Transgenic Expression of the Helicobacter pylori Virulence Factor CagA Promotes Apoptosis or Tumorigenesis through JNK Activation in Drosophila

CagA expression in the Drosophila wing causes apoptosis and epithelial disruption

In order to examine the effects of expressing the H. pylori effector protein CagA on an intact epithelium, we used the GAL4/UAS system to drive its expression in the wing imaginal disc. The Drosophila wing begins to form during early larval life when it exists as a primordial sac which contains both a simple columnar epithelium and the squamous epithelium of the peripodial membrane [23]. Cells within the wing imaginal disc proliferate extensively in larval stages followed by disc evagination during pupation, resulting in the adult wing structure. This developmental process is distinct from that of the eye imaginal disc used to model CagA pathogenesis previously [7]–[10], which undergoes systematic differentiation during larval stages. In addition, the fate of imaginal disc cells is specified early in development [24] which allowed us to express CagA in distinct regions of the wing disc (Figure 1A).

We expressed CagA with various GAL4 drivers specific to the wing (Figure S1A), and determined that both the level of CagA protein and the region in which it is expressed affect the resulting larval and adult wing phenotypes (Figure S1B–S1L). We focused our subsequent analysis on two different GAL4 drivers which express CagA either in a subset of wing cells or throughout the wing imaginal disc: beadex-GAL4 (bx-GAL4) is expressed specifically in cells of the columnar epithelium that give rise to the dorsal surface of the wing blade (Figure 1B), and 765-GAL4 is expressed ubiquitously throughout the wing. A membrane-localized GFP construct (mGFP) was used to visualize the expression domain. Expressing CagA with the 765-GAL4 ubiquitous wing driver did not cause any observable phenotype (Figure 1C). However, expressing CagA with the bx-GAL4 dorsal wing driver caused clusters of apoptotic cells to form near the center of the expression domain in wing imaginal discs from third instar larvae (Figure 1D). This phenotype was dose-dependent, since expressing two copies of CagA increased both the size and number of apoptotic clusters formed (Figure 1E). A similar phenotype has been shown to result from localized JNK pathway activation in the wing imaginal disc epithelium but does not occur upon more ubiquitous activation [25].

Interestingly, although expressing one copy of CagA^EPISA with the bx-GAL4 driver did not cause a phenotype (Figure 1F), expressing two copies induced formation of small apoptotic clusters within the expression domain (Figure 1G). This reduction in apoptosis induction suggests that the phenomenon does not require phosphorylated CagA, but that CagA^EPISA is a less potent activator of cell death. This observation is consistent with data obtained from transgenic expression of CagA^EPISA in the eye imaginal disc epithelium, where less severe phenotypes were shown to result from differential cellular localization of the phosphorylation-resistant form of CagA. Whereas wild type CagA was highly enriched at the apical membrane in eye imaginal disc epithelial cells, CagA^EPISA was expressed diffusely throughout the cytoplasm. We propose that the inability of phosphorylation-resistant CagA to localize apically within an epithelium influences its interactions with host cell proteins and their resulting effects on the epithelial tissue [9].

Cells within the apoptotic clusters generated by CagA expression were extruded from the basal surface of the wing imaginal disc epithelium. Further examination of this tissue revealed an enrichment of matrix metalloproteinases, which break down basement membrane, specifically in cells located directly apical to the apoptotic clusters (Figure 1H). This observation indicates that apoptotic cells generated by CagA expression are actively removed from the wing epithelium and not passively lost during development of the imaginal disc.

Many complex cellular interactions are required during wing disc development to ensure proper formation of the adult wing structure (Figure 1I). While this process did not appear to be disrupted by ubiquitous expression of CagA in the wing (Figure 1J), CagA expression specifically in the dorsal wing caused a dose-dependent disruption of the imaginal disc epithelium (Figure S1M and S1N) which affected the overall appearance of the adult wing (Figure 1K and 1L). This phenomenon also did not require phosphorylated CagA since expression of CagA^EPISA caused a less severe dose-dependent disruption of the adult wing (Figure 1M and 1N). The observation that ubiquitous expression of CagA in the wing does not cause apoptosis or epithelial disruption suggests that wild type cells surrounding those which express CagA are required to produce both phenotypes. This is consistent with the previous observation that JNK-dependent apoptosis is only triggered when aberrant cells within an epithelium are surrounded by wild type cells [26]. Taken together, these data prompted us to examine a potential role for JNK signaling in the apoptosis and epithelial disruption phenotypes resulting from localized expression of CagA in the wing imaginal disc.

CagA-induced apoptosis occurs through activation of the JNK signaling pathway

Several aspects of the apoptosis phenotype caused by CagA expression in the wing imaginal disc suggested an interaction between CagA and the JNK pathway. In order to determine the nature of this potential interaction, we examined the effects of expressing several forms of Bsk, the Drosophila homolog of JNK, on the CagA-induced wing phenotype. Ectopic overexpression of wild type Bsk with the bx-GAL4 dorsal wing driver generated small apoptotic clusters (Figure 2A), indicating that the presence of excess JNK in the wing can phenocopy CagA expression. Furthermore, the cell death phenotype caused by CagA expression in the wing was dramatically enhanced by coexpression with wild type Bsk (Figure 2B). Coexpression of Bsk with CagA^EPISA also caused a substantial amount of apoptosis in the wing imaginal disc, suggesting that this interaction is not dependent on phosphorylated CagA (Figure 2C). As expected, expression of a dominant-negative form of Bsk (Bsk^DN) alone did not cause apoptosis in the wing imaginal disc (Figure 2D). Significantly, coexpression of Bsk^DN with CagA almost completely suppressed the apoptosis phenotype caused by CagA expression (Figure 2E), indicating that blocking JNK signaling suppresses CagA-dependent cell death in the wing. These data suggest that CagA expression triggers wing imaginal disc apoptosis through JNK pathway activation.

We also examined the effects of JNK pathway modulation on the epithelial disruption phenotype caused by CagA expression. Although ectopic overexpression of wild type Bsk with bx-GAL4 caused only a minor adult wing phenotype in the form of extra vein material (Figure 2F), coexpression of Bsk with CagA dramatically enhanced the epithelial disruption phenotype (Figure 2G). Ectopic overexpression of Bsk with CagA^EPISA was not sufficient to induce epithelial disruption (Figure 2H). Expression of Bsk^DN also gave rise to only subtle vein defects in an otherwise normal adult wing (Figure 2I). Interestingly, Bsk^DN expression was not able to rescue but instead enhanced the epithelial disruption caused by CagA expression (Figure 2J). One explanation for this apparent contradiction is that blocking JNK signaling prevents the induction of apoptosis that is required to remove aberrant CagA-expressing cells from within the epithelium, which are then allowed to accumulate and lead to a more severe disruption of the adult structure. We tested this hypothesis using the apoptosis inhibitor p35, a baculovirus-derived suicide substrate for effector caspases. Overexpressing p35 alone with bx-GAL4 did not produce a phenotype (Figure S2A and S2C), while coexpressing p35 with CagA effectively blocked apoptosis but enhanced disruption of the adult wing epithelium (Figure S2B and S2D). This observation is consistent with the inhibition of apoptosis resulting in more severe CagA-dependent adult phenotypes. Enhancement and suppression of CagA-induced apoptosis in the wing imaginal disc was quantified using a method we developed to measure the percentage of the expression domain that is caspase-positive. These quantitative data showed that both the enhancement of CagA-induced apoptosis seen with coexpression of ectopic Bsk, and its suppression upon expression of Bsk^DN were statistically significant (Figure 2K).

In order to further examine the genetic interaction between CagA and JNK signaling, we used a lacZ reporter allele of puckered (puc), the main component of a negative feedback loop in the JNK pathway. This construct has been used extensively as a readout for JNK pathway activation in Drosophila tissue using antibody staining for β-galactosidase (β-gal). Expressing CagA in combination with puc-lacZ in the dorsal wing imaginal disc demonstrated that cells adjacent to those undergoing apoptosis are activating JNK signaling (Figure 2L). Upregulation of puc-lacZ correlated with phosphorylation of JNK, verifying that specific activation of JNK signaling results from CagA expression (Figure S2E). These data provide additional evidence that CagA expression activates JNK signaling in the wing imaginal disc epithelium.

Loss of neoplastic tumor suppressors and the TNF homolog Eiger enhances CagA-induced apoptosis

JNK signaling is activated by a complex set of signals including TNF and loss of epithelial polarity (Figure 2M). To examine the mechanism through which CagA activates JNK signaling, we used the bx-GAL4 driver to express CagA in combination with RNAi-mediated knockdown of known epithelial polarity determinants and examined wing imaginal discs for enhancement of the apoptosis phenotype (Figure 3A). We tested a panel of polarity proteins, many of which caused apoptosis when knocked down in the absence of CagA expression (Table S1). We chose to target a protein from each of the previously described complexes whose localization and function establish epithelial cell polarity [27], and to simplify our analysis we selected polarity proteins that did not cause an apoptosis phenotype when knocked down on their own (Figure S3A–S3C). When tested in combination with CagA expression, we found that RNAi-mediated knockdown of neither the junctional protein Bazooka (Baz), nor the apical protein Crumbs (Crb) enhanced apoptosis (Figure S3D and S3E). In addition, knockdown of Par1, which has been shown to interact with CagA in tissue culture cells [28], did not enhance the apoptosis phenotype caused by CagA expression in this context (Figure S3F).

Interestingly, RNAi-mediated knockdown of the basolateral protein Discs Large (Dlg) did not cause a significant phenotype (Figure 3B) but markedly enhanced the apoptosis caused by CagA expression (Figure 3C). The same effect was seen with knockdown of Lethal Giant Larvae (Lgl), another basolateral protein (Figure S3G and S3H). The genes encoding these polarity proteins are known as neoplastic tumor suppressor genes (nTSGs) because their loss causes tumor formation in Drosophila [29], and generating clones of cells which lack this specific class of polarity determinants has been shown to trigger JNK-dependent apoptosis in imaginal discs [30]. Our data suggest that nTSGs normally suppress CagA-mediated JNK pathway activation and subsequent apoptosis in the wing imaginal disc.

Disruption of the nTSGs activates JNK signaling through endocytosis of the TNF homolog Egr [31]. Homozygous egr mutant animals are viable and, as expected, no apoptosis was observed in their wing imaginal discs (Figure S3I). Conversely, ectopic overexpression of wild type Egr in the dorsal wing imaginal disc caused a severe apoptosis phenotype (Figure S3J), consistent with previous data showing Egr to be a potent activator of cell death in Drosophila epithelia [32]. We made the unexpected observation that expression of CagA in the dorsal wing disc of an egr mutant animal enhanced the apoptosis phenotype (Figure 3D). Interestingly, RNAi-mediated knockdown of Egr alone in the dorsal wing with bx-GAL4 did not cause a phenotype (Figure S3K) or enhance apoptosis when coexpressed with CagA (Figure S3L). This observation suggests that loss of Egr in wild type cells surrounding the CagA expression domain is responsible for the enhanced apoptosis phenotype seen in the wing imaginal discs of egr mutant animals expressing CagA.

Recent data has demonstrated that loss of nTSGs in clones of imaginal disc cells causes Egr-dependent activation of nonapoptotic JNK signaling in their wild type neighbors. JNK activation in surrounding wild type cells leads to induction of a phagocytic pathway which triggers engulfment of polarity-deficient cells within the clone [30]. A similar mechanism can be invoked to explain the enhancement of CagA-induced apoptosis seen in egr mutant wing imaginal discs. Loss of Egr in the wild type cells surrounding the expression domain may prevent engulfment of CagA-expressing cells. This would increase the number of aberrant cells available to undergo apoptosis upon CagA-mediated activation of JNK signaling via another parallel upstream pathway. We hypothesize that multiple cellular consequences of CagA expression can activate JNK signaling combinatorially. Supporting this view, we demonstrated that CagA-induced apoptosis was enhanced by ectopic overexpression with a wild type form of the small GTPase Rho1 (Figure 3E), another upstream activator of the JNK pathway that did not cause a phenotype when overexpressed alone (Figure S3M), and which our group has shown is activated by CagA [9].

Enhancement of CagA-induced apoptosis in the wing imaginal disc was quantified using the previously described method. These data showed significant enhancement of apoptosis with coexpression of CagA and knockdown of nTSGs, ubiquitous loss of Egr or overexpression of Rho1. Knockdown of several other polarity proteins or Egr in CagA-expressing cells did not enhance the apoptosis phenotype (Figure 3F). Overexpression of Rho1, ubiquitous or localized loss of Egr and knockdown of the other polarity proteins alone did not induce significant apoptosis in the wing imaginal disc (Figure S3N). These observations suggest that specific polarity protein complexes within the cell, as well as other upstream activators are responsible for transducing the signals that lead to JNK pathway activation upon CagA expression in the wing imaginal disc (Figure 3G).

CagA expression enhances the growth and invasion of tumors generated by expression of oncogenic Ras through JNK pathway activation

The finding that CagA activates the JNK pathway is intriguing in light of recent evidence indicating that activation of JNK signaling can switch from proapoptotic to progrowth in the presence of oncogenic Ras [19]. In order to examine a potential role for CagA-mediated JNK pathway activation in promoting tumorigenesis, we used a slight variation of a previously established Drosophila metastasis model to create whole eye clones expressing an activated form of the Ras oncogene (Ras^V12) in epithelial cells of the eye imaginal disc using the eyeless (ey) driver with the FLP/FRT system to generate primary tumors [33]. We then evaluated the size of GFP-marked tumors in whole larvae (Figure 4A) and dissected cephalic complexes (Figure 4B) in order to determine whether coexpression of CagA could enhance the growth and invasive potential of these tumor cells through activation of the JNK signaling pathway.

Expression of Ras^V12 alone in whole eye clones caused overgrowth of eye imaginal disc cells which resulted in tumor formation (Figure 4C), as previously described [34]. Although generating whole eye clones expressing either GFP alone (Figure S4A) or with CagA (Figure S4B) was not tumorigenic, coexpression of CagA enhanced the growth of tumors generated by Ras^V12 expression (Figure 4D). Whole eye clones expressing CagA^EPISA were also not tumorigenic (Figure S4C), and when combined with Ras^V12 expression caused only a minor enhancement of tumor growth (Figure 4E). As expected, coexpression of Bsk^DN did not affect the growth of tumors generated by Ras^V12 expression alone (Figure 4F). However, Bsk^DN expression caused a severe reduction in the growth of tumors expressing both Ras^V12 and CagA (Figure 4G). Quantification of these data was accomplished by determining the size of dissected cephalic complexes of each genotype and showed a significant growth enhancement with combined expression of Ras^V12 and CagA, which was suppressed by coexpression of Bsk^DN (Figure 4H). These data demonstrate that expression of CagA can enhance the growth of tumors generated by expression of Ras^V12 in a JNK-dependent manner.

Generating whole eye clones that express Ras^V12 alone most commonly caused either a mildly invasive phenotype characterized by the migration of a small number of GFP-positive cells along one edge of the ventral nerve cord (VNC), or a noninvasive phenotype in which cells within the optic lobe approached but did not migrate into the VNC (Figure 5A). Whole eye clones expressing either GFP alone (Figure S5A) or with CagA (Figure S5B) were not invasive, but coexpression of CagA with Ras^V12 resulted in a much larger number of GFP-positive tumor cells migrating from both optic lobes into the VNC (Figure 5B). These cells were not terminally differentiated, as indicated by a lack of staining with the neuron-specific ElaV antibody, and phalloidin staining showed a morphology distinct from other cells in the VNC (Figure S5D). Expressing CagA^EPISA in whole eye clones also did not produce an invasive phenotype (Figure S5C), and coexpression of CagA^EPISA with Ras^V12 caused a less pronounced enhancement of the mild invasion caused by expression of Ras^V12 alone (Figure 5C), suggesting that the phosphorylation-resistant form of CagA is less effective at promoting tumor progression. Coexpression of Bsk^DN did not affect the invasive phenotype generated by Ras^V12 expression alone (Figure 5D), but Bsk^DN expression caused a dramatic reduction in the invasive capacity of tumors expressing both Ras^V12 and CagA (Figure 5E). These data show that CagA expression can enhance the invasion of Ras^V12-expressing tumor cells through JNK activation.

In order to determine the significance of CagA's enhancement of invasion, we used a previously described method [35] to categorize invasive phenotypes into four distinct classes which represent a progression from non-invasive to severe invasion of the VNC (Figure 5F). Quantitation of the percentage of cephalic complexes exhibiting each class of VNC invasion showed a significant difference between expression of Ras^V12 alone and in combination with CagA, which was suppressed by coexpression of Bsk^DN (Figure 5G).

Distribution of CagA within an epithelium can affect manipulation of host proteins and intercellular interactions

Infection of tissue culture cells with H. pylori has been shown to activate JNK signaling, but a role for CagA in this process remains controversial [36]–[38]. Additionally, these experiments were performed in nonpolar AGS cells, so if polarity disruption plays a role in JNK pathway activation downstream of CagA, as our data suggest, these cell culture models may not reveal this interaction. JNK pathway activation has also been shown to result from infection with several pathogenic bacteria in epithelial cell culture models of infection [39]. Interestingly, the enteroinvasive bacterium Shigella flexneri was shown to activate JNK and upregulate TNFα expression in both infected and adjacent uninfected epithelial cells in culture [40], similar to our data showing that JNK-mediated tissue responses to CagA expression involve a cell-nonautonomous requirement for TNF/Egr. The distribution of H. pylori during infection of the gastric epithelium is known to be heterogeneous [2]. We therefore hypothesize that interactions between cells containing CagA protein and uninfected neighboring cells could also be important for pathogenesis of H. pylori.

Our data suggest that CagA is an important mediator of JNK pathway activation during H. pylori infection, and identify several host proteins involved in this process. We observe genetic interaction between CagA and nTSGs, but not junctional proteins involved in polarity. This is consistent with recent data from tissue culture cells which demonstrated that CagA-positive strains of H. pylori specifically disrupt apicobasal polarity in a polarized monolayer prior to affecting the integrity of cellular junctions [11]. Disruption of nTSGs has been shown to cause JNK-dependent apoptosis, and more recent data indicates that elimination of polarity-deficient cells is dependent on their location within the wing imaginal disc due to varying levels of dMyc throughout the tissue [41]. The extent of aberrant cell removal differs significantly with respect to established gradients of Wnt/Wingless, dMyc and Hippo-Salvador-Warts pathway activation that ensure proper development of the wing [42]. We propose that the extent of variation observed upon CagA expression in the wing with different GAL4 drivers is due to spatial variation in these host cell signaling pathways. Our data also suggest that CagA can activate JNK-dependent apoptosis through multiple upstream pathways. The observation that overexpression of Rho1 enhances CagA-dependent apoptosis in the wing imaginal disc epithelium is consistent with previous data from our group demonstrating a role for CagA in activating the Rho pathway to disrupt epithelial patterning [9].

Use of the unique genetic tools available in Drosophila has provided important insight into potential interactions between CagA-expressing cells and neighboring wild type cells. Our observation that loss of TNF/Egr in wild type cells surrounding those expressing CagA can enhance apoptosis, presumably by reducing engulfment of CagA-expressing cells, indicates that the genetic state of uninfected cells may also play a role in H. pylori pathogenesis. This finding is important with respect to the established function of TNF/Egr-dependent JNK activation in cell competition induced by intrinsic tumor suppression. Our data suggest that the presence of CagA protein induces changes in signaling and morphology which cause an epithelial cell to be outcompeted by its wild type neighbors through a local mechanism that requires TNF/Egr in the neighboring epithelial cells. Interestingly, Drosophila immune cells known as hemocytes have also demonstrated the ability to remove polarity-deficient cells from an epithelium through a more global extrinsic tumor suppression mechanism that is TNF/Egr-dependent [43]. Although we have not explored a role for hemocytes in removal of CagA-expressing wing epithelial cells, it is possible that a related mechanism may occur during H. pylori infection of the human stomach through immune surveillance mediated by TNF. Although this specific cytokine is an important component of the initial immune response to infection with a pathogen, TNF is also known to promote tumor progression specifically in the context of chronic inflammation or in the presence of activated Ras [43], [44]. We hypothesize that TNF functions to suppress tumor initiation resulting from the presence of CagA protein in gastric epithelial cells through several mechanisms, but that the inflammatory environment created by prolonged infection with H. pylori and the emergence of oncogenic mutations over time cause TNF to promote progression of gastric cancer.

Genetic changes in host cells can alter the downstream effects of CagA signaling during long-term association with H. pylori

Since it was first discovered, JNK has been demonstrated to have both pro-tumorigenic and tumor suppressor functions in different cell types and organs. Studies in Drosophila have helped shed light on the genetic contexts in which JNK activation functions to promote tumor progression, namely in the presence of oncogenic Ras [45]. Recently, JNK was shown to be required for activated KRas-induced lung tumor formation in mice [46], suggesting a conserved function of JNK activation in cooperating with activated Ras to promote tumorigenesis in mammals. A potential role for JNK pathway activation has also been explored in mammalian gastric cancer. Activation of JNK signaling has been detected in human gastric cancer samples, and mice lacking JNK1 exhibit a decrease in gastric apoptosis and an attenuation of gastric tumor development induced by the chemical carcinogen N-methyl-N-nitrosourea [47]. A role for H. pylori in the context of mammalian gastric cancers induced by cooperation between JNK and Ras signaling has not been explored.

Our finding that CagA expression can induce JNK-dependent apoptosis in a polarized epithelium is interesting with respect to data suggesting that JNK signaling has evolved as a cell editing mechanism to remove aberrant cells from within an epithelium [18]. Activation of JNK signaling could represent a host response aimed at removing cells containing CagA protein from the gastric epithelium. Similarly, P. aeruginosa-mediated activation of JNK signaling in the intestinal epithelium of Drosophila can trigger epithelial renewal as a host defense mechanism. However, this process can become pathogenic and lead to dramatic overproliferation of intestinal cells in animals harboring oncogenic Ras mutations [21]. In H. pylori infection, which can persist for many years before the development of gastric cancer, JNK-mediated apoptosis could be an effective mechanism to limit pathogenic effects on the gastric epithelium. However, this process of tissue editing can also increase cell turnover, contributing to accumulation of genetic mutations in host cells. Our data show that acquisition of an oncogenic mutation in host epithelial cells experiencing CagA-mediated JNK pathway activation can promote tumor progression, suggesting that this potential host defense strategy can become tumorigenic in certain genetic contexts (Figure 6B).

Transgenic expression of CagA was recently found to cause neoplastic transformation in a mouse model, providing evidence for CagA's role as a bacterial oncoprotein in mammals [48]. The low incidence and delayed development of gastrointestinal tumors in these mice was attributed to lower expression of CagA in the surviving animals, as higher expression was assumed to be lethal during embryogenesis. Additionally, secondary mutations were identified in the tumors, but their potential cooperation with host cell signaling pathways activated by CagA expression was not addressed [48]. Infection with CagA-positive H. pylori is also known to induce an invasive phenotype in tissue culture cells [49], but potential effects of the oncogenic mutations present in these immortalized cell lines is unknown. Although we did not demonstrate the sufficiency of CagA to induce tumor phenotypes in our Drosophila model, our data support a crucial role for CagA in promoting tumor progression in combination with oncogene activation. We believe that using an inducible expression system in Drosophila allowed us to bypass the toxicity observed upon CagA expression in mice and cell culture models, thus revealing novel interactions between CagA and host cell proteins with downstream effects on apoptosis and tumorigenesis.

Although half the world's population is thought to be infected with H. pylori, a small percentage of those individuals will develop gastric cancer [2]. This observation indicates that, in addition to the presence of the cag PAI in more virulent strains, host genetics must also play a crucial role in determining the outcome of H. pylori infection. Our results suggest that a change in host genetics during long-term association with H. pylori could cause JNK activation to switch from conferring a protective function against CagA-induced cellular changes to enabling tumor progression. Data collected from tissue biopsies indicate that Ras mutation may play a role in the development of gastric cancer in human patients [50], and our data put forward the idea that enhanced tumorigenic potential created by cooperation between JNK pathway activation via the bacterial genetic factor CagA and sporadic activation of Ras in host cells could drive gastric cancer formation in a subset of H. pylori infections.

Fly strains and generation of whole eye clones

The following fly stocks were used: UAS-CagA, UAS-CagA^EPISA [7]; bx-GAL4, sd-GAL4, ap-GAL4, en-GAL4, ptc-GAL4, hs-FLP, Act>y⁺>GAL4, UAS-GFP.S65T, UAS-mCD8::GFP (mGFP), UAS-bsk.B (Bsk¹), UAS-bsk.A-Y (Bsk²), UAS-bsk.K53R (Bsk^DN), egr ^MB06803 (egr ^−/−), UAS-Dlg-RNAi, UASp-FLAG.Rho1 (Rho1), UAS-Ras85D.V12 (Ras^V12), UAS-Crb-RNAi, UAS-Patj-RNAi, UAS-Cora-RNAi, UAS-Cdc42-RNAi (from Bloomington Stock Center); 765-GAL4 (provided by Ross Cagan, Mount Sinai School of Medicine); puc ^E69 (puc-lacZ), Regg1^GS9830 (UAS-Egr) (provided by Michael Galko, MD Anderson Cancer Center); UAS-Lgl-RNAi, UAS-Baz-RNAi, UAS-Par1-RNAi, UAS-Scrib-RNAi, UAS-Par6-RNAi, UAS-aPKC-RNAi, UAS-Mir-RNAi (provided by Chris Doe, University of Oregon); UAS-Egr-RNAi (from Vienna Drosophila Resource Center); ey-FLP; Act>y⁺>GAL4, UAS-GFP (provided by Tory Herman, University of Oregon). Flies were raised at 25°C using standard methods. Whole eye clones were generated as previously described [33] without the GAL80 repressor to express transgenes in all cells that give rise to the eye-antennal disc. FLP-out clones were generated by subjecting each 4–6 hour collection of embryos to one hour of heat-shock at 37°C, then dissecting wing discs approximately 96–120 hours later.

Histology

Larval tissues were fixed and stained using standard protocols. The following primary antibodies were used: rabbit anti-active caspase-3 (1∶200; BD Pharmingen), mouse anti-Mmp1 (1∶50; Developmental Studies Hybridoma Bank), mouse anti-β-galatosidase (1∶500; Sigma) rat anti-ElaV (1∶10; Developmental Studies Hybridoma Bank), rabbit anti-β-galatosidase (1∶200; MP Biomedicals) and mouse anti-phospho-SAPK/JNK (1∶100; Cell Signaling Technology). Both Cy3 and Cy5-conjugated secondary antibodies were used (1∶200; Jackson ImmunoResearch), as well as Alexa Fluor 546 and Alexa Fluor 633 phalloidin (1∶40; Molecular Probes). Intact adult wings were mounted in a 1∶1 mixture of lactic acid and ethanol.

Image analysis and quantitation

Adult wings, intact larvae and whole cephalic complexes were visualized using light microscopy or GFP fluorescence on a Zeiss dissecting microscope. Wing imaginal discs, ventral nerve cords and cephalic complexes were visualized on a Nikon confocal microscope. Images were processed using Adobe Photoshop, where levels were adjusted to optimize the signal-to-noise ratio in each color channel while maintaining similar levels of background noise and desired signal between channels and images. Adult wing images were removed from their background using the Extract filter in Adobe Photoshop. XZ confocal planes were created using the Reslice function in Image J. Projections of confocal cross sections were created using the Merge to HDR command in Adobe Photoshop. Apoptosis was quantified by selecting the single confocal cross section of each wing imaginal disc exhibiting the highest level of active caspase-3 staining and manually tracing the expression domain, then determining the percentage of this domain showing active caspase-3 staining using the Threshold function in Image J. Cephalic complex size was quantified using the Threshold function in Image J to determine the area of the tissue in µm². Graphs were created with GraphPad Prism software, which was also used to calculate two-tailed p values using the unpaired t test with Welch's correction for apoptosis quantitation. The statistical significance of differences in metastatic potential for each genotype was calculated using Excel to determine two-tailed p values using the unpaired t test.