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First published online 7 February 2007
doi: 10.1242/dev.02802
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Institute for Stem Cell Research, Centre Development in Stem Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JQ, UK.
Author for correspondence (e-mail:
Brian.Hendrich{at}ed.ac.uk)
Accepted 9 January 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Pluripotency, ES cells, Epiblast, Epigenetics, Mbd3, NuRD
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). At the morula
stage, the embryo begins to compact and by the blastocyst stage the outer
cells form an extra-embryonic cell layer called the trophectoderm, while the
inside cells form the inner cell mass (ICM) that will eventually give rise to
all cells of the foetus and some further extra-embryonic structures. Proper
segregation of the ICM and trophectoderm lineages at this stage requires the
activity of the transcription factors Cdx2 and Oct4 (Pou5f1)
(Nichols et al., 1998;
Niwa et al., 2005;
Strumpf et al., 2005). At 3.5
days post-coitus (dpc), the ICM contains two cell populations, one expressing
Pou5f1 and Nanog that is apparently fated to become
epiblast, the other expressing Pou5f1 and Gata6 and is
likely to become primitive endoderm
(Chazaud et al., 2006). At
late blastocyst stages (around 4.5 dpc) and immediately prior to implantation,
the two cell populations become spatially resolved with the primitive endoderm
forming a layer of cells facing the blastocyst cavity, while the early
epiblast retains the potential to derive all embryonic cell types, a property
known as pluripotency. Immediately after implantation, the early epiblast
begins to expand and form a columnar epithelium with a proamniotic cavity, a
structure that we define here as `late epiblast'. This transition is
accompanied by a dramatic increase in cell proliferation, resulting in
expansion of the pluripotent cell population from 
25 cells at 4.5 dpc to
660 cells by 6.5 dpc (Snow and
Bennett, 1978). In addition to having a shorter cell cycle, the
late epiblast also differs from early epiblast in terms of gene expression
(Pelton et al., 2002).
However, little is known of the differentiation events from 3.5 dpc ICM to
early epiblast and late epiblast, owing to the small size and inaccessibility
of relevant tissues.
One of the striking differences between the pluripotent cell populations in
3.5 dpc ICMs, early epiblast and late epiblast, is the ability to produce
embryonic stem (ES) cells (Evans and
Kaufman, 1981; Martin,
1981; Smith,
2001). ES cells, which can be maintained indefinitely in culture
while maintaining pluripotency, can be derived from cells of the ICM and early
epiblast, but not from late epiblast
(Brook and Gardner, 1997). ES
cells share many properties with ICM/early epiblast cells, and are able to
differentiate into all embryonic cell types including the germ line
(Bradley et al., 1984).
However, ES cells can be derived from only 30% of ICMs at best with
conventional methods (Brook and Gardner,
1997), and the molecular mechanisms underlying the derivation of
ES cells, and the molecular differences between ES cells and ICM/early
epiblast cells, are largely unknown (Zwaka
and Thomson, 2005).
The difference between early embryonic pluripotent cells and the
lineage-committed cells derived from them, or their transformed derivatives in
vitro (i.e. ES cells), is, by definition, epigenetic. Therefore, it stands to
reason that epigenetic silencing factors will play important roles in this
process. Several proteins involved in epigenetic silencing have been shown to
be important for early embryonic viability
(Bernstein et al., 2003;
Cowley et al., 2005;
Dannenberg et al., 2005;
David et al., 2003;
Dodge et al., 2004;
Hendrich et al., 2001;
O'Carroll et al., 2001;
Rayasam et al., 2003),
although it is not always clear why the mutant embryos do not survive. ES
cells appear to contain an unusual epigenetic profile consisting of both
active and silencing histone methylation marks, which tend to resolve into one
or the other upon differentiation (Azuara
et al., 2006; Bernstein et al.,
2006). Many genes which encode developmentally important
transcription factors are occupied by the Polycomb repressive complexes PRC1
and PRC2 in ES cells, and mouse ES cells deficient for PRC components fail to
maintain silencing at several of these loci
(Azuara et al., 2006;
Boyer et al., 2006;
Lee et al., 2006). Although
the details of epigenetic modifications in pluripotent cells in vivo have been
lacking owing to the scarcity of relevant tissue, a recent study has revealed
that ICMs have more intense silencing marks than do ES cells, at in least some
genes (O'Neill et al.,
2006).
The NuRD (Nucleosome Remodeling and Histone Deacetylation) co-repressor
complex is an abundant and biochemically well-characterised co-repressor which
is implicated in silencing in a number of contexts and in a variety of
organisms, including mammals, flies, nematodes and plants
(Ahringer, 2000;
Bowen et al., 2004). In
mammals, NuRD-mediated silencing has been implicated in cell fate decisions
during ES cell differentiation (Kaji et
al., 2006) and haematopoietic development
(Fujita et al., 2004;
Hong et al., 2005;
Hutchins et al., 2002;
Rodriguez et al., 2005;
Williams et al., 2004). One of
the core components of NuRD is the Mbd3 protein
(Wade et al., 1999;
Zhang et al., 1999). Mbd3 was
originally identified in mice and humans as a protein containing a region with
high homology to the methyl-CpG-binding domain (MBD) of MeCP2, but which did
not bind methylated DNA (Hendrich and
Bird, 1998). Recently, we generated Mbd3-/- ES
cells by gene targeting and showed that Mbd3 is required for stable formation
of the NuRD complex in ES cells (Kaji et
al., 2006). Mbd3-/- ES cells are viable and
maintain expression of genes associated with pluripotent cells such as Oct4
and Nanog, but fail to differentiate or silence expression of Oct4 upon
withdrawal of LIF in vitro or in chimeric embryos.
Mice lacking a functional Mbd3 gene die prior to midgestation
(Hendrich et al., 2001), but
why these embryos fail to develop, and what role is played by Mbd3/NuRD in
early embryos has not been described. In this study, we have characterised the
function of Mbd3 in peri-implantation development. We find that Mbd3 is
required for ICM cells to develop into late epiblast after implantation, for
proliferation of epiblast cells in culture, and for the proper function of
extra-embryonic tissues immediately after implantation. We further identify a
number of gene expression differences between ICMs from 3.5 dpc embryos, 4.5
dpc embryos and ES cells, many of which show misregulation in mutant embryos
prior to the onset of morphological abnormalities. Together, these data
demonstrate that Mbd3/NuRD plays a key role in the transition of pluripotent
ICM cells towards an epiblast fate during peri-implantation development, and
for the acquisition of an ES cell fate.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) were
backcrossed to C57Bl/6 mice for more than 10 generations. Hex-GFP mice
(Rodriguez et al., 2001) were
kindly provided by Joshua Brickman and used to generate
Mbd3-/- Hex-GFP embryos. Embryonic diapause was induced as
described (Buehr and Smith,
2003). To generate GFP-ES cell/Mbd3 mutant embryo chimeras,
Tau-GFP ES cells (Pratt et al.,
2000) were aggregated with morulae obtained from Mbd3 heterozygote
intercrosses. After overnight culture, embryos were transferred to the uteri
of embryonic day (E) 2.5 pseudopregnant females and collected after 3
days.
Immunofluorescence and RNA in situ hybridisation
Embryos were fixed with 4% paraformaldehyde and permeabilised with 0.25%
Triton X-100. After blocking with 1% skimmed milk and 0.05% Tween 20 in PBS,
embryos were stained with primary antibodies: anti-Mbd3 (sc-9402, Santa Cruz),
anti-Oct4 (sc-8628, sc-5279, Santa Cruz and Becton Dickinson), anti-Nanog
(provided by Ian Chambers, Institute for Stem Cell Research, Edinburgh, UK),
anti-Gata4 (sc-1237, Santa Cruz), anti-
7 integrin (provided by Ann
Sutherland, University of Virginia, Charlottesville, VA)
(Klaffky et al., 2001),
anti-activated caspase-3 (AF-835, R&D Systems), anti-Ki67 (MAB4062,
Chemicon), followed by the appropriate secondary antibodies. Nuclei were
stained with DAPI (Molecular Probes). Images were captured with a confocal
microscope (Leica TSC SP2) and Leica confocal software. Whole-mount RNA in
situ hybridisation was performed as described
(Rosen and Beddington, 1993).
Staining reactions were carried out for 2 days. The in situ probes for
Cdx2 and Mash2 have been described previously
(Buehr et al., 2003;
Nichols et al., 1998). Embryos
were genotyped by PCR.
Immunosurgery and ICM culture
Immunosurgery was carried out as described
(Solter and Knowles, 1975),
with collection of trophectoderm lysates for genotype determination by PCR.
ICMs were cultured in the presence of 2000 U/ml of LIF and 20% FCS in GMEM on
gelatin-coated dishes.
Single-ICM and single-cell PCR
cDNA from single ICMs (30-40 cells) or single cells was amplified as
described (Iscove et al.,
2002), except for the following modifications. The sequence of the
primer used for cDNA amplification is
5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGTACT30-3'.
SuperRNaseIn (Ambion) and SuperScript III (Invitrogen) were used as RNase
inhibitor and reverse transcriptase, respectively. PCR was carried out with
ExTaq (Takara). The genotype of individual ICMs was confirmed by PCR with
trophectoderm lysates. cDNAs from seven 3.5-dpc ICMs, six 4.5-dpc ICMs, or
five single ES cells, respectively, were combined and the amount of cDNA was
normalised for Gapdh expression by PCR. See
Table 1 for information on
gene-specific primers. All PCRs for specific genes were performed with an
annealing temperature of 60°C.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To
investigate the role played by Mbd3/NuRD in pluripotent cells in vivo, we
sought to determine the function of Mbd3 in pre- and peri-implantation
embryos. In wild-type embryos, Mbd3 expression was observed in all cells from
morulae to the egg cylinder stage at 6.5 dpc, and appeared exclusively nuclear
(Fig. 1A). Maternal Mbd3
protein was detected in morulae but not in blastocysts, as judged by
immunofluorescence of Mbd3-/- embryos produced by
intercrossing Mbd3 heterozygote parents
(Fig. 1B). At both 3.5 dpc and
4.5 dpc, Mbd3-/- embryos were morphologically
indistinguishable from wild-type littermates, showed normal expression of Oct4
at 3.5 dpc and 4.5 dpc (Fig.
2A), and normal segregation of early epiblast (Oct4-Nanog
double-positive cells; Fig. 2B)
and hypoblast or primitive endoderm (Gata4-expressing cells, some of which
also express Oct4; Fig. 2B) at
4.5 dpc. Null embryos were recovered at mendelian frequencies at both 3.5 dpc
and 4.5 dpc (Table 2).
|
). By contrast, Oct4-expressing cells in
Mbd3-/- embryos were gradually lost without ever forming a
proamniotic cavity (Fig. 2C)
while the ectoplacental cone continued to grow during early postimplantation
stages, demonstrating that Mbd3 is not required for growth of all cell types.
Whereas null embryos were recovered at sub-mendelian frequencies at 5.5 dpc
and 6.5 dpc, by 8.5 dpc the number of null embryos recovered reverted to the
expected 25%. This indicates that our failure to isolate some
Mbd3-/- embryos immediately after implantation is due to
their very small size, whereas by 8.5 dpc, null conceptuses have grown a large
ectoplacental cone and are easily identified
(Table 2 and
Fig. 2C).
|
). No
Oct4-caspase-3 double-positive cells were found in any
Mbd3-/- embryos (n=3), and were found only rarely
in wild-type embryos (8 of 16 embryos) (see Fig. S1A in the supplementary
material). To determine whether loss of Mbd3 results in quiescence of late
epiblast cells, embryos were stained for Ki67 (Mki67), a marker of
proliferating cells (Gerdes,
1990). Ki67-expressing cells are detectable in both the
extra-embryonic tissues and in the Oct4-expressing cells of both wild-type and
Mbd3-/- embryos at 5.5 dpc (see Fig. S1B in the
supplementary material). Thus, we find no evidence for death or quiescence of
ICM descendents in the absence of Mbd3 at 5.5 dpc.
|
).
The epiblast, extra-embryonic ectoderm, and primitive endoderm show a complex
interdependence during normal development, such that a defect in one tissue
can result in developmental abnormalities in an adjacent tissue
(Guzman-Ayala et al., 2004;
Murray and Edgar, 2000).
Therefore, we next assessed whether other cell types were formed properly in
mutant peri-implantation embryos. Double-staining of
Mbd3-/- 5.5 dpc embryos for Oct4 and 7 integrin, a
marker of extra-embryonic ectoderm and trophectoderm
(Klaffky et al., 2001), showed
that although the 7 integrin-expressing cells are distinct from and
surround the Oct4-expressing cells, mutant embryos do not form an organised
extra-embryonic ectoderm structure (Fig.
3A, bottom panels). Similarly, Mash2 (Ascl2 -
Mouse Genome Informatics) expression, which normally marks the developing
ectoplacental cone, was detectable over the entire mutant embryo
(Fig. 3B). Cdx2, which
is normally expressed in trophoblast stem cells and in extra-embryonic
ectoderm (Strumpf et al.,
2005), was detectable in only a few cells by whole-mount in situ
hybridisation in 5.5 dpc Mbd3-/- embryos, again consistent
with a lack of extra-embryonic ectoderm organisation
(Fig. 3B). The fact that
Mbd3-null embryos could form an ectoplacental cone
(Fig. 2C) and were able to
implant, indicates that some trophectoderm lineage cells could proliferate and
function in the absence of Mbd3. Nevertheless, we saw no evidence for
expansion of the Cdx2-expressing cell population or formation of
extra-embryonic ectoderm in mutant embryos after implantation.
Staining embryos for Oct4 and Gata4, an endoderm marker, revealed that
although Mbd3-/- embryos show normal endoderm
specification at 4.5 dpc (Fig.
2B), by 5.5 dpc mutant embryos have failed to form the organised
visceral endoderm epithelium seen in wild-type embryos at this stage
(Fig. 3C). In addition to the
Gata4-positive cells and the Oct4-positive cells, presumably indicating
primitive endoderm and primitive ectoderm, respectively, mutant embryos also
contained Gata4-Oct4 double-positive cells (n=3)
(Fig. 3C). Oct4-Gata4
double-positive cells were never observed in wild-type embryos at this stage
(n=12), but were characteristic of early primitive endoderm cells in
the ICMs of 4.5 dpc embryos (e.g. Fig.
2B). This persistence of Oct4-Gata4 double-positive cells in 5.5
dpc Mbd3-/- embryos, as well as the lack of robust
expression of Hex (Hhex - Mouse Genome Informatics), a
marker of distal visceral endoderm in 5.5 dpc embryos
(Rodriguez et al., 2001) (see
Fig. S2 in the supplementary material), indicates that primitive endoderm
formation is also abnormal in these embryos.
|
). Mbd3-/- embryo/ES cell
chimeras contained a significant number of GFP-expressing, ES-derived cells at
5.5 dpc; however, they formed neither a cavitated epiblast nor an organised
extra-embryonic ectoderm (Fig.
3D; n=3). Thus, a wild-type, ES cell-derived epiblast was
not able to rescue the Mbd3-/- embryonic phenotype at 5.5
dpc (Fig. 3D) or 6.5 dpc (data
not shown). We therefore conclude that Mbd3 is necessary for normal
development of both the embryonic and extra-embryonic tissues in
peri-implantation embryos.
Mbd3 is required for epiblast expansion in vivo and ex vivo
Given the different developmental capacity of Mbd3-/-
ES cells and ICM cells in embryos, we sought to characterise more fully the
ICM phenotype of Mbd3-/- embryos independently of the
extra-embryonic phenotype. In order to determine whether the loss of
Oct4-expressing cells seen in postimplantation Mbd3-/-
embryos is due to an autonomous defect, or to some developmental or
environmental cue, the preimplantation period was extended by subjecting
blastocysts to diapause (Buehr and Smith,
2003). In this context, epiblast cells and primitive endoderm
cells are spatially distinct, with the former expressing both Oct4 and Nanog
while the latter express Oct4 and Gata4
(Fig. 4A). As in non-delayed
5.5 dpc embryos, Mbd3-/- blastocysts subjected to diapause
for 2 days (i.e. at 5 days after fertilisation) generally contained few, if
any, Oct4/Nanog-expressing epiblast cells, whereas all embryos contained a
Gata4-expressing primitive endoderm layer that was comparable in size to that
see in wild-type or heterozygous littermates
(Fig. 4A). Thus, we conclude
that the loss of epiblast cells in Mbd3-null embryos occurs independently of
the process of implantation.
To exclude any possible influence of the trophectoderm on the growth of
mutant epiblasts, we next investigated the growth potential of isolated
Mbd3-/- ICMs ex vivo. ICMs from 3.5 dpc blastocysts were
isolated away from the surrounding trophectoderm by immunosurgery and cultured
in the presence of LIF. In most cases, the outgrowths from
Mbd3-/- ICMs were distinguishable from those of wild-type
or Mbd3+/- ICMs from the first or second day of culture,
producing a smaller cell mass and tighter cell junctions between outgrowing
endoderm cells (Fig. 4B). After
3 days in culture, only 32% of Mbd3-/- outgrowths
contained a recognisable cell mass as compared with 79% or 86% of wild-type or
heterozygous outgrowths, respectively (Fig.
4C). Mbd3-/- cell mass outgrowths were
invariably smaller than those produced by wild-type or heterozygous ICMs, and
contained few if any Oct4-positive cells
(Fig. 4B,D). By contrast, the
endoderm produced by Mbd3-/- outgrowths expressed Gata4
and Sparc, a marker of differentiated parietal endoderm
(Holland et al., 1987) (data
not shown). Thus, we conclude that although dispensable for primitive endoderm
growth, Mbd3 is required for the expansion of Oct4-expressing cells during ICM
culture that is essential for ES cell derivation
(Buehr and Smith, 2003).
|
).
Therefore, the failure of Mbd3-/- ICMs to expand their
pluripotent cell population is likely to be due to aberrant gene expression
patterns in the ICMs of Mbd3-null preimplantation embryos. We reasoned that
gene expression changes resulting in the observed embryonic phenotypes, rather
than occurring as a secondary consequence of them, should be present in
Mbd3-null ICMs after the loss of maternally-derived Mbd3 protein, but prior to
the onset of morphological abnormalities, i.e. in 3.5 dpc and 4.5 dpc ICMs. To
identify such genes, we monitored gene expression in wild-type and
Mbd3-/- 3.5 dpc and 4.5 dpc ICMs by RT-PCR
(Fig. 5). To make cDNA from
individual ICMs, we used a single-cell cDNA amplification method that includes
multiple amplification steps (see Materials and methods for details). In order
to control for possible threshold effects and/or amplification bias, cDNA was
also made from single ES cells and compared with ES cell cDNA prepared using a
conventional method. As shown in Fig.
5, the amplified cDNA produced identical results to those obtained
using conventional cDNA for a number of control genes (Mbd3, Gapdh, Oct4,
Nanog, Pramel4, Pramel5). It is noteworthy that the reported decrease in
Nanog expression in 4.5 dpc ICMs
(Chambers et al., 2003;
Saba-El-Leil et al., 2003) is
detectable in our ICM cDNAs, and indicates some degree of normal ICM
development between 3.5 and 4.5 dpc in the absence of Mbd3
(Fig. 5).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Mbd3-/- embryos can be recovered at each of these stages
that contain Oct4-Nanog double-positive cells; however, this presumptive
epiblast precursor population fails to expand in vivo or to outgrow in
culture. This finding is surprising given the fact that Mbd3 is not required
for viability or self-renewal of ES cells
(Kaji et al., 2006). These
data, along with our identification of differential gene expression between
3.5 dpc ICMs, 4.5 dpc ICMs and ES cells, demonstrate that although each of
these cell types are pluripotent, they differ from each other in terms of gene
expression and requirements for expansion; ES cells, which in practice are
most often derived from 3.5 dpc ICMs, are most similar to 4.5 dpc ICMs. In
addition, we show that Mbd3/NuRD is necessary for proper gene expression in
3.5 dpc and 4.5 dpc ICMs, and is important for development of the ICM-derived
cell population in peri-implantation embryos.
Several epigenetic silencing proteins have been reported to be important
for early murine development (Bernstein et
al., 2003; Cowley et al.,
2005; Dannenberg et al.,
2005; David et al.,
2003; Dodge et al.,
2004; Hendrich et al.,
2001; O'Carroll et al.,
2001; Rayasam et al.,
2003), and often a requirement in ES cells is inferred from the
failure of blastocysts to outgrow in culture. Here, we show that Mbd3, a
component of the NuRD co-repressor complex, is required for the initial step
of ES cell derivation, namely expansion of the early epiblast cell population,
despite being dispensable for ES cell self-renewal. This requirement in early
embryonic development and for the growth of ICM cells in vitro, but not for ES
cell maintenance, has been reported for only one other epigenetic silencing
protein, namely Dicer, a protein important in RNAi-mediated gene silencing
(Bernstein et al., 2003;
Kanellopoulou et al., 2005;
Murchison et al., 2005).
Recently, O'Neill et al. identified differences in histone acetylation
patterns between ICMs and ES cells, possibly indicating epigenetic changes
important for ES cell derivation from ICMs
(O'Neill et al., 2006).
Together, these data indicate that epigenetic silencing plays an important
role in the developmental and gene expression changes that occur during the
development of pluripotent cells in vivo and for ES cell derivation.
Mbd3/NuRD is required for programmed maturation of pluripotent ICM cells and development of extra-embryonic tissues
Maternal Mbd3 protein is lost in embryos by 3.5 dpc, at which point altered
gene expression patterns are detectable in ICM cells, followed by the
appearance of morphological abnormalities within 48 hours.
Mbd3-/- embryos at 5.5 dpc lack a mature late epiblast,
proper spatial organisation and distinct extra-embryonic ectoderm and visceral
endoderm structures, although they do contain cells expressing markers for
each of these lineages. Epiblast cells in 5.5 dpc Mbd3-/-
embryos display characteristics of 3.5 dpc ICM cells, such as a lack of
expansive growth or cavitation, proximal localisation and the presence of
Oct4-Gata4 double-positive cells. Mbd3-null embryos lose Oct4-expressing cells
by 7.5 dpc, while the ectoplacental cone continues to grow. Given that we
could find no evidence that the Oct4-expressing cells either die by apoptosis
or undergo cell cycle arrest, it may be that they fail to make the transition
from ICM cells to become late epiblast, and instead all gradually lose Oct4
expression while activating Gata4 expression and become primitive
endoderm.
|
),
whereas embryos lacking Elf5, a transcription factor belong to the Ets
superfamily, can develop a cavitated epiblast while lacking an extra-embryonic
ectoderm (Donnison et al.,
2005), indicating that extra-embryonic ectoderm is not strictly
required for epiblast growth in vivo. We cannot rule out the possibility that
the defect we see in Mbd3-/- ICM cells is actually caused
by some defect in very early trophectoderm cells. However, we feel this is
very unlikely given the fact that ES cell derivation is possible from
Cdx2-null embryos, which lack any detectable trophectoderm functions
(Meissner and Jaenisch, 2006),
demonstrating that early failure of trophectoderm lineages does not preclude
ICM outgrowth in vitro. Although Cdx2-null blastocysts appear to have
trophectoderm-like cells, these cells express Nanog and thus may not actually
be trophectoderm (Strumpf et al.,
2005).
It is also unlikely that the Mbd3-null embryonic phenotype is exclusively
due to an epiblast defect, as wild-type ES cells were unable to rescue the
mutant phenotype in chimeric embryos, and embryos lacking an epiblast due to
mutation in Foxd3, a member of the forkhead family of transcriptional
regulators, are able to form extra-embryonic ectoderm
(Hanna et al., 2002). In
addition, whereas most Mbd3-/- ICMs were able to give rise
to adherent primitive endoderm cells expressing Gata4, even these cells showed
a more compact growth morphology than did wild-type primitive endoderm
outgrowths, indicating some defect in proliferation and/or adhesion consistent
with the disorganised visceral endoderm seen in mutant embryos at 5.5 dpc.
Further analysis of Mbd3-deficient extra-embryonic tissues and of Mbd3-null
trophectoderm stem cells and endoderm stem cells
(Kunath et al., 2005;
Tanaka et al., 1998) will be
important for defining the tissue-specific and general requirements for
Mbd3/NuRD during early postimplantation development.
Transcriptional misregulation in Mbd3-/- ICMs
Although it is becoming clear that in 3.5 dpc ICMs there may be distinct
cell populations fated to be either epiblast or primitive endoderm with
distinct gene expression patterns (Chazaud
et al., 2006; Kurimoto et al.,
2006), gene expression changes that accompany the maturation of
epiblast cells upon implantation remain largely uncharacterised. We identified
11 genes that show altered expression patterns in 3.5 and/or 4.5 dpc ICMs in
the absence of Mbd3/NuRD. Sohlh2, Sfrp1, Aes and Dazl show
increased expression immediately after loss of maternally-derived Mbd3 protein
at 3.5 dpc, and misregulation persists in 4.5 dpc ICMs and in ES cells, making
these genes good candidates to be predominantly and directly regulated by
Mbd3/NuRD. Sfrp1 is a secreted antagonist of the Wnt signaling pathway
(Kawano and Kypta, 2003); Aes
is a naturally occurring dominant-negative molecule alleviating
transcriptional repression by TLE/GRG proteins, which bind and repress various
transcription factors including Tcf proteins, the nuclear mediators of
canonical Wnt signaling (Brantjes et al.,
2001). There are several Wnt pathway molecules expressed in
preimplantation embryos that can function in both canonical and non-canonical
Wnt pathways (Kemp et al.,
2005). Therefore, it will be interesting to determine whether
misregulation of Sfrp1 and Aes, alone or in combination, can
affect the normal development of pluripotent cells.
The two other genes showing early misexpression, Dazl, an
RNA-binding translational regulator, and Sohlh2, a basic
helix-loop-helix (bHLH) protein, are both exclusively expressed in germ cells
(Ballow et al., 2006;
Ruggiu et al., 1997). By
contrast, expression of another germ cell-specific gene, Dppa3 (also
called Stella or Pgc7), is not disturbed in
Mbd3-/- ICMs despite the fact that it is strongly
downregulated in Mbd3-/- ES cells
(Fig. 5)
(Kaji et al., 2006). Thus, it
is unlikely that misregulation of Dazl and Sohlh2 in
Mbd3-/- ICMs reflects some commitment towards the germ
cell lineage; however, it remains possible that their overexpression in
Mbd3-/- ICMs and ES cells interferes with normal
development and/or lineage commitment of pluripotent cells.
The Pramel (PRAME-like) genes encode proteins similar to the human
preferentially expressed antigen in melanoma, or PRAME, protein
(Bortvin et al., 2003). PRAME
is an antigen expressed in a wide variety of human tumours
(van Baren et al., 1998) and
was recently shown to act as a dominant repressor of retinoic acid receptor
signalling and to inhibit retinoic acid-induced differentiation of mouse
embryonal carcinoma cells (Epping et al.,
2005). Although the function of the Pramel proteins is not known,
their preimplantation-specific expression pattern in mouse
(Fig. 5), and the existence of
families of Pramel genes in the genomes of other mammals, Xenopus
tropicalis and Gallus gallus, might indicate that they play some
conserved role in early vertebrate development
(Birtle et al., 2005) (B.H.,
unpublished observations).
Molecular and transcriptional differences between ICM cells and ES cells
ES cells are derived from ICMs, inheriting their pluripotency and acquiring
the ability for indefinite self-renewal
(Brook and Gardner, 1997). ES
cell derivation remains an inefficient process, the efficiency of which varies
between different inbred mouse strains and is currently not possible in other
rodent species (Buehr and Smith,
2003; Robertson,
1987). Despite the growing popularity of ES cell research,
surprisingly little is known about the molecular differences between
ICM/epiblast cells and ES cells. In addition to identifying Mbd3-dependent
gene expression patterns in the ICMs of preimplantation stage embryos and ES
cells, we also found seven genes (Pramel4, Pramel5, Pramel6, Pramel7,
Ppp2c2r, 2410076I21Rik and Sohlh2) downregulated and 2 genes
(Htra1 and Pak1) upregulated between 3.5 dpc and 4.5 dpc in
wild-type ICMs (Fig. 5). The
expression of most of these genes in wild-type ES cells is more similar to
that in 4.5 dpc ICMs than in 3.5 dpc ICMs. These molecular data support the
assertion that the origin of ES cells is early epiblast rather than 3.5 dpc
ICM (Brook and Gardner, 1997).
This indicates that during ES cell derivation, pluripotent cells in 3.5 dpc
ICMs must first mature in culture to a 4.5 dpc-like state before undergoing
the necessary epigenetic changes that enable them to become ES cells
(Fig. 6). ICMs at 4.5 dpc,
which include the early epiblast, also show some gene expression differences
when compared with ES cells. Nanog is robustly expressed in ES cells,
but its mRNA levels are decreasing in 4.5 dpc ICMs
(Chambers et al., 2003), and
Pak1 expression is also lower than in ES cells
(Fig. 5). By contrast,
expression of Pramel7, 2410076I21Rik and Htra1 is higher in
4.5 dpc ICMs than in ES cells. Two genes (Pak1 and Dppa3)
showed no significant misregulation in Mbd3-/- ICMs,
although these genes are misregulated in Mbd3-/- ES cells
(Fig. 5)
(Kaji et al., 2006),
indicating a difference in control of epigenetic silencing between ICM cells
and ES cells.
The effect of loss of Mbd3 is different in 4.5 dpc ICMs than in ES cells.
Pak1 and Dppa3 expression is misregulated in
Mbd3-/- ES cells, but not in Mbd3-/-
4.5 dpc ICMs. Furthermore, whereas Mbd3-/- ICM cells fail
to initiate the rapid proliferation characteristic of late epiblast and do not
maintain expression of Oct4, Mbd3-/- ES cells show only a
modest growth defect and robustly express Oct4 even when induced to
differentiate (Kaji et al.,
2006). While we cannot exclude the possibility that some of the
differences observed here come from the presence of primitive endoderm cells
in 4.5 dpc ICMs, these data further illuminate the differences between ICM
cells and ES cells. Although the molecular events that occur during ES cell
derivation are largely unknown, here we show that Mbd3 is necessary for ICM
cells to gain ES cell-like properties of increased proliferation and an
appropriate gene expression profile necessary for both in vivo development and
ES cell derivation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/6/1123/DC1
|
ACKNOWLEDGMENTS |
|---|
7 integrin
antibody, Ian Chambers for the anti-Nanog antibody, Josh Brickman and Korinna
Henseleit for Hex-GFP mice, and Austin Smith, Val Wilson, Tilo Kunath, Laura
Batlle-Morera and Nicola Reynolds for advice, discussions and comments on the
manuscript. K.K. was the recipient of a postdoctoral fellowship from the
Japanese Society for the Promotion of Science. This work was funded by the
Wellcome Trust, the UK Medical Research Council and by a BBSRC UK-Japan
Partnering Award. |
Footnotes |
|---|
|
REFERENCES |
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