Somatic cell lineage is required for differentiationand not maintenance of germline stem cells inDrosophila testesJaclyn G. Y. Lima and Margaret T. Fullera,b,1

Departments of aDevelopmental Biology and bGenetics, Stanford University School of Medicine, Stanford, CA 94305

Contributed by Margaret T. Fuller, September 23, 2012 (sent for review May 29, 2012)

Adult stem cells are believed to be maintained by a specializedmicroenvironment, the niche, which provides short-range signalsthat either instruct stem cells to self-renew or inhibit execution ofpreprogrammed differentiation pathways. In Drosophila testes, so-matic cyst stem cells (CySCs) and the apical hub form the niche forneighboring germline stem cells (GSCs), with CySCs as the proposedsource of instructive self-renewal signals [Leatherman JL, Dinardo S(2010) Nat Cell Biol 12(8):806–811]. In contrast to this model, weshow that early germ cells with GSC characteristics can be main-tained over time after ablation of CySCs and their cyst cell progeny.Without CySCs and cyst cells, early germ cells away from the hubfailed to initiate differentiation. Our results suggest that CySCs donot have a necessary instructive role in specifying GSC self-renewaland that the differentiated progeny of CySCs provide an environ-ment necessary to trigger GSC differentiation. This work highlightsthe complex interaction between different stem cell populations inthe same niche and how the state of one stem cell population caninfluence the fate of the other.

The ability of a stem cell niche to maintain a population of stemcells ensures the continued availability of adult stem cells to

replenish and repair specific tissues throughout the lifetime of anorganism (1, 2). Failure of a niche to maintain its appropriate stemcell population may lead to degeneration, aging, or an inability torepair tissue damage (3). Conversely, failure of a niche to properlyregulate differentiation versus proliferation may contribute to thegenesis of cancer in adult stem cell lineages (4). A comprehensiveunderstanding of how the local microenvironment of the stem cellniche functions suggests strategies for expansion of adult stemcell populations in vitro, facilitates design of artificial niches fortransplantation, and provides ideas for increasing maintenanceand functionality of endogenous adult stem cell populations usedfor regenerative medicine.The Drosophila testis stem cell niche, a key model for un-

derstanding how themicroenvironment regulates stem cell behavior(5–7), supports two distinct adult stem cell populations—germlinestem cells (GSCs) and cyst stem cells (CySCs)—both ofwhich attachto a cluster of postmitotic somatic cells that form theapical hub (Fig.1A). GSCs and CySCs normally divide with oriented spindles toproduce daughters that remain next to the hub and self-renew anddaughters displaced away from the hub that initiate differentiation(8, 9). GSCs give rise to gonialblasts (Gb) and CySCs give rise topostmitotic cyst cells (10), a pair of which encapsulates each Gb toform a cyst. The encapsulated Gb undergoes four rounds of syn-chronous transit-amplifying (TA) divisions before entering meiosisand terminal differentiation (Fig. 1A).Both the apical hub and the CySCs influence the GSC state. A

cytokine-like signal from the hub activates the JAK-STAT sig-naling pathway in both GSCs and CySCs (11, 12). Although JAK-STAT signaling is required cell autonomously for CySC mainte-nance, it is not necessary to retain GSCs in their stem cell state.Rather, activity of Stat in the germline is essential for continuedattachment of GSCs to the hub and retains GSCs in their niche(13). Several lines of evidence suggest that CySCs provide a nichefor maintenance of GSCs (13–15). Consistent with this model, it

has been proposed that self-renewal of GSCs is specified by in-structive signal(s) from the CySCs, with a likely candidate beingTGF-β signaling (13).Here we show that early germ cells can bemaintained next to the

hub in testes in which CySCs and cyst cells had been permanentlyablated. We further show that the progeny of GSC-like cells dis-placed from the hub failed to initiate the TA program in the ab-sence of CySCs and cyst cells, and instead continued to proliferateas undifferentiated cells. Our findings suggest that CySCs do notplay a required instructive role in GSC self-renewal and that cystcells, the differentiated progeny of CySCs, are required for properonset of the germline differentiation.

ResultsSomatic CySCs and cyst cells in the testis were ablated by forcedexpression of the apoptotic activator Grim (16) (Fig. 1 C–H).Flies carrying the temperature-sensitive Gal4 repressor, tub-Gal80ts; the somatic-specific driver, c587Gal4; and UAS Grimwere grown to adulthood at 18 °C to avoid lethality during de-velopment, then shifted to 30 °C to induce Grim expression.Before the shift, immunofluorescence using antibodies againstthe transcription factor Traffic Jam (Tj) showed densely stainednuclei of CySCs flanking GSCs next to the hub (arrowheads),cyst cells (arrows), and lighter stained nuclei in the apical hub(Fig. 1B). By 1 d postshift to 30 °C, CySCs had been completelyablated in most testes (80%, n = 84 testes) as marked by theabsence of Tj positive nuclei and presence of only Vasa-positivecells next to the hub (Fig. 1 C and H). Because prolonged ex-pression of Grim caused mortality, flies were shifted back topermissive temperature after 1 d at 30 °C to allow the effects ofloss of CySCs to be followed over time.

Early Germ Cells Can Be Maintained After Ablation of CySCs and CystCells. Ablation of CySCs and cyst cells had profound but un-expected effects on germ cell behavior. Consistent with previousobservations (13), 49% (n = 59) of testes that lacked CySCs or cystcells had no detectable early germ cells 7 d after pulse expression ofGrim (Fig. 1D andH).However, early germ cells were present nextto the hub in the remaining 51% (Fig. 1 E and H). Similar pro-portions of testes that lacked CySCs but retained germ cells werealso observed at 14 d (57%, n = 94 testes) and 21 d (49%, n = 81testes) after pulse expression of Grim (Fig. 1H), suggesting thatearly germ cells were stably maintained next to the hub in the ab-sence of CySCs and cyst cells. In wild-type testes, GSCs around thehub were physically separated from one another by cytoplasmicextensions fromCySC cell bodies (Fig. 1B). In contrast, early germcells present next to the hub 7 d after ablation of CySCs and cyst

Author contributions: J.G.Y.L. designed research; J.G.Y.L. performed research; J.G.Y.L. andM.T.F. analyzed data; and J.G.Y.L. and M.T.F. wrote the paper.

The authors declare no conflict of interest.1To whom correspondence should be addressed. E-mail: mtfuller@stanford.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1215516109/-/DCSupplemental.

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cells were arranged in a tightly packed collar (Fig. 1E). In additionto collar germ cells next to the hub, a second population of earlygerm cells accumulated away from the hub over time in testes inwhich CySCs and cyst cells had been ablated (Fig. 1 F and G).Depletion of CySCs and cyst cells by RNAi knockdown of Stat inthese cells showed similar results as observed after ablation ofCySCs and cyst cells by pulse expression of Grim (Fig. S1 A–E).

Collar Germ Cells Maintained Next to the Hub in the Absence of CySCsand Cyst Cells Had GSC Characteristics. Collar germ cells maintainednext to the hub in the absence of CySCs and cyst cells retainedseveral GSC characteristics over time.Hub cells send a short-rangecytokine-like signal, Unpaired (Upd), that activates the JAK-STAT signaling pathway in neighboring GSCs (11, 12). Like con-trol GSCs (arrowheads, Fig. 2A), collar germ cells were Stat-pos-itive (arrowheads, Fig. 2B), suggesting that they are respondingto the Upd signals from the hub. However, Stat protein was not

detected in the mass of germ cells away from the hub (Fig. 2B),suggesting that these cells do not receive or respond to hub signalsand were in a different state than germ cells maintained next to thehub. Similar results were observed after CySCs and cyst cells weredepleted by RNAi knockdown of Stat (Fig. S2A).Collar germ cells maintained in the absence of CySCs and cyst

cells also displayed oriented centrosomes (arrows, Fig. 2C andD),a GSC characteristic that programs spindle orientation and theasymmetric outcome of stem cell division (8, 17). When the du-plicated centrosomes separate in the G2 phase of the cell cycle inwild-type Drosophila male GSCs, one normally remains near theGSC–hub interface, whereas the other migrates to the oppositeside of the cell (8, 18). In testes lackingCySCs and cyst cells, 92%ofcollar germ cells with two centrosomes (n = 78) displayed normalcentrosome orientation, comparable to the 94% observed in con-trol GSCs (n = 77) (Fig. 2D). Similar results were observed afterCySCs and cyst cells were depleted by RNAi knockdown of Stat inthese somatic cells (Fig. S2 C and D).Collar germ cells also continued to proliferate long term in the

absence of CySCs and cyst cells. Immunofluorescence using themitotic marker phospho-histone H3 (PH3) showed that collargerm cells next to the hub divided as individual cells (Fig. 2E) anddid so at a rate similar to GSCs in control testes; 2.8% (n = 12/425) for collar germ cells compared with 2.6% (n = 13/497) forcontrol GSCs. Collar germ cells also had dot fusomes (arrows,Fig. 2F), similar to wild-type GSCs and Gbs, and unlike thebranched fusomes typical of TA cells (Fig. 1A, arrows in Fig. 3A).

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Fig. 1. Early germ cells can be maintained after ablation of CySCs and cystcells. (A) Diagram of Drosophila spermatogenesis at the apical tip of thetestis. (Red) fusomes; (green) Bam protein expression. (B–G) Immunofluo-rescence images of c587Gal4; UAS Grim; tubGal80ts testes stained with anti-FasIII (white, hub), anti-Vasa (red, germ cells), and anti-Tj (green) nucleiof hub, CySCs, and cyst cells). (B) Newly eclosed flies before shift to 30 °C.(Arrowheads) CySCs; (arrows) cyst cells. (C) Flies shifted to 30 °C for 1 d. (D)Flies shifted to 30 °C for 1 d and back to 18 °C for 7 d. (E) Flies shifted to 30 °Cfor 1 d and back to 18 °C for 7 d. (F) Flies shifted to 30 °C for 1 d and backto 18 °C for 14 d. (G) Flies shifted to 30 °C for 1 d and back to 18 °C for 21 d.(H) Bar graph depicting phenotype distribution at different time points.(Blue bar) Testes with CySCs and/or cyst cells (incomplete ablation); (red bar)testes with early germ cells but lacked CySCs or cyst cells; (green bar) testeslacking early germ cells, CySCs, and cyst cells. No significant difference inphenotype distribution was observed among the 7-, 14-, and 21-d timepoints. (Scale bar: B–G, 10 μm.)

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Fig. 2. Collar germ cells maintained next to the hub in the absence of CySCsand cyst cells had GSC characteristics. Immunofluorescence images of testesstained with anti-Vasa (red, germ cells) from (A) +; UAS Grim control flies and(B, C, E, and F) c587Gal4; UAS Grim; tubGal80ts experimental flies shifted to30 °C for 1 d and back to 18 °C for 21 d. (A and B) Staining with anti-Stat(green) and anti-FasIII (white, hub). (Arrowheads) Early germ cells next to thehub; (*) Stat present at lower level in a Gb in control testes. (C) Staining withanti-γ tubulin (green, centrosomes) and anti-E-cadherin (white, hub). (Arrows)Oriented centrosomes in germ cell next to the hub. (D) Bar graph depictingcentrosome orientation in +; UAS Grim flies and c587Gal4; UAS Grim; tub-Gal80ts flies. (Blue bar) Germ cells next to the hub that had two orientedcentrosomes; (red bar) germ cells next to the hub that had two misorientedcentrosomes. (E) Staining with anti-PH3 (green, mitotic marker) and anti-FasIII(white, hub). (F) (Green) Anti-spectrin (fusome) and anti-FasIII (hub). (Arrows)Dot fusomes in germ cells next to the hub. (Scale bar: A–C, E, and F; 10 μm.)

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Without CySCs or Cyst Cells, Mass Germ Cells Away From the HubFailed to Properly Enter the TA Program. Although CySCs and cystcells were not required for maintenance of GSC-like cells next tothe hub, these somatic cells were required for germ cells to properlyenter theTAprogram, an early step of differentiation inwhich stemcell daughters execute finite rounds of mitotic division before en-tering terminal differentiation. Control TA spermatogonia dividedwith incomplete cytokinesis and remained interconnected byfusome, a membranous structure that normally forms a branchednetwork within each cyst (Fig. 1A, arrows in Fig. 3A). In contrast,most mass germ cells had dot (arrowheads, Fig. 3B) or dumbbellshaped fusomes (arrows, Fig. 3B), reminiscent of GSCs orgonialblasts. TA cells within a cyst also undergo mitosis in syn-chrony; in control testes, 26% of early germ cell divisions awayfrom the hub occurred as single cells, 25% as 2-cell clusters and49%as either 4- cell or 8- cell clusters (n= 53 divisions, Fig. 3C andE). However, 90% of mass germ cell divisions occurred as singlecells (n= 59 divisions, Fig. 3D andE). Even thoughmass germ cellsdivided as single cells, they did so at a lower rate compared withGSCs; 0.72% (39/5393) for mass germ cells compared with 2.8%and 2.6% for collar germ cells and control GSCs respectively.Similar results were observed after CySCs and cyst cells were de-pleted by RNAi knockdown of Stat (Fig. S2 B and F).TA spermatogonial cells turn on expression of bag-of-marbles

(Bam), a gene necessary for germline differentiation (19) by the four-cell stage (Fig. 1A) (20). In control testes, expression of GFP froma Bam-GFP fusion protein (21) was detected in 4-, 8-, and 16-cellspermatogonial cysts, but not in GSCs or Gbs (Fig. 4A). However,mass germ cells that accumulated in testes depleted of CySCs andcyst cells showed no detectable expression of Bam-GFP (Fig. 4B).

Immunofluorescence using the antibodies against phosphoSMAD(pSMAD) revealed that pSMAD levels were highly elevated in massgerm cells (Fig. 4D), even though pSMADwas not detected inGSCsand spermatogonia in control testes (Fig. 4C).

DiscussionOur observation that GSCs can be maintained long term in theDrosophila testis without CySCs indicates that CySCs are notrequired for GSC self-renewal as previously proposed (13). GSCsmay have an intrinsic capacity to undergo prolonged self-renewalwithout external environmental stimuli, as has been suggested formouse ES cells (22). It is also possible that GSC maintenancecould be specified by other unidentified signal(s) from the hub.Our finding that cyst cell function is required for early germ cells

to properly enter the TA program suggests that a key role of thesesomatic support cells is to promote GSC differentiation, likely byactive differentiation inputs and/or by down-regulation of the re-sponse of germ cells toTGF-β signaling (Fig. 4F). A requirement forproper cyst cell function for germ cells to initiate differentiation isconsistentwith previousfindings that activity of theEGF receptor orits downstream effector Raf in somatic cells in the testis is requiredfor male germ cells to enter the TA program (23, 24). The TGF-βpathway has been shown to be required cell-autonomously for GSCmaintenance (25–27). The high levels of pSMAD observed inmass germ cells suggest that the somatic support cells may nor-mally function in either physically restricting TGF-β signalingfrom reaching germ cells away from the hub or in providing cuesthat down-regulate the ability of germ cells to respond to TGF-βsignaling. However, we note that elevated pSMADexpression wasalso observed in ectopic GSC-like cells depleted of Stat (13), eventhough somatic cells were still present and seemed to functionnormally in promoting germ cell differentiation.The absence of Bamexpression in testes inwhichCySCs and cyst

cells had been ablated could be attributed to the high levels ofTGF-β signaling in mass germ cells. Consistent with this observa-tion, TGF-β signals from niche cells repress the expression of Bamin GSCs in the female germline (7). Unlike the female germlinehowever, function of Bam and deactivation of TGF-β signaling isnot necessary for male GSCs to enter the TA program (19, 27),suggesting that other factors are involved in instructingmale GSCsto initiate differentiation. Our finding that cyst cells are requiredfor germ cells to enter the TA divisions raises the possibility thatcyst cells may be the source of such factors.CySCs may normally provide an environment that allows GSC

maintenance because CySCs are kept in a state in which they areunable to induce germ cell differentiation. We posit that Chinmoand the transcriptional repressor Zfh1, which function cell au-tonomously to maintain CySC identity, may also act to block ex-pression of cyst cell programs that instruct germ cells to initiatedifferentiation. This model accounts for the results that forcedoverexpression of either Zfh1 or Chinmo in the cyst cell lineageresulted in continued proliferation of undifferentiated early germcells (14, 15).We note that in some cases germ cells were not maintained after

the ablation of the somatic cell lineage. The absence of germ cellsin 49% of testes depleted of CySCs and cyst cells may be due toleaky expression of the c587Gal4 driver in the germline, compro-mised hub function or both. Low GFP expression had been occa-sionally detected inGSCs in c587Gal4; UASGFP flies (∼1–2GSCsfrom 20 testes), suggesting that the driver may have low activity inthe germline. Ablation of somatic cells may also have negativeconsequences on the hub, because hub cells are derived from thesame somatic lineage (28). Thus, even though the hub is physicallypresent in testes in which CySCs and cyst cells had been ablated, itmay not be fully functional in some testes and therefore fail topromote germ cell attachment to the hub.Different aspects of the male GSC program appear to be

controlled by multiple inputs from the local microenvironment at

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Fig. 3. Without CySCs or cyst cells, mass germ cells away from the hub failedto properly enter the TA program. Immunofluorescence images of testesstained with anti-Vasa (red, germ cells) from (A, C) +; UAS Grim control fliesand (B and D) c587Gal4; UAS Grim; tubGal80ts experimental flies shifted to30 °C for 1 d and back to 18 °C for 21 d. (A and B) (Green) Anti-spectrin(fusome) and anti-FasIII (hub). (A) (Arrows) Branched fusomes in TA sper-matogonia. (B) (Arrowheads) Dot fusomes; (arrows) dumbbell-shapedfusomes. (C and D) Staining with anti-PH3 (green, mitotic marker) and anti-FasIII (white, hub). (Arrow) Synchronous spermatogonial division; (arrow-head) single cell division. (E) Bar graph depicting types of divisions of earlygerm cells away from the hub. (Blue bar) Dividing single cell; (red bar) di-viding two-cell cysts; (green bar) dividing four- and/or eight-cell cyst. (Scalebar: A–D; 10 μm.)

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the testis apical tip, indicating a complex niche. In addition toinfluences exerted by CySCs, two key characteristics of GSCsappear to be governed directly by activation of the JAK-STATsignaling pathway in GSCs by signals from the hub: attachmentto the hub and orientation of centrosomes with respect to theGSC–hub interface, resulting in oriented stem cell division.The function of Stat has been shown to be cell-autonomouslyrequired for continued attachment of GSCs to the hub (13).Orientation of centrosomes toward the hub in GSCs is alsodependent on action of the JAK-STAT signaling pathway;centrosomes became misoriented in germ cells next to the hubsoon after Stat was depleted in all cells by shifting statts mutantflies to restrictive temperature (13). Our finding that the collargerm cells maintained in absence of CySCs have orientedcentrosomes indicates that the centrosome orientation programcharacteristic of GSCs is likely an effect of Stat activation in theGSCs by Upd signals from the hub rather than an indirectconsequence of signals from the CySCs.Although the model that niches serve primarily to promote

stem cell self-renewal may be true for many adult stem cell sys-tems (7, 29, 30), our results indicate that components of themicroenvironment may play key roles that can trigger stem celldifferentiation. Indeed, as we come to understand the workings ofmore complex niches in mammalian systems, we may find com-binations of these two opposing influences, with some areas of theniche promoting self-renewal of stem cells, whereas neighboringregions drive differentiation of stem cell daughters. A balancebetween these competing regulatory machineries may serve as animportant defense against stem cell overproliferation or loss.Likewise, loss or subversion of the ability of local stromal cells topromote differentiation may contribute to abnormal proliferationof undifferentiated cells in tumors.

Materials and MethodsFly Husbandry and Stocks. Flieswere raised on standard cornmealmolasses agarmedium. Fly strains used in this study are c587Gal4 (S. Hou, National Cancer In-stitute-Frederick, Frederick,MD), traffic jamGal4 (Kyoto Stock Center, DGRC no.104055), eyaA3 Gal4 (S. Dinardo, University of Pennsylvania, Philadelphia), UASGrim (D. Bennett, University of Liverpool, Liverpool, UK), UAS stat92E RNAi(Vienna Drosophila Resource Center [VDRC] 106980), and tubGal80ts (Bloo-mington Drosophila Stock Center 7018). Bam expression was analyzed usinga transgenic BamGFP protein fusion reporter (D. McKearin, Chevy Chase, MD).

To ablate CySC and cyst cells, virgin female c587Gal4; Sco/Cyo; tubGal80ts

flies were crossed to w/Y; UAS Grim males and the progeny were grownto eclosion at 18 °C. Newly eclosed (0–1 d old) males were shifted to 30 °C for24 h, and then returned to 18 °C. In parallel, virgin female ywflieswere crossedto w/Y; UAS Grim males to generate control flies; the progeny were grown asdescribed previously. Either c587 Gal4; Sco or Cyo/UAS Grim; tubGal80ts ex-perimental flies, or yw; +/UAS Grim control flies were analyzed at indicatedtime points by whole mount immunofluorescence and confocal microscopy.

To induce RNAi of Stat in CySCs and cyst cells, w; tj Gal4; tubGa80ts werecrossed to w/Y; UAS stat92E RNAi (VDRC 106980) and the progeny wasgrown to eclosion at 18 °C. Newly eclosed males were shifted to 30 °C. Inparallel, virgin female yw flies were crossed to w/Y; UAS stat92E RNAi malesand were grown as described previously to generate control flies. Either w;tj Gal4/UAS stat92E RNAi; tub-Gal80ts/+ experimental, or y w; UAS stat92ERNAi/+ control flies were analyzed at indicated time points by whole mountimmunofluorescence and confocal microscopy.

Whole Mount Immunofluorescence. Testes were dissected in 1× PBS and fixedwith 4% (vol/vol) formaldehyde diluted in 1× PBS for 20 min at roomtemperature. Testes were then permeabilized for 2 h in 1× PBS with 0.6%(vol/vol) Triton-X 100 and 0.6% (wt/vol) sodium deoxycholate at roomtemperature, then washed once with 1× PBS with 0.1% (vol/vol) Triton-X100 (PBST). Primary and secondary antibody incubations were performed in1× PBST with 3% (wt/vol) BSA; overnight at 4 °C for primary and 2 h in thedark at room temperature for secondary. Testes were washed with 1× PBSTthree times at room temperature after each antibody incubation andmounted in Vectashield mounting media with DAPI (Vector Labs). Primaryantibodies used were guinea pig anti-Tj (a gift from D. Godt, Toronto;1:5,000); mouse anti-Fasciclin III (7G10; Developmental Studies HybridomaBank [DSHB]; 1:10); mouse anti-DE-cadherin (DCAD2; DSHB; 1:40); mouseanti-Alpha Spectrin (3A9; DSHB; 1:5); goat anti-Vasa (dC-13; Santa CruzBiotechnology; 1:100); rabbit anti-GFP (Invitrogen; 1:3,000); rabbit anti-STAT92E(a gift from D. Montell, Baltimore; 1:1,000); rabbit anti-STAT92E (a gift fromE. Bach, New York; 1:1,000); rabbit anti-PhosphoHistone3 Thr3 (Upstate Bio-technology/Millipore; 1:200);mouse anti-γ tubulin (Sigma; 1:100);mouse anti-Bam(DSHB; 1:10); and rabbit anti-pSMAD (gift from E. Laufer, New York; 1:1,000).DyLight conjugated donkey secondary antibodies were used at 1:500(Jackson ImmunoResearch Laboratories). Images were taken using a LeicaSP2 AOBS Confocal Laser Scanning microscope and processed with AdobePhotoshop CS4. Early germ cells were defined to be Vasa-positive cells thatwere <10 μm in diameter and GSCs were scored as Vasa-positive cells incontact with the hub. For centrosome orientation counts, only GSCs thatcontained two centrosomes were scored in the analysis. Centrosomes werescored as oriented if at least one of them was at the GSC-hub interface. Forthe purpose of determining the mitotic index of mass germ cells, Z-stackimages were first taken using the Leica TCS SP5 system. The images werethen compiled into 3D renditions and the percentage of PH3 (green) cellsthat were both Vasa- (red) and DAPI- (blue) positive were measured usingVolocity 3D Image Analysis Software.

ACKNOWLEDGMENTS. This work was supported by grants from the StarrStanford Graduate Fellowship and the Genentech Graduate Fellowship (toJ.G.Y.L.) and National Institutes of Health Grant R01GM080501 (to M.T.F.).The support of the Reed-Hodgson Professorship in Human Biology (M.T.F.)made part of this work possible.

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Fig. 4. TGF-β signaling was elevated in mass germ cells. Immunofluores-cence images of testes stained with anti-Vasa (red, germ cells) from (A and C)+; UAS Grim control flies and (B and D) c587Gal4; UAS Grim; tubGal80ts

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