1 Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO
64110, USA
2 Department of Anatomy and Cell Biology, University of Kansas School of
Medicine, 3901 Rainbow Blvd, Kansas City, KS 66160, USA
* Author for correspondence (e-mail: tgx{at}stowers-institute.org)
Accepted 3 April 2003
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SUMMARY |
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Key words: Somatic stem cells, Ovary, Drosophila, wingless
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INTRODUCTION |
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The Drosophila ovary is an excellent system in which to study two
different stem cell types that are responsible for egg production during adult
life (Lin, 1998;
Xie and Spradling, 2001
). The
adult ovary contains 12-16 ovarioles, each with a germarium at the tip, in
which GSCs and somatic stem cells (SSCs) are located
(Fig. 1A). Two or three GSCs,
surrounded by three groups of somatic cells at the germarial tip [terminal
filament cells (TFs), cap cells (CPCs) and inner germarial sheath (IGS) cells]
produce all germline cells in the ovariole. These stem cells directly contact
cap cells and are posterior to terminal filament cells. After a GSC divides,
the daughter still in contact with cap cells remains a stem cell, whereas the
daughter that is more distant from cap cells differentiates into a cystoblast.
However, if both daughters remain in contact with cap cells, they both become
stem cells (Xie and Spradling,
2000
). Consistent with the existence of niches, terminal filament,
cap cells and IGS cells express several genes that are important for GSC
function (Cox et al., 1998
;
King and Lin, 1999
;
Xie and Spradling, 1998
;
Xie and Spradling, 2000
). In
addition, the failure of GSCs to stay in their niches, because of defects in
DE-cadherin-mediated cell adhesion, results in stem cell loss
(Song et al., 2002
).
|
In mammals, increasing evidence shows that Wnt/ß-catenin signaling is
important for regulating epithelial cells
(Oshima et al., 2001;
Taylor et al., 2000
;
Watt, 2001
). Two populations
of stem cells are localized in the basal cell layer and the deep rete ridges.
Recent evidence indicates that long-lived stem cells reside apically within
the bulge region and are responsible for re-populating matrix stem cells and
replacing epidermal stem cells (Oshima et
al., 2001
; Taylor et al.,
2000
). The dermal papilla seems to be a key niche component,
producing signals that stimulate matrix stem cell activity. Signals such as
fibroblast growth factor (FGF)-7, ß1-integrin, bone morphogenetic protein
(BMP)-4 and shh have been shown to regulate different aspects of hair
follicle cell proliferation and differentiation
(Spradling et al., 2001
;
Watt and Hogan, 2000
).
Furthermore, increasing evidence shows that Wnt/ß-catenin signaling is
important for regulating epithelial cells in mammals. For example,
Wnt/ß-catenin signaling is required for hair follicle cells to form and
for matrix-derived cells to differentiate into follicular rather than
epidermal keratinocytes (DasGupta and
Fuchs, 1999
; Huelsken et al.,
2001
). Constitutive activation of Wnt signaling increases hair
follicle and skin tumor formation in mice and humans
(Chan et al., 1999
;
Gat et al., 1998
). Defective
maintenance of crypts in the small intestines of Tcf-4 knockout mice suggests
that Wnt signaling also regulates intestinal stem cell activity
(Korinek et al., 1998
).
In Drosophila, Wg signal is transduced by Frizzled receptors,
Frizzled (Fz) and Frizzled 2 (Fz2), resulting in the phosphorylation of the
cytoplasmic protein Dishevelled (Dsh), inhibition of Shaggy (Sgg) and Axin
(Axn), stabilization of Armadillo (Arm) and activation of target gene
expression (reviewed by Peifer and
Polakis, 2000). wg is involved in regulating many
different cellular processes during Drosophila development, and it is
transcribed in cells close to GSCs and SSCs, but its role in stem cell
regulation in the Drosophila has not been previously demonstrated
(Forbes et al., 1996b
). Here,
we report that wg signaling directly regulates SSC maintenance and
that constitutive wg signaling causes over-proliferation and improper
differentiation of their progeny in the Drosophila ovary.
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MATERIALS AND METHODS |
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Generating mutant somatic stem cell clones
Clones of mutant SSCs were generated by FLP-mediated mitotic recombination.
To generate the stocks for arm, dsh or sgg clonal analysis,
FRT18A sggM1-1/FM7, FRT18A
dshVA135/FM7, FRT18A
arm2/FM7, FRT18A arm8/FM7,
FRT18A arm3/FM7, FRT18A
arm4/FM7 and FRT18A + virgin females were mated
with males FRT18A armadillo-lacZ; hs-FLP,
respectively. To generate the stocks for Axn clonal analysis,
FRT82B AxnS044230/TM3 Sb and FRT82B
+ males were mated with virgin females yw hs-FLP; FRT82B
armadillo-lacZ, respectively. One- or two-day-old adult non-FM7 or
non-Sb females carrying an armadillo-lacZ transgene in trans to the
mutant-bearing chromosome were heat-shocked six times at 37°C for 1 hour
(8-12 hour intervals). The females were transferred to fresh food daily at
room temperature, and ovaries were removed from them one, two or three weeks
after the last heat-shock treatment and then further processed for antibody
staining. To determine stem cell maintenance, the percentages of the ovarioles
carrying a marked SSC clone at different time points were calculated by
dividing the number of germaria carrying marked follicles by the total number
of germaria examined.
Overexpression of wg downstream components in the ovary
To construct the stocks for over-expressing wg downstream
components in the ovary, the hs-Gal4 virgin females were crossed with
males carrying UAS-Fz2, UAS-dsh and UAS-armS10.
The females that did not carry balancer chromosomes were heat-shocked eight
times at 37°C for 1 hour (8-12 hour intervals for 4 days). The females
were further cultured at room temperature for 1 day before their ovaries were
isolated for analysis.
Immunohistochemistry
The following antisera were used: monoclonal anti-Hts antibody 1B1 (1:4),
monoclonal anti-Fas3 antibody 7G10 (1:4), monoclonal anti-Wg antibody 4D4
(1:2), rabbit polyclonal anti-ß-galactosidase (1:150; Molecular Probes)
and rabbit poly anti-Vasa (1:1000) antibodies.
To detect secreted Wg protein, we followed the published procedures
(Strigini and Cohen, 2000). To
detect other proteins, ovaries were processed according to the published
procedures (Song et al.,
2002
). All micrographs were taken using a Leica SPII confocal
microscope.
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RESULTS |
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To directly determine whether wg regulates somatic follicle cell
production, we studied the mutant wg ovary using a
temperature-sensitive allele wgts. The
wgts flies can survive to adulthood at 18°C
(Bejsovec and Martinez Arias,
1991), which allows us to examine the role of wg in the
adult ovary. One week after the shift to a restrictive temperature (30°C),
the ovaries were immunostained with an anti-Huli tai shao (Hts) antibody. The
Hts protein is present not only on spectrosomes in GSCs, cystoblasts and
fusomes in early germline cysts, but also on the membranes of somatic follicle
cells (Lin et al., 1994
). Both
wgts and wild-type females were raised under the same
conditions (incubation at the restrictive temperature, 30°C, for one
week). The wgts mutant germarium carried many more
germline cysts (20.0±3.8; n=40) in comparison with the
wild-type one (7.9±1.2; n=49)
(Fig. 1C,D). Sometimes, in the
wgts mutant germarium, two 16-cell cysts were packed into
an egg chamber because of over-crowded germline cysts or defects in follicle
cells (Fig. 1E). These swollen
germaria could result from over-production of germline cysts, reduction of
somatic follicle cell production or defects in follicle cell differentiation.
wg signaling does not seem to directly regulate the maintenance or
division of GSCs (Song et al.,
2002
) (data not shown). This raises the possibility that
wg could regulate proliferation and differentiation of follicle
cells.
To further determine whether wg signaling is capable of regulating
follicle cell production, we over-expressed Fz2, dsh and activated
arm in the adult ovary using transgenes UAS-Fz2, UAS-dsh and
UAS-armS10. These transgenes have been used to increase
wg signaling by increasing the amount of downstream components using
the GAL4-driven UAS target gene expression system
(Brand and Perrimon, 1993;
Axelrod et al., 1998
;
Cadigan et al., 1998
;
Pai et al., 1997
). The
hs-gal4 transgene allows us to induce transcription of target genes
in the adult Drosophila ovary by heat-shock treatments. Normally, a
row of 5-7 stalk cells link two adjacent egg chambers
(Fig. 2A). After four days of
pulsed heat-shock treatment, over-expression of Fz2, dsh and the
activated arm consistently produced more follicle cells that
accumulated between egg chambers (Fig.
2B-D). These extra follicle cells also formed long stalks with
multiple rows of cells instead of one row in wild type. These follicle cell
over-proliferation phenotypes are similar to, but weaker than, those caused by
increasing hh and Notch signaling activities
(Forbes et al., 1996b
;
Larkin et al., 1996
;
Zhang and Kalderon, 2000
).
These results raise the possibility that wg signaling modulates
somatic follicle cell production in the adult Drosophila ovary.
|
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Unlike dsh and arm, Axn and sgg are downstream
negative regulators of wg signaling, and removal of their function
from SSCs can cause constitutive wg signaling.
AxnS044230 is a transposon-induced loss-of-function allele
in which a P-element is inserted in the first exon, 135bp downstream of the
transcription start site (Hamada et al.,
1999); sggM1-1 is a strong loss-of-function
allele (Heslip et al., 1997
).
Surprisingly, mutant sgg and Axn SSC clones were also lost
much faster than the marked wild-type SSC clones. Three weeks after clone
induction, 98% of mutant AxnS044230 and
sggM1-1 SSC clones were lost, in contrast to the 44% loss
of marked wild-type control clones (Table
1). These results indicate that constitutive wg signaling
also shortens the SSC lifespan.
In the germaria carrying both wild-type and mutant SSCs, marked (lacZ-negative) mutant Axn or sgg, follicle cells appeared more in number than wild-type (lacZ-positive) ones (Fig. 3E), indicating that mutant Axn or sgg SSCs and/or follicle progenitor cells are over-proliferative. In other germaria carrying only lacZ-negative mutant sgg or Axn follicle cells (Fig. 3F), which is because of the natural loss of lacZ-positive wild-type SSCs, more mutant follicle cells accumulated. Interestingly, mutant sgg or Axn follicle cells appeared to express higher levels of Hts than wild-type follicle cells. In the germaria, where most or all follicle cells were mutant for sgg or Axn, germline cysts could not undergo normal morphological changes from ball-like structures in region 2a to lens-shape cysts in region 2b, and thus still remained in ball-like structures in region 2b (Fig. 3E,F). These results indicate that constitutive wg signaling can modulate proliferation and differentiation of SSCs and/or follicle cell progenitors.
sgg and Axn mutant follicle cells over-proliferate
and accumulate between egg chambers
To further investigate whether wg signaling regulates follicle
cell proliferation, we examined mutant dsh, arm, sgg and Axn
follicle cell clone size. During follicle epithelial development, a marked
early progenitor proliferates and generates a population of follicle cells
that stay together throughout development. Thus, the sizes of follicle cell
clones mutant for a particular gene can be used to determine whether its
function is required for the proliferation of follicle cells. Follicle cell
clones mutant for either arm or dsh formed big patches
similar to those formed by marked wild-type clones
(Fig. 4A-C). The mutant
arm2 and arm8 follicle cell clones had
normal morphology in comparison with neighboring wild-type follicle cells
(Fig. 4A). The mutant
arm4 or arm3 follicle cells lost
wild-type regular columnar shape, and there was abnormal accumulation of Hts
proteins on apical membranes instead of on lateral membranes in wild type
(Fig. 4B). These results
indicate that DE-cadherin-mediated cell adhesion, but not wg
signaling, is very important for maintaining the integrity of follicle cell
epithelia. Consistent with the idea that wg signaling does not affect
morphology of follicle cells, mutant dshVA135 follicle
cells had normal morphology and expression of Hts in their apical membranes
and normal sizes of mutant dsh or arm clones in the
germarium (Fig. 4C). To further
determine whether dsh is required for proliferation of follicle cells
in egg chambers, we generated twin clones derived from the same follicle cell
during the follicle cell development. Because the twin clones were produced at
the same time, sizes of homozygous mutant clones and corresponding wild-type
twin clones could be used to demonstrate the requirement of a particular gene
in cell proliferation. dsh mutant clones and their corresponding
wild-type twin clones had similar sizes
(Fig. 4D), supporting that
wg signaling does not regulate the proliferation of follicle cells in
egg chambers. These results demonstrate that wg signaling is not
essential for the proliferation of follicle cells in egg chambers.
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Follicle cell clones mutant for patched (ptc) or
protein kinase A (pka), two negative regulators of the
hh pathway, have similar mutant phenotypes to those of mutant
Axn or sgg follicle cells in the Drosophila ovary
(Forbes et al., 1996a;
Zhang and Kalderon, 2001
).
Some mutant ptc or pka follicle cells can adopt polar cell
fates. Normally, one pair of polar cells situates at each end of an egg
chamber and expresses high levels of Fas3. Polar cells play an important role
in regulating egg chamber budding and generating different follicle cell types
by expressing important signaling molecules such as unpaired
(Xi et al., 2003
). To
investigate whether mutant Axn or sgg follicle cells in egg
chambers can adopt polar cell fates, we examined Fas3 expression in mutant
Axn or sgg follicle clones. Mutant Axn or
sgg follicle cells expressed higher levels of Fas3 than wild-type
ones in the germaria and stage 2 chambers
(Fig. 6E,F). Extra mutant
Axn or sgg follicle cells that accumulated between egg
chambers also continuously expressed high levels of Fas3
(Fig. 6G,H). In this respect,
the mutant follicle cells do not behave like stalk cells because stalk cells
do not normally express high levels of Fas3. However, mutant Axn or
sgg follicle cells that were integrated into late egg chambers did
not express high levels of Fas3, similar to their neighboring wild-type
follicle cells (Fig. 6H-J).
Only two pairs of polar cells located at both ends of an egg chamber
continuously expressed high levels of Fas3
(Fig. 6I,J). Interestingly, in
some cases a few mutant follicle cells adjacent to wild-type polar cells
expressed slightly higher levels of Fas3 than wild-type follicle cells
neighboring polar cells (Fig.
6I), suggesting that mutant Axn follicle cells on egg
chambers also do not differentiate normally. All these results indicate that
mutant Axn or sgg follicle cells have differentiation
defects.
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DISCUSSION |
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Similarly, constitutively active ß-catenin in mice and humans disrupts
hair follicle differentiation, resulting in the formation of skin cancers
(DasGupta and Fuchs, 1999;
Huelsken et al., 2001
). No
Wnt-like molecules have been directly implicated in hair follicle stem cell
regulation. Because several aspects of epithelial cell regulation have been
conserved from Drosophila to humans
(Jordan et al., 2000
;
Zhang and Kalderon, 2001
), our
study suggests that the ability of Wnt-like signaling to regulate maintenance
of epithelial stem cells and the differentiation of their progeny may be
conserved.
wg signaling regulates the maintenance of somatic stem
cells
Wg produced from terminal filament and cap cells may reach SSCs at a
distance of a few cells by either diffusion or active transport, and then
directly controls SSC maintenance. Furthermore, correct intermediate levels of
wg signaling seem to be important for maintaining SSCs in the
Drosophila ovary. We show that reduction of wg signaling in
SSCs by removal of positive regulators such as arm and dsh
causes rapid SSC loss, as does constitutive wg signaling in SSCs by
removal of negative regulators such as Axn and sgg. wg
signaling maintains SSCs through several possible mechanisms. First,
wg signaling could be required for SSC self-renewal and/or survival.
Second, it could maintain the association of SSCs with IGS cells. Finally,
both mechanisms could work simultaneously. DE-cadherin-mediated cell adhesion
has been shown to be important for keeping SSCs in their niche
(Song and Xie, 2002), and also
shares arm as a common component with wg signaling
(Peifer and Polakis, 2000
).
wg signaling is known to regulate levels of arm, which are
also important for DE-cadherin-mediated cell adhesion. Thus, it is possible
that wg signaling regulates cell adhesion between SSCs and their
niches. In addition, our arm mutant clonal analysis strongly argues
that wg signaling must also directly regulate SSC self-renewal and/or
survival. arm2 mutant SSC clones are lost very quickly
over time in comparison with wild-type SSC clones, and the
arm2 mutation primarily affects wg signaling but
does not disrupt DE-cadherin-mediated cell adhesion. Therefore, wg
signaling controls SSC maintenance through regulating SSC
self-renewal/survival and/or cell adhesion between SSCs and their niche cells.
The temperature-sensitive allele of wg gives very mild phenotypes in
follicle cell production, however, removal of wg downstream
components has a dramatic impact on SSC maintenance. In Drosophila,
there are six other wg-related genes. This raises an interesting
possibility that other wg-like molecules could also be involved in
regulating SSC maintenance.
In addition to wg signaling, hh signaling is also
essential for SSC maintenance and proliferation. Hyperactive hh
signaling causes follicle cell over-proliferation and abnormal differentiation
of follicle cells (Forbes et al.,
1996a; Zhang and Kalderon,
2000
). Disrupting hh signaling in SSCs by removing the
function of hh downstream components such as smoothen and
Cubitus interruptus results in rapid SSC loss
(Zhang and Kalderon, 2001
).
Similarly, reduction or elimination of wg signaling also causes rapid
SSC loss. Removal of patched, a negative regulator of the hh
pathway, stabilizes SSCs (Zhang and
Kalderon, 2001
). However, SSCs mutant for negative regulators for
the wg pathway, sgg and Axn, are destabilized. All
the evidence indicates that wg and hh may use different
mechanisms to regulate SSCs in the Drosophila ovary.
Constitutive wg signaling causes over-proliferation and
abnormal differentiation of follicle cells
Constitutive wg signaling increases the division rates of early
follicle cell progenitors in the germarium. When Fz2, dsh and
activated arm are over-expressed, extra follicle cells accumulate in
the ovarioles, suggesting that hyper-activation of wg signaling
causes over-proliferation of follicle cells. Furthermore, sgg or
Axn mutations cause over-proliferation of follicle cells, resulting
in the formation of extra follicle cells that accumulate outside egg chambers.
These cells are not mitotically active and usually assume some stalk cell
characteristics. These results suggest that production of extra follicle cells
by excessive wg signaling is because of higher mitotic activities of
progenitors and/or SSCs in the germarium. It is important to note that
sgg mutations are more potent than Axn in stimulating the
proliferation of follicle cell progenitors. The different potencies may be
because of differences in how these mutations affect wg signaling.
Alternatively, because sgg negatively regulates hh signaling
(Jia et al., 2002),
sgg could be involved in negatively regulating both hh and
wg signaling in the ovary. It has been demonstrated that excessive
hh signaling causes extra follicle cells to accumulate outside egg
chambers (Forbes et al.,
1996a
; King et al.,
2001
; Zhang and Kalderon,
2001
). Therefore, it might be probable that sgg is
involved in regulating both hh and wg signaling pathways in
follicle cells of the Drosophila ovary.
This study also demonstrates that constitutive wg signaling disrupts the normal differentiation of somatic follicle cells. Mutant Axn or sgg follicle cells in and outside the germarium express higher levels of Hts in their membranes and tend to accumulate between egg chambers. In ovarioles that contain a majority of mutant follicle cells, germline cysts fail to undergo normal morphological changes necessary for proper encapsulation by follicle cells, although they are wild type, suggesting that the mutant follicle cells are defective in their interactions with germ cells. Although some of them are recruited to egg chambers, these mutant follicle cells have abnormal morphologies (e.g. smaller and irregular sizes). This phenotype may be because of abnormal levels of Hts, which may prevent follicle cells from shape changes and growth. The extra mutant follicle cells accumulating outside egg chambers express Lamin C and do not divide similar to stalk cells. However, unlike stalk cells, they express high levels of Fas3. Similar to the mutant follicle cells in the germarium, the mutant follicle cells that are recruited to egg chambers also express high levels of Hts. Unlike the follicle cells in the germarium, the cells fail to express high levels of Fas3. These results indicate that constitutive wg signaling in follicle cells disrupts proper follicle cell differentiation.
Follicle cells in the Drosophila ovary could potentially be
a great model to study epithelial stem cells and skin cancers
In mice and humans, excessive hh signaling disrupts the normal
differentiation processes of hair follicles, resulting in the formation of
skin cancers (Johnson et al.,
1996; Oro et al.,
1997
). In the Drosophila ovary, excessive hh
signaling disrupts the normal differentiation of somatic follicle cells and
produces extra follicle cells (Forbes et
al., 1996a
; King et al.,
2001
; Zhang and Kalderon,
2000
). hh also directly regulates the maintenance and
number of SSCs in the Drosophila ovary
(Zhang and Kalderon, 2001
). In
humans and mice, constitutively active ß-catenin also disrupts hair
follicle cell differentiation, causing skin cancer formation
(Chan et al., 1999
;
Gat et al., 1998
;
Zhu and Watt, 1999
). Here we
show that wg signaling directly controls SSC maintenance, whereas
constitutive wg signaling disrupts somatic follicle cell
differentiation, resulting in extra follicle cell production and abnormal
morphological changes. Notch signaling has been implicated in regulating
somatic follicle cell differentiation in the Drosophila ovary and
epithelial cells in the mouse skin (Jordan
et al., 2000
; Lowell et al.,
2000
). All of the evidence suggests that mechanisms regulating the
maintenance of epithelial stem cells and differentiation of their progeny
could be conserved from Drosophila to human. Therefore, the
Drosophila ovarian follicle epithelial cells represent a model system
in which to study epithelial stem cell regulation, and possibly skin cancer
formation in humans by identifying major signaling pathways and their
targets.
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ACKNOWLEDGMENTS |
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