergent extension movements of gastrulation (unpublished data).
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Discussion |
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What can one conclude from the observation that a known activator of PCP signaling, XDshDIX, also activates three effectors of WntCa2+ signaling? Moreover, what is the relevance of finding that interfering with the function of Dsh reduces the activity of at least one of these effectors, PKC? Answers to these questions, based on the available literature, suggest that either Dsh functions in three vertebrate WntFz signaling pathways, i.e., the Wntß-catenin, PCP, and WntCa2+ pathways, or that the PCP and WntCa2+ pathways are overlapping. We favor this latter possibility because in Xenopus and zebrafish embryos, both WntCa2+ signaling and vertebrate orthologues of the Drosophila PCP pathway modulate convergent extension movements during gastrulation. Moreover, a mutant form of XDsh lacking the DIX domain activates both the PCP and WntCa2+ pathways. Further supporting the overlap of these pathways, zebrafish Prickle, a component of the PCP pathway, stimulates Ca2+ signaling in embryos, albeit not as rapidly as some Wnts or Fzs (Veeman et al., 2003). That the PCP and WntCa2+ pathways were discovered independently, but may overlap mechanistically, is likely attributable to their being initially described by genetic versus nongenetic approaches, and in different organisms.
Besides asking whether an activator of PCP signaling can activate the WntCa2+ pathway, it is worth considering the reciprocal question of whether effectors of the WntCa2+ pathway are known to be involved in PCP signaling. XDsh has been reported to transduce Wnt-11/Fz7 signals to affect convergent extension movements during vertebrate gastrulation, perhaps via activation of the PCP pathway (Djiane et al., 2000; Heisenberg et al., 2000; Tada and Smith, 2000). As Wnt-11 and Fz7 have both been shown to activate the WntCa2+ pathway (Kühl et al., 2000a; Medina et al., 2000) and we have shown here that XDsh is both necessary and sufficient for activation of PKC and other effectors of the WntCa2+ pathway, this raises the question as to whether at least PKC is a part of the PCP pathway. Supportingly, in the present study, we found that a mutant form of Dsh that should inhibit the PCP pathway, XDEPGFP, at least partially inhibits Fz-mediated activation of ectopic PKC. Interestingly, both Dsh and PKC are required downstream of Drosophila Wnt-4 and Fz2 to regulate focal adhesion contacts during ovarian morphogenesis (Cohen et al., 2002), though this may not involve the PCP pathway. PKC activity is also required for the ability of Dsh to regulate amyloid precursor protein processing (Mudher et al., 2001), which at least further links Dsh with PKC. During Xenopus tissue separation, however, activation of PKC has been reported to be independent of XDsh (Winklbauer et al., 2001), and we have found that the Dsh1 mutation, which prevents activation of PCP signaling by Dsh in Drosophila, does not prevent XDshDIX from relocalizing XPKC in Xenopus (unpublished data).
The GTPase Cdc42 has a potential, though unresolved, role in PCP signaling and a more apparent role in cell polarity. In regulation of cell polarity, it is interesting that Cdc42 acts through an atypical PKC complex (Etienne-Manneville and Hall, 2003). It is also interesting that vertebrate Cdc42 may be regulated by PKC and the WntCa2+ pathway (Choi and Han, 2002). In both flies and vertebrates, activation of the PCP pathway by Dsh leads to activation of JNK (for review see Adler and Lee, 2001), and Cdc42 is a known activator of JNK (Levi et al., 1998). It is important to note, however, that activation of JNK by Dsh can occur independently of Cdc42 (Li et al., 1999).
Our data show that the WntCa2+ pathway, like other Fz-mediated pathways, involves Dsh. Whether the WntCa2+ and PCP pathways substantially overlap will still require further study, as will elucidating the mechanisms by which Dsh promotes Ca2+ release and activation of PKC and CamKII.
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Materials and methods |
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Western blot analysis of PKC and Dsh levels
Xenopus embryos were injected with RNAs or MO, as described, and cultured to stage 8. 20 animal caps from injected embryos were cut and lysed in 20 µl extraction buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100, 50 µM NaF, 10 µg/ml PMSF). Lysates were separated by SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes. The membranes were blocked in TBS with 0.1% Tween 20 and 5% dried milk, probed with anti-myc antibody (1:250 dilution) or antiDvl-1 (1:1,000 dilution), followed by an HRP-conjugated secondary antibody (1:5,000 dilution), and exposed using the ECL Western blotting detection system (Amersham Biosciences).
MO injections into Xenopus embryos
XDshMO oligonucleotide was obtained from Gene Tools, LLC and resuspended in sterile water. The sequences are as follows: XDshMO, 5'-TCACTTTAGTCTCCGCCATTCTGCG-3' (sequence corresponding to the start codon of XDsh is underlined); control MO, 5'- CTAAACTTGTGGTTCTGGCGGATA-3'. For biochemistry and cell biology experiments, 48 ng MO oligonucleotide was injected into one-cell Xenopus embryos plus 0.51 ng Xfz7, 0.51 ng PKCmyc, and either 12 ng XDshGFP or 12 ng GFP RNA. For phenotypic analysis of XDshMO, a total of 50 ng was injected into all four of the vegetal blastomeres of eight-cell Xenopus embryos.
PKC activity assays
Two- to four-cell embryos were injected as described in MO injections. 20 stage animal caps from stage-8 embryos were homogenized in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100, 50 µM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin) and assayed for activity using the following reagents: [32P]-ATP (NEN Life Science Products) and a biotinylated substrate peptide (Promega), phosphatidyl-L-serine (Avanti Polar Lipids, Inc.) as a cofactor, and GF109203X (BIOMOL Research Laboratories, Inc.) as a specific inhibitor. Standard assay conditions were as follows: 20 mM Tris, pH 7.5, 10 µM ATP, 20 mM MgCl2, 200 mM free Ca2+ (calculated using the CHELATE computer program), 100 µM biotinylated peptide substrate, 0.3% mixed micelles with 3% Triton X-100 and 50 µg/ml phosphatidyl-L-serine, 1 µM autoinhibitory peptide, and 10 µM KN93 (both CamKII inhibitors) for a final volume of 25 µl incubated for 10 min at 30°C and then spotted onto SAM2 Biotin Capture Membranes (Promega). [32P]
-ATP not incorporated into substrate peptide was washed away before counting membranes in a scintillation counter. Samples were run in duplicate for each experiment.
In situ hybridization
Whole mount in situ hybridization with TnIc cDNA (provided by P. Krieg, University of Arizona, Tucson, AZ) was performed using a standard protocol (Drysdale et al., 1994).
CamKII activity assays
Embryos were injected at the two-cell stage with RNAs encoding Rfz2 (1 ng), XDshGFP (0.75 ng), XDshDIXGFP (0.75 ng), or PTX A protomer (0.45 ng) and then processed for an in vitro CamKII activity assay as previously described (Kühl et al., 2000a).
Calcium flux measurements in zebrafish embryos
Zebrafish embryos were microinjected with a pressure injector with RNA mixed with Fura-2conjugated dextran (10,000 Mr; Molecular Probes) at the one-cell stage. RNA solutions included 1040 pg wt Dsh or Dsh
DIX, and 510 pg of A protomer, as previously described (Slusarski et al., 1997a). The Dsh constructs were cleaved with ClaI to remove the GFP domain before in vitro transcription to prevent interference with the Fura channels. For experiments with unilateral expression, Fura-injected embryos were injected again at the 816-cell stage with RNA mixed with lineage marker Texas reddextran.
Injected embryos were transferred to a coverslip bottomed heated chamber on an inverted epifluorescent microscope. Embryos with equal fluorescence intensity were selected for analysis and oriented in a lateral position. Image pairs were collected at 340- and 380-nm excitation wavelength (510-nm emission) at 15-s intervals. The ratio image, a pixel by pixel match of both excitation wavelengths, was calculated by computer software (RatioTool; Inovision), and the sequence of ratio images was processed. Ca2+ fluxes (transients) were determined by a subtractive analogue, as previously described (Slusarski and Corces, 2000), compiled for the duration of the time course (5075 min), and represented as a two-dimensional topographical image. The spatial distribution of the Ca2+ transients was mapped along the surface of the embryo. The peak height and color represent the total number of transients that occurred in that location, where a low peak, purple color, represents a small number (1) and a high peak, red color, represents a high number (25) of Ca2+ transients. When needed, Texas red distribution was collected.
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Acknowledgments |
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Submitted: 20 November 2002
Revised: 8 April 2003
Accepted: 8 April 2003
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References |
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