* Howard Hughes Medical Institute and Department of Pharmacology, Department of Biochemistry, University of Washington
School of Medicine, Seattle, Washington 98195; and § Lawrence Berkeley National Laboratory,
Department of Molecular and
Cell Biology, University of California at Berkeley, Berkeley, California 94720
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
-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
-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
-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
-catenin. Steadystate levels and nuclear accumulation of
-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
-catenin levels before the mid-blastula stage. As
greater levels and nuclear accumulation of
-catenin on
the future dorsal side of the embryo correlate with the
induction of specific dorsal genes, our data suggest that
early asymmetries in
-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
-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 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 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 While these reports indicate a role for 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 cDNA Constructs and Embryo Microinjection
cDNAs encoding Xenopus Wnt-8 (Xwnt)1, Xwnt-5A, BVg1, noggin, prolactin, and Confocal Microscopic Localization of 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
Embryos were viewed using a confocal laser scanning microscope
(model 1024; BioRad Labs, Hercules, CA) using rhodamine filters to collect the To determine the effects of Xwnt-8 or Xwnt-5A on endogenous
Enzyme-linked Whole-Mount Immunolocalization
of Where indicated, embryos were processed for whole-mount immunolocalization of c-myc-tagged ectopic Western Blot Analyses
To compare potential dorso-ventral differences in the steady-state levels
of endogenous
To monitor endogenous and ectopic Dorso-ventral Asymmetries in We first conducted a confocal microscopical analysis of
the early Xenopus embryo to investigate whether there
were asymmetries in To determine the earliest time at which asymmetries in
endogenous Staining of eight-cell embryos with control antibodies
for membrane skeleton protein 4.1 (Fig. 3 A), Strikingly, by the 16-cell stage To test whether the apparent differences in the spatial
distribution of endogenous Since UV irradiation of the vegetal pole during the first
cell cycle blocks formation of the dorsal axis (Gerhart et al., 1989 Wnt Signaling, but Not BVg1 or Noggin, Stabilizes
We next tested whether we could detect dorso-ventral
asymmetries in the accumulation of ectopically expressed
Table I.
Modulation of ). 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.
; 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
).
-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
).
-Catenin meets a number of
reasonable criteria for playing a role in specification of the
dorso-ventral axis in vertebrate embryos.
-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
-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
-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
-catenin to induce a
secondary axis (Molenaar et al., 1996
). These data collectively support the hypothesis that dorsal
-catenin interacts with architectural transcription factors to regulate the
expression of dorsal genes required for dorso-ventral axis
specification.
-catenin in
dorso-ventral axis formation, the following two key issues
need to be resolved: (a) Is endogenous
-catenin expressed appropriately to play a role in axis specification?
If
-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
-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
-catenin
levels? Since
-catenin is required for the formation of the
endogenous axes (Heasman et al., 1994
), and since
-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
-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
-catenin levels.
-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
-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
-catenin is regulated in a Wnt-independent manner.
Materials and Methods
-galactosidase were obtained and transcribed in vitro as described (Torres et al., 1996
), as were wild-type and point mutant Xenopus
-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.
-Catenin
; 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
-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
-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 -catenin in
early Xenopus embryos. Whole-mount staining with antibody to
-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
-catenin in the periphery of the animal dorsal blastomeres (A and C) and vegetal
dorsal blastomeres (B and D). Ventral blastomeres stain for
-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 -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
-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
-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,
-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
-catenin. At the blastula stage,
-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
-catenin staining, even when
blastula were bisected before immunolabeling to assure access of the antibodies to the inner blastomeres. Specific
-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 -catenin.
To assess whether the patterns
of
-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
-catenin (Fig. 1, A and B),
membrane skeleton protein 4.1 (A),
-spectrin (B), and cytokeratin (C) antibodies show no dorsal enrichment. The
-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
-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
-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 -catenin. (A)
The dorso-ventral asymmetry in
-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
-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
-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
-catenin
throughout the 32-cell embryo (d). (B) Injection of a
mixture of prolactin and
-catenin-myc RNAs leads to
the accumulation of
-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
-catenin-myc similar to
controls (right embryo) is evident when Xgsk-3 RNA is
injected with the
-catenin-
myc RNA (b). Inhibition of
endogenous Xgsk-3 by injecting dnXgsk-3 RNA increases
and expands the accumulation of ectopic
-catenin-
myc to the entire embryonic
marginal zone (c). Treatment with lithium also expands the
domain of
-catenin-myc accumulation in embryos overexpressing prolactin (d) or
Xgsk-3 (e). Expression of
pt
-catenin-myc (which encodes a form of
-catenin that is not phosphorylated by Xgsk-3; Yost et al., 1996
) with control prolactin
results in the accumulation of pt
-catenin-myc everywhere in the embryo (f). In these experiments 1 ng of
-catenin-myc or pt
-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
-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
-catenin by Western blot analysis. Lane 1, Uninjected controls do not express
-catenin-myc; lane 2, embryos injected with
-catenin-myc RNA express the encoded protein; lane 3, injection of
-catenin-myc RNA as in lane 2 followed by treatment with LiCl leads to greater accumulation of
-catenin-myc.
[View Larger Version of this Image (49K GIF file)]
-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
-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
-catenin in cleavage stage embryos
(Fagotto and Gumbiner, 1994
; Schneider et al., 1996
).
-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-
-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
-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
-catenin.
[View Larger Version of this Image (55K GIF file)]
-Catenin
-catenin using previously described
methods for localizing c-myc (Christian and Moon, 1993
). Where indicated, embryos were injected with various RNAs before localization of
-catenin-myc.
-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-
-catenin
polyclonal antibody (Yost et al., 1996
), a rabbit anti-
-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
-catenin,
-spectrin, and tubulin were quantitated by use of densitometry (model UA-5 Absorbance/Fluorescence detector; Isco, Lincoln, NE). Dorsal and ventral
-catenin levels were compared after normalization for
-spectrin or tubulin, to control for
variability in protein content. Dorsal
-catenin values were set at 100%
and ventral
-catenin levels were expressed as a ratio: (ventral
-catenin/
dorsal
-catenin) × 100%.
Fig. 4.
Western blot analysis of microdissected 32-cell Xenopus embryos reveals a dorso-vegetal enrichment of -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
-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
-catenin as the DVM zones. The
VVM portions dissected from UV-irradiated embryos contained
more
-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
-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
-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
-catenin,
-spectrin, and tubulin. Ventral levels of
-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
-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
-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)]
-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-
-catenin or anti-c-myc antibodies, respectively (above and Yost et al., 1996
).
Endogenous
-catenin signals were normalized to
-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
-catenin quantitation or as described (Yost et al.,
1996
) for ectopic
-catenin-myc quantitation.
-catenin levels present in control samples were set at 100% and experimental samples were expressed
as a ratio: (experimental
-catenin/control
-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
-Catenin in Cleavage
Stage Embryos
-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
-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.
-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
-catenin in the peripheral cytoplasm (94%, n = 17 embryos; Fig. 2 A), with
the elevated
-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,
-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
-catenin (Fig. 2 K) when fluorescence sensitivity is increased to levels that obscure the
dorso-ventral differences.
-spectrin
(Fig. 3 B), or cytokeratin (Fig. 3 C) did not reveal any dorsal enrichment for these proteins, demonstrating that the
dorsal enrichment of
-catenin is not simply a reflection of
the patterns of expression of structural proteins. Moreover,
-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.
-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
-catenin in
the dorsal (Fig. 2 I) relative to ventral (Fig. 2 H) blastomeres. At the blastula stage, nuclei stain for
-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
-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
-catenin-stained nucleus in Fig. 2 I (data
not shown).
-catenin in the early Xenopus
embryo reflect dorso-ventral differences in steady-state
levels of
-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
-catenin
present in the dorsal vegetal region (DVM) (n = 5 experiments, P < 0.005 by Student's t test; Fig. 4 B) after normalizing
-catenin to the levels of the membrane skeleton
protein
-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
-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
-catenin
present in the dorsal animal region (DAM) (n = 5 experiments, P < 0.05 by Student's t test) after normalization to
-spectrin. The comparable dorso-ventral distribution of
-catenin relative to tubulin in the animal hemisphere suggests that the dorsal enrichment of
-catenin observed by
confocal microscopy is shared by tubulin, while in the vegetal hemisphere
-catenin is dorsally enriched compared to both
-spectrin and tubulin. Although the
-spectrin
and
-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).
), we tested its effects on the dorso-ventral steadystate levels of
-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
-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
-catenin remain relatively unchanged after UV irradiation sufficient to ventralize embryos; further
UV irradiation reduces
-catenin levels nonspecifically
(not shown). We do not know why the greater level of
-catenin on the ventral side of UV-irradiated embryos does
not cause this side to become dorsal, since ectopic expression of
-catenin in UV-irradiated embryos restores axis
formation (Gruger and Gumbiner, 1995; Yost et al., 1996
).
It is possible that the absolute level of
-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
-catenin such that it is no longer
functional.
-Catenin in Early Embryos
-catenin as a prelude to asking which signaling factors
might modulate this accumulation. We injected a mixture
of c-myc-epitope-tagged
-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
-catenin levels relative to uninjected embryos (data
not shown). In these control embryos, the
-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
-catenin occurs on the prospective dorsal half of the
developing embryo. These results establish that the ectopic tracer
-catenin-myc accumulates in a manner similar to the spatial pattern of endogenous
-catenin (Fig. 2).
Although
-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
-galactosidase
RNA (not shown). The localized accumulation of
-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
-catenin for degradation (Yost et al., 1996
).
Fig. 5.
Wnts, but not BVg1 or noggin, are able to modulate ectopic -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
-catenin-myc and prolactin display a dorsal accumulation of ectopic
-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
-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
-catenin-myc accumulation on the ventral side, while
-catenin-myc accumulates on the dorsal side similarly to control embryos. (G)
Xwnt-5A RNA does not alter the pattern of
-catenin-myc accumulation during the early cleavage stages, but causes
-catenin-myc to
accumulate around the entire marginal zone after MBT (H). In these experiments, 1 ng of
-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)]
-catenin-myc Accumulation
Since both endogenous and ectopic -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
-catenin on the ventral sides of embryos. We injected
-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
-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
-catenin-myc before MBT when compared to the pattern in control-injected embryos (Fig 5 C),
nor did they promote increased accumulation of
-catenin-myc after MBT (Table I). In contrast, Xwnt-8 consistently expanded the domain of
-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
-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
-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 -catenin accumulation, we injected embryos
with RNAs encoding
-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-
-catenin antibodies to monitor
the steady state levels of ectopic and endogenous
-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
-catenin-myc an average of 231% when compared to embryos injected with
-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
-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
-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
-catenin-myc accumulation, consistent with their lack of
effect on
-catenin-myc staining in intact embryos (Fig. 5).
Turning to the effects of these secreted factors on endogenous -catenin, Western blots of embryos injected
with Xwnt-8 versus prolactin RNA reveal that Xwnt-8 elevated endogenous
-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
-catenin-
myc (Fig. 6 A). In these same experiments, Xwnt-5A, BVg1, and noggin RNAs did not reproducibly elevate endogenous
-catenin after normalization of protein levels
to
-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
-catenin.
We have previously shown that increasing the steadystate levels of endogenous -catenin by inhibiting Xgsk-3
activity in Xenopus embryos results in a greater nuclear
accumulation of
-catenin (Yost et al., 1996
). This led us
to test whether overexpression of Xwnt-8 would also lead
to the nuclear accumulation of endogenous
-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
-catenin. We injected embryos with Xwnt-8, Xwnt-5A, or prolactin RNA and stained animal cap explants of pre-MBT
embryos for endogenous
-catenin. Since pre-MBT explants were used, this is a time when Xwnt-8 but not Xwnt5A stabilizes ectopic
-catenin (Fig. 5). Control prolactinexpressing cells had nuclear staining of
-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
-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
-catenin (Fig. 7 C; 2% of
cells; n = 423 cells).
Implication of Localized Xgsk-3 Activity in Generating
Asymmetry in -Catenin
If the observed dorso-ventral differences in -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
-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
-catenin by confocal microscopy at the 32-cell stage. UVirradiated embryos (100%, n = 12) do not display dorsal
enrichment of
-catenin (Fig. 8 A, a) relative to control embryos (Fig. 8 A, b). Some UV-irradiated embryos showed
suppressed levels of
-catenin as we observed on Western
blots with high levels of irradiation, and some irradiated embryos showed an increase in ventral relative to dorsal
-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
-catenin in cleavage
stage embryos.
Recent data have demonstrated a postfertilization mechanism for regulating the stability of -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
-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
-catenin and allows the accumulation of
-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
-catenin in 2-32-cell embryos, then one would predict that active Xgsk-3 should antagonize this Wnt pathway and reduce dorsal
-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
-catenin
staining when overexpressed ventrally. Consistent with
these predictions, active Xgsk-3 reduces endogenous dorsal
-catenin staining in 32-cell embryos (Fig. 8 A, c, compared to controls, Fig. 2 E), while a dnXgsk-3 increases ventral
-catenin staining (Fig. 8 A, d).
Xgsk-3 activity also alters the spatial pattern of accumulation of ectopic -catenin by the blastula stage, both before and after MBT. Injection of
-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
-catenin-myc (Table I, Indistinguishable). Consistent with its effects on endogenous
-catenin at the 32-cell stage (Fig. 8 A), expressing a
dnXgsk-3 causes ectopic
-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
-catenin accumulation in a manner similar to Xwnt-8 and dnXgsk-3. Consistent with our expectations, lithium treatment expanded the domain of
-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
-catenin-myc
when analyzed by Western blots (Fig. 8 C, lane 3 relative
to 2). Finally, expression of point mutation (pt)
-catenin-
myc, which lacks the NH2-terminal conserved Xgsk-3
phosphorylation site required for efficient degradation of
-catenin (Yost et al., 1996
), resulted in the accumulation of pt
-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
-catenin.
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
-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
-catenin. The
present study investigated two heretofore unresolved issues: whether endogenous
-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
-catenin levels. We report that endogenous
-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
-catenin, while Vg1 and noggin do not.
-Catenin, Axis Specification, and the Definition of the
Nieuwkoop Center Activity
There is a remarkable agreement between our observed
patterns of enrichment of -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
-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
-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
-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
-catenin during the cleavage stages.
The Wnt Pathway, but Not Vg1 or Noggin,
Modulate -Catenin
Given the data presented here indicating that there are
greater levels of -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 -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
-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
-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
-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 -catenin assayed before the onset of zygotic
expression, but when sibling embryos are cultured further
and assayed for
-catenin levels after MBT, they now demonstrate much higher levels of newly synthesized
-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
-catenin. Despite the interesting effects of Xwnt-5A
on
-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
-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 -catenin on the dorsal side? While we have
not investigated regional synthesis of
-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
-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
-catenin
to allow us to follow newly synthesized
-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
-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
-catenin throughout the embryo, followed by
selective degradation of
-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
-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
-catenin, as well as increasing its steady-state levels. Third, mutating the amino-terminal site of
-catenin required for phosphorylation by Xgsk-3 (Yost et al., 1996
) overrides the endogenous mechanism that promotes the dorsal accumulation of
-catenin, such that the mutant
-catenin accumulates on
both the ventral and dorsal sides.
We propose that the dorsal-ventral asymmetry in -catenin in embryos before MBT is attributable to the broad
synthesis of
-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
-catenin in
vitro and since deletion of the major in vitro phosphorylation site blocks most in vivo phosphorylation and leads to
increased accumulation of
-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
-catenin. Further investigation is required to determine how the postfertilization cortical rotation may promote such differences.
In conclusion, we have demonstrated that -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
-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
-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 -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
-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,
-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
-catenin (Brannon and Kimelman, 1996
). Dorsal
-catenin may also
function in other capacities, since
-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
-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.
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.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.
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;
pt-catenin-myc, point mutant in
-catenin-myc;
VAM, ventral animal marginal;
VVM, ventral vegetal marginal;
Xgsk-3, Xenopus glycogen synthase kinase-3;
Xwnt, Xenopus Wnt.