1 Division of Developmental Biology, Cincinnati Children's Hospital Research
Foundation, Cincinnati, OH 45229, USA
2 Department of Pathology, The University of Iowa Roy J. and Lucille A. Carver
College of Medicine, Iowa City, IA 52242, USA
* Author for correspondence (e-mail: christopher.wylie{at}chmcc.org)
Accepted 25 September 2003
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SUMMARY |
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Key words: Mouse, Bax, Cell death, Primordial germ cells
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Introduction |
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Cell death is also a feature of germ cells that do arrive successfully in
the genital ridges. Gonadal male germ cells undergo a wave of apoptosis
between E13.5 and E17.0 (Coucouvanis et
al., 1993), followed by a second wave of cell death around the
time of birth, which depletes the number of male gonadal germ cells by around
50% (Roosen-Runge and Leik,
1968
). Female gonadal germ cells also die in two phases: at E13.5,
and between E15.5 and birth (Bakken and
McClanahan, 1978
; Beaumont and
Mandl, 1962
; Borum,
1961
; Gondos,
1978
). Cell death in the mouse germ line is due to apoptosis
(Coucouvanis et al., 1993
;
Ratts et al., 1995
;
Wang et al., 1998
). It is not
known whether the mechanism of cell death of ectopic primordial germ cells
that fail to arrive in the genital ridges is the same as that occurring later
in germ cell differentiation.
Several studies have implicated the Steel/Kit interaction in survival of
migrating primordial germ cells. In vivo, mutations in either gene lead to
decreased numbers of germ cells arriving in the gonads
(Bennett, 1956;
Mintz and Russell, 1957
), and
in culture, withdrawal of Steel factor causes decreased survival of explanted
germ cells (Dolci et al.,
1991
; Godin et al.,
1991
; Matsui et al.,
1991
), which die by apoptosis
(Pesce et al., 1993
). The
pathway downstream of the Kit receptor in primordial germ cells is unknown. In
addition, other secreted factors have been reported to increase the survival
of cultured germ cells, including LIF (De
Felici, 2000
; Koshimizu et
al., 1996
) and IL4 (Cooke et
al., 1996
).
Bcl2 family members have been implicated in later stages of intragonadal
germ cell apoptosis. Mice homozygous for mutations in the pro-apoptotic family
member Bax exhibit hyperplasia in the spermatogonial layers of the
seminiferous tubules, and the germ cells fail to differentiate
(Knudson et al., 1995). Female
Bax-/- mice possess three times as many primordial
follicles as Bax+/+ equivalents, and these follicles last
for a greater proportion than usual of the life-cycle
(Perez et al., 1999
). In
gonadal germ cells, the requirement for Bax for germ cell apoptosis is
balanced by a requirement for the anti-apoptotic family member Bcl2l
(previously known as Bclx) for their survival. Mice carrying two copies of a
hypomorphic allele of Bcl2l have reduced numbers of germ cells in the gonads
of both sexes, and this phenotype was rescued by removing Bax, indicating that
a balance of these two pro- and anti-apoptotic Bcl2 family members controls
the numbers of germ cells in the developing gonads
(Rucker et al., 2000
).
However, the numbers of germ cells that colonize the gonads are normal in
embryos carrying the hypomorphic allele of Bcl2l
(Rucker et al., 2000
),
indicating that Bcl2l is not required before this time. If this anti-apoptotic
protein was required during migration, more of the migrating germ cells would
die, and fewer would colonize the gonads.
In previous work, using a living marker of germ cells, we have shown that
germ cells leave the hindgut between E9.0 and E9.5, and migrate laterally
towards each gonad (Molyneaux et al.,
2001; Molyneaux et al.,
2003
). By E10.5, the embryos has grown significantly in size, and
a mesentery has formed that displaces the hindgut from the dorsal body wall,
and increases the distance of migration. Germ cells are left at all points
along the path from the hindgut to the genital ridges. Movies of slice
cultures of embryos at E10.5 showed that those in the midline, or in the
mesentery of the gut, at E10.5, do not migrate to the genital ridges. Instead,
they disintegrate and die, while the germ cells closest to the genital ridges
migrate into it and survive (Molyneaux et
al., 2001
). This mechanism generates a tight cluster of germ cells
in and around the genital ridges by E11.5, with few ectopic germ cells.
The mechanism by which the germ cells left in the middle of the embryo are
eliminated is unknown. It is likely to be different from the mechanism that
maintains germ cell numbers later in the gonads, as the anti-apoptotic Bcl2l
protein is not required for normal numbers of germ cells to colonize the
genital ridges. To initiate germ cell death, a pro-apoptotic gene has to be
activated. Bax has not previously been shown to be present during germ cell
migration, but is a likely candidate for a pro-apoptotic function, as it has a
role later in development in the germline. In light of the above studies, we
bred mice containing a targeted allele of Bax
(Knudson et al., 1995) into a
line of mice carrying the Oct4
PE:GFP transgene
(Anderson et al., 2000
), which
allows direct observation of germ cell behavior in living embryos or embryo
slices (Molyneaux et al.,
2001
). We report a significantly increased number of ectopic germ
cells in Bax-/- embryos. The ectopic germ cells are
developmentally delayed. They retain expression of early PGC markers and
retain motility. This shows that signals from the gonad regulate expression of
PGC markers and inhibit their motility. Bax-/- ectopic
germ cells occupy many positions in the embryo. However, they do not grow, and
their numbers dwindle until by E18.5 very few can be found. As these mice have
not been reported to have an increased incidence of germline tumors, the data
suggest that mice have a back-up mechanism for removing embryonic migratory
germ cells in ectopic locations. We also show that inactivation of Bax
protects germ cells against rapid cell death in culture, and against removal
of the Steel/Kit signaling interaction in culture. This shows that Bax is
downstream of the Kit receptor. However, protection against cell death in
culture is a short-term effect, showing that other apoptotic pathways exist in
germ cells.
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Materials and methods |
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Organ culture
Embryos were obtained by euthanizing pregnant females with C02,
followed by cervical dislocation, and immediate extraction of the uterus from
the female. Embryos were removed from the uterus and extra-embryonic tissue in
1xPBS + 2% FCS, and then placed in Hepes-buffered DMEM/F-12 medium plus
100 U/ml penicillin/streptomycin (Gibco BRL), which we designate as DF-12
media. Slices were cut from the trunk of embryos using a scalpel. Slices in
50 µl of DF-12 were placed into millicell CM organ culture chambers
(Millipore) pre-coated with collagen IV (Becton Dickinson). The millicell
organ chambers were placed into 50 mm glass-bottomed culture dishes (Willco
Wells, the Netherlands), and the dishes were filled with
2-3 ml DF-12
medium. Filming was carried out with a Zeiss LSM510 confocal system attached
to a Zeiss axiovert inverted scope. Images were captured during filming every
7 minutes for 700 minutes. Organ cultures were maintained at 37°C during
filming by placing the culture dish in a heating stage (Zeiss), and creating a
humidity chamber with wet paper towels placed in a 100 mm culture dish
fastened over the organ culture dish.
Wholemounts, cryosections and antibody staining
Gonads were extracted from pregnant females as stated above. Whole embryos
were fixed in 4% PFA (Sigma) overnight at 4°C, then washed twice with PBS.
For cryosections, embryos were submerged in 20% sucrose and embedded in OCT
medium. For wholemounts, gonads or partial embryos were placed in PBS + 0.1%
TritonX100 (Fisher) overnight at 4°C. SSEA1 antibody (The Developmental
Studies Hybridoma Bank, University of Iowa), an IgM mouse monoclonal antibody,
was used at a 1:100 dilution (4°C overnight). Samples were washed five
times for 1 hour in PBS+0.1% Tx-100 and exposed to the secondary antibody, an
anti-mouse IgM Cy5 conjugate (Jackson ImmunoResearch) at a 1:100 dilution
(4°C overnight). Samples were washed as described above and mounted in 80%
glycerol. Alkaline phosphatase staining followed the protocol outlined by
Hogan et al. (Hogan et al.,
1994). Whole gonads were exposed to AP staining solution for 12
minutes prior to being washed in PBS and cleared overnight in 80% glycerol.
AP-stained gonads were viewed with the Cy3 channel on the LSM510 confocal
microscope.
Cell culture
Feeder layers consisted of either irradiated MEF cells or irradiated STO
cells plated in a 12-well organ culture plate (Becton Dickinson) at 80%
confluency in 1.25 ml of DMEM (Gibco BRL) + 10% FCS (Gibco BRL) + 2 mM
reconstituted glutamine (Gibco BRL) + 1x antimycotic/antibiotic (Gibco
BRL), which we designated MEFs Medium. For the E10.5 PGC culture experiments,
the area between forelimb and hindlimb buds was isolated using a scalpel, and
the neural tube and notochord were excised and discarded. In the E12.5
experiments, whole gonads were isolated along with the dorsal aorta and
midline mesenchymal tissue. Hindlimb and tail tissue was frozen for
genotyping. PGC-containing tissue was trypsinized (Gibco BRL) for a 12 minute
interval, triturated and added to medium-laden feeder cell plates (one embryo
per well). Initial PGC counts were calculated using a hemacytometer (Fisher).
All counts were done on a Zeiss Axiovert 100M inverted fluorescence
microscope. Genotyping of embryos was carried out after the experiment and all
counts had been concluded. ACK-2 (22.5 mg/ml stock) and J2.1 (20.2 mg/ml
stock) were a kind gift from Dr Fred Finkelman (Veterans Hospital, Cincinnati,
OH). ACK-2 and J1.2 were diluted to the respective amounts in MEFs medium and
added directly to the feeder cells prior to addition of trypsinized gonads. AP
staining of cultured PGCs was done with incubation of cell culture wells in AP
staining solution (described above) for 4 minutes, and images were taken with
the LSM510 confocal microscope. AP staining was visualized with the Cy3
channel on the LSM 510 confocal microscope.
RT-PCR
PGC isolation and reverse transcription was performed as previously
described (Molyneaux et al.,
2003). PGC and somatic cDNAs were diluted 1:10 and 1 µl used
for PCR (25 µl). RedMix Plus (PGC Scientific) was used as the source of
Taq, buffer and dNTPs. Final primer concentrations were 0.4 µM. PCR
consisted of an initial denaturing step of 5 minutes at 95°C; followed by
5 cycles of 30 seconds at 95°C, 1 minute at 65°C, 30 seconds at
72°C; followed by 35 cycles of 30 seconds at 95°C, 1 minute at
60°C, 30 seconds at 72°C; followed by a 10 minute extension step at
72°C. Bax primers (Rucker at al.,
2000
) generate a 162 bp product: BaxF,
5'-ATGCGTCCACCAAGAAGCTGAG-3'; BaxR,
5'-CCCCAGTTGAAGTTGCCATCAG-3'.
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Results |
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Slice cultures taken from Oct4PE:GFP embryos have been useful in
studying the behavior of living germ cells during migration
(Molyneaux et al., 2001
).
Using this method, we found previously that during the E10.5 to E11 period,
germ cells have already emerged from the hindgut, and migrate laterally both
singly and in aggregating clusters, to the gonadal ridges; while germ cells
remaining distant from the genital ridges, in the midline or in the mesentery,
fragment and die (Molyneaux et al.,
2001
). To identify the role of Bax in this process, we filmed
slices dissected from Bax-/-, Bax+/-
and Bax+/+ littermates. We crossed
Bax+/- Oct4DPE:GFP+/+ animals, harvested
embryos at E10.5 and cut two transverse 200 µm slices through each embryo.
Slices from an entire litter were filmed simultaneously for a 12 hour period.
The cranial end of each embryo was genotyped by PCR. The genetic constitution
of one such litter was: 2xBax+/+,
3xBax+/- and 4xhomozygote
Bax-/-. Fig.
2 shows images from different time-points of movies from the
wild-type (Fig. 2A,B) and
Bax-/- (Fig.
2C-F) slices. In the wild type, germ cells that failed to migrate
with the main group of germ cells, and instead remained in the mesentery and
midline area, fragmented and disappeared during the 700 minute time period
(4/4 slices), leaving the central area of the embryo clear of germ cells.
Those in the Bax-/- slices survived (7/8 slices). Movies
are available online at
http://dev.biologists.org/supplemental/.
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Loss of Bax causes temporary, but not permanent, protection from cell
death of germ cells when explanted into culture
Primordial germ cells explanted into culture are extremely sensitive to the
absence of survival signals, and will only survive for short periods in
culture, and require specific types of feeder layer to do so
(Donovan et al., 1986). As Bax
is required for the death of germ cells seen in the mesentery and midline
during migration at E10.5, we hypothesized that Bax-/-
germ cells explanted into culture, and thus removed from their normal signals,
might be rescued from the high levels of cell death normally seen. Germ cells
were isolated from E10.5 and E12.5 embryos, and cultured on either STO cells,
or primary cultures of mouse embryonic fibroblasts (MEFs), both of which have
been shown to promote germ cell survival. In each experiment, each embryo from
a single litter was taken, the region containing the genital ridges dissected
as described previously (Cooke et al.,
1993
) and the rest of the embryo used for genotyping. The genital
ridges were disaggregated, and the number of germ cells in each separate
embryo determined by hemocytometer counting. Harvested cells from each embryo
were then cultured separately on either STO of MEFs and the germ cells counted
at different time intervals under the fluorescent microscope. Because the germ
cells express GFP, the same living cultures can be scored for germ cell number
at different time-points. In order to formally exclude the possibility that
other cell types express the Oct4/GFP transgene in culture, we co-stained one
culture with alkaline phosphatase to confirm that the GFP positive cells were
germ cells (data not shown). Fig.
5 and Table 1 show
data from two E10.5 litters and one E12.5 litter. First, the numbers of germ
cells present in the cultures at 48 hours (which we have defined as `survival'
in Fig. 5 and
Table 1, although the numbers
will reflect cell division as well as failure to die) was variable from
experiment to experiment; a greater percentage of germ cells survived in the
second batch of embryos. We routinely find this in culture experiments, and it
is the reason for the experimental design used here: to make an
embryo-by-embryo comparison within single litters. The variability from
experiment to experiment may be due to the health of the MEFs, or the
stringency with which embryo dissections from different experiments are
trypsinized. Second, it shows that large numbers of germ cells died in
culture, even when seeded onto feeder layers that promote their survival. This
is also a common observation. Third, it is clear that there is a significant
difference between the percentage survival of Bax-/- germ
cells, compared with both Bax+/+ and heterozygous germ
cells. There is also a significant difference between germ cell survival
between Bax+/+ and heterozygous embryos, indicating a gene
dosage effect of Bax. The same result was seen with E12.5 embryos. It has
previously been reported that fewer germs cells survive from E12.5 embryos
than from E10.5 embryos (Donovan et al.,
1986
), and this is seen again in
Fig. 5. However, as at E10.5,
loss of Bax function has a rescuing effect on the germ cells.
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Interaction between Bax and Steel/Kit signaling
As Steel factor is a known survival factor for early mouse germ cells, we
tested whether cell death following inhibition of Steel factor is mediated by
Bax. The germ cell-containing regions of individual E10.5 embryos from
Bax+/-,Oct4DPE:GFP+/+ crosses were dissected as
described previously (Cooke et al.,
1993). The germ cell number in each cell suspension was counted on
a hemocytometer, and the cell suspensions were then split and seeded onto MEF
or STO cell feeder layers in the presence of either the Kit blocking antibody
Ack-2 (Okada et al., 1991
) or
an isotype-matched control immunoglobulin J1.2. The Ack-2 antibody has been
previously shown to block the survival of cultured germ cells
(Matsui et al., 1991
).
Fig. 6 and
Table 2 show that the absence
of Bax protected germ cells against cell death by of the Steel/Kit
interaction, and therefore that Bax is an essential downstream component of
germ cell death following loss of the Steel/Kit signaling interaction.
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Discussion |
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The visible survival of ectopic germ cells in the midline of the embryo
slices suggests that there is a defined range of survival signals being
released by the genital ridges, that the mesentery and midline body wall are
outside of this range, and that the absence of such signals activates a
Bax-mediated cell death event. Such a mechanism is elegant, because as the
embryo grows (it grows twofold in size between E9.5 and 11.5), and if the
range of survival signals stays constant, it progressively restricts the
survival of germ cells to a smaller and smaller component of the embryo, and
acts as a back-up mechanism to that of directed cell migration. There is
strong evidence that the survival signal is Steel factor. It has been
previously shown to be essential to germ cell survival in vivo, and in vitro.
In this study, we show that withdrawal of Steel signaling using a blocking
antibody against Kit, which has been previously shown to rapidly kill cultured
germ cells, is not as effective in killing Bax-/- germ cells,
showing that Bax acts downstream of the Kit receptor. This observation fits
nicely with those of Felici et al. (Felici
et al., 1999), who showed that Bax protein is upregulated in germ
cells undergoing apoptosis in culture, and that addition of Steel factor
prevented this rise.
Although we cannot exclude the possibility that the later disappearance of ectopic germ cells is due to loss of expression of the GFP marker, perhaps by differentiation, the failure of Bax-/- germ cells to survive for long periods in culture suggests that there is an alternative mechanism for their cell death. This could be due to the replacement later in development of the Steel/Kit/Bax mechanism by different survival factors and/or proapoptotic proteins. It also suggests that Bax functions specifically to remove germ cells that are out of range of early survival signals that support the mechanism of directional migration of germ cells to the genital ridges. If germ cells survive this early mechanism of elimination, it is replaced by another one later in development. The fact that the Bax-/- mouse does not have an increased incidence of germline tumors supports this idea. In order to gain insights into the etiology of extragonadal germline tumors, it will be necessary to identify this later cell death mechanism.
An interesting finding in this study is that the large number of ectopic PGCs in the Bax-/- embryos continue to be motile, and continue to express surface markers of their earlier migratory stage. These data suggest that contact with the somatic cells of the gonad, rather than an inherent timing mechanism, is required to switch off the early migratory phenotype of the germ cells. It is likely that germ line tumors arise from ectopic germ cells that have escaped these signals.
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ACKNOWLEDGMENTS |
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Footnotes |
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