3627RESEARCH ARTICLE

INTRODUCTIONSustained gamete production during adulthood depends on germlinestem cell (GSC) self-renewal, which is also restrained to its properposition to avoid the risk of tumorigenesis. The Drosophila ovaryprovides an excellent system to study GSC fate determinationbecause its unique anatomical layout makes lineage tracingrelatively easy in a complex cell context (for reviews, see Li and Xie,2005; Spradling et al., 2001). Each ovary consists of 16-20 ovariolesthat are basically parallel ‘assembly lines’ of oocyte production. Atthe anterior tip of each ovariole (Fig. 1A), two to three GSCs arerestricted in a space called a niche, the somatic components of whichinclude terminal filament (TF) cells, cap cells and escort stem cells(ESCs) (Decotto and Spradling, 2005; Xie and Spradling, 2000). Tokeep egg production going, each GSC divides to generate twodaughter cells: one remains attached to the cap cells in the niche,thus retaining GSC fate; the other moves out of the niche andbecomes a differentiated cystoblast, which becomes the start of theegg-production line. The early germ cells in the germarium arereadily distinguished by the morphology of a germline-specific andspectrin-rich structure known as a fusome.

BMP morphogens promote GSC fate by preventing theexpression of Bag of marbles (Bam), a key differentiation factor, sothat Bam is repressed in GSCs but expressed in cystoblasts (Chenand McKearin, 2003a; Chen and McKearin, 2003b; McKearin andSpradling, 1990; Ohlstein and McKearin, 1997; Song et al., 2004).Transcriptional silencing of bam is established by a protein complex

including BMP downstream effectors (Chen and McKearin, 2003a;Pyrowolakis et al., 2004; Song et al., 2004) and a nuclear envelopecomponent Ote (Jiang et al., 2008). The direct upstream signals areapparently BMP morphogens, which have been proposed to beprovided by the somatic niche cells, or more specifically by the capcells (Kai and Spradling, 2003; Song et al., 2004; Xie and Spradling,1998). The high BMP signaling is precisely restricted to GSCs in theniche and is turned down in the immediately adjacent cystoblast.Clearly, this represents a typical short-range BMP signaling. AsDecapentaplegic (Dpp), one of the Drosophila BMP homologs, hasbeen shown to act as a long-range morphogen in manydevelopmental contexts (Entchev et al., 2000; Muller et al., 2003;Teleman and Cohen, 2000), how is the steep gradient of BMPresponse achieved in a span of only two cells? Casanueva et al.suggested that Dpp proteins are present throughout the anteriorgermarium, and Bam negatively feeds back to Dpp signaling, thusmaintaining the differentiated state of germ cells in the germariumoutside the niche (Casanueva and Ferguson, 2004). However, thisdoes not explain how the differential expression of Bam isestablished in the first place. Nevertheless, even if BMP moleculesare highly expressed in the niche, it is still difficult to understandhow BMP molecules evoke an ‘on-or-off’ outcome in terms of Bamexpression and specifying distinct cell fate between the adjacentGSCs and cystoblasts.

Division abnormally delayed (Dally) is a glypican member ofheparan sulphate proteoglycan (HSPG) and all glypicans possess aglycosylphosphatidylinositol (GPI) moeity, a hydrophobicmodification anchoring the core protein to the cell surface (Nakatoet al., 1995; Selleck, 2000; Tsuda et al., 1999). Dally has beendemonstrated 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 physicalinteraction between Dally and Dpp was detected (Akiyama et al.,2008). Misexpression of the cell-surface-anchored, but not the

The glypican Dally is required in the niche for themaintenance of germline stem cells and short-range BMPsignaling in the Drosophila ovaryZheng 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 aremaintained in a structure called a niche at the anterior tip of the ovary. The somatic niche cells surrounding the GSCs includeterminal filament cells, cap cells and escort stem cells. Mounting evidence has demonstrated that BMP-like morphogens are theimmediate upstream signals to promote GSC fate by preventing the expression of Bam, a key differentiation factor. In contrast totheir morphogenic long-range action in imaginal epithelia, BMP molecules in the ovarian niche specify GSC fate at single-cellresolution. How this steep gradient of BMP response is achieved remains elusive. In this study, we found that the glypican Dally isessential for maintaining GSC identity. Dally is highly expressed in cap cells. Cell-specific Dally-RNAi, mutant clonal analysis and cell-specific rescue of the GSC-loss phenotype suggest that Dally acts in the cap cells adjacent to the GSCs. We confirmed that Dallyfacilitated BMP signaling in GSCs by examining its downstream targets in various dally mutants. Conversely, when we overexpressedDally in somatic cells outside the niche, we increased the number of GSC-like cells apparently by expanding the pro-GSCmicroenvironment. 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 ensureshigh-level BMP signaling in the ovarian niche and thus female GSC determination.

KEY WORDS: Glypican, Germline stem cell, Niche, BMP, Short-range signaling

Development 136, 3627-3635 (2009) doi:10.1242/dev.036939

1Key Laboratory of Molecular and Developmental Biology, Institute of Genetics andDevelopmental Biology, Chinese Academy of Sciences, Beichen Xilu #1, Beijing100101, P.R. China. 2Graduate School, Chinese Academy of Sciences, 19 YuquanRoad, Beijing 100039, P.R. China.

*Author for correspondence (zhwang@genetics.ac.cn)

Accepted 1 September 2009 DEVELO

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secreted form, of Dally enhances Dpp signaling, and the secretedDally has rather a weak dominant-negative effect (Takeo et al.,2005). Immobilized Dally seems to have the ability toconcentrate/stabilize Dpp or increase the cellular response to Dpp(Akiyama et al., 2008).

Here we present genetic evidence to support the argument thatDally is responsible for establishing the steep gradient of BMPresponse from GSC to cystoblast. By altering the pattern of Dallyexpression, we were able to manipulate the number and position ofthe GSC-like cells. We also demonstrate that Dally defines adistinction between germline and somatic cells in their sensitivity toBMP signals.

MATERIALS AND METHODSFly geneticsP{PZ}dally06464, bab1-GAL4 (BL#6803), en-GAL4, ptc-GAL4, nos-Gal4on III, UAS-GFP and balancers were obtained from the BloomingtonStock Center; c587GAL4 from Ting Xie (Kawase et al., 2004); FRT2Adally80 from Xinhua Lin (Belenkaya et al., 2004); dallygem, dally305, UAS-TM-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

(Crickmore and Mann, 2007); Dad-lacZ from Rongwen Xi (Tsuneizumiet al., 1997; Zhao et al., 2008); esg-lacZ from Shigeo Hayashi. UASp-Dally was constructed by PCR cloning Dally cDNA from UASt-Dally andinserting the cDNA to UASp vector. None of the dally alleles used in thisstudy is a strict null or amorph. All stocks and crosses except theoverexpression experiments were cultured at 25°C. Overexpression ofDally and dallyRNAi were set up at 29°C.

Generation of female germline clonesFLP–FRT-mediated mitotic recombination was used to generate dally80

mutant GSC clones. To generate GSC clones and avoid TF and Cap cellsclones in adult ovaries, 3-day-old females (w hs-flp; FRT2A histone-GFP/FRT2A dally80) were heated at 37°C for 1 hour twice a day for 5consecutive days. w hs-flp; FRT2A hisGFP/FRT2A flies were treated inparallel as controls. Ovaries were dissected at day 2 and day 10 post-heatinduction. GSC clones were identified by the lack of GFP and the presenceof the anterior-positioned dot fusome (spectrosome) in the germline cells(Yang et al., 2007).

X-gal staining of -gal activityAdult ovaries and testes were fixed with 0.5% glutaraldehyde in PBS for 2minutes, and then subjected to the standard X-gal color reaction for 12 hoursat 37°C.

BrdU labelingOvaries were dissected in PBS, incubated for 1 hour in PBS containing 100g/ml BrdU (Sigma) at 25°C, then fixed. The rest of the procedure wasdescribed previously (Li et al., 2007).

Cell death assaysFor TUNEL assay, after fixation in 4% formaldehyde/PBS, samples wereincubated in the mixture of Enzyme and Label solutions (Roche Kit, 1 684795) at room temperature for at least 3 hours.

For Acridine Orange staining, samples were incubated in 1.6 MAcridine Orange/PBS for 20 minutes at room temperature.

For Caspase detection, rabbit anti-Active Caspase-3 (CM1, from BDPharmingen) was used at 1:5000.

Immunohistochemistry and microscopyAll 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 anti-Dlp (developed by P. A. Beachy, DSHB) at 1:50; mouse anti-FasIII(developed by Corey Goodman, DSHB); mouse anti-BamC at 1:4000 andrabbit 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, ColumbiaUniversity, New York, NY) (Crickmore and Mann, 2007); rabbit anti-GFPat 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-Fluor-conjugated secondary antibodies were used at 1:4000 (Molecular Probes,Invitrogen). Fluorescent images were collected using a Zeiss ApoTomemicroimaging system. 63� Oil Plan-Apochromat lens was used to visualizethe details in germarium.

RESULTSdally is required in the somatic niche cells for theovarian GSC maintenanceWe discovered the glypican Dally as one of the candidates that maybe involved in enhancing the BMP response in the female GSCniche when we examined the expression pattern of an enhancer traplacZ in the dally gene (P{PZ}dally06464) (Fig. 1B). In germarium,Dally--gal is expressed mainly in the cap cells, to which the GSCsare attached in the niche (Fig. 1C). Unlike Dally, another Drosophilaglypican, Dally-like (Dlp), is ubiquitous in the germarium, as shownin the immunostaining of the protein distribution (see Fig. S1A inthe supplementary material).

RESEARCH ARTICLE Development 136 (21)

Fig. 1. Dally is expressed in the cap cells of the GSC niche in theDrosophila ovary. Unless otherwise specified, all images are orientedwith anterior to the left. (A)Based on previous findings and especiallythe descriptions in Decotto and Spradling (Decotto and Spradling,2005), we use a cartoon to illustrate the anatomical layout of differentcell types at the ovarian tip: light blue, terminal filament (TF) cells; darkblue, 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 structuretermed the fusome in the germline cells is in red. The morphology andposition of this structure is used to distinguish different stages of theearly germ cells. Note that the fusome dots in GSCs are locatedtowards the cap cells. (B)X-gal staining (blue) reveals the-galactosidase expression in cap cells in the fly ovary carrying a lacZenhancer-trap construct at the dally locus (P{PZ}dally06464). Only thegermarium is shown. (C)Co-immunostaining with the protein markerEngrailed (En, present in both TF and cap cells) confirms the expressionof the dally-lacZ enhancer trap line in the cap cells. Co-localization ofEn, dally-lacZ and DNA appears white in the three-channel mergedimage (arrowheads point to the cap cells).

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To determine if dally is involved in GSC fate regulation, wecounted the GSC numbers in the ovaries of various dally mutantalleles. In wild-type ovaries at adult day 10, two to three GSCs werenormally present in the niche and germaria containing none or oneGSC were rarely found (Table 1). In the homozygous or trans-heterozygous mutants of dally, GSC loss was obvious after 10 daysinto adulthood, and was consistent in all mutants, although atdifferent severity (Fig. 2; Table 1). As early as day 3 after eclosion,complete germ cell loss due to lack of GSC renewal was observedin the strong dally mutant alleles (Fig. 2F,H; Table 1).

The loss of GSC was unlikely to be a result of cell death, becauseadvanced egg chambers were present in the same ovariolesdisplaying GSC loss (Fig. 2B). Using assays to evaluate DNAfragmentation or caspase activation for the signs of cell death, nodifference between the wild type and dally mutant was found (Fig.2I,J). In both wild type and mutant when germ cells were still presentin the germarium, programmed cell death was detected in a fewsporadic somatic cells in the germarial region in which the eggchamber starts to form but not in the cap cells or germline (Fig. 2I,J;see also Fig. S2 in the supplementary material), similar to thatreported previously (Decotto and Spradling, 2005). Additionally,dally mutant clones in GSCs remained in the niche 10 days afterclone induction (see Fig. S3 in the supplementary material),indicating that Dally is not autonomously required in the germlinefor the viability of GSC.

As Dally is highly expressed in the cap cells and required for GSCmaintenance, we wonder if Dally is essential for the viability and/oridentity of the cap cells. We therefore compared the cap cells in wildtype and dally mutant by Lamin C staining, which labels the TF andcap 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 (n46) in the wildtype, and 5.6 (n41) in the dally mutant.

To check whether Dally acts specifically in the somatic nichecells, we tried to rescue dally mutant phenotype by expressingDally in these cells. We took advantage of two Gal4 lines, whichdrive targeted expression in a slightly different range in the niche(see Fig. S4 in the supplementary material). bab1Gal4 is strong inboth TF and cap cells (Fig. 2K; see also Fig. S4 in thesupplementary material) (Bolivar et al., 2006), and its inductionof Dally significantly restored the GSCs in dally mutant (Fig. 2E;Table 1). Different from bab1Gal4, enGal4 is highly expressed inTF cells but was barely detected in the cap cells (Fig. S4 in thesupplementary material) (and personal communications withDahua Chen and A. Gonzalez-Reyes), and its ability to rescueGSC loss in the dally mutant is less than that of bab1Gal4 (Table1). Nevertheless, the cell-specific rescue of dally mutantphenotype implicates that Dally acts in the somatic niche tomaintain GSC fate.

To confirm the action of Dally in the niche, we also employedcell-specific RNAi to reduce Dally levels in somatic niche cells.bab1Gal4-driven Dally RNAi led to a complete GSC loss on day 10in ~30% of the germaria scored (Table 1, n122). On the contrary,Dlp RNAi in the same cells did not have any effect on GSC numberin the niche (see Fig. S1A in the supplementary material and morethan 100 ovarioles scored). Thus, data obtained from various dallymutations, cell-specific RNAi, germline mutant clones, and cell-specific rescue of dally mutations indicate that glypican Dally isrequired for GSC maintenance and probably it exerts this functionin somatic niche cells.

3629RESEARCH ARTICLEDally enhances BMP response in female GSC niche

Table 1. Scoring of GSC-like cells in dally mutants and Dally-RNAi flies

Dissection time after eclosion

Day 3 Day 10*

Genotype 2-3 GSCs/niche 1 GSC/niche 0 GSCs/niche 2-3 GSCs/niche 1 GSC/niche 0 GSCs/niche

126 (100%) 0 0 210 (98%) 2 (1%) 1 (1%)w1118

n=126 n=213126 (77%) 20 (12%) 17 (11%) 37 (23%) 34 (21%) 91(56%)dally305

n=163 n=16247 (16%) 19 (7%) 222(77%) 25 (8%) 3 (1%) 288 (91%)dallygem

n=288 n=31614 (31%) 11 (24%) 20 (45%)dally80

n=45ND (rarely survive to day 10)

52 (84%) 1 (2%) 9 (14%) 16 (39%) 1 (2%) 24 (59%)dallyPZ/dally80

n=62 n=4151 (20%) 10 (4%) 184 (76%) 35 (18%) 2 (1%) 147 (80%)dallygem/dallygem, UAS-dally

n=245 n=184107 (56%) 11 (6%) 73 (38%) 97 (48%) 5 (2%) 99 (50%)en-GAL4; dallygem/dallygem, UAS-

dally n=191 n=201248 (80%) 14 (4%) 51 (16%) 220 (74%) 11 (4%) 65 (22%)bab1-GAL4, dallygem/dallygem,

UAS-dally n=313 n=296251 (81%)† 2 (1%) 57 (18%) 119 (78%)‡ 3 (2%) 30 (20%)C587-GAL4;;dallygem/dallygem,

UAS-dally n=310 n=15266 (66%) 14 (14%) 21 (20%) 17 (26%) 3 (5%) 44 (69%)dallygem, DadP1883

n=101 n=64 (day 6)85 (94%) 2 (2%) 3 (4%) 79 (65%) 7 (6%) 36 (29%)bab1-GAL4>UAS-dallyRNAi

n=90 n=122 (day 10-12)98 (84%) 17 (14%) 2 (2%)bab1-GAL4>UAS-sec-dally

NDn=117 (day 10-13)

*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. D

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dally is essential for BMP signaling in germlinestem cellsDally has been demonstrated to regulate the gradient of Dpp, Hh(Hedgehog) and Wg (Wingless) in the imaginal epithelia (forreviews, see Lin, 2004; Nybakken and Perrimon, 2002). In theovary, the niche-restricted response of Dpp but not Hh or Wg playsa major role in maintaining GSC fate (King et al., 2001; Li and Xie,2005). The contrast in BMP response between GSC and cystoblastcan be revealed by the BMP-downstream targets such as pMad(phosphorylated Mad) or Bam. Mad is the intracellular effector ofBMP signaling and is phosphorylated upon ligand-receptor binding.Bam is required for germ-cell differentiation, and its transcription inGSC is directly repressed by the protein complex containing BMP-pathway effectors (Jiang et al., 2008).

To clarify whether the GSC loss in dally mutants is due to thecompromised BMP signaling, we examined the status of pMad andBam in germarium. In GSCs, the presence of pMad and the absenceof Bam reflect the activation of BMP signaling. These two eventsare normally associated with the GSC fate in the niche (Fig. 3A,B)(dally+/–; bamGFP, a bam promoter fused with GFP). In dally mutantovaries, pMad staining was barely above background and bamtranscription was de-repressed in the GSCs or germ cells occupyingthe niche (Fig. 3A,B) (dally–/–), indicating the downregulation ofBMP signaling in these cells.

To obtain genetic evidence confirming the association of Dallyand BMP signaling in GSC determination, we generated a doublemutant of dally and Dad (Daughters against dpp). Dad is anantagonist of Dpp signaling and its expression is turned on by Dpp

(Tsuneizumi et al., 1997). We found that the Dad mutation partiallyrescued the GSC loss phenotype of dally mutant (Fig. 3C; Table 1;dally Dad double mutant). Additionally, every double mutant femalelaid eggs, whereas the homozygous female of this particular dallyallele (dallygem) rarely did. The genetic interaction between dally andDad provides evidence for their functional interaction in oogenesis.Further, restoration of GSCs in dally mutant by expressing theactivated Tkv (a BMP receptor) in the early germline cells alsosupports the idea that Dally regulates BMP response in the germarialniche (Fig. 3D).

Bam-independent GSC differentiation has been reportedpreviously (Chen and McKearin, 2005; Szakmary et al., 2005; Xi etal., 2005). Mutations in pumilio and pelota lead to GSC loss withoutaffecting Bam expression, and pelota also modulates BMPsignaling. To exclude the possibility that Dally is involved in theBam-independent pathway in addition to the BMP-Bam pathway,we generated dally bam double mutant. dally bam double mutantproduced GSC-like over-proliferation in the germarium, aphenotype indistinguishable from that in the bam single mutant (Fig.3E). Thus, we genetically illustrate that Dally acts in the Bam-dependent pathway. Again, GSC-like cell accumulation in dally bamdouble mutant ovaries also supports the argument against thepossibility that dally mutations cause GSC death. No difference inTUNEL and Caspase3 signals was detectable between bam anddally bam mutants (see Fig. S2E-H in the supplementary material).

To further demonstrate that the lack of pMad signal in dallymutant is not simply due to the absence of GSCs, we took advantageof the dally bam double mutant in which GSC-like cells accumulate

RESEARCH ARTICLE Development 136 (21)

Fig. 2. dally is required in the somatic niche cells for ovarian GSCmaintenance. Vas, a germline-specific marker; Spectrin, a fusomecomponent used to distinguish GSC and the differentiated germ cells.(A)An ovariole (dally+/–) normally contains numerous Vas-positivegermline cells in the germarium (yellow dotted line) and egg chambersof nearly consecutive stages. (B)Ovarioles of dally homozygous mutant(dally–/–, image of dallygem allele) within 3 days after eclosion exhibit thetypical GSC loss phenotype: a tiny germarium missing germ cells(yellow dotted line, note the absence of Vas staining) at the anteriorend, as well as developmentally advanced egg chambers presentposteriorly with large stage gaps. (C)When viewed at highermagnifications, in dally+/– ovaries normally two to three GSCs are visiblein the niche and each GSC contains a single Spectrin dot positionedanteriorly (arrowheads). Germ cells mitotically dividing before reachingthe 16-cell stage are interconnected by branched Spectrin in thegermarium (dally+/–, Vas-positive cell clusters). (D)In the dallyhomozygous mutant (dally–/–, image of dally305 allele), only two germcells are left and the posterior one is apparently moving away from theniche (note the Spec staining between the two green germ cells).Ovaries were dissected from 3-day-old flies. (E)A germarium ofgenotype bab1-Gal4>dally,dallygem 10 days after eclosion. bab1-Gal4 isactive specifically in cap cells and TF cells (see bab1>GFP and cap cellstaining in Fig. S4 in the supplementary material). (F)An example ofdallygem with more severe GSC loss within 3 days after eclosion; lack ofVas-staining indicates that no germline cell was present in thegermarium. (G,H)Lamin C (LamC) labels the TF and cap cells of thegermarial niche. The number of cap cells remained similar between thecontrol (G) and dallygem (H). (I,J)The TUNEL assay detects programmedcell death, which was not detected in cap cells or germline cells ineither control (I) or dallygem (J) germaria. (K)bab1-Gal4,UAS- GFP.(L)Dally Knockdown by bab1-Gal4-controlled RNAi non-autonomouslyled to the empty-germarium phenotype, i.e. no Vas-positive cells wereobserved in the germarium. The effect of RNAi is variable and one ofthe most severe cases is shown here.

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in the germarium. Under both genetic conditions (bam and dallybam mutants), GSC-like cells containing spherical, not branched,fusomes occupied the germarium, and no differentiated germ cellswere observed (Fig. 3E). However, a molecular readout such aspMad exposed the difference between bam and dally bam mutants.pMad was present in the germ cells within the niche in bam singlemutant (Fig. 3F). By contrast, pMad was undetectable in the dallybam double mutant germarium (Fig. 3F). Consistently, bamGFP inanteriormost germ cells was repressed in bam but was derepressedin dally bam, indicating that BMP signaling was compromised whendally was disrupted (Fig. 3G). Taken together, these multiple piecesof evidence illustrate that dally is essential for BMP signaling inGSCs.

Overexpression of membrane-bound Dallyposterior to the niche induces GSC-like expansionand activation of BMP signalingTo see whether Dally expression in somatic cells outside the nicheis sufficient to induce BMP response and more GSC-like cells, weoverexpressed 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 thegermarium (Fig. 4, C587>dally; Table 2, C587>dally). Suchphenotype is similar to that of bam mutant. This was confirmedby the targeted expression in the somatic cells under the controlof a different Gal4 (see Fig. S4 in the supplementary material;Table 2, ptcGAL4>UAS-dally). Notably, it takes time for theGSC-like cells to accumulate and become evident (Table 2)(C587GAL4>UAS-dally and ptcGAL4>UAS-dally, day 5 versusday 15). These GSC-like cells hardly differentiated, leaving bigstage-gaps between the germarium and the adjacent egg chamber(Fig. 4D). In TF cells, Dally overexpression did not show anyeffect on GSC number or position (see Fig. S4 in thesupplementary material; Fig. 4A, en>dally+GFP; Table 2,enGAL4>UAS-dally). Furthermore, Dally expression in germlinecells also induced more GSC-like cells in the germarium (Fig. 4H;Table 2, nos-GAL4VP16>UASp-dally). Thus, overexpression ofDally outside niche causes GSC-like expansion.

To get a clue of whether GSC-like expansion induced by Dallyexpression in somatic germarium is simply due to an increase inGSC production at the anterior tip, or due to more cell divisionselsewhere in the escort cell-surrounded space, the mitotic activity ofthe GSC-like cells was evaluated by BrdU labeling (Fig. 4B). Wild-type germaria often contain BrdU-positive germ cells in a cluster offour or eight cells in the middle of the germarium, reflectingsynchronized division of cystocytes (Fig. 4B, wild type).Interestingly, we observed single cells or two-cell clusters activelydividing along the germarial periphery (Fig. 4B) (C587>dally). Itseems that cell proliferation posterior to the niche at least partiallyaccounts for the GSC-like cell expansion induced by Dally fromescort cells.

As Bam promotes differentiation and is repressed in the GSCs,we asked whether this protein is silenced in the GSC-like cellsinduced by Dally expansion. Bam proteins were present in a fewcystocytes of almost every control germarium (Fig. 4C)(C587>GFP). Not surprisingly, Bam was drastically reduced in thegermaria actively expressing Dally in the escort cells, and even inthe germaria in which GSC-like cells had not completely replacedthe differentiated germ cells (Fig. 4C,E) (C587>dally+GFP).Consistently, using bam-promoter-driven GFP as a reporter,C587Gal4 overexpression of Dally repressed the bamGFPexpression in all GSC-like cells (Fig. 4F).

3631RESEARCH ARTICLEDally enhances BMP response in female GSC niche

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 stainedas a readout of BMP signaling. In dally+/–, pMad was readily detected inthe GSC and is highlighted in a separate channel for pMad (inset,arrowhead indicates the faint signal in the GSC slightly off the focalplane). 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 (theanterior three cells labeled with dot-like Hts staining). In dally–/–,however, bamGFP is upregulated in the GSC-like cell at the anterior tipof the germarium (arrowhead, identified by the single-dot Hts-staininglocated at the anterior pole of the cell). (C)In comparison with dally–/– inFig. 2D,F, the dally–/– Dad–/– double mutant partially restored GSCmaintenance (also see scores in Table 1). (D)The constitutively activatedTkv (tkv*) driven by nos-Gal4 restored the germline cells in the dallygem

germarium. Samples were dissected from 5-day-old flies. (E) Thedally–/–

bam–/– double mutant has the same phenotype as bam–/– in terms ofGSC-like overproliferation, revealed by the mutant germarium filledwith germ cells containing dot-like Spec. (F)pMad signal in theanterior-most germ cells was easily detected in the bam–/– mutant buthardly in the dally–/– bam–/– double mutant. pMad labeling alone isshown in the insets. (G)Similar to that in wild type, bamGFP isrepressed in the most anterior GSC-like cells of the bam–/– mutant(yellow bracket), but is expressed in those of the dally–/– bam–/– doublemutant (yellow bracket). D

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To determine whether BMP signaling is indeed elevated in theGSC-like cells induced by ectopic Dally expression, we examinedthe activation of Dad, another downstream effector of BMPsignaling. In the wild-type controls, Dad expression revealed byDad--gal was strong in the GSCs next to the cap cells and weakerin cystoblasts (Fig. 4G) (C587>GFP). As we predicted, expandedDally expression was sufficient to enhance the Dad--gal signal inmost of the GSC-like cells (Fig. 4G).

Full-length Dally is anchored on the cell surface through theGPI addition at its C-terminus, and Dally’s ability to regulate Dppgradient depends on its membrane-anchor (Takeo et al., 2005).Nakado and colleagues had constructed the secreted form ofDally by deleting the GPI-linked C-terminus (sec-Dally), and thenfused the secreted Dally to a transmembrane domain to makeit membrane-tethered (TM-Dally) (Takeo et al., 2005). Toaddress how Dally acts from cap cells to maintain BMP signalingin the niche thus ensuring the GSC fate, we tried to expressdifferent 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 Dallyexhibited similar though weaker effect than the wild-typeDally when expressed in escort cells (Table 2, compare C587-GAL4>UAS-TM-dally and C587GA14>UAS-dally). By contrast,

the expression of the sec-Dally in somatic cells did not cause anydetectable change in GSC number or position (Table 2, compareC587-GAL4>UAS-sec-dally and C587-GAL4>UAS-dally). Takentogether, we suggest that membrane-anchored Dally caninduce GSC-like cell expansion when expressed outside theniche.

Dally is required for the short-range BMPsignaling in germline but not in somatic cellsIn the process of studying the collaborative effect of Dally and BMPsignaling on GSC-fate determination, we discovered a difference inBMP sensitivity between germline and somatic cells in the dallymutant carrying a Dad-lacZ reporter (Fig. 5). As a downstreamreadout of BMP response, Dad--gal signal is normally detected inthe GSCs and to a lesser degree in cystoblasts (Fig. 4G; Fig. 5A),and could also be induced in the more differentiated germ cells inthe niche after Bam overexpression (Fig. 5C). In dally mutants,when GSCs were completely lost in the germarium, the somaticescort 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 inthe absence of Dally, whereas the somatic cells around this GSCdisplayed obvious Dad--gal expression in the niche (Fig. 5D).

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Fig. 4. Overexpression of Dally induces GSC-like expansion andenhanced BMP signaling. C587Gal4 is expressed in somatic cells,including the escort cells and is visualized by UAS-GFP. (A)In the controlpanel, enGal4 is highly expressed in TF cells (see also Fig. S4 in thesupplementary material). No abnormal fusome (Spec) pattern wasdetected in the ovaries where enGal4 drove the overexpression of Dally(en>dally+GFP). C587Gal4 expands Dally expression to escort cells inaddition to cap cells, and this expression of Dally induced theaccumulation of GSC-like cells in the germarium. Note the Spec dots inthe GSC-like cells contacting escort cells at the germarial periphery.(B)BrdU labeling reveals cell division activities. In wild type, it is frequentlypresent in a cluster of cystocytes dividing synchronously, and is never seenin a cluster of two cells outside the space occupied by GSC andcystoblast. In the germarium, where Dally was expressed by C587Gal4,BrdU signal was observed in multiple two-cell clusters posterior anddistant from the niche. (C,E) Owing to technical difficulties, Bam andSpec cannot be stained simultaneously, but samples of the same age(day 5) were stained separately for either Bam or Spec. (C)Bam proteinstaining was present in some cystocytes of almost every controlgermarium (C587>GFP). In the germarium carrying Dally expressed byC587Gal4, Bam signal was reduced dramatically (C587>dally+GFP, onlybackground staining seen in the Bam channel). (D)In the germarium of a20-day-old C587>dally fly, no egg chambers differentiated out betweenthe germarium and the adjacent stage-10 egg chamber (partial view ofan egg chamber on the right). (E)C587>dally germarium of the same ageas that in C contained differentiated cystocytes (arrowhead indicatesbranched fusome). (F)Consistent with the observation of Bam protein inC, transcription reflected in bam-promoter-driven GFP shows a dramaticreduction in C587>dally fly. bamGFP expression remained in thedifferentiated cells bearing the branched fusome. (G)Germaria of 10-day-old c587>GFP and c587>dally+GFP. DadP1883 serves as a lacZ reporter inthese flies. Dad--gal expression was dramatically upregulated in theGSC-like cells encapsulated by the C587>dally escort cells. Note howinflated the space of C587>dally+GFP is relative to the control on theright, owing to the overproliferation of GSC-like cells. (H)nosGal4 isspecifically active in the early germline cells including GSCs (left panel).Dally expressed by nosGal4 induced an increase of GSC-like cells in thegermarium (right panel, 3 days after eclosion; the yellow arrowheadpoints to one of the GSC-like cells). Scale bars: 10m in D; 10m in Afor all other panels.

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Using pMad to reveal BMP pathway activation, we also observedBMP response in the somatic cells of dally mutant germaria (Fig.5E,F). The most likely interpretation of this observation is that Dallyis required for short-range BMP response in germline but not insomatic cells.

DISCUSSIONTo understand how a steep gradient of BMP response is establishedand thus determines cell fate at single-cell resolution in the GSCniche of Drosophila ovary, we have taken genetic approaches toexamine the role of the glypican Dally in the process. Based on ourcurrent data, we propose a model of how the expression pattern ofDally shapes the BMP-signaling range and consequently determinesdistinct cell fates in the ovarian niche (Fig. 6).

Female GSC fate requires high BMP signaling, which is providedin the ovarian niche. Dally is highly expressed in the cap cells thatcontact the GSCs. Cap-cell-localized and membrane-bound Dallyeither stabilizes/concentrates BMP molecules, or enhances BMPsensitivity to ensure that only the germ cells in contact with cap cellsbecome GSC (Fig. 6, wild type). Removal of Dally, BMPconcentration at the niche or BMP sensitivity in the germ cellsadjacent to cap cells dissipates, and GSCs cannot be maintained andsubsequently differentiate (Fig. 6, dally–/–). Conversely, when Dallyis ectopically overexpressed in the escort cells posterior to the niche,BMP signaling or sensitivity increases in all germ cells encapsulatedby these escort cells, and GSC-like cells accumulate in thegermarium (Fig. 6, C587>dally).

Consistent with this model, the secreted form of Dallyexpressed from cap cells caused GSC loss, possibly by competingwith endogenous membrane-anchored Dally for binding withBMP molecules or by interfering with BMP signaling (see Fig. S5in the supplementary material; Table 1). Because secreted Dallyexpressed from somatic cells in addition to cap cells did not causeGSC expansion as the membrane-anchored Dally did (Table 2), itfurther supports the idea that Dally’s function in the GSC nichedepends on the cap-cell-specific expression and membraneanchoring.

When somatic cells displace the differentiating germ cells in theniche and become close to or in contact with cap cells where the BMPmorphogen 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 isDally dependent or not distinguishes the germline and somatic cells.

The molecular aspects of how Dally modulatesshort-range BMP responseWe have noticed that it took 15 days for Dally overexpression insomatic cells to make all germ cells GSC-like in the germarium(Table 1), although C587Gal4 was active since stage larval 3 at thelatest. One possible explanation is that Dpp is limited and Dallystabilizes Dpp. This possibility is supported by a recent reportdemonstrating that Dally and Dpp physically interact with eachother in the cultured S2 cells and that Dally stabilizes Dpp on the cellsurface 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 nothave the activity that the full-length Dally possesses in terms ofenhancing 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 theendogenous Dally is produced had a weak dominant-negative effect(Table 1, bab1GAL4>UAS-sec-dally). By contrast, the secretedDally expressed elsewhere did not have any detectable effect onGSC, suggesting that it did not compete with the endogenous Dallyexpressed from the cap cells. These results imply that the anteriortip of the germarium contains the main source of BMP molecules,which the secreted Dally from cap cells has a better chance to catchthan that from elsewhere.

Dally-dependent short-range BMP signaling: theunique feature in germline cellsIn the imaginal epithelia, glypican Dally and Dlp are essential forDpp gradient formation but not for short-range Dpp signalingbecause one to two rows of cells in the glypican double mutant clonewere able to respond to the nearby Dpp signals (Belenkaya et al.,2004). Similarly, in dally mutant ovary, in which the germarium wasemptied due to GSC loss, we observed BMP response in the escortcells getting close to the cap cells, where the BMP source issupposed to be (Fig. 5B). However, the germ cell surrounded by theBMP-responsive somatic cells was refractory to BMP morphogen

3633RESEARCH ARTICLEDally enhances BMP response in female GSC niche

Table 2. Targeted overexpression of Dally and its effect on GSC number

Dissection time after eclosion

Day 5 Day 15

Genotype <7 spherical fusomes ≥7 spherical fusomes <7 spherical fusomes ≥7 spherical fusomes

141 (73%) 53 (27%) 0 231 (100%)C587GA14> UAS-dallyn=194 n=231

104 (88%) 14 (12%) 46 (75%) 15 (25%)C587GAL4>UAS-TM-dallyn=118 n=61

196 (100%) 0 285 (100%) 0C587GAL4>UAS-sec-dallyn=196 n=285

54 (57%) 40 (43%) 9 (29%) 22 (71%)ptcGAL4>UAS-dallyn=94 n=31

203 (100%) 0 115 (100%) 0enGAL4>UAS-dallyn=203 n=115

196 (99%) 2 (1%) 191 (85%) 34 (15%)bab1-GAL4>UAS-dallyn=198 n=225

58 (48%) 63 (52%) 12 (16%) 62 (84%)nos-GAL4VP16>UASP-dallyn=121 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 maximalnumber 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 thetotal (n) examined at the same stage of adulthood.sec, secreted; TM, transmembrane.

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in exactly the same circumstances (Fig. 5D). It appears that whenDally is compromised, the germline is less sensitive to BMPsignaling and Dally either recruits more ligands to the adjacent germcell or somehow enhances its response to BMP. Contrarily, thesomatic cells do not require Dally to sense and respond to BMPmorphogen in short range. Whether Dlp is essential for germarialsomatic cells in BMP response is unclear. What accounts for thedistinction in BMP sensitivity between germline and somatic cellsremains 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 thesupplementary material), we wondered whether dally is alsoinvolved in male GSC determination. We did observe a decrease inGSC number in the testis (see Fig. S6 in the supplementary material)(dally80; and data not shown for dallyPZ and dallygem), but we couldnot exclude the possibility that such GSC loss was due to the

malformation of dally mutant testis (see Fig. S6B in thesupplementary material). Because the tubular structure of the testisand the external reproductive organ do not properly form withoutDally (Nakato et al., 1995; Tsuda et al., 1999), this ‘no-exit’ cavityis crowded with continuously developing sperm cells. There is astrong possibility that the GSC loss around the hub was a result ofthe increased physical pressure inside the concealed testis. Inaddition, when Dally was overexpressed in the hub, cyst or earlygerm cells, no detectable effect was seen (our unpublished data).Due to the pleiotropic defects of dally mutants, Dally’s function inspermatogenesis remains to be clarified.

Controlling germline stem cell fate bymanipulating glypican DallyDally belongs to the glypican family of HSPG, part of theextracellular matrix involved in the signaling of many growthfactors. We have provided evidence supporting the idea that Dallyacts in trans to promote short-range BMP signaling and thus GSCfate in the Drosophila ovary. We also demonstrated that GSC fatecan be manipulated by simply altering Dally expression patterns(Fig. 4; Table 2). Although targeting Dally to germline cells canincrease the number of GSC-like cells in the germaria (Fig. 4H), wecould not exclude the possibility that Dally acts in trans fromneighboring germ cells instead of autonomously. Regardless ofDally acting autonomously or not, if Dally is localized on the GSCsurface to enhance BMP signaling, it would be difficult to switchfrom high to low BMP response in the presence of ligands. Perhapsthis is why Dally is abundant on the somatic niche cells but not onGSCs, which would shut down BMP signaling by simply leaving aDally-rich environment.

Notably, being Dally dependent or not for short-range BMPsignaling is different for germline and somatic cells. Thisphenomenon could be potentially valuable for in vitro manipulationof germ cells. Tremendous interest and efforts have been focused onthe in vitro manipulation of cell fate, which often involves the

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Fig. 5. Dally is required for short-range BMP signaling ingermline, but not in somatic, cells. (A-D)dallygem and DadP1883 wereused, and DadP1883 serves as a lacZ reporter (DadZ) for BMP signaling.Samples were dissected 3 days after eclosion (A,B,D-F) or after heatshock (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 islocated 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 cellsin contact with, or close to, the cap cells expressed Dad--gal.Arrowhead points to one of these somatic cells in the niche. (D)Inanother dally mutant sample, only one GSC was left in the niche(identified by its large size and single-dot Spec staining; encircled bydotted line) and Dad--gal was barely detected within it. By contrast,Dad--gal was expressed in the somatic cells next to this germ cell inthe 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 thedally mutant, germline cells were lost and pMad was observed insomatic cells (two of them are indicated by arrowheads). TF cells arehighlighted by dotted lines in F. Scale bar: 10m for all panels.

Fig. 6. Dally acts in the germarial niche to promote GSC fatedetermination. Summary of how the fate of germ cells alters indifferent genetic conditions. The somatic cells in the illustrations are: TF(light blue), cap cells (dark blue) and escort cells (green and purpletriangles). In wild type, cap-cell-expressed Dally induces a high BMPresponse in a single-cell range to ensure GSC fate in the niche. Removalof Dally (dally–/–) reduces the BMP concentration in the niche andprevents germ cells from evoking a BMP response, and consequentlyGSCs are lost. Somatic escort cells, however, can elicit a BMP responsein the absence of Dally (in dally–/–, the purple triangular cell next to thecap cells). When Dally is overexpressed beyond the niche (C587>dally),GSC-like cells are accumulated in the germarial spaces in addition tothe niche. It appears that extra Dally can expand the pro-GSCmicroenvironment and such activity of Dally requires membraneanchoring (see data in Table 2).

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addition of growth factors. Applying specific HSPG in trans tomodulate short-range cell response and to specify cell fate would bemore amenable and probably impose fewer side effects than addingmore growth factors.

AcknowledgementsWe are grateful to Dahua Chen, Michael A. Crickmore, Shigeo Hayashi, E.Laufer, Xinhua Lin, Hiroshi Nakato, Rongwen Xi and Ting Xie for sharing fliesand reagents; Rui Zhao for technical help; and Dahua Chen and Rongwen Xifor critical reading of the manuscript. The Bloomington Stock Center andDevelopmental Studies Hybridoma Bank provided invaluable tools. This work issupported by National Basic Research Program of China (2007CB947503),National Science Foundation China (30771061), and the BaiRen fund fromChinese Academy of Sciences.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/136/21/3627/DC1

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