1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University
Avenue, Toronto M5G 1X5, Canada
2 Department of Molecular and Medical Genetics, University of Toronto, Toronto
M5S 1A8, Canada
3 Unité de Génétique Moléculaire Murine, URA 2578
CNRS, Institut Pasteur, 25 rue du Docteur Roux, Paris 75015, France
4 Mammalian Development Laboratory, Department of Zoology, University of Oxford,
South Parks Road, Oxford, OX1 3PS, UK
5 CNRS UMR218, Curie Institute, 26 rue d'Ulm, Paris 75005, France
Authors for correspondence (e-mail:
rossant{at}mshri.on.ca
and
pavner{at}pasteur.fr)
Accepted 27 January 2005
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SUMMARY |
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Key words: Primitive endoderm, X-inactivation, Chimeras, Embryonic stem cells
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Introduction |
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Described here is the characterization of cell lines representative of the extra-embryonic endoderm lineage that can be reproducibly derived from mouse blastocysts. They express a molecular profile particular to this lineage and, importantly, they are developmentally restricted to form extra-embryonic endoderm in vivo. Our first use of this novel system was to investigate the status and mechanism of X-chromosome inactivation in female XEN cells. We show XEN cells exhibit paternally imprinted X-inactivation, and that they show a novel combination of epigenetic modifications on the imprinted X, suggesting that trophoblast and extra-embryonic endoderm lineages may differ in their imprinting maintenance mechanisms.
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Materials and methods |
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ES and TS cell cultures
HP3.10 female ES cell line have been described previously
(Clerc and Avner, 1998). F3
trophoblast stem cell line was isolated from F1 129/Sv Hprt-4 Pgk1a x
129/Sv embryos using a published protocol
(Tanaka et al., 1998
) and
maintained in RPMI 1640 (Gibco) with 20% FBS (Gibco), 1 mM sodium pyruvate, 2
mM L-glutamine, 50 mg/ml each of penicillin/streptomycin (all from Gibco), 100
µM ß-mercaptoethanol (Sigma), 25 ng/ml hrFGF4 and 1 µg/ml heparin
(both from Sigma).
DNA transduction of XEN cells
IM8A1 cells were co-electroporated with pCX-EGFP and pPGK-puro plasmids.
IM8A1 cells (5x106 cells at passage 39) were resuspended in
PBS (0.8 ml) and transferred to a GenePulser Cuvette (0.4 cm electrode, BioRad
catalog number 165-2088). ScaI-linearized pCX-EGFP plasmid (18 µg)
(Hadjantonakis et al., 1998)
and EcoRI-linearized pPGK-Puro plasmid (2 µg) were added to the
cell suspension and electroporated (0.25 V, 500 µF). The cells were
incubated on ice (20 minutes) and plated in 70% EMFI-CM (10 ml) on a
gelatinized 100 mm plate. Drug selection (puromycin, 1 µg/ml) was started 1
day later, and at 12 days the colonies were passaged and expanded as a pool
before FACS-sorting for GFP-positive cells (see below).
FACS analysis and subclonal lines
GFP-IM8A1 cells at one passage after electroporation (see above) were
FACS-sorted for GFP fluorescence with an argon ion laser (488 nm).
Approximately 800,000 GFP-positive cells were sorted into 70% EMFI-CM + G418
(90 µg/ml) + Puro (1 µg/ml) medium and plated on 0.1% gelatin in a 60 mm
dish. To derive subclonal lines, 192 GFP-positive cells were single-cell
sorted directly into 96-well plates.
PCR sexing
XEN cells (per single well of a four-well plate) were lysed in 100 µl of
0.1 mg/ml Proteinase K (Boehringer Mannheim) in a solution of 50 mM KCl, 10 mM
Tris.HCl (pH 8.3), 2 mM MgCl2, 0.1 mg/ml gelatin (Sigma), 0.45%
Nonidet P-40, and 0.45% Tween-20. PCR genotyping was performed for the
X-chromosome-specific Xist gene and Y chromosome-specific
Zfy1 gene. The primers are Xist
(5'-TTGCGGGATTCGCCTTGATT-3') and
(5'-TGAGCAGCCCTTAAAGCCAC-3'); Zfy1
(5'-GCATAGACATGTCTTAACATCTGTCC-3') and
(5'-CCTATTGCATGGACAGCAGCTTATG-3'). PCR was performed at an
annealing temperature of 65°C in 1.5 mM MgCl2 for 35 cycles.
The first four cycles had a 4 minute melting time at 95°C, while the next
31 cycles had a 1 minute melting time. The predicted Xist PCR product is 207
bp and Zfy1 is 183 bp. The IM5A1, IM9C4, GHP7/3 and GHP7/9 lines were
genotyped as female and the XEN1-3 and GHP7/7 lines were genotyped as male
(Table 1).
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Scanning electron microscopy
XEN cells (IM8A1 at passage 29) were plated on round, plastic Thermanox
coverslips (Nunc, Mississauga, Canada) in four-well plates in 70% EMFI-CM and
cultured for 2 days. The cells were washed with PBS and fixed in 2%
glutaraldehyde in 0.1 M phosphate buffer at 4°C overnight. The specimens
were then post-fixed in 1% osmium tetroxide in the same buffer, rinsed with
0.1 M phosphate buffer and dehydrated in a graded ethanol series. The
coverslips were then critical-point dried in a Bal-Tec CPD 030, mounted on
aluminum stubs and sputter coated with gold in a Denton Desk II. Imaging was
carried out on an FEI XL30 ESEM under standard high vacuum conditions. Most
images were taken at 0° (perpendicular to the mounting stage) and some
were taken at a 60° angle.
RT-PCR analysis
Total RNA was isolated from XEN cells, ES cells and embryoid bodies using
the Qiagen RNeasy midi kit (Qiagen, Santa Clarita, CA) according to
manufacturer's instructions. cDNA was prepared by annealing a
dT12-18 primer (1.0 µl of 0.5 µg/µl) to 460 ng (11 µl)
of total RNA at 65°C for 5 minutes. 5 x RT buffer (4 µl,
Invitrogen), 0.1 M DTT (2 µl), 10 mM dNTPs (1 µl) and Superscript II
reverse transcriptase (1 µl, Invitrogen) were added and incubated at
42°C for 1 hour. The resulting cDNA was analyzed for the following
markers: Afp, Gata4, Sox7, Hnf4, Foxa2, Oct4 and ß-actin (see
Table S1 in the supplementary material).
Affymetrix analysis
Three biological replicates of XEN cell RNA were submitted to the Centre
for Applied Genomics at the Hospital for Sick Children (Toronto, Canada) for
preparation of cRNA and hybridization to the mouse U74Av2 Affymetrix gene
array (Liu et al., 2003).
Qiagen RNeasy midi kit (Qiagen, Santa Clarita, CA) was used to extract total
RNA from all samples according to manufacturer's instructions. The samples
were: (1) XEN1-3 cells at passage 18 (ICR strain, male) cultured on gelatin
with 70% EMFI-CM; (2) IM8A1 cells at passage 27 (PO strain) cultured on
gelatin with 70% EMFI-CM; and (3) IM8A1 cells at passage 27 cultured on tissue
culture plastic in RPMI 1640 (Gibco) supplemented with 20% FBS (CanSera,
Rexdale, Canada), 1 mM sodium pyruvate, 2 mM L-glutamine, 50 mg/ml each of
penicillin/streptomycin (all from Gibco), 100 µM ß-mercaptoethanol
(Sigma) for 4 days. RNA was also obtained from R1 ES cells grown in the
absence of EMFIs in standard conditions
(Nagy et al., 1993
) and
subjected to the above analysis. A threshold significance value of
P<0.04 was used to consider a gene expressed or `present' and
P>0.06 for genes not expressed or `absent' and marginal values
were 0.04<P<0.06. Ratio data were obtained using Affymetrix MAS
5.0 software. GEO Accession Number is GSE2204.
Production of XEN cell chimeras
GFP-IM8A1 cells from a GFP-sorted population (passage 43) and from a
subclonal line (GFP-IM8A1-4, passage 46) were injected into E3.5 ICR
blastocysts. Between 10 and 15 XEN cells were injected per embryo and
transferred to the uterus of E2.5 pseudopregnant ICR females. Dissections were
performed at E6.5, E7.5 and E8.5 with special attention taken to keep the
parietal yolk sac intact. X-gal staining of embryos was performed as
previously described (Rossant et al.,
1991). Experimental animals were treated according to guidelines
approved by the Canadian Council for Animal Care.
Allelic quantitative real-time RT-PCR
Quantitative real-time PCR measurements of Xist cDNA were carried
out using TaqMan fluorescent probes and TaqMan Universal PCR Mix and an ABI
Prism 7700 (Perkin Elmer Applied Biosystem) as previously described
(Morey et al., 2001).
Xist cDNA quantitations were internally standardized against the
endogenous 18s rDNA gene. Quantitative real-time PCR measurements of
Nap1l2 cDNA were performed using SYBR Green Universal Mix. Two
informative SNPs identified between the 129 and Pgk alleles were used to
design allele-specific primers: Nap1l2-129-F
(5'-GCACCGTCTTTTATCCCCACT-3'), which is specific for the 129
allele; and Nap1L2-Pgk-F (5'-GCACCATCTTTTATCCCCACG-3'), which is
specific for the Pgk allele. Both were used in conjunction with a universal
reverse primer: Nap1L2-129/Pgk-R (5'-ACAGCAGATGCGCGATGAT-3').
Nap1L2 cDNA quantitations were standardized against Rrm2
cDNA (Morey et al., 2004
).
RNA FISH and immunostaining
Primary antibodies used have recently been described
(Okamoto et al., 2004): rabbit
polyclonals detecting H3 acK9, H3 di-meK4, H3 di-meK9 (Upstate biotechnology);
mouse monoclonal anti-H3 di/tri-meK27 (7B11); rabbit polyclonal anti-Enx1
(Ezh2); and mouse monoclonal anti-Eed
(Sewalt et al., 1998
;
Hamer et al., 2002
). TS and
XEN cells cultured on gelatin-coated coverslips were fixed in 3%
paraformaldehyde for 15 minutes at room temperature. Permeabilization was
performed on ice in PBS containing 0.5% Triton X-100 and 2 mM Vanadyl
Ribonucleoside Complex (VRC, Biolabs) for 3.5 minutes. After rinsing in PBS,
preparations were blocked in 1% BSA (Gibco) and 0.4 U/ml RNAguard
(Amersham/Pharmacia) in PBS for 15 minutes, incubated with primary antibody
(diluted in blocking buffer) for 40 minutes, then washed in PBS four times, 5
minutes each, and incubated with secondary antibody (Alexa Fluor 568 goat
anti-rabbit or anti-mouse, Molecular Probes) in blocking buffer for 40 minutes
at room temperature. After washing in PBS, preparations were post-fixed in 3%
paraformaldehyde for 10 minutes at room temperature and rinsed in 2
xSSC. For RNA FISH, the Xist probe used was a 19 kb genomic
fragment derived from a lambda clone (510) that covers most of the
Xist gene. The probe was labeled by nick translation (Vysis) with
spectrum green-dUTP (Vysis), and was hybridized (0.1 mg of probe with 10 mg of
salmon sperm DNA per coverslip) in 50% formamide, 2 xSSC, 20% dextran
sulfate, 1 mg/ml BSA (NEB), 200 mM VRC, overnight at 37°C. After three
washes in 50% formamide/2 xSSC and three washes in 2 xSSC at
42°C, DNA was counterstained for 2 minutes in 0.2 mg/ml DAPI, followed by
a final wash in 2 xSSC. Samples were mounted in 90% glycerol, 0.1
xPBS, 0.1% p-phenylenediamine (Aldrich) (pH 9). A Leica DMR fluorescence
microscope with a Cool SNAP fx camera (Photometrics) and Metamorph software
(Roper) were used for image acquisition.
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Results |
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In summary, XEN cell lines can be consistently derived from blastocysts or ICMs from different genetic backgrounds. Derivation from ICMs was more efficient than from intact blastocysts. However, once derived all XEN cell lines could be maintained in EMFI-CM on gelatin without supplementation with additional growth factors.
XEN cell morphology and behavior
In contrast to the coherent colonies formed by ES and TS cells, XEN cells
maintain very little cell-cell contact when grown at low density. The cultures
contain at least two cell morphologies; a rounded, highly refractile cell type
and a more stellate epithelial-like cell type
(Fig. 1B). At higher densities
XEN cells could form epithelial sheets
(Fig. 1C) and often formed a
lattice-type structure (Fig.
1D). When XEN cells were removed from gelatin and plated on tissue
culture plastic without EMFI-CM, many cells became large and vacuolated within
6 days (Fig. 1E) and the cell
lines could no longer be passaged. Although there was some variability
observed between different cell lines in terms of proliferation rates and
ratios of round versus epithelioid cell types, the basic morphology of the
cultures was consistent and easily recognizable.
To investigate whether the presence of the two cell morphologies
represented two cell types in the starting cultures, we FACS-sorted single
GFP-labeled XEN cells directly into 96-well plates and subclonal cell lines
were derived. Cell viability was 50% and 16% (31/192) of the subclonal
cultures could be passaged. Importantly, all subclonal lines exhibited both
round and epithelial-like cell morphologies. This suggested that the two cell
morphologies observed in the parental cell line was not due to a mixture of
two distinct cell populations that were being co-cultured. Videomicroscopy
showed that XEN cells are highly motile and that single cells made transitions
between round and epithelioid cell types without cell division (see Movie 1 in
the supplementary material). Twenty-five individual XEN cells were followed
for a 9-hour period by videomicroscopy and 76% (19/25) underwent reversible
phenotypic changes (round-to-epithelioid-to-round or
epithelioid-round-epithelioid) without cell division. Thus, the two cell
morphologies represent different phases of the dynamic behavior of XEN cells
in culture.
During these studies, we observed a large, unusual cell process being transiently produced by 25% of XEN cells in their rounded, but not their epithelioid, phase. The cellular process was rapidly extruded and retracted (within 6-10 minutes) and was often longer than a cell diameter and as wide as 30-40% of the thickness of the cell. Scanning electronmicroscopy (SEM) revealed that although both the rounded and epithelioid XEN cell surfaces were very dense with microvilli (Fig. 2A,B), the large pseudopodium was completely devoid of them (Fig. 2C-E). SEM images taken at a 60° angle showed that the pseudopodia were not attached to the cell culture substrate, but were raised above it (Fig. 2E). PE and VE of E5.0-E6.5 embryos were also analyzed by SEM and very similar pseudopodia were found protruding from the distal and anterior VE regions of E5.25-E5.75 embryos (Fig. 2F). These protrusions were not observed on PE cells or other regions of the VE, and disappeared completely by E6.5. Thus, although the overall morphology of the XEN cultures resembles PE, the presence of pseudopodia is a property more typical of VE.
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Several genes implicated, by mutant analysis, in visceral endoderm function
were also expressed in XEN cells. These genes are, however, are also expressed
in primitive and parietal endoderm and may be more representative of primitive
endoderm. They include Gata4 and Gata6
(Arceci et al., 1993;
Koutsourakis et al., 1999
;
Narita et al., 1997
),
Disabled2 (Morris et al.,
2002
; Yang et al.,
2002
), Tcf2 (vHnf1)
(Barbacci et al., 1999
) and
Vegfa (Miquerol et al.,
1999
). Markers specific to VE are often difficult to identify
because of the lack of comparable expression data for parietal endoderm. From
published expression data where both PE and VE were investigated, several
VE-specific genes have been identified. A number of these genes were expressed
in XEN cells and they include Foxa2
(Dufort et al., 1998
),
Ihh (Becker et al.,
1997
) and type I Acvr1 (Gu et
al., 1999
). Other VE-specific genes, such as Afp, Hnf4
and uPA (Plau) were not detected
(Table 2). However,
Afp can be induced in differentiated XEN cell cultures and
Hnf4 is indeed detectable by RT-PCR
(Fig. 3). Genes representative
of TS cells, Cdx2, Eomes and Esrrb
(Tanaka et al., 1998
), and the
ES cell markers, Oct4, Nanog and Rex1, were not detected in
XEN cells (Table 2). We
conclude, based on our marker studies, that XEN cells derive from the
primitive endoderm of the blastocyst and represent derivatives of this
lineage.
As well as examining the expression of known extra-embryonic endoderm markers, we also used a comparison of ES cell and XEN cell Affymetrix expression data to seek those genes specifically enriched in XEN cells. The three XEN cell data sets described above were each compared with a single data set from undifferentiated ES cells and three expression ratios were obtained for each gene. These ratios were averaged and rank-ordered, starting with the largest ratio. Within the list of 40 genes with the highest positive expression ratios, at least 13 genes are known extra-embryonic endoderm markers (see Table S3 in the supplementary material). This was in contrast to the list of genes with the largest negative expression ratios, which was highly enriched with known ES cell-specific genes (see Table S4 in the supplementary material).
XEN cell chimeras
We investigated the developmental potential of XEN cells by generating
chimeras with a GFP/lacZ cell line and one of its subclonal
derivatives (Table 3). XEN
cells (10-15) were injected into wild-type ICR blastocysts and analyzed for
contributions at E6.5, E7.5 and E8.5. GFP/lacZ XEN cells were never
found to contribute to the embryo proper, yolk sac mesoderm or the trophoblast
lineage. The overwhelming number of chimeras (49/50) had XEN cell
contributions restricted to parietal endoderm. At E6.5, XEN cells were usually
observed as dispersed cells lining the distal part of the parietal yolk sac
(Fig. 4A,B). At later stages,
chimeras could be found with large numbers of scattered XEN cells in their
parietal yolk sacs (Fig. 4C-E).
This pattern was typical of parietal endoderm growth in vivo
(Hogan and Newman, 1984). A
single chimera exhibited a contribution of XEN cells in the visceral endoderm
layer (Fig. 4F), but not in the
parietal yolk sac (not shown). The nature of this clone was very different
from the contributions to the parietal region, as the XEN cells formed a
coherent epithelial sheet without intermingling with host cells. This is
consistent with the coherent growth characteristics of VE in vivo
(Gardner and Cockroft,
1998
).
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When RNA FISH was carried out on female XEN cells, a Xist domain was found
in the majority of cells analyzed (Fig.
6A). A pinpoint signal was not found to be associated with the
active X chromosome and neither domain nor pinpoint Xist signals were found in
the male XEN cell line (GHP7/7). Chromatin modifications associated with the
inactive X chromosome in females were examined by Immuno-RNA FISH in female
XEN cells. Female TS cell lines were concurrently examined to explore possible
differences between the two extra-embryonic lineages. Unlike TS cells, which
show an enrichment of Eed and Ezh2 (Mak et
al., 2002), the Xist domain in XEN cells show no enrichment for
these polycomb group proteins (compare Fig.
6A with 6B). Histone H3 dimethylated K4 (H3 di-meK4), which is
normally associated with active euchromatin, is excluded from the Xist domain
in both XEN and TS cells, as is histone H3 acetylated K9 (H3 acK9)
(Fig. 6; see Fig. S1 in the
supplementary material). Histone H3 di-meK9, which has been associated with
the inactive X chromosome in differentiating ES cells and somatic cells
(Heard et al., 2001
), appears
only weakly enriched, if at all, in XEN and TS cells (see Fig. S1 in the
supplementary material). Histone H3 tri-meK27, which has recently also been
associated with the inactive X in both TS cells and differentiating ES cells
(Silva et al., 2003
), is
enriched over the Xist domain in only a small proportion of XEN cells. This is
consistent with the absence or low levels of Ezh2 on the X chromosome in XEN
cells, as Ezh2 is thought to be the histone methyltransferase responsible for
the H3 tri-meK27 mark (Erhardt et al.,
2003
). Thus, XEN cells exhibit a unique combination of histone
modifications on their inactive X chromosome which includes
hypoacetylation of H3 K9, hypomethylation of H3 K4 and little or no
methylation of H3 K27 and K9. The stability of the inactive state of the X
chromosome in these cells, despite the absence of H3 K9 and H3 K27
methylation, suggests that other epigenetic marks are involved. This is
different from the situation in ES cells, where the lack of Ezh2 accumulation
and H3 K27 methylation results in significant reactivation of X-linked genes
(Silva et al., 2003
).
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Discussion |
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XEN cell maintenance
XEN cell cultures are robust and easily maintained, but their precise
signaling requirements are not known. After derivation, they can be grown on
gelatin in medium supplemented with EMFI-CM. The FGF signaling pathway
required for TS cells is not important for XEN cell maintenance and indeed
none of the four FGF receptors is expressed in XEN cells (see Table S2 in the
supplementary material). The LIF-STAT pathway may be playing a role in XEN
cell maintenance. Several components (LIFR, gp130, JAK1, JAK2, STAT1 and
STAT3) of this pathway are expressed in XEN cells (see Table S2 in the
supplementary material), and their derivation and maintenance on EMFIs or
EMFI-CM would provide an adequate source of LIF
(Smith et al., 1992).
XEN cells show properties of both parietal and visceral endoderm
The previously reported mouse PEC and rat RE1 cell lines are similar, if
not identical, to the XEN cells described here. The two distinct morphologies
of XEN cells in culture rounded highly refractile and flattened
epithelioid have also been reported
(Fowler et al., 1990;
Notarianni and Flechon, 2001
).
These cell lines are considered representative of parietal endoderm based on
their morphology and the secretion of ECM proteins abundantly found in
Reichert's membrane. Our Affymetrix expression analysis of XEN cells supports
the idea that PE properties are highly represented in such cell culture
models. PE-associated genes, such as tPA (Plat),
thrombomodulin, Snail and Pdgfra, were strongly expressed.
Genes encoding for basement membrane proteins (perlecan, type IV collagen,
laminin and nidogen), abundant in Reichert's membrane, were also expressed at
high levels in XEN cells.
XEN cells, however, also expressed several VE-associated genes in all
cultures (Table 2). The high
expression of Sox7, Sox17 and Cited1 were of particular
interest, given their restricted expression in VE overlying the
extra-embryonic ectoderm and in the marginal zone VE
(Dunwoodie et al., 1998;
Kanai-Azuma et al., 2002
). The
reduction of Sox7 and increase of Afp expression in
differentiated XEN cell cultures suggests that XEN cells may have some
properties of extra-embryonic VE when cultured in standard conditions, but can
be differentiated to cells more related to the VE overlying the epiblast in
other conditions. Furthermore, Sox7 expression suggests that
definitive endoderm is not present in XEN cell cultures, as this gene, unlike
Sox17, is not detectable in definitive endoderm in vivo
(Kanai-Azuma et al.,
2002
).
The single-cell clonal analysis and videomicroscopy studies of XEN cells
have indicated that the rounded and epithelial-like cell types are
lineage-related and reversibly interchangeable. The morphology of marginal
zone VE cells in vivo is similar to the flattened, epithelial-like cells
observed in culture. The marginal zone cells have been described as
mesenchymal with ruffled cell membranes reminiscent of lamellipodia
(Hogan and Newman, 1984). The
unusual pseudopodia we observed in XEN cells were also observed in the AVE
region of early postimplantation embryos in vivo
(Fig. 2F), suggesting that XEN
cells may also transiently become AVE like. Taken together, the gene
expression studies and cell morphology/behavior suggest that clonal XEN cell
cultures exhibit properties of PE, marginal zone VE, AVE and extra-embryonic
VE, but not of embryonic VE. However, the induction of an embryonic VE marker,
Afp, in differentiated cultures of XEN cells
(Fig. 3) suggests that this VE
subtype could be induced in defined conditions.
XEN cell chimeras
XEN cell lines retain the capacity to contribute to primitive endoderm
derivatives in vivo in chimeras, attesting to their primitive endoderm nature
and the stability of their ex vivo phenotype. However, they exhibited a strong
bias to form parietal endoderm in chimeras, with only one visceral endoderm
clone observed. This does not necessarily indicate their full potential. The
blastocyst injection procedure used to generate chimeras may have provided an
environment that promotes PE and hinders VE differentiation. XEN cells
injected into the blastocoel are likely to end up in the superficial layer of
the primitive endoderm as it forms
(Gardner, 1985) or associated
with the TE away from the ICM. PrE or VE cells that lose contact with epiblast
and extra-embryonic ectoderm default to a PE phenotype or are instructed by
the trophectoderm to become PE by a combination of TE basement membrane and
the PTHrP/cAMP signaling pathway
(Verheijen et al., 1999a
).
This observation is supported by chimera experiments with primitive endoderm
cells and nascent VE cells directly isolated from embryos. Blastocyst
injection of PrE or early VE cells also resulted in chimeras with mostly PE
contributions (Gardner, 1982
;
Cockroft and Gardner, 1987
).
Although the donor PrE and VE cells clearly have the potential to make VE,
their behavior in chimeras did not reflect this. Given this intrinsic bias, an
alternative method for producing chimeras, such as morula or ICM injections,
may be required to observe the full potential of XEN cells.
Imprinted X-inactivation in XEN cells
The extra-embryonic trophoblast and endoderm lineages of the mouse undergo
imprinted inactivation of the paternal X chromosome (Xp)
(Takagi and Sasaki, 1975).
Initiation of X inactivation requires expression of the non-coding RNA,
Xist, and accumulation of this transcript on the inactive X
chromosome in cis (Penny et al.,
1996
). In agreement with their proposed extra-embryonic endodermal
origin, XEN cells maintain imprinted X inactivation of the Xp and exhibit
Xist accumulation on one X chromosome. This Xist domain also exhibits
other epigenetic marks of being inactive, such as exclusion of histone H3
acetylated K9 and dimethylated K4, both hallmarks of active euchromatin
(Heard et al., 2001
;
Boggs et al., 2002
).
Surprisingly, accumulation of the polycomb group proteins, Eed and Ezh2, was
not observed in XEN cells, as is seen in TS cells
(Fig. 6)
(Mak et al., 2002
). Mutant
analysis revealed an important role for Eed in the formation of
trophoblast giant cells, but not extra-embryonic endoderm, during development
(Wang et al., 2002
). The
proposed substrate of Ezh2, histone H3 K27, appears to be weakly
di/trimethylated in some XEN cells, suggesting a transient association of
Ezh2. Thus, the mechanisms to maintain X inactivation in XEN cells, TS cells
and ES cells differ at the levels of polycomb group proteins and histone
modifications. The HAT/6TG studies indicate that the inactive state of the Xp
in XEN cells is stable, despite the absence of H3 meK9 enrichment and low
levels of H3 K27 methylation. This suggests XEN cells may have different
mechanisms for maintenance of their inactive X chromosome and that other
epigenetics marks likely account for the stability of the inactive state.
These results illustrate the cell lineage-dependent variation in mechanisms of
X-inactivation maintenance that has also been observed in other systems
(Plath et al., 2003
).
XEN cells from ES cells
The XEN cell lines described here were derived de novo from blastocysts.
However, two reports suggest that similar cell lines can be derived directly
from established ES cell lines. Overexpression of the GATA factors, GATA4 or
GATA6, in ES cells induced uniform differentiation into extra-embryonic
endoderm cells (Fujikura et al.,
2002). In terms of marker analysis and cell morphology, these
cells appear identical to XEN cells. Fujikura et al., hypothesize that a
repressor of Gata4/6 expression is required to maintain ES cells
pluripotent. The recently identified pluripotent transcription factor, Nanog,
may fulfill this role (Chambers et al.,
2003
; Mitsui et al.,
2003
). Nanog/ ES cells express
many markers of extra-embryonic endoderm (Gata6, Tcf2 and
Ihh) and are also morphologically very similar to XEN cells
(Mitsui et al., 2003
). This
implicates Nanog as a general repressor of the extra-embryonic endoderm
lineage and it may function, in part, through repression of key regulatory
genes, such as Gata6.
XEN cell lines provide a unique model for an early mammalian lineage that will complement the established ES and TS cell lines. Through the study of essential genes and signaling requirements for this cell culture system, insights will be gained about the developmental program of the extra-embryonic endoderm lineage. In addition, in vitro combinations of ES, TS and XEN cells may help model the in vivo interactions between embryonic and extra-embryonic lineages important for embryonic patterning.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/7/1649/DC1
* Present address: Institute for Stem Cell Research, University of Edinburgh,
King's Buildings, West Mains Road, Edinburgh, EH9 3JQ, UK
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