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First published online 13 June 2007
doi: 10.1242/dev.005611
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1 Laboratory for Mammalian Germ Cell Biology, Center for Developmental Biology,
RIKEN Kobe Institute, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe, 650-0047,
Japan.
2 Laboratory of Molecular Cell Biology and Development, Graduate School of
Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto
606-8502, Japan.
3 Department of Biosystems Science, Graduate School of Science and Technology,
Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan.
4 Cell Resource Center for Biomedical Research, Institute of Development, Aging
and Cancer, Tohoku University, 4-1 Seiryo-cho, Aoba-ku, Sendai 980-8575,
Japan.
5 Department of Genetics and Division of Mammalian Development, National
Institute of Genetics, SOKENDAI, 1111 Yata, Mishima, Shizuoka 411-8540,
Japan.
6 Department of Cell Biology, Institute for Virus Research, Kyoto University,
Shogoin Kawara-cho, Kyoto 606-8507, Japan.
7 Precursory Research for Embryonic Science and Technology, Japan Science and
Technology Agency, 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan.
* Author for correspondence (e-mail: saitou{at}cdb.riken.jp)
Accepted 10 May 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Primordial germ cells (PGCs), Histone modifications, Reprogramming, Cell cycle, Epigenetics, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and covalent
modifications of histone N-terminal tails
(Martin and Zhang, 2005;
Peters and Schubeler, 2005),
which are often mitotically heritable. Thus, each cell type in our body
acquires a specific and stable epigenetic signature, often referred to as
`cellular memory'. The genome of the germ cell lineage, the sole source for
the next generation, however, must be maintained in an epigenetically
`reprogrammable' state in order for the creation of new generations to
continue. Therefore, the germline, from its outset, must be endowed with a
certain mechanism to ensure this crucial function.
There are essentially two modes, which are known as `preformation' and
`epigenesis', respectively, for the specification of germ cell fate during the
development of multicellular organisms
(Extavour and Akam, 2003). The
preformation, which is seen in model organisms such as Caenorhabditis
elegans and Drosophila melanogaster, involves localized maternal
determinants present in the egg, often referred to as the germ plasm, for the
specification of germ cell fate. The molecular components of the germ plasm
and the epigenetic properties of the early germline cells that inherit it have
been analyzed, leading to a general concept that the germ plasm imposes
transient yet robust transcriptional quiescence on the genome at the outset of
the germ cell lineage with appropriate epigenetic modifications
(Seydoux and Braun, 2006).
By contrast, in the `epigenesis', which is seen in many organisms,
including mammals, a potentially equivalent population of cells at a
relatively late stage of development is induced to form either germ cells or
somatic mesoderm in response to signaling molecules from adjacent tissues
(Lawson et al., 1999;
McLaren, 2003;
Ohinata et al., 2006). This
implies that cells recruited for the germline may have to undergo `epigenetic
reprogramming' from a somatic to a potentially totipotent germline phenotype.
Blimp1 (Prdm1 - Mouse Genome Informatics), a potent transcriptional repressor
with a PR domain and five zinc fingers, which is initially expressed in a few
cells of the most proximal epiblast cells at embryonic day (E) 6.25, has been
identified as a crucial requirement for the birth of this lineage
(Ohinata et al., 2005).
Blimp1-positive lineage-restricted precursors of PGCs increase in
number and exclusively go on to form stella (Pgc7, Dppa3)-positive
founder PGCs at around E7.25 that repress the somatic mesodermal program,
including Hox gene expression, and reactivate the pluripotency-associated gene
network (Ohinata et al., 2005;
Saitou et al., 2002;
Sato et al., 2002;
Yabuta et al., 2006).
Subsequently, from around E7.5 onwards, these cells initiate the migration
towards future genital ridges through the visceral and definitive hindgut
endoderm.
There has been a dearth of information and investigation on what is
happening at the cellular and molecular levels in the migrating PGCs, although
it has been established that PGCs undergo extensive epigenetic reprogramming,
including erasure of parental imprints and reactivation of inactive X
chromosome, when they colonize the genital ridges
(Li, 2002;
McLaren, 2003;
Surani, 2001). Our previous
study demonstrated that PGCs have already initiated a genome-wide epigenetic
reprogramming in their migration period
(Seki et al., 2005); they
undergo a significant loss of both DNA methylation and H3K9me2, two repressive
modifications with higher stability, from their genome at around E8.0 and
instead acquire high levels of H3K27me3, another repressive modification with
apparent plasticity, at around E9.0, suggesting that this reprogramming might
be essential for the potential totipotency of PGCs. However, the mechanisms of
this epigenetic reprogramming are unclear, and their clarification could
provide essential information on the reprogramming of somatic cell nuclei
generally. In this report, we provide a precise analysis of the cellular
dynamics associated with the epigenetic reprogramming in migrating PGCs.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Tachibana et al., 2005). The
rabbit anti-stella antibody was generated by GST fusion of the N-terminal half
of stella as an antigen, affinity-purified, and used at a dilution of
1:1000. The following secondary antibodies from Molecular Probes were used at a 1/500 dilution: Alexa Fluor 488 goat anti-rat IgG; Alexa Fluor 488 anti-rabbit IgG; Alexa Fluor 568 anti-rabbit IgG; Alexa Fluor 568 anti-mouse IgG; Alexa Fluor 568 anti-mouse IgM; and Alexa Fluor 633 anti-rabbit IgG.
Embryo isolation and staging
All the animals were treated with appropriate care according to the RIKEN
ethics guidelines. Embryos were isolated in Dulbecco's Modified Eagle's Medium
(DMEM) (Invitrogen) supplemented with 10% FCS (Stem Cell Science). Noon of the
day when the vaginal plugs of mated females were identified was scored as
E0.5. For a more accurate staging, embryos younger than E8.0 were classified
according to the morphological landmarks
(Downs and Davies, 1993), and
we corresponded embryonic days to embryonic stages as follows: E6.5, early
streak (ES); E6.75, mid streak (MS); E7.0, late streak (LS); E7.25, early bud
(EB); E7.5, late bud (LB); E7.75, early head fold (EHF); E8.0, late head fold
(LHF). Embryos older than E8.25 were staged according to the somite numbers
(St) as follows: E8.25, 2-4 St; E8.5, 6-8 St; E8.75, 10-12 St; E9.0, 14-16 St;
E9.25, 18-20 St; E9.5, 22-24 St; E9.75, 26-28 St; E10.0, 30-32 St; E10.25,
34-36 St; E10.5, 38-40 St.
Transgenic animals and Nanos3 knockout mice
Blimp1-mEGFP transgenic mice were generated as described
previously (Ohinata et al.,
2005), and backcrossed to C57Bl/6 background at least five times.
stella-EGFP transgenic mice on a C57Bl/6 background were generated by
pronuclear injection of a bacterial artificial chromosome bearing the stella
locus with the EGFP sequence fused to the end of the stella sequence
(Payer et al., 2006) (a kind
gift of Dr M. Azim Surani, Gurdon Institute, Cambridge, UK). Nanos3
knockout mice (Tsuda et al.,
2003) were maintained on a C57Bl/6/DBA background.
Whole-mount immunohistochemistry and confocal microscopic analyses
Female CD1 mice were mated with male Blimp1-mEGFP or stella-EGFP
transgenic lines, and were sacrificed at the appropriate stages to recover
embryos. Embryonic fragments from extra-embryonic mesoderm (E7.0), the base of
allantois (E7.25-8.0), hindgut epithelium (E8.25-9.25), hindgut epithelium and
mesentery (E9.5-10.0), and urogenital ridges (E10.25-10.5) were dissected out
and cut into smaller pieces to facilitate antibody permeabilization. They were
washed with PBS containing 0.1% BSA, fixed in 4% paraformaldehyde (PFA) in PBS
at 4°C for 30 minutes and subjected to three 15 minute washes with PBS
containing 1% Triton-X (PBS-T). The fragments were incubated with primary
antibodies in PBS-T containing 1 mg/ml BSA for 4 days at 4°C, washed with
PBS-T, incubated with the Alexa Fluor secondary antibodies in PBS-T containing
1 mg/ml BSA and 1 µg/ml Hoechst 33342 for 2 days at 4°C, washed with
PBS-Triton, mounted in Vectashield (Vector Laboratories) and observed under a
confocal microscope (Zeiss or Olympus). Immunofluorescence images were
acquired through the z-axis of the fragments at an interval of 4
µm.
PGC counts
To count the PGC number, Blimp1-mEGFP and stella-EGFP
transgenic embryos (C57Bl/6/CD1 F1) were used for E7.0-7.75 and for E8.0-10.5,
respectively. Embryonic fragments were collected as described above and used
for immunofluorescence staining with the anti-GFP antibody. All the fragments
were processed for confocal analysis as described above.
BrdU labeling
We used a BrdU labeling and detection Kit (Roche) for analyzing the
incorporation of Bromo-deoxyuridine (BrdU) into PGCs. Pregnant females mated
either with Blimp1-mEGFP or stella-EGFP males were injected
intraperitoneally with BrdU (10 mg/kg body weight) at appropriate stages.
After 6 hours, the embryos were isolated and fragments containing PGCs were
dissected out, cut into smaller pieces, fixed with 70% EtOH in 50 mM glycine
for 4 hours and washed in PBS. These pieces were incubated with anti-BrdU and
anti-GFP antibodies in incubation buffer at 37°C for 3 hours, washed with
PBS, incubated with the Alexa Fluor 568 secondary antibodies in PBS containing
1 µg/ml Hoechst 33342 at 4°C for 2 hours, and then washed again with
PBS, mounted in Vectashield and observed under a confocal microscope.
BrUTP labeling
At E8.75, hindgut epithelia containing PGCs were isolated, incubated with
0.25% trypsin, 0.5 mM EDTA in PBS for 10 minutes and dissociated into single
cells by pipetting. Dissociated cells were allowed to adhere to slides by
incubating in DMEM supplemented with 10% FCS at room temperature for 20
minutes. Cells were then incubated with Tris-glycerol buffer [20 mM Tris-HCl,
pH 7.4, 5 mM MgCl2, 25% glycerol, 0.5 mM
phenylmethylsulphonylfluoride (PMSF), 2 mM 4-(2-aminoethyl) benzenesulfonyl
fluoride hydrochloride (AEBSF)], permeabilized in the same buffer with 5
µg/ml digitonin at room temperature for 3 minutes, and then incubated with
transcription buffer [50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.5 mM
EGTA, 100 mM KCl, 25% glycerol, 0.025 mM S-adenosyl methionine, 0.75 mM BrUTP,
0.5 mM PMSF, 25 U/ml (1 µl) RNasin, 0.5 mM each nucleotide triphosphate, 4
mM AEBSF] for 30 minutes at 37°C. At the end of the transcription
reaction, cells were gently rinsed with PBS, fixed in 4% paraformaldehyde in
PBS and processed for immunofluorescence staining with anti-BrUTP and stella
antibodies.
Fluorescence-activated cell sorting (FACS) analysis
Embryonic fragments containing PGCs of E7.25 Blimp1-mEGFP
(visceral endoderm removed), E7.75, 8.25, 8.75, 9.75 and 10.5 stella-EGFP
transgenic embryos were dispersed into single cells by incubating with 0.25%
trypsin, 0.5 mM EDTA in PBS and fixed with 3.7% PFA in PBS at 4°C for 30
minutes. Cells were collected by centrifugation (3000 rpm, 5 minutes), washed
in PBS containing 1% BSA, incubated with 50 µg/ml propidium iodide
(Molecular Probes), 0.005% saponin (SIGMA) and 0.25 mg/ml RNase A at 37°C
for 20 minutes and immediately analyzed on a flow cytometer (FACS Canto;
Becton Dickinson). In total, over 10,000 events were collected and gated
through doublet discrimination to analyze the DNA content of single cells.
More than 40 EGFP-positive cells were analyzed in each experiment. The numbers
of recovered EGFP-positive cells/dissected embryos were 43/5, 42/8, 61/3,
127/5, 58/2, 177/3, and 748/3 for E7.25, 7.75, 8.25, 8.75, 9.25, 9.75 and
10.5, respectively.
Single-cell cDNA analysis
Single-cell cDNAs from EB and EHF stage embryos were generated, and
real-time quantitative PCR (Q-PCR) analyses were performed as described
previously (Yabuta et al.,
2006). The primer sequences used are listed in
Table 1.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), we explored the dynamics of this event using both
Blimp1-mEGFP (Ohinata et al.,
2005) and stella-EGFP transgenic mice
(Payer et al., 2006), as well
as anti-stella and anti-SSEA1 (Fox et al.,
1981; Gomperts et al.,
1994) antibody.
Blimp1-positive cells gradually acquire stella protein expression
To examine the manner of stella protein expression among the
Blimp1-positive cells, we stained Blimp1-mEGFP embryos with
anti-stella antibody. Consistent with the previous findings, we first detected
that about 20% of the Blimp1-positive cells were weakly positive for
stella at the LS stage (see Materials and methods for the staging of embryos)
(see Fig. S1 in the supplementary material). At the EB stage, we detected
stella in approximately 50% of Blimp1-positive cells. The number of
stella-positive cells among Blimp1-positive cells increased
progressively, and at the EHF stage as many as 
80% of
Blimp1-positive cells showed stella expression (see Fig. S1 in the
supplementary material) (Ohinata et al.,
2005). The stella protein levels were, however, still very
divergent, with Blimp1-positive cells migrating apart from the
cluster generally bearing higher stella expression. All the stella-positive
cells were Blimp1-positive. The numbers of stella-positive cells
detected using antibodies and the stella-EGFP transgene were similar after the
EHF stage. We therefore decided to use Blimp1-mEGFP embryos for
detecting PGC precursors and PGCs mainly by the EHF stage and stella-EGFP
embryos after the LHF stage.
Chromatin modifications in lineage-restricted precursors of PGCs
We first examined the global chromatin modification states of
Blimp1-positive PGC precursors at the ES-LS stages by whole-mount
immunohistochemistry using antibodies against specific modifications of
histone N-terminal tails. We found that the staining patterns of these cells
for the modifications H3K4me2, H3K4me3, H3K9Ac, H3K9me1, H3K9me2, H3K9me3,
H3K27me2 and H3K27me3 (see Fig. S2 in the supplementary material and data not
shown) were indistinguishable from those of their somatic mesodermal
neighbors, some of which should share common precursors with the germ cell
lineage. It is of note that the lineage-restricted PGC precursors in female
embryos showed prominent accumulation of H3K27me3 in a single spot (see Fig.
S2 in the supplementary material), most likely the inactive X chromosome
(Erhardt et al., 2003;
Plath et al., 2003;
Silva et al., 2003),
indicating that the initiation of the X-inactivation process in the germline
is similar to that in the somatic lineages.
Epigenetic reprogramming occurs progressively in migrating PGCs
We next set out to determine the precise onset and the completion of the
erasure and upregulation of H3K9me2 and H3K27me3, respectively, in the
developing PGC populations after the EB stage. As shown in
Fig. 1A, PGCs showed similar
levels of genome-wide H3K9me2 compared to their somatic neighbors at the EB
stage. We first observed the PGCs with low H3K9me2 levels at the LB stage, and
at the slightly later EHF stage nearly half of the PGCs appeared to show
reduced H3K9me2 levels (Fig.
1A,C,E). Low H3K9me2 was more frequently seen in PGCs that
initiated migration than in PGCs residing in a cluster (data not shown). At
E8.25, more than 80% of the stella-positive PGCs showed low H3K9me2, and some
of them had highly reduced levels of this modification. At E8.75, nearly all
of the PGCs showed very low H3K9me2 levels
(Fig. 1E). These findings
indicate that H3K9me2 demethylation is initiated in some PGCs at around E7.5,
that the ratio of H3K9me2 low PGCs increases along with development, and that
the degree of demethylation in individual PGCs also increases along with
development, reaching highly reduced levels at around E8.75. Regarding the
upregulation of H3K27me3, we first identified stella-positive PGCs with higher
levels of H3K27me3 at around E8.25. Both the percentage of PGCs with high
levels of H3K27me3 and the degree of H3K27me3 upregulation in individual PGCs
increased, as the development of PGCs proceeded
(Fig. 2B,D,E), indicating that
H3K27me3 upregulation in PGCs proceeds in a progressive, cell-by-cell manner,
depending on their developmental maturation. We additionally found that PGCs
at around E8.75 showed low levels of not only H3K9me2 but also H3K9me1 (see
Fig. S3 in the supplementary material), indicating that the reprogramming
event in PGCs effectively erases at least the mono- and di-methylated states
of H3K9.
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). H5 recognizes phosphorylated Ser2 of the C-terminal domain
(CTD) of RNA polymerase II (RNAP II), which is tightly associated with
transcriptional elongation (Phatnani and
Greenleaf, 2006) and is a good marker for the global
transcriptional activity of a wide range of cell types. We first examined CTD
Ser2 phosphorylation in PGCs at E8.75. Contrary to our expectation, PGCs at
this stage showed extremely low levels of this modification
(Fig. 2A). This was not due to
the PGC-specific downregulation of RNAP II itself, as the levels of RNAP II
protein detected in PGCs were comparable to those detected in their somatic
neighbors by the same immunohistochemical method
(Fig. 2B). We then examined the
global distribution and the levels of H3K4me2 and H3K4me3 that were associated
with transcriptionally permissive/active states and found that the
distribution and levels of these modifications in PGCs were similar to those
in their somatic neighbors (Fig.
2C), suggesting that PGCs at E8.75 repress RNAP II-dependent
transcription by a mechanism independent of their chromatin modification
states.
|
). We found that, in contrast to their somatic neighbors, PGCs
were again essentially negative for Ser5 phosphorylation
(Fig. 2D), indicating that the
global transcription in migrating PGCs is halted at a step before the
transition from the initiation complex formation to promoter clearance. To further monitor the transcription state of PGCs, we performed BrUTP incorporation experiments to examine nascent RNA synthesis at E8.75. As shown in Fig. 2E, somatic cells negative for stella showed strong BrUTP incorporation in their nucleoplasm, whereas approximately 90% of stella-positive PGCs exhibited weak nucleoplasmic BrUTP incorporation, except for a few intensive foci that were negative for DAPI and most likely nucleoli, indicating that migrating PGCs at E8.75 specifically repress RNAP II-dependent transcription with the RNAP I-dependent ribosomal RNA transcription kept constant.
We next examined the onset and the duration of the loss of CTD Ser2 phosphorylation in PGCs (Fig. 2F). Blimp1-positive PGC precursors at the ES-LS stages showed indistinguishable staining of CTD phosphorylation compared to their somatic neighbors and epiblast cells, indicating that these cells undergo active transcription, consistent with the finding that PGC precursors showed decent levels of H3K4me2, me3 and H3K9ac (see Fig. S2 in the supplementary material). Up until the EHF stage, most PGCs showed CTD phosphorylation. However, at the LHF stage (E8.0), many PGCs lost this modification (Fig. 2F,G). The percentage of PGCs negative for CTD phosphorylation increased gradually during development, and at E8.5-8.75, most PGCs detected by stella-EGFP lacked this modification (Fig. 2F,G), as we initially observed. At E9.25, some PGCs regained this modification, and by E10.5, staining for CTD phosphorylation was apparent in almost all the PGCs (Fig. 2F,G). These findings indicate that PGCs turned off RNAP II-dependent transcription transiently and in a cell-by-cell manner during their migration period, and this was presumably achieved through a mechanism independent from chromatin-based silencing.
Relationship between chromatin remodeling and transcriptional quiescence
Our timecourse analyses demonstrated that the reduction of H3K9me2 was
first observed as early as E7.75 (EHF stage) in 54% of PGCs, whereas loss of
transcriptional activity was still not evident in most PGCs at the same stage.
By contrast, the upregulation of H3K27me3 commenced as early as E8.25, when
PGCs were progressively losing RNAP II CTD phosphorylation, which was
recovered in some PGCs only at E9.09.25. These findings indicate that a
cascade of events takes place in individual PGCs, with the reduction of
H3K9me2 occurring first, and the loss of RNAP II CTD phosphorylation second,
followed by upregulation of H3K27me27 and subsequent resumption of RNAP
II-dependent transcription. To confirm this, we performed whole-mount triple
labeling, which demonstrated that some PGCs in which the reduction of H3K9me2
had already started at E8.0 had not yet established the transcriptional
quiescence (Fig. 2H, white
arrowhead). We also found that the upregulation of genome-wide H3K27me3
preceded the restoration of RNAP II-dependent transcriptional activity in PGCs
at E8.75 (Fig. 2H, pink
arrowhead), consistent with the proposed sequence of events in PGCs. The
emergence of PGCs with hyper-H3K27me3 from H5-negative PGCs indicated that de
novo transcription driven by RNA polymerase II was not required for
hyper-H3K27me3 in PGCs.
Epigenetic reprogramming proceeds in the prolonged G2 phase of the cell cycle
We next explored the cell cycle state of the migrating PGCs when all the
observed events were apparently occurring in an ordered manner. To this end,
we first examined the increase of PGC number using Blimp1-mEGFP (from
LS to EHF stage) and stella-EGFP embryos as well as anti-stella antibody
staining (from E8.0 onwards). As shown in
Fig. 3A (see also Table S1 in
the supplementary material), although the number of PGCs varied considerably
even among embryos at similar developmental stages, the increase of overall
PGC number between E8.0 and E9.09.25, coincident with the epigenetic
reprogramming, seemed slower than that after E9.5.
We therefore performed BrdU labeling to assess the S-phase entry of PGCs at each stage of development. We injected the pregnant females with BrdU intraperitoneally and isolated their embryos 6 hours later to evaluate the incorporation of BrdU into embryonic cells. We found that Blimp1-positive PGC precursors and PGCs in isolated embryos at the LS stage showed approximately 50% BrdU incorporation (Fig. 3A,B), indicating that 50% of Blimp1-positive cells had entered into S-phase in the previous 6 hours. Subsequently, however, the percentage of the BrdU-positive Blimp1-positive cells decreased to as low as 15% at the EHF stage, and the percentage of BrdU-positive, stella-positive PGCs remained low until around E9.0 (Fig. 3A,B). By contrast, more than half of the surrounding somatic cells incorporated BrdU throughout the stages examined. Consistent with our counting of PGC number, stella-positive PGCs that incorporated BrdU increased after E9.25 and reached approximately 50% at E9.75, which remained constant until at least E10.5 (Fig. 3A,B). These results indicate that a majority of PGCs from about E7.5 up until E9.0 did not enter into S-phase and may be blocked at a certain stage of the cell cycle.
To explore the cell cycle phase of the migrating PGCs, we analyzed the
expression of cyclin B1, which accumulates at high levels in the cytoplasm at
the late G2 phase of the cell cycle (Pines
and Hunter, 1991). At the LB to EHF stage, about 20% of the
Blimp1-positive PGCs showed a high concentration of cyclin B1 in
their cytoplasm (Fig. 3A,C).
Subsequently, after E8.0 onwards, the percentage of PGCs that were strongly
positive for cyclin B1 increased progressively, and it reached as high as 80%
at E8.5 to 8.75. The high percentage of PGCs showing strong positivity for
cyclin B1 persisted up until E9.5 and sharply decreased after E9.75
(Fig. 3A,C). Collectively,
these findings suggest that a majority of migrating PGCs are blocked at the G2
phase of the cell cycle.
Finally, we examined the DNA content of PGCs by FACS analysis. As shown in
Fig. 3D, embryonic cells
negative for Blimp1-mEGFP or stella-EGFP showed a clear G1 peak with
a broad S-phase and a less prominent G2 peak. This cell cycle distribution of
embryonic somatic cells resembles that of embryonic stem cells
(Fujii-Yamamoto et al., 2005)
and indicates a rapidly cycling cell population. We found that
Blimp1-positive cells at E7.25 showed a similar cell cycle
distribution. By contrast, however, a majority of stella-positive cells from
E7.75 to 8.75 (approximately 60%) were found to be at the G2 phase, consistent
with the BrdU incorporation and cyclin B1 immunostaining analyses. At E10.5,
stella-negative cells showed a typical somatic cell cycle distribution with a
very prominent G1 peak, whereas stella-positive PGCs were distributed almost
equally in the G1 (29.7%), S (34.4%) and G2 (35.4%) phases, indicating that
PGCs at this stage are rapidly cycling. Collectively, our findings indicate
that a majority of PGCs enter the G2 arrest of the cell cycle at some point
between E7.75 and 8.75, which is when H3K9me2 demethylation, transcriptional
quiescence and H3K27me3 upregulation proceed.
Nanos3 is not involved in the epigenetic reprogramming and transcriptional quiescence
To gain insight into the molecular mechanisms underlying the epigenetic
reprogramming, we first analyzed the genome-wide epigenetic properties of
Nanos3-deficient PGCs. Nanos is an evolutionally conserved protein
essential for germ cell development
(Kobayashi et al., 1996;
Subramaniam and Seydoux, 1999;
Wang and Lehmann, 1991;
Tsuda et al., 2003) and is
required to maintain low H3K4 methylation levels (C. elegans and
D. melanogaster) and to prevent premature zygotic transcription
(D. melanogaster) in PGCs
(Schaner et al., 2003). In the
mouse, Nanos3 is expressed in PGCs after E7.25
(Yabuta et al., 2006) and is
indispensable for their subsequent development
(Tsuda et al., 2003), making
it a good candidate for involvement in the epigenetic reprogramming in
migrating PGCs. However, Nanos3 mutant PGCs appeared to show the
proper H3K9me2 demethylation at E8.75 and H3K27me3 upregulation at E9.5
(Fig. 4A,B). The level of
H3K4me2 in Nanos3 mutant cells also looked indistinguishable from
that of wild-type PGCs (Fig.
4C). Moreover, quiescence of RNAP II-dependent transcription
seemed to occur normally in the absence of Nanos3
(Fig. 4D). These findings
indicate that the genome-wide epigenetic reprogramming and transcriptional
quiescence in mouse PGCs are independent from Nanos function.
|
).
Therefore, we examined the expression of all the members of the JmjC
domain-containing genes in the mouse genome by Q-PCR using single-cell cDNAs
prepared from PGCs and their somatic neighbors at the EB and the EHF stages
(Yabuta et al., 2006).
Although this analysis identified that many members of this family of proteins
including Jmjd1a (Jhdm2a), a demethylase for H3K9me1 and
me2, were indeed expressed in PGCs at these stages, none of them were specific
to PGCs as seen for genes such as Blimp1 and stella
(Fig. 5; see Fig. S4 in the
supplementary material), suggesting that PGCs may possess an additional
mechanism to direct genome-wide demethylation of H3K9.
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), was
downregulated as early as E7.25 specifically in PGCs
(Yabuta et al., 2006) and was
the only gene among the SET-domain-containing genes in the mouse genome that
showed enrichment in somatic neighbors (data not shown). GLP was shown to form
a complex with G9a and to be essential for the stability and the histone
methyltransferase activity of this complex. Thus, lack of either GLP or G9a
alone is considered to lead to the loss of function of this enzyme complex,
leading us to speculate that downregulation of Glp might be crucial
for the genome-wide demethylation of H3K9me2 in PGCs. To address this point,
we examined the expression of GLP and G9a proteins in embryos by whole-mount
immunofluorescence analysis. Consistent with our previous single-cell Q-PCR
analysis, at the EHF stage, Blimp1-positive cells, especially those
migrating toward the endoderm, already repressed GLP expression
(Fig. 6A). By contrast, somatic
neighbors expressed GLP almost uniformly at this stage. It is of note that
some of the Blimp1-positive cells forming a cluster at the base of
the allantois showed GLP expression (Fig.
6A), suggesting that GLP is repressed progressively in migrating
PGCs. Thereafter, we could not detect GLP expression in PGCs until at least
E10.5. By contrast, G9a was detected at E7.75 but was significantly reduced in
PGCs at E9.5 (Fig. 6B). These
findings indicate that PGCs downregulate GLP at around E7.57.75 and G9a
at around E9.0, resulting in the loss of two essential enzymes for genome-wide
H3K9me2.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Tam and Snow, 1981), we found
that the number of PGCs does not increase at a constant rate. This finding was
possible due to the high sensitivity of the PGC reporter mice used in this
study, which enabled more accurate counting of PGC numbers in the early stages
(E8.25) than the classical AP staining, which has been shown to
frequently underestimate the PGC number in the early stages
(Lawson et al., 1999). The
increase of PGC number from around E8.0 to 9.0, when most PGCs are migrating
in the hindgut endoderm, was relatively slower, and a majority (60%) of
stella-positive PGCs were in the G2 phase of the cell cycle. Epigenetic
reprogramming proceeds progressively in this period in individual PGCs. It
would therefore be likely that the progressive removal of H3K9me2,
transcriptional repression and H3K27me3 upregulation all occur in the G2 phase
of one particular cell cycle. As the decrease of H3K9me2 immunoreactivity
seemed greater than twofold (Fig.
1C), this process would not be explained by a simple passive
dilution associated with the doubling of chromosomes.
|
) were
indeed expressed in PGCs, but their expressions were similarly seen in somatic
neighbors, suggesting that PGCs might have additional mechanisms allowing
genome-wide removal of H3K9me2. As one such mechanism, we identified that PGCs
specifically repress an essential H3K9 methyltransferase, GLP, at around the
LB stage just before the reduction of H3K9me2 in some PGCs, suggesting that
repression of an active enzyme triggers genome-wide loss of H3K9me2, either
through a turnover of methyl groups or a replacement of the entire H3 with
unmodified H3. Consistently, a mass spectrometry analysis of H3K9 methyl
groups differentially pulse-labeled by a heavy isotope demonstrated that
H3K9me2 turns over without replication
(Fodor et al., 2006).
Furthermore, it was recently reported that chromatin-associated histones and
non-chromatin-associated histones are continually exchanged in
Xenopus oocytes, and maintenance of H3K9 methylation at a specific
site requires the continual presence of an H3K9 histone methyltransferase
(Stewart et al., 2006). Thus,
repression of an essential enzyme may shift the equilibrium between
methylation and demethylation toward demethylation, leading to the erasure of
H3K9me2, possibly through these mechanisms, in migrating PGCs. This is
consistent with our additional finding that PGCs also showed low H3K9me1,
another modification mediated by G9a-GLP complex
(Tachibana et al., 2005). Another finding of note is that, after the onset of H3K9me2 demethylation, PGCs appeared to paradoxically repress RNAP II-dependent transcription. Although the functional significance of this phenomenon is currently unknown, it may be crucial to protect the cells from deregulated transcription in the absence of major chromatin-based repressive mechanisms during epigenetic reprogramming. Consistently, this transcriptional repression ensues until PGCs acquire high levels of H3K27me3 and is relieved gradually afterwards, which seems concomitant with their release from the G2 arrest and rapid proliferation. Further studies will be needed to clarify the significance of this event as well as to identify the mechanism by which H3K27me3 is specifically upregulated.
A comparison with the cellular dynamics of PGCs in other organisms
This study makes it possible to compare the cellular dynamics of PGCs in
mice with those of other organisms that specify the germ cell lineage with the
`preformation' mode. In C. elegans, the germline precursor
blastomeres P1 to P4 repress RNAP II-dependent transcription
(Seydoux and Dunn, 1997).
Although they exhibit genome-wide H3K4 methylation at a similar level to their
somatic neighbors, they specifically express PIE-1, which prevents
phosphorylation of RNAP II CTD by competing with the CDK-9-cyclin T complex
(Zhang et al., 2003). P4
blastomeres divide to form Z2 and Z3 cells, the lineage-restricted PGCs, which
then degrade PIE-1 and become positive for the phosphorylation of RNAP II CTD
(Schaner et al., 2003).
However, these cells lose their genome-wide H3K4 methylation and appear to
transcribe only a few genes and to become mitotically quiescent until the
embryo hatches (Schaner and Kelly,
2006).
In Drosophila, pole cells, the lineage-restricted PGCs also
exhibit transcriptional quiescence with no RNAP II CTD phosphorylation, low
H3K4 methylation and high H3K9 methylation levels
(Schaner et al., 2003;
Seydoux and Dunn, 1997). This
transcriptional repression depends on pgc
(Deshpande et al., 2004;
Martinho et al., 2004;
Nakamura et al., 1996). After
asynchronous zero to two divisions, they become mitotically quiescent at the
G2 phase (Su et al., 1998) and
undergo migration. It therefore seems to be the case that PGC precursors and
PGCs in C. elegans and PGCs in Drosophila show no or very
low levels of transcription with initial chromatin-independent and subsequent
chromatin-dependent mechanisms. Interestingly, in both C. elegans and
Drosophila, Nanos activity is required to maintain the low H3K4
methylation levels in PGCs (Schaner et
al., 2003).
By contrast, in mice, we showed that Blimp1-positive PGC
precursors and stella-positive PGCs are transcriptionally active with
phosphorylated RNAP II CTD by E7.75, consistent with the activation of many
specific genes associated with germ cell specification
(Yabuta et al., 2006).
Nonetheless, we found that migrating PGCs transiently exhibit repression of
RNAP II-dependent transcription, an event that appears to be similar to those
observed in C. elegans and Drosophila. However, this seems
to be associated with epigenetic reprogramming and is not likely to be a
mechanism to prevent somatic gene expression in PGCs. Furthermore, we showed
that neither chromatin modifications nor transcriptional quiescence were
affected in the absence of Nanos3, suggesting that this protein is not
involved in these events in mice. Our observation that most migrating PGCs in
mice are in the G2 phase of the cell cycle may suggest a similarity to the
situation in Drosophila pole cells. Although the functional
significance of the G2 arrest in migrating pole cells is unclear, the G2
arrest of migrating PGCs in mice again seems tightly associated with the
epigenetic reprogramming. Therefore, repression of RNAP II-dependent
transcription and G2 arrest of the cell cycle in mice may be independently
acquired events for the epigenetic reprogramming to occur efficiently,
although these events are commonly observed in three distinct organisms.
Perspective
This study clarified, for the first time, a sequence of unique events
occurring in migrating PGCs in mice that undergo genome-wide epigenetic
reprogramming, which may have general implications for somatic cell
reprogrammings of any kind. Recently, it was shown that PGCs elevate H4R2 and
H2AR3 methylation at around E8.5 through an action of the Blimp1-Prmt5 complex
(Ancelin et al., 2006),
demonstrating that PGCs undergo yet another level of complex epigenetic
programming in their migration. These events precede reactivation of the
inactive X chromosome in female PGCs and erasure of parental imprints, both of
which occur when PGCs colonise the genital ridges
(Li, 2002;
McLaren, 2003;
Surani, 2001). The precise
relevance of the earlier events reported in this study to those in the genital
ridges is, however, currently unknown. A crucial step to resolve this issue
would be to determine the target sequences from which H3K9me2 is removed and
upon which H3K27me3 is imposed in migrating PGCs. There is currently only a
limited amount of information available regarding the genome-wide distribution
of these two modifications in mammalian cells in general
(Bernstein et al., 2007). We
consider it an essential challenge to develop an experimental system that
allows genome-wide analysis of specific histone modifications from a small
amount of biological samples, as in the case for PGCs.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/14/2627/DC1
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