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Proc Natl Acad Sci U S A. Nov 20, 2007; 104(47): 18543–18548.
Published online Nov 13, 2007. doi:  10.1073/pnas.0709021104
PMCID: PMC2141813
Developmental Biology

Soluble factors mediate competitive and cooperative interactions between cells expressing different levels of Drosophila Myc

Abstract

When neighboring cells in the developing Drosophila wing express different levels of the transcription factor, dMyc, competitive interactions can occur. Cells with more dMyc proliferate and ultimately overpopulate the wing, whereas cells with less dMyc die, thereby preventing wing overgrowth. How cells sense dMyc activity differences between themselves and the nature of the process leading to changes in growth and survival during competition remain unknown. We have developed a cell culture-based assay by using Drosophila S2 cells to investigate the mechanism of cell competition. We find that in vitro coculture of S2 cells that express different levels of dMyc leads to cellular interactions that recapitulate many aspects of cell competition in the developing wing. Our data indicate that both cell populations in the cocultures participate in and are required for the competitive process by releasing soluble factors into the medium. We demonstrate that the response of naive cells to medium conditioned with competitive cocultures depends on their potential to express dMyc: Cells that can express high levels of dMyc gain a survival advantage and proliferate faster, whereas cells with lower dMyc levels are instructed to die. We suggest that the ability of cells to perceive and respond to local differences in Myc activity is a cooperative mechanism that could contribute to growth regulation and developmental plasticity in organs and tissues during normal development and regeneration.

Keywords: apoptosis, cell competition, growth, proliferation

Local interactions between cells in developing tissues directly influence their growth and contribution to the mature organ. In the developing Drosophila wing, interactions between cells growing at different rates allow fast-growing populations to disproportionately occupy the tissue by competitively eliminating the slow-growing cells. The mechanism underlying this process, called “cell competition,” is not known, but its existence suggests that neighboring cells in a growing tissue sense one another's growth rate, a property that could contribute to the control of overall growth (1, 2). Competition and the elimination of slow-growing cells could ensure optimal cell fitness during normal development and tissue regeneration (3).

Recently, cell competition was observed to occur between cells expressing different levels of dMyc, the Drosophila homolog of the c-myc transcription factor and protooncogene (2, 4). In this process, cells that express less dMyc become losers and die by apoptosis. Their death requires the nearby presence of winner cells that express more dMyc (2, 4). Loser cells sense the presence of winner cells at a distance of ≤10 cell diameters away in the wing disc, suggesting that the two cell populations do not require physical interactions (2). However, how information about dMyc status is communicated between the cells is unclear. One possibility is that fast-growing, dMyc-expressing winner cells exhaust the local environment of developmental factors important for growth and survival, such as the patterning morphogen Dpp, limiting its availability to loser cells (4). This possibility is difficult to reconcile with several observations, including that activity differences in other potent growth regulators, such as phosphatidylinositide 3-kinase or cyclin D, do not induce cell competition (2). An alternative mechanism is that a short-range signaling process may be used to allow neighboring cells to compute and respond to local differences in dMyc expression.

The difficulty of distinguishing between these mechanisms in vivo led us to design a cell culture-based model of cell competition by using Drosophila S2 cells. Remarkably, despite the lack of developmental context, competitive interactions are induced between cultured S2 cells that express different levels of dMyc. Cell competition in this system occurs with characteristics similar to competition in the developing wing. We demonstrate that when grown in cocultures, winner cells, which express an inducible dMyc transgene, induce the death of loser cells, which express dmyc only from the endogenous locus. In turn, the presence of loser cells promotes proliferation of the winner cells. We demonstrate that this competition/cooperation between winner and loser cell populations does not require physical contact and can be induced with medium conditioned by competing cells. Our results indicate that production of the soluble, active factors requires the presence of both cell types during the process, each contributing to endow winner and loser status.

Results

We established a cell culture-based model for cell competition by constructing stably transfected lines of highly adherent Drosophila S2 cells that contained a metallothionein (MT)-inducible dMyc transgene or expressed GFP constitutively under control of the Actin5C promoter (see Materials and Methods). We optimized growth and induction conditions for each cell line and found 125 μM CuSO4 to be optimal for induction of MT-dMyc (Fig. 1B). Under these conditions, maximal induction of dMyc expression occurred by 4 h after CuSO4 treatment (data not shown). dMyc expression induced cell-autonomous apoptosis of expressing cells at low frequency for the first day of the growth period; longer incubation times and higher concentrations of CuSO4 increased this autonomous cell death (Fig. 1B). Optimization experiments were also carried out for a cell line containing an MT-inducible transgene encoding dp110, the catalytic subunit of the phosphatidylinositide 3-kinase (Fig. 1C).

Fig. 1.
Growth and survival of stable S2 cell lines. (A–C) Single cultures of GFP cells (A), dMyc cells (B), or dp110 cells (C) ± CuSO4 for 2, 4, 12, or 24 h. Hours indicate length of CuSO4 induction before fixation of the cells. Cell death was ...

The parental S2 cells, from which each of the stable transfectants was derived, and the GFP-expressing cells (GFP cells) proliferated at similar rates, doubling every 22.5 ± 0.37 h (Fig. 1D). Both dMyc-expressing S2 cells (dMyc cells) and dp110-expressing S2 cells (dp110 cells) proliferated slightly faster, doubling every 21.3 ± 0.24 h (dMyc cells) and 21.1 ± 0.27 h (dp110 cells) even in the absence of inducer (Fig. 1D). Induction of dMyc or dp110 with 125 μM CuSO4 accelerated cell proliferation further, yielding doubling times of 20.1 ± 0.22 h (dMyc cells) and 19.2 ± 0.21 h (dp110 cells). The faster proliferation of dMyc and dp110 cells in the presence of inducer was due to the increased expression of these regulators because addition of 125 μM CuSO4 to GFP cells did not alter their proliferation rate (Fig. 1D and data not shown).

Competition Occurs Specifically Between Cells Expressing Different Levels of dMyc.

To test whether dMyc and GFP cells competed with each other when grown in the same dish, we carried out coculture assays. The two cell lines were mixed, plated together, and treated with CuSO4 to induce expression of dMyc for specific lengths of time [supporting information (SI) Fig. 6]. Cell competition can be detected in the wing imaginal disc by the non-cell-autonomous induction of apoptosis by using an antibody against the cleaved form of caspase-3 (2). Thus, we examined the cultures for apoptosis occurring in GFP cells, specifically when cocultured with dMyc cells. Within 3 h of CuSO4-induced dMyc expression, there was a significant increase in apoptosis of GFP cells (Fig. 2A). This rate increased steadily over time, with the number of dying cells reaching 6-fold above controls after 12 h of CuSO4 treatment (Fig. 2 B and C). In contrast, the number of dMyc cells in the same culture dishes that expressed cleaved caspase-3 did not increase and was actually lower than when dMyc cells were cultured alone for similar periods of time (Figs. 1B and and22B). Control cocultures of GFP and parental S2 cells did not lead to cell death in either population regardless of the presence of CuSO4. Thus, the cell death was not induced spuriously by CuSO4 or by the coculture conditions per se (Fig. 2 D–F). These results indicate that when cocultured with dMyc cells, GFP cells died at significantly higher rates than either dMyc or GFP cells in control cocultures, where all cells expressed similar levels of dMyc. In addition, they suggest that, when grown in cocultures with GFP cells, dMyc cells were protected from death.

Fig. 2.
Cell death increases specifically in GFP cells during coculture with dMyc-expressing cells. (A–C) dMyc cells cocultured with GFP cells. CuSO4 was added at the concentrations indicated, and cells were fixed and immunostained for cleaved caspase-3 ...

We considered the possibility that, because of their faster growth rate, dMyc cells depleted the growth medium of factors necessary for the survival of GFP cells (5). Therefore, as a control for potential deleterious effects of fast-growing cells, we cocultured GFP cells with dp110 cells. Although addition of 125 μM CuSO4 induced faster growth of dp110 cells (Fig. 1D), this did not lead to an increase in apoptosis of GFP cells in cocultures (SI Fig. 7 E and F). This result and the fact that the death of GFP cells during coculture with dMyc cells occurred rapidly, within 3 h of induction of dMyc expression, suggest that growth and survival factors were not depleted from the medium. Because GFP cells died specifically when cocultured with dMyc cells, but not with dp110 or parental S2 cells, the experiments suggest that the dMyc-dependent death of GFP cells in these assays and the heightened survival of dMyc cells were the result of competitive interactions between the cell populations, as has been documented in vivo in the wing imaginal disc (2).

Direct Contact Between Cell Populations Is Not Required for Competition.

Previous in vivo experiments suggested that, although proximity to dMyc-expressing cells enhanced the effect of cell competition, direct cell–cell contact was not necessary (2). To test whether the competitive interactions between dMyc cells and GFP cells in vitro required direct cell–cell contact, we used an indirect coculture assay. In this assay, the two cell types were separated during the culture period by a membrane filter (SI Fig. 8A). The membrane was sufficiently porous to allow large proteins to pass, but prevented cross-mixing of the two cell populations. In addition, the distance between the filter and the cells at the bottom of the well kept them physically separate (SI Fig. 8A) (see Materials and Methods). GFP cells were seeded on glass coverslips within the well, and dMyc cells were seeded at the same density (adjusted to the smaller size of the insert) directly on the inserts. The cells were then cocultured in wells for defined periods of time in the presence or absence of CuSO4 to induce dMyc expression. The GFP cells on the glass coverslips were then examined for cleaved caspase-3; dMyc cells on the membrane were stained as a control. Remarkably, even in the absence of direct physical contact between dMyc and GFP cells, coculture of these two cell types led to GFP cell death. In this assay, death of GFP cells increased significantly within 2 h of CuSO4 treatment, and by 12 h 17.5% of the GFP-positive cells were undergoing apoptosis, 8-fold higher than background (SI Fig. 8B). In contrast, death of dMyc cells did not increase above background (data not shown). These experiments demonstrate that competition between the dMyc and GFP cells does not require direct cell–cell contact and support the hypothesis that soluble factors produced during the coculture period mediate the competitive process.

Conditioned Medium (CM) Contains Soluble Factors That Induce Cell Competition.

To further test the idea that soluble factors convey the competitive information in the cocultures, we asked whether the growth medium was conditioned with competition-inducing factors after coculture of dMyc and GFP cells. We collected CM from direct cocultures of competing (dMyc + GFP) cells and noncompeting, control (parental S2 + GFP) cells after 4, 12, or 24 h of conditioning in the presence or absence of CuSO4. The CM was then incubated with single cultures of either naive GFP or dMyc cells. As shown in Fig. 3A, only CM from the competing cocultures, in which dMyc and GFP cells were grown with CuSO4, induced a significant increased in cell death in naive GFP cells (Fig. 3A). In contrast, neither competitive nor control CM induced cell death in naive dMyc cells (Fig. 3B). Instead, the frequency of death of naive dMyc cells was significantly reduced compared with that in single cultures of these cells (compare Fig. 1B with Fig. 3B).

Fig. 3.
CM from competing cocultures kills naive GFP cells. (A and B) Cell competition is induced in naive cells with CM. Graphs show the percentage of naive GFP cells (A) or dMyc cells (B) that are cleaved caspase-3-positive after incubation with CM and collected ...

We carried out several control CM assays to determine the specificity of the competing CM. CM from any single culture of GFP cells, or of dMyc cells with or without CuSO4, did not induce cell death in either naive GFP or dMyc cells (Fig. 3 C–F). These experiments indicate that CM from single cultures was not sufficient to induce cell competition, suggesting that both cell types must be present for production of the active factors in the CM. Because CM from control cocultures grown in the absence of CuSO4 also was not sufficient to induce death of naive cells, we infer that, for the CM to be competitive, the two cell populations must recognize that each expresses different levels of dMyc. Therefore, our results suggest that signaling between the two cell populations leads to the release of soluble factors from each population into the medium, which, when incubated with naive cell populations, functions to kill GFP cells while promoting the survival of dMyc cells.

If each cell population actively participates in the competitive process, it might be predicted that one population is required to signal its presence first. We tested this possibility by using single cultures of each cell type to successively condition growth medium. For example, GFP cells were cultured alone (Cx1M); the resulting GFP cell CM was then used to grow single cultures of dMyc cells (Cx2M). The Cx2M from this sequential conditioning was then used as growth medium for cultures of naive GFP cells. CM from both conditioning sequences, GFP cells → dMyc cells or dMyc cells → GFP cells, was able to induce death of a significant number of naive GFP cells (Fig. 3 G and I) and enhance the survival of naive dMyc cells (Fig. 3 H and J). These data indicate that both cell populations produce factors necessary for competitive CM, but the order of their production is not important. However, when CM generated from independently grown single cultures of GFP and dMyc cells was subsequently mixed together ex vitro, the combined CM was unable to induce the death of naive GFP cells (Fig. 3 K and L). This result implies that the winner and loser cell populations must sense each other's presence before active, competitive CM is produced.

Proliferation of dMyc Cells Is Accelerated by Competitive CM.

During cell competition in vivo, the elimination of the loser cells is accompanied by an overproliferation of the winner cells. Our previous results indicated that dMyc cells died less frequently when cocultured with GFP cells, suggesting that competitive CM promoted dMyc cell proliferation. Therefore, we followed the proliferation of naive dMyc and GFP cells by using competitive CM conditioned for 24 h with cocultures of GFP and dMyc cells (Fig. 4, +CuSO4). Noncompetitive CM was used as a control (Fig. 4, −CuSO4). Remarkably, competitive CM dramatically increased the proliferation rate of naive dMyc cells, essentially converting these cells into winners (Fig. 4C). Naive dMyc cells doubled every 16.5 ± 0.23 h, compared with every 20.6 ± 0.60 h in the absence of CM. Competitive CM also converted naive GFP cells (and naive parental S2 cells) into loser cells as proliferation slowed dramatically in these cells, with doubling times increasing from 23.1 ± 0.50 to 27.8 ± 1.02 h after treatment with CM (Fig. 4 C and D). These cells proliferated slowly because of a significant increase in apoptosis; dsRNA against the proapoptotic genes hid or reaper blocked most of the cell death induced by competitive CM and led to their faster growth (Fig. 5A and data not shown).

Fig. 4.
Competing CM accelerates proliferation of naive dMyc cells. Competing or control cocultures conditioned growth medium (±CuSO4) for 24 h, and the CM was collected. Naive parental S2 (A and D), GFP (B and E), or dMyc cells (C and F) were seeded ...
Fig. 5.
The potential to express dMyc determines the response of naive cells to CM. (A) RNAi-mediated knockdown experiments. Graph shows percentage of cleaved caspase-3 (+) cells in competitive (filled bars) and noncompetitive (open bars) CM-treated, naive GFP ...

dMyc Expression Potential Determines a Cell's Response to Competition.

The ability of CM to convert completely naive cells into either winners or losers correlated with the level of dMyc expression the cells could achieve. In our experiments, cells carrying the inducible dMyc transgene invariably responded to competitive CM by growing faster, whereas cells that did not carry this transgene responded by dying more frequently. dMyc protein is destabilized by the Fbw7/hCDC4 homolog, Archipelago (Ago), and mutations in ago promote dMyc stability, increasing its level in the cell (6). To test whether deregulating dMyc levels protected naive GFP cells from the killing effects of competitive CM, we treated them with ago dsRNA. This treatment increased dMyc protein in the cells (data not shown) and significantly reduced the number of CM-induced apoptotic cells, whereas a control dsRNA did not (Fig. 5A). Moreover, knockdown of ago allowed them to proliferate faster than controls in response to competitive CM (Fig. 5B). Although ago dsRNA did not convert naive GFP cells into winners as strong as naive dMyc cells, these results suggest that the level of dMyc in the cell determines whether it survives and proliferates, or dies, in response to CM. Consistent with these results, reduction of dmyc expression in naive GFP cells with dmyc dsRNA significantly increased apoptosis in response to competitive CM and reduced their proliferation rate (Fig. 5 A and B). Interestingly, although reducing dmyc levels also slowed the growth of GFP cells grown in control, noncompetitive CM, it did not increase the frequency of their death, indicating that apoptosis was a specific response to competitive CM (Fig. 5 A, D, and E). Together these results indicate that the propensity of a cell to become a winner or loser cell in response to competitive CM depends on its potential for dMyc expression: Cells capable of inducing high levels of dMyc gain a survival advantage and proliferate faster, whereas cells that have a relatively low ability to express dMyc tend to induce the apoptotic program and die.

Discussion

We have demonstrated that the hallmarks of cell competition, extra proliferation of winner cells at the expense and ultimate death of loser cells, can be induced during the coculture of Drosophila S2 cell lines that express different levels of dMyc. Using CM from cellular cocultures, we find that cell competition is mediated by soluble factors produced in response to the culture of high dMyc expressors (dMyc cells) with low dMyc expressors (GFP or S2 cells). CM made from cultures of either cell type alone is not sufficient to induce the killing activity, nor is CM conditioned sequentially from single cultures and subsequently mixed. These results imply that both the high- and low-dMyc cells cooperate, each contributing to the production of the active factors. Our experiments were unable to determine whether signaling from one cell type is required first to initiate a response from the second cell type. We also do not know the identity of the factors. However, several models for their production are possible. One possibility is that each cell type synthesizes a factor upon sensing the presence of the other cell type, the combination of the two factors leading to an active complex. Alternatively, both cell types may express a common factor, which, upon recognition of differences in dMyc expression levels between cells, is shed into the medium and activated. Regardless of how they are produced or activated, the presence of these putative factors in CM provides a basis for mechanistic studies.

dMyc Activity Determines the Cellular Response to CM.

Our results indicate that cells can have radically different responses to the activity present in competitive CM. The response appears to be dMyc dose-dependent: Cells that can express high levels of dMyc, such as cells expressing the inducible dmyc transgene or GFP cells in which dMyc protein levels are enhanced because of knockdown of ago, have a better chance of becoming winners. In contrast, cells with limited potential for dmyc expression tend to commit suicide in response to CM. The proapoptotic genes hid and reaper are required for the death of loser cells in our in vitro assays because hid or reaper dsRNA abolishes most of the apoptosis induced in response to competitive CM (Fig. 5A). A dose-response, death-or-growth outcome also exists in vivo between low- and high-dMyc cells in the wing disc. There, loser cells that express less dMyc than their neighbors activate expression of hid, which drives the cell to kill itself through apoptosis (2). The developmental importance of this relationship is made clear by genetic experiments, where loss of hid function prevents all competition-induced cell death and leads to loss of wing size regulation (2). Interestingly, winner cells in the wing disc also express hid during competition, but, like winners in our in vitro assays, are protected by an unknown mechanism from its death-inducing effects.

A defining feature of cell competition, which allows wing size control in Drosophila by accommodating unevenly proliferating cells, is that winner cells proliferate more when loser cells die. Whether the rate of proliferation is faster when this event occurs has not been clear because of technical difficulties, including developmental delay of animals in the experiments (2, 4, 7, 8). Here our experiments demonstrate that CM from competitive cocultures promotes the survival of naive dMyc-expressing cells and, remarkably, converts them into winners that proliferate significantly faster than normal. Our data clearly indicate that winner cells provide instructive death cues to loser cells. However, whether the extra growth of winners is permitted indirectly as a result of loser death or the death of losers is actually instructive to winners remains to be determined. In some circumstances, dying cells have been implicated in the stimulation of growth of neighboring cells in the fly, although how this is accomplished is unknown (912). Nevertheless, in our experiments, the presence of both winner and loser cells is required for either process to occur, indicating that the cellular interactions are not only competitive, but also cooperative.

How Do Cells Compute dMyc Expression Differences?

Our data suggest that Drosophila cells have an inherent ability to sense and compute differences in dMyc expression in their neighbors. Given the conservation of Myc function between flies and mammals, this finding has several potentially broad implications for the control of growth during development, regenerative therapy, and cancer (3). For example, c-Myc is important for stem cell identity (1315); thus, such a computational ability might be used to optimize fitness within the niche. In other tissues, comparisons of Myc expression between cells could contribute to developmental plasticity during growth. Finally, c-Myc expression is significantly deregulated in many tumors, and the winning properties of those cells may boost tumor progression. Thus, identification of the factors that mediate winner–loser interactions could provide a basis for therapeutic studies.

Materials and Methods

Establishment of Stable S2 Cell Lines.

Full-length ORFs of dMyc and dp110 tagged at their N terminus with either HA or c-myc epitope tags and cloned into pMT expression vectors (Invitrogen, Carlsbad, CA) (pMT-HA-dMyc and pMT-c-myc-dp110). c-myc-tagged EGFP was cloned into pAc5.1 expression vector (pAc-c-myc-EGFP). S2 cells were cotransfected with target (pMT-HA-dMyc, pMT-c-myc-dp110, or pAc-c-myc-EGFP) and selection (pCoHygro) plasmids with Cellfectin (Invitrogen). Positive cells were cloned and selected by using hygromycin (Sigma–Aldrich, St. Louis, MO), and stable cell lines were established. Lines were subsequently selected for their ability to adhere well to coverslips to facilitate experimental analysis.

Single and Coculture Assays.

For single-culture or naive cell culture assays, cells were seeded at a concentration of 8 × 105 cells per ml in 48-well plates at 0.25 ml per well, in 24-well plates at 0.5 ml per well, or in 12-well plates at 1 ml per well. For direct coculture assay, two kinds of cell lines (GFP + dMyc cells, GFP + S2 cells or dp110 + S2 cells) were mixed seeded at a concentration of (4 + 4) × 105 cells per ml per well in 12-well plates. For indirect coculture assay, Corning transwell plates (#3401; Costar, Cambridge, MA) were used. The 8 × 105 cells per ml per well of GFP cells were seeded in lower plates, and the 4 × 105 cells per 0.5 ml per well of dMyc cells or parent S2 cells (numbers adjusted to the size of the insert) were seeded in the upper transwell inserts. Cells were precultured for 16 h and then treated ± CuSO4 for the indicated times. Several experiments were carried out with comparable results by using three independently derived cell lines.

CM Assay.

CM from direct cocultures with or without 125 μM CuSO4 for indicated times was centrifuged at 200 × g for 3 min, and the supernatant of CM was collected. Then 125 μM CuSO4 was added to CM without CuSO4 to control for inducer effects and then added to precultured naive cells for the indicated times.

Cell Proliferation Analysis.

For proliferation assays, cells were seeded at 1.2 × 104 cells per well in 48-well plates and precultured for 24 h before addition of 125 μM CuSO4. Total cell number in triplicate wells was counted for each time point in at least two independent experiments. Cell-doubling times were calculated with the formula DT (h) = log2(T1−T0)/logN1−N0 (T, growth in h; n, number cells). Statistics were carried out by using two-tailed Student's t tests with unequal variance.

dsRNA Synthesis.

Full-length cDNA for dMyc was subcloned into pBluescript II SK(+) (Stratagene, La Jolla, CA). pBSSK-Hid, pBSSK-DIAP1, and pBSSK-Caenorhabditis elegans CED-9, used as a control, were gifts of M. Miura (University of Tokyo, Tokyo, Japan), and pBSSK-Ago was a gift of K. Moberg (Emory University, Atlanta, GA). dsRNA was synthesized as in ref. 16.

dsRNA Assays.

RNAi assays by using dsRNA were carried out as described (17). Cells were washed with serum-free medium (Invitrogen), and dsRNA was added directly to the medium to a final concentration of 20 μg/ml. The cells were incubated for 30 min at room temperature, and then two volumes of fresh or conditioned Schneider's medium containing 10% FBS were added. The final working concentration of serum in the dsRNA assays was 6.7%.

Additional details are included in SI Materials and Methods.

Supplementary Material

Supporting Information:

Acknowledgments

We thank E. Yoshida for technical assistance; M. Miura and K. Moberg for reagents; and R. Mann, G. Struhl, and A. Tomlinson for advice and encouragement. This work was supported by Uehara Memorial Foundation grants (to N.S.-M.), the National Institutes of Health (L.A.J.), and the Rita Allen Foundation (L.A.J.). L.A.J. is a Rita Allen Scholar.

Abbreviations

CM
conditioned medium
MT
metallothionein.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0709021104/DC1.

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