Stit Is Required for Wing Growth

stit mutants die as pupae with melanized abdomens, and Stit protein was detected in several developing imaginal epithelial tissues (the wing, thorax, leg discs, and abdominal histoblasts), but was absent in eye-antennal disc (Figure S1A,B and unpublished data) [24]. To uncover a potential Stit function in epithelial tissue development, we focused on the wing imaginal discs. The larval wing discs generate a dorsal and a ventral epithelial cell layer, which appose each other during pupation to form the adult wing blade. We first made FRT stit^Exel9056 and stit^Ex266 mutant clones in the future dorsal surface of the wing using apterous (ap)-GAL4 to drive UAS-FLPase. This resulted in strong upwardly bent wings with no apparent defects in vein patterning or hair orientation. This phenotype suggested that Stit controls the balanced growth of the dorsal and ventral wing compartments (Figure 1A). As stit mutant cells showed residual immunoreactivity during larval stages (Figure S1A), we additionally used a transgene encoding a kinase defective variant of Stit (Stit^KD) to acutely disrupt endogenous Stit as well as stit RNAi-expressing trangenes (stit-IR). Broadly expressed Stit^KD by daughterless (da)-GAL4 caused pupal lethality resembling the stit mutant phenotype. Expression of Stit^KD or stit-IR in the dorsal portion of the wing using either ap-GAL4 or MS1096-GAL4 reproducibly caused upwardly bent wings, but the bending was more severe than that caused by the mutant clones (Figures 1A and 2A). Similarly, expression of Stit^KD and stit-IR using engrailed (en)-GAL4 resulted in bending of the posterior wing part, suggesting that Stit coordinates shape and growth in the entire tissue (Figure 1D and not shown). Overexpression of wild-type stit (ap>stit) could rescue the wing phenotypes caused by either Stit^KD or stit-IR (unpublished data), suggesting that both Stit^KD and stit-IR can act as potent and specific inhibitors of Stit function. In conclusion, clones of stit cells and compartmentalized stit inactivation in the wing resulted in growth defects leading to tissue shape alterations.

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Figure 1. Stit is required for optimal growth of the wing epithelium.

(A) Stit inactivation in the dorsal wing compartment using ap-GAL4-induced FRT stit mutant clones or expression of either Stit^KD or stit-IR caused an upwardly bent adult wing. (B) Wing cell area decreased in stit mutant clones compared to wild-type clones. (C) The expression of Stit^KD in the dorsal wing compartment led to a 33% reduction of the dorsal/ventral (D/V) cell number ratio (>15,000 cells from 10 Stit^KD pupal wings counted) relative to GFP expressing controls (8 animals, >15,000 cells counted). Student's t test, p<0.005. (D) Expression of stit-IR in the posterior compartment (en-GAL4) caused a backwardly bent wing. (E) The posterior compartment of en>stit-IR wings was reduced in size due to a reduction in both the total cell number and wing cell area relative to GFP-expressing control wings. (F) hs-flp;actin>CD2,stop>GAL4 (AFG4)-generated clones revealed a general reduction of proliferation and an increase in cell doubling time (CDT) when insulin (InR^DN) or amino acid signaling (RagA^DN) was reduced relative to clones expressing GFP alone (48 h after heatshock). A similar effect was observed in Stit^KD-expressing clones, whereas simultaneous reduction of Stit and InR signaling led to a further reduction in proliferation. The number of clones examined (n) for each genotype is indicated. (G) Analysis of Phospho-histone 3 (PH3)-positive mitotic profiles in third instar larval wing discs revealed a general (dorsal, D, and ventral, V, in graph) reduction in the number of mitotically active cells within ap>Stit^KD wing discs compared to control, Student's t test, p<0.005 when comparing either control compartment with either Stit^KD compartment. The number of discs examined (n) is indicated. Scale bar, 50 µm. (H) 10 min of EdU incorporation did not show a compartment-specific change in cells entering into S-phase in ap>Stit^KD discs versus ap>GFP control discs. However, the overall (both dorsal and ventral) labeling was more sparse in Stit^KD wings, although the D/V ratio was close to 1. The number of wing/discs examined (n) is indicated. All error bars indicate standard deviation. The nonautonomous compensatory growth effect is explored further in Figure S1.

https://doi.org/10.1371/journal.pbio.1001612.g001

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Figure 2. Genetic interactions of stit with mutations affecting the PI3K-I/TORC1 pathway.

(A) InR receptor inactivation in the dorsal compartment (ap>InR^DN) reduced total wing size and caused a minor wing bending primarily around the margin. Co-reduction of Stit activity (by Stit^KD) further reduced wing size and gave an upwardly bent wing in addition to the bending at the margin. The stit-IR wing bending phenotypes could be suppressed by co-expression of a constitutively active form of the InR (InR^CA), Rheb, Akt, and constitutively active dS6K (dS6K^STQE), indicating that stit acts via or in parallel to the PI3K-I/TORC1 cassette. Dorsal expression of Rheb led to a downwardly bent wing, indicative of overgrowth. This phenotype was also observed upon mild expression of wild-type stit (see Figure S2). (B) Overexpression of stit by MS1096-GAL4 led to a severe crumpled wing phenotype. This could be further enhanced by co-expression of PI3K-I or suppressed by co-expression of PTEN. See also Table S1.

https://doi.org/10.1371/journal.pbio.1001612.g002

Stit Is Required for Cellular Proliferation During Tissue Growth

To determine the cellular defects leading to wing shape changes upon Stit disruption, we first analyzed stit mutant clones in adult wings. We generated wings composed of forked (f)-marked wild-type cells or f-marked stit mutant cells opposing unmarked minute cells to assess the growth and competitiveness of stit mutant cells. In this setting, wild type almost completely outgrew the minute clones. We found a small, but significant reduction in both the size (wing cell area) and total number of cells in stit clones compared to wild-type clones (Figures 1B and S1C). The overall size of wings containing stit mutant clones was also slightly reduced compared to wings composed of f-marked wild type cells. Similarly, stit inactivation by expression of stit-IR^v1091 or stit-IR^D158 in the posterior compartment by en-GAL4 resulted in a 20%–30% reduction in cell numbers, a significant cell size reduction and an overall size reduction of the posterior wing compartment (Figure 1D,E). This indicated that Stit controls cell growth and/or proliferation in the wing imaginal discs. To directly assess imaginal epithelial growth, we co-expressed GFP with Stit^KD or stit-IR in the dorsal compartment using ap-GAL4. We labeled discs with anti-DE-Cadherin and determined cell numbers and their ratios in the juxtaposed dorsal and ventral epithelial layers at 20–40 h after pupal formation (APF) (Figures 1C and S1D). Wing discs from ap>GFP control pupae had a D/V cell ratio close to the expected value 1. ap>Stit^KD, however, showed a D/V cell ratio of 0.67, indicating a 33% reduction of cells within the dorsal compartment compared to the ventral or compared to the wild-type control ratio. The shape of pupal wing cells expressing Stit^KD was different than control wing cells. They were shorter than their ventral counterparts, or conversely their ventral counterparts appeared taller (Figure S1D, X–Z section). This D/V cell shape difference was not observed in the control and suggests a compensatory mechanism, where either the dorsal cells stretch to cover the larger ventral area or conversely the ventral cells compact to accommodate a reduced dorsal surface. Thus, stit mutant clones or localized expression of Stit^KD and stit-IR caused a decrease in cell numbers at pupal stages and a reduction in both cell size and number in the adult wing. We graded the impact of the genetic manipulations to cell numbers in the order: Stit^KD>stit-IR>stit null mutant. This suggested a prominent Stit protein perdurance in the null mutant clones in the wing and prompted us to focus our analysis on the phenotypes generated by the stit RNAi and the dominant negative transgenes.

We first assessed whether the reduction in cell numbers was due to an increase in cell death or a decrease in cell proliferation. Expression of stit-IR and Stit^KD in the dorsal compartment of wing discs did not lead to increase in cell death as assessed by TUNEL labeling or Caspase 3 staining (Figure S1G). Moreover, concurrent expression of p35, a baculoviral caspase inhibitor, together with stit-IR or Stit^KD did not ameliorate the wing bending caused by stit inactivation (unpublished data), suggesting that Stit does not influence apoptosis. To examine its role in cell proliferation, we inactivated stit in clones using hs-flp;Act>CD2>GAL4 (AFG4) to drive Stit^KD and GFP expression. In parallel, we used the same approach to express InR^DN or RagA^DN as positive controls. We determined the number of GFP positive cells in individual clones 48 h after clone induction. We plotted the percentage of clones within defined size intervals and calculated the mean cell doubling time (CDT) for each genotype. The cell numbers in GFP expressing control clones produced a bell-shaped distribution, where the majority (40%) of clones were comprised of 11–15 cells. This resulted in a CDT of 12.9 h (Figure 1F), in close agreement with previous findings [25]. The expression of InR^DN or RagA^DN shifted this bell-shaped curve to the left, indicating an increase in the frequency of clones comprised of fewer cells and a corresponding increase in mean CDT to 14.2 and 17.3 h, respectively. Similarly, Stit^KD expression reduced the number of cells per clone, resulting in a mean CDT of 14.9 h, suggesting that Stit is required for epithelial cell proliferation.

To pinpoint a potential function of Stit in cell proliferation, we labeled ap>Stit^KD or ap>stit-IR discs with anti-phosphohistone H3 (PH3), EdU (a BrdU analog), and anti-dGeminin. We observed a strong reduction in the labeling of all three markers in ap>Stit^KD or ap>stit-IR discs compared to the control discs (Figures 1G, 1H, S1E and unpublished data). Surprisingly, the 3-fold decrease of anti-PH3 positive cells was not confined in the dorsal compartment but was evident in the entire wing pouch of ap>Stit^KD. The EdU and Geminin stainings also showed a general reduction in labeling cells upon stit inactivation compared to control discs (Figure S1E). This generalized decrease of proliferative markers in the wing discs in response to localized Stit inactivation is in congruence with a nonautonomous mechanism that coordinates wing growth in response to local perturbations [26]. We labeled discs with the G2/M phase marker Cyclin B. Cyclin B expression is dynamic during the cell cycle, accumulating from the end of S-phase, through G2, abating during mitosis. ap>Stit^KD expressing wing discs showed increased Cyclin B levels in the ventral compartment, suggesting a compensatory prolongation or an arrest in G2 therein (Figure S1F). Thus, apart from the global reduction of mitotic markers and the reduction of the wing size upon Stit inactivation, we did not detect any selective block in the cell cycle. Presumably, the decrease in dorsal proliferation occurs early and continuously during development, while the ventral compensation follows in response. We conclude that stit is required for normal levels of cell proliferation during wing development. Rather than playing a direct role controlling the cell cycle, Stit is more likely required for cellular growth.

Stit and InR Function in Parallel to Promote Growth through TORC1

Inactivation of stit in the dorsal compartment either by MS1096>stit-IR or by ap>Stit^KD generated strong upwardly bent wings resembling the defects caused by the disruption of the InR and other growth regulators (Figures 1A, 2A and Table S1) [27],[28]. ap-GAL4-driven InR^DN caused a strong reduction in wing size and bending primarily around the margin (Figure 2A). This contrasted the bending of the entire wing blade generated by ap>Stit^KD expression (Figure 1A). The difference in wing bending at the margin caused by InR^DN versus the bending of the entire wing blade caused by Stit^KD suggests a spatial control of Stit and InR activation in the discs. Co-expression of Stit^KD and InR^DN in the dorsal compartment further reduced wing size and increased the bending compared to the defects caused by InR^DN alone (Figure 2A). These results suggest that Stit and InR function in parallel to control wing growth. To further examine the effect of inactivating both receptors on wing cell proliferation, we generated hs-flp;Act>CD2>GAL4 clones expressing both Stit^KD and InR^DN and determined the number of cells per clone. The fraction of clones composed of fewer cells increased, leading to a marked increase in CDT (17.5 h) compared to the effect caused by inactivation of either receptor alone. This suggests that both Stit and InR are required for optimal tissue growth (Figure 1F). We therefore examined potential genetic interactions of stit with mutations affecting the PI3K-I/TORC1 signaling cassette. Overexpression of Stit by MS1096>stit causes severe crumpling of the whole blade (Figure 2B). Milder overexpression in the dorsal surface by ap>stit at 18°C caused a gentle bend of the wing downwards, indicative of an overgrowth of the dorsal surface (Figure S2A). A similar downward bending was generated by overexpression of the growth activator Rheb in the dorsal compartment using MS1096>Rheb (Figure 2A). The wing overgrowth phenotypes caused by the overexpression of Stit in the dorsal compartment were accompanied by an increase of BrdU and anti-PH3 in the dorsal compartment (Figure S2B). Thus, the changes in wing shape provide a sensitive assay for Stit function in tissue growth. We tested whether the defects caused by stit inactivation or overexpression can be modified by an array of loss-of-function alleles and overexpression constructs of genes regulating cell death, cell cycle control, and growth (Table S1). We did not detect any interactions with mutations affecting cell cycle progression. However, MS1096-GAL4-driven co-expression of stit-IR together with activating components of the PI3K-I/TORC1 pathway—PI3K-I, Akt, and Rheb—suppressed the stit bent wing phenotype (Figure 2A). Similarly, simultaneous inactivation of stit and PTEN (PTEN-IR) gave a flatter wing, suppressing the effect of stit knock down (Table S1). Furthermore, expression of an activated form of dS6K kinase (the Drosophila ortholog of p70 S6K, a direct TORC1 target) was sufficient to suppress the stit inactivation wing-bending phenotype, indicating that increased TORC1 signaling at any level of the intracellular pathway is sufficient to compensate for a lack of stit. Conversely, the wing phenotype resulting from stit overexpression was suppressed by expression of PTEN, a negative regulator of the pathway and could be enhanced by increasing levels of positive regulators or effectors of the TORC1 pathway, PI3K-I, Akt, Rheb, and dS6K (Figure 2B, Table S1). This implied that Stit activates either the PI3K-I pathway or a novel TORC1 regulatory pathway.

Stit Drives the PI3K-I/TORC1 Pathway to Support Growth and Suppress Autophagy

To investigate the postulated role of Stit in cellular growth, we turned to the larval fat body, which is composed of endoreplicating cells growing without cellular division and thereby facilitates the analysis of cellular growth. Stit is not expressed in the fat body and its inactivation by Stit^KD expression in this tissue did not lead to any discernible cell growth phenotype (Figure S3B,C). To investigate if Stit can induce PI3K-I activation and cellular growth, we overexpressed stit and UAS-RFP in clones in larvae expressing the GFP-PH (tGPH) reporter, which is recruited by PIP3 at the plasma membrane, thus reflecting PI3K-I activity. In parallel, we generated clones overexpressing a membrane targeted PI3K-I (PI3K-CaaX) and Stit^KD as positive and negative controls (Figures 3 and S3). As expected, clonal overexpression of PI3K-CaaX caused a pronounced cell overgrowth and induced membrane accumulation of GFP, while Stit^KD had no effects (Figures 3A and S3B,C). stit overexpressing cells showed an accumulation of the GFP-PH signal but not significant overgrowth compared to their wild-type neighbors (Figure 3B). Upon 24 h starvation, however, PI3K-CaaX- or stit-expressing cells were clearly larger and maintained a strong cortical GFP-PH signal compared to their neighbors (Figure 3A,B). This analysis suggests that Stit, like InR signaling, can activate PI3K-I and spare fat body cells from starvation-induced size reduction [19],[20]. To investigate the potential regulatory role of Stit in starvation-induced autophagy, a TORC1-regulated process, we assessed the accumulation of a Cherry-tagged Atg8a autophagy reporter in clones overexpressing Stit. A starvation period of 5 h induced a punctate Ch::Atg8a accumulation in wild-type cells. This increase in Ch::Atg8a was not evident in cells overexpressing either stit or PI3K-I (Figure 3C, 3D, and 3G). Similarly, the developmental wave of programmed autophagy (P.A.) observed in fed larvae just before pupation was blocked as efficiently by Stit as by PI3K-I (Figure 3E, 3F, and 3G) [20]. Thus, Stit overexpression can spare fat body cells from starvation-induced cell size reduction and autophagy. These observations implicate TORC1 as a downstream effector of Stit. To test this hypothesis we reared larvae overexpressing stit or PI3K-CaaX with 50 µM rapamycin (a potent inhibitor of TORC1 activity) and investigated their ability to block Ch::Atg8a accumulation. Rapamycin treatment for 24 h abolished the inhibitory effect of stit or PI3K-I on Ch::Atg8a accumulation upon starvation (Figure 3E, 3F, and 3G), thus indicating that Stit-mediated suppression of autophagy requires TORC1 activation.

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Figure 3. Stit activates PI3K-I to support growth and suppress starvation-induced autophagy.

(A) Clonal overexpression in the fat body (marked by RFP, asterisk) of PI3K-CaaX led to increased recruitment of GFP-tagged PH probe (tGPH) to the fat body cell plasma membrane and cell enlargement. This was more evident under starvation conditions, where membrane-bound levels of GFP-PH declined in neighboring cells. (B) Clones expressing stit were slightly enlarged, more rounded, and had higher membrane-bound GFP-PH levels than neighbor cells. This persisted upon starvation. (C) Clonal overexpression of PI3K-CaaX or (D) stit (marked by GFP, asterisk) in the fat body of larvae expressing a Cherry-tagged Atg8a reporter expressed under the control of a fat body promoter showed that both PI3K-CaaX and Stit can block the starvation-induced punctate accumulation of Ch::Atg8a. Quantified in (G). (E) Clonal overexpression of either PI3K-CaaX (E) or stit (F) in larvae during programmed autophagy (P.A.) demonstrated that both can block Ch::Atg8a accumulation in the expressing cells. Quantified in (G). Feeding the TORC1 inhibitor rapamycin to larvae expressing PI3K-CaaX (E) or stit (F) in clones reverted the Stit-mediated block of Ch::Atg8a puncta accumulation. Quantified in (G). (G) The intensity of Ch::Atg8a in AFG4-positive cells was measured and compared to the nearest neighbor cells to calculate fold changes where a value of 1 (red hatched line) indicates no difference to the normal autophagy response to each condition (starved, fed, P.A./developmental, or rapamycin induced) observed in wild-type neighbor cells. Stit and PI3K-I could both suppress programmed and starvation-induced Ch::Atg8a accumulation/intensity increase. * indicates Student's t test scores of significance (p<0.005) between overexpressing cells and wild-type neighbor cells, while inset Student's t test scores indicate p values of the difference in response of stit-expressing cells/wild-type neighbor cells between conditions—that is, starved and fed. The number of transgene-expressing cells/wild-type neighbor cells where Ch::Atg8a intensity was measured (n) is indicated. Error bars indicate standard deviation. Scale bar, 50 µm. See also Figure S3.

https://doi.org/10.1371/journal.pbio.1001612.g003

To assay TORC1 activity, we examined the phosphorylation of the translational repressor 4E-BP by TORC1, upon stit overexpression. We generated clones of cells in the fat body of fed and starved larvae overexpressing stit, RagA^DN, or PI3K-CaaX together with GFP and labeled the tissues with an antibody against p-4E-BP (Figures 4 and S4A) [22]. RagA^DN expression, known to lower TORC1 activity, caused a reduction in p-4E-BP labeling in clones of fed larvae compared to neighboring cells (Figures 4K and S4A) [3]. Conversely, cells expressing PI3K-CaaX showed a stronger p-4E-BP signal than surrounding cells upon starvation (Figure 4A,K). As such, anti-p-4E-BP reflects the nutritional status and TORC1 activity levels of the cell.

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Figure 4. The PI3K-I/TORC1signalling cassette is required for Stit-dependent protection against starvation.

Clonal expression (cells marked by GFP/hatched line) of stit and/or transgenes interfering with TORC1 signaling components in the fat body of starved (5 h) larvae expressing Ch::Atg8a under the control of a fat body promoter (A) PI3K-I-expressing cells have larger nuclei, are protected from starvation-induced Ch::Atg8a puncta formation, and maintain high p-4E-BP levels compared to wild-type neighbors. (B) stit-expressing cells behave like PI3K-I-expressing cells in (A). (C) PI3K-I^DN-expressing cells are smaller and show Ch::Atg8a autophagic puncta and low levels of p-4E-BP (indistinguishable from neighbor cells). (D) stit and PI3K-I^DN co-expression resulted in smaller cells with autophagic puncta and low levels of p-4E-BP following starvation, indicating PI3K-I signaling lies downstream of Stit. (E) Akt-IR, (G) RagA^DN, and (I) TOR^TED-expressing cells are smaller, readily form autophagic puncta, and had lower or similar p-4E-BP levels to neighbor cells. (F) Akt-IR, (H) RagA^DN, or (J) TOR^TED co-expression in cells expressing stit inhibited the increase in cell size, resistance to starvation-induced autophagy (Ch::Atg8a puncta), or maintenance of p-4E-BP levels, indicating that these members of the PI3K-I/TORC1 signaling cassette are required for Stit-dependent starvation resistance. Scale bars, 25 µm. (K) Plot of the ratios of p-4E-BP labeling intensities in cells expressing the transgene to wild-type neighbor cells. The number of overexpressing cells/wild-type nearest neighbor cells counted (n) is given. * indicates Student's t test p values <0.005 between transgene-expressing and wild-type neighbor cells. Co-expression of RagA^DN together with stit blocked stit-supported growth under starvation. This effect was so strong on p-4E-BP levels that it reduced levels far beyond the starved wild-type cell levels, giving significant differences. Inset p values compare differences in preservation of p-4E-BP signal between stit-expressing cells/wild-type neighbor cells and stit and transgene co-expressing cells/wild-type cells. Error bars indicate standard deviation.

https://doi.org/10.1371/journal.pbio.1001612.g004

Clonal Stit overexpression in fed larvae did not greatly affect p-4E-BP signal intensity (Figure 4K) but, like PI3K-CaaX, caused a robust increase of the signal in stit-expressing cells compared to wild-type neighbors following starvation (Figure 4A, B, and K). This indicates that stit, like InR, activation is sufficient to activate TORC1-mediated p-4E-BP phosphorylation and to protect cells against the starvation-induced drop in TORC1 activity. Next, we investigated whether dS6K, another well-characterized TORC1 effector, can be phosphorylated by Stit overexpression upon starvation. We analyzed fat body cell clones overexpressing RagA^DN or stit with a p-dS6K antibody. RagA^DN expression strongly reduced the cytoplasmic punctate p-dS6K staining observed in neighboring cells lacking RagA^DN expression (Figure S4B and G). To assess the specificity of the cytoplasmic p-dS6K labeling, we overexpressed dS6K in fat body cell clones and subjected the larvae to a 5-h starvation period. We found that the p-dS6K signal was selectively enhanced in cells overexpressing dS6K compared to surrounding cells with endogenous dS6K levels (Figure S4C and G). This indicated that the p-dS6K antibody can faithfully recognize the cytoplasmic punctate accumulations of p-dS6K. To investigate the nature of these puncta, we labeled larvae expressing the late endosomal/lysosomal marker, GFP-Lamp1, with anti-p-dS6K. During the P.A. wave that clears the larval tissues at pupation GFP-Lamp1 puncta become enlarged and decorated by p-dS6K. This p-dS6K localization supports the observation that a major site of TORC1 activity is on late endosomal/lysosomal structures (Figure S4E) [5]. Clonal overexpression of stit in the fat body (Figure S4D and G) also resulted in the increase of p-dS6K puncta in starved larvae. This analysis indicates that Stit can induce the accumulation p-dS6K and p-4E-BP, two well-characterized TORC1 targets upon starvation.

We then monitored p-4E-BP accumulation, cell growth, and the autophagy reporter in clones of fat body cells expressing stit to ask if Stit acts through the conventional PI3K-I-TORC1 pathway to support growth and suppress starvation-induced autophagy. As previously established, clonal expression of PI3K^DN, Akt-IR, and RagA^DN in fat body cells all led to reduction of cell growth and p4E-BP levels and entry into autophagy (Figure 4C, E, and G) [3],[19]. Also, expression of Tor^TED led to a strong cell size reduction, rendered cells equal to neighbors in respect to p4E-BP levels, while it induced a high Ch::Atg8a accumulation (Figure 4I). Co-expression of either PI3K^DN, Akt-IR, RagA^DN, and Tor^TED together with stit reversed the effect of Stit in suppressing autophagy as well as its effect in sustaining growth and p-4E-BP levels upon starvation (Figure 4B, D, F, H, J, and K).

Collectively, the analysis of stit overexpression in endoreplicating tissues indicates that Stit can signal through the conventional PI3K-I/TORC1 pathway to sustain TORC1 signaling levels and block autophagy upon starvation.

Stit Controls TORC1 Activity During Wing Development

To address whether Stit controls the growth of proliferating epithelial wing discs through the PI3K-I pathway, we first expressed PI3K-CaaX in clones using hs-flp;Act>CD2>GAL4 (AFG4) and recorded the recruitment of the GFP-PH probe at the cell cortex. We detected a marked increase of the GFP-PH signal in the PI3K-CaaX cells, marked with RFP compared to adjacent nonexpressing cells under starvation conditions. This indicated that GFP-PH reliably reflects PI3K-I activation upon starvation in the wing (Figure 5A). Similarly to PI3K-CaaX, overexpression of stit by MS1096-GAL4 resulted in an increase of the GFP-PH signal along the membranes of the Stit overexpressing cells compared to their neighbors expressing endogenous levels (Figure 5B). This indicated that Stit could activate the PI3K-I pathway in the wing discs. However, neither the clonal inactivation of Stit nor interference with InR signaling by the expression of the dominant negative constructs was sufficient to induce a change in the intensity or the localization of the PI3K-I activity reporter (Figure 5C,D). By contrast, the concurrent expression of both Stit^KD and InR^DN using the same driver lead to a marked decrease in the GFP-PH intensity in the cells expressing both constructs compared to their neighbors (Figure 5E). This was most evident at the interface of cells expressing both Stit^KD and InR^DN. This suggests that Stit and InR co-operate to activate PI3K-I in the wing.

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Figure 5. Stit and the insulin receptor cooperate to activate the PI3K-I pathway.

(A) Clones of cells (marked with RFP) expressing PI3K-CaaX in the larval wing discs led to an increased recruitment of the tubulin::GFP-PH probe (tGPH) to the plasma membrane detectable following 24-h starvation. Inset X–Z section (3–4 µm) and line intensity graph is through/over the indicated region in merged panels. RFP (red) and GFP (green) intensities are represented as line intensity graphs. Peaks represent membrane-localized GFP, while troughs correspond to cytoplasmic signal. GFP-PH membrane-associated intensity in the PI3K-CaaX-expressing region is higher than the nonexpressing region. (B) MS1096 expression of stit (red) causes increased GFP-PH membrane recruitment in the expressing cells. (C) Clones of cells expressing Stit^KD or (D) InR^DN alone did not decrease membrane GFP-PH localization. (E) Clones expressing both Stit^KD and InR^DN had lower levels of membrane-localized GFP-PH than wild-type neighbors. Arrowheads denote clone boundaries. Insets show X–Z projections spanning the clones. All images are thin (8 µm) confocal projections. Scale bars, 10 µm.

https://doi.org/10.1371/journal.pbio.1001612.g005

We further investigated the interplay of Stit and InR during wing growth by monitoring TORC1-dependent dS6K phosphorylation. We first asked whether in situ staining with the p-dS6K antibody provides a reliable readout for the detection of dS6K activation in the wing discs. We expressed dS6K in the dorsal compartment of the wing discs and stained for p-dS6K. We detected an increase of the p-dS6K signal selectively in the basal side of the epithelial cells expressing dS6K (Figure S5A). Conversely, dS6K inactivation by expression dS6K-IR in the posterior compartment of wing discs resulted in the reduction of the pdS6K basal signal in the posterior cells (Figure S5B). This prompted us to use the p-dS6K antibody for the in situ analysis of dS6K activation upon localized inactivation of Stit and members of the InR pathway in the wing discs. We expressed RagA^DN, raptor-IR (an RNAi construct directed against the TORC1 component raptor), InR^DN, stit-IR, and Stit^KD together with GFP in the dorsal wing compartment and assessed their effects on dS6K phosphorylation. As expected, InR^DN, RagA^DN, or raptor-IR lead to a marked reduction in the intensity of p-dS6K labeling compared to the ventral compartment (Figure 6A, B, C, D, and L). Conversely, Rheb expression resulted in an increase in the intensity of p-dS6K labeling (Figure 6J,L). This indicates that the detected dS6K phosphorylation was responsive to the level of TORC1 activity in the compartment. The expression of Stit^KD or stit-IR with the same driver resulted in a reduction of p-dS6K labeling to similar levels as the inactivation of InR pathway components (Figure 6D, E, F, and L). In accord with the in situ analysis of p-dS6K, we detected a modest but reproducible reduction of p-dS6K levels in Western blots of wing disc protein extracts deriving from stit mutant larvae compared to wild-type controls (Figure 6M). The reduction in dS6K phosphorylation upon Stit or InR pathway component inactivation is in close agreement with the adult wing morphology defects caused by the same constructs. This indicates that Stit and InR in parallel control epithelial tissue growth by inducing dS6K phosphorylation.

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Figure 6. Stit and InR cooperatively govern TORC1 activity.

Anti-p-dS6K labeling of third instar larval wing discs revealed discrete puncta lying basally within cells. See Figure S5 for control experiments addressing labeling specificity. Transgenes were expressed by ap-GAL4 and expressing cells were identified by GFP (A–H and J) or by Stit detection (I, K). Arrows or arrowheads mark the D/V compartment boundary. D/V ratios of p-dS6K intensities are quantified in (L). (A) Expression of RagA^DN led to a reduction in basal p-dS6K puncta in the dorsal compartment. X–Z sections of the boxed area are shown in (B). (B–F) X–Z sections of discs expressing RagA^DN, raptor-IR, InR^DN, stit-IR, or Stit^KD in the dorsal compartment. The basal p-dS6K signal of interest is boxed in (B) and the dorsal (D) and ventral (V) compartments indicated. Dividing cells at the top of the panel also label strongly (see Figure S6). The dorsal compartment lies to the right in all X–Z sections and arrowheads mark the compartment borders. (G) Expression of stit caused overgrowth of the dorsal region of the disc accompanied by a change in disc morphology, preventing analysis of basal p-dS6K levels. (H) Co-expression of Stit^KD and InR^DN reduced the p-dS6K signal, to a similar extent as either inactivation alone; see (L) for quantification. (I) Co-expression of stit with Stit^KD leads to a reduction of the stit overgrowth phenotype but gave excessively high p-dS6K levels precluding quantification. (J) Rheb expression increased p-dS6K levels within the dorsal region. (K) Co-expression of Rheb with Stit^KD reverted the decrease in p-dS6K levels resulting from Stit inactivation (see L). Scale bar, 50 µm. (L) The intensity of basal p-dS6K within both dorsal and ventral compartments was measured and the D/V ratio for each genotype calculated. Student's t test showed p<0,001 when wild-type D/V ratios were compared with all genotypes except when compared to ap>Stit^KD>Rheb (p<0.01). ap>Stit^KD>Rheb was significantly different from ap>Stit^KD (p = 0.0015). (M) Immunoblots of total lysates prepared from third instar larval wing discs of wild-type, wild-type starved, and stit mutant larvae. The ratio of p-dS6k/dS6K was reproducibly lower (40% average reduction (45% in M), 15% standard error, 5 independent experiments, 7 samples) in stit mutant discs compared to wild-type fed animals. Arrowhead indicates the band recognized by the anti p-dS6K antibody.

https://doi.org/10.1371/journal.pbio.1001612.g006

To address possible intersection points of InR and Stit signaling, we overexpressed Rheb and inactivated Stit in the same cells using ap-GAL4. UAS-Rheb expression ameliorated the decrease in p-dS6K labeling caused by Stit inactivation (Figure 6F, K, and L). The restoration of p-dS6K staining intensity to 80% of wild-type together with the strong suppression of the adult wing-bending phenotype caused by Rheb expression in Stit-deficient cells indicate that Stit acts through Rheb to activate TORC1 during wing development (Figures 6K,L and 2A). We concluded that Stit and InR collectively activate PI3K-I and regulate TORC1 levels in the wing imaginal disc to control tissue growth.

Stit and InR Cooperatively Prevent the Catabolic Switch to Autophagy

InR and Stit control TORC1 activity during wing growth while Stit can block autophagy in endoreplicating larval tissues in response to starvation. To directly monitor starvation-induced autophagy in the wing, we used the Ch::Atg8a reporter under the control of its endogenous promoter. We first expressed either GFP alone, or TOR^TED, or RagA^DN or InR^DN or PTEN together with GFP using ptc-GAL4. While GFP alone had no effect on the induction of autophagy (Figures S6A and 7G), both TOR^TED and PTEN caused a marked increase in the number of Ch::Atg8a puncta forming within the GFP marked expression domain (Figure 7A, B, and G), indicating that the PI3K-I/TORC1 axis functions to suppress autophagy in the wing. Surprisingly, RagA^DN and InR^DN or InR-IR showed little or no accumulation of Ch::Atg8a puncta in the ptc expression domain (Figures 7C,G and S6B) despite each causing a substantial decrease in the levels of TOR target p-dS6K (Figure 6 and unpublished data). This argues that p-dS6K phosphorylation and the activation of autophagy markers respond to different levels of InR and TORC1 activity in the wing. This contrasts the analysis of TORC1 activation in the fat body, where InR signaling is the dominant receptor activating PI3K-I. In this tissue, InR mutant cells or cells expressing InR-IR or InR^DN both restrict growth and activate the catabolic process of autophagy (Figure S3E, F) [19]. Like the InR, Stit inactivation by either Stit^KD or stit-IR using ptc-GAL4 (Figure 7D, G and unpublished data) did not increase Ch::Atg8a puncta formation. This analysis indicates that although inactivation of TORC1 alone leads to autophagy, inactivation of either of its upstream receptors is not sufficient to induce the catabolic response in epithelial proliferating cells.

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Figure 7. Stit and InR are interchangeably sufficient to block autophagy.

ptc-GAL4 was used to drive transgenes and GFP in the wing discs of third-instar larvae expressing a Cherry-tagged Atg8a autophagy reporter under the control of its own promoter (Ch::Atg8a). GFP expression did not affect the formation of Ch::Atg8a puncta (see Figures S6A and 7G). (A) Expression of a dominant negative form of TOR (TOR^TED) led to a robust induction of autophagic puncta compared to wild-type neighboring tissue. Quantified in (G). (B) Expression of PTEN induced the formation of autophagic puncta, indicating PI3K-I signaling normally holds the autophagic machinery dormant. Expression of either (C) InR-IR or (D) Stit^KD in the ptc domain did not trigger an increase in punctate Ch::Atg8a accumulation. (E) Co-expression of InR-IR together with Stit^KD led to a robust increase in the formation of autophagic Atg8a puncta. Comparable levels of autophagic puncta were observed upon co-reduction of InR/Stit signaling via double RNAi or double dominant negative approaches (see G and Figure S6). (F) Rearing Stit^KD-expressing animals on nutrient-restricted (N.R.) low-energy food led to a notable induction of autophagic puncta, quantified in (G). Scale bar, 50 µm. (G) Graph displaying the ratios obtained from comparison of Ch::Atg8a intensities within the ptc-GFP stripe with the flanking tissue. Student's t test scores for significant differences to control are indicated.

https://doi.org/10.1371/journal.pbio.1001612.g007

We hypothesized that InR and Stit signaling may cooperatively drive TORC1 activation, and hence only a reduction of both pathways would mimic the effect of TOR^TED on autophagy. We introduced either Stit^KD InR^DN or Stit-IR InR-IR or Stit^KD InR-IR into the ptc-GAL4 Ch::Atg8a background to examine the effect of inactivating both receptors on autophagy (Figures 7E, G and S6). Each manipulation resulted in a striking increase in the levels of Ch::Atg8a puncta comparable to the one caused by TOR^TED expression. Finally we challenged ptc>Stit^KD animals with prolonged nutrient restriction on low-energy food. This regime delays development by 2–3 d but is sufficient to support larval development to adulthood. Remarkably, constant nutrient restriction gave an increase in autophagy specifically within the ptc domain upon reduction of Stit signaling (Figure 7F, G). We conclude that Stit and InR are interchangeably required to sustain TORC1 activity and to prevent a catabolic switch in wing discs (Figure 8). Thereby, they endow proliferating imaginal epithelial tissues with a two-tiered control of growth and autophagy.

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Figure 8. A model for the different modes of autophagy regulation in tissues that express stit.

(A) A model depicting Stit and its intersection with the InR/PI3K-I/TORC1 pathway. As Stit is required and sufficient for PI3K-I activation, we place it in parallel to the InR. While InR is known to signal through Chico (IRS) and Lnk, the RTK adaptor that couples Stit to PI3K-I signaling is unknown (X). (B) The presence of InR or stit is sufficient to block autophagy in the wing, while both are required for optimal wing growth. When reared on low-energy food, Stit is required for repressing autophagy. These findings predict a two-tiered model for the switch between anabolism to catabolism in epithelial tissues.

https://doi.org/10.1371/journal.pbio.1001612.g008

Fly Cultivation and Stocks

Flies were cultivated at 25°C on our standard lab fly medium consisting of, per liter, 32.7 g dried potato powder, 60 g sucrose, 27.3 g dry yeast, 7.3 g agar, 4.55 ml propionic acid, and 2 g nipagin, giving a final concentration of 15.3 g/l protein and 6 g/l sugar. Low-energy food consisted of 35 g dried potato powder, 10 g glucose, and 8 g agar per liter. For starvation experiments, larvae were transferred to PBS Agar (1%) for defined periods. Rapamycin (Santa Cruz Biotech, sc-3504A resuspended in Methanol) was diluted to 100 uM in 20% Sucrose PBS and mixed to a paste 1∶1 with dry yeast, which was added to PBS agar vials to which larvae were placed for defined periods.

The fly stocks w¹¹¹⁸, ap-GAL4/CyO, MS1096-GAL4 [38], en-GAL4, da-GAL4, UAS-InR^DN (K1409A), UAS-InR-CA(R418P), UAS-InR-IR, UAS-Rheb, UAS-PTEN, UAS-PTEN-IR, raptor-IR, UAS-Akt, UAS-PI3K-CaaX, UAS-PI3K-I, UAS-dS6K alleles (wild type and constitutive active, S6K^TE, S6K^STDE, or S6K^STDETE, which are intrinsically active due multiple serine/threonine to acidic amino acid substitutions [39]), UAS-TOR^TED, UAS-CycB, UAS-CycE.L, CycE^AR95, dap⁴, UAS-FLP, FRT82B Ubi-GFP, UAS-GFP, hs-flp; FRT82B, yw hs-flp;Dr/Tm3,Sb(1), y,w, hs-flp; Act>CD2>GAL4; UAS-GFP, and UAS-p35 were from Bloomington, while UAS-stit-IR (1091 and 8401) and UAS-dS6K-IR (18126) were from VDRC. UAS-Stit^KD, UAS-stit, stit-α-gfp, stit^ex266, and stit^EXEL9056 were described previously [24]. The pWIZ RNAi vector was used to generate the UAS-stit-IR^D158 that targets the third exon of stit. Other stocks included hs-flp,UAS-RFP (kind gift from D. Hipfner), UAS-RagA^T16N, designated RagA^DN, act>CD2>GAL4/CyO;tGFP-PH (gift from Stephen M. Cohen) [40], pmCh::Atg8a/CyO [41], yw FLPf36a; FRT82B UbqGFP 83f+ 87D M(3)95A/Tm6b, Tb(1) (gift from Dr. Fernando Roch), and hsp70-Flp; UAS-Dicer; r4-mCh::Atg8a, act>CD2>GAL4, and UAS-GFPnls (generously provided by Thomas Neufeld). The last stock has a leaky heat shock promoter requiring no heat shock, otherwise 20 or 75 min heat shock in a 37°C water bath was applied for fat body or wing clones, respectively. hs-flp; act >CD2>GAL4–generated clones are denoted as AFG4.

Immmunohistochemistry and Microscopy

Larval fat body and/or discs were dissected from larvae, fixed in 4% formaldehyde/PBS (either Sigma F1635 or polysciences #18814 ultrapure) for 20 min, and labeled immediately afterward following standard protocols (PBSBT containing 0.5% BSA). Primary anti-sera included rabbit anti-GFP (Invitrogen, 1/300), rabbit anti-p-dS6K and p-4E-BP (Cell signaling #9209 and #2855, each diluted 1/100), rabbit anti-PH3 (sigma, 1/200), rabbit anti-CycB (D. Glover, 1/750), rat anti-dGem (H. Richardson, 1/300), mouse anti-Fas3 (1/100), and rat anti-DE-Cad (1/30) from DSHB and guinea-pig anti-Stit (1/5,000) [24]. Secondary antibodies were from Jackson Immunolab and Molecular probes. TUNEL and EdU assay kits were from Roche and performed as detailed therein. Samples were mounted in Vectashield H-1000 (vectorlabs) for imaging on either Zeiss LSM510, 710, or 780 confocal microscopes or Zeiss Axioplan2. Western blotting was performed using HRP-conjugated antibodies (Jackson Immunolabs). Rabbit p-dS6K (as above) was used at 1/1,000, mouse anti-tubulin (sigma, T5168) was used at 1/100,000, while rabbit anti-dS6K (kind gift of T. Neufeld) was used at 1/1,000. Twenty larval wing discs per well were run on 10% mini-PROTEAN TGX gels (Biorad). Wild-type animals were starved for 48 h on PBS agar. Densitometric analysis was performed using ImageJ and Image Studio Lite Western Blot Analysis Software (LI-COR).

Image Analysis

Image analysis was performed using ImageJ. For DE-Cad pupal wing labeling and PH3 mitotic cell counting in the wing, a cell counting grid was applied to a projection of the confocal stack to aid manual counting. In the case of pupal wing cell quantification, projections of the dorsal or ventral apical cell surfaces immediately overlaying each other were analyzed. GAL4-driven expression of GFP or RFP allowed distinction of cells of interest in all cases. For intensity measurements (e.g., p-dS6K), thin (10 µm) projections of stacks were analyzed. In brief, the threshold function was applied to images and the dorsal and ventral regions (or in the fat body cells expressing GFP versus their immediate neighbors) were manually highlighted and the intensities measured using the analyzed particles function. Care was taken to ensure that control cells or regions of tissues from different samples and genotypes gave similar background values to which test cells or regions were compared. From this the intensity relative to area was determined for both background and test regions. For AFG4 clones, the number of GFP-positive cells within a clone was manually counted from confocal stacks spanning the entire disc. CDT was calculated using the formula (log2/logn)h, where n = median number of cells per clone and h = age of the clone [25]. The adult wing hair measurements were aided by the image J. In Figure S1C, the posterior region is defined as the area from L4 to posterior wing margin and the anterior region is defined as the area from L3 to anterior wing margin. Error bars in all figures represent standard deviation from the mean. All p values were generated by student's t test.