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Supplementary Information

Supplementary Figure 1. JAK/STAT in early wing development (a-f) Wing primordia of second instar larvae of the indicated genotypes labeled to visualize expression of upd mrna (purple, a), Wingless protein (Wg, red or white, b-f), Unpaired (green or white, b, c, in upd-gal4, UASmyrGFP larvae), activity of the JAK/STAT pathway (green or white, d-f, in 10xSTAT-GFP larvae), and DAPI (blue, b-f). (g, h) Adult thoraxes of male individuals expressing the indicated transgenes under the control of the sd-gal4 driver. Endogenous nota (N) and duplicated nota territories (N ) are marked. Red arrows point to duplicated nota. (i, j) Late third instar wing primordium (i) or adult fly (j) expressing hop::myc under the control of the en-gal4 driver. The wing primordium was labeled to visualize expression of Ci (green), Nub (red), and DAPI (blue). Endogenous wing (W), endogenous notum (N) and ectopic wing structures (W ) are marked. Red arrows in j point to ectopic wings arising from the mesothorax. Higher magnification of an ectopic wing is shown in the inset. (k, l) Late third instar wing disc expressing the indicated transgenes under the control of the ptc-gal4 driver and stained for Wg (red or white), GFP (green) and Nub (blue). Wing territory (W), the endogenous notum (N) and the ectopic wing territory (W ) are marked. (m) Early third instar larval wing primordia with clones of cells lacking stat92e activity and marked by the presence of two copies of RFP. Note that clones induce ectopic expression of upd (upd-gal4, UAS-myrGFP, green or white, red arrows). Scale bars, 20 µm (a-f) and 50 µm (i, k, l, m).

Supplementary Figure 2. JAK/STAT maintains the size of the P compartment (a-g) Mature larval wing primordia expressing the indicated transgenes under the control of the ci-gal4 (a), sd-gal4 (b, c) or en-gal4 (d-g) drivers and labeled to visualize activity of the JAK/STAT pathway (10xSTAT- GFP, green or white, a), expression of Ci (red, a, b, d-g, or green, c), En (green, b), Nub (red, c) and GFP (green, d-g). The white dashed line in c marks the AP boundary and the remaining P compartment maintains the wing identity monitored by Nub expression. (h-m) Mature larval leg (h, j, l) and haltere (i, k, m) discs from wild type larvae (h, i) or from larvae expressing hop RNAi under the control of hh-gal4 (j, k) or ci-gal4 (l, m) labeled to visualize expression of Ci (green) and En (red). (n-r) Early (n, o) and late (p-r) third instar larval wing primordia expressing GFP (n, p) or dome DN (o, q, r) under the control of the hh-gal4 driver and labeled to visualize expression of Ci (green) and En (red). Larvae were raised at 29 ºC until dissection (n-q) or raised

at 29 ºC and transferred to 18 ºC in early third instar and dissected in late third instar (r). The reduction in P size is largely restored after 4 days at the permissive temperature (18 ºC). Scale bars, 50 µm. (s) Graphic representation showing the average size of the P versus the A compartment (P/A ratio) of the wing primordia represented in n-r. P/A ratios: n: 0.4 ± 0.06 (n = 23); o: 0.13 ± 0.06 (n = 26); p: 0.63 ± 0.03 (n = 17); q: 0.21 ± 0.09 (n = 34); r: 0.5 ± 0.06 (n = 42). Error bars (vertical lines) represent standard deviation. P-values: Student s t-test, *** p<0.001. Supplementary Figure 3. JAK/STAT, growth promoting pathways and cell cycle regulation (a-h) Mature larval wing primordia of the indicated genotypes labeled to visualize expression of GFP (green, a-d, f), Ci (green, e, g, h), Spalt (Sal, red or white, a), expanded-lacz (antibody to β-gal, red or white, b), dmyc (red or white, c), CycE (red or white, d), CycA (red or white, e, f) and CycB (red or white, g, h). Scale bars, 50 µm. (i) Scatter plot showing the size of the P versus the A compartment (P/A ratio) of late third instar wing primordia expressing the indicated transgenes under the control of the en-gal4 driver. P/A ratios from left to right: 0.56 ± 0.04 (n = 36); 0.16 ± 0.09 (n = 47); 0.16 ± 0.06 (n = 25). Error bars (vertical lines) represent standard deviation and long horizontal lines mark the average values. P-values: Student s t-test, ns = not significant; *** p<0.001.

Supplementary Figure 4. JAK/STAT promotes survival of P cells through diap1 (a-g) Late third instar wing primordia from individuals of the indicated genotypes labeled to visualize expression of diap1 (red or white, a), En (red, b-d) and Ci (green) proteins, and apoptotic cells by TUNEL staining (red, e-g). The contour of the wing discs is marked by a white line in e-g. Scale bars, 50 µm. (h, i) Scatter plots showing the size of the P versus the A compartment (P/A ratio) of late third instar wing primordia expressing the indicated transgenes under the control of the en-gal4 driver. In h, P/A ratios from left to right: 0.56 ± 0.04 (n = 36); 0.2 ± 0.09 (n = 43); 0.36 ± 0.07 (n = 28). In i, P/A ratios from left to right: 0.56 ± 0.04 (n = 36); 0.15 ± 0.06 (n = 87); 0.28 ± 0.07 (n = 45). Error bars (vertical lines) represent standard deviation and long horizontal lines mark the average values. P-values: Student s t-test, *** p<0.001.

Supplementary Figure 5. JAK/STAT counteracts the effects of Engrailed on tissue growth (a, b) Late third instar wing primordia from individuals of the indicated genotypes labeled to visualize expression of Ci (green, a), Engrailed (green, b), Myristoylated-Tomato (TMT, red, b) and DAPI (blue). Scale bars, 50 µm. (c, d) Scatter plots showing the size of the P versus the A compartment (P/A ratio) of late third instar wing primordia expressing the indicated transgenes under the control of the en-gal4 driver. In c, P/A ratios from left to right: 0.56 ± 0.04 (n = 36); 0.13 ± 0.05 (n = 78); 0.16 ± 0.05 (n = 45). In d, P/A ratios from left to right: 0.56 ± 0.04 (n = 36); 0.16 ± 0.09 (n = 66); 0.21 ± 0.08 (n = 70). Error bars represent standard deviation. P-values: Student s t-test, *** p<0.001. (e) Scatter plot showing the absolute number of apoptotic cells in the P compartment of late third instar wing primordia from individuals of the indicated genotypes. Values from left to right: 233 ± 94 (n = 19); 49 ± 25 (n = 29). Error bars (vertical lines) represent standard deviation and long horizontal lines mark the average values. P-values: Student s t-test, *** p<0.001.

Supplementary Table Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Genotype a vn-gal4, UAS-GFP/+ c upd-gal4, UAS-myrGFP/+ d 10xSTAT-GFP/+ e, h, i wild type f, j, k sd-gal4/y; dome RNAi-VDRC /+; UAS-dcr2/+ g sd-gal4/y; dome RNAi-VDRC /+; UAS-dcr2/+, sd-gal4/y; UAS-dome RNAi-VDRC /wg CX4 ; UAS-dcr2/+, sd-gal4/y; UAS-dome RNAi-VDRC /EGFR F2 ; UAS-dcr2/+, and sd-gal4/y; UAS-dome RNAi-VDRC /+; UAS-dcr2/iro EGP7 a ptc-gal4, UAS-GFP/UAS-sgg; tub-gal80 TS /+ b ptc-gal4, UAS-GFP/UAS-dome RNAi-VDRC c, h mirror-lacz/+ d, i, j sd-gal4/y; UAS-dome RNAi-VDRC /+; UAS-dcr2/mirror-lacZ e, n vn-lacz/+ f, g, o, p sd-gal4/y; UAS-dome RNAi-VDRC /+; UAS-dcr2/vn-lacZ k ap-lacz/+ l, m sd-gal4/y; UAS-dome RNAi-VDRC /ap-lacz; UAS-dcr2/+ a ptc-gal4, UAS-GFP/+; mirror-lacz/tub-gal80 TS b-f ptc-gal4, UAS-GFP/UAS-upd; mirror-lacz/tub-gal80 TS g ptc-gal4, UAS-GFP/+; UAS-vn:aos/+ h 10xSTATGFP/+ i j ci-gal4, UAS-GFP/UAS-stat92E RNAi-VDRC ; mirror-lacz/+ k ci-gal4, UAS-GFP/UAS-dome RNAi-VDRC l ci-gal4, UAS-GFP/+; UAS-dome DN /+ m ci-gal4/uas-dome RNAi-VDRC ; UAS-dome DN /+ o wild type and ci-gal4, UAS-GFP/UAS-dome RNAi-VDRC a wild type b, c sd-gal4/+;; UAS-dome DN /+ d hop 27 /Y e ; UAS-dome DN /+ f hs-flp/+;; FRT82B/FRT82B M(3)95A2 UbiGFP g h ci-gal4, UAS-myrTomato, 10xSTAT-GFP/UAS-dome RNAi-VDRC hs-flp/+;; FRT82B stat92e 85c9 /FRT82B M(3)95A2 UbiGFP hs-flp/+;; FRT82B/FRT82B M(3)95A2 UbiGFP, and hs-flp/+;; FRT82B stat92e 85c9 /FRT82B M(3)95A2 UbiGFP j k a-c hop 27 /Y d, f wild type e diap1-lacz/+ hs-flp/+;; FRT82B stat92e 85c9 /FRT82B arm-lacz UAS-FLP/ubi-FRT-stop-FRT-GFP; UAS-dome DN /hh-gal4

Figure 5 Figure 6 Figure 7 Figure 8 Figure S1 g ; UAS-hop::myc/diap1-lacZ h i ; UAS-hop RNAi-TRiP /+ j en-gal4, UAS-GFP/UAS-GFP; UAS-dome DN /+ k en-gal4, UAS-GFP/UAS-dIAP1; UAS-dome DN /+ l ; UAS-hop RNAi-TRiP /diap1-lacz en-gal4, UAS-GFP/UAS-GFP; UAS-dome DN /+ and en-gal4, UAS-GFP/UAS-dIAP1; UAS-dome DN /+ m ; UAS-dome DN /+ n en-gal4, UAS-GFP/UAS-GFP; UAS-dome DN /+, and en-gal4>uas-gfp/uas-cyca; UAS-dome DN /+ a, c en-gal4, UAS-GFP/UAS-en RNAi-VDRC ; UAS-dome DN /+ b en-gal4, UAS-GFP/UAS-GFP; UAS-dome DN /+ d e f g h-j, l en-gal4, UAS-GFP/UAS-GFP; UAS-dome DN /+, and en-gal4, UAS-GFP/UAS-en RNAi-VDRC ; UAS-dome DN /+ en-gal4, UAS-GFP/UAS-RFP en-gal4, UAS-GFP/UAS-en RNAi-VDRC en-gal4, UAS-GFP/UAS-RFP, and en-gal4, UAS-GFP/UAS-en RNAi-VDRC hh-gal4, tub-gal80 TS /UAS-en k UAS-GFP/+; hh-gal4, tub-gal80 TS /+ m UAS-GFP/+; hh-gal4, tub-gal80 TS /+, and hh-gal4, tub-gal80 TS /UAS-en a dpp-lacz/+ b, c sd-gal4/+;; UAS-dome DN /dpp-lacz d e f UAS-GFP/+; hh-gal4/uas-dome DN UAS-hh::GFP/+; hh-gal4/uas-dome DN UAS-GFP/+; hh-gal4/uas-dome DN, UAS-hh::GFP/+; hh-gal4/uas-dome DN, and UAS-dIAP1/+; hh-gal4/uas-dome DN a upd-gal4, UAS-myrGFP/+ b 10xSTAT-GFP/+ c, i wild type d ci-gal4, UAS-GFP/+; dome DN /+ e dpp-lacz/+ f-h, j, k a wild type b upd-gal4, UAS-myrGFP/+ c d 10xSTAT-GFP/+ e wg CX3 /wg CX4 ; 10XSTAT-GFP/+ f hth-gal4, UAS-GFP/UAS-dome DN upd-gal4, UAS-myrGFP/+; wg CX3 /wg CX4 sd-gal4/uas-egfr λtop4.2 ; 10XSTAT-GFP/+

Figure S1 Figure S2 Figure S3 Figure S4 Figure S5 g sd-gal4/y;; UAS-dcr2/UAS-hop RNAi-TRiP h I, j ; UAS-hop::myc/+ k sd-gal4/y; UAS-stat92E RNAi-VDRC /+; UAS-dcr2/+ ptc-gal4, UAS-GFP/UAS-stat92E RNAi-VDRC l ptc-gal4, UAS-GFP/UAS-stat92E RNAi-VDRC ; UAS-wg RNAi-TRiP /+ m hs-flp/upd-gal4, UAS-myrGFP;; FRT82B ubi-rfp, stat92e 85c9 /FRT82B a ci-gal4, UAS-myrTomato, 10xSTAT-GFP; UAS-dome DN /+ b sd-gal4/+;; UAS-dcr2/UAS-dome RNAi-TRiP c sd-gal4/+;; UAS-dome DN /+ d UAS-dcr2/+; en-gal4, UAS-GFP/UAS-dome RNAi-VDRC e UAS-dcr2/+; ; UAS-dome RNAi-TRiP /+ f UAS-dcr2/+; ; UAS-hop RNAi-TRiP /+ g UAS-dcr2/+; ; UAS-stat92E RNAi-TRiP /+ h, i UAS-GFP/UAS-GFP; hh-gal4/+ j, k UAS-GFP/+; hh-gal4/uas-hop RNAi-TRiP l, m ci-gal4, UAS-GFP/+; UAS-hop RNAi-TRiP /+ n, p UAS-GFP/+; hh-gal4, tub-gal80 TS /+ o, q, r hh-gal4, tub-gal80 TS /UAS-dome DN s UAS-GFP/+; hh-gal4, tub-gal80 TS /+, and hh-gal4, tub-gal80 TS /UAS-dome DN a, c, d, h ; UAS-dome DN /+ b e wild type f, g ci-gal4, UAS-GFP/+; UAS-dome DN /+ i ; UAS-dome DN /ex-lacz en-gal4, UAS-GFP/UAS-GFP; UAS-dome DN /+, and ; UAS-dome DN /UAS-cycB:HA a ci-gal4, UAS-GFP/+; UAS-dome DN /+ b ; UAS-dome DN /+ c en-gal4, UAS-p35/+; UAS-dome DN /+ d, g ; UAS-dome DN /UAS-dronc RNAi-VDRC e en-gal4, UAS-GFP/UAS-GFP; UAS-dome DN /+ f en-gal4, UAS-GFP/UAS-dIAP1; UAS-dome DN /+ h i, ; UAS-dome DN /+, and en-gal4, UAS-p35/+; UAS-dome DN /+ en-gal4, UAS-GFP/UAS-GFP; UAS-dome DN /+ and ; UAS-dome DN /UAS-dronc RNAi-VDRC a ci-gal4/uas-smo DN ; UAS-dome DN /+ b ap-gal4, UAS-myrTomato/UAS-en RNAi-VDRC c ; UAS-dome DN /+, and

Figure S5 d e en-gal4, UAS-GFP/Df(2)en E ; UAS-dome DN /+ en-gal4, UAS-GFP/UAS-GFP; UAS-dome DN /+, and ; UAS-dome DN /UAS-en RNAi-TRiP en-gal4, UAS-GFP/UAS-GFP; UAS-dome DN /+, and en-gal4, UAS-GFP/UAS-en RNAi-VDRC ; UAS-dome DN /+ Supplementary Table 1. List of fly genotypes

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