Calcium signaling regulates apoptosis-induced proliferation in Drosophila

Komal Panchal Suthar; Caitlin Hounsell; Yun Fan; Andreas Bergmann

doi:10.1371/journal.pbio.3003607

. 2026 Jan 20;24(1):e3003607. doi: 10.1371/journal.pbio.3003607

Calcium signaling regulates apoptosis-induced proliferation in Drosophila

Komal Panchal Suthar ¹, Caitlin Hounsell ², Yun Fan ², Andreas Bergmann ^1,^*

Editor: Nicolas Tapon³

PMCID: PMC12829959 PMID: 41557739

Abstract

Caspases, traditionally viewed as mediators of apoptosis and tumor suppressors, have also been shown to promote cell proliferation and to contribute to tumor growth. For example, the initiator caspase Dronc (the Drosophila orthologue of Caspase-9) can trigger apoptosis-induced proliferation (AiP), a process where apoptotic cells generate mitogenic signals for compensatory proliferation independently of their apoptotic function. AiP is crucial for homeostatic cell turnover, wound healing, and tissue regeneration. Previously, we established that Dronc activates the NADPH oxidase DUOX at the plasma membrane, resulting in the production of extracellular reactive oxygen species (ROS) which are required for AiP. However, the mechanism by which Dronc activates DUOX has remained elusive. Here, we identified Dronc-dependent Ca²⁺ entry into the cytosol as a significant factor for DUOX activation and AiP. Three cell surface Ca²⁺ channels of the TRP family mediate Ca²⁺ influx in a non-redundant fashion. Additionally, calcium-induced calcium release (CICR) from the ER was identified as another source of cytosolic Ca²⁺ during AiP. Notably, DUOX itself acts as a Ca²⁺ effector in AiP, requiring Ca²⁺ binding for its activation. These findings highlight the importance of Ca²⁺ signaling in AiP and provide insights into how similar signaling mechanisms might operate in vertebrates.

The initiator caspase Dronc can trigger apoptosis-induced proliferation (AiP), a specific form of compensatory proliferation. This study shows that Dronc-dependent Ca2+ influx into the cytosol is an important contributor of AiP and activation of the NADPH oxidase DUOX in Drosophila, and that this process may be conserved in vertebrates.

Introduction

Apoptosis is a physiological form of cell death that accounts for most cell death in eukaryotes [1,2] and is essential for normal development, homeostasis, and immune defence by elimination of damaged, unnecessary, or potentially harmful cells from an organism [3]. Alterations of apoptosis can result in various diseases. Apoptotic resistance can lead to uncontrolled cell proliferation such as cancer [4,5] and autoimmune diseases [6], while exaggerated apoptosis contributes to neurodegenerative disorders and immunodeficiency [7]

A specific class of Cys proteases, termed Caspases, are the main executioners of apoptosis which cleave key intracellular substrates to induce cell death [8–10]. There are two types of caspases: initiator caspases such as Caspase-9 and effector (or executioner) caspases such as Caspase-3 and Caspase-7. Initiator caspases play a crucial role in the early stages of apoptosis as they are responsible for activating downstream effector caspases, which carry out the apoptotic cell death process by cleaving a large number of intracellular substrates [3,5,8,11].

The fruit fly, Drosophila melanogaster, has emerged as a powerful model organism for studying apoptosis [3,12]. In Drosophila, apoptosis is orchestrated by homologs of mammalian caspases. Of the seven caspases in Drosophila, only Dronc, a caspase-9-like initiator caspase, and DrICE and Dcp-1, caspase-3-like effector caspases, are critical for apoptosis [12,13]. Dronc cleaves and activates DrICE and Dcp-1, which perform the final dismantling of the cell [12,13]. Furthermore, the inhibitor of apoptosis proteins (IAPs), most notably Drosophila IAP1 (DIAP1), and their antagonists, Reaper, Hid, Grim, Sickle, and Jafrac-2 regulate apoptosis in Drosophila by modulating caspase activity [14–17]. These genes ensure that apoptosis occurs only under appropriate conditions to maintain cellular homeostasis.

However, while Caspases are best-known for their role in apoptosis, they also perform several non-apoptotic functions that are crucial for normal cellular processes, for example, caspase-mediated signaling required for tissue regeneration through a process called apoptosis-induced proliferation (AiP) [9,18–21]. AiP is a particular subtype of compensatory proliferation in which apoptotic cells, instead of being passively removed, actively signal to neighboring cells to proliferate [19,22–26]. This process plays a significant role in tissue regeneration, wound healing, and cancer development [19,27,28], demonstrating that apoptosis is not solely a mechanism for cellular death but also a trigger for compensatory growth. The initiator caspase Dronc plays a key role in AiP by promoting the release of mitogens, such as Wingless (Wg), Epidermal growth factor (EGF), and Decapentaplegic (Dpp), which stimulate cell proliferation in surviving neighboring cells [29–34].

A classic model of examining AiP in Drosophila uses the expression of the baculoviral protein p35, which acts as a potent inhibitor of the effector caspases Drice and Dcp-1, but not of the initiator caspase Dronc [35–37]. If p35 expression is combined with expression of the IAP antagonist hid, “undead” cells are produced, which are immortalized and continue to produce signals for the proliferation of neighboring cells, resulting in overgrowth of the undead tissue [34]. Thus, undead cells are key experimental tools for studying AiP, as they uncouple the apoptotic and proliferative functions of the initiator caspase Dronc. In Drosophila, AiP has been studied extensively in imaginal discs—larval epithelial tissues that give rise to adult structures during metamorphosis such as eyes, wings, legs, etc. If undead cells are created in the eye antennal imaginal discs using ey-Gal4 (denoted as ey>hid,p35), adult flies are recovered, which display a range of overgrown heads (Fig 1A) [34,38].

**(See also** S1 **and** S2 Figs). Yellow dotted lines highlight the *ey-Gal4*-expressing area of the eye discs. The disc outline is marked by white dashed lines. Scale bars: 100 μm (A, C–G), 50 μm (C′–G′, C′′-G′′). **(A)** Representative examples of adult fly heads, depicting either a wild-type head or a range of overgrown phenotypes categorized from weak to moderate to severe. The severe phenotype is characterized by overgrowth of the head, including amorphic head capsule tissue (purple arrows), numerous additional bristles (up to 15) (black arrows), duplicated ocelli (yellow arrows), and often a reduction of the eye size (due to the expansion of the head capsule area into the posterior eye field during development) (red arrows). Moderate overgrowth is characterized by head enlargement with fewer extra bristles (up to 6) (black arrow), duplicated ocelli (yellow arrows), and smaller eye size (red arrow). Weak overgrowth is characterized by only mild head enlargement with one to two additional bristles (black arrow). **(B)** Quantification of the suppression of head overgrowth of adult *ey > hid,p35* flies across the indicated genetic backgrounds. Flies were scored as wild type (wt) (black bars), weak (green bars), moderate (blue bars) or severe overgrown (red bars) according to the classification in Fig 1A. n = 100 flies counted per genotype in three independent experiments. **(C)** Wild-type fly with normal head morphology. **(D)** Severely overgrown head of *ey>hid,p35* flies expressing mock (*Luciferase)* RNAi. Arrows point to amorphic tissues, additional bristles and ocelli, and reduced eye tissue. **(E, F)** Representative examples of suppressed overgrowth of *ey>hid,p35* flies expressing *UAS*-*Duox* RNAi **(E)**, or *Duox-gRNA;UAS-Cas9.P2* **(F)**. Arrows point to one or two extra bristles indicating mild overgrowth. **(G)** Representative examples of completely suppressed overgrowth of *ey>hid,p35* flies in the background of *Duox*^EFm/+. **(C′–G′)** Confocal images showing third instar larval eye imaginal discs of control (*ey-Gal4*) (C′), and *ey>hid,p35* discs expressing mock (*Luciferase)* RNAi (D′), *UAS*-*Duox* RNAi (E′), *Duox-gRNA;UAS-Cas9.P2* (F′), and *Duox*^EFm/+ (H′) labeled for ROS with dihydroethidium (DHE) dye. The yellow arrow in (I) indicates DHE-positive cells. (C″–G″) Confocal images showing hemocyte labeling using the plasmatocyte-specific anti-NimC antibody in eye imaginal discs of control (*ey-Gal4*) (C″), and *ey>hid,p35* discs expressing mock (*Luciferase*) RNAi (D″), *UAS-Duox* RNAi (E″), *Duox-gRNA;UAS-Cas9.P2* (F″), and *Duox*^EFm/+ (H″). In *ey-Gal4* control discs, hemocytes are present as cell aggregates in the antennal portion of the disc and posterior to the morphogenetic furrow at the eye disc (C″). At undead *ey>hid,p35* discs expressing mock (*luciferase*) RNAi, an increased number of hemocytes is present in overgrown areas (yellow arrows) (D″). This increased number of the hemocytes at undead eye discs is strongly suppressed by expressing *UAS*-*Duox* RNAi, *Duox-gRNA;UAS-Cas9.P2* and *Duox*^EFm/+ (E″–G″). **(H)** Quantification of DHE fluorescence in (C′–G′). Data from n = 9 (*ey-Gal4*), 10 (*mock* RNAi), 11 (*UAS*-*Duox* RNAi), 7 (*Duox-gRNA;UAS-Cas9),* and 16 (*Duox*^EFm/+) discs were analyzed in three independent experiments. A.U.—arbitrary units. **(I)** Quantification of the number of hemocytes per disc in (C″-G″). Data from n = 13 (*ey-Gal4*), 10 (*mock* RNAi), 13 (*UAS*-*Duox* RNAi), 13 (*Duox-gRNA;UAS-Cas9),* and 25 (*Duox*^EFm/+) discs were analyzed in three independent experiments. Levels of significance are depicted by asterisks in the figures: ****p < 0.0001. The data underlying the graphs shown in this figure can be found in S1 Data.

Genetic screening in Drosophila for suppressors of ey>hid,p35-induced overgrowth identified several genes that regulate AiP. This work revealed a number of key signaling events for AiP to occur. First, Dronc is transported to the basal side of the plasma membrane by Myo1D, an unconventional class 1 myosin, and LimK1, a stabilizer of actin filaments [39,40]. At the membrane, Dronc either directly or indirectly activates the NADPH oxidase DUOX, leading to the production of extracellular reactive oxygen species (ROS) [38]. ROS attract and activate macrophage-like immune cells termed hemocytes which release signaling factors, including the TNF homolog Eiger to stimulate JNK signaling in undead cells [38,41,42]. JNK then drives the expression of endogenous hid and reaper genes, creating an amplification loop that sustains AiP signaling in undead cells [38,43]. Additionally, JNK induces expression of the mitogenic factors Wg, EGF, and Dpp, which promote the proliferation of surviving neighboring cells [29,30,44].

Calcium ions (Ca²⁺) function as versatile intracellular secondary messengers in a wide range of physiological processes [45–49]. While Ca²⁺ signaling has been best studied in excitable cells such as muscles and neurons, it is also essential in non-excitable cells such as epithelial cells [50–57] where it controls numerous physiological processes such as cell proliferation, differentiation, cellular migration, barrier function, secretion, immune responses, wound healing, and apoptosis [58–62]. The range of Ca²⁺ signaling in epithelial cells and its role in the regulation of epithelial characteristics are poorly understood.

Cytosolic Ca²⁺ levels are tightly regulated by Ca²⁺ channels, pumps, and exchangers located in the plasma membrane and intracellular organelles such as the endoplasmic reticulum (ER) and mitochondria [63,64]. Upon receiving external or internal stimuli, Ca²⁺ can be released into the cytosol, where it binds to target proteins and activates effector pathways [64]. Often, there is interplay between extracellular and intracellular Ca²⁺ sources to ensure precise control over cytosolic Ca²⁺ levels. For example, calcium-induced calcium release (CICR) from the ER amplifies the initial elevations of intracellular Ca²⁺ levels from the plasma membrane [65,66], and contributes to flashes and waves of Ca²⁺ activity [67]. Ca²⁺ flashes are a distinctive feature of epithelial Ca²⁺ signaling and are involved in processes such as gene expression, proliferation, and wound healing where Ca²⁺ waves propagate across epithelial layers to coordinate cell migration and tissue repair [57,59,68]. These flashes result from the interplay between Ca²⁺ influx, release, and re-uptake mechanisms that generate temporal Ca²⁺ patterns [69,70].

A key unresolved question in AiP research is the mechanism of DUOX activation in undead cells. DUOX contains two canonical Ca²⁺ binding EF-hand motifs on an intracellular loop, where cytosolic Ca²⁺ binding is essential for DUOX activation in both mammals and Drosophila [68,71–73]. For example, during embryonic wound repair in Drosophila, Ca²⁺ influx mediates activation of DUOX [68]. Furthermore, upon bacterial infection in the Drosophila intestine, Ca²⁺ binding activates DUOX for ROS generation, which serves as an antibacterial response [74,75]. Moreover, in zebrafish, tissue injury triggers early recruitment of leukocytes to the wound and it also induces an inflammatory response through activation of the NF-κB signaling pathway via activation of DUOX1, which induces the production of H₂O₂ and modulates the in vivo inflammatory response [76].

Here, we investigated the role of Ca²⁺ signaling for DUOX activation in AiP regulation. Our studies revealed that mutation of the EF-hand motifs of DUOX abolished ROS production and blocked AiP. Consistent with these findings, we detected Dronc-dependent cytosolic Ca²⁺ signaling in undead cells. Rather than maintaining steady-state levels, Ca²⁺ signaling in undead tissue occurred as flashes. We identified two distinct sources of Ca²⁺ influx: extracellular Ca²⁺ entry through three TRP family Ca²⁺ channels in a non-redundant manner, and CICR from the ER through the Ryanodine Receptor (RyR). The non-redundant roles of TRP channels in Ca²⁺ influx, RyR-mediated CICR, and DUOX’s Ca²⁺-dependent ROS production exemplify a complex yet robust system that enables cells to respond effectively to damage and stress. These findings provide valuable insights into conserved Ca²⁺ signaling pathways and their potential as therapeutic targets in diseases involving impaired tissue repair or dysregulated proliferation.

Results

A Duox mutant unable to bind Ca²⁺ suppresses all AiP phenotypes

Co-expression of hid and p35 under ey-Gal4 control (ey>hid,p35) generates a range of AiP-induced overgrowth phenotypes in the undead head capsule, categorized as weak, moderate, or severe (Fig 1A). Quantitative analysis reveals that the majority of ey>hid,p35 flies (79%) display severe overgrowth, while the remaining 21% show moderate overgrowth phenotypes (Fig 1B).

Previously, we demonstrated that Duox knockdown by RNA interference (RNAi) moderately suppresses the AiP-induced overgrowth phenotype of ey>hid,p35 animals [38] (Fig 1B–1E). To further validate DUOX’s requirement for AiP, we performed tissue-specific CRISPR/Cas9-mediated targeting of Duox in eye-antennal discs of ey>hid,p35 animals, expressing UAS-Cas9 under ey-Gal4 control. This approach similarly resulted in moderately strong suppression of ey>hid,p35-induced overgrowth (Fig 1B and 1F).

To specifically examine the role of Ca²⁺ binding in DUOX regulation, we targeted two highly conserved glutamate residues, Glu879 and Glu915, which occupy the canonical “Z” position within adjacent EF-hand motifs. In EF-hand Ca²⁺-binding loops, this terminal glutamate provides critical bidentate coordination of Ca²⁺, and substitution with glutamine is known to markedly reduce or abolish Ca²⁺ affinity without disrupting overall protein folding [77,78]. Guided by this precedent, we replaced Glu879 and Glu915 with glutamine (E879Q, E915Q), thereby generating a mutant allele of endogenous Duox, referred to as Duox^EFm, that is predicted to selectively disrupt Ca²⁺ chelation while preserving protein integrity. Heterozygously, Duox^EFm/+ suppressed ey>hid,p35-induced overgrowth to a similar extent as Duox RNAi or CRISPR/Cas9-mediated targeting of Duox (Fig 1B and 1G). The ability of Duox^EFm/+ to dominantly suppress the undead overgrowth of ey>hid,p35 animals indicates that intact EF-hand motifs, and thus Ca²⁺-dependent activation of DUOX, are essential for its function in undead AiP signaling.

Consistently, Duox^EFm/+ suppressed multiple AiP markers in ey>hid,p35 background. First, ROS production, the primary function of DUOX and a characteristic marker of undead signaling [38], was reduced to levels comparable to Duox RNAi treatment (Fig 1C′–1G′; quantified in Fig 1H). Second, hemocyte recruitment was similarly decreased, matching the reduction seen with Duox RNAi (Fig 1C″–1G″; quantified in Fig 1I). Finally, similar to expression of Duox RNAi, Duox^EFm/+ in the ey>hid,p35 background suppressed both JNK pathway activation, as measured by the marker MMP1, and Wg expression (S1 Fig).

To further probe the requirement of the EF-hand motifs, we expressed a Duox transgene that lacks these motifs (UAS-Duox^ΔEF) in ey>hid,p35 background. Like Duox^EFm, expression of UAS-Duox^ΔEF suppressed ey>hid,p35-induced overgrowth (S2A and S2B Fig), suggesting that it acts as a dominant-negative mutant. Similarly, expression of UAS-Duox^ΔEF reduced ROS production, decreased hemocyte recruitment, and suppressed both JNK activation (MMP1) and Wg expression (S2 Fig).

Together, these results demonstrate that the EF-hand motifs of DUOX are essential for both ROS generation and for subsequent ROS-dependent events in undead AiP signaling.

Release of Ca²⁺ during AiP in a Dronc-dependent manner

Because the only known function of EF-hand motifs is Ca²⁺ binding [72,73], we investigated the involvement of Ca²⁺ in AiP signaling using the cytosolic Ca²⁺ reporter GCaMP6s [79]. Control eye imaginal discs (ey-Gal4 and ey>p35) showed very low GCaMP6s activity anterior to the morphogenetic furrow where ey-Gal4 is expressed (Fig 2A and 2B; quantified in Fig 2D). In contrast, undead (ey>hid,p35) eye imaginal discs displayed very strong GCaMP6s activity in this region (Fig 2C, white arrows; quantified in Fig 2D), indicating significantly increased Ca²⁺ signaling. Time-lapse imaging revealed highly dynamic GCaMP6s activity in undead discs (S1–S3 Movies). Moreover, we detected irregular Ca²⁺ flashes in undead discs that were absent in controls (Fig 2E–2G; quantified in Fig 2H and S1–S3 Movies). These flashes varied between discs, persisted for seconds (Fig 2G and S3 Movie; for complete individual recordings of the Ca²⁺ traces see S3 Fig), and were restricted to the undead (ey-Gal4-expressing) domain. Within this domain, however, the flashes occurred without an obvious spatial pattern. Together, these data demonstrate that Ca²⁺ signaling in undead (ey>hid,p35) eye imaginal discs occurs as dynamic oscillations.

Importantly, Ca²⁺ signaling in undead tissue depends on the initiator caspase Dronc. RNAi targeting Dronc suppressed both the overall GCaMP6s fluorescence of undead (ey>hid,p35) discs (Fig 2I and 2J; quantified in Fig 2K) and the Ca²⁺ flashes of undead (ey>hid,p35) discs (Fig 2L and 2M; quantified in Fig 2N and S4–S5 Movies and S3 Fig). These data demonstrate that Ca²⁺ release and its flashes are direct consequences of hid,p35-induced undead signaling.

Identification and characterization of Ca²⁺ transporters involved in AiP

Next, we sought to identify the Ca²⁺ channel(s) that mediate cytosolic Ca²⁺ influx. Since Dronc localizes to the plasma membrane in undead cells [39,80], where it might directly or indirectly control Ca²⁺ influx, we focused on channels that transport Ca²⁺ across the plasma membrane. Transient receptor potential (TRP) channels represent one major class of plasma membrane Ca²⁺ transporters that respond to various extra- and intracellular signals potentially generated by undead cells [81,82]. Among the 13 TRP channels encoded in the D. melanogaster genome, RNAi screening identified three of them (TrpM, TrpA1, Pkd2) as moderately strong suppressors of undead overgrowth (Figs 3A, 3B, and S4A). Their suppression levels matched those observed with Duox inactivation or the Duox^EFm mutant (compare Fig 1B to Fig 3B). We validated these findings using CRISPR/Cas9-mediated gene inactivation for TrpM and TrpA1, and additionally confirmed the role of TrpA1 using a null mutant allele (TrpA1^ins) (S5A and S5B Fig).

Consistent with the function of these TRP channels as Ca²⁺ transporters, inactivation of TrpM, TrpA1, and Pkd2 in undead (ey>hid,p35) background blocked the GCaMP6s signal in eye imaginal discs (Fig 3C–3F; quantified in Fig 3S). Notably, inactivating any single channel alone strongly abolishes GCaMP6s signaling, suggesting that these channels operate in an interdependent non-redundant manner. Furthermore, RNAi targeting any of these three TRP channels effectively suppressed the Ca²⁺ flashes typically observed in undead discs (Fig 3G–3J; quantified in Fig 3T and S4 and S6–S8 Movies; for complete individual recordings of the Ca²⁺ traces see S6 Fig).

A key potential function of Ca²⁺ signaling in undead cells is the activation of DUOX through binding to the EF-hand motifs, leading to ROS generation. Using dihydroethidium (DHE) as a ROS indicator, we found that RNAi of either TrpM, TrpA1, or Pkd2 channels significantly impacted ROS generation in undead (ey>hid,p35) imaginal discs (Fig 3K–3N; quantified in Fig 3U). Consistently also, given that DUOX-generated ROS are essential for hemocyte recruitment to undead discs [38,41], inactivation of either TrpM, TrpA1, or Pkd2 substantially prevented the recruitment of hemocytes to the undead disc (Fig 3O–3R; quantified in Fig 3V). Furthermore, knockdown of TrpM, TrpA1, and Pkd2 in undead cells significantly suppressed both JNK activity and Wg expression (S4B–S4K Fig).

Additionally, the TrpA1 null mutant allele, TrpA1^ins, dominantly suppressed the GCaMP6s signal, and Ca²⁺ flashes in undead (ey>hid,p35) eye imaginal discs (S5C–S5H and S6F–S6G Figs and S4 and S10 Movies). Consistently, TrpA1^ins/+ suppressed all AiP markers including ROS levels, hemocyte recruitment, JNK activity, and Wg expression in undead eye imaginal discs (S5I–S5T Fig).

These findings demonstrate that disrupting Ca²⁺ signaling produces phenotypes identical to those observed with Duox^EFm and UAS-Duox^ΔEF expression in undead discs, suggesting that a primary function of Ca²⁺ is to activate DUOX through binding to its EF-hand motifs, thereby enabling ROS generation.

Identification and characterization of RyR as intracellular Ca²⁺ channel

CICR from intracellular stores, particularly the ER, serves as an essential mechanism for amplifying calcium signals in many cell types [65,66]. This process is initiated when low levels of Ca²⁺ bind to and activate the RyR at the ER membrane, triggering the release of additional Ca²⁺ from ER stores [83]. Through CICR, cells can rapidly amplify Ca²⁺ signals and extend their reach to distal regions within large cells, including muscle fibers, neurons, and also in epithelial cells. RyR-mediated Ca²⁺ release typically manifests as transient Ca²⁺ flashes [84]. Because we observed a similar Ca²⁺ dynamics in undead cells (S3 and S4 Movies), we investigated the role of the single Drosophila RyR gene in Ca²⁺ signaling within undead cells.

RyR RNAi moderately suppressed the undead overgrowth of ey>hid,p35 animals (Fig 4A and 4B). We also observed a significant reduction in GCaMP6s signaling (Fig 4C and 4D; quantified in Fig 4E) as well as Ca²⁺ flashes by RyR RNAi in ey>hid,p35 imaginal discs (Fig 4F and 4G; quantified in Fig 4H and S6 Fig and S4 and S9 Movies).

The reduction in Ca² ⁺ signaling by RyR RNAi has cascading effects on downstream processes. RyR knockdown strongly suppressed ROS generation in undead discs (Fig 4I and 4J; quantified in Fig 4K), consequently preventing hemocyte recruitment to these discs (Fig 4L and 4M; quantified in Fig 4N). Moreover, loss of RyR abolished both JNK and Wg signaling, key components of the AiP network (S7 Fig). Collectively, these findings demonstrate that RyR and CICR are essential for proper calcium signaling in undead tissue during AiP.

To further examine the functional roles of these Ca²⁺ channels in undead tissue, we determined whether they are transcriptionally regulated by measuring their mRNA expression using qRT-PCR (Fig 4O). Relative to GAPDH normalization, TrpM, TrpA1, and RyR transcripts were significantly upregulated by 1.7, 1.8, and 3.6 fold, respectively, in undead discs (ey>hid,p35) compared to both control conditions (ey-Gal4 and ey>p35) (Fig 4O). Among these, RyR exhibited the strongest induction, consistent with its functional requirement in sustaining AiP-associated signaling. These transcriptional changes support our genetic findings, demonstrating that the upregulation of these Ca²⁺ channels coincides with their critical role in mediating Ca²⁺ influx, DUOX activation, and downstream AiP responses.

TrpA1 and RyR contribute to regeneration in the DE^ts>hid “genuine” AiP model

Given that TrpM, TrpA1, Pkd2, and RyR are essential for Ca² ⁺ influx and DUOX activation in the undead AiP model (Figs 3 and 4), we tested whether these channels are also involved in “genuine” (P35-independent) regeneration using the DE^ts>hid model [34]. In this system, hid expression is spatially restricted to the dorsal eye disc by Dorsal Eye-Gal4 (DE-Gal4) [85] and temporally controlled by Gal80^ts [86] through a transient 12-hour temperature shift to 30 °C (Fig 5A). This model co-induces GFP to label hid-expressing cells. Compared to controls, 12-hour hid expression causes strong apoptosis (Fig 5C) and tissue loss [34]. However, 72 hours post-temperature shift (R72h), discs have fully recovered their shape and normal photoreceptor pattern as evaluated by ELAV labeling (Fig 5D). This recovery results from increased proliferation in the dorsal eye disc [34].

Fig 5 — **(See also** S8 Fig). (A) Experimental outline of the ablation/regeneration protocol. Crosses were incubated at 18 °C until 2nd larval instar. Tissue ablation was induced by *hid* expression through temperature shift to 30 °C for 12 hours. Subsequently, larvae were returned to 18 °C and allowed to recover for either 24 h (R24h) or 72 h (R72h) before dissection of imaginal discs. **(B)** Control DE^ts>GFP disc at R24h. Application of the experimental protocol in (A) induces GFP expression (green) in the dorsal half of the eye imaginal disc **(B)**, but does not trigger caspase activation as visualized by cDcp1 labeling (red in B, gray in B’). Scale bar: 100 μm. **(C)** DE^ts>*hid* disc at R24h. Application of the experimental protocol in (A) triggers extensive caspase activity (cDcp1; red in C, gray in C′) in the dorsal half of the eye imaginal disc (arrow in C′). **(D)** DE^ts>*hid* disc at R72h. The photoreceptor pattern as visualized by ELAV labeling (red in D, gray in D′) develops normally despite strong induction of apoptosis earlier in development according to the protocol in **(A)**. Only 2 out of 20 DE^ts>*hid* discs showed incomplete regeneration. Scale bar: 50 μm. **(E–G)** DE^ts>*hid* discs at R72h expressing *UAS*-*TrpA1* RNAi (E), TrpA1ins/+ (F), and UAS-RyR RNAi (G). A high percentage of the DE^ts>*hid* discs expressing *UAS*-*TrpA1* RNAi (n = 16 out of 20), *TrpA1*^ins (n = 17 out of 20), and *UAS*-*RyR* RNAi (n = 15 out of 20) do not completely recover after 72 h. Arrows in the prime panels highlight incomplete ELAV patterns in the dorsal half of the disc (compare to the ventral half which was not subject to *hid* expression), indicating that the regeneration response was partially impaired by reduction of Ca²⁺ activity. **(H)** Quantification of dorsal-to-ventral area ratios revealed a significant reduction in experimental genotypes (*TrpA1* RNAi/*TrpA1*^ins/*RyR* RNAi) compared to controls. Data were analyzed using one-way ANOVA with Tukey’s multiple comparisons test in GraphPad Prism. Results are shown as mean ± SEM. ****p < 0.0001. The data underlying the graph shown in this panel can be found in S1 Data.

In contrast, RNAi-mediated knockdown of TrpA1 or RyR during the apoptosis-inducing phase impaired regeneration, resulting in only partial recovery of disc structure with defective photoreceptor patterning as revealed by ELAV staining (Fig 5E and 5G; quantified in Fig 5H). The relatively modest effect likely reflects the limited 12-hour RNAi activity during the 30 °C pulse, with channel function restored shortly upon return to 18 °C. Nevertheless, DE^ts>hid discs in a heterozygous TrpA1^ins mutant background also showed incomplete regeneration, confirming the requirement for TrpA1 function (Fig 5F; quantified in Fig 5H). Knockdown of these receptors alone using DE-Gal4 does not affect photoreceptor patterning (S8A–S8D Fig). By contrast, TrpM and Pkd2 knockdown did not affect DE^ts>hid-induced regeneration (S8E and S8F Fig).

Together, these findings demonstrate that TrpA1 and RyR play significant roles in tissue regeneration following DE^ts>hid-induced ablation. Furthermore, these data highlight the utility of the simpler “undead” AiP model as a genetic screening platform to identify regulators with broader roles in genuine regenerative responses.

Discussion

Calcium (Ca²⁺) serves as a dynamic and versatile messenger system in excitable cells, such as neurons and muscles as well as in non-excitable cells, including epithelial cells. In the latter, Ca²⁺ signaling modulates essential processes from stem cell proliferation to immune defence and tissue repair [68,87–89]. Here, we showed that Ca² ⁺ signaling also has a crucial role in AiP. A Duox mutant lacking essential Glu residues in the EF-hand motifs (Duox^EFm) displayed reduced ROS levels and suppressed all AiP markers. Because the only known function of the EF-hand motif is Ca²⁺ binding [72,73], it is very likely that they mediate the activation of Duox by Ca²⁺ for the production of ROS during AiP.

Similarly, a previous study on wound repair in Drosophila embryos demonstrated that Ca²⁺ is necessary for DUOX activation and ROS generation [68]. In both cases, wound repair and AiP, the DUOX-generated ROS are needed to recruit hemocytes to wound sites to aid in tissue regeneration [38,68]. Together, these findings illustrate the essential role of Ca²⁺-activated DUOX across cell types and biological processes, highlighting its capacity to drive cellular responses to damage and stress by modulating ROS production and downstream repair mechanisms.

As source(s) of cytosolic Ca²⁺, we identified three specific TRP channels (TrpM, TrpA1, and Pkd2) as well as RyR-mediated CICR which are critical for maintaining Ca²⁺ levels in undead cells, where they are necessary for DUOX activation and ROS generation. Interestingly, the function of these TRP channels in AiP is non-redundant, as inactivating any one channel disrupts Ca²⁺ signaling, blocking ROS production and AiP.

The observation that TrpM, TrpA1, and Pkd2 each appear strictly required for Ca²⁺ signaling in undead discs was unexpected, and suggests that these channels act in a highly interdependent manner. Importantly, TrpM, TrpA1, and Pkd2 belong to different sub-families of the TRP channels with unique structural and functional properties [90], indicating that their requirement may be non-redundant due to unique contributions from each channel sub-family. For example, each channel may respond to distinct inputs generated in undead tissue, such as redox cues, mechanical stress, or metabolic changes, such that all inputs must converge to reach the threshold for CICR and DUOX activation. Another possibility is that they form a functional complex or signaling network in which the activity of each component is required to sustain Ca²⁺ entry. Therefore, disruption of a single channel may destabilize this network, leading to a collapse of Ca²⁺ signaling. Alternatively, the channels may operate in a sequential or feedback-dependent manner, or each may contribute only part of the total Ca²⁺ influx necessary to reach the threshold for DUOX activation. Removing one channel may reduce Ca²⁺ entry below this threshold, which would effectively shut down the cascade. Finally, activation of the RyR could depend on the combined input from all TRP channels to initiate CICR and generate Ca²⁺ flashes.

These possibilities indicate that the TRP channels create a robust but fragile signaling system that collapses when any single component is lost. Such interdependence may ensure that AiP is tightly regulated and triggered only under specific conditions, thereby preventing inappropriate activation.

Although the precise mechanism in undead discs remains to be elucidated, similar cases of non-redundant TRP channel function have been described in other systems, and our findings add to this emerging concept. In vertebrates including humans, TRP channels can play similarly specialized non-redundant roles. For example, TrpV4, TrpC7, and several TrpM family members (TrpM2, TrpM4, TrpM8) are upregulated in ovarian cancer, where their expression negatively correlates with patient prognosis [91]. In the trabecular meshwork of the eye, TrpM4 and TrpV4 cooperate to regulate intraocular pressure [92], while in mouse osteoblasts, combined regulation of Ca²⁺ influx by TrpM3 and TrpV4 controls bone remodeling [93]. Together, these examples illustrate that functional division of labor within the TRP family is evolutionarily conserved. The cooperative activation of multiple TRP channels may thus provide both specificity and robustness to Ca²⁺ signaling in complex tissues, ensuring that cells respond appropriately to diverse internal and external cues. [82,90,94–97]

This diversity of TRP function may particularly be beneficial for cells in regenerative contexts, as previously shown in the wound healing in embryos, where TrpM and possibly TrpA1 channels regulate Ca²⁺ influx to control wound repair in response to damage [68]. Thus, TRP channels collectively support dynamic and adaptive Ca²⁺ signaling, essential for both AiP and broader tissue repair mechanisms.

While our data show that Dronc is required for Ca²⁺ release through TRP channels (Fig 2I–2N), the underlying mechanism remains unknown. A direct activation seems unlikely, as caspase-mediated cleavage typically inactivates rather than activates proteins. It therefore remains to be determined whether Dronc acts through its proteolytic activity or via non-proteolytic mechanisms such as scaffolding, binding interactions, or indirect signaling pathways that modulate TRP channel function. Defining this mechanistic link represents an important direction for future research.

CICR, mediated by RyR channels, amplifies Ca²⁺ signaling in cells, a mechanism essential for rapid and sustained cellular responses in vertebrate muscle and neuronal cells [83,98]. We showed here that the RyR channel facilitates CICR in non-excitable cells, amplifying TRP-mediated Ca²⁺ entry to produce Ca²⁺ flashes that drive prolonged AiP signaling. RNAi targeting RyR in undead cells effectively suppresses these Ca²⁺ flashes, disrupting ROS production and hemocyte recruitment, underscoring the essential role of RyR in sustaining Ca²⁺-dependent regenerative signaling.

To compare the roles of the TRP and RyR channels across different contexts of AiP, we also tested their requirement in a genuine AiP model (DE^ts>hid) (Fig 5). Interestingly, while both the undead and genuine AiP paradigms share a core dependence on TrpA1 and RyR, the other two TRP channels identified in this study, TrpM and Pkd2, are required only in undead AiP. The additional involvement of TrpM and Pkd2 in undead AiP likely reflects the chronic nature of this system, where sustained caspase activity and prolonged stress signaling necessitate broader channel engagement to maintain elevated cytosolic Ca²⁺ levels. By contrast, genuine AiP represents a transient and self-limiting regenerative response that relies primarily on the TrpA1-RyR axis to generate a short but sufficient Ca²⁺ signal. Despite these differences, the overall mechanism linking Ca²⁺ signaling and undead/regenerative proliferation appears largely conserved between the two AiP models.

Our data collectively support a signaling model involving several key steps (Fig 6). The roles of TRP channels, RyR, and DUOX in AiP and tissue repair highlight a complex but integrated system of Ca²⁺ signaling that supports regenerative processes across diverse biological contexts. In undead cells, DUOX relies on Ca²⁺ binding to produce ROS, which activates downstream pathways essential for AiP. TRP channels ensure sustained calcium influx, with each channel offering a unique response to environmental stimuli, thus providing a non-redundant mechanism for Ca²⁺-dependent signaling. The RyR amplifies these signals via CICR, generating Ca²⁺ flashes that prolong and stabilize the regenerative response.

Apoptosis, AiP, and Ca²⁺ signaling are interconnected processes that play essential roles in development, homeostasis, and regeneration. AiP challenges the traditional view of cell death as a pure destructive process by demonstrating how apoptotic cells can promote tissue regeneration. Ca²⁺ signaling further complicates this picture by acting as both a promoter of apoptosis and a regulator of survival and proliferation. Understanding how these processes are coordinated in Drosophila offers valuable insights that are relevant to human health, including cancer biology, regenerative medicine, and neurodegenerative diseases. By understanding the interplay between apoptosis and Ca²⁺ signaling in the context of AiP, we can uncover new strategies for therapeutic interventions that promote regeneration while preventing unwanted cell death.

Materials and methods

Fly stocks and genetics

The following transgenic and mutant stocks were used: ey-Gal4, ey>p35 (exact genotype: ey-Gal4 UAS-p35/CyO), ey>hid,p35 (UAS-hid; ey-Gal4 UAS-p35/CyO,tub-Gal80) [34]; UAS-Duox RNAi (#44), UAS-Duox^ΔEF (gifted by Won-Jae Lee, Seoul National University, South Korea) [89], TrpA1^ins (gifted from Dr. Paul A. Garrity, Brandeis University, USA) [99], DE-Gal4; tub-Gal80^ts [85]. The following strains were obtained from the Bloomington Drosophila Stock Center: UAS-Luciferase RNAi (BL#31603), Duox-gRNA (BL#77305), UAS-Cas9.P2 (BL#58986), 20XUAS-IVS-GCaMP6s (Chrm II, BL#42746) (Chrm III, BL#42749) [79], UAS-TrpM RNAi (BL#35581), UAS-TrpA1 RNAi (BL#31504), UAS-Pkd2 RNAi (BL#51502), UAS-RyR RNAi (BL#28919), UAS-Trpl RNAi (BL#26722), UAS-Pain RNAi (BL#61299), UAS-TRP RNAi (BL#31650), UAS-Trpγ RNAi (BL#31298), UAS pyx RNAi (BL#31297), UAS-wtrw RNAi (BL#31292), UAS-Nompc RNAi (BL#31689), UAS-iav RNAi (BL#25865), UAS-nan RNAi (BL#31295), UAS-Trpml RNAi (BL#602188). The following strain was obtained from the Vienna Drosophila Resource Center (VDRC): UAS-Dronc RNAi (v100424).

The Duox^EFm mutant (this study) changes two invariant Glu residues in the EF hand motifs of DUOX to Gln (E879Q and E915Q) which disrupt coordination of Ca²⁺. It was generated by CRISPR/Cas9-mediated homology-directed repair (HDR) by WellGenetics (Taiwan) in the endogenous Duox gene. A single guide RNA (gRNA) was designed to target exon 7 of Duox near the desired mutation sites. The donor construct consisted of ~1 kb upstream and downstream homology arms flanking two engineered substitutions (E879Q and E915Q), encoded by GAG to CAG and GAA to CAA nucleotide changes, respectively. To facilitate genetic screening, the donor plasmid also carried a PBacDsRed cassette containing a 3xP3-DsRed selection marker, flanked by piggyBac terminal repeats, allowing marker excision by piggyBac transposase. Silent PAM mutations were incorporated into the donor to prevent re-cutting by Cas9. The construct was injected into w¹¹¹⁸ embryos expressing Cas9, and transgenic progeny were identified by DsRed eye fluorescence. Correctly edited alleles were validated by PCR amplification and Sanger sequencing across the targeted region. After marker excision, sequencing confirmed the presence of both E879Q and E915Q mutations in the endogenous Duox locus. The Duox^EFm mutant is homozygous lethal.

Flies were reared on standard cornmeal-molasses medium unless noted otherwise. Crossed flies were transferred into fresh food vials every 2 days. The conditional knockdown of different genes was achieved with the UAS-GAL4 system [100]. The ey>hid,p35 stock was crossed to UAS-luciferase RNAi as control. For CRISPR/Cas9 crosses, the gRNA and UAS-Cas9 transgenes were first crossed together, before crossing them with ey>hid,p35.

Fly head phenotype screening and quantification

Adult fly heads were imaged using a ZEISS SteREO Discovery.V8 microscope.

All ey>hid,p35 animals exhibited head overgrowth phenotypes of varying severity. We classified overgrowth into three categories based on the following criteria: severe cases are characterized by amorphic head cuticle tissue (purple arrows, Fig 1A), numerous additional bristles (up to 15) (black arrows, Fig 1A), extra ocelli (yellow arrows, Fig 1A), and loss of eye tissue (red arrows, Fig 1A). Moderate cases displayed head enlargement with fewer additional bristles (up to 6), duplicated ocelli, and smaller eye size. Weak cases showed mild head enlargement with one to two additional bristles (Fig 1A).

We scored offspring of the RNAi crosses with ey>hid,p35 for suppression of head capsule overgrowth using the criteria described above. The results of the head phenotype screening are presented as the percentage of adult flies displaying wild-type morphology (black bar), weak (green bar), moderate (blue bar), and severe (red bar) overgrowth (see Fig 1B for example).

ROS staining

ROS staining was performed using DHE dye following a published protocol by [41]. Briefly, unfixed eye-antennal imaginal discs from third instar larvae were dissected in fresh Drosophila Schneider’s medium (Thermofisher Scientific #21720024), and incubated in DHE solution (Invitrogen #D23107, final concentration 30 μM) for 5 min. Following staining with DHE, eye discs were washed 3X in 1X PBS and subsequently mounted in Vectashield mounting media. Imaging was done immediately using a Zeiss LSM700 confocal microscope.

Immunofluorescence labeling

Immunofluorescence labeling of eye imaginal discs was performed by following standard protocols [38]. Briefly, eye-antennal imaginal discs were dissected from third instar larvae in cold 1X PBS, then fixed with 4% paraformaldehyde (PFA) for 30 min at RT, rinsed three times in 1X PBS with 0.3% Triton X-100 for 5 min, blocked with Normal Donkey Serum, and stained with primary antibodies overnight at 4 °C. The following primary antibodies were used: mouse anti-NimC (1:100, P1a,P1b; a kind gift from István Andó) [101]; mouse anti-MMP1 (1:50, 3A6B4), rat anti-ELAV (1:50, 7E8A10), mouse anti- Wg (1:200, 4D4) (all from the Developmental Studies Hybridoma Bank (DSHB)), and rabbit anti-cDcp1 (1:500, Cell Signaling Technology #9578).

After incubation with the primary antibodies, imaginal discs were washed three times in 0.3% PBST and incubated with secondary antibodies and Hoechst 33342 (1:1,000, Cat#3570, Invitrogen) in PBST for 2.5 hr in the dark at RT. Secondary antibodies were anti-mouse IgGs conjugated to Alexa488 and anti-rat IgGs conjugated to Alexa647 (used at 1:20 and 1:30 dilution, respectively, Molecular Probes). Eye discs were counter-labeled with the nuclear dye Hoechst 33342 solution to visualize tissue outline. Discs were then washed 3× in PBST, followed by two times in 1× PBS and mounted in Vectashield.

Images of eye imaginal discs were captured either using a Zeiss LSM700 or a Nikon Eclipse Ti2 confocal microscope. A Z-stack of 20–40 images covering the eye imaginal discs was acquired and shown in maximum intensity projection.

Imaging cytosolic Ca²⁺ with GCaMP6s reporters

Cytosolic Ca²⁺ in third instar larval eye imaginal disc was monitored ex vivo using UAS-GCaMP6s as a Ca²⁺ marker [79]. UAS-GCaMP6s was crossed into the experimental background for imaging. Eye imaginal discs were dissected and handled in 1× External Saline Solution (ESS), pH 7.2 (1.2M NaCl, 0.04M MgCl₂.6H₂O, 0.03M KCl, 0.10M NaHCO₃, 0.10M Glucose, 0.10M Sucrose, 0.10M Trehalose, 0.05M TES, 0.10M HEPES, 1.5 mM CaCl₂) (gifted by Dr. Yang Xiang) at RT, and captured with a Zeiss LSM700 confocal microscope. To quantify eye imaginal discs with high Ca²⁺ levels in ey-Gal4-expressing regions, Z-stack images were acquired and shown in Z-max projection (Fig 2A–2C). For the recording of Ca²⁺ flashes, eye imaginal discs were dissected in 1X ESS and immersed in ESS for time lapse video using a Zeiss LSM700 confocal microscope. One layer of eye imaginal disc was recorded every 1s for 10 min resulting in 600 frames.

Quantification of Ca²⁺ flashes

Ca²⁺ flashes were manually quantified from time-lapse GCaMP6s recordings consisting of 600 frames per disc (10-min duration). For each recording, fluorescence intensity was examined across the entire movie. Ca²⁺ flashes were defined as transient, sharp increases in GCaMP6s fluorescence that was clearly distinguishable from background fluctuations. These events manifest as brief, high-amplitude peaks in intensity traces and short-lived bright spots in the imaging field. Due to variability in spontaneous flash amplitudes across discs, no fixed intensity threshold was applied. Instead, flashes were identified based on morphological criteria: rapid rise in fluorescence, short duration, and return to baseline. Minor fluctuations or slow drifts in signal were excluded. The total number of visually identifiable flashes was recorded for each disc.

Tissue ablation and recovery using the genuine DE^ts>hid AiP model

For tissue ablation using the genuine DE^ts>hid model, we adapted the protocol developed by [34]. Briefly, larvae of the genotype UAS-hid/+; UAS-GFP/+; DE-Gal4 tub-Gal80^ts/+, either alone (control) or combined with transgenes expressing TrpA1 and RyR RNAi or the TrpA1^ins/+ mutant, were raised at 18 °C. Egg laying was allowed for 48h at 18 °C, followed by 5.5 days of larval development at the same temperature and a subsequent 12 h temperature shift to 30 °C to induce hid expression. After the heat pulse, larvae were returned to 18 °C for recovery. Imaginal discs were dissected at 24 hours (R24h) or 72 hours (R72h) post-recovery and processed for ELAV or cDcp1 immunolabeling as described above.

Quantification and statistical analysis

All confocal images were analyzed with Zen3.5 (blue edition) imaging software (Carl Zeiss) and quantified with NIH Fiji software. For quantification of confocal images, the region of interest (ey-Gal4-expressing area of eye-antennal imaginal discs) was outlined for each disc and mean fluorescence signal intensity was determined using NIH Fiji software. The Ca²⁺ flashes were quantified using MATLAB (R2022a) software. Data are presented as mean ± SEM from at least three independent experiments. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparison test (Figs 1B, 3B, and S4A) or a two-tailed unpaired Student t test (Figs 4B, S2B, and S5B). For GCaMP6s, DHE, NimC, MMP1, and Wg labelings, fluorescence intensity of the maximum intensity projections was measured. At least three biological repeats were performed for each experiment. Analysis and graph preparation was done using GraphPad Prism 10. Statistical analysis for GCaMP6s, DHE, NimC, MMP1, and Wg fluorescence intensity was performed using one-way ANOVA with Tukey’s multiple comparisons test, except for Dronc, RyR RNAi and TRPA1^ins experiments, for which statistical analysis was performed using two-tailed unpaired Student T test. Data are represented as the mean ± SEM of aggregated data collected from the specified number of samples in each experiment. qRT-PCR results were quantified using one-way ANOVA with Tukey’s multiple comparisons test. Graph plotted as Mean ± SD. All figures were assembled with Adobe Photoshop (26.4.1). Levels of significance are depicted by asterisks in the figures: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Supporting information

S1 Fig. Knockdown of Duox suppresses JNK activity and wingless (wg) expression in ey>hid,p35 discs.

(Related to Fig 1). Disc boundaries are outlined with white dashed lines, and yellow dotted lines delineate ey-Gal4-expressing areas of the eye discs. Scale bars represent 50 μm (A–H). (A–D) Confocal images of third instar larval eye imaginal discs of control ey-Gal4 (A, A′), undead (ey>hid,p35) discs expressing mock (Luciferase) RNAi (B, B′), UAS-Duox RNAi (C, C′) and Duox^EFm mutant (D, D′) immunolabeled with MMP1 (a JNK activity marker) and ELAV antibodies. The suppression of the overgrowth phenotype by UAS-Duox RNAi and Duox^EFm (Fig 1) correlates with reduced JNK activity (MMP1; green in A–D; gray in A′, D′; see yellow arrow) and normalization of eye disc patterning as visualized by ELAV labeling (red). (E–H) Confocal images of third instar larval eye imaginal discs of control ey-Gal4 (E, E′), undead (ey>hid,p35) discs expressing mock (Luciferase) RNAi (F, F′), UAS-Duox RNAi (G, G′), and Duox^EFm mutant (H, H′) immunolabeled with Wingless (Wg) and ELAV antibodies. The suppression of the overgrowth phenotype by UAS-Duox RNAi and Duox^EFm (Fig 1) correlates with reduced Wg expression (green in E–H; gray in E′–H′; see yellow arrow) and eye disc patterning was normalized as seen by ELAV labeling (red). (I) Quantification of the MMP1 fluorescence levels in (A–D). Data from n = 11 (ey-Gal4), 14 (mock RNAi), 15 (UAS-Duox RNAi), and 19 (Duox^EFm) discs were analyzed in three independent experiments. A.U.—arbitrary units. (J) Quantification of the Wg fluorescence levels in (E–H). Data from n = 10 (ey-Gal4), 16 (mock RNAi), 11 (UAS-Duox RNAi), and 19 (Duox^EFm) discs were analyzed in three independent experiments. A.U.—arbitrary units. The data underlying the graphs shown in this figure can be found in S1 Data.

(TIF)

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S2 Fig. Loss of the EF-hands of DUOX suppresses the overgrowth of undead heads and all AiP markers.

(Related to Fig 1). Disc boundaries are outlined with white dashed lines, and yellow dotted lines delineate ey-Gal4-expressing areas of the eye discs. Scale bars: 100 μm (B) and 50 μm (C, D, F, G, I, J, K, and L). (A) Representative examples of a severely overgrown head of ey>hid,p35 flies expressing mock (Luciferase) RNAi (left) and the suppressed overgrowth of ey<hid,p35 flies expressing UAS-Duox RNAi (right). Arrows point to amorphic tissues (purple arrow), additional bristles (black arrows), and ocelli (yellow arrow), and reduced eye tissue (red arrow). (B) Quantification of the suppression of head overgrowth of adult ey>hid,p35 flies by expression of UAS-Duox^ΔEF. Progeny was scored as wild type (WT) (black bars), weak (green bars), moderate (blue bars), or severely overgrown (red bars) according to the classification in Fig 1A. n = 100 flies counted per genotype in three independent experiments. (C, D) Confocal images of third instar larval ey>hid,p35 discs expressing mock (Luciferase) RNAi (C) and UAS-Duox^ΔEF (D) labeled for ROS with dihydroethidium (DHE) dye. The yellow arrows indicate DHE-positive cells. (E) Quantification of the DHE fluorescence levels in (C, D). Data from n = 11 (mock RNAi) and 18 (UAS-Duox^ΔEF) discs were analyzed in three independent experiments. A.U.—arbitrary units. (F, G) Confocal images showing hemocytes labeled with the plasmatocyte-specific anti-NimC antibody in third instar larval ey>hid,p35 discs expressing mock (Luciferase) RNAi (F) and UAS-Duox^ΔEF (G). Yellow arrows indicate hemocytes. (H) Quantification of the number of hemocytes in (F, G). Data from n = 12 (mock RNAi) and 19 (UAS-Duox^ΔEF) discs were analyzed in three independent experiments. (I, J) Confocal images of third instar larval eye imaginal discs of undead (ey>hid,p35) discs expressing mock (Luciferase) RNAi (I, I′), and UAS-Duox^ΔEF (J, J′) immunolabeled with MMP1 (a JNK activity marker) and ELAV antibodies. The suppression of the overgrowth phenotype by UAS-Duox^ΔEF (panels A and B) correlates with reduced JNK activity (MMP1; green in I, J; gray in I′, J′; see yellow arrow) and the normalization of eye disc patterning as seen by ELAV labeling (red). (K, L) Confocal images of third instar larval eye imaginal discs of ey>hid,p35 discs expressing mock (Luciferase) RNAi (K, K′), and UAS-Duox^ΔEF (L, L′) immunolabeled with Wg and ELAV antibodies. The suppression of the overgrowth phenotype by UAS-Duox RNAi and UAS-Duox^ΔEF (panels A and B) correlates with reduced Wg expression (green in K, L; gray in K′, L′; see yellow arrow) and the normalization of eye disc patterning as seen by ELAV labeling (red). (M) Quantification of the MMP1 fluorescence levels in (I, J). Data from n = 15 (mock RNAi), and 12 (UAS-Duox^ΔEF) discs were analyzed in three independent experiments. A.U.—arbitrary units. (N) Quantification of the Wg fluorescence levels in (E–H). Data from n = 15 (mock RNAi) and12 (UAS-Duox^ΔEF) discs were analyzed in three independent experiments. A.U.—arbitrary units. The data underlying the graphs shown in this figure can be found in S1 Data.

(TIF)

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S3 Fig. Individual time-lapse recordings of Ca²⁺ traces in GCaMP6s-expressing eye imaginal discs.

(Related to Fig 2E, 2F, 2G, 2L, and 2M). Shown are the complete time-lapse recordings of the Ca²⁺ traces of the GCaMP6s-expressing eye imaginal discs corresponding to those presented in Fig 2E, 2F, 2G, 2L, and 2M. Each trace depicts the fluorescence intensity over time in a single eye disc where peaks in panels (G) and (L) represent the Ca²⁺ flashes occurring in the tissue. Notably, the fluorescence intensity in the undead (ey>hid,p35) eye discs is significantly increased compared to controls. Images were acquired for 600 consecutive frames at 1-second intervals (10 min total acquisition time). All recordings were performed under identical imaging conditions to ensure comparability across samples. Genotypes: (A) ey-Gal4>GCaMP6s (control) (n = 5); (B) ey-p35>GCaMP6s (n = 8); (C) ey>hid,p35/GCaMP6s (n = 8); (D) ey>hid,p35/GCaMP6s/Luciferase RNAi (n = 8); (E) ey>hid,p35/GCaMP6s/Dronc RNAi (n = 8).

(TIF)

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S4 Fig. Knockdown of three TRP channels suppresses JNK activity and Wg expression.

(Related to Fig 3). (A) Summary of the suppression screen targeting all 13 TRP channels in the D. melanogaster genome. Quantification of overgrowth suppression of adult ey>hid,p35 fly heads includes RNAi knockdown of control mock (Luciferase), TRP, TRPL, TRPγ, TrpA1, Painless (Pain), Pyrexia (pyx), Water witch (wtwr), NompC, Inactive (iav), Nanchung (nan), TrpM, Pkd2, and TRPML genes. Progeny was classified as wild type (wt) (black bars), weak (green bars), moderate (blue bars), or severe overgrown (red bars) based on criteria in Fig 1A. n = 100 flies counted per genotype in three independent experiments. TrpM, TrpA1, and Pkd2 showed the strongest suppression and were selected for further characterization. Disc boundaries are outlined with white dashed lines, and yellow dotted lines delineate ey-Gal4-expressing areas of the eye discs (B–E and F–I). In all panels, Scale bars are 50 μm. (B–E) Confocal images of undead third instar larval (ey>hid,p35) eye discs expressing mock (Luciferase) RNAi (B, B′), UAS-TrpM RNAi (C, C′), UAS-TrpA1 RNAi (D, D′), and UAS-Pkd2 RNAi (E, E′) immunolabeled with MMP1 (JNK marker) and ELAV antibodies. The strong MMP1 labeling in ey>hid,p35 discs (A, A′; yellow arrow) is strongly suppressed by inactivation of either TRP channel (B–D; B′–D′). ELAV labeling (red) indicates normalization of eye disc patterning upon TRP channel knockdown (B–E, red). (F–I) Confocal images of undead third instar larval (ey>hid,p35) eye discs expressing mock (Luciferase) RNAi (F, F′), UAS-TrpM RNAi (G, G′), UAS-TrpA1 RNAi (H, H′), and UAS-Pkd2 RNAi (I, I′) immunolabeled with Wg (green in F–I; gray in F′–I′; see yellow arrow) and ELAV antibodies (red in F–I). (J) Quantification of the MMP1 fluorescence levels in (A–D). Data from n = 8 (mock RNAi), 12 (UAS-TrpM RNAi), 12 (UAS-TrpA1 RNAi), and 11 (UAS-Pkd2 RNAi) discs were analyzed in three independent experiments. A.U.—arbitrary units. (K) Quantification of the Wg fluorescence levels in (E–H). Data from n = 14 (mock RNAi), 13 (UAS-TrpM RNAi), 12 (UAS-TrpA1 RNAi), and 15 (UAS-Pkd2 RNAi) discs were analyzed in three independent experiments. A.U.—arbitrary units. The data underlying the graphs shown in this figure can be found in S1 Data.

(TIF)

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S5 Fig. A TrpA1 null mutant suppresses overgrowth of undead heads and all AiP markers.

(Related to Fig 3). Disc boundaries are outlined with white dashed lines, and yellow dotted lines delineate ey-Gal4-expressing areas of the eye discs (B–E and F–I). Scale bars: 100 μm (A) and 50 μm (C, D, I, J, L, M, O, P, Q, and R). (A) Representative examples of a severely overgrown head of ey>hid,p35 flies expressing mock (Luciferase) RNAi and the suppressed overgrowth by TrpA1^ins/+. Arrows point to amorphic tissues (purple arrow), additional bristles (black arrows) as well as ocelli (yellow arrow) and loss of eye tissue (red arrows) in ey>hid,p35 expressing mock (Luciferase) RNAi, while black arrows in TrpA1^ins/+ point to one or two extra bristles. (B) Quantification of the dominant suppression of head overgrowth of adult ey>hid,p35 flies by heterozygous TrpA1^ins/+. Progeny was scored as wild type (wt) (black bars), weak (green bars), moderate (blue bars), or severe overgrowth (red bars) according to the classification in Fig 1A. n = 100 flies counted per genotype in three independent experiments. (C, D) Confocal images of third instar larval ey>hid,p35 eye imaginal discs expressing the Ca²⁺ reporter GCaMP6s with and without TrpA1^ins/+. White arrows point to high Ca²⁺ levels. (E) Quantification of the cytosolic Ca²⁺ levels in (C, D) via measuring GCaMP6s fluorescence intensity. Data from n = 17 (mock RNAi) and 14 (TrpA1^ins/+) discs were analyzed in three independent experiments. A.U.—arbitrary units. (F, G) Representative Ca²⁺ traces of eye imaginal discs expressing GCaMP6s obtained by time-lapse confocal imaging (600 frames, 1-second intervals). Each line represents an independent disc (numbered). For complete individual recordings of the Ca²⁺ traces, see S6 Fig. The numbers of Ca²⁺ flashes are strongly reduced by TrpA1^ins/+ (S4 and S10 Movies). Quantification shown in (H). Genotypes: (F) ey>hid,p35/GCaMP6s/Luciferase RNAi (n = 6); (G) ey>hid,p35/GCaMP6s/TrpA1^ins/+ (n = 9). (H) Quantification of Ca²⁺ flashes in (E, F). Data from n = 7 (mock RNAi) and 11 (TrpA1^ins/+) discs were analyzed from three independent experiments. (I, J) Confocal images of third instar larval ey>hid,p35 eye imaginal discs with and without TrpA1^ins/+ labeled for ROS with dihydroethidium (DHE) dye. Yellow arrows indicate DHE-positive cells. (K) Quantification of the DHE fluorescence levels in (H, I). Data from n = 13 (mock RNAi) and 10 (TrpA1^ins/+) discs were analyzed in three independent experiments. A.U.—arbitrary units. (L, M) Confocal images showing hemocytes labeled with the plasmatocyte-specific anti-NimC antibody attached to third instar larval mock (Luciferase) RNAi expressing ey>hid,p35 discs (K) and in TrpA1^ins/+ background (L). Yellow arrows indicate hemocytes. (N) Quantification of the number of hemocytes per disc in (K,L). Data from n = 25 (mock RNAi) and 21 (TrpA1^ins/+) discs were analyzed in three independent experiments. (O, P) Confocal images showing third instar larval eye imaginal discs of control undead (ey>hid,p35) expressing mock (Luciferase) RNAi (N, N′) and together with TrpA1^ins/+ (O, O′), labeled with MMP1 and ELAV antibodies. MMP1 labeling (green in N, O; gray in N′, O′; see yellow arrow) is strongly reduced by TrpA1^ins/+. ELAV labeling (red) indicates normalization of eye disc patterning. (Q, R) Confocal images of third instar larval eye imaginal discs of control undead (ey>hid,p35) expressing mock (Luciferase) RNAi (P, P′) and together with TrpA1^ins/+ (Q, Q′), immunolabeled with Wg and ELAV antibodies. Wg labeling (green in P, Q; gray in P′, Q′; see yellow arrow) is strongly reduced by TrpA1^ins/+. ELAV labeling (red) indicates normalization of eye disc patterning. (S) Quantification of the MMP1 fluorescence levels in (N, O). Data from n = 10 (mock RNAi) and 11 (TrpA1^ins/+) discs were analyzed in three independent experiments. A.U.—arbitrary units. (T) Quantification of the Wg fluorescence levels in (P, Q). Data from n = 10 (mock RNAi) and 11 (TrpA1^ins/+) discs were analyzed in three independent experiments. The data underlying the graphs shown in this figure can be found in S1 Data.

(TIF)

pbio.3003607.s005.tif^{(53MB, tif)}

S6 Fig. Individual time-lapse recordings of Ca²⁺ traces in GCaMP6s-expressing eye imaginal discs.

(Related to Figs 3G–3J, 4F, 4G, and S5F–S5G). Shown are the complete time-lapse recordings of the Ca²⁺ traces of the GCaMP6s-expressing eye imaginal discs corresponding to those presented in Figs 3G–3J, 4F, 4G, S4F, and S4G. Each trace depicts the fluorescence intensity over time in a single eye disc where peaks in panels (3G), (4F), and (S5A) represent the Ca²⁺ flashes occurring in the tissue. Notably, the fluorescence intensity in the undead (ey>hid,p35) eye discs is significantly increased compared to discs with reduced TRP or RyR function. Images were acquired for 600 consecutive frames at 1-second intervals (10 min total acquisition time). All recordings were performed under identical imaging conditions to ensure comparability across samples. Genotypes: (A) ey>hid,p35/GCaMP6s/Luciferase RNAi (n = 7); (B) ey>hid,p35/GCaMP6s/TrpM RNAi (n = 5); (C) ey>hid,p35/GCaMP6s/TrpA1 RNAi (n = 9); (D) ey>hid,p35/GCaMP6s/Pkd2 RNAi (n = 7); (E) ey>hid,p35/GCaMP6s/RyR RNAi (n = 7); (F) ey>hid,p35/GCaMP6s/Luciferase RNAi (n = 6); (G) ey>hid,p35/GCaMP6s/TrpA1^ins/+ (n = 9).

(TIF)

pbio.3003607.s006.tif^{(72.5MB, tif)}

S7 Fig. Loss of RyR abolished both JNK and Wg signaling in undead discs.

(Related to Fig 4). Disc boundaries are outlined with white dashed lines, and yellow dotted lines delineate ey-Gal4-expressing areas of the eye discs (A, B, A′, B′, and C, D, C′, D′). In all panels, Scale bars represent 50μm. (A, B) Confocal images of third instar larval eye imaginal discs of undead (ey>hid,p35) discs expressing mock (Luciferase) RNAi (A, A′) and UAS-RyR RNAi (B, B′) immunolabeled with MMP1 and ELAV antibodies. MMP1 labeling (green in A, B; gray in A′, B′; see yellow arrow) is strongly reduced by UAS-RyR RNAi. ELAV labeling (red) indicates normalization of eye disc patterning by UAS-RyR RNAi. (C, D) Confocal images showing third instar larval eye imaginal discs of undead (ey>hid,p35) discs expressing mock (Luciferase) RNAi (C, C′) and UAS-RyR RNAi (D, D’′ labeled with Wingless (Wg) and ELAV antibodies. Wg labeling (green in C, D; gray in C′, C′; see yellow arrow) is strongly reduced by UAS-RyR RNAi. ELAV labeling (red) indicates normalization of eye disc patterning. (E) Quantification of the MMP1 fluorescence levels in (A, B). Data from n = 8 (mock RNAi) and 13 (UAS-RyR RNAi) discs were analyzed in three independent experiments. A.U.—arbitrary units. (F) Quantification of the Wg fluorescence levels in (C, D). Data from n = 14 (mock RNAi), and 11 (UAS-RyR RNAi) discs were analyzed in three independent experiments. A.U.—arbitrary units. The data underlying the graphs shown in this figure can be found in S1 Data.

(TIF)

pbio.3003607.s007.tif^{(14.4MB, tif)}

S8 Fig. TrpA1 RNAi, TrpA1^ins/+ ,and RyR RNAi do not affect photoreceptor development.

(Related to Fig 5). (A) Control DE^ts>GFP disc at R72h. Following the 12 h temperature shift that induces GFP expression (green) in the dorsal half of the eye imaginal discs (see experimental protocol in Fig 5A), photoreceptor patterning appears normal as shown by ELAV labeling (red in A, gray in A′). Scale bar: 50 μm. (B) DE^ts>GFP,TrpA1 RNAi disc at R72h. TrpA1 RNAi does not affect photoreceptor development following 12 h induction at 30 °C and 72 h recovery at 18 °C (Fig 5A). ELAV staining appears normal (red in B, gray in B′). GFP expression (green) indicates that transgenes have been induced. (C) DE^ts>GFP; TrpA1^ins disc at R72h. TrpA1^ins/+ does not affect photoreceptor development following 12 h incubation at 30 °C and 72 h recovery at 18 °C (Fig 5A). ELAV staining appears normal (red in B, gray in B′). (D) DE^ts>GFP,RyR RNAi disc at R72h. RyR RNAi does not affect photoreceptor development following 12 h induction at 30 °C and 72 h recovery at 18 °C (Fig 5A). ELAV staining appears normal (red in D, gray in D′). GFP expression (green) indicates that transgenes have been induced. (E, F) DE^ts>hid eye discs at R72h expressing UAS-TrpM RNAi (E) and UAS-Pkd2 RNAi (F) show complete recovery, with all examined discs (n = 20 for E; n = 10 for F) displaying restored ELAV expression (red in E, F; gray in E′,F′). GFP expression (green) confirms induction of transgenes.

(TIF)

pbio.3003607.s008.tif^{(24.9MB, tif)}

S1 Data. The data underlying the graphs.

(XLSX)

pbio.3003607.s009.xlsx^{(43.4KB, xlsx)}

S1 Movie. SM1. ey-Gal4.

A total of 10 supplementary movies (SM) were uploaded. Each movie consists of 600 frames in 1-second intervals, i.e., totaling 10 min. The movies are either in MP4 formats and can be watched with Media Player or Elmedia Video Player.

(MP4)

Download video file^{(16.9MB, mp4)}

S2 Movie. SM2. ey>p35 (ey-Gal4 UAS-p35).

(MP4)

Download video file^{(16.3MB, mp4)}

S3 Movie. SM3. ey>hid,p35 (EHP).

(MP4)

Download video file^{(30.6MB, mp4)}

S4 Movie. SM4. EHP + mock (Luciferase RNAi).

(MP4)

Download video file^{(31.5MB, mp4)}

S5 Movie. SM5. EHP + UAS-dronc RNAi.

(MP4)

Download video file^{(9.9MB, mp4)}

S6 Movie. SM6. EHP + UAS-TrpM RNAi.

(MP4)

Download video file^{(14.4MB, mp4)}

S7 Movie. SM7. EHP + UAS-TrpA1 RNAi.

(MP4)

Download video file^{(10MB, mp4)}

S8 Movie. SM8. EHP + UAS-Pkd22 RNAi.

(MP4)

Download video file^{(19.5MB, mp4)}

S9 Movie. SM9. EHP + UAS-RyR RNAi.

(MP4)

Download video file^{(9.5MB, mp4)}

S10 Movie. SM10. EHP + TrpA1^ins/+.

(MP4)

Download video file^{(17.7MB, mp4)}

Acknowledgments

We would like to thank Dr. Fei Wang and Dr. Yang Xiang for their invaluable help with the Ca²⁺ assays; Dr. Paul Garrity, Dr. Won-Jae Lee and Dr. István Andó for fly stocks and antibodies; the Bloomington Drosophila Stock Center (supported by NIH grant 5P40OD018537-10) and the Vienna Drosophila Resource Center for fly stocks; and the Developmental Studies Hybridoma Bank (DSHB) for antibodies.

Abbreviations

AiP: apoptosis-induced proliferation
CICR: calcium-induced calcium release
DHE: dihydroethidium
Dpp: Decapentaplegic
EGF: epidermal growth factor
ER: endoplasmic reticulum
IAPs: inhibitor of apoptosis proteins
RNAi: RNA interference
ROS: reactive oxygen species
RyR: Ryanodine Receptor
TRP: transient receptor potential
Wg: Wingless

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

This work was supported by a grant from the National Institute of General Medical Sciences (NIGMS) under award number R35GM118330 to AB, and the UK Research and Innovation (UKRI) BBSRC under award number BB/Z516971/1 to YF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. https://www.nigms.nih.gov/ https://www.ukri.org/councils/bbsrc/.

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PLoS Biol. doi: 10.1371/journal.pbio.3003607.r001

Decision Letter 0

Ines Alvarez-Garcia

7 Mar 2025

Dear Andreas,

Thank you for submitting your manuscript entitled "Role of Calcium Signaling in Apoptosis-induced Proliferation in Drosophila" for consideration as a Short Report by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

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Ines

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

PLoS Biol. doi: 10.1371/journal.pbio.3003607.r002

Decision Letter 1

Ines Alvarez-Garcia

8 May 2025

Dear Andreas,

Thank you for your patience while your manuscript entitled "Role of Calcium Signaling in Apoptosis-induced Proliferation in Drosophila" was peer-reviewed at PLOS Biology as a Short Report. Please also accept my apologies for the delay in providing you with our decision. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by two independent reviewers.

The reviews are attached below. As you will see, the reviewers find the conclusions potentially interesting, but they also raise several concerns that would need to be addressed before we can consider the manuscript further for publication. Reviewer 1 notes that all the experiments have been performed using the ‘undead’ cell system, but that in this system AiP is prolonged and extended beyond physiological settings, thus the reviewer thinks that experiments should be performed in which apoptosis is induced by a pulse rather than being sustained by ‘undead’ cells; the wild type and TRPA1 mutants should then be compared to confirm the conclusions. Reviewer 2 points out that the full screen has not been included in the paper and it should be added. This reviewer also thinks that it’s unclear how Dronc activates the specific calcium channels and that experiments should be repeated using different controls for different experiments. In addition, the reviewer suggests exploring why there is more GCaMP6s activity in the antennal region of the disc upon generation of undead cells, and quantify Wg and Mmp1 in antennal discs to strengthen the conclusions.

Based on the reviewers’ specific comments and following discussion with the Academic Editor, it is clear that a substantial amount of work would be required to meet the criteria for publication in PLOS Biology. However, given our and the reviewer interest in your study, we would be open to inviting a comprehensive revision of the study that thoroughly addresses all the reviewers' comments. Given the extent of revision that would be needed, we cannot make a decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript would need to be seen by the reviewers again, but please note that we would not engage them unless their main concerns have been addressed.

In addition to these revisions, you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests shortly.

We appreciate that these requests represent a great deal of extra work, and we are willing to relax our standard revision time to allow you 6 months to revise your study. Please email us (plosbiology@plos.org) if you have any questions or concerns, or envision needing a (short) extension.

At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may withdraw it.

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Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Ines

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

-----------------------------------------

Reviewers' comments

Rev. 1: Paulo Ribeiro - note that this reviewer has signed the review

The report by Suthar and Bergmann addresses the molecular mechanisms regulating the process of apoptosis-induced proliferation (AiP). Building on previous work from the Bergmann laboratory, where several seminal discoveries of the basic biology regulating AiP, the authors now explore the mechanisms leading to the activation of Duox, one of the critical regulators of AiP. The authors show that, in agreement with its protein and domain structure, Duox is activated by Ca2+ and this activation is dependent on Dronc function. The authors also show that the Ca2+ is mobilised from different sources. Ca2+ is internalised from extracellular sources via specific Ca2+ channels and mobilised from the ER to activate Duox. The authors suggest that Ca2+ is therefore essential for AiP.

In general, experiments and data are well presented and appropriately controlled. The results obtained by the authors provide an extra layer of complexity regarding the process of AiP and will be of interest to groups studying apoptosis and regeneration, among others. Comments on the manuscript are shown below.

Major points:

Figure 2: Regarding the analysis of Ca2+ signalling, is there a pattern in the distribution of the Ca2+ flashes? Do they primarily occur in the ey-Gal4-expressing areas or are the surrounding areas also affected?

Related to the point above and given that there is an amplification loop of AiP signalling, is there a role for Duox in the regulation of Ca2+ flashes or AiP-mediated Ca2+ signalling?

Regarding the role of Ca2+ channels, is there an additive or synergistic effect of modulating extracellular Ca2+ channels and RyR regarding AiP phenotypes? Is there a way to test the epistatic relationship between the different Ca2+ channel types (i.e. those controlling extracellular Ca2+ and RyR)?

All the experiments have been performed using the "undead" cell system, which has been exploited at length in the field of AiP. However, by its nature, this is a system where AiP is prolonged and extended beyond what would normally occur in a physiological setting. Given that the authors have a TRPA1 mutant fly stock, would it be possible to conduct experiments where apoptosis is induced in a pulse (i.e. using a FLPout system) rather than sustained using "undead" cells and to compare the extent of AiP in the control situation vs the TRPA1 mutant (where AiP will be affected)? Whilst not strictly necessary, having an experiment conducted in a system beyond the "undead" model would enhance the conclusions of the study.

Related to the point above, authors should comment on the fact that TRPA1 mutants seem to suppress the AiP phenotypes in heterozygosity. Is 50% of gene dose not sufficient to maintain TRPA1 function?

Minor points:

Figure 1A, Figure 3A, Figure S3A, Figure 4A: If possible, statistical analysis of the comparison between the genotypes should be added.

Related to Figure 1, it would be helpful to provide more information regarding the criteria to classify the different phenotypes as weak, moderate or severe overgrowth. This could potentially be included in the relevant Materials and Methods section.

Figure 1C-H, Figure 1I-N and Figure S1: Have the quantifications of the intensity of the different stainings been normalised to tissue size? Moreover, given that AiP affects neighbouring tissue, have the authors analysed and quantified the stainings in the different areas (ey-Gal4-expressing and surrounding areas) separately?

Are both EF hand motifs required? Have the authors created Duox transgenes where only one EF hand has been mutated? If so, do these have intermediate phenotypes?

Related to the action of the Ca2+ channels, is it possible for the authors to specifically inhibit the different channels using chemical inhibitors to further confirm their role and potentially explain the non-redundancy issue? Alternatively, is it possible for authors to over-express (or deplete) Ca2+ regulators with the opposite effect of RyR, TRPA1, TRPM and PKD2 to further validate their results?

Line 215: Typo. S1I should be replaced with S2I.

Line 216: Typo S1J should be replaced with S2J.

Figure 4G: x-axis labels are covered in figure panel.

Line 554: Reference 39 is listed as in press but the correct citation should be provided.

Rev. 2:

The manuscript titled "Role of Calcium Signaling in Apoptosis-induced Proliferation in Drosophila" by Suthar and Bergmann explores the role of cytosolic calcium influx as a crucial event for DUOX activation during apoptosis-induced proliferation (AiP), using the Drosophila eye disc as a model system. This study builds upon previous work demonstrating that activation of the caspase Dronc leads to DUOX activation in "undead" cells, which produces reactive oxygen species (ROS), which recruits immune cells to activate JNK signaling and downstream mitogenic factors that promote proliferation of surrounding cells. In this work, the authors identify the calcium channels - TRPA1, TRPM, PKD2, and RyR - that allow Ca2+ entry into the cytosol when apoptosis is inhibited by p35, activating DUOX via its EF-hand calcium-binding motifs to drive AiP.

To establish the role of these channels, the authors employ a comprehensive suite of genetic tools, including RNA interference, CRISPR-Cas9-mediated knockouts, and mutants. These complementary approaches offer compelling evidence linking calcium channel activity to DUOX activation. However, the authors point out that calcium regulation of DUOX has been previously described, and identification of these channels does not explain how the caspase Dronc activates this pathway. While this manuscript is essentially a genetic screen paper, the screen itself is not presented. Furthermore, the duplication of controls throughout the manuscript raises concerns about the data itself. Thus, substantial work remains before this manuscript is suitable for a journal with a broad readership such as Plos Biology.

Major concerns:

1. Line 192-194: The authors mention a screen was conducted of 13 TRP channel genes. This paper is a genetic screen paper in which the screen itself is not presented. The authors should present the complete screen. How do we know that perturbation of any calcium channel will not have the same effect, and that the ones presented are unique in their role in AiP?

2. The new finding in this paper is identification of the specific calcium channels that act downstream of Dronc to promote Duox activity, as the fact that calcium activates Duox is known. This finding is assuming that presentation of the full screen as requested in point 1 will confirm that the effect on DUOX activity is specific to these four channels. Identification of how Dronc activates these channels would substantially increase the broad applicability of these findings and interest in this work.

3. The controls presented in the figures are identical for each figure - the same disc images are used throughout, sometimes tilted and sometimes not, and the graphs make it clear that the same measurements are graphed for the controls for different experiments. Thus, it appears the controls were done only once, and the numbers re-used for subsequent experiments without running the control side-by-side with the mutant or knockdown being tested. This would render the result invalid, as controls must be performed alongside the experimental condition for immunofluorescence or DHE staining.

a. DHE: 1D, 3N, 4H, S3H, are the same disc. 1H, 3D, 4J, S3J use the same data for the mock RNAi genotype.

b. NimC: 1J, 3R, 4K, S3K are the same disc, slightly rotated. 1N, 3E, S3M use the same data for the mock RNAi genotype.

c. GCamp: 2I, 3F, 4B, S3B are the same disc. 2K and 3B may be the same data plotted slightly differently. 2K, 4D, and S4D are the same data for the mock RNAi genotype.

d. Mmp1: S1B, S2A, S3N, and S4A are the same disc. S1K, S2I, S3R, and S4E are the same data for the mock RNAi genotype.

e. Wg: S1F, S2E, S3P, and S4C are the same disc. S1J, S2J, S3S, and S4S have the same data for the mock RNAi genotype.

Therefore, these experiments should all be repeated with side-by-side controls to confirm the results.

4. In Figure 1B, the four panels depict the extent of the overgrown phenotype, which includes a range of phenotypes relating to bristles, ocelli, eye size etc. The arrows should be changed to different markers pointing at different phenotypes. Importantly, each specific phenotype should be separately quantified to make its prevalence more apparent.

5. Line 199-206: The authors show an interesting finding, that inactivating any single

channel results in loss of GCaMP6s activity, suggesting these channels are non-redundant. However, no experiments were designed to probe the reason behind the fact that all appear necessary and none appear sufficient to activate Duox. The explanation provided in the discussion is unconvincing. If these channels have their own unique inputs, why would inactivating any one channel abolish all or most cytosolic calcium?

6. In the discussion (line 285-287), the authors mentioned some TRP channels were upregulated under certain conditions like cancer. Are they upregulated in the context of AiP as well?

7. In Fig 2C, there seems to be more GCaMP6s activity in the antennal region of the disc upon generation of undead cells, despite the system being restricted to ey+ cells. A similar effect is seen in the RyR RNAi disc (Fig 4C). The authors should explore why this is happening.

8. In Figure S2, Wg and MMP1 also seem to be upregulated in the antennal region, despite the undead cells restricted to the eye disc anterior to the furrow. The authors should quantify Wg and Mmp1 in the antennal discs, and if there is truly elevated expression non-autonomously, explain why.

Minor comments:

1. Line 53, add a period after caspases.

2. In figure 1A, a control for ey>hid,p35 without a mock RNAi should be added, to ensure activating the RNAi machinery does not itself cause any overgrowth.

3. Figure 1A-B: The examples of the phenotypes should be presented before the quantifications.

4. Figure 1: The figure legend stated that the scale bars for C-G are 50 microns and the scale bars for I-M are 100 microns, but the discs in I-M do not look half the size of the discs in C-G. This same concern exists in several other figures.

5. In Figure 1H and N, some bars are incorrectly labeled as "Doux". The labels are too small to read here and in all graphs throughout the other figures.

6. Figure 1H: The legend states that 6 discs were obtained from three experiments. Two discs per experiment is a very low yield - can the authors explain?

7. Figure 2 E-G and other similar panels - does each line represent a different disc? The Y-axis is not labeled (eg Fig2E-G, L etc.)

8. Figure 3 - show the examples of the phenotypes and then show the quantification. Otherwise, the figure panels are called out of order in the text.

9. Figure S3 was called before Figure S2; re-arrangements need to be done accordingly.

10. Figure 4, The images have no scale bars, and it does not seem accurate that K-L are a different scale from H-I.

11. Figure 4G the bottom of the graph is masked or incomplete.

12. Figure S3: The images have no scale bars.

13. Figure S3: the number 1055 is hovering above panel D

14. Videos should all be in MP4 format for universal access

PLoS Biol. 2026 Jan 20;24(1):e3003607. doi: 10.1371/journal.pbio.3003607.r003

Author response to Decision Letter 2

14 Oct 2025

Attachment

Submitted filename: Response to the reviewers comments.docx

pbio.3003607.s022.docx^{(35.8KB, docx)}

PLoS Biol. doi: 10.1371/journal.pbio.3003607.r004

Decision Letter 2

Ines Alvarez-Garcia

2 Dec 2025

Dear Andreas,

Thank you for your patience while we considered your revised manuscript entitled "Role of Calcium Signaling in Apoptosis-induced Proliferation in Drosophila" for consideration as a Short Report at PLOS Biology. Your revised study has now been evaluated by the PLOS Biology editors, the Academic Editor and the two original reviewers.

In light of the reviews, which you will find at the end of this email, we are pleased to offer you the opportunity to address the remaining points from Reviewer 2 in a revision that we anticipate should not take you very long. We will then assess your revised manuscript and your response to the reviewers' comments with our Academic Editor aiming to avoid further rounds of peer-review.

In addition to these revisions, you may need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests shortly. If you do not receive a separate email within a few days, please assume that checks have been completed, and no additional changes are required.

We expect to receive your revised manuscript within 1 month. Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension.

At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we withdraw the manuscript.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

*NOTE: In your point-by-point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

3. Resubmission Checklist

When you are ready to resubmit your revised manuscript, please refer to this resubmission checklist: https://plos.io/Biology_Checklist

Please make sure to read the following important policies and guidelines while preparing your revision and fulfil the editorial requests:

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Sincerely,

Ines

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

----------------------------------------------------------------

Reviewers' comments

Rev. 1: Paulo Ribeiro

The report by Suthar et al. addresses the molecular mechanisms regulating the process of apoptosis-induced proliferation (AiP). Building on previous work from the Bergmann laboratory, where several seminal discoveries of the basic biology regulating AiP, the authors now explore the mechanisms leading to the activation of Duox, one of the critical regulators of AiP. The authors show that, in agreement with its protein and domain structure, Duox is activated by Ca2+ and this activation is dependent on Dronc function. The authors also show that the Ca2+ is mobilised from different sources. Ca2+ is internalised from extracellular sources via specific Ca2+ channels and mobilised from the ER to activate Duox. The authors suggest that Ca2+ is therefore essential for AiP.

The revised manuscript is greatly improved, and the authors strived to address all the major points raised by the reviewers. Importantly, the new data provided enhances and further supports the hypothesis that Ca2+ signalling plays an important role in AiP. The use of a genetic mutant of Duox that is predicted to be unable to bind Ca2+ constitutes an additional genetic system to address the role of Duox and provides additional evidence beyond the use of the dominant-negative version of Duox. The new Duox allele further supports the proposed molecular mechanism presented by the authors. Moreover, the authors also present data in a genetic system that does not result in the long-term accumulation of "undead" cells and their experiments reveal that the mechanism uncovered in this report is likely to be important in more physiological conditions, due to the fact that there was incomplete tissue regeneration when Ca2+ channel function was affected.

Experiments and data are well presented and appropriately controlled. New figure panels are more extensive and provide indication of statistical analyses proposed by the reviewers. Methods include additional information important for clarification of specific points that could be confusing to a more general audience.

In general, the manuscript is vastly improved, includes further evidence supporting the main conclusions of the report and the level of evidence provided is now supported by additional statistical analyses. The data will be of interest to research groups studying apoptosis and regeneration, among others. I support its publication in PLoS Biology.

Rev. 2:

1. The authors re-used the same control discs in figures 3 and 4 and figures S4 and S7, given that the data are from the same experiment. However, this image re-use gives the impression that there may be only one control disc from each experiment that fits the narrative. Given that the data from these images are quantified, there must be additional control discs that can be used to avoid the re-use.

2. The initial review requested quantification of the individual phenotypes in the adult heads, which the authors declined to do, maintaining their semiquantitative categories of weak, moderate, and strong. It would be less subjective to quantitate the number of extra bristles per head, percent of heads with ectopic ocelli, percent of heads with reduced eyes, etc.

3. Figure 3B and S4A - it is unclear which experiments and controls were conducted at the same time. To be specific, the data for TrpMRNAi, TrpA1RNAi , and Pkd2RNAi in S4A - the original screen - look identical to the data for TrpMRNAi, TrpA1RNAi , and Pdk2RNAi in 3B - the follow-up experiment that tested multiple ways of reducing TrpM and TrpA1 expression. The data for the controls for these two experiments are not identical, but it looks like the data for the three RNAi lines was re-used.

4. Figure 5 E,F, and G - the phenotype should be quantified across sufficient sample size rather than just showing one disc, especially since the phenotype is so subtle. One method of quantification could be the difference in the number of rows of ommatidia between the dorsal and ventral halves of each eye disc.

5. The authors should include a more detailed explanation of the methodology used to quantify the calcium flashes. For example, what constitutes a flash? Signal over a specific magnitude or of a specific duration? This question is raised because in Figure 2H, for the ey>p35 genotype, there is a disc that had 9 flashes and one that had 4 flashes, but none of the traces in 2F/S3B appear to have flashes like those in 2G/S3C, even though the quantification claims that two of them did.

6. It is unclear why the mock RNAi is the appropriate control for the Cas9 knockouts or overexpression of Duox deltaEF and other non-RNAi conditions.

7. The authors use jargon that lacks precision when describing experimental conditions, such as the terms "undead head capsule overgrowth" line 164, "moderately strong suppressed undead overgrowth" line 259, "undead head" line 904, etc.

8. Line 299 describes data not shown - if PLOS Biology has a policy that all data must be shown, these data should be included.

9. For the DuoxEFm mutation, it is unclear if it is heterozygous or homozygous in the experiments. For example, in Line 928 it is written as heterozygous, but is not indicated as heterozygous throughout Figure 1 or S1 labels or line 931, 934, 938, 946, 1131, 1136, 1137, 1141, 1144.

10. Figure 3 Legend A and B are reversed. Similar for S2 Legend A and B.

11. Line 1138 - is Wg activity or expression being monitored?

12. Line1328 should perhaps read 12h temperature shift.

13. Figure 1H-R label each panel with the genotype.

14. Figure 3S-V the notations of statistical significance are too small to see when printed, especially the n.s. in panel V.

15. Figure 4 swapping panels A and B will show the phenotypes before the quantification.

16. Figure 5 A - note the time of egg laying and the time of the temperature shift on the schematic.

PLoS Biol. 2026 Jan 20;24(1):e3003607. doi: 10.1371/journal.pbio.3003607.r005

Author response to Decision Letter 3

26 Dec 2025

Attachment

Submitted filename: Point-by-point response to reviewers comments.docx

pbio.3003607.s023.docx^{(30.4KB, docx)}

PLoS Biol. doi: 10.1371/journal.pbio.3003607.r006

Decision Letter 3

Ines Alvarez-Garcia

7 Jan 2026

Dear Dr Bergmann,

Thank you for the submission of your revised Research Article entitled "Calcium signaling regulates apoptosis-induced proliferation in Drosophila" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Nic Tapon, I am delighted to let you know that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

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Many congratulations and thanks again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study.

Sincerely,

Ines

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Knockdown of Duox suppresses JNK activity and wingless (wg) expression in ey>hid,p35 discs.

(TIF)

pbio.3003607.s001.tif^{(28.2MB, tif)}

S2 Fig. Loss of the EF-hands of DUOX suppresses the overgrowth of undead heads and all AiP markers.

(TIF)

pbio.3003607.s002.tif^{(35.6MB, tif)}

S3 Fig. Individual time-lapse recordings of Ca²⁺ traces in GCaMP6s-expressing eye imaginal discs.

(TIF)

pbio.3003607.s003.tif^{(85.9MB, tif)}

S4 Fig. Knockdown of three TRP channels suppresses JNK activity and Wg expression.

(TIF)

pbio.3003607.s004.tif^{(53.1MB, tif)}

S5 Fig. A TrpA1 null mutant suppresses overgrowth of undead heads and all AiP markers.

(TIF)

pbio.3003607.s005.tif^{(53MB, tif)}

S6 Fig. Individual time-lapse recordings of Ca²⁺ traces in GCaMP6s-expressing eye imaginal discs.

(TIF)

pbio.3003607.s006.tif^{(72.5MB, tif)}

S7 Fig. Loss of RyR abolished both JNK and Wg signaling in undead discs.

(TIF)

pbio.3003607.s007.tif^{(14.4MB, tif)}

S8 Fig. TrpA1 RNAi, TrpA1^ins/+ ,and RyR RNAi do not affect photoreceptor development.

(TIF)

pbio.3003607.s008.tif^{(24.9MB, tif)}

S1 Data. The data underlying the graphs.

(XLSX)

pbio.3003607.s009.xlsx^{(43.4KB, xlsx)}

S1 Movie. SM1. ey-Gal4.

(MP4)

Download video file^{(16.9MB, mp4)}

S2 Movie. SM2. ey>p35 (ey-Gal4 UAS-p35).

(MP4)

Download video file^{(16.3MB, mp4)}

S3 Movie. SM3. ey>hid,p35 (EHP).

(MP4)

Download video file^{(30.6MB, mp4)}

S4 Movie. SM4. EHP + mock (Luciferase RNAi).

(MP4)

Download video file^{(31.5MB, mp4)}

S5 Movie. SM5. EHP + UAS-dronc RNAi.

(MP4)

Download video file^{(9.9MB, mp4)}

S6 Movie. SM6. EHP + UAS-TrpM RNAi.

(MP4)

Download video file^{(14.4MB, mp4)}

S7 Movie. SM7. EHP + UAS-TrpA1 RNAi.

(MP4)

Download video file^{(10MB, mp4)}

S8 Movie. SM8. EHP + UAS-Pkd22 RNAi.

(MP4)

Download video file^{(19.5MB, mp4)}

S9 Movie. SM9. EHP + UAS-RyR RNAi.

(MP4)

Download video file^{(9.5MB, mp4)}

S10 Movie. SM10. EHP + TrpA1^ins/+.

(MP4)

Download video file^{(17.7MB, mp4)}

Attachment

Submitted filename: Response to the reviewers comments.docx

pbio.3003607.s022.docx^{(35.8KB, docx)}

Attachment

Submitted filename: Point-by-point response to reviewers comments.docx

pbio.3003607.s023.docx^{(30.4KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting information files.

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PERMALINK

Calcium signaling regulates apoptosis-induced proliferation in Drosophila

Komal Panchal Suthar

Caitlin Hounsell

Yun Fan

Andreas Bergmann

Roles

Abstract

Introduction

Fig 1. Loss of EF-hand function of DUOX suppresses undead overgrowth and all AiP markers.

Results

A Duox mutant unable to bind Ca2+ suppresses all AiP phenotypes

Release of Ca2+ during AiP in a Dronc-dependent manner

Identification and characterization of Ca2+ transporters involved in AiP

Identification and characterization of RyR as intracellular Ca2+ channel

Fig 4. The Ryanodine Receptor (RyR) is required for cytosolic Ca2+ entry and AiP.

TrpA1 and RyR contribute to regeneration in the DEts>hid “genuine” AiP model

Fig 5. TrpA1 and RyR are required for complete regeneration in the “genuine” AiP model DEts>hid.

Discussion

Fig 6. Summary model.

Materials and methods

Fly stocks and genetics

Fly head phenotype screening and quantification

ROS staining

Immunofluorescence labeling

Imaging cytosolic Ca2+ with GCaMP6s reporters

Quantification of Ca2+ flashes

Tissue ablation and recovery using the genuine DEts>hid AiP model

Quantification and statistical analysis

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Ines Alvarez-Garcia

Roles

Decision Letter 1

Ines Alvarez-Garcia

Roles

Author response to Decision Letter 2

Decision Letter 2

Ines Alvarez-Garcia

Roles

Author response to Decision Letter 3

Decision Letter 3

Ines Alvarez-Garcia

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

A Duox mutant unable to bind Ca²⁺ suppresses all AiP phenotypes

Release of Ca²⁺ during AiP in a Dronc-dependent manner

Identification and characterization of Ca²⁺ transporters involved in AiP

Identification and characterization of RyR as intracellular Ca²⁺ channel

Fig 4. The Ryanodine Receptor (RyR) is required for cytosolic Ca²+ entry and AiP.

TrpA1 and RyR contribute to regeneration in the DE^ts>hid “genuine” AiP model

Fig 5. TrpA1 and RyR are required for complete regeneration in the “genuine” AiP model DE^ts>hid.

Imaging cytosolic Ca²⁺ with GCaMP6s reporters

Quantification of Ca²⁺ flashes

Tissue ablation and recovery using the genuine DE^ts>hid AiP model