Department of Medicine (Hematology-Oncology) and Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, 364 Clinical Research Building, 415 Curie Blvd, Philadelphia, PA 19104, USA
* Author for correspondence (e-mail: pklein{at}mail.med.upenn.edu)
Accepted 29 July 2003
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SUMMARY |
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Key words: Wnt/ß-catenin, Protein phosphatase 2A, B56, Xenopus, Embryo
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Introduction |
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Activation of the canonical Wnt/ß-catenin pathway leads to
stabilization and accumulation of ß-catenin, which in turn activates
transcription of Wnt target genes. In the absence of Wnts, cytosolic
ß-catenin binds to the axin complex, where it is phosphorylated and
targeted for rapid proteosomal degradation. Binding of Wnts to the coreceptors
frizzled and arrow/LRP inhibits ß-catenin phosphorylation and turnover,
allowing ß-catenin to accumulate and thus to activate TCF/Lef-mediated
transcription. In Drosophila, this pathway requires Dsh, a
cytoplasmic protein of unclear function, which has been placed downstream of
the receptor and upstream of the degradation complex
(Moon et al., 2002). This
pathway plays several roles during vertebrate development. Wnt signaling prior
to gastrulation is required for primary axis formation in vertebrates
(De Robertis et al., 2000
;
Harland and Gerhart, 1997
;
Heasman, 1997
;
Liu et al., 1999
). At a later
stage, the canonical Wnt pathway inhibits development of anterior structures,
including the head (De Robertis et al.,
2000
; Harland and Gerhart,
1997
; Heasman,
1997
; Mukhopadhyay et al.,
2001
). In addition, this pathway is required for
midbrain-hindbrain formation (McGrew et
al., 1999
; McMahon et al.,
1992
) and neural crest development (reviewed by
Wu et al., 2003
).
Protein phosphatase 2A (PP2A) is a heterotrimeric complex consisting of a
catalytic subunit (C), a structural subunit (A), and a variable regulatory
subunit (B), including B55, B56 and PR72, which are believed to regulate the
specificity and activity of PP2A (McCright
and Virshup, 1995). PP2A has been proposed to be involved in the
Wnt/ß-catenin pathway, based on the observations that the C and B56
subunits physically interact with Wnt pathway components and that
overexpression of PP2A subunits antagonizes Wnt/ß-catenin signaling. In
addition, treatment of HEK 293 cells with okadaic acid, an inhibitor of PP2A,
results in elevated ß-catenin protein levels
(Seeling et al., 1999
). The
epsilon isoform of B56 (PP2A:B56
) interacts with dsh and with the
adenomatous polyposis coli (APC) protein in yeast two hybrid assays;
furthermore, overexpression of PP2A:B56
inhibits
Wnt/ß-catenin signaling in tissue culture and in Xenopus embryos
(Gao et al., 2002
;
Li et al., 2001
;
Ratcliffe et al., 2000
;
Seeling et al., 1999
),
although the mechanism of this inhibition remains unclear. Analysis of the in
vivo function of PP2A:B56 subunits during embryonic development by
loss-of-function approaches has been hampered in part by the apparent
requirement for these subunits in cell survival; loss of PP2A:B56 family
members in fly causes early embryonic lethality
(Hannus et al., 2002
), and
depletion of PP2A:B56, by RNA interference (RNAi), in S2 cells
induces apoptosis (Li et al.,
2002
). Recently, however, widerborst, a fly mutant for
PP2A:B56, was identified in a screen for genes in the PCP pathway.
The widerborst phenotype includes formation of multiple wing hairs
and abnormal hair polarity in the wing blade. Further characterization placed
widerborst upstream of dsh and flamingo
(fla) in the PCP pathway. In zebrafish, knocking down
widerborst function inhibited convergent extension
(Hannus et al., 2002
), a type
of cell movement during vertebrate gastrulation that is probably regulated by
the PCP pathway (Tree et al.,
2002
). Depletion of widerborst also altered the
expression domain of goosecoid, a dorsally restricted organizer gene,
an effect that could represent disruption of either canonical or noncanonical
(PCP) Wnt signaling (Hannus et al.,
2002
). Therefore, a role for PP2A:B56 in canonical
Wnt/ß-catenin signaling has not yet been addressed directly by
loss-of-function analyses in any species.
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Materials and methods |
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Plasmid construction and morpholino
pCS2-PP2A:B56 was constructed by cloning a PCR fragment containing
the PP2A:B56
(AF298157, a gift from M. J. Ratcliffe) opening
reading frame into pCS2-FLAG. pCS2-PP2A:B56
-c was generated by
site-directed mutagenesis using the Quick-Change kit (Stratagene). All
constructs were verified by sequencing.
Morpholinos were ordered from Gene Tools (Corvallis, OR). The sequence of
the morpholino oligonucleotide directed against PP2A:B56 was:
5'-GAGGAGTGGTTGGTGCTGAGGACAT-3'; The mismatched control morpholino
had the sequence 5'-GAcGAcTGGTTGcTGCTGAcGAgAT-3'; and the standard
control morpholino had the sequence
5'-CCTCTTACCTCAGTTACAATTTATA-3'.
RT-PCR and whole-mount in situ hybridization
RNA extraction and RT-PCR methods were as described previously
(Yang et al., 2002). For host
transfer experiments, two embryos were included in each sample. For routine
experiments, five embryos were included in each sample. Whole-mount in situ
hybridization was performed as described previously
(Deardorff et al., 1998
).
Western blots
For western blot analysis, embryos or oocytes were homogenized (10 µl
per embryo) in modified RIPA buffer (159 mM NaCl, 50 mM Tris, pH 8, 1% NP40,
0.5% deoxycholate, 2 mM EDTA, 20 µg/ml aprotinin, 40 µg/ml leupeptin, 4
µg/ml pepstatin, 0.75 mM PMSF, 25 mM ß-glycerophosphate, 1 mM
Na3Vo4, 100 mM NaF). Lysates were centrifuged three
times at 14,000 g for 10 minutes in a tabletop centrifuge at
4°C. Supernatant was collected. For cytoplasmic ß-catenin analysis
(Fagotto et al., 1999), 50
µl lysate was incubated with 50 µl ConA-agarose beads (Calbiochem) at
4°C with rotation. Beads were removed 2 hours later by centrifuging at 500
g for 5 minutes at 4°C. Protein loading buffer was
included in each sample. Each sample was boiled at 100°C for 5 minutes and
separated by 7.5% SDS-PAGE electrophoresis. Western blots were performed
according to standard protocol with anti-Xenopus ß-catenin
(rabbit serum, 1:1000), anti-EGFP (Clontech, 1:1000), anti-Myc (9E10, Santa
Cruz, 1:1000), anti-FLAG (M2, Sigma, 1:1000) and anti-hnRNP (1:2000; gift from
Gideon Drefuss, University of Pennsylvania).
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Results |
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PP2A:B56 is maternally expressed (not shown) and uniformly
distributed in the embryo prior to midblastula transition (MBT)
(Fig. 1A,B). At the late
blastula stage, zygotic expression of PP2A:B56
begins in the
dorsal marginal zone (Fig. 1C),
partially overlapping the expression domain of two organizer-specific genes,
goosecoid (gsc) (Cho et
al., 1991
) and cerberus
(Bouwmeester et al., 1996
)
(data not shown). The expression of PP2A:B56
expands to the
lateral and ventral marginal zone shortly after the onset of gastrulation
(Fig. 1D). During the early
neurula stage, it is expressed exclusively in the neural ectoderm throughout
its anterior-posterior extent (Fig.
1E,F). Little if any expression can be detected in the underlying
mesoderm. The expression of PP2A:B56
persists in the anterior
region until the tadpole stage, when strong expression is detected in the eyes
and branchial arches (Fig. 1G).
The dynamic expression pattern of PP2A:B56
suggests that it may
play important roles during early embryonic development.
|
|
Depletion of maternal PP2A:B56 resulted in a severe defect
in dorsal development. Host-transfer embryos previously injected with the
PP2A:B56
morpholino did not form a dorsal blastopore lip
(Fig. 2C), a morphological
marker of the Spemann organizer (Harland
and Gerhart, 1997
), while sibling controls formed a normal dorsal
lip at stage 10 (Fig. 2B).
Later development could not be assessed in PP2A:B56
morpholino-injected embryos derived from host transfer, as the embryos died in
early neurula stages. This embryonic lethality does not appear to be a
nonspecific effect of morpholino injection or the host transfer technique, as
oocytes injected with 40 ng of control morpholino or 10 ng of a control
mismatched morpholino (similar in sequence to the PP2A:B56
morpholino, but containing 5 point mutations) developed relatively normally
when carried through the host-transferred procedure.
To confirm that depletion of maternal PP2A:B56 interfered
with dorsal development, host-transfer embryos were harvested at the
midgastrula stage and expression of dorsal-specific genes was analyzed by
RT-PCR (Fig. 2D). The
expression of the dorsal-specific genes noggin
(Smith and Harland, 1992
)
gsc, cerberus, dkk1 (Glinka et
al., 1998
), siamois
(Lemaire et al., 1995
) and
Xnr3 (Smith et al.,
1995
) was markedly reduced in PP2A:B56
knockdown embryos
while the expression of the ventrally restricted genes sizzled
(Salic et al., 1997
) and
Xvent2 (Onichtchouk et al.,
1996
) was increased, consistent with the loss of the dorsal
blastopore lip. Since the expression of all dorsal-specific genes analyzed in
these experiments was reduced, we conclude that maternal PP2A:B56
function is required for dorsal gene expression and organizer formation in
Xenopus embryos.
This loss of dorsal development and dorsal gene expression in embryos
lacking maternal PP2A:B56 is highly similar to depletion of maternal
ß-catenin, which is required for dorsal axis specification
(Heasman et al., 1994
;
Heasman et al., 2000
;
Xanthos et al., 2002
).
Therefore, we directly compared gene expression profiles in gastrulae derived
from embryos in which maternal PP2A:B56
or ß-catenin had been
depleted. Consistent with previous observations
(Heasman et al., 1994
;
Heasman et al., 2000
;
Xanthos et al., 2002
),
interfering with maternal ß-catenin function resulted in down-regulation
of all dorsal markers analyzed at this stage, including noggin, gsc,
cerberus, dkk1, siamois and Xnr3, similar to the PP2A:B56
knockdown embryos. Furthermore, the expression of sizzled, and
Xvent2, two ventral genes, was increased in both PP2A:B56
and
ß-catenin knockdown embryos, while Xvent1, sox17, mixer, msx1,
and zygotic PP2A:B56
did not change significantly in either
group (Fig. 2D).
This loss of dorsal development after knockdown of maternal PP2A:B56
and the striking similarity to embryos lacking maternal ß-catenin raises
the intriguing possibility that PP2A:B56
is required for maternal
Wnt/ß-catenin signaling. To explore this possibility further, we examined
maternal PP2A:B56
knockdown embryos at earlier stages of development.
The Xenopus nodal-related genes Xnr5 and Xnr6
require maternal ß-catenin for expression
(Takahashi et al., 2000
), and
both genes are transcribed prior to the midblastula transition (MBT) in a
ß-catenin-dependent manner (Yang et
al., 2002
). Expression of Xnr5 and Xnr6 was
reduced in PP2A:B56
knockdown embryos at the 1,000 cell stage (two cell
divisions prior to MBT) (Fig.
2E). In addition, post-MBT expression of Xnr3 and
siamois, which also requires maternal ß-catenin, was severely
reduced at the early gastrula stages (stage 10) in PP2A:B56
knockdown
embryos (Fig. 2E). Thus, the
early dorsal expression of multiple genes known to be targets of
Wnt/ß-catenin signaling requires maternal PP2A:B56
.
To test whether the repression of dorsal gene expression by morpholino
injection is specific to PP2A:B56 loss-of-function, we rescued dorsal
gene expression by coinjection of oocytes with the PP2A:B56
morpholino and an mRNA (PP2A:B56
-c) lacking the
morpholino target sequence. Transferred embryos were harvested at stage 9 for
gene expression analysis. PP2A:B56
-c mRNA completely
rescued the expression of Xnr3, Xnr5 and Xnr6 in a
dose-dependent manner, and weakly rescued siamois expression at this
stage (Fig. 2F). In addition,
dorsal gene expression in host-transferred embryos injected with 10 ng of
mismatched morpholino was relatively normal (data not shown). This suggests
that the reduction of dorsal gene expression in PP2A:B56
maternal
depleted embryos is specific to PP2A:B56
loss-of-function.
PP2A:B56 activity is required for ß-catenin stability
The effect of PP2A:B56 depletion on the expression of targets of
ß-catenin signaling suggests that PP2A:B56
may be required for
Wnt/ß-catenin signaling. To address this directly, we compared the
accumulation of endogenous ß-catenin protein, a well-established measure
of canonical Wnt signaling, in control and maternal PP2A:B56
-depleted
embryos at the late blastula stage (stage 9), when Wnt/ß-catenin
signaling is maximally activated in Xenopus embryos. Consistent with
the reduction in expression of ß-catenin-regulated genes, cytosolic
ß-catenin protein was significantly decreased in maternal
PP2A:B56
depleted embryos (Fig.
3A). In addition, depletion of PP2A:B56
did not affect
ß-catenin protein levels in settings where Wnt signaling is not
activated, such as oocytes or ventral blastomeres of maternally depleted
embryos (data not shown). Therefore, we conclude that maternal PP2A:B56
is required for Wnt-dependent accumulation of ß-catenin protein.
|
PP2A:B56 is required for midbrain-hindbrain boundary
formation
The above observations suggest that maternal PP2A:B56 is required for
Wnt/ß-catenin signaling during early development. To investigate whether
PP2A:B56
is required for Wnt/ß-catenin signaling at later stages,
we explored the function of PP2A:B56
in the neural ectoderm. It has been
shown extensively that Wnt/ß-catenin regulates formation of the
midbrain-hindbrain boundary (MHB) in vertebrates
(Joyner, 1996
;
McMahon et al., 1992
).
engrailed 2 (en2), which marks this boundary during early
stages of neural development
(Hemmati-Brivanlou et al.,
1991
; Hemmati-Brivanlou and
Harland, 1989
), is a well-characterized target of the
Wnt/ß-catenin pathway (Joyner,
1996
; McGrew et al.,
1999
). We therefore analyzed whether PP2A:B56
function is
required for en2 expression and subsequent MHB formation. The
PP2A:B56
morpholino was injected at the 8-cell stage into the dorsal
animal blastomeres that give rise to neural ectoderm
(Moody, 1987
), and expression
of en2, as well as other neural markers, was analyzed by whole mount
in situ hybridization and RT-PCR.
Expression of the MHB markers en2
(Fig. 4A) and wnt1
(Christian et al., 1991)
(Fig. 4A) was severely reduced
in morpholino-injected embryos at the midneurula stage (stage 16). In
addition, the anterior stripe of krox20
(Bradley et al., 1993
), which
marks rhombomere 3, was also down-regulated. In contrast, the posterior stripe
of krox20 was only weakly reduced
(Fig. 4A). Otx2, a
forebrain marker (Lamb, 1993
)
and hoxB9 (Fig. 4A), a
spinal cord marker (Sharpe et al.,
1987
), were expressed normally. These changes in neural marker
expression appear to be a specific consequence of PP2A:B56 loss-of-function,
as co-injection of PP2A:B56-c mRNA rescued both en2 and
krox20 (Fig. 4B)
expression.
|
|
PP2A:B56 is required for neural tube closure and proper head
formation
In addition to the loss of en2 expression and subsequent defects
in later midbrain-hindbrain formation, PP2A:B56 knockdown embryos
exhibited dramatic defects in neural tube closure
(Fig. 6B compare with 6A). The
failure of neural tube closure in the anterior region occurred in all embryos
injected with 2.5 ng morpholino, while defective posterior neural tube closure
was observed at a lower frequency (varying from 10% to 30% in different
clutches of eggs). The neural tube in most embryos injected with 2.5 ng of
morpholino eventually closed 2 to 3 hours later than control embryos. (At a
higher dose of morpholino (5 ng), the anterior neural tube failed to close,
leading to lethality at the tadpole stage; data not shown.) Morpholino
injection also delayed cement gland formation. At mid-neurula stage, cement
gland formation could be seen as condensed pigmentation below the anterior
neural ectoderm in controls (Fig.
6D); in morpholino-injected embryos, this pigmentation was reduced
in intensity and was present in a broader region below the edge of the open
anterior neural tube (Fig. 6E).
Interestingly, pigmentation was completely absent in embryos with more severe
neural tube defects (data not shown). At the swimming tadpole stage,
morpholino-injected embryos had smaller heads and either cyclopic eyes or no
eyes (Fig. 6H). These
phenotypes appeared to be due to the lack of PP2A:B56
in the neural
ectoderm, as 20 pg of PP2A:B56
-c mRNA rescued these
phenotypes (Fig. 6C,F,I),
including the lethal phenotype caused by 5 ng of morpholino injection (data
not shown). Embryos injected with the same dose of mismatched morpholino
developed relatively normally (data not shown). Therefore, we conclude that
PP2A:B56
is required for neural tube closure and proper formation of
head structures.
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Discussion |
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PP2A:B56 is required for Wnt/ß-catenin signaling
Loss-of-function analysis in Drosophila and zebrafish suggested
that PP2A:B56 family members play roles in the PCP pathway upstream of dsh
(Hannus et al., 2002).
Although Frizzled and Dsh function in both the PCP pathway and the canonical
Wnt/ß-catenin pathway (Mlodzik,
2002
), the requirement for PP2A:B56
upstream of Dsh in
Wnt/ß-catenin signaling has not previously been shown.
The loss-of-function analysis of PP2A:B56 presented here directly
supports the hypothesis that PP2A:B56
is required for Wnt/ß-catenin
signaling. PP2A:B56
is required for the expression of multiple
Wnt/ß-catenin target genes, including Xnr5, Xnr6 during pre-MBT
stages, Xnr3, siamois, noggin, goosecoid, cerberus and
Xdkk1, during the gastrula stage, and en2 during
neurulation. Furthermore, PP2A:B56
is required for ß-catenin
accumulation in cells that respond to endogenous Wnt signaling, such as dorsal
blastomeres in blastula-stage embryos and in the neural ectoderm of early
neurulae. In contrast, the constitutive turnover of ß-catenin in ventral
blastomeres and in oocytes is not sensitive to PP2A:B56
depletion (not
shown). The requirement for PP2A:B56
in canonical Wnt signaling is
further supported by epistatic analysis. Dorsal gene expression is rescued by
dsh and downstream Wnt signaling components but not by Wnt8b. Based on these
observations, we conclude that PP2A:B56
is required for
Wnt/ß-catenin signaling downstream of the Wnt ligand and upstream of
dsh.
In contrast to our observations with depletion of PP2A:B56,
overexpression of PP2A:B56 family members inhibits Wnt/ß-catenin
signaling (Gao et al., 2002
;
Li et al., 2001
;
Ratcliffe et al., 2000
;
Seeling et al., 1999
). Other
B56 family members, such as B56
, may have different functions in the
Wnt/ß-catenin pathway. For example, we found that overexpression of
PP2A:B56
in dorsal blastomeres leads to anterior truncation of
embryos without altering dorsal gene expression (see Fig. S1 at
http://dev.biologists.org/supplemental/),
a phenotype that is distinct from the strong ventralization caused by
PP2A:B56
overexpression (Li, 2001). These observations are
consistent with the suggestion that the two isoforms have different functions
in the Wnt pathway. However, overexpressed PP2A:B56
also inhibits
secondary dorsal axis induction by positive modulators of Wnt/ß-catenin
pathway (Ratcliffe et al.,
2000
) (see Table S1 at
http://dev.biologists.org/supplemental/).
To explain this, PP2A:B56
could regulate multiple steps in Wnt
signaling, and loss-of-function at an upstream step may obscure an inhibitory
role at a downstream step in the pathway. Alternatively, overexpression of
PP2A:B56 or other subunits that interact with the PP2A complex could sequester
essential components of the phosphatase complex and interfere with its normal
function. It also remains unclear whether PP2A:B56
regulates Wnt
signaling through modulation of PP2A activity. Further experiments are needed
in order to understand the mechanism by which PP2A:B56
regulates the
Wnt/ß-catenin pathway.
Similar to observations that B56 family members are required for
convergence and extension movements in zebrafish gastrulation
(Hannus et al., 2002), we find
that PP2A:B56
is required for neural tube closure in Xenopus.
The PCP pathway has been proposed to regulate the cell movements involved in
neural tube closure in Xenopus and mouse
(Hamblet et al., 2002
;
Wallingford and Harland, 2001
;
Wallingford and Harland,
2002
). Since PP2A:B56
is required for Wnt signaling upstream
of dsh, it would be of interest to test whether PP2A:B56
is a common
component in both non-canonical and canonical Wnt/frizzled signaling upstream
of dsh.
PP2A:B56 is required for early embryonic development
Consistent with the PP2A:B56 expression pattern during early
embryonic development, our loss-of-function analysis suggests that maternal
PP2A:B56
is required for dorsal-ventral patterning. Maternal depletion
of PP2A:B56
inhibits dorsal lip formation, a morphological marker for
the Spemann organizer. The expression of multiple dorsal-specific genes is
reduced in maternal PP2A:B56
-depleted embryos while ventral gene
expression is increased. This gene expression profile in maternal
PP2A:B56
-depleted embryos is reminiscent of embryos lacking maternal
ß-catenin (Heasman et al.,
1994
; Heasman et al.,
2000
; Xanthos et al.,
2002
). Our data, together with previous work showing that maternal
Xenopus frizzled7 is required for dorsal development
(Sumanas et al., 2000
),
suggest that components of the Wnt/ß-catenin pathway, upstream of dsh are
involved in primary dorsal axis specification during Xenopus
embryonic development.
The inhibition of dorsal development as well as dorsal gene expression
appears to be specific to PP2A:B56 loss-of-function, as PP2A:B56
-c
mRNA, which lacks the morpholino target sequence, rescues the expression of
most dorsal genes. Interestingly, siamois expression was not rescued
efficiently by PP2A:B56
-c. In addition, the PP2A:B56
morpholino causes an embryonic lethal phenotype, similar to homozygous
widerborst mutants (Hannus et
al., 2002
), which was not seen in embryos injected with control
morpholinos or the ß-catenin morpholino. Although dorsal gene expression
and dorsal lip formation were rescued with PP2A:B56
-c mRNA, we were
unable to rescue the embryonic lethality, and thus we cannot rule out the
possibility that the morpholino causes some additional non-specific defects at
a later stage in host transfer experiments.
In addition to the role of maternal PP2A:B56 in dorsal-ventral
patterning, PP2A:B56
is also required in neural ectoderm for expression
of the Wnt target gene en2. Depletion of PP2A:B56
blocks the
expression of en2 in the early neurula (stage 14), but not other
early MHB markers, including fgf8, wnt1, pax2.1 and XHR1,
suggesting that PP2A:B56
is specifically required for en2
expression but not for the specification of the MHB lineage. Interestingly,
the phenotype of morpholino-injected embryos becomes more severe at the
mid-neurula stage (stage 16), with reduction of MHB markers (wnt1 and
en2) and an anterior hindbrain marker (the anterior stripe of
krox20). In contrast, the anterior marker otx2
(Lamb et al., 1993
), the
spinal cord marker hoxB9 and the posterior stripe of krox20
appear to be normal in PP2A:B56
morpholino-injected embryos. This
phenotype is reminiscent of the remarkably restricted phenotype observed in
the mouse wnt1 knockout (McMahon
et al., 1992
) and the ß-catenin conditional knockout in
wnt1-expressing cells (Brault et
al., 2001
), and further supports the requirement of
Wnt/ß-catenin for en2 expression and for the maintenance of the
MHB. The PP2A:B56
morpholino did not reduce slug expression, a
target of Wnt/ß-catenin signaling in the neural crest lineage (not
shown), suggesting that other B56 family members may function redundantly
during embryonic development.
Interfering with PP2A:B56 also causes phenotypes that are not clearly
related to impaired Wnt/ß-catenin signaling. For example, depletion of
PP2A:B56
in dorsal-animal blastomeres leads to delayed neural tube
closure at the neurula stage, and causes lethality at the tadpole stage, which
is not seen with depletion of ß-catenin. In addition, cement gland
formation in PP2A:B56
knockdown embryos is delayed, in contrast to the
enlarged cement gland phenotype observed with downstream Wnt antagonists
(Itoh et al., 1995
;
Kofron et al., 2001
). These
observations are consistent with additional roles for PP2A:B56
independent of the Wnt pathway.
In summary, our data directly support the requirement of PP2A:B56 for
Wnt/ß-catenin signaling. PP2A:B56
is required in several
developmental processes regulated by Wnt/ß-catenin signaling, including
primary dorsal axis specification, formation of the midbrain-hindbrain
boundary, and closure of the neural tube. PP2A:B56
may thus function
during early embryonic development through the canonical Wnt pathway
dependent, as well as through Wnt-independent pathways.
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ACKNOWLEDGMENTS |
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Footnotes |
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