Author response:

Reviewer #1 (Public review):

Summary:

This work by Govorunova et al. identified three naturally blue-shifted channelrhodopsins (ChRs) from ancyromonads, namely AnsACR, FtACR, and NlCCR. The phylogenetic analysis places the ancyromonad ChRs in a distinct branch, highlighting their unique evolutionary origin and potential for novel applications in optogenetics. Further characterization revealed the spectral sensitivity, ionic selectivity, and kinetics of the newly discovered AnsACR, FtACR, and NlCCR. This study also offers valuable insights into the molecular mechanism underlying the function of these ChRs, including the roles of specific residues in the retinal-binding pocket. Finally, this study validated the functionality of these ChRs in both mouse brain slices (for AnsACR and FtACR) and in vivo in Caenorhabditis elegans (for AnsACR), demonstrating the versatility of these tools across different experimental systems.

In summary, this work provides a potentially valuable addition to the optogenetic toolkit by identifying and characterizing novel blue-shifted ChRs with unique properties.

Strengths:

This study provides a thorough characterization of the biophysical properties of the ChRs and demonstrates the versatility of these tools in different ex vivo and in vivo experimental systems. The mutagenesis experiments also revealed the roles of key residues in the photoactive site that can affect the spectral and kinetic properties of the channel.

We thank the Reviewer for his/her positive evaluation of our work.

Weaknesses:

While the novel ChRs identified in this work are spectrally blue-shifted, there still seems to be some spectral overlap with other optogenetic tools. The authors should provide more evidence to support the claim that they can be used for multiplex optogenetics and help potential end-users assess if they can be used together with other commonly applied ChRs. Additionally, further engineering or combination with other tools may be required to achieve truly orthogonal control in multiplexed experiments.

To demonstrate the usefulness of ancyromonad ChRs for multiplex optogenetics as a proof of principle, we co-expressed AnsACR with the red-shifted cation-conducting ChR Chrimson and measured net photocurrent generated by this combination as a function of the wavelength. We found that it is hyperpolarizing in the blue region of the spectrum, and depolarizing at the red region. In the revision, we added a new panel (Figure 1D) showing these results and the following paragraph to the main text:

“To test the possibility of using AnsACR in multiplex optogenetics, we co-expressed it with the red-shifted CCR Chrimson (Klapoetke et al., 2014) fused to an EYFP tag in HEK293 cells. We measured the action spectrum of the net photocurrents with 4 mM Cl^- in the pipette, matching the conditions in the neuronal cytoplasm (Doyon, Vinay et al. 2016). Figure 1D, black shows that the direction of photocurrents was hyperpolarizing upon illumination with λ<500 nm and depolarizing at longer wavelengths. A shoulder near 520 nm revealed a FRET contribution from EYFP (Govorunova, Sineshchekov et al. 2020), which was also observed upon expression of the Chrimson construct alone (Figure 1D, red)”.

In the C. elegans experiments, partial recovery of pharyngeal pumping was observed after prolonged illumination, indicating potential adaptation. This suggests that the effectiveness of these ChRs may be limited by cellular adaptation mechanisms, which could be a drawback in long-term experiments. A thorough discussion of this challenge in the application of optogenetics tools would prove very valuable to the readership.

We added the following paragraph to the revised Discussion:

“One possible explanation of the partial recovery of pharyngeal pumping that we observed after 15-s illumination, even at the highest tested irradiance, is continued attenuation of photocurrent during prolonged illumination (desensitization). However, the rate of AnsACR desensitization (Figure 1 – figure supplement 4A and Figure 1 – figure supplement 5A) is much faster than the rate of the pumping recovery, reducing the likelihood that desensitization is driving this phenomenon. Another possible reason for the observed adaptation is an increase in the cytoplasmic Cl^- concentration owing to AnsACR activity and hence a breakdown of the Cl^- gradient on the neuronal membrane. The C. elegans pharynx is innervated by 20 neurons, 10 of which are cholinergic (Pereira, Kratsios et al. 2015). A pair of MC neurons is the most important for regulation of pharyngeal pumping, but other pharyngeal cholinergic neurons, including I1, M2, and M4, also play a role (Trojanowski, Padovan-Merhar et al. 2014). Moreover, the pharyngeal muscles generate autonomous contractions in the presence of acetylcholine tonically released from the pharyngeal neurons (Trojanowski, Raizen et al. 2016). Given this complexity, further elucidation of pharyngeal pumping adaptation mechanisms is beyond the scope of this study.”

Reviewer #2 (Public review):

Summary:

Govorunova et al present three new anion opsins that have potential applications in silencing neurons. They identify new opsins by scanning numerous databases for sequence homology to known opsins, focusing on anion opsins. The three opsins identified are uncommonly fast, potent, and are able to silence neuronal activity. The authors characterize numerous parameters of the opsins.

Strengths:

This paper follows the tradition of the Spudich lab, presenting and rigorously characterizing potentially valuable opsins. Furthermore, they explore several mutations of the identified opsin that may make these opsins even more useful for the broader community. The opsins AnsACR and FtACR are particularly notable, having extraordinarily fast onset kinetics that could have utility in many domains. Furthermore, the authors show that AnsACR is usable in multiphoton experiments having a peak photocurrent in a commonly used wavelength. Overall, the author's detailed measurements and characterization make for an important resource, both presenting new opsins that may be important for future experiments, and providing characterizations to expand our understanding of opsin biophysics in general.

We thank the Reviewer for his/her positive evaluation of our work.

Weaknesses:

First, while the authors frequently reference GtACR1, a well-used anion opsin, there is no side-by-side data comparing these new opsins to the existing state-of-the-art. Such comparisons are very useful to adopt new opsins.

GtACR1 exhibits the peak sensitivity at 515 nm and therefore is poorly suited for combination with red-shifted CCRs or fluorescent sensors, unlike blue-light-absorbing ancyromonad ACRs. Nevertheless, we conducted side-by-side comparison of ancyromonad ChRs, GtACR1 and GtACR2, the latter of which has the spectral maximum at 470 nm. The results are shown in the new Figures 1E and F, and the new multipanel Figure 1 – figure supplement 4 added in the revision. We also added the following text, describing these results, to the revised Results section:

“Figures 1E and F show the dependence of the peak photocurrent amplitude and reciprocal peak time, respectively, on the photon flux density for ancyromonad ChRs and GtACRs. The current amplitude saturated earlier than the time-to-peak for all tested ChRs. Figure 1 – figure supplement 4A-E shows normalized photocurrent traces recorded at different photon densities. Quantitation of desensitization at the end of 1-s illumination revealed a complex light dependence (Figure 1, Figure Supplement 4F). Figure 1 – figure supplement 5 shows normalized photocurrent traces recorded in response to a 5-s light pulse of the maximal available intensity and the magnitude of desensitization at its end.”

Next, multiphoton optogenetics is a promising emerging field in neuroscience, and I appreciate that the authors began to evaluate this approach with these opsins. However, a few additional comparisons are needed to establish the user viability of this approach, principally the photocurrent evoked using the 2p process, for given power densities. Comparison across the presented opsins and GtACR1 would allow readers to asses if these opsins are meaningfully activated by 2P.

We carried out additional 2P experiments in ancyromonad ChRs, GtACR1 and GtACR2 and added their results to a new main-text Figure 6 and Figure 6 – figure supplement 1. We added the new section describing these results, “Two-photon excitation”, to the main text in the revision:

“To determine the 2P activation range of AnsACR, FtACR, and NlCCR, we conducted raster scanning using a conventional 2P laser, varying the excitation wavelength between 800 and 1,080 nm (Figure 6 – figure supplement 1). All three ChRs generated detectable photocurrents with action spectra showing maximal responses at ~925 nm for AnsACR, 945 nm for FtACR, and 890 nm for NlCCR (Figure 6A). These wavelengths fall within the excitation range of common Ti:Sapphire lasers, which are widely used in neuroscience laboratories and can be tuned between ~700 nm and 1,020-1,300 nm. To assess desensitization, cells expressing AnsACR, FtACR, or NlCCR were illuminated at the respective peak wavelength of each ChR at 15 mW for 5 seconds. GtACR1 and GtACR2, previously used in 2P experiments (Forli, Vecchia et al. 2018, Mardinly, Oldenburg et al. 2018), were included for comparison. The normalized photocurrent traces recorded under these conditions are shown in Figure 6B-F. The absolute amplitudes of 2P photocurrents at the peak time and at the end of illumination are shown in Figure 6G and H, respectively. All five tested variants exhibited comparable levels of desensitization at the end of illumination (Figure 6I).”

Reviewer #3 (Public review):

Summary:

The authors aimed to develop Channelrhodopsins (ChRs), light-gated ion channels, with high potency and blue action spectra for use in multicolor (multiplex) optogenetics applications. To achieve this, they performed a bioinformatics analysis to identify ChR homologues in several protist species, focusing on ChRs from ancyromonads, which exhibited the highest photocurrents and the most blue-shifted action spectra among the tested candidates. Within the ancyromonad clade, the authors identified two new anion-conducting ChRs and one cation-conducting ChR. These were characterized in detail using a combination of manual and automated patch-clamp electrophysiology, absorption spectroscopy, and flash photolysis. The authors also explored sequence features that may explain the blue-shifted action spectra and differences in ion selectivity among closely related ChRs.

Strengths:

A key strength of this study is the high-quality experimental data, which were obtained using well-established techniques such as manual patch-clamp and absorption spectroscopy, complemented by modern automated patch-clamp approaches. These data convincingly support most of the claims. The newly characterized ChRs expand the optogenetics toolkit and will be of significant interest to researchers working with microbial rhodopsins, those developing new optogenetic tools, as well as neuro- and cardioscientists employing optogenetic methods.

We thank the Reviewer for his/her positive evaluation of our work.

Weaknesses:

This study does not exhibit major methodological weaknesses. The primary limitation of the study is that it includes only a limited number of comparisons to known ChRs, which makes it difficult to assess whether these newly discovered tools offer significant advantages over currently available options.

We conducted side-by-side comparison of ancyromonad ChRs and GtACRs, wildly used for optical inhibition of neuronal activity. The results are shown in the new Figures 1E and F, and the new multipanel Figure 1 – figure supplement 4 and Figure 1 – figure supplement 5 added in the revision. We also added the following text, describing these results, to the revised Results section:

“Figures 1E and F show the dependence of the peak photocurrent amplitude and reciprocal peak time, respectively, on the photon flux density for ancyromonad ChRs and GtACRs. The current amplitude saturated earlier than the time-to-peak for all tested ChRs. Figure 1 – figure supplement 4A-E shows normalized photocurrent traces recorded at different photon densities. Quantitation of desensitization at the end of 1-s illumination revealed a complex light dependence (Figure 1, Figure Supplement 4F). Figure 1 – figure supplement 5 shows normalized photocurrent traces recorded in response to a 5-s light pulse of the maximal available intensity and the magnitude of desensitization at its end.”

Additionally, although the study aims to present ChRs suitable for multiplex optogenetics, the new ChRs were not tested in combination with other tools. A key requirement for multiplexed applications is not just spectral separation of the blue-shifted ChR from the red-shifted tool of interest but also sufficient sensitivity and potency under low blue-light conditions to avoid cross-activation of the respective red-shifted tool. Future work directly comparing these new ChRs with existing tools in optogenetic applications and further evaluating their multiplexing potential would help clarify their impact.

As a proof of principle, we co-expressed AnsACR with the red-shifted cation-conducting CCR Chrimson and demonstrated that the net photocurrent generated by this combination is hyperpolarizing in the blue region of the spectrum, and depolarizing at the red region. In the revision, we added a new panel (Figure 1D) showing these results and the following paragraph to the main text:

“To test the possibility of using AnsACR in multiplex optogenetics, we co-expressed it with the red-shifted CCR Chrimson (Klapoetke et al., 2014) fused to an EYFP tag in HEK293 cells. We measured the action spectrum of the net photocurrents with 4 mM Cl^- in the pipette, matching the conditions in the neuronal cytoplasm (Doyon, Vinay et al. 2016). Figure 1D, black shows that the direction of photocurrents was hyperpolarizing upon illumination with λ<500 nm and depolarizing at longer wavelengths. A shoulder near 520 nm revealed a FRET contribution from EYFP (Govorunova, Sineshchekov et al. 2020), which was also observed upon expression of the Chrimson construct alone (Figure 1D, red)”.

Reviewing Editor Comments:

The reviewers suggest that direct comparison to GtACR1 is the most important step to make this work more useful to the community.

We followed the Reviewers’ recommendations and carried out side-by-side comparison of ancyromonad ChRs and GtACR1 as well as GtACR2 (Figure 1E and F, Figure 1 – figure supplement 4, Figure 1 – figure supplement 5, and Figure 6). Note, however, that GtACR1’s spectral maximum is at 515 nm, which makes it poorly suitable for blue light excitation. Also, ChRs are known to perform very differently in different cell types and upon expression of their genes in different vector backbones, so our results cannot be generalized for all experimental systems. Each ChR user needs to select the most appropriate tool for his/her purpose by testing several candidates in his/her own experimental setting.

Reviewer #1 (Recommendations for the authors):

(1) The figure legend for Figure 2D-I appears to be incomplete. Please provide a detailed explanation of the panels.

In the revision, we have expanded the legend of Figure 2 to explain all individual panels.

(2) The meaning of the Vr shift (Y-axis in Figure 2H-I) should be clarified in the main text to aid reader understanding.

In the revision, we added the phrase “which indicated higher relative permeability to NO₃ than to Cl^-“ to explain the meaning of the Vr shift upon replacement of Cl^- with NO₃-.

(3) Adding statistical analysis for the peak and end photocurrent values in Figure 2D-F would strengthen the claim that there is minimal change in relative permeability during illumination.

In the revision, we added the V_r values for the peak photocurrent to Figure 2H-I, which already contained the V_r values for the end photocurrent, and carried out a statistical analysis of their comparison. The following sentence was added to the text in the revision:

“The V_r values of the peak current and that at the end of illumination were not significantly different by the two-tailed Wilcoxon signed-rank test (Fig. 2G), indicating no change in the relative permeability during illumination.”

(4) Figure 4H and I seem out of place in Figure 4, as the title suggests a focus on wild-proteins and AnsACR mutants. The authors could consider moving these panels to Figure 3 for better alignment with the content.

As noted below, we changed the panel order in Figure 4 upon the Reviewer’s request. In particular, former Figure 4I is Figure 4C in the revision, and former Figure 4H is now panel C in Figure 3 – figure supplement 1 in the revision. We rearranged the corresponding section of the text (highlighted yellow in the manuscript).

(5) The characterization section could be strengthened by including data on the pH sensitivity of FtACR, which is currently missing from the main figures.

Upon the Reviewer’s request, we carried out pH titration of FtACR absorbance and added the results as Figure 4B in the revision.

(6) The logic in Figure 4A-G appears somewhat disjointed. For example, Figure 4A shows pH sensitivity for WT AnsACR and the G86E mutant, while Figure 4 B-D shifts to WT AnsACR and the D226N mutant, and Figure 4E returns to the G86E mutant. Reorganizing or clarifying the flow would improve readability.

We followed the Reviewer’s advice and changed the panel order in Figure 4. In the revised version, the upper row (panels A-C) shows the pH titration data of the three WTs, the middle row (panels D-F) shows analysis of the AnsACR_D226N mutant, and the lower row (panels G-I) shows analysis of the AnsACR_G88E mutant. We also rearranged accordingly the description of these panels in the text.

(7) In Figure 5A, "NIACR" should likely be corrected to "NlCCR".

We corrected the typo in the revision.

(8) The statistical significance in Figure 6C and D is somewhat confusing. Clarifying which groups are being compared and using consistent symbols would improve interoperability.

In the revision, we improved the figure panels and legend to clarify that the comparisons are between the dark and light stimulation groups within the same current injection.

(9) The authors pointed out that at rest or when a small negative current was injected, the neurons expressing Cl- permeable ChRs could generate a single action potential at the beginning of photostimulation, as has been reported before. The authors could help by further discussing if and how this phenomenon would affect the applicability of such tools.

We mentioned in the revised Discussion section that activation of ACRs in the axons could depolarize the axons and trigger synaptic transmission at the onset of light stimulation, and this undesired excitatory effect need to be taken into consideration when using ACRs.

Reviewer #2 (Recommendations for the authors):

Govorunova et al present three new anion opsins that have potential applications in silencing neurons. This paper follows the tradition of the Spudich lab, presenting and rigorously characterizing potentially valuable opsins. Furthermore, they explore several mutations of the identified opsin that may make these opsins even more useful for the broader community. In general, I feel positively about this manuscript. It presents new potentially useful opsins and provides characterization that would enable its use. I have a few recommendations below, mostly centered around side-by-side comparisons to existing opsins.

(1) My primary concern is that while there is a reference to GtACR1, a highly used opsin first described by this team, they do not present any of this data side by side.

When evaluating opsins to use, it is important to compare them to the existing state of the art. As a potential user, I need to know where these opsins differ. Citing other papers does not solve this as, even within the same lab, subtle methodological differences or data plotting decisions can obscure important differences.

As we explained in the response to the public comments, we carried out side-by-side comparison of ancyromonad ChRs and GtACRs as requested by the Reviewer. The results are shown in the new Figures 1E and F, and the new multipanel Figure 1 – figure supplement 4 and Figure 1 – figure supplement 5, added in the revision. However, we would like to emphasize a limited usefulness of such comparative analysis, as ChRs are known to perform very differently in different cell types and upon expression of their genes in different vector backbones, so our results cannot be generalized for all experimental systems. Each ChR user needs to select the most appropriate tool for his/her purpose by testing several candidates in his/her own experimental setting.

(2) Multiphoton optogenetics is an emerging field of optogenetics, and it is admirable that the authors address it here. The authors should present more 2p characterization, so that it can be established if these new opsins are viable for use with 2P methods, the way GtACR1 is. The following would be very useful for 2P characterization:

Photocurrents for a given power density, compared to GtACR1 and GtACR2.

The new Figure 6 (B-F) added in the revision shows photocurrent traces recorded from the three ancyromonad ChRs and  two GtACRs upon 2P excitation of a given power density.

Comparing NICCR and FtACR's wavelength specificity and photocurrent. If these opsins are too weak to create reasonable 2P spectra, this difference should be discussed.

The new Figure 6A shows the 2P action spectra of all three ancyromonad ChRs.

A Trace and calculated photocurrent kinetics to compare 1P and 2P. This need not be the flash-based absorption characterization of Figure 3, but a side-by-side photocurrent as in Figure 2.

As mentioned above, photocurrent traces recorded from ancyromonad ChRs and GtACRs upon 2P excitation are shown in the new Figure 6 (B-F). However, direct comparison of the 2P data with the 1P data is not possible, as we used laser scanning illumination for the former and wild-field illumination for the latter.

Characterization of desensitization. As the authors mention, many opsins undergo desensitization, presenting the ratio of peak photocurrent vs that at multiple time points (probably up to a few seconds) would provide evidence for how effectively these constructs could be used in different scenarios.

We conducted a detailed analysis of desensitization under both 1P and 2P excitation. The new Figure 1 – figure supplement 4 and Figure 1 – figure supplement 5 show the data obtained under 1P excitation, and the new Figure 6 shows the data for 2P conditions.

I have to admit, that by the end of the paper, I was getting confused as to which of the three original constructs had which property, and how that was changing with each mutation. I would suggest that a table summarizing each opsin and mutation with its onset and offset kinetics, peak wavelength, photocurrent, and ion selectivity would greatly increase the ability to select and use opsins in the future.

In the revision, we added a table of the spectroscopic properties of all tested mutants as Supplementary File 2. This study did not aim to analyze other parameters listed by the Reviewer. We added the following sentence referring to this table to the main text:

“Supplementary File 2 contains the λ values of the half-maximal amplitude of the long-wavelength slope of the spectrum, which can be estimated more accurately from the action spectra than the λ of the maximum.”

It may be out of the scope of this manuscript, but if a soma localization sequence can be shown to remove the 'axonal spiking' (as described in line 441), this would be a significant addition to the paper.

Our previous study (Messier et al., 2018, doi: 10.7554/eLife.38506) showed that a soma localization sequence can reduce, but not eliminate, the axonal spiking. We plan to test these new ACRs with the trafficking motifs in the future.

NICCR appears to have the best photocurrents of all tested opsins in this paper. It seems odd that it was omitted from the mouse cortical neurons experiments.

We have not included analysis of NlCCR behavior in neurons because we are preparing a separate manuscript on this ChR.

Figure 6 would benefit from more gradation in the light powers used to silence and would benefit from comparison to GtACR. I suggest using a fixed current with a series of illumination intensities to see which of the three opsins (or GtACR) is most effective at silencing. At present, it looks binary, and a user cannot evaluate if any of these opsins would be better than what is already available.

In the revision, we added the data comparing the light sensitivity of AnsACR and FtACR with previously identified GtACR1 and GtACR2 (new Figure 1E and F) to help users compare these ACRs. Although they are less sensitive to light comparing to GtACR1 and GtACR2, they could still be activated by commercially available light sources if the expression levels are similar. Less sensitive ACRs may have less unwanted activation when using with other optogenetic tools.

Reviewer #3 (Recommendations for the authors):

Suggested Improvements to Experiments, Data, or Analyses:

(1) Line 25: "significantly exceeding those by previously known tools" and Line 408: "NlCCR is the most blue-shifted among ancyromonad ChRs and generates larger photocurrents than the earlier known CCRs with a similar absorption maximum." As noted in the public review, this statement applies only to a very specific subgroup of ChRs with spectral maxima below 450 nm. If the goal was to claim that NlCCR is a superior tool among a broader range of blue-light-activated ChRs, direct comparisons with state-of-the-art ChRs such as ChR2 T159C (Berndt et al., 2011), CatCh (Kleinlogel et al., 2014), CoChR (Klapoetke et al., 2014), CoChR-3M (Ganjawala et al., 2019), or XXM 2.0 (Ding et al., 2022) would be beneficial. If the goal was to demonstrate superiority among tools with spectra below 450 nm, I suggest explicitly stating this in the paper.

The Reviewer correctly inferred that we emphasized the superiority of NlCCR among tools with similar spectral maxima, not all blue-light-activated ChRs available for neuronal photoexcitation, most of which exhibit absorption maxima at longer wavelengths. To clarify this, we added “with similar spectral maxima” to the sentence in the original Line 25. The sentence in Line 408 already contains this clarification: “with a similar absorption maximum”.

(2) Lines 111-113: "The absorption spectra of the purified proteins were slightly blue-shifted from the respective photocurrent action spectra (Figure 1D), likely due to the presence of non-electrogenic cis-retinal-bound forms." I would be skeptical of this statement. The spectral shifts in NlCCR and AnsACR are small and may fall within the range of experimental error. The shift in FtACR is more apparent; however, if two forms coexist in purified protein, this should be reflected as two Gaussian peaks in the absorption spectrum (or at least as a broader total peak reflecting two states with close maxima and similar populations). On the contrary, the action spectrum appears to have two peaks, one potentially below 465 nm. Generally, neither spectrum appears significantly broader than a typical microbial rhodopsin spectrum. This question could be clarified by quantifying the widths of the absorption and action spectra or by overlaying them on the same axis. In my opinion, the two spectra seem very similar, and just appearance of the "bump" in the action spectum shifts the apparent maximum of the action spectrum to the red. If there were two states, then they should both be electrogenic, and the slight difference in spectra might be explained by something else (e.g. by a slight difference in the quantum yields of the two states).

As the Reviewer suggested, in the revision we added a new figure (Figure 1 – figure supplement 2), showing the overlay of the absorption and action spectra of each ancyromonad ChR. This figure shows that the absorption spectra are wider than the action spectra (especially in AnsACR and FtACR), which confirms our interpretation (contribution of the non-electrogenic blue-shifted cis-retinal-bound forms to the absorption spectrum). Note that the presence of such forms explaining a blue shift of the absorption spectrum has been experimentally verified in HcKCR1 (doi: 10.1016/j.cell.2023.08.009; 10.1038/s41467-025-56491-9). Therefore, we revised the text as follows:

“The absorption spectra of the purified proteins (Figure 1C) were slightly blue-shifted from the respective photocurrent action spectra (Figure 1 – figure supplement 3), likely due to the presence of non-electrogenic cis-retinal-bound forms. The presence of such forms, explaining the discrepancy between the absorption and the action spectra, was verified by HPLC in KCRs (Tajima et al. 2023, Morizumi et al., 2025).”

(3) Lines 135-136: "The SyncroPatch enables unbiased estimation of the photocurrent amplitude because the cells are drawn into the wells without considering their tag fluorescence." While SyncroPatch does allow unbiased selection of patched cells, it does not account for the fraction of transfected cells. Without a method to exclude non-transfected cells, which are always present in transient transfections, the comparison of photocurrents may be affected by the proportion of untransfected cells, which could vary between constructs. To clarify whether the statistically significant difference in the Kolmogorov-Smirnov test could indicate that the fraction of transfected cells after 48-72h differs between constructs, I suggest analyzing only transfected cells or reporting fractions of transfected cells by each construct.

The Reviewer correctly states that non-transfected cells are always present in transiently transfected cell populations. However, his/her suggestion to “exclude non-transfected cells” is not feasible in the absence of a criterion for such exclusion. As it is evident from our data, transient transfection results in a continuum of the amplitude values, and it is not possible to distinguish a small photocurrent from no photocurrent, considering the noise level. We would like, however, to emphasize that not excluding any cells provides an estimate of the overall potency of each ChR variant, which depends on both the fraction of transfected cells and their photocurrents. This approach mimics the conditions of in vivo experiments, when non-expressing cells also cannot be excluded.

(4) Line 176: "AnsACR and FtACR photocurrents exhibited biphasic rise." The fastest characteristic time is very close to the typical resolution of a patch-clamp experiment (RC = 50 μs for a 10 pF cell with a 5 MΩ series resistance). Thus, I am skeptical that the faster time constant of the biphasic opening represents a protein-specific characteristic time. It may not be fully resolved by patch-clamp and could simply result from low-pass filtering of a specific cell. I suggest clarifying this for the reader.

The Reviewer is right that the patch clamp setup acts as a lowpass filter. Earlier, we directly measured its time resolution (~15 μs) by recording the ultrafast (occurring on the ps time scale) charge movements related to the trans-cis isomerization (doi: 10.1111/php.12558). However, the lowpass filter of the setup can only slow the entire signal, but cannot lead to the appearance of a separate kinetic component (i.e. a monophasic process cannot become biphasic). Therefore, we believe that the biphasic photocurrent rise reflects biphasic channel opening rather than a measurement artifact. Two phases in the channel opening have also been detected in GtACR1 (doi: 10.1073/pnas.1513602112) and CrChR2 (10.1073/pnas.1818707116).

(5) Line 516: "The forward LED current was 900 mA." It would be more informative to report the light intensity rather than the forward current, as many readers may not be familiar with the specific light output of the used LED modules at this forward current.

We have added the light intensity value in the revision:

“The forward LED current was 900 mA (which corresponded to the irradiance of ~2 mW mm^-2)…”

(6) Lines 402-403: "The NlCCR ... contains a neutral residue in the counterion position (Asp85 in BR), which is typical of all ACRs. Yet, NlCCR does not conduct anions, instead showing permeability to Na+." This is not atypical for CCRs and has been demonstrated in previous works of the authors (CtCCR in Govorunova et al. 2021, ChvCCR1 in Govorunova et al. 2022). What is unique is the absence of negatively charged residues in TM2, as noted later in the current study. However, the absence of negatively charged residues in TM2 appears to be rare for ACRs as well. Not as a strong point of criticism, but to enhance clarity, I suggest analyzing the frequency of carboxylate residues in TM2 of ACRs to determine whether the unique finding is relevant to ion selectivity or to another property.

The Reviewer is correct that some CCRs lack a carboxylate residue in the D85 position, so this feature alone cannot be considered as a differentiating criterion. However, the complete absence of glutamates in TM2 is not rare in ACRs and is found, for example, in HfACR1 and CarACR2. We have discussed this issue in our earlier review (doi: 10.3389/fncel.2021.800313) and do not think that repeating this discussion in this manuscript is appropriate.

Recommendations for Writing and Presentation:

(1) Some figures contain incomplete or missing labels:

Figure 2: Panels D to I lack labels.

In the revision, we have expanded the legend of Figure 2 to explain all individual panels.

Figure 3 - Figure Supplement 1: Missing explanations for each panel.

In the revision, we changed the order of panes and explained all individual panels in the legend.

Figure 5 - Figure Supplement 1: Missing explanations for each panel.

No further explanation for individual panels in this Figure is needed because all panels show the action spectra of various mutants, the names of which are provided in the panels themselves. Repeating this information in the figure legend would be redundant.

(2) In Figure 2, "sem" is written in lowercase, whereas "SEM" is capitalized in other figures. Standardizing the format would improve consistency.

In the revision, we changed the font of the SEM abbreviation to the uppercase in all instances.

(3) Line 20: "spectrally separated molecules must be found in nature." There is no proof that they cannot be developed synthetically; rather, it is just difficult. I suggest softening this statement, as the findings of this study, together with others, will probably allow designing molecules with specified spectral properties in the future.

In the revision, we changed the cited sentence to the following:

“Multiplex optogenetic applications require spectrally separated molecules, which are difficult to engineer without disrupting channel function”.

(4) Line 216-219: "Acidification increased the amplitude of the fast current ~10-fold (Figure 4F) and shifted its Vr ~100 mV (Figure 3 - figure supplement 1D), as expected of passive proton transport. The number of charges transferred during the fast peak current was >2,000 times smaller than during the channel opening, from which we concluded that the fast current reflects the movement of the RSB proton." The claim about passive transport of the RSB proton should be clarified, as typically, passive transport is not limited to exactly one proton per photocycle, and the authors observe the increase in the fast photocurrents upon acidification.

We thank the Reviewer for pointing out the confusing character of our description. To clarify the matter, we added a new photocurrent trace to Figure 4I in the revision recorded from AnsACR_G86E at 0 mV and pH 7.4. We have rewritten the corresponding section of Results as follows:

“Its rise and decay τ corresponded to the rise and decay τ of the fast positive current recorded from AnsACR_G86E at 0 mV and neutral pH, superimposed on the fast negative current reflecting the chromophore isomerization (Figure 4I, upper black trace). We interpret this positive current as an intramolecular proton transfer to the mutagenetically introduced primary acceptor (Glu86), which was suppressed by negative voltage (Figure 4I, lower black trace). Acidification increased the amplitude of the fast negative current ~10-fold (Figure 4I, black arrow) and shifted its V_r ~100 mV to more depolarized values (Figure 4 – figure supplement 2A). This can be explained by passive inward movement of the RSB proton along the large electrochemical gradient.”

Minor Corrections:

(1) Line 204: Missing bracket in "phases in the WT (Figure 4D."

The quoted sentence was deleted during the revision.

(2) Line 288: Typo-"This Ala is conserved" should probably be "This Met is conserved."

We mean here the Ala four residues downstream from the first Ala. To avoid confusion, we changed the cited sentence to the following:

“The Ala corresponding to BR’s Gly122 is also found in AnsACR and NlCCR (Figure 5A)…”

(3) Lines 702-704: Missing Addgene plasmid IDs in "(plasmids #XXX and #YYY, respectively)."

In the revision, we added the missing plasmid IDs.

How do you make a kite fly?

Why do humans use bubbles to relax?

Why do bees buzz?

What's the secret to a good bedtime story?

Do humans serve plates as actual meals?

Do mice really live in grandfather clocks?

Why do some preschool channels' names end in 'Jr'?

Are champs distinct cousins of chimps?

Why is Bluey different from every other preschool show?

Can you use a dustpan and brush to brush your hair?

If there's a lollipop lady, can I eat the lollipops?

Are animal crackers made out of real animals?

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Reply to the reviewers

Note to all Reviewers:

We would like to thank all the reviewers for their time and insightful feedback. In response to the comments and points raised, we have performed major revisions to our manuscript. We have expanded our analysis on the role of TP53 loss of function in BM activation (Figure 3), investigating human LUAD datasets as well as murine LUAD models. We show that TP53 pathway is significantly negatively correlated with BM, and that loss of TP53 leads to the acquisition of the basal-like phenotype regardless of the type of driver oncogene present (KRAS/EGFR). Furthermore, we added a new figure (Figure 7), where we demonstrate that type I interferon can promote BM activation in LUAD harboring TP53 mutations but not in those with wild type TP53. With this, we propose a mechanism of action of how a subset of LUAD tumors (TP53-mut) upregulate BM, become more aggressive and resistant to therapies.

Finally, we have made the manuscript clearer and transparent by improving the presentation of plots, as well as including source data files and Rmarkdown files for reproducibility.

Reviewer 1:

Major comments

R1-Comment 1: The authors did not submit with the manuscript all the results that they have obtained from their analysis, on which they based their claims. I suggest that the authors submit a SourceData file in Excel format. This file should contain the values and the relevant information for each of the plots presented in the main and supplementary figures. For example, in case of box plots, the five-number summary should be provided. Further, the p-values and the test used for their calculations should be also mentioned. The file could be organized in a way that the data and relevant information for each figure panel are presented in separated data sheets in order that the reader can easily navigate through the file and find the information for each figure panel fast. Similarly, the authors should provide access to the scripts that they have developed or adapted from published scripts to perform the analysis of the datasets and obtain the results presented in the manuscript. The access to the scripts used in the manuscript is important to reproduce analysis. The scripts can be deposited at github, for example.

Reply: We thank the reviewer for their advice in making the presentation of our results and methods more transparent and reproducible. We have now provided the source data file (supplementary file 2), which contains relevant data for each figure. We have also uploaded Rmarkdown files to github and R Markdown HTML reports are compiled in Supplementary File 3, this shows how the analyses were performed and how each figure generated. All datasets required to reproduce the analyses and figures have also been added to Zenodo (10.5281/zenodo.16964654) and will be published when the article is in press.

R1-Comment 2: The results and their interpretations are mainly done based on in silico analysis from publicly available transcriptomic datasets. The confirmation of the results obtained by the in silico analysis is limited to the last figure, in which the authors show results obtained by multiplexed immunohistochemistry and histo-cytometry of tissue microarrays from a curated cohort of FFPE samples. The relevance of the results obtained by the in silico analysis may increase if the authors could present results either in a conditional (lung-specific) Kras mutant mouse model, or patient-derived xenograft (PDX) mouse model of lung cancer. The PDX mouse model will be more suitable in case that access to genetically modified mouse models is not given and/or the time for the experiments is limited. In both cases, the hyperactivation of the small GTPase KRAS should expand the BM gene expression signature in the mouse lung in a Sox9-dependent manner, thereby leading to lung tumors. Further, Sox9 loss-of-function experiments should reduce the BM gene expression signature and favor the ALV gene expression signature. These results would strongly support the interpretation of the in silico results by the authors in the present manuscript, and would significantly increase the impact of the manuscript in the scientific field of lung cancer.

Reply: We thank the reviewer for their insightful feedback on how to improve the impact of our study through further functional validation of in silico findings. To address this comment, we have performed additional analyses, including data and experiments from both murine and human LUAD model systems to elucidate a novel mechanism of BM activation in LUAD. We appreciate the reviewer’s suggestion to pursue analysis of Sox9 involvement in regulating BM activation and agree that both KRAS and SOX9 activation are likely to be involved in at least some elements of the process of disease progression we described in this manuscript. Indeed, previous studies have completed the experiments suggested, demonstrating Sox9 knock-out reduced Kras driven tumour progression and morphological grade in vivo (PMID: 37258742 and 34021911); and was associated with loss of AT2 lineage identity (PMID: 37468622).

Our analysis of human LUAD using scRNA-seq data has demonstrated that this differentiation spectrum in fact extends beyond loss of lineage fidelity and in a subset of cells leads to transdifferentiation to a basal-like cell state. In our revised manuscript, we have more clearly elucidated the role of KRAS and TP53 in these two events during LUAD progression, demonstrating that while oncogenic KRAS (and likely downstream SOX9 activation) can lead to the loss of lineage commitment in LUAD cells, mutations in TP53 are required for acquisition of the basal-like phenotype. We have also expanded on this mechanism identifying a novel role for type-1 interferon signaling in the presence of TP53 loss-of-function as a mechanism that can lead to BM activation and acquisition of a basal-like cell state in LUAD. The data related to these analyses are now presented in figures 3 and 7.

In accordance with the 3Rs principles for ethical use of animals in research we have taken advantage of publicly available data from previous experiments analyzing conditional (lung-specific) Kras mutant mouse model to validate our in-silico findings. This confirmed our in silico analysis of human LUAD, demonstrating an important role for TP53 loss of function in regulating BM activation (presented in Figure 3E&F and Figure S3F&G)

We also showed that the type I interferon signaling is capable of driving BM activation in LUAD but only in the context of TP53 loss-of-function. These experiments were performed using 3D organotypic cultures of H441 cells (human adenocarcinoma cell line with mutant TP53) and A549 cells (human adenocarcinoma cell line with wild-type TP53). These 3D cultures were treated with IFN-alpha, both BM and basal-like marker upregulation (MKI67, CDC20, TOP2A, S100A9, S100A2, SOX9 and KRT17) was observed only in LUAD cells carrying a mutation in TP53. These data are now presented in Figure S7D.

R1-Comment 3: In general, the description of the results in the corresponding section of the manuscript can be improved to facilitate the understanding of the results presented.

As an example, the figure 1B is described on page 13 as follows: "...we first used a publicly available microarray dataset [9] to identify genes differentially expressed between epithelial cells engaged in BM (embryonic day 14 [E14]) or ALV (embryonic day 19 [E19]) (Figure 1B and TABLE S1)." By looking at the plot in figure 1B, this description is not sufficient to understand what the authors present in this figure panel, not even after reading the corresponding figure legend.

Reply: We thank the reviewer for their advice on making our manuscript clearer. Throughout the manuscript we have now edited the result descriptions, we have also provided further detail to the methods sections, figure legends and axes labels to enhance clarity and facilitate understanding of the analyses performed.

In the example cited we have edited the sections referenced above as follows:

“To test this hypothesis, we identified genes that were differentially expressed in epithelial cells engaged in active BM (corresponding to embryonic day 14) vs active ALV (corresponding to embryonic day 19), using a publicly available microarray dataset.”

We have also changed the Y axis label of Figure 1B to: “log2(FC E19 [ALV] – E14[BM])”.

The description in the figure legend has also been modified to provide more context: “Dot plot showing the identification of genes differentially expressed by epithelial cells during murine developmental-BM (embryonic day 14) and ALV (embryonic day 19) [1]. Genes with the highest Fold Change of expression between day 14 (BM) and 19 (ALV) of murine lung development are coloured green or red, respectively. These genes were used to generate ALV/BM signatures [9]”

R1-Comment 5: Another example is the description of the figure 3B on page 16: "This showed low levels of BM activation in tumour cells from residual disease (RD) that was significantly increased in samples with recurrent progressive disease (PD) (Figure 3B)." By looking figure 3B and the corresponding figure legend, one cannot find the group "residual disease (RD)".

__Reply: __We thank the reviewer for their diligent reading and have now corrected the figures to provide clearer labelling of axes and maintain consistency throughout. In the example cited, we have corrected the axis label to Residual disease (RD) and partial response (PR).

R1-Comment 6: Another example is the description of the figure 3C and 3D on page 16: "Single-cell analysis showed that both ALV-BM- and ALV-BM+ LUAD cells were increased in samples from recurrent progressive disease (Figure 3C,D)." By looking at figure 3D and the corresponding legend, I do not find the explanation of "TRUE" and "FALSE". The same is for figures 3J and 3M.

Reply: For this example (Figure 3 in the original manuscript is now figure 4), TRUE/FALSE labels have been replaced by PR (partial response) and PD (progressive disease) in panel D; replaced by “Responder (R)” or “Non-responder (NR)” in panels J&M.

R1-Comment 7: Other figure panels were also poorly described in the results section and in the corresponding legends. Further, the presentation of the results in the main and supplementary figures has to be improved. For example, labeling of the Y-axis in the figures 1H to 1J, 2C, 2D, 2G, 2H, 3B, 3C, 3J, 3L, etc. has to be improved. As a point of reference, I would suggest checking how other authors present similar results in life science journals. These deficiencies in the presentation and description of the results make it difficult for the readers to understand the manuscript.

Reply: These axes labels have been changed throughout to provide more information. “BM” changed to “BM (ssGSEA score)” or “BM (module score)” and “ALV” changed to “ALV (ssGSEA score)” or “ALV (module score)” for figures 1H, 1I, 1J, S1H-L, 2C, 2D, 3B, 3F, S3E, S3F, S3G, 4B, 4C, 4J, 4L, S4C, S4D, S5A, 6A, S6A, S6B, ssGSEA score was applied to bulk RNAseq samples, and modules scores were calculated for single cells.

Additionally:

S2A, S2B – OS label changed to Survival probability/OS probability.

S4H – y axis label changed to PDL1 (RPPA).

S3B – y axis label changed to “Tumour mutational burden (mut/mB).

S3C – y axis label changed to “Tobacco smoking (SBS mutational signature)”.

4F – y axis label changed to “DFS (proportion)”.

4H – y axis label changed to “PFS (proportion)”.

R1-R1-Comment 8: The authors write on page 18 "Despite AT2 cells being well described as the cell of origin for LUAD, this population was significantly less abundant in LUAD samples compared to control, demonstrating a high degree of transcriptomic plasticity within LUAD epithelium (Figure 4D)." How can the authors show that these results are not produced by the process of integration of the four scRNA-seq NSCLC datasets, the implementation of a specific machine learning classifier for the cell type-classification, or the manually filtration to exclude doublets? For example, will the authors achieve the same (or similar) results using a different machine learning classifier? If yes, please include the results in the manuscript.

Reply: The integration was performed using the method described by Stuart et al. (PMID: 31178118), implemented in the Seurat package. The term “machine learning classifier” has now been replaced by “label transfer” to clarify the method used and avoid confusion. Label transfer was only used to identify major cell types in the datasets used, i.e. the whole epithelial population. Doublet removal was performed as follows (and described in the methods section): epithelial cells were clustered using the shared nearest neighbor (SNN) modularity optimization algorithm implemented by the FindClusters function in the Seurat R package, based on 30 principal components and setting the resolution parameter to 0.1. This clustering solution identified multiple small clusters with divergent expression profiles to the majority of cells that were initially classified as epithelial (in the label transfer analysis). Manual examination of the marker genes for these small clusters showed they were characterized by expression of epithelial genes alongside canonical markers for either B cells (CD79A), macrophages (CD68, SPP1, APOE, CD14, MARCO) or Tcells/NK cells (CD3D, NKG7, CXCR4). These cells were therefore classed as heterotypic doublets and excluded from further analysis. All other cell types from the integrated datasets were analyzed in the same way, and no further epithelial clusters (that were not small clusters of doublets) were identified.

Further clustering to identify epithelial subpopulations was performed on the integrated dataset and the results presented from this analysis represent the clustering solution that ensures all subpopulations were identified across datasets to mitigate any potential batch-effects not resolved by the integration process. Furthermore, our results showing that LUAD cells exhibit a high degree of transcriptomic plasticity were also confirmed by the lineage fidelity analysis (Figure 5G&I), which demonstrates this observation is not dependent on a single clustering, integration or machine learning algorithm. This observation is also supported by other studies that have described loss of lineage commitment during LUAD tumorigenesis, where tumour cells become transcriptionally and phenotypically distinct from healthy AT2 cells.

Reviewer 1:

Minor comments:

__R1-Comment 9: __Please introduce the abbreviation for alveogenesis the first time that is used in the abstract, as it was done for branching morphogenesis.

__Reply: __Abbreviation for alveogenesis has now been added to the abstract.

R1-Comment 10: On page 18 the author write: "Consistent with the analyses presented above, pseudo bulk expression profiles for each sample showed that ALV and BM scores were significantly negatively correlated (r = -0.68, p = 4.1e-09)." Where are these results shown? I was not able to find these results. If they are not in the current version of the manuscript, please include the results

Reply: Scatter plot showing the negative correlation has now been added as Figure S5A.

__R1-Comment 11: __The authors should submit a supplementary table containing a list of the different data sets that were used for this manuscript. The table should include accession numbers and links to the different depositories, in which the data sets can be found. This will improve the overview of the datasets used in the study, as well as facilitate the finding of the datasets by the readers.

Reply: The list of all datasets used in this study, together with accession numbers and links are now in Supplementary file1.

R1-Comment 12: In figure 1G, change the color for FALSE in the legend.

Reply: Color for FALSE changed in Figure 1G and Figure S1E.

R1-Comment 13: Provide the complete list of mutated genes for Figure S2C.

Reply: Figure S2C has been replaced by figures 3C (top mutated genes in LUAD-BM) and S3A (top mutated genes in LUAD-ALV).

Reviewer 1 (Significance (required)):

__R1-Comment 14: __Conceptually, Bienkowska KJ et al. propose that LUAD tumors undergo reversion from an alveogenic to branching morphogenic phenotype during disease progression, generating inflamed or basal-like cell states that are variably persistent following TKI or ICB treatments. This concept is in line with reports using murine models of Kras-driven LUAD. In addition, there are parallels with findings in idiopathic pulmonary fibrosis (IPF, another hyperproliferative lung disease), in which KRT5-/KRT17+ basaloid cells were transiently found, like the basal-like phenotype that Bienkowska KJ identified in human LUAD. In other words, the concept proposed by the authors is novel and in line with previous publication in LC and IPF.

Response: We are glad the reviewer found our results novel and appreciated how they provide a linkage of previously defined mechanisms seen in murine developmental models to human cancer progression, and how they may be relevant for other diseases such as IPF.

__R1-Comment 15: __The in silico analysis of publicly available transcriptomic datasets presented by Bienkowska KJ et al. is original and comprehensive. It is an interesting contribution to the cancer research field. However, the impact of their findings to this scientific field will significantly increase if the authors could confirm the interpretation of their results using other experimental systems in addition to the one used in the las figure. For example, the experiments that I suggested in point 2., using either conditional Kras transgenic mice or a PDX mouse model for lung cancer will not only confirm the concpet proposed by the authors, but it will also provide further mechanistic insides related to this model at cellular and molecular level.

Response: We thank the reviewer for describing our analysis as original and comprehensive and their suggestion to develop the manuscript further with additional mechanistic analyses. We have comprehensively examined the mechanisms responsible for regulating BM activation using a combination of in vivo models and 3D organotypic cultures, elucidating a novel role for type-1 interferon signaling in the presence of TP53 loss-of-function as a mechanism that can lead to BM activation and acquisition of a basal-like cell state in LUAD. For further information regarding these additions to the revised manuscript, we direct the reviewer to the response provided to R1-comment 2 (above).

__R1-Comment 16: __Overall, the manuscript by Bienkowska KJ et al. addresses topics that are relevant to the field of lung cancer, the leading cause of cancer-related deaths worldwide. The bioinformatic methods implemented are cutting-edge. However, the text of the manuscript and the presentation of the results in the figures have to be improved to better exploit the potential of their findings. In addition, further experiments should be performed to confirm (and perhaps complement) the interpretation of their findings. I hope that my comments support the authors to improve the manuscript to reach the standard of manuscripts recently published at renowned journals in Review COMMONS. I recommend a major revision of the manuscript before publication.

__Reply: __We are pleased to read that the reviewer found the methods implemented by us to be cutting-edge, and that they recognized the relevance of this topic to the lung cancer field.

We thank the reviewer for their comments, which have helped us to significantly improve our manuscript.

We have made changes to how we present our data (as described in responses above) and performed further analyses to support our original findings. We have also now performed further in silico and functional analyses to expand and complement our original findings.

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

R2-Comment 1: __The study is novel and interesting, but the mechanisms how the dysregulation of developmental program was driven by specific oncogene and how to link these signatures to therapy were also not clear. __

__Reply: __We are pleased that the reviewer finds our study to be novel and interesting. We appreciate the reviewer pointing out the need to clarify the role of specific oncogenes to BM activation and response to therapies.

We have now added further analyses and edited the text to examine and explain how the ALV and BM signatures are driven by different oncogenes (Figure 3 and results section “TP53 loss of function is required for BM activation”), which showed that common oncogenic drives (e.g. KRAS and EGFR) can drive reduced ALV signature expression but TP53 mutations (or deletion in murine models) was critical for driving BM activation. Implications for therapy response are shown in (Figure 4). We have shown that BM activation is a key determinant of tyrosine kinase inhibitor (TKI) resistance in LUAD, representing a frequently activated off-target mechanism of resistance that supersedes the presence of an actionable oncogenic driver in terms of response rates; and that the BM signature also identified patients that, although positive for immune checkpoint blockade (ICB) response biomarkers, will likely fail to respond to this treatment. In the manuscript we have now thoroughly revised these sections of the results to clarify the details associated with these conclusions (results sections: “BM activation is associated with targeted-therapy resistance in lung adenocarcinomas” and “BM activation predicts poor response to immune-checkpoint blockade”).

We have also added further data to the manuscript elucidating the molecular mechanisms regulating BM activation (Figure 7), which has identified an important role for aberrant type-I interferon signaling in the context of mutant TP53.

Reviewer #2 (Significance (Required)):

__R2-Comment 2: __The authors in this manuscript aimed to examine the role of developmental programmes, alveogenesis and branching morphogenesis (BM), in regulating phenotypic diversity in NSCLC. They demonstrated that developmental programmes (ALV and BM) frequently become

dysregulated in NSCLC, with BM activation identifying aggressive LUAD that were resistant

to multiple therapies, including TKIs and ICB. They found that BM activation in LUAD was associated with TP53 pathway mutations and required AT2 cells to lose their alveolar identity, acquiring a basallike state. The study is very intriguing, and the findings may pave a link to the disease progression and therapy resistance in LUAD.

__Response: __We are pleased the reviewer found the study intriguing and with the potential to better understand LUAD progression and resistance to therapies.

__R2-Comment 3: __The current results presented, although comprehensively presented, is still many an association study, the mechanisms how these dysregulations of developmental programmes driven by the driver oncogenes or carcinogens are still unknown.

Response: We thank the reviewer for challenging us to further examine the molecular mechanisms underpinning our initial observations. As described above (see response to Reviewer #1 comment 2), we have performed additional in silico and mechanistic experimental analyses, which identified a novel role for type-I IFN signaling and TP53 loss-of-function in the activation of the BM program in LUAD. We hope these additions have enhanced the significance of the manuscript presented.

__R2-Comment 4: __The NSCLC is a heterogeneous disease, LUAD and LUSC are two different diseases in terms of oncogenesis, driver mutations and response to treatment. The manuscript may either just focused on LUAD or describe results carefully to include both LUAD and LUSC. For example, in the result of abstract, only LUAD was described, there was no mention of LUSC.

__Response: __We agree with the reviewer that NSCLC is a heterogeneous and complex disease. Indeed, this was in part what motivated us to investigate the role played by developmental processes in these distinct oncogenic processes. Our analyses showed that LUSC tumors were generally high for the BM signature (Figure 1I), which likely contributed to why this signature did not stratify survival rates for LUSC (Figure S2B). As a result, we opted to focus on LUAD as we found that BM activation was predictive of disease progression and survival in this NSCLC subtype. However, we did not completely remove LUSC from our manuscript to examine the degree to which LUAD tumors upregulating BM become “LUSC-like” and evaluate whether histological transformations occurred in LUAD cases with BM activation (as described in Figure 5 and the “BM activation in LUAD is associated with a basal-like phenotype” results section).

We have also now added a description of results from both LUAD and LUSC analyses to the abstract to clarify these points.

__R2-Comment 5: __The most common driver mutation of LUAD was EGFR, the authors also try to link the BM activation link to TKI resistance. I assumed that the TKIs most of the patients used were EGFR TKI, but the study did not examine the role of EGFR in the dysregulation of developmental programmes.

__Response: __We would like to thank the reviewer for highlighting an important aspect of how our work fits with current clinical practice in LUAD management. Our analyses were carried out over multiple cohorts that include different patient demographics, which have varied prevalence for specific oncogenic driver mutations (with EGFR mutations typically being more prevalent in Asian cohorts and KRAS mutations generally being the most common oncogenic driver in Western cohorts). To examine these two common oncogenic drivers impact on BM activation, we now include a direct analysis of BM level in cases harboring these mutations (Figure S3D-E). This showed that that irrespective of oncogenic driver mutations TP53 loss of function was associated with increased BM. Our new analysis of KRAS driven mouse models has also showed that KRAS activation is sufficient to induce reduced expression of the ALV signature but failed to elicit increased BM activation. Given our analysis of human tumours showed that EGFR mutant LUAD cases with wild-type TP53 had low levels of BM activation (Figure S3D), we have no reason to suspect that EGFR mutations alone would be sufficient to elicit BM activation.

__R2-Comment 6: __The TKI resistance was very complicated, not just EGFR T790M, the results and discussion regarding the activation of BM and TKI resistance seems not adequate. The mouse model used by Dr. Chang was mainly KRAS driven mouse lung cancer model (mice carrying RosatdT, Sox2EGFP, ShhCre, Sox9CKO, Fgfr2CKO, RosamTmG, Sox9CreER, Nkx2.1CreER, and KrasLSL-G12D alleles). It is not clear whether the EGFR driven (the most common driver of LUAD) mouse model also has same genetic signature. At least, the authors should describe or discuss these discrepancies.

__Response: __We thank the reviewer for their comments and advice on making our manuscript clearer. We have now revised the description of BM activation and TKI resistance in the results section (titled “BM activation is associated with targeted-therapy resistance in lung adenocarcinomas”).

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Referee #2

Evidence, reproducibility and clarity

The study is novel and interesting, but the mechanisms how the dysregulation of developmental program was driven by specific oncogene and how to link these signatures to therapy were also not clear.

Significance

The authors in this manuscript aimed to examine the role of developmental programmes, alveogenesis and branching morphogenesis (BM), in regulating phenotypic diversity in NSCLC. They demonstrated that developmental programmes (ALV and BM) frequently become dysregulated in NSCLC, with BM activation identifying aggressive LUAD that were resistant to multiple therapies, including TKIs and ICB. They found that BM activation in LUAD was associated with TP53 pathway mutations and required AT2 cells to lose their alveolar identity, acquiring a basallike state.

The study is very intriguing and the findings may pave a link to the disease progression and therapy resistance in LUAD. The current results presented, although comprehensively presented, is still many an association study, the mechanisms how these dysregulations of developmental programmes driven by the driver oncogenes or carcinogens are still unknown. The NSCLC is a heterogeneous disease, LUAD and LUSC are two different diseases in terms of oncogenesis, driver mutations and response to treatment. The manuscript may either just focused on LUAD or describe results carefully to include both LUAD and LUSC. For example, in the result of abstract, only LUAD was described, there was no mention of LUSC. The most common driver mutation of LUAD was EGFR, the authors also try to link the BM activation link to TKI resistance. I assumed that the TKIs most of the patients used were EGFR TKI, but the study did not examine the role of EGFR in the dysregulation of developmental programmes. The TKI resistance was very complicated, not just EGFR T790M, the results and discussion regarding the activation of BM and TKI resistance seems not adequate. The mouse model used by Dr. Chang was mainly KRAS driven mouse lung cancer model (mice carrying RosatdT, Sox2EGFP, ShhCre, Sox9CKO, Fgfr2CKO, RosamTmG, Sox9CreER, Nkx2.1CreER, and KrasLSL-G12D alleles). It is not clear whether the EGFR driven (the most common driver of LUAD) mouse model also has same genetic signature. At least, the authors should describe or discuss these discrepancies.

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 #1

Evidence, reproducibility and clarity

Summary:

Bienkowska KJ et al show in this manuscript a compilation of bioinformatic analysis of publicly available microarray datasets, bulk RNA sequencing (RNA-seq) datasets and single cell RNA sequencing (scRNA-seq) datasets. One transcriptomic data set from mouse (Chang DR et al., Proc Natl Acad Sci USA, 2013) was analyzed in this manuscript to determine the gene expression signatures specific for the developmental processes alveogenesis (ALV) and branching morphogenesis (BM). The rest of the transcriptomic data sets that were analyzed for this manuscript were selected based on different parameters including the involvement of non-small cell lung cancer (NSCLC), lug adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) and various cohorts with different characteristics related to lung cancer (LC), such as mutations related to LC (in EGFR, ALK, BRAF, ROS1 and KRAS), and/or treatments/resistance of LC patients with/to tyrosine kinase inhibitors (TKI), anti-PD1 strategies, immune checkpoint blockade, (ICB) among others. In the last figure, the authors present results obtained by multiplexed immunohistochemistry and histo-cytometry of tissue microarrays from a curated cohort of archival formalin-fixed paraffin-embedded (FFPE) samples to confirm their interpretation of the results obtained by the transcriptomic analysis.

The findings and/or claims of the authors could be summarized in the following bullet points:

• After defining the ALV and BM gene expression signatures, the authors determined the expression of these signatures in bulk RNA-seq data sets from The Cancer Genome Atlas (TCGA). The ALV signature was significantly downregulated in LUAD and LUSC tumour subtypes compared to control samples, whereas BM was upregulated. These findings were validated using a Laser Capture Micro-Dissected (LCMD) microarray dataset (Lin J et al., AM J Pathol, 2014) comparing the epithelial compartment from non-tumour alveoli, non-tumour bronchi, LUAD and LUSC. Interestingly, ALV was suppressed and BM was activated in all LUSC tumours analyzed; whereas in LUAD tumours were heterogeneous for BM activation with some cases exhibiting ALV expression comparable to control tissue. In summary, the mutually antagonistic regulation of ALV and BM was found to account for a significant proportion of transcriptomic variance in human NSCLC bulk tissue datasets.
• BM activation was associated with poor overall survival rates in five independent LUAD cohorts (p=2.04e-13); and was significantly prognostic for resistance to TKIs (p=0.003) and ICBs (p=0.014), in pre-treatment biopsies.
• ScRNA-seq analysis revealed that malignant LUAD cells with loss of alveolar lineage fidelity predominantly acquired inflamed or basal-like cellular states, which were variably persistent in samples from TKI and ICB recurrence.
• The authors conclude from their analysis that LUAD tumours undergo reversion from an alveogenic to branching morphogenic phenotype during disease progression, generating inflamed or basal-like cell states that are variably persistent following TKI or ICB treatments.

Major comments:

1. The authors did not submit with the manuscript all the results that they have obtained from their analysis, on which they based their claims. I suggest that the authors submit a SourceData file in Excel format. This file should contain the values and the relevant information for each of the plots presented in the main and supplementary figures. For example, in case of box plots, the five-number summary should be provided. Further, the p-values and the test used for their calculations should be also mentioned.

The file could be organized in a way that the data and relevant information for each figure panel are presented in separated data sheets in order that the reader can easily navigate through the file and find the information for each figure panel fast.

Similarly, the authors should provide access to the scripts that they have developed or adapted from published scripts to perform the analysis of the datasets and obtain the results presented in the manuscript. The access to the scripts used in the manuscript is important to reproduce analysis. The scripts can be deposited at github, for example. 2. The results and their interpretations are mainly done based on in silico analysis from publicly available transcriptomic datasets. The confirmation of the results obtained by the in silico analysis is limited to the last figure, in which the authors show results obtained by multiplexed immunohistochemistry and histo-cytometry of tissue microarrays from a curated cohort of FFPE samples.

The relevance of the results obtained by the in silico analysis may increase if the authors could present results either in a conditional (lung-specific) Kras mutant mouse model, or patient-derived xenograft (PDX) mouse model of lung cancer. The PDX mouse model will be more suitable in case that access to genetically modified mouse models is not given and/or the time for the experiments is limited. In both cases, the hyperactivation of the small GTPase KRAS should expand the BM gene expression signature in the mouse lung in a Sox9-dependent manner, thereby leading to lung tumors. Further, Sox9 loss-of-function experiments should reduce the BM gene expression signature and favor the ALV gene expression signature. These results would strongly support the interpretation of the in silico results by the authors in the present manuscript, and would significantly increase the impact of the manuscript in the scientific field of lung cancer. 3. In general, the description of the results in the corresponding section of the manuscript can be improved to facilitate the understanding of the results presented. As an example, the figure 1B is described on page 13 as follows:

"...we first used a publicly available microarray dataset [9] to identify genes differentially expressed between epithelial cells engaged in BM (embryonic day 14 [E14]) or ALV (embryonic day 19 [E19]) (Figure 1B and TABLE S1)."

By looking at the plot in figure 1B, this description is not sufficient to understand what the authors present in this figure panel, not even after reading the corresponding figure legend.

Another example is the description of the figure 3B on page 16:

"This showed low levels of BM activation in tumour cells from residual disease (RD) that was significantly increased in samples with recurrent progressive disease (PD) (Figure 3B)."

By looking figure 3B and the corresponding figure legend, one cannot find the group "residual disease (RD)".

Another example is the description of the figure 3C and 3D on page 16:

"Single-cell analysis showed that both ALV-BM- and ALV-BM+ LUAD cells were increased in samples from recurrent progressive disease (Figure 3C,D)."

By looking at figure 3D and the corresponding legend, I do not find the explanation of "TRUE" and "FALSE". The same is for figures 3J and 3M.

Other figure panels were also poorly described in the results section and in the corresponding legends.

Further, the presentation of the results in the main and supplementary figures has to be improved. For example, labeling of the Y-axis in the figures 1H to 1J, 2C, 2D, 2G, 2H, 3B, 3C, 3J, 3L, etc. has to be improved. As a point of reference, I would suggest checking how other authors present similar results in life science journals.

These deficiencies in the presentation and description of the results make it difficult for the readers to understand the manuscript. 4. The authors write on page 18

"Despite AT2 cells being well described as the cell of origin for LUAD, this population was significantly less abundant in LUAD samples compared to control, demonstrating a high degree of transcriptomic plasticity within LUAD epithelium (Figure 4D)."

How can the authors show that these results are not produced by the process of integration of the four scRNA-seq NSCLC datasets, the implementation of a specific machine learning classifier for the cell type-classification, or the manually filtration to exclude doublets? For example, will the authors achieve the same (or similar) results using a different machine learning classifier? If yes, please include the results in the manuscript.

Minor comments:

1. Please introduce the abbreviation for alveogenesis the first time that is used in the abstract, as it was done for branching morphogenesis.
2. On page 18 the author write:

"Consistent with the analyses presented above, pseudo bulk expression profiles for each sample showed that ALV and BM scores were significantly negatively correlated (r = -0.68, p = 4.1e-09)."

Where are these results shown? I was not able to find these results. If they are not in the current version of the manuscript, please include the results 7. The authors should submit a supplementary table containing a list of the different data sets that were used for this manuscript. The table should include accession numbers and links to the different depositories, in which the data sets can be found. Thiy will improve the overview of the datasets used in the study, as well as facilitate the finding of the datasets by the readers. 8. In figure 1G, change the color for FALSE in the legend. 9. Provide the complete list of mutated genes for Figure S2C

Significance

Conceptually, Bienkowska KJ et al. propose that LUAD tumors undergo reversion from an alveogenic to branching morphogenic phenotype during disease progression, generating inflamed or basal-like cell states that are variably persistent following TKI or ICB treatments. This concept is in line with reports using murine models of Kras-driven LUAD. In addition, there are parallels with findings in idiopathic pulmonary fibrosis (IPF, another hyperproliferative lung disease), in which KRT5-/KRT17+ basaloid cells were transiently found, like the basal-like phenotype that Bienkowska KJ identified in human LUAD. In other words, the concept proposed by the authors is novel and in line with previous publication in LC and IPF.

The in silico analysis of publicly available transcriptomic datasets presented by Bienkowska KJ et al. is original and comprehensive. It is an interesting contribution to the cancer research field. However, the impact of their findings to this scientific field will significantly increase if the authors could confirm the interpretation of their results using other experimental systems in addition to the one used in the las figure. For example, the experiments that I suggested in point 2., using either conditional Kras transgenic mice or a PDX mouse model for lung cancer will not only confirm the concpet proposed by the authors, but it will also provide further mechanistic insides related to this model at cellular and molecular level.

Overall, the manuscript by Bienkowska KJ et al. addresses topics that are relevant to the field of lung cancer, the leading cause of cancer-related deaths worldwide. The bioinformatic methods implemented are cutting-edge. However, the text of the manuscript and the presentation of the results in the figures have to be improved to better exploit the potential of their findings. In addition, further experiments should be performed to confirm (and perhaps complement) the interpretation of their findings. I hope that my comments support the authors to improve the manuscript to reach the standard of manuscripts recently published at renowned journals in Review COMMONS. I recommend a major revision of the manuscript before publication.

This is a major point! It's all data to an AI or a computer. True or false has no meaning to it. That involves a higher level of thought/ sentience that it doesn't and can't have.

I feel that this is miss leading, they are generating answers based on a give data set that is explained higher up in the article. So It's a flawed data that causes false information, it extrapolates and publishes what makes sense from it's "education"

La fundación de unas 500 colonias griegas fue clave para la expansión del mundo helénico. alrededor del 40% de los griegos vivía en estos nuevos territorios, lo que fortaleció el comercio, difundió la cultura griega y amplió su influencia política y económica.

There are two types of errors; 1. Syntax Error (i.e code not following the rules of the language) 2. Logic Error (i.e the program executes but generates wrong results) <- These types of runtime errors are also called exceptions.

more like a Jensen Huang-like stunt

El comercio mejoró porque los barcos mercantes permitían transportar más productos de forma segura y el camino Diolkos en Corinto facilitaba acortar rutas evitando rodear toda la península.

El prestamo marítimo era un credito usado en el comercio: si el barco llegaba se pagaba con intereses, y si naufragaba no se devolvía.

este recurso era tan valioso para los griegos que era el único que controlaba el estado, servía para poder alimentar a la población, sobre todo en épocas de sequia

Acá se demuestra lo importante que fue el comercio para los griegos, no solo para lo económico, sino también en lo cultural.

Debido a su ubicación geográfica que conectaba a Atenas con Europa, Asia y áfrica, este se volvió un punto de encuentro clave para el comercio.

3000 a.C. hasta el 1200 a.C. Fue un periodo marcado por el uso de bronce para herramientas y armas, y la exportacion de aceite de oliva, vino, ceramica y productos artesanales. El crecimiento economico facilito la acumulacion de riqueza y el desarrollo de una clase mercantil.

We have been lied to about legal tender since Nixon ended the gold standard

This tries in with the sacred nature of the CIicle = Cáim

Does he mean TRIPLE? If so, this is Druidic in nature - the three spirals on Newgrange

I wondert is this the same as Needs and Offers as in HSC platform? It also sounds similar to our Du2chas gudielienss where we call what used to be a 'decision' an acceptancecommitment

I wonder can this be our Cosmunity Balance Sheet too?

This links to our sovereignty learnings and the estabishment of e-states and dual process of citizienry and sovereignty in Eire and the legal fiction state of Ireland

This is pure Archon wiziardry which has been in play for millenia

We also make space for the Gaelix language and we use the original terms to design our systems. English is a language od deception and lies.

We can link this to Quantum Balance Sheet and to Cohesion Dashboard too

Financial Sovereignty for Cosmunities !!

Wholenessing

Brehon Law is one such tradition and we are blessed to have much of it in written form. Thank you to the monks who wrote it down.

Yes, the debt is a source in itself We have been lied to for generations so it it requires us to change the narrative. To tell a new ending to the STONE SOUP STORY

Our primary message is that people ARE their own liquidity and this may be hard for people to believe becase they have been colonised for millenia

We will need simple use cases and examples they can do on their phone instantly and that we can set up with bakcks of ebvelopes or USSD technology on a nokie 6310 :-)

thank you. What is the business model of CYCLES though? Are you just another middle man? An ENTRE -PRENEUR??

Yay!! Sovereignty

The coherecne of greed is quote clear to me It is as ancient as in the gnostic texts where Sophia emanated Yeldabaoth who considered itself the only god.

A holomovement as per David Bohm

It can be the core of the D of DAGDA - Dúchas - Meitheal - Mutual Trust and Commitment Pooling Ssytem

Cohesion is accurate. Coherent thinks can produce language which can produce liquidity - creating value without harm

How close is this to commitment pooling?

This is a massive boost for cosmunities as a Cumann / Commons

Eirú sent us this potential to free the lawful peoples of the world - starting in Eire and then sharing it will all oppressed peoples.

Archonic instruction from their Yeldabaoth overlord - their demiurge

Eirúnomics is our Gaelic way of responding

It is desgned this way by Archons

Exactly, the people are the value, the relationships are the value - just like Brehon Law

We have been calling to the Universe for just such a system. We believe Eirú sent us Reinier to steward our techgnosis from which this will emanate

We have been dealing with Will Ruddick in Grassroots Econommics - but it is not market ready yet as fas as we are concerned. We love Will's work, and we are part of a community of practice very Wednesday for learning.

We say the very same thing to The Wheel in our proposition to them for us to start a mutual credit system to ease the burden on social enterprises

Ireland is an economy where 95% of people work in SME's The EU definition ofg an SME is revenue under €48m per annum

I was introduced to Tomaz by Dil Green. We met on Zoom. We agreed after an hour that we are a good fity and we ar both exited to co-create together. This is his white paper. I sent him Eirunomics google document https://docs.google.com/document/d/1xBijLaSwLdVa1YqhjXpBwZe7tlG7ypS3/edit?usp=sharing&ouid=112345107592419723130&rtpof=true&sd=true

and also the proposal I sent to The Wheel which is an association of 2400 social enterprises.

Tomas asked us to present to his technology team who will be in ireland in October so that they can offer us a case study platform to use in a small group on a no cost basis

Once we get to know the platform, we can offer it commercially to Chambers of Commerce and to Credit UNions for a revenue share to CYCLES

I immedtaly sent the white paper to Reinier so that he and I can review it together in advance of meeting Tomaz and his technology team in Ireland

They ahd chosen Ireland to do their retreat - this is why he was looking for a contact in Ireland to do a pilot

Thank you DIL!!

This is an important statement, as it guides us to let go of our own feelings about our biases. This allows us to look at them objectively without shame or guilt.

This seems like wasted legislation efforts. How can you legislate against an unknown unconscious force?

🌿 Diana de los bosques

Adriano no habla de la diosa literalmente, sino como símbolo de la caza y de un contacto intenso con la naturaleza. Diana es la divinidad de los bosques, los animales salvajes y la luna. Relacionarse con ella es relacionarse con lo salvaje, lo femenino y lo misterioso.

🐗 La caza del jabalí

En la adolescencia, la caza era rito de paso.

Para Adriano, matar un jabalí no era solo deporte, sino un primer contacto con el mando (dirigir a los cazadores, perros, organizar) y con el peligro real (un animal que podía matarlo).

La experiencia mezcla erotismo (“ser amado”), violencia y poder.

⚔️ Formación del carácter

Se entregaba con “furor”, es decir, con pasión casi dionisíaca.

Sus excesos provocaron que el propio Trajano —su tutor y futuro padre adoptivo— lo reprendiera.

En clave literaria, esto muestra a un joven impulsivo, con sed de riesgo, que debía ser domesticado para convertirse en emperador.

For backup copy simple mode (Enterprise plugins) we cannot disable/enable encryption while job is running currently. To enable/disable encryption we have to disable the job, change state and enable again

If we enable/disable encryption for already created application backup job backup became encrypted after next incremental or full backup (new storage is created) We do not force Full backup here

If password changed for policy it's applied right after next time VACM is generated (let's say new storage is created) , no UI messages here afaik

This isn't subtle. It's intuitive and obvious, being presented as if it's non-obvious. I kept waiting for the punchline.

I guess you can get here if your main mode of thinking is dealing in opaque "best practices" like "use references because it makes your code faster".

If password changed for policy it's applied right after next time VACM is generated (let's say new storage is created) , no UI messages here afaik

If we enable/disable encryption for already created application backup job backup became encrypted after next incremental or full backup (new storage is created) We do not force Full backup here

If password changed for policy it's applied right after next time VACM is generated (let's say new storage is created) , no UI messages here afaik

If we enable/disable encryption for already created application backup job backup became encrypted after next incremental or full backup (new storage is created) We do not force Full backup here

Why does temperature exacerbate ocean acidification in some cases but negate it in others? Does location make a difference? Type of habitat? Weather patterns? Local human activity?

Why does changing the temperature from ambient to warming cause such drastic behavioral changes in the infauna colonizing the different species of mussels?

etetye

ttt

test 2

test 1

eLife Assessment:

In this revised version, the authors provide a thorough investigation of the interaction of megakaryocytes (MK) with their associated extracellular matrix (ECM) during maturation; they provide compelling evidence that the existence of a dense cage-like pericellular structure containing laminin γ1 and α4 and collagen IV is key to fixing the perisinusoidal localization of MK and preventing their premature intravasation. Adhesion of MK to this ECM cage is dependent on integrin beta1 and beta3 expressed by MK. This strong conclusion is based on the use of state-of-the art techniques such f primary murine bone marrow MK cultures, mice lacking ECM receptors, namely integrin beta1 and beta3 null mice, as well as high-resolution 2D and 3D imaging. The study provides valuable insight into the role of cell-matrix interactions in MK maturation and provides an interesting model with practical implications for the fields of hemostasis and thrombosis.

Reviewer #1 (Public review):

The authors report on a thorough investigation of the interaction of megakaryocytes (MK) with their associated ECM during maturation. They report convincing evidence to support the existence of a dense cage-like pericellular structure containing laminin γ1 and α4 and collagen IV, which interacts with integrins β1 and β3 on MK and serve to fix the perisinusoidal localization of MK and prevent their premature intravasation. As with everything in nature, the authors support a Goldilocks range of MK-ECM interactions - inability to digest the ECM via inhibition of MMPs leads to insufficient MK maturation and development of smaller MK. This important work sheds light into the role of cell-matrix interactions in MK maturation, and suggests that higher-dimensional analyses are necessary to capture the full scope of cellular biology in the context of their microenvironment. The authors have responded appropriately to the majority of my previous comments.

Reviewer #2 (Public review):

Summary:

This study makes a significant contribution to understanding the microenvironment of megakaryocytes (MKs) in the bone marrow, identifying an extracellular matrix (ECM) cage structure that influences MK localization and maturation. The authors provide compelling evidence for the presence of this ECM cage and its role in MK homeostasis, employing an array of sophisticated imaging techniques and molecular analyses.

The authors have addressed most of the concerns raised in the previous review, providing clarifications and additional data that strengthen their conclusions

More broadly, this work adds to a growing recognition of the ECM as an active participant in haematopoietic cell regulation in the bone marrow microenvironment. This work could pave the way to future studies investigating how the megakaryocytes' ECM cage affects their function as part of the haematopoietic stem cell niche, and by extension, influences global haematopoiesis.

Author response:

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

Minor Issues:

(1) As the authors mention, MKs have been suggested to mature rapidly at the sinusoids, and both integrin KO and laminin KO MKs appear mislocalized away from the sinusoids. Additionally, average MK distances from the sinusoid may also help separate whether the maturation defects could be in part due to impaired migration towards CXCL12 at the sinusoid. Presumably, MKs could appear mislocalized away from the sinusoid given the data presented suggesting they are leaving the BM and entering circulation. Additional commentary on intrinsic (ex-vivo) MK maturation phenotypes may help strengthen the author's conclusions

Thank you for your insightful suggestion regarding intrinsic MK maturation defects in integrin KO and laminin KO mice. This indeed could be the case. We have now addressed this possibility in the revised discussion section (page 14; lines 14-15), acknowledging intrinsic maturation defects as a potential contributor to observed maturation issues.

(2) It would be helpful if the authors could comment as to whether MKs are detectable in blood.

We appreciate the opportunity to clarify this point. Intact Itgb1^-/-/Itgb3^-/- MKs were not detected in the peripheral blood by either flow cytometry or blood smear analysis. This indicates that megakaryocytes do not normally circulate in the systemic bloodstream. Instead, we observed large MK nuclei trapped specifically within the lung capillaries, consistent with their known physical retention in the pulmonary circulation during platelet release. This explanation is now better explained on page 10, lines 14-19.

(3) Supplementary Figure 6 - shows no effect on in vitro MK maturation and proplt, or MK area - But Figures 6B/6C demonstrate an increase in total MK number in MMP-inhibitor treated mice compared to control. This discrepancy should be better discussed.

We have now expanded the discussion in the revised manuscript to address the different results obtained in vitro and in vivo, emphazing that the in vitro model may not fully recapitulate the complex and dynamic bone marrow ECM niche. Additionally, differences in the source and regulation of MMPs likely contribute to the differing outcomes, underlining the importance of studying these processes within their physiological context. For instance, non-megakaryocytic sources of MMPs and paracrine regulatory mechanisms may play a critical role within the physiological microenvironment, ultimately affecting MK proliferation and maturation in a manner not observed in simplified culture systems. This clarifications can be found on page 12, lines 6-17.

(4) A function of the ECM discussed relates to MK maturation but in the B1/3 integrin KO mice, the presence of the ECM cage is reduced but there appears to be no significant impact upon maturation (Supplementary Figure 4). By contrast, MMP inhibition in vivo (but not in vitro) reduces MK maturation. These data could be better clarified in the text.

Thank you for raising this important point. While Suppl. Figure 4 shows normal size and ploidy in DKO MK, a critical defect is revealed at the ultrastructural level. Mature DKO MKs exhibit severe dysplasia of the demarcation membrane system (DMS), characterized by extensive membrane accumulation and abnormal archirecture, with no typical platelet territories visible. This DMS defect directly impairs MK maturation and explains the thrombocytopenia observed in these mice. Increased emperipolesis further indicated disrupted maturation processes. These observations confirm the essential role of the ECM cage in supporting proper DMS organization and overall MK maturation in vivo, consistent with findings from MMP inhibition experiments. We have clarified and emphasized the significance of these DMS abnormalities in the revised manuscripts, including updated results (Page 9, lines 17-21) and a new EM image in Suppl. Figure 4.

Reviewer #1 (Public review):

The authors report on a thorough investigation of the interaction of megakaryocytes (MK) with their associated ECM during maturation. They report convincing evidence to support the existence of a dense cage-like pericellular structure containing laminin γ1 and α4 and collagen IV, which interacts with integrins β1 and β3 on MK and serve to fix the perisinusoidal localization of MK and prevent their premature intravasation. As with everything in nature, the authors support a Goldilocks range of MK-ECM interactions - inability to digest the ECM via inhibition of MMPs leads to insufficient MK maturation and development of smaller MK. This important work sheds light into the role of cell-matrix interactions in MK maturation, and suggests that higher-dimensional analyses are necessary to capture the full scope of cellular biology in the context of their microenvironment. The authors have responded appropriately to the majority of my previous comments.

We sincerely thank the reviewer for their insightful comments.

Some remaining points:

In a previous critique, I had suggested that "it is unclear how activation of integrins allows the MK to become "architects for their ECM microenvironment" as the authors posit. A transcriptomic analysis of control and DKO MKs may help elucidate these effects". The authors pointed out the technical difficulty of obtained sufficient numbers of MK for such analysis, which I accept, and instead analyzed mature platelets, finding no difference between control and DKO platelets. This is not necessarily surprising, since mature circulating platelets have no need to engage an ECM microenvironment, and for the same reason I would suggest that mature platelet analyses are not representative of MK behavior as regards ECM interactions.

We fully agree with the reviewer that platelet analyses do not accurately reflect the behavior of MKs in the context of interactions with the ECM. This understanding is also one of the reasons why we chose not to include RT-PCR data on platelets in our manuscript. Instead, we emphasize the role of integrins as essential regulators of ECM remodeling, as they transmit traction forces that can significantly influence this process. We also report reduced RhoA activation in DKO MK, which is likely to affect ECM organization. We believe that these explanations contribute to a clearer understanding of how integrin activation enables megakaryocytes to act as "architects" of their ECM microenvironment.

Reviewer #2 (Public review):

This study makes a significant contribution to understanding the microenvironment of megakaryocytes (MKs) in the bone marrow, identifying an extracellular matrix (ECM) cage structure that influences MK localization and maturation. The authors provide compelling evidence for the presence of this ECM cage and its role in MK homeostasis, employing an array of sophisticated imaging techniques and molecular analyses.The authors have addressed most of the concerns raised in the previous review, providing clarifications and additional data that strengthen their conclusion.

More broadly, this work adds to a growing recognition of the ECM as an active participant in haematopoietic cell regulation in the bone marrow microenvironment. This work could pave the way to future studies investigating how the megakaryocytes' ECM cage affects their function as part of the haematopoietic stem cell niche, and by extension, influences global haematopoiesis.

We thank this reviewer for providing such constructive feedback.

that could well be a short-term effect

tets

test 5 - notatka page notes

This statement succinctly captures a key genetic and biochemical observation: increasing NPY1 expression results in phosphorylation changes that inhibit PIN function and, in turn, suppress pid null mutants. It supports a model where NPY1 acts to modulate PIN-mediated auxin transport, with downstream phenotypic consequences on the pid background.

eLife Assessment

This valuable study concerns a highly interesting and biologically relevant topic, the regulation of the PIN auxin transporter, which is of broad interest to the plant biology community. The authors propose NPY1 to act downstream of PID in auxin-mediated development by modulating PIN phosphorylation, which, if experimentally solidified, would expand our understanding of PIN regulation. While the genetic evidence is solid, the mechanistic role of NPY1 and the functional relevance of phosphorylated PIN residues are still uncertain. There are also concerns regarding experimental rigor and methodological transparency.

Reviewer #1 (Public review):

Summary:

The authors of this study propose a model in which NPY family regulators antagonize the activity of the pid mutation in the context of floral development and other auxin-related phenotypes. This is hypothesized to occur through regulation of or by PID and its action on the PIN1 auxin transporter.

Strengths:

The findings are intriguing.

Weaknesses and Major Comments:

(1) While the findings are indeed intriguing, the mechanism of action and interaction among these components remains poorly understood. The study would benefit from significantly more thorough and focused experimental analyses to truly advance our understanding of pid phenotypes and the interplay among PID, NPYs, and PIN1.

(2) The manuscript appears hastily assembled, with key methodological and conceptual details either missing or inconsistent. Although issues with figure formatting and clarity (e.g., lack of scale bars and inconsistent panel layout) may alone warrant revision, the content remains the central concern and must take precedence over presentation.

(3) Given that fertile progeny are obtained from pid-TD pin1/PIN1 and pid NPY OE lines, it would be important to analyze whether mutations and associated phenotypes are heritable. This is especially relevant since CRISPR lines can be mosaic. Comprehensive genotyping and inheritance studies are required.

(4) The Materials and Methods section lacks essential information on how the lines were generated, genotyped, propagated, and scored. There is also generally no mention of how reproducible the observations were. These genetic experiments need to be described in detail, including the number of lines analyzed and consistency across replicates.

(5) The nature of the pid alleles used in the study is not described. This is essential for interpretation.

(6) The authors measure PIN1 phosphorylation in response to NPY overexpression and conclude that the newly identified phosphorylation sites are inhibitory because they do not overlap with known activating sites. This conclusion is speculative without functional validation. Functional assays are available and must be included to substantiate this claim.

(7) Figure 5 implies that NPY1 acts downstream of PID, but there is no biochemical evidence supporting this hierarchy. Additional experiments are needed to demonstrate the epistatic or regulatory relationship.

(8) The authors should align their genetic observations with cell biological data on PIN1, PIN2, and PID localization and distribution.

Reviewer #2 (Public review):

Summary:

The study is well-conducted, revealing that NPY1, with previously less-characterized molecular functions, can suppress pid mutant phenotypes with a phosphorylation-based mechanism. Overexpression of NPY1 (NPY1-OE) results in PIN phosphorylation at unique sites and bypasses the requirement for PID for this event. Conversely, a C-terminal deleted form of NPY1 (NPY1-dC) fails to rescue pid despite promoting a certain phospho-profile in PIN proteins.

Strengths:

(1) The careful genetic analyses of pid suppression by NPY1-OE and the inability of NPY1dC to do the same.

(2) Phospho-proteomics approaches reveal that NPY1-OE induces phosphorylation of PINs at non-canonical sites, independent of PID.

Weaknesses:

(1) The native role of NPY1 is not tested by phospho-proteomics in loss-of-function npy1 mutants. Such analysis would be crucial to demonstrate that NPY1 is required for the observed phosphorylation events.

(2) The functional consequences of the newly identified phosphorylation sites in PINs remain speculative. Site-directed mutagenesis (phospho-defective and phospho-mimetic) would help clarify their physiological roles.

(3) The kinase responsible for NPY1-mediated phosphorylation remains unidentified. Since NPY1 is a non-kinase protein, a model involving recruitment of partner kinases (e.g., PIN-phosphorylating kinases other than PID) should be considered or discussed.

Reviewer #3 (Public review):

Summary:

This manuscript from Mudgett et al. explores the relative roles of PID and NPY1 in auxin-dependent floral initiation in Arabidopsis. Micro vectorial auxin flows directed by PIN1 are essential to flower initiation, and loss of PIN1 or two of its regulators, PID and NPY1 (in a yucca-deficient background) phenocopies the pinformed phenotype. This group has previously shown that PID-PIN1 interactions and function are dosage-dependent. The authors pick up this thread by demonstrating that a heterozygote containing a CRISPR deletion of one copy of PIN1 can restore quasi-wild type floral initiation to pid.

The authors then show that overexpression of NPY1 is sufficient to more or less restore wild-type floral initiation to the pid mutant. The authors claim that this result demonstrates that NPY1 functions downstream of PID, as this ectopic abundance of NPY1 resulted in phosphorylation of PIN1 at sites that differ from sites of action of PID. The authors pursue evidence that PID action via NPY1 is analogous to the mode of action by which phot1/2 act on NPH3 in seedling phototropism. Such a model is supported by the evidence presented herein that the C terminus of NPY1, which has abundant Ser/Thr content, is phosphorylated, and that the deletion of this domain prevents overexpression compensation of the pinformed phenotype.<br /> While the results presented support evidence in the literature that PID acts on NPY1 to regulate PIN1 function, it is also possible that NPY1 overexpression results in limited expansion of phosphorylation targets observed with other AGC kinases. And if the phot model is any indication, there may be other PID targets that modulate PIN1-dependent floral initiation.

However, overexpression of the NPY1 C-terminal deletion construct resulted in phosphorylation of both PIN1 and PIN2 and agravitropic root growth similar to what is observed in pin2 mutants. This suggests that direct PID phosphorylation of PINs and action via NPY1 can be distinguished by phosphorylation sites and by growth phenotypes.

Strengths:

A very important effort that places NPY1 downstream of PID in floral initiation.

Weaknesses:

As PID has been shown to act on sites that regulate PIN protein polarity as well as PIN protein function, it would be useful if the authors consider how their results would fit/not fit with a model where combinatorial function of NPY1 and PID regulate PIN1 in a manner similar to the way that PID appears to function combinatorially with D6PK on PIN3.

Author response:

Reviewer #1 (Public review):

Summary:

The authors of this study propose a model in which NPY family regulators antagonize the activity of the pid mutation in the context of floral development and other auxin-related phenotypes. This is hypothesized to occur through regulation of or by PID and its action on the PIN1 auxin transporter.

Strengths:

The findings are intriguing.

We are pleased that the reviewer found the work interesting!

Weaknesses and Major Comments:

(1) While the findings are indeed intriguing, the mechanism of action and interaction among these components remains poorly understood. The study would benefit from significantly more thorough and focused experimental analyses to truly advance our understanding of pid phenotypes and the interplay among PID, NPYs, and PIN1.

Elucidating the mechanism of action and interaction among these components will require years of additional research. As key steps toward these goals, our work clearly established that 1) NPY1 functions downstream of PID, as overexpression of NPY1 completely suppressed pid phenotypes. This is surprising because the predominant model is that PID functions by directly phosphorylating and activating PINs without the need of NPY1 involvement.  2) In the absence of PID, NPY1 protein accumulated less in the NPY1 OE lines, suggesting that PID plays a role in affecting NPY1 stability/degradation/accumulation. We are not sure what are the exact experiments this reviewer is proposing.

Regarding pid phenotypes, pid is completely sterile in our conditions, while the suppression by NPY1 OE is very clear and the lines are fertile.

(2) The manuscript appears hastily assembled, with key methodological and conceptual details either missing or inconsistent. Although issues with figure formatting and clarity (e.g., lack of scale bars and inconsistent panel layout) may alone warrant revision, the content remains the central concern and must take precedence over presentation.

We did not include scale bars in our figures because the phenotype of interest is presence/absence of flowers. Readers should compare the mutants with the rescued plants and the WT plants.

(3) Given that fertile progeny are obtained from pid-TD pin1/PIN1 and pid NPY OE lines, it would be important to analyze whether mutations and associated phenotypes are heritable. This is especially relevant since CRISPR lines can be mosaic. Comprehensive genotyping and inheritance studies are required.

We only use stable, heritable, Cas9-free mutants in our studies.  We genotype our mutants in every generation.  More details have been added to the Materials and Methods section. We provide the genetic materials we use to the scientific community when requested to enable verification and extension of our results. 

(4) The Materials and Methods section lacks essential information on how the lines were generated, genotyped, propagated, and scored. There is also generally no mention of how reproducible the observations were. These genetic experiments need to be described in detail, including the number of lines analyzed and consistency across replicates.

More details have been added to the Materials and Methods section

The criticism is not fully accurate. For example, we stated in the main text: “We genotyped T2 progenies from two pid-c1 heterozygous T1 plants (#68 and # 83) for the presence of pid-c1 and for pid-c1 zygosity. We used mCherry signal, which was included in the NPY1 OE construct, as a proxy to determine the presence and absence of the NPY1 transgene. For each line, we identified T2 plants without the NPY1 transgene and without the pid-c1 mutation (called WT-68 and WT-83, respectively). We also isolated T2 plants that contained the NPY1 overexpression construct, but did not have the pid-c1 mutation (called NPY1 OE #68 in WT, and NPY1 OE #83 in WT). Finally, we identified T2 plants that were pid-c1 homozygous and that had the NPY1 transgene (called NPY1 OE #68 in pid-c1 and NPY1 OE #83 in pid-c1). These genetic materials enabled us to compare the same NPY1 OE transgenic event in different genetic backgrounds.”

The genetic materials used are freely available to the scientific community.  We would like to point out that we used several pin1 and pid alleles to make sure that the phenotypes are caused by the genes of interest.

(5) The nature of the pid alleles used in the study is not described. This is essential for interpretation.

The mutants were described in a previous paper (M. Mudgett, Z. Shen, X. Dai, S.P. Briggs, & Y. Zhao, Suppression of pinoid mutant phenotypes by mutations in PIN-FORMED 1 and PIN1-GFP fusion, Proc. Natl. Acad. Sci. U.S.A. 120 (48) e2312918120, https://doi.org/10.1073/pnas.2312918120 (2023).  We have added the relevant information to Materials and Methods.

(6) The authors measure PIN1 phosphorylation in response to NPY overexpression and conclude that the newly identified phosphorylation sites are inhibitory because they do not overlap with known activating sites. This conclusion is speculative without functional validation. Functional assays are available and must be included to substantiate this claim.

We concluded that the phosphorylation of PINs in NPY1 OE is inhibitory on the basis of the following: 1) pid is suppressed in pin1 heterozygous backgrounds and by PIN1-GFP_HDR, demonstrating that partial loss of function of PIN1 or a decrease in PIN1 gene dosage, which decreases PIN1 protein expression, caused the suppression of pid. 2) pid is completely suppressed by NPY1 OE, which caused an increase of PIN phosphorylation, suggesting that phosphorylation of PINs in NPY1 OE lines is inhibitory.  It is true that we do not have biochemical data to support the conclusion. We would like to point out that the phosphorylation sites in PINs identified in this work do overlap with previously identified sites.

PIN activity assays are conducted in heterologous systems that do not include NPY proteins. Since NPY is important for PIN activities, we believe that these assays may provide misleading results. Moreover, PIN1 is likely part of a large protein complex.  Without knowing the composition of the complex, functional assays in heterologous systems will not be interpretable.

(7) Figure 5 implies that NPY1 acts downstream of PID, but there is no biochemical evidence supporting this hierarchy. Additional experiments are needed to demonstrate the epistatic or regulatory relationship.

We show that overexpression of NPY1 completely suppressed the pid phenotype, and this epistatic relationship indicates that NPY1 functions downstream of PID. Moreover, we report that PID is required for NPY1 accumulation, indicating that PID is upstream of NPY1.

(8) The authors should align their genetic observations with cell biological data on PIN1, PIN2, and PID localization and distribution.

We are hesitating in using traditional PIN1-GFP, PIN2-GFP lines, as they are not stable in our hands. Localization of PID is still not clear. We have generated PID-GFP_HDR lines, but we could not detect any fluorescent signals (unpublished results).  In addition, maize PINOID (BIF2) localizes to the nucleus, cytoplasm and cell periphery (Skirpan, A., Wu, X. and McSteen, P. (2008), Genetic and physical interaction suggest that BARREN STALK1 is a target of BARREN INFLORESCENCE2 in maize inflorescence development. The Plant Journal, 55: 787-797. https://doi.org/10.1111/j.1365-313X.2008.03546.x)

We would rather wait for the proper genetic materials before devoting our effort to this.

Reviewer #2 (Public review):

Summary:

The study is well-conducted, revealing that NPY1, with previously less-characterized molecular functions, can suppress pid mutant phenotypes with a phosphorylation-based mechanism. Overexpression of NPY1 (NPY1-OE) results in PIN phosphorylation at unique sites and bypasses the requirement for PID for this event. Conversely, a C-terminal deleted form of NPY1 (NPY1-dC) fails to rescue pid despite promoting a certain phospho-profile in PIN proteins.

Strengths:

(1) The careful genetic analyses of pid suppression by NPY1-OE and the inability of NPY1dC to do the same.

(2) Phospho-proteomics approaches reveal that NPY1-OE induces phosphorylation of PINs at non-canonical sites, independent of PID.

Thank you for having accurately summarized the main findings

Weaknesses:

(1) The native role of NPY1 is not tested by phospho-proteomics in loss-of-function npy1 mutants. Such analysis would be crucial to demonstrate that NPY1 is required for the observed phosphorylation events.

This is an excellent point and we agree with the reviewer that analyzing loss-of-function npy mutants is important. The challenge is that we need to knockout NPY1, NPY3, and NPY5 to phenocopy pid. We will also need to find a way to suppress the npy triple mutants, which are sterile, so that we can have meaningful comparisons.

(2) The functional consequences of the newly identified phosphorylation sites in PINs remain speculative. Site-directed mutagenesis (phospho-defective and phospho-mimetic) would help clarify their physiological roles.

We agree with the reviewer on this point as well. However, this is not trivial, as we have uncovered so many phosphorylation sites.

(3) The kinase responsible for NPY1-mediated phosphorylation remains unidentified. Since NPY1 is a non-kinase protein, a model involving recruitment of partner kinases (e.g., PIN-phosphorylating kinases other than PID) should be considered or discussed.

we will add a sentence to mention D6PK and other kinases in the Discussion in the revised version.  We are hoping that the kinases will come out of future forward genetic screens.

Reviewer #3 (Public review):

Summary:

This manuscript from Mudgett et al. explores the relative roles of PID and NPY1 in auxin-dependent floral initiation in Arabidopsis. Micro vectorial auxin flows directed by PIN1 are essential to flower initiation, and loss of PIN1 or two of its regulators, PID and NPY1 (in a yucca-deficient background) phenocopies the pinformed phenotype. This group has previously shown that PID-PIN1 interactions and function are dosage-dependent. The authors pick up this thread by demonstrating that a heterozygote containing a CRISPR deletion of one copy of PIN1 can restore quasi-wild type floral initiation to pid.

The authors then show that overexpression of NPY1 is sufficient to more or less restore wild-type floral initiation to the pid mutant. The authors claim that this result demonstrates that NPY1 functions downstream of PID, as this ectopic abundance of NPY1 resulted in phosphorylation of PIN1 at sites that differ from sites of action of PID. The authors pursue evidence that PID action via NPY1 is analogous to the mode of action by which phot1/2 act on NPH3 in seedling phototropism. Such a model is supported by the evidence presented herein that the C terminus of NPY1, which has abundant Ser/Thr content, is phosphorylated, and that the deletion of this domain prevents overexpression compensation of the pinformed phenotype.

While the results presented support evidence in the literature that PID acts on NPY1 to regulate PIN1 function, it is also possible that NPY1 overexpression results in limited expansion of phosphorylation targets observed with other AGC kinases. And if the phot model is any indication, there may be other PID targets that modulate PIN1-dependent floral initiation.

However, overexpression of the NPY1 C-terminal deletion construct resulted in phosphorylation of both PIN1 and PIN2 and agravitropic root growth similar to what is observed in pin2 mutants. This suggests that direct PID phosphorylation of PINs and action via NPY1 can be distinguished by phosphorylation sites and by growth phenotypes.

Strengths:

A very important effort that places NPY1 downstream of PID in floral initiation.

We thank the reviewer for the comments.

Weaknesses:

As PID has been shown to act on sites that regulate PIN protein polarity as well as PIN protein function, it would be useful if the authors consider how their results would fit/not fit with a model where combinatorial function of NPY1 and PID regulate PIN1 in a manner similar to the way that PID appears to function combinatorially with D6PK on PIN3

We agree with the reviewer that we do not have a complete picture of how NPY, PID, PIN work together to control flower initiation. Some aspects of our results are difficult to reconcile with the model of PIN1 and PID acting in tandem, i.e., by PID directly phosphorylating and activating PIN1. Indeed, our results suggest that PIN1 and PID have opposite effects on organogenesis. For example, heterozygous pin1 (or PIN1-GFP_HDR, which is presumably less active than wild type PIN1) suppresses the pid phenotype.  Moreover, pid and pin1 have opposite effects on cotyledon number and true leaf number. Mutations in PID lead to more cotyledons and more true leaves than WT whereas pin1 mutants make fewer cotyledons and fewer true leaves than WT (Bennett SRM, Alvarez J, Bossinger G, Smyth DR (1995) Morphogenesis in pinoid mutants of Arabidopsis thaliana. The Plant Journal 8: 505-520).  We have elaborated on this point in the last paragraph of the Discussion.

The genetic materials we have generated may allow us to uncover additional components in the pathway from forward genetic screens, which may eventually lead to a clear picture.

eLife Assessment

This study provides new and interesting findings that SCoR2 acts as a denitrosylase to control cardioprotective metabolic reprogramming and prevent injury following ischemia/reperfusion. The compelling evidence is supported by a novel multi-omics approach, but questions remain regarding the stability and human relevance of BDH1 as well as the sufficiency of SCoR2. Overall, the work will be of interest to cardiovascular researchers and provides useful information to the field, though some mechanistic aspects require further clarification.

Reviewer #1 (Public review):

Summary:

This study shows a novel role for SCoR2 in regulating metabolic pathways in the heart to prevent injury following ischemia/reperfusion. It combines a new multi-omics method to determine SCoR2 mediated metabolic pathways in the heart. This paper would be of interest to cardiovascular researchers working on cardioprotective strategies following ischemic injury in the heart.

Strengths:

(1) Use of SCoR2KO mice subjected to I/R injury.

(2) Identification of multiple metabolic pathways in the heart by a novel multi-omics approach.

Comments on revisions:

Authors have addressed all concerns raised in the previous round of review. Substantial modifications have been made in response to those concerns. There are no further comments.

Reviewer #2 (Public review):

Summary:

This manuscript addresses the gap in knowledge related to the cardiac function of the S-denitrosylase SNO-CoA Reductase 2 (SCoR2; product of the Akr1a1 gene). Genetic variants in SCoR2 have been linked to cardiovascular disease, yet its exact role in heart remains unclear. This paper demonstrates that mice deficient in SCoR2 show significant protection in a myocardial infarction (MI) model. SCoR2 influenced ketolytic energy production, antioxidant levels, and polyol balance through the S-nitrosylation of crucial metabolic regulators.

Strengths:

Addresses a well-defined gap in knowledge related to the cardiac function of SNO-CoA Reductase 2. Besides the in-depth case for this specific player, the manuscripts sheds more light on the links between S-nytrosylation and metabolic reprogramming in heart.

Rigorous proof of requirement through the combination of gene knockout and in vivo myocardial ischemia/reperfusion

Identification of precise Cys residue for SNO-modification of BDH1 as SCoR2 target in cardiac ketolysis

Weaknesses:

The experiments with BDH1 stability were performed in mutant 293 cells. Was there a difference in BDH1 stability in myocardial tissue or primary cardiomyocytes from SCoR2-null vs -WT mice? Same question extends to PKM2.

In the absence of tracing experiments, the cross-sectional changes in ketolysis, glycolysis or polyol intermediates presented in Figures 4 and 5 are suggestive at best. This needs to be stressed while describing and interpreting these results.

The findings from human samples with ischemic and non-ischemic cardiomyopathy do not seem immediately or linearly in line with each other and with the model proposed from the KO mice. While the correlation holds up in the non-ischemic cardiomyopathy (increased SNO-BDH1, SNO-PKM2 with decreased SCoR2 expression), how do the Authors explain the decreased SNO-BDH1 with preserved SCoR2 expression in ischemic cardiomyopathy? This seems counterintuitive as activation of ketolysis is a quite established myocardial response to the ischemic stress. It may help the overall message clarity to focus the human data part on only NICM patients.

(partially linked to the point above) an important proof that is lacking at present is the proof of sufficiency for SCoR2 in S-Nytrosylation of targets and cardiac remodeling. Does SCoR2 overexpression in heart or isolated cardiomyocytes reduce S-nitrosylation of BDH1 and other targets, undermining heart function at baseline or under stress?

Comments on revisions:

Some of my points have been addressed. However, the points related to 1) BDH1 stability effect in cardiomyocytes; 2) human relevance of SNO-BDH1; 3) SCoR2 sufficiency remain unclear. That said, this manuscript will provide useful information to the field as such.

Reviewer #3 (Public review):

Summary:

This manuscript demonstrates that mice lacking the denitrosylase enzyme SCoR2/AKR1A1 demonstrate a robust cardioprotection resulting from reprogramming of multiple metabolic pathways, revealing<br /> widespread, coordinated metabolic regulation by SCoR2.

Strengths:

The extensive experimental evidence provided the use of the knockout model

Weaknesses:

No direct evidence for the underlying mechanism.

The mouse model used is not a tissue-specific knock-out.

Author response:

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

Reviewer #1 (Public review): 

Summary: 

This study shows a novel role for SCoR2 in regulating metabolic pathways in the heart to prevent injury following ischemia/reperfusion. It combines a new multi-omics method to determine SCoR2 mediated metabolic pathways in the heart. This paper would be of interest to cardiovascular researchers working on cardioprotective strategies following ischemic injury in the heart. 

Strengths:

(1) Use of SCoR2KO mice subjected to I/R injury. 

(2) Identification of multiple metabolic pathways in the heart by a novel multi-omics approach.

We thank the Reviewer for the positive review of our manuscript.

Weaknesses:

(1) Use of a global SCoR2KO mice is a limitation since the effects in the heart can be a combination of global loss of SCoR2. 

(2) Lack of a cell type specific effect. 

We agree that global KOs limit the cell type-specific mechanistic conclusions that can be drawn. Global knockouts are nonetheless informative in their own right and serve to identify phenotypes worthy of further study.

Reviewer #2 (Public review):

Summary: 

This manuscript addresses the gap in knowledge related to the cardiac function of the S-denitrosylase SNOCoA Reductase 2 (SCoR2; product of the Akr1a1 gene). Genetic variants in SCoR2 have been linked to cardiovascular disease, yet their exact role in the heart remains unclear. This paper demonstrates that mice deficient in SCoR2 show significant protection in a myocardial infarction (MI) model. SCoR2 influenced ketolytic energy production, antioxidant levels, and polyol balance through the S-nitrosylation of crucial metabolic regulators. 

Strengths: 

(1) Addresses a well-defined gap in knowledge related to the cardiac function of SNO-CoA Reductase 2. Besides the in-depth case for this specific player, the manuscript sheds more light on the links between Snitrosylation and metabolic reprogramming in the heart.

(2) Rigorous proof of requirement through the combination of gene knockout and in vivo myocardial ischemia/reperfusion. 

(3) Identification of precise Cys residue for SNO-modification of BDH1 as SCoR2 target in cardiac ketolysis 

We thank the Reviewer for their kind words.

Weaknesses: 

(1) The experiments with BDH1 stability were performed in mutant 293 cells. Was there a difference in BDH1 stability in myocardial tissue or primary cardiomyocytes from SCoR2-null vs -WT mice? The same question extends to PKM2. 

We have not assessed BDH1 stability directly in cardiomyocytes. However, S-nitrosylation increased BDH1 stability in HEK293 cells, and BDH1 expression was increased in (injured) hearts of SCoR2KO mice, together with increased SNO-BDH1. 

For PKM2, there is a wealth of published evidence from us and others that S-nitrosylation does not regulate protein stability but rather inhibits tetramerization required for full activity.  

(2) In the absence of tracing experiments, the cross-sectional changes in ketolysis, glycolysis, or polyol intermediates presented in Figures 4 and 5 are suggestive at best. This needs to be stressed while describing and interpreting these results. 

We now acknowledge this limitation in the ‘Limitations’ section of the manuscript and in edits made to the text. 

(3) The findings from human samples with ischemic and non-ischemic cardiomyopathy do not seem immediately or linearly in line with each other and with the model proposed from the KO mice. While the correlation holds up in the non-ischemic cardiomyopathy (increased SNO-BDH1, SNO-PKM2 with decreased SCoR2 expression), how do the authors explain the decreased SNO-BDH1 with preserved SCoR2 expression in ischemic cardiomyopathy? This seems counterintuitive as activation of ketolysis is a quite established myocardial response to ischemic stress. It may help the overall message clarity to focus the human data part on only NICM patients. 

We find it interesting and important that SNO-BDH1 is readily detected in human heart tissue and its level is correlated to disease state. Our findings suggest conservation of this mechanism in human heart failure. However, we caution against drawing further conclusions related to NICM or ICM. Our animal model (based on a single time point) cannot faithfully recapitulate patients with chronic heart disease or differences between NICM and ICM. 

(4) This is partially linked to the point above. An important proof that is lacking at present is the proof of sufficiency for SCoR2 in S-nitrosylation of targets and cardiac remodeling. Does SCoR2 overexpression in the heart or isolated cardiomyocytes reduce S-nitrosylation of BDH1 and other targets, undermining heart function at baseline or under stress? 

The Reviewer proposes to test the effect of SCoR2 overexpression on cardioprotection. This is an interesting experiment for future study with the following caveats. First, it presupposes that native expression of SCoR2 is insufficient to control basal steady state S-nitrosylation of SNO-BDH1 and SNO-PKM2 (this does not seem to be the case). Second, overexpressed SCoR2 may be mislocalized within cells or associated with unnatural targets. Thank you.

Reviewer #3 (Public review): 

Summary: 

This manuscript demonstrates that mice lacking the denitrosylase enzyme SCoR2/AKR1A1 demonstrate a robust cardioprotection resulting from reprogramming of multiple metabolic pathways, revealing widespread, coordinated metabolic regulation by SCoR2. 

Strengths: 

(1) The extensive experimental evidence. 

(2) The use of the knockout model. 

We thank the Reviewer for identifying strengths in our work.

Weaknesses: 

(1) The connection of direct evidence for the mechanism. 

We believe we have identified a novel mechanism for cardioprotection entailing coordinate reprogramming of multiple metabolic pathways and suggesting a widescale role for SCoR2 in metabolic regulation. This is the key message we convey. While genetic dissection of individual pathways may be worthwhile, these investigations will have their own limitations. 

(2) The mouse model used is not tissue-specific. 

Please see our response to Reviewer 1, above. 

Reviewer #1 (Recommendations for the authors):

In the study, titled "The denitrosylase SCoR2 controls cardioprotective metabolic reprogramming", Grimmett ZW et al., describe a role for SNO-CoA Reductase 2 (SCoR2) in promoting cardioprotection via metabolic reprogramming in the heart after I/R injury. Authors show that loss SCoR2 coordinates multiple metabolic pathways to limit infarct size. Overall, the hypothesis is interesting, however there are some limitations as described below: 

(1) It is unclear whether SCoR2 mice are global or cardiomyocyte specific. 

We apologize for any confusion. These are global SCoR2^-/- mice. This is now stated in the Results when first identifying the strain, as well as in the Methods.  

(2) Can the authors clarify how divergent metabolic pathways such as Ketone oxidation, glycolysis, PPP and polyol metabolism work downstream of SCoR2 to impact cardioprotection in mice with I/R. 

The metabolic pathways of ketone oxidation, glycolysis, PPP and polyols appear to converge to support ischemic cardioprotection in SCoR2^-/- mice, as depicted in the model shown in Fig. 5L. Subsequent to SNO-PKM2 blockade of flux through glycolysis (detailed in this manuscript and in Zhou et al, 2019, PMID: 30487609, as well as by others), substrates of ketolysis and glycolysis are funneled into the PPP, producing the antioxidant NADPH and energy precursor phosphocreatine, which are well-known to be cardioprotective. This occurs more readily in SCoR2^-/- mice due to elevated SNO-BDH1 (detailed in this manuscript). 

Polyols, thought to be products of the PPP carbohydrate intermediates arabinose, ribulose, xylulose (among others), have recently been shown to be harmful to cardiovascular health in humans. These polyols are uniformly downregulated in SCoR2^-/- mice. We suggest this is likely the result of S-nitrosylation of SCoR2-substrate enzymes that form polyols (SCoR2/Akr1a1 is unable to directly reduce carbohydrates to their corresponding polyols). Regulation of endogenous polyol production in humans is a new concept and the mechanisms whereby these compounds increase risk of cardiac events are a subject of active investigation. This is detailed in the final paragraph of both the Results and Discussion sections, and in Fig. 5L. 

(3) The only functional outcome of SCoR2 loss in echocardiography and measurements for apoptosis. However, it would be important to determine whether the cardioprotective effect persists. It seems cardiac function was recorded 24hours post injury and whether the benefit remains till later time point such as 2 or 4 weeks is not shown. Without this time point, loss of SCoR2 only leads to an acute increment in function. 

Loss of SCoR2 reduced post-MI mortality at 4 hr; cardiac functional changes (plus troponin, LDH, and apoptosis) were studied in surviving animals at 24 hr post-MI. Cardiac response to acute injury and to chronic injury (weeks post-MI) are not the same metabolically. This is well elucidated in the literature and exemplified by the role of PKM2, which is protective in the chronic response to MI (28 days post-MI; PMID: 32078387), but implicated in injury at shorter timepoints post-MI (PMID: 33288902, 28964797). All that said, functional changes at 2-4 weeks will be important to determine in the future, as the Reviewer indicates. 

Reviewer #2 (Recommendations for the authors): 

(1) The last paragraph of the Results section should be divided into the statement related to Table S2 in the Results section, and the rest of the paragraph should be put somewhere in the Discussion. 

Thank you for this suggestion, which we have taken. 

(2) The number of mice alive/dead should be reported in the histogram in Figure 1G. 

Done.

(3) A concise Graphical Abstract will be useful to grasp the overall logic and message of the manuscript from the beginning. 

We thank you for this suggestion and have added a graphical abstract to the manuscript.

Reviewer #3 (Recommendations for the authors): 

I would suggest having more evidence on the effect of metabolic reprogramming on which cell type. The use of a global knockout is a major limitation, and probably some in vitro experiments with shRNA knockdown in endothelial cells and fibroblasts would provide more insights. 

The reviewer suggests one direction for future study. We identify a novel mechanism for cardioprotection entailing coordinate reprogramming of multiple metabolic pathways and suggesting a widescale role for SCoR2 in metabolic regulation. This is the message we wish to convey. The role of cardiomyocytes vs contributing cell types is a thoughtful direction for future study. Thank you. 

Editor's additional comment:

The editors wish to highlight a critical issue concerning the characterization of the SCoR2−/− mice employed in this study. 

In the Methods section (page 20), the manuscript states that "SCoR2+/− mice were made by Deltagen, Inc. as described previously (33)." However, reference 33 does not describe SCoR2−/− mice; instead, it refers to other genetically modified strains, including Akr1a1+/−, eNOS−/−, and PKM2−/− mice, with no mention of a SCoR2-targeted model. 

The editors fully acknowledge that the authors may be using the term "SCoR2" as a functional synonym for Akr1a1, based on its described role as a mammalian homologue of yeast SCoR. If this is the case, such equivalence should be explicitly stated in the manuscript to prevent potential confusion. Moreover, considering that the genetic deletion of Akr1a1 (i.e., SCoR2) underlies the key mechanistic findings presented, it is essential that the manuscript include a clear and comprehensive description of the generation and validation of the mouse model used. 

We therefore ask the authors to (1) clarify the nomenclature and relationship between "SCoR2" and Akr1a1, and (2) provide full details on the generation of the knockout mice, including the targeting strategy and the genotyping procedures. This information is necessary not only to ensure transparency and reproducibility but also to allow readers to fully appreciate the biological relevance of the findings.

Thank you for identifying this inconsistency. We have adjusted the manuscript text accordingly to clearly state that SCoR2 is a functional name for the product of the Akr1a1 gene and that these SCoR2^-/- mice are the same as Akr1a1^-/- mice described in Ref 33. We have augmented the Methods text to describe the generation and genotyping of these SCoR2/Akr1a1 knockout mice.

I strongly agree with this principle and have noticed that teachers often tend to skip this step when using digital tools. However, being able to facilitate learner progress and provide feedback is an essential requirement for any digital tool. If teachers cannot see their students’ work or give feedback on the process, then the tool is not suitable for classroom use.

Throughout the entire letter, it's obvious that Jourdan doesn't fully trust his old master, but he still holds some respect for him. He makes demands to his former master to ensure that his entire family would be safe if he came back down to work for him. I think it's important to point this line out in the letter because Jourdan is explicitly saying that he has legal documentation stating that he is free. He is making sure that his old master knows that he can't trap him into becoming a slave again if he were to go back down. I think that he did a good job in conveying in his letter how free he was and how much he and his family have been able to grow due to it.

Test, 1, 2, 3... Piergiorgio, do you see this?

eLife Assessment

This paper is important in demonstrating a requirement for sulfation in organizing apical extracellular matrix (aECM) during tubulogenesis in Drosophila melanogaster. The authors identify and characterize the organization of some of the first known components of the non-chitinous aECM in the Drosophila salivary gland tube, and these findings are supported by convincing data. This study would be of interest to developmental and cell biologists.

[Editors' note: this paper was reviewed by Review Commons.]

Reviewer #1 (Public review):

Summary:

There is growing appreciation for the important of luminal (apical) ECM in tube development, but such matrices are much less well understood than basal ECMs. Here the authors provide insights into the aECM that shapes the Drosophila salivary gland (SG) tube and the importance of PAPSS-dependent sulfation in its organization and function.

The first part of the paper focuses on careful phenotypic characterization of papss mutants, using multiple markers and TEM. This revealed reduced markers of sulfation and defects in both apical and basal ECM organization, Golgi (but not ER) morphology, number and localization of other endosomal compartments, plus increased cell death. The authors focus on the fact that papss mutants have an irregular SG lumen diameter, with both narrowed regions and bulged regions. They address the pleiotropy, showing that preventing the cell death and resultant gaps in the tube did not rescue the SG luminal shape defects and discussing similarities and differences between the papss mutant phenotype and those caused by more general trafficking defects. The analysis uses a papss nonsense mutant from an EMS screen - I appreciate the rigorous approach the authors took to analyze transheterozygotes (as well as homozygotes) plus rescued animals in order to rule out effects of linked mutations. Importantly, the rescue experiments also demonstrated that sulfation enzymatic activity is important.

The 2nd part of the paper focuses on the SG aECM, showing that Dpy and Pio ZP protein fusions localize abnormally in papss mutants and that these ZP mutants (and Np protease mutants) have similar SG lumen shaping defects to the papss mutants. A key conclusion is that SG lumen defects correlate with loss of a Pio+Dpy-dependent filamentous structure in the lumen. These data suggest that ZP protein misregulation could explain this part of the papss phenotype.

Overall, the text is very well written and clear. Figures are clearly labeled. The methods involve rigorous genetic approaches, microscopy, and quantifications/statistics and are documented appropriately. The findings are convincing.

Significance:

This study will be of interest to researchers studying developmental morphogenesis in general and specifically tube biology or the aECM. It should be particularly of interest to those studying sulfation or ZP proteins (which are broadly present in aECMs across organisms, including humans).

This study adds to the literature demonstrating the importance of luminal matrix in shaping tubular organs and greatly advances understanding of the luminal matrix in the Drosophila salivary gland, an important model of tubular organ development and one that has key matrix differences (such as no chitin) compared to other highly studied Drosophila tubes like the trachea.

The detailed description of the defects resulting from papss loss suggests that there are multiple different sulfated targets, with a subset specifically relevant to aECM biology. A limitation is that specific sulfated substrates are not identified here (e.g. are these the ZP proteins themselves or other matrix glycoproteins or lipids?); therefore, it's not clear how direct or indirect the effects of papss are on ZP proteins. However, this is clearly a direction for future work and does not detract from the excellent beginning made here.

Reviewer #2 (Public review):

Summary

This study provides new insights into organ morphogenesis using the Drosophila salivary gland (SG) as a model. The authors identify a requirement for sulfation in regulating lumen expansion, which correlates with several effects at the cellular level, including regulation of intracellular trafficking and the organization of Golgi, the aECM and the apical membrane. In addition, the authors show that the ZP proteins Dumpy (Dpy) and Pio form an aECM regulating lumen expansion. Previous reports already pointed to a role for Papss in sulfation in SG and the presence of Dpy and Pio in the SG. Now this work extends these previous analyses and provides more detailed descriptions that may be relevant to the fields of morphogenesis and cell biology (with particular focus on ECM research and tubulogenesis). This study nicely presents valuable information regarding the requirements of sulfation and the aECM in SG development.

Strengths:

- The results supporting a role for sulfation in SG development are strong. In addition, the results supporting the involvement of Dpy and Pio in the aECM of the SG, their role in lumen expansion, and their interactions, are also strong.

- The authors have made an excellent job in revising and clarifying the many different issues raised by the reviewers, particularly with the addition of new experiments and quantifications. I consider that the manuscript has improved considerably.

- The authors generated a catalytically inactive Papss enzyme, which is not able to rescue the defects in Papss mutants, in contrast to wild type Papss. This result clearly indicates that the sulfation activity of Papss is required for SG development.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review):

Summary:

There is growing appreciation for the important of luminal (apical) ECM in tube development, but such matrices are much less well understood than basal ECMs. Here the authors provide insights into the aECM that shapes the Drosophila salivary gland (SG) tube and the importance of PAPSS-dependent sulfation in its organization and function.

The first part of the paper focuses on careful phenotypic characterization of papss mutants, using multiple markers and TEM. This revealed reduced markers of sulfation and defects in both apical and basal ECM organization, Golgi (but not ER) morphology, number and localization of other endosomal compartments, plus increased cell death. The authors focus on the fact that papss mutants have an irregular SG lumen diameter, with both narrowed regions and bulged regions. They address the pleiotropy, showing that preventing the cell death and resultant gaps in the tube did not rescue the SG luminal shape defects and discussing similarities and differences between the papss mutant phenotype and those caused by more general trafficking defects. The analysis uses a papss nonsense mutant from an EMS screen - I appreciate the rigorous approach the authors took to analyze transheterozygotes (as well as homozygotes) plus rescued animals in order to rule out effects of linked mutations. Importantly, the rescue experiments also demonstrated that sulfation enzymatic activity is important.

The 2nd part of the paper focuses on the SG aECM, showing that Dpy and Pio ZP protein fusions localize abnormally in papss mutants and that these ZP mutants (and Np protease mutants) have similar SG lumen shaping defects to the papss mutants. A key conclusion is that SG lumen defects correlate with loss of a Pio+Dpy-dependent filamentous structure in the lumen. These data suggest that ZP protein misregulation could explain this part of the papss phenotype.

Overall, the text is very well written and clear. Figures are clearly labeled. The methods involve rigorous genetic approaches, microscopy, and quantifications/statistics and are documented appropriately. The findings are convincing.

Significance:

This study will be of interest to researchers studying developmental morphogenesis in general and specifically tube biology or the aECM. It should be particularly of interest to those studying sulfation or ZP proteins (which are broadly present in aECMs across organisms, including humans).

This study adds to the literature demonstrating the importance of luminal matrix in shaping tubular organs and greatly advances understanding of the luminal matrix in the Drosophila salivary gland, an important model of tubular organ development and one that has key matrix differences (such as no chitin) compared to other highly studied Drosophila tubes like the trachea.

The detailed description of the defects resulting from papss loss suggests that there are multiple different sulfated targets, with a subset specifically relevant to aECM biology. A limitation is that specific sulfated substrates are not identified here (e.g. are these the ZP proteins themselves or other matrix glycoproteins or lipids?); therefore, it's not clear how direct or indirect the effects of papss are on ZP proteins. However, this is clearly a direction for future work and does not detract from the excellent beginning made here.

Comments on revised version:

Overall, I am pleased with the authors' revisions in response to my original comments and those of the other reviewers

Reviewer #2 (Public review):

Summary

This study provides new insights into organ morphogenesis using the Drosophila salivary gland (SG) as a model. The authors identify a requirement for sulfation in regulating lumen expansion, which correlates with several effects at the cellular level, including regulation of intracellular trafficking and the organization of Golgi, the aECM and the apical membrane. In addition, the authors show that the ZP proteins Dumpy (Dpy) and Pio form an aECM regulating lumen expansion. Previous reports already pointed to a role for Papss in sulfation in SG and the presence of Dpy and Pio in the SG. Now this work extends these previous analyses and provides more detailed descriptions that may be relevant to the fields of morphogenesis and cell biology (with particular focus on ECM research and tubulogenesis). This study nicely presents valuable information regarding the requirements of sulfation and the aECM in SG development.

Strengths

-The results supporting a role for sulfation in SG development are strong. In addition, the results supporting the involvement of Dpy and Pio in the aECM of the SG, their role in lumen expansion, and their interactions, are also strong.

-The authors have made an excellent job in revising and clarifying the many different issues raised by the reviewers, particularly with the addition of new experiments and quantifications. I consider that the manuscript has improved considerably.

-The authors generated a catalytically inactive Papss enzyme, which is not able to rescue the defects in Papss mutants, in contrast to wild type Papss. This result clearly indicates that the sulfation activity of Papss is required for SG development.

Weaknesses

-The main concern is the lack of clear connection between sulfation and the phenotypes observed at the cellular level, and, importantly, the lack of connection between sulfation and the Pio-Dpy matrix. Indeed, the mechanism/s by which sulfation affects lumen expansion are not elucidated and no targets of this modification are identified or investigated. A direct (or instructive) role for sulfation in aECM organization is not clearly supported by the results, and the connection between sulfation and Pio/Dpy roles seems correlative rather than causative. As it is presented, the mechanisms by which sulfation regulates SG lumen expansion remains elusive in this study.

-In my opinion the authors overestimate their findings with several conclusions, as exemplified in the abstract:

"In the absence of Papss, Pio is gradually lost in the aECM, while the Dpy-positive aECM structure is condensed and dissociates from the apical membrane, leading to a thin lumen. Mutations in dpy or pio, or in Notopleural, which encodes a matriptase that cleaves Pio to form the luminal Pio pool, result in a SG lumen with alternating bulges and constrictions, with the loss of pio leading to the loss of Dpy in the lumen. Our findings underscore the essential role of sulfation in organizing the aECM during tubular organ formation and highlight the mechanical support provided by ZP domain proteins in maintaining luminal diameter."

The findings leading to conclude that sulfation organizes the aECM and that the absence of Papss leads to a thin lumen due to defects in Dpy/Pio are not strong. The authors certainly show that Papss is required for proper Pio and Dpy accumulation. They also show that Pio is required for Dpy accumulation, and that Pio and Dpy form an aECM required for lumen expansion. However, the absence of Pio and Dpy do not fully recapitulate Papss mutant defects (thin lumen). I wonder whether other hypothesis and models could account for the observed results. For instance, a role for Papss affecting secretion, in which case sulfation would have an indirect role in aECM organization. This study does not address the mechanical properties of Dpy in normal and mutant salivary glands.

-Minor issues relate to the genotype/phenotype analysis. It is surprising that the authors detect only mild effects on sulfation in Papss mutants using an anti-sulfoTyr antibody, as Papss is the only Papss synthathase. Generating germ line clones (which is a feasible experiment) would have helped to prove that this minor effect is due to the contribution of maternal product. The loss of function allele used in this study seems problematic, as it produces effects in heterozygous conditions difficult to interpret. Cleaning the chromosome or using an alternative loss of function condition (another allele, RNAi, etc...) would have helped to present a more reliable explanation.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Overall, I am pleased with the authors' revisions in response to my original comments and those of the other reviewers. The addition of the sulfation(-) mutant to Fig. 1 is particularly nice. I have just a few additional suggestions for text changes to improve clarity/precision.

(1) The current title of this manuscript is quite broad, making it sound like a review article. I recommend adding sulfation and salivary gland to the title to convey the main points more clearly. e.g. Sulfation affects apical extracellular matrix organization during development of the Drosophila salivary gland tube.

Thank you for the suggestion. We agree and have changed the title of the paper as suggested.

(2) Figure 1B shows very striking enrichment of papss expression in the salivary gland compared to other tubes like the trachea that also contain Pio and Dpy. To me, this implies that the key substrate(s) of Papss are likely to be unique, or at least more highly enriched, in the salivary gland aECM compared to the tracheal aECM (e.g. probably not Pio or Dpy themselves). I suggest that the authors address the implications of this apparent SG specificity in the discussion (paragraph beginning on p. 21, line 559).

Yes, we agree that there may be other key substrates of Papss in the SG, such as mucins, which play an important role in organizing the aECM and expanding the lumen. We have included a discussion.

(3) p. 15, lines 374-376 "The Pio protein is known to be cleaved, at one cleavage site after the ZP domain by the furin protease and at another cleavage site within the ZP domain by the matriptase Notopleural (Np) (Drees et al., 2019; Drees et al., 2023; Figure 5B)." As far as I can see, the Drees papers show that Pio is cleaved somewhere in the vicinity of a consensus furin cleavage site, but do not actually establish that the cleavage happens at this exact site or is done by a furin protease (this is just an assumption). Please word more carefully, e.g. "at one cleavage site after the ZP domain, possibly by a furin protease".

Thank you for pointing this out. We have edited the text.

Reviewer #2 (Recommendations for the authors):

Throughout the paper, I find a bit confusing the description of the lumen phenotype and their interpretations.

Papss mutants produce SG that are either "thin" or show "irregular lumen with bulges". Do the authors think that these are two different manifestations of the same effect? or do they think that there are different causes behind?

The thin lumen phenotype appears to occur when the Pio-Dpy matrix is significantly condensed. When this matrix is less condensed in one region of the lumen than in other regions, the lumen appears irregular with bulges.

Are the defects in Grasp65 mutants categorized as "irregular lumen with bulges" similar to those in Papss mutants? Why do these mutants don't show a "thin lumen" defect?

Grasp65 mutant phenotypes are milder than those of Papss mutants. Multiple mutations in several Golgi components that more significantly disrupt Golgi structures and function may cause more severe defects in lumen expansion and shape.

How the defects described for Pio ("multiple constrictions with a slight expansion between constrictions") and Dpy mutants ("lumen with multiple bulges and constrictions") relate to the "irregular lumen with bulges" in Papss mutants?

pio and dpy mutants show more stereotypical phenotypes, while Papss mutants exhibit more irregular and random phenotypes. The irregular lumen phenotypes in Papss mutants are associated with a condensed Pio-Dpy matrix.

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eLife Assessment

This useful paper examined the mechanism of planar cell polarity (PCP) using Drosophila pupal wing, investigating how 'cellular level', 'molecular level' and 'tissue level' mechanisms intersect to establish PCP. This represents a progress for the field, and the conclusions are mostly backed up by solid data. Whereas the manuscript is sound overall, the reviewers found remaining concerns, which can mostly be addressed by textual clarification of the concepts used in the manuscript.

[Editors' note: this paper was reviewed by Review Commons.]

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:

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 #2 (Public Review):

This paper aims to dissect the relative importance of the various cues that establish PCP in the wing disc of Drosophila, which remains a prominent and relevant model for PCP. The authors suggest that one must consider cues at three scales (molecular, cell and tissue) and specifically design tests for the importance of cell-level cues, which they call non-local cell scale signalling. They develop clever experimental approaches that allow them to track complex stability and also to induce polarity at experimentally defined times. In a first set of experiments, they restore PCP after the global cues have disappeared (de novo polarisation) and conclude from the results that another (cell scale) cue must exist. In another set of experiments, they show that de novo repolarization is robust to the dosage of various components of core PCP, leading them to conclude that there must be an underlying cell scale polarity, which, apparently, has nothing to do with microtubule or cell shape polarity. They then describe nice evidence that de novo polarisation is relatively short range both in a polarised and unpolarised field. They conclude that there is a strong cell-intrinsic polarity that remains to be characterised.

Major concerns:

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

(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.

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. 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.

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:

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.

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:

Summary

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 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:

Overview

This paper aims to dissect the relative importance of the various cues that establish PCP in the wing disc of Drosophila, which remains a prominent and relevant model for PCP. The authors suggest that one must consider cues at three scales (molecular, cell and tissue) and specifically design tests for the importance of cell-level cues, which they call non-local cell scale signalling. They develop clever experimental approaches that allow them to track complex stability and also to induce polarity at experimentally defined times. In a first set of experiments, they restore PCP after the global cues have disappeared (de novo polarisation) and conclude from the results that another (cell scale) cue must exist. In another set of experiments, they show that de novo repolarization is robust to the dosage of various components of core PCP, leading them to conclude that there must be an underlying cell scale polarity, which, apparently, has nothing to do with microtubule or cell shape polarity. They then describe nice evidence that de novo polarisation is relatively short range both in a polarised and unpolarised field. They conclude by there is a strong cell-intrinsic polarity that remains to be characterised.

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:

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' repolarization. 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

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.

Yea, she's racist

both

its impact and perceived value

Almost all information here except for the FGD number is shared across the FGDs. To make this table more informative, it could use more demographic information about the participants themselves such as Age, sex, and maybe academic performance (since it is used for maximum variation). Additionally, if the data is available, prior education (governmental or private schooling), or previous experience with SET (multiple experiences vs one, for instance) would be some demographic information shaping the participants'' views and the readers' interpretation of it

good

Were these from the same sample?

Good

Exclusion criteria are typically those that disqualify participants who could be included

Good

In need of references

Would be useful to include more information

Kindly reference

examples would be great

Could use some referencing

Recommend to separate the objectives into bullet points

As much as it could be true, recommend adding a reference to support this argument

Referencing some examples of studies in this section might strengthen it

Any references you could add to support this?

Well-written and informative

Inductive or deductive?

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Series data is organized into data groups. In case of more series, the data can be registered in repeating field groups.

Ezt töröljük, ki! A hivatalos leírásban nem szerepelhet olyan, hoyg TBD.

Fixed currency exchange rate can be specified for valuing a series with a currency different from the portfolio's base currency.

End date of series validity

Start date of series validity

In the age of the Ai and automation of menial jobs - is the emphasis still on the discouragement of reflection and critical thinking? What are the traits needed for those jobs that cannot be automated?

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Janie's willing to do what Tea Cake wants because she gets something out of it as well, something she's always yearned for, love.

Maybe Janie is still traumatized

Foreshadowing

Suppose \(x \neq 0\). Why is \(x^2 > 0\)?

Here the everyday negative number \(-0.8\) is represented as

$$ -1.20000\dots $$

We could also have used a signed model, where \(\sigma = \pm 1\) was added to the representation. This would make the model agree with everyday notation.

Love changed her so much that she started changing

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bbc reith lecture ＂polanyi＂ bbc - Google Search (9_1_2025 9：26：13 AM).html

This report came up in conversation. Compares 20yrs of EU digital policy to everything open. -[ ] lees report digital commons na 20jr EU digipol #geonovumtb

knowing explained

.4 -compare-etymology-understand&comprehend

The Reith Lectures

Both Kling and Stanford believe that there's very little between workers and their employers.

This can be observed within our current day in age too, pretty much every country has free trade with the rest of the world. It quite clear that this can be applied efficient in the real world.

This isn't something I would really expect to hear, but it does make sense. It does make me wonder though, how far could you go without professional education in the field of economics?

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No Liquidity Pool

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This quote stuck out to me because I think a lot of people forget that intentions don't always match the outcome. Just because you didn't mean to hurt someone doesn't mean you dint hurt them. I've seen situations where someone says something they thought was a joke but ended up really effecting the other person. This line is a reminder that we need to be more aware on how we come across and be willing to own up to it when we mess up, it's not about being perfect, no its about being respectable and showing others that their feelings do matter.

This quote struck me because most of the time, people are focused on themselves without even realizing it, of course it's natural to care about your own problems first, but compassion is about taking a step back and thinking about how someone else might be struggling too. The part about "dethroning ourselves" really stuck with me, it's a reminder that the world doesn't just revolve around us, and sometimes we need to put others first, even a small thing like helping a classmate or checking in on a friend can make a big difference.

I really connect with this because I feel a lot of people forget how important it is to treat others the way you want to be treated, and I always stood on that with the Golden Rule. But it could be easy to get caught up in your own thoughts and feelings, but we don't always stop to think what about someone else might be going through. This quote reminds me that compassion isn't just about being kind when its easy, it's about making the effort to understand people, even when we don't agree with them or when we're in a tough situation.

I've thought about this a few times in the past, but never really looked into it at all. I'll look deeper and figure out just how much of this statement is true.

Machines are such a crucial part of our world nowadays, even before the steam age we had rudimental machines and that does make me wonder, what would a world without advanced machinery look like for us?

I think this is something to think about, if economists are no longer using critical thinking skills then this could be very dangerous for corporations in the long run

What does happiness look like to Tin Tin?

Una primera definicion

Though these could be useful, these were not included in the earlier parts of the paper such as the methodology or results

It was also mentioned earlier in the thesis that the researcher only relied on focus groups due to resources

Could either include more information about these sources of data in earlier sections or omit parts related to journals/forums