SUPPLEMENTARY INFORMATION

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1 Supplementary Figure 1. Ras V12 expression in the entire eye-antennal disc does not cause invasive tumours. a, Eye-antennal discs expressing Ras V12 in all cells (marked with GFP, green) overgrow moderately but do not cause invasive tumours. Compare to Ras V12 clones (Fig. 1b) and Ras V12 //scrib - tumours (Fig. 1e). 1

2 Supplementary Figure 2. Interclonal cooperation between Ras V12 and lgl - causes tumours. Ras V12 clones confronted with lgl - clones cause tumours that overgrow and invade the VNC, similar to Ras V12 //scrib - tumours (Fig. 1e). 2

3 Supplementary Figure 3. Reduction of growth and invasion in Ras V12 scrib -, Ras V12 //scrib - and Ras V12 Upd tumours by mutation of stat92e. a, Growth and invasion of the VNC in Ras V12 scrib - tumors are partially suppressed by a loss-of-function mutation in stat92e, encoding the Drosophila STAT transcriptional activator. b, Growth of Ras V12 //scrib - is partially suppressed and VNC invasion completely abrogated when Ras V12 cells are mutant for stat92e. c, stat92e - partially suppresses the growth of Ras V12 Upd tumours and completely suppresses their invasion of the VNC. More efficient suppression by Dome DN may indicate a Stat92E-independent function of Domeless 1, a Domeless-independent function of Stat92E 2, or hypomorphy of the stat92e mutation. 1 Zeidler, M. P., Bach, E. A. & Perrimon, N. The roles of the Drosophila JAK/STAT pathway. Oncogene 19, (2000). 2 Li, W. X. Canonical and non-canonical JAK-STAT signaling. Trends Cell Biol 18, (2008). 3

4 Supplementary Figure 4. Cooperation between Upd overexpression and scrib - clones is not sufficient to cause tumours. a, scrib - clones overexpressing Upd marked by GFP (green) in eye-antennal discs. No tumour formation is observed. b, Wild-type clones overexpressing Upd (green) adjacent to scrib - clones. No tumour formation is observed either. 4

Supplementary Figure 5. Involvement of upd genes in growth and invasion of Ras V12 lgl - tumours. a, Ras V12 lgl - clones develop into tumours similar to Ras V12 scrib - tumours 1.

5 Supplementary Figure 5. Involvement of upd genes in growth and invasion of Ras V12 lgl - tumours. a, Ras V12 lgl - clones develop into tumours similar to Ras V12 scrib - tumours 1. b, Ras V12 lgl - clones expressing an interfering RNA against upd (upd IR ). Partial suppression of tumour is observed in 43% of examined animals. c, Ras V12 lgl - clones generated in a upd2 Δ3-62 mutant larva. upd2 Δ3-62 is a null mutation in the upd2 gene 2. No suppression is observed. d, Ras V12 lgl - clones expressing upd IR generated in a upd2 Δ3-62 mutant larva. The extent of upd IR suppression is increased: suppression of tumour is achieved in all animals, with 60% showing stronger suppression. These results suggest that upregulation of each of the upd genes contributes to tumour development in an additive and partially redundant way, consistent with their overlapping functions in normal development 2. 1 Pagliarini, R. A. & Xu, T. A genetic screen in Drosophila for metastatic behavior. Science 302, (2003). 2 Hombria, J. C., Brown, S., Hader, S. & Zeidler, M. P. Characterisation of Upd2, a Drosophila JAK/STAT pathway ligand. Dev Biol 288, (2005). 5

Supplementary Figure 6. Inhibition of cell death in Ras V12 - or Upd-expressing clones does not recapitulate Ras V12 Upd cooperation in tumour formation.

6 Supplementary Figure 6. Inhibition of cell death in Ras V12 - or Upd-expressing clones does not recapitulate Ras V12 Upd cooperation in tumour formation. a, Clones co-overexpressing Ras V12 and p35 marked by GFP (green) in eye-antennal discs. No tumour formation is observed. Compare to Fig. 2i. b, Clones co-overexpressing Upd and p35. No tumour formation is observed either. 6

Supplementary Figure 7. Feed-forward propagation of JNK activity in wounds. a, Propagation of JNK activity (puc-lacz reporter, green) in wing discs wounded in the posterior (P) compartment.

7 Supplementary Figure 7. Feed-forward propagation of JNK activity in wounds. a, Propagation of JNK activity (puc-lacz reporter, green) in wing discs wounded in the posterior (P) compartment. A yellow asterisk marks the wounding site. The blue arrowhead points to puc-lacz expression in cells of the anterior (A) compartment. b, puclacz expression in discs expressing the JNK-phosphatase Puc under the control of ptc- GAL4 in A compartment cells next to the P compartment (red cells, expressing RFP), wounded as in a. Puc is both a downstream target and a negative regulator of JNK. Propagation of puc-lacz expression into the A compartment is prevented (blue hollow arrowhead). A magnified view of the central region of the disc (bracket) is shown in b. Discs were dissected 24 hours after wounding. 7

Supplementary Figure 8. Inhibition of cell death in wounded wing discs does not recapitulate the tumour overgrowth of wounded Ras V12 wing discs.

8 Supplementary Figure 8. Inhibition of cell death in wounded wing discs does not recapitulate the tumour overgrowth of wounded Ras V12 wing discs. a, b, Wing discs of a larva whose right disc was wounded by repeated pinching of the wing blade region expressing the apoptosis inhibitor p35 under the control of nub-gal4. Expression of RFP driven by nub-gal4 marks the wing blade region (red). Cell nuclei stained with DAPI (blue). Compare to Fig 4a-d. c, Quantification of the size difference between wounded and unwounded discs in wild-type (n=21 pairs of discs), p35- expressing discs (n=16) and Ras V12 -expressing discs (n=16). The wounded/unwounded size ratios (small circles), were calculated for each pair of discs by dividing the area of the region expressing nub-gal4 in the wounded left disc by the area of the same region in the unwounded control right disc of the same animal. Plots represent the 25 th -75 th percentile range as a box, the median as a horizontal line dividing the box, the mean as a black square and the range of the central 95% of the data as a vertical bar. 8

doi: 10.1038/nature08702 stained with DAPI (blue). Compare to Fig 4a-d.

9 doi: /nature08702 stained with DAPI (blue). Compare to Fig 4a-d. c, Quantification of the size difference between wounded and unwounded discs in wild-type (n=21 pairs of discs), p35expressing discs (n=16) and RasV12-expressing discs (n=16). The wounded/unwounded size ratios (small circles), were calculated for each pair of discs by dividing the area of the region expressing nub-gal4 in the wounded left disc by the area of the same region in the unwounded control right disc of the same animal. Plots represent the 25th-75th percentile range as a box, the median as a horizontal line dividing the box, the mean as a black square and the range of the central 95% of the data as a vertical bar. Supplementary Figure 9. Requirement of JAK-STAT signaling for compensatory proliferation in the eye. a, b, stat92e- clones marked in white by absence of red pigment. c, d, Eyes containing scrib- clones (red) confronted with stat92e- cells (white). The size of the eye is reduced, same as Fig. 4h. e, Eyes where clones of wild-type cells (white) confront slower-growing M+/- mutant cells 1 (red). M+/- cells are completely absent. The size of the eye is normal. f-h, Eyes containing clones of stat92e- cells (white) confronted with M+/- cells (red). M+/cells are completely absent. The size of the eye is severely reduced. i, Eyes where cells confronting wild-type cells (light orange) have been eliminated through the GMR Hid/cell lethal system 2. GMR-Hid/cell lethal cells (red) are completely absent and the size of the eye is normal. j-l, Eyes where stat92e- cells (light orange) confront GMR- 9

10 confronting wild-type cells (light orange) have been eliminated through the GMR- Hid/cell lethal system 2. GMR-Hid/cell lethal cells (red) are completely absent and the size of the eye is normal. j-l, Eyes where stat92e - cells (light orange) confront GMR- Hid/cell lethal cells (red). GMR-Hid/cell lethal cells (red) are completely absent. The size of the eye is severely reduced. m, Graph representing the size of adult eyes containing wild-type or stat92e - clones confronted to wild-type, M +/- or GMR-Hid/cell lethal cells. n, Graph representing the portion of the eye that is white in eyes containing control wildtype and stat92e - clones marked by loss of red pigmentation (a, b and Fig. 4e, g). Error bars in m and n represent 95% confidence intervals. n 10 in all cases. 1 Morata, G. & Ripoll, P. Minutes: mutants of drosophila autonomously affecting cell division rate. Dev Biol 42, (1975). 2 Stowers, R. S. & Schwarz, T. L. A genetic method for generating Drosophila eyes composed exclusively of mitotic clones of a single genotype. Genetics 152, (1999). 10

11 Supplementary Figure 10. Requirement of upd for compensatory proliferation in the eye. a, Eye in which scrib - clones were induced. b, Eye in which scrib - clones expressing an interfering RNA against upd (upd IR ) were induced. c, Quantification of the size of eyes containing scrib - clones (n=13) and scrib - clones expressing upd IR (n=11). p-value obtained from a two-tailed t-test. Error bars represent 95% confidence intervals. 11

effects on organ development. a-f, Eye and wing discs with clones of ε j2b10 show no

Supplementary Figure 1. Loss of function clones of 14-3-3 or 14-3-3 show no significant effects on organ development. a-f, Eye and wing discs with clones of 14-3-3ε j2b10 show no obvious defects in Elav