1 Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, MO
64110, USA
2 Department of Anatomy and Cell Biology, University of Kansas School of
Medicine, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA
* Author for correspondence (e-mail: tgx{at}stowers-institute.org)
Accepted 5 October 2005
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SUMMARY |
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Key words: Germline stem cells, Self-renewal, Pelota, Bmp, Differentiation, Drosophila
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Introduction |
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In the Drosophila ovary, GSCs reside in a structure called the
germarium, which is at the anterior end of an ovariole
(Lin, 2002;
Xie and Spradling, 2001
). At
the anterior tip of the germarium, three types of somatic cells (terminal
filament cells, cap cells and inner sheath cells) constitute a niche that
supports two or three GSCs (Lin,
2002
; Xie and Spradling,
2001
; Xie and Spradling,
2000
) (Fig. 1A).
One GSC divides to generate two daughter cells: the daughter cell maintaining
contact with the cap cells through DE-cadherin-mediated cell adhesion renews
itself as a stem cell, while the daughter cell moving away from the cap cells
differentiates into a cystoblast (Song et
al., 2002
). The cystoblast divides four times with incomplete
cytokinesis to form a 16-cell cyst, in which one cell becomes an oocyte and
the rest become nurse cells (Spradling,
1993
). Bmp signals produced by the niche, Dpp and Gbb, have
essential roles in controlling GSC self-renewal, as reduction of Bmp signaling
activity results in the loss of GSCs by differentiation and overexpression of
dpp in the germarium produces GSC-like tumors
(Song et al., 2004
;
Xie and Spradling, 1998
). Bmps
from the cap cells function as short-range signals that directly repress the
transcription of bam in GSCs to maintain their self-renewal, and also
allow cystoblasts lying one cell diameter away to differentiate
(Chen and McKearin, 2003a
;
Song et al., 2004
).
bam is necessary and sufficient for germ cell differentiating in the
Drosophila ovary (Ohlstein and
McKearin, 1997
). In addition, two other genes, Yb and
piwi, function in the somatic niche cells to control GSC
(Cox et al., 2000
;
King et al., 2001
).
Yb encodes a novel protein and directly regulates expression of
piwi and hh in TFs; hh signaling also modulates GSC
self-renewal though it is not essential
(King et al., 2001
).
piwi encodes a family of conserved RNA-binding proteins and is
required in the niche cells for controlling GSC self-renewal and inside GSCs
for their division (Cox et al.,
1998
; Cox et al.,
2000
). Two recent studies have shown that piwi also
maintains GSC self-renewal by repressing bam expression through
regulation of either the Bmp signaling pathway or a Bmp-independent signaling
pathway (Chen and McKearin,
2005
; Szakmary et al.,
2005
). However, it remains unclear how piwi controls GSC
division intrinsically.
Two translational repressors, Nanos (Nos) and Pumilio (Pum), have been
shown to be required for the maintenance of ovarian GSCs by preventing
differentiation (Forbes and Lehmann,
1998; Wang and Lin,
2004
). Pum/Nos repress differentiation of PGCs and GSCs through a
Bmp-independent pathway, as their expression is not regulated by Bmp signaling
and their mutations cannot suppress hyperactive Bmp signaling-induced PGC
proliferation (Gilboa and Lehmann,
2004
). It is likely that Nos and Pum are involved in repressing
translation gene products that are important for germ cell differentiation and
thereby for controlling GSC self-renewal. To identify further intrinsic
factors that are required for Bmp-mediated GSC self-renewal, we performed a
genetic screen to identify dominant suppressors of the dpp-induced
GSC-like tumor phenotype (C.D. and T.X., unpublished). One of the suppressors
is pelota (pelo), which has been studied for its role in the
regulation of Drosophila male meiosis. Cellular and molecular
analysis showed that pelo is required for the progression through
meiosis in spermatogenesis and encodes an evolutionarily conserved protein
that contains an eukaryotic release factor 1
(eRF1
)-like domain
at its C terminus (Eberhart and Wasserman,
1995
). The studies on the yeast pelo homolog
dom34 suggest that Pelo is involved in translational regulation.
Deletion of Dom34 causes growth retardation, defective sporulation and reduced
polyribosomes (Davis and Engebrecht,
1998
). dom34 has a strong genetic interaction with
RPS30A, which encodes ribosomal protein S30A; overexpression of
RPS30A rescues the growth defects and reduced polyribosomes of dom34
mutants (Davis and Engebrecht,
1998
). Moreover, Dom34 specifically interacts with Hbs1, a small
GTPase that is also implicated in translational regulation
(Carr-Schmid et al., 2002
). It
has been recently shown that pelo knockout mice exhibit early
embryonic lethality with defects in cell division and proliferation
(Adham et al., 2003
). Taken
together, pelo may be involved in the regulation of meiosis and
mitosis possibly through regulating translation. In this study, we have
revealed an unexpected new role of Pelo in the control of GSC self-renewal and
division in the Drosophila ovary, possibly through regulating
translation.
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Materials and methods |
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Fly stocks and markers
Unless otherwise stated, flies were reared at 25°C on standard
molasses-based food. The fly stocks used for this study include:
pelo1, a strong P-element-induced allele
(Eberhart and Wasserman,
1995); bam86, a null allele
(McKearin and Ohlstein, 1995
);
Sec61
-GFP (ZCL0488) (Kelso et al.,
2004
); Dad-lacZ
(Tsuneizumi et al., 1997
);
bam-GFP (Chen and McKearin,
2003b
); nosgal4VP16
(Van Doren et al., 1998
); and
c587-gal4 (Song et al.,
2004
).
Generating marked clones
Marked clones were generated using the FLP-mediated FRT recombination
technique according to the published procedures
(Song et al., 2002;
Xie and Spradling, 1998
).
These marked clones were analyzed and quantified 3 days, 10 days, 17 days and
24 days after clone induction (ACI). For examination of Dad-lacZ and
bam-GFP expression in the marked pelo1 mutant
GSCs, the ovaries were analyzed 14 days ACI. The genotypes used for clonal
analysis in this study are shown as follows:
Immunohistochemistry
Antibody staining of ovaries was performed using our published protocol
(Song et al., 2002). The
following antisera and dilutions were used: rabbit anti-ß-galactosidase
(1:100; Molecular Probes); mouse anti-ß-galactosidase (1:50; Molecular
Probes); rabbit anti-GFP (1:100; Molecular Probes); monoclonal anti-Hts 1B1
(1:4; DSHB); monoclonal anti-HtsRC (1:4; DSHB); monoclonal anti-Orb (1:30;
DSHB); and rabbit anti-Vasa antibody (1:1000; a gift of Dr Paul Lasko).
Secondary antibodies including goat anti-rabbit or anti-mouse IgG conjugated
to Alexa 488 or Alexa 568 (Molecular Probes) were used at a dilution of
1:200.
ApopTag staining and BrdU labeling
Ovaries from 2-day-old females for ApopTag staining and BrdU labeling were
dissected in Grace's medium. ApopTag in situ apoptosis detection kit
(Serologicals, Clarkston, GA) was performed according to the manufacture's
manual. For BrdU labeling, the ovaries were incubated in the medium at a final
concentration at 75 µg/ml at room temperature for 1 hour. Fixation and BrdU
detection were described previously (Zhu
and Xie, 2003).
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Results |
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|
|
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pelo is required intrinsically for controlling GSC self-renewal
Pelo could control GSC self-renewal by acting either inside the GSC, in the
niche or both. Our mRNA in situ hybridization and gene expression profiles of
agametic ovaries show that pelo mRNAs were ubiquitously expressed at
lower levels throughout the germarium, suggesting that pelo could
function in GSCs or the somatic niche cells, or both (data not shown). We used
FLP-FRT-mediated mitotic recombination to determine whether Pelo functions
inside GSCs for controlling their self-renewal
(Xu and Rubin, 1993). The
FLP-mediated FRT recombination has been used to generate marked mutant GSCs
and determine their loss rates for deducing the role of a particular gene in
GSC maintenance (Xie and Spradling,
1998
). According to published experimental procedures
(Xie and Spradling, 1998
;
Song et al., 2002
), the
ovaries of the females of appropriate genotypes were dissected at 3, 10, 17
and 24 days after clone induction (ACI) mediated by heat-shock treatments, and
marked wild-type and pelo mutant GSCs were identified by the lack of
arm-lacZ expression and the presence of an anteriorly anchored
spectrosome. In the wild-type control, 55.0% of the marked GSCs (from 52.8% of
the germaria carrying one or more marked control GSCs at 3 days ACI to 28.9%
of the germaria carrying one or more marked control GSCs at 24 days ACI) were
still maintained for 3 more weeks (Xie and
Spradling, 1998
) (Fig.
2A,B). By contrast, 97% of the marked pelo mutant GSCs
(from 48.6% of the germaria carrying one or more marked mutant GSCs at 3 days
ACI to 1.7% of the germaria carrying one or more marked mutant GSCs at 24 days
ACI) were lost during the same 3- week period
(Table 2;
Fig. 2C,D). These results
demonstrate that pelo is required in GSCs for controlling their
self-renewal. It appears that these marked pelo mutant GSCs are lost
slower than the GSCs in the homozygous pelo mutant ovaries. This
could be explained by the possibility that Pelo is very stable or has
functions in both GSCs and soma. If Pelo is very stable, it takes longer for
residual wild-type Pelo that is made before clone induction to be degraded in
the marked pelo mutant GSCs. Later, we will try to address whether
pelo has a function in the soma to control GSC maintenance.
|
Pelo has no obvious role in the somatic cells of the germarium including SSCs
Although pelo is required intrinsically for controlling GSC
self-renewal, it does not rule out the possibility that a somatic function of
pelo is also involved in regulating GSC self-renewal as it is also
expressed in the somatic cells of the germarium. First, we examined whether
the pelo mutation affects the survival of cap cells. A
hh-lacZ enhancer trap line used in this study has been used to label
terminal filament cells and cap cells in the Drosophila ovary
(Forbes et al., 1996). In the
pelo homozygous germaria carrying one or no GSCs, the cap cell number
appeared to be normal, ranging from five to seven
(Fig. 2G,H), indicating that
Pelo function is at least not required for the formation or survival of cap
cells. However, pelo could still be required in the surrounding
somatic cells for controlling GSC function through regulating production of
signals. To further test whether pelo has a somatic function in GSC
regulation, we used the c587-gal4 driver, which is expressed in IGS
cells and follicle progenitor cells, and the same UASp-pelo transgene
to test whether somatic expression pelo can rescue the pelo
mutant GSC loss phenotype. However, c587-driven pelo expression in
the somatic cells could only confer very limited rescue of the pelo
mutant GSC loss phenotype, in addition to the partial rescue conferred by the
UAS-pelo transgene alone, suggesting that pelo has little
role in IGS cells and follicle cell progenitors for GSC self-renewal
(Fig. 2E). As we have not
tested whether pelo expression in terminal filaments/cap cells can
mitigate the GSC loss phenotype of the pelo mutant ovaries, our
results could not completely rule out the possibility that Pelo has a function
in somatic cells for controlling GSC self-renewal.
As pelo is expressed in all the somatic cells, including SSCs, we
then determined whether pelo has a role in SSC maintenance using
FLP-mediated FRT recombination to generate marked pelo mutant SSC
clones. The marked SSCs were identified as the arm-lacZ-negative
somatic cells residing at the 2a/2b boundary and generating
arm-lacZ-negative (marked) follicle cells in the germarium and egg
chambers, according to our previous studies
(Song and Xie, 2002;
Song and Xie, 2003
). In the
control, 68% of marked wild-type SSCs were maintained for 3 weeks, supporting
the fact that SSCs have a slow natural turnover
(Table 2). Similarly, 62% of
the marked pelo SSCs were maintained for 3 weeks, indicating that
pelo plays little or no role in SSC maintenance
(Table 2). The marked
pelo1 mutant follicle cell clones exhibited a very minor
phenotype: they appeared slightly thinner compared with wild-type follicle
cells (data not shown). Although pelo is ubiquitously expressed
throughout the germarium, the main function of pelo is primarily
restricted to GSCs and their progeny in the ovary.
Pelo protein is localized to the cytoplasm for controlling GSC self-renewal
Drosophila Pelo has a putative nuclear localization signal
sequence (PRKRK) at its N terminus
(Eberhart and Wasserman, 1995;
Nair et al., 2003
); this
sequence is perfectly conserved from Drosophila to human, raising an
interesting possibility that Pelo is a nuclear protein. If Pelo indeed
functions in the nucleus, we would expect that the disruption of the putative
nuclear localization sequence leads to loss of its function. To directly test
the idea, we generated a mutant version of Pelo with the replacement of PRKRK
by RSRS as ablation of helix-breaking residue proline and reduction of basic
residues can abolish the function of the nuclear localization signal
(Conti et al., 1998
). In S2
cells, the mutant Pelo protein tagged with 3xFlag and 6xMyc at its N terminus
[F-M-Pelo(nls*)] was localized in the cytoplasm in the same way as
the wild-type version tagged with the same tags at its N terminus (F-M-Pelo)
(data not shown). To further determine whether the NLS of Pelo is important
for Pelo function in controlling GSC self-renewal in vivo, we generated
transgenic flies carrying either UASp-F-M-Pelo or
UASp-F-M-Pelo(nls*). Two independent insertion lines for
UAS-F-M-Pelo could fully rescue the pelo mutant GSC loss phenotype
when they were driven to be expressed specifically in the germ cells by
nos-gal4VP16, indicating that FLAG and MYC tags do not interfere with
Pelo function (Fig. 2E). Similarly, two independent transgenic lines of the NLS mutated version of Pelo
could also fully rescue the pelo GSC loss phenotype
(Fig. 2E), further supporting
that the putative NLS is not important for Pelo function in GSCs and that Pelo
functions in the cytoplasm to control GSC self-renewal
(Fig. 2E). Interestingly, one
of the transgenic lines [UASp-FM(nls*)#1] showed complete rescue
for the pelo GSC loss phenotype with the nosgal4VP16 driver,
whereas it exhibited little rescue for the GSC loss phenotype of the
pelo mutant ovaries with the c587 driver or without any
gal4 driver, further supporting our earlier conclusion that the
mutation in the pelo gene is responsible for the GSC loss
(Fig. 2E).
|
|
pelo modulates dpp pathway activity in GSCs
As discussed earlier, pelo was identified in a genetic screen
looking for genes that can suppress dpp overexpression-induced
GSC-like tumors, suggesting that pelo must somehow genetically
interact with the dpp/Bmp pathway. To further reveal the relationship
between pelo and Bmp signaling, we carefully examined the dose effect
of pelo on dpp-induced GSC-like tumor formation. As reported
previously (Xie and Spradling,
1998; Song et al.,
2004
), all the ovarioles overexpressing dpp by the
c587 gal4 driver (n=77) contained only single germ cells
resembling GSCs (Fig. 4A).
Among the dpp-overexpressing ovarioles also carrying one copy of the
pelo1 mutation, 36% of them (n=206) showed the
same tumor phenotype, but the rest of the ovarioles contained differentiated
germline cysts, developing egg chambers and even mature eggs
(Fig. 4B), which could explain
why pelo was identified in our suppressor screen. Among the
dpp-overexpressing ovarioles also carrying two copies of the
pelo1 mutations, only 13.8% of them (n=261)
contained only GSC-like single germ cells, 49.8% of them had a mixture of
single germ cells and developing cysts
(Fig. 4C). Interestingly, the
rest (36.4%) were reminiscent of the pelo GSC loss phenotype only
(Fig. 4D). These results
suggest that pelo functions as one of the Bmp downstream components
or in a pathway parallel to the Bmp signaling pathway to control GSC
self-renewal.
|
pelo represses a bam-independent differentiation pathway in ovarian GSCs
bam is both essential and sufficient for cystoblast
differentiation (McKearin and Ohlstein,
1995; Ohlstein and McKearin,
1997
). Our observation that pelo1 mutant GSCs
are lost because of differentiation but do not upregulate bam
expression suggests that pelo mutant GSCs differentiate using a
bam-independent pathway. If so, we should expect bam mutant
germ cells to be able to differentiate in the absence of pelo
function. To test this idea, we investigated the genetic relationship between
bam and pelo. The pelo1 homozygous GSCs
that were heterozygous for bam
86, a
deletion allele of bam, were still lost rapidly as in the
pelo1 mutant GSCs (data not shown); the
bam
86 homozygous germ cells that were also
heterozygous for pelo1 still failed to differentiate, as
did the bam
86 mutant one
(Fig. 5A). Interestingly, in
pelo1; bam
86 double
homozygous germaria, most of the germ cells were cysts with branched fusomes,
and some of them still retained a round spectrosome
(Fig. 5B). The round
spectrosomes in the remaining single germ cells were unusually larger than
those in bam mutant single germ cells, suggesting the single germ
cells could be growth-arrested cystoblasts but can continue to grow their
spectrosome. The morphology of the branched fusomes of the double mutant cysts
appeared abnormal. To further determine whether the oocytes form in these
pelo; bam mutant cysts, we examined the expression of Orb
protein in the double mutant germaria. Orb normally starts to accumulate in
newly formed wild-type oocytes (Fig.
5C); however, no obvious Orb expression was detected in the
pelo; bam mutant germaria, indicating that there is no
oocyte formation in the double mutant cysts
(Fig. 5D). As pelo
mutant cysts can still form the oocyte, bam is probably required late
for oocyte formation. These findings show that pelo mutant
cystoblasts can differentiate without functional bam, and further
suggest that pelo must repress a bam-independent
differentiation pathway to maintain GSC self-renewal.
pelo is also required for survival and/or growth of germline cysts during mid-oogenesis
We also noticed that the pelo mutant females had some fertility
after eclosion and quickly lost their fertility. Interestingly, in the
2-day-old pelo mutant ovaries, early egg chambers looked largely
normal, and even a few mature eggs were present
(Fig. 6A). By contrast, in the
7-day-old ovaries, egg chambers older than stage 9 were rarely observed, and
even those early stage egg chambers exhibited condensed nurse cell DNA, which
suggest that they are in the process of undergoing apoptosis
(Fig. 6B,C). The egg chamber
degeneration is not due to oocyte formation defect because in these egg
chambers the oocyte was present (Fig.
6B). In addition, the size of some egg chambers exhibiting
condensed nurse cell DNA was also smaller than normal
(Fig. 6C). The Pelo function
also appeared to be germ-cell specific in egg chambers, as marked mutant
pelo germ cells failed to grow to the normal size and became
apoptotic (Fig. 6D). These
results indicate that pelo is also required in the germ cells for
their survival or normal differentiation during later oogenesis.
|
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Discussion |
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Pelo protein functions in the cytoplasm possibly as a translational regulator to control GSC self-renewal
Drosophila Pelo belongs to a family of evolutionarily conserved
proteins with their primary function in the regulation of cell division. In
the yeast, dom34 (pelo) mutant cells grow slowly and have
defects in the entry of meiosis, indicating that it is required for mitosis
and meiosis. In mice, disruption of the pelo gene causes early
embryonic lethality and defects in cell cycle progression
(Adham et al., 2003). Although
pelo is ubiquitously expressed throughout Drosophila
development (Eberhart and Wasserman,
1995
), the pelo mutants survive to adulthood without
obvious defects in the body. In Drosophila, pelo has been shown to be
required to control meiotic cell cycle progression in male germ cells. In this
study, we show that pelo is required intrinsically for controlling
self-renewal and division of GSCs but not SSCs in the ovary, which is
supported by rescue and stem cell clonal analysis experiments. Even though
Pelo members are required for regulating cell cycle progression from yeast to
mammals, it remains unclear how they accomplish this function.
The only clue to the potential cellular function of Pelo comes from its
high homology to the translation release factor 1. Its likely function as a
translational regulator is further complicated by the presence of a highly
conserved NLS sequence (Eberhart and
Wasserman, 1995). Using an epitope-tagged Pelo that can rescue
pelo mutants, we demonstrate that Pelo is mainly localized to the
cytoplasm of both S2 cells and germ cells. Furthermore, the pelo gene
with a mutated putative NLS is still fully functional. The yeast Pelo, Dom34,
is also localized to the cytoplasm (Davis
and Engebrecht, 1998
; Huh et
al., 2003
). These findings support the idea that Pelo proteins
function in the cytoplasm as translational regulators in different organisms.
If Pelo truly functions as a translational release factor, they must directly
interact with ribosomes that are either associated with ER or in the
cytoplasm. Consistent with this idea, some Pelo proteins are associated with
ER membranes though the majority of Pelo proteins are not associated with ER
membranes. In yeast, dom34 mutants have dramatically reduced
polyribosomes and can be rescued by a high-copy of the ribosomal protein
S30A gene, indicating that Dom34 is involved in translation
(Davis and Engebrecht, 1998
).
In addition, expression of Drosophila pelo in dom34 mutants
can rescue growth defects, indicating its conserved function during evolution.
Therefore, Pelo is also likely a translational regulator in
Drosophila, and is involved in regulating translation of a specific
class of mRNAs that are important for germ cell function. In the future, it
will be important to investigate whether Pelo is indeed involved in
translational regulation and to identify its targets in germ cells.
|
In the Drosophila ovary, one of the ways in which Bmp signaling
controls GSC self-renewal is to directly repress bam expression in
GSCs. bam is necessary and sufficient for cystoblast differentiation
in the Drosophila ovary (McKearin
and Spradling, 1990; Ohlstein
and McKearin, 1997
). In this study, we show that Pelo is essential
for controlling GSC self-renewal but is not involved in repressing
bam expression. pelo mutant GSCs have normal bam
repression but their progeny can still differentiate without bam,
suggesting that pelo maintains GSCs by repressing a
bam-independent pathway. During the preparation of this manuscript,
two studies were published showing that pum controls GSC self-renewal
by repressing a bam-independent pathway
(Chen and McKearin, 2005
;
Szakmary et al., 2005
). Pum is
known to work together with Nos, which is also essential for
Drosophila ovarian GSC self-renewal
(Gilboa and Lehmann, 2004
;
Wang and Lin, 2004
), to
repress gene translation in the embryo
(Barker et al., 1992
;
Forbes and Lehmann, 1998
). As
Pum/Nos does not participate in Bmp signaling
(Gilboa and Lehmann, 2004
) and
Pelo is a translational release factor-like protein, we propose that Pelo
works in a parallel genetic pathway with Pum in repressing the same or
different Bam-independent differentiation pathways through regulating
translation (Fig. 7). Although
it is essential for repressing a Bam-independent pathway(s) in GSCs, Pelo is
not so sufficient for doing so as Bmp signaling is for repressing bam
as overexpression of pelo has no effect on the GSC maintenance and
differentiation. In the future, it will be important to molecularly and
genetically characterize the Bam-independent pathway repressed by Pelo and to
further understand how Pelo represses it in relation to Pum.
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ACKNOWLEDGMENTS |
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