1 Department of Molecular Cell Biology, Research Institute for Microbial
Diseases, Osaka University, Osaka 565-0871, Japan
2 Department of Biochemistry, Akita University School of Medicine, Akita
010-8543, Japan
3 Department of Laboratory Sciences for Animal Experimentation, Research
Institute for Microbial Diseases, Osaka University, Osaka 565-0871,
Japan
4 Samuel Lunenfeld Research Institute, Toronto, Ontario M5G 1X5, Canada
5 Amgen Research Institute, Ontario Cancer Institute, and University of Toronto,
Toronto, Ontario M5G 2C1, Canada
* Author for correspondenc (e-mail: tnakano{at}biken.osaka-u.ac.jp)
Accepted 14 January 2003
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SUMMARY |
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Key words: PTEN, Germ cells, Teratoma, EG cells, Mouse, Human
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INTRODUCTION |
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PTEN contains a protein tyrosine phosphatase domain that is similar to
those of the dual-specificity phosphatases, which dephosphorylate both
phosphotyrosine and serine/threonine residues
(Li and Sun, 1997;
Myers et al., 1997
). In spite
of the homology with dual-specificity phosphatases, the in vitro protein
phosphatase activity of PTEN is not high, and PTEN is very active on a highly
acidic substrate. Both in vitro and in vivo analyses indicate that the major
substrate of PTEN is the lipid phosphatidylinositol (3,4,5)-triphosphate
[PtdIns(3,4,5)P3], which is a direct product of PI3-kinase
(PI3K) (Maehama and Dixon,
1998
; Myers et al.,
1998
; Stambolic et al.,
1998
). This substrate specificity has been confirmed by the
crystal structure of PTEN (Lee et al.,
1999
). Loss of PTEN function in embryonic stem (ES) cells and
human cancer cell lines results in PtdIns(3,4,5)P3
accumulation and the activation of its downstream signaling molecule, Akt/PKB
(Li and Sun, 1998
;
Li et al., 1998
;
Ramaswamy et al., 1999
;
Sun et al., 1999
;
Wu et al., 1998
).
Subsequently, activation of the PI3K/Akt pathway by the loss of PTEN
stimulates various biological functions, such as cell cycle progression, cell
survival and cell migration.
Several studies, including ours, have sought to elucidate the in vivo role
of PTEN in development and tumorigenesis. PTEN is crucial for normal
development, as homozygous Pten-null mice show overgrowth of cells
and disorganized cell layers in the cephalic and caudal regions, and die
during early embryogenesis at E6.5-9.5 (Di
Cristofano et al., 1998;
Podsypanina et al., 1999
;
Suzuki et al., 1998
). In
addition, mice that are heterozygous for the Pten-null mutation
develop a broad range of tumors. The Cre-LoxP conditional gene-targeting
system was adopted to analyze the in vivo function of PTEN in development,
differentiation and tumorigenesis (Backman
et al., 2001
; Groszer et al.,
2001
; Kwon et al.,
2001
; Li et al.,
2002
; Marino et al.,
2002
; Suzuki et al.,
2001
). Somatic cell lineage-specific Pten-null mice have
shown distinct functions of PTEN in different organs. For example,
T-cell-specific deficiencies lead to defects in immunological tolerance and
subsequent autoimmune disease (Suzuki et
al., 2001
). Nerve cell-specific loss leads to increased
proliferation and self-renewal of neural stem and/or progenitor cells
(Groszer et al., 2001
).
Primordial germ cells (PGCs) are germ cell precursors that emerge around
E7.5 and exist transiently in embryos
(Wylie, 1999). PGCs migrate
into the genital ridges and eventually differentiate into eggs and sperm.
Although PGCs are committed to the germ cell lineage, two lines of evidence
suggest that mammalian PGCs can de-differentiate into cells that have broader
developmental potential. First, PGCs can give rise to testicular teratomas
with various types of differentiated cells when grafted to adult testis
(Stevens, 1967
;
Stevens, 1984
). Second,
pluripotent embryonic germ (EG) cells can be established by culturing PGCs in
vitro in the presence of SCF (Stem cell factor), LIF (leukemia inhibitory
factor) and bFGF (basic fibroblast growth factor)
(Matsui et al., 1992
;
Resnick et al., 1992
).
However, it is likely that unknown mechanisms operate to prevent PGC
de-differentiation, as testicular teratoma is rare in normal mice
(Stevens, 1967
) and PGCs
cannot be incorporated into normal development even if injected into
blastocysts (Labosky et al.,
1994a
). In addition, clinical testicular teratomas are known to
develop from PGCs (Jiang and Nadeau,
2001
; Looijenga and
Oosterhuis, 1999
).
In order to analyze the role of PTEN in germ cell differentiation, we
produced conditional Pten knockout mice by crossing
Ptenflox/flox females with
Pten+/ males that carried a single
TNAP/Cre locus, in which Cre was knocked into the
PGC-specific TNAP (tissue-non-specific alkaline phosphatase) gene
(Lomeli et al., 2000;
Suzuki et al., 1998
;
Suzuki et al., 2001
). We
discovered that deletion of the Pten gene in PGCs caused testicular
teratomas in all male newborn mice, and that EG cell production was increased
in both sexes. These observations underscore the essential role of PTEN in
germ cell differentiation.
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MATERIALS AND METHODS |
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Histology, immunohistochemistry and the TUNEL assay
The testes, embryoid bodies and teratomas were fixed with 4%
paraformaldehyde and embedded in either methyl methacrylate or OCT compound.
The sections were stained with Hematoxylin and Eosin using standard
procedures.
For immunohistochemistry, the sections were stained with the following
primary antibodies: anti-PTEN (1:50; Neomarkers, Fremont, CA);
anti-phosphorylated Akt (1:50; NEB, Beverly, MA); anti-mvh (1:1000)
(Toyooka et al., 2000); and
the monoclonal antibodies TRA98 (1:500)
(Tanaka et al., 1997
) and 4C9
(1: 500) (Yoshinaga et al.,
1991
). The Vectastain ABC kit (Vector Lab, Burlingame, CA) was
used for the detection of PTEN. Signals for mvh and TRA98 were visualized
using the appropriate Alexa-Fluor-conjugated secondary antibodies (Molecular
Probes, Eugene, OR). The 4C9 and phosphorylated Akt proteins were detected
using biotin-conjugated antibodies (Vector Lab), followed by
streptavidin-peroxidase or streptavidin-Texas Red (Invitrogen, Carlsbad,
CA).
For TNAP staining, whole embryos were fixed with 4% paraformaldehyde and embedded in OCT compound. Serial transverse sections were collected for every three sections, and stained using the Alkaline Phosphatase Staining Kit (Sigma Diagnostics, St Louis, MO). Ectopic PGCs were also stained for 4C9, as described above. The TUNEL assay was carried out using the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany), according to the manufacturer's recommendations.
PGC culture
Genital ridges were obtained from E11.5 embryos and dissociated into single
cells by incubation with 0.05% trypsin, 0.02% EDTA in PBS. Dispersed
suspensions of PGC-containing tissues were cultured on Sl/Sl4-m220
feeder cells in DMEM that was supplemented with 15% fetal bovine serum, 2 mM
glutamine, 1 mM sodium pyruvate and non-essential amino acids, in the presence
or absence of LIF and bFGF (Koshimizu et
al., 1996; Matsui et al.,
1992
). The feeder cells were treated with 5 µg/ml mitomycin C
for 2 hours and plated at 2x105 cells/well in 24-well plates
1 day before use. PGCs and EG cells were fixed with 4% paraformaldehyde and
visualized by alkaline phosphatase staining, as described above. The number of
adherent PGCs 8 hours after seeding was defined as the number of seeded PGCs.
Multi-layered colonies that contained more than 20 cells were counted as EG
cell colonies, as described previously
(Koshimizu et al., 1996
). The
sex of the E11.5 embryos was determined using sex chromosome-specific PCR
(Chuma and Nakatsuji,
2001
).
Analysis of EG cells
EG cells (5x103 cells/ml) were plated as single cell
suspensions in DMEM medium that contained 1.0% methylcellulose to induce the
differentiation of embryoid bodies. At day 8 after induction, embryoid bodies
were collected and total RNA was extracted using the RNeasy Mini Kit (Qiagen,
Valencia, CA). One microgram of total RNA was reverse-transcribed with the
ThermoScript RT-PCR System (Invitrogen). Semi-quantitative PCR was performed
for 1 cycle at 95°C for 1 minute, followed by amplification cycles of
95°C for 30 seconds, 60°C for 30 seconds, 72°C for 40 seconds and,
finally, 72°C for 7 minutes in a PCR System 9700 (PE Applied Biosystems,
Foster City, CA). PCR amplification of the cDNA remained linear for 30 cycles
(data not shown). The amplification cycles for each gene were as follows:
Wnt1, 27 cycles; collagen IV, 21 cycles; T, 24 cycles; and
ß-actin, 17 cycles. The PCR products were analyzed by 2.0% agarose gel
electrophoresis, and visualized using ethidium bromide staining. The following
primer pairs were used for PCR amplification: Wnt1,
5'-GATTGCGAAGATGAACGCTGTTTC-3' and
5'-TCCTCCACGAACCTGTTGACGG-3'; collagen IV,
5'-CAAGCATAGTGGTCCGAGTC-3' and
5'-AGGCAGGTCAAGTTCTAGCG-3'; T,
5'-ATGCCAAAGAAAGAAACGAC-3' and
5'-AGAGGCTGTAGAACATGATT-3'; and ß-actin,
5'-GTGACGAGGCCCAGAGCAAGAG-3' and
5'-AGGGGCCGGACTCATCGTACTC-3'.
For teratoma induction, control and mutant EG cells (106 cells) were suspended in PBS and subcutaneously injected into the flanks of nude mice. After 2-4 weeks, the mice were sacrificed and the tumors were processed for pathological analysis.
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RESULTS |
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|
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The apoptosis of germ cells was examined by the TUNEL method
(Fig. 3A). The percentages of
apoptotic cells in mvh (mouse vasa homologue)-positive germ cells were
calculated (Toyooka et al.,
2000). As shown in Fig.
3B, no significant differences were observed between the control
and Pten-null mice at E13.5. No apoptotic germ cells were observed in
the control male gonads after E14.5. However, a significant percentage of the
germ cells in the gonads of Pten-null mice showed apoptosis after
E14.5.
|
PGCs in control mice began to express mvh and TRA98 at E11.5, and expression was sustained until birth (Fig. 4A). By contrast, expression of the pluripotent cell marker 4C9 was undetectable by E16.5. Similarly, all of the 4C9-positive PGCs in the Pten mutant embryos were mvh-positive at E13.5, which indicates that the PGCs in the mutant mice acquired the characteristics of germ lineage cells (Fig. 4B). TRA98 expression was also activated in the PGCs of the mutants. Taken together, the results show that all immature PGCs differentiated into relatively mature PGCs, even in the PTEN-null condition (Fig. 4B). However, at around E16.5, TRA98-positive cells, which were either mvh-negative or weakly positive for mvh, emerged (Fig. 4B, arrowheads) and outgrowth of these cells was observed (Fig. 4B, arrow). Finally, large teratomas were observed at P0. Those teratomas consisted of the cells with the marker expression similar to EG cells, namely, mvh-negative, weakly positive for TRA98 and 4C9-positive. PTEN null teratomas would emerge by proliferation of relatively immature PGCs or de-differentiation from germ cells. In either case, loss of PTEN caused significant effects on the differentiation state of germ lineage cells.
Increased numbers of ectopic PGCs in the Pten-null mice
PGCs first appear as a TNAP-positive population posterior to the primitive
streak at the gastrula stage (E7.5), in the base of the allantoic diverticulum
(Gomperts et al., 1994;
Wylie, 1999
). The cells become
incorporated into the developing hindgut and then migrate out of the hindgut
into the surrounding connective tissue at E9.5. The PGCs leave the gut
endoderm and transverse the dorsal mesentery toward the coelomic angles.
Finally, the first PGCs reach the genital ridges through the mesonephros
region by E11.0-11.5. The vast majority of PGCs colonize the gonad primordia
by E13.
PGC distribution at E12.5 was examined by TNAP staining (Fig. 5). The number of PGCs in the genital ridges of normal mice was comparable with that of Pten-null mice. By contrast, the numbers of ectopic PGCs that were localized outside the genital ridges in control and Pten-null mice differed significantly. The ectopic PGCs were positioned mainly along the path of PGC migration. However, low numbers of ectopic PGCs were detected in an irrelevant region (designated as `out of route' in Fig. 5A,C). Thereafter, the ectopic PGCs disappeared in both the control and Pten-null mice by E15.5. Although the cell adhesion activities of the Pten-null PGCs were extensively examined, they were not significantly different from those of control PGCs (data not shown). In addition, the percentage of PGCs with motile morphology in vitro was not increased (data not shown). These observations imply that the increased numbers of ectopic PGCs could be caused by the increased survival rather than the altered migratory capacity, but further analysis is necessary to discriminate these two possibilities.
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DISCUSSION |
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Our observations indicate deregulated maintenance of PGCs in
Pten-null male mice. Two main abnormalities were detected in
Pten-null male gonads after E14.5, i.e. increased numbers of mitotic
figures and higher apoptotic ratios (Figs
2,
3). One of the important roles
of PTEN is to induce G1 arrest through the suppression of the PI3K/Akt pathway
(Li and Sun, 1998;
Ramaswamy et al., 1999
;
Sun et al., 1999
). It seems
reasonable that a significantly higher proportion of the Pten-null
germ cells show mitotic figures. Although PTEN is known to cause apoptosis in
many cell types (Li et al.,
1998
; Myers et al.,
1998
; Stambolic et al.,
1998
; Sun et al.,
1999
), loss of PTEN induced apoptosis of germ cells. This
phenomenon seems ostensibly paradoxical, but might be explained as follows.
Isolated activation of the PI3K/Akt pathway without concomitant activation of
the other pathways may be perceived as abnormal by the cells and thereby
trigger apoptosis. The mechanism by which some proportion of PTEN null germ
cells escaped apoptosis and continued to grow remains unknown at present.
Meanwhile, it seems unlikely that additional genetic alteration took place for
teratoma formation, because the onset of teratoma was quite early. Selection
by local environmental factors or epigenetic events of germ lineage cells
might be involved in the survival and subsequent teratoma formation.
Overall, significant numbers of male germ cells die by apoptosis; some
cells survive and keep growing, and eventually, teratoma develops from the
surviving cells in the Pten-null mice. These phenotypes are similar
to those of 129/Sv-ter mutant mice, in which both testicular teratoma
and germ cell loss occur (Noguchi et al.,
1996; Noguchi and Stevens,
1982
; Stevens,
1984
). PGC mitotic activity correlates strongly with the incidence
of teratomas in 129/Sv-ter mice
(Noguchi and Stevens, 1982
).
Pten mutant mice show impaired mitotic arrest, as do
129/Sv-ter mice, which suggests that mitotic quiescence is the
critical event in the establishment of male germ cell commitment. The
Pten and ter loci are on chromosomes 19 and 18, respectively
(Asada et al., 1994
;
Hansen and Justice, 1998
;
Sakurai et al., 1994
);
however, common molecular mechanisms for teratoma formation and loss of germ
cells may exist.
The in vitro survival and proliferation properties of PGCs were enhanced by
the Pten-null mutation, which resembles testicular teratoma
formation, and EG cell colony formation was also increased significantly (Figs
6,
7). The potential of EG cell
formation of normal mice is very high in migratory phase PGCs at E8.5, then
declined gradually, and eventually disappeared in E13.5 gonadal PGCs
(Labosky et al., 1994a;
Labosky et al., 1994b
;
Matsui et al., 1992
). The high
level of EG cell formation in the E11.5 Pten mutants resembles that
of immature PGCs, and may be attributed partly to the high proliferative
activities of PGCs. However, it is unlikely that the abnormalities are caused
simply by increased proliferation, as cells that are derived from PTEN-null
PGCs attain much broader differentiation potential as teratomas or EG cells.
It is reasonable to assume that PTEN loss induces the de-differentiation of
germ lineage-committed cells or sustains the immature state of PGCs longer
than in control mice.
Two lines of evidence support this hypothesis. The first comes from the
study of cell lines, in which PI3K signaling is involved in de-differentiation
(Kobayashi et al., 1999;
Singh et al., 2002
). The
second is derived from tissue-specific PTEN-deficient mice. PTEN-deficient
neural stem cells exhibit increased proliferation and self-renewal capacities
(Groszer et al., 2001
). In
addition, B-cell-specific PTEN-null mice have significantly increased numbers
of B1 B cells, i.e. a subpopulation of B cells that resides mainly in the
pleural and peritoneal cavities, and possess self-renewing activities
(Suzuki et al., 2003
). Taken
together with our present data, the tumor suppressor PTEN negatively regulates
self-renewal and proliferation in various stem cell systems. In addition to
self-renewal and proliferation, our results suggest that PTEN is involved in
the normal differentiation of PGCs. Mutant PTEN is frequently involved in
tumor proliferation, and may enable tumor cells to regain pluripotency
(Cantley and Neel, 1999
;
Di Cristofano and Pandolfi,
2000
; Simpson and Parsons,
2001
; Yamada and Araki,
2001
). Tumors often originate through the transformation of stem
cells, and it has been hypothesized that the physiological and pathological
properties of stem cells are regulated by the same signaling pathway
(Penninger and Woodgett, 2001
;
Reya et al., 2001
). It seems
likely that PTEN negatively regulates not only self-renewal and proliferation,
but also the maintenance of immaturity of both tumor cells and stem cells.
The receptors for various growth factors that activate the PI3K signaling
pathway, e.g. SCF, LIF and bFGF, are expressed in gonadal PGCs
(Cheng et al., 1994;
Pesce et al., 1997
;
Resnick et al., 1998
). As Akt
was hyper-activated in Pten mutant PGCs, it is likely that the loss
of PTEN enhanced the PI3K signaling pathway, which then resulted in the
deregulated differentiation of PGCs. Although EG cell formation was enhanced
by the null mutation of Pten, no EG cell formation was observed
without the addition of LIF or bFGF. Accordingly, the loss of PTEN function
itself is not sufficient for enhanced EG colony formation, but probably
increases the signals for PI3K, which is one of the downstream molecules for
signaling via SCF, LIF and bFGF.
In conclusion, PTEN (PI3K) signaling plays a critical role in germ cell differentiation. Teratoma formation and increased EG colony formation suggest that PtdIns(3,4,5)P3 signaling is involved in the maintenance of stem cell characteristics. Further analysis of PtdIns(3,4,5)P3 signaling may provide new insights into stem cell biology.
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ACKNOWLEDGMENTS |
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