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First published online 15 August 2007
doi: 10.1242/dev.005967
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1 Molecular Embryology Group, MRC Clinical Sciences Centre, Imperial College
London, Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, UK.
2 Transgenic Facility, MRC Clinical Sciences Centre, Imperial College London,
Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, UK.
3 Molecular Developmental Biology Section, Laboratory of Reproductive and
Developmental Toxicology, National Institute of Environmental Health Sciences,
Research Triangle Park, NC 27709, USA.
* Author for correspondence (e-mail: tristan.rodriguez{at}csc.mrc.ac.uk)
Accepted 18 July 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Neural induction, Bmpr1a, BMP signalling
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In
mouse, although a large number of BMPs have been identified, only Bmp2 and
Bmp4 have been shown to have roles during early embryonic development.
Analysis of null mutations for Bmp2 and Bmp4 has revealed multiple roles for
these proteins during the early post-implantation period, for example in
primordial germ cell (PGC) induction
(Lawson et al., 1999),
mesoderm and node formation, primitive streak morphogenesis
(Fujiwara et al., 2001;
Winnier et al., 1995),
extra-embryonic mesoderm development
(Zhang and Bradley, 1996) and
left-right patterning (Fujiwara et al.,
2001). The fact that embryos carrying mutations in either type I
or type II BMP receptors display phenotypes that are more severe than those of
Bmp2 or Bmp4, suggests that functional redundancy exits between BMP proteins
(Mishina, 2003).
In Xenopus, chick and zebrafish, BMP signalling has been shown to
have a central role in neural induction
(Harland, 2000). Although
there is much debate regarding the precise mechanism for this process, two
main models have been proposed. The first, based primarily on experiments
performed in Xenopus, argues that BMP signalling prevents a `default'
neural differentiation of the naïve ectoderm. Central to this model is
the idea that secretion of BMP antagonists protects the presumptive neural
plate from the action of BMPs and allows neural differentiation to occur
(Vonica and Brivanlou, 2006).
Experiments in both chick and Xenopus embryos have challenged this
idea and have led to the proposal that inhibition of BMP signalling alone is
not sufficient for neural induction and that other signals are required
(Stern, 2005;
Wilson and Edlund, 2001).
Recent reports have indicated that fibroblast growth factors (FGFs) may
constitute one of these signals, as inhibition of FGF signalling blocks neural
induction in chick (Streit et al.,
2000; Wilson et al.,
2000) and Xenopus
(Delaune et al., 2005). The
emerging picture of how FGFs promote neural differentiation seems to be
complex. FGF signalling can repress BMP mRNA expression
(Wilson et al., 2000) and
downregulate BMP signalling intracellularly
(Pera et al., 2003), but, in
addition to this, FGF signalling is required for neural induction
independently of BMPs (Aubin et al.,
2004; Delaune et al.,
2005; Linker and Stern,
2004). In zebrafish, it has been proposed that both BMP inhibition
and FGF signalling can act as neural inducers, with BMP antagonism inducing
anterior and FGFs inducing posterior neural fates
(Kudoh et al., 2004).
In the mouse embryo, a role for BMPs in neural induction has not been
shown. Single mutations in BMPs or BMP antagonist do not affect the initial
size of the neural plate and overlapping activities have been proposed as an
explanation for this lack of neural induction phenotype. More surprisingly,
double mutants for the BMP antagonists chordin and noggin are able to form a
neural plate although fail to maintain forebrain markers at later stages
(Bachiller et al., 2000). In
mouse embryonic stem (ES) cells, BMP proteins have been implicated in
maintaining pluripotency. In ES cells, BMPs are required to induce the
expression of the inhibitor of differentiation (Id) genes, an essential step
for ES self-renewal (Ying et al.,
2003a). Bmp4 has also been shown to be required for ES cell
self-renewal by inhibiting mitogen-activated kinase pathways
(Qi et al., 2004). The
induction of Id genes in ES cells specifically blocks neural differentiation.
The role of BMPs in maintaining ES cell self-renewal while blocking neural
differentiation suggests that in the embryo, BMP-mediated inhibition of neural
fate might be part of a mechanism to maintain epiblast pluripotency. In the
mouse embryo, anterior neural markers are not expressed until 7.25 days
post-coitum (dpc) (Yang and Klingensmith,
2006) and the epiblast remains pluripotent until this stage
(Beddington, 1982;
Beddington, 1983;
Diwan and Stevens, 1976;
Lawson et al., 1991). However,
little is known about the mechanisms that maintain this pluripotency.
We have analysed the role of BMP signalling in patterning the epiblast of
the mouse embryo. Three type I receptors have been shown to act downstream of
BMPs: Acvr1, Bmpr1a and Bmpr1b (also known as Alk2,
Alk3 and Alk6). Acvr1 expression is restricted to the
visceral endoderm at 6.5 dpc. At 7.5 dpc, Acvr1 is expressed in the
extra-embryonic mesoderm, extra-embryonic ectoderm, in the mesoderm and in the
visceral endoderm (de Sousa Lopes et al.,
2004; Gu et al.,
1999; Kishigami et al.,
2004; Roelen et al.,
1994). Expression of Bmpr1b has not been detected prior
to 9.5 dpc (Dewulf et al.,
1995). The ubiquitous expression of Bmpr1a after
implantation makes it the only type I BMP receptor expressed in the epiblast
(Mishina et al., 1995;
Roelen et al., 1994). For this
reason, to address the role of BMP signalling in the epiblast we have used a
null mutation and an epiblast-specific deletion of Bmpr1a.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and Bmpr1afx/fx mice
(Mishina et al., 2002) were
maintained on a 129SvCC genetic background. The Bmpr1a+/-;
Sox2Cre+/- mice were kept on a mixed 129SvCC/CBA/J/C57BL6
background. Embryos were genotyped after whole-mount in situ hybridisation or
alkaline phosphatase staining as described
(Martinez Barbera et al.,
2000) using primers that are specific for the Bmpr1a-null
mutation (Mishina et al.,
1995), the Bmpr1afx/fx mutation
(Mishina et al., 2002) and
Sox2Cre transgene (Hayashi et
al., 2002).
Immunohistochemistry and whole-mount in situ hybridisation (WISH)
Staining for pSmad1/5/8 (a gift from Ed Laufer, Columbia University, New
York, NY) was performed as described
(Persson et al., 1998;
Yang and Klingensmith, 2006).
WISH was carried out following standard procedures
(Thomas and Beddington,
1996).
Western blotting
Western blots were carried out following manufacturers conditions for the
pSmad1/5/8 antibody (Cell Signaling)
Inhibition of FGF signalling
For inhibition of FGF signalling, embryos from
Bmpr1a+/- inter-cross litters or from
Bmpr1afx/fxxBmpr1a+/-;
Sox2Cre+/- crosses were dissected in M2 medium and cultured
for 24 or 48 hours in 1:1 DMEM:rat serum plus 80 µM SU5402 (Sigma) or plus
DMSO alone at 37°C, 5% CO2, fixed in 4% paraformaldehyde (PFA),
dehydrated through a methanol series and processed for WISH.
Blastocyst outgrowth assays and ES cell derivation
Blastocysts were harvested from Bmpr1a+/- intercrosses
and cultured in gelatin-coated 48-well plates in ES cell medium containing 20%
serum, or in serum-free medium with Bmp4 alone (ESGRO Clonal Grade, Chemicon)
or supplemented with 200 ng/ml Bmp7 (R&D Systems), 20 ng/ml activin
(Sigma), or 200 ng/ml activin for either 5 or 10 days. Blastocysts were then
stained for alkaline phosphatase activity according to manufacturer's
instructions (Sigma) or trypsinised and used to derive
Bmpr1a-/- ES cells following standard feeder-free
conditions (Nichols et al.,
1990). Given that we were not aware that it was possible to derive
Bmpr1a-/- ES cells in the absence of MAP kinase inhibition
prior to our outgrowth experiments, Bmpr1a-/- lacZ ES
cells were generated from parental ES cells homozygous for the floxed
Bmpr1a allele and heterozygous for the ROSA26lacZ allele by
infection of Cre-expressing adenovirus in the presence of the SB203580 MAP
kinase inhibitor as previously described
(Qi et al., 2004).
Generation of chimeric embryos
Chimeras were generated by blastocyst injection as described
(Bradley and Robertson, 1986)
and stained for lacZ expression following standard procedures
(Hogan et al., 1994).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A previous characterisation of the Bmpr1a mutant phenotype found
that mutant embryos fail to gastrulate and do not express the mesoderm marker
brachyury (T), suggesting that severe patterning defects are present
(Mishina et al., 1995). In ES
cells, BMP signalling has been implicated in the maintenance of pluripotency
(Qi et al., 2004;
Ying et al., 2003a). We
therefore studied the state of differentiation of the epiblast of
Bmpr1a mutants. For this we analysed the expression of Oct4
(also known as Pou5f1 - Mouse Genome Informatics), Nanog and
Fgf5. Oct4 is expressed throughout the epiblast at 5.5 and 6.5 dpc
(Rosner et al., 1990). At 5.5
dpc, we observed no difference in the level of Oct4 expression in
Bmpr1a mutant embryos when compared with controls
(Fig. 2A,B). However, at 6.5
dpc, half of the Bmpr1a mutant embryos displayed a clear
downregulation of Oct4 expression
(Fig. 2C,D; n=4/8).
Nanog is required for ES and inner cell mass pluripotency
(Chambers et al., 2003;
Mitsui et al., 2003). At 6.5
dpc, Nanog expression was observed in the proximal-posterior epiblast
(Fig. 2E). In all
Bmpr1a-/- embryos analysed at 6.5 dpc, Nanog
expression was completely lost from the epiblast
(Fig. 2F). Fgf5 is a
primitive-ectoderm marker and is expressed throughout the epiblast at 6.5 dpc
(Fig. 2G)
(Kaji et al., 2006;
Sun et al., 1999). When we
examined Fgf5 expression in Bmpr1a mutant embryos we
observed a severe downregulation or loss of expression of this gene
(Fig. 2H). The downregulation
or loss of Oct4, Nanog and Fgf5 expression indicates that
BMP signalling is required to maintain epiblast pluripotency.
Bmpr1a is required to inhibit premature neural differentiation of the epiblast
The downregulation of pluripotency markers suggests that a premature
differentiation of the epiblast is occurring in Bmpr1a mutant
embryos. In other vertebrates, inhibition of BMP signalling has been proposed
as a central step in neural induction
(Harland, 2000;
Vonica and Brivanlou, 2006).
For this reason we investigated the possibility that the epiblast of
Bmpr1a-/- embryos is undergoing neural differentiation. At
7.5 dpc, markers of the anterior neural ectoderm are expressed in a discrete
patch of the anterior epiblast (Yang and
Klingensmith, 2006). We analysed the expression of the anterior
neural markers Hesx1 and Six3 and of the pan-neural marker
Sox1 and found that at 7.5 dpc, these genes are expressed throughout
the epiblast of Bmpr1a mutant embryos
(Fig. 2J,L,N; n=7/8,
4/6 and 7/8), indicating that ectopic neural differentiation of the epiblast
is occurring in these embryos. Foxg1 is a forebrain marker that is
first expressed at the three-somite stage
(Xuan et al., 1995). At 7.5
dpc, a distinct patch of Foxg1 expression could be observed in
Bmpr1a-/- embryos at the boundary between the embryonic
and extra-embryonic regions (Fig.
2P; n=4/6), indicating that, at least to some degree, the
ectopic neural tissue is regionalised in Bmpr1a mutants. We next
analysed the expression of midbrain marker engrailed 1
(Davis and Joyner, 1988). In
Bmpr1a-/- embryos, no engrailed 1 expression was observed
at 7.5 dpc (data not shown, n=7), suggesting that there is only
premature expression of anterior neural genes in these embryos.
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Ectopic neural induction is accompanied by suppression of mesoderm in Bmpr1a-/- embryos
A necessary step for neural induction to occur is the suppression of
mesoderm and mesoderm-inducing signals in the prospective neural plate region.
To test whether the loss of pluripotency in the epiblasts of
Bmpr1a-/- embryos leads to any mesoderm differentiation we
analysed the expression of Fgf8, T and eomesodermin. At 6.5 dpc,
Fgf8 is expressed in the posterior epiblast and visceral endoderm
(Fig. 3A)
(Crossley and Martin, 1995)
and both eomesodermin and T are expressed in the posterior epiblast
and extra-embryonic ectoderm (Fig.
3C,E) (Perea-Gomez et al.,
2004; Russ et al.,
2000). In Bmpr1a mutant embryos, Fgf8 expression
was lost from the epiblast, but could still be observed in the visceral
endoderm (Fig. 3B) and
expression of both eomesodermin and T was lost from the epiblast and
strongly downregulated in the extra-embryonic ectoderm
(Fig. 3D,F).
Two signalling molecules that have been shown to be essential for mesoderm
specification are Nodal (Brennan et al.,
2001) and Wnt3 (Liu et al.,
1999). At 6.5 dpc, Nodal and the Nodal
co-receptor Cripto (also known as Tdgf1 - Mouse Genome
Informatics) (Gritsman et al.,
1999) were found to be expressed in the posterior epiblast
(Fig. 3G,I), but expression of
both genes was lost in Bmpr1a mutants
(Fig. 3H,J). Wnt3 is
normally expressed in the posterior epiblast and adjacent visceral endoderm
(Fig. 3K), but in embryos
lacking Bmpr1a, expression of Wnt3 was strongly
downregulated or lost from both these tissues
(Fig. 3L). The lack of
Fgf8, eomesodermin, T, Nodal, Cripto and Wnt3
expression in Bmpr1a -/- embryos indicates that the
ectopic neural differentiation observed in these embryos is accompanied by a
suppression of mesoderm and mesoderm-inducing signals.
BMP signalling is required in the epiblast to inhibit neural differentiation
Bmpr1a is expressed in all three tissues of the early
post-implantation embryo: the epiblast, the visceral endoderm and the
extra-embryonic ectoderm (Mishina et al.,
1995; Roelen et al.,
1994). To test whether the defects observed in
Bmpr1a-/- embryos are due to requirements of BMP
signalling in the epiblast, we deleted Bmpr1a specifically from this
tissue. We used Bmpr1afx/fx mice that carry a conditional
mutation in Bmpr1a (Mishina et
al., 2002), and the epiblast-specific Cre-driver mouse line
Sox2Cre+/- (Hayashi et
al., 2002).
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Bmp2/4 signal via Bmpr1a to maintain pluripotency
We next asked which BMPs signal via Bmpr1a to maintain
pluripotency. In Drosophila, the Bmpr1a homologue
thickveins preferentially transduces dpp signals (reviewed
by Podos and Ferguson, 1999),
and in vertebrates the Dpp homologues Bmp2 and Bmp4 bind with high affinity to
Bmpr1a (ten Dijke et al.,
1994). We therefore tested whether Bmpr1a is required for
Bmp2/4 signalling in the early embryo. We first analysed the ability of Bmp4
to stimulate the phosphorylation of Smad1/5/8 in vitro. We found that in
wild-type ES cells, Bmp4 can stimulate phosphorylation of Smad1/5/8. By
contrast, in Bmpr1a mutant ES cells, Bmp4 was unable to simulate
Smad1/5/8 phosphorylation (Fig.
5A). We further tested the ability of Bmp2/4 to signal via
Bmpr1a by using an in vitro blastocyst outgrowth assay. Wild-type and
Bmpr1a-/- blastocysts were cultured on gelatin-coated
dishes in serum-free conditions in the presence of Bmp4 and assayed for
alkaline phosphatase activity, a characteristic marker of pluripotent cells.
When grown for 10 days in these conditions, wild-type and heterozygous
blastocysts had formed large outgrowths that displayed strong levels of
alkaline phosphatase activity (Fig.
5G and Fig. 6I). By
contrast, Bmpr1a-/- blastocysts showed hardly any
proliferation during the 10 days in culture and exhibited variable levels of
alkaline phosphatase activity (Fig.
5H and Fig. 6J),
indicating that Bmp4 was unable to sustain self-renewal of the inner cell mass
in these embryos. The loss of pSmad1/5/8 expression observed in
Bmpr1a-/- mutant embryos at 5.5 dpc, coupled with the
inability of Bmp4 to signal in Bmpr1a mutant embryos and ES cells,
indicates that Bmp2/4 signalling via Bmpr1a is the BMP pathway that
maintains pluripotency prior to gastrulation.
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Bmp7 is a BMP of the 60A subgroup that is first expressed in mouse at the
start of gastrulation (Solloway and
Robertson, 1999). In both Drosophila and vertebrates,
BMPs of the 60A subgroup have been shown to signal preferentially via the type
I BMP receptor Acvr1 (Podos and
Ferguson, 1999; ten Dijke et
al., 1994). For this reason, we first tested whether Bmp7 could
signal in Bmpr1a-/- cells. We found that in contrast to
Bmp4, Bmp7 was capable of stimulating phosphorylation of Smad1/5/8 in
Bmpr1a-/- ES cells
(Fig. 5A). Interestingly, the
ability of Bmp7 to signal in Bmpr1a mutant ES cells correlated with
an upregulation of Acvr1 (Fig.
5B), but not of Bmpr1b, in these cells (data not shown),
suggesting that Bmp7 was signalling preferentially via this type I
receptor.
Given the ability of Bmp7 to signal in Bmpr1a mutant ES cells, we analysed whether Bmp7 could rescue the defects of Bmpr1a-/- cells in vivo. For this we carried out blastocyst outgrowth assays. When Bmpr1a-/- blastocysts were cultured in the presence of Bmp7 they formed outgrowths that were equivalent both in size and alkaline phosphatase activity to control outgrowths (Fig. 5I,J). Together, these results suggest that prior to gastrulation, Bmp2/4 signalling via Bmpr1a is the main source of BMP signalling that maintains epiblast pluripotency, but as gastrulation commences, signalling by BMPs of the 60A subgroup via Acvr1 cooperates with Bmp2/4 signalling to inhibit neural differentiation of the epiblast.
activin/Nodal signalling can rescue the pluripotency defects of Bmpr1a mutant embryos
Nodal signalling has been shown to be required to maintain
pluripotency and inhibit neural differentiation in the mouse embryo
(Camus et al., 2006;
Mesnard et al., 2006). To
determine whether at least part of the defects of
Bmpr1a-/- embryos are due to an absence of Nodal
signalling, we analysed how early Nodal expression is lost in
Bmpr1a mutant embryos. At 4.5 dpc, Nodal is expressed in the
epiblast and primitive endoderm (Takaoka
et al., 2006) and at 5.5 dpc in the epiblast and overlying
visceral endoderm (Varlet et al.,
1997). In Bmpr1a mutant embryos, we found no difference
in the expression of Nodal at 4.5 dpc
(Fig. 6A,B), but a clear
downregulation relative to wild-type embryos at 5.5 dpc
(Fig. 6C,D). We next analysed
the expression of the Nodal co-receptor Cripto and of the
Tgfß Gdf3 at 5.5 dpc. At this stage, both these markers are
normally expressed throughout the epiblast
(Chen et al., 2006;
Ding et al., 1998).
Interestingly, although expression of Gdf3 was normal,
Cripto expression was downregulated in Bmpr1a-/-
embryos (Fig. 6F,H). These
observations indicate that epiblast patterning defects are present in
Bmpr1a mutants as early as 5.5 dpc, and that BMP signalling is
required to maintain Nodal signalling in the early embryo.
We next tested whether Nodal signalling was capable of rescuing the pluripotency defects of Bmpr1a-/- embryos. For this, we cultured Bmpr1a-/- blastocysts in serum-free conditions in the presence of 20 ng/ml or 200 ng/ml activin (also known as inhibin) for 10 days. Interestingly, we found that although 200 ng/ml activin did not rescue the proliferation or pluripotency defects of Bmpr1a-/- embryos (n=0/6; data not shown), 20 ng/ml activin did rescue both these defects (n=5/7; Fig. 6L). Therefore, Nodal signalling can, at least in part, rescue the pluripotency defects of Bmpr1a-/- embryos.
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;
Wilson et al., 2000) and
Xenopus embryos (Delaune et al.,
2005), and in mouse have been shown to act antagonistically to BMP
signalling during mesoderm development
(Miura et al., 2006). We
tested wether FGF signalling is responsible for the precocious and ectopic
neural induction observed in Bmpr1a mutant embryos. In agreement with
previous studies (Corson et al.,
2003), we found that 80 µM SU5402 could efficiently inhibit FGF
signalling within 1 hour in cultured 6.0 dpc embryos (data not shown). Given
that Bmpr1a-/fx; Sox2Cre+/- embryos do not show
precocious neural marker expression at 6.5 dpc, we used these embryos to test
whether inhibition of FGF signalling during gastrulation could block the
expression of the anterior neural markers. 6.5 dpc control and
Bmpr1a-/fx; Sox2Cre+/- embryos were cultured
for 24 hours with DMSO or 80 µM SU5402 and assayed for Hesx1
expression. After 24 hours of culture, only a small proportion of DMSO-treated
controls (2/30) or Bmpr1a-/fx; Sox2Cre+/- (2/7)
embryos had initiated Hesx1 expression in the epiblast
(Fig. 7A,C). Surprisingly,
although SU5402 inhibition did not significantly change the proportion of
control embryos expressing Hesx1 (5/26;
Fig. 7B), it led to a drastic
increase in the proportion of Bmpr1a-/fx;
Sox2Cre+/- embryos displaying ectopic Hesx1
expression (11/13; Fig. 7D).
These results indicate that inhibition of FGF signalling during gastrulation
stages does not inhibit neural specification in Bmpr1a-/fx;
Sox2Cre+/- embryos and suggest that BMP and FGF signalling
might act synergistically to inhibit neural differentiation at these
stages.
To test whether FGF signalling may be acting prior to gastrulation to
induce neural fate, we inhibited FGF signalling by culturing 5.5 dpc
Bmpr1a-/fx; Sox2Cre+/- and
Bmpr1a-/- embryos for 48 hours in the presence of SU5402.
No control (0/33) or Bmpr1a-/fx; Sox2Cre+/-
(0/6) embryos and only 55% of Bmpr1a-/- embryos (5/9)
showed Hesx1 expression after 48 hours culture in DMSO
(Fig. 7E,G,I). By contrast, 29%
of control (15/52), 80% of Bmpr1a-/fx;
Sox2Cre+/- (4/5) and 90% of Bmpr1a-/-
embryos (10/11) displayed ectopic Hesx1 expression after 48 hours
culture with SU5402 (Fig.
7F,H,J). Interestingly, the extra-embryonic region of these
SU5402-treated embryos was much reduced compared with controls, most probably
owing to the effects of FGF inhibition on the trophoblast stem cells
(Tanaka et al., 1998). These
observations indicate that the ectopic neural induction seen in
Bmpr1a mutants is not mediated by FGF signalling and also suggest
that FGFs are not acting as direct neural inducers in the early
post-implantation mouse embryo.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The role of BMP signalling in maintaining epiblast pluripotency
Strong evidence exists that right up to 7.5 dpc, a proportion of cells from
the epiblast remain pluripotent
(Beddington, 1982;
Beddington, 1983;
Diwan and Stevens, 1976;
Lawson et al., 1991). The
maintenance of this progenitor population is crucial for the allocation of
cells to the three germ layers: mesoderm, endoderm and ectoderm. A limited
number of factors have been identified as required to maintain epiblast
pluripotency, including Oct4
(Nichols et al., 1998),
Nanog (Chambers et al.,
2003; Mitsui et al.,
2003), Sox2 (Avilion
et al., 2003), Foxd3
(Hanna et al., 2002) and
Fgf4 (Feldman et al.,
1995). However, given that mutations in these factors cause
lethality at peri-implantation stages, it has not been possible to determine
whether these genes affect pluripotency after implantation.
A number of observations indicate that BMP signalling via Bmpr1a
plays a crucial role in maintaining epiblast pluripotency. Bmpr1a is
expressed in inner cell mass-derived ES cells
(Ying et al., 2003a) and was
highly enriched in a screen aimed at identifying stem cell markers
(Sato et al., 2003). In
Bmpr1a-/- embryos, we find a downregulation or loss of
expression of genes required for epiblast pluripotency, such as Oct4,
Nanog, Nodal and Cripto. Furthermore, we show that
Bmpr1a-/- embryos exhibit self-renewal defects in
blastocyst outgrowth assays. In ES cells, BMP signalling cooperates with the
Lif/Stat pathway to drive self-renewal
(Ying et al., 2003a). BMP
signalling is therefore required both in vitro and in vivo to maintain
pluripotency.
Inhibition of BMP signalling and neural induction
A key aspect of the role of BMPs in maintaining epiblast pluripotency is
the inhibition of precocious neural differentiation. In ES cells, Bmp4
maintains pluripotency by specifically blocking the neural differentiation
pathway (Ying et al., 2003a).
Similarly, we find that Bmpr1a mutant embryos show precocious and
ectopic expression of anterior neural markers coupled to the loss of
pluripotency markers. Inhibition of BMP signalling has been shown to be a
central step for neural induction in a number of vertebrate species
(Harland, 2000). Mainly based
on experiments in Xenopus, a model has been proposed in which BMPs
act prior to gastrulation to inhibit a `default' neural differentiation of the
ectoderm. According to this model, neural specification occurs when the onset
of expression of BMP antagonists allows differentiation to take place in the
prospective neural ectoderm (Vonica and
Brivanlou, 2006). The precocious expression of neural markers
observed in Bmpr1a-/- embryos indicates that as in
Xenopus, inhibition of BMP signalling in the mouse embryo is
occurring prior to gastrulation; therefore, a crucial step in neural induction
must be the relief of BMP inhibition.
Our results also suggest that BMP inhibition of neural fate is required
continuously in the epiblast rather than at a single specific stage of
development. Support for this comes from the comparison of
Bmpr1a-/- and Bmpr1a-/fx;
Sox2Cre+/- mutant phenotypes. In Bmpr1a-/-
embryos, a complete loss of pSmad1/5/8 expression occurs in the epiblast as
early as 5.5 dpc and this correlates with the precocious and ectopic
expression of neural markers at 6.5 dpc. By contrast, a significant decrease
in BMP signalling only occurs in Bmpr1a-/fx;
Sox2Cre+/- embryos from 6.5 dpc and ectopic neural markers are
not seen until 7.5 dpc. Therefore, the timing of the loss of BMP signalling
correlates with the timing of the onset of neural differentiation in two
separate mutations. This observation suggests that in the wild-type embryo,
BMP signalling is required to maintain epiblast pluripotency from the time of
implantation until 7.5 dpc, when neural differentiation takes place. It is
likely that this neural differentiation occurs when BMPs are antagonised
(Fig. 8), first by factors
secreted by the anterior visceral endoderm (AVE) and later by the axial
mesendoderm, the two signalling centres required for neural specification
(Beddington and Robertson,
1999).
Which BMPs can inhibit neural fate? In Xenopus embryos, ubiquitous
neural induction throughout the ectoderm only occurs after knock-down of four
BMP family proteins: BMP2, BMP4, BMP7 and ADMP
(Reversade and De Robertis,
2005). By contrast, we find in mouse that Bmp2/4 signalling via
Bmpr1a is the main source of BMP signalling that inhibits neural fate
prior to gastrulation. The loss of pSmad1/5/8 expression in
Bmpr1a-/- embryos coupled with the fact that Bmp2/4 cannot
signal in Bmpr1a-/- ES cells but other BMPs can, supports
this view. Interestingly, in mouse it is only as gastrulation commences that
signalling by BMPs of the 60A subgroup via Acvr1 cooperates with
Bmp2/4 signalling to reinforce this inhibition of neural differentiation
(Fig. 8). Two observations
support this. First, members of the 60A subgroup only start to be expressed at
the onset of gastrulation (Solloway and
Robertson, 1999). Secondly, we find that Bmp7 can rescue the
self-renewal defects of Bmpr1a-/- embryos in blastocyst
outgrowth assays. Therefore, in mouse, although Bmp2 and Bmp4 signalling are
initially sufficient to inhibit neural fate, as development proceeds multiple
BMPs signalling via different receptor complexes are required to block neural
differentiation.
One candidate pathway for acting downstream of BMP signalling is the
Nodal signalling pathway. BMPs have been shown to act upstream of
Nodal during left-right patterning
(Fujiwara et al., 2002;
Piedra and Ros, 2002;
Schlange et al., 2002) and to
amplify Nodal expression in the epiblast
(Ben-Haim et al., 2006).
Nodal mutant embryos display similar pluripotency defects and
precocious neural differentiation to those we observe in Bmpr1a
mutant embryos (Camus et al.,
2006; Mesnard et al.,
2006). In Bmpr1a mutant embryos, we find a loss of
expression of both Nodal and its co-receptor Cripto just
prior to the time when we observe the precocious expression of neural markers.
Furthermore, we find that activin/Nodal signalling can rescue the
pluripotency defects of Bmpr1a mutant embryos in blastocyst outgrowth
assays. These lines of evidence suggest that BMP signalling might be
regulating the levels of Nodal signalling in the early mouse embryo
and that, at least in part, the defects in Bmpr1a mutant embryos are
owing to the lack of Nodal signalling activity.
Are FGFs neural inducers in the mouse embryo?
Recent experiments in both chick and Xenopus embryos have
challenged the idea that inhibition of BMP signalling is sufficient for neural
induction and have suggested that FGF signalling might be an inductive signal
during this process. These studies found that inhibition of FGF signalling by
the small molecule SU5402 blocks neural induction in both chick
(Streit et al., 2000;
Wilson et al., 2000) and
Xenopus (Delaune et al.,
2005) embryos and that this role of FGFs is, at least in part,
independent of antagonising BMP signalling. However, in contrast to these
observations, we find that FGF signalling is not responsible for the ectopic
neural induction observed in Bmpr1a-/- embryos. Inhibition
of FGF signalling using SU5402 24 hours before gastrulation did not inhibit
the expression of Hesx1 in the epiblast of either
Bmpr1a-/fx or Bmpr1a-/fx;
Sox2Cre+/- embryos. Furthermore, inhibition of FGF signalling
led to an expansion of anterior neural tissue in a proportion of wild-type
embryos. Given that an expansion of neural tissue is also observed in
Fgf8-null mutants (Sun et al.,
1999) and in Fgfr1 chimeras
(Ciruna et al., 1997) and that
Fgf2 has been shown to inhibit neural fate in mouse ectoderm explants
(Burdsal et al., 1998), it
seems unlikely that the primary role of FGF signalling in the early
post-implantation mouse embryo is to act as a direct neural inducer, but
instead is required to promote mesoderm formation and migration. In ES cells,
inhibiting FGF signalling using SU5402 has been shown to block neural
differentiation (Ying et al.,
2003b). It is therefore possible that FGFs are required during the
pre-implantation period for a maturation step in the epiblast that makes this
tissue competent for neural specification, but once this step has taken place
it is inhibition of BMP signalling that is the main event required for the
induction of neural marker expression. It is also possible that, as has been
suggested in Xenopus (Pera et
al., 2003), a role for FGF signalling during early
post-implantation development could be to attenuate BMP signalling and drive
neural specification in this way.
In conclusion, we have found that Bmp2/4 signalling via Bmpr1a is inhibiting neural differentiation of the epiblast prior to gastrulation and that these BMPs cooperate with members of the 60A subgroup during gastrulation. We have also shown that, in contrast to what seems to occur in chick, in the early mouse post-implantation embryo inhibition of FGF signalling does not block neural specification and that it is relief of BMP inhibition that is the critical step for neural induction to occur.
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ACKNOWLEDGMENTS |
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