The miRNA bantam regulates growth and tumorigenesis by repressing the cell cycle regulator tribbles

This work identifies the cell cycle regulator tribbles as a target of the miRNA bantam involved in the growth regulatory and oncogenic functions of bantam in Drosophila epithelia.

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Full guidelines are available on our Instructions for Authors page, http://www.life-sciencealliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** In their paper Gerlach and colleagues determine that the cell cycle regulator tribbles is a new target of the miRNA bantam, which in Drosophila has extensively been shown to control growth. The author use the wing imaginal disc system to show that a sensor that includes predicted bantam sites present in the tribbles 3'UTR is controlled by bantam and that tribbles modulation affects proliferation downstream of bantam and the hippo pathway component yorkie (YAP in humans).
The finally present evidence tribbles is part of a circuit regulate by yorkie and bantam that controls tissue growth by coordinating cell cycling and death decisions.
The genetic control of growth is central to tissue development, homeostasis and their alterations, which are observed in cancer and congenital syndromes. The fly system has played a key role in discovering most of the conserved growth regulators, such as components of the Tor and Hippo pathway among others. Since it serendipitous discovery, which sparked the entire miRNA field, bantam regulation of growth has represented a paradigm of posttranscriptional regulation. The work presented in the manuscript builds on previous discovery of the same authors and of others and represent an important step in revealing mechanisms that control coordination of cell cycle and cell death regulation. The manuscript presents an elegant set of genetic experiments which are solid, well controlled and properly quantified. It is clearly written and figure displays are logically organized. I only have few minor comments.
1-Some of the effect presented have been observed before in the same or in other systems (tumor suppressor acitvity of yorkie, cell cycle regulation by tribbles). These would probably make more sense as supplementary figures.
2-Bantam was reported to regulate many targets. However the authors have shown that 2 of them (tribbles and hid) are sufficient to recap most of the phenotype. Would you speculate in the discussion about the function of other targets? Is it tissue specificity? Is it other functions behond growth?
3-The first part of the discussion essentially restate most of what has been presented in the intro. Authors show streamline that part.
Reviewer #2 (Comments to the Authors (Required)): In this manuscript, Gerlach et al use the Drosophila wing epithelium to identify genes required for tissue growth downstream of the Yki target gene bantam. The authors identify the G2/M regulator Tribbles as a target of the bantam miRNA. Although tribbles downregulation did not result in overgrowth of the wing imaginal primordium, the authors nicely show that together with hid downregulation, tribbles depletion is capable of driving overgrowth to a similar extent as bantam does. The authors suggest that Yki promotes proliferation and apoptosis inhibition both directly by regulating cell cycle regulators and Diap1 as well as indirectly through bantam, which represses both tribbles and hid. Additionally, the authors demonstrate that tribbles can act as a tumour suppressor in other tumorigenic conditions such as cytokinesis blockage.
Overall, the work is of a high standard and provides interesting insights into bantam-dependent growth regulation during normal Drosophila wing disc development as well as in some tumorigenic conditions. The authors convincingly demonstrate regulation of tribbles by bantam and show that together with hid is the minimal combination that can explain most of the overgrowth that bantam overexpression drives. However, there are several important issues as detailed below that the authors should address prior to publication of this manuscript.
Major points: 1. Figure 1 requires quantification of disc size since the bantam+CF and Yki+CF conditions seem to have large variability (compare Figure 1 with Figure 2 examples). Besides, the authors specify in the methods section that "larvae were kept for 3 to 5 days at 29{degree sign} C" which can cause large differences in tissue size. Quantification of several examples would be helpful to support authors statement that "The effect of bantam in discs with cytokinesis failure was similar to the one observed by Yki expression (Fig 1C,I;and Fig 2G,H)." Figure 7H-M also needs quantification. Figure 3H-I the authors show bantam mutant clones in a Minute/+ background to demonstrate that endogenous bantam regulates the trbl 3'UTR sensor. Since whether endogenous bantam levels regulate tribbles is an important point in the manuscript, I believe that is necessary to show that Minute heterozygous cells do not shown changes in the trbl-sensor (wild type clones in a minute/+ background).

In
3. In Figure 3D-I it would be important to show whether the regulation of the trbl 3'UTR sensor results in increased levels of Trbl protein (either with antibody staining or the trbl-GFP line).
4. To link trbl with Hippo pathway perturbation, the authors should check if the trbl sensor and/or trbl-GFP are reduced in wts or hpo mutant clones. 5. Does combined loss of trbl and hid rescue the bantam clonal undergrowth phenotype? If so, it would strengthen the idea that these are key downstream targets of ban. Figure 4 what is the effect of trbl depletion on cell number/tissue size? 7. In Figure 4, the FACS data show that trbl overexpression causes cells to accumulate in G2 (increase number of cells in G2 and decrease number of cells in G1). Does this result in fewer mitotic cells? Please show PH3 staining in this condition. I also wonder how the authors explain that this manipulation does not affect tissue size, especially since it was shown that trbl overexpression in the eye and wing imaginal discs shows defects (see Figure 5D in Price et al., 2002 andFigure 1 in Das et al., 2014). They mention Mata et al., 2000 to support the fact that trbl overexpression does not cause size defects, however that observation still lacks proper quantification. In fact, Mata et al., 2000 shows that trbl overexpression does affects cell size and cell number in the wing disc. I believe that authors should clarify this issue since it is important to validate their rescue experiment in the EGFR+bantam tumorigenic condition.

In
8. Figure 6F-G requires quantification. Although the trbl-RNAi+hid-RNAi wing size result is quite convincing, the number of apoptotic cells seems to be very low to explain the fact that trbl knock down did not result in tissue overgrowth. The same holds true for the example shown in FigS3A, in which many cas3-act positive cells seem to be in the ventral compartment. The increase in cell death associated with changes in cell proliferation cited in Neufeld et al., 1998 is much stronger than the one authors observe with Trbl downregulation. The authors should further test whether hid-RNAi rescues the apoptosis induced by trbl-RNAi. 9. In Figure 6H-L, authors use UAS-p35 to show that suppression of apoptosis in trbl-RNAi led to tissue overgrowth. I would strongly encourage the authors to carry out the same experiment using another way to block apoptosis (such as UAS-diap1). Overexpression of p35 leads to the presence of "undead cells" that are known to secrete several growth-promoting factors such as Wg and this might be the explanation for the fact that the p35+trbl-RNAi gives much higher relative size than the hid-RNAi+trbl-RNAi. Using UAS-diap1 would be more similar to the situation of hid-RNAi (no undead cells).
10. The authors suggest that apoptosis in response to trbl depletion can be used as a homeostatic mechanism to offset tissue overgrowth. Is this specific to trbl or can it be mimicked by dMyt1-RNAi or Stg?
Minor points: 1. Authors should discuss the possible reasons for the difference between EGFR-activation and CF in the ability of trbl depletion to drive tumorigenesis (in CF, UAS-p35 is required). 2. The authors consistently (several times in the text as well as in Figure 7 title) claim that trbl acts as a tumour suppressor in the wing imaginal disc. However, this conclusion might be interpreted as if depletion of trbl on its own results in tumour formation, which clearly is not the case. I feel that authors should be careful in specifying that trbl acts as a tumour suppressor in the wing imaginal disc only in EGFR activation condition. Moreover, the case with CF is quite different, because trbl depletion alone is not capable of driving tumorigenesis in a CF background unless p35 is expressed as well. 3. From the observations in Figures 4 and 5 the authors say that "... these results suggest that bantam acts through trbl to stimulate tissue growth". I would be slightly more conservative and say "partially through trbl" since the rescue does not seem to be complete (show statistics between bantam+trbl and control in Figure 5G).

Methods section:
-Please specify the product number of each antibody listed as well as the concentrations at which each one was used.
-Please specify which secondary antibodies were used as well as the respectively concentrations.
-Describe how the flow cytometry-based cell cycle analysis was performed.
Typos: -In the summary blurb: "...which is involved in the bantam..." Reviewer #3 (Comments to the Authors (Required)): The submitted article demonstrates that bantam regulates growth by repressing tribbles. Previous work from the same lab has shown that coexpression of the Yki targets string and DIAP1 can promote tumorigenesis in wing discs with defective cytokinesis. Now, they identify the string regulator tribbles as a direct bantam target gene and show that tribbles regulation by bantam is central in controlling tissue growth and tumorigenesis. The study is well designed and performed, and provides valuable insight on the very complex mechanistic bases of bantam's role in development and disease. I recommend publication.
Considering/addressing the following points would result in a much more solid and rigorous article.
1-I really do not see the need for the CF model in this work. A great deal of the value of the main finding reported in the submitted manuscript is that it may be expected to apply to all processes that require cells to successfully cycle. That includes normal development as much as tumour growth, which itself includes CF-related tumours as much as any other type of tumour. Thus, quite frankly, the paper would be just as important -and much more clear-if it was limited to Figs 5, 6, S1 and S2, which are the ones that make the point. Reference to CF tumours do not contribute anything to this story.
As a matter of fact, the other figures are fairly dispensable. Demonstrating that tissue overgrowth can be limited by compromising cell cycle progression is rather trivial as trivial is showing the opposite. More so when what goes for Yki, CF, etc is likely to go for any other tumour condition. This is important because headings like "Trbl represses tumorigenesis in cells with cytokinesis failure" can be misleading if the point is not made clear that, more than likely, Trbl represses any kind of tumorigenesis.
2-Somewhat related to the above, the entire section "trbl antagonizes G2/M transition in the wing disc " is unnecessary because it quite simply confirms the obvious. Could be supplemental if anything.
3-The Becam et al., 2011 manuscript is cited, but not properly discussed. There, the authors showed that expression of a dsRNA form of tribbles did not rescue the defects in D/V boundary formation caused by ectopic expression of bantam and concluded that bantam targets other molecular effectors involved in the maintenance of the DV affinity boundary. The submitted results open a question mark on these results: what effect could be expected by expressing dsRNA tribbles in cells that overexpress bantam (tribbles would be downregulated any how!) I was surprised to find out that this point is not discussed at all -despite numerous references to the Becam paper-.
4-"Aneuploidy is common in human cancers, reviewed in (Gordon et al., 2012) ". Aneuploidy is indeed very common in some human cancers, but it also not common at all in others that are just as malignant. There is ample published evidence in this regard that is not referred to, all too often. If the authors wish to discuss aneuploidy and cancer, they should provide the reader with a more comprehensive view of the subject. If I am not mistaken what Dekanty and colleagues claimed was that "chromosomal instability leads to an apoptotic response", i.e. in Drosophila, aneuploidy triggers cell death, which is not quite the same. Please, correct this mistake. Please, cite bibliography showing that aneuploidy does not trigger tumorigenesis in wing discs ( Poulton et al. 2014 10.1016/j.devcel.2014.08.007, and others). Also, please, cite the very relevant recent paper from the Oliveira lab (Mirkovic 2019: https://doi.org/10.1371/journal.pbio.3000016) showing that other Drosophila cells respond differently to wing disc cells.
6-"the signals driving tumorigenesis in tetraploid cells". Do the authors know for certain that tumourigenesis starts in tetraploid cells? In the Gerlach et al., 2018 article they found that "tumours induced by yki in a context of flawed cytokinesis were heterogeneous and composed of aneuploid cells of different sizes (Gerlach et al., 2018)" (i.e. not just tetraploids). Indeed, cytokinesis failure causes tetraploid cells to begin with, and all sorts of polyploidy and aneuploid karyotypes later on. In the absence of solid evidence to substantiate that tumours start from tetraploid cells only, I would suggest avoiding statements that take that as a proven fact.

Reviewer #1 (Comments to the Authors (Required)):
In their paper Gerlach and colleagues determine that the cell cycle regulator tribbles is a new target of the miRNA bantam, which in Drosophila has extensively been shown to control growth. The author use the wing imaginal disc system to show that a sensor that includes predicted bantam sites present in the tribbles 3'UTR is controlled by bantam and that tribbles modulation affects proliferation downstream of bantam and the hippo pathway We show the effect of yki overexpression in Fig 3E. Even though we agree with the reviewer that this has been observed before, presenting it allows a direct comparison with discs that coexpress yki and trbl. We therefore believe it is important showing an example, as well as the size quantification, of those genetic manipulations in a main figure.
Previous studies have shown that trbl overexpression (gain of function approach -GOF) targets stg to degradation and therefore limits G2/M progression in the wing disc (Mata et al., 2000 -PMID: 10850493). However, the function of endogenous trbl in the proliferating wing epithelium has not yet been established. Here, we find that trbl downregulation leads, indeed, to faster G2/M transition. Given that GOF experiments can lead to non-specific effects, loss of function (LOF) analyses are central to determine gene function in a specific organ. We believe that the analysis of the trbl LOF, and the results obtained from that, are novel and relevant, and therefore merits being presented in a main figure. Besides, it supports other observations reported in this manuscript indicating that bantam regulates cell cycle progression by controlling G2/M transition the wing disc by reducing trbl levels.
2-Bantam was reported to regulate many targets. However the authors have shown that 2 of them (tribbles and hid) are sufficient to recap most of the phenotype. Would you speculate in the discussion about the function of other targets? Is it tissue specificity? Is it other functions behond growth?
In the discussion part we mention that depletion of trbl and hid is the minimum combination leading to tissue overgrowth but we cannot rule out that other bantam target genes might partially contribute to the growth regulatory role of bantam.
In the discussion it can be found as: "Our results establish the smallest combination of bantam targets that, when depleted, drive tissue overgrowth. We provide a new example whereby defects in the cell cycle and apoptosis, biological processes commonly dysregulated in cancer, lead to tissue hyperplasia. In addition to trbl and hid, bantam represses other negative growth regulators (Brennecke et al., 2003;Herranz and Cohen, 2010;Herranz et al., 2012a;Herranz et al., 2012b). Although individual depletion of those elements does not result in tissue hyperplasia, we cannot exclude them as important bantam targets in growth control that, together with trbl and hid, might contribute to the growth-promoting role of bantam." Given that we do not have experimental evidence about potential role related to tissue specificity and/or other function different from growth regulation, we prefer not to discuss about this -it would mere speculation without any solid evidence supporting it.
3-The first part of the discussion essentially restate most of what has been presented in the intro. Authors show streamline that part.
We thank the reviewer for the suggestion. The mentioned first part of the discussion is rewritten and streamlined now.

Reviewer #2 (Comments to the Authors (Required)):
In Besides, the authors specify in the methods section that "larvae were kept for 3 to 5 days at 29{degree sign}C" which can cause large differences in tissue size. Quantification of several examples would be helpful to support authors statement that "The effect of bantam in discs with cytokinesis failure was similar to the one observed by Yki expression (Fig 1C, I; and Fig 2G, H)." Figure 7H-M also needs quantification.
As suggested by the referee #3, we have eliminated the analysis of bantam modulation in the model of cytokinesis failure and therefore old Fig 1 and Fig 2 are  3. In Figure 3D-I it would be important to show whether the regulation of the trbl 3'UTR sensor results in increased levels of Trbl protein (either with antibody staining or the trbl-GFP line).
We have not been able to obtain an aliquot of anti-Trbl. Pernille Rørth developed antisera against Trbl for their "Mata et al, 2000" paper but they ran out of antibody a long time ago.
We have used the Trbl-GFP to analyze trbl levels in bantam mutants. Trbl-GFP is inserted in left arm of the 3 rd chromosome (cytological location 77, 3-47%). bantam is located in the tip of the same chromosome arm (3-0,5%). This did not allow us to make this analysis in bantam mutant clones because bantam mutant clones would have two copies of trbl-GFP as compared to the surrounding heterozygous tissue that would only have one.
We agree with the reviewer that this is an important issue to address. Therefore, we have measured have measured Trbl-GFP levels in bantam mutant larvae by western blot , using an anti-GFP. Bantam mutant larvae (transheterozygous for the bantam alleles bantam-delta1 and bantam EP3622) showed a robust increase in the levels of Trbl-GFP as compared to control larvae. This is shown in Fig S1.

To link trbl with Hippo pathway perturbation, the authors should check if the trbl sensor
and/or trbl-GFP are reduced in wts or hpo mutant clones.
Trbl-sensor is downregulated in wts mutant clones. This is shown in Fig 1J, K. In the text: "The Hippo pathway regulates the expression of bantam, which is central mediating the Yki growth regulatory role (Nolo et al., 2006;Thompson and Cohen, 2006).

Hippo activation results in Warts (Wts) phosphorylation, which acts with its cofactor Mats
to phosphorylate and inactivate Yki. wts mutant tissue upregulates Yki and overgrows, (reviewed in (Pan, 2010). As observed in discs upregulating bantam, wts mutant cells downregulated the trbl-sensor (Fig 1J, K). trbl 3'UTR is thus sensitive to Hippo pathway activity." 5. Does combined loss of trbl and hid rescue the bantam clonal undergrowth phenotype? If so, it would strengthen the idea that these are key downstream targets of ban.

Bantam mutant cells are eliminated by cell competition. Cell competition compares cellular fitness between different cells and triggers the elimination of the less fit population of cells.
The competitive characteristics of clones of cells are defined not only by their proliferative potential, but also by other cellular mechanisms such as cellular polarity, cell size, metabolic status, mechanical tension, etc. Bantam mutant cells deregulate multiple targets that might compromise the competitive properties of those cells.
To analyze the contribution of the bantam target genes trbl and hid to the growth regulatory role of bantam, we compared disc size of discs depleting bantam in the dorsal compartment with discs depleting bantam+hid+trbl (there is not cell competition between different compartments in the wing disc). We used a UAS-bantam-sponge to downregulate bantam specifically in the dorsal compartment. Expression of bantam-sponge leads to a reduction in tissue size. Depletion of hid and trbl in discs expressing bantam-sponge rescued the growth defects induced by bantam depletion. This strengthens the idea that these are key bantam targets. These results are shown in Fig 4I, K.
In the test: "Next, we studied the consequences of downregulating trbl and hid in discs with reduced bantam. miRNA sponges controlled by UAS sequences allow spatiallycontrolled downregulation of miRNAs under Gal4 control (Loya et al., 2009). The expression of a UAS-bantam-sponge transgene causes a reduction in tissue size (Herranz et al., 2012a). Notably, discs expressing simultaneously UAS-bantam-sponge, UAS-trbl-RNAi, and UAS-hid-RNAi were increased in size (Fig 4I-K). This suggests that codepletion of trbl and hid is sufficient to drive tissue overgrowth, even when bantam levels are reduced." Figure 4 what is the effect of trbl depletion on cell number/tissue size?

In
We have performed that analysis, which is shown in Fig 2H-K and Fig S4. This is described text as: "We used the adult wing to analyze how trbl affected cell number and tissue size. In the adult wing, each hair-like structure corresponds to a trichome originating from a single epithelial cell. Trichome density can hence be used as a proxy of cell size and number. trbl depletion did not affect cell or wing size. trbl overexpression caused a mild reduction in wing size. These wings showed an increase in cell size revealed by reduced trichome density, indicating that those wings had fewer cells (Fig 2H-K, Fig S4).

The increase cell size observed upon trbl upregulation is characteristic of cells with delayed
cell cycle progression (Neufeld et al., 1998) and is consistent with the PH3 staining and the FUCCI-FACS analysis." 7. In Figure 4, the FACS data show that trbl overexpression causes cells to accumulate in G2 (increase number of cells in G2 and decrease number of cells in G1). Does this result in fewer mitotic cells? Please show PH3 staining in this condition. I also wonder how the authors explain that this manipulation does not affect tissue size, especially since it was shown that trbl overexpression in the eye and wing imaginal discs shows defects (see Figure   5D in PH3 staining is shown in Fig 2E-G. Described in the text as: "We used anti-Phospho-Histone H3 (PH3) as a specific marker of cells in mitosis to study whether trbl overexpression affected the rate of cell proliferation of the wing disc. We detected a reduction in the number of PH3-positive cells in discs expressing UAS-trbl when compared control discs (Fig 2E-G). These observations are consistent with previous reports (Mata et al., 2000;Reis and Edgar, 2004)  In both cases, we find consistent results. Again, the magnitude is different to the one observed in Das et al., 2014, but the trend is the same and we can not find any inconsistency between their and our results.
8. Figure 6F-G requires quantification. Although the trbl-RNAi+hid-RNAi wing size result is quite convincing, the number of apoptotic cells seems to be very low to explain the fact that trbl knock down did not result in tissue overgrowth. The same holds true for the example shown in FigS3A, in which many cas3-act positive cells seem to be in the ventral compartment. The increase in cell death associated with changes in cell proliferation cited in Neufeld et al., 1998 is much stronger than the one authors observe with Trbl downregulation. The authors should further test whether hid-RNAi rescues the apoptosis induced by trbl-RNAi.
We have added the requested quantification (see new Fig 4F-H).
hid-RNAi did not completely rescue the levels of apoptosis observed in discs depleting UAStrbl-RNAi. This is shown in Fig S5G. In the text can be found as "hid depletion did not completely rescue the induction of apoptosis observed in discs expressing UAS-trbl-RNAi (Fig S5), suggesting that other proapoptotic genes, in addition to hid, might contribute to the apoptotic response to trbl knock down." 9. In Figure 6H-L, authors use UAS-p35 to show that suppression of apoptosis in trbl-RNAi led to tissue overgrowth. I would strongly encourage the authors to carry out the same experiment using another way to block apoptosis (such as UAS-diap1). Overexpression of p35 leads to the presence of "undead cells" that are known to secrete several growthpromoting factors such as Wg and this might be the explanation for the fact that the p35+trbl-RNAi gives much higher relative size than the hid-RNAi+trbl-RNAi. Using UAS-diap1 would be more similar to the situation of hid-RNAi (no undead cells).
We have used a UAS transgene expressing a miRNAs that simultaneously inhibits the proapoptotic genes rpr, hid, and grim (UAS-miR-RHG), and has been proven efficient blocking cell death (see, for example, PMID: 20346676; PMID: 30080872). Expression of this miRNA together with trbl-RNAi results in the formation of overgrown discs, which is consistent with the results we have obtained expressing p35+trbl-RNAi or hid-RNAi+trbl-RNAi. We have therefore obtained comparable results in 3 independent conditions: 1) trbl-RNAi + UAS-p35; 2) trbl-RNAi + UAS-hid-RNAi; and 3) trbl-RNAi + UAS-miR-RHG. This is now shown in Fig S5. 10. The authors suggest that apoptosis in response to trbl depletion can be used as a homeostatic mechanism to offset tissue overgrowth. Is this specific to trbl or can it be mimicked by dMyt1-RNAi or Stg?
The effect of trbl downregulation can be mimicked by stg overexpression. This result is shown in Fig S6, and can be found in the text as, "Similar results were obtained when we overexpressed cdc25-stg as an alternative way of forcing G2/M transition (Fig S6)." Minor points: 1. Authors should discuss the possible reasons for the difference between EGFR-activation and CF in the ability of trbl depletion to drive tumorigenesis (in CF, UAS-p35 is required).
We agree with the point of the reviewer and added this part to the discussion. It can be found as: "trbl downregulation is sufficient to drive tumorigenesis in discs expressing the oncogene EGFR. However, discs with cleavage defects also require suppression of apoptosis for tumor formation. Interestingly, EGFR pathway promotes cell survival through hid downregulation by Ras (Bergmann et al., 1998;Kurada and White, 1998). The role of EGFR in cell survival might explain why EGFR-driven tumors are not dependent on additional blockage of apoptosis, while discs downregulating pnut and trbl require suppression of apoptosis for tumor formation." 2. The authors consistently (several times in the text as well as in Figure 7 title) claim that trbl acts as a tumour suppressor in the wing imaginal disc. However, this conclusion might be interpreted as if depletion of trbl on its own results in tumour formation, which clearly is not the case. I feel that authors should be careful in specifying that trbl acts as a tumour suppressor in the wing imaginal disc only in EGFR activation condition. Moreover, the case with CF is quite different, because trbl depletion alone is not capable of driving tumorigenesis in a CF background unless p35 is expressed as well.
We have carefully addressed that. In the revised version of the manuscript we have been more precise stating the specific context in which we have observed trbl behaving as a tumor suppressor. Figures 4 and 5 the authors say that "... these results suggest that bantam acts through trbl to stimulate tissue growth". I would be slightly more conservative and say "partially through trbl" since the rescue does not seem to be complete (show statistics between bantam+trbl and control in Figure 5G).

From the observations in
As suggested by the reviewer, we have added that.

Methods section:
-Please specify the product number of each antibody listed as well as the concentrations at which each one was used.
-Please specify which secondary antibodies were used as well as the respectively concentrations.
As suggested by the reviewer, we have specified the primary and secondary antibodies as well as the product number and the concentrations at which each one was used.
-Describe how the flow cytometry-based cell cycle analysis was performed.
A description is included in the revised version of the manuscript.

Reviewer #3 (Comments to the Authors (Required)):
The submitted article demonstrates that bantam regulates growth by repressing tribbles.
Previous work from the same lab has shown that coexpression of the Yki targets string and DIAP1 can promote tumorigenesis in wing discs with defective cytokinesis. Now, they identify the string regulator tribbles as a direct bantam target gene and show that tribbles regulation by bantam is central in controlling tissue growth and tumorigenesis. The study is well designed and performed, and provides valuable insight on the very complex mechanistic bases of bantam's role in development and disease. I recommend publication.
Considering/addressing the following points would result in a much more solid and rigorous article.
1-I really do not see the need for the CF model in this work. A great deal of the value of the main finding reported in the submitted manuscript is that it may be expected to apply to all processes that require cells to successfully cycle. That includes normal development as much as tumour growth, which itself includes CF-related tumours as much as any other type of tumour. Thus, quite frankly, the paper would be just as important -and much more clear-if it was limited to Figs 5, 6, S1 and S2, which are the ones that make the point.
Reference to CF tumours do not contribute anything to this story. We rather focus in the role of bantam in growth control and tumorigenesis, and not in the functions of the bantam-trbl interaction in the generation and/or maintenance of the DV boundary. We cited that paper as an example of bantam target genes identified in the past but we prefer not to discuss this paper because the regulation of boundary formation is out of the scope of our manuscript. Aneuploidy is indeed very common in some human cancers, but it also not common at all in others that are just as malignant. There is ample published evidence in this regard that is not referred to, all too often. If the authors wish to discuss aneuploidy and cancer, they should provide the reader with a more comprehensive view of the subject.
We fully agree with the reviewer in that, although aneuploidy is commonly observed in human cancers, it remains still unclear whether it is a cause or consequence of cancer.
Given that the main focus of this work is describing the role of the gene tribbles as relevant bantam target in growth control and tumorigenesis, we have removed that part. showing that other Drosophila cells respond differently to wing disc cells.
As mentioned in the previous point, that part has been removed in the revised version of the manuscript. Indeed, cytokinesis failure causes tetraploid cells to begin with, and all sorts of polyploidy and aneuploid karyotypes later on. In the absence of solid evidence to substantiate that tumours start from tetraploid cells only, I would suggest avoiding statements that take that as a proven fact.
We have removed that statement in the revised version of the manuscript. Thank you for submitting your revised manuscript entitled "The miRNA bantam regulates growth and tumorigenesis by repressing the cell cycle regulator tribbles". As you will see, the reviewers appreciate the introduced changes, and we would thus be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines: -please add a callout to Fig 5H,I,J in the manuscript text.
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Full guidelines are available on our Instructions for Authors page, http://www.life-sciencealliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. **Submission of a paper that does not conform to Life Science Alliance guidelines will delay the acceptance of your manuscript.** **It is Life Science Alliance policy that if requested, original data images must be made available to the editors. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original data images prior to final submission.** **The license to publish form must be signed before your manuscript can be sent to production. A link to the electronic license to publish form will be sent to the corresponding author only. Please take a moment to check your funder requirements.** **Reviews, decision letters, and point-by-point responses associated with peer-review at Life Science Alliance will be published online, alongside the manuscript. If you do want to opt out of having the reviewer reports and your point-by-point responses displayed, please let us know immediately.** Thank you for your attention to these final processing requirements. Please revise and format the manuscript and upload materials within 7 days.
Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance.