![](x1_files/shim.gif) Science, Vol 303, Issue 5658, 666-669 , 30 January
2004
[DOI: 10.1126/science.1092674]
Reactivation of the Paternal X Chromosome in Early Mouse Embryos
Winifred Mak,1
Tatyana B. Nesterova,1 Mariana de
Napoles,1 Ruth Appanah,1
Shinya Yamanaka,2 Arie P.
Otte,3 Neil Brockdorff1*
It is generally accepted that paternally imprinted X
inactivation occurs exclusively in extraembryonic
lineages of mouse embryos, whereas cells of the embryo
proper, derived from the inner cell mass (ICM), undergo
only random X inactivation. Here we show that imprinted X
inactivation, in fact, occurs in all cells of early
embryos and that the paternal X is then selectively
reactivated in cells allocated to the ICM. This contrasts
with more differentiated cell types where X inactivation
is highly stable and generally irreversible. Our
observations illustrate that an important component of
genome plasticity in early development is the capacity to
reverse heritable gene silencing decisions.
1 X inactivation group, MRC Clinical Sciences Centre,
ICSM, Hammersmith Hospital, London, W12 0NN, UK. 2
Laboratory of Animal Molecular Technology, Research and Education
Center for Genetic information, Nara Institute of Science and
Technology, Nara 630-0192, Japan. 3 Swammerdam
Institute of Life Sciences, University of Amsterdam, Plantage
Muidergracht 12, 1018 TV Amsterdam, Netherlands.
* To whom correspondence
should be addressed. E-mail: neil.brockdorff@csc.mrc.ac.uk
X inactivation is the developmentally regulated silencing of
one of the two X chromosomes in female mammals, providing
the mechanism for dosage compensation of X-linked genes
relative to XY males. In mouse embryos, there is
imprinted X inactivation of the paternal X chromosome
(Xp) in the extraembryonic trophectoderm (TE) and
primitive endoderm (PE). In all other cells, X inactivation
is random. Establishment of these patterns has been
thought to occur in a lineage-specific manner, with
imprinted X inactivation initiated only in TE and PE
cells as they differentiate at the blastocyst stage. In
contrast, ICM cells, which give rise to the embryo
proper, have been thought to retain both X chromosomes in
the active state until they differentiate and undergo random
X inactivation during early postimplantation development (1–5).
Paradoxically, Xist RNA, the cis-acting signal that
initiates X inactivation, is expressed from Xp as early
as the two-cell stage (6,
7).
This has been rationalized by supposing that cells of the
early embryo cannot respond to Xist RNA (6).
In a recent study, we have shown that recruitment of the
Eed-Ezh2 Polycomb-Group (PcG) complex to the inactive X
(Xi), is required to establish trimethylation of histone
H3 lysine-27 (H3-K27) in postimplantation embryos (8).
In the course of analyzing localization of Eed-Ezh2 on Xi
in preimplantation embryos, we found that PcG
localization occurs in all cells of early- and mid-stage
XX blastocysts, including those corresponding morphologically
to the ICM (Fig.
1A). In contrast, localization to Xi was no longer
detectable in the ICM region of late-stage blastocysts,
despite high levels of the Eed-Ezh2 proteins in the nuclei (Fig.
1B). Scoring data demonstrate that only early-stage
blastocysts have Eed-Ezh2 foci in 100% of cells (Fig.
1C). In dual staining experiments, Ezh2 was seen to
colocalize with Eed at all stages analyzed and Eed-Ezh2
foci colocalized with Xist RNA domains (fig. S1, A
to C).
Fig. 1. Eed-Ezh2 localization in
ICM cells. Combined optical sections through (A) early-
and (B) late-stage XX blastocysts immunostained for Eed
(green). Presumptive ICM and TE are indicated with arrows.
(C) Scoring data illustrating the proportion of cells
with Xi associated Eed foci in individual XX embryos between
morula and late blastocyst. (D to F) Combined
optical sections illustrating (D) early-, (E) mid- and (F)
late-stage XX blastocysts stained for Eed (green) and Nanog
(red). Arrow in (F) indicates Nanog-positive cells without Eed
foci. [View
Larger Version of this Image (47K GIF file)]
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Eed-Ezh2 localization to Xi in the ICM was confirmed using
dual staining for Eed and the recently described
homeodomain protein Nanog, expressed specifically in ICM
cells (9,
10).
Eed foci were detectable in all Nanog-positive cells in
early- and mid-stage blastocysts (Fig.
1, D and E) but were progressively lost at later
stages (Fig.
1F). Interestingly, loss of Eed foci in the ICM
region of maturing blastocysts occurs specifically in
Nanog-positive cells, representing precursors of the
embryo proper, but does not occur in Nanog-negative
cells, which represent the primitive endoderm
lineage.
Our observations led us to consider that imprinted X
inactivation may occur in all cells of early blastocysts
and may then be selectively reversed in the ICM,
establishing the ground state for subsequent random X
inactivation. To determine whether early ICM cells
exhibit other markers of X inactivation, we carried out
dual labeling for Eed and specific modifications on histone
N-termini, namely trimethylation of H3-K27, deacetylation
of H3-K9, loss of methylation at H3-K4, and deacetylaton
of H4 (8,
11–13)
(fig. S2, A to D).
Eed localization to Xi in XX embryos is first detectable in
morula (8).
At this stage, H3-K27 methylation on Xi was only
detectable in some cells with Eed foci (Fig.
2, A, B, and D). The proportion increased rapidly
thereafter, and by blastocyst stage H3-K27 methylation on
Xi was detected in the majority of cells, including in
the ICM region (Fig.
2, C and D). Hypoacetylation of H3-K9 and loss of
H3-K4 methylation, which are early markers of X
inactivation (11),
were detectable in all cells with Eed localization to Xi,
even at the morula stage (Fig.
2, E and F). Also, H4 hypoacetylation, a later marker
for the X inactivation process (8,
11,
13),
was seen underlying Eed foci at morula and subsequent
stages (Fig.
2G). These results demonstrate that X inactivation
initiates earlier than previously thought, at or even
before morula stage, and that markers of X inactivation
other than Eed-Ezh2 localization are established in ICM
cells of early blastocysts.
Fig. 2. Inactive X chromatin
modification in early embryos. (A and B) Optical
sections illustrating examples of cells in XX morula-stage
embryos showing Eed (red) localization to Xi (arrows), either
(A) with or (B) without associated tri-meH3-K27 (green).
(C) Combined optical sections through a mid-stage XX
blastocyst illustrating colocalization of Eed and tri-meH3-K27
in all cells. (D) Scoring data showing the proportion
of Xi Eed foci with associated tri-meH3-K27 in individual XX
embryos between morula and late blastocyst. (E to
G) Scoring data for individual XX embryos between
morula and late blastocyst stage, showing association of Xi
Eed foci with hypoacetylation of (E) H3-K9, loss of
dimethylation at (F) H3-K4, and (G) hypoacetylation of H4.
Optical sections illustrating Eed (green) foci and absence of
histone modification (red) are provided under each scatter
plot. [View
Larger Version of this Image (35K GIF file)]
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Taken together, our results show that paternally imprinted X
inactivation is established in all cells in early XX
preimplantation embryos, implying that there is a
subsequent reactivation event in cells of the ICM.
Because Xp Xist expression is progressively
extinguished in ICM cells during blastocyst maturation (6,
7),
we infer that this may be the mechanism of X reactivation.
This contrasts with differentiated somatic cells in which
maintenance of X inactivation is Xist-independent
(14,
15).
Analysis of Eed-Ezh2 and H3-K27 methylation supports this
interpretation. First, using immunoRNA-FISH (fluorescence
in situ hybridization) analysis, we found that Eed-Ezh2
complexes dissociate rapidly from Xi as presumptive ICM
cells begin to extinguish Xp Xist RNA expression
(Fig.
3A). However, loss of tri-meH3-K27 staining on Xi
disappeared more gradually. Thus, immunostaining for both
Eed and meH3-K27 revealed cells in which Eed foci were
lost, but meH3-K27 staining was still clearly detectable
(Fig.
3B). In all instances, cells showing this pattern
also had high overall levels of Eed staining in the
nucleus, indicating that they represent ICM cells (Fig.
1B). In the ICM of mature blastocysts, both Eed and
meH3-K27 staining of Xi were no longer detectable (Fig.
3C). At 5.5 days post coitum (dpc), as epiblast cells
begin to undergo random X inactivation, localization of
Eed-Ezh2 and associated tri-meH3-K27 were again
detectable (Fig.
3D). This sequential loss and reestablishment—first
of Eed-Ezh2 localization and then of
tri-meH3-K27—provides a direct illustration of X
reactivation in ICM cells.
Fig. 3. Reactivation of Xp
during ICM maturation. (A) Single optical section of
ICM region of early XX blastocyst illustrating immunoRNA-FISH
detection of Xist RNA (red) and Ezh2 (green).
Counterstained with 4',6'-diamidino-2-phenylindole (DAPI)
(blue). Arrows indicate an example of a single cell where
Xist RNA has been down-regulated [appearance of two
pinpoint Xist RNA signals (6)]
as well as the absence of Ezh2 localization. (B)
Optical section through a mid-stage XX blastocyst,
highlighting two cells (shown by arrows a and b; insets,
expanded view), where Eed (red) foci are absent but
tri-meH3-K27 (green) on Xi is still detectable. (C)
Optical section through a mature (4.5 dpc) XX blastocyst
illustrating that ICM cells have high Eed and meH3-K27 levels
but no Xi foci. (D) Single optical section through a
5.5-dpc XX embryo illustrating reappearance of tri-meH3-K27
and Eed foci in the embryonic ectoderm region (EE), where
random X inactivation is being initiated. Strong foci are seen
in the extraembryonic ectoderm (EXE), where imprinted X
inactivation has been maintained. [View
Larger Version of this Image (46K GIF file)]
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In light of evidence that chromosome-wide features of X
inactivation are established in all cells of early
embryos, we were interested to investigate allelic
expression of individual X-linked genes. Embryos were
obtained from crosses between Mus musculus domesticus
and PGK strains, providing expressed single-nucleotide
polymorphisms for quantitative allele specific assays (16).
For early blastocyst and subsequent stages, XX and XY
embryo pools were separated using a green fluorescent
protein (GFP) transgene inherited on the paternal X
chromosome (fig. S3A).
We first analyzed Xist, which is expressed only from Xi.
Consistent with previous studies, expression was
exclusively from Xp during preimplantation development,
with up-regulation of the Xm allele first detectable at
the onset of random X inactivation at 5.5 to 6.5 dpc (Fig.
4A).
Fig. 4. Gene expression in early
embryogenesis. Quantitative allelic expression assays for
embryo stages from 2.5 to 6.5 dpc for (A)
Xist,(B) Pgk-1, and (C)
Smc1l1. The 3.5- to 6.5-dpc samples were from sexed
(XX) embryos. Samplese from 6.5 dpc represent embryonic
ectoderm regions of individual XX embryos. Examples show
duplicate loadings of Xp (129) and Xm (PGK) alleles for each
stage (129 denotes generic Mus musculus domesticus).
Single nucleotide primer extension (SNuPE) analysis of
polymerase chain reaction products from PGK x 129 F1 genomic DNA
(genomic) was used to normalize the data. Graphs illustrate
mean values (± standard deviation where applicable) for % of
Xm (Xist) or Xp(Pgk-1 and Smc1l1) transcripts
obtained from a number of independent experiments (table S1).
For control samples (6.5-dpc embryonic ectoderm and genomic
DNA), Xm and Xp notation does not apply. [View
Larger Version of this Image (57K GIF file)]
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We then analyzed expression of the Pgk-1 gene (Fig.
4B), which is known to be subject to X inactivation,
and also the Smc1l1 gene (Fig.
4C), which is located at the distal end of the X
chromosome and is also subject to X inactivation (Fig.
S3B). At 2.5 dpc (eight-cell to early morula stage), both
genes were biallelically expressed, consistent with
activity of Xp. However, Xp transcript levels were
relatively low, particularly at the Pgk-1 locus.
(In part, this may relate to the fact that both male and
female embryos were included in the 2.5 dpc pool.) At 3.5
and 4.5 dpc (blastocyst stage), Xp Pgk-1 levels were
further reduced to approximately 5%, whereas levels of
Smc1l1 were relatively unchanged. In
postimplantation embryos, Xp transcript levels increased
again, and in embryonic ectoderm of 6.5 dpc XX embryos
(after the onset of random X inactivation), the ratios of
Xp and Xm alleles are essentially equivalent.
Analysis of the Pgk-1 locus indicates inactivation of Xp
begins as early as the eight-cell stage. The further
marked reduction in Xp Pgk-1 RNA at the early
blastocyst stage is consistent with inactivation
occurring in all cells, as indicated by our analysis with
chromosome-wide markers. In contrast, the Smc1l1
gene shows only partial X inactivation at the blastocyst
stage, suggesting that the rate of X inactivation for
individual genes may vary across the chromosome. Similar
results, analyzing Pgk-1 and other X-linked loci,
have been reported in previous studies (2,
17),
leading to the idea that there may be a gradient of X
inactivation in early embryos, centered on the Xist
locus.
In summary, our results demonstrate that imprinted X
inactivation of the paternal X chromosome, assayed at the
level of chromosome-wide histone modifications and also
repression of at least some loci, occurs in all cells of
early mouse embryos, and that it is then reversed
selectively in ICM cells after extinction of Xp Xist
RNA expression. This latter observation indicates that
heritability of X inactivation in early embryos requires
ongoing Xist RNA expression, unlike XX somatic
cells in which loss of Xist has little or no
effect (14,
15).
Reversible Xist-dependent silencing has also been
reported to occur in response to inducible Xist
transgene expression in undifferentiated ES cells (18).
Thus, our findings provide an in vivo corollary for this
observation.
Reversibility of facultative heterochromatin in early
embryos and ES cells is mirrored in the capacity of these
cell types to reactivate the X chromosome in a somatic
cell nucleus in ES cell fusion hybrids (19)
or after nuclear transfer (20).
Indeed, our results help to understand these findings.
First, repression of Xp Xist occurs specifically
in Nanog-positive cells at the time they are first
allocated, suggesting that this is a property inherent to
the pluripotent ICM lineage. The same activity in ES
cells could result in repression of the somatic Xi
Xist allele in ES-somatic cell hybrids. This then
would lead to X reactivation in the ES nuclear environment,
where heritability of X inactivation is strictly
Xist-dependent. In the case of nuclear transfer,
the Xi from the donor somatic cell is also the Xi in TE
and PE lineages, but random X inactivation occurs in the
embryo proper (20).
This would be explained again if repression of
Xist occurs specifically in ICM cells. TE and PE
lineages would inactivate in response to maintained expression
of the somatic Xi Xist allele, whereas ICM cells would
repress Xist, establishing the ground state for
random X inactivation in the embryo proper.
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We thank colleagues for helpful
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This work was supported by the Medical Research Council, UK. |
Supporting Online Material
www.sciencemag.org/cgi/content/full/303/5658/666/DC1
Materials and Methods
Figs. S1 to S3
Table S1
References
17 October 2003; accepted 29 December
2003 10.1126/science.1092674 Include this information when
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Volume 303, Number 5658, Issue of 30 Jan 2004, pp. 666-669.
Copyright © 2004 by The American Association for the
Advancement of Science. All rights reserved.
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