Xrp1 drives damage-induced cellular plasticity of enteroendocrine cells in the adult Drosophila midgut

  1. Qingyin Qian
  2. Hiroki Nagai
  3. Yuya Sanaki
  4. Makoto Hayashi
  5. Kenichi Kimura
  6. Yu-ichiro Nakajima
  7. Ryusuke Niwa

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Abstract

Cellular plasticity, the ability of a differentiated cell to adopt another phenotypic identity, is restricted under basal conditions, but can be elicited upon damage to facilitate regeneration. Such damage-induced cellular plasticity restores homeostasis and prevents pathology, yet its underlying molecular basis remains largely unexplored. Here, we reported damage-induced cellular plasticity of secretory enteroendocrine cells (EEs) in the adult Drosophila midgut. We found that ionizing radiation enhanced EE plasticity such that it promoted EEs to dedifferentiate into ISCs and subsequently re-differentiate towards ECs. We identified that radiation induced the production of a stress-inducible transcription factor Xrp1 in EE lineages, and its upregulation was necessary for EE plasticity. Single-cell RNA sequencing of guts with EE-specific Xrp1 overexpression revealed ectopic expression of progenitor-specific genes in EEs, which was necessary for Xrp1 to drive EE plasticity. Our work provides a mechanistic framework for understanding cellular plasticity and suggests its potential role in damage-induced responses.

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    Reply to the reviewers

    Manuscript number: RC-2025-03111

    Corresponding author(s): Qingyin Qian and Ryusuke Niwa

    1. General Statements [optional]

    We would like to thank reviewers for their feedback on our initial submission. Changes in figures were noted in the point-to-point reply. For submission of our current revised manuscript, we provide two Word files, which are the “clean” and “Track-and-Change” files. Page and line numbers described below correspond to those of the “clean” file. The “Track-and-Change” file might be helpful for Reviewers to find what we have changed for the current revision.

    In the revised manuscript, major changes in the text were tracked, while minor edits in figure numbers and legends were not tracked. In the Discussion, the section “Xrp1-mediated EE plasticity…” was moved before “Xrp1, a transcription factor …”, to follow the order of the Results, and was split into two: “EE plasticity …” and “Xrp1-mediated EE plasticity …”.

    2. Description of the planned revisions

    - The authors should investigate the regenerative growth of the adult midgut after irradiation. Is there an impact on ISCs proliferation or cell turn over. Is Xrp1 in EEs required in this adaptive response. It would be elegant to use the recently generated tracing method by Tobias Reiff lab to observe overall impact on tissue renewal (rapport-tracing esglexReDDM esg-lexA, 13xLexAop2-CD8::GFP, 13xLexAop2-H2B::mCherry::HA, tub-Gal80ts on the second chromosome. It can be combined with any EEs Gal4-driver (see Nat Commun 2025, https://doi.org/10.1038/s41467-024-55664-2, the stock is already existing, see table1). This reviewer thinks that it is a key experiment to support the proposed model.

    2.1. Author response:

    We will conduct the following experiments to answer these criticisms.

    (1) We will investigate the ISC behavior, proliferation and differentiation, after 100 Gy of radiation by examining changes in the number of progenitor cells and their progenies, using esgtsF/O (esg-Gal4,* UAS-GFP, tub-Gal80ts; Act>Cd2>Gal4, UAS-Flp) generated in the study (Jiang et al. Cell 2009 DOI: 10.1016/j.cell.2009.05.014) or esgReDDM (esg-Gal4, UAS-CD8::GFP; UAS-H2B::RFP, tubGal80ts*) generated in the study (Antonello et al. EMBO J. 2015 DOI: 10.15252/embj.201591517). Flies will have progenitor cell lineages traced for 7 days, irradiated on day 6, and examined at different time points after radiation, following the design shown in Fig. 2A. Based on the previous findings (Sharma et al. Sci. Rep. 2020 DOI: 10.1038/s41598-020-75867-z; Pyo et al. Radiat. Res. 2014 DOI: 10.1667/RR13545.1), we anticipate that radiation compromises ISCs’ proliferation and differentiation. Should this be the case, our results can be interpreted in relation to those earlier studies.

    (2) In parallel, we will examine whether Xrp1 expression in EEs affects radiation-induced ISC behaviors. As suggested, we will use “EE Rapport” (esg-lexA, 13xLexAop2-CD8::GFP, 13xLexAop2-H2B::mCherry::HA, tub-Gal80ts; Rab3-Gal4) generated in the study (Zipper et al. Nat. Commun. 2025 DOI: 10.1038/s41467-024-55664-2) and compare control flies to flies with *Xrp1 *knocked down in EEs to assess the impact on ISC behaviors.

    - Is p53 required for Xrp1 induction in the gut after irradiation?

    2.2. Author response:

    To answer this point, we will perform immunostaining of anti-Xrp1 antibody to examine whether p53 is required for Xrp1 induction in irradiated flies with *p53 *knocked down in EEs.

    - Xrp1 over expression has been shown to induce upd3 ligand and nutrient-driven dedifferentiation of enteroendocrine cells is occuring by activation of the JAK-STAT pathway (DOI: 10.1016/j.devcel.2023.08.022). Could the authors test the function of this signaling pathway during irradiation (upd3-lacZ and Stat-GFP can be used in parallel of upd3 RNAi and UAS Dome-DN.

    2.3. Author response:

    We will conduct the following experiments to answer these points.

    (1) We will examine the cell type in which upd3 ligand induction occurs after radiation by using the upd3.1-LacZ reporter generated in the study (Jiang et al. Cell Stem Cell 2011 DOI: doi.org/10.1016/j.stem.2010.11.026).

    (2) One possibility is that upd3.1-LacZ is detected in EEs. In this case, we will examine the requirement of upd3 in EEs for radiation-induced EE plasticity by knocking down upd3. Another possibility is that upd3.1-LacZ is detected in non-EE cells. If so, we will examine the requirement of the JAK-STAT pathway in EEs by overexpressing dome[△cyt] generated in the study (Brown et al. Curr. Biol. 2001 DOI: 10.1016/s0960-9822(01)00524-3) or knocking down Stat92E in EEs. Because these conditions are not mutually exclusive, both approaches may be pursued, with the latter relating our results to nutrient-driven EE dedifferentiation.

    - Xrp1 is known for its role in cell competition and elimination of looser cells by induction of apoptosis. It would be interesting to check for induction of cell death and/or caspase activation in the fly gut after irradiation and verify a non apoptotic role of DRONC activation in this context using a Dronc RNAi (as proposed by Bergmann lab (https://doi.org/10.1038/s41598-021-81261-0) or Baena-Lopez lab (DOI: 10.15252/embr.201948892)). Overexpression of Xrp1 could be combined with UAS-p35.

    2.4. Author response:

    To address these points, we will investigate apoptosis induction following radiation with anti-cleaved Dcp-1 immunostaining. Based on the previous finding (Sharma et al. Sci. Rep. 2020 DOI: 10.1038/s41598-020-75867-z), we anticipate seeing increased cleaved Dcp-1 signals in all cell types after radiation. We intend to clarify whether radiation increases the ratio of apoptotic EEs among EEs; however, we cannot yet be certain whether it will be feasible.

    Regarding Dronc activation, we previously requested the antibody used in the study (Wilson et al. Nat. Cell Biol. 2002 DOI: 10.1038/ncb799; Lindblad et al. Sci. Rep. 2021 DOI: 10.1038/s41598-021-81261-0) and tested it in our context, after radiation and by Xrp1-S O/E in EEs. We present our data below. In the anterior midgut, anti-Dronc signals were not observed under both control conditions. After radiation and by Xrp1-S O/E in EEs, anti-Dronc signals were seen in part of past EEs (#2 past) and progenitor cells (#3 prgn), implying their EB identity. However, anti-Dronc signals were never observed in current EEs (#1 current), suggesting Dronc does not act directly downstream to* Xrp1*.

    We will address UAS-p35 in 3.3. Author response and Dronc-RNAi in 4.2. Author response.

    - The authors do not justify or explain why they used 100 Gy of radiation. This is higher than doses used in comparable regeneration studies in adult Drosophila (e.g., PMID25959206, PMID: 28925355). The authors should clarify why this dose was chosen.

    2.5. Author response:

    Our initial rationale was based on the paper (Sharma et al. Sci. Rep. 2020 DOI: 10.1038/s41598-020-75867-z), where the authors claimed that ISC proliferation was inhibited and the ISC number was decreased by 100 Gy of radiation.

    Nevertheless, we understand the reviewer’s concern and will examine 50 Gy of radiation as used in the papers the reviewer listed. We will examine radiation-induced changes in EE lineages and ISC behaviors. Depending on the results, we will evaluate whether and how they should be incorporated into the manuscript.

    - Fig. 2C, the number of past EE’s increased transiently so that baseline number is restored at 18 hr after IR. The authors conclude that fate plasticity is a transient event. Can they rule out loss due to cell death?

    2.6. Author response:

    In our system, past EEs were detected transiently but did not persist. We agree that we cannot distinguish whether the transient appearance of past EEs reflects transient adoption of another identity that ends in cell death or reversible plasticity.

    To partially address this criticism, as noted in 2.4. Author response, we will examine the apoptosis marker cleaved Dcp-1, which also tests whether cleaved Dcp-1-positive cells can be past EEs. However, regardless of detecting apoptosis markers in past EEs, we have changed “transient” into “temporary” to describe a short-lived cell state (see Page 8, Line 178; Page 15, Line 338).

    - They authors interpret fate-conversion as beneficial for tissue repair but never test whether blocking this process impairs recovery or organismal survival or whether promoting it improves outcomes.

    2.7. Author response:

    We have removed this potentially misleading interpretation (see Page 4, removed the last part of the previous introduction, “and propose the possibility that such plasticity contributes to tissue repair”). We present below the data showing a severe reduction of the ISC number in 7-day post-radiation guts, suggesting the inability of tissue repair. We will add this to the manuscript together with results from the following experiments.

    (1) We will examine if the blockage of radiation-induced EE plasticity, via knocking down Xrp1 in EEs, alters the epithelial cell number and cell junction protein localization.

    (2) To complement the result of plasticity inhibition, we attempt to promote plasticity by overexpressing Xrp1 in EEs, to test whether this rescues ISC loss or restores junctions.

    Should knockdown worsen ISC loss and junction integrity, or overexpression rescue them, we will describe EE plasticity as beneficial; otherwise, we will present it as a radiation-induced response without inferring benefits, while noting our limitations.

    We will address organismal survival in 4.3. Author response.

    - Related to the above, it would be helpful to know if fate-converted cells function as true ISCs or ECs (e.g., through proliferation or absorption assays).

    2.8. Author response:

    To partially answer this criticism, we will examine whether EE-derived ISCs are proliferative by examining whether they can be positive for the mitotic marker phospho-histone 3.

    We will address absorption assays in 4.4. Author response.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    - It is surprising to observe EEs dedifferentiation at a steady state during homeostasis, a condition in which Xrp1 is not detected in the gut. Can the authors comment this point in the discussion?

    3.1. Author response:

    We have added our thoughts in terms of Xrp1 being not detectable in homeostatic EE lineages (see Page 15, Line 350 - 356). We have also added our thoughts regarding observation of EE plasticity in homeostatic guts (see Page 14, Line 322 - 332).

    - Xrp1 is existing as a short of long isoforms. The short form has been recently proposed to be required for cell competition (https://doi.org/10.1101/2025.06.15.659587) whereas Xrp1 long isoform may be responsible for reduced cell growth. Could the authors test which isoform is induced in the gut after irradiation? Is the overexpression of Xrp1 long isoform having the same effect that the short isoform used by the authors.

    3.2. Author response:

    We have added data on the effect of Xrp1 long isoform overexpression on EE plasticity (see Fig. 5A - 5B, Page 12, Line 276 - 278), showing that overexpression of the* Xrp1* long isoform caused a similar increase in past EEs. In addition, we have changed Xrp1 O/E to *Xrp1-S *O/E in the contents related to Figs 4, 5, S4, and S5.

    We will address radiation-induced Xrp1 isoforms in 4.1. Author response.

    - Xrp1 is known for its role in cell competition and elimination of looser cells by induction of apoptosis. It would be interesting to check for induction of cell death and/or caspase activation in the fly gut after irradiation and verify a non apoptotic role of DRONC activation in this context using a Dronc RNAi (as proposed by Bergmann lab (https://doi.org/10.1038/s41598-021-81261-0) or Baena-Lopez lab (DOI: 10.15252/embr.201948892)). Overexpression of Xrp1 could be combined with UAS-p35.

    3.3. Author response:

    We have added data regarding p35 O/E combined with Xrp1 O/E, showing that p35 O/E did not further increase the number of past EEs, thereby suggesting that* Xrp1*-driven EE plasticity has a non-apoptotic nature (see Fig. 5C - 5D, Page 13, Line 293 - 297).

    - Line 221: fig S3E should be S3F

    - Line 230: fig S3F-G should be S3G-H

      • Line 230, Fig S3F-G should be Fig S3G-H.*

    3.4. Author response:

    We have fixed this error.

    - The posterior gut region R4 is more proliferative than the anterior part and is usually used for testing regenerative growth. What is happening there after irradiation?

    3.5. Author response:

    We present below radiation-induced changes in EE lineages and ISC number in the R4bc gut region. Radiation did not alter the proportion of past EEs among EE lineages but reduced the ISC number. We acknowledge differences between anterior and posterior gut regions, but we do not plan to further analyze regional differences or underlying mechanisms.

    - The authors’ explanation for cells with weak GFP in Figure 1 is not convincing. Induction of GFP is an all or nothing event as it results from Pros-driven FLPase and a recombination that removes the transcription stop signals to express GFP from a Ubi promotor. Once that happens, it should not matter how strong or weak Pros is, GFP should be the same. So, another explanation is needed. Nuclear staining of cell #2 in Fig 1B resembles a metaphase chromosome arrangement. Nuclear GFP may appear ‘weak’ in mitosis as the nuclear envelope breaks down. It is positive for the purple Pros/Dl stain, which makes it hard to tell if it is Pros+ or Pros- even though the authors state that cells with weak GFP are Pros- in line 104 (see the point above regarding confusing same-color stain for ISC and EE markers). Could cell #2 be a pre-EE that is undergoing mitosis since the lineage tracer marks both EE and pre-EE cells (line 119)? Or do the authors mean recombination on one or both homologs? This should not be possible since the cells are heterozygotes for the Ubi-GFP locus.

    3.6. Author response:

    For cell #5, RFP- GFPweak may result from the leakiness of the G-TRACE system. We have added our observations of the G-TRACE strains and changed our previous explanation (see Fig. S1B - S1C, Page 5, Line 94 - 97, 103 - 106).

    For cell #2, we agree that RFP+ GFPweak cells may either be a cell turning on pros expression just before sample preparation or a pre-EE undergoing mitosis. Nevertheless, it is not a past EE that has lost the EE marker Pros, so it is considered a current EE. We have removed our previous interpretation of cell #2 (see Page 5, removed “which likely had not yet fully activated recombination”), and changed the image to avoid confusion (see Fig. 1C).

    - Fig. 2C, if past-EE’s increased in number while current EE’s stayed the same, where are new past-EE’s coming from? There cannot be compensatory proliferations since EE’s are post-mitotic. For fate conversion, one would expect the generation of each past-EE to accompany loss of one current EE.

    3.7. Author response:

    We agree that the generation of one past EE should be accompanied by the loss of one current EE. We do not have a clear answer to this question. Our data showed cell numbers per ROI rather than the total cell number across the whole gut. To address this, we have changed the number to the proportion, calculated from [past EE] / ([past EE] + [current EE]), in experiments examining damage-induced EE plasticity, which provides a more informative measure for EE fate conversion (see Fig. 2C, also Fig. S2B and 3E).

    - Fig. 2E. Dl+ past-EE cell number declined at 14 and 18 h after IR and because cell sized increased, the authors conclude that EE cells that de-differentiated into ISCs subsequently re-differentiated into EC’s. To reach this conclusion, the authors should count past-EEs that are positive for EC markers. Cell size alone is insufficient evidence.

    3.8. Author response:

    We have added data quantifying the proportion of past EEs that are positive for the EC marker Pdm1, showing that past EEs were more likely to be ECs in guts examined 14 h after radiation (see Fig. 2F - 2G, Page 9, Line 189).

    - Fig. 6. Where are the % numbers for ISC, EB and EE’s coming from? And wouldn’t these change with time after IR, etc?

    3.9. Author response:

    The numbers came from the calculation of the percentage of the absolute values of control and 14 h post-IR conditions from Fig. 2E. These numbers changed with time after radiation. We realized that the precise numbers were misleading. We therefore have removed such illustration and instead added phrases “more current EEs → past EEs, more past EEs being ISCs → past EEs being ECs” to describe the increase in past EE cell number and the shift in the composition of past EEs (see Fig. 6).

    - Improve Figure 1B: Pros and Dl are shown in the same color, creating confusion. If both are stained together, different colors or clearer labeling should be used. Clarify how cells are identified as Pros+ vs Dl+.

    3.10. Author response:

    Anti-Pros and anti-Dl antibodies were produced from the same host species and were detected with the same secondary antibody, so they were in the same color. We have stated that solid nuclear staining indicates Pros, whereas punctate cytoplasmic staining indicates Dl (see Page 5, Line 100, 102, and 103). Such staining has been reported in previous studies (for example, Fig. 2A - 2B, Veneti et al. Nat. Commun. 2024 DOI: 10.1038/s41467-024-46119-9).

    - Why is Dl (supposed to be cytoplasmic) overlapping with nuclear GFP in cells #3 and 4 in Fig. 1B?

    3.11. Author response:

    Because Dl signals were located apically to DAPI/GFP signals, the overlap was likely due to Z-projection from stacked slices. We present below orthogonal slices along the z-axis, from top to bottom by row, and composite and individual color channels, from left to right by columns, for cell #3 (left) and cell #4 (right).

    For cell #3, Dl signals were present in slices 1/8 and 2/8 and disappeared in slice 3/8, whereas DAPI signals appeared from slice 2/8. For cell #4, Dl signals surrounded DAPI signals when viewed separately. In addition, we realized that nuclear GFP signals slightly outgrew DAPI signals, despite our confirmation that the GFP channel was not saturated.

    We have included separate color channels for DAPI signals and Pros, Dl and DAPI merged channels, showing that Dl signals were absent from the nucleus. For cell #3, in which the nuclear DAPI and cytoplasmic Dl cannot be distinguished in the stacked view, we show the images from a single orthogonal slice in the main panel, and the image from stacked slices as insets (see Fig. 1C).

    - Fig. S1E and F. Very hard to see what the authors describe about Arm and Cora. One problem is that cell boundaries are not visible, just the nuclei, so it is hard to know whether cell-cell interactions the authors describe as normal are really normal. Another problem is the overlap of Arm (supposed to be cytoplasmic) with the nuclear GFP signal. What is that?

    3.12. Author response:

    Regarding the invisibility of cell boundaries, we have improved the image of anti-Cora staining and added anti-Mesh staining and a separate color channel for DAPI signals to reinforce junction integrity (see Fig. S1H - S1I).

    Regarding the overlap of Arm signals with nuclear GFP signals, we realized similar problems as those noted in 3.11. Author response. We present below orthogonal slices along the z-axis and combined and individual color channels, for cell #2 (left) and cell #3 (right). For both cells, Arm signals did not overlap with DAPI signals. We have adjusted the maximum intensity projection to include slices 1-4 instead of 1-8 and added a separate color channel for DAPI signals to avoid the signals appearing to overlap (see Fig. S1G).

    - Include a simple schematic of ISC to EE/EC lineages for readers unfamiliar with Drosophila gut biology.

    3.13. Author response:

    We have included a schematic (see Fig. 1A). Although not requested, we have also improved Fig. 1B to enhance clarity.

    - Discuss the regional difference in Xrp1 efficacy (R2a vs R2b). Is there something known about gene expression differences in different gut regions that can explain the results?

    3.14. Author response:

    At present, we do not have an explanation for these results. We have refined our discussion regarding such regional differences (see Page 16 - 17, Line 381 - 390).

    - Consider moving scRNAseq (Fig. S1G) into main paper: this is a central part of the conclusion.

    3.15. Author response:

    We have moved Fig. S1G, as well as Fig. S1H and S1I, into the main figure (see Fig. 1G - 1I).

    4. Description of analyses that authors prefer not to carry out

    - Xrp1 is existing as a short of long isoforms. The short form has been recently proposed to be required for cell competition (https://doi.org/10.1101/2025.06.15.659587) whereas Xrp1 long isoform may be responsible for reduced cell growth. Could the authors test which isoform is induced in the gut after irradiation? Is the overexpression of Xrp1 long isoform having the same effect that the short isoform used by the authors.

    4.1. Author response:

    We prefer not to distinguish whether the long or short Xrp1 isoform is induced in the gut after radiation. This presents technical challenges and falls outside the scope of the present study. As noted in 3.2. Author response, we instead report in the revised manuscript that both isoforms similarly promote EE plasticity.

    - Xrp1 is known for its role in cell competition and elimination of looser cells by induction of apoptosis. It would be interesting to check for induction of cell death and/or caspase activation in the fly gut after irradiation and verify a non apoptotic role of DRONC activation in this context using a Dronc RNAi (as proposed by Bergmann lab (https://doi.org/10.1038/s41598-021-81261-0) or Baena-Lopez lab (DOI: 10.15252/embr.201948892)). Overexpression of Xrp1 could be combined with UAS-p35.

    4.2. Author response:

    We prefer not to perform Dronc-RNAi, because we did not observe Dronc activation downstream to Xrp1, as shown in 2.4. Author response.

    - They authors interpret fate-conversion as beneficial for tissue repair but never test whether blocking this process impairs recovery or organismal survival or whether promoting it improves outcomes.

    4.3. Author response:

    We prefer not to examine organismal survival. We agree that organismal survival would be informative, but our study focuses on epithelial cell number, which will be tested as noted in 2.7. Author response. We will not mention broad claims at the organismal level.

    - Related to the above, it would be helpful to know if fate-converted cells function as true ISCs or ECs (e.g., through proliferation or absorption assays).

    4.4. Author response:

    We prefer not to perform absorptive assays due to technical challenges. We will instead test proliferation, as noted in 2.8. Author response, and note our limitations.

  2. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    Qian and colleagues report a study on radiation induced cell fate plasticity in the intestine of Drosophila. Using lineage tracing to mark pre-EE and EE cells, the authors how that these cells can lose EE/pre-EE marker Pros and express ISC or EC markers, indicating fate conversion. Single cell RNAseq analysis showed that even under basal conditions, ISC/EB cell population includes those with EE/pre-EE lineage tracer, confirming fate conversion. The same analysis showed that fate converted ISC/EB cells express transcription factor Ets21C, which is associated with regeneration but not normal development. Exposure to ionizing radiation (IR) increases the frequency of fate conversion and accompanies the induction of Xrp1 (which is not expressed normally in the EE's). Xrp1 knock down reduced IR-induced fate conversion, demonstrating necessity. Xrp1 is also sufficient because overexpression of it resulted in increased fate conversion without IR. scRNAseq analysis showed that overexpression of Xrp1 in pre-EE/EE cells (without IR) resulted in the induction of ISC/progenitor state genes such as esg and Sox homologs. Functional testing of the latter group of genes demonstrated their essential role in cell fate plasticity induced by Xrp1.

    Major comments

    • The authors do not justify or explain why they used 100 Gy of radiation. This is higher than doses used in comparable regeneration studies in adult Drosophila (e.g., PMID25959206, PMID: 28925355). The authors should clarify why this dose was chosen.
    • The authors' explanation for cells with weak GFP in Figure 1 is not convincing. Induction of GFP is an all or nothing event as it results from Pros-driven FLPase and a recombination that removes the transcription stop signals to express GFP from a Ubi promotor. Once that happens, it should not matter how strong or weak Pros is, GFP should be the same. So, another explanation is needed. Nuclear staining of cell #2 in Fig 1B resembles a metaphase chromosome arrangement. Nuclear GFP may appear 'weak' in mitosis as the nuclear envelope breaks down. It is positive for the purple Pros/Dl stain, which makes it hard to tell if it is Pros+ or Pros- even though the authors state that cells with weak GFP are Pros- in line 104 (see the point above regarding confusing same-color stain for ISC and EE markers). Could cell #2 be a pre-EE that is undergoing mitosis since the lineage tracer marks both EE and pre-EE cells (line 119)? Or do the authors mean recombination on one or both homologs? This should not be possible since the cells are heterozygotes for the Ubi-GFP locus.
    • Fig. 2C, if past-EE's increased in number while current EE's stayed the same, where are new past-EE's coming from? There cannot be compensatory proliferations since EE's are post-mitotic. For fate conversion, one would expect the generation of each past-EE to accompany loss of one current EE.
    • Fig. 2C, the number of past EE's increased transiently so that baseline number is restored at 18 hr after IR. The authors conclude that fate plasticity is a transient event. Can they rule out loss due to cell death?
    • Fig. 2E. Dl+ past-EE cell number declined at 14 and 18 h after IR and because cell sized increased, the authors conclude that EE cells that de-differentiated into ISCs subsequently re-differentiated into EC's. To reach this conclusion, the authors should count past-EEs that are positive for EC markers. Cell size alone is insufficient evidence.
    • Fig. 6. Where are the % numbers for ISC, EB and EE's coming from? And wouldn't these change with time after IR, etc?
    • They authors interpret fate-conversion as beneficial for tissue repair but never test whether blocking this process impairs recovery or organismal survival or whether promoting it improves outcomes.
    • Related to the above, it would be helpful to know if fate-converted cells function as true ISCs or ECs (e.g., through proliferation or absorption assays).

    Minor comments

    • Improve Figure 1B: Pros and Dl are shown in the same color, creating confusion. If both are stained together, different colors or clearer labeling should be used. Clarify how cells are identified as Pros+ vs Dl+.
    • Why is Dl (supposed to be cytoplasmic) overlapping with nuclear GFP in cells #3 and 4 in Fig. 1B?
    • Fig. S1E and F. Very hard to see what the authors describe about Arm and Cora. One problem is that cell boundaries are not visible, just the nuclei, so it is hard to know whether cell-cell interactions the authors describe as normal are really normal. Another problem is the overlap of Arm (supposed to be cytoplasmic) with the nuclear GFP signal. What is that?
    • Include a simple schematic of ISC to EE/EC lineages for readers unfamiliar with Drosophila gut biology.
    • Discuss the regional difference in Xrp1 efficacy (R2a vs R2b). Is there something known about gene expression differences in different gut regions that can explain the results?
    • Consider moving scRNAseq (Fig. S1G) into main paper: this is a central part of the conclusion.
    • Line 230, Fig S3F-G should be Fig S3G-H.

    Significance

    Xrp1 is known to have a role in DNA Damage Responses and in cell competition and to function in the context of the p53 network, but this is the first time its role in fate conversion has been demonstrated. For the most part, the data are convincing and include strong genetic evidence from loss- and gain-of-function approaches that demonstrate a role for Xrp1 in activating progenitor gene expression and fate conversion. However, there are several experimental and presentation issues that need to be addressed first as outlined in the previous sections.

    The work highlights how mature cells may revert to stem-like states in response to injury, a theme with broad relevance in regenerative medicine.

    My field of expertise lies in DNA damage responses in Drosophila and human cancer models.

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    Referee #1

    Evidence, reproducibility and clarity

    Adult tissue homeostasis refers to the process by which tissues maintain a stable and functional state over time. This usually depends on stem cell activity and the balance between cell proliferation and differentiation to ensure that tissues can repair damage, replace old or dead cells, and maintain their structure and function.

    Damage-induced plasticity plays an important role in restoring tissue homeostasis. Cellular plasticity is the ability of differentiated cells to acquire alternative phenotypic identities. It is typically constrained under homeostatic conditions but can be activated in response to tissue damage to support regeneration. In this study entitled "Xrp1 drives damage-induced cellular plasticity of enteroendocrine cells in the adult Drosophila midgut", Qian Q. et al., describe damage-induced plasticity of secretory enteroendocrine cells (EEs) in the adult Drosophila midgut. They found that ionizing radiation enhances EE plasticity, enabling EEs to dedifferentiate into intestinal stem cells (ISCs), which subsequently re-differentiate into absorptive enterocytes (ECs). Mechanistically, radiation triggers the expression of Xrp1, a stress-responsive transcription factor, within EE lineages. Xrp1 upregulation is necessary for initiating EE plasticity by expressing progenitor specific genes (like escargot for example), as verified by single-cell RNA sequencing of midguts with EE-specific Xrp1 overexpression. This is suggesting that Xrp1 reprograms EEs by promoting progenitor-like transcriptional states.

    The authors nicely describe the dedifferentiation of EEs using the G-TRACE system in response to irradiation and the role of Xrp1 in this process. Yet, the authors need to show the requirement of the EEs dedifferenciation during regenerative growth.

    Major comments:

    • The authors should investigate the regenerative growth of the adult midgut after irradiation. Is there an impact on ISCs proliferation or cell turn over. Is Xrp1 in EEs required in this adaptive response. It would be elegant to use the recently generated tracing method by Tobias Reiff lab to observe overall impact on tissue renewal (rapport-tracing esglexReDDM esg-lexA, 13xLexAop2-CD8::GFP, 13xLexAop2-H2B::mCherry::HA, tub-Gal80ts on the second chromosome. It can be combined with any EEs Gal4-driver (see Nat Commun 2025, https://doi.org/10.1038/s41467-024-55664-2, the stock is already existing, see table1). This reviewer thinks that it is a key experiment to support the proposed model.
    • It is surprising to observe EEs dedifferentiation at a steady state during homeostasis, a condition in which Xrp1 is not detected in the gut. Can the authors comment this point in the discussion?

    Minor comments:

    • Is p53 required for Xrp1 induction in the gut after irradiation?
    • Xrp1 is existing as a short of long isoforms. The short form has been recently proposed to be required for cell competition (https://doi.org/10.1101/2025.06.15.659587) whereas Xrp1 long isoform may be responsible for reduced cell growth. Could the authors test which isoform is induced in the gut after irradiation? Is the overexpression of Xrp1 long isoform having the same effect that the short isoform used by the authors.
    • Xrp1 over expression has been shown to induce upd3 ligand and nutrient-driven dedifferentiation of enteroendocrine cells is occuring by activation of the JAK-STAT pathway (DOI: 10.1016/j.devcel.2023.08.022). Could the authors test the function of this signaling pathway during irradiation (upd3-lacZ and Stat-GFP can be used in parallel of upd3 RNAi and UAS Dome-DN.
    • Xrp1 is known for its role in cell competition and elimination of looser cells by induction of apoptosis. It would be interesting to check for induction of cell death and/or caspase activation in the fly gut after irradiation and verify a non apoptotic role of DRONC activation in this context using a Dronc RNAi (as proposed by Bergmann lab (https://doi.org/10.1038/s41598-021-81261-0) or Baena-Lopez lab (DOI: 10.15252/embr.201948892)). Overexpression of Xrp1 could be combined with UAS-p35.
    • Line 221: fig S3E should be S3F
    • Line 230: fig S3F-G should be S3G-H
    • The posterior gut region R4 is more proliferative than the anterior part and is usually used for testing regenerative growth. What is happening there after irradiation?

    Significance

    Altogether, the paper present compiling lines of evidence supporting the proposed model. The experiments are well designed and are convincing. The papers is interesting and relevant for a broad audience.

  4. Version published to 10.1101/2025.07.05.662934 on bioRxiv