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First published online 7 February 2007
doi: 10.1242/dev.003392
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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 Medical Center,
3901 Rainbow Boulevard, Kansas City, KS 66160, USA.
Author for correspondence (e-mail:
tgx{at}stowers-institute.org)
Accepted 3 January 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Notch, Stem cell, Germ line, Drosophila, Ovary, Niche
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Spradling et al., 2001;
Watt and Hogan, 2000). The
signals from niche cells work with intrinsic factors to control stem cell
self-renewal, proliferation and differentiation
(Molofsky et al., 2004;
Xi et al., 2005). Although the
identification of stem cells remains challenging, due to their rarity and lack
of unique molecular markers in mammalian systems, several niches are roughly
defined based on their proximity to stem cells
(Calvi et al., 2003;
Nishimura et al., 2002;
Tumbar et al., 2004;
Zhang et al., 2003). However,
little is known about how niche formation is genetically controlled, even
though niche structure is defined. In this study, we show that Notch
(N) signaling directly controls formation of the germline stem cell
(GSC) niche in the Drosophila ovary.
The Drosophila ovary is one of the best-studied stem cell systems
because of its easily identified stem cells and powerful genetic tools
(Xie et al., 2005). There
reside three types of stem cells: GSCs, somatic stem cells (SSCs) and newly
identified escort stem cells (ESCs), which are responsible for generating
differentiated germ cells, follicle cells and escort cells, respectively
(Decotto and Spradling, 2005;
Lin and Spradling, 1993;
Margolis and Spradling, 1995;
Wieschaus and Szabad, 1979).
GSCs have been shown to be situated in the niche, which is composed of cap
cells, and possibly terminal filament (TF) cells and ESCs
(Cox et al., 1998;
Decotto and Spradling, 2005;
Kretzschmar et al., 1999;
Xie and Spradling, 1998;
Xie and Spradling, 2000)
(Fig. 1A). Recent findings show
that the number of cap cells is closely correlated with the GSC number in the
normal ovary (Xie and Spradling,
2000) and that GSCs must be anchored to cap cells in order to be
maintained as stem cells through DE-cadherin-mediated cell adhesion
(Song et al., 2002). This
supports the idea that cap cells are an important component of the GSC niche.
dpp, gbb, Yb [fs(1)Yb - FlyBase],
piwi and hh, known to be important for GSC maintenance, are
expressed not only in cap cells but also in TFs and/or inner germarial sheath
(IGS) cells (Cox et al., 1998;
Cox et al., 2000;
Kiger and Fuller, 2001;
King and Lin, 1999;
King et al., 2001;
Song et al., 2004;
Song et al., 2002;
Xie and Spradling, 1998).
These findings point to a crucial function of cap cells in the GSC niche, but
it remains unclear how cap cell formation is genetically controlled.
N signaling plays an important role in regulating proliferation
and differentiation of many different cell types
(Artavanis-Tsakonas et al.,
1999; Lai, 2004).
In the Drosophila ovary, it was first shown to be required for
maintaining follicle cells in their precursor stage and for specification of
polar cells that mark the ends of the egg chamber
(Grammont and Irvine, 2001;
Larkin et al., 1996;
Xu et al., 1992). During late
oogenesis, N signaling is required for the switch from the mitotic
cycle to the endocycle and differentiation of follicle cells by negatively
regulating the cut gene
(Shcherbata et al., 2004;
Sun and Deng, 2005), and it is
also required for patterning the anterior egg shell
(Dobens et al., 2005). In this
study, we have shown, for the first time to our knowledge, that N
signaling is necessary and sufficient for controlling formation of the GSC
niche.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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); UAS-Dl30B, UAS-Dl36 and
hs-gal4 (Bloomington Drosophila Stock Center); two
UAS-Nint lines, UAS-NB2A2 and
UAS-N33c3 (kindly provided by Dr Gary Struhl, Columbia
University, New York City, NY); N264-39, N54l9,
Nts1 and hh-lacZ (Bloomington Drosophila
Stock Center); UAS-DlDN
(Parks et al., 2000);
UAS-mamH and UAS-mamN
(Helms et al., 1999);
Dl-lacZ and E(spl)-CD2 (kindly provided by
Dr Leonard Dobens, University of Missouri, Kansas City, MO).
Generation of the marked IGS cells overexpressing dpp and an activated N in the adult Drosophila ovary
IGS cells overexpressing dpp or NICD were
generated using a technique that combines the FLP-FRT and UAS-GAL4 systems
(Ito et al., 1997).
hs-flp; AyGal4 (act>>y>>gal4) UAS-GFP/CyO
virgin females were crossed with either UAS-dpp/TM3,
UAS-NB2A2/CyO or UAS-N33C3/TM3 males,
respectively. Clones were induced by two 1-hour heat shock treatments of
2-day-old females at 37°C separated by an interval of 5 hours. The
heat-shock-treated females were cultured at room temperature for 1 week with
daily supplied fresh food, and their ovaries were dissected out and processed
for immunostaining with monoclonal anti-Hts (1B1) and rabbit anti-GFP
antibodies as described previously (Xie
and Spradling, 1998).
BrdU labeling of germline stem cells
The 2-day-old hh-lacZ/+ control, c587-gal4/+;
UAS-Dl30B/+;hh-lacZ/+, c587-gal4/+; UAS-Dl36/hh-lacZ,
c587-gal4/+;UAS-NB2A2/+;hh-lacZ/+, c587-gal4/+;
UAS-N33c3/hh-lacZ females were fed on wet yeast paste
mixed with 20 mg/ml BrdU solution for 3 consecutive days with fresh BrdU yeast
paste each day. The ovaries from these flies were processed for immunostaining
with anti-BrdU, anti-Hts and anti-ß-galactosidase (ß-gal) antibodies
according to our published procedures
(Song et al., 2002). The rest
of the flies were then transferred to fresh food with yeast flakes containing
no BrdU every day for 3 consecutive weeks, and their ovaries were processed
for immunostaining with the same antibodies.
Immunohistochemistry
Immunostaining of the Drosophila ovaries was performed according
to previously published procedures (Song
et al., 2002). Primary antibodies used in this study are as
follows: rabbit anti-ß-galactosidase antibody (1:200, Cappel), rabbit
anti-GFP antibody (1:200, Molecular Probes), mouse monoclonal anti-CD2
antibody (1:100, Serotec), mouse monoclonal anti-Hts antibody, 1B1 (1:4,
DSHB), a mouse monoclonal anti-Dl antibody, c594.9B (1:3, DSHB), two mouse
monoclonal anti-N antibodies, F461.3B and c458.2H (1:3, DSHB), rabbit
anti-Vasa antibody (1:1000, a gift from Dr Paul Lasko, McGill University,
Montreal, Canada), rat anti-DE-cadherin (1:4, DSHB) and sheep anti-BrdU
antibody (1:100, Capralogies). The secondary antibodies used in this study are
the Alexa 568-, Alexa 468- and Alexa 596-conjugated goat or donkey anti-mouse,
rabbit, rat or sheep antibodies (1:200, Molecular Probes). All the images were
taken using a Leica TCS SP2 confocal microscope.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Lai, 2004),
we sought to test whether N signaling regulates the formation of cap
cells in the Drosophila ovary by manipulating the signaling pathway
in somatic cells of developing female gonads using the GAL4-UAS system
(Brand and Perrimon, 1993).
Ectopic expression of a truncated N intracellular domain
(Nint) or its ligand Delta (Dl) can
activate N signaling in ectopic locations
(Struhl et al., 1993), and the
c587-gal4 driver can drive a UAS-GFP specifically in most, but not
all, of the somatic cells of developing female gonads
(Zhu and Xie, 2003)
(Fig. 1B,B'). A
hedgehog (hh)-lacZ line (the bacterial
lacZ gene inserted in the hh gene) is highly expressed in
five to seven cap cells and eight to ten TF cells
(Forbes et al., 1996a)
(Fig. 1C). GSCs are identified
by their direct association with cap cells and the presence of an anteriorly
anchored spherical spectrosome (Fig.
1C). Their immediate daughters, cystoblasts, also contain a
spherical spectrosome but are positioned away from the cap cells, while other
further differentiated progeny, germ cell cysts, can be identified by the
presence of a branched fusome (Fig.
1C). The spectrosome and the branched fusome are identified by
their expression of Hu-li tai-shao (Hts)
(de Cuevas et al., 1997). By
contrast to the five to seven cap cells in a normal germarium
(Fig. 1C), we observed more
lacZ-positive cap cells at the tip of either Dl- or
Nint-overexpressing germaria using two independent
transgenic UAS-Dl or UAS-Nint lines
(Fig. 1D,D'). Note that
overexpression of Dl and Nint gave similar
phenotypes, although overexpression of the latter generated a stronger
phenotype than that of the former. In the germaria with increased cap cells,
spectrosome-containing single germ cells, which were later shown to behave
like GSCs, also increased at the germarial tip
(Fig. 1D,D'). This result
shows that N signaling is sufficient to induce cap cell formation and
supports the idea that cap cells are a key niche component for controlling GSC
self-renewal. In addition to increased cap cells at the germarial tip, we frequently observed one or more patches of strongly lacZ-positive somatic cells away from the germarial tip when Dl or Nint was overexpressed in the developing gonads (Fig. 1E-F'). These lacZ-positive somatic cells appeared to be functional cap cells at ectopic locations, as spectrosome-containing single germ cells (later shown to be GSCs) were closely associated with them (Fig. 1E-F'). The ectopic GSCs associated with the ectopic cap cells also anchored their spectrosome on the side that contacts cap cells, as observed in a normal GSC context. Some of these ectopic cap cells were surrounded by IGS cells (Fig. 1E,E') or somatic follicle cells (Fig. 1F,F'), and it appeared that both types of ectopic cap cells could sufficiently maintain GSCs. In rare cases of Nint overexpression, lacZ-positive cap cells completely occupied the anterior half of the germaria instead of IGS cells, and consequently, GSCs were everywhere in the anterior half of the germarium (Fig. 1G,G'). Germ cells moving away from the cap cells could still differentiate, as indicated by the presence of the branched fusomes. These observations further indicate that signals from the GSC niche directly repress differentiation of GSCs close to cap cells, allowing germ cells moving away from the cap cells to differentiate, because they are beyond the influence of short-range signals from the cap cells. The ectopic lacZ-positive cells and their associated GSCs could persist for at least 5 weeks (the longest time we had tested), suggesting that these expanded or ectopic cap cells are stable and sustain GSCs like normal cap cells. Together, these results demonstrate that N signaling is sufficient to induce cap cell formation. Furthermore, our observation that ectopic cap cells without TF cells or IGS cells are able to sustain GSC self-renewal indicates that cap cells are a key component to establish the niche for sustaining GSC self-renewal.
To further verify that the ectopic hh-lacZ-positive cap cells
exhibit known properties of normal cap cells, we examined the expression of
other markers for cap cells. A wingless (wg) enhancer trap
line, wg-lacZ, is known to be expressed in some but not all cap cells
(Forbes et al., 1996b) (see
Fig. S1A in the supplementary material). In agreement with our hypothesis,
some of these ectopic cap cells expressed wg-lacZ whether they were
surrounded by IGS cells or somatic follicle cells (see Fig. S1B,C in the
supplementary material). Nuclear lamin C is expressed highly in nuclear
membranes of TF cells and cap cells (Xie
and Spradling, 2000) (see Fig. S1D in the supplementary material),
and the cap cells in normal or ectopic locations highly express this marker
(see Fig. S1E in the supplementary material). Cap cells also express and
accumulate DE-cadherin at their junction with GSCs in keeping GSCs in the
niche (Song et al., 2002).
Indeed, DE-cadherin proteins significantly accumulated between ectopic cap
cells and their associated GSCs (see Fig. S1F in the supplementary material),
which might also function to anchor GSCs. Therefore, our molecular evidence
strongly indicates that these ectopic cap cells behave like normal cap
cells.
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;
Kai and Spradling, 2003;
Song et al., 2004). As in the
wild-type niche (Fig. 2A), the
spectrosome-containing single germ cells associated with those ectopic cap
cells did not express bam-GFP, which resembles the property of normal
GSCs (Fig. 2B,C).
Interestingly, germ cells lying one cell away from ectopic GSCs were often
germline cysts, as they contained branched fusomes, indicating that the
progeny of the ectopic GSCs probably undergo normal differentiation
(Fig. 2B,C). However, too many
cap cells (more than seven) at the normal location
(Fig. 2D) or at an ectopic site
(Fig. 2E) often caused the
accumulation of spectrosome-containing single germ cells located two or more
cell diameters away, and bam expression was also repressed in those
single cells, indicating that these extra single cells also resemble GSCs.
Normally, bam is only repressed in GSCs due to the short-range BMP
signal, which may be caused by a limited amount of DPP protein produced by
five to seven cap cells in a wild-type germarium. As we have previously
demonstrated that dpp overexpression in the germarium causes the
accumulation of single cells that are negative for bam expression
(Song et al., 2004;
Xie and Spradling, 1998),
these observations further suggest that cap cells are the major source of BMP,
and more cap cells can produce more BMP, which could help it diffuse farther
than the normal distance.
A short-range BMP signal from cap cells specifically activates its
signaling cascade in GSCs to activate expression of Dad
(Casanueva and Ferguson, 2004;
Kai and Spradling, 2003;
Song et al., 2004)
(Fig. 2F). As expected, all the
GSCs that directly contact the expanded cap cells at the germarial tip highly
expressed Dad-lacZ, indicating that the expanded cap cells have the
capacity to produce a BMP signal in a similar manner to normal cap cells
(Fig. 2G). Interestingly, many
spectrosome-containing single germ cells not directly associated with the
expanded cap cells (more than ten cap cells) also expressed Dad-lacZ,
further supporting the idea that the increased number of cap cells leads to
production of more diffusible BMP (Fig.
2G). When the ectopic cap cells are surrounded by IGS cells or
near follicle cells, their associated GSCs also highly expressed
Dad-lacZ as in normal GSCs (Fig.
2H), confirming the idea that ectopic cap cells can also emit the
BMP signal like normal cap cells. These results suggest that cap cells are the
source of active BMP and that the spectrosome-containing single germ cells
associated with expanded or ectopic cap cells resemble GSCs.
|
). In our earlier experiments, we observed that many cases of
ectopic GSCs carried an elongated spectrosome, indicating that those ectopic
GSCs are probably capable of dividing and generating differentiated germ
cells. To further confirm that these extra spectrosome-containing germ cells
behave like GSCs, we labeled them with BrdU, a nucleotide analog, for 3 days
and chased for 3 weeks. As a control, 96.4% of the germaria (n=57) of
the hh-lacZ/+ heterozygotes contained one or more BrdU-labeled GSCs
after 3 days of BrdU feeding, while 84.5% of the total GSCs (n=136)
were labeled by BrdU. Consequently, most of the control germaria contained two
or three labeled GSCs (Fig.
3A). After the females were fed for 3 more weeks on normal food
without BrdU, the GSCs in the control germaria (n=35) completely lost
their BrdU label (Fig. 3B),
indicating that the BrdU label is completely diluted out as the labeled GSCs
continuously divide for 3 weeks. Similarly, 98.8% of the germaria (n=85) (developed from the female gonads overexpressing Dl or Nint) carried one or more BrdU-labeled GSCs close to the cap cells at the normal location or at ectopic cap cells (Fig. 3C,D). Among them, 94.1% of the expanded niches (more than seven cap cells carrying four or more GSCs; n=51) at the normal location (close to TFs) carried one or more BrdU-labeled GSCs (Fig. 3C,C'), while 96.0% of the ectopic niches (n=25) carried one or more BrdU-labeled GSCs (Fig. 3D), indicating that extra GSCs in the expanded niches as well as in the ectopic niches are mitotically active like normal GSCs. After the 3 week chase, 85.9% of the germaria (n=120) did not carry any BrdU-labeled GSCs at the normal location or at ectopic sites (Fig. 3E), whereas 14.1% of them carried one or more BrdU-labeled GSCs (Fig. 3F), also indicating that the expanded or ectopic GSCs remain active for over 3 weeks. It also appeared that those GSCs did not divide as frequently as normal GSCs. Perhaps this is due to their excessive number at each niche. These results strongly suggest that the GSCs at the expanded niche or at the ectopic niche can continuously divide and generate differentiated germ cells like normal GSCs.
Notch signaling induces formation of ectopic cap cells only during the late third-instar larval and early pupal stages
Cap cells normally form during the larval-pupal transition and in the early
pupal stage (Zhu and Xie,
2003). To further determine when ectopic cap cells form, we used
an hs-gal4 transgene (the gal4 gene under the control of the
heat shock protein 70 promoter) to drive the expression of Dl during
gonadal development by heat shock treatments. When Dl expression was
induced during the second instar larval stage or after the mid-pupal stage,
about 98% of the germaria carried five to seven cap cells in the normal
location, just like in the wild type (n=603), whereas about 2% of the
germaria had ectopic cap cells positioned away from TFs, as detected in the
heat-shocked hsgal4; hh-lacZ control germaria (n=606). Also,
overexpression of Dl in adult ovaries did not increase cap cell
numbers and induce ectopic cap cells (n=212). Interestingly, when
Dl was ectopically expressed during the third instar and early pupal
stages, the number of cap cells at the tip of some germaria was increased
(Fig. 4A,A'), and about
10% of these germaria carried ectopic cap cells (n=234)
(Fig. 4B,B'), indicating
that elevated N signaling can induce cap cell formation in the normal
location as well as in the ectopic sites. In some of the germaria
(n=27), cap cells were formed away from TFs and still maintained
GSCs, but no GSCs were associated with TFs
(Fig. 4B,B'), indicating
that TFs alone are not sufficient to sustain GSC self-renewal. These results
suggest that the IGS precursors in the developing gonad are competent to form
cap cells in response to ectopic N signaling from the late
third-instar larval stage to the early pupal stage. Note that these ectopic
cap cells no longer expressed Dl or had the activated N
signaling in the adult ovary but were stable and able to maintain GSCs,
suggesting that ectopic activated N signaling by c587-gal4
is not directly involved in controlling GSC self-renewal.
|
).
Interestingly, when dpp was induced to be expressed in several of the
IGS cells of adult Drosophila ovaries, many spectrosome-containing
single germ cells accumulated, and those GFP-positive dpp-expressing
IGS cells still maintained their normal morphology, suggesting that
dpp-expressing IGS cells may sufficiently create ectopic GSC niches
without being transformed into cap cells (see Fig. S2A in the supplementary
material). By contrast, overexpression of the activated N in the
GFP-positive IGS cells did not affect the differentiation status of the
underlying germ cells, indicating that activation of N signaling in
adult IGS cells alone does not sufficiently create ectopic GSC niches, unlike
dpp overexpression (see Fig. S2B,C in the supplementary material). In
light of the evidence we have presented so far, we conclude that ectopic
expression of the activated N during niche formation leads to
formation of ectopic niches and GSCs, but the continuous expression of the
activated N in adult IGS cells might not directly contribute to the
maintenance of ectopic GSCs.
Dl on newly formed TF cells activates N signaling in their adjacent somatic cells to induce cap cell formation
To gain further insight into how N signaling is involved in
controlling cap cell formation, we examined N and Dl
expression in the developing gonads from the late third instar larval stage to
the early pupal stage. A Dl-lacZ line (the lacZ gene
inserted in the Dl locus to recapitulate its expression) and an
anti-Dl antibody were used to determine Dl expression in the
developing female gonads (Grossniklaus et
al., 1989). Dl was primarily expressed in newly formed
TFs and other somatic cells anterior to the primordial germ cell zone (PGCZ)
at high levels but not in primordial germ cells (PGCs) during the late
third-instar larval stage or the larval-pupal transitional stage
(Fig. 4C-D').
Interestingly, N protein was expressed at high levels in TFs and cap cells as
well as in the somatic cells that are mingled with PGCs at lower levels, but
not in PGCs (Fig. 4E,E').
N signaling is known to regulate expression of
E(spl) complex genes
(Bailey and Posakony, 1995); an
E(spl)mß-CD2 transgene has been used to
monitor N signaling activity in several tissue types
(Cooper and Bray, 1999;
de Celis and Bray, 1997).
Consistent with the idea that TFs and cap cells are capable of activating
N signaling due to presence of both Dl and N,
E(spl)mß-CD2 was expressed almost exclusively
in TFs and cap cells but not in the somatic cells that are mixed with PGCs
(Fig. 4F,F'). These
expression results suggest that developing TFs and cap cells are active in
N signaling, which may be dependent on Dl in TFs.
Our earlier observation that the somatic cells that mingle with PGCs express N raises the interesting possibility that activated N signaling transforms those somatic cells normally destined to form IGS cells and/or follicle cells into cap cells. To further investigate this possibility, we used the E(spl)mß-CD2 as a marker to study whether ectopic expression of Dl or Nint could induce expression of E(spl)mß-CD2 in the somatic cells that are mingled with PGCs. When Nint or Dl was expressed by c587-gal4 in most of the somatic cells in the gonad, clusters of the somatic cells that were also closely associated with PGCs started to express E(spl)mß-CD2 (Fig. 4G,G'). This suggests that ectopic N signaling in somatic cells, which is normally devoid of N signaling, induces them to assume cap cell fate.
|
), we examined the cap cell number in 2-day-old
heterozygotes carrying strong or null N mutants,
N54l9 and N264-39, using
hh-lacZ to label TFs and cap cells. Because there is no or little cap
cell and GSC turnover in 2-day old Drosophila females, the number of
cap cells and GSCs should truly reflect the number of cap cells that form
during the pupal stage. By contrast, with 2-day-old wild-type germaria with
5.5±1.3 cap cells and 2.5±0.6 GSCs (n=121), the
2-day-old N54l9 heterozygous germaria carried
4.1±1.5 cap cells and 1.8±0.6 GSCs (n=131), while the
N264-39 heterozygous germaria had 4.2±1.4 cap cells
and 1.8±0.6 GSCs (n=97), indicating that decreased N
signaling can significantly reduce cap cell number (P<0.001) and
consequently GSC number (P<0.001). Moreover, about 36% of the
N heterozygous germaria had three or fewer cap cells, by contrast to
the five to seven cap cells of the wild-type germaria
(Fig. 4H,H'). To gain
further evidence supporting the idea that N signaling is required for
cap cell formation, we used the c587-gal4 driver to overexpress
dominant-negative mutants for N pathway components, such as
Dl and mastermind (mam). The 2-day-old germaria
developing from female gonads that overexpressed a dominant-negative
Dl had an average of 3.3±1.6 cap cells and 1.6±0.6 GSCs
(total 62 germaria examined), while the germaria developing from female gonads
that overexpressed two dominant-negative forms of mam,
mamH and mamN, carried an average of
3.2±1.2 cap cells and 1.6±0.6 GSCs (total 162 germaria examined)
as well as 2.8±1.2 cap cells and 1.4±0.6 GSCs (total 128
germaria examined), respectively. As the dominant-negative forms of
Dl and mam have been shown to specifically block N
signaling in imaginal discs (Helms et al.,
1999; Parks et al.,
2000), our results further support the idea that N
signaling is required for controlling cap cell formation.
|
). Among
the 2-day-old agametic ovoD1rS1 mutant germaria, many
still contained one to five cap cells, although some completely lacked cap
cells, which could have degenerated during the pupal period
(Fig. 4I-J'). In the
osk mutant agametic germaria, cap cells appear to form and persist
(Margolis and Spradling,
1995). Together, these findings argue that TF-expressed Dl is
responsible for cap cell formation in the agametic germarium, and possibly in
the normal germarium as well.
Notch signaling is required for the maintenance of the niche and GSCs in the adult ovary
To investigate whether N signaling is also involved in regulation
of niche and GSC functions in the adult ovary, we examined the expression of
Dl using the Dl-lacZ line mentioned earlier. By contrast
with the fact that Dl expression is restricted to the somatic cells
anterior to the PGC zone in the third-instar larval stage, including newly
formed TFs, Dl was also observed to be expressed at low levels in
germ cells of adult germaria, including GSCs, in addition to its expression in
TFs (see Fig. S3A in the supplementary material).
E(spl)m7, a gene in the E(spl)
complex, is a response gene of N signaling
(Bray, 2006).
E(spl)m7-lacZ was used to monitor N
signaling activity in the germarium. Interestingly,
E(spl)m7-lacZ was mainly detected in cap cells,
possibly in GSCs at very low levels, suggesting that N signaling
remains active in cap cells of the adult germarium (see Fig. S3B in the
supplementary material). To further investigate whether or not N
signaling is required for the maintenance or function of the GSC niche, we
used a well-studied temperature-sensitive N allele
(Nts1) to determine the role of N signaling in
the adult ovary. One previous study using this N allele showed that
N signaling is required for proper differentiation of follicle cells
and thus proper formation of egg chambers
(Xu et al., 1992). As a
control, the germaria from the wild-type females cultured at 29°C for 1 or
2 weeks had 2.4±0.5 (n=33) and 2.2±0.7 (n=39)
GSCs, respectively, and germ cysts differentiated normally and egg chambers
formed normally as well (see Fig. S3C,D in the supplementary material). By
contrast, the germaria from the Nts1 mutant females
cultured at 29°C for 1 or 2 weeks were generally small, and contained
1.1±1.0 (n=27) and 0.5±0.8 (n=36) GSCs,
respectively. By contrast with 6.0±0.9 (1 week) and 5.8±1.0 (2
weeks) cap cells for the wild-type ovaries, the Nts1
mutant ovaries from the females cultured at 29°C for 1 or 2 weeks carried
4.6±1.3 and 2.7±1.4 cap cells, respectively. As complete loss of
GSCs does not quickly eliminate cap cells
(Xie and Spradling, 2000), our
results suggest that GSC loss in the N mutant ovaries is probably due
to loss of cap cells. Indeed, the germaria from the females cultured at
29°C for 1 week had more cap cells than the ones cultured for 2 weeks (see
Fig. S3E-H in the supplementary material), and in some of the germaria, cap
cells completely disappeared (see Fig. S3H in the supplementary material). In
light of the evidence that N signaling activity in the adult ovary is
restricted to cap cells, these results further suggest that N
signaling is required for maintaining cap cells and thus GSCs.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), it remains
unclear what constitutes a functional niche and how its formation is
genetically controlled. In this study, we have revealed one novel molecular
mechanism underlying GSC niche formation in the Drosophila ovary
(Fig. 5). The newly formed TFs
express Dl, which is probably responsible for activating N
signaling in their neighboring somatic cells and for inducing niche formation.
Ectopic N signaling is sufficient to induce niche formation in a
developmental stage-dependent manner, and reduced N signaling results
in reduction of cap cell and GSC numbers, demonstrating that N
signaling is important for controlling GSC niche formation. As ectopic niche
cells in different locations are stable and able to sustain GSC self-renewal,
this leads us to conclude that the niche does not have to function in a fixed
position. Finally, N signaling is also required for the survival of niche
cells in adults (see Fig. S3 in the supplementary material). By analogy,
N signaling may control niche formation in other systems, including
mammals.
N signaling is required for formation of the GSC niche
At the onset of the larval-pupal transition, all of the 16 to 20 TF stacks
have formed and initiate ovariole formation
(Godt and Laski, 1995;
King, 1970), while another
group of somatic cells, cap cells, start to occupy a position between the TFs
and the germ cells (Zhu and Xie,
2003). The PGCs in direct contact with cap cells are further
anchored through E-cadherin and are further expanded through symmetric
division and develop into permanent GSCs in the adult ovary
(Song et al., 2002;
Zhu and Xie, 2003).
Actin-filament regulator, Cofilin/ADF, and ecdysone signaling, are required
for TF formation (Chen et al.,
2001; Hodin and Riddiford,
1998). However, no studies have been carried out to investigate
the formation of cap cells, which are a key component of the GSC niche. In
this study, we have investigated the role of N signaling in
controlling cap cell formation.
N signaling controls cell fate determination in a variety of
organisms (Artavanis-Tsakonas et al.,
1999; Kadesch,
2004; Lai, 2004).
In this study, we show that in late third-instar larval female gonads, Dl is
expressed in newly formed TFs, while the N receptor is expressed in all the
somatic cells, including TFs and cap cells. Consequently, N signaling
is active in newly formed TFs and cap cells and its activation is sufficient
to induce cap cell formation, suggesting that TF-expressed Dl
activates N signaling to induce cap cell formation. To further
support the idea that N signaling specifies cap cell fate, reduction
of N signaling results in a reduced number of cap cells. Induction of
cap cells by N signaling can only take place during the late third
instar and early pupal stages, suggesting that active N signaling
only promotes cap cell formation along with other factors provided at
particular stages. Cap cells can still form without germ cells. This suggests
that Dl is unlikely to be required in germ cells for cap cell formation.
Therefore, we conclude that N signaling, activated probably by Dl
from newly formed cap cells, specifies cap cell fate through direct induction.
In this study, we also show that N signaling is required for
maintaining the GSC niche in the adult ovary, as loss of N function
results in rapid loss of cap cells and GSCs. Taken together, the results of
this study demonstrate that N signaling is important for controlling
niche formation as well as niche maintenance.
Expanded and ectopic niches are sufficient for controlling GSC self-renewal
Although niches have been defined for GSCs in the Drosophila ovary
and testis, as well as in several tissue types of the mammalian systems, it
remains unclear whether they still function properly for controlling stem cell
self-renewal after their location and size are changed. In this study, we have
provided two pieces of experimental evidence supporting the idea that expanded
niches are functional for controlling GSC self-renewal. First, increased cap
cells in the normal location express all known cap cell markers, such as
hh-lacZ, wg-lacZ, Lamin C and E-cadherin, and behave like normal cap
cells. Second, these expanded cap cells can support self-renewal of extra
GSCs, which behave similarly to normal ones based on Dad-lacZ and
bam-GFP expression, and their ability to self-renew and generate
differentiated germ cells. Even when cap cells cover the anterior half of the
germarium, the GSCs associated with the cap cells also appear to be capable of
self-renewing and generating differentiated germ cells. Our findings show that
GSC niche size can be expanded by adding more niche cells.
This study has also demonstrated that the GSC niche could function in
ectopic locations. Ectopic cap cells, which are surrounded by IGS cells or
follicle cells, also express known cap cell markers and sufficiently support
functional GSCs, supporting the idea that TFs and IGS cells are not essential
components of the GSC niche. This is consistent with early published studies
showing that the numbers of cap cells and GSCs are closely correlated and that
TFs and cap cells express the genes important for GSC self-renewal such as
piwi, Yb, hh and dpp/gbb
(Chen and McKearin, 2005;
Cox et al., 2000;
King et al., 2001;
Song et al., 2004;
Szakmary et al., 2005;
Xie and Spradling, 1998). In
light of the recent evidence that ESCs in direct contact with cap cells and
GSCs are required for maintaining GSCs
(Decotto and Spradling, 2005),
it remains formally possible that some unidentified ESC cells associated with
expanded or ectopic cap cells contribute to niche function. In any case, this
study demonstrates that the size and location of the GSC niche can be
genetically manipulated while it maintains its functions. Our ability to
manipulate niche location and size will further increase our capacity to
investigate how niche formation is controlled and how the niche controls stem
cell function in general.
Limited amount of active BMP produced by five to seven cap cells may explain its short-range effect on GSC self-renewal
One of the major unsolved issues for the GSC niche in the
Drosophila ovary is how BMPs function as a short-range signal to
control GSC self-renewal and allow the GSC daughter adjacent to the GSC to
differentiate at the same time. Several previous studies from us and others
have shown that BMP signaling activity is primarily restricted to the GSC
based on Mad phosphorylation and Dad expression, two indicators of
BMP signaling (Casanueva and Ferguson,
2004; Kai and Spradling,
2003; Song et al.,
2004). Our early work has also shown that dpp is
primarily expressed in TF and cap cells, while gbb is expressed in TF
and cap cells as well as in IGS cells
(Song et al., 2004). In this
study we show that BMP signaling activity can spread two or more cell
diameters based on expression of Dad-lacZ and bam-GFP when
more cap cells exist. Furthermore, when more cap cells accumulate in ectopic
sites, the GSCs associated with the cap cells as well as the germ cells lying
two or three cells away are capable of activating BMP signaling and repressing
bam expression. One of the explanations for these observations is
that cap cells are the source of BMP and more cap cells would produce more BMP
to diffuse further away to repress differentiation of germ cells lying two or
more cell diameters away. Another explanation is that the ratio of cap cells
to ESCs or escort cells increases so that BMP inhibitors, such as Sog,
produced by ESCs or escort cells, are diluted or deterred by more cap cells,
and consequently more active BMP is available for reaching and activating
cells lying more than two cell diameters away. In Xenopus
gastrula-stage embryos, an effective BMP-4 activity gradient is established,
not by diffusion of BMP-4 protein but by the long-range effects of two BMP-4
inhibitors, Noggin and Chordin (Jones and
Smith, 1998). Finally, it is also possible that a combination of
both mechanisms contributes to restriction of BMP signaling activity to one
cell diameter in the GSC niche. Our observations from this study have
suggested that a limited amount of active BMP produced by cap cells is
probably responsible for its short-range effect on GSC self-renewal in the GSC
niche.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/6/1071/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J.
(1999). Notch signaling: cell fate control and signal integration
in development. Science
284,770
-776.
Bailey, A. M. and Posakony, J. W. (1995).
Suppressor of hairless directly activates transcription of enhancer of split
complex genes in response to Notch receptor activity. Genes
Dev. 9,2609
-2622.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118,401 -415.[Abstract]
Bray, S. J. (2006). Notch signalling: a simple pathway becomes complex. Nat. Rev. Mol. Cell Biol. 7, 678-689.[CrossRef][Medline]
Calvi, L. M., Adams, G. B., Weibrecht, K. W., Weber, J. M., Olson, D. P., Knight, M. C., Martin, R. P., Schipani, E., Divieti, P., Bringhurst, F. R. et al. (2003). Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425,841 -846.[CrossRef][Medline]
Casanueva, M. O. and Ferguson, E. L. (2004).
Germline stem cell number in the Drosophila ovary is regulated by
redundant mechanisms that control Dpp signaling.
Development 131,1881
-1890.
Chen, D. and McKearin, D. M. (2003). A discrete
transcriptional silencer in the bam gene determines asymmetric division of the
Drosophila germline stem cell. Development
130,1159
-1170.
Chen, D. and McKearin, D. (2005). Gene circuitry controlling a stem cell niche. Curr. Biol. 15,179 -184.[CrossRef][Medline]
Chen, J., Godt, D., Gunsalus, K., Kiss, I., Goldberg, M. and Laski, F. A. (2001). Cofilin/ADF is required for cell motility during Drosophila ovary development and oogenesis. Nat. Cell Biol. 3,204 -209.[CrossRef][Medline]
Cooper, M. T. and Bray, S. J. (1999). Frizzled regulation of Notch signaling polarizes cell fate in the Drosophila eye. Nature 397,526 -530.[CrossRef][Medline]
Cox, D. N., Chao, A., Baker, J., Chang, L., Qiao, D. and Lin,
H. (1998). A novel class of evolutionarily conserved genes
defined by piwi are essential for stem cell self-renewal.
Genes Dev. 12,3715
-3727.
Cox, D. N., Chao, A. and Lin, H. (2000). piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells. Development 127,503 -514.[Abstract]
de Celis, J. F. and Bray, S. (1997). Feed-back mechanisms affecting Notch activation at the dorsoventral boundary in the Drosophila wing. Development 124,3241 -3251.[Abstract]
de Cuevas, M. and Spradling, A. C. (1998). Morphogenesis of the Drosophila fusome and its implications for oocyte specification. Development 125,2781 -2789.[Abstract]
de Cuevas, M., Lilly, M. A. and Spradling, A. C. (1997). Germline cyst formation in Drosophila. Annu. Rev. Genet. 31,405 -428.[CrossRef][Medline]
Decotto, E. and Spradling, A. C. (2005). The Drosophila ovarian and testis stem cell niches: similar somatic stem cells and signals. Dev. Cell 9, 501-510.[CrossRef][Medline]
Dobens, L., Jaeger, A., Peterson, J. S. and Raftery, L. A. (2005). Bunched sets a boundary for Notch signaling to pattern anterior eggshell structures during Drosophila oogenesis. Dev. Biol. 287,425 -437.[Medline]
Forbes, A. J., Lin, H., Ingham, P. W. and Spradling, A. C. (1996a). hedgehog is required for the proliferation and specification of ovarian somatic cells prior to egg chamber formation in Drosophila. Development 122,1125 -1135.[Abstract]
Forbes, A. J., Spradling, A. C., Ingham, P. W. and Lin, H. (1996b). The role of segment polarity genes during early oogenesis in Drosophila. Development 122,3283 -3294.[Abstract]
Godt, D. and Laski, F. A. (1995). Mechanisms of cell rearrangement and cell recruitment in Drosophila ovary morphogenesis and the requirement of bric a brac.Development 121,173 -187.[Abstract]
Grammont, M. and Irvine, K. D. (2001). fringe and Notch specify polar cell fate during Drosophila oogenesis. Development 128,2243 -2253.
Grossniklaus, U., Bellen, H. J., Wilson, C. and Gehring, W. J. (1989). Pelement-mediated enhancer detection applied to the study of oogenesis in Drosophila. Development 107,189 -200.[Abstract]
Helms, W., Lee, H., Ammerman, M., Parks, A. L., Muskavitch, M. A. and Yedvobnick, B. (1999). Engineered truncations in the Drosophila mastermind protein disrupt Notch pathway function. Dev. Biol. 215,358 -374.[CrossRef][Medline]
Hodin, J. and Riddiford, L. M. (1998). The ecdysone receptor and ultraspiracle regulate the timing and progression of ovarian morphogenesis during Drosophila metamorphosis. Dev. Genes Evol. 208,304 -317.[CrossRef][Medline]
Ito, K., Awano, W., Suzuki, K., Hiromi, Y. and Yamamoto, D. (1997). The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells. Development 124,761 -771.[Abstract]
Jones, C. M. and Smith, J. C. (1998). Establishment of a BMP-4 morphogen gradient by long-range inhibition. Dev. Biol. 194,12 -17.[CrossRef][Medline]
Kadesch, T. (2004). Notch signaling: the demise of elegant simplicity. Curr. Opin. Genet. Dev. 14,506 -512.[CrossRef][Medline]
Kai, T. and Spradling, A. (2003). An empty
Drosophila stem cell niche reactivates the proliferation of ectopic
cells. Proc. Natl. Acad. Sci. USA
100,4633
-4638.
Kiger, A. A. and Fuller, M. T. (2001). Male germ-line stem cells. In Stem Cell Biology (ed. D. R. Marshak, R. L. Gardner and D. Gottlieb), pp. 149-188. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
King, F. J. and Lin, H. (1999). Somatic signaling mediated by fs(1)Yb is essential for germline stem cell maintenance during Drosophila oogenesis. Development 126,1833 -1844.[Abstract]
King, F. J., Szakmary, A., Cox, D. N. and Lin, H. (2001). Yb modulates the divisions of both germline and somatic stem cells through piwi- and hh-mediated mechanisms in the Drosophila ovary. Mol. Cell 7, 497-508.[CrossRef][Medline]
King, R. C. (1970). Ovarian Development in Drosophila melanogaster. New York: Academic Press.
Kretzschmar, M., Doody, J., Timokhina, I. and Massague, J.
(1999). A mechanism of repression of TGFbeta/Smad signaling by
oncogenic Ras. Genes Dev.
13,804
-816.
Lai, E. C. (2004). Notch signaling: control of
cell communication and cell fate. Development
131,965
-973.
Larkin, M. K., Holder, K., Yost, C., Giniger, E. and Ruohola-Baker, H. (1996). Expression of constitutively active Notch arrests follicle cells at a precursor stage during Drosophila oogenesis and disrupts the anterior-posterior axis of the oocyte. Development 122,3639 -2650.[Abstract]
Li, L. and Xie, T. (2005). Stem cell niche: structure and function. Annu. Rev. Cell Dev. Biol. 21,605 -631.[CrossRef][Medline]
Lin, H. and Spradling, A. C. (1993). Germline stem cell division and egg chamber development in transplanted Drosophila germaria. Dev. Biol. 159,140 -152.[CrossRef][Medline]
Margolis, J. and Spradling, A. (1995). Identification and behavior of epithelial stem cells in the Drosophila ovary. Development 121,3797 -3807.[Abstract]
Molofsky, A. V., Pardal, R. and Morrison, S. J. (2004). Diverse mechanisms regulate stem cell self-renewal. Curr. Opin. Cell Biol. 16,700 -707.[CrossRef][Medline]
Nishimura, E. K., Jordan, S. A., Oshima, H., Yoshida, H., Osawa, M., Moriyama, M., Jackson, I. J., Barrandon, Y., Miyachi, Y. and Nishikawa, S. (2002). Dominant role of the niche in melanocyte stem-cell fate determination. Nature 416,854 -860.[CrossRef][Medline]
Parks, A. L., Klueg, K. M., Stout, J. R. and Muskavitch, M. A. (2000). Ligand endocytosis drives receptor dissociation and activation in the Notch pathway. Development 127,1373 -1385.[Abstract]
Rodesch, C., Geyer, P. K., Patton, J. S., Bae, E. and Nagoshi, R. N. (1995). Developmental analysis of the ovarian tumor gene during Drosophila oogenesis. Genetics 141,191 -202.[Abstract]
Shcherbata, H. R., Althauser, C., Findley, S. D. and
Ruohola-Baker, H. (2004). The mitotic-to-endocycle switch in
Drosophila follicle cells is executed by Notch-dependent regulation
of G1/S, G2/M and M/G1 cell-cycle transitions.
Development 131,3169
-3181.
Song, X., Zhu, C. H., Doan, C. and Xie, T.
(2002). Germline stem cells anchored by adherens junctions in the
Drosophila ovary niches. Science
296,1855
-1857.
Song, X., Wong, M. D., Kawase, E., Xi, R., Ding, B. C.,
McCarthy, J. J. and Xie, T. (2004). Bmp signals from niche
cells directly repress transcription of a differentiation-promoting gene, bag
of marbles, in germline stem cells in the Drosophila ovary.
Development 131,1353
-1364.
Spradling, A., Drummond-Barbosa, D. and Kai, T. (2001). Stem cells find their niche. Nature 414,98 -104.[CrossRef][Medline]
Struhl, G., Fitzgerald, K. and Greenwald, I. (1993). Intrinsic activity of the Lin-12 and Notch intracellular domains in vivo. Cell 74,331 -345.[CrossRef][Medline]
Sun, J. and Deng, W. M. (2005). Notch-dependent
downregulation of the homeodomain gene cut is required for the mitotic
cycle/endocycle switch and cell differentiation in Drosophila
follicle cells. Development
132,4299
-4308.
Szakmary, A., Cox, D. N., Wang, Z. and Lin, H. (2005). Regulatory relationship among piwi, pumilio, and bag-of-marbles in Drosophila germline stem cell self-renewal and differentiation. Curr. Biol. 15,171 -178.[CrossRef][Medline]
Tumbar, T., Guasch, G., Greco, V., Blanpain, C., Lowry, W. E.,
Rendl, M. and Fuchs, E. (2004). Defining the epithelial stem
cell niche in skin. Science
303,359
-363.
Watt, F. M. and Hogan, B. L. (2000). Out of
Eden: stem cells and their niches. Science
287,1427
-1430.
Wieschaus, E. and Szabad, J. (1979). The development and function of the female germ line in Drosophila melanogaster: a cell lineage study. Dev. Biol. 68, 29-46.[CrossRef][Medline]
Xi, R., Kirilly, D. and Xie, T. (2005). Molecular mechanisms controlling germline and somatic stem cells: similarities and differences. Curr. Opin. Genet. Dev. 15,381 -387.[CrossRef][Medline]
Xie, T. and Spradling, A. C. (1998). decapentaplegic is essential for the maintenance and division of germline stem cells in the Drosophila ovary. Cell 94,251 -260.[CrossRef][Medline]
Xie, T. and Spradling, A. C. (2000). A niche maintaining germ line stem cells in the Drosophila ovary. Science 290,328 -330.
