Establishment of the Dorso-ventral Axis in Xenopus Embryos Is Presaged by Early Asymmetries in beta -Catenin That Are Modulated by the Wnt Signaling Pathway

Carolyn A. Larabell,§ Monica Torres,* Brian A. Rowning,*§par Cynthia Yost,Dagger Jeffrey R. Miller,* Mike Wu,par David Kimelman,Dagger and Randall T. Moon*

* Howard Hughes Medical Institute and Department of Pharmacology, Dagger  Department of Biochemistry, University of Washington School of Medicine, Seattle, Washington 98195; and § Lawrence Berkeley National Laboratory, par  Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720

Abstract
Materials and Methods
Results
Discussion
Summary
Footnotes
Acknowledgements
Abbreviations used in this paper
References


Abstract

Eggs of Xenopus laevis undergo a postfertilization cortical rotation that specifies the position of the dorso-ventral axis and activates a transplantable dorsal-determining activity in dorsal blastomeres by the 32-cell stage. There have heretofore been no reported dorso-ventral asymmetries in endogenous signaling proteins that may be involved in this dorsal-determining activity during early cleavage stages. We focused on beta -catenin as a candidate for an asymmetrically localized dorsal-determining factor since it is both necessary and sufficient for dorsal axis formation. We report that beta -catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with beta -catenin accumulating in dorsal but not ventral nuclei by the 16- to 32cell stages. We then investigated which potential signaling factors and pathways are capable of modulating the steady-state levels of endogenous beta -catenin. Steadystate levels and nuclear accumulation of beta -catenin increased in response to ectopic Xenopus Wnt-8 (Xwnt-8) and to the inhibition of glycogen synthase kinase-3, whereas neither Xwnt-5A, BVg1, nor noggin increased beta -catenin levels before the mid-blastula stage. As greater levels and nuclear accumulation of beta -catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, our data suggest that early asymmetries in beta -catenin presage and may specify dorso-ventral differences in gene expression and cell fate. Our data further support the hypothesis that these dorso-ventral differences in beta -catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3.


Our understanding of how the embryonic axes are specified during the early development of vertebrates is progressing towards an understanding at the cellular and biochemical level. To a large degree, the discussion now centers on establishing the interplay between specific biochemical pathways and cellular events. The rate of these theoretical advances was stimulated by the discovery that ectopic expression of a member of the Wnt family in the ventral marginal zone of Xenopus embryos is sufficient to elicit a complete duplication of the embryonic axes (McMahon and Moon, 1989). This observation raised the possibility that an endogenous Wnt pathway might normally be involved in axis formation, but the observation that the unrelated signaling factors noggin (Smith and Harland, 1992) and Vg1 (Dale et al., 1993; Thomsen and Melton, 1993) can also induce complete axes suggests that further research is needed to distinguish which, if any of these factors, are normally involved in axis formation.

Any attempt to evaluate secreted factors that may be involved in specifying the dorso-ventral axis in Xenopus embryos should take into account known observations regarding the cellular basis for axis specification. Specifically, the postfertilization cortical rotation of Xenopus is important in determining the position of the future dorsal axis (for reviews see Gerhart et al., 1989; Larabell et al., 1996). Suggesting that dorsal-determining information is present in the vegetal pole before cortical rotation, removal of this region blocks axis formation (Sakai, 1996), and injection of vegetal pole cytoplasm into host embryos has the capacity to induce an ectopic axis (Fujisue et al., 1993; Holowacz and Elinson, 1993). After cortical rotation, this dorsal-determining activity is displaced to the future dorsal side of the embryo, and transplantation of dorsal cells or cytoplasm to the ventral side of a host embryo elicits formation of a secondary dorsal axis (Gimlich, 1986; Kageura, 1990; Yuge et al., 1990; Fujisue et al., 1993). While there are presently no data showing dorso-ventral differences in the localization or activity of endogenous secreted factors that correlate with this dorsal-determining activity of the egg and early embryo, candidate molecules include Wnts (for reviews see Cui et al., 1995; Torres et al., 1996), Vg1 (Dale et al., 1993; Thomsen and Melton, 1993), and noggin (Smith and Harland, 1992).

Given the lack of evidence of a dorsal enrichment in expression or activity of any of these secreted factors, it is likely that a greater understanding of the signal transduction cascades stimulated by these factors would contribute to an understanding of which of these signaling pathways, if any, are actually used by Xenopus embryos to initiate formation of the endogenous axis. With regard to candidate cytoplasmic signaling factors, attention justifiably should focus on beta -catenin, a multifunctional protein that is involved in cell adhesion at adherens junctions and in cytoplasmic and nuclear signal transduction events (for review see Miller and Moon, 1996). beta -Catenin meets a number of reasonable criteria for playing a role in specification of the dorso-ventral axis in vertebrate embryos. beta -Catenin is maternally expressed at the RNA and protein level (DeMarais and Moon, 1992), and when ectopically expressed, it is sufficient to mimic the endogenous dorsal-determining activity by inducing the formation of complete secondary axes in Xenopus (Funayama et al., 1995; Guger and Gumbiner, 1995) and in zebrafish (Kelly et al., 1995). Moreover, depletion of maternal beta -catenin transcripts from Xenopus oocytes prevents formation of the endogenous axis (Heasman et al., 1994) and disruption of the gene in mice prevents mesoderm formation (Haegel et al., 1995). It is likely that the ability of beta -catenin to alter gene expression and cell fate involves its interaction with architectural HMG box transcription factors (Behrens et al., 1996; Molenaar et al., 1996). Importantly, injection of a mutant form of one of these factors, XTcf-3, into the dorsal side of Xenopus embryos blocks formation of the endogenous dorsal axis and blocks the ability of ectopic beta -catenin to induce a secondary axis (Molenaar et al., 1996). These data collectively support the hypothesis that dorsal beta -catenin interacts with architectural transcription factors to regulate the expression of dorsal genes required for dorso-ventral axis specification.

While these reports indicate a role for beta -catenin in dorso-ventral axis formation, the following two key issues need to be resolved: (a) Is endogenous beta -catenin expressed appropriately to play a role in axis specification? If beta -catenin were involved in dorso-ventral axis specification, it should be dorsally enriched and/or have greater dorsal activity by the 32-cell stage. However, previous investigations of beta -catenin localization have reported either no dorso-ventral differences in embryos (Fagotto and Gumbiner, 1994) or dorso-ventral differences after the mid-blastula transition (Schneider et al., 1996)---too late to play a role in the dorsal-determining activity defined by cytoplasm and blastomere transplantation assays using cleavage stage embryos. (b) Can any or all of the secreted factors that induce an ectopic axis also modulate beta -catenin levels? Since beta -catenin is required for the formation of the endogenous axes (Heasman et al., 1994), and since beta -catenin steady-state levels and nuclear accumulation rise in response to modulation of cytoplasmic components of the Wnt signaling pathway in the gastrula (Yost et al., 1996), it is important to directly determine whether any of the axisinducing secreted factors modulate levels of beta -catenin in the cleavage stage embryo. Such assays would determine whether all of the axis-inducing factors operate through the same intracellular signaling pathway and which are candidates for normally playing a role in modulating beta -catenin levels.

The present study resolves both of these issues in a manner consistent with the embryological data on dorso-ventral axis formation. Using optimized immunocytochemistry and Western blot methods, we demonstrate that endogenous beta -catenin is enriched in dorsal regions of the early embryo, becoming pronounced by the 16- to 32-cell stages, when it accumulates in dorsal nuclei and cytoplasm. We then show that the Wnt signaling pathway, but not ectopic noggin or Vg1, regulates steady-state beta -catenin levels in the early embryo. Our concluding model for specification of the embryonic dorso-ventral axis takes into account these data, as well as the possibility that endogenous beta -catenin is regulated in a Wnt-independent manner.


Materials and Methods

cDNA Constructs and Embryo Microinjection

cDNAs encoding Xenopus Wnt-8 (Xwnt)1, Xwnt-5A, BVg1, noggin, prolactin, and beta -galactosidase were obtained and transcribed in vitro as described (Torres et al., 1996), as were wild-type and point mutant Xenopus beta -catenin tagged with a c-myc epitope (Yost et al., 1996), wild-type and kinase-dead Xenopus glycogen synthase kinase-3 (Xgsk-3) (Pierce and Kimelman, 1995), and Xwnt-II (Ku and Melton, 1993). Embryos were injected with these RNAs and cultured as described (Torres et al., 1996; Yost et al., 1996) with further details in the figure legends.

Confocal Microscopic Localization of beta -Catenin

For Figs. 1, 2, 3, and 8 A, whole-mount confocal immunocytochemistry was carried out on untreated embryos, and embryos were irradiated at the vegetal pole with UV light to prevent cortical rotation (e.g., Gerhart et al., 1989; Yost et al., 1996). Embryos were fixed overnight in 4% paraformaldehyde, 0.1% gluteraldehyde, 100 mM KCl, 3 mM MgCl2, 10 mM Hepes, 150 mM sucrose, and 0.1% Triton X-100, pH 7.6. Fertilization envelopes were manually removed from all embryos and blocking of nonspecific binding was carried out using 0.1% Triton X-100 in Super Block (Pierce, Rockford, IL). Embryos were incubated overnight with an antibody raised in rabbit (Yost et al., 1996) against a bacterial fusion protein derived from the amino-terminal region of Xenopus beta -catenin (kindly provided by P. McCrea, M.D. Anderson Cancer Center, Houston, TX). Control antibodies (see Fig. 3) were polyclonal antibodies against membrane skeleton protein 4.1 (Spencer et al., 1990), polyclonal antibodies against alpha -spectrin (Giebelhaus et al., 1987), or a commercial pan cytokeratin mouse monoclonal antibody (No. C2931; Sigma Chemical Co., St. Louis, MO). Embryos were rinsed for 1 h in Super Block plus 0.1% Triton X-100 with three changes and then incubated overnight in TRITC-conjugated antirabbit or anti-mouse (Sigma Chemical Co.) secondary antibody, with rotation, followed by three additional rinses in Super Block. Embryos were bisected relative to pigmentation and/or the sperm entry point under a dissecting microscope and cleared in a 1:2 mixture of benzyl alcohol/benzyl benzoate.


Fig. 1. Immunolocalization of beta -catenin in early Xenopus embryos. Whole-mount staining with antibody to beta -catenin demonstrates greater staining on the dorsal side of embryos (to the right in all panels) at the 8- (A and B) and 16-cell (C and D) stages. Immunolabeled embryos were cut along the equator separating the animal (A and C) from vegetal (B and D) blastomeres and examined with a confocal microscope. Optical sections through these blastomeres demonstrate an arc of cytoplasmic beta -catenin in the periphery of the animal dorsal blastomeres (A and C) and vegetal dorsal blastomeres (B and D). Ventral blastomeres stain for beta -catenin at the cell surface but not in the cytoplasm.
[View Larger Version of this Image (66K GIF file)]


Fig. 2. Spatial and temporal distribution of beta -catenin during early development of the Xenopus embryo. Embryos were bisected along the dorso-ventral axis after immunolabeling so that each half contained dorsal and ventral blastomeres. Each half was imaged from the bisected surface in the confocal microscope. Embryos are oriented with dorsal on the right, ventral on the left, animal hemisphere at the top, and vegetal hemisphere at the bottom, in A-F. Immunocytochemistry reveals an increase in cytoplasmic beta -catenin (orange) on the dorsal side of the embryo from the two-cell (A), to four-cell (B), to eight-cell (C) stages. At the 16-cell stage, intense beta -catenin is observed in the cytoplasm (D, arrow denotes nucleus) as well as in the nucleus (G, arrow) of the dorsal vegetal blastomeres. At the 32-cell stage, beta -catenin is enriched in the cytoplasm (E, arrows denote nuclei) and nuclei (I, arrow) of all dorsal blastomeres, while ventral blastomeres have lower cytoplasmic staining (E) but neglible nuclear staining (H, arrow). Overall, 93% of the embryos analyzed (n = 120) from the 2-32-cell stages displayed this dorsal enrichment of beta -catenin. At the blastula stage, beta -catenin is detected in the cytoplasm of both dorsal and ventral cells (F), as well as in ventral (J) and dorsal (L) nuclei, though the dorsal signal was stronger in both cellular compartments. Primarily the outer layers of blastomeres demonstrate intense cytoplasmic beta -catenin staining, even when blastula were bisected before immunolabeling to assure access of the antibodies to the inner blastomeres. Specific beta -catenin staining is detectable in these inner vegetal blastomeres (K) when fluorescence sensitivity is increased to the point where the dorsal signal becomes saturated.
[View Larger Version of this Image (86K GIF file)]


Fig. 3. Immunolocalization of proteins unrelated to beta -catenin. To assess whether the patterns of beta -catenin observed in the eight-cell embryo (Fig. 1) were unique or were shared by other proteins, we undertook a comparable analysis of eight-cell embryos stained with other antibodies, optically sectioned near the equator, and viewed at low magnification to monitor dorsoventral patterns (A-C) or viewed at the membrane at high magnification (D-F). Dorsal is to the right for A, B, and C as in Fig. 1. While eight-cell embryos show a clear dorsal enrichment of beta -catenin (Fig. 1, A and B), membrane skeleton protein 4.1 (A), alpha -spectrin (B), and cytokeratin (C) antibodies show no dorsal enrichment. The alpha -spectrin image in B is somewhat more pigmented in the animal hemisphere than the other images, hence the ventral blastomeres appear somewhat darker than the dorsal blastomeres (right) owing to pigmentation differences. The cytokeratin image is yellow because of the extensive overlap of the green autofluorescence signal with the red cytoplasmic staining of the antibody (see F for cytoplasmic stain at higher magnification). While beta -catenin staining extends extensively from the membrane through the cytoplasm at the eight-cell stage (Fig. 1, A and B), both protein 4.1 (D) and alpha -spectrin (E) are restricted to the plasma membrane, which is consistent with their known functions in the membrane skeleton, while the cytokeratin staining extends into the cytoplasm (F) as filaments as previously noted (Klymkowsky et al., 1987), though our fixation methods also retain nonpolymeric protein and increase nonfilamentous cytokeratin staining.
[View Larger Version of this Image (63K GIF file)]


Fig. 8. Effects of Xgsk-3 and LiCl on the spatial accumulation of beta -catenin. (A) The dorso-ventral asymmetry in beta -catenin requires the postfertilization cortical rotation and is regulated by Xgsk-3. When the postfertilization cortical rotation is prevented by UV-irradiation of the vegetal pole during the first cell cycle (a), 32-cell embryos do not display dorsal enrichments in beta -catenin observed in control embryos (b), and some show ventral increases. Injection of 4 ng of RNA encoding Xgsk-3 into the dorsal marginal zone of four- to eight-cell embryos also blocks the dorsal increase in beta -catenin staining at the 32-cell stage (c). Conversely, injection of 4 ng of RNA encoding a dominant negative Xgsk-3 into the ventral marginal zone promotes accumulation of beta -catenin throughout the 32-cell embryo (d). (B) Injection of a mixture of prolactin and beta -catenin-myc RNAs leads to the accumulation of beta -catenin-myc on the future dorsal side of the embryo (a), though the RNAs are injected and expressed throughout the embryo. Decreased staining (left embryo) or staining for beta -catenin-myc similar to controls (right embryo) is evident when Xgsk-3 RNA is injected with the beta -catenin- myc RNA (b). Inhibition of endogenous Xgsk-3 by injecting dnXgsk-3 RNA increases and expands the accumulation of ectopic beta -catenin- myc to the entire embryonic marginal zone (c). Treatment with lithium also expands the domain of beta -catenin-myc accumulation in embryos overexpressing prolactin (d) or Xgsk-3 (e). Expression of ptbeta -catenin-myc (which encodes a form of beta -catenin that is not phosphorylated by Xgsk-3; Yost et al., 1996) with control prolactin results in the accumulation of ptbeta -catenin-myc everywhere in the embryo (f). In these experiments 1 ng of beta -catenin-myc or ptbeta -catenin-myc RNA mixed with 3 ng of prolactin, Xgsk-3, or dnXgsk-3 RNA was injected into the marginal zone of each blastomere of fourcell embryos. Arrowheads denote dorsal (single arrowhead) or dorsal and ventral (two arrowheads) staining. (C) Lithium treatment increases steady-state levels of beta -catenin-myc expressed from injected RNA. Embryos were injected and treated with LiCl as in Materials and Methods. At stage 6.5-7, embryo extracts were prepared and probed for the c-myc epitope-tagged beta -catenin by Western blot analysis. Lane 1, Uninjected controls do not express beta -catenin-myc; lane 2, embryos injected with beta -catenin-myc RNA express the encoded protein; lane 3, injection of beta -catenin-myc RNA as in lane 2 followed by treatment with LiCl leads to greater accumulation of beta -catenin-myc.
[View Larger Version of this Image (49K GIF file)]

Embryos were viewed using a confocal laser scanning microscope (model 1024; BioRad Labs, Hercules, CA) using rhodamine filters to collect the beta -catenin signal (orange) superimposed on the blastomeres, which were imaged using fluorescein filters to excite the inherent autofluorescence (green). The threshold of fluorescence was set so that the greatest fluorescence was not saturated, with one consequence being that the images shown do not show the beta -catenin, which is evident at plasma membranes, albeit at lower levels. The small, dorsal blastomeres were easily distinguished from the larger, ventral blastomeres. Confirmation of dorsoventral polarity was also achieved by monitoring blastomere pigmentation, and in one- to four-cell embryos, by following the sperm entry point. Secondary antibody alone did not stain embryos (not shown), and preblocking the primary antibody with the fusion protein used as immunogen blocks all fluorescent staining (Yost et al., 1996). These fixation and visualization methods differ substantially from those that heretofore did not reveal dorso-ventral differences in beta -catenin in cleavage stage embryos (Fagotto and Gumbiner, 1994; Schneider et al., 1996).

To determine the effects of Xwnt-8 or Xwnt-5A on endogenous beta -catenin localization in the nucleus (see Fig. 7), embryos were fertilized and injected in the animal poles at the two-cell stage with 5-10 ng of RNA encoding Xwnt-8 (Christian et al., 1992), Xwnt-5A (Moon et al., 1993), or bovine prolactin. Animal caps were cut from stage 6.5-7 embryos and fixed in 4% paraformaldehyde-PBS overnight at 4°C. Animal caps were dehydrated by washing once in PBT (PBS, 0.2% Triton X-100), once in 75% methanol (MeOH)/75% PBT, and twice in 100% MeOH. Animal caps were then bleached overnight in 50% MeOH/30% H202/20% DMSO at room temperature. Explants were rehydrated by washing once in 75% PBT/25% MeOH and twice in PBT, followed by incubation overnight at 4°C in anti-beta -catenin antibody (Yost et al., 1996; 1:500 in PBT with 10% normal goat serum). After three washes in PBT, animal caps were incubated in CY-3-conjugated antibody (1:250 in PBT, with 10% normal goat serum; Jackson Laboratories, Bar Harbor, ME). After washing three times in PBT, animal caps were mounted under coverslips in Vectashield (Vector Laboratories, Burlingame, CA) and viewed under a confocal microscope (model MRC; BioRad Labs). Nuclear counts were performed on stacked z-series, and images shown in Fig. 7 are single optical sections.


Fig. 7. Xwnt-8, but not Xwnt-5A, increases the accumulation of endogenous beta -catenin in nuclei. The animal pole of each blastomere at the two-cell stage was injected with RNA encoding prolactin (A), Xwnt-8 (B), or Xwnt-5A (C). At stage 6.5-7 animal cap explants were isolated, fixed, and processed for immunolocalization of endogenous beta -catenin.
[View Larger Version of this Image (55K GIF file)]

Enzyme-linked Whole-Mount Immunolocalization of beta -Catenin

Where indicated, embryos were processed for whole-mount immunolocalization of c-myc-tagged ectopic beta -catenin using previously described methods for localizing c-myc (Christian and Moon, 1993). Where indicated, embryos were injected with various RNAs before localization of beta -catenin-myc.

Western Blot Analyses

To compare potential dorso-ventral differences in the steady-state levels of endogenous beta -catenin on protein blots, control embryos and embryos vegetally irradiated with UV light (e.g., as in Gerhart et al., 1989; Yost et al., 1996) were fixed in 3.7% formaldehyde in 1× modified Barth's solution (MBS; 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM Hepes, pH 7.5, 0.82 mM MgSO4·7H2O, 0.33 mM Ca(NO3)2·4H2O, CaCl2·6H2O) for 18 min at room temperature, followed by manual dissection as diagrammed in Fig. 4 A. Protein was extracted in lysis buffer (10 mM Tris, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% NP-40, 4 mM PMSF, pH 7.5) and 1,1,2-trichlorotrifluoroethane (Sigma Chemical Co.). Protein samples equivalent to five dissected portions were analyzed by SDSPAGE, and immunoblots were probed with a rabbit anti-GST-beta -catenin polyclonal antibody (Yost et al., 1996), a rabbit anti-alpha -spectrin polyclonal antibody (Giebelhaus et al., 1987), or a rabbit anti-chicken tubulin antibody, which was generated as in Giebelhaus et al. (1987) by immunizing rabbits with polyacrylamide gel slices containing chicken erythroid cell tubulin. Immunoblots were then probed with an HRP-conjugated goat anti- rabbit secondary antiserum (BioRad Labs). The HRP signal was visualized by enhanced chemiluminescence (Amersham, Corp., Arlington Heights, IL). Signals for beta -catenin, alpha -spectrin, and tubulin were quantitated by use of densitometry (model UA-5 Absorbance/Fluorescence detector; Isco, Lincoln, NE). Dorsal and ventral beta -catenin levels were compared after normalization for alpha -spectrin or tubulin, to control for variability in protein content. Dorsal beta -catenin values were set at 100% and ventral beta -catenin levels were expressed as a ratio: (ventral beta -catenin/ dorsal beta -catenin) × 100%.


Fig. 4. Western blot analysis of microdissected 32-cell Xenopus embryos reveals a dorso-vegetal enrichment of beta -catenin. (A) Schematic diagram of how embryos were dissected into dorsal animal marginal (DAM; light gray), ventral animal marginal (VAM; black), dorsal vegetal marginal (DVM; pattern), and ventral vegetal marginal (VVM; dark gray) portions. (B) Dorsal steady-state levels of endogenous beta -catenin are greater than ventral levels in untreated but not in UV-ventralized embryos. On average, the VVM portions from untreated embryos contained 77% (normalized to tubulin, three experiments) and 64% (normalized to spectrin, five experiments) as much beta -catenin as the DVM zones. The VVM portions dissected from UV-irradiated embryos contained more beta -catenin relative to their dorsal counterparts; 146% (normalized to tubulin, four experiments) and 172% (normalized to spectrin, four experiments). Spectrin-normalized steady-state levels of beta -catenin in the VAM zone were also lower than the DAM region in untreated embryos (78%, five experiments). These differences were eliminated with UV treatment (four experiments for both tubulin and spectrin normalization). The asterisks indicate that the difference in beta -catenin content between the dorsal and ventral marginal zones is statistically significant, as determined by Student's t-test (P < 0.05 for a single asterisk, P < 0.005 for a double asterisk). The error bars represent the standard error. (C) Representative Western blot detecting endogenous beta -catenin, alpha -spectrin, and tubulin. Ventral levels of beta -catenin are lower in the dissected VAM (lane 2) relative to the DAM (lane 1) regions, and in the VVM (lane 4) relative to DVM (lane 3) regions. It is worth noting that the dissected quadrants of the embryo do not precisely correspond to the areas of maximal beta -catenin staining determined by confocal microscopy. Moreover, the thresholds of fluorescence chosen for Figs. 1 and 2 were set high, so as not to saturate the dorsal-ventral differences, and as a result these images do not show the detectable lower levels of cytoplasmic and membrane-associated beta -catenin, which nevertheless would contribute to the signals on Western blots. Thus, one cannot quantitatively compare the dorso-ventral differences monitored by the dissections vs. confocal microscopy.
[View Larger Version of this Image (31K GIF file)]

To monitor endogenous and ectopic beta -catenin in injected and LiCltreated embryos, protein was extracted as described above 2-3 h after RNA injection (stage 5-6) and analyzed by Western blot using anti-beta -catenin or anti-c-myc antibodies, respectively (above and Yost et al., 1996). Endogenous beta -catenin signals were normalized to alpha -spectrin and tubulin bands from the same Western blot to control for protein content and gel loading (above). All Westerns were subsequently processed as described above for endogenous beta -catenin quantitation or as described (Yost et al., 1996) for ectopic beta -catenin-myc quantitation. beta -catenin levels present in control samples were set at 100% and experimental samples were expressed as a ratio: (experimental beta -catenin/control beta -catenin) × 100%. Where described, eight-cell embryos were incubated for 12 min at room temperature in 0.3M LiCl in 0.1× MMR (0.1 M NaCl, 2 mM KCl, 1 mM MgSO4·7H2O, 2 mM CaCl2, 5 mM Hepes, pH 7.8) immediately after RNA microinjection.


Results

Dorso-ventral Asymmetries in beta -Catenin in Cleavage Stage Embryos

We first conducted a confocal microscopical analysis of the early Xenopus embryo to investigate whether there were asymmetries in beta -catenin along the prospective dorso-ventral axis. Optical equatorial sections of eight-cell embryos demonstrate distinct peripheral cytoplasmic staining on the dorsal side of the embryo, monitored in both the animal (Fig. 1 A) and vegetal hemispheres (Fig. 1 B). Cytoplasmic beta -catenin is also more pronounced on the dorsal side of 16-cell embryos, in both the animal (Fig. 1 C) and vegetal (Fig. 1 D) hemispheres.

To determine the earliest time at which asymmetries in endogenous beta -catenin could be detected, and to determine whether it persisted through to the 32-cell stage when dorsal vegetal blastomeres can be transplanted and exhibit axis-inducing activity (Gerhart et al., 1989, Kageura, 1990), we analyzed embryos beginning at the two-cell stage. To allow simultaneous comparison of animal and vegetal regions, we undertook the analysis using optical sectioning along the dorso-ventral, rather than equatorial, axes. Twocell embryos exhibit an asymmetry in beta -catenin in the peripheral cytoplasm (94%, n = 17 embryos; Fig. 2 A), with the elevated beta -catenin predominantly forming opposite the visible sperm entry point (not shown), and thus corresponding to the prospective dorsal side. This asymmetry is also evident through the 4- (Fig. 2 B), 8- (C), 16- (D), and 32-cell (E) stages. By the blastula stage, beta -catenin is still elevated on the dorsal relative to ventral side with greatest staining of outer ectodermal layers (Fig. 2 F), as previously reported (Schneider et al., 1996). However, internal blastomeres also stain for beta -catenin (Fig. 2 K) when fluorescence sensitivity is increased to levels that obscure the dorso-ventral differences.

Staining of eight-cell embryos with control antibodies for membrane skeleton protein 4.1 (Fig. 3 A), alpha -spectrin (Fig. 3 B), or cytokeratin (Fig. 3 C) did not reveal any dorsal enrichment for these proteins, demonstrating that the dorsal enrichment of beta -catenin is not simply a reflection of the patterns of expression of structural proteins. Moreover, alpha -spectrin strongly stained plasma membranes between blastomeres and at the periphery of the embryo (Fig. 3 B), demonstrating that these internal regions were accessible to all reagents for immunolocalization.

Strikingly, by the 16-cell stage beta -catenin is no longer restricted to the cytoplasm but is also observed in nuclei of dorsal vegetal blastomeres (Fig. 2 G). At the 32-cell stage there is a pronounced enrichment of nuclear beta -catenin in the dorsal (Fig. 2 I) relative to ventral (Fig. 2 H) blastomeres. At the blastula stage, nuclei stain for beta -catenin on both dorsal (Fig. 2 L) and ventral (Fig. 2 J) sides, though with greater staining on the dorsal side. Previously, Schneider et al. (1996) observed nuclear beta -catenin only in dorsal regions of blastulas, but the discrepancy is likely due to the greater sensitivity of confocal microscopy. The structures examined at the 32-cell stage were confirmed to be nuclei by using the nuclear stain Bo-Pro-3 as in Molenaar et al. (1996), which labeled all nuclei, each of which resembled the beta -catenin-stained nucleus in Fig. 2 I (data not shown).

To test whether the apparent differences in the spatial distribution of endogenous beta -catenin in the early Xenopus embryo reflect dorso-ventral differences in steady-state levels of beta -catenin, we extracted proteins from the dorsal and ventral regions of 32-cell stage embryos (Fig. 4 A) and performed immunoblot analyses as in Fig. 4 C. We found that the ventral vegetal region (VVM) of the embryo contained a statistically significant average of 64% of the beta -catenin present in the dorsal vegetal region (DVM) (n = 5 experiments, P < 0.005 by Student's t test; Fig. 4 B) after normalizing beta -catenin to the levels of the membrane skeleton protein alpha -spectrin (the same antibody as used in Fig. 3). Consistent with these results, we found the VVM region contained an average of 77% of the beta -catenin present in the DVM zone after normalization to tubulin (n = 3 experiments). Smaller dorso-ventral differences were detected in the animal hemisphere (Fig. 4 B), where the ventral animal region (VAM) contained 78% of the beta -catenin present in the dorsal animal region (DAM) (n = 5 experiments, P < 0.05 by Student's t test) after normalization to alpha -spectrin. The comparable dorso-ventral distribution of beta -catenin relative to tubulin in the animal hemisphere suggests that the dorsal enrichment of beta -catenin observed by confocal microscopy is shared by tubulin, while in the vegetal hemisphere beta -catenin is dorsally enriched compared to both alpha -spectrin and tubulin. Although the alpha -spectrin and beta -catenin antibodies were also used in immunolocalization studies (tubulin could not be used owing to high background), the extent of the dorso-ventral differences measured by confocal microscopy and Western blots cannot be compared quantitatively (see Fig. 4 legend).

Since UV irradiation of the vegetal pole during the first cell cycle blocks formation of the dorsal axis (Gerhart et al., 1989), we tested its effects on the dorso-ventral steadystate levels of beta -catenin. Prospective dorsal and ventral sides were assigned before dissection with reference to blastomere size (ventral cells are larger) and pigmentation differences (ventral cells are darker). As shown in Fig. 4 B, UV-irradiated embryos dissected at the 32-cell stage no longer show a dorsal enrichment of beta -catenin in either the animal or vegetal hemispheres, and instead displayed greater relative ventral levels, especially in the VVM region. We emphasize that the higher bar heights in VVM and VAM in UV-treated embryos are due to normalization to dorsal DVM and DAM and do not imply increases in absolute levels above those seen dorsally in controls. Indeed, Western blot analysis of entire embryos shows that total amounts of beta -catenin remain relatively unchanged after UV irradiation sufficient to ventralize embryos; further UV irradiation reduces beta -catenin levels nonspecifically (not shown). We do not know why the greater level of beta -catenin on the ventral side of UV-irradiated embryos does not cause this side to become dorsal, since ectopic expression of beta -catenin in UV-irradiated embryos restores axis formation (Gruger and Gumbiner, 1995; Yost et al., 1996). It is possible that the absolute level of beta -catenin on the ventral side of UV-irradiated embryos is too low to promote axis formation or that the UV irradiation leads to subsequent damage to some beta -catenin such that it is no longer functional.

Wnt Signaling, but Not BVg1 or Noggin, Stabilizes beta -Catenin in Early Embryos

We next tested whether we could detect dorso-ventral asymmetries in the accumulation of ectopically expressed beta -catenin as a prelude to asking which signaling factors might modulate this accumulation. We injected a mixture of c-myc-epitope-tagged beta -catenin and control prolactin RNAs into the marginal zone of each blastomere at the fourcell stage, followed by anti-c-myc immunostaining of the embryos at different stages of development. Control Western blots show this leads to three- to fourfold increases in total beta -catenin levels relative to uninjected embryos (data not shown). In these control embryos, the beta -catenin-myc staining is evident on the dorsal side when stained either before mid-blastula transition (MBT) (Fig. 5 C, relative to uninjected embryos, A) or after MBT (Fig. 5 D, relative to uninjected embryos, B). When embryos were cultured to stage 10 (not shown), the enrichment of anti-c-myc staining colocalized with the dorsal lip in 100% of the embryos analyzed (n = 38), indicating that the accumulation of ectopic beta -catenin occurs on the prospective dorsal half of the developing embryo. These results establish that the ectopic tracer beta -catenin-myc accumulates in a manner similar to the spatial pattern of endogenous beta -catenin (Fig. 2). Although beta -catenin-myc does not accumulate on the ventral side of embryos, proteins from injected RNAs are indeed expressed, as shown by ventral injection and expression of green fluorescent protein RNA and beta -galactosidase RNA (not shown). The localized accumulation of beta -catenin-myc on the prospective dorsal side, despite the RNA being injected on both dorsal and ventral sides, is consistent with the proposed greater activity of Xgsk-3 on the ventral side (Pierce and Kimelman, 1995), which targets beta -catenin for degradation (Yost et al., 1996).


Fig. 5. Wnts, but not BVg1 or noggin, are able to modulate ectopic beta -catenin-myc accumulation both before and after MBT, as assayed by anti-c-myc immunostaining. Uninjected controls do not stain for c-myc when assayed before (A) or after (B) MBT. Embryos expressing beta -catenin-myc and prolactin display a dorsal accumulation of ectopic beta -catenin-myc both before (C) and after (D) MBT, while the ventral side shows negligible staining. (E) Xwnt-8 RNA expands the domain of accumulation of beta -catenin-myc when assayed before MBT (shown, 100 pg Xwnt-8 RNA was injected for the embryo on the left, and 3 ng RNA for the embryo on the right) or after MBT (see Table I). (F) Neither BVg1 (pre-MBT, shown; and post-MBT, Table I) nor noggin RNA (see Table I) are able to elevate beta -catenin-myc accumulation on the ventral side, while beta -catenin-myc accumulates on the dorsal side similarly to control embryos. (G) Xwnt-5A RNA does not alter the pattern of beta -catenin-myc accumulation during the early cleavage stages, but causes beta -catenin-myc to accumulate around the entire marginal zone after MBT (H). In these experiments, 1 ng of beta -catenin RNA and 3 ng of prolactin, Xwnt-8 (100 pg in left embryo of panel E), Xwnt-5A, BVg1, or noggin RNA were injected into the marginal zone of each blastomere of four cell stage embryos, followed by anti-c-myc immunostaining before and after MBT. Arrowheads denote dorsal (single arrowhead) or dorsal and ventral (double arrowhead) staining.
[View Larger Version of this Image (119K GIF file)]

Table I. Modulation of beta -catenin-myc Accumulation

[View Table]

Since both endogenous and ectopic beta -catenin accumulate on the prospective dorsal side of the embryo, we then asked whether overexpression of Wnts (for review see Du et al., 1995), BVg1 (a modified version of Vg1 which is efficiently processed; Thompsen and Melton, 1993), or noggin (Smith and Harland, 1992), all of which induce ectopic dorsal axes, would stabilize ectopic beta -catenin on the ventral sides of embryos. We injected beta -catenin-myc RNA with Xwnt-8, Xwnt-5A, BVg1, or noggin RNA into the marginal zone of each blastomere at the four-cell stage and assayed for ectopic beta -catenin accumulation by anti-c-myc immunostaining. We found that neither ectopic BVg1 (Fig. 5 F; Table I) nor noggin (Table I) altered the dorso-ventral accumulation of beta -catenin-myc before MBT when compared to the pattern in control-injected embryos (Fig 5 C), nor did they promote increased accumulation of beta -catenin-myc after MBT (Table I). In contrast, Xwnt-8 consistently expanded the domain of beta -catenin accumulation to span the entire marginal zone, when assayed either before MBT (Fig. 5 E, relative to control, C) or after MBT (Table I). Pointing to differences between distinct Xwnts, overexpression of Xwnt-5A before MBT did not alter the dorsally enriched pattern of ectopic beta -catenin accumulation (Fig. 5 G, relative to control, C; Table I), which is consistent with the inability of ectopic Xwnt-5A to induce dorsal structures in developing embryos (Moon et al., 1993; Du et al., 1995). However, anti-c-myc immunostaining after MBT revealed that Xwnt-5A RNA injected at the fourcell stage is capable of stabilizing ectopic beta -catenin after the onset of zygotic transcription (Fig. 5 H, relative to control, D; Table I), suggesting that at certain periods in development, Xwnt-8 and Xwnt-5A might act in part through a common pathway.

To further investigate whether Wnts are capable of modulating beta -catenin accumulation, we injected embryos with RNAs encoding beta -catenin-myc along with RNAs encoding various secreted factors and then analyzed total embryo extracts by Western blots. The blots were probed with anti-c-myc and anti-beta -catenin antibodies to monitor the steady state levels of ectopic and endogenous beta -catenin, respectively (Yost et al., 1996). We found that Xwnt-8 (Fig. 6 A, lane 4) is able to increase the steady-state level of ectopic beta -catenin-myc an average of 231% when compared to embryos injected with beta -galactosidase (Fig. 6 A, lane 2; n = 4 experiments, SE = 42%, P < 0.05 by Student's t test). Similarly, Xwnt-8b, a maternal Wnt gene with axis-inducing activity (Cui et al., 1995), also increased steady-state levels of ectopic beta -catenin (data not shown), while Xwnt-11 (functionally similar to Xwnt-5A; Du et al., 1995; Torres et al., 1996) was much less effective at stabilizing ectopic beta -catenin (Fig. 6 A, lane 3 relative to 4). Neither BVg1 (Fig. 6 A, lane 7 relative to control, lane 6) nor noggin (Fig. 6 A, lane 8) had a reproducible effect on beta -catenin-myc accumulation, consistent with their lack of effect on beta -catenin-myc staining in intact embryos (Fig. 5).


Fig. 6. Xwnt-8, but not BVg1 or noggin, is able to increase the accumulation of both ectopic and endogenous beta -catenin as assayed by Western blots. (A) In multiple independent experiments (see text), probing Western blots for c-myc, microinjection of 100 pg of beta -catenin-myc RNA with 200 pg of beta -galactosidase (lane 2), Xwnt-11 (lane 3), or Xwnt-8 (lane 4) reveals that only Xwnt-8 is able to increase beta -catenin levels (compare lanes 2 and 4). Uninjected embryos (lane 1) do not express the c-myc epitope. In separate experiments (lanes 5-8), neither BVg1 (lane 7) nor noggin (lane 8) increases the accumulation of beta -catenin above control-injected levels (lane 6), while uninjected embryos do not express beta -catenin-myc (lane 5). (B) Microinjection of 5-10 ng of control prolactin (lane 1), Xwnt-5A (lane 2), Xwnt-8 (lane 3), BVg1 (lane 4), or noggin (lane 5) RNA demonstrates that Xwnt-8 increases the steady state levels of endogenous beta -catenin (compare lanes 1 and 3), as shown above for ectopic beta -catenin. In these experiments, the animal poles of two-cell embryos were injected with RNA, followed by the extraction of protein before stage 7. To control for protein content and even loading, all endogenous beta -catenin bands were normalized to both alpha -spectrin and tubulin signals from the same Western blot.
[View Larger Version of this Image (33K GIF file)]

Turning to the effects of these secreted factors on endogenous beta -catenin, Western blots of embryos injected with Xwnt-8 versus prolactin RNA reveal that Xwnt-8 elevated endogenous beta -catenin (Fig. 6 B, lane 3 relative to control lane 1) an average of 254% relative to prolactin controls (n = 6 experiments, SE = 66%, P < 0.1 by Student's t test), similar to its effects on ectopic beta -catenin- myc (Fig. 6 A). In these same experiments, Xwnt-5A, BVg1, and noggin RNAs did not reproducibly elevate endogenous beta -catenin after normalization of protein levels to alpha -spectrin or tubulin (Fig. 6 B). We conclude that Wnts, but not BVg1 or noggin, reproducibly increase the steadystate levels of both endogenous and ectopic beta -catenin.

We have previously shown that increasing the steadystate levels of endogenous beta -catenin by inhibiting Xgsk-3 activity in Xenopus embryos results in a greater nuclear accumulation of beta -catenin (Yost et al., 1996). This led us to test whether overexpression of Xwnt-8 would also lead to the nuclear accumulation of endogenous beta -catenin, which would be consistent with the idea that an axis-inducing Wnt can suppress the activity of endogenous Xgsk-3 and thereby promote the nuclear accumulation of beta -catenin. We injected embryos with Xwnt-8, Xwnt-5A, or prolactin RNA and stained animal cap explants of pre-MBT embryos for endogenous beta -catenin. Since pre-MBT explants were used, this is a time when Xwnt-8 but not Xwnt5A stabilizes ectopic beta -catenin (Fig. 5). Control prolactinexpressing cells had nuclear staining of beta -catenin in a low percentage of cells (Fig. 7 A; 15% of cells scored, n = 324 cells), while ectopic Xwnt-8 led to strong nuclear staining of beta -catenin (Fig. 7 B; 50% of cells scored, n = 537 cells). Embryos injected with Xwnt-5A RNA, which does not induce a secondary axis (Du et al., 1995; Torres et al., 1996), did not display elevated nuclear beta -catenin (Fig. 7 C; 2% of cells; n = 423 cells).

Implication of Localized Xgsk-3 Activity in Generating Asymmetry in beta -Catenin

If the observed dorso-ventral differences in beta -catenin were important in the known ability of transplanted dorsal vegetal blastomeres (Gerhart et al., 1989; Kageura, 1990) and cytoplasm (Holowacz and Elinson, 1993; Fujisue et al., 1993) to induce a complete embryonic axis, then treatments of the embryo that block formation of this axis- inducing activity should affect the spatial pattern of beta -catenin. We irradiated embryos with UV light at the vegetal pole in the first cell cycle to block both cortical rotation and the formation of the axis-inducing activity (Gerhart et al., 1989) and then analyzed the spatial pattern of beta -catenin by confocal microscopy at the 32-cell stage. UVirradiated embryos (100%, n = 12) do not display dorsal enrichment of beta -catenin (Fig. 8 A, a) relative to control embryos (Fig. 8 A, b). Some UV-irradiated embryos showed suppressed levels of beta -catenin as we observed on Western blots with high levels of irradiation, and some irradiated embryos showed an increase in ventral relative to dorsal beta -catenin as observed analyzing populations of embryos on Western blots (Fig. 4). These data establish a linkage between the postfertilization cortical rotation and the formation of the dorsal enrichment in beta -catenin in cleavage stage embryos.

Recent data have demonstrated a postfertilization mechanism for regulating the stability of beta -catenin in Xenopus embryos, as well as its accumulation in nuclei. According to this model, in the absence of a Wnt signal, Xgsk-3 directly phosphorylates beta -catenin, targeting it for rapid degradation in a pathway that also involves the tumor suppressor protein adenomatosis polyposis coli (for review see Yost et al., 1996). Inhibition of Xgsk-3, which may occur in response to a Wnt signal or which theoretically could occur through a Wnt-independent mechanism, increases the stability of beta -catenin and allows the accumulation of beta -catenin in nuclei (Yost et al., 1996). Thus, if an endogenous Wnt signaling pathway were a candidate for establishing the observed dorso-ventral differences in levels and spatial distribution of beta -catenin in 2-32-cell embryos, then one would predict that active Xgsk-3 should antagonize this Wnt pathway and reduce dorsal beta -catenin levels to those observed ventrally (Yost et al., 1996). Conversely, a dominant negative (kinase-dead) Xgsk-3 (dn Xgsk-3) should mimic a Wnt signal and increase beta -catenin staining when overexpressed ventrally. Consistent with these predictions, active Xgsk-3 reduces endogenous dorsal beta -catenin staining in 32-cell embryos (Fig. 8 A, c, compared to controls, Fig. 2 E), while a dnXgsk-3 increases ventral beta -catenin staining (Fig. 8 A, d).

Xgsk-3 activity also alters the spatial pattern of accumulation of ectopic beta -catenin by the blastula stage, both before and after MBT. Injection of beta -catenin-myc and Xgsk-3 RNA into each blastomere at the four-cell stage results in patterns of c-myc immunostaining (right embryo in Fig. 8 B, b; Table I) that resemble prolactin controls (Fig. 8 B, a) or results in background levels of staining for c-myc (Fig. 8 B, b, left embryo). As many of the embryos injected with Xgsk-3 had background levels of staining, this RNA produced the highest percentage of embryos classified as having no dorso-ventral polarity in beta -catenin-myc (Table I, Indistinguishable). Consistent with its effects on endogenous beta -catenin at the 32-cell stage (Fig. 8 A), expressing a dnXgsk-3 causes ectopic beta -catenin to accumulate to high levels everywhere at the blastula stage (Fig. 8 B, c, compared to B, a; Table I).

As treatment of embryos with lithium mimics Wnt signaling (for review see Christian and Moon, 1993), in part by inhibiting the activity of endogenous Xgsk-3 (Klein and Melton, 1996), we tested whether lithium treatment could modulate ectopic beta -catenin accumulation in a manner similar to Xwnt-8 and dnXgsk-3. Consistent with our expectations, lithium treatment expanded the domain of beta -catenin-myc accumulation in both prolactin (Fig. 8 B, d; compared to control B, a; Table I) and Xgsk-3-treated embryos (Fig. 8 B, e, compared to Xgsk-3 alone, in B, b). Lithium also increased the accumulation of beta -catenin-myc when analyzed by Western blots (Fig. 8 C, lane 3 relative to 2). Finally, expression of point mutation (pt)beta -catenin- myc, which lacks the NH2-terminal conserved Xgsk-3 phosphorylation site required for efficient degradation of beta -catenin (Yost et al., 1996), resulted in the accumulation of ptbeta -catenin-myc everywhere in the embryo (Fig. 8 B, f; Table I). These data are consistent with a requirement for active Xgsk-3 in establishing the observed dorso-ventral asymmetry in endogenous and ectopic beta -catenin.


Discussion

Establishment of the embryonic axes of Xenopus laevis begins with the postfertilization cortical rotation and is elaborated during the cleavage stages, as evidenced by the ability of transplanted dorsal cytoplasm and dorsal cells to induce formation of a new dorsal axis in recipient embryos (reviewed in introduction). The multifunctional protein beta -catenin is required for formation of the endogenous dorsal axis in Xenopus (Heasman et al., 1994; for review see Miller and Moon, 1996), strongly suggesting that if a secreted signaling factor or downstream elements of its signal transduction cascade were involved in axis formation, then these pathways should impinge upon beta -catenin. The present study investigated two heretofore unresolved issues: whether endogenous beta -catenin is enriched in dorsal blastomeres by the 32-cell stage, when blastomere and cytoplasm transfer data demonstrate a dorsal determining activity is present in these cells, and whether any of the secreted signaling factors that can mimic this dorsal-determining activity can also modulate beta -catenin levels. We report that endogenous beta -catenin is indeed expressed at higher levels in dorsal relative to ventral blastomeres of cleavage stage embryos and that it accumulates in dorsal nuclei by the 16- to 32-cell stages. These results provide the first demonstration that an endogenous protein with a dorsaldetermining activity is asymmetrically localized along the dorso-ventral axis in the cleavage stage of a vertebrate embryo. Our data further show that ectopic Wnt ligands and Xgsk-3, a serine/threonine kinase known to function in the Wnt pathway, can modulate the dorso-ventral levels of beta -catenin, while Vg1 and noggin do not.

beta -Catenin, Axis Specification, and the Definition of the Nieuwkoop Center Activity

There is a remarkable agreement between our observed patterns of enrichment of beta -catenin on the dorsal side of cleavage stage embryos and the ability of transplanted dorsal blastomeres or cytoplasm to initiate formation of a new axis. Specifically, cytoplasm and blastomere transfer experiments show that both dorsal vegetal and dorsal animal cells of 32-cell stage embryos, but not ventral cells, can induce a new axis when transplanted to the ventral side of a host embryo (reviewed in introduction). Strikingly, we show that beta -catenin is enriched in both the cytoplasm and nuclei of both dorsal vegetal and dorsal animal cells by the 32-cell stage, and this persists through the blastula stage when zygotic transcription commences. This timing and localization of beta -catenin expression is consistent with a likely role in the Nieuwkoop Center activity of the embryo.

The Nieuwkoop Center activity of Xenopus embryos has been operationally defined as the ability of transplanted cells or injected factors to elicit formation of a gastrula organizer in a non-cell autonomous manner (e.g., Smith and Harland, 1992). Thus, when noggin, BVg1, some Wnts, and beta -catenin RNAs are injected into prospective ventral vegetal cells, which will physically become part of the endoderm, the overlying cells develop with properties of the gastrula organizer, leading to a new axis with an ectopic notochord, somites, and neural structures. These observations have led some in the field to consider that the Nieuwkoop Center activity is a discrete physical entity and/or that it is present only in the dorsal vegetal region of embryos. However, this is not consistent with blastomere transplantation experiments showing an axis-inducing activity extending into the animal hemisphere (see introduction), nor does it readily explain the mesoderm-forming ability of isolated or rotated eight-cell animal pole cells (Cardellini, 1988; Grunz, 1994; Li et al., 1996). To account for these data, we have previously suggested (Kimelman et al., 1992; Moon and Christian, 1992) that the Nieuwkoop Center comprises a large region of the dorsal side of the embryo, spreading from the dorsal animal to the dorsal vegetal cells, with maximum activity in the dorsal vegetal cells. This is the same localization as we report here for beta -catenin during the cleavage stages.

The Wnt Pathway, but Not Vg1 or Noggin, Modulate beta -Catenin

Given the data presented here indicating that there are greater levels of beta -catenin on the dorsal side relative to the ventral side by the 32-cell stage, it is important to ask how this asymmetry arises. As a theoretical device for allowing rational discussion, one can divide the possibilities into mechanisms that require a secreted signaling factor and mechanisms that do not.

If a secreted signaling factor were involved in the mechanism that establishes the observed dorso-ventral asymmetry in beta -catenin, then this signaling factor should be able to mimic the Nieuwkoop Center activity. Secreted factors meeting these criteria include a subset of the Xwnt gene family, specifically Xwnt-1, Xwnt-3A, Xwnt-8, and Xwnt-8b (for reviews see by Du et al., 1995; Torres et al., 1996), as well as the unrelated factors noggin (Smith and Harland, 1992) and Vg1 (Dale et al., 1993; Thomsen and Melton, 1993). Of the Xwnts mentioned, only Xwnt-8b is maternally expressed and thus a candidate for an endogenous regulator of beta -catenin in cleaving embryos (Cui et al., 1995; Du et al., 1995). In the present study, we found that ectopic expression of Xwnt-8, and the highly related maternal Xwnt-8b, but not noggin (Smith and Harland, 1992) or BVg1 (Thomsen and Melton, 1993), consistently promotes the accumulation of beta -catenin in embryos, as monitored by immunocytochemistry and Western blot analyses. Thus, of the small set of candidates proposed to be initiators of dorsal development, only Xwnts were able to regulate steady-state levels of beta -catenin in embryos. However, there are no loss of function data showing that any Xwnt is actually required for axis specification. Since ectopic expression of a dominant negative Xwnt-8 blocks formation of ectopic but not endogenous axes (Hoppler et al., 1996) and since overexpression of Xwnt-5A blocks ectopic axis formation by Xwnt-8 and Xwnt-8b but not the endogenous axes (Torres et al., 1996), available data support the conclusion that even if Wnt ligands are involved in endogenous axis formation, they are not strictly required.

Somewhat unexpectedly, we found that overexpression of Xwnt-5A does not promote the stabilization of newly synthesized beta -catenin assayed before the onset of zygotic expression, but when sibling embryos are cultured further and assayed for beta -catenin levels after MBT, they now demonstrate much higher levels of newly synthesized beta -catenin relative to control embryos. We speculate that this might be explained by the expression of new Wnt receptors (Bhanot et al., 1996; Yang-Snyder et al., 1996) after the midblastula stage, with these new receptors allowing the Xwnt-5A to activate an intracellular pathway that stabilizes beta -catenin. Despite the interesting effects of Xwnt-5A on beta -catenin levels after MBT, our data suggest that neither Xwnt-5A nor the functionally similar Xwnt-11 (Du et al., 1995; Torres et al., 1996) are involved in stabilizing beta -catenin levels during the cleavage stages when cytoplasm and blastomere transplantation experiments have demonstrated the presence of a localized dorsal-determining activity.

Do any data discriminate between greater synthesis versus stability of beta -catenin on the dorsal side? While we have not investigated regional synthesis of beta -catenin owing to technical difficulties in such studies, experiments reported here and elsewhere (Domingues et al., 1995; He et al., 1995; Pierce and Kimelman, 1995; Yost et al., 1996) strongly argue for Xgsk-3 regulating the stability of newly synthesized beta -catenin such that it is degraded on the prospective ventral side and stable on the dorsal side. Specifically, we injected RNA encoding c-myc-epitope-tagged beta -catenin to allow us to follow newly synthesized beta -catenin. This injection protocol leads to the RNA being distributed throughout the entire marginal zone on the dorsal and ventral sides. Despite the widespread distribution of the RNA (and uniform synthesis of unrelated proteins such as beta -galactosidase and green fluorescent protein, data not shown), we see the accumulation of the c-myc epitope only on the future dorsal side. This almost certainly involves synthesis of beta -catenin throughout the embryo, followed by selective degradation of beta -catenin on the ventral but not the dorsal side. We have presented three results in further support of this conclusion. First, coinjection of a dnXgsk-3 results in the accumulation of beta -catenin on the ventral as well as dorsal side. Second, treating embryos with LiCl, which inhibits Xgsk-3 (Klein and Melton, 1996), similarly promotes the ventral accumulation of epitope-tagged beta -catenin, as well as increasing its steady-state levels. Third, mutating the amino-terminal site of beta -catenin required for phosphorylation by Xgsk-3 (Yost et al., 1996) overrides the endogenous mechanism that promotes the dorsal accumulation of beta -catenin, such that the mutant beta -catenin accumulates on both the ventral and dorsal sides.

We propose that the dorsal-ventral asymmetry in beta -catenin in embryos before MBT is attributable to the broad synthesis of beta -catenin followed by its being targeted for degradation by Xgsk-3 on the ventral side to a greater extent than on the dorsal side. This is likely a very direct effect since Xgsk-3 directly phosphorylates beta -catenin in vitro and since deletion of the major in vitro phosphorylation site blocks most in vivo phosphorylation and leads to increased accumulation of beta -catenin (Yost et al., 1996). A prediction of this model is that there are dorsal-ventral differences in Xgsk-3 activities, or differences in other components of the catabolic pathway for beta -catenin. Further investigation is required to determine how the postfertilization cortical rotation may promote such differences.


Summary

In conclusion, we have demonstrated that beta -catenin accumulates in the cytoplasm and nuclei of dorsal blastomeres of cleavage stage embryos beginning at the two-cell stage, in a manner dependent upon the postfertilization cortical rotation and regulated by Xgsk-3, a kinase in the Wnt signaling pathway. We have shown that ectopic expression of specific Xwnts , and inhibition of Xgsk-3, elevates levels of beta -catenin before MBT, but the axis-inducing factors BVg1 and noggin do not. This leads us to conclude that neither endogenous Vg1 nor noggin is likely to play a role in establishing the observed asymmetries in endogenous beta -catenin in cleavage stage embryos, while Xgsk-3 working directly or in response to Wnt signaling is further implicated in axis specification. These data also suggest that Xwnts stimulate ectopic axis formation through a pathway that is distinct from BVg1 and noggin.

Since elevation of steady-state levels and nuclear accumulation of dorsal beta -catenin correlates with its ability to induce gene expression (Funayama et al., 1995, Kelly et al., 1995, Yost et al., 1996; Schneider et al., 1996), likely through interacting directly with HMG box architectural transcription factors (Behrens et al., 1996; Molenaar et al., 1996), we propose that greater levels of beta -catenin in nuclei of dorsal blastomeres of cleavage stage embryos presage and influence dorso-ventral differences in gene expression at the blastula stage, when zygotic transcription commences. Thus, beta -catenin is likely to play a key role in remodeling zygotic chromatin in dorsal blastomeres before the initiation of zygotic transcription. When zygotic transcription begins, these chromatin effects may work in a combinatorial manner with other growth factor pathways (for review see by Kimelman et al., 1992) to promote the expression of specific zygotic dorsal regulatory genes such as siamois (Lemaire et al., 1995), which is responsive to Wnt (Carnac et al., 1996; Yang-Snyder et al., 1996) and beta -catenin (Brannon and Kimelman, 1996). Dorsal beta -catenin may also function in other capacities, since beta -catenin mimics the ability of Xwnt-8 to enhance gap junctional coupling in the absence of transcription (Guger and Gumbiner, 1995). Remaining challenges include determining whether the postfertilization cortical rotation of Xenopus eggs establishes the dorso-ventral differences in beta -catenin levels through regulation of Xgsk-3 activity, and if so, whether this kinase is regulated in response to egg activation in a Wnt-independent or -dependent manner.


Footnotes

Carolyn Larabell and Monica Torres contributed equally to this work.

Received for publication 6 September 1996 and in revised form 5 November 1996.

   M. Torres, J. Miller, and R. Moon were supported by the Howard Hughes Medical Institute, and B. Rowning (HD27525 and HD 29360 to R.T. Moon), C. Yost (GM 07270), and D. Kimelman (HD27262) were supported by Public Health Service Awards from the National Institutes of Health. C. Larabell was supported by the Office of Health and Environmental Research, U.S. Department of Energy.
   Note Added in Proof. beta -Catenin was translated in vitro and labeled with [35S]methionine (as in Yost et al., 1996), then comparable amounts were injected into either dorsal or ventral blastomeres of four-cell embryos. Monitoring the injected beta -catenin by SDS-PAGE and autoradiography demonstrates that it is more stable on the dorsal side of the embryo, as predicted by our experiments presented here and in Yost et al. (1996).
   Address all correspondence to Randall T. Moon, Howard Hughes Medical Institute, Campus Box 357370, Room K536C Health Sciences Building, University of Washington School of Medicine, Seattle, WA 98195. Tel.: (206) 543-1722. Fax: (206) 543-0858.rtmoon{at}u.washington.edu

We thank M. Vo Sum (Howard Hughes Medical Institute, Seattle, WA) for assistance in preparing figures and J. Gerhart (University of California, Berkeley, CA) for discussions.


Abbreviations used in this paper

DAM, dorsal animal marginal; dnXgsk-3, dominant negative (kinase-dead) Xenopus glycogen synthase kinase-3; DVM, dorsal vegetal marginal; MBT, mid-blastula transition; MeOH, methanol; ptbeta -catenin-myc, point mutant in beta -catenin-myc; VAM, ventral animal marginal; VVM, ventral vegetal marginal; Xgsk-3, Xenopus glycogen synthase kinase-3; Xwnt, Xenopus Wnt.


References

  1. Behrens, J., J.P. von Kries, M. Kuhl, L. Bruhn, D. Wedlich, R. Grosschedl, and W. Birchmeier. 1996. Functional interaction of beta -catenin with the transcription factor LEF-1. Nature (Lond.) 382: 638-642
  2. Bhanot, P., M. Brink, C.H. Samos, J.-C. Hsieh, Y. Wang, J.P. Macke, D. Andrew, J. Nathans, and R. Nusse. 1996. A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature (Lond.) 382: 225-230
  3. Brannon, M., and D. Kimelman. 1996. Activation of Siamois by the Wnt pathway. Dev. Biol. 180: 344-347
  4. Cardellini, P.. 1988. Reversal of dorsal ventral polarity in Xenopus laevis embryos by 180° rotation of the animal micromeres at the eight cell stage. Dev. Biol. 128: 428-434
  5. Carnac, G., L. Kodjabachian, J.B. Gurdon, and P. Lemaire. 1996. The homeobox gene Siamois is a target of the Wnt dorsalization pathway and triggers organizer activity in the absence of mesoderm. Development (Camb.) 122: 2055-3065 .
  6. Christian, J.L., and R.T. Moon. 1993. Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus. Genes Dev. 7: 13-28 [Abstract].
  7. Christian, J.L., D.J. Olson, and R.T. Moon. 1992. Xwnt-8 modifies the character of mesoderm induced by bFGF in isolated Xenopus ectoderm. EMBO (Eur. Mol. Biol. Organ.) J. 11: 33-41 [Abstract].
  8. Cui, Y., J.D. Brown, R.T. Moon, and J.L. Christian. 1995. Xwnt 8b: a maternally expressed Xenopus Wnt gene with a potential role in establishing the dorsoventral axis. Development (Camb.) 121: 2177-2186 [Abstract/Free Full Text].
  9. Dale, L., G. Matthews, and A. Colman. 1993. Secretion and mesoderm-inducing activity of the TGFbeta -related domain of Xenopus Vg1. EMBO (Eur. Mol. Biol. Organ.) J. 12: 4471-4480 [Abstract].
  10. DeMarais, A., and R.T. Moon. 1992. The armadillo homologs beta -catenin and plakoglobin are differentially expressed during early development of Xenopus laevis. Dev. Biol. 153: 337-346
  11. Domingues, I., K. Itoh, and S.Y. Sokol. 1995. Role of glycogen synthase kinase 3-beta as a negative regulator of dorsoventral axis formation in Xenopus embryos. Proc. Natl. Acad. Sci. USA. 92: 8498-8502 [Abstract].
  12. Du, S.J., S. Purcell, J.L. Christian, L.L. McGrew, and R.T. Moon. 1995. Identification of distinct classes and functional domains of Wnts through expression of wild-type and chimeric proteins in Xenopus embryos. Mol. Cell. Biol. 15: 2625-2634 [Abstract].
  13. Fagotto, F., and B.M. Gumbiner. 1994. beta -catenin localization during Xenopus embryogenesis: accumulation at tissue and somite boundaries. Development (Camb.) 120: 3667-3679 [Abstract/Free Full Text].
  14. Fujisue, M., Y. Kobayakawa, and K. Yamana. 1993. Occurrence of dorsal axisinducing activity around the vegetal pole of an uncleaved Xenopus egg and displacement to the equitorial region by cortical rotation. Development (Camb.) 118: 163-178 [Abstract/Free Full Text].
  15. Funayama, N., F. Fagotto, P. McCrea, and B.M. Grumbiner. 1995. Embryonic axis induction by the armadillo repeat domain of beta -catenin: evidence for intracellular signaling. J. Cell Biol. 128: 959-968 [Abstract].
  16. Giebelhaus, D.H., B.D. Zelus, S.K. Henchman, and R.T. Moon. 1987. Changes in the expression of alpha -fodrin during embryonic development of Xenopus laevis. J. Cell Biol. 105: 843-853 [Abstract].
  17. Gerhart, J.C., M. Danilchick, T. Doniach, S. Roberts, B. Rowning, and R. Stewart. 1989. Cortical rotation of the Xenopus egg: consequences for the anteroposterior pattern of embryonic dorsal development. Development (Camb.). 107(Suppl.):37-51.
  18. Gimlich, R.L.. 1986. Acquisition of developmental autonomy in the equatorial region of the Xenopus embryo. Dev. Biol. 115: 340-352
  19. Grunz, H.. 1994. The four animal blastomeres of the eight-cell stage of Xenopus laevis are intrinsically capable of differentiating into dorsal mesodermal derivatives. Int. J. Dev. Biol. 38: 69-76
  20. Guger, K.A., and B.M. Gumbiner. 1995. beta -Catenin has Wnt-like activity and mimics the Nieuwkoop signaling center in Xenopus dorsal-ventral patterning. Dev. Biol. 172: 115-125
  21. Haegel, H., L. Larue, M. Ohsugi, L. Fedorov, K. Herrenknecht, and R. Kemler. 1995. Lack of beta -catenin affects mouse development at gastrulation. Development (Camb.) 121: 3529-3537 [Abstract/Free Full Text].
  22. He, X., J.P. Saint, Jeannet, J.R. Woodgett, H.E. Varmus, and I.B. Dawid. 1995. Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos. Nature (Lond.) 374: 617-622
  23. Heasman, J., A. Crawford, K. Goldstone, P. Garner, Hamrick, B. Gumbiner, P. McCrea, C. Kintner, C.Y. Noro, and C. Wylie. 1994. Overexpression of cadherins and underexpression of beta -catenin inhibit dorsal mesoderm induction in early Xenopus embryos. Cell. 79: 791-803
  24. Holowacz, T., and R.P. Elinson. 1993. Cortical cytoplasm, which induces dorsal axis formation in Xenopus, is inactivated by UV irradiation of the oocyte. Development (Camb.) 119: 277-285 [Abstract/Free Full Text].
  25. Hoppler, S., J.D. Brown, and R.T. Moon. 1996. Expression of a dominant negative Wnt blocks induction of MyoD in Xenopus embryos. Genes Dev. 10: 2805-2817 [Abstract].
  26. Kageura, H.. 1990. Spatial distribution of the capacity to initiate a secondary embryo in the 32-cell embryo of Xenopus laevis. Dev. Biol. 142: 432-438
  27. Kelly, G.M., D.F. Erezyilmaz, and R.T. Moon. 1995. Induction of a secondary embryonic axis in zebrafish occurs following the overexpression of beta -catenin. Mech. Dev. 53: 261-273
  28. Kimelman, D., J.L. Christian, and R.T. Moon. 1992. Synergistic principles of development: overlapping patterning systems in Xenopus mesoderm induction. Development (Camb.) 116: 1-9 [Abstract/Free Full Text].
  29. Klein, P.S., and D.A. Melton. 1996. A molecular mechanism for the action of lithium during early development. Proc. Natl. Acad. Sci. USA. 93: 8455-8459 [Abstract/Free Full Text].
  30. Klymkowsky, M.W., L.M. Maynell, and A.G. Polson. 1987. Polar asymmetry in the organization of the cortical cytokertain system of Xenopus laevis oocytes and embryos. Development (Camb.) 100: 543-557 [Abstract].
  31. Ku, M., and D.A. Melton. 1993. Xwnt-11: maternally expressed Xenopus Wnt gene. Development (Camb.) 119: 1161-1173 [Abstract/Free Full Text].
  32. Larabell, C.A., B.A. Rowning, J. Wells, M. Wu, and J.C. Gerhart. 1996. Confocal microscopy analysis of living Xenopus eggs and the mechanism of cortical rotation. Development (Camb.) 122: 1281-1289 [Abstract/Free Full Text].
  33. Lemaire, P., N. Garrett, and J.B. Gurdon. 1995. Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. Cell. 81: 85-94
  34. Li, S.H., Z.R. Mao, S.Y. Yan, and H. Grunz. 1996. Isolated dorsal animal blastomeres of Xenopus laevis are capable to form mesodermal derivatives, while the ventral animal blastomeres differentiate into ciliated epidermis only. Zool. Sci. 13: 125-131 .
  35. McMahon, A.P., and R.T. Moon. 1989. Ectopic expression of the proto-oncogene int-1 in Xenopus embryos leads to duplication of the embryonic axis. Cell. 58: 1075-1084
  36. Miller, J.R., and R.T. Moon. 1996. Signal transduction through beta -catenin and specification of cell fate during embryogenesis. Genes Dev. 10: 2527-2539
  37. Molenaar, M., M. van de Wetering, M. Oosterwegel, J. Peterson-Maduro, S. Godsave, V. Korinek, J. Roose, O. Destree, and H. Clevers. 1996. XTcf-3 transcripton factor mediates beta -catenin-induced axis formation in Xenopus embryos. Cell. 86: 391-399
  38. Moon, R.T., and J.L. Christian. 1992. Competence modifiers synergize with growth factors during mesoderm induction and patterning in Xenopus. Cell. 71: 709-712
  39. Moon, R.T., R.M. Campbell, J.L. Christian, L.L. McGrew, J. Shih, and S. Fraser. 1993. Xwnt-5A, a maternal Wnt that affects morphogenetic movements after overexpression in embryos of Xenopus laevis. Development (Camb.) 119: 97-111 [Abstract/Free Full Text].
  40. Pierce, S.B., and D. Kimelman. 1995. Regulation of Spemann organizer formation by the intracellular kinase Xgsk-3. Development (Camb.) 121: 755-765 [Abstract/Free Full Text].
  41. Sakai, M.. 1996. The vegetal determinants required for the Spemann organizer move equatorially during the first cell cycle. Development (Camb.) 122: 2207-2214 [Abstract/Free Full Text].
  42. Schneider, S., H. Steinbeisser, R.M. Warga, and P. Hausen. 1996. beta -Catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos. Mech. Dev. 57: 191-198
  43. Smith, W., and R.M. Harland. 1992. Expression cloning of noggin, a new dorsalizing factor localized in the Spemann organizer in Xenopus embryos. Cell. 70: 829-840
  44. Sokol, S., and D.A. Melton. 1992. Interaction of Wnt and activin in dorsal mesoderm induction in Xenopus. Dev. Biol. 154: 348-355
  45. Spencer, M., D.H. Giebelhaus, G.M. Kelly, J. Bicknell, S.K. Florio, A.H. Milam, and R.T. Moon. 1990. Membrane skeleton protein 4.1 in developing Xenopus: expression in postmitotic cells of the retina. Dev. Biol. 139: 279-291
  46. Thomsen, G.H., and D.A. Melton. 1993. Processed Vg1 is an axial mesoderm inducer in Xenopus. Cell. 74: 433-441
  47. Torres, M.A, J.A. Yang-Snyder, S.M. Purcell, A.A. DeMarais, L.L. McGrew, and R.T. Moon. 1996. Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A class and by a dominant negative cadherin in early Xenopus development. J. Cell Biol. 133: 1123-1137 [Abstract].
  48. Yang-Snyder, J., J.R. Miller, J.D. Brown, C.-J. Lai, and R.T. Moon. 1996. A frizzled homolog functions in a vertebrate Wnt signaling pathway. Curr. Biol. 6: 1302-1306
  49. Yost, C., M. Torres, J.R. Miller, E. Huang, D. Kimelman, and R.T. Moon. 1996. The axis-inducing activity, stability, and subcellular distribution of beta -catenin is regulated in Xenopus embryos by glycogen synthase kinase 3.  Genes Dev. 10: 1443-1454 [Abstract].
  50. Yuge, M., Y. Kobayakawa, M. Fujisue, and K. Yamana. 1990. A cytoplasmic determinant for dorsal axis formation in an early embryo of Xenopus laevis. Development (Camb.) 110: 1051-1056 [Abstract].

Copyright © 1997 by The Rockefeller University Press.