cái này khác với câu "d" ở trên là vì cái này cò dấu "." là dấu thập phân, mà thập phân thì giữ luôn số 0

Author response:

The following is the authors’ response to the original reviews

We would like to express our sincere gratitude to the reviewers for their thorough analysis of the manuscript and their extremely helpful comments. We have taken all the suggestions into consideration and conducted a range of additional experiments to address the points raised. We have also extensively revised the manuscript to clarify descriptions, correct inaccuracies and remove inconsistencies. We have modified the figures for clarity and content.

Overall, we expanded the description of the EBH structure to emphasise its dimeric nature and the impact of the two binding sites on interpreting the binding data, including cooperativity. Using ITC, we tested the effect of the pre-SxIP residues on the binding affinity with additional peptides. We found that these residues had a significant effect, albeit much smaller than that of the post-SxIP residues. We analysed the binding of the 11MACF-VLL mutant with EBH-ΔC and evaluated the exchange rates. In agreement with our model, we found that the EBH affinity for the SxIP peptide from CK5P2 (KKSRLPRILIKRSR), which has a C-terminal sequence similar to that of the 11MACF-VLLRK mutant, is 21nM, which is similar to the affinity of the mutant itself. This demonstrates the significant variation in affinity observed among natural SxIP ligands, as predicted by our study. Our responses to the specific points raised by the reviewers are provided below.

Reviewer #1 (Public Review):

There is no direct experimental evidence for independent dock and lock steps. The model is certainly plausible given their structural data, but all titration and CEST measurements are fully consistent with a simple one-step binding mechanism. Indeed, it is acknowledged that the results for the VLL peptide are not consistent with the predictions of this model, as affinity and dissociation rates do not co-vary. The model may still be a helpful way to interpret and discuss their results, and may indeed be the correct mechanism, but this has not yet been proven.

Unfortunately, it is not possible to obtain direct experimental evidence because the folding of the C-terminus is too fast to influence the NMR parameters. However, as the reviewer pointed out, our structural data support the two-step model, since folding of the C-terminus is only possible once the ligand containing the post-SxIP residues has bound. By adopting a mechanistically supported model, we can analyse the contributions to binding and relate them to the structural characteristics of the complex. This provides a clearer insight into the roles of the various regions in the interaction and allows to modify them rationally to enhance the ligand affinity.

In the revised version, we restate the equations in terms of comparing the on-rates. This provides a clearer view of the effect of the additional stage, which cannot increase the overall on-rate since the two stages are sequential. If the forward rate of the second stage is comparable to or slower than the off-rate of the first stage, the overall on-rate decreases. Conversely, if the forward rate is much faster, the overall on-rate remains unchanged. For the wild-type 11MACF peptide, we observed that the presence of the EBH C-terminus does not affect the on-rate of binding, which is in perfect agreement with the two-step model and indicates that the C-terminus folds very quickly.

Additionally, we evaluated the binding of the 11MACF-VLL mutant to EBH-ΔC and observed a twofold decrease in Kd compared to WT 11MAC, primarily due to an increase in the on-rate. Interestingly, this rate is approximately twice as low as the overall on-rate for EBH/11MACF-VLL binding, contradicting the sequential two-step model. This suggests a more complex binding process where binding is accelerated by additional hydrophobic interactions with the unfolded C-terminus. However, given the difficulty of quantifying very slow exchange rates, it is more likely that the discrepancy is due to the accuracy of the rate measurements. Therefore, the model allows the rational analysis of changes in binding parameters due to mutations.

There is little discussion of the fact that binding occurs to EBH dimers -  either in terms of the functional significance of this or in the  acquisition and analysis of their data. There is no discussion of  cooperation in binding (or its absence), either in the analysis of NMR  titrations or in ITC measurements. Complete ITC fit results have not  been reported so it is not possible to evaluate this for oneself.

We added information about the dimer to the introduction, emphasising its role in enhancing interaction with microtubules (MTs) and its structural role in SxIP binding. The ITC data do not exhibit any biphasic behaviour and can be fitted to a single-site model with 1:1 stoichiometry relative to the EB1c monomer. This corresponds to two independent binding sites in the dimer. We have added the stoichiometry to Table 1 and the description. The NMR titration data for the 11MACF and 11MACF-VLL interactions were fitted to the TITAN dimer model, which includes cooperativity parameters. For WT 11MACF, both cooperativity parameters were zero, corresponding to independent binding sites in the ITC model. For 11MACF-VLL, the fitting suggests weak negative cooperativity, with a ~3-fold increase in Kd for binding to the second site and no change in the off-rate. This difference in Kd is likely to be too small to induce a biphasic shape to the ITC curve. As the cooperativity effect on the NMR spectra is small and absent in the ITC, we used the independent sites model for data analysis, as there is insufficient justification for introducing extra parameters into the model. Crucially, fitting to this model did not alter the off-rate value obtained by NMR or affect the conclusions. We added a description of cooperativity to the results and discussion.

Three peptides are used to examine the role of C-terminal residues in SxIP motifs: 4-MACF (SKIP), 6-MACF (SKIPTP), and 11-MACF (KPSKIPTPQRK). The 11-mer demonstrates the strongest binding, but this has added residues to the N-terminal as well. It has also introduced charges at both termini, further complicating the interpretation of changes in binding affinities. Given this, I do not believe the authors can reasonably attribute increased affinities solely to post-SxIP residues.

We tested the 9MACF peptide SKIPTPQRK, which has the same N-terminus as the 4- and 6-MACF peptides, and found that its binding affinity is ~10-fold weaker than that of 11MACF. This demonstrates the contribution of both the pre- and post-SxIP residues. This is likely due to electrostatic interactions between the positively charged N-terminus and the negatively charged EBH surface, similar to those involving the positive charges at the peptide C-terminus. Although significant, the contribution of the N-terminal peptide region is approximately one order of magnitude lower than that of the post-SxIP residues, meaning the post-SxIP region is the main affinity modulator. We have added the binding data on 9MACF and a discussion of the contributions to the manuscript.

Experimental uncertainties are, with exceptions, not reported.

Uncertainties added to the number in Table 1 and the text. Information on how uncertainties were calculated added to Table 1.

Reviewer #1 (Recommendations For The Authors):

(1) Have you tested the binding of the WT dimer in your cell model?

We haven’t tested the WT dimer because it has already been reported in the 2009 Cell paper by Honappa et al. In the cell experiments, our main focus was on recruiting the high-affinity mutant to MTs. The low level of recruitment, despite the mutant's high affinity, highlights the importance of dimerisation or additional contributions to binding.

(2) Please deposit all NMR dynamics measurements (relaxation rates and derived model-free parameters) alongside structural data in the BMRB.

The relaxation data have been submitted to BMRB, IDs 53187 and 53188

(3) Please report complete fitting results, e.g. for ITC, including stoichiometries. Clarify what this means for binding to a dimer, and if there is any evidence of cooperativity. Figure 3C, right hand panel, shows an unusual stoichiometry, can the authors comment on this?

We have added more information on stoichiometry and cooperativity; please refer to our response to the above comment for details. We repeated the titration for the VLLRK mutant using fresh peptide stock. As expected, the stoichiometry was close to 1:1 relative to the EB1c monomer. The new data are now included in the table and figure.

(4) Please report uncertainties for all measurements of Kd, koff, kon, ∆G, ∆H, ∆S, and explain whether these are determined from statistical analysis, technical or biological repeats (and where reported, clarify between standard deviation/standard error). Please also be aware of standard guidelines for reporting significant figures for data with uncertainties, as these have not been followed in Table 1.

Uncertainties added to the number in Table 1 and the text. Information on how uncertainties were calculated added to Table 1.

(5) The construct design for the cell model is unclear - given the importance of flanking residues, please report and discuss how the sequences are attached to venus: which termini is attached, and what is the linker composition?

We cloned the peptides at the C-terminus of mTFP, after the GS linker of the vector. The peptide itself contains a GS sequence at the N-terminus, creating a highly flexible GSGS linker that separates the SxIP region from mTFP and minimises the potential effect of mTFP on binding. We followed the design of Honappa et al. to enable direct comparison with the published results. We have added this information to the 'Methods' section..

(6) Which HSQC pulse sequence was used for 2D lineshape analysis? The authors mention non-linear chemical shift changes, presumably associated with the dimer interface - this would be useful to expand upon and clarify.

For the lineshape analysis, we used the standard Bruker sequence hsqcfpf3gpphwg with soft-pulse watergate water suppression and flip-back. This sequence is included in the TITAN model. We added the description of the non-linear chemical shift changes and connection of these changes to the allosteric effect of the binding to the supplementary information describing details of the lineshape analysis.

(7) Figure 1A could usefully highlight the dimer interface in the surface representation also.

We believe that including the interface would make the figure too complicated. The dimer configuration is shown in different colours for the two subunits, clearly demonstrating their involvement in forming the binding site.

(8) Figures 1C and 1D could usefully show a secondary structure schematic to assist the reader. The x-axis in these figures is not linear and this should be corrected. The calculation of combined chemical shift perturbations should be described.

Thank you for the helpful suggestion. We changed the scale of the figures and added the diagram of the secondary structure.

(9) Units are missing from many figure axes.

We added missing units to the axes. Thank you for highlighting this.

(10) What peptide concentrations are used in Figure 1C? Presumably, these should be reported at saturation for this to be a fair comparison, this should be clarified.

The protein concentration was 50 µM. Peptides 4MACF and 6MACF were added at a 100-fold molar excess and peptide 11MACF was added at a 4-fold excess. Saturation was achieved for 11MACF. This was impossible for the short peptides due to their mM affinity. This information has been added to the figure legend. The figure's main aim is to illustrate the differences in the chemical shift perturbation profiles, which can be achieved even if full saturation is not attained. Although the absolute value of the chemical shifts is proportional to the degree of saturation, the distribution of the largest chemical shift changes is independent of this degree. Therefore, we can draw conclusions about the distribution of changes by comparing under non-saturation conditions.

(11) The presentation of raw peak intensities in Figure 1D shows primarily the flexibility of the C-terminal region associated with high intensities. Beyond this, when comparing the binding of peptides it would be much more informative to show relative peak intensities. Residues around 210-225 appear to show strong broadening in the presence of peptide, but this is masked by the low initial intensity. Can the authors clarify and discuss this? Also, what peptide concentrations were used for this comparison? For a fair comparison, it should be close to saturation - particularly to exclude exchange broadening contributions.

The protein concentration was 50 µM. 6MACF and 6MACF peptides were added at a 100-fold excess and 11MACF at a 4-fold excess. Saturation was achieved for 11MACF. This was impossible to achieve for the short peptide due to its mM affinity. This information has been added to the figure legend. Upon checking the data, we found a small systematic offset in the coiled-coil region of some of the complexes, as the integral intensity had been used in the initial plot. While this does not change the conclusion regarding the high dynamics of the C-terminus, it does create an inaccurate perception of the relative intensities of the folded regions in the different complexes, as noted by the reviewer. We have now plotted the amplitudes at the maximum of the peaks, which do not exhibit any systematic offset as they are much less susceptible to baseline distortions. We are grateful to the reviewer for highlighting this apparent discrepancy.

(12) Figure 2 - the scale for S2 order parameters appears to be backwards, given the caption, but its range should be indicated. Similarly, the range of values for Rex should also be indicated. These data should also be tabulated/plotted in supporting information.

We have corrected the figure legend and added S2 and Rex plots to the supplementary material. The figure aims to highlight regions of increased mobility, while the plots provide full quantitative information on the values. We thank the reviewer for pointing out the error in the figure legend and for the suggestions regarding the plots.

(13) The scale in Figure 3B is illegible. Indeed, the whole structure is quite small and could usefully be expanded.

We increased the size of the structure panels and added a scale.

(14) Figure 4 does not show a decrease in exchange rates, as per the caption - no comparison of exchange rates is shown, only thermodynamic information in panel E. Panel C shows CEST measurements, but it is not clear what system this is for - please clarify, and consider showing the comparable data for the ∆C construct for comparison.

We have amended the figure legend to clarify that the figure shows binding parameters. We added information about the CEST profiles for the EBH/11MACF interaction to the figure legend (Figure 4C). Exchange with the ∆C construct is too fast for CEST measurements. We used lineshape analysis to evaluate the exchange rates for this construct.

(15) The schematics shown in Figure 4D, and elsewhere, are really quite difficult to understand. They may pose additional challenges to colourblind readers. Please consider ways that this could be clarified.

We simplified the colour scheme in the model to make the colours easier to see and to highlight SxIP and non-SxIP regions. We believe that this improved the clarity of the figure.

(16) Figures S1D/E - the x-axes are unclear and units are missing from the y-axes.

We re-labelled the axes to clarify the scale and units. Thank you for pointing this.

Reviewer #2 (Public Review):

The C-terminal tail of EB1, which is adjacent to EBH and is not analyzed in this study, is highly acidic and plays an important role in protein interactions. If the authors discuss the C-terminus of EB1, they should analyze the whole C-terminus of EB1, which would strengthen the conclusion they have made.

Honapa et al., Cell, 2009, reported chemical shift perturbations (CSPs) on the peptide binding for the full EB1c fragment, which includes the negatively charged C-terminus. Similar to our study, they observed significant CSPs in the FVIP region but negligible CSPs at the negatively charged EEY end. They concluded that the final eight EB1c residues did not contribute to binding and used a truncated EB1c construct for their structural analysis. Building on that study, we used the same EEY-truncated construct to analyse the contribution of the C-terminus in more detail. We believe that conducting additional experiments with the full C-terminus with respect to SxIP binding would be superfluous, as it would merely replicate the findings of Honapa EA. We have added the rationale for selecting the truncated EB1c construct to the text, referencing Honapa et al.

Reviewer #2 (Recommendations For The Authors):

(1) Figure 2C: The authors can analyze the 11MACF peptide as well, to provide more assurance to their argument. It would be easier to distinguish the sequences of "SKIP" and "FVIP" by changing their colors.

Our relaxation analysis (Fig. 2C) focuses on the dynamics of the unstructured C-terminal region in both the free and complex forms. Further relaxation analysis of the peptide would not provide additional information on this, and would be complicated by the presence of free peptide in solution.

(2) Figure 3B: Acidic residues in EBH should be labeled.<br /> Page 6, line 11: If the authors insist that the acidic patch will influence the interactions between EB1 and the peptide, the data of the analysis using the entire EB1 C-terminus should be included, given that the C-terminal tail of EB1 is highly acidic.

To test the contribution of charge to binding, we conducted an ITC experiment at increasing salt concentrations. We observed a significant increase in Kd values when the concentration of NaCl increased from 50 to 150 mM, which supports our conclusion regarding the significant electrostatic contribution. This conclusion is independent of the presence or absence of the C-terminus.

As we explained earlier, Honapa et al., Cell 2009, conducted an NMR experiment on the full EB1c and observed no CPSs in the EEY region, indicating a negligible contribution from the EEY region to SxIP binding. Therefore, we think that additional experiments involving the entire C-terminus are unnecessary, as they would simply replicate the results of Honapa et al. We have added the rationale for selecting the truncated EB1c to the text, referencing Honapa et al.

It would be very difficult to label the acidic residues without enlarging 3B considerably. However, we do not think this is necessary as we are not discussing any specific residues. The current figure shows the distribution of the surface charge, which is sufficient for our purposes.

(3) Figure 2B (Page 4, line 27): The side chain of S5477 should be drawn. The authors should include a figure of the crystal structure of EBH and SxIP as a comparison (Honnappa et al., Cell, 2009). In their paper, Honnappa et al. performed chemical shift perturbation titrations by NMR. From their analysis, I imagine that the EB1 tail may not be critical for the EB1 C-terminus:SxIP interactions, since the signals in the tail are not significantly perturbed. The authors should cite this paper.

We are grateful to the reviewer for highlighting this. CSP analysis of the Honapa EA revealed significant changes in the FVIP region, which we also observed. They also reported negligible CSPs at the EEY end, demonstrating that this part of the tail is non-critical and can be removed. We have added text to the manuscript to highlight the similarity between CSPs and those observed in Honapa EA. Figure 2B shows the side chains for the residues with the strongest detected contacts. These do not include S5477.

(4) Figure 3C (ITC data): The stoichiometric ratios in the ITC data look strange. EBH vs KPSKIPVLLRKRK, is it 1:1?

We repeated the ITC experiments using a new stock of the peptide and a new batch of the protein, checking the concentrations using UV spectroscopy. The new experiments produced a stoichiometry close to 1, as shown in the table.

(5) Page 10, line 27: "The TPQ sequence of 11MACF is not optimal...": What is the meaning of "optimal"? The transient interaction between EB1 and its binding partner is responsible for the dynamics of the microtubule cytoskeleton. In a sense, the relatively weak interaction is "optimal" for the system. The authors should rephrase the word.

We agree that weak interactions are optimal from a functional perspective, as they have been selected through evolution. In our case, 'optimal' refers to the hydrophobic interaction with the C-terminus. We replaced 'optimal' with 'ideal' to draw more attention to the second part of the sentence, which clarifies the context.

(6) Page 11, line 2: "small number of comets enriched in the peptide that were too faint for the quantitative analysis, comparable to the reported previously (Honnappa, Gouveia et al. 2009)." Honnappa et al. used EGFP-fusion constructs in their study: EGFP forms a weak dimer, which presumably gave different results from the authors' mTFP-constructs. The authors can note this point in the text.

We are grateful to the reviewer for highlighting this. This aligns well with our conclusion that dimerisation is important for localisation to comets. We have added this point to the text.

(7) Page 10, line 21: The authors calculate the free energy of complex formation between EBH and MACF peptide and explain in the text, but it is hard to follow.

We simplified and clarified the description of the energy contributions by focusing on the SxIP and non-SxIP regions of the peptide, as well as the EBH C-terminus.

Minor points:

Page 2, line 9: IP motifs are not usually located in the C-terminus. For example, SxIP in Tastin is located in the N-terminal region, and SxIPs in CLASP are in the middle.

We corrected this statement, removing C-terminal.

Page 3, line 4: The authors should note the residue numbers of SKIP.

We think that in this context the residue number of the SxIP region are not important and would be distracting.

Figure 3D and Figure S3F: Make the colors and the order the same between the two figures.

We changed the colour scheme and the order of ITC parameters in S3F to match the main figure.

Figure 1A, 2B, Figure S5: Change the color of SKIP from other residues in the same chain, otherwise the readers cannot distinguish. Likewise, change the color of FVIP in Figure 2B.

We think that changing the colours will complicate the figures unnecessary. The corresponding residues are clearly labelled in the figures.

Figure 3, Figure S5, S6, S7: Box the letters of SKIP for clarity.

We boxed the SxIP region in S5 (new S6) and underlined in S6 (new S7). In S7 (new S8) the location of SxIP is very clear from the homology.

Figure 3B; Figure S2: Hard to recognize the peptide (MACF in green).

We increased the size of 3D and S2, making it easier to see the peptide.

Figure 1C and D: Make the residual numbers of the x-axes the same between the two graphs.

We made new plots with a linear scale for the residue numbers.

Figure 2A: The structures shown are not EB1. It should be described as EBH or EB1(191-260 a.a.).

Corrected.

Page 5, line 17: "the S2 values of the C-terminus" should be "the S2 values of the C-terminal loop in EBH", otherwise it is confusing.

Corrected.

Page 6, line 27; Figure S3C and S6: Please indicate the assignments of the resonances from "253FVI255" in the Figures.

We labelled the peaks corresponding to the 253FVI255 region in figure S6 (new S7). Figure S3 shows EBH-ΔC that does not include this region.

Page 7, line 25: Figure S7 should be S8.

Corrected

Page 12, line 6: "sulfatrahsferases" must by a typo.

Corrected.

• Informativo 1142
• ADI 7496 MC-Ref / GO
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. DIAS TOFFOLI
• Julgamento: 21/06/2024 (Virtual)
• Ramo do Direito: Processual Penal, Constitucional Matéria: Foro por prerrogativa de função; medidas cautelares; autorização prévia para investigação criminal/ Repartição de competências; direito penal e processual penal; direitos e garantias fundamentais

Tribunal de justiça e foro por prerrogativa de função: apreciação de medidas cautelares de natureza criminal

Resumo

• É inconstitucional — por violar a competência privativa da União para legislar sobre direito penal e processual penal (CF/1988, art. 22, I), o sistema acusatório e o princípio da isonomia (CF/1988, art. 5º, caput e LIII) — norma de Constituição estadual que condiciona à prévia autorização judicial, mediante decisão fundamentada da maioria absoluta do órgão especial do respectivo tribunal de justiça, o pedido de medida cautelar para fins de investigação criminal ou instrução processual penal em desfavor de autoridades com foro por prerrogativa de função.

• A norma impugnada, ao regular o foro por prerrogativa de função, não poderia dispor diversamente ou desbordar dos limites estabelecidos no modelo federal que, no caso, estão contidos no próprio Regimento Interno do STF (art. 21, XV). Conforme disposto na referida norma, que possui status de lei ordinária, a competência para supervisão judicial dos atos investigatórios de autoridades com prerrogativa de foro deve ser conferida ao relator, não havendo, portanto, necessidade de deliberação colegiada (1).

• A razão jurídica que justifica a necessidade de supervisão judicial dos atos investigatórios de autoridades com prerrogativa de foro no STF aplica-se, por simetria, às autoridades com prerrogativa de foro nos tribunais de segundo grau de jurisdição. Ademais, conforme jurisprudência desta Corte, a competência do respectivo tribunal para a supervisão judicial nesses casos não torna obrigatória a deliberação do respectivo órgão colegiado, sendo <u>suficiente</u> decisão do ministro ou desembargador relator (2).

• Nesse contexto, a exigência de controle judicial prévio por deliberação de órgão colegiado do tribunal de justiça local, além de conferir tratamento diferenciado aos seus detentores de foro por prerrogativa de função, destoa da lógica estabelecida por outras importantes disposições do RISTF (art. 21, IV e V, §§ 5º e 8º, e art. 230-C, § 2º).

• Com base nesses e em outros entendimentos, o Plenário, por unanimidade, converteu o referendo da medida cautelar em julgamento definitivo de mérito e, confirmando-a, julgou a ação parcialmente procedente para (i) declarar a inconstitucionalidade da expressão “mediante decisão fundamentada tomada pela maioria absoluta do órgão especial previsto no inciso VI do art. 93 da Constituição da República”, contida na alínea “p” do inciso VIII do art. 46 da Constituição do Estado de Goiás, com redação dada pela EC estadual nº 77/2023 (3); e (ii) dar à parte remanescente do referido dispositivo interpretação conforme a Constituição, a fim de esclarecer que “o Desembargador Relator pode apreciar monocraticamente as medidas cautelares penais requeridas durante a fase de investigação ou no decorrer da instrução processual nos casos de urgência e, ainda, quando a sigilosidade se mostrar necessária para assegurar a efetivação da diligência pretendida, ressalvada a obrigatoriedade de referendo pelo órgão colegiado competente, em momento oportuno, sobretudo quando resultar em prisão cautelar, mas sempre sem comprometer ou lhe frustrar a execução”.

(1) Regimento Interno do STF/1980: “Art.21. São atribuições do Relator: (...) XV – determinar a instauração de inquérito a pedido do Procurador-Geral da República, da autoridade policial ou do ofendido, bem como o seu arquivamento, quando o requerer o Procurador-Geral da República, ou quando verificar: (...)”

(2) Precedentes citados: ADI 6.732, ADI 7.083 e ADI 5.331.

(3) Constituição do Estado de Goiás: “Art. 46. Compete privativamente ao Tribunal de Justiça: (...) VIII - processar e julgar originariamente: (...) p) o pedido de medida cautelar para fins de investigação criminal ou instrução processual penal, quando o investigado ou o processado for autoridade cujos atos estejam sujeitos diretamente à sua jurisdição, mediante decisão tomada pelo voto da maioria absoluta do órgão especial previsto no inciso XI do art. 93 da Constituição da República;”

• Informativo STF

• Edição 1185/2025 19 de agosto de 2025

• Emissão de parecer prévio como condição para a Assembleia Legislativa apreciar as contas prestadas pelo governador - ADPF 434/AL

• RESUMO: A ausência de parecer prévio do Tribunal de Contas estadual não impede o julgamento das contas do governador pela Assembleia Legislativa. Entendimento contrário configuraria restrição desproporcional à autonomia do Poder Legislativo.

Conforme jurisprudência desta Corte (1), o referido parecer possui caráter meramente opinativo, tendo em vista a exclusividade da prerrogativa do Poder Legislativo para apreciar as contas em todas as esferas da Federação. Trata-se de opinião não vinculativa e cuja falta não enseja aprovação tácita das contas.

Na espécie, ainda que a Assembleia Legislativa alagoana sustente haver reiterada inércia do Tribunal de Contas local, com a passagem de anos sem o envio do parecer técnico, não há qualquer óbice para que o Poder Legislativo estadual exerça sua competência constitucional (CF/1988, arts. 49, IX; 71, I; e 75). Por outro lado, a competência legislativa para disciplinar sobre direito penal e processual penal é privativa da União (CF/1988, arts. 22, I; e 85, parágrafo único) (2) (3). O texto constitucional não prevê penalidade para essa omissão. Apesar disso, a Constituição do Estado de Alagoas tipifica a omissão do Presidente do Tribunal de Contas como crime de responsabilidade (art. 97, I), medida que viola o princípio da simetria (4).

Com base nesses entendimentos, o Plenário, por unanimidade, julgou improcedente a arguição e, por maioria – ante a abertura da causa de pedir – declarou a inconstitucionalidade da expressão “sob pena de crime de responsabilidade do Presidente do Tribunal” contida no art. 97, I, da Constituição do Estado de Alagoas (5).

• Informativo STF

• Edição 1185/2025 19 de agosto de 2025

• Vinculação remuneratória no âmbito estadual: equiparação do salário de empregados públicos ao vencimento de titulares de cargo efetivo – ADI 7.746/GO Relator: Ministro Cristiano Zanin

• RESUMO: É inconstitucional – por desobedecer ao disposto no art. 37, XIII, da Constituição Federal – a vinculação da remuneração de empregados públicos aos vencimentos de servidores efetivos, pois resultaria em equiparação remuneratória entre agentes públicos pertencentes a categorias diferentes.

O texto constitucional impede que determinadas categorias de servidores tenham seus vencimentos automaticamente majorados em decorrência do aumento concedido a carreiras diversas, já que cada uma deve ter estrutura remuneratória própria, estabelecida em lei específica, para que não haja aumentos salariais sem o devido processo legislativo.

Conforme jurisprudência desta Corte (1), a atual redação do dispositivo acima citado estabelece ampla vedação quanto à vinculação ou equiparação da remuneração de servidores públicos, de forma a evitar que o aumento na remuneração concedida a determinados servidores públicos aplique-se de forma automática a outras categorias.

Na espécie, o dispositivo impugnado dispõe que os empregados públicos que pertenceram aos quadros da Agência Goiana de Transportes e Obras (AGETOP – atual GOINFRA) fariam jus ao salário correspondente ao valor do vencimento fixado para o cargo efetivo equivalente. Essa medida permite que o aumento de vencimentos gere reflexos automáticos no vencimento dos empregados públicos da GOINFRA, de forma inconstitucional.

Com base nesses entendimentos, o Plenário, por maioria, julgou parcialmente procedente a ação para conferir interpretação conforme a Constituição Federal ao art. 7º, § 3º, I, a, da Lei nº 15.665/2006 do Estado de Goiás (2), de modo a preservar o valor nominal da remuneração vigente na data da publicação da ata deste julgamento, vedados reajustes automáticos futuros decorrentes da vinculação remuneratória declarada inconstitucional.

• Informativo STF
• Edição 1185/2025, 19 de agosto de 2025
• Programa de auxílio aos desempregados e contratação temporária no âmbito municipal – RE 1.551.780/SP
• RESUMO: É constitucional – pois concretiza o princípio da dignidade da pessoa humana e não ofende o princípio do concurso público (CF/1988, arts. 1º, III, e 37, II) – lei municipal que autoriza o chefe do Poder Executivo a criar programa de auxílio ao desempregado, de caráter assistencial, com o objetivo de dar ocupação, renda e qualidade profissional aos desempregados residentes no município.

Esta Corte já reconheceu a constitucionalidade de normas que instituem programas sociais com a finalidade de integrar pessoas em situação de vulnerabilidade ao mercado de trabalho (1). Na espécie, a pretensão da lei municipal impugnada é conferir dignidade a pessoas em estado de vulnerabilidade (2), inexistindo falta de razoabilidade no programa questionado, pois o caráter assistencial em favor de pessoas desempregadas prevalece em relação ao interesse da Administração Pública municipal em reorganizar os quadros de seus servidores públicos.

Ademais, não há que se falar em aplicação do que foi decidido no julgamento do processo paradigma do Tema 612 da repercussão geral, em que se fixaram os parâmetros de validade da contratação temporária de servidores públicos, a fim de impedir que ela seja utilizada como subterfúgio para o poder público se evadir da obrigação de realizar concurso público.

Com base nesse entendimento, o Plenário, por unanimidade, negou provimento ao recurso para manter o acórdão recorrido, o qual julgou improcedente a ação direta de inconstitucionalidade estadual movida contra a Lei nº 1.937/2023 do Município de Rubiácea/SP.

Aposentadoria especial de guardas municipais – ADPF 1.095/DF - Relatores: Ministro Gilmar Mendes - DIREITO ADMINISTRATIVO SERVIDOR PÚBLICO; GUARDA MUNICIPAL; APOSENTADORIA ESPECIAL - RESUMO: As guardas municipais fazem parte do Sistema Único de Segurança Pública (SUSP), mas não possuem direito à aposentadoria especial, visto que o rol constitucional de categorias com direito a esse benefício é taxativo e não as contempla.

Esta Corte, embora tenha reconhecido que as guardas municipais fazem parte do SUSP, não lhes conferiu integral isonomia com os demais órgãos de segurança pública, na medida em que há peculiaridades relevantes quanto ao regime jurídico desses órgãos (1).

A EC nº 103/2019, por sua vez, estabeleceu rol taxativo das categorias em que se pode instituir idade e tempo de contribuição diferenciados mediante lei complementar (2). Como as guardas municipais não f iguram de modo expresso nessa listagem, os respectivos entes federados ficam impedidos de conceder aposentadoria especial para essas carreiras (3).

Também é inaplicável a regra de aposentadoria especial do art. 40, § 4º-C do texto constitucional (4). Isso porque não se admite presunção de exposição a agentes nocivos à saúde pelo mero enquadramento profissional ou ocupacional. Ao contrário, é indispensável que se comprove a efetiva exposição a agentes químicos, físicos e/ou biológicos (5).

Por fim, eventual concessão da aposentadoria especial às guardas municipais sem a elaboração de plano próprio que contenha a devida indicação de fonte de custeio e de medidas compensatórias configura desobediência ao art. 195, § 5º da CF/1988 (6). Com base nesses e em outros entendimentos, o Plenário, por maioria, julgou improcedente a arguição.

• Informativo nº 859
• 26 de agosto de 2025.
• PRIMEIRA TURMA Processo: Processo em segredo de justiça, Rel. Ministro Gurgel de Faria, Primeira Turma, por maioria, julgado em 19/8/2025.

Ramo do Direito DIREITO ADMINISTRATIVO, DIREITO PROCESSUAL CIVIL

TemaPaz, Justiça e Instituições Eficazes <br /> Ação de Improbidade administrativa. Defensoria pública. Ilegitimidade ativa.

Destaque - A Defensoria Pública não possui legitimidade para propor a ação de improbidade administrativa.

Informações do Inteiro Teor - A controvérsia volta-se ao debate acerca da legitimidade ativa da Defensoria Pública para a ação de improbidade administrativa.

• A Lei n. 11.448/2007 alterou o art. 5º da Lei n. 7.347/1985 para incluir a Defensoria Pública como legitimada ativa para a propositura da ação civil pública em sentido largo; mas, podendo, não alterou a legitimidade para a propositura de ação civil pública regida pela Lei n. 8.429/1992 (Lei de Improbidade Administrativa - LIA), cujo objeto específico é a condenação pela prática de atos ímprobos.

• Isto é, a escolha do legislador operou-se mediante "silêncio eloquente", excluindo da Defensoria Pública a legitimidade para propor ação civil pública cujo pedido seja de aplicar as sanções previstas no art. 12 da Lei n. 8.429/1992.

• Note-se que, embora ambas as ações civis públicas (a geral da Lei n. 7.347/1985 e a de improbidade administrativa da Lei n. 8.429/1992) tenham algum ponto de aproximação, notadamente por serem instrumentos de proteção a direito transindividual, pelo que integram, em caráter global, o microssistema da tutela coletiva, elas diferenciam-se bastante no aspecto ontológico. É que as ações de improbidade são revestidas de caráter punitivo/sancionador próprio, sem equivalente na ação civil pública geral, e, por isso, aquela é regida por regras especiais, inclusive no que concerne à legitimidade ativa.

• Compreende-se que essa distinção entre a ação civil pública geral e a ação voltada a condenação por atos ímprobos também se extrai da opção do legislador ordinário, que resolveu concentrar exclusivamente no Ministério Público a legitimidade para propor esta última (art. 17, caput, da LIA, com a redação atual).

• Não se desconhece que o STF, após a ADI 7042, declarou a inconstitucionalidade parcial, com interpretação conforme sem redução de texto, do caput e dos §§ 6º-A e 10-C do art. 17 da Lei n. 8.429/1992, na redação dada pela Lei n. 14.230/2021, de modo a restabelecer a existência de legitimidade ativa concorrente e disjuntiva entre o Ministério Público e as pessoas jurídicas interessadas para a propositura da ação por ato de improbidade administrativa e para a celebração de acordos de não persecução civil.

• Contudo, no que se refere à ação de improbidade, esse julgamento somente admitiu a legitimidade ativa concorrente entre o Ministério Público e a pessoa jurídica supostamente lesada pelo ato ímprobo, sem que tenha sido estendida a ampliação da legitimidade à Defensoria Pública.

• Destarte, a legitimidade para propor a ação civil com fundamento na Lei n. 7.347/1985 não confere, em absoluto, a mesma legitimidade para propor a ação de improbidade da Lei n. 8.429/1992, sendo, portanto, a Defensoria Pública parte ilegítima para propor a ação de improbidade administrativa.

Reviewer #1 (Public review):

This paper presents a computational model of the evolution of two different kinds of helping ("work," presumably denoting provisioning, and defense tasks) in a model inspired by cooperatively breeding vertebrates. The helpers in this model are a mix of previous offspring of the breeder and floaters that might have joined the group, and can either transition between the tasks as they age or not. The two types of help have differential costs: "work" reduces "dominance value," (DV), a measure of competitiveness for breeding spots, which otherwise goes up linearly with age, but defense reduces survival probability. Both eventually might preclude the helper from becoming a breeder and reproducing. How much the helpers help, and which tasks (and whether they transition or not), as well as their propensity to disperse, are all evolving quantities. The authors consider three main scenarios: one where relatedness emerges from the model, but there is no benefit to living in groups, one where there is no relatedness, but living in larger groups gives a survival benefit (group augmentation, GA), and one where both effects operate. The main claim is that evolving defensive help or division of labor requires the group augmentation; it doesn't evolve through kin selection alone in the authors' simulations.

This is an interesting model, and there is much to like about the complexity that is built in. Individual-based simulations like this can be a valuable tool to explore the complex interaction of life history and social traits. Yet, models like this also have to take care of both being very clear on their construction and exploring how some of the ancillary but potentially consequential assumptions affect the results, including robust exploration of the parameter space. I think the current manuscript falls short in these areas, and therefore, I am not yet convinced of the results.

In this round, the authors provided some clarity, but some questions still remain, and I remain unconvinced by a main assumption that was not addressed.

Based on the authors' response, if I understand the life history correctly, dispersers either immediately join another group (with 1-the probability of dispersing), or remain floaters until they successfully compete for a breeder spot or die? Is that correct? I honestly cannot decide because this seems implicit in the first response but the response to my second point raises the possibility of not working while floating but can work if they later join a group as a subordinate. If it is the case that floaters can have multiple opportunities to join groups as subordinates (not as breeders; I assume that this is the case for breeding competition), this should be stated, and more details about how.

So there is still some clarification to be done, and more to the point, the clarification that happened only happened in the response. The authors should add these details to the main text. Currently, the main text only says vaguely that joining a group after dispersing " is also controlled by the same genetic dispersal predisposition" without saying how.

In response to my query about the reasonableness of the assumption that floaters are in better condition (in the KS treatment) because they don't do any work, the authors have done some additional modeling but I fail to see how that addresses my point. The additional simulations do not touch the feature I was commenting on, and arguably make it stronger (since assuming a positive beta_r -which btw is listed as 0 in Table 1- would make floaters on average be even more stronger than subordinates). It also again confuses me with regard to the previous point, since it implies that now dispersal is also potentially a lifetime event. Is that true?

Meanwhile, the simplest and most convincing robustness check, which I had suggested last round, is not done: simply reduce the increase in the R of the floater by age relative to subordinates. I suspect this will actually change the results. It seems fairly transparent to me that an average floater in the KS scenario will have R about 15-20% higher than the subordinates (given no defense evolves, y_h=0.1 and H_work evolves to be around 5, and the average lifespan for both floaters and subordinates are in the range of 3.7-2.5 roughly, depending on m). That could be a substantial advantage in competition for breeding spots, depending on how that scramble competition actually works. I asked about this function in the last round (how non-linear is it?) but the authors seem to have neglected to answer.

More generally, I find that the assumption (and it is an assumption) floaters are better off than subordinates in a territory to be still questionable. There is no attempt to justify this with any data, and any data I can find points the other way (though typically they compare breeders and floaters, e.g.: https://bioone.org/journals/ardeola/volume-63/issue-1/arla.63.1.2016.rp3/The-Unknown-Life-of-Floaters--The-Hidden-Face-of/10.13157/arla.63.1.2016.rp3.full concludes "the current preliminary consensus is that floaters are 'making the best of a bad job'."). I think if the authors really want to assume that floaters have higher dominance than subordinates, they should justify it. This is driving at least one and possibly most of the key results, since it affects the reproductive value of subordinates (and therefore the costs of helping).

Regarding division of labor, I think I was not clear so will try again. The authors assume that the group reproduction is 1+H_total/(1+H_total), where H_total is the sum of all the defense and work help, but with the proviso that if one of the totals is higher than "H_max", the average of the two totals (plus k_m, but that's set to a low value, so we can ignore it), it is replaced by that. That means, for example, if total "work" help is 10 and "defense" help is 0, total help is given by 5 (well, 5.1 but will ignore k_m). That's what I meant by "marginal benefit of help is only reduced by a half" last round, since in this scenario, adding 1 to work help would make total help go to 5.5 vs. adding 1 to defense help which would make it go to 6. That is a pretty weak form of modeling "both types of tasks are necessary to successfully produce offspring" as the newly added passage says (which I agree with), since if you were getting no defense by a lot of food, adding more food should plausibly have no effect on your production whatsoever (not just half of adding a little defense). This probably explains why often the "division of labor" condition isn't that different than the no DoL condition.

• Informativo nº 859
• 26 de agosto de 2025.
• RECURSOS REPETITIVOS
• Processo: REsp 2.148.059-MA, Rel. Ministro Luis Felipe Salomão, Corte Especial, por unanimidade, julgado em 20/8/2025. (Tema 1306).

REsp 2.148.580-MA, Rel. Ministro Luis Felipe Salomão, Corte Especial, por unanimidade, julgado em 20/8/2025 (Tema 1306).

REsp 2.150.218-MA, Rel. Ministro Luis Felipe Salomão, Corte Especial, por unanimidade, julgado em 20/8/2025 (Tema 1306).

Ramo do Direito DIREITO PROCESSUAL CIVIL

TemaPaz, Justiça e Instituições Eficazes <br /> Fundamentação por referência (per relationem ou por remissão). Ato decisório. Técnica de fundamentação. Cabimento. Tema 1306.

Destaque - 1) A técnica da fundamentação por referência (per relationem) é permitida desde que o julgador, ao reproduzir trechos de decisão anterior, documento e/ou parecer como razões de decidir, enfrente, ainda que de forma sucinta, as novas questões relevantes para o julgamento do processo, dispensada a análise pormenorizada de cada uma das alegações ou provas.

• 2) O § 3º do artigo 1.021, do CPC não impede a reprodução dos fundamentos da decisão agravada como razões de decidir pela negativa de provimento de agravo interno quando a parte deixa de apresentar argumento novo para ser apreciado pelo colegiado.

Informações do Inteiro Teor - Cinge-se a controvérsia a definir se a fundamentação por referência (per relationem ou por remissão) - na qual são reproduzidas as motivações contidas em decisão judicial anterior como razões de decidir - resulta na nulidade do ato decisório, à luz do disposto nos artigos 489, § 1º, e 1.022, parágrafo único, inciso II, do Código de Processo Civil de 2015.

• Segundo a doutrina, a obrigatoriedade da fundamentação das decisões judiciais, sob pena de nulidade, consubstancia, "a um só tempo, princípio processual, dever do juiz, direito individual da parte e garantia da Administração Pública".

• Tal obrigatoriedade - de justificação da convicção do magistrado em decisões judiciais - encontra-se prevista na Constituição Federal de 1988 (art. 93, inciso IX), tendo relação intrínseca com a definição da República Federativa do Brasil como Estado Democrático de Direito.

• Cuida-se de direito fundamental do jurisdicionado - consectário da garantia do devido processo legal - que subordina todos os integrantes do Poder Judiciário, aos quais é vedado proferir decisões arbitrárias, ou seja, pronunciamentos jurisdicionais que não se coadunem com o conceito democrático do exercício do poder, que exige a justificação - dialógica, racional e inteligível - do ato decisório de modo a viabilizar o seu "controle interno" pela parte e pelas instâncias judiciais subsequentes, bem como o seu "controle externo e difuso" pela sociedade, o que revela uma dupla função dessa obrigatoriedade.

• O Código de Processo Civil de 2015 inseriu o dever de fundamentação das decisões judiciais entre as "normas fundamentais do processo civil" (artigo 11), determinando ainda que: (i) em regra, "não se proferirá decisão contra uma das partes sem que ela seja previamente ouvida" (artigo 9º, caput); e (ii) "o juiz não pode decidir, em grau algum de jurisdição, com base em fundamento a respeito do qual não se tenha dado às partes oportunidade de se manifestar, ainda que se trate de matéria sobre a qual deva decidir de ofício" (artigo 10).

• No capítulo que versa sobre a "sentença" (lato sensu), o artigo 489 do CPC enumera os elementos essenciais do ato decisório, bem como hipóteses - exemplificativas - de "decisões não fundamentadas". Do referido dispositivo se extrai que o dever de fundamentação da decisão judicial considera-se adequadamente atendido quando o magistrado explicita as razões fáticas e jurídicas consideradas determinantes para a resposta oferecida no processo dentre outras conclusões possíveis.

• Ademais, nos termos do rol previsto no § 1º do artigo 489 do CPC, a fundamentação da decisão judicial deve ainda conter: a) explicação sobre o vínculo entre a norma jurídica - considerada aplicável à espécie - e a causa ou a questão decidida nos autos (inciso I); b) especificação do motivo concreto para o emprego de conceito jurídico indeterminado (inciso II); c) exame da situação concreta submetida ao crivo do Judiciário, revelando-se insuficiente a invocação de motivos que se prestariam a justificar qualquer outra decisão (inciso III); d) enfrentamento de "todos os argumentos deduzidos no processo capazes de, em tese, infirmar a conclusão adotada pelo julgador" (inciso IV); e) juízo de conformação entre a ratio decidendi de precedente - ou de enunciado de súmula - aplicado na decisão e o caso concreto (inciso V); f) indicação das diferenças fáticas que justificam a não aplicação de precedente obrigatório ao caso concreto (inciso VI, primeira parte); e g) informação sobre a superação de precedente obrigatório invocado nos autos (inciso VI, parte final).

• Assim, à luz do disposto no parágrafo único do artigo 1.022 do CPC, considera-se omissa - e, portanto, impugnável por embargos de declaração - a decisão que: (i) deixa de se manifestar sobre tese firmada em julgamento de casos repetitivos ou em incidente de assunção de competência aplicável à espécie; ou (ii) incorre em qualquer das hipóteses de ausência de fundamentação descritas no § 1º do artigo 489.

• Com as alterações promovidas em 2010 no Decreto-Lei n. 4.657/1942 (Lei de Introdução às Normas do Direito Brasileiro - LINDB), também passou a ser exigido que as consequências práticas - postas no debate judicial e que tenham lastro probatório nos autos - constem da fundamentação da decisão que, nas esferas administrativa, controladora ou judicial, decretar a invalidação de ato, contrato, ajuste, processo ou norma administrativa (artigos 20 e 21).

• Diante desse cenário normativo, discute-se se a utilização da técnica da fundamentação por referência - por remissão ou per relationem - é compatível com o dever de fundamentação imposto a todos os órgãos do Poder Judiciário, cuja inobservância resulta na nulidade do ato decisório. Trata-se de técnica discursiva na qual são reproduzidas as motivações contidas em decisão judicial anterior - ou em documento outro, a exemplo de parecer do Ministério Público - como razões de decidir.

• De acordo com a doutrina, a fundamentação por referência apresenta duas formas habituais: (i) a exclusiva (ou pura); e (ii) a integrativa (ou moderada). A utilização da "fundamentação por referência exclusiva ou pura" - ou seja, aquela consubstanciada na mera remissão ou transcrição integral dos fundamentos de outra peça processual sem análise específica dos argumentos trazidos pela parte - <u>implica violação ao direito fundamental ao contraditório</u> e vai de encontro às disposições contidas no § 1º do artigo 489 do CPC. Por outro lado, é válida a "fundamentação por referência integrativa ou moderada", na qual a transcrição de decisão ou parecer anterior é acompanhada de análise própria (do julgador) que dialoga com os argumentos levantados pela parte em sua impugnação.

• Ao tratar da matéria (sob o enfoque constitucional), o Supremo Tribunal Federal reconheceu a validade da fundamentação por referência - como técnica de motivação da decisão judicial - quando verificada "a compatibilidade entre o que alegado e o entendimento fixado pelo órgão julgador", ficando dispensado "o exame detalhado de cada argumento suscitado" (RE 1.397.056 ED-AgR/MA, Ministra Rosa Weber, Tribunal Pleno, DJe de 28/3/2023).

• Essa mesma exegese encontra-se retratada na jurisprudência do Superior Tribunal de Justiça (inclusive das Turmas de Direito Penal).

• Revela-se importante destacar, outrossim, que, em relação à norma inserta no § 3º do artigo 1.021 do CPC - segundo a qual "é vedado ao relator limitar-se à reprodução dos fundamentos da decisão agravada para julgar improcedente o agravo interno" - a jurisprudência do STJ firmou-se no sentido da necessidade de interpretação do referido comando em conjunto com a regra do inciso IV do § 1º do artigo 489, que somente reputa nula a decisão judicial que deixa de "enfrentar todos os argumentos deduzidos no processo capazes de, em tese, infirmar a conclusão adotada pelo julgador".

• Não obstante, é certo que já foram constatadas, por esta Corte, hipóteses de utilização da técnica de fundamentação por referência com flagrante violação dos artigos 489, § 1º, 1.021, § 3º, e 1.022, parágrafo único, inciso II, do CPC. Nesses casos, em virtude do uso inadequado da referida técnica discursiva, determinou-se o retorno dos autos à origem para rejulgamento de embargos de declaração das partes.

• Nesse contexto doutrinário e jurisprudencial, sendo pacífica a possibilidade de utilização da técnica de fundamentação da decisão por remissão, mas com cautela para garantir o contraditório e o direito à defesa, fixam-se as seguintes teses para fins dos artigos 1.036 a 1.041 do CPC:

• 1) A técnica da fundamentação por referência (per relationem) é permitida desde que o julgador, ao reproduzir trechos de decisão anterior, documento e/ou parecer como razões de decidir, enfrente, ainda que de forma sucinta, as novas questões relevantes para o julgamento do processo, dispensada a análise pormenorizada de cada uma das alegações ou provas.

• 2) O § 3º do artigo 1.021, do CPC não impede a reprodução dos fundamentos da decisão agravada como razões de decidir pela negativa de provimento de agravo interno quando a parte deixa de apresentar argumento novo para ser apreciado pelo colegiado.

• Informativo nº 859
• 26 de agosto de 2025.
• SEGUNDA TURMA
• Processo: REsp 2.218.969-SP, Rel. Ministro Afrânio Vilela, Segunda Turma, por unanimidade, julgado em 19/8/2025.

Ramo do Direito DIREITO ADMINISTRATIVO, DIREITO PROCESSUAL CIVIL

Tutela do patrimônio histórico-cultural. Imóvel tombado. Restauração. Meras intenções e atos administrativos convergentes com a pretensão judicial. Perda de objeto. Inocorrência. Necessidade de atendimento integral do pedido. Condução estrutural da fase executória.

Destaque - A mera intenção ou mesmo o início das obras de restauração de bem tombado não caracteriza por si só a perda de interesse processual, uma vez que o cumprimento integral da obrigação judicial é necessário para a extinção do processo por perda do objeto.

Informações do Inteiro Teor - A questão em discussão consiste em saber se o início das obras de restauração do bem tombado pelo município caracteriza perda de interesse processual, tornando desnecessária a continuidade da demanda.

• No caso, trata-se de ação civil pública ajuizada pelo Ministério Público contra um município, visando à restauração do Galpão da Oficina de Locomotivas, patrimônio tombado por Lei Municipal. A sentença condenou o ente federativo a executar as obras no prazo de seis meses, sob pena de multa e a apelação foi desprovida.

• A deterioração do bem é registrada desde a década de 1980, o tombamento ocorreu nos anos 1990, o imóvel está interditado desde 2009 e a municipalidade manifesta reiteradamente, ao longo de décadas, suas melhores intenções de devolver o bem à coletividade, sem efetivá-lo.

• No que tange ao interesse de agir do Ministério Público, o município recorrente defende sua inexistência porque teria conduzido a matéria administrativa de maneira adequada, com licitação e início das obras para restaurar o bem tombado.

• A pretensão de que seja reconhecida a perda de objeto dita unilateral depende de que a parte ré entregue ao autor o bem da vida integralmente demandado em juízo. No caso, a parte apenas manifesta a intenção de entregar parte do bem da vida demandado.

• Nas situações envolvendo o Poder Público, essa pretensão de reconhecimento da perda de objeto deve ser tratada com ainda maior critério. Isso porque, nos termos da doutrina, "Atores governamentais com frequência usam a perda de objeto para evadirem-se de precedentes desfavoráveis.".

• Por isso, a mera intenção ou mesmo início das obras de restauração não caracteriza perda de objeto, pois o cumprimento <u>integral</u> da obrigação judicial é necessário para a extinção do interesse processual.

• O cumprimento da obrigação disposta na sentença, portanto, somente poderá ser verificado na fase executória do provimento. É o juízo da execução que poderá considerar de modo efetivo os atos e esforços concretos da municipalidade que atendem de forma mais eficiente ao provimento judicial, inclusive com eventual modulação de prazos e multas, que não devem ser afastados de plano.

• Além disso, do cenário descrito, depreende-se a <u>natureza estrutural da demanda</u>. Assim, a abordagem da causa, ainda que tardiamente, já em sua fase executória, pode e deve ser feita pela lente dos processos estruturais.

• É certo que o caso trata de uma "nanoinstitucionalidade", uma situação de violação sistemática dos direitos da coletividade à cultura e à memória bastante delimitada, com um provimento jurisdicional bem específico, condição que nem sempre é entendida como matéria estrutural, dado seu limitado alcance.

• Mas isso não impede que, no momento da execução da sentença, práticas, métodos e princípios típicos do processo estrutural sejam adotados pelo juízo exequente, conforme necessários e adequados. Nesse sentido, recentemente o Conselho Nacional de Justiça - CNJ aprovou a Recomendação n. 163 de 16/6/2025, norma incentivadora da condução estrutural de processos judiciais.

• Ademais, considerando que se trata de patrimônio histórico e cultural de uma municipalidade, a adoção de técnicas estruturais de condução do feito atende, ainda, à concretização do princípio da participação comunitária, conforme reconhecido desde 1967 pelos Estados membros da Organização dos Estados Americanos - OEA,

• Nessa linha, e conforme disposto pelo Fórum Permanente de Processualistas Civis, como boa prática na condução do feito estrutural em instância recursal, afigura-se adequado dar indicações concretas ao juízo de execução sobre os parâmetros de atuação nessa circunstância (Boa Prática n. 22/FPPC), nos termos já adotados, inclusive como paradigmas desse agir, por esta Corte (REsp n. 1.854.842/CE, relatora Ministra Nancy Andrighi, Terceira Turma, julgado em 2/6/2020, DJe de 4/6/2020).

• Assim, recomenda-se ao magistrado encarregado da execução, resguardada sua independência funcional, a adoção de, entre outras, de medidas de natureza estruturante, tais como: i) estabelecimento de comitê de condução e monitoramento do projeto de restauração, inclusive com a participação de entidades da sociedade civil representantes do setor de cultura e memória, órgãos especializados de suporte, como o CREA, e representantes do Legislativo, além das partes e representante do juízo; ii) a eventual dilação do prazo de conclusão das obras, inclusive com suspensão temporária das multas condicionada ao cumprimento de eventual cronograma acordado pelas partes; iii) determinação de publicação no portal do Poder Executivo Municipal de relatórios periódicos, em intervalos de não mais que 45 dias, de execução do projeto de restauração, com os itens mínimos que entender necessários; e iv) realização de audiência pública prévia ao encerramento da obra, na sua iminência, para coleta de manifestações da sociedade sobre o alcance dos objetivos da sentença de conhecimento e prestação de contas pelos réus.

• Recomenda-se, ainda, ao Tribunal respectivo que providencie o apoio institucional necessário ao magistrado singular na implementação dessas medidas, tudo orientado pelo princípio maior de cooperação.

• Por fim, ao contrário do que alega a municipalidade, se a sentença confirma sua intenção administrativa, a imposição judicial pode destravar as diversas amarras burocráticas e políticas impostas a seus próprios gestores.

• A sentença não será vazia, mas catalisadora dos efeitos concretos da política pública de proteção ao patrimônio histórico-cultural que a própria Administração não só é obrigada por lei a implementar, como assim também o deseja há tantas décadas.

• Será, ainda, obrigação específica, decorrente de título judicial executivo, que vinculará não só a gestão atual como as futuras, de forma impessoal e para além de voluntarismos, como exige a situação degradante enfrentada pelo bem municipal tombado.

• Por isso, o provimento judicial resguardará tanto a pretensão do autor como as intenções do réu, de modo a concretizá-las a ambas as partes. Desse modo, o objeto jurídico, que deve ser entendido como a devolução à coletividade do bem histórico-cultural que verdadeiramente lhe pertence, permanece íntegro.

Observe que o destinatário desta multa é o agravado, isto é, a parte contrária, e não ao fundo de aparelhamento do judiciário, como reiterado pelo REsp 1.846.734-RS (Info 666)

Jurisprudência em teses - EDIÇÃO 182 - AGRAVO INTERNO

1) O agravo interposto contra decisão monocrática do Tribunal de origem, com o objetivo de exaurir a instância recursal ordinária, a fim de permitir a interposição de recurso especial e do extraordinário, não é manifestamente inadmissível ou infundado, o que torna inaplicável a multa prevista no art. 557, § 2º, do Código de Processo Civil. (Tese julgada sob o rito do art. 543-C do CPC/73 - Tema n. 434)

2) Em regra, descabe a imposição da multa (art. 1.021, § 4º, do CPC) em razão do não provimento do agravo interno em votação unânime, pois é necessária a configuração da manifesta inadmissibilidade ou improcedência do recurso para autorizar sua incidência.

3) Dever ser paga à parte contrária a multa do art. 1.021, § 4º, do Código de Processo Civil aplicada na hipótese de agravo interno manifestamente inadmissível ou improcedente.

• Informativo nº 857
• 12 de agosto de 2025.
• RECURSOS REPETITIVOS
• Processo: REsp 2.043.826-SC, Rel. Ministro Mauro Campbell Marques, Corte Especial, por unanimidade, julgado em 6/8/2025 (Tema 1201).

Multa prevista no art. 1.021, § 4º, do CPC. Critérios de aplicação. Manifesta improcedência ou inadmissibilidade. Agravo interno. Precedentes qualificados. Demonstração da distinção. Possibilidade. Revisão do Tema 434/STJ.

Destaque - 1) O agravo interposto contra decisão do Tribunal de origem, ainda que com o objetivo de exaurir a instância recursal ordinária, a fim de permitir a interposição de recurso especial e/ou extraordinário, quando apresentado contra decisão baseada em precedente qualificado oriundo do STJ ou do STF, autoriza a aplicação da multa prevista no art. 1.021, § 4º, do CPC (revisão do Tema 434/STJ);

• 2) A multa prevista no art. 1.021, § 4º, CPC, não é cabível quando (i) alegada fundamentadamente a distinção ou a superação do precedente qualificado oriundo do STJ ou do STF ou (ii) a decisão agravada estiver amparada em julgado de tribunal de segundo grau;

• 3) Excetuadas as hipóteses supra, caberá ao órgão colegiado verificar a aplicação da multa, considerando-se as peculiaridades do caso concreto.

Informações do Inteiro Teor - A primeira questão afetada para julgamento tem o seguinte teor: aplicabilidade da multa prevista no § 4º do art. 1.021 do CPC quando a decisão recorrida se baseia em precedente qualificado (art. 927, III, do CPC).

• No regime do CPC/73, o Superior Tribunal de Justiça pacificou orientação no sentido de que o agravo interposto contra decisão monocrática do Tribunal de origem, com o objetivo de exaurir a instância recursal ordinária, a fim de permitir a interposição de recurso especial e do extraordinário, não é manifestamente inadmissível ou infundado, motivo pelo qual inaplicável a multa que era prevista no art. 557, § 2º, do Código revogado. Essa orientação foi adotada em sede de recurso especial submetido ao regime dos recursos repetitivos (Tema 434/STJ).

• Ainda que o escopo do agravo interno seja viabilizar a interposição de recurso aos Tribunais Superiores (exaurimento de instância), não se pode olvidar que os recursos especial ou extraordinário terão seguimento negado quando o acórdão recorrido coincidir com a orientação do tribunal superior, proferida em sede de recurso extraordinário ou especial submetido ao regime dos recursos repetitivos.

• Consequentemente, o simples argumento de que se pretende o exaurimento de instância, por si só, não é suficiente para que seja reconhecida a ilegalidade da multa. Diante desse quadro, revela-se necessária a revisão do referido Tema Repetitivo.

• Por outro lado, o precedente qualificado autoriza tanto a improcedência liminar do pedido (pelo juízo singular) quanto o julgamento monocrático pelo relator (no âmbito dos tribunais). Admitir-se a interposição de recurso em face de decisão baseada em precedente qualificado - especialmente quando não há nenhuma sinalização de alteração do precedente - implica negar a própria finalidade da sua criação. Assim, o respeito ao precedente qualificado é regra de observância obrigatória.

• Entretanto, reconhecer que inexiste ilegalidade ao se declarar manifestamente improcedente agravo interno que impugna decisão baseada em precedente qualificado, para fins de aplicação da multa prevista no § 4º do art. 1.021 do CPC, não impõe que o órgão colegiado sempre declare manifestamente improcedente agravo interno interposto contra esse tipo de decisão. Em se tratando de penalidade, a aplicação deve ser proporcional.

• A título de exemplo, é comum em questões tributárias o questionamento de acórdão proferido sob o regime dos recursos repetitivos, no Superior Tribunal de Justiça, perante o Supremo Tribunal Federal. Trata-se de matéria amplamente regulamentada pela Constituição Federal, admitindo, ao menos em tese, a viabilização do recurso extraordinário. Nesse cenário, reconhecida a existência de repercussão geral, não é recomendável a imposição da penalidade em sede de agravo interno, ainda que a decisão recorrida esteja amparada em acórdão vinculante do Superior Tribunal de Justiça.

• Nesse cenário, pode-se afirmar que inexiste ilegalidade ao se declarar manifestamente improcedente agravo interno que impugna decisão baseada em precedente qualificado, para fins de aplicação da multa prevista no § 4º do art. 1.021 do CPC, ressalvados os casos em que a aplicação da multa não é recomendada (v.g. acórdão vinculante proferido pelo Tribunal local e recurso especial repetitivo pendente no STJ; acórdão vinculante proferido pelo STJ e pendência de julgamento de repercussão geral no STF).

• Por sua vez, a segunda questão afetada para julgamento tem o seguinte teor: possibilidade de se considerar manifestamente inadmissível ou improcedente (ainda que em votação unânime) agravo interno cujas razões apontam a indevida ou incorreta aplicação de tese firmada em sede de precedente qualificado.

• Como se constata, a questão guarda relação com o instituto da distinção, que é tratado especialmente nos arts. 489, V e VI e 1.037, §§ 9º a 13, do CPC.

• Segundo a doutrina, "o distinguishing expressa a distinção entre os casos para o efeito de se subordinar, ou não, o caso sob julgamento ao precedente", ou seja, "o distinguishing revela a demonstração entre as diferenças fáticas entre os casos ou a demonstração de que a ratio do precedente não se amolda ao caso sob julgamento, uma vez que os fatos de um e outro são diversos".

• Acerca do tema, é esclarecedor o Enunciado 306 do Fórum Permanente de Processualistas Civis (FPPC): "O precedente vinculante não será seguido quando o juiz ou tribunal distinguir o caso sob julgamento, demonstrando, fundamentalmente, tratar-se de situação particularizada por hipótese fática distinta, a impor solução jurídica diversa".

• A Segunda Turma do STJ, em recente precedente, afirmou que "o pedido (ou requerimento) de distinção deve ser apresentado na forma do art. 1.037, § 8º e seguintes do CPC. Nesse regime, tal pedido deve ser interposto na primeira oportunidade, após a determinação de sobrestamento, quando este ocorre em Tribunal Superior" (PDist no AgInt nos EDcl no AREsp n. 2.360.573/PE, relator Ministro Mauro Campbell Marques, Segunda Turma, julgado em 14/5/2024, DJe de 17/5/2024).

• Destarte, em se tratando de decisão do relator baseada em precedente qualificado, a alegação de distinção deve ser formulada na primeira oportunidade, assim como ocorre com pedido de distinção previsto no art. 1.037, § 8º e seguintes do CPC.

• Cabe ressaltar que é ônus da parte demonstrar a existência de distinção, em consonância com o Enunciado 9 da ENFAM. Essa demonstração deve ocorrer de forma fundamentada, de modo que é descabida a simples alegação de indevida ou incorreta aplicação de tese firmada em sede de precedente qualificado.

• Assim, o agravo interposto contra decisão do Tribunal de origem, ainda que com o objetivo de exaurir a instância recursal ordinária, a fim de permitir a interposição de recurso especial e/ou extraordinário, quando apresentado contra decisão baseada em precedente qualificado oriundo do STJ ou do STF, autoriza a aplicação da multa prevista no art. 1.021, § 4º, do CPC (revisão do TR 434/STJ).

• Além disso, a multa prevista no art. 1.021, §4º, CPC não é cabível quando (i) alegada de forma fundamentada a distinção ou superação do precedente qualificado oriundo do STJ ou do STF ou (ii) a decisão agravada estiver amparada em julgado de tribunal de segundo grau.

• Por fim, excetuadas as hipóteses supra, caberá ao órgão colegiado verificar a aplicação da multa, considerando-se as peculiaridades do caso concreto.

• Informativo nº 859
• 26 de agosto de 2025.
• TERCEIRA TURMA
• Processo: * Processo em segredo de justiça, Rel. Ministra Nancy Andrighi, Terceira Turma, por unanimidade, julgado em 17/6/2025, DJEN 27/6/2025.

Ação de exoneração de alimentos. Alimentos entre ex-cônjuges. Pagamento de pensão alimentícia por mais de duas décadas após o termo final da obrigação. Liberalidade. Expectativa legítima de continuidade da prestação. Supressio configurada.

Destaque - É possível a manutenção do pagamento de pensão alimentícia por prazo indeterminado, na hipótese em que o ex-marido, mesmo exonerado, optou voluntariamente por continuar realizando o pagamento de alimentos por duas décadas, em razão da configuração dos institutos da supressio para o alimentante, que deixou de exercer seu direito de cessar os pagamentos, e da surrectio para a alimentanda diante da expectativa de que o direito de exoneração dos alimentos não mais seria reivindicado pelo ex-cônjuge.

Informações do Inteiro Teor - Cinge-se a controvérsia em decidir se o pagamento de pensão alimentícia pelo ex-marido, por mais de duas décadas após o termo final da obrigação, configura a incidência do instituto da supressio, fazendo nascer para a ex-esposa a expectativa legítima de continuidade da prestação, em homenagem à boa-fé objetiva.

• A confiança, no contexto das relações privadas, desempenha papel fundamental ao assegurar proteção qualificada ao comportamento humano, sendo expressão concreta da solidariedade social constitucionalmente albergada. Essa confiança impõe a todos o dever jurídico de não frustrar, injustificadamente, as legítimas expectativas de terceiros. No âmbito das relações familiares, a noção de confiança deve ser especialmente protegida, de forma que as condutas contrárias à confiança serão, em regra, também contrárias à boa-fé objetiva.

• A tutela da confiança assume relevância ética nas relações privadas ao proibir comportamentos contraditórios (venire contra factum proprium) e ao reconhecer efeitos decorrentes da inércia prolongada (supressio) ou da prática constante (surrectio). Tais figuras jurídicas operam como mecanismos de estabilização das expectativas, impedindo mudanças abruptas de conduta que contrariem a confiança anteriormente depositada.

• Identifica-se a supressio como a perda de determinada faculdade jurídica em razão do não exercício prolongado desse direito, o que leva ao seu esvaziamento. Em contrapartida, a surrectio consiste no surgimento de uma vantagem para determinada pessoa, justamente porque a outra parte deixou de exercer o direito ao qual faria jus, criando, assim, a expectativa de que esse direito não mais seria reivindicado futuramente.

• A supressio aproxima-se, sem dúvida, do venire contra factum proprium, pois ambas as figuras atuam como fatores de preservação da confiança alheia. Mas dele se diferencia primordialmente pois, enquanto no venire, a expectativa do outro decorre de uma conduta ativa anterior, que não pode ser desmentida posteriormente; na supressio, a expectativa nasce da omissão prolongada do titular do direito, cuja inércia, associada a elementos objetivos que indiquem o desuso, conduz à convicção de que tal direito não será mais exercido.

• Assim, a inércia prolongada do credor de alimentos em promover a execução da pensão em débito pode gerar, no devedor, a legítima expectativa de que a prestação não é mais necessária, conduzindo à estabilização da situação de inadimplemento. Em sentido inverso, o alimentante que, mesmo exonerado, opta voluntariamente por continuar realizando os pagamentos, conduz ao alimentando a expectativa de continuidade da prestação, a qual pode tornar-se juridicamente relevante, especialmente diante da reiterada e sistemática manifestação de vontade.

• A aplicação da boa-fé no âmbito do Direito de Família reforça a dimensão ética e funcional da confiança, reafirmando seu papel como vetor interpretativo e integrativo. A eventual violação de justa expectativa deverá ser verificada na situação em concreto, devendo o julgador buscar a melhor forma de concretização das expectativas e esperanças criadas no ambiente familiar.

• O caráter de transitoriedade dos alimentos entre ex-cônjuges parece traduzir o conteúdo da boa-fé objetiva, uma vez que deve a obrigação alimentar garantir o fornecimento de auxílio material ao cônjuge depreciado em razão de sua vulnerabilidade social e econômica, até que possa retomar sua autonomia financeira.

• Os alimentos transitórios não serão cabíveis, entretanto, quando as necessidades são permanentes, em decorrência da incapacidade perene do alimentando de promover seu próprio sustento.

• A jurisprudência do Superior Tribunal de Justiça tem admitido a perenidade da obrigação de prestar alimentos entre ex-cônjuges em situações excepcionais, como na impossibilidade prática de reinserção do alimentando no mercado de trabalho; em hipótese de idade avançada do alimentando; ou de condição de saúde fragilizada.

• Dessa forma, constatando-se, na espécie, a incapacidade laboral do alimentando, saúde fragilizada, idade avançada ou qualquer impossibilidade prática de inserção no mercado de trabalho, ou de adquirir autonomia financeira, a pensão alimentícia entre ex-cônjuges poderá ser fixada por prazo indeterminado.

• No caso, é incontroverso que as partes se encontram divorciadas há mais de 30 (trinta anos), tendo firmado acordo para pagamento de pensão alimentícia pelo ex-marido à ex-esposa, correspondente a 5% dos seus rendimentos líquidos, além de pagamento de plano de saúde, pelo prazo de um ano. Referido acordo fora homologado judicialmente em 1993.

• Dois anos depois, as partes peticionaram nos autos da ação de divórcio requerendo a alteração do acordo, para que o pagamento da pensão alimentícia fosse prorrogado por prazo indeterminado. Embora não tenha o juízo conhecido do pedido, em razão da necessidade de ajuizamento de ação própria, o ex-marido permaneceu alcançando a pensão alimentícia à ex-esposa por mais de 25 (vinte e cinco) anos, até o ajuizamento da ação de exoneração, em julho de 2018.

• O fato de a ex-esposa ter recebido pensão alimentícia por mais de 25 (vinte e cinco) anos, no entanto, não demonstra sua inércia em retomar a independência financeira. Do contrário, a inércia do ex-marido em permanecer realizando os pagamentos mensais acordados por longo período, mesmo que exonerado, provocou na alimentanda a expectativa de que o direito de exoneração não seria mais por ele exercida.

• Portanto, evidencia-se, da conduta do alimentante, o instituto da supressio, visto que deixou de exercer seu direito de cessar o pagamento dos alimentos por mais de duas décadas, conduzindo à estabilização da situação de fato. Lado outro, surge para a alimentanda a surrectio, diante da expectativa de que o direito de exoneração dos alimentos não mais seria reivindicado pelo ex-marido.

• Com efeito, o alimentante que, mesmo exonerado, opta voluntariamente por continuar realizando os pagamentos, conduz ao alimentando a expectativa de continuidade da prestação, a qual pode tornar-se juridicamente relevante, especialmente diante da reiterada e sistemática manifestação de vontade.

• Some-se a isso o fato de que a ex-esposa teve de abdicar de seu trabalho em razão de mudança da família para a cidade de Petrópolis, em função do emprego do ex-marido. A realidade vivenciada pelo casal ao tempo da constância da sociedade conjugal deve ser considerada quando da fixação da pensão alimentícia.

• Ademais, tendo em vista que a alimentanda é pessoa idosa, possui doença grave e se encontra impossibilitada de se reinserir no mercado de trabalho; e o alimentante aufere renda suficiente para permanecer cumprindo a obrigação constituída; deve-se manter o pagamento da pensão alimentícia por prazo indeterminado.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

This study examined the effect of blood pressure variability on brain microvascular function and cognitive performance. By implementing a model of blood pressure variability using an intermittent infusion of AngII for 25 days, the authors examined different cardiovascular variables, cerebral blood flow, and cognitive function during midlife (12-15-month-old mice). Key findings from this study demonstrate that blood pressure variability impairs baroreceptor reflex and impairs myogenic tone in brain arterioles, particularly at higher blood pressure. They also provide evidence that blood pressure variability blunts functional hyperemia and impairs cognitive function and activity. Simultaneous monitoring of cardiovascular parameters, in vivo imaging recordings, and the combination of physiological and behavioral studies reflect rigor in addressing the hypothesis. The experiments are well-designed, and the data generated are clear. I list below a number of suggestions to enhance this important work:

(1) Figure 1B: It is surprising that the BP circadian rhythm is not distinguishable in either group. Figure 2, however, shows differences in circadian rhythm at different timepoints during infusion. Could the authors explain the lack of circadian effect in the 24-h traces?

The circadian rhythm pattern is apparent in Figure 2 (Active BP higher than Inactive BP), where BP is presented as 12hour averages. When the BP data is expressed as one-hour averages (rather than minute-to-minute) over 24hours, now included in the revised manuscript as Supplemental Figure 3C-D, the circadian rhythm becomes noticeable. In addition, we have included one-hour average BP data for all mice in the control and BPV groups, Supplemental Figure 3A-B.

Notably, the Ang-II induced pulsatile BP pattern remains evident in the one-hour averages for the BPV group, Supplemental Figure 3B. To minimize bias and validate variability, pump administrations start times were randomized for both control and BPV groups, Supplemental Figure 3A-B. Despite these adjustments, the circadian rhythm profile of BP is consistently maintained across individual mice and in the collective dataset, Supplemental Figure 3C-D.

(2) While saline infusion does not result in elevation of BP when compared to Ang II, there is an evident "and huge" BP variability in the saline group, at least 40mmHg within 1 hour. This is a significant physiological effect to take into consideration, and therefore it warrants discussion.

Thank you for this comment. The large variations in BP in the raw traces during saline infusion reflects transient BP changes induced by movement/activity, which is now included in Figure 1B (maroon trace). The revised manuscript now includes Line 222 “Note that dynamic activity-driven BP changes were apparent during both saline- and Ang II infusions, Figure 1B”.

(3) The decrease in DBP in the BPV group is very interesting. It is known that chronic Ang II increases cardiac hypertrophy, are there any changes to heart morphology, mass, and/or function during BPV? Can the decrease in DBP in BPV be attributed to preload dysfunction? This observation should be discussed.

The lower DBP in the BPV group was already present at baseline, while both groups were still infused with saline, and was a difference beyond our control. However, this is an important and valid consideration, particularly considering the minimal yet significant increase in SBP within the BPV group (Figure 1D). Our goal was to induce significant transient blood pressure responses (BPV) and investigate the impact on cardiovascular and neurovascular outcomes in the absence of hypertension. We did not anticipate any major cardiac remodeling at this early time point (considering the absence of overt hypertension) and thus cardiac remodeling was not assessed and this is now discussed in the revised manuscript (Line 443-453).

(4) Examining the baroreceptor reflex during the early and late phases of BPV is quite compelling. Figures 3D and 3E clearly delineate the differences between the two phases. For clarity, I would recommend plotting the data as is shown in panels D and E, rather than showing the mathematical ratio. Alternatively, plotting the correlation of ∆HR to ∆SBP and analyzing the slopes might be more digestible to the reader. The impairment in baroreceptor reflex in the BPV during high BP is clear, is there any indication whether this response might be due to loss of sympathetic or gain of parasympathetic response based on the model used?

We appreciate the reviewer’s suggestion and have accordingly generated new figures displaying scatter plots of SBP vs HR with linear regression analysis (Figure 3D-G). Our goal is to further investigate which branch of the autonomic nervous system is affected in this model. The loss of a bradycardic response suggests either an enhancement of sympathetic activity, a reduction in parasympathetic activity, or a combination of both. This is briefly discussed in the revised manuscript (Line 486-496).

Heart rate variability (HRV) serves as an index of neurocardiac function and dynamic, non-linear autonomic nervous system processes, as described in Shaffer and Ginsber[1]. However, given that our data was limited to BP and HR readings collected at one-minute intervals, our primary assessment of autonomic function is limited to the bradycardic response. Further studies will be necessary to fully characterize the autonomic parameters influenced by chronic BPV.

(5) Figure 3B shows a drop in HR when the pump is ON irrespective of treatment (i.e., independent of BP changes). What is the underlying mechanism?

We apologize for any lack of clarity. These observed heart rate (HR) changes occurred during Ang II infusion, when blood pressure (BP) was actively increasing. In the control group, the pump solution was switched to Ang II during specific periods (days 3-5 and 21-25 of the treatment protocol) to induce BP elevations and a baroreceptor response, allowing direct comparisons between the control and BPV group.

To clarify this point, we have revised Line 260-263 of the manuscript: “To compare pressure-induced bradycardic responses between BPV and control mice at both early and later treatment stages, a cohort of control mice received Ang II infusion on days 3-5 (early phase) (Supplemental Figure 4) and days 21-25 (late phase) thereby transiently increasing BP”.

Additionally, a detailed description has been added to the Methods section (Line 96-101): “Controls receiving Ang II: To facilitate between-group comparisons (control vs BPV), a separate cohort of control mice were subjected to the same pump infusion parameters as BPV mice but for a brief period receiving Ang II infusions on days 3-5 and 21-25 for experiments assessing pressure-evoked responses, including bradycardic reflex, myogenic response, and functional hyperemia at high BP.”

(6) The correlation of ∆diameter vs MAP during low and high BP is compelling, and the shift in the cerebral autoregulation curve is also a good observation. I would strongly recommend that the authors include a schematic showing the working hypothesis that depicts the shift of the curve during BPV.

Thank you for this insightful comment. The increase in vessel reactivity to BP elevations in parenchymal arterioles of BPV mice suggests that chronic BPV induces a leftward shift and a potential narrowing of the cerebral autoregulation range (lower BP thresholds for both the upper and lower limits of autoregulation). This has been incorporated (and discussed) into the revised manuscript (see Figure 5N).

One potential explanation for these changes is that the absence of sustained hypertension, a prominent feature in most rodent models of hypertension, limits adaptive processes that protect the cerebral microcirculation from large BP fluctuations (e.g., vascular remodeling). While this study does not specifically address arteriole remodeling, the lack of such adaptation may reduce pressure buffering by upstream arterioles, thereby rendering the microcirculation more vulnerable to significant BP fluctuations.

The unique model allows for measurements of parenchymal arteriole reactivity to acute dynamic changes in BP (both an increase and decrease in MAP). Our findings indicate that chronic BPV enhances the reactivity of parenchymal arterioles to BP changes—both during an increase in BP and upon its return to baseline, Supplemental Figure 5C, F. The data suggest an increased myogenic response to pressure elevation, indicative of heightened contractility, a common adaptive process observed in rodent models of hypertension[2-4]. However, our model also reveals a notable tendency for greater dilation when the BP drops, Supplemental Figure 5F. This intriguing observation may suggest ischemia during the vasoconstriction phase (at higher BP), leading to enhanced release of dilatory signals, which subsequently manifest as a greater dilation upon BP reduction. This phenomenon bears similarities to chronic hypoperfusion models[5,6], where vasodilatory mechanisms become more pronounced in response to sustained ischemic conditions. Future studies investigating the effects of BPV on myogenic responses and brain perfusion will be a priority for our ongoing research.

(7) Functional hyperemia impairment in the BPV group is clear and well-described. Pairing this response with the kinetics of the recovery phase is an interesting observation. I suggest elaborating on why BPV group exerts lower responses and how this links to the rapid decline during recovery.

Based on the heightened reactivity of BPV parenchymal arterioles to intravascular pressure (Figure 5), we anticipate that the reduction of sensory-evoked dilations results from an increased vasoconstrictive activity and/or a decreased availability of vasodilatory signaling pathways (NO, EETs, COX-derived prostaglandins)[7,8]. Consequently, the magnitude of the FH response is blunted during periods of elevated BP in BPV mice.

Additionally, upon termination of the stimulus-induced response−when vasodilatory signals would typically dominate−vasoconstrictive mechanisms are rapidly engaged (or unmasked), leading to quicker return to baseline. This shift in the balance between vasodilatory and vasoconstrictive forces favors vasoconstriction, contributing to the altered recovery kinetics observed in BPV mice. This has been included in the Discussion section of the revised manuscript.

(8) The experimental design for the cognitive/behavioral assessment is clear and it is a reasonable experiment based on previous results. However, the discussion associated with these results falls short. I recommend that the authors describe the rationale to assess recognition memory, short-term spatial memory, and mice activity, and explain why these outcomes are relevant in the BPV context. Are there other studies that support these findings? The authors discussed that no changes in alternation might be due to the age of the mice, which could already exhibit cognitive deficits. In this line of thought, what is the primary contributor to behavioral impairment? I think that this sentence weakens the conclusion on BPV impairing cognitive function and might even imply that age per se might be the factor that modulates the various physiological outcomes observed here. I recommend clarifying this section in the discussion.

We thank the reviewer for this comment. Clinical studies have demonstrated that patients with elevated BPV exhibit impairments across multiple cognitive domains, including declines in processing speed[9] and episodic memory[10]. To evaluate memory function, we utilized behavioral tests: the novel object recognition (NOR) task to assess episodic memory[11] and the spontaneous Y-maze to evaluate short-term spatial memory[12].

Previous research indicates that older C57Bl6 mice (14-month-old) exhibit cognitive deficits compared to younger counterparts (4- and 9-month-old)[13]. To ensure rigorous selection for behavioral testing, we conducted preliminary NOR assessment, evaluating recognition memory at the one-hour delay but observing failures at the four-, and 24-hour delays, indicating age-related deficits. Based on these results, animals failing recognition criteria were excluded from subsequent behavioral assessment. However, because no baseline cognitive testing was conducted for the spontaneous Y-maze, it is possible that some mice with aged-related deficits were included in this test, which may have influenced data interpretation.

Additionally, the absence of differences in the Y-maze performance may suggest that short-term spatial memory remains intact following 25 days of BPV, a point that is now discussed in the revised manuscript.

(9) Why were only male mice used?

We appreciate this comment and acknowledge the importance of conducting experiments in both male and female mice. Studies involving female mice are currently ongoing, with telemetry data collection approximately halfway completed and two-photon imaging studies on functional hyperemia also partially completed. However, using middleaged mice for these experiments has proven challenging due to high mortality rates following telemetry surgeries. As a result, we initially limited our first cohort to male mice.

(10) In the results for Figure 3: "Ang II evoked significant increases in SBP in both control and BPV groups;...". Also, in the figure legend: "B. Five-minute average HR when the pump is OFF or ON (infusing Ang II) for control and BPV groups...." The authors should clarify this as the methods do not state a control group that receives Ang II.

Please refer to response to comment 5.

Reviewer #2 (Public review):

Summary:

Blood pressure variability has been identified as an important risk factor for dementia. However, there are no established animal models to study the molecular mechanisms of increased blood pressure variability. In this manuscript, the authors present a novel mouse model of elevated BPV produced by pulsatile infusions of high-dose angiotensin II (3.1ug/hour) in middle-aged male mice. Using elegant methodology, including direct blood pressure measurement by telemetry, programmable infusion pumps, in vivo two-photon microscopy, and neurobehavioral tests, the authors show that this BPV model resulted in a blunted bradycardic response and cognitive deficits, enhanced myogenic response in parenchymal arterioles, and a loss of the pressure-evoked increase in functional hyperemia to whisker stimulation.

Strengths:

As the presentation of the first model of increased blood pressure variability, this manuscript establishes a method for assessing molecular mechanisms. The state-of-the-art methodology and robust data analysis provide convincing evidence that increased blood pressure variability impacts brain health.

Weaknesses:

One major drawback is that there is no comparison with another pressor agent (such as phenylephrine); therefore, it is not possible to conclude whether the observed effects are a result of increased blood pressure variability or caused by direct actions of Ang II.

We acknowledge this limitation and have attempted to address the concern by introducing an alternative vasopressor, norepinephrine (NE), Figure 4. A subcutaneous dose of 45 µg/kg/min was titrated to match Ang II-induced transient BP pulse (Systolic BP ~150-180 mmHg), Figure 4A. Similar to Ang II treated mice, NE-treated mice exhibited no significant changes in average mean arterial pressure (MAP) throughout the 20-day treatment period (Figure 4B). Although there was a trend (P=0.08) towards increased average real variability (ARV) (Figure 4C left), it did not reach statistical significance. The coefficient of variation (CV) (Figure 4C right) was significantly increased by day 3-4 of treatment (P=0.02).

Notably, unlike the bradycardic response observed during Ang II-induced BP elevations, NE infusions elicited a tachycardic response (Figure 4A), likely due to β-1 adrenergic receptor activation. However, significant mortality was observed within the NE cohort: three of six mice died prematurely during the second week of treatment, and two additional mice required euthanasia on days 18 and 20 due to lethargy, impaired mobility, and tachypnea.

While we recognize the importance of comparing results across vasopressors, further investigation using additional vasopressors would require a dedicated study, as each agent may induce distinct off-target effects, potentially generating unique animal models. Alternatively, a mechanical approach−such as implanting a tethered intra-aortic balloon[14] connected to a syringe pump−could be explored to modulate blood pressure variability without pharmacological intervention. However, such an approach falls beyond the scope of the present study.

Ang II is known to have direct actions on cerebrovascular reactivity, neuronal function, and learning and memory. Given that Ang II is increased in only 15% of human hypertensive patients (and an even lower percentage of non-hypertensive), the clinical relevance is diminished. Nonetheless, this is an important study establishing the first mouse model of increased BPV.

We agree that high Ang II levels are not a predominant cause of hypertension in humans, which is why it is critical that our pulsatile Ang II dosing did not cause overt hypertension, (no increase in 24-hour MAP). Ang II was solely a tool to produce controlled, transient increases in BP to yield a significant increase in BPV.

Regarding BPV specifically, prior studies indicate that primary hypertensive patients with elevated urinary angiotensinogen-to-creatinine ratio exhibit significantly higher mean 24-hour systolic ARV compared to those with lower ratios[15]. However, the fundamental mechanisms driving these harmful increases in BPV remain poorly defined. A central theme across clinical BPV studies is impaired arterial stiffness, which has been proposed to contribute to BPV through reduced arterial compliance and diminished baroreflex sensitivity. Moreover, increased BPV can exert mechanical stress on arterial walls, leading to arterial remodeling and stiffness−ultimately perpetuating a detrimental feed-forward cycle[16].

In our model, male BPV mice exhibited a minimal yet significant elevation in SBP without corresponding increases in DBP, potentially reflecting isolated systolic hypertension, which is strongly associated with arterial stiffness[17,18]. Our initial goal was to establish controlled rapid fluctuations in BP, and Ang II was selected as the pressor due to its potent vasoconstrictive properties and short half-life[19].

We appreciate the reviewer’s insightful comment and acknowledge the necessity of exploring alternative mechanisms underlying BPV, and independent of Ang II. It is our long-term goal to investigate these factors in further studies.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

(1) How was the dose of Ang II determined? It seems that this dose (3.1ug/hr) is quite high.

The Ang II dose was titrated in a preliminary study to one that induced a significant and transient BP response without increasing 24-hour blood pressure (i.e. no hypertension).

Ang II was delivered subcutaneously at 3.1 μg/hr, a concentration comparable to high-dose Ang II administration via mini-osmotic pumps (~1700 ng/kg/min)[20], with one-hour pulses occurring every 3-4 hours. With 6 pulses per day, the total daily dose equates to 18.6 µg/day in a ~30 gram mouse.

For comparison, if the same 18.6 µg/day dose were administered continuously via a mini-osmotic pump (18.6 µg/0.03kg/1440min), the resulting dosage would be approximately 431 ng/kg/min[21,22], aligning with subpressor dose levels. Thus, while the total dose may appear high, it is not delivered in a constant manner but rather intermittently, allowing for controlled, rapid variations in blood pressure.

(2) Were behavioral studies performed on the same mice that were individually housed? Individual housing causes significant stress in mice that can affect learning and memory tasks (PMC6709207). It's not a huge issue since the control mice would have been housed the same way, but it is something that could be mentioned in the discussion section.

Behavioral studies were performed on mice that were individually housed following the telemetry surgery. The study was started once BP levels stabilized, as mice required several days to achieve hemodynamic stability post-surgery. Consequently, all mice were individually housed for several days before undergoing behavioral assessment.

To account for potential cognitive variability, earlier novel object recognition (NOR) tests were conducted to established cognitive capacity, and mice that did not meet criteria were excluded from further behavioral testing. However, we acknowledge that individual housing induces stress, which can influence learning and memory, and this is a factor we were unable to fully control. Given that both experimental and control groups experienced the same housing conditions, this stress effect should be comparable across cohorts. A discussion on this limitation is now included in the text.

(3) It looks like one control mouse that was included in both Figures 1 and 2 (control n=12) but was excluded in Table 1 (control n=11), this isn't mentioned in the text - please include the exclusion criteria in the manuscript.

We apologize for the typo−12 control animals were consistently utilized across Figure 1-2, Table 1, Supplemental Table 1, Figure 6C, and Supplemental Figure 2B. Since the initial submission, one control mouse was completed and included into the telemetry control cohort. Thus, in the updated manuscript, we have corrected the control sample size to 13 mice across these figures ensuring consistency.

Additionally, exclusion criteria have now been explicitly included in the manuscript (Line 173-175). Mice were excluded from the study if they died prematurely (died prior to treatment onset) or mice exhibited abnormally elevated pressure while receiving saline, likely due to complications from telemetry surgery.

(4) Please include a statement on why female mice were not included in this study.

As discussed in our response to Reviewer #1, our initial intention was to include both male and female mice in this study. However, high mortality rates following telemetry surgeries significantly constrained our ability to advance all aspects of the study. As a result, we limited our first cohort to males to establish the basics of the model. A statement is now included in the manuscript, Line 50-53: “Female mice were not included in the present study due to high post-surgery mortality observed in 12-14-month-old mice following complex procedures. To minimized confounding effects of differential survival and to establish foundational data for this model, we restricted the investigation to male mice.”

Potential sex differences might be complex and warrants a separate future research to comprehensively assess sex as a biological variable, which are currently ongoing.

(5) On page 14, "experiments from control vs experimental mice were not equally conducted in the same season raising the possibility for a seasonal effect" - does this mean that control experiments were not conducted at the same time as the Ang II infusions in BPV mice? This has huge implications on whether the effects observed are induced by treatment or just batch seasonal effects.

We fully acknowledge the reviewer’s concern, and our statement aims to provide transparency regarding the study’s limitations. Several challenges contributed to this outcome, including high mortality rates following surgeries (primarily telemetry implantation) and technical issues related to instrumentation, particularly telemetry functionality.

Differences between BPV and saline mice emerge primarily due to mortality or telemetry failures−some mice did not survive post-surgery, while others remain healthy but had non-functional telemeters. This issue was particularly pronounced in 14-month-old mice, as their fragile vasculature occasionally prevented proper BP readings.

Each experiment required a minimum of two and a half months per mouse to complete, with a cost (also per mouse) exceeding $1500 USD ($300 pump, $175 mouse, $900 telemeters, per diem, drugs, reagents etc.). Despite our best effort to ensure comparable seasonal/batch data, these logistical and technical constraints prevented perfect synchronization.

To evaluate whether seasonal differences influenced our results, we incorporated additional telemetry data into the control cohort. Of the seven included control mice, six underwent the same treatment but were allocated to a separate branch of the study, which endpoints did not require a chronic cranial window. We found no significant differences in 24-hour average MAP during the baseline period between control mice with or without a cranial window, Supplemental Figure 2A. Additionally, we grouped mice into seasonal categories based on Georgia’s climate: “Spring-Summer” (May-September) and “Fall-Winter” (October-April) but observed no BP differences between these periods, Supplemental Figure 2B.

Given the absence of seasonal effects on BP and the fact that mice were sourced from two independent suppliers (Jackson Laboratory and NIA), we anticipate that the observed results are driven by treatment rather than seasonal or batch effects.

(6) Methods, two-photon imaging: did the authors mean "retro-orbital" instead of "intra-orbital" injection of the Texas red dye? Also, is this a Texas red-dextran? If so, what molecular weight?

Thank you for this comment. The correct terminology is “retro-orbital” rather than “intra-orbital” injection. Additionally, we utilized Texas Red-dextran (70 kDa, 5% [wt/vol] in saline) for the imaging experiments. These details have now been incorporated into the Methods section.

(1) Shaffer F, Ginsberg JP. An Overview of Heart Rate Variability Metrics and Norms. Front Public Health. 2017;5:258. doi: 10.3389/fpubh.2017.00258

(2) Pires PW, Jackson WF, Dorrance AM. Regulation of myogenic tone and structure of parenchymal arterioles by hypertension and the mineralocorticoid receptor. Am J Physiol Heart Circ Physiol. 2015;309:H127-136. doi: 10.1152/ajpheart.00168.2015

(3) Iddings JA, Kim KJ, Zhou Y, Higashimori H, Filosa JA. Enhanced parenchymal arteriole tone and astrocyte signaling protect neurovascular coupling mediated parenchymal arteriole vasodilation in the spontaneously hypertensive rat. J Cereb Blood Flow Metab. 2015;35:1127-1136. doi: 10.1038/jcbfm.2015.31

(4) Diaz JR, Kim KJ, Brands MW, Filosa JA. Augmented astrocyte microdomain Ca(2+) dynamics and parenchymal arteriole tone in angiotensin II-infused hypertensive mice. Glia. 2019;67:551-565. doi: 10.1002/glia.23564

(5) Kim KJ, Diaz JR, Presa JL, Muller PR, Brands MW, Khan MB, Hess DC, Althammer F, Stern JE, Filosa JA. Decreased parenchymal arteriolar tone uncouples vessel-to-neuronal communication in a mouse model of vascular cognitive impairment. GeroScience. 2021. doi: 10.1007/s11357-020-00305-x

(6) Chan SL, Nelson MT, Cipolla MJ. Transient receptor potential vanilloid-4 channels are involved in diminished myogenic tone in brain parenchymal arterioles in response to chronic hypoperfusion in mice. Acta Physiol (Oxf). 2019;225:e13181. doi: 10.1111/apha.13181

(7) Tarantini S, Hertelendy P, Tucsek Z, Valcarcel-Ares MN, Smith N, Menyhart A, Farkas E, Hodges EL, Towner R, Deak F, et al. Pharmacologically-induced neurovascular uncoupling is associated with cognitive impairment in mice. J Cereb Blood Flow Metab. 2015;35:1871-1881. doi: 10.1038/jcbfm.2015.162

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(9) Sible IJ, Nation DA. Blood Pressure Variability and Cognitive Decline: A Post Hoc Analysis of the SPRINT MIND Trial. Am J Hypertens. 2023;36:168-175. doi: 10.1093/ajh/hpac128

(10) Epstein NU, Lane KA, Farlow MR, Risacher SL, Saykin AJ, Gao S. Cognitive dysfunction and greater visit-to-visit systolic blood pressure variability. Journal of the American Geriatrics Society. 2013;61:2168-2173. doi: 10.1111/jgs.12542

(11) Antunes M, Biala G. The novel object recognition memory: neurobiology, test procedure, and its modifications. Cognitive processing. 2012;13:93-110. doi: 10.1007/s10339-011-0430-z

(12) Kraeuter AK, Guest PC, Sarnyai Z. The Y-Maze for Assessment of Spatial Working and Reference Memory in Mice. Methods Mol Biol. 2019;1916:105-111. doi: 10.1007/978-1-4939-8994-2_10

(13) Singhal G, Morgan J, Jawahar MC, Corrigan F, Jaehne EJ, Toben C, Breen J, Pederson SM, Manavis J, Hannan AJ, et al. Effects of aging on the motor, cognitive and affective behaviors, neuroimmune responses and hippocampal gene expression. Behav Brain Res. 2020;383:112501. doi: 10.1016/j.bbr.2020.112501

(14) Tediashvili G, Wang D, Reichenspurner H, Deuse T, Schrepfer S. Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta. J Vis Exp. 2018. doi: 10.3791/56477

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Trata-se de presunção relativa e não absoluta, cabendo à Administração executar as diligências previstas no § 2º, conforme acórdão do TCU:

1. O critério definido no art. 59, § 4º, da Lei 14.133/2021 conduz a uma presunção <u>relativa</u> de inexequibilidade de preços, devendo a Administração, nos termos do art. 59, § 2º, da referida lei, dar à licitante a oportunidade de demonstrar a exequibilidade de sua proposta.

(TCU, Acórdão 465/2024 Plenário, Representação, Relator Ministro-Substituto Augusto Sherman - Boletim nº 478)

• 1. O critério de desempate por localidade (art. 60, § 1º, inciso I, da Lei 14.133/2021) não se aplica a licitações realizadas no âmbito da Administração Pública Federal, por ausência de expressa previsão legal. A preferência por empresas estabelecidas no território do promotor do certame é restrita às licitações realizadas por órgãos e entidades dos entes subnacionais.

(TCU, Acórdão 1733/2025, Plenário, Representação, Relator Ministro Benjamin Zymler)

Obs.: Ou seja, propostas empatadas poderão ser decididas, se em igualdade de condições, por critério territorial. No entanto, observe que somente é aplicável tal critério aos entes subnacionais.

Com isso, o TCU entendeu que é indevido o desempate por critérios territoriais em licitações federais por expressa falta de previsão legal, considerando a expressa delimitação da abrangência da norma. Ou seja, aplicar-se critérios territoriais em licitações federais viola o princípio da legalidade e da isonomia.

@noemie @caovy Quand on aura terminé de rédiger la partie précédente : quels sont nos besoins, quelles solutions veut-on explorer, quelles méthodes va-t-on tester, que va-t-on développer et partager, il faudra produire une synthèse par le biais de listes ou de schémas qui permettront d'imprimer dans la rétine du lecteur l'essentiel de notre discours.

Reviewer #1 (Public review):

Summary:

Meijer et al. sought to investigate the role of cortical layer 6b (L6b) neurons in modulating sleep-wake states and cortical oscillations under baseline and sleep deprived conditions and in response to orexin A and B. Using chronic EEG recordings in mice with silencing of Drd1a+ neurons (via constitutive Cre-dependent knockout of SNAP25), the authors report that while overall baseline sleep-wake architecture and response to sleep deprivation minimal/unchanged, "L6b silencing" leads to a slowing of theta activity during wakefulness and REM sleep, and a reduction in EEG power during NREM sleep. Additionally, orexin B-induced increases in theta activity were attenuated in L6b silenced mice, which the authors state suggests a modulatory role for L6b in orexin-mediated arousal regulation. The manuscript is generally well written with clarity and transparency. However, a major concern is the lack of specificity in the genetic manipulation, which targets Drd1a+ neurons not exclusive to L6b, undermining the attribution of observed effects solely to L6b. Verification of neuronal silencing is also unclear, and statistical inconsistencies between the main text and figures/tables make it difficult to effectively evaluate the text and stated outcomes.

Strengths:

(1) The text is well written.

(2) The authors are transparent about methodological details.

(3) The stated sleep, circadian, and orexin infusion experiments appear to be well designed, executed, and analyzed (with the exceptions of some statistical analyses detailed below).

Weaknesses:

(1) All outcomes are attributed specifically to L6b neurons, but the genetic manipulation is not specific to L6b neurons. The authors acknowledge this as a limitation, but in my view, this global manipulation is more than a limitation - it affects the overall interpretations of the data. The Hoerder-Suabedissen et al., 2018 paper shows sparse, but also dense, expression of Drd1a+ neurons in brain regions outside of the L6b. Given this issue, the results are largely overstated throughout the paper.

(2) It is not clear to me that the "silencing" of Drd1a+ neurons was verified.

(3) There were various discrepancies (and potentially misattributions) between the stated significant differences in Supplementary Table T1 data and Figure 3a & S2 spectral plots. This issue makes it difficult to effectively evaluate the main text and stated outcomes.

Related, the authors stated that post hoc comparisons of EEG spectral frequency bins were not corrected for multiple testing. Instead, significance was only denoted if changes in at least two consecutive frequency bins were significant. However, there are multiple plots in which a single significance marker is placed over an isolated bin (i.e., 4c, 6, S5, S6). Unless each marker is equivalent to 2 consecutive frequency bins, these markers should be removed from the plots. Otherwise, please define the frequency and size of these markers in the main text.

(4) A rainbow color scale, as in Figure 3, we've now learned, can be misleading and difficult to interpret. The viridis color scale or a different diverging color scale are good alternatives.

(5) How much time elapsed between vehicle/orexin A & B infusions?

(6) For Figure 6, there are statistical discrepancies between the main text and the plots (pg. 10):

a) The text claims post hoc differences for relative ORXA frontal EEG, but there are no significance markers on the plot.<br /> b) The text states that there were no post hoc differences for the relative ORXA occipital EEG, but significance markers are on the plot.<br /> c) The main test for the relative ORXB frontal EEG was not significant, but there are post hoc significance markers on the plot.<br /> d) For relative ORXB occipital EEG, there are significant markers on the plot outside of the stated range in the text.

(7) Some important details are only available in figure captions, making it difficult to understand the main text. For example, when describing Figure 3c in the main text on page 7, it is not clear what type of transitions are being discussed without reading the figure caption. Likewise, a "decrease," "shift," and "change" are mentioned, but relative to what? Similar comment for the EEG theta activity description on pages 7 - 8. Please add relevant details to the main text.

(8) Statistical comparisons for data in Figure 3e, post hoc analyses for data in Figure S7a-b REM data, and post hoc analyses for Figure S7c (not b) occipital EEG should be included to support differences claims. Please denote these differences on the respective plots.

(9) In the subsection titled "Layer 6b mediates effects of orexin on vigilance states (pg. 8)," there does not seem to be any stated differences between control and L6b silenced mice. A more accurate subtitle is needed.

Author response:

Public Reviews:

Reviewer #1 (Public review):

(1) All outcomes are attributed specifically to L6b neurons, but the genetic manipulation is not specific to L6b neurons. The authors acknowledge this as a limitation, but in my view, this global manipulation is more than a limitation - it affects the overall interpretations of the data. The Hoerder-Suabedissen et al., 2018 paper shows sparse, but also dense, expression of Drd1a+ neurons in brain regions outside of the L6b. Given this issue, the results are largely overstated throughout the paper.

We appreciate the reviewer’s careful reading and concern that some of our statements may have overstated the implications of our data. The Drd1a Cre mouse model used (FK164) has a relatively selective expression of Drd1a Cre in cortex, especially in layer 6b, but indeed some expression is seen in layer 6a and subcortically. We will nuance our claims throughout the paper to ensure that the conclusions are supported by our findings, and further discuss the impact of this limitation on the overall interpretation of our results. Specifically, we will discuss the potential contribution of relevant subcortical areas and layer 6a in the effects we observed.

(2) It is not clear to me that the "silencing" of Drd1a+ neurons was verified.

In our previous publications, we showed confirmation of the loss of regulated synaptic vesicle release from the Cre positive neuronal population (Marques-Smith et al., 2016; Hoerder-Suabedissen et al., 2018; Messore et al., 2024), which validates our approach to “silence” cortical neurons. We will discuss this further in the revised manuscript.

(3) There were various discrepancies (and potentially misattributions) between the stated significant differences in Supplementary Table T1 data and Figure 3a & S2 spectral plots. This issue makes it difficult to effectively evaluate the main text and stated outcomes.

We thank the reviewer for spotting the inconsistencies in how the statistical comparisons were presented: indeed, in the text we described two-way ANOVAs with posthoc tests but in the figures significance markers were positioned based on multiple t-tests. We have revised Supplementary Table T1, Figure 3a and S2 to ensure that all statistics are presented consistently throughout the manuscript, i.e. with two-way ANOVAs and accompanying posthoc tests.

Related, the authors stated that post hoc comparisons of EEG spectral frequency bins were not corrected for multiple testing. Instead, significance was only denoted if changes in at least two consecutive frequency bins were significant. However, there are multiple plots in which a single significance marker is placed over an isolated bin (i.e., 4c, 6, S5, S6). Unless each marker is equivalent to 2 consecutive frequency bins, these markers should be removed from the plots. Otherwise, please define the frequency and size of these markers in the main text.

In line with the previous comment, we have adjusted markers to reflect the results from posthoc tests after two-way ANOVAs in Figures 6 and supplementary figures S5 and S6. 

We thank the reviewer for pointing out that in our comparisons of EEG spectra, in some cases single isolated frequency bins, where p-value reached 0.05 were shown as significantly different, which indeed could have occurred by chance given that, in line with previous literature, we have not employed multiple testing comparison. In the revised manuscript we will use an unbiased approach by plotting actual p-values for all bins, and moderate our conclusions accordingly, while giving the readers the opportunity to evaluate the magnitude and extent of the differences directly, rather than relying on an arbitrary threshold for significance.

(4) A rainbow color scale, as in Figure 3, we've now learned, can be misleading and difficult to interpret. The viridis color scale or a different diverging color scale are good alternatives.

Thank you for pointing this out, we have adjusted the colour scale.

(5) How much time elapsed between vehicle/orexin A & B infusions?

There were 2-4 non-infusions days between infusions. We will add this information to methods when revising the manuscript.

(6) For Figure 6, there are statistical discrepancies between the main text and the plots (pg. 10):

a) The text claims post hoc differences for relative ORXA frontal EEG, but there are no significance markers on the plot.

b) The text states that there were no post hoc differences for the relative ORXA occipital EEG, but significance markers are on the plot.

c) The main test for the relative ORXB frontal EEG was not significant, but there are post hoc significance markers on the plot.

d) For relative ORXB occipital EEG, there are significant markers on the plot outside of the stated range in the text.

Thank you for your careful observations, these issues reflect the same inconsistency as raise above, where the text describes two-way ANOVAs and the figures refers to results obtained with multiple t tests. We shall adjust the markers in the figures to be only shown when the ANOVA is significant and show the results of posthoc tests after ANOVAs instead of the results of multiple t tests.

(7) Some important details are only available in figure captions, making it difficult to understand the main text. For example, when describing Figure 3c in the main text on page 7, it is not clear what type of transitions are being discussed without reading the figure caption. Likewise, a "decrease," "shift," and "change" are mentioned, but relative to what? Similar comment for the EEG theta activity description on pages 7 - 8. Please add relevant details to the main text.

We will adjust the wording in the main text to reflect more precisely which comparisons are shown in the figures.

(8) Statistical comparisons for data in Figure 3e, post hoc analyses for data in Figure S7a-b REM data, and post hoc analyses for Figure S7c (not b) occipital EEG should be included to support differences claims. Please denote these differences on the respective plots.

We have added the statistical comparisons for Figure 3e to the results section.

We have added the statistical comparisons for Figure S7A to the results section.

We have added the statistical comparison for Figure S7b to the results section.

In Figure S7c, there was an overall genotype difference, but there was not a time x genotype interaction, so we have not performed posthoc tests and did not plot posthoc significance markers for this figure. We have adjusted the wording in the results section to make this clearer.

We have adjusted the reference to the figure S7c which was incorrect, thank you for your careful attention.

(9) In the subsection titled "Layer 6b mediates effects of orexin on vigilance states (pg. 8)," there does not seem to be any stated differences between control and L6b silenced mice. A more accurate subtitle is needed.

We shall change the subtitle to: “The effects of orexin on vigilance states in L6b silenced mice”. The main finding described in this section is that the increase in EEG theta frequency after ORXB infusion is attenuated in L6b silenced mice, so a statement summarizing this finding could be an alternative title. However, then it would not accurately reflect other, less conspicuous, yet potentially important findings described in this section (during NREM sleep, only in L6b silenced animals there is an increase in power in the lower frequency bins in the frontal derivation; in the occipital derivation, levels of relative SWA during NREM sleep after ORXA infusion were lower in L6b silenced than in control animals).

Reviewer #2 (Public review):

Weaknesses:

(1) Although the authors used a highly selective approach to silence layer 6b neurons, the observed changes in EEG oscillations cannot be solely attributed to layer 6b neurons because of the ICV route for orexin administration.

We completely agree, and did not want to imply that orexin administered through the ICV route reaches cortical Drd1a Cre expressing neurons only. We will re-word the corresponding sentences accordingly throughout the manuscript.

(2) The rationale for using only male rats is not provided.

We agree that this is an important limitation and will acknowledge and discuss it further in the revised manuscript. Unfortunately, our experimental protocol precluded the possibility of monitoring accurately the oestrous cycle, which as well-known has an influence on sleep-wake architecture, brain oscillations as well as orexin signalling and receptor abundance. We therefore decided to use male mice only for the current study, but planning to use both sexes in our follow up work.

eLife Assessment

This paper reports a valuable discovery that specific-mode electroacupuncture (EA) transiently opens the blood-brain barrier (BBB) in rats. The evidence is solid but lacks functional validation of BBB permeability changes. The work will be of interest to medical scientists working in the field of electroacupuncture and drug delivery.

Reviewer #1 (Public review):

Summary:

The work from this paper successfully mapped transcriptional landscape and identified EA-responsive cell types (endothelial, microglia). Data suggest EA modulates BBB via immune pathways and cell communication. However, claims of "BBB opening" are not directly proven (no permeability data).

Strengths:

First scRNA-seq atlas of EA effects on BBB, revealing 23 cell clusters and 8 cell types. High cell throughput (98,338 cells), doublet removal, and robust clustering (Seurat, SingleR). Comprehensive bioinformatics (GO/KEGG, CellPhoneDB for ligand-receptor interactions). Raw data were deposited in GEO (GSE272895) and can be accessed.

Weaknesses:

(1) No in vivo/in vitro assays confirm BBB permeability changes (e.g., Evans blue leakage, TEER).

(2) Only male rats were used, ignoring sex-specific BBB differences.

(3) Pericytes and neurons, critical for the BBB, were not captured, likely due to dissociation artifacts.

(4) Protein-level validation (Western blot, IHC) absent for key genes (e.g., LY6E, HSP90).

(5) Fixed stimulation protocol (2/100 Hz, 40 min); no dose-response or temporal analysis.

Reviewer #2 (Public review):

Summary:

This study uses single-cell RNA sequencing to explore how electroacupuncture (EA) stimulation alters the brain's cellular and molecular landscape after blood-brain barrier (BBB) opening. The authors aim to identify changes in gene expression and signaling pathways across brain cell types in response to EA stimulation using single-cell RNA sequencing. This direction holds promise for understanding the consequences of noninvasive methods of BBB opening for therapeutic drug delivery across the BBB.

Strengths:

(1) The study addresses an emerging and potentially important application of noninvasive stimulation methods to manipulate BBB permeability.

(2) The dataset provides broad transcriptional profiling across multiple brain cell types using single-cell resolution, which could serve as a valuable community resource.

(3) Analyses of receptor-ligand signaling and cell-cell communication are included and have the potential to offer mechanistic insight into BBB regulation.

Weaknesses:

(1) The work falls short in its current form. The experimental design lacks a clear justification, and readers are not provided with sufficient background information on the extent, timing, or regional specificity of BBB opening in this EA model. These details, established in prior work, are critical to understanding the rationale behind the current transcriptomic analyses.

(2) Further, the results are often presented with minimal context or interpretation. There is no model of intercellular or molecular coordination to explain the BBB-opening process, despite the stated goal of identifying such mechanisms. The statement that EA induces a "unique frontal cortex-specific transcriptome signature" is not supported, as no data from other brain regions are presented. Biological interpretation is at times unclear or inaccurate - for instance, attributing astrocyte migration effects to endothelial cell clusters or suggesting microglial tight junction changes without connecting them meaningfully to endothelial function.

(3) The study does include analyses of receptor-ligand signaling and cell-cell communication, which could be among its most biologically rich outputs. However, these are relegated to supplementary material and not shown in the leading figures. This choice limits the utility of the manuscript as a hypothesis-generating resource.

(4) Overall, while the dataset may be of interest to BBB researchers and those developing technologies for drug delivery across the BBB, the manuscript in its current form does not yet fulfill its interpretive goals. A more integrated and biologically grounded analysis would be beneficial.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The work from this paper successfully mapped transcriptional landscape and identified EA-responsive cell types (endothelial, microglia). Data suggest EA modulates BBB via immune pathways and cell communication. However, claims of "BBB opening" are not directly proven (no permeability data).

(1) No in vivo/in vitro assays confirm BBB permeability changes (e.g., Evans blue leakage, TEER).  

(2) Only male rats were used, ignoring sex-specific BBB differences.

(3) Pericytes and neurons, critical for the BBB, were not captured, likely due to dissociation artifacts.

(4) Protein-level validation (Western blot, IHC) absent for key genes (e.g., LY6E, HSP90).

(5) Fixed stimulation protocol (2/100 Hz, 40 min); no dose-response or temporal analysis.

(1) We sincerely apologize for the oversight regarding the description of changes in blood-brain barrier permeability. In fact, our team conducted a series of preliminary studies that verified this aspect, but we did not provide a more detailed introduction in the introduction section. We will address and improve this in the revised manuscript. (2) We are very grateful to the reviewers for pointing out the important and meaningful issue of "gender-specific BBB differences." We will make this a focal point in our future research.

(2) As for pericytes and neurons, we acknowledge their importance in the function of the blood-brain barrier. We acknowledge the importance of pericytes and neurons in the blood-brain barrier. However, neurons are absent because our sample processing method involves dissociation. During the dissociation procedure, neuronal axons, which are relatively long, are filtered out during the frequent cell suspension steps and cannot enter the downstream microfluidic system for analysis, so they are not present in our data. Since this experiment is primarily focused on non-neuronal cells, we did not choose to use nucleus extraction for sample processing. As for pericytes, we believe they are not captured because their proportion in our samples is extremely low, which is why they are not present in the data. Further research may require single-nucleus transcriptomics or the separate isolation of these two cell types for study. Of course, in our current mechanistic studies, we are also fully considering the important roles these two cell types play in BBB function.

(3) In addition, for verification at the protein level, we have recently conducted some experiments and will include these results in the revised version.

(5) Lastly, regarding our electroacupuncture intervention model, we actually conducted a series of parameter optimization experiments during the preliminary exploration phase. This part is indeed lacking in our current introduction, and we will add it to the research background and introduction.

Reviewer #2 (Public review):

Summary:

This study uses single-cell RNA sequencing to explore how electroacupuncture (EA) stimulation alters the brain's cellular and molecular landscape after blood-brain barrier (BBB) opening. The authors aim to identify changes in gene expression and signaling pathways across brain cell types in response to EA stimulation using single-cell RNA sequencing. This direction holds promise for understanding the consequences of noninvasive methods of BBB opening for therapeutic drug delivery across the BBB.

(1) The work falls short in its current form. The experimental design lacks a clear justification, and readers are not provided with sufficient background information on the extent, timing, or regional specificity of BBB opening in this EA model. These details, established in prior work, are critical to understanding the rationale behind the current transcriptomic analyses.

(2) Further, the results are often presented with minimal context or interpretation. There is no model of intercellular or molecular coordination to explain the BBB-opening process, despite the stated goal of identifying such mechanisms. The statement that EA induces a "unique frontal cortex-specific transcriptome signature" is not supported, as no data from other brain regions are presented. Biological interpretation is at times unclear or inaccurate - for instance, attributing astrocyte migration effects to endothelial cell clusters or suggesting microglial tight junction changes without connecting them meaningfully to endothelial function.<br /> (3) The study does include analyses of receptor-ligand signaling and cell-cell communication, which could be among its most biologically rich outputs. However, these are relegated to supplementary material and not shown in the leading figures. This choice limits the utility of the manuscript as a hypothesis-generating resource.

(4) Overall, while the dataset may be of interest to BBB researchers and those developing technologies for drug delivery across the BBB, the manuscript in its current form does not yet fulfill its interpretive goals. A more integrated and biologically grounded analysis would be beneficial.

(1) It was indeed our mistake that we did not pay attention to the importance of research background factors such as the degree, timing, or regional specificity of BBB opening for the rationale and purpose of this experimental design. In our revision, we will thoroughly elaborate on the relevant previous studies.

(2) Our current study is actually based on previous findings that electroacupuncture can open the BBB, with a more pronounced effect observed in the frontal lobe (this aspect should be further described in the research background). Building on this foundation, our aim is to delineate the potential biological mechanisms involved. Therefore, we selected frontal lobe tissue as our primary choice for sequencing and have not yet investigated differences across other brain regions, although this may become a focus of future research. Additionally, we recognize that the mechanism underlying BBB opening is complex, and at present, we cannot determine whether it is driven by a single direct factor or by coordinated actions between cells or molecules. As such, our results are presented only briefly for now, and we will carefully consider whether to supplement our findings by incorporating insights from other studies.

(3) Thank you very much for bringing this to our attention. We will include the key results of the receptor-ligand signaling and cell-cell communication analysis in the main manuscript.

(4) Indeed, our current dataset and analysis tend to present objective data results. We are also conducting a series of validations that may be related to the biology of the blood-brain barrier, and we look forward to sharing and discussing any future research findings with you and everyone.

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Reply to the reviewers

Proposed revision plan

Based on the below reviews, we propose the following revision plan. Briefly:

• We will remove the functional data on TGFβ signaling and mechanical loading/mechanosensing. We agree with the reviewers that we would need to generate additional histological and molecular data from conditional knockout mice, antibody and (ant)agonist treatments and the optogenetic model to determine their exact involvement in lining macrophage maturation. These experiments require significant time and other resources.
• We would therefore like to uncouple this question for a follow-on manuscript.We will re-focus the manuscript on the developmental data providing a molecular and cellular blueprint of lining macrophage development. This will include our data on CSF1 as a key signal. The novelty and relevance of our developmental data have been highlighted by all three reviewers, and they have also praised the rigor of these experiments and their interpretation. We thus believe that this re-focus will improve the manuscript message.
• To further enhance this, we are proposing to include additional data delineating the developmental dynamics of synovial fibroblasts. We have generated an in-depth single cell RNAsequencing dataset but did not include fibroblast-specific analyses in the original manuscript. This is not a change proposed by the reviewers, but we are proposing this because we believe this would be an impactful addition to a revised version of our study, providing data also on the maturation of the synovial (lining) macrophage niche.
• We will otherwise respond to all individual reviewer comments and implement the requested changes, unless technically not possible. Please find below detailed point-by-point answers.

Reviewer #1

Evidence, reproducibility and clarity

In their manuscript entitled "The synovial lining macrophage layer develops in the first weeks of life in a CSF1- and TGFβ-dependent but monocyte-independent process," the authors explore the developmental trajectory of synovial lining macrophages. They demonstrate that the formation of this specialized macrophage layer is age-dependent and governed by a distinct developmental program that proceeds independently of circulating monocytes. Through scRNA-Seq, the authors show that synovial lining macrophages originate locally from Aqp1⁺ macrophages and are marked by the expression of Csf1r, Tgfbr, and Piezo1. Notably, genetic ablation of each of these factors impaired the development of lining macrophages to varying degrees, suggesting differential contributions of CSF1, TGFβ, and PIEZO1 signaling pathways to their maturation and maintenance.

The manuscript is well written, and the data quality and representation is of a high standard. The authors have employed a sophisticated array of state-of-the-art mouse models and cutting-edge technologies to elucidate the developmental origin of synovial lining macrophages. Notably, the supporting scRNA-Seq datasets are of excellence and provide valuable insights that will likely be of significant interest to researchers in the field of immunology and joint biology. Accordingly, the experimental approach and interpretations regarding macrophage origin are well-founded and compelling. However, in the eye of the reviewer, the section addressing the underlying molecular mechanisms is a bit less convincing. This part of the study appears slightly underdeveloped, and some of the mechanistic claims lack sufficient experimental clarity. A more rigorous experimental investigation would be essential to reinforce the manuscript's conclusions, particularly concerning the data related to Tgfbr and Piezo1, where the current evidence appears insufficiently substantiated.

We thank the reviewer for their positive and constructive evaluation of our manuscript. We agree with them (and the other reviewers) that our functional data on the involvement of TGFβ signaling and mechanical loading/mechanosensing are comparably less convincing and substantiated than our developmental data. We are very grateful for their (and the other reviewers’) suggestions to provide more support for the involvement of these factors in lining macrophage development. However, we think that carrying this out to the same high standard will require substantial time and other resources. We have therefore decided to uncouple this from the developmental data and pursue this in follow-up work. We will re-focus the current manuscript on the developmental data. We have proposed to the editors to instead include additional data on synovial fibroblast development, to complement our macrophage data and also delineate the maturation of their niche, thereby providing a conclusive developmental atlas.

Major point:

1. The numbers of VSIG4⁺ macrophages appear either unaffected or only minimally altered in both Csf1rMerCreMer Tgfbr2floxed and Fcgr1Cre Piezo1floxed mouse models, respectively. This raises an important question: was the gene deletion efficiency sufficient in each model? Accordingly, the authors are encouraged to include quantitative data on gene deletion efficiency for both mouse models, as this information is critical for interpreting the observed phenotypic outcomes and validating the conclusions regarding gene function. Furthermore, to better assess the impact of Tgfbr2 and Piezo1 disruption, the authors should provide more comprehensive flow cytometry analyses and histological data for these mouse models. Given the apparent homogeneity of VSIG4⁺ macrophages (as shown by the authors themselves), bulk RNA-Seq of sorted Tgfbr2- and Piezo1-deficient VSIG4⁺ macrophages (or from TGFβ-treated animals) would offer valuable insights into both the effectiveness of gene deletion and the molecular pathways governed by TGFβ and PIEZO1 in lining macrophages.

As outlined above, we have decided to uncouple our functional data on TGFβ, Piezo1 and mechanical loading. The points raised here are all very valid, and we will implement your suggestions in our follow-up functional work focusing on signaling events regulating lining macrophage development. On the suggestion to perform bulk RNA sequencing for VSIG4+ macrophages: This is a good one in principle – although we will not be able to use this strategy where we want to assess the consequences of experimental treatments or genetic models on lining macrophage maturation, because acquisition of VSIG4 is a key maturation event that might be impaired in these conditions.

Minor points:

Consistent usage of Cx3cr1-GFP+ nomenclature (for instance: Fig. S1 legend "adult mouse synovial tissue, showing PDGFRα⁺ fibroblasts (yellow) and CX3CR1-GFP⁺ cells (cyan)." versus Fig. 1 legend "Automated spot detection highlights Cx3cr1-GFP⁺ macrophages)".

We will implement these changes.

Unclear Fig. 3 legend: "Representative immunofluorescence images of synovial tissue from Clec9aCre:Rosa26lsl-tdT mice at 3 weeks and in adulthood, showing and tdTomato (yellow) and stained for DAPI (blue), VSIG4 (cyan)" Check 'showing and tdTomato.'

We will implement these changes.

For greater clarity, it would have been helpful if the transcript names had been directly included within Figures 3C, S3A, and S3C.

We will implement these changes.

Page 24: "(Mki67CreERT2:Rosa26lsl-tdT)" Last bracket not superscript.

We will implement these changes.

Page 25: "we again leveraged our scRNAsequencing dataset" Missing punctuation.

We will implement these changes.

Page 27: Fig. 5C legend: " of synovial tissue of 1 week-old, 3 weeks-old and adult mice." Please specify and change to 'adult Csf1rΔFIRE/ΔFIRE mice'.

We will implement these changes.

Page 30: The outcome observed in the Acta1-rtTA:tetO-Cre:ChR2-V5fl mouse model appears to be inconclusive: "This approach resulted in an increased density of VSIG4+ and total (F4/80+) macrophages in the exposed leg of some 5 days-old pups, but others showed the opposite trend (Figure S5D)." This variability may reflect low efficiency of the model or other technical limitations (e.g. muscle contractions frequency or time point of analysis). Given this ambiguity, it is worth reconsidering whether the data are sufficiently robust to warrant inclusion. Should the authors choose to include these findings, further experimentation of appropriate depth and precision is required to allow a conclusive interpretation (either it increases the density of VSIG4+ macrophages or not). The same applies to the Yoda1-treated mice, for which additional data are needed to determine whether VSIG4⁺ macrophage density is truly affected.

We have decided to remove the data on the optogenetic mouse model and Yoda1 treatment and follow-on separately, implementing these suggestions, including proof of concept data for optogenetically induced muscle contractions.

Significance

General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed? This is a well-designed study that uses cutting-edge methodologies to investigate the developmental trajectory of synovial lining macrophages under homeostatic conditions. The authors present robust experimental evidence and compelling interpretations concerning synovial macrophage origin, which are both well-substantiated and impactful. Nonetheless, from the reviewer's perspective, the section exploring the molecular mechanisms underlying macrophage differentiation is comparatively less convincing. This section appears somewhat underdeveloped, as some of the mechanistic claims lack sufficient depth and experimental rigor to fully substantiate the conclusions.

Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field: In contrast to earlier studies (PMID: 31391580, 32601335), the inclusion of fate-mapping experiments adds an important dimension, offering novel insight into the ontogeny of synovial macrophages. This expanded perspective may prove particularly valuable in advancing our understanding of joint immunology, especially regarding the local origins and lineage relationships of macrophage populations.

Furthermore, the authors present novel insights into the molecular pathways underlying the differentiation and development of synovial lining macrophages. By demonstrating previously unrecognized regulatory mechanisms, this work significantly deepens our understanding of the cellular and transcriptional programs that drive macrophage specialization within the joint microenvironment.

Place the work in the context of the existing literature (provide references, where appropriate): This study builds upon previous work characterizing the macrophage compartment in the joint (PMID: 31391580, 32601335), yet provides a substantially more comprehensive dataset that spans multiple developmental time points and data on the origin of this specialized macrophage subset.

State what audience might be interested in and influenced by the reported findings: Immunologist, clinicians

Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. This study falls well within the scope of the reviewer's expertise in innate immunity.

Reviewer #2

Evidence, reproducibility and clarity

In the manuscript „The synovial lining macrophage layer develops in the first weeks of life in a CSF1- and TGFβ- dependent but monocyte-independent process", Magalhaes Pinto and colleagues carefully employ a wide range of technologies including single cell profiling, imaging and an exceptional combination of fate mapping models to characterize the ontogeny and development of lining macrophages in the joint, thus dissecting their maturation during postnatal development. Over the last decade, several landmark studies highlighted the imprinting of tissue-resident macrophages by a combination of ontogenetic and tissue-specific niche factors during development. So far, the ontogeny and the tissue niche factors governing the development and maturation of lining macrophages have not been described. Therefore, the results of this study offers insights on a small highly adapted macrophage population with relevance in many disease settings in the joint. Furthermore, the findings are nicely showcasing how macrophages are specializing to even very small tissue niches across development within one bigger anatomical compartment to serve dedicated functions within this niche.

This manuscript is beautifully written and highlights many novel, highly relevant findings on lining macrophage biology and the authors employ a wide range of different technologies to carefully dissect the postnatal development of lining macrophages.

In particular, the combination of scRNA-seq and fate mapping is providing a unique the link of transcriptional programs to ontogeny within the tissue niche. Furthermore, the integrative use of distinct fate mapping strategies, transgenic mouse lines, and treatment paradigms to elucidate key niche factors guiding the development and maturation of lining macrophages provides many interesting findings and data that are highly relevant to the field. I really enjoyed reading this manuscript.

Thank you for your complimentary and constructive assessment of our manuscript, and the detailed comments below, which are very helpful. Please find point-by-point responses below.

Major points:

The authors show dynamic regulation of VSIG4 in lining macrophages during development, therefore VSIG4 is maybe not an ideal choice for gating strategies to define lining macrophages or to show as a single markers in immunofluorescence (IF) stainings to demonstrate their abundance across development (even though it is clear that this is the reason why the F4/80 staining is shown next to it). To demonstrate the increase of lining macrophages during development in IF, it would be more helpful if the authors would show quantifications of all F4/80+ cells and additionally VSIG4+ as a proportion of F4/80+ cells (or VSIG4+ F4/80+ and all F4/80+ in a stacked bar plot). We agree with the assessment of VSIG4 not being ideal since this is a key marker of mature lining macrophages only.

We will provide these additional analyses.

In Figure 1C, the authors nicely demonstrate that the lining macrophages get closer in their distance across development to build the epithelial-like macrophage structure along the adult lining. Is the close proximity between lining macrophages already fully "matured" at 3 weeks of age and comparable to adults? Please quantify the distance in adult linings.

We will provide data for adult joints.

Can the authors explain how the grouping was performed between the analyzed human fetal joints? It is not clear why the cut was chosen between the groups at 16/17 weeks of age. Maybe it would be also beneficial if the authors would consider not grouping these samples but rather show the specific quantifications for each samples individually and estimate via linear regression the expansion over time across human development. Furthermore, can the authors give additional information about the distancing of lining macrophages in the human fetal samples, it would be great to see if they follow the same dynamics as in mouse. Maybe comparison to human juvenile/adult joints would also add on to substantiate the findings in human samples (if possible).

We will show samples ungrouped and perform linear regression analysis as suggested.

The scRNA-seq analysis leaves several questions open and some conclusions and workflows cannot be easily followed.

We appreciate this comment and the complexity of the data, and will implement the below recommendations, and clarify the issues raised.

It is not clear how and especially why the signature genes to define macrophages vs. monocytes were chosen. Especially as the signature genes for monocytes would not include patrolling monocytes and the macrophage signature genes seem to be highly regulated during development, see also Apoe expression in NB vs. adult in Figure S2e. Why did the authors not take classical markers such as Itgam, Fcgr1a, Csf1r?

Can dendritic cell signatures be excluded? Cluster 11 and 12 show indeed some DC markers, are these really macrophages?

The authors provide several figure panels showing TOP marker genes or key marker genes for the identified clusters, however it is not clear if these are TOP DE genes or if the genes were hand chosen. Somehow, the authors give the impression that the clusters were chosen and labeled not based on DE genes, but more on existing literature that previously reported these macrophage populations. DE gene lists for all annotated cell types and macrophage clusters need to be provided within the manuscript.

The authors claim that Clusters 1 and 4 are "developing" macrophages. How is this defined? Why are these developing cells compared to other clusters? And why are these clusters later on not considered as progenitors of Aqp1 macrophages and Vsig4 macrophages? Why are Aqp1+ macrophages not labeled as developing when they are later on in the manuscript shown as potential intermediate progenitors of lining macrophages?

Furthermore, it is again confusing that markers are used throughout Figure 2 which are labeled as "key marker genes" for a population and then later on they are claimed to be regulated during development within this population, see for example Figure 2D and 2H.

It is appreciated that the authors distinguished cycling clusters such as 8, 9, and 10 based on their cycling gene signature. Here it would be very exciting to see a cell cycle analysis across all clusters and time points to see when exactly the cells are expanding during development; this would also substantiate the data later shown for the Mki67-CreERT2 mouse model.

Can the authors identify certain gene modules during development of lining macrophages (and/or their progenitors) which are associated with certain functions (e.g. GO terms, GSEA enrichment)?

To determine the actual presence of the identified macrophage clusters from the scRNA-seq as macrophage populations in the joint, the authors should perform IF or FACS for key markers. Especially, Aqp1+ macrophages should be shown in the developing joint.

We will provide additional data, but would also like to reference a study by collaborators currently in revision at Immunity, which characterizes the Aqp1+ population in detail. We are hoping to have a doi available during our revision process.

The authors used a wide range of fate mapping models, which is quite unique and highly appreciated. The obtained results and the conclusions made from the models raise a couple of questions: Whereas contribution of HSC-derived/monocyte-derived macrophages to the lining compartment seems to be minor, there is still labeling across different models. Various aspects would need to be clarified.

We will clarify these data throughout as per below suggestions.

For example, the authors employ Ms4a3-Cre as a tracing model for GMP-derived monocytes, however all quantifications of the labeling efficiency are not normalized to the labeling in monocytes or another highly recombined cell population. This should be shown, similar to the other fate mapping models (Figure 3 F-I).

Labelling efficacy for Ms4a3-Cre is near complete for GMP-derived monocytes (and neutrophils) with the Rosa-lsl-tdT (aka Ai14) reporter we have used (see also PMID: 31491389 and doi: 10.1101/2024.12.03.626330); but we will include normalized data as requested.

Please show Ms4a3 expression across clusters across time points, to exclude expression in fetal-derived clusters.

We will include this in the revised supplementary information, but there is indeed very little at birth (in line with the original report for other tissues PMID: 31491389).

In line with the question raised above, if the authors can exclude a development of the Egfr1+ and Clec4n+ developing macrophages into Aqp1+ macrophages and subsequently into Vsig4 lining macrophages, the obtained data from the Ms4a3-Cre model highly suggests a correlative labeling across these clusters what could implicate a relation. However, the authors do not discuss throughout the manuscript the role of these developing macrophages. It is highly encouraged to include this into the manuscript and it would be of high relevance to understand lining macrophage development.

This is an interesting point and we agree it deserves consideration in the revised manuscript. Indeed, our trajectory analyses do not predict differentiation of the Egfr1+ and Clec4n+ developing macrophages into Aqp1+ macrophages, and hence, ultimately lining macrophages. Conversely, Aqp1+ cells might also convert into Egfr1+ and Clec4n+ developing macrophages. We will elaborate on this more in the revised manuscript.

The authors conclude from the pseudo bulk transcriptomic profiling of the different macrophage clusters that TdT+ and TdT- macrophages do not differ in their gene expression profile and that this is due to niche imprinting rather than origin imprinting. Even though the data supports that conclusion, the authors should verify if inkling cells early during development also show this similar gene expression profile and gene expression should be compared at the different developmental time points. Tissue niche imprinting is happening within the niche during development, most likely in a stepwise progress, and therefore there should be differences in the beginning.

This is another important point that we will address in the revised manuscript by performing additional differential gene expression analyses at the different developmental time points, including the earliest stages, as suggested.

The trajectorial analysis using different pseudotime pipelines is very interesting and nicely points out the potential role of Aqp1 macrophages as intermediates of Vsig4 lining macrophages. From my point of view, all trajectories seem to suggest that Egfr1 developing macrophages and Clec4n developing macrophages might differentiate into Aqp1 macrophages, however the authors are not exploring this further and the role of both developing macrophage clusters is not further discussed (see also comments above).

We will address and discuss this in the revised manuscript.

How was the starting point of the trajectorial analyses defined and is it the same for each pipeline used?

We will clarify this in the revised manuscript.

Are there potentially two trajectories? It looks like there is one in the beginning of postnatal life and a second one appearing from the monocyte-compartment later in life. If this is true, that would rather speak for a dual ontogeny of Vsig4+ macrophages, wouldn't it?

We will discuss this in the revised manuscript.

A heatmap (transcriptional shift) of trajectories between more clusters should be shown at least for Cluster 0,1,2, and 3. It is not sufficient to demonstrate this only between two clusters.

We will add these analyses during revision.

To show the similarity between Aqp1 macrophages and proliferating macrophage clusters, the authors should remove the cycling signature and compare these clusters to show that the cycling cells might be Aqp1 macrophages or earlier developing macrophage progenitors aka Clec4n or Egfr1 macrophages.

We will address this in the revised manuscript.

The conclusions made from the Mki67-CreERT2 data are a bit difficult to understand, whereas all progenitors (monocyte progenitors and macrophage progenitors will proliferate at the neonatal time point and no conclusions can be made if the cells expand in the niche. The authors should employ Confetti mice or other models (Ubow mice) to analyze clonal expansion in the niche.

We agree that interpretation of the Mki67-CreERT2 data is complicated by labeling of other cells, and notably, labeling observed in BM-derived cells. We will highlight this better in the revised manuscript. We have tried using Ubow mice to address this issue, but the recombination efficacy we yielded was too low to draw conclusions. We will address this during revision.

All predicted cell-cell interactions between macrophages and fibroblasts should be provided in a supplementary table. Are the interactions shown in Figure 5 chosen interactions or the TOP predicted ones? Whereas the authors show different numbers of interactions, it is most likely hand-picked and therefore biased.

We will provide a full list of all predicted interactions in the revised supplementary material in addition to a list of the full differential gene expression analysis.

The authors further aim to dissect the factors involved in the developmental niche imprinting of lining macrophages. Even though it is highly appreciated that the authors used so many experimental setups to show the reliance of lining macrophages on Csf1 and TGF-beta as well as mechanosensation, the wide range of models the different methods used and selected developmental time points make it very difficult to really interpret the data. The authors should carefully choose time points and methods (either FACS analysis across all models or IF across all, or both). Often deletion efficiencies for transgenic models and proof of concept that the inhibitors and agonists are working in the treatment paradigm are not provided. For example, Csf1rMer-iCre-Mer Tgfbr2fl/fl mice are used but no deletion efficiency is shown or different time points of analysis, maybe the macrophages are not properly targeted in the set up.

We have decided to uncouple our experimental data on Tgfb, Piezo1 and mechanosensing/mechanical loading, but are taking this into consideration for revision. In many cases, we have in fact performed flow cytometry and imaging analyses, and agree, we should be showing this consistently.

The authors have shown the role of Csf1 and Tgfbr2 only for lining macrophages, is this specific in the joint to this population of are subliming macrophages affected in a similar manner.

We will include data on sublining macrophages in the revised figure (for CSF1; Tgfb data will be uncoupled from this current manuscript).

Can the authors confirm their results in CSF1R-FIRE mice with anti-Csf1 injections or in Csf1op/op mice?

We will expand our discussion of the Csf1 findings, and will consider including anti-CSF1 data during revision. Phenotypes on other Csf1(r) deficient mice are published, if not with the same developmental resolution as our time course in Csf1rFIRE knockout mice and with simpler readouts. Csf1op/op mice are indeed deficient in synovial lining macrophages, from 2 days of age onwards (PMID: 8050349), and lining macrophages are also absent from 2-weeks-old and adult Csf1r-/- mice (PMID: 11756160).

The setup in Figure S5G is very interesting to test the role of movement and mechanical load on the joint, however, there is basically no data on the model provided showing the efficiency of the induced optogenetic muscle contractions, and only one time point is shown.

Data on mechanical loading will be uncoupled from the current manuscript and substantiated in a separate follow-up.

The results regarding the role of Piezo1 and mechanosensation vary a lot. Could it be that analyses were done too early or that actually proper weight load on the joint must be applied for the maturation of the macrophages? The authors should test this to.

We will uncouple these data from the current manuscript during revision. However, this is a possibility that we have discussed. In fact, the most appropriate experimental approach to address the involvement of mechanical loading, onset of walking and specifically, weight bearing would be a loss-of-function approach (i.e. paralysis at the newborn stage), for which we unfortunately could not obtain ethics approval from the UK Home Office.

The Rolipram experiment is shown in Figure S5G, but is not described in the result section. It only appears at some point in the discussion part. The authors should move it to results or remove it from the manuscript.

We will incorporate these data with the revised section on developing synovial macrophage populations.

Minor points:

Please reference the Figure panels in numeric order throughout the text.

We will change this where not the case.

Figure 2a and 2b are a bit out of the storyline, it is not obvious why this is shown here and maybe it would be good to move it to the supplements. Gating strategy is also not used for scRNA-seq. Therefore, it would better fit to the later analysis of joint macrophages across different transgenic mouse models and treatment paradigms. The gating strategies are changing across different experiments throughout the figures, it would be nice to have a similar gating strategy for all experiments, see also Figure 3 where the defining markers for joint macrophages are changing between models.

We will revise Figures 2, 3 and the related supplementary figures.

A lot of figure panels have very small labeling that is basically unreadable. Axes at FACS plots for example. Sometimes, it is even impossible to distinguish cluster labels especially when they have similar colors.

We will revise this, thanks for pointing it out.

In the text on page 14, many markers are named which are specifically regulated during development in lining macrophages, but these factors are not labeled anywhere in the volcano plot. It would be good to showcase at least some of these named genes in the figure panel, e.g. Trem2.

We will do this for revision.

Figure 2F and Figure S2F are really nicely showing the percentage of cells per cluster in each analyzed biological sample. Maybe the authors could additionally consider to show a stacked bar plot with the mean percentage of cells per cluster and how the clusters are distributed across time points?

We will include this in the revised manuscript.

Figure 3A: IF for adult lining macrophages and the quantification are missing.

This will be included in the revised version.

Significance

This manuscript highlights novel, highly relevant findings on lining macrophage biology and the authors employ a wide range of different technologies to carefully dissect the postnatal development of lining macrophages. Furthermore, this study showcases in a very elegant and detailed way the adaptation of macrophage progenitors to a highly specific anatomical tissue niche.

The manuscript is of high interest to basic scientists focussing on macrophage biology and immune cell development and clinicians and clinician scientists focussing on joint diseases such as RA.

Therefore the manuscript is of interest to a wide community working in immunology.

Reviewer #3

Summary:

Magalhaes Pinto, Malengier-Devlies, and co-authors investigated the developmental origins and maturation of synovial (lining and sublining) macrophages across embryonic, newborn, and postnatal stages in mouse. The authors used multiple transgenic reporter lines, lineage tracing, scRNA-seq, 2D confocal and 3D lightsheet imaging, and perturbations to delineate the macrophage states and ontogeny. They propose a model in which the majority of the joint lining macrophages has a fetal (EMP-derived) origin and a small proportion has a definitive HSC-derived monocyte origin, which both seed and mature within the synovial space in the postnatal period in the first 3 weeks of life. Using cell-cell communication analysis on their scRNA-seq data, they identified Fgf2, Csf1, and Tgfb as candidate signaling pathways that support (lining) macrophage development and maturation. Functional experiments indicate that the process is CSF1 and TGFb-dependent and also partly dependent on mechanosensing through Piezo1.

The key conclusions on the composition of the synovial macrophages are convincing based on the presented results, and are carefully phrased. The study is very comprehensive, yet the description and organization of the results of the different mouse models could be altered to improve the storyline. Several refinements in data presentation, formulation, and minor validation experiments would further improve the clarity of the story, as well as summary recaps of the major findings throughout the text.

We thank this reviewer for their detailed review. We will be implementing the requested changes wherever technically feasible.

Major comments:

Generally, the story could be more streamlined by introducing earlier reporter lines and lineage-origin logic. Clearly state which reporter/CreERT2 lines and acrosses are used. It was unclear in Figure 2 that cells of the cross of the Cx3cr1-GFP and Ms4a3Cre:Rosa26lsl-tdT reporter lines were used for the scRNA-seq. The principle that there are fetal-derived and bone marrow (GMP)-derived monocytes and macrophages doesn't need to be "hidden" until Figure 3. For example, also the imaging of Ms4a3Cre could be introduced before the scRNA-seq.

We will revise the structure and order of the manuscript during revision.

Figure 1 could benefit from a cartoon visualizing the anatomy of the knee joint. The terms "sublining" and "synovium" are now a bit unclear, as it appears that sometimes the synovium is indicated as sublining and vice versa. Additionally, a schematic developmental timeline could be added to indicate the parallels between mouse and human development (fetal and postnatal development in mouse versus gestational age in human). Also, the various waves of hematopoiesis could be indicated in this timeline, which would be particularly helpful for Figure 3 for the lineage-tracing readouts. Lastly, the authors could end the manuscript (a new Figure 6) with a general cartoon summarizing all the results presented.

We will include illustrations as suggested.

Figure 1 could be rearranged: first introduce the markers CX3CR1 and VSIG4 (Figure 1D) and then present the quantifications (Figure 1B/E). Where possible, co-visualization CX3CR1-GFP and VSIG4 on tissue sections to strengthen the claims on the relationship between these 2 markers. Tying the scRNA-seq insights (Figure 2) to the imaging would be elegant. Moreover, it would be informative to represent the CX3CR1+ and VSIG4+ macrophages as a percentage of F4/80+ macrophages (Figure 1B/E). Similarly, for the flow cytometry data in Figure 2, the relationship between the markers CX3CR1 and VSIG4 on macrophages could be more clearly displayed and discussed.

Thanks for this remark. We will endeavor to show co-localization and analysis of both markers wherever possible. However, where we did not use Cx3cr1gfp mice, co-staining was limited by antibody choice.

The 3D imaging of the joint is a nice addition to the manuscript, as it provides more context to the anatomical structure; however, while the text suggests several newborn joints were imaged, Figure 1F visualizes (again) the knee joint. Could other joints also be represented by 3D imaging? If the knee joint is the only joint available for imaging, and previous confocal imaging focused specifically on the meniscus in the knee joint, could the meniscus also be highlighted in the lightsheet imaging?

Apologies if this was not clear from the original manuscript text, but we have only imaged the knee joint in 3D. We will clarify this during revision and consider inclusion of additional imaging data.

Clarification is requested regarding the imaging quantification representation. The M&M section under "Statistical analysis and reproducibility" states that individual data points are displayed, and bars represent the mean. However, some of the Figure legends (e.g., Figures 1B and S1C) specify that each dot corresponds to an individual mouse, with quantification based on 2-3 sections per mouse. While this appears to be a very reasonable representation of the data, does this mean that for each dot, the mean value from the 2-3 sections per mouse was calculated and plotted?

We will clarify this.

It is not clear how the differential expression analysis was performed on the Vsig4+ cells. Please specify if Cluster 0 was used for analysis, or all Vsig4-expressing cells? Not all cells in Cluster 0 have Vsig4+ expression. The authors described the expression dynamics of Aqp1 as intriguing, but lack a reasoning on why this is interesting.

We will revise this section.

Figure S3E: In line with the previous comment, can the authors justify that the tdTomato+/- comparisons are not biased by scRNA-seq dropout (scRNA-seq is zero-inflated, so some tdTomato- cells could be false negatives), and provide methodological details (thresholds, ambient RNA correction, etc.) to support this?

We will clarify this and include additional representations of the tdTomato transcript data.

Although the sex-related differences in macrophage composition and the absence of differential expression are interesting, they distract from the manuscript's main messages. Moreover, the Discussion does not elaborate on how these observations relate to joint (disease) biology. Consider removing this section or integrating it clearly into the relevant biological context.

We will remove this section as suggested.

CreERT2 transgenic lines are often not 100% efficient in recombination, also depending on whether tamoxifen or 4-OHT is used. Could the authors report the percentage of tdTomato+ cells in the joints and compare them to the recombination efficiencies in the monocytes/microglia under the same tamoxifen or 4-OHT conditions? This would help clarify how the interpret the macrophage labeling %'s.

We will report labelling efficacies and/or show normalized data in the revised manuscript.

Could the authors draw parallels between the observations in the mouse knee joint macrophage populations and literature on other joints in mouse and the knee joint in human (for example, as described in Alivernini et al., 2020 and in the very recent Raut et al., 2025)?

We will include a section on this in the revised manuscript.

Minor comments:

In general, the authors should clarify in the Results what each marker used for imaging, flow cytometry, or in the mouse reporter lines delineates. For example, mention that F4/80 is a marker for tissue-resident macrophages (correct?) in immunofluorescence, that IBA1 is a marker for macrophages on human tissue sections (Figure S1), and PDPN is GP38 (Figure S2 - align usage of marker reference across main text and figures).

We will implement this request.

For clarity in the microscopy representation, the single channels should be represented in a grey scale.

We will revise image presentation.

Figure S1B: Is CX3CR1 also restricted to the lining macrophages in human? Could a co-staining with IBA1 be performed to strengthen the species similarities?

To our knowledge, there is no antibody available that works for imaging of human CX3CR1. Moreover, CX3CR1 is only limited to the lining population in adult joints, in fetal and newborn (mouse) joints, all macrophages express this receptor, as do fetal progenitors to macrophages. However, Alivernini and colleagues have reported that TREM2high macrophages are the human counterpart of the mouse CX3CR1+ lining population (PMID: 32601335).

Adipocyte diameter quantification: Avoid plotting individual adipocytes from 2 mice without per-mouse visualization. Instead, report the mean adipocyte diameter per mouse and plot those means.

We will implement this change.

A little typo was spotted in the "Statistical analysis and reproducibility" section: it is Dunn's, not Bunn's multiple-comparison correction.

Thanks for spotting this.

Figure 2A: The gating strategy for the CX3CR1-GFP cells is missing.

We will provide this in the revised manuscript or supplementary material.

Improve the visualization of some plots. For example, Figure 2F is hard to read because of the big dot size. The dots seem to add no information to the graph and could be removed. Additionally, for comparing the clusters across the different time points, one could project the cells from the other time points in grey in the background.

We will revise the presentation of these data.

Figure S2: The dotplot is more informative than the heatmap, consider removing the heatmap.

We will do that.

Figure 3A: If technically feasible, image and visualize both the GFP and tdTomato expression. It would be informative to see the Cx3cr1+ and Ms4a3-derived cells in the same specimen.

We will thrive to show this in the revised manuscript.

Figure 3C: Highlight that tdTomato expression is visualized here.

We will do that.

Figure 3G,F: The authors should place the schematics and graphs next to each other, so the data points can be more easily compared.

We aim to do this in the revised manuscript.

Figure 4B: Which co-staining was performed for the immunofluorescence to quantify the % of tdTomato+ cells?

We co-stained for F4/80 and assessed localization in the lining or sublining. This will be clarified in the revised Figure legend.

Figure 4C: The trajectory analysis appears to have an arrow pointing from the Ccr2+ macrophages to the Ly6c+ monocytes. Please verify this directionality, as its seems against the known biology.

This will be addressed during revision.

Figure 5 mentions that the Csfr1 levels were reduced in a tissue-specific manner, but it is unclear how this tissue specificity was achieved.

We apologize for this misunderstanding. Csfr1FIRE mice are not tissue-specific knockouts, but they are more specific than global knockout mice, since only a (myeloid-specific) enhancer is affected. We will clarify this in the relevant section.

For the TGFb perturbations (Tgfbr2 KO and systemic TGFb depletion): did the authors validate reduced TGFb pathway activity in the macrophages, for example, reduced pSMAD2/3 levels? This would validate the effectiveness of the perturbations. This is an important point, and assessing signaling events downstream of TGFb is a very good suggestion.

As per above comment, we have decided to uncouple the functional data with exception of CSF1 from the revised version of the current manuscript, but we will be taking this into account for substantiating our functional data in follow-up work.

Figure 5F could benefit from a timeline of the treatment.

As for the previous point raised, we will be taking this into account for follow-up work on the uncoupled functional data.

The Methods mention that Gene Ontology analysis was performed on the single-cell data, but the results are not plotted in a figure. It would be informative to include this GO/pathway analysis in the appropriate figure(s).

We will include this in the revised (supplementary) information.

Significance:

This work provides a high temporal-resolution and "spatial" resolution reference map of the ontogeny and maturation of the synovial lining macrophages in the knee joint. It complements existing literature that demonstrated the presence of tissue-resident macrophages in the synovial space and lining (Culemann, et al., 2019 and others) by charting the embryonic-to-postnatal emergence of lining and sublining subsets. In particular, this mouse work identified some key signaling pathways in shaping this tissue compartment. This dataset serves as a robust, steady-state reference for joint pathology and can be implemented with human studies on disease biology of the knee joint (e.g., Alivernini et al., 2020; Raut et al., 2025). Insights into the exact developmental origins, mechanisms contributing to diverse or seemingly similar cell types, and distinct maturation processes are crucial to understanding disease biology, in which developmental processes can be hijacked/reactivated.

These findings will interest researchers in joint disease biology (osteoarthritis and immune-mediated arthritides such as RA and psoriasis), macrophage development (tissue-resident vs monocyte-derived lineages), the bone/joint microenvironment, and joint mechanobiology.

The reviewer's expertise is in developmental biology, mesoderm, bone biology, hematopoiesis, and monocyte/macrophage biology in disease

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

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

Summary:

Magalhaes Pinto, Malengier-Devlies, and co-authors investigated the developmental origins and maturation of synovial (lining and sublining) macrophages across embryonic, newborn, and postnatal stages in mouse. The authors used multiple transgenic reporter lines, lineage tracing, scRNA-seq, 2D confocal and 3D lightsheet imaging, and perturbations to delineate the macrophage states and ontogeny. They propose a model in which the majority of the joint lining macrophages has a fetal (EMP-derived) origin and a small proportion has a definitive HSC-derived monocyte origin, which both seed and mature within the synovial space in the postnatal period in the first 3 weeks of life. Using cell-cell communication analysis on their scRNA-seq data, they identified Fgf2, Csf1, and Tgfb as candidate signaling pathways that support (lining) macrophage development and maturation. Functional experiments indicate that the process is CSF1 and TGFb-dependent and also partly dependent on mechanosensing through Piezo1. The key conclusions on the composition of the synovial macrophages are convincing based on the presented results, and are carefully phrased. The study is very comprehensive, yet the description and organization of the results of the different mouse models could be altered to improve the storyline. Several refinements in data presentation, formulation, and minor validation experiments would further improve the clarity of the story, as well as summary recaps of the major findings throughout the text.

Major comments:

1. Generally, the story could be more streamlined by introducing earlier reporter lines and lineage-origin logic. Clearly state which reporter/CreERT2 lines and acrosses are used. It was unclear in Figure 2 that cells of the cross of the Cx3cr1-GFP and Ms4a3Cre:Rosa26lsl-tdT reporter lines were used for the scRNA-seq. The principle that there are fetal-derived and bone marrow (GMP)-derived monocytes and macrophages doesn't need to be "hidden" until Figure 3. For example, also the imaging of Ms4a3Cre could be introduced before the scRNA-seq.
2. Figure 1 could benefit from a cartoon visualizing the anatomy of the knee joint. The terms "sublining" and "synovium" are now a bit unclear, as it appears that sometimes the synovium is indicated as sublining and vice versa. Additionally, a schematic developmental timeline could be added to indicate the parallels between mouse and human development (fetal and postnatal development in mouse versus gestational age in human). Also, the various waves of hematopoiesis could be indicated in this timeline, which would be particularly helpful for Figure 3 for the lineage-tracing readouts. Lastly, the authors could end the manuscript (a new Figure 6) with a general cartoon summarizing all the results presented.
3. Figure 1 could be rearranged: first introduce the markers CX3CR1 and VSIG4 (Figure 1D) and then present the quantifications (Figure 1B/E). Where possible, co-visualization CX3CR1-GFP and VSIG4 on tissue sections to strengthen the claims on the relationship between these 2 markers. Tying the scRNA-seq insights (Figure 2) to the imaging would be elegant. Moreover, it would be informative to represent the CX3CR1+ and VSIG4+ macrophages as a percentage of F4/80+ macrophages (Figure 1B/E). Similarly, for the flow cytometry data in Figure 2, the relationship between the markers CX3CR1 and VSIG4 on macrophages could be more clearly displayed and discussed.
4. The 3D imaging of the joint is a nice addition to the manuscript, as it provides more context to the anatomical structure; however, while the text suggests several newborn joints were imaged, Figure 1F visualizes (again) the knee joint. Could other joints also be represented by 3D imaging? If the knee joint is the only joint available for imaging, and previous confocal imaging focused specifically on the meniscus in the knee joint, could the meniscus also be highlighted in the lightsheet imaging?
5. Clarification is requested regarding the imaging quantification representation. The M&M section under "Statistical analysis and reproducibility" states that individual data points are displayed, and bars represent the mean. However, some of the Figure legends (e.g., Figures 1B and S1C) specify that each dot corresponds to an individual mouse, with quantification based on 2-3 sections per mouse. While this appears to be a very reasonable representation of the data, does this mean that for each dot, the mean value from the 2-3 sections per mouse was calculated and plotted?
6. It is not clear how the differential expression analysis was performed on the Vsig4+ cells. Please specify if Cluster 0 was used for analysis, or all Vsig4-expressing cells? Not all cells in Cluster 0 have Vsig4+ expression. The authors described the expression dynamics of Aqp1 as intriguing, but lack a reasoning on why this is interesting.
7. Figure S3E: In line with the previous comment, can the authors justify that the tdTomato+/- comparisons are not biased by scRNA-seq dropout (scRNA-seq is zero-inflated, so some tdTomato- cells could be false negatives), and provide methodological details (thresholds, ambient RNA correction, etc.) to support this?
8. Although the sex-related differences in macrophage composition and the absence of differential expression are interesting, they distract from the manuscript's main messages. Moreover, the Discussion does not elaborate on how these observations relate to joint (disease) biology. Consider removing this section or integrating it clearly into the relevant biological context.
9. CreERT2 transgenic lines are often not 100% efficient in recombination, also depending on whether tamoxifen or 4-OHT is used. Could the authors report the percentage of tdTomato+ cells in the joints and compare them to the recombination efficiencies in the monocytes/microglia under the same tamoxifen or 4-OHT conditions? This would help clarify how the interpret the macrophage labeling %'s.
10. Could the authors draw parallels between the observations in the mouse knee joint macrophage populations and literature on other joints in mouse and the knee joint in human (for example, as described in Alivernini et al., 2020 and in the very recent Raut et al., 2025)?

Minor comments:

1. In general, the authors should clarify in the Results what each marker used for imaging, flow cytometry, or in the mouse reporter lines delineates. For example, mention that F4/80 is a marker for tissue-resident macrophages (correct?) in immunofluorescence, that IBA1 is a marker for macrophages on human tissue sections (Figure S1), and PDPN is GP38 (Figure S2 - align usage of marker reference across main text and figures).
2. For clarity in the microscopy representation, the single channels should be represented in a grey scale.
3. Figure S1B: Is CX3CR1 also restricted to the lining macrophages in human? Could a co-staining with IBA1 be performed to strengthen the species similarities?
4. Adipocyte diameter quantification: Avoid plotting individual adipocytes from 2 mice without per-mouse visualization. Instead, report the mean adipocyte diameter per mouse and plot those means.
5. A little typo was spotted in the "Statistical analysis and reproducibility" section: it is Dunn's, not Bunn's multiple-comparison correction.
6. Figure 2A: The gating strategy for the CX3CR1-GFP cells is missing.
7. Improve the visualization of some plots. For example, Figure 2F is hard to read because of the big dot size. The dots seem to add no information to the graph and could be removed. Additionally, for comparing the clusters across the different time points, one could project the cells from the other time points in grey in the background.
8. Figure S2: The dotplot is more informative than the heatmap, consider removing the heatmap.
9. Figure 3A: If technically feasible, image and visualize both the GFP and tdTomato expression. It would be informative to see the Cx3cr1+ and Ms4a3-derived cells in the same specimen.
10. Figure 3C: Highlight that tdTomato expression is visualized here.
11. Figure 3G,F: The authors should place the schematics and graphs next to each other, so the data points can be more easily compared.
12. Figure 4B: Which co-staining was performed for the immunofluorescence to quantify the % of tdTomato+ cells?
13. Figure 4C: The trajectory analysis appears to have an arrow pointing from the Ccr2+ macrophages to the Ly6c+ monocytes. Please verify this directionality, as its seems against the known biology.
14. Figure 5 mentions that the Csfr1 levels were reduced in a tissue-specific manner, but it is unclear how this tissue specificity was achieved.
15. For the TGFb perturbations (Tgfbr2 KO and systemic TGFb depletion): did the authors validate reduced TGFb pathway activity in the macrophages, for example, reduced pSMAD2/3 levels? This would validate the effectiveness of the perturbations.
16. Figure 5F could benefit from a timeline of the treatment.
17. The Methods mention that Gene Ontology analysis was performed on the single-cell data, but the results are not plotted in a figure. It would be informative to include this GO/pathway analysis in the appropriate figure(s).

Significance

This work provides a high temporal-resolution and "spatial" resolution reference map of the ontogeny and maturation of the synovial lining macrophages in the knee joint. It complements existing literature that demonstrated the presence of tissue-resident macrophages in the synovial space and lining (Culemann, et al., 2019 and others) by charting the embryonic-to-postnatal emergence of lining and sublining subsets. In particular, this mouse work identified some key signaling pathways in shaping this tissue compartment. This dataset serves as a robust, steady-state reference for joint pathology and can be implemented with human studies on disease biology of the knee joint (e.g., Alivernini et al., 2020; Raut et al., 2025). Insights into the exact developmental origins, mechanisms contributing to diverse or seemingly similar cell types, and distinct maturation processes are crucial to understanding disease biology, in which developmental processes can be hijacked/reactivated.

These findings will interest researchers in joint disease biology (osteoarthritis and immune-mediated arthritides such as RA and psoriasis), macrophage development (tissue-resident vs monocyte-derived lineages), the bone/joint microenvironment, and joint mechanobiology.

The reviewer's expertise is in developmental biology, mesoderm, bone biology, hematopoiesis, and monocyte/macrophage biology in disease

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

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

In the manuscript „The synovial lining macrophage layer develops in the first weeks of life in a CSF1- and TGFβ- dependent but monocyte-independent process", Magalhaes Pinto and colleagues carefully employ a wide range of technologies including single cell profiling, imaging and an exceptional combination of fate mapping models to characterize the ontogeny and development of lining macrophages in the joint, thus dissecting their maturation during postnatal development. Over the last decade, several landmark studies highlighted the imprinting of tissue-resident macrophages by a combination of ontogenetic and tissue-specific niche factors during development. So far, the ontogeny and the tissue niche factors governing the development and maturation of lining macrophages have not been described. Therefore, the results of this study offers insights on a small highly adapted macrophage population with relevance in many disease settings in the joint. Furthermore, the findings are nicely showcasing how macrophages are specializing to even very small tissue niches across development within one bigger anatomical compartment to serve dedicated functions within this niche.

This manuscript is beautifully written and highlights many novel, highly relevant findings on lining macrophage biology and the authors employ a wide range of different technologies to carefully dissect the postnatal development of lining macrophages.

In particular, the combination of scRNA-seq and fate mapping is providing a unique the link of transcriptional programs to ontogeny within the tissue niche. Furthermore, the integrative use of distinct fate mapping strategies, transgenic mouse lines, and treatment paradigms to elucidate key niche factors guiding the development and maturation of lining macrophages provides many interesting findings and data that are highly relevant to the field. I really enjoyed reading this manuscript.

Major points:

1) The authors show dynamic regulation of VSIG4 in lining macrophages during development, therefore VSIG4 is maybe not an ideal choice for gating strategies to define lining macrophages or to show as a single markers in immunofluorescence (IF) stainings to demonstrate their abundance across development (even though it is clear that this is the reason why the F4/80 staining is shown next to it). To demonstrate the increase of lining macrophages during development in IF, it would be more helpful if the authors would show quantifications of all F4/80+ cells and additionally VSIG4+ as a proportion of F4/80+ cells (or VSIG4+ F4/80+ and all F4/80+ in a stacked bar plot).

2) In Figure 1C, the authors nicely demonstrate that the lining macrophages get closer in their distance across development to build the epithelial-like macrophage structure along the adult lining. Is the close proximity between lining macrophages already fully "matured" at 3 weeks of age and comparable to adults? Please quantify the distance in adult linings.

3) Can the authors explain how the grouping was performed between the analyzed human fetal joints? It is not clear why the cut was chosen between the groups at 16/17 weeks of age. Maybe it would be also beneficial if the authors would consider not grouping these samples but rather show the specific quantifications for each samples individually and estimate via linear regression the expansion over time across human development. Furthermore, can the authors give additional information about the distancing of lining macrophages in the human fetal samples, it would be great to see if they follow the same dynamics as in mouse. Maybe comparison to human juvenile/adult joints would also add on to substantiate the findings in human samples (if possible).

4) The scRNA-seq analysis leaves several questions open and some conclusions and workflows cannot be easily followed.

a. It is not clear how and especially why the signature genes to define macrophages vs. monocytes were chosen. Especially as the signature genes for monocytes would not include patrolling monocytes and the macrophage signature genes seem to be highly regulated during development, see also Apoe expression in NB vs. adult in Figure S2e. Why did the authors not take classical markers such as Itgam, Fcgr1a, Csf1r?

b. Can dendritic cell signatures be excluded? Cluster 11 and 12 show indeed some DC markers, are these really macrophages?

c. The authors provide several figure panels showing TOP marker genes or key marker genes for the identified clusters, however it is not clear if these are TOP DE genes or if the genes were hand chosen. Somehow, the authors give the impression that the clusters were chosen and labeled not based on DE genes, but more on existing literature that previously reported these macrophage populations. DE gene lists for all annotated cell types and macrophage clusters need to be provided within the manuscript.

d. The authors claim that Clusters 1 and 4 are "developing" macrophages. How is this defined? Why are these developing cells compared to other clusters? And why are these clusters later on not considered as progenitors of Aqp1 macrophages and Vsig4 macrophages? Why are Aqp1+ macrophages not labeled as developing when they are later on in the manuscript shown as potential intermediate progenitors of lining macrophages?

e. Furthermore, it is again confusing that markers are used throughout Figure 2 which are labeled as "key marker genes" for a population and then later on they are claimed to be regulated during development within this population, see for example Figure 2D and 2H.

f. It is appreciated that the authors distinguished cycling clusters such as 8, 9, and 10 based on their cycling gene signature. Here it would be very exciting to see a cell cycle analysis across all clusters and time points to see when exactly the cells are expanding during development; this would also substantiate the data later shown for the Mki67-CreERT2 mouse model.

g. Can the authors identify certain gene modules during development of lining macrophages (and/or their progenitors) which are associated with certain functions (e.g. GO terms, GSEA enrichment)?

5) To determine the actual presence of the identified macrophage clusters from the scRNA-seq as macrophage populations in the joint, the authors should perform IF or FACS for key markers. Especially, Aqp1+ macrophages should be shown in the developing joint.

6) The authors used a wide range of fate mapping models, which is quite unique and highly appreciated. The obtained results and the conclusions made from the models raise a couple of questions: Whereas contribution of HSC-derived/monocyte-derived macrophages to the lining compartment seems to be minor, there is still labeling across different models. Various aspects would need to be clarified.

a. For example, the authors employ Ms4a3-Cre as a tracing model for GMP-derived monocytes, however all quantifications of the labeling efficiency are not normalized to the labeling in monocytes or another highly recombined cell population. This should be shown, similar to the other fate mapping models (Figure 3 F-I).

b. Please show Ms4a3 expression across clusters across time points, to exclude expression in fetal-derived clusters.

c. In line with the question raised above, if the authors can exclude a development of the Egfr1+ and Clec4n+ developing macrophages into Aqp1+ macrophages and subsequently into Vsig4 lining macrophages, the obtained data from the Ms4a3-Cre model highly suggests a correlative labeling across these clusters what could implicate a relation. However, the authors do not discuss throughout the manuscript the role of these developing macrophages. It is highly encouraged to include this into the manuscript and it would be of high relevance to understand lining macrophage development.

d. The authors conclude from the pseudo bulk transcriptomic profiling of the different macrophage clusters that TdT+ and TdT- macrophages do not differ in their gene expression profile and that this is due to niche imprinting rather than origin imprinting. Even though the data supports that conclusion, the authors should verify if inkling cells early during development also show this similar gene expression profile and gene expression should be compared at the different developmental time points. Tissue niche imprinting is happening within the niche during development, most likely in a stepwise progress, and therefore there should be differences in the beginning.

7) The trajectorial analysis using different pseudotime pipelines is very interesting and nicely points out the potential role of Aqp1 macrophages as intermediates of Vsig4 lining macrophages. From my point of view, all trajectories seem to suggest that Egfr1 developing macrophages and Clec4n developing macrophages might differentiate into Aqp1 macrophages, however the authors are not exploring this further and the role of both developing macrophage clusters is not further discussed (see also comments above).

8) How was the starting point of the trajectorial analyses defined and is it the same for each pipeline used?

9) Are there potentially two trajectories? It looks like there is one in the beginning of postnatal life and a second one appearing from the monocyte-compartment later in life. If this is true, that would rather speak for a dual ontogeny of Vsig4+ macrophages, wouldn't it?

10) A heatmap (transcriptional shift) of trajectories between more clusters should be shown at least for Cluster 0,1,2, and 3. It is not sufficient to demonstrate this only between two clusters.

11) To show the similarity between Aqp1 macrophages and proliferating macrophage clusters, the authors should remove the cycling signature and compare these clusters to show that the cycling cells might be Aqp1 macrophages or earlier developing macrophage progenitors aka Clec4n or Egfr1 macrophages.

12) The conclusions made from the Mki67-CreERT2 data are a bit difficult to understand, whereas all progenitors (monocyte progenitors and macrophage progenitors will proliferate at the neonatal time point and no conclusions can be made if the cells expand in the niche. The authors should employ Confetti mice or other models (Ubow mice) to analyze clonal expansion in the niche.

13) All predicted cell-cell interactions between macrophages and fibroblasts should be provided in a supplementary table. Are the interactions shown in Figure 5 chosen interactions or the TOP predicted ones? Whereas the authors show different numbers of interactions, it is most likely hand-picked and therefore biased.

14) The authors further aim to dissect the factors involved in the developmental niche imprinting of lining macrophages. Even though it is highly appreciated that the authors used so many experimental setups to show the reliance of lining macrophages on Csf1 and TGF-beta as well as mechanosensation, the wide range of models the different methods used and selected developmental time points make it very difficult to really interpret the data. The authors should carefully choose time points and methods (either FACS analysis across all models or IF across all, or both). Often deletion efficiencies for transgenic models and proof of concept that the inhibitors and agonists are working in the treatment paradigm are not provided. For example, Csf1rMer-iCre-Mer Tgfbr2fl/fl mice are used but no deletion efficiency is shown or different time points of analysis, maybe the macrophages are not properly targeted in the set up.

15) The authors have shown the role of Csf1 and Tgfbr2 only for lining macrophages, is this specific in the joint to this population of are subliming macrophages affected in a similar manner.

16) Can the authors confirm their results in CSF1R-FIRE mice with anti-Csf1 injections or in Csf1op/op mice?

17) The setup in Figure S5G is very interesting to test the role of movement and mechanical load on the joint, however, there is basically no data on the model provided showing the efficiency of the induced optogenetic muscle contractions, and only one time point is shown.

18) The results regarding the role of Piezo1 and mechanosensation vary a lot. Could it be that analyses were done too early or that actually proper weight load on the joint must be applied for the maturation of the macrophages? The authors should test this to

19) The Rolipram experiment is shown in Figure S5G, but is not described in the result section. It only appears at some point in the discussion part. The authors should move it to results or remove it from the manuscript.

Minor points:

1) Please reference the Figure panels in numeric order throughout the text.

2) Figure 2a and 2b are a bit out of the storyline, it is not obvious why this is shown here and maybe it would be good to move it to the supplements. Gating strategy is also not used for scRNA-seq. Therefore, it would better fit to the later analysis of joint macrophages across different transgenic mouse models and treatment paradigms. The gating strategies are changing across different experiments throughout the figures, it would be nice to have a similar gating strategy for all experiments, see also Figure 3 where the defining markers for joint macrophages are changing between models.

3) A lot of figure panels have very small labeling that is basically unreadable. Axes at FACS plots for example. Sometimes, it is even impossible to distinguish cluster labels especially when they have similar colors.

4) In the text on page 14, many markers are named which are specifically regulated during development in lining macrophages, but these factors are not labeled anywhere in the volcano plot. It would be good to showcase at least some of these named genes in the figure panel, e.g. Trem2.

5) Figure 2F and Figure S2F are really nicely showing the percentage of cells per cluster in each analyzed biological sample. Maybe the authors could additionally consider to show a stacked bar plot with the mean percentage of cells per cluster and how the clusters are distributed across time points?

6) Figure 3A: IF for adult lining macrophages and the quantification are missing

Significance

This manuscript highlights novel, highly relevant findings on lining macrophage biology and the authors employ a wide range of different technologies to carefully dissect the postnatal development of lining macrophages. Furthermore, this study showcases in a very elegant and detailed way the adaptation of macrophage progenitors to a highly specific anatomical tissue niche.

The manuscript is of high interest to basic scientists focussing on macrophage biology and immune cell development and clinicians and clinician scientists focussing on joint diseases such as RA

Therefore the manuscript is of interest to a wide community working in immunology.

Reviewer #3 (Public review):

Summary:

The manuscript by Atchou et al. investigates the role of the microtubule cytoskeleton in sporozoites of Plasmodium berghei, including possible functions of microtubule post-translational modifications (tyrosination and polyglutamylation) in the development of sporozoites in the liver. They also assessed the development of sporozoites in the mosquito. Using cell culture models and in vivo infections with parasites that contain tubulin mutants deficient in certain PTMs, they show that may aspects of the life cycle progression are impaired. The main conclusion is that microtubule PTMs play a major role in the differentiation processes of the parasites.

However, there are a number of major and minor points of criticism that relate to the interpretation of some of the data.

Comments:

(1) The first paragraph of "Results" almost suggests that the presence of a subpellicular MT-array in sporozoites is a new discovery. This is not the case, see e.g. the recent publication by Ferreira et al. (Nature Communications, 2023).

(2) Why were HeLa cells and not hepatocytes (as in Figure 3) used for measuring infection rates of the mutants in Figure 5H and 5L? As I understand, HeLa cells are not natural host cells for invading sporozoites. HeLa cells are epithelial cells derived from a cervical tumour. I am not an expert in Plasmodium biology, but is a HeLa infection an accepted surrogate model for liver stage development?

(3) The tubulin staining in Figures 1A and 1B is confusing and doesn't seem to make sense. Whereas in 1A the antibody nicely stains host and parasite tubulin, in 1B, only parasite tubulin is visible. If the same antibody and the same host cells have been used, HeLa cytoplasmic microtubules should be visible in 1B. In fact, they should be the predominant antigen. The same applies to Figure 2, where host microtubules are also not visible.

(4) In Figures 2A and B, the host nuclei appear to have very different sizes in the DMSO controls and in the drug-treated cells. For example, in the 20 µM (-) image (bottom right), the nuclei are much larger than in the DMSO (-) control (top left). If this is the case, expansion microscopy hasn't worked reproducibly, and therefore, quantification of fluorescence is problematic. The scalebar is the same for all panels.

(5) I don't quite follow the argument that spindles and the LSPMB are dynamic structures (e.g., lines 145, 174). That is a trivial statement for the spindle, as it is always dynamic, but beyond that, it has only been shown that the structure is sensitive to oryzalin. That says little about any "natural" dynamic behaviour. Any microtubule structure can be destroyed by a particular physical or chemical treatment, but that doesn't mean all structures are dynamic. It also depends on the definition of "dynamic" in a particular context, for example, the time scale of dynamic behaviour (changes within seconds, minutes, or hours).

(6) I am not sure what part in the story EB1 plays. The data are only shown in the Supplements and don't seem to be of particular relevance. EB1 is a ubiquitous protein associated with microtubule plus ends. The statement (line 192) that it "may play a broader role..." is unsubstantiated and cannot be based merely on the observation that it is expressed in a particular life cycle stage.

(7) Line 196 onwards: The antibody IN105 is better known in the field as polyE. Maybe that should be added in Materials and Methods. Also, the antibody T9028 against tyrosinated tubulin is poorly validated in the literature and rarely used. Usually, researchers in this field use the monoclonal antibody YL1/2. I am not sure why this unusual antibody was chosen in this study. In fact, has its specificity against tyrosinated α-tubulin from Plasmodium berghei ever been shown? The original antigen was human and had the sequence EGEEY. The Plasmodium sequence is YEADY and hence very different. It is stated that the LSPMB is both polyglutamylated and tyrosinated. This is unusual because polyglutamylated microtubules are usually indicative of stable microtubules, whereas tyrosinated microtubules are found on freshly polymerised and dynamic microtubules. However, a co-localisation within the same cell has not been attempted. This is, however, possible since polyE is a rabbit antibody and T9028 is a mouse antibody. I suspect that differences or gradients along the LSPMB would have been noticed. Also, in lines 207/208, it is said that tyrosination disappears after hepatocyte invasion, which is shown in Figure 3. However, in Figure 3A, quite a lot of positive signals for tyrosination are visible in the 54 and 56 hpi panels.

(8) In line 229, it is stated that tyrosination "has previously been associated with stable microtubule in motility". This statement is not correct. In fact, none of the cited references that apparently support this statement show that this is the case. On the contrary, stable microtubules, such as flagellar axonemes, are almost completely detyrosinated. Therefore, tyrosination is a marker for dynamic microtubules, whereas detyrosinated microtubules are indicative of stable microtubules. This is an established fact, and it is odd that the authors claim the opposite.

(9) Line 236 onwards: Concerning the generation of tubulin mutants, I think it is necessary to demonstrate successful replacement of the wild-type allele by the mutant allele. I am sure the authors have done this by amplification and subsequent sequencing of the genomic locus using PCR primers outside the plasmid sequences. I suggest including this information, e.g., by displaying the chromatograph trace in a supplementary figure. Or are the sequences displayed in Figure S3B already derived from sequenced genomic DNA? This is not described in the Legend or in Materials and Methods. The left PCR products obtained for Figure S3 B would be a suitable template for sequencing.

(10) It is also important to be aware of the fact that glutamylation also occurs on β-tubulin. This signal will also be detected by polyE (IN105). Therefore, it is surprising that IN105 immunofluorescence is negative on the C-term Δ cells (Figure S3 D). Is there anything known about confirmed polyglutamylation sites on both α- and β-tubulins in Plasmodium, e.g., by MS? In Toxoplasma, both α- and β-tubulin have been shown to be polyglutamylated.

(11) Figure S3 is very confusing. In the legend, certain intron deletions are mentioned. How does this relate to posttranslational tubulin modifications? The corresponding section in Results (lines 288-292) is also not very helpful in understanding this.

(12) Figure 4E doesn't look like brightfield microscopy but like some sort of fluorescent imaging. In Figure 4C, were the control (NoΔ) cells with an integrated cassette, but no mutations, or non-transgenic cells?

(13) It is difficult to understand why the TyΔ and the CtΔ mutants still show quite a strong signal using the anti-tyrosination antibody. If the mutants have replaced all wild-type alleles, the signal should be completely absent, unless the antibody (see my comment above concerning T9028) cross-reacts with detyrosinated microtubules. Therefore, the quantitation in Figures 5F and 5G is actually indicative of something that shouldn't be like that. The quantitation of 5F is at odds with the microscopy image in 5D. If this image is representative, the anti-Ty staining in TyΔ is as strong as in the control NoΔ.

(14) The statement that the failure of CtΔ mutants to generate viable sporozoites is due to the lack of microtubule PTMs (lines 295-296) is speculative. The lack of the entire C-terminal tail could have a number of consequences, such as impaired microtubule assembly or failure to recruit and bind associated proteins. This is not necessarily linked to PTMs. Also, it has been shown in yeast that for microtubules to form properly and exquisite regulation (proteostasis) of the ratio between α- and β-tubulin is essential (Wethekam and Moore, 2023). I am not sure, but according to Materials and Methods (line 423), the gene cassettes for replacing the wild-type tubulin gene with the mutant versions contain a selectable marker gene for pyrimethamine selection. Are there qPCR data that show that expression levels of mutant α-tubulin are more or less the same as the wild-type levels?

(15) In the Discussion, my impression is that two recent studies, the superb Expansion Microscopy study by Bertiaux et al. (2021) and the cryo-EM study by Ferreira et al. (2023), are not sufficiently recognised (although they are cited elsewhere in the manuscript). The latter study includes a detailed description of the microtubule cytoskeleton in sporozoites. However, the present study clearly expands the knowledge about the structure of the cytoskeleton in liver stage parasites and is one of the few studies addressing the distribution and function of microtubule post-translational modifications in Plasmodium.

(16) I somewhat disagree with the statement of a co-occurrence of polyglutamylated and tyrosinated microtubules. I think the resolution is too low to reach that conclusion. As this is a bold claim, and would be contrary to what is known from other organisms, it would require a more rigorous validation. Given the apparent problems with the anti-Ty antibody (signal in the TyΔ mutant), one should be very cautious with this claim.

(17) In the Discussion (lines 311 and 377), it is again claimed that tyrosinated microtubules are "a well-known marker of stable microtubules". This statement is completely incorrect, and I am surprised by this serious mistake. A few lines later, the authors say that polyglutamylated is "commonly associated with dynamic microtubule behaviour". Again, this is completely incorrect and is the opposite of what is firmly established in the literature. Polyglutamylation and detyrosination are markers of stable microtubules.

(18) In line 339, the authors interpret the residual antibody staining after the introduction of the mutant tubulin as a compensatory mechanism. There is no evidence for this. More likely explanations are firstly the quality of the anti-Ty-antibody used (see comment above), and the fact that also β-tubulin carries C-terminal polyglutamylation sites, which haven't been investigated in this study. PTMs on β-tubulin are not compensatory, but normal PTMs, at least in all other organisms where microtubule PTMs have been investigated.

Author response:

Public Reviews:

Reviewer #1 (Public review):

The authors try to investigate how the population of microtubules (LSPMB) that originate from sporozoite subpellicular microtubules (SSPM) and are remodelled during liver-stage development of malaria parasites. These bundles shrink over time and help form structures needed for cell division. The authors have used expansion microscopy, live-cell imaging, genetically engineered mutants, and pharmacological perturbation to study parasite development with liver cells.

A major strength of the manuscript is the live cell imaging and expansion microscopy to study this challenging liver stage of parasite development. It gives important knowledge that PTMs of α-tubulin, such as polyglutamylation and tyrosination/detyrosination, are crucial for microtubule stability. Mutations in α-tubulin reduce the parasite's ability to move and proliferate in the liver cells. The drug oryzalin, which targets microtubules, also blocks parasite development, showing how important dynamic microtubules are at this stage.

The major problem in the manuscript was the way it flows, as the authors keep shifting from the liver stage to the sporogony stages and then back to the liver stages. It was very confusing at times to know what the real focus of the study is, whether sporozoite development or liver stage development. The flow of the manuscript could be improved. Some of the findings reported here substantiate the previous electron microscopy.

Overall, the study represents an important contribution towards understanding cytoskeletal remodelling during liver stage infection. The study suggests that tubulin modifications are key for the parasite's survival in the liver and could be targets for new malaria treatments. This is also the stage that has been used for vaccine development, so any knowledge of how parasites proliferate in the liver cells will be beneficial towards intervention approaches.

We would like to express our sincere gratitude to Reviewer #1 for the positive and encouraging feedback on our manuscript. We are delighted that the reviewer found our experimental design and methodologies appropriate and that our study represents an important contribution to understanding cytoskeletal remodelling during liver stage infection, a critical phase for vaccine development. We are also grateful to the reviewer for highlighting the issue with the manuscript's flow. We acknowledge this limitation and will significantly improve the narrative structure and logical progression in the revised manuscript to ensure clarity and avoid any potential confusion. Thank you again for your thoughtful and constructive comments.

Reviewer #2 (Public review):

Summary:

The authors investigated microtubule distribution and their possible post-translational modifications (PTM) in Plasmodium berghei during development of the liver stage, using either hepatocytes or HeLa cells as models. They used conventional immunofluorescence assays and expansion microscopy with various antibodies recognising tubulin and, in the second part of the work, its candidate PTMs, as well as markers of Plasmodium, in addition to live imaging with a fluorescent marker for tubulin. In the third part of the study, they generated 3 mutants deprived of either the last four residues or the last 11 residues, or where a candidate polyglutamylation site was substituted by an alanine residue.

Strengths:

In the first part, microtubules are monitored by a combination of two approaches (IFA and live), revealing nicely the evolution of the sporozoite subpellicular microtubules (SSPM, the sporozoite is the developmental stage present in salivary glands of the mosquitoes and that infects hepatocytes) into a different structure termed liver-stage parasite microtubule bundle (LSPMB). The LSPMB shrinks during the course of parasite development and finally disappears while hemi-spindles emerge over time. Contact points between these two structures are observed frequently in live cells and occasionally in fixed cells, suggesting the intriguing possibility that tubulin might be recycled from the LSPMB to contribute to hemi-spindle formation.

In the second part, antibodies recognising (1) the final tyrosine found at the C-terminal tail and (2) a stretch of 3 glutamate residues in a side chain are used to monitor these candidate PTMs. Signals are positive at the SSPM, and while it remains positive for polyglutamylation, it becomes negative for the final tyrosine at the LSPM, while a positive signal emerges at hemi-spindles at later stages of development.

In the last part, the three mutants are fed to mosquitoes, where they show reduced development, the one lacking the alpha-tubulin tail even failing to reach the salivary glands. However, the two other mutants infect HeLa cells normally, whereas sporozoites with the C-terminal tail deletion recovered from the haemolymph did not develop in these cells.

The first part provides convincing evidence that microtubules are extensively remodelled during the infection of hepatocytes and HeLa cells, in agreement with the spectacular Plasmodium morphogenetic changes accompanying massive and rapid proliferation. The third part brings further confirmation that the C-terminal tail of alpha-tubulin is essential for multiple stages of parasite development, in agreement with previous work (50). Since it is the region where several post-translational modifications take place in other organisms (detyrosination, polyglutamylation, glycylation), it makes sense to propose that the essential function is related to these PTMs also in Plasmodium.

Weaknesses:

The significance of tubulin PTM relies on two antibodies whose reactivity to Plasmodium tubulins is unclear (see below). The interpretation of the literature on detyrosination and polyglutamylation is confusing in several places, meaning that the statements about the possible role of these PTMs need to be carefully revisited.

The authors use the term "tyrosination" but the alpha1-tubulin studied here possesses the final tyrosine when it is synthesised, so it is "tyrosinated" by default. It could potentially be removed by a tyrosine carboxypeptidase of the vasoinhibin family (VASH) as reported in other species. After removal, this tyrosine can be added again by a tubulin-tyrosine ligase (TTL) enzyme. It is therefore more appropriate to talk about detyrosination-retyrosination rather than tyrosination (this confusion is unfortunately common in the literature, see Janke & Magiera, 2020).

The difficulty here is that there is so far no evidence that detyrosination takes place in Plasmodium. Neither VASH nor TTL could be identified in the Plasmodium genome (ref 31, something we can confirm with our unsuccessful BLAST analyses), and mass spectrometry studies of purified tubulin, albeit from blood stages, did not find evidence for detyrosination (reference 43). Western blots using an antibody against detyrosinated tubulin did not produce a positive signal, neither on purified tubulin, nor on whole parasites (43). Of course, the situation could be different in liver stages, but the question of the detyrosinating enzyme is still there. The existence of a unique Plasmodium system for detyrosination cannot be formally ruled out but given the high degree of conservation of these PTMs and their associated enzymes, it sounds difficult to imagine.

The fact that the anti-tyrosinated antibody still produced a signal in the cell line where the final tyrosine is deleted raises issues about its specificity. A cross-reactivity with beta-tubulin is proposed, but the Plasmodium beta-tubulin does not carry a final tyrosine, further raising concerns about antibody specificity.

The interpretation of these results should therefore be considered carefully. There also seems to be some confusion in the function of detyrosination cited from the literature. It is said in line 229 that "tyrosination has been associated with stable microtubules" (33, 34, 50, 55). References 33 and 34 actually show that tyrosinated microtubules turn over faster in neurons or in epithelial cells, respectively, while references 50 and 55 do not study de/retyrosination. The general consensus is that tyrosinated microtubules are more dynamic (see reference 24).

The situation is a bit different for polyglutamylation since several candidate poly- or mono-glutamylases have been identified in the Plasmodium genome, and at least mono-glutamylation of beta-tubulin has been formally proven, still in bloodstream stages (ref 43). The authors propose that the residue E445 is the polyglutamylation site. To our knowledge, this has not been demonstrated for Plasmodium. This residue is indeed the favourite one in several organisms such as humans and trypanosomes (Eddé et al., Science 1990; Schneider et al., JCS, 1997), and it is tempting to propose it would be the same here. However, TTLLs bind the tubulin tails from their C-terminal end like a glove on a finger (Garnham et al., Cell, 2015), and the presence of two extra residues in Plasmodium tubulins would mean that the reactive glutamate might be in position E447 rather than E445. This is worth discussing.

On the positive side, it is encouraging to see that signals for both anti-tyrosinated tail and poly-glutamylated side chain are going down in the various mutants, but this would need validation with a comparison for alpha-tubulin signal.

Line 316: polyglutamylation "is commonly associated with dynamic microtubule behavior (78-80)". Actually, references 78 and 79 show the impact of this PTM on interaction with spastin, and reference 80 discusses polyglutamylation as a marker of stable microtubules in the context of cilia and flagella. The consensus is that polyglutamylated microtubules tend to be more stable (ref24).

Conclusion:

The first and the third parts of this manuscript - evolution of microtubules and importance of the C-terminal tails for Plasmodium development - are convincing and well supported by data. However, the presence and role of tubulin PTM should be carefully reconsidered.

Plasmodium tubulins are more closely related to plant tubulins and are sensitive to inhibitors that do not affect mammalian microtubules. They therefore represent promising drug targets as several well-characterised compounds used as herbicides are available. The work produced here further defines the evolution of the microtubule network in sporozoites and liver stages, which are the initial and essential first steps of the infection. Moreover, Plasmodium has multiple specificities that make it a fascinating organism to study both for cell biology and evolution. The data reported here are elegant and will attract the attention of the community working on parasites but also on the cytoskeleton at large. It will be interesting to have the feedback of other people working on tubulin PTMs to figure out the significance of this part of the work.

We thank Reviewer #2 for the thoughtful and detailed evaluation of our manuscript. We are pleased that the reviewer found our study elegant and believe it will attract the attention of the broader scientific community, both those working on parasites and those focused on cytoskeleton biology. We also acknowledge the concerns raised regarding the specificity of the antibodies used to detect tubulin post-translational modifications (PTMs), as well as the interpretation of their signals and the current lack of identified detyrosination enzymes in the Plasmodium genome. We agree that these are important limitations, and we will address them thoroughly in the revised manuscript. This includes clarifying our interpretation of tyrosination versus detyrosination, adjusting our claims regarding polyglutamylation sites, and carefully revisiting the literature cited to ensure accurate contextualization of PTM function in microtubule stability.

We are grateful for the reviewer’s close reading and critical feedback, which will help us substantially improve the clarity, precision, and strength of our manuscript.

Reviewer #3 (Public review):

Summary:

The manuscript by Atchou et al. investigates the role of the microtubule cytoskeleton in sporozoites of Plasmodium berghei, including possible functions of microtubule post-translational modifications (tyrosination and polyglutamylation) in the development of sporozoites in the liver. They also assessed the development of sporozoites in the mosquito. Using cell culture models and in vivo infections with parasites that contain tubulin mutants deficient in certain PTMs, they show that may aspects of the life cycle progression are impaired. The main conclusion is that microtubule PTMs play a major role in the differentiation processes of the parasites.

However, there are a number of major and minor points of criticism that relate to the interpretation of some of the data.

We thank Reviewer #3 for the overall positive assessment of our study and for recognizing its contribution to advancing our understanding of Plasmodium biology and malaria pathogenesis. We appreciate the reviewer’s constructive feedback, particularly regarding the interpretation of some of our data. These comments have been very helpful in guiding our revisions, and we have worked to improve both the clarity of our presentation and the precision of our interpretations in the revised manuscript.

Below, we respond in detail to each of the reviewer’s points.

Comments:<br /> (1) The first paragraph of "Results" almost suggests that the presence of a subpellicular MT-array in sporozoites is a new discovery. This is not the case, see e.g. the recent publication by Ferreira et al. (Nature Communications, 2023).

We thank the reviewer for pointing this out and fully agree that the subpellicular microtubule (SPM) array in sporozoites is well established, as documented in earlier work (e.g., Cyrklaff et al., 2007) and more recently by Ferreira et al. (Nat. Commun., 2023). Our intention was not to suggest that the existence of the SSPM is a novel finding. Rather, our study builds on this existing knowledge by demonstrating that these sporozoite-derived microtubules are not disassembled upon hepatocyte entry but are repurposed into a newly described structure, the liver stage parasite microtubule bundle (LSPMB). This reorganization, its persistence into liver stage development, and its dynamic role in microtubule remodeling and nuclear division are, to our knowledge, novel observations. We will revise the manuscript to make this distinction clearer in the introduction and the results section.

(2) Why were HeLa cells and not hepatocytes (as in Figure 3) used for measuring infection rates of the mutants in Figure 5H and 5L? As I understand, HeLa cells are not natural host cells for invading sporozoites. HeLa cells are epithelial cells derived from a cervical tumour. I am not an expert in Plasmodium biology, but is a HeLa infection an accepted surrogate model for liver stage development?

We appreciate the opportunity to clarify our experimental model. While HeLa cells are not the natural host cells, they are a well-established and validated in vitro model for studying Plasmodium berghei liver stage development in our lab and others. In this system, the parasite completes its full development and generates infectious merozoites. Numerous studies have successfully used HeLa cells as a liver stage infection model, with key findings subsequently validated in primary hepatocytes or in vivo, confirming its utility as a representative model. We employed this cell line primarily to reduce animal usage in accordance with the 3Rs principles (Replacement, Reduction, Refinement). Importantly, to ensure the biological relevance of our discoveries in HeLa cells, we validated our key findings in primary mouse hepatocytes, as shown in Figure 3. Furthermore, we confirmed the in vivo infectivity of mutant parasite lines that produced typical salivary gland sporozoites through an in vivo infection assay, presented in Figure S4C.

(3) The tubulin staining in Figures 1A and 1B is confusing and doesn't seem to make sense. Whereas in 1A the antibody nicely stains host and parasite tubulin, in 1B, only parasite tubulin is visible. If the same antibody and the same host cells have been used, HeLa cytoplasmic microtubules should be visible in 1B. In fact, they should be the predominant antigen. The same applies to Figure 2, where host microtubules are also not visible.

We thank the reviewer for this careful observation regarding the α-tubulin staining in Figures 1A and 1B. The same host cell type (HeLa) and α-tubulin antibody were indeed used in both experiments. Figure 1A shows results from conventional immunofluorescence assays, where both host and parasite microtubules are clearly stained. In contrast, Figure 1B shows the outcome of ultrastructure expansion microscopy (U-ExM), where parasite microtubules appear prominently, while host microtubules are less visible.

This effect appears to be a technical outcome of the U-ExM protocol, which can differentially preserve or reveal microtubule epitopes. We consistently observed stronger parasite signal across various cell types, including primary hepatocytes (Figure 3A,B). The lack of visible host microtubules in some U-ExM images does not reflect their absence, but rather reduced signal intensity relative to the parasite structures. This is not observed with all antibodies, e.g., host microtubules stain strongly with anti-tyrosinated α-tubulin (Figure 3B), likely reflecting their high tyrosination state.

To overcome this limitation, we employed PS-ExM and combined PS-ExM/U-ExM approaches (as described in reference 56), which allowed simultaneous high-resolution visualization of both host and parasite microtubule networks. These combined methods are now being used in follow-up studies to investigate host–parasite microtubule interactions in more detail.

We will clarify this point in the revised manuscript to avoid confusion.

(4) In Figures 2A and B, the host nuclei appear to have very different sizes in the DMSO controls and in the drug-treated cells. For example, in the 20 µM (-) image (bottom right), the nuclei are much larger than in the DMSO (-) control (top left). If this is the case, expansion microscopy hasn't worked reproducibly, and therefore, quantification of fluorescence is problematic. The scalebar is the same for all panels.

The expansion microscopy methods used in this study have been rigorously validated for both reproducibility and isotropicity. However, as the reviewer rightly notes, host cell nuclei can vary in size due to several factors, including cell cycle stage, infection status, and the extent of parasite development, all of which can influence host nuclei morphology and size.

Importantly, the quantifications relevant to our conclusions were focused specifically on parasite structures. We did not rely on host nuclear size or host fluorescence intensity as a quantitative readout in this context. While we acknowledge the observed variability in host nuclear dimensions, it does not compromise the accuracy or reproducibility of the parasite specific measurements central to our study.

We will clarify this point in the revised figure legend and manuscript.

(5) I don't quite follow the argument that spindles and the LSPMB are dynamic structures (e.g., lines 145, 174). That is a trivial statement for the spindle, as it is always dynamic, but beyond that, it has only been shown that the structure is sensitive to oryzalin. That says little about any "natural" dynamic behaviour. Any microtubule structure can be destroyed by a particular physical or chemical treatment, but that doesn't mean all structures are dynamic. It also depends on the definition of "dynamic" in a particular context, for example, the time scale of dynamic behaviour (changes within seconds, minutes, or hours).

We agree that sensitivity to chemical depolymerization alone does not necessarily indicate dynamic behavior, particularly in the absence of data on turnover kinetics or temporal changes.

Our interpretation was based on two observations: first, that the LSPMB, which derives from the highly stable sporozoite subpellicular microtubules (known to be drug-resistant), becomes susceptible to depolymerization during the liver stage; and second, that the LSPMB gradually shrinks over time during parasite development. These features suggested a transition toward a more dynamic state compared to its origin. However, we fully agree that “dynamic” is a context-dependent term and that direct evidence such as turnover rates or structural changes on short time scales, is required to rigorously define microtubule dynamics.

We will revise the manuscript to clarify our use of this term and explicitly acknowledge the need for further studies to characterize the timescale and mechanisms underlying LSPMB remodeling.

(6) I am not sure what part in the story EB1 plays. The data are only shown in the Supplements and don't seem to be of particular relevance. EB1 is a ubiquitous protein associated with microtubule plus ends. The statement (line 192) that it "may play a broader role..." is unsubstantiated and cannot be based merely on the observation that it is expressed in a particular life cycle stage.

We agree that EB1 is a ubiquitous microtubule plus-end binding protein and that its presence alone does not imply a novel function. Previous studies (e.g., Maurer et al., 2023; Yang et al., 2023; Zeeshan et al., 2023) have focused on its role during Plasmodium sexual stages, while its expression during liver and mosquito stages has not been previously documented.

Our data extend this knowledge by showing that EB1 is also expressed during liver stage development, particularly during the highly mitotic schizont phase. While we agree that this observation alone does not prove functional involvement, it raises the possibility of a broader role for EB1 in regulating microtubule dynamics beyond sexual stages. To avoid overinterpretation, we have presented these findings in the supplementary material and will revise the manuscript to tone down speculative statements and clearly frame this as a preliminary observation that warrants further investigation.

(7) Line 196 onwards: The antibody IN105 is better known in the field as polyE. Maybe that should be added in Materials and Methods. Also, the antibody T9028 against tyrosinated tubulin is poorly validated in the literature and rarely used. Usually, researchers in this field use the monoclonal antibody YL1/2. I am not sure why this unusual antibody was chosen in this study. In fact, has its specificity against tyrosinated α-tubulin from Plasmodium berghei ever been shown? The original antigen was human and had the sequence EGEEY. The Plasmodium sequence is YEADY and hence very different. It is stated that the LSPMB is both polyglutamylated and tyrosinated. This is unusual because polyglutamylated microtubules are usually indicative of stable microtubules, whereas tyrosinated microtubules are found on freshly polymerised and dynamic microtubules. However, a co-localisation within the same cell has not been attempted. This is, however, possible since polyE is a rabbit antibody and T9028 is a mouse antibody. I suspect that differences or gradients along the LSPMB would have been noticed. Also, in lines 207/208, it is said that tyrosination disappears after hepatocyte invasion, which is shown in Figure 3. However, in Figure 3A, quite a lot of positive signals for tyrosination are visible in the 54 and 56 hpi panels.

First, we acknowledge that the IN105 antibody is more widely known as "polyE" in the field. We will update the Materials and Methods section accordingly to reflect this nomenclature.

Regarding the use of the T9028 antibody against tyrosinated α-tubulin: we agree that this monoclonal antibody is less commonly used than YL1/2, and we appreciate the reviewer drawing attention to this. The original antigen for T9028 is based on the mammalian C-terminal sequence EGEEY, which differs from the Plasmodium α1-tubulin sequence (YEADY). Like many in the field, we face the challenge that most available antibodies are raised against mammalian epitopes, and specificity in Plasmodium can vary. Nonetheless, the literature (e.g., Hirst et al., 2022; Fennell et al., 2008) has demonstrated that tyrosination occurs in Plasmodium α1-tubulin, using anti-tyrosination antibodies including YL1/2.

Following the reviewer’s excellent suggestion, we are currently repeating the key experiments using the YL1/2 antibody to compare staining patterns directly with those obtained using T9028. We will include these results in the revised manuscript.

Concerning the potential co-localization of polyglutamylation and tyrosination on the LSPMB: we agree that this is an interesting and testable hypothesis. In the current manuscript, Figures 3A and 3B were generated from independent experiments, and thus co-localization was not assessed. However, as the reviewer correctly notes, polyE and T9028 antibodies are raised in rabbit and mouse, respectively, making co-staining feasible. We will follow up on this experimentally and, if feasible within our revision timeline, include data in the revised version or highlight this as a future direction.

Finally, with regard to Figure 3 and the observation that tyrosination appears to persist at 54 and 56 hpi (Figure 3B): the reviewer is correct that tyrosination signal is still detectable at these time points. Our statement that tyrosination “disappears after hepatocyte invasion” was intended to refer to an overall decrease in signal intensity during early liver stage development, with a reappearance at later stages (e.g., cytomere formation). We will rephrase this section for greater clarity and ensure that figure annotations and legends unambiguously reflect the dynamics observed.

(8) In line 229, it is stated that tyrosination "has previously been associated with stable microtubule in motility". This statement is not correct. In fact, none of the cited references that apparently support this statement show that this is the case. On the contrary, stable microtubules, such as flagellar axonemes, are almost completely detyrosinated. Therefore, tyrosination is a marker for dynamic microtubules, whereas detyrosinated microtubules are indicative of stable microtubules. This is an established fact, and it is odd that the authors claim the opposite.

We fully agree that in canonical eukaryotic systems, tyrosinated microtubules are generally markers of dynamic microtubule populations, whereas detyrosinated microtubules are typically associated with stability particularly in structures such as flagellar axonemes.

Our original statement will be corrected. In our study, we observed that tyrosinated microtubules are prevalent in invasive stages (sporozoites and merozoites), while detyrosinated forms become more prominent during intracellular liver stage development. This pattern is consistent with the established link between tyrosination and dynamic microtubules.

What is particularly intriguing in Plasmodium is the apparent cycling of tyrosination despite the absence of known tubulin tyrosine ligase (TTL) homologs in the genome. This suggests either a highly divergent enzyme or the involvement of host cell factors, a hypothesis supported by the reappearance of tyrosinated microtubules during liver stage schizogony (Figure 3B).

We will revise the relevant text and the Discussion section to reflect these mechanistic considerations more accurately and to avoid misrepresenting established principles of microtubule biology.

(9) Line 236 onwards: Concerning the generation of tubulin mutants, I think it is necessary to demonstrate successful replacement of the wild-type allele by the mutant allele. I am sure the authors have done this by amplification and subsequent sequencing of the genomic locus using PCR primers outside the plasmid sequences. I suggest including this information, e.g., by displaying the chromatograph trace in a supplementary figure. Or are the sequences displayed in Figure S3B already derived from sequenced genomic DNA? This is not described in the Legend or in Materials and Methods. The left PCR products obtained for Figure S3 B would be a suitable template for sequencing.

Indeed, these data are presented in Figure 4B and the corresponding sequence data are shown in Figure S3B. We appreciate the reviewer’s suggestion, which will help improve the transparency and reproducibility of our methodology.

(10) It is also important to be aware of the fact that glutamylation also occurs on β-tubulin. This signal will also be detected by polyE (IN105). Therefore, it is surprising that IN105 immunofluorescence is negative on the C-term Δ cells (Figure S3 D). Is there anything known about confirmed polyglutamylation sites on both α- and β-tubulins in Plasmodium, e.g., by MS? In Toxoplasma, both α- and β-tubulin have been shown to be polyglutamylated.

Indeed, polyglutamylation is known to occur not only on α-tubulin but also on β-tubulin in many organisms, including Toxoplasma gondii, and the polyE (IN105) antibody is expected to detect polyglutamylation on both tubulin isoforms.

The parasites shown in Figure S3D correspond to mutant lines originally generated by Spreng et al. (2019): the IntronΔ mutant (with deletion of introns in the Plasmodium α1-tubulin gene) and the C-termΔ mutant (with deletion of the final three C-terminal residues: ADY). As the reviewer correctly notes, this particular C-terminal deletion does not include the predicted polyglutamylation site (E445 or E447, depending on alignment), and thus should not abolish all polyglutamylation. However, in our experiments, the IN105 signal is substantially reduced in this mutant. This may suggest that structural alterations in the tubulin tail affect accessibility of the polyglutamylation epitope or influence the modification itself though we cannot exclude other possibilities, including changes in antibody recognition.

To date, polyglutamylation sites in Plasmodium tubulins have not been definitively confirmed by mass spectrometry. However, a recent MS-based study (reference 43) detected monoglutamylation on β-tubulin in blood stage parasites. Direct MS evidence for polyglutamylation of either α- or β-tubulin in Plasmodium liver stages is still lacking. We will clarify these points in the revised manuscript to avoid potential confusion and to highlight the need for future biochemical validation of PTM sites.

(11) Figure S3 is very confusing. In the legend, certain intron deletions are mentioned. How does this relate to posttranslational tubulin modifications? The corresponding section in Results (lines 288-292) is also not very helpful in understanding this.

The parasite lines shown in Figure S3D were originally generated by Spreng et al. (2019) and are not directly part of the main set of PTM-targeted mutants described in our study. Specifically, the IntronΔ line carries deletions in introns of the Plasmodium α1-tubulin gene, while the C-termΔ line lacks the final three C-terminal residues (ADY). These lines were included for comparative purposes to explore whether structural changes in α-tubulin could impact polyglutamylation signal, as detected by the polyE (IN105) antibody.

We acknowledge that the figure legend and corresponding text (lines 288–292) did not adequately explain the rationale for including these control lines. We will revise both the legend and Results section to more clearly describe the origin, purpose, and relevance of these mutants to the overall study.

(12) Figure 4E doesn't look like brightfield microscopy but like some sort of fluorescent imaging. In Figure 4C, were the control (NoΔ) cells with an integrated cassette, but no mutations, or non-transgenic cells?

The reviewer is absolutely correct: Figure 4E shows a fluorescent image acquired using widefield microscopy and not a brightfield image. We will revise the figure legend accordingly to avoid confusion. The “BF” (brightfield) label applies only to the left panel in Figure 4C, which depicts oocysts imaged using transmitted light.

Regarding the controls labeled "NoΔ" in Figure 4C, we confirm that these parasites contain the integrated selection cassette but do not harbor any mutations in the target gene. They serve as proper integration controls, allowing us to distinguish the effects of the point mutations or deletions introduced in the experimental lines.

(13) It is difficult to understand why the TyΔ and the CtΔ mutants still show quite a strong signal using the anti-tyrosination antibody. If the mutants have replaced all wild-type alleles, the signal should be completely absent, unless the antibody (see my comment above concerning T9028) cross-reacts with detyrosinated microtubules. Therefore, the quantitation in Figures 5F and 5G is actually indicative of something that shouldn't be like that. The quantitation of 5F is at odds with the microscopy image in 5D. If this image is representative, the anti-Ty staining in TyΔ is as strong as in the control NoΔ.

We agree that the persistence of anti-tyrosination signal in the TyΔ and CtΔ mutant lines is unexpected, given that all wild-type alleles were replaced. This discrepancy has led us to further investigate the specificity of the T9028 antibody, as raised in the reviewer’s earlier comment. To address this concern, we are currently repeating the key experiments using the well-established YL1/2 monoclonal antibody, which is widely accepted for detecting tyrosinated α-tubulin in other systems.

We also acknowledge that Figure 5F shows residual tyrosination signal, and the reviewer is correct that this should not occur if the modified residues are the exclusive PTM sites. One possible explanation is that adjacent residues or even alternative tubulin isoforms may serve as substrates. While α1-tubulin is the dominant isoform in Plasmodium, low-level expression of α2-tubulin has been detected in liver stages based on transcriptomic data, and it may contribute to the observed signal.

Regarding the apparent discrepancy between the quantification in Figure 5F and the representative image in Figure 5D, we will revise the figure legend to clarify that image selection aimed to show detectable signal, not necessarily the average phenotype. We will also reassess and, if needed, repeat the quantification with improved image sets to ensure accuracy and consistency.

We will revise the manuscript to reflect these points and include a more nuanced interpretation of the residual staining in the mutant lines.

(14) The statement that the failure of CtΔ mutants to generate viable sporozoites is due to the lack of microtubule PTMs (lines 295-296) is speculative. The lack of the entire C-terminal tail could have a number of consequences, such as impaired microtubule assembly or failure to recruit and bind associated proteins. This is not necessarily linked to PTMs. Also, it has been shown in yeast that for microtubules to form properly and exquisite regulation (proteostasis) of the ratio between α- and β-tubulin is essential (Wethekam and Moore, 2023). I am not sure, but according to Materials and Methods (line 423), the gene cassettes for replacing the wild-type tubulin gene with the mutant versions contain a selectable marker gene for pyrimethamine selection. Are there qPCR data that show that expression levels of mutant α-tubulin are more or less the same as the wild-type levels?

We agree that attributing the developmental failure of the CtΔ mutants solely to the absence of microtubule post-translational modifications (PTMs) is speculative. As the reviewer rightly points out, deletion of the entire C-terminal tail may have multiple effects, including impaired microtubule assembly, altered α/β-tubulin stoichiometry, or disruption of interactions with essential microtubule-associated proteins (MAPs). These consequences may arise independently of PTMs.

That said, we note that PTMs particularly polyglutamylation, can modulate MAP binding by altering the surface charge of microtubules (Genova et al., 2023; Mitchell et al., 2010). Therefore, while PTM loss may be a contributing factor, we acknowledge that the phenotype likely results from a combination of mechanisms. We will revise the relevant section of the manuscript to present a more cautious and balanced interpretation.

Regarding the reviewer’s question on expression levels: although the replacement constructs include a pyrimethamine resistance cassette, we have not yet quantified α-tubulin transcript levels by qPCR. In the interim, the study by Spreng et al. (2019) (reference 50) on a related α1-tubulin nutations provides valuable insight. They observed no difference in mRNA levels in day 12 oocysts, yet reported fainter microtubule staining and shorter sporozoites, suggesting a post-transcriptional mechanism affecting protein expression or function in later stages. Furthermore, the phenotypic spectrum across their mutant panel (Suppl. Fig. 3 D and E) implies that robust α-tubulin regulation is highly sensitive to specific sequences.

We acknowledge this as a current limitation in our study and will address it in the revised manuscript, noting that direct measurement of transcript levels is a key area for future investigation.

(15) In the Discussion, my impression is that two recent studies, the superb Expansion Microscopy study by Bertiaux et al. (2021) and the cryo-EM study by Ferreira et al. (2023), are not sufficiently recognised (although they are cited elsewhere in the manuscript). The latter study includes a detailed description of the microtubule cytoskeleton in sporozoites. However, the present study clearly expands the knowledge about the structure of the cytoskeleton in liver stage parasites and is one of the few studies addressing the distribution and function of microtubule post-translational modifications in Plasmodium.

Indeed, our work builds upon the established knowledge from Bertiaux et al. (2021) and the cryo-EM study by Ferreira et al. (2023), as rightly mentioned by the reviewer. We agree that these foundational studies, combined with our findings, will significantly expand the understanding of Plasmodium biology and cytoskeleton dynamics across its life cycle and will open the door for further investigations. We are grateful for this suggestion and will ensure these key studies are appropriately acknowledged in the revised manuscript.

(16) I somewhat disagree with the statement of a co-occurrence of polyglutamylated and tyrosinated microtubules. I think the resolution is too low to reach that conclusion. As this is a bold claim, and would be contrary to what is known from other organisms, it would require a more rigorous validation. Given the apparent problems with the anti-Ty antibody (signal in the TyΔ mutant), one should be very cautious with this claim.

This is a very important point to clarify. As mentioned previously, the initial experiments for these modifications were performed independently. It is established that sporozoite subpellicular microtubules exhibit both tyrosination and polyglutamylation. We will revise the manuscript to temper this statement and clearly indicate that the co-occurrence of these PTMs remains a hypothesis that requires more rigorous validation. As suggested, we are now conducting additional co-staining experiments using the better validated YL1/2 antibody to re-express and directly compare the distribution of both PTMs within the same cell. These follow-up experiments will help clarify whether both modifications occur simultaneously on the same microtubule structures in Plasmodium liver stages.

(17) In the Discussion (lines 311 and 377), it is again claimed that tyrosinated microtubules are "a well-known marker of stable microtubules". This statement is completely incorrect, and I am surprised by this serious mistake. A few lines later, the authors say that polyglutamylated is "commonly associated with dynamic microtubule behaviour". Again, this is completely incorrect and is the opposite of what is firmly established in the literature. Polyglutamylation and detyrosination are markers of stable microtubules.

Indeed, in canonical eukaryotic systems, tyrosinated microtubules are generally considered markers of dynamic microtubule populations, whereas detyrosinated and polyglutamylated microtubules are more commonly associated with stability.

We acknowledge this mistake and will revise the Discussion to correct these statements accordingly. In the context of Plasmodium, our observations suggest an unusual regulation of microtubule dynamics, which may reflect parasite-specific adaptations. For example, we observed tyrosinated α-tubulin in the stable subpellicular microtubules of sporozoites structures typically known for their exceptional stability. This atypical association implies either non-canonical roles for tyrosination or parasite-specific mechanisms for modulating microtubule properties. Additionally, the presence of both PTMs at different stages of development and on different microtubule populations suggests tightly regulated spatial and temporal modulation of microtubule function.

We will carefully revise the relevant sections of the manuscript to remove incorrect generalizations and ensure accurate representation of the current consensus in the field, while emphasizing the possibility of Plasmodium-specific adaptations that merit further study.

(18) In line 339, the authors interpret the residual antibody staining after the introduction of the mutant tubulin as a compensatory mechanism. There is no evidence for this. More likely explanations are firstly the quality of the anti-Ty-antibody used (see comment above), and the fact that also β-tubulin carries C-terminal polyglutamylation sites, which haven't been investigated in this study. PTMs on β-tubulin are not compensatory, but normal PTMs, at least in all other organisms where microtubule PTMs have been investigated.

As mentioned above, we are currently repeating the key experiments with the [YL1/2] antibody, as suggested. Furthermore, we fully agree with the reviewer's point regarding polyglutamylation on β-tubulin. The C-terminal tail of β-tubulin does indeed contain polyglutamylation sites. As we noted in the manuscript (Lines 340-352), this aspect has not been investigated in the present study, and we acknowledge it as a valuable direction for future research. We will revise the text accordingly to avoid overinterpretation and to more accurately reflect the limitations of our current data.

DOI: 10.1038/s44318-025-00532-y

Resource: RRID:Addgene_53117

Curator: @dhovakimyan1

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DOI: 10.1186/s12877-025-06237-5

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2848250

What is this?

DOI: 10.1177/15347354251368410

Resource: (Cell Signaling Technology Cat# 15101, RRID:AB_2722591)

Curator: @scibot

SciCrunch record: RRID:AB_2722591

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DOI: 10.1126/sciimmunol.adt6660

Resource: (BioLegend Cat# 302326, RRID:AB_893283)

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DOI: 10.1101/2025.08.17.670702

Resource: (Vector Laboratories Cat# BA-1000, RRID:AB_2313606)

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DOI: 10.1038/s44318-025-00547-5

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DOI: 10.1016/j.devcel.2025.07.023

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Curator: @scibot

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What is this?

* get in = interponerte. * fuck of = vete a la mierda

Entiendo que aquí empieza la relación cohesión y migración, pero lo haría más explicito en el topic sentence. Tipo: Los niveles y formas de la cohesión social dependen fuertemente de la inmigración. Algo así

Esto podría fusionarse pues dicen lo mismo

Unificar parrafos en caracterizacion de la pob migrante

On July 2, 1839, enslaved Africans aboard La Amistad revolted against their captors

"source" i made it up

C'est encore largement comme cela aujourd'hui. Une personne qui veut avoir de l'attention doit aller à Paris pour "faire carrière". C'est valable pour le business comme pour la recherche ou la littérature. Cela paraît dingue pour tout le monde, mais la centralisation parisienne est encore trop ancrée dans nos gênes.

Reviewer #2 (Public review):

Summary

Calcium ions play a key role in synaptic transmission and plasticity. To improve calcium measurements at synaptic terminals, previous studies have targeted genetically encoded calcium indicators (GECIs) to pre- and postsynaptic locations. Here, Chen et al. improve these constructs by incorporating the latest GCaMP8 sensors and a stable red fluorescent protein to enable ratiometric measurements. In addition, they develop a new analysis platform, 'CaFire', to facilitate automated quantification. Using these tools, the authors demonstrate favorable properties of their sensors relative to earlier constructs. Impressively, by positioning postsynaptic GCaMP8m near glutamate receptors, they show that their sensors can report miniature synaptic events with speed and sensitivity approaching that of intracellular electrophysiological recordings. These new sensors and the analysis platform provide a valuable tool for resolving synaptic events using all-optical methods.

Strengths:

The authors present a rigorous characterization of their sensors using well-established assays. They employ immunostaining and super-resolution STED microscopy to confirm correct subcellular targeting. Additionally, they quantify response amplitude, rise and decay kinetics, and provide side-by-side comparisons with earlier-generation GECIs. Importantly, they show that the new sensors can reproduce known differences in evoked Ca²⁺ responses between distinct nerve terminals. Finally, they present what appears to be the first simultaneous calcium imaging and intracellular mEPSP recording to directly assess the sensitivity of different sensors in detecting individual miniature synaptic events.

Weaknesses:

Major points:

(1) While the authors rigorously compared the response amplitude, rise, and decay kinetics of several sensors, key parameters like brightness and photobleaching rates are not reported. I feel that including this information is important as synaptically tethered sensors, compared to freely diffusible cytosolic indicators, can be especially prone to photobleaching, particularly under the high-intensity illumination and high-magnification conditions required for synaptic imaging. Quantifying baseline brightness and photobleaching rates would add valuable information for researchers intending to adopt these tools, especially in the context of prolonged or high-speed imaging experiments.

(2) In several places, the authors compare the performance of their sensors with synthetic calcium dyes, but these comparisons are based on literature values rather than on side-by-side measurements in the same preparation. Given differences in imaging conditions across studies (e.g., illumination, camera sensitivity, and noise), parameters like indicator brightness, SNR, and photobleaching are difficult to compare meaningfully. Additionally, the limited frame rate used in the present study may preclude accurate assessment of rise times relative to fast chemical dyes. These issues weaken the claim made in the abstract that "...a ratiometric presynaptic GCaMP8m sensor accurately captures .. Ca²⁺ changes with superior sensitivity and similar kinetics compared to chemical dyes." The authors should clearly acknowledge these limitations and soften their conclusions. A direct comparison in the same system, if feasible, would greatly strengthen the manuscript.

(3) The authors state that their indicators can now achieve measurements previously attainable with chemical dyes and electrophysiology. I encourage the authors to also consider how their tools might enable new measurements beyond what these traditional techniques allow. For example, while electrophysiology can detect summed mEPSPs across synapses, imaging could go a step further by spatially resolving the synaptic origin of individual mEPSP events. One could, for instance, image MN-Ib and MN-Is simultaneously without silencing either input, and detect mEPSP events specific to each synapse. This would enable synapse-specific mapping of quantal events - something electrophysiology alone cannot provide. Demonstrating even a proof-of-principle along these lines could highlight the unique advantages of the new tools by showing that they not only match previous methods but also enable new types of measurements.

(4) For ratiometric measurements, it is important to estimate and subtract background signals in each channel. Without this correction, the computed ratio may be skewed, as background adds an offset to both channels and can distort the ratio. However, it is not clear from the Methods section whether, or how, background fluorescence was measured and subtracted.

(5) At line 212, the authors claim "... GCaMP8m showing 345.7% higher SNR over GCaMP6s....(Fig. 3D and E) ", yet the cited figure panels do not present any SNR quantification. Figures 3D and E only show response amplitudes and kinetics, which are distinct from SNR. The methods section also does not describe details for how SNR was defined or computed.

(6) Lines 285-287 "As expected, summed ΔF values scaled strongly and positively with AZ size (Fig. 5F), reflecting a greater number of Cav2 channels at larger AZs". I am not sure about this conclusion. A positive correlation between summed ΔF values and AZ size could simply reflect more GCaMP molecules in larger AZs, which would give rise to larger total fluorescence change even at a given level of calcium increase.

(7) Lines 313-314: "SynapGCaMP quantal signals appeared to qualitatively reflect the same events measured with electrophysiological recordings (Fig. 6D)." This statement is quite confusing. In Figure 6D, the corresponding calcium and ephys traces look completely different and appear to reflect distinct sets of events. It was only after reading Figure 7 that I realized the traces shown in Figure 6D might not have been recorded simultaneously. The authors should clarify this point.

(8) Lines 310-313: "SynapGCaMP8m .... striking an optimal balance between speed and sensitivity", and Lines 314-316: "We conclude that SynapGCaMP8m is an optimal indicator to measure quantal transmission events at the synapse." Statements like these are subjective. In the authors' own comparison, GCaMP8m is significantly slower than GCaMP8f (at least in terms of decay time), despite having a moderately higher response amplitude. It is therefore unclear why GCaMP8m is considered 'optimal'. The authors should clarify this point or explain their rationale for prioritizing response amplitude over speed in the context of their application.

Reviewer #1 (Public review):

Summary:

This study probes the role of the NF-κB inhibitor IκBa in the regulation of pluripotency in mouse embyronic stem cells (mESCs). It follows from previous work that identified a chromatin-specific role for IκBa in the regulation of tissue stem cell differentiation. The work presented here shows that a fraction of IκBa specifically associates with chromatin in pluripotent stem cells. Using three Nfkbia-knockout lines, the authors show that IκBa ablation impairs the exit from pluripotency, with embryonic bodies (an in vitro model of mESC multi-lineage differentiation) still expressing high levels of pluripotency markers after sustained exposure to differentiation signals. The maintenance of aberrant pluripotency gene expression under differentiation conditions is accompanied by pluripotency-associated epigenetic profiles of DNA methylation and histone marks. Using elegant separation of function mutants identified in a separate study, the authors generate versions of IκBa that are either impaired in histone/chromatin binding or NF-κB binding. They show that the provision of the WT IκBa, or the NF-κB-binding mutant can rescue the changes in gene expression driven by loss of IκBa, but the chromatin-binding mutant can not. Thus the study identifies a chromatin-specific, NF-κB-independent role of IκBa as a regulator of exit from pluripotency.

Strengths:

The strengths of the manuscript lie in:<br /> (a) the use of several orthogonal assays to support the conclusions on the effects of exit from pluripotency;<br /> (b) the use of three independent clonal Nfkbia-KO mESC lines (lacking IκBa), which increase confidence in the conclusions; and<br /> (c) the use of separation of function mutants to determine the relative contributions of the chromatin-associated and NF-κB-associated IκBa, which would otherwise be very difficult to unpick.

Weaknesses:

No notable weaknesses remain in this revised version.

Falsche Figur. Sie ist das Original vom Figure 2 und soll mit sie verbindet werden da wir englische Übersetzung wollten. Hier Figure 18

ll

https://www.youtube.com/watch?v=trdxrBVknXg

Synthèse : Le Malaise de l'Éducation Nationale Française Ce dossier de briefing examine les multiples facettes du malaise qui ronge l'Éducation Nationale française, révélant une institution en crise, tiraillée entre violence, surcharge de travail, manque de reconnaissance, inégalités et un rôle éducatif élargi qui dépasse largement sa mission d'instruction.

Le suicide tragique de Christine Renon, directrice d'école, sert de catalyseur pour explorer ces problèmes systémiques.

I. Une Institution Ébranlée : Violence, Incivilités et Perte de Repères

L'éducation nationale, pilier de la République française, est confrontée à une perte de repères.

La violence et les incivilités, bien que rares en leur forme extrême (comme l'incident filmé dans un lycée de Seine-et-Marne), contribuent à ce sentiment.

Violences et Incivilités : Le document s'ouvre sur l'image choc d'un élève violentant un personnel enseignant, un "geste rare mais pas isolé". Ce phénomène n'est pas la seule raison du malaise, mais il est significatif.

Au collège Côrousse en REP (Réseau d'Éducation Prioritaire) à Chambéry, les mesures de sécurité sont renforcées : "ils doivent présenter leur carnet à chaque entrée et à chaque sortie, enlever capuche et tout ce qui pourrait couvrir leur visage".

Perception du Public : Un sondage Ipsos révèle que "94 % des parents souhaitent le retour de l'autorité à l'école", illustrant une inquiétude généralisée.

Compréhension des Codes : Les enseignants en REP se heurtent à la difficulté que "les élèves ici n'ont pas les codes", conduisant à des malentendus profonds.

Isabelle Dumet, professeure d'histoire-géographie, cite un élève lui ayant dit "Madame tu me casses les couilles", et un autre qui a explosé de colère après qu'elle lui ait dit "arrête de m'aboyer dessus", l'interprétant comme une insulte grave : "Vous me dites que je suis un chien mais qu'est-ce que c'est ça ?".

II. Surcharge de Travail, Isolement et Souffrance des Directeurs d'École

Le suicide de Christine Renon, directrice d'école primaire à Pantin en septembre 2019, est l'illustration la plus tragique de la surcharge et de l'isolement des directeurs.

Sa lettre, envoyée à ses collègues, dépeint un tableau accablant : "je n'ai pas confiance au soutien et à la protection que devrait nous apporter notre institution et pour finir je me demande si je ne ferai pas une petite déprime".

Un "Geste Politique" : Sabine, syndicaliste, affirme que le suicide de Christine est "un geste politique, il est marqué par la lourdeur de ses conditions de travail, par son épuisement général lié à son à son travail".

Poste "d'Hommes et de Femmes à Tout Faire" : Les directeurs d'école primaire sont décrits comme des "hommes et des femmes à tout faire sans équipe pour les épauler comme dans le secondaire", ce qui génère un profond "isolement".

Missions Débordantes : La fonction de directeur dépasse largement la seule mission d'instruction. L'école devient un "point de ressources pour les familles" qui viennent chercher de l'aide pour "remplir des papiers", résoudre des "problèmes de logement". Sandrine Delmas, directrice à Pantin, confirme : "tout ça ça fait partie aussi des choses en plus des questions de l'école qu'on retrouve dans ce quartier".

L'Effet Miroir de la Lettre : La lettre de Christine a eu un "effet miroir" pour de nombreux collègues, les aidant à prendre conscience de la surcharge de travail et de la solitude.

Sandrine exprime ce sentiment : "notre métier c'est tout ça c'est tout ça et c'est pas tout à fait normal que tout ça pèse sur les épaules d'une seule personne".

Réponse Insuffisante de l'Institution : Deux ans après le suicide de Christine, "peu de choses ont changé" selon Sabine.

Le rectorat a tenté d'"étouffer l'affaire" et les "améliorations" promises pour alléger les tâches administratives ne se sont pas concrétisées.

III. Inégalités Territoriales et de Moyens

Le système éducatif français est paradoxal : il revendique l'égalité des chances tout en présentant de profondes inégalités de moyens.

Pantin, Ville à Deux Vitesses : Pantin, en Seine-Saint-Denis (93), est coupée en deux : d'un côté "les quartiers pauvres, parfois insalubres" où "les 3/4 des familles sont au chômage, certaines parlent à peine le français", et de l'autre "les quartiers bobo" en pleine expansion. Sur 11 écoles primaires, six sont en REP, "c'est énorme".

L'Origine Sociale : "La France est l'un des pays développés où l'origine sociale pèse le plus sur les trajectoires scolaires", résumant la situation par "dis-moi quel est le salaire de tes parents et je te dirai si tu réussiras".

Budgets Inégaux : Annabelle, ancienne directrice d'école, dénonce les "inégalités au niveau des territoires" et les budgets "qui varient du simple au double au triple" par enfant.

Elle souligne : "l'égalité des chances elle passe aussi parce qu'on va apporter en terme de moyen à l'école".

"Mixité Sociale" : Cédric Pusser, jeune directeur à Saint-Paul (Haute-Vienne), dans une école privilégiée, s'interroge sur la préparation de ses élèves à la vie en dehors de leur "environnement tellement protégé" et souligne l'importance d'une "mixité sociale".

IV. Désenchantement de la Profession Enseignante

Les conditions de travail et la faible reconnaissance salariale contribuent à un désenchantement croissant et à une crise des vocations.

Salaires Insuffisants : Avec un Bac+5, un instituteur débutant gagne en moyenne 1800 € net par mois, comparé au SMIC à 1200 € net. La France est en "20e position des pays développés" pour la revalorisation des salaires. Les témoignages révèlent une stagnation, voire une baisse du pouvoir d'achat : "j'ai pas vu bouger mon salaire du tout j'ai vu mon pouvoir d'achat baisser".

Crise des Vocations et Démissions : "Qui veut être enseignant aujourd'hui en France ? Je crois ceux qui ont la foi, ceux qui ont la vocation". Cependant, de plus en plus de personnes "rentrent dans le métier qui n'y restent pas, qui démissionnent, qui s'en vont parce que les conditions de travail ne sont pas forcément celle auxquelles ils s'attendaient".

En 2018, "1400 enseignants ont démissionné, c'est quatre fois plus qu'il y a 10 ans".

Non-Remplacement des Enseignants : Le document pointe un problème grave : "entre la rentrée des vacances des grandes vacances [...] jusqu'aux vacances d'automne, on a quand même 107 jours de classes qui n'ont pas été remplacés", ce qui représente "au moins un jour sans enseignant dans la classe".

Manque de Reconnaissance : Sandrine exprime le sentiment que les parents ne voient pas les enseignants comme des "professionnels de l'enseignement", attribuant cette perception aux "politiques éducatives" et aux clichés sur les fonctionnaires "toujours en vacances".

Elle insiste sur le "travail qu'on ne voit pas mais qui se fait à la maison".

V. Entre Instruire et Éduquer : Un Débat Central

Le débat entre "instruire ou éduquer" est au cœur des préoccupations et de la campagne présidentielle. Si la droite insiste sur le "retour au savoirs fondamentaux", la réalité du terrain est plus complexe.

La Priorité en REP : La directrice du collège Côrousse affirme que "quand on travaille en collège en REP la priorité c'est l'éducation".

Elle va plus loin en disant : "l'éducation nationale n'a jamais été aussi bien nommée que maintenant éducation nationale".

Pour elle, l'instruction n'est qu'un "prétexte" pour "poser les fondamentaux d'un citoyen éclairé autonome".

Former le Futur Citoyen : La mission du collège est de former "un futur citoyen", de lui donner un "esprit critique, une liberté" et de lui "remplir leur besace" pour qu'il puisse "choisir ce qu'ils en font ou pas".

Le Conseil de Discipline comme Acte Éducatif : Le conseil de discipline de Karim, un élève ayant dégradé une porte, est présenté comme un "acte éducatif" plutôt qu'une simple sanction.

La directrice explique à l'élève : "l'objectif du jour c'est de poser un acte éducatif c'est pas de sanctionner pour sanctionner c'est que tu comprennes ce que tu as fait".

Elle insiste sur la gravité de l'acte en comparant la situation à un "début de tribunal".

VI. L'Omerta et le Déni des Responsables Politiques

Le document met en lumière une résistance des autorités à aborder les problèmes de front.

Refus de Coopération : Le rectorat de Créteil a "refusé d'entrer dans les écoles de Pantin", le ministre de l'Éducation nationale a "décommandé une interview à la dernière minute", et la mairie de Pantin n'a "jamais répondu à nos sollicitations".

Sandrine Delmas pense que c'est lié à "la peur de d'encore étiqueter le 93 comme un département compliqué" et au "suicide de Christine" qui a suscité des "paroles qui ont dérangé l'institution".

Promesses Électorales Récurrentes : Les candidats à la présidentielle s'emparent de la question de l'éducation nationale avec des promesses de réformes, mais le document souligne que c'est une constante "à chaque élection présidentielle depuis 20 ans".

Conclusion

Le système éducatif français est à un point de rupture. Entre la violence en milieu scolaire, la détresse des personnels due à la surcharge et au manque de reconnaissance, les inégalités criantes entre territoires et la crise des vocations, l'urgence est réelle.

L'école, traditionnellement un lieu d'instruction, est devenue un acteur social et éducatif majeur, souvent sans les moyens nécessaires.

Le silence et le déni des autorités face à ces problématiques exacerbent le sentiment d'abandon des acteurs de terrain.

Le suicide de Christine Renon n'est pas un cas isolé, mais le symbole d'un "malaise bien plus répandu qu'on l'imagine".

Si "l'institution n'est pas encore en danger, il y a urgence".

Bonjour ! je n'ai bien compris pourquoi dans le code css dans la correction le selecteur "cta", on lui ajoute du PADDING : 20PX 30PX alors que ce n'est pas demandé dans l'exercice de Robbie Lens

Reviewer #2 (Public review):

General comments

We thank the reviewers and editor for their thoughtful feedback. We are glad that the minor comments appear resolved. In this revision, we added subject-specific analyses, further FC comparisons, and clarified our rationale for stimulation parameters. We acknowledge that two concerns remain: (1) the 1 mA-2 mA sequence may introduce confounds, and (2) electric field modeling was not included due to technical limitations. We now explicitly note these as limitations in the manuscript and provide justification and discussion accordingly.

Major comments

R.2.1. For the anesthetized monkeys, the anode location differs between subjects, with the electrode positioned to stimulate the left DLFPC in monkey R and the right DLPFC in monkey N. The authors mention that this discrepancy does not result in significant differences in the electric field due to the monkeys' small head size. However, this is incorrect, as placing the anode on the left hemisphere would result in a much lower EF in the right DLPFC than placing the anode on the right side. Running an electric field simulation would confirm this. Additionally, the small electrode size suggested by the Easy cap configuration for NHP appears sufficient to stimulate the targeted regions focally. If this interpretation is correct, the authors should provide additional evidence to support their claim, such as a computational simulation of the EF distribution.

R.2.1 Authors' answer: We thank the Reviewer for the comments. First, regarding the reviewer's statement that placing the anode on the left hemisphere would result in a much lower EF in the right DLPFC than placing the anode on the right side, we would like to clarify that we did not use a typical 4 x 1 concentric ring high-definition setup (which consists of a small centre electrode surrounded by four return electrodes), but a two-electrode montage, with one electrode over the left or right PFC and the other one over the contralateral occipital cortex. According to EF modelling papers, a 4 x 1 high-definition setup would produce an EF that is focused and limited to the cortical area circumscribed by the ring of the return electrodes (Datta et al. 2009; Alam et al. 2016). Therefore, targeting the left or right DLPFC with a 4 x 1 setup would produce an EF confined to the targeted hemisphere of the PFC. In contrast, we expect the brain current flow generated with our 2-electrode setup to be broader, despite the small size of the electrodes, because there is no constraint from return electrodes. Thus, with our setup, the current is expected to flow between the PFC and the occipital cortex (see also our responses to comments R3.3., R.E.C.#2.1. and R.E.C.#2.2.).

Second, we would like to point out that in awake experiments, in which we stimulated the right PFC of both monkeys, there was no gross evidence of left or right asymmetry in the computed functional connectivity patterns (Figure 3A, Figure 3 - figure supplement 2A; Figure 5A). These results, showing that our stimulation montages did not induce asymmetric dynamic FC changes in NHPs, support the idea that our setups did not generate EFs that were spatially focused enough to alter brain activity in one hemisphere substantially more than the other.

Third, it is also worth noting that current evidence suggests that human brains are significantly more lateralized than those of macaques. Macaque monkeys have been found to have some degree of lateralized networks, but these are of lower complexity, and the lateralization is less pronounced and functionally organized than in humans. (Whey et al., 2014; Mantini et al., 2013). This suggests that, even if the stimulation were focal enough to stimulate the left or the right part of the PFC only, the behavioural effects would likely be similar.

Follow-up comment: Thank you for the detailed response and for referencing both experimental data and prior literature. While I appreciate the discussion on the lack of functional asymmetry and reduced lateralization in macaques, my original concern was about the physical distribution of the electric field (EF) due to different anode placements. Functional connectivity outcomes do not necessarily reflect EF symmetry, and without EF modeling, it's difficult to determine whether the stimulation affected both hemispheres equally. I understand the challenges of NHP-specific modeling, but even a simplified simulation or acknowledgment of this limitation in the manuscript would help clarify the interpretability of your results.

R.2.2. For the anesthetized monkeys, the authors applied 1 mA tDCS first, followed by 2 mA tDCS. A 20-minute stimulation duration of 1 mA tDCS is strong enough to produce after-effects that could influence the brain state during the 2 mA tDCS. This raises some concerns. Previous studies have shown that 1 mA tDCS can generate EF of over 1 V/m in the brain, and the effects of stimulation are sensitive to brain state (e.g., eye closed vs. eye open). How do the authors ensure that there are no after-effects from the 1 mA tDCS? This issue makes it challenging to directly compare the effects of 1 mA and 2 mA stimulation.<br /> R.2.2 Authors' answer: We agree with the reviewer's comment that 1 mA tDCS may induce aftereffects, as has been observed in several human studies (e.g., (Jamil et al. 2017, 2020). Although the differences between the 1 mA post-stimulation and baseline conditions were not significant in our analyses, it's still possible that the stimulation produced some effects below the threshold of significance that may contribute, albeit weakly, to the changes observed during

Follow-up comment: Thank you for the clarification and for acknowledging the potential for 1 mA after-effects. While I appreciate the authors' transparency and the amendment to the manuscript, I still find it important that the limitation be clearly stated in the Discussion section. The fact that 2 mA stimulation always followed 1 mA introduces a potential confound, making it difficult to attribute observed changes uniquely to 2 mA. If a counterbalanced design was not feasible, I would recommend explicitly noting this as a limitation in the interpretation of dose-dependent effects.

R.2.3. The occurrence rate of a specific structural-functional coupling pattern among random brain regions shows significant effects of tDCS. However, these results seem counterintuitive. It is generally understood that non-invasive brain stimulation tends to modulate functional connectivity rather than structural or structural-functional connectivity. How does the occurrence rate of structural-functional coupling patterns provide a more suitable measure of the effectiveness of tDCS than functional connectivity alone? I would recommend that the authors present the results based on functional connectivity itself. If there is no change in functional connectivity, the relevance of changes in structural-functional coupling might not translate into a meaningful alteration in brain function, making it unclear how significant this finding is without corresponding functional evidence.

R.2.3. Authors' answer: First of all, we would like to make it clear that the occurrence rate of patterns as a function of their SFC is not intended to be used or seen as a 'better' measure of the efficacy of tDCS. Instead, it is one aspect of the effects of tDCS on whole-brain functional cortical dynamics, obtained from refined measures (phase-coherences), that specifically addresses the coupling between structure and function. This type of analysis is further motivated by its increasing use in the literature due to its suspected relationship to wakefulness (e.g., (Barttfeld et al. 2015, Demertzi et al. 2019; Castro et al. 2023)). Also, in our analysis, the structure is kept constant: the connectivity matrix used to correlate the functional brain states is always the same (CoCoMac82). Thus, the influence of tDCS on the structure-function side can only be explained by modulating the functional aspects, as suggested by intuition and previous results.

Then, we agree with the reviewer that studying the functional changes induced by tDCS alone could be valuable. However, usual metrics used in FC analysis are usually done statistically: FC-states are either computed through averaging spatial correlations over time, then analyzed through graph-theoretical properties for instance (or by just directly computing the element-wise differences), or either by considering the properties of the different visited FC-states by computing spatial correlations over a sliding time-window, and then similar analysis can be done as previously explained. But these are static metrics, if the states visited are essentially the same (which is expected from non-invasive neuromodulations that haven't already demonstrated strong and/or characteristic impact), but the dynamical process of visiting said states changes, one would see no difference in that regard. As such, in the case of resting-state fMRI, differences in FCs are hard to interpret given that between-sessions within-condition differences are usually found with some degree of variance for the respective conditions. Trying then to interpret between-condition differences is quite tricky in the case of subtle modulations of the system's activity. On the other hand, more subtle differences can be captured by considering more detailed analysis, such as using phase-based methods like we did, by incorporating some statistical learning component with regard to the dynamicity of the system (supervised learning for instance like we did followed by temporal & transition-based methodology), and by adding some dimensions along which one will be able to give some interpretation to the analysis. In our case we were interested in characterizing resting-state differences between stimulation conditions, which have nuanced and subtle interactions with the biological system. As such, classical measures of differences between FC states are likely to not be refined and precise enough. In fact, we propose additional files investigating those classically used measures such as differences in average FC matrices, or changes in functional graph properties (like modularity, efficiency and density) of the visited FC states. These figures show that, for the first case, comparing region-to-region specific FCs provides very few statistically significant results. With respect to the second part, we show that virtually no differences are observed in the properties of the functional states visited. These results suggest, as expected, that the actual brain states visited across the different stimulation conditions are topologically quite similar, and that only very few region-specific pairwise functional connectivities are particularly modulated by specific tDCS montages while, on the other hand, the actual dynamical process dictating how the brain activity passes from one state to another is in fact being influenced as shown by the dynamical analysis presented in the main figures in a more apparent and meaningful way (in that it is dependent on the montage, somewhat consistent with regard to the post-stimulations conditions, and can be made sense of by considering the theoretical effect of near-anodal versus near-cathodal neuromodulatory effects).

Actions in the text: We have added new supplementary files showing the effects of the stimulations on FC matrices and on classical functional graph properties in awake and anesthesia datasets (Supplementary Files 3 & 4). We have added new sentences about these new analyses on the effects of the stimulations on FC matrices and on classical functional graph properties in the Results section:<br /> Follow-up comment: Thank you for the detailed and comprehensive response. The clarification regarding the use of SFC dynamics and the additional analyses provided are convincing.

R2.4. The authors recorded data from only two monkeys, which may limit the investigation of the group effects of tDCS. As the number of scans for the second monkey in each consciousness condition is lower than that in the first monkey, there is a concern that the main effects might primarily reflect the data from a single monkey. I suggest that the authors should analyze the data for each monkey individually to determine if similar trends are observed in both subjects.

R.2.4. Authors' answer: We agree that the small number of subjects is a limitation of our study. However, we have already addressed these aspects by reporting statistical analyses that consider them, using linear models of such variables, and running them through ANOVA tests. In addition, we experimentally ensured that we recorded a relatively high number of sessions over a period of several years. Regardless, we agree that our study would benefit from further investigation into this matter. We have therefore prepared complementary figures showing the main analysis performed separately for the two monkeys as proposed, as well as further investigations into the inter-condition variability outmatching the inter-individual variability, itself being also outmatched by intra-individual changes.

Actions in the text: We have added a supplementary file showing the main analyses performed separately for the two monkeys (Supplementary File 2) and further investigations into the inter-condition variability (Supplementary Files 3 & 4). We have added new sentences about these analyses performed separately for the two monkeys in the Results section:

Follow-up comment: Thank you for addressing this concern and for providing the individual monkey analysis. The additional figures and statistical explanations are helpful and appreciated.

R2.5. Anodal tDCS was only applied to anesthetized monkeys, which limits the conclusion that the authors are aiming for. It raises questions about the conclusion regarding brain state dependency. To address this, it would be better to include the cathodal tDCS session for anesthetized monkeys. If cathodal tDCS changes the connectivity during anesthesia, it becomes difficult to argue that the effects of cathodal tDCS vary depending on the state of consciousness as discussed in this paper. On the other hand, if cathodal tDCS would not produce any changes, the conclusion would then focus on the relationship between the polarity of tDCS and consciousness. In that case, the authors could maintain their conclusion but might need to refine it to reflect this specific relationship more accurately.

R.2.5. Authors' answer: We agree with the reviewer that it would have been interesting to investigate the effects of cathodal tDCS in anesthetized monkeys. However, due to the challenging nature of the experimental procedures under anesthesia, we had to limit the investigations to only one stimulation modality. We chose to deliver anodal stimulation because, from a translational point of view, we aimed to provide new information on the effects of tDCS under anesthesia as a model for disorders of consciousness. It also made much more sense to increase the cortical excitability of the prefrontal cortex in an attempt to wake up the sedated monkeys rather than doing the opposite.

Actions in the text: We have added a new sentence in the Results section:

"Due to the challenging nature of the experimental procedures under anesthesia, we limited the investigations to only one stimulation modality. We chose to deliver anodal stimulation to provide new information on the effects of tDCS under anesthesia as a model for disorders of consciousness and to increase the cortical excitability of the PFC in an attempt to wake up the sedated monkeys."

Follow-up comment: Thank you for clarifying the rationale behind applying only anodal stimulation under anesthesia. While I appreciate the experimental constraints and the translational motivation, I would still encourage the authors to explicitly acknowledge in the Discussion that the absence of a cathodal condition under anesthesia limits the ability to dissociate polarity-specific effects from state-dependent effects. This clarification would help temper the conclusions and better reflect the scope of the current dataset.

Reviewer #3 (Public review):

Summary:

This study used transcranial direct current stimulation administered using small 'high definition' electrodes to modulate neural activity within the non-human primate prefrontal cortex during both wakefulness and anaesthesia. Functional magnetic resonance imaging (fMRI) was used to assess neuromodulatory effects of stimulation. The authors report on modification of brain dynamics during and following anodal and cathodal stimulation during wakefulness and following anodal stimulation at two intensities (1 mA, 2 mA) during anaesthesia. This study provides some support that prefrontal direct current stimulation can alter neural activity patterns across wakefulness and sedation in monkeys.

Strengths and Weaknesses:

A key strength of this work is the use of fMRI-based methods to track changes in brain activity with good spatial precision. Another strength is the exploration of stimulation effects across wakefulness and sedation, which has the potential to provide novel information on the impact of electrical stimulation across states of consciousness. The authors should be commended for undertaking this challenging and important work.

The lack of a sham stimulation condition is a limitation of the study, as it somewhat restricts the certainty with which the results can be attributed to the active stimulation as opposed to other external factors such as drowsiness or fatigue as a result of the experimental procedure? Nevertheless, I acknowledge the demanding nature of performing this work in non-human primates and that only runs with high fixation rates were included, which should have helped reduce any fatigue-related effects.

In the anaesthesia condition, the authors investigated the effects of two intensities of stimulation (1 mA and 2 mA). However, it is possible that the initial 1 mA stimulation block might have caused some level of plasticity-related changes in neural activity that could have potentially interfered with the following 2 mA block due to the lack of a sufficient wash-out period. This potentially limits the findings from the 2 mA block as they cannot be interpreted as completely separate to the initial 1 mA stimulation period, given that they were administered consecutively. However, I do acknowledge the author's point that differences between the 1 mA post-stimulation and baseline conditions were not significantly different, which provides some evidence against this possibility.

The different electrode placement for the two anaesthetised monkeys (i.e., Monkey R: F3/O2 montage, Monkey N: F4/O1 montage) is potentially problematic, as it might have resulted in stimulation over different brain regions. Electric field models of brain current flow for the monkeys would really be needed to understand with reasonable certainty, however, I recognise that these models are generally designed for human studies making their integration into non-human primate studies challenging.

Finally, the sample size is obviously small. However, I realise this is often a limitation in non-human primate research, which can be both expensive and labour intensive.

Assessment:

This manuscript presents some novel insights into the effects of transcranial direct current stimulation on functional brain dynamics in awake and anaesthetised monkeys using MRI-based connectivity indices. Overall, the study presents several interesting and potentially impactful findings regarding stimulation-induced changes in brain activity. There are some limitations, such as the small sample size, lack of a sham stimulation control, and lack of electric field models, which, if included, would have, in my view, further helped improve the rigour of the study. Nevertheless, the manuscript presents several important findings, which warrant further analysis in future work.

Author response:

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

Reviewer #1 (Public review): 

Summary: 

In this work, the authors apply TDCS to awake and anesthetized macaques to determine the effect of this modality on dynamic connectivity measured by fMRI. The question is to understand the extent to which TDCS can influence conscious or unconscious states. Their target was the PFC. During the conscious states, the animals were executing a fixation task. Unconsciousness was achieved by administering a constant infusion of propofol and a continuous infusion of the muscle relaxant cisatracurium. They observed the animals while awake receiving anodal or cathodal hd-TDCS applied to the PFC. During the cathodal stimulation, they found disruption of functional connectivity patterns, enhanced structure-function correlations, a decrease in Shannon entropy, and a transition towards patterns that were more commonly anatomically based. In contrast under propofol anesthesia anodal hd-TDCS stimulation appreciably altered the brain connectivity patterns and decreased the correlation between structure and function. The PFC stimulations altered patterns associated with consciousness as well as those associated with unconsciousness.

Strengths: 

The authors carefully executed a set of very challenging experiments that involved applying tDCS in awake and anesthetized non-human primates while conducting functional imaging.

We thank the Reviewer for summarising our study and for his appreciation of the highly challenging experiments we performed.

Weaknesses:

The authors show that tDCS can alter functional connectivity measured by fMRI but they do not make clear what their studies teach the reader about the effects of tDCS on the brain during different states of consciousness. No important finding is stated contrary to what is stated in the abstract. It is also not clear what the work teaches us about how tDCS works nor is it clear what are the "clinical implications for disorders of consciousness." The deep anesthesia is akin to being in a state of coma. This was not discussed.  

While the authors have executed a set of technically challenging experiments, it is not clear what they teach us about how tDCS works, normal brain neurophysiology, or brain pathological states such as disorders of consciousness.

We thank the reviewer for his comments. We agree that we could better highlight the value and implications of our work, and we take this opportunity to improve our manuscript according to the suggestions.

Actions in the text: We have added several new paragraphs in the Discussion section, considering these comments and other related remarks from the Reviewing Editor (see below our answer to the first comment of the Reviewing Editor: REC#1).

Reviewer #2 (Public review): 

General comments: 

The authors investigated the effects of tDCS on brain dynamics in awake and anesthetized monkeys using functional MRI. They claim that cathodal tDCS disrupts the functional connectivity pattern in awake monkeys while anodal tDCS alters brain patterns in anesthetized monkeys. This study offers valuable insight into how brain states can influence the outcomes of noninvasive brain stimulation. However, there are several aspects of the methods and results sections that should be improved to clarify the findings.

We thank the Reviewer for the summary and appreciation of our study.  

Major comments 

For the anesthetized monkeys, the anode location differs between subjects, with the electrode positioned to stimulate the left DLFPC in monkey R and the right DLPFC in monkey N. The authors mention that this discrepancy does not result in significant differences in the electric field due to the monkeys' small head size. However, this is incorrect, as placing the anode on the left hemisphere would result in a much lower EF in the right DLPFC than placing the anode on the right side. Running an electric field simulation would confirm this. Additionally, the small electrode size suggested by the Easy cap configuration for NHP appears sufficient to stimulate the targeted regions focally. If this interpretation is correct, the authors should provide additional evidence to support their claim, such as a computational simulation of the EF distribution.

We thank the Reviewer for the comments. First, regarding the reviewer’s statement that placing the anode on the left hemisphere would result in a much lower EF in the right DLPFC than placing the anode on the right side, we would like to clarify that we did not use a typical 4 x 1 concentric ring high-definition setup (which consists of a small centre electrode surrounded by four return electrodes), but a two-electrode montage, with one electrode over the left or right PFC and the other one over the contralateral occipital cortex. According to EF modelling papers, a 4 x 1 high-definition setup would produce an EF that is focused and limited to the cortical area circumscribed by the ring of the return electrodes (Datta et al. 2009; Alam et al. 2016). Therefore, targeting the left or right DLPFC with a 4 x 1 setup would produce an EF confined to the targeted hemisphere of the PFC. In contrast, we expect the brain current flow generated with our 2-electrode setup to be broader, despite the small size of the electrodes,  because there is no constraint from return electrodes. Thus, with our setup, the current is expected to flow between the PFC and the occipital cortex (see also our responses to comments R3.3., R.E.C.#2.1. and R.E.C.#2.2.). 

Second, we would like to point out that in awake experiments, in which we stimulated the right PFC of both monkeys, there was no gross evidence of left or right asymmetry in the computed functional connectivity patterns (Figure 3A, Figure 3 - figure supplement 2A; Figure 5A). These results, showing that our stimulation montages did not induce asymmetric dynamic FC changes in NHPs, support the idea that our setups did not generate EFs that were spatially focused enough to alter brain activity in one hemisphere substantially more than the other.

Third, it is also worth noting that current evidence suggests that human brains are significantly more lateralized than those of macaques. Macaque monkeys have been found to have some degree of lateralized networks, but these are of lower complexity, and the lateralization is less pronounced and functionally organized than in humans. (Whey et al., 2014; Mantini et al., 2013). This suggests that, even if the stimulation were focal enough to stimulate the left or the right part of the PFC only, the behavioural effects would likely be similar.

We strongly agree with the reviewer that conducting an EF simulation would be valuable to confirm our expectations and to gain a comprehensive view of the characteristics of the EFs generated with our different setups in NHPs. However, the challenge is in the fact that EF computational models have been developed for humans, and their use in NHPs is not straightforward due to significant anatomical differences. For example, macaque monkeys are distinct from humans in terms of brain size, shape and cortical organisation, skull thickness, and the presence of muscles, as well as different tissue conductivities (Lee et al. 2015; Datta et al.2016; Mantell et al. 2023). We plan to address this in future work.

Actions in the text: In the Materials and Methods section, we have modified the sentence: “Because of the small size of the monkey's head and because we did not use return electrodes to restrict the current flow (as is achieved with typical high-definition montages (Datta et al. 2009; Alam et al. 2016)), we expected that tDCS stimulation with the two symmetrical montages would result in nearly equivalent electric fields across the monkey’s head and produce roughly similar effects on brain activity.” 

We also added a new sentence about EF simulation: 

“This would need to be confirmed by running an electric field simulation. However, computational electric field models have been developed for humans, and their use in NHPs is not straightforward due to anatomical specificities. Indeed, monkeys differ from humans in terms of brain size, shape and cortical organization, skull thickness, tissue conductivities and the presence of muscles (Lee et al. 2015; Datta et al. 2016; Mantell et al. 2023). Modelling of EFs generated with the specific tDCS montages employed in this study will be performed in future work.”

For the anesthetized monkeys, the authors applied 1 mA tDCS first, followed by 2 mA tDCS. A 20-minute stimulation duration of 1 mA tDCS is strong enough to produce after-effects that could influence the brain state during the 2 mA tDCS. This raises some concerns. Previous studies have shown that 1 mA tDCS can generate EF of over 1 V/m in the brain, and the effects of stimulation are sensitive to brain state (e.g., eye closed vs. eye open). How do the authors ensure that there are no after-effects from the 1 mA tDCS? This issue makes it challenging to directly compare the effects of 1 mA and 2 mA stimulation.

We agree with the reviewer's comment that 1 mA tDCS may induce aftereffects, as has been observed in several human studies (e.g., (Jamil et al. 2017, 2020). Although the differences between the 1 mA post-stimulation and baseline conditions were not significant in our analyses, it's still possible that the stimulation produced some effects below the threshold of significance that may contribute, albeit weakly, to the changes observed during and after 2 mA stimulation. We have, therefore, amended the paper in line with the reviewer's comments.

Actions in the text: We have added the following text in the Result section: 

“While several human studies have reported that 1 mA transcranial stimulation induces aftereffects (e.g., (Jamil et al. 2017, 2020; Monte-Silva et al. 2010), the differences between the 1 mA post-stimulation and baseline conditions were not significant in our analyses. However, it is still possible that the 1 mA stimulation produced some effects below the threshold of significance that may contribute to the changes observed during and after the 2 mA stimulation.”

The occurrence rate of a specific structural-functional coupling pattern among random brain regions shows significant effects of tDCS. However, these results seem counterintuitive. It is generally understood that noninvasive brain stimulation tends to modulate functional connectivity rather than structural or structural-functional connectivity. How does the occurrence rate of structural-functional coupling patterns provide a more suitable measure of the effectiveness of tDCS than functional connectivity alone? I would recommend that the authors present the results based on functional connectivity itself. If there is no change in functional connectivity, the relevance of changes in structural-functional coupling might not translate into a meaningful alteration in brain function, making it unclear how significant this finding is without corresponding functional evidence.

First, of all, we would like to make it clear that the occurrence rate of patterns as a function of their SFC is not intended to be used or seen as a ‘better’ measure of the efficacy of tDCS. Instead, it is one aspect of the effects of tDCS on whole-brain functional cortical dynamics, obtained from refined measures (phase-coherences), that specifically addresses the coupling between structure and function. This type of analysis is further motivated by its increasing use in the literature due to its suspected relationship to wakefulness (e.g., (Barttfeld et al. 2015, Demertzi et al. 2019; Castro et al. 2023)). Also, in our analysis, the structure is kept constant: the connectivity matrix used to correlate the functional brain states is always the same (CoCoMac82). Thus, the influence of tDCS on the structure-function side can only be explained by modulating the functional aspects, as suggested by intuition and previous results.

Then, we agree with the reviewer that studying the functional changes induced by tDCS alone could be valuable. However, usual metrics used in FC analysis are usually done statistically: FC-states are either computed through averaging spatial correlations over time, then analyzed through graph-theoretical properties for instance (or by just directly computing the element-wise differences), or either by considering the properties of the different visited FC-states by computing spatial correlations over a sliding time-window, and then similar analysis can be done as previously explained. But these are static metrics, if the states visited are essentially the same (which is expected from non-invasive neuromodulations that haven’t already demonstrated strong and/or characteristic impact), but the dynamical process of visiting said states changes, one would see no difference in that regard. As such, in the case of resting-state fMRI, differences in FCs are hard to interpret given that between-sessions within-condition differences are usually found with some degree of variance for the respective conditions. Trying then to interpret between-condition differences is quite tricky in the case of subtle modulations of the system’s activity. On the other hand, more subtle differences can be captured by considering more detailed analysis, such as using phase-based methods like we did,  by incorporating some statistical learning component with regard to the dynamicity of the system (supervised learning for instance like we did followed by temporal & transition-based methodology), and by adding some dimensions along which one will be able to give some interpretation to the analysis.  In our case we were interested in characterizing resting-state differences between stimulation conditions, which have nuanced and subtle interactions with the biological system. 

As such, classical measures of differences between FC states are likely to not be refined and precise enough. In fact, we propose additional files investigating those classically used measures such as differences in average FC matrices, or changes in functional graph properties (like modularity, efficiency and density) of the visited FC states. These figures show that, for the first case, comparing region-to-region specific FCs provides very few statistically significant results. With respect to the second part, we show that virtually no differences are observed in the properties of the functional states visited. 

These results suggest, as expected, that the actual brain states visited across the different stimulation conditions are topologically quite similar, and that only very few region-specific pairwise functional connectivities are particularly modulated by specific tDCS montages while, on the other hand, the actual dynamical process dictating how the brain activity passes from one state to another is in fact being influenced as shown by the dynamical analysis presented in the main figures in a more apparent and meaningful way (in that it is dependent on the montage, somewhat consistent with regard to the post-stimulations conditions, and can be made sense of by considering the theoretical effect of near-anodal versus near-cathodal neuromodulatory effects).

Actions in the text: We have added new supplementary files showing the effects of the stimulations on FC matrices and on classical functional graph properties in awake and anesthesia datasets (Supplementary Files 3 & 4).

We have added new sentences about these new analyses on the effects of the stimulations on FC matrices and on classical functional graph properties in the Results section:

“In addition, we performed the main analyses separately for the two monkeys, explored the inter-condition variability (Supplementary File 2), and computed classical measures of functional connectivity such as average FC matrices and functional graph properties (modularity, efficiency and density) of the visited FC states (Supplementary File 3).... In contrast, classical FC metrics did not show significant differences across stimulation conditions, highlighting the value of dynamic FC metrics to capture the neuromodulatory effects of tDCS.”

“Analyses of the two monkeys separately showed that the changes in slope and Shannon entropy were bigger in one of the two monkeys but went in the same direction (Supplementary File 2), while classical FC metrics did not capture any statistical differences between the different stimulation conditions (Supplementary File 3).”

The authors recorded data from only two monkeys, which may limit the investigation of the group effects of tDCS. As the number of scans for the second monkey in each consciousness condition is lower than that in the first monkey, there is a concern that the main effects might primarily reflect the data from a single monkey. I suggest that the authors should analyze the data for each monkey individually to determine if similar trends are observed in both subjects.

We agree that the small number of subjects is a limitation of our study. However, we have already addressed these aspects by reporting statistical analyses that consider them, using linear models of such variables, and running them through ANOVA tests. In addition, we experimentally ensured that we recorded a relatively high number of sessions over a period of several years. Regardless, we agree that our study would benefit from further investigation into this matter. We have therefore prepared complementary figures showing the main analysis performed separately for the two monkeys as proposed, as well as further investigations into the inter-condition variability outmatching the inter-individual variability, itself being also outmatched by intra-individual changes. 

Actions in the text: We have added a supplementary file showing the main analyses performed separately for the two monkeys (Supplementary File 2) and further investigations into the inter-condition variability (Supplementary Files 3 & 4).

We have added new sentences about these analyses performed separately for the two monkeys in the Results section:

“In addition, we performed the main analyses separately for the two monkeys, explored the inter-condition variability (Supplementary File 2), and computed classical measures of functional connectivity such as average FC matrices and functional graph properties (modularity, efficiency and density) of the visited FC states (Supplementary File 3). The separate analyses showed that the changes in slope and Shannon entropy were substantially more pronounced in one of the two monkeys, corroborating some of the effects captured in the ANOVA tests.”

“Analyses of the two monkeys separately showed that the changes in slope and Shannon entropy were bigger in one of the two monkeys but went in the same direction (Supplementary

File 2)”.

Anodal tDCS was only applied to anesthetized monkeys, which limits the conclusion that the authors are aiming for. It raises questions about the conclusion regarding brain state dependency. To address this, it would be better to include the cathodal tDCS session for anesthetized monkeys. If cathodal tDCS changes the connectivity during anesthesia, it becomes difficult to argue that the effects of cathodal tDCS vary depending on the state of consciousness as discussed in this paper. On the other hand, if cathodal tDCS would not produce any changes, the conclusion would then focus on the relationship between the polarity of tDCS and consciousness. In that case, the authors could maintain their conclusion but might need to refine it to reflect this specific relationship more accurately. 

We agree with the reviewer that it would have been interesting to investigate the effects of cathodal tDCS in anesthetized monkeys. However, due to the challenging nature of the experimental procedures under anesthesia, we had to limit the investigations to only one stimulation modality. We chose to deliver anodal stimulation because, from a translational point of view, we aimed to provide new information on the effects of tDCS under anesthesia as a model for disorders of consciousness. It also made much more sense to increase the cortical excitability of the prefrontal cortex in an attempt to wake up the sedated monkeys rather than doing the opposite.

Actions in the text: We have added a new sentence in the Results section:

“Due to the challenging nature of the experimental procedures under anesthesia, we limited the investigations to only one stimulation modality. We chose to deliver anodal stimulation to provide new information on the effects of tDCS under anesthesia as a model for disorders of consciousness and to increase the cortical excitability of the PFC in an attempt to wake up the sedated monkeys.”

Reviewer #3 (Public review): 

Summary: 

This study used transcranial direct current stimulation administered using small 'high-definition' electrodes to modulate neural activity within the non-human primate prefrontal cortex during both wakefulness and anaesthesia. Functional magnetic resonance imaging (fMRI) was used to assess the neuromodulatory effects of stimulation. The authors report on the modification of brain dynamics during and following anodal and cathodal stimulation during wakefulness and following anodal stimulation at two intensities (1 mA, 2 mA) during anaesthesia. This study provides some possible support that prefrontal direct current stimulation can alter neural activity patterns across wakefulness and sedation in monkeys. However, the reported findings need to be considered carefully against several important methodological limitations. 

Strengths: 

A key strength of this work is the use of fMRI-based methods to track changes in brain activity with good spatial precision. Another strength is the exploration of stimulation effects across wakefulness and sedation, which has the potential to provide novel information on the impact of electrical stimulation across states of consciousness.

We thank the Reviewer for the summary and for highlighting the strengths of our study. 

Weaknesses: 

The lack of a sham stimulation condition is a significant limitation, for instance, how can the authors be sure that results were not affected by drowsiness or fatigue as a result of the experimental procedure?

We agree with the reviewer that adding control conditions could have strengthened our study. Control conditions usually consist of a sham condition or active control conditions. However, as mentioned in response to one of Reviewer 2 comments (R.2.5), we had to make choices as we could not perform as many experiments due to their demanding nature, especially under anesthesia. 

In the awake state, we acquired data with two experimental conditions; the monkeys were exposed to either anodal (F4/O1) or cathodal (O1/F4) PFC tDCS. As anodal tDCS of the PFC induced only minor changes in brain dynamics, it could be considered as an active control condition for the cathodal condition, which had striking effects on the cortical dynamics. It is also worth noting that doubts have been raised about the neurobiological inertia of certain sham protocols. Indeed, different sham protocols have been employed in the literature, some of which may produce unintended effects (Fonteneau et al. 2019). Therefore, active control conditions, such as reversing the polarity of the stimulation or targeting a different brain region, have been proposed to provide better control (Fonteneau et al. 2019). Furthermore, in the context of experiments performed under anesthesia, the relevance of a sham control condition typically used to achieve adequate blinding is questionable. 

With regard to drowsiness and fatigue as a result of the experimental procedure, we agree with the reviewer that this is a common problem in functional imaging due to the length of the recording sessions. We assumed, as was done in previous work (Uhrig, Dehaene, and Jarraya 2014; Wang et al. 2015), that the monkeys' performance on the fixation task during acquisition would capture these periods of fatigue. Therefore, only sessions with fixation rates above 85% were included in our analysis. 

Actions in the text: We have now specified, in the Materials and Methods section, the fact that only runs with a high fixation rate (> 85%) were included in the study: 

“To ensure that the results were not biased by fatigue or drowsiness due to the lengthy

In the anaesthesia condition, the authors investigated the effects of two intensities of stimulation (1 mA and 2 mA). However, a potential confound here relates to the possibility that the initial 1 mA stimulation block might have caused plasticity-related changes in neural activity that could have interfered with the following 2 mA block due to the lack of a sufficient wash-out period. Hence, I am not sure any findings from the 2 mA block can really be interpreted as completely separate from the initial 1 mA stimulation period, given that they were administered consecutively. Several previous studies have shown that same-day repeated tDCS stimulation blocks can influence the effects of neuromodulation (e.g., Bastani and Jaberzadeh, 2014, Clin Neurophysiol; Monte-Silva et al., J. Neurophysiology). 

We agree with the reviewer’s comment that the initial 1 mA stimulation block might have induced changes in neural activity and that the 20-minute post 1 mA block would not be long enough to wash out these changes. This comment is very similar to the second comment made by Reviewer 2 (R.2.2). Although our experimental data do not support this possibility (as the differences between the 1 mA post-stimulation and baseline conditions were not significant), it is still conceivable that the stimulation produced some effects below the threshold of significance and that these might weakly contribute to the changes observed during and after the 2 mA stimulation. 

Actions in the text: We have modified the paper according to the reviewers' comments (please see our answer and actions in the text to R.2.2.).

The different electrode placement for the two anaesthetised monkeys (i.e., Monkey R: F3/O2 montage, Monkey N: F4/O1 montage) is problematic, as it is likely to have resulted in stimulation over different brain regions. The authors state that "Because of the small size of the monkey's head, we expected that tDCS stimulation with these two symmetrical montages would result in nearly equivalent electric fields across the monkey's head and produce roughly similar effects on brain activity"; however, I am not totally convinced of this, and it really would need E-field models to confirm. It is also more likely that there would in fact be notable differences in the brain regions stimulated as the authors used HD-tDCS electrodes, which are generally more focal.

We thank the Reviewer for the remark, which is very similar to the second comment from Reviewer 2. Please see our answer to the first comment of Reviewer 2 

Actions in the text: We have modified the paper according to the reviewers' comments (please see the actions taken in response to R.2.1.).

Given the very small sample size, I think it is also important to consider the possibility that some results might also be impacted by individual differences in response to stimulation. For instance, in the discussion (page 9, paragraph 2) the authors contrast findings observed in awake animals versus anaesthetised animals. However, different monkeys were examined for these two conditions, and there were only two monkeys in each group (monkeys J and Y for awake experiments [both male], and monkeys R and N [male and female] for the anaesthesia condition). From the human literature, it is well known that there is a considerable amount of inter-individual variability in response to stimulation (e.g., Lopez-Alonso et al., 2014, Brain Stimulation; Chew et al., 2015, Brain Stimulation), therefore I wonder if some of these differences could also possibly result from differences in responsiveness to stimulation between the different monkeys? At the end of the paragraph, the authors also state "Our findings also support the use of tDCS to promote rapid recovery from general anesthesia in humans...and suggest that a single anodal prefrontal stimulation at the end of the anesthesia protocol may be effective." However, I'm not sure if this statement is really backed-up by the results, which failed to report "any behavioural signs of awakening in the animals" (page 7)?

We thank the Reviewer for this comment. Because working with non-human primates is expensive and labor intensive, the sample sizes in classical macaque experiments are generally small (typically 2-4 subjects per experiment). Our sample size (i.e. 2 rhesus macaques in awake experiments and 2 macaques under sedation, 11 +/- 9 scan sessions per animal, 288 and 136 runs in the awake and anesthesia state, respectively) is comparable to other previous work in non-human primates using fMRI (Milham et al. 2018; Yacoub et al. 2020; Uchimura, Kumano, and Kitazawa 2024). In addition, we would like to point out that the baseline cortical dynamics we found before stimulation, whether in the awake or sedated state, are comparable to previous studies (Barttfeld et al. 2015; Uhrig et al. 2018; Tasserie et al. 2022). This suggests our results are reproducible across datasets, despite the small sample size.

That being said, we agree with the reviewer that inter-individual variability in response to stimulation can be considerable, as shown by a large body of literature in the field. It seems possible that the two monkeys studied in each condition responded differently to the stimulation. But even if that’s the case, our results suggest that at least in one of the two monkeys, cathodal PFC stimulation in the awake state and anodal PFC stimulation under propofol anesthesia induced striking changes in brain dynamics, which we believe is a significant contribution to the field. 

In fact, supplementary analysis, as proposed by Reviewer 2 (cf R2.4), investigating how the different measurables we’ve used were differently affected by tDCS show that indeed monkey Y’s case is more apparent and significant than monkey J’s. Still, the effects observed in monkey J’s case are still congruent with what is observed in monkey Y’s and at the population level (though less flagrant). We also show that these inter-individual variabilities are outmatched by the inter-condition variability, (as indicated by our initially strong statistical results at the population levels), thus showing that, even though we have different responses depending on the subject, the effects observed at the population level cannot be only accounted for by the differences in subjects’ specificities.

Lastly, the Reviewer questioned whether our results support that a single anodal prefrontal stimulation at the end of the anesthesia protocol could effectively promote rapid recovery from general anesthesia, because the stimulation did not wake the animals in our experiments. It should be emphasized that in our case, the monkeys were stimulated while they were still receiving continuous propofol perfusion. In contrast, during the recovery process from anesthesia, the delivery of the anesthetic drug is stopped. It is therefore conceivable that anodal PFC tDCS, which successfully enriched brain dynamics in sedated monkeys in our experiments, may accelerate the recovery from anesthesia when the drug is no longer administered. 

Actions in the text: We have added a line in the Materials and Methods to compare to other studies:

“Our sample size is comparable to previous work in NHP using fMRI (Milham et al. 2018; Yacoub et al. 2020; Uchimura, Kumano, and Kitazawa 2024).”

Reviewing Editor Comments: 

In some cases, authors opt to submit a revised manuscript. Should you choose to do so, please be aware that the reviewers have indicated that their appraisal is unlikely to change unless some of the suggested field modelling is incorporated into the work. This may change the evaluation of the strength of evidence, but the final wording will be subject to reviewer discretion. Details for responding to the reviews are provided at the bottom of this email.

Reviewer #1 (Recommendations for the authors): 

The work should discuss the implications of their experiments for using tDCS to arouse a patient from a coma. The anesthetized animal is effectively in a drug-induced coma. While they observed connectivity changes, these changes did not map nicely onto behavioral changes. 

I would suggest that the authors spell out more clearly what they view as the clinical implications of their work in terms of new insights into how tDCS may be used to either understand and or treat disorders of consciousness.

We thank the Reviewer for his thoughtful comments. We appreciate the opportunity to clarify and expand on the key findings and implications of our work, particularly regarding the new insights into how tDCS can be used to understand and treat disorders of consciousness. We therefore provide a broader perspective on the clinical implications of our experiments regarding coma and disorders of consciousness. We also agree with the Reviewer that the absence of behavioral changes but the presence of functional differences should be more clearly addressed. 

Actions in the text: We have added a few lines about the relevance of anesthesia as a model for disorders of consciousness in the Introduction part:

“Anesthesia provides a unique model for studying consciousness, which, similarly to DOC, is characterized by the disruption or even  the loss of consciousness (Luppi 2024). Additionally, anesthesia mechanisms involve several subcortical nuclei that are key components of the brain's sleep and arousal circuits (Kelz and Mashour 2019).”

In the Discussion section, we have modified and expanded a paragraph about the effects of tDCS in DOC patients and how this technique could be further used to study consciousness: From another clinical perspective, our results demonstrating that 2 mA anodal PFC tDCS decreased the structure-function correlation and modified the dynamic repertoire of brain patterns during anesthesia (Figures 6 and 7) are consistent with the beneficial effects of such stimulation in DOC patients (Thibaut et al., 2014; Angelakis et al., 2014; Thibaut et al., 2017; Zhang et al., 2017; Martens et al., 2018; Cavinato et al., 2019; Wu et al., 2019; Hermann et al., 2020; Peng et al., 2022; Thibaut et al., 2023). Although some clinical trials investigated the effects of stimulating other brain regions, such as the motor cortex (Martens et al., 2019; Straudi et al., 2019) or the parietal cortex (Huang et al., 2017; Guo et al., 2019; Zhang et al., 2022; Wan et al., 2023; Wang et al., 2020), the DLPFC appears to be the most effective target for patients with a minimally conscious state (Liu et al., 2023). In terms of neuromodulatory effects in DOC patients, DLPFC tDCS has been reported to increase global excitability (Bai et al., 2017), increase the P300 amplitude (Zhang et al., 2017; Hermann et al., 2020), improve the fronto-parietal coherence in the theta band (Bai et al., 2018), enhance the putative EEG markers of consciousness (Bai et al., 2018; Hermann et al., 2020) and reduce the incidence of slow-waves in the resting state (Mensen et al., 2020). Our findings further support the PFC as a relevant target for modulating consciousness level and align with growing evidence showing that the PFC plays a key role in conscious access networks (Mashour, Pal, and Brown 2022; Panagiotaropoulos 2024). Nevertheless, we hypothesize that other brain targets for tDCS may be of interest for consciousness restoration, potentially using multi-channel tDCS (Havlík et al., 2023). Among transcranial electrical stimulation techniques, tDCS has the great advantage of facilitating either excitation or inhibition of brain regions, depending on the polarity of the stimulation (Sdoia et al., 2019) exploited this advantage to investigate the causal involvement of the DLPFC in conscious access to a visual stimulus during an attentional blink paradigm. While conscious access was enhanced by anodal stimulation of the left DLPFC compared to sham stimulation, opposite effects were found with cathodal stimulation compared to sham over the same locus. Finally, this literature and our findings suggest that tDCS constitutes a non-invasive, reversible, and powerful tool for studying consciousness.”

We have added a new paragraph about patients with cognitive-motor dissociation and dissociation between consciousness and behavioral responsiveness:

“Changes in the state of consciousness are generally closely associated with changes in behavioural responsiveness, although some rare cases of dissociation have been described. Cognitive-motor dissociation (CMD) is a condition observed in patients with severe brain injury, characterized by behavior consistent with unresponsive wakefulness syndrome or a minimally conscious state minus (Thibaut et al., 2019). However, in these patients, specific cortical brain areas activate in response to mental imagery tasks (e.g., imagining playing tennis or returning home) in a manner indistinguishable from that of healthy controls, as shown through fMRI or EEG (Thibaut et al., 2019; Owen et al., 2006; Monti et al., 2010; Bodien et al., 2024). Thus, although CMD patients are behaviorally unresponsive, they demonstrate cognitive awareness that is not outwardly apparent. It is worth noting that both the structure-function correlation and the rate of the pattern closest to the anatomy were shown to be significantly reduced in unresponsive patients showing command following during mental imagery tasks compared to those who do not show command following (Demertzi et al., 2019). These observations would be compatible with our findings in anesthetized macaques exposed to 2 mA anodal PFC tDCS. The richness of the brain dynamics would be recovered (at least partially, in our experiments), but not the behaviour. This hypothesis also fits with a recent longitudinal fMRI study on patients recovering from coma (Crone et al., 2020). The researchers examined two groups of patients: one group consisted of individuals who were unconscious at the acute scanning session but regained consciousness and improved behavioral responsiveness a few months later, and the second group consisted of patients who were already conscious from the start and only improved behavioral responsiveness at follow-up. By comparing these two groups, the authors could distinguish between the recovery of consciousness and the recovery of behavioral responsiveness. They demonstrated that only initially conscious patients exhibited rich brain dynamics at baseline. In contrast, patients who were unconscious in the acute phase and later regained consciousness had poor baseline dynamics, which became more complex at follow-up. Complete recovery of both consciousness and responsiveness under general anesthesia is possible through electrical stimulation of the central thalamus (Redinbaugh et al., 2020; Tasserie et al., 2022).”

Reviewer #2 (Recommendations for the authors): 

Method 

(1) The authors mentioned that they used HD-tDCS in their experiments; however, they used 1 x 1 tDCS, which is not HD-tDCS but rather single-channel tDCS.

We thank the Reviewing Editor for pointing out this ambiguous wording. We understand that "HD-tDCS", which we used in our paper to refer to high-density 1x1 tDCS (because we used small carbon electrodes instead of the large sponge electrodes employed in conventional tDCS), may cause some confusion with high-definition tDCS, which uses compact ring electrodes and most commonly refers to a 4x1 montage (1 active central electrode over the target area and 4 return electrodes placed around the central electrode).

Therefore, to avoid any confusion, we will use the term "tDCS" rather than “HD-tDCS” to qualify the technique used in this paper and suppress mentions of high-density or high-definition tDCS.

Actions in the text: We have replaced the abbreviation “HD-tDCS” with “tDCS” throughout the paper. We have also suppressed the sentence about high-definition tDCS in the Introduction (“While conventional tDCS relies on the use of relatively large rectangular pad electrodes, high-density tDCS (HD-tDCS) utilizes more compact ring electrodes, allowing for increased focality, stronger electric fields, and presumably, greater neurophysiological changes (Datta et al. 2009; Dmochowski et al. 2011)”) and the two related citations in the References section.

(2) Please provide the characteristics of electrodes, including their size, shape, and thickness.

We thank the Reviewing Editor for this recommendation. We now provide the complete characteristics of the tDCS electrodes used in the paper.

Actions in the text: We have added a sentence describing the characteristics of the tDCS electrodes in the Materials and Methods section:

“We used a 1x1 electrode montage with two carbon rubber electrodes (dimensions: 1.4 cm x 1.85 cm, 0.93  cm thick) inserted into Soterix HD-tES MRI electrode holders (base diameter: 25 mm; height: 10.5 mm), which are in contact with the scalp. These electrodes (2.59 cm2) are smaller than conventional tDCS sponge electrodes (typically 25 to 35 cm²).”

(3) Could the authors clarify why they chose to stimulate the right DLPFC? Is there a specific rationale for this choice? Additionally, could the authors explain how they ensured that the stimulation targeted the DLPFC, given that the monkey cap might differ from human configurations? In many NHP studies, structural MRI is used to accurately determine electrode placement. Considering that a single channel F4 - O2 montage was used, even a small displacement of the frontal electrode laterally could result in the electric field not adequately covering the DLPFC. Could the authors provide structural MRI images and details of electrode positioning to help readers better understand targeting accuracy?

We thank the Reviewing Editor for the thoughtful comments and recommendations. We appreciate the opportunity to further clarify our rationale for stimulating the right DLPFC and also the suggestion to provide structural MRI images and details of electrode positioning, which we think will improve the quality of the paper by showing targeting accuracy.

First, we would like to clarify that our initial decision to stimulate the right PFC in most animals was driven by experimental constraints. Indeed, we had limited access to the left PFC in three of the four macaques, either due to the presence of cement (spreading asymmetrically from the centre of the head) used to fix the head post in awake animals or due to a scar in one of the two animals studied under anesthesia. 

Second, we agree with the Reviewing Editor on the importance of showing details of electrode positioning and evidence of targeting accuracy across MRI sessions. Therefore, we now provide structural images showing the positions of anodal and cathodal electrodes in almost all acquired sessions: 10 sessions (out of 10) under anesthesia and 30 sessions in the awake state (out of 34 sessions, because we could not acquire structural images in four sessions). These images show that, in anesthesia experiments, the anodal electrode was positioned over the dorsal prefrontal cortex and the cathodal electrode was placed over the contralateral occipital cortex (at the level of the parieto–occipital junction) in both monkeys. In the awake state, the montage still targeted the prefrontal cortex and the occipital cortex, but with a slightly different placement. One of the electrodes was placed over the prefrontal cortex, closer to the premotor cortex than in anesthesia experiments, while the other one was placed over the occipital cortex (V1), slightly more posterior than in anesthesia experiments. These images therefore show that the placement was relatively accurate across sessions and reproducible between monkeys in each of the two arousal conditions.

Actions in the text: We have added a supplementary file showing electrode positioning in 40 of the 44 acquired MRI sessions (Supplementary File 1). We have also added a new supplement figure (Figure 1 - figure supplement 1) showing electrode positioning in representative MRI sessions of the awake and anesthetized experiments in the main manuscript. 

We added a few sentences referring to these figures in the Result section: 

“Representative structural images showing electrode placements on the head of the two awake monkeys are shown in Figure 1 - figure supplement 1A). Supplementary File 1 displays the complete set of structural images, showing that the two electrodes were accurately placed over the prefrontal cortex and the occipital cortex in a reproducible manner across awake sessions.”

Figure 1 - figure supplement 1. Structural images displaying electrode placements on the head of monkeys. A) Awake experiments. Representative sagittal, coronal and transverse MRI sections, and the corresponding skin reconstruction images showing the position of the prefrontal and the occipital electrodes on the head of monkeys J. and Y. B) Anesthesia experiments. Representative sagittal, coronal and transverse MRI sections, and the corresponding skin reconstruction images showing the position of the prefrontal and occipital electrodes over the occipital cortex on the head of monkeys R. and N.

Supplementary File 1 (see attached file). Structural images showing the position of the tDCS electrodes on the monkey's head across sessions. Sagittal, coronal and transverse MRI sections, and corresponding skin reconstruction images showing the position of the prefrontal and occipital electrodes on the monkey's head for each MRI session (except for 4 sessions in which no anatomical scan was acquired). The two electrodes were accurately placed over the prefrontal cortex and the occipital cortex in a reproducible manner across sessions and between the two monkeys studied in each arousal state. In anesthesia experiments, the anodal electrode was placed over the dorsal prefrontal cortex, while the cathodal electrode was positioned over the parieto-occipital junction. In awake experiments, the prefrontal electrode was positioned over the dorsal prefrontal cortex/pre-motor cortex, while the occipital electrode was placed over the visual area 1. The position of the two electrodes differed slightly between the anesthetized and awake experiments due to different body positions (the prone position of the sedated monkeys prevented a more posterior position of the occipital electrode) and also due to the presence of a headpost on the head of the two monkeys in awake experiments (the monkeys we worked with in anesthesia experiments did not have an headpost).

(4) If the authors did not analyze the data for the passive event-related auditory response, it may be helpful to remove the related sentence to avoid potential confusion for readers.

We thank the Reviewing Editor for the comment. Although we understand the reviewer’s point of view, we decide to keep this information in the paper to inform the reader that the macaques were passively engaged in an auditory task, as this could have some influence on the brain state. In the Materials and Methods section, we already mentioned that the analysis of the cerebral responses to the auditory paradigm is not part of the paper. We have modified the sentence to make it clearer and to avoid potential confusion for readers.

Actions in the text: We have modified the sentence referring to the passive event-related auditory response in the Materials and Methods section:

“All fMRI data were acquired while the monkeys were engaged in a passive event-related auditory task, the local-global paradigm, which is based on local and global deviations from temporal regularities (Bekinschtein et al. 2009; Uhrig, Dehaene, and Jarraya 2014). The present paper does not address how tDCS perturbs cerebral responses to local and global deviants, which will be the subject of future work.”

(5) Could the authors clarify what x(t) represents in the equation? Additionally, it would be better to number the equations.

We apologize for the confusion,  x(t) represents the evolution of the BOLD signals over time. We have numbered the equations as suggested. 

Actions in the text: We have added explanations about the notation and numerotation of equations.

(6) It would be much better to provide schematic illustrations to explain what the authors did for analyzing fMRI data.

We thank the Reviewing Editor for the suggestion and now provide a new figure as suggested.  

Actions in the text: We have added a new figure (Figure 2) graphically showing the overall analysis performed. We have added a sentence about the new Figure 2 in the Results section:  “A graphical overview of the overall analysis is shown in Figure 2.” We have renumbered Figure 2 - supplement figures accordingly.

Figure 2. fMRI Phase Coherence analysis. A) Left) Animals were scanned before, during and after PFC tDCS stimulation in the awake state (two macaques) or under deep propofol anesthesia (two macaques). Right) Example of Z-scored filtered BOLD time series for one macaque, 111 time points with a TR of 2.4 s. B) Hilbert transform of the z-scored BOLD signal of one ROI into its time-varying amplitude A(t) (red) and the real part of the phase φ (green). In blue, we recover the original z-scored BOLD signal as A(t)cos(φ). C) Example of the phase of the Hilbert transform for each brain region at one TR. D) Symmetric matrix of cosines of the phase differences between all pairs of brain regions. E) We concatenated the vectorized form of the triangular superior of the phase difference matrices for all TRs for all participants, in all the conditions for both datasets separately obtaining using the K-means algorithm, the brain patterns whose statistics are then analyzed in the different conditions.

Results 

(1) In Figures 3A, 5A, and 6A showing brain connectivity, it is difficult to relate the connectivity variability among the brain regions. Instead of displaying connection lines for nodes, it would be more effective if the authors highlighted significant, strong connectivity within specific brain regions using additional methods, such as bootstrapping.

We thank the Reviewing Editor for the comment and suggestion. The connection lines indeed represent all the synchronizations above 0.5 and all the anti-synchronization below -0.5 between all pairs of brain regions. As suggested, another element we haven’t addressed is the heterogeneity in coherences between individual brain regions. We hence propose additional supplementary figures showing, for all centroids mentioned in main figures, the variance in phase-based connectivity of the distributions of coherence of all brain regions to the rest of the brain. High value would then indicate a wide range of values of coherence, while low would indicate the different coherence a region has with the rest of the brain have similar values. Thus, a brain with uniform color would indicate high homogeneity in coherence among brain regions, while sharp changes in colors would reveal that certain regions are more subject to high variance in their coherence distributions. We expect this new figure to more clearly expose the connectivity variability among the brain regions.

Actions in the text: We have added new figures showing, for all centroids mentioned in the main figures, the variances in phase-based connectivity of the distributions of coherence  (Figure 3 - figure supplement 3;  Figure 5 - figure supplement 2; Figure 6 - figure supplement 3; Figure 7 - figure supplement 2). One of them is shown below for the only awake analysis (Figure 3 - figure supplement 3).

Figure 3 - figure supplement 3. Variance in inter-region phase coherences of brain patterns. Low values (red and light red) indicate that the distribution of synchronizations between a brain region and the rest of the brain has relatively low variance, while high values (blue and light blue) indicate relatively high variance. Are displayed both supra (top) and subdorsal (bottom) views for each brain pattern from the main figure, ordered similarly as previously: from left (1) to right (6) as their respective SFC increases. 

We added a few sentences about variances in phase-based connectivity of the distributions of coherence in the Result section: 

“Further investigation of the variances in inter-region phase coherences of brain patterns, presented in Figure 3 - figure supplement 3, revealed two main findings. First, all the patterns exhibited some degree of lateral symmetry. Second, except for the pattern with the highest SFC, most patterns displayed high heterogeneity in their coherence variances and striking inter-pattern differences. These observations reflect both the segmentation of distinct functional networks across patterns and a topological organization within the patterns themselves: some regions showed a broader spectrum of synchrony with the rest of the brain, while others exhibited narrower distributions of coherence variances. For instance, unlike other brain patterns, pattern 5 was characterized by a high coherence variance in the frontal premotor areas and low variance in the occipital cortex, whereas pattern 3 had a high variance in the frontal and orbitofrontal regions. In addition, we performed the main analyses separately for the two monkeys, explored the inter-condition variability (Supplementary File 2), and computed classical measures of functional connectivity such as average FC matrices and functional graph properties (modularity, efficiency and density) of the visited FC states (Supplementary File 3).”

“The variance in inter-regional phase coherence across brain patterns showed notably that pattern 4, in contrast to most other patterns, was characterized by a high variance in frontal premotor areas and a low variance in the occipital cortex (Figure 5 - figure supplement 2)." 

“The variance in inter-region phase coherences of the brain patterns is displayed in Figure 6 - figure supplement 3 and showed a striking heterogeneity between the patterns. For example, pattern 5 had a low overall variance (except in the frontal cortex), while pattern 1 was the only pattern with a high variance in the occipital cortex.”

“The variance in inter-region phase coherences of brain patterns is displayed in Figure 6 - figure supplement 2.”

(2) For both conditions, only 2 to 3 out of 6 patterns showed significant effects of tDCS on the occurrence rate. Is it sufficient to claim the authors' conclusion?

We thank the Reviewer Editor for the comment. We would like to point out that similar kinds of differences in the occurrence rates of specific brain patterns (particularly in patterns at the extremities of the SFC scale) have already been reported previously. Prior works in patients suffering from disorders of consciousness, in healthy humans or in non-human primates,  have shown, by using a similar method of analysis, that not all brain states are equally disturbed by loss of consciousness, even in different modalities of unconscious transitioning (Luppi et al. 2021; Z. Huang et al. 2020; Demertzi et al. 2019; Castro et al. 2023; Golkowski et al. 2019; Barttfeld et al. 2015). Therefore, yes we believe that our conclusions are still supported by the results.

(3) If the authors want to assert that the brain state significantly influences the effects of tDCS as discussed in the manuscript, further analysis is necessary. First, it would be great to show the difference in connectivity between two consciousness conditions during the baseline (resting state) to see how resting state connectivity or structural connectivity varies. Second, demonstrating the difference in connectivity between the awake and anesthetized conditions (e.g., awake during cathodal vs. anesthetized cathodal) to show how the connectivity among the brain regions was changed by the brain state during tDCS. This would strengthen the authors' conclusion.

We thank the reviewer for this comment. Firstly, we’d like to clarify that the structural connectivity doesn’t change from one session to another in the same animal and minimally between subjects. Secondly, we agree with the Reviewing Editor that it is informative to show the differences between the baselines and this is what we have done. The results are shown in Figures 5 and 7. Regarding the comparison of the stimulating conditions across arousal levels, the only contrast that we could make is to compare 2 mA anodal awake with 2 mA anodal anesthetized (during and post-stimulation). However, as 2 mA anodal stimulation in the awake state did not affect the connectivity much (compared to the awake baseline), the results would be almost similar to the comparison of the awake baseline with 2 mA anodal anesthetized, which is shown in Figure 7. Therefore, we believe that this would result in minimal informative gains and even more redundancy. 

Reviewer #3 (Recommendations for the authors): 

Introduction, par 2: HD-tDCS does not necessarily produce stronger electric fields (E-fields) in the brain. The E-field is largely montage-dependent, and some configurations such as the 4x1 configuration can actually have weaker E-fields compared to conventional tDCS designs (i.e., with two sponge electrodes) as electrodes are often closer together resulting in more current being shunted by skull, scalp, and CSF. I would consider re-phrasing this section.

We agree with the Reviewer Editor that high-definition tDCS does not necessarily produce stronger electric fields in the brain and apologize for the confusion caused by our use of HD-tDCS to refer to high-density tDCS. To avoid any confusion, we have removed the sentence mentioning that HD-tDCS produces stronger electric fields. 

Actions in the text: We have removed the sentence about high-definition tDCS in the Introduction (“While conventional tDCS relies on the use of relatively large rectangular pad electrodes, high-density tDCS (HD-tDCS) utilizes more compact ring electrodes, allowing for increased focality, stronger electric fields, and presumably, greater neurophysiological changes (Datta et al. 2009; Dmochowski et al. 2011)”) and the two related citations in the References section.

Reviewer #1 (Public review):

In this study, the authors identified an insect salivary protein LssaCA participating viral initial infection in plant host. LssaCA directly bond to RSV nucleocapsid protein and then interacted with a rice OsTLP that possessed endo-β-1,3-glucanase activity to enhance OsTLP enzymatic activity and degrade callose caused by insects feeding. The manuscript suffers from fundamental logical issues, making its central narrative highly unconvincing.

(1) These results suggested that LssaCA promoted RSV infection through a mechanism occurring not in insects or during early stages of viral entry in plants, but in planta after viral inoculation. As we all know that callose deposition affects the feeding of piercing-sucking insects and viral entry, this is contradictory to the results in Fig. S4 and Fig 2. It is difficult to understand callose functioned in virus reproduction in 3 days post virus inoculation. And authors also avoided to explain this mechanism.

(2) Missing significant data. For example, the phenotypes of the transgenic plants, the RSV titers in the transgenic plants (OsTLP OE, ostlp). The staining of callose deposition were also hard to convince. The evidence about RSV NP-LssaCA-OsTLP tripartite interaction to enhance OsTLP enzymatic activity is not enough.

(3) Figure 4a, there was the LssaCA signal in the fourth lane of pull-down data. Did MBP also bind LsssCA? The characterization of pull-down methods was rough a little bit. The method of GST pull-down and MBP pull-down should be characterized more in more detail.

Author response:

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

Reviewer #1 (Public Review):

In this study, the authors identify an insect salivary protein participating viral initiate infection in plant host. They found a salivary LssaCA promoting RSV infection by interacting with OsTLP that could degrade callose in plants. Furthermore, RSV NP bond to LssaCA in salivary glands to form a complex, which then bond to OsTLP to promote degradation of callose.

The story focus on tripartite virus-insect vector-plant interaction and is interesting. However, the study is too simple and poor-conducted. The conclusion is also overstated due to unsolid findings.

We thank the reviewer for their constructive feedback. We have conducted additional experiments to strengthen our results and conclusions as detailed below:

(1) The comparison between vector inoculation and microinjection involves multiple confounding factors that could affect the experimental results, including salivary components, RSV inoculation titers, and the precision of viral deposition. The differential outcomes could be attributed to these various factors rather than definitively demonstrating the necessity of salivary factors. Therefore, we have removed this comparison from the revised manuscript and instead focused on elucidating the specific mechanisms by which LssaCA facilitates viral infection.

(2) We conducted new experiments to assess the function of LssaCA enzymatic activity in mediating RSV infection. Additional experiments revealed that OsTLP enzymatic activity is highly pH-dependent, with increased activity as pH decreases from 7.5 to 5.0 (Fig. 3H). However, the LssaCA-OsTLP interaction at pH 7.4 significantly enhanced OsTLP enzymatic activity without requiring pH changes. These results demonstrate that LssaCA-OsTLP protein interactions are crucial for mediating RSV infection. In contrast to pH-dependent mechanisms, our study demonstrated that LssaCA's biological function in mediating RSV infection is at least partially, if not completely, independent of its enzymatic activity. We have added these new resulted into the revised manuscript (Lines 220-227). We have also added a comprehensive discussion comparing the aphid CA mechanism described by Guo et al. (2023 doi.org/10.1073/pnas.2222040120) with our findings in the revised manuscript (Lines 350-371).

(3) We have repeated majority of callose deposition experiments, providing clearer images (Figures 5-6). In addition to aniline blue staining, we quantified callose concentrations using a plant callose ELISA kit to provide more precise measurements (Figure 5A, I, 6A, C and S8A). We utilized RT-qPCR to measure callose synthase expression in both feeding and non-feeding areas, confirming that callose synthesis was induced specifically in feeding regions, leading to localized callose deposition (Figures 5D-G and S8B-E). For sieve plate visualization, we examined longitudinal sections, which revealed callose deposition in sieve plates during SBPH feeding and RSV infection (Figure S7).

(4) We generated OsTLP mutant rice seedlings (ostlp) and use this mutant to directly demonstrate that LssaCA mediates callose degradation in planta through enhancement of OsTLP enzymatic activity (Lines 288-302 and Figure 6).

(5) We produced LssaCA recombinant proteins in sf9 cells to ensure full enzymatic activity and constructed a comprehensive CA mutant protein, in which all seven residues constituting the enzymatic active center mutated (LssaCA^H111D,LssaCA^N139H,LssaCA^H141D, LssaCA^H143D, LssaCA^E153H, LssaCA^H166D, LssaCA^T253E) (Fig. S1B). This LssaCA mutant protein demonstrated complete loss of enzymatic activity (Fig. 1C).

Major comments:

(1) The key problem is that how long the LssCA functioned for in rice plant. Author declared that LssCA had no effect on viral initial infection, but on infection after viral inoculation. It is unreasonable to conclude that LssCA promoted viral infection based on the data that insect inoculated plant just for 2 days, but viral titer could be increased at 14 days post-feeding. How could saliva proteins, which reached phloem 12-14 days before, induce enough TLP to degrade callose to promote virus infection? It was unbelievable.

We appreciate your insightful comment and acknowledge that our initial description may have been unclear. We agree that salivary proteins would not present in plant tissues for two weeks post-feeding or post-injection. Our intention was to clarify that when salivary proteins enhance RSV infection, this initial enhancement leads to sustained high viral loads. We measured viral burden at 14 days post-feeding or post-injection because this is the common measurement time point when viral titers are sufficiently high for reliable detection by qRT-PCR or western blotting. We have clarified this rationale in the revised manuscript (Lines 155-157).

To determine the actual persistence of LssaCA in plant tissues, we conducted additional experiments where insects were allowed to feed on a defined aera of rice seedlings for two days. We then monitored LssaCA protein levels at 1 and 3 days after removing the insects. Western blotting analysis revealed that LssaCA protein levels decreased post-feeding and remained detectable at 3 days post-feeding. These results are presented in Figure 2H and described in detail in Lines 184-193.

(2) Lines 110-116 and Fig. 1, the results of viruliferous insect feeding and microinjection with purified virus could not conclude the saliva factor necessary of RSV infection, because these two tests are not in parallel and comparable. Microinjection with salivary proteins combined with purified virus is comparable with microinjection with purified virus.

We thank the reviewer’s insightful comment. We agree that “the results of viruliferous insect feeding and microinjection with the purified virus could not conclude the saliva factor necessary of RSV infection”. However, due to the technical difficulty in collecting sufficient quantities of salivary proteins to conduct the microinjection experiment, we have removed these results from the revised manuscript.

(3) The second problem is how many days post viruliferous insect feeding and microinjection with purified virus did author detect viral titers? in Method section, authors declared that viral titers was detected at 7-14 days post microinjection. Please demonstrate the days exactly.

We thank the reviewer’s insightful comment. We typically measured RSV infection levels at both 7- and 14-days post-microinjection. However, since the midrib microinjection experiments have been removed from the revised manuscript, this methodology has also been removed accordingly.

(4) The last problem is that how author made sure that the viral titers in salivary glands of insects between two experiments was equal, causing different phenotype of rice plant. If not, different viral titers in salivary glands of insects between two experiments of course caused different phenotype of rice plant.

We thank the reviewer’s comment. When we compared the effects of LssaCA deficiency on RSV infection of rice plants, we have compared the viral titers in the insect saliva and salivary glands. The results indicated that the virus titers in both tissues have not changed by LssaCA deficiency, suggesting that the viruses inoculated into rice phloem by insects of different treatments were comparable. Please refer to the revised manuscript Figures 2D-G and Lines 161-173.

(5) The callose deposition in phloem can be induced by insect feeding. In Fig. 5H, why was the callose deposition increased in the whole vascular bundle, but not phloem? Could the transgenic rice plant directional express protein in the phloem? In Fig. 5, why was callose deposition detected at 24 h after insect feeding? In Fig. 6A, why was callose deposition decreased in the phloem, but not all the cells of the of TLP OE plant? Also in Fig.6A and B, expression of callose synthase genes was required.

We thank the reviewer for these insightful comments.

(1) Figure 5. The callose deposition increased in multiple cells within the vascular bundle, including sieve tubes, parenchymatic cells, and companion cells. While callose deposition was detected in other parts of the vascular bundle, no significant differences were observed between treatments in these regions, indicating that in response to RSV infection and other treatments, altered callose deposition mainly occurred in phloem cells. Please refer to the revised 5B, 5J, 6B, and 6D.

(2) Transgenic plant expression. The OsTLP-overexpressing transgenic rice plants express TLP proteins in various cells under the control of CaMV 35S promoter, rather than being directionally expressed in the phloem. However, since TLP proteins are secreted, they are potentially transported and concentrated in the phloem where they can degrade callose.

(3) Figure 5. The 24-hour time point for callose deposition detection was selected based on established protocols from previous studies. According to Hao et al. (Plant Physiology 2008), callose deposition increased during the first 3 days of planthopper infestation and decreased after 4 days. Additionally, Ellinger and Voigt (Ann Bot 2014) demonstrated that callose visualization typically begins 18-24 hours after treatment, making 24 hours an optimal detection time point.

(4) Figure 6, Phloem-specific changes. Similar to Figure 5, while callose deposition was detected in other parts of vascular bundle, significant differences between treatments were mainly observed in phloem cells, indicating that RSV infection specifically affects callose deposition in phloem tissue.

(5) Callose synthase gene expression. We performed RT-qPCR analysis to measure the expression levels of callose synthase genes. The results indicated that OsTLP overexpression did not significantly alter the mRNA levels of these genes, regardless of RSV infection status in SBPH.

Reviewer #2 (Public Review):

There is increasing evidence that viruses manipulate vectors and hosts to facilitate transmission. For arthropods, saliva plays an essential role for successful feeding on a host and consequently for arthropod-borne viruses that are transmitted during arthropod feeding on new hosts. This is so because saliva constitutes the interaction interface between arthropod and host and contains many enzymes and effectors that allow feeding on a compatible host by neutralizing host defenses. Therefore, it is not surprising that viruses change saliva composition or use saliva proteins to provoke altered vector-host interactions that are favorable for virus transmission. However, detailed mechanistic analyses are scarce. Here, Zhao and coworkers study transmission of rice stripe virus (RSV) by the planthopper Laodelphax striatellus. RSV infects plants as well as the vector, accumulates in salivary glands and is injected together with saliva into a new host during vector feeding.

The authors present evidence that a saliva-contained enzyme - carbonic anhydrase (CA) - might facilitate virus infection of rice by interfering with callose deposition, a plant defense response. In vitro pull-down experiments, yeast two hybrid assay and binding affinity assays show convincingly interaction between CA and a plant thaumatin-like protein (TLP) that degrades callose. Similar experiments show that CA and TLP interact with the RSV nuclear capsid protein NT to form a complex. Formation of the CA-TLP complex increases TLP activity by roughly 30% and integration of NT increases TLP activity further. This correlates with lower callose content in RSV-infected plants and higher virus titer. Further, silencing CA in vectors decreases virus titers in infected plants.

(1) Interestingly, aphid CA was found to play a role in plant infection with two non-persistent non-circulative viruses, turnip mosaic virus and cucumber mosaic virus (Guo et al. 2023 doi.org/10.1073/pnas.2222040120), but the proposed mode of action is entirely different.

We appreciate the reviewer’s insightful comment and have carefully examined the cited publication. The study by Guo et al. (2023) elucidates a distinct mechanism for aphid-mediated transmission of non-persistent, non-circulative viruses (turnip mosaic virus and cucumber mosaic virus). In their model, aphid-secreted CA-II in the plant cell apoplast leads to H⁺ accumulation and localized acidification. This trigger enhanced vesicle trafficking as a plant defense response, inadvertently facilitating virus translocation from the endomembrane system to the apoplast.

In contrast to these pH-dependent mechanisms, our study demonstrated that LssaCA’s biological function in mediating RSV infection is, if not completely, at least partially independent of its enzymatic activity. We performed additional experiments to reveal that OsTLP enzymatic activity is highly pH-dependent and exhibits increased enzymatic activity as pH decreases from 7.5 to 5.0 (Fig. 3H); however, the LssaCA-OsTLP interaction occurring at pH 7.4 significantly enhanced OsTLP enzymatic activity without any change in buffer pH (Fig. 3G). These results demonstrate the crucial importance of LssaCA-OsTLP protein interactions, rather than enzymatic activity alone, in mediating RSV infection.

We have incorporated these new experimental results and added a comprehensive discussion comparing the aphid CA mechanism described by Guo et al. (2023) with our findings in the revised manuscript. Please refer to Figures 3G-H, Lines 220-227 and 350-371 for detailed information.

(2) While this is an interesting work, there are, in my opinion, some weak points. The microinjection experiments result in much lower virus accumulation in rice than infection by vector inoculation, so their interpretation is difficult.

We acknowledge the reviewer's concern regarding the lower virus accumulation observed in microinjection experiments compared to vector-mediated inoculation. We have removed these experiments from the revised manuscript. To address the core question raised by these experiments, we have conducted new experiments that directly demonstrate the importance of LssaCA-OsTLP protein-protein interactions in mediating RSV infection. These results demonstrate the crucial importance of LssaCA-OsTLP protein interactions, rather than enzymatic activity alone, in mediating RSV infection. Additionally, we have incorporated a comprehensive discussion examining carbonic anhydrase activity, pH homeostasis, and viral infection. Please refer to the detailed experimental results and discussion in the sections mentioned in our previous response (Figures 3G-H, Lines 220-227 and 350-371).

(3) Also, the effect of injected recombinant CA protein might fade over time because of degradation or dilution.

We appreciate the reviewer’s insightful comment. This is indeed a valid concern that could affect the interpretation of microinjection results. To address the temporal dynamics of CA protein presence in planta, we conducted time-course experiments to monitor the retention of naturally SBPH-secreted CA proteins in rice plants. Our analysis at 1- and 3- days post-feeding (dpf) revealed that CA protein levels decreased progressively following SBPH feeding, but could also been detected at 3dpf (Fig. 2H). Please refer to Figures 2H and lines 184-193 for detailed information.

(4) The authors claim that enzymatic activity of CA is not required for its proviral activity. However, this is difficult to assess because all CA mutants used for the corresponding experiments possess residual activity.

We appreciate the reviewer’s insightful comment. We constructed a comprehensive CA mutant protein in which all seven residues constituting the enzymatic active center mutated (LssaCA^H111D, LssaCA^N139H, LssaCA^H141D, LssaCA^H143D, LssaCA^E153H, LssaCA^H166D, LssaCA^T253E) (Fig. S1B). This LssaCA mutant protein demonstrated complete loss of enzymatic activity (Fig. 1C). However, since we have removed the recombinant CA protein microinjection experiments from the revised manuscript, we lack sufficient direct evidence to definitively demonstrate that CA enzymatic activity is dispensable for its proviral function. To address the core question raised by these experiments, we have conducted new experiments that provide direct evidence for the importance of LssaCA-OsTLP protein-protein interactions in mediating RSV infection. Additionally, we have incorporated a comprehensive discussion examining carbonic anhydrase activity, pH homeostasis, and viral infection. Please refer to the detailed experimental results and discussion in the sections mentioned in our previous response (Figures 3G-H, Lines 220-227 and 350-371).

(5) It remains also unclear whether viral infection deregulates CA expression in planthoppers and TLP expression in plants. However, increased CA and TLP levels could alone contribute to reduced callose deposition.

We have compared LssaCA mRNA levels in RSV-free and RSV-infected L.striatellus salivary glands, which indicated that RSV infection does not significantly affect LssaCA expression (Figure 1J). By using RSV-free and RSV-infected L.striatellus to feed on rice seedlings, we clarified that RSV infection does not affect TLP expression in plants (Figure 5H).

Reviewer #1: (Recommendations For The Authors):

Other comments:

(1) Most data proving viral infection and LssaCA expression were derived from qPCR assays. Western blot data are strongly required to prove the change at the protein level.

We agree that western blot data are required to prove the change at the protein level. In the revised manuscript, we have added western-blotting results (Figures 1F, 1I, 2C, 2J, and S6).

(2) Line 145, data that LssaCA was significantly downregulated should be shown.

Thank you and the data has been added to the revised manuscript. Please refer to Line 165 and Figure 2D.

(3) Lines 159-161, how did authors assure that the dose of recombinant LssCA was closed to the release level of insect feeding, but not was excessive? How did author exclude the possibility of upregulated RSV titer caused by excessive recombinant LssCA?

We appreciate this important concern regarding dosage controls. While microinjection of recombinant proteins typically yields viral infection levels significantly lower than those achieved through natural insect feeding, higher protein concentrations are often required to achieve high viral infection levels. In this experiment, we compared RSV infection levels following microinjection of BSA+RSV versus LssaCA+RSV, with the expectation that any observed upregulation in RSV titer would be specifically attributable to recombinant LssaCA rather than excessive protein dosing. However, given the low RSV infection levels observed with viral microinjection, we have removed their corresponding results from the revised manuscript.

(4) Lines 124-125, recombinantly expressed LssaCA protein should be underlined, but not the LssaCA protein itself.

We have clearly distinguished recombinantly expressed LssaCA from endogenous LssaCA protein throughout the manuscript, ensuring that all references to recombinant proteins are properly labeled as such.

(5) LssaCA expression in salivary glands of viruliferous and nonviruliferous insects is required. LssaCA accumulation in rice plant exposed to viruliferous and nonviruliferous insects is also required.

We have measured LssaCA mRNA levels in salivary glands of viruliferous and nonviruliferous insects (Figure 1J), and protein levels in rice plant exposed to viruliferous and nonviruliferous insects (Figure 1I).

(6) Fig. 4G, the enzymatic activities of OsTLP were too low compared with that in Fig. 4E and Fig. 7E. Why did the enzymatic activities of the same protein show so obvious difference?

We apologize for the error in Fig. 4G. The original data presented relative fold changes between OsTLP+BSA and OsTLP+LssaCA treatment, with OsTLP+BSA normalized to 1.0 and OsTLP+LssaCA values expressed as fold changes relative to this baseline. However, the Y-axis was incorrectly labeled as “β-1,3-glucanase (units mg^-1)”, which suggested absolute enzymatic activity values. We have now corrected the figure (revised Figure 3G) to display the actual absolute enzymatic activity values with the appropriate Y-axis label “β-1,3-glucanase (units mg^-1)”.

(7) Fig. 7E, was the LssaCA + NP and LssaCA + GST quantified?

Yes, all proteins were quantified, and enzymatic activity values were calculated and expressed as units per milligram of proteins (units mg^-1).

Minor comments:

(1) The keywords: In fact, the LssaCA functioned during initial viral infection in plant, but not viral horizontal transmission.

We appreciate the reviewer’s insightful comment. We have revised the manuscript title to “Rice stripe virus utilizes an Laodelphax striatellus salivary carbonic anhydrase to facilitate plant infection by direct molecular interaction” and changed the keyword from “viral horizontal transmission” to “viral infection of plant”.

(2) Fig. 2A, how about testes? Was this data derived from female insects? Fig. 2C, is the saliva collected from nonviruliferous insects? Fig. 2E, what is the control?

We appreciate the reviewer’s insightful comments.

(1) Fig. 2A: The data present mean and SD calculated from three independent experiments, with 5 tissue samples per experiment. Since 3^rd instar nymphs were used for feeding experiments in this study, we also used 3^rd instar RSV-free nymphs to measure gene expression in guts, salivary glands and fat bodies. R-body represents the remaining body after removing these tissues. Female insects were used to measure gene expression in ovaries, and gene expression in testes was also added. We have added this necessary information to the revised manuscript (please refer to new Figure 1F and Lines 402-403).

(2) Fig. 2C: Yes, saliva was collected from nonviruliferous insects.

(3) Fig. 2E: The control consisted of 100 mM PBS, as described in the experimental section (Lines 643-644): “A blank control consisted of 2 mL of 100 mM PBS (pH 7.0) mixed with 1 mL of 3 mM p-NPA.” In the revised manuscript, we recombinantly expressed LssaCA and its mutant proteins in both sf9 cells and E.coli. Therefore, we have used the mutant proteins as controls to demonstrate specific enzymatic activity. Please refer to Figure 1C, Lines 115-122 and 621-635 for detailed information.

(3) Some figure labeling appeared unprofessional. For example, "a-RSV", "loading" in Fig. 1, "W-saliva", "G-saliva" in Fig. 2, and so on, the related explanations were absent.

We appreciate the reviewer’s insightful comments. We have thoroughly reviewed all figures to ensure professional labels. Specifically, we have:

(1) Used proper protein names to label western blots and clearly explained the antibodies used for protein detection.

(2) Provided comprehensive explanations for all abbreviations used in figures within the corresponding figure legends.

(3) Ensured consistent and clear labeling throughout all figures.

Please refer to the revised Figures 1-3 for these corrections.

(4) Lines 83-84, please cite references on callose preventing viral movement. I do not think the present references were relevant.

We have added a more relevant reference (Yue et al., 2022, Line 82), which revealed that palmitoylated γb promotes virus cell-to-cell movement by interacting with NbREM1 to inhibit callose deposition at plasmodesmata.

(5) The background of transgenic plants of OsTLP OE should be characterized. And the overexpression of OsTLP should be shown. Which generation of OsTLP OE did authors use?

The background of transgenic plants of OsTLP OE and its generation used have been shown in the “Materials and methods” section (Line 782-786) and has been mentioned in the main text (Line 214). T² lines have been selected for further analysis (Line 789).

(6) Fig. 5A, the blank, which derived from plants without exposure to insect, was absent.

We appreciate the reviewer’s insightful comments. We have added the non- fed control in the revised Figure 5A-C.

(7) Fig. 7A, the nonviruruliferous insects were required to serve as a control.

Immunofluorescence localization of RSV and LssaCA in uninfected L. striatellus salivary glands have been added to the revised manuscript (Figure S2).

(8) The manuscript needs English language edit.

The manuscript has undergone comprehensive English language editing to improve clarity, grammar, and overall readability.

Reviewer #2 (Recommendations For The Authors):

(1) The first experiment compares vector inoculation vs microinjection of RSV in tissue. I am not sure that your claim (saliva factors are necessary for inoculation) holds, because the vector injects RSV directly into the phloem, whereas microinjection is less precise and you cannot control where exactly the virus is deposed. However, virus deposited in other tissues than the phloem might not replicate, and indeed you observe, compared to natural vector inoculation, highly reduced virus titers.

We appreciate the reviewer’s insightful comments. We agree that the comparison between vector inoculation and microinjection involves multiple confounding factors that could affect the experimental results, including salivary components, RSV inoculation titers, and the precision of viral deposition. As the reviewer correctly points out, the differential outcomes could be attributed to these various factors rather than definitively demonstrating the necessity of salivary factors. Therefore, we have removed this comparison from the revised manuscript and instead focused on elucidating the specific mechanisms by which LssaCA facilitates viral infection.

(2) Next the authors show that a carbonic anhydrase (CA) that they previously detected in saliva is functional and secreted into rice. I assume this is done with non-infected insects, but I did not find the information. Silencing the CA reduces virus titers in inoculated plants at 14 dpi, but not in infected planthoppers. At 1 dpi, there is no difference in RSV titer in plants inoculated with CA silenced planthoppers or control hoppers. To see a direct effect of CA in virus infection, purified virus is injected together with a control protein or recombinant CA into plants. At 14 dpi, there is about double as much virus in the CA-injected plants, but compared to authentic SBPH inoculation, titers are 20,000 times lower. Actually, I believe it is not very likely that the recombinant CA is active or present so long after initial injection.

We appreciate the reviewer’s insightful comments.

(1) Our previous study identified the CA proteins from RSV-free insects. We have added this information to the revised manuscript (Line 110).

(2) We acknowledge the reviewer's concern regarding the lower virus accumulation observed in microinjection experiments compared to vector-mediated inoculation. We have removed these experiments from the revised manuscript and instead focused on elucidating the specific mechanisms by which LssaCA facilitates viral infection.

(3) We didn’t intend to suggest that LssaCA proteins presented for 14 days post-injection. We measured viral titers at 14 days post-feeding or post-injection because this is the common measurement time point when viral titers are sufficiently high for reliable detection by RT-qPCR or western blotting. We have clarified this rationale in the revised manuscript (Lines 155-157). To determine the actual persistence of LssaCA in plant tissues, we monitored LssaCA protein levels at 1 and 3 dpf. Western blotting analysis revealed that LssaCA protein levels decreased post-feeding and remained detectable at 3 dpf. These results are presented in Figure 2H and described in detail in Lines 184-193.

(3) Then the authors want to know whether CA activity is required for its proviral action and single amino acid mutants covering the putative active CA site are created. The recombinant mutant proteins have 30-70 % reduced activity, but none of them has zero activity. When microinjected together with RSV into plants, RSV replication is similar as injection with wild type CA. Since no knock-out mutant with zero activity is used, it is difficult to judge whether CA activity is unimportant for viral replication, as claim the authors.

We appreciate the reviewer’s insightful comment. We constructed a comprehensive CA mutant protein in which all seven residues constituting the enzymatic active center mutated (LssaCA^H111D, LssaCA^N139H, LssaCA^H141D, LssaCA^H143D, LssaCA^E153H, LssaCA^H166D, LssaCA^T253E) (Fig. S1B). This LssaCA mutant protein demonstrated complete loss of enzymatic activity (Fig. 1C). However, since we have removed the recombinant CA proteins microinjection experiments from the revised manuscript, we lack sufficient direct evidence to definitively demonstrate that CA enzymatic activity is dispensable for its proviral function. To address the core question raised by these experiments, we have conducted new experiments that provide direct evidence for the importance of LssaCA-OsTLP protein-protein interactions in mediating RSV infection. Additionally, we have incorporated a comprehensive discussion examining carbonic anhydrase activity, pH homeostasis, and viral infection. Please refer to the detailed experimental results and discussion in the sections mentioned in our previous response (Figures 3G-H, Lines 220-227 and 350-371).

(4) Next a yeast two hybrid assay reveals interaction with a thaumatin-like rice protein (TLP). It would be nice to know whether you detected other interacting proteins as well. The interaction is confirmed by pulldown and binding affinity assay using recombinant proteins. The kD is in favor of a rather weak interaction between the two proteins.

We have added a list of rice proteins that potentially interact with LssaCA (Table S1) and have measured interactions with additional proteins (unpublished data). Despite the relatively weak binding affinity, the functional significance of the LssaCA-OsTLP interaction in enhancing TLP enzymatic activity is substantial.

(5) Then the glucanase activity of TLP is measured using recombinant TLP-MBP or in vivo expressed TLP. It is not clear to me which TLP is used in Fig. 4G (plant-expressed or bacteria-expressed). If it is plant-expressed TLP, why is its basic activity 10 times lower than in Fig. 4F?

Fig. 4G is the Fig. 3G in the revised manuscript. A E. coli-expressed TLP protein has been used. We apologize for the error in our original Fig. 4G. The original data presented relative fold changes between OsTLP+BSA and OsTLP+LssaCA treatment, with OsTLP+BSA normalized to 1.0 and OsTLP+LssaCA values expressed as fold changes relative to this baseline. However, the Y-axis was incorrectly labeled as “β-1,3-glucanase (units mg^-1)”, which suggested absolute enzymatic activity values. We have now corrected the figure to display the actual absolute enzymatic activity values with the appropriate Y-axis label “β-1,3-glucanase (units mg^-1)”.

(6) There is also a discrepancy in the construction of the transgenic rice plants: did you use TLP without signal peptide or full length TLP? If you used TLP without signal peptide, you should explain why, because the wild type TLP contains a signal peptide.

We cloned the full-length OsTLP gene including the signal peptide sequence (Line 782 in the revised manuscript).

(7) The authors find that CA increases glucanase activity of TLP. Next the authors test callose deposition by aniline blue staining. Feeding activity of RSV-infected planthoppers induces more callose deposition than does feeding by uninfected insects. In the image (Fig. 5A) I see blue stain all over the cell walls of xylem and phloem cells. Is this what the authors expect? I would have expected rather a patchy pattern of callose deposition on cell walls. Concerning sieve plates, I cannot discern any in the image; they are easier to visualize in longitudinal sections than in transversal section as presented here.

We appreciate the reviewer’s insightful comment.

(1) Callose deposition pattern: While callose deposition was detected in other parts of the vascular bundle, significant differences between treatments were mainly observed in phloem cells, indicating that phloem-specific callose deposition is the primary response to RSV infection and SBPH feeding (Figures 5B and 5J).

(2) Sieve plate visualization: We have examined longitudinal sections to visualize sieve plates, which revealed callose deposition in sieve plates during SBPH feeding and RSV infection (Figure S7).

(3) Quantitative analysis: In addition to aniline blue staining, we quantified callose concentrations using a plant callose ELISA kit to provide more precise measurements (Figure 5A, 5I and S8A).

(4) Gene expression analysis: We utilized RT-qPCR to measure callose synthase expression in both feeding and non-feeding areas, confirming that callose synthesis was induced specifically in feeding regions, leading to localized callose deposition (Figures 5D-H).

These experimental results collectively demonstrate that RSV infection induces enhanced callose synthesis and deposition, with this response occurring primarily in phloem cells, including sieve plates, within feeding sites and their immediate vicinity.

(8) I do not quite understand how you quantified callose deposition (arbitrary areas?) with ImageJ. Please indicate in detail the analysis method.

We have added more detailed information for the methods to quantify callose deposition (Lines 673-678).

(9) More callose content is also observed by a callose ELISA assay of tissue extracts and supported by increased expression of glucanase synthase genes. Did you look whether expression of TLP is changed by feeding activity and RSV infection? Silencing CA in planthoppers increases callose deposition, which is inline with the observation that CA increases TLP activity.

We measured OsTLP expression following feeding by RSV-free or RSV-infected SBPH and found that gene expression was not significantly affected by either insect feeding or RSV infection. These results have been added to the revised manuscript (Lines 275-277 and Figure 5H).

(10) Next, callose is measured after feeding of RSV-infected insects on wild type or TLP-overexpressing rice. Less callose deposition (after 2 days) and more virus (after 14 days) is observed in TLP overexpressors. I am missing a control in this experiment, that is feeding of uninfected insects on wild type or TLP overexpressing rice, where I would expect intermediate callose levels.

We appreciate the reviewer’s insightful comment and fully agree with the prediction. In the revised manuscript, we have constructed ostlp mutant plants and conducted additional experiments to further clarify how callose deposition is regulated by insect feeding, RSV infection, LssaCA levels, and OsTLP expression. Specifically: 

(1) Both SBPH feeding and RSV infection induce callose deposition, with RSV-infected insect feeding resulting in significantly higher callose levels compared to RSV-free insect feeding (Fig. 5A-C).

(2) LssaCA enhances OsTLP enzymatic activity, thereby promoting callose degradation (Fig. 5I-K).

(3) OsTLP-overexpressing (OE) plants exhibit lower callose levels than wild-type (WT) plants, while ostlp mutant plants show higher callose levels than WT (Fig. 6A-B).

(4) In ostlp knockout plants, LssaCA no longer affects callose levels, indicating that OsTLP is required for LssaCA-mediated regulation of callose (Fig. 6C-D).

These additional data address the reviewer’s concern and support the conclusion that OsTLP plays a central role in modulating callose levels in response to RSV infection and insect feeding.

(11) Next the authors test for interaction between virions and CA. Immunofluorescence shows that RSV and CA colocalize in salivary glands; in my opinion, there is partial and not complete colocalization (Fig. 7A).

We agree with the reviewer’s observation. CA is primarily produced in the small lobules of the principal salivary glands, while RSV infects nearly all parts of the salivary glands. In regions where RSV and CA colocalize within the principal glands, the CA signal appears sharper than that of RSV, likely due to the relatively higher abundance of CA compared to RSV in these areas. This may explain the partial, rather than complete, colocalization observed in our original Figure 7A. In the revised manuscript, please refer to Figure 1A.

(12) Pulldown experiments with recombinant RSV NP capsid protein and CA confirm interaction, binding affinity assays indicate rather weak interaction between CA and NP. Likewise in pull-down experiments, interaction between NP, CA and TLP is shown. Finally, in vitro activity assays show that activity of preformed TLP-CA complexes can be increased by adding NP; activity of TLP alone is not shown.

We performed two independent experiments to confirm the influence on TLP enzymatic activity by LssaCA or by the LssaCA-RSV NP complex. In the first experiment, we compared the enhancement of TLP activity by LssaCA using TLP alone as a control (Figure 3G). In the second experiment examining the LssaCA-RSV NP complex effect on TLP activity, we used the LssaCA-TLP combination as the baseline control rather than TLP alone (Figure 4B), since we had already established the LssaCA enhancement effect in the previous experiment.

(13) For all microscopic acquisitions, you should indicate the exact acquisition conditions, especially excitation and emission filter settings, kind of camera used and objectives. Use of inadequate filters or of a black & white camera could for example be the reason why you observe a homogeneous cell wall label in the aniline blue staining assays. Counterstaining cell walls with propidium iodide might help distinguish between cell wall and callose label.

Thank you for your insightful suggestions. We have added the detailed information to the revised manuscript (Lines 656-659 and 673-678).

(14) You should provide information whether CA is deregulated in infected planthoppers, as this could also modify its mode of action.\

We have compared LssaCA mRNA levels in RSV-free and RSV-infected L.striatellus salivary glands. The results indicated that RSV infection does not significantly affect LssaCA expression (Figure 1J).

(15) You should show purity of the proteins used for affinity binding measurements.

We have included SDS-PAGE results of purified proteins in the revised manuscript (Figure S3).

(16) L 39: Not all arboviruses are inoculated into the phloem.

Thank you. We have revised this description (Lines 40, 73, 95 and 97).

(17) L 76: Watery saliva is also injected in epidermis and mesophyll cells.

Thank you. We have revised this description (Line 73).

(18) L 79: What do you mean by "avirulent gene"?

Thank you for your valuable comments. We have revised this description as “certain salivary effectors may be recognized by plant resistance proteins to induce effector-triggered immunity”. Please refer to Lines 76-77 for detail.

(19) L 128: Please add delivery method.

Thank you. We have added the delivery methods (Line 134).

(20) L 195: Please explain "MST".

Explained (Line 124). Thank you.

(21) L 203: Please add the plant species overexpressing TLP.

Added (Line 214). Thank you.

(22) L 213: Callose deposition has also a role against phloem-feeding insects.

We appreciate the reviewer’s insight comment. We have added this information to the revised manuscript (Line 252).

(23) L 626: What is a "mutein"?

"mutein" is an abbreviation for mutant proteins. Since the recombinant protein microinjection experiments have been removed from the revised manuscript, the term “mutein” has also been removed. For all other instances, we now use the full term “mutant proteins”.

(24) Fig. 1E: what is "loading"? You should rather show here and elsewhere (or add to supplement) complete protein gels and Western blot membranes and not only bands of interest.

Thank you for your valuable suggestion. Although Figure 1E has been removed from the revised manuscript, we have carefully reviewed all figures to ensure that the term “loading” has been replaced with the specific protein names where appropriate.

(25) Fig. 2C: Please indicate which is the blot and which is the silver stained gel and add mass markers in kDa to the silver stained gel.

Thank you for your suggestion. We have revised figure to include labeled silver-stained gels with indicated molecular weight markers (Figure 1H in the revised manuscript).

Reviewer #1 (Public review):

Summary:

This study aimed to investigate the effects of optically stimulating the A13 region in healthy mice and a unilateral 6-OHDA mouse model of Parkinson's disease (PD). The primary objectives were to assess changes in locomotion, motor behaviors, and the neural connectome. For this, the authors examined the dopaminergic loss induced by 6-OHDA lesioning. They found a significant loss of tyrosine hydroxylase (TH+) neurons in the substantia nigra pars compacta (SNc) while the dopaminergic cells in the A13 region were largely preserved. Then, they optically stimulated the A13 region using a viral vector to deliver the channelrhodopsine (CamKII promoter). In both sham and PD model mice, optogenetic stimulation of the A13 region induced pro-locomotor effects, including increased locomotion, more locomotion bouts, longer durations of locomotion, and higher movement speeds. Additionally, PD model mice exhibited increased ipsilesional turning during A13 region photoactivation. Lastly, the authors used whole-brain imaging to explore changes in the A13 region's connectome after 6-OHDA lesions. These alterations involved a complex rewiring of neural circuits, impacting both afferent and efferent projections. In summary, this study unveiled the pro-locomotor effects of A13 region photoactivation in both healthy and PD model mice. The study also indicates the preservation of A13 dopaminergic cells and the anatomical changes in neural circuitry following PD-like lesions that represent the anatomical substrate for a parallel motor pathway.

Strengths:

These findings hold significant relevance for the field of motor control, providing valuable insights into the organization of the motor system in mammals. Additionally, they offer potential avenues for addressing motor deficits in Parkinson's disease (PD). The study fills a crucial knowledge gap, underscoring its importance, and the results bolster its clinical relevance and overall strength.

The authors adeptly set the stage for their research by framing the central questions in the introduction, and they provide thoughtful interpretations of the data in the discussion section. The results section, while straightforward, effectively supports the study's primary conclusion-the pro-locomotor effects of A13 region stimulation, both in normal motor control and in the 6-OHDA model of brain damage.

Weaknesses:

(1) Anatomical investigation. I have a major concern regarding the anatomical investigation of plastic changes in the A13 connectome (Figures 4 and 5). While the methodology employed to assess the connectome is technically advanced and powerful, the results lack mechanistic insight at the cell or circuit level into the pro-locomotor effects of A13 region stimulation in both physiological and pathological conditions. This concern is exacerbated by a textual description of results that doesn't pinpoint precise brain areas or subareas but instead references large brain portions like the cortical plate, making it challenging to discern the implications for A13 stimulation. Lastly, the study is generally well-written with a smooth and straightforward style, but the connectome section presents challenges in readability and comprehension. The presentation of results, particularly the correlation matrices and correlation strength, doesn't facilitate biological understanding. It would be beneficial to explore specific pathways responsible for driving the locomotor effects of A13 stimulation, including examining the strength of connections to well-known locomotor-associated regions like the Pedunculopontine nucleus, Cuneiformis nucleus, LPGi, and others in the diencephalon, midbrain, pons, and medulla. Additionally, identifying the primary inputs to A13 associated with motor function would enhance the study's clarity and relevance.

The study raises intriguing questions about compensatory mechanisms in Parkinson's disease a new perspective with the preservation of dopaminergic cells in A13, despite the SNc degeneration, and the plastic changes to input/output matrices. To gain inspiration for a more straightforward reanalysis and discussion of the results, I recommend the authors refer to the paper titled "Specific populations of basal ganglia output neurons target distinct brain stem areas while collateralizing throughout the diencephalon from the David Kleinfeld laboratory." This could guide the authors in investigating motor pathways across different brain regions.

(2) Description of locomotor performance. Figure 3 provides valuable data on the locomotor effects of A13 region photoactivation in both control and 6-OHDA mice. However, a more detailed analysis of the changes in locomotion during stimulation would enhance our understanding of the pro-locomotor effects, especially in the context of 6-OHDA lesions. For example, it would be informative to explore whether the probability of locomotion changes during stimulation in the control and 6-OHDA groups. Investigating reaction time, speed, total distance, and even kinematic aspects during stimulation could reveal how A13 is influencing locomotion, particularly after 6-OHDA lesions. The laboratory of Whelan has a deep knowledge of locomotion and the neural circuits driving it so these features may be instructive to infer insights on the neural circuits driving movement. On the same line, examining features like the frequency or power of stimulation related to walking patterns may help elucidate whether A13 is engaging with the Mesencephalic Locomotor Region (MLR) to drive the pro-locomotor effects. These insights would provide a more comprehensive understanding of the mechanisms underlying A13-mediated locomotor changes in both healthy and pathological conditions.

(3) Figure 2 indeed presents valuable information regarding the effects of A13 region photoactivation. To enhance the comprehensiveness of this figure and gain a deeper understanding of the neurons driving the pro-locomotor effect of stimulation, it would be beneficial to include quantifications of various cell types:

• cFos-Positive Cells/TH-Positive Cells: it can help determine the impact of A13 stimulation on dopaminergic neurons and the associated pro-locomotor effect in healthy condition and especially in the context of Parkinson's disease (PD) modeling.

• cFos-Positive Cells /TH-Negative Cells: Investigating the number of TH-negative cells activated by stimulation is also important, as it may reveal non-dopaminergic neurons that play a role in locomotor responses. Identifying the location and characteristics of these TH-negative cells can provide insights into their functional significance.<br /> Incorporating these quantifications into Figure 2 would enhance the figure's informativeness and provide a more comprehensive view of the neuronal populations involved in the locomotor effects of A13 stimulation.

(4) Referred to Figure 3. In the main text (page 5) when describing the animal with 6-OHDA the wrong panels are indicated. It is indicated in Figure 2A-E but it should be replaced with 3A-E. Please do that.

Summary of the Study after revision

The revised manuscript reflects significant efforts to improve clarity, organization, and data interpretation. The refinements in anatomical descriptions, behavioral analyses, and contextual framing have strengthened the manuscript considerably. However, the study still lacks direct causal evidence linking anatomical remodeling to behavioral improvements, and the small sample size in the anatomical analyses remains a concern. The authors have addressed many points raised in the initial review, but further acknowledgement of the exploratory nature of these findings would enhance the scientific rigor of the work.

Key Improvements in the Revision

The revised manuscript demonstrates considerable progress in clarifying data presentation, refining behavioral analyses, and improving the contextualization of anatomical findings. The restructuring of the anatomical section now provides greater precision in describing motor-related pathways, integrating terminology from the Allen Brain Atlas. The addition of new figures (Figures 4 and 5) strengthens the accessibility of these findings by illustrating key connectivity patterns more effectively. Furthermore, the correlation matrices have been adjusted to improve interpretability, ensuring that the presented data contribute meaningfully to the overall narrative of the study.

The authors have also made significant improvements in their behavioral analyses, particularly in the organization and presentation of locomotor data. Figure 3 has been revised to distinctly separate results from 6-OHDA and sham animals, providing a clearer comparison of locomotor outcomes. Additional metrics, such as reaction time, locomotion bouts, and movement speed, further enhance the granularity of the analysis, making the results more informative.

The discussion surrounding anatomical connectivity has also been strengthened. The revised manuscript now places greater emphasis on motor-related pathways and refines its analysis of A13 efferents and afferents. A newly introduced figure provides a concise summary of these connections, improving the contextualization of the anatomical data within the study's broader scope. Moreover, the authors have addressed the translational relevance of their findings by acknowledging the differences between optogenetic stimulation and deep brain stimulation (DBS). Their discussion now better situates the findings within existing literature on PD-related motor circuits, providing a more balanced perspective on the potential implications of A13 stimulation.

Remaining Concerns

Despite these substantial improvements, a number of critical concerns remain. The anatomical findings, though insightful, remain largely correlative and do not establish a causal link between structural remodeling and locomotor recovery. While the authors argue that these data will serve as a reference for future investigations, their necessity for the core conclusions of the study is not entirely clear. Additionally, while the anatomical data offer an interesting perspective on A13 connectivity, their direct relevance to the study's primary goal-demonstrating the role of A13 in locomotor recovery-remains uncertain. The authors emphasize that these data will be valuable for future research, yet their integration into the study's main narrative feels somewhat supplementary. Based on this last thought of the authors it is even more relevant another key limitation lying in the small sample size used for connectivity analyses. With only two sham and three 6-OHDA animals included, the statistical confidence in the findings is inherently limited. The absence of direct statistical comparisons between ipsilesional and contralesional projections further weakens the conclusions drawn from these anatomical studies. The authors have acknowledged that obtaining the necessary samples, acquiring the data, and analyzing them is a prolonged and resource-intensive process. While this may be a valid practical limitation, it does not justify the lack of a robust statistical approach. A more rigorous statistical framework should be employed to reinforce the findings, or alternative techniques should be considered to provide additional validation. Given these constraints, it remains unclear why the authors have not opted for standard immunohistochemistry, which could provide a complementary and more statistically accessible approach to validate the anatomical findings. Employing such an approach would not only increase the robustness of the results but also strengthen the study's impact by providing an independent confirmation of the observed structural changes.

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Reply to the reviewers

Reply to the reviewers

We would like to thank the reviewers for their overall positive evaluations of our manuscript and for their invaluable suggestions that will allow us to reinforce our conclusions. We acknowledge that there is some work to be done and are ready to address most of the reviewers' comments as detailed in our replies below.

Reviewer #1

1. The findings that mmDicer is proviral in bat cells relies exclusively on the observation that the depletion of Dicer in M. myotis cells leads to a reduced accumulation of SFV and SINV at the RNA and protein levels (figure 2). Heterologous expression of mmDicer in HEK 293T NoDice doesn't lead to an increase permissivity to viral infections (figure 1) and the accumulation of Dicer foci is only observed in M. myotis cells but not when mmDicer is expressed in HEK 293 NoDice cells (figure 6). Given that the key finding of this manuscript relies on these knockdown experiments, the authors should ensure that the impact on viral infections is due to the specific silencing of mmDicer and not caused by off-target effects of their siRNA-mediated approach. The authors designed a siRNA pool to efficiently knock-down mmDicer. They should validate their findings by using individual Dicer siRNA and verify whether the decrease SFV/SINV accumulation is observed with at least two individual siRNAs targeting Dicer. It would also strengthen their findings if they could show a complementation experiment in which a mmDicer (designed to not be affected by the siRNA-mediated silencing) is introduced exogenously in Dicer-depleted cells and show that it rescues the observed decrease in viral accumulation to demonstrate that the proviral role is strictly dependent on mmDicer. Alternatively, the authors could consider a CRISPR/Cas9 genome editing approach to knockout Dicer in bat cells to test whether this proviral effect is confirmed.

Reply: We agree with this reviewer that it is important to provide evidence for the specificity of the knock-down and to rule out any off-target effect of the siRNAs. This is the reason for using the siTool technology, which relies on the use of a pool of 30 siRNAs that are transfected at a final concentration of 3 nM. This means that each individual siRNA in the pool is at a concentration of 0.1 nM, so the possibility of off-target effect is largely avoided and the efficiency of silencing is boosted by the cooperative activity of many siRNAs (see https://www.sitoolsbiotech.com/documents/sipools/siPOOLBrochure2019_Web.pdf for more details). This being said, we agree that it would be better to confirm that the observed effect can be recapitulated using a single siRNA and that a complementation experiment would definitely strengthen our findings. For this reason, we will test two individual siRNAs targeting the 3' UTR of mmDicer, which will allow us to complement the knock-down by transfecting a cDNA construct. Regarding the CRISPR/Cas9 genome editing approach, we will give it a try, but Dicer is notoriously difficult to knock-out, so we cannot be sure that this will be successful.

Figure 2: the authors knock-downed Dicer in M. myotis nasal epithelial cells and carried out infections with SINV-GFP and SFV. The authors conclude that Dicer is proviral as its depletion causes a decrease in SINV-GFP and SFV accumulation. While this conclusion is supported by the decrease levels of viral RNA and protein levels upon Dicer depletion (figure 2D, 2E, 2G), the effect on the viral titers is non-significant for both viruses (Figure 2C and 2F) based on the statistical analysis. This reviewer appreciates that the titers are lower upon Dicer knockdown, which support the authors' findings at the viral RNA and protein levels. However, as these results are central to the core message of the manuscript, the authors should provide evidence that this proviral effect observed is statistically significant on viral titers by perhaps providing additional repeats and/or comment on this observation.

Reply: Indeed, we agree that even if the effect of Dicer knockdown results in a lowering of the viral titer, it would be better to have a statistically significant effect. We will repeat the experiment to increase the number of replicates and the power of the statistical test.

a) *In figure 4 and 5, the authors nicely show that mmDicer accumulate to cytoplasmic foci in M. myotis cells upon infection with SFV and SINV and these foci co-localise with double-stranded RNA. The authors used a commercial polyclonal antibody against Dicer (A301-937A, Bethyl according to the Material and Methods section) which is specific to human Dicer to carry out their immunostaining in bat cells. The authors should provide evidence that this antibody indeed recognises/crossreacts with mmDicer as well and that the staining shown is indeed specific to mmDicer localisation especially because the heterologous expression of HA-tagged version of mmDicer in HEK 293T NoDice cells did not show this accumulation of cytoplasmic foci. The authors should verify the specificity of their mmDicer immunostaining by performing the same labelling in bat cells in which Dicer is knock-downed (or knock out) by individual and validated siRNA against mmDicer. The decrease signal of bat Dicer staining using the anti-human Dicer antibody would indicate specificity. *

Reply: the reviewer is correct in its assertion and it is important to provide evidence that the protein that is detected by the anti-human Dicer antibody in bat cells is indeed Dicer. We will perform the suggested experiment and do an immunostaining using the Dicer antibody in bat cells upon Dicer knockdown.

b) Another complementary approach would be to test their Dicer staining between HEK NoDice cells (no Dicer present) versus NoDice complemented with either mmDicer or human Dicer constructs, which would then indicate how much the anti-human Dicer antibody recognises bat Dicer.

Reply: this complementary approach should yield even cleaner result than the previous one as there will be no expression of Dicer at all in the HEK NoDice cells. Therefore, we should be able to measure the increase of signal in the IF upon expression of either human or bat Dicer. We will perform this experiment together with the other one suggested above. In addition, since the constructs are tagged, we might be able to do a double-staining and verify the colocalization of the two signals.

c) In addition, the authors should overexpress HA-tagged mmDicer in M. myotis nasal epithelial cells and test whether HA-mmDicer accumulate into foci upon infection using an anti-HA immunostaining. This would confirm that these accumulation into foci indeed is specific to mmDicer but also would reinforce the authors' findings that host factors within bat cells are important for this formation into foci since mmDicer expression in HEK 293T No Dice cells didn't show this phenotype upon infection (figure 6). OPTIONAL: it would be interesting to overexpress HA-tagged human Dicer into M. myotis nasal epithelial cells as well to then test using anti-HA staining whether human Dicer in presence of host factors from the bat can accumulate into cytoplasmic foci or not upon viral infection.

Reply: we could perform the suggested experiment, but we might face the issue that transfected cells might mount an immune response, which makes them resistant to the infection. We have observed indeed that we needed to use a higher MOI to infect cells after they have been transfected. Since we will have controls in place, this might not be too much of a problem, but we will have to keep it in mind. Alternatively, we will perform a lentiviral transduction of the cells.

This reviewer appreciates that this might be judged as beyond the scope of this study since it is focused on the role of Dicer in M. myotis. However, the observation that mmDicer accumulates into foci containing as well viral dsRNA is very interesting and it would significantly improve the manuscript if the authors would provide further indications that this phenotype is related to the lack of antiviral activity of mmDicer compared to what has been previously shown in other bat species (P.alecto and T. brasiliensis). In other words, is this accumulation of mmDicer into foci responsible for its different impact on virus infection? It would therefore be insightful to compare Dicer localisation upon infection in M. myotis versus P.alecto and/or T. brasiliensis bat cells in which Dicer was shown to be antiviral and test whether this accumulation in foci is only observed in bat cells in which Dicer is proviral (M. myotis) but not in the other bat cells in which Dicer is antiviral (P.alecto and/or T. brasiliensis).

Reply: this is something that we have been wondering about and we have therefore started to look for the cell lines that have been described in the two published studies. While it proved difficult to find the PaKi cells from P. alecto bats, which is not commercially available, we have obtained the Tblu cells from T. brasiliensis and will look at Dicer localization in this model. However, we have to pay attention to the fact that the published data reported a contribution of RNAi in this cell line upon SARS-CoV-2 infection and that we will be using SINV. In addition, we do not know yet whether the anti-Dicer antibody will cross react with the T. brasiliensis Dicer protein.

OPTIONAL: Given the difference between the provial role of mmDicer compared to the antiviral activity of Dicer in cells from P.alecto and T. brasiliensis bat cells, it would strengthen the authors' findings. if additional experiments would be conducted in parallel using M. myotis, P.alecto and/or T. brasiliensis cells. Notably knocking down Dicer in both M. myotis, P.alecto and/or T. brasiliensis cells, compare the impact on viral infections with SINV, SFV, VSV and correlate any observed difference in phenotype with putative variations in the formation of foci.

Reply: it would indeed be really nice to be able to do the Dicer knockdown experiment in several bat cell lines and to correlate the phenotype with the formation of foci. This experiment might take a long time and we are not sure to be able to realize it in a reasonable amount of time. It could however be the subject of another manuscript further down the line.

*Minor comments *

• • Figure 2I: The authors performed a knockdown of Dicer in M. myotis nasal epithelial cells and monitor the impact on VSV-GFP infection. They found that knocking down Dicer leads to an increase in GFP protein and RNA levels suggesting an antiviral role of Dicer while, in contrast, no effect is observed on the production of infectious particles (figure 2H). On the western blot there is only a slight/weak increase of GFP protein level observed upon Dicer knockdown. Yet, the quantification of the band intensity shows a 4-fold increase relative to tubulin and compared to cells treated with siRNA control. This 4-fold increase seems exaggerated given the low increase in the intensity shown on the blot. This discrepancy is most likely due to the lower intensity of tubulin in the western blot analysis of siDicer-treated cells compared to siNeg-treated cells. The authors should reload their western blot with equal amount of protein extract loaded to ensure that the results shown on the western blot are in line with the quantification.*

Reply: the signal quantification for this experiment was done across several replicates, but we agree that the observed effect seems exaggerated when compared to the signal seen on the blot. We observed important variations between replicates, but we will make sure that this was not due to a problem in the analysis and reload the western blot if needed.

• • Figure 3D: the authors mention that in both HEK293T cells and M. myotis nasal epithelial cells infected with SINV-GFP, there was an enrichment of 22-nucleotides (nt) paired positive and negative sense reads that overlapped with a 2-nt overhang, typical of Dicer cleavage. In Figure 3D, the data shows indeed that the duplexes are enriched for reads of 22-nt but it is unclear how this analysis reveals a 3' 2nt overhang within these duplexes. Can the authors clarify this point and if the data provided in that particular analysis indeed doesn't allow to detect these overhangs, please rephrase accordingly or provide additional analysis to support that point. *

Reply: In Figure 3D, the graphs show the probability of pairing of all 22 nucleotides sequence mapping either to the plus or the minus strand of the viral RNA. Thus, for each sequence mapping to the plus strand, the number of sequences mapping to the minus strand with a full or partial overall is counted. A corresponding probability of pairing and Z score is calculated for each number of overlapping nucleotides (for more information on the calculation see Antoniewski (2014) Computing siRNA and piRNA Overlap Signatures. In Animal Endo-SiRNAs: Methods and Protocols, Werner A (ed) pp 135-146. New York, NY: Springer). The Z score peaks for an overlap of 20 nt in both HEK293T and M. myotis nasal epithelial cells infected with SINV. This means that there is a higher probability of two 22 nt sequence to pair along 20 nt, and thus that there are two unpaired nucleotides at the extremities of the duplexes. This higher Z score at 20 nt is not seen in VSV-infected cells. We will rephrase the text in the manuscript to make this point clearer.

• • Typo: page 5, line 152: the authors mention that Dicer knock down had an antiviral effect against VSV-GFP infection at the RNA and protein levels. However, the data in Figure 2I and 2J show an increase in both GFP RNA and proteins levels upon knockdown of Dicer. Although this data suggests that Dicer is antiviral against VSV, the knockdown of Dicer itself is not antiviral but rather proviral/increase virus accumulation. Please rephrase this sentence to avoid confusions. *

Reply: thank you for spotting this typo. We have corrected it accordingly.

Reviewer #2.

1. Figure 1 relies on transduction of cells and antibiotic selection to obtain mmDicer-expressing cells. Although we would expect that every cell expresses the construct of interest, this is not always the case, depending on the cell type and toxicity of the construct. As the constructs are tagged, I suggest that the authors use flow cytometry to measure expression levels in a single cell manner. While doing so, they can infect with SINV-GFP and correlate GFP signal with construct expression in each cell, providing a more accurate measurement of mmDicer effect on viral infection. Alternatively, the authors could use live microscopy, as done in Fig 2, to obtain similar data.

Reply: the reviewer is correct that we did not go for monoclonal selection of our mmDicer-expressing cells and therefore that there could be some cell-to-cell variation in expression. However, we have done immunostaining of Dicer in these cells and did not see drastic differences in expression, so we do not think this should impact SINV-GFP expression in a major way. We will provide these images and a quantification of the Dicer signal as a supplementary figure.

For Fig 1C and 1F, it would be great to have growth curves with two different MOIs, instead of a single time point, to ensure that a putative antiviral effect is not missed. Same goes for Fig 2C, especially when the authors document quite a big defect on GFP expression (a proxy for SINV infection) when Dicer is knocked down (Fig 2B). There may be a bigger difference in titers at earlier time points. This matter runs throughout the manuscript. I do not suggest that the authors should provide growth curves every time viral titers are measured, but it is still worth doing it for the 2-3 key experiments of the paper.

Reply: we will perform growth curves of virus infection for the key experiments in the manuscript as suggested. We already have done kinetic measurements of GFP accumulation at different MOIs, which we can provide as supplementary data, but we agree with the reviewer that GFP signal should not been used as the only proxy for the infection and that measuring viral titers by plaque assay is important as well.

Figure 4, could the authors provide a proof that the Dicer antibody is specific in the bat context? This can be done by staining Dicer in bat cells knocked down for Dicer and infected with SINV. The apparition of foci upon anti-Dicer antibody staining should be abbrogated or severely impaired by the knock-down.

Reply: see our reply to point 3 of Reviewer 1.

Fig 5C, please provide a quantification of the images.

Reply: these microscopy images have not been quantified because they have been obtained with an epifluorescence microscope. Indeed, the Pearson correlation coefficient can only be obtained using a confocal microscope. In fact, we have tried to use a confocal microscope to take pictures of these FISH images, but the SINV gRNA signal was too weak or the dots too small to be properly visualized. Furthermore, there is a very large difference in signal intensity between HEK293T and M. myotis cells, making it difficult to define a signal threshold compatible for both cell lines.

l.263, when comparing this work with the recent publications on bat antiviral RNAi, the authors could also provide the percentage identity between Dicers from different species.

Reply: this is a valid point, we have looked at the percentage identity between Dicer proteins from different bat species but we did not include this in our manuscript. We will provide this analysis in the revised version together with a comparison of Dicer from other mammals as a reference point.

Reviewer 3.

1. • Without direct comparison to the other bat species Dicers (especially where RNAi activity has been suggested as antiviral in previous publications) there is little in this paper that can be concluded about global aspects of bat dicer/RNAi.*

Reply: see our reply to point 4 of Reviewer 1. We are planning to look at least in Tblu cells whether there is also a relocalization of Dicer upon SINV infection. So far, we could not obtain PaKi cells, but we are still looking and should we get those, we will test them as well.

*Minor *

What rules out that the mmDicer re-localization observed in the immortalized mm nasal epithelial is due simply to greater expression levels over the NoDice cells heterologously expressing mmDicer?

Reply: we will provide an immunoblot to show the level of Dicer expression between HEK NoDice + mmDicer and M. myotis nasal epithelial cells as suggested below to address this point.

• Although partially addressed in the text stating the generally long half-life of miRNAs, it seems the simplest explanation for this observation is due to some activity of a shorter-lived miRNA is required for optimal alphavirus replication is the mm nasal epithelial cells. *

Reply: this is an interesting hypothesis that would prove difficult to test in a reasonable amount of time. We thank the reviewer and will mention this possibility in the discussion of the revised manuscript.

*Suggestions that could enhance the magnitude of conclusions that can be drawn from this work. *

*Major *

• • Making NoDice cells expressing other bat species Dicers, including those with claims that RNAi is antiviral, would address how universal these current observations are to bats/cell lines.*

Reply: this could be an alternative to the use of P. alecto or T. brasiliensis cell lines that we have mentioned above. We will try to clone Dicer from the Tblu cells that we have in the laboratory. Since we do not have PaKi cells at the moment, it will be more complicated for the Pteropus Dicer, but one possibility could be to synthesize it. However, Dicer is a big gene so it could prove tricky.

• • Including an immunoblot showing that mm cells express mmDicer no more abundantly than the heterologous NoDice cells would allow ruling out the trivial explanation that foci occur at a certain critical mass of Dicer*

Reply: yes, we will provide this piece of data as stated in reply to point 2.

*Minor *

• • I believe line 151 " In contrast, Dicer * *knock down had an ANTIVIRAL effect against VSV-GFP infection at the RNA and protein *

*levels, but no difference in titers was found (Fig. 2H-J)." should be " In contrast, Dicer *

*knock down had an PROVIRAL effect against VSV-GFP infection at the RNA and protein *

*levels, but no difference in titers was found (Fig. 2H-J)." *

Reply: thank you for spotting this error, which was also mentioned by Reviewer 1, we have corrected this in the text.

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Referee #1

Evidence, reproducibility and clarity

Bats acts a reservoir for many viruses. While some of these viruses can be pathogenic for humans and other animals, infected bats tolerate these viruses and show little to no pathogenesis. It is therefore key to characterise which immune pathways are active in bats and how do they differ from other mammals to understand how bats can sustain these virus infections. RNA interference (RNAi) acts as an antiviral mechanism in plants, invertebrates and was recently shown to be active in a cell type-dependent manner as a defence mechanism in mammals. Notably, recent findings show that antiviral RNAi activity is high in cells lines from two bats species (P.alecto and T. brasiliensis) and that this pathway might play an important role in bat viral tolerance. In this study, the authors investigate the antiviral role of Dicer in another bat species, Myotis myotis. First they express M. myotis Dicer (mmDicer) or human Dicer (hDicer) in a human epithelial kidney (HEK) 293T cell line knockout for Dicer (NoDice cells) and show that, in a human cell line, expression of mmDicer or hDicer doesn't restrict infections with either Sindbis virus (SINV) or vesicular stomatitis virus (VSV). The authors then tested the role of endogenous bat Dicer in M. myotis nasal epithelial cells and found that mmDicer has a proviral activity since its knockdown reduced the replication of SINV and Semliki Forest virus (SFV), but not of VSV. The authors also show by small RNA deep sequencing analysis that there was only a modest RNAi signature in both HEK293T and M. myotis infected with SINV suggesting that mmDicer does not have increased RNAi activity compared to human cells. Interestingly, the authors then found that in M. myotis cells infected with SINV, SFV but not VSV, mmDicer accumulates into cytoplasmic foci, which also contain double-stranded RNA (dsRNA) derived from viral replication. Finally, the authors showed that this relocalisation of mmDicer into foci was dependent on host factors from M. myotis cells as there was no change in localisation in SINV-infected HEK 293T NoDice cells complemented with mmDicer.

Major comments

• The findings that mmDicer is proviral in bat cells relies exclusively on the observation that the depletion of Dicer in M. myotis cells leads to a reduced accumulation of SFV and SINV at the RNA and protein levels (figure 2). Heterologous expression of mmDicer in HEK 293T NoDice doesn't lead to an increase permissivity to viral infections (figure 1) and the accumulation of Dicer foci is only observed in M. myotis cells but not when mmDicer is expressed in HEK 293 NoDice cells (figure 6). Given that the key finding of this manuscript relies on these knockdown experiments, the authors should ensure that the impact on viral infections is due to the specific silencing of mmDicer and not caused by off-target effects of their siRNA-mediated approach. The authors designed a siRNA pool to efficiently knock-down mmDicer. They should validate their findings by using individual Dicer siRNA and verify whether the decrease SFV/SINV accumulation is observed with at least two individual siRNAs targeting Dicer. It would also strengthen their findings if they could show a complementation experiment in which a mmDicer (designed to not be affected by the siRNA-mediated silencing) is introduced exogenously in Dicer-depleted cells and show that it rescues the observed decrease in viral accumulation to demonstrate that the proviral role is strictly dependent on mmDicer. Alternatively, the authors could consider a CRISPR/Cas9 genome editing approach to knockout Dicer in bat cells to test whether this proviral effect is confirmed.
• Figure 2: the authors knock-downed Dicer in M. myotis nasal epithelial cells and carried out infections with SINV-GFP and SFV. The authors conclude that Dicer is proviral as its depletion causes an decrease in SINV-GFP and SFV accumulation. While this conclusion is supported by the decrease levels of viral RNA and protein levels upon Dicer depletion (figure 2D, 2E, 2G), the effect on the viral titers is non-significant for both viruses (Figure 2C and 2F) based on the statistical analysis. This reviewer appreciates that the titers are lower upon Dicer knockdown, which support the authors' findings at the viral RNA and protein levels. However, as these results are central to the core message of the manuscript, the authors should provide evidence that this proviral effect observed is statistically significant on viral titers by perhaps providing additional repeats and/or comment on this observation.
• In figure 4 and 5, the authors nicely show that mmDicer accumulate to cytoplasmic foci in M. myotis cells upon infection with SFV and SINV and these foci co-localise with double-stranded RNA. The authors used a commercial polyclonal antibody against Dicer (A301-937A, Bethyl according to the Material and Methods section) which is specific to human Dicer to carry out their immunostaining in bat cells. The authors should provide evidence that this antibody indeed recognises/crossreacts with mmDicer as well and that the staining shown is indeed specific to mmDicer localisation especially because the heterologous expression of HA-tagged version of mmDicer in HEK 293T NoDice cells did not show this accumulation of cytoplasmic foci. The authors should verify the specificity of their mmDicer immunostaining by performing the same labelling in bat cells in which Dicer is knock-downed (or knock out) by individual and validated siRNA against mmDicer. The decrease signal of bat Dicer staining using the anti-human Dicer antibody would indicate specificity. Another complementary approach would be to test their Dicer staining between HEK NoDice cells (no Dicer present) versus NoDice complemented with with either mmDicer or human Dicer constructs, which would then indicate how much the anti-human Dicer antibody recognises bat Dicer. In addition, the authors should overexpress HA-tagged mmDicer in M. myotis nasal epithelial cells and test whether HA-mmDicer accumulate into foci upon infection using an anti-HA immunostaining. This would confirm that these accumulation into foci indeed is specific to mmDicer but also would reinforce the authors' findings that host factors within bat cells are important for this formation into foci since mmDicer expression in HEK 293T No Dice cells didn't show this phenotype upon infection (figure 6). OPTIONAL: it would be interesting to overexpress HA-tagged human Dicer into M. myotis nasal epithelial cells as well to then test using anti-HA staining whether human Dicer in presence of host factors from the bat can accumulate into cytoplasmic foci or not upon viral infection.
• This reviewer appreciates that this might be judged as beyond the scope of this study since it is focused on the role of Dicer in M. myotis. However, the observation that mmDicer accumulates into foci containing as well viral dsRNA is very interesting and it would significantly improve the manuscript if the authors would provide further indications that this phenotype is related to the lack of antiviral activity of mmDicer compared to what has been previously shown in other bat species (P.alecto and T. brasiliensis). In other words, is this accumulation of mmDicer into foci responsible for its different impact on virus infection? It would therefore be insightful to compare Dicer localisation upon infection in M. myotis versus P.alecto and/or T. brasiliensis bat cells in which Dicer was shown to be antiviral and test whether this accumulation in foci is only observed in bat cells in which Dicer is proviral (M. myotis) but not in the other bat cells in which Dicer is antiviral (P.alecto and/or T. brasiliensis).
• OPTIONAL: Given the difference between the provial role of mmDicer compared to the antiviral activity of Dicer in cells from P.alecto and T. brasiliensis bat cells, it would strengthen the authors' findings. if additional experiments would be conducted in parallel using M. myotis, P.alecto and/or T. brasiliensis cells. Notably knocking down Dicer in both M. myotis, P.alecto and/or T. brasiliensis cells, compare the impact on viral infections with SINV, SFV, VSV and correlate any observed difference in phenotype with putative variations in the formation of foci.

Minor comments

• Figure 2I: The authors performed a knockdown of Dicer in M. myotis nasal epithelial cells and monitor the impact on VSV-GFP infection. They found that knocking down Dicer leads to an increase in GFP protein and RNA levels suggesting an antiviral role of Dicer while, in contrast, no effect is observed on the production of infectious particles (figure 2H). On the western blot there is only a slight/weak increase of GFP protein level observed upon Dicer knockdown. Yet, the quantification of the band intensity shows a 4-fold increase relative to tubulin and compared to cells treated with siRNA control. This 4-fold increase seems exaggerated given the low increase in the intensity shown on the blot. This discrepancy is most likely due to the lower intensity of tubulin in the western blot analysis of siDicer-treated cells compared to siNeg-treated cells. The authors should reload their western blot with equal amount of protein extract loaded to ensure that the results shown on the western blot are in line with the quantification.
• Figure 3D: the authors mention that in both HEK293T cells and M. myotis nasal epithelial cells infected with SINV-GFP, there was an enrichment of 22-nucleotides (nt) paired positive and negative sense reads that overlapped with a 2-nt overhang, typical of Dicer cleavage. In Figure 3D, the data shows indeed that the duplexes are enriched for reads of 22-nt but it is unclear how this analysis reveals a 3' 2nt overhang within these duplexes. Can the authors clarify this point and if the data provided in that particular analysis indeed doesn't allow to detect these overhangs, please rephrase accordingly or provide additional analysis to support that point.
• Typo: page 5, line 152: the authors mention that Dicer knock down had an antiviral effect against VSV-GFP infection at the RNA and protein levels. However, the data in Figure 2I and 2J show an increase in both GFP RNA and proteins levels upon knockdown of Dicer. Although this data suggests that Dicer is antiviral against VSV, the knockdown of Dicer itself is not antiviral but rather proviral/increase virus accumulation. Please rephrase this sentence to avoid confusions.

Significance

The findings from this study are interesting as they provide further insights into the role of RNAi towards virus infections. Notably, it highlights a putative proviral role of Dicer in M. myotis bat cells in contrast to the antiviral role in mammals (including other bat species) as well as in plants and invertebrates. Another exciting finding of this study is the observation that mmDicer accumulates in cytoplasmic foci upon viral infection and that these foci also contain viral dsRNA replication intermediates. These accumulation of Dicer into foci only appear in bat cells infected with viruses producing large amounts of dsRNA such as SFV and SINV but not with VSV infection where no dsRNA was detected.

While these findings are novel and interesting, this study, as it stands, is rather descriptive and doesn't provide mechanistic insights into the proviral activity of mmDicer and its localisation into cytoplasmic foci upon infections. The importance of the authors' findings would greatly improve if there were some experiments addressing whether this localisation of mmDicer into foci is responsible or at least correlate with its proviral activity/its lack of antiviral activity. Comparative studies between M. myotis cells in which Dicer is proviral and/or P.alecto and T. brasiliensis cells where RNAi was previously shown to be antiviral would likely provide key mechanistic insights.

DOI: 10.1016/j.ccell.2025.07.021

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DOI: 10.1016/j.scr.2025.103812

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DOI: 10.1016/j.isci.2025.113301

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DOI: 10.1016/j.celrep.2025.116188

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What is this?

ES à la pace de SP

ES à la place de SP

ES à la place de SP

We are natural humans; we have our weaknesses and strengths. I accept myself for falling down, but I believe I can get up. We all have fearance, at first, I just ran away to escape it. Sooner or later, I am aware that you will know yourself better. I'm still scared to study English 1B cause I give myself a bunch of reasons. Fighting to achieve your goal as a dental hygienist, that is my slogan to chase away my laziness. That was what I learned from English 1A for a psychological hint. I'm excited to learn something, especially writing strategies, from all of you.

Como orden, pondría: 1. Contexto (primer parrafo) 2. Barreras y desafios que contrae 3. Percepción ciudadana 4. Cómo se relaciona la migración con la cohesión horizontal (conceptual o literatura previa + datos elsoc)

Los descriptivos y resultados de la prueba t cambiarán al no tener semilla de reproductibilidad en el código.

Creo que este subtítulo del ejercicio 3 quedó como título en la página del práctico

Los valores no coinciden con los de la tabla anterior

j

j

m

Altadena Town Council

https://www.sidecca.com/<br /> Sidecca

Altadena apparel

This paragraph should be on the same page as the graph below. (remove the page break)

Por qué hay ? en la matriz? si no hay datos entonces filtrar

Pero la corr policórica se usa cuando tenemos dos variables ordinales, no cuando tenemos un índice (asumo continuo) y otra ordinal

?

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Reply to the reviewers

Manuscript number: RC-2025-02946

Corresponding author(s): Margaret, Frame

Roza, Masalmeh

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Reviewer #1

Evidence, reproducibility and clarity

Review of Masalmeh et al. Title: "FAK modulates glioblastoma stem cell energetics..."

Previous studies have implicated FAK and the related tyrosine kinase PYK2 in glioblastoma growth, cell migration, and invasion. Herein, using a murine stem cell model of glioblastoma, the authors used CRISPR to inactivate FAK, FAK-null cells selected and cloned, and lentiviral re-expression of murine FAK in the FAK-null cells (termed FAK Rx) was accomplished. FAK-/- cells were shown to possess epithelial characteristics whereas FAK Rx cells expressed mesenchymal markers and increased cell migration/invasion in vitro. Comparisons between FAK-/- and FAK Rx cells showed that FAK re-expressed increased mitochondrial respiration and amino acid uptake. This was associated with FAK Rx cells exhibiting filamentous mitochondrial morphology (potentially an OXPHOS phenotype) and decreased levels of MTFR1L S235 phosphorylation (implicated in mito morphology fragmentation). Mito and epithelial cell morphology of FAK-/- cells was reversed by treatment with Rho-kinase inhibitors that also increased mito metabolism and cell viability. Last, FAK-dependent glioblastoma tumor growth was shown by comparisons of FAK-/- and FAK Rx implantation studies.

The studies by Masalmeh provide interesting findings associating FAK expression with changes in mitochondrial morphology, energy metabolism, and glutamate uptake. According to the authors model, FAK expression is supporting a glioblastoma stem cell like phenotype in vitro and tumor growth in vivo. What remains unclear is the mechanistic connection to cell changes and whether or not these are be dependent on intrinsic FAK activity or as the Frame group has previously published, potentially FAK nuclear localization. The associations with MTFR1L phosphorylation and effects by Rho kinase inhibition are likely indirect and remind this reviewer of long-ago studies with FAK-null fibroblasts that exhibit epithelial characteristics, still express PYK2, exhibited elevated RhoA GTPase activity. Some of these phenotypes were linked to changes in RhoGEF and RhoGAP signaling with FAK and/or Pyk2. At a minimum, it would be informative to know whether Pyk2 signaling is relevant for observed phenotypes and whether the authors can further support their associations with FAK-targeted or FAK-Pyk2-targeted inhibitors or PROTACs.

Some questions that would enhance potential impact. 1. Cell generation. Please describe the analysis of FAK-/- clones in more detail. The "low viability" phenotype needs further explanation with regard to clonal expansion and growth characteristics?

Response:

• We included a better description and a supplementary figure in our revised manuscript to indicate that we have examined several FAK -/- clones and confirmed that our observations were not due to clonal variation; multiple clones displayed similar morphological changes (Figure S1D). We also show that the elongated mesenchymal-like morphology was observed at 48 h after nucleofecting the cells with the FAK‑expressing vector, before beginning G418 selection to enrich for cells expressing FAK (Figure S1C). We also included experiments to acutely modulate FAK signalling (detaching and seeding cells on fibronectin) (Figure S2D, E, F and Figure S3) to exclude the possibility that the profound effects are due to protocols/selection we used for generating FAK-deleted cells.
• Regarding the term "low viability", we have clarified in the text that there is no significant difference in cell number (Figure S1A) or 'cell viability' when it is assessed by trypan blue exclusion (a non-mitochondria-dependent read-out) (Figure S1B) between FAK-expressing FAK Rx and FAK-/- cells cultured for three days under normal conditions. Therefore, we agree the term 'cell viability' in this context could be confusing and have replace "cell viability" with "metabolic activity as measured by Alamar Blue." in Figure 1D and Figure 5B, and the corresponding text in the original manuscript. This wording more accurately reflects the data.

Figure 1F: need further support of MET change upon FAK KO and EMT reversion.

Response: We have added a heatmap (Figure S1E) illustrating the changes in protein expression of core-enriched EMT/MET genes products (by proteomics) after FAK gene deletion (EMT genes as defined in Howe et al., 2018) ; this strengthens the conclusion that the MET reversion morphological phenotype is accompanied by recognised MET protein changes.

Fig. 2: Need further support if FAK effects impact glycolysis or oxidative phosphorylation in particular as implicated by the stem cell model.

Response: We show that FAK impacts both glycolysis (Figure 2A, 2E, and 2F) and mitochondrial oxidative phosphorylation on the basis of the oxygen consumption rate (OCR) (Figure 2B, and 2D), showing both are contributing pathways to FAK-dependent energy production. We have clarified this in the text.

Is there a combinatorial potential between FAKi and chemotherapies used for glioblastoma. Need to build upon past studies.

Response: Yes, previous studies suggest that inhibiting FAK can sensitize GBM cells to chemotherapy (Golubovskaya et al., 2012; Ortiz-Rivera et al., 2023). We have included a paragraph in the discussion section to make sure this is clearer. Although it is not the subject of this study, we appreciate it is useful context.

The notation of changes in glucose transporter expression should be followed up with regard to the potential that FAK-expressing cells may have different uptake of carbon sources and other amino acids. Altered uptake could be one potential explanation for increase glycolysis and glutamine flux.

Response: We agree with the reviewer that glucose uptake could be contributing and we include data that 2 glucose transporters are indeed FAK-regulated namely Glucose transporter 1 (GLUT1, encoded by Slc2a1 gene) and Glucose transporter 3 (GLUT 3, encoded by Slc2a3 gene) (shown in Figure S2B and C).

It would be helpful to support the confocal microscopy of mitos with EM.

Response:

We are concerned (and in our experience) that Electron microscopy (EM) may introduce artefacts during sample preparation. In contrast, immunofluorescence sample preparation is less susceptible to artefacts. The SORA system we used is not a conventional point-scanning confocal microscope, but is a super-resolution module based on a spinning disk confocal platform (CSU-W1; Yokogawa) using optical pixel reassignment with confocal detection. This method enhances resolution in all dimensions with resolution in our samples measured at 120nm. This has been instructive in defining a new level of changes in mitochondrial morphology upon FAK gene deletion.

Lack of FAK expression with increased MTFR1 phosphorylation is difficult to interpret.

Response: We do not directly show that this phosphorylation event is causal in our experiments; however, we think it important to document this change since it has been published that phosphorylation of MTFR1 has been causally linked to the mitochondrial morphology we observed in other systems (Tilokani et al., 2022).

Need to have better support between loss of FAK and the increase in Rho signaling. Use of Rho kinase inhibitors is very limited and the context to FAK (and or Pyk2) remains unclear. Past studies have linked integrin adhesion to ECM as a linkage between FAK activation and the transient inhibition of RhoA GTP binding. Is integrin signaling and FAK involved in the cell and metabolism phenotypes in this new model?

Response: To better support the antagonistic effect of FAK on Rho-kinase (ROCK) signalling, we included a new experiment in which the integrin-FAK signalling pathway has been disrupted by treating FAK WT cells with an agent that causes detachment from the substratum, Accutase, and growing the cells in suspension in laminin-free medium. We present ROCK activity data, as judged by phosphorylated MLC2 at serine 19 (pMLC2 S19), relating this to induced FAK phosphorylation at Y397 (a surrogate for FAK activity) that is supressed after integrin disengagement. These measurements have been compared with conditions whereby integrin-FAK signalling is activated by growing the cells on laminin coated surfaces. We observed a time-dependent decrease in pFAK(Y397) levels (normalised to total FAK) in suspended cells compared to those spread on laminin, while pMLC2(S19) levels increased in a reciprocal manner over time in detached cells relative to spread cells (S4A and B). There is therefore an inverse relationship between integrin-FAK signalling and ROCK-MLC2 activity, consistent with findings from FAK gene deletion experiments. In the former case, we do not rely on gene deletion cell clones.

Significance

The studies by Masalmeh provide interesting findings associating FAK expression with changes in mitochondrial morphology, energy metabolism, and glutamate uptake. According to the authors model, FAK expression is supporting a glioblastoma stem cell like phenotype in vitro and tumor growth in vivo. What remains unclear is the mechanistic connection to cell changes and whether or not these are be dependent on intrinsic FAK activity or as the Frame group has previously published, potentially FAK nuclear localization. The associations with MTFR1L phosphorylation and effects by Rho kinase inhibition are likely indirect and remind this reviewer of long-ago studies with FAK-null fibroblasts that exhibit epithelial characteristics, still express PYK2, exhibited elevated RhoA GTPase activity. Some of these phenotypes were linked to changes in RhoGEF and RhoGAP signaling with FAK and/or Pyk2. At a minimum, it would be informative to know whether Pyk2 signaling is relevant for observed phenotypes and whether the authors can further support their associations with FAK-targeted or FAK-Pyk2-targeted inhibitors or PROTACs.

__Response: __

Deleting the gene encoding FAK in mouse embryonic fibroblasts leads to elevated Pyk2 expression (Sieg, 2000). However, in the GBM stem cell model we used here, Pyk2 was not expressed (determined by both transcriptomics and proteomics). We have included Figure S1E to show that PYK2 expression was undetectable in FAK -/- and FAK Rx cells at the RNA level (Figure S1F). We conclude that there is no compensatory increase in Pyk2 upon FAK loss in these cells. In the transformed neural stem cell model of GBM, we do not consistently or robustly detect nuclear FAK.

Review #2

Masalmeh and colleagues employ a neural stem/progenitor cell-based glioma model (NPE cells) to investigate the role of Focal Adhesion Kinase (FAK) in GBM, with a focus on potential links between the regulation of morphological/adhesive and metabolic GBM cell properties. For this, the authors employ wt cells alongside newly generated FAK-KO and -reexpressing cells, as well as pharmacological interventions to probe the relevance of specific signaling pathways. The authors´ main claims are that FAK crucially modulates glioma cell morphology, cell-cell and cell-substrate interactions and motility, as well as their metabolism, and that these effects translate to changes to relevant in vivo properties such as invasion and tumor growth.

My main issues are with the model chosen by the authors.

As per the methods section, generation of FAK-KO and -"Rx" NPE cells entailed protracted selection/expansion processes, which may have resulted in inadvertent selection for cellular/molecular properties unrelated to the desired one (loss or gain of FAK expression) and which may have had cascading effects on NPE cells. The authors nonetheless repeatedly claim the parameters they quantify, such as mitochondrial or cytoskeletal properties or metabolic features, to have directly resulted from FAK loss or reintroduction. Examples of such causal inferences are to be found in lines 123, 134/135, 165, 181. Such causal claims are, in my view, unsupported.

Acute perturbation of FAK expression/activity, genetically or pharmacologically, followed by a rapid assessment of the processes under investigation, would be needed to begin to assess causality, even if acute genetic perturbations may be technically challenging as sufficient gene expression reduction or restoration to physiologically relevant levels may be hard to achieve.

Response:

We would like to first comment on the model we used here, which we think will clarify the validity of our approach. The model is a transformed stem cell model of GBM that was published in (Gangoso et al., Cell, 2021) and is now used regularly in the GBM field. As mentioned in the response to Reviewer 1, we have added text (page 4 and 5 in the revised manuscript) and a new supplementary figure (Figure S1D) clarifying that the morphological changes we observed were consistent across multiple FAK -/- clones, showing this was not due to any inter-clonal variability. We also added images showing that the morphological changes were apparent at 48 h after nucleofecting FAK -/- cells with the FAK‑expressing vector specifically (not the empty vector), prior to starting G418 selection to enrich for FAK‑expressing cells (Figure S1C), addressing the worry that clonal variation and selection was the cause of the FAK-dependent phenotypes we observed. We believe that our model provides a type of well controlled, clean genetic cancer cell system of a type that is commonly used in cancer cell biology, allowing us to attribute phenotypes to individual proteins.

We have also carried out a more acute treatment by using the FAK inhibitor VS4718 to perturb FAK kinase activity and assessed the effects on glycolysis and glutamine oxidation after 48h treatment (Figure S2D, E and F). We found that treating the transformed neural stem cells (parental population) with FAK inhibitor (300nM VS4718) decreases glucose incorporation into glycolysis intermediates and glutamine incorporation into TCA cycle intermediates, consistent with a role for FAK's kinase activity in maintaining glycolysis and glutamine oxidation.

The employed pharmacological modulation of ROCK activity is the only approach that, given the presumably acute nature of the treatment, may have allowed the authors to probe the proposed functional links. The methods section of the manuscript does not however comprise details as to the duration of these treatments, which leaves open the possibility of long-term treatment having been carried out (data shown in Figure 5B refers to 72hr treatment).

__Response: __

We have added the duration of the treatment to the Methods section and Figure Legends, to clarify that cells were treated with ROCK inhibitors for 24h, before assessing the effects on mictochondria (Figure 4C, D, S4C and D) and glutamine oxidation (Figure 5A, and S5). For metabolic activity by AlamarBlue assay, cells were treated with ROCK inhibitors for 72h (Figure 5B).

Even in the case of ROCK inhibitor experiments, it is however unclear if and how the effects on cell morphology and adhesion, mitochondrial organization and metabolic activity may be connected to each other and, if at all, to FAK expression.

Given the above uncertainties due to the nature of the model and experimental approaches, it is hard to assess the reliability and thus the relevance of the findings.

Response:

FAK suppresses ROCK activity (as judged by pMLC2 S19, Figure 4A and B). Treating FAK -/- cells with two different ROCK inhibitors restored mesenchymal-like cell morphology, mitochondrial morphology and glutamine oxidation. As mentioned above, to strengthen our evidence for the antagonistic role of FAK in ROCK-MLC2 signalling, we have now introduced an experiment whereby integrin-FAK signalling was disrupted through treatment with a detachment agent (Accutase), and subsequently maintaining the cells in suspension in laminin-free medium. We assessed pMLC2 S19 levels (a measure of ROCK activity) relating this to FAK phosphorylation that is supressed after integrin disengagement. These results were evaluated relative to spread wild type cells growing on laminin where Integrin-FAK signalling was active (Figure S4A and B). We observed an inverse relationship between Integrin-FAK signalling and ROCK-MLC2 activity in keeping with our conclusions (Figure 4A and B).

Experimental support for the ability of cell-substrate interaction modulation to concomitantly impact cellular metabolism and motility/invasion would be significant both in terms of advancing our understanding of glioma cell biology and of its translational potential, but the evidence being provided is at best compatible with the proposed model.

Response: We carried out a new experiment to support the ability of cell-substrate interaction modulation to impact metabolism; specifically, we inhibited cell-substrate interactions by plating the cells on Poly-2-hydroxyethyl methacrylate (Poly 2-HEMA)-coated dishes. This suppressed FAK phosphorylation at Y397, as expected, with concomitant reduction in glutamine utilisation in the TCA cycle (Figure S3A, B and C).

My background/expertise is in developmental and adult neurogenesis, in vivo modelling of gliomagenesis and cell fate control/reprogramming, with a focus on molecular mechanisms of differentiation and quantitative aspects of lineage dynamics; molecular details of the control of cellular metabolism, cell-cell adhesion and cytoskeletal dynamics are not core expertise of mine.

We appreciate this reviewer's expertise are not necessarily in the cancer cell biology and genetic intervention aspects of our study. We hope that the explanations we have provided satisfy the reviewer that our conclusions are valid.

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Reply to the reviewers

RESPONSE TO REVIEWERS

We thank the reviewers for their thoughtful and constructive feedback, which has been instrumental in improving the overall quality of our manuscript.

In response, we have undertaken a substantial revision that includes new experimental data, refined analyses, and clearer presentation of our findings. Specifically, we have addressed concerns about RNAi efficiency and protein-level validation, expanded our genetic models to include loss-of-function contexts, and clarified the interpretation of mitochondrial morphology using both confocal and electron microscopy. We also incorporated new data on Cyclin E regulation and mitochondrial membrane potential to strengthen the mechanistic link between dPGC1 depletion and Yki-driven tumorigenesis. These revisions not only address the specific points raised by the reviewers but also enhance the coherence and impact of the study. We are confident that the revised manuscript presents a more robust and compelling case for the role of dPGC1 as a context-dependent tumor suppressor and that it will be of broad interest to the fields of developmental biology, cancer metabolism, and mitochondrial dynamics.

Reviewer #1 (Evidence, reproducibility and clarity (Required)): Sew et al. examine the master regulator of mitochondrial biogenesis, dPGC1, in the context of Drosophila wing and larval development. They primarily use confocal imaging to probe the interplay between dPGC1 and an overactive Hippo pathway, driven by overexpression of the main effector protein, Yki. In their study, they find that tumors, driven by overactivity of Yki grow larger when dPGC1 is downregulated, implicating the mitochondrial biogenesis pathway in tumor suppression. Furthermore, in the context of Yki overexpression, they find that levels of Mfn or Opa1 modulate tumor size. Lastly, they show a role of cyclin E in controlling the size of tumors formed by Yki OE + dPGC1 RNAi. The potential role of dPGC1 as a tumor suppressor is interesting because it highlights an emerging recognition of mitochondria in the aetiology of cancer. However, before publication, much of the data in this manuscript should be strengthened by a refinement in the methods/analysis and an increase in orthogonal approaches.

We addressed concerns regarding RNAi efficiency and wing development by incorporating data from a dPGC1 mutant allele and using a ubiquitous driver for qPCR validation of transgene efficiency. We clarified the rationale for EM use. The manuscript now avoids overinterpretation of mitochondrial morphology and focuses on fusion-specific regulators. We also revised the narrative arc to maintain coherence and added loss-of-function models to support our conclusions.

Below, we address each of the reviewer’s points in detail.

Major comments:

The authors indicate that for example, in lines127-28, that neither downregulating or overexpressing dPGC1 affects wing size. However, the quantification in Fig. 1C shows a significant decrease in wing size following RNAi treatment. This decrease is modest, but it is nevertheless significant. It is worth pointing out, too, that the efficiency of the RNAi in Fig. S1C suggests that the conclusions drawn are premature. While a roughly 55% drop in mRNA levels may be statistically significant, it is unclear whether this drop in transcripts corresponds to a commensurate depletion of protein. Moreover, it is unclear, in this context, how much dPGC1 may indeed be necessary to drive a relatively normal program of mitochondrial biogenesis in wing development. To obtain a clear result, it is necessary to show significant depletion of the dPGC1 protein. (Ultimately, if it is the case that dPGC1 is unnecessary for wing development and function, a more coherent line of inquiry would be to find out the reason for this rather than to pivot the story to studying tumorigenesis in larva.)

We agree that the interpretation of the RNAi efficiency data requires clarification.

The qPCR analysis shown in former Fig. S1C was performed using wing discs from flies expressing UAS-dPGC1-RNAi under the control of the MS1096-Gal4 driver. However, as shown in current Fig. 1C, MS1096-Gal4 is not expressed uniformly across the wing disc. Some regions remain RFP-negative, indicating that the RNAi construct is not active in all cells. As a result, the measured mRNA levels likely underestimate the true knockdown efficiency. This is because the qPCR includes mRNA from both RNAi-expressing and non-expressing cells, diluting the apparent reduction in transcript levels.

To address this limitation and more accurately assess RNAi efficiency, we repeated the qPCR analysis using a ubiquitous driver (actin-Gal4) to ensure uniform expression of the RNAi construct. Under these conditions, we observed a more substantial knockdown, with dPGC1 mRNA levels reduced to approximately 25% of control levels (this is shown in current Fig S2). This result indicates that the RNAi line is more effective than initially suggested by the MS1096-Gal4-based analysis.

To complement our RNAi-based analysis, we additionally used a mutant strain carrying a characterized allele of dPGC1 (dPGC11, also known as dPGC1KG08646; see FlyBase: https://flybase.org/reports/FBal0148128). This genetically distinct approach allowed us to validate and strengthen our findings regarding dPGC1 function. Flies homozygous for this allele exhibited a modest but statistically significant reduction in both wing disc and adult wing size. These results support the conclusion that dPGC1 is required for normal wing growth and development. The new data are now included in Figure 1 and referenced in the main text (lines 144-153).

Additionally, as suggested by the reviewer, we have revised the relevant section to maintain a coherent line of inquiry. The updated text can be found in lines 163–172.

In Figure 3H-K, it is not clear why the authors used electron microscopy to evaluate mitochondrial morphology. The very good confocal images in Figure 3C-G show a clear change in mitochondrial morphology following the knockdown of Mfn, Opa1, and Miro. While it is clear from the electron micrographs in Figure H that the mitochondria are enlarged, it is not obvious that this increase in length is a result of increased mitochondrial fusion. Indeed, if the mean form factor were used to quantify the shape, it is likely that in both conditions, the value would be close to 1, indicating more of a round object, and it not obvious whether there would be a difference between the Yki OE versus the YkI OE + dPGC1 RNAi. Therefore, from this data alone, it cannot be concluded that the YkI OE + dPGC1 RNAi condition leads to mitochondrial hyperfusion.

Our rationale for including electron microscopy (EM) was to overcome specific limitations in imaging mitochondrial morphology within the main epithelium of the wing disc, where Yki-driven tumors arise. These tumors were generated using ap-Gal4, which drives expression specifically in the main epithelium and is not active in the peripodial membrane. This is an important distinction, as the peripodial membrane—used in Figures 3C–G—has a squamous architecture and larger cytoplasmic volume, making it ideal for high-resolution confocal imaging and for assessing the effects of manipulating dMfn, Opa1, and miro. However, because ap-Gal4 is not expressed in the peripodial membrane, this tissue could not be used to analyze mitochondrial morphology in the actual tumorous context.

To directly evaluate mitochondria in the main epithelium, we employed EM, which provides the resolution necessary to visualize ultrastructural changes that are not easily captured by confocal microscopy in this densely packed tissue. While EM does not directly measure fusion events, it allowed us to detect changes in mitochondrial size and shape that support our broader findings.

We acknowledge that mitochondrial enlargement alone does not definitively demonstrate hyperfusion. However, the EM data were interpreted alongside additional evidence: the upregulation of mitochondrial fusion genes (dMfn and Opa1) in Yki + dPGC1-RNAi tumors, and functional data showing that overexpression of these genes promotes fusion in the peripodial membrane. Together, these findings suggest that dPGC1 depletion enhances mitochondrial fusion in Yki-driven tumors.

To further clarify this point, we also imaged mitochondria in the main epithelium using confocal microscopy. However, the resolution was considerably lower than that achieved with EM, limiting our ability to assess fine mitochondrial structures. We have prepared a representative figure for the reviewer (below), showing representative confocal images of wing discs from three genotypes: (A) ap-Gal4, UAS-GFP (control), (B) ap-Gal4, UAS-Yki, and (C) ap-Gal4, UAS-Yki, UAS-dPGC1-RNAi. We used anti-ATP-synthase (Abcam, ab14748, dilution 1:200), to label the mitochondria for this Figure. Despite the lower resolution, mitochondria in the Yki + dPGC1-RNAi tumors appear elongated (yellow arrows) compared to those in the other conditions, consistent with the changes observed by EM. We believe this example illustrates the limitations of confocal imaging in this tissue and reinforces the need for EM to accurately assess mitochondrial morphology in the tumorous epithelium.

While our EM analyses reveal mitochondrial enlargement in wing discs co-expressing Yki and PGC1-RNAi, we acknowledge that these structural features alone do not conclusively demonstrate mitochondrial hyperfusion. To address this, we have revised the manuscript to avoid overinterpreting the EM data and instead emphasize the functional relevance of mitochondrial fusion regulators such as dMfn and Opa1 in promoting tumor growth.

Taken together, the EM analysis provides structural validation in the tumorous epithelium (Fig 4), while the confocal imaging and functional manipulation of fusion genes in the peripodial membrane offer mechanistic insight (Fig 3). This integrated approach strengthens the conclusion that PGC1 depletion in a Yki-overexpressing context promotes changes in mitochondrial morphology and contributes to tumorigenesis, independent of whether these changes reflect hyperfusion.

Figure 4. refers to changes in mitochondrial fusion and fission in tumor formation; however, the authors do not attempt to alter mitochondrial fission factors, so it is not accurate to mention a role of mitochondrial fission, in this context.

As we did not directly manipulate fission-related factors in our experiments, we agree that it would be inappropriate to draw conclusions about the role of mitochondrial fission in this context. Our revised figure (current Fig 5) and accompanying text now focus exclusively on the effects of mitochondrial fusion and the genes directly involved in regulating this process.

It must be noted, too, that the authors have not demonstrated that their genetic interventions have actually affected mitochondrial morphology in these experiments. As noted in the previous figure, the Yki OE + dPGC1 RNAi condition showed enlarged mitochondria, but not necessarily hyperfused organelles. Therefore, the downregulation of Mfn or Opa1 in this set of experiments may not necessarily have altered mitochondrial morphology. Perhaps suppression of Mfn or Opa1 would normalize the areas of these evidently swollen mitochondria, but this is unclear without images. Furthermore, it should be appreciated that both Opa1 and Mfn exhibit pleiotropic attributes - e.g., Opa1 not only regulates IMM fusion, but it also modulates the shape and tightness of cristae membranes, specialized sites of oxidative phosphorylation as well as sequestration of cytochrome c, the release of which influences apoptosis (Frezza et al., 2006). At least in mammalian cells, Mfn2 is thought to regulate contacts between mitochondria and endoplasmic reticulum (Naon et al., 2023), which may serve other functions than OMM fusion, such as stabilization of the MAM.

To directly address this point, we performed EM to assess mitochondrial ultrastructure in Yki + dPGC1-RNAi wing disc tumors, with and without dMfn1 downregulation, the most upregulated mitochondrial fusion gene in this tumor context. In Yki + dPGC1-RNAi tumors, mitochondria appeared more elongated, consistent with increased fusion. Upon dMfn1 depletion, we observed a dramatic shift in mitochondrial morphology: mitochondria became larger and more rounded, with disrupted cristae and onion-like structures, indicative of compromised mitochondrial integrity and function (see current Fig. 4).

As the reviewer rightly notes, these morphological changes are consistent with the pleiotropic roles of Mfn and Opa1, which extend beyond outer and inner membrane fusion to include regulation of cristae architecture and ER-mitochondria contacts (Frezza et al., 2006; Naon et al., 2023). We now discuss these broader roles in the revised manuscript (lines 493–497). Taken together, our EM and confocal analyses, combined with targeted genetic manipulations, provide evidence that mitochondrial morphology is indeed altered in response to dPGC1 depletion and fusion gene deregulation in the wing disc.

Figure 5 highlights a connection between dysregulation of mitochondria and Cyclin E, which allows cells to prematurely enter S phase. The data presented here do not offer clarity on whether the enlargement of the tumors results from increase cellular proliferation and/or cell size. The role of the cell cycle adds a layer of complexity to these results, because it is thought that mitochondria undergo fragmentation during the cell cycle to promote an even distribution of the organelle population after mitosis (Taguchi et al., 2007); however, in this manuscript, the authors contend that the downregulation dPGC1 is promoting mitochondrial hyperfusion. It is unclear how and whether cellular division and proliferation would proceed at an accelerated rate in a situation with mitochondrial hyperfusion.

To address this point, we started by analyzing whether Yki + dPGC1-RNAi tumors exhibit increased proliferation compared to tumors expressing Yki alone. We quantified mitotic activity using the phospho-Histone H3 (PH3) marker of mitotic cells and observed a significant increase in PH3-positive cells in the Yki + dPGC1-RNAi condition. These results indicate an elevated proliferation rate in these tumors and are now presented in Fig 2O–Q. In the text, can be found in lines 221-228.

We agree with the reviewer that our findings challenge the conventional view that mitochondrial fragmentation is a prerequisite for mitosis, as we observe increased expression of gene promoting mitochondrial fusion in the context of dPGC1 downregulation alongside signs of accelerated cell cycle entry. It is important to note that we also show that the levels of the oncogene Cyclin E, a key driver of cell cycle progression and S-phase entry, were elevated in Yki + dPGC1-RNAi tumors compared to those expressing Yki alone, suggesting that the increased proliferation observed is at least in part driven by enhanced cycle activity. To further probe Cyclin E’s role, we used the CycE-05306 heterozygous mutant allele, which reduces Cyclin E levels by ~50% without affecting normal development. Notably, this partial reduction strongly suppressed tumor growth in the Yki + dPGC1-RNAi background (Fig 6), underscoring Cyclin E’s functional importance in supporting oncogenic growth in this context.

These findings support the notion that defects in the expression of mitochondrial genes involved in mitochondrial morphology induced by dPGC1 depletion do not impair but rather coincide with accelerated cell division.

Minor comments:

Lines 69-72 contrast the roles of PGC1α and β. It is not clear whether the comparison is of their respective roles in cancer or in normal physiology. In either case, it is important to note that PGC1β has been shown to drive mitochondrial fusion as well as biogenesis through its control of MFN2, among other factors (Liesa et al., 2008).

In response, we have clarified the comparison between PGC1α and PGC1β in the introduction to specify that it refers to their roles in cancer. Additionally, we now acknowledge that PGC1β has been shown to promote mitochondrial biogenesis and fusion, notably through the regulation of MFN2, as demonstrated by Liesa et al. (2008). This reference has been added to provide a more balanced and accurate representation of PGC1β’s functions. In the text it can be found in lines 77-81.

Although this study focuses on PGC1, the authors do not seem to site the original literature from the Spiegelman lab.

In response to the reviewer’s comment, we have added a new section in the introduction that cites key foundational studies from the Spiegelman lab. This addition can be found in the introduction in lines 68-73.

There are 10-20 grammatical errors throughout the text.

We apologize for this. We have carefully revised the text, and we are very confident those errors have been corrected.

**Referee Cross-commenting**

There is agreement among the referees that the potential role of PGC1 as a tumor suppressor is interesting and significant. However, various aspects of this work require attention prior to publication. For example, there needs to be a complete knock down of PGC1 to come to any conclusion as to its role in wing development. The methods for analyzing mitochondrial morphology need to be clarified and be consistent with standards in the field of mitochondrial dynamics. Also, the authors need to quantify their Western blots to obtain accurate assessments of protein levels. Generally, the study relies too heavily on overexpression experiments; understanding the potential role of mitochondria in regulating the Hippo pathway should include various knockdown and/or knockout models.

Reviewer #1 (Significance (Required)):

Overall, the authors show an interesting dampening effect of dPGC1 on growth of Yki-driven tumors. This data could be relevant for elucidating how dysregulation of the Hippo signalling pathway can underlie tumorigenesis.

The narrative arc of the study, however, appears to lack a focused line of inquiry. Figure 1 highlights an attempt to modulate Drosophila wing size and/or structure by downregulating dPGC1, but to no effect. Although examination of the efficiency of the RNAi revealed that the transcripts were still present in significant quantities; so, the conclusion that dPGC1 is dispensable for wing formation is premature. To have clarity on this point, it would be necessary to completely knockdown the gene, preferably by showing a total loss of protein. This should be feasible for the authors, since they showed Western blotting in Figure 5A. In any event, it seems that this negative data led the authors to study the Hippo pathway in the larval stage. This transition from Figure 1 to 2 seemed somewhat arbitrary and leads to a rather disjointed sense of the main line of inquiry around dPGC1.

It is important to note, too, that the authors highlight a role of mitochondrial dynamics in the pathway of Yki-driven tumor formation; however, they only directly evaluate mitochondrial dynamics in this context in a single assay, namely, Figure 3H-K, and this quantification is likely inaccurate because the mitochondria in the Yki OE + dPGC1 RNAi condition seem to be substantially enlarged, circular structures. It is critical to keep in mind that mitochondrial enlargement does not necessarily stem from hyperfusion. It could come from a decrease in the activity of Drp1 or result from an imbalance between mitochondrial biogenesis and mitophagy.

As noted in our responses above, we have addressed these concerns by clarifying the limitations of our mitochondrial morphology analysis. Additionally, we have expanded the discussion (lines 498-504) to explicitly acknowledge that mitochondrial enlargement does not necessarily indicate hyperfusion. In that paragraph, we consider alternative explanations such as reduced fission or imbalances in mitochondrial biogenesis and mitophagy, and we outline the need for future studies using dynamic assays and additional markers to more precisely dissect mitochondrial remodeling in Yki-driven tumors.

A marked limitation of this study is the overuse of rather artificial manipulations of transcriptional regulatory pathways. The study would benefit a lot from investigation of the loss of function of components of the Hippo pathway rather than just OE of Yki.

We performed additional experiments using Warts (Wts) mutant clones to assess the role of dPGC1 in a loss-of-function context within the Hippo pathway. While our initial analyses were based on Yki overexpression, which allowed us to robustly probe the interaction between Yki and dPGC1, we agree that this approach may not fully reflect physiological conditions. By generating Wts mutant clones, which endogenously activate Yki through loss of upstream inhibition, we were able to evaluate the impact of dPGC1 depletion in a more physiologically relevant setting. These new results confirm and extend our previous findings, showing that dPGC1 limits tissue overgrowth even when Yki is activated through loss of Wts, thereby strengthening the biological relevance of our conclusions.

These results are presented in Fig 2F-I. In the text, those results are presented in lines 181-189.

My expertise is in mitochondrial biology, with specialization in super-resolution imaging, mitochondrial dynamics and membrane architecture. I have also worked in the interface between mitochondrial physiology and cancer. With this perspective, I think that the authors uncover a potentially interesting role of PGC1 as a tumor suppressor.

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

Summary In this manuscript the authors the investigate the role of the mitochondrial regulatory transcription factor dPGC1 in tissue growth and oncogenic transformation. They show that dPGC1 limits hyperplasia mediated by overexpression of Yki in the Drosophila wing disc, while having no effect on normal growth. dPGC1 depletion in discs overexpressing Yki results neoplastic overgrowth and hyperfused mitochondria, which was dependent on the increased expression of genes involved in promoting mitochondrial fusion. Additionally, the authors show that dPGC1 limits CycE levels post-transcriptionally in Yki tumors.

In the revised version of our manuscript, we have clarified the relationship between our findings and prior work by Nagaraj et al., including new experiments that demonstrate the specificity of dPGC1’s role in Yki-driven growth. Specifically, we show that dPGC1 depletion does not enhance tissue overgrowth in EGFR or InR contexts, nor does it affect Yki expression or activity. Furthermore, we tested dPGC1 overexpression in Yki-overexpressing tissues and observed no significant changes in growth or mitochondrial fusion gene expression. Additional controls confirmed that Cyclin E upregulation is specific to the Yki + dPGC1 depletion condition, reinforcing the context-dependent nature of our findings.

Each of the reviewer’s comments is addressed below.

Major comments 1) The authors mention several times in passing in the results a manuscript from the Banerjee lab (Nagaraj et al 2012), which shows that many of the genes the authors of the present manuscript show are upregulated upon Yki overexpression + dPGC1-RNAi compared with Yki overexpression alone are in fact upregulated upon Yki overexpression alone compared with control (dMfn/marf, opa1, miro - while interestingly dPGC1 itself is not affected). Nagaraj et al further show that Yki-overexpressing discs have longer mitochondria suggesting increased fusion even in the absence of dPGC1 depletion. The findings from Nagaraj et al should be mentioned explicitly in the introduction and the relationship between this manuscript and the present work clearly outlined in the discussion.

In the revised manuscript, we have now explicitly referenced the findings of Nagaraj et al. (2012) in the Introduction (lines 106-118), Results (lines 355-360) and Discussion (lines 466-468) sections.

In the revised Introduction, we summarize their key observations that Yki overexpression alone upregulates mitochondrial fusion genes such as dMfn and Opa1, and leads to mitochondrial elongation, while not affecting dPGC1 expression.

In the revised Results section, we mention that, building on that work, our study demonstrates that dPGC1 depletion further amplifies this effect, leading to enhanced mitochondrial elongation and tumor growth.

In the revised Discussion, we now explicitly reference the findings by Nagaraj et al. (2012), which demonstrated that Yki overexpression promotes mitochondrial fusion and upregulates key fusion genes. We build upon this work by showing that dPGC1 depletion in a Yki-overexpressing background further enhances mitochondrial fusion gene expression and tumor growth. This supports a model in which dPGC1 acts as a safeguard against Yki-induced mitochondrial remodeling and oncogenesis, reinforcing its role as a context-dependent tumor suppressor.

Importantly, we show that this effect is context-dependent and not observed in otherwise normal tissues, highlighting a sensitized mitochondrial response to Yki activation when dPGC1 is lost. These additions help delineate the novel contribution of our study in identifying dPGC1 as a critical modulator of mitochondrial dynamics and tumorigenesis downstream of Yki.

2) Given that Yki overexpression alone induces mitochondrial fusion and that dMfn/marf and opa1 depletion suppresses Yki-induced overgrowth (Nagaraj et al), does dPGC1 overexpression also suppress Yki-induced overgrowth?

If so, is this correlated with reduction in dMfn/marf and opa1 compared with Yki overexpression alone?

In response, we performed additional experiments to assess whether dPGC1 overexpression influences Yki-driven overgrowth. We also analyzed the expression of mitochondrial fusion genes (dMfn and Opa1) in this context. As shown in new Fig. S8, dPGC1 overexpression in Yki-overexpressing wing discs did not significantly affect tissue growth, nor did it alter the mRNA levels of key fusion regulators, dMfn and Opa1. These findings suggest that the transcriptional upregulation of mitochondrial fusion genes observed upon dPGC1 depletion is not a general consequence of altered dPGC1 levels, but rather a specific response that emerges in the context of Yki activation. We now present and discuss these results in the revised manuscript (lines 278-285), highlighting the sensitized nature of mitochondrial remodeling in an oncogenic environment driven by Yki signaling.

3) One important question raised by this study is: how specific is the effect of dPGC1 depletion to Yki-driven overgrowth? As Yki-driven overgrowth already have increased mitochondrial length, it is possible that Yki-expressing cells are already sensitised to the effects of dPGC1 depletion. Interestingly, Nagaraj et al show that mitochondrial morphology is not affected upon EGFR activation (hyperplasia) or upon scrib and avl depletion (neoplasia). The authors should therefore test if dPGC1 depletion can potentiate the growth of other hyperplasia drivers such as activated EGFR and InR in the wing disc.

We tested whether the growth-suppressive effect of dPGC1 depletion was specific to Yki-driven overgrowth or could also potentiate tissue growth in other oncogenic contexts. Specifically, we downregulated dPGC1 in wing discs overexpressing either EGFR or InR. In both cases, we did not observe any enhancement of tissue overgrowth upon dPGC1 depletion, in contrast to what we observed in Yki-overexpressing discs. These results suggest that the sensitivity to dPGC1 depletion is specific to Yki-driven overgrowth and is not a general feature of hyperplastic growth induced by other oncogenes.

These results are shown in Fig S4 and in lines 195-202.

4) There are a few simple control experiments the authors should provide to clarify the relationship between Yki and dPGC1: - Are Yki levels affected by dPGC1 depletion?

To address the potential regulation of Yki by dPGC1, we performed quantitative PCR (qPCR) analysis to measure the expression levels of yki and its well-established transcriptional targets—Cyclin E, Diap1, and bantam—in wing discs depleted of dPGC1. As shown in Fig. S3, we did not detect significant changes in the transcript levels of yki or its target genes, suggesting that the enhanced phenotype observed upon dPGC1 depletion is unlikely to be driven by increased Yki expression or activity. These results indicate that dPGC1 does not strongly influence Yki expression or activity. These new results are presented in lines 190-194.

• Does dPGC1 knockdown alone modify the expression of the genes tested in Fig.3A? In other words, is this upregulation specific of the Yki-overexpression context?

We have conducted this analysis, and the results are now presented in new Fig S7. While the trend is similar to that observed in tumors with both Yki depletion and dPGC1 depletion, the magnitude of change is smaller compared to the context of Yki overexpression. This is described in the text in lines 273-277.

• Does dPCG1 knockdown also stabilise CycE in the absence of Yki overexpression or does the stabilisation of CycE occur only in Yki tumors?

To address this, we examined Cyclin E levels in wing imaginal discs mutant for dPGC1 alone. Our analysis did not reveal any detectable changes in Cyclin E levels under these conditions. These findings suggest that the upregulation of Cyclin E is not a general consequence of dPGC1 loss, but rather a feature specific to the context of Yki overactivation. The corresponding data are now included in Fig S14 of the revised manuscript. In the text, it can be found in lines 442-448.

5) Figure 3C-G: it is not clear how the authors can quantify the length of 3D structures like mitochondria from 2D TEM images (unless they have done volume reconstruction from consecutive sections) and no details are provided in the methods. The quantification of mitochondrial length has to be performed rigorously as it is a key part of the paper.

We agree that TEM provides only 2D profiles of 3D mitochondrial structures, and that this does not allow for precise volumetric reconstruction. In our study, we measured the longest axis of mitochondria visible in thin TEM sections, which is a commonly used 2D proxy for mitochondrial length in the literature (e.g., PMID: 36367943 and PMID: 38637532). To avoid misunderstandings, we have clarified in the Material and Methods section that the reported values represent apparent mitochondrial length in 2D sections, not true 3D length. To enhance the accuracy of these estimates, we measured more than three tissues per genotype, multiple regions per tissue, several cells per region, and various fields of view per cell.

Minor Comments:

1) Line 51: "Mitochondria are highly dynamics organelles." should be "Mitochondria are highly dynamic organelles."

We have corrected that mistake. Thanks!

2) Introduction: the authors should summarise the known physiological functions of PGC1α in order to put their findings in context.

We have added a section in the introduction (lines 66-81) summarizing the known physiological functions of PGC1α

3) lines: 121-3: "...depletion of dPGC1...did not have a major impact on adult wing size and shape (Fig 1B, C)." There is a small but statistically significant difference so the authors should state this in the text.

We have revised the text to acknowledge that dPGC1 depletion leads to a modest but statistically significant reduction in wing size. In addition to the original analysis, we have now included further experiments to strengthen this point. Specifically, we analyzed wings from flies homozygous for the dPGC11 allele (also known as dPGC1KG08646; see FlyBase: https://flybase.org/reports/FBal0148128) and confirmed a small but significant reduction in both wing disc and adult wing size compared to controls (this can be found in Fig. 1 and Fig. S1). These results support the conclusion that, although dPGC1 is dispensable for viability and gross morphology, it contributes to normal wing growth. These new results can be found in lines 144-153.

4) Figure 5A (Cyclin E western blot): the authors should show molecular weight markers. In the revised version of our manuscript, we are including the molecular markers as indicated by the reviewer. These can be found in Fig S12.

Reviewer #2 (Significance (Required)):

The manuscript by Sew et al builds on the previous work by Nagaraj et al to explore the role of mitochondrial function in tumors driven by disruption of the Hippo pathway. In particular, the authors identify dPGC1 as a transcription factor that limits Yki-driven mitochondrial fusion and tissue growth. Interestingly, they further show that Yki/PGC1-depleted tumors are highly sensitive to Cyclin E levels, due to post-transcriptional Cyclin E increase. These results further our knowledge of how Yki drives growth and how mitochondria participate in oncogenic transformation. With appropriate revision as outlined above (for example exploring whether the mechanism proposed is Yki-specific), the manuscript will be of broad interest to developmental and cancer biologists.

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

The manuscript presents compelling evidence that dPGC1 acts as a context-dependent tumor suppressor in Drosophila by modulating mitochondrial dynamics and limiting Yorkie (Yki)-induced oncogenic growth. By leveraging the Drosophila wing imaginal disc as a model, the authors investigate how dPGC1 depletion exacerbates Yki-driven tissue overgrowth, mitochondrial hyperfusion, Cyclin E upregulation, and DNA damage, leading to tumorigenesis. The study provides valuable insights into the interplay between mitochondrial dynamics and cancer, with implications for understanding metabolic regulation in oncogenesis. While the findings are significant and well-aligned with the field, certain aspects of the experimental design, data presentation, and mechanistic insights require further attention to enhance clarity, reproducibility, and impact. Below, I outline my major concerns and recommendations.

We addressed concerns about RNAi efficiency and protein-level validation with new qPCR data and mutant analysis. We provided EM and confocal evidence of mitochondrial changes. We clarified non-autonomous effects and quantified Mmp1 and F-actin and added data on miro and Opa1 manipulations. Cyclin E quantification was expanded using multiple Western replicates and a validated mutant allele, and we included new data on mitochondrial membrane potential to assess functional consequences.

Our detailed responses to each point raised by the reviewer are provided below.

Major Points

1. One point is the knock-down efficiency of dPGC1 on the mRNA level, which is between 30 to >50% (Fig. S1C). This is not too strong, so the question arises how severly the protein levels are affected. If possible, an antibody staining with quantification should be performed. From these data it cannot be concluded dPGC1 is not required for normal development, half the dose could be sufficient. How do wings look like when the ap-GAL4 driver is used for dPGC1 knock-down, as this is the driver used in the subsequent experiments? Reviewer 1 also raised concerns about the potential inefficiency of the RNAi treatment in revealing a function during normal wing growth. We agree with both reviewers that the interpretation of the RNAi efficiency data requires clarification.

The qPCR analysis shown in former Fig. S1C was performed using wing discs from flies expressing UAS-dPGC1-RNAi under the control of the MS1096-Gal4 driver. However, as shown in current Fig. 1C, MS1096-Gal4 is not expressed uniformly across the wing disc. Some regions remain RFP-negative, indicating that the RNAi construct is not active in all cells. As a result, the measured mRNA levels likely underestimate the true knockdown efficiency. This is because the qPCR includes mRNA from both RNAi-expressing and non-expressing cells, diluting the apparent reduction in transcript levels.

To address this limitation and more accurately assess RNAi efficiency, we repeated the qPCR analysis using a ubiquitous driver (actin-Gal4) to ensure uniform expression of the RNAi construct. Under these conditions, we observed a more substantial knockdown, with dPGC1 mRNA levels reduced to approximately 25% of control levels (this is shown in current Fig S2). This result indicates that the RNAi line is more effective than initially suggested by the MS1096-Gal4-based analysis.

To complement our RNAi-based analysis, we additionally used a mutant strain carrying a characterized allele of dPGC1 (dPGC11, also known as dPGC1KG08646; see FlyBase: https://flybase.org/reports/FBal0148128). This genetically distinct approach allowed us to validate and strengthen our findings regarding dPGC1 function. Flies homozygous for this allele exhibited a modest but statistically significant reduction in both wing disc and adult wing size. These results support the conclusion that dPGC1 is required for normal wing growth and development. The new data are now included in Figure 1 and referenced in the main text (lines 144-151).

Unfortunately, we cannot perform antibody staining due to the unavailability of antibodies against dPGC1.

How does the wing disc look like when dPGC1 is overepressed together with Yki?

In response, we performed additional experiments to assess whether dPGC1 overexpression influences Yki-driven overgrowth. We also analyzed the expression of mitochondrial fusion genes (dMfn and Opa1) in this context. As shown in new Fig. S8, dPGC1 overexpression in Yki-overexpressing wing discs did not significantly affect tissue growth, nor did it alter the mRNA levels of key fusion regulators, dMfn and Opa1. These findings suggest that the transcriptional upregulation of mitochondrial fusion genes observed upon dPGC1 depletion is not a general consequence of altered dPGC1 levels, but rather a specific response that emerges in the context of Yki activation. We now present and discuss these results in the revised manuscript (lines 278-285), highlighting the sensitized nature of mitochondrial remodeling in an oncogenic environment driven by Yki signaling.

In Fig 2D (but also in Fig. 2C) not only cells in the dorsal but also in the ventral comparmtent seem to overproliferate. Either this is a mis-conception or it is a non-autonomous effect from interfering with Yki and dPGC1 in the vertrnal compartment. In either cases, this has to be clarified.

Ventral cells are not labelled by GFP. Fig 3D shows a tumor in which GFP-negative cells are not present, suggesting that they are not overproliferating but rather being eliminated. This phenomenon is consistent with cell competition, a well-characterized process in which transformed or tumorigenic cells outcompete and eliminate neighboring wild-type cells. We have previously described this behavior in wing disc tumors (PMID: 26853367; DOI: 10.1016/j.cub.2015.12.042), and it likely contributes to the expansion of the tumor mass by removing surrounding normal tissue also in this context.

In Fig. 2F-H quantification of Mmp1 and F-actin is missing. Mmp1 is a JNK target, so the authors could do in addition an anti-phospho JNK antibody staining.

In response, we have performed those quantifications. They are now included in Fig 2M, N.

In Fig. 3: how does the mitochondrial network look like in the wing disc periopodial epithelium using the Gug>Yki+dPGC1 genotype? Is it similar to Gug>dMfn or Gug>miro?

We attempted to perform this analysis; however, Yki overexpression under the control of Gug-GAL4 resulted in larval lethality, likely due to GAL4 activity in essential tissues such as the central nervous system. As a result, we were only able to induce transgene expression for 24 hours before lethality occurred.

At this early point, no detectable changes in mitochondrial morphology were observed in the peripodial membrane, likely because the duration of transgene expression was insufficient to elicit phenotypic alterations in this specific tissue. Therefore, while we aimed to compare this genotype to Gug>dMfn and Gug>miro, the technical limitations prevented a conclusive analysis.

We have prepared a representative figure for the reviewer (below), showing representative confocal images of wing discs showing mito-GFP and Dapi in the three genotypes indicated in the Fig.

In Fig. 3I: what is really the mitochondrion? It would be good to outline the region(s) that was/were measured.

To improve clarity, we have repeated the electron microscopy (EM) analysis and now provide representative images that more clearly illustrate mitochondrial morphology in the different genotypes analyzed. These updated images presented in Fig 4 better highlight the structural alterations observed upon genetic manipulation and help clarify the basis for our morphological assessments.

We have extended our analysis and have assessed mitochondrial ultrastructure in Yki + dPGC1-RNAi wing disc tumors, with and without dMfn1 downregulation—the most upregulated mitochondrial fusion gene in this tumor context. In Yki + dPGC1-RNAi tumors, mitochondria appeared more elongated, consistent with increased fusion. Upon dMfn1 depletion, we observed a dramatic shift in mitochondrial morphology: mitochondria became larger and more rounded, with disrupted cristae and onion-like structures, indicative of compromised mitochondrial integrity and function (see new Fig 4).

A quantification of RNAi and overexpression efficiencies of the different transgenes in Fig. 3 is required.

To assess the efficiency of RNAi-mediated knockdown and transgene overexpression, we performed quantitative PCR (qPCR) using the ubiquitous Actin-Gal4 driver. While we acknowledge that this driver does not replicate the spatial specificity of the periodic membrane Gal4 driver used in the experiments shown in Figure 3 (Gug-Gal4), the latter targets a very limited number of cells within the imaginal disc, making reliable qPCR quantification unfeasible.

Using Actin-Gal4 allows us to obtain a relative and informative measure of transgene efficiency across the different constructs. These data confirm effective knockdown and overexpression of the relevant genes and are now included in Figure S2.

In Fig. 4: what is the phenotype when miro is over-expressed in combination with Yki? Or when it is knocked down in the ap>Yki-dPGC1 background? This was the gene tested in Fig. 3 with a clear mitochondrial phenotype

To address whether miro contributes to Yki-mediated tumor growth, we performed the requested experiments and now include the results in the revised manuscript (see updated Results section, lines 374-377, and new Fig. S11).

Our data show that overexpression of miro in combination with Yki does not lead to a significant increase in tissue growth or tumor-like phenotypes, in contrast to the effects observed with dMfn or Opa1 overexpression. Similarly, knockdown of miro in the ap>Yki-dPGC1-RNAi background did not suppress tumor growth, indicating that miro is not required for the enhanced proliferation observed in this context.

These findings suggest that, although miro influences mitochondrial morphology in normal wing discs (as shown in Fig. 3), its role in tumorigenesis is distinct from that of dMfn and Opa1. We have revised the manuscript to clarify the gene-specific contributions of mitochondrial fusion regulators to Yki-driven tumorigenesis. This distinction underscores the complexity of mitochondrial dynamics and highlights that not all fusion-related genes exert the same functional impact in oncogenic settings.

How does the mitochondrial morphology in the wing disc peripodial epithelium look like in Gug>Opa1RNAi or Gug>Opa1 discs?

To assess the impact of Opa1 on mitochondrial morphology in the peripodial epithelium of the wing disc, we used the Gug-GAL4 driver to either overexpress or knock down Opa1. Our analysis revealed that Opa1 overexpression led to slightly elongated mitochondria, but did not result in extensive network formation, suggesting a modest enhancement of inner membrane fusion. In contrast, Opa1 knockdown caused clear mitochondrial fragmentation, closely resembling the phenotype observed upon dMfn depletion. These results shown in Fig 3 are consistent with the distinct roles of Opa1 and dMfn in regulating mitochondrial fusion: Opa1 primarily modulates inner membrane fusion and cristae architecture, while dMfn drives outer membrane fusion and network connectivity.

The corresponding data are presented in Figure 3F, G, and quantified in Figure S9, alongside experiments manipulating other genes involved in mitochondrial dynamics.

Why have the authors switched between the ap>Yki+dPGCRNAi and the ap>Yki+dPGC1shRNA lines? It would be important to have this series of experiments in the same backgrounds, as KD efficiencies are different (Fig. S1C).

The primary reason for switching between the dPGC1-RNAi and dPGC1-shRNA lines was practical: the chromosomal insertion sites of the transgenes made certain genetic combinations more feasible with one line over the other. This flexibility significantly facilitated our experimental design and analysis.

To address concerns regarding knockdown efficiency, we performed a comparative analysis using the ubiquitous actin-GAL4 driver, rather than MS1096-GAL4, which exhibits patchy and dynamic expression in the wing imaginal disc. This allowed us to obtain a more consistent and interpretable measure of mRNA downregulation for both transgenes. Our results show that both lines achieve comparable levels of knockdown, as shown in Figure S2.

Fig. 5A: proper quantification of Western Blot signals is required. I do not agree that Cyclin E protein levels are elevated in ap>Yki or ap>Yki+dPGC1 discs. Even at the mRNA levels the increase in expression is rather weak. From these results nothing can be concluded.

We have repeated the Western blot analysis using seven independent membranes to ensure robust quantification of Cyclin E levels in ap>Yki and ap>Yki+dPGC1-RNAi wing discs (Fig 6).

Although the increase in Cyclin E protein levels is subtle, it is consistent across replicates and statistically significant. We have now included the quantification of these Western blot signals in the revised Figure 6, which supports the conclusion that Cyclin E levels are elevated in ap>Yki+dPGC1 discs.

We hope this additional data addresses the reviewer’s concern and strengthens the interpretation of our results.

Knock-down efficiencies for dap and CycE needs to be quantifiec (Fig. 5H-N). Although the rescue experiment with CycE knock down is from the phenotype convincing, it is nonetheless puzzling, as CycE is accodring to Fig. 5A+B hardly upregulated. An independent CycE RNAi line would be useful.

We have quantified the knockdown efficiency of the dap-RNAi line, and the results are included in Figure S13.

Regarding Cyclin E, we would like to clarify that we did not use an RNAi line in this experiment. Instead, we employed the CycE-05306 mutant allele in a heterozygous background, which is expected to reduce Cyclin E levels by approximately 50%. The CycE-05306 allele in Drosophila melanogaster is a loss-of-function allele of the Cyclin E gene. This allele carries a P-element insertion in the first intron of the CycE gene, which disrupts normal transcription and reduces Cyclin E expression. In a heterozygous background, as used in your experiments, CycE-05306/+ is expected to reduce Cyclin E levels by approximately 50%, which is typically sufficient to observe genetic interactions or sensitized phenotypes without affecting normal development. This makes it a valuable tool for studying gene dosage effects, particularly in tumor models where Cyclin E activity may be rate-limiting.

Importantly, this partial reduction does not impair normal tissue growth, but it strongly limits tumor growth in the context of Yki overexpression combined with dPGC1 downregulation, as shown in Figure 6. This selective sensitivity highlights the functional importance of Cyclin E in supporting oncogenic growth driven by Yki and dPGC1 depletion. We believe this provides compelling evidence for Cyclin E’s role in this tumor model.

Reviewer #3 (Significance (Required)):

Strengths and Limitations of the Study Strengths Innovative Focus on Mitochondrial Dynamics and Oncogenesis: The study provides compelling evidence linking mitochondrial dynamics, particularly hyperfusion, to tumorigenesis in Drosophila. The identification of dPGC1 as a context-dependent tumor suppressor adds novel insights into the interplay between metabolism and oncogenesis. Comprehensive Use of Drosophila as a Model System: The study leverages the genetic tractability of Drosophila, allowing precise manipulation of mitochondrial regulators and signaling pathways. The use of wing imaginal discs as a model for tumor growth is well-established and appropriate. Integration of Morphological and Genetic Data: The manuscript combines confocal imaging, electron microscopy, and genetic tools to demonstrate the role of dPGC1 in regulating mitochondrial dynamics, Cyclin E levels, and tissue overgrowth. Relevance to Cancer Biology: The findings address key hallmarks of cancer, including deregulated metabolism, genomic instability, and cell cycle misregulation. The study's exploration of these processes in a simple model organism provides a strong basis for translating findings to mammalian systems.

Limitations Validation of RNAi and Overexpression Efficiency: The knockdown efficiency of dPGC1 on the mRNA level is only moderate (30-50%), and protein-level validation is missing. Without this, the study cannot conclusively demonstrate the role of dPGC1 in normal development or tumorigenesis. Incomplete Mechanistic Insights: The manuscript identifies Cyclin E as a potential driver of tumor growth but does not adequately explore how mitochondrial hyperfusion leads to Cyclin E regulation (e.g., post-transcriptional mechanisms or protein stability). Inconsistencies in Experimental Backgrounds: The study uses different RNAi/shRNA lines and driver combinations inconsistently across experiments, making it difficult to compare results directly. This variability undermines the robustness of the conclusions. Limited Functional Analysis of Mitochondria: While mitochondrial morphology is well-characterized, functional assays (e.g., membrane potential or ATP production) are missing. These would confirm the impact of hyperfusion on cellular energetics and oncogenesis.

In the revised manuscript, we have addressed each of the concerns raised.

In addition to that, in the revised version of the manuscript, we have included new experiments to assess mitochondrial functionality in tumors co-expressing Yki and dPGC1-RNAi. Specifically, we analyzed the Mitochondrial Membrane Potential (MMP). We used TMRE staining to evaluate MMP, a key indicator of mitochondrial integrity and oxidative phosphorylation capacity. Our analysis revealed no significant differences in MMP between Yki tumors and Yki + dPGC1-RNAi tumors, suggesting that mitochondrial membrane potential is preserved despite the observed morphological abnormalities. These results are shown in Fig S6. In the text it is discussed in lines 233-243.

Contribution to Existing Literature The study makes a significant contribution to the growing body of literature on the metabolic regulation of cancer by identifying dPGC1 as a tumor suppressor modulating mitochondrial dynamics. Previous work has established the dual roles of mammalian PGC1α in promoting or suppressing cancer depending on context. This study adds depth by demonstrating similar context-dependent effects in a simpler model organism, facilitating further exploration of the molecular mechanisms involved.

By linking mitochondrial fusion, Yki signaling, and Cyclin E regulation, the manuscript aligns with and expands upon research on Hippo pathway regulation, cancer metabolism, and mitochondrial biology. The findings highlight the importance of integrating metabolic and signaling networks in understanding oncogenesis.

Community Selection The current form of the manuscript is best suited for a specialized audience, particularly mitochondrial biologists, Drosophila researchers, and Hippo pathway specialists. To engage a broader community, additional work linking these findings to mammalian models or human cancer biology would be necessary.

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

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

The manuscript presents compelling evidence that dPGC1 acts as a context-dependent tumor suppressor in Drosophila by modulating mitochondrial dynamics and limiting Yorkie (Yki)-induced oncogenic growth. By leveraging the Drosophila wing imaginal disc as a model, the authors investigate how dPGC1 depletion exacerbates Yki-driven tissue overgrowth, mitochondrial hyperfusion, Cyclin E upregulation, and DNA damage, leading to tumorigenesis. The study provides valuable insights into the interplay between mitochondrial dynamics and cancer, with implications for understanding metabolic regulation in oncogenesis. While the findings are significant and well-aligned with the field, certain aspects of the experimental design, data presentation, and mechanistic insights require further attention to enhance clarity, reproducibility, and impact. Below, I outline my major concerns and recommendations.

Major Points

1. One point is the knock-down efficiency of dPGC1 on the mRNA level, which is between 30 to >50% (Fig. S1C). This is not too strong, so the question arises how severly the protein levels are affected. If possible, an antibody staining with quantification should be performed. From these data it cannot be concluded dPGC1 is not required for normal development, half the dose could be sufficient. How do wings look like when the ap-GAL4 driver is used for dPGC1 knock-down, as this is the driver used in the subsequent experiments?
2. How does the wing disc look like when dPGC1 is overepressed together with Yki?
3. In Fig 2D (but also in Fig. 2C) not only cells in the dorsal but also in the ventral comparmtent seem to overproliferate. Either this is a mis-conception or it is a non-autonomous effect from interfering with Yki and dPGC1 in the vertrnal compartment. In either cases, this has to be clarified.
4. In Fig. 2F-H quantification of Mmp1 and F-actin is missing. Mmp1 is a JNK target, so the authors could do in addition an anti-phospho JNK antibody staining.
5. In Fig. 3: how does the mitochondrial network look like in the wing disc periopodial epithelium using the Gug>Yki+dPGC1 genotype? Is it similar to Gug>dMfn or Gug>miro?
6. In Fig. 3I: what is really the mitochondrion? It would be good to outline the region(s) that was/were measured.
7. A quantification of RNAi and overexpression efficiencies of the different transgenes in Fig. 3 is required.
8. In Fig. 4: what is the phenotype when miro is over-expressed in combination with Yki? Or when it is knocked down in the ap>Yki-dPGC1 background? This was the gene tested in Fig. 3 with a clear mitochondrial phenotype. How does the mitochondrial morphology in the wing disc peripodial epithelium look like in Gug>Opa1RNAi or Gug>Opa1 discs? Why have the authors switched between the ap>Yki+dPGCRNAi and the ap>Yki+dPGC1shRNA lines? It would be important to have this series of experiments in the same backgrounds, as KD efficiencies are different (Fig. S1C).
9. Fig. 5A: proper quantification of Western Blot signals is required. I do not agree that Cyclin E protein levels are elevated in ap>Yki or ap>Yki+dPGC1 discs. Even at the mRNA levels the increase in expression is rather weak. From these results nothing can be concluded.
10. Knock-down efficincies for dap and CycE needs to be quantifiec (Fig. 5H-N). Although the rescue experiment with CycE knock down is from the phenotype convincing, it is nonetheless puzzling, as CycE is accodring to Fig. 5A+B hardly upregulated. An independent CycE RNAi line would be useful.

Significance

Strengths and Limitations of the Study

Strengths

Innovative Focus on Mitochondrial Dynamics and Oncogenesis: The study provides compelling evidence linking mitochondrial dynamics, particularly hyperfusion, to tumorigenesis in Drosophila. The identification of dPGC1 as a context-dependent tumor suppressor adds novel insights into the interplay between metabolism and oncogenesis. Comprehensive Use of Drosophila as a Model System: The study leverages the genetic tractability of Drosophila, allowing precise manipulation of mitochondrial regulators and signaling pathways. The use of wing imaginal discs as a model for tumor growth is well-established and appropriate. Integration of Morphological and Genetic Data: The manuscript combines confocal imaging, electron microscopy, and genetic tools to demonstrate the role of dPGC1 in regulating mitochondrial dynamics, Cyclin E levels, and tissue overgrowth. Relevance to Cancer Biology: The findings address key hallmarks of cancer, including deregulated metabolism, genomic instability, and cell cycle misregulation. The study's exploration of these processes in a simple model organism provides a strong basis for translating findings to mammalian systems.

Limitations

Validation of RNAi and Overexpression Efficiency: The knockdown efficiency of dPGC1 on the mRNA level is only moderate (30-50%), and protein-level validation is missing. Without this, the study cannot conclusively demonstrate the role of dPGC1 in normal development or tumorigenesis. Incomplete Mechanistic Insights: The manuscript identifies Cyclin E as a potential driver of tumor growth but does not adequately explore how mitochondrial hyperfusion leads to Cyclin E regulation (e.g., post-transcriptional mechanisms or protein stability). Inconsistencies in Experimental Backgrounds: The study uses different RNAi/shRNA lines and driver combinations inconsistently across experiments, making it difficult to compare results directly. This variability undermines the robustness of the conclusions. Limited Functional Analysis of Mitochondria: While mitochondrial morphology is well-characterized, functional assays (e.g., membrane potential or ATP production) are missing. These would confirm the impact of hyperfusion on cellular energetics and oncogenesis.

Contribution to Existing Literature

The study makes a significant contribution to the growing body of literature on the metabolic regulation of cancer by identifying dPGC1 as a tumor suppressor modulating mitochondrial dynamics. Previous work has established the dual roles of mammalian PGC1α in promoting or suppressing cancer depending on context. This study adds depth by demonstrating similar context-dependent effects in a simpler model organism, facilitating further exploration of the molecular mechanisms involved.

By linking mitochondrial fusion, Yki signaling, and Cyclin E regulation, the manuscript aligns with and expands upon research on Hippo pathway regulation, cancer metabolism, and mitochondrial biology. The findings highlight the importance of integrating metabolic and signaling networks in understanding oncogenesis.

Community Selection

The current form of the manuscript is best suited for a specialized audience, particularly mitochondrial biologists, Drosophila researchers, and Hippo pathway specialists. To engage a broader community, additional work linking these findings to mammalian models or human cancer biology would be necessary.

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

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Summary

In this manuscript the authors the investigate the role of the mitochondrial regulatory transcription factor dPGC1 in tissue growth and oncogenic transformation. They show that dPGC1 limits hyperplasia mediated by overexpression of Yki in the Drosophila wing disc, while having no effect on normal growth. dPGC1 depletion in discs overexpressing Yki results neoplastic overgrowth and hyperfused mitochondria, which was dependent on the increased expression of genes involved in promoting mitochondrial fusion. Additionally, the authors show that dPGC1 limits CycE levels post-transcriptionally in Yki tumors.

Major comments

1. The authors mention several times in passing in the results a manuscript from the Banerjee lab (Nagaraj et al 2012), which shows that many of the genes the authors of the present manuscript show are upregulated upon Yki overexpression + dPGC1-RNAi compared with Yki overexpression alone are in fact upregulated upon Yki overexpression alone compared with control (dMfn/marf, opa1, miro - while interestingly dPGC1 itself is not affected). Nagaraj et al further show that Yki-overexpressing discs have longer mitochondria suggesting increased fusion even in the absence of dPGC1 depletion. The findings from Nagaraj et al should be mentioned explicitly in the introduction and the relationship between this manuscript and the present work clearly outlined in the discussion.
2. Given that Yki overexpression alone induces mitochondrial fusion and that dMfn/marf and opa1 depletion suppresses Yki-induced overgrowth (Nagaraj et al), does dPGC1 overexpression also suppress Yki-induced overgrowth? If so, is this correlated with reduction in dMfn/marf and opa1 compared with Yki overexpression alone?
3. One important question raised by this study is: how specific is the effect of dPGC1 depletion to Yki-driven overgrowth? As Yki-driven overgrowth already have increased mitochondrial length, it is possible that Yki-expressing cells are already sensitised to the effects of dPGC1 depletion. Interestingly, Nagaraj et al show that mitochondrial morphology is not affected upon EGFR activation (hyperplasia) or upon scrib and avl depletion (neoplasia). The authors should therefore test if dPGC1 depletion can potentiate the growth of other hyperplasia drivers such as activated EGFR and InR in the wing disc.
4. There are a few simple control experiments the authors should provide to clarify the relationship between Yki and dPGC1:
   • Are Yki levels affected by dPGC1 depletion?
   • Does dPGC1 knockdown alone modify the expression of the genes tested in Fig.3A? In other words, is this upregulation specific of the Yki-overexpression context?
   • Does dPCG1 knockdown also stabilise CycE in the absence of Yki overexpression or does the stabilisation of CycE occur only in Yki tumors?
5. Figure 3C-G: it is not clear how the authors can quantify the length of 3D structures like mitochondria from 2D TEM images (unless they have done volume reconstruction from consecutive sections) and no details are provided in the methods. The quantification of mitochondrial length has to be performed rigorously as it is a key part of the paper.

Minor Comments:

1. Line 51: "Mitochondria are highly dynamics organelles." should be "Mitochondria are highly dynamic organelles."
2. Introduction: the authors should summarise the known physiological functions of PGC1α in order to put their findings in context.
3. lines: 121-3: "...depletion of dPGC1...did not have a major impact on adult wing size and shape (Fig 1B, C)." There is a small but statistically significant difference so the authors should state this in the text.
4. Figure 5A (Cyclin E western blot): the authors should show molecular weight markers.

Significance

The manuscript by Sew et al builds on the previous work by Nagaraj et al to explore the role of mitochondrial function in tumors driven by disruption of the Hippo pathway. In particular, the authors identify dPGC1 as a transcription factor that limits Yki-driven mitochondrial fusion and tissue growth. Interestingly, they further show that Yki/PGC1-depleted tumors are highly sensitive to Cyclin E levels, due to post-transcriptional Cyclin E increase. These results further our knowledge of how Yki drives growth and how mitochondria participate in oncogenic transformation. With appropriate revision as outlined above (for example exploring whether the mechanism proposed is Yki-specific), the manuscript will be of broad interest to developmental and cancer biologists.

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

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

Sew et al. examine the master regulator of mitochondrial biogenesis, dPGC1, in the context of Drosophila wing and larval development. They primarily use confocal imaging to probe the interplay between dPGC1 and an overactive Hippo pathway, driven by overexpression of the main effector protein, Yki. In their study, they find that tumors, driven by overactivity of Yki grow larger when dPGC1 is downregulated, implicating the mitochondrial biogenesis pathway in tumor suppression. Furthermore, in the context of Yki overexpression, they find that levels of Mfn or Opa1 modulate tumor size. Lastly, they show a role of cyclin E in controlling the size of tumors formed by Yki OE + dPGC1 RNAi. The potential role of dPGC1 as a tumor suppressor is interesting because it highlights an emerging recognition of mitochondria in the aetiology of cancer. However, before publication, much of the data in this manuscript should be strengthened by a refinement in the methods/analysis and an increase in orthogonal approaches.

Major comments:

The authors indicate that for example, in lines127-28, that neither downregulating or overexpressing dPGC1 affects wing size. However, the quantification in Fig. 1C shows a significant decrease in wing size following RNAi treatment. This decrease is modest, but it is nevertheless significant. It is worth pointing out, too, that the efficiency of the RNAi in Fig. S1C suggests that the conclusions drawn are premature. While a roughly 55% drop in mRNA levels may be statistically significant, it is unclear whether this drop in transcripts corresponds to a commensurate depletion of protein. Moreover, it is unclear, in this context, how much dPGC1 may indeed be necessary to drive a relatively normal program of mitochondrial biogenesis in wing development. To obtain a clear result, it is necessary to show significant depletion of the dPGC1 protein. (Ultimately, if it is the case that dPGC1 is unnecessary for wing development and function, a more coherent line of inquiry would be to find out the reason for this rather than to pivot the story to studying tumorigenesis in larva.)

In Figure 3H-K, it is not clear why the authors used electron microscopy to evaluate mitochondrial morphology. The very good confocal images in Figure 3C-G show a clear change in mitochondrial morphology following the knockdown of Mfn, Opa1, and Miro. While it is clear from the electron micrographs in Figure H that the mitochondria are enlarged, it is not obvious that this increase in length is a result of increased mitochondrial fusion. Indeed, if the mean form factor were used to quantify the shape, it is likely that in both conditions, the value would be close to 1, indicating more of a round object, and it not obvious whether there would be a difference between the Yki OE versus the YkI OE + dPGC1 RNAi. Therefore, from this data alone, it cannot be concluded that the YkI OE + dPGC1 RNAi condition leads to mitochondrial hyperfusion.

Figure 4. refers to changes in mitochondrial fusion and fission in tumor formation; however, the authors do not attempt to alter mitochondrial fission factors, so it is not accurate to mention a role of mitochondrial fission, in this context. It must be noted, too, that the authors have not demonstrated that their genetic interventions have actually affected mitochondrial morphology in these experiments. As noted in the previous figure, the Yki OE + dPGC1 RNAi condition showed enlarged mitochondria, but not necessarily hyperfused organelles. Therefore, the downregulation of Mfn or Opa1 in this set of experiments may not necessarily have altered mitochondrial morphology. Perhaps suppression of Mfn or Opa1 would normalize the areas of these evidently swollen mitochondria, but this is unclear without images. Furthermore, it should be appreciated that both Opa1 and Mfn exhibit pleiotropic attributes - e.g., Opa1 not only regulates IMM fusion, but it also modulates the shape and tightness of cristae membranes, specialized sites of oxidative phosphorylation as well as sequestration of cytochrome c, the release of which influences apoptosis (Frezza et al., 2006). At least in mammalian cells, Mfn2 is thought to regulate contacts between mitochondria and endoplasmic reticulum (Naon et al., 2023), which may serve other functions than OMM fusion, such as stabilization of the MAM.

Figure 5 highlights a connection between dysregulation of mitochondria and Cyclin E, which allows cells to prematurely enter S phase. The data presented here do not offer clarity on whether the enlargement of the tumors results from increase cellular proliferation and/or cell size. The role of the cell cycle adds a layer of complexity to these results, because it is thought that mitochondria undergo fragmentation during the cell cycle to promote an even distribution of the organelle population after mitosis (Taguchi et al., 2007); however, in this manuscript, the authors contend that the downregulation dPGC1 is promoting mitochondrial hyperfusion. It is unclear how and whether cellular division and proliferation would proceed at an accelerated rate in a situation with mitochondrial hyperfusion.

Minor comments:

Lines 69-72 contrast the roles of PGC1α and β. It is not clear whether the comparison is of their respective roles in cancer or in normal physiology. In either case, it is important to note that PGC1β has been shown to drive mitochondrial fusion as well as biogenesis through its control of MFN2, among other factors (Liesa et al., 2008).

Although this study focuses on PGC1, the authors do not seem to site the original literature from the Spiegelman lab.

There are 10-20 grammatical errors throughout the text.

Referee Cross-commenting

There is agreement among the referees that the potential role of PGC1 as a tumor suppressor is interesting and significant. However, various aspects of this work require attention prior to publication. For example, there needs to be a complete knock down of PGC1 to come to any conclusion as to its role in wing development. The methods for analyzing mitochondrial morphology need to be clarified and be consistent with standards in the field of mitochondrial dynamics. Also, the authors need to quantify their Western blots to obtain accurate assessments of protein levels. Generally, the study relies too heavily on overexpression experiments; understanding the potential role of mitochondria in regulating the Hippo pathway should include various knockdown and/or knockout models.

Significance

Overall, the authors show an interesting dampening effect of dPGC1 on growth of Yki-driven tumors. This data could be relevant for elucidating how dysregulation of the Hippo signalling pathway can underlie tumorigenesis.

The narrative arc of the study, however, appears to lack a focused line of inquiry. Figure 1 highlights an attempt to modulate Drosophila wing size and/or structure by downregulating dPGC1, but to no effect. Although examination of the efficiency of the RNAi revealed that the transcripts were still present in significant quantities; so, the conclusion that dPGC1 is dispensable for wing formation is premature. To have clarity on this point, it would be necessary to completely knockdown the gene, preferably by showing a total loss of protein. This should be feasible for the authors, since they showed Western blotting in Figure 5A. In any event, it seems that this negative data led the authors to study the Hippo pathway in the larval stage. This transition from Figure 1 to 2 seemed somewhat arbitrary and leads to a rather disjointed sense of the main line of inquiry around dPGC1.

It is important to note, too, that the authors highlight a role of mitochondrial dynamics in the pathway of Yki-driven tumor formation; however, they only directly evaluate mitochondrial dynamics in this context in a single assay, namely, Figure 3H-K, and this quantification is likely inaccurate because the mitochondria in the Yki OE + dPGC1 RNAi condition seem to be substantially enlarged, circular structures. It is critical to keep in mind that mitochondrial enlargement does not necessarily stem from hyperfusion. It could come from a decrease in the activity of Drp1 or result from an imbalance between mitochondrial biogenesis and mitophagy.

A marked limitation of this study is the overuse of rather artificial manipulations of transcriptional regulatory pathways. The study would benefit a lot from investigation of the loss of function of components of the Hippo pathway rather than just OE of Yki.

My expertise is in mitochondrial biology, with specialization in super-resolution imaging, mitochondrial dynamics and membrane architecture. I have also worked in the interface between mitochondrial physiology and cancer. With this perspective, I think that the authors uncover a potentially interesting role of PGC1 as a tumor suppressor.

Súmula nº 264/TST. Hora suplementar. Cálculo

• A remuneração do serviço suplementar é composta do valor da hora normal, integrado por parcelas de natureza salarial e acrescido do adicional previsto em lei, contrato, acordo, convenção coletiva ou sentença normativa.

OJ-SDI1-97. Horas extras. Adicional noturno. Base de cálculo (inserida em 30.05.1997).

• O adicional noturno integra a base de cálculo das horas extras prestadas no período noturno.

[Observe que somente a hora extra noturna será calculada com base no adicional noturno] -> Ex.: Pessoa que trabalha até às 21h, porém trabalha, como hora extra, até às 22h52 min. Contendo ai 2 horas extras, uma normal e outra em adicional noturno, o cálculo se dará de forma a ser uma extra prestada normalmente e outra com a incidência do adicional noturno de 20%

Súmula nº 291/ TST HORAS EXTRAS. HABITUALIDADE. SUPRESSÃO. INDENIZAÇÃO - A supressão total ou parcial, pelo empregador, de serviço suplementar prestado com habitualidade, durante pelo menos 1 (um) ano, assegura ao empregado o direito à indenização correspondente ao valor de 1 (um) mês das horas suprimidas, total ou parcialmente, para cada ano ou fração igual ou superior a seis meses de prestação de serviço acima da jornada normal. O cálculo observará a média das horas suplementares nos últimos 12 (doze) meses anteriores à mudança, multiplicada pelo valor da hora extra do dia da supressão.

Obs.: O empregador pode, unilateralmente, suprimir horas extras habituais. No entanto, deverá indenizar o empregado se essa habitualidade for <u>superior</u> a 1 ano.

O cálculo se dá a partir da consideração da média de horas extras prestadas nos últimos 12 meses anteriores a supressão. Verificada qual a média mensal, a indenização será essa média multiplicada pelo valor da última hora extra vigente.

A cada 1 ano de prestação de hora extra habitual significará o valor de 1 mês de horas extras habituais.

Súmula nº 363/TST - CONTRATO NULO. EFEITOS - A contratação de servidor público, após a CF/1988, sem prévia aprovação em concurso público, encontra óbice no respectivo art. 37, II e § 2º, somente lhe conferindo direito ao pagamento da contraprestação pactuada, em relação ao número de horas trabalhadas, respeitado o valor da hora do salário mínimo, e dos valores referentes aos depósitos do FGTS.

Obs.: Apesar da patente nulidade de contrato de servidor admitido sem concurso público, ao menos lhe garante a contraprestação e o FGTS

Súmula nº 430/TST - ADMINISTRAÇÃO PÚBLICA INDIRETA. CONTRATAÇÃO. AUSÊNCIA DE CONCURSO PÚBLICO. NULIDADE. ULTERIOR PRIVATIZAÇÃO. CONVALIDAÇÃO. INSUBSISTÊNCIA DO VÍCIO - Convalidam-se os efeitos do contrato de trabalho que, considerado nulo por ausência de concurso público, quando celebrado originalmente com ente da Administração Pública Indireta, continua a existir após a sua privatização.

Obs.: Muito embora nulo o contrato por falta de concurso público, ele poderá ser convalidado na hipótese de privatização.

Súmula nº 455/TST - EQUIPARAÇÃO SALARIAL. SOCIEDADE DE ECONOMIA MISTA. ART. 37, XIII, DA CF/1988. POSSIBILIDADE. - À sociedade de economia mista não se aplica a vedação à equiparação prevista no art. 37, XIII, da CF/1988, pois, ao admitir empregados sob o regime da CLT, equipara-se a empregador privado, conforme disposto no art. 173, § 1º, II, da CF/1988.

Obs.: Logo, estatais por deverem ter regime jurídico próprio de empresas privadas, podem ter que equiparar salários por adotarem o regime celetista.

OJ-SDI1-366 - ESTAGIÁRIO. DESVIRTUAMENTO DO CONTRATO DE ESTÁGIO. RECONHECIMENTO DO VÍNCULO EMPREGATÍCIO COM A ADMINISTRAÇÃO PÚBLICA DIRETA OU INDIRETA. PERÍODO POSTERIOR À CONSTI-TUIÇÃO FEDERAL DE 1988. IMPOSSIBILIDADE - Ainda que desvirtuada a finalidade do contrato de estágio celebrado na vigência da Constituição Federal de 1988, é inviável o reconhecimento do vínculo empregatício com ente da Administração Pública direta ou indireta, por força do art. 37, II, da CF/1988, bem como o deferimento de indenização pecuniária, exceto em relação às parcelas previstas na Súmula nº 363 do TST, se requeridas.

Obs.: Via de regra, o desvirtuamento de estágio possa gerar o reconhecimento vínculo empregatício, não é admissível essa hipótese para a Administração Pública em decorrência do Princípio do Concurso Público. No entanto, isso não impede o reconhecimento, se o caso, do pagamento do previsto em contrato + FGTS (Sum 363)

OJ-SDI2-128 - AÇÃO RESCISÓRIA. CONCURSO PÚBLICO ANULADO POSTERIORMENTE. APLICAÇÃO DA SÚMULA Nº 363 DO TST - O certame público posteriormente anulado equivale à contratação realizada sem a observância da exigência contida no art. 37, II, da Constituição Federal de 1988. Assim sendo, aplicam-se à hipótese os efeitos previstos na Súmula nº 363 do TST.

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Plantea es que la Psicología, tal como se ha configurado históricamente, tiene raíces conservadoras e individualistas. Eso significa que gran parte de la disciplina se ha centrado en el individuo aislado, en sus procesos internos, sin atender con suficiente fuerza a lo que Marx llamaría las condiciones materiales de existencia. El artículo propone recuperar la perspectiva crítica y liberadora que Ignacio Martín-Baró desarrolló, precisamente porque él entendía que la psicología debía situarse en la vida concreta de las personas y en su contexto histórico, social y político.

Para llegar a ese punto, el texto dialoga con otros autores que también han trabajado desde marcos marxistas, como Reich, que analiza cómo la ideología penetra hasta en la vida psíquica y en la organización del deseo, o Ratner, que retoma a Vygotsky para insistir en que no hay subjetividad fuera de un entramado sociohistórico. Laraia, desde la antropología, recuerda que el ser humano es producto del medio cultural en el que se socializa. Todo esto converge en una misma dirección: si la Psicología se desentiende del contexto social y cultural, se convierte en una ciencia mutilada, incapaz de comprender plenamente a su objeto.

El aporte central de Martín-Baró —que el artículo recupera— es la idea de una Psicología Social de la liberación. Esto supone considerar de forma seria las contradicciones de clase, los procesos de deshumanización que genera el capitalismo y, al mismo tiempo, las luchas sociales que abren horizontes de emancipación. El marxismo aquí no se presenta como un dogma, sino como una herramienta crítica para interrogar tanto la práctica científica como la praxis social.

Juan Pablo Patriglia tiene la capacidad de situar el proyecto intelectual y político de García Linera en una clave que excede la pura reconstrucción bibliográfica. Comprende las publicaciones editadas desde la Vicepresidencia de Bolivia como un ejercicio de interpretación del marxismo, en el sentido gramsciano y benjaminiano del término, como un proceso de reapropiación, de actualización y de disputa de sentidos. El autor consigue mostrar cómo, bajo la gestión de García Linera, la edición y circulación de textos marxistas fueron parte de una estrategia de producción teórica que buscó enlazar la tradición crítica con las experiencias de lucha de Bolivia y de América Latina. Me parece especialmente interesante la manera en articula las tres dimensiones de su estudio: la obra propia de García Linera, la recuperación de intelectuales latinoamericanos como Echeverría, Veraza o Dussel, y la edición de inéditos de Marx. Cada uno de estos apartados es tratado como partes de un mismo esfuerzo de traducción que rompe con el eurocentrismo y contribuye a un marxismo vivo, situado y latinoamericano. Allí radica, creo, la virtud de este trabajo: hacer visible la densidad teórica y política que tuvo el proyecto editorial de la Vicepresidencia de Bolivia al mando de García Linera. El artículo da cuenta de un momento relevante de la historia intelectual reciente de América Latina. Ofrece un análisis sólido, bien documentado y con una clara apuesta interpretativa.

DOI: 10.1038/s41586-019-1213-4

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

「Claude Codeに生成させたもの」なのでコメントしても仕方なさそうですが、69:ログメッセージをフォーマットしてロガーに渡さない に従って、%演算子を使って記述した方がよいと思いました。(他の記述も同様)

Q

Ja aber das muss dann bei effect size angepasst werden (zB Dummies müssen dann bei Effektgröße auf 0/1 gleich sein (zB employed/not employed), etc., wichtig für Vergleichbarkeit.

Q

Ja aber das muss dann bei effect size angepasst werden (zB Dummies müssen dann bei Effektgröße auf 0/1 gleich sein (zB employed/not employed), etc., wichtig für Vergleichbarkeit.

Q

Ja gerne, würd ich eventuell sogar mal auslassen.

Q

Ja hier eventuell DiD/RDD/ähnliche zusammenfasen. Hir müssen wir aber diskutieren ob wir hier auf Kausalität, Endogenität, etc abzielen – analog diskutieren wie zu WL-PL Paper.

Ganz wichtig: DiD in spirit vs. DiD richtig (würde in spirit nicht zu DiD tun)

Q

Ja versteh ich, Vorschlag klingt gut. Weiß nicht inwiefern wir diese Variable eigentlich verwenden

Q

Ja wage compensation muss in Wage compensation variable (muss geändert werden in Excel)

Q

Ja brauchen wir für Partialkorrelationen

Q

Australien & Afrika find ich auch spannend – für Afrika ähnlich wie WL-PL Paper. Kann man vermutlich irgendwie erklären (Daten, Systeme, Agrarisch geprägt teilweise, etc.). Eventuell noch eher was für South Africa vorhanden da gabs zB auch 4 day work week trials und vor kurzem mögliche Senkung auf 40 Stunden) aber aktuell macht es keinen Sinn nach mehr Paper zu suchen. Vielleicht für Zukunft. Australien haben wir andere Studien aber keine ökonometrischen Ergebnisse für employment soweit ich weiß.

I find this very intriguing that a 3 sylable word can have so much meaning.

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

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Reply to the reviewers

Manuscript number: RC- 2025-03073

Corresponding author(s): Shaul Yogev

1. General Statements [optional]

We kindly thank our reviewers for their enthusiasm, thoughtful feedback, and constructive suggestions on how to strengthen our manuscript. Below, we provide a point-by-point response to reviewer comments and outline the experiments we will do to address every concern that has been raised.

2. Description of the planned revisions

• *

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

This interesting study uses an unbiased genetic screen in C. elegans to identify SAX-1/NDR kinase as a regulator of dendritic branch elimination. Loss of SAX-1 results in an excess branching phenotype that is striking and highly penetrant. The authors identify several additional regulators of branch elimination (SAX-2, MOB-1, RABI-1, RAB-11.2) by using a candidate genetic screen aimed at factors that interact physically or genetically with SAX-1. They propose that SAX-1 acts by promoting membrane retrieval based on the nature of these interactors and the results of an imaging-based in vivo assay for endocytic puncta.

Major comments.

1. My biggest concern is that the phenotypes are only observed in temperature-sensitive dauer-constitutive mutant backgrounds, and not in wild-type dauers. That is, wild-type animals exiting dauer do not require SAX-1 for dendrite elimination. While this does not undermine the importance of the results, it does require more explanation. The authors write that "the requirement for sax-1... relies on specific physiological states of the dauer stage," but I do not understand what this means. Are they saying that daf-7 and daf-2 dauers are in a different "physiological state" than wild-type dauers? In what way? What is the evidence for this? A more rigorous explanation is needed. We agree that this is puzzling, and we thank the reviewer for recognizing that this does not undermine the importance of the results. There is ample evidence that daf-2 and daf-7 differ from starvation-induced dauers. For example, a recent preprint finds that the transcriptomes of these two mutants at dauer cluster much closer to each other than to starvation-induced dauers (Corchado et al. 2024). Older work has noted other differences, such as the time the dauer entry decision is made (Swanson and Riddle 1981), the synchronicity of dauer exit, the ability to force dauer entry in daf-d mutants, as well as additional dauer-unrelated phenotypes (reviewed in Karp 2018). We agree with the reviewer that this merits further clarifications and will perform the experiments suggested by the reviewer below:

To me, the simplest genetic explanation is that daf-7 and daf-2 are partially required for branch retraction in a manner redundant with sax-1, and the ts mutants are not fully wild-type at 15C. Thus, the sax-1 requirement is revealed only in these mutant backgrounds. Can the authors examine starvation-induced dauers of daf-7 or daf-2 raised continuously at 15C?

We will do this experiment.

daf-7 and daf-2 ts strains can form "partial dauers" that have a dauer-like appearance but are not SDS resistant. Could the difference between partial dauers and full dauers account for the difference in sax-1-dependence? The authors could use SDS selection of the daf-7 strain at 25C to ensure they are examining full dauers.

We tested daf-7 mutants with 1% SDS when we set up the system – they are fully dauer at 25°C and are SDS sensitive after exit. We will repeat this important control with daf-7; sax-1 double mutants.

The Bargmann lab has created a daf-2 FLP-OUT strain (ky1095ky1087) that allows cell-type-specific removal of daf-2. Could this be used to test for a cell-autonomous role of daf-2 in IL2Q related to branch elimination?

We can attempt this experiment. However, since IL2 promoters turn on prior to dauer, the interpretation would not be straightforward – it would be hard to exclude that a cell autonomous defect in dauer entry does not account for the IL2 dauer exit phenotype, even if branching appears normal.

These ideas are not a list of specific experiments the authors need to complete, rather they are meant to illustrate some possible approaches to the question. Whatever approach they use, it is important for them to more rigorously explain why SAX-1 is not required for branch removal in wild-type animals.

We completely agree. We will carry out the 15°C experiment, examine morphological characteristics and test SDS resistance. In addition, we will test neuronal markers that differ between dauers and non-dauers to determine whether the mutants are full or partial dauers at the relevant timepoints.

The SAX-2 localization (Fig. 4) and endocytosis assay (Fig. 6) results were not clear to me from the data shown. Overall a more rigorous analysis and presentation of the data would be important to make these conclusions convincing. This may involve refining the data presentation in the figures, modifying the claims (e.g., "we propose" vs "we find"), or saving some of the data to be more fully explored in a future paper. In my view, these figures are the biggest weak point of the manuscript and also are not important for the central conclusions (which are well supported and convincing), indeed these results are barely mentioned in the Abstract or last paragraph of Introduction.

We agree that the analysis and presentation of Figures 4 and 6 need to be improved. The presentation has already been updated, and the figures are clearer now. In the revision, we will increase sample size to provide stronger conclusions, consolidate some of the analysis and further improve presentation. While we agree with the reviewer that conclusions from these figures are not as strong as those drawn from genetic experiments, they do complement and support the conclusions of those other figures.

• In Fig. 4D, why is SAX-2 visible throughout the entire neuron and why is the "punctum" marked with an arrow also seen in the tagRFP channel? One gets the impression that some of the puncta may be background, bleed-through, or artifacts due to cell varicosities.

There is no bleed-through: this is most evident by looking at the brightest signals in the cell body (now labelled with an asterisk in a zoomed-out image) and noting that they do not bleed between channels. In sax-1 mutants, the SAX-2::GFP puncta are very obvious and distinguishable from the tagRFP channel. In control, SAX-2::GFP is very faint in the dendrite, so we increased the contrast to allow visualization. The reviewer is correct that under these conditions, some puncta look like the cytosolic fill. In the revision, we will re-analyze the data and will not consider these as bona-fide SAX-2 puncta, but rather cytosolic SAX-2 that accumulates due to constrictions and varicosities in the dendrite.

• Related to both Fig. 4 and Fig. 6, where does SAX-1 localize in IL2Q in dauer and post-dauer? Does its expression or localization change during branch retraction? Does it co-localize with SAX-2 or endocytic puncta?

We generated an endogenously tagged sax-1 with a 7xspGFP11 tag; however, this was below detection in the IL2s. For the revisions, we can test an overexpressed cDNA construct.

**Referee cross-commenting**

I think we all touched on similar points. I wanted to follow up on Reviewer 3's comment, "Is the failure to eliminate branches an indication of incomplete dauer recovery? Do sax-1 mutants retain additional characteristics of dauer morphology in post dauer adults." I thought this was an excellent point. It made me wonder if that might explain why the defect is only seen in daf-7 and daf-2 mutant backgrounds - maybe these strains retain partial dauer traits even after exit. Is there a specific experiment that they could do? Did you have specific characteristics of dauer morphology in mind for them to check? (Ideally something in the nervous system that can be scored quantitatively.)

Please see response to point #1 regarding experiments we will do to confirm the “dauer state” of daf-7 and daf-7; sax-1 double mutants.

Reviewer #1 (Significance (Required)):

A major strength of this work is the pioneering use of a novel system to study neuronal branch retraction. C. elegans has provided a powerful model for studying how dendrite branches form, but much less attention has been paid to how excess neuronal branches are removed. The post-dauer remodeling of IL2Q neurons provides an exciting and dramatic physiological example to explore this question.

This paper is notable for taking the first steps towards developing this innovative model. It does exactly what is needed at the outset of a new exploration - a forward genetic screen to discover the main regulators of the process. Using a combination of classical and modern genetic approaches, the authors bootstrap their way to a sizeable list of factors and a solid understanding of the properties of this system, for example that retraction of higher vs lower order dendrites show different genetic requirements.

We thank the reviewer for recognizing the novelty and significance of our work.

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

In this manuscript, the authors establish C. elegans IL2 neurons as a system in which to study dendrite pruning. They use the system to perform a genetic screen for pruning regulators and find an allele of sax-1. Unexpectedly sax-1 is only required for post-dauer pruning in two different genetic backgrounds that induce dauer formation, but not starvation-induced dauer formation. Sax-1/NDR kinase reduction has previously been associated with increased outgrowth and branching in other systems, so this is a new role for this protein. However, the authors show that proteins that work with Sax-1 in other systems, like sax-2/fry, also play a role in this pathway. The genetic experiments are beautiful and the findings are all clearly explained and strongly supported. The authors also examine sax-2 localization, which localizes sax-1 in other systems, and show it in puncta in dendrites that increase with dauer exit, consistent with function at the time of pruning. They also show that membrane trafficking regulators associated with NDR kinases function in the same pathway here, hinting that endocytosis may play a role during pruning as in Drosophila. The link to endocytosis was a little weak (see Major point below). Overall, this study describes a new system to study pruning and identifies NDR/fry/Rabs as regulators of pruning during dauer exit. The work is very high quality and both the imaging and genetics are extremely well done.

We thank the reviewer for their positive assessment of the manuscript.

Major points

1. The only place where there were any questions about the data was the last figure (6G and I). Here they use uptake of GFP secreted from muscle as a readout of endocytosis in IL2 neurons. They nicely show that more internalized puncta accumulate as animals exit dauer. The claim that this is reduced in sax-1 mutants doesn't seem to match the images shown well. In the image there are many more puncta in the GFP channel and much more accumulation of the RFP-tagged receptor everywhere. It seems like some additional analysis of this data is important to fully capture what is going on and whether this really represents an endocytic defect. We agree and will provide additional data in Figure 6. The specific discrepancy between the image and the quantification is because we showed a single focal plane rather than a projection. This does not capture all the puncta in a neurite. The current version shows a projection, making it evident that the mutants has fewer puncta compared to the control.

Reviewer #2 (Significance (Required)):

Neurite pruning is important in all animals with neurons. Genetic approaches have primarily been applied to the problem using Drosophila, so identifying a new model system in which to study it is an important step. Using this system, a pathway known to function in a different context is linked to pruning. Thus the study provides new insights into both pruning and this pathway.

We thank the reviewer for the positive assessment of our study’s significance.

__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

Summary: Figueroa-Delgado et al. use a C. elegans neuro plasticity model to examine how dendrites are eliminated upon recovery from the stress induced larval stage, dauer. The authors performed a mutagenesis screen to identify novel regulators of dendrite elimination and revealed some surprising results. Branch elimination mechanism varies between 2{degree sign}, 3{degree sign}, and 4{degree sign} branches. The NDR kinase, SAX-1 and it's interactors (SAX-2 and MOB-2) are required for elimination of second and third order branches but not fourth order branches. Interestingly they showed that branch elimination varies depending on the stimulus of dendrite outgrowth such that the NDR kinase is required for branch elimination after genetically inducing the dauer stage but is not required if dauers are produced through food deprivation. The authors go a step further to include a small candidate screen looking at various pathways of membrane remodeling and identify additional regulators of dendrite elimination related to membrane trafficking including RABI-1, RAB-8, RAB-10, and RAB-11.2.

We thank the reviewer for their time and suggestions below

Major comments:

• While I find the data promising and exciting, several of the experiments have concerningly low sample sizes. Fig 3G, Fig 4G, Fig 5J and L, and Fig 6I all contain data sets that are fewer than 10 animals. Sample sizes should be stated specifically in the figure legends for all data represented in the graphs. We thank the reviewer for finding the data exciting. We agree that the sample sizes in some panels is low and will increase it in the revised version. Sample sizes are now specifically listed in the figure legends.

• All statements based on data not shown should be amended to include the data as a supplemental figure or edited to omit the statement based on withheld data. We agree. Some “not shown” data are already added to the current version of the manuscript and the rest will be added to the fully revised version, or the statements will be omitted.

• Rescue experiments (Fig 2J) should demonstrate failure to rescue from neighboring tissue types (hypodermis and muscle) to conclude cell autonomous rescue rather than a broadly acting factor. Thank you for the suggestion. We will use a hypodermal promoter and a muscle promoter driving SAX-1 cDNA expression to strengthen the claim of cell autonomy.

• Fig 4 needs quantification of higher order branches and SAX-2 proximity to branch nodes as these are discussed in the text. We will add this quantification.

Minor comments:

• Fig 1C-F, It appears like the shy87 allele produces animals of significantly different body sizes. It would improve rigor to normalize the dendrite coverage to body size in the quantification. We do not see a biologically meaningful size difference between shy87 and control, it may be the specific image shown. We will confirm this by measuring animal size for the final revision.

• Is the failure to eliminate branches an indication of incomplete dauer recovery? Do sax-1 mutants retain additional characteristics of dauer morphology in post dauer adults. This important point was also raised by Reviewer 1. We will test SDS sensitivity, morphological markers, and molecular markers to determine the dauer “state” of the mutants used in this study. The results will be included in the final revision.

• The text references multiple transgenic lines tested in Fig 2I-J but only one line is shown. Additional lines were visually examined under a fluorescent compound microscope but not imaged or quantified. We will add this quantification to the final revision.

• Fig 4F, Additional timepoints would enhance the sax-1 localization result and might provide insight into mechanism of action for sax-1. We will add the localization in post-dauer adults.

• Fig 6I Control and sax-1(ky491) example images should be provided in the supplement. We will add these images to the final revision.

**Referee cross-commenting**

I agree that we shared many of the same concerns.

There are several general assays for dauer characteristics that could be used here to determine if the post-dauer animals retain other characteristics of the dauer stage in addition to IL2 branches (SDS resistance, alae remodeling, pharyngeal bulb morphology, nictation behavior). The nictation behavior has been connected very nicely with IL2 neurons (Junho Lee's group). Additionally, FLP dendrites occupy the same space as the IL2 branches and outgrowth in post-dauers occurs in coordination with IL2 branch elimination - this might be another optional experiment, to check if FLP growth is impeded by persistent IL2 branches. All of these could be quantified similar to how the authors have already established with their IL2 model (FLP dendrite branches) or with a binary statistic.

Please see responses to Reviewer 1 and 3 above for the list of experiments to determine whether the animals fail to completely enter or exit dauer.

Reviewer #3 (Significance (Required)):

SIGNIFICANCE ============ These results describe a new role for the NDR kinase complex in dendrite pruning that has clinical significance to our understanding of human brain development and human health concerns in which pruning is dysregulated, such as observed in the case of autism. The authors use an established neuro-plasticity, C. elegans model (Schroeder et al. 2013) which provides a tractable and reproduceable platform for discovering the mechanism of dendrite pruning. These results would influence future work in the fields of cell biology of the neuron and disease models of brain development.

My expertise is in the field of C. elegans neuroscience and stress biology and have sufficient expertise to evaluate all aspects of this work.

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

Reviewer #1

• In Fig. 4C, the distinction between puncta in the primary or higher-order dendrites is not clear to me, and several puncta that I would have scored as primary are marked as higher-order.

We apologize for a mistake in the arrowhead color and overall presentation of this figure. It has been fixed in the current version.

• Related to this, in Fig. 4B are the two arrows meant to be white as in the top panel, or yellow as in the bottom panel?

We thank Reviewer #1 for their observation, and we apologize for our oversight. We fixed this in the current version.

• In Fig. 4, where in the head are we looking? It would help to show a more low-magnification view of the entire cell.

We added zoomed-out images and indicated where the zoomed in insets are taken from. We thank the reviewer for helping us improve the clarity of the data.

• The main sax-1 phenotype is increased SAX-2 puncta in dauer, but the branch retraction defect is in post-dauers. How is this relevant to the phenotype?

This is a very good point. The increase in SAX-2 puncta in sax-1 mutants is stronger during dauer-exit than in dauer, consistent with this being the time when SAX-1 functions. We agree that some earlier activity of SAX-1 cannot be excluded, and we do not assume that the effect on SAX-2 completely accounts for the pruning defects. This is now acknowledged in the text. However, given that both proteins function together in pruning, and given that the effect is strongest during dauer exit, we do believe that this data is informative and worth showing.

• The number of SAX-2 puncta in sax-1 mutants decreases almost to normal in post dauers. Is there a correlation between the number of remaining branches and the number of SAX-2 puncta? That is, do the many wild-type animals with "excess" SAX-2 puncta also fail to retract branches?

There is no correlation. In other words, the number of SAX-2 puncta does not instruct the extent of pruning. Please note the quantifications underestimate the number of SAX-2 puncta in the mutants, since they were only done on the primary dendrite. This is necessary because the mutant and control have different arbor size, so only branch order that can be appropriately compared are primary dendrites.

• The control post-dauer data in Fig. 4F and 4H are identical (re-used data) but the corresponding control dauer data in Fig. 4F and 4G are different. What is going on here?

We thank the reviewer for raising this point and apologize for the oversight in data presentation. In the revised manuscript, we now show all control and experimental data integrated into a single graph, ensuring that each dataset is represented accurately to provide a comparison between dauer and post dauer recovery conditions.

• Why are sample sizes so small for both strains in Fig. 4G compared to Fig. 4F and 4H?

We sincerely apologize for this mistake, some of the data was erroneously grouped in the original submission. The revised version contains an updated number of neurons, presented on the same graph, and in the final revision we will further increase sample size. We apologize again for this error.

• In Fig. 6C, why are the tagRFP (blue) puncta larger than the neurite? Aren't these meant to represent vesicles inside the surrounding neurite? One gets the impression that this is bleed-through from the GFP channel.

Based on EM, both an endocytic punctum and the diameter of the neuron are smaller than a single pixel. The apparent difference in size in fluorescence microscopy is because the puncta are brighter (they contain more membrane) and thus appear larger. In the current version, the improved presentation of the figure contains zoomed out images that clearly show that there is no bleed-through.

• In Fig. 6E and 6F, why are there no tagRFP (blue) puncta? Is CD8 not endocytosed at all if it lacks the nanobody sequence? One would expect the tagRFP (blue) signal to be the same in both strains and simply to lack yellow if the nanobody is not present.

CD8 lacks clear endocytosis motifs, which is why it is advantageous for labelling neurites and testing endocytosis when paired with an endocytic signal (Lee and Luo 1999; Kozik et al. 2010). Conversely, extracellular GFP binding to a membrane GFP antibody can induce endocytosis (for example, see (Tang et al., 2020)), likely by inducing clustering, although we are not familiar with work that explored the mechanism. In the updated version we included a rare example of an mCD8 punctum.

• The authors report a decrease in endocytic events in sax-1, but qualitatively it looks like there are vastly more puncta inside the neuron in Fig. 6H than in 6G.

We apologize for the presentation in the original version of Figure 6. This impression was because we showed single focal planes that only captured some of the signal. In the revised version we show projections, which makes it evident that there are fewer endocytic events in the mutant.

• In Fig. 6E and 6H, why are there so many GFP (yellow) puncta outside the neuron? What are these structures and why are they absent in the strain with the nanobody?

These puncta are secreted or muscle-associated GFP that has not been internalized by IL2Q neurons. They are present in all strains in this figure, this can be clearly seen in the zoomed-out images that have been added to the updated figure.

• What is the large central blue structure in Fig. 6H - is this the soma? - and why are puncta in this region not counted?

This is indeed the soma. In the updated version this can be clearly seen in the zoom-out. The large puncta in the soma were not counted because they may arise from the fusion of an unknown number of smaller puncta, and their precise number cannot be determined at the resolution of fluorescence microscopy.

• minor: there is text reading "40-" in the bottom panel of Fig. 6H. It is visible when printed but not on screen - adjust levels in Photoshop to reveal it.

We thank the reviewer for catching this oversight, it is now fixed.

Minor points:

1. At several points the authors emphasize the relationship of neurite remodeling to stress, e.g. Abstract and Discussion: "we adapted C. elegans IL2 sensory dendrites as a model [of...] stress-mediated dendrite pruning". It seems unnecessary and potentially misleading to treat this as a neuronal stress response. First, it conflates organismal and cellular stress - there is no reason to think that IL2 neurons are under cellular stress in dauer. In fact parasitic nematodes go through dauer-like stages as part of healthy development and probably have similar remodeling of IL2. Second, dendrite pruning occurs during dauer exit, which is the opposite of a stress response - it reflects a return to favorable conditions. We agree. We modified the abstract and discussion to avoid conflating organismal stress (the alleviation of which is relevant for triggering pruning) and cellular stress. Thank you for pointing this out.

In Fig. 1A, C. elegans is shown going directly from L1 to dauer in response to unfavorable conditions, which is incorrect. Animals proceed through L2 (in many cases actually an alternative L2d pre-dauer) and then molt into dauer (an alternative L3 stage) after completing L2.

We updated the schematic to include the L2d stage where commitment to dauer entry or resumption to reproductive development is made.

In Fig. 1B, please check if it is correct that hypodermis contacts the pharynx basement membrane as drawn. The schematic in the top panel makes it look like there is a single secondary branch and the quaternary branches are similar in length to the primary dendrite. The schematic in the bottom panel makes it look like the entire neuron is a small fraction of the length of the pharynx. Could these be drawn closer to scale?

The hypodermis does contact the pharynx basement membrane. We redrew the schematic for clarity.

Reviewer #2

For context, it might be helpful to know whether branching of other dendrites is increased in sax-1 mutants (as expected based on phenotypes in other animals) or decreased like IL2 neurons.

We examined the branching pattern of PVD, a polymodal nociceptive neuron (new Supplemental Figure 3). We find no significant difference between control and sax-1 or sax-2 mutants, suggesting that these genes function in the context of pruning. Recent work (Zhao et al. 2022) confirms that sax-1 is not required for PVD branching.

Minor:

"shy87 mutant dauers showed a minor reduction in secondary and tertiary branches compared to control (Figure 1G). These results indicate that shy87 is specifically required for the elimination of dauer-generated dendrite branches." Maybe temper the specificity claim some as the reduction in branches is definitely there.

We agree, the claim was tempered.

"three complimentary approaches" should be complementary

Thank you for noticing. We fixed this.

"In control animals, SAX-2 was mostly concentrated in the cell body (data not shown)" It might be nice to include some overview images that show the cell body for completeness.

We added zoomed-out images to the revised figure, thank you for the suggestion.

Reviewer #3

Minor comments:

• Fig 1G-H, are shy87 second and third order branch counts statistically different between dauer and post dauer adults? This comparison would strengthen the claim that these order branches fail to eliminate all together rather than undergo a partial elimination. We added this to Figure S2. The shy87 mutants show a complete failure in eliminating secondary branches (i.e. no difference between dauer and post-dauer) and a strong but incomplete defect in eliminating tertiary branches.

• Fig 4B-E Indicate branch order in the images, this is unclear and a point that is focused on in the text. Done.

• Discussion of Fig 1G from the text claims that shy87 is specifically required for branch elimination yet the data shows significant defects in branch outgrowth as well. This raises the question, are the branches abnormally stabilized that results in early underdevelopment and late atrophy? Authors should acknowledge alternative hypotheses. We agree and will revise the text accordingly. The difference between shy87 and control dauers, while statistically significant, is relatively minor and can only be detected by careful quantification, it is not apparent from looking at the images (in contrast for example to rab-8 and rab-10 mutants, where we acknowledge in the text that their branching defects might affect subsequent pruning.

• Authors reference a branch elimination process but don't outline what this would entail and where their results fit in. We apologize for being unclear. Given that sax-1 and sax-2 function together, one would intuitively expect to see SAX-2 being reduced in sax-1 mutants, yet the opposite is observed. On potential explanation is that SAX-1 does not directly control SAX-2 abundance, but that clearance of SAX-2 is part of the pruning process that both proteins regulate. This would explain the enrichment of SAX-2 in sax-1 mutants. However, additional models cannot be excluded, and we acknowledge this in the revised text.

References:

Corchado, Johnny Cruz, Abhishiktha Godthi, Kavinila Selvarasu, and Veena Prahlad. 2024. “Robustness and Variability in Caenorhabditis Elegans Dauer Gene Expression.” Preprint, bioRxiv, August 26. https://doi.org/10.1101/2024.08.15.608164.

Karp, Xantha. 2018. “Working with Dauer Larvae.” WormBook, August 9, 1–19. https://doi.org/10.1895/wormbook.1.180.1.

Kozik, Patrycja, Richard W Francis, Matthew N J Seaman, and Margaret S Robinson. 2010. “A Screen for Endocytic Motifs.” Traffic (Copenhagen, Denmark) 11 (6): 843–55. https://doi.org/10.1111/j.1600-0854.2010.01056.x.

Lee, T., and L. Luo. 1999. “Mosaic Analysis with a Repressible Cell Marker for Studies of Gene Function in Neuronal Morphogenesis.” Neuron 22 (3): 451–61.

Swanson, M. M., and D. L. Riddle. 1981. “Critical Periods in the Development of the Caenorhabditis Elegans Dauer Larva.” Developmental Biology 84 (1): 27–40. https://doi.org/10.1016/0012-1606(81)90367-5.

Tang, Rui, Christopher W Murray, Ian L Linde, et al. n.d. “A Versatile System to Record Cell-Cell Interactions.” eLife 9: e61080. https://doi.org/10.7554/eLife.61080.

Zhao, Ting, Liying Guan, Xuehua Ma, Baohui Chen, Mei Ding, and Wei Zou. 2022. “The Cell Cortex-Localized Protein CHDP-1 Is Required for Dendritic Development and Transport in C. Elegans Neurons.” PLOS Genetics 18 (9): e1010381. https://doi.org/10.1371/journal.pgen.1010381.

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

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Referee #1

Evidence, reproducibility and clarity

This interesting study uses an unbiased genetic screen in C. elegans to identify SAX-1/NDR kinase as a regulator of dendritic branch elimination. Loss of SAX-1 results in an excess branching phenotype that is striking and highly penetrant. The authors identify several additional regulators of branch elimination (SAX-2, MOB-1, RABI-1, RAB-11.2) by using a candidate genetic screen aimed at factors that interact physically or genetically with SAX-1. They propose that SAX-1 acts by promoting membrane retrieval based on the nature of these interactors and the results of an imaging-based in vivo assay for endocytic puncta.

Major comments.

1. My biggest concern is that the phenotypes are only observed in temperature-sensitive dauer-constitutive mutant backgrounds, and not in wild-type dauers. That is, wild-type animals exiting dauer do not require SAX-1 for dendrite elimination.

While this does not undermine the importance of the results, it does require more explanation. The authors write that "the requirement for sax-1... relies on specific physiological states of the dauer stage," but I do not understand what this means. Are they saying that daf-7 and daf-2 dauers are in a different "physiological state" than wild-type dauers? In what way? What is the evidence for this? A more rigorous explanation is needed.

To me, the simplest genetic explanation is that daf-7 and daf-2 are partially required for branch retraction in a manner redundant with sax-1, and the ts mutants are not fully wild-type at 15C. Thus, the sax-1 requirement is revealed only in these mutant backgrounds. Can the authors examine starvation-induced dauers of daf-7 or daf-2 raised continuously at 15C?

daf-7 and daf-2 ts strains can form "partial dauers" that have a dauer-like appearance but are not SDS resistant. Could the difference between partial dauers and full dauers account for the difference in sax-1-dependence? The authors could use SDS selection of the daf-7 strain at 25C to ensure they are examining full dauers.

The Bargmann lab has created a daf-2 FLP-OUT strain (ky1095ky1087) that allows cell-type-specific removal of daf-2. Could this be used to test for a cell-autonomous role of daf-2 in IL2Q related to branch elimination?

These ideas are not a list of specific experiments the authors need to complete, rather they are meant to illustrate some possible approaches to the question. Whatever approach they use, it is important for them to more rigorously explain why SAX-1 is not required for branch removal in wild-type animals. 2. The SAX-2 localization (Fig. 4) and endocytosis assay (Fig. 6) results were not clear to me from the data shown. Overall a more rigorous analysis and presentation of the data would be important to make these conclusions convincing. This may involve refining the data presentation in the figures, modifying the claims (e.g., "we propose" vs "we find"), or saving some of the data to be more fully explored in a future paper. In my view, these figures are the biggest weak point of the manuscript and also are not important for the central conclusions (which are well supported and convincing), indeed these results are barely mentioned in the Abstract or last paragraph of Introduction.

• In Fig. 4, where in the head are we looking? It would help to show a more low-magnification view of the entire cell.
• In Fig. 4D, why is SAX-2 visible throughout the entire neuron and why is the "punctum" marked with an arrow also seen in the tagRFP channel? One gets the impression that some of the puncta may be background, bleed-through, or artifacts due to cell varicosities.
• In Fig. 4C, the distinction between puncta in the primary or higher-order dendrites is not clear to me, and several puncta that I would have scored as primary are marked as higher-order.
• Related to this, in Fig. 4B are the two arrows meant to be white as in the top panel, or yellow as in the bottom panel?
• The main sax-1 phenotype is increased SAX-2 puncta in dauer, but the branch retraction defect is in post-dauers. How is this relevant to the phenotype?
• The number of SAX-2 puncta in sax-1 mutants decreases almost to normal in post dauers. Is there a correlation between the number of remaining branches and the number of SAX-2 puncta? That is, do the many wild-type animals with "excess" SAX-2 puncta also fail to retract branches?
• The control post-dauer data in Fig. 4F and 4H are identical (re-used data) but the corresponding control dauer data in Fig. 4F and 4G are different. What is going on here?
• Why are sample sizes so small for both strains in Fig. 4G compared to Fig. 4F and 4H?
• In Fig. 6C, why are the tagRFP (blue) puncta larger than the neurite? Aren't these meant to represent vesicles inside the surrounding neurite? One gets the impression that this is bleed-through from the GFP channel.
• In Fig. 6E and 6F, why are there no tagRFP (blue) puncta? Is CD8 not endocytosed at all if it lacks the nanobody sequence? One would expect the tagRFP (blue) signal to be the same in both strains and simply to lack yellow if the nanobody is not present.
• In Fig. 6E and 6H, why are there so many GFP (yellow) puncta outside the neuron? What are these structures and why are they absent in the strain with the nanobody?
• What is the large central blue structure in Fig. 6H - is this the soma? - and why are puncta in this region not counted?
• The authors report a decrease in endocytic events in sax-1, but qualitatively it looks like there are vastly more puncta inside the neuron in Fig. 6H than in 6G.
• minor: there is text reading "40-" in the bottom panel of Fig. 6H. It is visible when printed but not on screen - adjust levels in Photoshop to reveal it.
• Related to both Fig. 4 and Fig. 6, where does SAX-1 localize in IL2Q in dauer and post-dauer? Does its expression or localization change during branch retraction? Does it co-localize with SAX-2 or endocytic puncta?

Minor points:

1. At several points the authors emphasize the relationship of neurite remodeling to stress, e.g. Abstract and Discussion: "we adapted C. elegans IL2 sensory dendrites as a model [of...] stress-mediated dendrite pruning". It seems unnecessary and potentially misleading to treat this as a neuronal stress response. First, it conflates organismal and cellular stress - there is no reason to think that IL2 neurons are under cellular stress in dauer. In fact parasitic nematodes go through dauer-like stages as part of healthy development and probably have similar remodeling of IL2. Second, dendrite pruning occurs during dauer exit, which is the opposite of a stress response - it reflects a return to favorable conditions.
2. In Fig. 1A, C. elegans is shown going directly from L1 to dauer in response to unfavorable conditions, which is incorrect. Animals proceed through L2 (in many cases actually an alternative L2d pre-dauer) and then molt into dauer (an alternative L3 stage) after completing L2.
3. In Fig. 1B, please check if it is correct that hypodermis contacts the pharynx basement membrane as drawn. The schematic in the top panel makes it look like there is a single secondary branch and the quartenary branches are similar in length to the primary dendrite. The schematic in the bottom panel makes it look like the entire neuron is a small fraction of the length of the pharynx. Could these be drawn closer to scale?

Referee cross-commenting

I think we all touched on similar points. I wanted to follow up on Reviewer 3's comment, "Is the failure to eliminate branches an indication of incomplete dauer recovery? Do sax-1 mutants retain additional characteristics of dauer morphology in post dauer adults." I thought this was an excellent point. It made me wonder if that might explain why the defect is only seen in daf-7 and daf-2 mutant backgrounds - maybe these strains retain partial dauer traits even after exit. Is there a specific experiment that they could do? Did you have specific characteristics of dauer morphology in mind for them to check? (Ideally something in the nervous system that can be scored quantitatively.)

Significance

A major strength of this work is the pioneering use of a novel system to study neuronal branch retraction. C. elegans has provided a powerful model for studying how dendrite branches form, but much less attention has been paid to how excess neuronal branches are removed. The post-dauer remodeling of IL2Q neurons provides an exciting and dramatic physiological example to explore this question.

This paper is notable for taking the first steps towards developing this innovative model. It does exactly what is needed at the outset of a new exploration - a forward genetic screen to discover the main regulators of the process. Using a combination of classical and modern genetic approaches, the authors bootstrap their way to a sizeable list of factors and a solid understanding of the properties of this system, for example that retraction of higher vs lower order dendrites show different genetic requirements.

Also deine Motivation für deine Arbeit ist es die Arbeit in nem geilen Format zu schrieben so dass es jeder versteht?:D Ich würd das vielleicht eher auf das Framework verallgemeinern? Also deine Motivation war es ja nen Framework zu bauen was self-documenting workflow hat.

Es gibt ja nicht eine methode um Hadron physics zu machen oder? Also würde nochmal speziell auf Scattering theory approach und partial wave decomposition eingehen.

Es un poco rara la numeración de figuras y subfiguras

Me gustaría conocer más detalles de estas decisiones. ¿Cuál fue la tasa de aprendizaje y por qué? ¿Cómo es el esquema de validación?

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