Reviewer #1 (Public review):

The authors use inducible Fz::mKate2-sfGFP to explore "cell-scale signaling" in PCP. They reach several conclusions. First, they conclude that cell-scale signaling does not depend on limiting pools of core components (other than Fz). Second, they conclude that cell-scale signaling does not depend on microtubule orientation, and third, they conclude that cell-scale signaling is strong relative to cell to cell coupling of polarity.

There are some interesting inferences that can be drawn from the manuscript, but there are also some significant challenges in interpreting the results and conclusions from the work as presented. I suggest that the authors 1) define "cell-scale signaling," as the precise meaning must be inferred, 2) reconsider some premises upon which some conclusions depend, 3) perform an essential assay validation, and 4) explain some other puzzling inconsistencies.

Major concerns (first round of review):

The exact meaning of cell-scale signaling is not defined, but I infer that the authors use this term to describe how what happens on one side of a cell affects another side. The remainder of my critique depends on this understanding of the intended meaning.

The authors state that any tissue wide directional information comes from pre-existing polarity and its modification by cell flow, such that the de novo signaling paradigm "bypasses" these events and should therefore not be responsive to any further global cues. It is my understanding that this is not a universally accepted model, and indeed, the authors' data seem to suggest otherwise. For example, the image in Fig 5B shows that de novo induction restores polarity orientation to a predominantly proximal to distal orientation. If no global cue is active, how is this orientation explained? The 6 hr condition, that has only partial polarity magnitude, is quite disordered. Do the patterns at 8 and 10 hrs become more proximally-distally oriented? It is stated that they all show swirls, but please provide adult wing images, and the corresponding orientation outputs from QuantifyPolarity to help validate the notion that the global cues are indeed bypassed by this paradigm.

It is implicit that, in the de novo paradigm, polarization is initiated immediately or shortly after heat shock induction. However, the results should be differently interpreted if the level of available Fz protein does not rise rapidly and then stabilize before the 6 hr time point, and instead continues to rise throughout the experiment. Western blots of the Fz::mKate2-sfGFP at time points after induction should be performed to demonstrate steady state prior to measurements. Otherwise, polarity magnitude could simply reflect the total available pool of Fz at different times after induction. Interpreting stability is complex, and could depend on the same issue, as well as the amount of recycling that may occur. Prior work from this lab using FRAP suggested that turnover occurs, and could result from recycling as well as replenishment from newly synthesized protein.

From the Fig 3 results, the authors claim that limiting pools of core proteins do not explain cell-scale signaling, a result expected based on the lack of phenotypes in heterozygotes, but of course they do not test the possibility that Fz is limiting. They do note that some other contributing protein could be.

In Fig 3, it is unclear why the authors chose to test dsh1/+ rather than dsh[null]/+. In any case, the statistically significant effect of Dsh dose reduction is puzzling, and might indicate that the other interpretation is correct. Ideally, a range including larger and smaller reductions would be tested. As is, I don't think limiting Dsh is ruled out.

The data in Fig 5 are somewhat internally inconsistent, and inconsistent with the authors' interpretation. In both repolarization conditions, the authors claim that repolarization extends only to row 1, and row 1 is statistically different from non-repolarized row 1, but so too is row 3. Row 2 is not. This makes no sense, and suggests either that the statistical tests are inappropriate and/or the data is too sparse to be meaningful. For the related boundary intensity data in Fig 6, the authors need to describe exactly how boundaries were chosen or excluded from the analysis. Ideally, all boundaries would be classified as either meido-lateral (meaning anterior-posterior) or proximal-distal depending on angle.

If the authors believe their Fig 5 and 6 analyses, how do they explain that hairs are reoriented well beyond where the core proteins are not? This would be a dramatic finding, because as far as I know, when core proteins are polarized, prehair orientation always follows the core protein distribution. Surprisingly, the authors do not so much as comment about this. The authors should age their wings just a bit more to see whether the prehair pattern looks more like the adult hair pattern or like that predicted by their protein orientation results.

Reviewer #3 (Public Review):

The manuscript by Carayon and Strutt addresses the role of cell-scale signaling during the establishment of planar cell polarity (PCP) in the Drosophila pupal wing. The authors induce locally the expression of a tagged core PCP protein, Frizzled, and observe and analyze the de novo establishment of planar cell polarity. Using this system, the authors show that PCP can be established within several hours, that PCP is robust towards variation in core PCP protein levels, that PCP proteins do not orient microtubules, and that PCP is robust towards 'extrinsic' re-polarization. The authors conclude that the polarization at the cell-scale is strongly intrinsic and only weakly affected by the polarity of neighboring cells.

Major comments (first round of review):

The data are clearly presented and the manuscript is well written. The conclusions are well supported by the data. 

(1) The authors use a system to de novo establish PCP, which has the advantage of excluding global cues orienting PCP and thus to focus on the cell-intrinsic mechanisms. At the same time, the system has the limitation that it is unclear to what extent de novo PCP establishment reflects 'normal' cell scale PCP establishment, in particular because the Gal4/UAS expression system that is used to induce Fz expression will likely result in much higher Fz levels compared with the endogenous levels. The authors should briefly discuss this limitation.

(2) Fig. 3. The authors use heterozygous mutant backgrounds to test the robustness of de novo PCP establishment towards (partial) depletion in core PCP proteins. The authors conclude that de novo polarization is 'extremely robust to variation in protein level'. Since the authors (presumably) lowered protein levels by 50%, this conclusion appears to be somewhat overstated. The authors should tune down their conclusion.

Significance: 

The manuscript contributes to our understanding of how planar cell polarity is established. It extends previous work by the authors (Strutt and Strutt, 2002,2007) that already showed that induction of core PCP pathway activity by itself is sufficient to induce de novo PCP. This manuscript further explores the underlying mechanisms. The authors test whether de novo PCP establishment depends on an 'inhibitory signal', as previously postulated (Meinhardt, 2007), but do not find evidence. They also test whether core PCP proteins help to orient microtubules (which could enhance cell intrinsic polarization of core PCP proteins), but, again, do not find evidence, corroborating previous work (Harumoto et al, 2010). The most significant finding of this manuscript, perhaps, is the observation that local de novo PCP establishment does not propagate far through the tissue. A limitation of the study is that the mechanisms establishing intrinsic cell scale polarity remain unknown. The work will likely be of interest to specialists in the field of PCP.

Summary of comments from the Reviewing Editor on the revised version:

In the introduction, when you refer to Figure 1, the definition of Molecular, cellular, tissue scale is indeed not too clear to outside readers. For example, when you first refer to 'cell scale' you define it 'non-local', but probably it is not clear to many readers 'non-local' means 'the mechanism that cannot be explained by 'molecular scale'. (because 'molecular scale = local' is only inferred).

The 'conclusion paragraph' at the end of the Introduction does not have conclusion (only explained 'which question was tested by which method').

Minor comments that can easily be addressed by textual edits:

– they do not explain why gene dosage affects constitutive but not de novo polarization. It seems to me that one would expect de novo to be at least as sensitive if not more.

– Unconventional nomenclature for tissue axes - mediolateral, horizontal - are frequently used. These are sometimes difficult to parse. Please stick with universally accepted anterior, posterior, proximal and distal.

Author response:

(1) General Statements

Our manuscript studies mechanisms of planar polarity establishment in vivo in the Drosophila pupal wing. Specifically we seek to understand mechanisms of ‘cell-scale signalling’ that is responsible for segregating core pathway planar polarity proteins to opposite cell edges. This is an understudied question, in part because it is difficult to address experimentally.

We use conditional and restrictive expression tools to spatiotemporally manipulate core protein activity, combined with quantitative measurement of core protein distribution, polarity and stability. Our results provide evidence for a robust cell-scale signal, while arguing against mechanisms that depend on depletion of a limited pool of a core protein or polarised transport of core proteins on microtubules. Furthermore, we show that polarity propagation across a tissue is hard, highlighting the strong intrinsic capacity of individual cells to establish and maintain planar polarity.

The original manuscript received three fair and thorough peer-reviews, which raised many important points. In response, we decided to embark on a full revision that attempts to answer all of the points. We have included new data to support our conclusions in Supplemental Figures 1, 2 and 5.

Additionally in response to the reviewers we have revised the manuscript title, which is now ‘Characterisation of cell-scale signalling by the core planar polarity pathway during Drosophila wing development’.

(2) Point-by-point description of the revisions

We thank all of the reviewers for their thorough and thoughtful review of our manuscript. They raise many helpful points which have been extremely useful in assisting us to revise the manuscript.

In response we have carried out a major revision of the manuscript, making numerous changes and additions to the text and also adding new experimental data. Specific changes are listed after our detailed response to each comment.

Reviewer #1:

[…] Major points:

The exact meaning of cell-scale signaling is not defined, but I infer that the authors use this term to describe how what happens on one side of a cell affects another side. The remainder of my critique depends on this understanding of the intended meaning.

As the reviewer points out, it is important that the meaning of the term ‘cell-scale signalling’ is clear to the reader and in response to their comment we have had another go at defining it explicitly in the Introduction to the manuscript.

Specifically, we use the term ‘cell-scale signalling’ to describe possible intracellular mechanisms acting on core protein segregation to opposite cell membranes during core pathway dependent planar polarisation. For example, this could be a signal from distal complexes at one side of the cell leading to segregation of proximal complexes to the opposite cell edge, or vice versa. See also our response to Reviewer #2 regarding the distinction between ‘molecular-scale’ and ‘cell-scale’ signalling. 

Changes to manuscript: Revised definition of ‘cell-scale signalling’ in Introduction.

The authors state that any tissue wide directional information comes from pre-existing polarity and its modification by cell flow, such that the de novo signaling paradigm "bypasses" these events and should therefore not be responsive to any further global cues. It is my understanding that this is not a universally accepted model, and indeed, the authors' data seem to suggest otherwise. For example, the image in Fig 5B shows that de novo induction restores polarity orientation to a predominantly proximal to distal orientation. If no global cue is active, how is this orientation explained?

We assume that the reviewer’s point is that it is not universally accepted that de novo induction after hinge contraction leads to uncoupling from global cues (rather than that it is not accepted that hinge contraction remodels radial polarity to a proximodistal pattern). We are (we believe) the only lab that has used de novo induction as a tool, and we’re not aware of any debate in the literature about whether this bypasses global cues. Nevertheless, we accept that it is hard to prove there is no influence of global cues, when the nature of those cues and the time at which they act remain unclear. Below we summarise the reasons why we believe there are not significance effects of global cues in our experiments that would influence the interpretation of our results.

First, our reading of the literature supports a broad consensus that an early radial core planar polarity pattern is realigned by cell flow produced by hinge contraction beginning at around 16h APF (e.g. Aigouy et al., 2010; Strutt and Strutt, 2015; Aw and Devenport, 2017; Butler and Wallingford, 2017; Tan and Strutt, 2025). Taken at face value, this suggests that there are ‘radial’ cues present prior to hinge contraction, maybe coming from the wing margin – arguably these radial cues could be Ft-Ds or Wnts or both, given they are expressed in patterns consistent with such a role (notwithstanding the published evidence arguing against roles for either of these cues). It then appears that hinge contraction supercedes these cues to convert a radial pattern to a proximodistal pattern – whether the radial cues that affect the core pathway earlier remain active after hinge contraction is unclear, although both Ft-Ds and Wnts appear to maintain their ‘radial’ patterns beyond the beginning of hinge contraction (e.g. Merkel et al., 2014; Ewen-Campen et al., 2020; Yu et al., 2020).

We think that the reviewer is proposing the presence of a proximodistal cue that is active in the proximal region of the wing that we use for our experiments shown e.g. in Fig.5, and that this cue orients core polarity here (but not elsewhere in the wing) in a time window after 18h APF. Ft-Ds and Wnts do not seem to be plausible candidates as they are still in ‘radial’ patterns. This leaves either an unknown proximodistal cue (a gradient of some unknown signalling molecule?), or possibly some ability of hinge contraction to align proximodistal polarity specifically in this wing region but not elsewhere. We cannot definitively rule out either of these possibilities, but neither do we think there is sufficient evidence to justify invoking their existence to explain our observations.

In particular, the reason that we don’t think there is a proximodistal cue in the proximal part of the wing after 18h APF, is that work from our lab shows that induction of Fz or Stbm expression at times around or after the start of hinge contraction (i.e. >16 h APF) results in increasing levels of trichome swirling with polarity not being coordinated with the tissue axis either proximally or distally (Strutt and Strutt, 2002; Strutt and Strutt 2007). Our simplest interpretation for this is that induction at these stages fails to establish the early radial pattern of core pathway polarity and hence hinge contraction cannot reorient radial to proximodistal. If hinge contraction alone could specify proximodistal polarity in the absence of the earlier radial polarity, then we would not expect to see swirling over much of the proximal wing (where the forces from hinge contraction are strongest (Etournay et al., 2015)).

In this manuscript, our earliest de novo experiments begin with Fz induction at 18h APF (de novo 10h), then at 20h APF (de novo 8h) and at 22h APF (de novo 6h). The image in Fig. 5B, referred to by the reviewer, is of a wing where Fz is induced de novo at 22 h APF. In these wings, as expected, the core proteins localise asymmetrically in stereotypical swirling patterns throughout the wing surface (see Fig. 2M and also Strutt and Strutt, 2002; Strutt and Strutt 2007), but – usefully for our experiments – they broadly localise along the proximal-distal axis in the region analysed in Fig. 5B. Given the strong swirling in surrounding regions when inducing at >20h APF, we feel reasonably confident in assuming that the pattern is not due to a proximodistal cue present in the proximal wing.

We appreciate that the original manuscript did not show images including the trichome pattern in adjacent regions, so this point would not have been clear, but we now include these in Supplementary Fig. 5. We have also added a note in the legend to Fig. 5B to clarify that the proximodistal pattern seen is local to this wing region. We apologise for this oversight and the confusion caused and appreciate the feedback.

The 6 hr condition, that has only partial polarity magnitude, is quite disordered. Do the patterns at 8 and 10 hrs become more proximally-distally oriented? It is stated that they all show swirls, but please provide adult wing images, and the corresponding orientation outputs from QuantifyPolarity to help validate the notion that the global cues are indeed bypassed by this paradigm.

In all three ‘normal’ de novo conditions (6h, 8h and 10h), regardless of the time of induction, the polarity orientation patterns of Fz-mKate2 in pupal and adult wings are very similar in the experimentally analysed region (Fig. S5B-E). The strong local hair swirling agrees with the previous published data (Strutt and Strutt, 2002; Strutt and Strutt 2007). Overall, we don’t see any evidence that the 10h de novo induction results in more proximodistally coordinated polarity than the 8h or 6h conditions. This is consistent with our contention that there is no global cue present at these stages, which presumably would have a stronger effect when core pathway activity was induced at earlier stages.

Changes to manuscript: Added additional explanation of the ‘de novo induction’ paradigm and why we believe the resulting polarity patterns are unlikely to be influenced by any global signals in Introduction and Results section ‘Induced core protein relocalisation…’. Added quantification of polarity in the experiment region proximal to the anterior cross-vein in pupal wings (Fig.S5E-E’’’) and zoomed-out images of the surrounding region in adult wings showing that the polarity pattern does not become more proximodistal when induction time is longer, and also that there is not overall proximodistal polarity in proximal regions of the wing (Fig.S5B-D), arguing against an unknown proximodistal polarity cue at these stages of development.

In the de novo paradigm, polarization is initiated immediately or shortly after heat shock induction. However, the results should be differently interpreted if the level of available Fz protein does not rise rapidly and then stabilize before the 6 hr time point, and instead continues to rise throughout the experiment. Western blots of the Fz::mKate2-sfGFP at time points after induction should be performed to demonstrate steady state prior to measurements. Otherwise, polarity magnitude could simply reflect the total available pool of Fz at different times after induction. Interpreting stability is complex, and could depend on the same issue, as well as the amount of recycling that may occur. Prior work from this lab using FRAP suggested that turnover occurs, and could result from recycling as well as replenishment from newly synthesized protein. 

The reviewer raises an important point, which we agree could confound our experimental interpretations. As suggested we have now carried out western blotting and quantitation for Fz::mKate2-sfGFP levels and added these data to Fig.S1 (Fig. S1C,D). Quantified Fz is not significantly different between the three de novo polarity induction timings and not significantly different compared to constitutive Fz::mKate2-sfGFP expression (although there is a trend towards increasing Fz::mKate2-sfGFP protein levels with increasing induction times). These data are consistent with Fz::mKate2-sfGFP being at steady state in our experiments and that levels are sufficient to achieve normal polarity (as constitutive Fz::mKate2-sfGFP does so). Therefore it is unlikely that differing protein levels explain the differing polarity magnitudes at the different induction times. Interestingly, Fz::mKate2-sfGFP levels are lower than endogenous Fz levels, possibly due to lower expression or increased turnover/reduced recycling.

Changes to manuscript: Added western blot analysis of Fz::mKate2-sfGFP expression under 10h, 8h and 6h induction conditions vs endogenous Fz expression and constitutive Fz::mKate2sfGFP expression (Fig.S1C-D) and discussed in Results section ‘Planar polarity establishment is…’.

From the Fig 3 results, the authors claim that limiting pools of core proteins do not explain cellscale signaling, a result expected based on the lack of phenotypes in heterozygotes, but of course they do not test the possibility that Fz is limiting. They do note that some other contributing protein could be. 

Previously published results from our lab (Strutt et al., 2016 Cell Reports; Supplemental Fig. S6E) show that in a heterozygous fz mutant background, Fz protein levels are not affected by halving the gene dosage when compared to wt, suggesting that Fz is most likely produced in excess and is not normally limiting, but that protein that cannot form complexes may be rapidly degraded. We have now added this information to the text.

Changes to manuscript: Added explanation in text that Fz levels had previously been shown to not be dosage sensitive in Results section ‘Planar polarity establishment is…’ and also added a caveat to the Discussion about not directly testing Fz.

In Fig 3, it is unclear why the authors chose to test dsh1/+ rather than dsh[null]/+. In any case, the statistically significant effect of Dsh dose reduction is puzzling, and might indicate that the other interpretation is correct. Ideally, a range including larger and smaller reductions would be tested. As is, I don't think limiting Dsh is ruled out. 

Concerning the choice of dsh allele, we appreciate the query of the reviewer regarding use of dsh[1] instead of a null, as there might be a concern that dsh[1] would give a less strong phenotype. The answer is that over more than two decades we and others have never found any evidence that dsh[1] does not act as a ‘null’ for planar polarity in the pupal wing, and furthermore use of dsh[1] preserves function in Wg signalling – and we would prefer to rule out any phenotypic effects due to any potential cross-talk between the two pathways that might be seen using a complete null. To expand on this point, dsh[1] mutant protein is never seen at cell junctions (Axelrod 2001; Shimada et al., 2001; our own work), and by every criteria we have used, planar polarity is completely disrupted in hemizygous or homozygous mutants e.g. see quantifications of polarity in (Warrington et al., 2017 Curr Biol).

In terms of the broader point, whether we can rule out Dsh being limiting, we were very careful to be clear that we did not see evidence for Dsh (or other core proteins) being limiting in terms of ‘rates of core pathway de novo polarisation’. When the reviewer says ‘the statistically significant effect of Dsh dose reduction is puzzling’ we believe they are referring to the data in Fig. 3J, showing a small but significantly different reduction in stable Fz in de novo 6h conditions (also seen in 8h de novo conditions, Fig. S3I). As Dsh is known to stabilise Fz in complexes (Strutt et al., 2011 Dev Cell; Warrington et al., 2017 Curr Biol), in itself this result is not wholly surprising. Nevertheless, while this shows that halving Dsh levels does modestly reduce Fz stability, it does not alter our conclusion that halving Dsh levels does not affect Fz polarisation rate under either 6h or 8h de novo conditions.

Unfortunately, we do not have available to us a practical way of achieving consistent intermediate reductions in Dsh levels (e.g. a series of verified transgenes expressing at different levels). Levels of all the core proteins could be dialled down using transgenes, to see when the system breaks, and indeed we have previously published that lower levels of polarity are seen if Fmi levels are <<50% or if animals are transheterozygous for pk, stbm, dgo or dsh, pk, stbm, dgo simultaneously (Strutt et al., 2016 Cell Reports). However, it seems to be a trivial result that eventually the ability to polarise is lost if insufficient core proteins are present at the junctions. For this reason we have focused on a simple set of experiments reducing gene dosage singly by 50% under two de novo induction conditions, and have been careful to state our results cautiously. The assays we carried out were a great deal of work even for just the 5 heterozygous conditions tested.

We believe that the experiments shown effectively make the point that there is no strong dosage sensitivity – and it remains our contention that if protein levels were the key to setting up cell-scale polarity, then a 50% reduction would be expected to show an effect on the rate of polarisation. We further note that as Fz::mKate2-sfGFP levels are lower than endogenous Fz levels (see above), the system might be expected to be sensitised to further dosage reductions, and despite this we failed to see an effect on rate of polarisation.

We note that Reviewer #3 made a similar point about whether we can rule out dosage sensitivity on the basis of 50% reductions in protein level. To address the comments of both reviewers we had now added some further narrative and caveats in the text.

In a similar vein, Reviewer #2 requested data on whether dosage reduction altered protein levels by the expected amount. We have now added further explanation/references and western blot data to address this.

Changes to manuscript: Added more explanation of our choice of dsh[1] as an appropriate mutant allele to use in Results section ‘Planar polarity establishment is…’. Added some narrative and caveats regarding whether lowering levels more than 50% would add to our findings in the Discussion. Revised conclusions to be more cautious including altering section title to read ‘Planar polarity establishment is not highly sensitive to variation in protein levels of core complex components’.

Also added westerns and text/references showing that for the tested proteins there is a reduction in protein levels upon removal of one gene dosage in Results section ‘Planar polarity establishment is…’ and Fig.S2.

The data in Fig 5 are somewhat internally inconsistent, and inconsistent with the authors' interpretation. In both repolarization conditions, the authors claim that repolarization extends only to row 1, and row 1 is statistically different from non-repolarized row 1, but so too is row 3. Row 2 is not. This makes no sense, and suggests either that the statistical tests are inappropriate and/or the data is too sparse to be meaningful. 

As we’re sure the reviewer appreciates, this was an extremely complex experiment to perform and analyse. We spent a lot of time trying to find the best way to illustrate the results (finally settling on a 2D vector representation of polarity) and how to show the paired statistical comparisons between different groups. Moreover, in the end we were only able to detect generally quite modest (statistically significant) changes in cell polarity under the experimental conditions.

However, we note that failure to see large and consistent changes in polarity is exactly the expected result if it is hard to repolarise from a boundary – and this is of course the conclusion that we draw. Conversely, if repolarisation were easy, which was our expectation at least under de novo conditions without existing polarity, then we would have expected large and highly statistically significant changes in polarity across multiple cell rows. Hence we stand by our conclusion that ‘it is hard to repolarise from a boundary of Fz overexpression in both control and de novo polarity conditions’.

Overall, we were trying to establish three points:

(1) to demonstrate that repolarisation occurs from a boundary of overexpression i.e. from boundary 0 to row 0

(2) to establish whether a wave of repolarisation occurs across rows 1, 2 and 3

(3) to determine if in repolarisation in de novo condition it is easier to repolarise than in repolarisation in the control (already polarised) condition Taking each in turn:

(1) To detect repolarisation from a boundary relative to the control condition, we have to compare row 0 in repolarisation condition (Fig.5G,K) vs control condition (Fig.5F,J). This comparison shows a significative repolarisation (p=0.0014). From now, row 0 in repolarisation condition is our reference for repolarisation occurring.

(2) To determine if there is a wave of repolarisation in the repolarisation condition we have to compare row 0 vs row 1 to 3 in the repolarisation condition (Fig.5K). Row 1 is not significantly different to row 0, but rows 2 and 3 are different and the vectors show obviously lower polarity than row 0. Hence no wave of repolarisation is detected over rows 1 to 3.

(3) To determine if it is easier to repolarise in the de novo condition, our reference for establishment of a repolarisation pattern is the polarisation condition in rows 0 to 3. So, we compare repolarisation condition vs repolarisation in de novo condition, row 0 vs row 0, row 1 vs row 1, row 2 vs row 2 and row 3 vs row 3 – in each case no significative difference in polarity is detected, supporting our conclusion that it is not easier to repolarise in the de novo condition.

We agree that the variations in row 3 are puzzling, but there is no evidence that this is due to propagation of polarity from row 0, and so in terms of our three questions, it does not alter our conclusions.

Changes to manuscript: We have extensively revised the text describing the results in Fig.5 to hopefully make the reasons for our conclusions clearer and also be more cautious in our conclusions in Results section ‘Induced core protein relocalisation…’. 

For the related boundary intensity data in Fig 6, the authors need to describe exactly how boundaries were chosen or excluded from the analysis. Ideally, all boundaries would be classified as either meido-lateral (meaning anterior-posterior) or proximal-distal depending on angle. 

We thank the reviewer for pointing out that this was not clear.

All boundaries were classified following their orientation compared to the Fz over-expression boundary using hh-GAL4 expressed in the wing posterior compartment. Horizontal junctions were defined as parallel to the Fz over-expression boundary (between 0 and 45 degrees) and mediolateral junctions as junctions linking two horizontal boundaries (between 45 and 90 degrees).

Changes to manuscript: The boundary classification detailed above has been added in the Materials and Methods.

If the authors believe their Fig 5 and 6 analyses, how do they explain that hairs are reoriented well beyond where the core proteins are not? This would be a dramatic finding, because as far as I know, when core proteins are polarized, prehair orientation always follows the core protein distribution. Surprisingly, the authors do not so much as comment about this. The authors should age their wings just a bit more to see whether the prehair pattern looks more like the adult hair pattern or like that predicted by their protein orientation results.

Again the reviewer makes an interesting point, and we agree that this is something that we should have more directly addressed in the manuscript.

There are three reasons why we might expect adult trichomes to show a different effect from the measured core protein polarity pattern seen in our experiments:

(i) we are assaying core protein polarity at 28h APF, but trichomes emerge at >32h APF, so there is still time for polarity to propagate a bit further from the boundary. We now have added data showing that by the point of trichome initiation, the wave of polarisation extends 3-4 cell rows (Fig.S5A).

(ii) it has long been known that a strong localisation of core proteins at a cell edge is not required for polarisation of trichome polarity from a boundary. For instance, in Strutt & Strutt 2007 we show clones of cells overexpressing Fz causing propagation through pk[pk-sple] mutant tissue where there is no detectable core protein polarity. We were following up prior observations of Adler et al., 2000 in the wing and Lawrence et al., 2004 in the abdomen.

(iii) there is evidence to suggest that the polarity of adult trichomes is locally coupled, possibly mechanically. This point is hard to prove without live imaging taking in both initial core protein localisation, the site of actin-rich trichome initiation and then the final orientation of the much larger microtubule filled trichome, and we’re not aware that such data exist. However, Wong & Adler 1993 (JCB) showed that over a number of hours trichomes become much larger and move towards the centre of the cell, presumably becoming decoupled from any core protein cue. The images in Guild … & Tilney, 2005 (MBoC)  are also interesting to look at in this regard. Finally, septate junction proteins have been implicated in local alignment of trichomes, independently of the core pathway (Venema … & Auld, 2004 Dev Biol).

Changes to manuscript: Added new data in Fig.S5A showing where trichomes initiate under 6h de novo induction conditions, for comparison to core protein localisation and adult trichome data in Fig.5. Added some text explaining why adult trichome repolarisation might be stronger than the observed effects on core protein localisation in Discussion. 

Minor points:

As the authors know, there is a model in the literature that suggests microtubule trafficking provides a global cue to orient PCP. The authors' repolarization data in Fig 4 make a reasonably convincing case against a role for no role for microtubules in cell-scale signaling, but do not rule out a role as a global cue. The authors should be careful of language such as "...MTs and core proteins being oriented independently of each other" that would appear to possibly also refer to a role as a global cue. 

Thank you for pointing out that this was not clear. We have now modified the text to hopefully address this.

Changes to manuscript: Text updated in Results section ‘Microtubules do not provide…’.

Significance:

There are two negative conclusions and one positive conclusion made by the authors. Provided the above points are addressed, the negative conclusions, that core proteins are not limiting and that microtubules are not involved in cell-scale signaling are solid. The positive conclusion is more nebulous - the authors say that cell-scale signaling is strong relative to cell-cell signaling - but how strong is strong? Strong relative to their prior expectations? I'm not sure how to interpret such a conclusion. Overall, we learn something from these results, though it fails to reveal anything about mechanism. These results will be of some interest to those studying PCP.

The reviewer raises an interesting point, which is how do you compare the strength of two different processes, even if both processes affect the same outcome (in this case cell polarity). Repolarisation from a boundary has not been carefully studied at the level of core protein localisation in any previous study to our knowledge – this is one of the important novel aspects of this study. Hence there is not a baseline for defining strong repolarisation. Similarly, there has been no investigation of the nature of ‘cell-scale signalling’. This was a considerable challenge for us in writing the manuscript, and we have done our best to find appropriate language that hopefully conveys our message adequately. Minimally our work may provide a baseline for helping to define the ‘strengths’ of these processes in future studies.

One of our main points is that we can generate an artificial boundary of Fz expression, where Fz levels are at least several fold higher than in the neighbouring cell (e.g. compare Fig.4N’ and O’) and only two rows of cells show a significant change in polarity relative to controls. Even when the tissue next to the overexpression domain is still in the process of generating polarity (de novo condition) then the boundary has little effect on polarity in neighbouring cell rows. This was a result that surprised us, and we tried to convey that by using language to suggest cell-scale signalling was stronger than cell-cell signalling i.e. stronger in terms of the ability to define the final direction of polarity.

Changes to manuscript: In the revised manuscript we have reviewed our use of language and now avoid saying ‘strong’ but instead use terms such as ‘effective’ and ‘robust’ in e.g. Results section ‘Induced core protein relocalisation…’, the Discussion and we have also changed the title of the manuscript to avoid claiming a ‘strong’ signal.

Reviewer #2:

[…] Critique

The experiments described in this paper are of high quality with a sophisticated level of design and analysis. However, there needs to be some recalibration of the extent of the conclusions that can be drawn (see below). Moreover, a limitation of this paper is that, despite the quality of their data, they cannot give a molecular hint about the nature of their proposed cell-scale signal. Below are a two key points that the authors may want to clarify.

(1) The first set of repolarisation experiment is performed after the global cell rearrangements that have been shown to act as global signal. However, this approach does not exclude the possible contribution of an unknown diffusible global signal.

A similar point was raised by Reviewer 1. For the convenience of this reviewer, we’ll summarise the arguments against such an unknown cue again below. More broadly, both reviewers asking a similar question indicates that we have failed to lay out the evidence in sufficient detail. In our defence, we have used the same ‘de novo’ paradigm in three previous publications (Strutt and Strutt 2002, 2007; Brittle et al 2022) without attracting (overt) controversy. We have now added text to the Introduction and Results that goes into more detail, as well as more experimental evidence (Fig.S5).

Firstly, it is worth noting that the global cues acting in the wing are poorly understood, with mostly negative evidence against particular cues accruing in recent years. This makes it a hard subject to succinctly discuss. Secondly, we accept that it is hard to prove there is no influence of global cues, when the nature of those cues and the time at which they act remain unclear. Below we summarise the reasons why we believe there are not significance effects of global cues in our experiments that would influence the interpretation of our results.

First, our reading of the literature supports a broad consensus that an early radial core planar polarity pattern is realigned by cell flow produced by hinge contraction beginning at around 16h APF (e.g. Aigouy et al., 2010; Strutt and Strutt, 2015; Aw and Devenport, 2017; Butler and Wallingford, 2017; Tan and Strutt, 2025). Taken at face value, this suggests that there are ‘radial’ cues present prior to hinge contraction, maybe coming from the wing margin – arguably these radial cues could be Ft-Ds or Wnts or both, given they are expressed in patterns consistent with such a role (notwithstanding the published evidence arguing against roles for either of these cues). It then appears that hinge contraction supercedes these cues to convert a radial pattern to a proximodistal pattern – whether the radial cues that affect the core pathway earlier remain active after hinge contraction is unclear, although both Ft-Ds and Wnts appear to maintain their ‘radial’ patterns beyond the beginning of hinge contraction (e.g. Merkel et al., 2014; Ewen-Campen et al.,2020; Yu et al., 2020).

We think that the reviewers are proposing the presence of a proximodistal cue that is active in the proximal region of the wing that we use for our experiments shown e.g. in Fig.5, and that this cue orients core polarity here (but not elsewhere in the wing) in a time window after 18h APF. Ft-Ds and Wnts do not seem to be plausible candidates as they are still in ‘radial’ patterns. This leaves either an unknown proximodistal cue (a gradient of some unknown signalling molecule?), or possibly some ability of hinge contraction to align proximodistal polarity specifically in this wing region but not elsewhere. We cannot definitively rule out either of these possibilities, but neither do we think there is sufficient evidence to justify invoking their existence to explain our observations.

In particular, the reason that we don’t think there is a proximodistal cue in the proximal part of the wing after 18h APF, is that work from our lab shows that induction of Fz or Stbm expression at times around or after the start of hinge contraction (i.e. >16 h APF) results in increasing levels of trichome swirling with polarity not being coordinated with the tissue axis either proximally or distally (Strutt and Strutt, 2002; Strutt and Strutt 2007). Our simplest interpretation of this is that induction at these stages fails to result in the early radial pattern of core pathway polarity being established and hence a failure of hinge contraction to reorient radial to proximodistal. If hinge contraction alone could specify proximodistal polarity in the absence of the earlier radial polarity, then we would not expect to see swirling over much of the proximal wing (where the forces from hinge contraction are strongest, Etournay et al., 2015).

In this manuscript, our earliest de novo experiments begin at 18h APF (de novo 10h), then at 20h APF (de novo 8h) and at 22h APF (de novo 6h). The image in Fig. 5B referred to by Reviewer 1, is of a wing where Fz is induced de novo at 22 h APF. In these wings, as expected, the core proteins localise asymmetrically in stereotypical swirling patterns throughout the wing surface (see Fig. 2M and also Strutt and Strutt, 2002; Strutt and Strutt 2007), but – usefully for our experiments – they broadly localise along the proximal-distal axis in the region analysed in Fig. 5B. Given the strong swirling in surrounding regions when inducing at >20h APF, we feel reasonably confident in assuming that the pattern is not due to a proximodistal cue present in the proximal wing. We appreciate that the original manuscript did not show images including the trichome pattern in adjacent regions, so this point would not have been clear, but we now include these in Supplementary Fig.S5. We have also added a note in the legend to Fig. 5B to clarify that the proximodistal pattern seen is local to this wing region.

Changes to manuscript: Text extended in Introduction and Results to better explain why we believe the de novo conditions that we use most likely result in a polarity pattern that is not significantly influenced by ‘global cues’. Now show zoomed-out images of the surrounding region around the experiment region proximal to the anterior cross-vein region in adult wings, showing that the polarity pattern does not become more proximodistal when induction time is longer, and also that there is not overall proximodistal polarity in proximal regions of the wing, arguing against an unknown proximodistal polarity cue at these stages of development (Fig.S5B-E’’’).

(2) The putative non-local cell scale signal must be more precisely defined (maybe also given a better name). It is not clear to me that one can separate cell-scale from molecular-scale signal.

Local signals can redistribute within a cell (or membrane) so local signals are also cell-scale. Without a clear definition, it is difficult to interpret the results of the gene dosage experiments. The link between gene dosage and cell-scale signal is not rigorously stated. Related to this, the concluding statement of the introduction is too cryptic.

We thank the reviewer for raising this, as again a similar comment was made by Reviewer 1, so we are clearly falling short in defining the term. We have now had another attempt in the Introduction.

To more specifically answer the point made by the reviewer regarding molecular vs cellular, we are essentially being guided here by the prior computational modelling work, as at the biological level the details are still being worked out. A specific class of previous models only allowed ‘signals’ between core proteins to act ‘locally’, meaning within a cell junction, and within the models there was no explicit mechanism by which proteins on other junctions could ‘detect’ the polarity of a neighbouring junction (e.g. Amonlirdviman et al., 2005; Le Garrec et al., 2006; Fischer et al., 2013). Other models implicitly or explicitly encode a mechanism by which cell junctions can be influenced by the polarity of other junctions (e.g. Meinhardt, 2007; Burak and Shraiman, 2009; Abley et al., 2013; Shadkhoo and Mani, 2019), for instance by diffusion of a factor produced by localisation of particular planar polarity proteins.

We agree with the reviewer that a cell-scale signal will depend on ‘molecules’ and thus could be called ‘molecular-scale’, but here by ‘molecular-scale’ we mean signals that at the range of the sizes of molecules i.e. nanometers, rather than cell-scale signals that act at the size of cells i.e. micrometers. A caveat to our definition is that we implicitly include interactions that occur locally on cell junctions (<1 µm range) within ‘molecular-scale’, but this is a shorter range than ‘cellular-scale’ which requires signals acting over the diameter of a cell (3-5 µm). Nevertheless, we think the concept of ‘molecular-scale’ vs ‘cell-scale’ is a helpful one in this context, and have attempted to address the issue through a more careful definition of the terms.

Changes to manuscript: Text revised in Introduction and legend to Fig.1 to more carefully define ‘cell-scale signalling’ and to distinguish it from ‘molecular-scale signalling’. Final sentence of Introduction also altered so we no longer cryptically speculate on the nature of the cell-scale signal but leave this to the Discussion.

Minor comments. 

Some of the (clever) genetic manipulation may need more details in the text. For example:

- Need to specify if the hs-flp approach induces expression throughout the tissue.

We apologise for the lack of clarity. In all the experiments, the hs-FLP transgene is present in all cells, and heat-shock results in ubiquitous expression. 

Changes to manuscript: We have clarified this in the Results and Materials and Methods.

- Need to specify in the text that in the unpolarised condition the tissue is both dsh and fz mutant.

The reviewer is of course correct and we have updated this point in the text. The full genotype for the unpolarised condition is: w dsh¹ hsFLP22/y;; Act>>fz-mKate2sfGFP, fz^P21/fz^P21 (see Table S1). So this line is mutant for dsh and fz with induced expression of Fz-mKate2sfGFP. 

Changes to manuscript: We have clarified this in the relevant part of the Results.

- Need to specify in the text that the experiment illustrated in Fig 5 is with hh-gal4. 

As noted by the reviewer, we continued to use the same hh-GAL4 repolarisation paradigm as in Fig.4 and this info was in the legend to Fig.5 legend. However, we agree it is helpful to be explicit about this in the main text.

Changes to manuscript: We have added this to this section of the Results.

- Need to address a possible shortcoming of the hh experiment, that the AP boundary is a region of high tension.

It is true that the AP boundary is under high tension in the wing disc (e.g. Landsberg et al., 2009). But we are not aware of any evidence that this higher tension persists into the pupal wing. In separate studies we have labelled for Myosin II in pupal wings (Trinidad et al 2025 Curr Biol; Tan & Strutt 2025 Nature Comms), and as far as we have noticed have not seen preferentially higher levels on the AP boundary. We think if tension were higher, the cell boundaries would appear straighter than in surrounding cells (as seen in the wing disc) and this is not evident in our images.

- Need to dispel the possibility that there is no residual polarisation (e.g. of other components) in fz1 mutant (I assume this is the case).

We use the null allele fz[P21] through this work, and we and others have consistently reported a complete loss of polarisation of other core proteins or downstream components in this background. The caveat to this is that core proteins that persist at cell junctions always appear at least slightly punctate in mutant backgrounds for other core proteins, and so any automated detection algorithm will always find evidence of individual cell polarity above a baseline level of uniform distribution. Hence we tend to use lack of local coordination of polarity (variance of cell polarity angle) as an additional measure of loss of polarisation, in addition to direct measures of average cell polarity. (We discuss this in the QuantifyPolarity manuscript Tan et al 2021 e.g. Fig.S6).

Changes to manuscript: We now include in the Materials and Methods section ‘Fly genetics…’ a much more extensive explanation of the evidence for specific mutant alleles being ‘null’ for planar polarity function (including dsh1 as raised by Reviewer 1), specifically that they result in no detectable planar polarisation of either other core proteins or downstream effectors, and added appropriate references.

- Need to provide evidence that 50% gene dosage commensurately affect protein level. 

This is a good suggestion. In the case of Stbm, we have already published a western blot showing that a reduction in gene dosage results in reduced protein levels (Strutt et al 2016, Fig.S6). We have now performed western blots to quantify protein levels upon reduction of fmi, pk and dgo levels (we actually used EGFP-dgo for the latter, as we don’t have antibodies that can detect endogenous Dgo on western blots).

Changes to manuscript: When presenting the dosage reduction experiments, we now refer back to Strutt et al., 2016 explicitly for Stbm, and have added western blot data for Fmi, Pk and EGFPDgo in new Fig.S2.

- I am surprised that the relationship with microtubule polarity was never investigated. Is this true? 

We agree this is a point that needed further clarification, as Reviewer 1 made a related point regarding the two possible roles for microtubules, one being as a mediator of a global cue upstream of the core pathway, and the second (which we investigate in this manuscript) as a mediator of a cell-scale signal downstream of the core pathway.

Both the Uemura and Axelrod groups have published on potential upstream function as a global cue mediator in the Drosophila wing (e.g. Shimada et al., 2006; Harumoto et al., 2010; Matis et al., 2014).

Both groups have also looked out whether core pathway components could affect orientation of microtubules (Harumoto et al., 2010; Olofsson at al., 2014; Sharp and Axelrod 2016). Notably Harumoto et al., 2010 observed that in 24h APF wings, loss of Fz or Stbm did not alter microtubule polarity from a proximodistal orientation consistent with the microtubules aligning along the long cell axis in the absence of other cues. However, this did not rule out an instructive effect of Fz or Stbm on microtubule polarity during core pathway cell-scale signalling. The Axelrod lab manuscripts saw interesting effects of Pk protein isoforms on microtubule polarity, albeit not throughout the entire wing, which hinted at a potential role in cell-scale signalling. Taken together this prior work was the motivation for our directed experiments to specifically test whether the core pathway might generate cell-scale polarity by instructing microtubule polarity.

Changes to manuscript: We have revised the Results section ‘Microtubules do not…’ to make a clearer distinction regarding possible ‘upstream’ and ‘downstream’ roles of microtubules in Drosophila core pathway planar polarity and the motivation for our experiments investigating the latter.

- The authors suggest that polarity does not propagate as a wave. And yet the range measured in adult is longer than in the pupal wing. Explain. 

Again an excellent point, also made by Reviewer 1, which we have now addressed explicitly in the manuscript. For the convenience of this reviewer, we lay out the reasons why we think the propagation of polarity seen in the adult is further than seen for core protein localisation.

There are three reasons why we might expect adult trichomes to show a different effect from the measured core protein polarity pattern seen in our experiments:

(i) we are assaying core protein polarity at 28h APF, but trichomes emerge at >32h APF, so there is still time for polarity to propagate a bit further from the boundary. We now have added data showing that by the point of trichome initiation, the wave of polarisation extends 3-4 cell rows (Fig.S5A).  

(ii) it has long been known that a strong localisation of core proteins at a cell edge is not required for polarisation of trichome polarity from a boundary. For instance, in Strutt & Strutt 2007 we show clones of cells overexpressing Fz causing propagation through pk[pk-sple] mutant tissue where there is no detectable core protein polarity. We were following up prior observations of Adler et al 2000 in the wing and Lawrence et al 2004 in the abdomen.

(iii) there is evidence to suggest that the polarity of adult trichomes is locally coupled, possibly mechanically. This point is hard to prove without live imaging taking in both initial core protein localisation, the site of actin-rich trichome initiation and then the final orientation of the much larger microtubule filled trichome, and we’re not aware that such data exist. However, Wong & Adler 1993 (JCB) showed that over a number of hours trichomes become much larger and move towards the centre of the cell, presumably becoming decoupled from any core protein cue. The images in Guild … & Tilney, 2005 (MBoC)  are also interesting to look at in this regard. Finally, septate junction proteins have been implicated in local alignment of trichomes, independently of the core pathway (Venema … & Auld, 2004 Dev Biol).

Changes to manuscript: Added new data in Fig.S5A showing where trichomes initiate under 6h de novo induction conditions, for comparison to core protein localisation and adult trichome data in Fig.5. Added some text explaining why adult trichome repolarisation might be stronger than the observed effects on core protein localisation in Discussion. 

- The discussion states that the cell-intrinsic system remains to be fully characterised, implying that it has been partially characterised. What do we know about it? 

As the reviewer probably realises, we were attempting to side-step a long speculative discussion about the various hints and ideas in the literature by grouping them under the umbrella of ‘remaining to be fully characterised’. We would argue that this current manuscript is the first to attempt to systematically investigate the nature of ‘cell-scale signalling’. The lack of prior work is probably due to two factors (i) pioneering theoretical work showed that a sufficiently strong global signal coupled with ‘local’ (i.e. confined to one cell junction) protein interactions was sufficient to polarise cells without the need to invoke the existence of a cell-scale signal; (ii) there is no easy way to identify cell-scale signals as their loss results in loss of polarity which will also occur if other (i.e. more locally acting) core pathway functions are compromised.

The main investigation of the potential for cell-scale signalling has been another set of theory studies (Burak and Shraiman 2009; Abley et al., 2013; Shadkhoo and Mani 2019) which have considered the possibility of diffusible signals. In our present work we have further considered the possibility of a ‘depletion’ model, based on the pioneering theory work of Hans Meinhardt, and as discussed above the possibility that microtubules could mediate a cell-scale signal.

Changes to manuscript: We have revised the Discussion to hopefully be clearer about the current state of knowledge.

Reviewer #3:

[…] Major comments

The data are clearly presented and the manuscript is well written. The conclusions are well supported by the data. 

(1) The authors use a system to de novo establish PCP, which has the advantage of excluding global cues orienting PCP and thus to focus on the cell-intrinsic mechanisms. At the same time, the system has the limitation that it is unclear to what extent de novo PCP establishment reflects 'normal' cell scale PCP establishment, in particular because the Gal4/UAS expression system that is used to induce Fz expression will likely result in much higher Fz levels compared with the endogenous levels. The authors should briefly discuss this limitation. 

We apologise if this wasn’t clear. We only used GAL4/UAS overexpression when we were generating an artificial boundary of Fz expression with hh-GAL4 to induce repolarisation. The de novo induction system involves Fz::mKate2-sfGFP being expressed directly under an Act5C promoter without use of GAL4/UAS. In response to a comment from Reviewer 1 we have now carried out western blot analysis which shows that Fz::mKate2-sfGFP levels under Act5C are actually lower than endogenous Fz levels. As we achieve normal levels of polarity, similar to what we measure in wild-type conditions when measured using QuantifyPolarity, we assume that therefore Fz levels are not limiting under these conditions. However, we note that lower than normal levels of Fz might sensitise the system to perturbation, which in fact would be advantageous in our study, as it might for instance have been expected to more readily reveal dosage sensitivity of other components.

Changes to manuscript: We now describe the levels of expression achieved using the de novo induction system (Fig.S1C-D) and discuss possible consequences in the relevant Results sections and Discussion.

(2) Fig. 3. The authors use heterozygous mutant backgrounds to test the robustness of de novo PCP establishment towards (partial) depletion in core PCP proteins. The authors conclude that de novo polarization is 'extremely robust to variation in protein level'. Since the authors (presumably) lowered protein levels by 50%, this conclusion appears to be somewhat overstated. The authors should tune down their conclusion. 

Reviewer 1 makes a similar point about whether we can argue that the lack of sensitivity to a 50% reduction in protein levels actually rules out the depletion model. To address the comments of both reviewers we had now added some further narrative and caveats in the text.

We nevertheless believe that the experiments shown effectively make the point that there is no strong dosage sensitivity – and it remains our contention that if protein levels were the key to setting up cell-scale polarity, then a 50% reduction would be expected to show an effect on the rate of polarisation. We further note that as Fz::mKate2-sfGFP levels are lower than endogenous Fz levels, the system might be expected to be sensitised to further dosage reductions, and despite this we fail to see an effect on rate of polarisation.

In a similar vein, Reviewer 2 requested data on whether dosage reduction altered protein levels by the expected amount. We have now added further explanation/references and western blot data to address this.

Changes to manuscript: Added some narrative and caveats regarding whether lowering levels more than 50% would add to our findings in the Discussion. Revised conclusions to be more cautious including altering section title to read ‘Planar polarity establishment is not highly sensitive to variation in protein levels of core complex components.

Also added westerns and text/references showing that for the tested proteins there is a reduction in protein levels upon removal of one gene dosage in Results section ‘Planar polarity establishment is…’ and Fig.S2.

Minor comments :

(1) Page 3. The authors mention and reference that they used the PCA method to quantify cell polarity magnification and magnitude. It would help the unfamiliar reader, if the authors would briefly describe the principle of this method. 

Changes to manuscript: More details have been added in Materials & Methods.

Significance:

The manuscript contributes to our understanding of how planar cell polarity is established. It extends previous work by the authors (Strutt and Strutt, 2002,2007) that already showed that induction of core PCP pathway activity by itself is sufficient to induce de novo PCP. This manuscript further explores the underlying mechanisms. The authors test whether de novo PCP establishment depends on an 'inhibitory signal', as previously postulated (Meinhardt, 2007), but do not find evidence. They also test whether core PCP proteins help to orient microtubules (which could enhance cell intrinsic polarization of core PCP proteins), but, again, do not find evidence, corroborating previous work (Harumoto et al, 2010). The most significant finding of this manuscript, perhaps, is the observation that local de novo PCP establishment does not propagate far through the tissue. A limitation of the study is that the mechanisms establishing intrinsic cell scale polarity remain unknown. The work will likely be of interest to specialists in the field of PCP.

For the smooth comparison (i.e., for comparing the effects of time) this is a bit more tricky. Model comparison is a common option, but the problem is that this requires fitting models with ML rather than fREML, and then it is not possible to model autocorrelation any longer).

The other option is ordered factors. Camera would be coded as an ordered factor and the smooth needs to be "duplicated" (so to speak) in order to have a reference smooth and a difference smooth. HOWEVER, this can produce different results (and different p-values) depending on which level is chosen as the reference. You could arguably use the lower-quality camera as the reference, because you'd like to estimate that level as well as you can (and estimating via a difference is more uncertain). More conservatively, you could do it both ways and only take a p-value as significant when it is significant in both models.

The fact that the ordered factor approach can lead to different results is why I prefer to conduct onset detection from a model with unordered factors (i.e., to estimate a separate smooth in each level).

Your hypotheses don't currently refer directly to the analysis of timecourse differences (via difference smooths), but maybe this could be added? i.e., that you expect a different timecourse in the two camera conditions, as revealed by different time smooths. Then the onset estimation would be a particular aspect of that timecourse.

Here I would set it up in a different way. I've seen "this way" before (IIRC, Dan Mirman has models like this in his book). However, the issue with this model is the mutual exclusivity between responses (because looks to the cohort cant be looks to unrelated, and vice-versa). One could say that this kind of perfect correlation then gets captured by the model, but even so, this would only work if certain random effects are included. Worse, the sample size ends up not being right either because the model sees 2 counts for every binomial occurrence ( based on one 1 and one 0 for every "real" observation).

Instead, I treat looks to one or the other object in the display as part of the dependent variable (conceptually, this also seems more correct, because objects aren't quite manipulations, but part of the outcome). So the model will have a DV like cbind(fix_cohort, fix_unrelated), which means that only the webcam condition predictor is necessary, with no interaction. I.e., there'll be 2 smooths, one for each camera condition, and each smooth reflects cohort-versus-unrelated proportions. The "onsets" are the earliest points at which a cohort-versus-unrelated preference is detected--which is hypothesised to be earlier in one webcam condition than the other.

(note that in the example dataset from Ito et al. in our preprint, we do have cohort and unrelated as levels of a condition predictor; but that's because that dataset was a target-absent design with different displays in each condition)

here maybe cite the van Rij paper (though it's on pupillometry, but all the same)

in our previous work we added two steps about here: (1) binarization, i.e., within each time bin, only one data point in each trial is counted. The main reason for this was not to overstate the data, which would happen when counting multiple samples within each bin. Naturally, this intersects with "bin size" (as i mentioned above, we've used pretty small bins, but then tried to prevent the overstating of evidence by modelling autocorrelation). When you say "resampled into 100-ms bins", I suppose this means this "binarization" also takes place (as part of downsampling)? Still, the fact that you end up with 1 count within each participant x trial x bin combination could be made explicit. (2) aggregation. Given that we ended up with 1 count per participant x trial x bin, we then summed counts across trials (i.e., items) in each participant x bin combination. The main purpose of this was again to avoid large autocorrelation values, which in by-trial data could be as high as .99. Another advantage is that we end up with smaller (more parsimonious) models without item random effects (though this could also be seen as a disadvantage). Still another advantage is that both the model fitting and the onset detection are extremely fast.

in our previous work, we've used 20ms attempting to get a compromise between temporal resolution while not overstating the evidence (essentially, the combination of bin size, binarization within each bin, subject aggregation, and modelling of autocorrelation all matter in some way for this balance). I imagine that in the low-quality condition, the sampling rate will not be enough for something like that, but is something like 50ms possible?

There's a question here about whether to "chop off" a bit of time from the analysis (100ms if starting at word onset, or 300ms if also accounting for saccade planning/execution), or instead, modelling the whole curve in the GAM including that initial part. Given that the images are previewed, I'd probably fit the whole curve (or chop off 100ms). On top of that, the onset detection method also allows defining a window, which can be smaller than the time window in the GAM. But we probably dont need to touch that.

Here there is an explicit reference to noise but then effect sizes and timing are mentioned as the result, perhaps as a consequence of the reduced noise? This is not super clear to me. My immediate feeling is that they're separate notions and I'm not sure that reduced noise would lead to all these effects.

On the other hand: (1) In the proportions/binomial case, I suppose more variation could be linked to lower proportions of looks. E,g,, maximum variation between trials/participants would be a mixture of 0% and 100% of looks, so 50% overall, and minimum variation could be that oinly a certain image was looked at, which would correspond to 100% off looks (although it could also be 0% of looks, that would also be minimum variation) ... but I dont know if you had something like this in mind?

(2) Our onset detection method can be sensitive to amount of noise and indeed might end up detecting less noisy effects as being earlier. This is both good and bad. I discuss this ore below.

Still, despite these points, it seems to me that the link between noise and the other aspects needs to be made more explicitly.

"Effect size" is a bit of a tricky term because it can refer to both standardized or unstandardized effects, i.e., taking into account the noise/uncertainty/variation or not. In this case, I think you mean, e.g., proportion of looks, irrespective of noise, but maybe that can be made more explicit.

It makes me think about how teachers might overestimate abilities in certain areas. Recognizing both strengths and gaps is important for effective teaching.

Reviewer #1 (Public Review):

In this study, Li et al. aim to determine the effect of navigational experience on visual representations of scenes. Participants first learn to navigate within simple virtual environments where navigation is either unrestricted or restricted by an invisible wall. Environments are matched in terms of their spatial layout and instead differ primarily in terms of their background visual features. In a later same/different task, participants are slower to distinguish between pairs of scenes taken from the same navigation condition (i.e. both restricted or both unrestricted) than different navigation conditions. Neural response patterns in the PPA also discriminate between scenes from different navigation conditions. These results suggest that navigational experience influences perceptual representations of scenes. This is an interesting study, and the results and conclusions are clearly explained and easy to follow. There are a few points that I think would benefit from further consideration or elaboration from the authors, which I detail below.

First, I am a little sceptical of the extent to which the tasks are able to measure navigational or perceptual experience with the scenes. The training procedure seems like it wouldn't require obtaining substantial navigational experience as the environments are all relatively simple and only require participants to follow basic paths, rather than encouraging more active exploration of a more complex environment. Furthermore, in the same/different task, all images show the same view of the environment (meaning they show the exact same image in the "same environment" condition). The task is therefore really a simple image-matching task and doesn't require participants to meaningfully extract the perceptual or spatial features of the scenes. An alternative would have been to present different views of the scenes, which would have prevented the use of image-matching and encouraged further engagement with the scenes themselves. Ultimately, the authors do still find a response time difference between the navigation conditions, but the effect does appear quite small. I wonder if the design choices could be obscuring larger effects, which might have been better evident if the navigational and perceptual tasks had encouraged greater encoding of the spatial and perceptual features of the environment. I think it would be helpful for the authors to explain their reasons for not employing such designs, or to at least give some consideration to alternative designs.

Figure 1B illustrates that the non-navigable condition includes a more complicated environment than the navigable condition, and requires following a longer path with more turns in it. I guess this is a necessary consequence of the experiment design, as the non-navigable condition requires participants to turn around and find an alternative route. Still, this does introduce spatial and perceptual differences between the two navigation conditions, which could be a confounding factor. What do the response times for the "matched" condition in the same/different task look like if they are broken down by the navigable and non-navigable environments? If there is a substantial difference between them, it could be that this is driving the difference between the matched and mismatched conditions, rather than the matching/mismatching experience itself.

In both experiments, the authors determined their sample sizes via a priori power analyses. This is good, but a bit more detail on these analyses would be helpful. How were the effect sizes estimated? The authors say it was based on other studies with similar methodologies - does this mean the effect sizes were obtained from a literature search? If so, it would be good to give some details of the studies included in this search, and how the effect size was obtained from these (e.g., it is generally recommended to take a lower bound over studies). Or is the effect size based on standard guidelines (e.g., Cohen's d ≈ 0.5 is a medium effect size)? If so, why are the effect sizes different for the two studies?

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__Reviewer #1 __

Major comments

1. The manuscript posits that the loss of function of MASh components (Ogc1 and Aralar) decreases adrenergic-stimulated lipolysis by altering the cytosolic NAD⁺/NADH ratio, with AMPK/ACC mentioned as possible mediators. However, this remains speculative. Please provide mechanistic data directly linking MASh-dependent NAD⁺/NADH changes to the regulation of lipolysis in brown adipocytes during adrenergic stimulation. Answer 1) The reviewer raises an important point regarding the direct assessment of cytosolic NAD⁺/NADH redox changes as a mechanistic link for altered lipolysis in brown adipocytes lacking MASh components. To address this point, we added new data to the revised manuscript showing lactate/pyruvate ratio as measured by metabolomics. This is a well-established surrogate marker to monitor changes in redox balance. Notably, under basal (non-stimulated) conditions, the lactate/pyruvate ratio did not display any significant differences between Aralar 1 KD and control cells, suggesting preservation of cytosolic NAD⁺/NADH levels in the absence of functional MASh under these conditions. This finding is consistent with reports showing the robustness of NAD⁺ regeneration via multiple shuttles and the possibility of metabolic compensation when one shuttle is compromised (PMID: 40540398; PMID: 37647199).

The results have been added as new supplementary Figure 1 as following:

Our new metabolomics data also revealed substantial reductions in the aspartate/glutamate ratio in Aralar 1 knockdown cells, serving as a metabolomic signature of impaired MASh function and reduced exchange of these amino acids between the cytosol and mitochondria. Given that the MASh is a major mechanism for exporting cytosolic reducing equivalents into the mitochondria under high metabolic demand, its loss would be expected to impact redox homeostasis, particularly under adrenergic stimulation when glycolytic flux and lipolytic activity are elevated (PMID: 40540398).

Importantly, although our redox surrogate marker did not detect alterations, this may be explained by activation of compensatory pathways, most notably the glycerol phosphate shuttle (GPSh), which is highly expressed and active in brown adipocytes. Indirect support for this compensation comes from data shown in figure 4I showing reduced glycerol release in Aralar 1 KD cells upon norepinephrine stimulation and blocked lipolysis. This suggests a redirection of glycolytically derived G3P away from release and toward enhanced cycling within the GPSh, supporting cytosolic NAD⁺ regeneration via mitochondrial FAD-dependent G3PDH and cytosolic NAD⁺-dependent G3PDH activity. This is consistent with studies documenting that the combined action of MASh and GPSh maintains NAD redox homeostasis in brown adipocytes especially during non-thermogenic conditions (PMID: 168075; PMID: 40540398; PMID: 37647199). We have included a discussion about this possibility at page 9, third paragraph as follows:

*“Previous studies have shown that BAT exhibits high activity of mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (mG3PDH), which functions as an electron sink to sustain low cytosolic NADH levels essential for continuous glycolytic flux [11]. Accordingly, suppression of the MASh, either genetically or pharmacologically, is likely to induce a compensatory upregulation of the GPSh. This adaptation would enhance G3P turnover, contributing to the maintenance of cytosolic NAD redox balance. Moreover, the increased flux through the GPSh could favor fatty acid esterification and triglyceride synthesis or re-esterification, consistent with our findings in Ogc and/or Aralar 1 KD cells, where (i) triglyceride content rises (Fig. 3), (ii) overall respiratory rates remain largely unaltered (Figs. 2D–G), and (iii) glycerol release declines significantly (Fig. 4I). Notably, the decrease in glycerol release persists even when lipolysis is blocked by ATGlistatin, suggesting that the available G3P pool is rerouted from dephosphorylation and extracellular release toward oxidation to DHAP by mG3PDH to regenerate cytosolic NAD+ under MASh-deficient conditions. We propose that interference with the MASh does not directly impact lipolysis but instead alters the cellular balance between DHAP and G3P owing to enhanced activity of the GPSh. This metabolic shift would favor the esterification of G3P with free fatty acids, thereby promoting triglyceride synthesis. These results support the notion that, even during adrenergic stimulation—when long-chain unsaturated fatty acids and their CoA esters strongly inhibit mG3PDH activity [11]—the residual flux through the glycerophosphate shuttle remains critical for sustaining cytosolic NAD redox equilibrium [11,19,32].” *

• *

At the mechanistic level, adrenergic stimulation in brown adipocytes activates robust lipolysis and thermogenic gene programs, generating high NADH that must be efficiently reoxidized to sustain flux through glycolysis and lipolysis-linked pathways. Our findings are consistent with a model in which the loss of MASh does not prevent cytosolic NAD⁺ regeneration or lipolytic flux during acute adrenergic stimulation, due to compensatory upregulation of the GPSh, as suggested by the glycerol release changes. Thus, while MASh normally acts as a conduit for NADH export and aspartate/glutamate exchange, in its absence, the GPSh maintains cytosolic redox balance, thereby sustaining glycolytic and lipolytic capacity.

We agree that future studies should employ direct measurements of cytosolic NAD⁺/NADH ratios (e.g., genetically-encoded redox sensors) during adrenergic stimulation and specific pharmacological inhibition of both shuttles to dissect these relationships in greater detail. We sincerely appreciate the reviewer's input, which has prompted us to clarify the indirect but robust evidence supporting a role for compensatory redox shuttle activity in preserving brown adipocyte lipolysis in the setting of MASh impairment.

We have further added a new paragraph in the discussion section (page 10)::

*“Mechanistically, the connection between the MASh and lipolysis appears to involve regulation of the cytosolic NAD⁺/NADH redox balance. MASh activity facilitates the regeneration of NAD⁺ from NADH in the cytosol, primarily through the reduction of oxaloacetate to malate by cytosolic malate dehydrogenase (Fig. 1G-H). Despite the theoretical expectation that reductions in MASh activity would disturb redox homeostasis, our metabolomic data show that the lactate/pyruvate ratio remains unchanged under conditions of MASh impairment, indicating that the overall cytosolic NAD⁺/NADH ratio is maintained (Figure S1A-C). While direct measurements of cytosolic NAD⁺/NADH were not performed, the preserved lactate/pyruvate ratio in Aralar 1 KD cells under basal conditions strongly suggests redox stability, likely due to compensatory activity by alternative mitochondrial shuttles or metabolic adaptations that maintain NAD redox homeostasis despite MASh impairment [18,33]. *

Previous evidence indicates that BAT exhibits high activity of mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (G3PDH), which acts as an electron sink to sustain low cytosolic NADH levels critical for glycolysis [34]. In this sense, it is conceivable that genetic or pharmacological suppression of MASh triggers compensatory enhancement of the G3P shuttle, increasing G3P availability and facilitating the maintenance of cytosolic NAD redox balance. This adaptation could also promote fatty acid esterification and triglyceride synthesis or re-esterification, aligning with our observations that in Ogc and/or Aralar 1 KD cells: (i) triglyceride levels increase (Fig. 3); (ii) overall respiratory rates are preserved (Figs. 2D–G); and (iii) glycerol release is significantly reduced (Fig 4I).”

• *

__ The absence of in vivo analysis of lipid-droplet size in MASh loss-of-function models is a major concern. In vitro results could be confounded by differences in differentiation stage between groups. Please document equivalent adipogenesis across groups (e.g., Pparg/Cebpa/Plin1/Fabp4 expression).__

Answer 2) We thank the reviewer for the thoughtful and constructive comment regarding potential confounding by differences in differentiation stage, and for highlighting the importance of documenting equivalence between experimental groups. We appreciate the opportunity to clarify and provide additional assurance on this point.

As detailed in our manuscript, we have performed qPCR analysis of multiple well-established markers of brown adipocyte differentiation, including Ucp1, Elovl3, Prdm16, Pparg, Cebpa, Plin1, and Fabp4, in both scramble, aralar1 KD, and Ogc KD cells (see Fig. S1A and accompanying text). Our results show no apparent effect of these genetic interventions on overall differentiation, as the expression levels of these key markers were consistently unaltered across groups. Furthermore, adenoviral-mediated knockdown of Ogc achieved an approximate 80% reduction in Ogc mRNA (see Fig. S1B), yet most differentiation markers remained unaffected. We did observe significant increases in Atgl, Pgc1α, and Tfam mRNA levels, which may indicate a degree of pathway reprogramming without affecting the general differentiation profile. We propose that interference with the MASh does not directly impact lipolysis but instead alters the cellular balance between DHAP and G3P owing to enhanced activity of the GPSh. This metabolic shift would favor the esterification of G3P with free fatty acids, thereby promoting triglyceride synthesis.

Additional experimental support for equivalent differentiation can be drawn from our respirometry data presented in Figures 2E and 2G. These figures demonstrate that respiratory rates upon norepinephrine stimulation, which is a sensitive indicator of brown adipocyte thermogenic capacity, were essentially identical in scramble, aralar1 KD, and Ogc KD cells. Since norepinephrine-stimulated respiration requires both functional mitochondria and the full differentiation of brown adipocytes, these results strongly support the conclusion that silencing either MASh component does not impair the fundamental ability of cells to undergo brown adipocyte differentiation or achieve functional thermogenic competence.

This is consistent with published findings showing that norepinephrine triggers robust respiration and thermogenic activation only in fully differentiated and functional brown adipocytes, making such measurements a widely accepted proxy for differentiation status and mitochondrial integrity. Thus, the equivalent respiratory responses observed in all groups further validate that differentiation was not compromised by the genetic interventions.

We hope this clarifies that equivalent adipogenesis was carefully documented and that any observed phenotypes are unlikely to be attributable to differences in differentiation stages. Thank you again for your rigorous assessment and for helping to ensure the robustness of our study.

__ Please include rescue experiments (add-back OGC1 and Aralar) to rule out siRNA/shRNA off-target effects and verify that the phenotype stems from MASh loss of function.__

Answer 3) We thank the reviewer for this important suggestion regarding the inclusion of rescue experiments with add-back of Ogc and Aralar to definitively exclude off-target effects of the siRNA/shRNA-mediated knockdowns.

We would like to kindly point out that although we did not perform add-back rescue experiments directly, the consistency of phenotypes observed across two independent genetic interventions—aralar 1 KD and Ogc KD—strongly argues against off-target effects being responsible for the observed metabolic and functional alterations. Specifically, both knockdowns yielded remarkably similar phenotypes in multiple assays, including respirometry analyses, mitochondrial morphology, lipid droplet homeostasis, and lipid metabolism, supporting the conclusion that these effects stem from MASh loss of function rather than nonspecific silencing.

Furthermore, our new supplementary data (new Supplementary Figure 1A-F) reveals a significant reduction in the aspartate/glutamate ratio in Aralar 1 KD cells, a compelling functional readout for MASh impairment. This molecular evidence corroborates that our genetic interventions effectively disrupted MASh activity as intended.

We sincerely appreciate the reviewer’s thorough evaluation and understand the importance of rescue experiments. While recognizing their value, we believe the convergent genetic, metabolic, and functional evidence presented across two different MASh components provides strong and consistent support that the phenotypes observed are due to specific loss of MASh function.

__ Please expand on physiological significance: What is the importance of MASh regulation of BAT lipolysis in long-term adaptive thermogenesis?__

Answer 4) This is a very interesting aspect, and we have included a new paragraph in the discussion section (page 14) to address it as follows:

“Our results, supported by recent literature, strongly indicate that the malate–aspartate shuttle (MASh) plays a key role in facilitating fatty acid–dependent thermogenesis in brown adipocytes. Specifically, BAT-targeted overexpression of GOT1 has been shown to enhance β-oxidation and support acute cold-induced thermogenesis (PMID: 40540398). Interestingly, genetic ablation of GOT1—and thus MASh inhibition—preserves cold-induced thermogenesis by promoting a metabolic shift from fatty acid to glucose oxidation. Our findings corroborate and extend these observations by demonstrating that MASh impairment sustains overall respiratory activity in norepinephrine-stimulated brown adipocytes (Figures 2D–2G), while concurrently impairing lipolysis and resulting in an accumulation of small lipid droplets (Figures 3 and 4). Collectively, these data suggest that MASh not only modulates substrate preference towards fatty acid oxidation but also facilitates lipolysis, an essential upstream step that enables lipid oxidation and supports thermogenic heat production.”

Minor comments

1. __ Fig. 4 legend/title contains a typo ("lypolysis" → lipolysis).__ Answer 1) Corrected

__ In Fig. 2 legend line: "Adevirus-mediated" → Adenovirus-mediated; "OCAR" → OCR.__

Answer 2) Corrected

__ For lipolysis imaging, you already show Forskolin/Atglistatin/Etomoxir controls; add a vehicle-only time course overlay in the main figure (currently in text/legend) to aid visual comparison.__

Answer 3) We thank the reviewer for pointing this out. To improve clarity, we have updated the labeling in Figures 3 and 4: “basal” now clearly refers to the unstimulated/untreated condition, and the previously labeled “UT” condition has been clarified as “untransduced.” These changes make the figure legends and data presentation more consistent and easier to interpret.

__ Ensure consistent gene symbols (Atgl/Pnpla2), and protein capitalization.__

Answer 4) Corrected.

__Reviewer #2 __

Major points:

1. __ In the current manuscript, mitochondrial morphology (area, aspect ratio, and roundness) was analyzed in OGC1 KD cells using TMRE, whereas MitoTracker Deep Red (MTDR) was used in Aralar1 KD cells. Notably, TMRE is a ΔΨm-dependent probe. The signal intensity can change, or the distribution may reflect alterations in membrane potential rather than true morphological changes. Therefore, the observed differences in OGC1 KD cells based on TMRE staining may be confounded by the dye's functional dependence, potentially biasing the conclusions. It is recommended to evaluate mitochondrial morphology with consistent trackers across conditions. In addition, in the subsequent OCR analysis, mitochondrial area was used for normalization. Please clarify which staining method was employed, and provide justification for its suitability.__ Answer 1) We thank the reviewer for this insightful comment. Indeed, TMRE is a membrane potential-sensitive dye and could therefore potentially affect measurements of mitochondria.

We would like to point out that mitochondrial morphology was quantified based on mitochondrial area rather than fluorescence intensity. To create an accurate binary map of mitochondria, we used a low threshold, which allowed us to include even weakly stained mitochondria and thereby detect them independently of their membrane potential. In all imaged cells, TMRE signal was sufficient to reliably identify mitochondrial pixels. Moreover, these images were acquired using a confocal microscope, where the risk of pixel expansion due to higher fluorescence intensity is minimized. Lastly, given that overall mitochondrial oxygen consumption in these cells remains largely intact, we do not expect a substantial loss of membrane potential, although minor effects cannot be entirely excluded.

We opted to use TMRE for imaging Ogc KD cells because the scramble control for these shRNA viruses carries an mKate fluorescent tag, which overlaps with the MTDR signal. Since accurate assessment of transduction efficiency relied on detecting mKate, MTDR could not be used in these experiments. Importantly, we only compare mitochondrial morphology within the same staining condition and do not draw conclusions across cells stained with different dyes.

To ensure transparency, we have added a new section at the discussion (page 17, 2nd paragraph) highlighting the potential influence of ΔΨm-dependent dyes on morphological measurements as follows:

“It is also important to note that mitochondrial morphology was quantified using MTDR in Aralar 1 KD cells and TMRE in Ogc KD cells due to experimental constraints (see Methods). TMRE is a membrane potential–dependent dye, which could potentially influence morphology measurements. To minimize this risk, we used confocal microscopy, which reduces the likelihood of pixel expansion due to higher fluorescence intensity, and set thresholds to detect even weakly stained mitochondria. Nonetheless, we cannot fully exclude the possibility that the differences in morphology observed between Aralar 1 and Ogc KD are influenced by the use of different dyes; however, statistical comparisons were never performed across samples stained with different dyes.”

Also, we have expanded the Methods section (page 22, 2nd paragraph) to include a rationale for using these dyes and describe the analysis protocol as following:

“TMRE was used for Ogc KD cells because the scramble control for the shRNA viruses carries an mKate fluorescent tag, which overlaps with MTDR fluorescence, preventing its use. MTDR was used for Aralar KD cells. Image Analysis was performed in FIJI (ImageJ, NIH). For the quantification of mitochondrial morphology and area, images stained with TMRE or MTDR were analyzed. Thresholds were adjusted to ensure that even weakly stained mitochondria were detected and included in the analysis. Only the mitochondrial area was evaluated, independent of fluorescence intensity.”

Minor points:

1. __ In the introduction, the authors state that "LDH activity increases in the context of BAT activation". This point is important for the logic of the manuscript, reference [10] cited here is not sufficient to support this claim. It is recommended to provide appropriate references to support this statement.__ Answer 1) We have substantially changed this paragraph in the revised manuscript to better explain why LDH would not act as a major player in contributing to NAD redox balance in the context of BAT thermogenesis, as follows:

“In mammalian cells, cytosolic NAD⁺ is regenerated through lactate dehydrogenase (LDH), the glycerol-3-phosphate shuttle (GPSh), or the malate-aspartate shuttle (MASh). In BAT, however, lactate production rises only slightly with adrenergic activation and most lactate is oxidized via the TCA cycle, suggesting that LDH primarily consumes NAD⁺ rather than regenerating it [PMID: 30456392; PMID: 37337122; PMID: 30456392; PMID: 37802078; PMID: 40982723]. Consequently, mitochondrial redox shuttles become critical for sustaining cytosolic NAD⁺ supply”.

We have also provided additional references to support this new section at the introduction.

__ In Fig. 1A and B-D, there are inconsistencies and duplications in the abbreviation labels. Please check and revise accordingly. __

Answer 2) We thank the reviewer for this comment. We would like to clarify that Figure 1A is a schematic overview of the system, while Figures 1B–D show protein expression in specific contexts: whole BAT (B), whole liver (C), and BAT mitochondria (D). In Figures 1B and 1C, all components are shown because both cytosolic (MDH1 and GOT1) and mitochondrial proteins (MDH2, GOT2, Aralar 1 and 2 and OGC) are present. In contrast, Figure 1D shows only mitochondrial components (OGC, Aralar1, MDH2, and GOT2). Although Aralar2 is a mitochondrial protein, it was not detected in this study (Forner et al., 2009). Similarly, cytosolic components such as MDH1 and GOT1 are not shown in Figure 1D because they are absent in the mitochondrial fraction. We have revised the figure legend to make these distinctions clearer.

__ In Fig. S1, the number of n indicated does not match the number of data points shown. Please clarify whether these represent technical replicates or biological replicates, and provide a detailed description of the statistical methods used throughout the manuscript.__

Answer 3) We thank the reviewer for catching this and allowing us to correct our mistakes. In the revised version, we have corrected the figure legend of Supplementary Figure 1 so that the number of n matches the data points shown.

__ Please provide details on the normalization strategy used in the BODIPY-C12/BODIPY-493 staining analysis, such as whether fluorescence intensity was quantified as mean or integrated values, and whether the analysis was normalized to lipid droplet area, cell number, or baseline. Since lipolytic stimulation can reduce droplet size and increase droplet number, these factors may bias the results. __

Answer 4) We thank the reviewer for this important comment and apologize for the lack of detail regarding this analysis. The analysis of BODIPY-C12 and BODIPY-493 was performed by quantifying the mean fluorescence intensity of BODIPY-C12 detected within a mask generated from the BODIPY-493 signal. This approach allowed us to define all lipid droplets and measure the release of previously esterified C12. To account for variability across samples, the data were normalized to each sample’s individual baseline at time point 0 and expressed as fold change relative to this baseline. In the revised manuscript we have included this description in the Methods section (page 18, last paragraph) for clarity and reproducibility, as following:

“Lipid Droplet area was defined based on Bodipy 493/503 signal, which was used to generate a mask identifying all lipid droplets. Within this mask, the mean fluorescence intensity of BODIPY C12 was quantified over time to monitor the release of previously esterified C12. To account for variability between samples, data were normalized to each sample’s individual baseline at time point 0 and expressed as fold change relative to this baseline.”

__ The manuscript notes that the unexpected result in Fig. 3K-M in parallel with increased Atgl mRNA expression might be because it does not reflect protein levels or enzymatic activity. To strengthen this point, it is recommended to include data on ATGL and phosphorylation ATGL. __

Answer 5) We thank the reviewer for this constructive comment. We have clarified these aspects in the revised Results and Discussion sections to reflect this interpretation more accurately as follows:

“Notably, Atgl mRNA measurement in our study was primarily used as a marker of brown adipocyte differentiation, rather than as a direct indicator of ATGL protein abundance or enzymatic activity. We detected increased Atgl expression only in Ogc KD cells (Fig. S1H), but not in Aralar 1 KD cells (Fig. S1G). This likely does not reflect a major difference in differentiation status, as other brown adipocyte markers and norepinephrine-stimulated respiration were comparable between scramble and knockdown cells (Fig. 2D-G and 2N-O and S1G-H). Although lipolysis was not evaluated in Ogc KD cells, in Aralar 1 KD cells basal lipolysis remained unchanged (Fig. 4D-E and 4G-I), whereas norepinephrine-stimulated lipolysis was delayed or partially inhibited. Notably, the enhanced fatty acid esterification observed in Ogc KD cells despite elevated Atgl expression is not contradictory, since in brown adipocytes lipolysis and re-esterification occur concurrently to sustain high lipid turnover [34].

__ Red-on-black is not a great color code for IMFs, how about black-and-white? __

Answer 6) We have changed color text for white on figures 2H and K as suggested.

__Reviewer #3 __

Major points;

1. __ Although in the manuscript Veliova and coworkers demonstrated that MAS is functional in brown adipocytes showing kinetic parameters equivalent to that previously described in other tissues, surprisingly, when its components are downregulated, no effect, or very little, on mitochondrial respiration is found (figure 2). This is an intriguing result since MAS disruption has been widely reported to impair respiration in different cell types and tissues. However, since no direct evidence of MAS dysfunction is provided, it is possible that MAS may still remain partially or fully functional under the conditions used by the authors, and therefore this point needs to be clarified to validate these results.__ Answer 1) We thank the reviewer for the insightful comment and the opportunity to clarify these important points regarding MASh dysfunction validation in our study. We acknowledge the reviewer’s observation that mitochondrial respiration was largely unaffected by MASh component knockdown, which is indeed intriguing. Importantly, as already indicated in our responses to Reviewer 1, we have provided new data showing direct molecular evidence of MASh impairment through substantial reductions in the aspartate/glutamate ratio in Aralar 1 KD cells (new Supplementary Figure S1F). This ratio is a well-established functional readout reflecting MASh activity and amino acid exchange between cytosol and mitochondria, as demonstrated in original experimental studies of MASh function in multiple tissues including brown adipocytes (PMID: 4436323). The reduction in the aspartate/glutamate ratio directly confirms loss of MASh functionality even though respiratory rates remained unchanged, likely due to metabolic compensation by robust glycerol phosphate shuttle (GPSh) activity, as further supported by our data showing reduced glycerol release upon norepinephrine stimulation in Aralar 1 KD cells cells (Figure 4I). This metabolic rerouting maintains cytosolic NAD⁺ regeneration and partially preserves respiration and energy metabolism under these experimental conditions (PMID: 168075; PMID: 40540398; PMID: 37647199). Thus, the combination of metabolomic, respirometry, and functional lipid data strongly indicates that MASh activity was disrupted specifically and effectively by our genetic interventions. This molecular evidence was already signposted in our original manuscript and responses, underscoring that MASh loss of function—and not residual or compensatory MASh activity—is responsible for the phenotypes reported. We greatly appreciate the reviewer’s insightful attention to this critical mechanistic issue and hope this provides clear reassurance that MASh impairment was indeed achieved and functionally validated within our study framework.

Furthermore, strategies used to downregulate MAS components produce only a partial reduction in mRNA levels, about 70 %, but its outcome on protein levels has not been determined. and the remaining protein level could be sufficient to maintain shuttle activity. Therefore, the effect of silencing at protein level should be analyzed, because as authors also point out on page 16; "mRNA levels may not reflect actual protein levels or activity".

Answer 2) We thank the reviewer for this important point. Our knockdowns resulted in ~70–80% reduction in mRNA levels. While not complete, this represents a substantial decrease and is sufficient to produce strong functional effects. At the time the experiments were performed, we did not have access to suitable antibodies, and the available antibodies did not provide reliable signals in our samples, which is why we used qPCR to estimate knockdown efficiency. Importantly, we observed clear phenotypic changes in both knockdowns (Aralar and OGC), and both showed very similar phenotypes. This suggests that the level of knockdown was sufficient to significantly impair MAS activity. In the revised version we added new data which further validated the functional impact of Aralar KD (given that this protein has an alternative isoform, as pointed out by the reviewer). We performed metabolomics experiments measuring aspartate and glutamate levels. Our new data shows that the aspartate-to-glutamate ratio is significantly reduced in Aralar KD cells. This ratio serves as a proxy for glutamate catabolism, and the observed decrease suggests reduced glutamate catabolism, likely due to impaired MAS activity. Therefore, the reduced whole-cell aspartate/glutamate ratio serves as a metabolic signature of MAS impairment, consistent with Aralar KD. These data indicate that Aralar is sufficiently downregulated to produce a functional effect, supporting our conclusion that MAS activity is impaired. The results have been added as new supplementary Figure 1 as follows:

__ In the case of aspartate/glutamate carriers (AGCs) the role of citrin/slc25a13, the second AGC paralog, should also be analyzed. This AGC isoform is discarded based on proteomic data from brown adipose tissue, but, as it is shown in figure 1B, its levels are similar those of Aralar/slc25a12, the only AGC silenced. Besides, primary brown adipocytes differentiated for 7 days are used here, and it is possible that factors such as culture conditions or differentiation itself could alter AGC levels. Therefore, it is necessary to determine the protein levels of citrin/AGC2, and, if necessary, downregulate it together with the Aralar/AGC1 isoform. citrin/AGC2 activity may be responsible for the observed difference between the OGC and Aralar/AGC1 KD adipocytes.__

Answer 3) We thank the reviewer for this important point. We chose Aralar1 because it is the isoform predominantly expressed in brown adipose tissue (PMID: 23436904). We acknowledge, however, that compensatory increases in Citrin/AGC2 upon Aralar1 knockdown are possible. To address this, we have included new metabolomics data in the revised manuscript (added as Supplementary Figure 1), which provides additional support that downregulation of Aralar1, even if not complete, is sufficient to cause a metabolic change reflected by a reduced aspartate/glutamate ratio in these cells. This functional change supports that the knockdown of Aralar1 alone is sufficient to study its role in brown adipocytes, although minor compensation by Citrin/AGC2 cannot be entirely excluded.

To address this explicitly, we have added a paragraph to the discussion (page 13, 2nd paragraph) highlighting the potential for partial compensation by Citrin/AGC2 and explaining why the observed metabolic effects are still attributable to Aralar 1 knockdown, as follows:

“Phenotypes observed in Aralar 1 KD cells closely resemble those in Ogc KD cells, particularly in terms of lipid metabolism alterations and energy expenditure. The main difference lies in mitochondrial morphology, which is altered in Ogc KD cells but remains unchanged in Aralar 1-silenced cells (Fig. 2J,M). Unlike Ogc, which lacks an alternative isoform, Aralar 1 has a paralog Aralar 2 (Citrin, or SLC25A13) that may partially compensate for its loss. This potential compensation might explain the preservation of mitochondrial morphology in Aralar 1 KD cells. Nonetheless, our metabolomics data demonstrate that downregulation of Aralar 1 alone significantly reduces the aspartate/glutamate ratio (Fig. S1D-F). Since this ratio reflects glutamate catabolism, its decrease indicates impaired malate-aspartate shuttle activity and reduced glutamate catabolism. Therefore, although compensation by Aralar 2 cannot be entirely excluded, Aralar 1 KD alone suffices to cause substantial impairment of malate-aspartate shuttle function”.

• *

__ OGC and Aralar/AGC1 silencing is associated with the accumulation of smaller lipid droplets and impaired norepinephrine-induced lipolysis, but no mechanistical evidence is provided. The authors discuss a role for AMPK signaling associated with the redox unbalance generated by MAS disfunction but neither of them is proven.__

Answer 4) We thank the reviewer for this insightful question, which was also raised by Reviewer 1 (see Reviewer 1, Question 1 above). Here, we aim to clarify the mechanistic basis by which MASh may regulate lipolysis in BAT in a complementary and refined manner.

Our new data directly addresses this issue by examining cytosolic redox status through the lactate/pyruvate ratio, a well-established indicator of NAD⁺/NADH balance. Under basal conditions, Aralar 1 KD cells showed no change in this ratio compared to controls, indicating preserved cytosolic NAD⁺ regeneration despite reduced MASh activity. This observation is consistent with previous studies demonstrating the resilience of cellular redox homeostasis through overlapping NAD⁺-regenerating systems (PMID: 40540398; PMID: 37647199). The new results are shown in Supplementary Figure 1.

At the same time, we detected a marked decrease in the aspartate/glutamate ratio in Aralar 1 KD cells, confirming impaired MASh function and reduced amino acid exchange between cytosol and mitochondria. The lack of redox imbalance likely reflects compensatory mechanisms, most notably the GPSh, which is highly active in brown adipocytes. Supporting this view, Aralar 1 KD cells displayed significantly reduced glycerol release upon norepinephrine stimulation (Fig. 4I), suggesting enhanced metabolic cycling of G3P through mitochondrial and cytosolic G3PDH, thereby sustaining NAD⁺ regeneration and redox equilibrium.

We therefore propose that, although MASh normally facilitates NADH export and aspartate/glutamate exchange, its loss activates GPSh-mediated compensation that preserves cytosolic NAD⁺/NADH balance and maintains lipolytic flux during adrenergic stimulation. These findings refine our mechanistic understanding of how redox shuttle interplay supports glycolytic and lipolytic processes in BAT. Future studies employing NAD⁺/NADH sensors and simultaneous blockade of both shuttles will be essential to dissect these compensatory mechanisms in greater detail.

Minor points;

1. __ Is pyruvate present in respiration medium? If so, no effect on respiration is expected as pyruvate reverses the respiratory defects caused by MAS inactivation. __ Answer 1) Thanks for this important insight. In fact, as indicated in the methods section (page 17, last paragraph) all respirometry experiments were carried out in the absence of pyruvate in the media. Therefore, preserved overall respiratory rates in Aralar 1 and Ogc KD cannot be explained by compensatory pyruvate oxidation present in the media.

__ In figure 4, only data from Aralar KD cells in relation to norepinephrine-stimulated lipolysis are shown. What happens when OGC is silenced? __

Answer 2) This is a very interesting and relevant question. We did not perform the norepinephrine-stimulated lipolysis experiments in Ogc-silenced cells, since in most of the other experiments presented in the manuscript Ogc and Aralar 1 silencing converged to very similar, if not identical, phenotypes. Based on these consistent overlaps, we anticipate that Ogc KD would likely lead to comparable effects on lipolysis as observed in Aralar 1 KD cells. Nonetheless, we fully agree that direct assessment of lipolysis upon Ogc KD would strengthen this conclusion, and we consider this an important aspect for future studies.

__ Nomenclature used for mitochondrial carriers is confusing. Please do not use OGC1 as there is only one isoform. Furthermore, different names for OGC are used in the manuscript; oxoglutarate carrier, malate-ketoglutarate carrier or OGC1/SLC25A11. In the case of citrin/AGC2, Aralar2 is used and is a uncommon designation.__

Answer 3) We corrected all OGC naming in the revised manuscript. We also changed “aralar 2” for “citrin” since this was more commonly used in the literature.

__ Some panels of figures 3 and 4 should be improved. Panels 3J, 3L and 4G are difficult to see. In panel 3J please clarify UT line from untreated/NE, are they not transduced? No equivalents conditions are assayed in Aralar KD and OGC KO cells.__

Answer 4) We thank the reviewer for giving us the opportunity to improve this figure and apologize for the confusing labeling. In the revised version, we have clarified the labels in panels 3J, 3L, and 4G to improve visibility, and we have added descriptions of all abbreviations to the figure legends, accordingly.

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Referee #3

Evidence, reproducibility and clarity

In this manuscript, Veliova and coworkers explore the contribution of the malate-aspartate NADH shuttle (MAS) to energy metabolism in brown adipose tissue. This work done by a group expert in mitochondrial metabolism, continues an interesting previous one (Veliova, 2020) where it was shown that the inhibition of the mitochondrial pyruvate carrier caused an increase in energy expenditure mediated by the activation of MAS in BAT. Here, the authors have explored the consequences of the lack of MAS activity on BAT metabolism by the silencing of the metabolite transporters that are part of MAS in cultured primary brown adipocytes. Using this loss-of-function approach, the role for MAS in the regulation of lipid homeostasis in BAT is analyzed. The results could be interesting, but in my opinion, they are not sufficiently proven. Much more evidence should be provided to confirm MAS deficiency and the mechanisms involved in the alteration of lipid homeostasis.

Major points

1. Although in the manuscript Veliova and coworkers demonstrated that MAS is functional in brown adipocytes showing kinetic parameters equivalent to that previously described in other tissues, surprisingly, when its components are downregulated, no effect, or very little, on mitochondrial respiration is found (figure 2). This is an intriguing result since MAS disruption has been widely reported to impair respiration in different cell types and tissues. However, since no direct evidence of MAS dysfunction is provided, it is possible that MAS may still remain partially or fully functional under the conditions used by the authors, and therefore this point needs to be clarified to validate these results. Furthermore, strategies used to downregulate MAS components produce only a partial reduction in mRNA levels, about 70 %, but its outcome on protein levels has not been determined. and the remaining protein level could be sufficient to maintain shuttle activity. Therefore, the effect of silencing at protein level should be analyzed, because as authors also point out on page 16; "mRNA levels may not reflect actual protein levels or activity".
2. In the case of aspartate/glutamate carriers (AGCs) the role of citrin/slc25a13, the second AGC paralog, should also be analyzed. This AGC isoform is discarded based on proteomic data from brown adipose tissue, but, as it is shown in figure 1B, its levels are similar those of Aralar/slc25a12, the only AGC silenced. Besides, primary brown adipocytes differentiated for 7 days are used here, and it is possible that factors such as culture conditions or differentiation itself could alter AGC levels. Therefore, it is necessary to determine the protein levels of citrin/AGC2, and, if necessary, downregulate it together with the Aralar/AGC1 isoform. citrin/AGC2 activity may be responsible for the observed difference between the OGC and Aralar/AGC1 KD adipocytes.
3. OGC and Aralar/AGC1 silencing is associated with the accumulation of smaller lipid droplets and impaired norepinephrine-induced lipolysis, but no mechanistical evidence is provided. The authors discuss a role for AMPK signaling associated with the redox unbalance generated by MAS disfunction but neither of them is proven.

Minor points

1. Is pyruvate present in respiration medium? If so, no effect on respiration is expected as pyruvate reverses the respiratory defects caused by MAS inactivation.
2. In figure 4, only data from Aralar KD cells in relation to norepinephrine-stimulated lipolysis are shown. What happens when OGC is silenced?
3. Nomenclature used for mitochondrial carriers is confusing. Please do not use OGC1 as there is only one isoform. Furthermore, different names for OGC are used in the manuscript; oxoglutarate carrier, malate-ketoglutarate carrier or OGC1/SLC25A11. In the case of citrin/AGC2, Aralar2 is used and is a uncommon designation.
4. Some panels of figures 3 and 4 should be improved. Panels 3J, 3L and 4G are difficult to see. In panel 3J please clarify UT line from untreated/NE, are they not transduced? No equivalents conditions are assayed in Aralar KD and OGC KO cells.

Significance

General assessment: The robust part of this study is its analysis of some aspects related to lipid metabolism in cultured primary cells derived from brown adipose tissue. The participating teams are well-versed in this topic and the approaches used are correct. However, no data in animal models supporting these results are provided and this fact rests interest.

Advance: This manuscript is the "logical" continuation of a previous study, Veliova et al., (2020) EMBO Rep, more relevant in my opinion. Also, recently, it has been also proposed using animal models, either by overexpression or using deficient mice for GOT1 a cytosolic protein component of MAS, a role for MAS in BAT thermogenesis (Park et al., Cell Rep. 2025). The novelty in this manuscript is the analysis of deficient cells in the metabolite transporter that regulate the direction of NADH shuttling. However, since no evidence is provided its effect on NAD+/NADH ratio, the conclusions related to the role of MAS, or the mitochondrial carriers silenced, in the regulation of lipolysis in BAT and its involvement in thermogenesis are not convinced.

Audience: These results could be of interest to the audience interested in basic research, but could also be useful in the translational/clinical area because they address metabolic aspects in adipose tissue.

My expertise is focus on mitochondrial metabolism, specifically in the function of a subtype of mitochondrial carriers regulated by cytosolic calcium and how they participate in the control of different mitochondrial functions, such as respiration, calcium buffering, cell proliferation. Some of these transporters are components of MAS such as Aralar/AGC1 or citrin/AGC2.

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Referee #1

Evidence, reproducibility and clarity

The paper makes a clear, well-supported case that the malate-aspartate shuttle (MAS) is active in brown adipocytes and supports adrenergically stimulated lipolysis. The combination of a functional MAS assay, targeted carrier knockdowns, and multi-modal lipolysis measurements is a strong package. The reconstituted mitochondrial assay paired with live-cell lipolysis imaging is technically elegant and broadly reusable. The main gap is the limited in-vitro scope relative to in-vivo cold adaptation.

Major comments

1. The manuscript posits that the loss of function of MASh components (Ogc1 and Aralar) decreases adrenergic-stimulated lipolysis by altering the cytosolic NAD⁺/NADH ratio, with AMPK/ACC mentioned as possible mediators. However, this remains speculative. Please provide mechanistic data directly linking MASh-dependent NAD⁺/NADH changes to the regulation of lipolysis in brown adipocytes during adrenergic stimulation.
2. The absence of in vivo analysis of lipid-droplet size in MASh loss-of-function models is a major concern. In vitro results could be confounded by differences in differentiation stage between groups. Please document equivalent adipogenesis across groups (e.g., Pparg/Cebpa/Plin1/Fabp4 expression)
3. Please include rescue experiments (add-back OGC1 and Aralar) to rule out siRNA/shRNA off-target effects and verify that the phenotype stems from MASh loss of function.
4. Please expand on physiological significance: What is the importance of MASh regulation of BAT lipolysis in long-term adaptive thermogenesis?

Minor comments

1. Fig. 4 legend/title contains a typo ("lypolysis" → lipolysis).
2. In Fig. 2 legend line: "Adevirus-mediated" → Adenovirus-mediated; "OCAR" → OCR.
3. For lipolysis imaging, you already show Forskolin/Atglistatin/Etomoxir controls; add a vehicle-only time course overlay in the main figure (currently in text/legend) to aid visual comparison.
4. Ensure consistent gene symbols (Atgl/Pnpla2), and protein capitalization.

Referees cross-commenting

In my view, the feedback offered by all three reviewers has been highly constructive, as each of them has contributed thoughtful and meaningful criticism that can help improve the quality, clarity, and overall impact of the work.

Significance

Advance - how it fits the literature and what kind of advance.

Relative to prior work linking MASh (often via GOT1) to fuel preference and redox during thermogenesis, this study fills a mechanistic gap by showing that carrier-level MASh disruption (Aralar1/OGC1) specifically impairs adrenergic lipid mobilization upstream of β-oxidation, while respiration per cell can be buffered by compensatory mitochondrial biogenesis (lower OCR per mitochondrion). Conceptual/fundamental advance: it sharpens the redox - lipolysis axis in BAT and clarifies why changes in fuel availability (lipolysis) may limit thermogenesis even when bulk OCR looks preserved.

Audience - who will be interested/influenced.

Specialized but cross-cutting: adipose biology & thermogenesis, mitochondrial/redox metabolism, lipid-droplet and lipolysis communities, and metabolic-disease researchers exploring strategies to modulate BAT fuel handling.

Reviewer expertise

Adipose tissue and systemic energy metabolism; mitochondrial bioenergetics; thermogenic mechanisms in BAT/beige fat; transcriptional and metabolic control of lipid mobilization. Not a specialist in membrane-carrier biophysics.

A few weeks ago, I watched one of my favorite movie in a US theater. And I posted some videos about that movie on Chinese TikTok since the movie was not available in China. But as more and more people watched my video, it was suddenly deleted by the platform. At that moment, I realized that I violated the copyright of the movie company. This incident made me feel the existence of content moderation and how capable algorithms are. And I think content moderation is needed to protect the rights of users and companies.

Reviewer #1 (Public review):

Summary:

The authors investigate the role of H3K115ac in mouse embryonic stem cells. They report that H3K115ac localizes to regions enriched for fragile nucleosomes, CpG islands, and enhancers, and that it correlates with transcriptional activity. These findings suggest a potential role for this globular domain modification in nucleosome dynamics and gene regulation. If robust, these observations would expand our understanding of how non-tail histone modifications contribute to chromatin accessibility and transcriptional control.

Strengths:

(1) The study addresses a histone PTM in the globular domain, which is relatively unexplored compared to tail modifications.

(2) The implication of a histone PTM in fragile nucleosome localization is novel and, if substantiated, could represent a significant advance for the field.

Weaknesses:

(1) The absence of replicate paired-end datasets limits confidence in peak localization.

(2) The analyses are primarily correlative, making it difficult to fully assess robustness or to support strong mechanistic conclusions.

(3) Some claims (e.g., specificity for CpG islands, "dynamic" regulation during differentiation) are not fully supported by the analyses as presented.

(4) Overall, the study introduces an intriguing new angle on globular PTMs, but additional rigor and mechanistic evidence are needed to substantiate the conclusions.

Reviewer #2 (Public review):

Summary:

Kumar et al. aimed to assess the role of the understudied H3K115 acetylation mark, which is located in the nucleosomal core. To this end, the authors performed ChIP-seq experiments of H3K115ac in mouse embryonic stem cells as well as during differentiation into neuronal progenitor cells. Subsequent bioinformatic analyses revealed an association of H3K115ac with fragile nucleosomes at CpG island promoters, as well as with enhancers and CTCF binding sites. This is an interesting study, which provides important novel insights into the potential function of H3K115ac. However, the study is mainly descriptive, and functional experiments are missing.

Strengths:

(1) The authors present the first genome-wide profiling of H3K115ac and link this poorly characterized modification to fragile nucleosomes, CpG island promoters, enhancers, and CTCF binding sites.

(2) The study provides a valuable descriptive resource and raises intriguing hypotheses about the role of H3K115ac in chromatin regulation.

(3) The breadth of the bioinformatic analyses adds to the value of the dataset

Weaknesses:

(1) I am not fully convinced about the specificity of the antibody. Although the experiment in Figure S1A shows a specific binding to H3K115ac-modified peptides compared to unmodified peptides, the authors do not show any experiment that shows that the antibody does not bind to unrelated proteins. Thus, a Western of a nuclear extract or the chromatin fraction would be critical to show. Also, peptide competition using the H3K115ac peptide to block the antibody may be good to further support the specificity of the antibody. Also, I don't understand the experiment in Figure S1B. What does it tell us when the H3K115ac histone mark itself is missing? The KLF4 promoter does not appear to be a suitable positive control, given that hundreds of proteins/histone modifications are likely present at this region.

It is important to clearly demonstrate that the antibody exclusively recognizes H3K115ac, given that the conclusion of the manuscript strongly depends on the reliability of the obtained ChIP-Seq data.

(2) The association of H3K115ac with fragile nucleosomes based on MNase-Sensitivity and fragment length, which are indirect methods and can have technical bias. Experiments that support that the H3K115ac modified nucleosomes are indeed more fragile are missing.

(3) The comparison of H3K115ac with H3K122ac and H3K64ac relies on publicly available datasets. Since the authors argue that these marks are distinct, data generated under identical experimental conditions would be more convincing. At a minimum, the limitations of using external datasets should be discussed.

(4) The enrichment of H3K115ac at enhancers and CTCF binding sites is notable but remains descriptive. It would be interesting to clarify whether H3K115ac actively influences transcription factor/CTCF binding or is a downstream correlate.

(5) No information is provided about how H3K115ac may be deposited/removed. Without this information, it is difficult to place this modification into established chromatin regulatory pathways.

At the very least, the authors should acknowledge these limitations and provide additional validation of antibody specificity.

Reviewer #3 (Public review):

Summary:

Kumar et al. examine the H3K115 epigenetic mark located on the lateral surface of the histone core domain and present evidence that it may serve as a marker enriched at transcription start sites (TSSs) of active CpG island promoters and at polycomb-repressed promoters. They also note enrichment of the H3K115ac mark is found on fragile nucleosomes within nucleosome-depleted regions, on active enhancers, and CTCF-bound sites. They propose that these observations suggest that H3K115ac contributes to nucleosome destabilization and so may serve as a marker of functionally important regulatory elements in mammalian genomes.

Strengths:

The authors present novel observations suggesting that acetylation of a histone residue in a core (versus on a histone tail) domain may serve a functional role in promoting transcription, in CPG islands and polycomb-repressed promoters. They present a solid amount of confirmatory in silico data using appropriate methodology that supports the idea that the H3K115ac mark may function to destabilize nucleosomes and contribute to regulating ESC differentiation.

Weaknesses:

Additional experiments to confirm antibody specificity are needed. The authors use synthetic peptides for other markers (e.g., H3K122) to support the claim that the antibody is specific, but ChIP-ChIP assays are performed under cross-linked, non-denatured conditions, which preserve structure and epitope accessibility differently than synthetic peptides used for dot blots. Does the antibody give a single band in western blots of histones, and can the H3K115ac peptide block western and immunofluorescence signals of the antibody? Given that the antibody is a rabbit polyclonal, specificity is not a trivial consideration.

Author response:

Reviewer 1:

Comment 1. The reviewer was under the impression that that we did not perform biological replicates of our ChIP-seq experiments. All ChIP-seq (and ATAC-seq) experiments were performed with biological replicates and the Pearson’s correlations (all >0.9) between replicates were provided in Supplementary Table 1. We had indicated this in the text and methods but will try to make this even clearer.

Reviewer 2:

Comment 2. The reviewer states that our claim of H3K115ac being associated with fragile nucleosomes is based solely on MNase sensitivity and fragment length. This is not correct. Figure 3C and D show the results of sucrose gradient sedimentation experiments, followed by ChIP-seq clearly showing that H3K115ac fractionates with chromatin particles that are enriched for fragile nucleosomes and subnucleosomes. By contrast, H3K115ac is not enriched in stable mononucleosome

Comment 3. The reviewer states that our H3K122ac and H3K64ac comparison rely on publicly available datasets. We would emphasize that these are our own datasets generated and published previously (Pradeepa et. al., 2016) but using exactly the same native MNase ChIP protocol as used here for H3K115ac and processed with identical computational pipelines.

Reviewer 3:

Reviewer 3 is mistaken in thinking our ChIP experiments are performed under cross-linked conditions. As clearly stated in the main text and methods, all our ChIP-seq for histone modifications is done on native MNase-digested chromatin – with no cross-linking. This includes the spike-in experiment shown in Fig S1B to test H3K115ac antibody specificity against the bar-coded SNAP-ChIP® K-AcylStat Panel from Epicypher. We could not include H3K115ac bar-coded nucleosomes in that experiment since they are not available in the panel. 

Following that, we would propose to make minor revisions in response to specific reviewer recommendations before posting a version of record. These would include:

(1) Figure 2: title needs change: "H3K115ac marks CpG island promoters poised for activation". this is to make sure it reads with the title for the corresponding section in the main text. Also see: Reviewer 1 comment 7 in Recommendations part. 

(2) Figure S2B: legend should read: "Gene ontology analysis for the set of genes analysed in Figure 2C"

(3) Figure F4D: Provide the replicates for western blot 

(4) Figure 4A,B: Corrected formatting issues.

Author response:

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

Reviewer #1 (Public review): 

The manuscript by Bru et al. focuses on the role of vacuoles as a phosphate buffering system for yeast cells. The authors describe here the crosstalk between the vacuole and the cytosol using a combination of in vitro analyses of vacuoles and in vivo assays. They show that the luminal polyphosphatases of the vacuole can hydrolyse polyphosphates to generate inorganic phosphate, yet they are inhibited by high

concentrations. This balances the synthesis of polyphosphates against the inorganic phosphate pool. Their data further show that the Pho91 transporter provides a valve for the cytosol as it gets activated by a decline in inositol pyrophosphate levels. The authors thus demonstrate how the vacuole functions as a phosphate buffering system to maintain a constant cytosolic inorganic phosphate pool. 

This is a very consistent and well-written manuscript with a number of convincing experiments, where the authors use isolated vacuoles and cellular read-out systems to demonstrate the interplay of polyphosphate synthesis, hydrolysis, and release. The beauty of this system the authors present is the clear correlation between product inhibition and the role of Pho91 as a valve to release Pi to the cytosol to replenish the cytosolic pool. I find the paper overall an excellent fit and only have a few issues, including: 

(1) Figure 3: The authors use in their assays 1 mM ZnCl2 or 1mM MgCl2. Is this concentration in the range of the vacuolar luminal ion concentration? Did they also test the effect of Ca2+, as this ion is also highly concentrated in the lumen? 

The concentrations inside vacuoles reach those values. However, given that polyP can chelate divalent metal ions, what would matter are the concentrations of free Zn²⁺ or Mg²⁺ inside the organelle. These are not known. This is not critical since we use those two conditions only as a convenient tool to differentiate Ppn1 and Ppn2 activity in vitro. In our initial characterisation of Ppn2 (10.1242/jcs.201061), we had also tested Mn, Co, Ca, Ni, Cu. Only Zn and Co supported activity. Ca did not. Andreeva et al. (10.1016/j.biochi.2019.06.001) reached similar conclusions and extended our results.

(2) Regarding the concentration of 30 mM K-PI, did the authors also use higher and lower concentrations? I agree that there is inhibition by 30 mM, but they cannot derive conclusions on the luminal concentration if they use just one in their assay. A titration is necessary here. 

The concentration of 30 mM was not chosen arbitrarily. It is the luminal Pⁱ concentration that the vacuoles reached through polyP synthesis and hydrolysis when they entered a plateau of luminal Pⁱ. We consider this as an upper limit because polyP kept increasing which luminal Pⁱ did not. Thus, there is no physiological motivation for trying higher values. We have nevertheless added a titration to the revised version (new Fig. 3A).

(3) What are the consequences on vacuole morphology if the cells lack Pho91? 

We had not observed significant abnormalities during a screen of the genome-wide deletion collection of yeast (10.1371/journal.pone.0054160), nor in other experiments with pho91 mutants, which we have not included in this manuscript due to a lack of effect.

(4) Discussion: The authors do not refer to the effect of calcium, even though I would expect that the levels of the counterion should affect the phosphate metabolism. I would appreciate it if they would extend their discussion accordingly. 

The situation is much more complex because Ca2+ is not the only counterion. Major pools of counterions (up to hundreds of mM) are constituted by vacuolar lysine, arginine, polyamines, Mg, Zn etc. Their interplay with polyP is probably complex and worth to be treated in a dedicated project. If we wanted to limit the discussion of this complexity not to the simple statement that it is not understood, which is not very useful, we would have to engage in a lot of speculation. We feel that this would make the discussion lose focus and not contribute concrete insights.

(5) I would appreciate a brief discussion on how phosphate sensing and control are done in human cells. Do they use a similar lysosomal buffer system? 

Mammalian cells have their Pi exporter XPR1 mainly on a lysosome-like compartment (10.1016/j.celrep.2024.114316). Whether and how it functions there for Pi export from the cytosol is not entirely clear. We have addressed this situation in the revised discussion section.

Reviewer #2 (Public review): 

Summary: 

This manuscript presents a well-conceived and concise study that significantly advances our understanding of polyphosphate (polyP) metabolism and its role in cytosolic phosphate (Pi) homeostasis in a model unicellular eukaryote. The authors provide evidence that yeast vacuoles function as dynamic regulatory buffers for Pi homeostasis, integrating polyP synthesis, storage, and hydrolysis in response to cellular metabolic demands. The work is methodologically sound and offers valuable insights into the conserved mechanisms of phosphate regulation across eukaryotes. 

Strengths: 

The results demonstrate that the vacuolar transporter chaperone (VTC) complex, in conjunction with luminal polyphosphatases (Ppn1/Ppn2) and the Pi exporter Pho91, establishes a finely tuned feedback system that balances cytosolic Pi levels. Under Pi-replete conditions, inositol pyrophosphates (InsPPs) promote polyP synthesis and storage while inhibiting polyP hydrolysis, leading to vacuolar Pi accumulation. 

Conversely, Pi scarcity triggers InsPP depletion, activating Pho91-mediated Pi export and polyP mobilization to sustain cytosolic phosphate levels. This regulatory circuit ensures metabolic flexibility, particularly during critical processes such as glycolysis, nucleotide synthesis, and cell cycle progression, where phosphate demand fluctuates dramatically. 

From my viewpoint, one of the most important findings is the demonstration that vacuoles act as a rapidly accessible Pi reservoir, capable of switching between storage (as polyP) and release (as free Pi) in response to metabolic cues. The energetic cost of polyP synthesis-driven by ATP and the vacuolar proton gradient-highlights the evolutionary importance of this buffering system. The study also draws parallels between yeast vacuoles and acidocalcisomes in other eukaryotes, such as Trypanosoma and Chlamydomonas, suggesting a conserved role for these organelles in phosphate homeostasis. 

Weaknesses: 

While the manuscript is highly insightful, referring to yeast vacuoles as "acidocalcisome-like" may warrant further discussion. Canonical acidocalcisomes are structurally and chemically distinct (e.g., electrondense, in most cases spherical, and not routinely subjected to morphological changes, and enriched with specific ions), whereas yeast vacuoles have well-established roles beyond phosphate storage. A comment on this terminology could strengthen the comparative analysis and avoid potential confusion in the field.  

Yeast vacuoles show all major chemical features of acidocalcisomes. They are acidified, contain high concentrations of Ca, polyP (which make them electron-dense, too), other divalent ions, such as Mg, Zn, Mn etc, and high concentrations of basic amino acids. Thus, they clearly have an acidocalcisome-like character. In addition, they have hydrolytic, lysosomelike functions and, depending on the strain background, they can be larger than acidocalcisomes described e.g. in protists. We have elaborated on this point in the introduction of the revised version.

Reviewer #3 (Public review): 

Bru et al. investigated how inorganic phosphate (Pi) is buffered in cells using S. cerevisiae as a model. Pi is stored in cells in the form of polyphosphates in acidocalcisomes. In S. cerevisiae, the vacuole, which is the yeast lysosome, also fulfills the function of Pi storage organelle. Therefore, yeast is an ideal system to study Pi storage and mobilization. 

They can recapitulate in their previously established system, using isolated yeast vacuoles, findings from their own and other groups. They integrate the available data and propose a working model of feedback loops to control the level of Pi on the cellular level. 

This is a solid study, in which the biological significance of their findings is not entirely clear. The data analysis and statistical significance need to be improved and included, respectively. The manuscript would have benefited from rigorously testing the model, which would also have increased the impact of the study. 

It is not clear to us what the reviewer would see as a more rigorous test of the model.  

Reviewer #1 (Recommendations for the authors): 

(1) Figure 2: Why do the authors label the blue curve in A and B as BY and in C and D as WT? Is this a different genetic background they used here? This should be specified in the legend. 

No, it is the same background. The figures had been reshuffled before submission and we overlooked to replace "BY" by "WT". This has been corrected. Now we consistently use WT in all figures

(2) Figure 4 has different scaling for the two panels, which should be labeled as A and B. I am aware that the authors do this for comparison, but it is rather confusing at first glance. I recommend having them at the same scale. 

We chose this representation on two separate scales because this figure shall primarily illustrate that the shift between pho91 and WT curves vanishes in the presence of IP7. We now highlight in the figure legend that the scales are different to avoid confusion.  

(3) Figure 8: I would appreciate a model with normal and low Pi concentrations in comparison, as this is what the authors worked out. 

We have modified the figure. It now compares Pi-rich and Pi-limited scenarios.

(4) Minor issue: Wouldn't it make more sense to show the molar concentration in the Figures rather than the nmol of Pi/ug of protein? I am aware that this would require information on the vacuole volume rather than the reaction volume, and the authors do this calculation later on. 

It depends. We often chose this representation because it illustrates the price to pay (metabolic input in terms of protein that must be dedicated to this task) to sequester a certain quantity of Pⁱ. But, as we provide the corresponding Pⁱ concentration in the text, this information is accessible to the reader, too.

Reviewer #2 (Recommendations for the authors): 

As stated above in the weaknesses section, while functional parallels exist, canonical acidocalcisomes are structurally and chemically distinct, typically smaller, electron-dense, and enriched with cations. Whereas yeast vacuoles are larger, multifunctional organelles with well-established roles beyond phosphate storage. Explicitly addressing these differences would strengthen the comparative framework and prevent potential confusion in interpreting the evolutionary relationships between these organelles. 

We agree to some degree, which is the reason why we refer to vacuoles as acidocalcisome-like organelles. In fact, vacuoles share virtually all defining chemical traits of acidocalcisomes. They just have a second functional domain as hydrolytic, lysosome-like organelles. Given the plasticity of endo-lysosomal compartments, and acidocalcisomes belong to this group because of their biogenesis through the AP3 pathway, this is not shocking to us. But the reviewer's comment made us realize that it is better to explicitly address this point. We have added a section to the introduction to do this.

Reviewer #3 (Recommendations for the authors): 

(1) Page 8: It is unclear why the authors only estimated the Pi concentration in wild-type vacuoles. This should also be done for vacuoles from other strains. 

This information is inherent in Figure 2. PolyP hyperaccumulating strains show the same plateau as the wildtype, meaning that they also reach around 30 mM luminal Pi concentration, whereas vtc4 vacuoles reach only around 1/10th of that increase, indicating that they remain at 3 mM. We mention this now in the text.

(2) The attempts of the localization of Pho91 through tagging are not satisfactory. The author described different localizations for Pho91 depending on whether it was tagged on the N- or C-terminus or when Nterminally tagged and overexpressed using two strong promoters. While it is not uncommon that proteins show different localization patterns, depending on where the tag is inserted, it is possible that one of the tags would reflect the localization of the endogenous protein. There is an easy way to test this, in particular when Pho91 is endogenously tagged. pho91∆ has reported phenotypes such as abnormal vacuolar morphology or increased autophagy. They could also measure PI content in vacuoles. The authors could compare the phenotypes of the endogenously tagged strains with WT and a pho91∆ strain. 

Indeed, the attempts to localise the protein through fluorescent tags are unsatisfactory, in our hands as in the hands of others. We would not have created a series of many different tagged versions (we present only a selection of these in the manuscript) if the creation of a faithful reporter for Pho91 localisation were so straightforward. Expression from the endogenous promoter yields quite low signals (which is why others have overexpressed their GFP fusion from strong promotors). But overexpression brings at least a significant part of the protein to the cell surface, where it can then function as Pi importer and suffice to restore much of the maximal Pi uptake capacity that genuine plasma membrane transporters provide and support normal growth of the cells (Wykoff & O’Shea, 2001). But the localisation pattern of Pho91-GFP, likewise overexpressed from a strong promotor, does not reflect this plasma membrane localisation (see the references that the reviewer mentioned under (3)). The published overexpressed GFP-fusions localise only to the vacuole, suggesting that even in this case the GFP tag may create an artefact. Therefore, we went through a large variety of Pho91 gene fusions, which led us to the conclusion that the protein is very sensitive to tags at both ends and that fusion proteins hence are unlikely to reliably report the correct location of the protein. Given this, we resorted to quantitative proteomics to clarify the issue. This quantitative experiment goes beyond previously published proteomics analyses that the reviewer mentions under (3), which found the protein in the vacuolar fraction but did not calculate the enrichment factors, which is crucial. 

A strong phenotype of abnormal vacuolar morphology is not apparent in our cultures. 

(3) Moreover, Pho91 has been identified as a component enriched in vacuolar-mitochondria contact sites (vCLAMP), and this localization was confirmed with GFP-Pho91 (PMID: 25026036). Likewise, PMID: 35175277 also detected Pho91 by mass spectrometry as a vacuolar protein and showed endogenously tagged GFP-Pho91 on the vacuole (co-staining with Vph1). The authors may request the strains from the authors of these papers and use them for their experiments. PMID: 17804816, the oldest of the three reports (from 2007) reports a GFP-Pho91 under either TEF or ADH promoter that localizes to the vacuole. They also showed that the fusion protein is functional. These and other experiments led them to conclude that Pho91 exports phosphate from the vacuolar lumen to the cytoplasma. 

We have now included these references. As argued above, we have analysed also the strains from PMID17804816. The observed clear localisation of the fusion protein to vacuoles is only visible upon overexpression, not upon expression from the endogenous locus. Apparently also this construct is unlikely to report Pho91 localisation reliably (though, by chance, overexpression leads it to the correct location). Thus, we maintain our conclusion that C- or N-terminally GFP-tagged versions of Pho91 are unreliable tools for localising the protein.

(4) The impact of pho91∆ on Pho4-GFP nuclear localization is modest at best (increase from 5% of cells showing Pho4-GFP in the nucleus in WT vs 10% in pho91∆), and only somewhat stronger in ppn1∆/ppn2∆. This means 90% of pho91∆ cells do not respond, and Pho4-GFP stays cytoplasmic. It is unclear how the author can derive a meaningful conclusion from these data. Moreover, are these data really supporting the model, or do these data rather indicate that there are additional factors/pathways needed? What is the biological significance of the marginal increase from 5% to 10% of cells that would respond? What happens to the cells that cannot respond? Will they die or at least have a growth disadvantage? It would be useful to provide some functional studies. 

We should have explained the nature of the assay better. The experiment exploits the fact that dividing yeast cells transiently fall into a state of Pi scarcity during S-phase. Since S-phase is less than a quarter of the cell cycle, only a small fraction of the cells transiently activates the PHO pathway. These cannot be well characterised by ensemble assays, but microscopy circumvents this background of the whole population and picks them up very clearly, allowing to quantify them. We have adapted the respective chapter in the results section to improve the description of this experiment.

(5) The quantification of the data is suboptimal, as in most assays the mean and standard error of the mean (SEM) are given. SEM is not really appropriate in these cases because it gives only the error of the mean and not of the entire data. Therefore, the standard deviation (SD) is needed, which reports on the variability of the data, and which is usually much larger than the SEM. Using the SD, would also allow the authors to do proper statistical analysis, which is missing entirely in this manuscript. 

SEM also comprises the variability of the data. It is linked with the SD (SEM=SD/SQRT(n)), but SEM also considers the number of the experiments n. The main goal is to compare the means, and SEM is an appropriate and frequently used tool for this because it illustrates how well the arithmetic mean may estimate the true mean of the population. Therefore, we kept the SEM but have added tests of significance for the differences shown.

(6) Statistical testing in Figure 7 is essential as the effects are very small. Again, are these changes big enough for a biologically meaningful response? The authors should at least discuss this. 

Our previous time course analyses of InsPP dynamics, performed under comparable conditions as in this study, showed that InsP8 decreases by around 50% in the first 30 min after transfer to Pi starvation (DOI: https://doi.org/10.7554/eLife.87956) and that this decline is already sufficient to trigger the PHO starvation program, as assessed by Pho4-GFP translocation into the nucleus. Thus, a 50% decrease, which is observed in ppn1 ppn2 mutants, is functionally significant. We have now also evaluated statistical significance in Fig. 7, which is given for the 50% reduction of InsP8 and 1-InsP7 in ppn1 ppn2. 

Minor points: 

(1) There are a number of smaller edits (use of italic or better the absence thereof, lacking information in the reference list, and some typos). 

Thank you. We have corrected those.

(2) The exact n should be given in the Figure legend. 

Corrected.

(3) Page 8, line 8: it would be nice to have a picture of the wild-type vacuoles and what you measured. 

We now present a sample image in the new Suppl. Fig. 1.

(4) PMID: 11779791 showed already that Pho91 cannot rescue the absence of the plasma membrane Pi transporters. This study should be at least cited. 

This is not quite correct. The study that the reviewer mentions showed that Pho91 supports slower growth and the authors concluded that "A synthetic lethal phenotype was observed when (all) five phosphate transporters were inactivated...". We had cited the same group and the same first author, just using their later study (Wykoff et al., 2007) that had recapitulated the results from PMID11779791 and showed in addition quite good growth of the PHO91 expressing strain on YPD (Suppl. Fig. 2). We had obtained the strains from this group. In reproducing their experiments, we noticed that the growth of Pho91 that these authors had observed is due to incomplete repression of Pho84. They had overexpressed Pho84 from a galactose inducible promotor to generate a background with a regulatable Pi transporter. This trick allowed them to conveniently manipulate the strain and reduce (but not abolish) Pho84 expression by transferring the cells from galactose to glucose for their experiments. Therefore, we chose a more rigorous plasmid shuffling strategy to test the individual P_i transporter, which allows an assessment without the leaky background expression of Pho84 on glucose. In contrast to O'Shea and colleagues, we observed zero growth of a strain expressing only PHO91. We have revised the results section to make this discrepancy more evident and provide a better motivation for our experiment.

(5) It would be nice to see the actual data in Figure 6; not only a quantification. 

We illustrate the phenotype of nuclear Pho4-GFP in panel A. Showing all the images necessary to appreciate the differences between the strains would require including many dozens of images into the figure, which would not be useful.

Reviewer #2 (Public review):

Summary:

Majeed and colleagues aimed to evaluate whether the metabolic effects of NMN in the context of a high-fat diet are SIRT1 dependent. For this, they used an inducible SIRT1 KO model (SIRT1 iKO), allowing them to bypass the deleterious effects of SIRT1 ablation during development. In line with previous reports, the authors observed that NMN prevents, to some degree, diet-induced metabolic damage in wild-type mice. When doing similar tests on SIRT1 iKO mice, the authors see that some, but not all, of the effects of NMN are abrogated. The phenotypic studies are complemented by plasma proteomic analyses evaluating the influence of the high-fat diet, SIRT1, and NMN on circulating protein profiles.

Strengths:

The mechanistic aspects behind the potential health benefits of NAD+ precursors have been poorly elucidated. This is in part due to the pleiotropic actions of NAD-related molecules on cellular processes. While sirtuins, most notably SIRT1, have been largely hypothesized to be key players in the therapeutic actions of NAD+ boosters, the proof for this in vivo is very limited. In this sense, this work is an important contribution to the field.

Weaknesses:

While the authors use a suitable methodology (SIRT1 iKO mice), the results show very early that the iKO mice themselves have some notable phenotypes, which complicate the picture. The actions of NMN in WT and SIRT1 KO mice are most often presented separately. However, this is not the right approach to evaluate and visualize SIRT1 dependency. Indeed, many of the "SIRT1-dependent" effects of NMN are consequent to the fact that SIRT1 deletion itself has a phenotype equivalent to or larger than that induced by NMN in wild-type mice. This would have been very evident if the two genotypes had been systematically plotted together. Consequently, and despite the value of the study, the results obtained with this model might not allow for solidly established claims of SIRT1 dependency on NMN actions. The fact that some of the effects of SIRT1 deletion are similar to those of NMN supplementation also makes it counterintuitive to propose that activation of SIRT1 is a major driver of NMN actions. Unbiasedly, one might as well conclude that NMN could act by inhibiting SIRT1. The fact that readouts for SIRT1 activity are not explored makes it also difficult to test the influence of NMN on SIRT1 in their experimental setting, or whether compensations could exist.

A second weak point is that the proteomic explorations are interesting, yet feel too descriptive and disconnected from the overall phenotype or from the goal of the manuscript. It would be unreasonable to ask for gain/loss-of-function experiments based on the differentially abundant peptides. Yet, a deeper exploration of whether their altered presence in circulation is consistent with changes in their expression - and, if so, in which tissues - and a clearer discussion on their link to the phenotypes observed would be needed, especially for changes related to SIRT1 and NMN.

Impact on the field and further significance of the work:

Despite the fact that, in my opinion, the authors might not have conclusively achieved their main aim, there are multiple valuable aspects in this manuscript:

(1) It provides independent validation for the potential benefits of NAD+ boosters in the context of diet-induced metabolic complications. Previous efforts using NR or NMN itself have provided contradicting observations. Therefore, additional independent experiments are always valuable to further balance the overall picture.

(2) The metabolic consequences of deleting SIRT1 in adulthood have been poorly explored in previous works. Therefore, irrespective of the actions of NMN, the phenotypes observed are intriguing, and the proteomic differences are also large enough to spur further research to understand the role of SIRT1 as a therapeutic target.

(3) Regardless of the influence of SIRT1, NMN promotes some plasma proteomic changes that are very well worth exploring. In addition, they highlight once more that the in vivo actions of NMN, as those of other NAD+ boosters, are pleiotropic. Hence, this work brings into question whether single gene KO models are really a good approach to explore the mechanisms of action of NAD+ precursors.

Author response:

Reviewer #1 (Public review):

Summary:

This manuscript investigates the effects of oral supplementation with nicotinamide mononucleotide (NMN) on metabolism and inflammation in mice with diet-induced obesity, and whether these effects depend on the NAD⁺-dependent enzyme SIRT1. Using control and inducible SIRT1 knockout mice, the authors show that NMN administration mitigates high-fat diet-induced weight gain, enhances energy expenditure, and normalizes fasting glucose and plasma lipid profiles in a largely SIRT1-dependent manner. However, reductions in fat mass and adipose tissue expansion occur independently of SIRT1. Comprehensive plasma proteomic analyses (O-Link and mass spectrometry) reveal that NMN reverses obesity-induced alterations in metabolic and immune pathways, particularly those related to glucose and cholesterol metabolism. Integrative network and causal analyses identify both SIRT1-dependent and -independent protein clusters, as well as potential upstream regulators such as FBXW7, ADIPOR2, and PRDM16. Overall, the study supports that NMN modulates key metabolic and immune pathways through both SIRT1-dependent and alternative mechanisms to alleviate obesity and dyslipidemia in mice.

Strengths:

Well-written manuscript, and state-of-the-art proteomics-based methodologies to assess NMN and SIRT1-dependent effects.

We thank the reviewer for highlighting that state-of-the-art proteomic research methods used, and we report for the first time on significant changes in plasma proteomics in mice after NMN supplementation in both wild-type and SIRT1-KO mice using a combination of DIA mass spectrometry and Olink.

Weaknesses:

Unfortunately, the study design, as well as the data analysis approach taken by the authors, are flawed. This limits the authors' ability to make the proposed conclusions.

We agree that the administration of tamoxifen, along with the associated weight loss, could affect the obesity phenotype. For this reason, we ensured that both Cre-positive and Cre-negative mice received tamoxifen. Importantly, after the tamoxifen 'washout', the two groups weighed essentially the same. Going forward, we plan to address this comment by performing additional statistical tests on all six experimental groups to gain insights into dependencies. Based on your suggestions, we will clarify the limitations of the study design and improve the data analysis approaches to provide stronger support for our conclusions in the revised version of the paper.

Reviewer #2 (Public review):

Summary:

Majeed and colleagues aimed to evaluate whether the metabolic effects of NMN in the context of a high-fat diet are SIRT1 dependent. For this, they used an inducible SIRT1 KO model (SIRT1 iKO), allowing them to bypass the deleterious effects of SIRT1 ablation during development. In line with previous reports, the authors observed that NMN prevents, to some degree, diet-induced metabolic damage in wild-type mice. When doing similar tests on SIRT1 iKO mice, the authors see that some, but not all, of the effects of NMN are abrogated. The phenotypic studies are complemented by plasma proteomic analyses evaluating the influence of the high-fat diet, SIRT1, and NMN on circulating protein profiles.

Strengths:

The mechanistic aspects behind the potential health benefits of NAD+ precursors have been poorly elucidated. This is in part due to the pleiotropic actions of NAD-related molecules on cellular processes. While sirtuins, most notably SIRT1, have been largely hypothesized to be key players in the therapeutic actions of NAD+ boosters, the proof for this in vivo is very limited. In this sense, this work is an important contribution to the field.

We thank the reviewer for acknowledging the importance of this work to the field. In this report, we provide in vivo evidence of the action of NAD+ boosting, and hope to delineate the action of Sirt1, as well as the pleiotropic effects of NAD-related molecules on cellular and metabolic processes.

Weaknesses:

While the authors use a suitable methodology (SIRT1 iKO mice), the results show very early that the iKO mice themselves have some notable phenotypes, which complicate the picture. The actions of NMN in WT and SIRT1 KO mice are most often presented separately. However, this is not the right approach to evaluate and visualize SIRT1 dependency. Indeed, many of the "SIRT1-dependent" effects of NMN are consequent to the fact that SIRT1 deletion itself has a phenotype equivalent to or larger than that induced by NMN in wild-type mice. This would have been very evident if the two genotypes had been systematically plotted together. Consequently, and despite the value of the study, the results obtained with this model might not allow for solidly established claims of SIRT1 dependency on NMN actions. The fact that some of the effects of SIRT1 deletion are similar to those of NMN supplementation also makes it counterintuitive to propose that activation of SIRT1 is a major driver of NMN actions. Unbiasedly, one might as well conclude that NMN could act by inhibiting SIRT1. The fact that readouts for SIRT1 activity are not explored makes it also difficult to test the influence of NMN on SIRT1 in their experimental setting, or whether compensations could exist.

We thank the reviewer for raising this point and acknowledge the limitations of using Sirt1 iKO mice. However, inducing Sirt1 KO in adulthood is a better alternative than using a homozygous Sirt1 KO mouse model, as the latter leads to embryonic lethality and many other developmental defects (1, 2). The proteomics analysis can provide insight into the effects of SIRT1 deletion under chow and high-fat diet (HFD) conditions, as well as the effects of diet in the presence or absence of nicotinamide mononucleotide (NMN). We will discuss these limitations and present the results for the two genotypes together, as suggested.

A second weak point is that the proteomic explorations are interesting, yet feel too descriptive and disconnected from the overall phenotype or from the goal of the manuscript. It would be unreasonable to ask for gain/loss-of-function experiments based on the differentially abundant peptides. Yet, a deeper exploration of whether their altered presence in circulation is consistent with changes in their expression - and, if so, in which tissues - and a clearer discussion on their link to the phenotypes observed would be needed, especially for changes related to SIRT1 and NMN.

First, we presented the data in this manner as a proof of concept, to demonstrate the effect of the diet on the plasma proteome and corroborate our findings with those published in the literature. We then investigated the effects of NAD boosting and Sirt1 KO in order to identify significant changes. We agree with the reviewer that it would be unreasonable to validate all the differentially abundant proteins. However, we will choose key proteins and assess their expression in different tissues, such as the liver, white adipose tissue (WAT) and muscles, and attempt to connect these changes with the phenotypes.

Impact on the field and further significance of the work:

Despite the fact that, in my opinion, the authors might not have conclusively achieved their main aim, there are multiple valuable aspects in this manuscript:

(1) It provides independent validation for the potential benefits of NAD+ boosters in the context of diet-induced metabolic complications. Previous efforts using NR or NMN itself have provided contradicting observations. Therefore, additional independent experiments are always valuable to further balance the overall picture.

(2) The metabolic consequences of deleting SIRT1 in adulthood have been poorly explored in previous works. Therefore, irrespective of the actions of NMN, the phenotypes observed are intriguing, and the proteomic differences are also large enough to spur further research to understand the role of SIRT1 as a therapeutic target.

(3) Regardless of the influence of SIRT1, NMN promotes some plasma proteomic changes that are very well worth exploring. In addition, they highlight once more that the in vivo actions of NMN, as those of other NAD+ boosters, are pleiotropic. Hence, this work brings into question whether single gene KO models are really a good approach to explore the mechanisms of action of NAD+ precursors.

We thank the reviewer for their analysis in highlighting the valuable aspects of the manuscript and we hope the revised manuscript will further strengthen the key results.

References:

(1) McBurney   MW, Yang   X, Jardine   K, Hixon   M, Boekelheide   K, Webb   JR, Lansdorp   PM, Lemieux   M. The mammalian SIR2alpha protein has a role in embryogenesis and gametogenesis. Mol Cell Biol  2003; 23:38–54.

(2) Cheng   HL, Mostoslavsky   R, Saito   S, Manis   JP, Gu   Y, Patel   P, Bronson   R, Appella   E, Alt   FW, Chua   KF. Developmental defects and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice. Proc Natl Acad Sci U S A  2003; 100:10794–10799.

Interesting to see companies vary in their est of how AI will impact workforce. A third expects reduction (but not much, about 3%), 13% an increase (AI related hiring), 43% no change.

The paper shows that it's not a percentage of training data that needs to be poisoned for an attack, but an almost fixed number of documents (250!) which is enough across large models too.

Paper concludes most benchmarks used for LLMs to establish progress are mistargeted / leave out aspects that matter.

How spinal cord stay in its normal position when cbf and meninges ballooned out?

This section has data that can help build an argument for proposal. Basically this section states that not only is intervention important for the demographic, but also hypothesizes that interventions would be well received.

Reviewer #1 (Public review):

Summary:

Fungal survival and pathogenicity rely on the ability to undergo reversible morphological transitions, which are often linked to nutrient availability. In this study, the authors uncover a conserved connection between glycolytic activity and sulfur amino acid biosynthesis that drives morphogenesis in two fungal model systems. By disentangling this process from canonical cAMP signaling, the authors identify a new metabolic axis that integrates central carbon metabolism with developmental plasticity and virulence.

Strengths:

The study integrates different experimental approaches, including genetic, biochemical, transcriptomic, and morphological analyses, and convincingly demonstrates that perturbations in glycolysis alter sulfur metabolic pathways and thus impact pseudohyphal and hyphal differentiation. Overall, this work offers new and important insights into how metabolic fluxes are intertwined with fungal developmental programs and therefore opens new perspectives to investigate morphological transitioning in fungi.

Weaknesses:

A few aspects could be improved to strengthen the conclusions. Firstly, the striking transcriptomic changes observed upon 2DG treatment should be analyzed in S. cerevisiae adh1 and pfk1 deletion strains, for instance, through qPCR or western blot analyses of sulfur metabolism genes, to confirm that observed changes in 2DG conditions mirror those seen in genetic mutants. Secondly, differences between methionine and cysteine in their ability to rescue the mutant phenotype in both species are not mentioned, nor discussed in more detail. This is especially important as there seem to be differences between S. cerevisiae and C. albicans, which might point to subtle but specific metabolic adaptations.

The authors are also encouraged to refine several figure elements for clarity and comparability (e.g., harmonized axes in bar plots), condense the discussion to emphasize the conceptual advances over a summary of the results, and shorten figure legends.

Reviewer #3 (Public review):

This study investigates the connection between glycolysis and the biosynthesis of sulfur-containing amino acids in controlling fungal morphogenesis, using Saccharomyces cerevisiae and C. albicans as model organisms. The authors identify a conserved metabolic axis that integrates glycolysis with cysteine/methionine biosynthetic pathways to influence morphological transitions. This work broadens the current understanding of fungal morphogenesis, which has largely focused on gene regulatory networks and cAMP-dependent signaling pathways, by emphasizing the contribution of metabolic control mechanisms. However, despite the novel conceptual framework, the study provides limited mechanistic characterization of how the sulfur metabolism and glycolysis blockade directly drive morphological outcomes. In particular, the rationale for selecting specific gene deletions, such as Met32 (and not Met4), or the Met30 deletion used to probe this pathway, is not clearly explained, making it difficult to assess whether these targets comprehensively represent the metabolic nodes proposed to be critical. Further supportive data and experimental validation would strengthen the claims on connections between glycolysis, sulfur amino acid metabolism, and virulence.

Strengths:

(1) The delineation of how glycolytic flux regulates fungal morphogenesis through a cAMP-independent mechanism is a significant advancement. The coupling of glycolysis with the de novo biosynthesis of sulfur-containing amino acids, a requirement for morphogenesis, introduces a novel and unexpected layer of regulation.

(2) Demonstrating this mechanism in both S. cerevisiae and C. albicans strengthens the argument for its evolutionary conservation and biological importance.

(3) The ability to rescue the morphogenesis defect through exogenous supplementation of sulfur-containing amino acids provides functional validation.

(4) The findings from the murine Pfk1-deficient model underscore the clinical significance of metabolic pathways in fungal infections.

Weaknesses:

(1) While the link between glycolysis and sulfur amino acid biosynthesis is established via transcriptomic and proteomic analysis, the specific regulation connecting these pathways via Met30 remains to be elucidated. For example, what are the expression and protein levels of Met30 in the initial analysis from Figure 2? How specific is this effect on Met30 in anaerobic versus aerobic glycolysis, especially when the pentose phosphate pathway is involved in the growth of the cells when glycolysis is perturbed?

(2) Including detailed metabolite profiling could have strengthened the metabolic connection and provided additional insights into intermediate flux changes, i.e., measuring levels of metabolites to check if cysteine or methionine levels are influenced intracellularly. Also, it is expected to see how Met30 deletion could affect cell growth. Data on Met30 deletion and its effect on growth are not included, especially given that a viable heterozygous Met30 strain has been established. Measuring the cysteine or methionine levels using metabolomic analysis would further strengthen the claims in every section.

(3) In comparison with the previous bioRxiv (doi: https://doi.org/10.1101/2025.05.14.654021) of this article in May 2025 to the recent bioRxiv of this article (doi: https://doi.org/10.1101/2025.05.14.654021), there have been some changes, and Met30 deletion has been recently included, and the chemical perturbation of glycolysis has been added as new data. Although the changes incorporated in the recent version of the article improved the illustration of the hypothesis in Figure 6, which connects glycolysis to Sulfur metabolism, the gene expression and protein levels of all genes involved in the illustrated hypothesis are not consistently shown. For example, in some cases, the Met4 expression is not shown (Figure 4), and the Met30 expression is not shown during profiling (gene expression or protein levels) throughout the manuscript. Lack of consistency in profiling the same set of key genes makes understanding more complicated.

(4) The demonstrated link between glycolysis and sulfur amino acid biosynthesis, along with its implications for virulence in C. albicans, is important for understanding fungal adaptation, as mentioned in the article; however, the Met4 activation was not fully characterized, nor were the data presented when virulence was assessed in Figure 4. Why is Met4 not included in Figure 4D and I? Especially, according to Figure 6, Met4 activation is crucial and guides the differences between glycolysis-active and inactive conditions.

(5) Similarly, the rationale behind selecting Met32 for characterizing sulfur metabolism is unclear. Deletion of Met32 resulted in a significant reduction in pseudohyphal differentiation; why is this attributed only to Met32? What happens if Met4 is deleted? It is not justified why Met32, rather than Met4, was chosen. Figure 6 clearly hypothesizes that Met4 activation is the key to the mechanism.

(6) The comparative RT-qPCR in Figure 5 did not account for sulfur metabolism genes, whereas it was focused only on virulence and hyphal differentiation. Is there data to support the levels of sulfur metabolism genes?

(7) To validate the proposed interlink between sulfur metabolism and virulence, it is recommended that the gene sets (illustrated in Figure 6) be consistently included across all comparative data included throughout the comparisons. Excluding sulfur metabolism genes in Figure 5 prevents the experiment from demonstrating the coordinated role of glycolysis perturbation → sulfur metabolism → virulence. The same is true for other comparisons, where the lack of data on Met30, Met4, etc., makes it hard to connect the hypothesis. It is also recommended to check the gene expression of other genes related to the cAMP pathway and report them to confirm the cAMP-independent mechanism. For example, gap2 deletion was used to confirm the effects of cAMP supplementation, but the expression of this gene was not assessed in the RNA-seq analysis in Figure 2. It would be beneficial to show the expression of cAMP-related genes to completely confirm that they do not play a role in the claims in Figure 2.

(8) Although the NAC supplementation study is included in the new version of the article compared to the previous version in BioRxiv (May 2025), the link to sulfur metabolism is not well characterized in Figure 5 and their related datasets. The main focus of the manuscript is to delineate the role of sulfur metabolism; hence, it is anticipated that Figure 5 will include sulfur-related metabolic genes and their links to pfk1 deletion, using RT-PCR measurements as shown for the virulence genes.

(9) The manuscript would benefit from more information added to the introduction section and literature supports for some of the findings reported earlier, including the role of (i) cAMP-PKA and MAPK pathways, (ii) what is known in the literature that reports about the treatment with 2DG (role of Snf1, HXT1, and HXT3), as well as how gpa2 is involved. Some sentences in the manuscripts are repetitive; it would be beneficial to add more relevant sections to the introduction and discussion to clarify the rationale for gene choices.

Tumblr used to be a fun place for entertainment like jokes, memes, online hot trends and small online communities. But after new owners made big changes, many people left. The site lost the special feeling it once had. The article says Tumblr could get better again if it focuses on creativity and community like before.

I learned more about spam, which refers to the act of sending a large amount of unsolicited information through the Internet or communication platforms for advertising, fraud or other purposes. However, it is not only found in emails but also widely present on social media, text messages, forums and other platforms. Moreover, spam information has had a very negative impact on user experience and the online environment. Therefore, we need to formulate more regulations to restrict it.

Author response:

Reviewer #1 (Public review):

Summary:

The study by Yu et al investigated the role of protein N-glycosylation in regulating T-cell activation and functions is an interesting work. By using genome-wide CRISPR/Cas9 screenings, the authors found that B4GALT1 deficiency could activate expression of PD-1 and enhance functions of CD8+ T cells both in vitro and in vivo, suggesting the important roles of protein N-glycosylation in regulating functions of CD8+ T cells, which indicates that B4GALT1 is a potential target for tumor immunotherapy.

Strengths:

The strengths of this study are the findings of novel function of B4GALT1 deficiency in CD8 T cells.

Weaknesses:

However, authors did not directly demonstrate that B4GALT1 deficiency regulates the interaction between TCR and CD8, as well as functional outcomes of this interaction, such as TCR signaling enhancements.

We are very sorry that we did not highlight our results in Fig. 5f-h enough. In those figures, we demonstrated the interaction between TCR and CD8 increased significantly in B4GALT1 deficient T-cells, by FRET assays. To confirm the important role of TCR-CD8 interaction in mediating the functions of B4GALT1 in regulating T-cell functions, such as in vitro killing of target cells, we artificially tethered TCR and CD8 by a CD8β-CD3ε fusion protein and tested its functions in both WT and B4GALT1 knockout CD8⁺ T-cell. Our results demonstrate that such fusion protein could bypass the effect of B4GALT1 knockout in CD8⁺T-cells (Fig. 5g-h). Together with the results that B4GALT1 directly regulates the galactosylation of TCR and CD8, those results strongly support the model that B4GALT1 modulates T-cell functions mainly by galactosylations of TCR and CD8 that interfere their interaction.

Reviewer #2 (Public review):

Summary:

In this study, the authors identify the N-glycosylation factor B4GALT1 as an important regulator of CD8 T-cell function.

Strengths:

(1) The use of complementary ex vivo and in vivo CRISPR screens is commendable and provides a useful dataset for future studies of CD8 T-cell biology.

(2) The authors perform multiple untargeted analyses (RNAseq, glycoproteomics) to hone their model on how B4GALT1 functions in CD8 T-cell activation.

(3) B4GALT1 is shown to be important in both in vitro T-cell killing assays and a mouse model of tumor control, reinforcing the authors' claims.

Weaknesses:

(1) The authors did not verify the efficiency of knockout in their single-gene KO lines.

Thank reviewer for reminding. We verified the efficiency of some gRNAs by FACS and Surveyor assay. We will add those data in supplementary results in revised version later.

(2) As B4GALT1 is a general N-glycosylation factor, the phenotypes the authors observe could formally be attributable to indirect effects on glycosylation of other proteins.

please see response to reviewer #1.

(3) The specific N-glycosylation sites of TCR and CD8 are not identified, and would be helpful for site-specific mutational analysis to further the authors' model.

Thank reviewer for suggestion! Unfortunately, there are multiple-sites of TCR and CD8 involved in N-glycosylation (https://glycosmos.org/glycomeatlas). We worry that mutations of all these sites may not only affect glycosylation of TCR and CD8 but also other essential functions of those proteins.

(4) The study could benefit from further in vivo experiments testing the role of B4GALT1 in other physiological contexts relevant to CD8 T cells, for example, autoimmune disease or infectious disease.

Thank reviewer for this great suggestion to expand the roles of B4GALT1 in autoimmune and infection diseases. However, since in current manuscript we are mainly focusing on tumor immunology, we think we should leave these studies for future works.

benny morris was once known as a new historian who challenged zionist myths, gaining recognition for uncovering evidence that around 700,000 palestinians were expelled in 1948 and that israeli forces committed massacres and rapes--acts he linked to ben guirion's implicit message of transfer. however, as baruch kimmerling points out, morris made a drastic ideological shift in 2004 interview, arguing that "under some circumstances explusion is not a war crimes" and that ben guiron's mistake was not expelling all the palestinians. kimmerling criticizes this reversal, noting morris's own earlier work showed the refugee crisis to be the root of the conflict

I thought this was such an interesting response that is so important to the way that we currently consume and spread media and news. Sometimes people get so wrapped up in associating authors, styles of writing, or certain topics in extreme categories such "Zionist, Post-Zionist, or Anti-Zionist". These categories actually end up restricting and limiting people from reading other opinions or perspectives on issues and historical recounts because they are so wrapped up in the categorization of the work. People then choose to solely consume the things they think they should agree with and judge the others instead of learning from and witnessing multiple views and piecing together their own thoughts. These quick judgements or firm categorizations gather so much attention that they can actually take focus and discussion away from the actual important issues.

This one sentence makes the argument of language less about culture and more about politics.

Term + my working definition:

Role of language = how language functions as a tool of identity, power, inclusion/exclusion.

Why this matters: Frames the essay’s lens for analyzing Englishes.

Theres definitely a few things to consider in terms of reasons for this, as well as criticisms of the decision. Comments can be constructive but often people with complaints may argue them in a hateful or at least unproductive way. They may be insightful, though that likely accounts for very few. They may be entertaining, but that falls under unproductive and is of no benefit to the news organization.

I think this is a great idea and judging by how much I enjoyed reading this I think being able to research about more dialects and study them, but then write in them I don't think it would be just hard but also fun and interesting to learn more about.

this helps define what code meshing is and what it can be used for, but also explains that it's not just some small academic concept and that many people all over the world do it.

this sentence directly states when ideology the author is fighting against and clears any confusion in the text.

Implication: Raises audience-design and clarity concerns that the synthesis will address (how to mesh codes while ensuring mutual intelligibility).

Why this matters for my synthesis: I’ll weigh inclusivity/justice against practical comprehension and gatekeeping.

Vernacular as agency: Young’s code meshing aligns with Heller’s claim that AAVE conveys identity, confidence, and critique; both position vernacular forms as tools of voice and resistance.

What the example demonstrates: Journalistic prose mixing formal description with vernacular insertions—live code meshing in print.

How I will connect it later: identity/voice via vernacular (Young) / AAVE-as-agency (Heller).

Why this quote matters to my theme: Reframes “deficits” as listener prejudice, not speaker dialect.

Signal phrase I might use: Young contends, “It’s ATTITUDES.”

This whole section provides a direct solution to writers block but also provides a relatable experience about writers block making the text more entertaining to read and make the author seem more human. It also stands out compared to the rest of the text, while most of the text is spent identifying problems this section gives you a solution while also identifying the problems.

I this sentence hold a lot of power because the text brings the reader into it by saying that you hold the tools to align with the modern day standards but also have your own individuality when writing.

What certain scores mean. Healthy or unhealthy score depending on the number scored.

BJP now second largest party

giving more concessions to hindus but not letting the muslims back in

This supports the hypothesis, but it isn't a strong correlation, again.

courts still acknowledged that there was no historical hindu temple and that the acts commited to destroy the mosque were wrong, but still permitted the construction

building a temple in a place where a historical mosque was located till it was burned down by hindu mobs

This book covers topics in language education like heritage and minority languages, multicultural teaching, and how culture and identity tie into schooling. While most contributors are based in Western Australia, their research also looks at places like Singapore, Hong Kong, and Vietnam, studying languages from English and Chinese to Japanese, Malay, Tamil, Cantonese, an African musical language, and Aboriginal English.

The book is easy to read and uses lots of examples, but it doesn’t give many specific tips for teachers. Readers may finish the book still wondering exactly how to use translanguaging in real classrooms.

There aren’t strict rules for teaching with translanguaging, but the authors suggest things like pairing students by skill level, grouping them by language background, using words that sound similar in both languages, and using different ways to communicate. They stress the importance of being open, taking risks, and focusing on fairness and justice in the classroom. However, schools rarely teach or test translanguaging directly.

The authors explain that the way people think about language has changed over time. They mention that languages are not natural categories but are created by society, often to support things like colonialism or national identity we see even today.

García and Wei base their ideas on newer theories about language. They disagree with older thinkers who saw language as fixed and separate. Instead, they support the view that language is always changing and shaped by its social context

I totally agree. This part really stood out to me too. I’ve read this story before, but it still makes me think about how something as simple as a “male or female” button can show the limits built into technology. It’s crazy how designs like that assume everyone fits into just two options, when that’s not the case at all. It really opened my eyes to how bias can exist in everyday things like airport scanners.

So far, my research has involved tabling ingredients in ancient recipes. There is a certain level of extra granularity I need to provide to account for vague descriptions, but my initial model of separating ingredients created an absolute mess.

I think this intro sets up an important point we've talked about in class: SUDs are polygenic, so they are influenced my multiple genes at once, but their effects depend a lot on the environment. The same genotype can lead to different phenotypes depending on factors like stress, trauma, and other environmental exposures, partly through epigenetic changes (like methylation which we learned about in class) that turn certain genes up, down, on, or off. This makes me wonder how many high-risk alleles for SUDs only become problematic and take affect in specific environments, and how focusing on genetics alone gives an incomplete picture of who is actually vulnerable to SUDs.

This part really stood out to me because it shows how students find strength and creativity in resisting norms. Even with all the pressure from schools and families, they still create spaces of support. It also made me think about how acceptance depends not just on schools, but on home and community reactions too.

He states that schools need to pay attention to both racism and homophobia. McCready explains that some students face unfair treatment because of their race and because they are part of the LGBTQ+ community. Schools often focus on one issue but forget that these problems can happen together. He believes that teachers should work on stopping both racism and homophobia so that every student feels respected and safe in their school environment.

This shows how students' struggles aren't just because of her disability, but because of how others treat her and how little support she receives. The real problem is the way the school environment excludes her and fails to provide all the help she needs. Inclusion and proper support systems are so important and essential for students with disabilities to feel comfortable and accepted in schools.

Time can help bring change, but it’s not enough by itself. Real change needs action, patience, and constant effort. Every small step matters if we want hope to become reality.

Laws should protect everyone equally. If a law discriminates against a group, it stops being fair and must be challenged. When a minority has no voice, an unfair law can seem right, but it remains morally wrong.

By suppressing protests, people show that deep down they know they are wrong, but their pride stops them from admitting it. When someone feels superior, it becomes hard to recognize their own mistakes and try to change.

I can understand what he says: sometimes people judge demonstrations without worrying about their causes or the reasons that led to them. Especially because these protests are nonviolent, so they are not hurting anyone, they are just trying to protect their rights.

ok so this is a lot, i get where hes coming from but this is a ton of conjecture regarding the feeling of the baby. we can't ask this baby what's going on and what he thinks. the expression on one's face as they fall asleep is hardly enough evidence to propose a meaningful connection

i would like to know where he's getting this information. my instinct is not to believe him because ive never heard of tthis before. i mean, i can imagine it, but he's like "It Is Known That This Occurs" and im like , i didn't know! Nobody talks about this!! and again yes the cultural stigma. of course. but still

This was not well-known to me! well.. okay! really important here that freud uses the word unscrupulous to point out that the nurses here are doing A Bad Thing. this increases my trust in his pov here; not that i thought he was morally questionable simply for writing this essay--lord knows im interested in the topic myself--but like. You kinda, you wanna know! You wanna know where he stands

ok so sometimes sexual stuff breaks free from the latency period, and that i presume is what child sexual behavior looks like. im struggling to understand the second part i think... hes saying that educators dismiss child sexuality as a vice and deems these children ineducable but "we" (psychologists?) will turn to the topic without fear to search for the answer to the origin of sexuality in brains

period of latency = children havent hit puberty yet and cannot act on sexual feelings the way older people can. but the urges still exist, they just appear in alternate ways

This means Black LGBTQ youth may encounter racism and bullying at similar rates to other groups, but their treatment may be more severe. They simultaneously experience racism and homophobia, creating a more complex identity. Schools must better understand and improve how they support and protect these students.

Both require clear organization but differ in purpose (inform vs. argue).

Clarifies difference from other analytical methods.

Definition of synthesis; emphasizes purposeful combination and thesis focus.

Blend ideas from different sources into one big picture

This sentence distinguishes synthesis from other analytical methods like classification, comparison, contrast and division.

it may feel like an extra step along the way but the final draft process is ay easier when you prep for it.

make your voice the loudest in the text. you should have enough research and understanding of the topic to construct a thesis-driven synthesis

not just comparing the sources but connecting them and finding common themes between them.

Use this to refer back to

The study began with 75 patients but lost 25 (a third of the initial sample) to attrition. This high dropout rate is a notable methodological limitation.

This identifies the exact "gap in the literature" that my research project is trying to fill. They know they're linked, but not how they develop.

The argument then develops by introducing a psychoanalytic framework, drawing on Homi Bhabha to analyze the treatment of Fokir's "indigenous canny" within the nation-state's dominant cultural discourse. Nayar suggests that Fokir's intimate, local knowledge is the psychic reality of "repression," meaning it is hidden or submerged beneath official narratives. Conversely, "surmounting" is the term used for the process where the postcolonial nation acts through its "cultural unconscious" to erase and reject this indigenous canny. This erasure is seen as a form of "postcolonial colonization" driven by forces like development and technologization that cause displacement.

This section establishes the physical setting of the novel as the foundation for Nayar's argument about the postcolonial uncanny. The core idea here is that the Sundarbans, with its constant fluidity, being "now-land, now-water", and its inherent dangers from predators, fundamentally prevents any settlement from becoming a true "home." ​The key takeaway is a definition of home as requiring stability, security, and freedom from fear, conditions which the Sundarbans actively denies its inhabitants. It is precisely this impossibility of dwelling, the tension between the desire for home and the reality of homelessness in a physically unstable environment, that gives rise to the postcolonial uncanny. The region itself is inherently "unhomely," making any presence within it uncanny.

I think that the content review by social media platforms is not only for quality control, but also for legal and security considerations. They must remove illegal content, and platforms like YouTube have always been controversial over the balance between copyright enforcement and the rights of creators. At the same time, the review mechanism also helps to reduce cyber violence and thus maintain the user base of the platform.

I totally agree what it said, social media platform have to prohibit the trolling, scam posts for every users. Also, nowadays, not only the posts, but also the comments under the posts needs to control. For example, My friend posted some thoughts praising celebrities on her social media, but some of the celebrities' fans disagreed and started attacking her in the comments. The system then automatically deletes some of these comments. I think this is a great way to protect posters from attacks and it makes the social media environment better.

They're acknowledging it here. It's not "bad parents" causing it, but the challenges of parenting an ADHD child that strains the bond.

This finding shows insecure attachment isn't universal in ADHD, but is a key factor when anxiety is also present. This refines my project by suggesting insecure (avoidant) attachment may be a critical variable that mediates the link between ADHD and anxiety.

This is the likely goal of my project. I had some weak foundational material to begin with, but simply making the attempt and getting over a want of total success might help me get a better perspective on my research and on me as a person.

LCGs are not the only type of PRNGs. They have some good qualities, they have pretty good randomness qualities, they're fast, and they don't require much memory, but they're not good enough for certain computing operations, such as cryptography or encryption, and certain types of simulation problems, and for those applications, stronger PRNGs are used

Let’s add mod to our formula. But we’ll use mod 13 (instead of mod 12).

For example, if we had a PRNG doing X sub i is 10, then X sub i plus 1 is 10 times 2 plus 1 is 21, then mod 13 gives us 8. If X sub i is 8, X sub i times 2 plus 1 is 17 mod 13 is 4. If we take 4 as our input, we get 9 and then 6 and then 0, so forth, so if we continue in this way, we will produce another sequence of numbers.

The numbers jump around seemingly randomly between 0 and 11. But notice that the sequence repeats when it hits 10, so it repeats after a very short time. And, it can’t generate a 12. It just wouldn't be a very good PRNG.

Clock Arithmetic

In a 12 hour clock (not 24 hour clock)

9:00 o’clock + 4 hours = 1:00 o’clock (not 13 o’clock)

9 + 4 = 13 - 12 = 1

11:00 o’clock + 5 hours = 4:00 o’clock (not 16 o’clock)

11 + 5 = 16 - 12 = 4

In a 12 hour clock (not 24 hour clock)

11:00 o’clock + 20 hours = ?

11 + 20 = 31 31 - 12 = 19, but no 19 o’clock, so 19 - 12 = 7

[[Julia Ravanis]] in [[Skönheten i Kaos by Julia Ravanis]] is here said to argue that a way of moving past 'quantum mechanics does not make sense' is by letting go of default (visual) metaphors and using other metaphors that can embrace ambiguity. This sounds somewhat like [[Is het nieuwe uit te leggen in taal van het oude 20031104104340]] or even [[Avoid greedy reductionism 20041114065928]] accusation levelled here at Einstein.

A correlation in higher suicide rates and increased hyper-methylation of serotonin receptors, another essential neurotransmitter, is not only extremely scary, but also does make sense because of serotonin's key functions as a communicator. Specifically, it is associated with mood correlation, learning and memory (see annotation above), and sleep. Going off of the data presented in above paragraphs, this evidence strengthens the claim that Schizophrenia is heavilly associated with memory and learning blockage.

The correlation with a memory BDNF gene being methylated and the expression of not even just Schizophrenia, but many other mental disorders in general, raises questions of just how important the ability to learn and maintain information is relevant to dangerous mental disorders. This also makes me question what other genes associated with memory, learning, and neuronal development could be impacting the development of diseases such as Schizophrenia.

A large issue for me, as my network analysis and visualization all depend on me establishing a good data table that is accurate. An incorrect translation of the source or a typo on my end could be equally culpable for false results.

While there are some questions I still have over the different terminologies, Network Navigator simply displays what data I provide it. Given that I am preparing all the data myself, it is entirely transparent to me. You paste it in and hit a button, and it shows it all as a connected chart. I appreciate the tooltips for the different views, as it increases the accessibility significantly. Those data tables, however, are another issue of complexity entirely.

I'm essentially using a calculator to summarize a data table and show me a graph. It serves a function in that it allows me to tackle the broader subject, but it does not "think" for itself.

Perhaps I should learn to prepare a memo, write a brief, or design a proposal forms of research concept. It could be essential, but difficult.

I suspect the essential feedback, but these reviews can be consistent to my findings.

It is often determined whether the numerous database matches the information. But if I do understand how to present the support to my decisions, it will be shown to my comprehension.

It might be worth a description, but I do know about Gibbs and Johnson's evidence about Lighton's knowledge.

The academic research essay can be challenging, but important as a result. I should help myself to practice many skills what I learned to be a expert reader.

I could admire, even I doubt to require to synthesize a large amount of sources comes from conversations that needed to be taken.

Questions or issues were important, but first off, the factor is either reliable or not reliable, and second, the consideration could be challenging to determine whether the source converges the new information.

It could be a major identical issues, but rather than perfect spot for numbers.

Controversial question can be important to my academic research.

Any flickr download link is available in account settings, but for a limited time.

for - metaphor - pinky and thumb - connectedness

for - language - nondual - as the reference system

Description of moving away from Spotify to Qobuz. Spotify is Swedish but in the spotlight for hosting ICE ads in the USA at the moment. Qobuz is French, but hosts their stuff on AWS.

the authors acknowledge sampling limits but still emphasize real-world diagnostic overlap relevant for psychotherapy and medication management.

this helps to clarify that narcissistic features in ADHD emerge from the impulsivity/hyperactivity dimension rather than from attention deficits.

the core finding - It separates narcissistic behavior (planned, "proactive") from ADHD's core impulsivity ("reactive").

holy worship sites are here its very important

for - search - Google - Can we unlearn language? - https://www.google.com/search?sca_esv=869baca48da28adf&sxsrf=AE3TifMGTNfpTekWWBdYUA96_PTLS9T00A:1762658867809&q=can+we+unlearn+language%3F&source=lnms&fbs=AIIjpHxU7SXXniUZfeShr2fp4giZ1Y6MJ25_tmWITc7uy4KIegmO5mMVANqcM7XWkBOa06dn2D9OWgTLQfUrJnETgD74qUQptjqPDfDBCgB_1tdfH756Z_Nlqlxc3Q5-U62E4zbEgz3Bv4TeLBDlGAR4oTnCgPSGyUcrDpa-WGo5oBqtSD7gSHPGUp_5zEroXiCGNNDET4dcNOyctuaGGv2d44kI9rmR9w&sa=X&ved=2ahUKEwj4_LP9j-SQAxVYXUEAHVT8FfMQ0pQJegQIDhAB&biw=1920&bih=911&dpr=1 search results returned - interesting - to - article - Can You Unlearn A Language? - IFLScience It's definitely possible to lose fluency in your native language, but research suggests you're unlikely to forget it altogether. - https://hyp.is/MdiWar0dEfC4ajvO0fJCkA/www.iflscience.com/can-you-unlearn-a-language-70874 - from - Linkedin post - John Vervaeke - https://hyp.is/pIMO8rzIEfCPtcvbQ8nTxg/www.linkedin.com/feed/update/urn:li:activity:7392196128005537792/

new search prompt - This prompt did not give me the results I was looking for - Need to refine the prompt - Can an adult who has learned language experience pre-linguistic reality like an infant who hasn't learned language yet? - to - new search prompt - can an adult who has learned language experience pre-linguistic reality like an infant who hasn't learned language yet? -

Method/evidence: Essayistic argument using experiential and anecdotal evidence (teaching tenure, professional roles, stories).

What this lets the author prove (and what it can’t): It shows practitioner perspective and patterns he’s observed, but not generalized causal proof from empirical studies.

I feel like the author is so close to the point, but is still missing it juts a tad because of his delivery through out the paper.

There is always a time and place for certain attitude, way of speaking, etc. The way the lady reponded to him was unprofessional, she should have caught that before she sent it.

You have to decide for yourself which end you would rather be on.

As stated at the beginning of the paper, the author mentioned SAE others have called it racist, however I have to agree, it's not the language it is who uses it and how it is used that can been seen that way. As well as who created SAE and why.

I'm not sure how I feel about this. I suppose the author wrote "Educated American's" because that is the subject of the article, but again in my opinion, it is starting to sound harsh.

Does he really understand the importance of this claim? The author says he "sympathises" but then goes on to write about (what I view) as imposing Standard English onto others.

The author aims to rebut anti-SAE arguments and advocate for requiring SAE mastery.

yes, please

Totally fair--BUT, should a religious extremist have the same right, re: wedding cakes and Plan B prescriptions?

I'm not sure about this one! But I'm probably being defensive!

But what about accountability and keeping receipts?

But how can you predict?

Wow--should that matter? But I guess it does because people can use the materials to create (more) harm

Main focus should be you but it should be broader than that with the ability for your readers to relate as well

reply to u/SlumberCrow at https://reddit.com/r/typewriters/comments/1orwxqq/type_writer_leaving_small_divets_in_paper_when/

A new platen will certainly help, but it's also a question of having a proper ring and cylinder adjustment across the length of your platen and segment. Often letters that punch through tend to be the . , and o which are at the extreme end of the segment. Some machines have adjustment screws at either end of the carriage and the adjustment should be checked at not only the center of the platen but both ends. If you don't have an experienced mechanic who knows how to do all of this properly you can easily get issues which will most often show up at the far ends of the the segment/platen.

Beyond a proper adjustment, it's also the case that the surface area of the . and , are smaller than other characters and so they tend to get more force even when actuated by the weaker fingers on the right hand when touch typing. Some older manuals and training films will suggest putting less pressure on these keys when typing. This is likely even more important for those who hunt-and-peck and are likely using the full force of their index fingers.

Unless your ribbon is obviously dry or marginal, replacing your ribbon isn't likely to help much. Slugs are made out of hardened steel and you'd have to do something incredibly drastic to damage the slugs, so don't sweat that too much. Backing sheet will help as a stop-gap particularly on machines with older/hardened platens, but there's only so much help that will do without a good platen and a properly adjusted machine.

reply to question about tension control at https://reddit.com/r/typewriters/comments/1orxtvt/strange_lever/

Joe Van Cleave has a great video on this with respect to the Smith-Corona 5 series that will give one an idea on the entirety of adjustment points that are at play in some typewriters. https://www.youtube.com/watch?v=OYOXgqiHBmg

Personally, I've yet to run across a vintage Series 5 machine whose user-facing control lever was adjusted in a way such that it did anything at all because the linkages were so far out of whack. I suspect this may be the case on a lot of vintage machines.

On some machines the adjustment isn't controlling the amount of finger force one must apply, but it's controlling springs relating more to the return of the typebars and the slugs so that touch typers can type much faster without having issues with typebar return jamming things up.

Further, on many machines the dynamic range of forces involved is so narrow that most hobbyist and occasional typists aren't going to really notice a significant difference. This may be different for those who are more experienced and used to typing on a manual machine for several hours a day.

for - adjacency - learning - unlearning - ritual - language - BEing journey - question - Could we apply ritual to unlearn language? - quote - Your brain is incredible at pattern recognition. But this superpower has a dark side: - Once you see a pattern, it becomes incredibly hard to "unsee" it. - You become trapped in your own mental models - John Vervaeke

adjacency - learning - unlearning - ritual - language - BEing journey - Could we apply ritual to break the pattern of language? This could be an interesting BEing journey!

Richard Sutton says that in the long run, general-purpose, compute-driven learning methods beat hand-crafted, human-designed rules. This pushes us to think of modern AI success as a systems engineering achievement rather than a clever human knowledge design achievement. Why is this a "bitter lesson"? because it hurts our ego. We love to inject expert knowledge, handcrafted features, tricks, etc. but historically (e.g. chess, Go, speech recognition, NLP, and more) whenever the field matured, the handcrafted approaches got crushed by large-scale compute-heavy general algorithms that learned directly from data.

From this discussion, it’s clear that using Allegro Pay isn’t inherently bad, but it can be disadvantageous in certain situations — especially if you plan to apply for a mortgage or larger bank loan soon. Here are the key takeaways:

🟢 When using Allegro Pay is fine

• If you repay everything on time (e.g., within 30 days) and don’t have many active purchases at once.
• If you use it occasionally, for example to avoid freezing your cash or to grab a promotion (like a 30 PLN coupon).
• If you’re not planning any major loans soon, small transactions usually don’t make much difference.

🔴 When you should AVOID Allegro Pay

• If you plan to take out a mortgage or consumer loan within the next few months – banks see these purchases in your credit report (BIK) as short-term loans (BNPL), sometimes even classified as payday loans.
• Each purchase can appear as a separate entry in your BIK, so frequent usage might look like a large number of small loans, which can lower your credit score and borrowing capacity.
• Some banks (e.g., BNP Paribas, Pekao, Millennium – according to users’ reports) have automated systems that reduce creditworthiness if multiple BNPL loans are active.

⚖️ Summary

• For a one-time promotion (e.g., 30 PLN coupon) – it’s okay to use, but repay immediately after the purchase.
• Avoid regular use if you’re planning a mortgage or major loan within the next 12–24 months.
• Allegro Pay is visible in BIK, so even small transactions leave a footprint in your credit history.

👉 Conclusion: If you want to maintain a spotless credit profile — avoid Allegro Pay. If you’re not planning any big loans — occasional use is perfectly fine, as long as you repay on time

The focus is not on content creation and management

but articulation/formulation of ideas externalization of human peron-first interpersonal intellect individually and co-laboratively scaling autonomous Human Mutual Learning symmathesy sacling significance, salient synthesis and reach

regular expression

Just from what I am reading on this section it seems that the right wing is trying to take a bigger grasp on school by removing certain things that are essential but do not align with their plans.

This quote highlights the explicit linking of national identity and religion in the document’s vision: it doesn’t merely propose religious freedom, but privileges a specific religious identity (Christianity) as foundational to society and governance. The blending of “America must remain Christian” with “the government must take steps to ensure this” suggests a reversal of church-state separation, pushing toward a state endorsed religious culture.

They are not only just trying to get rid of DEI programs but they are also trying silence those who do not agree with their ideas. The jobs of those who dare speak against them are at risk just based on their opinions.

In today's world of increasing globalization, more countries are establishing their roles in the international community more so than ever before. In the past the world was ruled by empires that took up large territories in comparison to today being ruled by many countries with varying amounts of power and so there is more cooperation between countries. However, to become a superpower, the same things apply today as at any point in history. Two of the most important resources in being a global superpower is having a large population and territory which Russia most definitely has both. The value of having a large territory and population would separate Russia ahead from most countries in the world and in the same category as the U.S, China, and others.

Spasskiy’s idea of Russia using the approach of being its own island on the world stage is how to deal with the 21st century world. As foreign affairs without the hegemony of two superpowers like in the cold war, makes the world more unpredictable as there are not two big sides to pick but several powers both big and small. Spasskiy writes how Russia was a global superpower as the Soviet Union but for much of its history Russia was not a superpower. Today Russia has many advantages in becoming a superpower in the 21st century like its natural resources but still faces a number of struggles such as its demographic problem. With Russia acting as an island, they can modernize while still maintaining the status as a global power that they already have.

AGAIN NOT A NEW WAY TO USE - THE BOTS ALSO ALREADY DO THIS

The title, "Let the best story win," is a strong instance of overgeneralization and implies a direct causal link. The data only supports a correlation—that highly-cited articles tend to have strong narratives. Other confounds, such as the journal's prestige, the seniority of the authors, or the simple age of the paper, are also correlated with citation count, but the title and likely the conclusion prioritize narrative as the key driving factor for success.It is a classic mistake of mistaking correlation for causation. While a compelling story might be present in a highly-cited work, it is impossible to claim it caused the citation count without a controlled study (which is impossible in this context). The overgeneralization suggests that any well-told story will become highly cited, ignoring the foundational requirement that the story must also be historically accurate, logically argued, and published in a reputable venue.

This study inherently suffers from Selection Bias because the researchers deliberately chose to study only the most cited articles. By pre-selecting for success (high citations), they cannot draw conclusions about the factors that distinguish successful papers from unsuccessful ones across the whole population. Their findings are limited to the characteristics shared among successful papers, confirming what is already accepted, rather than challenging the metrics of success themselves.

The bias affects the findings by reinforcing the status quo within the field. If "storytelling" is a trait shared by the most-cited papers, the study's conclusion will encourage future researchers to focus on storytelling, potentially at the expense of methodological innovation or empirical depth. This creates a self-fulfilling prophecy and could lead to a field where narrative appeal is prioritized over scientific substance, simply because the study did not analyze a random sample of all published works.

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

Learn more at Review Commons

Reply to the reviewers

Response to Reviewer’s Comments

We thank all three reviewers for their thoughtful and detailed comments, which will help us to improve the quality and clarity of our manuscript.

__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __ Summary: In this work, Tripathi et al address the open question of how the Fat/Ds pathway affects organ shape, using the Drosophila wing as a model. The Fat/Ds pathway is a conserved but complex pathway, interacting with Hippo signalling to affect growth and providing planar cell polarity that can influence cellular dynamics during morphogenesis. Here, authors use genetic perturbations combined with quantification of larval, pupal, and adult wing shape and laser ablation to conclude that the Ft/Ds pathway affects wing shape only during larval stages in a way that is at least partially independent of its interaction with Hippo and rather due to an effect on tissue tension and myosin II distribution. Overall the work is clearly written and well presented. I only have a couple major comments on the limitations of the work.

Major comments: 1. Authors conclude from data in Figures 1 and 2 that the Fat/Ds pathway only affects wing shape during larval stages. When looking at the pupal wing shape analysis in Figure 2L, however, it looks there is a difference in wt over time (6h-18h, consistent with literature), but that difference in time goes away in RNAi-ds, indicating that actually there is a role for Ds in changing shape during pupal stages, although the phenotype is clearly less dramatic than that of larval stages. No statistical test was done over time (within the genotype), however, so it's hard to say. I recommend the authors test over time - whether 6h and 18h are different in wild type and in ds mutant. I think this is especially important because there is proximal overgrowth in the Fat/Ds mutants, much of which is contained in the folds during larval stages. That first fold, however, becomes the proximal part of the pupal wing after eversion and contracts during pupal stages to elongate the blade (Aiguoy 2010, Etournay 2015). Also, according to Trinidad Curr Biol 2025, there is a role for Fat/Ds pathway in pupal stages. All of that to say that it seems likely that there would be a phenotype in pupal stages. It's true it doesn't show up in the adult wing in the experiments in Fig 1, but looking at the pupal wing itself is more direct - perhaps the very proximal effect is less prominent later, as there is potential for further development after 18hr before adulthood and the most proximal parts are likely anyway excluded in the analysis.

Response: Our main purpose in examining pupal wing shape was to emphasize that wings lacking ds are visibly abnormal even at early pupal stages. The reviewer makes the point that the change in shape from 6h to 18h APF is greater in control wings than in RNAi-ds wings. We have added quantitation of this to the revised manuscript as suggested. This difference could be interpreted as indicating that Ds-Fat signaling actively contributes to wing shape during pupal morphogenesis. However, given the genetic evidence that Ds-Fat signaling influences wing shape only during larval growth, we favor the interpretation that it reflects consequences of Ds-Fat action during larval stages – eg, overgrowth of the wing, particularly the proximal wing and hinge as occurs in ds and fat mutants, could result in relatively less elongation during the pupal hinge contraction phase. This wouldn’t change our key conclusions, but it is something that we discuss in a revised manuscript.

I think there needs to be a mention and some discussion of the fact that the wing is not really flat. While it starts out very flat at 72h, by 96h and beyond, there is considerable curvature in the pouch that may affect measurements of different axis and cell shape. It is not actually specified in the methods, so I assume the measurements were taken using a 2D projection. Not clear whether the curvature of the pouch was taken into account, either for cell shape measurements presented in Fig 4 or for the wing pouch dimensional analysis shown in Fig 3, 6, and supplements. Do perturbations in Ft/Ds affect this curvature? Are they more or less curved in one or both axes? Such a change could affect the results and conclusions. The extent to which the fat/ds mutants fold properly is another important consideration that is not mentioned. For example, maybe the folds are deeper and contain more material in the ds/fat mutants, and that's why the pouch is a different shape? At the very least, this point about the 3D nature of the wing disc must be raised in discussion of the limitations of the study. For the cell shape analysis, you can do a correction based on the local curvature (calculated from the height map from the projection). For the measurement of A/P, D/V axes of the wing pouch, best would be to measure the geodesic distance in 3D, but this is not reasonable to suggest at this point. One can still try to estimate the pouch height/curvature, however, both in wild type and in fat/ds mutants.

Response: The wing pouch measurements were done on 2D projections of wing discs that were already slightly flattened by coverslips, so there is not much curvature outside of the folds. We will revise the methods to make sure this is clear. While we recognize that the absolute values measured can be affected by this, our conclusions are based on the qualitative differences in proportions between genotypes and time points, and we wouldn’t expect these to differ significantly even if 3D distances were measured. Obtaining accurate 3D measures is technically more challenging - it requires having spacers matching the thickness of the wing disc, which varies at different time points and genotypes, and then measuring distances across curved surfaces. What we propose to address this is to do a limited set of 3D measures on wild-type and dsmutant wing discs at early and late stages and which we expect will confirm our expectation that the conclusions of our analysis are unaffected, while at the same time providing an indication of how much curvature affects the values obtained. We will also make sure the issue of wing disc curvature and folds is discussed in the text.

Minor comments: 1. The analysis of the laser ablation is not really standard - usually one looks at recoil velocity or a more complicated analysis of the equilibrium shape using a model (e.g Shivakumar and Lenne 2016, Piscitello-Gomez 2023, Dye et al 2021). One may be able to extract more information from these experiments - nevertheless, I doubt the conclusions would change, given that that there seems to be a pretty clear difference between wt and ds (OPTIONAL).

Response: We will add measurements of recoil velocities to complement our current analysis of circular cuts.

Figure 7G: I think you also need a statistical test between RNAi-ds and UAS-rokCA+RNAi-ds.

Response: We include this statistical test in the revised manuscript (it shows that they are significantly different).

In the discussion, there is a statement: "However, as mutation or knock down of core PCP components, including pk or sple, does not affect wing shape... 59." Reference 59 is quite old and as far as I can tell shows neither images nor quantifications of the wing shape phenotype (not sure it uses "knockdown" either - unless you mean hypomorph?). A more recent publication Piscitello-Gomez et al Elife 2023 shows a very subtle but significant wing shape phenotype in core PCP mutants. It doesn't change your logic, but I would change the statement to be more accurate by saying "mutation of core PCP components has only subtle changes in adult wing shape"

Response: Thank-you for pointing this out, we have revised the manuscript accordingly.

**Referee cross-commenting**

Reviewer2: Reviewer 2 makes the statement: "The distance along the AP boundary from the pouch border to DV midline is topologically comparable to the PD length of the adult wing. The distance along the DV boundary from A border to P border is topologically comparable to the AP length of the adult wing."

I disagree - the DV boundary wraps around the entire margin of the adult wing (as correctly drawn with the pink line in Fig 2A). It is not the same as the wide axis of the adult wing (perpendicular to the AP boundary). It is not trivial to map the proximal-distal axis of the larval wing to the proximal-distal axis of the adult, due to the changes in shape that occur during eversion. Thus, I find it much easier to look at the exact measurement that the authors make, and it is much more standard in the field, rather than what the reviewer suggests. Alternatively, one could I guess measure in the adult the ratio of the DV margin length (almost the circumference of the blade?) to the AP boundary length. That may be a more direct comparison. Actually the authors leave out the term "boundary" - what they call AP is actually the AP boundary, not the AP axis, and likewise for the DV - what they measure is DV boundary, but I only noticed that in the second read-through now. Just another note, these measurements of the pouch really only correspond to the very distal part of the wing blade, as so much of the proximal blade comes from the folds in the wing disc. Therefore, a measurement of only distal wing shape would be more comparable.

Response: We thank Reviewer 1 for their comments here. In terms of the region measured, we measure to the inner Wg ring in the disc, the location of this ring in the adult is actually more proximal than described above (eg see Fig 1B of Liu, X., Grammont, M. & Irvine, K. D. Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge. Developmental Biology 228, 287–303 (2000)), and this defines roughly the region we have measured in adult wings (with the caveat noted above that the measurements in the disc can be affected by curvature and the hinge/pouch fold, which we will address).

Reviewer 2 states that authors cannot definitively conclude anything about mechanical tension from their reported cutting data because the authors have not looked at initial recoil velocity. I strongly disagree. __The wing disc tissue is elastic on much longer timescales than what's considered after laser ablation (even hours), and the shape of the tissue after it equilibrates from a circular cut (1-2min) can indeed be used to infer tissue stresses (see Dye et al Elife 2021, Piscitello-Gomez et al eLife 2023, Tahaei et al arXiv 2024).__ In the wing disc, the direction of stresses inferred from initial recoil velocity are correlated with the direction of stresses inferred from analysing the equilibrium shape after a circular cut. Rearrangements, a primary mechanism of fluidization in epithelia, does not occur within 1'. Analysing the equilibrium shape after circular ablation may be more accurate for assessing tissue stresses than initial recoil velocity - in Piscitello-Gomez et al 2023, the authors found that a prickle mutation (PCP pathway) affected initial recoil velocity but not tissue stresses in the pupal wing. Such equilibrium circular cuts have also been used to analyze stresses in the avian embryo, where it correlates with directions of stress gathered from force inference methods (Kong et al Scientific Reports 2019). The Tribolium example noted by the reviewer is on the timescale of tens to hundreds of minutes - much longer than the timescale of laser ablation retraction. It is true the analysis of the ablation presented in this paper is not at the same level as those other cited papers and could be improved. But I don't think the analysis would be improved by additional experiments doing timelapse of initial retraction velocity.

Response: Thank-you, we agree with Reviewer 1 here.

Reviewer 2 states "If cell anistropy is caused by polarized myosin activity, that activity is typically polarized along the short edges not long edges" Not true in this case. Myosin II accumulates along long boundaries (Legoff and Lecuit 2013). "Therefore, interpreting what causes the cell anistropy and how DS regulates it is difficult," Agreed - but this is well beyond the scope of this manuscript. The authors clearly show that there is a change of cell shape, at least in these two regions. Better would be to quantify it throughout the pouch and across multiple discs. Similar point for myosin quantifications - yes, polarity would be interesting and possible to look at in these data, and it would be better to do so on multiple discs, but the lack of overall myosin on the junctions shown here is not nothing. Interpreting what Ft/Ds does to influence tension and myosin and eventually tissue shape is a big question that's not answered here. I think the authors do not claim to fully understand this though, and maybe further toning down the language of the conclusions could help.

Response: We agree with Reviewer 1 here and will also add quantitation of myosin across multiple discs and will include higher magnification myosin images and polarity tests.

Reviewer 3: I agree with many of the points raised by Reviewer 3, in particular that relevant for Fig 1. The additional experiments looking at myosin II localization and laser ablation in the other perturbations (Hippo and Rok mutants/RNAi) would certainly strengthen the conclusions.

Response: Reviewer 3 comment on Fig 1 requests Ab stains to assess recovery of expression after downshift, which we will do.

We will add examination of myosin localization in hpo RNAi wing discs, and in the ds/rok combinations. We note that the effects of Rok manipulations on myosin and on recoil velocity have been described previously (eg Rauskolb et al 2014).

Reviewer #1 (Significance (Required)): I think the work provides a clear conceptual advance, arguing that the Ft/Ds pathway can influence mechanical stress independently of its interaction with Hippo and growth. Such a finding, if conserved, could be quite important for those studying morphogenesis and Fat function in this and other organisms. For this point, the genetic approach is a clear strength. Previous work in the Drosophila wing has already shown an adult wing phenotype for Ft/Ds mutations that was attributed to its role in the larval growth phase, as marked clones show aberrant growth in mutants. The novelty of this work is the dissection of the temporal progression of this phenotype and how it relates to Hippo and myosin II activation. It remains unclear exactly how Ft/Ds may affect tissue tension, except that it involves a downregulation of myosin II - the mechanism of that is not addressed here and would involve considerable more work. I think the temporal analysis of the wing pouch shape was quite revealing, providing novel information about how the phenotype evolves in time, in particular that there is already a phenotype quite early in development. As mentioned above, however, the lack of consideration of the wing disc as a 3D object is a potential limitation. While the audience is likely mostly developmental biologists working in basic research, it may also interest those studying the pathway in other contexts, including in vertebrates given its conservation and role in other processes.

__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ The manuscript begins with very nice data from a ts sensitive period experiment. Instead of a ts mutation, the authors induced RNAi in a temperature dependent manner. The results are striking and strong. Knockdown of FT or DS during larval stages to late L3 changed shape while knockdown of FT or DS during later pupal stages did not. This indicates they are required during larval, not pupal stages of wing development for this shape effect. They did shift-up or shift-down at "early pupa stage" but precisely what stage that means was not described anywhere in the manuscript. White prepupal? Time? Likewise a shift-down was done at "late L3" but that meaning is also vague. Moreover, I was surprised to see they did not do a shift-up at the late L3 stage, to give completeness to the experiment. Why?

Response: We have added more precise descriptions of the timing, and we will also add the requested late L3 shift-up experiment.

Looking at the "shape" of the larval wing pouch they see a difference in the mutants. The pouch can be approximated as an ellipse, but with differing topology to the adult wing. Here, they muddled the analysis. The adult wing surface is analogous to one hemisphere of the larval wing pouch, ie., either dorsal or ventral compartment. The distance along the AP boundary from the pouch border to DV midline is topologically comparable to the PD length of the adult wing. The distance along the DV boundary from A border to P border is topologically comparable to the AP length of the adult wing. They confusingly call this latter metric the "DV length" and the former metric the "AP length" , and in fact they do not measure the PD length but PD+DP length. Confusing. Please change to make this consistent with earlier analysis of the adult and invert the reported ratio and divide by two.

Then you would find the larval PD/AP ratio is smaller in the FT and DS mutants than wildtype, which resembles the smaller PD/AP ratio seen in the mutant adult wings. Totally consistent and also provides further evidence with the ts experiments that FT and DS exert shape effects in the larval phase of life.

Response: As noted by Reviewer 1 in cross-referencing, some of the statements made by Reviewer 2 here are incorrect, eg “The distance along the DV boundary from A border to P border is topologically comparable to the AP length of the adult wing.” They are correct where they note that the A-P length we measure in the discs is actually equivalent to 2x the adult wing length, since we are measuring along both the dorsal and ventral wing, but this makes no difference to the analysis as the point is to compare shape between time points and genotypes, not to make inferences based on the absolute numbers obtained. The numerical manipulations suggested are entirely feasible but we think they are unnecessary.

The remainder of the manuscript has experimental results that are more problematic, and really the authors do not figure out how the shape effect in larval stages is altered. I outline below the main problems.

1. They compare the FT DS shape phenotypes to those of mutants or knockdowns in Hippo pathway genes (Hippo is known to be downstream of FT and DS). They find these Hippo perturbations do have shape effects trending in same direction as FT and DS effects. Knockdown reduces the PD/AP ratio while overexpressing WARTS increases the PD/AP ratio. The effect magnitudes are not as strong, but then again, they are using hypomorphic alleles and RNAi, which often induces partial or hypomorphic phenotypes. The effect strength is comparable when wing pouches are young but then dissipates over time, while FT and DS effects do not dissipate over time. The complexity of the data do not negate the idea that Hippo signaling is also playing some role and could be downstream of FT and DS in all of this. But the authors really downplay the data to the point of stating "These results imply that Ds-Fat influences wing pouch shape during wing disc growth separately from its effects on Hippo signaling." I think a more expansive perspective is needed given the caveats of the experiments.

Response: Our results emphasize that the effects of Ds-Fat on wing shape cannot be explained solely by effects on Hippo signaling, eg as we stated on page 7 “These observations suggest that Hippo signaling contributes to, but does not fully explain, the influence of ds or fat on adult wing shape.” We also note that impairment of Hippo signaling has similar effects in younger discs, but very different effects in older discs, which clearly indicates that they are having very different effects during disc growth; we will revise the text to make sure our conclusions are clear.

              The reviewer wonders whether some of the differences could be due to the nature of the alleles or gene knockdown. First, the *ex*, *ds*, and *fat* alleles that we use are null alleles (eg see FlyBase), so it is not correct to say that we use only hypomorphic alleles and RNAi. We do use a hypomorphic allele for wts, and RNAi for hpo, for the simple reason that null alleles in these genes are lethal, so adult wings could not be examined. A further issue that is not commented on by the reviewer, but is more relevant here, is that there are multiple inputs into Hippo signaling, so of course even a null allele for ex, ds or fat is not a complete shutdown of Hippo signaling. Nonetheless, one can estimate the relative impairment of Hippo signaling by measuring the increased size of the wings, and from this perspective the knockdown conditions that we use are associated with roughly comparable levels of Hippo pathway impairment, so we stand by our results. We do however, recognize that these issues could be discussed more clearly in the text, and will do so in a revised manuscript.

Puzzlingly, this lack of taking seriously a set of complex results does not transfer to another set of experiments in which they inhibit or activate ROK, the rho kinase. When ROK is perturbed, they also see weak effects on shape when compared to FT or DS perturbation. This weakness is seen in adults, larvae, clones and in epistasis experiments. The epistasis experiment in particular convincingly shows that constitutuve ROK activation is not epistatic to loss of DS; in fact if anything the DS phenotype suppresses the ROK phenotype. These results also show that one cannot simply explain what FT and DS are doing with some single pathway or effector molecule like ROK. It is more complex than that.

What I really think was needed were experiments combining FT and DS knockdown with other mutants or knockdowns in the Hippo and Rho pathways, and even combining Hippo and Rho pathway mutants with FT or DS intact, to see if there are genetic interactions (additive, synergistic, epistatic) that could untangle the phenotypic complexity.

Response: We’re puzzled by these comments. First, we never claimed that what Fat or Ds do could be explained simply by manipulation of Rok (eg, see Discussion). Moreover, examination of wings and wing discs where ds is combined with Rho manipulations is in Fig 7, and Hippo and Rho pathway manipulation combinations are in Fig S5. We don’t think that combining ds or fat mutations with other Hippo pathway mutations would be informative, as it is well established that Ds-Fat are upstream regulators of Hippo signaling.

Laser cutting experiments were done to see if there is anisotropy in tissue tension within the wing pouch. This was to test a favored idea that FT and DS activity generates anisotropy in tissue tension, thereby controlling overall anisotropic shape of the pouch. However there is a fundamental flaw to their laser cutting analysis. Laser cutting is a technique used to measure mechanical tension, with initial recoil velocity directly proportional to the tissue's tension. By cutting a small line and observing how quickly the edges of the cut snap apart, people can quantify the initial recoil velocity and infer the stored mechanical stress in the tissue at the time of ablation. Live imaging with high-speed microscopy is required to capture the immediate response of the tissue to the cut since initial recoil velocity occurs in the first few seconds. A kymograph is created by plotting the movement of the tissue edges over this time scale, perpendicular to the cut. The initial recoil velocity is the slope of the kymograph at time zero, representing how fast the severed edges move apart. A higher recoil velocity indicates higher mechanical tension in the tissue. However, the authors did not measure this initial recoil velocity but instead measured the distance between the severed edges at one time point: 60 seconds after cutting. This is much later than the time point at which the recoil usually begins to dissipate or decay. This decay phase typically lasts a minute or two, during which time the edges continue to separate but at a progressively slower rate. This time-dependent decay of the recoil reveals whether the tissue behaves more like a viscous fluid or an elastic solid. Therefore, the distance metric at 60 seconds is a measurement of both tension and the material properties of the cells. One cannot know then whether a difference in the distance is due to a difference in tension or fluidity of the cells. If the authors made measurements of edge separation at several time points in the first 10 seconds after ablation, they can deconvolute the two. Otherwise their analysis is inconclusive. Anisotropy in recoil could be caused by greater tissue fluidity along one axis. Observing a gradient of cell fluidity in a tissue along one axis of a tissue has been observed in the amnioserosa of Tribolium for example. (Related and important point - was the anisotropy of recoil oriented along the PD or AP axis or not oriented to either axis, this key point was never stated)..

The authors cannot definitiviely conclude anything about mechanical tension from their reported cutting data.

Response: As noted by Reviewer 1 in cross-commenting, there is no fluidity on a time scale of 1 minute in the wing disc, and circular ablations are an established methods to investigate tissue stress. We choose the circular ablation method in part because it interrogates stress over a larger area, whereas cutting individual junctions is subject to more variability, particularly as the orientation of the junction (eg radial vs tangential) impacts the tension detected in the wing disc. Nonetheless, we will add recoil measurements to the revised manuscript to complement our circular ablations, which we expect will provide independent confirmation of our results and address the Reviewer’s concern here.

They measured the eccentricity of wing pouch cells near the pouch border, and found they were highly anisotropic compared to DS mutant cells at comparable locations. Cells were elongated but again what if either axis (PD or AP) they were elongated along was never stated. If cell anistropy is caused by polarized myosin activity, that activity is typically polarized along the short edges not long edges. Thus, recoil velocity after laaser cutting would be stonger along the axis aligned with short cell edges. It looks like the cutting anisotropy they see is greater along the axis aligned with long cell edges. Of course, if the cell anisotropy is caused by a pulling force exerted by the pouch boundary, then it would stretch the cells. This would in fact fit their cutting data. But then again, the observed cell anisotropy could also be caused by variation in the fluid-solid properties of the wing cells as discussed earlier. Compression of the cells then would deform them anisotropically and produce the anisotropic shapes that were observed, Therefore, interpreting what causes the cell anistropy and how DS regulates it is difficult,

Response: As noted by Reviewer 1 in cross-commenting, it is well established that tension and myosin are higher along long edges in the proximal wing. However, we acknowledge that we could do a better job of making the location and orientation of the regions shown in these experiments clear and, we will address this in a revised manuscript.

The imaging and analysis of the myosin RLC by GFP tagging is also flawed. SQH-GFP is a tried and true proxy for myosin activity in Drosophila. Although the authors image the wing pouch of wildtype and DS mutants. they did so under low magnification to image the entire pouch. This gives a "low-res" perspective of overall myosin but what they needed to do was image at high magnification in that proximal region of the pouch and see if Sqh-GFP is polarized in wildtype cells along certain cell edges aligned with an axis. And if such a polarity is observed, is it present or absent in the DS mutant. From the data shown in Figure 5, I cannot see any significant difference between wildtype and knocked down samples at this low resolution. Any difference, if there is any, is not really interpretable.

Response: We agree that examination of myosin localization at high resolution to see if it is polarized is a worthwhile experiment. We did in fact do this, and myosin (Sqh:GFP) appeared unpolarized in ds mutants. However, the levels of myosin were so low that we didn’t feel confident in our assessment, so we didn’t include it. We now recognize that this was a mistake, and we will include high resolution myosin images and assessments of (lack of) polarity in a revised manuscript to address this comment.

In conclusion, the manuscript has multiple problems that make it imposiible for the authors to make the claims they make in the current manuscript. And even if they calibrated their interpretations to fit the data, there is not much of a simple clear picture as to how FT and DS regulate pouch eccentricity in the larval wing.

Response: We think that the legitimate issues raised are addressable, as described above, while some of the criticisms are incorrect (as noted by Reviewer 1).

Reviewer #2 (Significance (Required)): This manuscript describes experiments studying the role that the protocadherins FAT and DACHSOUS play in determining the two dimensional "shape" of the fruit fly wing. By "shape", the manuscript really means how much the wing's outline, when approximated as an ellipse, deviates from a circle. The elliptical approximations of FT and DS mutant wings more closely resemble a circle compared to the more eccentric wildtype wings. This suggests the molecules contribute to anisotropic growth in some way. A great deal of attention has been paid on how FT and DS regulate overall organ growth and planar cell polarity, and the Irvine lab has made extensive contributions to these questions over the years. Somewhat understudied is how FT and DS regulate wing shape, and this manuscript focuses on that. It follows up on an interesting result that the Irvine lab published in 2019, in which mud mutants randomized spindle pole orientation in wing cells but did not change the eccentricity of wings, ruling out biased cell division orientation as a mechanism for the anisotropic growth.

__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __ Summary The authors investigate the mechanisms underlying epithelial morphogenesis using the Drosophila wing as a model system. Specifically, they analyze the contribution of the conserved Fat/Ds pathway to wing shape regulation. The main claim of the manuscript is that Ds/Fat controls wing shape by regulating tissue mechanical stress through MyoII levels, independently of Hippo signaling and tissue growth.

Major Comments To support their main conclusions, the authors should address the following major points and consider additional experiments where indicated. Most of the suggested experiments are feasible within a reasonable timeframe, while a few are more technically demanding but would substantially strengthen the manuscript's central claims.

Figure 1: The authors use temperature-sensitive inactivation of Fat or Ds to determine the developmental window during which these proteins regulate wing shape. To support this claim, it is essential to demonstrate that upon downshift during early pupal stages, Ds or Fat protein levels are restored to normal. For consistency, please include statistical analyses in Figure 1P and ensure that all y-axis values in shape quantifications start at 1.

Response: We will do the requested antibody stains for Fat (Ds antibody is unfortunately no longer available, but the point made by the reviewer can be addressed by Fat as the approach and results are the same for both genes). We have also added the requested statistical analysis to Fig 1P, and adjusted the scales as requested.

Figure 2: The authors propose that wing shape is regulated by Fat/Ds during larval development. However, Figure 2L suggests that wing elongation occurs in control conditions between 6 and 12 h APF, while this elongation is not observed upon Ds RNAi. The authors should therefore perform downshift experiments while monitoring wing shape during the pupal stage to substantiate their main claim. In addition, equivalent data for Fat loss of function should be included to support the assertion that Fat and Ds act similarly.

Response: As noted in our response to point 1 of Reviewer 1, we agree that there does seem to be relatively more elongation in control wings than in ds RNAi wings, but we think this likely reflects effects of ds on growth during larval stages, and we will revise the manuscript to comment on this.

We will also add the suggested examination of fat RNAi pupal wings.

The suggested examination of pupal wing shape in downshift experiments is unfortunately not feasible. Our temperature shift experiments expressing ds or fat RNAi are done using the UAS-Gal4-Gal80tssystem. We also use the UAS-Gal4 system to mark the pupal wing. If we do a downshift experiment, then expression of the fluorescent marker will be shut down in parallel with the shut down of ds or fat RNAi, so the pupal wings would no longer be visible.

Figure 3: The authors state that "These observations indicate that Ds-Fat signaling influences wing shape during the initial formation of the wing pouch, in addition to its effects during wing growth." This conclusion is not fully supported, as the authors only examine wing shape at 72 h AEL. At this stage, fat or ds mutant wings already display altered morphology. The authors could only make this claim if earlier time points were fully analyzed. In fact, the current data rather suggest that Ds function is required before 72 h AEL, as a rescue of wing shape is observed between 72 and 120 h AEL.

Response: First, I think we are largely in agreement with the Reviewer, as the basis for our saying that DS-Fat are likely required during initial formation of the wing pouch is that our data show they must be required before 72 h AEL. Second, 72 h is the earliest that we can look using Wg expression as a marker, as at earlier stages it is in a ventral wedge rather than a ring around the future wing pouch + DV line (eg see Fig 8 of Tripathi, B. K. & Irvine, K. D. The wing imaginal disc. Genetics (2022) doi:10.1093/genetics/iyac020.). We can revise the text to make sure this is clear.

Figure 4: The authors state that "The influence of Ds-Fat on wing shape is not explained by Hippo signaling." However, this conclusion is not supported by their data, which show that partial loss of ex or hippo causes clear defects in wing shape. In addition, the initial wing shape is affected in wts and ex mutants, and hypomorphic alleles were used for these experiments. Therefore, the main conclusion requires revision. It would be useful to include a complete dataset for hippo RNAi, ex, and wts conditions in Figure S1. The purpose and interpretation of the InR^CA experiments are also unclear. While InR^CA expression can increase tissue growth, Hippo signaling has functions beyond growth control. Whether Hippo regulates tissue shape through InR^CA-dependent mechanisms remains to be clarified.

Response: As noted in our response to point 1 of Reviewer 2 - our results emphasize that the effects of Ds-Fat on wing shape cannot be explained solely by effects on Hippo signaling, eg as we stated on page 7 “These observations suggest that Hippo signaling contributes to, but does not fully explain, the influence of ds or fat on adult wing shape.” We also note that impairment of Hippo signaling has similar effects in younger discs, but very different effects in older discs, which clearly indicates that they are having very different effects during disc growth. We will make some revisions to the text to make sure that our conclusions are clear throughout.

While we used a hypomorphic allele for wts, because null alleles are lethal, the ex allele that we used is described in Flybase as an amorph, not a hypomorph, and as noted in our response to Reviewer 2, we will add some discussion about relative strength of effects on Hippo signaling.

In Fig S1, we currently show adult wings for ex[e1] and RNAi-Hpo, and wing discs for wts[P2]/wts[x1], and for ex[e1]. The wts combination does not survive to adult so we can’t include this. We will however, add hpo RNAi wing discs as requested.

              The purpose of including InR^CA experiments is to try to separate effects of Hippo signaling from effects of growth, because InR signaling manipulation provides a distinct mechanism for increasing growth. We will revise the text to try to make sure this is clearer.

Figure 5: This figure presents images of MyoII distribution, but no quantification across multiple samples is provided. Moreover, the relationship between changes in tissue stress and MyoII levels remains unclear. Performing laser ablation and MyoII quantification on the same samples would provide stronger support for the proposed conclusions.

Response: We will revise the quantitation so that it presents analysis of averages across multiple discs, rather than representative examples of single discs.

Both the myosin imaging, and the laser ablation were done on the same genotypes (wildtype and ds) at the same ages (108 h AEL) so we think it is valid to directly compare them. Moreover, the imaging conditions for laser ablation and myo quantification are different, so it’s not feasible to do them at the same time (For ablations we do a single Z plane and a single channel (has to include Ecad, or an equivalent junctional marker) on live discs, so that fast imaging can be done. For Myo imaging we do multiple Z stacks and multiple channels (eg Ecad and Myo), which is not compatible with the fast imaging needed for analysis of laser ablations).

Figure 6: It is unclear when Rok RNAi and Rok^CA misexpression were induced. To substantiate their claims, the authors should measure both MyoII levels and mechanical tension under the different experimental conditions in which wing shape was modified through Rok modulation (i.e. the condition shown in Fig. 7G). For comparison, fat and ds data should be added to Fig 6H. Overall, the effects of Rok modulation appear milder than those of Fat manipulation. Given that Dachs has been shown to regulate tension downstream of Fat/Ds, it would be informative to determine whether tissue tension is altered in dachs mutant wings and to assess the relative contribution of Dachs- versus MyoII-mediated tension to wing shape control. It would also be interesting to test whether Rok activation can rescue dachs loss-of-function phenotypes.

Response: In these Rok experiments there was no separate temporal control of Rok RNAi or Rok^CA expression, they were expressed under nub-Gal4 control throughout development.

We will add examination of myosin in combinations of ds RNAi and rok manipulation as in Fig 7G to a revised manuscript.

Data for fat and ds comparable to that shown in Fig 6H is already presented in Fig 3D, and we don’t think its necessary to reproduce this again in Fig 6H.

We agree that the effects of Rok manipulations are milder than those of Fat manipulations; as we try to discuss, this could be because the pattern or polarity of myosin is also important, not just the absolute level, and we will add assessment of myosin polarity.

The suggestion to also look at dachs mutants is reasonable, and we will add this. In addition, we plan to add an "activated" Dachs (a Zyxin-Dachs fusion protein previously described in Pan et al 2013) that we anticipate will provide further evidence that the effects of Ds-Fat are mediated through Dachs. We will also add the suggested experiment combining Rok activation with dachs loss-of-function.

Figure 7: The authors use genetic interactions to support their claim that Fat controls wing shape independently of Hippo signaling. However, these interactions do not formally exclude a role for Hippo. Moreover, previous work has shown that tissue tension regulates Hippo pathway activity, implying that any manipulation of tension could indirectly affect Hippo and growth. To provide more direct evidence, the authors should further analyze MyoII localization and tissue tension under the various experimental conditions tested (as also suggested above).

Response: As discussed above, our data clearly show that Fat has effects independently of Hippo signaling that are crucial for its effects on wing shape, but we did not mean to imply that the regulation of Hippo signaling by Fat makes no contribution to wing shape control, and we will revise the text to make this clearer. We will also add additional analysis of Myosin localization , as described above.

Reviewer #3 (Significance (Required)): How organ growth and shape are controlled remains a fundamental question in developmental biology, with major implications for our understanding of disease mechanisms. The Drosophila wing has long served as a powerful and informative model to study tissue growth and morphogenesis. Work in this system has been instrumental in delineating the conserved molecular and mechanical processes that coordinate epithelial dynamics during development. The molecular regulators investigated by the authors are highly conserved, suggesting that the findings reported here are likely to be of broad biological relevance.

Previous studies have proposed that anisotropic tissue growth regulates wing shape during larval development and that such anisotropy induces mechanical responses that promote MyoII localization (Legoff et al., 2013, PMID: 24046320; Mao et al., 2013, PMID: 24022370). The Ds/Fat system has also been shown to regulate tissue tension through the Dachs myosin, a known modulator of the Hippo/YAP signaling pathway. As correctly emphasized by the authors, the respective contributions of anisotropic growth and mechanical tension to wing shape control remain only partially understood. The current study aims to clarify this issue by analyzing the role of Fat/Ds in controlling MyoII localization and, consequently, wing shape. This represents a potentially valuable contribution. However, the proposed mechanistic link between Fat/Ds and MyoII localization remains insufficiently explored. Moreover, the role of MyoII is not fully discussed in the broader context of Dachs function and its known interactions with MyoII (Mao et al., 2011, PMID: 21245166; Bosveld et al., 2012, PMID: 22499807; Trinidad et al., 2024, PMID: 39708794). Most importantly, the experimental evidence supporting the authors' conclusions would benefit from further strengthening. It should also be noted that disentangling the relative contributions of anisotropic growth and MyoII polarization to tissue shape and size remains challenging, as MyoII levels are known to increase in response to anisotropic growth (Legoff et al., 2013; Mao et al., 2013), and mechanical tension itself can modulate Hippo/YAP signaling (Rauskolb et al., 2014, PMID: 24995985).

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Referee #3

Evidence, reproducibility and clarity

Summary

The authors investigate the mechanisms underlying epithelial morphogenesis using the Drosophila wing as a model system. Specifically, they analyze the contribution of the conserved Fat/Ds pathway to wing shape regulation. The main claim of the manuscript is that Ds/Fat controls wing shape by regulating tissue mechanical stress through MyoII levels, independently of Hippo signaling and tissue growth.

Major Comments

To support their main conclusions, the authors should address the following major points and consider additional experiments where indicated. Most of the suggested experiments are feasible within a reasonable timeframe, while a few are more technically demanding but would substantially strengthen the manuscript's central claims.

Figure 1:

The authors use temperature-sensitive inactivation of Fat or Ds to determine the developmental window during which these proteins regulate wing shape. To support this claim, it is essential to demonstrate that upon downshift during early pupal stages, Ds or Fat protein levels are restored to normal. For consistency, please include statistical analyses in Figure 1P and ensure that all y-axis values in shape quantifications start at 1.

Figure 2:

The authors propose that wing shape is regulated by Fat/Ds during larval development. However, Figure 2L suggests that wing elongation occurs in control conditions between 6 and 12 h APF, while this elongation is not observed upon Ds RNAi. The authors should therefore perform downshift experiments while monitoring wing shape during the pupal stage to substantiate their main claim. In addition, equivalent data for Fat loss of function should be included to support the assertion that Fat and Ds act similarly.

Figure 3:

The authors state that "These observations indicate that Ds-Fat signaling influences wing shape during the initial formation of the wing pouch, in addition to its effects during wing growth." This conclusion is not fully supported, as the authors only examine wing shape at 72 h AEL. At this stage, fat or ds mutant wings already display altered morphology. The authors could only make this claim if earlier time points were fully analyzed. In fact, the current data rather suggest that Ds function is required before 72 h AEL, as a rescue of wing shape is observed between 72 and 120 h AEL.

Figure 4:

The authors state that "The influence of Ds-Fat on wing shape is not explained by Hippo signaling." However, this conclusion is not supported by their data, which show that partial loss of ex or hippo causes clear defects in wing shape. In addition, the initial wing shape is affected in wts and ex mutants, and hypomorphic alleles were used for these experiments. Therefore, the main conclusion requires revision. It would be useful to include a complete dataset for hippo RNAi, ex, and wts conditions in Figure S1. The purpose and interpretation of the InR^CA experiments are also unclear. While InR^CA expression can increase tissue growth, Hippo signaling has functions beyond growth control. Whether Hippo regulates tissue shape through InR^CA-dependent mechanisms remains to be clarified.

Figure 5:

This figure presents images of MyoII distribution, but no quantification across multiple samples is provided. Moreover, the relationship between changes in tissue stress and MyoII levels remains unclear. Performing laser ablation and MyoII quantification on the same samples would provide stronger support for the proposed conclusions.

Figure 6:

It is unclear when Rok RNAi and Rok^CA misexpression were induced. To substantiate their claims, the authors should measure both MyoII levels and mechanical tension under the different experimental conditions in which wing shape was modified through Rok modulation (i.e. the condition shown in Fig. 7G). For comparison, fat and ds data should be added to Fig 6H.<br /> Overall, the effects of Rok modulation appear milder than those of Fat manipulation. Given that Dachs has been shown to regulate tension downstream of Fat/Ds, it would be informative to determine whether tissue tension is altered in dachs mutant wings and to assess the relative contribution of Dachs- versus MyoII-mediated tension to wing shape control. It would also be interesting to test whether Rok activation can rescue dachs loss-of-function phenotypes.

Figure 7:

The authors use genetic interactions to support their claim that Fat controls wing shape independently of Hippo signaling. However, these interactions do not formally exclude a role for Hippo. Moreover, previous work has shown that tissue tension regulates Hippo pathway activity, implying that any manipulation of tension could indirectly affect Hippo and growth. To provide more direct evidence, the authors should further analyze MyoII localization and tissue tension under the various experimental conditions tested (as also suggested above).

Significance

How organ growth and shape are controlled remains a fundamental question in developmental biology, with major implications for our understanding of disease mechanisms. The Drosophila wing has long served as a powerful and informative model to study tissue growth and morphogenesis. Work in this system has been instrumental in delineating the conserved molecular and mechanical processes that coordinate epithelial dynamics during development. The molecular regulators investigated by the authors are highly conserved, suggesting that the findings reported here are likely to be of broad biological relevance.

Previous studies have proposed that anisotropic tissue growth regulates wing shape during larval development and that such anisotropy induces mechanical responses that promote MyoII localization (Legoff et al., 2013, PMID: 24046320; Mao et al., 2013, PMID: 24022370). The Ds/Fat system has also been shown to regulate tissue tension through the Dachs myosin, a known modulator of the Hippo/YAP signaling pathway. As correctly emphasized by the authors, the respective contributions of anisotropic growth and mechanical tension to wing shape control remain only partially understood. The current study aims to clarify this issue by analyzing the role of Fat/Ds in controlling MyoII localization and, consequently, wing shape. This represents a potentially valuable contribution. However, the proposed mechanistic link between Fat/Ds and MyoII localization remains insufficiently explored. Moreover, the role of MyoII is not fully discussed in the broader context of Dachs function and its known interactions with MyoII (Mao et al., 2011, PMID: 21245166; Bosveld et al., 2012, PMID: 22499807; Trinidad et al., 2024, PMID: 39708794). Most importantly, the experimental evidence supporting the authors' conclusions would benefit from further strengthening. It should also be noted that disentangling the relative contributions of anisotropic growth and MyoII polarization to tissue shape and size remains challenging, as MyoII levels are known to increase in response to anisotropic growth (Legoff et al., 2013; Mao et al., 2013), and mechanical tension itself can modulate Hippo/YAP signaling (Rauskolb et al., 2014, PMID: 24995985).

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Referee #2

Evidence, reproducibility and clarity

The manuscript begins with very nice data from a ts sensitive period experiment. Instead of a ts mutation, the authors induced RNAi in a temperature dependent manner. The results are striking and strong. Knockdown of FT or DS during larval stages to late L3 changed shape while knockdown of FT or DS during later pupal stages did not. This indicates they are required during larval, not pupal stages of wing development for this shape effect. They did shift-up or shift-down at "early pupa stage" but precisely what stage that means was not described anywhere in the manuscript. White prepupal? Time? Likewise a shift-down was done at "late L3" but that meaning is also vague. Moreover, I was surprised to see they did not do a shift-up at the late L3 stage, to give completeness to the experiment. Why?

Looking at the "shape" of the larval wing pouch they see a difference in the mutants. The pouch can be approximated as an ellipse, but with differing topology to the adult wing. Here, they muddled the analysis. The adult wing surface is analogous to one hemisphere of the larval wing pouch, ie., either dorsal or ventral compartment. The distance along the AP boundary from the pouch border to DV midline is topologically comparable to the PD length of the adult wing. The distance along the DV boundary from A border to P border is topologically comparable to the AP length of the adult wing. They confusingly call this latter metric the "DV length" and the former metric the "AP length" , and in fact they do not measure the PD length but PD+DP length. Confusing. Please change to make this consistent with earlier analysis of the adult and invert the reported ratio and divide by two. Then you would find the larval PD/AP ratio is smaller in the FT and DS mutants than wildtype, which resembles the smaller PD/AP ratio seen in the mutant adult wings. Totally consistent and also provides further evidence with the ts experiments that FT and DS exert shape effects in the larval phase of life.

The remainder of the manuscript has experimental results that are more problematic, and really the authors do not figure out how the shape effect in larval stages is altered. I outline below the main problems.

1. They compare the FT DS shape phenotypes to those of mutants or knockdowns in Hippo pathway genes (Hippo is known to be downstream of FT and DS). They find these Hippo perturbations do have shape effects trending in same direction as FT and DS effects. Knockdown reduces the PD/AP ratio while overexpressing WARTS increases the PD/AP ratio. The effect magnitudes are not as strong, but then again, they are using hypomorphic alleles and RNAi, which often induces partial or hypomorphic phenotypes. The effect strength is comparable when wing pouches are young but then dissipates over time, while FT and DS effects do not dissipate over time. The complexity of the data do not negate the idea that Hippo signaling is also playing some role and could be downstream of FT and DS in all of this. But the authors really downplay the data to the point of stating "These results imply that Ds-Fat influences wing pouch shape during wing disc growth separately from its effects on Hippo signaling." I think a more expansive perspective is needed given the caveats of the experiments.

Puzzlingly, this lack of taking seriously a set of complex results does not transfer to another set of experiments in which they inhibit or activate ROK, the rho kinase. When ROK is perturbed, they also see weak effects on shape when compared to FT or DS perturbation. This weakness is seen in adults, larvae, clones and in epistasis experiments. The epistasis experiment in particular convincingly shows that constitutuve ROK activation is not epistatic to loss of DS; in fact if anything the DS phenotype suppresses the ROK phenotype. These results also show that one cannot simply explain what FT and DS are doing with some single pathway or effector molecule like ROK. It is more complex than that.

What I really think was needed were experiments combining FT and DS knockdown with other mutants or knockdowns in the Hippo and Rho pathways, and even combining Hippo and Rho pathway mutants with FT or DS intact, to see if there are genetic interactions (additive, synergistic, epistatic) that could untangle the phenotypic complexity. 2. Laser cutting experiments were done to see if there is anisotropy in tissue tension within the wing pouch. This was to test a favored idea that FT and DS activity generates anisotropy in tissue tension, thereby controlling overall anisotropic shape of the pouch. However there is a fundamental flaw to their laser cutting analysis. Laser cutting is a technique used to measure mechanical tension, with initial recoil velocity directly proportional to the tissue's tension. By cutting a small line and observing how quickly the edges of the cut snap apart, people can quantify the initial recoil velocity and infer the stored mechanical stress in the tissue at the time of ablation. Live imaging with high-speed microscopy is required to capture the immediate response of the tissue to the cut since initial recoil velocity occurs in the first few seconds. A kymograph is created by plotting the movement of the tissue edges over this time scale, perpendicular to the cut. The initial recoil velocity is the slope of the kymograph at time zero, representing how fast the severed edges move apart. A higher recoil velocity indicates higher mechanical tension in the tissue. However, the authors did not measure this initial recoil velocity but instead measured the distance between the severed edges at one time point: 60 seconds after cutting. This is much later than the time point at which the recoil usually begins to dissipate or decay. This decay phase typically lasts a minute or two, during which time the edges continue to separate but at a progressively slower rate. This time-dependent decay of the recoil reveals whether the tissue behaves more like a viscous fluid or an elastic solid. Therefore, the distance metric at 60 seconds is a measurement of both tension and the material properties of the cells. One cannot know then whether a difference in the distance is due to a difference in tension or fluidity of the cells. If the authors made measurements of edge separation at several time points in the first 10 seconds after ablation, they can deconvolute the two. Otherwise their analysis is inconclusive. Anisotropy in recoil could be caused by greater tissue fluidity along one axis. Observing a gradient of cell fluidity in a tissue along one axis of a tissue has been observed in the amnioserosa of Tribolium for example. (Related and important point - was the anisotropy of recoil oriented along the PD or AP axis or not oriented to either axis, this key point was never stated)..

The authors cannot definitiviely conclude anything about mechanical tension from their reported cutting data. 3. They measured the eccentricity of wing pouch cells near the pouch border, and found they were highly anisotropic compared to DS mutant cells at comparable locations. Cells were elongated but again what if either axis (PD or AP) they were elongated along was never stated. If cell anistropy is caused by polarized myosin activity, that activity is typically polarized along the short edges not long edges. Thus, recoil velocity after laaser cutting would be stonger along the axis aligned with short cell edges. It looks like the cutting anisotropy they see is greater along the axis aligned with long cell edges. Of course, if the cell anisotropy is caused by a pulling force exerted by the pouch boundary, then it would stretch the cells. This would in fact fit their cutting data. But then again, the observed cell anisotropy could also be caused by variation in the fluid-solid properties of the wing cells as discussed earlier. Compression of the cells then would deform them anisotropically and produce the anisotropic shapes that were observed, Therefore, interpreting what causes the cell anistropy and how DS regulates it is difficult, 4. The imaging and analysis of the myosin RLC by GFP tagging is also flawed. SQH-GFP is a tried and true proxy for myosin activity in Drosophila. Although the authors image the wing pouch of wildtype and DS mutants. they did so under low magnification to image the entire pouch. This gives a "low-res" perspective of overall myosin but what they needed to do was image at high magnification in that proximal region of the pouch and see if Sqh-GFP is polarized in wildtype cells along certain cell edges aligned with an axis. And if such a polarity is observed, is it present or absent in the DS mutant. From the data shown in Figure 5, I cannot see any significant difference between wildtype and knocked down samples at this low resolution. Any difference, if there is any, is not really interpretable.

In conclusion, the manuscript has multiple problems that make it imposiible for the authors to make the claims they make in the current manuscript. And even if they calibrated their interpretations to fit the data, there is not much of a simple clear picture as to how FT and DS regulate pouch eccentricity in the larval wing.

Significance

This manuscript describes experiments studying the role that the protocadherins FAT and DACHSOUS play in determining the two dimensional "shape" of the fruit fly wing. By "shape", the manuscript really means how much the wing's outline, when approximated as an ellipse, deviates from a circle. The elliptical approximations of FT and DS mutant wings more closely resemble a circle compared to the more eccentric wildtype wings. This suggests the molecules contribute to anisotropic growth in some way. A great deal of attention has been paid on how FT and DS regulate overall organ growth and planar cell polarity, and the Irvine lab has made extensive contributions to these questions over the years. Somewhat understudied is how FT and DS regulate wing shape, and this manuscript focuses on that. It follows up on an interesting result that the Irvine lab published in 2019, in which mud mutants randomized spindle pole orientation in wing cells but did not change the eccentricity of wings, ruling out biased cell division orientation as a mechanism for the anisotropic growth.

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Referee #1

Evidence, reproducibility and clarity

Summary:

In this work, Tripathi et al address the open question of how the Fat/Ds pathway affects organ shape, using the Drosophila wing as a model. The Fat/Ds pathway is a conserved but complex pathway, interacting with Hippo signalling to affect growth and providing planar cell polarity that can influence cellular dynamics during morphogenesis. Here, authors use genetic perturbations combined with quantification of larval, pupal, and adult wing shape and laser ablation to conclude that the Ft/Ds pathway affects wing shape only during larval stages in a way that is at least partially independent of its interaction with Hippo and rather due to an effect on tissue tension and myosin II distribution. Overall the work is clearly written and well presented. I only have a couple major comments on the limitations of the work.

Major comments:

1. Authors conclude from data in Figures 1 and 2 that the Fat/Ds pathway only affects wing shape during larval stages. When looking at the pupal wing shape analysis in Figure 2L, however, it looks there is a difference in wt over time (6h-18h, consistent with literature), but that difference in time goes away in RNAi-ds, indicating that actually there is a role for Ds in changing shape during pupal stages, although the phenotype is clearly less dramatic than that of larval stages. No statistical test was done over time (within the genotype), however, so it's hard to say. I recommend the authors test over time - whether 6h and 18h are different in wild type and in ds mutant. I think this is especially important because there is proximal overgrowth in the Fat/Ds mutants, much of which is contained in the folds during larval stages. That first fold, however, becomes the proximal part of the pupal wing after eversion and contracts during pupal stages to elongate the blade (Aiguoy 2010, Etournay 2015). Also, according to Trinidad Curr Biol 2025, there is a role for Fat/Ds pathway in pupal stages. All of that to say that it seems likely that there would be a phenotype in pupal stages. It's true it doesn't show up in the adult wing in the experiments in Fig 1, but looking at the pupal wing itself is more direct - perhaps the very proximal effect is less prominent later, as there is potential for further development after 18hr before adulthood and the most proximal parts are likely anyway excluded in the analysis.
2. I think there needs to be a mention and some discussion of the fact that the wing is not really flat. While it starts out very flat at 72h, by 96h and beyond, there is considerable curvature in the pouch that may affect measurements of different axis and cell shape. It is not actually specified in the methods, so I assume the measurements were taken using a 2D projection. Not clear whether the curvature of the pouch was taken into account, either for cell shape measurements presented in Fig 4 or for the wing pouch dimensional analysis shown in Fig 3, 6, and supplements. Do perturbations in Ft/Ds affect this curvature? Are they more or less curved in one or both axes? Such a change could affect the results and conclusions. The extent to which the fat/ds mutants fold properly is another important consideration that is not mentioned. For example, maybe the folds are deeper and contain more material in the ds/fat mutants, and that's why the pouch is a different shape? At the very least, this point about the 3D nature of the wing disc must be raised in discussion of the limitations of the study. For the cell shape analysis, you can do a correction based on the local curvature (calculated from the height map from the projection). For the measurement of A/P, D/V axes of the wing pouch, best would be to measure the geodesic distance in 3D, but this is not reasonable to suggest at this point. One can still try to estimate the pouch height/curvature, however, both in wild type and in fat/ds mutants.

Minor comments:

1. The analysis of the laser ablation is not really standard - usually one looks at recoil velocity or a more complicated analysis of the equilibrium shape using a model (e.g Shivakumar and Lenne 2016, Piscitello-Gomez 2023, Dye et al 2021). One may be able to extract more information from these experiments - nevertheless, I doubt the conclusions would change, given that that there seems to be a pretty clear difference between wt and ds (OPTIONAL).
2. Figure 7G: I think you also need a statistical test between RNAi-ds and UAS-rokCA+RNAi-ds.
3. In the discussion, there is a statement: "However, as mutation or knock down of core PCP components, including pk or sple, does not affect wing shape... 59." Reference 59 is quite old and as far as I can tell shows neither images nor quantifications of the wing shape phenotype (not sure it uses "knockdown" either - unless you mean hypomorph?). A more recent publication Piscitello-Gomez et al Elife 2023 shows a very subtle but significant wing shape phenotype in core PCP mutants. It doesn't change your logic, but I would change the statement to be more accurate by saying "mutation of core PCP components has only subtle changes in adult wing shape"

Referee cross-commenting

Reviewer2:

Reviewer 2 makes the statement: "The distance along the AP boundary from the pouch border to DV midline is topologically comparable to the PD length of the adult wing. The distance along the DV boundary from A border to P border is topologically comparable to the AP length of the adult wing."

I disagree - the DV boundary wraps around the entire margin of the adult wing (as correctly drawn with the pink line in Fig 2A). It is not the same as the wide axis of the adult wing (perpendicular to the AP boundary). It is not trivial to map the proximal-distal axis of the larval wing to the proximal-distal axis of the adult, due to the changes in shape that occur during eversion. Thus, I find it much easier to look at the exact measurement that the authors make, and it is much more standard in the field, rather than what the reviewer suggests. Alternatively, one could I guess measure in the adult the ratio of the DV margin length (almost the circumference of the blade?) to the AP boundary length. That may be a more direct comparison. Actually the authors leave out the term "boundary" - what they call AP is actually the AP boundary, not the AP axis, and likewise for the DV - what they measure is DV boundary, but I only noticed that in the second read-through now. Just another note, these measurements of the pouch really only correspond to the very distal part of the wing blade, as so much of the proximal blade comes from the folds in the wing disc. Therefore, a measurement of only distal wing shape would be more comparable.

Reviewer 2 states that authors cannot definitively conclude anything about mechanical tension from their reported cutting data because the authors have not looked at initial recoil velocity. I strongly disagree. The wing disc tissue is elastic on much longer timescales than what's considered after laser ablation (even hours), and the shape of the tissue after it equilibrates from a circular cut (1-2min) can indeed be used to infer tissue stresses (see Dye et al Elife 2021, Piscitello-Gomez et al eLife 2023, Tahaei et al arXiv 2024). In the wing disc, the direction of stresses inferred from initial recoil velocity are correlated with the direction of stresses inferred from analysing the equilibrium shape after a circular cut. Rearrangements, a primary mechanism of fluidization in epithelia, does not occur within 1'. Analysing the equilibrium shape after circular ablation may be more accurate for assessing tissue stresses than initial recoil velocity - in Piscitello-Gomez et al 2023, the authors found that a prickle mutation (PCP pathway) affected initial recoil velocity but not tissue stresses in the pupal wing. Such equilibrium circular cuts have also been used to analyze stresses in the avian embryo, where it correlates with directions of stress gathered from force inference methods (Kong et al Scientific Reports 2019). The Tribolium example noted by the reviewer is on the timescale of tens to hundreds of minutes - much longer than the timescale of laser ablation retraction. It is true the analysis of the ablation presented in this paper is not at the same level as those other cited papers and could be improved. But I don't think the analysis would be improved by additional experiments doing timelapse of initial retraction velocity.

Reviewer 2 states "If cell anistropy is caused by polarized myosin activity, that activity is typically polarized along the short edges not long edges" Not true in this case. Myosin II accumulates along long boundaries (Legoff and Lecuit 2013). "Therefore, interpreting what causes the cell anistropy and how DS regulates it is difficult," Agreed - but this is well beyond the scope of this manuscript. The authors clearly show that there is a change of cell shape, at least in these two regions. Better would be to quantify it throughout the pouch and across multiple discs. Similar point for myosin quantifications - yes, polarity would be interesting and possible to look at in these data, and it would be better to do so on multiple discs, but the lack of overall myosin on the junctions shown here is not nothing. Interpreting what Ft/Ds does to influence tension and myosin and eventually tissue shape is a big question that's not answered here. I think the authors do not claim to fully understand this though, and maybe further toning down the language of the conclusions could help.

Reviewer 3:

I agree with many of the points raised by Reviewer 3, in particular that relevant for Fig 1. The additional experiments looking at myosin II localization and laser ablation in the other perturbations (Hippo and Rok mutants/RNAi) would certainly strengthen the conclusions.

Significance

I think the work provides a clear conceptual advance, arguing that the Ft/Ds pathway can influence mechanical stress independently of its interaction with Hippo and growth. Such a finding, if conserved, could be quite important for those studying morphogenesis and Fat function in this and other organisms. For this point, the genetic approach is a clear strength. Previous work in the Drosophila wing has already shown an adult wing phenotype for Ft/Ds mutations that was attributed to its role in the larval growth phase, as marked clones show aberrant growth in mutants. The novelty of this work is the dissection of the temporal progression of this phenotype and how it relates to Hippo and myosin II activation. It remains unclear exactly how Ft/Ds may affect tissue tension, except that it involves a downregulation of myosin II - the mechanism of that is not addressed here and would involve considerable more work. I think the temporal analysis of the wing pouch shape was quite revealing, providing novel information about how the phenotype evolves in time, in particular that there is already a phenotype quite early in development. As mentioned above, however, the lack of consideration of the wing disc as a 3D object is a potential limitation. While the audience is likely mostly developmental biologists working in basic research, it may also interest those studying the pathway in other contexts, including in vertebrates given its conservation and role in other processes.

This email does not belong to the listed business. It belongs to a similar business in Charlotte Massachusetts, same name and same type of business, but not in New Brunswick, Canada.

I generally access media such as television through my laptop, using Hulu or Pluto because it's cheaper as a college student, but I think people from older generations would keep using classic channels because it's what they're used to, and it's familiar.

I own a 1949 Dodge truck which doesn't have an original radio, but has an original heater. It's in great condition but again, it's lacking a radio, not that it would work anyways in current day.

I agree, I think in higher education yea, sometimes AI can be very useful but it stops us from actually making mistakes and learning from them. Now, some students might just be relying on AI and not learning anything really.

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Reply to the reviewers

Overall Response.

We would like to thank the reviewers for their analysis of the manuscript. From their comments it is clear that our manuscript was not. We completely rewrote the manuscript to focus on the central core question which was how does Adam13 regulates gene expression in general and TFap2a in particular leading to the expression of Calpain8 a protein required for CNC migration.

The following model will be the central line of our story. It will address all of the proteins involved and mechanistical evidences that link Adam13 to one of its proven effector target Calpain8.

• *

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

In this manuscript, Pandey et al. show that the ADAM13 protein modulates histone modifications in cranical neural crest and that the Arid3a protein binds the Tfap2a promoter in an Adam13-dependent manner and has promoter-specific effects on transcription. Furthermore, they show that the Adam13 and human ADAM9 proteins associated with histone modifiers as well as proteins involved in RNA splicing. Although the manuscript is mostly clearly written and the figures well assembled, it reads like a couple of separate and unfinished stories.*

I believe that our story line was not clear and that the overarching questions was not well stated. We have made every effort to change this in the revised manuscript. I would like to include a figure that explains the story.

In short:

1 We knew that Adam13 could regulate gene expression in CNC via its cytoplasmic domain.

2 We also knew that this required Adam13 interaction with Arid3a and that a direct target with the transcription factor TFAP2a which in turn regulates functional targets that we had identified including the protocadherin PCNS and the protease Calpain8.

Our goal was to understand the mechanism allowing Adam13 to regulate gene expression.

3 This first part of this manuscript shows how Adam13 modulates Histone modification in vivo in the CNC globally as well as specifically on the Tfap2a promoter. This results I an Open chromatin.

4 Using Chip we show that Adam13 and Arid3a both bind to the Tfap2a promoter and that Arid3a binding to the first ATG depends on Adam13.

5 Using Luciferase reporter we show that both Adam13 and Arid3a can induce expression at the first ATG.

*They show using immunocytochemistry and qPCR that ADAM13 knockouts in CNCs afffects histone modifications. Here ChIP-seq or Cut-n-Run experiments would be more appropriate and would result in a more comprehensive understanding of the changes mediated. *

I agree but we did not have the fund and now I have nobody working in the lab to do this experiment. These are also likely to overlap with the RNAseq data that we have and would simply add more open leads. We selected to go after the only direct target that we know which is TFAP2a and focus on this gene to understand the mechanism.

We believe that the Chip PCR experiment are sufficient for this story.

*The immunohistochemistry assays should at least be verified further using western blotting or other more quantiative methods. *

Immunofluorescence and statistical analysis is a valid quantification method. Western blot of CNC explants is not trivial and requires a large amount of material. Given the small overall change we also would not expect to be able to detect the change over the noise of western blot. The Chip PCR confirms our finding in a completely independent manner.

*The authors then show that ADAM13 interacts with a number of histone modifiers such as KDM3B, KDM4B and KMT2A but strangely they do not follow up this interesting observation to map the interactions further (apart from a co-ip with KMT2A), the domains involved, the functional role of the interactions or how they mediate the changes in chromatin modifications. *

We selected KMT2a because it is expressed in the Hek293T cells. KMT2D has been shown to regulate CNC development in Xenopus and is responsible for the Kabuki syndrome in human. We used aphafold to predict interaction and found that Adam13 interact with the Set domain. In addition we see multiple Set- containing domain protein in our mass spec data. The mass spec is done on Human hek293T cells that express a subset of KMT proteins. We now include evidence that Adam13 interact with the KMT2D SET domain (new figure 5D)

The authors then show that ADAM13 affects expression of the TFAP2a gene in a promoter specific manner - affecting expression from S1 but not S2.

It is the S1 but not S3. Adam13 has no effect on S2.

• They further show that ADAM13 affects the binding of the Arid3 transcription fator to the S1-promoter but not to the S3 promoter. However, ADAM13 was present at both promoters. Absence of ADAM13 resulted in increased H3K9me2/3 and decreased H3K4me3 at the S1 promoter whereas only H3K4me3 was changed at the S2 promoter*

S3 not S2*. Unfortunately, they do not show how this is mediated or through which binding elements this takes place. Why is ADAM13 present at both promoters but only affects Arid3 binding at S1? *

We agree this is a very interesting question that could be the subject of an entire publication. Promoter deletion and mutation to identify which site are bound by and modulated by Adam13/Arid3a is not trivial.

*The authors claim that transfecting Arid3a and Adam13 together further increases expression from a reporter (Fig 4E) but this is not true as no statistical comparison is done between the singly transfected and double transfected cells. *

This is correct, there is a small increase that is not significant with both. The fact that both proteins can induce the promoter suggest but does not prove that they can have additive roles. The loss of function experiment shows that the human Arid3a expressed in Hek293T cells is important for Adam13 increases of S1. It is possible that the dose of the endogenous Arid3a is sufficient to get full activity of Adam13.* Then the authors surprisingly start investigating association of proteins with the two isoforms of TFAP2a which in the mind of this reviewer is a different question entirely. *

We agree and have removed this part of the manuscript.

*They find a number of proteins involved in splicing. And the observation that ADAM13 also interacts with splicing factors is really irrelevant in terms of the story that they are trying to tell. Transcription regulation and splicing are different processes and although both affect the final outcome, mRNA, they need to be investigated separately. The link is at least not very clear from the manuscript. Again, the effects on splicing are not further investigated through functional analysis and as presented the data presented is too open-ended and lacking in clarity. *

We agree that beside the different activities of the TFap2a isoform, the rest of the splicing regulation could be a separate study. We were interested to understand how these two isoforms could activate Calpain8 so differently this is why we looked at LC/MS/MS. We have removed this part of the story from the manuscript.*

Additional points: 1. In the abstract they propose that the ADAMs may act as extracellular sensors. This is not substantiated by the results. *

As an extracellular protein translocating into the nucleus it is a possibility that we propose, but I agree this is not investigated in this manuscript. We will modify the text.* 2. Page 5, line 16: what is referred to by 6 samples 897 proteins? Were 6 samples analyzed for each condition? The number of repeats for the mass spec analysis is not clear from the text nor are the statistical parameters used to analyse the data. This is also true for the mass spec presented in the part on TFAP2aL-S1 and Adam13 regulate splicing. Statistics and repeats are not presented. *

In general we provide biological triplicate and use the statistical function of Scaffold to identify proteins that are significantly enriched or absent in each samples.

When we specify 6 samples it means 6 independent proteins samples were analyzed and used for our statistic. We use Scafold T-test with a p value less than 0.05. Peptides were identified with 95% confidence and proteins with 99% confidence.* 3. Page 6, line 19: set domain should be SET domain. *

Yes* 4. The number of repeats in the RNA sequencing of the CNCs is not clear from the text. *

Three biological replicates (Different batch of embryos from different females).* 5. The explanation of Figure C is a bit lacking. There are two forms of TFAP2a, L and S, but only one is presented in the figure. Do both forms have the extra S1-3 exons? Also, at the top of the figure it is not clear that the boxes are part of a continuous DNA sequence. Also, it is not clear which codon is not coding. *

Xenopus laevis are pseudo tetraploid giving in most cases L and S genes in addition to the 2 alleles from being diploid. The TFAP2a gene structure is conserved between both aloalleles and is similar to the human gene. For promoter analysis and Chip PCR we chose one of the alloallele (L), given that the RNAseq data showed that both genes and variant behave the same in response to Adam13. This only becomes important in loss of function experiment in which both L and S version need to be knock down or Knock out.

* In the sashimi plot there are green and pink shaded areas. What do they denote? What exactly is lacking in the MO13 mutant - seems that a particular exon is missing suggesting skipping?*

MO13 is a morpholino that bocks the translation of Adam13 (Already characterized with >90% of the protein absent) but does not affect Adam13 mRNA expression.* 7. Page 11, line 9: „with either MbC or MbC and MO13" needs to be rephrased. *

Will do *8. Page 11, line 19: „the c-terminus of....and S3) and" should be „the C-terminus of...and S3 and". ** 9. Page 15, line 10: substrateS 10. Page 16, line 23: the sentence „increases H3K9 to the promoter of the most upstream" needs revision. 11. Page 26, line 12: Here the authors say: „for two samples two-tail unpaired". What does this mean? Statistics should not be performed on fewer than three samples. In legnd to Figure 6 it indicates that T-test was performed on two samples. 12. The discussion should be shortened and simplified. 13. Figure 1 legend. How many images were quantitated for each condition? *

At least 3 images per condition. For 3 independent experiments. (9 images per condition).* 14. Figure 2 has a strange order of panels where G is below B. 15. Figure 6 legend, line 12. „proteins that were significantly enriched in either of the 2 samples" is not very clear. What exactly does this mean?

Reviewer #1 (Significance (Required)):

If the authors follow up on either the transcription-part of the story, or the splicing part of the story, they are likely to have important results to present. However, in the present format the paper is lacking in focus as both issues are mixed together without a clear end-result. *

We have entirely changed the paper according to these comments.

*

• *

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

Panday et al seeks to determine the function of ADAM13 in regulating histone modifications, gene expression and splicing during cranial neural crest development. Specifically, the authors tested how Adam13, a metalloprotease, could modify chromatin by interaction with Arid3a and Tfap2a and RNA splicing and gene expression. They then utilize knockouts in Xenopus and HEK293T cells followed by immunofluorescence, IPs, BioID, luciferase assays, Mass spec and RNA assays. Although there is some strong data in the BioID and luciferase experiments, the manuscript tells multiple stories, linking together too many things to make a compelling story. The result is a paper that is very difficult to read and understand the take home message. In addition, some of the conclusions are not supported by the data. This unfortunately means it is not ready for publication. However, I have added below some suggestions that would strengthen the manuscript. My comments are below: *

Clarity is clearly an issue here. The new version is entirely re-written.

Here is the take home message:

We knew that Adam13 could regulate gene expression via its cytoplasmic domain. One of the key targets was identified as Calpain8, a protein critical for CNC migration. We subsequently showed that Adam13 and Arid3a regulated Tfap2a expression which in turn regulated Calpain8.

In this manuscript we investigated 1) how Adam13 regulates TFAP2a and 2) how Tfap2a controls Calpain8 expression.

The take home message is that Adam13 bind to Histone methyl transferase and changes the histone methylation code overall in the CNC and in particular at the TFAP2a promoter. This results in more open chromatin. We further find that Adam13 binds to the Tfap2a promoter in vivo and is important for Arid3a binding to the first start. Tfap2a that include this N-terminus sequence regulates Capn8 expression.*

Major comments: 1. I think it would be better to split out the chromatin modification function from the splicing in two separate papers. While there is a connection, having it all together makes the story difficult to follow. *

Agree but I believe that the S1 vs S3 story of Tfap2a is important for the overall story. The new paper does not emphasize splicing.* 2. The immunofluorescence of H3K9me2/3, in Figure 1, 2, 3 following Adam13 knockdown is not convincing. There seems to be a strong edge effect especially in Figure 2 and 3. *

The statistical analysis shows that the results, while modest, are significant (Three independent experiments using 3 different females and 3 explants for each condition were analyzed). The edge effect observed is eliminated by the mask that we use that normalize the expression to either DAPI or Snai2. The edge effect is seen in both control and KD as well. These are further confirmed by the Chip PCR on one direct target.

Similarly the Arid3a expression in Supp Figure 1 if anything seems increased.

We have previously shown that Arid3a expression is not affected by Adam13 KD (Khedgikar et al). Our point here is simply that the difference in Tfap2a cannot be explained by a decrease in Arid3a expression. It is not a critical figure and was eliminated in the new manuscript.

*It would be better to quantify by western blot and not by fluorescent intensity since it is difficult to determine what a small change in fluorescent intensity means in vivo. *

Not all antibodies used here work by western blot and the quantity of material required for western blot is much larger than IF. Given the small overall changes and the variability observed in Western blot it is not a viable alternative.

IF is a quantitative method that has been used widely to assert increase or decrease of protein level or post translational modification. The fact that the same post translational modification that we see in cranial neural crest explants can also be seen by ChipPCR on the Tfap2a promoter confirm this observation.

*Also, it does not say in the text or the figure legend what these are, Xenopus explants of CNC? *

These are CNC explants. It is now clearly stated in the figure legend.* 3. The rationale for isolating KMT2A from the other chromatin modifiers in the dataset is not clear. *

The new manuscript is clarifying that point. Because we are using Hek293T cells in this assay, which are human embryonic kidney derived instead of Xenopus Cranial neural crest cells, we are not interested in a specific protein but rather a family of protein that can modify histones (KMT and KDM). Our rational is if Adam13 can bind to KMT2 via the SET domain, it is likely to interact with KTM2 that are expressed in the CNC. KMT2A and D are expressed in the CNC. This is why we selected KMT2a here (Hek293T). We now include 1 co-IP with the Set domain of Xenopus KMT2D (new figure 5D)

From the RNA-seq in Supp Figure 2 it is not changed as much as likely some of the others.

The new manuscript addresses this point. We did not show or expect that the loss of Adam13 would affect mRNA expression of Kmt2.

*Also, the arrow seems to indicate that it is right above the cutoff. What about other proteins with ATPase activity? That is the top hit in the Dot plot nuclear function. Would be helpful to write out Adam13 cytoplasm/nucleus here. *

We have used another set of proteomics data that does not include the cytoplasmic/nuclear extract to simplify the results. We hope that the changes make it more obvious.

Given that we are looking at Chromatin remodeling enzyme here we did not chose to investigate further in this report the ATPase. This is such a wide category that it could lead us away from the main story here.* 4. The splicing information, while interesting would be better as a different manuscript. The sashimi plot requires more explanation as written. *

We agree and think that a simple representation of the fold change of the different isoform is more obvious. It is now a minor part of figure 1 and the legend has been improved to describe the method here.

How do you tell if the interactions are changed from this?

I do not understand this question. The sashimi plot indicate the read through from the mRNA that goes from one exon to the next quantifying the specific exon usage. It can therefore be quantified and compared between different conditions.

• The authors argue there is a reduction of Tfap2a in Figure 3H but half the explant is not expressing sox9 in the Adam13 knockdown. How is this kind of experiment controlled when measure areas that don't have any fluorescence because of the nature of the explants? *

We have removed this figure as we had already shown previously by western blot that Tfap2a protein decreased in MO13 embryos. As noted on the histogram, the fluorescence is only measured in Sox9 positive cells in each explant. Three independent experiments with 3 explants for each. We also have seen a decrease by Western blot and mRNA expression (Both RNAseq and realtime PCR). In most of our explants, the vast majority of the cells are positive for Snai2 and Sox9, while those that are negative are positive for Sox3 (data not shown here). There is always less signal in the center of the explant possibly due to the penetration of antibody or interference with the signal by the cells pigment or yolk autofluorescence. Our control explants have the same effect so our quantification is valid.* 5. The use of a germ line Xenopus mutant for Adam13 is great but how were these knockouts validated? *

All of the KO were validated by sequencing, RNAseq and protein expression. These are now included in the supplemental figure 1.

*More information is required here. The Chip-qPCR has a lot of variability between the samples, especially in the H3K9me2/3. *

All ChipPCR were performed on Xenopus embryos. The variability is tested by statistical analysis and is either significant or not.

Because these are in cell lines, this should be more consistent.

They are not in cell lines but in Xenopus embryos.

• In addition, it is difficult to understand what this means for cranial neural crest cells when assaying in HEK293T cells with the luciferase assay. *

We use Luciferase assay in Hek293T cells to test if Xenopus protein can induce a specific reporter (Gain of function). We also use luciferase reporter in Xenopus to test if they can perceive the loss of a specific protein (For example Adam13).

Our result show that Adam13 or Arid3a expression in Hek293T cells can induce the TFAP2S1 reporter. * 6. The migration assay shows only an example of what it looks like to have defective migration. But it would be better to show control embryos, embryos with Adam13 knockdown and what the rescues look like so the reader can make their own conclusion.*

We can certainly include this but have published this assay in multiple publication before. The picture is a single example, the histogram shows that statistical validation.

• The argument from the section above suggests the S1 isoform is the primary one but S3 in this assay also rescues, please explain what this result means since it seems to suggest that even though these isoforms have different activity the function is similar in terms of the ability to rescue defective migration. *

The result in Hek293T cells shows that only TFAP2aS1 can induce Calpain8, while both S1 and S3 can partially rescue CNC migration in embryos lacking Adam13. The issue here is the dose of mRNA injected for each variant might be too high. Adam13 proteolytic activity is also critical, so we do not expect a complete rescue. The fact that S1 is significantly better at rescuing than S3 is relevant here. It is possible that if we were to decrease the dose of each mRNA we would find one in which S3 no longer rescues but S1 does.

* The next section again talks about yet another protein Calpain-8. Here the authors use MO13 for luciferase assays instead of HEK293 cells. The authors do not explain why they decided to switch from cells to MO.*

Calpain8 is one of the validate target of Adam13 that can rescue CNC migration (Cousin et al Dev Cell). We use the luciferase reporter corresponding to the Xenopus Capn8 reporter to show 1 in vivo that loss of Adam13 reduce its expression (Similar to the Capn8 gene). We then went in vitro using Hek293T cells for gain of function experiment that shows that only the Tfaps2S1 variant can induce it while S3 does not.

We hope that the graphical summary and the new manuscript make this clear.* 8. The experiment to IP RNA supports only the correlation that Adam9 and Adam13 bind RNA and RNA binding proteins to regulate splicing. This conclusion presented is not supported by the data presented here. While there is a sentence about why Adam9 was chosen here, it would be preferred to focus on Adam13 as the rest of the manuscript is focused on Adam13. The conclusions are generalized to all ADAMs, but ADAM13 and ADAM9 are the only ADAMs investigated in the manuscript *

This figure is no longer included. For each of the protein classes that we identify by Masspec we try to find a validation. RNA-IP is simply a validation that Adam13 and Adam9 can bind to complexes that include RNA in a cytoplasmic domain dependent fashion. The conclusion that Adam13 and possibly ADAM9 might be involved in regulating splicing is 1) that the protein associated with Adam13 are include multiple splicing factors, 2) that the RNAseq analysis shows abnormal splicing in CNC missing Adam13 and 3) that the form of TFAP2a induced by Adam13 (S1) associate significantly more with splicing factor than the S3 isoform.

We agree that the generalization to other ADAM is not demonstrated here but only suggested. We selected ADAM9 and ADAM19 because we have shown that they can each rescue Adam13 function in the CNC. Unfortunately there are no ADAM19 antibody that work by IP on the market. We have tested multiple company and multiple cell lines.

We believe that the ADAM9 experiment is critical to show that the protein associated with Adam13 are not simply the result of overexpressing a different species protein sin ADAM9 is the endogenous protein.*

Minor comments 1. The manuscript using a lot of abbreviations (PCNS, NI, MO, SH3) and lingo that are unclear to a general reader. Please define acronyms when first used, as well as be clear on which model is being used in each experiment. *

We have corrected this* 2. Similarly, the figures are not labeled such that a reader would be able to understand ie MO13 should be Adam13 knockdown etc. *

We have corrected this in the legend

• Please identify the genes on the heatmap and some highlighted genes from volcano plot from the RNA-seq.*

The volcano plot is from MS/MS not RNAseq. We have list of all of the genes and/or proteins corresponding to each figure in tables

We now have a figure from the RNAseq and a subset of genes of interest are show. *4. Why use the flag tag in Figure 5? *

We used Flag-tagged construct to only immunoprecipitated the variants and not the endogenous TFPA2a in these experiments. Also we used RFP-Flag to eliminate any protein that bound to the tag or the antibody.

This figure is no longer in the manuscript.* 5. Is the data in figure 4A-D the same as Supp. Figure 4A-D? *

These are independent biological replicates of the same experiment.* 6. Please italicize gene symbols - e.g. "key transcription factors that exemplify CNC, such as the SOX9, FOXD3, SNAI1, SNAI2, and TFAP2 family". *

We clearly have missed some, we are using italicized for gene, and regular for proteins. It might not be clear in the text when we are referring to genes and proteins. We will correct this in the rewrite. 7. Please review the manuscript for grammatical and typographical errors. * We have used all available software including Word and Grammarly. We will try to improve on the next version. **Cross-commenting**

I think the two reviewers on one the same page on this manuscript.

Reviewer #2 (Significance (Required)):

If more solid, would be a conceptual advance in role of Adam13 in mediating chromatin modification and transcription factors, adds to exiting work from this lab, good for a specialize audience, my expertise is in in neural crest development, non-mammalian modes, epigenetic regulators.*

• *

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

Panday et al seeks to determine the function of ADAM13 in regulating histone modifications, gene expression and splicing during cranial neural crest development. Specifically, the authors tested how Adam13, a metalloprotease, could modify chromatin by interaction with Arid3a and Tfap2a and RNA splicing and gene expression. They then utilize knockouts in Xenopus and HEK293T cells followed by immunofluorescence, IPs, BioID, luciferase assays, Mass spec and RNA assays. Although there is some strong data in the BioID and luciferase experiments, the manuscript tells multiple stories, linking together too many things to make a compelling story. The result is a paper that is very difficult to read and understand the take home message. In addition, some of the conclusions are not supported by the data. This unfortunately means it is not ready for publication. However, I have added below some suggestions that would strengthen the manuscript. My comments are below:

Major comments:

1. I think it would be better to split out the chromatin modification function from the splicing in two separate papers. While there is a connection, having it all together makes the story difficult to follow.
2. The immunofluorescence of H3K9me2/3, in Figure 1, 2, 3 following Adam13 knockdown is not convincing. There seems to be a strong edge effect especially in Figure 2 and 3. Similarly the Arid3a expression in Supp Figure 1 if anything seems increased. It would be better to quantify by western blot and not by fluorescent intensity since it is difficult to determine what a small change in fluorescent intensity means in vivo. Also, it does not say in the text or the figure legend what these are, Xenopus explants of CNC?
3. The rationale for isolating KMT2A from the other chromatin modifiers in the dataset is not clear. From the RNA-seq in Supp Figure 2 it is not changed as much as likely some of the others. Also, the arrow seems to indicate that it is right above the cutoff. What about other proteins with ATPase activity? That is the top hit in the Dot plot nuclear function. Would be helpful to write out Adam13 cytoplasm/nucleus here.
4. The splicing information, while interesting would be better as a different manuscript. The sashimi plot requires more explanation as written. How do you tell if the interactions are changed from this? The authors argue there is a reduction of Tfap2a in Figure 3H but half the explant is not expressing sox9 in the Adam13 knockdown. How is this kind of experiment controlled when measure areas that don't have any fluorescence because of the nature of the explants?
5. The use of a germ line Xenopus mutant for Adam13 is great but how were these knockouts validated? More information is required here. The Chip-qPCR has a lot of variability between the samples, especially in the H3K9me2/3. Because these are in cell lines, this should be more consistent. In addition, it is difficult to understand what this means for cranial neural crest cells when assaying in HEK293T cells with the luciferase assay.
6. The migration assay shows only an example of what it looks like to have defective migration. But it would be better to show control embryos, embryos with Adam13 knockdown and what the rescues look like so the reader can make their own conclusion. The argument from the section above suggests the S1 isoform is the primary one but S3 in this assay also rescues, please explain what this result means since it seems to suggest that even though these isoforms have different activity the function is similar in terms of the ability to rescue defective migration.
7. The next section again talks about yet another protein Calpain-8. Here the authors use MO13 for luciferase assays instead of HEK293 cells. The authors do not explain why they decided to switch from cells to MO.
8. The experiment to IP RNA supports only the correlation that Adam9 and Adam13 bind RNA and RNA binding proteins to regulate splicing. This conclusion presented is not supported by the data presented here. While there is a sentence about why Adam9 was chosen here, it would be preferred to focus on Adam13 as the rest of the manuscript is focused on Adam13. The conclusions are generalized to all ADAMs, but ADAM13 and ADAM9 are the only ADAMs investigated in the manuscript

Minor comments

1. The manuscript using a lot of abbreviations (PCNS, NI, MO, SH3) and lingo that are unclear to a general reader. Please define acronyms when first used, as well as be clear on which model is being used in each experiment.
2. Similarly, the figures are not labeled such that a reader would be able to understand ie MO13 should be Adam13 knockdown etc.
3. Please identify the genes on the heatmap and some highlighted genes from volcano plot from the RNA-seq.
4. Why use the flag tag in Figure 5?
5. Is the data in figure 4A-D the same as Supp. Figure 4A-D?
6. Please italicize gene symbols - e.g. "key transcription factors that exemplify CNC, such as the SOX9, FOXD3, SNAI1, SNAI2, and TFAP2 family".
7. Please review the manuscript for grammatical and typographical errors.

Cross-commenting

I think the two reviewers on one the same page on this manuscript.

Significance

If more solid, would be a conceptual advance in role of Adam13 in mediating chromatin modification and transcription factors, adds to exiting work from this lab, good for a specialize audience, my expertise is in in neural crest development, non-mammalian modes, epigenetic regulators

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 #1

Evidence, reproducibility and clarity

In this manuscript, Pandey et al. show that the ADAM13 protein modulates histone modifications in cranical neural crest and that the Arid3a protein binds the Tfap2a promoter in an Adam13-dependent manner and has promoter-specific effects on transcription. Furthermore, they show that the Adam13 and human ADAM9 proteins associated with histone modifiers as well as proteins involved in RNA splicing.

Although the manuscript is mostly clearly written and the figures well assembled, it reads like a couple of separate and unfinished stories. They show using immunocytochemistry and qPCR that ADAM13 knockouts in CNCs afffects histone modifications. Here ChIP-seq or Cut-n-Run experiments would be more appropriate and would result in a more comprehensive understanding of the changes mediated. The immunohistochemistry assays should at least be verified further using western blotting or other more quantiative methods. The authors then show that ADAM13 interacts with a number of histone modifiers such as KDM3B, KDM4B and KMT2A but strangely they do not follow up this interesting observation to map the interactions further (apart from a co-ip with KMT2A), the domains involved, the functional role of the interactions or how they mediate the changes in chromatin modifications.

The authors then show that ADAM13 affects expression of the TFAP2a gene in a promoter specific manner - affecting expression from S1 but not S2. They further show that ADAM13 affects the binding of the Arid3 transcription fator to the S1-promoter but not to the S3 promoter. However, ADAM13 was present at both promoters. Absence of ADAM13 resulted in increased H3K9me2/3 and decreased H3K4me3 at the S1 promoter whereas only H3K4me3 was changed at the S2 promoter. Unfortunately, they do not show how this is mediated or through which binding elements this takes place. Why is ADAM13 present at both promoters but only affects Arid3 binding at S1? The authors claim that transfecting Arid3a and Adam13 together further increases expression from a reporter (Fig 4E) but this is not true as no statistical comparison is done between the singly transfected and double transfected cells.

Then the authors surprisingly start investigating association of proteins with the two isoforms of TFAP2a which in the mind of this reviewer is a different question entirely. They find a number of proteins involved in splicing. And the observation that ADAM13 also interacts with splicing factors is really irrelevant in terms of the story that they are trying to tell. Transcription regulation and splicing are different processes and although both affect the final outcome, mRNA, they need to be investigated separately. The link is at least not very clear from the manuscript. Again, the effects on splicing are not further investigated through functional analysis and as presented the data presented is too open-ended and lacking in clarity.

Additional points:

1. In the abstract they propose that the ADAMs may act as extracellular sensors. This is not substantiated by the results.
2. Page 5, line 16: what is referred to by 6 samples 897 proteins? Were 6 samples analyzed for each condition? The number of repeats for the mass spec analysis is not clear from the text nor are the statistical parameters used to analyse the data. This is also true for the mass spec presented in the part on TFAP2aL-S1 and Adam13 regulate splicing. Statistics and repeats are not presented.
3. Page 6, line 19: set domain should be SET domain.
4. The number of repeats in the RNA sequencing of the CNCs is not clear from the text.
5. The explanation of Figure C is a bit lacking. There are two forms of TFAP2a, L and S, but only one is presented in the figure. Do both forms have the extra S1-3 exons? Also, at the top of the figure it is not clear that the boxes are part of a continuous DNA sequence. Also, it is not clear which codon is not coding.
6. In the sashimi plot there are green and pink shaded areas. What do they denote? What exactly is lacking in the MO13 mutant - seems that a particular exon is missing suggesting skipping?
7. Page 11, line 9: „with either MbC or MbC and MO13" needs to be rephrased.
8. Page 11, line 19: „the c-terminus of....and S3) and" should be „the C-terminus of...and S3 and".
9. Page 15, line 10: substrateS
10. Page 16, line 23: the sentence „increases H3K9 to the promoter of the most upstream" needs revision.
11. Page 26, line 12: Here the authors say: „for two samples two-tail unpaired". What does this mean? Statistics should not be performed on fewer than three samples. In legnd to Figure 6 it indicates that T-test was performed on two samples.
12. The discussion should be shortened and simplified.
13. Figure 1 legend. How many images were quantitated for each condition?
14. Figure 2 has a strange order of panels where G is below B.
15. Figure 6 legend, line 12. „proteins that were significantly enriched in either of the 2 samples" is not very clear. What exactly does this mean?

Significance

If the authors follow up on either the transcription-part of the story, or the splicing part of the story, they are likely to have important results to present. However, in the present format the paper is lacking in focus as both issues are mixed together without a clear end-result.

This statement suggests a mechanistic advantage for peptides, but it is somewhat broad. A more critical approach would consider whether these interactions have been quantified in vivo or only in vitro. Are there limitations in tissue penetration for larger peptides or specific types of receptors that could affect efficacy?

Have you considered incorporating an upfront screening or triage model to distinguish patients who present with advanced disease and are at high risk of early mortality from those likely to survive beyond the first months after diagnosis? Such a gating classifier—for example, predicting high versus low short-term (e.g., 6-month) progression risk—could enable a two-stage framework in which rapid-progression cases are identified separately, while the proposed dynamic longitudinal model is applied primarily to patients expected to survive beyond this early window. This may reduce late-diagnosis bias and preserve information about rapid deteriorators without excluding them from analysis.

In the article I could not seem to find any information any where that went into who funded the study but it does say that the instution, Innovation Research Interchange, did have a role in the study. With looking into the bibliographies of the authors it can be seen that they all have past research in research article and some of them have even done their own lectures.

I feel as this study could of had partial bias just because the researchers did not take into account how different global companies can differ from one another. So, without doing this then only these results can be predicted for Chinese based companies. But, still using the information recovered is still very vital because it can be an overview for future work that can look into different cultures across the world and what impact it has on companies and their AI usage.

The results from the study was that firms would best be able to use AI by using it in conjunction with the capabilities that they already have. So, in the firms the best use of AI would be to use it with the pre-existing conditions and standards that are already in place.

Wearing Even Realities’ $599 smart glasses, the reporter finds the tech useful for quick tasks like on-the-fly translation, prompts, and notifications, but the bigger story is social. People repeatedly ask if they’re being recorded—even though these glasses have no cameras—and conversations often turn awkward. Highly reflective waveguides make bystanders think the wearer is reading something, so friends and even family feel ignored.The piece argues that discomfort isn’t irrational, since microphones and growing camera-glasses in general raise real privacy worries. Still, the author notes this may ease if the devices become common, as tech firms push AR eyewear from Meta and Google toward everyday use. As he puts it, “Like it or not, Big Tech is coming for our glasses,” and “it’s going to take some getting used to.”