(A) ATAC-seq chromatin accessibility traces at the asperous (aspr) locus, showing the computationally detected DRMS peak that was experimentally validated as a DRMS enhancer (DRMSaspr, dark green box), (B) Ectopic expression of an epitope tagged Aspr (Aspr::HA, green) in the posterior compartment using en-GAL4. The protein is cytoplasmic and is mostly excluded from nuclei. DE-cadherin (gray). DAPI: blue. Yellow line indicates plane of image in (C), (C) Z-section though the disc shown in (B), showing mostly apical localization of Aspr::HA in the expressing cells (green), and presence of Aspr::HA between the disc proper (DP) and peripodial epithelium (PE), suggesting extracellular localization. DE cadherin shows cell membranes (gray), (D) High magnification imaging of an apical disc section of a disc expressing Aspr::HA (green) under the control of hh-GAL4, showing punctae of staining away from the expressing cells at a level between the peripodial membrane and the disc proper epithelium, (E–F) RNA in situ hybridization detects weak aspr expression in the ventral and lateral areas of the disc in early L3 (E) and late L3 (F) discs, but is mostly absent from the pouch, (G–J) RNA in situ hybridization detecting aspr in early L3 (G and I) or late L3 (H and J) discs following ablation with egr using rnts> (G–H) or DUAL Control (I–J). In both cases, aspr is upregulated in blastema cells in the pouch upon ablation in early L3 discs, but only weakly in late L3, (K–N) Expression of an aspr GFP MiMIC reporter line (green) in early L3 (K and M) and late L3 discs (L and N) during normal development (K–L) and following ablation with rnts>egr (M–N). Consistent with the RNA in situ expression data, aspr reporter activity is mostly absent during normal development, being activated by damage in early L3 discs, with reduced activity in late L3 discs, (O–P) Expression of cut during normal development in early L3 (O) and late L3 (P) wing discs. Cut protein (red) is detected in cells of the notum, including myoblasts and trachea, at both developmental time points, and becomes upregulated at the D-V boundary in late L3 in response to Notch signaling, (Q) Knockdown of aspr in the posterior compartment with en-GAL4 driving aspr RNAi delays onset of cut expression, as shown by a lack of Cut at mid L3 (open arrowhead) when it usually extends across the entire posterior compartment. Ci: Gray, (R–S) Cut protein (red) and a Notch reporter NRE-GFP (green) in mid-L3 wing discs, showing expression of both extending across the entire D-V boundary in wild type discs (R), while discs expressing aspr RNAi in the posterior compartment using hh-GAL4 have delayed expression of Cut and weaker NRE-GFP activity (S, open arrowheads). Ci (gray) demarcates A-P compartment boundary (yellow dotted line), (T–U) Mid L3 discs ablated with rpr using DUAL Control DVE>>GAL4 and driving a control RNAi (T) or aspr RNAi (U). Cut protein (red) is quickly reestablished following rpr ablation with a control RNAi, and extends across the D-V boundary (T). Knockdown of aspr in the entire pouch with DVE>>GAL4 prevents reestablishment of Cut during regeneration (U, open arrowhead). DCP1: gray, DAPI: blue, (V) Knockdown of aspr using two different RNAi lines, or heterozygosity for a presumed aspr mutant (Mi[MIC]CG9572), in discs ablated with rpr using DUAL Control DVE>>GAL4 reduces regeneration compared to wRNAi control, as assessed by wing size. *** p = 0.0007 (asprRNAi (1)), **** p <0.0001, Fisher exact test. Scale bars in (B–D) = 20 μm, other scale bars = 50 μm.