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Address correspondence to Diane C. Slusarski, Dept. of Biological Sciences, 312 Biology Building, University of Iowa, Iowa City, IA 52242. Tel.: (319) 335-3229. Fax: (319) 335-1069. email: diane-slusarski{at}uiowa.edu
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Abstract |
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Key Words: dorsalventral patterning; calcium; zebrafish; morphogenesis; signal transduction
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Introduction |
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The common element of noncanonical Wnt signaling is that this class (including Wnt-5A, -4, and -11) appears to be ß-cateninindependent (Kuhl et al., 2000b). Noncanonical Wnt activity can be viewed as a complex network with several cellular outputs identified by calcium (Ca2+) modulation and polarized cell movement (Wnt/Ca2+ and planar cell polarity [PCP]; Mlodzik, 2002). Stimulation of the WntCa2+ pathway triggers the release of Ca2+ (Slusarski et al., 1997a, b), activating Ca2+ sensitive proteins including PKC (Sheldahl et al., 1999), Ca2+/calmodulin-dependent kinases (CaMKII; Kuhl et al., 2000a) and calcineurin-dependent nuclear factor of activated T cells (Saneyoshi et al., 2002). More recently, a PCP-specific component, Prickle, has been shown to modulate cell movement and stimulate Ca2+ release in zebrafish, and a PCP-specific form of dsh has been shown to activate the Wnt/Ca2+ cascade in Xenopus and zebrafish (Sheldahl et al., 2003; Veeman et al., 2003). This work raises the intriguing possibility that Wnt/Ca2+ and PCP either substantially overlap or are part of the same signaling network.
In zebrafish, overexpression of Xwnt-5A is antagonistic to the Wnt/ß-catenin class in that coinjection of RNA encoding Wnt-8 with Wnt-5A inhibits the dorsalizing effects of Wnt-8 overexpression. Stimulating Ca2+ release with activated serotonin receptor also antagonized Wnt-8 induced expansion of the dorsal domain (Slusarski et al., 1997b), suggesting that Wnt-5 antagonism of Wnt/ß-catenin is downstream of the receptorligand interaction and mediated by Ca2+ release. Consistent with an antagonistic role, expression of antisense Wnt-5A in mammalian cell lines mimics Wnt-1 mediated transformation (Olson and Gibo, 1998). Furthermore, Drosophila Wnt 4 (Dwnt-4) is antagonistic to wg as injection of antisense Dwnt-4 RNA resembles wg gain-of-function mutations and Dwnt-4 antagonizes wg in Xenopus axis-inducing assays (Gieseler et al., 1999; Buratovich et al., 2000). Dwnt-4 also functions in cell movement (Cohen et al., 2002). Misexpression of zebrafish Wnt-5 and Wnt-4 alters morphogenetic movements (Ungar and Moon, 1995; Slusarski et al., 1997b) and genetic mutations in zebrafish Wnt-5/pipetail (ppt) and Wnt-11/silberblick (slb) have been shown to influence convergence extension movements during gastrulation (Heisenberg et al., 2000; Kilian et al., 2003). Thus, in both vertebrates and invertebrates, noncanonical Wnts have dual functions; they are antagonistic to canonical Wnt signaling and modulate cell movement/polarity.
Although there is genetic and biochemical evidence for how the Wntß-catenin pathway works in both vertebrates and invertebrates, there is little genetic evidence for how noncanonical Wnt signaling pathways work in axis formation in vertebrates. Our paper provides a genetic demonstration of a maternal requirement for a Wnt in vertebrate dorsal-ventral (D-V) patterning. We identify zebrafish Wnt/Ca2+ class members (Wnt-4, -5, and -11) by virtue of their ability to stimulate Ca2+ release and demonstrate genetic interaction between Wnt-5/ppt and Wnt-11/slb. We show that manipulation of Wnt-5 activity by either gain-of-function or loss-of function results in changes in endogenous Ca2+ activity, and we can obtain partial rescue of the Wnt-5/ppt mutant with overexpression of a proposed downstream Ca2+-sensitive protein, Ca2+/calmodulin-dependent kinase (CaMKII). Through loss-of-function analyses, our data demonstrate an increase in ß-catenin accumulation and activation of downstream genes supporting a functional role for Wnt/Ca2+ antagonism of Wnt/ß-catenin activity in vertebrate development.
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Results |
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The slb mutation affects forebrain patterning and homozygous slbtz216 mutant embryos often have incomplete separation of the eyes (Fig. 3 B, arrow pointing to a fused lens) compared with wild-type (Fig. 3 A). Homozygous pptti265 mutant embryos display shortened body length and undulating notochords but most commonly tail defects (deformed tip of tail resembling a pipe; Fig. 3 C, arrow). To test for genetic interaction, we generated adult fish doubly heterozygous for pptti265 and slbtz216 and crossed to obtain doubly homozygous mutant embryos, confirmed by PCR of genomic DNA. The double homozygous mutant embryo phenotype is markedly more severe than the additive of the single mutant combinations. The eye anlagen fuse more frequently in the double mutant embryos than in slb-/- embryos (Fig. 3 D). Double homozygous mutant embryos also have more severe tail and trunk defects than observed in ppt-/- (Fig. 3 D).
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Alterations in cell movement have been described in slb mutant embryos (Heisenberg and Nusslein-Volhard, 1997; Heisenberg et al., 2000) and in embryos homozygous for an allele of ppt that replaces one conserved cysteine (pptta89 ) leaving the rest of the Wnt-5 sequence intact (Heisenberg et al., 2000; Kilian et al., 2003). In addition to the prototypical ppt tail defect (Fig. 3 C), we also observe some epiboly defects in the pptti265 allele genetic background. As the pptti265 is the most severe allele identified thus far, we believe it uncovers additional roles for Wnt-5 in cell movement. Consistent with altered Ca2+ modulation in ppt-/- embryos contributing to the mutant phenotypes is the fact that we reproduce, in part, the phenotypic defects in Ca2+ release suppressed embryos. Late treatments with phosphoinositide cycle inhibitors (after 128256 cell stage) result in anterior brain defects and eye fusions similar to those described for slb (Westfall et al., 2003). We target phosphoinositide cycle turnover with L-690,330 and block Ca2+release from inositol 1,4,5-triphosphate receptor (IP3R) channels with Xestospongin C (XeC; Atack et al., 1993; Gafni et al., 1997). Inhibitor-treated embryos were analyzed for in vivo Ca2+ release dynamics and we selected a dose that approximately mimics the reduced Ca2+ release frequency observed in ppt-/- embryos. In both Ca2+ releaseinhibited (Fig. 3 E) and in ppt-/- (Fig. 3 F) embryos, we observe cell movement defects resulting in split somites encircling the yolk typically fusing back together at the most posterior tip of the tail. We also observe twisted posterior notochord defects, thus, contorting the tail similar to ppt-/- phenotypes and also generated embryos with trunk and tail defects similar to ppt-/-;slb-/- phenotypes (Fig. 3 G).
Maternal depletion of Wnt-5 (ppt) reveals a necessary role in ventral patterning
Strong inhibition of IP3R function with blocking antibodies leads to expanded dorsal structures in Xenopus (Kume et al., 1997). Consistent with the Xenopus studies, inhibition of either IP3R or phosphoinositide cycle turnover results in hyperdorsalization in zebrafish (Westfall et al., 2003). In comparison, we use relatively mild doses of inhibitor (XeC and L-690,330) to match the reduced Ca2+ release frequency in the zygotic ppt-/- embryos. This raises the possibility that there may be residual Wnt-5 in these zygotic ppt-/- embryos. In support of this notion, Wnt-5 transcript is maternally deposited and ubiquitously expressed in zebrafish (Blader et al., 1996) but protein distribution is unknown. Antisense morpholino knockdown of Wnt-5 generates phenotypes similar to the zygotic mutant (Lele et al., 2001). It should be noted that the antisense approach does not eliminate maternal protein. Therefore, in order to determine if maternally supplied Wnt-5 is contributing to early patterning, we set out to generate ppt-/- females by gene product rescue.
Assuming that maternal product would be present in embryos from a heterozygous ppt cross, we injected Wnt-5 DNA (200 pl of 1012 ng/ul) at a concentration that activates mild yet sustained Ca2+ release. We score rescue by suppression of the ppt morphological phenotypes. Injection sets with less than 10% ppt-like phenotypes, compared with 25% phenotypes in clutch controls, were allowed to develop. A small number of the rescued embryos developed swim bladders, a necessary organ for zebrafish viability and were raised to adulthood. Mature ppt-/- females were PCR genotyped.
Consistent with maternal effect mutations, embryos collected from ppt-/- females exhibit phenotypes regardless of the paternal genotype. Embryos from ppt-/- females crossed to heterozygous ppt+/- males fell into two general phenotypic groups. One class, accounting for >50% of the defects, is similar to the tail defects observed in mutant embryos from heterozygous females (Fig. 4 B), however, embryos from ppt-/- females have pointedly more severe defects with extreme shortened axis, undulating notochord and tail defects (Fig. 4 C). This class of phenotypes was scored as ppt-zygoticlike. The other phenotypic group (38%, 193/506) demonstrated dorsalized mutant phenotypes similar to those described in Mullins et al. (1996), as well as axis duplication phenotypes. These include expansion of somites, shortened and twisted tail (piggy-taillike; Fig. 4 D) and severe curling of the tail over the trunk (snail-houselike; Fig. 4 F) instead of straight extension off of the yolk as in wild-type (Fig. 4, A and E). The ppt-/- embryo in Fig. 4 G has a partial secondary axis with an ectopic pair of otic vesicles associated with a duplicated beating heart. Organ duplication was verified in several maternal-zygotic ppt-/- (mzppt) embryos by whole mount in situ with a cardiac specific probe (Fig. 4 I; Chen and Fishman, 1996). Based on morphology, the hyperdorsalized and duplicated axis defect frequency of 38% is consistent, yet lower than the expected 50% frequency for embryos depleted of both maternal and zygotic product. The lower frequency could be the result of a maternal effect that is not fully penetrant or that we were conservative in scoring a phenotype as dorsalized and instead classified it as a severe ppt-zygoticlike defect. To distinguish between these two possibilities, we employed a more sensitive molecular analysis of dorsal patterning.
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Maternal loss of Wnt-5 (ppt) results in ß-catenin stabilization
One mechanism leading to the dorsalization phenotypes described in the previous paragraph is activation of the canonical Wnt path leading to accumulation of ß-catenin protein. Although there is no evidence for a maternal canonical Wnt ligand in zebrafish, there is nuclear accumulation of ß-catenin protein on the future dorsal side (Schneider et al., 1996). To determine if there is a change at the level of ß-catenin, embryos are analyzed for ß-catenin protein distribution. In wild-type embryos, we typically observe nuclear ß-catenin spanning 20% the circumference of the embryo when observed from the animal pole. Fig. 5 A represents one frame of a confocal series at lower magnification with the regions of nuclear ß-catenin localization (dots) confirmed at higher magnification. A representative frame from images collected around the circumference of a wild-type embryo at higher magnification is shown in Fig. 5 C with an arrowhead marking a cell with nuclear ß-catenin. In wild type, we observe an average of 11 ß-catenin-positive nuclei/embryo, (n = 10 embryos). In contrast, ß-catenin-positive nuclei in embryos from ppt-/- females span >50% the embryo circumference, in some embryos, the domains are opposite the putative endogenous dorsal domain (Fig. 5 B, dots). Embryos from ppt-/- females demonstrate a dramatically higher number of cells with nuclear ß-catenin (Average of 48 nuclei/embryo, n = 10 embryos) with a noticeable increase in the overall level of protein (Fig. 5 D, arrows denoting a few of the cells with nuclear ß-catenin). The individual panels of wild type at higher magnification typically have 12 nuclei/frame in the dorsal domain, whereas ppt panels can have upwards of 610 nuclei/frame.
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Discussion |
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Wnt-5 loss-of-function mimics activation of Wnt/ß-catenin signaling most likely by relieving negative regulation of some component(s) of the Wntß-catenin pathway (Fig. 8). We observe an accumulation of ß-catenin protein and ectopic activation of target genes. Although further studies in the mz-ppt embryos are needed to confirm if the impact on ß-catenin and target genes is a direct effect of Wnt-5/Ca2+ activity, antagonism of ß-catenin levels is consistent with Wnt-5 misexpression resulting in reduced chordin expression domains. As there has not been a clear demonstration of a maternally provided canonical Wnt, the level of antagonism may lie within the cell. Recent demonstration of dsh activating Wnt/Ca2+ provides one candidate for maternal influence (Sheldahl et al., 2003). The cellular response to Ca2+ release most likely involves a network of proteins activating multiple components. Data suggests an antagonistic role for CaMKII, interfering with Xenopus gastrulation movements (Kuhl et al., 2001) and by CaMKII-dependent activation of a ß-catenin/Tcf inhibiting nemolink kinase (Ishitani et al., 2003). The partial rescue of the ppt-/- phenotype suggests that although CaMKII is a downstream responder to Wnt/Ca2+ activation, other Ca2+-sensitive components may also be required. In particular, PKC (Kuhl et al., 2001) and naked cuticle (Zeng et al., 2000; Yan et al., 2001) proteins, which could lead to ß-catenin accumulation or those that could influence ß-catenin nuclear import (Tolwinski et al., 2003), are perhaps required.
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In summary, Wnt/Ca2+ functions in D-V axis specification. Our in vivo studies reveal that changes in intracellular Ca2+ concentrations as a result of Wnt-5 modulation can lead to rapid and sustained events by establishing feedback loops to sharpen boundaries or to coordinate cell movement. Wnt-5 is maternally required and its biological effects are in part due to negative regulation of Wnt/ß-catenin signaling. Because activation of ß-catenin signaling has been implicated in cancer, our work provides genetic evidence that the WntCa2+ pathway may be a tumor suppressor pathway. This is further supported by the recent demonstration that Ca2+ activity suppresses ß-catenin in human colon carcinomas (Chakrabarty et al., 2003). Knowledge of the developmental processes of Wnt/Ca2+ signaling will lay the foundation for understanding complex developmental events, as well as the oncogenic role of the Wnt signaling family.
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Materials and methods |
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Mutant identification
Stock families of heterozygous adults for pptti265 (Hammerschmidt et al., 1996), slbtz216 (Heisenberg et al., 1996), and pptti265;slbtz216 are maintained under standard conditions. Mutants are identified by test crosses back to pptti265;slbtz216, as well as by PCR analysis. Genomic DNA isolated from single embryos or fin-snipped adults were proteinase Ktreated (2 mg/ml in PCR buffer + Tween + NP-40). The PCR primers used for pptti265 in exon 4 (5'-CTACACCATCAGTATATTTCACC-3' and 5'-CTATCAACGGCACACGTACAC-3') identify an A to T transition at position 1045 and the primers specific for slbtz216 identify the truncation at glycine 155 (5'-AGCGTTTGTG GTTTCTCTGG-3' and 5'-TCCTCATTGGTGCATCTGAG-3'). The PCR products were isolated and sequenced by standard techniques.
Pharmacological reagents
L-690,330 (Tocris), an inositol monophosphatase inhibitor (Atack et al., 1993), was injected to 1.52 ng/embryo after the 8-cell stage. XeC (Calbiochem), a membrane-permeable blocker of IP3-mediated Ca2+ release (Gafni et al., 1997), was used at 12 µM doses. For the cell movement defects, treatment was typically after the 128-cell stage.
Whole mount in situ hybridization
For all the manipulations, embryos at the appropriate developmental stage, sphere/dome or 50% epiboly, were placed in 4% PFA/PBS fixative. Digoxygenin-UTP RNA probes (Roche) were synthesized from linearized templates. Single probe hybridizations were done as described in Thisse et al. (1993) and double label in situ as described in Long and Rebagliati (2002). After probe detection, embryos were mounted and photographed.
ß-Catenin immunolocalization
Embryos were fixed in 4% PFA/1x PBS at the sphere/dome stage. Overnight incubation with antiß-catenin (P14L, provided by Dr. T. Kurth, MPI für Entwicklungsbiologie, Tübingen, Germany); Schneider et al., 1996), followed by secondary antibody conjugated with a fluorescent label (Texas-red or Alexa633; Molecular Probes). Nuclei were identified by counter stain with the 5 µM Sytox Green (Molecular Probes). Whole mount embryos in an animal pole orientation were optically sectioned using two-channel imaging on a scanning laser confocal microscope system (20x/0.7 Plan Apo and 63x/1.2 water objectives; model TCS-NT; Leica). The image stacks collected at 4-µm intervals were evaluated and nuclear ß-catenin in nonoverlapping cells was counted as positive.
Calcium image analysis
The ratiometric Ca2+-sensing dye Fura-2 (dextran conjugated; Molecular Probes) was injected into 1-cell zebrafish embryos. Indicated RNAs or pharmacological reagents were either coinjected with the Fura-2 at the 1-cell stage or unilaterally injected at the 816 cell stage mixed with dextran-conjugated Texas red lineage tracer (Molecular Probes) and imaged from 32 to 64 cell stage until beyond dome/early epiboly stages. Embryos collected from heterozygous ppt crosses were injected with Fura-2. The genotype of individual embryos was determined by PCR. Details of data collection and analysis are as described in Slusarski and Corces (2000). Ca2+ fluxes are determined by a subtractive analogue and represented as either a surface plot of Ca2+ activity across the embryo or a graphical representation of the number of new transients per image as a function of time.
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Acknowledgments |
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This work was supported by March of Dimes (MOD) (grant 5-FY99-806) and the American Cancer Society (grant IN-122V) administered through the Holden Comprehensive Cancer Center at the University of Iowa. D.C. Slusarski is a MOD Basil O'Conner Research Scholar.
Submitted: 17 March 2003
Accepted: 2 July 2003
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