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First published online 3 August 2006
doi: 10.1242/dev.02523
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1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of
Toronto, 600 University Avenue, Toronto, Ontario, M5G 1X5, Canada.
2 Department of Molecular and Medical Genetics, University of Toronto, 600
University Avenue, Toronto, Ontario, M5G 1X5, Canada.
Author for correspondence (e-mail:
claire.chazaud{at}inserm.u-clermont1.fr)
Accepted 27 June 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Mouse embryogenesis, Axis formation, Wnt, Chimera, Asymmetry, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Tam and Steiner, 1999) and by
expression of organizer-related genes, such as Gsc and Foxa2
(Blum et al., 1992;
Ang and Rossant, 1994;
Filosa et al., 1997). However,
axis induction by the EGO or node is incomplete with no anterior structures
being formed (Beddington, 1994;
Tam and Steiner, 1999). More
recently, the anterior visceral endoderm (AVE) has been shown to be important
for head induction in the embryo (Thomas
and Beddington, 1996;
Beddington and Robertson,
1999; Tam and Steiner,
1999), and interactions between AVE, the node and PS signals are
required to establish the full AP axis
(Tam and Steiner, 1999).
Markers of the AVE, such as Hex, are first expressed in the distal
part of the visceral endoderm (DVE) at 5.5 dpc (days post coitum)
(Thomas et al., 1998;
Rivera-Perez et al., 2003;
Srinivas et al., 2004). Thus,
by 5.5 dpc, the first molecular asymmetries are established in the embryo,
setting up a proximodistal (PD) axis. Subsequently, the cells of the DVE move
towards the anterior side (Thomas and
Beddington, 1996; Thomas et
al., 1998; Kimura et al.,
2000; Rivera-Perez et al.,
2003; Srinivas et al.,
2004), becoming the AVE. The AVE produces molecules, such as Cer1
and Lefty1, that act to inhibit posterior fate and establish the AP axis
(Kimura et al., 2000;
Perea-Gomez et al., 2001).
After the AVE has moved, genes such as brachyury (T) are expressed
proximally in the epiblast and the extraembryonic ectoderm (ExE)
(Liu et al., 1999;
Brennan et al., 2001;
Kimura et al., 2001;
Beck et al., 2002;
Perea-Gomez et al., 2004;
Thomas et al., 1998). Multiple
signaling pathways are involved in these events of molecular asymmetry and
axis formation. Most notably, the activity of the Nodal/CFC signaling pathway
has been shown to be crucial for several aspects of mesendoderm development
and axial patterning in mice, including formation and migration of the AVE
(Ding et al., 1998;
Brennan et al., 2001) with
Cer1 and Lefty1 acting as localized Nodal antagonists ensuring repression of
anterior fates (Perea-Gomez et al.,
2002).
Wnt/ß-catenin signaling has also been shown to be involved in the
earliest asymmetries that are associated with axis development in different
vertebrates (Harland and Gerhart,
1997; Yamanaka et al.,
1998; Fekany et al.,
1999; Roeser et al.,
1999). In the canonical Wnt/Wingless signaling pathway,
Wnt/Wingless activates Frizzled receptors, which then act through Dishevelled
to increase free ß-catenin levels by preventing its degradation by the
proteasome pathway (Cadigan and Nusse,
1997). ß-Catenin is then translocated to the nucleus, where
it can cooperate with members of the Lef/Tcf transcription factor family to
activate gene transcription (Huelsken and
Behrens, 2002). In the absence of a Wnt/Wingless signal,
ß-catenin is phosphorylated by Gsk3ß
(Rubinfeld et al., 1996;
van Es et al., 2003) and
released from a complex that promotes its ubiquitination and degradation. The
tumor suppressor protein adenomatous polyposis coli (Apc) is a key component
of this complex, acting as a scaffold to which ß-catenin, Axin1 and Axin2
can bind (Polakis, 2001;
van Es et al., 2003), and can
also behave as a shuttle between the nucleus and cytoplasm for ß-catenin
(Bienz, 2002). Given this mode
of action of the Wnt signaling pathway, mutations in different components can
lead to both gain and loss of signaling activity, providing different insights
into the role of Wnt/ß-catenin signaling in development. Loss-of-function
mutations in Wnt proteins, Frizzled proteins, ß-catenin and the
Lef/Tcf genes should lead to reduced Wnt signaling, while mutations
in Apc or axin will lead to nuclear accumulation of ß-catenin and to
constitutive activation of the Wnt signaling pathway
(van Es et al., 2003).
A number of different mutations implicate Wnt/ß-catenin signaling in
patterning around gastrulation in the mouse. Of the different Wnt proteins
expressed around gastrulation, mutation of Wnt3 results in the
earliest phenotype. Embryos mutant for Wnt3 completely lack a
primitive streak and mesendoderm formation
(Liu et al., 1999),
demonstrating a clear role for Wnt signaling in mesendoderm development.
However, genes expressed in the AVE are correctly localized
(Liu et al., 1999), suggesting
that Wnt3-/- embryos initiate AP axis formation and that
Wnt3 acts downstream of an earlier AP induction signal.
ß-Catenin mutant embryos (Haegel et
al., 1995; Huelsken et al.,
2000) also fail to form mesendoderm and they completely lack
expression of mesoderm markers such as T and goosecoid
(Gsc). However, they also show defects in the AVE not shown by
Wnt3 mutants. They fail to express Hex in the visceral
endoderm (VE) while other AVE markers, such as Cer1 and
Lhx1, remain distal. ß-Catenin signaling has indeed recently
been shown to have a role in AVE migration
(Kimura-Yoshida et al., 2005).
Thus, ß-catenin is required for both mesendoderm induction and
transformation of the PD to AP axis via movement of the AVE. Recent studies on
microarray profiling of ß-catenin mutant embryos have identified cripto,
a Nodal co-receptor, as a likely ß-catenin target gene
(Morkel et al., 2003). As
cripto is known to be required for movement of the AVE
(Ding et al., 1998), this
suggests that the loss of AVE movement in ß-catenin mutants may be a
secondary effect of reduced Nodal/cripto signaling.
Activation of Wnt signaling in the embryo also implicates Wnt pathways in
axis formation in mice. Misexpression of chick Wwnt8c in transgenic
lines produces a range of defects including ectopic embryonic axes
(Popperl et al., 1997).
Axin1 mutations (Zeng et al.,
1997) and a hypomorphic mutation in Apc
(Ishikawa et al., 2003) also
lead to embryos with ectopic posterior axis development, phenocopying the
chick Wnt8c transgenic embryos. Thus, ectopic activation of the Wnt
pathway can interfere with axis development around gastrulation. However, in
both the Axin1 mutation and the hypomorphic Apc allele,
ß-catenin activity is not completely deregulated
(Zeng et al., 1997;
Ishikawa et al., 2003). In
other words, the earliest effects of constitutive activation of the
Wnt-ß-catenin pathway are not being assessed. Several different
Apc mutations have been made by gene targeting in mice
(Shibata et al., 1997;
Kielman et al., 2002;
Ishikawa et al., 2003) with
different levels of activity. However, the original spontaneous
ApcMin mutation (Moser
et al., 1990; Su et al.,
1992) appears to be the most severe
(Kielman et al., 2002). The
ApcMin mutation was first identified by its heterozygous
effect in promoting intestinal neoplasia
(Su et al., 1992). It produces
a stable truncated protein of 850 amino acids that lacks all ß-catenin
binding and other regulatory motifs (Su et
al., 1992), leading to complete inability to bind and downregulate
ß-catenin, thus producing constitutively active ß-catenin signaling.
Complete loss of function of the Apc gene in
ApcMin/Min embryos results in block of development at the
5.5-6.5 dpc stage and mutant embryos fail to form a pro-amniotic cavity or a
primitive streak (Moser et al.,
1995). This suggests that the patterning of the earliest phases of
PD axis formation may also be susceptible to elevated ß-catenin activity.
ApcMin/Min ES cells fail to differentiate into teratomas
in vivo (Kielman et al.,
2002), suggesting that elevated ß-catenin could lead to a
general block in differentiation. This has been postulated to result from
induction of cell death by high levels of nuclear ß-catenin
(Kim et al., 2000), rather
than a specific result of disturbed Wnt signaling.
We have re-examined the phenotype of ApcMin/Min embryos to determine whether non-specific induction of cell death or specific effects on embryo patterning are the cause of early lethality. We see no obvious induction of cell death but find very specific effects on expression of genes involved in early PD and AP patterning. Known Wnt target genes, normally proximally restricted, such as T, are expressed throughout the epiblast. In addition, the DVE fails to form, as assessed by expression of genes such as Hex. These effects of activated ß-catenin signaling are autonomous to the epiblast, as some DVE marker expression can be rescued in mutant VE when the epiblast is wild type. However, these DVE markers are no longer restricted to the distal tip of the embryo, suggesting that ß-catenin activity in the extraembryonic lineages may drive expansion of the DVE. We suggest that localized Wnt-ß-catenin signaling is required initially to establish proximodistal identity, restricting the formation of the DVE to the distal tip, prior to its known role in posteriorizing the proximal epiblast.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
markers staining as follows: 3.75 dpc, late blastocyst; 4.0 dpc, just before
implantation (smooth trophoblast); 4.5 dpc, implanted embryo with PrE
epithelium; 4.75 dpc, induction of ExE (thickening of the polar trophectoderm,
induction of Pem ExE expression), PrE epithelium is rounder; 5.0 dpc,
bowl-shaped epiblast and PrE, just before amniotic cavitation; 5.5 dpc, DVE
induction distally; 5.75 dpc, AVE has moved anteriorly; 5.75-6.0 dpc, onset of
mesendoderm induction (T expression); 6.25-6.5 dpc, onset of
gastrulation movements.
Litters from heterozygous crosses were processed for in situ hybridization
or immunohistology and finally genotyped by PCR. The whole embryo was lyzed in
PCR tissue homogenizing buffer (Nagy et
al., 2003) and 10% of the extract was used for PCR reactions:
wild-type allele, 5'-TAAAGACCAGGAAGCCTTGT-3' with
5'-AATACCTCGCTCTCTCTCCA-3', the annealing temperature was
67°C; ApcMin allele,
5'-TGAGAAAGACAGAAGTTA-3' with
5'-TTCCACTTTGGCATAAGGC-3', the annealing temperature was 50°C.
In figures, +/+ embryos are either wild type or heterozygous as there was no
difference in the phenotype.
Whole-mount fluorescent in situ hybridization
Double whole-mount fluorescent in situ hybridization was adapted from
Ciruna and Rossant (Ciruna and Rossant,
2001) and was performed according to Chazaud et al.
(Chazaud et al., 2006) (see
also
www.sickkids.ca/rossant/protocols/doubleFluor.asp).
Immunohistology
After dissection in PBS, embryos were fixed in 4% paraformaldehyde for 3
hours to overnight. They were washed in PBST (PBS, 0.1% Triton), twice for 5
minutes. Embryos were preblocked in PBST with 10% FBS and incubated overnight
with the antibodies (rabbit anti-ß-catenin, 1/500, Sigma; rat
anti-uvomorulin, 1/500, Sigma; mouse anti-Dab-2, 1/400, BD Transduction
Laboratories). After five 20-minute washes in PBST they were incubated with
the second antibody coupled to a fluorophore (Jackson Laboratories). After two
washes in PBST, nuclei were stained for 10-30 minutes with YOYO-1 (Molecular
Probes, 1/1000 in PBST with 200 µg/ml RNAse A) or propidium iodide
(Molecular Probes, 1 µg/ml) and washed three times for 20 minutes in PBST.
Embryos were observed under Zeiss confocal microscope (LSM510, 20x
air-objective with NE 0.75). In combined staining with in situ hybridization,
the immunolocalisation was processed after the probe hybridization.
Lefty1 was hybridized with a probe recognizing both Lefty1
and Lefty2.
Chimeras
Eight-cell embryos were collected from heterozygous
ApcMin/+ crosses and aggregated to ES cells constitutively
expressing YFP, using the standard morula aggregation technique
(Nagy et al., 2003). Chimeras
were transferred to pseudopregnant recipients and embryos were dissected out
at different stages and processed for in situ hybridization or/and
immunostaining as described above. Wild-type cells were visualized with a
riboprobe for EGFP. Mutant embryos were identified either by T
expression, by PCR genotyping of the extraembryonic ectoderm or by analyzing
nuclear ß-catenin immunostaining after in situ hybridization.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunostaining at 5.75 dpc showed high levels of ß-catenin in both the PrE and the epiblast of ApcMin/Min mutants (Fig. 1G). The staining in PrE cells was clearly localized to the cell membrane and the nucleus whereas the epiblast cells accumulated ß-catenin throughout the cell, including the nucleus, with variable expression levels among cells. The remaining ExE cells did not show obvious nuclear accumulation of ß-catenin. As early as 4.75 dpc, when no morphological phenotype was apparent, nuclear accumulation of ß-catenin was observed in the epiblast and PrE of most mutant embryos (Fig. 1H-J, n=4/5). At 3.5 dpc, patterns of ß-catenin localization were similar in wild-type and mutant embryos, with only membrane localized ß-catenin apparent (data not shown, n=3). Staining with the nuclear dye, YOYO-1, did not reveal any obvious apoptotic or pycnotic nuclei in mutants at any stage. TUNEL staining did not show any increase in cell death even in the ExE between 5.0 and 5.5 (n=3, data not shown).
Mispatterning of epiblast and PrE in ApcMin/Min mutants
Given the clear accumulation and nuclear translocation of ß-catenin in
the early epiblast of mutant embryos, we asked whether genes associated with
later domains of Wnt activity were ectopically activated in
ApcMin/Min mutants. We first analyzed the PS/mesendoderm
marker brachyury (T), as it has been shown to be a direct target of
the Wnt pathway (Yamaguchi et al.,
1999; Arnold et al.,
2000). In ApcMin/Min mutants, the entire
epiblast expressed T by 5.75 dpc, a stage when it could not be
detected in the wild-type embryos with our fluorescent procedure
(Fig. 2A-C). Despite T
expression, Oct4 (Rosner et al.,
1990) (Fig. 2A,B,
n=4) was still expressed in the epiblast. We then examined earlier
stages and observed that T was co-expressed with Oct4 in the
entire epiblast as early as 4.75 dpc (Fig.
2D,E, n=10) before any morphological phenotype could be
seen. At late blastocyst stage (3.75 dpc), ectopic expression of T
was seen only in a subset of embryos (Fig.
2F,G), suggesting that the onset of ectopic gene activation is
around 4.0 dpc.
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), a
marker of the middle streak, was expressed ectopically in a few epiblast cells
in a transient manner (Fig. 3D,
n=2). Snail (Snai1), which is involved in
epithelium-mesenchyme transition (Nieto et
al., 1992; Carver et al.,
2001), was also expressed ectopically in the mutant epiblast
(Fig. 3I, n=3),
demonstrating that the epiblast acquired a mesendoderm identity. Expression of
Otx2, a marker of distal and then anterior ectoderm
(Ang et al., 1994), was absent
in mutant epiblast (Fig. 3E,
n=3), consistent with a respecification of the fate of the epiblast
from distal/anterior to proximal/posterior. Interestingly, Lhx1 and
Cer1 (Barnes et al.,
1994; Shawlot and Behringer,
1995), which mark later AVE and organizer tissues, were
ectopically expressed in the epiblast at 5.5 dpc
(Fig. 3G,H; n=5 and
n=3, respectively). Although T expression was induced widely
in the epiblast, this did not reflect a broad induction of PS markers. Levels
and patterns of expression of other PS/mesendoderm markers such as
Fgf8 (Crossley and Martin,
1995) (Fig. 3F,
n=3), Mml (Pearce and
Evans, 1999; Robb et al.,
2000) (n=2 and data not shown) and Wnt3
(Liu et al., 1999) remained
unchanged in the epiblast of mutant embryos. Cripto, a proposed Wnt target
gene (Morkel et al., 2003), is
normally expressed throughout the epiblast at 5.5 dpc and was not obviously
altered in expression pattern at this stage in mutants
(Fig. 3J, n=2). The
epiblast-wide weak expression of Nodal at this stage was also not
affected (Fig. 3K,
n=3).
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)
(Fig. 3A, n=3) and
Foxa2 (Ang et al.,
1993; Belo et al.,
1997) (Fig. 3L,
n=4), but it was not patterned in a PD manner. Indeed, all the genes
normally expressed in the DVE/AVE that we have tested, such as Gsc
(Fig. 3B, n=6),
Fgf8 (Fig. 3F,
n=3), Lhx1 (Fig.
3G, n=5), Eomes
(Fig. 3D, n=2),
Cer1 (Fig. 3H,
n=3), Otx2 (Fig.
3E, n=3) and Hex
(Fig. 3C, n=2), failed
to be expressed at all in the PrE of Apc mutants at 5.5 dpc. These
results suggest that the main effect of activated ß-catenin signaling in
ApcMin/Min mutants is a proximalization of the epiblast
and concomitant failure to induce the DVE, such that the PD axis of the early
egg cylinder fails to be specified.
Chimeric analysis of ApcMin/Min cells
To determine whether the effect of activated ß-catenin on PD axis
formation was intrinsic to embryonic or extraembryonic lineages, wild-type ES
cells constitutively expressing YFP were aggregated with embryos from
heterozygous ApcMin/+ matings. Chimeras with contributions
from homozygous mutant embryos were identified by the presence of cells with
high ß-catenin immunolocalization in the VE or ectopic T
expression in the epiblast. Low and high contribution of ES cells produced
chimeras with a mix of wild-type and mutant cells in the epiblast or a fully
wild-type epiblast respectively. In all cases, the extraembryonic tissues (VE
and ExE) were derived from the mutant embryo as ES cells cannot differentiate
into these tissues in vivo (Beddington and
Robertson, 1989). In mixed wild-type and mutant epiblasts,
T expression was seen only in the mutant cells, indicating that the
ectopic activation of this gene in epiblast is cell-autonomous
(Fig. 4A).
In chimeras in which the entire epiblast was wild type, no ectopic
T or Gsc expression was observed in the epiblast (not
shown), and the pro-amniotic cavity was rescued
(Fig. 4B-D). Thus, the
Apc mutation does not affect the ability of the VE to induce
cavitation in the epiblast (Coucouvanis
and Martin, 1995; Coucouvanis
and Martin, 1999), but rather it interferes with the ability of
the epiblast to respond to the cavitation-inducing signals. As the egg
cylinder looked more normal than in ApcMin/Min mutants
alone, we asked whether wild-type epiblast could rescue the proximodistal
patterning of the ApcMin/Min VE. Markers of the DVE/AVE,
which were not expressed at all in ApcMin/Min mutants,
were examined in chimeras where the epiblast was wild type. Gsc and
Hex expression were rescued in the VE of the 5.5 dpc mutant chimeras
(Fig. 4B,C; n=4). This
chimera analysis showed that the ApcMin/Min mutant VE is
still competent to express some DVE genes, once the epiblast is wild type.
However, expression of these DVE markers was not restricted distally, but was
induced in the whole VE. By contrast, Cer1 and Lefty1, which
are known to regulate DVE size
(Perea-Gomez et al., 2002;
Yamamoto et al., 2004), were
not expressed in the mutant VE in the chimeras
(Fig. 4D, n=3/3).
Although the epiblast appeared more normal in the rescued chimeras, they could
not proceed to gastrulate and showed no expression of T at 6.5 dpc
(n=3, not shown).
Molecular asymmetry in implanting embryo
The analysis of the early development of the ApcMin/Min
mutants suggests that highly restricted activation of ß-catenin is
normally required to initiate PD axis development. Expression patterns of the
components of the Wnt pathway do not necessarily indicate actual sites of
active Wnt signaling. In other species, localized nuclear ß-catenin has
been observed in association with active sites of signaling
(Lemaire and Kodjabachian,
1996; Harland and Gerhart,
1997). We have sought evidence of early asymmetric activation of
ß-catenin in the normal embryo to identify possible sites of Wnt
signaling. We have used fluorescent antibody staining and confocal imaging on
all stages from 3.5 to 5.5 dpc and have been unable to detect any nuclear
ß-catenin with the exception of a transient phase around 4.5 dpc, during
implantation and PrE differentiation. ß-Catenin nuclear localization was
detected in one cell of the trophectoderm facing the PrE
(Fig. 5A,B,E,G) in about 15% of
the embryos examined (n=13/86). We noticed that ß-catenin was
not restricted to the nucleus but was found throughout the cell, as reported
previously in other tissues (Merrill et
al., 2001). E-cadherin, its binding partner in the plasma
membrane, remained at the cell surface
(Fig. 5B). A few hours later,
by 4.75 dpc, non-membrane-bound localization of ß-catenin was never
observed.
If the nuclear localization of ß-catenin observed transiently at the
blastocyst is causally involved in patterning, we would predict that other
genes would be expressed asymmetrically at the same time or subsequent to
ß-catenin nuclear localization. The homeobox gene Hex has been
reported to be expressed in the whole PrE at 4.5 dpc and later to be
restricted to the DVE (Thomas et al.,
1998). Pem is another homeobox gene expressed in the PrE
and ExE during implantation (Lin et al.,
1994) (and data not shown). Hex and Pem were
perfectly co-expressed in the PrE, with stronger expression in one or a few
cells adjacent to the trophectoderm (Fig.
5C,H). This asymmetric expression was found in 75%
(n>25) of the embryos analyzed, but was transient: a few hours
later, by 4.75 dpc, Hex was expressed uniformly in all the cells at
the periphery of the PrE, in proximity to the trophectoderm
(Fig. 5D), and Pem was
expressed throughout the PrE. By analyzing triple staining for ß-catenin,
Hex and the nucleus, we found that Hex and ß-catenin
asymmetric expression do not colocalize but are rather on opposite sides
(Fig. 5E-H;
n=4/4).
|
). In an attempt to compare Lefty1 expression with
the asymmetric markers described above, we carried out in situ hybridization
in 4.5 to 5.0 dpc embryos. To identify all the PrE cells we co-stained the
embryos with Dab-2 antibody (Yang et al.,
2002). In 4.5-4.75 dpc embryos, Lefty1 was always
expressed symmetrically within the PrE compared with Dab-2 expression
(Fig. 5I,J). Interestingly,
owing to different levels of the sensitivity of the experiment, varying
degrees of Lefty1 expression could be observed ranging from no
staining (n=3/16), one cell expression at the center of the PrE
(Fig. 5I, n=5/16), two
cells expression at the center of the PrE (n=1/16), to a fully
stained PrE (n=7/16), with a gradual decreasing expression from the
center towards the periphery in some cases
(Fig. 5J). Thus,
Lefty1 is expressed in a gradient with expression higher at the
center of the PrE and lower at the periphery. We were not able to detect any
Lefty1 RNA in 4.75-5.0 dpc embryos, just before amniotic cavitation
(Fig. 5K). This contradiction
with the results of Takaoka and collaborators could be due to differing
interpretation of morphology and differing staining techniques. Indeed,
between 4.5 and 5.0 dpc, the embryo goes through morphological changes
probably resulting from ExE asymmetrical growth and anchoring
(Smith, 1985;
Rossant and Tam, 2004). Thus,
the center of the PrE might appear asymmetric (arrowhead in
Fig. 5K) when analyzing embryos
with transmission light. Moreover, ß-galactosidase protein activity lasts
longer than native mRNA and could explain why Takaoka and collaborators
detected lacZ staining after 4.75 dpc; it could also reflect the
clonal growth of previously Lefty1-expressing cells, shortly after
symmetrical expression of Lefty1 mRNA. Accordingly, it is interesting
that such clonal growth is asymmetric, perhaps depending on morphological
changes in the embryo during ExE growth
(Rossant and Tam, 2004). |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and a series of Apc alleles of differing severity
was shown to affect differentiation capacity of ES cells in a dose-dependent
manner (Kielman et al., 2002).
The ApcMin mutation leads to a truncation of the APC
protein so that no binding to ß-catenin is possible and other regulatory
domains are deleted. Homozygous ApcMin/Min ES cells show
the highest levels of activation of ß-catenin reporter constructs and
nuclear accumulation of ß-catenin
(Kielman et al., 2002). They
were also severely impaired in their differentiation capacity in vitro and
were unable to form teratomas. As all other alleles produced some teratomas,
it was suggested that excess nuclear ß-catenin in the mutant ES cells
might be promoting cell death (Kielman et
al., 2002), as observed in other cell types
(Kim et al., 2000). The
reported phenotype of ApcMin/Min mutant embryos was a
block in development prior to gastrulation and failure to form a normal
elongated egg cylinder (Moser et al.,
1995). This early block in development could imply non-specific
apoptotic effects of excess ß-catenin. Here, we show that
ApcMin/Min embryos do not show any early evidence of
excess apoptosis. Rather, they show a complex series of alterations in
expression of embryonic patterning genes that can explain the phenotypes
observed and are consistent with a role for APC in regulating the activity of
the Wnt/ß-catenin pathway in the early embryo.
The early mouse embryo, even as early as the blastocyst stage, has been
shown to express most components of the downstream canonical Wnt signaling
response (Hamatani et al.,
2004; Wang et al.,
2004), although there is no previous evidence that the pathway can
actually be activated at these stages. Disruption of the complex that normally
shunts ß-catenin to the proteosome for degradation by mutation of
Apc demonstrates clearly that the pathway can be activated in both
embryonic and extraembryonic lineages, and that the Apc complex is necessary
to prevent ectopic Wnt/ß-catenin signaling from disrupting development.
When APC is lost, cells of the epiblast and PrE show accumulation of
non-membrane-bound ß-catenin in the cytoplasm and the nucleus from 4.75
dpc. Consistent with the appearance of nuclear ß-catenin, some direct Wnt
target genes were ectopically activated in ApcMin/Min
embryos. Notably, brachyury (T), which has been shown to be a direct
Wnt target in embryos and ES cells
(Yamaguchi et al., 1999;
Arnold et al., 2000), is
ectopically expressed throughout the epiblast in
ApcMin/Min mutants and is even expressed in the late
blastocyst stage, long before its usual expression. This ectopic expression is
cell-autonomous, as only mutant cells misexpress T in chimeric
embryos. However, T expression is not seen in the trophoblast or VE
of ApcMin/Min mutants. T is normally turned on in
the ExE and proximal epiblast region
(Perea-Gomez et al., 2004)
(C.C. and J.R., unpublished), and then moves to the PS, where its expression
is known to be regulated by Wnt signaling
(Liu et al., 1999). Thus,
constitutive activation of the Wnt signaling pathway in the epiblast seems to
lead to proximalization of epiblast identity. Consistent with this,
Otx2, which is expressed distally and then later anteriorly
(Simeone et al., 1993;
Rhinn et al., 1998;
Kimura et al., 2001), is
repressed in ApcMin/Min mutants. Snai1, which is
involved in epithelial-mesenchyme transition and required for mesendoderm
formation (Nieto et al., 1992;
Carver et al., 2001), as well
as Eomes, another marker of the primitive streak, are also
ectopically expressed. Not all markers of later primitive streak show ectopic
expression in the mutant epiblast, and expression of the key signaling
molecule Nodal and its co-receptor Cripto is not altered - both are expressed
throughout the early epiblast of wild-type and mutant embryos.
Activation of ß-catenin throughout the embryo is sufficient to block
the normal induction of patterning in the overlying VE. No VE expression of
DVE markers, such as Hex, Cer1 and Gsc, was observed in
ApcMin/Min mutants. This effect is cell-autonomous to the
epiblast, as some DVE gene expression can be rescued in chimeras where the
epiblast is wild type. This suggests that proximalization of the epiblast by
activated ß-catenin overrides the Nodal signal that would normally induce
DVE marker gene expression (Fig.
6B). Nodal signal from the epiblast is required for DVE induction,
as no DVE marker gene expression is seen in Nodal mutants
(Brennan et al., 2001).
|
;
Thomas et al., 1998) was
turned on in the mutant VE. However, expression of these markers was no longer
restricted to the distal tip but was found throughout the VE overlying the
epiblast. Thus, rescue of the mutant epiblast reveals a second role for
ß-catenin signaling in the extraembryonic lineages. Unlike the mutant
embryos, where no DVE forms at all, the rescued epiblast allows the induction
of some DVE markers, but constitutive activation of ß-catenin in the
extraembryonic lineages leads to expansion of these markers over the entire
epiblast (Fig. 6C). Although
these AVE markers, such as Hex, were present and expanded in the
mutant VE of these chimeras, expression of the Nodal inhibitors
Lefty1 and Cer1 was absent. Lefty1 and Cer1 are known to
regulate DVE migration (Yamamoto et al.,
2004) and size, because, in Cer1/Lefty1 double mutants,
Gsc and Hex expression domains are expanded
(Perea-Gomez et al., 2002).
Thus, the absence of Cer1 and Lefty1 in the mutant VE in the
ApcMin/Min chimeras is consistent with the expansion of
the Gsc and Hex domains.
It is not clear exactly how such expansion occurs. ß-Catenin
overexpression in the extraembryonic lineages could inhibit Lefty1
and Cer1 expression in the VE directly. Indeed, some microarray data
have shown an increase of Cer1 expression in ß-catenin 6.0 dpc
mutant embryos (Morkel et al.,
2003). It is also possible that ß-catenin overexpression in
the ExE disrupts the normal signals to the epiblast and VE that establish
proximal identity. Indeed, embryos with surgically removed ExE can show an
expansion of expression of some AVE genes
(Rodriguez et al., 2005;
Richardson et al., 2006), but
only under specific culture conditions
(Georgiades and Rossant,
2006), suggesting that ExE could be involved in the restriction of
DVE genes expression distally. In chimeras, Nodal signaling would then not be
restricted in its action to the distal region and DVE markers would be induced
throughout the overlying VE (Fig.
6C) (Ang and Constam,
2004). Whatever the mechanism, the expansion of the DVE leads to
an anteriorization of the epiblast, as judged by the absence of T
expression and normal gastrulation in the rescued chimeras. Thus, the loss of
Apc in the epiblast and the extraembryonic lineages separately give
essentially opposite phenotypes, indicating the complex interactions between
ß-catenin signaling activity in the different components of the
embryo.
Loss of Apc in both epiblast and extraembryonic lineages can
influence the development of the PD axis before 5.5 dpc, suggesting that
localized Wnt/ß-catenin activity may be important for the events that
initiate the PD axis. Loss-of-function Wnt mutations or
ß-catenin mutations have not previously indicated such early roles for
the Wnt signaling pathway. In addition, a mutation in ß-catenin that
should lead to constitutive activation by removing its ability to be
phosphorylated leads to disruption of patterning at later stages than observed
with ApcMin/Min mutants
(Morkel et al., 2003), as does
mutation in Axin1 (Zeng et al.,
1997). Another mutation stabilizing ß-catenin leads to
defects in the epiblast before gastrulation, although less severe than the
ApcMin/Min mutants
(Kemler et al., 2004). This
mutation does not impair ExE early development, as seen in
ApcMin/Min mutants. This might suggest that some of
ApcMin/Min phenotypes may result from some
ß-catenin-independent roles of Apc such as in cell adhesion or
in microtubule function (Bienz,
2002). Although this cannot be completely ruled out, the phenotype
and gene expression patterns in ApcMin/Min mutants are
largely consistent with an ectopic activation of the canonical Wnt signaling
pathway. Presence of maternal ß-catenin
(Haegel et al., 1995) and
redundant action of multiple Wnts and Axins could obscure early roles for
regulated ß-catenin activity in other Wnt pathway mutants.
Clearly, the mouse embryo is primed for canonical Wnt signaling as early as
the blastocyst stage and removing the restraint imposed by APC has major
effects on embryonic patterning. As the ß-catenin pathway can be
activated as early as the blastocyst stage, this leaves open the possibility
that localized Wnt activity could be important at these early stages to
initiate the events that lead to induction of the PD and then the AP axis. We
have found some tantalizing evidence of localized nuclear ß-catenin in
restricted cells in the trophoblast of 4.5 dpc embryos, in a region in close
proximity to the PrE. We have also shown that the PrE shows morphological and
molecular asymmetries by the same stage. These data are circumstantial
evidence that Wnt signaling and asymmetries leading to later axis development
may begin as early as 4.5 dpc and might be driven by extraembryonic tissues.
Combined with recent evidence that the growth of the PrE from the blastocyst
is asymmetric with relation to the PD axis of the postimplantation embryo
(Weber et al., 1999) and that
the DVE is asymmetric before moving anteriorly
(Yamamoto et al., 2004) (C.C.
and J.R., unpublished), this suggests that there may be complex interactions
between embryonic and extraembryonic lineages by the late blastocyst stage
leading to later axial asymmetries
(Rossant and Tam, 2004;
Ang and Constam, 2004).
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
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|
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