JNK signaling-mediated exocytosis coordinates epithelial cell-turnover in Drosophila ribosomal protein mutants

  1. Nanami Akai
  2. Tatsushi Igaki
  3. Shizue Ohsawa

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Abstract

Robust tissue growth is orchestrated by the precise coordination of cell death and cell proliferation. Our previous study found that in the developing wing pouches of Drosophila Minute /+ animals, both cell death and compensatory cell proliferation are dramatically increased, which contributes to robust growth of mutant tissue. The induction of this cell-turnover depends on activation of JNK signaling, although the mechanism by which JNK activation induces cell-turnover remained unclear. Here, we show that JNK-mediated elevation of exocytosis in dying cells is crucial for triggering cell-turnover in M/+ wing morphogenesis. Mechanistically, elevated JNK signaling in dying cells upregulates exocytosis-related genes and Wingless (Wg), leading to enhanced Wg secretion. Furthermore, this exocytosis-mediated Wg secretion generally occurs downstream of JNK signaling, regardless of the genetic background. Overall, our findings provide mechanistic insights into robust tissue growth through the orchestration of cell-turnover, which is primarily governed by JNK-mediated exocytosis during Drosophila Minute/+ wing morphogenesis.

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  1. Version published to 10.1101/2023.12.27.573431v3 on bioRxiv
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    Reply to the reviewers

    Reply to the Reviewers

    We would like to thank the reviewers for their insightful comments, which will significantly guide us in enhancing our manuscript. We are capable of addressing most of the concerns raised by the reviewers. However, we encounter limitations in addressing Reviewer 2's comment regarding the delineation between cell autonomy and non-autonomy. As highlighted by the reviewer, ideally, we would dissect the mechanisms underlying cell turnover within the M/+ wing pouch, particularly in terms of cell autonomy and non-autonomy. Unfortunately, clonal analysis is not feasible due to the 'salt and pepper' distribution pattern of cell death and subsequent proliferation within the M/+ wing pouch. Despite these challenges, we commit to making efforts to address these issues to the best of our ability. Furthermore, we will aim to present our findings with greater precision and without speculation, an approach we believe will significantly enhance the quality of our manuscript.

    In this manuscript, we extend the insights gained from our previous study (Akai et al., PLOS Genetics, 2021) by uncovering a novel mechanism within the context of the M/+ mutant. We demonstrate that JNK-dependent exocytosis in dying cells is crucial for cell turnover in the M/+ wing pouch. This phenomenon of cell turnover, initiated by cell death and the following proliferation, is fundamental to a variety of processes in multicellular organisms, such as normal development, tissue homeostasis, wound healing, tumor development, and potentially tumor recurrence. While our analysis is specific to the M/+ mutant, the underlying mechanisms of cell-cell communication between dying and proliferating cells, which are still not fully understood, may have broader implications. Our findings offer significant contributions to the field of cell competition and suggest a framework for understanding the operating principles of multicellular communities through cell-cell communications. Although it is yet to be determined how these insights apply in various contexts, they possess the potential to guide future research.

    Referee #1

    It would be important to check how much JNK is sufficient to trigger the exocytosis upregulation. Is the accumulation of Cyt1 and CD63 vesicles in apoptotic cell common to any JNK dependent death or does it require the Minute background? Could the authors check whether clones expressing HepCA transiently in WT background also accumulate the same vesicles?

    Response:

    Following the reviewer's suggestion, we plan to examine whether the accumulation of Syt1- and CD63-positive vesicles in apoptotic cells is a general characteristic of JNK-dependent cell death or requires the Minute background. Specifically, we intend to check whether mitotic clones expressing HepCA in the wild-type eye-antennal disc to see if these vesicles also accumulate. Additionally, we will assess mitotic clones expressing Eiger in the wild-type eye-antennal disc, which activates JNK signaling. This is due to concerns that HepCA may induce cell death too strongly, potentially resulting in clones too small for effective analysis. Moreover, we also plan to express HepCA transiently only in the posterior region of the wing disc to further explore its effects.

    1. It would be relevant to have the status of JNK in Minute disc upon downregualtion of exocytosis. One could imagine some positive feedback between JNK activation, exocytosis, cell death and further JNK activation.

    Response:

    We thank the reviewer for the comment. As pointed out by the reviewer, there could be a positive feedback loop involving JNK activation, exocytosis, cell death, and further JNK activation. Our observations lend support to this hypothesis, as we specifically noted a significant reduction in the number of JNK-activating cells following targeted rab3 knockdown in these cells using puc-gal4 driver within the RpS3/+ wing disc (Fig R1, below). To further confirm the positive feedback loop between JNK activation and exocytosis, we plan to investigate the effects of downregulating unc-13 on JNK activation within the M/+ wing pouch, employing the JNK reporter TRE-DsRed for this purpose.

    Additionally, we need to elucidate the molecular mechanism linking cell death and exocytosis. We are currently exploring two potential relationships between JNK activation, caspase signaling, and exocytosis, as depicted in Fig R2 below. To assess these possibilities, we aim to investigate if inhibiting apoptosis-either by introducing the H99/+ mutation or by overexpressing DroncDN or mirRHG-can affect JNK-mediated exocytosis in the RpS3/+ wing pouch.

    So far, the evidence for the epistatic link between exocytosis, Wg and cell turnover is mostly based on colocalization and the similarity of the phenotype but I believe this may need some additional evidences. Ideally one would need to be able to enhance exocytosis and test whether Wg downregulation suppress the phenotype, but I am not sure that upregulation of core exocytosis genes will be sufficient to do this. Alternatively, if Wg is indeed downstream of the upregulation of exocytosis, the reduction of cell turnover upon Wg flattening (e.g. : ftz2-/+ background) should not be enhanced by the reduction of exocytosis. Moreover, could the authors test the status of Wg downstreams targets upon inhibition of exocytosis in the Minute background (for instance, do they see a supression of the nmo-LacZ upregulation that they previously characterised in Minute wing disc in 2021) ?

    Response:

    We thank the reviewer for the comment. In line with the reviewer's suggestion, exploring the enhancement of exocytosis would be valuable to elucidate the epistatic relationship between exocytosis, Wg signaling, and cell turnover. Therefore, we plan to attempt the overexpression of exocytosis-related genes to check if we can indeed enhance exocytosis. Additionally, in response to the reviewer's comment, we intend to investigate whether the reduction in cell turnover observed upon Wg attenuation (ftz2-/+background or Wg-/+ background) is not exacerbated by further reducing exocytosis. Furthermore, we intend to investigate the status of the Wg downstream target, specifically using nmo-lacZ, when exocytosis is inhibited in the RpS3/+ wing pouch.

    Since Cyt1 and CD63 seem to mostly accumulate in apoptotic cells, it would be interesting to check their status in Minute wing disc upon apoptosis inhibition (e.g. : with H99 or mirRHG).

    Response:

    We thank the reviewer for the comment. Following the reviewer's suggestion, we intend to investigate whether inhibiting apoptosis, either through the introduction of H99/+ or by overexpressing DroncDN or mirRHG, could suppress the increase in CD63- or Sty1-positive vesicles in the M/+ wing pouch.

    I would remain cautious about some of the statements, notably in the abstract, since some of them are mostly speculative and not really based on any experiments. For instance, the statement "This interaction between dying cells and their neighboring living cells is pivotal in determining cell fate, dictating which cells will undergo apoptosis and which cells will proliferate" is not backed up by any experiment (which would require to show that exocytosis and Wg from the dying cell specifically is required for the survival and proliferation of their neighbours, and/or showing that cell death occurs specifically in cells with local differences in Wg signaling). I would recommend to be more cautious here and us a clear conditional statement.

    • *Response:

    We thank the reviewer for their insightful comment. In response, we aim to present our findings with greater precision and to avoid speculative interpretations. At this stage, we have focused on revising the Abstract to include clear conditional statements, as suggested. Furthermore, we plan to comprehensively update the remaining sections of the manuscript to reflect the additional experiments requested by the reviewers. These updates will ensure a more cautious approach throughout the paper, aligning with the reviewer's recommendations.

    __(page 2, line 34-39 in the "Abstract") __

    "Our data also suggest a potential role for the Wg receptor Frizzled-2 (Fz2) in inducing cell-turnover within the M/+ wing pouch. Overall, our findings provide mechanistic insights into robust tissue growth through the orchestration of cell-turnover, which is primarily governed by JNK-mediated exocytosis in the context of Drosophila Minute/+ wing morphogenesis."

    Other minor point:

    The authors document Wg localisation in Minute wing disc upon expression of P35. It would be interesting to describe what is the status of Wg in Rps3+/- compared to WT without p35 (if I am correct, this was done in their previous article, and in that case it would be relevant to describe these former results in the main text).

    Response:

    We thank the reviewer for the comment. In the RpS3/+ wing pouch without p35, we were unable to detect any upregulation of Wg using the anti-Wg antibody (Fig R3, below). However, we observed an increase in GFP-Wg-positive puncta originating from a knock-in allele (McGough et al., Nature, 2020) within areas of massive cell death in the RpS3/+ wing pouch lacking p35, compared to the wild-type control (Fig 3B, compared to Fig 3A in the transferred manuscript). This increase is similar to the phenotype observed in the RpS3/+ wing pouch expressing p35, where Wg-positive puncta are significantly elevated (Fig R4B below, corresponding to Fig 3B in the original manuscript). Moreover, the increase in GFP-Wg-positive puncta in regions of massive cell death in the RpS3/+ wing pouch becomes more pronounced when utilizing a membrane-tethered anti-GFP nanobody (Vhh4-CD8) (McGough et al., Nature, 2020) (Fig 3G, compared to Fig 3F in the transferred manuscript). These observations indicate that Wg-positive puncta are indeed upregulated in the RpS3/+ wing pouch without p35 compared to the wild-type control.

    In the transferred manuscript, we have now made modifications to Fig. 3 by replacing images of the RpS3/+ wing pouch overexpressing p35, which were stained with the anti-Wg antibody, with new images. These new images depict the RpS3/+ wing pouch that harbors the GFP-Wg knock-in allele, which was stained with both anti-GFP and anti-cDCP1 antibodies.

    To clarify this point, we have now modified the sentence as follows:

    (page 7, line 204-210)

    Interestingly, we found that the RpS3/+ wing pouch expressing the cell death inhibitor p35 (which allows dying RpS3/+ cells to survive) exhibited elevated levels of Wg protein, compared to the wild-type control (Fig 3A and 3B). This increase in Wg protein was significantly diminished by overexpressing the JNK inhibitor Puc (Fig 3C), suggesting that Wg expression is upregulated via JNK signaling in the M/+ wing pouch, similar to apoptotic cells in which JNK signaling induces the production of secreted growth factor, including Wg (18, 53, 54). In addition, ____W____e found t____hat GFP-Wg-positive puncta, derived from a knock-in allele ____(34)____, ____were more abundant in the RpS3/+ wing pouch compared to the wild-type control (Fig 3A and 3B). This increase in GFP-Wg-positive puncta, ____especially in the area with massive cell death within the RpS3/+ wing pouch (Fig S3C-S3D'')____, was more evident when using a membrane-tethered anti-GFP nanobody (Vhh4-CD8), which immobilizes GFP-Wg on the cell surface ____(34)____ (Fig 3F and 3G, quantified in Fig 3J).

    Referee #2

    Primary Concerns:

    A significant challenge arises concerning the delineation of cell autonomy/non-autonomy. This study focuses on two distinct cell types, namely dying cells and proliferating cells. However, the consistent use of nub-gal4, a wing pouch driver, and the heterozygous minute mutant throughout the paper impedes the ability to conclusively analyze the autonomy of events. The authors previously posited that caspase-induced cell death triggers non-autonomous proliferation, but recent studies also suggested caspase-induced autonomous proliferation in both flies and mammals (Yosefzon et al. Mol. Cell 2018, Shinoda et al., PNAS 2019). Therefore, a meticulous distinction between autonomous and non-autonomous events, particularly through experimentation involving clones, is imperative. This necessity is particularly evident in Fig 4.

    Response:

    • *As pointed out by the reviewer, it is ideal to dissect the mechanisms underlying cell turnover within the *M/+ *wing pouch, especially concerning cell autonomy and non-autonomy. However, clonal analysis is not feasible due to the pattern of cell death and subsequent proliferation occurring in a 'salt and pepper' distribution within the M/+ wing pouch. In response to this challenge, we found that almost dying cells do not undergo proliferation, as assessed by staining with the M phase marker phospho-Histone H3 (Fig R5, below).

    We also plan to investigate whether JNK-activating cells similarly refrain from proliferating, which will be assessed by staining with the anti-phospho-Histone H3 antibody.

    Additionally, about Fig 4, considering the evidence presented in this study, which demonstrates that dying cells increase Wg secretion through JNK-dependent exocytosis (Fig 3G-3H'), and given that Fz2 is expressed in adjacent cells within the M/+ wing pouch (Fig 4B-C'' and Fig S4B-C''), it is plausible that these neighboring cells could receive Wg via Fz2, leading to the upregulation of Wg signaling in these cells in the M/+ wing pouch. This upregulation of Wg signaling is supported by Fig S3A in the transferred manuscript. However, we were unable to delineate the specific role of Fz2, particularly in terms of cell autonomy and non-autonomy. In response to this challenge, we aim to explore whether cell turnover can be inhibited by enhancing Wg signaling exclusively in JNK-activated cells through the overexpression the active form of ArmadilloS10 (Baena-Lopez LA et al., Sci Signal., 2009), utilizing the puc-gal4 driver. Should cell turnover be inhibited under these conditions, it would indicate that differences in Wg signaling activity between dying cells and their neighboring cells drive cell turnover. While the exact mechanism of Fz2 remains unclear, the inhibition of cell turnover under these conditions clearly demonstrates that differences in Wg signaling activity play a significant role in cell turnover. Additionally, in our response to Reviewer 2's comment No. 2, we outline our intention to investigate whether the increase in Wg signaling could be induced by JNK-dependent exocytosis. Accordingly, we plan to assess the effects of downregulating JNK signaling or exocytosis on the elevated Wg signaling activity observed in the RpS3/+ wing pouch.

    Furthermore, we intend to present our findings with greater precision, steering clear of speculative interpretations. At this stage, we have focused on revising the Abstract to include clear conditional statements, as detailed below. We plan to comprehensively update the remaining sections of the manuscript to reflect the additional experiments requested by the reviewers.

    (page 2, line 34-39 in the "Abstract")

    "Our data also suggest a potential role for the Wg receptor Frizzled-2 (Fz2) in inducing cell-turnover within the M/+ wing pouch. Overall, our findings provide mechanistic insights into robust tissue growth through the orchestration of cell-turnover, which is primarily governed by JNK-mediated exocytosis in the context of Drosophila Minute/+ wing morphogenesis."

    Closely tied to the issue of cell autonomy/non-autonomy is the question of how cells differentiate Wg from dying cells and the dorsal-ventral boundary.

    Response:

    Wg is expressed at the dorsal-ventral boundary in both wild-type and M/+ wing discs. However, we observed in our previous study that Wg signaling activity was significantly more elevated in the RpS3/+ pouch compared to the localized activation in wild-type controls at the same developmental stage, as assessed by the nmo-lacZ reporter (Fig 3C-D' in Akai et al., PLOS Genetics, 2021, as also shown in Fig R6A-B' below). Interestingly, the areas of massive cell death in the RpS3/+ wing pouch always corresponded to the areas of relatively lower Wg signaling activity (Fig S3A in the transferred manuscript). Moreover, our previous study revealed that decreasing or increasing Wg signaling activity, thus reducing the aberrant Wg signaling gradient, significantly inhibited cell death in the M/+ wing pouch (Fig 3I-K in Akai et al., PLOS Genetics, 2021, as also shown in Fig R6C-E' below). This suggest that the aberrant Wg signaling gradient is crucial for massive cell-turnover in the M/+ wing pouch. As also described above, considering the evidence presented in this study, which demonstrates that dying cells increase Wg secretion through JNK-dependent exocytosis (Fig 3G-3H'), and given that Fz2 is expressed in adjacent cells (Fig 4B-C'' and Fig S4B-C'') within the M/+ wing pouch, it is plausible that these neighboring cells could receive Wg via Fz2, leading to the upregulation of Wg signaling in these cells within the M/+ wing pouch. To further investigate whether the increase in Wg signaling could be induced by JNK-dependent exocytosis, we plan to examine the effects of downregulating JNK signaling or exocytosis on the elevated Wg signaling activity observed in the RpS3/+ wing pouch. Should Wg signaling activity decrease as a result of these genetic interventions, it would imply that the enhanced Wg signaling in the M/+ wing pouch depends on JNK-mediated exocytosis.

    In Fig 1, the authors interpret the upregulation of exocytosis-related genes as indicative of increased exocytosis. However, this interpretation lacks direct evidence and overlooks the possibility of opposing effects. For example, autophagosome accumulation means either activation or inhibition of autophagy. Or, in case of Dilp secretion, absence of vesicles indicates upregulation of secretion. To substantiate their claim, the authors must provide more conclusive evidence of increased exocytosis. Fig 2 suggests that inhibiting exocytosis-related genes suppresses caspase activation, favoring the proposition that exocytosis is upregulated. However, demonstrating a direct increase in exocytosis in minute cells would bolster their argument. Higher resolution imaging of the exosome marker, with overlayed images, would enhance clarity too.

    Response:

    We have demonstrated an increase in the number of vesicles positive for EGFP-CD63 (an exosome marker) and Syt1-EGFP (a vesicle marker) in the RpS3/+ wing pouch compared to the wild-type control (Fig 1A, 1B, 1G, and 1H in the transferredmanuscript). To more directly investigate whether exocytosis is indeed elevated in the M/+ wing pouch, we plan to assess if cells activating JNK signaling upregulate the production of extracellular vesicles. This will be done by specifically expressing the EGFP-CD63 probe in JNK-activating cells, utilizing the puc-gal4 driver for targeted expression. Additionally, following the reviewer's suggestion, we plan to prepare high resolution images of the exosome marker, with overlayed images in the revised manuscript.

    Fig 3 introduces ambiguity regarding the relationship between cell death and exocytosis. The authors assert that dying cells exhibit elevated Wg protein levels compared to the wild-type control but omit an important comparison to the minute disc. Moreover, while Figs 1-2 propose a signaling cascade involving minute>JNK>exocytosis>cell death, Fig 3 implies that cell death regulates exocytosis. The coherence of their model and logic requires clarification - specifically, elucidating the molecular coupling mechanism between cell death and exocytosis.

    Response:

    We observed an increase in GFP-Wg-positive puncta originating from a knock-in allele (McGough et al., Nature, 2020) in the area of massive cell death within the RpS3/+ wing pouch, compared to the wild-type control (Fig 3A and 3B in the transferredmanuscript). However, as pointed out by the reviewer, it is challenging to determine whether the GFP-Wg-positive puncta are emanating from dying cells, given that Wg is secreted from the cells that produce it. To address this issue, we employed a membrane-tethered anti-GFP nanobody (Vhh4-CD8 morphotrap) designed to capture GFP-Wg on the cell surface, thereby preventing the diffusion of GFP-Wg from its producing cells. By utilizing the Vhh4-CD8 morphotrap, we found that GFP-Wg levels are indeed elevated in areas of cell death compared to adjacent regions within the RpS3/+ wing pouch (Fig 3G in the transferred manuscript).

    To clarify this point, we have now modified the sentence as follows:__ __

    (page 7, line 204-210)

    In addition, ____W____e found t____hat GFP-Wg-positive puncta, derived from a knock-in allele ____(34)____, ____were more abundant in the RpS3/+ wing pouch compared to the wild-type control (Fig 3A and 3B). This increase in GFP-Wg-positive puncta, ____especially in the area with massive cell death within the RpS3/+ wing pouch (Fig S3C-S3D'')____, was more evident when using a membrane-tethered anti-GFP nanobody (Vhh4-CD8), which immobilizes GFP-Wg on the cell surface ____(34)____ (Fig 3F and 3G, quantified in Fig 3J).

    Additionally, as pointed out by the reviewer, we need to elucidate the molecular mechanism linking cell death and exocytosis. We are currently exploring two potential relationships between JNK activation, caspase signaling, and exocytosis, as depicted in Fig R2 below. To assess these possibilities, we aim to investigate if inhibiting apoptosis-either by introducing the H99/+ mutation or by overexpressing DroncDN or mirRHG-can affect JNK-mediated exocytosis in the RpS3/+ wing pouch.

    Specific Points:

    In Fig S1D, contrary to the authors' claim, cadp2 is not upregulated in a JNK-dependent manner.

    Response:

    We apologize for any confusion caused. We found that Cadps expression in the RpS3/+ wing pouch was increased compared to both the wild-type control (2.29-fold increase, RpS3/+ compared to wild-type) and the RpS3/+ wing pouch expressing Puckered (Puc), driven by the nub-gal4 driver (3.33-fold increase, RpS3/+ compared to RpS3/+ + Puckered). This suggests that the increase in Cadps expression in the RpS3/+ wing pouch is dependent on JNK signaling. We have now revised Fig. S1D for clearer representation. We also have made modifications in the transferred manuscript as follows:

    (page 5, line 107-113)

    "Mining the list of genes differentially expressed in the RpS3/+ wing pouch cells dependent on JNK signaling (Fig S1C and S2 Table), we noticed that among the genes associated with the "secretion by cell" GO term (Fig S1B), the evolutionarily conserved exocytosis-related genes unc-13, SNAP25, and cadps (Calcium-dependent secretion activator) were upregulated in a JNK-dependent manner (2.29-fold increase, RpS3/+ compared to wild-type; 3.33-fold increase, RpS3/+ compared to RpS3/+ +____ Puckered____)____ (Fig S1D)____."

    When detecting multiple proteins in the same tissue, it is advisable for the authors to present overlayed images to enhance the clarity of their findings. Many pictures require higher magnification too.

    Response:

    Following the reviewer's suggestion, we have overlayed images in Fig 1A-D, Fig 1G-J, and Fig S1E in the transferredmanuscript. Additionally, following the reviewer's suggestion, we plan to prepare high resolution images in the revised manuscript.

    In Fig 1A, the observed upregulation of EGFP-CD63 and Syt1-EGFP may potentially result from an artifactual effect of apoptosis. To validate their findings are specific, the authors should include negative controls that do not exhibit upregulation in dying cells.

    Response:

    Following the reviewer's suggestion, we used the CD8-PARP-Venus probe as a negative control. We observed that CD8-PARP-Venus-positive puncta were minimally present (Fig R7, below), indicating that the upregulation of EGFP-CD63 and Syt1-EGFP in the RpS3/+ wing pouch is indeed occurring, rather than being an artifactual effect of apoptosis. We intend to incorporate this negative control into the revised manuscript.

    Referee #3

    Major comments:

    Figure 1A-H, S1E. The authors used EGFP-CD63 and Syt1-EGFP as markers of exocytosis. They see an increased number of puncta in apoptotic cells. Is this a specific effect in the dying cells in M mutant discs, or is it a general effect in apoptotic cell death? This should be examined in a condition where apoptosis is induced independently of M mutants such as nub-reaper or nub-hid.

    Response:

    We thank the reviewer for the comment. To ascertain whether the increase in EGFP-CD63-/Sty1-EGFP-positive puncta is specific to dying cells in M/+ mutants or a general characteristic of apoptotic cell death, we examined the presence of EGFP-CD63-positive puncta in the wing pouch, where Reper (Rpr) was expressed under the control of the nub-gal4 driver. Unfortunately, the expression of Rpr driven by nub-gal4 resulted in significant cell death, preventing us from drawing a definitive conclusion (Fig R8, below). Nonetheless, we did observe EGFP-CD63-positive puncta under these conditions, as indicated by the arrowheads in Fig R8 below. To further investigate, we plan to induce temporary expression of Rpr in the wing pouch using a combination of the temperature-sensitive Gal80 and the nub-gal4 driver.

    Is exocytosis actually upstream or downstream of cell death, or both? The authors are kind of vague about it. On one hand, they say the dying cells induce exocytosis and secrete Wg. On the other hand, unc13RNAi can suppress cell death (Figure 1D', J'). Please clarify.

    Response:

    As pointed out by the reviewer, we need to clarify whether exocytosis actually occurs upstream or downstream of cell death. We are currently exploring two potential relationships between JNK activation, caspase signaling, and exocytosis, as depicted in Fig R2 below. To assess these possibilities, we aim to investigate if inhibiting apoptosis-either by introducing the H99/+ mutation or by overexpressing DroncDN or mirRHG-can affect JNK-mediated exocytosis in the RpS3/+ wing pouch.

    Figure 1L-P. The observation of Ca++ flashes is very interesting. However, are they important for exocytosis, cell death and compensatory proliferation? Right now, this is just a stand-alone observation. Can mutants affecting Ca++ signaling block exocytosis, cell death and comp prol?

    Response:

    We thank the reviewer for the comment. we plan to investigate whether the downregulation of Ca++ signaling impacts exocytosis, cell death, and compensatory proliferation in the M/+ wing pouch. This will be achieved by downregulating genes essential for sensing intracellular calcium concentrations (Rizo J and Rosenmund C, Nat Struct Mol Biol., 2008; Sudhof TC, Annu Rev Neurosci., 2004) and genes encoding voltage-gated calcium channels (Kuromi H et al., Neuron, 2004).

    Figure 3B'. Can you visualize aberrant wg expression in RpS3/+ wing discs only in the presence of p35? The expression of p35 in apoptotic cells generates undead cells which by itself induce Wg expression in a JNK-dependent manner (Perez-Garijo et al 2004; Ryoo et al 2004). Also, in Figure 3B', Wg is upregulated in the ventral half of the pouch, whereas in other figures (4B for example) apoptosis is strongly induced in the dorsal half. Does that make sense?

    Response:

    We thank the reviewer for the comment. In the RpS3/+ wing pouch without p35, we were unable to detect any upregulation of Wg using the anti-Wg antibody (Fig R3, below). However, we observed an increase in GFP-Wg-positive puncta, derived from a knock-in allele (McGough et al., Nature, 2020), in areas of massive cell death within the RpS3/+ wing pouch without p35, relative to the wild-type control (Fig 3B, compared to Fig 3A in the transferred manuscript). This increase is similar to the phenotype observed in the RpS3/+ wing pouch expressing p35, where Wg-positive puncta are significantly elevated (Fig R4B below, corresponding to Fig 3B in the original manuscript). Moreover, the increase in GFP-Wg-positive puncta in regions of massive cell death in the RpS3/+ wing pouch becomes more pronounced when utilizing a membrane-tethered anti-GFP nanobody (Vhh4-CD8) (McGough et al., Nature, 2020) (Fig 3G, compared to Fig 3F). These observations indicate that Wg-positive puncta are indeed upregulated in the RpS3/+ wing pouch without p35 compared to the wild-type control.

    In the transferred manuscript, we have now made modifications to Fig. 3 by replacing images of the RpS3/+ wing pouch overexpressing p35, which were stained with the Wg antibody, with new images. These new images depict the RpS3/+ wing pouch that harbors the GFP-Wg knock-in allele, which was stained with both anti-GFP and anti-cDCP1 antibodies.

    To clarify this point, we have now modified the sentence as follows:

    (page 7, line 204-210)

    Interestingly, we found that the RpS3/+ wing pouch expressing the cell death inhibitor p35 (which allows dying RpS3/+ cells to survive) exhibited elevated levels of Wg protein, compared to the wild-type control (Fig 3A and 3B). This increase in Wg protein was significantly diminished by overexpressing the JNK inhibitor Puc (Fig 3C), suggesting that Wg expression is upregulated via JNK signaling in the M/+ wing pouch, similar to apoptotic cells in which JNK signaling induces the production of secreted growth factor, including Wg (18, 53, 54). In addition, ____W____e found t____hat GFP-Wg-positive puncta, derived from a knock-in allele ____(34)____, ____were more abundant in the RpS3/+ wing pouch compared to the wild-type control (Fig 3A and 3B). This increase in GFP-Wg-positive puncta, ____especially in the area with massive cell death within the RpS3/+ wing pouch (Fig S3C-S3D'')____, was more evident when using a membrane-tethered anti-GFP nanobody (Vhh4-CD8), which immobilizes GFP-Wg on the cell surface ____(34)____ (Fig 3F and 3G, quantified in Fig 3J).

    Additionally, we apologize for any confusion caused by Fig 3B'. It is noteworthy that cell death sometimes occurs more prominently in the ventral half of the RpS3/+ wing pouch than in the dorsal half (Fig R9, below). Furthermore, the increase in Wg level can be more pronounced on the ventral side, or at times, it is equally strong on both the dorsal and ventral sides (Fig S3C-D in the transferred manuscript).

    Can wg RNAi in cells destined to die (puc-Gal4 UAS-wgRNAi) suppress apoptosis and comp prol?

    Response:

    Following the reviewer's suggestion, we intend to investigate whether expressing Wg-RNAi in JNK activated cells, using the puc-gal4 driver, could suppress apoptosis and compensatory proliferation.

    Figure 3D,E. Use cDcp1 as apoptotic marker instead of CD63-mCherry which is not an apoptotic marker.

    Response:

    Following the reviewer's suggestion, we have now utilized the cDCP1 antibody as an apoptotic marker in Fig S3B-D'' in the transferred manuscript, as described in the Reviewer 3's comment No.4.

    Figure 4A' and B'. I am not sure there is much of a difference in the expression of fz2-lacZ in wild-type and RpS3/+ discs. The quantification in 4F shows there is no difference in the dorsal half of the pouch where massive apoptosis occurs. Can you generate a condition in which only one compartment (anterior or posterior) is mutant for RpS3/+, while the other compartment is wt? That would allow direct side-by-side comparison. Comparisons between different discs is problematic.

    Response:

    We thank the reviewer for the comment. Following the reviewer's suggestion, we conducted a more detailed investigation into the differences in fz2-lacZ expression levels between wild-type and RpS3/+ wing discs. We found that the expression level of fz2-lacZwas indeed elevated in the RpS3/+ wing pouch. This elevation in expression is demonstrated by the results of the genetic rescue experiment in the posterior compartment of RpS3/+ wing discs, where overexpression of RpS3 using the engrailed-gal4 driver led to a reduction in fz2-lacZ expression compared to the anterior RpS3/+ control (Fig R10, below).

    After conducting statistical analysis, we plan to include Fig R10 in the revised manuscript.

    There is some inconsistency in the presence of apoptotic cells and presence of cells which secrete Wg. Apoptotic cells occur in large cell clusters (Fig. 2G, 3H', S3A', S4B), while markers of exocytosis and Wg are present in individual puncta (Fig 3D', E', S3C,E). That does not seem to fit with the authors' conclusion that dying cells secrete Wg by exocytosis. Are all dying cells secreting Wg through exocytosis? Please explain.

    Response:

    We thank the reviewer for the comment. As highlighted by the reviewer, it appears that not all dying cells secrete Wg through exocytosis. Beyond exosomes, various mechanisms for Wg/Wnt transport have been proposed, including those mediated by lipoprotein particles, the cell-surface proteoglycan Dally-like protein (Dlp), and filopodia-like cellular extensions known as cytonemes. Therefore, it's plausible that dying cells may also utilize these additional mechanisms for Wg transport alongside exosomes. We have incorporated this explanation into the transferred manuscript as follows:

    (page 9, line 272-286)

    Previous studies have identified exosomes as carriers of Wnt/Wg in the extracellular space of both mammalian and Drosophila cells, including wing disc cells (28, 49, 50, 51, 52, 64). Concurrently, alternative mechanisms for Wg transport, such as those involving lipoprotein particles or a lipocalin Swim in the Drosophila wing disc, have been reported (29, 65). Additionally, the cell-surface proteoglycan Dally-like-protein (Dlp) has been reported to enable long-range signaling of the palmitoylated Wg ____(51)____.____ Intriguingly, Wnt/Wg transport has also been observed through filopodia-like cellular extensions known as cytonemes (66, 67, 68, 69). In our study of the ____M/+____ wing disc, a model characterized by massive cell-turnover, we observed partial colocalization of Wg-GFP puncta with exosomal markers such as CD63-mCherry and Hrs.____ Our data also suggest that Wg secretion through exocytosis may not uniformly occur among all dying cells within this context. It is noteworthy that whereas dying cells frequently form large clusters, exosomal markers and Wg typically localize within individual puncta. This disparity suggests that while exosomes from dying cells significantly contribute to Wg transport within the ____M/+____ wing pouch, other pathways may also be operative.

    I was a bit confused by the conclusion of the authors about the results in Figure 4G-I. What do they mean by "difference in Fz2 expression"? Are they referring to the gradient that they described in their previous work? Or is it just the absolute level of Fz2 that determines apoptosis and proliferation? If the latter, is the gradient still present (at least in fz2/+), just at a lower level?

    Response:

    I apologize for any confusion caused by the way we presented our conclusions regarding the results depicted in Figure 4G-I in the original manuscript.

    In our previous study, we observed that Wg signaling activity was elevated much more broadly in the RpS3/+ pouch compared to the localized activation observed in the wild-type control at the same developmental stage, as assessed by the nmo-lacZ reporter(Fig 3D in Akai et al., PLOS Genetics, 2021, as also shown Fig. R6 below). Interestingly, the areas of massive cell death in the RpS3/+ wing pouch always corresponded to the areas of relatively lower Wg signaling activity (as also shown in Fig S3A in the transferred manuscript). Moreover, our previous study revealed that decreasing or increasing Wg signaling activity, thus reducing the aberrant Wg signaling gradient, significantly inhibited cell death in the M/+ wing pouch (Fig 3I-K in Akai et al., PLOS Genetics, 2021, as also shown in Fig R6C-E' below). This suggest that the aberrant Wg signaling gradient is crucial for massive cell-turnover in the M/+ wing pouch.

    Considering the evidence presented in this study, which demonstrates that dying cells increase Wg secretion through JNK-dependent exocytosis (Figure 3G-3H'), and given that Fz2 is expressed in adjacent cells within the M/+ wing pouch (Figure 4B-C'' and Fig S4B-C''), it is plausible that these neighboring cells could receive Wg via Fz2, leading to the upregulation of Wg signaling in these cells in the M/+ wing pouch.

    However, we were unable to delineate the specific role of Fz2, particularly in terms of cell autonomy and non-autonomy as highlighted by Reviewer 2, through clonal analysis due to the occurrence of cell death and subsequent proliferation in a salt-and-pepper pattern within the M/+ wing pouch. In response to this challenge, we aim to explore whether cell turnover can be inhibited by enhancing Wg signaling exclusively in JNK-activated cells through the overexpression the active form of ArmadilloS10(Baena-Lopez LA et al., Sci Signal., 2009), utilizing the puc-gal4 driver. Should cell turnover be inhibited under these conditions, it would indicate that differences in Wg signaling activity between dying cells and their neighboring cells drive cell turnover. While the exact mechanism of Fz2 remains unclear, the inhibition of cell turnover under these conditions clearly demonstrates that differences in Wg signaling activity play a significant role in cell turnover. Additionally, in our response to Reviewer 2's comment No.2, we outline our intention to investigate whether the increase in Wg signaling could be induced by JNK-dependent exocytosis. Accordingly, we plan to assess the effects of downregulating JNK signaling or exocytosis on the elevated Wg signaling activity observed in the RpS3/+ wing pouch.

    Furthermore, we intend to present our findings with greater precision, steering clear of speculative interpretations. At this stage, we have focused on revising the Abstract to include clear conditional statements, as detailed below. We plan to comprehensively update the remaining sections of the manuscript to reflect the additional experiments requested by the reviewers.

    (page 2, line 34-39 in the "Abstract")

    "Our data also suggest a potential role for the Wg receptor Frizzled-2 (Fz2) in inducing cell-turnover within the M/+ wing pouch. Overall, our findings provide mechanistic insights into robust tissue growth through the orchestration of cell-turnover, which is primarily governed by JNK-mediated exocytosis in the context of Drosophila Minute/+ wing morphogenesis."

    Regarding point 8, given that reduction of Fz2 can suppress apoptosis in RpS3/+ wing discs, how does it regulate apoptosis when it is down-regulated anyway in apoptotic cells (Fig 4B', S4B')?

    Response:

    As described in our response to point 9, our previous study suggested that we observed that Wg signaling activity was elevated much more broadly in the RpS3/+ pouch compared to the localized activation observed in the wild-type control at the same developmental stage, as assessed by the nmo-lacZ reporter (Fig 3D in Akai et al., PLOS Genetics, 2021, as also shown Fig. R6 above). This elevation leads to the formation of an ectopic cell population with high Wg signaling activity in the wing pouch, potentially causing non-autonomous cell death among cells exhibiting lower Wg signaling activity, a process akin to cell competition. Notably, the significant reduction of apoptosis in RpS3/+ wing discs following Fz2 downregulation suggests that Fz2 may play a crucial role in the massive cell turnover, through a mechanism similar to cell competition, even though the exact mechanism remains unclear. We plan to incorporate such discussions into the discussion section of the revised manuscript.

    Figure S1D. It seems that cadps is further up-regulated if JNK signaling is inhibited in RpS3/+ cells. Why did the authors select this gene?

    Response:

    We apologize for any confusion caused. We found that Cadps expression in the RpS3/+ wing pouch was increased compared to both the wild-type control (2.29-fold increase, RpS3/+ compared to wild-type) and the RpS3/+ wing pouch expressing Puc, driven by the nub-gal4 driver (3.33-fold increase, RpS3/+ compared to RpS3/+ +Puc). This suggests that the increase in Cadpsexpression in the RpS3/+ wing pouch is dependent on JNK signaling. We have now revised Fig S1D for clearer representation. We also have made modifications in the transferred manuscript as follows:

    (page 5, line 107-113)

    "Mining the list of genes differentially expressed in the RpS3/+ wing pouch cells dependent on JNK signaling (Fig S1C and S2 Table), we noticed that among the genes associated with the "secretion by cell" GO term (Fig S1B), the evolutionarily conserved exocytosis-related genes unc-13, SNAP25, and cadps (Calcium-dependent secretion activator) were upregulated in a JNK-dependent manner (2.29-fold increase, RpS3/+ compared to wild-type; 3.33-fold increase, RpS3/+ compared to RpS3/+ +____ Puckered____)____ (Fig S1D)____."

    Figure S1H'. I don't see that Hrs is upregulated in this panel.

    Response:

    We thank the reviewer for the comment. Our quantitative analysis showed an increase in Hrs-positive puncta in the RpS3/+ wing pouch compared to the wild-type control (Fig S1I). We have substituted the relevant Figure with new images that more clearly demonstrate the elevation of Hrs-positive puncta (Fig S1G-H' in the transferred manuscript).

    Minor points:

    Describe in more detail, what are unc13, SNAP25 and cadps.

    Response:

    We thank the reviewer for the comment. We have now included an explanation for unc-13, SNAP25 and cadps in the transferredmanuscript as follows:

    (page 5, line 117-126)

    "It has been shown that unc-13, SNAP25, and cadps collectively regulate the docking process of secretory vesicles to the plasma membrane during Ca2+-mediated exocytosis ____((21, 23, 24), Reviewed in (22)). ____Specifically, UNC-13, a conserved presynaptic protein with calcium-binding domains, interacts with syntaxin to prime vesicles for fusion, crucial for calcium-regulated exocytosis (____23____). SNAP-25, in conjunction with syntaxin-1 and synaptobrevin, forms the pivotal SNARE complex for neuronal exocytosis, assembling into a four-helix bundle that is essential for drawing vesicle and plasma membranes close together to enable membrane fusion (____24, 25____). Like UNC-13, CAPS possesses conserved C-terminal domains that are instrumental in the assembly of SNARE complexes, thus priming vesicles for Ca2+-induced exocytosis (____26)____.____"

    I wondered about the use of Hrs as exosome marker. To my knowledge, it is an endosomal marker. Same with Alix. Please clarify.

    Response:

    As pointed out by the reviewer, ESCRT-0 component Hrs is also localized to endosome. However, Hrs is also used as an exosome marker in the previous manuscript (McGough IJ et al., Nature, 2020), alongside Alix and Tsg101, which are widely recognized as exosome markers (Willms E et al., Sci Rep., 2016; Dear JW et al., Proteomics, 2013). Indeed, Hrs is a key factor for mediating ESCRT-dependent exosome secretion in mammals and flies (Tamai K et al., Biochem Biophys Res Commun., 2010; Gross JC et al., Nat Cell Biol., 2012; Colombo M et al., *J Cell Sci., 2013; Vietri M et al., Nat Rev Mol Cell Biol., *2020). We have incorporated this explanation into the revised manuscript as follows:

    (page 6, line 139-141)

    "____Furthermore, we noted an increase in vesicles positive for the ESCRT protein Hrs, ____an additional exosome marker crucial for exosome secretion (____34, 35____), in the RpS3/+ wing pouch relative to the wild-type control (Fig S1G and S1H, quantified in Fig S1I)."

    In the context of references 16 and 17, Huh et al. (2004), Current Biology needs also to be cited.

    Response:

    We thank the reviewer for the comment. We have referenced the paper by Huh et al. (2004) published in Current Biology in the transferred manuscript as follows:

    (page 3, line 68-71)

    "Apoptotic cells, for example, can secrete mitogens su____ch as Wingless ____(Wg; a Wnt homolog), dpp (a BMP homolog), and Hh, which could promote the proliferation of nearby cells in the *Drosophila *epithelium (15, ____16____, 17, 18, 19)."

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    Referee #3

    Evidence, reproducibility and clarity

    Summary: In previous work, the authors showed that in the pouch of wing imaginal discs in Minute (M) mutants apoptosis and compensatory proliferation are dramatically increased in a Wingless (Wg) and JNK-dependent manner. Here, they show that JNK-induced exocytosis is mediating the secretion of Wg in apoptotic cells. Wg in turn stimulates the Fz2 receptor in neighboring surviving cells promoting their proliferation.

    Major comments:

    1. Figure 1A-H, S1E. The authors used EGFP-CD63 and Syt1-EGFP as markers of exocytosis. They see an increased number of puncta in apoptotic cells. Is this a specific effect in the dying cells in M mutant discs, or is it a general effect in apoptotic cell death? This should be examined in a condition where apoptosis is induced independently of M mutants such as nub-reaper or nub-hid.
    2. Is exocytosis actually upstream or downstream of cell death, or both? The authors are kind of vague about it. On one hand, they say the dying cells induce exocytosis and secrete Wg. On the other hand, unc13RNAi can suppress cell death (Figure 1D', J'). Please clarify.
    3. Figure 1L-P. The observation of Ca++ flashes is very interesting. However, are they important for exocytosis, cell death and compensatory proliferation? Right now, this is just a stand-alone observation. Can mutants affecting Ca++ signaling block exocytosis, cell death and comp prol?
    4. Figure 3B'. Can you visualize aberrant wg expression in RpS3/+ wing discs only in the presence of p35? The expression of p35 in apoptotic cells generates undead cells which by itself induce Wg expression in a JNK-dependent manner (Perez-Garijo et al 2004; Ryoo et al 2004). Also, in Figure 3B', Wg is upregulated in the ventral half of the pouch, whereas in other figures (4B for example) apoptosis is strongly induced in the dorsal half. Does that make sense?
    5. Can wg RNAi in cells destined to die (puc-Gal4 UAS-wgRNAi) suppress apoptosis and comp prol?
    6. Figure 3D,E. Use cDcp1 as apoptotic marker instead of CD63-mCherry which is not an apoptotic marker.
    7. Figure 4A' and B'. I am not sure there is much of a difference in the expression of fz2-lacZ in wild-type and RpS3/+ discs. The quantification in 4F shows there is no difference in the dorsal half of the pouch where massive apoptosis occurs. Can you generate a condition in which only one compartment (anterior or posterior) is mutant for RpS3/+, while the other compartment is wt? That would allow direct side-by-side comparison. Comparisons between different discs is problematic.
    8. There is some inconsistency in the presence of apoptotic cells and presence of cells which secrete Wg. Apoptotic cells occur in large cell clusters (Fig. 2G, 3H', S3A', S4B), while markers of exocytosis and Wg are present in individual puncta (Fig 3D', E', S3C,E). That does not seem to fit with the authors' conclusion that dying cells secrete Wg by exocytosis. Are all dying cells secreting Wg through exocytosis? Please explain.
    9. I was a bit confused by the conclusion of the authors about the results in Figure 4G-I. What do they mean by "difference in Fz2 expression"? Are they referring to the gradient that they described in their previous work? Or is it just the absolute level of Fz2 that determines apoptosis and proliferation? If the latter, is the gradient still present (at least in fz2/+), just at a lower level?
    10. Regarding point 8, given that reduction of Fz2 can suppress apoptosis in RpS3/+ wing discs, how does it regulate apoptosis when it is down-regulated anyway in apoptotic cells (Fig 4B', S4B')?
    11. Figure S1D. It seems that cadps is further up-regulated if JNK signaling is inhibited in RpS3/+ cells. Why did the authors select this gene?
    12. Figure S1H'. I don't see that Hrs is upregulated in this panel.

    Minor points:

    1. Describe in more detail, what are unc13, SNAP25 and cadps.
    2. I wondered about the use of Hrs as exosome marker. To my knowledge, it is an endosomal marker. Same with Alix. Please clarify.
    3. In the context of references 16 and 17, Huh et al. (2004), Current Biology needs also to be cited.

    Referees cross-commenting

    I think all three reviewers agree in their assessment of this manuscript. Some of the comments by the reviewers address the same concerns. I liked the comment by reviewer 2 about the autonomy/non-autonomy of the signaling events in this model. I was thinking that, too, but didn't express it in my review. So, thanks reviewer 2, for bringing this up.

    Significance

    General assessment. A major strength of the work is that the experiments are done in a very good manner. The authors used multiple assays to come to the same conclusions. Limitations and weaknesses are mentioned in "major comments".

    Advance. In my mind, one problem is novelty of this work. The authors showed before that Wg is secreted by dying cells in Minute mutants. It was also shown that Wg can be secreted by exocytosis in many studies from different authors. Here, the authors basically combine both observations and show that Wg is secreted by exosomes in Minute mutants.

    Audience. This work likely addresses a specialized audience involved in Minute-induced cell competition. I don't think it will be of interest beyond this specific field.

    Background of the reviewer. I have an interest in cell competition, apoptosis and neurodegeneration.

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    Referee #2

    Evidence, reproducibility and clarity

    The authors initiate their study by illustrating that minute cells undergo an augmentation in exocytosis-related proteins and calcium flashes (Fig 1). Subsequently, they establish a correlation between JNK-mediated exocytosis and the regulation of caspase activation in minute cells (Fig 2). Further, the authors unveil a dependency of Wg secretion on exocytosis, elucidated in Fig 3. Finally, they discern the involvement of one of the Wg receptors, Fz2, in the cell death/proliferation phenotype of minute cells. Despite the formulation of an intriguing hypothesis, the presented data falls short of robustly supporting their assertions.

    Primary Concerns:

    1. A significant challenge arises concerning the delineation of cell autonomy/non-autonomy. This study focuses on two distinct cell types, namely dying cells and proliferating cells. However, the consistent use of nub-gal4, a wing pouch driver, and the heterozygous minute mutant throughout the paper impedes the ability to conclusively analyze the autonomy of events. The authors previously posited that caspase-induced cell death triggers non-autonomous proliferation, but recent studies also suggested caspase-induced autonomous proliferation in both flies and mammals (Yosefzon et al. Mol. Cell 2018, Shinoda et al., PNAS 2019). Therefore, a meticulous distinction between autonomous and non-autonomous events, particularly through experimentation involving clones, is imperative. This necessity is particularly evident in Fig 4.
    2. Closely tied to the issue of cell autonomy/non-autonomy is the question of how cells differentiate Wg from dying cells and the dorsal-ventral boundary.
    3. In Fig 1, the authors interpret the upregulation of exocytosis-related genes as indicative of increased exocytosis. However, this interpretation lacks direct evidence and overlooks the possibility of opposing effects. For example, autophagosome accumulation means either activation or inhibition of autophagy. Or, in case of Dilp secretion, absence of vesicles indicates upregulation of secretion. To substantiate their claim, the authors must provide more conclusive evidence of increased exocytosis. Fig 2 suggests that inhibiting exocytosis-related genes suppresses caspase activation, favoring the proposition that exocytosis is upregulated. However, demonstrating a direct increase in exocytosis in minute cells would bolster their argument. Higher resolution imaging of the exosome marker, with overlayed images, would enhance clarity too.
    4. Fig 3 introduces ambiguity regarding the relationship between cell death and exocytosis. The authors assert that dying cells exhibit elevated Wg protein levels compared to the wild-type control but omit an important comparison to the minute disc. Moreover, while Figs 1-2 propose a signaling cascade involving minute>JNK>exocytosis>cell death, Fig 3 implies that cell death regulates exocytosis. The coherence of their model and logic requires clarification - specifically, elucidating the molecular coupling mechanism between cell death and exocytosis.

    Specific Points:

    1. In Fig S1D, contrary to the authors' claim, cadp2 is not upregulated in a JNK-dependent manner.
    2. When detecting multiple proteins in the same tissue, it is advisable for the authors to present overlayed images to enhance the clarity of their findings. Many pictures require higher magnification too.
    3. In Fig 1A, the observed upregulation of EGFP-CD63 and Syt1-EGFP may potentially result from an artifactual effect of apoptosis. To validate their findings are specific, the authors should include negative controls that do not exhibit upregulation in dying cells.

    Referees cross-commenting

    I agree with comments by other reviewers.

    Significance

    The authors initiate their study by illustrating that minute cells undergo an augmentation in exocytosis-related proteins and calcium flashes (Fig 1). Subsequently, they establish a correlation between JNK-mediated exocytosis and the regulation of caspase activation in minute cells (Fig 2). Further, the authors unveil a dependency of Wg secretion on exocytosis, elucidated in Fig 3. Finally, they discern the involvement of one of the Wg receptors, Fz2, in the cell death/proliferation phenotype of minute cells. Despite the formulation of an intriguing hypothesis, the presented data falls short of robustly supporting their assertions.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this study, Akai and colleagues study the cellular mechanism leading to high cell turnover in the Minute mutant background in Drosophila wing disc. Previously, the same group characterised an unexpected high rate of proliferation in the wing pouch of the Minute heterozygous larvae, which have been long known to have slower development time. This high rate of proliferation is driven by high apoptosis rate, JNK activation, the release of Dilp8 hormone, and the upregulation of Wg signaling (downstream of JNK) necessary for increased apoptosis and proliferation ( Akai et al., Plos Genetics 2021). Here, the authors address now the molecular link connecting JNK activation and the increased turnover. Using RNAseq and comparing WT, M+/- wing disc with M+/- wing disc upon JNK inhibition, they found a number of genes associated with exocytosis that were upregulated in Minute disc which was lost upon JNK inhibition. The authors first confirm the existence of higher number of exocytic vesicles in Minute wing disc as well as higher calcium activity that were all reduced upon JNK inhibition. Moreover, downregulation of several components of the exocytosis machinery abolished the high turnover rate of Minute wing disc (reducing both apoptosis and proliferation) while leading to the appearance of morphological defects in adult wing, while having no effect in a WT background. Interestingly, Wg accumulates in these exocytosis vesicles, and this accumulation relies on JNK and core exocytosis machinery. The role of Wg is confirmed by reducing the concentration (using heterozygous mutant or RNAi) of the Wg receptor Fz2, which is also specifically downregulated in the cluster of apoptotic cells. Altogether, this study suggests that upregulation of exocytosis by JNK activation is a central regulator of the higher cell turnover in Minute background.

    The article is well written and the data overall convincing, including a lot of genetic backgrounds confirming the impact of exocytosis, as well as all the necessary quantifications. Some additional epistatic experiments may help to clearly test to which extend exocytosis is the major downstream target of JNK, and additional control may also help to clarify the sufficiency of JNK for generating such phenotype. Finally, I believe the epistatic link between exocytosis and Wg would deserve more experiments to be definitly proven.

    Major comments:

    1. It would be important to check how much JNK is sufficient to trigger the exocytosis upregulation. Is the accumulation of Cyt1 and CD63 vesicles in apoptotic cell common to any JNK dependent death or does it require the Minute background ? Could the authors check whether clones expressing HepCA transiently in WT background also accumulate the same vesicles ?
    2. It would be relevant to have the status of JNK in Minute disc upon downregualtion of exocytosis. One could imagine some positive feedback between JNK activation, exocytosis, cell death and further JNK activation.
    3. So far, the evidence for the epistatic link between exocytosis, Wg and cell turnover is mostly based on colocalization and the similarity of the phenotype but I believe this may need some additional evidences. Ideally one would need to be able to enhance exocytosis and test whether Wg downregulation suppress the phenotype, but I am not sure that upregulation of core exocytosis genes will be sufficient to do this. Alternatively, if Wg is indeed downstream of the upregulation of exocytosis, the reduction of cell turnover upon Wg flattening (e.g. : ftz2-/+ background) should not be enhanced by the reduction of exocytosis. Moreover, could the authors test the status of Wg downstreams targets upon inhibition of exocytosis in the Minute background (for instance, do they see a supression of the nmo-LacZ upregulation that they previously characterised in Minute wing disc in 2021) ?
    4. Since Cyt1 and CD63 seem to mostly accumulate in apoptotic cells, it would be interesting to check their status in Minute wing disc upon apoptosis inhibition (e.g. : with H99 or mirRHG).
    5. I would remain cautious about some of the statements, notably in the abstract, since some of them are mostly speculative and not really based on any experiments. For instance, the statement "This interaction between dying cells and their neighboring living cells is pivotal in determining cell fate, dictating which cells will undergo apoptosis and which cells will proliferate" is not backed up by any experiment (which would require to show that exocytosis and Wg from the dying cell specifically is required for the survival and proliferation of their neighbours, and/or showing that cell death occurs specifically in cells with local differences in Wg signaling). I would recommend to be more cautious here and us a clear conditional statement.

    Other minor point:

    The authors document Wg localisation in Minute wing disc upon expression of P35. It would be interesting to describe what is the status of Wg in Rps3+/- compared to WT without p35 (if I am correct, this was done in their previous article, and in that case it would be relevant to describe these former results in the main text).

    Referees cross-commenting

    I overall agree with all the other comments. It seems that the overall assessment and criticisms (cell autonomy, better characterisation of the status of exocytsosi) are along the same line

    Significance

    This article is mostly a follow up of a former study published by the same authors in Plos Genetics in 2021. The high turnover (high proliferation and high apoptosis rate) in the minute background was the most surprising observations that was performed previously by the authors, and the role of JNK and Wg was already quite extensively explored in this previous article. The main novelty here is to provide a systematic analysis of expression profile of Minute disc upon JNK inhibition and identify the strong contribution of exocytosis for increased apoptosis and proliferation in Minute wing disc downstream of JNK. In that sense, I believe these are interesting results, but novelty remains a bit limited (at least relative to their previous study). Still, these results could be interesting for the community studying cell competition, ribosomopathies, and growth regulation specially in Drosophila (so mostly for a specialised readership).

    I have expertise in epithelial cell death, apoptosis, and cell competition, specially in Drosophila. I feel confident to evaluate every part of the article.

  6. Version published to 10.1101/2023.12.27.573431v2 on bioRxiv
  7. Version published to 10.1101/2023.12.27.573431v1 on bioRxiv