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Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity):

Summary<br /> The authors have set out to study the Drosophila immune response against the fungus Aspergillus fumigatus. They found that Aspergillus fumigatus kills Drosophila Toll pathway mutants. The fungus does this without invasion because its dissemination is blocked by melanization. They suggest that there is a role for Toll in host defense distinct from resistance. The findings are interesting, and looks like the mycotoxins play a role. It also seems that there is some role of the Bomanins here, but I find that in particular Figure4 experiments are not convincing enough to provide a mechanistic insight as to what is going on. I think the authors need to think through what their results mean, and also, explain better (especially regarding Fig 4) their ideas and how the data fits them.

We thank the reviewer for scrutinizing our manuscript as well as for suggestions to improve it.

The role of mycotoxins is demonstrated:

i) the fungus does not proliferate nor disseminate, also in Toll pathway mutant flies: thus, it must kill through diffusible substances, in as much as these immuno-deficient flies exhibit tremors toward the end of the infection;

ii) a fungal strain devoid of the capacity to produce secondary metabolites is no longer virulent, even in Toll pathway mutant flies.

The role of Bomanins is also demonstrated: the finding of a susceptibility of Bom__D__55C deletion flies to A. fumigatus and to mycotoxin challenges clearly shows that at least one or several Bomanin genes are required in the host defense against these challenges. The observation that this susceptibility can be rescued by the genetic overexpression of specific Bomanins indicates which ones are likely to mediate protection. The novel data we have included with the protection from mycotoxin action in neurons point clearly to BomS6 being the major mediator of protection against verruculogen action since it is the only one of two Bom genes to be induced in the head and with a proven potential for rescue of the Bom__D__55C phenotype.

As regards the concept of the article, it is simple: we show that the Toll pathway does not control A. fumigatus infection by directly attacking the fungus but does so by neutralizing the effects of secreted virulence factors such as restrictocin and verruculogen. We further identify some of the relevant effectors such as Bomanins by using a genetic complementation strategy. To make our point clearer, we have now included additional data in which we show that BomS6 and BomS4 are the only Bomanins induced in the head of flies upon the injection of these two toxins. We next determine that BomS6 and not BomS4 expression in the nervous system dominantly protects the flies from the deleterious effects of verruculogen injection, both in terms of recovery from tremors and survival. Mechanistically, the Toll pathway protects the host from the action of verruculogen by expressing and likely secreting BomS6 from neurons.

Major comments:<br /> Page 5: .."the fungal burden did not increase much in MyD88 flies challenged with 50 conidia (Fig. 1B)" - What do you mean did not increase much? There is a clear increase in Myd88 mutants compared to controls; would you expect a bigger increase (e.g. log scale induction)? Explain.

When the injected dose is higher than 50 injected colonies, the fungal burden remains very close to that of the injected inoculum (Fig. EV1_F, J_). As for other pathogens regulated by the Toll pathway, it has been published that the microbial burden increases by log factors for filamentous fungi (Huang et al.., in revision), pathogenic yeasts (e.g., work from our laboratory Quintin et al. Journal of Immunology, 2013), bacteria (e. g., Duneau et al., eLife 2017; Huang et al., in revision). The pathogens usually proliferate exponentially in immuno-deficient hosts, which is clearly not the case of A. fumigatus, the first example we know of.

Page 6: "the SPZ/Toll/MyD88 cassette is required for host defense against A. fumigatus infections, even though this pathogen only mildly stimulates the Toll pathway." - Should you rather say that A. fumigatus only mildly induces the Toll pathway target gene Drosomycin?

The answer is negative. Fig. EV1_C_ clearly shows that BomS1 is also modestly induced as compared to an infection with E. faecalis. The promoter of BomS1 contains a canonical Dif-response element (Busse et al., EMBO J., 2007_)_. For a more thorough discussion of this point, please, see reply to Reviewer 2, Major Comment 2.

Page 6: "...we tested Hayan mutant flies defective for this arm of innate immunity (Nam et al., 2012)." - elaborate this, which arm/which pathway?

The title of the paragraph is “Drosophila melanization curbs A. fumigatus invasion”. The full first sentence of the paragraph actually read: “As melanization is a host defense of insects effective against fungal infections, we tested Hayan mutant flies defective for this arm of innate immunity”.

This has not been introduced in the introduction. Explain.

We have now added a couple of lines (82-83) to introduce melanization for the nonspecialist reader.

Can you really draw this conclusion: "We conclude that melanization limits the proliferation and the dissemination of A. fumigatus injected into wild-type flies yet does not eradicate it at the injection site, where a melanization plug forms." Maybe you can based on the function/importance of the pathway to melanization, but you need to explain.

Melanization is mediated by the Hayan protease and three phenol oxidases (two in adults) that catalyze the enzymatic reactions leading to melanin production (for Drosophila, please see Nam et al. EMBO J. (2012), Bingelli et al., PLoS Pathogen (2014), Dudzic et al., BMC Biology (2015), Cell Reports, 2019). Thus, finding that there is an increased proliferation and dissemination in null Hayan mutants is a strong indication for a role of melanization. The identification of a similar phenotype for PPO2 and PO1-PPO2 mutants demonstrates that melanization is curbing A. fumigatus. Our sentence is therefore fully justified.

Page 10: "The cleavage of the 18S RNA was however much less pronounced in wild-type flies as compared to MyD88" - I am not sure what this means. Do you mean 28S?

We thank the reviewer for pointing out this mistake that has now been corrected.

And that the 28S peak is lower? Is this a quantitative method?

The technique is liquid electrophoresis on a microchip. It is both a qualitative and quantitative technique that replaces traditional agarose or polyacrylamide gels.

Fig. legend: "Arrows show the position of the 28S RNA sarcin fragment" - there are three arrows in both Fig 4E and F; specify which arrows point what.

The thick arrow is now indicated in the figure legend to correspond to the much smaller sarcin fragment whereas the thin arrows on the graph clearly specify the position of the 28S RNA peaks.

Based on the results, I am not convinced about the conclusion, that "restrictocin is able to inhibit translation to a detectable degree in vivo, likely through the cleavage of the ribosomal 28S a-sarcin/ricin loop as described in vitro." <- Do you draw this conclusion before doing the actual in vitro experiment, which is described next in the text (The rabbit reticulocute assay, S2 cells)?

The existing literature (line 259 for a few selected references) has largely proven that restrictocin cleaves 28S RNA in vitro. We are demonstrating that this also happens in vivo in flies based on the generation of the alpha-sarcin fragment as well as the decreased 28S peaks. Our transgenic approach also indicates that restrictocin blocks translation in vivo. The in vitro approach has been implemented so that we could test the effect of synthetic BomS1 and BomS3 in cell culture. As to our knowledge, no one had demonstrated that restrictocin blocks translation in Drosophila cultured cells. It was therefore important to demonstrate it in cell culture using well-characterized in vitro techniques mastered by AT and FM.

4H: Not sure what should be seen here, is it the darkest band at 0 uM that disappears?

We have improved the figure and added an arrow to point out to the relevant band on the gel.

HI & J need more explanation than what is now included in the text or Figure legend, is the conclusion that there is no difference? Write the stats above the Figs 4I & J (n.s.?).

We have added NS on the figures and made our conclusion clearer (lines 295-298).

Minor comments:

It would have helped commenting if the manuscript contained line numbers

We apologize for having initially provided a version in which lines were not numbered. At the prompting of Review Commons we immediately provided such a version, that was actually used by Reviewer 2.

Why do you have the title "Hayan" on top of Fig 1F; you don't have this marking system in the other survival curves

This point has now been addressed and the survival experiments checked for consistency.

Fig 2A: Can you speculate why MyD88 flies die rapidly at day 10 if you inject PBST (your control)? What would happen to uninjected controls in otherwise the same conditions? (you could include an uninjected control here?)

We suspect that this is linked to the trauma induced by the injection. Trauma has been shown to impact the homeostasis of the midgut epithelium (Lee & Miura, Current Topics Developmental Biology 2014, Chakrabarti et al., PLoS Genetics (2016)), and we suspect that it may lead to a leakiness of the gut allowing the passage of some bacteria from the gut microbiota that can proliferate in the hemocoel. Hence, we checked axenic and antibiotics-treated MyD88 flies to exclude that the limited sensitivity to trauma was not significantly contributing to the phenotypes we describe. It is also linked to the thickness of the needle and the problem is alleviated by using thinner needles.

The uninjected control is now shown in Fig. EV8_E_.

Please, see also the answer to Reviewer 2 Major comment 1.

Fig 2E: Not sure what would be the best way of presenting the curves - different colors, dotted lines or something? Now if there are too many lines, they are hard to tell apart. because the symbols are not that visible. Like in 2E if you want to compare the light red/orange colored lines.

We agree with the reviewer that the lines are hard to tell apart. This is however not a significant issue since the glip mutants display curves similar to that of the wt A. fumigatus control strain.

For consistency add the caption also to Fig 3D (I assume it is the same as 3C)

The caption was present in our version and is present in the revised version.

For consistency, should you add Verruculogen on top of Fig 3F?

Same reply as for the previous comment.

Chronologically, how it is explained in the text, Figs 4A and B are in the wrong order.

We fully agree with the reviewer. This problem has been addressed in the revised version.

The quality of Fig 4 is not great, the text is hard to read (too small) and becomes blurry upon magnification.

We fully agree with the reviewer. This problem has been addressed in the revised version.

Page 12; "These data then suggest that a process akin to the immune surveillance of core cellular processes first described in C. elegans may also exist in Drosophila" - I think this sentence belongs to the discussion, this is not directly drawn from the results.

We have followed the reviewer suggestion and have now developed our Discussion paragraph now entitled “Induction of the expression of specific Bomanin genes upon mycotoxin challenge”

Referees cross-commenting

I think we share many thoughts among all the reviewers.

The main problem is that the manuscript language is quite strong; from the results many times it is not ok to make such strong statements. Some experiments need further analysis and clarification.

I think in most cases, this could be achieved by softening the statements and adding more discussion, and not by making new experiments (some may be needed).

We respectfully disagree with the reviewer on this point. There were obviously some misunderstandings that might be traced to the short format of the initial version. We have now developed the Discussion to clarify our conclusions as suggested by the reviewer.

Minor things are that experiments are not advancing in a logical order between the text and the figures and there are problems with resolution in some figures.

Statistics in some figures needs to be added.

Please, see above.

Reviewer #1 (Significance):

The nature of the work is conceptual for the field, to understand the role of the Toll pathway and Bomanins in particular, in this fungal infection model. The work is interesting to a somewhat limited audience, mainly immunologists and in particular, people interested in the Drosophila model for immunity. The work may be interesting conceptually in understanding fungal infections.

We are not certain that immunologists represent a limited audience. We agree that work on fungal infections is insufficiently funded with respect to the medical importance of these infections, as highlighted in our introduction and Perspective section of the Discussion.

My expertise: I am a Drosophila immunity researcher with nearly 20 years of experience in working with fly immunity, in particular the Toll and the Imd pathways.

Reviewer #2 (Evidence, reproducibility and clarity):

Summary:

Xu et al. describe how A. fumigatus kills Toll-deficient fruit flies not by hyperproliferation, but more likely by virulence factors. Melanization is important for suppressing fungal spread. The Bomanin genes have an unknown function, and here the data suggest a reasonably convincing role for Toll in resilience. Overall the manuscript is thorough and presents a diversity of approaches that show Toll and the Bomanins in particular contribute to this resilience effect. The idea that Toll effectors are essential for resilience is interesting as other fly stress response pathways like JAK-STAT are better known for helping the fly cope with damages, while Toll is better known as an antifungal response.

I believe the study, with some careful considerations added, would add a valuable series of observations to understanding how the host immune system promotes survival after infection. Overall I am quite positive about the results, and the authors have made a significant effort.

We thank the reviewer for the positive evaluation of our work that actually spans many years of research on the Aspergillus fumigatus Drosophila infection model that is a major topic of our work at the Sino-French Hoffmann Institute of Guangzhou Medical University.

Any experiment suggestions I make are strictly to improve the confidence in the interpretations of the results, but the language could alternately be softened to address those concerns. My major critique is that the authors repeatedly extend beyond what is shown, and occasionally in defiance of what is shown (if I understand the results correctly).

We have chosen to perform additional experiments when needed. We have also clarified points where there were obvious misunderstandings by expanding our text that had been written under a very concise format.

It is not thoroughly clear what the reviewer has in mind when using the word defiance. We suppose it refers to the work of Scott Lindsay with whom we are in contact. He actually attempted to monitor the C. glabrata burden but did not pursue this line of investigations as he already saw a difference after one hour and he thought that the Toll pathway cannot be induced so rapidly. Actually, David Duneau mentions a time of two to three hours for the Toll pathway to control E. faecalis infections (eLife, 2017) and Sandrine Uttenweiler-Joseph already saw by MALDI-TOF MS an induction of Bomanins and other DIMs at the earliest point tested, six hours (PhD thesis). There is absolutely no critique of the work of the Wasserman laboratory who has greatly contributed to our understanding of Bomanin functions. Some of our unpublished data clearly point out to an AMP role for at least one Bomanin gene against E. faecalis and we certainly do not exclude an AMP role for BomS against C. glabrata. This however does not dismiss the possibility that Bomanins may also have other roles in dealing with microbial toxins. We have been studying Candida infections in Drosophila for many years and have documented the host defense against C. glabrata (Quintin et al., JI, 2013). We do suspect that C. glabrata likely secretes virulence factors that have not been identified so far. We mention this as a possibility and certainly not as a truth. One should remember that investigators were unaware for a long period of the role of Candidalysin, a pore-forming toxin, in C. albicans infections.

Finally, a dual role as AMP and protecting from secreted toxins has been clearly shown in the case of alpha-mammalian Defensins that we now are describing in our Revised Discussion (Kudryashova,Immunity, 2014).

Comments below.

Major comments:

1) The language is too strong. Specifically the use of the phrase "anti-toxin" is too generalist, especially as the authors show that their candidate Bomanin does not bind to the toxin directly.

We have checked all of the submitted documents: the term anti-toxin was never used (just found “anti” in antimicrobial, antifungal, antibiotics..), in this manuscript as well as in the companion article. and we have never excluded an indirect effect, quite to the contrary because of the in vitro experiment with restrictocin mentioned by the reviewer and other observations now included (see further below). We use the terms “protection” or “counteract”, which have not such a meaning. It is burdensome for the reader to read each time “counteract or protect from the actions of the toxins or the effects of the toxin.

Instead, Toll mutants seem susceptible to damage/stress caused by injury/toxins. MyD88 even show general susceptibility to vehicle controls in Fig3C-D.

The effects of stress related to the infection conditions and injury are clearly distinct from the much stronger ones exerted by the toxins themselves. As requested by the reviewer further below, we have submitted wild-type and immuno-deficient flies to several stresses such as heat or the injection of hydrogen peroxide or salt solution (Fig. EV8_B-E_). While the latter did not reveal any difference, MyD88 flies succumbed slightly faster to a strong 37°C stress; in contrast, they survived better to a 29°C exposure, the temperature at which we perform most experiments. However, the difference started to be visible only after some 15 days whereas the time frame in which flies succumb to A. fumigatus or toxin challenges is definitely much shorter by some 10 days. We also note that Bom__D__55C mutant flies behave like the isogenized wild-type controls in these assays, further excluding a potential role for general stress sensitivity as a contributor to the effect of toxins.

As regards DMSO, there is indeed a general mild sensitivity of flies to DMSO, but not specifically affecting MyD88 mutant (Rebuttal Fig. 1J). We find that this effect is lessened when using thinner needles. Thus, the problem has become minor as we became more experienced. We had checked axenics- and antibiotics-treated flies to exclude a contribution from the microbiota. Finally, to uncouple the effects of verruculogen from those of DMSO, we have also challenged flies directly by introducing the powder, using a technique similar to that of the septic injury. While it is quantitatively less accurate, it clearly proves that verruculogen produces the reported effects (Fig. 3C) and was useful to measure Bom and Drosomycin expression by digital PCR in the heads of challenged flies, e.g., Fig. EV6_J-K_ and Figs EV_11&12_.

Toll is important for development, so it may be expected that Toll flies could have development defects impacting resilience even if/when Toll flies can survive to adulthood. I don't say this to be too negative on the findings, which are quite convincing. But I am not sure that the phrase "anti-toxin" is right for what is shown.

We fully agree with the reviewer on this point. We have failed to find RNAi lines that are efficient enough to mimic the Toll pathway phenotype when expressed ubiquitously at the adult stage. However, Bom__D__55C mutants do not seem to display a developmental phenotype and display a phenotype similar to that of MyD88 flies. Furthermore, our rescue experiments of the Bom__D__55C sensitivity phenotype to mycotoxin challenge is achieved by the overexpression of specific Boms that are induced only at the adult stage, making it unlikely that this sensitivity phenotype reflects a developmental problem, as had been shown to be the case for 18-wheeler that had initially been proposed to encode the IMD pathway receptor.

A very interesting recent study shows Dif has a role in the synapse of neurons to protect from alcohol sensitivity. Could secreted Bomanins participate? This emphasizes a mechanism through which Toll mutants likely have defective neural development, which could make them stress response defective, especially to things like neurotoxins. See: https://pubmed.ncbi.nlm.nih.gov/35273084/

We are aware of this study first presented at the 2019 Fly Meeting in Dallas and this author did discuss with the authors of the study. However, we have found that Dif (and Dorsal) mutants are not sensitive to A. fumigatus infections nor to injected mycotoxins, as was the case already for C. glabrata (Quintin et al., JI, 2013).

Lin et al. (2019) also showed lack of Bomanin secretion from the fat body in Bombardier mutants causes loss of tolerance (resilience?). So does Bomanin disruption increase susceptibility to stresses more generally, rather than specifically fungal toxins? And is this a development role, rather than an immune response role?

The authors could try to use other stresses (NaCl, oxygen, heat, alcohol) to test the contribution of Bomanins to this resilience, which may reflect defective neural development rather than a role for secreted systemic immune-response peptides.

Please, see replies above.

2) The authors present a paradox. On the one hand, A. fumigatus hardly induces Drs/Bomanins (Fig. S1). Yet on the other, they propose that inducible Bomanins protect the fly from mycotoxins. Why do the authors say Toll is hardly induced by A. fumigatus at the start of the study (Fig S1), but later use the same data to argue that Bomanin induction underlies the resilience phenotype (Fig5).

The reviewer raises an interesting point. Of note, we have added new data in Fig. EV2_B_ that document that all 55C Bomanin genes, BomS4-_excepted, are induced by a systemic infection. There is indeed somewhat of a paradox. The _Bom__D__55C deletion phenotype clearly establishes that Bomanins play a major role in the protection against mycotoxins and A. fumigatus. The rescue experiments rely on ectopic expression and therefore establish that specific Bomanins can mediate the protective effect. Our data on verruculogen suggest that there might be local inductions, e. g., in the head of BomS6 and BomS4. The brain represents a compartment that is separated from the hemocoel by the blood-brain-barrier. We have not been able to generate BomS6 null mutants so far. In this case, the relevant response may not be systemic. We only detect a weak signal for BomS peptides in the hemolymph of unchallenged flies, making it unlikely that a basal expression is important, at least as regards a systemic infection. We cannot however exclude local inductions at the level of tissues. This would not rely on hemocytes as “hemoless” flies are not susceptible to A. fumigatus or toxin challenges. This topic definitely warrants further investigations.

In Fig 5, it looks like DMSO is nearly identical to A. fumigatus, so can the authors really suggest that equal induction to DMSO is relevant?

We had stated that an induction of the Bomanins by the injection of DMSO alone precluded us from analyzing the effects of verruculogen on Bom gene expression. We have now bypassed this difficulty through direct challenges by the undissolved powder (Fig. 6_J-K,_ Fig. EV11).

The authors' discussion of these points would benefit from considering Vaz et al. (2019; Cell Rep) to frame how much PAMP is injected given equal numbers of fungal cells vs. bacterial cells. To me the lower induction by injecting a few fungal cells with much lower surface area to volume ratio means equal microbe mass has exponentially less PAMP in fungal conidia cell walls (2-3um diameter) vs. equal mass of bacteria (0.5-1um diameter).

We fully agree with the reviewer and now mention that C. glabrata also led to a milder induction of the Toll-mediated humoral response (Quintin et al. JI, 2013). In addition, it has been shown previously that ß-(1-3)-glucans, which are sensed by GNBP3 in Drosophila (Gottar et al., Cell, 2006), are concealed by the cell wall (germinating conidia) or hydrophobins (Wheeler et al., PLoS Pathogens, 2006; Aimanianda et al., Nature, 2009) . In the case of yeasts, these glucans are accessible only at the budding scar (Gantner et al., EMBO J., 2003).

Fig S1O is not convincing that Boms alone are present. There is significant noise near Drs in FigS1 infected, which likely saturates the detector before Drs can fly to it. I say this because DIM4 (Daisho) indicates that Toll is strongly induced. The authors should show a larger mass range on the x-axis including peaks of other Toll-induced peptides like the BaramicinA DIM10, DIM12 and DIM13 peptides of their companion paper and DIM14 (Daisho), which are closer in mass to the Bomanins and less likely disrupted by the noise at 4300 m/z. The maldi-tof calibration to correct ranges is critical for arguments of quantification.

We provide the primary data in the Rebuttal figures at the end of this document. These are the results obtained from three single flies (Files A29683PBUG22, A29684PBUG23 and A29684PBUG24). The first three spectra correspond to the full scale based on the major peaks observed (DIM4/BomS5) in two out of three spectra. At this scale, no signal is visible for Drosomycin at 4891 and the “noise” at 4278 is modest. Next, the multi-spectra report allows to put all three samples on the same sheet, this time zooming on the peaks of interests in the region 4300 (“noise”) and 4891 (Drosomycin). Finally, the next two pages zoom in on the BomS peptide signals and the next page keeps the same scale to document the 4300-5000 region. On the last page, it is obvious that the signal around 4300 is very modest and too distant to influence the Drosomycin ion, thereby excluding any effect of suppression. Of note, in the systemic immune response, Drosomycin is the most induced AMP with a concentration estimated to be around 0.3µM, an order of magnitude higher than other AMPs. Finally, these experiments have been performed by PB who initially developed the technique (Uttenweiler-Joseph, PNAS, 1998) and has been using and developing it ever since.

Combined with comments in Major Concern 1, I am not convinced that the -inducible- Bomanin response mediates the resilience phenotype.

Besides our replies above, we do hope that the new data we have included in Fig. 6 that document an induction of only two BomS genes in the heads of Drosophila upon verruculogen and the finding that BomS6 expression in the nervous system protects the fly from the effects of verruculogen will convince this reviewer.

3) The author's language is very strong to disregard a possible antimicrobial activity.

As noted above, this is a misunderstanding that we hope is dispelled in the revised discussion (see also above and replies to Reviewer 1).

Previous studies showed increased Candida growth and decreased hemolymph killing activity in Bom55C flies (Lindsay et al. 2018 and Hanson et al. 2019).

Please, see reply above. Factually, Lindsay et al. did not study the C. glabrata titer in vivo but using collected hemolymph. The killing activity likely requires a cofactor regulated by the Toll pathway. Hanson investigated the burden of the dimorphic C. albicans pathogen that in flies is filamentous and not C. glabrata.

Also see minor concern (i).<br /> I grant that the data are consistent with a resilience role. However the authors found no binding of Bomanin to restrictocin, countering their idea of a -direct- anti-toxin effect.

We are surprised by this comment. We certainly did not favor this idea nor developed it in the original manuscript, even though we cannot formally exclude it at this stage. Future experiments will focus on BomS6 potential interactions with these two mycotoxins.

At present the authors cannot rule out a direct antimicrobial role, or even the possibility of two different roles for the same peptides (ex: one in resilience, one antimicrobial). For instance, it is difficult to explain the loss of killing activity of Bom-deficient hemolymph ex vivo from Lindsay et al. if Bomanins are strictly anti-toxins. Surely they must also do something generalist?

Please, see our replies above and the paragraph dedicated to this topic in the Discussion.

4) In most figures, the authors do not compare flies with shared genetic backgrounds.

The MyD88 allele we are using is a transposon insertion from the Exelixis collection and we are using the wA5001 strain that was used to generate the collection of insertion (Thibault et al., Nat. Genetics 2004). We thank the reviewer for this comment as we realized we had forgotten to mention the Bom__D__55C strain. Lines 603-604 state that the deficiency line has been isogenized in the wA5001 background.

The phenotypes are usually strong so I am not concerned.

However the rescue effect of Bom transgenes in Fig 5C-D is based on smaller differences. Were these genetic backgrounds controlled?

Yes, as much as we reasonably could. The fact that most BomS transgenes did not rescue gives further confidence in the data.

Were transgenes inserted at the same site?

We used the strategy for overexpression developed by the Basler laboratory (Bishof et al., Development 2013, Nat. Protocols 2014) that relies on insertions at the same site.

The authors seemingly used a heat shock to express transgenes.

Heat-shocks are usually a short exposure to higher temperatures, usually 37°C. Here, we have used the inducible Gal4-Gal80ts system developed by McGuire and Davis (Trends in Genetics, 2004). The Gal80 repressor inhibits Gal4 function at the permissive temperature (18°C) and becomes inactive at the restrictive temperature (29°C). Thus, we use a temperature shift and not a bona fide heat shock.

Given a resilience effect is being studied, this heat stress approach is sub-optimal. Earlier experiments showing effect/no effect of Bomanin on heat shock resilience would improve confidence here. I would recommend assaying temperatures that can kill wild-type in order to confirm that Bom do not succumb earlier (ex. up to 37'C).

The results have been discussed above and show that 29°C is not a concern for Bom__D__55C and not much of a significant problem as regards MyD88.

In Fig5C the time resolution is poor, and the effect inconsistent across Bomanins. What are the differences in the Bomanins that the authors suspect could cause this? And how consistent are the experiments?

We provide all the primary survival data in Rebuttal Fig.1 A-H. The partial protection effects of BomBc1, BomS3 and BomS6 against restrictocin are consistent in the three independent experiments (Fig. 5D and Rebuttal Fig. 1 A-B). As regards the seven independent experiments performed with verruculogen, we observed a strong protection conferred by BomS6 expression in six experiments whereas we detected a milder protection conferred by BomS1 in four out of seven experiments and no protection in the three other ones. The effects were always there after 24 hours, in keeping with our novel data showing that BomS6 expression allows a faster recovery, around 10 hours, from verruculogen-induced tremors (Fig. 6E-F).

Since the effect is finished by 24h, perhaps a boxplot of percent survival at this time would better show the consistency across experiments.

Given the argument presented just above and considering that this rebuttal letter will be published alongside the article, this may not be needed.

Minor concerns:

i) The authors say the fungal burden of Bom55C flies remains low in Fig 5B, but they never measure flies that are near death when fungal load is greatest, or FLUD like in other figures. Given low mortality at the following time points, it seems likely that A. fumigatus would grow beyond initial loads in those individuals and kill them. I grant that these loads are less than what is seen in Hayan mutants. I just might suggest a more careful consideration of the time points used and what can be said about the trends shown here.

This is certainly a relevant point. The FLUD data are now presented in Fig. EV8_A_ and do not reveal any additional growth.

ii) Could the authors comment somewhere about the levels of toxin they were required to inject to get a phenotype vs. the level of toxins the authors expect are found in the fly during infection? I appreciate that toxin injection likely requires much higher doses, but it would be good to know just how far the authors have pushed their experimental system beyond its natural range.

This a question that is difficult to answer accurately as we are not sure the techniques exist to measure toxin levels in these small flies. We have tested a range of concentrations. It is clear that we push the system and likely use concentrations that are higher than those actually secreted by A. fumigatus during infection. Indeed, the mutant strains defective for the production of verruculogen or restrictocin display only a mildly reduced virulence in MyD88 flies. This makes it even more remarkable that wild-type flies are able to withstand these high, unphysiological concentrations, an argument for an indirect effect independent of the dose as pointed out now in the Discussion. How fungal pathogens balance the expression of the hundreds of secreted virulence factors, proteins and secondary metabolites, is a major frontier for future investigations be them plant or animal pathogenic fungi/

Again regarding toxins vs. general stresses, one could manage to inject salt into the hemolymph and show a stress-sensitized fly would succumb at lower doses than wild-type, emphasizing the relevance of defining concentrations.

We feel that just monitoring the survival of flies after a challenge that produces an effect is sufficient (Fig. EV8_C_).

The authors could also write toxin concentrations clearly in the figure/legend per experiment.

Corrected.

iii) Throughout the manuscript, the order that figures/panels are cited is inconsistent. Perhaps the text could be re-written so the reader can follow the figures more intuitively while going through the text?

Corrected.

iv) There are a few points where run-on sentences, involving many commas, make it hard to follow the logic. I might suggest a careful reading to break up long sentences into two sentences to ensure clarity.

We hope to have addressed this concern.

v) Line 279-281: this is the first and only mention of the immune surveillance hypothesis in nematodes. This is strange, given the authors are effectively describing an analogous idea exists in flies? Perhaps this could be added somewhere in the introduction or discussion.

We have followed the advice of the reviewers and now discuss this point more fully in the Discussion under its own subheading.

Small points

• What timepoints are the gene expression data from? Could the authors indicate this in figures/legends?

Done

• Line 133-135: "We conclude that MyD88 flies succumb to a low A. fumigatus burden..." - could the authors cite a figure panel here to emphasize what evidence they're referring to.

Done

• Line 151-152: Dudzic et al. (2019- Cell Reports Figure 3) showed that PPO2 was regulated by Hayan, while PPO1 by Sp7. This relevant study should be cited here or in the introduction/discussion.

Excellent suggestion, this was indeed an important study. Done

• Line 179-180: could the authors define the gliotoxin mutant strain here in the text for clarity?

Done

• Line 196: Fig. 4A-B should be Fig. **S4 A-B?

Corrected.

• Fig4A: perhaps the authors could reduce the x-axis to focus on the early time points? If I understand correctly, aspf1 has slightly delayed killing compared to akuB (˜50% difference at 2 days), but both kill 100% by 3 days.

Done

• Fig4G: can the authors define the GFP transgene on pg10? Not clear what this is, or what this means. Brain? Fat body? The legend of Fig4G and the key in the top left... it's not easy to quickly understand what is shown in Fig4G.

Done

• Line 247: I would drop the "at the intracellular level" part. I'm not sure this is robustly shown given the use of an in vitro model where there is no closed extracellular environment. The data are convincing of the effect, this is just a semantic point.

We agree that there is no closed extracellular environment and that therefore any signal emitted by the cells might get too diluted. However, the addition of EGF will activate the Toll intracellular through the chimeric EGFR-Toll receptor. As restrictocin is known to act intracellularly, one might have though that there might be some intracellular effectors mediating the Toll-dependent protection against restrictocin. Our sentence excludes this possibility.

• Line 257-258: Cohen et al. (2020- Front Imm) never used Bomanin mutants. Did the authors mean to cite Hanson et al. (2019) here, which seems to fit their described citation re: Bom55C vs. Toll mutant flies (Fig. 2)? Given Hanson et al. infected Toll mutant and Bom55C flies with many bacteria/fungi including A. fumigatus, it's strange this study is not discussed currently.

The reviewer is correct. Cohen et al. did use A. fumigatus, but on Daisho mutants and MyD88 and not Bom__D__55C as a control. We are now citing Hanson et al., 2019 in lines 443-449 (Discussion).

• Fig5C-D: the labeling is difficult to follow.

This is difficult to address unless multiplying EV figures. We feel this is not needed: the important curves are in color and each such curve is seen on the graphs.

• Line 318: a -possible- AMP role of Bomanins was proposed because of the aforementioned killing activity of wt but not Bom mutant hemolymph, alongside rescue by single Bom genes. To say this was based only on survival experiments is incorrect.

The paragraph has been rewritten and expanded to dispel any misunderstanding.

• Line 324-328: could the authors cite appropriate references after "inhibition of calcium-activated K+ channels" ?

Done

• comment re-Line 334: Toll10b flies have melanotic tumors and are in general in a stressed state. Might their rescue be due to increased stress tolerance by pre-activated stress responses?

This is a developmental effect occurring during larval stages, also observed for Cactus mutants. Here, we use a UAS-Tl10B transgene that is induced only at the adult stage using the Gal4-Gal80ts system. Thus, any stress is minimized as much as possible. Furthermore, we can phenocopy this phenotype to a large extent using a UAS-BomS6 driver, even though the phenotypes are subtly different as regards the protection against verruculogen-induced tremors.

Referees cross-commenting

Yes I agree that the data themselves are not the issue, nor even the direction of the results. But there are many overly-strong statements that go so far as to refute ideas which are supported by other studies, and for which the authors here do not provide any contradictory evidence.

We hope that this revised, extended version has clarified any misunderstanding in the initial version.

As per my review, I would be happy with a re-write that softened the language overall. I genuinely wonder if these Bomanin mutants simply have poor development, and so they are susceptible to neurotoxins/stress because their nervous system/development leaves them less resilient in general. Experiments testing their resilience to different stresses would greatly elevate the ability to make confident insights in the present manuscript. Currently the authors have only investigated one type of phenotype and interpreted it as if that is evidence of the evolved purpose of the peptides. This approach does not account for many other possible (and reasonable) explanations.

We have performed the experiments suggested by the reviewer. While we see a modest effect of heat on MyD88, it is not found in Bom__D55C flies, which display essentially the same phenotype as MyD88 with regards to the sensitivity to A. fumigatus or some of its secreted mycotoxins_._

Reviewer #2 (Significance):

This paper should be of broad interest to the study of immunology, where roles for effectors are typically thought of as cytokines. In fruit flies and other invertebrates that lack adaptive immunity, immune effectors are more thought of as direct actors likely with antimicrobial properties. The finding that Toll might mediate resilience is interesting, and implicating well known Toll effectors provides an important step forward towards a mechanistic basis behind this resilience effect.

We thank the reviewer for his appraisal of the significance of our work.

My expertise is in insect and innate immunity.

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Referee #2

Evidence, reproducibility and clarity

Summary:

Xu et al. describe how A. fumigatus kills Toll-deficient fruit flies not by hyperproliferation, but more likely by virulence factors. Melanization is important for suppressing fungal spread. The Bomanin genes have an unknown function, and here the data suggest a reasonably convincing role for Toll in resilience. Overall the manuscript is thorough and presents a diversity of approaches that show Toll and the Bomanins in particular contribute to this resilience effect. The idea that Toll effectors are essential for resilience is interesting as other fly stress response pathways like JAK-STAT are better known for helping the fly cope with damages, while Toll is better known as an antifungal response.

I believe the study, with some careful considerations added, would add a valuable series of observations to understanding how the host immune system promotes survival after infection. Overall I am quite positive about the results, and the authors have made a significant effort. Any experiment suggestions I make are strictly to improve the confidence in the interpretations of the results, but the language could alternately be softened to address those concerns. My major critique is that the authors repeatedly extend beyond what is shown, and occasionally in defiance of what is shown (if I understand the results correctly). Comments below.

Major comments:

1. The language is too strong. Specifically the use of the phrase "anti-toxin" is too generalist, especially as the authors show that their candidate Bomanin does not bind to the toxin directly. Instead, Toll mutants seem susceptible to damage/stress caused by injury/toxins. MyD88 even show general susceptibility to vehicle controls in Fig3C-D. Toll is important for development, so it may be expected that Toll flies could have development defects impacting resilience even if/when Toll flies can survive to adulthood. I don't say this to be too negative on the findings, which are quite convincing. But I am not sure that the phrase "anti-toxin" is right for what is shown.<br /> A very interesting recent study shows Dif has a role in the synapse of neurons to protect from alcohol sensitivity. Could secreted Bomanins participate? This emphasizes a mechanism through which Toll mutants likely have defective neural development, which could make them stress response defective, especially to things like neurotoxins. See: https://pubmed.ncbi.nlm.nih.gov/35273084/<br /> Lin et al. (2019) also showed lack of Bomanin secretion from the fat body in Bombardier mutants causes loss of tolerance (resilience?). So does Bomanin disruption increase susceptibility to stresses more generally, rather than specifically fungal toxins? And is this a development role, rather than an immune response role?<br /> The authors could try to use other stresses (NaCl, oxygen, heat, alcohol) to test the contribution of Bomanins to this resilience, which may reflect defective neural development rather than a role for secreted systemic immune-response peptides.
2. The authors present a paradox. On the one hand, A. fumigatus hardly induces Drs/Bomanins (Fig. S1). Yet on the other, they propose that inducible Bomanins protect the fly from mycotoxins. Why do the authors say Toll is hardly induced by A. fumigatus at the start of the study (Fig S1), but later use the same data to argue that Bomanin induction underlies the resilience phenotype (Fig5). In Fig 5, it looks like DMSO is nearly identical to A. fumigatus, so can the authors really suggest that equal induction to DMSO is relevant?<br /> The authors' discussion of these points would benefit from considering Vaz et al. (2019; Cell Rep) to frame how much PAMP is injected given equal numbers of fungal cells vs. bacterial cells. To me the lower induction by injecting a few fungal cells with much lower surface area to volume ratio means equal microbe mass has exponentially less PAMP in fungal conidia cell walls (2-3um diameter) vs. equal mass of bacteria (0.5-1um diameter).<br /> Fig S1O is not convincing that Boms alone are present. There is significant noise near Drs in FigS1 infected, which likely saturates the detector before Drs can fly to it. I say this because DIM4 (Daisho) indicates that Toll is strongly induced. The authors should show a larger mass range on the x-axis including peaks of other Toll-induced peptides like the BaramicinA DIM10, DIM12 and DIM13 peptides of their companion paper and DIM14 (Daisho), which are closer in mass to the Bomanins and less likely disrupted by the noise at 4300 m/z. The maldi-tof calibration to correct ranges is critical for arguments of quantification.<br /> Combined with comments in Major Concern 1, I am not convinced that the -inducible- Bomanin response mediates the resilience phenotype.
3. The author's language is very strong to disregard a possible antimicrobial activity. Previous studies showed increased Candida growth and decreased hemolymph killing activity in Bom55C flies (Lindsay et al. 2018 and Hanson et al. 2019). Also see minor concern (i).<br /> I grant that the data are consistent with a resilience role. However the authors found no binding of Bomanin to restrictocin, countering their idea of a -direct- anti-toxin effect. At present the authors cannot rule out a direct antimicrobial role, or even the possibility of two different roles for the same peptides (ex: one in resilience, one antimicrobial). For instance, it is difficult to explain the loss of killing activity of Bom-deficient hemolymph ex vivo from Lindsay et al. if Bomanins are strictly anti-toxins. Surely they must also do something generalist?
4. In most figures, the authors do not compare flies with shared genetic backgrounds. The phenotypes are usually strong so I am not concerned.<br /> However the rescue effect of Bom transgenes in Fig 5C-D is based on smaller differences. Were these genetic backgrounds controlled? Were transgenes inserted at the same site? The authors seemingly used a heat shock to express transgenes. Given a resilience effect is being studied, this heat stress approach is sub-optimal. Earlier experiments showing effect/no effect of Bomanin on heat shock resilience would improve confidence here. I would recommend assaying temperatures that can kill wild-type in order to confirm that Bom do not succumb earlier (ex. up to 37'C).<br /> In Fig5C the time resolution is poor, and the effect inconsistent across Bomanins. What are the differences in the Bomanins that the authors suspect could cause this? And how consistent are the experiments? Since the effect is finished by 24h, perhaps a boxplot of percent survival at this time would better show the consistency across experiments.

Minor concerns:

• i) The authors say the fungal burden of Bom55C flies remains low in Fig 5B, but they never measure flies that are near death when fungal load is greatest, or FLUD like in other figures. Given low mortality at the following time points, it seems likely that A. fumigatus would grow beyond initial loads in those individuals and kill them. I grant that these loads are less than what is seen in Hayan mutants. I just might suggest a more careful consideration of the time points used and what can be said about the trends shown here.
• ii) Could the authors comment somewhere about the levels of toxin they were required to inject to get a phenotype vs. the level of toxins the authors expect are found in the fly during infection? I appreciate that toxin injection likely requires much higher doses, but it would be good to know just how far the authors have pushed their experimental system beyond its natural range. Again regarding toxins vs. general stresses, one could manage to inject salt into the hemolymph and show a stress-sensitized fly would succumb at lower doses than wild-type, emphasizing the relevance of defining concentrations. The authors could also write toxin concentrations clearly in the figure/legend per experiment.
• iii) Throughout the manuscript, the order that figures/panels are cited is inconsistent. Perhaps the text could be re-written so the reader can follow the figures more intuitively while going through the text?
• iv) There are a few points where run-on sentences, involving many commas, make it hard to follow the logic. I might suggest a careful reading to break up long sentences into two sentences to ensure clarity.
• v) Line 279-281: this is the first and only mention of the immune surveillance hypothesis in nematodes. This is strange, given the authors are effectively describing an analogous idea exists in flies? Perhaps this could be added somewhere in the introduction or discussion.

Small points

• What timepoints are the gene expression data from? Could the authors indicate this in figures/legends?
• Line 133-135: "We conclude that MyD88 flies succumb to a low A. fumigatus burden..." - could the authors cite a figure panel here to emphasize what evidence they're referring to.
• Line 151-152: Dudzic et al. (2019- Cell Reports Figure 3) showed that PPO2 was regulated by Hayan, while PPO1 by Sp7. This relevant study should be cited here or in the introduction/discussion.
• Line 179-180: could the authors define the gliotoxin mutant strain here in the text for clarity?
• Line 196: Fig. 4A-B should be Fig. **S4 A-B?
• Fig4A: perhaps the authors could reduce the x-axis to focus on the early time points? If I understand correctly, aspf1 has slightly delayed killing compared to akuB (˜50% difference at 2 days), but both kill 100% by 3 days.
• Fig4G: can the authors define the GFP transgene on pg10? Not clear what this is, or what this means. Brain? Fat body? The legend of Fig4G and the key in the top left... it's not easy to quickly understand what is shown in Fig4G.
• Line 247: I would drop the "at the intracellular level" part. I'm not sure this is robustly shown given the use of an in vitro model where there is no closed extracellular environment. The data are convincing of the effect, this is just a semantic point.
• Line 257-258: Cohen et al. (2020- Front Imm) never used Bomanin mutants. Did the authors mean to cite Hanson et al. (2019) here, which seems to fit their described citation re: Bom55C vs. Toll mutant flies (Fig. 2)? Given Hanson et al. infected Toll mutant and Bom55C flies with many bacteria/fungi including A. fumigatus, it's strange this study is not discussed currently.
• Fig5C-D: the labeling is difficult to follow.
• Line 318: a -possible- AMP role of Bomanins was proposed because of the aforementioned killing activity of wt but not Bom mutant hemolymph, alongside rescue by single Bom genes. To say this was based only on survival experiments is incorrect.
• Line 324-328: could the authors cite appropriate references after "inhibition of calcium-activated K+ channels" ?
• comment re-Line 334: Toll10b flies have melanotic tumors and are in general in a stressed state. Might their rescue be due to increased stress tolerance by pre-activated stress responses?

Referees cross-commenting

Yes I agree that the data themselves are not the issue, nor even the direction of the results. But there are many overly-strong statements that go so far as to refute ideas which are supported by other studies, and for which the authors here do not provide any contradictory evidence.

As per my review, I would be happy with a re-write that softened the language overall. I genuinely wonder if these Bomanin mutants simply have poor development, and so they are susceptible to neurotoxins/stress because their nervous system/development leaves them less resilient in general. Experiments testing their resilience to different stresses would greatly elevate the ability to make confident insights in the present manuscript. Currently the authors have only investigated one type of phenotype and interpreted it as if that is evidence of the evolved purpose of the peptides. This approach does not account for many other possible (and reasonable) explanations.

Significance

This paper should be of broad interest to the study of immunology, where roles for effectors are typically thought of as cytokines. In fruit flies and other invertebrates that lack adaptive immunity, immune effectors are more thought of as direct actors likely with antimicrobial properties. The finding that Toll might mediate resilience is interesting, and implicating well known Toll effectors provides an important step forward towards a mechanistic basis behind this resilience effect.

My expertise is in insect and innate immunity.

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Referee #1

Evidence, reproducibility and clarity

Summary

The authors have set out to study the Drosophila immune response against the fungus Aspergillus fumigatus. They found that Aspergillus fumigatus kills Drosophila Toll pathway mutants. The fungus does this without invasion because its dissemination is blocked by melanization. They suggest that there is a role for Toll in host defense distinct from resistance. The findings are interesting, and looks like the mycotoxins play a role. It also seems that there is some role of the Bomanins here, but I find that in particular Figure4 experiments are not convincing enough to provide a mechanistic insight as to what is going on. I think the authors need to think through what their results mean, and also, explain better (especially regarding Fig 4) their ideas and how the data fits them.

Major comments:

Page 5: .."the fungal burden did not increase much in MyD88 flies challenged with 50 conidia (Fig. 1B)" - What do you mean did not increase much? There is a clear increase in Myd88 mutants compared to controls; would you expect a bigger increase (e.g. log scale induction)? Explain.

Page 6: "the SPZ/Toll/MyD88 cassette is required for host defense against A. fumigatus infections, even though this pathogen only mildly stimulates the Toll pathway." - Should you rather say that A. fumigatus only mildly induces the Toll pathway target gene Drosomycin?

Page 6: "...we tested Hayan mutant flies defective for this arm of innate immunity (Nam et al., 2012)." - elaborate this, which arm/which pathway? This has not been introduced in the introduction. Explain. Can you really draw this conclusion: "We conclude that melanization limits the proliferation and the dissemination of A. fumigatus injected into wild-type flies yet does not eradicate it at the injection site, where a melanization plug forms." Maybe you can based on the function/importance of the pathway to melanization, but you need to explain.

Page 10: "The cleavage of the 18S RNA was however much less pronounced in wild-type flies as compared to MyD88" - I am not sure what this means. Do you mean 28S? And that the 28S peak is lower? Is this a quantitative method? Fig. legend: "Arrows show the position of the 28S RNA sarcin fragment" - there are three arrows in both Fig 4E and F; specify which arrows point what.<br /> Based on the results, I am not convinced about the conclusion, that "restrictocin is able to inhibit translation to a detectable degree in vivo, likely through the cleavage of the ribosomal 28S a-sarcin/ricin loop as described in vitro." <- Do you draw this conclusion before doing the actual in vitro experiment, which is described next in the text (The rabbit reticulocute assay, S2 cells)?

4H: Not sure what should be seen here, is it the darkest band at 0 uM that disappears? HI & J need more explanation than what is now included in the text or Figure legend, is the conclusion that there is no difference? Write the stats above the Figs 4I & J (n.s.?).

Minor comments:

It would have helped commenting if the manuscript contained line numbers

Why do you have the title "Hayan" on top of Fig 1F; you don't have this marking system in the other survival curves

Fig 2A: Can you speculate why MyD88 flies die rapidly at day 10 if you inject PBST (your control)? What would happen to uninjected controls in otherwise the same conditions? (you could include an uninjected control here?)

Fig 2E: Not sure what would be the best way of presenting the curves - different colors, dotted lines or something? Now if there are too many lines, they are hard to tell apart. because the symbols are not that visible. Like in 2E if you want to compare the light red/orange colored lines.

For consistency add the caption also to Fig 3D (I assume it is the same as 3C)

For consistency, should you add Verruculogen on top of Fig 3F?

Chronologically, how it is explained in the text, Figs 4A and B are in the wrong order.

The quality of Fig 4 is not great, the text is hard to read (too small) and becomes blurry upon magnification.

Page 12; "These data then suggest that a process akin to the immune surveillance of core cellular processes first described in C. elegans may also exist in Drosophila" - I think this sentence belongs to the discussion, this is not directly drawn from the results.

Referees cross-commenting

I think we share many thoughts among all the reviewers. The main problem is that the manuscript language is quite strong; from the results many times it is not ok to make such strong statements. Some experiments need further analysis and clarification. I think in most cases, this could be achieved by softening the statements and adding more discussion, and not by making new experiments (some may be needed).

Minor things are that experiments are not advancing in a logical order between the text and the figures and there are problems with resolution in some figures. Statistics in some figures needs to be added.

Significance

The nature of the work is conceptual for the field, to understand the role of the Toll pathway and Bomanins in particular, in this fungal infection model. The work is interesting to a somewhat limited audience, mainly immunologists and in particular, people interested in the Drosophila model for immunity. The work may be interesting conceptually in understanding fungal infections.

My expertise: I am a Drosophila immunity researcher with nearly 20 years of experience in working with fly immunity, in particular the Toll and the Imd pathways.

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Reply to the reviewers

We thank the reviewers for their time in evaluating of our manuscript and for the useful feedback. We are grateful that reviewers acknowledged that our study is important because it “sheds much needed light on this less documented early stage of cancer development”. The reviewers were overall positive in their assessment and, as reviewer #3 noted, our study “advances this field conceptually by highlighting the importance of targeting the cell signaling and chromatin regulation together”. The common criticism of all reviewers relates to writing style, some textual interpretation and ensuring that the number of replicates, statistical analysis, and cell culture type were appropriately mentioned. We felt these were valid points and have taken onboard all these comments. A shared concern between two of the reviewers was related to the logic behind the timepoints we chose to analyse cells in the different assays. We are confident that we have addressed this, and all other comments as detailed below.<br /> Please find below a point-by-point reply to the reviewer’s comments.

Reviewer #1 (Evidence, reproducibility and clarity):

This study aimed to identify events that happens early in malignant transformation of breast cancer (BC) cells that are driven by HER2 oncogene. Constructing a 3D inducible model to study impact of HER2 protein level on BC cell and assessment of gross morphological changes, protein phosphorylation and chromatin accessibility at different time points of HER2 activation.

Using a controllable in vitro model is a good approach although it is not novel. Also the method used to assess HER2 protein positivity is not standardized nor clinically relevant. Positivity of HER2 in clinical practice is assessed either through immunohistochemistry (IHC 3+ or 2+ with gene amplification), however the author did not mention any control for positivity except western blot which is not used in clinical practice.

We agree with the reviewer that we should have included our comparison of HER2 protein levels for our cells with a positive control. We have tested this, and the data will be included in the revised version of the manuscript. Briefly, both western blot (WB) and IHC are very useful methods with different benefits: WB is less cost effective but more quantitative, while IHC gives a better overview of tissue heterogeneity. Indeed, due to higher sample processing costs, WB is not used in clinical practice to assess HER2 but it has been shown that there is a high concordance (in 95% of over 300 tumours analysed) between the two methods as both techniques showed prognostic significance R. Molina et al., 1992 (PMID: 1363511). We performed comparison of HER2 protein expression levels of our subpopulations (low, medium, and high HER2 expressing cells) versus two patients’ samples that were already known to be HER2 positive using IHC 3+ or 2+. We were able to demonstrate that HER2 protein levels as measured by western blotting showed that the low HER2 expressing cells expressed less HER2 protein compared to IHC 3+ or 2+ and may be comparable to patients with IHC 1+, which are considered HER2 negative and do not qualify for anti-HER2 therapies such as Trastuzumab.

There is difference between early HER2 positive BC and HER2 low BC. As the earlier is driven by HER2 oncogenic signalling pathway, but the latter is not.<br /> Identification of molecular changes that occur at HER2 low BC seems very important and clinically relevant, however HER 2 low is not fully characterized, yet. And the only definition available is either HER2 1+ or 2+ without gene amplification. The author was not very clear about threshold he followed to call the model HER2 low. Is it positive with lower limit of positivity or just small amount of protein). He also concluded that BC with sub-threshold of HER2 protein behave more aggressive than HER2 positive BC. What is the threshold and was it correlated with IHC or gene amplification level to be reliable?

The HER2 positive population in our in vitro inducible system was determined by flow cytometry, we separated the overall (bulk) HER2 positive cells into three different subpopulations and selected the bottom 20% of HER2 expressing cells as the “low HER2” and the top 20% of HER2 expressing cells as “high HER2”. We show in figure 4C the different thresholds for low, med, high HER2 protein expression by flow cytometry. We have modified the figure and the figure legend (figure 4C) to better indicate the different subpopulations. Through western blotting we compared these population of cells with patients’ samples that had IHC 3+ or 2+ and showed that low HER2 population expressed less protein than IHC 2+, whereas the high HER2 was relatively comparable to IHC 3+ sample.

The status of oestrogen and progesterone receptors were not highlighted. Triple negative breast cancer, for instance, is more aggressive than HER2 positive BC, this may be the reason for the worse behaviour.

We have modified our main text in the manuscript, line 68-69, to better reflect the fact the MCF10A cells are both oestrogen (ER) and progesterone (PR) negative, this has been already characterised by Qu, Y et al., 2015 (PMID: 26147507). However, importantly, we do not think that ER and PR status is the reason these cells are relatively more aggressive, as normal MCF10A cells without HER2 expression did not display any transformative characteristics in our molecular analysis and/or in vitro functional assays, despite being ER and PR negative.

At line 130, "The low levels of HER2 protein activation at early time point may closely mimic at least partially the signalling changes occurring in HER2 positive BC patients". This claim is not quite true, as low levels of HER2 protein activation doesn't activate HER2 oncogenic signalling pathway as HER2 positive does.

We thank you for this insightful comment, we have modified our main text to better reflect our view (line 132-133). However, we were not sure which published data the reviewer was referring to in this case. In particular, if low HER2 levels can still form dimerisation with its family members and induce signalling via its family partners such as HER1, HER3 or HER4.

The author aimed to study the signalling changes accompanying low levels of HER2 induction by lowering significance threshold to log2fold > 0.5. Lowering the threshold for significance will increase the total number of phosphorylated protein (both at low HER2 levels and high levels). So, studying the whole significant proteins at whole time points will not be exclusive for low HER2 levels and this was evident through activation of MAPK cascade which is one of downstream signalling pathway of HER2 positive BC.

We agree that a log2fold change > 0.5 would increase the total number of significantly phosphorylated proteins. We first performed the analysis on a more stringent cut-off value of log2fold change > 1.5 p-value, <0.05 as shown in figure 2B. In the supplementary we also show the reduced stringency of log2fold change > 0.5, p-value <0.05, for the following reasons: when it comes to proteins, it is conceivable that a log2fold change > 0.5 is sufficient to induce molecular changes; secondly, our study investigates changes that occur just half an hour, and up to 7 hours, after HER2 protein induction. At such early time-points, proteins would be beginning to be phosphorylated and the extent of it may not be pronounced (especially in a small subset of the population); finally, we thought it is important to share this supplementary analysis with the scientific community to have access to this data so that they may further interrogate it from different perspectives.

Combining HER2 protein level (both IHC and Western blot) to different time points will give better understanding of events associated with HER2 low, early positive or late positive.

As above, IHC is routinely performed for clinical diagnosis because it is cost effective. Although, western blotting is laborious and expensive, it is more quantitative compared to IHC.

Reviewer #1 (Significance):

This work provides good evidence to changes that happen at early HER2 positive breast cancer transformation and introducing a chromatin opening and accessibility as a new target of treatment of HER2 positive breast cancer patients.

We thank reviewer #1 for their thoughtful feedback and for their appreciation of our work.

Reviewer #2 (Evidence, reproducibility and clarity):

HER2 amplification is associated with poor prognosis of breast cancer. Despite it has been extensively studied, it deserves thorough study how HER2 amplification alters downstream signaling pathways, chromatin structure and gene expression, and how cells overcome the hurdles in order to transform. In this study, Hayat et al used doxycycline-induced HER2 expression in MCF10A cells to recapitulate the very early stage of HER2 expression and HER2-induced mammary epithelial cell transformation. The authors performed global phosphoproteomic, ATAC-seq and single-cell RNA-seq, and propose sub-threshold low level HER2 expression activates signaling pathways and increases chromatin accessibility required for cell transformation, while high HER2 expression level in early stages results in decreased chromatin accessibility.

Major comments:<br /> 1. Although it is not clearly described, it seems that phosphoproteomic and single-cell RNA-seq were performed using 2D-cultured cells, while ATAC-seq was performed using 2D (FACS sorted cells based on HER2 expression levels) or 3D (time course)-cultured cells. Cells cultured on 2D and 3D are significantly different on cell signaling, chromatin structure and gene expression, and therefore cannot be compared.

We agree that there are differences between 2D and 3D cell cultures, which may impact on the multi-omics experiments performed in this study. In an ideal world we would have preferred to be able to conduct all experiments in 3D cell cultures, including the phosphoproteomics experiments. However, this is not feasible because the phosphoproteomics experiment requires 500ug of total protein which corresponds to approximately 10 million cells for each condition and replicates in 3D matrices. 3D structures would have also presented with accessibility issues since doxycycline might not have reached all cells equally at the 30 minutes timepoint. Since we were analysing early timepoints for phosphoproteomics, homogeneity in induction was important. We performed ATAC-seq in 3D cell culture because it was feasible as it only required 25,000-50,000 cells to be grown in small 3D cell cultures and is indeed superior for physiological relevance. We therefore had to compromise and worked with the assumption that immediate signaling events will not be fundamentally different in 2D vs 3D. We have modified the main text to better reflect this and have indicated which experiments were performed in 2D vs 3D in the figure legends and the methods section.

1. Phosphoproteomic (0.5, 4 and 7 hours), ATAC-seq (1, 4, 7, 24 and 48 hours) and single-cell RNA-seq (7, 24, 48 and 72 hours) were performed on cells at different time points after doxycycline treatment. The authors need to clearly explain the rationale why such time points were chosen for each experiment in the text.

There are indeed differences in the time-points analysed between the different multi-omics analysis. However, as mentioned above, the reason for selecting such early time points for the phosphoproteomic experiment was that signalling changes are rapid and we were focused on characterising the early signalling dynamics. With regards to the ATAC-seq and scRNA-seq, there are several shared time-points such as the 7h, 24h, and the 48h. Additionally, as the chromatin changes would be slower acting as compared to signalling changes, two later time-points were selected including the 48h (ATAC-seq) and 72h (scRNA-seq) to capture some late changes during cellular transformation.

1. Change on chromatin accessibility does not necessarily mean change on gene expression levels. RNA-seq needs to be performed and analyzed along with ATAC-seq data.

We agree that chromatin accessibility does not necessarily correlates with gene expression changes and the need to perform RNA-seq to make such a conclusion. This is the reason we performed single cell RNA-seq, which looks at changes in high temporal and cellular resolution. This is particularly useful for the heterogenous cell population that we worked in to better understand the differences between cell types.

1. Analyses on multi-omics data are quite preliminary. Clustering analysis on the time course of phosphoproteomic, ATAC-seq and single-cell RNA-seq will help characterize the dynamics of cell signaling and gene expression. Integrated analyses on multi-omics data and construction of regulatory network are necessary to identify the key signaling node and key epigenetic regulators/machinery that facilitate or prevent cell transformation. Integrated analyses, of course, need to be performed on data obtained from cells cultured in the same conditions.

We think our study is an important work and provides a strong foundation for a comprehensive, integrative multi-omics study using primary human breast cells with parallel analysis performed on the same population of cells using the latest techniques such as scATAC and RNA-seq or scNMT-seq. We are indeed in the process to apply for funding in a larger analysis that involves in vivo work and clinical samples, using this study as a foundation.

1. The authors picked up several genes from the analyses, and discussed the potential importance in cell transformation without functional validation. It is important to show data demonstrating altered expression of certain genes and/or altered activity of certain signaling pathway/epigenetic regulators is indeed important for cell transformation in low HER2-expressing condition or preventing cell transformation in high HER2-expressing condition.

We agree that this is important. The scope of this study is to report on the result that low HER2 was unexpectedly more aggressive compared to high HER2, which was a highly reproducible observation, and identified a molecular explanation for this behaviour (dedifferentiation and predominant chromatin opening). In terms of cross validation, we found the MUC1 protein expression to be low in low HER2 expressing cells, indicating that they are more stem-like (figure 4B). We confirmed and validated this finding in our scRNA-seq data shown in figure 4F. The pathway analysis from phosphoproteomic study shows that MAPK pathway is highly activated upon HER2 protein overexpression. To validate this claim, we performed western blotting analysis that confirm this as the ERK protein was hyperphosphorylated in HER2 expressing cells compared to controls. Thus, our resource study provides many candidates that can be tested to further explore the biology.

1. HER2 expression in MCF10A cells is insufficient in inducing tumor formation in vivo, although HER2 expression results in disrupted acini structure and colony formation in vitro (e.g. Alajati et al. 2013 Cancer Res, 73:5320-5327 cited in the manuscript). It is interesting to investigate whether this is due to the mechanisms identified in this study.

MFC10A cells are generally difficult to transform in vivo. It is possible that mechanisms identified in our study might be responsible for lower tumourigenicity in vivo with WT HER2 compared to HER2 variants, since our study suggests activated checkpoints in high HER2 cells. It would be interesting to compare the differential impact on chromatin for the two HER2 variants too. In our system, we think the reasons why cells form abnormal morphological changes and grow colonies in vitro is a result of HER2 overexpression, which induces aberrant signalling, and this may be leading to loss of cell-to-cell contact and disruption of adhesion molecules. However, the objective of this study was to understand the early signaling to chromatin changes in in vitro cellular transformation, and changes in cell morphology are a consequential part of the process.

1. In Figure 2C, two replicates are completely separated and replicates of each time points are not clustered together.

We agree that the two replicates are separated into two separate groups, this was demonstrated by the PCA analysis (Supplementary Fig 1F). We grouped these samples into “early” (0h, 1h, 4h, and 7h time-points) or “late” (24h and 48h time-points) based on them clustering well into these two groups. The subsequent analysis were performed based on these groups that clustered together. However, we still showed each replicate in figure 2C to appreciate the dynamics of chromatin accessibility between each time-point, which shows clear differences in HER2 versus Control.

Minor comments:<br /> 1. Essential experimental information, e.g. whether cells were cultured in 2D or 3D, needs to be clearly and accurately described in main text, figure legends and experimental procedures.

The figure legends in the manuscript have now been modified to include information on cell culture type.

1. Statistic methods are not provided. In Fig. 4D, HER2-med and HER2-high need to be compared to HER2-low group.

Statistical analyses have been added to figure 4D and HER2-med and HER2-high have been compared to HER2-low group.

Reviewer #2 (Significance):

The authors propose sub-threshold low level HER2 expression activates signaling pathways and increases chromatin accessibility, which facilitates mammary epithelial cell transformation, while high HER2 expression in early stages results in decreased chromatin accessibility via unknown feedback mechanisms. It is interesting to identify which signaling and epigenetic regulators are essential to cell transformation, which feedback mechanisms prevent the transformation of HER2-amplified mammary epithelial cells, whether inactivation of such feedback mechanism indeed occurs in tumorigenesis of HER2-amplified breast cancer, and whether it is a potential therapeutic target for HER2-amplified breast cancer.

Expertise of review: breast cancer, cell signaling, tumor microenvironment.

We thank reviewer #2 for their time and for providing such useful feedback on our work.

Reviewer #3 (Evidence, reproducibility and clarity):

In this paper Hayat et.al study the early transformational events that follow the activation of the oncogenic HER2 signaling pathway and its crosstalk with chromatin opening. Using an inducible in vitro model of HER2+ breast cancer they have identified that the overexpression of HER2 transforms non-tumorigenic breast epithelial cells via chromatin regulation. The study also shows that the transformative potential of the cells is inversely related their HER2 expression where the low HER2 expressing cells obtain a stem-cell like signature and increased chromatin accessibility leading to an increased transformative potential.

Major comments:

While the key conclusions of the paper are convincing, here are the parts of the study that need further clarification or supporting data from the authors.

1. In Figure 1C the authors show that MCF10AHER2 cells formed complex transformed masses when grown in 3 dimensional cultures. From the figure it is evident that that the transformative potential of the HER overexpression is far more pronounced at the Day 6 and Day 9 mark. Therefore, one wonders why these time points weren’t used as the “late timepoint” in any of the sequencing studies moving forward. Can the authors comment on this choice and perform additional experiments to address the molecular changes that lead to the dramatic transformations seen at this timepoint? Since the authors have a well-established protocol in place, looking at an additional time point could be potentially feasible, provided the cells/samples have been frozen down at this stage. If unable to do so, could the authors comment on the molecular changes they would expect to see at this time point.

In our study we primarily focused on the early events upon HER2 overexpression because the changes appear to be much more dynamic, and we hypothesised that these events are the cause of the subsequent, more pronounced featured later on. The rationale behind employing an inducible system and capturing the early changes was to identify aberrant molecular events at the earliest time possible. Indeed, numerous studies have investigated the differences between normal versus cancer cells (many of which are at later time points, that have missed the foremost aberrant molecular changes). Based on our ATAC-seq analysis at late-timepoints, 24h and 48h time-point, the number of changes in chromatin accessibility become relatively more stable as compared to early time points (supplementary figure 2A).

1. Fig 1D the authors conclude that the overexpression of HER2 causes increased cell invasion based on the results seen in a collagen coated plate. How to the authors explain the lack of any such significant change in a Matrigel coated plate?

To test the invasiveness of the HER2 overexpressing cells, collagen is used to increase stiffness to Matrigel. Stiffness is relevant for the type of invasion seen in these 3D cultures because it activates pathways important for invasion. We added the references to the text for clarity (PMID: 15838603 and PMID: 16472698).

1. In Supp Fig 1D the authors use the DAVID Bioinformatics tool to identify the various signaling pathways enriched in the HER2 induced system. In addition to the MAPK pathway this analysis also shows other common cancer-related pathways (eg. The Mtor pathway) being enriched to a similar or higher extent. Can authors address why only the MAPK pathways was pursed in detail?

HER2 is major receptor that can signal through various signalling pathways. We highlighted the MAPK pathway because it has been previously shown that MAPK cascades can modify chromatin through transcription factors and chromatin regulators Clayton and Mahadevan., 2010 (PMID:19948258). We think that when HER2 is overexpressed, it primarily signals down the MAPK pathway, resulting in the activation of transcription factors and chromatin regulators that lead to a highly accessible chromatin and ultimately contributes to transformation. To confirm this result, we did perform western blotting control analysis and found that indeed, HER2 overexpression consistently activates the MAPK pathway that shows phosphorylation of ERK but does not influence AKT phosphorylation. We can include this data in the manuscript.

1. Figure 4B and supplementary figure 3E only show that percentage of the cells have either MUC1-ve or EpCAMlow or CD24low expression. However, Figure 4A and the corresponding text indicates that that breast stem cells are defined by a combination of MUC1-ve, EpCAMlow, and CD24low expression. If this is the case, the authors need to show the percentage of the cells within each population have an overlap of all these expression signatures, to support the claim of low HER2 expressing cells showing a more de-differentiated stem-cell like property.

Our results confirm that upon HER2 overexpression, cells become MUC1-ve, EpCAMlow and CD24-ve, acquiring the breast stem cell signature. We did not show the CD24 expression because all the cells that were MUC1-ve and EpCAMlow were also 100% CD24-ve. We have now modified figure 4B and the figure legend to reflect this change, additionally, we added another figure (supplementary figure 4) that shows how the analysis was performed systematically.

1. The authors also state 'other biological effects being responsible for the lower capacity in anchorage-independent growth of high HER2 expressing cells' that is shown in fig 4d. While an experimental investigation of these effects may be out of the scope of this study, the authors may consider commenting (and referencing additional literature) on the other biological effects they think may result in this phenomenon.

We have modified the manuscript (lines 294-296) and added further explanation as to what other biological effects may be responsible for lack of colony growth in high HER2 expressing cells in lines.

1. The authors do a great job providing details about all statistical analyses performed, however the details regarding the experimental replicates are only provided for some experiments making it difficult to infer if the experiments have been adequately replicated before concluding results. Can the authors please add the n - value for all applicable experiments in the figure legend or the methods section?

The number of replicates has now been added to the respective figure legends.

1. What is the scope for validation of these findings in vivo and in human samples? Could the authors please comment on this in the discussion section of the manuscript.

The primary goal of this study was to understand the early transformational events in a simple in vitro, yet a robust model that is highly accessible. We have analysed some human samples to compare the HER2 protein expression levels. However, the findings from this manuscript could be validated in more precious models such as primary human cells, human tumours samples and in vivo in animals. We have modified the end of discussion to address these points (lines 394-399).

Minor comments:

1. In figure 1B the authors show a western blot analysis for HER2 expression over time while using GAPDH as a loading control. However, GADPH control seems to be unequal, especially in the 1ug/ml Dox lane. This needs to be addressed.

We agree that there is a slight difference in the GAPDH levels in this western blot. We have carried out densitometry analysis which could be added to the supplementary data if required, to show that even though the GAPDH appears to be slightly less in the 1ug/ml of dox (last lane), it shows that HER2 levels are even greater than what appears on the blot, thereby confirming the trend we have observed in the current western blot.

1. In figure 1C, it is unclear if the images shown are representative of the exact same spot over a 9-day period or of different spots.

In figure 1C, the morphological regions are representative of the whole well in which the cells were growing but not the exact same spot. This is because nearly all the cells (>90%) transformed from round, organised acini to the fibroblastic, invasive morphology by day 9. We have captured multiple images of different areas in the well using confocal microscopy, and this can be added in the supplementary data.

1. In Supplementary figure 3E, labeling the y-axis on the figure as opposed to just in the legends would make it easy for the reader.

The figure has now been appropriately labelled.

1. With respect to presentation: In figures involving single cell RNA sequencing and phosphoproteome analyses, highlighting the specific genes that are focused in detail on the manuscript would aid the reading process. The current format makes it difficult for the reader to spot the specific genes that are the points of focus within each heat map.

We modified the figures concerning the phosphoproteomic analysis and scRNA-seq and have highlighted important genes for readers’ ease.

Reviewer #3 (Significance):

I have close to a decade's experience in working on breast cancer. In the past I focused on studying intratumor genetic heterogeneity and cell signaling pathway interactions. I am currently working on identifying novel therapeutic targets for the treatment of ER+ breast cancer. My expertise lies in understanding molecular biology of the disease. While I have worked with and understand most techniques used in this study, I would like to indicate that I do not have sufficient expertise in ATAC seq and am unable to evaluate the intricacies of this technique.

While molecular changes that occur in HER2+ breast cancer have been highly investigated, the changes that occur at an early pre-cancerous stage of the disease aren't as well documented. The work by Hayat et al., sheds much needed light on this less documented early stage of cancer development. The past decade has shown an increased focus on epigenetic therapy with more chromatin targeting drugs entering clinic (Siklos et al., 2022). There has also been increased clinical evidence underlining the efficiency of combining epigenetic therapy and with hormonal and other anticancer therapies in solid tumors (Jin et al., 2021). Phase II clinical trials combining HDAC inhibitors with aromatase inhibitor have shown to improve clinical outcomes in patients (Yardley et al., 2013). Similarly, pre-clinical studies have shown that combination therapy with BET inhibitors improved treatment efficacy and circumvented drug resistance in fulvestrant (Feng et al., 2014) and everolimus (Bihani et al., 2015) treatments. Conclusions from the work by Hayat et.al, although based on in vitro analyses, advances this field conceptually by highlighting the importance of targeting the cell signaling and chromatin regulation together. If validated in in vivo models and clinical samples, this may open up potential possibilities of combining anti-HER2 therapies with epigenetic therapies. Additionally, the study also makes an interesting observation that low HER2 expression could result in increased tumorigenicity of cells which is in contrary to current clinical norm of looking at increased HER2 expression as a sign of aggressive disease. These findings are of interest to the scientific and clinical community working on discovering novel therapeutic targets and biomarkers for treatment of HER2+ breast cancer.

We thank reviewer #3 for his/her overall assessment and for appreciating this work. There is a significant focus regarding low HER2 positive breast cancers in the field. Approximately 50-60% of breast cancers have "low" HER2 expression and in many cases, this low HER2 is seen together with metastatic cancer. The FDA has very recently approved fam-trastuzumab deruxtecan-nxki aka Enhertu, which appears to target these cancers with low HER2 well and is shown to be relatively effective in a phase 3 clinical trial known as Destiny Breast-04. However, it is not yet clear how low HER2 expressing cells drive the metastatic spread of breast cancers or why they are so aggressive. Our work sheds a light that increased chromatin accessibility could be a route of transformation in low HER2 cancers. Therefore, providing an alternative platform to target these cancers and why it is crucial that this work reaches the clinical and scientific community as soon as possible.

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Referee #3

Evidence, reproducibility and clarity

In this paper Hayat et.al study the early transformational events that follow the activation of the oncogenic HER2 signaling pathway and its crosstalk with chromatin opening. Using an inducible in vitro model of HER2+ breast cancer they have identified that the overexpression of HER2 transforms non-tumorigenic breast epithelial cells via chromatin regulation. The study also shows that the transformative potential of the cells is inversely related their HER2 expression where the low HER2 expressing cells obtain a stem-cell like signature and increased chromatin accessibility leading to an increased transformative potential.

Major comments:

While the key conclusions of the paper are convincing, here are the parts of the study that need further clarification or supporting data from the authors.

1. In Figure 1C the authors show that MCF10AHER2 cells formed complex transformed masses when grown in 3 dimensional cultures. From the figure it is evident that that the transformative potential of the HER overexpression is far more pronounced at the Day 6 and Day 9 mark. Therefore, one wonders why these time points weren't used as the "late timepoint" in any of the sequencing studies moving forward. Can the authors comment on this choice and perform additional experiments to address the molecular changes that lead to the dramatic transformations seen at this timepoint? Since the authors have a well-established protocol in place, looking at an additional time point could be potentially feasible, provided the cells/samples have been frozen down at this stage. If unable to do so, could the authors comment on the molecular changes they would expect to see at this time point.
2. Fig 1D the authors conclude that the overexpression of HER2 causes increased cell invasion based on the results seen in a collagen coated plate. How to the authors explain the lack of any such significant change in a Matrigel coated plate?
3. In Supp Fig 1D the authors use the DAVID Bioinformatics tool to identify the various signaling pathways enriched in the HER2 induced system. In addition to the MAPK pathway this analysis also shows other common cancer-related pathways (eg. the mTOR pathway) being enriched to a similar or higher extent. Can authors address why only the MAPK pathways was pursed in detail?
4. Figure 4B and supplementary figure 3E only show that percentage of the cells have either MUC1-ve or EpCAMlow or CD24low expression. However, Figure 4A and the corresponding text indicates that that breast stem cells are defined by a combination of MUC1-ve, EpCAMlow, and CD24low expression. If this is the case, the authors need to show the percentage of the cells within each population have an overlap of all these expression signatures, to support the claim of low HER2 expressing cells showing a more de-differentiated stem-cell like property.
5. The authors also state 'other biological effects being responsible for the lower capacity in anchorage-independent growth of high HER2 expressing cells' that is shown in fig 4d. While an experimental investigation of these effects may be out of the scope of this study, the authors may consider commenting (and referencing additional literature) on the other biological effects they think may result in this phenomenon.
6. The authors do a great job providing details about all statistical analyses performed, however the details regarding the experimental replicates are only provided for some experiments making it difficult to infer if the experiments have been adequately replicated before concluding results. Can the authors please add the n - value for all applicable experiments in the figure legend or the methods section?
7. What is the scope for validation of these findings in vivo and in human samples? Could the authors please comment on this in the discussion section of the manuscript.

Minor comments:

1. In figure 1B the authors show a western blot analysis for HER2 expression over time while using GAPDH as a loading control. However, GADPH control seems to be unequal, especially in the 1ug/ml Dox lane. This needs to be addressed.
2. In figure 1C, it is unclear if the images shown are representative of the exact same spot over a 9-day period or of different spots.
3. In Supplementary figure 3E, labeling the y-axis on the figure as opposed to just in the legends would make it easy for the reader.
4. With respect to presentation: In figures involving single cell RNA sequencing and phosphoproteome analyses, highlighting the specific genes that are focused in detail on the manuscript would aid the reading process. The current format makes it difficult for the reader to spot the specific genes that are the points of focus within each heat map.

Significance

I have close to a decade's experience in working on breast cancer. In the past I focused on studying intratumor genetic heterogeneity and cell signaling pathway interactions. I am currently working on identifying novel therapeutic targets for the treatment of ER+ breast cancer. My expertise lies in understanding molecular biology of the disease. While I have worked with and understand most techniques used in this study, I would like to indicate that I do not have sufficient expertise in ATAC seq and am unable to evaluate the intricacies of this technique.

While molecular changes that occur in HER2+ breast cancer have been highly investigated, the changes that occur at an early pre-cancerous stage of the disease aren't as well documented. The work by Hayat et al., sheds much needed light on this less documented early stage of cancer development. The past decade has shown an increased focus on epigenetic therapy with more chromatin targeting drugs entering clinic (Siklos et al., 2022). There has also been increased clinical evidence underlining the efficiency of combining epigenetic therapy and with hormonal and other anticancer therapies in solid tumors (Jin et al., 2021). Phase II clinical trials combining HDAC inhibitors with aromatase inhibitor have shown to improve clinical outcomes in patients (Yardley et al., 2013). Similarly, pre-clinical studies have shown that combination therapy with BET inhibitors improved treatment efficacy and circumvented drug resistance in fulvestrant (Feng et al., 2014) and everolimus (Bihani et al., 2015) treatments. Conclusions from the work by Hayat et.al, although based on in vitro analyses, advances this field conceptually by highlighting the importance of targeting the cell signaling and chromatin regulation together. If validated in in vivo models and clinical samples, this may open up potential possibilities of combining anti-HER2 therapies with epigenetic therapies. Additionally, the study also makes an interesting observation that low HER2 expression could result in increased tumorigenicity of cells which is in contrary to current clinical norm of looking at increased HER2 expression as a sign of aggressive disease. These findings are of interest to the scientific and clinical community working on discovering novel therapeutic targets and biomarkers for treatment of HER2+ breast cancer.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #2

Evidence, reproducibility and clarity

HER2 amplification is associated with poor prognosis of breast cancer. Despite it has been extensively studied, it deserves thorough study how HER2 amplification alters downstream signaling pathways, chromatin structure and gene expression, and how cells overcome the hurdles in order to transform. In this study, Hayat et al used doxycycline-induced HER2 expression in MCF10A cells to recapitulate the very early stage of HER2 expression and HER2-induced mammary epithelial cell transformation. The authors performed global phosphoproteomic, ATAC-seq and single-cell RNA-seq, and propose sub-threshold low level HER2 expression activates signaling pathways and increases chromatin accessibility required for cell transformation, while high HER2 expression level in early stages results in decreased chromatin accessibility.

Major comments:

1. Although it is not clearly described, it seems that phosphoproteomic and single-cell RNA-seq were performed using 2D-cultured cells, while ATAC-seq was performed using 2D (FACS sorted cells based on HER2 expression levels) or 3D (time course)-cultured cells. Cells cultured on 2D and 3D are significantly different on cell signaling, chromatin structure and gene expression, and therefore cannot be compared.
2. Phosphoproteomic (0.5, 4 and 7 hours), ATAC-seq (1, 4, 7, 24 and 48 hours) and single-cell RNA-seq (7, 24, 48 and 72 hours) were performed on cells at different time points after doxycycline treatment. The authors need to clearly explain the rationale why such time points were chosen for each experiment in the text.
3. Change on chromatin accessibility does not necessarily mean change on gene expression levels. RNA-seq needs to be performed and analyzed along with ATAC-seq data.
4. Analyses on multi-omics data are quite preliminary. Clustering analysis on the time course of phosphoproteomic, ATAC-seq and single-cell RNA-seq will help characterize the dynamics of cell signaling and gene expression. Integrated analyses on multi-omics data and construction of regulatory network are necessary to identify the key signaling node and key epigenetic regulators/machinery that facilitate or prevent cell transformation. Integrated analyses, of course, need to be performed on data obtained from cells cultured in the same conditions.
5. The authors picked up several genes from the analyses, and discussed the potential importance in cell transformation without functional validation. It is important to show data demonstrating altered expression of certain genes and/or altered activity of certain signaling pathway/epigenetic regulators is indeed important for cell transformation in low HER2-expressing condition or preventing cell transformation in high HER2-expressing condition.
6. HER2 expression in MCF10A cells is insufficient in inducing tumor formation in vivo, although HER2 expression results in disrupted acini structure and colony formation in vitro (e.g. Alajati et al. 2013 Cancer Res, 73:5320-5327 cited in the manuscript). It is interesting to investigate whether this is due to the mechanisms identified in this study.
7. In Figure 2C, two replicates are completely separated and replicates of each time points are not clustered together.

Minor comments:

1. Essential experimental information, e.g. whether cells were cultured in 2D or 3D, needs to be clearly and accurately described in main text, figure legends and experimental procedures.
2. Statistic methods are not provided. In Fig. 4D, HER2-med and HER2-high need to be compared to HER2-low group.

Significance

The authors propose sub-threshold low level HER2 expression activates signaling pathways and increases chromatin accessibility, which facilitates mammary epithelial cell transformation, while high HER2 expression in early stages results in decreased chromatin accessibility via unknown feedback mechanisms. It is interesting to identify which signaling and epigenetic regulators are essential to cell transformation, which feedback mechanisms prevent the transformation of HER2-amplified mammary epithelial cells, whether inactivation of such feedback mechanism indeed occurs in tumorigenesis of HER2-amplified breast cancer, and whether it is a potential therapeutic target for HER2-amplified breast cancer.

Expertise of review: breast cancer, cell signaling, tumor microenvironment.

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Referee #1

Evidence, reproducibility and clarity

This study aimed to identify events that happens early in malignant transformation of breast cancer (BC) cells that are driven by HER2 oncogene. Constructing a 3D inducible model to study impact of HER2 protein level on BC cell and assessment of gross morphological changes, protein phosphorylation and chromatin accessibility at different time points of HER2 activation.

Using a controllable in vitro model is a good approach although it is not novel. Also the method used to assess HER2 protein positivity is not standardized nor clinically relevant. Positivity of HER2 in clinical practice is assessed either through immunohistochemistry (IHC 3+ or 2+ with gene amplification), however the author did not mention any control for positivity except western blot which is not used in clinical practice.<br /> There is difference between early HER2 positive BC and HER2 low BC. As the earlier is driven by HER2 oncogenic signalling pathway, but the latter is not.

Identification of molecular changes that occur at HER2 low BC seems very important and clinically relevant, however HER 2 low is not fully characterized, yet. And the only definition available is either HER2 1+ or 2+ without gene amplification. The author was not very clear about threshold he followed to call the model HER2 low. Is it positive with lower limit of positivity or just small amount of protein). He also concluded that BC with sub-threshold of HER2 protein behave more aggressive than HER2 positive BC. What is the threshold and was it correlated with IHC or gene amplification level to be reliable?

The status of oestrogen and progesterone receptors were not highlighted. Triple negative breast cancer, for instance, is more aggressive than HER2 positive BC, this may be the reason for the worse behaviour.<br /> At line 130, "The low levels of HER2 protein activation at early time point may closely mimic at least partially the signalling changes occurring in HER2 positive BC patients". This claim is not quite true, as low levels of HER2 protein activation doesn't activate HER2 oncogenic signalling pathway as HER2 positive does.<br /> The author aimed to study the signalling changes accompanying low levels of HER2 induction by lowering significance threshold to log2fold > 0.5. Lowering the threshold for significance will increase the total number of phosphorylated protein (both at low HER2 levels and high levels). So, studying the whole significant proteins at whole time points will not be exclusive for low HER2 levels and this was evident through activation of MAPK cascade which is one of downstream signalling pathway of HER2 positive BC.

Combining HER2 protein level (both IHC and Western blot) to different time points will give better understanding of events associated with HER2 low, early positive or late positive.

Significance

This work provides good evidence to changes that happen at early HER2 positive breast cancer transformation and introducing a chromatin opening and accessibility as a new target of treatment of HER2 positive breast cancer patients.

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Reply to the reviewers

Reviewer #1:

We thank the Reviewer for stating that “Overall the article is well structured, the experiments are clearly and logically described. The data is convincing and there does not seem to be a sticking point”, and also for pointing to the fact that “This manuscript will therefore be of interest to people working in the field of readthrough, therapeutic approaches and genetic diseases, but also more generally to people studying gene translation and expression”.

Specific comments:

In chapter "Serum starvation increased APC nonsense-mutation readthrough in CRC cell lines", last line please replace "sratvation" by "starvation".

The mistake has been corrected.

In chapter "Torin-1 increases antibiotic-mediated nonsense codon readthrough" , 6 lines before the end please replace "Totin-1" by "Torin-1"

The mistake has now been corrected.

The following sentence in the discussion has to be rewritten because NMD degrades RNA and not proteins: "In many cases, the cancer cells express a truncated APC protein that is not degraded by the NMD as most of the nonsense mutations occur in a hotspot within the last APC exon, thus they are not recognized by the exon junction complex method of NMD [55] ".

The sentence has been corrected and rephrased to say: “Mutated APC transcripts are often NMD-resistant as most of the nonsense mutations occur in a hotspot within the last APC exon and therefore not recognized by the exon junction complex that induces NMD”.

Change "combitation" into "combination" 7 lines from the end of the discussion.

The mistake has been corrected.

Figure 5, the authors analyze the effect of an inhibition of the activity of eIF4E using the small molecule 4EGl-1. They are testing for an endogenous nonsense mutation in the APC gene in COLO320 cells. To be consistent with Figure 4, the authors should also show the same effect on SW403 cells.

The requested missing experiment has been added to Figure 5 (Fig.5D) and the results are discussed.

Reviewer #2:

We thank the Reviewer for acknowledging the “nice flow of the paper” and that “The involvement of mTOR pathway in PTC RT is interesting”. We have addressed the Reviewer’s comments and added the requested experiments as follows:

Major comments -

1- My major concern is about the concentration of G418 that authors used in their PTC RT experiments. G418 at 1.5 mg/ml is extremely high and usually very toxic in many cell types. We have observed that in the presence of high levels of aminoglycosides, PTC RT enhances significantly at the cost of severe toxicity. Authors should be careful to avoid such toxicity. Showing the viability (live cells as well as apoptosis levels, both) in cells (stable and CRC cells) at 24 and also 48 hours post treatment (G418, Torin-1, Rapamycin and their combination) must be performed as an indicator of cellular health.

We thank the Reviewer for raising this point. In the revised manuscript the vast majority of experiments were conducted with a lower dose of antibiotics (500ug/ml). We have used the G418 at 1.5 mg/ml only when comparing it to our previous results showing the effects of serum starvation on readthrough, where this high concentration was used [1] (Fig. 1 & Fig. S1) and when using immunofluorescent experiments on colo320 and SW403 cells (Fig. 4D). In all other experiments 500ug/ml G818 was used. We have now tested cell viability under the different treatments, using the 500ug/ml dose (Fig. S3) and demonstrate that cell survival is between 60%-100% under the different conditions. This point has now been emphasized in the revised manuscript (results section - Torin-1 increases antibiotic-mediated nonsense codon readthrough).

2- Control cell lines (a CRC cell line without APC mutation to show WT levels of APC, and a CRC cell line with APC mutations other than PTC as negative control) must be included to the experiments. It is much better to report the level of PTC readthrough relative to WT rather than untreated mutant cells. Regarding the low level of PTC RT enhancement in combination treatment it is good to know whether these levels have any biological significance when compared to normal APC levels.

We have now added the requested missing control cells to the manuscript (Fig. 1C): HCT116

which harbor an b-catenin mutation (and wt APC) and SW48 expressing an APC gene with a missense mutation. In these cell lines, APC is mostly unaffected by the enhancing readthrough treatment. Please note that the endogenous expression levels of APC in these cells are higher than those achieved by restoring APC levels in Colo320 cells. Importantly, although the induced APC restoration is relatively minor, the effect on reducing active b-catenin levels is significant. The levels of induced readthrough depend on different factors such as the type of the stop codon, the surrounding sequence and the gene itself [2, 3]. As the Reviewer stated, it is important to determine what is the minimal levels of full-length protein induced by the readthrough treatment that has therapeutic effects. It has been shown that in each protein and disease, this level is different. For example, in lysosomal storage disease, even 1 % of normal protein function may restore a near-normal or clinically less severe phenotype [4, 5]. For cystic fibrosis 10 to 35 % of CFTR activity might be needed to significantly alleviate pulmonary morbidity [7] and in DMD – 1-30% of the full-length dystrophin is needed [6]. Similarly, our results indicate that even if we can restore only relatively low amounts of the APC protein [1], these_ levels may _have beneficial therapeutic effects [8]. This important point has now been added to the introduction of the revised manuscript.

3- In the introduction section the authors mentioned that "there is increasing evidence that APC truncations may exert dominant functions contributing to colon tumorigenesis. These include enhancement of cell migration, interference with spindle formation, and induction of chromosome instability [35-38]." Usually in the course of PTC readthrough the truncated protein is also increased (Baradaran-Heravi et al, 2016, Nucleic Acids Research). In this study, in addition to full length APC authors need to show the truncated form in the CRC cell lines and find out whether this form also increases during mTOR inhibition and G418 treatment. Since the dominant function of APC truncation contributes to colon tumorigenesis, would increase in truncated protein during PTC readthrough be considered as an adverse side effect?

We have now conducted the missing experiment. In revised Figs. 1B and S1 we show that the increase in full length APC following nonsense mutation-induced readthrough is not observed in the truncated APC protein product. Truncated APC is known to be NMD-resistant [9] and thus accumulates in cancers that originate from APC-premature termination codons. p53, on the other hand, is highly affected by NMD (as discussed in Baradaran-Heravi et al, 2016, Nucleic Acids Research) and thus nonsense mutation readthrough, which leads to prolonged ribosomal protection of the p53 transcripts, could affect the low levels of the truncated p53 protein product.

4- I am wondering how the authors reconcile diminished translation initiation and increased PTC readthrough? What is the author's proposed model?

We agree that this is a very important point. Our results show that 4EG1-1 that affects translation initiation, enhanced-PTC readthrough only in the presence of aminoglycosides (Fig. 5). Aminoglycosides exert their PTC readthrough activity by binding at the decoding center of the eukaryotic ribosome and reducing the ability of translation termination factors to accurately recognize the PTC [10, 11]. Similarly to our results, It has been shown that other compounds such as the small molecules CDX5-1 [12] or the drug mefloquine [13], that do not show readthrough activity when used as single agents, potentiate the readthrough activity of aminoglycoside possibly by directly targeting the translation machinery although the exact mechanism is still unclear and should be further studied. Another interesting possibility is that the effect of 4EGI-1 on PTC readthrough arises from its inhibitory effect on mTORC1 signaling which may be independent of its role in cap-dependent translation initiation [14]. This important point has now been discussed in the revised manuscript (discussion paragraph) and, although beyond the scope of the current report, we are currently conducting additional experiments to understand the exact mechanism of enhancing the activity of aminoglycosides on nonsense mutation readthrough.

5- In figure 2C, can authors induce Gentamicin related PTC RT in TSC-/- cells by treating them with Torin-1 or Rapamycin or 4EGI-1? Please show the results.

The requested missing data has been added to the Figure (Fig. 2) and corresponding text.

6- Please show the APC mRNA levels in CRC cell lines and discuss its changes in different treatment combinations.

We have now measured the APC mRNA levels under the different treated combinations and have added the results to the revised manuscript (Fig. S5). These results have been discussed (in the result section -_ Rapamycin increases antibiotic-mediated nonsense codon readthrough) _as follows: "Interestingly, although mutated APC transcripts are relatively stable, a slight increase in mRNA levels was observed in treated Colo320 cells as opposed to SW403 where mRNA transcripts were unaffected by readthrough or readthrough enhancement".

7- It would be nice to see the effect of combination treatment on PTC RT response in other CRC cell lines they discussed in Figure 2A.

We have now added two CRC cell lines, chosen from the group of cells where serum starvation enhances readthrough. These cells respond to the combination treatment on PTC readthrough (SW837 and SW620; Fig. S4).

Minor comments-

1- It would be nice to explain in more detail the GFP-BFP cell line when the authors mention it for the first time.

A detailed explanation on the_ GFP-BFP _reporter plasmin has been added to the revised manuscript (in the results section, under the paragraph - The mTOR pathway may regulate antibiotic-induced nonsense mutation readthrough).

2- In figure 2A, how many proteins did they end up analyzing? Please mention the number.–

We tested 214 proteins where we had the data for all 8 cell lines examined. Out of these proteins, 8 were statistically significantly different. Out of these proteins, 4EPB-1 and its three phosphorylated forms had the most statistical significance. This information has now been added to the text.

3- Authors mentioned that "As can be seen, Totin-1 induced APC restoration in both cell lines, though the re-expression of full-length APC was more complete in COLO320 cells". What do they mean by "complete" when they do not have WT levels of APC to compare with? Do they mean "more efficient" ?

We apologize for the confusing terminology. We compared the readthrough activity to the null condition and not the wild-type expression. The sentence has been completely rephrased in the discussion paragraph.

4- Please provide the full image of APC western blots to better visualize to full length and truncated forms in one blot.

Figures 1C and S1 now show both full-length-APC and truncated APC in untreated and treated cells. Technically, due to the differences in protein sizes (90-160kDa for the truncated APC protein product in the different cell lines and full-length APC-312kDa) and the poor quality of the available antibodies, both APC forms cannot be detected on the same blot and were thus analyzed on separate gels.

5- In figure 5, please add 4EGI-1 treatment (alone) lane for both panels. Also, please add quantification of active b-catenin for panel B.

The experiments have been repeated and this missing data has been added to the figure and corresponding text.

6- In the discussion it is said "As all the CRC cells that responded to mTOR inhibition<br /> by increased PTC readthrough show high levels of 4E-PB1 we conclude that inhibiting<br /> cap-dependent protein translation initiation enhances antibiotic mediated PTC<br /> readthrough". This statement is not accurate. The authors have tested only one cell line, COLO320, which has high 4E-PB1 expression and responds to mTOR inhibition in terms of increased PTC RT.

This statement has been changed and corrected to state that: "As CRC cells that responded to mTOR inhibition by increased PTC readthrough show high levels of 4EPB-1 (Figs. 3-4 and data not shown)"

*Referees cross-commenting*

I appreciate reviewer #1 and #3 comments and I also agree about the nice flow of the paper. We routinely study G418 effect on PTC readthrough in many different cell lines. My major reservation is about the concentration of G418 that authors used in their PTC RT experiments. G418 at 1.5 mg/ml is extremely high and usually very toxic in many cell types. We have observed that in the presence of high levels of aminoglycosides, PTC RT enhances significantly at the cost of severe toxicity. Authors should be careful to avoid such toxicity. Showing the viability (live cells as well as apoptosis levels, both) in cells (stable and CRC cells) at 24 and also 48 hours post treatment (G418, Torin-1, Rapamycin and their combination) must be performed as an indicator of cellular health.

Thank you for this comment. As described above in response to Reviewer #2 comments, the majority of our experiments were now conducted with a lower dose of antibiotics (500ug/ml). Although Reviewer #3 mentioned that “some cell types can tolerate those doses”, we have now tested the survival of the various treatments, using the 500ug/ml dose (Fig. S3) and demonstrated that cell death did not exceed 40% under any condition. Only viable cells were used in our experiments.

The involvement of mTOR pathway in PTC RT is interesting; however, I am not sure about the biological value of this finding as mTOR inhibition marginally enhances aminoglycoside induced PTC RT (2-2.5-fold in COLO320 cells). Also, the number of cell lines tested in this manuscript is limited to only two CRC cell lines which makes the interpretation of the results more difficult.

To address this important point additional CRC cell lines have now been used throughout the manuscript. As different studies show that increasing nonsense mutation readthrough levels and inducing some restoration of the full-length protein, even by small amounts could have beneficial value (please see our response to Reviewer # 2, point 2) we suggest that enhancing nonsense mutation readthrough by inhibiting the mTOR pathway may have therapeutic value. We have now emphasized in the manuscript that the different strategies for inducing readthrough (including ours) do not achieve wild-type levels and that this point needs to be considered when evaluating the therapeutic potential of this treatment strategy.

Reviewer #3 :

We thank the Reviewer for stating that “ This is an important finding”. We have addressed the specific Reviewer’s comments as follows:

1) In the first paragraph of the Results Section, you use serum starvation to enhance readthrough. However, I could not find how long you maintained serum starvation, whether it was added before or concurrently to aminoglycoside addition, etc. Please clarify this point.

We apologize for omitting this point. The treatment conditions of serum starvation have now been added to the results section and to the legend (cells were incubated for 24 h in a medium containing 10% or 1% serum supplemented with 1.5mg/ml G418).

2) Fig. S1: I can't read the x-axis labels. Please fix this.

The figure has been corrected (currently Fig. S2).

3) First paragraph in the torin-1 section: you don't refer the reader to Fig 3B and 3C. I suggest that you revise the text as follows: "Next, the effect of mTOR inhibition on antibiotic-mediated endogenous APC readthrough in the CRC cell lines COLO320 (Fig. 3B) and SW403 (Fig. 3C) was examined where aminoglycosides induced relatively high levels of APC restoration”.

The text has been revised and corrected.

4) In figs. 3, 4 and 5, you label the panels using the cell lines COLO320 (panel B) and SW403 (panel C), but not for the APC R1450X line (panel A). The reason for this omission is not clear, but it would help the reader follow your work if you added it.

The missing panels have now been labeled correctly.

5) You don't mention Fig. S3 in the text of the manuscript. Please add a sentence to the last paragraph of the Results since it is important to note that 4EGI-1 does not induce readthrough alone.

The Figure has now been mentioned and the finding that 4EGI-1 does not induce readthrough alone is now shown in Fig. 5 (please see our response to Reviewer #2, point 5, minor points section).

*Referees cross-commenting*

I agree that 1.5 mM G418 sounds high, but some cell types can tolerate those doses. Controls to examine toxicity seem appropriate and won't take too long. In addition, one panel showing that the mTOR inhibition also stimulate readthrough at a lower G418 dose would help to allay this concern.

Please see our response to this point above (Reviewer #2, point 1). In the current manuscript all experiments except Fig. 1 & Fig.S1 were conducted with 500ug/ml G418.

References

[1] A. Wittenstein, M. Caspi, Y. David, Y. Shorer, P.T. Nadar-Ponniah, R. Rosin-Arbesfeld, Serum starvation enhances nonsense mutation readthrough, J Mol Med (Berl), 97 (2019) 1695-1710.

[2] C. Floquet, I. Hatin, J.P. Rousset, L. Bidou, Statistical analysis of readthrough levels for nonsense mutations in mammalian cells reveals a major determinant of response to gentamicin, PLoS Genet, 8 (2012) e1002608.

[3] L. Martorell, V. Cortina, R. Parra, J. Barquinero, F. Vidal, Variable readthrough responsiveness of nonsense mutations in hemophilia A, Haematologica, 105 (2020) 508-518.

[4] I. Maire, Is genotype determination useful in predicting the clinical phenotype in lysosomal storage diseases?, J Inherit Metab Dis, 24 Suppl 2 (2001) 57-61; discussion 45-56.

[5] I. Nudelman, D. Glikin, B. Smolkin, M. Hainrichson, V. Belakhov, T. Baasov, Repairing faulty genes by aminoglycosides: development of new derivatives of geneticin (G418) with enhanced suppression of diseases-causing nonsense mutations, Bioorg Med Chem, 18 (2010) 3735-3746.

[6] M. Dabrowski, Z. Bukowy-Bieryllo, E. Zietkiewicz, Advances in therapeutic use of a drug-stimulated translational readthrough of premature termination codons, Mol Med, 24 (2018) 25.

[7] E. Kerem, Pharmacologic therapy for stop mutations: how much CFTR activity is enough?, Curr Opin Pulm Med, 10 (2004) 547-552.

[8] R. Kariv, M. Caspi, N. Fliss-Isakov, Y. Shorer, Y. Shor, G. Rosner, E. Brazowski, G. Beer, S. Cohen, R. Rosin-Arbesfeld, Resorting the function of the colorectal cancer gatekeeper adenomatous polyposis coli, Int J Cancer, 146 (2020) 1064-1074.

[9] R.G. Lindeboom, F. Supek, B. Lehner, The rules and impact of nonsense-mediated mRNA decay in human cancers, Nat Genet, 48 (2016) 1112-1118.

[10] B. Francois, R.J. Russell, J.B. Murray, F. Aboul-ela, B. Masquida, Q. Vicens, E. Westhof, Crystal structures of complexes between aminoglycosides and decoding A site oligonucleotides: role of the number of rings and positive charges in the specific binding leading to miscoding, Nucleic Acids Res, 33 (2005) 5677-5690.

[11] N. Garreau de Loubresse, I. Prokhorova, W. Holtkamp, M.V. Rodnina, G. Yusupova, M. Yusupov, Structural basis for the inhibition of the eukaryotic ribosome, Nature, 513 (2014) 517-522.

[12] A. Baradaran-Heravi, A.D. Balgi, C. Zimmerman, K. Choi, F.S. Shidmoossavee, J.S. Tan, C. Bergeaud, A. Krause, S. Flibotte, Y. Shimizu, H.J. Anderson, V. Mouly, E. Jan, T. Pfeifer, J.B. Jaquith, M. Roberge, Novel small molecules potentiate premature termination codon readthrough by aminoglycosides, Nucleic Acids Res, 44 (2016) 6583-6598.

[13] M.W. Ferguson, C.A.N. Gerak, C.C.T. Chow, E.J. Rastelli, K.E. Elmore, F. Stahl, S. Hosseini-Farahabadi, A. Baradaran-Heravi, D.M. Coltart, M. Roberge, The antimalarial drug mefloquine enhances TP53 premature termination codon readthrough by aminoglycoside G418, PLoS One, 14 (2019) e0216423.

[14] H. Wang, F. Huang, J. Wang, P. Wang, W. Lv, L. Hong, S. Li, J. Zhou, The synergistic inhibition of breast cancer proliferation by combined treatment with 4EGI-1 and MK2206, Cell Cycle, 14 (2015) 232-242.

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Referee #3

Evidence, reproducibility and clarity

In this study, the authors show that inhibition of the translation initiation-controlled by the cap-dependent (eIF4E) branch of the mTOR pathway enhances antibiotic-mediated nonsense mutation readthrough mediated by aminoglycosides. Interestingly, inhibition of this pathway in the absence of mTOR inhibitors has no effect on readthrough. These studies suggest that inhibition of this pathway may be used to enhance readthrough of disease-causing mutations.

I suggest that the authors consider the following points to improve the manuscript:

1. In the first paragraph of the Results Section, you use serum starvation to enhance readthrough. However, I could not find how long you maintained serum starvation, whether it was added before or concurrently to aminoglycoside addition, etc. Please clarify this point.
2. Fig. S1: I can't read the x-axis labels. Please fix this.
3. First paragraph in the torin-1 section: you don't refer the reader to Fig 3B and 3C. I suggest that you revise the text as follows: "Next, the effect of mTOR inhibition on antibiotic-mediated endogenous APC readthrough in the CRC cell lines COLO320 (Fig. 3B) and SW403 (Fig. 3C) was examined where aminoglycosides induced relatively high levels of APC restoration. Next, the effect of mTOR inhibition on antibiotic-mediated endogenous APC readthrough in the CRC cell lines COLO320 and SW403 was examined where aminoglycosides induced relatively high levels of APC restoration."
4. In figs. 3, 4 and 5, you label the panels using the cell lines COLO320 (panel B) and SW403 (panel C), but not for the APC R1450X line (panel A). The reason for this omission is not clear, but it would help the reader follow your work if you added it.
5. You don't mention Fig. S3 in the text of the manuscript. Please add a sentence to the last paragraph of the Results since it is important to note that 4EGI-1 does not induce readthrough alone.

Referees cross-commenting

I agree that 1.5 mM G418 sounds high, but some cell types can tolerate those doses. Controls to examine toxicity seem appropriate and won't take too long. In addition, one panel showing that the mTOR inhibition also stimulate readthrough at a lower G418 dose would help to allay this concern.

Significance

Overall, this manuscript demonstrates that inhibition of mTOR-dependent translation initiation by various means (serum starvation, the mTOR inhibitors torin-1 or rapamycin, or 4EGI-1) all stimulate nonsense suppression. This is an important finding.

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Referee #2

Evidence, reproducibility and clarity

This study evaluates the involvement of mTOR pathway in premature termination codon (PTC) readthrough (RT) using cell-based assays. Initially the authors claim that similar to their previous finding serum starvation enhances aminoglycoside induced PTC RT in several cancer cell lines with APC nonsense mutations. They found association between enhanced PTC RT in serum starved cells and increased expression level of 4E-BP using DepMap data and speculated about the role of mTOR in PTC RT. Furthermore, they claim that Torin-1 or Rapamycin treatment of a stable cell line expressing an exogenous PTC construct as well as two colorectal cancer cell lines with APC nonsense mutations increased aminoglycoside induced PTC RT and suppressed active beta-catenin in CRC cells. Finally, they observed enhancement of aminoglycoside induced PTC RT by chemically inhibition of translation initiation factor eIF4E.

Major comments:

1. My major concern is about the concentration of G418 that authors used in their PTC RT experiments. G418 at 1.5 mg/ml is extremely high and usually very toxic in many cell types. We have observed that in the presence of high levels of aminoglycosides, PTC RT enhances significantly at the cost of severe toxicity. Authors should be careful to avoid such toxicity. Showing the viability (live cells as well as apoptosis levels, both) in cells (stable and CRC cells) at 24 and also 48 hours post treatment (G418, Torin-1, Rapamycin and their combination) must be performed as an indicator of cellular health.
2. Control cell lines (a CRC cell line without APC mutation to show WT levels of APC, and a CRC cell line with APC mutations other than PTC as negative control) must be included to the experiments. It is much better to report the level of PTC readthrough relative to WT rather than untreated mutant cells. Regarding the low level of PTC RT enhancement in combination treatment it is good to know whether these levels have any biological significance when compared to normal APC levels.
3. In the introduction section the authors mentioned that "there is increasing evidence that APC truncations may exert dominant functions contributing to colon tumorigenesis. These include enhancement of cell migration, interference with spindle formation, and induction of chromosome instability [35-38]."

Usually in the course of PTC readthrough the truncated protein is also increased (Baradaran-Heravi et al, 2016, Nucleic Acids Research). In this study, in addition to full length APC authors need to show the truncated form in the CRC cell lines and find out whether this form also increases during mTOR inhibition and G418 treatment. Since the dominant function of APC truncation contributes to colon tumorigenesis, would increase in truncated protein during PTC readthrough be considered as an adverse side effect?<br /> 4. I am wondering how the authors reconcile diminished translation initiation and increased PTC readthrough? What is the author's proposed model?<br /> 5. In figure 2C, can authors induce Gentamicin related PTC RT in TSC-/- cells by treating them with Torin-1 or Rapamycin or 4EGI-1? Please show the results.<br /> 6. Please show the APC mRNA levels in CRC cell lines and discuss its changes in different treatment combinations.<br /> 7. It would be nice to see the effect of combination treatment on PTC RT response in other CRC cell lines they discussed in Figure 2A.

Minor comments:

1. It would be nice to explain in more detail the GFP-BFP cell line when the authors mention it for the first time.
2. In figure 2A, how many proteins did they end up analyzing? Please mention the number.
3. Authors mentioned that "As can be seen, Totin-1 induced APC restoration in both cell lines, though the re-expression of full-length APC was more complete in COLO320 cells". What do they mean by "complete" when they do not have WT levels of APC to compare with? Do they mean "more efficient" ?
4. Please provide the full image of APC western blots to better visualize to full length and truncated forms in one blot.
5. In figure 5, please add 4EGI-1 treatment (alone) lane for both panels. Also, please add quantification of active beta-catenin for panel B.
6. In the discussion it is said "As all the CRC cells that responded to mTOR inhibition<br /> by increased PTC readthrough show high levels of 4E-PB1 we conclude that inhibiting<br /> cap-dependent protein translation initiation enhances antibiotic mediated PTC<br /> readthrough". This statement is not accurate. The authors have tested only one cell line, COLO320, which has high 4E-PB1 expression and responds to mTOR inhibition in terms of increased PTC RT.

Referees cross-commenting

I appreciate reviewer #1 and #3 comments and I also agree about the nice flow of the paper. We routinely study G418 effect on PTC readthrough in many different cell lines. My major reservation is about the concentration of G418 that authors used in their PTC RT experiments. G418 at 1.5 mg/ml is extremely high and usually very toxic in many cell types. We have observed that in the presence of high levels of aminoglycosides, PTC RT enhances significantly at the cost of severe toxicity. Authors should be careful to avoid such toxicity. Showing the viability (live cells as well as apoptosis levels, both) in cells (stable and CRC cells) at 24 and also 48 hours post treatment (G418, Torin-1, Rapamycin and their combination) must be performed as an indicator of cellular health.

Significance

The involvement of mTOR pathway in PTC RT is interesting; however, I am not sure about the biological value of this finding as mTOR inhibition marginally enhances aminoglycoside induced PTC RT (2-2.5 fold in COLO320 cells). Also, the number of cell lines tested in this manuscript is limited to only two CRC cell lines which makes the interpretation of the results more difficult.

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Referee #1

Evidence, reproducibility and clarity

The manuscript by Wittenstein et al. aims to demonstrate that an optimization of the efficiency of readthrough induced by aminoglycosides can be obtained by inhibiting the mTOR pathway. The results presented in the manuscript show that serum starvation, inhibition of the mTOR pathway using Torin-1 or rapamycin leads to an increase in the efficiency of readthrough induced by G418. These results were shown on mRNA from a transfected construct and on endogenous mRNA coding for APC in cancer cells carrying a nonsense mutation in the APC gene. All results show an increase in readthrough induced by G418 when the mTOR pathway is impacted. Overall the article is well structured, the experiments are clearly and logically described. The data is convincing and there does not seem to be a sticking point. I would only have minor points which would make it possible to improve the reading of the manuscript and a possible additional experience in order to make figures 4 and 5 homogeneous.

Minor comments:

In chapter "Serum starvation increased APC nonsense-mutation readthrough in CRC cell lines" , last line please replace "sratvation" by "starvation"

In chapter "Torin-1 increases antibiotic-mediated nonsense codon readthrough" , 6 lines before the end please replace "Totin-1" by "Torin-1"

The following sentence in the discussion has to be rewritten because NMD degrades RNA and not proteins: "In many cases, the cancer cells express a truncated APC protein that is not degraded by the NMD as most of the nonsense mutations occur in a hotspot within the last APC exon, thus they are not recognized by the exon junction complex method of NMD [55].".

Change "combitation" into "combination" 7 lines from the end of the discussion.

Figure 5, the authors analyze the effect of an inhibition of the activity of eIF4E using the small molecule 4EGl-1. They are testing for an endogenous nonsense mutation in the APC gene in COLO320 cells. To be consistent with Figure 4, the authors should also show the same effect on SW403 cells.

Significance

The study described here shows that the efficiency of readthrough by aminoglycosides can be regulated by different parameters (serum concentration; translation efficiency). Few data are available on the regulation of this mechanism whose interest in generating new therapeutic approaches in genetic diseases is increasingly growing. This manuscript will therefore be of interest to people working in the field of readthrough, therapeutic approaches and genetic diseases, but also more generally to people studying gene translation and expression.

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Reply to the reviewers

Response to Reviewers:

1. General Statements

We thank the reviewers for the comments and the suggestions. We hope that we have addressed all the queries raised by the reviewers in the revised manuscript. We provide a point-by-point response below. Please note that the line numbers indicated in parentheses correspond to the pdf file without the track changes display.

2. Point-by-point description of the revisions

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Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary: Srinivasan and co-workers developed an alternative screening method for defining the ability of FtsZ inhibitor to affect FtsZ polymerization. This alternative assay was defined considering the expertise of the authors on the topic, and they use Schizosaccharomyces pombe as a model for studying the effect of PC190723, sanguinarine and berberine on FtsZ assembly. The use of a heterologous expression system is useful for the evaluation of FtsZ coming from different strains, both Gram - and Gram +. The same model could gain insights also on the capability of FtsZ inhibitors to affect eukaryotic cell physiology. Finally, authors resulted also in suggesting a possible cause to suspected resistance to PC190723 from Gram - strains as E. coli.

Major comments: • The conclusions are included in the discussion section and are quite convincing, for a general audience.

We thank the reviewer for the positive comments.

In my opinion, the authors should define which could be the limits of their method, since no data on the possible weaknesses are reported.

RESPONSE: We have discussed the limitations of the methods as well. The discussion has been modified and the following sentences have been now included in the revised manuscript.

“However, one of the major disadvantages of using fission yeast could be the need to use much higher concentrations of drugs than normally used for mammalian cell cultures to achieve an inhibitory effect. This could probably be due to the poor permeability of certain drugs in fission yeast because of its thick cell wall (Benko et al. 2017; Pérez and Ribas 2004). A similar effect of toxicity might arise at much lower concentrations in other eukaryotic cells, such as human cells. Consistently, while sanguinarine and berberine are known to affect the eukaryotic microtubules at 10 μΜ – 20 μM concentrations (Lopus and Panda 2006; Wang et al. 2016; Raghav et al. 2017), morphological effects on yeast cells were observed only at concentrations > 100 μM. However, yeast microtubules were not affected by berberine and sanguinarine. Differences in membrane lipid profiles and MDR efflux pumps between yeasts and mammalian cells might also contribute to differential resistance to the drugs being tested (Balzi and Goffeau 1991). Conversely, an inhibitory effect in yeast cells may not necessarily translate into toxicity in a human cell. These and the permeability of drugs in yeast cells represent an important caveat in using such heterologous expression systems for the screening of compounds against target molecules.”

[Lines 498-513]

As suggested in the later sections, we have also elaborated on the pros and cons of various methods including the yeast-based screening methods. [Lines 462-523]

• No additional experiments are required to support the claims.

• The suggested experiments could be quite easy to be realized for authors working in the microbiological field, and familiar with protein expression and purification, as well as bacteria and yeast growth.

• From my side, even if I am not so expert in microbiology and plasmid/protein purification, the methods presented could be reproduced with no significant doubt.

• Statistical analysis was done and seems to be adequate.

RESPONSE: We thank the reviewer for these encouraging comments.

Minor comments: • Prior studies should be deepened, especially for the state of art authors referred to. Additional paper, both reviews on the possible methods for evaluating FtsZ inhibition, as well as research papers on FtsZ inhibitors targeting E. coli and other Gram negative strains should be mentioned, since, in my opinion, these could move authors in changing a little bit the overall text of the manuscript.

RESPONSE: We have now elaborated the state-of-art methods used for evaluation of FtsZ inhibition and cited the relevant papers and reviews. We have also included papers on development of FtsZ inhibitors, especially the ones similar to PC190723, targeting Gram-negative bacteria. The following sentences have been included in the revised manuscript.

“Several approaches have been used to screen small molecules targeting bacterial cell division and FtsZ. While in vitro methods such as NMR (Domadia et al. 2007; Sun et al. 2014; Araújo‑Bazán et al. 2019) and crystallography (Läppchen et al. 2008; Fujita et al. 2017) are valuable and offer information on distinct binding sites, these are not efficient for screening. Electron microscopic examination can distinguish the effects of the compounds being tested on the FtsZ protofilament assembly and lateral associations (Nova et al. 2007; Kaul et al. 2012; Anderson et al. 2012; Sun et al. 2014; Huecas et al. 2017; Kumar et al. 2011; Park et al. 2014). Other techniques that are routinely used include fluorescence anisotropy (Ruiz‑Avila et al. 2013; Park et al. 2014), 90º light-scattering assay (Mukherjee and Lutkenhaus 1999) and dynamic light scattering (Hou et al. 2012; Di Somma et al. 2020) for assessing inhibition of FtsZ assembly (Kaul et al. 2012; Nova et al. 2007; Lui et al. 2019; Anderson et al. 2012, (Irwin et al. 2015). Other easily scalable high-throughput assays include FCS/FCCS and FRET-based methods (Hernández‑Rocamora et al. 2015; Mikuni et al. 2015; Reija et al. 2011).

In vivo assays relying on cell filamentation phenotype coupled with the localization of Z-ring might be a good indicator of FtsZ being the direct target. However, since bacteria can undergo cell filamentation and not assemble FtsZ rings in response to a variety of conditions, including DNA damage (Mukherjee et al. 1998) and disruption of membrane potential (Strahl and Hamoen 2010), the in vivo assay is not so useful unless combined with the in vitro assays mentioned above. Finally, the isolation of resistance mutants in FtsZ to the drug can provide strong evidence of FtsZ being the direct target.

Reconstitution systems are powerful and provide excellent control over the system, but they are emerging technologies and are technically challenging. Reconstitution systems include a variety of methods, such as the use of membrane nanodiscs, microbeads of different materials, supported bi-layer membranes (SLBs) and biomimetic systems that provide cell-like environments (Monterroso et al. 2013; Rivas et al. 2014).”

[Lines 462-487]

“Several compounds have been evaluated for their activity against FtsZ from both Gram-positive bacteria and Gram-negative bacteria. Although many exhibited only weak activity in vivo against Gram-negative bacteria, derivatives could be promising. These include benzamides (Haydon et al. 2008; Adams et al. 2011; Straniero et al. 2017, 2020a), trisubstituted benzimidazoles (Kumar et al. 2011), 4-bromo-1H-indazole derivatives (Wang et al. 2015), cinnamaldehyde and its derivatives (Domadia et al. 2007; Li et al. 2015), curcumin (Rai et al. 2008), heterocyclic molecules like guanidinomethyl biaryl compounds (Kaul et al. 2012), pyrimidine-quinuclidine scaffolds (Chan et al. 2013), 3-phenyl substituted 6,7-dimethoxyisoquinoline (Kelley et al. 2012), thiazole orange derivatives (Sun et al. 2017), viriditoxin (Wang et al. 2003), N-heterocycles such as zantrins and derivatives (Margalit et al. 2004; Nepomuceno et al. 2015).”

[Lines 69-80]

“Several efforts have been made to target Gram-negative bacteria with derivatives of benzamide. Examples include difluorobenzamides, substituted benzodioxanes, heterocyclic and non-heterocyclic derivatives (Straniero et al. 2017; Chai et al. 2020; Straniero et al. 2020a, 2020b). Although many exhibited promising activity in vitro, most were substrates for the AcrAB class of efflux pumps (Chai et al. 2020; Kaul et al. 2014; Straniero et al. 2020a, 2020b; Casiraghi et al. 2020). Thus, the poor membrane permeability, signature outer membrane, particularly lipopolysaccharide (LPS) structure (Wang et al. 2021), the presence of multiple efflux pumps in species such as E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa (Piddock 2006), and differences in FtsZ sequences in the binding-site (Kaul et al. 2013b; Miguel et al. 2015) have been cited as reasons for lack of susceptibility of Gram-negative bacteria to benzamide derivatives (Casiraghi et al. 2020). More recently, two molecules, TXA6101 and TXY6129, with substituted 2,6-difluorobenzamide scaffold, have been shown to inhibit the polymerization of both E. coli and Klebsiella pneumoniae FtsZ. Moreover, despite being substrates for efflux pumps, TXA6101 induced morphological changes in K. pneumoniae (Rosado‑Lugo et al. 2022). Studies in the past on the effects of PC190723 on E. coli have been confusing, with a few reports suggesting an effect on FtsZ polymerization resulting in cell filamentation (Kaul et al. 2014), while others did not find any effect on EcFtsZ (Andreu et al. 2010; Anderson et al. 2012; Khare et al. 2019)⁠. The outer membrane has been shown to be a permeability barrier for PC190723 in E. coli (Khare et al. 2019; Chai et al. 2020). In addition, the Resistance-Nodulation-Division (RND) family of efflux pumps has been attributed to resistance against 2,6-difluorobenzamide derivatives, including TX436 (a prodrug of PC190723) in Gram-negative bacteria (Kaul et al. 2014).”

[Lines 527-550]

The whole text requires a deep check for grammar and word choice. Some sentences should be re-written since it is not so easy to understand their meaning. Figures are clear, even if I am not so convinced on the need of including Figure 1.

RESPONSE: We have now deleted Figure 1 and 2 (as also suggested by Reviewer #2), revised the manuscript and have re-written certain long sentences. We have used Grammarly to check for grammatical errors. We hope the manuscript is easier to follow with these changes.

Reviewer #1 (Significance (Required)):

• In my opinion, the outcome coming from this work could move researchers in evaluating an alternative method for assessing FtsZ inhibition. Nevertheless, the actual state of art, a few reviews of the last years confirm this, already underlined a huge number of possible assays, both microbiological, biochemical, biophysical, physiological, or other. As a result, the authors did not result in convincing me about the importance of their methods, when compared to others. They may include some other possible assays and comment of the differences, pros and cons.

RESPONSE: Several alternative methods have been evaluated and several excellent reviews published in the recent past have underlined the importance of these multiple methods to screen and validate small molecules targeting FtsZ. As suggested by the reviewer here and above, we have now discussed these methods including the yeast-based assay we describe, their advantages and limitations in the revised manuscript.

The following lines have now been included in Introduction.

“Several methods have been used to ascertain FtsZ as the target of the drug, and the various approaches have been reviewed in detail by many (Kusuma et al. 2019; Silber et al. 2020; Zorrilla et al. 2021; Andreu et al. 2022). Andreu et al. (2022) have recently proposed a streamlined experimental protocol for the screening and characterization of FtsZ inhibitors.”

Introduction – [Lines 113-117]

The following paragraphs, including ones as mentioned above have included in the discussion sections of the revised manuscript.

“Several approaches have been used to screen small molecules targeting bacterial cell division and FtsZ. While in vitro methods such as NMR (Domadia et al. 2007; Sun et al. 2014; Araújo‑Bazán et al. 2019) and crystallography (Läppchen et al. 2008; Fujita et al. 2017) are valuable and offer information on distinct binding sites, these are not efficient for screening. Electron microscopic examination can distinguish the effects of the compounds being tested on the FtsZ protofilament assembly and lateral associations (Nova et al. 2007; Kaul et al. 2012; Anderson et al. 2012; Sun et al. 2014; Huecas et al. 2017; Kumar et al. 2011; Park et al. 2014). Other techniques that are routinely used include fluorescence anisotropy (Ruiz‑Avila et al. 2013; Park et al. 2014), 90º light-scattering assay (Mukherjee and Lutkenhaus 1999) and dynamic light scattering (Hou et al. 2012; Di Somma et al. 2020) for assessing inhibition of FtsZ assembly (Kaul et al. 2012; Nova et al. 2007; Lui et al. 2019; Anderson et al. 2012, (Irwin et al. 2015). Other easily scalable high-throughput assays include FCS/FCCS and FRET-based methods (Hernández‑Rocamora et al. 2015; Mikuni et al. 2015; Reija et al. 2011).

In vivo assays relying on cell filamentation phenotype coupled with the localization of Z-ring might be a good indicator of FtsZ being the direct target. However, since bacteria can undergo cell filamentation and not assemble FtsZ rings in response to a variety of conditions, including DNA damage (Mukherjee et al. 1998) and disruption of membrane potential (Strahl and Hamoen 2010), the in vivo assay is not so useful unless combined with the in vitro assays mentioned above. Finally, the isolation of resistance mutants in FtsZ to the drug can provide strong evidence of FtsZ being the direct target.

Reconstitution systems are powerful and provide excellent control over the system, but they are emerging technologies and are technically challenging. Reconstitution systems include a variety of methods, such as the use of membrane nanodiscs, microbeads of different materials, supported bi-layer membranes (SLBs) and biomimetic systems that provide cell-like environments (Monterroso et al. 2013; Rivas et al. 2014). While in vitro biochemical assays and reconstitution systems are useful to find molecules that directly target FtsZ, they are cumbersome and need to be performed at optimal physiological pH and ionic conditions, which can be considerably variable among FtsZ from different species.

Our results on the ability of sanguinarine and berberine to specifically affect the assembly of FtsZ and not MreB in fission yeast highlight the utility of the heterologous expression system as a platform to identify molecules that specifically affect FtsZ polymerization. The yeast platform offers a cellular context mimicking the cytoplasm for cytoskeletal assembly. The system is simple to replicate in any laboratory, including those focused on chemical synthesis with minimum microbiological expertise and can be easily reproduced and scaled up as well. However, one of the major disadvantages of using fission yeast could be the need to use much higher concentrations of drugs than normally used for mammalian cell cultures to achieve an inhibitory effect. This could probably be due to the poor permeability of certain drugs in fission yeast because of its thick cell wall (Benko et al. 2017; Pérez and Ribas 2004). A similar effect of toxicity might arise at much lower concentrations in other eukaryotic cells, such as human cells. Consistently, while sanguinarine and berberine are known to affect the eukaryotic microtubules at 10 μΜ – 20 μM concentrations (Lopus and Panda 2006; Wang et al. 2016; Raghav et al. 2017), morphological effects on yeast cells were observed only at concentrations > 100 μM. However, yeast microtubules were not affected by berberine and sanguinarine. Differences in membrane lipid profiles and MDR efflux pumps between yeasts and mammalian cells might also contribute to differential resistance to the drugs being tested (Balzi and Goffeau 1991). Conversely, an inhibitory effect in yeast cells may not necessarily translate into toxicity in a human cell. These and the permeability of drugs in yeast cells represent an important caveat in using such heterologous expression systems for the screening of compounds against target molecules. However, notwithstanding this caveat, the heterologous system provides significant advantages in assessing the direct effects of the drug on FtsZ assembly. Moreover, fission yeast-based high-throughput platform screening methods using imaging have been successfully adapted to the screening of drugs against HIV-1 proteases by large-scale screening facilities such as the NIH Molecular Libraries Probe Production Centers Network in the Molecular Libraries Program, leading to several candidate drugs (Benko et al. 2017, 2019).”

Discussion - [Lines 462-519]

“A powerful emerging technique based on cytological profiling has been successfully used to identify the cellular pathways targeted by the inhibitors (Nonejuie et al. 2013; Martin et al. 2020), including cell division inhibition by FtsZ (Araújo‑Bazán et al. 2016). The recent advances in computational image analysis and deep learning approaches (von Chamier et al. 2021; Spahn et al. 2022) could further advance image-based screening for FtsZ inhibitors (Andreu et al. 2022).”

Discussion – [Lines 581-586]

As I mentioned before, there are a lot of reviews including the possible tests to perform for assessing FtsZ inhibition. A recent one was not cited, but, from my side, it should be mentioned (10.3390/antibiotics10030254).

The suggested article is an excellent review that in addition to providing an overview of the state-of-art methods currently in practice for screening drugs targeting FtsZ, also suggests other emerging technologies suitable for assay development. We had cited this article (Zorrilla et al., 2021; doi: 10.3390/antibiotics10030254) in other contexts in our original manuscript but inadvertently missed in the text while mentioning the methods for screening.

We have now cited Zorrilla et al., 2021 at all appropriate places in the revised manuscript. In addition, we have also cited (Monterroso 2013; https://doi.org/10.1016/j.ymeth.2012.12.014); (Rivas 2014; https://doi.org/10.1016/j.cbpa.2014.07.018); Kusuma 2019 (doi: 10.1021/acsinfecdis.9b00055); Schaffner-Barbero 2012 (doi: 10.1021/cb2003626); Silber et al 2020 (doi: 10.2217/fmb-2019-0348); Li et al., 2015 (doi: 10.1016/j.ejmech.2015.03.026); Casiraghi et al 2020 (doi: 10.3390/antibiotics9020069); Andreu et al., 2022 (10.3390/biomedicines10081825)

Moreover, I think authors should reconsidered novel research papers, in which researchers evaluated the reason behind the apparent inactivity of benzamide derivatives, similar to PC190723, towards Gram negative strains.

RESPONSE: Several novel papers that have reported reason for the inactivity of benzamide derivatives towards Gram-negative bacteria, including PC190723 have now been cited. The following sentences have been now included in the revised manuscript.

“Several efforts have been made to target Gram-negative bacteria with derivatives of benzamide. Examples include difluorobenzamides, substituted benzodioxanes, heterocyclic and non-heterocyclic derivatives (Straniero et al. 2017; Chai et al. 2020; Straniero et al. 2020a, 2020b). Although many exhibited promising activity in vitro, most were substrates for the AcrAB class of efflux pumps (Chai et al. 2020; Kaul et al. 2014; Straniero et al. 2020a, 2020b; Casiraghi et al. 2020). Thus, the poor membrane permeability, signature outer membrane, particularly lipopolysaccharide (LPS) structure (Wang et al. 2021), the presence of multiple efflux pumps in species such as E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa (Piddock 2006), and differences in FtsZ sequences in the binding-site (Kaul et al. 2013b; Miguel et al. 2015) have been cited as reasons for lack of susceptibility of Gram-negative bacteria to benzamide derivatives (Casiraghi et al. 2020). More recently, two molecules, TXA6101 and TXY6129, with substituted 2,6-difluorobenzamide scaffold, have been shown to inhibit the polymerization of both E. coli and Klebsiella pneumoniae FtsZ. Moreover, despite being substrates for efflux pumps, TXA6101 induced morphological changes in K. pneumoniae (Rosado‑Lugo et al. 2022). Studies in the past on the effects of PC190723 on E. coli have been confusing, with a few reports suggesting an effect on FtsZ polymerization resulting in cell filamentation (Kaul et al. 2014), while others did not find any effect on EcFtsZ (Andreu et al. 2010; Anderson et al. 2012; Khare et al. 2019)⁠. The outer membrane has been shown to be a permeability barrier for PC190723 in E. coli (Khare et al. 2019; Chai et al. 2020). In addition, the Resistance-Nodulation-Division (RND) family of efflux pumps has been attributed to resistance against 2,6-difluorobenzamide derivatives, including TX436 (a prodrug of PC190723) in Gram-negative bacteria (Kaul et al. 2014).”

[Lines 527-550]

Researchers working on FtsZ inhibitors could be interested in this paper, especially microbiologists.

I specifically work on the design, synthesis and evaluation of the microbiological assays performed by others on my compounds.

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Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Dr. Srinivasin and colleagues previously developed a system where they expressed bacterial FtsZ in yeast and showed that it could assemble into polymers related to the Z rings. Here they develop this system further as a way to assay for drugs that may poison FtsZ, which would be candidates for new antibiotics. They test three drugs against three species of FtsZ. The results suggest that this system should be useful in screening new drugs that may target FtsZ. I would recommend publication after addressing a number of concerns and apparent contradictions.

Fig. 1 showing chemical formulas of the drugs, and Fig. 2 showing a schematic of the yeast expression system, are probably not needed.

RESPONSE: Reviewer #1 had also made a similar suggestion and we have now deleted these two figures (Fig. 1 and Fig. 2 in the older version).

The authors make a point that sanguinarine and berberine inhibit eukaryote cell morphology. In fact, what they show is that they affect yeast cell morphology. This may or may not extend to other eukaryotes. Also, other eukaryotic cells may be more sensitive to drugs than yeast. They should me more conservative in this claim that the system also screens for drugs effects on eukaryotes.

RESPONSE: We agree with the reviewer’s suggestions here that other eukaryotic cells may be more sensitive to drugs than yeast. We have modified the statements pertaining to these claims in the revised manuscript.

We have made the following changes in the revised version.

The title of the manuscript has been now modified as “A salt bridge-mediated resistance mechanism to FtsZ inhibitor PC190723 revealed by a cell-based screen”.

Lines 23-24 in the abstract has been modified to read as “The strategy also allows for simultaneous assessment of the toxicity of the drugs to eukaryotic yeast cells.”

Other sentences modified in the revised version are:

“We find that although sanguinarine and berberine affected FtsZ polymerization, they also affected yeast cell physiology”. [Lines 146-147]

“In this study, we have attempted to develop a cell-based assay using fission yeast (S. pombe) as a heterologous expression host, which would enable the screening of compounds that could directly affect FtsZ polymerization as well as identify potential toxicity to yeast (or eukaryotic) cells simultaneously”. [Lines 444-447]

“However, one of the major disadvantages of using fission yeast could be the need to use much higher concentrations of drugs than normally used for mammalian cell cultures to achieve an inhibitory effect. This could probably be due to the poor permeability of certain drugs in fission yeast because of its thick cell wall (Benko et al. 2017; Pérez and Ribas 2004). A similar effect of toxicity might arise at much lower concentrations in other eukaryotic cells, such as human cells”. [Lines 498-503]

“Conversely, an inhibitory effect in yeast cells may not necessarily translate into toxicity in a human cell. These and the permeability of drugs in yeast cells represent an important caveat in using such heterologous expression systems for the screening of compounds against target molecules”. [Lines 510-513]

Fig. 3 has some new structural data that should be explored more quantitatively. My quick measurement gave 0.5 and 0.8 µm for the outside diameters of Ec and Sa rings. The spirals of Hp seem to be 0.8 µm outside diameter, similar to SA rings. These spirals may be related to those reported by Popp and by Andreu under certain buffer conditions. This should be explored and referenced.

RESPONSE: We have now quantitatively measured the diameters of the rings formed by EcFtsZ and SaFtsZ and the diameter and pitch of the spiral polymers of HpFtsZ. These have been now included in the results section and presented as a graph in a new figure (Supplementary Fig. S2). Please also note that the scale bar in Figure 1 (previously Figure 3) was erroneously marked as 5 µm. This has been corrected in the revised version to 2.5 µm.

Also, the possibility that these spiral polymers may be related to those described by Popp and Andreu have been discussed. We included the following sentences in the discussion.

“Previous studies have shown that various factors such as molecular crowding, variable C-terminal regions and bound nucleotide state lead to the formation of supramolecular structures like twisted helical structures, toroids and rings similar to those that have been observed in vivo (Popp et al. 2009; Huecas et al. 2017). Thus, the molecular crowding due to the dense cytoplasm of the yeast cells could have possibly induced the spiral and ring-like assembly of FtsZ polymers (Erickson et al. 2010).”

[Lines 456-461]

But Fig. 4 presents a contradiction. Here the Hp control cells show long smooth polymers, not helical. This seems an important difference and needs to be addressed. Are the polymers sometimes straight and sometimes helical? After finishing the paper I see that in some experiments the HP is helical, and in others the polymers are straight and smooth. I think it would be important to determine what favors the two forms. If this remains a mystery, at least address it openly.

RESPONSE: This was definitely an oversight from the authors. We should have clearly mentioned this in the manuscript but completely missed the description of different polymers assembled by HpFtsZ.

We have now described this clearly in the results and added a new Figure (Supplementary Fig. S1) showing a time course for the appearance of spiral and linear polymers. We have also replaced the images in Figure 5E.

We have modified the results to read as:

“Interestingly, HpFtsZ assembled into linear cable-like structures as well as twisted polymers that were curled and spiral in appearance (Fig. 1D). The spiral filaments were more clearly visualized by deconvolution of the images (Fig. 1D iii and 1E). Further, super-resolution imaging using 3D-SIM clearly revealed that HpFtsZ assembles into spiral filaments in fission yeast (Fig. 1F).”

[Lines 171-175]

We have also added the following lines in the results section:

“Spiral polymers appeared early, at 16 – 18 hours after induction of expression (absence of thiamine), and linear cables appeared later at 20 – 22 hours (Fig. S1). The smooth linear polymers possibly arise from lateral association and bundling of FtsZ filaments (Monahan et al. 2009), but the factors determining the two forms in yeast cells remain unclear.”

[Lines 175-179]

I am concerned that the quantitation of drug inhibition in Fig 4, 5 is flawed. Visually from 4A it looks like ~90-100% of control cells have polymers, and sang reduces polymers by 70% for Sa and Ec and 100% for Hp: this is based on the number of spots and filaments I see in Fig. 4 Aii. But the quantitation in D shows only 17-23% reduction for all three. These numbers were based on determining the fraction of cells that showed polymer (spots or lines) vs diffuse. It seems that cells are counted as containing polymer even if they had a great reduction in spots or lines, but still had a few. E.g., 4Aii Sa has 4 cells, two of them with no spots, one with only 2, and one with ~7, which totals ~1/3 the spots in control cells. Categorizing cells with only a couple of spots as polymerized, seems to be a poor way to quantitate. Would it not be better to count all spots in all cells, or measure the total length of line polymers, as a measure of inhibition.

RESPONSE: We agree with the reviewer here that number of spots or the length of the polymers would be a better quantitative measure of the effect of the drugs than the percentage of cells presented. In the revised manuscript, we now present quantified data as suggested.

We have quantitated the number of spots per cell for SaFtsZ and total polymer length per cell for HpFtsZ to elucidate the effect of drugs on FtsZ polymers. The number of spots per cell were counted using built-in ImageJ macro OPS threshold IJ1 script which combines the otsu thresholding method and analyse particles plugin. The total polymer length per cell in the case HpFtsZ, was measured using used the lpx-plugins as described by Higaki (Higaki et al., 2017).

In addition, using the lpx-plugins, we also quantify density, a measure of the amount cytoskeleton per unit area in a given cell (Henty-Ridilla et al., 2014; Higaki et al., 2017). We had previously used this measure successfully to quantify assembly of Spiroplasma citri MreB in fission yeast (Pande et al., 2022).

The methodology has been described in detail in the Materials and Methods section under the heading – “Quantitation of the number of spots, polymer length and density”

Lines [665-689]

The new data has been included in the results (lines 207-231 and 275-284) and new Figures (Fig. 2 E, G and Fig. 3 G, H) have been added.

Fig. 5 makes a convincing case that PC19 accelerates or enhances the polymerization of Sa and Hp. Fig. S2 shows that the structures of polymers are not changed when PC19 is added at 20 hrs, after polymers have already formed. It would have been nice to see for both 5A and S2A that the round spots had holes in the center, when imaged by SIM. Again the quantitation of cells as polymer vs diffuse seems ill suited, because it misses cells with a reduced number of spots.

RESPONSE: We have imaged the FtsZ polymers of Sa and Hp in the presence of PC190723 using SIM and included these images as new panels in the figures. Figure 3C, 3F and Figure S4 in the revised manuscript.

Again, for Figure 5 (Fig. 3 in the revised version), we have provided the quantitation as number of spots per cell, polymer length per cell and density (amount of cytoskeleton per unit area) as described above (new Figures - Fig. 3 G, H) in the revised manuscript.

[Lines 275-284]

Fig. 6 uses FRAP to show that PC reduces the dynamic exchange of Sa polymers by a factor of 3. It is remarkable to me that rapid exchange is not completely eliminated by PC. Regardless, it would be very important to reference the previous study of Adams..Errington 2011, where they showed the same thing for Foci in Bacillus. PC19 reduced the exchange from 3 to 10 s, but the foci were still very dynamic.

RESPONSE: We had referenced this work in the original submission in the discussion section – “These results are also consistent with the earlier findings that PC190723 acts to induce FtsZ polymerization and stabilize FtsZ filaments (Andreu et al. 2010; Elsen et al. 2012; Miguel et al. 2015; Fujita et al. 2017) and its derivative compound, 8j acting to slow down FtsZ-ring turnover by 3-fold in B. subtilis (Adams et al. 2011).”

[Lines 563-567] in revised manuscript

We have now added the following statement and referenced Adams et al., 2011 in the results section as well.

“Interestingly, compound 8j, a related benzamide derivative, has been shown to slow down FtsZ-ring turnover by 3-fold in B. subtilis (Adams et al. 2011).”

[Lines 324-326]

The analysis of the salt bridge as opposed to a single Arg or His being the cause of resistance to PC19 is an interesting addition to the study. In Fig. 8D some numbers do not agree between the caption and figure (R309/7; S226/7). The whole figure should be carefully checked.

RESPONSE: We thank the reviewer for pointing to these. We have corrected these errors now in the revised version (Fig. 6).

I am not familiar with the Gram -ve and Gram +ve nomenclature. Why not simply gram- and gram+?

RESPONSE: We agree that Gram -ve / +ve are not standard notations and inappropriate.

We have now written them as Gram-negative and Gram-positive throughout the text.

The Discussion is quite long largely because it repeats items from Results and Introduction. It is also redundant to hype the value of this system in both Introduction and Discussion; The Introduction should be sufficient. The Discussion should be pared down by eliminating repetition and focusing on relating results to previous literature, in particular items that have not been referenced previously in the paper. Also, I think we don't need the final "In summary" paragraph. That is already nicely presented in the Abstract.

RESPONSE: We have omitted the repetitive statements from the discussion. We have also deleted the final summary paragraph. We had added new paragraphs [lines 462-519] pertaining to previous literature (also suggested by Reviewer #1) to the discussion section in the revised manuscript.

The authors should probably provide references to other studies that have used yeast expression to study assembly of FtsZ. I am thinking in particular of papers from the Osteryoung lab looking at chloroplast FtsZ.

RESPONSE: We have now referenced other papers that have used yeast expression to study assembly of FtsZ.

The following statement has been added to the introduction:

“Moreover, the dynamics of chloroplast FtsZs have also been successfully studied using the heterologous fission yeast expression system (TerBush and Osteryoung 2012; Yoshida et al. 2016; TerBush et al. 2018).”

Lines [132-134]

NO PAGE NUMBERS. Authors should be penalized a week delay for submitting a mss without page numbers.

RESPONSE: We sincerely apologise for this gross error and oversight and thank the reviewer for patiently reading through and reviewing a manuscript with no page numbers and line numbers. We are truly sorry for having submitted a manuscript as such and have now included page numbers and line numbers in the manuscript.

Reviewer #2 (Significance (Required)):

This work should be of interest to the broad field of research on FtsZ. The authors present it as a new platform for assaying drugs targeting FtsZ, and researchers in this area will certainly be interested. It will also be of broader interest for the novel assay of assembly and exchange dynamics and how they may be modulated by small molecules.

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Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary: The authors established a proof-of-concept assay to investigate the bacterial cytoskeletal protein FtsZ in fission yeast, and this heterologous yeast system is useful for compounds identification targeting FtsZ. The authors used this system to understand the mechanism of FtsZ's resistance to drug PC190723. Major comments: 1. From the study, the pombe seems to be a good system for investigating the bacterial cytoskeleton proteins and testing the drugs for them. However, to my knowledge it is not convincing that this is the proper system can be used to assessing the eukaryotic toxicity, since no toxicity to pombe does not mean no toxicity to human cells and vice versa.

RESPONSE: We agree with the reviewer that toxicity to S. pombe cannot be directly extended to assessing toxicity to other eukaryotic cells such as human cells. As suggested by Reviewer#2 as well, we have modified these claims in the revised manuscript, discussed the possibilities and limited the scope of this work to assessing toxicity in yeast cells.

We have made the following changes in the revised version.

The title of the manuscript has been now modified as “A salt bridge-mediated resistance mechanism to FtsZ inhibitor PC190723 revealed by a cell-based screen”.

Lines 23-24 in the abstract has been modified to read as “The strategy also allows for simultaneous assessment of the toxicity of the drugs to eukaryotic yeast cells.”

Other sentences modified in the revised version are:

“We find that although sanguinarine and berberine affected FtsZ polymerization, they also affected yeast cell physiology”. [Lines 146-147]

“In this study, we have attempted to develop a cell-based assay using fission yeast (S. pombe) as a heterologous expression host, which would enable the screening of compounds that could directly affect FtsZ polymerization as well as identify potential toxicity to yeast (or eukaryotic) cells simultaneously”. [Lines 444-447]

“However, one of the major disadvantages of using fission yeast could be the need to use much higher concentrations of drugs than normally used for mammalian cell cultures to achieve an inhibitory effect. This could probably be due to the poor permeability of certain drugs in fission yeast because of its thick cell wall (Benko et al. 2017; Pérez and Ribas 2004). A similar effect of toxicity might arise at much lower concentrations in other eukaryotic cells, such as human cells”. [Lines 498-503]

“Conversely, an inhibitory effect in yeast cells may not necessarily translate into toxicity in a human cell. These and the permeability of drugs in yeast cells represent an important caveat in using such heterologous expression systems for the screening of compounds against target molecules”. [Lines 510-513]

From figure 4A to 4C, there seems no big difference of cell morphology between control and drug treatment, except for Berberine treatment of SaFtsZ-GFP. Under the low concentration of Sanguinarine (20 µM) and Berberine (53.791 µm), the FtsZ polymerization was disrupted and seems no effect on cell morphology. Why would the authors use much higher Sanguinarine (135.95 µM) and Berberine (134.45 µM) to prove there two drugs are toxic to pombe cells?

RESPONSE: Earlier reports had shown that sanguinarine and berberine affect mammalian microtubules (Lopus and Panda 2006 - DOI: 10.1111/j.1742-4658.2006.05227.x; Raghav et al., 2017 - DOI: 10.1021/acs.biochem.7b00101). While, we did not observe any growth defect in yeast cells, earlier studies have suggested that yeasts possibly require higher concentrations of certain drugs than used for mammalian cells due to the presence of the cell wall, particularly S. pombe (Perez and Ribas 2004 - https://doi.org/10.1016/j.ymeth.2003.11.020; Benko et al., 2017 - DOI: 10.1186/s13578-016-0131-5). We had thus explored the possibility of cell toxicity to yeast cells at higher concentrations of the drugs.

The following lines have thus been added to the results section in the revised manuscript.

“Although we did not observe any growth defect in yeast cells at lower concentrations of the drugs, earlier studies have suggested that yeast cells possibly require higher concentrations of drugs than used for mammalian cells due to the presence of the cell wall, which is particularly thick in S. pombe (Benko et al. 2017; Pérez and Ribas 2004). We thus explored the possibility of cell toxicity to yeast cells at higher concentrations of the drugs.”

Lines [234-239]

Sanguinarine and Berberine are FtsZ disruption drugs, do these drugs have effect on microtubule?

RESPONSE: We have now examined the effect of Sanguinarine and Berberine on yeast microtubules as well and did not find any visible differences between the control and inhibitor (either low or high concentrations) treated cells. This data has been added as a new figure (Supplementary Fig. S3 A and B) in the revised manuscript and the following line added to the results.

“However, even at higher concentrations, neither of the drugs showed any visible effect on yeast microtubules (Fig. S3 A and B).”

[Lines 241-242]

The discussion has been modified as follows:

“However, one of the major disadvantages of using fission yeast could be the need to use much higher concentrations of drugs than normally used for mammalian cell cultures to achieve an inhibitory effect. This could probably be due to the poor permeability of certain drugs in fission yeast because of its thick cell wall (Benko et al. 2017; Pérez and Ribas 2004). A similar effect of toxicity might arise at much lower concentrations in other eukaryotic cells, such as human cells. Consistently, while sanguinarine and berberine are known to affect the eukaryotic microtubules at 10 μΜ – 20 μM concentrations (Lopus and Panda 2006; Wang et al. 2016; Raghav et al. 2017), morphological effects on yeast cells were observed only at concentrations > 100 μM. However, yeast microtubules were not affected by berberine and sanguinarine. Differences in membrane lipid profiles and MDR efflux pumps between yeasts and mammalian cells might also contribute to differential resistance to the drugs being tested (Balzi and Goffeau 1991). Conversely, an inhibitory effect in yeast cells may not necessarily translate into toxicity in a human cell. These and the permeability of drugs in yeast cells represent an important caveat in using such heterologous expression systems for the screening of compounds against target molecules.”

[Lines 498-513]

There are very few SaFtsZ-GFP dot structure in fig 5B, and this is inconsistent with the SaFtsZ-GFP dot structure in fig 4A. Fig 5D has the same issue compare to Fig 4Ci

RESPONSE: We had probably not made it very clear the experimental differences between Figure 4 and 5 (Figure 2 and 3 in the revised manuscript), which has led to this apparent inconsistency.

The strong nmt1 promoter (thiamine repressible) takes about 18 hours for full-induction in the absence of thiamine (Forsburg 1993 - https://doi.org/10.1093/nar/21.12.2955). We have utilised the medium strength nmt41 promoter in our studies and hence, in Figure 2, expression of FtsZ-GFP fusions were allowed for longer periods of time (22 – 24 hours) in the experiments concerning sanguinarine and berberine treatments.

This has been now clearly mentioned in the revised version of the manuscript in the results section (lines 196-199) as well as in figure legends.

In contrast the very few dot structures or polymers in Figure 3 (revised manuscript) is because of a shorter period of expression of FtsZ-GFP (12 – 14 hours in the absence of thiamine). The shorter period of expression time in these experiments allowed us to test if PC190723 indeed induced the polymerisation of FtsZ, at a stage when the control cells still exhibited diffuse fluorescence and had minimal FtsZ assembly. Thus, the cultures were allowed to express FtsZ for a shorter period of time and imaged in the case of experiments presented in Figure 3.

This has been now clearly mentioned in the results (lines 259-263) as well as in figure legends in the revised manuscript.

We hope that we have now made these experimental differences clear and provide more clarity. We have also included this information (hours of induction) in the figure panel.

The concentration of PC190723 the author used is 20 µg/ml, which is enough for disrupting FtsZ function, however according to the Sanguinarine and Berberine experiments, the author may use higher concentration of PC190723 to assess its toxicity to pombe cells. Same as Sanguinarine and Berberine, does PC190723 has effect on microtubule?

RESPONSE: As suggested by the reviewer, we have tested the effect of PC190723 at a higher concentration (140.6 µM) similar to that of Sanguinarine and Berberine. We did not find any morphological changes in yeast upon treatment with higher concentrations of PC190723. Also, the drug did not seem to affect the yeast microtubules. These have been now included in the results section and new images have been added in the figure (Supplementary Fig. S3).

The following lines have been added in the revised manuscript to the results section:

“Earlier studies had reported that PC190723 was non-toxic to eukaryotic cells, including budding yeast (Haydon et al. 2008). We further tested if PC190723 resulted in morphological defects in S. pombe, like sanguinarine and berberine, at higher concentrations. However, consistent with the earlier reports, PC190723 was inactive against S. pombe at both 56.2 μM and 140.6 μM and did not cause any morphological changes (Fig. 2H iv). Further, PC190723 did not disrupt the yeast microtubules at either of the concentrations (Fig. S3 A iv and B iv).”

[Lines 294-300]

The authors mentioned much higher concentrations of drugs than normally used for mammalian cell cultures have to be used for fission yeast. Is there any criterion for this?

RESPONSE: In the discussion section, we had mentioned that “Much higher concentrations of drugs than normally used for mammalian cell cultures have to be used for fission yeast probably due to permeability issues because of the presence of a thick cell wall (Benko 2017 - DOI: 10.1186/s13578-016-0131-5).

This has now been mentioned in the results as well in the revised manuscript.

“Although we did not observe any growth defect in yeast cells at lower concentrations of the drugs, earlier studies have suggested that yeast cells possibly require higher concentrations of drugs than used for mammalian cells due to the presence of the cell wall, which is particularly thick in S. pombe (Benko et al. 2017; Pérez and Ribas 2004). We thus explored the possibility of cell toxicity to yeast cells at higher concentrations of the drugs.”

[Lines 234-239]

The following lines in the discussion have been modified in the revised manuscript to read as – “However, one of the major disadvantages of using fission yeast could be the need to use much higher concentrations of drugs than normally used for mammalian cell cultures to achieve an inhibitory effect. This could probably be due to the poor permeability of certain drugs in fission yeast because of its thick cell wall (Benko et al. 2017; Pérez and Ribas 2004). A similar effect of toxicity might arise at much lower concentrations in other eukaryotic cells, such as human cells.”

[Lines 498-503]

Minor comments: 1. There are two units used for drug concentration µM for Sanguinarine and Berberine and µg/ml for PC190723, I think they should be consistent.

We have now used µM for all drugs.

Check the units (µM and µg/ml) italic in text and figure legend.

We have now used µM for all drugs and corrected the italics. We apologise for the erroneous usage of italics in the text for µM.

Reviewer #3 (Significance (Required)):

The authors provided a proof-of-concept assay for studying bacterial cytoskeleton proteins in yeast cells. This idea will facilitate people to investigate the bacterial cytoskeleton proteins and also find compounds targeting them without affecting the yeast cells. This study will provide different perspectives to people who study cell biology and secondary metabolites discovery.

We hope that we have satisfactorily addressed all the concerns raised by the reviewers in the revised manuscript.

Thanking you,

With Regards

Dr. Ramanujam Srinivasan

Dr. Pananghat Gayathri

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Referee #3

Evidence, reproducibility and clarity

Summary:

The authors established a proof-of-concept assay to investigate the bacterial cytoskeletal protein FtsZ in fission yeast, and this heterologous yeast system is useful for compounds identification targeting FtsZ. The authors used this system to understand the mechanism of FtsZ's resistance to drug PC190723.

Major comments:

1. From the study, the pombe seems to be a good system for investigating the bacterial cytoskeleton proteins and testing the drugs for them. However, to my knowledge it is not convincing that this is the proper system can be used to assessing the eukaryotic toxicity, since no toxicity to pombe does not mean no toxicity to human cells and vice versa.
2. From figure 4A to 4C, there seems no big difference of cell morphology between control and drug treatment, except for Berberine treatment of SaFtsZ-GFP. Under the low concentration of Sanguinarine (20 µM) and Berberine (53.791 µm), the FtsZ polymerization was disrupted and seems no effect on cell morphology. Why would the authors use much higher Sanguinarine (135.95 µM) and Berberine (134.45 µM) to prove there two drugs are toxic to pombe cells? Sanguinarine and Berberine are FtsZ disruption drugs, do these drugs have effect on microtubule?
3. There are very few SaFtsZ-GFP dot structure in fig 5B, and this is inconsistent with the SaFtsZ-GFP dot structure in fig 4A. Fig 5D has the same issue compare to Fig 4Ci
4. The concentration of PC190723 the author used is 20 µg/ml, which is enough for disrupting FtsZ function, however according to the Sanguinarine and Berberine experiments, the author may use higher concentration of PC190723 to assess its toxicity to pombe cells. Same as Sanguinarine and Berberine, does PC190723 has effect on microtubule?
5. The authors mentioned much higher concentrations of drugs than normally used for mammalian cell cultures have to be used for fission yeast. Is there any criterion for this?

Minor comments:

1. There are two units used for drug concentration µM for Sanguinarine and Berberine and µg/ml for PC190723, I think they should be consistent.
2. Check the units (µM and µg/ml) italic in text and figure legend.

Significance

The authors provided a proof-of-concept assay for studying bacterial cytoskeleton proteins in yeast cells. This idea will facilitate people to investigate the bacterial cytoskeleton proteins and also find compounds targeting them without affecting the yeast cells.<br /> This study will provide different perspectives to people who study cell biology and secondary metabolites discovery.

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Referee #2

Evidence, reproducibility and clarity

Dr. Srinivasin and colleagues previously developed a system where they expressed bacterial FtsZ in yeast and showed that it could assemble into polymers related to the Z rings. Here they develop this system further as a way to assay for drugs that may poison FtsZ, which would be candidates for new antibiotics. They test three drugs against three species of FtsZ. The results suggest that this system should be useful in screening new drugs that may target FtsZ. I would recommend publication after addressing a number of concerns and apparent contradictions.

Fig. 1 showing chemical formulas of the drugs, and Fig. 2 showing a schematic of the yeast expression system, are probably not needed.

The authors make a point that sanguinarine and berberine inhibit eukaryote cell morphology. In fact, what they show is that they affect yeast cell morphology. This may or may not extend to other eukaryotes. Also, other eukaryotic cells may be more sensitive to drugs than yeast. They should me more conservative in this claim that the system also screens for drugs effects on eukaryotes.

Fig. 3 has some new structural data that should be explored more quantitatively. My quick measurement gave 0.5 and 0.8 µm for the outside diameters of Ec and Sa rings. The spirals of Hp seem to be 0.8 µm outside diameter, similar to SA rings. These spirals may be related to those reported by Popp and by Andreu under certain buffer conditions. This should be explored and referenced.

But Fig. 4 presents a contradiction. Here the Hp control cells show long smooth polymers, not helical. This seems an important difference and needs to be addressed. Are the polymers sometimes straight and sometimes helical? After finishing the paper I see that in some experiments the HP is helical, and in others the polymers are straight and smooth. I think it would be important to determine what favors the two forms. If this remains a mystery, at least address it openly.

I am concerned that the quantitation of drug inhibition in Fig 4, 5 is flawed. Visually from 4A it looks like ~90-100% of control cells have polymers, and sang reduces polymers by 70% for Sa and Ec and 100% for Hp: this is based on the number of spots and filaments I see in Fig. 4 Aii. But the quantitation in D shows only 17-23% reduction for all three. These numbers were based on determining the fraction of cells that showed polymer (spots or lines) vs diffuse. It seems that cells are counted as containing polymer even if they had a great reduction in spots or lines, but still had a few. E.g., 4Aii Sa has 4 cells, two of them with no spots, one with only 2, and one with ~7, which totals ~1/3 the spots in control cells. Categorizing cells with only a couple of spots as polymerized, seems to be a poor way to quantitate. Would it not be better to count all spots in all cells, or measure the total length of line polymers, as a measure of inhibition.

Fig. 5 makes a convincing case that PC19 accelerates or enhances the polymerization of Sa and Hp. Fig. S2 shows that the structures of polymers are not changed when PC19 is added at 20 hrs, after polymers have already formed. It would have been nice to see for both 5A and S2A that the round spots had holes in the center, when imaged by SIM. Again the quantitation of cells as polymer vs diffuse seems ill suited, because it misses cells with a reduced number of spots.

Fig. 6 uses FRAP to show that PC reduces the dynamic exchange of Sa polymers by a factor of 3. It is remarkable to me that rapid exchange is not completely eliminated by PC. Regardless, it would be very important to reference the previous study of Adams..Errington 2011, where they showed the same thing for Foci in Bacillus. PC19 reduced the exchange from 3 to 10 s, but the foci were still very dynamic.

The analysis of the salt bridge as opposed to a single Arg or His being the cause of resistance to PC19 is an interesting addition to the study. In Fig. 8D some numbers do not agree between the caption and figure (R309/7; S226/7). The whole figure should be carefully checked.

I am not familiar with the Gram -ve and Gram +ve nomenclature. Why not simply gram- and gram+?

The Discussion is quite long largely because it repeats items from Results and Introduction. It is also redundant to hype the value of this system in both Introduction and Discussion; The Introduction should be sufficient. The Discussion should be pared down by eliminating repetition and focusing on relating results to previous literature, in particular items that have not been referenced previously in the paper. Also, I think we don't need the final "In summary" paragraph. That is already nicely presented in the Abstract.

The authors should probably provide references to other studies that have used yeast expression to study assembly of FtsZ. I am thinking in particular of papers from the Osteryoung lab looking at chloroplast FtsZ.

NO PAGE NUMBERS. Authors should be penalized a week delay for submitting a mss without page numbers.

Significance

This work should be of interest to the broad field of research on FtsZ. The authors present it as a new platform for assaying drugs targeting FtsZ, and researchers in this area will certainly be interested. It will also be of broader interest for the novel assay of assembly and exchange dynamics and how they may be modulated by small molecules.

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Referee #1

Evidence, reproducibility and clarity

Summary:

Srinivasan and co-workers developed an alternative screening method for defining the ability of FtsZ inhibitor to affect FtsZ polymerization. This alternative assay was defined considering the expertise of the authors on the topic, and they use Schizosaccharomyces pombe as a model for studying the effect of PC190723, sanguinarine and berberine on FtsZ assembly. The use of a heterologous expression system is useful for the evaluation of FtsZ coming from different strains, both Gram - and Gram +. The same model could gain insights also on the capability of FtsZ inhibitors to affect eukaryotic cell physiology. Finally, authors resulted also in suggesting a possible cause to suspected resistance to PC190723 from Gram - strains as E. coli.

Major comments:

• The conclusions are included in the discussion section and are quite convincing, for a general audience.
• In my opinion, the authors should define which could be the limits of their method, since no data on the possible weaknesses are reported.
• No additional experiments are required to support the claims.
• The suggested experiments could be quite easy to be realized for authors working in the microbiological field, and familiar with protein expression and purification, as well as bacteria and yeast growth.
• From my side, even if I am not so expert in microbiology and plasmid/protein purification, the methods presented could be reproduced with no significant doubt.
• Statistical analysis was done and seems to be adequate.

Minor comments:

• Prior studies should be deepened, especially for the state of art authors referred to. Additional paper, both reviews on the possible methods for evaluating FtsZ inhibition, as well as research papers on FtsZ inhibitors targeting E. coli and other Gram negative strains should be mentioned, since, in my opinion, these could move authors in changing a little bit the overall text of the manuscript.
• The whole text requires a deep check for grammar and word choice. Some sentences should be re-written since it is not so easy to understand their meaning. Figures are clear, even if I am not so convinced on the need of including Figure 1.

Significance

• In my opinion, the outcome coming from this work could move researchers in evaluating an alternative method for assessing FtsZ inhibition. Nevertheless, the actual state of art, a few reviews of the last years confirm this, already underlined a huge number of possible assays, both microbiological, biochemical, biophysical, physiological, or other. As a result, the authors did not result in convincing me about the importance of their methods, when compared to others. They may include some other possible assays and comment of the differences, pros and cons.
• As I mentioned before, there are a lot of reviews including the possible tests to perform for assessing FtsZ inhibition. A recent one was not cited, but, from my side, it should be mentioned (10.3390/antibiotics10030254). Moreover, I think authors should reconsidered novel research papers, in which researchers evaluated the reason behind the apparent inactivity of benzamide derivatives, similar to PC190723, towards Gram negative strains.
• Researchers working on FtsZ inhibitors could be interested in this paper, especially microbiologists.
• I specifically work on the design, synthesis and evaluation of the microbiological assays performed by others on my compounds.

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Reply to the reviewers

1. Description of the planned revisions

Suggested by reviewer 3:

1. • The authors showed the importance of the 3'UTR of Ldh in regulating Ldh translation under hypoxia, and they mention in the discussion that further investigation is needed to understand the nature of this regulation. Mutational analysis of the 3'UTR sequence could help to extend the paper and enhance its impact. *

*A mutational analysis of Ldh mRNA 3’UTR is currently under way and will be included in the revised version of the manuscript. *

• *

2.One obvious shortcoming of this paper is that the functions of the Ldh 3' UTR and or eIF4EHP are not connected by experimental tests. Experiments aimed at determining the functional relation of the 3' UTR and eIF4EHP could enhance the paper and deliver a more complete story about the mechanism of selective translation under hypoxia.

We believe that data presented in figure 4 do functionally connect eIF4EHP and Ldh 3’UTR since we show that a reporter mRNA containing the Ldh mRNA 3’UTR is specifically recruited on polysome in hypoxic control cells but not in eIF4EHP-depleted cells. However, we plan to further strengthen this demonstration by analyzing Ldh mRNA 3’UTR mutants in polysomes of WT or eIF4EHP-deficient cells.

• *

*3.The authors refer to human cell studies showing that HIF2a is involved in mRNA translation in hypoxic conditions. They mentioned in the discussion that Drosophila Sima has not been identified to interact with eIF4EHP. To test whether Sima regulates Ldh translation, the authors could test the involvement of Sima in experiments from Fig 6. *

As suggested, we plan to inhibit Sima expression by CRISPR/Cas9 in S2 cells and if viable, evaluate the recruitment of eiF4EHP on polysomes of hypoxic Sima-KO cells. The interaction of Ldh mRNA with eiF4EHP in absence of Sima could also be tested. However, the later experiment is most probably impossible as the transcriptional induction of Ldh by hypoxia is strongly reduced upon Sima depletion in S2 cells (Dekanty A et al 2010, Plos Genet 6(6) e1000994) and Drosophila larvae (Wang et al. 2016, ELife:e18126).

2.Description of the revisions that have already been incorporated in the transferred manuscript* *

Reviewer 1

• * *5.The authors should integrate the emerging concept of adaptive cap-binding translation factors in the discussion. It is still generally assumed that inhibition of eIF4E results automatically to cap-independent protein synthesis. The discovery that eIF3D, 4E2 and 4E3, amongst others, can initiate stimuli-specific translation should be discussed in the context of the work from this paper. *

The discussion now includes the emerging concept of diverse non-canonical cap-dependent translation mechanisms.

• *

Reviewer 2

Minor comments:

• • Similarly, in Fig 6C it would be good to show the equivalent CLIP data for 21% oxygen. Presumably, since eIF4HP is not in polysomes in the normoxic condition, there should be no enrichment (or little enrichment) for the Ldh mRNA. *

Fig. 6C has been revised to indicate more clearly that the data were obtained in cells under hypoxia. The method section has been modified to include a reference protocol. A statistical test has been included. Ldh mRNA is expressed at very low level in normoxic S2 cells and is strongly induced upon hypoxic exposure (>500 fold). Therefore, the comparison of eiF4EHP binding to Ldh mRNA between normoxic and hypoxic conditions is not relevant due to this strong difference in Ldh mRNA abundance.

• *

*For Fig 7B - it's a bit confusing to label the y-axis as "flies escaping" to mean flies that climb past a certain limit. I suggest relabeling the axis to something like "flies climbing past threshold". *

The proposed modification has been introduced in Fig.7B.

Reviewer 3

• *

• In Fig 3A-B, induction of LDH by hypoxia is almost completely blocked by eiF4EHP knockdown in fly heads but not in S2 cells. We wonder whether this indicates tissue specific regulation of LDH translation, such that there might be alternative cap-binding proteins in S2 cells. Please comment.*

Expression data from flyRNAi and our RNAseq experiments (de Toeuf et al, Scientific Reports, 2018) indicate that only eiF4E1, eiF4E6 and eiF4EHP are expressed in S2 cells in normoxia and hypoxia, all the other members (eIF4E3, 4, 5, 7) being very weakly or not expressed at all. We show that eiF4E6 KO does not impair Ldh mRNA association to polysomes in S2 under hypoxia (Fig.3E,F), thereby suggesting that this member of the family is not involved in Ldh mRNA hypoxic translation. Therefore, the difference in LDH suppression resulting from eiF4EHP KO in S2 versus fly heads cannot be explained by the involvement of alternative eiF4E family members in S2 cells*. *

• *

• In Fig 3E, the authors present results of puromycin incorporation with eIF4EHP KD under hypoxia. It is actually not clear what the function of eIF4EHP is under normoxic conditions. The authors shall include a control of puromycin incorporation with eIF4EHP KD under 21% O2. In addition, the authors should include statistical tests for the comparisons.*

• These data are now presented in fig. 3G. We have increased the number of replicates (now n=12) and, as suggested, we compared the incorporation of puromycin in normoxic and hypoxic condition. We observed a stronger reduction in puromycin incorporation in eIF4EHP KO cells as compared to controls when cells are exposed to hypoxic conditions. Our results therefore suggest that the positive effect of eiF4EHP on protein synthesis is predominant under hypoxic conditions. *

• *

• In Fig 4C, the authors did Western Blots to detect eIF4EHP, and find that there is protein signal under the condition with eIF4EHP KD + D. persimilis eIF4EHP overexpression. It is unclear whether the antibody detects both eIF4EHPs in Drosophila melanogaster and D. persimilis, or whether, alternatively, expressing the D. persimilis version induces cells to express endogenous eIF4EHP. Please comment. * The antibody detects both eiF4EHP, with DpeiF4EHP-V5 being slightly heavier. The difference of band intensity between WT+ DpeiF4EHP and KO + DpeiF4EHP most probably results from a difference of DpeiF4EHP construct transfection efficiency in the two cell lines. This difference of expression has no influence on results shown in Fig.4D,E.

The antibody used in Fig.4C and its cross-reactivity to dpeiF4EHP have been specified in the figure legend.

• *

• In Fig 7A, we don't see the point of including results of flies carrying balancers as control. Direct comparisons with mCherry-RNAi should suffice. Also, presenting the percentage of hatched embryos can be misleading. We would suggest the authors present the absolute numbers of embryos examined and indicate the number of larvae that hatched.*

We believe that our experimental protocol is valid and supports our conclusions. The presence of the balancer chromosome provides an internal control for each cross. *The balancer being transmitted in 50% of the embryos we can directly compare for each individual cross, after eclosion, the number of individuals bearing or not the dominant balancer marker. We have performed an additional experiment to increase the sample size for hypoxic conditions and we now provide a statistical analysis. *

• In addition (Fig 7) "birth" is not an appropriate term for insects. Please state whether the numbers indicated larvae "hatched" from eggs, or adult flies "eclosed" from pupae. Please use these terms in the text, figure and figure legends. *

These terms have been replaced in the revised manuscript.

• In Fig 6C, a statistical test is missing. *

A statistical test has been included*. *

• *

• The authors sometimes refer to knockdown as KO, please be accurate.*

This point has been revised. We define KO upon gene disruption by CRISPR/cas9 as performed in S2 cells and KD upon Knock down by RNAi as performed in flies.

Reviewer 4

1. • In human cells eIF4E2 (the eIF4EHP orthologue) can activate translation in hypoxia by forming a complex with HIF2alpha, RBM4, and eIF4G3. The paper states in the discussion that inhibition of the Drosophila eIF4G3 counterparts eIF4G2 and NAT1 does not affect translation under hypoxia. However, the data behind this important conclusion are not shown, nor are details given as to how eIF4G2 and NAT1 were 'inhibited', whether or not both were inhibited at the same time, and whether the fly RBM4 orthologue Lark was investigated. While the mechanism of how eIF4EHP activates translation must be different in Drosophila from that in human cells because of the absence of HIF2alpha, not much more can be concluded than that in the absence of explicit experimental data. *

We tested the role of eIF4G2 and NAT1 by CRISPR/cas9 inactivation independently in S2 cells under hypoxia. We did not observe any modification in LDH synthesis and Ldh mRNA distribution in polysomes. These experiments are mentioned in the discussion. We tested Lark KO in S2 and Lark KD in flies. We observed in both settings that Lark inactivation is lethal, thereby precluding the study of Lark in hypoxic translation by gene inactivation*. *

• Fig 3B shows a large increase in LDH levels under hypoxic conditions in eIF4EHP KO cells, which is inconsistent with the narrative description of this result and the paper's conclusions. Ratios of LDH in normoxic and hypoxic conditions for eIF4EHP KO and control cells should be quantitated from multiple experiments, compared, and tested for statistical significance. * *The results now shown in Fig.3C,D are crucial and are consistent with the main conclusion of the paper that eiF4EHP activates Ldh mRNA translation in a 3’UTR-dependent manner. *

*Ratios of LDH/Actin levels have now been measured in 7 independent experiments and tested for statistical significance. We observed a significant decrease of hypoxia induced LDH production in eIFEHP KO cell line as compared to Cas9 control cell line. *

• Statistical significance should also be shown for the data in Fig 3E. I note that the empty vector control reduces puromycin incorporation by quite a lot. *

*The experiment (now presented in fig. 3G) has been reproduced (from n=3 to n=6). A statistical analysis has been performed. The cell line transfected with the empty vector is the adequate control and reduction of puromycin incorporation in these cells in comparison to untransfected cells might results from cas9 expression. The figure was modified to present comparisons between KO and control cell lines both in hypoxic and normoxic conditions. *

• The number of survivors is very low for both control and eIF4EHP KD flies in Fig 7A. Are these comparisons statistically significant? *

This experiment was repeated to increase the number of flies in the hypoxic group. Statistical significance is now indicated and the legend was modified accordingly.

• *

• • It should be explained why eIF4E6 KO was included in Fig 3. *

This explanation is now included in the text. 2.

• I assume that 6% oxygen was used for the viability experiment in Fig 7A because the lower concentration of 1% used in all other experiments would be lethal to both control and KD flies. This reasoning should be made explicit. *

This is in fact the reason for using 6% oxygen. It is indicated in the revised manuscript.

3. There is a great deal of sloppiness about nomenclature. The FlyBase term for the molecule of interest is eIF4EHP. Within this manuscript I found it referred to as eIF4EHP, eiF4EHP, eIF4HP, eiF4HP, and ei4EHP. This requires correction. * The nomenclature has been revised according to FlyBase terms.*

• The references are also sloppy and presented in several different formats*.

The reference list has been revised and is now homogenously formatted.

2. Description of analyses that authors prefer not to carry out

Experiments suggested by Reviewer 1:

*1.The work focuses mainly on LDH. It would be a missed opportunity to show the effect of eIF4EHP in hypoxic Drosophila on puromycin incorporation. *

This experiment has been performed. However, puromycin incorporation is only detectable in normoxic larvae. Puromycin labeling is undetectable in hypoxic larvae or adult flies. This is most likely due to 2 distinct parameters that make the experiment irrelevant. First, translational activity is inhibited in hypoxia (the measured parameter) and second, the feeding activity of larvae and adults is also strongly reduced or totally stopped in hypoxia, therefore drastically reducing the uptake of puromycin compared to normoxic individuals and making the comparison of the 2 conditions (normoxia vs hypoxia) impossible.

*We decided not to further pursue this approach. *

*2.In mammalian cells, blocking de novo transcription does not affect protein accumulation of eIF4E2 translated mRNA, even if these mRNA do not increase during hypoxia. It would be important to silence sima and test if this could block increased translation of ldh or other target in hypoxic conditions. This would suggest that sima is both a transcription and translation factor that evolved in HIF1a and HIF2a, the latter being a hypoxic translation regulator. This can be compared to cells treated with a general transcription inhibitor. These experiments would broaden the impact of the work. *

*Here, reviewer 1 is most certainly referring to figure 1 of Uniacke et al. 2012 (Nature 486:126) where the authors observed that human EGFR protein accumulates in hypoxic human U87MG cells treated or not with actinomycin D in a HIF2-dependent manner. Supplementary Fig.3 from the same paper clearly shows that EGFR mRNA is constitutively present in U87MG independently of hypoxic exposure or actinomycin D treatment. The same type of experiment cannot be transposed in for LDH synthesis in Drosophila cells. Indeed, Ldh mRNA is strongly induced at the transcriptional level upon hypoxia. Therefore, inhibiting Sima expression or treating cells will Actinomycin D will block Ldh mRNA synthesis making it impossible to analyze further its translational status. *

*3.Fig 5C. It is unclear what are the conclusions of the authors on the lack of co-localization between poly(A)RNA and eIF4EHP. In principle, eIF4EHP should co-localize with poly(A) mRNA. Perhaps a proximity ligation assay should be done to clarify this question. The data shown in F5C is not convincing one way or the other and needs clear cut experiments. Idem for 5A and B. PLA would provide convincing results. *

*We observed a reduced puromycin incorporation in eIF4EHP KO cells under hypoxic conditions (Fig. 3E), revealing a decrease in protein synthesis activity in hypoxic cells depleted of eiF4EHP. Therefore, our observations support a model in which eiF4HP is promoting translation of specific mRNA targets under hypoxic conditions rather than acting as a global translational inhibitor. In this context, it is expected, as confirmed in figure 5C, that eiF4EHP will not colocalize with poly(A)+ mRNA in hypoxic S2 cells as most of these mRNA are not translated under these conditions (Fig1 A,B). The paragraph describing fig. 5C has been modified to clearly specify this point. *

• *

• Fig 6C is important but the data is hard to understand. First, the Y-axis is "enrichment relative to input". Is this hypoxia vs normoxia? Is the Y-axis log (probably)? The best would be to show that normoxia and hypoxia and see if eIF4EHP binds to ldh mRNA in both conditions perhaps acting as a translational repressor in normoxia and translational activator in hypoxia. Also, the authors could pool monosome and polysome fractions and see in which fractions eIF4EHP binds to Ldh mRNA. Finally, do the authors think that rpl32 remains associated with ldh mRNA in normoxia and hypoxia? *

*Fig.6C (linear Y-axis) shows that Ldh mRNA is specifically bound by eiF4EHP in S2 cells under hypoxia as it is immunoprecipitated with eiF4EHP in WT cells and not in eIF4EHP KO S2. The Ldh mRNA/eiF4EHP interaction is specific as rpl32 mRNA is not immunoprecipitated in the same conditions. *

*Ldh mRNA is barely expressed in normoxia, rendering the analysis of Ldh mRNA binding to eiF4EHP technically difficult and introducing a strong bias to the relative comparison of this association in hypoxia versus normoxia. *

Our data showing that Ldh mRNA is massively associated to polysomes under hypoxia (fig.1D,E) and that this association is strongly impaired in eiF4EHP KO cells (Fig. 3C,D) combined to Ldh mRNA direct association to eiF4EHP in hypoxia (Fig.6C) strongly support the role of eiF4EHP in Ldh mRNA translation. Association of Ldh mRNA to eiF4EHP in polysomes versus monosomes would not significantly contribute to our conclusions.

• *

Experiments suggested by Reviewer 2:

Minor comments:

• • Fig 2A - it would be good to show the equivalent luciferase assays at 21% oxygen, to test whether the elevated activity of the Ldh 3'UTR is something specific to the hypoxic condition, or whether this is always the case, also under non-stressed conditions.*

*The reporter gene assays of fig2A have been performed with luciferase reporters placed downstream of the Ldh gene promoter whose activity is very low under normoxia. This strategy was used to avoid luciferase expression and accumulation prior to exposure to hypoxia and potential masking of the Ldh 3’UTR effect on luciferase activity. The corresponding paragraphs have been modified to explicitly describe that reporter constructs used in this experiment are hypoxia-inducible. *

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Referee #4

Evidence, reproducibility and clarity

Summary:

The manuscript by Liang et al. shows that, while overall translation is reduced under hypoxic conditions, translation of Ldh mRNA substantially increases. This increase is demonstrated to depend upon the Ldh 3' UTR and the variant translation factor eIF4EHP. The manuscript further shows that eIF4EHP associates with polysomal fractions and with Ldh mRNA under hypoxic conditions and that it is enriched in cytoplasmic foci apparently distinct from stress granules and P bodies in hypoxia. Finally, the paper provides evidence that loss of eIF4EHP worsens the survival rate of flies in reduced oxygen conditions.

Major comments:

The experimental data largely support the conclusions that are drawn but I have some important reservations.

1. In human cells eIF4E2 (the eIF4EHP orthologue) can activate translation in hypoxia by forming a complex with HIF2alpha, RBM4, and eIF4G3. The paper states in the discussion that inhibition of the Drosophila eIF4G3 counterparts eIF4G2 and NAT1 does not affect translation under hypoxia. However, the data behind this important conclusion are not shown, nor are details given as to how eIF4G2 and NAT1 were 'inhibited', whether or not both were inhibited at the same time, and whether the fly RBM4 orthologue Lark was investigated. While the mechanism of how eIF4EHP activates translation must be different in Drosophila from that in human cells because of the absence of HIF2alpha, not much more can be concluded than that in the absence of explicit experimental data.

2. Fig 3B shows a large increase in LDH levels under hypoxic conditions in eIF4EHP KO cells, which is inconsistent with the narrative description of this result and the paper's conclusions. Ratios of LDH in normoxic and hypoxic conditions for eIF4EHP KO and control cells should be quantitated from multiple experiments, compared, and tested for statistical significance.

3. Statistical significance should also be shown for the data in Fig 3E. I note that the empty vector control reduces puromycin incorporation by quite a lot.

4. The number of survivors is very low for both control and eIF4EHP KD flies in Fig 7A. Are these comparisons statistically significant?

Minor comments:

1. It should be explained why eIF4E6 KO was included in Fig 3.

2. I assume that 6% oxygen was used for the viability experiment in Fig 7A because the lower concentration of 1% used in all other experiments would be lethal to both control and KD flies. This reasoning should be made explicit.

3. There is a great deal of sloppiness about nomenclature. The FlyBase term for the molecule of interest is eIF4EHP. Within this manuscript I found it referred to as eIF4EHP, eiF4EHP, eIF4HP, eiF4HP, and ei4EHP. This requires correction.

4. The references are also sloppy and presented in several different formats.

Significance

As mentioned above, it is established in human cells that the orthologue of eIF4EHP can activate translation under hypoxic conditions. So the basic result in this manuscript simply confirms that the same phenomenon occurs in flies. As mentioned in the previous section, in human cells eIF4EHP activates translation in a 3' UTR dependent manner as part of a complex containing HIF2alpha, RBM4, and eIF4G3. While a different mechanism is likely to exist in Drosophila, it is not elucidated by the experimental data presented in this paper. Therefore I find the work to be of limited significance.

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Referee #3

Evidence, reproducibility and clarity

Exposure to hypoxia induces dramatic metabolic changes in metazoan cells, and a major reprogramming of gene expression occurs to adapt to decreased energy production, such as global repression of protein synthesis, with exception of a select subset of genes whose functions are required during hypoxia. Inspired by work from a mammalian study, the authors of this manuscript investigated how a certain mRNA, in this case Ldh, is selectively translated under hypoxia in Drosophila melanogaster. The authors discovered that the 3'UTR of Ldh mediates its translational activation in hypoxia. Furthermore, they identify eIF4EHP as a critical component for this hypoxia induced translation, and show that its function is important for fly survival and development under hypoxic conditions. The authors present extensive, compelling results to support their conclusions. However, we still find this work can be improved in several aspects:

1. The authors showed the importance of the 3'UTR of Ldh in regulating Ldh translation under hypoxia, and they mention in the discussion that further investigation is needed to understand the nature of this regulation. Mutational analysis of the 3'UTR sequence could help to extend the paper and enhance its impact.

2. One obvious shortcoming of this paper is that the functions of the Ldh 3' UTR and o eIF4EHP are not connected by experimental tests. Experiments aimed at determining the functional relation of the 3' UTR and eIF4EHP could enhance the paper and deliver a more complete story about the mechanism of selective translation under hypoxia.

3. The authors refer to human cell studies showing that HIF2a is involved in mRNA translation in hypoxic conditions. They mentioned in the discussion that Drosophila Sima has not been identified to interact with eIF4EHP. To test whether Sima regulates to Ldh translation, the authors could test the involvement of Sima in experiments from Fig 6.

4. In Fig 3A-B, induction of LDH by hypoxia is almost completely blocked by eiF4EHP knockdown in fly heads but not in S2 cells. We wonder whether this indicates tissue specific regulation of LDH translation, such that there might be alternative cap-binding proteins in S2 cells. Please comment.

5. In Fig 3E, the authors present results of puromycin incorporation with eIF4EHP KD under hypoxia. It is actually not clear what the function of eIF4EHP is under normoxic conditions. The authors shall include a control of puromycin incorporation with eIF4EHP KD under 21% O2. In addition, the authors should include statistical tests for the comparisons.

6. In Fig 4C, the authors did Western Blots to detect eIF4EHP, and find that there is protein signal under the condition with eIF4EHP KD + D. persimilis eIF4EHP overexpression. It is unclear whether the antibody detects both eIF4EHPs in Drosophila melanogaster and D. persimilis, or whether, alternatively, expressing the D. persimilis version induces cells to express endogenous eIF4EHP. Please comment.

7. In Fig 7A, we don't see the point of including results of flies carrying balancers as control. Direct comparisons with mCherry-RNAi should suffice. Also, presenting the percentage of hatched embryos can be misleading. We would suggest the authors present the absolute numbers of embryos examined and indicate the number of larvae that hatched.

8. In addition (Fig 7) "birth" is not an appropriate term for insects. Please state whether the numbers indicated larvae "hatched" from eggs, or adult flies "eclosed" from pupae. Please use these terms in the text, figure and figure legends.

9. In Fig 6c, a statistical test is missing.

10. The authors sometimes refer to knockdown as KO, please be accurate.

Significance

Exposure to hypoxia induces dramatic metabolic changes in metazoan cells, and a major reprogramming of gene expression occurs to adapt to decreased energy production, such as global repression of protein synthesis, with exception of a select subset of genes whose functions are required during hypoxia. Inspired by work from a mammalian study, the authors of this manuscript investigated how a certain mRNA, in this case Ldh, is selectively translated under hypoxia in Drosophila melanogaster. The authors discovered that the 3'UTR of Ldh mediates its translational activation in hypoxia. Furthermore, they identify eIF4EHP as a critical component for this hypoxia induced translation, and show that its function is important for fly survival and development under hypoxic conditions. These results are unique and significant, and should be of interest to researchers who work on hypoxia, stress responses in general, and mRNA translation. As noted above (#1, #2) the paper could go further mechanistically, to determine the relative roles of the LDH 3' UTR and eIF4EHP. But it already has an extensive array of impressive translation data.

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Referee #2

Evidence, reproducibility and clarity

Summary:

When cells are stressed, such as during hypoxia, most of the canonical mRNA translation is shut off. Nonetheless, stress-response genes need to be translated. One such example is the lactate dehydrogenase (Ldh) mRNA, which encodes for an enzyme needed by cells to resist hypoxia. Therefore, these mRNA employ non-canonical mechanisms of translation to escape the general repression. Here, Liang et al. study the molecular mechanism how the Ldh mRNA is translated in Drosophila during hypoxia. They discover that the Ldh 3'UTR contains sequences that enable efficient translation. They identify the non-canonical initiation factor eIF4HP as a factor required for Ldh translation, and show that it binds the Ldh mRNA. Interestingly, they show that the ability of the Ldh 3'UTR to promote translation during hypoxia depends on eIF4HP. They go on to show that unlike other canonical translation initiation factors, during hypoxia eIF4HP remains in polysomes and does not form translationally-repressed aggregates such as stress granules or P-bodies. Finally, Liang et al show that eIF4HP is required in flies to efficiently resist hypoxia.

Major comments:

This is a very nice and solid study. Overall, I found the data and the conclusions very convincing. Also nice is how they cover the topic broadly, starting at the molecular level with the 3'UTR of the Ldh mRNA and ending with physiological assays such as resistance of flies to hypoxia. In particular I found the experiments presented in Fig 2C and 4A to be very nice, showing that the 3'UTR is responsible for the resistance to translation inhibition, and that this is mediated by eiF4EHP. I have only a few minor comments (below) to strengthen further the study, but I think it could be published also without these additional experiments.

Minor comments:

I have only two minor suggestions for experiments that would further strengthen the conclusions presented in the paper:

• Fig 2A - it would be good to show the equivalent luciferase assays at 21% oxygen, to test whether the elevated activity of the Ldh 3'UTR is something specific to the hypoxic condition, or whether this is always the case, also under non-stressed conditions.

• Similarly, in Fig 6C it would be good to show the equivalent CLIP data for 21% oxygen. Presumably, sinc eIF4HP is not in polysomes in the normoxic condition, there should be no enrichment (or little enrichment) for the Ldh mRNA.

• For Fig 7B - it's a bit confusing to label the y-axis as "flies escaping" to mean flies that climb past a certain limit. I suggest relabeling the axis to something like "flies climbing past threshold".

Significance

• The mechanisms of translation that one can read when opening a standard molecular biology textbook are the canonical mechanisms that take place in cells when they are not stressed. Cells, however, often experience stress. For instance, animals in the wild are exposed to heat or cold stress, cancer cells in a tumor are exposed to hypoxia, low amino acids, low sugar, etc. In such conditions, the canonical mRNA translation systems are shut down, and non-canononical mechanisms are remain (or are turned on). So it is a very interesting topic to understand how these non-canonical mRNA translation mechanisms function. This study contributes significantly to this topic by identifying the molecular mechanism how Ldh is translated during hypoxia. Ldh is an important gene because it is the last step of anaerobic glycolysis in animals, needed for cells to produce ATP when oxidative phosphorylation in mitochondria is incapable of functioning. Hence, understanding how the Ldh mRNA is translated is important for cell metabolism, cell viability, and organismal physiology. Furthermore, discovering that a non-canonical form of eIF4E, called eIF4EHP in Drosophila, or eIF4E2 in humans, is responsible for this translation is an important finding that opens up new avenues for future research, asking more broadly which parts of the translatome depend on this factor for their translation. The fact that eIF4EHP knockdown flies fare less well than control flies in response to hypoxia shows that eIF4EHP is playing an important role.

• I think these findings will be interesting for a broad audience studying mRNA translation, hypoxia, cell stress responses, cell and organismal metabolism, and organismal physiology.

• My expertise is in Drosophila development, mRNA translation and tissue growth.

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Referee #1

Evidence, reproducibility and clarity

The manuscript by Gueydan and collaborators examines the role of eiF4EHP in Drosophila. The major conclusion of the paper is that eIF4EHP believed to be a translation repressor in fact drives protein synthesis during hypoxia. The bulk of the study is focused on the lactate dehydrogenase (LDH) mRNA, a mRNA that undergoes efficient translation during hypoxia thereby going against the grain of the general protein synthesis inhibition by low oxygen tension. While the paper is somewhat confirmatory of work published by other groups that eIF4E2 (homolog of eIF4EHP) drives hypoxic translation, the work shown here is done in whole organism that adds what I consider to be another significant layer of evidence to the story. The paper also provides additional evidence that cells are equipped with multiple stimuli-specific cap-binding translation initiation factors that produce adaptive translatomes. The work is convincing, the paper well-written and the demonstration of eIF4EHP role in whole organism is important. Nonetheless, I do have a few comments that should be addressed to strengthen the conclusions of the authors.

1. The work focuses mainly of LDH. It would be a missed opportunity to show the effect of eIF4EHP in hypoxic Drosophila on puromycin incorporation. The work on S2 cells shown in Fig. 3E, while convincing, only reproduces what is already known. Puromycin incorporation in larvae.

2. In mammalian cells, blocking de novo transcription does not affect protein accumulation of eIF4E2 translated mRNA, even if these mRNA do not increase during hypoxia. It would be important to silence sima and test if this could block increased translation of ldh or other target in hypoxic conditions. This would suggest that sima is both a transcription and translation factor that evolved in HIF1a and HIF2a, the latter being a hypoxic translation regulator. This can be compared to cells treated with a general transcription inhibitor. These experiments would broaden the impact of the work

3. Fig 5C. It is unclear what are the conclusions of the authors are on the lack of co-localization between poly(A)RNA and eIF4EHP. In principle, eIF4EHP should co-localize with poly(A)mRNA. Perhaps a proximity ligation assay should be done to clarify this question. The data shown in F5C is not convincing one way or the other and needs clear cut experiments. Idem for 5A and B. PLA would provide convincing results.

4. Fig 6C is important but the data is hard to understand. First, the Y-axis is "enrichment relative to input". Is this hypoxia vs normoxia? Is the Y-axis log (probably)? The best would be to show that normoxia and hypoxia and see if eIF4EHP binds to ldh mRNA in both conditions perhaps acting as a translational repressor in normoxia and translational activator in hypoxia. Also, the authors could pool monosome and polysome factions and see in which fractions eIF4EHP binds to ldh mRNA. Finally, do the authors think that rpl32 remains associated with ldh mRNA in normoxia and hypoxia?

5. The authors should integrate the emerging concept of adaptive cap-binding translation factors in the discussion. It is still generally assumed that inhibition of eIF4E results automatically to cap-independent protein synthesis. The discovery that eIF3D, 4E2 and 4E3, amongst others, can initiate stimuli-specific translation should be discussed in the context of the work from this paper.

Significance

While the paper is somewhat confirmatory of work published by other groups that eIF4E2 (homolog of eIF4EHP) drives hypoxic translation, the work shown here is done in whole organism that adds what I consider to be another significant layer of evidence to the story

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Reply to the reviewers

Manuscript number: RC-2022-01680

Corresponding author(s): Woo Jae, Kim

1. General Statements The goal of this study is to provide the groundwork for future studies of genetically controlled neuronal regulation of ‘interval timing’ through the provision of a behavioral paradigm. Interval timing, or the sense of time in the seconds to hours range, is important in foraging, decision making, and learning in humans via activation of cortico-striatal circuits. Interval timing requires completely distinct brain processes from millisecond or circadian timing. In summary, interval timing allows us to subjectively sense the passage of physical time, allowing us to integrate action sequences, thoughts, and behavior, detect developing trends, and predict future consequences.

Many researchers have tried to figure out how animals, including humans, can estimate time intervals with such precision. However, most investigations have been conducted in the realm of psychology rather than biology thus far. Because the study of interval timing was limited in its ability to intervene in the human brain, many psychologists concentrated on developing convincing theoretical models to explain the known occurrence of interval timing.

To overcome the limits of studying interval timing in terms of genetic control, we have reported that the time investment strategy for mating in Drosophila males can be a suitable behavioral platform to genetically dissect the principle of brain circuit mechanism for interval timing. For example, we previously reported that males prolong their mating when they have previously been exposed to rivals (Kim, Jan & Jan, "Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals" Nature Neuroscience, 2012), and this behavior is regulated by visual stimuli, clock genes, and neuropeptide signaling in a subset of neurons (Kim, Jan & Jan, “A PDF/NPF Neuropeptide Signaling Circuitry of Male Drosophila melanogaster Controls Rival-Induced Prolonged Mating” Neuron, 2013).

Throughout their lives, all animals must make decisions in order to optimize their utility function. Male reproductive success is determined by how many sperms successfully fertilize an egg with a restricted number of investment resources. To optimize male reproductive fitness, a time investment strategy has been devised. As a consequence, we believe that the flexible responses of mating duration to different environmental contexts in Drosophila males might be an excellent model to investigate neural circuits for interval timing.

One of the most well-known features of human interval timing is the association of different sensory inputs with perception of time intervals, which influences our estimate of time intervals. Therefore, the first step toward comprehending the neural regulation of interval timing is to dissect the role that numerous sensory inputs play in determining the time duration. In this article, we discuss a different time-investment strategy adopted by males, called "Shorter-Mating-Duration" (SMD). According to our findings, male Drosophila with more sexual experience had shorter mating duration. During our investigation into the sensory inputs for SMD behavior, we found a small number of cells that express sugar receptors and pheromone receptors (ppk25 and ppk29) and thus transmit the multisensory information from females in order to generate memories of sexual experiences, which will determine the final decision of mating duration.

Our discovery of sensory integration mechanisms associated with complex behavioral trait in male Drosophila at the brain circuit and genetic network levels will be a huge step forward in our knowledge of interval timing behavior.

Description of the planned revisions

REVIEWER #1

1. • Overall I think this would be difficult for a general audience as the rationale and explanation of experiments needs to be clearer. * Answer: During the revision process, we will make our text more legible for wide audiences.

REVIEWER #2

1. • 'The knockdown of LUSH, an odorant-binding protein' Lush is expressed in trichoid sensilla in olfactory organs , from the beginning, they exclude the role of olfaction and later one they said 'suggesting that the expression of the pheromone sensing proteins LUSH and Snmp1 in Gr5a-positive gustatory neurons is critical for generating SMD behavior.' ? Therefore, I recommend If available, please provide a reference for the statement in the Methods section that the Orco1 line was "validated via electrophysiology", or include the electrophysiology data itself in this manuscript as supplementary figure. Ideally, positive behavioral controls for this line would also be included in the manuscript. * Answer: We value the reviewer's concern. LUSH has been discovered as an odorant-binding protein; nevertheless, current research suggests that LUSH may be involved in the sensing of additional pheromones to cVA, implying the presence of a lush-independent cVA detection mechanism [1]. Billeter et al. demonstrated in their paper that LUSH detects a female stimulatory chemical and modifies male mating latency (Fig. 2 of Billeter at al.). As Billeter et al. stated, our present understanding of pheromonal recognition in Drosophila is insufficient, and we concur. As a result, we attempted to validate the expression of Snmp1 in the male leg by experiments (Fig. 7I-J) performing sncRNA seq analysis on the Fly SCope dataset, as shown in Fig. 12. As demonstrated in Fig.12, Snmp1 and LUSH is higly expressed fly leg and wing system. Future study will look at the roles of Snmp1 and LUSH in female pheromone sensing, as well as PPK receptors.

Following the reviewer's advice, we will repeat the electrophysiologically validated Orco2 mutant phenotype with proper control and attach it when we submit the complete revision to the journal.

• What is this (GustDx6)? I suggest using Poxn mutant line. *

Answer: We value the reviewer's recommendation. We believe we have previously demonstrated that the Gr5a-mediated gustatory pathway is essential for the generation of sensory input for SMD behavior, but we will test the Poxn mutant and Poxn-RNAi to replace the GustDx6 mutant result.

Description of the revisions that have already been incorporated in the transferred manuscript

REVIEWER #1

2. My copy of this ms does not have page numbers or line numbers, this makes it extremely difficult to identify where I am making queries/ suggestions. I don't know whether this is a decision of the journal or authors, but please change this in the future.* Answer: We put page numbers and line numbers.

3. A general point, there is simply too much in this ms. It covers too much ground and so doesn't give proper descriptions, discuss the consequences of the data fully or integrate properly with existing literature. Quantity does not equal impact. *

Answer: We appreciate the reviewer's insight. We have previously separated this document from our original preprint [2] in response to a prior reviewer's advice; we believe we have included too much data, which may confuse readers. As a result, we will delete all of the mechanosensory/thermosensory receptor screening data from our present paper and write a second manuscript on sensory integration for the production of SMD behavior. We also removed the most of sncRNA seq data analysis except Fig.12 which confirms our finding in a single diagram.

• Results paragraph 1 says that white mutant background had no effect "unlike that of LMD behavior as reported previously", ignoring that there has been a contrary report that extension of mating duration after exposure to a rival does not involve visual cues and so is not affected by the white mutation (Bretman et al 2011 Curr Biol). *

Answer: We recognize that there is a conflicting report concerning white mutation on LMD behavior, however because we are now reporting SMD rather than LMD behavior, we deleted the statement comparing white mutant results to earlier reports, as shown below;

“thus suggesting that the effect of the white mutant genetic background was not evident.” (line 97)

• A general point in the methodology, it's not very helpful just to say "as in a previous study" without giving at least a brief idea of what that was (e.g. the explanation of egg counting procedures).

A "sperm depletion" assay is described in the results that I cannot find any methodology for. *

Answer: We thank the reviewer for allowing us to clarify our lacking methodologies for a better comprehension of our manuscript.

We included the egg counting procedure to the EXPERIMENTAL PROCEDURES section to further illustrate our approach of egg laying assay as below;

“In short, wild type females mated with naïve or experienced males were transferred to a fresh new vial and allowed to lay eggs for 24 hr at 25°C. After 24 hr of egg laying, number of eggs were counted under the stereomicroscope. After we count the number of eggs, we kept vials in 25°C incubator and counted the total number of progenies ecolsed from them.” (line 956-960)

We included “Sperm Depletion from Males” section in EXPERIMENTAL PROCEDURES as below;

“To deplete sperm from males, 40 virgin Defexel6234 females which lacks SPR and shows multiple mating with males (Yang 2009) were placed in a vial containing four CS males for indicated time (2 h, 4 h, 8 h, and 24 h).” (line 880)

• Was the "excessive mating" with SPR females actually observed, or inferred from previous work? Needs to be clear. In what way do virgins expressing fruitless behave like mated females? It is so unclear how all the evidence in this paragraph leads to the conclusion that both cues from females and successful copulation. Especially as in the next paragraph experience with feminized females (with which the focal males cannot copulate) elicits the response.

It might be helpful to combine the results into a table, so it is easy to see under which conditions males reduce mating duration. *

Answer: We modified the sentence describing SPR mutant female experiment and added references as below;

“Sexual experiences with sex peptide receptor (SPR) mutant females which exhibit a delayed post-mating response and multiple mating with males [3] had no additional effect on SMD (Fig. 2I).” (line 135)

We clarify in which extent, fru>UAS-mSP virgin females behave like mated females as below;

“Virgin females behave like mated females by expressing a membrane-bound version of male sex-peptide in fruitless-positive neurons, hence rejecting the male's copulation attempt.” (line 136)

In the instance of feminized males, we assume that these feminine males can give adequate signals for inducing SMD and eliminated the term "successful copulation" since we are unsure if males can copulate these feminized males or not, despite the fact that males can mount and mate with them (Fig. 2O-P).

Tables S1 and S2 describe the conditions, genotypes, and descriptions of an experiments illustrated in Fig. 2. We believe that these tables may assist general audiences in comprehending our experimental design.

• Why are no statistics reported in the results? Identifying sig diffs on figures is not sufficient. I'm very sceptical that "mating duration of males showed normal distribution" for all comparisons, but then it's also difficult to identify which were analysed in this way (if statistics were properly reported this would not be an issue). *

Answer: We described our statistical analysis with mating duration previously [4–7] and followed the statistical analysis of copulation duration assay reported by Crickmore et al., published in CELL (2013) and NEURON (2020) [8,9]. To further validate our statistical analysis, we added estimation statistics which focuses on the effect size of one's experiment/intervention, as opposed to significance testing [10]. We already described our statistical analysis in EXPERIMENTAL PROCEDURES section in details. We also described our statistical analysis for mating duration will be same in all other figures in the Fig.1 legend.

We appreciate the reviewer's recommendation that the normal distribution of our mating duration data be validated. As a consequence, we performed the normailty test with Graphpad prism and added the histogram and QQ plot results to Fig. S1M and N. Table S3 also contains the results of the normality and lognormality tests.

• Gr5a/ Gr66a mediate acceptance/ avoidance of what? Why would you hypothesise these in particular to be involved? *

Answer: We accidentally left out the citation for that phrase and updated it with Wang et al.'s CELL (2004) paper. Wang et al. wrote in their article about taste representations in the Drosophila brain, “Our behavioral studies reveal that Gr5a cells recognize sugars and mediate acceptance/attractive behaviors whereas Gr66a cells recognize bitter compounds and mediate avoidance…. This suggests that Gr5a cells may be “acceptance” cells rather than “sweet” cells…. Our expression and behavioral studies reveal that Gr5a marks cells that recognize sugars and mediate taste acceptance, whereas Gr66a marks cells that recognize bitter compounds and mediate avoidance.” [11]

As a result, we hypothesize that Gr5a and Gr66a-positive cells influence acceptance or avoidance of "taste." We also changed certain sentences to make them clearer, as seen below;

“Of the various gustatory receptors, Gr5a marks cells that recognize sugars and mediate taste acceptance, whereas Gr66a marks cells that recognizes bitter compounds and mediates avoidance.” (line 173)

• As Orco was not found to affect the behaviour, why test Or67d? *

Answer: We appreciate the reviewer bringing this to our attention. We omitted the Or67d result from the present manuscript to simplify it and make it easier for readers to grasp.

• "Mate guarding" suddenly appears in the modelling section. Can a difference of a couple of minutes in a mating duration of 15-20min really be considered mate guarding? A similar variation in response to rival males is not considered mate guarding, but is linked to adjustments in ejaculate expenditure (admittedly not in a very straight forward way). Surely in a system like this the benefits arise more from how many females the male can mate with in a given time? How does this model relate to any of the previous models of mate guarding?

In this section the work of Linklater et al 2007 is important, they showed progeny declined over successive matings, and related this to exhaustion of Acps rather than sperm. I would urge the authors to consider that what they observe does not necessarily have an adaptive explanation. *

Answer: We have defined “mate guarding” in the text now. The costs and benefits of mate guarding have been extensively studied in insects and demonstrated to shape the optimal mating duration of males. In our experiment, we cannot specify whether the shortened mating duration was caused by the adjustments in ejaculate expenditure or a shorted stay after the ejaculation. Instead, our model has a general assumption that the costs of mate guarding increase linearly at the same rate in both pre- and post-ejaculation periods, which is highlighted in the model text.

There exist many models for the optimal mating duration (earlier models include Grafen and Ridley, 1983. A model of mate guarding. J. Theor. Biol. 102: 549 – 567 [12]). While our model was not built upon a novel theoretical approach (it was built based on the classical Charnov’s marginal value theorem equation), our model was developed specifically for generating testable predictions for the observed SMD behaviors.

We have rephrased the text as follow;

“This model assumes that (i) the shortened (or prolonged) mating duration is controlled by males and shaped by a trade-off between the benefit of mate guarding (remaining with the female both before and after the sperm ejaculation) and opportunistic costs (e.g. searching for another mate).” (line 970)”

• I can't find a data accessibility statement. *

Answer: We added it in the manuscript.

• That said, a current grand challenge in understanding behaviour is discovering the mechanisms that enable individuals to respond plastically to changing environments. This speaks directly to that challenge. However, this behavioural observation is not novel, as claimed. Generally the idea of refractoriness is widely known, and specifically the reduction in mating duration over successive matings in D. melanogaster was shown by Linklater et al 2007 Evolution. Moreover, the time between exposure to females has been shown to be important. Linklater et al 2007 gave males mating attempts in quick succession and observed the decrease in mating duration, whereas given recovery time of 3 days, males either mate equally as long, or even longer across their life course (Bretman et al 2011 Proc B, Bretman et al 2013 Evolution). These papers should be discussed, and more broadly the work understood in the light of previous knowledge. The behaviour does not need to be novel for this manuscript to make a significant contribution to the field. *

Answer: We believe the reviewer highlighted relevant past research that examined the influence of female experiences on mating duration. We agree with the reviewer that SMD behavior does not have to be original in order to contribute significantly to the field. As a result, we examined past reports and updated the introduction as follows;

“It has been reported that previous sexual experience with females influences the mating duration of male D. melanogaster [15,16,34]; however, the neural circuits and physiology underlying this behavior have not been deeply investigated. Here, we report the sensory integration mechanisms by which sexually experienced males exhibit plastic behavior by limiting their investment in copulation time; we refer to this behavior as "shorter mating duration (SMD)."” (line 85)

• Both in the introduction and discussion the extended mating duration in response to rivals is raised. A great deal of work has been done on this plasticity and yet the way this is written implies just two papers from these authors (whilst referencing others elsewhere). *

Answer: We agree with the reviewer. In the introductory and discussion sections, we cited as many key publications explaining the plastic responses of male mating duration as we could.

__REVIEWER #2

__

1. • Summary: The submitted manuscript reports that Drosophila melanogaster males use information derived from their previous sexual experiences from multiple sensory inputs to optimize their investment in mating. They refer to this plasticity as 'shorter-mating duration (SMD)'. SMD requires sexually dimorphic taste neurons. They identified several neurons in the male foreleg and midleg that express specific sugar, pheromone and mechanosensory receptors. Unfortunately, several aspects of the study design and methods used are inappropriate. Although the statistical approaches used are appropriate, the results are questionable. The discussion and conclusions are therefore too speculative in my view and overstretch the implications of the results as presented. Below I explain each one of these concerns about the study design, methods and results in detail as follows.* Answer: We appreciate the reviewer's assessment, especially the statement that our statistical approaches were appropriate. We will revise our manuscript in response to the reviewer's suggestions.

2. The conclusions (as the authors point out) hinge on small (often extremely small) effect sizes. This is not an insurmountable problem, so long as the assays are robust across trials. Unfortunately, they are not-the variation in the baseline for control replicates is often as large as, or larger than, the effects from which the conclusions are derived. Given the extreme experimental challenges of small effect size combined with large intertrial variability, it is notable that the authors do not report any likely false negative or false positive data, as would be frequently expected under these conditions. One explanation for the reproducibility of statistical effect seen across many experiments despite these experimental hurdles is manipulation of sample size. The authors acknowledge the extreme variability in sample size offer seemingly harmless explanations, but a closer look shows how problematic this practice is. For example, see Figure 1 (I, J, L) there is a big different between naive and experience males? *

Answer: We value the reviewer's feedback. Several research have been conducted to investigate the mating duration of male fruit fly. For example, our lab [2,13–15] and others [13–30] have regularly reported that previous rival exposure increases male fruit fly mating duration. Bretman A et al. utilized 49-59 males in their studies to compare the variations in mating duration between circumstances. Crickmore et al. also reported the effect of mating duration differences caused by genetic or experimental modification [8]. They utilized 10-18 male flies in their study to compare the variations in mating duration across circumstances, as shown in Figs. 1G (n=15-18) and 2A (n=10-27). All of these findings indicate that our mating duration sample size is sufficient to examine the effect size variations between the naive and experienced conditions. To confirm our statistical analysis further, we incorporated estimate statistics, which focus on the effect size of one's experiment/intervention rather than significance tests [10]. We have already detailed our statistical analyses under the EXPERIMENTAL PROCEDURES section. We conducted hundreds of mating duration assays using this configuration and confirmed that all of our results are reproducible in a blind test. As a result, we believe our mating duration assay has been validated by other groups' findings, several analytic tools, and numerous blind tests conducted by us. We appreciate the reviewers' concerns, but our data meets the reproducibility requirements.

• I am not sure if you keep using the same control with different experiments (that is okay if those exp is done in the same time) as in figure 1 B, I,J,K,L.But I don't think you did Fig 1B in the same time with Fig 1I, J, K,L. *

Answer: We appreciate the reviewer's feedback. Yes, all of our tests comparing the differences in mating duration between naive and experienced conditions were conducted under the same conditions and at the same time. We replaced Fig.1B with new data (n=49-51) obtained lately in a new lab in China. As previously stated, SMD behavior could be reproduced by the same Canton S genotype in different locations by different experimenters.

• It will be clear if you mention in the text how much reduction in percent happened in copulation duration when the males had previous sexual experience? *

Answer: We appreciate the reviewer’s suggestion and added in the manuscript as follow;

“We found that the mating duration of various wild-type and w1118 naïve males are significantly longer (wild type 15.7~15.8%, w1118 12.4%) than that of sexually experienced males (Fig. 1B-D, Fig. S1A)” (line 99)

• 'Drosophila simulans, the sibling species of D. melanogaster also exhibits SMD, thus suggesting that SMD is conserved between close species of D. melanogaster (Fig. S1B).'. If you want come with this conclusion, you need to test D. erecta, D. sechelia and D. yakuba. *

Answer: We appreciate the reviewer's feedback. We removed the D. simulans data because it is not required for the conclusion of this manuscript. In future research, we will look on the conservation of SMD behavior between species.

• The authors mention that Gr66a is salt. This is not 100% correct. GR66a is expressed in many bitter sensing neurons and is required for the physiological and behavioral responses to many bitter compounds. check this reference DOI:https://doi.org/10.1016/j.cub.2019.11.005. *

Answer: We made the following changes and cited the article reviewer's suggestion.

“Of the various gustatory receptors, Gr5a marks cells that recognize sugars and mediate taste acceptance, whereas Gr66a marks cells that recognizes bitter compounds and mediates avoidance (Wang et al, 2004; Dweck & Carlson, 2020).” (line 180)

• Drosophila melanogaster mating duration is between 21- 23 mins. I never saw copulation duration in normal condition (control) 10-15 mins as in figure fig 2E, Fig 7 C,E,F, Fig 8 E and fig 12 G . To the best of my knowledge, of all of the papers on copulation duration, the only one that ascribes a shortened duration to manipulations of the female is Rideout...Goodwin Nature Neuroscience 2010, who argue that this shortening results from markedly increased female activity/agitation during mating, leading the male to terminate early. *

Answer: We appreciate the reviewer's feedback. Copulation duration in Drosophila melanogaster male is extremely variable and has been reported to be approximately 20 minutes. However, as other groups documented, male copulation duration can range from 10-15 minutes depending on sperm completion (Fig. 1a-c of Bretman A et al.) [30] and genetic background (Fig. 1C, Fig. 2E, Fig. 5D, and Fig. 7A and E of Crickmore et al) [8]. And, as previously stated, males dominate copulation duration [8,30], not females, and we always utilized the same genotype of females for mating duration experiment. As a result, we believe that these rather short mating duration outcomes are the product of a distinct genetic background. Because we employed the same genotype of males while altering the female experience condition, we believe our mating duration results are all equivalent and comparable.

• In some experiments, the authors test very few number of replicates which is not convinced me to their conclusion as example Fig 2F and Fig 12 E. Why you test 100, 103 replicates in this exp fig 10 F? How you compare 47 replicates against 9 replicates in fig S10 I? *

Answer: We appreciate the reviewer's input. As we previously stated in response to Reviewer Question 2, the n number of males exhibited in Figs. 2F and 12E is statistically significant. To corroborate findings with replication, we examined 100, 103 duplicates of Fig. 10F, which represents pyx-RNAi screening results. The results of Fig. S10I are screening data, and we cannot rule out the possibility that TrpA1 knockdown in Gr5a neurons affects the mating success of sexually experienced males. We only placed it there because it was screening results and the differences between naive and experienced conditions were substantial despite the small sample size. However, we deleted Fig. 10F and Fig. S10I data from the current paper in response to Reviewer #1's advice, thus it will not be an issue for the manuscript's conclusion.

• 'Next, to decipher whether DEG/NaC channel-expressing pheromone sensing neurons require the function of OBP, we expressed lush-RNAi using ppk23-, ppk25- and ppk29-GAL4 drivers to knockdown LUSH in each channel-expressing neuron. The knockdown of LUSH in ppk25- and ppk29-GAL4 labeled cells, but not in ppk23-GAL4 labeled cells, led to a disturbance in SMD behavior, thus suggesting that LUSH functions in ppk25- and ppk29-positive neurons to detect pheromones and elicit SMD behavior (Fig. 9G-I). The knockdown of SNMP1 in ppk29-GAL4- labeled neurons also inhibited SMD behavior (Fig. 9J), thus suggesting that SNMP1 also functions in ppk29-positive neurons to induce SMD behavior.' What about ppk25? **

*

Answer: As indicated by the reviewer, we included ppk25-GAL4/snmp1-RNAi data in Fig. S9I, indicating that snmp1 expression in ppk25-positive cells is similarly implicated in SMD behavior.

• There are no page or line numbers throughout the ms! *

Answer: We included page and line numbers.

• The use of subheadings in the results section makes reading much easier.*

Answer: We added subheadings in the results section.

• 'We found that the mating duration of various wild-type and w 1118 naïve males are significantly longer than that of sexually experienced males (Fig. 1B-D, Fig. S1A)' . I think you should change various wild type to CS and WT Berlin as in legend and figure 1B,C .*

Answer: The revised sentence is as follows:

“We found that the mating duration of Canton S, WT-Berlin, Oregon-R, and w1118 naïve males are significantly longer (wild type 15.7~15.8%, w1118 12.4%) than that of sexually experienced males (Fig. 1B-D, Fig. S1A)” (line 102)

• Suggested exp , Fig S1E-H , they might test 2,6, 12 hours males separation from females to test exactly when this behavior change over time. *

Answer: We value the reviewer's recommendation. As seen in Fig. S4B of Kim et al., we have previously conducted experiments for examining the memory circuit of SMD [6]. Briefly, the male with a shorter mating duration recovers completely after 12 to 24 hours of isolation from females. As we are currently preparing the memory section of the SMD study, this information will be included in a future manuscript.

• General comment in figures, you could remove the common y axis as example in figure 1 B,C,D , difference between means and mating duration. *

Answer: We welcome the reviewer's idea, however in this situation we believe that the y axis of each data set is independent from one another and will thus retain the originals. We feel this would be more useful for the general audiences.

• You might move the number of replicates to the legend. *

Answer: We appreciate the reviewer's idea, however we feel that adding more information to the graphic will aid the general audience in comprehending our statistics.

• Latin name should be italic as example Drosophila simulans.*

Answer: We fixed it.

Description of analyses that authors prefer not to carry out

N/A

References

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2. Kim WJ, Lee SG, Schweizer J, Auge A-C, Jan LY, Jan YN. Sexually experienced male Drosophila melanogaster uses gustatory-to-neuropeptide integrative circuits to reduce time investment for mating. Biorxiv. 2016; 088724. doi:10.1101/088724
3. Yang C, Rumpf S, Xiang Y, Gordon MD, Song W, Jan LY, et al. Control of the Postmating Behavioral Switch in Drosophila Females by Internal Sensory Neurons. Neuron. 2009;61: 519–526. doi:10.1016/j.neuron.2008.12.021
4. Kim WJ, Jan LY, Jan YN. Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals. Nat Neurosci. 2012;15: 876–883. doi:10.1038/nn.3104
5. Kim WJ, Jan LY, Jan YN. A PDF/NPF Neuropeptide Signaling Circuitry of Male Drosophila melanogaster Controls Rival-Induced Prolonged Mating. Neuron. 2013;80: 1190–1205. doi:10.1016/j.neuron.2013.09.034
6. Kim WJ, Lee SG, Auge A-C, Jan LY, Jan YN. Sexually satiated male uses gustatory-to-neuropeptide integrative circuits to reduce time investment for mating. Biorxiv. 2016; 088724. doi:10.1101/088724
7. Wong K, Schweizer J, Nguyen K-NH, Atieh S, Kim WJ. Neuropeptide relay between SIFa signaling controls the experience-dependent mating duration of male Drosophila. Biorxiv. 2019; 819045. doi:10.1101/819045
8. Crickmore MA, Vosshall LB. Opposing Dopaminergic and GABAergic Neurons Control the Duration and Persistence of Copulation in Drosophila. Cell. 2013;155: 881–893. doi:10.1016/j.cell.2013.09.055
9. Thornquist SC, Langer K, Zhang SX, Rogulja D, Crickmore MA. CaMKII Measures the Passage of Time to Coordinate Behavior and Motivational State. Neuron. 2020;105: 334-345.e9. doi:10.1016/j.neuron.2019.10.018
10. Claridge-Chang A, Assam PN. Estimation statistics should replace significance testing. Nat Methods. 2016;13: 108–109. doi:10.1038/nmeth.3729
11. Wang Z, Singhvi A, Kong P, Scott K. Taste Representations in the Drosophila Brain. Cell. 2004;117: 981–991. doi:10.1016/j.cell.2004.06.011
12. Grafen A, Ridley M. A model of mate guarding. J Theor Biol. 1983;102: 549–567. doi:10.1016/0022-5193(83)90390-9
13. Kim WJ, Jan LY, Jan YN. A PDF/NPF Neuropeptide Signaling Circuitry of Male Drosophila melanogaster Controls Rival-Induced Prolonged Mating. Neuron. 2013;80: 1190–1205. doi:10.1016/j.neuron.2013.09.034
14. Kim WJ, Jan LY, Jan YN. Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals. Nat Neurosci. 2012;15: 876–883. doi:10.1038/nn.3104
15. Wong K, Schweizer J, Nguyen K-NH, Atieh S, Kim WJ. Neuropeptide relay between SIFa signaling controls the experience-dependent mating duration of male Drosophila. Biorxiv. 2019; 819045. doi:10.1101/819045
16. Bretman A, Fricke C, Chapman T. Plastic responses of male Drosophila melanogaster to the level of sperm competition increase male reproductive fitness. Proc Royal Soc B Biological Sci. 2009;276: 1705–1711. doi:10.1098/rspb.2008.1878
17. Bretman A, Westmancoat JD, Chapman T. Male control of mating duration following exposure to rivals in fruitflies. J Insect Physiol. 2013;59: 824–827. doi:10.1016/j.jinsphys.2013.05.011
18. Bretman A, Gage MJG, Chapman T. Quick-change artists: male plastic behavioural responses to rivals. Trends Ecol Evol. 2011;26: 467–473. doi:10.1016/j.tree.2011.05.002
19. Lizé A, Doff RJ, Smaller EA, Lewis Z, Hurst GDD. Perception of male–male competition influences Drosophila copulation behaviour even in species where females rarely remate. Biol Letters. 2012;8: 35–38. doi:10.1098/rsbl.2011.0544
20. Rouse J, Bretman A. Exposure time to rivals and sensory cues affect how quickly males respond to changes in sperm competition threat. Anim Behav. 2016;122: 1–8. doi:10.1016/j.anbehav.2016.09.011
21. Bretman A, Fricke C, Hetherington P, Stone R, Chapman T. Exposure to rivals and plastic responses to sperm competition in Drosophila melanogaster. Behav Ecol. 2010;21: 317–321. doi:10.1093/beheco/arp189
22. Rouse J, Watkinson K, Bretman A. Flexible memory controls sperm competition responses in male Drosophila melanogaster. Proc Royal Soc B Biological Sci. 2018;285: 20180619. doi:10.1098/rspb.2018.0619
23. Maguire CP, Lizé A, Price TAR. Assessment of Rival Males through the Use of Multiple Sensory Cues in the Fruitfly Drosophila pseudoobscura. Plos One. 2015;10: e0123058. doi:10.1371/journal.pone.0123058
24. Bretman A, Westmancoat JD, Gage MJG, Chapman T. COSTS AND BENEFITS OF LIFETIME EXPOSURE TO MATING RIVALS IN MALE DROSOPHILA MELANOGASTER. Evolution. 2013;67: 2413–2422. doi:10.1111/evo.12125
25. Bretman A, Fricke C, Westmancoat JD, Chapman T. Effect of competitive cues on reproductive morphology and behavioral plasticity in male fruitflies. Behav Ecol. 2016;27: 452–461. doi:10.1093/beheco/arv170
26. Price TAR, Lizé A, Marcello M, Bretman A. Experience of mating rivals causes males to modulate sperm transfer in the fly Drosophila pseudoobscura. J Insect Physiol. 2012;58: 1669–1675. doi:10.1016/j.jinsphys.2012.10.008
27. Bretman A, Westmancoat JD, Gage MJG, Chapman T. Males Use Multiple, Redundant Cues to Detect Mating Rivals. Curr Biol. 2011;21: 617–622. doi:10.1016/j.cub.2011.03.008
28. Fowler EK, Leigh S, Rostant WG, Thomas A, Bretman A, Chapman T. Memory of social experience affects female fecundity via perception of fly deposits. Bmc Biol. 2022;20: 244. doi:10.1186/s12915-022-01438-5
29. Dore AA, Rostant WG, Bretman A, Chapman T. Plastic male mating behavior evolves in response to the competitive environment*. Evolution. 2021;75: 101–115. doi:10.1111/evo.14089
30. Bretman A, Fricke C, Chapman T. Plastic responses of male Drosophila melanogaster to the level of sperm competition increase male reproductive fitness. Proc Royal Soc B Biological Sci. 2009;276: 1705–1711. doi:10.1098/rspb.2008.1878

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Referee #2

Evidence, reproducibility and clarity

Summary:

The submitted manuscript reports that Drosophila melanogaster males use information derived from their previous sexual experiences from multiple sensory inputs to optimize their investment in mating. They refer to this plasticity as 'shorter-mating duration (SMD)'. SMD requires sexually dimorphic taste neurons. They identified several neurons in the male foreleg and midleg that express specific sugar, pheromone and mechanosensory receptors. Unfortunately, several aspects of the study design and methods used are inappropriate. Although the statistical approaches used are appropriate, the results are questionable. The discussion and conclusions are therefore too speculative in my view and overstretch the implications of the results as presented. Below I explain each one of these concerns about the study design, methods and results in detail as follows.

Major comments:

1. The conclusions (as the authors point out) hinge on small (often extremely small) effect sizes. This is not an insurmountable problem, so long as the assays are robust across trials. Unfortunately, they are not-the variation in the baseline for control replicates is often as large as, or larger than, the effects from which the conclusions are derived. Given the extreme experimental challenges of small effect size combined with large intertrial variability, it is notable that the authors do not report any likely false negative or false positive data, as would be frequently expected under these conditions. One explanation for the reproducibility of statistical effect seen across many experiments despite these experimental hurdles is manipulation of sample size. The authors acknowledge the extreme variability in sample size offer seemingly harmless explanations, but a closer look shows how problematic this practice is. For example, see Figure 1 (I, J, L) there is a big different between naive and experience males?
2. I am not sure if you keep using the same control with different experiments (that is okay if those exp is done in the same time) as in figure 1 B, I,J,K,L.But I don't think you did Fig 1B in the same time with Fig 1I, J, K,L.
3. It will be clear if you mention in the text how much reduction in percent happened in copulation duration when the males had previous sexual experience?
4. 'Drosophila simulans, the sibling species of D. melanogaster also exhibits SMD, thus suggesting that SMD is conserved between close species of D. melanogaster (Fig. S1B).'. If you want come with this conclusion, you need to test D. erecta, D. sechelia and D. yakuba.
5. The authors mention that Gr66a is salt. This is not 100% correct. GR66a is expressed in many bitter sensing neurons and is required for the physiological and behavioral responses to many bitter compounds. check this reference DOI:https://doi.org/10.1016/j.cub.2019.11.005.
6. Drosophila melanogaster mating duration is between 21- 23 mins. I never saw copulation duration in normal condition (control) 10-15 mins as in figure fig 2E, Fig 7 C,E,F, Fig 8 E and fig 12 G . To the best of my knowledge, of all of the papers on copulation duration, the only one that ascribes a shortened duration to manipulations of the female is Rideout...Goodwin Nature Neuroscience 2010, who argue that this shortening results from markedly increased female activity/agitation during mating, leading the male to terminate early.
7. In some experiments, the authors test very few number of replicates which is not convinced me to their conclusion as example Fig 2F and Fig 12 E
8. Why you test 100, 103 replicates in this exp fig 10 F?
9. How you compare 47 replicates against 9 replicates in fig S10 I?
10. 'The knockdown of LUSH, an odorant-binding protein' Lush is expressed in trichoid sensilla in olfactory organs , from the beginning, they exclude the role of olfaction and later one they said 'suggesting that the expression of the pheromone sensing proteins LUSH and Snmp1 in Gr5a-positive gustatory neurons is critical for generating SMD behavior.' ? Therefore, I recommend If available, please provide a reference for the statement in the Methods section that the Orco1 line was "validated via electrophysiology", or include the electrophysiology data itself in this manuscript as supplementary figure. Ideally, positive behavioral controls for this line would also be included in the manuscript.
11. 'Next, to decipher whether DEG/NaC channel-expressing pheromone sensing neurons require the function of OBP, we expressed lush-RNAi using ppk23-, ppk25- and ppk29-GAL4 drivers to knockdown LUSH in each channel-expressing neuron. The knockdown of LUSH in ppk25- and ppk29-GAL4 labeled cells, but not in ppk23-GAL4 labeled cells, led to a disturbance in SMD behavior, thus suggesting that LUSH functions in ppk25- and ppk29-positive neurons to detect pheromones and elicit SMD behavior (Fig. 9G-I). The knockdown of SNMP1 in ppk29-GAL4- labeled neurons also inhibited SMD behavior (Fig. 9J), thus suggesting that SNMP1 also functions in ppk29-positive neurons to induce SMD behavior.' What about ppk25?

Minor comments:

1. There are no page or line numbers throughout the ms!
2. The use of subheadings in the results section makes reading much easier.
3. 'We found that the mating duration of various wild-type and w 1118 naïve males are significantly longer than that of sexually experienced males (Fig. 1B-D, Fig. S1A)' . I think you should change various wild type to CS and WT Berlin as in legend and figure 1B,C .
4. Suggested exp , Fig S1E-H , they might test 2,6, 12 hours males separation from females to test exactly when this behavior change over time.
5. What is this (GustDx6)? I suggest using Poxn mutant line.
6. General comment in figures, you could remove the common y axis as example in figure 1 B,C,D , difference between means and mating duration.
7. You might move the number of replicates to the legend.
8. Latin name should be italic as example Drosophila simulans.

Referees cross-commenting

I found the comments of the other reviewer reasonable and fair. I agree that the time for fixing all these comments is about six months.

Significance

The idea of the work is interesting, but the design of experiments in some places is inappropriate (see above). The discussion mainly depends on doi: 10.1016/j.neuron.2013.09.034. I study chemoreception and sexual communication in Drosophila and insect vectors of human disease.

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Referee #1

Evidence, reproducibility and clarity

My copy of this ms does not have page numbers or line numbers, this makes it extremely difficult to identify where I am making queries/ suggestions. I don't know whether this is a decision of the journal or authors, but please change this in the future.

Overall I think this would be difficult for a general audience as the rationale and explanation of experiments needs to be clearer.

Results paragraph 1 says that white mutant background had no effect "unlike that of LMD behavior as reported previously", ignoring that there has been a contrary report that extension of mating duration after exposure to a rival does not involve visual cues and so is not affected by the white mutation (Bretman et al 2011 Curr Biol).

A general point in the methodology, it's not very helpful just to say "as in a previous study" without giving at least a brief idea of what that was (e.g. the explanation of egg counting procedures).

A "sperm depletion" assay is described in the results that I cannot find any methodology for.

Was the "excessive mating" with SPR females actually observed, or inferred from previous work? Needs to be clear. In what way do virgins expressing fruitless behave like mated females? It is so unclear how all the evidence in this paragraph leads to the conclusion that both cues from females and successful copulation. Especially as in the next paragraph experience with feminized females (with which the focal males cannot copulate) elicits the response.

It might be helpful to combine the results into a table, so it is easy to see under which conditions males reduce mating duration.

Why are no statistics reported in the results? Identifying sig diffs on figures is not sufficient. I'm very sceptical that "mating duration of males showed normal distribution" for all comparisons, but then it's also difficult to identify which were analysed in this way (if statistics were properly reported this would not be an issue).

Gr5a/ Gr66a mediate acceptance/ avoidance of what? Why would you hypothesise these in particular to be involved?

As Orco was not found to affect the behaviour, why test Or67d?

"Mate guarding" suddenly appears in the modelling section. Can a difference of a couple of minutes in a mating duration of 15-20min really be considered mate guarding? A similar variation in response to rival males is not considered mate guarding, but is linked to adjustments in ejaculate expenditure (admittedly not in a very straight forward way). Surely in a system like this the benefits arise more from how many females the male can mate with in a given time? How does this model relate to any of the previous models of mate guarding?

In this section the work of Linklater et al 2007 is important, they showed progeny declined over successive matings, and related this to exhaustion of Acps rather than sperm. I would urge the authors to consider that what they observe does not necessarily have an adaptive explanation.

I can't find a data accessibility statement.

Significance

A general point, there is simply too much in this ms. It covers too much ground and so doesn't give proper descriptions, discuss the consequences of the data fully or integrate properly with existing literature. Quantity does not equal impact.

That said, a current grand challenge in understanding behaviour is discovering the mechanisms that enable individuals to respond plastically to changing environments. This speaks directly to that challenge.

However, this behavioural observation is not novel, as claimed. Generally the idea of refractoriness is widely known, and specifically the reduction in mating duration over successive matings in D. melanogaster was shown by Linklater et al 2007 Evolution. Moreover, the time between exposure to females has been shown to be important. Linklater et al 2007 gave males mating attempts in quick succession and observed the decrease in mating duration, whereas given recovery time of 3 days, males either mate equally as long, or even longer across their life course (Bretman et al 2011 Proc B, Bretman et al 2013 Evolution). These papers should be discussed, and more broadly the work understood in the light of previous knowledge. The behaviour does not need to be novel for this manuscript to make a significant contribution to the field.

Both in the introduction and discussion the extended mating duration in response to rivals is raised. A great deal of work has been done on this plasticity and yet the way this is written implies just two papers from these authors (whilst referencing others elsewhere).

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Reply to the reviewers

Reviewer #1:

Minor edits

1. Line 91. Is a bit misleading to say "many other vibrios" possess T3SS. This conveys that this is perhaps the majority, but T3SS in vibrios is at best 50/50. I think best just to delete this sentence.

We deleted this comment, as suggested.

1. Revised to "Thus, in this study, we set out to..." Since the entire paragraph starts with "recent study" I missed that this was summary of new data rather than preview of new results.

The sentence was revised as suggested.

1. Line 503. Correct "xxx-584" or more detail on what this means.

We Thank the reviewer for pointing out this typo._ This refers to the deletion made in tie1, in the region corresponding to nucleotides 485-584 of this gene. The text was corrected accordingly.

1. Line 603. Salmonella should be italicized.

Corrected.

1. The labelling of the figures is pretty complicated with the long genetic designations. Is it reasonable to for example name the ∆vprh/∆hns1 strain with an abbreviation (such as ∆VH)? Or instead create a strain name, common used approaches would be HC## (for Hadar Cohen) or TAU# for Tel Aviv University. If you go this route, be sure to update the strain list. The current method can be followed, the figures are just complicated.

We thank the reviewer for raising this concern. We acknowledge the difficulty in following the many different strains and mutations. Nevertheless, after considering the proposed modifications to the strain names, we believe that they will not add much clarity, and may even cause some confusion. Therefore, we respectfully decided to keep the current nomenclature in place.

Reviewer #2:

Minor edits

1. The authors used a hyperactive T6SS (HNS mutant) to investigate its toxicity. Would the authors be able to use a wild type strain to reproduce the function of T6SS?

We have yet to reveal the external cues that lead to full activation of T6SS3 in vitro. Therefore, in the current study we used genetic tools, such as hns deletion or Ats3 over-expression, to monitor the effect of this system on immune cells. We will dissect the activating conditions in future studies, but we believe that the use of genetic tools should not affect the validity of the results in the current study, nor their timely publication.

1. The authors showed that Tie1 and Tie2 are secreted by T6SS3. It is important to show if they are actually delivered into the host cells during infection. Otherwise it is hard to conclude that they are truly effectors. The primary concern is the lack of in vivo studies to show that Tie1 and Tie2 are actually effectors that play a role in activation of NLRP3 inflammasome._

We present 3 pieces of evidence that, when taken together, support the conclusion that Tie1 and Tie2 are T6SS3 effectors: 1) the proteins are secreted in a T6SS3-dependent manner; 2) their deletion does not hamper overall T6SS3 activity; and 3) their deletion causes the same loss of NLRP3-mediated inflammasome activation and pyroptosis as does inactivation of T6SS3 by deletion of its structural component, tssL3. Although we agree with the reviewer that directly showing delivery of Tie1 and Tie2 into host cells will further strengthen our conclusion, such experiments are quite challenging and difficult to interpret, especially with T6SS effectors that can use diverse mechanisms for secretion through the system. This point was also noted by reviewer #3: “…I believe they were suggesting to demonstrate secretion in host cells. Although this would be nice, it is non-standard and technically not feasible. These types of experiments require genetically fusing the effector with either an enzymatic moiety (e.g. Beta lactamase) or fragment of split GFP. Although such approaches have been previously performed, they often result in either blocked or aberrant secretion due to the presence of the added fragment."

Regarding the reviewer’s comment on the lack of in vivo studies: we agree that these are extremely important, yet they are beyond the scope of the current work, as concurred by reviewers #1 and #3:

Reviewer#1 with regard to Reviewer#2: "I don't think mouse (or aquatic animal) studies are essential for this study. The work contributes nicely to our understanding molecular mechanisms of this T6SS system. As noted in my review, there are many additional lines of study that can be pursued from this work, including animal studies, but this should not preclude publication of this work that is itself an intact unit."

Reviewer#3 regarding reviewer #1's comment on Reviewer#2: "I don't believe that reviewer #2 was suggesting to perform mouse or aquatic animal studies by suggesting in vivo demonstration of secretion…”

Reviewer #3:

Major comments:

1. If the authors believe that GSDME partially compensates in the absence of GSDMD, have they infected a GSDME/GSDMD double knockouts to see if there is an additive effect?

Indeed, this is a very interesting and specific question for the cell death field. We do not currently possess such a GSDME/GSDMD double knockout mouse, and generating one will be a long endeavor. Since its absence does not diminish the importance or the conclusions of the current work, we think that it should not warrant a delay in publication. We do plan to address this question in future studies.

1. It is clear that Ats3 regulates T6SS3, but not the T6SS1; however, there no evidence suggesting that Atg3 does not regulate other gene clusters. For example, have the authors performed RNA seq to compare the transcriptomes of WT and an Ats3 mutant? If not, the authors should refrain using the words "specific activation".

We thank the reviewer for this important note. Indeed, we lack additional data indicating that Ats3’s effect is indeed restricted only to T6SS3. Therefore, we modified the text accordingly and removed mentions of specific T6SS3 activation.

1. In figure 6B, it's unclear why the bacteria infecting cytochalasin D-treated cells grow more than the T6SS3 mutants in the absence of cytochalasin D.

The difference probably stems from the fact that phagocytosis, the major mechanisms by which BMDMs kill bacteria, is hampered in the presence of cytochalasin D, thus allowing bacteria to grow more than when the BMDMs phagocytose them. The results show that in the absence of cytochalasin D, an active T6SS3 counteracts the killing effect by BMDMs with functional phagocytosis.

Minor comments:

1. Figure 1A and other secretion assays: The Western blots include loading control (LC) blots. These are non-standard, non-informative, and not required with the inclusion of the western blots on the "cells" fraction. I would suggest removing these as they may confuse the reader.

We respectfully disagree. Loading controls are standard in bacterial secretion assays, and they are important since they confirm comparable loading and allow proper analysis of the results, especially since we aim to determine whether certain mutations affect the expression of T6SS components. Notably, some groups choose to blot for a cytoplasmic protein (e.g., RpoB in Allsop et al., PNAS, 2017; Liang et al., PLoS Pathogens, 2021) instead of showing overall loaded proteins, as shown in our figures.

1. Line 503: "xxx" should reflect the actual nucleotide nubmers_

We thank the reviewer for pointing out this typo._ This refers to the deletion made in tie1, in the region corresponding to nucleotides 485-584 of this gene. The text was corrected accordingly.

1. Since V. proteolyticus is an aquatic pathogen, have the authors tried to infect corals, fish, and crustaceans (or derived cells) with WT and effector mutants?

This is an interesting point, and indeed we are setting up such systems and we plan to perform such experiments in the future as part of follow up projects. However, these in vivo studies are beyond the scope of the current manuscript, as also noted by the reviewer in the cross-consultation comments: “…my previous comment on infecting aquatic animals or cells derived from them is non-standard and not necessary…”

1. Are the targeted host proteins in this study (performed with murine BMDM) conserved in the natural hosts for V. proteolyticus?

We hypothesize that the conservation is not in the pathway components that are activated upon infection, but rather in the ability of the host cell to sense danger (i.e., to sense the effect of T6SS3 effectors on the host cell or one of its components), which is the role of the NLRP3 inflammasome in mammalian cells. It is well documented that major differences in immune mechanisms exist between mammals and the potential natural marine hosts of V. proteolyticus (e.g., corals, arthropods, and fish); therefore, the conservation at the protein level is low. Nevertheless, basic signaling pathways, such as programed cell death, are conserved between the different phyla. For example, a caspase-1 homolog which was found in arthropods (Chu, B. et al. PLoS One (2014). doi:10.1371/journal.pone.0085343) probably induces an apoptotic-like cell death mechanism, similar to apoptosis in C. elegans. We now provide further discussion on this point in the text (lines 648-659).

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Referee #3

Evidence, reproducibility and clarity

Bacterial type VI secretion systems (T6SSs) are best-characterized in their ability to drive interbacterial competition; however, several systems target host cells and are important for pathogen-host interactions. Based on the presence of T6SS in almost a quarter of sequenced Gram-negative bacterial genomes, including many well-known pathogens, the T6SS is an understudied pathway that is worthy of further inspection. This manuscript by Cohen et al. investigates the role three T6SSs in Vibrio proteolyticus by 1) identifying two of these systems play a role in targeting host cells, 2) characterizing the host pyroptotic pathways targeted by these systems, and 3) identifying two secreted toxins responsible for the host response. Furthermore, this manuscript identifies a specific transcriptional regulator of the T6SS3 and suggests that the host cell responds with an alternative/compensatory pathway upon inhibition of the canonical pyroptosis pathway.

Major comments:

• If the authors believe that GSDME partially compensates in the absence of GSDMD, have they infected a GSDME / GSDMD double knockouts to see if there is an additive effect?
• It is clear that Ats3 regulates T6SS3, but not the T6SS1; however, there no evidence suggesting that Atg3 does not regulate other gene clusters. For example, have the authors performed RNA seq to compare the transcriptomes of WT and an Ats3 mutant? If not, the authors should refrain using the words "specific activation".
• In figure 6B, it's unclear why the bacteria infecting cytochalasin D-treated cells grow more than the T6SS3 mutants in the absence of cytochalasin D.

Minor comments:

• Figure 1A and other secretion assays: The Western blots include loading control (LC) blots. These are non-standard, non-informative, and not required with the inclusion of the the western blots on the "cells" fraction. I would suggest removing these as they may confuse the reader.
• Line 503: "xxx" should reflect the actual nucleotide nubmers
• Since V. proteolyticus is an aquatic pathogen, have the authors tried to infect corals, fish, and crustaceans (or derived cells) with WT and effector mutants?
• Are the targeted host proteins in this study (performed with murine BMDM) conserved in the natural hosts for V. proteolyticus?

Referees cross-commenting

Regarding Reviewer #1's comment on 2022-07-26, I don't believe that reviewer #2 was suggesting to perform mouse or aquatic animal studies by suggesting in vivo demonstration of secretion. (At least, I hope they weren't.) I believe they were suggesting to demonstrate secretion in host cells. Although this would be nice, it is non-standard and technically not feasible. These types of experiments require genetically fusing the effector with either an enzymatic moiety (e.g. Beta lactamase) or fragment of split GFP. Although such approaches have been previously performed, they often result in either blocked or aberrant secretion due to the presence of the added fragment.

Likewise, my previous comment on infecting aquatic animals or cells derived from them is non-standard and not necessary. It would however be interesting to note (either through a supplementary figure or in the text) if the targeted host proteins are conserved between mice and aquatic animals.

Significance

This manuscript provides both novel and innovative details of a previously uncharacterized secretion system. The interested audience includes readers who are interested in host-targeted secretion systems, bacterial effectors, and mechanisms of inflammasome activation. As with any key discovery, there are many avenues of investigation that will follow from this study.

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Referee #2

Evidence, reproducibility and clarity

The authors used a hyperactive T6SS (HNS mutant) to investigate its toxicity. Would the authors be able to use a wild type strain to reproduce the function of T6SS?<br /> The authors showed that Tie1 and Tie2 are secreted by T6SS3. It is important to show if they are actually delivered into the host cells during infection. Otherwise it is hard to conclude that they are truly effectors.<br /> The primary concern is the lack of in vivo studies to show that Tie1 and Tie2 are actually effectors that play a role in activation of NLRP3 inflammasome.

Significance

The authors demonstrated in vitro that T6SS of Vibrio plays an important role in inflammasome activation. This is potentially important as this may suggest that T6SS may be a virulence factor. However, as mentioned above, it is important to show in vivo data to demonstrate this.

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Referee #1

Evidence, reproducibility and clarity

This paper by Cohen et al described discovery of the function of novel genes in the T6SS operon of Vibrio proteolyticus, a Vibrio isolated from corals. V. proteolyticus also impacts other sea animals. The T6SS3 in particular is found to kill eukarytoic phagocytic cells following engulfment of bacteria into the phagocyte. This strategy of killing phagocytic cells following entry has been shown for other Vibrios. The net goal is protection of the population by the bystander effector. The study first shows that deletion of H-NS (a global negative regulator) stimulates T6SS facilitating ease of work by pushing the system to great cell killing. This allowed them to probe the mechanism of cell death and reveal it as NLRP3 dependent, capase 1 dependent pyroptosis via pore formation by Gasdermin D. Activation of the inflammasome is also linked to cleavage and release of IL-1beta. When GSDMD is absent, there was a slower cell killing by GSDME via capsase 3 activation. The stimulation of this system is additive by two newly recognized T6SS effectors Tie1 and Tie2.

The study is complete, the experiments are well conducted and well controlled. The experiments show reproducibility. The manuscript text is clear, Overall. I suggest no changes in the results or experiments and suggest only a few minor edits of the text.

Minor edits

Line 91. Is a bit misleading to say "many other vibrios" possess T3SS. This conveys that this is perhaps the majority, but T3SS in vibrios is at best 50/50. I think best just to delete this sentence.

Line 102. Revised to "Thus, in this study, we set out to..." Since the entire paragraph starts with "recent study" I missed that this was summary of new data rather than preview of new results.

Line 503. Correct "xxx-584" or more detail on what this means.

Line 603. Salmonella should be italicized.

Figures. The labelling of the figures is pretty complicated with the long genetic designations. Is it reasonable to for example name the ∆vprh/∆hns1 strain with an abbreviation (such as ∆VH)? Or instead create a strain name, common used approaches would be HC## (for Hadar Cohen) or TAU# for Tel Aviv University. If you go this route, be sure to update the strain list. The current method can be followed, the figures are just complicated.

Referees cross-commenting

With regard to Reviewer#2, I don't think mouse (or aquatic animal) studies are essential for this study. The work contributes nicely to our understanding molecular mechanisms of this T6SS system. As noted in my review, there are many additional lines of study that can be pursued from this work, including animal studies, but this should not preclude publication of this work that is itself an intact unit.

Significance

The work is significant in that it links T6SS to a eukaryotic killing system and discovers novel details regarding the mechanisms of death, that may impact our knowledge of other Vibrio T6SS (including V. cholerae) that also target eukaryotic cell actin. There are remaining questions that could be probed, but these are in my opinion major studies that would easily themselves comprise new papers if done properly and thus are not essential for this paper. These include the struture and biochemical activity of Tie1 and Tie2 and the mechanism of caspase-8 independent activation of caspase-3 to then cleave GSDME. Why NLRP3 is required for capase 3 activation is also an open question. I look forward to following this work for some time to come. The authors have revealed very interesting effectors and interesting cell biological process that will merit multiple years and multiple manuscripts to unravel. This work will be of interest to the community interested in bacterial toxin systems (microbial pathogenesis), the bacterial effector mechanism field (biochemistry and cell biology), and the inflammasome activation field (immune systems). The work will be of interest (with essentially no modification) directed at these fields of interest.

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Reply to the reviewers

Reviewer #1:

Major comments:

In general, the data support the conclusions. I cannot comment on the atomistic simulation experiment as it is outside of my expertise. I had some difficulties interpreting Figure 2 as the contrast in the colour panels made it difficult to assess the different staining patterns. I would recommend changing the blue to cyan for easier visibility. While I agree that there are some differences between Fig 2F and Fig 2G it is not simple for the non-expert to distinguish the gonadal mesoderm from the somatic mesoderm. I think the enlarged panels could do with also showing the overlap in staining, or at least a tracing of the different cell populations so that the gonadal mesoderm can be clearly defined. Please also add some scale bars to the figure. Figure 3 demonstrates clear differences in gonad morphology between male and female mutants but the contrast in the colour panels A-G could also be improved. Panels H-J are very clear.

Response: As suggested by Referees 1 and 3 we have modified the colour channels in all figures. We have also enlarged the figures taking away the uninformative region and focused around the enlarged gonads and added scale bars. For Fig 2F-G, we have added a close up of the region of interest both in colour and in black and white. These changes have increased the contrast and facilitate the data interpretation to non-expert readers.

The rescue experiment in Figure 4 is clearly presented but could the DLC3 mutants in the graph (panel b) please be named similarly to the schematic proteins shown in panel a.

Response: We have changed the names to maintain nomenclature uniformity.

I found the difference between the RhoGAP domain mutants and the StART domain mutants of Cv-c to be clearly defined, and correlate with DLC3 function. This is a very interesting result that indicates multiple molecular functions for the Cv-c /DLC family.

Response: The methods are well described, statistics adequate and the data well described._

Minor Comments:

My only suggestion for the text is to provide a more through description of the StART domain in the introduction.

Response: We have included the following paragraph in the introduction describing the StART domain:

“This family of proteins share different domains: besides the Rho GTPase Activating Protein domain (GAP), they present a protein-protein interacting Sterile Alpha Motif (SAM) at the N terminal end and a Steroidogenic Acute Regulatory protein (StAR)-related lipid transfer (StART) domain at the C terminal. StART domains have been shown in other proteins to be involved in lipid interaction, protein localization and function.”

Reviewer #2:

My only issue with the present study is to how well the present experimental findings in Drosophila translate to humans. As far as I can tell the present studies show that inactivating mutations in Cv-c in Drosophila result in failure of germ cell enclosure by somatic cells into the testis, resulting in sterility. In humans, and in experimental mouse transgenic lines, it has been well established that absence of germ cells does not of itself lead to failure of testis differentiation and onward development, nor does it lead automatically to sex reversal or impairment of masculinization. For the latter to occur, there must be impairment/failure of fetal Leydig cell function such that insufficient androgen is produced to effect genital/bodywide masculinization. Obviously, this will happen if no testis forms as appears to be the case in the new human DLC3 mutant reported in the present manuscript (although detail on this is unfortunately lacking). This appears to be different to the previous published DLC3/STARD8 mutant sisters, in whom the phenotype appears to reflect failure of steroidogenesis. Is the proposal that DLC3/STARD8 plays a role in both testis differentiation and in Leydig cell function (steroidogenesis) or is this due to different DLC3 genes? I think the authors need to address these key issues in their discussion, if only to highlight that there are at present many gaps in our understanding.

The reviewer says:

“As far as I can tell the present studies show that inactivating mutations in Cv-c in Drosophila result in failure of germ cell enclosure by somatic cells into the testis, resulting in sterility.”

Response: This sentence does not represent the spirit of our findings accurately and this probably reflects the fact that we stressed the interaction between somatic mesodermal cells and germ cells in Drosophila which probably concealed that the main defects in Cv-c mutants are caused by the abnormal interaction of the mesodermal cells with germ cells but also among themselves. Our study provides insights about a new conserved pathway required in the mesodermal cells for the maintenance of an already formed testis, and only indirectly can be considered to deal with sterility. We show that Cv-c is required in the mesodermal cells for the correct maintenance of the testis structure, that when it fails leads to the testis dysgenesis which, among other defects, releases the germ cells. We show that in the absence of Cv-c function in the testis, the mesodermal pigment cells do not form a continuous layer around the testis and the ECM surrounding the testis breaks. We also show that the interstitial gonadal cells fail to ensheath the germ cells and as a result of all these the germ cells become dispersed. These perturbations can be partially corrected by expression in the testis mesoderm of human DLC3 or Drosophila Cv-c that in both cases require a functional StART domain. Thus, our results suggest that Cv-c/DLC3 have a fundamental function on the mesodermal testis cells that has been conserved. These results indicate that, as in Drosophila, the primary cause for the gonadal dysgenesis in DLC3 human patients is due to the abnormal maintenance of the testis mesoderm cells, which include both Sertoli and Leydig cells. Thus, our proposal is that DLC3/STARD8 plays a role in testis maintenance through its function in mesodermal cells which will probably affects both Sertoli and Leydig cell function.

To clarify the issue raised by the referee we have modified both, the introduction and the discussion to highlight that although humans and Drosophila diverged millions of years ago there are similarities regarding gonad stabilisation.

We have modified the introduction to clarify this issue:

“Gonadogenesis can be subdivided into three stages: specification of precursor germ cells, directional migration towards the somatic gonadal precursors and gonad compaction. In mammals, somatic cells, i.e. Sertoli cells in male and Granulosa cells in females, play a central role in sex determination with the germ cells differentiating into sperms or oocytes depending on their somatic mesoderm environment. In humans, Primordial Germ Cells (PGCs) are formed near the allantois during gastrulation around the 4th gestational week (GW) and migrate to the genital ridge where they form the anlage necessary for gonadal development (GW5-6). Somatic mesodermal cells are required for both PGCs migration and the formation of a proper gonad. Once PGCs reach their destination, the somatic gonadal cells join them (around GW 7-8 in males, GW10 in females) and provide a suitable environment for survival and self-renewal until gamete differentiation {Jemc, 2011 #413}. Thus, mutations in genes regulating somatic Sertoli and Granulosa support cell function in humans are often associated with complete or partial gonadal dysgenesis in both sexes and sex reversal in males {Zarkower, 2021 #430; Knower, 2011 #418; Brunello, 2021 #399}. Other mesodermal cells, the Leydig cells, also play an important role in the testis by being the primary source of testosterone and other androgens and maintaining secondary sexual characteristics.”

Also we have added a paragraph in the discussion to emphasize this argument:

“We show that in the absence of Cv-c function in the testis, the mesodermal pigment cells do not form a continuous layer around the testis and the ECM surrounding the testis breaks. We also show that the interstitial gonadal cells fail to ensheath the germ cells and as a result of all these the germ cells become dispersed from the testis. These perturbations can be partially corrected by expression in the testis mesoderm of human DLC3 or Drosophila Cv-c that in both cases require a functional StART domain. Thus, our results suggest that Cv-c/DLC3 have a fundamental function on the mesodermal testis cells that has been conserved. These results indicate that, as in Drosophila, the primary cause for the gonadal dysgenesis in DLC3 human patients is due to the abnormal maintenance of the testis mesoderm cells, which include both Sertoli and Leydig cells”.

I would also suggest that the authors highlight another potentially more important spin-off from such studies, namely that understanding of the regulation of DLC3/STARD8 genes, and what might perturb their expression/action would appear to present a whole new area for exploration in relation to testicular dysgenesis/masculinization disorders.

Response: We have modified the last part of the discussion to introduce referee 2’s suggestion:

“Our work points to DLC3/Cv-c as a novel gene required specifically in testis formation. Adding DLC3 to the list of genes involved in 46X,Y complete dysgenesis opens up a new avenue to analyse the molecular and cellular mechanisms behind these disorders that could help in diagnosis and the development of future treatments”.

Reviewer #3 :

Major comments:

1. This study has shown the expression pattern of cv-c and the consequence of cv-c mutation on different aspects of gonad development. However, one major comment is there is no quantification of the expression levels as well as the scoring of the mutant phenotypes.
2. In Figure 2, for instance, I recommend that the authors display the quantification of the fluorescence intensity of the cv-c expression under all circumstances (in situ hybridization as well as protein-trap based GFP expression) to better depict the differences among the male vs female gonad.

Response: We don’t think quantifying the stainings will add much to the results. We believe that the changes performed increasing the images’ contrast and their amplification are sufficient to illustrate our statement about cv-c being expressed in testis but not in ovaries.

1. In Figure 3, the authors show the different gonad developmental defects associated with the cv-c mutation. Specifically, the authors show that the gonad mesoderm cells are displaced with the pigment cells failing to ensheath the germ cells. In addition, the authors also suggest that there is an increased frequency of germ cell blebbing, an indication of migratory activity. However, there is no quantification of these findings. I think the authors should display a quantitative estimation of % of the mutant gonad depicting these phenotypes vs the normal gonad to have a perspective of how penetrant the phenotypes are.

Response: As referee suggested, we have quantified bleb phenotype. The results are presented in figure 3, panel J.

1. In Figure 4, the authors attempt to rescue the Cv-C mutation linked gonadal defects by overexpressing different Cv-C protein variants. The rescue experiments are not very clear. The graph shows the % of normal testes under different genotypic combinations. It is not very clear what the authors mean by normal (in what context)? Since the mutation results in different defects of gonad development, I think recommend that represent the rescue in terms of these defects. It would be interesting to see for instance, what happens to the blebbing or germ cell ensheathment phenoype upon rescue. How many % of testes show the rescue as compared to cv-c mutants?

Response: The percentages are quantified considering if the testes have any germ cell outside the gonad. We have added a line to clarify this point in the figure legend: “…quantified as encapsulated gonads with all germ cells inside the testis as assessed by Fisher-test”.

Nevertheless, we are going to quantify the number of ECM breaks and show the results in the reviewed manuscript.

1. Did the authors try cell-specific depletion of cv-c and examined the consequence on gonad development?

Response: cv-c mutants are embryonic lethal because of Cv-c’s widespread requirement on various embryonic tissues during development. Induction of FRT clones in the embryonic testis mesoderm was unsuccessful because of the low number of divisions during embryogenesis. We also tried to knock down cv-c expression with 3 different RNAi lines. Unfortunately, overexpression of these RNAi with different testis Gal4 drivers did not decrease cv-c mRNA levels significantly in the mesoderm or in other tissues where cv-c is expressed. Despite these experiments unsatisfactory outcome, our finding that cv-c is expressed in the testis mesoderm cells, and the fact that we can rescue the testis phenotypes by expressing Cv-c with gonadal mesodermal specific Gal4 lines supports a testis mesoderm requirement of cv-c for its gonadal function.

1. Another major concern is the lack of mechanistic insight of cv-c. For example, how does loss of cv-c result in gonadal dysgenesis? The authors suggested that StART domains regulate via lipid binding. The authors could examine if StART domain function is dependent on lipid-mediated interactions.

Response: We agree with the referee that the molecular characterisation of the StART-mediated GAP-independent Cv-c function we have uncovered in this work is a very interesting finding that should be addressed by future work. However, such biochemical characterisation requires a complex approach to distinguish between the already known StART function regulating the GAP activity shown before (Sotillos Scientific Reports) and the new GAP-independent function we describe in the testes that falls beyond this work.

The central point of this manuscript is the demonstration that both DLC3/Cv-c are involved in male gonad formation, an important conserved function for both of them that had been overlooked by previous publication. Thus, DLC3 should be considered a new gene to be analysed in the future when studying gonadal dysgenesis. A second important point raised by our work is the demonstration that DLC3/Cv-c can perform RhoGAP independent functions, something that had never been described for these proteins.

Not withstanding this, in the revised version, we have added a new supplementary figure (1) related to the StART domain-lipid interaction analysed in-silico. The in-silico model shows that the DLC3-StART domain Ω1-loop structure displays the highest frequency of interaction with the membrane. This loop is conserved in the StART domains of several other STARD proteins and seems to modulate access to the ligand binding cavity. Ω-loops play multiple roles in protein function, often related to ligand binding, stability and folding. In this context, mutations in the proximity of the Ω1-loop, like the ones carried by the patients, may have drastic effects on overall protein stability that could affect the interaction between gonadal precursor cells.

1. Do the cv-c mutants survive to adulthood? If yes, then it would be interesting to know how the adult testis behaves in cv-c mutants. Does it result in sterility?

Response: Unfortunately, all studied cv-c mutants are embryonic lethal.

1. Ensheathment is required for proper germline development and defects in ensheathment can affect soma-germline communication and germline development. Germ cell ensheathment affects the proliferation of germ cells and display defective JAK/STAT signaling. It would be interesting to know if the germ cells in cv-c mutant gonad show the proliferation defect and impaired JAK/STAT signaling.

Response: This is an interesting suggestion. JAK/STAT signalling has a male specific function that could explain why cv-c gonadal defects are male specific. We are going to study how cv-c affects STAT signalling in the male gonad. We are currently preparing stocks combining 10XSTAT::GFP reporter with cv-c mutants and preparing samples for anti-STAT labelling. We will also analyse if embryos lacking STAT activation, activate cv-c expression in the testes.

1. I was also wondering if the authors have examined the number of germ cells in the mutant gonads.

Response: Yes, we have counted the number of germ cells in cv-c mutants and, if anything, there are more. We initially considered that an excess of GC proliferation could be the cause of gonad disruption. However, we have discarded this hypothesis as phospho-histone 3 stainings did not show a significant increase of GC divisions. Moreover, when we blocked cell proliferation in cv-c’ mutant gonads using UAS-p21, the testes phenotype was not rescued. We are unsure what could be responsible for the slight increase of germ cells observed.

1. In addition, I think the quality of the images should be improved.

Response: We have changed the colours used in the confocal images and amplified the relevant regions in all panels. We thank both referees for this suggestion as these changes have improved the figure contrast.

Minor comments:

1. cv-c mRNA in Figure 2 panels (Fig. 2D) should be in italics.

Response: We have changed it.

1. There is no scale bar in Figure panels. In addition, there is no scale bar in the zoomed images in Figure 2. Scale bars should be consistently put in the all the Figures, in particular on the first panels of the Figures.

Response: We have added scale bars to all panels.

1. In the line 677, the manuscript says "arrowhead". There are no arrowheads but the arrows.

Response: Corrected

1. Please be consistent with the labels in Figure panels: Vasa is shown in capital while Eya is not.

Response: Corrected

1. Please be consistent with the labeling of the Figure panels: Figure 3A vs Figure 4a.

Response: Corrected

1. What does the asterisk signify in Figure 2? There is no mention of asterisk in the Figure 2 legend.

Response: The meaning of the asterisk was explained in the figure legend.

1. There is no grey channel (sagittal view) for the panels Figure 3I and J.

Response: We have already included sagittal views in the figure.

1. Please be thorough in labeling the genotypes in Figures. For instance, Figure 4c depict the % of normal testis in cv-c delta StART. However, the correct genotype is twi>Cv-c StART. In addition, in Figure 4c graph, cv-c mut should be cv-cGAPmut.
2. Please be consistent with the depiction of the "START" domain of the protein throughout the manuscript. In figure 4c for instance, it is "START" in the graph while in the figure panel 4i, it is StART.
3. In Figure 4b, it is written DLC3-GA. Did the authors mean DLC3-S993N?
4. In line 723, it should be anti-beta catenin.

Response: As suggested, we have unified figure labelling.

1. The authors have shown two images to suggest that cv-c mutant gonad depict the germ cell blebbing (Figure 3I and J). I think it would be much better to put up a graph showing the number or percentage of cv-c mutant gonads displaying the germ cell blebbing than putting two images with the same information.

Response: We have already done the quantification and added the data as a graph in figure (3J).

1. The previous comment is also true for Figure 6H and I. In both the panels, the authors wish to show discontinuous ECM marked by Perlecan expression in cv-c mutant gonads. I think it would be better to display a score of the number of mutant gonads depicting the discontinuous ECM.

Response: We are repeating stainings to quantify Perlecan disruption in cv-c mutants and we will display the results as a graph in figure 6.

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Referee #3

Evidence, reproducibility and clarity

Summary:

This manuscript uses genetics, cell biology and confocal imaging to study the loss-of-function phenotypes of Cv-c, the Drosophila ortholog of DLC3. DLC3 mutation has been recently associated with male gonad dysgenesis in DSD patients. This work showed that Cv-c acts in the somatic gonadal cells to regulate male gonad development. Interestingly, the Cv-c mutant phenotypes result in testicular dysgenesis, marked by defective germ cell ensheathment and blebbing. Using different variants of the Cv-c protein and genetics based analyses, the authors further identified that the gonad development is dependent on StART domain but is independent of the GAP domain. Overall, these results should be of interest to researchers in disorders of sexual development, germ cell biology and developmental biology fields. However, at several places in order to reach the conclusions, more rigorous experiments should be performed (see revision details). In conclusion, this work has the potential but requires statistical quantification to support the central claims.

Major comments:

1. This study has shown the expression pattern of cv-c and the consequence of cv-c mutation on different aspects of gonad development. However, one major comment is there is no quantification of the expression levels as well as the scoring of the mutant phenotypes.
2. In Figure 2, for instance, I recommend that the authors display the quantification of the fluorescence intensity of the cv-c expression under all circumstances (in situ hybridization as well as protein-trap based GFP expression) to better depict the differences among the male vs female gonad.
3. In Figure 3, the authors show the different gonad developmental defects associated with the cv-c mutation. Specifically, the authors show that the gonad mesoderm cells are displaced with the pigment cells failing to ensheath the germ cells. In addition, the authors also suggest that there is an increased frequency of germ cell blebbing, an indication of migratory activity. However, there is no quantification of these findings. I think the authors should display a quantitative estimation of % of the mutant gonad depicting these phenotypes vs the normal gonad to have a perspective of how penetrant the phenotypes are.
4. In Figure 4, the authors attempt to rescue the Cv-C mutation linked gonadal defects by overexpressing different Cv-C protein variants. The rescue experiments are not very clear. The graph shows the % of normal testes under different genotypic combinations. It is not very clear what the authors mean by normal (in what context)? Since the mutation results in different defects of gonad development, I think recommend that represent the rescue in terms of these defects. It would be interesting to see for instance, what happens to the blebbing or germ cell ensheathment phenoype upon rescue. How many % of testes show the rescue as compared to cv-c mutants?
5. Did the authors try cell-specific depletion of cv-c and examined the consequence on gonad development?
6. Another major concern is the lack of mechanistic insight of cv-c. For example, how does loss of cv-c result in gonadal dysgenesis? The authors suggested that StART domains regulate via lipid binding. The authors could examine if StART domain function is dependent on lipid-mediated interactions.
7. Do the cv-c mutants survive to adulthood? If yes, then it would be interesting to know how the adult testis behaves in cv-c mutants. Does it result in sterility?
8. Ensheathment is required for proper germline development and defects in ensheathment can affect soma-germline communication and germline development. Germ cell ensheathment affects the proliferation of germ cells and display defective JAK/STAT signaling. It would be interesting to know if the germ cells in cv-c mutant gonad show the proliferation defect and impaired JAK/STAT signaling.
9. I was also wondering if the authors have examined the number of germ cells in the mutant gonads.
10. In addition, I think the quality of the images should be improved.

Minor comments:

1. cv-c mRNA in Figure 2 panels (Fig. 2D) should be in italics.
2. There is no scale bar in Figure panels. In addition, there is no scale bar in the zoomed images in Figure 2. Scale bars should be consistently put in the all the Figures, in particular on the first panels of the Figures.
3. In the line 677, the manuscript says "arrowhead". There are no arrowheads but the arrows.
4. Please be consistent with the labels in Figure panels: Vasa is shown in capital while Eya is not.
5. Please be consistent with the labeling of the Figure panels: Figure 3A vs Figure 4a.
6. What does the asterisk signify in Figure 2? There is no mention of asterisk in the Figure 2 legend.
7. There is no grey channel (sagittal view) for the panels Figure 3I and J.
8. Please be thorough in labeling the genotypes in Figures. For instance, Figure 4c depict the % of normal testis in cv-c delta StART. However, the correct genotype is twi>Cv-c StART. In addition, in Figure 4c graph, cv-c mut should be cv-cGAPmut.
9. Please be consistent with the depiction of the "START" domain of the protein throughout the manuscript. In figure 4c for instance, it is "START" in the graph while in the figure panel 4i, it is StART.
10. In Figure 4b, it is written DLC3-GA. Did the authors mean DLC3-S993N?
11. In line 723, it should be anti-beta catenin.
12. The authors have shown two images to suggest that cv-c mutant gonad depict the germ cell blebbing (Figure 3I and J). I think it would be much better to put up a graph showing the number or percentage of cv-c mutant gonads displaying the germ cell blebbing than putting two images with the same information.
13. The previous comment is also true for Figure 6H and I. In both the panels, the authors wish to show discontinuous ECM marked by Perlecan expression in cv-c mutant gonads. I think it would be better to display a score of the number of mutant gonads depicting the discontinuous ECM.

Significance

The significance of this paper is the identification of a conserved protein that has a conserved function in regulating spermatogenesis. The Drosophila embryonic testis is an ideal system to study the role of cv-c in gonadogenesis. It also sets the stage for studying the potential roles of cv-c in adult testes.

The existing published knowledge about somatic ensheathment of germ stem cells is sufficiently covered and cv-c is a new player in this process.

The audience for this study is developmental biologists and researchers studying disorders of sex development.

Our expertise is somatic sex identity in adult Drosophila gonads and DSD.

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Referee #2

Evidence, reproducibility and clarity

This is an interesting and generally convincing study demonstrating the potential role of DLC3 (STARD8)/Cv-c in testicular development, and in particular its role in early (fetal in human) germ cell development. The genetic manipulations and associated techniques involving Drosophila appear to be state of the art, although I would not class myself as expert enough in such techniques to be able to give a truly informed opinion.

My only issue with the present study is to how well the present experimental findings in Drosophila translate to humans. As far as I can tell the present studies show that inactivating mutations in Cv-c in Drosophila result in failure of germ cell enclosure by somatic cells into the testis, resulting in sterility. In humans, and in experimental mouse transgenic lines, it has been well established that absence of germ cells does not of itself lead to failure of testis differentiation and onward development, nor does it lead automatically to sex reversal or impairment of masculinization. For the latter to occur, there must be impairment/failure of fetal Leydig cell function such that insufficient androgen is produced to effect genital/bodywide masculinization. Obviously, this will happen if no testis forms as appears to be the case in the new human DLC3 mutant reported in the present manuscript (although detail on this is unfortunately lacking). This appears to be different to the previous published DLC3/STARD8 mutant sisters, in whom the phenotype appears to reflect failure of steroidogenesis. Is the proposal that DLC3/STARD8 plays a role in both testis differentiation and in Leydig cell function (steroidogenesis) or is this due to different DLC3 genes? I think the authors need to address these key issues in their discussion, if only to highlight that there are at present many gaps in our understanding.

I would also suggest that the authors highlight another potentially more important spin-off from such studies, namely that understanding of the regulation of DLC3/STARD8 genes, and what might perturb their expression/action would appear to present a whole new area for exploration in relation to testicular dysgenesis/masculinization disorders.

Significance

This represents a significant advance in our understanding and identifies model systems in which to gain further insight.

I would also suggest that the authors highlight another potentially more important spin-off from such studies, namely that understanding of the regulation of DLC3/STARD8 genes, and what might perturb their expression/action would appear to present a whole new area for exploration in relation to testicular dysgenesis/masculinization disorders in humans.

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Referee #1

Evidence, reproducibility and clarity

Summary:

The authors have utilised Drosophila to provide evidence that a DLC3 mutation identified in 46,XY patients with male gonadal dysgenesis is likely causative for the phenotype. They demonstrated that mutation of the Drosophila ortholog of DLC3, Cv-c, was associated with defects in embryonic testis development and that the phenotype could be rescued by expression of wildtype human DLC3 but not the patient variant. DLC3/Cv-c are members of the RhoGAP family of proteins but GAP activity was not required for in the testis while mutation of the StART domain disrupted gonad morphogenesis. Mutations in this domain were associated with human gonadal dysgenesis suggesting a conservation of function during gonad development.

Major comments:

In general, the data support the conclusions. I cannot comment on the atomistic simulation experiment as it is outside of my expertise. I had some difficulties interpreting Figure 2 as the contrast in the colour panels made it difficult to assess the different staining patterns. I would recommend changing the blue to cyan for easier visibility. While I agree that there are some differences between Fig 2F and Fig 2G it is not simple for the non-expert to distinguish the gonadal mesoderm from the somatic mesoderm. I think the enlarged panels could do with also showing the overlap in staining, or at least a tracing of the different cell populations so that the gonadal mesoderm can be clearly defined. Please also add some scale bars to the figure.<br /> Figure 3 demonstrates clear differences in gonad morphology between male and female mutants but the contrast in the colour panels A-G could also be improved. Panels H-J are very clear.<br /> The rescue experiment in Figure 4 is clearly presented but could the DLC3 mutants in the graph (panel b) please be named similarly to the schematic proteins shown in panel a.<br /> I found the difference between the RhoGAP domain mutants and the StART domain mutants of Cv-c to be clearly defined, and correlate with DLC3 function. This is a very interesting result that indicates multiple molecular functions for the Cv-c /DLC family.<br /> The methods are well described, statistics adequate and the data well described.

Minor Comments:

My only suggestion for the text is to provide a more through description of the StART domain in the introduction.

Referees cross-commenting

Quantification of dispersed low level punctate expression levels will be very difficult to achieve and I do not believe will alter the conclusions. I agree that quantification of the percentage of gonads that display the illustrated phenotypes would be beneficial. The manuscript nicely describes a phenotype that has been defined to be due to mutation of the StART domain. Defining the mechanism of how the StART domain regulates protein function would take the study to a new level but may not be necessary for publication of the findings.

Significance

The significance of this work is two-fold.

1. A demonstration that Cv-c and DLC3 have similar functions in regulation of gonad morphogenesis, and that the patient mutations almost certainly correlate with the observed phenotypes.
2. An intriguing demonstration that this family of proteins is not simply functioning as RhoGAPs, and that there is a specific roles for the StART domain in male gonad development.

DLC3 has already been shown to rescue Malpighian tubule defects associated with Cv-c mutants so the functional equivalence of these proteins has already been established. The novelty of the present study relates to a demonstration that the StART domain is specifically required for male gonad morphogenesis, and the utility of experiments in Drosophila to assist in associating a human mutation with a clinical phenotype.

This work should find a willing audience from clinical geneticists as an example of how model organism genetics can assist genetic diagnoses. It will also be of great interest to the field of reproductive biologists who wish to understand how the sex determination pathways are resolved by differential tissue development. The obvious next steps are to determine why mutations in Cv-c only affect male gonads and not females, and what is the specific role of the StART domain in this process.<br /> My field of study has focussed on adult gonad development and regeneration, and I found the manuscript presented in a style that was simple to follow with my suggestion that contrast of some of the colour panels could be improved.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

The rapid syncytial nuclear cycles that occur during the first ~2.5 hours of Drosophila embryogenesis and give rise to the blastoderm are supported by large amounts of maternally deposited histone proteins which are stored in the egg cytoplasm for deposition into replicating DNA during each round of S phase. Although the H2A/H2B storage chaperone Jabba was identified by Michael Welte's lab several years ago, maternal H3/H4 storage chaperones have not been identified. Tirgar et al provide evidence that the Drosophila NASP protein provides histone H3 and H4 storage function during these earliest stages of Drosophila embryogenesis. The data include genetic analyses that NASP function is required maternally, but not zygotically, and molecular analyses that NASP binds H3 and that H3 and H4 levels are reduced in the embryo and late-stage oocytes in the absence of NASP. These data are convincing and support the conclusion that NASP is a maternally acting H3/H4 storage chaperone needed in the early embryo.

Two additional lines of investigation would strengthen this conclusion and perhaps increase the impact and appeal of the manuscript.

The first is a microscopic analysis of the nuclear division cycles in eggs derived from NASP mutant mothers. The authors report DAPI staining and assessment of nuclear cycles, but do not show these data. In fact, the two embryos shown in Figure 4B do not look like DAPI stained embryos-there are no nuclei apparent in the images. Loss of maternal histone causes defects in chromosome morphology that result in characteristic defects such as lagging chromosomes and the failure of sister chromatid segregation leading to fused daughter nuclei (see PMID: 11157774 for an example). These defects should not be difficult to detect via DNA staining or even using fluorescently labeled H2 type histones. Characterizing such defects would lend support to the hypothesis and I think is important for this paper.

We thank the reviewer for their constructive review and feedback. We have switched to Propidium Iodide (PI) staining to increase the signal-to-noise for DNA staining in early embryos. Given the improved signal we see with PI over DAPI, we will be able to provide both improved images of nuclear staining and assay for defects in chromosome morphology as suggested. We will include this data in the revised version of the manuscript. Second, determining the location of NASP in the early embryo might provide further insight into the mechanism of storage. i.e. is NASP located in the cytoplasm rather than the nucleus, perhaps in association with lipid droplets like Jabba? Do the antibodies the authors developed work in IF experiments to ask this question? At the moment what is shown is that NASP is present in 0-2 hour embryos via western blot analysis, supporting the conclusion that it functions in the early embryo as a storage chaperone. This analysis would be nice to have but is not essential in my view.

We have tried to use our antibody to monitor the localization of NASP in the early embryo. Unfortunately, the staining has yet to work. We will continue to alter fixation and permeabilization conditions in the early embryo with the goal of including this data in the revised manuscript. We have, however, been able to monitor NASP localization in Drosophila S2 cultured cells with our antibody. If we are unable to get the antibody staining to work in embryos, we will include the NASP localization data in S2 cells in combination with EdU labeling to mark cells in S phase.

Small points: Is NASP really a maternal effect "lethal"? Some of the eggs do hatch, and so some develop to stages where maternal histones are no longer necessary and zygotic production takes over (i.e. cycle 15). Perhaps consider the language used here.

We see the reviewers point with respect to the term ‘lethal’. We do see a very small fraction of progeny laid by NASPmutant mothers make it to adulthood, although they die shortly after hatching. We’ve removed the term ‘lethal’ and refer to NASP solely as a maternal effect gene. On this point, do NASP mutant females lay the same number of eggs as wild type? i.e. is there a requirement for oogenesis/egg production (other than depositing H3/H4 into the egg), or just for the early zygotic cycles?

We have noticed that NASP mutant mothers have lower fecundity. We have included this data in the revised manuscript as Supplemental Figure 2A.

The first paragraph of the results is redundant with much of the introduction, which I think could do a better job at describing in more detail the syncytial cycles and the special needs they have for histone storage and chaperone function versus the post-blastoderm embryonic cycles and the rest of development. i.e. make a better distinction between the first two hours of embryogenesis versus the rest of embryogenesis, and the when the switch from maternal to zygotic control of development and histone production occurs (cycle 15 at 3-4 hours AED).

We appreciate the reviewer for this suggestion. The manuscript has been edited to be less redundant and include details of embryogenesis as suggested. CROSS-CONSULTATION COMMENTS Seems like all reviewers are in general agreement, particularly about providing additional data regarding chromosome/nuclear behavior in the NASP mutants and NASP localization in the early embryo to increase impact of the study. While rescue of the NASP mutant phenotype with a transgene would be nice, as suggested by referee #2, I don't think it's essential given the genetic approaches employed.

Reviewer #1 (Significance (Required)):

see above

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Tirgar et al. report on a functional characterization of the Drosophila homolog of the histone H3/H4 chaperone NASP. They generated a loss of function allele of NASP by CRISPR/Cas9, which induces a partial maternal effect embryo lethal phenotype. Using quantitative mass spectrometry, they demonstrate that NASP stabilizes reservoirs of H3 and H4 in the early embryo. The manuscript is very clear and confirms the functional importance of maternal NASP for the early embryo. Genetic analyses are well conducted (but see my comments below) and the impact of NASP maternal mutant on H3 and H4 stockpiles is convincingly established by both quantitative mass spectrometry and Western-blotting.

Major comments:

• Although the authors used two independent deficiencies of the NASP genetic region to characterize their NASP CRISPR alleles, it is relatively standard in this type of functional analyses to perform rescue experiments using a transgene expressing the WT protein.

We thank the reviewer for this suggestion. As discussed in the cross consultation, we agree that the use of the two different deficiency lines and the NASP1 CRISPR control are clear lines of evidence that the phenotypical data are due to lack of NASP.

• In WB analyses, NASP appears systematically shorter in the NASP[1]/Df genotype compared to WT. Can the authors comment on this?

While we reproducibly see this change in migration, we can only guess as to why this may be. One possible reason is that the NASP1 mutant protein could be missing a post-translational modification. Proteomic data from Krauchunas et al. (Dev Biol. 2012; PMC3441184) shows that NASP has the potential to be regulated by phosphorylation. Therefore, the NASP1 mutant protein could be missing a phosphorylation. Intriguingly, the 6bp insertion is next to a Thr residue that could affect its ability to be phosphorylated (if it is phosphorylated at all). Since we can only offer speculation, we do not feel comfortable adding this to the manuscript.

• The authors do not mention the centromeric histone H3 variant Cid in their analyses. Do they have evidence that it is not affected by loss of maternal NASP?

We thank the reviewer for raising this great point. Our mass spec data reveals that Cid levels stay the same in the absence of NASP in both embryos and stage 14 egg chambers. We have edited Figures 3D and 3E to include Cid. Unfortunately, we did not identify any Cid-specific peptides in our IP-mass spec data.

• The authors could have chosen to explore in more details the phenotypic defects of embryos derived from NASP mutant mothers. Instead, a single abnormal embryo is shown with no cytological details. This is a bit problematic since an earlier study (Zhang et al 2018, cited in the manuscript) actually provided more phenotypic details of embryos from NASP KD mothers.

This issue was also raised by Reviewer 1. We have switched to Propidium Iodide (PI) staining to increase the signal-to-noise for DNA staining in early embryos. Given the improved signal we see with PI over DAPI, we will be able to provide both improved images of nuclear staining and assay for defects in chromosome morphology as suggested. We will include this data in the revised version of the manuscript. - Similarly, the authors could have used their anti-NASP antibody to analyze the distribution of NASP during cleavage divisions. Does it behave like ASF1, for instance, which enters S phase nuclei at each cycle or does it remain in the cytoplasm? These are relatively simple experiments/analyses that could increase the significance of the study.

This point was also raised by Reviewer 1. We have tried to use our antibody to monitor the localization of NASP in the early embryo. Unfortunately, the staining has yet to work. We will continue to alter fixation and permeabilization conditions in the early embryo with the goal of including this data in the revised manuscript. We have, however, been able to monitor NASP localization in Drosophila S2 cultured cells with our antibody. If we are unable to get the antibody staining to work in embryos, we will include the NASP localization data in S2 cells in combination with EdU labeling to mark cells in S phase.

Minor comments:

• line 60: I suggest to introduce Drosophila in the next sentence, where it seems more appropriate (not all embryos develop "extremely rapidly").

We have edited the second sentence to state “the early Drosophila embryo”.

• line 68: the 50% estimation of free histones does not really make sense without defining the embryonic stage.

We have edited the manuscript to state the specific cell cycle in which there has been 50% free histones measured. - line 89: Are the authors specifically referring to Drosophila NASP?

Yes, we have edited the text to include Drosophila in this instance. - lines 99-106: I found this paragraph redundant with the introduction.

We appreciate this suggestion. It was also pointed out by Reviewer 1. We have made changes to the manuscript to address the redundancy.

• line 142: H3-H4

Thank you for noticing this. We have edited the text to include 4.

• line190-191: It seems to me that data of Figure S2C are already included in Fig. 2E.

The data in FigureS2C was performed with virgin females compared to the data in Figure 2E that was generated with non-virgin mothers. This was important to control the genotype of the embryos.

• line 232: it is surprising that the Zhang et al paper (reporting maternal KD of NASP) is only mentioned here. As a reader, I would certainly prefer to have it presented right from the introduction.

We have edited the manuscript to include this reference in the introduction.

• Figure 4B needs a scale bar.

Figure 4B will be replaced with better images of the embryo stained with PI. It will also include images of chromosome morphology/segregation. We will be sure to include scale bars.

• line 302: Mentioning the identity and function of known H3/H4 histone chaperones acting in the early embryo (ASF1, HIRA, CAF-1, ...) could provide perspective to the present study.

Thank you for this suggestion. We have edited the manuscript to include functions of other histone chaperones in the early embryo to provide context.

• line 304: in contrast to this statement, I found quite surprising and interesting that NASP is not absolutely essential for embryo development considering its role. This should be discussed.

In the absence of Jabba alone, upregulation of translation can compensate for the destabilization of H2A, H2B, and H2Av. It is only when translation is inhibited in embryos laid by Jabba mutant mothers that embryos die (Li.Z, et al. Curr Biol 2013). Therefore, it is possible that translation can partially compensate for the degradation of H3 and H4 in the absence of NASP. This may be why a fraction of embryos laid by NASP mutant mothers are able to hatch and why we still detect some H3 in embryos laid by NASP mutant mothers. We have edited the manuscript to discuss this more in depth.

CROSS-CONSULTATION COMMENTS I fully agree with the other reports. The NASP rescue experiment is just a suggestion but is not essential.

Reviewer #2 (Significance (Required)):

This work clarifies the identity and function of Drosophila NASP and clearly demonstrates that NASP is important for the stabilization of maternal stockpiles of H3 and H4 during early embryo development. The conservation of NASP function as a histone H3/H4 chaperone in Drosophila is not really a surprise but the merit of this study is to establish this assumption as a fact. It also establishes useful tools (mutant lines and antibody) for the fly community interested in this topic. The study however does not provide new insights about the dynamic distribution of NASP and the cytological consequences of its maternal depletion on the amplification of cleavage nuclei.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary: Rapid cell cycles in early embryogenesis is driven from maternally supplied stockpiles of RNA and protein, including histones H3 and H4. This study uses sequence homology searches, biochemical approaches (immunoprecipitation and mass spectrometry) and genetics to identify NASP (CG8223) as the H3-H4 chaperone in Drosophila. Using CRISPR technology, the authors generate a NASP mutant fly line and show using genetic crosses that NASP is a maternal lethal gene. Furthermore the study shows that NASP stabilises H3-H4 during oogenesis and embryogenesis and is required for early embryogenesis.

Major comments: The key conclusions of this study are very convincing. For example, the authors use multiple approaches to show H3-H4 specific interactions with NASP and that H3-H4 protein levels are reduced in mutants (Western analyses, quantitative MS). Analysis is carried out on two individual NASP mutant lines (one deletion that produces no protein, one insertion that still produces some protein acting as a control). All experiments are well controlled, executed and presented. Genetic crossing schemes are well presented and statistical analysis of progeny is clear.

• We thank the reviewer for their positive feedback of our manuscript. Minor comments: In Figure 1B - Authors could indicate amino acids shown or are they full length proteins?

We have edited the methods to include specific amino residues that are included for each structure.

In Figure 2B - Authors could (semi) quantify reduction in NASP1 mutant to show this is a gene dose effect?

We have now included the quantification of the Western blot in Figure 2B.

CROSS-CONSULTATION COMMENTS I agree with the other reports. Although I did not indicate it in my original report, I agree that more in depth analysis of nuclear or chromosomal defects in NASP mutant embryos would enhance the study.

Thank you for this suggestion. We are repeating the DNA staining in embryos and will include this new data in the revised version of the manuscript.

Reviewer #3 (Significance (Required)):

Excess soluble histones can be toxic and must be bound to chaperones. Until this study the chaperone responsible for H3-H4 stabilisation in rapidly cycling cells in Drosophila embryos was not known. Moreover, the NASP homolog had not yet been identified in Drosophila nor had its function been characterised. The findings are of interest to Drosophila researchers, the field of chromatin assembly, as well as those interested in early embryogenesis in animals.

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Referee #3

Evidence, reproducibility and clarity

Summary:

Rapid cell cycles in early embryogenesis is driven from maternally supplied stockpiles of RNA and protein, including histones H3 and H4. This study uses sequence homology searches, biochemical approaches (immunoprecipitation and mass spectrometry) and genetics to identify NASP (CG8223) as the H3-H4 chaperone in Drosophila. Using CRISPR technology, the authors generate a NASP mutant fly line and show using genetic crosses that NASP is a maternal lethal gene. Furthermore the study shows that NASP stabilises H3-H4 during oogenesis and embryogenesis and is required for early embryogenesis.

Major comments:

The key conclusions of this study are very convincing. For example, the authors use multiple approaches to show H3-H4 specific interactions with NASP and that H3-H4 protein levels are reduced in mutants (Western analyses, quantitative MS). Analysis is carried out on two individual NASP mutant lines (one deletion that produces no protein, one insertion that still produces some protein acting as a control). All experiments are well controlled, executed and presented. Genetic crossing schemes are well presented and statistical analysis of progeny is clear.

Minor comments:

In Figure 1B - Authors could indicate amino acids shown or are they full length proteins?

In Figure 2B - Authors could (semi) quantify reduction in NASP1 mutant to show this is a gene dose effect?

Referees cross-commenting

I agree with the other reports. Although I did not indicate it in my original report, I agree that more in depth analysis of nuclear or chromosomal defects in NASP mutant embryos would enhance the study.

Significance

Excess soluble histones can be toxic and must be bound to chaperones. Until this study the chaperone responsible for H3-H4 stabilisation in rapidly cycling cells in Drosophila embryos was not known. Moreover, the NASP homolog had not yet been identified in Drosophila nor had its function been characterised. The findings are of interest to Drosophila researchers, the field of chromatin assembly, as well as those interested in early embryogenesis in animals.

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Referee #2

Evidence, reproducibility and clarity

Tirgar et al. report on a functional characterization of the Drosophila homolog of the histone H3/H4 chaperone NASP. They generated a loss of function allele of NASP by CRISPR/Cas9, which induces a partial maternal effect embryo lethal phenotype. Using quantitative mass spectrometry, they demonstrate that NASP stabilizes reservoirs of H3 and H4 in the early embryo. The manuscript is very clear and confirms the functional importance of maternal NASP for the early embryo. Genetic analyses are well conducted (but see my comments below) and the impact of NASP maternal mutant on H3 and H4 stockpiles is convincingly established by both quantitative mass spectrometry and Western-blotting.

Major comments:

• Although the authors used two independent deficiencies of the NASP genetic region to characterize their NASP CRISPR alleles, it is relatively standard in this type of functional analyses to perform rescue experiments using a transgene expressing the WT protein.
• In WB analyses, NASP appears systematically shorter in the NASP[1]/Df genotype compared to WT. Can the authors comment on this?
• The authors do not mention the centromeric histone H3 variant Cid in their analyses. Do they have evidence that it is not affected by loss of maternal NASP?
• The authors could have chosen to explore in more details the phenotypic defects of embryos derived from NASP mutant mothers. Instead, a single abnormal embryo is shown with no cytological details. This is a bit problematic since an earlier study (Zhang et al 2018, cited in the manuscript) actually provided more phenotypic details of embryos from NASP KD mothers.
• Similarly, the authors could have used their anti-NASP antibody to analyze the distribution of NASP during cleavage divisions. Does it behave like ASF1, for instance, which enters S phase nuclei at each cycle or does it remain in the cytoplasm? These are relatively simple experiments/analyses that could increase the significance of the study.

Minor comments:

• line 60: I suggest to introduce Drosophila in the next sentence, where it seems more appropriate (not all embryos develop "extremely rapidly").
• line 68: the 50% estimation of free histones does not really make sense without defining the embryonic stage.
• line 89: Are the authors specifically referring to Drosophila NASP?
• lines 99-106: I found this paragraph redundant with the introduction.
• line 142: H3-H4
• line190-191: It seems to me that data of Figure S2C are already included in Fig. 2E.
• line 232: it is surprising that the Zhang et al paper (reporting maternal KD of NASP) is only mentioned here. As a reader, I would certainly prefer to have it presented right from the introduction.
• Figure 4B needs a scale bar.
• line 302: Mentioning the identity and function of known H3/H4 histone chaperones acting in the early embryo (ASF1, HIRA, CAF-1, ...) could provide perspective to the present study.
• line 304: in contrast to this statement, I found quite surprising and interesting that NASP is not absolutely essential for embryo development considering its role. This should be discussed.

Referees cross-commenting

I fully agree with the other reports.

The NASP rescue experiment is just a suggestion but is not essential.

Significance

This work clarifies the identity and function of Drosophila NASP and clearly demonstrates that NASP is important for the stabilization of maternal stockpiles of H3 and H4 during early embryo development. The conservation of NASP function as a histone H3/H4 chaperone in Drosophila is not really a surprise but the merit of this study is to establish this assumption as a fact. It also establishes useful tools (mutant lines and antibody) for the fly community interested in this topic. The study however does not provide new insights about the dynamic distribution of NASP and the cytological consequences of its maternal depletion on the amplification of cleavage nuclei.

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Referee #1

Evidence, reproducibility and clarity

The rapid syncytial nuclear cycles that occur during the first ~2.5 hours of Drosophila embryogenesis and give rise to the blastoderm are supported by large amounts of maternally deposited histone proteins which are stored in the egg cytoplasm for deposition into replicating DNA during each round of S phase. Although the H2A/H2B storage chaperone Jabba was identified by Michael Welte's lab several years ago, maternal H3/H4 storage chaperones have not been identified. Tirgar et al provide evidence that the Drosophila NASP protein provides histone H3 and H4 storage function during these earliest stages of Drosophila embryogenesis. The data include genetic analyses that NASP function is required maternally, but not zygotically, and molecular analyses that NASP binds H3 and that H3 and H4 levels are reduced in the embryo and late-stage oocytes in the absence of NASP. These data are convincing and support the conclusion that NASP is a maternally acting H3/H4 storage chaperone needed in the early embryo.

Two additional lines of investigation would strengthen this conclusion and perhaps increase the impact and appeal of the manuscript.

The first is a microscopic analysis of the nuclear division cycles in eggs derived from NASP mutant mothers. The authors report DAPI staining and assessment of nuclear cycles, but do not show these data. In fact, the two embryos shown in Figure 4B do not look like DAPI stained embryos-there are no nuclei apparent in the images. Loss of maternal histone causes defects in chromosome morphology that result in characteristic defects such as lagging chromosomes and the failure of sister chromatid segregation leading to fused daughter nuclei (see PMID: 11157774 for an example). These defects should not be difficult to detect via DNA staining or even using fluorescently labeled H2 type histones. Characterizing such defects would lend support to the hypothesis and I think is important for this paper.

Second, determining the location of NASP in the early embryo might provide further insight into the mechanism of storage. i.e. is NASP located in the cytoplasm rather than the nucleus, perhaps in association with lipid droplets like Jabba? Do the antibodies the authors developed work in IF experiments to ask this question? At the moment what is shown is that NASP is present in 0-2 hour embryos via western blot analysis, supporting the conclusion that it functions in the early embryo as a storage chaperone. This analysis would be nice to have but is not essential in my view.

Small points:

Is NASP really a maternal effect "lethal"? Some of the eggs do hatch, and so some develop to stages where maternal histones are no longer necessary and zygotic production takes over (i.e. cycle 15). Perhaps consider the language used here. On this point, do NASP mutant females lay the same number of eggs as wild type? i.e. is there a requirement for oogenesis/egg production (other than depositing H3/H4 into the egg), or just for the early zygotic cycles?

The first paragraph of the results is redundant with much of the introduction, which I think could do a better job at describing in more detail the syncytial cycles and the special needs they have for histone storage and chaperone function versus the post-blastoderm embryonic cycles and the rest of development. i.e. make a better distinction between the first two hours of embryogenesis versus the rest of embryogenesis, and the when the switch from maternal to zygotic control of development and histone production occurs (cycle 15 at 3-4 hours AED).

Referees cross-commenting

Seems like all reviewers are in general agreement, particularly about providing additional data regarding chromosome/nuclear behavior in the NASP mutants and NASP localization in the early embryo to increase impact of the study. While rescue of the NASP mutant phenotype with a transgene would be nice, as suggested by referee #2, I don't think it's essential given the genetic approaches employed.

Significance

see above

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

An exciting development in our knowledge about how the Arp2/3 complex controls the assembly of actin networks has come from the discovery that in addition to forming branched networks, Arp2/3 can nucleate linear filaments when it is activated by WISH/DIP/SPIN90. However, despite some excellent work largely done by the Nolen lab in yeast, many questions remain about how Arp2/3-mediated assembly of branched vs. linear actin filament. This is especially true in the complex environment of cells, were synergy and competition of different actin networks is used to control biological processes. Knowing the biochemical and physical properties of these different Arp2/3 assemblies will be key to figuring out how they work in cells. Here Cao et al. use an elegant microfluidics based single filament assay system to perform a comparative analysis of the stability of linear and branched Arp2/3 networks. They find interesting differences in how they respond to stabilizing and destabilizing factors. The most striking differences happens when force or aging is applied- both cause debranching of branched networks but have little effect on Spin90-Arp2/3 nucleated filaments.

We thank the reviewer for their positive comments.

Major comments:

As a comparative study on the stability of branched vs. linear Arp2/3 nucleated filaments, this manuscript is fairly complete. The key conclusions are well supported by rigorous experiments which can be reproduced by others based on the information provided. However, I am not seeing explicit information on performing biological replicates. This should be included in the manuscript. The use of statistics is largely fine; however I question the use of one statistical test on one figure (see minor comments below).

The revised manuscript is now explicit about biological replicates. We now specify the biological repeats of all our experiments in the figure legends, and we now show the results from new repeats in Fig 4 and Supp Fig S2 (please see also our response to the minor comments below, for more details).

I would not ask for additional experiments at this time. However, there is an analysis that would be important for interpreting the authors' claims- branch/filament length at the time of dissociation or destabilization of Arp2/3. This would help address if there was a physical tipping point for each type of structure that could explain potential differences they see. The authors should already have this data and the time to complete it would be negligible in delaying publication.

If we understand correctly, the “physical tipping point” mentioned by the reviewer would be a threshold force, where the Arp2/3-filament interface would become unstable. This is an interesting idea. Indeed, the applied force scales with the length of the filament (or branch), as well as with the flow velocity. In most of our experiments, however, the force applied to SPIN90-Arp2/3 and to branch junctions was kept constant and below 0.2 pN. This was done by exposing the filaments (or branches) to G-actin at the critical concentration, in order to minimize variations of their lengths. Therefore, by design, dissociation events in these experiments take place at the same length, ruling out the existence of a “tipping point”.

Our data provide another test of the reviewer’s hypothesis, thanks to the experiments where we specifically address the question of the impact of force (Fig 5 and Supp Fig S6), by varying length and flow rate. We found that the stability of SPIN90-Arp2/3 linear filaments was unaffected by force, and that debranching was steadily accelerated by force. In both cases, it thus appears that there is no detectable threshold.

One additional major comment is that the manuscript's title and abstract hint that this paper explores the differences in nucleation of branched vs. linear filaments by Arp2/3. However, the only figure that deals explicitly with nucleation in the paper is Figure 1, which is really just a confirmation that the mammalian proteins used in this study perform similarly to their yeast homologues (Balzer et al, Current Biology 2019). The authors might think about rewording the title/abstract to better reflect that paper really explores the differences in the stability of the two networks

This is a fair point. We have now modified the title into “Regulation of branched versus linear Arp2/3-generated actin filaments”.

Minor comments:

1 in 12 men and 1 in 200 women are red/green colorblind. Please change the coloring of the schematics and images so that they can be easily seen by all people. This is especially true of the schematics, which are important for understanding exactly what each assay is measuring.

We thank the reviewer for pointing this out. We have now made the schematics and images in Figs 1A, 2A, 2D and 4D colorblind-friendly.

The Introduction is a bit choppy and unfocused. It was difficult to deduce exactly where the paper was going from it. Please consider re-writing it for better clarity. The Discussion on the other hand was fantastic. Great job on interpreting your results in a larger context.

We have re-written large parts of the Introduction to make it clearer. We are glad the reviewer liked the Discussion, where we have nonetheless made some small changes in response to comments from the other reviewers.

Many figures- while the use of different lightness values of the same color is appreciated in conveying different concentrations of reagents used, there were several instances where it was very hard to read the one on the very bottom (ex. 2B, E; 3A; 5C, G).

We have now changed the colors in these figures, to make them clearer.

Figure 1- since this is a confirmation of previous results performed using the same proteins from other species, the title should reflect that (ex. VCA domains accelerate the nucleation of filaments by mammalian SPIN90-Arp2/3). Also, to me this figure is supplementary to the main message of the paper. The authors might think of moving it to Supplementary Information.

We have modified the title of Figure 1, now specifying “mammalian”, following the reviewer’s suggestion. However, we prefer to keep this figure as a main figure, rather than move it to Supplementary as proposed. Indeed, this figure does more than simply confirm previous results with mammalian proteins, since it compares different VCAs, which is new. These results are important because they are put in perspective with our results on the acceleration of linear filament detachment by different VCAs, later in the manuscript.

Figure 1- If the goal was to verify that G-actin recruitment by VCA was important for Spin90-Arp 2/3 nucleation by performing a competition experiment with profilin, why was the concentration of G-actin AND profilin increased between the experiments in 1B vs. 1C. It makes it hard to directly compare the results.

We now provide new data in Fig 1C, which can be directly compared to Fig 1B (only the profilin concentration was increased). It clearly shows that the effect of VCA disappears when the profilin concentration is increased.

Figure 4B-F- Here, it would be nice to see the distribution of all the individual results, which are hidden by the bar graph. Additionally, the Chi-square test is not the appropriate test for evaluating statistical significance between multiple groups. ANOVA followed by an appropriate post hoc test should be used here.

We now show the individual results in the bar graphs of figure 4. In this situation, we agree that the statistical significance should not be evaluated by a Chi-square test. We now indicate the p-values obtained from a paired t-test, which seems appropriate since we are comparing averages in pairs.

Figure 4G- Please quantify and show reproducibility.

We now show quantified repeats (shown in Fig 4, new panels H and I).

Figure 5- the piconewton forces used for these experiments is in line with measured forces that are applied to actin in cells (ex. Mehida et al, Nature Cell Biology 2021; Jiang et al, Nature 2003). The text would benefit if this was explicitly stated.

We now state this explicitly, when presenting these results.

Reviewer #1 (Significance (Required)):

The real significance of this work is in characterizing the differential stabilities of linear vs. branched Arp2/3 filaments in response to actin-binding proteins, mechanical stress, and aging. While both types of filaments respond similarly to actin-binding proteins, with nuanced differences, the most striking results came from applied force and aging experiments, with Spin90-Arp2/3 filaments being much more resistant to both. This has some very interesting implications for how these two types of assemblies might synergize in cells. Additionally, the results also have some exciting implications for the pointed-end regulation of actin filaments, which is still poorly understood in complex systems. Since the manuscript is A) more of a survey study on the factors that influence filament stability that does not go particularly deep into any particular mechanism of regulation and B) has no direct applicability to how the physical properties of branched and linear Arp2/3 nucleated actin filaments influence actin network activity in cells, the audience will likely by limited to actin enthusiasts. However, the work is still important in both what it reveals and implies.

We thank the reviewer for pointing out the novelty and the importance of our work. We agree that the significance of our paper lies in the characterization of the differential stabilities of linear vs. branched Arp2/3 filaments, in response to different physiological factors. One of the strengths of our approach is that we do not focus on one regulatory mechanism in particular. Rather, we reveal fundamental differences between the Arp2/3-generated filaments and how they can be regulated. Understanding these basic mechanisms is a prerequisite to understand the regulation of entire cytoskeletal networks.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

The quantitative analysis can be improved. It appears that most of the data results from single experiments, with rate values and errors resulting from fitting of single experiments without repetitions. In Fig. 1C legend (p.5) the authors state "These experiments were repeated three times, with similar results", but the data is not used in the analysis and other experiments do not mention this point. This is particularly important for comparisons among different VCAs that are rather similar in nature. In Fig. 1B. N-WASP is more efficient in nucleating SPIN90-Arp2/3 complex-linear filaments followed by WASP and then WASH. In Fig. 2 B,C, N-WASP is the most effective in dissociating SPIN90-Arp2/3 complex linear filaments followed by WASH and then WASP. But in Fig. 2 E,F, WASH is by far the most effective in dissociating branches followed by N-WASP and then WASP. Therefore, the conclusion in the Discussion (p.12) "While these regulatory proteins similarly affect branched and linear Arp2/3-generated filaments, they do so with clear quantitative differences" is not supported by quantification. To remedy this problem the authors should include at least 3 repeats of each experiment in data analysis. Also, they could include an analysis of sequence differences among VCAs and discuss how these may correlate with the observed differences. For instance, one WH2 in WASP vs. two in N-WASP.

Indeed, we argue that the two forms of activated Arp2/3 differ in their sensitivity to different VCA motifs, based on how these VCA motifs rank in their ability to destabilize branched and linear filaments (the VCA motifs also rank differently in their activation and co-activation of Arp2/3 to nucleate branches and linear filaments, but this result does not contribute to our discussion of how proteins interact with the activated Arp2/3). Following the reviewer’s suggestion, we now show repeats of these experiments (new Supp Fig S2), clearly showing that N-WASP is the most effective in dissociating linear filaments while the differences are milder for dissociating branches, with WASH being at least as effective as NWASP. We now also discuss how this observation could relate to differences in sequence between VCAs (Discussion section and new Supp Fig S9).

Also, please note that, following a suggestion from Reviewer 3, we have now performed experiments with the CA-domains of NWASP (new Supp Fig S4C and S4D), which show that the V-domain plays an important role in debranching but plays no role in destabilizing SPIN90-Arp2/3 at filament pointed ends. These new results reinforce our statement that VCA affects branched and linear Arp2/3-generated filaments differently.

Reviewer #2 (Significance (Required)):

Arp2/3 complex is a 7-protein complex implicated in actin filament nucleation and branching. Arp2/3 complex-nucleated branched networks are found at several locations in cells and are responsible for processes such as cell motility.

Cao et al. compare the effect of several proteins on the filament nucleation activity of Arp2/3 complex, and the stabilization or destabilization of actin filament branches as well as linear actin filaments nucleated by SPIN90-Arp2/3 complex. The proteins tested include the VCA regions of three NPFs (N-WASP, WASP, and WASH) that activate Arp2/3 complex, GMF (a debranching protein) and cortactin (a branch stabilizing protein). For the most part, the study uses a single method, microfluidics-TIRF microscopy.

The main findings are:

1. VCA domains enhance nucleation of linear filaments by SPIN90-Arp2/3 complex in the presence of actin monomers.
2. However, VCA domains can also destabilize existing SPIN90-Arp2/3 complex linear filaments and branches, and this effect depends on the presence of of V-domain (WH2 domain that binds actin monomers).
3. The debranching factor GMF also destabilizes SPIN90-Arp2/3 complex linear filaments. Both GMF and VCA generate free pointed ends by dissociating Arp2/3 complex from pointed ends and SPIN90.
4. SPIN90-Arp2/3 complex linear filaments are less susceptible to force and aging than filament branches.
5. Cortactin stabilizes SPIN90-Arp2/3 complex linear filaments to higher degree than it does branches. These are novel and very interesting new observations of significant interest to the actin cytoskeleton field. Therefore, I recommend publication of this paper in EMBO J.

We thank the reviewer for their positive evaluation of our work.

I have one recommendation and one suggestion for improvement:

Major:

1. The quantitative analysis can be improved. It appears that most of the data results from single experiments, with rate values and errors resulting from fitting of single experiments without repetitions. In Fig. 1C legend (p.5) the authors state "These experiments were repeated three times, with similar results", but the data is not used in the analysis and other experiments do not mention this point. This is particularly important for comparisons among different VCAs that are rather similar in nature. In Fig. 1B. N-WASP is more efficient in nucleating SPIN90-Arp2/3 complex-linear filaments followed by WASP and then WASH. In Fig. 2 B,C, N-WASP is the most effective in dissociating SPIN90-Arp2/3 complex linear filaments followed by WASH and then WASP. But in Fig. 2 E,F, WASH is by far the most effective in dissociating branches followed by N-WASP and then WASP. Therefore, the conclusion in the Discussion (p.12) "While these regulatory proteins similarly affect branched and linear Arp2/3-generated filaments, they do so with clear quantitative differences" is not supported by quantification. To remedy this problem the authors should include at least 3 repeats of each experiment in data analysis. Also, they could include an analysis of sequence differences among VCAs and discuss how these may correlate with the observed differences. For instance, one WH2 in WASP vs. two in N-WASP.

This comment is identical to the reviewer’s first paragraph. We copy our answer here again, for convenience:

Indeed, we argue that the two forms of activated Arp2/3 differ in their sensitivity to different VCA motifs, based on how these VCA motifs rank in their ability to destabilize branched and linear filaments (the VCA motifs also rank differently in their activation and co-activation of Arp2/3 to nucleate branches and linear filaments, but this result does not contribute to our discussion of how proteins interact with the activated Arp2/3). Following the reviewer’s suggestion, we now show repeats of these experiments (new Supp Fig S2), clearly showing that N-WASP is the most effective in dissociating linear filaments while the differences are milder for dissociating branches, with WASH being at least as effective as NWASP. We now also discuss how this observation could relate to differences in sequence between VCAs (Discussion section and new Supp Fig S9).

Also, please note that, following a suggestion from Reviewer 3, we have now performed experiments with the CA-domains of NWASP (new Supp Fig S4C and S4D), which show that the V-domain plays an important role in debranching but plays no role in destabilizing SPIN90-Arp2/3 at filament pointed ends. These new results reinforce our statement that VCA affects branched and linear Arp2/3-generated filaments differently.

Minor:

In GST-pull-down experiments (Fig. 4G), the amount of Arp2/3 complex bound is analyzed by Western, which is rather unprecise. Is the amount of Arp2/3 complex so little that it cannot be quantified using regular SDS-PAGE? If that is the case, this would suggest rather low affinity of SPIN90 for Arp2/3 complex. How does this affect the proposed mechanism and experiments in the microfluidics chamber?

Indeed, the amount of pulled-down Arp2/3 is low and difficult to quantify by SDS-PAGE. This is consistent with previous reports which indicate a low affinity of SPIN90 for the Arp2/3 complex (Wagner et al. Current Biology 2013, Balzer et al. eLife 2020). This does not affect our conclusions, which we now confirm by showing quantified repeats of our pull-down experiments (new panels H and I, in Figure 4). In spite of this low affinity, which makes it difficult to saturate SPIN90 with Arp2/3, the SPIN90-Arp2/3 interaction is very stable and allows us to carry out our experiments in the microfluidics chamber over several tens of minutes (as was already the case in our previous study, Cao et al. NCB 2020).

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary:

In this study, Cao and collaborators investigate the biochemical and mechanical differences between branched actin filaments nucleated by WASP-activated Arp2/3 complex and linear actin filaments nucleated by SPIN90-activated Arp2/3 complex. They use TIRF microscopy in a microfluidic chamber to show that the mammalian proteins, SPIN90 and WASP (or N-WASP or WAVE), like their yeast homologues, co-activate Arp2/3 complex to nucleate linear actin filaments. Using the same assays, they find the surprising result that the VCA segment of WASP proteins destabilizes the interaction between SPIN90 and Arp2/3 complex in linear actin filaments nucleated by Arp2/3 complex. They then show that VCA also destabilizes actin filament branches. The remainder of the study explores the influence of branch stabilizing/destabilizing proteins or mechanical stress on the stability of the interaction between SPIN90 and Arp2/3 complex on the pointed end of the actin filament. They find that like branch junctions, SPIN90-bound Arp2/3 is destabilized at the end of linear filaments by GMF and stabilized by cortactin. However, unlike branch junctions, SPIN90-Arp2/3 complex is not destabilized on filament ends by piconewton forces or by aging. They conclude that SPIN90- versus VCA-activated Arp2/3 complex adopt similar but non-identical conformations.

Overall, the paper is well written and the experiments, which are very challenging, are rigorously executed. The biochemical results are convincing, novel and unexpected. However, the work could be strengthened by more strongly connecting the biochemical observations to biological implications. In addition, there are some interpretations/conclusions that seem somewhat weakly supported, and the authors should consider revising. Nonetheless, given the quality of the work and the importance of the system, this manuscript will appeal to a broad audience.

We thank the reviewer for their positive comments. We have rewritten parts of the Discussion in order to better connect our observations to implications in cells. We address the concerns regarding our interpretations in the point-by-point, below.

Comments on evidence, reproducibility, clarity and significance:

The differences in the stability of SPIN90-Arp2/3 on linear filaments verses branch junctions led the authors to conclude that SPIN90- versus VCA-activated complexes adopt similar yet non-identical conformations. There are two problems with this conclusion:

1) This conclusion rests on the idea that the biochemical differences can only be due to differences in the "ground state" active conformations of the complex. Another possible scenario would be that the active conformations are the same, but the transition state or intermediate state structures within the debranching reactions are different, thus changing the kinetics of the debranching reactions.

We thank the reviewer for this remark, and we agree that conformational differences may also arise in the intermediate states, during dissociation (of the branch from the mother, or of the linear filaments from SPIN90). We now mention this possibility in our Discussion.

2.) There are already structural data showing conformational differences between the Dip1-bound Arp2/3 complex on the end of a linear filament and Arp2/3 complex at a branch junction. While there are some caveats to comparisons of the structures (e.g., the Dip1 structure includes the fission yeast SPIN90 protein (Dip1) and the fission yeast Arp2/3 complex while the branch junction contains mammalian proteins), these data offer much stronger evidence that the active states adopt (somewhat) different conformations than the data presented here.

We agree that the available structural data (in particular, Ding et al. PNAS 2022, which was not yet published when we submitted our manuscript, and which we now cite) provide a clear indication that active Arp2/3 adopts different conformations in branches and linear filaments. We have modified our text to make this point clearer.

The authors make comparisons between the Fäβler branch junction structure and the Shaaban Dip1-Arp2/3-filament structure. The Fäβler branch junction structure is a low resolution structure (9 angstroms) and should be interpreted with caution (see below). A much higher resolution of a branch junction structure was recently solved (Ding et al, PNAS 2022) and should be used for comparisons between the structures.

Ding et al. PNAS 2022 was not yet published when we submitted our manuscript. We now use it to compare the structures of active Arp2/3, and we have modified the text accordingly.

Pg 14 - The authors say differences between ARPC3-Arp2 and ARPC5-Arp2 contacts in the two structures are likely to cause the differences in interactions with GMF and VCA. Two concerns with this statement are: 1.) The basis for the conclusion that the ARPC5-Arp2 contacts are different (in Fäβler, et al.) is not solid (see Ding, et al) and 2.) The analysis is vague. To reasonably conclude that differences in the contacts would influence GMF and VCA interactions would require mapping out the structural connection between the ARPC3-Arp2 interaction site and the GMF or VCA binding sites. If there is no obvious connection between these sites, the conclusion that the differences in the ARPC3-Arp2 interface cause differences in VCA and GMF binding should be far more circumspect.

We have re-written this part of the Discussion section. In light of the new data by Ding et al., we agree with the reviewer that the conclusion that the ARPC5-Arp2 contacts are different is not solid. Our revised text makes it clear that we are not making any claims involving interactions within the Arp2/3 complex. Our point is simply that recent cryo-EM reports indicate conformational differences in Arp2 and Arp3 between the two activated forms of the Arp2/3 complex and that, since the CA-domain of NPFs bind to Arp2 and Arp3, it appears reasonable to make a connection with our results.

Pg 6. "These observations suggest that the ability of VCA to destabilize Arp2/3-nucleated filaments relies on the availability of its V-domain." It's possible that G-actin binding to V blocks the CA from accessing the branch junction. Therefore, it seems important to test whether N-WASP-CA can destabilize Arp2/3-nucleated actin filaments.

We thank the reviewer for this suggestion. We now present results from new experiments performed with the CA-domain of NWASP (new Supp Fig S4C,D). We find that the V-domain participates in the enhancement of debranching, but that it appears to play no role in the destabilization of SPIN90-Arp2/3 from the pointed end. It thus seems that the reviewer’s proposal is correct, and that G-actin binding to the V-domain blocks the CA-domain from accessing the branch junction. We now propose this interpretation in the text.

Pg 1 - The authors state that "It thus appears that linear and branched Arp2/3-generated filaments respond similarly to regulatory proteins, albeit with quantitative differences". It is worth considering if one should make a blanket statement that linear and branched filaments respond similarly to regulatory proteins when they have tested 3 in total.

We have rephrased this sentence. It now reads “… respond similarly to the regulatory proteins we have tested…”

Pg 3 - "More generally, the stability of SPIN90-Arp2/3 at the pointed end, which is important to understand the reorganization and disassembly of actin filament networks, remains to be established." In some ways this statement not quite accurate because Balzer et al previously showed that Dip1-Arp2/3 complex is very stable at the pointed end. Is the question here whether that stability is also conserved in mammalian systems? If so, that should be more directly stated.

We meant that, beyond observing that SPIN90 remains visible at the pointed end for some time (as in Balzer et al.), a lot remained unknown: its lifetime had not been quantified, and its sensitivity to the factors that affect branch junctions (proteins, aging, mechanical tension) had not been studied. We have rephrased the sentence in the manuscript to clarify this point.

The observation that VCA accelerates debranching and SPIN90-Arp2/3 dissociation is very interesting. However, it is uncertain if this biochemical activity has biological relevance, given that once nucleation occurs, Arp2/3 complex will move away from the membrane. While the authors mention in the discussion that debranching by VCA could be relevant when the network is compressed near the membrane, this argument is not particularly strong. Are there ways to strengthen this argument, or find another impact this finding might have on our understanding of Arp2/3 complex regulation?

We now mention another situation where branch junctions could encounter membrane-bound VCA domains: on the dorsal and ventral membrane surfaces of lamellipodia. We now cite the recent Kage et al. J Cell Science 2022 and Mehidi et al. NCB 2021, where WAVE has been observed in lamellipodia away from the leading edge.

The observation that SPIN90+Arp2/3-nucleated filaments are not sensitive to piconewton forces is also very interesting. The authors focus on the differences in the amount of surface area buried when discussing this result. However, if seems a key factor in the stability of the linear filaments would be the direction of the force relative to the complex and attached filament(s), which would be very different for a branch versus a linear filament. The authors should consider addressing this in their discussion.

The orientation of the applied force is an interesting point. In their study on debranching, Pandit et al. (PNAS 2020) report that their results are not affected by the angle of the applied force relative to the mother filament (their Fig S1D). We now specify this in our manuscript, when introducing our results on mechanical tension. Similarly, we found that anchoring SPIN90 to the coverslip surface by its N-terminus rather than its C-terminus, which likely affects the orientation of the applied force, had no impact on our results (Supp Fig S6A). We have now also added a sentence regarding this aspect in our manuscript, after presenting this result.

Fig 4, D-F: It is unclear how the authors determined which filaments were spontaneously nucleated versus those that were nucleated by SPIN90-Arp2/3 complex in these experiments. In reactions containing SPIN90 and Arp2/3 complex what fraction of the filaments will be spontaneously nucleated?

In our conditions, there is no detectable spontaneous nucleation. In control experiments where we flow in the same concentration of G-actin, in the absence of Arp2/3 or in the absence of SPIN90, we observe no filaments at all on the surface, over several fields of view, after 5 minutes. We now specify this in the Methods section.

Pg 9 - The observation that VCA negatively influences binding of SPIN90 to the complex is unexpected. What implications does this have for understanding how SPIN90 and VCA synergize to activate the complex?

It appears that the outcome depends on the context. The main role of VCA during co-activation of the Arp2/3 complex with SPIN90 seems to be to supply G-actin, as already proposed (Balzer, 2020) and confirmed by our results (Fig 1C). In the absence of G-actin, VCA is more likely to remove Arp2/3 from SPIN90 (Fig 4G,I). Similarly, when a filament is already formed, the presence of G-actin mitigates the removal of SPIN90-Arp2/3 from the pointed end by VCA (Supp Fig S4).

Fig 4B - Why is there greater nucleation when Arp2/3 complex and GMF are added together compared to renucleation in reactions that don't have any GMF? This is surprising, especially considering that GMF decreases binding of Arp2/3 complex to SPIN90.

Indeed, there is a small yet statistically significant difference in the re-nucleation fraction we measured in the presence of Arp2/3, with or without GMF (Fig 4B). This may be due to the different timescales of the two situations. In the absence of GMF, the detachment of filaments is slow and new filaments are nucleated from the initial Arp2/3 complexes, which remained bound to SPIN90 upon detachment of the first filaments. In contrast, in the presence of GMF, detachment is faster and accompanied by the departure of the initial Arp2/3, and a fresh Arp2/3 then binds to SPIN90 to nucleate a new filament. It is thus possible that, in the absence of GMF, a small fraction of the SPIN90 and/or their initially bound Arp2/3 complexes would denature over the time they spend at the bottom of the microchamber at 25°C, thereby leading to a slightly smaller re-nucleation fraction. A similar mechanism could be at play in the experiments with or without VCA, in addition to the enhancement of nucleation by VCA (Fig 4C).

Minor Corrections/Comments

Pg 3 "We show that Arp2/3 nucleation is similarly stabilized by cortactin and destabilized by GMF" Do the authors mean branches and linear filaments nucleated by Arp2/3 complex?

Yes, that is what we meant. This sentence has now been modified.

Pg 6- The cyan 3uM data and legend in figure 2B and E is probably too dim to see clearly.

The colors have been changed to improve readability.

Fig 4 B,C,E,F: It would be best to show the individual data points here if possible.

We now show individual data points in all these figure panels.

Pg 16 Please specify which antibody was used to anchor SPIN90.

The antibodies are Anti-GST for Nter anchoring of GST-SPIN90, and anti-His for Cter anchoring of SPIN90-His. We now specify this in the Methods section.

CROSS-CONSULTATION COMMENTS I agree with the points that the other reviewers raised.

Reviewer #3 (Significance (Required)):

Comments on significance are in the above section.

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Referee #3

Evidence, reproducibility and clarity

Summary:

In this study, Cao and collaborators investigate the biochemical and mechanical differences between branched actin filaments nucleated by WASP-activated Arp2/3 complex and linear actin filaments nucleated by SPIN90-activated Arp2/3 complex. They use TIRF microscopy in a microfluidic chamber to show that the mammalian proteins, SPIN90 and WASP (or N-WASP or WAVE), like their yeast homologues, co-activate Arp2/3 complex to nucleate linear actin filaments. Using the same assays, they find the surprising result that the VCA segment of WASP proteins destabilizes the interaction between SPIN90 and Arp2/3 complex in linear actin filaments nucleated by Arp2/3 complex. They then show that VCA also destabilizes actin filament branches. The remainder of the study explores the influence of branch stabilizing/destabilizing proteins or mechanical stress on the stability of the interaction between SPIN90 and Arp2/3 complex on the pointed end of the actin filament. They find that like branch junctions, SPIN90-bound Arp2/3 is destabilized at the end of linear filaments by GMF and stabilized by cortactin. However, unlike branch junctions, SPIN90-Arp2/3 complex is not destabilized on filament ends by piconewton forces or by aging. They conclude that SPIN90- versus VCA-activated Arp2/3 complex adopt similar but non-identical conformations.

Overall, the paper is well written and the experiments, which are very challenging, are rigorously executed. The biochemical results are convincing, novel and unexpected. However, the work could be strengthened by more strongly connecting the biochemical observations to biological implications. In addition, there are some interpretations/conclusions that seem somewhat weakly supported, and the authors should consider revising. Nonetheless, given the quality of the work and the importance of the system, this manuscript will appeal to a broad audience.

Comments on evidence, reproducibility, clarity and significance:

The differences in the stability of SPIN90-Arp2/3 on linear filaments verses branch junctions led the authors to conclude that SPIN90- versus VCA-activated complexes adopt similar yet non-identical conformations. There are two problems with this conclusion:

1. This conclusion rests on the idea that the biochemical differences can only be due to differences in the "ground state" active conformations of the complex. Another possible scenario would be that the active conformations are the same, but the transition state or intermediate state structures within the debranching reactions are different, thus changing the kinetics of the debranching reactions.
2. There are already structural data showing conformational differences between the Dip1-bound Arp2/3 complex on the end of a linear filament and Arp2/3 complex at a branch junction. While there are some caveats to comparisons of the structures (e.g., the Dip1 structure includes the fission yeast SPIN90 protein (Dip1) and the fission yeast Arp2/3 complex while the branch junction contains mammalian proteins), these data offer much stronger evidence that the active states adopt (somewhat) different conformations than the data presented here.

The authors make comparisons between the Fäβler branch junction structure and the Shaaban Dip1-Arp2/3-filament structure. The Fäβler branch junction structure is a low resolution structure (9 angstroms) and should be interpreted with caution (see below). A much higher resolution of a branch junction structure was recently solved (Ding et al, PNAS 2022) and should be used for comparisons between the structures.

Pg 14 - The authors say differences between ARPC3-Arp2 and ARPC5-Arp2 contacts in the two structures are likely to cause the differences in interactions with GMF and VCA. Two concerns with this statement are: 1.) The basis for the conclusion that the ARPC5-Arp2 contacts are different (in Fäβler, et al.) is not solid (see Ding, et al) and 2.) The analysis is vague. To reasonably conclude that differences in the contacts would influence GMF and VCA interactions would require mapping out the structural connection between the ARPC3-Arp2 interaction site and the GMF or VCA binding sites. If there is no obvious connection between these sites, the conclusion that the differences in the ARPC3-Arp2 interface cause differences in VCA and GMF binding should be far more circumspect.

Pg 6. "These observations suggest that the ability of VCA to destabilize Arp2/3-nucleated filaments relies on the availability of its V-domain." It's possible that G-actin binding to V blocks the CA from accessing the branch junction. Therefore, it seems important to test whether N-WASP-CA can destabilize Arp2/3-nucleated actin filaments.

Pg 1 - The authors state that "It thus appears that linear and branched Arp2/3-generated filaments respond similarly to regulatory proteins, albeit with quantitative differences". It is worth considering if one should make a blanket statement that linear and branched filaments respond similarly to regulatory proteins when they have tested 3 in total.

Pg 3 - "More generally, the stability of SPIN90-Arp2/3 at the pointed end, which is important to understand the reorganization and disassembly of actin filament networks, remains to be established." In some ways this statement not quite accurate because Balzer et al previously showed that Dip1-Arp2/3 complex is very stable at the pointed end. Is the question here whether that stability is also conserved in mammalian systems? If so, that should be more directly stated.

The observation that VCA accelerates debranching and SPIN90-Arp2/3 dissociation is very interesting. However, it is uncertain if this biochemical activity has biological relevance, given that once nucleation occurs, Arp2/3 complex will move away from the membrane. While the authors mention in the discussion that debranching by VCA could be relevant when the network is compressed near the membrane, this argument is not particularly strong. Are there ways to strengthen this argument, or find another impact this finding might have on our understanding of Arp2/3 complex regulation?

The observation that SPIN90+Arp2/3-nucleated filaments are not sensitive to piconewton forces is also very interesting. The authors focus on the differences in the amount of surface area buried when discussing this result. However, if seems a key factor in the stability of the linear filaments would be the direction of the force relative to the complex and attached filament(s), which would be very different for a branch versus a linear filament. The authors should consider addressing this in their discussion.

Fig 4, D-F: It is unclear how the authors determined which filaments were spontaneously nucleated versus those that were nucleated by SPIN90-Arp2/3 complex in these experiments. In reactions containing SPIN90 and Arp2/3 complex what fraction of the filaments will be spontaneously nucleated?

Pg 9 - The observation that VCA negatively influences binding of SPIN90 to the complex is unexpected. What implications does this have for understanding how SPIN90 and VCA synergize to activate the complex?

Fig 4B - Why is there greater nucleation when Arp2/3 complex and GMF are added together compared to renucleation in reactions that don't have any GMF? This is surprising, especially considering that GMF decreases binding of Arp2/3 complex to SPIN90.

Minor Corrections/Comments

Pg 3 "We show that Arp2/3 nucleation is similarly stabilized by cortactin and destabilized by GMF" Do the authors mean branches and linear filaments nucleated by Arp2/3 complex?

Pg 6- The cyan 3uM data and legend in figure 2B and E is probably too dim to see clearly.

Fig 4 B,C,E,F: It would be best to show the individual data points here if possible.

Pg 16 Please specify which antibody was used to anchor SPIN90.

Referees cross-commenting

I agree with the points that the other reviewers raised.

Significance

Comments on significance are in the above section.

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Referee #2

Evidence, reproducibility and clarity

The quantitative analysis can be improved. It appears that most of the data results from single experiments, with rate values and errors resulting from fitting of single experiments without repetitions. In Fig. 1C legend (p.5) the authors state "These experiments were repeated three times, with similar results", but the data is not used in the analysis and other experiments do not mention this point. This is particularly important for comparisons among different VCAs that are rather similar in nature. In Fig. 1B. N-WASP is more efficient in nucleating SPIN90-Arp2/3 complex-linear filaments followed by WASP and then WASH. In Fig. 2 B,C, N-WASP is the most effective in dissociating SPIN90-Arp2/3 complex linear filaments followed by WASH and then WASP. But in Fig. 2 E,F, WASH is by far the most effective in dissociating branches followed by N-WASP and then WASP. Therefore, the conclusion in the Discussion (p.12) "While these regulatory proteins similarly affect branched and linear Arp2/3-generated filaments, they do so with clear quantitative differences" is not supported by quantification. To remedy this problem the authors should include at least 3 repeats of each experiment in data analysis. Also, they could include an analysis of sequence differences among VCAs and discuss how these may correlate with the observed differences. For instance, one WH2 in WASP vs. two in N-WASP.

Significance

Arp2/3 complex is a 7-protein complex implicated in actin filament nucleation and branching. Arp2/3 complex-nucleated branched networks are found at several locations in cells and are responsible for processes such as cell motility.

Cao et al. compare the effect of several proteins on the filament nucleation activity of Arp2/3 complex, and the stabilization or destabilization of actin filament branches as well as linear actin filaments nucleated by SPIN90-Arp2/3 complex. The proteins tested include the VCA regions of three NPFs (N-WASP, WASP, and WASH) that activate Arp2/3 complex, GMF (a debranching protein) and cortactin (a branch stabilizing protein). For the most part, the study uses a single method, microfluidics-TIRF microscopy.

The main findings are:

1. VCA domains enhance nucleation of linear filaments by SPIN90-Arp2/3 complex in the presence of actin monomers.
2. However, VCA domains can also destabilize existing SPIN90-Arp2/3 complex linear filaments and branches, and this effect depends on the presence of of V-domain (WH2 domain that binds actin monomers).
3. The debranching factor GMF also destabilizes SPIN90-Arp2/3 complex linear filaments. Both GMF and VCA generate free pointed ends by dissociating Arp2/3 complex from pointed ends and SPIN90.
4. SPIN90-Arp2/3 complex linear filaments are less susceptible to force and aging than filament branches.
5. Cortactin stabilizes SPIN90-Arp2/3 complex linear filaments to higher degree than it does branches.

These are novel and very interesting new observations of significant interest to the actin cytoskeleton field. Therefore, I recommend publication of this paper in EMBO J. I have one recommendation and one suggestion for improvement:

Major:

1. The quantitative analysis can be improved. It appears that most of the data results from single experiments, with rate values and errors resulting from fitting of single experiments without repetitions. In Fig. 1C legend (p.5) the authors state "These experiments were repeated three times, with similar results", but the data is not used in the analysis and other experiments do not mention this point. This is particularly important for comparisons among different VCAs that are rather similar in nature. In Fig. 1B. N-WASP is more efficient in nucleating SPIN90-Arp2/3 complex-linear filaments followed by WASP and then WASH. In Fig. 2 B,C, N-WASP is the most effective in dissociating SPIN90-Arp2/3 complex linear filaments followed by WASH and then WASP. But in Fig. 2 E,F, WASH is by far the most effective in dissociating branches followed by N-WASP and then WASP. Therefore, the conclusion in the Discussion (p.12) "While these regulatory proteins similarly affect branched and linear Arp2/3-generated filaments, they do so with clear quantitative differences" is not supported by quantification. To remedy this problem the authors should include at least 3 repeats of each experiment in data analysis. Also, they could include an analysis of sequence differences among VCAs and discuss how these may correlate with the observed differences. For instance, one WH2 in WASP vs. two in N-WASP.

Minor:

1. In GST-pull-down experiments (Fig. 4G), the amount of Arp2/3 complex bound is analyzed by Western, which is rather unprecise. Is the amount of Arp2/3 complex so little that it cannot be quantified using regular SDS-PAGE? If that is the case, this would suggest rather low affinity of SPIN90 for Arp2/3 complex. How does this affect the proposed mechanism and experiments in the microfluidics chamber?

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Referee #1

Evidence, reproducibility and clarity

An exciting development in our knowledge about how the Arp2/3 complex controls the assembly of actin networks has come from the discovery that in addition to forming branched networks, Arp2/3 can nucleate linear filaments when it is activated by WISH/DIP/SPIN90. However, despite some excellent work largely done by the Nolen lab in yeast, many questions remain about how Arp2/3-mediated assembly of branched vs. linear actin filament. This is especially true in the complex environment of cells, were synergy and competition of different actin networks is used to control biological processes. Knowing the biochemical and physical properties of these different Arp2/3 assemblies will be key to figuring out how they work in cells. Here Cao et al. use an elegant microfluidics based single filament assay system to perform a comparative analysis of the stability of linear and branched Arp2/3 networks. They find interesting differences in how they respond to stabilizing and destabilizing factors. The most striking differences happens when force or aging is applied- both cause debranching of branched networks but have little effect on Spin90-Arp2/3 nucleated filaments.

Major comments:

As a comparative study on the stability of branched vs. linear Arp2/3 nucleated filaments, this manuscript is fairly complete. The key conclusions are well supported by rigorous experiments which can be reproduced by others based on the information provided. However, I am not seeing explicit information on performing biological replicates. This should be included in the manuscript. The use of statistics is largely fine; however I question the use of one statistical test on one figure (see minor comments below).

I would not ask for additional experiments at this time. However, there is an analysis that would be important for interpreting the authors' claims- branch/filament length at the time of dissociation or destabilization of Arp2/3. This would help address if there was a physical tipping point for each type of structure that could explain potential differences they see. The authors should already have this data and the time to complete it would be negligible in delaying publication.

One additional major comment is that the manuscript's title and abstract hint that this paper explores the differences in nucleation of branched vs. linear filaments by Arp2/3. However, the only figure that deals explicitly with nucleation in the paper is Figure 1, which is really just a confirmation that the mammalian proteins used in this study perform similarly to their yeast homologues (Balzer et al, Current Biology 2019). The authors might think about rewording the title/abstract to better reflect that paper really explores the differences in the stability of the two networks

Minor comments:

1 in 12 men and 1 in 200 women are red/green colorblind. Please change the coloring of the schematics and images so that they can be easily seen by all people. This is especially true of the schematics, which are important for understanding exactly what each assay is measuring.

The Introduction is a bit choppy and unfocused. It was difficult to deduce exactly where the paper was going from it. Please consider re-writing it for better clarity. The Discussion on the other hand was fantastic. Great job on interpreting your results in a larger context.

Many figures- while the use of different lightness values of the same color is appreciated in conveying different concentrations of reagents used, there were several instances where it was very hard to read the one on the very bottom (ex. 2B, E; 3A; 5C, G).

Figure 1- since this is a confirmation of previous results performed using the same proteins from other species, the title should reflect that (ex. VCA domains accelerate the nucleation of filaments by mammalian SPIN90-Arp2/3). Also, to me this figure is supplementary to the main message of the paper. The authors might think of moving it to Supplementary Information.

Figure 1- If the goal was to verify that G-actin recruitment by VCA was important for Spin90-Arp 2/3 nucleation by performing a competition experiment with profilin, why was the concentration of G-actin AND profilin increased between the experiments in 1B vs. 1C. It makes it hard to directly compare the results.

Figure 4B-F- Here, it would be nice to see the distribution of all the individual results, which are hidden by the bar graph. Additionally, the Chi-square test is not the appropriate test for evaluating statistical significance between multiple groups. ANOVA followed by an appropriate post hoc test should be used here.

Figure 4G- Please quantify and show reproducibility.

Figure 5- the piconewton forces used for these experiments is in line with measured forces that are applied to actin in cells (ex. Mehida et al, Nature Cell Biology 2021; Jiang et al, Nature 2003). The text would benefit if this was explicitly stated.

Significance

The real significance of this work is in characterizing the differential stabilities of linear vs. branched Arp2/3 filaments in response to actin-binding proteins, mechanical stress, and aging. While both types of filaments respond similarly to actin-binding proteins, with nuanced differences, the most striking results came from applied force and aging experiments, with Spin90-Arp2/3 filaments being much more resistant to both. This has some very interesting implications for how these two types of assemblies might synergize in cells. Additionally, the results also have some exciting implications for the pointed-end regulation of actin filaments, which is still poorly understood in complex systems. Since the manuscript is A) more of a survey study on the factors that influence filament stability that does not go particularly deep into any particular mechanism of regulation and B) has no direct applicability to how the physical properties of branched and linear Arp2/3 nucleated actin filaments influence actin network activity in cells, the audience will likely by limited to actin enthusiasts. However, the work is still important in both what it reveals and implies.

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Reply to the reviewers

We thank all three reviewers for their thoughtful and rigorous critique of our manuscript, which we feel has significantly improved the presentation of our work. Below we detail point-by-point responses to comments made by the three reviewers as well changes we have already made addressing the majority of minor and some major points.

Specification of the eye-field during gastrulation represents the earliest known stage of eye development. Using an optic-vesicle organoid model system, the overall goal of our work is to provide an unbiased characterisation of this critical, early developmental event in mammals and to gain insights into relevant gene regulatory mechanisms. A common theme to some of the reviewer comments is that this work doesn't provide much of an advance to the field and our findings are not particularly original. We feel that these comments are slightly harsh for the following reasons. Firstly, although some of our findings are not unexpected, to our knowledge, this is the first unbiased characterisation of the eye-field in a mammalian model system, and not based on knowledge gained through previous work in other non-mammalian vertebrate systems, e.g. Xenopus. Secondly, by generating both RNA-seq and ATAC-seq from a timecourse of organoid development we have been able to quantify dynamic patterns of gene-expression as the eye-field is established and simultaneously gain insights to the regulatory role of some of the key transcription factors, both of which are not present in the literature. Thirdly, by constructing careful, integrated analyses of our RNA-seq and ATAC-seq datasets we were able to generate specific hypotheses regarding cis-regulation of key genes, which we have then demonstrated are possible to efficiently test within the organoid system. In all, although we have been purposely careful not to overinterpret our results, we feel our work does represent a significant step towards understanding the mammalian eye field and additionally provides important datasets as well as an analysis framework to begin to quantitatively probe the regulatory mechanisms underlying the transition to an ocular fate. Given the relevance of this developmental event to clinical genetics research as well as to developmental biology we are confident that this work represents an important and significant advance to the literature.

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary Owen et al. characterize the transcriptome and chromatin accessibility of mouse retinal organoids at early stages during which eye field-like cells are specified. Since cell specification and differentiation in retinal organoids largely mimic those processes in vivo, retinal organoids are viable models for studying the mechanisms of early eye development. Owen et al. utilize a previously established Rx-GFP cell line, bulk RNA sequencing, and bulk ATAC sequencing to dissect the mechanisms of early eye development in mice. Their findings are generally consistent with previous studies. Overall, the study is interesting for the field, but its conceptual and technical advances are moderate. In addition, a few major points need to be clarified.

Major points 1. The authors did not show any analysis of retinal organoids at stages when Vsx2 is expressed. This is a significant weakness since the chemically defined medium (CDM) used in Owen et al.'s study was previously shown to induce rostral hypothalamic differentiation (Wataya et al., 2008). Related to this notion, several eye-field transcription factors, such as Rax and Six3, are also expressed in the hypothalamus. Therefore, Owen et al. need to demonstrate that organoids in their modified differentiation system efficiently produce Vsx2-positive retinal progenitors, and samples of organoids at stages when Vsx2 is expressed should be included for RNA sequencing. If Vsx2 is not efficiently expressed in their organoids, the interpretation of results will be very different.

We thank the reviewer for their important comments here. There are several reasons why we are confident that our data and conclusions regarding the organoid eye-field are robust. Firstly, our RNA-seq data, in particular the differences between GFP-positive and GFP-negative cells, clearly show a coordinated up-regulation of the set of canonical eye-field TFs (not individually), which previous studies in Xenopus have shown is a prerequisite for differentiation into anterior eye structures (including retina). Secondly, we have checked that some of the later (in development) eye markers, including Vsx2, are differentially up-regulated (DeSeq2, logfc>1.5, FDRIn all, we are very confident that our approach of using the optic-vesicle organoids and generating molecular data from an organoid developmental timecourse (including sorting), is unpicking the ocular-fate transition event that we are interested in.

1. The authors state that "two differentiation medias were used for this work due to the differentiation becoming unstable after the initial experiments had been performed. The organoids used for RNA and ATAC-seq were grown in CDM media and the organoids with mutations introduced in potential CREs were grown in KSR media". Why the differentiation becomes unstable after the initial experiments? Differences in the two media cause additional complexities. Related to this notion, "WT Rx-GFP" in Figure 4B and 4E appears to show a different expression pattern compared to that in Figure 1A.

We were unable to identify the reason behind the destabilisation of differentiation in CDM media after the cell lines had been through CRISPR despite thorough testing. The differentiation of these cell lines was stabilised enough using KSR media such that every batch of organoids grown contained some organoids that expressed GFP in a pattern similar to what we had seen before and we carried on our experiments using this. We recognise that using two different media adds complexity, however we see the same patterns of organoid growth and GFP expression when differentiating untransfected WT Rax-GFP cells in both of these medias. We have edited Fig.S1 to include representative images of organoids grown in KSR media which can be directly compared to those grown in CDM shown in Figure 1A.

The reviewer has pointed out that the WT Rx-GFP organoids in Figure 6B and 6E show a different expression pattern to those in figure 1A. With the addition of the supplemental figure mentioned above it becomes apparent that these differences are not due to the change of media. We have clarified in the text that these WT cells have also been transfected so as to act as appropriate controls that have been treated identically to the CRISPR edited cell lines and that this has affected their differentiation capacity.

1. Is the deletion of Rax and Six6 regulatory elements homozygous? Sanger sequencing or amplicon sequencing is needed to show the deletion.

The deletions are homozygous (we have stated this in the manuscript text) and as suggested we have added a supplementary figure showing the Sanger sequencing traces for the WT and mutant cell lines used in this study.

1. The deletion of Rax and Six6 regulatory elements appears to cause minor changes in the expression of Rax and Six6 (Figure 6C, F). Therefore, the impact of findings in bulk RNA seq and bulk ATAC seq in this study is still unclear.

We have added a sentence to the text underlining that developmental genes are expected to be regulated by multiple enhancers. Our expectation is therefore, that in perturbing a single putative regulatory element for Rax/Six6, we will very likely not see the complete ablation of Rax/Six6 expression.

1. Retinal organoids and sorted cells are composed of heterogeneous cell populations. Bulk RNA seq and bulk ATAC seq do not have the power to dissect the complexity of heterogeneous cell populations. Single-cell RNA seq and single-cell ATAC seq are more powerful for this study.

We agree with the referee about the fact that the organoids are likely composed of relatively heterogeneous cell populations. We have added this limitation of our generated datasets in a “limitations” paragraph in the discussion.

1. Numerous motifs in the JASPAR database are identified using in vitro assays and have not been validated using in vivo assays. Unexpected results in motif analysis could be due to the differences in DNA binding motifs between in vitro and in vivo conditions. This notion should be added in the discussion.

We have added a couple of sentences in the discussion section, highlighting that TF-motif and footprinting analyses of ATAC-seq data provide indirect evidence of TF binding, and to validate these findings experiments such as ChIP-seq or Cut&Run could be performed in the future.

Minor points

Numerous labels in figures are too small.

We have adjusted the size of a number of the figures to increase the size of the labels, which are now mostly the same size as the text in the corresponding figure captions. We are very happy to make further increases in the sizes of figure labels/text upon recommendation.

CROSS-CONSULTATION COMMENTS

My fellow reviewers identify similar major weaknesses and additional points. I agree with the other reviewers' comments.

Reviewer #1 (Significance (Required)): Nature and Significance of the advances In Owen et al.'s study, the Rx-GFP cell line and retinal differentiation protocol were established in previous studies (Wataya et al., 2008; Eiraku et al., 2011); bulk RNA sequencing and bulk ATAC sequencing are standard procedures. Although candidate regulatory elements for early eye development are identified, deletions of two prioritized elements using CRISPR/Cas9 only cause minor changes in the expression of targeted genes. Overall, conceptual and technical advances in Owen et al.'s study are moderate. Compare to existing published knowledge The datasets could be useful for the field, but conceptual and technical advances are moderate.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

The authors grow eye organoids from cells with a reporter driving GFP in the Rax locus, a gene that is expressed in the eye field in many animal model systems. They show that expression of GFP picks up by day 4 and performed FACS sorting of GFP+ cells on day 4 and day 5 organoids to compare gene expression by RNAseq comparing with earlier day organoids. The data shows 37 genes with a differential expression on days 4 and 5, compared to day 3, and enriched in GFP+ cells, which they define as EF-up genes. It is notable that some of these genes had already been identified as canonical eye field gene regulatory network transcription factors. In the same way, they identify a group of differentially expressed regulated genes, EF-down, and state that 'many' of them are involved in pluripotency. However, they do not mention how many, or the proportion of these genes in the whole list.

The number of EF-down genes with GO terms linked to pluripotency has now been added to the text.

It would be useful if they could provide the number to understand how many of these genes are related to pluripotency, the whole list of genes mentioned to be downregulated in a supplementary file.

We appreciate that this list was missing and will include it now as a supplemental file.

The authors also note that genes known to be required for eye specification like Sox2 and Otx2 are not differentially expressed across the day 3-4 timepoint (Ln 190). However, this is not surprising considering that both genes are broadly expressed in the anterior neural ectoderm and required for its specification, which should be noted by the authors.

We have amended the aforementioned sentence to reflect this: “It is noteworthy that Sox2 and Otx2, known to be crucial in eye development are not differentially expressed across this critical time-point (Fig.2A), consistent with these genes being more broadly expressed in the anterior neuroectoderm in vivo.”

The authors then go on and cluster the EF-up, EF-down and genes deferentially expressed between days 2 and 3, and identify 6 discreet trajectory groups. From this analysis, they identify a third group of genes which shows a peak on day 3 but whose expression falls on days 4 and 5. It is interesting to see that this group includes Wnt and Fgf morphogenes. The authors should provide a list of the genes in the different clusters for the readers to inspect and analyse.

We note that there was a typo in the original manuscirpt and the genes that were clustered were the EF-up and EF-down genes. This typo has been fixed and the requested information is now available in a supplementary file.

Aiming to generate insight into the cis-regulatory elements that regulate of the genes the authors found differentially expressed in their model system they performed a series of ATAC-seq experiments. When linking the genomic regions with differential ATAC-seq accessibility to gene locus using the GREAT analysis, they identified association to 22 of the EF-up and 161 of the EF-down genes. This suggests a functional link between the ATAC-seq genomic regions and the gene regulation of the differentially expressed genes.

The authors later screened the ATAC-seq regions of increased accessibility for TF binding motifs and found that these regions were enriched with motifs for EFTF genes Rax, Lhx2 and Pax6. When assessing motifs in the ATAC-seq regions in EF-up TADs, Rax and Lhx2 motifs scored highly associated to open chromatin positions. Authors also observe a positive gene expression-accessibility correlation between in Pax6, Lhx2, Six3 and Otx2, and suggest this could mean these genes activate transcription of the EF-up group of genes. The same analysis, but focusing on EF-down genes, suggests that EFTFs repress the expression of EF-down genes which include those involved in pluripotency.

Further interrogating the ATAC-seq data, the authors use TOBIAS footprinting analysis to identify changes in TF binding in EF-TADs and EF-up motifs. Remarkably, whole genome analysis reveals that the largest increase in motif binding corresponds to EF-up genes Rax, Pax6 and Lhx2. The authors then narrow down on specific gene regulation by studying the ATAC-seq data within the TAD of Rax and Six6. However, they do not explain the rationale for which these two genes were highlighted, and why Pax6 or Lhx2 were excluded. This explanation should be added to the manuscript.

We have expanded this section of the manuscript to explain that Rax and Six6 were prioritised due to the GFP readout of Rax expression and Six6 being located in a smaller and thus less complex TAD than Pax6, Six3 and Lhx2 after the initial analysis was performed for all five TADs.

The analysis identifies three regulatory elements in the Rax TAD and two for Six6. They then go on and study one putative regulatory element of each gene and generate CRISPR deletions in cell lines. The rationale for the choice of these particular elements is not clear, nor if the cell lines are the same used for the RNAseq experiments. This information should be explicit in the results and in the methods section.

The manuscript has been updated to include the rationale behind our choice of the regulatory elements deleted.

The authors mention that the CRISPR cell lines are "considerably more variable" (Ln 822) compared to the previously studied organoids and suggest that no conclusions can be driven from GFP expression or morphology alone. However, they do not specify which is the variable trait. This information should be added to the text.

We have amended the text to include that the organoids are more variable in terms of the OV like structures produced and GFP expression level.

The authors also miss out on specifying the time stage of the organoids in figure 6 which should be stated.

We thank the reviewer for pointing this out and have updated the manuscript to contain the stage of the organoids.

Regardless, the wildtype organoids in figure 6 and figure S7 show a very different morphology and GFP expression compared to those in figure 1, suggesting that the conclusions from this last set of experiments are not reliable or comparable to those in figure 1. This, together with the fact that different reagents were used to grow the organoids for the RNAseq and the CRISPR experiments, is a weakness of this work that must be addressed.

We recognise this weakness however our amendments detailed above in response to reviewer 1’s comments, including adding a figure showing WT organoids grown in the KSR media that closely resemble the organoids in Fig.1A, removes the uncertainty that it is the change in media producing these differences in morphology and GFP expression.

Our aim in this section was to specifically test the hypotheses regarding the regulatory nature of the distal genomic regions identified by our intra-TAD analyses of ATAC-seq data. To do this it was important to compare organoids derived from wildtype and mutant cells that had been subjected to the same growth conditions and genomic-editing protocols. The stress associated with the latter is what we expect has resulted in the differences in morphology and GFP expression compared with the original Fig1. organoids (which have not been through this procedure).

The last part of the results section belongs to the discussion as no results generated by the researchers are included.

Although no new data was generated for this section, we have used the data generated in our work, together with existing ChIP-seq datasets to construct a new plausible hypothesis regarding the activation of Rax-expression through changes in TF-binding at an enhancer displaying little/no change in accessibility. As this section ties in with previous results sections discussing the regulation of eye-field genes, we feel it belongs in the results section rather than in the discussion.

The discussion in this paper is a good opportunity to state the limitation of this study.

As requested, we have added a paragraph discussing the main limitations to our study in the discussion section.

Major comments to address

1. One of the main issues identified is that the morphology of the control conditions in the CRISPR experiments (Fig.6) do not look is that those used for the RNAseq experiments (Fig.1) and the authors should address this issue. The fact that CDM media was used on the RNA extraction and ATACseq experiments and then KSR media was used for the CRISPR experiments is worrying and makes one wonder whether the second set of experiments is at all comparable to the first. This should be somehow controlled carefully by at least replicating one set of RNA experiments with the KSR media.

We have addressed this in response to the reviewer’s summary above. Unfortunately, it is not possible for us to replicate the RNA experiments in the KSR media due to the research group closure upon Professor FitzPatrick’s retirement.

1. The requirement of Wnt signalling inhibition has been well established as a requirement for forebrain specification, including the eye field. Considering the link of the Wnt/beta-catenin pathway to eye specification and that TCFs, the transcription factors that mediate Wnt pathway transcription regulation, have known and well-studied DNA motifs, it is surprising that authors do not include the analysis of TCF motifs in their study. Also considering that TCF7l1 (TCF3, old nomenclature) has recently been shown to be cell-autonomously required for the expression of rx3 (Rax homologue) in zebrafish. One would expect TCFs to be included in the analysis as it was done with Sox2 and Otx2, which were studied due to the known relevance in forebrain specification rather than from the direct analysis of the differential gene expression experiments.

We thank the referee for their valuable comment here. Our current analyses indeed do not consider TCFs and are therefore likely incomplete. We plan to address this by further analysing our data to quantify the patterns and effects of the TCF genes, and will appropriately amend our manuscript to reflect our findings.

Minor comments to address

1. The authors should clearly state the day timepoint used in the organoids experiments in the results section and figure legends, not just in the methods.

We have updated the text and figure legends to include the time point of all organoids.

1. The report by Agnes et al Development 2022 should be cited in the introduction as it is an excellent paper related to this topic, including a comprehensive analysis of the EFTFs expression pattern.

We thank the reviewer for pointing us to this very interesting paper. Although we feel it doesn’t fit in with our introduction that is currently tailored to the set of genes that has historically defined the eye-field (and which was discovered in non-mammalian models), we do recognise that the 3D organisation of the eye-field and in particular the patterns of gene-expression defining different regions of this is important to disentangle in mammalian systems. We have therefore inserted a reference to the Agnes at al 2022 study on the dimorphic teleost in our extended discussion.

1. Ln 41. Mutations in these genes do not always cause severe bilateral eye malformations. Probably best to moderate and mention that they 'can' cause these malformations.

As suggested we have softened this sentence to: “ Mutations in at least three of the genes encoding orthologs of the Xenopus EFTF can cause severe bilateral eye malformations in humans (OTX2, PAX6 and RAX) (Fitzpatrick and van Heyningen, 2005).”

1. Ln 146. Authors mention that in vitro organoid systems "closely mimic the in vitro regulatory dynamics". This statement should be moderated as we do not know if this is true. In fact, one of the positive aspects of this study is that it contributes to supporting this statement.

We agree with the referee regarding the strength of this original statement. We have changed this to:

“We have exploited a reproducible, in vitro organoid model system enabling us to generate data from this cell-state transition and through computational analysis gain a quantitative understanding of the underlying regulatory mechanisms.”

1. Ln 150. Rax homologue Rx3 is also expressed in cells that give rise to the hypothalamus in zebrafish and cavefish, and probably in Xenopus too. It could well be the case in mice too.

We have corrected this to indicate that Rax is also expressed in the hypothalamus in mice.

1. I do not think the GO term data adds much to Figure 1. If possible, I would move it to the supplementary section.

We have moved the GO visualisations to supplementary, Fig.S2.

1. It should be made clear which set of experiments was performed as biological replicates and which did not.

We have added details on the number of replicates used in each experiment.

1. Based on the heatmap in Fig1A, expression of Rax is significant in GFP- cells at days 4 and 5. The authors should comment or discuss this.

We have amended the text and supplemental methods section to include more details of our FACS protocol. The limitations of our sorting procedure include the fact that cells are not sorted into pure GFP expressing and non-expressing populations. Rather the GFP negative sample may contain some cells with low Rax expression or cells that have just begun to express Rax that were not excluded by our sorting. Our aim was to collect sufficient numbers of cells for each condition and separate out cells that expressed GFP to get a more uniform population of cells to study. It is also of note that the heatmap shows Rax expression by day 3. Although it was not detectable by imaging there were around 100 cells per organoid that FACS marked as GFP positive but were retained within the day 3 sample to ensure we had a complete picture of the gene expression at this time point.

1. Ln 99 of materials and methods mentions that the sorting of GFP+ was performed "when possible". The authors should state the differences in the conditions in the different experiments.

This has been expanded to detail exactly how cells were sorted.

1. The sentence closing the first section of the results (Ln 270) is an overstatement and should be moderated. I cannot see how the results shown in this section on their own could reflect and drive solid conclusions on brain cell fate specification.

We agree with the referee and have changed this sentence to: “In summary, these first analyses of RNA-seq data generated from the timecourse of optic vesicle organoid development, show that this is a robust and relevant model system with which to study the gene dynamics underlying mammalian eye field specification.”

1. Appropriate citations should be added to back up the argument that opens the second part of the results section (starting Ln 279).

We have added several citations that discuss and review the current knowledge regarding gene regulation via TF-binding at accessible cis-regulatory elements.

1. Ln 342-343. I suggest being consistent and using the EF-up or EF-down nomenclature on the whole manuscript unless referring to a different subset of genes.

We have modified the text to consistently use “EF-up” or “EF-down” terminology.

1. Ln 692 Refers to Fig.S4F, but this figure has only panels A-D.

This was a typo and has been corrected in the text.

1. Figures 6B and E and the figure legend do not indicate the differences between the panels, or the time stage of the experiments.

The figure legend has been updated to include these details.

CROSS-CONSULTATION COMMENTS I agree with the comments and suggestions made by the other two reviewers, which identify similar and also specific issues in the manuscript. I believe they are all pertinent and should be acknowledged before re-submitting.

Reviewer #2 (Significance (Required)): The manuscript by Owen et al, presents the analysis of in vitro eye vesicle organoids derived from mouse ESCs at stages equivalent to when the eye field is specified in vivo. This work is pertinent and necessary as detailed data on gene expression in early eye organoids was missing in the field and is necessary for the interpretation of experiments in these systems.

Although the computational data provided in this manuscript is based on consensus TF motifs, the functional relevance of the specific motifs must be proven before being able to drive any significant conclusions, and one should be moderate about the conclusion that can be driven from this kind of analysis. Still, the analysis put forward is a good reference and starting point for future functional studies. One possible limitation of this study is that the quantification of the expression of genes is based on the RNAseq data, and the expression data should be further confirmed using a proper quantitative method like qPCR.

This study will be of interest to the audience studying eye development and disease in animal model systems and humans.

My lab studies the genetic, cellular, and molecular aspects of eye specification, development and disease in zebrafish, and study mutations identified patients with eye globe defects.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Studies in Xenopus embryos have established that the specification of the eye field requires a core set of transcription factors (TFs) that impose eye identity to anterior neural plate progenitors. In this manuscript the authors have used mouse embryonic stem cells-derived optic vesicle organoid to ask if the acquisition of mammalian eye identity requires the same set of TFs. They further use different genomic approaches to identify the cis-regulatory elements involved in the expression of these genes and analyses the consequences of altering the sequence of some of the identified regulatory elements. Their results confirm that in mammals the acquisition of eye field identity requires the upregulation of the expression of the same core set of TFs described in Xenopus, with a particularly important role for three of them: Rax, Pax6 and Lhx2. This upregulation is associated to the downregulation of pluripotency genes.

This is a generally well-performed study, that indeed involves a large amount work and adds the identification of several cis-regulatory elements controlling the expression of this core set of already identified eye field TFs. However, conceptually the study does not add much to what is already known and the authors do not offer any very original conclusion from their study. They have generated a large amount of information that likely could allow them to go beyond what is known. For example, they could enlarge the composition of the gene regulatory network that controls eye field specification, given than one of their argument is that their analysis can predict the composition of such a network. Perhaps, they could also address some of the questions that are posed in the discussion. This will strengthen the manuscript and valorize their work.

Additional points that could be taken into consideration are the following:

1) According to the text, the authors identify only 53 CREs with decreased chromatin accessibility (ATACseq signal) between the 3 day and 5 days timepoints, versus the 7752 CREs with increased signal. However, this contrasts with the proportion of genes upregulated/ downregulated in their RNAseq analysis (37 vs 448) and with the notion that specification of the eye field involves the concomitant repression of other neural fates. This also suggests that at least an important fraction of the dynamic ATACseq peaks associated with 161 of the 448 downregulated genes increase their accessibility and allow the recruitment of transcriptional repressors. However, the role of TF binding and chromatin accessibility dynamics on gene repression is poorly discussed and the authors need to provide some interpretation of these observations. Also, authors interpret the fact that the presence of BS for EF downregulated genes, such as En2 and GATA6, correlates with increased chromatin accessibility as a consequence of the fact that TFBS can be bound by different TF paralogs but do not seem to consider that these TFs have been reported to work as transcriptional repressors, so that their downregulation could well explain the changes in chromatin accessibility.

We thank the reviewer for their interesting comments here. We have added short discussions on both main points above (EF-down genes linked to peaks with increasing accessibility and En2/Gata as transcriptional repressors) in the text related to the analysis of our ATAC-seq data. The notion that a loss of repression leading to the activation of gene-expression is indeed a very exciting one and one that we have thought about in the context of the switch-on of the eye-field TFs. This certainly deserves further future work, however in the present study we wanted to be careful not to overinterpret our data. To robustly gain insights into the loss of repression, experiments such as En1/Gata6 ChIP-seq would be very useful, though we are unable to perform these in the near future.

2) ATACseq signal analysis is an indirect measure of TF binding. The authors demonstrate the predictive nature of this analysis of TF dynamics and have use an available Sox2 ChIP dataset. However, this does not allow assessing dynamic changes in the occupancy of this TF and its correlation with ATACseq. Therefore, at least for few of the TF stressed in this work (e.g. Sox2 and Otx2 and for which good antibodies exist) they could attempt ChIP-seq analysis. This would considerably strengthen the work and provide support to an idea that the authors have particularly emphasized in their manuscript.

We agree with the referee that not having generated ChIP-seq data does not allow us to validate some of the hypotheses and evidence provided by the computational analysis of our ATAC-seq data – we have added a discussion of this limitation in the discussion section of our manuscript. We do note however, as observed in Bentsen et al, 2020, that compared to simple TF-motif occurrence analyses, TF-footprinting analyses (such as those we have performed) yield results on putative TF binding that are much closer to more direct measurements of TF binding via e.g. ChIP-seq. We fully agree that it would be very interesting to perform ChIP-seq/Cut&Cut experiments on the organoid system for a set of interesting TFs identified in our study. Unfortunately, because the lab of Prof FitzPatrick has now closed, it is not possible for us to perform further wet-lab experiments in the very near future. However, we plan to further explore the literature to try to find additional publicly-available ChIP-seq datasets (including for Otx2) which would help reinforce some of the hypotheses we make, and will report any relevant findings in our final manuscript.

3) Previous studies (i.e. 10.1242/dev.067660; 10.1093/hmg/ddt562) have shown the importance of gene dosage in eye field specification and repression of other fates. These studies could be included in the discussion, which, in its current version is a quite brief and leaves out many of the reported analysis.

We thank the referee for pointing us to this very relevant question – we have added this to the further research questions in the discussion.

CROSS-CONSULATION COMMENTS

The comments from the other reviewers complement the aspects that we have underscored and should be fully considered as they will contribute to improve the manuscript.

Reviewer #3 (Significance (Required)): This is a generally well-performed study, that indeed involves a large amount work and adds the identification of several cis-regulatory elements controlling the expression of this core set of already identified eye field TFs. However, conceptually the study does not add much to what is already known and the authors do not offer any very original conclusion from their study. They have generated a large amount of information that likely could allow them to go beyond what is known.

Developmental neurobiologists, genome

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Referee #3

Evidence, reproducibility and clarity

Studies in Xenopus embryos have established that the specification of the eye field requires a core set of transcription factors (TFs) that impose eye identity to anterior neural plate progenitors. In this manuscript the authors have used mouse embryonic stem cells-derived optic vesicle organoid to ask if the acquisition of mammalian eye identity requires the same set of TFs. They further use different genomic approaches to identify the cis-regulatory elements involved in the expression of these genes and analyses the consequences of altering the sequence of some of the identified regulatory elements. Their results confirm that in mammals the acquisition of eye field identity requires the upregulation of the expression of the same core set of TFs described in Xenopus, with a particularly important role for three of them: Rax, Pax6 and Lhx2. This upregulation is associated to the downregulation of pluripotency genes.

This is a generally well-performed study, that indeed involves a large amount work and adds the identification of several cis-regulatory elements controlling the expression of this core set of already identified eye field TFs. However, conceptually the study does not add much to what is already known and the authors do not offer any very original conclusion from their study. They have generated a large amount of information that likely could allow them to go beyond what is known. For example, they could enlarge the composition of the gene regulatory network that controls eye field specification, given than one of their argument is that their analysis can predict the composition of such a network. Perhaps, they could also address some of the questions that are posed in the discussion. This will strengthen the manuscript and valorize their work. Additional points that could be taken into consideration are the following:

1. According to the text, the authors identify only 53 CREs with decreased chromatin accessibility (ATACseq signal) between the 3 day and 5 days timepoints, versus the 7752 CREs with increased signal. However, this contrasts with the proportion of genes upregulated/ downregulated in their RNAseq analysis (37 vs 448) and with the notion that specification of the eye field involves the concomitant repression of other neural fates. This also suggests that at least an important fraction of the dynamic ATACseq peaks associated with 161 of the 448 downregulated genes increase their accessibility and allow the recruitment of transcriptional repressors. However, the role of TF binding and chromatin accessibility dynamics on gene repression is poorly discussed and the authors need to provide some interpretation of these observations. Also, authors interpret the fact that the presence of BS for EF downregulated genes, such as En2 and GATA6, correlates with increased chromatin accessibility as a consequence of the fact that TFBS can be bound by different TF paralogs but do not seem to consider that these TFs have been reported to work as transcriptional repressors, so that their downregulation could well explain the changes in chromatin accessibility.
2. ATACseq signal analysis is an indirect measure of TF binding. The authors demonstrate the predictive nature of this analysis of TF dynamics and have use an available Sox2 ChIP dataset. However, this does not allow assessing dynamic changes in the occupancy of this TF and its correlation with ATACseq. Therefore, at least for few of the TF stressed in this work (e.g. Sox2 and Otx2 and for which good antibodies exist) they could attempt ChIP-seq analysis. This would considerably strenghen the work and provide support to an idea that the authors have particularly emphasized in their manuscript.
3. Previous studies (i.e. 10.1242/dev.067660; 10.1093/hmg/ddt562) have shown the importance of gene dosage in eye field specification and repression of other fates. These studies could be included in the discussion, which, in its current version is a quite brief and leaves out many of the reported analysis.

Referees cross-commenting

The comments from the other reviewers complement the aspects that we have underscored and should be fully considered as they will contribute to improve the manuscript

Significance

This is a generally well-performed study, that indeed involves a large amount work and adds the identification of several cis-regulatory elements controlling the expression of this core set of already identified eye field TFs. However, conceptually the study does not add much to what is already known and the authors do not offer any very original conclusion from their study. They have generated a large amount of information that likely could allow them to go beyond what is known.

Developmental neurobiologists, genome

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Referee #2

Evidence, reproducibility and clarity

The authors grow eye organoids from cells with a reporter driving GFP in the Rax locus, a gene that is expressed in the eye field in many animal model systems. They show that expression of GFP picks up by day 4 and performed FACS sorting of GFP+ cells on day 4 and day 5 organoids to compare gene expression by RNAseq comparing with earlier day organoids. The data shows 37 genes with a differential expression on days 4 and 5, compared to day 3, and enriched in GFP+ cells, which they define as EF-up genes. It is notable that some of these genes had already been identified as canonical eye field gene regulatory network transcription factors. In the same way, they identify a group of differentially expressed regulated genes, EF-down, and state that 'many' of them are involved in pluripotency. However, they do not mention how many, or the proportion of these genes in the whole list. It would be useful if they could provide the number to understand how many of these genes are related to pluripotency, the whole list of genes mentioned to be downregulated in a supplementary file. The authors also note that genes known to be required for eye specification like Sox2 and Otx2 are not differentially expressed across the day 3-4 timepoint (Ln 190). However, this is not surprising considering that both genes are broadly expressed in the anterior neural ectoderm and required for its specification, which should be noted by the authors.

The authors then go on and cluster the EF-up, EF-down and genes deferentially expressed between days 2 and 3, and identify 6 discreet trajectory groups. From this analysis, they identify a third group of genes which shows a peak on day 3 but whose expression falls on days 4 and 5. It is interesting to see that this group includes Wnt and Fgf morphogenes. The authors should provide a list of the genes in the different clusters for the readers to inspect and analyse. Aiming to generate insight into the cis-regulatory elements that regulate of the genes the authors found differentially expressed in their model system they performed a series of ATAC-seq experiments. When linking the genomic regions with differential ATAC-seq accessibility to gene locus using the GREAT analysis, they identified association to 22 of the EF-up and 161 of the EF-down genes. This suggests a functional link between the ATAC-seq genomic regions and the gene regulation of the differentially expressed genes.

The authors later screened the ATAC-seq regions of increased accessibility for TF binding motifs and found that these regions were enriched with motifs for EFTF gens Rax, Lhx2 and Pax6. When assessing motifs in the ATAC-seq regions in EF-up TADs, Rax and Lhx2 motifs scored highly associated to open chromatin positions. Authors also observe a positive gene expression-accessibility correlation between in Pax6, Lhx2, Six3 and Otx2, and suggest this could mean these genes activate transcription of the EF-up group of genes. The same analysis, but focusing on EF-down genes, suggests that EFTFs repress the expression of EF-down genes which include those involved in pluripotency.

Further interrogating the ATAC-seq data, the authors use TOBIAS footprinting analysis to identify changes in TF binging in EF-TADs and EF-up motifs. Remarkably, whole genome analysis reveals that the largest increase in motif binging corresponds to EF-up genes Rax, Pax6 and Lhx2. The authors then narrow down on specific gene regulation by studying the ATAC-seq data within the TAD of Rax and Six6. However, they do not explain the rationale for which these two genes were highlighted, and why Pax6 or Lhx2 were excluded. This explanation should be added to the manuscript. The analysis identifies three regulatory elements in the Rax TAD and two for Six6. They then go on and study one putative regulatory element of each gene and generate CRISPR deletions in cell lines. The rationale for the choice of these particular elements is not clear, nor if the cell lines are the same used for the RNAseq experiments. This information should be explicit in the results and in the methods section. The authors mention that the CRISPR cell lines are "considerably more variable" (Ln 822) compared to the previously studied organoids and suggest that no conclusions can be driven from GFP expression or morphology alone. However, they do not specify which is the variable trait. This information should be added to the text. The authors also miss out on specifying the time stage of the organoids in figure 6 which should be stated. Regardless, the wildtype organoids in figure 6 and figure S7 show a very different morphology and GFP expression compared to those in figure 1, suggesting that the conclusions from this last set of experiments are not reliable or comparable to those in figure 1. This, together with the fact that different reagents were used to grow the organoids for the RNAseq and the CRISPR experiments, is a weakness of this work that must be addressed.

The last part of the results section belongs to the discussion as no results generated by the researchers are included. The discussion in this paper is a good opportunity to state the limitation of this study.

Major comments to address

1. One of the main issues identified is that the morphology of the control conditions in the CRISPR experiments (Fig.6) do not look is that those used for the RNAseq experiments (Fig.1) and the authors should address this issue. The fact that CDM media was used on the RNA extraction and ATACseq experiments and then KSR media was used for the CRISPR experiments is worrying and makes one wonder whether the second set of experiments is at all comparable to the first. This should be somehow controlled carefully by at least replicating one set of RNA experiments with the KSR media.
2. The requirement of Wnt signalling inhibition has been well established as a requirement for forebrain specification, including the eye field. Considering the link of the Wnt/beta-catenin pathway to eye specification and that TCFs, the transcription factors that mediate Wnt pathway transcription regulation, have known and well-studied DNA motifs, it is surprising that authors do not include the analysis of TCF motifs in their study. Also considering that TCF7l1 (TCF3, old nomenclature) has recently been shown to be cell-autonomously required for the expression of rx3 (Rax homologue) in zebrafish. One would expect TCFs to be included in the analysis as it was done with Sox2 and Otx2, which were studied due to the known relevance in forebrain specification rather than from the direct analysis of the differential gene expression experiments.

Minor comments to address

1. The authors should clearly state the day timepoint used in the organoids experiments in the results section and figure legends, not just in the methods.
2. The report by Agnes et al Development 2022 should be cited in the introduction as it is an excellent paper related to this topic, including a comprehensive analysis of the EFTFs expression pattern.
3. Ln 41. Mutations in these genes do not always cause severe bilateral eye malformations. Probably best to moderate and mention that they 'can' cause these malformations.
4. Ln 146. Authors mention that in vitro organoid systems "closely mimic the in vitro regulatory dynamics". This statement should be moderated as we do not know if this is true. In fact, one of the positive aspects of this study is that it contributes to supporting this statement.
5. Ln 150. Rax homologue Rx3 is also expressed in cells that give rise to the hypothalamus in zebrafish and cavefish, and probably in Xenopus too. It could well be the case in mice too.
6. I do not think the GO term data adds much to Figure 1. If possible, I would move it to the supplementary section.
7. It should be made clear which set of experiments was performed as biological replicates and which did not.
8. Based on the heatmap in Fig1A, expression of Rax is significant in GFP- cells at days 4 and 5. The authors should comment or discuss this.
9. Ln 99 of materials and methods mentions that the sorting of GFP+ was performed "when possible". The authors should state the differences in the conditions in the different experiments.
10. The sentence closing the first section of the results (Ln 270) is an overstatement and should be moderated. I cannot see how the results shown in this section on their own could reflect and drive solid conclusions on brain cell fate specification.
11. Appropriate citations should be added to back up the argument that opens the second part of the results section (starting Ln 279).
12. Ln 342-343. I suggest being consistent and using the EF-up or EF-down nomenclature on the whole manuscript unless referring to a different subset of genes.
13. Ln 692 Refers to Fig.S4F, but this figure has only panels A-D.
14. Figures 6B and E and the figure legend do not indicate the differences between the panels, or the time stage of the experiments.

Referees cross-commenting

I agree with the comments and suggestions made by the other two reviewers, which identify similar and also specific issues in the manuscript. I believe they are all pertinent and should be acknowledged before re-submitting.

Significance

The manuscript by Owen et al, presents the analysis of in vitro eye vesicle organoids derived from mouse ESCs at stages equivalent to when the eye field is specified in vivo. This work is pertinent and necessary as detailed data on gene expression in early eye organoids was missing in the field and is necessary for the interpretation of experiments in these systems.

Although the computational data provided in this manuscript is based on consensus TF motifs, the functional relevance of the specific motifs must be proven before being able to drive any significant conclusions, and one should be moderate about the conclusion that can be driven from this kind of analysis. Still, the analysis put forward is a good reference and starting point for future functional studies. One possible limitation of this study is that the quantification of the expression of genes is based on the RNAseq data, and the expression data should be further confirmed using a proper quantitative method like qPCR.

This study will be of interest to the audience studying eye development and disease in animal model systems and humans.

My lab studies the genetic, cellular, and molecular aspects of eye specification, development and disease in zebrafish, and study mutations identified patients with eye globe defects.

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Referee #1

Evidence, reproducibility and clarity

Summary

Owen et al. characterize the transcriptome and chromatin accessibility of mouse retinal organoids at early stages during which eye field-like cells are specified. Since cell specification and differentiation in retinal organoids largely mimic those processes in vivo, retinal organoids are viable models for studying the mechanisms of early eye development. Owen et al. utilize a previously established Rx-GFP cell line, bulk RNA sequencing, and bulk ATAC sequencing to dissect the mechanisms of early eye development in mice. Their findings are generally consistent with previous studies. Overall, the study is interesting for the field, but its conceptual and technical advances are moderate. In addition, a few major points need to be clarified.

Major points

1. The authors did not show any analysis of retinal organoids at stages when Vsx2 is expressed. This is a significant weakness since the chemically defined medium (CDM) used in Owen et al.'s study was previously shown to induce rostral hypothalamic differentiation (Wataya et al., 2008). Related to this notion, several eye-field transcription factors, such as Rax and Six3, are also expressed in the hypothalamus. Therefore, Owen et al. need to demonstrate that organoids in their modified differentiation system efficiently produce Vsx2-positive retinal progenitors, and samples of organoids at stages when Vsx2 is expressed should be included for RNA sequencing. If Vsx2 is not efficiently expressed in their organoids, the interpretation of results will be very different.
2. The authors state that "two differentiation medias were used for this work due to the differentiation becoming unstable after the initial experiments had been performed. The organoids used for RNA and ATAC-seq were grown in CDM media and the organoids with mutations introduced in potential CREs were grown in KSR media". Why the differentiation becomes unstable after the initial experiments? Differences in the two media cause additional complexities. Related to this notion, "WT Rx-GFP" in Figure 4B and 4E appears to show a different expression pattern compared to that in Figure 1A.
3. Is the deletion of Rax and Six6 regulatory elements homozygous? Sanger sequencing or amplicon sequencing is needed to show the deletion.
4. The deletion of Rax and Six6 regulatory elements appears to cause minor changes in the expression of Rax and Six6 (Figure 6C, F). Therefore, the impact of findings in bulk RNA seq and bulk ATAC seq in this study is still unclear.
5. Retinal organoids and sorted cells are composed of heterogeneous cell populations. Bulk RNA seq and bulk ATAC seq do not have the power to dissect the complexity of heterogeneous cell populations. Single-cell RNA seq and single-cell ATAC seq are more powerful for this study.
6. Numerous motifs in the JASPAR database are identified using in vitro assays and have not been validated using in vivo assays. Unexpected results in motif analysis could be due to the differences in DNA binding motifs between in vitro and in vivo conditions. This notion should be added in the discussion.

Minor points

Numerous labels in figures are too small.

Referees cross-commenting

My fellow reviewers identify similar major weaknesses and additional points. I agree with the other reviewers' comments.

Significance

Nature and Significance of the advances

In Owen et al.'s study, the Rx-GFP cell line and retinal differentiation protocol were established in previous studies (Wataya et al., 2008; Eiraku et al., 2011); bulk RNA sequencing and bulk ATAC sequencing are standard procedures. Although candidate regulatory elements for early eye development are identified, deletions of two prioritized elements using CRISPR/Cas9 only cause minor changes in the expression of targeted genes. Overall, conceptual and technical advances in Owen et al.'s study are moderate.

Compare to existing published knowledge

The datasets could be useful for the field, but conceptual and technical advances are moderate.

Audience

Developmental biologists, stem cell biologists, vision researchers.

Your expertise

Developmental biology, stem cell biology, vision research

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Reply to the reviewers

The authors do not wish to provide a response at this time.

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Referee #3

Evidence, reproducibility and clarity

The authors have utilised single-cell RNA seq to profile cells in the Drosophila female germline cells and somatic cells within the ovary. Using a candidate approach, the authors assessed whether some of the candidate genes identified in this study play a role in germ cell differentiation. Amongst these, they found that an uncharacterized gene called eggplant is required for differentiation. On rich diets, Eggplant expression is reduced. Furthermore, Mmps and Timp are required to regulate Eggplant expression level.

Major comments:

Overall, the conclusions are supported by experimental evidence. The sc-RNA seq provides an important resource for the community, and some of the reagents generated such as the antibody against Eggplant or the tagged lines would provide valuable resource for future studies. The main criticism I have for this manuscript is that it lies half way between a resource paper and a mechanistic paper. If it was just a resource paper, then the sc data should suffice. Instead, the authors studied the effects of Eggplant and MMP/TIMP on GSC differentiation, however, not enough mechanism was gained through these studies.

1. It is not clear what kind of protein Eggplant is, and its mode of action is unclear, despite that it was shown by the authors that Eggplant is required for GSC differentiation. The authors further showed that animlas fed a high yeast diet promotes GSC proliferation and increased egg production by inhibiting the expression of eggpl. The authors suggested that this may lie downstream of insulin signaling, however no genetic experiments were conducted. In order to support this line of conclusions, essential experiments should include: is there a genetic interaction between Eggplant and insulin signaling? Is this happening autonomously within the GSCs or at the GSC niche? In the discussion the authors mentioned that since egg production can occur upon Eggpl knockdown even on normal diet, thus protein is not involved in this process. So, what is the mechanism?
2. Similarly, the authors mentioned that MMPs and TIMP are involved in GSC differentiation, and they discussed that it could be mediated via tissue stiffness. However, no experimental data was presented to support this. It would be good to offer some mechanistic insights. TIMP and Eggplant genetic interaction should be done.

Minor comments:

Line 376 and Line 437 are repetitive in explaining why the gene is called eggplant. I did not find the pictures in Figure7 F,G and Figure 8 B informative. Some quantifications should also proof the same point.

Referees cross-commenting

I also agree with the comments from both reviewers 1 and 2.

Significance

• The significance of this study lies in presentation of a thorough characterization of gene expression at the single cell level in the GSC and in identification of novel regulators of GSC differentiation.
• My field of expertise is Drosophila stem cells and nutrition/metabolism.

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Referee #2

Evidence, reproducibility and clarity

Brief summary

The authors conducted single cell RNA sequencing on adult fly ovaries to survey the transcriptomic profile of individual ovarian cells. The datasets resulted in classification of 24 discrete cellular populations, including different types of stem cells, progenitors and differentiated cells. Genes that differentially expressed during germline differentiation were selected and then examined with RNA interference to determine their specific roles in regulating germline differentiation. Meanwhile, using the single cell transcriptomic profiles acquired from specific types of germ cells, gene regulatory networks were further characterized. Among the genes examined, the gene eggplant (eggpl) was found to act as a novel regulator mediating germline differentiation with its protein expression predominantly detected in germline stem cells (GSCs), CBs and early cysts. Interestingly, since the protein expression of Eggplant was decreased upon rich dietary intake, eggpl may function as a novel molecular link coupling the nutritional status and the process of germline differentiation during fly oogenesis.

Specific comments

Major points:

1. The authors chose a specific group of differentially upregulated genes (in GSCs) and performed germline specific RNA interference (RNAi) to determine their function in regulating germline differentiation. The RNAi results were then compiled and presented in Figure 3B-C. However, details shall be provided for the readers/reviewers to understand the phenotypic analyses. Specifically, as stated in text that "we found that RNAi knockdown of 19 upregulated genes induced disruption of GSCs/CB homeostasis. Of these, 12 genes were classified as "changes to the number of CSC/CB", 6 genes as "empty germarium" and 4 genes exhibited "differentiation defects"... ", there was neither specific descriptions nor representative examples (with proper labelling) explaining the bases of such classification. It was particularly difficult to appreciate the assorted phenotypes presented in Figure 3C without proper descriptions. Not to mention that the immunostaining needs to be optimized in some of the representative pictures. Meanwhile, it was not clearly stated in the text about how 19 candidate genes were then classified into (12+6+4=22) genes. Along the same line, it will be helpful to include a supplementary table of summarizing the genes tested and their corresponding phenotypes.
2. The authors also performed germline specific RNAi to knockdown the expression of 21 out of 39 most highly expressing genes in GSCs to determine their function in regulating oogenesis. Some of the RNAi effects were presented in Figure S2B. Similarly, the effects of RNAi experiments will be better appreciated if some more descriptions on specific RNAi phenotypes are added. Also, inclusion of a supplementary table summarizing the RNAi lines utilized in this experiment is required.
3. The authors performed in situ hybridization and immunostaining to discover that the eggpl transcripts were enriched in GSCs and CBs (Bam-GFP- germ cells), while the Eggpl::GFP expression (in the knock-in line, KI) was detected throughout region 1 within germarium. The authors then concluded that protein level of Eggpl was "actually highest in cells that have no detectable eggpl transcripts". However, as shown in Figure 5F, the highest level of Eggpl::GFP appeared in the cell that contains a spectrosome, suggesting that is either a GSC or a CB obtaining highest Eggpl::GFP (KI) expression. Furthermore, as shown in Figure 4E, similar level of Eggpl protein expression indicated by immunostaining was found in GSCs and CBs, indicating a nice coupling between the mRNA and protein level of eggpl found in GSCs/CBs. Therefore, the authors need to simultaneously perform in situ hybridization and immunostaining of eggpl to visualize the expression of eggpl transcripts and protein within the same germarium and to directly determine whether the statement that "protein level of Eggpl was actually highest in cells that have no detectable eggpl transcripts" holds true.
4. The genetic experiments done by the authors show that disruption of eggpl led to an increase in the average number of Brdu+ cysts, the size of the ovary and ultimately the number of eggs produced. Interestingly, such phenotypes were reminiscent to the effects caused by rich dietary intakes. The authors interpret these results by proposing that eggpl negatively regulates GSC proliferation in regulating oogenesis. However, as shown in Figure 5J, the number of GSCs was not significantly altered upon eggpl RNAi but was increased when over-expressing Eggpl::GFP, indicating that eggpl promotes the proliferation of GSCs. To be able to determine the function of eggpl in regulating GSC proliferation during oogenesis and to support the stamen that "We also found that cell cycle in germ cell cysts was accelerated" (in introduction), the authors need to perform clonal analyses to monitor the progression rate of eggpl mutant cells (of both germline clones and follicle cell clones) compared to control cells. An accelerated rate of germline development shall be predicted by increased GSC proliferation. Moreover, to rule out the possibility that changes in the dynamics of germline apoptosis can affect the number of Brdu+ cysts observed upon eggpl loss, the authors should also perform experiments assaying the number of apoptotic cells in the germarium when manipulating the expression level of eggpl.
5. The authors examined the expression level of Eggpl::GFP (KI) in different feeding conditions and found a reverse correlation between Eggpl::GFP (KI) protein level and the quality of dietary condition. Moreover, the enlarged ovary seen upon eggpl loss was recapitulated when over-expressing Timp and Mmp1/2. Interestingly, the authors also found that overexpression of Timp and Mmp1/2 led to lower protein expression of Eggpl::GFP (KI). The authors interpret these results by proposing that eggpl mediates the GSC differentiation via MMP-dependent Timp regulation pathway. However, the authors should perform experiments to directly examine if eggpl and Timp (and Mmp1/2) interact genetically to regulate GSC proliferation/egg production while monitoring the dietary status.

Minor points:

1. Please clarify what you mean in this sentence in the introduction as the message is not clear: "Mei-P26 suppress transcripts that promote differentiation in CB by antagonizing miRNA pathway
2. In Figure 3B, for the genes that were listed for more than once (CG42250, CG10295, CG6904 and CG15845), please note that those are different RNAi lines.
3. In Figure S2A, for the genes that were listed for more than once, please note that those are different RNAi lines.
4. If applicable, more than one RNAi line of each candidate gene should be examined to validate the specific effects caused by gene KD.
5. As shown in Figure S2B, knocking down CG31666 and CG12743 led to impaired oogenesis. Please add their gene name (CG31666 (chinmo) and CG12743 (otu)) for acknowledging their well-documented role in regulating oogenesis.
6. How was the RNAi generally performed. Please add into material and method.
7. In Figure 7A, it is difficult to tell how many of the BrdU+ cysts was shown in Eggpl[1] mutant germarium and whether it is qualitatively/quantitively different from control.
8. The authors compared their scRNA-Seq dataset with other 4 public available Drosophila ovary scRNA-Seq datasets and concluded that comparable features were found among these datasets by UMAP analysis. The authors should also comment on what are the potential differences among these adult ovary scRNA-Seq datasets in the discussion section. Providing such information will not only highlight the importance of this dataset and will also be beneficial to the fly community for future research.

Referees cross-commenting

I also agree with the comments from both reviewers 1 and 3.

Significance

In summary, the characterization of single-cell atlas of adult fly ovary from this study adds additional datasets for future investigation on cell-specific differentiation in vivo. Meanwhile, the identification of a novel molecular link between nutritional status and germline differentiation is of broad interest to readers in the field of dietary/metabolic control on stem cell function. However, there are specific concerns about how the results were presented and interpreted while some important controls are missing. I believe this article would be much improved if the points mentioned can be effectively addressed.

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Referee #1

Evidence, reproducibility and clarity

In this manuscript, Sun et al. conduct single-cell RNAseq on germline stem cells in the Drosophila ovary to better understand the transcriptional profile of this small, but important, population of stem cells. This resulted in the identification of a subset of genes and gene networks proposed to be involved in early germ cell development and differentiation. A subsequent RNAi screen of these candidate genes indicated that an uncharacterized one (renamed eggplant) was expressed at the transcriptional level in GSCs and at the protein level in the germarium. RNAi-mediated knockdown or CRISPR/Cas9-mediated knockout of eggplant resulted in an accelerated cell cycle in germ cell cysts and flies with overall larger ovaries. These results were not observed, however, when flies were fed a rich yeast-based diet. The authors also draw a connection between the MMP-Timp pathway and the regulation of eggplant in ovarian germ cells. Collectively, the findings presented in this manuscript detail novel modulators of germ cell differentiation and proliferation that are regulated by nutrient availability. While the data presented are intriguing (especially in the first part of the manuscript), there are several major and minor issues that should be addressed prior to publication.

Major issues:

1. The bioinformatic analysis of the scRNA-seq data is elegant and well done. However, the data from the follow-up studies on eggplant are mostly observational, correlative, and not convincing. There is little to no mechanism described regarding the function of eggplant. This may be beyond the scope of this paper, but it should at least be addressed in more detail in the Discussion section.
2. Along the lines of point 1, what is the amino acid sequence of eggplant? What is its predicted molecular weight? Does it contain any predicted domains that may hint at its function in the germarium? The answers to these questions should be included in the manuscript.
3. There is no validation of the novel eggplant antibody that was generated. This needs to be demonstrated in order to be confident that the antibody is working as expected to interpret the immunofluorescence data.
4. In Figure 8, the connection between MMPs, TIMPs, and eggplant is very weak. It is unclear how the regulation is occurring. Is it direct or indirect? And by what mechanism? Also need to show representative images to go along with the graphs. But as is, this is the weakest data in the manuscript and needs more clarification.

Minor issues:

1. It is unclear what it is being shown in Figure 5M.
2. In Figure 7B, how was the egg-laying assay performed. This information was not included in the Materials and Methods section.
3. There are some grammar issues in the Introduction section.

Referees cross-commenting

My comments align closely with those of Reviewer #3. I also agree that the specific points raised by Reviewer #2 should be addressed to strengthen the manuscript.

Significance

The significance of this work lies in the transcriptional profiling of the Drosophila ovarian germline stem cells. These cells are scarce and are notoriously difficult to isolate and study. This work makes headway in that regard, laying the foundation for future studies, and as mentioned, the bioinformatic analysis of the scRNA-seq data is the strength of the study.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity):

Summary<br /> Authors show that overexpression of bHLH transcription factor Dpn in the medullary neurons of the Drosophila optic lobe results in the dedifferentiation of these neurons back into the NBs. These dedifferentiated NBs acquire and maintain mid-temporal identity, express Ey and Slp, and show delayed onset of tTF Tailless (Tll), leading to an excess of neurons of mid-temporal fate at the expense of late temporal fate neurons and glial cells. The dedifferentiated NBs are stalled in the cell cycle and fail to undergo terminal differentiation. Over expression of tTF Dicheate (D) or promoting G1/S transition pushed these NBs to late stages of the temporal series, partly rescuing the neuronal diversity and causing their terminal differentiation. They also show that the dedifferentiation of NBs by Notch hyper-activation also exhibited stalled temporal progression, which is restored by D overexpression.<br /> Authors suggest that cell cycle regulation and tTF are primary to the proliferation and termination profile of dedifferentiated NBs.<br /> Using these conclusions, the authors emphasize the need to recreate the right temporal profile and ensure appropriate cell cycle progression to use dedifferentiated NSC for regenerative purposes or prevent tumorigenesis originating from differentiated cell types.

Major comments:<br /> - Are the key conclusions convincing?<br /> Most conclusions are convincing; however, some issues are pointed out below.

• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

The authors have overexpressed Dpn and shown that medulla neurons dedifferentiate to NBs, similar to the loss of function phenotype seen for the Nerfin-1 of which Dpn is a target. They also show that temporal series progression defect is also seen in the case of dedifferentiated NB generated by Notch over-activation.<br /> Using these two examples, the authors suggest that for dedifferentiated NSC, which are to be used for the regenerative purpose, one needs to recreate the right temporal profile and ensure cell cycle progression occurs appropriately. Authors also claim that to prevent tumorigenesis originating from differentiated cell types, one needs to recreate the right temporal profile and ensure cell cycle progression occurs appropriately.

While I agree with this, I think this is an overreaching conclusion based on just these two examples. If they could show the same for one more method of dedifferentiation (For, e.g. Lola) happening in medulla neurons which happens by a mechanism independent of Nerfin-1, Dpn, Notch axis, the argument will become more convincing and broad.

We will characterise the temporal identity, termination and cellular identity of Lola-Ri induced ectopic neuroblasts. If these parameters are disrupted, we will overexpress D to assess whether this can trigger the progression of the temporal series.

Also when authors mention N mediated dedifferentiation, they need to inform that Dpn is a direct target of Notch in NBs (Doi. 10.1016/j.ydbio.2011.01.019), they do so in the discussion, but mentioning it here gives a broader context to the reader.

We will include that Dpn is a target of Notch when first mentioned.

Another important point that needs a mentioned here is that conclusions are based on dedifferentiation happening in the medulla neurons, which are considered less stable since they lack Prospero. Therefore whether this conclusion can be generalized for all the tumors arising from dedifferentiation in the CNS (eg, those arising from NICD activation in the central brain or thoracic region of the VNC) is another concern. Maybe authors can consider making a more conservative claim.<br /> Generalizing this conclusion to Prospero expressing NBs lies outside the scope of the current study and cannot be addressed here because central brain Type-I NBs use a different set of tTFs.

We will make a more conservative claim and clarify all of our conclusion are medulla neuron-specific.

Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.<br /> Experiments with Lola knockdown/mutants in medulla neurons can be done quickly, in my opinion, and will substantiate this claim.<br /> Another obvious question that comes to mind is if medulla neurons dedifferentiate on overexpression of Dpn, does the same happen in nerfin-1 mutant clones as well? And if yes, why has the author not done similar experiments for nerfin-1 mutants.

We will assess the temporal identity of neuroblasts in nerfin-1 mutant clones.

Please show Ey staining in Fig-2 if possible, it will also help to add a line on why Slp was used as marker for mid tTFs instead of Ey.

Ey is shown in Fig-2 (D-D’’) already. Slp is used as a marker of mid tTFs as Ey is expressed also in neurons thus would also be present in deep sections of control clones, whereas Slp is not expressed in neurons. We therefore used Slp as a proxy for mid-temporal identity throughout our study. We will include this text in our revision.

In Model shown in last figure Dpn is shown to repress D and activate Slp. Can authors show that Dpn overexpression represses D and activate Slp either by antibody staining or by RT PCR.

In Figure 2H, we have shown in clones that overexpression of Dpn induced a significant increase of Slp. In Figure S3B-B’’, we have shown that Dpn overexpression causes an upregulation of Slp at 6 hr APF. We can think we have pretty convincingly shown that Dpn overexpression activates Slp.

For Dichaete, our existing data shows that Dpn overexpression did not significantly alter D expression. To assess if using a stronger driver might allow us to see some changes, we will induced dedifferentiation via Dpn overexpression using the Eyeless-Gal4 driver. In this experiment, we will quantify the amount of D upon Dpn overexpression. Depending on this result, we will revise our conclusion on whether Dpn overexpression represses D.

Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.<br /> Experiments with Lola and nerfin-1 mutants can be done in a few months. I cannot comment on the cost involved.<br /> - Are the data and the methods presented in such a way that they can be reproduced?<br /> Yes

Are the experiments adequately replicated and statistical analysis adequate?<br /> Replication and statistical analysis are fine. The activated Notch experiments show only three data points in all the experiments. It will be good to increase this number.

We will repeat Notch experiments to increase the n number for these experiments.

Minor comments:<br /> - Specific experimental issues that are easily addressable.<br /> There is a problem with Fig-5F (both 5E and 5F have % EdU in clone/ % Mira in the clone as y-axis), I do not understand how the Fig-5F let them conclude that D overexpression increases the rate of neuronal production.

In the text we said: “We found that D overexpression did not significantly increase neuronal production, suggesting that it is likely that cell cycle progression lies upstream or in parallel to the temporal series, to promote the generation of neurons.”

In one place, the authors conclude, "Together, this data suggests that it is likely that cell cycle progression lies upstream of the temporal series, to promote the generation of neurons". Authors should consider adding "medulla NBs" at the end of the sentence since cell cycle progression being upstream of temporal series is already known in Type-I NBs, as pointed out by authors as well (Ameele and Brand 2019).

We will add “medulla NBs” to the end of this sentence.

In the discussion authors says that "Our data support the possible links between cell cycle progression and the expression of temporal regulators controlling NB proliferation and cellular diversity". This is new information, as the 2019 study did not show how cell diversity changes with a changed tTF profile. I think the authors should elaborate on this point to highlight how this is different from what is already known from the 2019 study (done in the context of Type-I NBs).<br /> Maybe they need to highlight that the cell cycle directs/regulates the progression of temporal series compared to the earlier observation where temporal series was shown to be downstream of the cell cycle.

We will expand in discussion to discuss the link between cell cycle/tTFs.

In fig-3J in clones even after 24 AHS, Dpn continues to be overexpressed but these cells undergo terminal differentiation, can authors comment why is it so?<br /> In one place authors say, "To better assess the cumulative effect of the neurons made throughout development, EyOK107-GAL4 was used to drive the expression of Dpn" maybe some background on why use this specific GAL4.<br /> Also a line about why GMR31HI08-GAL4 eyOK107-GAL4 and and eyR16F10-GAL4 were used.

While Dpn is overexpressed, it progresses through the temporal series at a slower pace due to a delay in cell cycle progression, as well as delayed onset of D, these NBs still eventually reach the terminal temporal identity, and are thus about to undergo terminal differentiation. We will include an additional piece of data that shows NBs induced by Dpn overexpression do eventually turn on Tll.

Are prior studies referenced appropriately ?<br /> Yes, but in a few places, some references can be added.<br /> An important point that needs to be mentioned for the context is the medulla neurons do not use Prospero for terminal differentiation and are thus considered less stable (DOI: 10.1242/dev.14134

We beg to disagree with the reviewer in terms of Pros is not required for terminal differentiation of medulla neuroblasts. Li et al., 2013 shows that nuclear Pros is found in the oldest NBs. We do agree that differentiated state of medulla neurons is less stable, possibly owing to absence of Pros, and we will include that in our discussion.

In discussion, the authors say that "It would be interesting to explore whether N similarly acts on these target genes to specify cell fate and proliferation profiles of dedifferentiated NBs." There is a study looking at Notch targets in NB hyperplasia (DOI: 10.1242/dev.126326); whether that study shows if any of the cell cycle genes are downstream of activated Notch, needs a mention here.<br /> Also, when authors mention N mediated dedifferentiation, they need to inform that Dpn is a direct target of Notch in NBs (Doi. 10.1016/j.ydbio.2011.01.019). They do so in the discussion, but mentioning it in the introduction or results will give a broader context to the reader.

We will discuss the study looking at N targets in NB hyperplasia in the discussion of the revised manuscript.

We will mention that Dpn is a target of Notch in the results section.

Another gene that needs a mention is "Brat", which regulates both Dpn and Notch, and causes dedifferentiation and tumors in CNS, I think this gene and its interaction with Dpn and Nerfin and Notch needs to be discussed either in the introduction or discussion.

We will comment on Brat in the discussion.

Are the text and figures clear and accurate?<br /> The main figures are not labeled. Therefore, it was very annoying to deduce the specific figure numbers.<br /> There are 1 or 2 places where figure calling is wrong in the text.<br /> The Image Fig-5I shows cycD and CDK4 at the G2-M transition; while the text says it supports G1/S, which is indeed the case, the figure needs modification.

We thank the reviewers for identifying these mistakes, and will correct them.

Do you have suggestions that would help the authors improve the presentation of their data and conclusions?<br /> The presentation is okay, in my opinion.

Reviewer #1 (Significance):

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

The factors leading to dedifferentiation of the neurons have been identified previously by groups of Chris Doe (mldc, DOI: 10.1242/dev.093781), Andrea brand (10.1016/j.devcel.2014.01.030.) as well as the authors of this paper (10.1101/gad.250282.114, 10.1016/j.celrep.2018.10.038.). However, many questions remained unaddressed regarding such NB generated from neuronal dedifferentiation. For example, whether these cells contribute to native cell diversity of the CNS, undergo timely differentiation or their progeny cells incorporated into appropriate circuits is not well understood. Successful execution of these phenomena is critical for generating functional CNS and such insights are crucial for understanding the origin of tumorigenesis in CNS or employing dedifferentiated NSC for regenerative purposes.

This study is an overexpression-based study, however, some of the results give significant conceptual insights into the tumors arising out of the dedifferentiation of the neurons. It also gives insights into the fact that the dedifferentiated cells need to be carefully examined for the temporal factor profile before they can be employed for regeneration or any therapy targeting them.<br /> However, in my opinion, they need to test this idea at least in one more system of neuronal dedifferentiation, preferably independent of the nerfin-1/Notch/Dpn axis to generalize this claim.

• Place the work in the context of the existing literature (provide references, where appropriate).<br /> Cerdic Maurange's group had looked at the role of temporal factors and identified the early phase of malignant susceptibility in Drosophila in 2016 (doi: 10.7554/eLife.13463). Andrea Brand's group has shown in a 2019 paper that cell cycle progression is essential for temporal transition in NBs (doi: 10.7554/eLife.47887). Both these studies were in the context of Type-I NBs, which express Prospero, which is crucial for the differentiation of the neurons.<br /> Previously the authors have studied type-I NBs and shown by Targeted DamID that Dpn is Nerfin-1 target. They also show that Nerfin-1 mutants show dedifferentiation of neurons. They follow up on this observation in medulla neurons, where they find that Dpn overexpression results in their dedifferentiation into medulla NBs. Medulla NBs differ from Type-I NBs in using a separate set of tTFs. Also, Type-I NB and neurons arising from them use Prospero for terminal differentiation, while medulla neurons do not express Prospero and are therefore considered less stable (DOI: 10.1242/dev.141341).

The importance of the study lies in the results that show that the NB arising out of dedifferentiation of medulla neurons takes up mid-temporal fate. These NBs are stalled in Slp expressing mid-temporal stage unless the cell cycle is promoted by overexpression of cell cycle genes regulating G1/S transition.<br /> Authors also show that overexpression of D promotes the progression of temporal series in these dedifferentiated NBs, which could partly rescue neuronal diversity and result in terminal differentiation. Thus D plays an important role in determining the type of neurons these NBs generated. This suggests that knowing the tTF profile of these types of dedifferentiated NBs is vital if these cells were to be used for regenerative purposes. Authors further claimed that cell cycle regulation and tTFs are critical determinants of the proliferation and termination profile of dedifferentiated NBs.

• State what audience might be interested in and influenced by the reported findings.<br /> The study will be of broader interest to researchers interested in central nervous system patterning, regeneration, and cancer biology.

• Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.<br /> Drosophila, central nervous system patterning and cell fate determination of neural stem cells.

Reviewer #2 (Evidence, reproducibility and clarity):

Stem cells can divide asymmetrically to self-renew the stem cell while generating differentiating sibling cells. To restrict the number and type of differentiating sibling cells, stem cells often undergo terminal differentiation. Terminally differentiated cells can dedifferentiate and revert to a stem cell like fate. However, the underlying molecular mechanisms are incompletely understood in vivo.<br /> Here, Veen et al., use Drosophila neural stem cells (called neuroblasts) to investigate how terminal differentiation is regulated. Neuroblasts faithfully produce the correct number and type of neuronal cells through temporal patterning and regulated terminal differentiation. The authors show that misexpression of the bHLH transcription factor Deadpan (Dpn) induces ectopic neuroblasts, which predominantly express mid-temporal transcription factors at the expense of late-temporal transcription factors. As a consequence, these ectopic neuroblasts also fail to produce Repo positive glial cells and are stalled in their cell cycle progression. The authors provide evidence that promoting cell cycle progression and overexpression of the transcription factor Dichaete (D) is sufficient to restore the temporal transcription factor series, neuronal diversity and timely neuroblast differentiation.

This is an interesting study that will be of interest to the stem cell field. However, I encourage the authors to consider the following critiques:

1. Explain the rationale for the three different neuronal/NB drivers (GMR31HI08-GAL4, eyOK107-GAL4, eyR16F10-GAL4. How are they expressed?

We will include an expression analysis of EyOK107-GAL4 and eyR16F10-GAL4. GMR31HI08-GAL4 expression analysis was previously published (Vissers et al., 2018). We will explain in the text the benefits of each driver.

1. The rationale for the Edu experiment (Figure S1I) is not clear. Why is this a measure for the production of neuronal progeny? For the correct interpretation of these results, the authors should also provide control clones or Edu experiments of regular neuroblasts.

We will repeat this experiment and mark the progeny with the neuronal marker Elav, to demonstrate that they are neurons. Additionally, we will add the control to this figure.

1. How was % of Mira (Figure 1K and below) or the % of tTFs (Figure 2H onward) quantified? For instance, Figure 2C-G often shows clonal signal that is not highlighted with the dashed lines and the corresponding tTF intensity does not match the intensity in the outlined clone (eg. Figure 2D-D'; a large optic lobe clone is negative for Ey. Figure 2E-E'; an unmarked clone is negative for Slp).<br /> Similarly, the Hth signal is very weak to begin with so it is unclear how this was quantified. How was determined what constitutes real signal vs. background noise?<br /> Additional explanations in the methods section is needed to assess the robustness of the data.

We will expand the methods section and mention that we used similar thresholding in antibody staining between control and uas dpn in all instances, so even if the antibody is weaker (eg hth) it is consistently quantified. Additionally, we can increase the intensity of Ey in Figure 2D-2D’, as it is expressed at low levels.

1. This sentence should be rephrased: 'As the tumour cell-of-origin can define the competence of tumour NBs to undergo malignancy (Farnsworth et al., 2015; Narbonne-Reveau et al., 2016), we next tested whether the temporal identity of the dedifferentiated NBs were conferred by the age of the neurons they were derived from.'<br /> The connection between tumorigenicity and temporal identity is not really clear and should be briefly reintroduced for this paragraph.

We will rephrase this sentence and further introduce this concept when talking about tumour cell of origin and competence.

1. Figure 2I-N: The experimental outline in I and J should be grouped with the corresponding images to clarify what is compared. Also, there are no images for the control clones, which make a comparison difficult. The images are also too small. I cannot really see the Hth, or Slp signal in the small clones shown in Figure 2K-L".

We will split figure 2 into two images. The first image including A-H and the control data. And the second including I-Q and the control data. This will increase the size of the images. Additionally, we will group I and J with corresponding data.

1. Figure 3H: It is not clear why there are only a small group of Nbs that are positive for Mira. Please explain.

Most NBs have terminated by this time point, we will explain this within the text.

1. Figure 3K-M: Please explain how the Toy signal was measured and quantified.

We will expand the methods section and explain how Toy quantification is made.

1. The TaDa data set is very interesting but the following might be an overstatement: "We found that Dpn directly binds to slp1 as well as the Sox-family TF dichaete (D) which is expressed in medulla NBs after slp1 (Li et al., 2013) (Figure S6 A-B)."<br /> More direct binding assays might be needed to show that Dpn directly binds to slp1 and D. If this is already shown, clarify the sentence to indicate what is published and what is extracted from the data shown here.<br /> Also, what is the rationale for this statement: "Consistent with the model that D represses Slp-1..."?

The DamID data do actually show that Dpn binds (i.e. there is a statistically significant peak at FDR<0.01) directly at these loci (see the TaDa supp fig A & B). Whether it’s doing anything functional or not, we can’t say, but our data shows that Dpn directly binds to slp1 and D. We will clarify the sentence to indicate this in our revision.

1. This might be an overinterpretation: D overexpression in UAS-Dpn NBs promoted their pre-mature cell cycle exit at 6 hrs APF using eyR16F10-GAL4. The data shows loss of Mira signal, which could occur through different mechanisms.

Our data already shows that these NBs express Tll, the terminal temporal transcription factor (Figure 4F). In addition, we show that there is an increase in Tll+ and Repo+ progeny (Figure 4K, L). Together, this suggests that D overexpression promotes the progression of the temporal series. However, it is possible that Mira+ cells can disappear via cell death. We will assess this possibility by staining for cell death marker Dcp1 at 6hr APF.

Reviewer #2 (Significance):

These appear to be novel and significant findings that will enhance our understanding of the temporal progression and terminal differentiation program of neural stem cells in vivo.<br /> I think the findings will be of interest to cell, developmental cell and stem cell biologists.

My primary expertise is in the cell biology of fly neural stem cells and asymmetric cell division of neuroblasts. Although I am not intimately familiar with the differentiation and differentiation literature, I consider the findings reported here relevant and impactful.

Reviewer #3 (Evidence, reproducibility and clarity):

The discoveries that the author describe in this manuscript are very specific to dedifferentiated neuroblasts created by UAS-dpn transgene overexpression. Dpn is endogenously expressed in optic lobe neuroblast throughout larval stage, which makes understanding how Dpn regulates gene expression based on the authors results (suppression of cell-cycle genes, and promotion of a specific temporal state) confusing.

Our data relate specifically to gene regulation by Dpn in a dedifferentiated context, and do not seek to understand Dpn regulation in wt neuroblasts. The reviewer is assuming our scope is greater here: we’re not trying to claim that we know what Dpn is doing in wt NBs, and it’s not surprising that ectopic effects in neurons may be different to wt NBs.

To assess whether the mechanisms described apply to more than Dpn overexpression, we will also assess whether the temporal series progression is affected in Lola RNAi and Nerfin-1 mutant.

Therefore, this manuscript does not advance our understanding of regulation of temporal identity and cell cycle progression in optic lobe neuroblasts during normal neurogenesis.<br /> The author's state:<br /> "However, beyond the fact that misexpression of these factors and pathways caused the formation of ectopic NBs, whether these dedifferentiated NBs faithfully produce the correct number and types of neurons or glial cells, or undergo timely terminal differentiation, has not been assessed. These characteristics are key determinants of overall CNS size and function, thus are important parameters when considering whether dedifferentiation leads to tumourigenesis or can be appropriately utilized for regenerative purposes."<br /> at the end of introduction. If this is a true primary goal of this study, the authors should describe it in abstract. Otherwise, readers will lose enthusiasm to read this manuscript in abstract and no longer read the following sections.

We will add this to the abstract.

Results<br /> 1. The authors should describe the expression pattern of all three of the Gal4 drivers used. While there are dotted outlines in the supplemental figure, there should be a description in the main text for the expression pattern of these lines which described with temporal state of NBs these lines are expressed in, and whether they are also expressed in the neurons or not.

We will include expression analysis of all three drivers in a supplementary figure and explain in the text the benefit of each driver.

1. The authors claim that overexpression of Dpn in the medulla region causes "dedifferentiation." The data provided however is not sufficient to conclude that dedifferentiation is occurring. The GAL4s used all drive in the NBs, and so it is unclear if the ectopic NBs ever became mature neurons. In addition, the lack of ectopic NBs in the clonal analysis 16hrs AHS does not prove that ectopic NBs at 24hrs AHS must have come from "mature neurons." To demonstrate dedifferentiation, the authors should use a driver system that is specific to mature neurons, and then overexpress dpn and look for mira+ cells. Currently, the authors data does not prove that mature neurons dedifferentiatiate into ectopic NBs upon Dpn OE.

We have conducted lineage tracing (G-Trace) analysis of the medulla neuron driver GMR31H08-GAL4 which we utilise in our study, this driver is predominantly expressed within the medulla neurons (real time) except for a few GMCs present in the lineage. Therefore, the Mira positive cells induced via Dpn overexpression are most likely from dedifferentiation (We will include this data in a supplemental figure in our revised manuscript).

To further support this, we will use GMR31H08-GAL4 with a Gal80ts, to restrict the timing to dedifferentiation induction to 3rd instar, so that the driver is restricted to neurons. Similar strategy to induce dedifferentiation was utilised in DOI: 10.1242/dev.141341 and DOI: 10.1016/j.devcel.2014.01.030.

1. What is a conclusion of fig 2C-H?

Fig 2C-H assess the expression of tTFs in UAS-dpn induced ectopic NBs. We will make these conclusions clearer in the text.

1. "As the tumor cell-of-origin can define the competence of tumor NBs to undergo malignancy identity of the dedifferentiated NBs were conferred by the age of the neurons they were derived from". This sentence is confusing. What are the authors investigating in the following experiment? Do they want to see ectopic NBs keep their early identity like Chinmo in ventral cord tumor NB? Or tll-positive NB's progenies can dedifferentiate to ectopic NB, but this ectopic neuroblast is not able to keep proliferation in pupal stage? It is hard to understand the connection of this sentence and the following experiment.

We will rephrase this sentence and further introduce this concept when talking about tumour cell of origin and competence. Additionally, we will make the connection to the experiments which follow it clearer.

1. The DamID experiment described used wor-gal4 as a driver, which means the Dpn binding profile generated is coming from not only optic lobe NBs, but central brain NBs and VNC NBs as well. In Magadi et al. (2020), the authors profiled Dpn binding in CNS hyperplasia, and found that dpn strongly bound Nerfin-1 and gcm. However, it does not bind cell cycle genes in this context. How do the authors know that the region that they claim are bound by dpn are bound in medulla NBs? The authors should also include tracks to show dpn binding at Nerfin-1, as well as the other tTFs (hth, ey, tll, and gcm). Providing this data will help to understand if Dpn binding is specific to the mid-temporal genes, as Dpn expression is known to be expressed in all medulla NBs regardless of temporal state.

We agree with the reviewer that the profile is not specific to medulla NBs. To assess Dpn binding profiles specifically in the medulla NBs, we will use the recently-published NanoDam technique (https://doi.org/10.1016/j.devcel.2022.04.008) for profiling GFP-fusion proteins, with a medulla specific driver (eyR16F10-GAL4) and Dpn-GFP (recombineered locus under endogenous control). This should inform us whether the target genes we have identified are relevant in the medulla.

We will include the tracks of the other transcription factors.

1. Currently, the DamID data does not help to interpret the Dpn overexpression phenotype at all. Inside of flip-out clone, some cells show Slp-1 expression while others showed D expression. The authors explain that Slp-1 and D suppress their expression to each other. But the DamID data indicate that both Slp-1 and D are Dpn target genes. If this is true, why did they observe the mosaic expression pattern inside of the same clone.

We observed that high levels of Slp-1 is correlated with low levels of D. This suggest to us that the initial stochastic differences accounts for where Slp-1 is high is where D is low, and vice versa.

1. The authors hypothesized if Dpn activated Slp-1directly. Does this mean that Dpn directly activate transcription of Slp-1? It is well known that Dpn is transcriptional repressor. Hes family proteins form a homodimer or heterodimer with another Hes protein and interacts Gro, which recruits a Histon deacetylase protein. The author's claim does not fit to the model what we currently believe. In addition, the authors claimed that Dpn inhibits cell cycle gene transcription directly. This is inconsistent to their claim that Dpn directly activate Slp-1 expression. If the authors want to claim that Dpn has two different functions in this context, the authors must demonstrate it by experimental results.

We will discuss these models in the Discussion, and make our claims more conservative, as we do not have direct experimental evidence to prove or disprove the model that Dpn is acting as an activator in this context.

1. Related to the above question, I wondered if the authors guess Dpn activate or repress D transcription by binding to D promoter region because they claimed that Dpn activate Slp-1, while suppress cell cycle genes.

We will make our claims more conservative, and discuss this point further in the Discussion.

1. I am confused to the claim that Dpn suppress cell cycle genes expression. Dpn overexpression induces dedifferentiation of neuron into NB and re-entry into the cell cycle. If Dpn suppress cell cycle genes how can the dedifferentiated cell re-enter into the cell cycle?

The data points towards that Dpn overexpression has two separate roles in regulating the cell cycle. Ofcourse dedifferentiation requires a commitment of neurons into the cell cycle (this we think is still happening), however, we think once these cells have turned on NB markers, they have limited ability to progress through the cell cycle. We will discuss this point in the Discussion.

1. Figure 6 looked redundant because we know Dpn is a direct target of Notch. It is obvious that an upstream factor overexpression can induce the identical phenotype to the phenotype induced by overexpression of a downstream factor.

A direct target does not necessarily infer the same phenotype. To assess whether the mechanisms apply to other dedifferentiation models, we will add Lola-RNAi and Nerfin-1 data to our revised manuscript.

Minor comments:<br /> 1. Typo in main text: "GMR31HI08-GAL4" should be "GMR31H08-GAL4"<br /> 2. In figure 1E-H the dotted line regions indicated the clones are not shown in the merge image. Please include<br /> 3. Typo in discussion paragraph 2: "temporal series was no sufficient to rescue cycle cycle progression"

We will correct these typos.

Reviewer #3 (Significance):

Insights into the developmental capacity of dedifferentiated stem cells will likely lead to novel strategy to replenish cells lost due to aging, injury and diseases in regenerative medicine.

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Referee #3

Evidence, reproducibility and clarity

The discoveries that the author describe in this manuscript are very specific to dedifferentiated neuroblasts created by UAS-dpn transgene overexpression. Dpn is endogenously expressed in optic lobe neuroblast throughout larval stage, which makes understanding how Dpn regulates gene expression based on the authors results (suppression of cell-cycle genes, and promotion of a specific temporal state) confusing. Therefore, this manuscript does not advance our understanding of regulation of temporal identity and cell cycle progression in optic lobe neuroblasts during normal neurogenesis.

The author's state:

"However, beyond the fact that misexpression of these factors and pathways caused the formation of ectopic NBs, whether these dedifferentiated NBs faithfully produce the correct number and types of neurons or glial cells, or undergo timely terminal differentiation, has not been assessed. These characteristics are key determinants of overall CNS size and function, thus are important parameters when considering whether dedifferentiation leads to tumourigenesis or can be appropriately utilized for regenerative purposes."<br /> at the end of introduction. If this is a true primary goal of this study, the authors should describe it in abstract. Otherwise, readers will lose enthusiasm to read this manuscript in abstract and no longer read the following sections.

Results

1. The authors should describe the expression pattern of all three of the Gal4 drivers used. While there are dotted outlines in the supplemental figure, there should be a description in the main text for the expression pattern of these lines which described with temporal state of NBs these lines are expressed in, and whether they are also expressed in the neurons or not.
2. The authors claim that overexpression of Dpn in the medulla region causes "dedifferentiation." The data provided however is not sufficient to conclude that dedifferentiation is occurring. The GAL4s used all drive in the NBs, and so it is unclear if the ectopic NBs ever became mature neurons. In addition, the lack of ectopic NBs in the clonal analysis 16hrs AHS does not prove that ectopic NBs at 24hrs AHS must have come from "mature neurons." To demonstrate dedifferentiation, the authors should use a driver system that is specific to mature neurons, and then overexpress dpn and look for mira+ cells. Currently, the authors data does not prove that mature neurons dedifferentiatiate into ectopic NBs upon Dpn OE.
3. What is a conclusion of fig 2C-H?
4. "As the tumor cell-of-origin can define the competence of tumor NBs to undergo malignancy identity of the dedifferentiated NBs were conferred by the age of the neurons they were derived from". This sentence is confusing. What are the authors investigating in the following experiment? Do they want to see ectopic NBs keep their early identity like Chinmo in ventral cord tumor NB? Or tll-positive NB's progenies can dedifferentiate to ectopic NB, but this ectopic neuroblast is not able to keep proliferation in pupal stage? It is hard to understand the connection of this sentence and the following experiment.
5. The DamID experiment described used wor-gal4 as a driver, which means the Dpn binding profile generated is coming from not only optic lobe NBs, but central brain NBs and VNC NBs as well. In Magadi et al. (2020), the authors profiled Dpn binding in CNS hyperplasia, and found that dpn strongly bound Nerfin-1 and gcm. However, it does not bind cell cycle genes in this context. How do the authors know that the region that they claim are bound by dpn are bound in medulla NBs? The authors should also include tracks to show dpn binding at Nerfin-1, as well as the other tTFs (hth, ey, tll, and gcm). Providing this data will help to understand if Dpn binding is specific to the mid-temporal genes, as Dpn expression is known to be expressed in all medulla NBs regardless of temporal state.
6. Currently, the DamID data does not help to interpret the Dpn overexpression phenotype at all. Inside of flip-out clone, some cells show Slp-1 expression while others showed D expression. The authors explain that Slp-1 and D suppress their expression to each other. But the DamID data indicate that both Slp-1 and D are Dpn target genes. If this is true, why did they observe the mosaic expression pattern inside of the same clone.
7. The authors hypothesized if Dpn activated Slp-1directly. Does this mean that Dpn directly activate transcription of Slp-1? It is well known that Dpn is transcriptional repressor. Hes family proteins form a homodimer or heterodimer with another Hes protein and interacts Gro, which recruits a Histon deacetylase protein. The author's claim does not fit to the model what we currently believe. In addition, the authors claimed that Dpn inhibits cell cycle gene transcription directly. This is inconsistent to their claim that Dpn directly activate Slp-1 expression. If the authors want to claim that Dpn has two different functions in this context, the authors must demonstrate it by experimental results.
8. Related to the above question, I wondered if the authors guess Dpn activate or repress D transcription by binding to D promoter region because they claimed that Dpn activate Slp-1, while suppress cell cycle genes.
9. I am confused to the claim that Dpn suppress cell cycle genes expression. Dpn overexpression induces dedifferentiation of neuron into NB and re-entry into the cell cycle. If Dpn suppress cell cycle genes how can the dedifferentiated cell re-enter into the cell cycle?
10. Figure 6 looked redundant because we know Dpn is a direct target of Notch. It is obvious that an upstream factor overexpression can induce the identical phenotype to the phenotype induced by overexpression of a downstream factor.

Minor comments:

1. Typo in main text: "GMR31HI08-GAL4" should be "GMR31H08-GAL4"
2. In figure 1E-H the dotted line regions indicated the clones are not shown in the merge image. Please include
3. Typo in discussion paragraph 2: "temporal series was no sufficient to rescue cycle cycle progression"

Significance

Insights into the developmental capacity of dedifferentiated stem cells will likely lead to novel strategy to replenish cells lost due to aging, injury and diseases in regenerative medicine.

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Referee #2

Evidence, reproducibility and clarity

Stem cells can divide asymmetrically to self-renew the stem cell while generating differentiating sibling cells. To restrict the number and type of differentiating sibling cells, stem cells often undergo terminal differentiation. Terminally differentiated cells can dedifferentiate and revert to a stem cell like fate. However, the underlying molecular mechanisms are incompletely understood in vivo.<br /> Here, Veen et al., use Drosophila neural stem cells (called neuroblasts) to investigate how terminal differentiation is regulated. Neuroblasts faithfully produce the correct number and type of neuronal cells through temporal patterning and regulated terminal differentiation. The authors show that misexpression of the bHLH transcription factor Deadpan (Dpn) induces ectopic neuroblasts, which predominantly express mid-temporal transcription factors at the expense of late-temporal transcription factors. As a consequence, these ectopic neuroblasts also fail to produce Repo positive glial cells and are stalled in their cell cycle progression. The authors provide evidence that promoting cell cycle progression and overexpression of the transcription factor Dichaete (D) is sufficient to restore the temporal transcription factor series, neuronal diversity and timely neuroblast differentiation.

This is an interesting study that will be of interest to the stem cell field. However, I encourage the authors to consider the following critiques:

1. Explain the rationale for the three different neuronal/NB drivers (GMR31HI08-GAL4, eyOK107-GAL4, eyR16F10-GAL4. How are they expressed?
2. The rationale for the Edu experiment (Figure S1I) is not clear. Why is this a measure for the production of neuronal progeny? For the correct interpretation of these results, the authors should also provide control clones or Edu experiments of regular neuroblasts.
3. How was % of Mira (Figure 1K and below) or the % of tTFs (Figure 2H onward) quantified? For instance, Figure 2C-G often shows clonal signal that is not highlighted with the dashed lines and the corresponding tTF intensity does not match the intensity in the outlined clone (eg. Figure 2D-D'; a large optic lobe clone is negative for Ey. Figure 2E-E'; an unmarked clone is negative for Slp).<br /> Similarly, the Hth signal is very weak to begin with so it is unclear how this was quantified. How was determined what constitutes real signal vs. background noise?<br /> Additional explanations in the methods section is needed to assess the robustness of the data.
4. This sentence should be rephrased: 'As the tumour cell-of-origin can define the competence of tumour NBs to undergo malignancy (Farnsworth et al., 2015; Narbonne-Reveau et al., 2016), we next tested whether the temporal identity of the dedifferentiated NBs were conferred by the age of the neurons they were derived from.'<br /> The connection between tumorigenicity and temporal identity is not really clear and should be briefly reintroduced for this paragraph.
5. Figure 2I-N: The experimental outline in I and J should be grouped with the corresponding images to clarify what is compared. Also, there are no images for the control clones, which make a comparison difficult. The images are also too small. I cannot really see the Hth, or Slp signal in the small clones shown in Figure 2K-L".
6. Figure 3H: It is not clear why there are only a small group of Nbs that are positive for Mira. Please explain.
7. Figure 3K-M: Please explain how the Toy signal was measured and quantified.
8. The TaDa data set is very interesting but the following might be an overstatement: "We found that Dpn directly binds to slp1 as well as the Sox-family TF dichaete (D) which is expressed in medulla NBs after slp1 (Li et al., 2013) (Figure S6 A-B)."<br /> More direct binding assays might be needed to show that Dpn directly binds to slp1 and D. If this is already shown, clarify the sentence to indicate what is published and what is extracted from the data shown here.<br /> Also, what is the rationale for this statement: "Consistent with the model that D represses Slp-1..."?
9. This might be an overinterpretation: D overexpression in UAS-Dpn NBs promoted their pre-mature cell cycle exit at 6 hrs APF using eyR16F10-GAL4. The data shows loss of Mira signal, which could occur through different mechanisms.

Significance

These appear to be novel and significant findings that will enhance our understanding of the temporal progression and terminal differentiation program of neural stem cells in vivo.<br /> I think the findings will be of interest to cell, developmental cell and stem cell biologists.

My primary expertise is in the cell biology of fly neural stem cells and asymmetric cell division of neuroblasts. Although I am not intimately familiar with the differentiation and differentiation literature, I consider the findings reported here relevant and impactful.

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Referee #1

Evidence, reproducibility and clarity

Summary

Authors show that overexpression of bHLH transcription factor Dpn in the medullary neurons of the Drosophila optic lobe results in the dedifferentiation of these neurons back into the NBs. These dedifferentiated NBs acquire and maintain mid-temporal identity, express Ey and Slp, and show delayed onset of tTF Tailless (Tll), leading to an excess of neurons of mid-temporal fate at the expense of late temporal fate neurons and glial cells. The dedifferentiated NBs are stalled in the cell cycle and fail to undergo terminal differentiation. Over expression of tTF Dicheate (D) or promoting G1/S transition pushed these NBs to late stages of the temporal series, partly rescuing the neuronal diversity and causing their terminal differentiation. They also show that the dedifferentiation of NBs by Notch hyper-activation also exhibited stalled temporal progression, which is restored by D overexpression.<br /> Authors suggest that cell cycle regulation and tTF are primary to the proliferation and termination profile of dedifferentiated NBs.<br /> Using these conclusions, the authors emphasize the need to recreate the right temporal profile and ensure appropriate cell cycle progression to use dedifferentiated NSC for regenerative purposes or prevent tumorigenesis originating from differentiated cell types.

Major comments:

• Are the key conclusions convincing?

Most conclusions are convincing; however, some issues are pointed out below.<br /> - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

The authors have overexpressed Dpn and shown that medulla neurons dedifferentiate to NBs, similar to the loss of function phenotype seen for the Nerfin-1 of which Dpn is a target. They also show that temporal series progression defect is also seen in the case of dedifferentiated NB generated by Notch over-activation.<br /> Using these two examples, the authors suggest that for dedifferentiated NSC, which are to be used for the regenerative purpose, one needs to recreate the right temporal profile and ensure cell cycle progression occurs appropriately. Authors also claim that to prevent tumorigenesis originating from differentiated cell types, one needs to recreate the right temporal profile and ensure cell cycle progression occurs appropriately.

While I agree with this, I think this is an overreaching conclusion based on just these two examples. If they could show the same for one more method of dedifferentiation (For, e.g. Lola) happening in medulla neurons which happens by a mechanism independent of Nerfin-1, Dpn, Notch axis, the argument will become more convincing and broad.<br /> Also when authors mention N mediated dedifferentiation, they need to inform that Dpn is a direct target of Notch in NBs (Doi. 10.1016/j.ydbio.2011.01.019), they do so in the discussion, but mentioning it here gives a broader context to the reader.

Another important point that needs a mentioned here is that conclusions are based on dedifferentiation happening in the medulla neurons, which are considered less stable since they lack Prospero. Therefore whether this conclusion can be generalized for all the tumors arising from dedifferentiation in the CNS (eg, those arising from NICD activation in the central brain or thoracic region of the VNC) is another concern. Maybe authors can consider making a more conservative claim.<br /> Generalizing this conclusion to Prospero expressing NBs lies outside the scope of the current study and cannot be addressed here because central brain Type-I NBs use a different set of tTFs.<br /> - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

Experiments with Lola knockdown/mutants in medulla neurons can be done quickly, in my opinion, and will substantiate this claim.<br /> Another obvious question that comes to mind is if medulla neurons dedifferentiate on overexpression of Dpn, does the same happen in nerfin-1 mutant clones as well? And if yes, why has the author not done similar experiments for nerfin-1 mutants.<br /> Please show Ey staining in Fig-2 if possible, it will also help to add a line on why Slp was used as marker for mid tTFs instead of Ey.<br /> In Model shown in last figure Dpn is shown to repress D and activate Slp. Can authors show that Dpn overexpression represses D and activate Slp either by antibody staining or by RT PCR.<br /> - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

Experiments with Lola and nerfin-1 mutants can be done in a few months. I cannot comment on the cost involved.<br /> - Are the data and the methods presented in such a way that they can be reproduced?

Yes<br /> - Are the experiments adequately replicated and statistical analysis adequate?

Replication and statistical analysis are fine. The activated Notch experiments show only three data points in all the experiments. It will be good to increase this number.

Minor comments:

• Specific experimental issues that are easily addressable.

There is a problem with Fig-5F (both 5E and 5F have % EdU in clone/ % Mira in the clone as y-axis), I do not understand how the Fig-5F let them conclude that D overexpression increases the rate of neuronal production.

In one place, the authors conclude, "Together, this data suggests that it is likely that cell cycle progression lies upstream of the temporal series, to promote the generation of neurons". Authors should consider adding "medulla NBs" at the end of the sentence since cell cycle progression being upstream of temporal series is already known in Type-I NBs, as pointed out by authors as well (Ameele and Brand 2019).

In the discussion authors says that "Our data support the possible links between cell cycle progression and the expression of temporal regulators controlling NB proliferation and cellular diversity". This is new information, as the 2019 study did not show how cell diversity changes with a changed tTF profile. I think the authors should elaborate on this point to highlight how this is different from what is already known from the 2019 study (done in the context of Type-I NBs).<br /> Maybe they need to highlight that the cell cycle directs/regulates the progression of temporal series compared to the earlier observation where temporal series was shown to be downstream of the cell cycle.

In fig-3J in clones even after 24 AHS, Dpn continues to be overexpressed but these cells undergo terminal differentiation, can authors comment why is it so?<br /> In one place authors say, "To better assess the cumulative effect of the neurons made throughout development, EyOK107-GAL4 was used to drive the expression of Dpn" maybe some background on why use this specific GAL4.<br /> Also a line about why GMR31HI08-GAL4 eyOK107-GAL4 and and eyR16F10-GAL4 were used.<br /> - Are prior studies referenced appropriately ?

Yes, but in a few places, some references can be added.<br /> An important point that needs to be mentioned for the context is the medulla neurons do not use Prospero for terminal differentiation and are thus considered less stable (DOI: 10.1242/dev.141341).<br /> In discussion, the authors say that "It would be interesting to explore whether N similarly acts on these target genes to specify cell fate and proliferation profiles of dedifferentiated NBs." There is a study looking at Notch targets in NB hyperplasia (DOI: 10.1242/dev.126326); whether that study shows if any of the cell cycle genes are downstream of activated Notch, needs a mention here.<br /> Also, when authors mention N mediated dedifferentiation, they need to inform that Dpn is a direct target of Notch in NBs (Doi. 10.1016/j.ydbio.2011.01.019). They do so in the discussion, but mentioning it in the introduction or results will give a broader context to the reader.<br /> Another gene that needs a mention is "Brat", which regulates both Dpn and Notch, and causes dedifferentiation and tumors in CNS, I think this gene and its interaction with Dpn and Nerfin and Notch needs to be discussed either in the introduction or discussion.<br /> - Are the text and figures clear and accurate?

The main figures are not labeled. Therefore, it was very annoying to deduce the specific figure numbers.<br /> There are 1 or 2 places where figure calling is wrong in the text.<br /> The Image Fig-5I shows cycD and CDK4 at the G2-M transition; while the text says it supports G1/S, which is indeed the case, the figure needs modification.<br /> - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

The presentation is okay, in my opinion.

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

The factors leading to dedifferentiation of the neurons have been identified previously by groups of Chris Doe (mldc, DOI: 10.1242/dev.093781), Andrea brand (10.1016/j.devcel.2014.01.030.) as well as the authors of this paper (10.1101/gad.250282.114, 10.1016/j.celrep.2018.10.038.). However, many questions remained unaddressed regarding such NB generated from neuronal dedifferentiation. For example, whether these cells contribute to native cell diversity of the CNS, undergo timely differentiation or their progeny cells incorporated into appropriate circuits is not well understood. Successful execution of these phenomena is critical for generating functional CNS and such insights are crucial for understanding the origin of tumorigenesis in CNS or employing dedifferentiated NSC for regenerative purposes.

This study is an overexpression-based study, however, some of the results give significant conceptual insights into the tumors arising out of the dedifferentiation of the neurons. It also gives insights into the fact that the dedifferentiated cells need to be carefully examined for the temporal factor profile before they can be employed for regeneration or any therapy targeting them.<br /> However, in my opinion, they need to test this idea at least in one more system of neuronal dedifferentiation, preferably independent of the nerfin-1/Notch/Dpn axis to generalize this claim.<br /> - Place the work in the context of the existing literature (provide references, where appropriate).

Cerdic Maurange's group had looked at the role of temporal factors and identified the early phase of malignant susceptibility in Drosophila in 2016 (doi: 10.7554/eLife.13463). Andrea Brand's group has shown in a 2019 paper that cell cycle progression is essential for temporal transition in NBs (doi: 10.7554/eLife.47887). Both these studies were in the context of Type-I NBs, which express Prospero, which is crucial for the differentiation of the neurons.<br /> Previously the authors have studied type-I NBs and shown by Targeted DamID that Dpn is Nerfin-1 target. They also show that Nerfin-1 mutants show dedifferentiation of neurons. They follow up on this observation in medulla neurons, where they find that Dpn overexpression results in their dedifferentiation into medulla NBs. Medulla NBs differ from Type-I NBs in using a separate set of tTFs. Also, Type-I NB and neurons arising from them use Prospero for terminal differentiation, while medulla neurons do not express Prospero and are therefore considered less stable (DOI: 10.1242/dev.141341).

The importance of the study lies in the results that show that the NB arising out of dedifferentiation of medulla neurons takes up mid-temporal fate. These NBs are stalled in Slp expressing mid-temporal stage unless the cell cycle is promoted by overexpression of cell cycle genes regulating G1/S transition.<br /> Authors also show that overexpression of D promotes the progression of temporal series in these dedifferentiated NBs, which could partly rescue neuronal diversity and result in terminal differentiation. Thus D plays an important role in determining the type of neurons these NBs generated. This suggests that knowing the tTF profile of these types of dedifferentiated NBs is vital if these cells were to be used for regenerative purposes. Authors further claimed that cell cycle regulation and tTFs are critical determinants of the proliferation and termination profile of dedifferentiated NBs.<br /> - State what audience might be interested in and influenced by the reported findings.

The study will be of broader interest to researchers interested in central nervous system patterning, regeneration, and cancer biology.<br /> - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

Drosophila, central nervous system patterning and cell fate determination of neural stem cells.

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Reply to the reviewers

We propose three revisions, that have not yet been included in the current manuscript:

1. All three reviewers comment on the data in figure 7, in which the application of the sensor is shown. We agree that the number of cells is low, and we plan to repeat this experiment to increase the number of cells, and better demonstrate the usefulness of the new probe. We note that the improved Cdc42 sensor is used in a recent preprint (see figures 7 and 9 of: https://www.biorxiv.org/content/10.1101/2022.06.22.497207v2.full), clearly showing the potential of the probe for detection of Cdc42 and increasing our confidence that we can generate higher quality data.
2. The ratio of expression of the different components was not quantified. We have these data and we will (re)analyze it and present the results (related to Reviewer #3, point2).
3. We will reanalyze the images to ensure that representative images are depicted in the manuscript (related to Reviewer #2, point 3).

   Reviewer #1

1) It is not clear why RhoA data were included in this manuscript (Fig. 1), since they seem irrelevant to the primary topic addressed.

We have cell-based data from our previous (published) work that we can use to check whether these results align with the mass-spec data. To make this point clearer we add “We looked first into GBDs for Rho, to compare the results of the mass spectrometry screen with the results of our cell-based assays”.

2) It is not clear what cell type was used when screening for p67phox. The expression of this component of the NADPH oxidase is restricted to a few specific cell types.

That’s a relevant point and therefore observation that p67phox is not detected is perhaps not surprising. We removed this statement.

3) There is precious little quantitation of the colocalization or translocation of the probes throughout the manuscript. It is difficult to assess the validity of the conclusions in the absence of analysis of the statistical significance of the colocalization.

In figure 2, which is an initial screen, there is only a qualitative assessment. However, for the promising candidates, there is a quantitative assessment in Figures 3B and 4 B as to which extent the candidates colocalize with the nuclear localized target. From the rank order and individual datapoints the best performing binder can be inferred.

4) It is not clear why translocation to mitochondria was used in some experiments and translocation to the nucleus in others.

To clarify, we have added text: ”We have previously used nuclear localized, constitutive active Rho GTPases, but these are not accessible for larger proteins that cannot enter the nucleus”

5) In the S1P experiments, it is difficult to ascertain whether increased fluorescence resulted from membrane folding/ruffling or is actually a consequence of localized activation of receptors. Why does the fluorescence decrease progressively over 1500 seconds? Isn't maximal receptor activation accomplished much sooner?

This experiment suffered from bleaching. We will redo the experiment to get higher number of cells and to improve the data.

Reviewer #2

Major comments

1. Statistical tests are missing in most of the figures. If the principal purpose of this work is to compare the performance of candidate peptides, the quantitative comparison is essential. If the purpose is just to report another relocation probe, then, more application data may be necessary.

We will improve the quality of the application data. As for statistics, we have added the effect size to figures 5C-F and figure 6A. To explain this (not so common) statistic we add to the materials and methods: “The effect size that quantifies the difference and its distribution was calculated with the web tool ‘PlotsofDifferences’”.

1. The criteria for selecting the best peptide should be clearly described. Is it just by inspection or based on any quantitative data? We know that quantification of colocalization is a difficult task. Therefore, it depends on the aim of this work whether the authors are asked to show quantitative data or not. If a strict comparison of peptides is aimed at, the expression level of each target peptide should be at a comparable level. It will be also required whether the design of each probe guarantees the proper folding to bind to GTPases.

There are two stages for the selection. First, we did a qualitative analysis of colocalization (shown in figure 2). Based on the results (“Candidates colocalizing with the mitochondrial tagged Rho GTPase were further tested for their potential as localization-based sensors”), we generated smaller biosensor candidates of which binding to a nuclear target was quantitatively analyze (figures 3B and 4B). As the expression level is an important factor, we ascertained potential candidates were expressed at roughly the same level in the nuclear accumulation assay.

1. About the images of cells: When a fluorescent image is presented, we assume it represents all other cells. Please check all images whether they are truly representing the data. For example, in Fig. S3 the nuclei of ABI1-expressing cells look weird, and the nucleus of CYRI-A is very large. If this is true, the reason why ABI1 and CYRI-A should be excluded from the candidate is not the relocation efficiency but the undesired effect on cell physiology. For the screening of the peptides, this information is also very important. With that, this paper becomes more valuable for scientists.

We agree that this is an important point. We will reanalyze the data as indicated in the ‘planned revisions’.

1. Please examine the order of panels. For example, the result of mScarlet is on the top in Fig3, but at the bottom in Fig4. Such inconsistency would disturb readers.

We thank the reviewer for this suggestion and we changed figure 4.

1. The label should be consistent throughout the paper. For example, in Fig. 5A, Lck-FRB-mTurquoise2 is labeled as Lck-FRB (without the fluorescent protein's name). WASp(CRIB)-mScarlet-I-WASp(CRIB) is labeled as WASp(CRIB)-mScar-WASp(CRIB) (with fluorescent protein's name). Moreover, the same peptide is labeled as mSca-1xWASp(CRIB) in Panel B. Such inconsistency is confusing.

We agree, we have updated figure 5A by adding the abbreviations of the fluorescent proteins. Please note that WASp(CRIB)-mSca-WASp(CRIB), mSca-1xWASp(CRIB) and mSca-2xWASp(CRIB) are three different constructs. In the first one the CRIB domains are sandwiching the fluorescent protein and in the third one they are in tandem downstream of the fluorescent protein.

1. Quantitative insight would improve this work. For example, in Fig. 7, the reason why the authors believe that the probe worked is the accumulation of probe at the tip of lamellipodia and the decrease in cytoplasmic intensity. This reviewer does not think the accumulation of the probe in the small area of the lamellipodia explains the massive decrease of cytoplasmic signals. Probably, a substantial amount of the probe is relocated to the plasma membrane, not limited to the lamellipodia.

Minor comments

We propose to repeat the experiment shown in figure 7 and to improve the quality of the data.

1. Introduction, "FRET signal is typically measured with a wide field microscope.": This reviewer does not agree with this statement. Confocal and two-photon microscopes have also been used widely.

Fair point. We changed the text to “when the FRET signal is measured with a wide field microscope”

Introduction, "G-protein activating proteins (GAP)": It should read as "GTPase-activating proteins (GAPs)"

Thanks, corrected.

TRIF should read as TIRF.

All instances have been corrected.

Fig.1: To the best of this reviewer's knowledge, PKN1 was first used as the RhoA target peptide by Yoshizaki et al in 2003. J Cell Biol 162, 223-232. They also examined mDia, Rhoteki, and Rhophilin as the target peptides. Pak1 was first used as the Rac1 probe by Kraynov et al. Science 290, 333-337, 2000. Use of Pak1 as the Cdc42 probe was reported by Itoh et al. Mol Cell Biol 22, 6582-659, 2002. This reviewer believes that the priority of the first report should be respected.

We changed part of the introduction to:

High scoring proteins for interacting with constitutively active RhoA(Q63L) included ANLN part of the AniRBD Rho location sensor (Piekny and Glotzer, 2000), PKN1 part of aRho FRET sensor (Yoshizaki et al., 2003) and RTKN part of the rGBD Rho location sensor (Benink and Bement, 2005; Mahlandt et al., 2021) (Fig. 1A,B). This suggested that proteins with a high score in the mass spectrometry screen are potentially suitable as Rho GTPase activity biosensor. Indeed, the GBDs used for Cdc42 location sensors from, PAK1 used in the PBD location sensor (Itoh et al., 2002; Petrie et al., 2012) and N-WASP similar to WASp used in the wGBD location sensor (Benink and Bement, 2005) showed a high score in the screen (Fig. 1A,B).

Discussion:

Another challenge is the Rho GTPase specificity of the relocation-based sensor. For example, Pak1(CRIB) was first used in a Rac1 FRET sensor (Kraynov et al., 2000)____. ThenPak1(CRIB) has been utilized in Cdc42 FRET sensors and in an intensiometric Cdc42 sensor (Hanna et al., 2014; Itoh et al., 2002; Kim et al., 2019). However, Pak1(CRIB), also named PBD sensor, has then been reintroduced by Weiner and colleagues as a Rac1 specific location-based sensor and is often used in neutrophil HL60 cells (Brunetti et al., 2022; Graziano et al., 2019; Le et al., 2021; Weiner et al., 2007).

We also updated the tables in Figure 1.

Fig. 1: Why do the authors omit other promising candidates shown in panel 1B? Please describe the reason for the choice.

We took into account the availability of plasmid DNA, as also explained in the manuscript: “candidate GBDs were selected from top 30 scores of the mass spectrometry screen, that were specific for one Rho GTPase and their DNA was available on addgene”

Fig. 1B: Be consistent to use either "Name" or "Uni Prot name" in Panel A.

We updated figure 1.

Fig. 2: Please include information on TOMM20. The readers may not read the paper by Gillingham et al.

We added an explanation: “To this end, a fusion with TOMM20 was used for mitochondrial localization.”

Fig3 and 4: The authors should show the images of control H2A.

We provide the data for control H2A in figures 3B and 4B.

In Fig3B and 4B, "Cdc42/Rac1 affinity" would be misleading, because the control dots represent their authentic localization rather than "Cdc42/Rac1 affinity".

We agree, we have updated figure 3B and 4B.

Fig. 4: More explanation of this figure is required.

We added text: “Hence, the sensor candidate can freely partition between Rac and Cdc42 binding.”

Fig. 5: More explanation about the FKBP-FRB system will be helpful.

We changed the text to: “The system used rapamycin induced heterodimerization of the two domains FRB and FKBP to recruit the DHPH domain of the Cdc42 specific GEF ITSN1 to the plasma membrane, where it induces activity of the endogenous Cdc42”

Fig. 6: It is rather surprising to see that control-mScarlet also responds to Rac1 activation. What is the explanation for this observation?

We agree and have no explanation.

Fig. 7: A single champion data may not be convincing to prove the usefulness of this probe.

We agree and propose to repeat the experiment.

Reviewer #3

1) The discussion comparing different types of biosensors missed important points. Although the advantages of localization biosensors listed by the authors are correct, they gave the impression that these should simply be an improved replacement for FRET biosensors. There are times when FRET biosensors provide clear advantages. Unlike other proteins, Rho GTPases are well suited for localization sensors because the activated conformation, and only the activated conformation, localizes to the membrane. For diffuse or 3D localization FRET can provide better quantification. Subtle features such as gradients are not easily quantified over a background of unattached domain. The authors state that localization biosensors have enhanced spatial resolution, but this is not explained.

We agree that our introduction is biased towards a preference for relocation based biosensors. However, having used both approaches, we see that both strategies have pro’s and cons. Therefore, we removed the claim for higher resolution and we added: “Still, the ratiometric mode of imaging FRET sensors is beneficial for detection of gradients or activity in 3D imaging”.

2) Throughout the paper, the ratio between the GTPase and the domain, and the overall expression level of each, was not sufficiently examined. The results in many cases would be dependent on both these factors (was a large excess of domain used? Was there insufficient domain to bind the GTPase and provide a signal? Did this vary for different domains, and therefore produce the differences observed? A lack of apparent binding specificity could be produced by high domain expression.)

This is an important point. We will re-analyze the data and include a figure where we add the binding efficiency versus the expression level.

3) In the nuclear exclusion assay, some GTPases were excluded from the nucleus and others not. This was true even without expression of the domains. When GTPases were excluded from the nucleus, domains were eliminated from contention, even when this was true without domain. The authors could at least mention that these domains may be viable.

Correct, and we have added this text: “we cannot exclude that these would be viable Cdc42 sensor candidates”

4) In the multiplexing experiment, only two cells were imaged. In one cell RhoA activity was inversely correlated with Cdc42 activity. In the other cell it was not. It seems there is insufficient information to reach firm conclusions.

We agree and in the revision plan we indicate that we will repeat this experiment to increase the number of cells.

Minor points:

• There appear to be errors in naming mutants. Q60L is used for constitutively active Rac, but Q61L is likely meant. H2A-mTurquoise2-Rac1(G12V)-ΔCaaX is used when it likely should be H2A-mTurquoise2-Rac1(Q61L)-ΔCaaX. There are other examples -- a careful check of these names throughout the manuscript would be valuable.

Thanks for spotting this. Q60L is changed to Q61L. Note that the Rac1(G12V) is correct as it also is a constitutive active Rac1.

• Intro-Paragraph 1-line 5: change present to presence

• Intro-Paragraph 5- line 7: use them instead of theme.

Thanks, both corrected.

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Referee #3

Evidence, reproducibility and clarity

The manuscript was clearly written, but the introduction or discussion could provide a more balanced view of the significance. There are important experiments that are required to support the conclusions regarding selectivity and differences between the domains, particularly the role of expression level and the ratio of expressed proteins. Our review is summarized here:

The authors set out to improve localization-based biosensors for Rac1 and Cdc42 by identifying domains that bind selectively to the activated conformation of Rac1 and Cdc42. Using screens based on mitochondrial and nuclear relocalization, together with mass spec and proximity biotinylation, several potential candidates were identified. The work concludes with the identification of a WASp CRIB domain that can be used as a useful Cdc42 relocalization biosensor. It was applied in a well-executed proof of principle study demonstrating multiplexed imaging of RhoA and Cdc42 localization biosensors.

1. The discussion comparing different types of biosensors missed important points. Although the advantages of localization biosensors listed by the authors are correct, they gave the impression that these should simply be an improved replacement for FRET biosensors. There are times when FRET biosensors provide clear advantages. Unlike other proteins, Rho GTPases are well suited for localization sensors because the activated conformation, and only the activated conformation, localizes to the membrane. For diffuse or 3D localization FRET can provide better quantification. Subtle features such as gradients are not easily quantified over a background of unattached domain. The authors state that localization biosensors have enhanced spatial resolution, but this is not explained.
2. Throughout the paper, the ratio between the GTPase and the domain, and the overall expression level of each, was not sufficiently examined. The results in many cases would be dependent on both these factors (was a large excess of domain used? Was there insufficient domain to bind the GTPase and provide a signal? Did this vary for different domains, and therefore produce the differences observed? A lack of apparent binding specificity could be produced by high domain expression.)
3. In the nuclear exclusion assay, some GTPases were excluded from the nucleus and others not. This was true even without expression of the domains. When GTPases were excluded from the nucleus, domains were eliminated from contention, even when this was true without domain. The authors could at least mention that these domains may be viable.
4. In the multiplexing experiment, only two cells were imaged. In one cell RhoA activity was inversely correlated with Cdc42 activity. In the other cell it was not. It seems there is insufficient information to reach firm conclusions.

Minor points:

• There appear to be errors in naming mutants. Q60L is used for constitutively active Rac, but Q61L is likely meant. H2A-mTurquoise2-Rac1(G12V)-ΔCaaX is used when it likely should be H2A-mTurquoise2-Rac1(Q61L)-ΔCaaX. There are other examples -- a careful check of these names throughout the manuscript would be valuable.
• Intro-Paragraph 1-line 5: change present to presence
• Intro-Paragraph 5- line 7: use them instead of theme.

Significance

The assays and approach to finding domains for biosensors were novel and interesting. The end result was not as surprising as one might have hoped, but the approach alone made the paper worthwhile.

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Referee #2

Evidence, reproducibility and clarity

Mahlandt et al report Rho GTPase relocation sensors. First, the authors picked up candidate peptides based on the Mass-Spec data reported by Sean Munro's laboratory. The authors repeated the experiments to confirm the binding of peptides to mitochondria-targeted Cdc42 and Rac1 and narrowed down the candidate peptides by binding to nuclear Cdc42. The specificity of binding to Rac1 and Cdc42 was also tested. Eventually, they concluded that dimeric Tomato-WASp(CRIB) is the best sensor for Cdc42, which could detect S1P-induced Cdc42 activation in primary endothelial cells. The effort to improve the relocation sensors should be evaluated highly. This reviewer has some suggestions to improve this paper.

Major comments:

1. Statistical tests are missing in most of the figures. If the principal purpose of this work is to compare the performance of candidate peptides, the quantitative comparison is essential. If the purpose is just to report another relocation probe, then, more application data may be necessary.
2. The criteria for selecting the best peptide should be clearly described. Is it just by inspection or based on any quantitative data? We know that quantification of colocalization is a difficult task. Therefore, it depends on the aim of this work whether the authors are asked to show quantitative data or not. If a strict comparison of peptides is aimed at, the expression level of each target peptide should be at a comparable level. It will be also required whether the design of each probe guarantees the proper folding to bind to GTPases.
3. About the images of cells: When a fluorescent image is presented, we assume it represents all other cells. Please check all images whether they are truly representing the data. For example, in Fig. S3 the nuclei of ABI1-expressing cells look weird, and the nucleus of CYRI-A is very large. If this is true, the reason why ABI1 and CYRI-A should be excluded from the candidate is not the relocation efficiency but the undesired effect on cell physiology. For the screening of the peptides, this information is also very important. With that, this paper becomes more valuable for scientists.
4. Please examine the order of panels. For example, the result of mScarlet is on the top in Fig3, but at the bottom in Fig4. Such inconsistency would disturb readers.
5. The label should be consistent throughout the paper. For example, in Fig. 5A, Lck-FRB-mTurquoise2 is labeled as Lck-FRB (without the fluorescent protein's name). WASp(CRIB)-mScarlet-I-WASp(CRIB) is labeled as WASp(CRIB)-mScar-WASp(CRIB) (with fluorescent protein's name). Moreover, the same peptide is labeled as mSca-1xWASp(CRIB) in Panel B. Such inconsistency is confusing.
6. Quantitative insight would improve this work. For example, in Fig. 7, the reason why the authors believe that the probe worked is the accumulation of probe at the tip of lamellipodia and the decrease in cytoplasmic intensity. This reviewer does not think the accumulation of the probe in the small area of the lamellipodia explains the massive decrease of cytoplasmic signals. Probably, a substantial amount of the probe is relocated to the plasma membrane, not limited to the lamellipodia.

Minor comments:

1. Introduction, "FRET signal is typically measured with a wide field microscope.": This reviewer does not agree with this statement. Confocal and two-photon microscopes have also been used widely.
2. Introduction, "G-protein activating proteins (GAP)": It should read as "GTPase-activating proteins (GAPs)"
3. TRIF should read as TIRF.
4. Fig.1: To the best of this reviewer's knowledge, PKN1 was first used as the RhoA target peptide by Yoshizaki et al in 2003. J Cell Biol 162, 223-232. They also examined mDia, Rhoteki, and Rhophilin as the target peptides. Pak1 was first used as the Rac1 probe by Kraynov et al. Science 290, 333-337, 2000. Use of Pak1 as the Cdc42 probe was reported by Itoh et al. Mol Cell Biol 22, 6582-659, 2002. This reviewer believes that the priority of the first report should be respected.
5. Fig. 1: Why do the authors omit other promising candidates shown in panel 1B? Please describe the reason for the choice.
6. Fig. 1B: Be consistent to use either "Name" or "Uni Prot name" in Panel A.
7. Fig. 2: Please include information on TOMM20. The readers may not read the paper by Gillingham et al.
8. Fig3 and 4: The authors should show the images of control H2A.
9. In Fig3B and 4B, "Cdc42/Rac1 affinity" would be misleading, because the control dots represent their authentic localization rather than "Cdc42/Rac1 affinity".
10. Fig. 4: More explanation of this figure is required.
11. Fig. 5: More explanation about the FKBP-FRB system will be helpful.
12. Fig. 6: It is rather surprising to see that control-mScarlet also responds to Rac1 activation. What is the explanation for this observation?
13. Fig. 7: A single champion data may not be convincing to prove the usefulness of this probe.

Significance

1. The authors have screened many peptides, which may serve as the relocation sensor for Rho-family GTPases.
2. There are precedent relocation sensors, a part of which is listed in Fig. 1A. This work discloses an improved relocation biosensor.
3. Cell biologists who is working on Cdc42 will be interested in this probe.
4. Expertise of this reviewer: Signal transduction, Fluorescence microscopy.

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Referee #1

Evidence, reproducibility and clarity

In this manuscript Mahlandt et al. report efforts to generate sensitive fluorescent biosensors to monitor the activation of Rac and Cdc42. While biosensors for these GTPases have been designed and used by others, some have poor specificity while others require the measurement of FRET, which is technically more complex and usually yields low signal to noise ratios. The efforts of Mahlandt et al. are therefore well justified and commendable, and follow on the heels of their successful development of a Rho-selective probe (J. Cell Sci., 2021).

The authors used existing data of interacting proteins to identify candidate domains that could be adapted for use as Rac or Cdc42 activation indicators. They proceeded to select the most promising candidates, assessed their specificity and tried to improve their efficiency by generating tandem constructs that are expected to have higher avidity. They identify CYRA-I as a positive Rac interactor, but the affinity and selectivity of this construct were not deemed to be sufficient and this line of enquiry was not pursued further. In contrast, the WASp-CRIB was found to be sufficiently Cdc42-specific and its avidity was improved by generating a construct tagged with dimeric Tomato fluorescent protein. Unfortunately, while data using overexpression of active Cdc42 are convincing and clear, the results obtained under physiological conditions -stimulating cells with S1P- show very modest recruitment. The general usefulness of the probe is therefore questionable.

There are also a number of specific issues:

1. It is not clear why RhoA data were included in this manuscript (Fig. 1), since they seem irrelevant to the primary topic addressed.
2. It is not clear what cell type was used when screening for p67phox. The expression of this component of the NADPH oxidase is restricted to a few specific cell types.
3. There is precious little quantitation of the colocalization or translocation of the probes throughout the manuscript. It is difficult to assess the validity of the conclusions in the absence of analysis of the statistical significance of the colocalization.
4. It is not clear why translocation to mitochondria was used in some experiments and translocation to the nucleus in others.
5. In the S1P experiments, it is difficult to ascertain whether increased fluorescence resulted from membrane folding/ruffling or is actually a consequence of localized activation of receptors. Why does the fluorescence decrease progressively over 1500 seconds? Isn't maximal receptor activation accomplished much sooner?

Significance

While the purpose and intent of the study are commendable, the results are far from convincing and the probes designed do not represent a sufficient improvement over existing biosensors. The lack of quantitative and statistical analyses is problematic, as is it is difficult to assess the significance of the results.

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Reply to the reviewers

1. General Statements [optional]

We thank the reviewers for fair and constructive comments, and for good suggestions for how to further improve the manuscript. We also appreciate the comments on novelty and potential importance for future clinical approaches for CADASIL. Below we provide our point-by-point responses to the reviewers’ comments. We start with a description of planned revisions, followed by describing changes already carried out in the transferred manuscript.

2. Description of the planned revisions

We plan to experimentally address the following points raised by the reviewers.

First, we will address the ASMA staining in the arterioles by isolating brain microvessels from the TgN3R182C150 mouse and WT mouse and strain for ASMA and perlecan/PDGFRb (stain pericytes and VSMC) to in more detail visualize that ASMA is only expressed in the arterioles and not in the pericytes in the capillaries (reviewer 1, point 4).

Second, we will assess the safety and toxicity of the active immunotherapy (reviewer 2, point 4). Specifically, we plan to analyse the structure and morphology of the kidneys from sham and vaccinated mice for renal damage by histology, H/E staining as well as creatinine levels in the serum. If the histology shows signs of damage (such as necrosis, apoptosis or granules in the tubules), we will stain the tissue with KIM1 for acute renal damage and caspase 3 for apoptosis. Moreover, we will analyse the inflammation C-Reactive Protein (CRP) marker in the serum by western blot analysis and/or ELISA and finally Neurofilament Light chain protein in the serum by SIMOA analysis to monitor if the vaccination is causing any neurodegeneration.

Third, we will improve the image quality for Figure 5A (reviewer 1, point 5).

Reviewer #1 (Evidence, reproducibility and clarity):

Summary:

This study have used immunogenic aggregates formed by recombinant Notch3 fragments EGF 1-5 that contain CADASIL NOTCH3 R133C and wild type NOTCH3 to inoculate a CADASIL mouse model TgN3R182C as an active immunisation therapy of CADASIL. The vaccinated mice showed a decreased deposition of NOTCH3 around brain capillaries, reduced blood NOTCH3 ECD and microglia activation, suggesting a potential novel therapy for future treatment of CADASIL. The lack of impact on retinal vasculature, body weight, and general behaviour of the treated mice indicate the safety of the therapy.

We thank the reviewer for the positive comments and for finding it to be a potential novel and safe therapy for CADASIL.

Major comments:

The results are interesting and the manuscript was carefully written and presented. However, the reduction of NOTCH3 in blood samples and the deposition around capillaries were so modest, plus there was no significant change of NOTCH3 deposition in arterioles. This questions the real effectiveness of this immunisation therapy when translating to clinical patients.

We thank the reviewers for the positive words on the manuscript and understand the concerns over the lack of effects in the arterioles. We still believe that observing NOTCH3 ECD reductions around capillaries and in the blood is a significant step forward and supports further endeavors in the area of active immunization, especially considering that the treatment is well tolerated with no overt NOTCH3-related toxicity effects. With this said, we of course realize that there would still be a long way towards clinical use, but all therapy developments have to start somewhere, and future studies (from us and others) can build on the data presented here. There are a number of possible explanations as to why there was not a significant reduction of NOTCH3 around the arterioles. It might be due to that the TgN3R182C150 model is a mild disease model which has a NOTCH3 accumulation onset around 6-7 months of age that starts in the capillaries and at later ages spreads to the arterioles, and that a different time axis or longer immunization period would also lead to a reduction of NOTCH3 in the arterioles, which can be addressed in future experiments. We however believe it was worthwhile to first explore an early immunization protocol, as it may pave the way to a treatment strategy that can be used early on the disease progress, maybe already at the pre-symptomatic phase.

Minor comments:

• Figure EV1 is redundant as Figure 2C has the same information.

We agree the figures are redundant and have deleted Figure EV1 in our new version and renamed EV2 and EV3 to EV1 and EV2, respectively.

• P5 last line "there was a prominent loss of monomeric NOTCH3 EGF1-5 when WT and R133C fragments were mixed as compared to incubating them separately (Fig 2C)". Why the aggregation is more obvious when mutant protein fragment mixed with wild type comparing mutant fragment alone?

The reviewer raises an interesting point. While we do not strictly know why a mixing of mutated and wildtype fragments promote aggregation, our observations are in line with previous reports. Duering et al. Hum. Mol. Genet, 2011 used a single-particle approach SIFT (scanning for intensely fluorescent targets) to study the co-aggregation of WT and mutant NOTCH3 EGF1-5 proteins and reported that mixtures of WT and mutant proteins showed dual colour high-intensity signals, representing de novo aggregates. They also observed WT/R133C multimer formation over three days as compared to five days used in our study. It is also of note that most CADASIL cases are heterozygous for the NOTCH3 mutation, which suggests that wildtypeand mutant NOTCH3 proteins co-exist in vivo, which also have been confirmed by co-immunoprecipitation experiments from cell lysates by Opherk et al. Hum. Mol. Genet. 2009. In the light of these previous reports, we performed a variety of ratio between WT and R133C NOTCH3 EGF1-5 proteins and opted for a 1:1 mixture since it showed the highest amount of multimers in relation to monomers after incubation.

• How was the dosage or concentration of aggregated protein (0.5 mg/ml) used for immunisation determined?

The dosage concentration was calculated by measuring the purified protein with a BCA absorbance assay. The protein was then mixed with the adjuvant or PBS to the desired concentration. This is now more clearly described in the Materials and Methods.

• P7 line 1-2, while using ASMA as a marker for arteries/arterioles, didn't the author see any expression of ASMA in pericytes (capillaries)?

We have used a direct fluorescent conjugated ASMA antibody for this mouse strain and another mouse strain with same mutation but higher NOTCH3 expression (described in Rutten et al, Acta Neuropathol Commun, 2015) as well as for the wildtype mice. In all cases, we find that pericytes have no or very low ASMA expression. This is in line with a report from Ghezali et al. Ann Neurol 2018, in which they used perlecan as a marker for pericytes in the rat Notch3 R169C mutation model, since ASMA did not stain the capillaries. The lack of ASMA staining in pericytes is in agreement with our previous large-scale single cell RNA-seq study of the mouse brain vasculature, where we find very little ASMA (ACTA2) gene expression in pericytes, while it is abundant in the vascular smooth muscle cells (Vanlandewijck et al., Nature 2018).

With this said, we will in the revised version include images on isolated brain microvessels from the N3TgR182C150 mouse model and WT that are stained with ASMA and perlecan/PDGFRb to clearly show that ASMA only stains the arterioles and not the capillaries, see Point 2 “Description of planned revisions” part above.

• In Figure 5A, the NOTCH3 ECD signal looks like similar between Shame and Vaccinated, although the quantifications seem significant in Figure 5B. The author may discuss the robustness of the quantitation method used and therefore the conclusion (i.e., active immunization with NOTCH3 EGF1-5 WT/R133C aggregates specifically reduces the amount of NOTCH3 aggregates around cerebral capillaries.).

We agree that Figure 5A is not clearly showing the differences between vaccinated and sham although highly significant. We have included more representative images and we have also improved the description of the quantification in the method part (page 18-19).

• In Figure 8B, what does the "% of microglia area" mean and how was it calculated?

The % of microglia area represents the percentage that the CD68 staining occupies per microglia area (stained by Iba1), meaning that the vaccinated mice contain more activated microglia compared to sham. This was calculated by measuring the area of the CD68 staining that colocalizes with the microglia. In the transferred version, we have improved the description of this in the text (page 18-19).

• Figure 9A doesn't seem to support the conclusion "There was also a trend towards more NOTCH3 ECD deposits inside or in close vicinity to microglia in vaccinated TgN3R182C150 mice compared to control or sham-vaccinated TgN3R182C150 mice, although the difference did not reach statistical significance", as it is not so convincing that the signals of the NOTCH3 ECD staining co-localise with microglia in the vaccinated sample. Besides, what's the meaning or functional significance about "% of microglia area", "number/1000 um2 microglia", and "average size per microglia"?

Thank you for the comments and we understand the concern, given the highly complex morphology and dynamics of microglia, which makes it difficult to describe potential differences in a straightforward manner. In the first graph in Figure 9B we show that the vaccinated mice had a higher percentage of microglia with NOTCH3 ECD deposits. However, these deposits did not occupy more area of the microglia (% microglia area, second graph), or showed a significant increase in the number of deposits per microglia area (number/1000 µm2 microglia, third graph). The average size of these deposits did not increase in a significant way per microglia area (average size per microglia µm2, fourth graph) in comparison to sham and non-vaccinated mice. We have rephrased the text to better indicate that the proportion of microglia with NOTCH3 deposits increased, while not the area or number of deposits within a microglial cell (Results, page 9).

Reviewer #1 (Significance):

CADASIL is the most common genetic small vessel disease that leads to cognitive defect and eventual vascular dementia. Current, there is no specific treatment available. Similar to a number of neurodegenerative conditions caused by protein accumulations like Alzheimer's disease, Parkinson disease and Huntington disease etc, NOTCH3 protein accumulation represents a key pathological change in CADASIL and therefore a drug target. The approach of active immunisation therapy described in this paper demonstrated a novel method for the treatment of this condition. Although the effectiveness of the therapy in the transgenic mouse model of CADASIL was yet highly impressive, this paper provides a proof-of-principle that the active immunisation is more or less functional and, most importantly, tolerable. One other advantage of this approach is that the immunisation therapy is not restricted to a specific NOTCH3 mutation. After further development this strategy could potentially benefit patients in the future.

This paper may be interested by researchers working on diseases that are caused by specific protein accumulation or aggregations.

My expertise is in the area of studying the molecular mechanisms of CADASIL and other genetic small vessel diseases.

We thank the reviewer for these comments and that the manuscript provides proof-of-principle for a novel strategy towards a safe and tolerable therapy for future treatment of CADASIL patients. As such, we believe the study will be a useful platform and resource for future studies from us and others.

Reviewer #2 (Evidence, reproducibility and clarity):

The article submitted by Daniel V. Oliveira et al to Review Commons and titled "NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model" describes a novel active immunization therapy against the aggregated NOTCH3 mutant protein associated to CADASIL pathology. This strategy induces a significant reduction in NOTCH3 deposition around brain capillaries, increase of microglia activation and lowering of serum levels of NOTCH3, that demonstrates the potential clinical value of the therapy. In addition, the authors report that the therapy is safe and tolerable.<br /> The study is well written, very clear and the results are very promising.

We thank the reviewer for positive comments on our work and pleased that you find the manuscript well and clearly written and containing promising data.

Same minor comments need to be clarified before publication.<br /> 1. In the introduction is indicated that ".....Aducanumab recently being approved for treatment of AD by the Food and Drug Association (Budd Haeberlein et al, 2017; Mintun et al, 2021; Tolar et al, 2020)." In the discussion, Page 10, "In the quest for AD therapies, emerging encouraging results suggest a clinically meaningful effect from immunotherapies aimed at clearing Ab-amyloid which recently also have led to the first FDA approval of a passive vaccine for treatment of AD (Demattos et al, 2012; Golde et al, 2009; Sevigny et al, 2016).<br /> References should be updated with recent clinical studies about the efficacy of Aducanumab.

This is a valid point. We have updated the manuscript accordingly, and included a recent study on the effect of Aducanumab (Knopman et al. Alzheimers Dement. 2021).

1. Schedule used to induce active immunization should be justified or referenced.

We appreciate this comment and realize that we did not explain the rationale for the immunization scheme in the original version. We used a similar immunization schedule as used by Kontsekova et al. Alzheimer's Research & Therapy 2014 in their study of active immunization of a tau peptide in a transgenic rat Alzheimer disease model. This is now mentioned in the text on page 6. The slight modifications of the Kontsekova et al., scheme are also described in the Materials and Methods, page 15 (we made some slight modifications in order to be compatible to mouse and also immunized with an aggregated protein of 25kD instead of a 2 kD tau peptide).

1. CADASIL is associated with high risk of stroke, dementia and migraine. Did the authors check the brain of TgN3R182C150 mice by MRI, for instance? This analysis could be interesting in order to increase the clinical impact and the translational value of the therapy.

Thank you for pointing this out. We have previously reported MRI data on a more severe CADASIL mouse model (TgN3R182C350), which harbors the same transgene but expressed at a 2.3 times higher level compared to the mouse model used in this study (Rutten et al. Acta Neuropathol. Commun. 2015 and Gravesteijn et al. Transl. Stroke Res. 2020). We did not observe any consistent differences on MRI or behavior between the TgN3R182C350 model and controls at 20 months of age. From this data we do not expect to see any differences on MRI in our milder model at 7 months of age. We have however included a short description of the previous MRI data in the transferred version on page 12.

1. It is indicated that the therapy did not affect to the vascular structure of the retina, suggesting that endogenous Notch signaling was not affected by vaccination and therefore the therapy is safe; however, additional tox analysis should be included to confirm the biocompatibility, such as blood pressure analysis, inflammation markers, renal and hepatic damage marker.

Thank you for pointing out this important issue regarding the safety aspects for our active immunotherapy. We will address this by additional experiments of the renal damage by histology and inflammation markers and neuronal damage markers in the serum with western blotting/ELISA and SIMOA, which we have written in more detail in Point 2 “Description of planned revisions” part above.

The suggestions for blood pressure and hepatic damage analysis are valid but would require that we restart novel series of experiments from scratch, as we currently do not have immunized mice up and running. Such an experiment would take several months, and with the subsequent analysis, most likely more than a year. We believe this time axis is too long, and we therefore respectfully suggest waiving these experiments. Also, we unfortunately did not collect livers from the first round of animals, so analysis of livers would have a similarly long time axis.

Reviewer #2 (Significance):

The study is well written, very clear and the results are very promising. Some references related with the effect of Aducanumab in AD should ne updated. Safety and Tox analysis of the therapy should be complemented with additional assays.

We thank the reviewer for these positive comments. We have updated the manuscript with a new reference regarding the effect of Aducanumab. For the safety and tox analysis we will do additional experimental analysis which we have addressed above in “Description of planned revisions”.

3. Description of the revisions that have already been incorporated in the transferred manuscript

Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

We deleted Figure EV1 in our new version and renamed EV2 and EV3 to EV1 and EV2, respectively. (Reviewer 1, point 1).

We provide an improved description of how the dosage concentration was calculated by measuring the purified protein with a BCA absorbance assay (Reviewer 1, point 3) (Materials and Methods, page 15).

We have improved the description of the quantification of the immunohistochemistry in the method part (Reviewer 1, point 5) (Materials and Methods, page 18-19).

We have improved the description of microglia in the quantification in the method part (Reviewer 1, point 6 and 7) (Materials and Methods, page 18-19).

We have rephrased the text to better indicate that the proportion of microglia with NOTCH3 deposits increased, while not the area or number of deposits within a microglial cell (Results, page 9).

We have updated the manuscript accordingly, and included a recent study on the effect of Aducanumab (Knopman et al. Alzheimers Dement. 2021). (Reviewer 2, point 1).

We have provided a rationale and a more detailed description of the immunization protocol. (Reviewer 2, point 2) (Materials and Methods, page 15).

We have included a short description of the previous MRI data in the transferred version. (Reviewer 2, point 3), (Discussion, page 12)

We have included the following new references (Vanlandewijck et al., Nature 2018, Knopman et al. Alzheimers Dement. 2021, Kontsekova et al. Alzheimer's Research & Therapy 2014, Gravesteijn et al. Transl. Stroke Res. 2020).

4. Description of analyses that authors prefer not to carry out

Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

The suggestions for blood pressure and hepatic damage analysis are valid but would require that we restart novel series of experiments from scratch, as we currently do not have immunized mice up and running. Such an experiment would take several months, and with the subsequent analysis, most likely more than a year. We believe this time axis is too long, and we therefore respectfully suggest waiving these experiments. Also, we unfortunately did not collect livers from the first round of animals, so analysis of livers would have a similar time axis.

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Referee #2

Evidence, reproducibility and clarity

The article submitted by Daniel V. Oliveira et al to Review Commons and titled "NOTCH3 active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model" describes a novel active immunization therapy against the aggregated NOTCH3 mutant protein associated to CADASIL pathology. This strategy induces a significant reduction in NOTCH3 deposition around brain capillaries, increase of microglia activation and lowering of serum levels of NOTCH3, that demonstrates the potential clinical value of the therapy. In addition, the authors report that the therapy is safe and tolerable.<br /> The study is well written, very clear and the results are very promising.

Same minor comments need to be clarified before publication.

1. In the introduction is indicated that ".....Aducanumab recently being approved for treatment of AD by the Food and Drug Association (Budd Haeberlein et al, 2017; Mintun et al, 2021; Tolar et al, 2020)." In the discussion, Page 10, "In the quest for AD therapies, emerging encouraging results suggest a clinically meaningful effect from immunotherapies aimed at clearing Ab-amyloid which recently also have led to the first FDA approval of a passive vaccine for treatment of AD (Demattos et al, 2012; Golde et al, 2009; Sevigny et al, 2016).<br /> References should be updated with recent clinical studies about the efficacy of Aducanumab.
2. Schedule used to induce active immunization should be justified or referenced.
3. CADASIL is associated with high risk of stroke, dementia and migraine. Did the authors check the brain of TgN3R182C150 mice by MRI, for instance? This analysis could be interesting in order to increase the clinical impact and the translational value of the therapy.
4. It is indicated that the therapy did not affect to the vascular structure of the retina, suggesting that endogenous Notch signaling was not affected by vaccination and therefore the therapy is safe; however, additional tox analysis should be included to confirm the biocompatibility, such as blood pressure analysis, inflammation markers, renal and hepatic damage marker, ....

Significance

The study is well written, very clear and the results are very promising. Some references related with the effect of Aducanumab in AD should ne updated. Safety and Tox analysis of the therapy should be complemented with additional assays.

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Referee #1

Evidence, reproducibility and clarity

Summary:

This study have used immunogenic aggregates formed by recombinant Notch3 fragments EGF 1-5 that contain CADASIL NOTCH3 R133C and wild type NOTCH3 to inoculate a CADASIL mouse model TgN3R182C as an active immunisation therapy of CADASIL. The vaccinated mice showed a decreased deposition of NOTCH3 around brain capillaries, reduced blood NOTCH3 ECD and microglia activation, suggesting a potential novel therapy for future treatment of CADASIL. The lack of impact on retinal vasculature, body weight, and general behaviour of the treated mice indicate the safety of the therapy.

Major comments:

The results are interesting and the manuscript was carefully written and presented. However, the reduction of NOTCH3 in blood samples and the deposition around capillaries were so modest, plus there was no significant change of NOTCH3 deposition in arterioles. This questions the real effectiveness of this immunisation therapy when translating to clinical patients.

Minor comments:

• Figure EV1 is redundant as Figure 2C has the same information.
• P5 last line "there was a prominent loss of monomeric NOTCH3 EGF1-5 when WT and R133C fragments were mixed as compared to incubating them separately (Fig 2C)". Why the aggregation is more obvious when mutant protein fragment mixed with wild type comparing mutant fragment alone?
• How was the dosage or concentration of aggregated protein (0.5 mg/ml) used for immunisation determined?
• P7 line 1-2, while using ASMA as a marker for arteries/arterioles, didn't the author see any expression of ASMA in pericytes (capillaries)?
• In Figure 5A, the NOTCH3 ECD signal looks like similar between Shame and Vaccinated, although the quantifications seem significant in Figure 5B. The author may discuss the robustness of the quantitation method used and therefore the conclusion (i.e., active immunization with NOTCH3 EGF1-5 WT/R133C aggregates specifically reduces the amount of NOTCH3 aggregates around cerebral capillaries.).
• In Figure 8B, what does the "% of microglia area" mean and how was it calculated?
• Figure 9A doesn't seem to support the conclusion "There was also a trend towards more NOTCH3 ECD deposits inside or in close vicinity to microglia in vaccinated TgN3R182C150 mice compared to control or sham-vaccinated TgN3R182C150 mice, although the difference did not reach statistical significance", as it is not so convincing that the signals of the NOTCH3 ECD staining co-localise with microglia in the vaccinated sample. Besides, what's the meaning or functional significance about "% of microglia area", "number/1000 um2 microglia", and "average size per microglia"?

Significance

CADASIL is the most common genetic small vessel disease that leads to cognitive defect and eventual vascular dementia. Current, there is no specific treatment available. Similar to a number of neurodegenerative conditions caused by protein accumulations like Alzheimer's disease, Parkinson disease and Huntington disease etc, NOTCH3 protein accumulation represents a key pathological change in CADASIL and therefore a drug target. The approach of active immunisation therapy described in this paper demonstrated a novel method for the treatment of this condition. Although the effectiveness of the therapy in the transgenic mouse model of CADASIL was yet highly impressive, this paper provides a proof-of-principle that the active immunisation is more or less functional and, most importantly, tolerable. One other advantage of this approach is that the immunisation therapy is not restricted to a specific NOTCH3 mutation. After further development this strategy could potentially benefit patients in the future.

This paper may be interested by researchers working on diseases that are caused by specific protein accumulation or aggregations.

My expertise is in the area of studying the molecular mechanisms of CADASIL and other genetic small vessel diseases.

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Reply to the reviewers

Author response (Tane at al: RC-2022-01646)

Reviewer #1 (Evidence, reproducibility and clarity (Required)): * Comments The work described in this manuscript starts with an in-silico analysis of the primary amino-acid sequence of CAP-H proteins that reveals the presence in vertebrate orthologs of an N-terminal extension of ~80 amino acids in length which contains 19 serine or threonine residues and also, in its centre, a stretch of conserved basic amino acids predicted to form a helix. These features suggest a regulatory module. Using xenopus egg extracts depleted of endogenous condensins and supplemented with recombinant condensin I holocomplexes, either wildtype or mutants, the authors show that deleting the N-terminal tail of CAP-H, or just the central helix (CH), increases the association condensin I with chromatin in mitotic egg extracts and accelerates the formation of mitotic chromosomes. Interestingly, they also show that deleting the N-tail enables a substantial amount of condensin I to associate with chromatin in interphase extracts and to form chromosome-like structures, while WT condensin I cannot. Using in vitro assays and naked DNA as substrate, the authors further show that removing the N-terminal tail of CAP-H improves both the topological (salt-resistant) association of condensin I with DNA and it loop extrusion activity. These experiments appear to me as are clear and robust. They convincingly reveal that N-tail of human CAP-H hinders the binding of condensin I to DNA and both its loop-extrusion and chromosome-shaping activities. However, the mechanism through which such hindrance is achieved remains elusive (see major comments 1-3). A complementary part of the work tackles the important question of the cell cycle control of such counteracting effect. Using newly-designed antibodies against two phospho-serine residues within the tail, the authors provide evidence that the tail is phosphorylated in mitosis-specific manner. This points towards phosphorylation as a biological mean to modulate the effect of the tail on condensin's binding during the cell cycle. In support to this idea, using non-phosphorylatable or phosphomimic substitutions of all the serine and threonine residues within the tail (n =19), including one substitution within the CH domain (Ser 70), the authors show that non-phosphorylatable mutations (H-N19A) or phosphomimic mutations (H-N19D) respectively reduce or improve condensin I binding to chromatin in mitotic egg extracts. This suggests that the phosphorylation of the N-terminal tail in mitosis might relieve its negative effect on condensin I binding to chromatin. The weaknesses I see in this part of the study concern (1) the validation of the phospho-antibodies, which appears to me as insufficiently described (major comment 4), (2) the possibility the bulk changes in amino acids (n=19 out of 80) could impact the folding of the central helix (minor comment X) and (3) that some substitutions could impact the binding of condensin I by different mechanisms (minor comment X).

Major comments:

1. On the model. The authors propose that the N-tail could stabilise an interaction between the N-terminal part of CAP-H and SMC2's neck, which would restrain the transient opening of a DNA entry gate within the ring, necessary for the topological engagement of DNA and loop formation. Although the model is sound, I see no direct data that support it in the manuscript. Such model predicts that a CAP-H protein containing or not the N-terminal tail (or the central helix) should exhibit different binding strengths to SMC2 in vitro. It seems to me that the authors could easily test this prediction using the recombinant proteins they produced in the context of this study. *

Response

We thank the reviewer for pointing out this important issue. To test whether the CAP-H N-tail indeed contributes to the stabilization of the SMC2-kleisin gate, we set up a highly sophisticated functional assay described by Hassler et al (2019). The authors used this assay to demonstrate that an N-terminal fragment of kleisin (engineered to be cleaved by TEV protease) is released from the rest of the condensin complex in an ATP-dependent (i.e., head-head engagement-dependent) manner. We reasoned that this assay is most powerful to prove our hypothesis in a mechanistically relevant context. We envisioned that the CAP-H fragment lacking its N-tail can readily be released whereas the CAP-H fragment retaining its N-tail is more difficult to be released (because of the postulated stabilization of the SMC2-CAP-H interaction). Despite substantial efforts in making TEV-cleavable constructs and in testing various releasing conditions, we have not been able to recapitulate the ATP-dependent release even with the holo(H-dN) construct. Thus, unfortunately, this trial enabled us to neither prove nor disprove our hypothesis.

We are fully aware that the full reconstitution of ATP-dependent and phosphorylation-stimulated gate-opening reaction in vitro is a very important direction in the future. It is beyond the scope of the current study, however.

2. On ATP-hydrolysis. Given the importance of ATP hydrolysis for the engagement of condensin into a topological mode of association with DNA and for its loop extrusion activity, I suggest that the authors measure the impact of the N-tail and of the CH domain on the rate of ATP hydrolysis by condensin I holocomplexes. I suppose that it can be relatively easily done (PMID: 9288743) using the recombinant WT and mutant versions they purified in the course of this study.

Response

We appreciate this constructive comment. In fact, we did a preliminary experiment and found that ATPase activities (either in the absence or presence of DNA) were not significantly different between holo(WT) and holo(H-dN). We were not surprised with this result because our previous study on condensin II indicated that enhanced ATP hydrolysis by a class of mutant complexes is not directly coupled to their enhanced association with chromosomes (Yoshida et al., 2022, eLife). We consider that other functional assays, such as the topological loading assay and the loop extrusion assay shown in the current manuscript, are more informative assays to address ATP-dependent activities of the condensin complexes.

3. A conundrum with previous work? In Kimura et al. Science 1998 (PMID: 9774278), the lab of Tatsuya Hirano has shown that xenopus condensin I purified from mitotic egg extracts induces the supercoiling of plasmid DNA in vitro, but fails to do so when it is purified from interphase egg extracts. This echoes the inhibitory effect of the N-tail of the topological binding of condensin I described in the current manuscript. However, using a gel shift assay, Kimura et al. 1998 also provide evidence that interphase and mitotic condensin I bind plasmid DNA in vitro with similar efficiencies. At first sight, this prior observation seems to contradict the idea that the N-tail of CAP-H restrains DNA binding unless it is phosphorylated in mitosis. Is it possible that the in vitro binding assays used in Kimura et al. 1998 and in this work might assess different modes of binding? I suggest that this apparent conundrum should to be discussed.

Response

We thank the reviewer for following our early studies. As discussed below, we are confident that our conclusion reported in the current study by no means contradicts our previous observations.

We reason that the confusion expressed by the reviewer stems from intrinsic, technical limitations of the gel-shift assay. Such limitations become apparent especially when it is applied to the functional analyses of complicated protein machines such as condensins. For instance, the DNA-binding activity of condensin I detected by the gel-shift assay is neither ATP-dependent nor phosphorylation-dependent (Kimura and Hirano, 1997; Kimura et al., 1998). It is fundamentally different from the ATP-dependent activities detected by the topological loading and loop extrusion assays reported in the current study (It remains unknown whether the two activities are stimulated by mitotic phosphorylation). Thus, the DNA-binding activity detected by the gel-shift assay does not reflect “productive” DNA interactions that depend on ATP hydrolysis in vitro. We therefore consider that gel-shift analyses of holo(WT) and holo(H-dN) would not produce any useful information.

*Related to that, could it be possible for the authors to assess the impact of the N-tail on the salt-sensitive binding of condensin to DNA, i.e. by reproducing the topological binding assay but omitting the high salt washes? I guess this information could be useful to fully apprehend the impact of the N-tail on the binding of condensin. *

Response

When we set up the topological loading assay, we actually tested a low-salt wash condition that the reviewer suggests here. Although a much higher level of DNA recovery was observed with the low-salt condition than with the high-salt wash condition, no nucleotide dependency was detectable with the low-salt condition. Moreover, no difference in DNA recovery between holo(WT) and holo(H-dN) was observed. Thus, the low-condition condition allowed us to detect the “bulk” DNA-binding activity that is equivalent to that detected by the gel-shift assay. These results were fully consistent with the discussion above.

4. Validation of phospho-antibodies and by extension showing the phosphorylation of the tail. The newly-designed phospho-serine antibodies used in this study to show that the N-tail is phosphorylated at serine 17 and serine 76 in mitosis (Fig. EV3) are, in my view, not characterized enough. This piece of data is key to substantiate the idea that the tail is phosphorylated in mitosis. Yet, showing that these antibodies detect epitopes on WT condensin I from mitotic egg extracts but not on the H-N19A counterpart, nor on WT condensin I from interphase extracts, does not demonstrate the phospho-specificity of such antibodies. I suggest that a PPase treatment should be conducted to assess the phospho-specificity of these antibodies. Moreover, since the lab of Tatsuya Hirano has the know-how to deplete Cdc2/CDK1 from xenopus egg extract, such strategy could/should be used to further validate the antibodies and assess to which extent the N-tail is phosphorylated in a Cdc2-dependent manner.

Response

We have performed two sets of experiments to confirm the specificity of the phosphoepitopes recognized by anti-hHP1 and anti-hHP2. In the first set, we performed a phosphatase treatment assay. Holo(WT) that had been preincubated with Dcond M-HSS was immunoprecipitated using an antibody against hCAP-G, treated with l protein phosphatase in the presence or absence of phosphatase inhibitors, and analyzed by immunoblotting using anti-hHP1 and anti-hHP2. The results (now shown in Supplementary Fig 3C) demonstrated that the epitopes recognized by anti-hHP1 and anti-hHP2 are sensitive to phosphatase treatment. In the second set, we performed a phosphopeptide competition assay. Holo(WT) that had been preincubated with Dcond M-HSS was immunoprecipitated and subjected to immunoblotting. The membranes were triplicated and probed with anti-hHP1 in the presence of no competing peptide, hHP1 or hHP2. Similarly, another set of triplicated membranes was probed with anti-hHP2 in the presence of no competing peptide, hHP1 or hHP2. We found that the signal recognized by anti-hHP1 competed with hHP1, but not with hHP2, and that the signal recognized by anti-hHP2 competed with hHP2, but not with hHP1. The results (now shown in Supplementary Fig 3D) demonstrated the sequence specificity of the phosphoepitopes recognized by the two antibodies. The procedures for these experiments have been described in Materials and Methods.

Because Cdk1 depletion from M-HSS creates an HSS equivalent to I-HSS, we do not consider that the suggested experiment will provide additional information.

*Minor comments:

1. The impact of the 19 mutations, A or D, introduced within the tail on the folding of the central helix? The idea that the negative effect of the N-tail is relieved by phosphorylation is based on the chromatin binding phenotypes exhibited by the H-N19D or H-N19A mutant holocomplexes, in which 19 amino-acids out of 80 have been modified, include one in the central helix. The authors also provide evidence that the central helix (CH) located within the tail plays a key role in the negative regulation of condensin I binding. Thus, I wonder to which extent the folding of the central helix could be impacted by the mutations introduced in the tail and notably the one within the central helix itself. Could the author assess the structure of mutated tails using Alpho-fold and/or discuss this point? *

Response

According to the reviewer’s suggestion, we performed structure predictions using Alphafold2, and found that neither the N19A nor N19D mutations alter the original prediction of helix formation that was made for the wild-type CH sequence. A conventional secondary structure prediction using Jpred4 reached the same conclusion.

2. Phosphorylation of serine 70 in the central helix by Aurora-B kinase? A prior study by Tada et al. (PMID: 21633354) has shown (1) that serine 70 of the N-tail of hCAP-H is phosphorylated by Aurora-B kinase, (2) that the mutation S70A reduces the binding of condensin I to chromatin in HeLa cells and (3) that hCAP-H interacts with histone H2A in an Aurora-B dependent manner. This draws a picture in which the phosphorylation of Ser70 by Aurora-B would improve condensin I binding to chromatin by promoting an interaction between hCAP-H and histone H2A/nucleosomes. Intriguingly, Ser 70 in Tada et al. correspond to the serine residue located within the conserved central helix analysed in this study, and this Ser70 residue is mutated in the H-N19D or H-N19A holocomplexes that show reduced chromatin binding in this study. This raises the question as what could be the contribution of the S70A or S70D substitution to the chromatin binding phenotypes shown by the H-N19D or H-N19A holocomplexes. This is not discussed in the manuscript, and the authors do not cite this earlier work (PMID: 21633354) in their manuscript. Is there any reason for that? I suggest it should be cited and discussed.

Response

We thank the reviewer for bringing up this issue. In many respects, we do not trust the data reported by Tada et al (2011) and the resultant model they proposed. Previous and emerging lines of evidence reported from our own and other laboratories indicate that histones compete with condensins for DNA binding, strongly arguing against the possibility that histone H2A acts as a “chromatin receptor” for condensins. We formally discussed and criticized the Tada 2011 model in our previous publications, which included Shintomi et al (2015) NCB, Shintomi et al (2017) Science, Hirano (2016) Cell and Kinoshita/Hirano (2017) COCB. We thought that those were enough. That said, we also consider that the reviewer is right. The current study demonstrates that the deletion of the CAP-H N-tail accelerates, rather than decelerates, condensin I loading, providing an additional line of evidence that argues against the Tada model. A critical comparison between the Tada model and our current model would benefit the readers. In the revised manuscript, we have added the following discussion:

In terms of the regulatory role of the CAP-H N-tail, it would be worthy to discuss the model previously proposed by Tada et al (2011). According to their model, aurora B-mediated phosphorylation of the CAP-H N-tail allows its direct interaction with the histone H2A N-tail, which in turn triggers condensin I loading onto chromatin. Accumulating lines of evidence, however, strongly argue against this model: (i) aurora B is not essential for single chromatid assembly in Xenopus egg extracts (MacCallum et al., 2002) or in a reconstitution assay (Shintomi et al., 2015); (ii) the H2A N-tail is dispensable for condensin I-dependent chromatid assembly in the reconstitution assay (Shintomi et al., 2015); (iii) even whole nucleosomes are not essential for condensin I-mediated assembly of chromatid-like structures (Shintomi et al., 2017). The current study demonstrates that the deletion of the CAP-H N-tail accelerates, rather than decelerates, condensin I loading, providing an additional piece of evidence against the model proposed by Tada et al (2011).

3. Other minor comments - Please provide a microscope image of DNA loop in Fig. 4D.

Response

In the revised manuscript, we have provided a set of time-lapse images of loop extrusion events catalyzed by holo(WT) and holo(H-dN) in Fig 4E.

*- The authors do not compare the kleisin of condensin I with the one of condensin II with respect to the features tackled in this work. Given the different behaviours condensin I and II, such comparison could be informative for the readers. *

Response

We thank the reviewer for this constructive comment. In the revised manuscript, we have added the following statement:

It should also be added that CAP-H2, the kleisin subunit of condensin II, lacks the N-terminal extension that corresponds to the CAP-H N-tail. Thus, the negative regulation by the kleisin N-tail reported here is not shared by condensin II.

*- The authors do not reference the work of Robellet et al. Genes & Dev (2015) (PMID: 25691469) on the regulation of condensin binding in budding yeast by an SMC4 phospho-tail. I suggest that the analogy should be discussed. *

Response

According to the reviewer’s comment, we have added the following statements at the beginning of Discussion.

Previous studies showed that mitotic phosphorylation of Cut3/SMC4 regulates the nuclear import of condensin in fission yeast (Sutani et al. 1999) and that phosphorylation of Smc4/SMC4 slows down the dynamic turnover of condensin on mitotic chromosomes in budding yeast (Robellet et al. 2015; Thadani et al. 2018). In the current study, we have focused on the phosphoregulation of vertebrate condensin I by its kleisin subunit CAP-H.

- In the introduction section, lane 5, the sentence "Many if not all eukaryotic species have two different condensin complexes" appears inappropriate since budding and fission yeast cells possess a single condensin complexes, similar to condensin I in term of primary amino-acid sequence.

Response

We thought that the original wording “Many if not all” was good enough to imply that some species, which include budding yeast and fission yeast, have only a single condensin complex. To make it clear, however, we have modified the sentence in the revised manuscript as follows:

Many eukaryotic species have two different condensin complexes although some species including fungi have only condensin I.

*- page 4; typo: motif I and V bind to the SMC neck and the SMC4 cap regions, respectively. Should read SMC2 neck. *

Response

The reviewer is right. It should read the SMC2 neck. Corrected.

*- Are the data and the methods presented in such a way that they can be reproduced? YES - Are the experiments adequately replicated and statistical analysis adequate? YES - Are prior studies referenced appropriately? Not all of them (see above) - Are the text and figures clear and accurate? YES

CROSS-CONSULTATION COMMENTS I consider the comments from all reviewers as helpful for the authors.

Reviewer #1 (Significance (Required)):

Summary Condensins are genome organisers of the family of SMC ATPase complexes and are best characterized as the drivers of mitotic chromosome assembly (condensation). It is acknowledged that condensins shape mitotic chromosomes by massively associating with DNA upon mitotic entry (loading step) and by folding chromatin fibres into arrays of loops, most likely through an ATP-dependent extrusion of DNA into loops, as seen in vitro. What remains unclear, however, are the mechanisms by which condensins load onto DNA and fold it into arrays of loops in vivo, and how these reactions are coupled with the cell cycle, i.e. restricted mostly to mitosis. Condensins are ring shaped pentamers that change conformation upon ATP-hydrolysis. In vitro studies suggest that condensins bind DNA via ATP-hydrolysis-independent, direct electrostatic contacts between condensin subunits and DNA. Such electrostatic contacts are salt-sensitive in in-vitro assays. Upon ATP-hydrolysis, condensins engage into an additional mode of binding that is resistant to high salt concentration and likely to be topological in nature. Both modes of association are necessary to form DNA loops. Vertebrates possess two types of condensin complexes, condensin I and II, each composed of a same SMC2-SMC4 ATPase core but associated with two different sets of three non-SMC subunits; a kleisin and two HEAT-repeat proteins. Condensin II is nuclear during interphase and stably binds chromatin upon mitotic entry, while condensin I is located in the cytoplasm during interphase and binds chromatin in a dynamic manner upon nuclear envelope breakdown. How the spatiotemporal control of condensin I and II is achieved remains poorly understood. Previous studies have shown that the phosphorylation of condensin I by mitotic kinases, such as CDK1, Aurora-B and Polo, play a positive role in its binding to chromatin and/or its functioning, but the underlying mechanisms remain to be characterised. In this manuscript, Shoji Tane and colleagues provide good evidence that the N-terminal tail of the human kleisin subunit of condensin I, hCAP-H, serves as a regulatory module for the cell-cycle control of condensin I binding to chromatin and chromosome shaping activity. The authors clearly show that the N-tail of CAP-H hinders the binding of condensin I to chromatin in xenopus egg extracts and, using in vitro assays, that the N-tail also hinders the topological association of condensin I with DNA and its loop extrusion activity. The authors provide additional data suggesting that the phosphorylation of the N-tail of CAP-H, in mitosis, relieves its inhibitory effect on condensin I binding. Based on their findings, Tane et al. propose a model suggesting that the N-terminal tail of CAP-H constitutes a gate keeper that maintains condensin-rings in a closed conformation that is unfavourable for topological binding to DNA, and whose locking effect is relieved in mitosis by phosphorylation.

Taken as a whole, this work has the potential to reveal a molecular basis for the cell cycle regulation of condensin I in vertebrate cells and as such to significantly improve our understanding of the integrated functioning condensin I. The characterisation of the inhibitory effect of the N-tail on condensin binding to chromatin and to naked DNA in vitro is well described, the data are clear and robust and the results convincing. On the other hand, some of the data on the phospho-regulation appear to me as are more debatable and I think that some of the results described here deserve to be discussed in the context of previous works. Finally, I see no data in the manuscript that directly supports the mechanistic model proposed by the authors, while it seems to me that such model could have been easily tested exprimentally. Thus, I suggest that Tane and colleagues should perform a couple of relatively easy experiments to strengthen their claims and that a few omitted prior studies on the topic should be referenced and discussed. *

Reviewer #2 (Evidence, reproducibility and clarity (Required)): * The manuscript reveals that the N-terminal region of CAPH could play a role in regulating condensin I activity, using a range of in vitro methods. They propose that the N-terminal region of CAPH inhibits complex activity, and this is turned off upon deletion or phosphorylation, by using truncations, phospho-mimics or phospho-deficient mutations. While the results are interesting to the field, and helps to address the question as to how condensin complexes are controlled in a cell cycle dependent manner, some key data and controls are necessary to ensure the conclusion is robust.

Main comments

• What is meant by "unperturbed I-HSS" on page 7, ie membrane containing versus membrane free or condensin depleted? *

Response

We apologize for having created unnecessary confusion. We meant that the “unperturbed I-HSS” is the “undepleted I-HSS”. As far as the issue of membrane-containing vs membrane-free is concerned, we explicitly mentioned that “we used membrane-free I-HSS in the following experiments” several lines above. In the revised manuscript, we have revised the statement accordingly.

In many of the protein gels eg figure 4B, the bands for SMC2 and 4 are more intense that the non-SMC components. The method for protein purification also does not include a size exclusion step to ensure sample homogeneity. Authors should perform some sort of quality control checks on samples such as analytical gel filtration or mass photometry to ensure stoichiometry/homogeneity. This is particularly important for samples eg the N19A, where activity is reduced compared to wild-type as poor protein behaviour could create false negative results.

Response

As the reviewer is fully aware, the reconstitution and purification of multiprotein complexes, such as condensins, is by no means an easy task. We notice that many groups in the field share common concerns about sample homogeneity and subunit stoichiometry, and that these concerns cannot completely be eliminated even after size exclusion chromatography. Because the current study handles a large number of mutant complexes, we consider that the purification by two-step column chromatography is the most practical approach. We do not notice any abnormal behaviors of holo(H-N19A) in the processes of expression and purification. It is also important to emphasize that the H-N19D mutations cause the completely opposite phenotype. Taken all together, we are confident of our current conclusions.

That said, in the revised manuscript, we have added the following statement in Results:

Although we cannot rule out the possibility that the introduction of multiple mutations into the N-tail causes unforeseeable adverse effects on protein conformations, these results supported the idea that ….

• Loop extrusion assays in figure 4D-G shows no example data i.e. no pictures or videos of loops being formed. These should also be included.*

Response

In the revised manuscript, we have provided a set of time-lapse images of loop extrusion events catalyzed by holo(WT) and holo(H-dN) in Fig 4E.

• Given the mutant holo(H-dN) has higher activity than wild-type, a negative control such as holo(H-dN) without ATP or holo(H-dN) ATPase deficient mutant should also be measured in loop extrusion assays, to ensure the activity is derived from the ATPase activity.*

Response

In the revised manuscript, we have added loop formation data for both holo(WT) and holo(H-dN) in the absence or presence of ATP (Supplementary Fig 5). We are confident that both complexes support loop extrusion strictly in an ATP-dependent manner.

• According to the methods, this work performs the same loop extrusion assay as described in Kinoshita et al, 2022, however, in Kinoshita et al, wild type condensin I makes loops in 30-50% of DNA molecules, where in this study the percentage is less than half that. Can the author please explain the discrepancy given the same method was used?*

Response

First of all, we wish to remind the reviewer that the holo(WT) constructs used in the two studies are not identical: CAP-H was N-terminally HaloTagged in all constructs used in Kinoshita et al (2022), whereas the same subunit was C-terminally HaloTagged in the pair of constructs used in the current study. Because we wanted to compare the activities between the full-length CAP-H and N-terminally deleted version of CAP-H (H-dN), we reasoned that it would be inappropriate to put the HaloTag to the N-terminus of CAP-H. The difference in the constructs could explain the observed discrepancy, even if it might not be the sole reason.

The design of the constructs was accurately described in each manuscript, but the statements were not very explicit about the positions of the HaloTag. To clarify this issue, we have added the following sentences in the revised manuscript:

Note that the HaloTag was fused to the C-terminus of CAP-H in the current study because we wanted to investigate the effect of the N-terminal deletion of CAP-H. We used N-terminally HaloTagged CAP-H constructs in our previous study (Kinoshita et al., 2022).

• In the concluding statement the author suggests "Upon mitotic entry, multisite phosphorylation of the N-tail relieves the stabilization, allowing the opening of the DNA entry gate, hence, the loading of condensin I onto chromosomes." This seems unlikely as fusion the N-terminus of the of the kleisin to the C-terminus of SMC2 is able to function for yeast (Shaltiel et al 2022) and condensin II (Houlard et al 2021), and equivalently in cohesin (Davidson et al 2019).*

Response

We appreciate the reviewer’s concern. In our view, however, the issue of the “DNA-entry gate” remains under debate in the SMC field (e.g., Higashi et al [2020] Mol Cell; Taschner and Gruber [2022] bioRxiv). For instance, Shaltiel et al (2022) demonstrated that neck-gate fusion constructs can support in vitro activities including topological loading under certain conditions, but also showed that such constructs greatly reduce the cell viability, leaving the possibility that the gate opening is required for some physiological functions.

That said, it is true that the data reported in the current manuscript do not exclude the possibility that the SMC2 neck-kleisin interface is not used as a DNA entry gate for condensin I loading. In the revised manuscript, we have added the following statement in Discussion:

Although our model predicts that the SMC2 neck-kleisin interface is used as a DNA entry gate, we are aware that several studies reported evidence arguing against this possibility (e.g., Houlard et al [2021]; Shaltiel et al [2022]). Our current data do not exclude other models.

*Reviewer #2 (Significance (Required)):

This is an interesting story that reveals new insights about condensin regulation.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

This paper reveals a role of an N-terminal extension of CAP-H in the regulation of condensin-I activity in Xenopus extracts using biochemical reconstitution experiments. The authors demonstrate that a motif in the N-terminal tail that is conserved in vertebrates acts as an inhibitor of condensin I activity. Using several mutant constructs, it is shown that the inhibition by this motif is in turn counteracted by the phosphorylation of neighbouring serine and threonine residues in mitosis, presumably at least in part by CdK. Mutants that have lost this inhibition are able to condense chromatin into chromatid-like structures more efficiently and to some degree even in interphase extracts. Moreover, one such mutant is characterized in detail by biochemical and biophysical experiments and shown to have increased capacity in salt-stable DNA loading and in DNA loop extrusion.

Major comments: This is a beautiful and thorough study that is presented in a clear and concise manner. The main conclusions are well justified. No additional experiments are needed to support them. Replication and statistical analysis appear adequate. The final model is however largely speculative. Recent work has indicated that loading of yeast condensin does not require gate opening. The authors may thus want to include alternative scenarios or remain more vague. *

Response

This comment is related to the last comment of Reviewer#2. See above for our response.

*The H-N19A mutant has a loss of function phenotype (possibly due to folding problem caused by 19 point mutations rather than lack of phosphorylation), the authors could consider to rescue the phenotype by also including the CH motif mutations in this construct (or make an explanatory statement in the text). *

Response

We understand the reviewer’s logic here, but overlaying additional mutations into the H-N19A mutations could cause an unforeseeable effect, potentially making the interpretation of the outcome complicated.

We also wish to point out that it may be inappropriate to regard the phenotype exhibited by holo(H-N19A) as a simple loss-of-function phenotype. This is because the opposite, accelerated loading phenotype exhibited by holo(H-dN) can be regarded as a consequence of loss of negative regulation. Like holo(H-dN), the phosphomimetic mutant complex holo(H-N19D) displayed an accelerated loading phenotype, fully supporting our conclusion. In the revised manuscript, we have added the following statement in Results:

Although we cannot rule out the possibility that the introduction of multiple mutations into the N-tail causes unforeseeable adverse effects on protein conformations, these results supported the idea that ….

*Albeit not necessary for the main conclusions, the authors could possibly significantly strengthen their study by testing for binding partners of the N-tail and the CH motif by running AlphaFold predictions against the condensin I subunits. *

Response

We appreciate this constructive comment. We attempted to predict possible interactions between SMC2 and a CAP-H fragment containing its N-tail and motif I using

ColabFold (Mirdita et al., 2022, Nat. Methods). The algorism excellently predicted the proper folding of the CAP-H motif I and its interaction with the SMC2 neck. Under this condition of predictions, however, the N-tail remained largely disordered (except for the CH), and no interaction with any part of SMC2 was predicted. The same was true when the N19D mutations were introduced in the N-tail sequence. Thus, this trial did not provide much information about the potential interaction target(s) of the CAP-H N-tail.

*The efficiency of depletion of condensin subunits from I-HSS extracts is not documented (in contrast to M-HSS extracts - figure EV1C). While any condensin remaining in these extracts might not be active (or interfering), the authors may want to at least comment on this in the text. *

Response

We check the efficiency of immunodepletion every time by immunoblotting and confirm that a high level of depletion is achieved from both M-HSS and I-HSS. According to the reviewer’s comment, the following statement was placed in Materials and Methods:

The efficiency of immunodepletion was checked every time by immunoblotting. An example of immunodepletion from M-HSS was shown in Supplemental figure 1C. We also confirmed that a similar efficiency of immunodepletion was achieved from I-HSS.

*The authors should include information on the quantification of chromatid morphology. Is the analysis based on chromatids taken from the same images/imaging session, from technical replicates, biological replicates? *

Response

In the revised manuscript, we have added statements on image presentation and experimental repeats in the appropriate figure legends and methods section. During the revision process, we repeated the experiments shown in Supplementary Fig 2, and obtained the same results. In the revised manuscript, the original set of data has been replaced with the new set of data along with panel C (Quantification of the intensity of mSMC4 per DNA area).

Minor comment: The colour scheme in Figure 5A is confusing. Use less colour? The orange and red colours are moreover quite similar.

Response

According to the reviewer’s comment, we have modified Figure 5A.

*Reviewer #3 (Significance (Required)):

The findings provide new insights into how condensin-I activity is restricted outside of mitosis. It was previously assumed that this regulation was (largely) due to the exclusion of condensin I from the nucleus prior to nuclear envelope breakdown. This study shows that another pathway is contributing to the regulation and implies that controlling condensin I activity is more important than previously appreciated. Whether all residual nuclear condensin I is inactivated, remains to be determined. The physiological impact of loss of autoinhibition on chromosome segregation and cell cycle progression also remains to be uncovered. The observed effects are robust and appear significant. Future research on condensin I using reconstitution will likely benefit from being able to control or eliminate the self-inhibition.

This reviewer has expertise on the biochemistry and structural biology of SMC protein complexes.

Reviewer #4 (Evidence, reproducibility and clarity (Required)):

Mitotic chromosome formation is a cell cycle-regulated process that is crucial for eukaryotic genome stability. The chromosomal condensin complex promotes chromosome condensation, but the temporal control over condensin function is only scantly understood. In this impressive manuscript, "Cell cycle-specific loading of condensin I is regulated by the N-terminal tail of its kleisin subunit", Tane and colleagues provide important new insight into the cell cycle-regulation of condensin. The authors identify a kleisin N-tail that acts as a negative regulator of condensin's DNA interactions. Removal of this N-tail, or its cell cycle-dependent phosphorylation, relieves inhibition and activates condensin. This is a simple, yet very important story, that advances our molecular understanding of chromosome formation. The experiments are performed to a very high technical standard and support the authors conclusions. This manuscript is highly suitable for publication in any molecular biology journal, once the authors have considered the following points.

1. Introduction. a) The authors could better explain their own prior work (Kimura et al. 1998), which has identified the condensin XCAP-D2 and XCAP-H as the targets of phosphoregulation. The current manuscript explains the role of XCAP-H phosphorylation. *

Response

According to the reviewer’s comment, we have added the following sentence in Introduction:

The major targets of mitotic phosphorylation identified in these studies included the CAP-D2 and CAP-H subunits.

1. b) Given the limited knowledge about condensin cell cycle regulation, it seems prudent to provide a brief summary of what is known. Fission yeast Smc4 phosphorylation regulates condensin nuclear import (Sutani et al. 1999), while budding yeast Smc4 phosphorylation slows down the dynamic turnover of the condensin complex on chromosomes (Robellet et al. 2015 and Thadani et al. 2018).

Response

We appreciate this constructive comment. According to the reviewer’s comment, we have added the following statements at the beginning of Discussion.

Previous studies showed that mitotic phosphorylation of Cut3/SMC4 regulates the nuclear import of condensin in fission yeast (Sutani et al. 1999) and that phosphorylation of Smc4/SMC4 slows down the dynamic turnover of condensin on mitotic chromosomes in budding yeast (Robellet et al. 2015 and Thadani et al. 2018). In the current study, we have focused on the phosphoregulation of vertebrate condensin I by its kleisin subunit CAP-H.

2. Extracts were mixed with mouse sperm nuclei. If there is a reason why mouse rather than Xenopus sperm nuclei were used, this would be interesting to know.

Response

The original motivation for introducing mouse sperm nuclei into Xenopus egg extracts was to test the functional contribution of nucleosomes to mitotic chromosome assembly. When mouse sperm nuclei are incubated with an extract depleted of the histone chaperone Asf1, the assembly of octasomes can be suppressed almost completely. Remarkably, we found that even under this “nucleosome-depleted” condition, mitotic chromosome-like structures can be assembled in a manner dependent on condensins (Shintomi et al., 2017, Science). Xenopus sperm nuclei cannot be used in this type of experiment because they endogenously retain histones H3 and H4 and are therefore competent in assembling octasomes even in the Asf1-depleted extract. During this study, we realized that the use of mouse sperm nuclei in Xenopus egg extracts provides additional and deep insights into the basic mechanisms of mitotic chromosome assembly. For instance, the functional contribution of condensin II to chromosome assembly could be observed more prominently when mouse sperm nuclei are used as a substrate than when Xenopus sperm nuclei are used (Shintomi et al., 2017, Science). We suspected that the slow kinetics of nucleosome assembly on the mouse sperm substrate creates an environment in favor of condensin II’s action. For these reasons, our laboratory now extensively uses mouse sperm nuclei for the functional analyses of condensin II (Yoshida et al., 2022. eLife) and other purposes (Kinoshita et al., 2022, JCB). Yoshida et al (2022) used experimental approaches analogous to the current study, and found that the deletion of the CAP-D3 C-tail, causes accelerated loading of condensin II. One of the long-term goals in our laboratory is to critically compare and contrast the actions of condensin I and condensin II in mitotic chromosome assembly. Thus, the use of the same substrate in the two complementary studies can be fully justified.

During the preparation of this response, we realized that the readers would benefit from a brief statement about the comparison between condensin I and condensin II. In the revised manuscript, we have added the following statement in Discussion:

It should also be added that CAP-H2, the kleisin subunit of condensin II, lacks the N-terminal extension that corresponds to the CAP-H N-tail. Thus, the negative regulation by the kleisin N-tail reported here is not shared by condensin II. Interestingly, however, a recent study from our laboratory has shown that the deletion of the CAP-D3 C-tail causes accelerated loading of condensin II onto chromatin (Yoshida et al., 2022). It is therefore possible that condensins I and II utilize similar IDR-mediated regulatory mechanisms, but they do so in different ways.

3. Page 5. "we next focused on the conserved helix (CH) [...], that is enriched with basic amino acids." Based on the provided sequence alignment, the helix contains an equal number of both basic and acidic residues. Is it correct to characterize this helix as positively charged?

Response

The reviewer is right. In the revised manuscript, we have used a more neutral expression as follows:

we next focused on the conserved helix (CH) [...], that contains conserved basic amino acids.

4. To prevent N-tail phosphorylation, the authors create a (H-N19A) allele, referring to Cdk promiscuity. Cdk cooperation with other mitotic kinases can also be expected. Nevertheless, in case the authors created a variant with only the 4 Cdk consensus sites mutated, it would be interesting to know its consequences.

Response

We consider that this is a reasonable question. In our early experiments, we noticed that introduction of multiple SP/TP sites in the non-SMC subunits of condensin I including CAP-H caused a relatively mild phenotype in mitotic chromosome assembly in Xenopus egg extracts. Then we found that the deletion of the CAP-H N-tail caused a very clear, accelerated loading phenotype, prompting us to focus on the regulatory function of the CAP-H N-tail. As the reviewer correctly points out, the current study does not pinpoint the number and position of target sites involved in the proposed phosphoregulation by the CAP-H N-tail. We wish to address this important issue in the near future, along with reconstitution of the phosphoregulation using purified components.

5. Fig EV3A, a second region of mitotic condensin phosphorylation is XCAP-D2. The authors state that XCAP-D2 phosphorylation does not impact on condensin function in their assays. This is very relevant to the current paper, so it would be good to see the Yoshida et al. 2022 Elife publication (in press) as an accompanying manuscript.

Response

We thank the reviewer for pointing out this issue, but it is not necessarily clear to us what the reviewer requests. In the original manuscript, we cited Yoshida et al (2022) in Discussion as follows:

Recent studies from our laboratory showed that the deletion of the CAP-D2 C-tail, which also contains multiple SP/TP sites (Supplementary Figure 3A), has little impact on condensin I function as judged by the same and related add-back assays using Xenopus egg extracts (Kinoshita et al, 2022; Yoshida et al, 2022).

We believe that the current statement is good enough.

6. One of the authors' most striking results is chromosome formation in interphase egg extracts using condensin (H-dN). At the same time, condensin (H-dN) is unable to support DNA supercoiling or chromosome reconstitution with recombinant components. More emphasis might be placed on this important piece of information, and possible reasons should be discussed. Can Cdk-treatment restore condensin (H-dN) biochemical activity? If not, then condensin (H-dN) might have lost more than just an inhibitory N-tail. The cohesin N-tail is thought to fulfil a positive role during DNA loading (Higashi et al. 2020). Could it be that the condensin N-tail encompasses both positive and negative roles?

Response

We were also surprised to find that holo(H-dN) gains the ability to assemble mitotic chromosome-like structures in interphase extracts. It should be stressed, however, that the formation of mitotic chromosome-like structures in I-HSS requires a much higher concentration (150 nM) than the standard concentration used in M-HSS (35 nM). Thus, the deletion of the CAP-H N-tail alone cannot make the condensin I complex fully active in I-HSS. We think that the negative regulation by the CAP-H N-tail is not the sole mechanism responsible for the very tight cell cycle regulation of condensin I function. We emphasize this important point by mentioning that “our results uncover one of the multilayered mechanisms that ensure cell cycle-specific loading of condensin I onto chromosomes” in Summary.

At the end of Discussion, we describe the limitations of the current study: “we have so far been unsuccessful in using these recombinant complexes to recapitulate positive DNA supercoiling or chromatid reconstitution, both of which require proper Cdk1 phosphorylation in vitro”. We are fully aware that full reconstitution of phosphorylation-dependent activation of condensin I in vitro is one of the most important directions in the future.

Although we currently do not have any evidence to suggest that the H N-tail has a positive role, we do not exclude such a possibility.

7. Here comes my main question for the authors (though I should stress that I do not expect an answer for publication in a Review Commons journal). The authors now have a unique opportunity to gain key mechanistic insight into condensin by answering the question, 'how does the kleisin N-tail inhibit condensin'? The authors allude to a model in which the N-tail interacts with Smc2 to close/obstruct the kleisin N-gate, through which the DNA likely enters the condensin ring. Can the authors biochemically recapitulate an interaction between an isolated N-tail (or N-terminal section of XCAP-H) and Smc2? Does Cdk phosphorylation alter this interaction?

Response

This comment is related to Comment #1 of Reviewer#1. See above for our response.

*Minor points. 8. The condensin loop extrusion results would benefit from a supplementary movie or time-series, to illustrate the comparison. Details of how loop rate, duration and sizes were assessed should be added to the methods section. *

Response

In the revised manuscript, we have provided a set of time-lapse images of loop extrusion events catalyzed by holo(WT) and holo(H-dN) in Fig 4E. We have also added the following explanations for how the parameters of loop extrusion reactions were assessed in Materials and Methods:

To determine the loop size, the fluorescence intensity of the looped DNA was divided by that of the entire DNA molecule for each image, and multiplied by the length of the entire DNA molecule (48.5 kb). The loop rate was obtained by averaging the increase in looped DNA size per second. The loop duration was calculated by measuring the time from the start of DNA loop formation until the DNA loop became unidentifiable.

9. Figure EV3A legend, "hHP4" should probably read "hHP2".

Response

The reviewer is right. It should read hHP2. Corrected.

*Reviewer #4 (Significance (Required)):

see above *

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Referee #4

Evidence, reproducibility and clarity

Mitotic chromosome formation is a cell cycle-regulated process that is crucial for eukaryotic genome stability. The chromosomal condensin complex promotes chromosome condensation, but the temporal control over condensin function is only scantly understood. In this impressive manuscript, "Cell cycle-specific loading of condensin I is regulated by the N-terminal tail of its kleisin subunit", Tane and colleagues provide important new insight into the cell cycle-regulation of condensin. The authors identify a kleisin N-tail that acts as a negative regulator of condensin's DNA interactions. Removal of this N-tail, or its cell cycle-dependent phosphorylation, relieves inhibition and activates condensin. This is a simple, yet very important story, that advances our molecular understanding of chromosome formation. The experiments are performed to a very high technical standard and support the authors conclusions. This manuscript is highly suitable for publication in any molecular biology journal, once the authors have considered the following points.

1. Introduction.

   • a) The authors could better explain their own prior work (Kimura et al. 1998), which has identified the condensin XCAP-D2 and XCAP-H as the targets of phosphoregulation. The current manuscript explains the role of XCAP-H phosphorylation.
   • b) Given the limited knowledge about condensin cell cycle regulation, it seems prudent to provide a brief summary of what is known. Fission yeast Smc4 phosphorylation regulates condensin nuclear import (Sutani et al. 1999), while budding yeast Smc4 phosphorylation slows down the dynamic turnover of the condensin complex on chromosomes (Robellet et al. 2015 and Thadani et al. 2018).
   • Extracts were mixed with mouse sperm nuclei. If there is a reason why mouse rather than Xenopus sperm nuclei were used, this would be interesting to know.
   • Page 5. "we next focused on the conserved helix (CH) [...], that is enriched with basic amino acids." Based on the provided sequence alignment, the helix contains an equal number of both basic and acidic residues. Is it correct to characterize this helix as positively charged?
   • To prevent N-tail phosphorylation, the authors create a (H-N19A) allele, referring to Cdk promiscuity. Cdk cooperation with other mitotic kinases can also be expected. Nevertheless, in case the authors created a variant with only the 4 Cdk consensus sites mutated, it would be interesting to know its consequences.
   • Fig EV3A, a second region of mitotic condensin phosphorylation is XCAP-D2. The authors state that XCAP-D2 phosphorylation does not impact on condensin function in their assays. This is very relevant to the current paper, so it would be good to see the Yoshida et al. 2022 Elife publication (in press) as an accompanying manuscript.
   • One of the authors' most striking results is chromosome formation in interphase egg extracts using condensin (H-dN). At the same time, condensin (H-dN) is unable to support DNA supercoiling or chromosome reconstitution with recombinant components. More emphasis might be placed on this important piece of information, and possible reasons should be discussed. Can Cdk-treatment restore condensin (H-dN) biochemical activity? If not, then condensin (H-dN) might have lost more than just an inhibitory N-tail. The cohesin N-tail is thought to fulfil a positive role during DNA loading (Higashi et al. 2020). Could it be that the condensin N-tail encompasses both positive and negative roles?
   • Here comes my main question for the authors (though I should stress that I do not expect an answer for publication in a Review Commons journal). The authors now have a unique opportunity to gain key mechanistic insight into condensin by answering the question, 'how does the kleisin N-tail inhibit condensin'? The authors allude to a model in which the N-tail interacts with Smc2 to close/obstruct the kleisin N-gate, through which the DNA likely enters the condensin ring. Can the authors biochemically recapitulate an interaction between an isolated N-tail (or N-terminal section of XCAP-H) and Smc2? Does Cdk phosphorylation alter this interaction?

Minor points.

1. The condensin loop extrusion results would benefit from a supplementary movie or time-series, to illustrate the comparison. Details of how loop rate, duration and sizes were assessed should be added to the methods section.
2. Figure EV3A legend, "hHP4" should probably read "hHP2".

Significance

see above

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Referee #3

Evidence, reproducibility and clarity

This paper reveals a role of an N-terminal extension of CAP-H in the regulation of condensin-I activity in Xenopus extracts using biochemical reconstitution experiments. The authors demonstrate that a motif in the N-terminal tail that is conserved in vertebrates acts as an inhibitor of condensin I activity. Using several mutant constructs, it is shown that the inhibition by this motif is in turn counteracted by the phosphorylation of neighbouring serine and threonine residues in mitosis, presumably at least in part by CdK. Mutants that have lost this inhibition are able to condense chromatin into chromatid-like structures more efficiently and to some degree even in interphase extracts. Moreover, one such mutant is characterized in detail by biochemical and biophysical experiments and shown to have increased capacity in salt-stable DNA loading and in DNA loop extrusion.

Major comments:

This is a beautiful and thorough study that is presented in a clear and concise manner. The main conclusions are well justified. No additional experiments are needed to support them. Replication and statistical analysis appear adequate. The final model is however largely speculative. Recent work has indicated that loading of yeast condensin does not require gate opening. The authors may thus want to include alternative scenarios or remain more vague.

The H-N19A mutant has a loss of function phenotype (possibly due to folding problem caused by 19 point mutations rather than lack of phosphorylation), the authors could consider to rescue the phenotype by also including the CH motif mutations in this construct (or make an explanatory statement in the text).

Albeit not necessary for the main conclusions, the authors could possibly significantly strengthen their study by testing for binding partners of the N-tail and the CH motif by running AlphaFold predictions against the condensin I subunits.

The efficiency of depletion of condensin subunits from I-HSS extracts is not documented (in contrast to M-HSS extracts - figure EV1C). While any condensin remaining in these extracts might not be active (or interfering), the authors may want to at least comment on this in the text.

The authors should include information on the quantification of chromatid morphology. Is the analysis based on chromatids taken from the same images/imaging session, from technical replicates, biological replicates?

Minor comment:

The colour scheme in Figure 5A is confusing. Use less colour? The orange and red colours are moreover quite similar.

Significance

The findings provide new insights into how condensin-I activity is restricted outside of mitosis. It was previously assumed that this regulation was (largely) due to the exclusion of condensin I from the nucleus prior to nuclear envelope breakdown. This study shows that another pathway is contributing to the regulation and implies that controlling condensin I activity is more important than previously appreciated. Whether all residual nuclear condensin I is inactivated, remains to be determined. The physiological impact of loss of autoinhibition on chromosome segregation and cell cycle progression also remains to be uncovered. The observed effects are robust and appear significant. Future research on condensin I using reconstitution will likely benefit from being able to control or eliminate the self-inhibition.

This reviewer has expertise on the biochemistry and structural biology of SMC protein complexes.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

The manuscript reveals that the N-terminal region of CAPH could play a role in regulating condensin I activity, using a range of in vitro methods. They propose that the N-terminal region of CAPH inhibits complex activity, and this is turned off upon deletion or phosphorylation, by using truncations, phospho-mimics or phospho-deficient mutations.

While the results are interesting to the field, and helps to address the question as to how condensin complexes are controlled in a cell cycle dependent manner, some key data and controls are necessary to ensure the conclusion is robust.

Main comments

• What is meant by "unperturbed I-HSS" on page 7, ie membrane containing versus membrane free or condensin depleted?
• In many of the protein gels eg figure 4B, the bands for SMC2 and 4 are more intense that the non-SMC components. The method for protein purification also does not include a size exclusion step to ensure sample homogeneity. Authors should perform some sort of quality control checks on samples such as analytical gel filtration or mass photometry to ensure stoichiometry/homogeneity. This is particularly important for samples eg the N19A, where activity is reduced compared to wild-type as poor protein behaviour could create false negative results.
• Loop extrusion assays in figure 4D-G shows no example data i.e. no pictures or videos of loops being formed. These should also be included.
• Given the mutant holo(H-dN) has higher activity than wild-type, a negative control such as holo(H-dN) without ATP or holo(H-dN) ATPase deficient mutant should also be measured in loop extrusion assays, to ensure the activity is derived from the ATPase activity.
• According to the methods, this work performs the same loop extrusion assay as described in Kinoshita et al, 2022, however, in Kinoshita et al, wild type condensin I makes loops in 30-50% of DNA molecules, where in this study the percentage is less than half that. Can the author please explain the discrepancy given the same method was used?
• In the concluding statement the author suggests "Upon mitotic entry, multisite phosphorylation of the N-tail relieves the stabilization, allowing the opening of the DNA entry gate, hence, the loading of condensin I onto chromosomes." This seems unlikely as fusion the N-terminus of the of the kleisin to the C-terminus of SMC2 is able to function for yeast (Shaltiel et al 2022) and condensin II (Houlard et al 2021), and equivalently in cohesin (Davidson et al 2019)

Significance

This is an interesting story that reveals new insights about condensin regulation.