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481
Development 128, 481-490 (2001)
Printed in Great Britain © The Company of Biologists Limited 2001
DEV3231
Stage-specific tissue and cell interactions play key roles in mouse germ cell
specification
Tomomi Yoshimizu1, Masuo Obinata2 and Yasuhisa Matsui1,*
1Department
of Molecular Embryology, Research Institute, Osaka Medical Center for Maternal and Child Health, 840, Murodo-cho,
Izumi, Osaka 594-1101, Japan
2Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, 4-1, Seiryo-machi, Sendai, Miyagi
980-8575, Japan
*Author for correspondence (e-mail: [email protected])
Accepted 7 December 2000; published on WWW 23 January 2001
SUMMARY
Primordial germ cells (PGCs) in mice have been
recognized histologically as alkaline phosphatase (AP)
activity-positive cells at 7.2 days post coitum (dpc) in
the extra-embryonic mesoderm. However, mechanisms
regulating PGC formation are unknown, and an
appropriate in vitro system to study the mechanisms has
not been established. Therefore, we have developed a
primary culture of explanted embryos at pre- and earlystreak stages, and have studied roles of cell and/or tissue
interactions in PGC formation. The emergence of PGCs
from 5.5 dpc epiblasts was observed only when they were
co-cultured with extra-embryonic ectoderm, which may
induce the conditions required for PGC formation within
epiblasts. From 6.0 dpc onwards, PGCs emerged from
whole epiblasts as did a fragment of proximal epiblast
that corresponds to the area containing presumptive
PGC precursors without neighboring extra-embryonic
ectoderm and visceral endoderm. Dissociated epiblasts
at these stages, however, did not give rise to PGCs,
indicating that interactions among a cluster of a specific
number of proximal epiblast cells is needed for PGC
differentiation. In contrast, we observed that dissociated
epiblast cells from a 6.5-b (6.5+15-16 hours) to 6.75 dpc
embryo that had undergone gastrulation gave rise to
PGCs. Our results demonstrate that stage-dependent
tissue and cell interactions play key roles in PGC
determination.
INTRODUCTION
timing of germ cell and somatic cell segregation in mouse
embryos. By genetically marking a blastomere of a four-cell
embryo (Kelly, 1977) or by marking cells of the inner cell mass
(ICM) of a blastocyst with retroviruses (Soriano and Jaenisch,
1986), single cells at these developmental stages have been
shown to contribute to both germ cells and somatic cells. A
single cell in the ICM, however, has been observed to
contribute only to germ cells in just a few cases, suggesting
that the germ cell lineage can occasionally be set aside in ICM
(Soriano and Jaenisch, 1986).
A perigastrula-stage mouse embryo consists of three major
tissues: the epiblast, the extra-embryonic ectoderm and the
visceral endoderm. Extra-embryonic ectoderm and visceral
endoderm originate from the trophectoderm and the most
parietal ICM, respectively, and they give rise to placenta and
other extra-embryonic tissues. In contrast, cup-shaped epiblast
originates from ICM and differentiates into all types of cells
found throughout the entire embryo, including germ cells. The
developmental potential of epiblast cells has been examined by
injecting those cells into blastocysts (Gardner and Rossant,
1979). The contribution of the donor epiblast cells in chimeric
mice was judged either by the presence of distinct GPI markers
In such organisms as Drosophila melanogaster,
Caenorhabditis elegans and Xenopus laevis, germ plasm (polar
plasm) containing germ cell determinants is maternally
accumulated and localized in eggs, and only blastomeres that
inherit germ plasm develop into germ cells. In contrast, in early
mammalian embryos, germ plasm and even the Nuage-like
structure that is characteristic of germ plasm (Eddy, 1975) have
not been found (Snow and Monk, 1983), and the restriction of
the germ and somatic cell lineage is thought to occur after a
mid-gastrula stage of development.
During mouse embryogenesis, primordial germ cells (PGCs)
are first identified histologically within the posterior extraembryonic mesoderm at 7.2 dpc as a cluster of cells expressing
tissue-nonspecific alkaline phosphatase (AP) (Chiquoine,
1954; Mintz and Russell, 1957; Ginsburg et al., 1990).
Thereafter, PGCs undergo migration from the base of the
allantois through the dorsal mesentery, and reach the genital
ridges by 11 dpc. Throughout these developmental stages,
PGCs continue to display AP activity.
Several experiments have been carried out to determine the
Key words: Primordial germ cell, Epiblast, Gastrulation, Mouse
482
T. Yoshimizu, M. Obinata and Y. Matsui
or by coat colors. The results indicated that at least two cells
in 4.5 dpc epiblast cells can give rise to both germ cells and
somatic cells (Gardner and Rossant, 1979).
The fates of later epiblast cells have been examined by
clonal analysis (Lawson and Hage, 1994). The results showed
that presumptive precursors of PGCs are scattered in a ring in
the most proximal region of the epiblast, adjacent to the extraembryonic ectoderm at 6.0-6.5 dpc. In these experiments, all
cells that gave rise to PGCs also differentiated into extraembryonic mesoderm, indicating that the allocation to PGCs
had not occurred at 6.5 dpc, which is the start of gastrulation.
Clonal analysis has also suggested that allocation to the germ
cell lineage occurs at 7.2 dpc, with the founding population of
PGCs consisting of 45 cells (Lawson and Hage, 1994). In
addition, the results of a heterotopic transplantation of epiblast
cells (Tam and Zhou, 1996) have demonstrated that even distal
epiblast cells have the ability to give rise to PGCs as proximal
cells when they are transplanted to a proximal region. This
observation suggests the existence of putative local cues in the
proximal regions of epiblasts that specify the germ cell
lineage.
After the onset of gastrulation at 6.5 dpc, totipotent epiblast
cells are destined for each somatic cell lineage after passing
through the primitive streak during gastrulation (Hogan et al.,
1994). The presumptive PGC precursors located within the
proximal epiblast may also pass through the posterior end
of the primitive streak and reside in the extra-embryonic
mesoderm. Although it has been suggested that cell movement
accompanied by gastrulation can lead to a determination of the
germ line fate in the new environment, probably at the posterior
primitive streak (Tam and Zhou, 1996), the requirement of
gastrulation for PGC formation is still unclear. Microsurgical
grafting experiments have indicated that posterior primitive
streak cells of 7 dpc embryos grafted orthotopically to the
same site in other embryos yield PGCs (Copp et al., 1986).
In contrast, lateral ectoderm/mesoderm cells yield somatic
derivatives (but not PGCs) after heterotopic grafting to the
posterior primitive streak site, indicating that the lateral
ectoderm/mesoderm at the early allantoic bud stage no longer
has the potential to give rise to PGCs.
As described above, the outline of PGC formation from
epiblasts has been reported, but the mechanisms that regulate
allocation to germ cell lineage are unknown. Although only
little evidence is available, it is thought that interactions
between tissues and/or cells play a critical role in the formation
of mammalian germ cells in relation to the differentiation and
patterning of somatic tissues. We have therefore designed a
primary culture of 5.5-6.75 dpc embryos to assay the
conditions required for PGC formation from epiblasts. The
results demonstrate the role of cell-cell communication within
proximal epiblasts and of the extra-embryonic ectoderm in
PGC formation.
MATERIALS AND METHODS
Source, recovery and staging of concepti
Concepti used throughout this study were obtained from female mice
of an outbred strain (CD-1) that were ICR-mated with male mice of
BDF1 (C57/B6j × DBA2). Those mice were exposed to light daily
between 08:00 and 20:00 hours. Taking the time of mating as the mid-
Fig. 1. Staging system used in this study. (A) 5.5 dpc. The newly
formed proamniotic cavity is seen only in the epiblast. The decidual
reaction is not complete. (B) 6.0 dpc. A proamniotic cavity is
continuous between an epiblast and extra-embryonic ectoderm.
(C) 6.25 dpc. The anterior-posterior axis can be recognized by signs
of bilateral asymmetry (arrowhead). (D) 6.5-a dpc. The length of the
primitive streak (as judged by the extent of mesoderm reaching down
the posterior side; bar) is 30% of the length of the epiblast. This
stage corresponds to the early-streak stage in Downs and Davies
(Downs and Davies, 1993). (E) 6.5-b dpc. The length of the primitive
streak is 50-70%. A small posterior amniotic fold is now visible.
Scale bar; 200 µm.
point of the dark period (noon on the day of plug is 0.5 days
postcoitum; dpc), pregnant females were sacrificed at 5.5 (5 days +
12 hours), 6.5-a (6 days + 12 hours), and 6.5-b (6 days + 15-16 hours)
dpc, and their uteri were placed in PB-1 medium (Quinn et al., 1982)
for isolating decidua. Concepti were staged by visual inspection,
according to the scheme shown in Fig. 1.
Isolation of explants is shown in Fig. 2. Concepti were dissected
out from decidua (Fig. 2A-a), and then Reichert’s membrane and
visceral endoderm were mechanically removed (Fig. 2A-b) with fine
forceps and a tungsten needle. Thereafter, extra-embryonic ectoderm
(Fig. 2A-c) and epiblast (Fig. 2A-d) were separated by a fine tungsten
needle. The boundary between extra-embryonic ectoderm and epiblast
was identified based on the circumferential constriction (Downs and
Davies, 1993).
Isolated epiblasts were further processed for each experiment as
follows. (1) Epiblasts were divided into sub-fragments using
tungsten needles and fine forceps (Fig. 2Af-h). (2) Epiblasts were
trypsinized for 2.5-3.5 minutes at room temperature and
subsequently dispersed by pipetting using a capillary micropipette
(Fig. 2Ai). (3) For recombining epiblasts and extra-embryonic
ectoderm at different developmental stages (asynchronous
recombination), two female mice, mated 24 hours apart, were
sacrificed at the same time (i.e. 5.5 and 6.5-a dpc), and extraembryonic ectoderm and distal epiblast were then separated (Fig.
2Ba-d) and used for culture (Fig. 2Be).
Explant culture and examination of PGC emergence
Isolated tissues were cultured on mitomycin C-treated STO
PGC specification from epiblast
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Fig. 2. Method of embryo preparation for culture. Embryos were cut into parts
at the positions indicated by solid lines. (A) Explant culture of a 6.5-a dpc
epiblast; (a) after removal of Reichert’s membrane, (b) after removal of
visceral endoderm. The embryo was divided into extra-embryonic ectoderm(c)
and epiblast (d). (e) The epiblast was opened up into a flat sheet and the sheet
was subdivided into proximal (f) and distal (g) fragments. (h) The proximal
fragment was cut into four pieces, or were dissociated with trypsin treatment
(i). (B) Recombination of epiblast and extra-embryonic ectoderm. Extraembryonic ectoderm (a,c) and fragments of epiblast (b,d) at 6.5-b dpc (a, b) or
at 5.5 dpc (c,d) were separated and tissue fragments were recombined in an
aggregation well (e).
fibroblast (Donovan et al., 1986) or Sl/Sl4 (Matsui et al., 1991) cells
plated at a density of 1×105 or 2×105 cells per well, respectively, in
24-well culture plates (Falcon) with DMEM medium (15% FCS,
0.22 mg/ml sodium pyruvate, 1.8 mg/ml L-glutamine, 100 units/ml
Penicillin, 100 µg/ml Streptomycin) at 37°C in the gas phase of
5.0% CO2 in air. In some cases, isolated epiblasts were cultured in
fibronectin-coated 24-well plates (CBP Bioproducts). The explants
were allowed to develop until the time corresponding to 9.5 dpc
(approximately 96 hours for 5.5 dpc, 84 hours for 6.0 dpc and 72
hours for 6.5-a and 6.5-b dpc).
For the recombination culture, a small hole was made in the bottom
of a four-well culture plate (Nunc) by means of pressure from a
Hungarian needle (Fig. 2Be; Ang and Rossant, 1993), and the culture
wells with holes were covered with a feeder layer of STO cells.
Thereafter, an extra-embryonic ectoderm and a distal epiblast were
placed together in the hole.
To examine the emergence of PGCs, alkaline-phosphatase (AP)
staining was carried out as described (Matsui et al., 1992). Each
experiment was repeated at least three times. PGCs were identified
based on high AP activity, 4C9 and Oct3/4 expression.
Immunohistochemistry for 4C9 and Oct3/4
Explants were fixed in 4% paraformaldehyde and were stained by
each antibody as described (Davis, 1993). After incubation with 4C9
monoclonal antibody (a kind gift from Dr T. Muramatsu; Yoshinaga
et al., 1991) or affinity-purified polyclonal antibody against Oct3/4 (a
kind gift from Dr H. Hamada; Shimazaki et al., 1993), explants were
incubated with FITC-conjugated anti-rat IgM (Zymed) or rhodamineconjugated anti-rabbit IgG (Reinco technology), respectively.
Thereafter, the same explants were stained for AP activity.
Immunohistochemistry for mesoderm markers
Explants were fixed in 4% paraformaldehyde, followed by AP staining.
Explants that contained PGCs were subsequently incubated with
monoclonal antibody against Flk-1 (Kdr; a kind gift from Dr S.-I.
Nishikawa; Kataoka et al., 1997), or with affinity-purified polyclonal
antibody against HNF-3β (Foxa2: a kind gift from Dr H. Sasaki; Yasui
et al., 1997) or Brachyury (a kind gift from Drs B. Hermann and H.
Koseki; Herrmann, 1991), followed by incubation with HRPconjugated anti-rat IgG (Flk-1) or HRP-conjugated anti-rabbit IgG
(HNF-3β and Brachyury).
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T. Yoshimizu, M. Obinata and Y. Matsui
RESULTS
Establishment of explant culture of epiblast at preand early-streak stages
We first established a primary culture system for mouse
epiblasts at pre- and early-streak (corresponding to 6.25 or 6.5a dpc in Fig. 1C,D, respectively) stages, in which the PGCs
developed. Staging of the embryos used in this study is
described in Fig. 1. Typical pictures of each embryo stage are
shown, and the staging is based on both morphological and
morphometrical features. Epiblasts were isolated free of
visceral endoderm and extra-embryonic ectoderm and were
opened up to form a flat sheet. Epiblast fragments before
culture are shown in Fig. 2, confirming complete removal of
extra-embryonic tissues. They were then cultured on a feeder
layer of STO fibroblasts until the time corresponding to 9.5
dpc, i.e. approximately 78 hours for 6.25 dpc embryos and 72
hours for 6.5-a dpc embryos.
After culture, AP-positive PGC-like cells were scattered
around outgrowing epiblast (Fig. 3A). In case of 6.25 dpc
explants, none of these cells were recognized after 24 hours in
culture, but by 48 hours, migratory PGC-like cells were
observed (data not shown), and the timing of PGC-like cell
emergence in culture corresponded well with that in vivo. To
judge whether the AP-positive PGC-like cells (Fig. 3A,B,D)
were PGCs, we examined the expression of markers such as
4C9 (Fig. 3C, Yoshinaga et al., 1991), Oct3 (Fig. 3E, Okamoto
et al., 1990) Oct4 (Schöler et al., 1990) by immunostaining. As
shown in Fig. 3, all AP-positive PGC-like cells also expressed
4C9 (Fig. 3B,C shows double staining of an explant with AP
and 4C9, respectively) and Oct3/4 (Fig. 3D,E shows double
staining of an explant with AP and Oct3/4). In addition, when
we cultured whole epiblasts, those marker positive cells
exhibited the typical morphology of migrating PGCs (Fig. 3C;
Donovan et al., 1986). However, only a part of the markerpositive cells from fragments of epiblasts or from dissociated
epiblast cells showed the morphology of migrating PGCs and
none of marker-positive cells was migratory when epiblasts
were cultured on fibronectin or a feeder layer of Sl/Sl4 cells.
We also cultured epiblasts of the Oct3/4 (GOF18/deltaPE)/GFP
transgenic mice (Yoshimizu et al., 1999), which express green
fluorescent protein (GFP) only in PGCs but not in epiblasts.
We found that AP-positive cells also expressed GFP (data not
shown). Thus, from all the available evidence, the cells were
judged to be PGCs. In contrast, no AP positive cells were seen
when extra-embryonic ectoderm was cultured in the same
condition.
Emergence of PGCs in response to culturing
isolated whole epiblasts at 6.0-6.5 dpc on STO
feeder cells or on fibronectin
Whole epiblasts obtained from 6.0-6.5-a dpc embryos were
cultured on either an STO feeder layer (Donovan et al., 1986)
or on a fibronectin (FN)-coated plate. Table 1 shows that
approximately half of the explants of 6.25 and 6.5-a dpc
epiblasts gave rise to PGC, both on STO cells and on FN. On
an STO feeder layer, they formed a packed cluster of cells (Fig.
3A), whereas only a small cluster was found on FN (data not
shown). However, 6.0 dpc epiblasts formed a cluster and gave
rise to PGCs at a similar frequency only when they were
cultured on the feeder layer (Table 1). They formed a
monolayer of cells (data not shown) and no PGCs on FN (Table
1). These observations suggest that 6.0 dpc epiblasts require
some external support or signals to develop the appropriate
conditions for PGC formation, which in the present study was
considered to be represented by a cluster of cells.
Fig. 3. Emergence of primordial germ cells (PGCs) from early-streak
epiblasts in primary culture. (A) An explant with AP-positive cells
(arrows). A whole epiblast isolated from a 6.5-a dpc embryo was
cultured on an STO feeder layer for 72 hours, and stained for AP
activity. AP-positive cells were also positive for other markers for
PGCs, 4C9 (C) and Oct3/4(E, arrowheads), and showed the typical
morphology of migrating PGCs (B,C). B and D show counterstaining
by AP staining for C and E, respectively. AP expression within a cell
mass of an explanted epiblast was downregulated at the time of
staining, but some Oct3/4-positive cells remained (upper left in E).
Scale bar; A, 200 µm; B,C, 30 µm; D, E, 60 µm.
Table 1. Derivation of PGCs from isolated whole epiblasts
cultured on a feeder layer of STO cells (STO) or
fibronectin (FN)-coated dishes
Culture
method
Stage
(dpc)
Explants with PGCs/
total explants (%)
Range of PGC
numbers
STO
5.5
6.0
6.25
6.5-a
0/15 (0)
6/17 (35)
13/21 (62)
10/20 (50)
0
2-16
3-27
4-37
FN
6.0
6.25
6.5-a
0/21 (0)
21/49 (43)
10/21 (48)
0
1-30
3-34
PGC specification from epiblast
485
Fig. 4. PGC emergence from 6.25 dpc epiblasts without the expression of mesoderm markers. Double staining with AP and antibodies against
mesoderm markers was carried out on explants cultured on an STO feeder layer for 72 hours. (A-D) AP staining of explants shows the
emergence of PGCs (arrowheads indicate PGCs derived from cell masses of explants). The same explants were stained with Flk-1 (E,F),
Brachyury (G) and HNF-3β (H) antibodies. The expression of these markers was negative, except Flk-1, which was occasionally positive (F).
Scale bars; 200 µm.
It seems likely that this explant culture also supports the
development of cells of other lineages, and for this reason the
expression of mesodermal markers in the cultured explants of
proximal epiblasts at 6.0 and 6.25 dpc was examined
immunohistologically. We have examined Flk-1, which, in
vivo, is first expressed in the allantois and later in endothelial
cell precursors (Yamaguchi et al., 1993) that share common
precursor cells with PGCs. We have also tested Brachyury,
which is expressed in the proximal region of prestreak-stage
epiblasts and later in the gastrulating primitive streak
(Herrmann, 1991), and HNF-3β, which is expressed in the
prechordal mesoderm and their derivatives, the node and head
process (Sasaki and Hogan, 1993). As shown in Fig. 4E,G,H
explants were found to be Flk-1, Brachyury and HNF-3β
negative, even when PGCs were observed (Fig. 4A,C,D).
Although Flk-1 expression was sometimes detected (Fig. 4F),
other markers were not. These results suggest that mesoderm
cell differentiation does not progress well in this explant
culture, even when PGCs are differentiated.
PGCs specifically emerge from a fragment of
proximal epiblast consisting of a specific number of
cells
A heterotopic transplantation experiment (Tam and Zhou,
1996) has revealed that the fate of epiblast cells depends on
their position in the epiblast, i.e. localization in the proximal
Table 2. Derivation of PGCs
A From fragments of epiblast cultured on a feeder layer of STO cells
6.0 dpc
6.25 dpc
6.5-a dpc
Region of explant
Proximal
Distal
Proximal
Distal
Proximal
Distal
Explants with PGCs/total explants
(%)
4/11
(36)
0/11
(0)
13/21
(62)
0/21
(0)
10/22
(46)
0/22
(0)
Range of PGC numbers
2-5
0
1-45
0
2-34
0
B From subdivided proximal epiblasts on a feeder layer of Sl/Sl4 cells
6.0 dpc
6.25 dpc
No. of cells in a subdivided fragment
40-60
20-40
50-100
40-60
20-40
1-10
Whole
proximal epiblast
Explants with PGCs/total number of explants
(%)
4/10
(40)
1/13
(8)
5/15
(33)
2/7
(29)
2/4
(50)
0/24
(0)
7/20
(35)
Range of PGC numbers
2-5
1
1-7
1-11
7-9
0
4-7
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T. Yoshimizu, M. Obinata and Y. Matsui
Fig. 5. (A,B) AP staining of explants of proximal and
distal fragments of 6.25 dpc epiblasts cultured for 72
hours on an STO feeder layer. Proximal epiblast (A)
gave rise to a cluster of densely packed cells (arrow) and
PGCs (arrowheads), while distal epiblast (B) formed a
monolayer of cells and never gave rise to PGCs.
(C-E) The development of PGCs from dissociated
epiblast derived from 6.5-a dpc embryos after 72 hours
in culture. AP staining showed six PGCs (C,
arrowheads). AP-positive cells were also positive for
4C9 (D, E). Scale bar; A-C,100 µm; D,E, 20 µm.
region is necessary for them to differentiate into PGCs. To
explicate local effects on PGC determination further, an
epiblast was cut into proximal and distal fragments, and each
fragment was cultured on an STO feeder layer. As shown
in Table 2A, in all stages tested, 36-62% of the proximal
fragments gave rise to PGCs (Fig. 5A), but no distal
fragments did so (Fig. 5B). These results indicate that the
proximal fragment of an epiblast is sufficient for PGC
formation in culture, and that the region-specific competence
of epiblast cells that is required for PGC determination
is already established at 6.0 dpc. Proximal and distal
explants were also distinct with regard to their morphology
after culture, i.e. proximal explants formed a packed
cluster of cells (Fig. 5A), while distal explants spread out
into a monolayer (Fig. 5B). The cluster of cells may
provide the environmental interaction required for PGC
differentiation.
We next cultured proximal epiblast fragments cut into up
to four pieces (Fig. 2A,f,h shows an example of four pieces)
on a feeder layer of Sl/Sl4 cells (Matsui et al., 1991), which
we had previously shown do not support proliferation of
PGCs. As shown in Table 2B, a proximal fragment (up to one
fifth of a proximal fragment) of 6.25 dpc epiblast cells could
yield PGCs at a similar frequency to those raised on an
STO feeder layer. The number of cells within these small
subfragments of epiblast were easily counted under an
inverted microscope, and only 20-40 cells were counted.
However epiblasts dissociated by trypsinization failed to give
rise to PGCs. After trypsinization, the cell suspensions
contained single cells as well as a small number of aggregates
consisting of less than 10 cells. With 6.0 dpc epiblasts, one
third of the fragments (found in 40-60 cells) gave rise to
PGCs, but the frequency of PGC formation from smaller
fragments was decreased from 40% to 8%. These results
indicate that PGC formation requires a rather small mass of
epiblast cells, consisting of 40-60 cells at the start of the
culture period, and that close cell interactions appear to be
essential. In addition, these results suggest that normal
gastrulation is not required for PGC determination.
Emergence of PGCs is independent of cell-cell
interaction after the onset of gastrulation
We further tested the requirement for cell interactions among
epiblast cells for PGC differentiation in later embryos (Table
3). After trypsinization, we cultured cell suspensions obtained
from 6.5-a, 6.5-b and 6.75 dpc embryos on an STO feeder
layer for 66-72 hours. As shown in Table 3, PGCs were never
formed from dissociated 6.5-a dpc epiblast, but thereafter the
frequency of PGC emergence gradually rose, with 29% and
70%, respectively, of dissociated epiblasts at 6.5-b and 6.75
dpc giving rise to PGCs. AP-positive cells (Fig. 5C) that
emerged in the cultures were also positive for 4C9 (Fig. 5D,E).
These results indicate that cell interactions among epiblast cells
becomes less important for PGC differentiation shortly after
the onset of gastrulation.
Table 3. Derivation of PGCs from dissociated early- to
mid-streak epiblasts
Stage
(dpc)
Explants with PGCs/
total explants (%)
Range of
PGC numbers
6.5-a
6.5-b
6.75
0/20 (0)
5/17 (29)
14/22 (70)
0
13-30
9-34
PGC specification from epiblast
487
Fig. 6. Recombination culture
of epiblast and extra-embryonic
ectoderm. Arrowheads indicate
PGCs. 5.5 dpc epiblasts were
cultured alone (A) or with
extra-embryonic ectoderm (B).
(C) A 6.5-b dpc distal epiblast
was asynchronously
recombined with 5.5 dpc extraembryonic ectoderm. (D) A 5.5
dpc distal epiblast was
asynchronously recombined
with 6.5-b dpc extra-embryonic
ectoderm. Scale bar; 200 µm.
The extra-embryonic ectoderm induces the proximal
epiblast-specific competence for PGC formation
before 6.0 dpc.
Isolated 5.5 dpc epiblasts free of extra-embryonic tissues never
gave rise to PGCs in culture (Table 1). In order to investigate
the effect of the extra-embryonic ectoderm on germline
development, 5.5 dpc epiblasts with or without extraembryonic ectoderm were isolated free of visceral endoderm
and cultured on an STO feeder layer. Explants were fixed after
90-96 hours in culture, and the emergence of PGCs was
examined by staining for AP activity (Fig. 6A,B). The
emergence of PGCs as well as overall cell proliferation and the
subsequent formation of packed cluster of cells appeared to
depend completely on the existence of the extra-embryonic
ectoderm (Fig. 6A,B; Tables 1, 4).
Table 4. PGC emergence from epiblasts recombined with
extraembryonic ectoderm
Type of explants
Non recombination
5.5 dpc extraembryonic ectoderm
+ 5.5 dpc whole epiblast
Synchronous recombination
5.5 dpc extraembryonic ectoderm
+ 5.5 dpc distal epiblast
6.5-b dpc extraembryonic ectoderm
+ 6.5-b dpc distal epiblast
6.75 dpc extraembryonic ectoderm
+ 6.75 dpc distal epiblast
Asynchronous recombination
5.5 dpc extraembryonic ectoderm
+ 6.5-b dpc distal epiblast
6.5-b dpc extraembryonic ectoderm
+ 5.5 dpc distal epiblast
Explants with PGCs/
total explants
(%)
11/30
(37)
Distal epiblasts at early-streak stages respond to
putative inducing signals from the extra-embryonic
ectoderm
As described above, we demonstrated that 5.5 dpc epiblasts can
respond to the extra-embryonic ectoderm, thereby acquiring
the ability to differentiate into PGCs. We next examined
whether even later epiblasts had the competence to respond to
the extra-embryonic ectoderm and whether even later extraembryonic ectoderm could induce competence in epiblasts. To
answer these questions, we isolated fragments of distal epiblast
at 5.5, 6.5-b and 6.75 dpc that could not alone give rise to PGCs
and recombined them with extra-embryonic ectoderm at the
same developmental stages (synchronous recombination). We
found PGC differentiation in all of these cultures (Table 4),
indicating that distal epiblast cells and extra-embryonic
ectoderm at 6.5-b and 6.75 dpc maintain competence and
induction ability, respectively. We next examined whether
recombination of distal epiblast and extra-embryonic
ectoderm at different developmental stages (asynchronous
recombination) could give rise to PGCs. PGC differentiation
was also observed in these cases (Table 4; Fig. 6C,D).
DISCUSSION
2/2
(100)
4/11
(36)
4/23
(17)
5/16
(32)
11/27
(41)
These results demonstrate that primordial germ cell (PGC)
differentiation from epiblast cells in mouse embryos is
accomplished by stage-specific cell or tissue interactions. At
the beginning of this study, we tried to culture isolated epiblasts
without any extra-embryonic tissue at an early-streak stage
(6.5-a dpc) on a feeder layer of STO cells known to support
proliferation of 8.5 dpc PGCs in primary culture (Donovan et
al, 1986). As shown in Fig. 3A, AP-positive cells crawling
out from an explant were observed. These AP-positive cells
were also positive for 4C9 (Fig. 3B,C) and Oct3/4 (Fig. 3D,E,
488
T. Yoshimizu, M. Obinata and Y. Matsui
arrowheads) as detected by immunostaining. Although the
expression of these markers was downregulated in explants,
epiblast cells were positive for all of these markers before
culture. Based on the above and the additional criteria
described below, we were able to confirm the identity of these
cells as PGCs. First, these marker-positive cells showed, in
many cases, the morphological features of migrating PGCs. In
addition, the cells were first observed after 2 days in culture,
which corresponded to the timing of the emergence of
migrating PGCs in vivo. Furthermore, we cultured epiblasts of
transgenic mice expressing GFP under the control of Oct3/4
gene promoter lacking proximal enhancer (GOF18-delta PE;
Yoshimizu et al., 1999) to identify PGCs in the culture.
As described previously, GFP expression was specifically
observed in PGCs but not in epiblast cells in the transgenic
mice. After culturing epiblasts of the transgenic mice, most of
the AP-positive cells also expressed GFP (data not shown),
indicating that they were PGCs but not epiblast cells.
When fragments of epiblasts or dissociated epiblast cells
from 6.5-b or 6.75 dpc were cultured, or when epiblasts were
cultured on a feeder layer of Sl/Sl4, AP-positive cells often
did not exhibit typical migratory morphology. It is unlikely,
however, that these cells represent undifferentiated epiblast
cells. For example, 6.5-a dpc dissociated epiblasts did not
produce any AP-positive cells (Table 3), suggesting that
dissociated epiblast cells are not maintained as AP-positive
undifferentiated cells unless differentiation to PGCs occurs
under these culture conditions. It has been observed that PGCs
do not have pseudopodia before they occur in hind gut
endoderm (Snow and Monk, 1983), suggesting that earlier
PGCs do not undergo active migration but instead passively
move into hind gut endoderm, along with a morphogenetic
expansion of embryonic tissues. Therefore, one possibility is
that tissues, including hind gut endoderm, in which PGCs
differentiate to become migratory, do not develop properly
from fragmented epiblasts either on a feeder layer of Sl/Sl4 or
under dissociated culture conditions.
We initially used a feeder layer for culturing isolated
epiblast, which raised the question of whether feeder cells
provide factors that support the emergence of PGCs. To clarify
this point, we cultured epiblast cells on fibronectin (FN)coated plates (Table 1). We found PGC differentiation on FN
was of a similar frequency to that on a feeder layer of STO
cells when epiblasts at 6.25 dpc onwards were cultured. The
same result was also obtained by culturing epiblast on
biologically non-active poly-D-lysine-coated plates (data not
shown). These observations indicate that the emergence of
PGCs from isolated epiblasts at 6.25 dpc onwards is
independent of the support of a feeder layer and that epiblast
can establish all conditions required for PGC formation by
themselves. In contrast, PGC differentiation from 6.0 dpc
epiblast appears to depend on the presence of a feeder layer.
We also found that the explants always formed packed clusters
of cells when PGCs emerge (Fig. 5A,B and data not shown).
These observations suggest that the feeder layer may provide
external signals that could help 6.0 dpc epiblasts to maintain
the three-dimensional tissue organization and/or simply the
cell proliferation that may be necessary for subsequent PGC
differentiation.
Our results also indicated that Steel factor, which is known
to be essential for PGC growth and survival (Dolci et al., 1991;
Godin et al., 1991; Matsui et al., 1991), is not necessary for
PGC determination because a feeder layer of Sl/Sl4 cells
lacking the Steel factor gene also supported PGC formation in
culture (Table 2B). Although the frequencies of explants with
PGCs on Sl/Sl4 and on STO feeder layers were similar, the
number of PGCs on STO cells was more than twice that on
Sl/Sl4 cells (Tables 1, 2). In culture, Steel factor may support
the growth of newly formed PGCs. This observation is
consistent with the phenotype of Sl or W mutant mice, which
are deficient in Steel factor or its receptor gene c-kit,
respectively (Williams et al., 1992). In these mutant mice, a
normal number of PGCs is observed in early embryos, but
these PGCs fail to increase in number (Mintz and Russell,
1957).
We observed PGC differentiation from proximal fragments
of epiblasts after 6.0 dpc, but distal fragments never gave
rise to PGCs (Table 2A), suggesting that cell populations in
proximal and distal regions have distinct character with regard
to PGC formation. In other words, the specific local cues or
competence required for differentiation into PGCs may be
established within the proximal region. In addition, the
differentiation of PGCs was not observed from trypsinized
epiblasts. PGCs arose from a small subfragment consisting of
a specific number of cells or of the two most proximal rows of
epiblast cells (Table 2B and data not shown). The minimum
number of cells required was found to be 40 and 20 at 6.0 and
6.25 dpc, respectively. Taken together, these observations
suggest that presumptive local cues within proximal epiblast
are maintained among a small but specific number of cells.
We have shown that the functions of the extra-embryonic
ectoderm are essential for PGC determination, and a study by
Lawson et al. has demonstrated that BMP4 expressed in the
extra-embryonic ectoderm may play a key role (Lawson et al.,
1999). A 5.5 dpc epiblast required extra-embryonic ectoderm
to form PGCs, while isolated proximal epiblasts without extraembryonic ectoderm at 6.0 dpc did give rise to PGCs (Tables
1, 2 and 4). These results indicate that the functions of the
extra-embryonic ectoderm before 6.0 dpc are critical for
establishing putative local cues and subsequent PGC
formation, and that BMP4 could be involved in this process.
BMP4 expression in the extra-embryonic ectoderm is,
however, observed even at later stages (Lawson et al., 1999),
suggesting that it has additional and/or alternative functions for
PGC formation at later stages. Consistent with this hypothesis,
we found that extra-embryonic ectoderm at 6.5-b and 6.75 dpc
is fully active in inducing PGCs (Table 4). The extraembryonic ectoderm stimulates the growth of epiblast in
culture (Fig. 6 and data not shown), which might consequently
support differentiation of the putative progenitors of PGCs in
the proximal region at later stages.
Previous experiments involving heterotopic transplantation
of epiblast cells have indicated that even distal epiblast cells,
which normally gave rise to neuroectoderm, have the ability to
differentiate into PGCs, but that the proximal localization of
cells is required (Tam and Zhou, 1996). Our result also showed
that isolated distal epiblast fails to give rise to PGCs, but in the
presence of extra-embryonic ectoderm, PGCs are formed
(Table 4). Together these observations suggest that the
proximal localization of cells is necessary for receiving signals
from extra-embryonic ectoderm, which may induce putative
local competence or cues within a population of totipotent
PGC specification from epiblast
epiblast cells. The recombination experiment also indicated
that distal epiblast after the onset of gastrulation (6.75 dpc) still
maintains the ability to respond to extra-embryonic ectoderm
and to give rise to PGCs (Table 4, Fig. 6) suggesting a
remarkable plasticity of gastrulating epiblast cells with regard
to the restriction of cell lineage, including germ line lineage.
Snow found that fragments of different parts of epiblast at 7.0
dpc follow the same fates in culture as in vivo, and that cells
differentiate autonomously into PGCs as well as into other cell
lineages, including mesoderm (Snow, 1981). In contrast, preand early-streak epiblasts failed to express mesoderm markers
such as Brachyury, Flk-1, and HNF-3β in our culture (Fig. 4).
The development of cells in 7.0 dpc epiblast may progress with
regard to the lineage restriction of somatic cells, which may
explain why they can follow various somatic cell fates, even in
culture. In contrast, the culture of pre- or early-streak epiblast
may not be able to fully reproduce gastrulation, which may result
in a failure to express mesodermal markers in culture. In
particular, recent studies have implicated Brachyury (Wilson et
al., 1993; Wilson et al., 1995) and FGF (Ciruna et al., 1997; Sun
et al., 1999) signaling in the control of migration of epiblast cells
through the primitive streak. If this is so then the failure to
express Brachyury further suggests that gastrulation does not
occur in our explant culture. Even if gastrulation did not occur
properly, however, PGCs did emerge in our culture, which is
consistent with observations of the emergence of AP-positive
cells in eed mutant embryos that fail to undergo gastrulation
(Faust et al., 1995). Taken together, these observations suggest
that PGC differentiation need not be accompanied by
gastrulation. The expression of Flk-1 should also be noted. Flk1 is normally expressed over the nascent extra-embryonic
mesoderm area after passing through the primitive streak
(Kataoka et al., 1997). Based on fate-map analysis, PGCs and
Flk-1-expressing mesoderm cells should be derived from
common precursors (Lawson and Hage, 1994). Therefore, if the
differentiation of PGCs and mesoderm cells is concomitantly
regulated, Flk-1 must always be expressed when PGCs emerge
in culture. This is not, however, the case.
Our explant culture described here provided novel
approaches to studying the mechanisms of mouse germline
formation. This culture system may also be useful for
identifying and/or analyzing the functions of molecules
involved in germline formation in future experiments.
We thank T. Muramatsu for 4C9 antibody, H. Hamada for Oct3/4
antidbody, S.-I. Nishikawa for Flk-1 antibody, B. Hermann and H.
Koseki for Brachyury antibody, H. Sasaki for HNF-3β antibody. This
work was supported in part by the grants-in-aid from the Ministry of
Education, Science, Sports and Culture of Japan, and by the Science
and Technology Agency of Japan, with Special coordinating funds for
Promoting Science and Technology.
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