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Origin of cellular asymmetries in the preimplantation mouse embryo: a hypothesis
Katsuyoshi Takaoka1,2 and Hiroshi Hamada1,2
rstb.royalsocietypublishing.org
Opinion piece
Cite this article: Takaoka K, Hamada H. 2014
Origin of cellular asymmetries in the preimplantation mouse embryo: a hypothesis.
Phil. Trans. R. Soc. B 369: 20130536.
http://dx.doi.org/10.1098/rstb.2013.0536
One contribution of 14 to a Theme Issue ‘From
pluripotency to differentiation: laying the
foundations for the body pattern in the
mouse embryo’.
Subject Areas:
developmental biology, cellular biology
Keywords:
cell fate, epigenetic, mouse embryo,
pre-implantation
Author for correspondence:
Hiroshi Hamada
e-mail: [email protected]
1
Developmental Genetics Group, Graduate School of Frontier Biosciences, and 2CREST, Japan Science and
Technology Corporation (JST), Osaka University, 1–3 Yamada-oka, Suita, Osaka 565-0871, Japan
The first cell fate decision during mouse development concerns whether a blastomere will contribute to the inner cell mass (ICM; which gives rise to the
embryo proper) or to trophectoderm (TE; which gives rise to the placenta).
The position of a cell within an 8- to 16-cell-stage embryo correlates with its
future fate, with outer cells contributing to TE and inner cells to the ICM. It
remains unknown, however, whether an earlier pre-pattern exists. Here, we
propose a hypothesis that could account for generation of such a prepattern and which is based on epigenetic asymmetry (such as in histone or
DNA methylation) between maternal and paternal genomes in the zygote.
1. Introduction: the first cell fate decision
The first cell fate specification during mouse development relates to the decision
whether a blastomere will contribute to the inner cell mass (ICM) or trophectoderm
(TE). The precise timing of and mechanism underlying this decision are fundamental aspects of early development, but they remain unknown. Asymmetric cell
division that occurs at the 8- to 16-cell stage inevitably generates two types of
cell: polar cells located outside of the embryo proper and apolar cells located
inside. Outer polar cells eventually become TE, whereas inner apolar cells will contribute to the ICM. Polar and apolar cells have intrinsic differences that establish the
outer–inner configuration [1,2]. The position of a cell within the embryo therefore
determines its future fate. An important question then arises as to whether the
mouse embryo is pre-patterned or not before the position-dependent cell specification that occurs after compaction. If no earlier pre-patterning exists, then the first
cell fate decision would be based solely on the differential positioning of cells
on the inside or outside of the embryo at the 8- to 16-cell stage [2,3]. Theoretically,
symmetry breaking can be initiated without a pre-patterning molecule by
stochastic disturbance of a self-organizing system, as previously proposed [4].
Alternatively, recent studies suggest the possibility that an earlier pre-pattern
may indeed exist in the mouse embryo [5]. Drosophila has been found to have a
pre-pattern in oocytes and zygotes. Several molecules that act as maternal determinants of the future body axis (for example, bicoid mRNA) are thus localized
asymmetrically in both oocytes and fertilized embryos of Drosophila [6]. No
such molecule has been identified in the mouse oocyte or embryo, although certain molecular and structural asymmetries have been described in unfertilized
mouse oocytes. For example, Par4 protein is asymmetrically localized in mouse
oocytes [7], but whether this protein is unevenly distributed among blastomeres
after fertilization remains unknown. In addition, transcriptome asymmetry in
terms of mRNA regionalization within mouse oocytes and zygotes has been
identified, but no such asymmetry was detected between blastomeres within
2- or 3-cell-stage embryos [8]. However, several recent studies suggest molecular
differences exist between blastomeres of the mouse embryo as early as the
4-cell stage. These molecular heterogeneities include the level of H3R17me2
and H3R26me2 (dimethylation of histone H3 at arginines 17 and 26, respectively)
[9] and the level of Cdx2 expression [10]. The notion that differences between
blastomeres arise as early as the 4-cell stage is supported by cell fate studies.
For example, the orientation and the order of cell division from the 2- to the
4-cell stage influence the fate of a blastomere [11]. Furthermore, genetic labelling
of cells at the 4-cell stage by the rainbow technique shows the presence of
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
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Two recent studies have revealed epigenetic differences
among blastomeres as early as the 4-cell stage, suggesting
that the onset of the first cell fate decision may take place
earlier than the 8- to 16-cell stage. First, global levels of
H3R17me2 and H3R26me2 were found to differ among blastomeres of 4-cell-stage embryos that have undergone
tetrahedral division patterns [9]. Those cells showing higher
levels of H3R17me2 and H3R26me2 preferentially contributed to the ICM, whereas those with lower levels were
biased to become TE. When an expression vector for Carm1
(the enzyme that mediates the transfer of methyl groups to
arginine residues, including H3R17 and H3R26) was injected
into one blastomere of a 2-cell embryo, the injected cell
exhibited upregulation of Nanog and Sox2 expression and
preferentially contributed to the ICM [13].
Another study reporting an epigenetic difference among
blastomeres was concerned with the behaviour of Oct3/4, a
key transcription factor required for pluripotency [14]. The
intracellular behaviour of Oct3/4 was examined by monitoring the kinetics of nuclear export, import, and retention of a
green fluorescent protein (GFP)::Oct3/4 fusion protein in
embryos at the 4- and 8-cell stages. Two types of Oct3/4
behaviour were observed, probably reflecting differences in
the access of this transcription factor to its binding sites
in the genome. One type of behaviour was characterized by
low rates of nuclear import and export as well as by immobility of Oct3/4 ( presumably as a result of Oct3/4 being
tightly bound to its target sites). Cells that manifested such
behaviour underwent asymmetric cell division, generating
inside cells and outside cells, and they preferentially contributed to the ICM and TE. The other type of behaviour was
characterized by high Oct3/4 mobility (Oct3/4 is presumably
not bound to its target sites in these cells). Cells manifesting
this pattern of behaviour underwent symmetric cell division,
generating only outside cells, and they showed a biased
contribution to TE.
Several important questions remain to be addressed. For
example, are these two epigenetic differences—in the levels
of H3R17me2 and H3R26me2 at the 4-cell stage and in
Oct3/4 behaviour at the 4- and 8-cell stages—related to
each other? How might low Oct3/4 mobility lead to asymmetric division? When and how is differential histone
modification (H3R17me2 and H3R26me2) generated among
blastomeres? If Carm1 is the only enzyme that methylates
arginine residues, we need to know its expression pattern
3. Epigenetic asymmetries between maternal
and paternal genomes
During gametogenesis and preimplantation development,
epigenetic asymmetry is apparent between maternal and
paternal genomes. Sperm DNA is tightly associated with protamines, but on fertilization, these are replaced by histones
that are acetylated, but not methylated, at H3K9 or H3K27.
By contrast, DNA and associated histones (such as H3) in
the oocyte are highly methylated. Histone methyltransferase
activity present in oocytes is responsible for H3 methylation
in these cells. On fertilization, the maternal genome exists
in a nucleosomal configuration with histone methylation
inherited from the oocyte.
Differential histone modification between parental
genomes in the preimplantation embryo includes monomethylation of histone H3 at lysine 9 (H3K9me) and H3
trimethylation at lysine 27 (H3K27me3). Given that the
maternal genome of the oocyte contains histone H3 methylated
at K9, whereas the paternal genome of the sperm does not
[16,17], fertilization gives rise to an asymmetry in H3K9me
between parental genomes, and this asymmetry persists at
the 2-cell stage. De novo methylation of H3K9 occurs in both
parental genomes at the 4-cell stage, however, which erases
the asymmetry in H3K9me by the 8-cell stage. As described
above, the level of H3R26me2 also exhibits asymmetry
among blastomeres of the 4-cell-stage embryo, being high in
those blastomeres fated to contribute to the ICM but low
in those fated to become mural TE [9]. The origin of this asymmetry in H3R26me2 is unknown, however. It may be generated
by differential de novo methylation or demethylation after
fertilization, or it may hark back to differential methylation
between parental genomes.
Parental genomes also show different levels of DNA
methylation [17,18]. The paternal genome in the male pronucleus undergoes rapid demethylation after fertilization, with
DNA methylation being largely lost before the first mitosis.
This active demethylation is mediated by Tet3, a dioxygenase
that converts 5-methlycytosine to 5-hydroxymethlycytosine
and which is present specifically in the male pronucleus [19].
By contrast, the maternal genome in the female pronucleus
remains highly methylated until cleavage stages, when methylation is passively lost over the entire genome. This loss of DNA
methylation is followed by active methylation throughout the
genome during the blastocyst stage. The pattern of DNA
methylation thus changes rapidly between fertilization and
the blastocyst stage, but asymmetry in this pattern exists
between the male and female pronuclei.
How is the paternal genome specifically demethylated by
Tet3, whereas the maternal genome is protected from Tet3mediated demethylation? Whereas Tet3 mRNA is abundant
in oocytes and zygotes, Tet3 protein is specifically enriched
in the male pronucleus (it is localized to the cytoplasm at
later preimplantation stages). How then is Tet3 protein
excluded from the female pronucleus? PGC7 is required for
2
Phil. Trans. R. Soc. B 369: 20130536
2. Epigenetic differences among early
blastomeres: a sign of a pre-pattern?
and distribution in oocytes, zygotes and cleavage-stage
embryos. Zygotic Carm1 mutants develop normally until
late embryonic stages [15]. However, Carm1 may function
as a maternal factor, so it will be interesting to characterize
embryos of maternal Carm1 knockout mice. Some of these
questions can be addressed with current technology.
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individuals that will contribute either to ICM or TE, in a
significant number of embryos [12].
Given that the molecular mechanism for the positiondependent cell specification in mouse embryos has been
extensively studied and discussed elsewhere, we address, in
this paper, the origin of pre-patterning, based on the assumption that the mouse embryo is indeed pre-patterned before
the position-dependent cell specification (while a growing
number of studies suggest the presence of pre-pattern in the
mouse embryo, one cannot completely exclude an alternative
possibility. Therefore, in this paper, we would like to maintain that the presence of pre-pattern in the mouse embryo is a
strong possibility).
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zygotic expression
begins
3
P
M
Figure 1. Hypothetical model for generation of an early embryonic pre-pattern based on epigenetic asymmetry between parental genomes. The maternal (M)
genome and its derivatives are shown in orange, whereas the paternal (P) genome and its derivatives are shown in blue. Asymmetric modification of parental
genomes (either DNA modification or histone modification) is represented by red dots on the maternal chromosome. In this model, asymmetric modification is
not inherited by a daughter chromosome after cell division. At the 4-cell stage, de novo modification (black dots) occurs equally on maternally and paternally
derived genomes. Given that zygotic transcription begins at the 2-cell stage, cells with the maternally derived modification (red dots) at the 2- and
4-cell stages express unknown regulatory genes (green) that will influence the outer – inner position and ICM– TE fate decision at later stages. The second
cell from the top at the 4-cell stage contains a low level of the gene products (light green) inherited from its mother cell, which may be sufficient to express
regulatory genes. (Online version in colour.)
this exclusion of Tet3 protein given that the latter is present in
both pronuclei of PGC7-deficient zygotes [20,21]. PGC7 is
thought to prevent recruitment of Tet3 to the female pronucleus
by binding to H3K9me2, a type of histone modification present
in the maternal genome, but absent from the paternal genome
[20]. If this is the case, then DNA methylation and histone
methylation are interconnected and may regulate each other.
Does parental epigenetic asymmetry influence cell fate
specification, in particular the decision to contribute to the
ICM or TE? There is currently no direct evidence to support
such an influence. PGC7 acts as a maternal factor essential
for preimplantation development [22,23]. Embryos lacking
maternal PGC7 rarely reach the blastocyst stage, with most
remaining at the 1-cell, 2-cell or morula stage [23], making
it difficult to examine whether the ICM –TE decision is
impaired in such mutant embryos. Many genes responsible
for the regulation of H3 methylation have been identified,
but their roles in preimplantation development and cell fate
specification have not been fully investigated. The histone
methyltransferases Suv39h1 and Suv39h2 are responsible for
parental asymmetry in H3K9me3 [24]. Interestingly, female
zygotic null mutants for Suv39h1 or Suv39h2 are infertile
[25]. The methyltransferases Prdm3 and Prdm16 are implicated in parental asymmetry of H3K9me [26]. Zygotic null
mutants for Prdm3 or Prdm16 do not survive beyond the late
gestation stage or manifest hematopoietic and immune defects
at the adult stage, respectively. The maternal role of these
proteins remains unknown, however.
4. A hypothesis: epigenetic asymmetry
between parental genomes can account
for pre-patterning
Epigenetic asymmetry between maternal and paternal genomes could theoretically generate an early pre-pattern. We
here propose a hypothetical model for generation of epigenetic and transcriptional differences among blastomeres as
early as the 2-cell stage (figure 1). In this model, the two
parental genomes exhibit asymmetric modification (either
DNA or histone methylation), with this modification existing in the maternal genome at the 1-cell stage. This
modification is maintained on the original chromosome but
is not inherited by a daughter chromosome after cell division.
Because zygotic transcription begins at the 2-cell stage, only
the blastomere that has inherited the modified maternal
genome will express at this stage an unknown regulatory
gene (or genes) that will influence the later ICM– TE fate
decision. Given a role for the plane of cell division at the
2-cell stage [11], blastomeres derived from the vegetal or
animal pole may preferentially inherit genomes in the
defined epigenetic states. At the 4-cell stage, de novo modification occurs equally on maternally and paternally derived
genomes, which may or may not erase the epigenetic asymmetry. Validation of this model will require investigation of
the maternal role of genes involved in asymmetric epigenetic
modification as well as identification of specific genes that are
Phil. Trans. R. Soc. B 369: 20130536
TE
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ICM
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regulated by asymmetric epigenetic modification and
influence the first cell fate specification.
The position of a cell within the mouse embryo at the 8- to 16cell stage determines its fate. Although it is unknown whether
an earlier pre-pattern contributes to the first cell fate decision,
epigenetic differences among blastomeres probably exist as
early as the 2- or 4-cell stage. Such epigenetic differences
among blastomeres could be generated by asymmetry in
Acknowledgements. We thank members of our laboratory and anonymous reviewers for their comments on the manuscript.
Funding statement. The work performed in our laboratory was supported by a grant from CREST (Core Research for Evolutional
Science and Technology) of the Japan Science and Technology Corporation and by a grant-in-aid from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
Conflict of interests. We have no conflict of interests.
2.
3.
4.
5.
6.
7.
8.
9.
Anani S, Bhat S, Honma-Yamanaka N, Krawchuk D,
Yamanaka Y. 2014 Initiation of Hippo signaling is
linked to polarity rather than to cell position in the
pre-implantation mouse embryo. Development 141,
2813–2824. (doi:10.1242/dev.107276)
Johnson MH, McConnell JML. 2004 Lineage
allocation and cell polarity during mouse
embryogenesis. Semin. Cell Dev. Biol. 15, 583–597.
(doi:10.1016/j.semcdb.2004.04.002)
Tarkowsk AK, Wróblewska J. 1967 Development of
blastomeres of mouse eggs isolated at 4- and 8-cell
stage. J. Embryol. Exp. Morphol. 18, 155–180.
Wennekamp S, Mesecke S, Nédélec F, Hiiragi T. 2013
A self-organization framework for symmetry
breaking in the mammalian embryo. Nat. Rev. Mol.
Cell Biol. 14, 452–459. (doi:10.1038/nrm3602)
Zernicka-Goetz M. 2013 Development: do mouse
embryos play dice? Curr. Biol. 23, R15–R17.
(doi:10.1016/j.cub.2012.10.032)
St Johnston D. 2005 Moving messages: the
intracellular localization of mRNAs. Nat. Rev. Mol.
Cell Biol. 6, 363–375. (doi:10.1038/nrm1643)
Szczepanska K, Maleszewski M. 2005 LKB1/PAR4 protein
is asymmetrically localized in mouse oocytes and
associates with meiotic spindle. Gene Express. Patterns
6, 86–93. (doi:10.1016/j.modgep.2005.04.013)
VerMilyea MD, Maneck M, Yoshida N, Blochberger I,
Suzuki E, Suzuki T, Spang R, Klein CA, Perry ACF.
2011 Transcriptome asymmetry within mouse
zygotes but not between early embryonic sister
blastomeres. EMBO J. 30, 1841 –1851. (doi:10.
1038/emboj.2011.92)
Torres-Padilla ME, Parfitt D-E, Kouzarides T,
Zernicka-Goetz M. 2007 Histone arginine
methylation regulates pluripotency in the early
mouse embryo. Nature 445, 214–218. (doi:10.
1038/nature05458)
10. Jedrusik A, Parfitt D-E, Guo G, Skamagki M,
Grabarek JB, Johnson MH, Robson P, Zernicka-Goetz
M. 2008 Role of Cdx2 and cell polarity in cell
allocation and specification of trophectoderm and
inner cell mass in the mouse embryo. Genes Dev.
22, 2692–2706. (doi:10.1101/gad.486108)
11. Bischoff M, Parfitt D-E, Zernicka-Goetz M. 2008
Formation of the embryonic–abembryonic axis of
the mouse blastocyst: relationships between
orientation of early cleavage divisions and pattern
of symmetric/asymmetric divisions. Development
135, 953 –962. (doi:10.1242/dev.014316)
12. Tabansky I et al. 2013 Developmental bias in
cleavage-stage mouse blastomeres. Curr. Biol. 23,
21 –31. (doi:10.1016/j.cub.2012.10.054)
13. Parfitt DE, Zernicka-Goetz M. 2010 Epigenetic
modification affecting expression of cell polarity and
cell fate genes to regulate lineage specification in
the early mouse embryo. Mol. Biol. Cell 21,
2649 –2660. (doi:10.1091/mbc.E10-01-0053)
14. Plachta N, Bollenbach T, Pease S, Fraser SE, Pantazis
P. 2011 Oct4 kinetics predict cell lineage patterning
in the early mammalian embryo. Nat. Cell Biol. 13,
337. (doi:10.1038/ncb0311-337)
15. Yadav N, Lee J, Kim J, Shen J, Hu MCT, Aldaz CM,
Bedford MT. 2003 Specific protein methylation
defects and gene expression perturbations in
coactivator-associated arginine methyltransferase
1-deficient mice. Proc. Natl Acad. Sci. USA 100,
6464 –6468.
16. Liu HL, Kim J-M, Aoki F. 2004 Regulation of histone
H3 lysine 9 methylation in oocytes and early preimplantation embryos. Development 131,
2269 –2280. (doi:10.1242/dev.01116)
17. Reik W, Santos F, Mitsuya K, Morgan H, Dean W.
2003 Epigenetic asymmetry in the mammalian
zygote and early embryo: relationship to lineage
18.
19.
20.
21.
22.
23.
24.
25.
26.
commitment? Phil. Trans. R. Soc. B 358,
1403– 1409. (doi:10.1098/rstb.2003.1326)
Shi LJ, Wu J. 2009 Epigenetic regulation in
mammalian preimplantation embryo development.
Reprod. Biol. Endocrinol. 7, 59. (doi:10.1186/14777827-7-59)
Gu TP et al. 2011 The role of Tet3 DNA dioxygenase
in epigenetic reprogramming by oocytes. Nature
477, 606. (doi:10.1038/nature10443)
Nakamura T et al. 2012 PGC7 binds histone
H3K9me2 to protect against conversion of 5mC to
5hmC in early embryos. Nature 486, 415–419.
(doi:10.1038/nature11093)
Nakamura T et al. 2007 PGC7/Stella protects against
DNA demethylation in early embryogenesis. Nat.
Cell Biol. 9, 64 –71. (doi:10.1038/ncb1519)
Bortvin A, Goodheart M, Liao M, Page DC. 2004
Dppa3/Pgc7/stella is a maternal factor and is not
required for germ cell specification in mice. BMC
Dev. Biol. 4, 2. (doi:10.1186/1471-213X-4-2)
Payer B et al. 2003 stella is a maternal effect gene
required for normal early development in mice.
Curr. Biol. 13, 2110–2117. (doi:10.1016/j.cub.2003.
11.026)
Puschendorf M et al. 2008 PRC1 and Suv39h specify
parental asymmetry at constitutive heterochromatin
in early mouse embryos. Nat. Genet. 40, 411 –420.
(doi:10.1038/ng.99)
Peters AH et al. 2001 Loss of the Suv39h
histone methyltransferases impairs mammalian
heterochromatin and genome stability. Cell
107, 323–337. (doi:10.1016/S0092-8674(01)
00542-6)
Pinheiro I et al. 2012 Prdm3 and Prdm16 are
H3K9me1 methyltransferases required for
mammalian heterochromatin integrity. Cell 150,
948–960. (doi:10.1016/j.cell.2012.06.048)
Phil. Trans. R. Soc. B 369: 20130536
References
1.
4
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5. Concluding remarks
histone or DNA modification between maternal and paternal
genomes. Verification of a role for such asymmetry in
embryonic pre-patterning awaits further study.