To identify regulators of Drosophila cell size, a library of approximately 22,000 double-stranded RNAs (dsRNAs) covering 91% of the Drosophila genome 14 was screened in 384-well plates. After 5 days of RNAi treatment, S2R+ cells were stained to visualize F-actin, microtubules and DNA and imaged by automated microscopy (Figure 1). The resulting images were computationally analyzed to identify dsRNAs that led to a reduction in average cell area. First, regions of each image containing a monolayer of adherent cells were identified using an algorithm that removes cell clumps and non-cellular background. Individual nuclei within this region were identified, and average cell area calculated by dividing the monolayer area by the nuclear count (Figure 1). Next, scores were normalized using the CellHTS package 19 within the online RNAi database FLIGHT 20 (Figure 1). Results were then filtered to remove dsRNAs that have a profound affect on cell number (Figure 1), which included many housekeeping genes. Finally, manual curation was used to filter out dsRNAs displaying secondary phenotypes, including defects in cell adhesion. The remaining scores were then ranked based on normalized mean cell area, to reveal the 15 dsRNAs that act most potently to reduce S2R+ cell size (Table 1 and Additional data file 1), all of which are conserved between fly and human.

Network analysis of this putative hit list in the database FLIGHT 20 revealed a core set of genes that participate in the Ras/MAPK signaling pathway (drk/Grb2, csw/SHP-2, Sos, Ras1, Dsor1/MEK and rl/ERK; Figure 1). In most cases, Ras/MAPK signaling is thought to be activated downstream of ligand binding to a receptor tyrosine kinase 21. It was notable, therefore, that the screen identified a single receptor tyrosine kinase gene, Pvr, that exhibited a strong reduced cell size phenotype (Table 1). Pvr is the sole member of the platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) family of receptors in Drosophila, and has previously been implicated in a range of cellular functions, including migration, proliferation and survival 22232425262728293031, but not thus far in the regulation of cell size.

Of the remaining putative hits, only Rheb, a component of the growth regulating Tor pathway 32, had a known signaling function. Novel hits were diverse in functions and included CG9306, which encodes a component of the electron transport machinery, Nup44A, which encodes a nuclear pore component, and several transcription factors. The screen also identified a large number of housekeeping genes, such as ribosomal components Rps8 and Rps18 and proteosomal components Prosalpha7 and Pomp, most of which led to a reduction in both cell size and number (Additional data file 1). However, given our focus on cell growth, we limited our further analysis to delineation of the signaling pathway by which Pvr and Ras1 regulate cell size.

In order to reduce the likelihood of false positives resulting from sequence-specific off-target effects, two non-overlapping dsRNAs were used to validate each putative hit identified in the screen 33. RNAi phenotypes for all components of the canonical Ras/MAPK signaling pathway (Figure 2a) were verified using a microscopy-based assay (Figure 2b) and by using an electronic cell counter to directly measure cell volumes (Figure 2c). This analysis revealed that dsRNAs targeting Pvr, Grb2, Sos, Ras1, ERK and ksr reduce cell size. Conversely, RNAi-induced silencing of Gap1, a Ras1 GTPase activating protein (GAP) that is a negative regulator of the pathway, led to a significant increase in cell size. However, RNAi-induced silencing of Raf and MEK, previously described as members of the Ras/MAPK signaling pathway, failed to generate equivalent changes in cell size, even when targeted using different dsRNAs (data not shown). Why this might be the case is explored below.

In order to identify the upstream signal(s) that trigger the Pvr-dependent increase in S2R+ cell size, we turned our attention to the previously described Pvr ligands Pvf1, Pvf2 and Pvf3 24. Since the genes for none of these three ligands were identified in the phenotypic screen (Additional data file 2), we tested for functional redundancy between the three ligands using RNAi to silence the expression of Pvfs in combination. Whilst silencing of individual Pvfs failed to induce a change in cell size, a significant reduction in cell size was observed when Pvf2 and Pvf3 were silenced together (Figure 2d), suggesting that these two ligands act redundantly to activate Pvr. No such synergy was seen with Pvf1 and the other ligands.

To verify this putative role for Pvf2 and Pvf3 in the control of S2R+ cell size, cells were transiently transfected with Pvf-containing plasmids. Pvf expression was then induced and cell volumes were measured using an automatic cell counter. Significantly, the expression of either Pvf2 or Pvf3 was sufficient to induce a significant increase in the average size of S2R+ cells relative to a green fluorescent protein (GFP) control (Figure 2e). By contrast, Pvf1 expression had no detectable effect on cell size (Figure 2e). Although it is unclear why one ligand should be non-functional in this context, previous studies have shown that different ligands operate in different settings in vivo 26272830. Importantly, the increase in cell size induced by Pvf2/3 was observed across the population, even though transfection efficiencies remained at approximately 20%. This implies that secreted Pvf2 and Pvf3 are able to diffuse in the culture medium to trigger cell signaling in a paracrine fashion, as has been previously suggested 29. To confirm that this effect of Pvfs on cell size was mediated by the Pvr receptor, an epistasis experiment was carried out in which Pvr RNAi cells were transfected with a construct expressing Pvf3 (Figure 2e), or a control plasmid. As expected, this eliminated significant differences in cell size between experimental and control populations, confirming that Pvfs act via Pvr to alter cell size.

Changes in cell size can occur in the absence of alterations in the rate of cell growth via an acceleration or delay of cell division 3435. Such effects were clearly seen in the screen, where the silencing of cdc25 (string) caused growing cells to arrest in G2, resulting in a large increase in cell size over time (yielding a mean cell area z-score of +13.51) and a concomitant reduction in cell number. Conversely, the acceleration of cell cycle progression induced by silencing a negative regulator of the cell cycle, wee, reduced cell size (yielding a mean cell area z-score of -1.53). Noticeably, however, this was not accompanied by a reduction in cell number like that seen following Pvr or Ras RNAi (data not shown) 2.

Because of this link between cell cycle progression and cell size, it was important to determine whether changes in cell cycle progression contribute to the effects of Pvr/Ras signaling on cell size. To do this, we used a FACS analysis to examine the cell cycle profile of cells compromised for Pvr/Ras signaling. This revealed a significant increase in the proportion of cells in G1 in cells treated with dsRNA targeting Pvr or Ras (Figure 3a). This could be the result of a delay in the progression of cells from G1 into S-phase or the arrest of a sub-population of cells at the G1/S transition. To determine which is likely to be the case, in a second experiment we used the incorporation of bromodeoxyuridine (BrdU) as a measure of the proportion of cycling cells. BrdU was added to Pvr, Ras and Rheb RNAi cells 3 days after dsRNA treatment. Cells were then fixed and permeabilized 24 hours later so that incorporated BrdU could be visualized (Figure 3b). In each case, the percentage of BrdU positive cells was similar to that of the GFP RNAi control (>50%). These data strongly suggest that Pvr/Ras silencing causes a shift in the relative timing of G1/S and G2/M progression, without inducing a cell cycle arrest.

We then combined dsRNA targeting Pvr or other components of the Ras/MAPK pathway (Sos, Ras1, ksr, Raf, MEK and ERK) with string dsRNA to determine whether Pvr/Ras is required for cell growth in S2R+ cells that are unable to cycle. In each case, the FACS profile revealed a large G2 peak (data not shown), and an accompanying reduction in BrdU incorporation between days 3-4 after dsRNA treatment (Figure 3b), as expected for a string dsRNA-induced G2/M arrest. Significantly, however, dsRNAs targeting components of the Pvr/Ras pathway caused a significant reduction in the size of string RNAi cells (Figure 3c), indicating that the pathway is required for cell growth in cells arrested in G2, as it is in cycling cells. Taken together, these data suggest that the Pvr/Ras pathway is rate-limiting for the growth (accumulation of mass) of S2R+ cells and, either directly or indirectly, affects the relative time cells spend in G1 and G2.

Since Pvr has not been previously reported to control cell size, we examined the role of established growth regulatory pathways in the S2R+ cell line. Previous studies have identified the protein kinase Tor as a key regulator of cell growth in a wide variety of eukaryotic systems 32. In the canonical Tor pathway, the small GTPase Rheb activates the Tor/Raptor complex, which phosphorylates ribosomal S6-kinase to stimulate cell growth 32. However, our genome-wide RNAi screen only identified a single member of the Tor pathway, Rheb, as a putative regulator of cell size. A closer examination of the screen data revealed that our failure to identify other components of the Tor signaling pathway was due in part to the stringent cut-off employed in the computational analysis to reduce the number of false positives. In fact, Tor, Raptor and S6k silencing was associated with a small, but measurable decrease in cell area (z less than -1.6 in each case), suggesting that the Tor pathway does indeed play a role in the control of cell growth in S2R+ cells. To confirm this, we generated non-overlapping dsRNAs for each pathway component and directly measured cell sizes using an electronic cell analyzer. All core members of the canonical Tor pathway displayed the expected RNAi phenotype. Silencing positive regulators of the pathway (Rheb, Tor, Raptor and S6k) led to a significant decrease in cell size (Figure 3d). Conversely, dsRNAs targeting either of the two negative regulators of the pathway, Tsc1 and Tsc2 (which together form a Rheb GAP), increased cell size (Figure 3d). Furthermore, FACS analysis revealed that Rheb or Tor RNAi leads to an increase in the proportion of cells in G1, similar to that seen in Pvr RNAi cells (Figure 3a). These results confirm that the canonical Tor pathway controls cell growth in S2R+ cells, as previously demonstrated in S2 cells 3637 and in vivo 10.

Tor has been shown to act downstream of insulin-induced receptor tyrosine kinase (RTK) signaling to control Drosophila cell growth in vivo 10. Moreover, insulin has been shown to stimulate the growth of Drosophila cells in vivo 38 and in fly cell culture 39. This signal is mediated by the insulin receptor (InR). The activated receptor recruits the insulin receptor substrate (IRS) adaptor protein, which binds the regulatory (p60) subunit of class I PI3K, enabling the catalytic (p110) subunit to convert the phospholipid PIP2 to PIP3 in the membrane. PIP3 then recruits several downstream targets, most notably PDK1, to the membrane, to induce the phosphorylation and activation of Akt/PKB, which goes on to inactivate Tsc1/2 to stimulate the Tor pathway. In analyzing the role of insulin signaling in the growth of S2R+ cells, we first verified that insulin is able to alter their growth. As expected for a cell line with an intact insulin signaling pathway, the addition of insulin to the medium of these cells increased the rate of proliferation and average cell volume (data not shown). This does not mean, however, that insulin signaling is required for normal S2R+ cell growth. To test whether or not this was the case, we measured cell size following RNAi-induced silencing of pathway components. Knocking down of the upstream components InR, IRS, p60 or p110 had no effect on cell size (Figure 3d), even though this was sufficient to fully (InR, IRS, p60) or partially (p110) 40 attenuate the insulin-induced phospho-Akt response (data not shown), while PDK1 or Akt silencing induced a small reduction in cell size (Figure 3d). These experiments suggest that while the insulin pathway is operational in S2R+ cells, it is not rate-limiting for size control in this cell line under normal cell culture conditions.

Having established important roles for Pvr and Ras1 in the regulation of S2R+ cell growth, an RNAi epistasis analysis was used in an attempt to delineate downstream signaling events in more detail, and to better understand the reason for the minor phenotypic consequences of using RNAi to deplete several well-established Ras targets. To begin this analysis we used negative regulators of cell growth signaling in this system, Gap1 and Tsc2, as genetic landmarks to position positive regulators within the pathway.

We began by using RNAi to modify the Gap1 phenotype. As expected, the Gap1 RNAi-induced increase in cell size could be suppressed by ERK RNAi, and reversed by dsRNA-mediated silencing of Ras1, and by reductions in the expression of downstream components of the Tor signaling pathway, Rheb, Tor and S6K (Figure 4a). Interestingly, however, direct targets of Ras1, Raf and p110 only partially suppressed the effects of Gap1 RNAi (Figure 4a), mirroring the results of single Raf and p110 RNAi experiments (Figures 2c and 3c). These results suggest that both the MAPK and PI3K pathways contribute to the communication of the growth signal downstream of Ras1.

We then repeated this epistasis analysis in a background in which the gene for the Rheb GAP Tsc2 was silenced, deregulating Rheb activity to increase cell growth (Figure 3c). Once again, although several dsRNAs (Ras1, p110 or Akt) reduced the extent of the cell size increase seen following Tsc2 RNAi, the Tsc2 phenotype dominated in each case (Figure 4b), placing these genes genetically upstream of Tsc2. Although different pathway members (for example, Pvr versus Ras1) exhibited minor differences in their ability to suppress the Tsc2 phenotype, we believe that this is likely to reflect the fact that epistasis experiments are inherently sensitive to gene-specific differences in the kinetics of RNAi knockdown. Only Rheb and S6k strongly attenuated the Tsc2 phenotype, implying that they function downstream of Tsc2, as previously reported 32. Taken together (compare Figure 4a and 4b), these results suggest that Pvr/Ras signaling is likely to operate upstream of the Tor pathway in controlling cell growth in S2R+ cells, although we cannot exclude the possibility that Ras and Tor signaling operate in parallel.

These results focused our attention on the function of intermediate pathway components that have a minor impact on cell growth when targeted using RNAi (Figures 2c and 3d). Since Ras has been shown to signal directly to both PI3K and Raf in other systems 41, we decided to use combinatorial RNAi experiments to test whether p110 and Akt might cooperate with Raf in relaying the growth signal downstream of Ras1 in S2R+ cells (Figure 4c). This analysis revealed a set of additive and synergistic interactions between components of the MAPK and PI3K pathways (Figure 4c, d). This was clearest for p110, since the reduction in cell size observed following silencing of p110 together with either Raf, ksr or ERK was equal to or greater than the sum of phenotypes observed in RNAi experiments targeting these genes independently (Figure 4c, d and data not shown). In addition, there was an additive effect of targeting Akt and these components of the MAPK pathway. Since InR RNAi failed to enhance the effect of Raf silencing (Figure 4c), this synergy between Raf and p110/Akt is unlikely to be the result of a parallel input from insulin signaling. Instead, because p110/Akt RNAi did not synergize with Pvr and Ras1 RNAi (Figure 4d), the PI3K pathway likely functions downstream of Ras1 in this growth assay, as has been described in other systems 41. Taken together, these results suggest that signals relayed by both the MAPK and PI3K pathways cooperate in growth signaling. Indeed, both Akt and ERK have been shown to phosphorylate and inactivate Tsc2 in mammalian systems 42434445. Thus, the Tsc1/Tsc2 complex may serve as a hub to integrate growth signals.

Having identified a Pvr signaling pathway that is rate-limiting for the growth of the S2R+ hemocyte-derived Drosophila cell line, we extended this analysis to investigate possible implications for the growth of other cells. First, we examined the effects of Pvr silencing in a variety of other Drosophila cell lines. Pvr showed a strong cell size phenotype in both the S2 hemocyte cell line and the neuronal cell line ML-DmBG3-c2 (Figure 5a), implying that Pvf/Pvr autocrine signaling is a common feature of the growth of Drosophila cell lines in culture.

To test whether Pvr might play a similar role in the regulation of cell size in vivo, we extracted hemocytes from Drosophila larvae containing Pvr or Ras RNAi constructs under the control of two different hemocyte drivers (Hml-Gal4 and Cg-Gal4). In both cases, we used upstream activation sequence (UAS)-GFP as a marker to confirm that transgenes were being expressed in these primary cells. In order to estimate cell size, mean cell area was measured after GFP-labeled cells had been given time to spread on an adhesive concanavilin A-coated surface. As controls, we also measured the spread area of cells lacking either the driver (data not shown) or the RNAi hairpin. In this experiment, Pvr silencing or the expression of a dominant negative Pvr or Ras construct led to a significant reduction in the size and number of hemocytes relative to control experiments (Figure 5b and data not shown). Although Ras1 over-expression has previously been shown to cause an increase in larval hemocyte number 46, which necessarily requires coincident cell growth and division, these data suggest that Ras/MAPK signaling also plays a role in mass accumulation. Thus, Pvr and Ras1 control the growth, proliferation 29 and viability 22 of Drosophila hemocytes in vivo.

S2R+ cells were grown in Schneider's medium (Invitrogen, Carlsbad, California, USA) or Shields and Sang M3 insect medium (Sigma-Aldrich, St Louis, Missouri, USA) with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, St Louis, Missouri, USA) and penicillin-streptomycin (Sigma-Aldrich). S2 cells were grown in InsectExpress media with L-Glutamine (PAA Laboratories, Pasching, Austria). ML-DmBG3-c2 cells were cultured in M3 media supplemented with fetal bovine serum, antibiotics and 10 μg/ml bovine insulin (Sigma-Aldrich, St Louis, Missouri, USA). Drosophila S2R+ cells were transiently transfected using the CellFectin (Invitrogen, Carlsbad, California, USA) lipid transfection reagent according to the manufacturer's protocol. Where necessary, gene expression was induced by addition of 1 mM CuSO₄ solution.

dsRNA templates were amplified by PCR from genomic DNA using pairs of gene-specific primers. dsRNA synthesis was performed using the T7 Megascript kit (Applied Biosystems, Foster City, California, USA). RNA preparations were purified using PCR96 cleanup plates (Millipore, Billerica, Massachusetts, USA) attached to a vacuum pump. Purified RNAs were resuspended in Tris-EDTA buffer (TE) and annealed by heating at 65°C for 10 minutes and cooling slowly. Typically, cells suspended in serum-free medium were mixed with dsRNA to give a final concentration of 30 μg/ml then plated into tissue culture dishes and incubated at 24°C for 30 minutes. Subsequently, three volumes of complete medium was added and cells were grown for 5-7 days at 24°C to allow for protein turnover 50.

Cells in 384-well plates were washed with phosphate buffered saline (PBS) and fixed for 10 minutes in 4% formaldehyde (Polyscience, Niles, Illinois, USA). After fixation cells were permeablized by washing with PBS containing 0.1% Triton-X-100 (PBS-T), then blocked with 5% bovine serum albumin (Sigma) in PBS-T for 20 minutes. For staining, cells were first incubated with 1:500 α-Tubulin antibody (Sigma) in PBS-T containing 1% bovine serum albumin overnight at 4°C. Cells were then washed twice with PBS-T and incubated with fluoro-isothiocyanate (FITC) anti-mouse IgG secondary antibody (The Jackson Laboratory, Bar Harbor, Maine, USA) combined with TRITC-Phalloidin (Sigma) and DAPI (Sigma) for 2 hours. For BrdU experiments, BrdU was added to the culture medium 3 days after the addition of RNAi. Cells were then fixed, acid washed and stained 24 hours later, using TRITC-labeled anti-BrdU antibodies to reveal the extent of BrdU incorporation into DNA. In each case, fluorescent images were acquired using an automated Nikon TE2000E microscope with a 20× objective and HTS (high throughput screening) MetaMorph software (Molecular Devices, Sunnyvale, California, USA) running an automated stage and shutter (Prior, Cambridge, UK), and a Roper CoolSNAP cooled-coupled device camera.

Image analysis was performed using the image analysis toolkit in Matlab (Mathworks, Natick, Massachusett, USA). F-actin and microtubule stained images were processed to remove cell clumps and background, leaving the cell monolayer. DNA stained images were segmented to identify nuclei in the cell monolayer. Mean cell area was calculated as the area of the monolayer divided by the number of nuclei. Raw scores from image analysis were normalized to correct for systematic differences between assay plates. Normalization was performed using the CellHTS package 19, part of the Bioconductor suite of biological data analysis packages for the R statistical computing environment. Briefly, mean cell area scores were normalized using median centering per plate, and screen z-scores were calculated using the screen median and the median absolute deviation (MAD). Replicate scores from different image sites in the same well were summarized using the closest to zero function (equivalent to taking the minimum, independent of sign) to calculate a single z-score for each screen well. The proportion of nuclei that had undergone division was established by computational image analysis of BrdU and DAPI images.

Cell volume was measured using a CASY cell counter and analysis system (Scharfe System, Reutlingen, Germany). Cells diluted 1:101 in CasyTon reagent (Scharfe System) were measured in triplicate. The mean cell volume for each treatment was calculated as the average peak volume from three independent readings. For each experiment the peak cell volume (the peak in the histogram of individual cell volumes) for at least ten control wells measured in triplicate was used to establish a solid baseline for comparison. Since control cell size varied between experiments, it was necessary to normalize scores for each experiment before summarization. Thus, volumes were converted to the percentage of mean control cell volume. Percentage of mean control cell volume from at least two independent experiments were averaged and used to construct bar charts. For cell cycle profiles, the cells were fixed in 70% ethanol at -20°C and subsequently resuspended in PBS containing 50 μg/ml propidium iodide and 60 μg/ml RNaseA. The profiles were acquired on a FACSanto anlyzer, using FACSDiva software (Becton Dickinson, Franklin Lakes, New Jersey, USA). All cells were in log growth phase during the course of the experiments.

Cg-Gal4, Hml-Gal4, UAS-GFP and UAS-RasDN lines were obtained from the Bloomington stock centre. The UAS-PvrDN line was a gift from P Rorth and Pvr RNAi lines were gifts from Benny Shilo. Late third instar larvae were washed and the integument was disrupted in the latero-posterior region without organ disruption. The circulating hemocytes were directly collected in M3 medium. In each case, pooled hemocytes from several larvae were plated on conconavalin A coated 384-well plates and allowed to spread flat on this substrate for 2 hours. Attached hemocytes were fixed, stained and imaged as above and cell area was measured computationally. UAS-GFP was used to confirm Gal4 expression in larval hemocytes.