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, male, Bmps
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Introduction |
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The Drosophila ovarian germline stem cells (GSCs) have become an
attractive system to study stem cells and their relationship with niches
(Xie and Spradling, 2001;
Lin, 2002
). Two or three GSCs
are located at the tip of the ovariole, also known as the germarium, and are
surrounded by terminal filament cells, cap cells and inner sheath cells that
form a niche for GSCs. GSCs and their progeny in the germarium can be reliably
identified and distinguished by a germ cell-specific structure, called the
fusome, which is rich in membrane skeletal proteins, such as Hu li tai shao
(Hts) and
-Spectrin (Lin et al.,
1994
; de Cuevas et al.,
1997
). In GSCs and their immediate differentiating daughters,
cystoblasts, the fusome is spherical in shape, and is also known as the
spectrosome. A cystoblast will undergo synchronous mitotic divisions with
incomplete cytokinesis to generate two-, four-, eight- and sixteen-cell cysts,
in which the fusome is branched to interconnect individual cystocytes
(Lin et al., 1994
). GSCs are
invariably anchored to cap cells through adherens junctions
(Song et al., 2002
). Loss of
adherens junctions between cap cells and GSCs causes GSCs to migrate away from
cap cells and undergo differentiation
(Song et al., 2002
). Upon GSC
division, the original GSC remains anchored to cap cells and retains stem cell
identity, whereas the cystoblast moves away from cap cells and undergoes
differentiation. As a GSC is lost, a neighboring GSC can generate two daughter
cells that both contact cap cells and remain as GSCs, thus replenishing a
vacant niche space (Xie and Spradling,
2000
). Functioning as a GSC niche, terminal filament/cap cells
express piwi, dpp, fs(1)Yb (also known as Yb) and
hedgehog (hh), which are essential for maintaining GSC
asymmetric cell division (Xie and
Spradling, 1998
; King and Lin,
1999
; Cox et al.,
2000
; Xie and Spradling,
2000
; King et al.,
2001
). Intrinsic factors in GSCs, including pumilio, nanos,
dpp receptors and downstream components, are also important for GSC
maintenance (Lin and Spradling,
1997
; Forbes and Lehmann,
1998
; Xie and Spradling,
1998
). Two intrinsic factors, bag of marbles
(bam) and benign gonial cell neoplasm (bgcn), are
required in cystoblasts for their proper differentiation
(McKearin and Spradling, 1990
;
McKearin and Ohlstein, 1995
;
Lavoie et al., 1999
). However,
in GSCs the interplay between genes involved in self-renewal versus
differentiation remains unclear.
The functions of dpp signaling and bam in the maintenance
of GSCs and the differentiation of cystoblasts seem to be directly opposing.
Loss of bam function completely eliminates cystoblast
differentiation, similar to that caused by dpp overexpression
(McKearin and Spradling, 1990;
Xie and Spradling, 1998
). By
contrast, forced overexpression of bam in GSCs causes their
elimination, similar to that observed when dpp signaling is disrupted
in GSCs (Ohlstein and McKearin,
1997
; Xie and Spradling,
1998
). These observations can be explained by a simple model
wherein dpp, functioning as a short-range signal, directly promotes
GSC self-renewal and suppresses bam expression in GSCs, while
allowing cystoblasts to express bam and differentiate.
Several studies have supported this model
(Xie and Spradling, 1998;
Chen and McKearin, 2003a
;
Kai and Spradling, 2003
).
bam mRNA is absent in GSCs, but quickly accumulates in cystoblasts
and mitotic cysts (McKearin and Spradling,
1990
). Overexpression of dpp completely suppresses the
expression of BamC protein in germ cells, thus preventing cystoblasts from
differentiating (Xie and Spradling,
1998
). A recent study by Kai and Spradling showed that
dpp signaling activity is restricted to GSCs and cystoblasts
(Kai and Spradling, 2003
).
The asymmetric distribution of bam between GSCs and cystoblasts
could be due to transcriptional regulation and/or mRNA stability. The recent
elegant bam promoter analysis has revealed that its transcription is
actively repressed through a silencer (Chen
and McKearin, 2003a). However, whether and how dpp
signaling directly represses bam transcription remains unknown. In
this study, we provide genetic and molecular evidence to support the model
proposing that Bmp signaling represses bam transcription through
binding of its downstream transcriptional effectors, Mad and Medea (Med), to
the defined bam silencer.
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Materials and methods |
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Most stocks were cultured at room temperature. To maximize their mutant phenotypes, dpp, gbb and punt mutant adult females were cultured at 29°C for 2-7 days. To achieve the uniform GSC-like phenotype, c587-gal4;UAS-dpp females were also cultured at 29°C for 7 days.
Generating mutant GSC clones and overexpression
Clones of mutant GSCs were generated by Flp-mediated mitotic recombination,
as described previously (Xu and Rubin,
1993; Xie and Spradling,
1998
). To generate the stocks for making mutant GSC clones and
examining bam-GFP expression, 2-day old hsFLP; bam-GFP/+;
FRT82B
punt135/FRT82B
armadillo-lacZ and hsFLP; bam-GFP/+;
FRT82B
Med26/FRT82B armadillo-lacZ
females were heat-shocked at 37°C for 3 consecutive days with two one-hour
heat-shock treatments separated by 8-12 hours. The ovaries were removed 3 days
after the last heat-shock treatment and then processed for antibody
staining.
To construct the stocks for overexpressing dpp or gbb, the females that carried hs-gal4, and either UAS-dpp or UAS-gbb, were heat-shocked at 37°C for different lengths of time for particular experiments, as indicated in the Results. The females that carried c587-gal4 and UAS-dpp or UAS-gbb were cultured at room temperature or at 29°C for 7 days. For examining the expression of bam-GFP in the ovary overexpressing dpp or gbb, the females that carried c587-gal4 or hs-gal4, and UAS-dpp or UAS-gbb, also carried a bam-GFP transgene.
Measuring GSC loss in gbb mutants and examining bam-GFP expression in gbb, dpp or punt mutant germaria
To measure stem cell loss in gbb mutant and control ovaries, the
germaria with different numbers of GSCs, ranging from three to none, were
counted from the ovaries of 2-day- and one-week-old bam-GFP
gbb4/gbbD4, bam-GFP
gbb4/gbbD20 or
bam-GFP (control) females. The 2-day-old control and gbb
mutant females were cultured at room temperature after they eclosed at
18°C, whereas the one-week-old control and gbb mutant females
were cultured at 29°C. Values are expressed as the average GSC number per
germarium and the percentage of germaria with no GSCs.
To examine bam-GFP expression in dpp, gbb or punt mutant germaria, we generated females with the following genotypes at 18°C: bam-GFP gbb4/gbbD4, bam-GFP gbb4/gbbD20, bam-GFP dpphr56/dpphr4, bam-GFP; punt10460/punt135 or bam-GFP (control) females. All the control and mutant females were cultured at 29°C for 4 days before their ovaries were isolated and immunostained, to compare bam-GFP expression under identical conditions.
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-galactosidase
antibody (1:100; Cappel); polyclonal anti-GFP antibody (1:200; Molecular
Probes); and 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.
Examining gene expression using the Affymetrix microarray
Total RNA from the ovaries of different genotypes or treatments was
isolated using Trizol (Invitrogen), and biotin-labeled cRNA probes were
produced using an RNA transcript labeling kit (Enzo BioArray). The
Drosophila GeneChips were purchased from Affymetrix, and were
hybridized, stained and detected according to the manufacturer's
instructions.
Detecting gene expression in purified component cells using RT-PCR
After sorting GFP-positive cells by using Cytomation MoFlo, total RNA was
prepared using Trizol (Invitrogen) from these isolated cells. The RNA samples
were further amplified using the GeneChip Eukaryotic Small Sample Target
Labeling Assay Version II (Affymetrix). After the RNA amplification, 100 ng of
total RNA was reverse-transcribed (RT) using the SuperScriptIII First-Strand
Synthesis System for RT-PCR, according to manufacturer's protocol
(Invitrogen). The following primers were used in this study:
PCR was performed as follows: 94°C for 2 minutes; 35 cycles of 94°C for 30 seconds, 45°C for 30 seconds and 72°C for 45 seconds; and 72°C for 7 minutes. RT-PCR products were electrophoresed on a 2% agarose gel in the presence of ethidium bromide.
Electrophoretic mobility shift assays for the binding of Mad and Med to the bam silencer
The GST-Mad construct was described previously
(Kim et al., 1997). Med was
PCR-amplified from its cDNA, with the introduction of XhoI sites at
both ends, then subcloned into a pGEX-4T2 vector (Amersham Pharmacia Biotech).
Its sequence was confirmed by sequencing. GST-Mad, GST-Med and GST proteins
were purified by affinity chromatography using Glutathione SepharoseTM 4B
according to the manufacturer's protocol (Amersham Pharmacia Biotech), and
confirmed by western blots.
A Cy5 5'-modified oligonucleotide containing the bipartite
bam silencer element (+17 to +54) was used as a probe. Binding
reactions were performed according to the published protocol
(Kim et al., 1997).
Specificity of binding was determined by the addition of 100-fold molar excess
of unlabeled competitor DNA corresponding to the bam silencer
element, with site A and/or B. DNA-protein complexes were resolved on a 5%
(w/v) non-denaturing polyacrylamide gel using 0.5xTBE running buffer at
150 V for 3 hours at 4°C. Gels were imaged on a Typhoon 8700 (Amersham
Biosciences).
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Results |
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dpp signaling is essential for repressing bam transcription in GSCs
The
dpphr56/dpphr4
temperature-sensitive mutant was chosen to investigate the expression of
bam-GFP in dpp mutant GSCs because it shows gradual loss of
GSCs within two weeks at a restrictive temperature (29°C)
(Xie and Spradling, 1998).
After bam-GFP and bam-GFP
dpphr56/dpphr4 females
were cultured at 29°C for 2, 4 or 7 days, the ovaries were immunostained
with anti-GFP and anti-Hts antibodies to visualize bam-GFP and
fusomes, respectively. In the germaria from the bam-GFP females, the
bam-GFP expression pattern was completely normal, and was absent in
GSCs even one week after being cultured at 29°C
(Fig. 2A). However, even two
days after being cultured at 29°C, 28% of the bam-GFP
dpphr56/dpphr4 germaria
that contained GSCs started to express bam-GFP in one or more GSCs
(n=283; Fig. 2B).
After 4 days and 7 days, 66% (n=35) and 89% (n=19) of the
mutant germaria that still had at least one GSC expressed bam-GFP in
one or more GSCs, respectively (Fig.
2C,D). To further confirm the role of dpp signaling in
repressing bam transcription, we also compared the levels of
bam mRNA in wild-type and dpp mutant ovaries using a
microarray approach. bam mRNA was dramatically upregulated in
dpphr4/dpphr56
mutant ovaries in comparison with wild type
(Table 1; samples were
normalized with an internal control, the Actin 42A gene). These
results demonstrate that the dpp signal is required to repress
bam transcription in GSCs.
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|
dpp overexpression is sufficient for repressing bam transcription in the cystoblast
Our previous study showed that overexpression of dpp throughout
the germarium completely inhibits cystoblast differentiation and causes the
accumulation of GSC-like cells that fail to express BamC
(Xie and Spradling, 1998). Our
experiments described above suggest that the dpp signal is likely to
be restricted to the tip of the germarium, adjacent to cap cells. To test
whether GSCs are competent to respond to dpp signaling outside their
niches, dpp was specifically overexpressed in somatic cells other
than cap cells, using the c587-gal4 line to drive a UAS-dpp
transgene. The c587-gal4 line can drive expression of a UAS-GFP
transgene in inner sheath cells and early follicle cells
(Fig. 3A). When
UAS-dpp expression was driven in inner sheath cells and follicle
cells, germaria were filled with single germ cells with a spectrosome,
suggesting that germ cells distant from their niche are still capable of
responding to dpp (Fig.
3B). To further test whether dpp overexpression is
sufficient to inhibit bam expression, we examined bam-GFP
expression in dpp-induced GSC-like tumors. In
dpp-overexpressing ovaries, bam-GFP was not expressed in the
single germ cells either close to (Fig.
3C) or away from (Fig.
3D) the germarial tip. These results indicate that dpp
signaling is sufficient to inhibit bam transcription.
|
gbb is expressed in the somatic cells of the germarium and is essential for maintaining GSCs and repressing bam transcription in GSCs in the Drosophila ovary
In addition to Dpp, another Bmp-like molecule, Glass bottom boat (Gbb),
exists in Drosophila and resembles human BMPs 5, 6, 7 and 8
(Wharton et al., 1991;
Doctor et al., 1992
). It has
been shown that synergistic signaling by dpp and gbb
controls wing growth and patterning in Drosophila
(Haerry et al., 1998
;
Khalsa et al., 1998
). To
investigate the possibility that gbb could also be involved in the
regulation of GSCs, we first used RT-PCR to determine whether gbb
mRNA was present in different cell types of the germarium. Inner sheath cells
and early follicle cells were isolated from c587-gal4;UAS-GFP females
using fluorescent-activated cell sorting (FACS). Agametic ovaries were
isolated from newly eclosed females that developed from
ovoD1rS1 homozygous embryos lacking germ cells
(Oliver et al., 1990
). The
agametic ovary is composed of terminal filament cells, cap cells and early
follicle cells but lacks inner sheath cells
(Margolis and Spradling,
1995
). Single germ cells, resembling GSCs, were isolated from
c587-gal4; vasa-GFP/UAS-dpp females using FACS. vasa is a
germ cell-specific gene (Hay et al.,
1988
; Lasko and Ashburner,
1988
), and vasa-GFP is specifically expressed in the germ
cells (Nakmura et al., 2001). dpp is expressed in the somatic cells
of the germarium but not in germ cells
(Xie and Spradling, 2000
).
vasa mRNA was present in germ cells but not in inner sheath cells and
agametic ovaries (Fig. 4A),
whereas dpp mRNA was present in inner sheath cells and agametic
ovaries but not in germ cells (Fig.
4A), indicating that the different cell types in germaria were
properly isolated. gbb mRNA was detected in inner sheath cells and
agametic ovaries but not in the GSC-like germ cells
(Fig. 4A), indicating that
gbb is expressed in the somatic cells. These results indicate that
gbb could be another somatic signal for controlling GSCs.
|
|
Having established that dpp is sufficient to repress bam
expression in GSCs, we then asked whether gbb overexpression was
sufficient to repress bam transcription in germ cells. Similarly,
bam-GFP expression was studied in germaria overexpressing
gbb using the C587 driver and the UAS-gbb transgene, which
has been used to effectively overexpress gbb in the wing disc
(Khalsa et al., 1998). The
germaria overexpressing gbb had the normal number of GSCs and cysts
(Fig. 4I), indicating that GSC
maintenance and division, and germ cell differentiation, appeared to be
normal. Similarly, the bam-GFP expression pattern was also normal in
the gbb-overexpressing germaria
(Fig. 4I). These results
suggest that gbb overexpression, unlike that of dpp, is not
sufficient to inhibit bam transcription.
Loss of gbb signaling results in a reduction of pMad in GSCs that is related to bam upregulation in GSCs
It appears that gbb uses the same downstream components as
dpp does in regulating wing development
(Haerry et al., 1998;
Khalsa et al., 1998
).
dpp signaling results in the production of pMad
(Newfeld et al., 1997
;
Tanimoto et al., 2000
). To
investigate whether gbb is also involved in the production of pMad in
GSCs, we examined pMad accumulation in gbb mutant GSCs, and the
relationship between pMad accumulation and bam transcription. As
expected, pMad and bam-GFP expression patterns in GSCs and
cystoblasts remained normal four days after the control females were cultured
at 29°C (Fig. 5A,A').
Four days after being cultured at 29°C, the expression of pMad in the GSCs
in both gbb4/gbbD4
and gbb4/gbbD20
females was generally reduced (Fig.
5B-F'). Some of the mutant gbb germaria that had
moderately reduced pMad expression in GSCs showed no bam-GFP
expression in GSCs (Fig.
5B-D'), whereas the germaria that had severely reduced
levels of pMad in GSCs showed significant bam-GFP upregulation in
GSCs (Fig. 5E-F'). There
appeared to be a good correlation between levels of pMad and bam-GFP
expression in gbb mutant GSCs. These results indicate that
gbb signaling also results in the phosphorylation of Mad and that
levels of pMad in GSCs seem to correlate with levels of bam
repression.
|
|
Mad and Med directly bind to the silencer in the bam promoter in vitro
We have so far shown that Bmp signaling mediated by Dpp and Gbb is
essential for repressing bam transcription in GSCs. This bam
transcriptional repression could be directly or indirectly controlled by Bmp
signaling. As shown recently by Chen and McKearin, a silencer located at the
5' UTR of the bam gene is both necessary and sufficient for
repressing bam transcription in GSCs
(Chen and McKearin, 2003a). In
Drosophila, the brinker (brk) gene is actively
repressed by dpp signaling through a transcriptional silencer
(Campbell and Tomlinson, 1999
;
Jazwinska et al., 1999
;
Minami et al., 1999
;
Marty et al., 2000
;
Muller et al., 2003
).
Interestingly, bam and brk silencers show remarkably similar
sequences: 13 out of 19 base pairs are identical in A and B sites
(Fig. 7A). The brk
silencer has been shown to be directly occupied by a complex containing Mad,
Med and Schnurri (Shn), and its repression requires shn and
functional dpp signaling (Muller
et al., 2003
). shn is known to be required in GSCs for
their maintenance, and loss of shn function results in GSC loss
(Xie and Spradling, 2000
). All
the evidence suggests that the bam silencer could be directly
occupied by a complex containing Mad, Med and possibly Shn.
|
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Discussion |
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Gbb is another niche signal that is essential for maintaining GSCs
In this study, a new function of gbb in the regulation of GSCs in
the Drosophila ovary is revealed. Loss of gbb function leads
to GSC differentiation and stem cell loss, similar to dpp mutants.
gbb is expressed in somatic cells but not in germ cells, suggesting
that gbb is another niche signal that controls GSC maintenance. Like
dpp, gbb contributes to the production of pMad in GSCs and also
functions to repress bam expression in GSCs. As in the wing imaginal
disc (Haerry et al., 1998;
Khalsa et al., 1998
;
Ray and Wharton, 2001
),
gbb also probably functions to augment the dpp signal in the
regulation of GSCs through common receptors in the Drosophila ovary.
In both dpp and gbb mutants, pMad accumulation in GSCs is
severely reduced but not completely diminished. As the dpp or
gbb mutants used in this study do not carry complete loss-of-function
mutations, it remains possible that complete elimination of either
dpp or gbb function is sufficient for eradicating pMad
accumulation in GSCs. Alternatively, both dpp and gbb
signaling are required independently for full pMad accumulation in GSCs, and
thus disrupting either one of them only partially diminishes pMad accumulation
in GSCs. The lethality of null dpp and gbb mutants, and the
difficulty in completely removing their function in the adult ovary, prevent
us from further testing these possibilities directly.
Interestingly, dpp overexpression results in complete suppression
of cystoblast differentiation and complete repression of bam
transcription in the germ cells, whereas gbb overexpression does not
have obvious effects on cystoblast differentiation or bam
transcription. Even though the UAS-gbb transgene and the
c587 driver for gbb overexpression have been demonstrated
previously to function properly (Khalsa et
al., 1998; Kai and Spradling,
2003
; Zhu and Xie,
2003
), it is possible that active Gbb proteins are not produced in
inner sheath cells and somatic follicle cells because of a lack of proper
factors that are required for Gbb translation and processing in those cells,
which could explain why the assumed gbb overexpression does not have
any effect on cystoblast differentiation. However, as active Dpp proteins can
be successfully achieved using the same expression method, and Dpp and Gbb are
closely related Bmps, it is unlikely that active Gbb proteins are not produced
in inner sheath cells and follicle cells. Alternatively, dpp and
gbb signals could have distinct signaling properties, and
dpp may play a greater role in regulating GSCs and cystoblasts.
Recent studies have indicated that Dpp and Gbb have context-dependent
relationships in wing development (Ray and
Wharton, 2001
). In the wing disc, duplications of dpp are
able to rescue many but not all of the phenotypes associated with gbb
mutants, suggesting that dpp and gbb have not only partly
redundant functions but also distinct signaling properties. In the wing and
ovary, gbb and dpp function through two Bmp type I
receptors, sax and tkv
(Khalsa et al., 1998
;
Xie and Spradling, 1998
) (this
study). The puzzling difference between gbb and dpp could be
explained by context-dependent modifications of Bmp proteins, which render
them different signaling properties in different cell types. It will be of
great interest to better understand what causes Bmps to have distinct
signaling properties in the future.
Dpp and Gbb function as short-range signals in the GSC niche
All the defined niches share a commonality, structural asymmetry, which
ensures stem cells and their differentiated daughters receive different levels
of niche signals (Watt and Hogan,
2000; Spradling et al.,
2001
). In order for a niche signal to function differently in a
stem cell and its immediately differentiating daughter cell that is just one
cell away, it has to be short-ranged and localized. This study, and a recent
study by Kai and Spradling (Kai and
Spradling, 2003
), show that Bmp signaling mediated by Dpp and Gbb
results in preferential expression of pMad and Dad in GSCs. In this study, we
show that Bmp signaling appears to elicit different levels of responses in
GSCs and cystoblasts, suggesting that the cap cells are likely to be a source
for active short-ranged Bmp signals. These observations support the idea that
Bmp signals are only active around cap cells. Consistently, when GSCs lose
contact with the cap cells following the removal of adherens junctions they
move away from the niche and then are lost
(Song et al., 2002
). As
gbb and dpp mRNAs are broadly expressed in the other somatic
cells of the germarium besides cap cells, localized active Bmp proteins around
cap cells could be generated by localized translation and/or activation of Bmp
proteins. As they can function as long-range signals
(Podos and Ferguson, 1999
), it
remains unclear how Dpp and Gbb act as short-range signals in the GSC
niche.
Bmp signals probably directly repress bam transcription in GSCs
Previous studies, and this study, have shown that Bmp signaling and
bam expression are directly opposing in Drosophila ovarian
GSCs (McKearin and Spradling,
1990; Ohstein and McKearin, 1997;
Xie and Spradling, 1998
).
bam is actively repressed in GSCs through a defined transcriptional
silencer (Chen and McKearin,
2003a
). These observations lead us to propose a model in which Bmp
signals from the niche maintain adjacent germ cells as GSCs by actively
suppressing bam transcription and thus preventing differentiation
into cystoblasts (Fig. 7E).
In this study, we show that the levels of pMad are correlated with the
amount of bam transcriptional repression in GSCs and cystoblasts. In
the wild-type germarium, pMad is highly expressed in GSCs and some cystoblasts
where bam is repressed. In other cystoblasts and differentiated
germline cysts, pMad is reduced to very low levels, and thus bam
transcriptional repression is relieved. In the GSCs mutant for dpp,
gbb or punt, pMad levels are severely reduced, and bam
begins to be expressed. The repression of bam transcription as a
result of dpp overexpression seems to be a rapid process as
bam mRNA was reduced to below detectable levels two hours after
dpp was overexpressed. This suggests that repression of bam
transcription by Bmp signaling could be direct. Furthermore, Med and Mad can
bind to the defined bam silencer in vitro, which also supports the
idea that Bmp signaling acts directly to repress bam transcription.
Similar results have been obtained in a recent study
(Chen and McKearin, 2003b).
Similarly, Dpp signaling has been shown to repress brk expression in
the wing imaginal disc and in the embryo
(Campbell and Tomlinson, 1999
;
Jazwinska et al., 1999
;
Minami et al., 1999
). The
repression of brk expression by Dpp signaling is mediated by the
direct binding of Mad and Med to a silencer element in the brk
promoter (Muller et al.,
2003
). As the brk silencer is very similar to the
bam silencer, our results suggest that bam repression in
GSCs is also mediated directly by Dpp and Gbb in a similar manner.
It remains unclear how the binding of Med and Mad to the bam
silencer results in bam transcriptional repression in GSCs. For the
brk silencer, Dpp signaling and Shn are both required to repress
brk expression in the Drosophila wing disc and embryo
(Marty et al., 2000;
Torres-Vazquez et al., 2001
;
Muller et al., 2003
). Mad and
Med belong to the Smad protein family, which are known to function as
transcriptional activators by recruiting co-activators with histone
acetyltransferase activity (reviewed by
Massague and Wotton, 2000
). In
the wing disc, Shn is proposed to function as a switch factor that converts
the activating property of Mad and Med proteins into a transcriptional
repressor property (Muller et al.,
2003
). Possibly, the Mad-Med complex could also recruit Shn to the
bam repressor element. Consistent with the possible role of Shn in
repressing bam expression in GSCs is the observation that GSCs that
lose shn function differentiate, and thus are lost
(Xie and Spradling, 2000
).
Also, it remains possible that Mad and Med could recruit a repressor other
than Shn when binding to the bam repressor element. In the future, it
will be very important to determine whether Shn itself is a co-repressor for
Mad/Med proteins or whether it directly recruits a co-repressor to repress
bam transcription in GSCs.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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