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First published online 11 February 2009
doi: 10.1242/dev.033340
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1 Spanish National Cancer Centre (CNIO), Melchor Fernández Almagro, 3.
E-28029 Madrid, Spain.
2 Department of Developmental Biology and Cancer Research, IMRIC, The Hebrew
University-Hadassah Medical School, Jerusalem 91120, Israel.
Author for correspondence (e-mail:
emoreno{at}cnio.es)
Accepted 15 January 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Dpp, Cell competition, Stem cells, Drosophila
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The fly ovary germline is a well-characterized stem cell system that allows
complex questions of stem cell behavior to be addressed. Approximately 16
ovarioles constitute an ovary. Each ovariole harbors two to three ovarian
germline stem cells (GSCs) at the tip of a germarium (reviewed by
Fuller and Spradling, 2007
),
which acts as a stem cell niche (Xie and
Spradling, 2000), a special environment where adult stem cells are
maintained. GSCs in this niche are anchored via E-cadherin-mediated adhesion
to stromal cap cells (Song et al.,
2002), which secrete the stem cell factor Dpp to the adjacent stem
cells to prevent GSC differentiation (Xie
and Spradling, 1998) (Fig.
1A). Phosphorylated Mad (pMad) transduces the Dpp signal in GSCs
and represses the differentiation factor bag of marbles
(bam) by direct binding to the bam promoter
(Chen and McKearin, 2003).
Stem cells divide asymmetrically and give rise to daughters with different
fates. The niche proximal daughter cell maintains GSC identity, cap cell
anchorage and high Dpp signaling levels, whereas the distal daughter
differentiates into a Bam-expressing cystoblast (CB). CBs undergo several
rounds of divisions with incomplete cytokinesis to form cysts of 16 cystocytes
connected by branched fusomes (Deng and
Lin, 1997). Alternatively, GSCs can also divide symmetrically to
substitute stem cells that are lost from the niche due to natural turnover
(Fig. 1A)
(Xie and Spradling, 2000).
Under certain conditions, even differentiated CBs up to 8-cell cysts can
revert to stem cells to repopulate an empty niche
(Kai and Spradling, 2004).
Mutations in both bam and bgcn (benign gonial cell
neoplasm) block the differentiation of GSCs and produce stem cell tumors in
homozygous flies. It has been recently reported that bam and
bgcn mutant stem cells, which express high levels of E-cadherin, are
able to gradually take over a niche shared with wild-type GSCs, suggesting a
competitive relationship among stem cells
(Jin et al., 2008).
Originally, cell competition has been discovered in fly wing imaginal discs
where clones heterozygous for mutations in ribosomal genes (Minutes)
were found to be outcompeted by wild-type cells without changing tissue
morphology (Morata and Ripoll,
1975; Lambertsson,
1998). Such Minute and wild-type cells grew normally in a
homotypic environment and only apposition of both cell types triggered
apoptosis of the loser cells. The unequal fates are thought to be mediated by
differential competition for the survival factor Dpp. Lower Dpp signaling
levels in Minutes allow the expression of brinker, which in
turn triggers JNK-dependent apoptosis
(Morata and Ripoll, 1975;
Moreno et al., 2002) (reviewed
by Moreno, 2008). More recent
studies demonstrated that dMyc-overexpressing cells act as supercompetitors
that outcompete surrounding wild-type cells in the wing epithelium
(Johnston et al., 1999;
de la Cova et al., 2004;
Moreno and Basler, 2004).
Because cells with lower levels of protein synthesis (Minutes) or
dMyc are outcompeted, cell competition was proposed to serve as a quality
control mechanism that improves organ function
(Diaz and Moreno, 2005). In
order to test this hypothesis, we turned to the Drosophila ovary
germline, a stem cell-based tissue where quality control is thought to be of
paramount importance. In the Drosophila ovary germline,
dmyc, the homolog of the human c-myc oncogene, is known to
be required for endoreplication of the differentiating cysts
(Maines et al., 2004), but its
role in GSCs is not known. Here, we show that differential expression of dMyc
in GSCs triggers competitive interactions. dMyc-mediated competition in the
germline is non-apoptotic and does not affect total stem cell numbers. In
addition, we present supporting data for a naturally ongoing level of
competition between high dMyc stem cells and low dMyc differentiating
daughters, which seems to favor efficient launching of the differentiation
program.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) transgenes. The bamP-GFP line (D.
McKearin, University of Texas, TX, USA) was used to monitor bam
expression. Clones in the GSCs were typically generated by heat-shocking 2-day-old flies twice per day. Flies were heat-shocked at 37°C for 1 hour, then allowed to recover for a period of 6-7 hours before they were subjected to a second identical heat-shock. Flies were then kept at 25°C and transferred to fresh food every 2 days. For the induction of tub>dmyc mutant GSC clones, females of genotype tub-FRT-CD2-FRT-dmyc were generated and heat-shocked for 30 minutes at 37°C.
We used 1-week-old adults of genotype dmycP0 FRT18A and
Dp (1;1) Co hsp70-Flp FRT18A to study the GSC niche. As a control
niche, we used arm LacZ FRT18A; hs-Flp38. For the induction of
dmycP0 and 4xdmyc (Dpmyc) mutant
GSC clones, females of genotype dmycP0 FRT18A/arm-lacZ FRT18A;
hs-Flp and yw Dp (1;1)Co hs-Flp FRT18A/arm-lacZ FRT18A were
generated, respectively. As a control, we generated FRT18A/arm-lacZ
FRT18A; hs-Flp. For the induction of other mutant GSCs, females of the
following genotypes were generated: hs-Flp Ubi-GFP
FRT40/Pten2L100 FRT40, hs-Flp Ubi-GFP FRT40/Pten2L117
FRT40, hs-Flp; Ubi-GFP FRT18A/mei-P261FRT18A, hs-Flp; Ubi-GFP
FRT40/lgl4FRT40 (Gateff
and Schneiderman, 1974), hs-Flp; Arm-Lacz
FRT42/ptcs2FRT42 (Simcox
et al., 1989), hs-Flp; Ubi-GFP
FRT80/sty
5FRT80
(Hacohen et al., 1998),
hs-Flp;Ubi-GFPFRT82/bam86FRT82,
hs-Flp;Ubi-GFP FRT82/savshrp6B21FRT82
(Kango-Singh et al., 2002),
and hs-Flp; Ubi-GFP FRT82/scrib1FRT82
(Bilder et al., 2000). To
analyze GSC competition in tkvACT germaria, flies of
genotypes Dp (1;1) Co/arm-lacZ FRT18A; UASp-tkvACT/+;
nanos-Gal4:VP16/+ and dmycP0FRT18A /arm-lacZ FRT18A;
UASp-tkvACT/+;nanos-Gal4:VP16/+ were generated
(Van Doren et al., 1998;
Johnston et al., 1999).
We used tub>dmyc>Gal4/Cyo adult females, and generated dmycP0 FRT18A/dm4, dmycPG45/dmycPG45; tub>dmyc>Gal4/+ and dmycPG45/dm4; tub>dmyc>Gal4/+. Ovaries were processed from 1-week-old flies. The dmyc sequence was amplified by PCR, subcloned into the pUASp vector and constructs sent to generate transgenic UASp dMyc flies (BestGene). They were used to overexpress dMyc in the germline using the nanosGal4 driver by generating dmycP0/dmycPG45; UASp Myc/+; nanosGal4 UASp tub-GFP flies.
Calculations
The percentage of GSCs inside a marked clone was quantified after
processing the corresponding samples for immunostaining with anti-β-Gal
and or anti-Hts antibodies. GSCs were identified by their attachment to cap
cells and by the presence of a round fusome.
The decay time of clones in the niche was calculated as follows. For homogenous niches, the number of GSCs was counted at 1-4 weeks, then data were modeled with a linear regression N=b0+b1*T (T in weeks and N is the number of GSCs. Example for dmycP0 homogenous niche. 2.4 (1 week), 2.1 (2 weeks), 1.53 (3 weeks), 1 (4 weeks), Regression b0=2.96 (P<1e-5), b1=–0.48 (P<1e-15), degrees of freedom=50. Decay time: time to reach half of the 1st week value=3.67 weeks. To quantify GSCs decay in mosaic niches, we modelled R1 scores with a linear or nonlinear regression for the following scenarios (y, R1; x, time in unit of days; goodness of fit scored by the norm of residuals). Example for dmycP0 mosaic niche. R1: 60.0% (4 days), 33.3% (1 week), 14.3% (2 weeks), 14.3% (3 weeks). Linear regression y=p1*x+p2, coefficients: p1=–0.024773, p2=0.58965, norm of residuals=0.18504. Decay time: time to reach half of the 4-day value=1.67 weeks (cubic regression 1.07 weeks). To test if differences between decays are significant, we performed t-tests. Complete calculations are available upon request.
To measure stem cell division rates, we used the same system as Xie and
Spradling (Xie and Spradling,
1998). We determined the relative number of wild-type and mutant
cysts in germaria that contained one control and one mutant stem cell. For a
given genotype, these values were similar at each time point, and the average
is presented in the text. Marked wild-type stem cells gave a value of 0.8.
Mutant 4xdmyc GSCs occupy the niche very early after clone
induction. In this case, the GSC division rate could only be scored at very
early time points.
GSC size was measured after staining with Alexa-conjugated phalloidin (Molecular Probes) to reveal the cortical actin that delineates cellular contours. Serial confocal images of 2 µm each were obtained. Cell size was calculated as the maximum area of the whole collection of sections per individual germarium by using the appropriate Leica Confocal Software (LCS) tool.
Immunohistochemistry and apoptosis assays
We used the following antibodies: monoclonal (Promega) or polyclonal
(Cappel) anti-β-Gal, anti-pMad (P. ten Dijke and G. Morata, University of
Leiden, The Netherlands), polyclonal or monoclonal anti-dMyc (R. Eisenman and
B. Edgar, Fred Hutchinson Cancer Research Center, WA, USA), anti-Hts (1B1;
Developmental Studies Hybridoma Bank; DSHB). All images were obtained with a
LEICA TCS-SP2-AOBS. For each germarium, six to ten image stacks of 2 µm
were obtained through the z dimension in order to analyze the whole
GSC population. For detection of apoptosis, we used anti-cleaved caspase-3
(Cell Signaling Tech), anti-Hid antibody (H. Steller, The Rockefeller
University, NY, USA) or analysis of nuclear fragmentation by DAPI staining. As
a positive control of apoptosis in the GSCs, we stained ovaries 7 hours after
the induction of apoptosis by heat-stress (45 minutes at 38°C).
In situ hybridization
FISH was performed using standard protocols. Briefly, ovaries were fixed in
4% formaldehyde (FA) and hybridized with a digoxigenin-labeled RNA probe
overnight or for 48 hours at 56°C. After washing, signal was developed by
using an anti-digoxigenin antibody (Jackson ImmunoResearch) and the Tyramide
Signal Amplification System (Invitrogen).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
However, dmyc transcripts were tightly downregulated in the
intermediate zone where cystoblast differentiation up to the 16-cell cyst
stage takes place. A similar pattern could be observed by immunohistochemistry
with an anti-dMyc antibody (Fig.
1C). dMyc was highly expressed in cells showing characteristic
features of GSCs: they expressed nanos, were negative for the
differentiation marker bam and expressed high levels of active
(phosphorylated) Mad (pMad) (Fig.
1C-F), indicating that they were transducing high levels of Dpp
(Kai and Spradling, 2004).
dMyc was also present in cells that had already lost niche contact but that
did not yet upregulate the differentiation factor Bam. Such pre-cystoblasts
(pre-CBs) (Gilboa et al.,
2003) showed intermediate levels of pMad
(Fig. 1E,F). In differentiating
cystoblasts, however, expression of dMyc protein was strongly reduced,
consistent with the pattern observed using mRNA in situ hybridization against
dmyc.
Because dMyc expression in the ovary appeared to be developmentally
regulated, we sought to determine how dMyc downregulation is established. The
F-box protein archipelago (ago) has been shown to regulate
dMyc levels in Drosophila wing and eye tissues
(Moberg et al., 2004),
therefore we tested whether it also modulated dMyc expression in the germline.
However, ago mutant GSCs gave rise to differentiating CBs that showed
normal dMyc downregulation (Fig.
1G). More recently, the cytoplasmic protein Brat was found to
regulate dMyc in the daughter cell of neuronal stem cells
(Betschinger et al., 2006).
Brat forms part of a family of tumor suppressors that includes the
gene mei-P26, which is known to produce ovarian tumors when mutated
(Page et al., 2000).
Interestingly, we found that progeny derived from mei-P261
mutant stem cells failed to downregulate dMyc
(Fig. 1H). This does not prove
that Mei-P26 is indeed regulating dMyc expression because mei-26
mutations might simply block GSC differentiation leading to an accumulation of
dMyc-positive GSC-like cells. However, recent findings demonstrate that
mei-p26 mutant GSCs are able to differentiate into cystocyte
marker-expressing cells, but that these mei-p26 mutant CBs later fail
to form oocytes because they proliferate indefinitely
(Neumüller et al., 2008).
We therefore consider it likely that Mei-p26, whose levels are low in stem
cells, but high in cysts, might control the expression of dMyc.
Stem cells with elevated levels of dMyc outcompete wild-type GSCs
In order to test whether dMyc can induce competitive interactions in the
niche, we manipulated dMyc levels by generating one unmarked GSC with higher
dMyc levels (Dpmyc/Dpmyc, with four copies of dmyc) next to
a resident marked stem cell using a heat-shock-inducible Flipase, which
mediates FRT mitotic recombination (Xie
and Spradling, 1998). Four days after clone induction (ACI), we
verified the presence of such mosaic niches. Already one week ACI, a subset of
4xdmyc-containing GCSs had replaced adjacent stem cells
(Dpmyc/+) and, after 2 to 3 weeks, 4xdmyc stem cells
constituted the entire germline (47.2% of the total GSCs were
4xdmyc-containing cells at 4 days ACI, but they expanded to
95.8% of the total GSCs after 4 weeks; Fig.
2A-C, Table 1,
Fig. 3A). Although the marked
control stem cells (which carried one lacZ copy) disappeared from the
niche, a constant number of GSCs was maintained, possibly by horizontal
division of the 4xdmyc GSCs (see
Fig. 1A). The elimination of
resident GSCs by 4xdmyc stem cells did not seem to be caused by
apoptosis, as we found no evidence of induction of the pro-apoptotic gene
hid (Grether et al.,
1995) (Fig. 2D,
inset shows positive control for hid induction after heat stress).
Furthermore, we did not detect activation of caspase-3, nuclear fragmentation
or aberrant mitochondrial function (in vivo imaging with mitotracker; data not
shown) suggesting that wild-type GSCs did not die, but were expelled from the
niche and differentiated.
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), we generated clones mutant for the negative regulator
Pten to test whether increased division rates of PTEN mutant
GSCs are sufficient to drive surrounding stem cells out of the niche.
Pten mutant GSCs showed increased proliferation (1.6 versus 0.8 in
the wild type, Table 1) but did
not extrude neighboring GSCs and co-existed with them instead
(Fig. 2E,F;
Table 1). Moreover,
dMyc-overexpressing GSCs showed only slightly elevated proliferation rates
compared with the wild-type GSCs (1 versus 0.8 in the wild-type,
Table 1). These results suggest
that the competitive advantage of dMyc-overexpressing GSCs does not rely on
increased proliferation. As a second possibility, we envisaged that larger
cells might displace smaller ones, as dmyc is implicated in cell size
control (Johnston et al.,
1999). GSCs that express higher levels of dMyc in a homogeneous
niche were slightly larger than wild type (4xdmyc GSCs
68±17 µm2 versus 60±10 µm2 in the
control, P=not significant). Previous studies have shown that Pten
negatively regulates cell size (Goberdhan
et al., 1999). Therefore, we used two alleles
(Oldham et al., 2002)
(Pten2L100 and Pten2L117) to generate
GSC mutant clones. Pten clearly affected GSC size
(Fig. 2G),
(Pten2L100 GSCs, 83±15;
Pten2L117 GSCs, 86±16 µm2; versus
54±18 µm2 in the wild type; P<0.001) but did
not eliminate neighboring stem cells, speaking against cell size as a factor
influencing stem cell competition. In a third scenario, dMyc might stimulate
the capture and/or transduction of extracellular factors like Dpp
(Moreno and Basler, 2004) that
are required to maintain GSC fate. Consistent with this idea,
4xdmyc GSCs showed higher levels of pMad than did control GSCs
in a mosaic niche (6/6, 4 days ACI; Fig.
2H-J).
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), we devised a second genetic setup to verify the
effect of dMyc levels on GSC competition. To this end, we heat-shocked hs-flp; tub>cd2stoP>dmyc transgenic flies for 30 minutes to induce a moderate amount of tub>dmyc clones, which could be visualized by the loss of the membrane marker CD2, next to tub>cd2 control cells. Five days ACI, a high percentage of germaria showed mosaic niches containing both a marked control and an unmarked tub>dmyc stem cell (Fig. 2K-O). After 2 weeks, the loss of CD2-positive GSCs became evident, concomitant with a decrease in number of mosaic niches (49.7% of the total GSCs were tub>dmyc-containing cells at 4 days ACI, but they had expanded to 62% of the total GSCs after 2 weeks). Three weeks ACI, a high proportion of germaria showed complete absence of CD2-positive germ cells (70% of the total GSCs were tub>dmyc-containing cells at 3 weeks ACI), indicating that dMyc-overexpressing cells had occupied the majority of niches (Fig. 2N,O; see also Table 1). The analysis of pMad expression in tub>cd2/tub>dmyc mosaic niches reinforced the previous results that dMyc-overexpressing GSCs typically showed higher levels of pMad. Differences in pMad levels were strongest at 5 days ACI (11/16 germaria, five germaria showed identical pMad levels) (Fig. 2L,M). Importantly, tub>dmyc GSCs were not differentiation-defective and produced normal cysts and oocytes, as verified by phalloidin and Hts staining (fusome marker; Fig. 2O; data not shown).
Stem cells expressing low levels of dMyc are expelled from the niche and differentiate
To investigate whether wild-type stem cells have a competitive advantage
over suboptimal GSCs, in this case GSCs expressing low levels of dMyc, we
first examined niches containing only dmyc mutant stem cells in flies
homozygous for the hypomorphic dmycP0 allele
(Johnston et al., 1999).
Although dmycP0 mutant stem cells express lower levels of
dMyc, this reduction does not affect the ability of dmycP0
GSCs to self-renew and generate differentiated progeny, ruling out the
possibility that such GSCs might be lost because they are not fully functional
(Fig. 4A). We then created
mosaic niches harboring unmarked dmycP0 mutant GSCs next
to marked dmycP0/+ control stem cells. Four days ACI, we
were able to detect such mosaic niches; however, after 1 week, half of the
homozygous mutant dmycP0 GSCs had abandoned the niche.
This elimination trend continued in later weeks (60.2% of the total GSCs were
dmycP0/dmycP0 cells at 4 days ACI, but they had
decreased to 6.2% of the total GSCs after 2 weeks)
(Table 1,
Fig. 3A,
Fig. 4B-D). As a second
measurement, we quantified the time it takes to halve the initial
dmycP0 homozygous stem cell population (decay time) in a
mosaic niche with control GSCs, and compared it with that exhibited in a
homogeneous niche of only dmycP0 GSCs. The decay time in
the latter niche was around four weeks but dropped to one week in a mosaic
niche (see Materials and methods). The fact that dmycP0
mutant cells were still present in the germaria 2 weeks ACI, forming part of
developing cysts outside the niche (Fig.
4D), suggested that suboptimal stem cells did not undergo
apoptosis, but had rather moved out of the niche to differentiate. By using
various methods to detect apoptotic cells, such as Caspase 3 activation, lack
of Hid staining (Grether et al.,
1995), absence of nuclear fragmentation and intact mitochondrial
function, we were unable to detect cell death of dmycP0
mutant GSCs even under intense monitoring. However, we could easily detect
activated Caspase 3 in wild-type GSCs after heat-shock-induced stress or
irradiation (Fig. 4E, inset;
data not shown). Finally, the elimination of mutant stem cells along the
differentiation pathway still took place when apoptosis was blocked in the
germline by overexpressing the apoptosis inhibitor dIAP-1
(Hay et al., 1995) from the
early germline nanos-Gal4 driver
(Rørth, 1998;
Van Doren et al., 1998)
(Fig. 4F). These experiments
show that suboptimal cells are reliably detected and driven out of the niche,
which suggests that dMyc-induced cell competition might represent a continuous
survey instrument used to maintain optimal stem cell pools at any time.
The competitive advantage of differentiation-defective stem cells was shown
to rely on their higher E-cadherin levels, which seem to confer physical
strength with which to push out competitor stem cells
(Jin et al., 2008). By
contrast, we did not observe noticeable differences in E-cadherin expression
between suboptimal dmycP0 and dmycP0/+
control cells (see Fig. S1 in the supplementary material). In order to test
whether the competitive advantages of stem cells depended on Dpp signaling
(higher pMad levels), we overexpressed a constitutively active version of the
Dpp receptor (tkvACT) in the germline and monitored mosaic
niches harboring stem cells with different dMyc levels. We found that
expression of tkvACT blocked not only the loss of
dmycP0 homozygous GSCs sharing the niche with wild-type
GSCs but also the expulsion of wild-type stem cells facing a
4xdmyc competitor (4/4 and 5/5, 3 weeks ACI, respectively;
Fig. 4G,H), suggesting that
tkvACT could equalize competition for Dpp in the niche.
However, the expression of tkvACT on its own causes the
accumulation of stem cell-like cells. Therefore, we cannot exclude that the
observed rescue of the loser GSCs might be partially caused by difficulties in
niche exit of such stem cells due to impaired differentiation.
dMyc-induced competition and other growth-promoting mutations
GSCs carrying dmyc duplication and Pten mutations might
have been expected to behave more similarly, as both genes are
growth-promoting and related to oncogenic transformation. We therefore decided
to test other mutations in oncogenic pathways to determine their invasive
potential in the fly ovary stem cell niche. To this purpose, we monitored
again mosaic niches and studied the integration of the mutant GSCs into the
stem cell niche. None of the tested mutations (Pten, scribble, salvador,
patched, ago, sprouty, lgl) induced competitive interactions that were as
potent as those observed for the dmyc duplication
(Fig. 3B, data not shown).
Although, salvador (sav) and sprouty (sty)
mutant stem cells also improved niche occupancy during the observed 20 days
ACI, they never exceeded a ratio of 0.5, where half of all GSCs in a clone are
still wild type. sav is a component of the Salvador-Warts-Hippo
pathway, which inhibits cell growth, whereas sty is a negative
regulator of receptor tyrosine kinase signaling. In the absence of strong
invasive behavior, we therefore classified such mutations as `settler'
mutations that successfully establish themselves in the germline, but co-exist
with resident GSCs (Fig. 3B and
Fig. 4I and 4K), despite higher
proliferation rates in the case of sav and Pten stem cells
(Table 1). Genetic lesions in
bam represent a second category of mutations that produced a `stem
cell plague' (Fig. 4J,M), as it
has been previously described. Such mutant stem cells expand by blocking the
differentiation of their daughters, thereby increasing the total number of
stem cell-like cells. Only stem cells with more dMyc behaved differently,
occupying the niche by eliminating and replacing normal stem cells without
affecting total stem cell numbers. Because of these characteristic
differences, we refer to the gain-of-function mutation in dmyc as a
`squatter' mutation to describe that mutant stem cells take over the space
formerly inhabited by resident stem cells
(Fig. 2A-C,
Fig. 4L).
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To examine this hypothesis, we altered the endogenous dMyc expression
pattern along the germarium. First, we added ubiquitous overexpression of dMyc
to the physiological levels by using a dmyc transgene under the
tubulin promoter (tub>dmyc). Under these
conditions, we observed an unusually high number of undifferentiated GSC-like
cells that resided in the anterior tip of the germarium, identified by a round
fusome, compared with the wild-type situation
(Fig. 5A-H). To further
minimize differences in dMyc between GSCs and their daughters, we next
expressed the tub>dmyc transgene in females mutant for
very strong lethal dmyc alleles
(dmycPG45/dmycPG45or
dmycPG45/dm4)
(Bourbon et al., 2002;
Pierce et al., 2004). We found
that even more daughter cells exhibited a GSC-like morphology at positions
that were very distal from the niche, until they finally entered
differentiation (Fig.
5I-P).
If changes in dMyc levels induced competition for Dpp uptake in the niche, one would predict to find more Dpp-transducing cells when dMyc differences, and therefore competition is eliminated. This is expected because stem cells would lose their competitive power to turn over most diffusible Dpp. As a consequence, the available Dpp could travel farther and could now be acquired by cells more distal from the niche. Indeed, our experiments showed that pMad activation expanded substantially in the dmycPG45/ dmycPG45; tub>dmyc/+ flies compared with in tub>dmyc flies, where differences in dMyc levels still exist (Fig. 5F,J,N; Table 2). Thus, minimizing the difference in dMyc levels between GSCs and differentiating CBs increases the range of the extracellular Dpp signal.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Non-apoptotic competition in GSCs induced by dMyc
In our analysis of mosaic niches, we found that stem cells mutant for the
hypomorphic allele dmycP0
(dmycP0/dmycP0) were gradually outcompeted by
neighboring GSCs with higher levels of dMyc (dmycP0/+).
The same effect was observed with stem cells overexpressing dMyc relative to
their counterparts, resulting in the niche conquest by such
dMyc-overexpressing stem cells. Comparison of mosaic niches with niches
harboring a homogenous population of stem cells regarding dMyc levels showed
that dMyc was able to induce competition, measurable in shorter decay times of
the outcompeted cells.
Our results contrast, however, with a recent study by Ting Xie and
colleagues, who also examined dmyc in GSC competition. They tested
the lethal dmyc alleles dm2 and
dm4, which behave genetically as null alleles and found
that such dMyc-deficient GSCs were not outcompeted by control GSCs
(Jin et al., 2008).
Strikingly, they also obtained different results with stem cells carrying two
copies of the nos-gal4VP16 driver to overexpress UASdmyc in
the germline. Such GSCs did not compete with control GSCs carrying only one
copy of nos-gal4VP16, rather they were lost slightly faster from the
niche.
Part of the explanation for the discrepancies might lay in differences in
experimental design and chosen dmyc alleles. In our hands, GSCs
carrying strong or null mutations in dmyc
(dmycPG45 or dm4, respectively) showed
poor viability and, in the few mosaic niches found, dmyc-deficient
stem cells were not lost from the niche. We therefore decided to study
hypomorphic dmycP0 stem cells that express dMyc, but at
lower levels – an ideal case of viable, but suboptimal stem cells. GSCs
devoid of any dmyc (dm4 deletion) may not enter competitive
interactions because some basal dMyc levels might be required to permit an
intercellular comparison of relative competitiveness. Wing pouch cells of the
imaginal disc have been proposed to be able to compare their Dpp signaling
levels with those of cells outside of the pouch
(Rogulja and Irvine, 2005).
Intriguingly, this differential behavior coincides with the dMyc expression
pattern (Johnston et al.,
1999). We also believe that dMyc-induced cell competition acts
only within a certain range of dMyc fluctuation, and will be overrun by
apoptosis as a consequence of the well-characterized effect of dMyc to induce
cell-autonomous apoptosis when expressed at high levels
(Hueber and Evan, 1998).
Both genetic systems employed here to raise dMyc levels (dmyc duplication and tub>cd2>dmyc flip-out cassette) are compatible with GSC viability. The overexpression of dMyc using Gal4 amplification systems with two copies of nos-gal4VP16 driver may not be so well tolerated by stem cells, compromising their competitiveness.
In contrast to studies that have focused on cell adhesion
(O'Reilly et al., 2008;
Jin et al., 2008) and
mechanical models of GSC extrusion from the niche, we provide evidence that
Dpp signaling and cell-cell communication play a role for dMyc-induced
competition. We do not completely understand how cells compare the Myc levels
of one another, but we present evidence that the ability to compete for
`stemness' factors, like Dpp, is important: when GSCs with more dMyc contact
GSCs with less dMyc, the cells with higher levels become more sensitive to Dpp
and accumulate pMad, which ensures a long-lasting stem cell fate, whereas the
cells with lower relative levels of dMyc lose responsiveness and eventually
differentiate. A possible connection between dMyc and the acquisition of an
extracellular ligand(s) has been proposed before. This relationship seems to
be indirect and is likely to involve the coordinated control of several target
genes, resulting in a gain of several aspects of biometabolic functions, e.g.
modifying rates of endocytosis (Moreno and
Basler, 2004).
The identification of paracrine and/or juxtacrine signals that are specific
to loser and/or winner cells is likely to become a fascinating line of future
research, as in the case of the apoptotic elimination of `loser' cells
(Ryoo et al., 2004;
Perez-Garijo et al., 2004;
Huh et al., 2004). In fact,
the possibility that cells can compare their relative Dpp signaling levels has
been postulated to explain both apoptotic cell competition
(Diaz and Moreno, 2005;
Senoo-Matsuda and Johnston,
2007; Moreno,
2008) and the regulation of cell proliferation
(Rogulja and Irvine, 2005),
but the molecules that mediate this comparison are unknown. Further studies
will also clarify how similar apoptotic and non-apoptotic cell competition
really are.
|
), which
would be consistent with our model that competitive stem cells show high pMad
levels and, hence, tightly repressed bam expression.
Although cell competition seems an excellent tool with which to select the
`fittest' stem cell when compromised GSCs are present, it bears the risk that
stem cells with modest dMyc overexpression are selected as being preferable
over the wild type. Niches occupied by stem cells harboring dMyc duplications
will give rise to differentiated tissue containing identical genetic
alterations. Such `pre-cancerous' fields are then more likely to accumulate
secondary or tertiary hits that will lead to tumor formation
(Braakhuis et al., 2005). Given
that stem cells are long-lived, we believe that the characteristics and
consequences of dMyc-induced competition are relevant for cancerous
transformation, especially for tissues with a high turnover
(Merlo et al., 2006). The most
notable features of niche occupancy by dMyc-overexpressing stem cells are (1)
the replacement of normal GSCs, which was not observed by other
proliferation-promoting mutations (Pten, sty), and (2) the absence of
tissue alteration. The property to outcompete wild-type stem cells renders
dMyc mutations potentially dangerous because they are capable of establishing
a mutant stem cell population that can remain long enough to accumulate
further cooperating mutations (Moreno,
2008; Rhiner and Moreno,
2009). This could be a reason why myc is the target of
early mutations in cancers and Pten inactivation occurs only at later
stages.
dMyc expression at the GSC/CB interface
So far, cell competition has been analyzed extensively using artificial
means to create mosaic tissues, but few attempts have been undertaken to
reveal competitive interactions in a more physiological situation. An
exception is the study on the competitive interactions of cells during liver
repopulation after hepatectomy (Oertel et
al., 2006).
In this study, we have tested the novel idea that developmentally regulated
expression of dMyc in the germline triggers competition for the stem cell
factor Dpp between high dMyc-expressing stem cells and low dMyc-expressing
progeny. This competition might be classified as `low level', compared with
stem cell-stem cell interactions in the niche, as the daughter cells compete
with the handicap of being located more distally from the source of Dpp than
are the stem cells. Nonetheless, our experiments, in which we perturbed the
physiological dMyc pattern and equalized GSCs and CBs in terms of dMyc levels,
strongly suggested a contribution of competition in the initial step of
differentiation. The ability of dMyc to activate a variety of genes encoding
components of protein synthesis pathways
(Grewal et al., 2005)
indicates that it may have the capacity to stimulate protein translation in a
coordinate manner. We propose a model in which high dMyc levels in stem cells
stimulate high metabolic rates, including increased protein synthesis and
endocytosis, through the activation of multiple target genes
(Fig. 6). This enables the stem
cells to compete efficiently and turn over a high amount of niche secreted
Dpp, resulting in elevated pMad levels, which in turn ensure a tight
repression of the differentiation gene bam. During the pre-CB to CB
transition, dMyc levels are downregulated, probably because of the oncoming
expression of Mei-P26, which lowers the efficiency of pre-CBs/CBs to take up
Dpp. The consequence is a steep decline of the Dpp gradient across the niche,
where remaining Dpp input in CBs is too low to activate pMad and Bam-mediated
differentiation is fully initiated. In the absence of competition, achieved by
imposing equivalent dMyc levels in all three cell types (see
Fig. 5), available Dpp
molecules distribute more uniformly over several cell diameters because cells
compete on a more similar basis. As a consequence, Dpp signaling thresholds
are still attained in cells distal from the niche
(Fig. 6, lower part), which
still present stem cell-like morphology owing to repressed bam
transcription by pMad. We do not intend to play down the role of Bam, which is
the main trigger for differentiation, but we suggest that dMyc-induced
competition for Dpp reinforces the tight repression of Bam in GSCs and the
efficient derepression of Bam in the differentiating daughter cells. If
competition is impaired, the differentiation process is delayed and less
defined, occasionally leading to the mixing of cystoblasts at different stages
of differentiation.
The competitive interaction between stem cells and their daughters containing different relative levels of dMyc described here is of particular interest because the interface along which the competition takes place is created through gene regulation. Therefore, we propose the term `programmed cell competition' to distinguish it from competitive interactions that arise as a result of genetic alterations. Programmed cell competition will occur along boundaries of gene expression that are epigenetically defined (i.e. by gene regulation) and does not require a mutational event, as in previously described forms of competition.
Because Myc proteins play important roles in the adult stem cells of
several mammalian niches (reviewed by
Murphy et al., 2005), it is
possible that the interactions described here are conserved, at least in
certain tissues (Muncan et al.,
2006). In tissues where stem cells are not grouped together within
the same niche, the process may be aided by migration of the stem cells from
one niche to the other, as has been recently described for the somatic stem
cell niches of the Drosophila ovary
(Nystul and Spradling, 2007).
More generally, the concept of competition among cells could be of use to
describe several aspects of development and homeostasis, an idea nicely
supported, for example, by the competition that occurs between the soma and
the germline for lipid phosphate uptake
(Renault et al., 2004). Stem
cell interactions such as those described here significantly contribute to the
balance between differentiation and self-renewal, and may be relevant for
diverse processes such as aging, the accumulation of pre-cancerous mutations
and the successful application of stem cell therapies.
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/136/6/995/DC1
* These authors contributed equally to this work 
Present address: Burnham Institute for Medical Research, La Jolla, San
Diego, CA, USA
Present address: Spanish National Biotechnology Centre, Consejo Superior de
Investigaciones Científicas (CSIC), 28049 Madrid, Spain
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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