NIP is required to sustain JNK signaling during late regeneration.

<p><b>(</b>A-H) Confocal images of fluorescence from the <i>TRE-red</i> reporter for JNK signaling in <i>w</i>^<i>1118</i> (A-D) and <i>mol</i>^{<i>e02670</i>}<i>/+</i> (E-H) regenerating discs at R0 (A,B), R24 (B,F), R48 (C,G) and R72 (D,H). (I) Quantification of fluorescence intensity of the <i>TRE-red</i> reporter in max projections of the confocal images at R0, because at this time point the epithelium cannot be distinguished from the debris. <i>w</i>^<i>1118</i> n = 10 discs, <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 14 discs. (J) Quantification of fluorescence intensity of the <i>TRE-red</i> reporter in single slices of the confocal images through the regenerating epithelium at R24, R48, and R72. R24 <i>w</i>^<i>1118</i> n = 11 discs, <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 11 discs. R48 <i>w</i>^<i>1118</i> = 14 discs, <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 15 discs. R72 <i>w</i>^<i>1118</i> n = 11 discs, <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 11 discs. (K) Regeneration assays using adult wing size to assess extent of regenerative growth in the imaginal discs in <i>w</i>^<i>1118</i>, <i>mol</i>^{<i>e02670</i>}<i>/+</i>, <i>puc</i>^<i>E69</i><i>/+</i>, and <i>mol</i>^{<i>e02670</i>}<i>/+;puc</i>^<i>E69</i><i>/+</i> animals. Two independent experiments, thus error bars are SD. <i>w</i>^<i>1118</i> n = 26 wings, <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 83 wings, <i>puc</i>^<i>E69</i><i>/+</i> n = 99 wings, and <i>mol</i>^{<i>e02670</i>}<i>/+;puc</i>^<i>E69</i><i>/+</i> n = 95 wings. p<0.0001 for all comparisons using a chi-squared test. Dashed blue line outlines the wing primordium. Scale bars are 100 μm. Error bars are SEM unless otherwise noted. *p<0.05, **p<0.001, ***p<0.0001.</p

Genetic assays demonstrating that differentially expressed genes have functional roles in regeneration.

<p><b>(</b>A) Representative examples of wings from damaged discs that are approximately <25%, 25%, 50%, 75%, and 100% of a normal wing. Scale bar is 1mm. (B-G) Sizes of adult wings after regeneration in control (<i>w</i>^<i>1118</i>) and heterozygous mutant animals. Three independent experiments each, error bars are SEM. (B) Sizes of adult wings after regeneration in <i>w</i>^<i>1118</i> and <i>Ets21C</i>^{<i>f03639</i>}<i>/+</i> animals. <i>w</i>^<i>1118</i> n = 318 wings, <i>Ets21C</i>^{<i>f03639</i>}<i>/+</i> n = 255 wings, p<0.0001 using a chi-squared test. (C) Sizes of adult wings after regeneration in <i>w</i>^<i>1118</i> and <i>CG9336</i>^{<i>MI03849</i>}<i>/+</i> animals. <i>w</i>^<i>1118</i> n = 374 wings, <i>CG9336</i>^{<i>MI03849</i>}<i>/+</i> n = 215 wings, p<0.0001 by a chi-squared test. (D) Sizes of adult wings after regeneration in <i>w</i>^<i>1118</i> and <i>Alp4</i>^<i>07028</i><i>/+</i> animals. <i>w</i>^<i>1118</i> n = 239 wings, <i>Alp4</i>^<i>07028</i><i>/+</i> n = 217 wings, p<0.0001 by a chi-squared test. (E) Sizes of adult wings after regeneration in <i>w</i>^<i>1118</i> and <i>Thor</i>^<i>06270</i><i>/+</i> animals. <i>w</i>^<i>1118</i> n = 224 wings, <i>Thor</i>^<i>06270</i><i>/+</i> n = 146 wings, p = 0.0021 by a chi-squared test. (F) Sizes of adult wings after regeneration in <i>w</i>^<i>1118</i> and <i>mol</i>^{<i>e02670</i>}<i>/+</i> animals. Three independent experiments, <i>w</i>^<i>1118</i> n = 356 wings, <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 183 wings, p = 0.00001 by a chi-squared test. (G) Sizes of adult wings after regeneration in <i>w</i>^<i>1118</i>, <i>Col4a1</i>^{<i>k00405</i>}<i>/+</i>, and <i>vkg</i>^{<i>k00236</i>}<i>/+</i> animals. <i>w</i>^<i>1118</i> n = 320 wings, <i>Col4a1</i>^{<i>k00405</i>}<i>/+</i> n = 71 wings, and <i>vkg</i>^{<i>k00236</i>}<i>/+</i> n = 134 wings, p<0.0001 by a chi-squared test.</p

Labeling and isolating regeneration blastema cells.

<p><b>(</b>A-B) Wing imaginal discs that are undamaged (A) or ablated and at 0 hrs recovery (R0) (B). Green = <i>rnGal4</i>, <i>UAS-EGFP</i>. Red = anti-Nub. Blue = DAPI. (C) Wing imaginal disc showing overlap of anti-Nub immunostaining (red) and expression of the <i>nub-GFP</i> MiMIC enhancer trap (green). (D-F) <i>nub-GFP</i> marks the wing pouch at 24 hrs (D) 48 hrs (E) and 72 hrs (F) after ablation. (G) <i>nub-GFP</i> (green) coincides with the regeneration blastema as defined by a zone of high EdU incorporation (red). (H) Schematic of the mRNA-seq procedure, from tissue ablation through cell dissociation and sort to sequencing and data analysis. Scale bars are100 μm.</p

Gene ontology (GO) analysis for enrichment of biological process terms.

<p>Gene ontology (GO) analysis for enrichment of biological process terms.</p

List of reactive oxygen species (ROS) regulating genes.

<p>List of reactive oxygen species (ROS) regulating genes.</p

Validation of genes identified as downregulated in the regeneration blastema.

<p>Undamaged (A-E) and regenerating (R24) (A’-E’) wing discs. (A-A’) <i>dve-lacZ</i> enhancer trap. (B-B’) Hr78-GFP protein trap. (C-C’) NC2β-GFP protein trap. (D-D’) <i>sm-lacZ</i> enhancer trap. (E-E’) <i>Cat-GFP</i> enhancer trap. Blue dashed lines outline the wing primordium. Scale bars are 100 μm.</p

<i>moladietz</i> is required for wing disc regeneration.

<p><b>(</b>A-D) DHE fluorescence (red) indicates the presence of ROS. <i>nub-GFP</i> (green) marks the regenerating wing pouch. (A-B) Confocal slices of a <i>w</i>^<i>1118</i> regenerating disc through the debris field (A,A’) and the disc epithelium (B,B’). Asterisks mark cellular debris in the debris field and in a few folds in the epithelium. Arrow points to the position of the regenerating wing pouch. (C-D) Confocal slices of a <i>mol</i>^{<i>e02670</i>}<i>/+</i> regenerating disc through the debris field (C,C’) and the regenerating epithelium (D,D’). Asterisk and arrow same as above. (E-F) Quantification of DHE fluorescence intensity in the debris fields of <i>w</i>^<i>1118</i> and <i>mol</i>^{<i>e02670</i>}<i>/+</i> regenerating discs (E) and in the regenerating epithelia of <i>w</i>^<i>1118</i> and <i>mol</i>^{<i>e02670</i>}<i>/+</i> regenerating discs and control undamaged discs (F). For R24, three independent experiments, for a total <i>w</i>^<i>1118</i> regenerating n = 12 discs, <i>mol</i>^{<i>e02670</i>}<i>/+</i> regenerating n = 18 discs, <i>w</i>^<i>1118</i> undamaged n = 11 discs. For R48, three independent experiments for a total <i>w</i>^<i>1118</i> regenerating n = 30 discs, <i>mol</i>^{<i>e02670</i>}<i>/+</i> regenerating n = 25 discs, <i>w</i>^<i>1118</i> undamaged n = 10 discs. (G,H) Quantification of GFP fluorescence from a <i>gstD-GFP</i> reporter for ROS-regulated transcription in regenerating <i>w</i>^<i>1118</i> and <i>mol</i>^{<i>e02670</i>}<i>/+</i> discs. For R24, <i>w</i>^<i>1118</i> n = 12 discs, <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 20 discs. For R48, <i>w</i>^<i>1118</i> n = 12 discs, <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 10 discs. (I) Adult wing area in <i>w</i>^<i>1118</i> and <i>mol</i>^{<i>e02670</i>}<i>/+</i> male and female wings from undamaged discs and after disc regeneration. Three independent experiments. Undamaged: <i>w</i>^<i>1118</i> females n = 125 wings, <i>w</i>^<i>1118</i> males n = 132 wings, <i>mol</i>^{<i>e02670</i>}<i>/+</i> females n = 82 wings, <i>mol</i>^{<i>e02670</i>}<i>/+</i> males n = 73 wings. Regenerated: <i>w</i>^<i>1118</i> females n = 226 wings, <i>w</i>^<i>1118</i> males n = 134 wings, <i>mol</i>^{<i>e02670</i>}<i>/+</i> females n = 128 wings, <i>mol</i>^{<i>e02670</i>}<i>/+</i> males n = 133 wings. (J-O) Anti-Nub marks the regenerating wing primordium at R0, R24 and R48 in <i>w</i>^<i>1118</i> and <i>mol</i>^{<i>e02670</i>}<i>/+</i> discs. (P) Quantification of the size of the regenerating wing primordium at R0, R24 and R48. R0 <i>w</i>^<i>1118</i> n = 26 and <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 29, R24 <i>w</i>^<i>1118</i> n = 42 and <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 41, R48 <i>w</i>^<i>1118</i> n = 29 and <i>mol</i>^{<i>e02670</i>}<i>/+</i> n = 42. Scale bars are 100 μm. Error bars are SEM. **p<0.05, *p<0.005, ***p<0.0002, ****p<0.0001.</p

Validation of genes identified as upregulated in the regeneration blastema.

<p>Undamaged (A-I) and regenerating (R24) (A’-I’) wing discs. (A-A’) <i>Alp4-lacZ</i> enhancer trap. (B-B’) Atf3-GFP protein trap. (C-C’) <i>chinmo-lacZ</i> enhancer trap. (D-D’) Ets21C-GFP protein trap. (E-E’) <i>mol-lacZ</i> enhancer trap. (F-F’) <i>fru-lacZ</i> enhancer trap. (G-G’) Lamin-GFP protein trap. (H-H’) <i>pigs-GFP</i> enhancer trap. (I-I’) 10xSTAT92E-GFP reporter for STAT activity. Blue dashed line outlines the wing primordium. Scale bars are 100μm.</p