Flamingo participates in multiple models of cell competition

  1. Pablo Sanchez Bosch
  2. Bomsoo Cho
  3. Jeffrey D Axelrod

  • Curated by eLife

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    eLife Assessment

    This study investigates the role of the Cadherin Flamingo (Fmi) in cell competition in developing tissues in Drosophila melanogaster. The findings are valuable in that they show that Fmi is required in winning cells in several competitive contexts. The evidence supporting the conclusions is solid, as the authors identify Fmi as a potential new regulator of cell competition, however, they don't delve into a mechanistic understanding of how this occurs.

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Abstract

The growth and survival of cells with different fitness, such as those with a proliferative advantage or a deleterious mutation, is controlled through cell competition. During development, cell competition enables healthy cells to eliminate less fit cells that could jeopardize tissue integrity, and facilitates the elimination of pre-malignant cells by healthy cells as a surveillance mechanism to prevent oncogenesis. Malignant cells also benefit from cell competition to promote their expansion. Despite its ubiquitous presence, the mechanisms governing cell competition, particularly those common to developmental competition and tumorigenesis, are poorly understood. Here, we show that in Drosophila , the planar cell polarity (PCP) protein Flamingo (Fmi) is required by winners to maintain their status during cell competition in malignant tumors to overtake healthy tissue, in early pre-malignant cells when they overproliferate among wildtype cells, in healthy cells when they later eliminate pre-malignant cells, and by supercompetitors as they compete to occupy excessive territory within wildtype tissues. ‘Would-be’ winners that lack Fmi are unable to overproliferate, and instead become losers. We demonstrate that the role of Fmi in cell competition is independent of PCP, and that it uses a distinct mechanism that may more closely resemble one used in other less well-defined functions of Fmi.

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  1. Version published to 10.7554/elife.98535.4 on eLife
  2. Version published to 10.7554/elife.98535 on eLife
  3. eLife

    eLife Assessment

    This study investigates the role of the Cadherin Flamingo (Fmi) in cell competition in developing tissues in Drosophila melanogaster. The findings are valuable in that they show that Fmi is required in winning cells in several competitive contexts. The evidence supporting the conclusions is solid, as the authors identify Fmi as a potential new regulator of cell competition, however, they don't delve into a mechanistic understanding of how this occurs.

  4. eLife

    Reviewer #1 (Public review):

    Summary:

    This paper is focused on the role of Cadherin Flamingo (Fmi) in cell competition in developing Drosophila tissues. A primary genetic tool is monitoring tissue overgrowths caused by making clones in the eye disc that expression activated Ras (RasV12) and that are depleted for the polarity gene scribble (scrib). The main system that they use is ey-flp, which make continuous clones in the developing eye-antennal disc beginning at the earliest stages of disc development. It should be noted that RasV12, scrib-i (or lgl-i) clones only lead to tumors/overgrowths when generated by continuous clones, which presumably creates a privileged environment that insulates them from competition. Discrete (hs-flp) RasV12, lgl-i clones are in fact out-competed (PMID: 20679206), which is something to bear in mind. They assess the role of fmi in several kinds of winners, and their data support the conclusion that fmi is required for winner status. However, they make the claim that loss of fmi from Myc winners converts them to losers, and the data supporting this conclusion is not compelling.

    Strengths:

    Fmi has been studied for its role in planar cell polarity, and its potential role in competition is interesting.

  5. eLife

    Reviewer #2 (Public review):

    Summary:

    In this manuscript, Bosch et al. reveal Flamingo (Fmi), a planar cell polarity (PCP) protein, is essential for maintaining 'winner' cells in cell competition, using Drosophila imaginal epithelia as a model. They argue that tumor growth induced by scrib-RNAi and RasV12 competition is slowed by Fmi depletion. This effect is unique to Fmi, not seen with other PCP proteins. Additional cell competition models are applied to further confirm Fmi's role in 'winner' cells. The authors also show that Fmi's role in cell competition is separate from its function in PCP formation.

    Strengths:

    (1) The identification of Fmi as a potential regulator of cell competition under various conditions is interesting.
    (2) The authors demonstrate that the involvement of Fmi in cell competition is distinct from its role in planar cell polarity (PCP) development.

  6. eLife

    Reviewer #3 (Public review):

    Summary:

    In this manuscript, Bosch and colleagues describe an unexpected function of Flamingo, a core component of the planar cell polarity pathway, in cell competition in Drosophila wing and eye disc. While Flamingo depletion has no impact on tumour growth (upon induction of Ras and depletion of Scribble throughout the eye disc), and no impact when depleted in WT cells, it specifically tunes down winner clone expansion in various genetic contexts, including the overexpression of Myc, the combination of Scribble depletion with activation of Ras in clones or the early clonal depletion of Scribble in eye disc. Flamingo depletion reduces proliferation rate and increases the rate of apoptosis in the winner clones, hence reducing their competitiveness up to forcing their full elimination (hence becoming now "loser"). This function of Flamingo in cell competition is specific of Flamingo as it cannot be recapitulated with other components of the PCP pathway, does not rely on interaction of Flamingo in trans, nor on the presence of its cadherin domain. Thus, this function is likely to rely on a non-canonical function of Flamingo which may rely on downstream GPCR signaling.

    This unexpected function of Flamingo is by itself very interesting. In the framework of cell competition, these results are also important as they describe, to my knowledge, one of the only genetic conditions that specifically affect the winner cells without any impact when depleted in the loser cells. Moreover, Flamingo do not just suppress the competitive advantage of winner clones, but even turn them in putative losers. This specificity, while not clearly understood at this stage, opens a lot of exciting mechanistic questions, but also a very interesting long term avenue for therapeutic purpose as targeting Flamingo should then affect very specifically the putative winner/oncogenic clones without any impact in WT cells.

    The data and the demonstration are very clean and compelling, with all the appropriate controls, proper quantifications and backed-up by observations in various tissues and genetic backgrounds. I don't see any weakness in the demonstration and all the points raised and claimed by the authors are all very well substantiated by the data. As such, I don't have any suggestions to reinforce the demonstration.

    While not necessary for the demonstration, documenting the subcellular localisation and levels of Flamingo in these different competition scenarios may have been relevant and provide some hints on a putative mechanism (specifically by comparing its localisation in winner and loser cells).

    Also, on a more interpretative note, the absence of impact of Flamingo depletion on JNK activation does not exclude some interesting genetic interactions. JNK output can be very contextual (for instance depending on Hippo pathway status), and it would be interesting in the future to check if Flamingo depletion could somehow alter the effect of JNK in the winner cells and promote downstream activation of apoptosis (which might normally be suppressed). It would be interesting to check if Flamingo depletion could have an impact in other contexts involving JNK activation or upon mild activation of JNK in clones.

    Strengths:

    - A clean and compelling demonstration of the function of Flamingo in winner cells during cell competition

    - One of the rare genetic conditions that affects very specifically winner cells without any impact in losers, and then can completely switch the outcome of competition (which opens an interesting therapeutic perspective on the long term)

  7. eLife

    Author response:

    The following is the authors’ response to the previous reviews.

    (1) We agreed that there was insufficient evidence for the authors' conclusion that Myc-overexpressing clones lacking Fmi become losers. We request that the authors change the text to discuss that suppression of Myc clone growth through Fmi depletion is reminiscent of a cell acquiring loser status, although at this point in the manuscript there is no clear demonstration whether this is mostly driven by growth suppression and/or an increase in apoptosis.

    We agree that at the point in the manuscript where we have only described the clone sizes, one cannot make firm conclusions about competition, so we have changed the language to reflect this. We argue that after showing our apoptosis data, those conclusions become firm. Please see the more lengthy responses to reviewers below.

    (2) We agreed that the apoptosis assay, data and interpretation need to be improved. The graphs in Fig. 4O and P should be better discussed in the text and in the legend. Additionally, the graphs are lacking the red lines that are written in the text.

    We regret that we did not adequately explain the data displayed in these two graphs. Supercompetition tends to cause apoptosis in both winners and losers, with the ratio between WT and super-competitor cells being critical in deciding the outcome of competition. We wanted to represent this visually but failed to properly explain our analysis. We have rewritten the figure legend and our discussion in the main text, hopefully making it clearer.

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    This paper is focused on the role of Cadherin Flamingo (Fmi) in cell competition in developing Drosophila tissues. A primary genetic tool is monitoring tissue overgrowths caused by making clones in the eye disc that expression activated Ras (RasV12) and that are depleted for the polarity gene scribble (scrib). The main system that they use is ey-flp, which make continuous clones in the developing eye-antennal disc beginning at the earliest stages of disc development. It should be noted that RasV12, scrib-i (or lgl-i) clones only lead to tumors/overgrowths when generated by continuous clones, which presumably creates a privileged environment that insulates them from competition. Discrete (hs-flp) RasV12, lgl-i clones are in fact out-competed (PMID: 20679206), which is something to bear in mind. They assess the role of fmi in several kinds of winners, and their data support the conclusion that fmi is required for winner status. However, they make the claim that loss of fmi from Myc winners converts them to losers, and the data supporting this conclusion is not compelling.

    Strengths:

    Fmi has been studied for its role in planar cell polarity, and its potential role in competition is interesting.

    Weaknesses:

    I have read the revised manuscript and have found issues that need to be resolved. The biggest concern is the overstatement of the results that loss of fmi from Myc-overexpressing clones turns them into losers. This is not shown in a compelling manner in the revised manuscript and the authors need to tone down their language or perform more experiments to support their claims. Additionally, the data about apoptosis is not sufficiently explained.

    We take issue with this reviewer’s framing of their criticism. First, the reviewer is selectively reporting the results published in PMID: 20679206. They correctly state that those authors show that small discreet clones of RasV12 lgl are eliminated (Fig. 3B), but they omit the fact that the authors also show that larger RasV12 lgl clones induce apoptosis in the surrounding wild type cells, and therefore behave as winners (Fig. 3C). Hence, the size of the clone appears to determine its winner/loser status. Of course, lgl is not scrib, and it is not a certainty that they would behave similarly, but they also show that large RasV12 scrib clones induce considerable apoptosis of the neighboring wild type cells.

    The reviewer then discusses “continuous” clones induced by ey-flp, as we use in our manuscript. Here, the term “continuous” is probably misleading; because ey is expressed ubiquitously in the disc from early in development, it is most likely the case that the majority of cells have flipped relatively early, resulting in ~half the cells becoming clone and the other ~half twin spot. The clone cells then likely fuse to make larger clones. We show that ey-flp induced RasV12 scrib clones also behave as winners. It is logical to conclude that this is because they are large. The reviewer talks about “a privileged environment that insulates them from competition,” but if they were insulated from competition, how could they become winners? Because they occupy more territory than the wild type cells, and because they induce apoptosis in the wild type neighbors, they are winners.

    Having shown that ey-flp induced RasV12 scrib clones behave as winners, we then remove Fmi from these clones, and show that they behave as losers by the same criteria: they occupy less area than the wild type cells (our Fig. 1 and Fig. 1 Supp 2), and they induce apoptosis in the wild type cells (our Fig 4A-H).

    With respect to the comment about additional experiments are needed to support the claim that loss of Fmi from Myc winners converts them to losers, we’re not sure what additional data the reviewer would want. As for the tumor clones, we show that >>Myc clones get bigger than the twin control clones (Fig. 2), and we measure similar low levels of apoptosis in each (Fig. 4I-K, O). In contrast >>Myc fmi clones are out-grown by wild type clones, and apoptosis is higher in the >>Myc fmi clones than in the wild type clones (Fig. 4L-N, P-S). We therefore believe it is correct to say that >>Myc clones become losers when Fmi is removed.

    In additional comments, the reviewer takes issue with using winner and loser language at the point in the manuscript where we have only shown the clone sizes but not yet the apoptosis data, and about this we agree. We have changed the language accordingly.

    Re explanation of the apoptosis data, see the response to reviewer #3.

    Reviewer #2 (Public review):

    Summary:

    In this manuscript, Bosch et al. reveal Flamingo (Fmi), a planar cell polarity (PCP) protein, is essential for maintaining 'winner' cells in cell competition, using Drosophila imaginal epithelia as a model. They argue that tumor growth induced by scrib-RNAi and RasV12 competition is slowed by Fmi depletion. This effect is unique to Fmi, not seen with other PCP proteins. Additional cell competition models are applied to further confirm Fmi's role in 'winner' cells. The authors also show that Fmi's role in cell competition is separate from its function in PCP formation.

    Strengths:

    (1) The identification of Fmi as a potential regulator of cell competition under various conditions is interesting.

    (2) The authors demonstrate that the involvement of Fmi in cell competition is distinct from its role in planar cell polarity (PCP) development.

    Weaknesses:

    (1) The authors provide a superficial description of the related phenotypes, lacking a mechanistic understanding of how Fmi regulates cell competition. While induction of apoptosis and JNK activation are commonly observed outcomes in various cell competition conditions, it is crucial to determine the specific mechanisms through which they are induced in fmi-depleted clones. Furthermore, it is recommended that the authors utilize the power of fly genetics to conduct a series of genetic epistasis analyses.

    We agree that it is desirable to have a mechanistic understanding of Fmi’s role in competition, but that is beyond the scope of this manuscript. Here, our goal is to report the phenomenon. We understand and share with the reviewer the interest in better understanding the relationship between Fmi and JNK signaling in competition. The role of JNK in competition, tumorigenesis and cell death is infamously complex. In some preliminary experiments, we explored some epistasis experiments, but these were inconclusive so we elected to not report them here. In the future, we will continue with additional analyses to gain a better understanding of the mechanism by which Fmi affects competition.

    Reviewer #3 (Public review):

    Summary:

    In this manuscript, Bosch and colleagues describe an unexpected function of Flamingo, a core component of the planar cell polarity pathway, in cell competition in Drosophila wing and eye disc. While Flamingo depletion has no impact on tumour growth (upon induction of Ras and depletion of Scribble throughout the eye disc), and no impact when depleted in WT cells, it specifically tunes down winner clone expansion in various genetic contexts, including the overexpression of Myc, the combination of Scribble depletion with activation of Ras in clones or the early clonal depletion of Scribble in eye disc. Flamingo depletion reduces proliferation rate and increases the rate of apoptosis in the winner clones, hence reducing their competitiveness up to forcing their full elimination (hence becoming now "loser"). This function of Flamingo in cell competition is specific of Flamingo as it cannot be recapitulated with other components of the PCP pathway, does not rely on interaction of Flamingo in trans, nor on the presence of its cadherin domain. Thus, this function is likely to rely on a non-canonical function of Flamingo which may rely on downstream GPCR signaling.

    This unexpected function of Flamingo is by itself very interesting. In the framework of cell competition, these results are also important as they describe, to my knowledge, one of the only genetic conditions that specifically affect the winner cells without any impact when depleted in the loser cells. Moreover, Flamingo do not just suppress the competitive advantage of winner clones, but even turn them in putative losers. This specificity, while not clearly understood at this stage, opens a lot of exciting mechanistic questions, but also a very interesting long term avenue for therapeutic purpose as targeting Flamingo should then affect very specifically the putative winner/oncogenic clones without any impact in WT cells.

    The data and the demonstration are very clean and compelling, with all the appropriate controls, proper quantifications and backed-up by observations in various tissues and genetic backgrounds. I don't see any weakness in the demonstration and all the points raised and claimed by the authors are all very well substantiated by the data. As such, I don't have any suggestions to reinforce the demonstration.

    While not necessary for the demonstration, documenting the subcellular localisation and levels of Flamingo in these different competition scenarios may have been relevant and provide some hints on a putative mechanism (specifically by comparing its localisation in winner and loser cells).

    While we did not perform a thorough analysis, our current revision of the manuscript shows Fmi staining results that do not support a change in subcellular localization of Fmi. In our images, Fmi seemed to localize similarly along the winner-loser clone boundaries, and inside and outside the clones. We cannot rule out that a subtle change in localization is taking place that could perhaps be detected with higher resolution imaging.

    Also, on a more interpretative note, the absence of impact of Flamingo depletion on JNK activation does not exclude some interesting genetic interactions. JNK output can be very contextual (for instance depending on Hippo pathway status), and it would be interesting in the future to check if Flamingo depletion could somehow alter the effect of JNK in the winner cells and promote downstream activation of apoptosis (which might normally be suppressed). It would be interesting to check if Flamingo depletion could have an impact in other contexts involving JNK activation or upon mild activation of JNK in clones.

    See our comment to Reviewer 2 regarding JNK.

    Strengths:

    A clean and compelling demonstration of the function of Flamingo in winner cells during cell competition

    One of the rare genetic conditions that affects very specifically winner cells without any impact in losers, and then can completely switch the outcome of competition (which opens an interesting therapeutic perspective on the long term) Weaknesses:

    The mechanistic understanding obviously remains quite limited at this stage especially since the signaling does not go through the PCP pathway.

    We agree that in the future, it will be desirable to gain a mechanistic understanding of Fmi’s role in competition.

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors):

    I have read the revised manuscript and have found issues that need to be resolved. The biggest concern is the overstatement of the results that loss of fmi from Myc-overexpressing clones turns them into losers. This is not shown in a compelling manner in the revised manuscript and the authors need to tone down their language or perform more experiments to support their claims.

    (1) I do not agree with the language used by the authors last paragraph of p. 4 stating loss of fmi from Myc supercompetitors (Fig. 2) makes them losers. At this point in the paper, they only use clone size as a readout. By definition, losers in imaginal discs die by apoptosis, which is not measured in this figure. As such, the authors do not prove that fmi-mutant Myc over-expressing clones are now losers at this point in the manuscript. The authors should discuss this in the results section regarding Fig. 2.

    We have modified the language in text and figure legend to acknowledge that the clone size data alone do not demonstrate competition.

    (2) Related to point #1, I do not agree with the language in the legend of Fig. 2H that the graph is measuring "supercompetition". They are only measuring clone ratios, not apoptosis. Growing to a smaller size does not make a clone have loser status without also assessing cell death.

    (a) I suggest that the authors remove the sentence "A ratio over 0 indicates supercompetition of nGFP+ clones, and below 0 indicates nGFP+ cells are losers." in the legend to Fig. 2H. Instead, they should describe the assay in times of clone ratios.

    The reviewer raises a valid point, as at this point in the manuscript we did not quantify cell death and proliferation. However, based on decades of knowledge of supercompetiton, Myc clones are classified as super-competitors in every instance they’ve been studied. (Myc clones show apoptosis when competing with WT cells, while at the same time they eliminate WT neighbors by apoptosis to become winners. Their faster proliferation rate may be what ultimately makes them winners.) We changed the language to address this distinction.

    (3) In Fig. 4, they do attempt to monitor apoptosis, which is the fate of bona fide losers in imaginal tissue. However, I have several concerns about these data (panels 4I-K, O and P have been added to the revised manuscript.)

    (a) In Fig. 4I-K, why is there no death of WT cells which would be expected based on de la Cova Cell 2004? The authors need to comment on this.

    (b) Cell death should also be observed in the Myc over-expressing clones but none is seen in this disc (see de la Cova 2004 and PMID: 18257071 Fig. 4). The authors need to comment on this.

    We do not understand why the reviewer raises these two points. We see some cell death in >Myc eye discs both in winners and losers, as displayed in the graph. In our hands, the levels were on average very low. The example shown is representative of the analysis and shows apoptosis both in WT and >Myc cells, highlighted by the arrows in 4J. We added a mention to the arrows in the figure legend to make it clearer. In the main text, we already compared our observations to the same publication the reviewer mentions (De la Cova 2004).

    (c) The data in panel 4O is not explained sufficiently in the legend or results section. What do the lines between the data points in the left side of the panel mean? Why is there a bunch of clustered data points in the right part of the Fig. 4O, when two different genotypes are listed below? I would have expected two clusters of points. The authors need to comment on this.

    We intended to convey as much information as possible in an informative manner in these graphs, and we regret not explaining better the analysis shown. We modified the legends for the apoptosis analysis to better explain the displayed data.

    (d) What is the sample size (n) for the genotypes listed in this figure? The authors need to comment on this and explicitly list the sample size in the legend.

    We added the n for both conditions to the figure.

    (e) In panels 4L-N, why is the death occurring in the apparent center of the fmiE59>>Myc clone. If these clones are truly losers as the authors claim, then apoptosis should be seen at the boundaries between the fmiE59>>Myc clone and the WT clones. The results in this figure are not compelling, yet this is the critical piece of data to support their claim that fmiE59>>Myc clone are losers. The authors need to comment on this.

    The majority of cell death in this example is observed 1-3 cells away from the clone boundary. In some cases, we observe cell death farther from the boundary, but those cells were not counted in our analyses. As described in our methods, we only considered for the analysis cells at the clone boundary or in the vicinity, as those are the ones that most probably have apoptosis triggered by the neighboring clone.

    (f) There is no red line in Fig. 4O and 4P, in contrast to what is written in the legend in the revised manuscript. This should be corrected.

    We thank the reviewer for catching the error about the line. We have now simplified the graph by removing the line at Y=0 and just leave one dashed line, representing the mean difference between WT and >>Myc cells.

    (4) On p. 10, the reference Harvey and Tapon 2007 to support hpo-/- supercompetitor status is incorrect. The references are Ziosi 2010 and Neto-Silva 2010. This should be changed.

    We thank the reviewer for the correction. While the review we provided discusses the role of the Hpo pathway in proliferation and cancer, it does not discuss competition. The reference we intended to include here was Ziosi 2010. We now cite both in the revised manuscript.

    (5) The legend for Fig. 3A-H is missing from the revised manuscript. This needs to be added.

    This was likely a copy-edit glitch. The missing parts of the legend have been restored.

    (6) Material and methods is missing details on the hs-induced clones. The authors need to specifically state when the clones were generated and when they were analyzed in hours after egg laying.

    The timing of the heat-shock and analysis was described in the methods: “Heat-shock was performed on late first instar and early second instar larvae, 48 hrs after egg laying (AEL). Vials were kept at 25ºC after heat-shock until larvae were dissected”. And additionally, in the dissection methods: “Third instar wandering larvae (120 hrs AEL) were dissected…” We have included in this revision the length of the heat-shock (15 min).

    I have read the rebuttal and some of my concerns are not sufficiently addressed.

    (8) I raised the point of continuously-generated clones becoming large enough to evade competition, and I disagree with the authors' reply. I think that competition of RasV12, scrib (or lgl) competition largely depends the size of the clone, which is de facto larger when generated by continuous expression of flp (such as eyeless or tubulin promoters used in this study). I think that at that point, we are at an impasse with respect to this issue, but I wanted to register my disagreement for the record. Related to this, one possible reason for the fragmentation of the fmimutant Myc overexpressing clones in the wing disc is because they were not continuously generated and hence did not merge with other clones.

    Please see the discussion above in the public comments. We remain unclear about what, exactly, the reviewer disagrees. As stated above, we think they are correct that the size of the clone is critical in determining winner vs loser status.

    Reviewer #2 (Recommendations for the authors):

    Although the authors have addressed some of my concerns, I still feel that a detailed mechanistic understanding is essential. I hope the authors will conduct additional experiments to solve this issue.

    We also consider the mechanism of interest and will pursue this in the future. To test our hypotheses we require a set of genetic mutants that are still in the making that will help us dissect the function and potential partners of Fmi, and we hope to have these results in a future publication.

    Reviewer #3 (Recommendations for the authors):

    - There is no clear demonstration that the relative decrease of clone size in UASMyc/Fmi mutant is mostly driven by either a context dependant suppression of growth and/or an increase of apoptosis (the latter being the more classic feature of loser phenotype).

    We believe that it is driven by both, and refrain from making assumptions about the magnitude of contribution from each. This question is something that we will be interested to explore in the future.

    The distribution of cell death in Fmi/UAS-Myc mutant is somehow surprising and may not fit with most of the competition scenarios where death is mostly restricted to clone periphery (although this may be quite variable and would require much more quantification to be clear).

    While we observe some cell death far from clone boundaries, most of the dying cells are a few cells away from a clone boundary. In other publications quantifying cell death, examples of cell death farther from the boundary are not rare (See for example Moreno and Basler 2004 Fig 6, De la Cova et al. Fig 2, Meyer et al 2014 Fig 2). We did not count cells dying far from clone boundaries in our analysis.

    I just noticed a few mistakes in the legend :

    Figure 3M legend is missing (it would be useful to know at which stage the quantification is performed)

    Another reviewer brought to our attention the problems with Fig 3 legend. We restored the missing parts.

    It would be good to give an estimate of the number of larvae observed when showing the representative cases in Figure 1 .

    This is a good point. We now include these numbers in the figure legend.

  8. Version published to 10.7554/elife.98535.3 on eLife
  9. Version published to 10.1101/2023.09.24.559197v4 on bioRxiv
  10. Version published to 10.7554/elife.98535.2 on eLife
  11. eLife

    eLife Assessment

    This study investigates the role of the Cadherin Flamingo (Fmi) in cell competition in developing tissues in Drosophila melanogaster. The findings are valuable in that they show that Fmi is required in winning cells in several competitive contexts. The evidence supporting the conclusions is solid, as the authors identify Fmi as a potential new regulator of cell competition, however, they don't delve into a mechanistic understanding of how this occurs.

  12. eLife

    Reviewer #1 (Public review):

    Summary:

    This paper is focused on the role of Cadherin Flamingo (Fmi) in cell competition in developing Drosophila tissues. A primary genetic tool is monitoring tissue overgrowths caused by making clones in the eye disc that expression activated Ras (RasV12) and that are depleted for the polarity gene scribble (scrib). The main system that they use is ey-flp, which make continuous clones in the developing eye-antennal disc beginning at the earliest stages of disc development. It should be noted that RasV12, scrib-i (or lgl-i) clones only lead to tumors/overgrowths when generated by continuous clones, which presumably creates a privileged environment that insulates them from competition. Discrete (hs-flp) RasV12, lgl-i clones are in fact out-competed (PMID: 20679206), which is something to bear in mind. They assess the role of fmi in several kinds of winners, and their data support the conclusion that fmi is required for winner status. However, they make the claim that loss of fmi from Myc winners converts them to losers, and the data supporting this conclusion is not compelling.

    Strengths:

    Fmi has been studied for its role in planar cell polarity, and its potential role in competition is interesting.

    Weaknesses:
    I have read the revised manuscript and have found issues that need to be resolved. The biggest concern is the overstatement of the results that loss of fmi from Myc-overexpressing clones turns them into losers. This is not shown in a compelling manner in the revised manuscript and the authors need to tone down their language or perform more experiments to support their claims. Additionally, the data about apoptosis is not sufficiently explained.

  13. eLife

    Reviewer #2 (Public review):

    Summary:
    In this manuscript, Bosch et al. reveal Flamingo (Fmi), a planar cell polarity (PCP) protein, is essential for maintaining 'winner' cells in cell competition, using Drosophila imaginal epithelia as a model. They argue that tumor growth induced by scrib-RNAi and RasV12 competition is slowed by Fmi depletion. This effect is unique to Fmi, not seen with other PCP proteins. Additional cell competition models are applied to further confirm Fmi's role in 'winner' cells. The authors also show that Fmi's role in cell competition is separate from its function in PCP formation.

    Strengths:

    (1) The identification of Fmi as a potential regulator of cell competition under various conditions is interesting.
    (2) The authors demonstrate that the involvement of Fmi in cell competition is distinct from its role in planar cell polarity (PCP) development.

    Weaknesses:

    (1) The authors provide a superficial description of the related phenotypes, lacking a mechanistic understanding of how Fmi regulates cell competition. While induction of apoptosis and JNK activation are commonly observed outcomes in various cell competition conditions, it is crucial to determine the specific mechanisms through which they are induced in fmi-depleted clones. Furthermore, it is recommended that the authors utilize the power of fly genetics to conduct a series of genetic epistasis analyses.

  14. eLife

    Reviewer #3 (Public review):

    Summary:

    In this manuscript, Bosch and colleagues describe an unexpected function of Flamingo, a core component of the planar cell polarity pathway, in cell competition in Drosophila wing and eye disc. While Flamingo depletion has no impact on tumour growth (upon induction of Ras and depletion of Scribble throughout the eye disc), and no impact when depleted in WT cells, it specifically tunes down winner clone expansion in various genetic contexts, including the overexpression of Myc, the combination of Scribble depletion with activation of Ras in clones or the early clonal depletion of Scribble in eye disc. Flamingo depletion reduces proliferation rate and increases the rate of apoptosis in the winner clones, hence reducing their competitiveness up to forcing their full elimination (hence becoming now "loser"). This function of Flamingo in cell competition is specific of Flamingo as it cannot be recapitulated with other components of the PCP pathway, does not rely on interaction of Flamingo in trans, nor on the presence of its cadherin domain. Thus, this function is likely to rely on a non-canonical function of Flamingo which may rely on downstream GPCR signaling.

    This unexpected function of Flamingo is by itself very interesting. In the framework of cell competition, these results are also important as they describe, to my knowledge, one of the only genetic conditions that specifically affect the winner cells without any impact when depleted in the loser cells. Moreover, Flamingo do not just suppress the competitive advantage of winner clones, but even turn them in putative losers. This specificity, while not clearly understood at this stage, opens a lot of exciting mechanistic questions, but also a very interesting long term avenue for therapeutic purpose as targeting Flamingo should then affect very specifically the putative winner/oncogenic clones without any impact in WT cells.

    The data and the demonstration are very clean and compelling, with all the appropriate controls, proper quantifications and backed-up by observations in various tissues and genetic backgrounds. I don't see any weakness in the demonstration and all the points raised and claimed by the authors are all very well substantiated by the data. As such, I don't have any suggestions to reinforce the demonstration.

    While not necessary for the demonstration, documenting the subcellular localisation and levels of Flamingo in these different competition scenarios may have been relevant and provide some hints on a putative mechanism (specifically by comparing its localisation in winner and loser cells).

    Also, on a more interpretative note, the absence of impact of Flamingo depletion on JNK activation does not exclude some interesting genetic interactions. JNK output can be very contextual (for instance depending on Hippo pathway status), and it would be interesting in the future to check if Flamingo depletion could somehow alter the effect of JNK in the winner cells and promote downstream activation of apoptosis (which might normally be suppressed). It would be interesting to check if Flamingo depletion could have an impact in other contexts involving JNK activation or upon mild activation of JNK in clones.

    Strengths:

    - A clean and compelling demonstration of the function of Flamingo in winner cells during cell competition

    - One of the rare genetic conditions that affects very specifically winner cells without any impact in losers, and then can completely switch the outcome of competition (which opens an interesting therapeutic perspective on the long term)

    Weaknesses:

    - The mechanistic understanding obviously remains quite limited at this stage especially since the signaling does not go through the PCP pathway.

  15. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Reviewer 1:

    Summary:

    This paper is focused on the role of Cadherin Flamingo (Fmi) - also called Starry night (stan) - in cell competition in developing Drosophila tissues. A primary genetic tool is monitoring tissue overgrowths caused by making clones in the eye disc that express activated Ras (RasV12) and that are depleted for the polarity gene scribble (scrib). The main system that they use is ey-flp, which makes continuous clones in the developing eye-antennal disc beginning at the earliest stages of disc development. It should be noted that RasV12, scrib-i (or lgl-i) clones only lead to tumors/overgrowths when generated by continuous clones, which presumably creates a privileged environment that insulates them from competition. Discrete (hs-flp) RasV12, lgl-i clones are in fact outcompeted (PMID: 20679206), which is something to bear in mind.

    We think it is unlikely that the outcome of RasV12, scrib (or lgl) competition depends on discrete vs. continuous clones or on creation of a privileged environment. As shown in the same reference mentioned by the reviewer, the outcome of RasV12, scrib (or lgl) tumors greatly depends on the clone being able to grow to a certain size. The authors show instances of discrete clones where larger RasV12, lgl clones outcompete the surrounding tissue and eliminate WT cells by apoptosis, whereas smaller clones behave more like losers. It is not clear what aspect of the environment determines the ability of some clones to grow larger than others, but in neither case are the clones prevented from competition. Other studies show that in mammalian cells, RasV12, scrib clones are capable of outcompeting the surrounding tissue, such as in Kohashi et al (2021), where cells carrying both mutations actively eliminate their neighbors.

    The authors show that clonal loss of Fmi by an allele or by RNAi in the RasV12, scrib-i tumors suppresses their growth in both the eye disc (continuous clones) and wing disc (discrete clones). The authors attributed this result to less killing of WT neighbors when Myc over-expressing clones lacking Fmi, but another interpretation (that Fmi regulates clonal growth) is equally as plausible with the current results.

    See point (1) for a discussion on this.

    Next, the authors show that scrib-RNAi clones that are normally out-competed by WT cells prior to adult stages are present in higher numbers when WT cells are depleted for Fmi. They then examine death in RasV12, scrib-i ey-FLP clones, or in discrete hsFLP UAS-Myc clones. They state that they see death in WT cells neighboring RasV12, scrib-i clones in the eye disc (Figures 4A-C). Next, they write that RasV12, scrib-I cells become losers (i.e., have apoptosis markers) when Fmi is removed. Neither of these results are quantified and thus are not compelling. They state that a similar result is observed for Myc over-expression clones that lack Fmi, but the image was not compelling, the results are not quantified and the controls are missing (Myc over-expressing clones alone and Fmi clones alone).

    We assayed apoptosis in UAS-Myc clones in eye discs but neglected to include the results in Figure 4. We include them in the updated manuscript. Regarding Fmi clones alone, we direct the reviewer’s attention to Fig. 2 Supplement 1 where we showed that fminull clones cause no competition. Dcp-1 staining showed low levels of apoptosis unrelated to the fminull clones or twin-spots.

    Regarding the quantification of apoptosis, we did not provide a quantification, in part because we observe a very clear visual difference between groups (Fig. 4A-K), and in part because it is challenging to come up with a rigorous quantification method. For example, how far from a winner clone can an apoptotic cell be and still be considered responsive to the clone? For UASMyc winner clones, we observe a modest amount of cell death both inside and outside the clones, consistent with prior observations. For fminull UAS-Myc clones, we observe vastly more cell death within the fminull UAS-Myc clones and modest death in nearby wildtype cells, and consequently a much higher ratio of cell death inside vs outside the clone. Because of the somewhat arbitrary nature of quantification, and the dramatic difference, we initially chose not to provide a quantification. However, given the request, we chose an arbitrary distance from the clone boundary in which to consider dying cells and counted the numbers for each condition. We view this as a very soft quantification, but we nevertheless report it in a way that captures the phenomenon in the revised manuscript.

    They then want to test whether Myc over-expressing clones have more proliferation. They show an image of a wing disc that has many small Myc overexpressing clones with and without Fmi. The pHH3 results support their conclusion that Myc overexpressing clones have more pHH3, but I have reservations about the many clones in these panels (Figures 5L-N).

    As the reviewer’s reservations are not specified, we have no specific response.

    They show that the cell competition roles of Fmi are not shared by another PCP component and are not due to the Cadherin domain of Fmi. The authors appear to interpret their results as Fmi is required for winner status. Overall, some of these results are potentially interesting and at least partially supported by the data, but others are not supported by the data.

    Strengths:

    Fmi has been studied for its role in planar cell polarity, and its potential role in competition is interesting.

    Weaknesses:

    (1) In the Myc over-expression experiments, the increased size of the Myc clones could be because they divide faster (but don't outcompete WT neighbors). If the authors want to conclude that the bigger size of the Myc clones is due to out-competition of WT neighbors, they should measure cell death across many discs of with these clones. They should also assess if reducing apoptosis (like using one copy of the H99 deficiency that removes hid, rpr, and grim) suppresses winner clone size. If cell death is not addressed experimentally and quantified rigorously, then their results could be explained by faster division of Myc over-expressing clones (and not death of neighbors). This could also apply to the RasV12, scrib-i results.

    Indeed, Myc clones have been shown to divide faster than WT neighbors, but that is not the only reason clones are bigger. As shown in (de la Cova et al, 2004), Myc-overexpressing cells induce apoptosis in WT neighbors, and blocking this apoptosis results in larger wings due to increased presence of WT cells. Also, (Moreno and Basler, 2004) showed that Myc-overexpressing clones cause a reduction in WT clone size, as WT twin spots adjacent to 4xMyc clones are significantly smaller than WT twin spots adjacent to WT clones. In the same work, they show complete elimination of WT clones generated in a tub-Myc background. Since then, multiple papers have shown these same results. It is well established then that increased cell proliferation transforms Myc clones into supercompetitors and that in the absence of cell competition, Myc-overexpressing discs produce instead wings larger than usual.

    In (de la Cova et al, 2004) the authors already showed that blocking apoptosis with H99 hinders competition and causes wings with Myc clones to be larger than those where apoptosis wasn’t blocked. As these results are well established from prior literature, there is no need to repeat them here.

    (2) This same comment about Fmi affecting clone growth should be considered in the scrib RNAi clones in Figure 3.

    In later stages, scrib RNAi clones in the eye are eliminated by WT cells. While scrib RNAi clones are not substantially smaller in third instar when competing against fmi cells (Fig 3M), by adulthood we see that WT clones lacking Fmi have failed to remove scrib clones, unlike WT clones that have completely eliminated the scrib RNAi clones by this time. We therefore disagree that the only effect of Fmi could be related to rate of cell division.

    (3) I don't understand why the quantifications of clone areas in Figures 2D, 2H, 6D are log values. The simple ratio of GFP/RFP should be shown. Additionally, in some of the samples (e.g., fmiE59 >> Myc, only 5 discs and fmiE59 vs >Myc only 4 discs are quantified but other samples have more than 10 discs). I suggest that the authors increase the number of discs that they count in each genotype to at least 20 and then standardize this number.

    Log(ratio) values are easier to interpret than a linear scale. If represented linearly, 1 means equal ratios of A and B, while 2A/B is 2 and A/2B is 0.5. And the higher the ratio difference between A and B, the starker this effect becomes, making a linear scale deceiving to the eye, especially when decreased ratios are shown. Using log(ratios), a value of 0 means equal ratios, and increased and decreased ratios deviate equally from 0.

    Statistically, either analyzing a standardized number of discs for all conditions or a variable number not determined beforehand has no effect on the p-value, as long as the variable n number is not manipulated by p-hacking techniques, such as increasing the n of samples until a significant p-value has been obtained. While some of our groups have lower numbers, all statistical analyses were performed after all samples were collected. For all results obtained by cell counts, all samples had a minimum of 10 discs due to the inherent though modest variability of our automated cell counts, and we analyzed all the discs that we obtained from a given experiment, never “cherry-picking” examples. For the sake of transparency, all our graphs show individual values in addition to the distributions so that the reader knows the n values at a glance.

    (5) Figure 4 - shows examples of cell death. Cas3 is written on the figure but Dcp-1 is written in the results. Which antibody was used? The authors need to quantify these results. They also need to show that the death of cells is part of the phenotype, like an H99 deficiency, etc (see above).

    Thank you for flagging this error. We used cleaved Dcp-1 staining to detect cell death, not Cas3 (Drice in Drosophila). We updated all panels replacing Cas3 by Dcp-1.

    As described above, cell death is a well established consequence of myc overexpression induced cell death and we feel there is no need to repeat that result. To what extent loss of Fmi induces excess cell death or reduces proliferation in “would-be” winners, and to what extent it reduces “would-be” winners’ ability to eliminate competitors are interesting mechanistic questions that are beyond the scope of the current manuscript.

    (6) It is well established that clones overexpressing Myc have increased cell death. The authors should consider this when interpreting their results.

    We are aware that Myc-overexpressing clones have increased cell death, but it has also been demonstrated that despite that fact, they behave as winners and eliminate WT neighboring cells. And as mentioned in comment (1), WT clones generated in a 3x and 4x Myc background are eliminated and removed from the tissue, and blocking cell death increases the size of WT “losers” clones adjacent to Myc overexpressing clones.

    (7) A better characterization of discrete Fmi clones would also be helpful. I suggest inducing hs-flp clones in the eye or wing disc and then determining clone size vs twin spot size and also examining cell death etc. If such experiments have already been done and published, the authors should include a description of such work in the preprint.

    We have already analyzed the size of discrete Fmi clones and showed that they did not cause any competition, with fmi-null clones having the same size as WT clones in both eye and wing discs. We direct the reviewer’s attention to Figure 2 Supplement 1.

    (8) We need more information about the expression pattern of Fmi. Is it expressed in all cells in imaginal discs? Are there any patterns of expression during larval and pupal development?

    Fmi is equally expressed by all cells in all imaginal discs in Drosophila larva and pupa. We include this information and the relevant reference (Brown et al, 2014) in the updated manuscript.

    (9) Overall, the paper is written for specialists who work in cell competition and is fairly difficult to follow, and I suggest re-writing the results to make it accessible to a broader audience.

    We have endeavored to both provide an accessible narrative and also describe in sufficient detail the data from multiple models of competition and complex genetic systems. We hope that most readers will be able, at a minimum, to follow our interpretations and the key takeaways, while those wishing to examine the nuts and bolts of the argument will find what they need presented as simply as possible.

    Reviewer 2:

    Summary:

    In this manuscript, Bosch et al. reveal Flamingo (Fmi), a planar cell polarity (PCP) protein, is essential for maintaining 'winner' cells in cell competition, using Drosophila imaginal epithelia as a model. They argue that tumor growth induced by scrib-RNAi and RasV12 competition is slowed by Fmi depletion. This effect is unique to Fmi, not seen with other PCP proteins. Additional cell competition models are applied to further confirm Fmi's role in 'winner' cells. The authors also show that Fmi's role in cell competition is separate from its function in PCP formation.

    We would like to thank the reviewer for their thoughtful and positive review.

    Strengths:

    (1) The identification of Fmi as a potential regulator of cell competition under various conditions is interesting.

    (2) The authors demonstrate that the involvement of Fmi in cell competition is distinct from its role in planar cell polarity (PCP) development.

    Weaknesses:

    (1) The authors provide a superficial description of the related phenotypes, lacking a comprehensive mechanistic understanding. Induction of apoptosis and JNK activation are general outcomes, but it is important to determine how they are specifically induced in Fmi-depleted clones. The authors should take advantage of the power of fly genetics and conduct a series of genetic epistasis analyses.

    We appreciate that this manuscript does not address the mechanism by which Fmi participates in cell competition. Our intent here is to demonstrate that Fmi is a key contributor to competition. We indeed aim to delve into mechanism, are currently directing our efforts to exploring how Fmi regulates competition, but the size of the project and required experiments are outside of the scope of this manuscript. We feel that our current findings are sufficiently valuable to merit sharing while we continue to investigate the mechanism linking Fmi to competition.

    (2) The depletion of Fmi may not have had a significant impact on cell competition; instead, it is more likely to have solely facilitated the induction of apoptosis.

    We respectfully disagree for several reasons. First, loss of Fmi is specific to winners; loss of Fmi has no effect on its own or in losers when confronting winners in competition. And in the Ras V12 tumor model, loss of Fmi did not perturb whole eye tumors – it only impaired tumor growth when tumors were confronted with competitors. We agree that induction of apoptosis is affected, but so too is proliferation, and only when in winners in competition.

    (3) To make a solid conclusion for Figure 1, the authors should investigate whether complete removal of Fmi by a mutant allele affects tumor growth induced by expressing RasV12 and scrib RNAi throughout the eye.

    We agree with the reviewer that this is a worthwhile experiment, given that RNAi has its limitations. However, as fmi is homozygous lethal at the embryo stage, one cannot create whole disc tumors mutant for fmi. As an approximation to this condition, we have introduced the GMR-Hid, cell-lethal combination to eliminate non-tumor tissue in the eye disc. Following elimination of non-tumor cells, there remains essentially a whole disc harboring fminull tumor. Indeed, this shows that whole fminull tumors overgrow similar to control tumors, confirming that the lack of Fmi only affects clonal tumors. We provide those results in the updated manuscript (Figure 1 Suppl 2 C-D).

    (4) The authors should test whether the expression level of Fmi (both mRNA and protein) changes during tumorigenesis and cell competition.

    This is an intriguing point that we considered worthwhile to examine. We performed immunostaining for Fmi in clones to determine whether its levels change during competition. Fmi is expressed ubiquitously at apical plasma membranes throughout the disc, and this was unchanged by competition, including inside >>Myc clones and at the clone boundary, where competition is actively happening. We provide these results as a new supplementary figure (Figure 5 Suppl 1) in the updated manuscript.

    Reviewer 3:

    Summary:

    In this manuscript, Bosch and colleagues describe an unexpected function of Flamingo, a core component of the planar cell polarity pathway, in cell competition in the Drosophila wing and eye disc. While Flamingo depletion has no impact on tumour growth (upon induction of Ras and depletion of Scribble throughout the eye disc), and no impact when depleted in WT cells, it specifically tunes down winner clone expansion in various genetic contexts, including the overexpression of Myc, the combination of Scribble depletion with activation of Ras in clones or the early clonal depletion of Scribble in eye disc. Flamingo depletion reduces the proliferation rate and increases the rate of apoptosis in the winner clones, hence reducing their competitiveness up to forcing their full elimination (hence becoming now "loser"). This function of Flamingo in cell competition is specific to Flamingo as it cannot be recapitulated with other components of the PCP pathway, and does not rely on the interaction of Flamingo in trans, nor on the presence of its cadherin domain. Thus, this function is likely to rely on a non-canonical function of Flamingo which may rely on downstream GPCR signaling.

    This unexpected function of Flamingo is by itself very interesting. In the framework of cell competition, these results are also important as they describe, to my knowledge, one of the only genetic conditions that specifically affect the winner cells without any impact when depleted in the loser cells. Moreover, Flamingo does not just suppress the competitive advantage of winner clones, but even turns them into putative losers. This specificity, while not clearly understood at this stage, opens a lot of exciting mechanistic questions, but also a very interesting long-term avenue for therapeutic purposes as targeting Flamingo should then affect very specifically the putative winner/oncogenic clones without any impact in WT cells.

    The data and the demonstration are very clean and compelling, with all the appropriate controls, proper quantification, and backed-up by observations in various tissues and genetic backgrounds. I don't see any weakness in the demonstration and all the points raised and claimed by the authors are all very well substantiated by the data. As such, I don't have any suggestions to reinforce the demonstration.

    While not necessary for the demonstration, documenting the subcellular localisation and levels of Flamingo in these different competition scenarios may have been relevant and provided some hints on the putative mechanism (specifically by comparing its localisation in winner and loser cells).

    Also, on a more interpretative note, the absence of the impact of Flamingo depletion on JNK activation does not exclude some interesting genetic interactions. JNK output can be very contextual (for instance depending on Hippo pathway status), and it would be interesting in the future to check if Flamingo depletion could somehow alter the effect of JNK in the winner cells and promote downstream activation of apoptosis (which might normally be suppressed). It would be interesting to check if Flamingo depletion could have an impact in other contexts involving JNK activation or upon mild activation of JNK in clones.

    We would like to thank the reviewer for their thorough and positive review.

    Strengths:

    - A clean and compelling demonstration of the function of Flamingo in winner cells during cell competition.

    - One of the rare genetic conditions that affects very specifically winner cells without any impact on losers, and then can completely switch the outcome of competition (which opens an interesting therapeutic perspective in the long term)

    Weaknesses:

    - The mechanistic understanding obviously remains quite limited at this stage especially since the signaling does not go through the PCP pathway.

    Reviewer 2 made the same comment in their weakness (1), and we refer to that response. In future work, we are excited to better understand the pathways linking Fmi and competition.

  16. Version published to 10.1101/2023.09.24.559197v3 on bioRxiv
  17. Version published to 10.7554/elife.98535.1 on eLife
  18. eLife

    Author response:

    We would like to thank the reviewers for their constructive feedback. We have thoroughly considered their concerns and comments and we aim to include some additional results in an updated version of this manuscript. In addition, we would like to address some of the comments, with which we respectfully disagree. Below is our point-by-point reply.

    Reviewer 1:

    Summary:

    This paper is focused on the role of Cadherin Flamingo (Fmi) - also called Starry night (stan) - in cell competition in developing Drosophila tissues. A primary genetic tool is monitoring tissue overgrowths caused by making clones in the eye disc that express activated Ras (RasV12) and that are depleted for the polarity gene scribble (scrib). The main system that they use is ey-flp, which makes continuous clones in the developing eye-antennal disc beginning at the earliest stages of disc development. It should be noted that RasV12, scrib-i (or lgl-i) clones only lead to tumors/overgrowths when generated by continuous clones, which presumably creates a privileged environment that insulates them from competition. Discrete (hs-flp) RasV12, lgl-i clones are in fact out-competed (PMID: 20679206), which is something to bear in mind.

    We think it is unlikely that the outcome of RasV12, scrib (or lgl) competition depends on discrete vs. continuous clones or on creation of a privileged environment. As shown in the same reference mentioned by the reviewer, the outcome of RasV12, scrib (or lgl) tumors greatly depends on the clone being able to grow to a certain size. The authors show instances of discrete clones where larger RasV12, lgl clones outcompete the surrounding tissue and eliminate WT cells by apoptosis, whereas smaller clones behave more like losers. It is not clear what aspect of the environment determines the ability of some clones to grow larger than others, but in neither case are the clones prevented from competition. Other studies show that in mammalian cells, RasV12, scrib clones are capable of outcompeting the surrounding tissue, such as in Kohashi et al (2021), where cells carrying both mutations actively eliminate their neighbors.

    The authors show that clonal loss of Fmi by an allele or by RNAi in the RasV12, scrib-i tumors suppresses their growth in both the eye disc (continuous clones) and wing disc (discrete clones). The authors attributed this result to less killing of WT neighbors when Myc over-expressing clones lacking Fmi, but another interpretation (that Fmi regulates clonal growth) is equally as plausible with the current results.

    See point (1) for a discussion on this.

    Next, the authors show that scrib-RNAi clones that are normally out-competed by WT cells prior to adult stages are present in higher numbers when WT cells are depleted for Fmi. They then examine death in RasV12, scrib-i ey-FLP clones, or in discrete hs-FLP UAS-Myc clones. They state that they see death in WT cells neighboring RasV12, scrib-i clones in the eye disc (Figures 4A-C). Next, they write that RasV12, scrib-I cells become losers (i.e., have apoptosis markers) when Fmi is removed. Neither of these results are quantified and thus are not compelling. They state that a similar result is observed for Myc over-expression clones that lack Fmi, but the image was not compelling, the results are not quantified and the controls are missing (Myc over-expressing clones alone and Fmi clones alone).

    We assayed apoptosis in UAS-Myc clones in eye discs but neglected to include the results in Figure 4. We will include them in the updated manuscript. Regarding Fmi clones alone, we direct the reviewer’s attention to Fig. 2 Supplement 1 where we showed that fminull clones cause no competition. Dcp-1 staining showed low levels of apoptosis unrelated to the fminull clones or twin-spots, and we will comment on this in the revised manuscript.

    Regarding the quantification of apoptosis, we did not provide a quantification, in part because we observe a very clear visual difference between groups (Fig. 4A-K), and in part because it is challenging to come up with a rigorous quantification method. For example, how far from a winner clone can an apoptotic cell be and still be considered responsive to the clone? For UAS-Myc winner clones, we observe a modest amount of cell death both inside and outside the clones, consistent with prior observations. For fminull UAS-Myc clones, we observe vastly more cell death within the fminull UAS-Myc clones and modest death in nearby wildtype cells, and consequently a much higher ratio of cell death inside vs outside the clone. Because of the somewhat arbitrary nature of quantification, and the dramatic difference, we initially chose not to provide a quantification. However, given the request, we chose an arbitrary distance from the clone boundary in which to consider dying cells and counted the numbers for each condition. We view this as a very soft quantification, but will report it in a way that captures the phenomenon in the revised manuscript.

    They then want to test whether Myc over-expressing clones have more proliferation. They show an image of a wing disc that has many small Myc overexpressing clones with and without Fmi. The pHH3 results support their conclusion that Myc overexpressing clones have more pHH3, but I have reservations about the many clones in these panels (Figures 5L-N).

    As the reviewer’s reservations are not specified, we have no specific response.

    They show that the cell competition roles of Fmi are not shared by another PCP component and are not due to the Cadherin domain of Fmi. The authors appear to interpret their results as Fmi is required for winner status. Overall, some of these results are potentially interesting and at least partially supported by the data, but others are not supported by the data.

    Strengths:

    Fmi has been studied for its role in planar cell polarity, and its potential role in competition is interesting.

    Weaknesses:

    (1) In the Myc over-expression experiments, the increased size of the Myc clones could be because they divide faster (but don't outcompete WT neighbors). If the authors want to conclude that the bigger size of the Myc clones is due to out-competition of WT neighbors, they should measure cell death across many discs of with these clones. They should also assess if reducing apoptosis (like using one copy of the H99 deficiency that removes hid, rpr, and grim) suppresses winner clone size. If cell death is not addressed experimentally and quantified rigorously, then their results could be explained by faster division of Myc over-expressing clones (and not death of neighbors). This could also apply to the RasV12, scrib-i results.

    Indeed, Myc clones have been shown to divide faster than WT neighbors, but that is not the only reason clones are bigger. As shown in (de la Cova et al, 2004), Myc-overexpressing cells induce apoptosis in WT neighbors, and blocking this apoptosis results in larger wings due to increased presence of WT cells. Also, (Moreno and Basler, 2004) showed that Myc-overexpressing clones cause a reduction in WT clone size, as WT twin spots adjacent to 4xMyc clones are significantly smaller than WT twin spots adjacent to WT clones. In the same work, they show complete elimination of WT clones generated in a tub-Myc background. Since then, multiple papers have shown these same results. It is well established then that increased cell proliferation transforms Myc clones into supercompetitors and that in the absence of cell competition, Myc-overexpressing discs produce instead wings larger than usual.

    In (de la Cova et al, 2004) the authors already showed that blocking apoptosis with H99 hinders competition and causes wings with Myc clones to be larger than those where apoptosis wasn’t blocked. As these results are well established from prior literature, there is no need to repeat them here.

    (2) This same comment about Fmi affecting clone growth should be considered in the scrib RNAi clones in Figure 3.

    In later stages, scrib RNAi clones in the eye are eliminated by WT cells. While scrib RNAi clones are not substantially smaller in third instar when competing against fmi cells (Fig 3M), by adulthood we see that WT clones lacking Fmi have failed to remove scrib clones, unlike WT clones that have completely eliminated the scrib RNAi clones by this time. We therefore disagree that the only effect of Fmi could be related to rate of cell division.

    (3) I don't understand why the quantifications of clone areas in Figures 2D, 2H, 6D are log values. The simple ratio of GFP/RFP should be shown. Additionally, in some of the samples (e.g., fmiE59 >> Myc, only 5 discs and fmiE59 vs >Myc only 4 discs are quantified but other samples have more than 10 discs). I suggest that the authors increase the number of discs that they count in each genotype to at least 20 and then standardize this number.

    Log(ratio) values are easier to interpret than a linear scale. If represented linearly, 1 means equal ratios of A and B, while 2A/B is 2 and A/2B is 0.5. And the higher the ratio difference between A and B, the starker this effect becomes, making a linear scale deceiving to the eye, especially when decreased ratios are shown. Using log(ratios), a value of 0 means equal ratios, and increased and decreased ratios deviate equally from 0.

    Statistically, either analyzing a standardized number of discs for all conditions or a variable number not determined beforehand has no effect on the p-value, as long as the variable n number is not manipulated by p-hacking techniques, such as increasing the n of samples until a significant p-value has been obtained. While some of our groups have lower numbers, all statistical analyses were performed after all samples were collected. For all results obtained by cell counts, all samples had a minimum of 10 discs due to the inherent though modest variability of our automated cell counts, and we analyzed all the discs that we obtained from a given experiment, never “cherry-picking” examples. For the sake of transparency, all our graphs show individual values in addition to the distributions so that the reader knows the n values at a glance.

    (5) Figure 4 - shows examples of cell death. Cas3 is written on the figure but Dcp-1 is written in the results. Which antibody was used? The authors need to quantify these results. They also need to show that the death of cells is part of the phenotype, like an H99 deficiency, etc (see above).

    Thank you for flagging this error. We used cleaved Dcp-1 staining to detect cell death, not Cas3 (Drice in Drosophila). We will update all panels replacing Cas3 by Dcp-1.

    As described above, cell death is a well established consequence of myc overexpression induced cell death and we feel there is no need to repeat that result. To what extent loss of Fmi induces excess cell death or reduces proliferation in “would-be” winners, and to what extent it reduces “would-be” winners’ ability to eliminate competitors are interesting mechanistic questions that are beyond the scope of the current manuscript.

    (6) It is well established that clones overexpressing Myc have increased cell death. The authors should consider this when interpreting their results.

    We are aware that Myc-overexpressing clones have increased cell death, but it has also been demonstrated that despite that fact, they behave as winners and eliminate WT neighboring cells. And as mentioned in comment (1), WT clones generated in a 3x and 4x Myc background are eliminated and removed from the tissue, and blocking cell death increases the size of WT “losers” clones adjacent to Myc overexpressing clones.

    (7) A better characterization of discrete Fmi clones would also be helpful. I suggest inducing hs-flp clones in the eye or wing disc and then determining clone size vs twin spot size and also examining cell death etc. If such experiments have already been done and published, the authors should include a description of such work in the preprint.

    We have already analyzed the size of discrete Fmi clones and showed that they did not cause any competition, with fmi-null clones having the same size as WT clones in both eye and wing discs. We direct the reviewer’s attention to Figure 2 Supplement 1.

    (8) We need more information about the expression pattern of Fmi. Is it expressed in all cells in imaginal discs? Are there any patterns of expression during larval and pupal development?

    Fmi is equally expressed by all cells in all imaginal discs in Drosophila larva and pupa. We will include this information in the updated manuscript.

    (9) Overall, the paper is written for specialists who work in cell competition and is fairly difficult to follow, and I suggest re-writing the results to make it accessible to a broader audience.

    We have endeavored to both provide an accessible narrative and also describe in sufficient detail the data from multiple models of competition and complex genetic systems. We hope that most readers will be able, at a minimum, to follow our interpretations and the key takeaways, while those wishing to examine the nuts and bolts of the argument will find what they need presented as simply as possible.

    Reviewer 2:

    Summary:

    In this manuscript, Bosch et al. reveal Flamingo (Fmi), a planar cell polarity (PCP) protein, is essential for maintaining 'winner' cells in cell competition, using Drosophila imaginal epithelia as a model. They argue that tumor growth induced by scrib-RNAi and RasV12 competition is slowed by Fmi depletion. This effect is unique to Fmi, not seen with other PCP proteins. Additional cell competition models are applied to further confirm Fmi's role in 'winner' cells. The authors also show that Fmi's role in cell competition is separate from its function in PCP formation.

    We would like to thank the reviewer for their thoughtful and positive review.

    Strengths:

    (1) The identification of Fmi as a potential regulator of cell competition under various conditions is interesting.

    (2) The authors demonstrate that the involvement of Fmi in cell competition is distinct from its role in planar cell polarity (PCP) development.

    Weaknesses:

    (1) The authors provide a superficial description of the related phenotypes, lacking a comprehensive mechanistic understanding. Induction of apoptosis and JNK activation are general outcomes, but it is important to determine how they are specifically induced in Fmi-depleted clones. The authors should take advantage of the power of fly genetics and conduct a series of genetic epistasis analyses.

    We appreciate that this manuscript does not address the mechanism by which Fmi participates in cell competition. Our intent here is to demonstrate that Fmi is a key contributor to competition. We indeed aim to delve into mechanism, are currently directing our efforts to exploring how Fmi regulates competition, but the size of the project and required experiments are outside of the scope of this manuscript. We feel that our current findings are sufficiently valuable to merit sharing while we continue to investigate the mechanism linking Fmi to competition.

    (2) The depletion of Fmi may not have had a significant impact on cell competition; instead, it is more likely to have solely facilitated the induction of apoptosis.

    We respectfully disagree for several reasons. First, loss of Fmi is specific to winners; loss of Fmi has no effect on its own or in losers when confronting winners in competition. And in the Ras V12 tumor model, loss of Fmi did not perturb whole eye tumors – it only impaired tumor growth when tumors were confronted with competitors. We agree that induction of apoptosis is affected, but so too is proliferation, and only when in winners in competition.

    (3) To make a solid conclusion for Figure 1, the authors should investigate whether complete removal of Fmi by a mutant allele affects tumor growth induced by expressing RasV12 and scrib RNAi throughout the eye.

    We agree with the reviewer that this is a worthwhile experiment, given that RNAi has its limitations. However, as fmi is homozygous lethal at the embryo stage, one cannot create whole disc tumors mutant for fmi. As an approximation to this condition, we have introduced the GMR-Hid, cell-lethal combination to eliminate non-tumor tissue in the eye disc. Following elimination of non-tumor cells, there remains essentially a whole disc harboring fminull tumor. Indeed, this shows that whole fminull tumors overgrow similar to control tumors, confirming that the lack of Fmi only affects clonal tumors. We will provide those results in the updated manuscript.

    (4) The authors should test whether the expression level of Fmi (both mRNA and protein) changes during tumorigenesis and cell competition.

    This is an intriguing point that we would like to validate. We are currently performing immunostaining for Fmi in clones to confirm whether its levels change during competition. We will provide these results in the updated manuscript.

    Reviewer 3:

    Summary:
    In this manuscript, Bosch and colleagues describe an unexpected function of Flamingo, a core component of the planar cell polarity pathway, in cell competition in the Drosophila wing and eye disc. While Flamingo depletion has no impact on tumour growth (upon induction of Ras and depletion of Scribble throughout the eye disc), and no impact when depleted in WT cells, it specifically tunes down winner clone expansion in various genetic contexts, including the overexpression of Myc, the combination of Scribble depletion with activation of Ras in clones or the early clonal depletion of Scribble in eye disc. Flamingo depletion reduces the proliferation rate and increases the rate of apoptosis in the winner clones, hence reducing their competitiveness up to forcing their full elimination (hence becoming now "loser"). This function of Flamingo in cell competition is specific to Flamingo as it cannot be recapitulated with other components of the PCP pathway, and does not rely on the interaction of Flamingo in trans, nor on the presence of its cadherin domain. Thus, this function is likely to rely on a non-canonical function of Flamingo which may rely on downstream GPCR signaling.

    This unexpected function of Flamingo is by itself very interesting. In the framework of cell competition, these results are also important as they describe, to my knowledge, one of the only genetic conditions that specifically affect the winner cells without any impact when depleted in the loser cells. Moreover, Flamingo does not just suppress the competitive advantage of winner clones, but even turns them into putative losers. This specificity, while not clearly understood at this stage, opens a lot of exciting mechanistic questions, but also a very interesting long-term avenue for therapeutic purposes as targeting Flamingo should then affect very specifically the putative winner/oncogenic clones without any impact in WT cells.

    The data and the demonstration are very clean and compelling, with all the appropriate controls, proper quantification, and backed-up by observations in various tissues and genetic backgrounds. I don't see any weakness in the demonstration and all the points raised and claimed by the authors are all very well substantiated by the data. As such, I don't have any suggestions to reinforce the demonstration.

    While not necessary for the demonstration, documenting the subcellular localisation and levels of Flamingo in these different competition scenarios may have been relevant and provided some hints on the putative mechanism (specifically by comparing its localisation in winner and loser cells).

    Also, on a more interpretative note, the absence of the impact of Flamingo depletion on JNK activation does not exclude some interesting genetic interactions. JNK output can be very contextual (for instance depending on Hippo pathway status), and it would be interesting in the future to check if Flamingo depletion could somehow alter the effect of JNK in the winner cells and promote downstream activation of apoptosis (which might normally be suppressed). It would be interesting to check if Flamingo depletion could have an impact in other contexts involving JNK activation or upon mild activation of JNK in clones.

    We would like to thank the reviewer for their thorough and positive review.

    Strengths:

    - A clean and compelling demonstration of the function of Flamingo in winner cells during cell competition.

    - One of the rare genetic conditions that affects very specifically winner cells without any impact on losers, and then can completely switch the outcome of competition (which opens an interesting therapeutic perspective in the long term)

    Weaknesses:

    - The mechanistic understanding obviously remains quite limited at this stage especially since the signaling does not go through the PCP pathway.

    Reviewer 2 made the same comment in their weakness (1), and we refer to that response. In future work, we are excited to better understand the pathways linking Fmi and competition.

  19. eLife

    eLife assessment

    This study investigates the role of the Cadherin Flamingo (Fmi) in cell competition in developing tissues in Drosophila melanogaster. The findings are valuable in that they show that Fmi is required in winning cells in several competitive contexts. The evidence supporting the conclusions is solid, as the authors identify Fmi as a potential new regulator of cell competition, however, they don't delve into a mechanistic understanding of how this occurs.

  20. eLife

    Reviewer #1 (Public Review):

    Summary:

    This paper is focused on the role of Cadherin Flamingo (Fmi) - also called Starry night (stan) - in cell competition in developing Drosophila tissues. A primary genetic tool is monitoring tissue overgrowths caused by making clones in the eye disc that express activated Ras (RasV12) and that are depleted for the polarity gene scribble (scrib). The main system that they use is ey-flp, which makes continuous clones in the developing eye-antennal disc beginning at the earliest stages of disc development. It should be noted that RasV12, scrib-i (or lgl-i) clones only lead to tumors/overgrowths when generated by continuous clones, which presumably creates a privileged environment that insulates them from competition. Discrete (hs-flp) RasV12, lgl-i clones are in fact out-competed (PMID: 20679206), which is something to bear in mind.

    The authors show that clonal loss of Fmi by an allele or by RNAi in the RasV12, scrib-i tumors suppresses their growth in both the eye disc (continuous clones) and wing disc (discrete clones). The authors attributed this result to less killing of WT neighbors when Myc over-expressing clones lacking Fmi, but another interpretation (that Fmi regulates clonal growth) is equally as plausible with the current results. Next, the authors show that scrib-RNAi clones that are normally out-competed by WT cells prior to adult stages are present in higher numbers when WT cells are depleted for Fmi. They then examine death in RasV12, scrib-i ey-FLP clones, or in discrete hs-FLP UAS-Myc clones. They state that they see death in WT cells neighboring RasV12, scrib-i clones in the eye disc (Figures 4A-C). Next, they write that RasV12, scrib-I cells become losers (i.e., have apoptosis markers) when Fmi is removed. Neither of these results are quantified and thus are not compelling. They state that a similar result is observed for Myc over-expression clones that lack Fmi, but the image was not compelling, the results are not quantified and the controls are missing (Myc over-expressing clones alone and Fmi clones alone). They then want to test whether Myc over-expressing clones have more proliferation. They show an image of a wing disc that has many small Myc overexpressing clones with and without Fmi. The pHH3 results support their conclusion that Myc overexpressing clones have more pHH3, but I have reservations about the many clones in these panels (Figures 5L-N). They show that the cell competition roles of Fmi are not shared by another PCP component and are not due to the Cadherin domain of Fmi. The authors appear to interpret their results as Fmi is required for winner status. Overall, some of these results are potentially interesting and at least partially supported by the data, but others are not supported by the data.

    Strengths:

    Fmi has been studied for its role in planar cell polarity, and its potential role in competition is interesting.

    Weaknesses:

    (1) In the Myc over-expression experiments, the increased size of the Myc clones could be because they divide faster (but don't outcompete WT neighbors). If the authors want to conclude that the bigger size of the Myc clones is due to out-competition of WT neighbors, they should measure cell death across many discs of with these clones. They should also assess if reducing apoptosis (like using one copy of the H99 deficiency that removes hid, rpr, and grim) suppresses winner clone size. If cell death is not addressed experimentally and quantified rigorously, then their results could be explained by faster division of Myc over-expressing clones (and not death of neighbors). This could also apply to the RasV12, scrib-i results.

    (2) This same comment about Fmi affecting clone growth should be considered in the scrib RNAi clones in Figure 3.

    (3) I don't understand why the quantifications of clone areas in Figures 2D, 2H, 6D are log values. The simple ratio of GFP/RFP should be shown. Additionally, in some of the samples (e.g., fmiE59 >> Myc, only 5 discs and fmiE59 vs >Myc only 4 discs are quantified but other samples have more than 10 discs). I suggest that the authors increase the number of discs that they count in each genotype to at least 20 and then standardize this number.

    (4) There is a typo when referring to Figures 3C-D. It should be Figure 2C-D.

    (5) Figure 4 - shows examples of cell death. Cas3 is written on the figure but Dcp-1 is written in the results. Which antibody was used? The authors need to quantify these results. They also need to show that the death of cells is part of the phenotype, like an H99 deficiency, etc (see above).

    (6) It is well established that clones overexpressing Myc have increased cell death. The authors should consider this when interpreting their results.

    (7) A better characterization of discrete Fmi clones would also be helpful. I suggest inducing hs-flp clones in the eye or wing disc and then determining clone size vs twin spot size and also examining cell death etc. If such experiments have already been done and published, the authors should include a description of such work in the preprint.

    (8) We need more information about the expression pattern of Fmi. Is it expressed in all cells in imaginal discs? Are there any patterns of expression during larval and pupal development?

    (9) Overall, the paper is written for specialists who work in cell competition and is fairly difficult to follow, and I suggest re-writing the results to make it accessible to a broader audience.

  21. eLife

    Reviewer #2 (Public Review):

    Summary:

    In this manuscript, Bosch et al. reveal Flamingo (Fmi), a planar cell polarity (PCP) protein, is essential for maintaining 'winner' cells in cell competition, using Drosophila imaginal epithelia as a model. They argue that tumor growth induced by scrib-RNAi and RasV12 competition is slowed by Fmi depletion. This effect is unique to Fmi, not seen with other PCP proteins. Additional cell competition models are applied to further confirm Fmi's role in 'winner' cells. The authors also show that Fmi's role in cell competition is separate from its function in PCP formation.

    Strengths:

    (1) The identification of Fmi as a potential regulator of cell competition under various conditions is interesting.

    (2) The authors demonstrate that the involvement of Fmi in cell competition is distinct from its role in planar cell polarity (PCP) development.

    Weaknesses:

    (1) The authors provide a superficial description of the related phenotypes, lacking a comprehensive mechanistic understanding. Induction of apoptosis and JNK activation are general outcomes, but it is important to determine how they are specifically induced in Fmi-depleted clones. The authors should take advantage of the power of fly genetics and conduct a series of genetic epistasis analyses.

    (2) The depletion of Fmi may not have had a significant impact on cell competition; instead, it is more likely to have solely facilitated the induction of apoptosis.

    (3) To make a solid conclusion for Figure 1, the authors should investigate whether complete removal of Fmi by a mutant allele affects tumor growth induced by expressing RasV12 and scrib RNAi throughout the eye.

    (4) The authors should test whether the expression level of Fmi (both mRNA and protein) changes during tumorigenesis and cell competition.

  22. eLife

    Reviewer #3 (Public Review):

    Summary:

    In this manuscript, Bosch and colleagues describe an unexpected function of Flamingo, a core component of the planar cell polarity pathway, in cell competition in the Drosophila wing and eye disc. While Flamingo depletion has no impact on tumour growth (upon induction of Ras and depletion of Scribble throughout the eye disc), and no impact when depleted in WT cells, it specifically tunes down winner clone expansion in various genetic contexts, including the overexpression of Myc, the combination of Scribble depletion with activation of Ras in clones or the early clonal depletion of Scribble in eye disc. Flamingo depletion reduces the proliferation rate and increases the rate of apoptosis in the winner clones, hence reducing their competitiveness up to forcing their full elimination (hence becoming now "loser"). This function of Flamingo in cell competition is specific to Flamingo as it cannot be recapitulated with other components of the PCP pathway, and does not rely on the interaction of Flamingo in trans, nor on the presence of its cadherin domain. Thus, this function is likely to rely on a non-canonical function of Flamingo which may rely on downstream GPCR signaling.

    This unexpected function of Flamingo is by itself very interesting. In the framework of cell competition, these results are also important as they describe, to my knowledge, one of the only genetic conditions that specifically affect the winner cells without any impact when depleted in the loser cells. Moreover, Flamingo does not just suppress the competitive advantage of winner clones, but even turns them into putative losers. This specificity, while not clearly understood at this stage, opens a lot of exciting mechanistic questions, but also a very interesting long-term avenue for therapeutic purposes as targeting Flamingo should then affect very specifically the putative winner/oncogenic clones without any impact in WT cells.

    The data and the demonstration are very clean and compelling, with all the appropriate controls, proper quantification, and backed-up by observations in various tissues and genetic backgrounds. I don't see any weakness in the demonstration and all the points raised and claimed by the authors are all very well substantiated by the data. As such, I don't have any suggestions to reinforce the demonstration.

    While not necessary for the demonstration, documenting the subcellular localisation and levels of Flamingo in these different competition scenarios may have been relevant and provided some hints on the putative mechanism (specifically by comparing its localisation in winner and loser cells).

    Also, on a more interpretative note, the absence of the impact of Flamingo depletion on JNK activation does not exclude some interesting genetic interactions. JNK output can be very contextual (for instance depending on Hippo pathway status), and it would be interesting in the future to check if Flamingo depletion could somehow alter the effect of JNK in the winner cells and promote downstream activation of apoptosis (which might normally be suppressed). It would be interesting to check if Flamingo depletion could have an impact in other contexts involving JNK activation or upon mild activation of JNK in clones.

    Strengths:

    - A clean and compelling demonstration of the function of Flamingo in winner cells during cell competition.

    - One of the rare genetic conditions that affects very specifically winner cells without any impact on losers, and then can completely switch the outcome of competition (which opens an interesting therapeutic perspective in the long term)

    Weaknesses:

    - The mechanistic understanding obviously remains quite limited at this stage especially since the signaling does not go through the PCP pathway.

  23. Version published to 10.1101/2023.09.24.559197v2 on bioRxiv
  24. Version published to 10.1101/2023.09.24.559197v1 on bioRxiv