RESEARCH ARTICLE 3627
Development 136, 3627-3635 (2009) doi:10.1242/dev.036939
The glypican Dally is required in the niche for the
maintenance of germline stem cells and short-range BMP
signaling in the Drosophila ovary
Zheng Guo1,2 and Zhaohui Wang1,*
The Drosophila ovary is an excellent system with which to study germline stem cell (GSC) biology. Two or three female GSCs are
maintained in a structure called a niche at the anterior tip of the ovary. The somatic niche cells surrounding the GSCs include
terminal filament cells, cap cells and escort stem cells. Mounting evidence has demonstrated that BMP-like morphogens are the
immediate upstream signals to promote GSC fate by preventing the expression of Bam, a key differentiation factor. In contrast to
their morphogenic long-range action in imaginal epithelia, BMP molecules in the ovarian niche specify GSC fate at single-cell
resolution. How this steep gradient of BMP response is achieved remains elusive. In this study, we found that the glypican Dally is
essential for maintaining GSC identity. Dally is highly expressed in cap cells. Cell-specific Dally-RNAi, mutant clonal analysis and cellspecific rescue of the GSC-loss phenotype suggest that Dally acts in the cap cells adjacent to the GSCs. We confirmed that Dally
facilitated BMP signaling in GSCs by examining its downstream targets in various dally mutants. Conversely, when we overexpressed
Dally in somatic cells outside the niche, we increased the number of GSC-like cells apparently by expanding the pro-GSC
microenvironment. Furthermore, in a genetic setting we revealed a BMP-sensitivity distinction between germline and somatic cells,
namely that Dally is required for short-range BMP signaling in germline but not in somatic cells. We propose that Dally ensures
high-level BMP signaling in the ovarian niche and thus female GSC determination.
INTRODUCTION
Sustained gamete production during adulthood depends on germline
stem cell (GSC) self-renewal, which is also restrained to its proper
position to avoid the risk of tumorigenesis. The Drosophila ovary
provides an excellent system to study GSC fate determination
because its unique anatomical layout makes lineage tracing
relatively easy in a complex cell context (for reviews, see Li and Xie,
2005; Spradling et al., 2001). Each ovary consists of 16-20 ovarioles
that are basically parallel ‘assembly lines’ of oocyte production. At
the anterior tip of each ovariole (Fig. 1A), two to three GSCs are
restricted in a space called a niche, the somatic components of which
include terminal filament (TF) cells, cap cells and escort stem cells
(ESCs) (Decotto and Spradling, 2005; Xie and Spradling, 2000). To
keep egg production going, each GSC divides to generate two
daughter cells: one remains attached to the cap cells in the niche,
thus retaining GSC fate; the other moves out of the niche and
becomes a differentiated cystoblast, which becomes the start of the
egg-production line. The early germ cells in the germarium are
readily distinguished by the morphology of a germline-specific and
spectrin-rich structure known as a fusome.
BMP morphogens promote GSC fate by preventing the
expression of Bag of marbles (Bam), a key differentiation factor, so
that Bam is repressed in GSCs but expressed in cystoblasts (Chen
and McKearin, 2003a; Chen and McKearin, 2003b; McKearin and
Spradling, 1990; Ohlstein and McKearin, 1997; Song et al., 2004).
Transcriptional silencing of bam is established by a protein complex
1
Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and
Developmental Biology, Chinese Academy of Sciences, Beichen Xilu #1, Beijing
100101, P.R. China. 2Graduate School, Chinese Academy of Sciences, 19 Yuquan
Road, Beijing 100039, P.R. China.
*Author for correspondence ([email protected])
Accepted 1 September 2009
including BMP downstream effectors (Chen and McKearin, 2003a;
Pyrowolakis et al., 2004; Song et al., 2004) and a nuclear envelope
component Ote (Jiang et al., 2008). The direct upstream signals are
apparently BMP morphogens, which have been proposed to be
provided by the somatic niche cells, or more specifically by the cap
cells (Kai and Spradling, 2003; Song et al., 2004; Xie and Spradling,
1998). The high BMP signaling is precisely restricted to GSCs in the
niche and is turned down in the immediately adjacent cystoblast.
Clearly, this represents a typical short-range BMP signaling. As
Decapentaplegic (Dpp), one of the Drosophila BMP homologs, has
been shown to act as a long-range morphogen in many
developmental contexts (Entchev et al., 2000; Muller et al., 2003;
Teleman and Cohen, 2000), how is the steep gradient of BMP
response achieved in a span of only two cells? Casanueva et al.
suggested that Dpp proteins are present throughout the anterior
germarium, and Bam negatively feeds back to Dpp signaling, thus
maintaining the differentiated state of germ cells in the germarium
outside the niche (Casanueva and Ferguson, 2004). However, this
does not explain how the differential expression of Bam is
established in the first place. Nevertheless, even if BMP molecules
are highly expressed in the niche, it is still difficult to understand
how BMP molecules evoke an ‘on-or-off’ outcome in terms of Bam
expression and specifying distinct cell fate between the adjacent
GSCs and cystoblasts.
Division abnormally delayed (Dally) is a glypican member of
heparan sulphate proteoglycan (HSPG) and all glypicans possess a
glycosylphosphatidylinositol (GPI) moeity, a hydrophobic
modification anchoring the core protein to the cell surface (Nakato
et al., 1995; Selleck, 2000; Tsuda et al., 1999). Dally has been
demonstrated to regulate the Dpp gradient in the imaginal epithelia
(Akiyama et al., 2008; Belenkaya et al., 2004; Crickmore and Mann,
2007; Fujise et al., 2003; Jackson et al., 1997), and physical
interaction between Dally and Dpp was detected (Akiyama et al.,
2008). Misexpression of the cell-surface-anchored, but not the
DEVELOPMENT
KEY WORDS: Glypican, Germline stem cell, Niche, BMP, Short-range signaling
3628 RESEARCH ARTICLE
Development 136 (21)
(Crickmore and Mann, 2007); Dad-lacZ from Rongwen Xi (Tsuneizumi
et al., 1997; Zhao et al., 2008); esg-lacZ from Shigeo Hayashi. UASpDally was constructed by PCR cloning Dally cDNA from UASt-Dally and
inserting the cDNA to UASp vector. None of the dally alleles used in this
study is a strict null or amorph. All stocks and crosses except the
overexpression experiments were cultured at 25°C. Overexpression of
Dally and dallyRNAi were set up at 29°C.
Generation of female germline clones
FLP–FRT-mediated mitotic recombination was used to generate dally80
mutant GSC clones. To generate GSC clones and avoid TF and Cap cells
clones in adult ovaries, 3-day-old females (w hs-flp; FRT2A histoneGFP/FRT2A dally80) were heated at 37°C for 1 hour twice a day for 5
consecutive days. w hs-flp; FRT2A hisGFP/FRT2A flies were treated in
parallel as controls. Ovaries were dissected at day 2 and day 10 post-heat
induction. GSC clones were identified by the lack of GFP and the presence
of the anterior-positioned dot fusome (spectrosome) in the germline cells
(Yang et al., 2007).
secreted form, of Dally enhances Dpp signaling, and the secreted
Dally has rather a weak dominant-negative effect (Takeo et al.,
2005). Immobilized Dally seems to have the ability to
concentrate/stabilize Dpp or increase the cellular response to Dpp
(Akiyama et al., 2008).
Here we present genetic evidence to support the argument that
Dally is responsible for establishing the steep gradient of BMP
response from GSC to cystoblast. By altering the pattern of Dally
expression, we were able to manipulate the number and position of
the GSC-like cells. We also demonstrate that Dally defines a
distinction between germline and somatic cells in their sensitivity to
BMP signals.
MATERIALS AND METHODS
Fly genetics
P{PZ}dally06464, bab1-GAL4 (BL#6803), en-GAL4, ptc-GAL4, nos-Gal4
on III, UAS-GFP and balancers were obtained from the Bloomington
Stock Center; c587GAL4 from Ting Xie (Kawase et al., 2004); FRT2A
dally80 from Xinhua Lin (Belenkaya et al., 2004); dallygem, dally305, UASTM-dally, UAS-Sec-dally from Hiroshi Nakato (Nakato et al., 1995;
Takeo et al., 2005); hs-bam, FRT2A histone-GFP, FRT2A, bamP-GFP,
UASp-Tkv* (constitutively active Tkv), and bamBG from Dahua Chen
(Chen and McKearin, 2003a; Chen and McKearin, 2003b; Jiang et al.,
2008); UAS-dally(strong) (Jackson et al., 1997), UAS-dallyRNAi (Vienna,
Austria) and UAS-dlpRNAi (Vienna, Austria) from Michael A. Crickmore
X-gal staining of -gal activity
Adult ovaries and testes were fixed with 0.5% glutaraldehyde in PBS for 2
minutes, and then subjected to the standard X-gal color reaction for 12 hours
at 37°C.
BrdU labeling
Ovaries were dissected in PBS, incubated for 1 hour in PBS containing 100
g/ml BrdU (Sigma) at 25°C, then fixed. The rest of the procedure was
described previously (Li et al., 2007).
Cell death assays
For TUNEL assay, after fixation in 4% formaldehyde/PBS, samples were
incubated in the mixture of Enzyme and Label solutions (Roche Kit, 1 684
795) at room temperature for at least 3 hours.
For Acridine Orange staining, samples were incubated in 1.6 M
Acridine Orange/PBS for 20 minutes at room temperature.
For Caspase detection, rabbit anti-Active Caspase-3 (CM1, from BD
Pharmingen) was used at 1:5000.
Immunohistochemistry and microscopy
All samples were dissected in PBS, fixed and stained as described previously
(Li et al., 2007). Primary antibodies were used at the following dilutions:
mouse anti--spectrin at 1:50 (a gift from Rongwen Xi) (Zhao et al., 2008);
mouse anti-Hts (1B1, developed by Howard D. Lipshitz, DSHB) at 1:100;
mouse anti-En (developed by Corey Goodman, DSHB) at 1:50; mouse antiDlp (developed by P. A. Beachy, DSHB) at 1:50; mouse anti-FasIII
(developed by Corey Goodman, DSHB); mouse anti-BamC at 1:4000 and
rabbit anti-Vasa at 1:5000 (gifts from Dahua Chen) (Jiang et al., 2008);
guinea pig anti-pMAD at 1:4000 (E. Laufer and T. Jessell, Columbia
University, New York, NY) (Crickmore and Mann, 2007); rabbit anti-GFP
at 1:5000 (Invitrogen); mouse anti--gal (JIE7, developed by T. L. Mason,
DSHB) at 1:200; and rabbit anti--gal at 1:10000 (Cappel). Alexa-Fluorconjugated secondary antibodies were used at 1:4000 (Molecular Probes,
Invitrogen). Fluorescent images were collected using a Zeiss ApoTome
microimaging system. 63⫻ Oil Plan-Apochromat lens was used to visualize
the details in germarium.
RESULTS
dally is required in the somatic niche cells for the
ovarian GSC maintenance
We discovered the glypican Dally as one of the candidates that may
be involved in enhancing the BMP response in the female GSC
niche when we examined the expression pattern of an enhancer trap
lacZ in the dally gene (P{PZ}dally06464) (Fig. 1B). In germarium,
Dally--gal is expressed mainly in the cap cells, to which the GSCs
are attached in the niche (Fig. 1C). Unlike Dally, another Drosophila
glypican, Dally-like (Dlp), is ubiquitous in the germarium, as shown
in the immunostaining of the protein distribution (see Fig. S1A in
the supplementary material).
DEVELOPMENT
Fig. 1. Dally is expressed in the cap cells of the GSC niche in the
Drosophila ovary. Unless otherwise specified, all images are oriented
with anterior to the left. (A)Based on previous findings and especially
the descriptions in Decotto and Spradling (Decotto and Spradling,
2005), we use a cartoon to illustrate the anatomical layout of different
cell types at the ovarian tip: light blue, terminal filament (TF) cells; dark
blue, cap cells; purple, GSCs; pink, cystoblast (containing one red dot)
and cystocytes (containing the red-branched structure); green, ESCs
(those encapsulating GSCs) and escort cells. The cytoskeletal structure
termed the fusome in the germline cells is in red. The morphology and
position of this structure is used to distinguish different stages of the
early germ cells. Note that the fusome dots in GSCs are located
towards the cap cells. (B)X-gal staining (blue) reveals the
-galactosidase expression in cap cells in the fly ovary carrying a lacZ
enhancer-trap construct at the dally locus (P{PZ}dally06464). Only the
germarium is shown. (C)Co-immunostaining with the protein marker
Engrailed (En, present in both TF and cap cells) confirms the expression
of the dally-lacZ enhancer trap line in the cap cells. Co-localization of
En, dally-lacZ and DNA appears white in the three-channel merged
image (arrowheads point to the cap cells).
Dally enhances BMP response in female GSC niche
RESEARCH ARTICLE 3629
To determine if dally is involved in GSC fate regulation, we
counted the GSC numbers in the ovaries of various dally mutant
alleles. In wild-type ovaries at adult day 10, two to three GSCs were
normally present in the niche and germaria containing none or one
GSC were rarely found (Table 1). In the homozygous or transheterozygous mutants of dally, GSC loss was obvious after 10 days
into adulthood, and was consistent in all mutants, although at
different severity (Fig. 2; Table 1). As early as day 3 after eclosion,
complete germ cell loss due to lack of GSC renewal was observed
in the strong dally mutant alleles (Fig. 2F,H; Table 1).
The loss of GSC was unlikely to be a result of cell death, because
advanced egg chambers were present in the same ovarioles
displaying GSC loss (Fig. 2B). Using assays to evaluate DNA
fragmentation or caspase activation for the signs of cell death, no
difference between the wild type and dally mutant was found (Fig.
2I,J). In both wild type and mutant when germ cells were still present
in the germarium, programmed cell death was detected in a few
sporadic somatic cells in the germarial region in which the egg
chamber starts to form but not in the cap cells or germline (Fig. 2I,J;
see also Fig. S2 in the supplementary material), similar to that
reported previously (Decotto and Spradling, 2005). Additionally,
dally mutant clones in GSCs remained in the niche 10 days after
clone induction (see Fig. S3 in the supplementary material),
indicating that Dally is not autonomously required in the germline
for the viability of GSC.
As Dally is highly expressed in the cap cells and required for GSC
maintenance, we wonder if Dally is essential for the viability and/or
identity of the cap cells. We therefore compared the cap cells in wild
type and dally mutant by Lamin C staining, which labels the TF and
cap cells of the niche. However, we did not detect a loss of cap cells
in dally mutant even when GSCs were completely lost (Fig. 2G,H).
The average cap cell number per ovariole was 5.5 (n46) in the wild
type, and 5.6 (n41) in the dally mutant.
To check whether Dally acts specifically in the somatic niche
cells, we tried to rescue dally mutant phenotype by expressing
Dally in these cells. We took advantage of two Gal4 lines, which
drive targeted expression in a slightly different range in the niche
(see Fig. S4 in the supplementary material). bab1Gal4 is strong in
both TF and cap cells (Fig. 2K; see also Fig. S4 in the
supplementary material) (Bolivar et al., 2006), and its induction
of Dally significantly restored the GSCs in dally mutant (Fig. 2E;
Table 1). Different from bab1Gal4, enGal4 is highly expressed in
TF cells but was barely detected in the cap cells (Fig. S4 in the
supplementary material) (and personal communications with
Dahua Chen and A. Gonzalez-Reyes), and its ability to rescue
GSC loss in the dally mutant is less than that of bab1Gal4 (Table
1). Nevertheless, the cell-specific rescue of dally mutant
phenotype implicates that Dally acts in the somatic niche to
maintain GSC fate.
To confirm the action of Dally in the niche, we also employed
cell-specific RNAi to reduce Dally levels in somatic niche cells.
bab1Gal4-driven Dally RNAi led to a complete GSC loss on day 10
in ~30% of the germaria scored (Table 1, n122). On the contrary,
Dlp RNAi in the same cells did not have any effect on GSC number
in the niche (see Fig. S1A in the supplementary material and more
than 100 ovarioles scored). Thus, data obtained from various dally
mutations, cell-specific RNAi, germline mutant clones, and cellspecific rescue of dally mutations indicate that glypican Dally is
required for GSC maintenance and probably it exerts this function
in somatic niche cells.
Table 1. Scoring of GSC-like cells in dally mutants and Dally-RNAi flies
Dissection time after eclosion
Day 3
Day 10*
2-3 GSCs/niche
1 GSC/niche
0 GSCs/niche
2-3 GSCs/niche
1 GSC/niche
0 GSCs/niche
w1118
126 (100%)
0
210 (98%)
126 (77%)
17 (11%)
37 (23%)
dallygem
47 (16%)
222(77%)
25 (8%)
2 (1%)
n=213
34 (21%)
n=162
3 (1%)
n=316
1 (1%)
dally305
dally80
14 (31%)
dallyPZ/dally80
52 (84%)
dallygem/dallygem, UAS-dally
51 (20%)
en-GAL4; dallygem/dallygem, UASdally
bab1-GAL4, dallygem/dallygem,
UAS-dally
C587-GAL4;;dallygem/dallygem,
UAS-dally
dallygem, DadP1883
107 (56%)
0
n=126
20 (12%)
n=163
19 (7%)
n=288
11 (24%)
n=45
1 (2%)
n=62
10 (4%)
n=245
11 (6%)
n=191
14 (4%)
n=313
2 (1%)
n=310
14 (14%)
n=101
2 (2%)
n=90
bab1-GAL4>UAS-dallyRNAi
bab1-GAL4>UAS-sec-dally
248 (80%)
251 (81%)†
66 (66%)
85 (94%)
ND
20 (45%)
91(56%)
288 (91%)
ND (rarely survive to day 10)
9 (14%)
16 (39%)
184 (76%)
35 (18%)
73 (38%)
97 (48%)
51 (16%)
220 (74%)
57 (18%)
119 (78%)‡
21 (20%)
17 (26%)
3 (4%)
79 (65%)
98 (84%)
1 (2%)
n=41
2 (1%)
n=184
5 (2%)
n=201
11 (4%)
n=296
3 (2%)
n=152
3 (5%)
n=64 (day 6)
7 (6%)
n=122 (day 10-12)
17 (14%)
n=117 (day 10-13)
24 (59%)
147 (80%)
99 (50%)
65 (22%)
30 (20%)
44 (69%)
36 (29%)
2 (2%)
*Unless otherwise specified, adult day 10 samples were used.
†About half of these ovarioles contained more than three GSCs.
‡Approximately 80% of these ovarioles contained more than three GSCs.
ND, not determined.
GSCs were identified by the morphology of fusome staining ( Spec or Hts). Numbers in the table are the counts of germarium and their percentage of the total (n)
germaria examined at the same stage of adulthood. Flies of RNAi experiments were raised at 29°C.
DEVELOPMENT
Genotype
3630 RESEARCH ARTICLE
Development 136 (21)
dally is essential for BMP signaling in germline
stem cells
Dally has been demonstrated to regulate the gradient of Dpp, Hh
(Hedgehog) and Wg (Wingless) in the imaginal epithelia (for
reviews, see Lin, 2004; Nybakken and Perrimon, 2002). In the
ovary, the niche-restricted response of Dpp but not Hh or Wg plays
a major role in maintaining GSC fate (King et al., 2001; Li and Xie,
2005). The contrast in BMP response between GSC and cystoblast
can be revealed by the BMP-downstream targets such as pMad
(phosphorylated Mad) or Bam. Mad is the intracellular effector of
BMP signaling and is phosphorylated upon ligand-receptor binding.
Bam is required for germ-cell differentiation, and its transcription in
GSC is directly repressed by the protein complex containing BMPpathway effectors (Jiang et al., 2008).
To clarify whether the GSC loss in dally mutants is due to the
compromised BMP signaling, we examined the status of pMad and
Bam in germarium. In GSCs, the presence of pMad and the absence
of Bam reflect the activation of BMP signaling. These two events
are normally associated with the GSC fate in the niche (Fig. 3A,B)
(dally+/–; bamGFP, a bam promoter fused with GFP). In dally mutant
ovaries, pMad staining was barely above background and bam
transcription was de-repressed in the GSCs or germ cells occupying
the niche (Fig. 3A,B) (dally–/–), indicating the downregulation of
BMP signaling in these cells.
To obtain genetic evidence confirming the association of Dally
and BMP signaling in GSC determination, we generated a double
mutant of dally and Dad (Daughters against dpp). Dad is an
antagonist of Dpp signaling and its expression is turned on by Dpp
(Tsuneizumi et al., 1997). We found that the Dad mutation partially
rescued the GSC loss phenotype of dally mutant (Fig. 3C; Table 1;
dally Dad double mutant). Additionally, every double mutant female
laid eggs, whereas the homozygous female of this particular dally
allele (dallygem) rarely did. The genetic interaction between dally and
Dad provides evidence for their functional interaction in oogenesis.
Further, restoration of GSCs in dally mutant by expressing the
activated Tkv (a BMP receptor) in the early germline cells also
supports the idea that Dally regulates BMP response in the germarial
niche (Fig. 3D).
Bam-independent GSC differentiation has been reported
previously (Chen and McKearin, 2005; Szakmary et al., 2005; Xi et
al., 2005). Mutations in pumilio and pelota lead to GSC loss without
affecting Bam expression, and pelota also modulates BMP
signaling. To exclude the possibility that Dally is involved in the
Bam-independent pathway in addition to the BMP-Bam pathway,
we generated dally bam double mutant. dally bam double mutant
produced GSC-like over-proliferation in the germarium, a
phenotype indistinguishable from that in the bam single mutant (Fig.
3E). Thus, we genetically illustrate that Dally acts in the Bamdependent pathway. Again, GSC-like cell accumulation in dally bam
double mutant ovaries also supports the argument against the
possibility that dally mutations cause GSC death. No difference in
TUNEL and Caspase3 signals was detectable between bam and
dally bam mutants (see Fig. S2E-H in the supplementary material).
To further demonstrate that the lack of pMad signal in dally
mutant is not simply due to the absence of GSCs, we took advantage
of the dally bam double mutant in which GSC-like cells accumulate
DEVELOPMENT
Fig. 2. dally is required in the somatic niche cells for ovarian GSC
maintenance. Vas, a germline-specific marker; Spectrin, a fusome
component used to distinguish GSC and the differentiated germ cells.
(A)An ovariole (dally+/–) normally contains numerous Vas-positive
germline cells in the germarium (yellow dotted line) and egg chambers
of nearly consecutive stages. (B)Ovarioles of dally homozygous mutant
(dally–/–, image of dallygem allele) within 3 days after eclosion exhibit the
typical GSC loss phenotype: a tiny germarium missing germ cells
(yellow dotted line, note the absence of Vas staining) at the anterior
end, as well as developmentally advanced egg chambers present
posteriorly with large stage gaps. (C)When viewed at higher
magnifications, in dally+/– ovaries normally two to three GSCs are visible
in the niche and each GSC contains a single Spectrin dot positioned
anteriorly (arrowheads). Germ cells mitotically dividing before reaching
the 16-cell stage are interconnected by branched Spectrin in the
germarium (dally+/–, Vas-positive cell clusters). (D)In the dally
homozygous mutant (dally–/–, image of dally305 allele), only two germ
cells are left and the posterior one is apparently moving away from the
niche (note the Spec staining between the two green germ cells).
Ovaries were dissected from 3-day-old flies. (E)A germarium of
genotype bab1-Gal4>dally,dallygem 10 days after eclosion. bab1-Gal4 is
active specifically in cap cells and TF cells (see bab1>GFP and cap cell
staining in Fig. S4 in the supplementary material). (F)An example of
dallygem with more severe GSC loss within 3 days after eclosion; lack of
Vas-staining indicates that no germline cell was present in the
germarium. (G,H)Lamin C (LamC) labels the TF and cap cells of the
germarial niche. The number of cap cells remained similar between the
control (G) and dallygem (H). (I,J)The TUNEL assay detects programmed
cell death, which was not detected in cap cells or germline cells in
either control (I) or dallygem (J) germaria. (K)bab1-Gal4,UAS- GFP.
(L)Dally Knockdown by bab1-Gal4-controlled RNAi non-autonomously
led to the empty-germarium phenotype, i.e. no Vas-positive cells were
observed in the germarium. The effect of RNAi is variable and one of
the most severe cases is shown here.
Dally enhances BMP response in female GSC niche
RESEARCH ARTICLE 3631
Fig. 3. dally is essential for BMP signaling in germline stem cells.
dally305 was used in A and B; dallygem and DadP1883 in C; dallygem in D;
dallygem and bamBG in E-G. (A)Phosphorylated Mad (pMad) was stained
as a readout of BMP signaling. In dally+/–, pMad was readily detected in
the GSC and is highlighted in a separate channel for pMad (inset,
arrowhead indicates the faint signal in the GSC slightly off the focal
plane). By contrast, pMad was hardly detectable in dally–/– germaria.
(B)In dally+/–, bam-promoter-controlled expression of GFP (bamGFP)
was repressed by BMP signaling in GSC and one of the cystoblasts (the
anterior three cells labeled with dot-like Hts staining). In dally–/–,
however, bamGFP is upregulated in the GSC-like cell at the anterior tip
of the germarium (arrowhead, identified by the single-dot Hts-staining
located at the anterior pole of the cell). (C)In comparison with dally–/– in
Fig. 2D,F, the dally–/– Dad–/– double mutant partially restored GSC
maintenance (also see scores in Table 1). (D)The constitutively activated
Tkv (tkv*) driven by nos-Gal4 restored the germline cells in the dallygem
germarium. Samples were dissected from 5-day-old flies. (E) Thedally–/–
bam–/– double mutant has the same phenotype as bam–/– in terms of
GSC-like overproliferation, revealed by the mutant germarium filled
with germ cells containing dot-like Spec. (F)pMad signal in the
anterior-most germ cells was easily detected in the bam–/– mutant but
hardly in the dally–/– bam–/– double mutant. pMad labeling alone is
shown in the insets. (G)Similar to that in wild type, bamGFP is
repressed in the most anterior GSC-like cells of the bam–/– mutant
(yellow bracket), but is expressed in those of the dally–/– bam–/– double
mutant (yellow bracket).
Overexpression of membrane-bound Dally
posterior to the niche induces GSC-like expansion
and activation of BMP signaling
To see whether Dally expression in somatic cells outside the niche
is sufficient to induce BMP response and more GSC-like cells, we
overexpressed Dally in the somatic cells posterior to the niche
(Fig. 4, C587GFP; see also Fig. S4 in the supplementary material)
and observed substantial accumulation of GSC-like cells in the
germarium (Fig. 4, C587>dally; Table 2, C587>dally). Such
phenotype is similar to that of bam mutant. This was confirmed
by the targeted expression in the somatic cells under the control
of a different Gal4 (see Fig. S4 in the supplementary material;
Table 2, ptcGAL4>UAS-dally). Notably, it takes time for the
GSC-like cells to accumulate and become evident (Table 2)
(C587GAL4>UAS-dally and ptcGAL4>UAS-dally, day 5 versus
day 15). These GSC-like cells hardly differentiated, leaving big
stage-gaps between the germarium and the adjacent egg chamber
(Fig. 4D). In TF cells, Dally overexpression did not show any
effect on GSC number or position (see Fig. S4 in the
supplementary material; Fig. 4A, en>dally+GFP; Table 2,
enGAL4>UAS-dally). Furthermore, Dally expression in germline
cells also induced more GSC-like cells in the germarium (Fig. 4H;
Table 2, nos-GAL4VP16>UASp-dally). Thus, overexpression of
Dally outside niche causes GSC-like expansion.
To get a clue of whether GSC-like expansion induced by Dally
expression in somatic germarium is simply due to an increase in
GSC production at the anterior tip, or due to more cell divisions
elsewhere in the escort cell-surrounded space, the mitotic activity of
the GSC-like cells was evaluated by BrdU labeling (Fig. 4B). Wildtype germaria often contain BrdU-positive germ cells in a cluster of
four or eight cells in the middle of the germarium, reflecting
synchronized division of cystocytes (Fig. 4B, wild type).
Interestingly, we observed single cells or two-cell clusters actively
dividing along the germarial periphery (Fig. 4B) (C587>dally). It
seems that cell proliferation posterior to the niche at least partially
accounts for the GSC-like cell expansion induced by Dally from
escort cells.
As Bam promotes differentiation and is repressed in the GSCs,
we asked whether this protein is silenced in the GSC-like cells
induced by Dally expansion. Bam proteins were present in a few
cystocytes of almost every control germarium (Fig. 4C)
(C587>GFP). Not surprisingly, Bam was drastically reduced in the
germaria actively expressing Dally in the escort cells, and even in
the germaria in which GSC-like cells had not completely replaced
the differentiated germ cells (Fig. 4C,E) (C587>dally+GFP).
Consistently, using bam-promoter-driven GFP as a reporter,
C587Gal4 overexpression of Dally repressed the bamGFP
expression in all GSC-like cells (Fig. 4F).
DEVELOPMENT
in the germarium. Under both genetic conditions (bam and dally
bam mutants), GSC-like cells containing spherical, not branched,
fusomes occupied the germarium, and no differentiated germ cells
were observed (Fig. 3E). However, a molecular readout such as
pMad exposed the difference between bam and dally bam mutants.
pMad was present in the germ cells within the niche in bam single
mutant (Fig. 3F). By contrast, pMad was undetectable in the dally
bam double mutant germarium (Fig. 3F). Consistently, bamGFP in
anteriormost germ cells was repressed in bam but was derepressed
in dally bam, indicating that BMP signaling was compromised when
dally was disrupted (Fig. 3G). Taken together, these multiple pieces
of evidence illustrate that dally is essential for BMP signaling in
GSCs.
3632 RESEARCH ARTICLE
Development 136 (21)
Fig. 4. Overexpression of Dally induces GSC-like expansion and
enhanced BMP signaling. C587Gal4 is expressed in somatic cells,
including the escort cells and is visualized by UAS-GFP. (A)In the control
panel, enGal4 is highly expressed in TF cells (see also Fig. S4 in the
supplementary material). No abnormal fusome (Spec) pattern was
detected in the ovaries where enGal4 drove the overexpression of Dally
(en>dally+GFP). C587Gal4 expands Dally expression to escort cells in
addition to cap cells, and this expression of Dally induced the
accumulation of GSC-like cells in the germarium. Note the Spec dots in
the GSC-like cells contacting escort cells at the germarial periphery.
(B)BrdU labeling reveals cell division activities. In wild type, it is frequently
present in a cluster of cystocytes dividing synchronously, and is never seen
in a cluster of two cells outside the space occupied by GSC and
cystoblast. In the germarium, where Dally was expressed by C587Gal4,
BrdU signal was observed in multiple two-cell clusters posterior and
distant from the niche. (C,E) Owing to technical difficulties, Bam and
Spec cannot be stained simultaneously, but samples of the same age
(day 5) were stained separately for either Bam or Spec. (C)Bam protein
staining was present in some cystocytes of almost every control
germarium (C587>GFP). In the germarium carrying Dally expressed by
C587Gal4, Bam signal was reduced dramatically (C587>dally+GFP, only
background staining seen in the Bam channel). (D)In the germarium of a
20-day-old C587>dally fly, no egg chambers differentiated out between
the germarium and the adjacent stage-10 egg chamber (partial view of
an egg chamber on the right). (E)C587>dally germarium of the same age
as that in C contained differentiated cystocytes (arrowhead indicates
branched fusome). (F)Consistent with the observation of Bam protein in
C, transcription reflected in bam-promoter-driven GFP shows a dramatic
reduction in C587>dally fly. bamGFP expression remained in the
differentiated cells bearing the branched fusome. (G)Germaria of 10-dayold c587>GFP and c587>dally+GFP. DadP1883 serves as a lacZ reporter in
these flies. Dad--gal expression was dramatically upregulated in the
GSC-like cells encapsulated by the C587>dally escort cells. Note how
inflated the space of C587>dally+GFP is relative to the control on the
right, owing to the overproliferation of GSC-like cells. (H)nosGal4 is
specifically active in the early germline cells including GSCs (left panel).
Dally expressed by nosGal4 induced an increase of GSC-like cells in the
germarium (right panel, 3 days after eclosion; the yellow arrowhead
points to one of the GSC-like cells). Scale bars: 10m in D; 10m in A
for all other panels.
the expression of the sec-Dally in somatic cells did not cause any
detectable change in GSC number or position (Table 2, compare
C587-GAL4>UAS-sec-dally and C587-GAL4>UAS-dally). Taken
together, we suggest that membrane-anchored Dally can
induce GSC-like cell expansion when expressed outside the
niche.
Dally is required for the short-range BMP
signaling in germline but not in somatic cells
In the process of studying the collaborative effect of Dally and BMP
signaling on GSC-fate determination, we discovered a difference in
BMP sensitivity between germline and somatic cells in the dally
mutant carrying a Dad-lacZ reporter (Fig. 5). As a downstream
readout of BMP response, Dad--gal signal is normally detected in
the GSCs and to a lesser degree in cystoblasts (Fig. 4G; Fig. 5A),
and could also be induced in the more differentiated germ cells in
the niche after Bam overexpression (Fig. 5C). In dally mutants,
when GSCs were completely lost in the germarium, the somatic
escort cells became in contact with cap cells and expressed Dad-gal, indicative of BMP response (Fig. 5B). In another sample,
however, the remaining GSC was unable to show such response in
the absence of Dally, whereas the somatic cells around this GSC
displayed obvious Dad--gal expression in the niche (Fig. 5D).
DEVELOPMENT
To determine whether BMP signaling is indeed elevated in the
GSC-like cells induced by ectopic Dally expression, we examined
the activation of Dad, another downstream effector of BMP
signaling. In the wild-type controls, Dad expression revealed by
Dad--gal was strong in the GSCs next to the cap cells and weaker
in cystoblasts (Fig. 4G) (C587>GFP). As we predicted, expanded
Dally expression was sufficient to enhance the Dad--gal signal in
most of the GSC-like cells (Fig. 4G).
Full-length Dally is anchored on the cell surface through the
GPI addition at its C-terminus, and Dally’s ability to regulate Dpp
gradient depends on its membrane-anchor (Takeo et al., 2005).
Nakado and colleagues had constructed the secreted form of
Dally by deleting the GPI-linked C-terminus (sec-Dally), and then
fused the secreted Dally to a transmembrane domain to make
it membrane-tethered (TM-Dally) (Takeo et al., 2005). To
address how Dally acts from cap cells to maintain BMP signaling
in the niche thus ensuring the GSC fate, we tried to express
different forms of Dally at different positions in the germarium.
In addition to the targeted expression of the wild-type Dally
(supposedly GPI-modified), the membrane-tethered Dally
exhibited similar though weaker effect than the wild-type
Dally when expressed in escort cells (Table 2, compare C587GAL4>UAS-TM-dally and C587GA14>UAS-dally). By contrast,
Dally enhances BMP response in female GSC niche
RESEARCH ARTICLE 3633
Table 2. Targeted overexpression of Dally and its effect on GSC number
Dissection time after eclosion
Day 5
Genotype
C587GA14> UAS-dally
Day 15
<7 spherical fusomes
≥7 spherical fusomes
<7 spherical fusomes
141 (73%)
53 (27%)
0
n=194
C587GAL4>UAS-TM-dally
104 (88%)
14 (12%)
196 (100%)
46 (75%)
0
54 (57%)
285 (100%)
40 (43%)
203 (100%)
9 (29%)
0
196 (99%)
115 (100%)
2 (1%)
58 (48%)
191 (85%)
34 (15%)
n=225
63 (52%)
n=121
0
n=115
n=198
nos-GAL4VP16>UASP-dally
22 (71%)
n=31
n=203
bab1-GAL4>UAS-dally
0
n=285
n=94
enGAL4>UAS-dally
15 (25%)
n=61
n=196
ptcGAL4>UAS-dally
231 (100%)
n=231
n=118
C587GAL4>UAS-sec-dally
≥7 spherical fusomes
12 (16%)
62 (84%)
n=74
Flies were raised at 29°C. The number of spherical fusomes is equal to the number of GSC-like cells and cystoblasts. ‘7’ is used because generally 6 is the maximal
number of spherical fusomes observed in wild type (three in GSCs and three in cystoblasts). Numbers in the table are the counts of germaria and their percentage of the
total (n) examined at the same stage of adulthood.
sec, secreted; TM, transmembrane.
DISCUSSION
To understand how a steep gradient of BMP response is established
and thus determines cell fate at single-cell resolution in the GSC
niche of Drosophila ovary, we have taken genetic approaches to
examine the role of the glypican Dally in the process. Based on our
current data, we propose a model of how the expression pattern of
Dally shapes the BMP-signaling range and consequently determines
distinct cell fates in the ovarian niche (Fig. 6).
Female GSC fate requires high BMP signaling, which is provided
in the ovarian niche. Dally is highly expressed in the cap cells that
contact the GSCs. Cap-cell-localized and membrane-bound Dally
either stabilizes/concentrates BMP molecules, or enhances BMP
sensitivity to ensure that only the germ cells in contact with cap cells
become GSC (Fig. 6, wild type). Removal of Dally, BMP
concentration at the niche or BMP sensitivity in the germ cells
adjacent to cap cells dissipates, and GSCs cannot be maintained and
subsequently differentiate (Fig. 6, dally–/–). Conversely, when Dally
is ectopically overexpressed in the escort cells posterior to the niche,
BMP signaling or sensitivity increases in all germ cells encapsulated
by these escort cells, and GSC-like cells accumulate in the
germarium (Fig. 6, C587>dally).
Consistent with this model, the secreted form of Dally
expressed from cap cells caused GSC loss, possibly by competing
with endogenous membrane-anchored Dally for binding with
BMP molecules or by interfering with BMP signaling (see Fig. S5
in the supplementary material; Table 1). Because secreted Dally
expressed from somatic cells in addition to cap cells did not cause
GSC expansion as the membrane-anchored Dally did (Table 2), it
further supports the idea that Dally’s function in the GSC niche
depends on the cap-cell-specific expression and membrane
anchoring.
When somatic cells displace the differentiating germ cells in the
niche and become close to or in contact with cap cells where the BMP
morphogen is localized (Kai and Spradling, 2003), these somatic
cells are able to respond to BMP when Dally is lacking (Fig. 5; Fig.
6, purple escort cell in dally–/–). Whether a cellular BMP response is
Dally dependent or not distinguishes the germline and somatic cells.
The molecular aspects of how Dally modulates
short-range BMP response
We have noticed that it took 15 days for Dally overexpression in
somatic cells to make all germ cells GSC-like in the germarium
(Table 1), although C587Gal4 was active since stage larval 3 at the
latest. One possible explanation is that Dpp is limited and Dally
stabilizes Dpp. This possibility is supported by a recent report
demonstrating that Dally and Dpp physically interact with each
other in the cultured S2 cells and that Dally stabilizes Dpp on the cell
surface in the wing imaginal epithelia (Akiyama et al., 2008).
Consistent with their theory of cell-surface-associated stabilization,
the secreted Dally, although retaining the ability to bind Dpp, did not
have the activity that the full-length Dally possesses in terms of
enhancing GSC proliferation (Table 2, C587GAL4>UAS-sec-dally).
It suggests that Dally can only stabilize Dpp at the cell surface.
Additionally, secreted Dally expressed in the same cells in which the
endogenous Dally is produced had a weak dominant-negative effect
(Table 1, bab1GAL4>UAS-sec-dally). By contrast, the secreted
Dally expressed elsewhere did not have any detectable effect on
GSC, suggesting that it did not compete with the endogenous Dally
expressed from the cap cells. These results imply that the anterior
tip of the germarium contains the main source of BMP molecules,
which the secreted Dally from cap cells has a better chance to catch
than that from elsewhere.
Dally-dependent short-range BMP signaling: the
unique feature in germline cells
In the imaginal epithelia, glypican Dally and Dlp are essential for
Dpp gradient formation but not for short-range Dpp signaling
because one to two rows of cells in the glypican double mutant clone
were able to respond to the nearby Dpp signals (Belenkaya et al.,
2004). Similarly, in dally mutant ovary, in which the germarium was
emptied due to GSC loss, we observed BMP response in the escort
cells getting close to the cap cells, where the BMP source is
supposed to be (Fig. 5B). However, the germ cell surrounded by the
BMP-responsive somatic cells was refractory to BMP morphogen
DEVELOPMENT
Using pMad to reveal BMP pathway activation, we also observed
BMP response in the somatic cells of dally mutant germaria (Fig.
5E,F). The most likely interpretation of this observation is that Dally
is required for short-range BMP response in germline but not in
somatic cells.
Fig. 5. Dally is required for short-range BMP signaling in
germline, but not in somatic, cells. (A-D)dallygem and DadP1883 were
used, and DadP1883 serves as a lacZ reporter (DadZ) for BMP signaling.
Samples were dissected 3 days after eclosion (A,B,D-F) or after heat
shock (C). (A,C)In the controls (dally+/–,DadZ/+ and hs>bam;DadZ/+),
Dad-lacZ expression was observed in germline cells close to the niche.
In the germarium of hs>bam;DadZ/+, even the differentiated germ cell
(containing branched Spec) showed Dad--gal activation when it is
located in the niche. Arrowheads in A point to the GSCs in the niche;
the arrowhead in C indicates a differentiated germ cell in the niche.
(B)In a germ-cell-less germarium of the dally mutant, the somatic cells
in contact with, or close to, the cap cells expressed Dad--gal.
Arrowhead points to one of these somatic cells in the niche. (D)In
another dally mutant sample, only one GSC was left in the niche
(identified by its large size and single-dot Spec staining; encircled by
dotted line) and Dad--gal was barely detected within it. By contrast,
Dad--gal was expressed in the somatic cells next to this germ cell in
the niche (one of them is indicated by the arrowhead).
(E,F)Immunostaining of pMad reflects BMP activation. In wild-type
(w1118) germarium, pMad was detected in GSCs (arrowheads). In the
dally mutant, germline cells were lost and pMad was observed in
somatic cells (two of them are indicated by arrowheads). TF cells are
highlighted by dotted lines in F. Scale bar: 10m for all panels.
in exactly the same circumstances (Fig. 5D). It appears that when
Dally is compromised, the germline is less sensitive to BMP
signaling and Dally either recruits more ligands to the adjacent germ
cell or somehow enhances its response to BMP. Contrarily, the
somatic cells do not require Dally to sense and respond to BMP
morphogen in short range. Whether Dlp is essential for germarial
somatic cells in BMP response is unclear. What accounts for the
distinction in BMP sensitivity between germline and somatic cells
remains to be investigated.
Is Dally involved in male GSC maintenance?
As in the testis the Dally--gal reporter is expressed in the hub cells
(an equivalent of the female niche; see Fig. S6A in the
supplementary material), we wondered whether dally is also
involved in male GSC determination. We did observe a decrease in
GSC number in the testis (see Fig. S6 in the supplementary material)
(dally80; and data not shown for dallyPZ and dallygem), but we could
not exclude the possibility that such GSC loss was due to the
Development 136 (21)
Fig. 6. Dally acts in the germarial niche to promote GSC fate
determination. Summary of how the fate of germ cells alters in
different genetic conditions. The somatic cells in the illustrations are: TF
(light blue), cap cells (dark blue) and escort cells (green and purple
triangles). In wild type, cap-cell-expressed Dally induces a high BMP
response in a single-cell range to ensure GSC fate in the niche. Removal
of Dally (dally–/–) reduces the BMP concentration in the niche and
prevents germ cells from evoking a BMP response, and consequently
GSCs are lost. Somatic escort cells, however, can elicit a BMP response
in the absence of Dally (in dally–/–, the purple triangular cell next to the
cap cells). When Dally is overexpressed beyond the niche (C587>dally),
GSC-like cells are accumulated in the germarial spaces in addition to
the niche. It appears that extra Dally can expand the pro-GSC
microenvironment and such activity of Dally requires membrane
anchoring (see data in Table 2).
malformation of dally mutant testis (see Fig. S6B in the
supplementary material). Because the tubular structure of the testis
and the external reproductive organ do not properly form without
Dally (Nakato et al., 1995; Tsuda et al., 1999), this ‘no-exit’ cavity
is crowded with continuously developing sperm cells. There is a
strong possibility that the GSC loss around the hub was a result of
the increased physical pressure inside the concealed testis. In
addition, when Dally was overexpressed in the hub, cyst or early
germ cells, no detectable effect was seen (our unpublished data).
Due to the pleiotropic defects of dally mutants, Dally’s function in
spermatogenesis remains to be clarified.
Controlling germline stem cell fate by
manipulating glypican Dally
Dally belongs to the glypican family of HSPG, part of the
extracellular matrix involved in the signaling of many growth
factors. We have provided evidence supporting the idea that Dally
acts in trans to promote short-range BMP signaling and thus GSC
fate in the Drosophila ovary. We also demonstrated that GSC fate
can be manipulated by simply altering Dally expression patterns
(Fig. 4; Table 2). Although targeting Dally to germline cells can
increase the number of GSC-like cells in the germaria (Fig. 4H), we
could not exclude the possibility that Dally acts in trans from
neighboring germ cells instead of autonomously. Regardless of
Dally acting autonomously or not, if Dally is localized on the GSC
surface to enhance BMP signaling, it would be difficult to switch
from high to low BMP response in the presence of ligands. Perhaps
this is why Dally is abundant on the somatic niche cells but not on
GSCs, which would shut down BMP signaling by simply leaving a
Dally-rich environment.
Notably, being Dally dependent or not for short-range BMP
signaling is different for germline and somatic cells. This
phenomenon could be potentially valuable for in vitro manipulation
of germ cells. Tremendous interest and efforts have been focused on
the in vitro manipulation of cell fate, which often involves the
DEVELOPMENT
3634 RESEARCH ARTICLE
addition of growth factors. Applying specific HSPG in trans to
modulate short-range cell response and to specify cell fate would be
more amenable and probably impose fewer side effects than adding
more growth factors.
Acknowledgements
We are grateful to Dahua Chen, Michael A. Crickmore, Shigeo Hayashi, E.
Laufer, Xinhua Lin, Hiroshi Nakato, Rongwen Xi and Ting Xie for sharing flies
and reagents; Rui Zhao for technical help; and Dahua Chen and Rongwen Xi
for critical reading of the manuscript. The Bloomington Stock Center and
Developmental Studies Hybridoma Bank provided invaluable tools. This work is
supported by National Basic Research Program of China (2007CB947503),
National Science Foundation China (30771061), and the BaiRen fund from
Chinese Academy of Sciences.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/21/3627/DC1
References
Akiyama, T., Kamimura, K., Firkus, C., Takeo, S., Shimmi, O. and Nakato, H.
(2008). Dally regulates Dpp morphogen gradient formation by stabilizing Dpp
on the cell surface. Dev. Biol. 313, 408-419.
Belenkaya, T. Y., Han, C., Yan, D., Opoka, R. J., Khodoun, M., Liu, H. and Lin,
X. (2004). Drosophila Dpp morphogen movement is independent of dynaminmediated endocytosis but regulated by the glypican members of heparan sulfate
proteoglycans. Cell 119, 231-244.
Bolivar, J., Pearson, J., Lopez-Onieva, L. and Gonzalez-Reyes, A. (2006).
Genetic dissection of a stem cell niche: the case of the Drosophila ovary. Dev.
Dyn. 235, 2969-2979.
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. (2003a). Dpp signaling silences bam transcription
directly to establish asymmetric divisions of germline stem cells. Curr. Biol. 13,
1786-1791.
Chen, D. and McKearin, D. M. (2003b). 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.
Crickmore, M. A. and Mann, R. S. (2007). Hox control of morphogen mobility
and organ development through regulation of glypican expression.
Development 134, 327-334.
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.
Entchev, E. V., Schwabedissen, A. and Gonzalez-Gaitan, M. (2000). Gradient
formation of the TGF-beta homolog Dpp. Cell 103, 981-991.
Fujise, M., Takeo, S., Kamimura, K., Matsuo, T., Aigaki, T., Izumi, S. and
Nakato, H. (2003). Dally regulates Dpp morphogen gradient formation in the
Drosophila wing. Development 130, 1515-1522.
Jackson, S. M., Nakato, H., Sugiura, M., Jannuzi, A., Oakes, R., Kaluza, V.,
Golden, C. and Selleck, S. B. (1997). dally, a Drosophila glypican, controls
cellular responses to the TGF-beta-related morphogen, Dpp. Development 124,
4113-4120.
Jiang, X., Xia, L., Chen, D., Yang, Y., Huang, H., Yang, L., Zhao, Q., Shen, L.
and Wang, J. (2008). Otefin, a nuclear membrane protein, determines the fate
of germline stem cells in Drosophila via interaction with Smad complexes. Dev.
Cell 14, 494-506.
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.
Kawase, E., Wong, M. D., Ding, B. C. and Xie, T. (2004). Gbb/Bmp signaling is
essential for maintaining germline stem cells and for repressing bam
transcription in the Drosophila testis. Development 131, 1365-1375.
RESEARCH ARTICLE 3635
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 hhmediated mechanisms in the Drosophila ovary. Mol. Cell 7, 497-508.
Li, C. Y., Guo, Z. and Wang, Z. (2007). TGFbeta receptor saxophone nonautonomously regulates germline proliferation in a Smox/dSmad2-dependent
manner in Drosophila testis. Dev. Biol. 309, 70-77.
Li, L. and Xie, T. (2005). Stem cell niche: structure and function. Annu. Rev. Cell
Dev. Biol. 21, 605-631.
Lin, X. (2004). Functions of heparan sulfate proteoglycans in cell signaling during
development. Development 131, 6009-6021.
McKearin, D. M. and Spradling, A. C. (1990). bag-of-marbles: a Drosophila gene
required to initiate both male and female gametogenesis. Genes Dev. 4, 22422251.
Muller, B., Hartmann, B., Pyrowolakis, G., Affolter, M. and Basler, K. (2003).
Conversion of an extracellular Dpp/BMP morphogen gradient into an inverse
transcriptional gradient. Cell 113, 221-233.
Nakato, H., Futch, T. A. and Selleck, S. B. (1995). The division abnormally
delayed (dally) gene: a putative integral membrane proteoglycan required for cell
division patterning during postembryonic development of the nervous system in
Drosophila. Development 121, 3687-3702.
Nybakken, K. and Perrimon, N. (2002). Heparan sulfate proteoglycan
modulation of developmental signaling in Drosophila. Biochim. Biophys. Acta
1573, 280-291.
Ohlstein, B. and McKearin, D. (1997). Ectopic expression of the Drosophila
Bam protein eliminates oogenic germline stem cells. Development 124, 36513662.
Pyrowolakis, G., Hartmann, B., Muller, B., Basler, K. and Affolter, M. (2004).
A simple molecular complex mediates widespread BMP-induced repression
during Drosophila development. Dev. Cell 7, 229-240.
Selleck, S. B. (2000). Proteoglycans and pattern formation: sugar biochemistry
meets developmental genetics. Trends Genet. 16, 206-212.
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.
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 selfrenewal and differentiation. Curr. Biol. 15, 171-178.
Takeo, S., Akiyama, T., Firkus, C., Aigaki, T. and Nakato, H. (2005). Expression
of a secreted form of Dally, a Drosophila glypican, induces overgrowth
phenotype by affecting action range of Hedgehog. Dev. Biol. 284, 204-218.
Teleman, A. A. and Cohen, S. M. (2000). Dpp gradient formation in the
Drosophila wing imaginal disc. Cell 103, 971-980.
Tsuda, M., Kamimura, K., Nakato, H., Archer, M., Staatz, W., Fox, B.,
Humphrey, M., Olson, S., Futch, T., Kaluza, V. et al. (1999). The cell-surface
proteoglycan Dally regulates Wingless signalling in Drosophila. Nature 400, 276280.
Tsuneizumi, K., Nakayama, T., Kamoshida, Y., Kornberg, T. B., Christian, J. L.
and Tabata, T. (1997). Daughters against dpp modulates dpp organizing
activity in Drosophila wing development. Nature 389, 627-631.
Xi, R., Doan, C., Liu, D. and Xie, T. (2005). Pelota controls self-renewal of
germline stem cells by repressing a Bam-independent differentiation pathway.
Development 132, 5365-5374.
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.
Xie, T. and Spradling, A. C. (2000). A niche maintaining germ line stem cells in
the Drosophila ovary. Science 290, 328-330.
Yang, L., Duan, R., Chen, D., Wang, J. and Jin, P. (2007). Fragile X mental
retardation protein modulates the fate of germline stem cells in Drosophila.
Hum. Mol. Genet. 16, 1814-1820.
Zhao, R., Xuan, Y., Li, X. and Xi, R. (2008). Age-related changes of germline
stem cell activity, niche signaling activity and egg production in Drosophila.
Aging Cell 7, 344-354.
DEVELOPMENT
Dally enhances BMP response in female GSC niche