Regeneration following tissue necrosis is mediated by non-apoptotic caspase activity

Reviewer #1 (Public review):

Summary:

In previous work, the authors described necrosis-induced apoptosis (NiA) as a consequence of induced necrosis. Specifically, experimentally induced necrosis in the distal pouch of larval wing imaginal discs triggers NiA in the lateral pouch. In this manuscript, the authors confirmed this observation and found that while necrosis can kill all areas of the disc, NiA is limited to the pouch and to some extent to the notum, but is excluded from the hinge region. Interestingly and unexpectedly, signaling by the Jak/Stat and Wg pathways inhibits NiA. Further characterization of NiA by the authors reveals that NiA also triggers regenerative proliferation which can last up to 64 hours following necrosis induction. This regenerative response to necrosis is significantly stronger compared to discs ablated by apoptosis. Furthermore, the regenerative proliferation induced by necrosis is dependent on the apoptotic pathway because RNAi targeting the RHG genes is sufficient to block proliferation. However, NiA does not promote proliferation through the previously described apoptosis-induced proliferation (AiP) pathway, although cells at the wound edge undergo AiP. Further examination of the caspase levels in NiA cells allowed the authors to group these cells into two clusters: some cells (NiA) undergo apoptosis and are removed, while others referred to as Necrosis-induced Caspase Positive (NiCP) cells survive despite caspase activity. It is the NiCP cells that repair cellular damage including DNA damage and that promote regenerative proliferation. Caspase sensors demonstrate that both groups of cells have initiator caspase activity, while only the NiA cells contain effector caspase activity. Under certain conditions, the authors were also able to visualize effector caspase activity in NiCP cells, but the level was low, likely below the threshold for apoptosis. Finally, the authors found that loss of the initiator caspase Dronc blocks regenerative proliferation, while inhibiting effector caspases by expression of p35 does not, suggesting that Dronc can induce regenerative proliferation following necrosis in a non-apoptotic manner. This last finding is very interesting as it implies that Dronc can induce proliferation in at least two ways in addition to its requirement in AiP.

Strengths:

This is a very interesting manuscript. The authors demonstrate that epithelial tissue that contains a significant number of necrotic cells is able to regenerate. This regenerative response is dependent on the apoptotic pathway which is induced at a distance from the necrotic cells. Although regenerative proliferation following necrosis requires the initiator caspase Dronc, Dronc does not induce a classical AiP response for this type of regenerative response. In future work, it will be very interesting to dissect this regenerative response pathway genetically.

Weaknesses:
No weaknesses were identified.

Reviewer #2 (Public review):

Summary / Strengths:

In this manuscript, Klemm et al., build on past published findings (Klemm et al., 2021) to characterize caspase activation in distal cells following necrotic tissue damage within the Drosophila wing imaginal disc. Previously in Klemm et al., 2021, the authors describe necrosis-induced-apoptosis (NiA) following the development of a genetic system to study necrosis that is caused by the expression of a constitutive active GluR1 (Glutamate/Ca2+ channel), and they discovered that the appearance of NiA cells were important for promoting regeneration.

In this manuscript, the authors aim to investigate how tissues regenerate following necrotic cell death. They find that:
(1) the cells of the wing pouch are more likely to have non-autonomous caspase activation than other regions within the wing imaginal disc (hinge and notum),
(2) two signaling pathways that are known to be upregulated during regeneration, Wnt (wingless) and JAK/Stat signaling, act to prevent additional NiA in pouch cells, and may explain the region specificity,
(3) the presence of NiA cells promotes regenerative proliferation in late stages of regeneration,
(4) not all caspase-positive cells are cleared from the epithelium (these cells are then referred to as Necrosis-induced Caspase Positive (NiCP) cells), these NiCP cells continue to live and promote proliferation in adjacent cells,
(5) the caspase Dronc is important for creating NiA/NiCP cells and for these cells to promote proliferation. Animals heterozygous for a Dronc null allele show a decrease in regeneration following necrotic tissue damage.

The study has the potential to be broadly interesting due to the insights into how tissues differentially respond to necrosis as compared to apoptosis to promote regeneration.

Weaknesses:

However, here are some of my current concerns for the manuscript in its current version:

(1) The presence of cells with activated caspase that don't die (NiCP cells) is an interesting biological phenomenon but is not described until Figure 5. How does the existence of NiCP cells impact the earlier findings presented? Is late proliferation due to NiA, NiCP, or both? Does Wg and JAK/STAT signaling act to prevent the formation of both NiA and NiCP cells or only NiA cells? Moreover, the authors are able to specifically manipulate the wound edge (WE) and lateral pouch cells (LP), but don't show how these manipulations within these distinct populations impact regeneration. The authors provide evidence that driving UAS-mir(RHG) throughout the pouch, in the LP or the WE all decrease the amount of NiA/NiCP in Figure 3G-O, but no data on final regenerative outcomes for these manipulations is presented (such as those presented for Dronc-/+ in Fig 7M). The manuscript would be greatly enhanced by quantification of more of the findings, especially in describing if the specific manipulations that impacted NiA /NiCP cells disrupt end-point regeneration phenotypes.

(2) How fast does apoptosis take within the wing disc epithelium? How many of the caspase(+) cells are present for the whole 48 hours of regeneration? Are new cells also induced to activate caspase during this time window? The author presented a number of interesting experiments characterizing the NiCP cells. For the caspase sensor GC3Ai experiments in Figure 5, is there a way to differentiate between cells that have maintained fluorescent CG3Ai from cells that have newly activated caspase? What is the timeline for when NiA and NiCP are specified? In addition, what fraction of NiCP cells contribute to the regenerated epithelium? Additional information about the temporal dynamics of NiA and NiCP specification/commitment would be greatly appreciated.

(3) The notum also does not express developmental JAK/STAT, yet little NiA was observed within the notum. Do the authors have any additional insights into the differential response between the pouch and notum? What makes the pouch unique? Are NiA/NiCP cells created within other imaginal discs and other tissues? Are they similarly important for regenerative responses in other contexts?

Reviewer #3 (Public review):

The manuscript "Regeneration following tissue necrosis is mediated by non-apoptotic caspase activity" by Klemm et al. is an exploration of what happens to a group of cells that experience caspase activation after necrosis occurs some distance away from the cells of interest. These experiments have been conducted in the Drosophila wing imaginal disc, which has been used extensively to study the response of a developing epithelium to damage and stress. The authors revise and refine their earlier discovery of apoptosis initiated by necrosis, here showing that many of those presumed apoptotic cells do not complete apoptosis. Thus, the most interesting aspect of the paper is the characterization of a group of cells that experience mild caspase activation in response to an unknown signal, followed by some effector caspase activation and DNA damage, but that then recover from the DNA damage, avoid apoptosis, and proliferate instead. Many questions remain unanswered, including the signal that stimulates the mild caspase activation, and the mechanism through which this activation stimulates enhanced proliferation.

The authors should consider answering additional questions, clarifying some points, and making some minor corrections:

Major concerns affecting the interpretation of experimental results:

Expression of STAT92E RNAi had no apparent effect on the ability of hinge cells to undergo NiA, leading the authors to conclude that other protective signals must exist. However, the authors have not shown that this STAT92E RNAi is capable of eliminating JAK/STAT signaling in the hinge under these experimental conditions. Using a reporter for JAK/STAT signaling, such as the STAT-GFP, as a readout would confirm the reduction or elimination of signaling. This confirmation would be necessary to support the negative result as presented.

Similarly, the authors should confirm that the Zfh2 RNAi is reducing or eliminating Zfh2 levels in the hinge under these experimental conditions, before concluding that Zfh2 does not play a role in stopping hinge cells from undergoing NiA.

EdU incorporation was quantified by measuring the fluorescence intensity of the pouch and normalizing it to the fluorescence intensity of the whole disc. However, the images show that EdU fluorescence intensity of other regions of the disc, especially the notum, varied substantially when comparing the different genetic backgrounds (for example, note the substantially reduced EdU in the notum of Figure 3 B' and B'). Indeed, it has been shown that tissue damage can lead to suppression of proliferation in the notum and elsewhere in the disc, unless the signaling that induces the suppression is altered. Therefore, the normalization may be skewing the results because the notum EdU is not consistent across samples, possibly because the damage-induced suppression of proliferation in the notum is different across the different genetic backgrounds.

The authors expressed p35 to attempt to generate "undead cells". They take an absence of mitogen secretion or increased proliferation as evidence that undead cells were not generated. However, there could be undead cells that do not stimulate proliferation non-autonomously, which could be detected by the persistence of caspase activity in cells that do not complete apoptosis. Indeed, expressing p35 and observing sustained effector caspase activation could help answer the later question of what percentage of this cell population would otherwise complete apoptosis (NiA, rescued by p35) vs reverse course and proliferate (NiCP, unaffected by p35).

It is unclear if the authors' model is that the NiCP cells lead to autonomous or non-autonomous cell proliferation, or both. Could the lineage-tracing experiments and/or the experiments marking mitosis relative to caspase activity answer this question?

Many of the conclusions rely on single images. Quantification of many samples should be included wherever possible.

Why does the reduction of Dronc appear to affect regenerative growth in females but not males?

Author response:

Reviewer #1 (Public Review):

Summary:

In previous work, the authors described necrosis-induced apoptosis (NiA) as a consequence of induced necrosis. Specifically, experimentally induced necrosis in the distal pouch of larval wing imaginal discs triggers NiA in the lateral pouch. In this manuscript, the authors confirmed this observation and found that while necrosis can kill all areas of the disc, NiA is limited to the pouch and to some extent to the notum, but is excluded from the hinge region. Interestingly and unexpectedly, signaling by the Jak/Stat and Wg pathways inhibits NiA. Further characterization of NiA by the authors reveals that NiA also triggers regenerative proliferation which can last up to 64 hours following necrosis induction. This regenerative response to necrosis is significantly stronger compared to discs ablated by apoptosis. Furthermore, the regenerative proliferation induced by necrosis is dependent on the apoptotic pathway because RNAi targeting the RHG genes is sufficient to block proliferation. However, NiA does not promote proliferation through the previously described apoptosis-induced proliferation (AiP) pathway, although cells at the wound edge undergo AiP. Further examination of the caspase levels in NiA cells allowed the authors to group these cells into two clusters: some cells (NiA) undergo apoptosis and are removed, while others referred to as Necrosis-induced Caspase Positive (NiCP) cells survive despite caspase activity. It is the NiCP cells that repair cellular damage including DNA damage and that promote regenerative proliferation. Caspase sensors demonstrate that both groups of cells have initiator caspase activity, while only the NiA cells contain effector caspase activity. Under certain conditions, the authors were also able to visualize effector caspase activity in NiCP cells, but the level was low, likely below the threshold for apoptosis. Finally, the authors found that loss of the initiator caspase Dronc blocks regenerative proliferation, while inhibiting effector caspases by expression of p35 does not, suggesting that Dronc can induce regenerative proliferation following necrosis in a non- apoptotic manner. This last finding is very interesting as it implies that Dronc can induce proliferation in at least two ways in addition to its requirement in AiP.

Strengths:

This is a very interesting manuscript. The authors demonstrate that epithelial tissue that contains a significant number of necrotic cells is able to regenerate. This regenerative response is dependent on the apoptotic pathway which is induced at a distance from the necrotic cells. Although regenerative proliferation following necrosis requires the initiator caspase Dronc, Dronc does not induce a classical AiP response for this type of regenerative response. In future work, it will be very interesting to dissect this regenerative response pathway genetically.

Weaknesses:

No weaknesses were identified.

We thank the reviewer for their positive evaluation and kind words.

Reviewer #2 (Public Review):

Summary / Strengths:

In this manuscript, Klemm et al., build on past published findings (Klemm et al., 2021) to characterize caspase activation in distal cells following necrotic tissue damage within the Drosophila wing imaginal disc. Previously in Klemm et al., 2021, the authors describe necrosis-induced-apoptosis (NiA) following the development of a genetic system to study necrosis that is caused by the expression of a constitutive active GluR1 (Glutamate/Ca2+ channel), and they discovered that the appearance of NiA cells were important for promoting regeneration.

In this manuscript, the authors aim to investigate how tissues regenerate following necrotic cell death. They find that the cells of the wing pouch are more likely to have non-autonomous caspase activation than other regions within the wing imaginal disc (hinge and notum),two signaling pathways that are known to be upregulated during regeneration, Wnt (wingless) and JAK/Stat signaling, act to prevent additional NiA in pouch cells, and may explain the region specificity, the presence of NiA cells promotes regenerative proliferation in late stages of regeneration, not all caspase-positive cells are cleared from the epithelium (these cells are then referred to as Necrosis-induced Caspase Positive (NiCP) cells), these NiCP cells continue to live and promote proliferation in adjacent cells, the caspase Dronc is important for creating NiA/NiCP cells and for these cells to promote proliferation. Animals heterozygous for a Dronc null allele show a decrease in regeneration following necrotic tissue damage.

The study has the potential to be broadly interesting due to the insights into how tissues differentially respond to necrosis as compared to apoptosis to promote regeneration.

Weaknesses:

However, here are some of my current concerns for the manuscript in its current version:

The presence of cells with activated caspase that don't die (NiCP cells) is an interesting biological phenomenon but is not described until Figure 5. How does the existence of NiCP cells impact the earlier findings presented? Is late proliferation due to NiA, NiCP, or both? Does Wg and JAK/STAT signaling act to prevent the formation of both NiA and NiCP cells or only NiA cells? Moreover, the authors are able to specifically manipulate the wound edge (WE) and lateral pouch cells (LP), but don't show how these manipulations within these distinct populations impact regeneration. The authors provide evidence that driving UAS-mir(RHG) throughout the pouch, in the LP or the WE all decrease the amount of NiA/NiCP in Figure 3G-O, but no data on final regenerative outcomes for these manipulations is presented (such as those presented for Dronc-/+ in Fig 7M). The manuscript would be greatly enhanced by quantification of more of the findings, especially in describing if the specific manipulations that impacted NiA /NiCP cells disrupt end-point regeneration phenotypes.

We thank the reviewer for their assessment and helpful suggestions to improve the manuscript. Regarding the presence of NiA and NiCP cells, and the proportion of each within a regenerating wing disc, unfortunately we are currently limited in our ability to distinguish each type of cell using available tools. This applies to both visualizing these cells via anti-cDcp-1 staining or the activity of GC3Ai, DBS-GFP and CasExpress, and detecting their function via their influence on proliferation. As such, although the identification of NiCP does not change any of the conclusions prior to Figure 5 in which NiCP are described, we are currently unable to comment on the contribution of NiA versus NiCP to late proliferation, or whether they are differently affected by Wg and JAK/STAT signaling. This issue is touched on in the discussion, but we will expand upon our commentary to better highlight these issues.

With respect to the reviewer’s suggestion to include evidence on whether blocking NiA/NiCP influences final regenerative outcomes, these data were published in our first paper on this work (Klemm et al., 2021, PMID: 34740246), which we will gladly reiterate in this work.

Finally, we agree that further quantification of our findings will strengthen the work, which is also suggested by Reviewer 3, and plan to add it where possible in a revised manuscript.

How fast does apoptosis take within the wing disc epithelium? How many of the caspase(+) cells are present for the whole 48 hours of regeneration? Are new cells also induced to activate caspase during this time window? The author presented a number of interesting experiments characterizing the NiCP cells. For the caspase sensor GC3Ai experiments in Figure 5, is there a way to differentiate between cells that have maintained fluorescent CG3Ai from cells that have newly activated caspase? What is the timeline for when NiA and NiCP are specified? In addition, what fraction of NiCP cells contribute to the regenerated epithelium? Additional information about the temporal dynamics of NiA and NiCP specification/commitment would be greatly appreciated.

Regarding the timing of apoptosis, Schott et al., 2017 (PMID:28870988) demonstrated that apoptotic GC3Ai-labeled cells in imaginal discs are extruded within 1 hr of labeling, the kinetics of which agree with previously published work on the temporal dynamics of apoptotic cell extrusion by Monier et al., 2015 (PMID:25607361). This is much faster than the continued labeling that we observe up to 64 hr post necrosis. We will include this information alongside a quantification of the percent of the wing pouch with GC3Ai-positive cells over time to better address whether the GC3Ai signal is maintained over time or if newly activated caspases account for the signal in late regenerating discs. We plan to include PH3 staining to distinguish between cells that have activated GC3Ai and are proliferating versus new caspase activity. Additionally, we plan to include new experimental evidence to evaluate the timing of GC3Ai-labelled apoptotic cell loss in our system.

The question of when NiA/NiCP are specified is difficult to address due to the issue of not being able to easily distinguish between these cell types. We previously attempted to answer this particular question, and also to determine what fraction of these cells contribute to the regenerated epithelium, using caspase-based lineage tracing with CasExpress. However, as shown in the paper, we are unable to label NiA/NiCP with CasExpress, either due to the lack of caspase activity level or subcellular localization. We are currently attempting to combine other caspase reporters with lineage tracing tools and examine late-stage wing discs to address these questions.

The notum also does not express developmental JAK/STAT, yet little NiA was observed within the notum. Do the authors have any additional insights into the differential response between the pouch and notum? What makes the pouch unique? Are NiA/NiCP cells created within other imaginal discs and other tissues? Are they similarly important for regenerative responses in other contexts?

As noted by Martin et al., 2017 (PMID:28935711), Martin & Morata, 2018 (PMID:29938762), and our own observations in Harris et al., 2016 (PMID:26840050), the notum does not respond to damage in a way that leads to regeneration, while the pouch does. As NiA/NiCP are a pro-regenerative response, we speculate that this intrinsic difference in regenerative capacity that is potentially caused by a different proliferative and genetic response to injury may account for the disparity in NiA/NiCP occurrence in the pouch vs the notum. A difference in the presence of the (currently unidentified) DAMPs or PRRs in notum vs pouch cells may also be responsible. Alternatively, since the hinge tissue is also refractory to NiA/NiCP due to the presence of genetic factors such as Wg and JAK/STAT, there may be an analogous pathway present in notum cells that acts to protect against the induction of pro-apoptotic factors. Indeed, caspase 3 activation does not seem to occur upon ablation of the notum (Bergantinos et al. 2010, PMID:20215351). We plan to add these points to the discussion.

Regarding the existence of NiA/NiCP in other contexts, we have additional data stemming from our clonal patch experiments (Figure S1) that demonstrates this phenomenon occurs in other imaginal discs, which we plan to include in the revised manuscript.

Reviewer #3 (Public Review):

The manuscript "Regeneration following tissue necrosis is mediated by non- apoptotic caspase activity" by Klemm et al. is an exploration of what happens to a group of cells that experience caspase activation after necrosis occurs some distance away from the cells of interest. These experiments have been conducted in the Drosophila wing imaginal disc, which has been used extensively to study the response of a developing epithelium to damage and stress. The authors revise and refine their earlier discovery of apoptosis initiated by necrosis, here showing that many of those presumed apoptotic cells do not complete apoptosis. Thus, the most interesting aspect of the paper is the characterization of a group of cells that experience mild caspase activation in response to an unknown signal, followed by some effector caspase activation and DNA damage, but that then recover from the DNA damage, avoid apoptosis, and proliferate instead. Many questions remain unanswered, including the signal that stimulates the mild caspase activation, and the mechanism through which this activation stimulates enhanced proliferation.

The authors should consider answering additional questions, clarifying some points, and making some minor corrections:

Major concerns affecting the interpretation of experimental results:

Expression of STAT92E RNAi had no apparent effect on the ability of hinge cells to undergo NiA, leading the authors to conclude that other protective signals must exist. However, the authors have not shown that this STAT92E RNAi is capable of eliminating JAK/STAT signaling in the hinge under these experimental conditions. Using a reporter for JAK/STAT signaling, such as the STAT-GFP, as a readout would confirm the reduction or elimination of signaling. This confirmation would be necessary to support the negative result as presented.

We thank the reviewer for their assessment and helpful suggestions to improve the manuscript. Although the knockdown of Stat92E using this RNAi line has been shown to produce phenotypes associated with loss of JAK/STAT signaling in previous papers (Monahan and Starz-Gaiano, 2014, 2016 PMID:26277564, 26993259), we agree it would be useful to demonstrate this in our hands and therefore plan to include these data.

Similarly, the authors should confirm that the Zfh2 RNAi is reducing or eliminating Zfh2 levels in the hinge under these experimental conditions, before concluding that Zfh2 does not play a role in stopping hinge cells from undergoing NiA.

We attempted to demonstrate the loss of Zfh2 using this RNAi line, but as noted by the reviewer the antibody staining appears mostly unchanged. A reduction in Zfh2 protein levels by this RNAi has previously been demonstrated in cells of the gut (Rojas Villa et al., 2019, PMID: 31841513), suggesting that the persistent Zfh2 staining we see could be due to perdurance of the Zfh2 protein, high levels of expression or high sensitivity of the Zfh2 antibody (or a combination of these factors). We plan to repeat the experiment using a longer knockdown duration prior to ablation to show a change in Zfh2, and/or test alternative RNAi lines. In the absence of these data, we will alter our conclusions to state that Zfh2 cannot be ruled out as playing a role in preventing NiA/NiCP formation in the hinge.

EdU incorporation was quantified by measuring the fluorescence intensity of the pouch and normalizing it to the fluorescence intensity of the whole disc. However, the images show that EdU fluorescence intensity of other regions of the disc, especially the notum, varied substantially when comparing the different genetic backgrounds (for example, note the substantially reduced EdU in the notum of Figure 3 B' and B'). Indeed, it has been shown that tissue damage can lead to suppression of proliferation in the notum and elsewhere in the disc, unless the signaling that induces the suppression is altered. Therefore, the normalization may be skewing the results because the notum EdU is not consistent across samples, possibly because the damage-induced suppression of proliferation in the notum is different across the different genetic backgrounds.

We agree with the reviewer that the use of EdU cannot distinguish between an increase in proliferation in the pouch versus a decrease in proliferation of the notum (or a combination of the two), since EdU incorporation by its nature is a relative rather than absolute measure of proliferation. However, we believe that the important finding is that a localized change in proliferation is observed late in necrosis, which is dependent on NiA/NiCP since blocking the formation of these cells prevents this change. While it is possible that this observed change is caused by a reduction in proliferation of the notum, with little or even no alteration in the pouch, this would imply that NiA/NiCP act to non-autonomously limit the proliferation of cells far from where they appear in the pouch, rather than causing localized proliferation in the immediately surrounding tissue that is representative of a blastema. Although we cannot rule this possibility out, our use of a different marker for proliferation in this work (fluorescent E2F) and a more objective proliferation marker, PH3, (Klemm et al., 2021, PMID: 34740246) agree with our observations made using EdU and suggest the formation of a localized blastema in the pouch. To attempt to address this issue, due to the variability of EdU staining between samples, we aim to quantify changes in EdU that are normalized to undamaged discs stained and mounted in the same sample, thus allowing a more objective background level of proliferation to be used for comparison.

The authors expressed p35 to attempt to generate "undead cells". They take an absence of mitogen secretion or increased proliferation as evidence that undead cells were not generated. However, there could be undead cells that do not stimulate proliferation non-autonomously, which could be detected by the persistence of caspase activity in cells that do not complete apoptosis. Indeed, expressing p35 and observing sustained effector caspase activation could help answer the later question of what percentage of this cell population would otherwise complete apoptosis (NiA, rescued by p35) vs reverse course and proliferate (NiCP, unaffected by p35).

While it is very possible that expression of P35 in NiA/NiCP could induce a previously uncharacterized type of undead cell that persists but does not secrete known AiP-related factors, the way in which P35 blocks activity of effector caspases (Drice and Dcp-1) precludes our ability to reliably detect and assay NiA/NiCP over time: P35 inactivates caspases by binding to their catalytic site, which causes cDcp-1 labeling to become weak and diffuse (Klemm et al 2021, PMID: 34740246), likely because the detectable epitope is in the catalytic site (Florentin & Arama, 2012. PMID: 22351928). Similarly, the GC3Ai reporter acts as a substrate for caspases and must be cleaved for fluorescence to occur (Zhang et al., 2013 PMID: 23857461). Thus, co-expressing P35 with GC3Ai actually reduces the number of NiA/NiCP cells labeled by GC3Ai and weakens cDcp-1 staining, preventing us from assaying their persistence or association with proliferative markers.

It is unclear if the authors' model is that the NiCP cells lead to autonomous or non-autonomous cell proliferation, or both. Could the lineage-tracing experiments and/or the experiments marking mitosis relative to caspase activity answer this question?

While we see GC3Ai-labeled NiA/NiCP in the same area of the pouch with high levels of proliferation (PH3), we observe a mixture of GC3Ai cells that overlapped the PH3 cells and GC3Ai cells that were adjacent to PH3(+) cells. Thus, we are unable to conclusively say whether proliferation is induced autonomously or non-autonomously. We have attempted to answer this question with lineage tracing, however NiA/NiCP are not labeled by the CasExpress tool, and we were unable to define a relationship between NiA/NiCP and proliferation through lineage tracing. However, we add further explanation of our findings to better clarify the proposed model of NiA/NiCP-induced proliferation.

Many of the conclusions rely on single images. Quantification of many samples should be included wherever possible.

As suggested by Reviewers 2 and 3 we plan to strengthen our findings by adding quantification of phenotypes where possible, in particular in Figure 2 as mentioned in the “Recommendations for the authors”.

Why does the reduction of Dronc appear to affect regenerative growth in females but not males?

We note that the effect on regenerative growth does appear to be present in males, but that the effect is not significant. We suspect that the lower n for this experiment is the cause, and are addressing this by repeating the experiment to increase the n.