Draper-mediated efferocytosis by Drosophila imaginal disc epithelial cells clears cellular debris during regeneration.

Regeneration, Drosophila wing discs , efferocytosis, Draper , hemocytes, 13 mmp2, cell debris, clearance, basement membrane 14 15 Short running head: Efferocytosis clears debris during regeneration 16 17 18 19 1 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Article summary 20 Tissue regeneration requires organized responses to damage, including clearance of 21 cellular debris. Using a genetic ablation system in Drosophila wing imaginal discs, we 22 show that most debris is cleared within two days despite the absence of immune cell 23 recruitment, which is restricted by the basement membrane. In the absence of immune 24 cells, debris clearance occurs through Draper -mediated efferocytosis and lysosomal 25 processing by epithelial cells . Disruption of this pathway delays debris removal and 26 impacts regeneration. Residual debris consists of a heterogeneous mix of cellular 27 components, indicating non-selective clearance. Together, our findings identify epithelial 28 cells as key non-professional phagocytes during regeneration. 29 30 31 32 33 34 35 36 37 38 39 2 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration

40 Regeneration is a coordinated process that restores tissue integrity following damage. 41 Following injury, tissues initiate early responses , including epithelial remodeling and 42 clearance of cellular debris. However, how debris clearance is coordinated with 43 regenerative growth to ensure efficient tissue repair remains poorly understood. To 44 address how early damage responses, particularly debris clearance, are coordinated with 45 regeneration, we used a genetic ablation system in Drosophila wing imaginal discs to 46 induce apoptosis in the pouch region. Targeted damage generates cellular debris that 47 localizes to both the apical and basal sides of the epithelium. We show that most cellular 48 debris is cleared within two days after damage, although some debris persists apical to 49 the regenerating epithelium . Notably, immune cells are not recruited to the damaged 50 tissue due to restricted access by an intact basement membrane. Instead, we discovered 51 that debris clearance is mediated by efferocytosis, whereby neighboring hinge epithelial 52 cells activate JNK signaling and engulf debris via lysosomal formation. Reduction of 53 efferocytosis by mutation of the phagocytic receptor Draper delays debris removal and 54 increases debris persistence. This impairment has a modest impact on regeneration, as 55 measured by adult wing size. Finally, our data indicate that residual debris consists of a 56 heterogeneous mixture of cellular components, suggesting no preferential targeting by 57 the clearance machinery. Together, our results reveal a previously unappreciated role for 58 epithelial cells as non-professional phagocytes for debris clearance during regeneration. 59 60 61 3 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration

62 Regeneration proceeds through multiple tightly regulated stages, including early damage-63 sensing responses that initiate regeneration, followed by proliferation and growth, and 64 ultimately repatterning and morphogenesis to restore tissue structure and function 65 (reviewed in Poss and Tanaka 2024; Li et al. 2025). The Drosophila wing imaginal disc is 66 a well- established model system for studying tissue regeneration and repair. These 67 epithelial structures possess robust regenerative capacity and high genetic tractability, 68 making them extensively characterized over the past several decades (reviewed in 69 Tripathi and Irvine 2022; Worley and Hariharan 2022) . Earlier studies primarily relied on 70 physical fragmentation and ex vivo culture of wing discs to investigate regeneration 71 (reviewed in Fox et al. 2020). More recently, the development of genetic ablation systems 72 has enabled precise and controlled induction of tissue damage, providing a powerful 73 approach to study regenerative responses (Smith-Bolton et al. 2009; Bergantiños et al. 74 2010). These systems involve targeted expression of pro- apoptotic factors, leading to 75 rapid, localized apoptosis within the epithelium and the generation of substantial amounts 76 of apoptotic cell debris (reviewed in Fox et al. 2020). Damaged wing discs activate wound-77 responsive mechanisms and multiple molecular pathways that are drivers of 78 regeneration. These drivers include reactive oxygen species (ROS) (Santabárbara-Ruiz 79 et al. 2015; Brock et al. 2017; Khan et al. 2017) , Wingless (Wg) signaling (Smith-Bolton 80 et al. 2009; Harris et al. 2016), Jun N-terminal kinase (JNK) signaling (Bosch et al. 2005; 81 Bergantiños et al. 2010), p38 MAPK activity (Santabárbara-Ruiz et al. 2015), JAK/STAT 82 signaling (Katsuyama et al. 2015; La Fortezza et al. 2016) , Dpp signaling (Herrera et al. 83 2013), and Yorkie (Yki) activity (Sun and Irvine 2010; Grusche et al. 2011) . While 84 4 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration apoptosis-induced damage activates these regenerative growth pathways, the extensive 85 cell death necessitates efficient clearance of apoptotic debris from the tissue. 86 87 Rapid and efficient clearance of apoptotic cells is a critical process that maintains tissue 88 homeostasis, suppresses inflammatory responses, and regulates immune signaling 89 (reviewed in Elliott and Ravichandran 2010). Failure to remove apoptotic cells in a timely 90 manner can lead to secondary necrosis and the uncontrolled release of harmful 91 intracellular contents (reviewed in Poon et al. 2014) . Thus, apoptosis must be tightly 92 coupled with the swift clearance of apoptotic debris. Clearance of dead or dying cells is 93 typically mediated by specialized immune cells called professional phagocytes (reviewed 94 in Melcarne et al. 2019). These include macrophages in mammals and plasmatocytes in 95 Drosophila, which serve as the primary cell types responsible for engulfing and removing 96 cellular debris (reviewed in Shklover et al. 2015; Melcarne et al. 2019) . In Drosophila, 97 plasmatocytes phagocytose caspase- activated cells within tumor microenvironments, 98 such as in malignant eye imaginal disc tumors induced by oncogenic mutations (Hirooka 99 et al. 2025) . Furthermore, clearance of apoptotic corpses by plasmatocytes during 100 Drosophila embryonic development represents a well -established paradigm of 101 phagocytosis (Weavers et al. 2016). 102 103 In addition to professional phagocytes, non- professional phagocytes, including tissue-104 resident neighboring cells, also play important roles in engulfing apoptotic material 105 through a process known as efferocytosis (reviewed in Shklover et al. 2015; Melcarne et 106 al. 2019) . While phagocytosis broadly refers to sensing and internaliz ing extracellular 107 5 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration particles larger than ~0.5µm, efferocytosis is a specialized form of phagocytosis 108 dedicated to the recognition and clearance of apoptotic cells (reviewed in Uribe -Querol 109 and Rosales 2020; Moon et al. 2023) . In Drosophila, this function is widely distributed 110 across non-professional phagocytes in multiple tissues. For example, glial cells contribute 111 to central nervous system remodeling during development by clearing apoptotic neurons 112 (Kurant et al. 2008; Etchegaray et al. 2016) , while ovarian follicle epithelial cells engulf 113 nurse cells during oogenesis (Meehan et al. 2016; Serizier and McCall 2017) . Similarly, 114 glial cells mediate debris clearance during larval axon pruning (Awasaki and Ito 2004) , 115 and epidermal cells remove degenerating dendrites (Han et al. 2014) . Notably, in 116 Drosophila eye imaginal disc epithelial cells, neighboring cells function as non-117 professional phagocytes and mediate engulfment of oncogenic cells in neoplastic tumor-118 suppressor mutant tissues, highlighting a broader role for epithelial tissues in cell 119 clearance (Ohsawa et al. 2011) . Whether similar non- professional phagocytic 120 mechanisms operate in regenerating wing imaginal discs remains unclear. 121 122 In this study, we investigated how apoptotic cell debris is cleared during wing imaginal 123 disc regeneration. We found that most debris generated by tissue ablation was eliminated 124 within two days post-injury, while a small portion of debris persisted. Notably, Drosophila 125 immune cell subtypes were not recruited to the damaged epithelium. To understand this 126 lack of immune cell involvement, we examined tissue accessibility and found that the 127 intact basement membrane of the regenerating wing disc serves as a physical barrier, 128 restricting immune cell entry. In the absence of professional phagocyte recruitment, we 129 show that neighboring undamaged epithelial cells in the hinge region of the wing disc 130 6 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration activate JNK signaling and act as non-professional phagocytes, engulfing and processing 131 apoptotic debris through lysosome formation. We impaired debris clearance by using a 132 mutation in the phagocytic receptor draper, resulting in increased debris persistence and 133 delayed lysosome formation. T his defective debris clearance had minimal impact on 134 overall regeneration, as assessed by adult wing size. Finally, we characterized the 135 composition of debris that persisted more than two days after damage and found it to be 136 a heterogeneous mixture of cellular components and organelles. Together, our results 137 reveal that apoptotic debris clearance is independent of immune cells and instead relies 138 on Draper-mediated efferocytosis by surrounding epithelial cells. 139 140 141 142 143 144 145 146 147 148 149 150 7 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration

151 Genetic ablation system used to induce tissue damage in wing imaginal discs 152 To understand the early damage response in the wing imaginal disc, we induced targeted 153 tissue damage specifically in the wing pouch, which gives rise to the adult wing blade. To 154 achieve this damage, we used our genetic ablation system (Smith-Bolton et al. 2009) , 155 which combines spatial and temporal control of ablation via GAL4-UAS-GAL80ts to induce 156 tissue damage (Figure 1a) . Apoptosis was induced in third- instar larvae by expressing 157 the pro-apoptotic transgene UAS-reaper in the rotund-GAL4-expressing wing pouch cells. 158 Briefly, the larvae were reared at 18°C, where a tubulin- GAL80ts inhibited apoptosis by 159 restraining GAL4 from binding and activating expression of UAS-reaper. On day 7 after 160 egg lay, the temperature was shifted to 30°C to relieve the inhibition by GAL80ts, enabling 161 GAL4-induced Reaper to drive cell death in the wing imaginal disc pouch cells for 24 162 hours. The animals were then shifted back to 18°C to permit regeneration, and the 163 subsequent recovery timepoints (0 to 72 hours post -damage) were used to assess the 164 process of regeneration in the wing discs, including early damage responses such as 165 debris clearance. Adult wing sizes were used as a readout of the regenerative capacity 166 of the larval wing imaginal discs. 167 168 To examine early responses to tissue damage, wing imaginal discs were characterized 169 morphologically using confocal microscopy (Figure 1b-d). We used double-sided tape as 170 spacers while mounting to preserve the 3- dimensional architecture of the fixed wing 171 imaginal disc (Aldaz et al. 2010). The epithelial organization of the wing imaginal disc was 172 8 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration resolved by orthogonal projection of z-stack images, revealing the squamous peripodium 173 and the columnar disc proper (Figure1b- d) (reviewed in Tripathi and Irvine 2022) . This 174

of imaging allowed us to observe distinctive folds in the columnar epithelium 175 between the notum, hinge, and wing pouch, denoted as Notum-Hinge (N-H), Hinge-Hinge 176 (H-H), and Hinge-Pouch (H-P) folds (Figure 1b, d). 177 178 Cellular debris is cleared during the first 48 hours of regeneration with some 179 persisting debris 180 Two of the initial steps in early regeneration are re- establishing tissue continuity and 181 clearing cellular debris (reviewed in Smith-Bolton 2016). To determine how cellular debris 182 is processed, we closely examined the dynamics of debris clearance during wing imaginal 183 disc regeneration. To label cellular debris, we expressed UAS -EYFP along with our 184 ablation system, which resulted in EYFP expression in all cells in which GAL4 induced 185 expression of reaper. As a result, the EYFP signal was observed in apoptotic cells and 186 the resulting cellular debris. To obtain a comprehensive view of debris clearance, we 187 examined orthogonal slices of confocal image stacks of undamaged and regenerating 188 wing imaginal discs (Figure 2, S1). 189 190 In the undamaged control, rotund-expressing cells were detected via EYFP signal in the 191 pouch, extending from the H-P fold to the end of the wing pouch (Figure 2a-a”). The EYFP 192 signal was also observed within the adult muscle precursor cells adjacent to the notum, 193 as marked by Cut expression (Figure S1a-a***), a marker for adult muscle precursor cells 194 9 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration (Sudarsan et al. 2001) . Following damage, at 0 hours of recovery (R0), an extensive 195 amount of cellular debris was detected adjacent to the remaining pouch. The EYFP -196 labelled cellular debris predominantly localized apically to the epithelium of the disc 197 proper, with a smaller portion extruded basally (Figure 2b- b”). This localization is 198 consistent with our previous observations after ablating most of the wing pouch (Brock et 199 al. 2017), but contrasts with debris localization after ablation of smaller areas of cells, 200 which primarily extrude basally (Bergantiños et al. 2010). Surprisingly, we also observed 201 some debris in the undamaged hinge epithelium marked by the H -H fold (Figure 2b-b’’). 202 3D renderings of regenerating wing imaginal discs at R0 that excluded the peripodium 203 confirmed this pattern of debris localization (Figure 2c-f). 204 205 To measure the amount of cell debris in the regenerating wing pouch over time, we 206 estimated debris volume throughout the regeneration time course by first quantifying the 207 planar area of debris using UAS-EYFP signal in the top- down (XY) images and then 208 multiplying it by debris height in orthogonal (YZ) cross -sections. By R4, the amount of 209 debris visible in the orthogonal (YZ) cross -sections was less than that at R0, with some 210 portion of debris still detected within the undamaged hinge epithelium (Figure 2g-g”). We 211 did not detect a statistically significant reduction in the volume of debris from R4 to R12 212 (Figures S1b-d”, 2k). At R16, we observed a noticeable drop in debris volume, marking a 213 second phase of clearance (Figure 2h-h’’, 2k). By R24, most basal EYFP-labeled debris 214 had been cleared, while the remaining apical debris was confined to the lumen between 215 the peripodium and the disc proper (Figure 2i -i”), consistent with our previous 216 observations at this time point (Brock et al. 2017). The overall debris volume, planar area, 217 10 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration and height remained unchanged through R36 (Figure 2k, S1e- g). By R48, most of the 218 cellular debris had been eliminated, although some remnants persisted on the apical side 219 of the columnar epithelium (Figure 2j-j”,k). Taken together, these results suggest that by 220 two days post -damage, cellular debris is largely cleared, although a residual fraction 221 persists in the regenerating wing disc. 222 223 Debris clearance during regeneration occurs independently of immune cell 224 recruitment 225 We next asked how cell debris is cleared in the regenerating wing imaginal disc. Previous 226 work from our lab showed robust Reactive Oxygen Species (ROS) in both cellular debris 227 and the regenerating epithelium, but no detectable hemocyte recruitment at 24 hours 228 post-damage (R24) (Khan et al. 2017). Because a major portion of cell debris is cleared 229 by R24, we reasoned that if hemocytes contribute to this process, their involvement would 230 occur earlier. Therefore, we examined earlier time points to determine whether any of the 231 three major Drosophila hemocyte classes, plasmatocytes, lamellocytes, or crystal cells 232 (reviewed in Mathey -Prevot and Perrimon 1998) , are recruited to the damaged 233 epithelium. 234 235 To assess plasmatocyte recruitment, we used the plasmatocyte- specific reporter 236 Hemolectin-RFP (Hml -RFP) (Makhijani et al. 2011) , and immunostained for the 237 plasmatocyte marker Nimrod (NimC) (Kurucz, Váczi, et al. 2007; Kurucz, Márkus, et al. 238 2007) (Figure S2). Although a small number of Hml-positive plasmatocytes were present 239 11 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration in the undamaged control discs, their numbers were considerably reduced at R0 and were 240 not significantly different than the controls at R24 (Figure S2a-d). To assess plasmatocyte 241 presence specifically in the regenerating pouch, we quantified Hml -positive cells in the 242 pouch region, marked using the UAS-EYFP debris reporter. This analysis revealed no 243 significant differences across conditions (Figure S2e). 244 245 Similar results were observed with immunostaining for anti -NimC, where we detected a 246 few NimC-positive cells in the undamaged control discs (Figure S2 f-f’). At R0, the number 247 of NimC-positive cells was significantly reduced and w as not significantly different than 248 controls at R24 (Figure S2 f-j), consistent with our previous report (Khan et al. 2017). 249 250 A recent report showed that the detection of hemocytes in the wing imaginal disc can be 251 influenced by immunostaining procedures, with hemocytes getting dislodged by rigorous 252 washing (Zhu et al. 2025) . Therefore, we repeated the NimC experiments without 253 subjecting the discs to vigorous washes after dissection and after incubation with primary 254 and secondary antibody (Figure 3). Although some NimC -positive plasmatocytes were 255 detected in the no- wash condition at both R0 and R24 in the damaged pouch, these 256 numbers were not significantly different from the respective undamaged controls at 0 and 257 24 hours after thermal shift (Figure 3a- e). The orthogonal YZ slices showed that NimC -258 positive plasmatocytes were attached to the basal side of the disc proper in the 259 undamaged discs . In contrast, at R0 and R24, plasmatocytes were predominantly 260 localized to the basal side of the peripodial epithelium, a region that lacks cell debris 261 12 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration localization (Figure 3f-h). The overall number of NimC-positive plasmatocytes across the 262 whole disc was not significantly different in the no- wash condition when comparing the 263 R0 and R24 regenerating discs to their respective controls (Figure S3a). Additionally, we 264 observed no significant differences in the number of NimC -positive plasmatocytes in the 265 undamaged pouch between the wash and no- wash conditions (Figure S3b) . While a 266 significantly higher number of NimC -positive cells was detected at R0 in the no- wash 267 condition compared to the wash condition (Figure S3b), the number was not significantly 268 different from the undamaged no wash condition. Together, these results show no 269 detectable increase in plasmatocytes in the regenerating wing imaginal disc, while 270 suggesting that the plasmatocytes present at damaged and undamaged discs at R0 may 271 be more easily dislodged. 272 273 We next sought to detect the presence of lamellocytes using established markers 274 (reviewed in Evans et al. 2014) : Atilla/L1 (Kurucz, Váczi, et al. 2007; Evans et al. 2014), 275 Atilla-MiET1, a GFP reporter element insertion which allows in vivo detection of 276 lamellocyte differentiation (Honti et al. 2009), ItgαPS4 (Irving et al. 2005; Krzemień et al. 277 2007), and MSNF9 (Tokusumi et al. 2009) . To validate Atilla antibody specificity for 278 lamellocytes, we immunostained third- instar larval hemolymph with Atilla antibody and 279 confirmed that it selectively labels lamellocytes (Figure S4a-a’), which are larger than 40 280 µm, consistent with previous reports (reviewed in Lan et al. 2020) . We examined 281 regenerating and control wing discs and found no Atilla- positive lamellocytes in the 282 undamaged, R0 or R24 discs. However, the antibody marked discs in a consistent 283 speckled pattern within the disc and debris (Figure S4b-d’). To confirm what lamellocytes 284 13 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration would look like in the wing imaginal discs, we used a model that overproduces 285 lamellocytes, caused by the gain- of-function allele hop[Tum] (Luo et al. 1995) , which 286 encodes a constitutively active form of the Hopscotch (Hop)-encoded Janus kinase (JAK). 287 Larvae carrying this mutation develop a melanotic tumor phenotype characterized by 288 extensive lamellocyte differentiation in a temperature- dependent manner and presence 289 of lamellocytes throughout the animal, including at the imaginal discs. We observed 290 lamellocytes in the hemolymph samples as well as on the wing imaginal discs from 291 hop[Tum] larvae by immunostaining with the Atilla antibody (Figure S4e-f’). No lamellocytes 292 were detected in undamaged or R0 discs under no- wash conditions using the anti -Atilla 293 antibody (Figure S4g- g′). We further examined lamellocyte markers αPS4- GFP and 294 MSNF9mo-GFP, confirming their specificity by co-staining with the Atilla antibody (Figure 295 S5a-b″). Consistent with these results, no lamellocytes were detected in undamaged or 296 R0 discs using αPS4 -GFP, MSNF9mo-GFP, or the Atilla-MiET1 reporter (Figure S5c -i). 297 Together, these findings indicate that lamellocytes are not recruited to damaged wing 298 discs for debris clearance. 299 300 We then assessed the presence of crystal cells using the crystal cell markers PPO1 301 (reporter line PPO1-mcherry.F6) (Tokusumi et al. 2017), and Lozenge (Lz) (Lebestky et 302 al. 2000) . A few PPO1 -mcherry.F6 positive cells were detected in both undamaged 303 control discs and regenerating discs at R0 (Figure S6a-b’). However, their numbers were 304 not significantly different between undamaged and R0 conditions in either the whole disc 305 or the regenerating pouch (Figure S6c-d). By contrast, neither the anti-Lz immunostaining 306 nor the GFP-tagged Lz protein detected any crystal cells in the regenerating discs (Figure 307 14 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration S6e-h’). Thus, crystal cells are not significantly increased in number in the regenerating 308 wing imaginal discs. 309 310 Finally, to confirm the absence of hemocyte recruitment to the debris region of the 311 regenerating wing imaginal discs, we tested two pan- hemocyte markers: Hemese (He) 312 (Kurucz et al. 2003), and Serpent (Srp) (reviewed in Evans et al. 2014). Immunostaining 313 with an anti-Hemese antibody revealed no significant difference between the number of 314 He-positive hemocytes, both in the pouch and throughout the disc, at R0 and R24 315 compared to the undamaged controls under the no- wash conditions (Figure S7a- e). To 316 further evaluate hemocyte presence, we verified the pan- hemocyte reporter srpHemo -317 H2A::3xmCh, where the srpHemo promoter drives a fusion of 124 amino acids of Histone 318 H2A linked to 3xmCherry, concentrating the fluorescence in the nucleus, and SrpHemo -319 3xmCh, where the srpHemo promoter drives a fusion of three copies of mCherry that 320 localizes to the cytoplasm (Gyoergy et al. 2018). These transgenes are reported to detect 321 all three hemocyte sub-classes: plasmatocytes, lamellocytes, and crystal cells. In the third 322 instar larval hemolymph, we found that plasmatocytes positive for NimC (white arrows, 323 Figure S8a-a’, a’’’) and crystal cells positive for Lz-GFP (yellow arrow, Figure S8a’’-a’’’) 324 expressed the srpHemo-H2A::3XmCh reporter. Interestingly, we also observed some srp-325 positive cells that were not positive for either NimC1 or Lz (orange arrows, Figure S8a”’). 326 We also detected lamellocytes double- positive for both Atilla and the srpHemo -327 H2A::3xmCh reporter (Figure S8b-b”’). Together, these results indicate that the srpHemo 328 reporters reliably label plasmatocytes, lamellocytes, and crystal cells, while also marking 329 a small population of mCherry-positive cells lacking these three hemocyte markers. 330 15 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration 331 On testing these reporters in regenerating wing discs, we detected srpHemo -332 H2A::3XmCh-positive nuclei at R0, R24, and R48, compared to very few in the 333 undamaged controls (Figure S8c-f). Given that none of the three immune cell types were 334 detected in our previous experiments, we hypothesized that the mCherry-positive nuclei 335 might represent primocytes, a distinct cell type that expresses srp but lacks mature 336 hemocyte marker expression (Fu et al. 2020) , or they might result from unexpected 337 reporter expression in the ablated cells or the regenerating epithelium. To test whether 338 primocytes were present in the regenerating disc, we stained R48 srpHemo-H2A::3xmCh 339 discs with an anti -Antennapedia antibody (Antp), which is a primocyte marker (Fu et al. 340 2020). We detected no Antp expression in the mCherry-positive nuclei, thereby ruling out 341 the presence of primocytes (Figure S8g-g’). 342 343 Orthogonal YZ slices of the srpHemo- H2A::3xmCh discs at R48 revealed mCherry -344 positive nuclei that colocalized within the regenerating epithelium marked by Discs-large 345 (Dlg) and DAPI (Figure S8h- h’). This unexpected finding prompted further investigation 346 into the identity of the cells containing the mCherry-positive nuclei. Given that hemocytes 347 and columnar epithelial cells are very different in shape, we used a srpHemo -348 moe::3xmCh transgene, in which the mCherry -tagged Moesin would mark the actin 349 cytoskeleton and reveal the morphology of the mCherry-expressing cells (Gyoergy et al. 350 2018). At R48, srpHemo-moe::3xmCh expression showed that these cells had a columnar 351 16 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration shape and were integrated into the regenerating epithelium, indicating that they are not 352 immune cells but disc proper cells expressing this reporter (Figure S8i-i’’). 353 Thus, we are likely observing regeneration- associated activation of the srp promoter 354 fragment rather than recruitment or persistence of srp -positive hemocytes in the 355 regenerating disc. Collectively, these findings indicate that immune cells are not recruited 356 for debris clearance in the regenerating wing imaginal discs. 357 358 Intact basement membrane restricts immune cell recruitment in the regenerating 359 epithelium 360 Given our previous analysis showing that immune cells are not recruited to regenerating 361 wing imaginal discs, we asked what factors restrict immune cell recruitment to this tissue. 362 In damaged eye discs, reactive oxygen species (ROS) recruit hemocytes to the site of 363 damage, which then serve as a stimulus to activate JNK signaling in the regenerating 364 epithelium (Fogarty et al. 2016). In regenerating wing discs, although we observe a similar 365 upregulation of ROS and JNK signaling (Khan et al. 2017) , we do not detect increased 366 recruitment of immune cells. This discrepancy prompted us to investigate how immune 367 cell recruitment differs between eye and wing discs, despite their overall structural 368 similarity. 369 370 A prior study in developing wing discs demonstrated that damage to the basement 371 membrane is sufficient to recruit hemocytes (Diwanji and Bergmann 2020) . Using eye 372 imaginal discs, the same group showed that damaged eye discs exhibit basement 373 17 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration membrane disruption (Diwanji and Bergmann 2020) . To test whether basement 374 membrane integrity similarly regulates immune cell recruitment in regenerating wing 375 discs, we overexpressed matrix metalloproteinase 2 ( Mmp2), an endopeptidase that 376 cleaves components of the basement membrane and extracellular matrix (reviewed in 377 Page-McCaw et al. 2007) . In undamaged and regenerating wing discs, the basement 378 membrane was visualized using Vkg-GFP, a protein trap including GFP fused to Collagen 379 IV encoded by the viking gene (Morin et al. 2001) (Figure 4a-f ). At R0, control 380 regenerating discs showed a continuous basement membrane in the pouch region, 381 although Vkg- GFP signal intensity appeared reduced compared to the pouch in 382 undamaged control discs (Figure 4a-b ’). In undamaged discs, NimC -positive 383 plasmatocytes were attached to the basal side of either the disc proper or the peripodial 384 epithelium in 11 out of 23 discs (Figure 3f, Figure 4a- a’, d). In the regenerating control 385 discs at R0, NimC- positive plasmatocytes were rarely detected, and, when present, 386 remained associated with the peripodium and did not invade the disc epithelium in 8 out 387 of 24 discs (Figure 3g , Figure 4b- b’,e). By contrast, upon mmp2 overexpression, we 388 observed disruption of basement membrane at R0, indicated by loss of Vkg- GFP 389 continuity, along with invasion of NimC -positive cells into the disc epithelium (Figure 4c -390 c’). The presence of immune cell clusters within the damaged pouch in 4 out of 8 discs 391 (Figure 4f) indicates that, in regenerating wing discs, an intact basement membrane 392 functions as a physical barrier to immune cell invasion, distinguishing their damage 393 response from that of damaged eye discs. 394 395 18 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Debris clearance during regeneration is associated with lysosome formation in 396 hinge epithelial cells 397 Having tested the role of hemocytes, the professional phagocytes in Drosophila, and 398 finding no evidence for their involvement in debris clearance, we next investigated 399 whether non- professional phagocytes might instead mediate debris clearance during 400 regeneration. Several Drosophila cell types can carry out efferocytosis, or phagocytosis 401 of apoptotic debris, including ovarian follicle epithelial cells (reviewed in Serizier and 402 McCall 2017) , eye imaginal disc epithelial cells that eliminate oncogenic neighbors 403 (Ohsawa et al. 2011), and embryonic glia (reviewed in Heron et al. 2023). As previously 404 observed, our UAS-EYFP labelled debris revealed debris from the damaged pouch within 405 the cells of the adjacent hinge epithelium fold (Figure 5a-a ”). This observation is 406 consistent with a role for neighboring hinge epithelial cells in engulfing apoptotic debris 407 generated during pouch ablation. To assess whether there is an increase in phagosomes 408 in the hinge that could be clearing debris, we stained regenerating wing imaginal discs 409 with LysoTracker, a marker of phagosome maturation that labels the completion of the 410 phagocytic engulfment process (Awasaki and Ito 2004; Kurant et al. 2008). We detected 411 very few LysoTracker-positive puncta in both top-down views and orthogonal YZ slices in 412 the undamaged discs (Figure 5b , Figure S9a -a″). Upon damage, LysoTracker -positive 413 puncta were detected in R0 discs, specifically in the hinge- pouch fold (Figure 5c- e’). 414 Quantification showed that damaged R0 discs contained significantly more LysoTracker-415 positive puncta compared to undamaged controls or R24 discs (Figure 5f-g). 416 417 19 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Closer examination of R0 discs, with the epithelium marked by Discs large (Dlg), revealed 418 that these lysosomes were confined to the neighboring hinge-pouch epithelial fold (Figure 419 5d-d’, e-e’). LysoTracker-positive puncta overlapped with the UAS -EYFP-labeled debris 420 and were colocalized within the Dlg-marked epithelium as seen by both top-down and YZ 421 orthogonal slices (Figure 5d’-e’, h-h”’). 422 423 To refine our understanding of the spatial and temporal dynamics of debris engulfment 424 and lysosome formation during regeneration, we analyzed additional regeneration time 425 points. At R4 and R8, LysoTracker-positive puncta were detected in the hinge epithelium, 426 where they colocalized with the EYFP -labeled debris (Figure S9b- c”). By R10 and R12, 427 puncta persisted primarily within the hinge-hinge and hinge-pouch epithelial folds (Figure 428 S9d-e”). By R24, we observed a significant reduction in the number of LysoTracker -429 positive puncta relative to R0 (Figure 5e-f, and S9f-f”), consistent with our previous finding 430 that cellular debris volume declines by this stage. By R48, LysoTracker -positive signal 431 was largely absent, as confirmed by orthogonal YZ imaging (Figure S9g-g”). 432 433 To confirm the efferocytosis function of the underlying hinge epithelial cells, we tested the 434 genetically encoded fluorescent reporter CharON (Caspase and pH -Activated Reporter, 435 Fluorescence ON), which enables tracking of emerging apoptotic debris and its clearance 436 by efferocytosis (Raymond et al. 2022). Although this reporter has previously been used 437 to detect clearance of apoptotic debris by phagocytes in the Drosophila embryo 438 (Raymond et al. 2022) , we applied it here to the regenerating wing imaginal discs. The 439 20 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration UAS-CharON dual reporter consists of a pH -tolerant pH -Caspase-GFP domain that 440 reports apoptosis and a pHlorina red- fluorescent pH sensor that exhibits increased 441 fluorescence upon acidification during apoptotic debris internalization (Raymond et al. 442 2022). At R0, we detected a pHlorina signal localized to the hinge-pouch fold, as observed 443 in YZ orthogonal sections (10/17 discs), where it colocalized with the pH -Caspase-GFP 444 signal (Figure S9h- h″), consistent with efferocytic processing of apoptotic debris . 445 However, signal intensity was variable when using the CharON reporter. 446 447 In eye imaginal discs, activation of JNK signaling in neighboring epithelial cells promotes 448 the elimination of neoplastic tumor cells by engulfment (Ohsawa et al. 2011) . To test 449 whether JNK signaling occurs in hinge- pouch epithelial cells during debris clearance in 450 regenerating wing discs, we used the transcriptional JNK signaling reporter TRE-RFP 451 (Chatterjee and Bohmann 2012) . At R0, we observed localization of both LysoTracker 452 signal and TRE -RFP expression within hinge- pouch fold epithelial cells (Figure 5i-i ”’). 453 Together, these results support a model in which, after tissue ablation, activation of JNK 454 signaling in neighboring hinge epithelial cells promotes lysosome formation and epithelial 455 engulfment, leading to efficient clearance of cellular debris. 456 457 Reduction of engulfment receptor Draper impairs cell debris clearance and 458 lysosome formation in regenerating wing discs 459 Next, we asked whether reducing the uptake of cellular debris affects its clearance and 460 subsequent regeneration of the damaged disc . We focused on the phagocytic receptor 461 21 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Draper ( Drpr), which is the Drosophila ortholog of CED -1, a transmembrane receptor 462 required for engulfment of neurons (Freeman et al. 2003) , bacteria (Cuttell et al. 2008) , 463 severed axons (MacDonald et al. 2006) , germline cells in the ovary (Etchegaray et al. 464 2012), and imaginal disc cells during cell competition (Li and Baker 2007) . Although 465 multiple engulfment receptors have been described in Drosophila, we focused on Draper 466 because of its well -established role in mediating engulfment across diverse 467 developmental and injury contexts (Serizier and McCall 2017). 468 469 We used the drprΔ5 null allele(Freeman et al. 2003) to impair Draper function. First, we 470 confirmed that Draper protein levels were reduced in a heterozygous drprΔ5 /+ background 471 by immunostaining with Draper antibody (Figure S10a- c). We then examined the effects 472 of Draper reduction on debris clearance during wing disc regeneration by quantifying the 473 volume of EYFP -labeled cellular debris at multiple regeneration time points (Figure 6a-474 g). At R0, regenerating drpr Δ5 /+ discs exhibited a significant increase in debris volume 475 compared to controls (Figure 6a- b’, g). While debris volume decreased over time in 476 control discs, drprΔ5 /+ discs showed a continued presence of debris at R24, indicating 477 impaired clearance (Figure 6c -d’, g). This defect persisted at R48, when debris was 478 largely eliminated in controls but remained elevated in drprΔ5 /+ discs, with no significant 479 reduction in volume compared to R0 levels (Figure 6e- f’, g). Consistent with these 480 findings, debris planar area was significantly increased in drpr Δ5 /+ discs compared to 481 controls at all three regeneration time points (Figure S10d). Debris height was 482 comparable between controls and drprΔ5 /+ mutants at R0; however, at both R24 and R48, 483 22 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration debris height was increased in the drprΔ5 /+ discs, suggesting stalled or delayed debris 484 clearance (Figure S10e). 485 486 We next examined whether impaired debris clearance in drpr Δ5 /+ discs was associated 487 with altered lysosome formation. In control regenerating discs, lysosome number was 488 highest at R0 and progressively decreased at R24 and R48 (Figure 6h- n). By contrast, 489 drprΔ5 /+ discs exhibited a marked reduction in lysosome number at R0 (Figure 6h-n and 490 S10f-g) followed by an increase at R24 (Figure 6n and S10h- i) and sustained elevated 491 lysosome numbers at R48 compared to controls (Figure 6n, S10j -k). Together, these 492 observations indicate that Draper reduction leads to impaired debris clearance 493 accompanied by a temporal shift in lysosome formation. 494 495 To determine whether defective debris clearance impacts regenerative capacity, we 496 assessed pouch regrowth by staining regenerating discs with the wing pouch marker 497 Nubbin (Nub) to quantify pouch area at R48 (Figure S11a-c). We observed no significant 498 difference in Nub-positive pouch area between drprΔ5 /+ discs and controls (Figure S11c), 499 indicating that growth through this time point is comparable. To understand the effect of 500 impaired debris clearance on regeneration, we quantified adult wing size following disc 501 regeneration (Figure S11d). The adult wings derived from drprΔ5 /+ revealed a difference 502 in the distribution of adult wing sizes between controls and drprΔ5 /+ animals, as observed 503 by chi- square analysis . The distribution of adult wing sizes in the mutant showed an 504 increased proportion of severely reduced (0- 25%) and reduced (50%) wings (Figure 505 23 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration S11d). Taken together, these results demonstrate that epithelial debris clearance during 506 regeneration is at least partially mediated by the phagocytic receptor Draper. Reduction 507 of this Draper-mediated efferocytosis has a modest impact on wing disc regeneration. 508 509 Persistent debris in the regenerating discs consists of heterogeneous cellular 510 components 511 Our findings thus far indicate that although the majority of cellular debris is cleared by 512 R48 during wing disc regeneration, small remnants of debris persist within the tissue. We 513 therefore sought to determine the composition of this persistent debris. Using UAS -514 mCherry (Ni et al. 2011) to label cell debris, we examined the colocalization of residual 515 debris with markers for different cellular components and organelles. We hypothesized 516 that selective engulfment or clearance might result in preferential persistence of specific 517 cellular constituents. 518 519 We did not observe persistence of distinct cellular components within the debris at R48 520 (Figure 7). Instead, the residual debris consisted of a heterogeneous mixture of cellular 521 materials. Nuclear material (DAPI) (Figure 7 a-a’ ), nucleolar components (Fibrillarin) 522 (Figure 7 b-b’), nuclear envelope nuclear lamina, as assessed by Lamin A immunostaining 523 (Figure 7c-c’), actin (phalloidin) (Figure 7d-d’), and plasma membrane (CellMask) (Figure 524 7e-e’) all colocalized with mCherry -labeled debris. Similarly, we examined intracellular 525 organelles, including Mitochondria, Endoplasmic Reticulum (ER), and Golgi, using 526 previously published transgenic EYFP reporters that are under the ubiquitous spaghetti 527 24 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration squash ( sqh) promoter (LaJeunesse et al. 2004) . Each of the reporters marking 528 mitochondria (Figure 7f-f”), endoplasmic reticulum (Figure g-g’), and Golgi (Figure 7h-h’) 529 colocalized with the mCherry -labelled debris. Collectively, these findings indicate that 530 persistent debris in the regenerating discs does not represent a selectively retained 531 subset of cellular components, but rather a heterogeneous mixture, suggesting no strong 532 preference in the uptake or clearance of specific debris constituents. 533 534 535 536 537 538 539 540 541 542 543 544 545 546 25 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration

547 In this study, we demonstrate that apoptotic cell debris generated during tissue ablation 548 in the wing imaginal disc is cleared in the absence of immune cell recruitment. Despite 549 robust activation of canonical damage responses, including Wnt/Wg signaling, ROS 550 production, and JNK activation, similar to those observed in damaged eye discs (Fan et 551 al. 2014; Fogarty et al. 2016; Diwanji and Bergmann 2020; Worley and Hariharan 2022), 552 the basement membrane acts as a barrier to immune cell infiltration. In contrast to the 553 eye disc, the regenerating wing disc maintains a continuous extracellular matrix that 554 physically restricts immune cell access to the epithelium. Consistent with this finding, both 555 our study and previous work (Harris et al. 2016) show that the basement membrane 556 remains intact during regeneration. This barrier function is further supported by our prior 557 transcriptional profiling of the regeneration blastema at 24 hours post -damage, which 558 revealed elevated expression of the Collagen IV gene viking (log₂FC = 5.45, p = 0.00027) 559 and reduced expression of mmp2 (log₂ FC = −1.30, p = 0.0002) (Khan et al. 2017). 560 Together, these transcriptional changes favor basement membrane stabilization over 561 degradation. Importantly, experimental disruption of basement membrane continuity was 562 sufficient to permit immune cell infiltration, directly linking extracellular matrix integrity to 563 immune cell access during epithelial regeneration. 564 565 We further show that most apoptotic cell debris generated during tissue ablation is 566 efficiently cleared from the wing imaginal disc within two days after damage by 567 neighboring hinge epithelial cells. This process represents a form of non- professional 568 efferocytosis. Efficient clearance of apoptotic debris is critical for maintaining tissue 569 26 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration homeostasis and enabling repair, a process well characterized in mammalian systems 570 (reviewed in Mehrotra and Ravichandran 2022) and during Drosophila melanogaster 571 embryogenesis and metamorphosis (reviewed in Adell et al. 2025) . When immune cell 572 access is restricted, this function must be carried out by non- professional phagocytes. 573 Thus, the spatial confinement of apoptotic cells within an intact epithelial and basement 574 membrane architecture during wing disc regeneration likely necessitates the uptake of 575 debris by neighboring epithelial cells. Our findings establish the regenerating wing 576 imaginal disc as a model for epithelial efferocytosis, enabling future study of the 577 mechanisms involved and how some debris is left behind . While JNK signaling is a key 578 driver of regenerative responses (reviewed in Tripathi and Irvine 2022), our data indicate 579 that its activation is not confined to the regeneration blastema but also occurs in 580 surrounding epithelial cells. Notably, our results show that JNK activity is elevated in 581 undamaged hinge cells at early time points (R0), whereas at later stages (R24), it is 582 enriched in the regenerating blastema (Brock et al. 2017; Khan et al. 2017) , suggesting 583 temporally and spatially distinct activation across undamaged and regenerating cell 584 populations. This early activation in hinge cells suggests that JNK may promote 585 efferocytosis in neighboring epithelial cells, analogous to its role in tumor cell engulfment 586 in eye imaginal discs (Ohsawa et al. 2011). 587 588 Delayed or defective clearance of apoptotic debris is associated with secondary necrosis 589 and can lead to pathological outcomes, including autoimmune and chronic inflammatory 590 disorders (reviewed in Kawano and Nagata 2018; Doran et al. 2019) . Draper, the 591 Drosophila homolog of Ced-1, is a well-established receptor required for the recognition 592 27 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration and clearance of apoptotic corpses and plays critical roles in multiple developmental 593 contexts (reviewed in Melcarne et al. 2019) . In regenerating wing discs, blocking debris 594 clearance with draper mutants led to delayed removal of debris and a delayed lysosome 595 formation. Despite these defects, prolonged debris persistence in the regenerating 596 epithelium had only a limited effect on overall regeneration, as assessed by adult wing 597 size. These findings suggest that debris clearance, while actively engaged, may be 598 dispensable during regeneration. However, because draper and the ablation system are 599 located on the same chromosome, these experiments were performed in a heterozygous 600 background, and the lack of an effect on adult wing sizes may reflect the incomplete loss 601 of Draper function. Alternatively, compensatory mechanisms involving other engulfment 602 receptors, such as Croquemort, NimC4/SIMU, and integrins ( αPS3βPS/βν), which can 603 also mediate apoptotic cell clearance (reviewed in Serizier and McCall 2017; Melcarne et 604 al. 2019), may partially compensate for the reduction in Draper during regeneration. In 605 the regenerating wing disc, persistence of apoptotic debris two days after damage ha d 606 minimal impact on regeneration of the disc and resulting adult wing sizes. 607 608 Residual debris comprises a heterogeneous mixture of cellular components, with no clear 609 bias toward retaining or preferentially clearing specific subcellular structures. In most 610 systems, efferocytic receptors recognize broadly conserved “eat-me” signals on apoptotic 611 material, which may be membrane-anchored or presented via soluble bridging molecules 612 (reviewed in Ravichandran 2010) . Phosphatidylserine (PS) is the best characterized of 613 these signals and, when externalized on apoptotic cells, promotes their recognition and 614 clearance (reviewed in Moon et al. 2023) . In Drosophila, Draper similarly responds to 615 28 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration damage-associated cues and PS exposure, enabling the uptake of diverse apoptotic 616 fragments (Tung et al. 2013) . Notably, these mechanisms are generally described in 617 terms of broad recognition rather than selective targeting of specific subcellular 618 components, and there is limited evidence supporting strict discrimination between 619 organelle types. Our observations supports a model in which debris clearance is largely 620 non-selective and may be driven by general engulfment mechanisms rather than targeted 621 degradation pathways. 622 623 Together, our work redefines debris clearance during regeneration, demonstrating that 624 epithelial cells can autonomously carry out efferocytosis in the absence of immune cell 625 involvement. The wing imaginal disc has not traditionally been considered a model for 626 studying efferocytosis. Our findings now establish apoptosis -induced damage and 627 regeneration in this system as a tractable context to investigate apoptotic debris 628 clearance and epithelial efferocytosis in vivo. 629 630

631 Tissue ablation 632 All experiments were performed using a protocol adapted from prior work (Smith-Bolton 633 et al. 2009). Egg lays were conducted at 25°C for 4 hours in the dark on grape juice agar 634 plates supplemented with yeast paste (defined as day 0), after which embryos were 635 incubated at 18°C. First instar larvae were collected on day 2 and transferred to Nutri-Fly 636 Bloomington food vials supplemented with yeast paste at a density of approximately 50 637 29 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration larvae per vial. To induce tissue damage, vials were transferred early on day 7 to a water 638 bath maintained at 30°C for 24 hours. Following heat treatment, vials were briefly cooled 639 in an ice-water bath for 1 minute and then returned to 18°C. Regenerating animals were 640 dissected at the indicated time points or allowed to develop to adulthood for quantification 641 of adult wing size. Undamaged control animals were maintained at 18°C and dissected 642 at the third instar crawling stage. In addition, temperature- shifted genetic controls were 643 subjected to the same 30°C heat treatment, but do not undergo ablation. 644 645 Drosophila strains 646 The fly stocks used for this study are as follows: 647 w1118 (isogenic line made in the Smith- Bolton lab) , w1118; rnGal4, UAS -rpr, 648 tubGal80ts/TM6B, tubGal80 (Smith-Bolton et al. 2009), UAS-2xEYFP (Halfon et al. 2002) 649 (RRID:BDSC_6659), HmlΔRFP (gift from K. Bruckner) (Makhijani et al. 2011) , hopTum 650 (Luo et al. 1995) (RRID:BDSC_8492), αPS4-GFP (Sarov et al. 2016) (VDRC_318086), 651 MSNF9-GFP (gift from S. Govind) (Tokusumi et al. 2009), Atilla-MiET1 GFP (Honti et al. 652 2009) (RRID:BDSC_23540), PPO1-mCherry.F6 (Tokusumi et al. 2017) 653 (RRID:BDSC_600219), lz-GFP (Kudron et al. 2018) , (RRID:BDSC_43954), srpHemo-654 3XmCherry/CyO (RRID:BDSC_78358), srpHemo-3XmCherry/TM3 655 (RRID:BDSC_78359), srpHemo-H2A.3XmCherry/TM3 (RRID:BDSC_78360), srpHemo-656 H2A.3XmCherry/CyO (RRID:BDSC_78361), srpHemo -Moe.3XmCherry 657 (RRID:BDSC_78362) , srpHemo-Moe.3XmCherry/CyO (RRID:BDSC_78363) (Gyoergy 658 et al. 2018) , vkg-GFP (Kyoto DGRC# 110626) (Morin et al. 2001) , UAS-Mmp2 659 30 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration (RRID:BDSC_58706) (Page-McCaw et al. 2003) , TRE-RFP (gift from D. Bohmann) 660 (Chatterjee and Bohmann 2012) , UAS-CHARON (gift from W. Wood) (Raymond et al. 661 2022), Df(3L)drpr[Delta5]/TM6B (Freeman et al. 2003) , (RRID:BDSC_67033), sqh-662 EYFP-Mito (RRID:BDSC_7194), sqh-EYFP-ER (RRID:BDSC_7195), sqh-EYFP-Golgi 663 (RRID:BDSC_7193) (LaJeunesse et al. 2004) , UAS-mCherry (Ni et al. 2011) 664 (RRID:BDSC_35787). 665 666 Immunofluorescence 667 Primary antibodies: Primary antibodies used were mouse anti-cut (1:150, DSHB Cat# 668 2b10, RRID: AB_528186), mouse anti-Nimrod (1:250, gift from I. Ando) (Kurucz, Márkus, 669 et al. 2007), mouse anti-AtillaL1abc (1:100, gift from I. Ando) (Kurucz, Váczi, et al. 2007), 670 mouse anti-Lozenge (1:10, DSHB, RRID: AB_528346) (Lebestky et al. 2000), mouse anti-671 Hemese (1:100) (gift from I. Ando) (Kurucz et al. 2003), mouse anti-Antennapedia (1:10, 672 DSHB Cat# 8C11, RRID: AB_528083), mouse anti-Discs large (1:100, DSHB Cat# 4F3, 673 RRID: AB_528203), mouse anti-Nubbin (1:100, DSHB Cat# 2D4, RRID:AB_2722119), 674 rabbit anti -Draper (1:500) (gift from M. Freeman) (Freeman et al. 2003), mouse anti-675 Fibrillarin (1:100; Abcam, ab4566), mouse anti-Lamin (1:100, DSHB Cat# ADL67.10, 676 RRID: AB_528336). 677 678 Secondary antibodies: Secondary antibodies used were Alexa Fluor 488, 555, 633, 647 679 (1:500; Invitrogen, A21424, A21245, A21240, A21071, A21052 and A21094). 680 31 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Stains/Dyes: Phalloidin (1:100, Invitrogen, Thermo Fisher Scientific, R415), DAPI (1:500, 681 Thermo Fisher Scientific, D1306), LysoTracker ™ Deep Red (1:250, Invitrogen, Thermo 682 Fisher Scientific, L12492), CellMask ™ Deep Red (1:500, Invitrogen, Thermo Fisher 683 Scientific, C10046). 684 685 Immunostaining 686 Immunostaining was performed using a protocol similar to that previously described 687 (Smith-Bolton et al. 2009) Briefly, dissected larval carcasses were fixed in 4% 688 paraformaldehyde (PFA) in 1× phosphate- buffered saline (PBS) for 20 minutes at room 689 temperature in 1.5 mL microcentrifuge tubes (paraformaldehyde, 16% w/v aqueous 690 solution, methanol-free; Thermo Fisher Scientific, Cat# 043368.9M). Samples were then 691 washed three times for 10 minutes each in 0.1% Triton X-100 in PBS (0.1% PBST) on a 692 nutator at room temperature (Triton X -100; Thermo Fisher Scientific, Cat# PRH5141). 693 Larvae were incubated overnight at 4 °C in primary antibodies diluted as indicated in 0.1% 694 PBST containing 5% normal goat serum (MP Biomedicals ™, Cat# MP92939154), 695 followed by three 10-minute washes in 0.1% PBST. Secondary antibodies were used at 696 a 1:500 dilution and incubated overnight at 4 °C. After secondary antibody incubation, 697 samples were washed three times in 0.1% PBST and equilibrated in 70% glycerol in 1× 698 PBS (glycerol, molecular biology grade; Fisher BioReagents™, Cat# BP229 -1). 699 The no-wash immunostaining protocol was adapted from Zhu et al, (Zhu et al. 2025) with 700 a few additions. Following dissections, larval carcasses were fixed in 10 µL of 4% 701 paraformaldehyde (PFA) at room temperature in a 72- well microwell plate (Nunc ™ 702 32 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration MicroWell™ MiniTrays; Thermo Fisher Scientific, Cat# 438733), with three larval 703 carcasses placed per well. After a 20- minute fixation, carcasses were removed using 704 forceps, briefly dabbed on filter paper (Whatman™ 1001-110 Filter Circles, Cat# 1001-705 110) to remove excess solution, and transferred to a new well containing 10 µL of 0.1% 706 PBST. 707 Carcasses were subsequently transferred between primary (1:50) and secondary 708 antibody solutions using repeated filter -paper dabbing in place of conventional wash 709 steps. Primary and secondary antibody incubations were performed overnight, with 710 intermediate 10-minute incubations in 0.1% PBST between antibody steps. Finally, larvae 711 were equilibrated overnight in 70% glycerol in the microwell plate and mounted 712 immediately in VECTASHIELD mounting medium to prevent desiccation due to the small 713 incubation volumes. 714 715 Wing discs mounting 716 Wing imaginal discs were then dissected and mounted on glass slides (plain microscope 717 slides, Fisher Scientific, Cat# 12550A3; coverslips, 24 × 50 mm, Fisher Scientific, Cat# 718 12541042) using VECTASHIELD anti-fade mounting medium (Vector Laboratories, Cat# 719 H-1000), and the edges were sealed with Sally Hansen® Quick Dry clear nail polish. 720 To obtain orthogonal optical sections, a double-sided tape mounting method was adapted 721 from Aldaz et al. (Aldaz et al. 2010) with modifications for fixed -wing imaginal discs. In 722 this modified approach, double- sided tape was used as a spacer. Briefly, double- sided 723 tape (3M 667 Scotch® Double- Sided Tape, Cat# 34-8724-5241-1) was cut into four 19 724 33 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration mm × 2 mm rectangular strips and affixed to a glass microscope slide to make a double-725 sided tape square. Wing imaginal discs were dissected and mounted at the center of a 726 square coverslip to position the sample as close to the coverslip as possible during 727 imaging (Corning® 18 × 18 mm square #1 cover glass, Cat# 2865- 18) in 2 µL of 728 VECTASHIELD mounting medium. The coverslip was then carefully inverted and placed 729 onto the tape- affixed slide. The edges were sealed with Sally Hansen® Quick Dry nail 730 polish to secure the mount and prevent desiccation of the sample. 731 732 Larval hemocytes extraction and immunostaining 733 Hemocytes were extracted from third instar larval hemolymph using a protocol adapted 734 from Hiroyasu et al. (Hiroyasu et al. 2018) . Briefly, a single third instar larva of the 735 indicated genotype was placed in a 2 µL drop of 1× PBS on a circular coverslip 736 (Fisherbrand™ circular cover glass, 18 mm diameter, Cat# 12541005CA) within a glass 737 dissection dish. Larvae were allowed to bleed into the PBS drop for approximately 5 738 minutes, after which the larval carcass was removed. The hemolymph droplet was then 739 allowed to air-dry on the coverslip for 5 minutes. Coverslips were transferred to a 12-well 740 plate (CytoOne® 12- well plates, Cat# CC7682 -7512) and fixed by completely covering 741 the coverslip with 4% paraformaldehyde. Samples were washed three times with PBST 742 on a nutator at room temperature. Primary and secondary antibody incubations were 743 performed overnight at 4 °C on a nutator. Following antibody incubation, coverslips were 744 washed three times for 10 minutes each in PBST, mounted on glass slides with 745 VECTASHIELD mounting medium, and sealed with nail polish. 746 34 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration 747 Confocal microscopy, image acquisition, and processing 748 Wing imaginal discs were imaged using a Zeiss LSM 880 or LSM 900 confocal 749 microscope. All images within a given experiment were acquired on the same microscope 750 to ensure consistency using a 20x/0.45 objective. For orthogonal YZ and XZ imaging, z-751 stacks were collected with a step size of 2 µm. Image acquisition speed was maintained 752 between 3 and 5, and all other imaging parameters, including laser power and detector 753 gain, were kept constant across experimental and control samples. 754 Orthogonal projections were generated using the Zeiss ZEN Blue software by selecting 755 the ortho view for representative slices. Image files were subsequently opened in 756 ImageJ/FIJI (Schindelin et al. 2012; Schneider et al. 2012) where maximum -intensity 757 projections (MIPs) were generated for downstream processing and figure preparation. 3D 758 reconstruction and surface rendering were performed using Imaris software (Bitplane AG, 759 Oxford Instruments, Zurich, Switzerland), excluding the peripodial epithelium from the z 760 stacks. For Nubbin pouch area quantifications, MIPs were generated, and the Nubbin-761 positive pouch was manually outlined using the Freehand Selection tool in ImageJ. The 762 enclosed area was then measured and recorded for analysis. 763 764 Cell debris volume quantification 765 To quantify the volume of cell debris labeled by UAS-EYFP, debris area and height were 766 measured from confocal image stacks. MIPs were generated from confocal z -stacks 767 using ImageJ/Fiji. Top-down XY projections were used to outline debris regions in the 768 35 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration UAS-EYFP (green) channel, and the debris area was quantified using the Measure 769 function. Debris height was determined from the same image stacks by generating 770 orthogonal YZ re- slices followed by maximum -intensity projection. Five independent 771 height measurements were obtained per sample using the Straight -Line tool in ImageJ, 772 and the average height within the debris region, as defined by the UAS-EYFP signal, was 773 used as the debris height value. At later regeneration time points, when epithelial regrowth 774 altered tissue morphology, height was measured from YZ projections, accounting for both 775 apical and basal debris relative to the epithelium. Debris volume was estimated by 776 multiplying the measured debris area by the corresponding average debris height for each 777 sample. 778 779 Hemocyte quantification 780 For quantification of hemocyte number, maximum intensity projections (MIPs) of top-781 down XY confocal images were generated. Hemocytes were manually counted either 782 across the entire wing disc (including notum, hinge, and pouch regions) or specifically 783 within the rn>EYFP-labeled cell debris in the pouch. For pouch-specific quantification, 784 the rn>EYFP-labelled cell debris was segmented, and immune cells within this domain 785 were counted separately. 786 787 Adult wing mounting and quantification 788 Adult wings were scored into approximate size categories of 0%, 25%, 50%, 75%, and 789 100% relative to a wild -type wing that did not undergo damage and regeneration as a 790 36 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration disc. Representative wings from each category were dissected and mounted on glass 791 slides using Gary’s Magic Mount. Images were obtained on an Echo Revolve R4 792 microscope and processed using ImageJ/FIJI. The mounting medium was prepared by 793 dissolving Canada balsam in methyl salicylate at a 5:4 ratio, then gently evaporating the 794 mixture on a medium -heat hot plate with slow stirring at room temperature (for up to 4 795 days) until most of the solvent had evaporated, leaving a viscous solution. The mounting 796 medium was protected from light during preparation, storage, and mounting. 797 798 Lysosome puncta quantification 799 Lysosome puncta were quantified using an image analysis pipeline adapted from a 800 previous image analysis pipeline (Gamarra et al. 2020) developed for translation site-801 specific foci detection and modified here for lysosome analysis. All image processing and 802 quantification were performed using ImageJ/Fiji. Briefly, Lysotracker confocal image 803 stacks were opened in ImageJ/Fiji and converted to grayscale. A region of interest (ROI) 804 encompassing the wing pouch and hinge was manually selected using the freehand 805 selection tool and duplicated, ensuring that all associated hyperstacks were retained. 806 Brightness and contrast were adjusted uniformly across samples to enhance visualization 807 of Lysotracker-positive puncta. A maximum-intensity projection (MIP) was generated from 808 all z-stacks. The MIP image was processed using Process - Filters - Convolve with a 5 x 809 5 kernel, with the central kernel value set to 48. A threshold was applied to the processed 810 (Image - Adjust - Threshold). Lysosome puncta were quantified using Analyze Particles, 811 with particle size restricted to 2- 15 µm² and circularity set to 1.0. Particle counts were 812 exported and plotted using GraphPad Prism. 813 37 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration 814 Statistical analysis 815 All statistical analyses were performed using GraphPad Prism. Comparisons between 816 two groups were conducted using Welch’s t -test. Adult wing size distributions were 817 analyzed using a chi -square test. All graphs and plots were generated using GraphPad 818 Prism. All error bars represent the standard error of the mean (SEM). 819 820 821 822 823 824 825 826 827 828 829 830 831 38 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 39 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Figure 1. Tissue Damage and Orthogonal Projections of the Wing Imaginal Disc 832 (a) Schematic representation of the genetic ablation system. (b) Schematic 833 representation of orthogonal projections of wing imaginal disc images , showing 834 morphology in the XZ projection through the pouch and the YZ projection through the 835 notum, hinge, and pouch. Purple cells are the pouch, orange cells are the hinge, yellow 836 cells are the remainder of the disc proper. Yellow arrow head: Notum-Hinge fold (N -H); 837 Orange arrowhead: Hinge-Hinge fold (H-H); Purple arrowhead: Hinge-Pouch fold (H-P). 838 (c) Phalloidin staining showing actin in the XZ projection of an undamaged wing disc 839 through the pouch. (d) Phalloidin staining showing actin in the YZ projection of an 840 undamaged wing disc through the notum, hinge, and pouch. Yellow arrow head: Notum-841 Hinge fold (N-H); Orange arrowhead: Hinge-Hinge fold (H-H); Purple arrowhead: Hinge-842 Pouch fold (H-P). Scale bars: 50 µm. 843 844 845 846 847 848 849 850 851 40 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 41 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Figure 2. Cellular debris localization and clearance during wing disc regeneration 852 (a-a″) Orthogonal YZ projection of undamaged wing imaginal disc expressing rn-GAL4 > 853 UAS-EYFP (rn>EYFP). rn>EYFP expression marks the pouch and adult muscle 854 precursor cells in the notum (blue arrow heads, a and a’’). Nuclei are labeled with DAPI 855 (a′-a″). For all panels, Yellow arrowhead: Notum-Hinge fold (N -H); Orange arrow head: 856 Hinge-Hinge fold (H-H); Purple arrowhead: Hinge-Pouch fold (H-P). (b-b″) Orthogonal YZ 857 projection of R0 wing disc expressing rn >EYFP (b,b’’) with DAPI staining (b′ -b″). Apical 858 debris is indicated by white arrowheads, and basal debris by blue arrowheads. (c-f) Three-859 dimensional reconstructions of R0 wing discs stained with DAPI and expressing rn>EYFP 860 to label cell debris. (c -e) Wing disc showing apical debris (white arrow head) and basal 861 debris (blue arrow head). (f) Orthogonal side view highlighting the apical and basal 862 localization of debris. (g- g″) Orthogonal YZ projection of R4 wing disc expressing 863 rn>EYFP (g,g’’) with DAPI staining (g′ -g”). (h-h″) Orthogonal YZ projection of R16 wing 864 disc expressing rn>EYFP (h,h’’) with DAPI staining (h′ -h”). Debris localized in the hinge 865 epithelium (blue dotted circle). (i-i″) Orthogonal YZ projection of R24 wing disc expressing 866 rn>EYFP (i,i’’) with DAPI staining (i′ -i”). (j-j″) Orthogonal YZ projection of R48 wing disc 867 expressing rn>EYFP (j,j’’) with DAPI staining (j′ -j”). (K) Quantification of total cell debris 868 volume across regeneration time points: R0 n = 11 discs, R4 n = 9 discs; ****P = 0.0001 869 compared to R0, R8 n = 7 discs, R10 n = 5 discs, R12 n = 8 discs, R16 n = 6 discs; **P 870 = 0.0036 compared to R4, R24 n = 10 discs, R36 n = 6 discs, and R48 n = 5 discs; **P = 871 0.0039 compared to R16. Pairwise comparisons showed no significant differences 872 between R4 and R8 ns, P = 0.6004, R4 and R10 ns, P = 0.5330, R4 and R12 ns, P = 873 42 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration 0.3507, R16 and R24 ns, P = 0.5961 or R16 and R36 ns, P = 0.3727. Statistical 874 significance was determined using Welch’s t-test. Error bars: SEM. Scale bars: 50 µm. 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 43 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 44 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Figure 3. Immune cell recruitment is not detected under no-wash conditions in the 892 regenerating wing disc 893 (a-d) Anti -Nimrod (NimC) (yellow arrowheads) and DAPI staining under no -wash 894 conditions of an undamaged wing disc at 0 hours (a-a’), a regenerating disc at R0 (b-b′), 895 an undamaged disc at 24 hours (c-c′), and a regenerating disc at R24 (d-d′). (e) 896 Quantification of NimC-positive plasmatocytes within the wing disc pouch under no-wash 897 conditions. Undamaged discs at 0 hours n=13 discs, regenerating discs at R0 n=18 discs, 898 ns, p=0.9158, undamaged discs at 24 hours n=7 discs, and regenerating discs at R24 899 n=14 discs, ns, p=0.7614. (f -h) Orthogonal YZ projections of discs stained with anti-900 Nimrod (NimC) to mark plasmatocytes, rn>EYFP to mark cell debris, and DAPI staining 901 to mark nuclei. (f) Undamaged disc at 0 hours. (g) Damaged discs at R0 ( h). Damaged 902 discs at R24. Yellow arrow heads indicate NimC- positive plasmatocytes. Statistical 903 significance was determined using Welch’s t-test. Error bars: SEM. Scale bars: 50 µm. 904 905 906 907 908 909 910 911 45 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 46 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Figure 4 . Basement membrane remains intact and restricts immune cell 912 recruitment 913 (a-c’) Orthogonal YZ projections of wing disc expressing Vkg- GFP and co- stained with 914 NimC and DAPI in control undamaged (w¹¹¹⁸) (a-a’), control regenerating R0 disc (b-b’), 915 and rn-GAL4 > UAS-mmp2 (rn>mmp2) (c-c′) discs. Yellow brackets indicate the wing disc 916 pouch. Blue arrow head indicates NimC-positive plasmatocytes. For all panels, y ellow 917 arrowhead: Notum-Hinge fold (N-H); Orange arrowhead: Hinge-Hinge fold (H-H); Purple 918 arrowhead: Hinge-Pouch fold (H-P). (d-f) Top-down views of undamaged control (d), R0 919 regenerating disc (e), and rn >mmp2 R0 disc (f). Undamaged control n= 23 discs, 11/23 920 discs were positive for NimC, R0 discs. w¹¹¹⁸ , n=24 discs, 8/24 discs were positive for 921 NimC, and rn>mmp2, n=8 discs, 4/8 discs were positive for NimC. Scale bars: 50 µm. 922 923 924 925 926 927 928 929 930 931 47 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 48 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Figure 5: Epithelial debris is cleared t hrough hinge-mediated efferocytosis and 932 lysosome formation 933 (a-a’’) Orthogonal YZ projection of R10 wing disc expressing rn>EYFP with DAPI staining. 934 Blue arrowheads indicate EYFP -labelled cell debris in the hinge epithelium. For all the 935 panels, yellow arrowhead: Notum-Hinge fold (N-H); Orange arrowhead: Hinge-Hinge fold, 936 purple arrow head: Hinge- Pouch fold (H -P). (b, c) Top -down single- slice views of 937 undamaged control and R0 wing discs stained with LysoTracker . Yellow dashed box - 938 higher magnification region of interest (d-e) Higher-magnification views of the R0 disc in 939 (c) (yellow dashed box) showing LysoTracker -positive puncta (d,d’,e’), anti-Discs large 940 (Dlg) to show epithelial membranes (d’) , and rn>EYFP-labeled debris (e,e’). Orange 941 arrowhead: Hinge-Hinge fold, blue arrowheads indicate lysosomes. (f) Top-down single-942 slice view of an R24 wing disc stained with LysoTracker. ( g) Quantification of lysosome 943 number (LysoTracker -positive puncta) across regeneration time points. Undamaged 944 control n=10 discs, R0 n=9 discs, ****P= 0.0001 compared to undamaged control , R24 945 n=11 discs, ****P=0.0001 compared to R0. ( h-h‴) YZ orthogonal projection of R0 disc 946 stained with LysoTracker (h,h’,h’’’), Dlg (h’), and expressing rn>EYFP to label debris (h’’). 947 The white dotted circle marks lysosomes localized to the hinge-pouch (H-P) fold. (i-i‴) YZ 948 orthogonal projection of R0 disc expressing rn>EYFP to mark the debris (i’) and TRE -949 RFP to mark JNK signaling (i’’,i’’’), and stained with LysoTracker (i,i’) and DAPI (i’’’). The 950 yellow dotted circle marks lysosomes localized in the H -P fold that are associated with 951 JNK signal activation. Error bars: SEM. Scale bars: 50 µm. 952 953 49 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 50 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Figure 6: Heterozygous draper mutants show impaired debris clearance and 954 altered lysosome dynamics 955 (a-f) Orthogonal YZ projections of rn>EYFP-expressing wing discs stained with DAPI in 956 w1118 controls (a,c,e) and draper∆5/+ mutants (b,d,f) at R0 (a-b), R24 (c-d), and R48 (e-f). 957 Yellow brackets indicate rn>EYFP-labeled cell debris. For all panels, yellow arrow head: 958 Notum-Hinge fold (N -H); Orange arrow head: Hinge -Hinge fold (H-H), and p urple 959 arrowhead: Hinge -Pouch fold (H -P). (g) Quantification of debris volume across 960 regeneration time points in w1118 controls (grey bars) and draper∆5/+ mutants (yellow bars). 961 w1118 R0 n=11 discs, w1118 R24 n=12 discs, w1118 R48 n=9 discs, draper∆5/+ R0 n=17 discs, 962 draper∆5/+ R24 n=12 discs, draper∆5/+ R48 n=10 discs. Pairwise comparisons showed 963 significant differences between control and draper∆5/+ R0 ***P= 0.0002, R24 ****P= 964 0.0001, and R48 ****P= 0.0001. No significant differences were found between draper∆5/+ 965 R0 and draper∆5/+ R24 ns, P=0.2749, and draper∆5/+ R24 and draper ∆5/+ R48 ns, 966 P=0.5491. (h-m) Orthogonal YZ projections of rn>EYFP expressing discs, co-stained with 967 LysoTracker and DAPI in w ¹¹¹⁸ controls (h,j,l) and draper∆5/+ mutants (i,k,m) at R0 (h,i), 968 R24 (j,k), and R48 (l,m). Yellow dotted circles mark lysosome-enriched regions within the 969 hinge and pouch. (n) Quantification of lysosome number (LysoTracker -positive puncta) 970 across regeneration time points in w1118 controls (grey bars) and draper∆5/+ mutant wing 971 discs (yellow bars). w 1118 R0 n=11 discs, w1118 R24 n=12 discs, w 1118 R48 n=9 discs, 972 draper∆5/+ R0 n=17 discs, draper∆5/+ R 24 n=12 discs, draper ∆5/+ R 48 n=10 discs. 973 Pairwise comparisons showed significant differences between the control and draper∆5/+ 974 at R0 ***P= 0.0003 , R24 ****P= 0.0001 , and R48 **P= 0.0090. Error bars: SEM. Scale 975 bars: 50 µm. 976 51 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 52 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Figure 7: Persistent d ebris at R48 contains mixed cellular components and 977 organelles 978 (a-h) Orthogonal YZ projections of R48 wing discs expressing rn >mCherry to label cell 979 debris. For all panels, yellow arrowhead: Notum-Hinge fold (N -H); Orange arrow head: 980 Hinge-Hinge fold (H -H); Purple arrow head: Hinge -Pouch fold (H -P). (a -a’) Discs were 981 stained with DAPI to mark DNA. 10/10 discs showed DNA in the debris. (b-b’) Discs were 982 stained with Fibrillarin to mark the nucleolus . 3/3 discs showed nucleolus material in the 983 debris. (c-c’) Discs were stained with Lamin A to mark the nuclear lamina. 3/3 discs 984 showed nuclear lamina in the debris. (d-d’) Discs were stained with phalloidin to label F-985 actin. 3/3 discs showed F-actin in the debris. (e-e’) Discs were stained with CellMask to 986 label the plasma membrane. 3/3 discs showed plasma membrane in the debris. (f-h) R48 987 discs expressing rn>mCherry to label debris were also imaged with transgenic organelle 988 markers. (f-f’) Discs were expressing sqh -EYFP-Mito to label mitochondria. 3/3 discs 989 showed mitochondria in the debris. (g-g’) Discs expressed sqh -EYFP-ER to label the 990 endoplasmic reticulum (ER). 6/6 discs showed ER in the debris. (h-h’) Discs expressed 991 sqh-EYFP-Golgi to label the Golgi apparatus . 10/10 discs showed Golgi in the debris . 992 Blue arrowheads indicate regions of overlap between rn>mCherry-labeled cell debris and 993 the indicated cellular components or organelle markers. Scale bars: 50 µm. 994 995 996 997 998 53 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 54 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Supplementary Figure S1. Temporal dynamics of d ebris clearance in the 999 regenerating wing disc 1000 (a–a***) Orthogonal YZ projections of an undamaged wing disc expressing rn >EYFP (a, 1001 a*, a***), co -stained with DAPI (a′, a**, a***) and anti -Cut antibody (a″–a***). Light blue 1002 arrowheads indicate the adult muscle precursor cells in the notum. (b-e) For all panels, 1003 yellow arrowhead: Notum–Hinge fold (N– H); Orange arrow head: Hinge–Hinge fold (H–1004 H); Purple arrow head: Hinge –Pouch fold (H –P). (b –e) Orthogonal YZ projections of 1005 regenerating discs at R8 (b-b”), R10 (c-c”), R12 (d-d”), and R36 (e-e”), with GFP labeling 1006 cell debris (b-e, and b”-e”) and DAPI marking the nuclei (b’-e’, and b”-e”) (f) Quantification 1007 of total debris area across regeneration time points. R0 n=10 discs, R4 n=9 discs, **P= 1008 0.0031 compared to R0, R8 n=7 discs, R10 n=10 discs, R12 n=8 discs, R16 n=6 discs, 1009 ns P=0.0696 compared to R4 , R24 n=10 discs, * P=0.0241 compared to R4, R36 n=5 1010 discs, R48 n=4 discs, ** P=0.0069 compared to R24. (g) Quantification of debris height 1011 across regeneration time points, with the same n as in (f). R0-R4 (****P<0.0001), R4-R16 1012 (*P=0.0204). Statistical significance was determined using Welch’s t-test. Scale bars: 50 1013 µm. Error bars: SEM. 1014 1015 1016 1017 1018 1019 55 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 56 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Supplementary Figure S2. Plasmatocytes are not recruited to the regenerating wing 1020 disc epithelium. 1021 (a-c′) Wing discs from larvae expressing Hml Δ-RFP co -stained with DAPI . (a-a′) 1022 Undamaged control. (b-b′) R0 disc. (c-c′) R24 disc. Yellow arrowheads indicate Hml-RFP-1023 positive plasmatocytes. (d) Quantification of Hml Δ-RFP-positive plasmatocytes 1024 throughout the whole wing disc. No damage 0 hours n=7 discs, R0 n=13 discs , 1025 *P=0.0231, R24 n=6 discs, ns P=0.1583 (e) Quantification of Hml Δ-RFP-positive 1026 plasmatocytes within the wing disc pouch region, same n as in (d) . No damage 0 hours-1027 R0, ns P=0.0506. No damage 0 hours -R24, ns P=0.0731. (f -h′) Wing discs co -stained 1028 with anti-Nimrod (NimC) and DAPI in undamaged controls (f -f′), at R0 (g-g′), and at R24 1029 (h-h′). Yellow arrow heads indicate NimC- positive plasmatocytes. (i) Quantification of 1030 NimC-positive plasmatocytes in the whole wing disc. No damage n=9 discs, R0 n=13 1031 discs, **** P<0.0001, R24 n=9 discs, ns P=0.5459. (j) Quantification of NimC -positive 1032 plasmatocytes within the pouch region. No damage n=9 discs compared to R0 n=13 1033 discs, ***P= 0.0002. No damage compared to R24 n=9 discs, ns P=0.1530. Statistical 1034 significance was determined using Welch’s t-test. Scale bars: 50 µm. Error bars: SEM. 1035 1036 1037 1038 1039 1040 57 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 58 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Supplementary Figure S3. Plasmatocyte quantification under wash and no- wash 1041 conditions. 1042 (a) Quantification of NimC-positive plasmatocytes in the whole wing disc under no- wash 1043 conditions. No damage 0 hours n=13 discs, R0 n=18 discs, ns P= 0.4662. Undamaged 1044 24 hours n=7 discs, and R24 n=14 discs, ns P=0.7715. (b) Quantification of NimC-positive 1045 plasmatocytes in wash and no- wash conditions within the wing disc pouch. Wash n o 1046 damage n= 9 discs, No wash no damage n=13 discs, ns P=0.0610 compared to no 1047 damage wash, wash R0 n=13 discs, no wash R0 n=18 discs, * P=0.0373 compared to 1048 wash R0, and ns P=0.9158 compared to no damage no wash. Statistical significance was 1049 determined using Welch’s t-test. Error bars: SEM. 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 59 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 60 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Supplementary Figure S4. Lamellocytes are not detected in regenerating wing 1061 discs. 1062 (a–a′) Hemolymph from a n undamaged larva containing circulating hemocytes, co-1063 stained with anti-Atilla and DAPI. Yellow arrowheads indicate Atilla-positive lamellocytes. 1064 (b–d′) Wing imaginal discs co- stained with anti -Atilla and DAPI . (b–b′) Undamaged 1065 controls, 0/15 discs contained lamellocytes. (c–c′) R0, 0/11 discs contained lamellocytes. 1066 (d–d′) R24, 0/13 discs contained lamellocytes . (e –e′) Undamaged hop [Tum] larval 1067 hemolymph sample co-stained with anti-Atilla and DAPI. The yellow arrowhead indicates 1068 an Atilla-positive lamellocyte. (f–f′) Undamaged hop[Tum] wing imaginal disc co-stained with 1069 anti-Atilla and DAPI. Yellow arrowheads indicate Atilla -positive lamellocytes (g). 1070 Undamaged disc stained with anti -Atilla under no -wash conditions, 0/3 discs contained 1071 lamellocytes. (h) R0 disc stained with anti-Atilla under no-wash conditions, 0/3 wing discs 1072 contained lamellocytes. Scale bars: 50 µm. 1073 1074 1075 1076 1077 1078 1079 1080 61 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 62 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Supplementary Figure S5. Multiple lamellocyte reporters confirm the absence of 1081 lamellocytes in regenerating wing discs. 1082 (a-a″) An undamaged larval hemolymph sample containing circulating hemocytes 1083 expressing α-PS4-GFP (a′, a″), co -stained with anti -Atilla (a, a″) and DAPI (a″). Yellow 1084 arrowheads indicate lamellocytes. (b- b″) Undamaged larval hemolymph sample 1085 expressing MSNF9mo-GFP (b′, b″), co-stained with anti-Atilla (b, b″) and DAPI (b″). Yellow 1086 arrowheads indicate lamellocytes. ( c-d) Top-down images of wing discs from larvae 1087 expressing α-PS4-GFP. (c) Undamaged disc, 0/6 discs contained lamellocytes. (d) R0 1088 disc, 0/6 discs contained lamellocytes. (e-f) Top-down images of wing discs from larvae 1089 expressing MSNF9mo-GFP. (e) Undamaged disc 0/7discs contained lamellocytes. (f) R0 1090 disc, 0/6 discs contained lamellocytes. (g-i) Top-down images of wing discs from larvae 1091 expressing Atilla-MiET1GFP. (g) Undamaged disc, 0/9 discs contained lamellocytes. (h) 1092 R0 disc, 0/6 discs contained lamellocytes. (i) R24 disc, 0/5 discs contained lamellocytes. 1093 Scale bars: 50 µm. 1094 1095 1096 1097 1098 1099 1100 1101 63 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 64 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Supplementary Figure S6. Crystal cells are not enriched in regenerating wing discs. 1102 (a-b′) Wing discs from larvae expressing PPO1- mCherry.F6 stained with DAPI . (a-a′) 1103 Undamaged control disc (b-b′) R0 disc. Yellow arrowheads indicate PPO1-mCherry.F6-1104 positive crystal cells. (c) Quantification of PP O1-mCherry.F6-positive crystal cells in the 1105 whole wing disc. No damage n=10 discs, R0 n=11 discs, ns, P=0.2118 (d) Quantification 1106 of PPO1-mCherry.F6-positive crystal cells within the pouch region. No damage- R0 ns, 1107 P=0.1147 with the same n as in (c). (e-f′) Top-down images of Wing discs co-stained with 1108 anti-Lozenge (Lz) and DAPI in undamaged controls (e-e′) and at R0 (f-f′). (g-h′) Top-down 1109 images of wing discs from larvae expressing Lozenge-GFP (Lz-GFP) stained with DAPI. 1110 (g-g′) Undamaged control disc. (h-h′) R0 disc. Scale bars: 50 µm. Statistical significance 1111 was determined using Welch’s t-test. Error bars: SEM. 1112 1113 1114 1115 1116 1117 1118 1119 1120 65 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 66 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Supplementary Figure S7. Hemese- positive hemocytes are not enriched in 1121 regenerating wing discs. 1122 (a-c′) Top-down images of w ing discs co -stained with anti -Hemese (He) and DAPI in 1123 undamaged controls (a-a′), at R0 (b-b′), and at R24 (c-c′). Yellow arrowheads indicate He-1124 positive hemocytes. (d) Quantification of He- positive hemocytes in the whole wing disc: 1125 no damage, n = 10 discs, R0 n = 6 discs, ns, P = 0.5212, and R24 n = 5 discs, ns, P = 1126 0.6466. (e) Quantification of He-positive hemocytes within the pouch region: no damage 1127 vs. R0 ns, P = 0.9000 , and no damage vs. R24 ns, P = 0.3434, with same n as in (d). 1128 Scale bars: 50 µm. Statistical significance was determined using Welch’s t-test. Error bars 1129 represent SEM. 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 67 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 68 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Supplementary Figure S8. srpHemo reporters reveal columnar epithelial 1140 expression during regeneration. 1141 (a-a‴) Hemolymph from an undamaged larva containing circulating hemocytes expressing 1142 srpHemo-H2A::3XmCh (a′, a‴) and Lz -GFP (a″, a‴), co -stained with anti -Nimrod (a, a‴). 1143 The yellow arrow head indicates a Lz-GFP-positive crystal cell, orange arrow heads 1144 indicate srpHemo- H2A::3XmCh-positive hemocytes, and white arrow heads indicate 1145 NimC-positive plasmatocytes. (b- b‴) Hemolymph from an u ndamaged larva expressing 1146 srpHemo::3XmCh co-stained with anti -Atilla and DAPI. Blue arrow heads indicate Atilla-1147 positive lamellocytes co- expressing srpHemo::3XmCh. (c -f) Wing discs from larvae 1148 expressing srpHemo-H2A::3XmCh. (c) Undamaged control disc. (d) R0 disc. (e) R24 disc. 1149 (f) R48 disc. (g-g′) Wing disc from larva expressing srpHemo-H2A::3XmCh at R48 co-1150 stained with anti -Antennapedia (Antp). Yellow arrow head indicates srpHemo-1151 H2A::3XmCh-positive cells. (h-h′) YZ orthogonal projections of R48 wing discs from larvae 1152 expressing srpHemo-H2A::3XmCh to mark putative immune cells and rn>EYFP to mark 1153 the cell debris co-stained with anti-Discs large (Dlg) to mark cell membranes and DAPI. 1154 Yellow arrowheads indicate srpHemo-H2A::3XmCh-positive cells within the regenerating 1155 epithelium. (i-i″) R48 wing disc from larvae co-expressing srpHemo-moe::3XmCh to mark 1156 membranes of putative immune cells and stained with anti-Nubbin (Nub) to mark the wing 1157 pouch and DAPI. Yellow arrow head labels columnar epithelial pouch cells expressing 1158 both the srpHemo-moe::3XmCh reporter and Nubbin. Scale bars: 50 µm. 1159 1160 1161 69 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 70 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Supplementary Figure S9. Cell debris clearance is followed by lysosome formation 1162 during regeneration. 1163 For panels a- f’’, dotted yellow circles mark regions of LysoTracker staining. Yellow 1164 arrowhead: Notum-Hinge fold (N-H); Orange arrowhead: Hinge-Hinge fold (H-H); Purple 1165 arrowhead: Hinge-Pouch fold (H -P). (a-a″) YZ orthogonal projection of an undamaged 1166 wing disc co -stained with LysoTracker (a- a″) and DAPI (a″). (b- b″) YZ orthogonal 1167 projection of an R4 wing disc expressing rn>EYFP-labeled cell debris (b′, b″), co-stained 1168 with LysoTracker (b-b″) and DAPI (b″). (c-c″) YZ orthogonal projection of an R8 wing disc 1169 expressing rn>EYFP-labeled debris (c′, c″), co-stained with LysoTracker (c-c″) and DAPI 1170 (c″). (d-d″) YZ orthogonal projection of an R10 wing disc expressing rn>EYFP-labeled 1171 debris (d′, d″), co- stained with LysoTracker (d- d″) and DAPI (d″). (e- e″) YZ orthogonal 1172 projection of a n R12 wing disc expressing rn>EYFP-labeled debris (e′, e″), co- stained 1173 with LysoTracker (e-e″) and DAPI (e″). (f-f″) YZ orthogonal projection of an R24 wing disc 1174 expressing rn>EYFP-labeled debris (f′, f″), co- stained with LysoTracker (f -f″) and DAPI 1175 (f″). (g-g″) YZ orthogonal projection of an R48 wing disc expressing rn >EYFP-labeled 1176 debris (g′, g″), co-stained with LysoTracker (g-g″) and DAPI (g″). (h-h″) rnGAL4>UAS-rpr, 1177 UAS-CHARON-expressing wing imaginal disc at R0 stained with DAPI (h″), showing 1178 pHlorina (h-h″) and pH-CaspGFP (h′, h″) signals. 10/17 discs were positive for phlorina 1179 signal in the hinge and pouch region. Dotted yellow circles mark regions of pHlorina 1180 expression. Scale bars: 50 µm. 1181 1182 1183 71 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 72 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Supplementary Figure S10. Heterozygous draper mutants show increased debris 1184 retention and delayed lysosome dynamics during regeneration. 1185 (a-b) Undamaged w 1118 (a) and draper Δ5/+ (b) wing discs stained with anti -Draper. (c) 1186 Quantification of total Draper fluorescence intensity in w 1118 and draperΔ5/+ wing discs. 1187 w1118 n=9, and draperΔ5/+ n=6, *P=0.0259. (d) Quantification of total debris area in w 1118 1188 (grey bars) and draperΔ5/+ (yellow bars) wing discs across regeneration time points. w1118 1189 R0 n=11 discs, draperΔ5/+ R0 n=17 discs, **P=0.0014, w1118 R24 n=12 discs, draperΔ5/+ 1190 R24 n=12 discs,****P<0.0001, draperΔ5/+ R0 and draperΔ5/+ R24 ns, P=0.0537, w1118 R48 1191 n=9 discs, draperΔ5/+ R48 n=10 discs, ****P<0.0001, and draperΔ5/+ R24 and draperΔ5/+ 1192 R48 ns, P=0.0718. (e) Quantification of debris height in w1118 (grey bars) and draperΔ5/+ 1193 (yellow bars) wing discs across regeneration time points. w1118 R0 n=11, and draperΔ5/+ 1194 R0 n=17 discs, ns, P=0.6836, w 1118 R24 n=12 discs, and draperΔ5/+ R24 n=12 discs , 1195 **P=0.0031, w1118 R48 n=9 discs, and draperΔ5/+ R48 n=10 discs, ****P<0.0001. Pairwise 1196 comparisons were not significant between draper Δ5/+ R0 and draper Δ5/+ R24, ns, 1197 P=0.9222, and draperΔ5/+ R24 compared to R48, ns, P=0.4409. (f-k) w1118 and draperΔ5/+ 1198 wing discs stained with LysoTracker at R0 (f, g), R24 (h, i), and R48 (j, k). Yellow dotted 1199 circles indicate lysosome-positive regions. Notum-Hinge fold (N-H), Hinge-Hinge fold (H-1200 H), and Hinge- Pouch fold (H -P) are marked in (f). Scale bars: 50 µm. Statistical 1201 significance was determined using Welch’s t-test. Error bars represent SEM. 1202 1203 1204 1205 73 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint 74 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Supplementary Figure S11. Heterozygous draper mutants impair overall 1206 regeneration 1207 (a-c) R48 wing discs stained with anti-Nubbin to mark the regenerating pouch in w1118 (a) 1208 and draperΔ5/+ (b) discs. ( c) Quantification of total Nubbin- positive pouch area in R48 1209 w1118 wing discs, n=11 discs, and draperΔ5/+ wing discs, n=14 discs, ns, P=0.0642). Dotted 1210 yellow circles indicate the Nubbin area. Statistical significance was determined using 1211 Welch’s t-test. (d) Quantification of adult wing size following disc regeneration in w1118 and 1212 draperΔ5/+ mutants. w1118 n=302 wings, draperΔ5/+ n=986 wings, *P=0.0142. Statistical 1213 significance was determined using chi- square test. Scale bars: 50 µm. Error bars 1214 represent SEM. 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 75 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration Acknowledgments: We thank Dr. Anish Bose, Dr. Felicity Hsu, Connor Powers, 1226 Nicholas Magdadaro, Devan Bianchini, Akash Patel, and Shrunali Amin for critical reading 1227 of the manuscript. We thank Dr. Katja Brückner, Dr. Shubha Govind, Dr. Dirk Bohmann, 1228 Dr. Will Wood, Dr. Marc Freeman, and Dr. Istvan Andó for providing reagents. We 1229 acknowledge the Bloomington Drosophila Stock Center (NIH P40OD018537), the 1230 Developmental Studies Hybridoma Bank (NICHD, The University of Iowa) , the Kyoto 1231 Stock Center, and FlyBase for fly stocks, reagents, and database resources . We also 1232 thank Dr. Glenn Fried, Dr. Austin Cybersmith, Dr. Kingsley Boateng, Dr. Duncan Nall, and 1233 Dr. Umnia Doha of the Core Facilities at the Carl R. Woese Institute for Genomic Biology 1234 for assistance with microscopy and image analysis. 1235 1236 Funding: This work was supported by the National Institutes of Health (R01GM107140 1237 and R35GM141741 to R.K.S.-B.) 1238 1239 Data and Resource availability: Raw data will be posted to the Illinois Databank (a URL 1240 will be generated upon acceptance). All other relevant resources and data can be found 1241 within this article and its supplementary information. 1242 1243 1244 1245 1246 76 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted May 7, 2026. ; https://doi.org/10.64898/2026.05.04.722804doi: bioRxiv preprint Efferocytosis clears debris during regeneration

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