DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 5087
Development 138, 5087-5097 (2011) doi:10.1242/dev.067850
© 2011. Published by The Company of Biologists Ltd
Self-maintained escort cells form a germline stem cell
differentiation niche
Daniel Kirilly*,‡, Su Wang* and Ting Xie§
SUMMARY
Stem cell self-renewal is controlled by concerted actions of niche signals and intrinsic factors in a variety of systems. In the
Drosophila ovary, germline stem cells (GSCs) in the niche continuously self-renew and generate differentiated germ cells that
interact physically with escort cells (ECs). It has been proposed that escort stem cells (ESCs), which directly contact GSCs, generate
differentiated ECs to maintain the EC population. However, it remains unclear whether the differentiation status of germ cells
affects EC behavior and how the interaction between ECs and germ cells is regulated. In this study, we have found that ECs can
undergo slow cell turnover regardless of their positions, and the lost cells are replenished by their neighboring ECs via selfduplication rather than via stem cells. ECs extend elaborate cellular processes that exhibit extensive interactions with
differentiated germ cells. Interestingly, long cellular processes of ECs are absent when GSC progeny fail to differentiate,
suggesting that differentiated germ cells are required for the formation or maintenance of EC cellular processes. Disruption of
Rho functions leads to the disruption of long EC cellular processes and the accumulation of ill-differentiated single germ cells by
increasing BMP signaling activity outside the GSC niche, and also causes gradual EC loss. Therefore, our findings indicate that ECs
interact extensively with differentiated germ cells through their elaborate cellular processes and control proper germ cell
differentiation. Here, we propose that ECs form a niche that controls GSC lineage differentiation and is maintained by a nonstem cell mechanism.
INTRODUCTION
Adult tissues are often maintained by a population of tissue-specific
stem cells, which have the capacity to self-renew and generate
differentiated cells throughout the organism’s lifetime. Many fast
turnover tissues, such as blood, skin, intestine and testis, are known
to be maintained by stem cells (Li and Xie, 2005; Morrison and
Spradling, 2008). Some slow turnover tissues are either maintained
by stem cells as in the brain or by self-duplication as for insulinsecreting  cells in the pancreas (Dor et al., 2004; Zhao et al.,
2008). These adult stem cells are maintained in the niche for longterm self-renewal (Li and Xie, 2005; Morrison and Spradling,
2008). However, it remains unclear whether stem cell lineage
differentiation is also controlled by a niche mechanism.
In the Drosophila ovary, two or three germline stem cells
(GSCs) are anchored physically to their niche, which is composed
of five to seven cap cells, through E-cadherin-mediated cell
adhesion at the tip of the germarium (Song et al., 2002). Recently,
a population of escort stem cells (ESCs), which directly contacts
GSCs and cap cells, has been proposed to generate the
differentiated escort cells (ECs) that accompany differentiated germ
cells to the middle region of the germarium where ECs undergo
apoptosis (Decotto and Spradling, 2005). The germ cells released
from ECs are subsequently surrounded by follicle cells, which are
Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO
64110, USA and Department of Cell Biology and Anatomy, University of Kansas
Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA.
*These authors contributed equally to this work
‡
Present address: Temasek Life Sciences Laboratory, 1 Research Link, National
University of Singapore, Singapore 117604
§
Author for correspondence ([email protected])
Accepted 30 September 2011
produced by two follicular stem cells (FSCs), to form individual
egg chambers (Margolis and Spradling, 1995; Song and Xie, 2002).
Therefore, ESCs have been proposed to behave similarly to cyst
progenitor cells in the Drosophila testis, which produce
differentiated somatic cells that wrap around differentiated germ
cells to support their differentiation (Gonczy et al., 1992; Kiger et
al., 2000; Tran et al., 2000; Schultz et al., 2002; Decotto and
Spradling, 2005). A previous study has shown that stem cell tumor
(stet) encodes a germ cell-specific protein related to Rhomboid,
which is required for the formation or maintenance of the long EC
cellular processes and germ cell differentiation (Schultz et al.,
2002). The Rhomboid family of transmembrane proteases is
involved in processing EGF (epidermal growth factor) ligands
(Urban et al., 2001). Indeed, another recent study showed that three
EGF ligands function in germ cells to activate EGF receptor
(EGFR) signaling in ECs, which in turn represses Dally expression
(Liu et al., 2010). Dally is a proteoglycan protein that binds bone
morphogenetic protein (BMP) and facilitates its signaling (Jackson
et al., 1997; Belenkaya et al., 2004; Akiyama et al., 2008). In
addition, Dally is also important for restricting BMPs inside the
GSC niche in the Drosophila ovary (Guo and Wang, 2009; Hayashi
et al., 2009). These findings suggest a model in which
differentiated germ cells activate EGFR signaling in ECs, which
prevents BMP diffusion from the GSC niche to ECs and thus
promotes germ cell differentiation. However, it remains unclear
how EC cellular process-mediated interactions between ECs and
germ cells are regulated. In this study, we show that ECs are
maintained by self-duplication instead of by ESCs. Furthermore,
EC cellular processes are dependent on differentiated germ cells,
and the physical interactions between ECs and germ cells are
essential for germ cell differentiation. Therefore, we propose that
self-maintained ECs form a niche that controls germ cell
differentiation.
DEVELOPMENT
KEY WORDS: Drosophila, Germline stem cell, Niche, Differentiation, Escort cell
MATERIALS AND METHODS
Drosophila strains and culture
Information about the Drosophila stocks used in this study is either
available from Flybase (http://www.flybase.org) or specified here: X-15-29
(Harrison and Perrimon, 1993), X-15-33 (Harrison and Perrimon, 1993),
UAS-mEGFP (a membrane-tethered GFP generated by a fusion of the Src
membrane signal peptide with GFP) (Kirilly et al., 2005), FRT52B (Kirilly
et al., 2005), UAS-EGFP, FRT19A, tubulin-gal80 (Lee et al., 2000), tubulingal4 (Lee et al., 2000), c587 (Song et al., 2004), hsFLP, UAS-RhoDN (a
dominant-negative Rho) (Strutt et al., 1997), PZ1444 (Margolis and
Spradling, 1995), UAS-dsRed, UAS-dcr2, UAS-dallyRNAi lines (on
chromosome 2 and 3; kindly provided by Dr Xinhua Lin, Cincinnati
Children’s Hospital Medical Center, Cincinnati, USA), UAS-dppRNAi lines
(TR00047A: 47A; TR00047R: 47R; kindly provided by Dr Norbert
Perrimon, Harvard Medical School, Boston, USA), UAS-capuRNAi
(VDRC#110404), dpphr4 and dpphr56. Flies were maintained and crossed at
room temperature on standard cornmeal/molasses/agar media unless
specified. For maximizing the effect of RNAi-mediated knockdown or
gene overexpression, newly eclosed flies were shifted to 29°C for a week
before the analysis of ovarian phenotypes.
Generating positively labeled EC clones
Three different positive marking systems were used for labeling ECs in the
Drosophila ovary: the lacZ-based positive labeling system (Harrison and
Perrimon, 1993), the UAS-GAL4-based positively marked mosaic lineage
(PMML) system (Kirilly et al., 2005) and the GAL80-based mosaic
analysis with a repressible cell marker (MARCM) system (Lee et al.,
2000). We generated mitotic clones according to our previously published
procedures for labeling the FSC lineage (Kirilly et al., 2005). One- to threeday-old females with genotypes hs-FLP; X-15-33/X-15-29 and hs-FLP
UAS-mEGFP; FRT52B/FRT52B UAS-EGFP and hs-FLP FRT19A gal80/
FRT19A; tub-gal4 UAS-GFP were subjected to incubation in a water bath
at 37°C for 30 or 60 minutes to induce FLP expression and FRT-mediated
mitotic recombination. Flies were transferred daily to the fresh yeastcontaining food, and the marked EC clones were detected one week, two
weeks and three weeks after the heatshock treatment.
BrdU labeling and retention assays
Two different types of BrdU incorporation assays were utilized to
investigate the proliferation patterns of ECs: two-hour BrdU labeling and
BrdU retention. For the two-hour BrdU labeling, the ovaries were
incubated in Grace’s medium containing 75 mM BrdU for 2 hours at 25°C.
For the BrdU retention assay, female flies were fed on food with yeast
paste containing BrdU (10 mg/ml) for three consecutive days, and then on
food with BrdU-free yeast paste for 14 days. The ovaries from these two
types of assays were then fixed and processed for BrdU label detection
along with other protein markers according to our previously published
procedures (Xie and Spradling, 1998).
Immunohistochemistry
Antibody staining was performed according to our previously published
procedures (Xie and Spradling, 1998; Song and Xie, 2002). The following
antibodies were used in this study: rabbit polyclonal anti--galactosidase
antibody (1:100, Cappel), rabbit polyclonal anti-GFP antibody (1:200,
Molecular Probes), mouse monoclonal anti-Fasciclin3 antibody [1:3,
Developmental Studies Hybridoma Bank (DSHB)], mouse monoclonal
anti-Hts antibody (1:3, DSHB), rat monoclonal anti-Vasa antibody (1:3,
DSHB) and monoclonal anti-BrdU antibody (1:20, Oncogene). All images
were taken with either a Leica TCS SP2 or a Leica TCS SP5 confocal
microscope.
RESULTS
ECs in different regions of the germarium
undergo slow cell turnover and proliferation
The ESC model predicts that only ESCs at the tip of the germarium
are mitotically active and that ECs undergo apoptosis at the 2a/2b
junction (Decotto and Spradling, 2005). To investigate whether
ECs die only at the 2a/2b junction, we used Apoptag labeling to
Development 138 (23)
detect apoptotic ECs. c587 is a GAL4 line that drives UAS-GFP
expression in ESCs, ECs and early follicle cell progenitors (Song
et al., 2004) (Fig. 1A). PZ1444 is a lacZ enhancer trap line that is
expressed in cap cells and ECs (Margolis and Spradling, 1995; Xie
and Spradling, 2000) (Fig. 1B). lacZ-positive ECs can be easily
identified in the germarium in which germ cells are labeled with
vasa-GFP (Sano et al., 2002) (Fig. 1B). Here, we used PZ1444,
c587-driven UAS-dsRed and vasa-GFP to assist in the
identification of ECs. One or more dying EC is present in the 2a/2b
junction area of 14.7% of the 8-day-old germaria (n116), whereas
only 4.3% of those harbor one or more dying ECs in region 1 or 2a
(Fig. 1C-F). These results indicate that ECs at different positions
can undergo slow turnover but with the highest turnover rate for
the most posterior ECs.
To investigate whether those lost ECs in regions 1 and 2a are
repopulated by their neighboring ECs, we performed two-hour
BrdU labeling of PZ1444 ovaries in vitro to detect proliferative
ECs. Following two hours of BrdU incorporation, 9.6% of the
germaria contain at least one BrdU-positive EC at the 2a/2b
boundary, and only 0.8% of them contain a BrdU-positive EC in the
region 1 or 2a (n125) (Fig. 1G). This result indicates that ECs are
slow cycling cells and that the ones at the 2a/2b boundary proliferate
faster than those in more anterior regions. To investigate further the
slow cycling property, we also performed the BrdU retention assay
by feeding PZ1444 or vasa-GFP flies for 3 days with BrdUcontaining food and then 15 days with BrdU-free food. In the vasaGFP germarium, ECs are reliably identified in the periphery of the
anterior half of the germarium by absence of GFP expression,
whereas ECs in the PZ1444 germarium are readily identified by
lacZ-expression (Fig. 1B). After 3 days of BrdU feeding, 40% the
GSCs in the PZ1444 germaria (n155 germaria) and 48.9% of the
GSCs in the vasa-GFP germaria become BrdU-positive because of
their fast proliferative nature (Fig. 1H,I). For ECs, only a small
number of them in either a PZ1444 germarium or a vasa-GFP
germarium are BrdU-positive, and most of those BrdU-positive ECs
are localized in the 2a/2b junction (Fig. 1H,I). After 15 days of
chase, no GSCs in either PZ1444 (n212 germaria) or vasa-GFP
(n154 germaria) germaria retain the BrdU label, indicating that fast
cycling GSCs and FSCs do not retain BrdU label (Fig. 1J-K). This
result is consistent with the notion that not all adult stem cells retain
BrdU label as is proposed for some adult stem cell types in
mammalian systems (Morrison and Spradling, 2008). For the
PZ1444 germaria, 34.3% of them harbor one or more BrdU-labeled
ECs in the 2a/2b junction area, and only 13.8% of them have one or
more BrdU-positive ECs in region 1 or 2a (n137; Fig. 1J). The
vasa-GFP germaria exhibit a similar BrdU labeling pattern among
the ECs (Fig. 1K,L). The previously defined ESCs are rarely BrdUpositive (Fig. 1L), suggesting that ESCs are unlikely to be
responsible for generating ECs. These results show that ECs
proliferate infrequently owing to slow cell turnover and most of the
proliferative cells are restricted to the 2a/2b junction area, which is
consistent with the EC turnover pattern.
ECs are not maintained by ESCs
In the previous study, the lacZ-based positive labeling system was
used to show that the most anterior ECs, which directly contact cap
cells and GSCs, were proposed to function as ESCs to generate
differentiated ECs (Decotto and Spradling, 2005). In this system,
the FLP-induced FRT mitotic recombination leads to the fusion
between the -galactosidase (lacZ) gene and the tubulin (tub)
promoter and, thereby, the reconstitution of tub-lacZ in mitotic
cells, and tub-lacZ expression further positively and permanently
DEVELOPMENT
5088 RESEARCH ARTICLE
Roles of escort cells
RESEARCH ARTICLE 5089
marks mitotic cells and their progeny (Harrison and Perrimon,
1993). This system has been used to label GSCs, FSCs, ESCs and
their progeny (Margolis and Spradling, 1995; Decotto and
Spradling, 2005). In the current study, to ensure that lacZ-labeled
somatic cells that surround differentiated germ cells are indeed
ECs, we focused on the germaria only carrying lacZ-labeled ESCs
or ECs and disregarded other germaria carrying lacZ-positive FSC
or GSC clones. To verify whether ESCs are indeed responsible for
producing ECs, we used the same system to repeat the lacZ-based
EC lineage tracing experiments using half-an-hour and one-hour
heatshock treatments. These germaria are also labeled for Fasciclin
3 (Fas3), which is expressed in follicle cell progenitors but not in
ECs (Zhang and Kalderon, 2001). As the previous study reported
(Decotto and Spradling, 2005), both heatshock regimes can
efficiently induce lacZ-labeled ESCs and ECs although the onehour treatment generates higher lacZ-labeling efficiencies than the
half-an-hour treatment (Fig. 2A,A⬘). For example, one week after
clone induction (ACI), 34.5% and 56.2% of the germaria carry at
least one lacZ-marked ESC after the half-an-hour and one-hour
heatshock treatments, respectively (Fig. 2A). These lacZ-labeled
ESCs are relatively stable three weeks ACI, which is consistent
with the prediction of the ESC model. In addition, 82.4% of the
germaria carrying one or more lacZ-marked ESCs also have one or
more lacZ-positive ECs for the half-an-hour heatshock treatment,
and 99.1% of the germaria carrying one or more lacZ-marked
ESCs have one or more lacZ-positive ECs for the one-hour
heatshock treatment (Fig. 2B). These observations could explain
why the previous study concludes that ECs are produced by ESCs
(Decotto and Spradling, 2005) (Fig. 2A,B). However, careful
analyses of the lacZ-marked ECs have revealed the serious inherent
problem of the lacZ labeling system. First, the percentages of
germaria carrying only marked ECs but not ESCs, which are
induced by either of the heatshock treatments, remain constant one
week, two weeks and three weeks ACI (Fig. 2A⬘,C). This result
indicates that lacZ-labeled ECs can be stably maintained in the
absence of lacZ-labeled ESCs. Second, some germaria carrying one
lacZ-labeled ESC do not contain any lacZ-positive ECs
(supplementary material Fig. S1A), suggesting that they might not
be responsible for EC production. Third, high lacZ labeling rates
for ESCs and ECs are not supported by our BrdU labeling results.
This system has recently been shown to generate lacZ-positive cells
even in mitotically inactive Drosophila polyploid intestinal cells
(Fox and Spradling, 2009). Our results also suggest that this system
can reconstitute the tub-lacZ gene in an FLP-dependent but
division-independent manner in ECs, and, therefore, is not a
suitable system for determining the presence of ESCs.
To investigate further whether ESCs are responsible for producing
ECs, we used two other FLP-FRT-mediated positive lineage labeling
systems, positively marked mosaic lineage (PMML) and mosaic
analysis with a repressible cell marker (MARCM), to generate
positive GFP-marked EC clones and study their behavior. In the
PMML system, FLP-induced FRT recombination can randomly
generate the fusion gene between the tubulin promoter and the yeast
gal4 gene, which can drive UAS-GFP expression in mitotic cells
DEVELOPMENT
Fig. 1. ECs at different positions can undergo apoptosis and cell proliferation. (A)c587 drives UAS-GFP expression in ESCs and ECs in
regions 1 and 2a, and also in early follicle cells (FCs) in region 2b. GSCs (dashed lines) and differentiated germ cells, including 16-cell cysts, can be
identified by presence of a spectrosome (SS) and branched fusome (FS), respectively. (B)PZ1444 can also label ESCs (arrow), ECs (arrowheads) and
cap cells (oval) in the germarium in which germ cells are labeled by Vasa-GFP. (C-F)ApopTag-positive ECs are found in region 2a (C,D, arrows) and
the 2a/2b boundary (E,F, arrows). (G)An EC at the 2a/2b boundary is positive for BrdU after two hours of BrdU incorporation. (H,I)GSCs (white
arrowheads), mitotic cysts and follicle cells in the germarium are positive for BrdU (green) one day after three days of BrdU feeding. Most of the ECs
(black arrowheads) are negative for BrdU, but some ECs (arrows) are BrdU-positive. (J-L)After two weeks of chase following three days of BrdU
feeding, all the GSCs and mitotic cysts are BrdU-negative, but some ECs (arrows) remain positive for BrdU. ESCs are normally negative for BrdU
(black arrowhead, K), but are rarely found to be positive (black arrowhead, L). Scale bars: 10mm in B-L.
5090 RESEARCH ARTICLE
Development 138 (23)
(Kirilly et al., 2005). In the MARCM system, the FLP-induced FRT
recombination can generate cells that lose the expression of tubgal80, a transcriptional repressor for GAL4, and consequently allow
actin-gal4 to drive UAS-GFP expression in mitotic cells (Lee et al.,
2000). In contrast with the lacZ-based labeling system, the two GFPbased labeling systems do not produce GFP-labeled GSCs and
mitotic germ cell cysts owing to the inability of the GAL4-UAS
system to be expressed in GSCs and early mitotic cysts. To ensure
that GFP-labeled somatic cells that surround differentiated germ cells
are indeed ECs, we focused on the germaria carrying only ESC and
ECs by ignoring the germaria carrying a FSC clone. Interestingly, the
MARCM and PMML systems produce much lower percentages of
GFP-positive ESCs than the lacZ-based system (Fig. 2D;
supplementary material Fig. S1B). For the PMML system, the halfan-hour and one-hour heatshock treatments can label one ESC with
GFP in 0.9% (n1482) and 2.5% (n1456) of the germaria,
respectively (the data from three time points are pooled together; Fig.
2D). These GFP-labeled ESCs are stably maintained in the germaria
with time. There are similar observations using the MARCM system
(supplementary material Fig. S1B). These results support the
findings from BrdU-labeling experiments that ESCs proliferate
rarely. In addition, among the total 76 GFP-positive ESCs examined
(produced by either MARCM or PMML), 66 do not have any GFPpositive ECs in the same germaria, indicating that these ESCs are not
responsible for producing ECs (Fig. 2E; supplementary material Fig.
S1D). All of these observations do not support the previously
established ESC model (Decotto and Spradling, 2005).
The MARCM and PMML systems also produce much lower
percentages of GFP-positive ECs than the lacZ-based system (Fig.
2D⬘; supplementary material Fig. S1C). Following the half-an-hour
and one-hour heatshock treatments, the PMML system can label at
least one EC with GFP in the absence of marked ESC labeling in
10.6% and 19.7% of the germaria one week ACI, respectively (Fig.
2D⬘,F,G). Although those germaria do not have any GFP-labeled
ESCs, the GFP-labeled ECs are stably maintained three weeks
ACI. Similar findings have been made for the MARCM-labeled
ECs (supplementary material Fig. S1C). These results indicate that
GFP-labeled ECs are not produced by GFP-labeled ESCs. Along
with the earlier results from the BrdU and TUNEL labeling
experiments, our findings strongly argue against the idea that the
previously defined ESCs are responsible for generating ECs, and
instead support the new model in which ECs undergo slow
turnover and lost ECs can be replenished by their proliferative
neighboring ECs. Recently, a live imaging study has also shown
that escort cells do not move along with differentiated cells and,
thus, are not maintained by stem cells (Morris and Spradling,
2011).
Long cellular processes of escort cells wrap up
differentiated germ cells
ECs have been shown to wrap around germ cells in the anterior
half of the germarium (Schultz et al., 2002; Decotto and Spradling,
2005), and these long cellular processes appear to be involved in
passing differentiated germline cysts posteriorly (Morris and
DEVELOPMENT
Fig. 2. ECs are not maintained by ESCs. (A,A⬘) Quantitative data showing that the percentages of the germaria carrying one or more lacZlabeled ESCs (A) and the percentages of the germaria carrying only lacZ-labeled ECs, but no lacZ-labeled ESCs (A⬘) remain largely constant one
week, two weeks and three weeks ACI. (B)A lacZ-positive ESC (arrow) contacts cap cells (oval) and a GSC (dashed circle), which is accompanied by
a few lacZ-positive ECs (arrowheads). (C)A lacZ-positive EC (arrowhead) is a few cells away from cap cells (oval) but without any lacZ-positive ESCs.
(D,D⬘) Quantitative data showing that the percentages of the germaria carrying one GFP-labeled ESC (D) and the percentages of the germaria
carrying only GFP-labeled ECs (D⬘), remain largely unchanged one week, two weeks and three weeks ACI. These GFP-labeled EC clones are induced
by PMML. (E)Only a GFP-positive ESC (arrow) contacts cap cells (oval) and a GSC (dashed circle) but no GFP-positive ECs are detected. (F,G)Only
GFP-positive ECs (arrowheads) can be found in region 2a (F) or at the 2a/2b boundary (G). The image in G represents overlaid confocal sections,
and the remaining images are single confocal sections. Scale bar: 10mm.
Roles of escort cells
RESEARCH ARTICLE 5091
Spradling, 2011). To investigate further to what extent germ cells
interact with ECs, we used c587-driven UAS-GFP expression to
label ECs with GFP, which allows the visualization of EC cellular
processes. In the whole-mount germaria immunostained for GFP
and Hts, the long cellular processes of ECs wrap around
differentiated germ cells, which are identified by the presence of
branched fusomes labeled by Hts protein (Fig. 3A). Hts protein is
rich in spherical spectrosomes in GSCs and cystoblasts (CBs) and
in branched fusomes in differentiated germ cells (Lin et al., 1994).
In cryosections of c587-gal4;UAS-GFP germaria labeled for GFP
and Vasa, EC long cellular processes are shown to separate
individual germ cells from one another (Fig. 3B). Vasa is a
commonly used germ cell-specific marker (Hay et al., 1988; Lasko
and Ashburner, 1988). Our results further support the previous
finding that individual early differentiated germline cysts interact
extensively with long EC cellular processes.
Previous studies were unable to determine the morphology of
individual ECs using nuclear lacZ-labeled ECs and c587-driven or
patch-gal4-driven UAS-GFP expression (Margolis and Spradling,
1995; Schultz et al., 2002; Song et al., 2004; Decotto and
Spradling, 2005). To investigate the EC morphology of individual
ECs, we used the PMML system described above to label one or a
few ECs with tub-gal4-driven expression of UAS-mGFP (a
membrane-targeted GFP; the fusion between the Src membrane
localization signal and GFP). In the ovariole that lacks a GFPlabeled FSC clone, the GFP-positive ECs in regions 1, 2a and
2a/2b can be readily identified (Fig. 3C-I). The most anterior GFPlabeled ECs, which correspond to the previously identified ESCs,
directly contact GSCs and wrap around GSCs with their cellular
processes as previously described (Decotto and Spradling, 2005)
(Fig. 3C). The ECs in region 1 that interact with CBs and mitotic
germ cysts have short cellular processes (Fig. 3D,E; supplementary
material Movie 1). In region 2a, where 16-cell cysts occupy half
the diameter of the germaria, individual ECs have slightly longer
cellular processes to encase individual 16-cell cysts (Fig. 3F,G;
supplementary material Movie 2). In the 2a/2b junction areas,
where germline cysts start to adopt a lens shape crossing the
germarium, the ECs extend their cellular processes across the
whole width of the germarium to wrap around individual 16-cell
cysts (Fig. 3H,I; supplementary material Movie 3). These results
show that ECs at different positions adopt distinct morphologies
based on the size and morphology of underlying differentiated
germ cell cysts, further suggesting that EC cellular processes are
likely to be regulated by the underlying differentiated germ cell
cysts.
EC cellular processes might be regulated by
underlying differentiated germ cells
Although EC long cellular processes are only associated with
differentiated germ cells, it remains unclear whether differentiated
germ cells are required for the formation of long cellular processes
in ECs. bag of marbles (bam) is required for germ cell
DEVELOPMENT
Fig. 3. Differentiated germ cells are wrapped by EC cellular processes. (A)Differentiated germ cell cysts (dashed lines) are encased by GFPpositive EC cellular processes. (B)A cryosectioned germarium showing that germ cell cysts (dashed lines) are separated from one another by GFPpositive EC cellular processes. (C-I)Germaria labeled for GFP (green) and -galactosidase (PZ1444, red, C), Hts (red, D) or Fas3 (red, differentiated
follicle cells, E-I) containing a GFP-positive EC (arrow) at the ESC position (C), region 1 (D,E), region 2a (F,G) or the 2a/2b boundary (H,I). The ECs in
D and E have short cellular processes encasing the cystoblast (CB) and mitotic cyst, respectively, whereas those in F-I have longer cellular processes
wrapping around 16-cell cysts. The images in C, E, F and H are one confocal section, whereas images in D, G and I are overlaid confocal sections.
Scale bar: 10mm.
5092 RESEARCH ARTICLE
Development 138 (23)
differentiation, and its mutant ovaries only contain undifferentiated
cystoblast-like single germ cells (McKearin and Spradling, 1990;
McKearin and Ohlstein, 1995). bamD86 (a bam deletion allele)
mutant germaria are filled with undifferentiated germ cells, which
are identified by spherical spectrosomes (Fig. 4A). Interestingly, in
bam mutant germaria in which ECs are labeled by c587-driven
UAS-GFP expression, the long cellular processes of most ECs fail
to form or to penetrate inside the germ cells (Fig. 4A). To rule out
the possibility of poor antibody penetration into tightly packed
germ cells, the c857; UAS-GFP; bamD86/ bamD86 germarial
cryosections are labeled for GFP and Vasa to detect EC cellular
processes and germ cells, respectively. Consistently, long EC
cellular processes are not observed, although some ECs have small
EC protrusions (Fig. 4B, arrows). These results suggest that
differentiated germ cells might be required for the formation or
maintenance of long EC cellular processes.
To determine further whether the formation of long EC cellular
processes is dependent on differentiated germ cells or Bam
function in germ cells, we examined the formation of long EC
cellular processes in germaria carrying Dpp overexpressioninduced GSC-like cells. c587-driven UAS-dpp results in the
formation of ovaries filled with GSC-like cells (Fig. 4C), as
previously reported (Song et al., 2004). In the dpp-induced
tumorous germaria, EC cellular processes, which are visualized by
c587-driven UAS-GFP expression, fail to penetrate into individual
GSC-like aggregates and to wrap around individual single germ
cells (Fig. 4C). As c587-driven dpp overexpression cannot
completely inhibit GSC differentiation owing to mosaic expression,
some differentiated cysts form but some GFP-positive cellular
processes can still be detected to wrap up the differentiated germ
cells (Fig. 4D). Along with the bam mutant results, these results
further support the idea that differentiated germ cells might support
the formation of EC cellular processes.
Rho functions in ECs to maintain cellular
processes and promote germ cell differentiation
The small GTPase Rho has been extensively studied in different
organisms for its role in the regulation of cell protrusion formation
and cell-cell adhesion (Jaffe and Hall, 2005). To investigate further
whether EC cellular process-mediated interactions with germ cells
are important for germ cell differentiation, we overexpressed a
dominant negative form of Rho (RhoDN) in ECs using c587-driven
UAS-RhoDN expression. UAS-RhoDN has previously been used to
disrupt specifically Rho function in different Drosophila cell types
(Strutt et al., 1997; Hacker and Perrimon, 1998; Prokopenko et al.,
1999; Billuart et al., 2001; Bloor and Kiehart, 2002; Magie et al.,
2002). Interestingly, in the germaria in which RhoDN is expressed
in ECs, differentiated germ cells are not wrapped up by EC cellular
processes, suggesting that Rho might regulate EC cellular process
formation or maintenance (Fig. 5A-C). In those germaria, spherical
spectrosome-containing single germ cells often accumulate (Fig.
5A,C). In contrast with the wild-type germaria, which contain ~5.5
spectrosome-containing single germ cells including both GSCs and
cystoblasts (n91), the RhoDN-expressing germaria contain an
average of 8.9 spectrosome-containing germ cells (n69). Also, the
disruption severity of EC cellular processes is related to the
increase in the number of spectrosome-containing single germ
cells, suggesting that long EC cellular processes might regulate
germ cell differentiation.
One of the potential mechanisms for regulation by Rho of EC
cellular processes is by modulation of physical interactions
between ECs and germ cells through regulating actin cytoskeleton
because Rho is known to regulate actin dynamics and cell-cell
interaction in different Drosophila tissues (Hacker and Perrimon,
1998; Prokopenko et al., 1999; Bloor and Kiehart, 2002; Magie et
al., 2002). To determine whether actin regulation is important for
EC function and germ cell differentiation, we sought to inactivate
the function of the Formin-like actin regulator cappuccino (capu)
in ECs using c587-driven UAS-capuRNAi expression. Capu is
known to regulate actin nucleation and the formation of actin
meshwork (Dahlgaard et al., 2007; Quinlan et al., 2007). Following
capu knockdown in ECs, the ECs located in the anterior half of the
germaria generally lack cellular processes, and the anterior part of
the germaria accumulate more spectrosome-containing single germ
cells, suggesting that actin regulation is crucial for maintaining EC
cellular process and, thus, CB differentiation (supplementary
material Fig. S2A,B). However, all of the germaria appear to be
normal in size, indicating that disruption of actin dynamics does
not affect EC survival. These results further support the idea that
EC cellular processes regulated by actin dynamics are important
for germ cell differentiation.
Because some germaria expressing RhoDN in ECs are small in
comparison with wild-type germaria (Fig. 5A,B), we used the
PZ1444 EC marker to examine whether the number of ECs
following RhoDN expression is indeed reduced. The control weekold PZ1444 germaria contain an average of 42 ECs (n31). Only
very few of the week-old RhoDN-expressing germaria have the
normal or close to normal number of ECs (Fig. 5D); most of them
DEVELOPMENT
Fig. 4. Differentiated germ cells might maintain EC
cellular processes. (A)c587;UAS-GFP; bamD86/bamD86
germarium showing that all the ECs (one by arrowhead) fail
to form cellular processes penetrating inside tumorous germ
cells. (B)c587;UAS-GFP; bamD86/bamD86 cryosection
showing that ECs can form some protrusions (arrowhead)
that fail to encase germ cells. (C)c587;UAS-dpp/UAS-GFP
germarium showing that ECs fail to form long cellular
processes wrapping single germ cells (dashed lines).
(D)c587;UAS-dpp/UAS-GFP germarium showing that EC
cellular processes wrap round a differentiated cyst (arrow)
but fail to penetrate inside the clusters (dashed lines)
containing single germ cells. Scale bar: 10mm.
Roles of escort cells
RESEARCH ARTICLE 5093
Fig. 5. Defective Rho signaling in ECs disrupts EC-germ cell interactions and germ cell differentiation. The c587;UAS-RhoDN/UAS-GFP
germaria (single confocal section) in A-C are labeled for GFP, Hts and DNA, whereas c587;UAS-RhoDN/PZ1444 germaria (overlaid confocal sections)
in D-F and H-I are labeled for -galactosidase (PZ1444), Hts and DNA, and ApopTag, -galactosidase (PZ1444) and DNA, respectively. (A)The GFPpositive cellular processes of RhoDN-expressing ECs fail to wrap around a differentiated cyst (dashed lines). (B)RhoDN expression in ECs leads to a
reduction of regions 1 and 2a of the germarium and a defect in wrapping a differentiated cyst (dashed lines) by EC cellular processes.
(C)Spectrosome-containing single germ cells and a 16-cell cyst (dashed lines) are clustered together and are not individually wrapped by RhoDNexpressing EC cellular processes. Arrowheads indicate spectrosomes. (D)Germarium contains a normal number of ECs but excess single germ cells
(arrowheads) located anteriorly to differentiated cysts (arrows). (E,F)Germaria show a moderate (E) or severe (F) reduction of ECs and excess
spectrosome-containing single germ cells (arrowheads). (G)RhoDN-expressing germaria have significantly fewer ECs than do control germaria.
P-value is indicated. (H)A control germarium contains no apoptotic ECs. (I)A RhoDN-expressing germarium contains an apoptotic EC (arrowhead).
Scale bar: 10mm.
expressing (n2213) ECs are positive for BrdU, respectively,
indicating that disruption of Rho function does not change EC
proliferation. By contrast, 1.8% and 4.2% of control (n4000) and
RhoDN-expressing (n2795) ECs are positive for ApopTag labeling,
respectively, indicating that disruption of Rho function increases
EC apoptosis (Fig. 5H,I). These results indicate that Rho is
required for EC survival but not for EC proliferation.
Defective Rho in ECs causes the germ cell
differentiation defect by upregulating BMP
signaling activity
To characterize further the identity of accumulated spectrosomecontaining single germ cells in the RhoDN-overexpressing germaria,
we examined expression of bam-GFP and Dad-lacZ in these extra
single germ cells. In the wild-type germarium, bam-GFP is a
reporter construct for monitoring bam transcription and is mainly
expressed in differentiated germ cells but is repressed in GSCs by
BMP signaling (Chen and McKearin, 2003), whereas Dad-lacZ is
a BMP signaling activity reporter and is activated in GSCs and
repressed in differentiated germ cells (Kai and Spradling, 2003;
Casanueva and Ferguson, 2004; Song et al., 2004). As expected,
GSCs close to cap cells have low bam-GFP and high Dad-lacZ
expression in control germaria (Fig. 6A,D). Most of the single
DEVELOPMENT
have fewer ECs (Fig. 5E,F). The week-old RhoDN-expressing
germaria have an average of 21 ECs (n50), significantly fewer
than those in control germaria (Fig. 5G). Those germaria with
fewer ECs tend to have more spectrosome-containing single germ
cells than the germaria with more ECs, suggesting that ECs are
important for germ cell differentiation. The RhoDN-expressing
germarium in Fig. 5D has a close to normal number of ECs, but
contains excess spectrosome-containing single germ cells lying
more posteriorly to GSCs, suggesting that disruption of EC cellular
processes could also contribute to the delay in germ cell
differentiation. Taken together, these results suggest that Rho is
required for EC-germ cell interactions and EC maintenance, and
that ECs are important for normal germ cell differentiation. The
exact role of long EC cellular processes in the regulation of germ
cell differentiation needs to be investigated further by identifying
molecule(s) involved in the EC-cyst interaction.
The reduction of EC number in the RhoDN-expressing
germarium could be due to EC proliferation or survival. To
determine whether Rho is required for EC proliferation or survival,
we used BrdU incorporation and ApopTag-labeling to quantify
proliferating and dying ECs in control and RhoDN-expressing
PZ1444 germaria, respectively. Following two hours of BrdU
incorporation, 0.97% and 1.0% of control (n5145) and RhoDN-
5094 RESEARCH ARTICLE
Development 138 (23)
germ cells express high levels of bam-GFP and low levels DadlacZ, suggesting that those single germ cells resemble CBs instead
of GSCs (Fig. 6B,C,E,F). However, some spectrosome-containing
single germ cells that are located several cells away from cap cells
express Dad-lacZ and repress bam-GFP expression (Fig. 6C,F).
This observation indicates that BMP signaling activity has
expanded outside the GSC niche and thereby interferes with germ
cell differentiation. Therefore, these results demonstrate that Rho
function is required in ECs to control CB differentiation possibly
by restricting BMP signaling to the GSC niche.
Rho in ECs helps restrict BMP signaling activity to
the GSC niche
Activation of EGFR signaling leads to phosphorylation of ERK
(pERK) in ECs, and defective EGFR signaling leads to the absence
of long EC cellular processes and defective germ cell differentiation
(Schultz et al., 2002). EGFR signaling in ECs promotes germ cell
differentiation by repressing the expression of dally, encoding a
protein important for Dpp diffusion and function, in ECs (Liu et al.,
2010). To investigate whether the disruption of Rho function affects
EGFR signaling in ECs, we examined the expression of pERK in the
ECs of RhoDN-expressing germaria. Our quantitative result indicates
that RhoDN-expressing ECs (n160) have significantly, though not
dramatically, less pERK expression than control ECs (n160) (Fig.
6G-I). This finding demonstrates that defective Rho function
moderately decreases EGFR signaling in ECs.
To determine whether the germ cell differentiation defect in
RhoDN-expressing germaria is caused by upregulation of BMP
signaling outside the GSC niche, we tested whether the removal of
a copy of the dpp gene could suppress the differentiation defect. In
contrast with the results that 65% of the RhoDN-expressing germaria
accumulate excessive spectrosome-containing single germ cells
outside the niche (Fig. 7A,B), only 15% and 20% of the RhoDNexpressing dpphr56 and dpphr4 heterozygous germaria, respectively,
have extra single germ cells (Fig. 7C,D). These results indicate that
upregulation of Dpp/BMP signaling in the RhoDN-expressing
germarium contributes to the germ cell differentiation defect. To
knock down specifically dally function in ECs, two independent
dally RNAi lines were used to be expressed in ECs using c587.
Interestingly, EC-specific knockdown of dally in the RhoDNexpressing germaria can also slightly suppress the differentiation
defect based on the accumulation of single germ cells, indicating that
downregulation of EGFR signaling makes a slight contribution to the
germ cell differentiation defect (Fig. 7C,E). Although EC-specific
knockdown of dpp mRNAs in ECs using two independent RNAi
lines produces no discernible phenotype in wild-type germaria, it
leads to strong suppression of the germ cell differentiation defect in
the RhoDN-expressing germaria, suggesting that RhoDN-expressing
DEVELOPMENT
Fig. 6. Defective Rho signaling in ECs delays cystoblast differentiation. The germaria in A-C are labeled for GFP, Hts and DNA, those in D-F
are labeled for -galactosidase (Dad), Hts and DNA, and those in G and H are labeled for pERK, Hts and DNA. (A)Two control GSCs (solid circles)
expressing low bam-GFP and differentiated cysts expressing high bam-GFP. (B,C)c587;UAS-RhoDN/bam-GFP germarium carrying many of
spectrosome-containing single germ cells, most of which express bam-GFP (cystoblast-like; dashed circles) and the remaining ones show low bamGFP expression (GSC-like; solid circle). (D)Two control GSCs (solid circles) expressing high levels of Dad-lacZ. (E,F)c587; UAS-RhoDN/Dad-lacZ
germaria carrying spectrosome-containing single germ cells, of which those further away from the niche (dashed circle) express lower Dad-lacZ than
do endogenous GSCs (solid circles) contacting the niche. Some single cells away from the niche (solid circle, F) express high levels of Dad-lacZ.
(G)Wild-type ECs (two indicated by arrowheads) are positive for pERK staining. (H)RhoDN-expressing ECs away from (some indicated by
arrowheads) or close to (dashed circle) single germ cells remain positive for pERK staining. (I)RhoDN-expressing ECs have significantly less pERK than
control ECs. Error bars represent s.d. Number above horizontal line represents P-value. Scale bar: 10mm.
Roles of escort cells
RESEARCH ARTICLE 5095
ECs might also upregulate dpp mRNAs via transcription or
stabilization (Fig. 7C,F). Taken together, Rho is required in ECs to
control CB differentiation by restricting BMP signaling activity to
the GSC niche.
DISCUSSION
In the Drosophila ovary, ECs are thought to be produced by a
population of ESCs and to move along with differentiated germ
cells and commit apoptosis after differentiated germ cell cysts are
surrounded by follicle cells (Decotto and Spradling, 2005). In this
study, we have used three positive labeling systems and BrdU
labeling experiments to demonstrate that ECs are maintained by
self-duplication but not by the previously identified ESCs (Fig.
7G). Furthermore, ECs exhibit extensive interactions with
differentiated germ cells through their long cellular processes, and
differentiated germ cells are likely to be responsible for
maintaining long cellular processes. Interestingly, EC cellular
process-mediated interactions are required for germ cell
differentiation by restricting BMP signaling to the GSC niche.
Therefore, we propose that ECs form a germ cell differentiation
niche by preventing BMP signaling from interfering with CB
differentiation (Fig. 7G).
In this study, we have provided three pieces of experimental
evidence demonstrating that ECs are maintained by self-duplication
but not by stem cells. First, contrary to one of the predictions by
the ESC model that ECs can only undergo apoptosis at the junction
between ECs and follicle cell progenitors, we show that ECs in any
position can undergo apoptosis although those at the junction die
relatively more frequently. Second, contrary to another prediction
of the ESC model that only ESCs can proliferate, we show that
ECs in any position can proliferate slowly although those at the
posterior divide relatively more frequently. Third, we have used
three independent lineage-labeling methods to demonstrate that the
previously defined ESCs do not produce new ECs (Fig. 7G).
Therefore, the ECs in the Drosophila ovary behave similarly to the
differentiated  cells in the pancreas that are maintained by selfduplication (Dor et al., 2004). Possibly, in other slow turnover
mammalian tissues, stem cells are not required for maintaining
tissue homeostasis.
Long EC cellular processes might help pass on a differentiated
germ cell cluster from one EC to other (Morris and Spradling,
2011). In this study, we propose that long cellular process-mediated
intimate interactions between ECs and differentiation are required
for proper germ cell differentiation probably by restricting BMP
DEVELOPMENT
Fig. 7. Rho contributes to the restriction of
BMP signaling to the GSC niche. The germaria
in A-F are labeled for Hts and DNA. (A,B)RhoDN
expression in ECs leads to the accumulation of
excessive spectrosome-containing single germ
cells. (C)The percentages of excessive single germ
cell-containing germaria in ovaries in which RhoDN
is specifically expressed in the ECs and which are
wild type, heterozygous for dpp (dpphr4 and
dpphr56), or co-expressed with RNAi constructs for
dally (one on chromosome 2 and one on
chromosome 3) and dpp (47A and 47R). In
addition to endogenous GSCs, three more
additional single cells outside the GSC niche are
considered to be excessive single germ cells
because a wild-type germarium contains one to
two CB-like single germ cells. (D-F)The germ cell
differentiation defect induced by RhoDN expression
can be dramatically suppressed by a heterozygous
dpphr4 mutation (D), by co-expression of dally
RNAi (E) or by co-expression of dpp RNAi (F).
(G)Working model for EC maintenance and Rho
function in ECs. When an EC dies, its neighbor EC
divides to generate a new EC to replace the lost
one. Rho works in ECs to restrict BMP signaling
within the GSC niche by preventing Dpp diffusion
via regulation of EGFR-Dally signaling and
repressing dpp mRNA accumulation. In addition,
Rho signaling is important for EC survival.
signaling activity to the GSC niche (Fig. 7G). EC cellular processes
extensively wrap around differentiated germ cells, and are modeled
according to the size, shape and differentiated status of the
underlying differentiated germ cells. Forced expression of a
dominant-negative Rho or knocking down expression of the actin
regulator capu causes defects in EC cellular extension and germ
cell differentiation. This is consistent with the finding in the
previous study that defective EGFR signaling causes loss of EC
cellular processes and germ cell differentiation defects (Schultz et
al., 2002). Our genetic results have revealed two possible ways for
Rho in ECs to control germ cell differentiation by restricting BMP
signaling within the GSC niche. One of them is to repress dally
expression in ECs via EGFR signaling and thereby prevent BMP
diffusion to outside the GSC niche because EGFR signalingmediated repression of dally expression has been shown to be
essential for preventing BMP diffusion and germ cell
differentiation (Guo and Wang, 2009; Hayashi et al., 2009; Liu et
al., 2010). The other is to prevent BMP expression in ECs by
repressing dpp transcription or degrading dpp mRNAs through
unknown mechanisms. In addition to the regulation of EC-germ
cell interaction (Ridley, 2006), this study also shows that Rho is
also required for promoting EC survival. Therefore, we propose
that ECs form a niche for promoting germ cell differentiation, and
that Rho functions in ECs to promote germ cell differentiation at
least partly by preventing BMP signaling in the differentiation
niche (Fig. 7G).
The differentiation niche is likely to be a conserved feature for
different adult stem cell systems. In the Drosophila testis, somatic
cyst cells, which are produced by cyst stem cells, encase
differentiated germ cells and move together to accompany the
differentiated germ cells (Gonczy and DiNardo, 1996). EGFR
signaling is required in somatic cyst cells, as it is in ECs, to control
germ cell differentiation because defective EGFR signaling in the
Drosophila testis causes germ cell differentiation defects (Kiger et
al., 2000; Tran et al., 2000). Although, unlike ECs, somatic cyst
cells are not stationary, they function like ECs to control germ cell
differentiation. Although equivalent cells for ECs in mammalian
adult stem cell systems have not been defined, they are likely to
exist and to play important roles in the regulation of lineagespecific differentiation because cultured adult stem cells often
require a cocktail of growth factors for their proper differentiation.
As easily defined stem cell niches in Drosophila contribute to a
better understanding of mammalian stem cell regulation (Li and
Xie, 2005; Morrison and Spradling, 2008), we anticipate that what
we will learn from studies on the Drosophila GSC differentiation
niche will be equally important for defining differentiation niches
and studying their functions in lineage differentiation in
mammalian systems.
Acknowledgements
We would like to thank L. Cai, X. Lin, N. Perrimon, D. McKearin,
Developmental Studies Hybridoma Bank and Bloomington Drosophila Stock
Center for reagents; the Xie laboratory members for stimulating discussions;
and S. Chen, M. Lewallen and C. Flournoy for critical comments.
Funding
This work is supported by the National Institutes of Health [R01GM64428 to
T.X.]; and the Stowers Institute for Medical Research [T.X.]. Deposited in PMC
for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Development 138 (23)
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.067850/-/DC1
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Roles of escort cells