Loss of Atrx Affects Trophoblast Development
and the Pattern of X-Inactivation
in Extraembryonic Tissues
David Garrick1, Jackie A. Sharpe1, Ruth Arkell2, Lorraine Dobbie3, Andrew J. H. Smith3, William G. Wood1,
Douglas R. Higgs1, Richard J. Gibbons1*
1 MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom, 2 MRC Mammalian Genetics Unit, Harwell,
Oxfordshire, United Kingdom, 3 Institute for Stem Cell Research, University of Edinburgh, Edinburgh, United Kingdom
ATRX is an X-encoded member of the SNF2 family of ATPase/helicase proteins thought to regulate gene expression by
modifying chromatin at target loci. Mutations in ATRX provided the first example of a human genetic disease
associated with defects in such proteins. To better understand the role of ATRX in development and the associated
abnormalities in the ATR-X (alpha thalassemia mental retardation, X-linked) syndrome, we conditionally inactivated
the homolog in mice, Atrx, at the 8- to 16-cell stage of development. The protein, Atrx, was ubiquitously expressed, and
male embryos null for Atrx implanted and gastrulated normally but did not survive beyond 9.5 days postcoitus due to
a defect in formation of the extraembryonic trophoblast, one of the first terminally differentiated lineages in the
developing embryo. Carrier female mice that inherit a maternal null allele should be affected, since the paternal X
chromosome is normally inactivated in extraembryonic tissues. Surprisingly, however, some carrier females
established a normal placenta and appeared to escape the usual pattern of imprinted X-inactivation in these tissues.
Together these findings demonstrate an unexpected, specific, and essential role for Atrx in the development of the
murine trophoblast and present an example of escape from imprinted X chromosome inactivation.
Citation: Garrick D, Sharpe JA, Arkell R, Dobbie L, Smith AJH, et al. (2006) Loss of Atrx affects trophoblast development and the pattern of X-inactivation in extraembryonic
tissues. PLoS Genet 2(4): e58. DOI: 10.1371/journal.pgen.0020058
ATR-X syndrome is a severe, nonprogressive form of X-
linked mental retardation that is frequently associated with
multiple congenital abnormalities . It is usually associated
with a mild form of a-thalassaemia, caused by reduced
expression of structurally intact a-globin genes, and charac-
terised by the presence of b-globin tetramers (haemoglobin H
inclusion bodies) in peripheral red blood cells. Carrier
females occasionally manifest haemoglobin H inclusions,
but are otherwise intellectually and physically normal. Studies
of X-chromosome inactivation in carrier females have
demonstrated preferential inactivation of the chromosome
bearing the abnormal allele in a variety of tissues , and this
skewing of X-inactivation is thought to explain the mild
phenotype observed in carriers.
The ATR-X syndrome is caused by mutations in a gene
(ATRX) that comprises 36 exons spanning 300 kb of genomic
DNA at Chromosome Xq13.3 . This gene encodes two
dominant protein isoforms (Figure 1). As well as the full-
length ATRX protein of ;280 kDa, which is encoded by a
transcript of ;10 kb, we recently demonstrated that a
truncated isoform called ATRXt (;200 kDa) is produced
from a transcript of around 7 kb, which arises when intron 11
fails to be spliced from the primary transcript and an
alternative intronic poly(A) signal is used . The mouse
homolog of the ATRX gene, Atrx, is also situated on the X
chromosome, and also gives rise to full-length (Atrx, ;280
kDa) and truncated (Atrxt, ;200 kDa) isoforms [4,5].
Disease-causing missense mutations are clustered in two
regions of the gene: a PHD-like zinc finger domain and a
SNF2-like ATPase domain (Figure 1) . The former motif is
thought to be involved in protein-protein interactions in
chromatin , and the latter is a feature of chromatin-
remodelling proteins, and the presence of disease-causing
mutations indicates the functional importance of these
domains. ATRX has been shown to remodel chromatin .
It also interacts with HP1 at heterochromatin  and is
recruited to promyelocytic leukemia nuclear bodies via an
interaction with Daxx . Furthermore, disruption of ATRX
leads to diverse changes in DNA methylation . Never-
theless, the role ATRX plays in gene expression remains
The consistent core of clinical and haematological features
observed in ATR-X patients suggests that, like the SWI2/SNF2
chromatin-remodelling protein, ATRX probably regulates
transcription of a discrete set of target genes. However,
although there are clearly others to be found, at present the
Editor: Wolf Reik, The Babraham Institute, United Kingdom
Received September 2, 2005; Accepted March 3, 2006; Published April 21, 2006
Copyright: ? 2006 Garrick et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: BrdU, bromodeoxyuridine; dpc, days postcoitus; ES cells,
embryonic stem cells; IAP, intracisternal A particle; rDNA, ribosomal DNA; TGC,
trophoblast giant cell; TUNEL, TdT-mediated dUTP nick end labeling; WMISH,
whole-mount in situ hybridisation
* To whom correspondence should be addressed. E-mail: richard.gibbons@
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a-globin genes remain the only confirmed targets for tran-
scriptional regulation by ATRX. Little is currently known
about the precise role of the ATRX protein during
mammalian development. To investigate the role of this
protein during mouse development, we generated a condi-
tionally deleted allele of the Atrx gene in mouse embryonic
stem (ES) cells, and used these cells to examine the effect of
ablating expression of the full-length Atrx protein in ES cells
and in mouse embryos.
Generation of ES Cells Lacking Full-Length Atrx
Like the human gene, the mouse Atrx gene is also X-linked,
such that a direct disruption of the single Atrx allele in male
ES cells would immediately give rise to the null state. No
targeted clones were recovered after attempted homologous
recombination in male E14TG2a ES cells using two different
vectors that removed exon 18 of the Atrx gene. Exon 18
encodes the first of the seven motifs composing the conserved
SNF2-like domain of Atrx (Figure 1); mutation of the
corresponding motif of the yeast SNF2 protein has been
shown to severely impair SWI/SNF-dependent gene expres-
sion . The failure to recover targeted clones with these
vectors suggested that Atrx may be important for normal ES
cell growth and expansion and that direct targeting of the
single locus may not be possible. We therefore adopted a
conditional strategy for targeting exon 18 (Figure 2) and
recovered two clones in which exon 18 has been flanked by
loxP recognition sites for the Cre recombinase (Atrxfloxallele
in Figure 2A) (Figure 2B). This allele also contains a loxP-
flanked MC1-neorcassette in intron 17 (Figure 2A). Northern
and Western blot analyses (Figure 2D and 2E) confirmed that
the Atrxfloxclones continued to express both full-length Atrx
protein and the truncated Atrxt isoform.
To generate the full deletion in ES cells, the Atrxfloxclones
(1/F12 and 1/G11) were transiently transfected with a Cre-
recombinase expression plasmid (pCAGGS-Cre-IRESpuro),
and subclones were recovered bearing an allele (AtrxD18Dneo
in Figure 2A) in which both exon 18 and the neorcassette had
been deleted by the Cre recombinase (resulting from the
recombination event labelled ‘‘C’’ in the Atrxfloxallele shown
in Figure 2A) (Figure 2C). Northern and Western blot
analyses (Figure 2D and 2E) revealed that the full-length
Atrx transcript and protein is completely abolished in the
AtrxD18Dneorecombinant clones, suggesting that deletion of
this region has a highly destabilising effect on the full-length
transcript. As expected, the truncated Atrxt isoform, the
transcript of which is terminated within intron 11 , is
unaffected by the deletion of exon 18 (Figure 2E). While the
function of Atrxt is not yet clear, this isoform, which contains
the PHD-like domain but not the SWI/SNF motifs (Figure 1),
is unlikely to be functionally equivalent to the full-length
protein. Thus, a conditional knockout strategy allowed the
isolation of ES cells that are null for full-length Atrx.
Perturbed Growth and Methylation Defects in AtrxnullES
AtrxnullES cells could be maintained in culture but were
generally slower growing than AtrxþES clones, and appeared
to undergo higher rates of spontaneous differentiation. We
investigated directly the effect of Atrx on ES cell growth by
comparing Atrxþand AtrxnullES cell clones in competition
Figure 1. Schematic Representation of the ATRX Isoforms
Shown at the top is the human ATRX cDNA. The boxes represent the 36 exons. The introns are not to scale. The alternative splicing of exons 6 and 7 is
indicated. Shown underneath are the two ATRX protein isoforms. Full-length ATRX (;280 kDa) is encoded by the largest open reading frame. The
positions of the principal features (the PHD-like domain and the seven SWI/SNF-like motifs) are indicated. Above full-length ATRX is shown the
truncated ATRXt isoform (apparent molecular weight of ;200 kDa) that arises through the failure to splice intron 11 and the use of an intronic poly(A)
signal. The intron-encoded region of ATRXt is indicated as a filled grey box. Locations of recombinant proteins (A2, FXNP5, and H-300) used to generate
antibodies are shown. The scale bar represents 200 amino acids.
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Trophoblast Defect in Mice Lacking Atrx
ATRX belongs to a class of proteins that may modify how DNA is
packaged into chromatin, altering the accessibility of other proteins
in the nucleus to DNA. In this way, ATRX is thought to influence
gene expression. Mutations in the ATRX gene, which is located on
the female sex chromosome (X), provided the first example of a
human disease (ATR-X syndrome) associated with defects in such
proteins. Affected males (XMUTY) have multiple developmental
abnormalities in a wide variety of systems. Currently, it is not
understood how proteins like ATRX influence cell biology. To
address this question, the authors deleted the version of the gene in
mice, Atrx. Although affected male mice (XMUTY) started to develop
normally, they died early in development because they failed to
form a normal placenta. In the placenta, female mice normally
inactivate the X chromosome that they inherit from their fathers
(Xp), so if females inherit from their mother an X chromosome (Xm)
that bears the abnormal copy of Atrx (XmMUTXp), one would predict
that, like affected males, they would fail to form a normal placenta.
The authors unexpectedly found this not to be so. They showed,
instead, that in such females the normal, paternally derived Atrx
gene is active. This study has therefore demonstrated an important
facet of X-chromosome imprinting.
cultures. Equal numbers of Atrxþ(bearing either an AtrxWTor
an Atrxfloxallele) and Atrxnull(bearing an AtrxD18Dneoallele) ES
cells were inoculated into cultures and the mixed cultures
were passaged (1:3 split) every 2 d for 8–10 d. The relative
abundance of the different alleles in the culture at each time
point was analysed by Southern blotting (Figure 3A). The
clone containing the AtrxD18Dneoallele was rapidly outgrown
by both AtrxWTES cells and cells bearing the Atrxfloxallele. In
Figure 2. Cre-Mediated Ablation of Full-Length Atrx Protein in ES Cells
(A) Strategy for targeted deletion of exon 18 of the Atrx gene. The top line shows the wild-type allele (AtrxWT) at the region surrounding exon 18. Below
is shown the targeting vector and the targeted allele (Atrxflox) resulting from homologous recombination. The loxP target sites of the Cre recombinase
are shown as black triangles, and the three possible recombination events that can be mediated by the Cre recombinase are indicated (labelled A, B,
and C in the Atrxfloxallele). At bottom is shown the Cre-recombined allele (AtrxD18Dneo) (resulting from recombination event C) in which both exon 18
and the MC1neopA selection cassette have been deleted. EcoRI (labelled E) and SacI (labelled S) sites present on the targeted 129 strain X chromosome
are indicated. Black bars indicate the positions of the probes used in Southern blots.
(B) Southern blot analysis of EcoRI-digested DNA from either wild-type ES cells (E14) or targeted ES cell clones bearing the Atrxfloxallele (1/F12 and
1/G11) hybridised with either the 20/27 (left blot) or Hae0.9 (right blot) probes. The EcoRI fragment of the AtrxWTallele (18.5 kb) has been replaced with
the expected fragments of 11.2 kb (20/27 probe) or 8.5 kb (Hae0.9 probe)
(C) Southern blot analysis of SacI-digested DNA from either wild-type ES cells (E14) or targeted ES cell clones bearing the Atrxfloxallele (1/F12 and 1/G11)
or Cre-recombinant clones derived from these (1/F12B1F12 and 1/G11D5). The membrane was hybridised with the intron 17 probe indicated in (A). The
expected bands of 6.2 (AtrxWT), 5.0 (Atrxflox), and 2.8 (AtrxD18Dneo) kb were observed.
(D) Northern blot analysis of RNA from ES cells shown in (C). The membrane was hybridised first to a probe from exon 10 of the Atrx gene (top blot) and
subsequently to a b-actin cDNA probe as loading control (bottom blot). The transcripts responsible for full-length Atrx (;10 kb) and the truncated Atrxt
isoforms (;7 kb) are indicated.
(E) Western blot analysis of whole-cell extracts from the clones shown in (C) using an anti-ATRX monoclonal antibody (23C, raised against peptide A2 of
the human ATRX protein shown in Figure 1). The full-length and truncated Atrx isoforms are indicated.
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Trophoblast Defect in Mice Lacking Atrx
Figure 3. Growth and Methylation Defects in AtrxnullES Cells
(A) Cultures were inoculated with equivalent numbers of ES cells bearing different Atrx alleles as indicated, and were serially passaged. After the
indicated days of coculture, DNA extracted from a sample of cells was analysed by Southern blot to detect the Atrx alleles. DNA was digested with SpeI,
and the membrane was hybridised with the 20/27 probe shown in Figure 2A. The expected sizes of the different alleles are indicated.
(B) Schematic diagram of the transcribed portion of the mouse rDNA repeat with the 18S, 5.8S, and 28S genes indicated. The positions of the limit-
digesting enzymes BamH (labelled B) and EcoRI (labelled E) and the probes (RIB3 and RIB4) used in the Southern blots shown in (C) are indicated. Below
are shown the locations of the methylation-sensitive enzymes (SmaI, PvuI, and MluI) whose methylation status has been analysed in the Southern blots
shown in (C).
(C) DNA from Atrx-positive (Atrxþ, bearing either an AtrxWTor Atrxfloxallele) or Atrxnull(bearing the AtrxD18Dneoallele) ES cells and 7-d embryoid bodies
were digested with the enzymes shown and analysed by Southern blotting using the probes indicated. Arrows indicate the fully methylated copies (cut
by only the limit-digesting enzyme). Phosphorimager quantitation of the blots are shown below. The y-axis shows the percentage of copies that are
undigested by the methylation-sensitive enzyme as a percentage of the total signal from cut and uncut rDNA. Mean values are indicated by horizontal
lines, and the significance of the differences between the Atrx-positive and Atrxnullpopulations are shown for each enzyme.
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Trophoblast Defect in Mice Lacking Atrx
a control competition experiment between different clones
bearing functional Atrx alleles (AtrxWTand Atrxflox), both
clones continued to be equally represented after 8 d of
cocultivation. Thus, although AtrxnullES cells could be
recovered and maintained in culture by a conditional
targeting approach, these cocultivation experiments sug-
gested that the absence of Atrx does negatively impact upon
normal ES cell growth.
To investigate a possible cell-cycle defect in the absence of
Atrx, we analysed the cell cycle distribution of bromodeox-
yuridine (BrdU)-pulsed ES cells by flow cytometry (Figure
S1A). Surprisingly, both AtrxnullES cell clones exhibited a cell
cycle profile that was indistinguishable from ES cells bearing
a functional Atrx allele (AtrxWTor Atrxflox). We also specifically
quantitated the mitotic index within each population by flow
cytometry after staining ES cells for phosphorylated (Ser10)
histone H3, a specific marker of mitosis (Figure S1B) .
Consistent with the normal cell-cycle profile observed above,
there was no depletion in the size of the mitotic population in
the AtrxnullES clones, despite their slow growth. Finally, we
investigated whether the growth defect in the AtrxnullES cells
was due to an up-regulation of apoptosis by staining cells with
Annexin V (Figure S2) and found that the proportion of
apoptotic cells was not significantly affected by the absence of
full-length Atrx. Thus, the growth defect observed in ES cells
lacking Atrx is not due to a specific cell cycle block or
significant induction of cell death. While the cause of the
proliferative delay is not yet clear, since AtrxnullES cells
appear to undergo higher rates of spontaneous differ-
entiation (unpublished data), it seems likely that the observed
growth defect reflects the spontaneous transition from fast-
cycling, undifferentiated ES cells into more slowly cycling,
differentiated cell types in these cultures.
It has been shown that disease-causing mutations in the
human ATRX gene give rise to changes in the normal pattern
of DNA methylation at several repetitive sequences within the
human genome . Notably, the transcribed region of the
ribosomal DNA (rDNA) repeat was found to be significantly
hypomethylated in ATR-X patients relative to normal
individuals. Using methylation-sensitive restriction enzymes,
we also observed significant hypomethylation at several sites
tested within the mouse rDNA repeats in AtrxnullES cells and
12-d embryoid bodies relative to ES cells and embryoid
bodies bearing a functional Atrx allele (AtrxWTor Atrxflox)
(Figure 3B and 3C). The observation that rDNA is hypome-
thylated in the absence of Atrx, even in ES cells, is consistent
with the finding that hypomethylation of the human rDNA
repeats is detectable from an early developmental stage in
ATR-X patients. Other mouse repetitive sequence elements
surveyed in ES cell DNA include the heterochromatic major
satellite (assayed with MaeII) and minor satellite (assayed with
HpaII) repeats, as well as interspersed retroviral repeats of
the intracisternal A particle (IAP) type and the Line 1 and
Sine B1 families (all assayed with HpaII). These repeats were
found to be moderately (Line 1 and Sine B1) or highly (IAP,
major satellite, minor satellite) methylated in wild-type ES
cells, and this methylation was not detectably perturbed by
the absence of Atrx (Figure S3 and unpublished data). Taken
together, these data indicate that the subtle interplay between
the ATRX protein and DNA methylation observed in human
patients is also present in mouse cells.
Early Embryonic Lethality in AtrxnullMale Mice
To investigate the role of the Atrx protein during mouse
development, we initially established lines of mice bearing the
Atrxfloxallele. Two independently targeted AtrxfloxES cell
clones with normal male karyotype were injected into C57BL/6
blastocysts to produce chimaeric mice, which were then used
to obtain germline transmission. Intercrosses between males
hemizygous (Atrxflox/Y) and females heterozygous (AtrxWT/flox)
for the floxed allele were also carried out to generate
homozygous females (Atrxflox/flox). Males hemizygous and
females heterozygous or homozygous for the Atrxfloxallele
were viable, appeared healthy, and bred normally, suggesting
that, as expected, the Atrxfloxallele was functionally normal. To
generate Atrxnullmice by Cre-mediated recombination, the
Atrxfloxmice were crossed with mice harboring a transgene in
which the Cre recombinase is expressed under the control of
the regulatory elements of the mouse GATA-1 gene (GATA1-
cre) . Widespread expression of the GATA1-cre transgene
has been demonstrated during early embryogenesis . We
more accurately defined the onset of GATA1-cre expression
using a ROSA26 reporter strain, in which a b-galactosidase/
neorfusion reporter gene is expressed only after Cre-
mediated excision of loxP-flanked transcription and trans-
lation termination signals . We found that the GATA1-cre
transgene was already active at the 16-cell morula stage of
development (0.5 days postcoitus [dpc]) (Figure 4A).
To generate Atrxnullmice, heterozygous floxed females
(AtrxWT/flox) were mated with homozygous GATA1-cre trans-
genic males (AtrxWT/Y;GATA1-creþ/þ). No Atrxnullmales
(Atrxnull/Y;GATA1-creþ/?) were recovered at birth, indicating
that the absence of Atrx results in embryonic lethality. This
finding was unexpected, since human ATR-X patients clearly
survive to adulthood (see Discussion). Embryos were dissected
at 7.5, 8.5, and 9.5 dpc and genotyped by PCR analysis of DNA
extracted from yolk sac or total embryo (Figure 4B and
Protocol S1). Atrxnullmales were present at expected
mendelian ratios (;25%) at both 7.5 dpc and 8.5 dpc (Table
1). However, by 9.5 dpc, depletion was observed both in the
number of Atrxnullmales (7%) and in the total number of
males recovered (31%). No Atrxnullmales were recovered
after 9.5 dpc. Thus the absence of Atrx gives rise to
embryonic lethality in mice before 9.5 dpc.
To investigate the morphology of Atrxnullembryos prior to
death, embryos from the above crosses were initially dissected
in their deciduas at 7.5 dpc, and paraffin sections were
stained with haematoxylin (Figure 5A) or with an anti-ATRX
antibody (Figure 5B–5E). Immunohistochemical staining
revealed that Atrx was widely expressed in wild-type 7.5 dpc
embryos (Figure 5B). Expression was highest in the embryonic
region (Figure 5C) and the chorion (Figure 5D). Detectable
but lower levels of expression were observed in the
ectoplacental cone (Figure 5D) and surrounding decidual
tissue. We also observed very high levels of Atrx expression in
trophoblast giant cells (TGCs) surrounding the Reichert’s
membrane (Figure 5E). Within the large nuclei of these TGCs,
the typical nuclear association of Atrx with blocks of
pericentromeric heterochromatin  was clearly observable.
Only background staining was seen in the corresponding
Atrxnullembryonic tissues (Figure 5B–5D), while expression
in the surrounding decidual tissue (of maternal origin) was
normal and served as an antibody staining control (unpub-
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Trophoblast Defect in Mice Lacking Atrx
lished data). Morphologically, 7.5 dpc Atrxnullembryos were
dramatically reduced in size and appeared developmentally
retarded relative to stage-matched wild-type embryos (Figure
5A and 5B). However, despite their reduced size, the general
morphology and organisation of embryonic structures in
Atrxnullconceptuses appeared grossly normal. The amnion
and chorion were clearly present and the amniotic, exocoe-
lomic, and ectoplacental cavities were distinguishable, as were
all three embryonic germ layers (Figure 5A–5C). At 8.5 dpc,
embryos were dissected free of deciduas, and observed in
whole mount. Individual conceptuses were genotyped by PCR
using DNA isolated from yolk sac as described in Protocol S1.
Consistent with observations at 7.5 dpc, the general mor-
phology of the embryo proper of Atrxnullconceptuses also
appeared grossly normal at 8.5 dpc. The head fold had clearly
formed, and expression of the early mesoderm marker
brachyury (T)  was detected in the primitive streak and
emerging notochord by whole-mount in situ hybridisation
(WMISH) (Figure 5F), indicating that Atrxnullembryos had
To investigate whether the reduced size of the Atrxnull
embryos was due to an increase in apoptosis, we analysed
sections of paraffin-embedded 7.5 dpc embryos by TdT-
mediated dUTP nick end labeling (TUNEL) assay (Figure 6A).
Very few apoptotic cells were detected in wild-type 7.5 dpc
embryos. In Atrxnullembryos, a slight increase in the
apoptotic population was evident. However, consistent with
our observation of a grossly normal apoptotic index in
AtrxnullES cells (Figure S2), the apoptotic response observed
in Atrxnullembryos was also not uniform, but was restricted
to a low number of scattered TUNEL-positive cells. Since this
small apoptotic response is unlikely to account fully for the
dramatic size deficit observed in Atrxnullembryos, a possible
proliferation defect was also investigated by immunohisto-
chemical staining of 7.5 dpc embryo sections for the mitosis
marker phosphorylated (Ser10) histone H3 . Relative to
the very high mitotic index observed in wild-type embryos,
the proportion of mitotic cells observed in Atrxnullembryos
at 7.5 dpc was dramatically reduced (Figure 6B). Taken
Table 1. Distribution of Atrx Genotypes in Timed Matings
Unspecified Total Wild TypeNulla
Atrx genotype AtrxWT/WT;
Expected mendelian %
7.5 dpc, number observed (%)
(n ¼ 39)
8.5 dpc, number observed (%)
(n ¼ 59)
9.5 dpc, number observed (%)
(n ¼ 71)
14 (24%)9 (15%) 5 (8%) 28 (47%)15 (25%) 14 (24%) 2 (3%)31 (52%)
31 (44%) 18 (25%)0 (0%) 49 (69%)15 (21%)5 (7%) 2 (3%) 22 (31%)
Crosses were carried out initially between AtrxWT/flox;GATA1-cre?/?females and AtrxWT/Y;GATA1-creþ/þmales. Litters were dissected at the times shown and genotyped by PCR as described
in Protocol S1. These crosses would be expected to yield wild-type females (AtrxWT/WT;GATA1-creþ/?), carrier females (AtrxWT/null;GATA1-creþ/?), wild-type males (AtrxWT/Y;GATA1-creþ/?), and
null males (Atrxnull/Y;GATA1-creþ/?) in a ratio of 1:1:1:1. Subsequently, breedings were also carried out between females carrying a single recombined allele (AtrxWT/D18Dneo) and wild-type
males (AtrxWT/Y), which would be expected to yield the same four Atrx genoptyes in a 1:1:1:1 ratio as shown above. The data from these two breedings have been combined in the table,
but the same trends were observed when these two crosses were considered separately.
aThe Atrxnullallele is a combination of the AtrxD18and the AtrxD18Dneoalleles (resulting from recombination events B and C in Figure 2A). Both alleles were recovered in combination with
the GATA1-Cre transgene and both are null for full-length Atrx protein (unpublished data).
bND indicates that the Atrx genotype could not be determined by PCR. Sex was determined as described in Figure 4B.
Figure 4. Timing of Onset of GATA1-Cre Expression and PCR Genotyping
of Atrx Alleles
(A) GATA1-creþ/þtransgenic males were crossed to females of the
ROSA26 reporter strain (ROSA26þ/?), and embryos were recovered at 0.5
dpc (;16-cell morula stage) and stained with X-gal. Cre-mediated
activation of the ROSA26 b-galactosidase reporter allele was detected in
all cells in embryos in which both alleles are coinherited.
(B) Top gel: PCR genotyping of Atrx alleles in embryos using primers
PPS1.15 (exon 17) and Mxnp30 (exon 20) as described in Protocol S1. The
sizes of PCR products from the different alleles are indicated. Both the
AtrxD18(resulting from recombination event B in Figure 2A) and the
AtrxD18Dneoallele (resulting from recombination event C in Figure 2A) are
null for full-length Atrx protein. The bottom gel shows products from a
PCR reaction (primers DG52/DG53) used to sex embryos as described in
Protocol S1. A 450-bp PCR product is amplified from a mouse Y
chromosome-specific satellite repeat.
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Trophoblast Defect in Mice Lacking Atrx
together, these results suggest that the size deficit observed in
Atrxnullembryos prior to lethality largely reflects a prolifer-
ative defect, with a minor but indirect contribution from
increased apoptosis. Although a growth defect was also
observed in AtrxnullES cells (Figure 3A), in contrast to the
Atrxnullembryos, the mitotic index of the ES cell population
(as measured with the same antibody) was not depleted
(Figure S1B). These observations suggest that the mitotic
defect observed in embryos is unlikely to be a direct, cell-
autonomous effect of the absence of Atrx, and is more likely
to be a secondary effect resulting from the failure to develop
a normal trophoblast (see below).
Trophectoderm Failure in AtrxnullEmbryos
Whole-mount observation of 8.5 dpc embryos revealed that,
in contrast to the basically normal although delayed morphol-
ogy of the embryo itself, the extraembryonic tissues of Atrxnull
conceptuses appeared highly disorganised. When embryos
were removed from deciduas, the surrounding trophectoderm
Figure 5. Morphology of AtrxnullEmbryos at 7.5 dpc and 8.5 dpc
Paraffin sections of wild-type or Atrxnull7.5 dpc embryos (dissected in
their deciduas) were stained with haematoxylin (A) or with an anti-ATRX
antibody (H-300, Figure 1) (B–E). Photomicrographs C–E show higher
magnification images (2003) of the stained sections shown in (B) (403).
Scale bars represent 200 lm (403 magnification) or 40 lm (2003
magnification). a, amnion; ac, amniotic cavity; c, chorion; e, epiblast; ec,
ectoplacental cavity; ecc, exocoelomic cavity; ep, ectoplacental cone; ne,
neural ectoderm; rm, Reichert’s membrane; tgc, trophoblast giant cell.
(F) Detection of brachyury (T) expression in Atrxnull8.5 dpc embryo (head
fold stage) by WMISH. The genotype was determined by PCR (as shown
in Protocol S1) using DNA extracted from yolk sac. hf, head fold; n,
emerging notochord; ps, primitive streak.
Figure 6. Analysis of Apoptosis and Mitosis in AtrxnullEmbryos
(A) Paraffin sections of wild-type or Atrxnull7.5 dpc embryos (dissected in
their deciduas) were analysed by TUNEL assay and apoptotic cells
labelled with fluorescein-dUTP. Sections were counterstained with DAPI.
(B) Paraffin sections of wild-type or Atrxnull7.5 dpc embryos were stained
with an antibody against the mitosis marker phosphorylated (Ser10)
histone H3. Sections were counterstained with haematoxylin. For both
(A) and (B), the presence or absence of Atrx in each embryo was
determined by staining adjacent sections with the anti-ATRX antibody
(H-300) as in Figure 5 (unpublished data).
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Trophoblast Defect in Mice Lacking Atrx
layer appeared dramatically reduced in Atrxnullembryos
relative to wild-type littermates, and the underlying ectopla-
cental cone appeared reduced and abnormally shaped (Figure
7A). Vacated deciduas surrounding 8.5 dpc wild-type and
Atrxnullembryos were bisected and analysed by WMISH for
expression of placental lactogen-1 (Pl-1), a marker of
terminally differentiated TGCs. The number of Pl-1-express-
ing cells attached to the decidual wall after removal of the
embryo is an indication of the density of trophoblast cells
surrounding each implantation site . We found that the
population of Pl-1-expressing cells was depleted in the
decidual implantation sites containing Atrxnullembryos
relative to those containing wild-type littermates (Figure
7B); this was also apparent at 7.5 dpc, as determined by
immunohistochemical staining of paraffin sections of em-
bryos in deciduas with an anti-Pl-1 antibody (Figure 7C). A
TGC deficiency in the absence of Atrx is consistent with the
observation that Atrx is highly expressed in giant cells
surrounding wild-type 7.5 dpc embryos (Figure 5E).
To investigate whether the trophoblast defect was re-
stricted to the production of secondary TGCs (produced by
diploid precursors in the ectoplacental cone and derived
originally from the polar trophectoderm overlying the inner
cell mass of the blastocyst) or also affected the production of
primary TGCs (resulting from differentiation of the mural
trophectoderm of the blastocyst), blastocysts (3.5 dpc) from
crosses between AtrxWT/floxfemales and GATA1-Cre homozy-
gous transgenic males (AtrxWT/Y;GATA1-Creþ/þ) were cultured
in vitro for 5 d to monitor outgrowth of the primary
trophoblast. After 5 d, individual blastocyst cultures were
scored for the extent of primary trophoblast outgrowth, and
the Atrx genotype and sex of the blastocyst were determined
by PCR. Most blastocysts hatched from the zona pellucida
within 24 h, and trophoblast cells spreading out from the
inner cell mass could usually be detected within 48 h of
culture. No difference was observed in the rate or extent of
trophoblast outgrowth over 5 d of culture between Atrxnull
blastocysts (Atrxnull/Y, n¼6) and blastocysts bearing an AtrxWT
allele (AtrxWT/WT, n ¼ 6; AtrxWT/null, n ¼ 6; AtrxWT/Y, n ¼ 6)
(examples shown in Figure 7D), suggesting that the defect
specifically involves the secondary giant cell compartment.
This is consistent with the observation that Atrxnullcon-
ceptuses implant successfully and survive to gastrulation.
Taken together, these data suggest that loss of Atrx results in
a defect in formation of the secondary trophoblast that is
apparent from 7.5 dpc. Despite initiating normal organisa-
tion in the embryo proper, Atrxnullconceptuses exhibit a
proliferative defect by 7.5 dpc and die by around 9.5 dpc,
Figure 7. Trophectoderm Defect in AtrxnullEmbryos
(A) 8.5 dpc embryos were dissected from surrounding decidual tissue and observed in whole mount. The genotype of each (indicated above) was
determined by PCR using DNA extracted from whole embryos after photography. In the left image, the wild-type female (three-somite stage, left) is
surrounded by trophoblast (t) while the trophoblast component surrounding the Atrxnullmales (at headfold/presomite [middle] and two-somite stages
[right], respectively) is severely depleted. In the right image, the trophoblast has been dissected away from the embryonic region of the wild-type
embryo, to reveal the small, abnormally shaped ectoplacental cone (epc) of the mutant littermates.
(B) WMISH to detect expression of Pl-1 (a marker of TGCs) at the implantation sites in vacated deciduas that had contained 8.5 dpc wild-type (AtrxWT/WT)
or Atrxnull(AtrxD18Dneo/Y) embryos. The genotype was determined by PCR using DNA extracted from whole embryos. TGCs are stained with Pl-1.
(C) Paraffin sections of wild-type or Atrxnull7.5 dpc embryos (dissected in their deciduas) were stained with an anti-Pl-1 antibody. The presence or
absence of Atrx in each embryo was determined by staining adjacent sections with the anti-ATRX antibody (H-300) as in Figure 5 (unpublished data).
(D) Examples of 5-d blastocyst outgrowth cultures. Extensive trophoblast outgrowing from the inner cell mass (icm) was observed in all genotypes. The
Atrx genotype and sex of the blastocyst indicated were determined by PCR.
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Trophoblast Defect in Mice Lacking Atrx
probably due to a nutritional deficit resulting from failure to
develop a normal trophoblast.
Escape from Imprinted Inactivation of the Paternally
Inherited AtrxWTAllele in Extraembryonic Tissues of Carrier
Female mice carrying an Atrxnullallele (AtrxWT/null;
GATA1-creþ/?) were detected at 9.5 dpc (Table 1) and
recovered at birth (unpublished data), although at both time
points the number of carrier females was lower than that of
wild-type (AtrxWT/WT;GATA1-creþ/?) females, suggesting that a
proportion of carrier female embryos died in utero. Surviv-
ing adult carrier female mice were not phenotypically normal
and exhibited mild behavioural abnormalities, although some
could reproduce. For all AtrxWT/nullcarrier female embryos
presented in Table 1, the AtrxWTallele was paternally derived,
while the Atrxnullallele was maternally derived. In the mouse,
X chromosome inactivation is subject to parental imprinting
in the trophectoderm and primitive endoderm lineages that
give rise to the extraembryonic tissues, resulting in inactiva-
tion of the paternal X chromosome (Xp) . In contrast, in
tissues of the embryo proper (derived from the inner cell
mass of the blastocyst) X-inactivation is random . Thus, in
the extraembryonic compartment of AtrxWT/nullcarrier
females, normal imprinted X-inactivation would be expected
to result in silencing of the paternally derived AtrxWTallele,
leaving only the Atrxnullallele on the active maternal X (Xm)
and thereby render the extraembryonic tissues null for full-
length Atrx protein. However, the absence of Atrx in the
extraembryonic compartment is lethal in Atrxnull/Y males.
This suggested the possibility of an escape from imprinted
inactivation of the paternally derived AtrxWTallele in the
extraembryonic compartment of a proportion of carrier
To investigate further, we crossed homozygous Atrxflox/flox
females and homozygous GATA1-cre transgenic males (AtrxWT/
Y;GATA1-creþ/þ), which would be expected to yield only
Atrxnullmales (Atrxnull/Y;GATA1-creþ/?) and carrier females
(AtrxWT/null;GATA1-creþ/?). In these carrier females, the AtrxWT
allele is paternally inherited. Embryos were dissected in their
deciduas at 7.5 dpc, and paraffin sections were stained with
anti-ATRX antibody, along with sections from a wild-type 7.5
dpc embryo for comparison (Figure 8). As described above,
Atrx expression was detected in every cell in the epiblast
(embryo proper) region of wild-type 7.5 dpc embryos (Figure
8B). In contrast, the epiblast region of carrier female embryos
was composed of a mosaic of small clusters of Atrx-positive
cells (in which the Atrxnullallele on Xm had been inactivated)
and Atrx-negative cells (in which the AtrxWTallele on Xp had
been inactivated), indicating that the Atrx gene was subject to
normal random X-inactivation in the epiblast. Remarkably,
clear Atrx expression could also be detected in the
extraembryonic tissues of carrier females, as shown in the
extraembryonic-derived chorionic ectoderm (Figure 8C).
Atrx expression could be detected in almost all cells of the
chorionic ectoderm. Atrx expression was also clearly detected
in other extraembryonic structures, including TGCs (unpub-
lished data). Escape from silencing of an Xp-inherited AtrxWT
allele was also observed in the chorionic ectoderm of carrier
females at 8.5 dpc (Figure S4). Thus, although random X-
inactivation occurs normally within the epiblast, the AtrxWT
allele (inherited on the Xp chromosome) escaped the normal
imprinted X-inactivation in the extraembryonic compart-
ment of some carrier females.
We investigated the role of the Atrx protein in mouse
development. By using a conditional knockout approach, we
ablated the full-length Atrx protein first in ES cells and
embryoid bodies, and then in developing mouse embryos.
Atrx in ES Cells
AtrxnullES cells could not be recovered by direct targeting
and were eventually generated by adopting a conditional
targeting approach. This is consistent with our observation
that Atrx is highly expressed in ES cells, and that the absence
of full-length Atrx imparts a growth disadvantage relative to
cells bearing a functional Atrx allele. At present, the cause of
the proliferative delay in AtrxnullES cells is not known.
Figure 8. Escape from Imprinted Inactivation of the Paternally Inherited
AtrxWTAllele in Carrier Females
Paraffin sections of wild-type (AtrxWT/Y) and carrier female (AtrxWT/null) 7.5
dpc embryos (dissected in their deciduas) were stained with the anti-
ATRX antibody (H-300). Scale bars represent 200 lm (403magnification)
or 20 lm (4003 magnification).
(A) Stained sections showing whole embryos at 403 magnification. a,
amnion; c, chorion; e, epiblast; ep, ectoplacental cone.
(B) Higher-magnification image (4003) of the epiblast regions of the
stained sections shown in (A).
(C) Higher-magnification image (4003) showing the extraembryonic
derived-chorionic ectoderm of the stained sections shown in (A). ce,
chorionic ectoderm; cm, chorionic mesoderm.
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Trophoblast Defect in Mice Lacking Atrx
Interestingly, we demonstrated that apoptosis is not signifi-
cantly up-regulated in ES cells lacking Atrx and is only mildly
elevated in Atrxnull7.5 dpc mouse embryos. In contrast, it was
recently shown that the loss of Atrx markedly increased the
apoptotic population in the differentiating cells of the
embryonic cortex and postnatal hippocampus, when Atrx
expression was ablated in the developing mouse forebrain
using the Atrxfloxallele described here . The human ATRX
protein has been shown to associate in a complex with Daxx
, a protein that has been implicated in multiple pathways
for the regulation of apoptosis . It is possible that
disruption of the mouse Atrx-Daxx complex (by ablation of
the Atrx protein) could have triggered a universal proapop-
totic response. However, our observations in ES cells
demonstrate that the induction of apoptosis is not an
automatic response triggered by the removal of Atrx in all
cell types, and suggest that the inappropriate apoptosis
observed in the Atrx-mutant forebrain may reflect a require-
ment for Atrx during terminal differentiation.
An Unexpected Role for Atrx in Development of the
We show that Atrxnullmale mice are not viable and
embryos die by around 9.5 dpc. Before death, Atrxnull
embryos exhibit a markedly reduced mitotic index, suggest-
ing a proliferative defect. Although the embryonic compart-
ment of the conceptus appears initially normal, by 7.5 dpc
Atrxnullembryos display abnormalities in development of the
trophoblast, including a depletion in the population of TGCs
surrounding the conceptus and a reduction in the size of the
ectoplacental cone, which contains the diploid giant cell
precursors . TGCs are highly differentiated, postmitotic
cells that ultimately form an epithelial layer at the periphery
of the placenta, which interfaces with the maternal cells of
the decidua . These highly invasive cells are important for
mediating initial invasion of the uterine tissue, but are also
involved in remodelling the maternal decidua after implan-
tation and in secreting hormones that regulate fetal and
maternal growth . Since Atrxnullembryos appear to
implant normally, lethality is likely to arise due to a failure
of TGC function later during development.
Embryonic lethality in mice in the absence of Atrx was a
surprising finding, as there had been no suggestion of foetal
loss in the human ATR-X syndrome. It is possible that the
role of Atrx in the trophoblast is specific to mice and that
ATRX has no role or is redundant in the human trophoblast.
Indeed, the birth weight of babies with ATR-X syndrome is
usually normal. An alternative explanation for the unexpect-
edly severe phenotype we observed in mice is that the
AtrxD18Dneodeletion generated by Cre recombination com-
pletely ablates full-length Atrx protein (Figure 2E). In
contrast, all disease-causing mutations characterised in
human ATR-X pedigrees appear to give rise to hypomorphic
alleles. Some full-length ATRX protein is detected in cases
predicted to have truncating mutations (RJG, unpublished
data), and residual ATPase activity in ATRX immunopreci-
pitates can be detected in Epstein-Barr virus-transformed
lymphocytes of all human patients analysed to date (A.
Argentaro and M. Mitson, unpublished data). The failure to
observe a truly null ATRX allele among human patients
strongly suggests that, as in the mouse, the complete absence
of ATRX protein is incompatible with human fetal survival.
While this study has revealed an unexpected role for Atrx
in the murine trophectoderm, as a result of the early lethality
observed in Atrxnullembryos it is not possible to rule out
other roles for Atrx at later developmental stages in tissues of
the embryo proper. Indeed, we show that Atrx is highly
expressed throughout the entire developing embryo at 7.5
dpc (Figure 5B), and it is likely that Atrx function will turn
out to be important for other differentiating tissues.
Tetraploid aggregation experiments (in which mutant em-
bryos are rescued with wild-type extraembryonic tissues)
might shed more light on the role of Atrx during later mouse
development, but these issues can be more subtly dissected by
combining the conditional Atrxfloxallele that we have
generated with different tissue-specific Cre transgenes. As
mentioned above, this approach has already revealed a
critical role for Atrx during neuronal differentiation in adult
mice . Further evidence that Atrx is also required at later
stages of mouse development is provided by the observed
dramatic skewing against Atrx-negative cells in some somatic
tissues of carrier female mice, whose tissues initially comprise
a mosaic of Atrx-positive and Atrx-negative cells as a result of
random X-inactivation (M. Muers, personal communication).
Atrx joins an expanding list of mouse genes for which
targeted disruption results in peri-implantation lethality as a
result of trophoblast or placental abnormalities (reviewed in
). Comparison with other phenotypes might provide some
insight into the role of Atrx in trophoblast development. Atrx-
mutant embryos progress further than embryos nullizygous
for factors involved in the initial specification of trophoblast
stem cells (such as Cdx2) or in stem cell maintenance and
proliferation (such as Eomes). Cdx2-mutant embryos fail to
implant and die between 3.5 and 5.5 dpc , while Eomes-
mutant blastocysts implant into the uterus, but arrest soon
after implantation without forming organised embryonic or
extraembryonic structures . In contrast, Atrx-mutant
embryos implant successfully and establish organised embry-
onic structures by 7.5 dpc. The Atrx-mutant phenotype closely
resembles that observed in mice nullizygous for the basic
helix-loop-helix transcription factor Hand1. Hand1-mutant
conceptuses arrest at around 7.5 dpc and display a normal
embryonic compartment, but, like Atrx-mutant embryos,
ablation of Hand1 causes a reduction in the size of the
ectoplacental cone and density of TGCs . As with Atrx
mutants, only arrested or resorbed Hand1-mutant concep-
tuses were recovered after 8.5 dpc. Also like Atrx, disruption
of Hand1 specifically affects secondary giant cell formation,
and primary trophoblast outgrowths from blastocysts ap-
peared normal. Hand1 is required for terminal differentia-
tion of secondary TGCs, and in its absence trophoblast cells
arrest at a precursor stage in the ectoplacental cone [17,28].
Given the similarity of the Atrx- and Hand1-mutant pheno-
types and the likelihood that Atrx acts as a transcriptional
regulator by modifying chromatin structure, it will be of
interest to determine whether Atrx is itself a regulator of
Hand1 expression, or alternatively whether it acts as a co-
regulator of one or more of the downstream transcriptional
targets of Hand1. It is noteworthy that, in the brain-specific
Atrx knockout mice, the defect was observed in terminally
differentiating neurons . The secondary TGCs affected in
the universal Atrx knockout reported here represent one of
the first terminally differentiated tissues in the developing
mouse, and this may point to the requirement for Atrx in the
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Trophoblast Defect in Mice Lacking Atrx
high-level expression of some tissue-specific genes during the
final stages of differentiation. Interestingly, the a-globin
genes, the only confirmed transcriptional targets of regu-
lation by human ATRX, are also highly expressed specifically
during terminal differentiation within the erythroid lineage.
Atrx Escapes Imprinted X-Inactivation in Extraembryonic
Tissues of Carrier Female Mice
Another surprising finding of this study is that, in carrier
female embryos, a paternally inherited AtrxWTallele appears
to escape the process of imprinted X-inactivation, which
ordinarily silences the Xp chromosome in the extraem-
bryonic compartment of female murine tissues . Silencing
of the AtrxWTallele on Xp should render these females null
for Atrx in the extraembryonic tissues, since the normally
active Xm chromosome carries the AtrxD18Dneoallele.
Although not phenotypically normal, some Atrx carrier
females developed to term and went on to reproduce. Thus,
the failure to correctly silence the paternally derived AtrxWT
allele in the extraembryonic tissues of carrier females is
consistent with our observations that in Atrxnullmales, the
Atrx protein plays an essential role in the development of the
trophoblast and is necessary for survival in utero in the
The survival of Atrx carrier females contrasts with the
phenotypes seen in carriers of mutations of other murine X-
linked genes known to be essential in the extraembryonic
compartment. For example, targeted disruption of the
dyskerin (Dkc1), glucose 6-phosphate dehydrogenase (G6PD),
and choroideremia (Chm) genes cause embryonic lethality in
null male embryos through defects of the extraembryonic-
derived tissues [29–31]. Female mice carrying mutations of
these genes on the maternally inherited X chromosome also
die in utero, whereas females that inherit the mutation on the
Xp chromosome survive. Thus, unlike Atrx, these genes and/or
their effects on cell growth are unable to circumvent the
processes that ultimately cause all cells in the extraembryonic
tissues to express only the maternally derived X chromosome.
How might expression of the paternal AtrxWTallele be
maintained in the extraembryonic tissues of the Atrx carrier
One possibility is that, like some other X-linked genes,
silencing of the Atrx gene on Xp is incomplete, such that there
is always a low-level, leaky output of Atrx from a normally
inactivated Xp chromosome in extraembryonic tissues.
However, it was recently demonstrated that the paternal Atrx
(called Xnp) allele is completely silenced in a normal mouse
trophoblast stem cell line , suggesting that Atrx does not
normally escape imprinted X-inactivation in the extraem-
bryonic tissues of wild-type females. Thus, the expression of
the Xp-linked AtrxWTallele that we observed is unique to
female carriers of the Atrxnullallele.
Perhaps a more likely explanation for this phenomenon
stems from experimental observations suggesting that im-
printed X-inactivation is not imposed on all precursors of the
mouse extraembryonic tissues: A subpopulation of cells may
escape this process and make a random ‘‘choice’’ of which X
chromosome will be inactivated. On average, 50% of the cells
in this randomly inactivating subpopulation would be
expected to maintain an active Xp chromosome. In support
of this hypothesis, it has been demonstrated that expression
of paternally transmitted X-linked lacZ [33,34] and GFP 
transgenes failed to be silenced in a small subpopulation of
extraembryonic cells. Further, it has been shown that in a
subpopulation of extraembryonic cells, it is the Xm rather
than the Xp that undergoes late replication, a molecular
correlate of the inactive state [18,36]. Although initially small
and quickly diluted in normal embryos, the cellular sub-
population that inactivates the Xm chromosome could
rapidly expand to replace the normally imprinted cells in
extraembryonic lineages if the normal silencing of Xp
compromises cell growth or differentiation. Interestingly, it
has been suggested that the size of the population that
initially escapes imprinting may range widely (from 0% to
30%), even between genetically identical embryos , and
this may account for the variable phenotype observed among
females bearing Xm-linked mutant alleles of genes essential
for normal extraembryonic development . Put simply,
carrier females bearing a small initial population of escaping
cells would be more severely affected than those bearing a
larger population. This could explain why we have observed
significant phenotypic variation among Atrx carrier females,
with some carriers dying in utero by 9.5 dpc (Table 1) and
others developing to term.
Another possible mechanism is that inactivation of the
paternal X proceeds normally in all cells, but subsequently
the Atrx gene within individual cells is reactivated. Alter-
natively, in the absence of Atrx, the paternal allele may
partially escape the normal process of silencing. In both of
these cases, other genes on the paternal X chromosome must
be inactivated and remain so, since blocking inactivation of
the entire Xp chromosome causes embryonic lethality due to
biallelic expression of X-linked genes in the trophoblast .
ATR-X syndrome is the first human genetic disease known
to be caused by mutations in a chromatin remodelling factor.
At present we do not know how ATRX influences gene
expression or what effect it has on cell behaviour. Never-
theless, we have previously noted that none of the natural
mutations causing ATR-X syndrome are nulls, which suggests
that it plays a critical role in normal development. Results of
conditional inactivation of Atrx in the developing mouse
forebrain, based on the Atrxfloxallele described here, shows
that Atrx exerts a major effect on terminally differentiating
neurons. Conditional inactivation of Atrx in other tissues is
underway. Here we have shown that animal-wide disruption
of the Atrx gene causes a severe embryonic-lethal phenotype,
revealing an essential role for Atrx in the formation of the
murine trophoblast. In addition, Atrx appears to escape
imprinted X-chromosome inactivation in the extraembryonic
tissues of some carrier female mice.
Materials and Methods
Generation of ES cells bearing the Atrxfloxallele. Briefly, the
targeting vector (shown in Figure 2A) places a loxP site within intron
18 and a loxP-flanked MC1neopA selection cassette in intron 17 of the
Atrx gene. A detailed description of the targeting construct is
provided in . Linearised plasmid (150 lg) was electroporated
into 1 3 108E14Tg2a ES cells, and colonies resistant to G418 and
ganciclovir were isolated. Homologous targeting events were identi-
fied by Southern blot of EcoRI-digested DNA and hybridisation with
a 59 probe (generated with primers PPS1.20 and PPS1.27) and a 39
probe (a 0.9-kb HaeIII fragment) as shown in Figure 2A and 2B. DNA
from correctly targeted clones was also digested with SacI and
analysed by Southern blot and hybridisation with a probe from
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Trophoblast Defect in Mice Lacking Atrx
within intron 17 (a PCR product generated with primers PPS1.15 and
Xnp46) to confirm that the loxP site within intron 18 had been
included within the crossed-over region (Figure 2A and 2C).
Sequences of primers are shown in Table S1.
Cre-recombination and characterisation of AtrxnullES cells and
embryoid bodies. ES cell clones bearing the Atrxfloxallele (1 3 107
cells) were transiently transfected with 50 lg of uncut Cre expression
plasmid (pCAGGS-Cre-IRESpuro) . Following transfection, cells
were plated at a range of clonal densities in complete medium
without G418, and isolated subclones were picked after 7 d. Subclones
were expanded and analysed for the presence of a recombinant locus
initially by PCR, to detect deletion of the MC1neopA cassette, and
then by Southern blot and hybridisation with the intron 17 probe
described above (Figure 2A and 2C). Northern blots were carried out
according to standard techniques using 20 lg of total RNA isolated
using TRI Reagent (Sigma-Aldrich, St. Louis, Missouri, United States).
The blot was hybridised with a probe from within exon 10 of the Atrx
gene (generated with primers Mxnp4 and Mxnp28 [Table S1]). After it
was stripped, the membrane was hybridised with a b-actin cDNA
probe (Clontech, Palo Alto, California, United States). Protein
extraction and detection of Atrx by Western blotting was performed
as described previously , using the mouse monoclonal anti-ATRX
antibody 23C . Analyses of cell cycle and apoptosis are described
in Protocol S1. Methylation of rDNA was analysed in DNA from ES
cell clones or from embryoid bodies recovered after 7 d of in vitro
differentiation as described previously . Genomic DNA was
digested with methylation-sensitive restriction enzymes as described
and analysed by Southern blotting. The RIB3 and RIB4 probes (which
were amplified from human DNA, but cross-react with the mouse
rDNA repeat) have been described previously . Oligonucleotide
probes to detect Line 1 and Sine B1 repeats have been described
previously . The minor satellite probe was a 27-mer oligonucleo-
tide (mCENT2). The major satellite probe was a 27-mer oligonucleo-
tide (DG27). The IAP probe was an ;400 bp PCR product (primers
14A and 13K) amplified from an IAP inserted into the mouse agouti
gene  and was a gift from Peter Warnecke and Tim Bestor. The
PCR product included the entire 59 LTR of the IAP. All oligonucleo-
tide sequences are shown in Table S1.
Generation of chimeras, floxed mice, and mutant mice. Targeted
AtrxfloxES cell clones were injected into C57BL/6 blastocysts and
transferred into 2.5 dpc pseudopregnant recipients by standard
techniques. Resulting chimeras were mated with C57BL/6 to establish
germline transmission. Offspring with the Atrxfloxallele were
identified by Southern blotting of SacI-digested tail DNA using the
intron 17 probe as shown in Figure 2A and 2C. For Cre-
recombination, Atrxfloxmice were crossed with GATA1-Cre transgenic
mice as described in the text. Recombinant alleles were detected by
Southern blotting as described above or by PCR as described in
Immunohistochemistry, in situ hybridisation, and TUNEL assay.
7.5 dpc decidual swellings were dissected away from maternal tissue
and fixed in 4% paraformaldehyde/PBS overnight at 4 8C. After
embryos were washed thoroughly in PBS, they were dehydrated
through an ethanol series and xylene, embedded in paraffin, and
sectioned at 5 lm. Sections were processed for immunohistochem-
istry using the ABC Staining System (Santa Cruz Biotechnology, Santa
Cruz, California, United States) according to the manufacturer’s
instructions. Sections were stained with rabbit polyclonal antibodies
against ATRX (H-300, Santa Cruz Biotechnology), Placental lactogen-
I (AB1288, Chemicon International, Temecula, California, United
States) and phospho (Ser10)-histone H3 (06–570, Upstate Biotechnol-
ogy, Waltham, Massachusetts, United States). Where appropriate,
adjacent sections were stained with haematoxylin. In some cases,
adjacent sections were also analysed to detect apoptotic cells by
TUNEL using the in situ cell death detection kit (Roche, Basel,
Switzerland). After labelling, these slides were mounted in Vecta-
shield containing DAPI (Vector Laboratories, Burlingame, California,
United States) and visualised by fluorescence microscopy. Whole-
mount in situ hybridisations were performed on 8.5 dpc embryos
(dissected away from maternal and extraembryonic tissues) using a
brachyury (T) riboprobe  and on bisected decidual implantation
sites from which embryos (8.5 dpc) had been removed using a
placental lactogen-1 (Pl-1) riboprobe (see Protocol S1 for details).
Blastocyst outgrowth cultures. Superovulated heterozygous female
mice (AtrxWT/flox) were mated to homozygous GATA1-creþ/þtransgenic
males, and blastocysts were flushed from uterine horns with M2
medium (Sigma) at 3.5 dpc. Individual blastocysts were cultured in
multiwell tissue cultures plates as described previously . Cultures
were inspected and photographed daily and the extent of outgrowth
scored. After 7 d, cultures were harvested and DNA extracted. The
Atrx genotype and sex of each culture was determined by PCR as
described in Protocol S1.
Figure S1. Cell Cycle Analysis of Atrx-Positive and AtrxnullES Cells
(A) Representative FACS profiles of BrdU-pulsed ES cells bearing
either functional (AtrxWTor Atrxflox) or null (AtrxD18Dneo) Atrx alleles
showing BrdU-FITC (y-axis, logarithmic scale) against propidium
iodide (x-axis, linear scale). The gated populations show cells in G1
(PIlow, BrdU-negative) (R1), S (BrdU-positive) (R2), and G2/M (PIhi,
BrdU-negative) (R3) phases of the cell cycle. Below is shown the
quantitation of gated populations, indicating the percentage of cells
in G1, S, and G2/M cell-cycle phases in each culture.
(B) FACS profiles of ES cells bearing either functional (AtrxWTor
Atrxflox) or null (AtrxD18Dneo) Atrx alleles stained for the mitosis marker
phosphorylated (Ser10) histone H3 (FITC, y-axis, logarithmic scale)
against propidium iodide (x-axis, linear scale). The size of the mitotic
population (phosphoH3S10-positive, PIhi)(gate R3) is indicated for
each profile. The ES cell clone analysed in each trace is indicated.
Found at DOI: 10.1371/journal.pgen.0020058.sg001 (3.2 MB PDF).
Figure S2. Analysis of Apoptosis in Atrx-Positive and AtrxnullES Cells
FACS analysis of ES cells bearing different Atrx alleles as shown, after
costaining for Annexin V (FITC, x-axis, logarithmic scale) and
propidium iodide (y-axis, logarithmic scale). The size of the early
apoptotic (Annexin-positive, PIlow) and late apoptotic or necrotic
(Annexin-positive, PIhi) populations is indicated for each genotype.
Found at DOI: 10.1371/journal.pgen.0020058.sg002 (223 KB PDF).
Figure S3. Normal DNA Methylation at Mouse Repetitive Elements in
DNA from Atrx-positive (bearing either an AtrxWTor Atrxfloxallele) or
Atrxnull(bearing the AtrxD18Dneoallele) ES cells was digested with
either a CpG-methylation-sensitive enzyme HpaII (H) or its methyl-
ation-insensitive isoschizomer MspI (M) as indicated, and digested
DNA was analysed by Southern blotting. Membranes were hybridised
with probes specific for the mouse Line 1 (A), Sine B1 (B), minor
satellite (C), and IAP (D) repeat elements. No significant loss of CpG-
methylation was observed at any of these repetitive elements in
AtrxnullES cells. As a positive control for loss of methylation, DNA
from ES cells lacking either the Dnmt1 (Dnmt1?/?) or both the
Dnmt3a and Dnmt3b (Dnmt3a3b?/?) DNA methyltransferases was
included in the analysis of Line 1 and Sine B1 repeats. Loss of CpG-
methylation was clearly observed at these repetitive elements in these
Found at DOI: 10.1371/journal.pgen.0020058.sg003 (1.8 MB PDF).
Figure S4. Escape from Imprinted Inactivation in 8.5 dpc Carrier
A cross was carried out between a carrier female (AtrxWT/null) and
wild-type male (AtrxWT/Y), and embryos were dissected in their
deciduas at 8.5 dpc. Any carrier female progeny of this cross will carry
an AtrxWTallele on the Xp chromosome and an Atrxnullallele on the
Xm chromosome. Transverse paraffin sections were stained with the
anti-ATRX antibody (H-300).
(A) High-magnification image (4003) of the neural fold region (nf)
(embryo proper) of a carrier female (AtrxWT/null) embryo. This tissue is
clearly comprised of a mosaic of Atrx-positive and Atrx-negative cells
due to random X-inactivation. This section was counterstained with
(B) High-magnification image (4003) of the same embryo depicted in
(A), showing the extraembryonic derived-chorionic ectoderm (ce) and
visceral endoderm (ve), two tissues that undergo imprinted X-
inactivation. Although Atrx is poorly expressed in the visceral
endoderm (even in wild-type females [unpublished data]), strong
expression of Atrx can be seen in the chorionic ectoderm, indicating
that the paternally-derived AtrxWTallele had escaped inactivation in
Found at DOI: 10.1371/journal.pgen.0020058.sg004 (1.7 MB PDF).
Protocol S1. Supplementary Methods
Found at DOI: 10.1371/journal.pgen.0020058.sd001 (42 KB DOC).
Table S1. Primers Used in This Study
Found at DOI: 10.1371/journal.pgen.0020058.st001 (20 KB DOC).
PLoS Genetics | www.plosgenetics.orgApril 2006 | Volume 2 | Issue 4 | e58 0449
Trophoblast Defect in Mice Lacking Atrx
The Online Mendelian Inheritance in Man (http://www.ncbi.nlm.nih.
gov/entrez/query.fcgi?db¼OMIM) accession number for ATR-X syn-
drome is 301040. The GeneID (www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db¼gene) for human ATRX is 546 and for mouse Atrx is 22589.
The GenBank (http://www.ncbi.nlm.nih.gov/) accession number for
the minor satellite probe mCENT2 is X14470 (nucleotides 75–101),
for the major satellite probe DG27 is M25032 (nucleotides 146–172),
and for the IAP probe is L33247.
We would like to thank Stu Orkin for providing access to the GATA1-
Cre transgenic mice, Gillian Morriss-Kay for advice, Peter Warnecke
and Tim Bestor for the IAP probe, En Li for the Dnmt knockout ES
cells, Frank Talamantes for the pRSV-mPL-1 plasmid, Ann Atzberger
for assistance with flow cytometry, and Deb Bogani and Terry Hacker
for assistance with whole-mount in situ hybridisation analyses and
Author contributions. DG, AJHS, WGW, DRH, and RJG conceived
and designed the experiments. DG, JAS, RA, and RJG performed the
experiments. DG, RA, WGW, DRH, and RJG analysed the data. LD
contributed reagents/materials/analysis tools. LD provided technical
assistance. DG wrote the paper.
Funding. This work was supported by the Medical Research
Council of the United Kingdom.
Competing interests. The authors have declared that no competing
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PLoS Genetics | www.plosgenetics.orgApril 2006 | Volume 2 | Issue 4 | e580450
Trophoblast Defect in Mice Lacking Atrx