Ectopic Dpp signaling promotes stem cell competition through EGFR signaling in the Drosophila testis

Stem cell competition could select the fittest stem cells and potentially control tumorigenesis. However, little is known about the underlying molecular mechanisms. Here, we find that ectopic Decapentaplegic (Dpp) signal activation by expressing a constitutively active form of Thickveins (TkvCA) in cyst stem cells (CySCs) leads to competition between CySCs and germline stem cells (GSCs) for niche occupancy and GSC loss. GSCs are displaced from the niche and undergo differentiation. Interestingly, we find that induction of TkvCA results in elevated expression of vein, which further activates Epidermal Growth Factor Receptor (EGFR) signaling in CySCs to promote their proliferation and compete GSCs out of the niche. Our findings elucidate the important role of Dpp signaling in regulating stem cell competition and tumorigenesis, which could be shed light on tumorigenesis and cancer treatment in mammals.

In this study, we investigate whether additional factors regulate stem cell competition in the testis niche. Interestingly, we find that ectopic expression of tkv CA in CySCs results in competition between CySCs and GSCs for niche occupancy and GSC loss. We demonstrate that CySC-GSC competition observed in tkv CA -expressing testis is caused by enhanced expression of the EGF vein (vn), which in turn activates EGFR/MAPK signaling in CySCs to promote CySCs to outcompete GSCs. Our data elucidate a novel mechanism of stem cell competition, which may shed light into the development of potential clinical treatment for cancer.

Results
Ectopic expression of tkv CA in CySCs leads to CySC-GSC competition and GSC loss. In order to search for new regulators of stem cell competition, we performed a large-scale screen using a c587 ts driver (c587Gal4, UAS-GFP; esg-lacZ, tubGal80 ts ) (data not shown) 53 . c587Gal4 is strongly expressed in CySCs and somatic cyst cells of the Drosophila testis (Fig. 1a). Our recent data show that Tkv acts as receptor trap to restrain Dpp signaling within the niche 53 . Surprisingly, we found that when a constitutively active form of tkv (tkv CA ) was expressed in CySCs (c587 ts > tkv CA ), all germline cells, including GSCs, were lost (Fig. 1b). The hub was tightly surrounded by a group of somatic cells, instead of GSCs ( Fig. 1b-d). These data indicate that ectopic expression of tkv CA may cause CySC-GSC competition. The observed phenotype was resulted from systemic expression of tkv CA in all CySCs, we wondered whether ectopic expression of tkv CA in single CySC or only a portion of CySCs could cause the same defect. We explored this possibility by using MARCM technique to generate CySC clones expressing tkv CA . Compared with FRT control CySC clones, we found that tkv CA -expressing CySC clones tightly attached to the hub, and the number of GSCs per testis was significantly decreased (Fig. 1e-h). These data indicate that ectopic tkv CA expression in CySCs causes stem cell competition. www.nature.com/scientificreports www.nature.com/scientificreports/ Mad (Mothers against dpp), a transducer of Dpp signaling, is phosphorylated when the Dpp pathway is activated. Therefore, the accumulation of phosphorylated Mad (pMad) can be used as a read-out of Dpp pathway activation 43,55,56 . Consistent with previous reports that ectopic expression of tkv CA induces ectopic Dpp signaling activation 43,55 , we found that Dpp signaling activation was greatly increased in the cyst cell lineage of c587 ts > tkv CA testis, using pMAD as a readout ( Supplementary Fig. 1). As Dpp signaling is highly activated upon ectopic expression of tkv CA in CySCs ( Supplementary Fig. 1), we examined whether the observed stem cell competition phenotype was a consequence of ectopic Dpp signaling. We used various functional RNAi lines to simultaneously deplete components downstream of Tkv in c587 ts > tkv CA testes 53,57 . Mad and Med (Medea) are components downstream of Tkv in the Dpp signaling pathway. When these RNAi constructs were co-expressed with tkv CA in CySCs, we found that further removal of either Mad or Med could successfully suppress stem cell competition observed in c587 ts > tkv CA testes ( Supplementary Fig. 2). In these testes, GSCs were restored and resided around the hub, and differentiating spermatogonia could be observed ( Supplementary Fig. 2). These data demonstrate that stem cell competition and GSC loss resulted from ectopic expression of tkv CA in CySCs is a consequence of ectopic Dpp signaling.
CySCs overproliferate and outcompete GSCs upon tkv CA expression. Next, we investigated the cell identity of the cells in c587 ts > tkv CA testes. We first examined c587 ts > tkv CA testes using the Zfh1 antibody, which labels CySCs and early cyst cells. The number of Zfh1 + cells was significantly increased compared to control testes, and CySCs tightly attached to the hub with their cell bodies, indicating that ectopic Dpp signaling in CySCs promotes CySC proliferation (Fig. 2a-c; data not shown). We then examined these c587 ts > tkv CA testes using esg-lacZ, which was highly expressed in the hub and GSCs, and at low levels in CySCs (Fig. 2d). Interestingly, no germline cells were observed in these testes (Fig. 2e,f). esg-lacZ could only be observed in the hub and CySCs, and the number of esg-lacZ + cells was dramatically increased (Fig. 2e,g). These data show that upon ectopic expression of tkv CA , CySCs continued to proliferate and occupied the whole niche, while the germline cells were completely lost.
GSCs are competed out of the niche and undergo differentiation upon tkv CA expression. As no germline cells were observed in these testes, we examined the fate of the germline cells, especially GSCs. The complete disappearance of germline cells, especially GSCs, may be caused by differentiation or cell death. To distinguish these two possibilities, we first performed time chase experiments. No differences were observed between the control and c587 ts > tkv CA testes at 6 hours and 24 hours after the flies were shifted from 18 °C to 29 °C ( Fig. 3; Supplementary Fig. 3). However, by the 2nd day, we found that some CySCs closely attached to the hub with their cell bodies in the c587 ts > tkv CA flies, and the number of GSCs per testis was decreased ( Fig. 3; Supplementary Fig. 3). By the 3rd day after shifting, we found that the hub was closely associated by CySCs and all GSCs were competed out of the niche ( Fig. 3; Supplementary Fig. 3). As time lapsed, GSCs were pushed further away from the hub by CySCs and underwent differentiation. By the 6th day, almost all germline cells were terminally differentiated, and fully differentiated spermatids could be observed at regions near the hub ( Fig. 3; Supplementary Fig. 3). CySCs closely attached to the hub kept proliferating, resulting in accumulation of CySCs ( Fig. 3; Supplementary Fig. 3). On the contrary, we did not find any significant increase of GSC/germline cell death (by active Caspase-3) in these testes ( Supplementary Fig. 4). These data indicate that ectopic activation of Dpp signaling in CySCs outcompetes GSCs from the niche by CySCs, and the outcompeted GSCs are lost due to differentiation.
Ectopic Dpp signaling in CySCs promotes Vn expression. Previous report found that increased expression of the adhesion protein integrin in socs36E mutant CySCs could promote CySC-GSC competition 16 . We examined whether CySC-GSC competition observed in c587 ts > tkv CA testes was due to elevated expression of integrin. However, no obvious change in integrin levels was observed (by βPS-integrin), indicating that CySC-GSC competition observed in c587 ts > tkv CA testes is unlikely mediated by integrin molecules ( Supplementary Fig. 5).
Previous studies found that elevated EGFR signaling in socs36E-and Madm-deficient CySCs was responsible for CySC-GSC competition 15,19 . Activation of EGFR by its extracellular ligands triggers a signal transduction cascade, mediated by the Ras/Raf/MEK cassette, which ultimately leads to dual phosphorylation and activation of the mitogen-activated protein kinase/extracellularly regulated kinase (MAPK/ERK), therefore, phosphorylated ERK (pERK) can be used as a read-out of EGFR pathway activation 58 . To investigate whether EGFR signaling is responsible for CySC-GSC competition observed in c587 ts > tkv CA testes, we examined the activation of EGFR signaling by detecting the levels of pERK in tkv CA -expressing CySCs. Interestingly, we found the levels of pERK was significantly increased in tkv CA -expressing CySCs than those in the control, indicating that ectopic Dpp signaling promotes the activation of EGFR signaling (Fig. 4a-c). To further confirm this, we examined the expression of kekkon (kek), a primary downstream target of EGFR signaling. kek-lacZ is an enhancer trap that reflects endogenous kek expression 59 . We found that kek-lacZ was expressed in the early cyst cells and differentiated cyst cells in wild type testis (Fig. 4d). The expression pattern of kek-lacZ is similar to that of pERK, indicating that kek-lacZ could be used as a readout of EGFR activation in testis (Fig. 4a,d) 60 . We found tkv CA induction significantly enhanced the expression levels of kek-lacZ (Fig. 4e,f). These data show that ectopic Dpp signaling significantly promotes EGFR signaling in the somatic cyst cells.
We then explored how EGFR signaling was activated by ectopic tkv CA expression. We reasoned that some components of the EGFR signaling pathway may be transcriptional upregulated by the activated MAD/MED complex, which in turn activate EGFR signaling. We thus investigated whether ectopic tkv CA expression promotes the transcription of EGFs. We examined the expression of EGFs (Spitz (Spi) and Vein (Vn)) in the testis using their enhancer traps. Consistently, we found that spi was expressed in the germline cells (by spi-lacZ) ( Supplementary Fig. 6) 61 . While vn was expressed in the early somatic cyst cells, including CySCs (by vn-lacZ) (Fig. 4g) 17,19 . As tkv CA was ectopically expressed in CySCs, therefore, we focused on vn for further examination. To explore the relationship between ectopic Dpp signaling and vn expression, we examined the expression levels of vn in c587 ts > tkv CA testes. We found vn expression was markedly increased upon tkv CA induction (Fig. 4h,i). These data suggest that ectopic Dpp signaling promotes vn expression, which in turn induces elevated EGFR signaling in the early cyst cells.
Ectopic Vn/EGFR/MAPK signaling is responsible for CySC-GSC competition. Therefore, we addressed whether elevated vn expression was responsible for CySC-GSC competition observed in c587 ts > tkv CA testes. When ectopically expressed in CySCs (c587 ts > vn EP ), we found that vn EP overexpression resulted in CySC-GSC competition, which mimics c587 ts > tkv CA testes (Fig. 5a-d). Consistently, the number of GSCs per testis was significantly reduced in c587 ts > vn EP testes (Fig. 5a-c), and the number of CySCs tightly attaching to the hub was greatly increased in c587 ts > vn EP testes compared with that of control testes (Fig. 5b,d). These data indicate that elevated vn expression promotes CySC-GSC competition. Furthermore, we found that expression of a constitutively active form of Ras (Ras V12 ) also resulted in CySC-GSC competition and GSC loss, phenocopying  www.nature.com/scientificreports www.nature.com/scientificreports/ tkv CA expression (data not shown). These data indicate that CySC-GSC competition observed in c587 ts > tkv CA testes is likely a consequence of ectopic EGFR/MAPK signaling.
To further confirm that elevated Vn/EGFR/MAPK signaling is responsible for CySC-GSC competition observed in c587 ts > tkv CA testes, we performed suppression experiments. No obvious defects was caused when vn was depleted in CySCs using a shRNA (TH03149.N) (Fig. 5f). When vn was compromised in c587 ts > tkv CA testes using this shRNA, the observed CySC-GSC competition and GSC loss defects were almost completely suppressed ( Fig. 5e-g). The number of GSCs per testis and the number of CySCs tightly attaching to the hub were almost completely reverted by simultaneous knockdown of vn (Fig. 5a-d). These results indicate that ectopic vn expression is responsible for CySC-GSC competition observed in c587 ts > tkv CA testes. To further confirm our conclusion, we targeted EGFR itself for suppression assay. As EGFR signaling is essential for spermatogonia differentiation, we selected a weak dsRNA against egfr (JF01368) to inhibit EGFR signaling 17,37,62 . Knockdown of egfr using this dsRNA resulted in no obvious defects (Fig. 5h). We found that the observed CySC-GSC competition and GSC loss defects were almost completely suppressed by simultaneous induction of this dsRNA in c587 ts > tkv CA testes (Fig. 5e,h,i). Consistently, the number of GSCs per testis and the number of CySCs tightly attaching to the hub were almost completely suppressed by co-inhibition of egfr (Fig. 5c,d). Together, these data demonstrate that ectopic Vn/EGFR/MAPK signaling is responsible for CySC-GSC competition and GSC loss resulted from tkv CA expression in CySCs.

Discussion
Fittest stem cells are selected through stem cell competition in the niche to maintain tissue homeostasis. However, the mechanisms underlying stem cell competition remain largely unknown. Here, we reveal that cell-autonomous activation of Dpp signaling in CySCs results in CySC-GSC competition and GSC loss, which is mediated by elevated Vn/EGFR/MAPK signaling. The mechanism we uncovered may be general features of stem cell systems in regulating stem cell competition 2,22,63 .
Stem cell competition emerges as a mechanism to select fit stem cells and control tumorigenesis [1][2][3][4][5] . Stem cell competition takes place in three steps. The competitive stem cells first become more fit, before they move and anchor to a defined niche, followed by proliferation and outcompetition of neighboring stem cells. However, the www.nature.com/scientificreports www.nature.com/scientificreports/ detailed mechanisms underlying stem cell competition in the Drosophila testis are poorly understood. Elucidating the mechanisms controlling stem cell competition will help to develop potential clinic treatments for cancer. The testis niche supports two groups of stem cells: GSCs and CySCs, making it an excellent model to study stem cell competition regulation. Previous studies found that CySCs compete with each other and with GSCs for niche occupancy 15,16,19,46 . Mutations that confer increased competitiveness to CySCs result in outcompetition of wild type resident stem cells by the mutant stem cells and their descendants. The first identified regulator of niche competition is Socs36E, a negative feedback inhibitor of the JAK/STAT pathway. The competitive behavior of socs36E mutant CySCs was first attributed to increased JAK/STAT signaling 16 . However, it was recently found that the competitiveness of socs36E mutant CySCs is likely due to elevated MAPK signaling 15 . Stem cell competition also occurs among CySCs, it was reported that CySCs with increased Hh or Yorkie (Yki) activity displaced neighboring wildtype CySCs from the niche before they outcompeted neighboring wild type GSCs, indicating that both intra-(CySC-CySC) and inter-lineage (CySC-GSC) competitions take place in the testis 46 . It was recently reported that Slit-Robo signaling only regulates intra-lineage competition among CySCs 54 .
Ectopic Dpp signaling in CySCs results in CySC-GSC competition for niche anchoring and GSC loss (Fig. 1). We found that ectopic Dpp signaling leads to elevated Vn expression, which in turn activates EGFR/MAPK signaling in CySCs to promote their proliferation and ability to outcompete GSCs for niche occupancy (Figs 4 and  5). Ectopic expression of vn in CySCs results in CySC-GSC competition, which mimics c587 ts > tkv CA . However, the GSC loss and the CySC overproliferation phenotype in c587 ts > vn EP is not as severe as the latter. The differences may be caused by the vn EP line used in this study, which may not produce sufficient vn transcripts as that of tkv CA expression. Nevertheless, the observed CySC-GSC competition upon tkv CA expression is almost completely suppressed by compromising EGFR signaling (Fig. 5). Our study here demonstrate that the niche signals must be tightly controlled to prevent CySC-GSC competition, thereby maintaining niche homeostasis. Interestingly, a recent study found that the novel tumor suppressor Mlf1-adaptor molecule (Madm) regulates www.nature.com/scientificreports www.nature.com/scientificreports/ CySC-GSC competition 19 . They found that Madm regulates CySC-GSC competition by suppressing the expression of integrin and EGFR ligand Vn 19 . Although tkv CA induction promotes vn expression, we found that, unlike loss of madm, tkv CA induction does not affect integrin expression levels, suggesting that the downstream events regulating stem cell competition in tkv CA and madm −/− CySCs are not identical ( Supplementary Fig. 5). It is established that EGFR/MAPK signaling is required in the somatic cyst cells for their proper differentiation and engulfment of the developing germline cells 17,37,62 . From recent studies on socs36E, Madm, and our study on tkv CA , we can conclude that EGFR/MAPK signaling in CySCs also plays a pivotal role in regulating CySC-GSC competition 15,19 . It will be interesting to investigate why BMP signaling is kept from being over-activated in CySCs under physiological conditions, and how different input signals are converged on the EGFR/MAPK signaling pathway to regulate CySC-GSC competition, which will help to understand the regulation of stem cell competition, tissue homeostasis, and tumorigenesis.

Materials and Methods
Fly lines and cultures. Flies were maintained on standard corn-meal cultural media at 25 °C. To inactivate

RNAi knock down and overexpression experiments.
To examine gene function in CySCs, c587 ts (c587Gal4, UAS-GFP, esg-lacZ, tubGal80 ts ) was used. Crosses were maintained at 18 °C. Progeny with the proper genotypes was collected 1-2 days after eclosion and maintained at 29 °C before examination. UAS-dsRNA and UAS-shRNA transgenic flies were used.

MARCM clone analyses.
CySC MARCM clones were generated by heat shock treatment 65 . 1-3 days old adult flies were heat-shocked at 37 °C for 60 minutes for 2 consecutive days. Flies were maintained at 25 °C and transferred to new vials every day. The clones were assayed at indicated time points after clone induction (ACI).

Data Availability
The number of GSCs and CySCs was counted manually. For fluorescence intensity of pERK and lacZ, all images were taken under the same confocal settings. Image Pro Plus 5.0 software was used to measure fluorescence intensity of pERK and lacZ (using the measure/count function). Statistical analysis was performed using the Student's t-test. PEMS 3.1 software was used for SEM analyses. The graphs were generated using SigmaPlot 10.0 software, and further modified using Adobe Photoshop and Illustrator.