1 Committee on Developmental Biology, University of Chicago, Chicago IL 60637,
USA
2 Department of Molecular Genetics and Cell Biology, University of Chicago,
Chicago IL 60637, USA
* Author for correspondence (e-mail: elfergus{at}midway.uchicago.edu)
Accepted 14 January 2004
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SUMMARY |
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Key words: Decapentaplegic, Dpp, Stem cell, GSC, Bam, Smurf, Drosophila, Germline
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Introduction |
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The anatomy of the germarium of the Drosophila ovariole is consistent with the existence of a niche that maintains the GSCs (Fig. 1A). Two or three GSCs lie at the anterior tip of the germarium in contact with somatic CpCs and in close apposition to somatic terminal filament (TF) cells. The plane of GSC division is perpendicular to the anteroposterior axis of the germarium such that the daughter cell that stays in contact with the CpCs remains a GSC, while the more posterior daughter cell becomes a cystoblast (CB). Each CB divides four times with incomplete cytokinesis, resulting in a germline cyst containing 16 interconnected cells, one of which will become the oocyte.
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Conversely, there is at least one gene, bag of marbles
(bam), whose activity in CBs is necessary to promote germline
differentiation. Females that lack bam activity have ovarioles that
contain an overproliferation of cells with GSC or CB-like morphology
(McKearin and Ohlstein, 1995).
The bam gene is repressed in GSCs under control of Dpp signaling
(Chen and McKearin, 2003a
), but
is expressed in CBs (McKearin and
Spradling, 1990
; Chen and
McKearin, 2003b
). Ectopic expression of bam in GSCs
results in their elimination (Ohlstein and
McKearin, 1997
). Bam function requires the activity of benign
gonadal cell neoplasm (bgcn), whose predicted protein product
shares several motifs with the DexH family of RNA helicases
(Lavoie et al., 1999
;
Ohlstein et al., 2000
),
suggesting that Bam and Bgcn act together to promote GSC differentiation by
post-transcriptional regulation of gene activity
(Ohlstein et al., 2000
). These
data suggest a model for control of the asymmetric GSC division in which the
anterior GSC daughter has a high level of Dpp signaling and maintains a GSC
fate by repressing bam transcription, while the posterior GSC
daughter has a lower level of Dpp signaling, thereby allowing bam
expression, which triggers a program leading to CB differentiation.
In this paper, we examine the mechanisms underlying the temporal and spatial control of Dpp signaling within the developing germline. We find that, although Dpp signaling in the somatic cells is not limited to the niche, the expression of the Dpp target gene Dad-lacZ in the germline is confined to GSCs and CBs. We demonstrate that one, but not the only, function of Bam is to downregulate Dpp signaling downstream of Dpp receptor activation, and that action of the ubiquitin protein ligase Smurf (Lack FlyBase) is functionally redundant with that of Bam in downregulation of Dpp signaling. These data provide potential insight into the mechanisms underlying the stable switch in developmental states that occurs during GSC differentiation.
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Materials and methods |
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Immunocytochemistry, fluorescence and confocal microscopy
Ovaries were dissected in EBR buffer and stained as described
(Lin et al., 1994). The
following primary antibodies were used: monoclonal anti-Orb antibody (1:10)
(Lantz et al., 1994
);
polyclonal anti-lacZ antibody (1:500, Cappel); monoclonal anti-Hts
antibody 1B1 (1:2.5) (Zaccai and Lipshitz,
1996
); monoclonal anti-Myc antibody 1-9E10 (1:100, Santa Cruz);
polyclonal anti-alpha spectrin antibody (1:400)
(Byers et al., 1987
);
polyclonal anti-Vasa antibody (1:1000)
(Liang et al., 1994
); rat
polyclonal anti-Tkv antibody (1:5)
(Teleman and Cohen, 2000
);
monoclonal anti-BamC antibody used as described by McKearin and Ohlstein
(McKearin and Ohlstein, 1995
);
and polyclonal anti-GFP (1:500, Abcam). The following secondary antibodies
were used: anti-rabbit, anti-mouse and anti-rat Alexa Fluor 488 (1:1000,
Molecular Probes); and anti-mouse and anti-rabbit Cy3 (1:1000, Jackson
Immunoresearch). DNA was visualized either by YOPRO1 (1 µM, Molecular
Probes) or by DAPI (0.3 µM, Molecular Probes) staining. Mounting of samples
was carried out in a 70%Tris-glycerol mixture pH 7.6 containing 2% DABCO
(Sigma). Fluorescent images were captured with a Zeiss Axiocam mounted on a
Zeiss Axioplan microscope equipped with a 20x Plan-Apo 1.4 NA objective.
Digital images of serial optical sections were collected with a BioRad 1024
Zeiss confocal microscope using either 25x or 63x objectives.
Images were merged using the LSM50 software and further processed using Adobe
Photoshop 5.0.
Determination of niche size in wild-type and mutant females
The number of Dad-lacZ-expressing germ cells was
determined in multiple ovarioles from females of each of the following
genotypes (separated by commas): P{lacZ}DadP1883/TM3,
P{lacZ}DadP1883 bamD86/bamD86,
Tp(2;2)DTD48/CyO; P{lacZ}DadP1883/TM2,
saxB18/CyO; P{lacZ}DadP1883/TM2,
saxB18 Tp(2;2)DTD48/CyO;
P{lacZ}DadP1883/TM2, saxB18
Tp(2;2)DTD48/CyO; bamD86/bamD86
P{lacZ}DadP1883, Smurf15C; bamD86
P{lacZ}DadP1883/TM2 and Smurf15C;
bamD86/bamD86 P{lacZ}DadP1883.
For each ovariole from single mutant females, the number of cells expressing
lacZ was counted using confocal microscopy. The statistical analyses
of the number of GSCs in wild-type and single mutant ovarioles were performed
using the GraphPad Prism program. We note that the survival to adulthood of
double mutant flies of genotypes Smurf15C;
bamD86/bamD86 P{lacZ}DadP1883
and saxB18 Tp(2;2)DTD48/CyO;
bamD86/bamD86 P{lacZ}DadP1883
was much lower than expected.
Phenotype of tumorous ovaries after Bam expression
Flies of genotypes P{hs-bam.O}; P{UAS.p-TkvAct)/TM3,Sb
and P{Gal4::VP16-nos.UTR} were mated at 25°C and transferred
every day to new bottles. In experimental but not control crosses, on the
seventh and following days after egg deposition, the F1 larvae/pupae were
subject to two 1-hour 37°C heat shocks, separated by 1 hour at room
temperature. After eclosion, experimental non-Sb F1 females were collected and
kept in vials with fresh yeast at 25°C and heat-shocked daily using the
same protocol. Dissected ovaries of F1 non-Sb control and experimental females
of the same age were examined with Nomarski or confocal optics. Some
experimental females were mated with wild-type males in small egg-laying cups
for 3 days to examine the follicular morphology of the eggs. Application of
the identical heat shock protocol caused females of genotype
P{hs-bam.O} to lose all germ cells within their germaria.
Nos and Dad-lacZ detection in tumorous ovaries after Bam expression
Flies of genotype P{nos-myc.V}/CyO; P{UAS.p-TkvAct}
P{lacZ}DadP1883/TM3,Sb were mated with flies of
genotype P{hs-bam.O}; P{GAL4::VP16-nos.UTR}/TM3,Sb.
In experimental but not control crosses, F1 larvae/pupae were heat shocked as
described above, beginning on day 9-10 of development and continuing for 1 or
2 days after eclosion. Ovaries from control and experimental non-Sb, non-Cy F1
females of the same age were stained at the same time, and image acquisition
of control and experimental samples was performed at the same session.
Specifically, control tumors were scanned first and the same acquisition
parameters were used to scan the experimental samples. The images of control
and experimental ovaries were subsequently processed identically. Each set of
experiments was repeated at least three times.
Expression of lacZ in bamD86 mutant ovaries
Flies of genotypes P{hs-bam.O}; bamD86/TM3,Sb
and P{UAS-lacZ.p}; bamD86
P{GAL4::VP16-nos.UTR}/TM3,Sb were crossed. For the experimental
but not control cross, F1 progeny were subject to heat shock using the
protocol described above. Ovaries from F1 non-Sb experimental and control
females were dissected 1-2 days after eclosion.
Epistatic analysis of Mad12 and bamD86
To determine the approximate half lives of wild-type or Mad mutant
GSCs, females of genotype
P{ry+,hs-neo,FRT}40A/CyO;
bamD86/TM2,Ubx (for wild-type GSCs) or
Mad12 P{ry+,hs-neo,FRT}40A/CyO;
bamD86/TM2,Ubx (for Mad mutant GSCs)
were mated with males of genotype P{hs-FLP}/Y; P{arm-lacZ}
P{ry+,hs-neo,FRT}40A;
bamD86/TM2,Ubx in bottles and transferred daily.
For both sets of crosses, the F1 progeny from the cross were heat-shocked
twice at 37°C for 1 hour separated by 8-12 hours at stage P4 pre-pupae or
P5 pupa, and ovaries from phenotypically Ubx F1 females were examined. Clones
of wild-type GSCs were present in 38% of ovarioles (n=138) in
5-day-old females and 48% of ovarioles in 10-day-old females (n=79).
GSCs mutant for Mad12 were present in 20% of ovarioles
(n=73) from 5-day-old females, but only 4% of ovarioles
(n=140) from 10-day-old females. Germline clones of wild-type or
Mad cells in a bam background were examined in ovaries of
non-Ubx F1 females from the above crosses.
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Results |
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Because Dpp is both necessary for GSC maintenance and can be sufficient to
cause overproliferation of cells with morphologies similar to GSCs, we wished
to determine which germ cells within the germarium are responsive to Dpp
signaling. To do so, we examined the spatial expression of lacZ
driven by a P-element enhancer trap inserted near the Dpp target gene,
Dad, which encodes an inhibitory Smad that in other developmental
contexts has been shown to be transcriptionally activated by Dpp signaling
(Tsuneizumi et al., 1997).
Previous analysis (Xie and Spradling,
1998
) had shown that GSCs lacking Dad have a longer
half-life than do wild-type GSC clones, indicating that Dad functions
within the germline. We found that lacZ is expressed only in
spectrosome-containing cells and is absent from all fusome-containing cells
(Fig. 1C). Moreover, putative
GSC cells at the anterior tip of the germarium have an elevated level of
lacZ expression compared with putative CB cells removed from the
anterior tip (Fig. 1C,E,F).
Using the criteria of cell position and level of lacZ expression, we
found that wild-type ovarioles have an average of 2.3±0.9 putative
GSCs, and 1.2±0.8 putative CBs (n=24). Moreover,
Dad-lacZ is not expressed within the developing cysts and
egg-chambers (Fig. 1G). Thus,
within the germline, Dpp signaling is strictly limited to GSCs and CBs, and
CBs appear to be less responsive to Dpp than their GSC sisters. Similar
results were also reported recently by Kai and Spradling
(Kai and Spradling, 2003
).
The pattern of Dad-lacZ expression also allowed us to
assay which somatic cells within the germarium are responsive to endogenous
Dpp signaling. Although lacZ expression in somatic cells varied
between individual preparations, in a significant fraction of wild-type
ovarioles Dad-lacZ expression was visible in CpCs, but also
in multiple somatic cells located in the same positions within regions 1 and
2A of the germarium as ISCs that express patched
(Forbes et al., 1996)
(Fig. 1D). This observation
indicates that the CpCs and the ISCs are both exposed to Dpp and responsive to
Dpp signaling, suggesting that Dpp is not limited to the GSC niche.
We then expressed a constitutively active form of the Dpp receptor
Thickveins (TkvAct) in germ cells using a Gal4-UAS system optimized for
germline expression (Rørth,
1998). When the P{UAS.p-TkvAct} construct was placed
initially under the control of a P{vas-GAL4} driver, no overt change
in morphological phenotype was observed. We assayed these ovarioles for Dpp
signaling, as evidenced by Dad-lacZ expression. While the
vasa promoter drives expression throughout the germline
(Fig. 1H), we observed that the
expression of the Dad-lacZ reporter gene in females of
genotype P{vas-GAL4}/+; P{UAS.p-TkvAct}
P{lacZ}DadP1883/+ + was not uniform. Specifically, in all
ovarioles examined (n=15), lacZ was expressed in the GSCs
and CBs and in the developing egg chambers (asterisks,
Fig. 1I), but was absent from
the developing cysts (bracket, Fig.
1I), suggesting that cyst cells are partially or completely
refractory to Dpp signaling. Taken together, these data argue against a model
in which the observed restriction of germline Dpp signaling to the GSC and CBs
is caused only by the limited exposure of GSCs to Dpp ligand. Rather, these
data strongly suggest that Dpp is present throughout the anterior germarium
and that cell-intrinsic mechanisms operating within the developing cysts play
an active role in downregulating Dpp signaling.
Constitutive Dpp signaling within the germline prevents GSC differentiation
Expression of the P{UAS.p-TkvAct) construct at higher levels,
through use of a P{Gal4::VP16-nos.UTR} driver, resulted in production
of `tumorous' ovarioles that did not contain any differentiating egg chambers
(Fig. 2A), but were filled with
cells with all characteristics of wild-type GSCs. Specifically, all germline
cells in these tumorous ovarioles contained spectrosomes
(Fig. 2B,C), expressed
Dad-lacZ (Fig.
2B), and stained for Nos-Myc
(Fig. 3D). Moreover, no
germline cells in any mutant ovariole (n=40) stained with the CB
marker, Bam-C (Fig. 2C), nor
expressed bam mRNA (not shown), as opposed to sibling wild-type
ovaries in which Bam-C was expressed in CBs and young cysts (not shown). Many
germ cells within these tumors are capable of undergoing an apparent
self-renewal division, as evidenced by staining with an anti-Histone H3
antibody that recognizes cells in M phase (not shown), and, as described
below, are capable of differentiation. Thus, using the spectrum of markers
available to us, we conclude that cell autonomous activation of Dpp signaling
in the germline produces cells with morphological, molecular and functional
characteristics indistinguishable from wild-type GSCs.
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These data demonstrate that Dpp has no additional, obligate role in somatic
cells for maintenance of GSC-like identity, and that Dpp signaling initially
can maintain GSC-like cells independent of the somatic niche. However, as the
majority of these germ cells are not in contact with the somatic niche, and
thus may not receive contact-dependent niche signals, these germ cells could
represent an intermediate state between a GSC and a CB. A similar hypothesis
was proposed recently by Gilboa et al.
(Gilboa et al., 2003). For
simplicity of nomenclature, though, we will refer to these cells in the
remainder of this paper as GSCs, but we recognize that a more extensive panel
of molecular markers will be required to determine whether these cells are in
fact identical to wild-type GSCs.
Bam expression promotes GSC differentiation in part by downregulation of Dpp signaling
We then wished to investigate whether Bam function would be sufficient to
promote GSC differentiation in the presence of constitutive Dpp signaling. To
do so, we heat shocked flies of genotype P{hs-bam.O}/+;
P{UAS.p-TkvAct}/P{Gal4::VP16-nos.UTR} daily starting at 7 days of
development and observed their ovaries at various periods after eclosion. Our
data indicate that expression of Bam is sufficient to completely overcome the
effects of constitutive Dpp signaling to promote normal germline
differentiation.
Examination of ovarioles 3 or 4 days after eclosion revealed significant rescue of the tumorous ovariole phenotype (Fig. 3A), with all ovarioles containing egg chambers with large, polyploid nuclei and germline expression of Orb. Although many of these egg chambers had a normal 15:1 nurse cell to oocyte ratio, some contained an abnormal number of germ cells (Fig. 3A and inset). Extension of the heat shock to 7 days post-eclosion and assay of the ovarian phenotype 3 days later resulted in the production of ovarioles with completely normal morphology (Fig. 3B). The germaria of many rescued ovarioles had a wild-type appearance with a small number of spectrosome-containing cells in the GSC niche (Fig. 3C). However, some germarium were devoid of germ cells, suggesting that germ cells in the GSC niche may be more, but not completely, resistant to the effects of Bam. Many females subject to this heat shock regiment laid eggs with normal follicular morphology; however, the eggs did not differentiate cuticle, possibly because of the dorsalizing effects of the TkvAct receptor on embryonic pattern.
We then determined whether ubiquitous ectopic expression of Bam in tumorous ovarioles caused the same pattern of changes in morphology and gene expression during initial GSC differentiation as was observed in wild-type ovarioles. Flies of genotype P{hs-bam.O}/+; P{nos-myc.V}/+; P{UAS.p-TkvAct} P{lacZ}DadP1883/P{GAL4::VP16-nos.UTR} were heat shocked daily starting at 9 or 10 days of development and their ovaries were examined 2 days after eclosion for the expression of the Nos-Myc and Dad-lacZ markers. Although Nos was present in all germ cells in control tumorous ovarioles not subject to heat shock (Fig. 3D), Nos displayed a dynamic pattern of expression in heat-shocked ovarioles that was identical to its pattern in the wild type (Fig. 3E). Specifically, Nos was expressed in all spectrosome-containing cells in the heat-shocked ovarioles (100%, n=87 cells), was absent in two- to eight-cell cysts (18%, n=17 cysts), and became upregulated in all 16-cell cysts (100%, n=15 cysts). These results demonstrate that the dynamic pattern of wild-type Nos expression is recapitulated after ectopic Bam expression, raising the possibility that Nos downregulation is necessary for cyst formation.
We were particularly interested in examining the pattern of Dad-lacZ expression in these heat-shocked tumorous ovarioles. If the spatial extent of Dpp signaling in wild-type germaria were controlled solely by ligand availability, then we would expect to see that germ cells in the heat-shocked ovarioles would continue to express Dad-lacZ. Alternatively, if Dpp signaling is actively downregulated during germ cell differentiation, then differentiating germ cells in the heat shocked ovarioles should not express Dad-lacZ.
Comparison of confocal projections of heat-shocked ovarioles to control, non heat-shocked ovarioles that were processed identically revealed a decrease in the amount of lacZ present in the germ cells of the heat-shocked ovarioles compared with the controls (78%, n=41 ovarioles). Significant downregulation (compare Fig. 3F to Fig. 3G) but not elimination (Fig. 3H) of Dad-lacZ levels occurred prior to downregulation of Nos during cyst formation and was specific to the germline, as Dad-lacZ expression remained at high levels in somatic cells (Fig. 3G). We correlated the expression of Dad-lacZ in these heat shocked ovarioles with two distinct markers for the state of germline differentiation, the presence of spectrosomes or fusomes in a given germ cell, and whether any surrounding germ cells expressed Orb. Strikingly, lacZ was never present in germ cells undergoing overt differentiation, either those that had fusomes (Fig. 3I; 0%, n=20 cysts), or expressed Orb (not shown). Moreover, the decrease in lacZ expression is apparent (67%, n=168 cells) in many spectrosome-containing cells (compare Fig. 3J with 3K). In general, downregulation of lacZ expression in spectrosome-containing cells was more evident in ovarioles with signs of overt differentiation, such as cyst formation. The downregulation of lacZ expression is not likely to be due to action of Bam on either the UAS-GAL4 system or the stability of lacZ, as flies of genotype P{hs-bam.O}/P{UAS.p-lacZ}; P{GAL4::VP16-nos.UTR} bamD86/bamD86, in which the same regulatory constructs were used to drive lacZ expression, displayed lacZ staining throughout the germline prior to (Fig. 3L), and after a heat shock expression of Bam that caused germline cyst production (Fig. 3M). Taken together, these data indicate that expression of Bam leads to a block in Dpp signaling downstream of Tkv activation, and that this block occurs prior to onset of overt GSC differentiation.
Bam plays an instructive role in germline differentiation
Previous experiments (Xie and
Spradling, 1998) have shown that loss of Dpp signaling in the
germline leads to failure to maintain a GSC fate. Conversely, Bam has been
shown to be both necessary and sufficient to promote CB differentiation
(McKearin and Spradling, 1990
;
Ohlstein and McKearin, 1997
).
The data presented above raise the possibility, however, that Bam could
promote CB differentiation solely by blocking Dpp signaling. To determine
whether Bam has additional functions during CB differentiation, we used the
FLP-FRT system (Xu and Rubin,
1993
) to make clones of germ cells doubly mutant for bam
and the Dpp signal transducer Mad.
Previous work (Xie and Spradling,
1998) indicated that GSCs homozygous for Mad12
are not maintained over time. We repeated these experiments and arrived at
similar conclusions (Materials and methods). Although the fates of GSCs
lacking Mad function were not ascertained in these experiments, in
both our analysis and that of Xie and Spradling
(Xie and Spradling, 1998
),
young Mad mutant cysts were found in the anterior germarium a
significant time after induction of mitotic recombination, suggesting they
were progeny of mutant GSCs that had undergone differentiation. Moreover, we
found that all such cysts (n=23) were phenotypically normal,
indicating Dpp signaling is not required for cyst differentiation.
We were unable to obtain either wild-type or Mad mutant germ cell clones by inducing mitotic recombination in adult bam females, suggestive of a very slow rate of germ cell division in the adult. However, small clones of germ cells doubly mutant for Mad and bam, containing on average one or two cells, could be obtained after induction of recombination in pupal stages. These doubly mutant cells contained round spectrosomes and were identical in morphology to bam single mutant germ cells (100%, n=30 ovarioles, compare Fig. 4B with 4A), indicating these cells did not form germline cysts. Because Dpp signaling is not required for cyst differentiation, these data indicate that Bam plays an essential role in CB differentiation independent of its function in downregulating Dpp signaling.
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We then determined how these classes of mutations affected the spatial extent of Dpp signaling within the GSC niche. Ovarioles from Smurf 15C mutant females had a significantly greater number (4.0±2.0, P<0.001, Neuman-Kuels test, n=20) of high-lacZ expressing cells than did wild-type ovarioles (Fig. 5C). Although the number of high-lacZ expressing cells in ovarioles of females carrying three copies of dpp+ (2.7±1.3, P>0.05, n=18) and in ovarioles from females carrying the dominant saxB18 allele (3.2±1.3, P>0.05, n=9) was not significantly different from wild type, ovarioles from females carrying both the dpp+ duplication and the saxB18 allele had a significantly greater number (4.0±1.7, P<0.001, n=25) of high-lacZ-expressing cells than did wild type (Fig. 5B). Thus, mutations that elevate or prolong Dpp signaling can increase the number of putative GSCs within the niche, suggesting mechanisms that control perdurance of Dpp signaling could also play a role in limiting Dpp signaling within the germline.
To determine whether either of these genotypes could synergize with bam mutations to result in a more extensive deregulation of Dpp signaling within the germline, we constructed bam mutant females that also carried the Smurf or sax mutations. We found that in many multiply mutant ovarioles the spatial extent of Dpp signaling was significantly expanded, so that Dad-lacZ expression was observed in germ cells throughout the entire ovariole. This phenotype was not uniform, however, and could vary even in ovarioles from a single female (e.g. Fig. 5D-F contains images from a single confocal section of ovarioles from one female). In general, ovarioles from females of these mutant genotypes fell into two phenotypic classes. The first class showed a substantial increase in the number (12.1±6.0, n=21 saxB18 Tp(2;2)DTD48/+ +; bamD86 ovarioles; 11.1±3.8, n=17 Smurf15C; bamD86 ovarioles) of germ cells expressing high levels of lacZ. In these ovarioles, lacZ-expressing germ cells were found associated with somatic cells and/or distributed in a salt-and-pepper fashion throughout the ovariole [Fig. 5D,E,G (saxB18 Tp(2;2)DTD48/+ +; bamD86 ovarioles), upper ovariole in Fig. 5H,J (Smurf15C; bamD86 ovarioles)]. Occasionally, we observed a Smurf15C; bamD86 ovariole in which most, if not all, germ cells within the tumor expressed lacZ (ranging from 40 to 60 germ cells, n=2) (Fig. 5I). The second class of ovarioles showed no significant increase in the number (3.3±0.6, n=12 saxB18 Tp(2;2)DTD48/+ +; bamD86 ovarioles; 3.0±1.2, n=12 Smurf15C; bamD86 ovarioles) of germ cells expressing Dad-lacZ, and the lacZ-expressing cells were confined to the anterior region of the tumor close to the terminal filament (Fig. 5F, a saxB18 Tp(2;2)DTD48/+ +; bamD86 ovariole; Fig. 5H lower ovariole, a Smurf15C; bamD86 ovariole). Our data thus indicate that in both double mutants, unlike any of the single mutants, germ cells throughout the ovariole can be responsive to Dpp signaling. We conclude that during wild-type development Bam downregulates Dpp signaling during germline differentiation, but that its activity is functionally redundant with certain components of the Dpp signaling pathway.
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Discussion |
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Model for control of Dpp signaling within the germline
If GSCs and CBs are exposed to equivalent amounts of Dpp protein, as is
suggested by both the transcription pattern of the Dpp gene
(Xie and Spradling, 2000) and
the expression of Dad-lacZ in the CpCs of the niche and the
ISCs posterior to the niche, then it is likely that the observed reduction in
Dad-lacZ expression between the GSC and the CB results from
intracellular modulation of the strength of the Dpp signal. One hallmark of
the GSC is its invariant plane of division. We propose that the differential
Dpp signaling between the GSC and CB sign results from an intracellular
modulation of Dpp signal strength between the two daughter cells, either by
the asymmetric segregation of one or more cellular components that modulate
Dpp signaling, or by loss of a contact-based niche signal that elevates Dpp
signaling preferentially within the GSCs. Removal of the CB cell from the
niche thus results in partial downregulation of Dpp signaling. A lower level
of Dpp signaling in the CB cell results in the transcription of Bam, which
plays multiple roles in CB differentiation, one of which is to cause the
daughters of the CB cell to become refractory to further Dpp signaling. Thus,
sequential regulatory mechanisms cooperate to ensure an irreversible change in
the fate of the GSC cell within two generations
(Fig. 6).
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Bam downregulates Dpp signaling downstream of receptor activation
The reduction in Dpp signaling between the GSC and the CB releases Bam from
Dpp-dependent transcriptional repression
(Chen and McKearin, 2003b), and
we have shown that one, but not the only, function of Bam is to downregulate
Dpp signaling downstream of receptor activation prior to overt GSC
differentiation. This is the first molecular action ascribed to Bam, and these
data could provide an entry point to elucidate the biochemical basis of the
function of Bam in CB differentiation. Further work will be necessary to
determine whether the action of Bam on the Dpp pathway is direct or indirect,
whether Bam action results in the reduction or complete elimination of Dpp
signaling in the developing cysts, and which step in the intracellular Dpp
signal transduction pathway or expression of Dpp target genes is affected by
Bam action. However, it is possible that initial insights into Bam function
can be made by comparing the thresholds for Dpp signaling readouts in the
developing wing disc of the larva to the data we and others have obtained in
the germarium. In the wing disc, Dpp diffuses from a limited source to form a
gradient throughout the disc that displays different thresholds for multiple
signaling readouts. Specifically, Dad-lacZ is transcribed in
response to high and intermediate levels of Dpp, but does not respond to the
lowest levels of ligand (Minami et al.,
1999
). An antibody exists that recognizes the active
phosphorylated form of Mad, pMad (Persson
et al., 1998
). In the wing disc, high level staining with the pMad
antibody is present in only a subset of cells that express high levels of
Dad-lacZ, suggesting that in this tissue the pMad antibody
is less sensitive to Dpp signaling than is Dad-lacZ
expression (Minami et al.,
1999
; Tanimoto et al.,
2000
; Teleman and Cohen,
2000
). Intriguingly, Gilboa et al.
(Gilboa et al., 2003
) recently
reported that in the ovariole pMad staining is visible in the GSCs, CBs and
the developing cysts. Because we never observe Dad-lacZ
expression in the developing cysts, these results could suggest that the
relative sensitivities of these two reagents are reversed within the germline.
Alternatively, if the reagents have the same relative sensitivities in the two
tissues, the data suggest that Bam could act, probably at a
post-transcriptional level, to downregulate Dpp signaling downstream of Mad
activation.
Functional redundancy in control of Dpp signaling in the germ line
We have shown that the pattern of Dad-lacZ expression
observed in the Smurf; bam and sax; bam double
mutant ovarioles is qualitatively different from that observed in any of the
single mutant ovarioles. Although Dad-lacZ expression is
only observed at the anterior tip of the germarium of each single mutant,
many, but not all, of the double mutant ovarioles contain germ cells
throughout the ovariole that express high levels of
Dad-lacZ. From these data, we conclude that two redundant
pathways downregulate Dpp signaling in the germline, and that in the single
mutants, the action of the remaining active pathway is sufficient to constrain
Dpp responsiveness to the anterior tip of the germarium. However, we note that
not all doubly mutant ovarioles display a spatial expansion of Dpp signaling,
and that this variability can even be observed in ovarioles from a single
female. We propose that the observed variability results because the
Smurf and sax mutations have modulatory effects on Dpp
signaling that are both dependent on the presence of ligand and are sensitive
to additional mechanisms that downregulate Dpp signaling. In both the
Smurf; bam and sax; bam ovarioles, the
germ cells that express Dad-lacZ are observed throughout the
ovariole, but are more likely to be near somatic cells. It is possible that
the variability in Dad-lacZ expression occurs because of a
non-uniform distribution of the Dpp ligand. Nevertheless, there is not a
consistent correlation between the domains of Dad-lacZ
expression in the somatic and germ cells, suggesting that there may be
additional germline intrinsic factors that affect Dpp signaling.
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ACKNOWLEDGMENTS |
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