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.

Figures and data