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• Motohiro Morikawa, 
• Tetsutaro Hayashi, 
• Yuko Ikegawa, 
• Tomomi Takano, 
• Kazuya Wata, 
• Hirohisa Kyogoku, 
• Mariko Kuse, 
• Mika Yoshimura, 
• Hiroshi Nishida, 
• Rahul Parit

x

Abstract

Citation: Morikawa M, Hayashi T, Ikegawa Y, Takano T, Wata K, Kyogoku H, et al. (2026) Heme acts as a metabolic brake on erebosis in the Drosophila gut. PLoS Biol 24(8): e3003942. https://doi.org/10.1371/journal.pbio.3003942

Academic Editor: Mathieu JM Bertrand, Universiteit Gent, BELGIUM

Received: October 26, 2025; Accepted: July 27, 2026; Published: August 19, 2026

Copyright: © 2026 Morikawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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

Funding: This work was supported by RIKEN Junior Research Associate Program to M.M.; JST FOREST (JPMJFR216F) to S.K.Y.; JST CREST (Grant Number JPMJCR21N6), Multilayered Stress Diseases in TMDU, Medical Research Center Initiative for High Depth Omics in TMDU, RIKEN TRIP initiative, the ‘Stage Transition’ project of RIKEN BDR to I.N. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: 1-ABT, 1-Aminobenzotriazole; 5-ALA, 5-aminolevulinic acid; CO, carbon monoxide; DEGs, differentially expressed genes; DHE, dihydroethidium; GO, gene ontology; Ho, Heme oxygenase; ISCs, intestinal stem cells; NAC, N-acetylcysteine; NGS, normal goat serum; PCA, principal component analysis; pH3, phopho-histone H3; pMad, phosphorylated Mad; PBO, piperonylbutoxide; PFA, paraformaldehyde; ROS, reactive oxygen species; TPM, transcripts per million.

Introduction

Dynamic tissue homeostasis of the gut is maintained by cell turnover, which is mediated by intestinal stem cells (ISCs) and enterocyte death. Since the discovery of ISCs in the Drosophila gut [1,2], there has been intensive research on the regulatory mechanisms controlling ISC activity [3–11]. Regarding enterocyte death, many studies have used experimental conditions that artificially induce apoptosis or necrosis, such as exposure to infection, DSS, paraquat, or bleomycin [12–15]. Genetic inhibition of genes that are essential for tissue maintenance, such as Rab21 or septate junction components, can trigger enterocyte apoptosis [16,17]. Inhibition of the unconventional myosin, Myo1D, also disturbs physiological function of enterocytes [18]. However, our understanding of how enterocyte death is regulated under normal physiological conditions remains very limited.

We recently demonstrated that gut cell death under the homeostatic condition is predominantly erebosis, a previously unrecognized mode of cell elimination in adult tissue homeostasis [19]. Erebosis is a distinct form of cell death that is nonapoptotic, nonautophagic, and nonnecrotic [20], which most frequently occurs in the R4 region. A recent report also demonstrates that enterocyte turnover is regulated in an apoptosis-independent manner [21], consistent with the notion that nonapoptotic cell elimination such as erebosis contributes to gut homeostasis. Interestingly, erebosis was suggested to exist in the mammalian brain [22]. Erebotic cells accumulate Ance (angiotensin-converting enzyme) while losing many other intracellular proteins, ultimately leading to the loss of organelles and the nucleus [19]. Despite phenotypic characterization of erebosis, its molecular mechanism remains to be solved. In this study, we sought to elucidate the molecular basis underlying erebosis.

Results

To investigate the mechanism of erebosis, we decided to perform single-cell RNA sequencing. We found that, following the cell dissociation process using trypsin or elastase, it was difficult to isolate erebotic cells. In addition, the tissue dissociation procedure, which lasts at least 1 hour at 37 °C, can induce stresses and alter stress-response-related gene expression [23]. We thus opted to use micromanipulation under a microscope to collect live cells directly from the gut, immediately lyse the collected cells, and perform RamDA-seq, a highly sensitive single-cell total RNA sequencing method [24].

Erebotic cells in live guts can be identified using the GFP/nlsRFP reporter [19]. In enterocytes expressing GFP and nlsRFP, GFP disappearance occurs concomitantly with Ance accumulation during erebosis, which is followed by the later loss of nlsRFP [19]. Especially, GFP negativity with preserved nlsRFP fluorescence marks early-to-intermediate erebosis, whereas loss of both GFP and nlsRFP represents either late-stage erebosis [19] or necrosis, during which both cytoplasmic and nuclear proteins are lost [25–27]. Apoptotic cells, which often become apically extruded, do not lose fluorescent proteins [16,17,28,29]. To roughly categorize cells as erebotic or normal based on their fluorescence, we expressed GFP and nlsRFP in enterocytes using the Myo1D-Gal4 driver. We picked up GFP− cells with intact nlsRFP fluorescence, which we phenotypically classified as erebotic but neither necrotic nor apoptotic. By taking advantage of the GFP/nlsRFP signals, we aimed to collect approximately equal numbers of normal enterocytes and erebotic cells.

We collected 8 GFP+ normal enterocytes and 8 GFP− erebotic cells from the R4 region [30], a region where erebosis readily occurs, of two guts, and performed RamDA-seq (S1A Fig). Of the 16 cells, only one failed the quality control analysis, and the remaining 15 were subjected to further analyses (S1B–S1D Fig). Despite the limited number of cells, we obtained high-quality data (GSE308217) in which enterocytes were grouped into three clusters (S1E Fig). Based on GFP fluorescence patterns and gene ontology (GO) analysis, we interpreted the three clusters as a normal cell cluster, an erebotic cluster, and a damaged cell cluster (S1E Fig). Classification of the normal cell cluster was straightforward because all enterocytes in this cluster were GFP+. The cluster we categorized as the erebotic cell cluster included both GFP+ and GFP− cells, suggesting that changes in gene expression patterns precede phenotypic manifestation such as GFP loss. In support of this interpretation, in situ hybridization revealed that GFP− erebotic cells expressed CG8745 and LManIII, which were specifically upregulated in this erebotic cell cluster (S2A, S2B Fig). In contrast, GFP+ normal enterocytes expressed Srg3/CG33926, which was upregulated in the normal cell cluster (S2A, S2B Fig). We also validated the classification with Fly Cell Atlas dataset [31] using module score analysis [32]. Module score of R4-specific genes [30] identified “Enterocyte of posterior adult midgut epithelium” and “Midgut large flat cell” clusters as R4 enterocytes in the Fly Cell Atlas data (S3A–S3C Fig). Module scores of top 20 genes in the normal cell cluster or the erebotic cell cluster (S4 Fig) were enriched in different populations of R4 enterocytes (S3D, S3E, S3G Fig), indicating that the cell population identified in our analysis exists distinctly in Fly Cell Atlas dataset. The cluster we categorized as the damaged cell cluster, composed entirely of GFP− cells, showed high activation of the Toll, IMD, and JAK/STAT pathways in the GO analysis (S2C Fig). These pathways are known to be activated by infection and tissue injuries [14,33], implying that this cluster was under such stresses. Consistently, cells in this cluster were isolated at the end of the collecting procedure, exposed to the nonphysiological environment for longer time. Moreover, we previously showed that the IMD pathway or Upd2 is not involved in erebosis [19]. Artificial activation of STAT using the Myo1D-Gal4 driver with tub-Gal80^ts (referred to as Myo1D-Gal4^ts) did not induce erebosis either (S2D Fig). Furthermore, Fly Cell Atlas data did not have a distinct cell population with an enrichment of top20 genes of the damaged cell cluster (S3F Fig). Thus, we assume that this cluster is not related to erebosis. However, we cannot completely exclude the possibility that it may represent a very late stage of erebosis, where gene expression patterns may become dysregulated.

Initially, we became interested in genes whose expression was altered in the erebotic cell cluster. We tested RNAi-mediated knockdown of differentially expressed genes (DEGs) in the erebotic cluster, such as CG8745 (S4 Fig). However, none of them affected erebosis. We speculate that many of the genes upregulated in this cluster are involved in the processes that occur after initiation of erebosis, but not in its initiation.

Next, we turned our attention to the normal cell cluster, hypothesizing that it might include “pre-erebosis” cells. GO analysis revealed strong enrichment of the proteasome degradation pathway in this cluster (S5A Fig). Further examination showed that three cells in this cluster particularly upregulated nearly all proteasome genes (S5B Fig). We also confirmed that proteasome inhibition suppresses erebosis, accumulating polyubiquitinated proteins (S5C Fig). We note that the effects of proteasome inhibition should be interpreted with caution, as the proteasome regulates a wide range of biological processes. For example, proteasome inhibition can induce apoptosis by preventing DIAP1-mediated degradation of caspases [34,35]. Thus, to better define the relevant mechanism, it was important to manipulate specific pathways correlated with proteasome activation. For that purpose, we decided to screen DEGs in the proteasome-high cells, which we interpreted as putative pre-erebotic cells.

For the screening, we focused on the number of GFP− nlsRFP+ enterocytes, as this state represents the most frequent early/intermediate stage of erebosis. We classified the extent of erebosis into three groups based on the number of erebotic cells per microscopic field (Fig 1A): class 0 – three or fewer erebotic cells; class 1 – between class 0 and 2; and class 2 – many (>30% of all enterocytes) erebotic cells.

As a result of the screening, we discovered that Heme oxygenase (Ho) RNAis reduced erebosis (Fig 1B). In guts expressing Ho RNAis, more than 50% were classified as class 0, whereas the control expressing mCherry RNAi showed less than 30% class 0 guts. Two independent RNAi stocks similarly reduced erebosis, indicating that the reduction is unlikely to be due to the off-target effect of RNAi. Knockdown efficiency was also confirmed by measuring mRNA levels (S6A Fig). Consistent with the design of the screening, Ho is upregulated in the pre-erebosis cells with proteasome enrichment (S5B Fig).

Ho is an enzyme that degrades heme, an iron-containing tetrapyrrole that plays essential roles in diverse biological functions, including oxygen transport, oxidative metabolism, and xenobiotic detoxification [36]. Heme can function by binding to proteins or, in its labile form, as an oxidant itself. Its degradation produces oxidative Fe²⁺ iron, as well as the antioxidants, bilirubin and carbon monoxide (CO).

Since Ho inhibition can affect many metabolites, including heme, bilirubin, Fe²⁺, and CO, the next step was to clarify which molecule regulates erebosis. We decided to test the effects of manipulating heme metabolism on erebosis. Heme is synthesized from glycine and succinyl-CoA via 5-aminolevulinic acid (5-ALA) [37](Fig 2A). It is degraded by Ho or further converted by Cox10 or Cchl [38–40]. In Drosophila, heme is exported out of the cell by Mrp5 [41]. We performed RNAi-mediated knockdown of genes involved in heme conversion or export. Inhibition of heme export with Mrp5 RNAi markedly reduced erebosis, while inhibition of heme conversion (Cox10, Cchl RNAi) showed only a mild trend of suppression (Fig 2B, 2C). As with Ho RNAi, these manipulations are expected to increase intracellular heme, indicating that heme itself, rather than its metabolites, influences erebosis. The stronger effects of Ho or Mrp5 suppression compared to conversion inhibition suggest that heme degradation and export are key processes limiting intracellular heme. Notably, Mrp5 is upregulated in pre-erebotic cells (S5B Fig), similar to Ho, which would be expected to further decrease the cytoplasmic heme. We confirmed that even under the conditions with reduced erebosis following RNAi treatment, GFP and Ance inversely correlate (Fig 2D), indicating that the persistence of GFP signals is not an artifactual effect and that heme controls erebosis.

We investigated whether Ho RNAi or Mrp5 RNAi, both of which achieved efficient gene knockdown (S6A, S6B Fig), truly affects intracellular heme levels. We visualized intracellular heme using the fluorescent heme sensor H-FluNox, which becomes fluorescent upon binding to heme in a live condition [42]. Imaging with H-FluNox revealed that these anti-erebotic RNAis increased intracellular heme levels in enterocytes (Fig 2E, 2F).

Besides the genetic approaches, we also increased the intracellular amount of heme nongenetically. Feeding of a heme precursor 5-ALA, which is known to elevate intracellular heme [42], also reduced erebosis (Fig 2G, 2H). From these results, we conclude that heme itself, rather than its metabolites, suppresses erebosis.

Transcriptomic analysis revealed that Ho is upregulated in pre-erebosis cells, suggesting that heme is decreased in the initiation process of erebosis. To investigate this possibility, we examined heme levels in enterocytes using the heme probe H-FluNox. Since signals of H-FluNox and GFP overlap, we sought for an alternative method to label erebotic cells. Although erebosis is distinct from necrosis, it is characterized by the acute and simultaneous occurrence of both cellular entry of extracellular Ance, which is normally located between enterocytes and the peritrophic membrane (Fig 3A), and loss of cytoplasmic proteins [19], due to transient membrane pore formation [43]. Feeding flies with Rhodamine B, a red fluorescent dye, prior to imaging enables selective labeling of erebotic cells (Fig 3B) [43]. Combining Rhodamine B-mediated pre-labeling of erebotic cells and heme imaging with H-FluNox, which was added at the time of imaging, we found that H-FluNox signals were weaker in Rhodamine+ erebotic cells compared to neighboring Rhodamine- normal enterocytes (Fig 3C). Quantitative analysis confirmed that erebotic cells significantly reduced H-FluNox signals, indicating that intracellular heme is reduced in these cells (Fig 3D).

To further test whether heme reduction induces erebosis, we artificially decreased intracellular heme in enterocytes by ectopic expression of Mrp5, a heme exporter. This manipulation led to an increase in erebosis (Fig 3E, 3F). GFP− erebotic cells exclusively accumulated Ance, again revealing that heme is upstream of GFP clearance and Ance incorporation (Fig 3G). These results indicate that the depletion of heme is a critical step for cells to initiate erebosis. Importantly, p35 expression did not reverse the effects of heme manipulation on erebosis (S6C, S6D Fig), indicating that these effects are not mediated by apoptosis.

Next, we explored how intracellular heme inhibits erebosis. Heme is known to serve multiple functions, such as acting as a coenzyme for cytochrome P450s and contributing to the generation of reactive oxygen species (ROS) [36]. Inhibition of heme oxygenase could potentially induce oxidative stress due to both the accumulation of oxidative labile heme and the decrease in reducing bilirubin. Since there are at least 85 cytochrome P450s in Drosophila [44], genetic knockdown may not be productive due to redundancy. Thus, we resorted to pharmacological approaches to inhibit cytochrome P450s. We treated flies with cytochrome P450 inhibitors 1-Aminobenzotriazole or Piperonyl butoxide, both of which are potent cytochrome p450 inhibitors and have been used in Drosophila adults [45,46]. Neither of them restored erebosis in enterocytes that expressed Ho RNAi or Mrp5 RNAi (Fig 4A), suggesting that cytochrome P450s are not responsible for heme-mediated regulation of erebosis. Next, we tested whether excess heme promotes ROS generation in the Drosophila midgut. Using dihydroethidium (DHE), we measured ROS levels in Mrp5 RNAi clones generated by the heat shock-induced flippase system, which enables sensitive detection of ROS levels in comparison to surrounding wildtype enterocytes. DHE levels were not changed between Mrp5 RNAi clones and surrounding control cells (Fig 4B). Additionally, a lipid peroxide marker Liperfluo signal was not increased by Ho RNAi or Mrp5 RNAi, whereas it was elevated by the positive control, bleomycin treatment (Fig 4C). Furthermore, feeding antioxidant N-acetylcysteine did not suppress erebosis (Fig 4D). Taken together, these results indicate that heme inhibits erebosis independent of cytochrome P450 activity or ROS generation.

To further explore molecular mechanisms by which heme suppresses erebosis, we employed RNA-seq analysis. We analyzed guts from two different RNAi lines for each of Ho RNAi and Mrp5 RNAi to minimize the off-target effects of RNAis. We discovered that both of Ho RNAi and Mrp5 RNAi upregulated Dad, a Dpp signaling marker gene [47] (Fig 5A). We also observed a modest trend toward increased expression of the Dpp ligands, Dpp and Gbb (S7A Fig). Dpp signaling regulates gut tissue homeostasis via its regulation of ISCs and enterocytes [8,48–51]. Consistent with the transcription data, erebotic cells showed weaker phosphorylated Mad signals (Fig 5B), indicating that Dpp signaling is suppressed during erebosis. RNAis for Dpp signaling components Tkv, Put, and Med increased erebosis (Fig 5C, 5D), while Dpp activation by Dpp or the constitutively active form of Tkv reduced it (Fig 5E). Moreover, Dpp activation reversed erebosis induction by Mrp5 overexpression, while Dpp inhibition suppressed erebosis inhibition by feeding of the heme precursor 5-ALA (Fig 5F, 5G). Dpp-mediated effects on erebosis were not suppressed by p35 (S7B, S7C Fig), indicating that the effects are independent of caspase activation. Taken together, heme positively regulates Dpp signaling, which in turn suppresses erebosis.

Finally, we investigated the role for heme-mediated regulation of erebosis in tissue homeostasis. Tissue homeostasis is maintained through a balance between cell death and proliferation [52]. To investigate whether erebosis is involved in this balance, we examined the effect of erebosis inhibition on ISC division in the midgut. ISC proliferation was assessed by a mitosis marker anti phospho-Histone H3 (pH3) staining. Quantification revealed a significant decrease of pH3+ ISCs when erebosis was suppressed (Fig 6A). In control guts, we observed 4–5 pH3+ ISCs per gut, consistent with previous reports [5]. In contrast, inhibition of erebosis by Ho RNAi or Mrp5 RNAi resulted in a marked reduction of mitosis, with fewer than one pH3+ ISC per gut on average, indicating that the decrease in ISC division counteracts the reduction in erebosis. As a result of this balanced mechanism between erebosis and mitosis, inhibition of erebosis did not change the gut size or the cell density (Fig 6B, 6C). Heme accumulation or depletion did not cause apoptosis or necrosis, as assessed using the GC3Ai sensor [53] and SYTOX (Figs 6D–6F, S8A–S8C). These results indicate that changes in erebosis are compensated for by ISC proliferation, rather than by other types of cell death. From these results, we conclude that erebosis cooperates with ISC proliferation and plays a crucial role in tissue homeostasis, maintaining a constant gut size.

Discussion

We found that heme decreases in erebotic cells, and experimentally induced heme reduction promotes erebosis. Conversely, heme accumulation inhibits erebosis. Downstream of heme, Dpp signaling suppresses erebosis. These findings indicate a causal relationship between intracellular heme levels and the initiation of erebosis via Dpp signaling. In pre-erebotic cells, upregulation of Ho and Mrp5 occurs. Taken together, we propose that heme reduction in enterocytes is a critical initial step in erebosis (Fig 6G).

Heme and its metabolism are well known to regulate the cellular oxidative–reductive state or modulate protein activities as a co-factor. Labile heme itself is pro-oxidative, while heme catabolism produces both antioxidants, bilirubin and CO, and prooxidant Fe²⁺. Due to the opposing roles for heme metabolism in redox regulation, both upregulation and downregulation of Ho have been implicated in cell death. Suppression of Ho or excess heme can promote multiple types of cell death, including apoptosis [54], autophagic cell death [55], and necrotic cell death [56–60], mainly due to oxidative stress due to increase of labile heme and/or decrease of bilirubin. On the other hand, Ho activation has also been associated with necrosis and ferroptosis [61–64], which is mainly attributable to prooxidant function of Fe²⁺ released from heme. Several studies also showed that excess heme inhibits apoptosis [65–67]. In contrast to these previous reports, heme regulates erebosis without inducing oxidative stress, as its manipulation did not change ROS and lipid peroxide levels. Furthermore, erebosis does not have any feature of apoptosis, necrosis, or autophagy [19], which phenotypically contrasts with the previously reported cell death controlled by heme metabolism. Thus, the relationship between heme and erebosis is unique and different from previously characterized heme-associated cell death.

Regarding signaling upstream of heme, since Ho is upregulated in proteasome-high pre-erebotic cells (S5B Fig) and proteasome inhibition suppresses erebosis (S5C Fig), we speculate that proteasome-mediated protein degradation plays a key role. Proteasome inhibition is expected to have a broad range of effects, and thus it will be important in future studies to identify the E2 and E3 ubiquitin ligases and their substrates that regulate erebosis. Downstream of heme, we found that heme enhances Dpp signaling, which suppresses erebosis. Dpp was reported to play critical roles in gut physiology by affecting both enterocytes and ISCs [8,48–51]. Dpp ligands are secreted by trachea [49], enterocytes [50], hemocytes [51], and visceral muscles [48]. At least, Dpp ligand transcription is mildly increased by heme increase (S7A Fig). How heme activates Dpp signaling in enterocytes, whether by affecting only Dpp ligands or also their receptors and downstream signaling cascade, remains to be clarified. Since our data suggest that heme itself suppresses erebosis independent of its metabolism and cellular oxidative stress, its binding to some proteins should be a key mechanism. Proteomics approaches, which recently uncovered numerous heme-interacting proteins besides traditional heme binding proteins [68,69], would elucidate regulatory mechanisms of heme-mediated Dpp signaling. In addition to identifying the upstream mechanism by which heme regulates Dpp signaling, how downstream signals of Dpp mediate erebosis needs to be clarified in the future study.

Regarding the role for heme-mediated erebosis in tissue homeostasis, we discovered that inhibition of erebosis also reduces ISC proliferation. It suggests that there is a mechanism that couples ISC proliferation and enterocyte erebosis. We have previously shown that ISCs are located in close proximity to erebotic cells, which possibly reflects the communication between enterocytes and ISCs [19]. Similarly, previous research also suggested a cross-talk mechanism between enterocytes and ISCs [18,70]. How ISCs sense erebotic enterocytes, either directly or indirectly, remains to be determined. Considering that Dpp ligands are transcriptionally increased under the conditions that suppress erebosis (S7A Fig), and that Dpp can inhibit ISC proliferation in some context [8,48–51], a parsimonious hypothesis is that Dpp signaling simultaneously regulates erebosis and ISC behavior, thereby coupling enterocyte loss with ISC proliferation. In addition, bulk RNA-seq analysis of guts under heme-increased conditions demonstrates changes in multiple potentially mitogenic secreted factors, including Wingless, EGF ligands, Drosophila insulin like peptides (Dilps) and Unpaired (S9 Fig), raising the possibility that these pathways may also contribute to intricate communication between erebotic cells and ISCs. Beyond biochemical autocrine or paracrine mechanisms, tissue-level biophysical properties such as intercellular tension may also influence ISC activity. Defining these intercellular communication mechanisms will be an important direction for future study.

Methods

Supporting information

Acknowledgments

We thank Elwyn Isaac for the Ance antibody, and Tatsushi Igaki, TRiP at Harvard Medical School, the Bloomington Stock Center and the VDRC stock center for fly stocks. We thank A. Matsushima for the management of IT infrastructure. We thank the Yoo lab members for helpful comments on the manuscript.

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