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First published online 25 May 2006
doi: 10.1242/dev.02413
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1 Ecole Polytechnique Fédérale de Lausanne EPFL-ISREC, Chemin des Boveresses 155, CH-1066, Switzerland.
* Author for correspondence (e-mail: daniel.constam{at}isrec.ch)
Accepted 20 April 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: TGFß, Oct4, Stem cells, Differentiation, Implantation, Axis specification, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Hanna et al.,
2002; Avilion et al.,
2003; Chambers et al.,
2003; Mitsui et al.,
2003). However, when the blastocyst implants in the uterus, a
subpopulation of ICM cells expresses the zinc finger transcription factor
Gata6 and congregates as a layer of primitive endoderm (PrE) facing the
blastocoelic cavity, followed by downregulation of Oct4
(Rossant et al., 2003). PrE
cells in contact with the ICM subsequently become visceral endoderm (VE) and
act as a substrate for the remaining pluripotent cells to firmly adhere and
form a rapidly expanding epithelial sheet, or epiblast, which gives rise to
the embryo proper. Meanwhile, outer trophectoderm cells abutting the ICM
undergo a burst of proliferation to form the extra-embryonic ectoderm (ExE)
and its derivatives in the fetal layers of the placenta. Coordinate growth
after implantation thus converts the mouse blastocyst within 24 hours into the
egg cylinder, a structure peculiar to rodents, with the epiblast and ExE
forming the inner layer of an elongated cylinder that is invested by an outer
VE layer.
Communication with the VE is essential to control the fate of pluripotent
cells in the epiblast, as distal visceral endoderm (DVE) moving along one side
of the epiblast restricts mesoderm and endoderm formation to the opposite pole
and thereby defines the position of the prospective anteroposterior (AP) axis
(Kimura et al., 2000;
Perea-Gomez et al., 2002) (for
a review, see Rossant and Tam,
2004). Moreover, the VE is essential for gas and nutrient exchange
and as a substratum to which the epiblast and ExE adhere (reviewed by
Bielinska et al., 1999).
However, lineage-tracing experiments have shown that VE cells do not colonize
embryonic tissues, and instead give rise to the outer layer of the visceral
yolk sac (Lawson and Pedersen,
1987). Irrespective of this common extra-embryonic fate, two
morphologically different populations of embryonic VE (EmVE) and
extra-embryonic VE (ExVE) cells are distinguished at the egg cylinder stage
based on their association with the epiblast or ExE, respectively
(Enders et al., 1978).
Accordingly, the DVE can be regarded as a specialized derivative of EmVE.
Alternatively, it may be set aside as a separate lineage before or in parallel
to EmVE.
By the time the DVE has moved anteriorly, cells in the epiblast are not
committed to specific fates (Tam and Zhou,
1996) and express Oct4 as well as Foxd3
(Hanna et al., 2002). However,
they have lost the capacity to give rise to self-renewing ES cells
(Rossant, 1977;
Beddington, 1983). Possibly
this transition reflects a change in the microenvironment caused by the DVE.
Alternatively, DVE formation and initiation of differentiation in the epiblast
may coincide because they are triggered by a common stimulus after
implantation. Consistent with the second hypothesis, both DVE and germ-layer
specification rely on the TGFß-related protein Nodal
(Zhou et al., 1993;
Conlon et al., 1994;
Brennan et al., 2001), and
prolonged activation of the Nodal pathway recently has been shown to impose a
mesendodermal differentiation program on cultured ES cells
(Tada et al., 2005). However,
depending on the context, Nodal may also provide anti-differentiation signals,
as it is essential to maintain Oct4 expression during gastrulation
(Brennan et al., 2001), and
activation of its signaling receptors prevents differentiation of cultured
human ES cells (James et al.,
2005). Nodal signaling in the ExE during gastrulation also
stimulates the expression of Bmp4 (Brennan
et al., 2001; Beck et al.,
2002), another factor inhibiting ES cell differentiation
(Ying et al., 2003). This
paradox indicates that Nodal may have distinct functions as its activity is
dynamically regulated in space and time. Indeed, after a wave of widespread
auto-induction throughout the epiblast and EmVE at the egg cylinder stage,
Nodal is swiftly silenced in the VE and anterior epiblast
(Collignon et al., 1996;
Norris and Robertson, 1999),
presumably by secreted feedback antagonists from the DVE such as Lefty1,
cerberus 1 homolog (Cer1), and Dkk1, whereas the posterior epiblast maintains
Nodal expression through positive feedback loops mediated by Wnt3,
Nodal itself, and its co-receptor Cripto (Cfc1-Mouse Genome Informatics) (for
a review, see Ang and Constam,
2004). This network of positive and negative feedback signals is
activated by the secreted proprotein convertases Furin and Pace4 (Pcsk6-Mouse
Genome Informatics), which are necessary for proteolytic maturation of the
Nodal precursor protein (Beck et al.,
2002). Thus, Furin-/-;
Pace4-/- compound mutants display a delayed onset of Nodal
expression and lack both DVE and AP asymmetry in the epiblast. Based on the
analysis of chimeric embryos, and as Furin and Pace4 during gastrulation are
specifically expressed in the ExE in a pattern that is complementary to that
of Nodal, we previously proposed that they act cell nonautonomously
to cleave Nodal after secretion (Beck et
al., 2002). Accordingly, we hypothesized that proNodal is more
efficiently processed in the proximal epiblast near the source of its
convertases than in distal regions. This model is consistent with the idea
that the level of Nodal processing may have to exceed a certain threshold
before it induces germ-layer formation. However, it does not explain how Furin
and Pace4 activities specify DVE at the apex of the egg cylinder. In one
possible scenario, Furin and Pace4 promote DVE formation only indirectly by
stimulating Nodal auto-induction, while DVE is actually induced by
uncleaved Nodal reaching maximal levels far from the source of proprotein
convertases. Alternatively, we hypothesized that DVE forms in response to
processed Nodal generated locally at a distance from the ExE.
The aim of this study was to determine whether Nodal and proprotein convertases already interact during implantation to pattern the VE, epiblast or ExE. We find that key components of the Nodal pathway are present from the time of implantation at embryonic day (E) 4.5, with Pace4 mRNA being localized to the ExE, and Furin and Nodal being expressed together in PrE. Furthermore, we show that Nodal signaling is required initially to specify EmVE and promote growth of the egg cylinder before it can induce DVE. In addition, analysis of Nodal mutants and embryo explants suggests that early Nodal signaling is also essential to sustain the expression of several molecular determinants of pluripotency. We conclude that Nodal activity is already required during implantation to control the fate of early progenitor cells in the epiblast and PrE lineages.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Mouse strains
Mice carrying the HexP-GFP reporter transgene
(Rodriguez et al., 2001) were
maintained pathogen-free in individually ventilated cages on a mixed genetic
background of C57BL6 x NMRI. Mice carrying the
NodalLacZ allele
(Collignon et al., 1996) were
maintained on a mixed genetic background of 129svEV x NMRI. Both strains
were inter-crossed to obtain males carrying the NodalLacZ
allele and homozygous for the HexP-GFP transgene. Outbred diabetes-resistant
NMRI mice were from Harlan or Janvier.
Isolation of embryos and explants
All embryos dissected on the fifth (E4.5) or sixth (E5.0-5.75) day
post-coitum were staged according to their size and expression of the HexP-GFP
transgene inherited from their HexP-GFP homozygous fathers. `Post-DVE' stage
corresponded to position of the AVE on the lateral side
(Rivera-Perez et al., 2003).
`DVE' corresponded to the stage when upregulation of GFP could be observed in
the distal tip of the visceral endoderm only. `Pre-DVE' stage corresponded to
younger embryos, in which GFP was only detected at reduced levels and with no
specific location. The presence and position of the DVE/AVE correlated with
the size of the embryo (Fig.
4A). Epiblast explants from staged heterozygous HexP-GFP
transgenic embryos were isolated and cultured essentially as described
(Beck et al., 2002) using
serum-free DMEM supplemented with glutamine and 15% knockout serum-replacement
(Invitrogen). Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Alexis) was used at
a concentration of 25 µmol/l. SB-431542 (Sigma) was used at a concentration
of 10 µmol/l. Nodal (R&D) was used at a concentration of 50
µg/ml.
Immunofluorescence, in-situ hybridization and ß-galactosidase staining
Whole-mount immunofluorescence was performed as described for blastocysts
(www.mshri.on.ca/rossant/protocols/immunoStain.html).
Rat anti-E-cadherin (Sigma) was used at a concentration of 1/200. Rabbit
anti-Laminin (Biodesign) was used at a concentration of 1/100.
ß-Galacosidase staining and RNA probes for whole-mount in-situ
hybridization have been described (Brennan
et al., 2001; Beck et al.,
2002). A Bmp2 antisense probe was derived from an
EagI-EcoRI 988-bp fragment of reference sequence NM_007553.
Additional RNA probes for Foxd3
(Hanna et al., 2002),
Hnf4a (Morrisey et al.,
1998), Nanog (Chambers
et al., 2003), Fgf5
(Hebert et al., 1994) and
Ttr (Makover et al.,
1989) have been described. Immunofluorescence images were acquired
on a Zeiss LSM510 confocal microscope. HexP-GFP and transmitted light images
were acquired on a Leica DC200 fluorescent microscope.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Despite the early presence of Nodal and its convertases, the DVE is not specified before E5.5
The early presence of Nodal and its convertases prompted us to monitor
transcription of other known agonists and antagonists of the Nodal signaling
pathway. The Nodal co-receptor Cripto was previously detected at the
blastocyst stage by RT-PCR and whole-mount staining of a lacZ
reporter allele (Kimura et al.,
2001). Using whole-mount in-situ hybridization, we confirmed that
Cripto is specifically expressed in the epiblast at all stages
between E4.5-5.5 (Fig. 1A). By
contrast, the Nodal antagonist Lefty1 was expressed in the PrE (E4.5). After
implantation (E5.0-5.25), Lefty1 expression became downregulated before it
resumed in the DVE at E5.5 (Fig.
1B). Prompted by this early expression pattern of Lefty1,
we next asked whether the DVE is already specified at E4.5. Contrary to this
idea, mRNA encoding the DVE marker Cer1 was not expressed above detectable
levels before E5.5. Moreover, the homeobox transcription factor Hex
(Hhex-Mouse Genome Informatics), which marks PrE and DVE
(Thomas et al., 1998) was
clearly induced at E4.5 and 5.5, but undetectable in the VE between E5.0 and
5.25 (Fig. 1C). This expression
pattern was confirmed by analyzing a GFP reporter transgene driven by the Hex
promoter (HexP-GFP) (Rodriguez et al.,
2001). In these transgenic embryos, GFP is induced in the PrE
(E4.5) and DVE (E5.5). However, at E5.25, HexP-GFP is only detected at reduced
levels throughout the entire VE, probably due to the perdurance of GFP protein
in descendants of the PrE. Taken together, the absence of significant
expression levels of Lefty1, Cer1 and Hex mRNAs between E5.0
and 5.25 strongly suggests that proximal-distal patterning in the visceral
endoderm is only determined around E5.5 at the time of DVE formation. This
interpretation is corroborated by the fact that a characteristic thickening of
DVE cells (Rivera-Perez et al.,
2003) is not observed until E5.5
(Fig. 1).
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).
However, we noticed that among embryos from NodallacZ/+
heterozygous intercrosses, homozygous mutants could already be identified at
E5.0 at the expected Mendelian frequency based on the absence of a distinct
ExE/epiblast boundary, an abnormally round shape, and frequent detachment of a
thickened VE from the epiblast (Fig.
2). In-situ hybridization of a Nodal antisense probe directed
against the deleted exon 2 confirmed that these abnormal embryos were
NodallacZ/lacZ null mutants
(Fig. 2A,B). Moreover,
ß-galactosidase staining revealed that
NodallacZ/lacZ homozygotes
expressed the lacZ reporter allele only in the epiblast, whereas
heterozygous littermates also induced lacZ in the EmVE
(Fig. 2C,D). This result is
consistent with the existing model that epiblast-derived Nodal activity is
essential to induce Nodal expression in the VE
(Brennan et al., 2001).
Moreover, immunofluorescent staining of E-cadherin revealed that cells in the
ExE and epiblast were abnormally heterogeneous in size, arranged irregularly,
and more rounded than the wild-type controls, especially near the boundary
between the epiblast and the ExE, which thus remained diffuse. In addition,
the epiblast epithelium of Nodal mutants only loosely adhered to the VE
(Fig. 2E,F). Adhesion of the VE
to the epiblast is known to depend on laminin alpha 1 in the basement membrane
(Smyth et al., 1999;
Miner et al., 2004). However,
laminin staining was unaffected in Nodal mutants
(Fig. 2G,H). Cell attachment
thus is not impaired due to a lack of basement membrane, but rather seems to
reflect a failure of VE cells to tightly adhere to it
(Fig. 2H,P).
Nodal signaling is required to confine ExVE to the extra-embryonic region
To define the early defect in the VE of Nodal mutants at the molecular
level, we first monitored the expression of Gata4, Hnf4 and
Ttr. During gastrulation, these genes are specifically expressed in
the ExVE (Makover et al.,
1989; Duncan et al.,
1994; Morrisey et al.,
1998). However, during implantation, they are initially
transcribed throughout the PrE and only become downregulated specifically in
the EmVE around E5.5 (see Fig. S2 in the supplementary material)
(Duncan et al., 1994). An
analogous expression pattern has been described for vHnf1ß
(Barbacci et al., 1999),
suggesting that markers of the ExVE generally become excluded from the
embryonic region at the egg cylinder stage. By contrast, in Nodal mutants,
these markers remained ectopically expressed at elevated levels in the
embryonic region (Fig. 2I-N).
Interestingly, Nodal mutants also displayed ectopic expression of
Furin, both in the VE and throughout the epiblast
(Fig. 2O,P). These results show
that Nodal signaling is essential to downregulate a subset of PrE markers,
including the proprotein convertase Furin, in the embryonic region and thus
restrict their expression to the ExVE.
Nodal defines an EmVE compartment before it can specify DVE
Nodal mutants may form ectopic ExVE in the embryonic region simply because
they lack a DVE. In this scenario, we expected that a functional DVE should
appear before the EmVE. Alternatively, the VE may first differentiate into
EmVE before it can form DVE. Consistent with the second hypothesis, we
identified a panel of EmVE markers that failed to be induced in Nodal mutants
at E5.0 long before the DVE would normally appear, including Lim1, Fgf5,
Fgf8, Bmp2, Otx2 and Hnf3b (Foxa2-Mouse Genome
Informatics) (Fig. 3, and data
not shown). Nodal, Fgf5 and Fgf8 mRNAs are also detected in the epiblast of
wild-type, but not of Nodal mutant embryos (Fig. 3; see Fig. S3 in the
supplementary material). These data reveal that Nodal is essential to impose
an embryonic identity on VE cells that are in contact with the epiblast.
Several possibilities can be considered why Nodal specifies EmVE before the
DVE can form at the apex of the egg cylinder. First of all, ExVE is unlikely
to be competent to express DVE markers because of dominant inhibitory signals
from the ExE. Direct support for this hypothesis comes from the fact that ExE
cells transplanted to distal epiblast repress DVE formation, whereas ablation
of the ExE leads to ectopic expression of DVE markers
(Rodriguez et al., 2005).
Based on this model, we expected that the DVE should only be induced once the
egg cylinder has reached a critical size. Confirming this prediction, we found
that HexP-GFP expression at E5.5 appeared only once the distance between ExE
and the apex of the egg cylinder was larger than 70 µm, and when the
conceptus reached a minimal length of 180 µm
(Fig. 4A). Moreover, in Nodal
mutants lacking DVE and EmVE, the average length of the egg cylinder at E5.5
was reduced by 24% (Fig. 4B).
Accordingly, Nodal may primarily be required as a permissive signal to support
egg cylinder growth and thereby overcome the prohibitive influence of the ExE.
Secondly, processed Nodal in addition may provide instructive cues to induce
DVE-specific genes. In this case, the conversion of PrE to EmVE and the
concomitant downregulation of Furin may also be important to prevent
ectopic DVE formation. To distinguish whether Nodal actively induces DVE
markers, or whether it only provides a permissive signal, we asked whether it
is necessary for HexP-GFP expression in embryo explants. As described, embryo
explants severed from the extra-embryonic region ectopically express the
HexP-GFP transgene throughout the EmVE
(Rodriguez et al., 2005).
However, induction of HexP-GFP was abolished in explants treated with
SB-431542, a pharmacological inhibitor of Nodal signaling
(Fig. 5). Moreover, incubation
with the Furin inhibitor decanoyl-RVKR-chloromethylketone (dec-RVKR-CMK) had a
similar effect at concentrations that block Nodal processing
(Le Good et al., 2005)
(Fig. 5). Likewise,
Lim1, a marker of EmVE and DVE, was lost when Nodal activity was
blocked. Thus, Nodal signaling is essential as an instructive cue even in the
absence of ExE to induce DVE.
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),
and cannot access the apical membrane of the VE. However, HexP-GFP was rescued
upon addition of recombinant activin, a Cripto-independent activator of the
Nodal pathway (Fig. 5).
Likewise, activin also restored expression of Lim1. These results confirm that
the SPC inhibitor dec-RVKR-CMK is specific and blocks expression of EmVE
markers by inhibiting Nodal processing.
Nodal is required before E5.5 to maintain pluripotency in the epiblast
Previous work suggests that Nodal processing is also essential for the
epiblast to maintain expression of Oct4 during gastrulation
(Beck et al., 2002). To assess
whether Nodal already influences pluripotency during implantation, we
monitored the expression of Oct4 and other markers of pluripotent
cells in early Nodal mutants and control littermates before E5.5. Whereas
Fgf4 and Sox2 were normally expressed, Oct4, Nanog,
Foxd3 and Cripto expression levels were already reduced or
undetectable at E5.0 (Fig. 6A),
suggesting that pluripotency is severely compromised or lost by that
stage.
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), raising the question of whether Nodal mutants cannot
maintain Oct4 expression simply because they downregulate
Bmp4 (Brennan et al.,
2001). Contrary to this idea, we found that Bmp4 remained
normally expressed in Nodal mutants at E5.25
(Fig. 6B). Likewise, the
proprotein convertases Furin and Pace4 were still present
(Fig. 2P,
Fig. 6B), suggesting that
markers of pluripotency are not simply lost at this early stage due to a lack
of ExE. Therefore, Nodal either directly induces Oct4 in the
epiblast, or maintains pluripotent cells indirectly by specifying the EmVE. To
distinguish between these possibilities, the effect of Nodal on the expression
of Oct4 was analyzed in cultured embryo explants. Epiblast explants
isolated at E5.75 without VE failed to maintain Nodal expression, due
to the absence of Nodal proprotein convertases and the resulting inhibition of
auto-induction (Guzman-Ayala et al.,
2004). Likewise, epiblast explants cultured without VE and ExE
also downregulated Oct4 (Fig.
6C). However, Oct4 expression could be restored in the
absence of VE by adding recombinant Nodal. This effect was specific, as it was
blocked by the Nodal receptor antagonist SB-431542
(Fig. 6C). Treatment of
epiblast explants with recombinant Nodal also restored expresson of
Cripto (Beck et al.,
2002) and Fgf4
(Guzman-Ayala et al., 2004).
It follows that Nodal signaling within the epiblast is sufficient to stimulate
Oct4 expression and several other markers of pluripotency
independently of the VE. During the course of these experiments, we observed that epiblasts isolated before E5.5 together with VE maintained expression of Oct4 for at least 30 hours. In addition, such explants also maintained significant expression levels of Nodal and Furin (Fig. 6D, top panels). However, in the presence of SB-431542 or the Furin inhibitor dec-RVKR-CMK, both Oct4 and Nodal were downregulated (Fig. 6D). We conclude that endogenous Nodal and Furin activities are necessary to maintain Nodal and Oct4 expression in embryo explants. However, once Nodal has been processed, its activity in the epiblast no longer depends on the VE (Fig. 6C).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Beck et al., 2002), which
defines the position of the future AP body axis
(Thomas et al., 1998;
Yamamoto et al., 2004). In
addition, Nodal signaling has recently been implicated in maintaining the
pluripotency of cultured human ES cells
(James et al., 2005). The
present study reveals two important novel functions for mouse Nodal that
pre-date DVE formation. First, we have shown that Nodal, Furin and Pace4 are
already expressed during implantation, and that early Nodal signaling is
essential to specify EmVE and confine the expression of Furin to the
extra-embryonic region. As Furin and Pace4 potentiate Nodal activity, this
conversion of PrE into EmVE is likely to be important to spatially regulate
Nodal processing during gastrulation. In addition, differentiation of the
EmVE, and adhesion of the epiblast are necessary to enlarge the egg cylinder
(Fassler and Meyer, 1995;
Coffinier et al., 1999;
Smyth et al., 1999;
Miner et al., 2004) and
thereby distance its apex from the source of dominant signals in the ExE that
prohibit DVE formation (Rodriguez et al.,
2005). Furthermore, our embryo explant experiments show that Nodal
and Furin activities are essential even in the absence of ExE to induce DVE
markers. In the simplest model, Furin expression in the VE of such explants is
necessary to activate proNodal, even though its loss in whole embryos is
partially compensated by ExE-derived Pace4
(Roebroek et al., 1998;
Beck et al., 2002). Thus, we
conclude that besides stimulating egg cylinder growth, processed Nodal also
provides an instructive signal to specify DVE. A second novel function for
early Nodal signaling uncovered before E5.5 is to maintain molecular markers
of pluripotency in the epiblast, including Oct4, Nanog, Foxd3 and
Cripto. Together, these results provide insights into the early
function of Nodal that are essential to define how inductive tissue
interactions after implantation control the fate of undifferentiated
progenitor cells in the mammalian embryo.
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; Thomas et al.,
1998). While our results do not exclude a role for Lefty1 in the
blastocyst, several observations argue against the idea that the DVE is
already induced at E4.5. First of all, targeted inactivation of Lefty1 does
not disrupt AP asymmetry (Meno et al.,
1998), because the DVE at later stages expresses the unrelated
Nodal antagonist Cer1, which is sufficient to repress posterior cell fates in
the adjacent epiblast (Perea-Gomez et al.,
2002). It follows that Lefty1 cannot be used as a reliable marker
to decide whether a functional DVE is present or not. However, Cer1
mRNA expression is undetectable before E5.5, even though in-situ hybridization
reveals a very robust signal once the DVE is induced. Together, these
observations argue against the idea that a functional DVE is established
before E5.5. Finally, and most importantly, Lefty1 and Hex mRNA are not
continuously expressed between E4.5-5.5, but instead are transiently
downregulated between E5.0 and 5.25. This intriguing on/down/on pattern
strongly suggests that the VE is not competent to form DVE before E5.5.
Previous work has shown that dominant inhibitory signals from the ExE are
sufficient to prevent induction of HexP-GFP and necessary at E5.5 to confine
DVE formation to the apex of the egg cylinder
(Rodriguez et al., 2005). As
the ExE arises during the time window between E5.0 and 5.25 when
Lefty1 and Hex are downregulated, this tissue is likely to
be responsible for repressing DVE formation before E5.5. Therefore, we propose
that DVE cannot form until the apex of the egg cylinder has reached a certain
distance from the ExE (Fig. 7).
According to this model, Nodal signaling in the VE is required before E5.5 to
impose an embryonic identity on the VE that is in contact with the epiblast.
In response to Nodal, we have found that the EmVE broadly expresses Fgf8 and
Fgf5 and provides an adhesive substratum that is essential for normal growth
of the epiblast (Smyth et al.,
1999). It is plausible that the resulting growth of the egg
cylinder sufficiently removes the ExE for distalmost cells to become competent
to form DVE. Moreover, our explant experiments show that, even in the absence
of ExE, the DVE does not arise by default. Instead, Nodal is essential both to
overcome the inhibitory influence of the ExE and as an instructive
signal to induce DVE-specific genes.
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;
Beck et al., 2002). Therefore,
we asked whether Oct4 or other known markers of pluripotent cells already
depend on Nodal during implantation. Indeed, analysis of Nodal mutants
revealed that expression of Oct4 and Foxd3 was already
downregulated before E5.5, whereas that of Sox2 and Fgf4 was
unaffected at that stage. However, Fgf4 expression is lost by E6.5
(Guzman-Ayala et al., 2004),
as expected if Oct4 protein is no longer present in sufficient amounts to
synergize with Sox2 and directly activate the Fgf4 promoter
(Yuan et al., 1995;
Nichols et al., 1998). Foxd3
is essential in the ICM to derive ES cell lines, and to sustain a normal
epiblast after implantation (Hanna et al.,
2002). In particular, while loss of Foxd3 apparently does not
affect DVE formation, it abolishes the expression of Nodal and its
targets in the epiblast during gastrulation, including Cripto
(Hanna et al., 2002). Foxd3
thus emerges as a key mediator of Nodal activities in the embryonic lineage.
However, the direct target genes of Foxd3 required in ES cells or during
gastrulation are unknown. One candidate is Foxa2 (previously Hnf3ß),
which is induced in the primitive streak but absent in ES cells, possibly
because a physical interaction with Oct4 can mask the DNA-binding domain of
Foxd3 (Guo et al., 2002).
Finally, progenitor cells in the ICM also depend on the homeobox transcription
factor Nanog (Mitsui et al.,
2003). Nanog is highly expressed in early blastocysts, but
downregulated during implantation (E4.5) when the ICM loses its potential to
generate self-renewing ES cells (Chambers
et al., 2003). During egg cylinder formation (E5.0-5.5), we
detected low levels Nanog mRNA in the epiblast of normal embryos, but
not in Nodal mutants. Even though the functional significance of Nanog at this
stage is unknown, this result together with the observed downregulation of
Foxd3 and Oct4 strongly suggests that pluripotent cells are prematurely lost
in Nodal mutants. Recently, Nodal signaling has also been implicated in
inhibiting the differentiation of cultured human ES cells
(James et al., 2005). By
contrast, mouse ES cells can be derived and cultured in the absence of Nodal
and its signal transduction machinery
(Varlet et al., 1997;
Gu et al., 1998;
Sirard et al., 1998;
Tremblay et al., 2000). It
follows that Nodal activity in the mouse embryo is required only after
implantation to sustain pluripotent cells. We have not analyzed the fate of
cells that prematurely downregulate markers of pluripotency, as
Nodal-deficient epiblasts do not adhere well to VE and become disorganized,
possibly as a secondary consequence of their exposure to ectopic ExVE. Nodal
mutants also ectopically express Furin in the epiblast, which may
contribute to premature or aberrant differentiation. Consistent with this
idea, forced expression of Furin in wild-type mouse ES cells blocks
self-renewal and triggers rapid differentiation (D.B.C., unpublished). It will
be interesting to determine in future experiments whether or not this effect
of ectopic Furin expression relies on Nodal processing, and to characterize
the resulting cell types.
Recently, the TGFß family member Gdf3 has been shown to play an
important role in embryonic stem cells and in peri-gastrulation mouse embryos.
By E6.5, Gdf3 null mutants share some defects of Nodal-/-
embryos (Chen et al., 2006).
However, it is controversial whether Gdf3 primarily acts as a Nodal/activin
family member (Chen et al.,
2006) or rather as a Bmp inhibitor
(Levine and Brivanlou, 2006).
To address this issue, it will be necessary to characterize in future studies
the expression of Nodal target genes in the EmVE and epiblast of Gdf3 null
mutants between E5.0 and 5.5.
Besides its role in the VE and epiblast, Nodal is also required to maintain
trophoblast stem cells in ExE during gastrulation
(Guzman-Ayala et al., 2004).
However, between E5.0 and 5.5, we did not observe impaired expression of ExE
markers in NodallacZ/lacZ
null mutant embryos. In particular, Bmp4, Furin and Pace4
and the trophoblast stem cell marker Cdx2 were unaffected, and the
differentiation marker Mash2 (Ascl2-Mouse Genome
Informatics) was not precociously upregulated (unpublished data). These
results suggest that the ExE does not depend on Nodal until after E5.5.
Overall, this study demonstrates that Nodal signaling in the mouse embryo
is already required during implantation to expand a population of pluripotent
progenitor cells in the epiblast, and to render visceral endoderm competent to
form DVE, before its known role during gastrulation and axis specification.
Secondly, it uncovers a novel role for Furin in potentiating Nodal signaling
locally within the primitive endoderm and embryonic visceral endoderm during a
limited time window, a function that has been masked in Furin mutants
(Roebroek et al., 1998) due to
the partially redundant activity of the related Nodal convertase Pace4
(Beck et al., 2002). Together,
these results suggest a model in which proNodal matures locally in the
embryonic hemisphere during implantation, before the expression of Furin and
Pace4 becomes restricted to the extra-embryonic region.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/13/2497/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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