LETTERS

Interaction between RasV12 and scribbled clonesinduces tumour growth and invasionMing Wu1*, Jose Carlos Pastor-Pareja1* & Tian Xu1,2

Human tumours have a large degree of cellular and genetic hete-rogeneity1. Complex cell interactions in the tumour and itsmicroenvironment are thought to have an important role intumorigenesis and cancer progression2. Furthermore, cooperationbetween oncogenic genetic lesions is required for tumour develop-ment3; however, it is not known how cell interactions contribute tooncogenic cooperation. The genetic techniques available in thefruitfly Drosophila melanogaster allow analysis of the behaviourof cells with distinct mutations4, making this the ideal modelorganism with which to study cell interactions and oncogeniccooperation. In Drosophila eye-antennal discs, cooperation betweenthe oncogenic protein RasV12 (ref. 5) and loss-of-function mutationsin the conserved tumour suppressor scribbled (scrib)6,7 gives rise tometastatic tumours that display many characteristics observedin human cancers8–11. Here we show that clones of cells bearingdifferent mutations can cooperate to promote tumour growth andinvasion in Drosophila. We found that the RasV12 and scrib2 muta-tions can also cause tumours when they affect different adjacentepithelial cells. We show that this interaction between RasV12 andscrib2 clones involves JNK signalling propagation and JNK-inducedupregulation of JAK/STAT-activating cytokines, a compensatorygrowth mechanism for tissue homeostasis. The development ofRasV12 tumours can also be triggered by tissue damage, a stresscondition that activates JNK signalling. Given the conservation ofthe pathways examined here, similar cooperative mechanisms couldhave a role in the development of human cancers.

Clones of mutant cells marked with green fluorescent protein (GFP)can be generated in the eye-antennal imaginal discs of Drosophila larvaeby mitotic recombination. Clones expressing RasV12, an oncogenicform of the Drosophila Ras85D protein, moderately overgrow12

(Fig. 1a, b). Clones mutant for scrib lose apico-basal polarity anddie6,13 (Fig. 1c). In contrast, scrib clones simultaneously expressingRasV12 grow into large metastatic tumours (Fig. 1d)8. To understandbetter the cooperation between these two mutations, we producedanimals in which cell division after a mitotic recombination eventcreates two daughter cells: one expressing RasV12 and the other mutantfor scrib. Discs containing adjacent RasV12 (GFP-positive) and scrib2

clones developed into large tumours, capable of invading the ventralnerve cord (Fig. 1e). This shows that RasV12 and scrib also cooperate fortumour induction when they occur in different cells. We will refer tothese tumours as RasV12//scrib2 tumours, to denote interclonal onco-genic cooperation and distinguish them from RasV12scrib2 tumours, inwhich cooperation occurs in the same cells intraclonally.

In the late stages of the development of RasV12//scrib2 tumours, mostcells in the tumour mass are RasV12 cells (Fig. 1h, i). scrib2 cells, as well asresidual wild-type cells, are almost completely absent from the tissue,similar to the absence of wild-type cells in late RasV12scrib2 tumours(Fig. 1f, g). To test the possibility that RasV12//scrib2 tumours are caused

by unopposed growth of RasV12 cells, we examined eye-antennal discswhere all cells expressed RasV12. Marked overgrowth or invasion did notoccur (Supplementary Fig. 1), showing that interaction between RasV12

and scrib2 cells is required for tumour development. Interclonal co-operation between RasV12 and lethal giant larvae (lgl) also producedtumours (Supplementary Fig. 2), indicating that other polaritymutations can cooperate interclonally with RasV12. Intrigued by thesefindings, we decided to investigate the mechanisms underlying

*These authors contributed equally to this work.

1Howard Hughes Medical Institute, Department of Genetics, Yale University School of Medicine, 295 Congress Avenue, New Haven, Connecticut 06519, USA. 2Fudan-Yale BiomedicalResearch Center, Institute of Developmental Biology and Molecular Medicine, School of Life Sciences, Fudan University, 220 Han Dan Road, Shanghai 20043, China.

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Figure 1 | Interclonal cooperation between RasV12 and scrib2

causes tumours. a–e, Clones of cells marked with GFP in the eye-antennaldiscs of third-instar larvae. Upper panels show the cephalic complex (CC),consisting of eye-antennal discs (EA), brain (B) and ventral nerve cord(VNC). Lower panels show the dissected ventral nerve cord. Compared towild-type clones (a), RasV12 clones overgrow moderately (b). scrib2 clonesare eliminated from the tissue (c). Double mutant RasV12scrib2 clones(d, intraclonal cooperation), as well as RasV12 clones confronted with scrib2

clones (e, interclonal cooperation), cause tumours that overgrow and invadethe ventral nerve cord. f–i, Confocal sections of the inner tumour mass inRasV12scrib2 (f, g) and RasV12//scrib- tumours (h, i) at day 6 and 14 after egglaying. Non-RasV12 cells (absence of GFP) are progressively eliminated fromthe tissue. Cell nuclei labelled with 49,6-diamidino-2-phenylindole (DAPI).Yellow arrowheads point to scattered remaining GFP-negative cells (insets ing and i).

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non-autonomous oncogenic cooperation and sustained growth inRasV12//scrib2 tumours.

JAK/STAT signalling promotes cell proliferation in different con-texts in mammals and flies14, including the overgrowth caused bymutation of several tumour suppressors15. In a complementaryDNA microarray analysis of RasV12scrib2 tumours, we discoveredupregulation of the unpaired genes (upd, upd2 and upd3; data notshown), which encode JAK/STAT-activating cytokines related tointerleukin 6 (refs 16–18). We confirmed the upregulation of theunpaired genes in RasV12scrib2 and RasV12//scrib2 tumours by real-time polymerase chain reaction with reverse transcription (RT–PCR;Fig. 2a). Furthermore, we observed elevated expression of the JAK/STAT reporter STAT–GFP19 in both RasV12scrib2 and RasV12//scrib2

tumours (Fig. 2b–e), thus correlating high expression of Upd cyto-kines with increased JAK/STAT activity.

To test the involvement of JAK/STAT signalling in the growth ofRasV12scrib2 and RasV12//scrib2 tumours, we used a dominant negativeform of the JAK/STAT receptor Domeless (DomeDN)20. Expression ofDomeDN achieved suppression of overgrowth and invasion of theventral nerve cord in RasV12scrib2 tumours (Fig. 2f). Furthermore,RasV12//scrib2 tumours were suppressed by expression of DomeDN

in RasV12 cells (Fig. 2g). A loss-of-function mutation in Stat92E, encod-ing the Drosophila STAT transcriptional activator, reduced growth andinvasiveness of RasV12scrib2 and RasV12//scrib2 tumours (Sup-plementary Fig. 3). From these experiments, we conclude that JAK/STAT signalling is required for the development of RasV12scrib2 andRasV12//scrib2 tumours.

The suppression of RasV12scrib2 and RasV12//scrib2 tumours byreducing JAK/STAT activity in RasV12 cells points to cooperationbetween Ras and JAK/STAT signalling as a cause of tumour growth.To confirm this, we generated clones of cells co-expressing Updcytokines and RasV12. Whereas Upd overexpression in wild-type cells(Fig. 2h), scrib2 cells or in wild-type cells adjacent to scrib2 cells(Supplementary Fig. 4) did not cause tumours, co-expression ofRasV12 and Upd produced large invasive tumours (Fig. 2i). Similarresults were obtained co-expressing RasV12 and Upd2 (Fig. 2j),whereas co-expression of RasV12 and Upd3 caused smaller,non-invasive tumours (Fig. 2k). Finally, RasV12Upd Upd2 tumours(overexpressing RasV12, Upd and Upd2) were larger than RasV12Upd

and RasV12Upd2 tumours (Fig. 2l), indicating an additive effect ofthe expression of different Upd cytokines (see also SupplementaryFig. 5).

Prevention of actual cell death in cells apoptotically stimulated hasbeen shown to potently promote overgrowth21. To assess a possibleinvolvement of apoptosis prevention in the synergy between Ras andJAK/STAT signalling, we co-expressed the apoptosis inhibitor p35with RasV12 or Upd. Neither condition produced tumours (Sup-plementary Fig. 6), indicating that cooperation between Ras andJAK/STAT involves a mechanism other than apoptosis prevention.RasV12Upd tumours were suppressed by expression of DomeDN

(Fig. 2m), thus confirming that their development requires JAK/STAT activity. In all, both loss- and gain-of-function experimentslead us to conclude that Ras and JAK/STAT signalling exhibit a strongsynergistic tumour-promoting interaction, responsible for thedevelopment of RasV12scrib2 and RasV12//scrib2 tumours.

Having established the involvement of JAK/STAT signalling in thegrowth of RasV12scrib2 and RasV12//scrib2 tumours, we decided toinvestigate how expression of the Upd cytokines is upregulated. Wepreviously showed that expression of the unpaired genes is elevated inwounds in a JNK-dependent manner22. It has also been shown thatJNK signalling can induce non-autonomous overgrowth23,24 and thatJNK signalling is upregulated in scrib2 clones13,25 and scrib2 discs22,which develop as tumours in scrib2 larvae6. To test the possibility thatJNK activation causes ectopic JAK/STAT signalling in scrib2 cells, wemonitored STAT–GFP expression in discs doubly mutant for scriband hep, coding for the Drosophila JNK-kinase Hemipterous. In thesediscs, STAT–GFP expression was reduced and overgrowth sup-pressed (Fig. 3a–c), showing that JAK/STAT elevation in scrib2 cellsdepends on JNK activity.

The induction of Upd cytokines by JNK in scrib2 cells can explainthe growth of RasV12scrib2 tumours, placing JAK/STAT signallingdownstream of JNK. In support of this, a dominant negative formof the Jun kinase Basket (BskDN) suppressed RasV12scrib2 tumours9

(Fig. 3d, e), but not RasV12Upd tumours (Fig. 3f, g). In the case ofRasV12//scrib2 tumours, few scrib2 cells remain in the tissue at latestages (Fig. 1i). Therefore, Upd induction in scrib2 cells cannot fullyaccount for tumour development. Indeed, expression of BskDN inRasV12 cells partially suppressed the growth of RasV12//scrib2

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Figure 2 | Synergy between Ras and JAK/STATsignalling promotes growth and invasion inRasV12scrib2 and RasV12//scrib2 tumours. a,Quantification by real-time RT–PCR of expressionof the upd genes—encoding the JAK/STAT-activating cytokines Upd, Upd2 and Upd3—ineye-antennal discs containing wild-type clones,RasV12-expressing clones, scrib2 clones (day 6 afteregg laying), RasV12scrib2 tumours and RasV12//scrib2 tumours (day 10 after egg laying).Expression is normalized to the housekeepinggene rp49. Error bars depict 95% confidenceintervals (1.96 3 s.e.m., n 5 3). b–e, Expression ofthe JAK/STAT reporter STAT–GFP (green, lowerpanels) in day 6 after egg laying eye-antennal discscontaining wild-type clones (b), RasV12 clones(c), RasV12scrib2 (d) and RasV12//scrib2 tumours(e). Wild-type, RasV12 and RasV12scrib2 clones aremarked with Myr–RFP (red) in upper panels.f, g, Suppression of RasV12scrib2 (f) and RasV12//scrib2 (g) tumours by expression of a dominantnegative form of the JAK/STAT receptor Domeless(DomeDN). h, Clones overexpressing Upd.i–l, Tumours caused by RasV12 clones co-overexpressing Upd (i), Upd2 (j), Upd3 (k) andboth Upd and Upd2 (l). m, RasV12Upd tumoursare suppressed by DomeDN. Upper panels showthe cephalic complex and lower panels the ventralnerve cord in f–m.

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tumours (Fig. 3h, i) and expression of the unpaired genes (Fig. 3j).This shows that in RasV12//scrib2 tumours expression of Upd cyto-kines downstream of JNK signalling also occurs in RasV12 cells.

scrib2 clones cause JNK activation both autonomously and non-autonomously25. Furthermore, in wing discs, wounding induces JNKactivation away from the site of wounding22,26, indicating that JNKactivity can propagate. To investigate this, we wounded wing discsand examined the expression of puckered (puc), a JNK downstreamgene encoding a JNK phosphatase that negatively regulates the path-way27. When wounds were induced in the anterior or posterior wingregions, JNK activation, revealed by puc-lacZ expression, wasobserved across the disc in the opposite compartment (Fig. 3k). Incontrast, overexpression of Puc in a central stripe of cells preventedexpansion of JNK to the opposite compartment (Fig. 3l andSupplementary Fig. 7). This indicates that JNK activity propagatesthrough a feed-forward loop and, together with previous findings,suggests that in RasV12//scrib2 tumours, scrib2 cells trigger JNKactivation and that this activation propagates to adjacent RasV12 cells.JNK-dependent upregulation of Upd cytokines in RasV12 cells, thus,can sustain tumour growth when the original source of JNK activity,the scrib2 cells, is no longer present.

The previous experiments reveal a central role for JNK in thecooperation of RasV12 and scrib2. Because both wounds and scrib2

induce JNK activation, we tested the possibility that tissue damagecould cooperate with RasV12 to promote tumour overgrowth. Wewounded larval right wing discs and examined them 48 h later. Inwild-type discs, compared to the unwounded left disc, woundingresulted in size reduction (Fig. 4a, b and Supplementary Fig. 8;wounded/unwounded size ratio (6s.d.) 5 0.70 6 0.18). In contrast,wounding of RasV12-expressing discs caused a marked increase inRasV12-induced overgrowth (Fig. 4c, d and Supplementary Fig. 8;1.46 6 0.31). No metastasis was detected in this experiment (notshown). Finally, the wounded/unwounded ratio in p35-expressingdiscs (1.09 6 0.14; Supplementary Fig. 8) shows that apoptosis

prevention by RasV12 cannot completely account for its cooperationwith mechanically-induced damage.

The fact that both scrib2 clones and tissue damage induce over-growth of RasV12 tissue indicates that compensatory proliferation inresponse to scrib2 cells could underlie cooperation in RasV12//scrib2

tumours. To test this, we studied the effect of confronting scrib2 cellswith cells mutant for Stat92E. When scrib2 clones are generated in eye-antennal discs, scrib2 cells in the adult eye are mostly absent13 and theeye appears normal in size (Fig. 4e, f). When Stat92E2 cells confrontscrib2 cells, in contrast, the eye is greatly reduced (Fig. 4g, h andSupplementary Fig. 9), showing that Stat92E2 cells cannot compen-sate for the loss of scrib2 cells. These data indicate a role for JAK/STATsignalling in tissue homeostasis through compensatory proliferation(see also Supplementary Figs 9 and 10). Therefore, a mechanism toensure recovery after damage explains the development ofRasV12scrib2 and RasV12//scrib2 tumours and can mediate interclonaloncogenic cooperation (Fig. 4i).

We have used Drosophila to investigate how oncogenic coopera-tion between different cells can promote tumour growth and inva-sion. Our experiments, addressed to understanding interclonalcooperation in RasV12//scrib2 tumours, uncovered a two-tier mech-anism by which scrib2 cells promote neoplastic development ofRasV12 cells: (1) propagation of stress-induced JNK activity fromscrib2 cells to RasV12 cells; and (2) expression of the JAK/STAT-activating Unpaired cytokines downstream of JNK. Our findings,therefore, highlight the importance of cell interactions in oncogeniccooperation and tumour development. We also show that stress-induced JNK signalling and epigenetic factors such as tissue damagecan contribute to tumour development in flies. Notably, tissuedamage caused by conditions such as chronic inflammation has beenlinked to tumorigenesis in humans28,29. Furthermore, expression ofthe Unpaired cytokines promotes tumour growth (this study) as wellas an antitumoural immune response22, which parallels the situationin mice and humans30. Future research into phenomena such as

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Figure 3 | JNK signalling drives oncogenic cooperation upstream of JAK/STAT. a–c, STAT–GFP expression in wing discs of wild-type larvae (a) andscrib2 larvae heterozygous (b) or hemizygous (male, c) for the JNK-kinaseloss-of-function mutation hepr75. Overgrowth and STAT–GFP upregulationare suppressed by hepr75. Arrowheads point to normal STAT–GFPexpression in the wing hinge. Discs stained with phalloidin (red).d–g, Expression of a dominant negative form of the Jun-kinase Basket(BskDN) suppresses RasV12scrib2 tumours (d, e), but not RasV12Updtumours (f, g). h, i, Expression of bskDN in RasV12 cells partially suppressesRasV12//scrib2 tumours. Upper panels show the cephalic complex and lowerpanels the ventral nerve cord in d–i. j, Quantification by real-time RT–PCR

of expression of the upd genes in RasV12//scrib2 and RasV12bskDN//scrib2

tumours (day 6 after egg laying). Error bars represent 95% confidenceintervals (n 5 3). k, Propagation of JNK activity (puc-lacZ reporter, green)into the posterior (P) compartment (arrowhead) in wing discs wounded inthe anterior (A) compartment (asterisk) 24 h after wounding. l, puc-lacZexpression in discs expressing the JNK phosphatase Puc under control ofptc-GAL4 (red cells, expressing RFP), wounded as in k. Puc is both adownstream target and a negative regulator of JNK. Propagation of puc-lacZexpression into the posterior compartment is not observed (openarrowhead).

NATURE | Vol 463 | 28 January 2010 LETTERS

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compensatory growth and interclonal cooperation in Drosophila willprovide valuable insights into the biology of cancer.

METHODS SUMMARYClones of mutant cells in the eye-antennal discs were generated as previously

described8. Detailed genotypes of the experimental individuals are described in

Supplementary Information. The following antibodies and dyes were used:

mouse monoclonal anti-bgal (1:500, Sigma), goat Alexa-488-conjugated anti-mouse IgG (1:200, Molecular Probes), phalloidin Texas red (Molecular Probes).

Wounds were performed with forceps in mid third-instar larvae as described

previously22.

Full Methods and any associated references are available in the online version ofthe paper at www.nature.com/nature.

Received 24 September; accepted 20 November 2009.Published online 13 January 2010.

1. Heppner, G. H. Tumour heterogeneity. Cancer Res. 44, 2259–2265 (1984).2. Hanahan, D. & Weinberg, R. A. The hallmarks of cancer. Cell 100, 57–70 (2000).3. Kinzler, K. W. & Vogelstein, B. Lessons from hereditary colorectal cancer. Cell 87,

159–170 (1996).4. Xu, T. & Rubin, G. M. Analysis of genetic mosaics in developing and adult

Drosophila tissues. Development 117, 1223–1237 (1993).5. Barbacid, M. ras genes. Annu. Rev. Biochem. 56, 779–827 (1987).

6. Bilder, D. & Perrimon, N. Localization of apical epithelial determinants by thebasolateral PDZ protein Scribble. Nature 403, 676–680 (2000).

7. Zhan, L. et al. Deregulation of scribble promotes mammary tumourigenesis andreveals a role for cell polarity in carcinoma. Cell 135, 865–878 (2008).

8. Pagliarini, R. A. & Xu, T. A genetic screen in Drosophila for metastatic behavior.Science 302, 1227–1231 (2003).

9. Igaki, T., Pagliarini, R. A. & Xu, T. Loss of cell polarity drives tumour growth andinvasion through JNK activation in Drosophila. Curr. Biol. 16, 1139–1146 (2006).

10. Uhlirova, M. & Bohmann, D. JNK- and Fos-regulated Mmp1 expressioncooperates with Ras to induce invasive tumours in Drosophila. EMBO J. 25,5294–5304 (2006).

11. Srivastava, A., Pastor-Pareja, J. C., Igaki, T., Pagliarini, R. & Xu, T. Basementmembrane remodeling is essential for Drosophila disc eversion and tumourinvasion. Proc. Natl Acad. Sci. USA 104, 2721–2726 (2007).

12. Karim, F. D. & Rubin, G. M. Ectopic expression of activated Ras1 induceshyperplastic growth and increased cell death in Drosophila imaginal tissues.Development 125, 1–9 (1998).

13. Brumby, A. M. & Richardson, H. E. scribble mutants cooperate with oncogenic Rasor Notch to cause neoplastic overgrowth in Drosophila. EMBO J. 22, 5769–5779(2003).

14. Zeidler, M. P., Bach, E. A. & Perrimon, N. The roles of the Drosophila JAK/STATpathway. Oncogene 19, 2598–2606 (2000).

15. Hariharan, I. K. & Bilder, D. Regulation of imaginal disc growth by tumour-suppressor genes in Drosophila. Annu. Rev. Genet. 40, 335–361 (2006).

16. Harrison, D. A., McCoon, P. E., Binari, R., Gilman, M. & Perrimon, N. Drosophilaunpaired encodes a secreted protein that activates the JAK signaling pathway.Genes Dev. 12, 3252–3263 (1998).

17. Agaisse, H., Petersen, U. M., Boutros, M., Mathey-Prevot, B. & Perrimon, N.Signaling role of hemocytes in Drosophila JAK/STAT-dependent response toseptic injury. Dev. Cell 5, 441–450 (2003).

18. Hombrıa, J. C., Brown, S., Hader, S. & Zeidler, M. P. Characterisation of Upd2, aDrosophila JAK/STAT pathway ligand. Dev. Biol. 288, 420–433 (2005).

19. Bach, E. A. et al. GFP reporters detect the activation of the Drosophila JAK/STATpathway in vivo. Gene Expr. Patterns 7, 323–331 (2007).

20. Brown, S., Hu, N. & Hombrıa, J. C. Identification of the first invertebrate interleukinJAK/STAT receptor, the Drosophila gene domeless. Curr. Biol. 11, 1700–1705 (2001).

21. Martın, F. A., Perez-Garijo, A. & Morata, G. Apoptosis in Drosophila: compensatoryproliferation and undead cells. Int. J. Dev. Biol. 53, 1341–1347 (2009).

22. Pastor-Pareja, J. C., Wu, M. & Xu, T. An innate immune response of blood cells totumours and tissue damage in Drosophila. Disease Models Mech. 1, 144–154 (2008).

23. Ryoo, H. D., Gorenc, T. & Steller, H. Apoptotic cells can induce compensatory cellproliferation through the JNK and the Wingless signaling pathways. Dev. Cell 7,491–501 (2004).

24. Uhlirova, M., Jasper, H. & Bohmann, D. Non-cell-autonomous induction of tissueovergrowth by JNK/Ras cooperation in a Drosophila tumour model. Proc. NatlAcad. Sci. USA 102, 13123–13128 (2005).

25. Igaki, T., Pastor-Pareja, J. C., Aonuma, H., Miura, M. & Xu, T. Intrinsic tumoursuppression and epithelial maintenance by endocytic activation of Eiger/TNFsignaling in Drosophila. Dev. Cell 16, 458–465 (2009).

26. Bosch, M., Serras, F., Martın-Blanco, E. & Baguna, J. JNK signaling pathwayrequired for wound healing in regenerating Drosophila wing imaginal discs. Dev.Biol. 280, 73–86 (2005).

27. Martın-Blanco, E. et al. puckered encodes a phosphatase that mediates a feedbackloop regulating JNK activity during dorsal closure in Drosophila. Genes Dev. 12,557–570 (1998).

28. Dvorak, H. F. Tumors: wounds that do not heal. Similarities between tumor stromageneration and wound healing. N. Engl. J. Med. 315, 1650–1659 (1986).

29. Balkwill, F. & Mantovani, A. Inflammation and cancer: back to Virchow? Lancet357, 539–545 (2001).

30. de Visser, K. E., Eichten, A. & Coussens, L. M. Paradoxical roles of the immunesystem during cancer development. Nature Rev. Cancer 6, 24–37 (2006).

Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank E. Bach, D. Harrison, J. Castelli-Gair Hombria,M. Zeidler, T. Adachi-Yamada, M. Mlodzik, E. Matunis, D. Montell, H. Agaisse, theBloomington Stock Center and the National Institute of Genetics (Kyoto) for flystrains, and T. Ni, S. Landrette and M. Rojas for comments. R. Pagliarini andS. Landrette helped with microarray analysis and RT–PCR. We thank T. Igaki fordiscussing the manuscript and providing FRT82B,tub-Gal80,scrib1/TM6B flies.M.W. is a Yale predoctoral fellow. J.C.P.-P. was funded by a Spanish Ministry ofEducation postdoctoral fellowship. This work was supported by a grant from NIH/NCI to T.X. T.X. is a Howard Hughes Medical Institute Investigator.

Author Contributions M.W., J.C.P.-P. and T.X. designed research, M.W. andJ.C.P.-P. performed experiments and analysed the data. M.W., J.C.P.-P. and T.X.wrote the manuscript.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Correspondence and requests for materials should be addressed to T.X.(tian.xu@yale.edu).

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Figure 4 | Tissue damage, compensatory growth and a model for interclonaloncogenic cooperation. a–d, Cooperation between RasV12 and tissue damage.Right (wounded) and left (unwounded) wing discs of a wild-type larva(a, b) and a larva expressing RasV12 under control of nub-GAL4 (c, d). Discswere wounded by repeated pinching and dissected 48 h later. Expression ofRFP driven by nub-GAL4 marks the wing blade region (red). Cell nuclei arestained with DAPI (blue). e–h, Requirement of JAK/STAT signalling incompensatory proliferation. Wild-type (e) and Stat92E2 (g) clones in adulteyes, marked by the absence of red pigment. In eyes containing scrib2 clonesconfronted with wild-type cells (f), scrib2 cells (red) are mostly absent andsize of the eye is largely normal. In eyes containing scrib2 clones (red)confronted with Stat92E2 cells (h), the size of the eye is reduced. i, Model forthe involvement of JNK and JAK/STAT signalling in intraclonal andinterclonal cooperation between RasV12 and scrib2. See text for details.

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METHODSStrains and culture. Cultures were maintained at 25 uC on standard medium.

Whenever staging of larvae was required, parental flies were placed in a fresh

culture vial and left there to lay eggs for 1 day; we considered the time of remov-

ing the flies from the vial 12 h (612) after egg laying. The following strains were

used in this study: (1) y w; FRT82B; (2) y w; FRT82B,scrib1/TM6B; (3) w;

UAS-RasV12 (II); (4) w; UAS-RasV12 (III); (5) y w,ey-Flp1;act.y1.GAL4,

UAS-GFP.S65T;FRT82B,tub-GAL80; (6) w; FRT82B, tub-GAL80,scrib1/TM6B;

(7) y w,ey-Flp1;act.y1.GAL4,UAS-myrRFP;FRT82B,tub-GAL80; (8) tub-

GAL80,FRT19A;eyFLP5,act.y1.GAL4,UAS-GFP; (9) y w,ey-Flp1;tub-GAL80,

FRT40A;act.y1.GAL4,UAS-GFP.S65T; (10) y w,ey-Flp1;FRT82B,ubi-GFP;

(11) w;10XSTAT-GFP.1 (II); (12) w; ey-Flp6 (III); (13) w; UAS-upd (II); (14)

w; UAS-upd2 (III); (15) w; UAS-upd3 (II); (16) w; UAS-upd-IR(R-1) (III); (17)

w,UAS-domeDcyt1.1; (18) w; UAS-domeDcyt2.1 (II); (19) y w upd2D3-62; (20) w,UAS-

bskDN; (21) w; UAS-bskDN (III); (22) y w hepr75/FM7i,act-GFP; (23) w; ptc-GAL;

(24) w; UAS-myr-RFP (II); (25) pucE69-lacZ,ry/TM3,Sb; (26) w; UAS-puc (III);

(27) w; nub-GAL4.K; (28) w; UAS-p35 (II); (29) w; UAS-p35 (III); (30) w;

FRT82B,Stat92E06346/TM6B; (31) w; FRT82B,Stat92E397/TM3,Sb; (32) w;FRT82B,Stat92E85C9/TM3,Sb; (33) w; FRT82B,ubi-GFP,RpS3Plac92/TM6C, Sb;

(34) y w; ey-GAL4,UAS-Flp; FRT82B,GMR-hid,CL3R/TM6B.

Real-time RT–PCR. Total RNA from wild-type and tumour discs was isolated

using Trizol (Invitrogen). cDNA was synthesized from 2 mg of RNA with the

SuperScriptIII First-Strand Synthesis System (Invitrogen). Resulting DNA was

subjected to real-time PCR with the SYBR green fast kit (Applied Biosystems)

according to the manufacturer’s instructions. Relative gene expression was com-

pared to rp49 as an internal control. Three experiments for each condition were

averaged. The following primers were used: upd, 59-TCCACACGCACAAC

TACAAGTTC-39 and 59-CCAGCGCTTTAGGGCAATC-39; upd2, 59-AGTG

CGGTGAAGCTAAAGACTTG-39 and 59-GCCCGTCCCAGATATGAGAA-39;

upd3, 59-TGCCCCGTCTGAATCTCACT-39 and 59-GTGAAGGCGCCCACG

TAA-39; rp49, 59-GGCCCAAGATCGTGAAGAAG-39 and 59-ATTTGTGCGA

CAGCTTAGCATATC-39.

Stainings and imaging. Images documenting tumour size and ventral nerve cord

invasion were taken in a Leica MZ FLIII fluorescence stereomicroscope with an

Optronics Magnafire camera. Antibody staining was performed according to

standard procedures for imaginal discs. The following antibodies and dyes were

used: mouse monoclonal anti-bgal (1:500, Sigma), goat Alexa-488-conjugated

anti-mouse IgG (1:200, Molecular Probes), phalloidin Texas red (Molecular

Probes). Samples imaged through confocal microscopy were mounted in

DAPI-Vectashield (Vector Labs). Confocal images were taken in a Zeiss

LSM510 Meta confocal microscope. Adult eyes were imaged with a Leica

DFC300FX camera in a Leica MZ FLIII stereomicroscope. Measurements of wing

blade size were performed from confocal pictures using NIH Image-J software.

Adult eye size measurements were performed for each genotype from pictures of

at least ten female flies collected 1–3 days after hatching using NIH Image-J

software.

doi:10.1038/nature08702

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