Dpp Signaling Silences bam Transcription Directly to Establish Asymmetric Divisions of Germline Stem Cells

Current Biology, Vol. 13, 1786–1791, October 14, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/j.cub.2003.09.033

Dpp Signaling Silences bam Transcription Directly to Establish Asymmetric Divisions of Germline Stem Cells Dahua Chen and Dennis McKearin* University of Texas Southwestern Medical Center at Dallas Dallas, Texas 75390-9148

Summary Stem cells execute self-renewing and asymmetric cell divisions in close association with stromal cells that form a niche [1]. The mechanisms that link stromal cell signaling to self-renewal and asymmetry are only beginning to be identified, but Drosophila oogenic germline stem cells (GSCs) have emerged as an important model for studying stem cell niches. A member of the Bone Morphogenetic Protein (BMP) ligand family, Decapentaplegic (Dpp), sustains ovarian GSCs by suppressing differentiation in the stem cell niche (Figure 1A). Dpp overexpression expands the niche, blocks germ cell differentiation, and causes GSC hyperplasty [2, 3]. Here, we show that the bag-of-marbles (bam) differentiation factor is the principal target of Dpp signaling in GSCs; ectopic bam expression restores differentiation even when Dpp is overexpressed. We show that the transcriptional silencer element in the bam gene integrates Dpp control of bam expression. Finally and most significantly, we demonstrate for the first time that Dpp signaling regulates bam expression directly since the bam silencer element is a strong binding site for the Drosophila Smads, Mad and Medea. These studies provide a simple mechanistic explanation for how stromal cell signals regulate both the self-renewal and asymmetric fates of the products of stem cell division. Results and Discussion GSCs divide in the anterior/posterior axis, and this division produces daughters with different fates [1]. The anterior cell of a GSC division retains contact with the stromal cap cells [4], maintains high levels of Dpp signaling [5], and continues as a stem cell. The posterior stem cell daughter dissociates from the cap cells and becomes a cystoblast (CB). The CB divides precisely four times with incomplete cytokinesis, giving rise to a cyst of 16 interconnected cells that differentiate further into 1 oocyte and 15 nurse cells (Figure 1A). The progress of cyst formation can be followed by monitoring the morphogenesis of a dynamic organelle, termed the fusome (Figure 1B), that grows and branches with each cyst cell mitosis [6, 7]. Cystoblasts require the product of the bag-of-marbles (bam) gene, which is both necessary and sufficient for differentiation [8, 9]. Germ cells lacking bam fail to differentiate into cystoblasts and continue to divide with full cytokinesis, producing germ cell hyperplasia [8, 10]. The *Correspondence: [email protected]

failure of these proliferating germ cells to differentiate can be recognized by following the fusome since it remains spherical (Figure 1CA) instead of growing into a branched structure [11]. Superficially, therefore, bam mutant cells behaved like GSCs, but molecular markers to determine the stage of arrest have been lacking. Our studies characterizing bam transcription demonstrated that bam is tightly regulated such that it is off in GSCs and on in CBs [10]. Thus, it was possible to determine if bam mutant cells are “stem cell-like” since the activity of a bam reporter transgene would distinguish GSCs from CBs precisely [10]. We examined GFP expression in bam mutant animals carrying a transgene with the bam promoter fused to GFP and observed that most germ cells were GFP positive (Figure 1CA). Thus, unlike GSCs [8, 10], germ cells lacking bam advanced to a state of differentiation sufficient to activate bam transcription. Xie and Spradling [2] showed that misexpression of Dpp, a BMP-like ligand [12], could also produce germ cell hyperplasia that superficially resembled the bam phenotype. Furthermore, ectopic dpp elicited activation of the dpp signal transduction pathway in the hyperplastic germ cells [5]. We asked if these germ cells, responding to ectopic dpp (hereafter referred to as ectopic dpp cells), expressed the bam reporter and found that they were GFP negative (Figure 1CC). Thus, dpp, when expressed broadly in the germarium, blocked the differentiation of GSC daughter cells and prevented bam transcriptional activation. In this sense, ectopic dpp cells were more stem cell-like than bam mutant cells. One of the mechanisms we considered to explain the repression of bam transcription in ectopic dpp cells was that bam transcriptional control elements might respond to dpp signal transduction. Transcriptional quiescence of bam in GSCs depends on a silencer element (SE) [10]. We tested if the SE could account for the sensitivity of {bamP-GFP} expression to dpp by using a bam reporter that lacked the SE (P{bamP⌬SE-GFP}). All hyperplastic germ cells, induced by heat shock induction of dpp expression and carrying the P{bamP⌬SE-GFP} reporter, were GFP positive (Figure 1CD), showing that the SE was required to suppress bam expression in response to dpp. We concluded that the bam SE was probably a direct target of dpp signal transduction. Dpp signal transduction requires phosphorylation of Mad, its nuclear translocation, and binding to DNA target sites [12, 13]. Several related Mad binding sites have been identified previously from a collection of Dpp target genes [14–16]. As the bam SE was a candidate for a direct dpp signaling target, we examined the SE for Mad binding sites and noted that Site B (Figure 2A) matched a strong Mad binding site (Mad-A site) found in the Dpp response elements of the Ubx gene [15]. Therefore, we tested the ability of the bam SE to act as a Mad binding site by using bacterial GST-MadN chimeric protein [14]. We verified that the GST-MadN protein was active by testing its binding (not shown) to a control oligonucleotide containing a previously characterized Dpp response

Smads Directly Regulate bam in the Stem Cell Niche 1787

Figure 1. The Role of dpp in GSC Maintenance and Regulating bam Transcription (A–C) The schematic in (A) summarizes previous data demonstrating that dpp was necessary to maintain the GSC fate [1]. Germ cells at the anterior of the germarium transduce the highest levels of dpp signaling [5], are prevented from differentiating, and remain a self-renewing population of GSCs. Genetic assays showed that proteins of the dpp signaling cassette, including the ligand Dpp, the receptors Punt and Thick veins, the DNA binding Mad, Medea, and Schnurri proteins [2, 3], are required for GSC maintenance. Germ cells that escape the niche activate bam transcription and execute the cystoblast program with successive incomplete divisions to form a syncytial cyst. (B) Antibodies against the Adducin-related Hts protein [25] reveal the progress from cystoblast to cyst by allowing us to follow the growth of the fusome from a single dot in GSCs (thin arrow) to a large, branched organelle in completed cysts (large arrowhead). The panels in (C) document the responsiveness of the bam SE to dpp signaling. Panel (CA) shows the expression of the P{bamP-GFP} reporter, which carries a fully functional bam promoter (P{bamP-GFP}; bam[⌬86] genotype), in bam mutant cells [10]. GFP antibodies show the cells expressing the reporter, while the Hts antibody reaction reveals fusomes and somatic cells’ cytoskeleton [25]. The ovariole is filled with germ cells that fail to differentiate and therefore contain spherical fusomes (two of the many are indicated with arrows). Panel (CB) shows the expression of the same reporter as in (CA) in control animals. These ovarioles include maturing cysts (white arrows) and germaria. The P{bamP-GFP} reporter first becomes active in the CB, one of which is indicated by the yellow arrow. Heat shock-induced misexpression of dpp caused germ cell hyperplasia (Panel C.C), similar to the bam mutant germarium in (CA), except that the bam reporter was not active. However, hyperplastic germ cells induced by dpp misexpression in females carrying a bam promoter lacking the silencer element expressed GFP (Panel BD). All images in (C) are oriented such that the anterior ends of germaria are to the left. GSC, germline stem cell; CB, cystoblast.

element from the Drosophila Ubx promoter [14]. A 31 bp oligonucleotide that contained the full bam SE (see the Experimental Procedures) was efficiently bound and shifted by MadN protein (Figure 2B, lane 2). Binding was specific, as it was competed by increasing amounts of unlabeled bam SE oligonucleotide (Figure 2B, lanes 3 and 4), but not by a 20-fold excess of an irrelevant oligonucleotide (Figure 2B, lane 5). Mad is required genetically to prevent GSC loss; mad inactivation causes GSCs to convert precociously into

CBs [2]. Our results indicate that in cells transducing high levels of Dpp signaling, such as those in the GSC niche [1] or in dpp-overexpressing germaria [5], activated Mad binds to the bam silencer element and recruits other components of a transcriptional silencing complex. Blocking bam expression would be sufficient to prevent the cell from differentiating as a CB [10]. Mad transcriptional regulation requires the co-Smad, Medea [12], and GSCs lacking medea also differentiate precociously [2]. We predicted, therefore, that Medea

Current Biology 1788

Figure 2. Mad Protein Binds to the bam SE (A–C) The sequence around the bam transcriptional start site (“⫹1”) is shown in (A). The sequences that correspond to the bipartite silencer element [10] are outlined in blue (Site A) and yellow (Site B). The minimal Medea consensus site is indicated (red underline) in Site A. The consensus core of Mad binding sites within Site B is underlined, while the entire Site B (yellow highlight) constitutes a consensus Mad-A binding site [14, 15]. Note that the Mad-A site contains three base pairs beyond the fragment that had been described as required to provide silencer activity [10]. The significance of those three base pairs had been obscured since transgenes testing the sufficiency of the bam silencer had reconstituted a 9/10 match for the full Mad-A site with three base pairs from vector sequences [10]. The mutations introduced into the Mad-A site (see text) are indicated. (B) A gel from EMSA reactions with MadN protein, the SE oligonucleotide, and several oligonucleotide competition assays. Lanes 3 and 4 contain 5⫻ and 20⫻, respectively, mass excesses of unlabeled SE. Lane 5 contains a 20-fold excess of an irrelevant oligonucleotide (“AT-rich”; Experimental Procedures), and lanes 6, 7, and 8 contain 5⫻, 20⫻, and 100⫻, respectively, mass excesses of unlabeled mSE. The SE oligonucleotide also binds GST-Med protein ([C], lane 2). Lanes 3 and 4, respectively, show the effects of adding unlabeled SE or mSE that lacks the Mad binding site to the assay extract. Gel-shift reactions were also challenged with oligonucleotides that lack Medea consensus sites (Ubx and AT-rich), and the results are shown in lanes 5 and 6, respectively.

would also be found in the complex of Silencer Element Binding Proteins. We noted that Site A in the SE contains the “AGAC” Smad consensus element (underlined, Figure 2A) that can serve as a Medea binding site [16]. Indeed, GST-Med protein bound to the SE oligonucleotide in standard gel-shift assays (Figure 2C, lane 2). Unlabeled SE oligonucleotides or SE oligos carrying mutations in the Mad binding site (Figure 2C, lanes 3 and 4) competed efficiently for Med binding. Oligos that did

not contain recognizable Med sites, however, did not affect the binding affinity of GST-Med and SE (Figure 2C, lanes 5 and 6). Thus, the bam SE contains a binding site for the Mad:Med complex. Inactivation of Dpp signaling and Bam misexpression caused GSC loss [2], while Dpp misexpression and Bam inactivation caused germ cell hyperplasia [2]. A likely interpretation of the reciprocal relationship between Dpp and Bam expression is that transcriptional silencing

Smads Directly Regulate bam in the Stem Cell Niche 1789

Figure 3. Ectopic Bam Suppresses Dpp Misexpression Phenotype (A) Ovaries from females misexpressing Dpp reacted with Hts antibodies to reveal fusome morphology as a marker for germ cell differentiation. Undifferentiated, hyperplastic germ cells fill the equivalent of germarial Region 1, while the remaining germarial regions are missing. (B) Ovaries from females simultaneously misexpressing Dpp and Bam have been incubated with Hts antibodies to determine the state of differentiation in these germ cells. Two of the several developing cysts with extended fusomes are labeled, and the reappearance of germarial Region 2, where complete cysts become surrounded by follicle cells, is indicated. The images in (A) and (B) were collected as a confocal Z-series and are shown here as a projection of multiple optical sections.

of bam in GSCs is the principal role of dpp signaling. If this is correct, misexpression of bam should reverse the effects of ectopic dpp on germ cell differentiation. We therefore compared the germ cell phenotypes of animals expressing Dpp alone or Dpp and Bam from heat shockinducible transgenes. Figure 3A shows an example of the germ cell hyperplasia induced by Dpp overexpression. Since cystoblasts fail to differentiate, all germ cells born during the time of misexpression (4 or 6 days; Experimental Procedures) accumulated as nondifferentiating, mitotically active cells and caused the germarium to swell. Germ cells that were already older than cystoblasts at the time of dpp misexpression continued to mature into cysts and leave the germarium. Consequently, the germarium region that normally contains maturing cysts (Region 2) was vacated and remained empty since no new cysts formed (116/119 germaria of genotype P{hs-Gal4} P{UAS-dpp} examined). Ovaries from flies that misexpressed both dpp and bam showed a distinctly different effect on ovarian development since these germaria now contained maturing cysts (103/135 germaria of genotype P{hs-Bam}

P{hs-Gal4} P{UAS-Dpp} examined). Frequently, these cysts could be recognized by the extended and branched fusomes that marked clusters of 8 or 16 interconnected cells (Figure 3B). Even more striking was the reemergence of germarial Region 2, which was filled with completed cysts in the process of becoming enveloped by follicle cells. Cysts at this stage of development would have arisen from cystoblasts that were born and differentiated during the time of Dpp misexpression (4 or 6 days; Experimental Procedures). The simplest explanation for these results is that dpp signaling suppressed CB differentiation by blocking bam transcription and that this restriction was bypassed when bam was expressed from a dpp-insensitive promoter. These data show that bam is the primary target of dpp signaling since simply supplying bam rescued CB differentiation. Conclusions Mad-Dependent Silencing of bam Controls GSC Maintenance and Differentiation Two features of GSC divisions demand molecular explanation: how is the anterior daughter of the GSC division

Current Biology 1790

Figure 4. A Molecular Mechanism for GSC Maintenance and Asymmetric Division The GSC resides in a niche [1] that supplies sufficient Dpp to activate the heterodimeric receptor (Put:Tkv) and phosphorylate cytoplasmic Mad (pMad). Activated Mad binds to its partner, Medea (Med), translocates to the nucleus, and binds to the bam SE, where the complex acts to silence bam transcription. pMad levels are lower in CBs, presumably reflecting decreased extracellular Dpp. The concomitant decrease in activated pMad:Med complexes leads to lower occupancy levels at the SE and eventual transcriptional activation of bam.

retained as a stem cell (self-renewal) and what causes the posterior daughter to differentiate into a CB (asymmetric division)? Stromal cells, including the cap and inner sheath cells, at the germarial tip express several signaling molecules [17] and are a likely source of dpp [3, 18]. Previous studies concluded that dpp signaling was required to maintain GSCs [2] and that transcriptional control over Bam distinguishes GSCs from CBs [10]. We can now link these two phenomena directly. In GSCs, in which dpp signaling and pMad levels are highest [5], the Mad:Med complex binds to the bam SE and prevents bam transcription (Figure 4). GSCs selfrenew because association of the anterior daughter with stromal cells permits sufficient dpp signaling to block CB differentiation by assembling a repressor complex on the bam SE element. This complex is likely to include other factors required for transcriptional antagonism, such as TGIF or Ski/Sno factors, which can recruit histone-modifying enzymes [19]. The complex may also contain Schnurri, a negatively acting Mad cofactor [20– 22], since shn mutant GSCs also differentiate precociously [3]. During division, a GSC daughter cell is displaced away from the cap cells and into a region of diminished dpp signaling [2, 5]. This cell, the CB precursor (pre-CB; [9]), expresses lower pMad levels [5] that would cause the concentration of Mad:Med complexes to fall. Declining occupancy levels of the bam SE would produce derepression of bam transcription and concomittant activation of the CB differentiation program (Figure 4). Embryonic stem cells are considered totipotent because they can populate any of the adult niches. Although the degree of adult stem cell plasticity is currently receiving much attention [1, 23], assembly of stem cells into signaling niches during postembryonic development might impose differentiation limits. What are the specific effects of stromal cell niches on captured stem cells? In the case of the Drosophila ovarian niche, GSCs

are maintained as “CBs-in-waiting” because a stromal cell signal represses the expression of one key factor (i.e., Bam). Perhaps other types of stem cells are similarly differentiated but blocked by stromal cell signals and require the expression of only one or a few key molecules to resume development. Experimental Procedures Fly Stocks Flies carrying P{HS-Bam}, P{HS-Gal4}, and P{UAS-dpp} transgenes were used to generate bam and dpp overexpression phenotypes by heat shock induction. Heat shocks were carried out over 4–7 days by applying two or three 1-hr heat shocks/day. The {bamPGFP}, {bamP⌬SE-GFP}, and {hs-bam}18d transgenes [9, 10] and ovary immunohistochemistry [24] were described previously. DNA Binding Assays and Oligos A PCR fragment encoding amino acids 1–241 of Mad [14] or 1–358 of Medea was cloned into pET-42a and purified as GST fusion proteins as described by the manufacturer (Pierce Biochemicals). Complementary DNA oligos were synthesized (IDT) and made doublestranded by incubation in annealing buffer (50 mM Tris-HCl [pH 7.4], 10 mM MgCl2, 100 mM NaCl) at 65⬚C for 5 min and 37⬚C for 60 min. After labeling by the Klenow fill-in reaction, EMSA was performed as described in [14]. Briefly, reagents were incubated in binding buffer (20 mM HEPES [pH 7.9], 100 mM KCl, 1 mM DTT, 0.3% BSA, 20% glycerol, 0.01% NP-40, 1 mM EDTA, and 2 ␮g poly dI/dC in 20 ␮l reaction) and were separated by 5% polyacrylamide gels in running buffer (25 mM Tris, 190 mM glycine, and 1 mM EDTA). The following oligonucleotides and their complements were used in EMSA (the lowercase letters indicate nucleotides changed by site-directed mutagenesis): SE: 5⬘-GGAATTCGCAGACAGCGTGGC GTCAGCGATT-3⬘; mSE: 5⬘-GGAATTCGCAGACAGCGTaattTCAG CGATT-3⬘; Ubx Mad-A: 5⬘-GGCTTTCTGGACTGGCGTCAGCGATT CCGAT-3⬘; AT-rich: 5⬘-GTCAATAAAATGATCAATCAATTTC-3⬘. Complementary oligonucleotides were synthesized such that each annealed pair would have two overhanging “G” residues for radioactive labeling by the Klenow fill-in reaction. Acknowledgments The authors thank Mary Kuhn for excellent technical assistance with immunohistochemistry and confocal microscopy. We are grateful to

Smads Directly Regulate bam in the Stem Cell Niche 1791

A. Laughon, T. Beres, and R. MacDonald, who contributed important reagents and advice for the gel-shift experiments. T. Xie, E. Ferguson, and O. Casanueva generously communicated results prior to publication. S. Cameron, L. Cooley, S. DiNardo, R. Hammer, H. Kra¨mer, B. Ohlstein, E. Olson, and the McKearin lab members graciously provided reviews and comments during the manuscript’s preparation. This work was supported by National Institutes of Health GM45820 to D.M. Received: June 23, 2003 Revised: August 26, 2003 Accepted: August 27, 2003 Published: October 14, 2003 References 1. Spradling, A., Drummond-Barbosa, D., and Kai, T. (2001). Stem cells find their niche. Nature 414, 98–104. 2. 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. 3. Xie, T., and Spradling, A.C. (2000). A niche maintaining germ line stem cells in the Drosophila ovary. Science 290, 328–330. 4. Song, X., Zhu, C.H., Doan, C., and Xie, T. (2002). Germline stem cells anchored by adherens junctions in the Drosophila ovary niches. Science 296, 1855–1857. 5. Kai, T., and Spradling, A.C. (2003). An empty Drosophila stem cell niche re-activates the proliferation of ectopic cells. Proc. Natl. Acad. Sci. USA 100, 4633–4638. 6. McKearin, D. (1997). The Drosophila fusome, organelle biogenesis and germ cell differentiation: if you build it. Bioessays 19, 147–152. 7. de Cuevas, M., and Spradling, A.C. (1998). Morphogenesis of the Drosophila fusome and its implications for oocyte specification. Development 125, 2781–2789. 8. 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, 2242–2251. 9. Ohlstein, B., and McKearin, D. (1997). Ectopic expression of the Drosophila Bam protein eliminates oogenic germline stem cells. Development 124, 3651–3662. 10. Chen, D., and McKearin, D.M. (2003). A discrete transcriptional silencer in the bam gene determines asymmetric division of the Drosophila germline stem cell. Development 130, 1159–1170. 11. McKearin, D., and Ohlstein, B. (1995). A role for the Drosophila bag-of-marbles protein in the differentiation of cystoblasts from germline stem cells. Development 121, 2937–2947. 12. Raftery, L.A., and Sutherland, D.J. (1999). TGF-beta family signal transduction in Drosophila development: from Mad to Smads. Dev. Biol. 210, 251–268. 13. Massague, J., and Wotton, D. (2000). Transcriptional control by the TGF-beta/Smad signaling system. EMBO J. 19, 1745–1754. 14. Kim, J., Johnson, K., Chen, H.J., Carroll, S., and Laughon, A. (1997). Drosophila Mad binds to DNA and directly mediates activation of vestigial by Decapentaplegic. Nature 388, 304–308. 15. Szuts, D., Eresh, S., and Bienz, M. (1998). Functional intertwining of Dpp and EGFR signaling during Drosophila endoderm induction. Genes Dev. 12, 2022–2035. 16. Knirr, S., and Frasch, M. (2001). Molecular integration of inductive and mesoderm-intrinsic inputs governs even-skipped enhancer activity in a subset of pericardial and dorsal muscle progenitors. Dev. Biol. 238, 13–26. 17. Forbes, A.J., Spradling, A.C., Ingham, P.W., and Lin, H. (1996). The role of segment polarity genes during early oogenesis in Drosophila. Development 122, 3283–3294. 18. 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 hh-mediated mechanisms in the Drosophila ovary. Mol. Cell 7, 497–508. 19. Wotton, D., and Massague´, J. (2001). Smad transcriptional corepressors in TGF␤ family signaling. In Transcriptional Corepressors: Mediators of Eukaryotic Gene Expression, Volume 254, M.L. Privalsky, ed. (New York: Springer-Verlag), pp. 145–164.

20. Marty, T., Muller, B., Basler, K., and Affolter, M. (2000). Schnurri mediates Dpp-dependent repression of brinker transcription. Nat. Cell Biol. 2, 745–749. 21. Dai, H., Hogan, C., Gopalakrishnan, B., Torres-Vazquez, J., Nguyen, M., Park, S., Raftery, L.A., Warrior, R., and Arora, K. (2000). The zinc finger protein schnurri acts as a Smad partner in mediating the transcriptional response to decapentaplegic. Dev. Biol. 227, 373–387. 22. 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. 23. Fuchs, E., and Segre, J.A. (2000). Stem cells: a new lease on life. Cell 100, 143–155. 24. Lavoie, C.A., Ohlstein, B., and McKearin, D.M. (1999). Localization and function of Bam protein require the benign gonial cell neoplasm gene product. Dev. Biol. 212, 405–413. 25. Zaccai, M., and Lipshitz, H.D. (1996). Differential distributions of two adducin-like protein isoforms in the Drosophila ovary and early embryo. Zygote 4, 159–166.