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First published online 19 November 2003
doi: 10.1242/dev.00870
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1 Department of Molecular Embryology, Research Institute, Osaka Medical Center
for Maternal and Child Health, 840, Murodo-cho, Izumi, Osaka, 594-1101,
Japan
2 Osaka University, Graduate School of Medicine, Suita, Osaka, Japan
3 CREST, JST, Saitama, Japan
4 Department of Molecular Cell Biology, Research Institute for Microbial
Diseases, Osaka University, Suita, Osaka, Japan
* Author for correspondence (e-mail: ymatsui{at}lab.mch.pref.osaka.jp)
Accepted 16 September 2003
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: Primordial germ cell, E-cadherin, Cell interaction, Cell fate
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
McLaren, 1999;
Zhao and Garbers, 2002;
Lawson and Hege, 1994;
Tam and Zhou, 1996). However,
the mechanisms of germ cell specification in mice remain unknown. Although
maternal germ cell determinants are critical for germ cell formation in
organisms such as Caenorhabditis, Drosophila and Xenopus,
they do not seem to be involved in mouse germ cell allocation. Cell lineage
analyses have revealed that a portion of proximal epiblast cells in pre- and
early streak mouse embryos are common precursors of primordial germ cells
(PGCs) and extra-embryonic mesoderm
(Lawson and Hage, 1994) and
that final cell fate is not yet determined at this time. In addition, after
transplantation into the proximal region of the epiblast, distal epiblast
cells that normally become ectoderm cells follow the fate of the proximal
epiblast and give rise to PGCs as well as extra-embryonic mesoderm
(Tam and Zhou, 1996). This
also indicates that proximal location in the epiblast is important in becoming
committed to PGCs.
We established a primary culture system that can mimic PGC differentiation
from the epiblast, and have shown that the extra-embryonic ectoderm induces
the conditions required for PGC formation within the adjacent proximal
epiblast (Yoshimizu et al.,
2001). In addition, studies of knockout mice have revealed that
BMP4/8b signaling is critical for this induction event
(Lawson et al., 1999;
Ying et al., 2000), and
recombinant BMPs can induce PGCs from epiblasts in culture
(Hayashi et al., 2002).
Signals from BMP might induce precursors of PGCs and of extra-embryonic
mesoderm, but may not be involved in actual PGC determination. Thus, a second
local signal at the posterior end of the embryo might regulate allocation to
the PGC lineage (Lawson et al.,
1999; Fujiwara et al.,
2001). The nature of this presumptive second signal remains
obscure. A gene known as fragilis/mil-1 that is potentially expressed
in PGC precursors (Saitou et al.,
2002; Tanaka and Matsui,
2002) encodes a transmembrane protein that belongs to the
interferon induced transmembrane protein family, and it might be involved in
cell-cell contact as well as cell growth control
(Deblandre et al., 1995). As
yet there is no evidence that this gene functions in PGC formation.
Cadherins are responsible for cell adhesion through calcium-dependent
homophilic interactions and have various functions including cell
differentiation (Takeichi,
1991; Gumbiner,
1996; Laprise et al.,
2002). For example, N-cadherin-dependent cell interactions in a
group of muscle progenitors plays a crucial role in promoting differentiation
into MyoD-expressing muscle in Xenopus embryos
(Holt et al., 1994). Although
high levels of E-cadherin are expressed in the epiblast but not in the
mesoderm, we show here that E-cadherin is expressed in the proximal
extra-embryonic mesoderm that contains the PGC precursors and that cell-cell
interaction mediated by E-cadherin is crucial for these precursors to be
allocated to the germ cell lineage.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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PE)-GFP
heterozygous male mice (Yoshimizu et al.,
1999) maintained in the C57Bl/6 background. Embryos were staged
before the experiments as described previously
(Downs and Davies, 1993): E6.5,
E6.75, E7.0, E7.25 and E7.5 embryos corresponding to early streak, mid-streak,
mid to late streak, early allantoic bud and late allantoic bud stages,
respectively.
Histological staining
Whole embryos and cultured explants were histologically stained as
described previously (Ciruna and Rossant,
2001). Primary antibodies were applied at the following
concentrations: 10 µg/ml for rat anti-E-cadherin (ECCD-2) (Takara Shuzo),
3.0 µg/ml for rat anti-Flk1 (AVAS12)
(Kataoka et al., 1997) and at
a 1:1000 dilution for rabbit anti-PGC7
(Sato et al., 2002). The
appropriate species-specific fluorophore-labeled secondary antibodies
(Molecular Probes) were applied at a 1:200 dilution. Nuclei were stained with
TOTO-3 iodide (Molecular Probes) at a 1:500 dilution in the presence of 100
µg/ml of RNase A. Staining was examined using a confocal microscope
(Leica). Specimens were stained with alkaline phosphatase (ALP) after
immunostaining as described (Donovan et
al., 1986).
Dissection of embryos and explant culture
Embryos were dissected out from decidua and then Reichert's membrane and
visceral endoderm were mechanically removed using fine forceps and a tungsten
needle to isolate epiblasts or extra-embryonic mesoderm
(Hogan et al., 1994). The
proximal quarter of epiblasts was dissected at E6.5 and E6.75 as proximal
epiblasts. To isolate extra-embryonic mesoderm at E6.75 and E7.0, boundaries
of extra-embryonic mesoderm, extra-embryonic ectoderm and epiblast were cut
with a tungsten needle. We also isolated the base of allantoic buds as
extra-embryonic mesoderm at E7.25 and E7.5. The fragments of extra-embryonic
mesoderm were dissociated into single cells by gentle pipetting in 0.025%
trypsin/0.75 mM EDTA. Extra-embryonic mesoderm explants were co-cultured with
200 µg/ml of ECCD-1, a blocking antibody for E-cadherin, or with the same
concentration of ECCD-2 as a control. Explants were cultured on a feeder layer
of STO fibroblast cells as described previously
(Yoshimizu et al., 2001).
Time-lapse analysis
Time-lapse images were analyzed using a Leica confocal microscope equipped
with a cell culture chamber. Images were captured every hour for 40 hours.
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Saitou et al., 2002), whereas
E6.75 explants did not (Table 1
and Fig. 1A-D). These findings
suggested that PGC precursors migrated out of the proximal epiblast between
E6.5 and E6.75. Because ALP-positive (Ginsberg et al., 1990) and
PGC7/stella-positive (Sato et al.,
2002; Saitou et al.,
2002) PGCs appear first in the extra-embryonic mesoderm at E7.25,
and fragilis/mil-1 (Saitou et
al., 2002; Tanaka and Matsui,
2002), which seems to be expressed in PGC precursors, is expressed
in the earlier (E6.75) extra-embryonic mesoderm, we cultured fragments of
isolated extra-embryonic mesoderm from E6.75 embryos under the same
conditions. All of these explants gave rise to PGCs, indicating that PGC
precursors are located within the extra-embryonic mesoderm at E6.75
(Table 1, Fig. 1E).
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).
Although GFP is also weakly expressed in somatic tissues before this stage,
the E6.75-E7.25 extra-embryonic mesoderm expressed more GFP than anywhere else
in the embryo, suggesting that cell populations containing PGC precursors can
be distinguished by intense GFP signals
(Fig. 2). Explants of E6.75
extra-embryonic mesoderm of the transgenic embryos did indeed express high
levels of GFP, and ALP-positive, PGC7/stella-positive PGCs migrated from a
cluster of intensely GFP-positive cells after culture
(Fig. 3), further indicating
that intense GFP expression in the extra-embryonic mesoderm marks a cell
population that includes forming PGCs. The intensely GFP-expressing cells
formed clusters within the explants before the PGCs started migration
(Fig. 3B-D and Supplemental
data:
http://dev.biologists.org/supplemental).
In addition, the cell clusters were formed at 9 hours in culture, but cells
were not PGC7 positive until 12 hours in culture
(Fig. 7; data not shown),
suggesting that cell contact is important in PGC formation before commitment.
To test the requirement for cell-cell interaction, we cultured extra-embryonic
mesoderm of E6.75 and E7.5 embryos after dissociating the cells by
trypsinization. No PGCs were evident in the dissociated E6.75 explants
(Table 2). The frequency of PGC
formation gradually increased from E6.75 to E7.5, when 100% of the dissociated
explants gave rise to PGCs (Table
2, Fig. 4),
suggesting an early requirement for cell contact that is relieved by E7.5.
Trypan Blue staining showed that over 80% of both E6.75 and E7.25 cells
survived after trypsinization. In addition, the total number of cells after
culture, determined by culturing embryos ubiquitously expressing GFP, was
86-90% of the number of plated cells, and no difference was found between
E6.75 and E7.25 cells. Thus, the rates of cell survival at E6.75 and E7.25 in
vitro were identical. Together, these results suggest that close cell-cell
interaction around E6.75 is critical for PGC differentiation from
extra-embryonic mesoderm.
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).
E-cadherin is a cell adhesion molecule that is involved in many aspects of
development, including organogenesis and cell differentiation
(Takeichi, 1991;
Gumbiner, 1996;
Laprise et al., 2002).
Although E-cadherin expression in early streak embryos is high in the epiblast
but undetectable in the mesoderm (Damjanov
et al., 1986), we found intense expression at E6.75 in a proximal
region of the extra-embryonic mesoderm adjacent to the epiblast
(Fig. 5B), and expression was
localized to the sites of cell contact. E-cadherin expression was not, however
detected in the more distal part of the extra-embryonic mesoderm, which was
developing into the allantois (Fig.
5B). By E7.0, cells that might be PGC precursors and that
expressed more intense GFP, formed a cluster in the most distal region of the
extra-embryonic mesoderm expressing E-cadherin
(Fig. 2F). This suggested that
E-cadherin is involved in clustering PGC precursors. PGC7/stella was not
expressed in these cells, indicating that PGCs had not yet differentiated from
precursors at E7.0 (Fig. 5E-H,
data not shown). Similar expression profiles persisted at E7.25, when
PGC7/stella-positive PGCs were obvious among cells expressing E-cadherin
(Fig. 5I-L). Therefore,
E-cadherin expression appears to define the cell population containing nascent
PGCs within the extra-embryonic mesoderm.
|
).
We cultured fragments of extra-embryonic mesoderm from E6.75 embryos with or
without the blocking antibody, ECCD-1. In the absence of antibody
(Fig. 6A-D) or in the presence
of the control antibody, ECCD-2 (Fig.
6I-L), a cluster of PGCs expressing Oct4-GFP and PGC7/stella
formed within explants after 15 hours in culture, but not with ECCD-1
(Fig. 6E-H). Although cells
often expressed very high levels of GFP in cultured explants in the presence
of ECCD-1, they were PGC7 negative (Fig.
6E-G). These cells may be PGC precursors that failed to commit to
a PGC fate.
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In contrast to its effect on PGC formation, ECCD-1 did not prevent the
expression of Flk1, a marker of extra-embryonic mesoderm and allantois
(Kataoka et al., 1997)
(Fig. 8). One report has
indicated that Flk1 is not expressed in E6.75 extra-embryonic mesoderm
(Yamaguchi et al., 1993), and
we confirmed that Flk1 expression was undetectable in the explant before
culture (data not shown), indicating that differentiation of extra-embryonic
mesoderm progressed in culture in the presence of ECCD-1. These results
indicate that blocking E-cadherin function inhibits PGC formation, but does
not affect the differentiation of extra-embryonic mesoderm cells. After 48-60
hours in culture, ECCD-1-dependent PGC formation was still inhibited in E6.75
explants, while E7.25 explants were not affected
(Table 3, Fig. 9), consistent with the
conclusion from the cell dissociation study that cell contact was no longer
required by E7.25 (Table
2).
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Discussion |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). The
expression profiles of fragilis/mil-1
(Saitou et al., 2002;
Tanaka and Matsui, 2002) and
Oct4-GFP (Yoshimizu et al.,
1999) as well as our culture experiments
(Table 1) suggest that these
precursor cells migrate to and remain in a region at the posterior of embryos
by E6.75. Our present results indicate that E-cadherin-mediated interactions
among such common precursor cells regulate their fates and are necessary for
their commitment to the germ cell lineage
(Fig. 10).
|
). Four Notch genes (Notch1-4) have been identified
in mice and we found by whole-mount in situ hybridization that only
Notch1 and Notch2 were uniformly expressed in epiblasts at
E6.5 (data not shown). However, not all of the Notch genes were expressed in
extra-embryonic mesoderm at E7.0 (Williams
et al., 1995) (data not shown). In addition, we examined
abnormalities of PGC development in homozygous Notch1 and
Notch2 mutant embryos by whole mount ALP staining, but the numbers
and localization of PGCs in the homozygous embryos did not significantly
differ from those of the heterozygous and wild type littermates at E8.5. Taken
together, we concluded that Notch signaling does not function in PGC
development. However, the possibility of redundancy in Notch1 and Notch2
functions cannot be ruled out, as in the roles they play in left-right
asymmetry determination (Krebs et al.,
2003).
Several mechanisms by which E-cadherin regulates PGC determination are
possible. The simplest model is that E-cadherin itself transmits instructive
signals for PGC determination. For example, homophilic interactions of
E-cadherin transmit intracellular signals by sequestering ß-catenin from
lymphoid enhancer factor/T cell factor (LEF/TCF)
(Hecht and Kemler, 2000)
transcription factors (Orsulic et al.,
1999). E-cadherin might also facilitate the clustering of
signaling molecules with growth factor receptors that are specifically
expressed within the cluster. For instance, VE-cadherin in endothelial cells
forms a complex consisting of ß-catenin, PI 3-kinase and VEGF receptor 2,
by which VEGF-A activates Akt (Carmeliet et
al., 1999). E-cadherin also stimulates the MAPK pathway through
ligand-independent activation of EGFR in epithelial cells (Pece et al., 2000).
Because PI 3-kinase is also involved in E-cadherin-dependent activation of
MAPK (Pece et al., 1999;
Laprise et al., 2002), we
examined its involvement in PGC formation by adding the potent PI 3-kinase
inhibitors, LY294002 and wortmanin to cultured E6.75 extra-embryonic mesoderm.
However, PGC formation was not significantly affected (data not shown). This
indicates that E-cadherin-mediated signals are transmitted to PGC precursors
via different signaling pathways. Fragilis/mil-1
(Saitou et al., 2002;
Tanaka and Matsui, 2002) is
likely to be associated with E-cadherin in signal transmission.
Fragilis/mil-1 is specifically expressed in a cell population in the
extra-embryonic mesoderm and in newly formed PGCs, and encodes a transmembrane
protein. A role in cell-cell interaction thus seems likely for
fragilis/mil-1, although clarification of its functions await further
study.
Alternatively, E-cadherin might play more permissive roles in transmitting
signals for PGC determination. E-cadherin may simply permit close enough
contact for other independent instructive signals such as juxtacrine signals
or signals via other adhesion molecules including Fragilis/Mil-1. In this
regard, E-cadherin could also function as an anchor that settles precursor
cells within niches for PGC differentiation. Cell adhesion mediated by
DE-cadherin is required in the Drosophila ovary to anchor germline
stem cells in niches for their renewal
(Song et al., 2002). In mouse
extra-embryonic mesoderm, E-cadherin expression is restricted to the proximal
region that is adjacent to the epiblast, which also expresses E-cadherin, and
is not expressed in the distal portion of extra-embryonic mesoderm or in the
allantois (Fig. 5B,F,J). This
spatial distribution of E-cadherin prompts the notion that homophilic
E-cadherin interactions prevent precursors from moving to the distal part of
the extra-embryonic mesoderm, where the allantois differentiates
(Downs and Bertler, 2000). The
continuous expression of E-cadherin in the proximal extra-embryonic mesoderm
might thus protect PGC precursors from allantois differentiation
(Fig. 10).
The present study provides evidence for the importance of E-cadherin-mediated cell interaction in germ cell determination. Signaling molecules that depend on E-cadherin function should be identified. The identification of such molecules will further increase understanding of the process of germ cell determination and help to identify novel germ cell determinants.
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ACKNOWLEDGMENTS |
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Footnotes |
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