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First published online March 23, 2006
doi: 10.1242/10.1242/dev.02317
1 Unité de Biologie du Développement et de la Reproduction, UMR
INRA-ENVA, Institut National de la Recherche Agronomique (INRA), Jouy-en-Josas
78352, France.
2 Laboratoire de Développement des Vertébrés, Institut
Jacques Monod, UMR 7592 CNRS, Université Paris 6 et 7, 2 place Jussieu,
75251 Paris, France.
Authors for correspondence (e-mail:
alice.jouneau{at}jouy.inra.fr;
jeanpaul.renard{at}jouy.inra.fr)
Accepted 9 February 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Nuclear transfer, Growth, Gastrulation, Mouse, Trophoblast, Chimaera
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Latham, 2004;
Solter, 2000).
Whatever the type of the donor cell used, nuclear transfer (NT) embryos can
easily be reprogrammed into blastocysts from which pluripotent embryonic stem
(ES) cells lines can be derived in vitro
(Munsie et al., 2000;
Wakayama et al., 2001). More
recently, it has been shown that ntES cell lines were very similar to
fertilised embryos-derived ES cells in terms of their ability to differentiate
in vitro and in vivo, and to colonise the germ-line in chimaera mice
(Barberi et al., 2003;
Rideout et al., 2002;
Wakayama et al., 2005). As NT
blastocysts derived from mouse somatic donor cells more often exhibit an
abnormal expression pattern of genes compared with NT blastocysts derived from
pluripotent ES nuclei (Boiani et al.,
2002; Bortvin et al.,
2003), these results indicate that in vitro culture is able to
either complete the reprogramming process in ICM cells from which ES cells are
derived or select the few pluripotent ones.
By contrast, only a few percent of those blastocysts give rise to live
pups, despite the fact that most of them can implant after transfer into
recipient females (Wakayama et al.,
1998; Wakayama et al.,
1999; Wakayama and
Yanagimachi, 2001; Zhou et
al., 2001). This is observed whatever the type of donor cell used,
although the post-implantation developmental arrest of ES-derived NT embryos
is lower than that of somatic cell-derived NT embryos
(Eggan et al., 2001;
Wakayama et al., 1999). In all
cases, the period of highest lethality starts after the blastocyst stage.
Several histological and molecular placental abnormalities considered to be
the major causes of foetal death have been identified after nuclear transfer
in different mammalian species (Heyman et
al., 2002; Hill et al.,
2000; Lee et al.,
2004; Wilmut et al.,
2002), including the mouse
(Suemizu et al., 2003;
Tanaka et al., 2001). In the
latter species, widespread differences in the expression profile of genes in
the liver of control pups, and premature death and obesity of surviving mice
have also been reported (Humpherys et al.,
2002; Ogonuki et al.,
2002; Tamashiro et al.,
2002)
Taken together, these data indicate that incomplete reprogramming after
nuclear transfer affects more severely the extra-embryonic tissues, which
directly participate in the post implantation development, than the embryonic
tissues, which remain able to provide at least in vitro pluripotent ES cells.
The fact that a high rate of derivation of ntES cell lines is not predictive
of a high rate of successful development
(Boiani et al., 2005) further
supports this view. Although this has practical important consequences for the
use of pluripotent ntES cells for therapeutic applications
(Jaenisch, 2004), it leaves
unresolved the issue of how reprogramming can occur differently in the two
compartments of the blastocyst and how this, in turn, will affect the
interaction between the different emerging tissues of the conceptus. The
concept of epiblast pluripotency encompasses the ability of these embryonic
tissues not only to differentiate into different cell lineages but also to
establish the embryonic axes and the body plan
(Tam et al., 2001). In the
mouse, the development of the embryo after the blastocyst stage relies on
close relationships between the conceptus and the maternal environment, and
within the conceptus, between the embryo proper and its surrounding
extra-embryonic tissues (Camus et al.,
2004). Thus, the fate of the epiblast of NT embryos has to be
characterised carefully in vivo to ascertain the impact of the cloning
procedure on the developmental competence of reconstructed embryos. Despite
its importance to development and the need of a basic understanding of
reprogramming processes, the early post-implantation period of NT embryos has
remained poorly characterised up until now. What is more, no attempt to track
down the origin of the defects observed at later stages in the placenta has
yet been reported. In the mouse, most of the cells that compose the placenta
come from the trophoblast and, more precisely, from the extra-embryonic
ectoderm that develops in close contact with the epiblast at early
post-implantation stages (Cross,
2005; Rossant and Cross,
2001).
Therefore, we have produced NT embryos and focused our analysis on the
development beyond the blastocysts stage. We devised previously specific
conditions allowing the production of a high rate of morphologically normal
blastocysts using ES cells as donors (Zhou
et al., 2001). It allowed us to create an experimental situation
where the blastocysts formed a homogenous and apparently normal population, at
least for several genes like Oct4 essential for the immediate
peri-implantation survival (Bortvin et
al., 2003; Nichols et al.,
1998). In this context, we could address clearly the question of
the potency of the NT epiblast in vivo. The aim of the present study was to
pinpoint the embryological origin of the developmental defects encountered
during the post-implantation period of NT embryos in terms of stage and tissue
affected.
Our results show that growth alteration at early post-implantation stages is the first manifestation of reprogramming defects. It first affects the embryonic tissue and then the trophoblast, but it does not affect the differentiation potential of epiblast cells.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The R26.6 ES cell line (129 background), derived
from ROSA26 mice, was kindly provided by Philip Soriano
(Friedrich and Soriano, 1993).
GFP-expressing mice were kindly obtained from Masaru Okabe. They are
transgenic mice (B6 background) ubiquitously expressing GFP under a
ß-actin promoter and cytomegalovirus enhancer
(Okabe et al., 1997).
Production of NT embryos from ES nuclei
Nuclear transfer of ES nuclei into enucleated oocytes was performed as
previously described (Zhou et al.,
2001). Briefly, mitotic cells were collected from R1 ES cells
treated with demecolcin. NT embryos were reconstituted by injection of
metaphase nuclei into enucleated oocytes from superovulated B6CB F1 females
and then activated in strontium containing CZB medium.
Embryos were either transferred at two-cell stage into pseudo pregnant B6CB F1 females or cultured in M16 medium (Sigma) up to the blastocyst stage. Recipients were sacrificed at different stages and the embryos dissected into PB1 medium containing 10% foetal calf serum. The recovered embryos or foetuses were fixed with 4% paraformaldehyde. They were then dehydrated through graded ethanol series and stored at 20°C. Controls consisted of in vivo or in vitro fertilised embryos (B6CB F2) cultured for at least 1 day before transfer into recipients.
Immunofluorescence studies on preimplantation embryos
Embryos were rinsed in PBS and fixed in 4% paraformaldehyde at room
temperature for 20 minutes. They were then permeabilised in PBS containing 10%
FCS and 0.4% Triton X100 for 20 min at room temperature. Incubation was
carried out for 1 hour at room temperature with anti-Oct4 monoclonal antibody
(Becton-Dickinson) diluted 1/100 in the same solution. After rinsing, the
embryos were incubated under the same conditions with anti-mouse IgG coupled
to FITC (Jackson). Nuclei were counterstained with propidium iodide (Sigma)
before the slides were mounted. Observations were made with an LSM 310
confocal microscope (Carl Zeiss, Germany).
Blastocyst outgrowths
NT or control embryos were cultured in M16 medium for 4 days. The zona
pellucida of the blastocysts was removed in acid tyrode and the embryos were
cultured in DMEM with 20% serum on gelatinised four-well plates. Outgrowth
formation was followed daily using an inverted microscope and photographed.
The areas occupied by the ICM outgrowths were measured on the digitalised
images.
RT-PCR analysis of gene expression on single embryos
Poly A+ RNA from each single NT embryo was isolated using a
Dynabeads mRNA DIRECT kit (DYNAL). The Oligo-(dT)25 covalently
bound to the biomagnetic dynabeads was used to capture mRNA and subsequently
as a primer for the reverse transcriptase (Superscript II, GIBCO) to
synthesise the first strand of cDNA at 42°C for 1 hour. PCR products were
separated on 1.5% agarose gel and visualised with ethidium bromide
fluorescence. Our RT-PCR assays were not quantitative; a positive signal shows
that the transcript of the gene is detected.
Volume and mitotic index analyses
Embryos from pre-streak to late streak stages were fixed with 4%
paraformaldehyde, then permeabilised with 0.4% Triton X100 before being
treated by RNase A (20 µg/ml) for 20 minutes at 37°C. The nuclei were
then stained with propidium iodide at 10 µg/ml in PBS before being mounted
on slide with Vectashield mounting medium (Vector). Optical sectioning was
carried out using a confocal microscope (LSM 310, Zeiss). From each series of
slices, we determined the volume of the epiblast and the extra-embryonic
ectoderm using Image Tool software. The number of mitotic figures was also
counted on each section. The initial determination of the correlation between
the number of cells and the volume was made on a subset of control and cloned
embryos using a method adapted from Power and Tam
(Power and Tam, 1993). For
these experiments, several small areas of the epiblast and the extra-embryonic
ectoderm were selected from the optical sections (4 µm) of each embryo. The
number of cells contained in these areas and the number of cells per unit
volume were both determined. The total cell number in each epiblast and
extra-embryonic ectoderm was obtained by dividing the volume of each region by
the number of cells per unit volume. The equations of the resulting linear
curves were then used to calculate the number of cells from each volume
subsequently determined.
Whole-mount in situ hybridisation
The probe-containing plasmids were transcribed in vitro using the Dig-RNA
labelling kit (Roche). Whole-mount in situ hybridisation was performed
essentially as previously described
(Wilkinson et al., 1990).
Proteinase K and RNAse treatments were omitted for embryos up to MS stage.
Alkaline phosphatase activity was detected by using BM purple AP substrate
(Roche). Embryos were observed and photographed through a SZX binocular
(Olympus) coupled to a camera.
Probes for the following genes were obtained as follows: Oct4, from A.
Smith; Bmp4, from B. Hogan (Winnier et
al., 1995); brachyury, from B. Herrmann
(Herrmann et al., 1990); Eomes
and Cdx2, from J. Rossant (Beck et al.,
1995; Ciruna and Rossant,
1999), and Nodal from E. Robertson
(Varlet et al., 1997).
Generation and analysis of chimaeras
Blastocysts developed in vitro from NT embryos were injected with 8-12
R26.6 ES cells and then transferred into pseudo-pregnant females. Conceptuses
were recovered at E7 or E19 then fixed and processed for X-gal staining as
described by Hogan et al. (Hogan et al.,
1994).
Embryos expressing GFP were produced after mating GFP-expressing homozygous
B6 males with B6CB F1 females. ICM were isolated by immunosurgery from
GFP-expressing blastocysts, as described by Solter and Knowles
(Solter and Knowles, 1975).
They were briefly incubated with 0.5% Pronase and injected into NT or control
blastocysts using an injection pipette with an internal diameter of 20 µm.
Injected blastocysts were then transferred into pseudo-pregnant
recipients.
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After dissection at E7, chimaeras expressing GFP were fixed with 4% paraformaldehyde, permeabilised and subjected to RNAseA treatment before nuclear staining with propidium iodide. They were then observed under the confocal microscope.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In the
present study, we examined whether the pluripotential gene marker
Oct4 was correctly expressed in a subset of these blastocysts.
Analysis of the expression of Oct4 in ES-cell NT blastocysts both at the RNA
(n=14) and protein (n=9) levels showed a normal pattern of
expression restricted to ICM (Fig.
1A-D).
Although blastocysts with `excellent morphology' may display abnormal gene
expression levels (Mann et al.,
2003), we concluded that most of the ES-cell derived embryos could
consistently develop to the blastocyst stage. To achieve a more functional
view of the developmental potential of these blastocysts, we assessed their
ability to grow in culture as an in vitro assay of the peri-implantation
period. We found that the ICM cells from almost all NT blastocysts (18 out of
20) grew and formed a typical mass of cells
(Fig. 1E,F), the areas of which
were not significantly different from those of controls after 2 and 3 days of
culture (Fig. 1G).
Finally, the ability of the NT blastocysts to implant and form deciduas was tested after transfer into pseudo-pregnant mice. Most of them (12 out of 14) were found to be pregnant at E7, with two-thirds of the transferred blastocysts implanted (67±19%, n=112). In vitro fertilised embryos used as controls have an implantation rate of 80% (n=40). Under our conditions, we thus conclude that most of the ES-cell derived blastocysts were indistinguishable from normal blastocysts in terms of cell allocation, ICM cell growth ability in vitro and high rate of implantation.
Main features of the post-implantation development of NT embryos
Because placental defects are considered to be the main cause of death of
clones, we first assessed the post-implantation survival of NT embryos after
E10 to cover the placental period of development
(Iguchi et al., 1993;
Rossant, 1995). NT embryos
were transferred at the two-cell stage into pseudo-pregnant females that were
dissected at E10, E13, E15 and E19 (Table
1).
|
)
(A.J., L.M., H. Jammes, V.B., Q.Z. and J.-P.R., unpublished). When present,
the foetuses were, however, very similar to controls, although slightly
heavier (Fig. 2D).
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Most NT embryos initiate gastrulation
Upon implantation, the rapid growth of the epiblast, the extra-embryonic
ectoderm (derived from the trophectoderm) and the overlaying visceral endoderm
(derived from the primitive endoderm) leads to the formation and elongation of
an egg-cylinder-shaped embryo (Camus et
al., 2004; Downs and Davies,
1993; Lewis and Rossant,
1982). The visceral endoderm and extra-embryonic ectoderm do not
contribute directly to the embryonic tissues but have an important role in
growth and patterning of the epiblast (Ang
and Constam, 2004; Beck et al.,
2002; Perea-Gomez et al.,
2004). In the epiblast, proliferation and morphogenetic movements
cooperate to generate the primitive streak and the three primitive germ layers
(Lawson et al., 1991;
Tam and Behringer, 1997). To
characterise the gastrulation pattern in NT embryos, we dissected recipient
females at E7 to E8.5 and recorded the length of the primitive streak and the
presence of either an anterior neural plate or a headfold, according to Downs
and Davis (Downs and Davis,
1993). The presence of somites signified the end of gastrulation.
We found that NT embryos were developmentally delayed compared with controls:
at E7.5 only a few had reached the neural plate stage, whereas more than half
of the controls had (see Table
2). One day later (at E8.5), fewer NT embryos had reached the
somite stage compared with controls (Table
2).
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;
Varlet et al., 1997) were
absent (data not shown). By contrast, three markers of the differentiation of
the visceral endoderm, the early marker Hesx1 and two late markers
Ttr and Afp (Barbacci et
al., 1999; Coffinier et al.,
1999; Thomas et al.,
1998), were expressed, suggesting that this tissue was
differentiating at least to some extent. The sizes of the other NT embryos were within the normal range at E7 and showed the presence of a clear limit between the extra-embryonic and embryonic regions. A closer morphological examination, however, revealed two abnormal phenotypes: one with an abnormal rounded rather than elongated shape, as illustrated in Fig. 3B. These embryos were, however, capable of initiating gastrulation, as half of them had a visible primitive streak (Fig. 3B, arrow on right). The second one, illustrated in Fig. 3D, was called the `large Exe' phenotype, and was found at all gastrulation stages in NT embryos. It was characterised by an abnormal ratio between the extra-embryonic and the embryonic regions. These two abnormal morphological phenotypes were the only ones observed at these stages (Table 3). These defects also occurred independently, as some NT embryos displayed both defects.
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|
).
However, in any case, the normal balance of the cell number in one compartment
in relation to the other is disrupted. Cell number determination for embryos
with the abnormal shape revealed that they were within the normal range
according to their stage of development (data not shown), suggesting that
their poorly elongated shape is not due to a specific proliferation defect but
rather to some perturbation of the control of the directed growth of the
conceptus along its proximodistal axis.
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In conclusion, this embryological analysis revealed that: (1) developmental defects in NT embryos arose early after implantation and affected some of the embryos recovered at E7 (Table 3); and (2) the types of defects observed were related to growth deregulation in one or both compartments of the conceptus.
Molecular pattern of gastrulation in NT embryos
Most of the defects described above did not prevent the initiation of
gastrulation. The formation of the primitive streak and the patterning of the
embryo require finely regulated crosstalk between the extra-embryonic ectoderm
and adjacent epiblast (Ang and Constam,
2004; Brennan et al.,
2001). Nodal in the epiblast, and Cdx2, Eomes and Bmp4 in the
extra-embryonic ectoderm are essential players in this process
(Fujiwara et al., 2002;
Russ et al., 2000;
Strumpf et al., 2005;
Vincent et al., 2003).
Brachyury, a T-box transcription factor, is first expressed in the
extra-embryonic ectoderm abutting the epiblast, then in the posterior epiblast
where the primitive streak forms and finally in the primitive streak cells
(Perea-Gomez et al., 2004;
Thomas et al., 1998). The
expression of this marker at the onset of gastrulation is indicative of the
correct establishment of the anteroposterior polarity. As an abnormal shape of
the egg cylinder or a disproportionate extra-embryonic ectoderm may perturb
this crosstalk and the establishment of morphogenetic gradients, we analysed
the expression of these different genes by whole-mount in situ hybridisation
in NT embryos displaying these different phenotypes
(Fig. 5).
Embryos with abnormal shapes had normal spatial patterns of expression of
brachyury (Fig. 5B) and
Eomes (Fig. 5J).
Embryos with the `Exe' phenotype expressed Nodal
(Fig. 5F) and Bmp4
(Fig. 5H), as in controls. In
the NT embryo expressing Bmp4, histological examination confirmed
that the size of the posterior amniotic fold in the extra-embryonic region was
indicative of a late-streak stage, whereas the elongation in the size of the
streak was that of a mid-streak stage (data not shown). This discrepancy
correlates the respective size of the extra-embryonic and embryonic regions of
the conceptus in such NT embryos with the `large Exe' phenotype. At the
advanced stage, this developmental gap was even larger, as illustrated by the
embryo in Fig. 5D. Brachyury
was still expressed in the primitive streak of the reduced embryonic region.
The presence of a fully expanded amniotic fold and an allantoic bud were
features of a neural plate embryo, whereas the epiblast was still at
late-streak stage (Downs and Davies,
1993). Localisation of Cdx2 in the extra-embryonic
ectoderm was correct in the different NT embryos analysed
(Fig. 5L).
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Normal ES or ICM cells do not rescue the developmental defects of NT embryos
The abnormal phenotypes that we characterised during the gastrulation of NT
embryos perhaps resulted from defective functioning of the extra-embryonic or
embryonic lineages (or both). Such defects, when occurring in the epiblast,
can be remedied by injecting normal ES cells into NT blastocysts. In the
resulting chimaeras, ES cells incorporate in the ICM and contribute mainly to
epiblast derivatives (Beddington and
Robertson, 1989). To confirm the contribution of ES cells to the
embryo, we used R26.6 ES cells that constitutively expressed the reporter
lacZ gene (Friedrich and Soriano,
1993). Injected blastocysts were transferred into recipients and
conceptuses recovered at different stages (Tables
4,
5;
Fig. 6A-D).
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Extra-embryonic tissues are composed of the trophoblast and the primitive
endoderm. Alteration in the latter could have negative consequences on the
growth of the embryo, because at early stages it provides the embryo with
nutrients (Bielinska et al.,
1999). As ICM cell derivatives are found in both the epiblast and
the primitive endoderm, we conducted chimaera experiments by injecting
GFP-expressing ICM cells from fertilised embryos into control or NT
blastocysts (Table 6). The
epiblast, its derivatives and the visceral endoderm were found to be chimaeric
(Fig. 6E). As for injected ES
cells, a partial rescue was observed, with a high rate of embryos found in the
deciduas at E7. The same morphological abnormalities were, however, observed
at similar rates (Table 6;
Fig. 6F). Altogether, these
results refute a primary source of abnormalities in the epiblast or the
visceral endoderm, and point to the extra-embryonic ectoderm lineage as the
defective tissue.
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). We checked for the presence of both types of
cell (GFP positive and negative) in the chimaeric embryos at morula stage
before transferring them into fosters. Upon recovery at E7, we found half of
the implanted embryos of each group (control and NT) to be chimaeric
(Table 7). Only a subset of
these chimaeras, however, had the expected contribution of GFP-expressing,
tetraploid cells in the extra-embryonic ectoderm. Most of them had normal
morphology, suggesting that the tetraploid extra-embryonic cells remedied the
defects (Fig. 7A,B;
Table 7). In some of the
embryos, the presence of non GFP-expressing, extra-embryonic ectoderm cells
indicated that NT derived cells had not been overcome by the tetraploid
cells.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
However, few if any results have been provided about the first stages and
tissues which are affected during development of NT embryos. Here, we provide
the first detailed analysis of the post-implantation development of NT
embryos.
We have chosen ES cells as donors for two reasons: first, a high rate of
blastocysts with both a normal morphology and allocation of cells to the first
two lineages can be obtained in an easy and reproducible manner
(Zhou et al., 2001), which is
not the case for somatic cells; second, ES-cell derived embryos faithfully
recapitulate the expression of Oct4 and related genes
(Bortvin et al., 2003). In the
present study, we have confirmed that all ICM cells of ES-cell derived
blastocysts express the protein Oct4 and we have shown, using an in vitro
outgrowth assay, that they can proliferate very similarly to controls. After
transfer into foster mice, the rate of implantation of NT embryos is high but
half of them resorb completely before E7. This result further confirms a
previous study which showed that the pattern of expression of Oct4 is not a
reliable indicator of developmental competence in vivo
(Boiani et al., 2005). We have
shown that this peri-implantation lethality could, however, be rescued
partially by the injection of wild-type ES or ICM cells into NT blastocysts,
as we found a higher number of deciduas containing embryos at the time of
dissection (E7). As trophectoderm proliferation is an essential process for
the initiation of implantation and is controlled by the ICM
(Chai et al., 1998;
Copp, 1978), our results are
indicative of a lack of production of some trophic factors by the embryonic
compartment (the ICM) of the NT embryos. No such proliferation defect was
observed in the in vitro outgrowth assays, indicating some compensation by
similar growth factors already present in the serum-containing medium. We
propose that a non-cell autonomous defect arising in the early epiblast
lineage of NT embryos is mainly responsible for the first peak of embryonic
resorption occurring around the time of implantation and before E7.
Once implanted, a fraction of the NT embryos exhibit a marked delay in
their growth, and their epiblast is severely compromised, as shown by the
simultaneous absence of expression of the key markers Oct4 and
Nodal. By contrast, their visceral endoderm shows some signs of
differentiation. These embryos can be considered as the remnants of those with
a proliferation defect in the ICM. The remaining ones gastrulate, and the
expression profile of a panel of gastrulation markers indicates no obvious
impairment of differentiation in the epiblast-derived tissues. This
observation is in agreement with the ability to derive and differentiate ES
cells in vitro from NT blastocysts (Munsie
et al., 2000; Wakayama et al.,
2001). Consistently, the NT embryo-derived cells are not excluded
from the embryonic lineages after the injection of normal ES or ICM cells into
NT blastocysts and formation of the chimaeric epiblast. However, and in spite
of an apparently normal initiation of gastrulation, anomalies in the growth of
the conceptus rapidly appear. The alteration in the ratio of
extra-embryonic/embryonic compartment sizes, which characterises a group of
implanted but obviously abnormal NT embryos ('large Exe' phenotype) is not
remedied by the inclusion of wild-type ES or ICM cells. By contrast, during
normal development, the growth of the conceptus is tightly regulated between
the two compartments during the rapid expansion of the egg cylinder
(Downs and Davies, 1993;
Lawson et al., 1991). As
development proceeds, abnormal growth of the trophoblast of NT embryos is
consistently observed during the foetal stages, leading to placentomegaly
(Fig. 2). In contrast to normal
development (Iguchi et al.,
1993), NT placentas continue to grow after E15 and some placentas
even keep growing after the disappearance of the foetus. We suggest that these
conceptuses that contain only trophoblast tissue come from embryos with the
`large Exe' phenotype. Trophoblast stem cells are thought to give rise to all
the differentiated trophoblast derivatives of the placenta
(Tanaka et al., 1998), and Uy
and Gardner (Uy and Gardner, 2002) have shown that only a few stem cells
reside in the extra-embryonic ectoderm in vivo. An increased proliferating
activity of this small compartment of trophoblast stem cells could be
sufficient to give the trophoblast a growth advantage in the NT embryos with
the `large Exe' phenotype. The growth characteristics of the trophoblast stem
cell of NT embryos are under investigation. Preliminary data indicate that
they have the ability to grow faster than controls during the early culture
period. Placentomegaly is a consistent feature of the development of NT
embryos, and has been observed both in ES and somatic NT embryos
(Suemizu et al., 2003;
Tanaka et al., 2001). Our
ongoing histological analysis of the ES NT-derived placenta shows several
similarities with that of somatic cell derived placentas
(Tanaka et al., 2001).
Moreover, the comparison of the transcriptome of NT placentas revealed that
more than 70% of the deregulated genes were common to both ES and somatic cell
NT placentas (Humpherys et al.,
2002). This suggests that the placental defects observed after
both somatic cell- and ES-derived NT have a common origin in the trophoblast
stem cell lineage.
The development of diploid/tetraploid aggregates to E7 indicates that the first abnormal morphological phenotypes arising in NT embryos are rescued by the presence of tetraploid extra-embryonic ectoderm cells. The use of tetraploid cells in the trophoblast would be useful in more deeply evaluating the developmental potential of the epiblast derivatives themselves at later (foetal) stages of the development of NT embryos.
Changes in the shape of the embryo should modify the establishment of
morphogenetic gradients and the crosstalk between the epiblast and the
extra-embryonic ectoderm. The robustness of developmental patterning
(Eldar et al., 2004) helps
them to develop further, and we have thus observed that they can initiate
gastrulation. However, as only half of the remaining foetuses are still alive
at E10, we suggest that they correspond to those with normal phenotypes at E7.
The rest are either degenerated (those that do not gastrulate) or dead (those
with morphological or growth defects).
Several recent studies have unveiled multiple roles of the crosstalk
between the extra-embryonic tissues and the epiblast in regulating their
growth and differentiation (Donnison et
al., 2005; Guzman-Ayala et
al., 2004; Rodriguez et al.,
2005; Strumpf et al.,
2005). In this regard, our analysis of the development after
nuclear transfer reveals a new aspect of this crosstalk: it appears that the
extra-embryonic ectoderm somehow controls the harmonious growth and shape of
the whole embryo and the relative number of cells in both regions. This
finding is in agreement with the analysis of the transcriptome of
extra-embryonic tissues from normal embryos, which revealed that a lot of
genes expressed specifically at E7.5 are cell cycle regulators and growth
factors (Hemberger et al.,
2001). We therefore hypothesise that the analysis of the
transcriptome of this tissue in NT embryos will help to confirm these new
functions for the extra-embryonic ectoderm.
Altogether, our results indicate that the epiblast is either not affected
or affected merely in its role of sustaining trophoblast proliferation. By
contrast, the trophoblast lineage rapidly becomes abnormal, leading to severe
deregulation of the growth of the embryo. As suggested by Boiani and
colleagues (Boiani et al.,
2005), the reprogramming process may follow different paths in the
two lineages. At least one epigenetic process, the remethylation of the genome
that occurs soon after the blastocyst stage, is indeed different in the two
regions of the conceptus (Monk et al.,
1987; Santos et al.,
2002). The result is an asymmetric pattern of methylation, with
the trophoblast lineage remaining hypomethylated in comparison with the
epiblast. Recent results in cattle show that, despite their low developmental
potential in vivo, NT blastocysts closely resemble in vivo, but not in vitro,
fertilised embryos in term of their global patterns of expressed genes
(Smith et al., 2005). This
also suggests that reprogramming errors mainly affecting the trophoblast
lineage must arise during the epigenetic modifications after the blastocyst
stage, and supports the view that reprogramming should be viewed essentially
as a multistep process.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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