Supporting InformationKatsuyama et al. 10.1073/pnas.1423074112SI Materials and MethodsFly Stocks. The transgenic fly lines used were pucE69-I-GAL4 (1),UAS-flp (8209; Bloomington Drosophila Stock Center at In-diana University), Act-FRT-STOP-FRT-GAL4 (2), UAS-GFP,10xSTAT92E-GFP (3), en-GAL4, tub-GAL80ts (4), UAS-do-meΔCYT (5), hepr75 (6), hop3 (7), wgl-12 (8), and dilp8MI00727 (9).The hop3 male larvae (hemizygotes) were available if cultured inuncrowded conditions at 25 °C. To obtain wgl-12 homozygousthird instar larvae, embryo collection and subsequent incubationwere performed at 15 °C. Oregon R and w1118 were used as controlstrains. For continuous GFP labeling of regenerating disc cells, weused pucE69-I-GAL4, UAS-GFP, UAS-flp, and Act-FRT-STOP-FRT-GAL4. Unless otherwise indicated, fly lines were obtainedfrom the Bloomington Drosophila Stock Center at Indiana Uni-versity or the Drosophila Genomic Resource Center.

Analysis of Microarray Data.Microarray data were preprocessed andanalyzed using R (10) and BioConductor (11). First, diagnostic plotswere produced and used to exclude spatial artifacts. Second, probeset-level intensities were background-subtracted and quantile–quantile-normalized using robust multichip average (12). Hierar-chical clustering analysis of expression values indicated the presenceof batch effects (Fig. S2A). Data were, therefore, adjusted for thissystematic error by performing a genewise one-way ANOVA usingPrediction Analysis of Microarrays for R (PAMR) (Fig. S2B) (13).Differential expression analysis was carried out by computingmoderated F statistics using limma (Linear Models for MicroarrayData) (14). Test P values were adjusted for multiple comparisonsusing the Benjamini–Hochberg correction (15), and genes withadjusted P < 0.001 were deemed significantly differentially ex-pressed in at least a core comparison (in the text). Differentiallyexpressed genes were clustered using k-means clustering (16).The number of clusters (k = 4 in our analysis) was selected byanalyzing the tradeoff between the within-cluster sum of squaresand the value of k (Fig. S2C). GO analysis was performed usingDAVID (17).

Histochemistry. To visualize proliferating cells in regeneratingdiscs, host flies were fed with BrdU medium from 24 h aftertransplantation. Discs were recovered at 48 h after fragmentation/cultivation. Immunohistochemistry of imaginal discs was per-formed as described in ref. 18. Primary and secondary antibodiesused were rat anti-BrdU [BU1/75 (ICR1); 1:200; Abcam] andAlexa Fluor 647-conjugated goat anti-rat IgG (Invitrogen/LifeTechnologies). All preparations were analyzed with a Leica TCSSP5 Confocal Microscope equipped with LAS AF software(Leica Microsystems).Detection of mRNAs by in situ hybridization was performed

as previously described (19). To generate digoxigenin-labeled RNAprobes, the template cDNAs were cloned into the pGEM-T Easyvector (Promega). The sequences of primer pairs for each probewere CG1092-forward (F)/reverse (R): ggttcgtatacagggctccat/caga-tccttcccaaactcaca; CG13215-F/R: atgaagtgcatcatatccatcg/ttatttgccaaag-ctgtcgg; CG17108-F/R: gttagtcagctgcaggcaaag/tttgtgcttgtggctaagacc;CG17124-F/R: atggcaaaaggataagcctg/tttccgctagcccttcattacagc;CG33099-F/R: atgggatagcagaggacaag/gattttcatatggctagagc; CG4520-F/R: tagcaca-catcggtcggaata/cgccaacaatttgtgtgattag; CG5002-F/R: aacatcgtgggtctgacc-ag/aatggagttgatctcctcgc; CG5958-F/R: gaacgctgaaaaaccctcaat/ag-tattgaaatggggccaatc; CG6749-F/R: gtccaaatgtgtttggagc/ttgtggacca-atttgctagc; CG8303-F/R: catagttaggccatcgattg/tgacttgtacttggcttgtg;dilp8-F/R: ccgctcgtgattatcaggta/cgatcagttgccgtattcg; Ets21C-F/R: cat-tctacagaatagccgcca/tcaaacttgtaggcataccgc; Igdf1-F/R: gcttgctcagtgtga-caagt/tgcgttctgcgacaacttcg; Mmp1-F/R: gaccttcaccaggaagacctc/acttgt-cgcccttgaagaagt; mthl2-F/R: cgatttctgtaaagcagcagg/acatacagggtttttcc-tcgg; puc-F/R: gagtgctgctgcactgccac/ttcactgctgctacttgacg; Tg-F/R: at-cgtacaacagattgcgtcc/tcaggaccacactgaagctct; upd-F/R: gcctaaacagtagc-caggac/cgagaacagagactatgtgc; upd2-F/R: ccacatgcagaccttcgtgg/aatag-tcgagttggcactgg; upd3-F/R: ttgaaccttcgccacgccca/tcgagaaaactgggtatc-tc; wg-F/R: gaagtgcaagctgtgtcggac/tacctacatggatggggtggt; and wun2-F/R: attcgtttgtgtgtctccgtc/cacaggtgaactcctcgatgt. In all histochemi-cal experiments, we examined at least five disc samples from re-peated several experiments.

1. Pastor-Pareja JC, Grawe F, Martín-Blanco E, García-Bellido A (2004) Invasive cell be-havior during Drosophila imaginal disc eversion is mediated by the JNK signalingcascade. Dev Cell 7(3):387–399.

2. Ito K, Awano W, Suzuki K, Hiromi Y, Yamamoto D (1997) The Drosophila mushroombody is a quadruple structure of clonal units each of which contains a virtuallyidentical set of neurones and glial cells. Development 124(4):761–771.

3. Bach EA, et al. (2007) GFP reporters detect the activation of the Drosophila JAK/STATpathway in vivo. Gene Expr Patterns 7(3):323–331.

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5. Brown S, Hu N, Hombría JC (2001) Identification of the first invertebrate interleukinJAK/STAT receptor, the Drosophila gene domeless. Curr Biol 11(21):1700–1705.

6. Glise B, Bourbon H, Noselli S (1995) hemipterous encodes a novel Drosophila MAPkinase, required for epithelial cell sheet movement. Cell 83(3):451–461.

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8. DiNardo S, Sher E, Heemskerk-Jongens J, Kassis JA, O’Farrell PH (1988) Two-tieredregulation of spatially patterned engrailed gene expression during Drosophila em-bryogenesis. Nature 332(6165):604–609.

9. Garelli A, Gontijo AM, Miguela V, Caparros E, Dominguez M (2012) Imaginal discssecrete insulin-like peptide 8 to mediate plasticity of growth and maturation. Science336(6081):579–582.

10. R Core Team (2013) R: A Language and Environment for Statistical Computing(R Foundation for Statistical Computing, Vienna).

11. Gentleman RC, et al. (2004) Bioconductor: Open software development for compu-tational biology and bioinformatics. Genome Biol 5(10):R80.

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14. Smyth GK (2005) limma: Linear models for microarray data. Bioinformatics andComputational Biology Solutions Using R and Bioconductor. Statistics for Biology andHealth, eds Gentleman R, Carey V, Huber W, Irizarry R, Dudoit S (Springer, New York),pp 397–420.

15. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: A practical andpowerful approach to multiple testing. J R Stat Soc Series B Stat Methodol 57(1):289–300.

16. Hastie T, Tibshirani R, Friedman J (2009) The Elements of Statistical Learning: DataMining, Inference, and Prediction (Springer, New York), 2nd Ed.

17. Huang W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis oflarge gene lists using DAVID bioinformatics resources. Nat Protoc 4(1):44–57.

18. Katsuyama T, Paro R (2013) Innate immune cells are dispensable for regenerativegrowth of imaginal discs. Mech Dev 130(2-3):112–121.

19. Niimi T, Seimiya M, Kloter U, Flister S, Gehring WJ (1999) Direct regulatory interactionof the eyeless protein with an eye-specific enhancer in the sine oculis gene during eyeinduction in Drosophila. Development 126(10):2253–2260.

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puc-GAL4, UAS-GFP, UAS-flp, Act-FRT-STOP-FRT-GAL4

puc-GAL4, UAS-GFP, UAS-flp, Act-FRT-GAL4

constitutive

transientG

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Fig. S1. Continuous labeling of JNK signaling activated cells and regenerated blastema formation. (A–F) In situ hybridization of puc transcripts in (A) intact legdisc or (B–F) regenerating P3/4 disc fragments at different time points (x = 0, 3, 6, 12, or 24 h cultured). (G) Genetic scheme for regenerating cell lineage tracing.

Legend continued on following page

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After a cell at the wound site activates JNK signaling, Flippase driven by puc-GAL4 excises the FRT cassette, and the Actin promoter (Act) -GAL4 maintainscontinuously GFP expression. (H–L) puc>>GFP labeling of regenerating P3/4 leg disc fragments at different time points after wounding: (H) 0, (I) 6, (J) 12, (K) 24,and (L) 48 h. (H′) Leg discs show the GFP expression at the hypodermal stalk and chordotonal sense organ precursor (white arrowhead and asterisk, re-spectively). (K″ and L″) BrdU-incorporated cells during 0–24 and 24–48 h, respectively, are shown. Anterior is to the left, and dorsal is up. Yellow arrowheadsindicate the fragmented positions. (Scale bar: 100 μm.)

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Fig. S2. Hierarchical clustering of microarray samples and the selection of cluster numbers in k-means clustering of differentially expressed genes. (A and B)The result of batch effect removal. All microarray samples were hierarchically clustered based on their expression profiles (A) before or (B) after batch effectremoval with PAMR. (B) The algorithm corrected the systematic error evident in A. Indeed, uncut and uncultured samples, uncut and cultured samples, and cutand cultured samples form three different clusters in B. In addition, good agreement between replicates was generally observed. (C) Within-clusters sum ofsquares as a function of the number of clusters k. The blue point corresponds to k = 4.

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Fig. S3. Gene expression heat map of genes encoding JAK/STAT signaling components. Drosophila JAK/STAT signaling consists of three ligand-coding genes(upd, upd2, and upd3), a single receptor Dome, a single JAK Hop, and a single STAT (Stat92E). Suppressor of cytokine signaling at 36E (Socs36E) and Proteintyrosine phosphatase 61F (Ptp61F) encode downstream negative feedback regulators. Expression values of each gene were color-coded as indicated in thecolor key. Among these components, only upd showed a significant up-regulation in regenerating cells within both anterior and posterior fragments, whereassimilar tendencies were barely visible in other components, including upd2 and upd3.

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Fig. S4. upd Expression in early regenerating discs was not influenced by halving Pc gene doses. (A and B) upd Expression in WT (A) C24P or (B) C72Pfragments. The wound site upd expression that up-regulated within 24 h was diminished by 72 h after cultivation. (C and D) In a heterozygote of a Pc null allele(Pc3/+), the upd up-regulation was neither enhanced nor prolonged, which was comparable with WT in A and B. Asterisk in D indicates the staining signal onthe attached debris on disc during staining procedure. In all images, anterior is to the left, and dorsal is up. Arrowheads indicate the fragmented positions.(Scale bar: 100 μm.)

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Fig. S5. The imaginal discs of hop3 JAK/STAT signaling mutant. (A) Leg and (B) wing discs of WT control at the third instar larval stage. In both discs, the weakpuc>>GFP expression was observed at hypodermal stalks (white arrowheads). (C) Leg and (D) wing discs of hop3 mutant third instar larvae. White arrowheadsand asterisks in puc>>GFP images indicate the abnormal activation of JNK signaling in hop3 mutant discs. (E) The activation of JNK signaling in regeneratinghop3 wing disc. The disc was cut at the anterior ventral sector, and P3/4 fragment was cultivated for 24 h. Yellow arrowheads indicate the wound-inducedpuc>>GFP. In all images, anterior is to the left, and dorsal is up. (Scale bar: 100 μm.)

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Fig. S6. JAK/STAT signaling is involved in cell proliferation in disc regeneration. (A) A pair of prothoracic leg discs of en-GAL4, UAS-GFP. The yellow lineindicates the position of the nick introduced. (B) Experimental design to examine the effect of JAK/STAT signaling inhibition on regenerative cell proliferation.The disc-donor larvae were grown at 18 °C until day 8 and then, shifted to 29 °C. Six hours later, the discs were fragmented and transplanted into an adult flyabdomen. The host flies were continuously incubated at 29 °C for 48 h. To check the proliferation capacities, the host flies were fed on BrdU-containing mediafor 24 h as indicated. (C and D) BrdU incorporation in the regenerating disc fragment. Either (C) solely GFP or (D) DomeΔCYT and GFP were expressed in theposterior compartment. (C′) Although the control disc showed BrdU incorporation (magenta) in posterior cells, (D′) the BrdU signals in the DomeΔCYT-expressing disc were exclusively observed in adjacent anterior cells. The yellow squares in C′ and D′ were three times magnified in C″ and D″, respectively. In allimages, anterior is to the left, and dorsal is up. Yellow arrowheads indicate the fragmented positions. (Scale bar: 100 μm.)

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A

B

Cluster I(151) 93 58 280

Colombani et al. (2012)Up-regulated genesRNAi-Ctrl at 116 hour AEL(338, adj.P < 1.00 x 10-5)

Cluster III(91) 26 16465

Colombani et al. (2012)Down-regulated genesRNAi-Ctrl at 116 hour AEL(190, adj.P < 1.00 x 10-5)

Fig. S7. Commonality in gene expression changes to disc fragmentation or genetic disc cell perturbation. The transcriptome data of genetically perturbedwing discs generated by Colombani et al. (1) were compared with clusters I and III genes. (A) The Venn diagram showed overlap between cluster I genes (151)and the up-regulated genes in the genetically impaired disc (338 genes; adjusted P < 1.0 × 10−5). (B) The Venn diagram showed overlap between cluster IIIgenes (91) and the down-regulated genes in the genetically impaired disc (190 genes; adjusted P < 1.0 × 10−5). AEL, after egg laying.

1. Colombani J, Andersen DS, Léopold P (2012) Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing. Science 336(6081):582–585.

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Fig. S8. Variance of wg expression in three sample replicates across biological conditions. Distribution of gene expression variances across biological con-ditions is shown by boxplots. Genes belonging to the same expression decile aswg were considered. The expression variance forwg was shown with red circles.A one-sided empirical P value for the observed variance of wg expression is indicated for each sample.

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Table S1. GO analysis with genes of each cluster

GO terms Counts Fold enrichment Benjamini

Cluster I (GO-assigned = 77)Defense response 19 9.993 7.77E-11Immune response 16 8.958 3.00E-08Innate immune response 12 11.834 4.82E-07Response to bacterium 9 10.699 1.66E-04Antibacterial humoral response 6 21.695 5.56E-04Defense response to bacterium 8 11.020 4.79E-04Humoral immune response 8 9.778 9.18E-04Defense response to Gram-negative bacterium 6 17.954 9.29E-04Antimicrobial humoral response 7 9.642 0.003716Defense response to fungus 5 17.356 0.007556Response to fungus 5 16.070 0.009359

Cluster II (GO-assigned = 351)Ribosome biogenesis 24 7.369 1.89E-11Respiratory electron transport chain 24 7.139 2.12E-11Electron transport chain 26 6.346 2.22E-11Mitochondrial ATP synthesis-coupled electron transport 23 7.298 2.39E-11ATP synthesis-coupled electron transport 23 7.062 4.21E-11Cellular respiration 27 5.411 2.21E-10Ribonucleoprotein complex biogenesis 26 5.380 6.24E-10Oxidative phosphorylation 29 4.639 1.24E-09Energy derivation by oxidation of organic compounds 27 4.942 1.43E-09Ribosomal RNA processing 16 7.810 4.39E-08Ribosomal RNA metabolic process 16 7.615 6.09E-08Generation of precursor metabolites and energy 33 3.141 9.93E-07RNA processing 40 2.720 1.19E-06Noncoding RNA processing 21 4.442 2.13E-06Noncoding RNA metabolic process 25 3.689 3.18E-06Mitochondrial electron transport, NADH to ubiquinone 11 6.755 1.71E-04Mitochondrial electron transport, cytochrome c to oxygen 7 11.105 9.94E-04Translation 35 2.214 0.001011

Cluster III (GO-assigned = 56)Instar larval or pupal development 15 4.760 7.21E-04Postembryonic development 15 4.566 5.93E-04Instar larval or pupal morphogenesis 13 5.036 9.99E-04Postembryonic morphogenesis 13 4.940 9.14E-04Metamorphosis 13 4.817 9.46E-04Regulation of transcription 19 3.114 0.001012Regulation of transcription, DNA-dependent 16 3.516 0.001606Imaginal disc morphogenesis 11 5.336 0.001772Postembryonic organ morphogenesis 11 5.336 0.001772Postembryonic organ development 11 5.068 0.002452Regulation of RNA metabolic process 16 3.140 0.004228Imaginal disc development 12 4.045 0.005725

Cluster IV (GO-assigned = 343)Regulation of transcription 73 1.953 2.29E-05Glucan metabolic process 7 15.152 0.001034Energy reserve metabolic process 7 15.152 0.001034Glycogen metabolic process 7 15.152 0.001034Cellular glucan metabolic process 7 15.152 0.001034Regulation of transcription, DNA-dependent 53 1.901 0.002949Neuron differentiation 41 2.124 0.002542Regulation of RNA metabolic process 56 1.794 0.005105Sensory organ development 35 2.185 0.005342Cell morphogenesis involved in differentiation 32 2.226 0.007945Cell morphogenesis 42 1.957 0.007006Transcription 44 1.872 0.011289

Other Supporting Information Files

Dataset S1 (XLSX)

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