1 Stowers Institute for Medical Research, 1000 East 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 9 December 2003
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SUMMARY |
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Key words: Germline, Stem cells, Bmp, Bam, Drosophila
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Introduction |
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The Drosophila testis has become one of the premier stem cell
systems to study molecular mechanisms governing stem cell self-renewal,
differentiation and proliferation (Kiger
et al., 2000; Tran et al.,
2000
; Kiger et al.,
2001
; Tulina and Matunis,
2001
). In the testis, there are two types of stem cells, GSCs and
somatic stem cells (also known as cyst progenitor cells), which are
responsible for producing differentiated germ cells and somatic cyst cells
that encapsulate differentiated germ cells, respectively
(Fig. 1A). Seven to nine GSCs
can be reliably identified by their attachment to hub cells (a group of
tightly packed somatic cells) and existence of a spectrosome
(Fig. 1A,B). The spectrosome is
a spherical fusome that is unique to GSCs and their early progeny, also known
as gonialblasts. The fusome is rich in cytoskeletal proteins such as Hu li tai
shao (Hts) and
-Spectrin (Lin et
al., 1994
; de Cuevas and Spradling, 1997). Once a GSC divides, one
daughter cell that is in contact with the hub cells retains stem cell
identity, whereas the other daughter cell that is not in contact with the hub
cells initiates differentiation and becomes a gonialblast. The gonialblast
then undergoes four rounds of synchronous cell division to generate a 16-cell
germline cluster in which individual germ cells are connected by ring canals
and a branched fusome. During the course of germ cell development from a
gonialblast to a 16-cell cyst, a pair of somatic cyst cells surrounds the
gonialblast, or a developing germ cell cluster, and control proper germ cell
proliferation and differentiation (Matunis
et al., 1997
).
|
The Drosophila ovary represents another attractive stem cell
system in which stem cells and their niche cells can be reliably identified
(Xie and Spradling, 2001;
Lin, 2002
). Germline stem
cells have first been demonstrated to be located in the niche, consisting of
terminal filament/cap cells and inner sheath cells
(Xie and Spradling, 2001
;
Lin, 2002
). fs(1)Yb
and piwi are expressed in the terminal filament/cap cells and are
essential for GSC maintenance (King and
Lin, 1999
; Cox et al.,
1998
; Cox et al.,
2000
; King et al.,
2001
). decapentaplegic (dpp), a
Drosophila Bmp member, is expressed in somatic cells such as cap
cells, and is essential for GSC maintenance and division in the
Drosophila ovary (Xie and
Spradling, 1998
; Xie and
Spradling, 2000
). Interestingly, another Bmp member, glass
bottom boat (gbb, also known as 60A), is highly
expressed in the male (Wharton et al.,
1991
; Doctor et al.,
1992
), but its role in spermatogenesis has not been investigated.
Here we show that both gbb and dpp, are expressed in the
somatic cells of the testis and act cooperatively on GSCs to control their
maintenance. In addition, gbb signaling is essential for repressing
bam expression in GSCs in Drosophila.
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Materials and methods |
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Generating mutant GSC clones and overexpression
Clones of mutant GSCs were generated by Flp-mediated mitotic recombination,
as described previously (Xie and
Spradling, 1998). To generate the stocks for stem cell clonal
analysis, 2-day-old adult males carrying an armadillo-lacZ transgene
in trans to the mutant-bearing chromosome were generated using standard
genetic crosses and then heat-shocked at 37°C for 3 consecutive days with
two one-hour heat-shock treatments daily separated by 8-12 hours. The males
were transferred to fresh food every day at room temperature, and the testes
were removed 2 days, 1 week and 2 weeks after the last heat-shock treatment,
and then processed for antibody staining.
To construct the stocks for overexpressing dpp or gbb, nanos-gal4VP16 virgins were crossed with UAS-dpp and UAS-gbb males, respectively. The males that carried nanos-gal4VP16 and UAS-dpp or UAS-gbb were cultured at room temperature, or at 29°C, for one week. For examining the expression of bam-GFP in the testes overexpressing dpp or gbb, the bam-GFP/CyO; nanos-gal4VP16 virgins were used in the crosses.
Measuring GSC loss in gbb mutants and marked GSCs, and examining bam-GFP expression in gbb, dpp and punt mutant testes
To determine loss of marked mutant GSC clones, GSCs were marked in 1- to
2-day-old males of the appropriate genotype. Subsequently, testes were
isolated from some of the males 2 days, 1 and 2 weeks later, and stained with
anti-Hts and anti-ß-Gal antibodies. The percentage of testes containing
one or more marked GSCs was determined by counts of 55-227 testes at each time
point.
To measure stem cell loss in gbb mutant testes, the testes with different numbers of GSCs were determined based on anti-Hts and anti-Fas3 antibody staining of gbb4/gbbD4 or gbb4/gbbD20 testes of different ages and different treatments. yw males carrying no gbb mutations served as a control. The 2-day-old control and gbb mutant males were cultured at different temperatures after they eclosed at 18°C. Values are expressed as the average GSC number per testis, and/or the percentage of testes carrying no GSCs.
To examine bam-GFP expression in dpp, gbb and punt mutant testes, we generated males with the following genotypes at 18°C: bam-GFP gbb4/gbbD4, bam-GFP gbb4/gbbD20, bam-GFP dpphr56/dpphr4 or punt10460/punt135; bam-GFP. bam-GFP males carrying no mutations for gbb, dpp or punt served as a control. All the control and mutant males were cultured at 29°C for 4 days before their testes were isolated, stained with antibodies and compared for bam-GFP expression at identical conditions.
The TUNEL cell death assay was performed on punt mutant testes (Intergen Company).
Immunohistochemistry
The following antisera were used: polyclonal anti-Vasa antibody (1:2000)
(Liang et al., 1994);
monoclonal anti-Hts antibody (1:3); polyclonal anti-ß-Gal antibody
(1:1000; Cappel); monoclonal anti-ß-Gal antibody (1:100; Promega);
polyclonal anti-GFP antibody (1:200; Molecular Probes); polyclonal anti-pMad
antibody (1:200) (Tanimoto et al.,
2000
). The immunostaining protocol used in this study was
described previously (Song et al.,
2002
). All micrographs were taken using a Leica SPII confocal
microscope.
Detecting gene expression in purified component cells using RT-PCR
The tips of the testes for the males of the appropriate genotype were
dissected off from the whole testes in Grace's media, and were dissociated
with collagenase II (Sigma) solution at a concentration of 6 mg/ml. After
sorting of GFP-positive cells, using Cytomation MoFlo, from testes with
GFP-marked hub cells, somatic cyst cells or germ cells, total RNA was prepared
using Trizol (Invitrogen). RNA samples were further amplified using the
GeneChip Eukaryotic Small Sample Target Labeling Assay Version II
(Affymetrix). After RNA amplification, 100 ng of total RNA was reverse
transcribed (RT) using SuperScriptIII First-Strand Synthesis System for RT-PCR
(Invitrogen). The following primers were used in this study:
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Results |
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Two temperature-sensitive allelic combinations,
dpphr4/dpphr56 and
dppe90/dpphr56 were used to investigate the
role of dpp in maintaining GSCs in the testis. Similarly,
dpp homozygous males were developed to adulthood at 18°C and were
then shifted to a restrictive temperature (29°C) for one week. In the
Drosophila ovary, both mutant combinations lose their GSCs very
rapidly at a restrictive temperature (Xie
and Spradling, 1998). Surprisingly, one week after being cultured
at the restrictive temperature, the testes from dpp mutants had no
significant GSC loss: dpphr4/dpphr56 and
dppe90/dpphr56 mutant testes had an average of
7.4 (n=44) and 8.8 (n=49) GSCs/testis, respectively
(Fig. 1E), which is in contrast
with the severe GSC loss phenotype in the dpp mutant ovary and in the
gbb mutant testis.
The two allelic dpp combinations used in this study represent very
weak dpp mutants. As there is a stringent requirement for
dpp during early Drosophila development, it is difficult to
examine GSC loss in stronger dpp mutants because they do not survive
to adulthood, even at 18°C. It is still possible that the role of
dpp in the maintenance of male GSCs can be revealed if gbb
signaling is comprised, as dpp and gbb could use the same
receptors and downstream components to transduce their signals
(Khalsa et al., 1998). To
further study the role of dpp in the regulation of male GSCs, we
constructed two mutant strains homozygous for two dpp allelic
combinations that were also heterozygous for gbb:
dpphr4/dpphr56 gbbD4 and
dppe90/dpphr56 gbbD4. The testes
from the heterozygous gbbD4, which were cultured at
29°C for one week, had a normal GSC number (8.6 GSCs/testis,
n=38). The testes from dpphr4/dpphr56
gbbD4 and dppe90/dpphr56
gbbD4 had an average of 3.0 (n=13) and 5.7
(n=56) GSCs/testis, respectively
(Fig. 1F), in comparison with
7.4 and 8.8 GSCs/testis for dpp mutants alone, suggesting that
partial removal of gbb function can enhance the dpp-mutant
GSC-loss phenotype in the Drosophila testis. These results indicate
that dpp and gbb function cooperatively to regulate male
GSCs in Drosophila.
To further confirm that Bmp signaling is essential for maintaining male
GSCs, we studied mutant phenotypes for one of the Bmp downstream components,
punt, which encodes a type II serine/threonine kinase receptor for
dpp, and also possibly for gbb
(Letsou et al., 1995;
Ruberte et al., 1995
). A
punt allelic combination,
punt10460/punt135, exhibits a
temperature-sensitive phenotype: developing to adulthood at 18°C and
showing mutant phenotypes at 29°C
(Theisen et al., 1996
).
Interestingly, punt10460/punt135 mutant males
had normal GSC numbers (8.5 GSCs/testis, n=20) after being cultured
at 22°C for a week (Fig.
2A). However, one week after shifting to 29°C, almost all the
mutant testes completely lost their GSCs (0.1 GSCs/testis, n=58;
Fig. 2B,C), although wild-type
testes still maintained normal GSC number under the same conditions (data not
shown). To exclude the possibility that Bmp signaling is important for GSC
survival, we applied the TUNEL labeling assay to look for dying GSCs in
punt mutant testes. During the one-week period at 29°C, no dying
GSCs were detected in the punt mutant testes, but some rare dying
cyst cells or differentiated germ cells were observed (n=38,
Fig. 2D), suggesting that GSC
loss is most likely caused by differentiation triggered by the lack of
sufficient Bmp signaling. This result further supports the idea that Bmp
signaling is essential for maintaining GSCs in the Drosophila
testis.
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|
To further test whether Dad could inhibit both gbb and
dpp signaling, we overexpressed Dad in germ cells using the
Gal4-UAS bipartite expression system (Brand
and Perrimon, 1993). A germline-specific nanos-gal4VP16
driver can express a target gene under the control of UAS promoter
specifically in germ cells (Van Doran et al., 1998), whereas a
UAS-Dad transgene can be used to produce Dad under a
gal4 driver to inhibit dpp signaling
(Tsuneizumi et al., 1997
).
When the UAS-Dad transgene was used to overexpress Dad in
germ cells by nanos-gal4VP16, all GSCs were lost in the testes before
adulthood (Fig. 3C), indicating
that blocking Bmp signaling causes GSC loss or prevents the formation of GSCs.
The GSC loss phenotype induced by Dad overexpression mimics that of
gbb mutants, suggesting that Dad overexpression probably
inhibits not only dpp signaling but also gbb signaling.
Thus, Dad-lacZ expression in GSCs may reflect the activities of both
dpp and gbb signaling pathways. Together, these results
suggest that Bmp signals appear to function as short-range signals to control
GSC maintenance through direct signaling to GSCs.
In Drosophila, Dpp brings type I receptors Tkv and Sax, and the
type II receptor Punt to form receptor complexes, which in turn phosphorylate
Mad (Brummel et al., 1994;
Nellen et al., 1994
;
Xie et al., 1994
;
Letsou et al., 1995
;
Ruberte et al., 1995
;
Newfeld et al., 1996
;
Newfeld et al., 1997
). The
phosphorylated Mad (pMad) is then associated with Medea (Med) and translocated
to the nucleus to function as transcriptional activators for
dpp-responsive genes (Das et al.,
1998
; Wisotzkey et al.,
1998
). pMad expression has been directly associated with
dpp signaling activity in responding cells
(Tanimoto et al., 2000
). To
further determine whether gbb signaling is responsible for pMad
expression in GSCs, we examined the pMad expression in wild-type, gbb
and punt mutant GSCs in the testis. pMad preferentially accumulated
in GSCs but was absent from gonialblasts and two-cell germ cell clusters
(Fig. 3D), which is in contrast
to Dad-lacZ expression in both GSCs and gonialblasts. This difference
could be due to the perdurance of lacZ mRNA and/or protein.
Alternatively, levels of pMad in gonialblasts are low and undetectable with
the existing anti-pMad antibody. In the gbb mutant testes that still
maintained some GSCs, pMad expression in the GSCs was severely reduced and
below the limits of detection (Fig.
3E). In the testes of
punt10460/punt135 mutant males cultured at
29°C, pMad levels in GSCs were severely reduced and were sometimes
difficult to detect (Fig. 3F). However, pMad expression in late 16-cell germ cell clusters remained high in
both the gbb and punt mutant testes (data not shown).
Therefore, gbb probably signals through common Bmp receptors, which
leads to phosphorylation of Mad and Dad transcription.
Bmp signals directly act on GSCs to control their maintenance
To definitely confirm that Bmp signals directly act on GSCs and control
their maintenance, we used the FLP-mediated FRT mitotic recombination to
generate marked GSC clones mutant for Bmp downstream components (Xie and
Spradling et al., 1998; Kiger et al.,
2001; Tulina and Matunis,
2001
). The armadillo-lacZ transgenes that are strongly
expressed in all the cells in the tip of the testis were used to mark mutant
GSC clones. The marked GSCs were induced in adult testes by heat-shock
treatments and identified as lacZ negative, spectrosome-containing
germ cells that are in direct contact with the hub cells. The percentages of
testes carrying one or more marked GSCs were determined at different time
points after clone induction. The rate of loss of GSCs mutant for different
Bmp downstream components can be used to determine how each Bmp downstream
component contributes to the regulation of GSCs.
GSC clones mutant for punt, tkv, sax, mad and Med were
generated as described previously (Xie and
Spradling, 1998), and their testes were examined 2 days later. Two
days after clone induction, 100% of the testes carried one or more marked
wild-type GSCs (Fig. 4A),
whereas 2 weeks after clone induction, 63% of the testes still carried one or
more marked wild-type GSCs (Fig.
4B, Table 1). Two
days after clone induction, over 80% of the testes still carried one or more
marked GSCs mutant for tkv, sax, punt, mad or Med
(Fig. 4C,E;
Table 1). In contrast to
wild-type clones, marked GSC clones mutant for punt, tkv, sax, mad
and Med were lost rapidly 2 weeks after clone induction
(Fig. 4D,F;
Table 1). For example, none of
the testes mutant for punt10460,
punt135, tkv8,
mad12 and Med26 had any GSCs left 2
weeks after clone induction. punt10460 is a moderate
allele, while the rest are strong or null alleles
(Brummel et al., 1994
;
Nellen et al., 1994
;
Xie et al., 1994
;
Letsou et al., 1995
;
Ruberte et al., 1995
;
Das et al., 1998
;
Wisotzkey et al., 1998
).
Interestingly, even though sax4 is a strong or null
sax allele (Brummel et al.,
1994
), 2 weeks after the clone induction, 6.3% of the testes
carried sax mutant GSCs, indicating that sax plays a less
important role in maintaining GSCs than tkv, the other type I
receptor. Previous studies suggest that gbb preferentially uses
sax to transduce its signal, whereas dpp prefers
tkv for its signal transduction
(Haerry et al., 1998
). Our
results argue that gbb preferentially uses tkv instead of
sax to transduce its signal in male GSCs. Therefore, we conclude that
Bmp signals directly act on GSCs and control their maintenance in the
Drosophila testis.
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To determine whether bam transcription is repressed in male GSCs,
we used a bam-GFP transgene (a bam promoter fused to the GFP
gene) to examine its transcription (Chen
and McKearin, 2003a). Interestingly, bam was transcribed
predominantly in the differentiated germ cells but not in GSCs and
gonialblasts in the Drosophila testis
(Fig. 5A), which is consistent
with the BamC expression pattern. Our result that Dad is expressed
only in GSCs and gonialblasts supports the idea that Bmp signaling suppresses
bam expression in GSCs and gonialblasts. If bam repression
in GSCs is mediated by Bmp signaling, we would predict that bam
expression in GSCs defective for Bmp signaling would be upregulated. To test
this hypothesis, we generated dpp, gbb or punt homozygous
mutant males that also carried the bam-GFP transgene to monitor
bam expression. As predicted, bam-GFP was not obviously
upregulated in dpphr56/dpphr4 mutant GSCs just
like in wild-type ones (data not shown), consistent with the fact that the
dpp mutations have little effect on the maintenance of male GSCs.
Interestingly, bam-GFP expression was elevated in the
gbb4/gbbD4 or
gbb4/gbbD20 mutant GSCs
(Fig. 5B), indicating that
gbb signaling is essential for repressing bam transcription
in GSCs. Furthermore, at 22°C, bam expression was undetectable in
the punt mutant GSCs, but it was elevated in punt mutant
GSCs at 29°C (Fig. 5C). To
further confirm this observation, we generated marked Med mutant GSCs
that carried the bam-GFP transgene. Consistently, 66% of 3-day-old
marked lacZ-negative Med mutant GSCs expressed
bam-GFP, while neighboring unmarked lacZ-positive wild-type
GSCs failed to express it (Fig.
5D). These results demonstrate that Bmp signaling is required to
suppress bam transcription in GSCs in the Drosophila
testis.
|
dpp or gbb overexpression cannot completely block the differentiation of GSCs and their progeny in the male
In the Drosophila ovary, dpp overexpression completely
blocks germ cell differentiation, resulting in the formation of GSC-like
tumors (Xie and Spradling,
1998). To determine whether dpp or gbb
overexpression can also prevent germ cell differentiation in the testis, we
overexpressed dpp or gbb using the nanos-gal4VP16
driver. In the testes overexpressing dpp, the hub appeared to be
bigger with more cells, and there were slightly more single germ cells with a
spectrosome around the hub cells (Fig.
6A), whereas the testes overexpressing gbb appeared to be
normal (Fig. 6B). In the
dpp-overexpressing testes, the gonialblasts could still differentiate
and divide but failed to stop after four rounds of cell division for normal
gonialblasts, resulting in the formation of the spermatogonial clusters with
more than 16 germ cells (data not shown). These results suggest that
overexpression of either dpp or gbb does not block
gonialblast differentiation. These observations suggest that the contribution
of dpp signaling to the regulation of the GSC lineage differentiation
is different in males and in females. It seems that dpp
overexpression directly or indirectly influences hub cell formation during
early development as the nanos-gal4VP16 driver is expressed in germ
cells during early gonadal development. Extra single germ cells in the
dpp-overexpressing testes are probably a consequence of more hub
cells, as the bigger hub could potentially produces more Upd molecules, which
are known to influence germ cell differentiation.
|
Both dpp and gbb are expressed in the somatic cells that are in close association with GSCs in the Drosophila testis
To determine the sources for Gbb and Dpp in the testis, we used RT-PCR to
study the presence of gbb and dpp mRNAs in the purified hub
cells, somatic cyst cells and germ cells using fluorescent-activated cell
sorting (FACS). The hub cells were marked by the upd-gal4 driven
UAS-GFP expression (Fig.
7A). The somatic cyst cells and somatic stem cells were marked by
the c587-gal4-driven UAS-GFP
(Fig. 7B). vasa is a
germline-specific gene (Lasko and
Ashburner, 1988; Hay et al.,
1988
). The germ cells were marked by a vasa-GFP transgene
(Nakamura et al., 2001
)
(Fig. 7C). The tips of the
testes were isolated and dissociated, and the GFP-positive cells were purified
from the dissociated testicular cells by FACS. As a control, vasa
mRNAs were present in the whole testis and isolated germ cells but were absent
in the somatic cyst cells and hub cells
(Fig. 7D). Interestingly,
gbb and dpp mRNAs were present in the hub cells and the
somatic cysts/somatic stem cells but were absent in the germ cells
(Fig. 7D). In addition,
dpp mRNAs appeared to be less abundant than gbb mRNAs in the
testis. These results indicate that both Dpp and Gbb are probably somatic
cell-derived Bmp signals that directly regulate GSC maintenance in the
testis.
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Discussion |
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In the gbb mutant testis, GSCs are lost very rapidly but
gonialblasts still develop into 16-cell cysts, suggesting that gbb
functions specifically to control GSC maintenance during germ cell development
in the testis. Surprisingly, mutations in dpp have very little effect
on GSC maintenance, which is in contrast to the role of dpp in the
ovary (Xie and Spradling,
1998). However, a mutation in one copy of the gbb gene
greatly enhances the stem cell loss phenotype of dpp mutants even
though heterozygous gbb males have normal GSC number, indicating that
dpp and gbb work cooperatively to control GSC maintenance.
In the Drosophila testis, dpp plays a less important role
than does gbb with regards to GSC regulation, which could be due to
much lower dpp expression. In the Drosophila ovary, both
dpp and gbb are equally important for maintaining GSCs and
repressing bam transcription in GSCs
(Xie and Spradling, 1998
;
Song et al., 2004
). Although
dpp overexpression in the ovary completely blocks cystoblast
differentiation and causes the accumulation of GSC-like germ cells
(Xie and Spradling, 1998
),
overexpression of either dpp or gbb has little effect on
differentiation of gonialblasts in the testis. These observations suggest that
Bmp signaling is essential for maintaining GSCs in both sexes but gbb
and dpp contribute differently.
Even though gbb has been shown to work synergistically with
dpp potentially through the use of common Bmp receptors in patterning
wing imaginal discs (Khalsa et al.,
1998), it is not known whether gbb signaling directly
contributes to the production of pMad. This study suggests that gbb
signals through previously defined dpp receptors to regulate the
phosphorylation of Mad. We show that pMad in gbb mutant GSCs is
severely reduced just like in punt mutant GSCs. Dad has been
established as a dpp-responsive gene in other developmental processes
(Tsuneizumi et al., 1997
). In
this study, we show that Dad-lacZ expression in GSCs and gonialblasts
is beyond detection in the gbb mutant testis. Interestingly, partial
removal of Dad function can also partially suppress the stem cell
loss phenotype of gbb mutants, suggesting that Dad
negatively regulates gbb signaling. However, Dad-lacZ
expression is only slightly reduced in dpp mutant GSCs and
gonialblasts. These results indicate that Dad is primarily a
gbb responsive gene in the Drosophila testis. They also
argue that gbb indeed signals through common dpp receptors,
promotes Mad phosphorylation and activates Dad transcription in GSCs
in the same way as dpp does.
Dpp can function as a long-range gradient, which elicits different
responses at different concentrations (reviewed by
Podos and Fugerson, 1999). In
the tip of the Drosophila testis, Gbb and Dpp appear to function as
short-range signals and their signaling activities are restricted to GSCs and
gonialblasts based on expression of Dad-lacZ and pMad. gbb
and dpp mRNAs appear to be expressed in both the hub cells and the
somatic cyst cells. In the ovary, Bmp signals also appear to function as
short-range signals (Kai and Spradling,
2003
; Song et al.,
2004
). Gbb and Dpp must be produced and/or activated around the
hub cells and the somatic stem cells in order for them to signal locally to
GSCs and gonialblasts. It would be very interesting to see whether Gbb and Dpp
are localized and/or activated around the hub cells.
Repression of bam transcription in GSCs by Bmp signaling may help maintain GSCs in the testis
Stem cells must remain undifferentiated and continue self-renewal at every
cell division. The relationship between the undifferentiated state and
self-renewal remains to be defined. Even though several signals have been
identified for stem cells in different systems, there is little known about
their direct target genes in stem cells, which could help us to understand how
these signals are translated into the self-renewal or undifferentiated state
of GSCs. In order for a stem cell to maintain its identity, it at least
requires the repression of the genes that are important for differentiation of
stem cell daughters. In this study, we show that Bmp signals from the niche
cells are involved in repressing bam transcription in GSCs in the
testis.
In the present study, we demonstrate that Bmp signaling mediated by Dpp and
Gbb is essential for maintaining GSCs in the testis. bam is known to
be both necessary and sufficient for the differentiation of a cystoblast in
the Drosophila ovary (McKearin
and Spradling, 1990; Ohlstein
and McKearin, 1997
). In the bam mutant testis, GSCs are
well maintained, and gonialblasts still differentiate but overproliferate into
clusters with more than 16 germ cells
(Gonczy et al., 1997
),
suggesting that bam is not necessary for the initial differentiation
of gonialblasts. In this study, we show that forced expression of bam
in GSCs causes them to be lost, which may be due to differentiation and/or
cell death. Normally, bam transcription is absent in GSCs, suggesting
that an active mechanism exists to repress bam expression in GSCs.
The mechanism appears to be mediated by Bmp signals that originate from the
surrounding somatic cells - the niche cells. In the testis, the GSCs mutant
for gbb, punt and Med have elevated bam
transcription. dpp overexpression leads to bam
transcriptional repression in all the germ cells of the testis. These results
demonstrate that Bmp signaling is essential for keeping bam repressed
in GSCs. In the Drosophila ovary, Bmp signaling appears to directly
repress bam expression in GSCs
(Chen and McKearin, 2003b
;
Song et al., 2004
). Whether
the repression of bam transcription in GSCs mediated by Bmp signaling
in the testis is direct remains to be determined. This study indicates that
niche signals maintain the undifferentiated or self-renewal state of stem
cells, at least in part, by repressing the expression of the genes that are
important for the differentiation of their progeny.
How are Bmp and Jak-Stat signaling pathways integrated in male GSCs?
Upd is another known signal for GSCs in the Drosophila testis, and
activates the Jak-Stat signal transduction cascade in GSCs to maintain their
stem cell identity (Kiger et al.,
2001; Tulina and Matunis,
2001
). upd overexpression disrupts normal differentiation
of gonialblasts, leading to the accumulation of stem cell-like germ cells in
the testis. As both Jak-Stat and Bmp signaling pathways are required in GSCs
for their maintenance, they must be integrated and interpreted collectively in
GSCs. There are several possible ways both signaling transduction pathways
could interact with each other. First, Jak-Stat and Bmp signaling pathways
regulate each other in GSCs. In the mammalian system, Bmps can regulate Stat
function by controlling the activity of the FRAP serine-threonine kinase in
neural stem cells (Rajan et al.,
2003
). This may also happen in the GSCs of the testis. It is
possible that Jak-Stat signaling regulates Bmp signaling through a yet
unidentified mechanism. Second, both signaling pathways activate their own
transcription factors, which together activate the expression of the genes
that are important for maintaining GSCs in the undifferentiated or
self-renewal state. Third, both signaling pathways could activate expression
of different genes that are important for maintaining GSCs while repressing
different genes that cause GSC differentiation. These different scenarios
await to be investigated in the future.
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ACKNOWLEDGMENTS |
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