Aquí conviene incluir alguna cita de DeepONet

1 Stroom komt uit in meerdere stromen. (Na het bestellen word èn het pakket ingepakt èn de factuur opgesteld.)

This defines the Bantu as groups of people who speak related languages characterized by the stem "ntu" and the prefix "ba-", meaning "people."

Aunque la abreviación EDP es muy común, si la vas a usar en el resumen conviene definirla aquí, algo así como "............ecuaciones diferenciales parciales (EDPs), .............."

Author response:

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

Reviewer #1 (Public review):  

(1) To distinguish autophagosomes from autolysosomes, the authors used vps16 RNAi cells, which are supposed to be fusion deficient. However, the extent to which fusion is actually inhibited by knockdown of Vps16A is not shown. The co-localization rate of Atg8 and Lamp1 should be shown (as in Figure 8). Then, after identifying pre-fusion autophagosomes and lysosomes, the localization of each should be analyzed.

Thank you for this insightful comment. We analyzed the colocalization of 3xmCherry-Atg8a and GFP-Lamp1, which label autophagic structures and lysosomes, respectively, in Vps16A RNAi fat body cells. As expected, Vps16A silencing markedly reduced the overlap between these two signals, indicating a strong block in autophagosome–lysosome fusion. Moreover, both 3xmCherry-Atg8a and GFP-Lamp1 became more perinuclearly localized compared to the control (luciferase RNAi) cells.

It is also possible that autophagosomes and lysosomes are tethered by factors other than HOPS (even if they are not fused). If this is the case, autophagosomal trafficking would be affected by the movement of lysosomes.  

Thank you for raising this possibility. While we cannot fully exclude that autophagosomes might be indirectly transported via tethering to lysosomes, we consider this unlikely. We believe that in Drosophila fat cells, autophagosomes and lysosomes rapidly fuse once in close proximity. Therefore, even if alternative tethering mechanisms exist, they are unlikely to permit prolonged joint trafficking without fusion.

(2) The authors analyze autolysosomes in Figures 6 and 7. This is based on the assumption that autophagosome-lysosome fusion takes place in cells without vps16A RNAi. However, even in the presence of Vps16A, both pre-fusion autophagosomes and autolysosomes should exist. This is also true in Figure 8H, where the fusion of autophagosomes and lysosomes is partially suppressed in knockdown cells of dynein, dynactin, Rab7, and Epg5. If the effect of fusion is to be examined, it is reasonable to distinguish between autophagosomes and autolysosomes and analyze only autolysosomes.  

Thank you for this careful observation. The 3xmCherry-Atg8a reporter is well suited to identify both autophagosomes and autolysosomes, as the mCherry fluorophore is resistant to degradation in the acidic environment of autolysosomes. Notably, mCherry-Atg8a–positive autolysosomes appear larger and brighter than pre-fusion autophagosomes, which are typically smaller and dimmer, especially under fusion-deficient conditions (e.g., Figure 4). Therefore, we use these morphological differences as a proxy to distinguish between the two.

To improve structural assignment, we incorporated endogenous Lamp1 staining (Figure 10) and a Lamp1-GFP reporter (Figure 10—figure supplement 1). Vesicles positive for mCherryAtg8a but negative for Lamp1 are considered pre-fusion autophagosomes. Structures double-positive for mCherry-Atg8a and Lamp1 represent autolysosomes, while Lamp1positive, Atg8a-negative vesicles correspond to non-autophagic lysosomes. To clarify these interpretations, we revised the Results section and explained these reporters in more detail.

(3) In this study, only vps16a RNAi cells were used to inhibit autophagosome-lysosome fusion. However, since HOPS has many roles besides autophagosome-lysosome fusion, it would be better to confirm the conclusion by knockdown of other factors (e.g., Stx17 RNAi).  

Thank you for this valuable suggestion. We initially considered using Syntaxin17 RNAi; however, our recent findings indicate that loss of Syx17 results in a HOPS-dependent tethering lock between autophagosomes and lysosomes (DOI: 10.1126/sciadv.adu9605). In this case, tethered vesicles would likely move together, confounding the interpretation of autophagosome-specific trafficking.

Therefore, we turned to other SNAREs such as Vamp7 and Snap29. One Snap29 RNAi was located on the appropriate chromosome needed for our genetic experiments. We generated a transgenic fly line expressing both Snap29 RNAi and the mCherry-Atg8a reporter under a fat body-specific R4 promoter. When we tested our key trafficking hits in this background, we observed similar autophagosome localization phenotypes as in Vps16A RNAi cells. These results, now included in the revised manuscript (see Figure 6), confirm that the observed transport phenotypes are not specific to Vps16A or HOPS complex loss.

(4) Figure 8: Rab7 and Epg5 are also known to be directly involved in autophagosomelysosome tethering/fusion. Even if the fusion rate is reduced in the absence of Rab7 and Epg5, it may not be the result of defective autophagosome movement, but may simply indicate that these molecules are required for fusion itself. How do the authors distinguish between the two possibilities?

Thank you for this important point. While Rab7 and Epg5 indeed participate in autophagosome–lysosome tethering and fusion, our data suggest they also contribute to autophagosome movement. This is evident from the distinct phenotypes observed upon Rab7 or Epg5 RNAi compared to Vps16A or SNARE RNAi. Depletion of Vps16A, Syx17, Vamp7, or Snap29 (factors involved specifically in fusion) results in perinuclear accumulation of autophagosomes. In contrast, Rab7 or Epg5 RNAi leads to a dispersed autophagosome pattern throughout the cytoplasm.

These differences suggest that Rab7 and Epg5 play additional roles in positioning autophagosomes. Supporting this, our co-immunoprecipitation experiments show that Epg5 interacts with dynein motors. Therefore, we propose that Rab7 and Epg5 influence both autophagosome fusion and their microtubule-based transport.

Reviewer #2 (Public review):  

One limitation of the study is the genetic background that serves as the basis for the screening. In addition to preventing autophagosome-lysosome fusion, disruption of Vps16A has been shown to inhibit endosomal maturation and block the trafficking of components to the lysosome from both the endosome and Golgi apparatus. Additional effects previously reported by the authors include increased autophagosome production and reduced mTOR signaling. Thus Vps16A-depleted cells have a number of endosome, lysosome, and autophagosome-related defects, with unknown downstream consequences. Additionally, the cause and significance of the perinuclear localization of autophagosomes in this background is unclear. Thus, interpretations of the observed reversal of this phenotype are difficult, and have the caveat that they may apply only to this condition, rather than to normal autophagosomes. Additional experiments to observe autophagosome movement or positioning in a more normal environment would improve the manuscript.  

Thank you for highlighting this limitation. We have tried to conduct time-lapse imaging of live fat body cells expressing 3xmCherry-Atg8a and GFP-Lamp1 to visualize the movement and fusion events of pre-fusion autophagosomes (3xmCherry-Atg8a positive and GFP-Lamp1 negative) and lysosomes (GFP-Lamp1 positive). Despite different experimental setups and durations of starvation, no vesicle movement was observed at all, so live imaging of larval Drosophila fat tissue will require time-consuming optimizations of in vitro culture conditions. Consistent with this, we did not find any literature data where organelle motility in fat body cells was successfully observed. Nuclear positioning in fat body cells was investigated in detail in an excellent study, however the authors were able to observe only very little movement of the nuclei by live imaging (Zheng et al. Nat Cell Biol. 2020 Mar;22(3):297-309. doi: 10.1038/s41556-020-0470-7), further highlighting the technical difficulties of live or timelapse imaging in this tissue type.

Specific comments  

(1) Several genes have been described that when depleted lead to perinuclear accumulation of Atg8-labeled vesicles. There seems to be a correlation of this phenotype with genes required for autophagosome-lysosome fusion; however, some genes required for lysosomal fusion such as Rab2 and Arl8 apparently did not affect autophagosome positioning as reported here. Thus, it is unclear whether the perinuclear positioning of autophagosomes is truly a general response to disruption of autophagosome-lysosome fusion, or may reflect additional aspects of Vps16A/HOPS function. A few things here would help. One would be an analysis of Atg8a vesicle localization in response to the depletion of a larger set of fusionrelated genes. Another would be to repeat some of the key findings of this study (effects of specific dynein, dynactin, rabs, effectors) on Atg8a localization when Syx17 is depleted, rather than Vps16A. This should generate a more autophagosome-specific fusion defect.  

Thank you for this insightful suggestion. We recently discovered that Syx17 depletion induces a HOPS-dependent tethering lock between autophagosomes and lysosomes (DOI: 10.1126/sciadv.adu9605), making it unsuitable for modeling autophagosome-specific fusion defects. In contrast, Vamp7 and Snap29 knockdowns do not appear to cause such tethering lock. We were able to generate a suitable Drosophila line using a Snap29 RNAi transgene located on a compatible chromosome. Upon testing key hits from our screen in this background, we found that autophagosomes redistributed similarly, supporting our conclusions. These new results have been included in the revised manuscript (see Figure 6)

Third, it would greatly strengthen the findings to monitor pre-fusion autophagosome localization without disrupting fusion. Such vesicles could be identified as Atg8a-positive Lamp-negative structures. The effects of dynein and rab depletion on the tracking of these structures in a post-induction time course would serve as an important validation of the authors' findings.  

Thank you for this helpful suggestion. As described above, we attempted time-lapse imaging of 3xmCherry-Atg8a and GFP-Lamp1-expressing fat body cells under various conditions to identify motile pre-fusion autophagosomes. However, we did not observe any vesicle movement, regardless of the starvation duration or experimental setup. As this likely reflects technical limitations of ex vivo fat body imaging, we were unable to achieve live tracking of autophagosome dynamics without introducing perturbations. This limitation is now discussed in the revised manuscript.

(2) The authors nicely show that depletion of Shot leads to relocalization of Atg8a to ectopic foci in Vps16A-depleted cells; they should confirm that this is a mislocalized ncMTOC by colabeling Atg8a with an MTOC component such as MSP300. The effect of Shot depletion on Atg8a localization should also be analyzed in the absence of Vps16A depletion.  

Thank you for this positive comment. We co-labeled Atg8a with the minus-end microtubule marker Khc-nod-LacZ in both shot single knockdown and shot; vps16A double knockdown cells. Ectopic Khc-nod-LacZ-positive MTOC foci were clearly visible in both conditions, and Atg8a-positive autophagosomes accumulated around these structures. These findings confirm that Shot depletion induces ectopic MTOC formation, which correlates with autophagosome relocalization. The new data have been incorporated into the revised manuscript (see Figure 1O-S).

(3) The authors report that depletion of Dynein subunits, either alone (Figure 6) or codepleted with Vps16A (Figure 2), leads to redistribution of mCherry-Atg8a punctae to the "cell periphery". However, only cell clones that contact an edge of the fat body tissue are shown in these figures. Furthermore, in these cells, mCherry-Atg8a punctae appear to localize only to contact-free regions of these cells, and not to internal regions of clones that share a border with adjacent cells. Thus, these vesicles would seem to be redistributed to the periphery of the fat body itself, not to the periphery of individual cells. Microtubules emanating from the perinuclear ncMTOC have been described as having a radial organization, and thus it is unclear that this redistribution of mCherry-Atg8a punctae to the fat body edge would reflect a kinesin-dependent process as suggested by the authors.  

Thank you for this detailed observation. We frequently observe autophagosomes accumulating in contact-free peripheral regions of dynein-depleted cells, resulting in an asymmetric distribution. While previous studies describe a radial microtubule organization in fat body cells, none of them directly label MT plus ends, the direction of kinesin-based transport.

To further explore this, we overexpressed a HA-tagged kinesin, Klp98A-3xHA, in both control and Vps16A RNAi backgrounds. Immunolabeling revealed that Klp98A localizes to the contact-free peripheral regions in both conditions, consistent with the distribution of autophagosomes in dynein knockdown cells. This supports our interpretation that kinesindependent transport drives autophagosome redistribution in the absence of dynein, and that fat body cells exhibit subtle asymmetries in MT polarity that influence this transport. These new results have been included in the revised manuscript (see Figure 3G, H).

(4) To validate whether the mCherry-Atg8a structures in Vps16A-depleted cells were of autophagic origin, the authors depleted Atg8a and observed a loss of mCherry- Atg8a signal from the mosaic cells (Figure S1D, J). A more rigorous experiment would be to deplete other Atg genes (not Atg8a) and examine whether these structures persist.  

Thank you for the suggestion to further validate our reporter. We depleted Atg1, a key kinase required for phagophore initiation, in the Vps16A RNAi background. This completely abolished the punctate mCherry-Atg8a distribution in knockdown cells (see Figure 1—figure supplement 1E, K), confirming that the labeled structures are indeed of autophagic origin.

(5) The authors found that only a subset of dynein, dynactin, rab, and rab effector depletions affected mCherry-Atg8a localization, leading to their suggestion that the most important factors involved in autophagosome motility have been identified here. However, this conclusion has the caveat that depletion efficiency was not examined in this study, and thus any conclusions about negative results should be more conservative.  

Thank you for this constructive feedback. We agree that negative results must be interpreted conservatively due to potential differences in knockdown efficiency. We have revised our conclusions accordingly, clarifying that the factors identified are key for autophagosome motility, while acknowledging the possibility of false negatives.

Reviewer #3 (Public review):  

Major concerns:

(1) The localization of EPG5 should be determined. The authors showed that EPG5 colocalizes with endogenous Rab7. Rab7 labels late endosomes and lysosomes. Previous studies in mammalian cells have shown that EPG5 is targeted to late endosomes/lysosomes by interacting with Rab7. EPG5 promotes the fusion of autophagosomes with late endosomes/lysosomes by directly recognizing LC3 on autophagosomes and also by facilitating the assembly of the SNARE complex for fusion. In Figure 5I, the EPG5/Rab7colocalized vesicles are large and they are likely to be lysosomes/autolysosomes.

Thank you for suggesting to improve our Epg5 localization data. We performed triple immunostaining for Atg8a, Lamp1-3xmCherry, and Epg5-9xHA in S2R+ cells. In addition to triple-positive structures—likely representing autolysosomes—we observed Atg8a and Epg59xHA double-positive vesicles that lacked Lamp1-3xmCherry signal, which likely correspond to pre-fusion autophagosomes. Based on these results, we propose that in addition to arriving via the endocytic route, Epg5 may also reach lysosomes through autophagosomes. These findings have been included in the revised manuscript (see Figure 5K).

(2) The experiments were performed in Vps16A RNAi KD cells. Vps16A knockdown blocks fusion of vesicles derived from the endolysosomal compartments such as fusion between lysosomes. The pleiotropic effect of Vps16A RNAi may complicate the interpretation. The authors need to verify their findings in Stx17 KO cells, as it has a relatively specific effect on the fusion of autophagosomes with late endosomes/lysosomes.  

Thank you for this valuable suggestion. We initially considered Syntaxin17 for validation; however, we recently found that loss of Syx17 leads to a HOPS-dependent tethering lock between autophagosomes and lysosomes, which would confound interpretation, as autophagosomes remain tethered to lysosomes (DOI: 10.1126/sciadv.adu9605). Therefore, Syntaxin17 loss is not suitable for our purpose. Among the remaining fusion SNAREs, one RNAi line targeting Snap29 was available on a compatible chromosome for generating Drosophila lines equivalent to those used in the screen. We established this Snap29 RNAicontaining tester line and crossed it with our top hits. We observed that autophagosome motility was comparable to that in the Vps16A RNAi background, further supporting our conclusions. These results have been incorporated into the revised manuscript (see Figure 6)

(3) Quantification should be performed in many places such as in Figure S4D for the number of FYVE-GFP labeled endosomes and in Figures S4H and S4I for the number and size of lysosomes.  

Thank you for pointing this out. We performed the suggested quantifications and statistical analyses for FYVE-GFP labeled endosomes, as well as for the number and size of lysosomes. The updated data are now presented in the revised Figure 5—figure supplement 1.

(4) In this study, the transport of autophagosomes is investigated in fly fat cells. In fat cells, a large number of large lipid droplets accumulate and the endomembrane systems are distinct from that in other cell types. The knowledge gained from this study may not apply to other cell types. This needs to be discussed.

Thank you for raising this important point. We agree that our findings may not be fully generalizable to all cell types. Given that the organization of the microtubule network depends on both cell function and developmental stage, it is plausible that the molecular machinery described here operates differently elsewhere. We now mention this limitation in the Discussion.

Minor concerns:  

(5) Data in some panels are of low quality. For example, the mCherry-Atg8a signal in Figure 5C is hard to see; the input bands of Dhc64c in Figure 5L are smeared.  

Thank you for pointing this out. We repeated the experiment shown in Figure 5C and replaced the panel with a clearer image. The smeared Dhc64C input bands in Figure 5L result from the unusually large size of this protein, which affects its electrophoretic migration. We mentioned this point in the corresponding figure legend.

(6) In this study, both 3xmCherry-Atg8a and mCherry-Atg8a were used. Different reporters make it difficult to compare the results presented in different figures.  

Thank you for this comment. Both 3xmCherry-Atg8a and mCherry-Atg8a are well-established reporters that behave similarly as autophagic markers. Nevertheless, to avoid confusion, we ensured that each figure uses only one type of reporter consistently, which is now clearly indicated in the revised manuscript.

(7) The small autophagosomes presented in Figures such as in Figure 1D and 1E are not clear. Enlarged images should be presented.  

Thank you for your suggestion. We repeated these experiments and replaced the relevant panels with higher-quality images, including enlarged insets to better visualize small autophagosomes. These updated figures are now included in the revised manuscript.

(8) The authors showed that Epg5-9xHA coprecipitates with the endogenous dynein motor Dhc64C. Is Rab7 required for the interaction?  

Thank you for this insightful question. We tested this by co-transfecting S2R+ cells with Epg5-9xHA and different forms of Rab7: wild-type, GTP-locked (constitutively active), and GDP-locked (dominant-negative). Our results indicate that the strength of Epg5-Dhc interaction does not change in the presence of either GTP-locked or GDP-locked Rab7. However, we believe that Epg5 and dynein are recruited to the vesicle membranes via Rab7 in vivo, so we did not include these results in the revised manuscript.

(9) The perinuclear lysosome localization in Epg5 KD cells has no indication that Epg5 is an autophagosome-specific adaptor.

Thank you for this important comment. Accordingly, we have toned down our statements about Epg5 functions throughout the revised manuscript.

Reviewer #1 (Recommendations for the authors):  

(1) Figure 6: What do "autolysosome maturation" and "small autolysosomes" mean? Do different numbers of lysosomes fuse to a single autophagosome?

Thank you for highlighting this point. We concluded that the formation of smaller autolysosomes—compared to controls—is likely due to a defect in autolysosome maturation, as is often the case. We had not explicitly considered whether a different number of lysosomes fuse with each autophagosome during this process. We clarified this issue in the revised manuscript.

(2) Figure 5A shows that the localization of endogenous Atg8 requires Epg5, but the data is not as clear as for mCherry-Atg8 (Figure 4B). Why the difference?  

Thank you for this question. The difference arises because the mCherry-Atg8a reporter strongly labels autolysosomes, as the mCherry fluorophore remains stable in acidic compartments. As a result, mCherry-Atg8a labels both autophagosomes and autolysosomes, but the strong autolysosomal signal originating from the surrounding GFP negative, nonRNAi cells can make accumulated autophagosomes appear fainter in fusion-defective cells (as in Figure 4). In contrast, endogenous Atg8a is degraded in lysosomes, and therefore labels only autophagosomes. This means that the appearance of these two experiments can be slightly different, but since in both cases autophagosomes no longer accumulate in the perinuclear region of Vps16A,Epg5 double RNAi cells we can conclude that Epg5 is required for autophagosome positioning. We explained this difference of the two methods in the revised manuscript where it first appears (Figure 1B and Figure 1—figure supplement 1A).

(3) Blue letters on the black micrographs are hard to see. Some of the other letters are also small and hard to read.  

Thank you for this suggestion. We improved the visibility and readability of the labels in the revised figures.

MAYBE

Serán noticias y sentimientos con la menor frecuencia posible / series temporales de aranceles (NO creo pero quizás). Para disminuir la dimensionalidad utilizar PCA.

IMPORTANTE

OJO, IMPORTANTE

OJO, MIRAR

pas sure que ça soit compréhensible, je pensais que le paragraphe d'avant voulait dire: ce serait idéal que chacun puisse être qui il est, avec ses bizzareries sans une normativité extreme, et pas: il y a un idiot du village et on l'accepte car il nous permet de faire groupe, je pense que ça peut être mal compris ça serait dommage

s'en remettre à la misanthropie et à l'attrait pour le néant et la mort ?

qui affirme ne plus avoir la possibilité, avec sa génération, de rêver de "fonder une famille", d'avoir des idéaux, des aspirations de métier, convaincu de vivre à la fin de l'histoire de l'humanité

cette phrase m'a surprise: pas sure que ça soit nécessaire on ne comprends pas directement le sens je trouve

peut être faire deux phrases: "La désaffection dont il est atteint ne le rend pas capable d'aimer quoi ni qui que ce soit, à commencer par lui-même. Elle fait écho à la désaffection dont témoignent notre silence et nos caméras de surveillance, révélatrices d'une société qui, elle non plus, ne semble plus capable de s'aimer et d'aimer."

peut être pas obligé de rajouter ça, s'arrêter à "l'intégrer."

peut être déjà de la destruction plutôt qu’une « inclination à »?

il faudra écrire quelque chose sur les ontologies ? notamment pour la description d'ensembles et sous-ensembles thématiques plus complexes ?

SUPER !!!!!!

et si on écrivait : "Les études aréales sont confrontées à la pauvreté ou la mauvaise qualité des référentiels à leur disposition pour la prise en compte des spécificités du domanie et du multilinguisme, se retrouvant souvent face à la difficulté d’alignement avec des référentiels existants. Ces référentiels ne font pas état, à l'heure actuelle, de la concaténation entre les langues, les orthographes et les translitérations qui ont pu varier dans l'espace et dans le temps."

j'ai ajouté ça

je propose de couper beaucoup cette partie et de proposer quelque chose comme ceci : "L'exemple des Cikh (...) qui n'a pas de terme de description propre ni en français ni en anglais et qui a moultes variantes dans les sociétés d’éleveurs d’Asie centrale et du monde turco-iranien, est écrit à partir de la même racine turcophone mais avec des écriture différentes ( çît et çîẍ (چیغ), selon les cas."

Ajouter la dimension multilangue et multécriture nécessaires à ce travail, ceci pourra se décliner en plusieurs langues et écritures (à l'instar du thésaurus des traducteurs de Frédéric ...)

@mariebizais je propose : "... anglais ou en allemand. Cette limitation néglige les correspondances avec le vocabulaire “local”, et contribue à rompre les liens à la fois avec les corpus et avec les experts qui travaillent aujourd'hui ...."

@mariebizais je mettrai "écriture car l'unicode est un système de codage de caractères (lettres, glyphes, etc.)

Il va falloir réduire cette liste : nous avons besoin de deux personnes qui soient motrices, sinon la thématique ne va pas être explorée. Barbara, Michaël et Lia ?

A-t-on vraiment besoin de ce paragraphe très général ? Il me semble que tout le CS d'HN est au courant de ces enjeux, non ?

globalization

replacer par "cette synergie" ?!!

Je propose : "Les participants à ce séminaire seront appelés à tour de rôle à rédiger un cr de la séance et à publier un billet sur le sujet dans le carnet distam."

remplacer par "la question traitée" ?!!

ajouter : "et dans la conduite d'un projet numérisue" ?!!

El presente artículo tiene como finalidad estructurar una visión general sobre el streaming de videojuegos como agente generador de empleo y nuevas carreras profesionales en el Ecuador. En la metodología se planteó un diseño no experimental de alcance descriptivo y correlacional, de enfoque mixto, empleando el método Delphi en el componente cualitativo, mientras que se ejecutaron encuestas para solventar el componente cuantitativo. En este sentido, se pudo observar una mayor participación de la generación Z y millennials en este tipo de actividades, lo que a su vez refleja el interés por formar parte del streaming de videojuegos, mientras que otros que ya son usuarios han percibido un aporte o beneficio económico por jugar o crear contenido. Además, se resalta al género masculino como el principal participante en estas plataformas. Se concluyó que hay relación entre las personas que dedican horas a los videojuegos y el deseo de obtener réditos económicos a través de este modelo de negocio. De igual forma se observa una tendencia de los gamers en profesionalizarse en carreras de marketing digital y producción audiovisual y multimedia, lo cual sirve para que las instituciones de educación superior ajusten y fortalezcan los programas académicos relacionados, así como enfocar de manera más efectiva sus estrategias de marketing hacia este público objetivo. [ABSTRACT FROM AUTHOR]

El objetivo principal de esta tesis es presentar un análisis de las aportaciones del anime de ciencia ficción, considerado tanto en sus aspectos narrativos como en su materialidad como imagen en movimiento, a la cuestión de las relaciones entre identidad, cuerpo y tecnología. A través de animes como Akira, Evangelion, Ghost in the Shell y Serial Experiments Lain, exploro de qué forma la tecnología pone en duda los límites de la identidad individual, y cómo es fundamento para la extensión de ciertas capacidades corporales, cognitivas y sociales del ser humano. A través de conceptos extraídos de las filosofías de Henri Bergson, Gilbert Simondon, Gilles Deleuze, Jacques Lacan y Donna Haraway, entre otros, exploro la relación entre elementos narrativos y extra-narrativos en estos animes. En estos trabajos se nos presentan no sólo narrativas complejas, sino también elementos que parecen escapar a la lógica narrativa. Deben tenerse debidamente en cuenta tanto los aspectos extra-narrativos "flotantes" como puntos específicos de ruptura de la narración. Además, pongo en relación éstas formas de narratividad con ciertas nociones de temporalidad, en las que se pone en entredicho lo que Bergson denominó el tiempo espacializado, un tiempo homogéneo y lineal, para hacer emerger formas de temporalidad más cercanas a la idea de proceso, es decir, heterogéneas y no ligadas a una lógica teleológica. Finalmente, comento de qué forma el objeto técnico anime constituye una forma de transducción material específica de ciertos procesos psíquicos, y como en éste se produce significación de forma transindividual y colectiva. Tener en cuenta el anime desde este punto de vista, como una transducción de ciertos esquemas psíquicos, nos permite poder hablar de él como dispositivo pensante.

chrome-extension://bjfhmglciegochdpefhhlphglcehbmek/pdfjs/web/viewer.html?file=file%3A%2F%2F%2FUsers%2Fprestontaylor%2FDesktop%2F61302_e-flux-journal-unglamorous-tasks-what-can-education-learn-from-its-political-traditions.pdf

Reviewer #3 (Public review):

Summary:

In their revised manuscript, the authors addressed most of the reviewers' concerns. One concern was the emphasis on increased MLEC-OST interactions during infection, which the authors toned down in the revision. They clarified that MLEC interaction with OST is maintained-rather than increased-during infection, while its interaction with other QC factors decreases. They also added context and discussion of the co-localization of viral proteins with ER and mitochondrial proteins, noting that both nsp2 and MLEC localize to mitochondria-associated membranes (MAMs), providing a plausible explanation for these interactions.

Another concern involved the effects of MLEC KD on the cellular environment. To address this, the authors analyzed stress pathway activation and glycosylation of endogenous proteins in MLEC KD cells. They found only modest upregulation of the HSF1 pathway and no changes in the UPR or other stress responses, suggesting MLEC KD does not broadly disrupt ER proteostasis. Additionally, glycopeptide profiling showed only minor changes in host protein glycosylation, supporting a more direct role for MLEC in viral replication rather than general host glycoprotein disruption.

However, some weaknesses remain. Direct interaction between MLEC and nsp2 during infection was not detected, and the identified viral glycopeptides were limited to only five Spike sites. Furthermore, the mechanism by which MLEC promotes viral replication is still unclear.

In summary, the authors strengthened the manuscript by addressing reviewers' concerns through additional data, clarified language, and expanded discussion. While the overall support for MLEC's pro-viral role is solid, its precise mechanism of action remains speculative. Future work will be needed to directly link MLEC's activity to specific steps in viral protein biogenesis and replication.

Original summary: In this study, Davies and Plate set out to discover conserved host interactors of coronavirus non-structural proteins (Nsp). They used 293T cells to ectopically express flag-tagged Nsp2 and Nsp4 from five human and mouse coronaviruses, including SARS-CoV-1 and 2, and analyzed their interaction with host proteins by affinity purification mass-spectrometry (AP-MS). To confirm whether such interactors play a role in coronavirus infection, the authors measured the effects of individual knockdowns on replication of murine hepatitis virus (MHV) in mouse Delayed Brain Tumor cells. Using this approach, they identified a previously undescribed interactor of Nsp2, Malectin (Mlec), which is involved in glycoprotein processing and shows a potent pro-viral function in both MHV and SARS-CoV-2. Although the authors were unable to confirm this interaction in MHV-infected cells, they show that infection remodels many other Mlec interactions, recruiting it to the ER complex that catalyzes protein glycosylation (OST). Mlec knockdown reduced viral RNA and protein levels during MHV infection, although such effects were not limited to specific viral proteins. However, knockdown reduced the levels of five viral glycopeptides that map to Spike protein, suggesting it may be affected by Mlec.

Strengths:

This is an elegant study that uses a state-of-the-art quantitative proteomic approach to identify host proteins that play critical roles in viral infection. Instead of focusing on a single protein from a single virus, it compares the interactomes of two viral proteins from five related viruses, generating a high confidence dataset. The functional follow-ups using multiple live and reporter viruses, including MHV and CoV2 variants, convincingly depict a pro-viral role for Mlec, a protein not previously implicated in coronavirus biology.

Weaknesses:

Although a commonly used approach, AP-MS of ectopically expressed viral proteins may not accurately capture infection-related interactions. The authors observed Mlec-Nsp2 interactions in transfected 293T cells (1C) but were unable to reproduce those in mouse cells infected with MHV (3C). EIF4E2/GIGYF2, two bonafide interactors of CoV2 Nsp2 from previous studies, are listed as depleted compared to negative controls (S1D). Most other CoV2 Nsp2 interactors are also depleted by the same analysis (S1D). Previously reported MERS Nsp2 interactors, including ASCC1 and TCF25, are also not detected (S1D). Furthermore, although GIGYF2 was not identified as an interactor of MHV Nsp2/4 in human cells (S1D), its knockdown in mouse cells reduced MHV titers about 1000 fold (S4). The authors should attempt to explain these discrepancies.

More importantly, the authors were unable to establish a direct link between Mlec and the biogenesis of any viral or host proteins, by mass-spectrometry or otherwise. Although it is clear that Mlec promotes coronavirus infection, the mechanism remains unclear. Its knockdown does not affect the proteome composition of uninfected cells (S15B), suggesting it is not required for proteome maintenance under normal conditions. The only viral glycopeptides detected during MHV infection originated from Spike (5D), although other viral proteins are also known to be glycosylated. Cells depleted for Mlec produce ~4-fold less Spike protein (4E) but no more than 2-fold less glycosylated spike peptides (5D), compounding the interpretation of Mlec effects on viral protein biogenesis. Furthermore, Spike is not essential for the pro-viral role of Mlec, given that Mlec knockdown reduces replication of SARS-CoV-2 replicons that express all viral proteins except for Spike (6A/B).

Any of the observed effects on viral protein levels could be secondary to multiple other processes. Interventions that delay infection for any reason could lead to imbalance of viral protein levels, because Spike and other structural proteins are produced at a much higher rate than non-structural proteins due to the higher abundance of their cognate subgenomic RNAs. Similarly, the observation that Mlec depletion attenuates MHV-mediated changes to the host proteome (S15C/D) can also be attributed to indirect effects on viral replication, regardless of glycoprotein processing. In the discussion, the authors acknowledge that Mlec may indirectly affect infection through modulation of replication complex formation or ER stress, but do not offer any supporting evidence. Interestingly, plant homologs of Mlec are implicated in innate immunity, favoring a more global role for Mlec in mammalian coronavirus infections.

Finally, the observation that both Nsp2 (3C) and Mlec (3E/F) are recruited to the OST complex during MHV infection neither support nor refute any of these alternate hypotheses, given that Mlec is known to interact with OST in uninfected cells and that Nsp2 may interact with OST as part of the full length unprocessed Orf1a, as it co-translationally translocates into the ER.

Therefore, the main claims about the role of Mlec in coronavirus protein biogenesis are only partially supported.

Comments on revisions:

Figure 7B should be revised to show that MLEC maintains interactions with rather than recruited to the OST.

Author response:

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

Reviewer #1 (Public Review):

In this manuscript, the authors employ a combined proteomic and genetic approach to identify the glycoprotein QC factor malectin as an important protein involved in promoting coronavirus infection. Using proteomic approaches, they show that the non-structural protein NSP2 and malectin interact in the absence of viral infection, but not in the presence of viral infection. However, both NSP2 and malectin engage the OST complex during viral infection, with malectin also showing reduced interactions with other glycoprotein QC proteins. Malectin KD reduce replication of coronaviruses, including SARS-COV2. Collectively, these results identify Malectin as a glycoprotein QC protein involved in regulating coronavirus replication that could potentially be targeted to mitigate coronavirus replication.

Overall, the experiments described appear well performed and the interpretations generally reflect the results. Moreover, this work identifies Malectin as an important pro-viral protein whose activity could potentially be therapeutically targeted for the broad treatment of coronavirus infection. However, there are some weaknesses in the work that, if addressed, would improve the impact of the manuscript.

Notably, the mechanism by which malectin regulates viral replication is not well described. It is clear from the work that malectin is a pro-viral protein in the work presented, but the mechanistic basis of this activity is not pursued. Some potential mechanisms are proposed in the discussion, but the manuscript would be strengthened if additional insight was included. For example, does the UPR activated to higher levels in infected cells depleted of malectin? Do glycosylation patterns of viral (or non-viral) proteins change in malectindepleted cells? Additional insight into this specific question would significantly improve the manuscript.

We concur with the reviewer that the mechanism by which Malectin regulates viral replication is an important point to elucidate further. Our proteomics data were able to offer additional insight into the questions posed here. We examined the upregulation of protein markers of the UPR and other stress response pathways in cells depleted of MLEC (Fig. S15D). We find that the UPR pathways are moderately but insignificantly upregulated, while the Heat Shock Factor 1 (HSF1) pathway is moderately and significantly upregulated. The fold change increase of these marker proteins are relatively small, so while upregulation of this pathway may contribute to the suppression of CoV replication, it may not fully explain the phenotype.

In addition, to address the second question, we compared the glycosylation patterns of endogenous proteins in MLEC-KD cells (Fig. S15E-G). We found that there is a small increase in abundance of glycopeptides associated with LAMP2, SERPHINH1, RDX, RPL3/5, CADM4, and ITGB1, however these fold changes are small and tested to be insignificant. These results indicate there is relatively little modification of endogenous glycoproteins upon MLEC-depletion. These findings support a more direct role for MLEC in regulating viral replication.

We added the following section to the manuscript text to discuss these results:

“In uninfected cells, MLEC KD leads to relatively little proteome-wide changes, with MLEC being the only protein significantly downregulated and no other proteins significantly upregulated, supporting the specificity of MLEC KD in MHV suppression (Fig.  S15C). To determine whether MLEC KD alters general host proteostasis, we further examined the levels of protein markers of stress pathways based on previous gene pathway definitions(Davies et al., 2023; Grandjean et al., 2019; Shoulders et al., 2013) (Fig. S15D). We find that there are modest but significant increases in protein levels associated with the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response (UPR) pathways are largely unmodified. 

We also probed the effect of MLEC KD on endogenous protein glycosylation. We find that there is only a small increase in abundance of glycopeptides, including those associated with the ribosome (Rpl3, Rpl5), a cytoskeletal protein (Rdx), the integrin Itgb1, and the ER-resident chaperone Serphinh1 (Fig. S15E-G).”

“Our proteomics data reveals that there is only a modest increase in the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response is relatively unchanged (Fig. S15D). In addition, there are only minor increases in endogenous glycopeptide levels (Fig. S15E-G). Together, these results indicate that while MLEC KD leads to some alterations in ER proteostasis and host glycosylation, these changes are modest and may not be the primary mechanism by which MLEC KD hinders CoV replication.”

Further, the evidence for increased interactions between OST and malectin during viral infection is fairly weak, despite being a major talking point throughout the manuscript. The reduced interactions between malectin and other glycoproteostasis QC factors is evident, but the increased interactions with OST are not well supported. I'd recommend backing off on this point throughout the text, instead, continuing to highlight the reduced interactions.

We agree that the fold change increase of OST interactions with malectin are small compared to the fold change decrease of other glycoproteostasis factors We have modified the text to less emphasize this point and instead highlight the reduced interactions:

“Further, MHV infection retains the association of MLEC with the OST complex while titrating off other interactors, potentially leading to more efficient glycoprotein biogenesis.”

I was also curious as to why non-structural proteins, nsp2 and nsp4, showed robust interactions with host proteins localized to both the ER and mitochondria? Do these proteins localize to different organelles or do these interactions reflect some other type of dysregulation? It would be useful to provide a bit of speculation on this point.

We also find these ER and mitochondrial protein interactions curious, which we initially reported on (Davies, Almasy et al. 2020 ACS Infectious Diseases). In this prior report, we found that when expressed in HEK293T cells, SARS-CoV-2 nsp2 and nsp4 have partial localization to mitochondrial-associated ER membranes (MAMs), as determined by subcellular fractionation. Given that malectin has also been shown to have MAMs localization (Carreras-Sureda, et al. 2019 Nature Cell Biology), we have added additional text in the Discussion to speculate on this point:

“Additionally, MLEC has also been shown to localize to ER-mitochondria contact sites (MAMs)(Carreras-Sureda et al., 2019), which regulate mitochondrial bioenergetics. We have previously shown that SARS-CoV-2 nsp2 and nsp4 can partially localize to MAMs(Davies et al., 2020), so these viral proteins may also dysregulate MLEC and MAMs activity to promote infection.”

Again, the overall identification of malectin as a pro-viral protein involved in the replication of multiple different coronaviruses is interesting and important, but additional insights into the mechanism of this activity would strengthen the overall impact of this work.

Thank you for this endorsement. We hope the additional analyses and discussion points in the revised manuscript further homed in on a direct mechanistic function for MLEC in modulating viral replication.

Reviewer #2 (Public Review):

Summary:

A strong case is presented to establish that the endoplasmic reticulum carbohydrate binding protein malectin is an important factor for coronavirus propagation. Malectin was identified as a coronavirus nsp2 protein interactor using quantitative proteomics and its importance in the viral life cycle was supported by using a functional genetic screen and viral assays. Malectin binds diglucosylated proteins, an early glycoform thought to transiently exist on nascent chains shortly after translation and translocation; yet a role for malectin has previously been proposed in later quality control decisions and degradation targeting. These two observations have been difficult to reconcile temporally. In agreement with results from the Locher lab, the malectininteractome shown here includes a number of subunits of the oligosaccharyltransferase complex (OST). These results place malectin in close proximity to both the co-translational (STT3A or OST-A) and post-translational (STT3B or OST-B) complexes. It follows that malectin knockdown was associated with coronavirus Spike protein hypoglycosylation.

Strengths:

Strengths include using multiple viruses to identify interactors of nsp2 and quantitative proteomics along with multiple viral assays to monitor the viral life cycle.

Weaknesses:

Malectin knockdown was shown to be associated with Spike protein hypoglycosylation. This was further supported by malectin interactions with the OSTs. However, no specific role of malectin in glycosylation was discussed or proposed.

We have emphasized our hypotheses on this point in the discussion and added a summary figure to highlight the specific role of malectin.

Given the likelihood that malectin plays a role in the glycosylation of heavily glycosylated proteins like Spike, it is unfortunate that only 5 glycosites on Spike were identified using the MS deamidation assay when Spike has a large number of glycans (~22 sites). The mass spec data set would also include endogenous proteins. Were any heavily glycosylated endogenous proteins hypoglycosylated in the MS analysis in Fig 5D?

Thank you for this suggestion. We compared the glycosylation patterns of endogenous proteins in MLEC-KD cells (Fig. S15E-G). We found that there is a small increase in abundance of glycopeptides associated with LAMP2, SERPHINH1, RDX, RPL3/5, CADM4, and ITGB1, however these fold changes are small and tested insignificant. These results indicate there is relatively little modification of endogenous glycoproteins upon MLEC-depletion. We added the following sections:

“We also probed the effect of MLEC KD on endogenous protein glycosylation. We find that there is only a small increase in abundance of glycopeptides, including those associated with the ribosome (Rpl3, Rpl5), a cytoskeletal protein (Rdx), the integrin Itgb1, and the ER-resident chaperone Serphinh1 (Fig. S15E-G).”

“Our proteomics data reveals that there is only a modest increase in the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response is relatively unchanged (Fig. S15D). In addition, there are only minor increases in endogenous glycopeptide levels (Fig. S15E-G). Together, these results indicate that while MLEC KD leads to some alterations in ER proteostasis and host glycosylation, these changes are modest and may not be the primary mechanism by which MLEC KD hinders CoV replication.”

The inclusion of the nsp4 interactome and its partial characterization is a distraction from the storyline that focuses on malectin and nsp2.

We believe the nsp4 comparative interactome and functional genomics data offers a rich resource for further functional investigation by others, if made public. While we found the malectin and nsp2 storyline the most compelling to pursue, we believe the inclusion of the nsp4 data strengthens the overall approach, in agreement with Reviewer #3’s comments.

Reviewer #3 (Public Review):

Summary:

In this study, Davies and Plate set out to discover conserved host interactors of coronavirus non-structural proteins (Nsp). They used 293T cells to ectopically express flag-tagged Nsp2 and Nsp4 from five human and mouse coronaviruses, including SARS-CoV-1 and 2, and analyzed their interaction with host proteins by affinity purification mass-spectrometry (AP-MS). To confirm whether such interactors play a role in coronavirus infection, the authors measured the effects of individual knockdowns on replication of murine hepatitis virus (MHV) in mouse Delayed Brain Tumor cells. Using this approach, they identified a previously undescribed interactor of Nsp2, Malectin (Mlec), which is involved in glycoprotein processing and shows a potent pro-viral function in both MHV and SARS-CoV-2. Although the authors were unable to confirm this interaction in MHVinfected cells, they show that infection remodels many other Mlec interactions, recruiting it to the ER complex that catalyzes protein glycosylation (OST). Mlec knockdown reduced viral RNA and protein levels during MHV infection, although such effects were not limited to specific viral proteins. However, knockdown reduced the levels of five viral glycopeptides that map to Spike protein, suggesting it may be affected by Mlec.

Strengths:

This is an elegant study that uses a state-of-the-art quantitative proteomic approach to identify host proteins that play critical roles in viral infection. Instead of focusing on a single protein from a single virus, it compares the interactomes of two viral proteins from five related viruses, generating a high confidence dataset. The functional follow-ups using multiple live and reporter viruses, including MHV and CoV2 variants, convincingly depict a pro-viral role for Mlec, a protein not previously implicated in coronavirus biology.

Weaknesses:

Although a commonly used approach, AP-MS of ectopically expressed viral proteins may not accurately capture infection-related interactions. The authors observed Mlec-Nsp2 interactions in transfected 293T cells (1C) but were unable to reproduce those in mouse cells infected with MHV (3C). EIF4E2/GIGYF2, two bonafide interactors of CoV2 Nsp2 from previous studies, are listed as depleted compared to negative controls (S1D). Most other CoV2 Nsp2 interactors are also depleted by the same analysis (S1D). Previously reported MERS Nsp2 interactors, including ASCC1 and TCF25, are also not detected (S1D). Furthermore, although GIGYF2 was not identified as an interactor of MHV Nsp2/4 in human cells (S1D), its knockdown in mouse cells reduced MHV titers about 1000 fold (S4). The authors should attempt to explain these discrepancies.

We acknowledge these limitations in AP-MS from ectopically expressed viral proteins and have addressed these discrepancies with further elaboration in the text:

“A limitation of our study is the initial overexpression of individual proteins for AP-MS, in which we find some variation between our data with other AP-MS studies. We sought to overcome these variations by focusing on conserved interactors and testing interactions in a live infection context.”

“We also found GIGYF2-KD strongly suppressed MHV infection, despite GIGYF2 not interacting with MHV nsp2 (Fig. S1D), highlighting the importance of proteostasis factors in infection regardless of direct PPIs.”

More importantly, the authors were unable to establish a direct link between Mlec and the biogenesis of any viral or host proteins, by mass-spectrometry or otherwise. Although it is clear that Mlec promotes coronavirus infection, the mechanism remains unclear. Its knockdown does not affect the proteome composition of uninfected cells (S15B), suggesting it is not required for proteome maintenance under normal conditions. The only viral glycopeptides detected during MHV infection originated from Spike (5D), although other viral proteins are also known to be glycosylated. Cells depleted for Mlec produce ~4-fold less Spike protein (4E) but no more than 2-fold less glycosylated spike peptides (5D), compounding the interpretation of Mlec effects on viral protein biogenesis. Furthermore, Spike is not essential for the pro-viral role of Mlec, given that Mlec knockdown reduces replication of SARS-CoV-2 replicons that express all viral proteins except for Spike (6A/B).

Thank you, these are all important points. We have acknowledged these compounding factors in the Discussion:

“Concurrently, knockdown of MLEC leads to impediment of nsp production and aberrant glycosylation of other viral proteins like Spike, though it should be noted that the decrease in Spike glycopeptides is compounded by the overall decrease in Spike protein. Given that MLEC is pro-viral in a SARS-CoV-2 replicon model lacking Spike (Fig. 6), MLEC can promote CoV replication independent of Spike production.”

Any of the observed effects on viral protein levels could be secondary to multiple other processes.Interventions that delay infection for any reason could lead to an imbalance of viral protein levels because Spike and other structural proteins are produced at a much higher rate than non-structural proteins due to the higher abundance of their cognate subgenomic RNAs. Similarly, the observation that Mlec depletion attenuates MHV-mediated changes to the host proteome (S15C/D) can also be attributed to indirect effects on viral replication, regardless of glycoprotein processing. In the discussion, the authors acknowledge that Mlec may indirectly affect infection through modulation of replication complex formation or ER stress, but do not offer any supporting evidence. Interestingly, plant homologs of Mlec are implicated in innate immunity, favoring a more global role for Mlec in mammalian coronavirus infections.

We examined the upregulation of protein markers of the UPR and other stress response pathways in cells depleted of MLEC (Fig. S15D). We find that the UPR pathways are moderately but insignificantly upregulated, while the Heat Shock Factor 1 (HSF1) pathway is moderately and significantly upregulated. The fold change increase of these marker proteins are relatively small, so while upregulation of this pathway may contribute to the suppression of CoV replication, it may not fully explain the phenotype. Please all see similar points brought up by reviewer 1 (comment 1). We added the following section to the manuscript text to discuss these results:

“In uninfected cells, MLEC KD leads to relatively little proteome-wide changes, with MLEC being the only protein significantly downregulated and no other proteins significantly upregulated, supporting the specificity of MLEC KD in MHV suppression (Fig.  S15C). To determine whether MLEC KD alters general host proteostasis, we further examined the levels of protein markers of stress pathways based on previous gene pathway definitions(Davies et al., 2023; Grandjean et al., 2019; Shoulders et al., 2013) (Fig. S15D). We find that there are modest but significant increases in protein levels associated with the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response (UPR) pathways are largely unmodified. 

“Our proteomics data reveals that there is only a modest increase in the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response is relatively unchanged (Fig. S15D). […] Together, these results indicate that while MLEC KD leads to some alterations in ER proteostasis and host glycosylation, these changes are modest and may not be the primary mechanism by which MLEC KD hinders CoV replication.”

Finally, the observation that both Nsp2 (3C) and Mlec (3E/F) are recruited to the OST complex during MHV infection neither support nor refute any of these alternate hypotheses, given that Mlec is known to interact with OST in uninfected cells and that Nsp2 may interact with OST as part of the full length unprocessed Orf1a, as it co-translationally translocates into the ER. Therefore, the main claims about the role of Mlec in coronavirus protein biogenesis are only partially supported.

We have acknowledged this point in the Discussion. 

“We find that nsp2 interacts with several OST complex members, including DDOST, STT3A, and RPN1, though whether this is as part of the uncleaved Orf1a polyprotein during co-translational ER translocation or as an individual protein is unclear.”

Reviewer #2 (Recommendations For The Authors):

What is the proof that MLEC is a type I membrane protein? If it is strictly sequence analysis, this conclusion should be tapered in the text.

Our response: We have added appropriate evidence on the biochemical characterization of MLEC topology from Galli et al., 2011, and cryo-EM structural characterization by Ramírez et al., 2019.

“As it was surprising that nsp2, a non-glycosylated, cytoplasmic protein, would interact with MLEC, an integral ER membrane protein with a short two amino acid cytoplasmic tail(Galli et al., 2011; Ramírez et al., 2019), we assessed a broader genetic interaction between nsp2 and MLEC.”

Validation of some of the nsp2 and malectin interactome components by pulldowns should be included.

Our response: The interactions between nsp2 and Ddost, Stt3A, and Rpn1 passed a stringent confidence filter in our AP-MS experiment (Fig. 3C) based on several replication. For this reason, we do not believe additional validation by Western blotting will offer much useful information.

NGI-1 inhibition of glycosylation looks to be very weak in Fig. 5B and Fig. S14B.

Our response: It is important to note that the NGI-1 inhibition assay used a suboptimal NGI-1 concentration to prevent full suppression of MHV infection, which we have found previously. We have added this justification in the Methods section and associated figure legend (Fig. S14A).

“The 5 uM NGI-1 dosage was chosen as it resulted in partial inhibition of glycosylation while not completely blocking MHV infection.”

“This dosage and timing were chosen to partially inhibit the OST complex without fully ablating viral infection, as NGI-1 has been shown previously to be a potent positive-sense RNA virus inhibitor(Puschnik et al., 2017)  (Fig. S14)”

Summary model figure at the end would help to communicate the conclusions.

Our response: Thank you for this suggestion. We agree and have added a summary model figure at the end as suggested.

Reviewer #3 (Public review):

Significance:

About 5% of metastatic castration-resistant prostate cancers (mCRPC) display genomic alterations in the transcriptional kinase CDK12. The mechanisms by which CDK12 alterations drive tumorigenesis in this molecularly-defined subset of mCRPC have remained elusive. In particular, some studies have suggested that CDK12 loss confers a homologous recombination deficiency (HRd) phenotype, However, clinical studies have not borne out the benefit to PARP inhibitors in patients with CDK12 alterations, despite the fact that these agents are typically active against tumors with HRd.

In this study, Frank et al. reconcile these findings by showing that: (1) tumors with biallelic CDK12 alterations do not have genomic features of HRd; (2) in vitro, HR gene downregulation occurs with acute depletion of CDK12 but is far less pronounced with chronic CDK12 loss; (3) CDK12-altered cells are uniquely sensitive to genetic or pharmacologic inhibition of CDK13.

Strengths:

Overall, this is an important study that reconciles disparate experimental and clinical observations. The genomic analyses are comprehensive and conducted with a high degree of rigor and represent an important resource to the community regarding the features of this molecular subtype of mCRPC.

Weaknesses:

(1) It is generally assumed that CDK12 alterations are inactivating, but it is noteworthy that homozygous deletions are comparatively uncommon (Figure 1a). Instead many tumors show missense mutations on either one or both alleles, and many of these mutations are outside of the kinase domain (Figure 1b). It remains possible that the CDK12 alterations that occur in some tumors may retain residual CDK12 function, or may confer some other neomorphic function, and therefore may not be accurately modeled by CDK12 knockout or knockdown in vitro. This would also reconcile the observation that knockout of CDK12 is cell-essential while the human genetic data suggest that CDK12 functions as a tumor suppressor gene.

(2) It is not entirely clear whether CDK12 altered tumors may require a co-occurring mutation to prevent loss of fitness, either in vitro or in vivo (e.g. perhaps one or more of the alterations that occur as a result of the TDP may mitigate against the essentiality of CDK12 loss).

Author response:

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

Reviewer #1 (Public Review):

Given that there are different mutations identified at different CDK12 sites as illustrated in Figure 1B it would be nice to know which ones have been functionally classified as pathogenic and for which ones that the pathogenicity has not been determined. This would be especially interesting to perform in light of the differences in the LOH scores and WES data presented - specifically, are the pathogenic mutations vs the mutations for which true pathogenicity is unknown more likely to display LOH or TD?

Alterations were classified as pathogenic when resulting in frameshift, nonsense, or cause an aminoacid change likely to alter function (according to ANNOVAR).  Four patients were called CDK12^BAL but were negative for TDP signatures. Three of these had CDK12 mutations downstream of the kinase domain, which may be less likely to ablate protein activity. Most functionally validated pathogenic mutations include disruption of the kinase domain (PMID: 25712099). We added a sentence to the Results section (under “Identification of genomic characteristics that associate with CDK12 loss in prostate cancer”) to highlight this caveat on pathogenic mutation calls.

For the cell inhibition studies with the CDK12/13 inhibitor, more details characterizing the specificity of this molecule to these targets would be useful. Additionally, could the authors perform short-term depletion studies with a PROTAC to the target or short shRNA or non-selected pool CRISPR deletion studies of CDK12 in these same cell lines to complement their pharmacological studies with genetic depletion studies? Also perhaps performing these same inhibitor studies in CDK12/13 deleted cells to test the specificity of the molecule would be useful.

We are not aware of a CDK12-specific PROTAC, and generate such as reagent is beyond the scope of the present study. Regarding the specificity of the CDK12/13 inhibitor molecules, additional information on the specificity and in vivo dose selection were added to the Results section (under “CDK13 is synthetic lethal in cells with biallelic CDK12 loss”). Cells with CDK12-KO did not tolerate CDK13-KO, so we were unable to generate double knockouts to test for CDK12/13 inhibitor non-specific effects. 

Additionally, expanding these studies to additional prostate cancer cell lines or organdies models would strengthen the conclusions being made. More information should be provided about the dose and schedule chosen and the rationale for choosing those doses and schedules for the in vivo studies proposed should be presented and discussed. Was there evidence for maximal evidence of inhibition of the target CDK12/13 at the dose tested given the very modest tumor growth inhibition noted in these studies.

With respect to additional acute CDK12 loss models, our Tet-inducible shCDK12 models show only minor growth slowdown and do not appear to phenocopy the strong arrest or apoptosis seen with CDK12 KO or inhibition, respectively. Future work is ongoing to generate CDK12-degron regulated cell lines. We added a new immunoblot panel showing that acute CRISPR/sgRNA targeting of CDK12 does indeed lead to BRCA2 and ATM protein decrease (Fig. S4g), providing some orthogonal genomic targeting evidence of the acute HR gene effect.  We are continuing efforts to collect and generate additional CDK12^BAL cell models, in both 2D and 3D culture systems, but none are presently available. We added a 3D culture drug dose curve with LuCaP189.4 exposed to THZ531 (Fig. S7m), which confirms heightened sensitivity vs two CDK12-intact lines. 

Regarding assessments of CDK12 targets; as we are not aware of any unique CDK12 substrates, it is fair to ask but difficult to measure precise CDK12 inhibition by the compounds in tumors. We dosed mice using the same protocol as detailed in the original report testing SR4835 in mice (PMID: 31668947). We performed immunoblots on lysates from 3 and 28 day treated PDX tumors and did not see any consistent decreases in pRBP1(Ser2) or ATM or increases in γH2A.X (data not shown). However, we did see increases in APA usage and downregulation of DNA repair transcripts with three day treatment (Fig. 6k-l), as would be expected from on target acute effects.

Reviewer #2 (Public review)

One caveat that continues to be unclear as presented, is the uncoupling of cell cycle/essentiality of CDK12/13 from HR-directed mechanisms. Is this purely a cell cycle arrest phenotype acutely with associated down-regulation of many genes?

In regard to untangling the effects of cell arrest on HR gene expression, this is a difficult question given that many HR genes, including BRCA2, are S/G2 linked. We attempted to account for those effects in the acute CDK12 inhibition experiment by including a palbociclib (CDK4/6i) control, which caused cell arrest and decreased BRCA1/2 RNA expression with no apparent 5/3’ transcript imbalance determined by qPCR (Fig. 4e,g). Though overall BRCA1 and BRCA2 mRNA levels are lower in the stable 22Rv1-CDK12-KO2 and KO5 lines, they do not show selective 3’ loss (Fig. 5c), suggesting the downregulation in these lines is mostly due to their slower growth (Fig. S4k) and not intronic polyA usage.

While the RAD51 loading ssRNA experiments are informative, the Tet-inducible knockdown of BRCA2 and CDK12 is confusing as presented in Figure 5, shBRCA2 + and -dox are clearly shown. However, were the CDK12_K02 and K05 also knocked down using inducible shRNA or a stable knockout? The importance of this statement is the difference between acute and chronic deletion of CDK12. Previously, the authors showed that acute knockdown of CDK12 led to an HR phenotype, but here it is unclear whether CDK12K02/05 are acute knockdowns of CDK12 or have been chronically adapted after single cell cloning from CRISPR-knockout. 

As a clarification, the 22Rv1-CDK12-KO2 and 22Rv1-CDK12-KO5 are stable CRISPR knockout clonal lines that were expanded from single cells. We added a new figure to include more validation of these lines (Fig. S5). We tried multiple times to reproduce the HRd phenotype and PARPi sensitivity with siRNA and inducible shRNA lines but were unable to see clear sensitivity differences, despite seeing the expected shifts with shBRCA2 controls (data not shown). It is possible the degree of knockdown (~80%), timing (8 days), or specific cell lines used in our experiments were not sufficient to expose the acute phenotype by this method.

However, we were able to see acute HR gene decreases by inhibitor treatment (Fig. 4) or acute CRISPR (Fig. S4g).

Given the multitude of lines, including some single-cell clones with growth inhibitory phenotypes and ex-vivo derived xenografts, the variability of effects with SR4835, ATM, ATR, and WEE1 inhibitors in different models can be confusing to follow. Overall, the authors suggest that the cell lines differ in therapeutic susceptibility as they may have alternate and diverse susceptibilities. It may be possible that the team could present this more succinctly and move extraneous data to the supplement.  

We appreciate the complexity of the data and attempted to use multiple models to report consistency and variability. We are not able to ascertain what data would be extraneous, and elected to present data we view as relevant in the main figures while moving supporting data in the supplement.

The in-vitro data suggests that SR4835 causes growth inhibition acutely in parental lines such as 22RV1. However, in vivo, tumor attenuation appears to be observed in both CDK12 intact and deficient xenografts, LuCAP136 and LuCaP 189.4 (albeit the latter is only nominally significant). Is there an effect of PARPi inhibition specifically in either model? What about the 22RV1-K02/05? Do these engraft? Given the role of CDK12/13 in RNAP II, these data might suggest that the window of susceptibility in CDK12 (mutant) tumors may not be that different from CDK12 intact tumors (or intact tissue) when using dual CDK12/13 inhibitors but rather represent more general canonical essential functions of CDK12 and CDK13 in transcription. From a therapeutic development strategy, the authors may want to comment in the discussion on the ability to target CDK13 specifically.

Though the response of the CDK12^BAL models to some compounds is variable, we believe those mixed results are important and future studies may be able to better explain why some show shifts in sensitivity while others do not. We hope future studies with additional models will help determine which sensitivities are more consistently true, and perhaps provide explanations for differences between models.

Regarding SR4835, we find, and others have reported, a toxic (i.e. apoptotic) effect for in vitro treatment with dual CDK12/13 inhibitors (Fig. 4f, S4e,f); in fact, that may be why previous studies have used short timepoints in cell culture assays with these dual inhibitors. In mice, SR4835 was tolerated well but only LuCaP 189.4 showed statistically significant decreases in tumor volume and weight (Fig. 6j). We did not test PARPi responses in the PDX models, nor did we attempt engrafting the 22Rv1-CDK12-KO cell lines, but both would be worthwhile experiments in the future. Beyond CDK12^BAL tumors, we agree that CDK12/13 inhibitors could be effective in cancer therapies more generally (e.g. triggering acute HRd, loss of RNAP2 phosphorylation). We added a line to the discussion section about ongoing efforts to combine PARPi and CDK12/13i, which we expect to be synergistic in CDK12-intact tumors due to the acute loss phenotype. We certainly agree that development of a specific CDK13 inhibitor would be the ideal therapeutic option for CDK12^BAL tumors. However, CDK12 and CDK13 are 43% conserved at the protein level (PMID: 26748711), with 92% conservation in the active site (PMID: 30319007), and there are no available pharmacologic inhibitors that discriminate between CDK12 and CDK13.

Reviewer #3 (Public review):

It is generally assumed that CDK12 alterations are inactivating, but it is noteworthy that homozygous deletions are comparatively uncommon (Figure 1a). Instead many tumors show missense mutations on either one or both alleles, and many of these mutations are outside of the kinase domain (Figure 1b). It remains possible that the CDK12 alterations that occur in some tumors may retain residual CDK12 function, or may confer some other neomorphic function, and therefore may not be accurately modeled by CDK12 knockout or knockdown in vitro. This would also reconcile the observation that knockout of CDK12 is cell-essential while the human genetic data suggest that CDK12 functions as a tumor suppressor gene.

Thank you for the feedback. It is a keen observation that homozygous deletions of CDK12 are not typical, though many mutations are upstream frameshifts that are expected to lead to loss of functional protein and mRNA via nonsense mediated decay. LuCaP189.4, our only natural mutant model, has two upstream frameshifts leading to complete protein loss (Fig 5b, S4h-i). We also added a caveat previously mentioned (in response to Reviewer 1) that mutations downstream of the kinase domain may be less likely to be fully pathogenic. For upstream missense mutations, the possibility of neuromorphic function remains an intriguing possibility that cannot be ruled out and would not be captured in our current models. Hopefully additional models can be developed, both natural and engineered, to help dissect that question in future studies.  

It is not entirely clear whether CDK12 altered tumors may require a co-occurring mutation to prevent loss of fitness, either in vitro or in vivo (e.g. perhaps one or more of the alterations that occur as a result of the TDP may mitigate against the essentiality of CDK12 loss).

We concur. Another caveat with the CRISPR models, beyond reliance on upstream frameshift mutations, is the simultaneous loss of alleles. In human tumors, there may be a period of single copy loss before the second hit that may provide a window for adaptation. It is possible that sequential loss is far easier for a cell to tolerate than acute bi-allelic inactivation. We agree that the question of what (if any) cooperating genetic alterations are required to tolerate CDK12 loss is an important one that we plant to further explore in future work.

Recommendations for Authors:

Reviewer #1 (Recommendations for Authors):

The authors have thoroughly addressed all issues of data availability, reagents, in vivo protocols, and animal approvals associated with the studies presented in this manuscript. Specific comments and experimental suggestions that in my opinion would strengthen the conclusions of this interesting and compelling manuscript are included above

Reviewer #2 (Recommendations for the authors):

The authors were thorough in their studies. As a general note, switching between the cell lines is often overwhelming in interpreting the data given cell-to-cell variability in response. If possible, consolidating the text/conclusions in results would improve the readability of the manuscript.

The variety of cell lines and models is perhaps expansive at times, but we hope the inclusion of these different models helps support the conclusions. 

Is it possible to knockout CDK12 acutely using a degron-based approach, instead of utilizing an inhibitor that targets both CDK12/13?

There is a HeLa cell line made with analog-sensitive CDK12 (Bartkowiak, Yan, and Greenleaf 2016) but we were unaware of any such prostate lines at the time of this work. We are attempting to develop engineered prostate lines with specific CDK12 degradation but do not yet have them available.

How do the authors address a lower BRCA1/2 level in for example 22RV1-K05, does this cell line have increased sensitivity to PARPi over its parental 22RV1 line? Could this be added to Figure 5h/i?

The lower BRCA2 levels in 22Rv1CDK12-KO5 is likely due to the slower growth rate (Fig. S4k), as BRCA2 expression is S/G2 linked. While the mRNA level of BRCA2 overall is lower in the KO5 line, we do not observe the 5’/3’ transcript imbalance (Fig. 5c). The 22Rv1-CDK12-KO lines did not show increased sensitivity to carboplatin, while inducible shBRCA2 did (Fig. S7a), so we do not believe this lower BRCA2 confers functional HRd. We did test the KO lines with olaparib (Fig. S7d) and saw a modest increased sensitivity compared to parental 22Rv1, but not to the extent measured in the BRCA1 mutant line UWB1.289.

What is the clonality of the LuCAP 189.4 lines upon derivation? Is biallelic CDK12 loss seen in all cells?

We do not know the exact clonality of the LuCAP 189.4 PDX or CL model, but we do see highly uniform CDK12 protein loss in these cells (quantified by IHC staining, data not shown).

The authors state that 22RV1-K02/05 has an increased growth arrest to CDK13 inhibition. However, in Figure 6h, it appears the viability is not significantly different compared to the parental 22RV1 line. Similar aspects noted in 189.4-vec/CDK12?

We found that 22Rv1 KO2/KO5 have growth arrest with sgCDK13 and cell death with CDK12/13 inhibitor. We did notice that SR4835 did not show the differential effects we anticipated (Fig. 6h), as was seen with THZ531 (Fig. 6i). SR4835 is a non-covalent inhibitor, while THZ531 is a covalent binder, so there are some functional differences between these compounds that might explain the lack of differential effects in the isogenic lines in a 4 day in vitro assay. We included the SR4835 in vitro data because it was used for the in vivo experiment. THZ531 is not suited for animal use.

Could the authors comment on SR4835 response in vivo as a function of tumor growth rate?

The in vivo SR4835 treated LuCaP189.4 did show signs of reduced proliferation with decreased Cell Cycle and DNA Replication in the RNA-seq signatures, but a more detailed investigation into cell cycle arrest vs apoptotic response has yet to be fully explored. We plan to conduct additional PDX experiments if we can obtain a selective CDK13 inhibitor. 

Do the authors explore TDPs in their isogenic cell lines?

We performed low coverage WGS on the 22Rv1 KO clones and added that to the paper (Fig. S5c). We did not see any obvious signs of TDP. We suspect the phenotype takes longer to accumulate and is not apparent within the ~20 passages our clones underwent in culture. This would be consistent with the tumor analysis (Fig. 2b) showing increase in TDs from primary to metastatic tumors, suggesting TDs accumulate over time.

A future study may allow for screening synthetic lethals in the context of CDK12 loss in the presence or absence of SR4835 inhibition.

We are actively pursuing experiments to identify new synthetic lethal targets by CRISPR and drug screens in CDK12 loss models and hope to report those in a future study.

Reviewer #3 (Recommendations for the authors):

As discussed above, the authors may wish to adjust their terminology to "CDK12-altered" rather than "CDK12 lost" or "CDK12-inactivated" to leave open the possibility that some tumors may retain residual CDK12 function or adopt neomorphic functions.

Thank you for the additional comments and feedback. The possibility of neomorphic CDK12 allele function is important. As our models were all complete protein loss mutations, we decided to retain “biallelic loss” as our preferred nomenclature, but the note is well taken.

The plots in Figures 1f-h are interesting and suggest that certain cancer genes (especially oncogenes) are recurrently gained in CDK12-altered tumors. It may be interesting to look at this on the individual level rather than the cohort level to see whether any groups of oncogenes tend to be gained together in an individual patient - this could inform whether certain combinations of cancer drivers cooperate with CDK12 alteration to drive oncogenesis.

Thank you for the idea of looking at the patient-level for TDP-enriched oncogenes. A preliminary assessment did not identify recurrent co-gained oncogenes. We will continue these analyses as additional patient tumors with CDK12 alterations are identified. 

The finding that AR gene or enhancer are recurrently gained with TDP is interesting and I am curious whether the authors have thoughts on whether these alterations can also be seen in the 1-2% of CDK12altered primary prostate cancers that are treatment naïve, and where AR pathway alterations are not as frequently seen.

We did not focus on CDK12 altered primary prostate cancers, but we did check if there is AR amplification enrichment in the 6 CDK12^BAL cases of the TCGA-PRAD dataset and did not identify enrichment. However, with such small numbers we would hesitate to draw any hard conclusions. 

It could be interesting to more comprehensively characterize some of the CDK12 KO-adapted lines in Figure 5 (e.g. by WES or WGS) to determine whether they exhibit the TDP and/or whether they have acquired any secondary mutations that allow them to adapt to CDK12 loss.

We are planning to do further genomics characterization of the CDK12-KO lines and will hopefully include that in a future study. Genomic analyses of the 22Rv1 clones (see copy number plots in Fig. S5c) did not identify a TDP. We plan to repeat the genomic assessments over additional cell passages and we have planned additional experiments designed to understand why some cells tolerate CDK12 loss and others do not.

Je propose : "Contacts épistolaires ou oraux avec le consortium et ses membres, participation à l'école d'été, émergence d'un projet numérique au détor d'une conversation, sont autant de motivations pouvant conduire à la demande d'un accompagnement numérique."

Je déplacerais ce paragraphe plus haut, l'insérant dans la phrase qui commence par "Elles offrent ..." Je propose : "Elles aident les chercheurs à acquerir des données ....., mais elles offrent aussi des espaces de travail et de rencontre, ...

quelles sont ces bases de référence et quels référentiels ? @mariebizais qu'entends-tu par ça ?

Author response:

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

Reviewer #1 (Public review):

Hurtado et al. show that Sox9 is essential for retinal integrity, and its null mutation causes the loss of the outer nuclear layer (ONL). The authors then show that this absence of the ONL is due to apoptosis of photoreceptors and a reduction in the numbers of other retinal cell types such as ganglion cells, amacrine cells, and horizontal cells. They also describe that Müller Glia undergoes reactive gliosis by upregulating the Glial Fibrillary Acidic Protein. The authors then show that Sox9+ progenitors proliferate and differentiate to generate the corneal cells through Sox9 lineage-tracing experiments. They validate Sox9 expression and characterize its dynamics in limbal stem cells using an existing single-cell RNA sequencing dataset. Finally, the authors argue that Sox9 deletion causes progenitor cells to lose their clonogenic capacity by comparing the sizes of control and Sox9-null clones. Overall, Hurtado et al. underline the importance of Sox9 function in retinal and corneal cells.

Strengths:

The authors have characterized a myriad of striking phenotypes due to Sox9 deletion in the retina and limbal stem cells which will serve as a basis for future studies.

Weaknesses:

Hurtado et al. investigate the importance of Sox9 in the retina and limbal stem cells. However, the overall experimental narrative appears dispersed.

(1) The authors begin by characterizing the phenotype of Sox9 deletion in the retina and show that the absence of the ON layer is due to photoreceptor apoptosis and a reduction in other retinal cell types. The authors also note that Müller glia undergoes gliosis in the Sox9 deletion condition. These striking observations are never investigated further, and instead, the authors switch to lineage-tracing experiments in the limbus that seem disconnected from the first three figures of the paper. Another example of this disconnect is the comparison of Sox9 high and Sox9 low populations using an existing scRNA-seq dataset and the subsequent GO term analysis, which does not directly tie in with the lineage-tracing data of the succeeding Sox9∆/∆ experiments.

We thank the reviewer for their thoughtful observations. We would like to clarify the rationale behind the structure of our study and how the different parts are conceptually connected.

Our central aim was to investigate the role of Sox9 in the adult eye. Given that Sox9 has been extensively studied during embryonic development, we specifically chose to use an inducible conditional knockout strategy (CAG-CreERTM) in order to assess its function postnatally, in the adult eye. This approach revealed a severe retinal phenotype, whereas the cornea showed no overt phenotype. A major strength of our experimental design is that it allowed us to examine the role of Sox9 specifically in the adult eye, avoiding confounding effects from embryonic development. Nevertheless, this approach entails an inherent limitation: the mosaic nature of the CAG-CreERTM system leads to substantial variability in both the extent and distribution of Sox9 inactivation among individual animals. We invested considerable effort over extended periods to obtain reliable and biologically meaningful data despite this variability. We did not proceed further because this mosaicism poses a significant limitation when attempting to dissect downstream mechanisms in a consistent and reproducible manner, making it extremely challenging to perform in-depth mechanistic studies.

Regarding the cornea, given the absence of a clear phenotype upon Sox9 deletion, we expanded our investigation by adding lineage-tracing and transcriptomic analyses to better understand Sox9’s potential role in adult limbal epithelial stem cells. These additional experiments provided valuable insight into Sox9 function in the adult cornea, even in the absence of gross morphological changes. Thus, while the retinal and corneal data stem from different experimental approaches, they are unified by a shared goal: understanding the celltype-specific and tissue-specific functions of Sox9 in the adult eye.

To ensure that other readers do not perceive this apparent disconnect, and overstate our conclusions, we have modified the manuscript.  In the Introduction section, we have included the main findings from studies conducted to date on the role of Sox9 in the cornea and retina, and we have removed the corresponding section from the Discussion. We believe it is now clear that our study focuses on the role of Sox9 in the adult eye, in contrast to previous studies, which focused on the developing eye.

In the Discussion section, we have added a new paragraph at the beginning and end that explicitly addresses the relationship between the retinal and limbal findings, illustrating how a single transcription factor can play distinct roles in different tissues within the same organ.

Regarding the reviewer’s comment that the scRNA-seq analyses appear disconnected from the lineage-tracing data, we respectfully disagree. These analyses provide independent transcriptional confirmation that Sox9 is a marker of limbal stem cells, reinforcing the conclusions drawn from our in vivo experiments. These approaches are complementary and they converge on the same biological insight: Sox9 marks a population with stem-like properties in the adult limbus. Nevertheless, we acknowledge the reviewer’s concern and have moderated the tone of our statements in the revised version of the manuscript to better reflect the supporting nature of the scRNA-seq data, without overstating its functional implications.

(2) A major concern is that a single Sox9∆/∆ limbal clone has a sufficiently large size, comparable to wild-type clones, as seen in Figure 6D. This singular result is contrary to their conclusion, which states that Sox9-deficient stem cells minimally contribute to the maintenance of the cornea.

We thank the reviewer for this important observation.

Ligand-independent activity of Cre-ER fusion proteins has been repeatedly reported in various mouse models (Vooijs et al., 2001; Kemp et al., 2004; Haldar et al., 2009). This basal recombinase activity is thought to arise from inappropriate nuclear translocation or proteolysis of the Cre-ER fusion protein, leading to low-level recombination even in the absence of tamoxifen. Consistent with this, prior studies using the same CAGG-CreERTM; R26R-LacZ system for clonal analysis in the cornea have observed sparse reporter expression before tamoxifen administration (Dorà et al., 2015).

In line with these findings, we also detected minimal background LacZ staining in Sox9Δ/ΔLacZ corneas (mean surface area: 0.85%; n = 8 eyes). This low-level staining likely reflects recombination events in transient amplifying or more differentiated cells, which are not expected to generate long-lived clones. However, in the rare instance of a large clone, as shown in Figure 6D, we believe that a spontaneous recombination event may have occurred in a bona fide limbal stem cell, giving rise to a sustained contribution. To rigorously address this potential artefact and assess the true contribution of Sox9-deficient stem cells, we conducted a comparative analysis of 8 control (Sox9Δ/+-LacZ) and 5 mutant (Sox9Δ/ΔLacZ) corneas. This analysis revealed a highly significant 8-fold reduction in the LacZpositive surface area in mutant samples (Sox9Δ/+-LacZ: 6.65 ± 1.77%; Sox9Δ/Δ-LacZ: 0.85 ± 0.85%; paired t-test, p = 0.00017; Figs. 6E and F; Table S12).

We chose to include the image of the large clone in the main figure precisely because it does not align with our working hypothesis. We believe that showing such exceptions transparently is scientifically important and may be valuable for other researchers using similar inducible systems. Nonetheless, based on previous literature, the number of samples analyzed, and the statistically significant reduction in clonal contribution, we maintain that the observed phenotype reflects a true biological effect of Sox9 loss, supporting our conclusion that Sox9-deficient stem cells contribute minimally to corneal maintenance. To make that point clearer, we have introduced the following sentence in lines 462-464 of the revised version of the manuscript.

“A possible explanation for this clone may be that spontaneous ligand-independent activity of Cre-ER fusion may have occurred in a bona fide limbal stem cell, as previously reported (Vooijs et al., 2001; Kemp et al., 2004; Haldar et al., 2009, Dorà et al., 2015).”

Reviewer #2(Public revciew):

Sox9 is a transcription factor crucial for development and tissue homeostasis, and its expression continues in various adult eye cell types, including retinal pigmented epithelium cells, Müller glial cells, and limbal and corneal basal epithelia. To investigate its functional roles in the adult eye, this study employed inducible mouse mutagenesis. Adult-specific Sox9 depletion led to severe retinal degeneration, including the loss of Müller glial cells and photoreceptors. Further, lineage tracing revealed that Sox9 is expressed in a basal limbal stem cell population that supports stem cell maintenance and homeostasis. Mosaic analysis confirmed that Sox9 is essential for the differentiation of limbal stem cells. Overall, the study highlights that Sox9 is critical for both retinal integrity and the differentiation of limbal stem cells in the adult mouse eye.

Strengths:

In general, inducible genetic approaches in the adult mouse nervous system are rare and difficult to carry out. Here, the authors employ tamoxifen-inducible mouse mutagenesis to uncover the functional roles of Sox9 in the adult mouse eye.

Careful analysis suggests that two degeneration phenotypes (mild and severe) are detected in the adult mouse eye upon tamoxifen-dependent Sox9 depletion. Phenotype severity nicely correlates with the efficiency of Cre-mediated Sox9 depletion.

Molecular marker analysis provides strong evidence of Mueller cell loss and photoreceptor degeneration.

A clever genetic tracing strategy uncovers a critical role for Sox9 in limbal stem cell differentiation.

Weaknesses:

(1) The Introduction can be improved by explaining clearly what was previously known about Sox9 in the eye. A lot of this info is mentioned in a single, 3-page long paragraph in the Discussion. However, the current study's significance and novelty would become clearer if the authors articulated in more detail in the Introduction what was already known about Sox9 in retina cell types (in vitro and in vivo).

We appreciate this insightful comment. Following the reviewer`s suggestion, we have reorganized the manuscript to provide a clearer scientific context in the Introduction. Specifically, we have moved the relevant background information on Sox9 in different retinal cell types—previously included in a single, extended paragraph in the Discussion—into the Introduction. This helps to better frame our study within the context of existing knowledge.

Additionally, we have emphasized more explicitly that our work does not focus on embryonic development, as most previous studies on Sox9 have done, but instead investigates its role in the adult mouse retina and limbus/cornea. We believe this represents an important and novel aspect of our study, as the mechanisms of retinal maintenance and limbal stem cell differentiation in the adult have been less extensively studied.

(2) Because a ubiquitous tamoxifen-inducible CreER line is employed, non-cell autonomous mechanisms possibly contribute to the observed retina degeneration. There is precedence for this in the literature. For example, RPE-specific ablation of Otx2 results in photoreceptor degeneration (PMID: 23761884). Have the authors considered the possibility of non-cell autonomous effects upon ubiquitous Sox9 deletion?

Given the similar phenotypes between animals lacking Otx2 and Sox9 in specific cell types of the eye, the authors are encouraged to evaluate Otx2 expression in the tamoxifen-induced Sox9 adult retina.

We appreciate the insightful comment of the reviewer regarding the potential contribution of non-cell autonomous mechanisms to the retinal degeneration observed upon ubiquitous Sox9 deletion. We agree that this is an important consideration, particularly in the context of findings showing that RPE-specific deletion of Otx2 results in secondary photoreceptor degeneration.

However, we would like to emphasize that RPE-specific deletion of Sox9 does not lead to photoreceptor loss or retinal degeneration, as previously shown (Masuda et al., 2014; Goto et al., 2018; Cohen-Tayar et al., 2018) [PMID: 24634209; PMID: 29609731; PMID: 29986868]. In addition, it was shown that Sox9 deletion in the RPE caused downregulation of visual cycle genes but did not compromise photoreceptor integrity or survival. Interestingly, Otx2 expression was found to be upregulated in the absence of Sox9, further supporting the view that Sox9 is not a simple upstream regulator of Otx2 in the adult RPE (Matsuda, 2014). These findings suggest that RPE dysfunction alone cannot account for the severe retinal phenotype we observe in our model.

In our study, we observed that photoreceptor degeneration correlates strongly with the depletion of Sox9 Müller glial cells. Given the well-established supportive and neuroprotective roles of Müller glia, we interpret the retinal degeneration in our model to be primarily a consequence of Müller cell dysfunction (confirmed by the loss of Müller glia markers, such as SOX8 and S100). This interpretation is further supported by previous studies showing that selective ablation of Müller glia can lead to photoreceptor degeneration through cell-autonomous mechanisms (Shen et al., 2012) [PMID: 23136411].

Nevertheless, we agree that this possibility deserves further investigation, and we have acknowledged it in the following paragraph that has been added to the Discussion section (lines 511-523 of the revised ms):

“An important consideration in our model is the potential contribution of non-cell autonomous mechanisms to photoreceptor degeneration. Sox9 is expressed in both MG and RPE cells, and both cell types are known to support photoreceptor viability (Poché et al., 2008; Masuda et al., 2014). Notably, Sox9 and Otx2 cooperate to regulate visual cycle gene expression in the RPE (Masuda et al., 2014), and loss of Otx2 specifically in the adult RPE leads to secondary photoreceptor degeneration through non-cell autonomous mechanisms (Housset et al., 2013). However, RPE-specific deletion of Sox9 does not induce retinal degeneration and in fact results in Otx2 upregulation (Masuda et al., 2014; Goto et al., 2018; Cohen-Tayar et al., 2018), suggesting that Sox9 is not an upstream regulator of Otx2 in this context. Further investigation into the molecular and cellular interactions between MG, RPE, and photoreceptors may help to clarify the indirect pathways contributing to degeneration in the absence of Sox9.”

Consistent with the above, a new citation has been included:

Housset M, Samuel A, Ettaiche M, Bemelmans A, Béby F, Billon N, Lamonerie T. 2013. Loss of Otx2 in the adult retina disrupts retinal pigment epithelium function, causing photoreceptor degeneration. J Neurosci 33:9890–904. doi:10.1523/JNEUROSCI.1099-13.2013.

(3) The most parsimonious explanation for the dual role of Sox9 in retinal cell types and limbal stem cells is that the cell context is different. For example, Sox9 may cooperate with TF1 in photoreceptors, TF2, in Mueller cells, and TF3 in limbal stem cells, and such cell typespecific cooperation may result in different outcomes (retinal integrity, stem cell differentiation). The authors are encouraged to add a paragraph to the discussion and share their thoughts on the dual role of Sox9.

We thank the reviewer for this thoughtful and constructive suggestion. In , we have added a paragraph at the end of the Discussion addressing the potential dual role of Sox9 in the cornea and retina. In this new section, we discuss how Sox9 might exert distinct functions depending on the cellular context, possibly through interactions with different transcriptional partners in specific cell types. This may help explain the contrasting roles of Sox9 in maintaining retinal integrity versus regulating stem cell differentiation in the limbal epithelium.

(4) One more molecular marker for Mueller glial cells would strengthen the conclusion that these cells are lost upon Sox9 deletion.

We thank the reviewer for this constructive suggestion. To reinforce our conclusion that most Müller glial cells are lost following Sox9 deletion, we analysed the expression of S100, a well-established cytoplasmic marker of Müller glia. As S100 is primarily localized to the innermost Müller cell processes and not restricted to cell bodies, direct cell counting was not feasible. Instead, we quantified the S100+ signal intensity across defined retinal surface areas. This analysis revealed a statistically significant reduction in S100 signal in Sox9^Δ/Δ retinas compared to controls. These new data, included in the revised Figure 1 (panels F and G), support and extend our previous observations using SOX8, further confirming the loss of Müller glial cells in Sox9-deficient retinas.

We have also modified the manuscript based on this new evidences as follows:

In the Results section, lines 168-177 of the revised ms, we have added the following paragraph: “To independently validate the loss of MG cells in Sox9-deficient retinas, we examined the expression of S100, a cytoplasmic marker that labels the processes of adult Müller cells. In control retinas, strong S100 immunoreactivity was observed across the inner retina, outlining the typical radial projections of Müller glia (Fig. 1F). In contrast, Sox9Δ/Δ retinas with an extreme phenotype exhibited a marked reduction in S100 signal (Fig. 1G). Given the diffuse cytoplasmic localization of S100, we quantified its expression by measuring the fluorescence signal within a defined surface area of the retina. This analysis revealed a statistically significant reduction in S100 signal intensity in mutant samples (including both mild and extreme phenotypes) compared to controls (Fig. 1G; Table S4), further supporting the loss of MG cells upon Sox9 deletion.”

In Methods, line 684 of the revised ms, the anti-S100 antibody reference and its working dilution have been added.

(5) Using opsins as markers, the authors conclude that the photoreceptors are lost upon Sox9 deletion. However, an alternate possibility is that the photoreceptors are still present and that Sox9 is required for the transcription of opsin genes. In that case, Sox9 (like Otx2) may act as a terminal selector in photoreceptor cells. This point is particularly important because vertebrate terminal selectors (e.g., Nurr1, Otx2, Brn3a) initially affect neuron type identity and eventually lead to cell loss.

We perfectly understand the reviewer’s point. However, we believe that the possibility that Sox9 regulates opsin gene expression without affecting photoreceptor survival is very unlikely in our model. The primary evidence comes from the histological analysis shown in Figure 1B, where hematoxylin and eosin staining clearly demonstrates the complete loss of the ONL in Sox9^Δ/Δ retinas exhibiting the extreme phenotype. Similarly, DAPI counterstain also evidences the lack of the ONL in many of our immunofluorescence images of these samples.  This morphological disappearance of the ONL strongly supports the conclusion that photoreceptor cells are not merely transcriptionally silent but are physically absent.

Furthermore, TUNEL assays in two retinas with a mild phenotype revealed extensive apoptosis within the ONL, suggesting a progressive degeneration process rather than a selective transcriptional effect. While we acknowledge that transcriptional regulation of opsin genes by Sox9 cannot be entirely ruled out, the observed phenotype is more consistent with a structural loss of photoreceptors rather than a change in their molecular identity alone. Therefore, our data support the interpretation that Sox9 is required for photoreceptor survival, likely through non-cell autonomous mechanisms related to Müller glia dysfunction, rather than acting as a terminal selector within photoreceptor cells themselves.

(6) Quantification is needed for the TUNEL and GFAP analysis in Figure 3.

We have quantified the GFAP immunofluorescence signal across defined surface areas of the retina and found a statistically significant increase in GFAP expression in Sox9^Δ/Δ mutants compared to controls (Mann-Whitney U test, P = 0.0240; n = 4 controls, 10 mutants). These quantification data are now included in the revised Figure 3.

Regarding the TUNEL assay, although extensive apoptosis was clearly observed in two Sox9<^Δ/Δ retinas with a mild phenotype (as shown in Figure 3A), this pattern was not consistent across the full study mouse cohort. Out of 15 mutant samples analyzed (5 of them previously analyzed and 10 additional ones that have been newly analyzed), only two exhibited this pronounced apoptotic pattern. However, in the remaining 13 mutants, we did observe a small but statistically significant increase in the number of TUNEL+ cells compared to controls (zero-inflated Poisson test, P = 0.028, n = 5 controls, 13 mutants). These results are now included in Figure 3 and in Tables S7 and S8.

This pattern likely reflects the transient nature of apoptosis in the degenerative process, which may occur rapidly and thus be difficult to capture consistently at a single time point. Nevertheless, the quantification supports our conclusion that Sox9 loss is associated with increased photoreceptor cell death.

Based on the above, we have included the following paragraphs in the Results section of the manuscript:

In lines 224-252 of the revised ms, the final version of the paragraph is as follows: “Since photoreceptors are absent in severely affected Sox9-mutant retinas, we conducted TUNEL assays to study the role of cell death in the process of retinal degeneration. In control samples (n=5), almost no TUNEL signal was observed in the retina. In contrast, Sox9^Δ/Δ mice (n=15) showed numerous TUNEL+ cells, mainly located in the persisting ONL, indicating that photoreceptor cells were dying (Fig. 3A). Although extensive TUNEL staining in the ONL was clearly observed in two Sox9^Δ/Δ retinas with mild phenotypes, this pattern was not consistently present across the full cohort. In the remaining 13 mutant retinas, we observed a modest but noticeable increase in the number of apoptotic cells compared to controls (Fig. 3B; Table S7). Despite a high frequency of zero counts (particularly among controls), the difference between groups reached statistical significance when analyzed using a zeroinflated Poisson model (P = 0.028; n = 5 controls, 13 mutants). These findings suggest that photoreceptor apoptosis following Sox9 deletion may occur acutely and within a narrow temporal window, making it challenging to capture the full degenerative process at a single time point”.

Lines 263-269 of the revised ms: “To support these observations quantitatively, we measured GFAP fluorescence intensity across defined retinal surface areas in control and Sox9^Δ/Δ mice (Fig. 3D; Table S8). This analysis revealed a statistically significant increase in GFAP signal in mutant retinas compared to controls (Mann-Whitney U test, P = 0.0240; n = 4 controls, 10 mutants). These results are consistent with a progressive gliotic  following Sox9 deletion and provide further evidence that MG cells become reactive in the absence of Sox9”.

Similarly, the section “Estimation of the percentage of tamoxifen-induced, Cre-mediated recombination” has been expanded as follows:

Lines 660-665 of the revised ms: “In parallel, to quantify GFAP expression as a measure of MG reactivity, we analyzed GFAP immunofluorescence intensity across defined retinal surface areas. Given the cytoplasmic distribution of GFAP within glial processes, direct cell counting was not feasible. Instead, fluorescence intensity was measured using ImageJ, within full-thickness retinal regions in 20x microphotographs of a retinal sections stained for GAFP. The total GFAP signal was normalized to the measured area for each section”.

(7) Line 269-320: The authors examined available scRNA-Seq data on adult retina. This data provides evidence for Sox9 expression in distinct cell types. However, the dataset does not inform about the functional role of Sox9 because Sox9 mutant cells were not analyzed with RNA-Seq. Hence, all the data that claim that this experiment provides insights into possible Sox9 functional roles must be removed. This includes panels F, G, and H in Figure 5. In general, this section of the paper (Lines 269-320) needs a major revision. Similarly, lines 442-454 in the Discussion should be removed.

We thank the reviewer for this important observation. We agree that the scRNA-Seq dataset used in this section does not include Sox9 mutant cells and therefore does not allow us to assess the consequences of Sox9 loss-of-function. However, we believe that this analysis still provides valuable complementary information. Specifically, it confirms that Sox9 is expressed in a distinct population of limbal stem cells, and that its expression dynamically changes along differentiation trajectories. Although we do not infer causality or phenotypic consequences, the ability to observe how gene expression programs shift as Sox9 is downregulated offers insights into potential transcriptional programs associated with Sox9 activity.

We have carefully revised Lines 269–320 to remove any overinterpretations, and eliminated the corresponding lines in the Discussion (Lines 442–454). However, we have retained Panels G, and H in Figure 5 with updated text that reflect the descriptive nature of these findings, specifically to illustrate that the Sox9-positive cell signature is consistent with a stem cell genetic program, and that when Sox9 is downregulated some gene pathways involved in stem cell differentiation are upregulated.

Reviewer #1 (Recommendations for the authors):

Major points

(1) Figure 1C shows the proportions of Sox9+cells that express Sox8 in control, mild and extreme phenotypes. However, as no quantitative classification of mild and extreme phenotypes is reported along with Figure 1A, the large standard deviation for Sox9∆/∆ mild retina might be due to a misclassification of the sample. Therefore, the authors must ascribe each sample to "mild" or "extreme" based on a quantitative metric.

We appreciate the reviewer’s suggestion to clarify the classification criteria used to distinguish “mild” and “extreme” phenotypes in Sox9^Δ/Δ retinas. As noted, our classification was based on a qualitative, phenotypic assessment of retinal morphology in hematoxylin/eosin-stained sections. Specifically, retinas were classified as “extreme” when the outer nuclear layer (ONL) was completely absent, and as “mild” when the ONL was present, although often reduced in thickness. This classification reflects the observable structural depletion of the ONL and aligns well with the extent of Sox9 loss in Müller glial cells, as shown in Figure 1. We acknowledge that some variability exists within the “mild” group, likely due to differences in recombination efficiency and the mosaic nature of tamoxifen-induced deletion.

The phenotypic classification of each individual sample is explicitly provided in Supplementary Table S1. We have also added a statement in the Results section clarifying that this classification was based on qualitative histological criteria rather than a numerical threshold.

Lines 104-113 of the revised ms: “We categorized Sox9^Δ/Δ retinas into “mild” and “extreme” phenotypes in order to facilitate interpretation of our data. Clasification was based on a qualitative assessment of ONL integrity in histological sections. Specifically, samples were classified as “extreme” when the ONL was completely depleted, and as “mild” when the ONL persisted, albeit variably reduced in thickness. This phenotypic classification reflects observable structural differences rather than a fixed quantitative threshold. Some variability exists within the “mild” group, likely due to differences in recombination efficiency and the mosaic nature of tamoxifen-induced Cre-mediated Sox9 deletion”

(2) The authors infer Sox9 high and Sox9 low groups of limbal stem cells using an existing scRNA-seq dataset. However, an immunohistology-based validation of this difference is missing. Given that limbal stem cells express Sox9, the authors must examine the heterogeneity in Sox9 levels within the Sox8+ population to demonstrate their claim: "...Sox9 expression decreases as transiently amplifying progenitors undergo progressive differentiation from limbal to peripheral corneal cells." in Line 292. Ideally, this must be further validated using differentiation markers corresponding to CB and ILB populations that show lower Sox9 expression according to the pseudotime graph.

To validate the Sox9 expression results obtained with scRNA-seq, we performed double immunofluorescence for Sox9 and P63, the latter expressed by the basal cells of the limbal epithelium, but not by transient amplifying cells covering the corneal surface (Pellegrini et al., 2001, https://www.pnas.org/doi/abs/10.1073/ pnas.061032098). These results can be observed in the new panel 5F. Accordingly we have included a new paragraph in lines 369-396 of the revised version of the ms:

“To validate these results, we decided to closely examine Sox9 expression in the limbus using immunofluorescence. Previous analyses revealed that the outer limbus is approximately 100 μm wide, while the inner limbus is wider, around 240 μm (Altshuler 2021). We observed that in the region corresponding to the OLB, most cells showed strong Sox9 expression. In the area corresponding to the ILB, this immunoreactivity appeared weaker in the basal layer (corresponding to the ILB proper), and no expression was detected in the suprabasal layers (flattened cells; Fig 5F left). Double immunofluorescence for SOX9 and P63, which is expressed in basal cells of the limbal epithelium, but not by transient amplifying cells covering the corneal surface (Pellegrini et al., 2001) revealed that Sox9 expression was restricted to P63-positive cells (Fig 5F right). These observations confirm that Sox9 is expressed in a basal cell population within both the OLB and ILB, and that its expression decreases in differentiated transient amplifying cells. ”

We also have deleted  “This expression pattern is consistent with our immunofluorescence observations" from line 356 of the revised ms.

(3) The authors' claim of "...Sox9-null cells cannot survive or proliferate as well as their wildtype neighbors, and are hence outcompeted over time, leading to an essentially wild-type cornea" does not seem very convincing in the light of Fig.6D and S3B where Sox9 deletion can still allow for a large LacZ+ clone. Their claim of wild-type cornea due to out-competing neighbors must be validated by increasing the number of Sox9-null progenitors, which can be tested by administering tamoxifen for a significantly longer duration, leading to a majority Sox9 deficient progenitor population, and then examining limbal and corneal defects.

As previously discussed, we observed only one instance of a large LacZ+ clone across 8 Sox9^Δ/Δ-LacZ eyes. Based on prior reports of ligand-independent Cre activity (Vooijs et al., 2001; Kemp et al., 2004; Haldar et al., 2009; Dorà et al., 2015), we believe this rare event likely resulted from spontaneous recombination in a bona fide limbal stem cell, independent of tamoxifen administration. For this reason, we do not expect that increasing the dose or duration of tamoxifen would eliminate such rare events. Furthermore, due to the mosaic and highly variable recombination efficiency of the CAGG-CreERTM system in the adult eye (see McMahon et al., 2008), attempting to increase the TX dosage would likely lead to systemic toxicity or lethality, without guaranteeing full inactivation of the gene in the limbus. Thus, this system is not well-suited for generating a fully Sox9-deficient limbal epithelium. To overcome this limitation, we crossed our mice with the R26R-LacZ reporter line to track the clonal behavior of Sox9-deficient cells. In control animals (Sox9Δ/+-LacZ), LacZ+ stripes originating from limbal stem cells are readily observed. In contrast, in Sox9Δ/Δ-LacZ mutants, these clones are either absent or drastically reduced. This suggests that Sox9-null cells have a severely impaired ability to form and sustain clones. To rigorously quantify this effect, we compared 8 control and 5 mutant corneas, revealing a highly significant 8-fold reduction in LacZ-positive area in the mutants (6.65 ± 1.77% vs. 0.85 ± 0.85%; p = 0.00017; Fig. 6F; Table S12; Supp. Fig. X???), supporting our claim that Sox9null cells cannot survive or proliferate as well as their wild-type neighbors, and are hence outcompeted over time, leading to an essentially wild-type cornea.

Minor points

(1) Quantification for Figure 2C and 2D is missing.

We have now included quantification of BRN3A+ retinal ganglion cells (Figure 2E) across control and Sox9^Δ/Δ retinas. Cell counts were performed on matched retinal sections, and the difference between groups was found to be statistically significant through Mann–Whitney U test (Table S5).

Regarding PAX6/AP2a, we quantified inner retinal neurons by analyzing AP2α+ amacrine cells and PAX6+/AP2α- horizontal cells as distinct subpopulations, rather than simply comparing total PAX6 or AP2α immunoreactivity. This approach allowed us to better resolve specific neuronal subtype changes. Both populations showed a statistically significant reduction in Sox9-deficient retinas relative to controls. The quantification for these analyses has now been incorporated into the revised Figure 2F and G (Table S6).

Consequently with the above, the following paragraph of the Results section (line 210 of the revised ms:

“We also studied the status of other retinal cell types. The transcription factor BRN3A was used to identify ganglion cells (Nadal-Nicolás et al., 2009), which were shown to decrease in number in the mutant retinas, compared to control ones (Fig. 2C). Similarly, double immunodetection of the transcription factors PAX6 and AP2A was used to identify both amacrine and horizontal cells, as previously described (Marquardt et al., 2001; Barnstable et al., 1985; Edqvist and Hallböök, 2004), showing a similar reduction in both cell types in degenerated retinas (Fig. 2D).”

Has been modified as follows:

“We also studied the status of other retinal cell types. The transcription factor BRN3A was used to identify ganglion cells (Nadal-Nicolás et al., 2009), which were shown to decrease in number in the mutant retinas, compared to control ones (Figs. 2C and 2D and Table S5; n = 5 controls, n = 12 mutants; Mann-Whitney U test, P = 3 × 10^-4). Similarly, double immunodetection of the transcription factors PAX6 and AP2A was used to identify both amacrine and horizontal cells (Fig. 2E), as previously described (Marquardt et al., 2001; Barnstable et al., 1985; Edqvist and Hallböök, 2004), showing a similar reduction in both cell types in degenerated retinas (Figs. 2F and 2G and Table S6; AP2α+ amacrine cells: n = 3 controls, n = 8 mutants;  2-sample T-tests P = 0.029; PAX6+/AP2α− horizontal cells: n = 3 controls, n = 8 mutants; Mann-Whitney U test P = 0.021). These findings indicate that the loss of Sox9 in the adult retina ultimately leads to the degeneration of multiple inner retinal neuronal populations, beyond the previously described effects on photoreceptors and Müller glia.

(2) Figure 4G & H: The authors must mention that the dashed lines enclose the limbal area.

Done

(3) The authors infer from an existing scRNA-seq dataset that OLB cells have high Sox9 expression as compared to ILB and corneal populations. However, Figures 4A and B do not indicate the anatomical positions of these cell types. The authors must label these for the reader's reference as they state that "[Sox9] expression pattern is consistent with our immunofluorescence observations" in Line 280.

As previously indicated, we have generated a new panel 5F and a corresponding paragraph to illustrate Sox9 expression pattern in the limbus. Accordingly, we have removed the sentence from line 280.

(4) Quantification for Figures 6A and 6B is missing.

We have quantified the number of Sox9 and P63 positive cells in the limbus between mutant and control corneas and found no difference in the number of positive cells. We have included these data in new panel 6C and Table S11.

Reviewer #2 (Recommendations for the authors):

Line 24: "synapsis" should be "synapses".

Done

(1) Consider starting a new paragraph after line 30.

Done

(2) Lines 42-48: make clear that this paragraph provides information only for HUMAN SOX9.

We now distinguish which studies were conducted in humans and which in mice.

(3) Line 55: explain to the non-expert reader what the "visual cycle" is.

Done (lines 64-65 of the revised ms)

(4) Line 66: consider "inactivate" instead of "suppress".

We substituted “suppress” with “inactivate”

(5) Line 90-92: ONLY PCR for the cGMP will provide formal evidence that this is not present in the mouse line.

We agree with the reviewer that PCR genotyping is the most straightforward method to exclude the presence of the Pde6^brd1 allele. Although retinal degeneration was never observed in untreated or control animals in our study, we have now removed the term “formal possibility” from the text to better reflect this limitation.

We have modified the following paragraph (page 116 in the revised version of the manuscript): “Retinal degeneration was never observed in mice that had not been tamoxifen-treated, nor any other controls, eliminating the formal possibility that the retinal degeneration allele of photoreceptor cGMP phosphodiesterase 6b (Pde6brd1) was present in our mice (Bowes et al., 1990).”

As follows: “Retinal degeneration was never observed in mice that had not been tamoxifentreated, nor any other control groups, making the presence of the retinal degeneration allele of photoreceptor cGMP phosphodiesterase 6b (Pde6^brd1) unlikely in our mice (Bowes et al., 1990). However, we acknowledge that definitive exclusion of this possibility would require PCR-based genotyping.”

(6) Line 160-166: This paragraph needs a conclusion.

We agree with the reviewer and have added the following sentence at the end of the paragraph:

“These findings indicate that the loss of Sox9 in the adult retina ultimately leads to the degeneration of multiple inner retinal neuronal populations, beyond the previously described effects on photoreceptors and Müller glia”

(7) Line: 240-265: This paragraph ends without a conclusion.

We have include the following conclusion:

“Thus, Sox9 is expressed in a basal limbal stem cell population with the ability to form two types of long-lived cell clones involved in stem cell maintenance and homeostasis.”

(8) In Results, it needs to be specified when exactly in adulthood the tamoxifen treatment started. This information is only provided in the Methods.

We have specified the age of the mice at the onset of tamoxifen treatment (two months)  and included it in the schemes of Figs 1A, 4C, 4H, 6E.

(9) Line 250: Because live imaging is not conducted, the word "dynamics" is not suitable.

We substituted “dynamics” with “contribution”

(10) Panel C in Figure 6 is nice and helpful. Consider adding a similar panel in Figure 1.

Done.

(11) Line 420: is this the human Sox9 enhancer?

Yes. It is a human enhancer. We have indicated it in the text.

(12) Line 459: typo "detected tissue".

Corrected

(13) Line 448 and 468: citations are needed.

Line 448 is deleted in the revised version of the ms.

(14) 479: typo "clones clones'.

Corrected.

Briefing : Les Violences Éducatives Ordinaires (VEO)

Résumé

Ce document de synthèse analyse les Violences Éducatives Ordinaires (VEO) en s'appuyant sur l'expertise de professionnels de l'enfance.

Il met en lumière le contexte historique, la définition, les impacts neuroscientifiques et les défis sociétaux liés à ces pratiques.

Les VEO, héritage d'une histoire millénaire de domination patriarcale, englobent non seulement les violences physiques (gifles, fessées) mais aussi des formes psychologiques et verbales (humiliations, chantage, cris) qui sont banalisées et profondément ancrées dans les schémas éducatifs.

La législation française n'a que très récemment, en 2019, interdit explicitement ces pratiques, marquant une rupture avec un passé où le "droit de correction" était légitimé.

L'impact des VEO sur l'enfant est désormais documenté par les neurosciences : loin de favoriser l'obéissance, le stress généré active les circuits cérébraux de la peur, inhibant les capacités de raisonnement et de coopération.

Cela compromet le "méta-besoin" fondamental de sécurité de l'enfant, essentiel à son développement.

Les parents actuels se trouvent dans une "période de transition éducative" complexe, cherchant à abandonner des modèles transmis sur des générations.

Il est crucial de distinguer l'éducation sans violence du laxisme : l'enjeu est de poser un cadre clair, prévisible et contenant, tout en instaurant un dialogue basé sur la confiance et le respect.

Ce document détaille ces concepts et recense les ressources disponibles pour accompagner les familles dans cette transition.

I. Contexte Historique et Sociétal : De la Domination aux Droits de l'Enfant

La notion de violences éducatives ordinaires est intrinsèquement liée à une longue histoire de domination et à l'évolution du statut de l'enfant dans la société.

A. L'Héritage Patriarcal

• Antiquité Romaine : Le concept du pater familias donnait au chef de famille un pouvoir absolu, y compris un droit de vie et de mort sur ses enfants et ses esclaves, afin de maintenir un ordre social fondé sur la domination.

• Code Civil Napoléonien (1804) : Cet héritage a été formalisé dans la loi française, qui a réaffirmé la "puissance paternelle" et le "droit de correction" du père sur ses enfants.

L'article 375 permettait même au père de faire enfermer sa progéniture au titre de la correction. Bien que datant de plus de deux siècles, ce code constitue encore la base du droit civil actuel.

• XIXe et début du XXe siècle : Le père conservait un pouvoir coercitif majeur, pouvant décider de l'enfermement d'un enfant jugé "rebelle" ou désobéissant dans des "maisons de correction" ou des "colonies pénitentiaires agricoles" (comme celle de Mettray en Indre-et-Loire), qui s'apparentaient davantage à des bagnes qu'à des lieux d'éducation.

B. L'Émergence Lente des Droits de l'Enfant

Le XXe siècle a vu une évolution progressive de la perception de l'enfant, qui passe d'un objet de correction à un sujet de droits.

• 1935 : Abolition de la "correction paternelle", mettant fin au droit d'enfermement parental.

• 1945 : L'ordonnance de 1945, dans le contexte de l'après-guerre, crée les juges pour enfants et pose les fondements d'une justice moderne pour les mineurs, axée sur la protection et l'éducation plutôt que sur la seule coercition.

• 1970 : La "puissance paternelle" est définitivement abolie et remplacée par l'autorité parentale, qui instaure des droits et devoirs égaux entre la mère et le père. C'est une étape majeure mais le droit de correction reste toléré dans la pratique.

En 1982, un juge en cour d'appel pouvait encore statuer que fessées et coups de règle ne constituaient pas une "brutalité excessive" s'ils ne laissaient pas de traces.

• 1989 : La Convention Internationale des Droits de l'Enfant (CIDE), ratifiée par la France en 1990, reconnaît enfin l'enfant comme un sujet de droit à part entière, devant être protégé de toute forme de violence.

C. La Loi de 2019 : Une Reconnaissance Tardive

Malgré la CIDE, la France a mis près de 30 ans à légiférer spécifiquement sur les VEO.

• 2015 : La France est condamnée par le Conseil de l'Europe pour l'absence d'une loi "suffisamment claire" interdisant les châtiments corporels.

• Juillet 2019 : Adoption de la loi, souvent surnommée péjorativement "loi anti-fessée". Cette loi, proposée par la députée Maud Petit, a fait l'objet de nombreuses moqueries et d'une forte résistance, illustrée par l'argument "j'ai pris des claques et je n'en suis pas mort".

• Contenu de la loi : Elle stipule de manière concise que "l'autorité parentale s'exerce sans violence physique ou psychologique".

L'introduction de la notion de violence psychologique est une avancée fondamentale, car elle reconnaît les impacts invisibles mais profonds de certaines pratiques éducatives.

II. Définition et Formes des Violences Éducatives Ordinaires (VEO)

Les VEO sont définies comme des pratiques punitives et coercitives, banalisées et courantes ("ordinaires"), utilisées au nom de l'éducation mais qui n'ont aucune valeur éducative et portent atteinte à la dignité et à l'intégrité de l'enfant.

Elles se classifient en trois grandes catégories.

Type de Violence Exemples Concrets Citées Violence Physique Gifles, fessées, tapes sur les mains, tirage de cheveux, pincements, secousses, jet d'objets, destruction de jouets, privation de nourriture, isolement forcé dans une pièce. Violence Psychologique Menaces ("tu vas voir..."), culpabilisation, chantage affectif, éducation par la peur, indifférence (ignorer l'enfant, notamment quand il pleure), créer un climat d'insécurité. Violence Verbale Humiliations, insultes, cris, dévalorisation ("tu es nul", "tu n'y arriveras jamais"), comparaisons (entre frères et sœurs ou avec d'autres enfants), moqueries.

Ces pratiques sont souvent des réactions automatiques de l'adulte face à un sentiment de débordement ou d'impuissance, et peuvent être la reproduction de schémas éducatifs subis durant sa propre enfance.

III. L'État des Lieux Actuel et la Perception Sociétale

Une enquête IFOP réalisée pour la Fondation pour l'enfance en 2024 révèle une évolution contrastée des mentalités depuis la loi de 2019. • Baisse des violences physiques : La loi "anti-fessée" semble avoir eu un impact positif, avec une diminution déclarée du recours aux châtiments corporels.

• Stagnation des violences psychologiques : Les violences psychologiques et verbales peinent à diminuer, voire augmentent pour certaines.

Cela traduit une difficulté à prendre conscience de la portée de ces actes et à modifier des modèles de communication profondément ancrés.

• Résistance parentale : Une part significative des parents interrogés exprime encore une réticence face à la loi, la percevant comme une ingérence de l'État dans la sphère privée ("de quoi se mêle l'État").

Cet argument de "l'intimité familiale" a historiquement freiné l'avancée de la législation.

IV. L'Impact des VEO sur le Développement de l'Enfant

Les neurosciences permettent de comprendre pourquoi les VEO sont non seulement néfastes, mais aussi contre-productives.

A. La Réponse Cérébrale à la Peur

Face à un adulte perçu comme menaçant (cris, gestes brusques), le cerveau de l'enfant active un mécanisme de survie.

1. Perception d'un danger : L'adulte devient une source de frayeur.

2. Court-circuit du raisonnement : Le signal de peur est traité directement par les zones archaïques du cerveau (système limbique), qui gèrent les émotions et le danger, en contournant le cortex préfrontal, siège de la réflexion et de l'apprentissage.

3. Réactions instinctives : Le cerveau déclenche l'une des trois réponses primaires au danger :

◦ L'attaque (Fight) : Rare envers un parent.

◦ La fuite (Flight) : Évitement.

◦ La sidération (Freeze) : L'enfant est "tétanisé", incapable d'agir ou de réagir.

C'est souvent interprété à tort par le parent comme de la provocation.

Ce processus s'accompagne d'une surproduction d'hormones de stress comme le cortisol, qui, en excès, est néfaste pour le développement cérébral.

B. L'Atteinte au Besoin Fondamental de Sécurité

Le "méta-besoin" de sécurité est le pilier du développement de l'enfant, comme l'a reconnu la loi de protection de l'enfance de mars 2016.

Ce besoin inclut :

• Les besoins physiologiques (sommeil, alimentation).

• La nécessité de relations affectives stables et prévisibles.

Les VEO créent une insécurité fondamentale : l'enfant perd confiance en son environnement et en les figures qui sont censées le protéger.

C. Les Risques à Long Terme

Bien qu'il n'y ait pas de causalité automatique, un enfant exposé de manière répétée aux VEO présente une vulnérabilité accrue à :

• Des difficultés scolaires et d'apprentissage : Un enfant en état d'alerte permanent n'est pas disponible pour apprendre.

• Une faible estime de soi : Les messages dévalorisants sont intériorisés.

• La reproduction des schémas de violence : Il peut devenir lui-même auteur de violences ou se retrouver en situation de victime à l'âge adulte (auto-maltraitance ou maltraitance subie).

V. Le Défi de la "Transition Éducative" pour les Parents

Les parents d'aujourd'hui sont à la charnière de deux modèles, ce qui crée une période de "transition éducative".

• Sortir de la reproduction : La plupart des adultes ont été éduqués avec des VEO. En situation de stress, la tendance est de reproduire inconsciemment ces schémas. Prendre conscience de cela est la première étape du changement.

• Réparer la relation : Il n'est jamais trop tard pour revenir sur un incident. Reconnaître son erreur devant l'enfant ("ma réaction était disproportionnée"), lui expliquer le contexte sans se défausser, permet de restaurer le lien de confiance et de lui montrer un modèle de gestion de conflit non-violent.

• Éduquer sans violence n'est pas du laxisme : C'est une confusion fréquente. L'enfant a un besoin essentiel de cadre.

Ce cadre doit être :

◦ **Clair et prévisible** : Les règles sont connues et cohérentes.

◦ Contenant : Il offre une sécurité affective qui permet à l'enfant d'explorer le monde.

◦ Adapté et évolutif : Il change avec l'âge et les compétences de l'enfant, et peut être discuté, notamment avec un adolescent.

• Sanction vs. Punition : La sanction, si elle est proportionnée à l'acte et à l'âge, peut être éducative si elle n'a pas pour but d'humilier ou de dominer, mais de poser une limite et de permettre une réparation.

VI. Cas Pratiques et Lignes Directrices

• Obliger à embrasser un proche : Cette pratique est une VEO car elle ne respecte pas le corps et le consentement de l'enfant.

C'est une occasion manquée d'enseigner le droit de dire "non", une compétence cruciale pour la prévention des abus. Il est préférable de proposer des alternatives ("tu peux dire bonjour d'une autre façon").

• Forcer à manger ou à goûter : Le forçage alimentaire peut générer des troubles du comportement alimentaire.

L'utilisation de la nourriture comme chantage (notamment le sucre comme récompense) crée une relation malsaine à l'alimentation.

Le rôle du parent est de proposer une alimentation variée, mais l'enfant doit rester maître de ce qu'il ingère.

• Refus de soins (médicaments, brossage de dents) :

La santé de l'enfant n'est pas négociable, et l'adulte doit poser un cadre ferme. Cependant, l'approche est essentielle : expliquer l'importance du soin, rester calme et convaincu, et utiliser des stratégies ludiques pour obtenir l'adhésion plutôt que de recourir à la force.

VII. Ressources et Soutien Disponibles

Il est essentiel pour les parents de ne pas rester isolés face à leurs difficultés. De nombreuses structures, gratuites et confidentielles, existent pour offrir écoute et accompagnement.

• Protection Maternelle Infantile (PMI) : Pour les parents d'enfants de 0 à 6 ans, dans les Maisons Départementales de la Solidarité.

• Lieux d'Accueil Enfants-Parents (LAEP) : Espaces de rencontre et de jeu pour les parents et enfants de 0 à 3 ans.

• Maison des Adolescents : Lieu dédié aux jeunes et à leurs parents.

• Espace Santé Jeune : Ligne d'écoute et accueil pour les 7-25 ans.

• Espaces Parents : Nouveaux lieux d'accueil, d'écoute et d'activités pour tous les parents.

• Associations spécialisées : Comme Les Établis, qui proposent prévention, écoute et orientation.

• Ressources en ligne : Le site stopveo.fr (Violences Éducatives Ordinaires) offre des articles, vidéos et témoignages.

L'Orientation Scolaire : Parcours, Psychologie et Stratégies

Synthèse Exécutive

L'orientation scolaire est présentée non pas comme une série de décisions ponctuelles et anxiogènes, mais comme un cheminement progressif et continu, un parcours singulier qui se construit tout au long de la vie.

Les psychologues de l'Éducation Nationale (PsyEN) jouent un rôle central dans cet accompagnement, en s'appuyant sur leur expertise en psychologie du développement de l'enfant et de l'adolescent pour aider les jeunes et leurs familles à naviguer dans ce processus.

Les points critiques qui émergent sont :

• La primauté de l'épanouissement : La réussite scolaire et professionnelle est directement conditionnée par l'épanouissement du jeune dans la voie choisie.

Le choix doit être en phase avec sa manière d'apprendre (théorique, pratique, par projet) et ses centres d'intérêt.

• La déconstruction des mythes : Il n'existe pas de "métier idéal" unique ni de trajectoire linéaire.

La voie professionnelle n'est pas une impasse et peut mener à des études supérieures.

L'apprentissage est une modalité d'excellence accessible à tous les niveaux, du CAP au diplôme d'ingénieur.

• La notion de "maturité vocationnelle" : La capacité à se projeter dans un avenir professionnel est une compétence cognitive qui mûrit progressivement, atteignant un pic vers 18 ans.

Il est donc normal pour un adolescent de ne pas avoir de projet défini. Le rôle des adultes est de rassurer, d'encourager et d'ouvrir des horizons.

• L'importance des expériences multiples : La construction du projet d'orientation se nourrit autant des temps formels (stages, séances en classe) que des expériences informelles (discussions en famille, loisirs, voyages).

"Se tromper" n'est pas un échec, mais une expérience qui affine la connaissance de soi.

• Parcoursup comme outil : Loin d'être un juge, Parcoursup est une plateforme de mise en relation entre les candidats et les établissements d'enseignement supérieur, qui conservent leurs propres critères de recrutement.

C'est également une ressource d'information précieuse, utilisable bien avant la classe de Terminale pour explorer les possibilités via sa "carte des formations".

En somme, l'approche préconisée est de dédramatiser l'orientation en la considérant comme un processus d'exploration, où la connaissance de soi, la flexibilité et l'ouverture aux opportunités sont les clés d'un parcours réussi et épanouissant.

Le Rôle Central du Psychologue de l'Éducation Nationale (PsyEN)

De Conseiller à Psychologue : Une Approche Globale

Le métier a évolué de "conseiller d'orientation" à "psychologue de l'Éducation Nationale" pour refléter un changement fondamental d'approche.

L'accompagnement proposé dépasse le simple conseil pour s'ancrer dans une compréhension profonde de la psychologie de l'enfant et de l'adolescent.

• Fondements psychologiques : Les entretiens s'appuient sur la connaissance du développement psychosocial, cognitif et affectif du jeune.

• Objectif : Aider le jeune à construire son projet d'orientation et à faire des choix éclairés lors des moments clés de sa scolarité.

• Citation clé : Géraldine Mignier précise : "Ce sont toutes ces notions sur lesquelles nous on appuie nos entretiens pour travailler avec le jeune son projet d'orientation, l'aider à faire des choix au moment où il doit prendre des décisions."

• Formation : Les PsyEN sont des psychologues de formation ayant suivi une année de spécialisation supplémentaire pour exercer au sein de l'Éducation Nationale.

Deux Spécialités pour un Accompagnement Adapté

Les PsyEN se divisent en deux spécialités pour couvrir l'ensemble du parcours scolaire :

1. EDA (Éducation, Développement, Apprentissage) :

Interviennent dans le premier degré, de l'école maternelle au CM2.

2. EDO (Éducation, Développement et Conseil en Orientation Scolaire et Professionnelle) :

Interviennent dans l'enseignement secondaire (collège, lycée) et supérieur. Ce sont ces professionnels qui sont au cœur du sujet de l'orientation.

Mission : Rassurer, Accompagner et Servir de Médiateur

Le PsyEN agit souvent comme une tierce personne neutre entre le jeune et ses parents, surtout lorsque les désirs de l'un ne correspondent pas aux attentes des autres.

Leur rôle est d'apaiser les angoisses, d'expliquer le fonctionnement du système et de permettre des prises de décision plus sereines.

Ils reçoivent les élèves, mais aussi les parents, que ce soit au sein des établissements scolaires ou dans les Centres d'Information et d'Orientation (CIO).

Les Parcours d'Orientation : Un Paysage aux Multiples Voies

L'orientation n'est pas limitée aux classes de 3ème et de Terminale. Elle est jalonnée de plusieurs moments de choix tout au long de la scolarité.

Au-delà de la 3ème et de la Terminale : Les Premiers Paliers

La question de l'orientation peut se poser bien avant les grandes échéances connues :

• Dès la 6ème : Des orientations adaptées peuvent être envisagées (SEGPA, dispositif ULIS).

• En 4ème ou 3ème : Des choix vers l'enseignement agricole ou une 3ème "prépa-métiers" sont possibles pour découvrir le monde professionnel plus tôt.

Après la 3ème : Choisir sa Modalité d'Apprentissage

Le choix post-3ème doit avant tout se fonder sur la manière dont l'élève apprend et s'épanouit.

L'épanouissement conditionne la réussite.

Voie Description Profil d'Apprenant Débouchés

Générale et Technologique (Seconde GT) Classe commune menant soit à un Bac Général (enseignements théoriques et généraux), soit à un Bac Technologique (apprentissage par projets).

Bac Général : À l'aise avec les apports théoriques. <br> Bac Technologique : Apprécie l'apprentissage par le biais de projets.

Nécessite une poursuite d'études supérieures.

Professionnelle

Prépare à deux diplômes : le CAP (en 2 ans) ou le Bac Pro (en 3 ans). Peut se faire sous statut scolaire (lycée professionnel) ou en apprentissage (entreprise).

Apprend de manière concrète, avec plus de pratique et d'expérience.

Permet une insertion professionnelle directe mais aussi une poursuite d'études supérieures. La voie pro n'est pas synonyme d'études courtes.

Focus sur l'Apprentissage : L'apprentissage n'est pas réservé aux filières courtes.

Il est possible de suivre un parcours complet en apprentissage, du CAP jusqu'au Bac+5 (Master, titre d'ingénieur), en alternant formation en école et en entreprise.

L'Enseignement Supérieur après le Baccalauréat

Le schéma des études supérieures n'est pas linéaire ni figé.

• Flexibilité des parcours : Il existe de nombreuses passerelles entre les filières (ex: de BTS/BUT vers une grande école ou un master universitaire).

• Trajectoires singulières : Les parcours sont propres à chacun et peuvent inclure des pauses (année de césure, service civique), des réorientations ou des reprises d'études.

• Diversité des diplômes : L'offre va du Bac+2 (BTS) au Bac+5 (Master) et au-delà (Doctorat), avec des durées et des modalités variées (alternance, etc.).

La Construction du Projet : Un Cheminement Personnel et Expérientiel

Le projet d'orientation s'élabore progressivement grâce à une multitude d'expériences qui nourrissent la réflexion.

• Expériences formelles : Visites d'entreprises, forums, stages (obligatoire en 3ème, puis en 2nde GT), mini-stages en lycée professionnel.

• Expériences informelles : Discussions sur le travail à la maison, voyages, loisirs, rencontres.

Il est crucial de permettre aux jeunes de se forger des représentations variées du monde du travail.

• Le stage : Il s'agit d'une étape de découverte du monde de l'entreprise (codes, fonctionnement, assiduité) et de soi-même, plus que d'une validation d'un choix de métier.

• La psychologie de l'adolescent et la "Maturité Vocationnelle" :

La capacité à définir un projet professionnel, appelée "maturité vocationnelle", se développe avec la maturation cognitive du cerveau, qui se poursuit bien après 18 ans.

Il est donc contre-productif de mettre la pression sur un jeune adolescent pour un choix de métier définitif.

Démystifier l'Orientation et Gérer l'Anxiété Une part importante de l'accompagnement consiste à rassurer les familles et à déconstruire certaines idées reçues.

• Le droit à l'erreur : Une réorientation n'est pas un échec mais une expérience enrichissante qui permet d'affiner son projet et d'acquérir de nouvelles compétences.

Comme le souligne une intervenante : "Ce ne sont que des expériences qui permettent d'alimenter la connaissance de soi."

• Le mythe du "métier idéal" : L'idée d'un métier unique et parfait pour une personne n'est pas réaliste. Les parcours professionnels sont aujourd'hui marqués par la reconversion et l'évolution.

• Un monde du travail en évolution : Avec l'émergence de l'IA et de l'écologie, de nombreux métiers de demain n'existent pas encore.

Il est donc plus pertinent de faire des choix basés sur ses intérêts actuels que de tenter de prédire l'avenir du marché du travail.

• Analyse Statistique : Environ 50% des jeunes n'exercent pas un emploi directement lié à leur formation initiale.

Cela ne signifie pas une erreur d'orientation, mais illustre la transférabilité des compétences (méthodologie, savoir-faire) acquises et l'évolution naturelle des trajectoires professionnelles.

Outils et Plateformes Clés : Le Cas de Parcoursup

Parcoursup : Une Plateforme de Candidature, pas de Décision

Parcoursup est souvent une source de stress, mais son rôle doit être bien compris :

• Mise en relation : C'est une plateforme qui met en relation les candidats avec les formations post-bac.

• Le recrutement par les écoles : Ce sont les établissements d'enseignement supérieur qui examinent les dossiers et effectuent le recrutement selon leurs propres critères (dossier scolaire, concours, entretiens, etc.).

Calendrier et Fonctionnement

• Période clé : La saisie des vœux se déroule généralement du 15 janvier au 13 mars.

• Importance de l'information : Il est essentiel que le jeune se renseigne en amont sur les "attendus" et les modalités de recrutement de chaque filière qui l'intéresse pour formuler des vœux ajustés à son profil.

Un Outil de Recherche Précieux dès le Collège

La plateforme n'est pas réservée aux élèves de Terminale. Sa "carte des formations" est un moteur de recherche accessible à tous, qui permet de :

• Explorer les formations par mots-clés ou diplômes au niveau national.

• Visualiser géographiquement les établissements.

• Consulter les spécificités de chaque formation, les critères de recrutement et les statistiques des années précédentes.

Lieux et Ressources pour S'orienter

Les Centres d'Information et d'Orientation (CIO)

Les CIO offrent des entretiens gratuits sur rendez-vous (environ 1 heure) avec des PsyEN, pour les jeunes seuls ou accompagnés de leur famille. Ils sont ouverts pendant les vacances scolaires.

• Sites en Indre-et-Loire : Tours (sur le site de Joué-lès-Tours), Joué-lès-Tours, Loches, Amboise, Chinon. Ressources Numériques de Référence Ces sites sont recommandés pour la fiabilité et l'actualisation de leurs informations :

• ONISEP : Informations sur les diplômes et filières. Propose un guide pour les parents sur l'onglet "Avenir(s)".

• Parcoursup.fr : Pour la carte des formations et les informations sur l'enseignement supérieur.

• Eduscol : Ressources pédagogiques de l'Éducation Nationale.

• Cléor : Informations sur les métiers et le monde économique régional.

• orientation.centre-valdeloire.fr : Calendrier des journées portes ouvertes et recherche de formations.

• CIDJ (Centre d'Information et de Documentation Jeunesse) : Fiches métiers détaillées avec les parcours d'études associés.

Acteurs Locaux en Indre-et-Loire

• Bureau Information Jeunesse (BIJ) : Situé à Tours, il propose accueil, écoute et ateliers. Des Points Information Jeunesse (PIJ) existent aussi en milieu rural.

• Maison de l'Orientation et de l'Insertion Professionnelle (MOIP) : Rattachée à l'Université, elle s'adresse principalement aux étudiants mais reçoit aussi des lycéens.

Author response:

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

Response to Reviewer #3:

We thank reviewer 3 for spending their valuable time on commenting on our revised paper.

We would like to reiterate the central conclusion of this work, which appears to have been missed by Reviewer 3. Using a BFP-expressing lineage tracer hPSC line for tracking LMX1A+ midbrain-patterned neural progenitors and their differentiated progeny, we discovered a loss of the LMX1A lineage during pluripotent stem cell differentiation into astrocytes, despite BFP+ neural progenitors were the dominant population at the onset of astrocyte induction.

Hence, the take-home message of this study is, as summarized in the abstract, ‘ the lineage composition of iPSC-derived astrocytes may not accurately recapitulate the founder progenitor population’ and that one should not take for granted that in vitro/stem cell-derived astrocytes are the descendants of the dominant starting neural progenitors (which is a general assumption in PSC publications as described in the paper and our response to reviewers).

Please find below our point-by-point response to reviewer comments. We have re-ordered the points according to their relative importance to our main conclusions.

‘ the lineage composition of iPSC-derived astrocytes may not accurately recapitulate the founder progenitor population’ and that one should not take for granted that in vitro/stem cell derived astrocytes are the descendants of the dominant starting neural progenitors (which is a general assumption in PSC publications as described in the paper and our response to reviewers).

Please find below our point-by-point response to their comments. We have re-ordered the points according to their relative importance to our main conclusions.

…. They used lineage tracing with a LMX1A-Cre/AAVS1-BFP iPSCs line, where the initial expression of LMX1A and Cre allows the long-lasting expression of BFP, yielding BFP+ and BFP- populations, that were sorted when in the astrocytic progenitor expansion. BFP+ showed significantly higher number of cells positive to NFIA and SOX9 than BFP- cells …

This is a misunderstanding by reviewer 3. As indicated in the first sentence of the second section, BFP- populations used for functional and transcriptomic analysis was not sorted BFP^- cells, but those derived from unsorted, BFP⁺ enriched populations. Our scRNAseq analysis indicated that they were transcriptomically aligned to human midbrain astrocytes. This finding is consistent with the fact that they are derived from midbrain-patterned neural progenitors, presumably minority LMX1A- progenitors.

Reviewer 3’s comments indicate that they misunderstood the primary aims of our study as a mere functional and transcriptomic comparison of the two astrocyte populations.

(9) BFP+ cells did not show higher levels of transcripts for LMX1A nor FOXA2. This fact jeopardizes the claim that these cells are still patterned. In the same line, there are not significant differences with cortical astrocytes, indicating a wider repertoire of the initially patterned cells, that seems to lose the midbrain phenotype. Furthermore, common DGE shared by BFP- and BFP+ cells when compared to non-patterned cells indicate that after culture, the pre-pattern in BFP+ cells is somehow lost, and coincides with the progression of BFP- cells.

The reviewer seems to assume that astrocytes derived from LMX1A+ ventral midbrain progenitors must retain LMX1A expression. We do not take this view and do not claim this in this study. Moreover, we have discussed in the paper that due to a lack of transcriptomic studies of in vivo track regional progenitors (such as LMX1A), it remains unknown whether and to what extent patterning gene expression is maintained in astrocytes of different brain regions.

Our findings on the lack of LMX1A and FOXA2 in BFP+ astrocytes are supported by several published single-cell transcriptomic studies of human midbrain astrocytes (La Manno et al. 2016; Agarwal et al. 2020; Kamath et al. 2022). We have a paragraph of discussion on this topic in both the original and updated versions of the paper with the relevant publications cited.

Other points raised by reviewer 3

(1) It is very intriguing that GFAP is not expressed in late BFP- nor in BFP+ cultures, when authors designated them as mature astrocytes.

We did not designate our cells as ‘mature’ astrocytes but ‘astrocytes’ based on their global gene expression with the human fetal and adult brain astrocytes as references.

Moreover, ‘mature’ only appeared once in the paper indicating that our cells lie in between the fetal and adult astrocytes in maturity.

(2) In Fig. 2D, authors need to change the designation "% of positive nuclei".

To be corrected in the version of record.

(3) In Fig. 2E, the text describes a decrease caused by 2APB on the rise elicited by ATP, but the graph shows an increase with ATP+2APB. However, in Fig. 2F, the peak amplitude for BFP+ cells is higher in ATP than in ATP+2APD, which is mentioned in the text, but this is inconsistent with the graph in 2E.

To be corrected in the version of record.

(4) The description of Results in the single-cell section is confusing, particularly in the sorted CD49 and unsorted cultures. Where do these cells come from? Are they BFP-, BFP+, unsorted for BFP, or non-patterned? Which are the "all three astrocyte populations"? A more complete description of the "iPSC-derived neurons" is required in this section to allow the reader to understand the type and maturation stage of neurons, and if they are patterned or not.

As previously reported in the reference cited, CD49 is a novel human astrocyte marker. This is independent of BFP expression. For all three astrocyte populations studied here (BFP+, BFP-, and non-patterned astrocytes), we included both CD49f+ sorted and unsorted samples to account for selection bias caused by FACS. iPSC-derived neurons were included in the sequencing study to provide a reference for cell-type annotation. They were generated following a GABAergic neuron differentiation protocol. However, their maturation stages and/or regional characteristics are not relevant to astrocytes.

(5) A puzzling fact is that both BFP- and BFP- cells have similar levels of LMX1A, as shown in Fig. S6F. How do authors explain this observation?

This figure panel shows that LMX1A, LMX1B and FOXA2 are essentially NOT expressed in these astrocytes.

(6) In Fig. 3B, the non-patterned cells cluster away from the BFP+ and BFP-; on the other hand, early and late BFP- are close and the same is true for early and late BFP+. A possible interpretation of these results is that patterned astrocytes have different paths for differentiation, compared to non-patterned cells. If that can be implied from these data, authors should discuss the alternative ways for astrocytes to differentiate.

Both BFP+ and BFP- astrocyte are from ventral midbrain patterned neural progenitors, while non-patterned neural progenitors are more akin to that of forebrain. Figure 3B is expected and confirms the patterning effect.

(7) Fig. 3D shows that cluster 9 is the only one with detectable and coincident expression of both S100B and GFAP expression. Please discuss why these widely-accepted astrocyte transcripts are not found in the other astrocytes clusters. Also, Sox9 is expressed in neurons, astrocyte precursors and astrocytes. Why is that?

S100B and GFAP are classic astrocyte markers in certain states. We are not relying only on two markers but the genome-wide expression profile as the criteria for astrocytes. As shown in the unbiased reference mapping to multiple human brain astrocyte scRNA-seq datasets, all our astrocyte clusters were mapped with high confidence to human astrocytes.

SOX9 is an important regulator for astrogenesis, so its expression is expected in precursors (doi.org/10.1016/j.neuron.2012.01.024). In addition, recent studies have uncovered that SOX9 expression is also reported in foetal striatal projection neurons and early postnatal cortical neurons, where SOX9 regulates neuronal synaptogenesis and morphogenesis (dois:10.1016/j.fmre.2024.02.019; 10.1016/j.neuron.2018.10.008). Therefore, the expression of SOX9 in multiple cell types was expected. Instead of using a few selected markers for cell-type annotation, we employed a genomic approach relying on an unbiased reference mapping approach and a combination of various markers to ascertain our annotation results.

(8) Line 337, Why authors selected a log2 change of 0.25? Typically, 1 or a higher number is used to ensure at least a 2-fold increase, or a 50% decrease. A volcano plot generated by the comparison of BFP+ with BFP- cells would be appropriate. The validation of differences by immunocytochemistry, between BFP+ and BFP-, is inconclusive. The staining is blur in the images presented in Fig. S8C. Quantification of the positive cells, without significant background signal, in both populations is required.

We used a lenient threshold owing to the following considerations: 1) High FC does not necessarily mean biological relevance, as gene expression does not necessarily translate to protein expression. Therefore, a smaller FC value could also be biologically meaningful. 2) Balance between noise and biological differences. Any threshold was chosen arbitrarily. 3) We are identifying a trend rather than pinpointing a specific set of

The quality was unfortunately reduced due to restrictions on file size upon submission. A high resolution Fig. S8C is available.

(10) For the GO analyses, How did authors select 1153 genes? The previous section mentioned 287 genes unique for BFP+ cells. The Results section should include a rationale for performing a wider search for the enriched processes.

GO enrichment using unique DEGS may not capture the wider landscape of the transcriptomic characteristics of BFP⁺ astrocytes. The 287 unique genes were only differentially expressed in BFP⁺ astrocytes. However, apart from these 287 genes, other genes among the 1187 DEGs were differentially expressed in BFP⁺ astrocytes and in one other population.

(11) For Fig. 4C and 4D, both p values and the number of genes should be indicated in the graph. I would advise to select the 10 or 15 most significant categories, these panels are very difficult to read. Whereas the listed processes for BFP+ have a relation to Parkinson disease, the ones detected for BFP- cells are related to extracellular matrix and tissue development. Does it mean that BFP+ cells have impaired formation of this matrix, or defective tissue development? This is in contradiction of enhanced calcium responses of BFP+ cells compared to BFP- cells.

Information on all DEGs, including p values and numbers, is provided in Supplementary data 1-5.

BFP+ astrocytes do have enrichment for GO terms related to extracellular matrix and tissue development, although not as obvious as BFP- astrocytes. Previous work have shown that both in vitro and in vivo derived astrocytes are functionally heterogeneous, containing functionally distinct subtypes exhibiting different GO enrichment profiles (doi: 10.1016/j.ygeno.2021.01.008; 10.1038/s41598-024-74732-7).

(12) Both the comparison between midbrain and cortical astrocytes in Fig. S8A, and the volcano plot in S8B do not show consistent changes. For example, RCAN2 in Fig. S8A has the same intensity for cortical and midbrain cells, but is marked as an enriched gene in midbrain in the p vs log2FC graph in Fig. S8B.

These are integrated analyses of published human datasets. S8A and S8B show the same data in different formats. The differences are better shown in the volcano plot/easier detected by the human eye.

These are integrated analysis of published human datasets. S8A and S8B are the same data shown in different format. Differences are better shown in volcano plot /easier detected by the human eye. RCAN2 had a higher average expression in the midbrain than in the telencephalon, albeit small, and the difference was statistically significant (as shown in the volcano plot).

The following is the authors’ response to the original reviews

Reviewer 1:

In vitro nature of this work being the fundamental weakness of this paper

We disagree with this statement. As explained in the provisional response, the aim of this study was to test the validity of a general concept applied in pluripotent stem cell research that pluripotent stem cell-derived astrocytes faithfully represent the lineage heterogeneity of their ancestral neural progenitors and hence preserve the regionality of such progenitors. Our genetic lineage study is justified for addressing this in vitro-driven question. However, we have highlighted the rationale where appropriate in the revised paper.

If regional identity is not maintained, so what? Don't we already know that this can happen? The authors acknowledge that this is known in the discussion.

Importance of regional identity: Growing evidence demonstrates the functional heterogeneity of brain astrocytes in health and disease. Therefore, for in vitro disease modeling, it is believed that one should use astrocytes represent the anatomy of disease pathology; for example, midbrain astrocytes for studying dopamine neurodegeneration and Parkinson’s disease. Understanding the dynamics of stem cell-derived astrocytes and identifying astrocyte subtypes is important for their biomedical applications.

Regional identity change/Discussion: It seems that the reviewer misunderstood the context in which the ‘identity change’ was discussed. The literature referred to (in the Discussion) concerns shifts in regional gene expression in bulk-cultured cells. In the days of pre-single-cell analysis/lineage tracking, one cannot distinguish whether this was due to a change in the transcriptomic landscape in progenies of the same lineage or alterations in lineage heterogeneity, but to interpret at face value as regional identity was not maintained. In the revised paper, we have made an effort to indicate that ‘regional identity’ is used broadly to refer to lineage relationships and/or traits rather than static gene expressioin.

validation of the markers/additional work

The scNAseq analysis performed in this study compared the profiles of astrocytes derived from LMX1A+ and LMX1A- ventral midbrain-patterned neural progenitors. Since it is not possible to perform genetic lineage tracking in humans and an analogous mouse lineage tracer line is not available, in vivo validation of these markers with respect to their lineage relationship is not currently feasible. However, we took advantage of abundant single-cell human astrocyte transcriptomic datasets and validated our genes in silico. We also validated the differential expression of selected markers in late BFP+ and BFP- astrocytes using immunocytochemistry, where reliable antibodies are available. The results of the additional analyses are presented in Figure S8 and Supplemental Data 5.

Knowledge gaps concerning astrocyte development

Reviewer 1 pointed out a number of knowledge gaps concerning astrocyte development, such as the transcriptomic landscape trajectories of midbrain floor plate cells as they progress towards astrocytes. Indeed, the limited knowledge on regional astrocyte molecule heterogeneity restricts the objective validation of in vitro-derived astrocyte subtypes and the development of novel approaches for their generation in vitro. We agree with the need for in-depth in vivo studies using model organisms, although these are beyond the scope of the current work.

Reviewer 2:

(1) The authors argue that the depletion of BFP seen in the unsorted population immediately after the onset of astrogenic induction is due to the growth advantage of the derivatives of the residual LMX1A- population. However, no objective data supporting this idea is provided, and one could also hypothesize that the residual LMX1A- cells could affect the overall LMX1A expression in the culture through negative paracrine regulation.

We acknowledge the lack of evidence-based explanation for the depletion of BFP+ cells in mixed cultures. We were unable to perform additional experiments because of resource limitations. The design of the LMX1A-Cre/AAVS1-BFP lineage tracer line determines that BFP is expressed irreversibly in LMX1A-expressing cells or their derivatives regardless of their LMX1A expression status. Therefore, the potential negative paracrine regulation of LMX1A by residual LMX1A- cells should not affect cells that have already turned on BFP. We have highlighted the working principles of the LMX1A tracer line in the revised manuscript.

(2) Furthermore, on line 124 it is stated that: "Interestingly, the sorted BFP+ cells exhibited similar population growth rate to that of unsorted cultures...". In the face of the suggested growth disadvantage of those cells, this statement needs clarification.

To avoid confusion, we have removed the statement.

(3) Regarding the fidelity of the model system, it is not clear to me how the TagBFP expression was detected in the BFP+ population supposedly in d87 and d136 pooled astrocytes (Fig S6C) while no LMX1A expression was observed in the same cells (Fig S6F).

The TagBFP tracer is expressed in the progenies of LMX1A+ cells, regardless of their LMX1A expression status. We have gone through the MS text to ensure that this information has been provided.

(4) The generated single-cell RNASeq dataset is extremely valuable. However, given the number of conditions included in this study (i.e. early vs late astrocytes, BFP+ vs BFP-, sorted vs unsorted, plus non-patterned and neuronal samples) the resulting analysis lacks detail. For instance, from a developmental perspective and to better grasp the functional significance of astrocytic heterogeneity, it would be interesting to map the identified clusters to early vs late populations and to the BFP status.

We performed additional bioinformatics analysis, which provided independent support for the relative developmental maturity suggested by functional assays. The additional data are now provided in the revised Figure 3B, C, E.

Moreover, although comprehensive, Figure S7 is complex to understand given that citations rather than the reference populations are depicted.

The information provided in the revised Figure S7.

(5) Do the authors have any consideration regarding the morphology of the astrocytes obtained in this study? None of the late astrocyte images depict a prototypical stellate morphology, which is reported in many other studies involving the generation of iPSC-derived astrocytes and which is associated with the maturity status of the cell.

The morphology of our astrocytes was not unique to the present study. Many factors may influence the morphology of astrocytes, such as the culture media and supplements used, and maturity status. Based on the functional assays and limited GFAP expression, our astrocytes were relatively immature.

• Informativo nº 847
• 15 de abril de 2025.
• RECURSOS REPETITIVOS
• Processo:

REsp 2.129.162-MG, Rel. Ministro Paulo Sérgio Domingues, Primeira Seção, por unanimidade, julgado em 9/4/2025. (Tema 1298).

REsp 2.131.059-MG, Rel. Ministro Paulo Sérgio Domingues, Primeira Seção, por unanimidade, julgado em 9/4/2025 (Tema 1298).

Ramo do Direito DIREITO ADMINISTRATIVO, DIREITO PROCESSUAL CIVIL

TemaPaz, Justiça e Instituições Eficazes

Ação de desapropriação por utilidade pública ou de constituição de servidão administrativa. Desistência. Honorários sucumbenciais. Limites percentuais do art. 27, § 1º, do DL n. 3.365/41. Incidência. Base de cálculo dos honorários. Valor atualizado da causa. Arbitramento por apreciação equitativa (Art. 85, § 8º, do CPC). Cabimento apenas quando o valor da causa é muito baixo. Tema 1298.

Destaque - Aplicam-se os percentuais do art. 27, § 1º, do DL n. 3.365/1941 no arbitramento de honorários sucumbenciais devidos pelo autor em caso de <u>desistência</u> de ação de desapropriação por utilidade pública ou de <u>constituição de servidão administrativa</u>, os quais terão como base de cálculo o valor atualizado da causa. Esses percentuais não se aplicam somente se o valor da causa for muito baixo, caso em que os honorários serão arbitrados por apreciação equitativa do juiz, na forma do art. 85, § 8º, do CPC.

Informações do Inteiro Teor - Cinge-se a controvérsia em definir se os limites percentuais previstos no art. 27, § 1º, do Decreto-Lei n. 3.365/1941 devem ser observados no arbitramento de honorários sucumbenciais em caso de desistência de ação de desapropriação por utilidade pública ou de constituição de servidão administrativa.

• A previsão do art. 27, § 1º, do DL n. 3.365/1941 veio para estabelecer normas especiais para os honorários advocatícios em ações expropriatórias seja quanto à base de cálculo de tal verba, seja quanto aos percentuais que devem incidir sobre a base arbitrada. Embora amalgamadas em um único preceito (texto), subsiste relativa independência entre as normas jurídicas contidas no dispositivo legal, de modo que alterações circunstanciais na base de cálculo não devem afastar, obrigatoriamente, a incidência da lex specialis relativa aos percentuais estabelecidos para o arbitramento dos honorários advocatícios.

• Assim, em havendo desistência da ação de desapropriação ou de constituição de servidão administrativa, é evidente que cai por terra a possibilidade de arbitramento dos honorários sucumbenciais tomando por base de cálculo a diferença entre o preço ofertado pelo expropriante e a indenização fixada na sentença, tal como previsto em norma especial inserida no texto do art. 27, § 1º, do DL n. 3.365/1941, uma vez que a sentença, nessa excepcional circunstância, não estabelecerá indenização alguma.

• Nesse cenário ocasional, embora não haja condenação, o princípio da causalidade impõe que o ente (não mais) expropriante seja declarado sucumbente de modo que os honorários correrão a sua conta, porque deu causa ao ajuizamento da demanda e dela desistiu (art. 90 do Código de Processo Civil).

• À falta de condenação ou de proveito econômico efetivo, já foi dito que não há suporte jurídico para o estabelecimento da base de cálculo dos honorários nos moldes do art. 27, § 1º, do DL 3.365/1941, de modo que essa base será fixada de acordo com norma jurídica supletiva prevista no art. 85, § 2º, do CPC, tomando-se em conta, então, o valor atribuído à causa.

• O socorro à norma supletiva do CPC faz-se porque não existe suporte jurídico para a aplicação da norma especial do DL 3.365/1941 apenas no que toca à base de cálculo dos honorários sucumbenciais. Ora, a desistência da ação não implica desaparecimento do suporte jurídico de aplicação dessa lex specialis, de modo que não há razão jurídica para se recorrer, quanto aos percentuais, a outras normas jurídicas que pudessem ser aplicadas de forma supletiva ou subsidiária.

• Dessarte, mesmo em caso de desistência da ação expropriatória, os percentuais a serem observados devem ser os estabelecidos no art. 27, § 1º, do DL3.365/1941.

• Ressalte-se, contudo, que haverá casos em que o valor da causa, mesmo que atualizado, corresponderá a valor ínfimo a implicar honorários irrisórios caso aquele valor seja mantido como base para a incidência das alíquotas do art. 27, § 1º, do DL n. 3.365/1941.

• Nessa excepcional hipótese, portanto, afasta-se completamente a aplicação do art. 27, § 1º, do DL n. 3.365/1941 para a fixação dos honorários sucumbenciais - seja quanto à base de cálculo estabelecida no preceito, seja quanto aos percentuais ali estabelecidos -, uma vez que a verba honorária será arbitrada pelo juiz por apreciação equitativa, com fundamento no art. 85, § 8º, do CPC, a fim de impedir que a verba honorária seja fixada em patamar incompatível com a dignidade do trabalho advocatício.

• Dessa forma, deve ser fixada a seguinte tese jurídica de eficácia vinculante: Aplicam-se os percentuais do art. 27, § 1º, do DL 3.365/41 no arbitramento de honorários sucumbenciais devidos pelo autor em caso de desistência de ação de desapropriação por utilidade pública ou desconstituição de servidão administrativa, os quais terão como base de cálculo o valor atualizado da causa. Esses percentuais não se aplicam somente se o valor da causa for muito baixo, caso em que os honorários serão arbitrados por apreciação equitativa do juiz, na forma do art. 85, § 8º, do CPC.

• Informativo nº 724
• 14 de fevereiro de 2022.
• PRIMEIRA SEÇÃO
• Compartilhe:
• Processo: CC 174.764-MA, Rel. Min. Mauro Campbell Marques, Primeira Seção, por unanimidade, julgado em 09/02/2022.

Ramo do Direito DIREITO ADMINISTRATIVO, DIREITO CONSTITUCIONAL, DIREITO PROCESSUAL CIVIL

TemaPaz, Justiça e Instituições Eficazes

Conflito negativo de competência. Juízos estadual e federal. Ação de improbidade administrativa ajuizada por ente municipal. Prestação de contas de verbas federais. Mitigação das súmulas 208/STJ e 209/STJ. Competência cível da Justiça Federal absoluta em razão da pessoa. Art. 109, I, da CF. Ausência de ente federal em qualquer dos polos da relação processual. Competência da Justiça Estadual.

Destaque - Nas ações de improbidade administrativa, a competência da Justiça Federal é definida em razão da presença das pessoas jurídicas de direito público previstas no art. 109, I, da Constituição Federal na relação processual, e não em razão da natureza da verba federal sujeita à fiscalização da Tribunal de Contas da União.

Informações do Inteiro Teor - No caso, o ente municipal ajuizou ação de improbidade administrativa, em razão de irregularidades na prestação de contas de verbas federais decorrentes de convênio.

• A competência para processar e julgar ações de ressarcimento ao erário e de improbidade administrativa, relacionadas à eventuais irregularidades na utilização ou prestação de contas de repasses de verbas federais aos demais entes federativos, estava sendo dirimida por esta Corte Superior sob o enfoque das Súmulas 208/STJ ("Compete à Justiça Federal processar e julgar prefeito municipal por desvio de verba sujeita à prestação de contas perante órgão federal") e 209/STJ ("Compete à Justiça Estadual processar e julgar prefeito por desvio de verba transferida e incorporada ao patrimônio municipal").

• O art. 109, I, da Constituição Federal prevê, de maneira geral, a competência cível da Justiça Federal, delimitada objetivamente em razão da <u>efetiva</u> presença da União, entidade autárquica ou empresa pública federal, na condição de autoras, rés, assistentes ou oponentes na relação processual. Estabelece, portanto, competência absoluta em razão da pessoa (ratione personae), configurada pela presença dos entes elencados no dispositivo constitucional na relação processual, independentemente da natureza da relação jurídica litigiosa.

• Por outro lado, o art. 109, VI, da Constituição Federal dispõe sobre a competência penal da Justiça Federal, especificamente para os crimes praticados em detrimento de bens, serviços ou interesse da União, entidades autárquicas ou empresas públicas. Assim, para reconhecer a competência, em regra, bastaria o <u>simples interesse</u> da União, inexistindo a necessidade da efetiva presença em qualquer dos polos da demanda.

• Nesse contexto, a aplicação dos referidos enunciados sumulares, em processos de natureza cível, tem sido mitigada no âmbito deste Tribunal Superior. A Segunda Turma afirmou a necessidade de uma distinção (distinguishing) na aplicação das Súmulas 208 e 209 do STJ, no âmbito cível, pois tais enunciados provêm da Terceira Seção deste Superior Tribunal, e versam hipóteses de fixação da competência em matéria penal, em que basta o interesse da União ou de suas autarquias para deslocar a competência para a Justiça Federal, nos termos do inciso IV do art. 109 da CF. Logo adiante concluiu que a competência da Justiça Federal, em matéria cível, é aquela prevista no art. 109, I, da Constituição Federal, que tem por base critério objetivo, sendo fixada tão só em razão dos figurantes da relação processual, prescindindo da análise da matéria discutida na lide (REsp 1.325.491/BA, Rel. Ministro Og Fernandes, Segunda Turma, julgado em 05/06/2014, DJe 25/06/2014).

• Assim, nas ações de ressarcimento ao erário e improbidade administrativa ajuizadas em face de eventuais irregularidades praticadas na utilização ou prestação de contas de valores decorrentes de convênio federal, <u>o simples fato das verbas estarem sujeitas à prestação de contas perante o Tribunal de Contas da União, por si só, não justifica a competência da Justiça Federal</u>.

• O Supremo Tribunal Federal já afirmou que o fato dos valores envolvidos transferidos pela União para os demais entes federativos estarem eventualmente sujeitos à fiscalização do Tribunal de Contas da União não é capaz de alterar a competência, pois a competência cível da Justiça Federal exige o efetivo cumprimento da regra prevista no art. 109, I, da Constituição Federal.

• Igualmente, a mera transferência e incorporação ao patrimônio municipal de verba desviada, no âmbito civil, não pode impor de maneira absoluta a competência da Justiça Estadual. Se houver manifestação de interesse jurídico por ente federal que justifique a presença no processo, (v.g. União ou Ministério Público Federal) regularmente reconhecido pelo Juízo Federal nos termos da Súmula 150/STJ, a competência para processar e julgar a ação civil de improbidade administrativa será da Justiça Federal.

• Em síntese, é possível afirmar que a competência cível da Justiça Federal é definida em razão da presença das pessoas jurídicas de direito público previstas no art. 109, I, da CF na relação processual, seja como autora, ré, assistente ou oponente e não em razão da natureza da verba federal sujeita à fiscalização da Corte de Contas da União.

• No caso, não figura em nenhum dos pólos da relação processual ente federal indicado no art. 109, I, da Constituição Federal, o que afasta a competência da Justiça Federal para processar e julgar a referida ação. Ademais, não existe nenhuma manifestação de interesse em integrar o processo por parte de ente federal e o Juízo Federal consignou que o interesse que prevalece restringe-se à órbita do Município autor, o que atrai a competência da Justiça Estadual para processar e julgar a demanda.

Obs.: Ou seja, para fins de fixação de competência, não basta a mera indicação da origem do recurso (se estadual ou se federal). Para haver competência da Justiça Federal, no âmbito cível, deve haver efetiva presença da União; não basta mero interesse da União como ocorre na seara penal. Importante destacar que as súmulas 208 e 209, com indicado, provêm da seção especializada em Direito Criminal.

Een causaal verband kan alleen worden aangetoond als buitenexperimentele factoren uitgeschakeld zijn dan wel onder controle Maatregelen: 1a. Manipulatie van de onafhankelijke variabele 1b. Vergelijkingsbasis creëren 2. Zorg voor een eerlijke vergelijking: randomiseren, matchen of counterbalancen

Ato da autoridade jurídica máxima poderá determinar a dispensa de análise jurídica prévia para licitações de baixo valor e complexidade em hipóteses previamente definidas.

Após a homologação, acaso haja documentos da fase preparatória não disponibilizados, serão publicados no PNCP mesmo que não integrante do edital e seus anexos.

DOI: 10.1101/2025.02.21.639590

Resource: (DSHB Cat# 3A9 (323 or M10-2), RRID:AB_528473)

Curator: @scibot

SciCrunch record: RRID:AB_528473

What is this?

DOI: 10.1016/j.ebiom.2025.105943

Resource: RRID:IMSR_JAX:000664

Curator: @scibot

SciCrunch record: RRID:IMSR_JAX:000664

What is this?

DOI: 10.1016/j.cmet.2025.08.009

Resource: (R and D Systems Cat# AB-450-NA, RRID:AB_354363)

Curator: @scibot

SciCrunch record: RRID:AB_354363

What is this?

explain the fast growth in this period will ya?

URLのjaありとなしが混在しているのでどっちかに統一して欲しい

The footnote text is very small and light grey on a white background that's all the way at the bottom of the iPhone homepage. This goes against the accessibility guideline of clearly distinguishing foreground from background. It is an example of bad practice.

el capital ve a la naturaleza como recurso ilimitado y explotable, pero es todo lo contrario, destruyendo la bases de los materiales. el capital depende de la naturaleza pero a la vez la destruye

cada innovación técnica busca ahorrar costos de trabajo humano y aumentar la productividad, esto provoca desempleo, inseguridad laboral. en vez de usarse como herramientas, se ocupa mas en la sustitución en el trabajo humano.

el capitalismo impulsa la competencia para crear innovación y eficiencia, esto mismo genera monopolios y concentración de mercado que eliminan la misma competencia

el capital depende del trabajo para generar plusvalia, pero el mismo capital buscar pagar menos por el trabajo. trabajador crea riqueza, el capitalista, se apropia la riqueza.

aqui el trabajo es el origen del valor, pero existe solamente si se expresa en dinero. es un mediador universal y eso es mas importante que el trabajo mismo.

lo humano queda subordinado a lo abstracto, lo cual genera una distancia bruta entre la riqueza y su reconocimiento.

valor de uso y valor de cambio. el valor como objetivo de satisfacer necesidades, solo lo que se domina es que tenga precio y se pueda vender

This is a good visualization as these are areas that can help contribute to teaching students more effectively by targeting what they know or don't know and they can learn them.

• Informativo nº 834
• 26 de novembro de 2024.
• SEGUNDA TURMA
• Compartilhe:
• Processo: AgInt no RMS 66.132-RS, Rel. Ministro Afrânio Vilela, Segunda Turma, por unanimidade, julgado em 12/11/2024, DJe 18/11/2024.

Ramo do Direito DIREITO ADMINISTRATIVO

TemaPaz, Justiça e Instituições Eficazes <br /> Servidor Público. Aposentadoria voluntária com proventos integrais. Regra de transição prevista no art. 3º da EC n. 47/2005. Data do ingresso no serviço público. Regime celetista em Fundação prestadora de serviço público. Não abrangência pela regra de transição.

Destaque - A regra de transição prevista no art. 3º, caput, da EC n. 47/2005, a qual garantiu aposentadoria com proventos integrais a servidor que tenha ingressado no serviço público anteriormente a 16/12/1998, não se aplica à prestação de serviço em fundação pública sob o <u>regime celetista</u> e por meio de <u>contrato administrativo</u>.

Informações do Inteiro Teor - A controvérsia cinge-se a aferir se o período laborado em Fundação prestadora de serviço público, como celetista, antes de ser ocupante de cargo efetivo, deve ser considerado como tempo de efetivo serviço para o fim de aposentadoria com proventos integrais, nos termos do previsto no art. 3º, caput, da Emenda Constitucional n. 47/2005.

• O caput do art. 3º da EC n. 47/2005, expressamente, garante o direito à aposentadoria com proventos integrais ao servidor "da União, dos Estados, do Distrito Federal e dos Municípios, incluídas suas autarquias e fundações" cujo ingresso no serviço público tenha ocorrido anteriormente a 16/12/1998.

• Não se contesta a natureza de serviço público prestado em Fundação estadual (pessoa jurídica de direito privado). O que se discute é a existência de vínculo efetivo, com o fim de integralizar a aposentadoria pelo regime próprio.

• Despicienda a natureza jurídica da Fundação estadual, cujo serviço prestado era inequivocamente de caráter público. O que importa para a solução da controvérsia é a natureza do vínculo empregatício.

• O trabalho junto à Fundação se deu por meio de contrato administrativo, regido pela CLT e com contribuição, portanto, ao Regime Geral da Previdência Social - RGPS. Embora o tempo laborado junto à Fundação seja computado para sua aposentadoria, a contribuição naquele período difere-se daquela como servidor público concursado e não é apta a integralizar a sua aposentadoria voluntária como almejado.

• Assim, conclui-se que a regra prevista no art. 3º da EC n. 47/2005 destina-se aos servidores ocupantes de <u>cargo efetivo</u>. A expressão "ingresso no serviço público" refere-se à investidura em cargo público, nos termos do art. 37 da Constituição Federal, que expressamente prevê, no inciso II, que "a investidura em cargo ou emprego público depende de aprovação prévia em concurso público".

Obs.: O precedente traz à discussão o direito de servidor poder se aposentar com proventos integrais. Mas a EC delimitou que esse direito somente é aplicável para o servidor efetivo que tenha ingressado no serviço público até 16 de dezembro de 1998, inclusive efetivos alocados em fundações e autarquias.

No entanto, o caso concreto julgado versa sobre pessoa que, embora tenha ingressado no serviço público anteriormente a 16/12/1998, não ingressou na qualidade de efetivo, mas sim como servidor regido pela CLT. Logo, descaracteriza-se o direito à aposentadoria por proventos integrais.

• Informativo 1.190
• RE 1469887 / AL - Tema 1.424
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. PRESIDENTE
• Julgamento: 12/09/2025 (Virtual)
• Ramo do Direito: Administrativo
• Matéria: Militar; Concurso Público; Requisitos Básicos para a Investidura; Altura Mínima

Polícia Militar: altura mínima para investidura em cargo da carreira Tese fixada - A exigência de altura mínima para ingresso em cargo do Sistema Único de Segurança Pública pressupõe a existência de lei e da observância dos parâmetros fixados para a carreira do exército (Lei federal nº 12.705/2012, 1,60m para homens e 1,55m para mulheres).

Resumo - É inconstitucional — por violar o princípio da razoabilidade — lei estadual que exige, como requisito para ingresso na Polícia Militar, altura mínima superior à prevista para ingresso nas carreiras do Exército.

• A imposição, pelo legislador estadual, de requisitos mais rigorosos do que os previstos na legislação federal para o Exército, sem qualquer justificativa relacionada às atribuições do cargo, configura afronta aos princípios da razoabilidade e da proporcionalidade (1).
• Na espécie, o Tribunal de Justiça do Estado de Alagoas manteve a eliminação de candidata em concurso público para a Polícia Militar por ela não possuir a altura mínima de 1,65m exigida pela legislação estadual (2).
• Com base nesses entendimentos, o Plenário, por unanimidade, reconheceu a existência de repercussão geral da questão constitucional suscitada (Tema 1.424 da repercussão geral) e, no mérito, por maioria: (i) reafirmou a jurisprudência dominante sobre a matéria (3); (ii) deu provimento ao recurso extraordinário para reformar o acórdão recorrido e determinar o prosseguimento da candidata no concurso público; e, por fim, (iii) fixou a tese anteriormente citada.

(1) Lei nº 12.705/2012: “Art. 2º A matrícula para o ingresso nos cursos de formação de oficiais e sargentos de carreira do Exército depende de aprovação prévia em concurso público, atendidos os seguintes requisitos, dentre outros estabelecidos na legislação vigente: (...) XIII - ter altura mínima de 1,60 m (um metro e sessenta centímetros) ou, se do sexo feminino, a altura mínima de 1,55 m (um metro e cinquenta e cinco centímetros).” (2) Lei nº 5.346/1992 do Estado de Alagoas: “Art. 7º O ingresso na Polícia Militar do Estado de Alagoas é facultado a todos os brasileiros, sem distinção de raça, sexo, cor ou credo religioso, mediante matrícula ou nomeação, após aprovação em concurso público de prova ou provas e títulos, desde que observadas as seguintes condições: (...) III – altura mínima de 1,65m (um metro e sessenta e cinco centímetros), se do sexo masculino, e 1,60m (um metro e sessenta centímetros), se do sexo feminino;” (3) Precedentes citados: ADI 5.044, ARE 1.459.395 AgR, RE 1.465.829 AgR e RE 1.480.201, bem como ARE 1.562.570, ARE 1.511.877 e RE 1.500.883 (decisões monocráticas).

Legislação: Lei nº 12.705/2012: art. 2º, XIII. Lei nº 5.346/1992 do Estado de Alagoas: art. 7º, III.

Precedentes: ADI 5.044, ARE 1.459.395 AgR, RE 1.465.829 AgR e RE 1.480.201, bem como ARE 1.562.570, ARE 1.511.877 e RE 1.500.883 (decisões monocráticas).

• Informativo 1163
• ADI 3516 / CE
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. EDSON FACHIN
• Julgamento: 13/12/2024 (Virtual)
• Ramo do Direito: Administrativo
• Matéria: Servidor Público; Inativos e Pensionistas; Gratificações; Proibição de Vinculação da Receita de Impostos; Atividades de Administração Tributária; Princípio da Eficiência

Impossibilidade de vinculação de receita de imposto a pagamento de Prêmio por Desempenho Fiscal a inativos e pensionistas

Resumo - São inconstitucionais — pois afrontam o art. 167, IV, da CF/1988 — dispositivos de lei estadual que vinculam a receita de impostos ao pagamento de Prêmio por Desempenho Fiscal (PDF) ou de gratificação a inativos e pensionistas.

• A ressalva contida no dispositivo acima citado (1) autoriza a vinculação da receita tributária ao pagamento do PDF apenas aos servidores <u>em atividade</u> na administração tributária. Ela tem respaldo no princípio da eficiência (CF/1988, art. 37, caput), na medida em que visa ao aumento da produtividade dos fiscais, e se fundamenta no incremento da arrecadação, no alcance de metas fixadas em regulamento, bem como na instituição de programas de qualidade e produtividade no serviço público, a ser viabilizado sob a forma de adicional ou prêmio de produtividade (2).

• À luz do caráter contributivo do sistema previdenciário, a concessão de vantagem remuneratória a servidores inativos sem o devido desconto da contribuição previdenciária também é inconstitucional, sob pena de desvirtuamento do equilíbrio atuarial e financeiro.

• Com base nesses e em outros entendimentos, o Plenário, por unanimidade, conheceu em parte da ação e, nessa extensão, a julgou parcialmente procedente para declarar a inconstitucionalidade dos arts. 1º, § 1º; 1º-A e 5º-A, da Lei nº 13.439/2004, com a redação da Lei nº 14.969/2011, ambas do Estado do Ceará (3).

(1) CF/1988: “Art. 167. São vedados: (...) IV - a vinculação de receita de impostos a órgão, fundo ou despesa, ressalvadas a repartição do produto da arrecadação dos impostos a que se referem os arts. 158 e 159, a destinação de recursos para as ações e serviços públicos de saúde, para manutenção e desenvolvimento do ensino e para realização de atividades da administração tributária, como determinado, respectivamente, pelos arts. 198, § 2º, 212 e 37, XXII, e a prestação de garantias às operações de crédito por antecipação de receita, previstas no art. 165, § 8º, bem como o disposto no § 4º deste artigo;”

(2) CF/1988: “Art. 39. (...) § 7º Lei da União, dos Estados, do Distrito Federal e dos Municípios disciplinará a aplicação de recursos orçamentários provenientes da economia com despesas correntes em cada órgão, autarquia e fundação, para aplicação no desenvolvimento de programas de qualidade e produtividade, treinamento e desenvolvimento, modernização, reaparelhamento e racionalização do serviço público, inclusive sob a forma de adicional ou prêmio de produtividade.”

(3) Lei nº 13.439/2004 do Estado do Ceará: “Art. 1º Fica instituído para os servidores públicos ativos, integrantes do Grupo Ocupacional Tributação, Arrecadação e Fiscalização - TAF, o Prêmio por Desempenho Fiscal - PDF, a ser concedido mensalmente, desde que implementadas as condições previstas para a sua concessão, nos valores e limites fixados nesta Lei, com o objetivo de estimular os aumentos de produtividade da Secretaria da Fazenda que impliquem no incremento. (Redação dada pela Lei n.º 14.969, de 01.08.11) I - da arrecadação tributária anual, inclusive multas e juros e outras receitas previstas na legislação tributária; II - de outros indicadores de desempenho referidos nesta Lei ou que venham a ser estabelecidos em regulamento. § 1º. O Prêmio por Desempenho Fiscal (PDF) de que trata o caput será extensivo a pensionistas de servidores fazendários, conforme disposto em regulamento. (...) Art. 1º-A Aos aposentados na data da publicação desta Lei e aos que estejam em processo de aposentadoria instaurados nesta mesma data, bem como aos pensionistas de ex-servidores fazendários é devida gratificação em substituição ao valor percebido no mesmo título, na data de vigência desta Lei, totalmente desvinculado da sistemática de apuração e distribuição prevista na Lei n° 13.439, de 16 de janeiro de 2004, correspondente a 97,34% (noventa e sete vírgula trinta e quatro por cento) do valor da 1ª Classe, referência ‘C’ da Tabela B, do anexo III, da Lei n° 13.778, de 6 de junho de 2006, com a redação dada pela Lei n° 14.350, de 19 de maio de 2009, e alterações posteriores, observando-se, para os pensionistas, a proporcionalidade da pensão, submetida exclusivamente à revisão geral dos servidores, a serem custeados com recursos do PDF, Grupo I, conforme disposição em regulamento. Parágrafo único. No prazo de 90 (noventa) dias, a contar da data da publicação da presente Lei, a Secretaria da Fazenda - SEFAZ, juntamente com a Secretaria de Planejamento e Gestão – SEPLAG, e Procuradoria Geral do Estado-PGE, deverão apresentar os atos normativos e legais necessários à realização dos ajustes dos atos de aposentadoria, concedidas até a data de publicação desta Lei. (redação dada pela Lei n.º 14.969, de 01.08.11) (Revogado pela Lei n.º 17.393, de 26/02/2021) (...) Art. 5º-A O Prêmio por Desempenho Fiscal - PDF, será devido ao servidor efetivo do grupo TAF que venha a se aposentar após a publicação desta Lei, nos seguintes termos: I – aos servidores que implementarem as regras dos arts. 3º ou 6º da Emenda Constitucional nº 41, de 19 de dezembro de 2003, ou do art. 3º da Emenda Constitucional nº 47, de 5 de julho de 2005, o Prêmio por Desempenho Fiscal – PDF, será calculado pela média aritmética simples de valores mensais percebidos, a esse título, pelo servidor fazendário nos 24 (vinte e quatro) meses anteriores ao pedido de aposentadoria; II – para os servidores que implementarem as regras dos arts. 3º ou 6º da Emenda Constitucional nº 41, de 19 de dezembro de 2003, ou do art. 3º da Emenda Constitucional nº 47, de 5 de julho de 2005, cujo período de percepção por ocasião do pedido de aposentadoria seja menor do que 24 (vinte e quatro) meses, será observada a média aritmética do período de percepção, multiplicado pela fração cujo numerador será o número correspondente ao total de meses trabalhado e o denominador será sempre o numeral 24; III – para os que implementarem os requisitos de aposentadoria previstos no art. 40, da Constituição Federal, com a redação dada pela Emenda Constitucional nº 41, de 19 de dezembro de 2003, nos termos da legislação federal. Parágrafo único. Nas hipóteses dos incisos I e II deste artigo, o PDF não poderá ser inferior ao limite mínimo definido no art. 4º-A desta Lei. (Redação dada pela Lei n.º 14.969, de 01.08.11)”

Legislação: CF/1988: art. 37, caput, XXII; art. 39, § 7º; art. 165, §§ 4º e 8º; art. 167, IV; art. 198, § 2º; art. 212. Lei nº 13.439/2004 do Estado do Ceará: art. 1º, § 1º; 1º-A e 5º-A.

• Informativo nº 862
• 16 de setembro de 2025.
• SEGUNDA TURMA
• Processo: REsp 2.026.929-ES, Rel. Ministro Marco Aurélio Bellizze, Segunda Turma, por unanimidade, julgado em 9/9/2025.

Ramo do Direito DIREITO CIVIL

TemaPaz, Justiça e Instituições Eficazes <br /> Protestos. Ausência de comunicação prévia às autoridades competentes. Paralisação de diversas vias de acesso. Dano moral coletivo. Caracterização.

Destaque - A realização de protestos sem comunicação prévia às autoridades e com obstrução de diversas vias públicas de acesso à capital do Estado por lapso temporal considerável configura dano moral coletivo in re ipsa.

Informações do Inteiro Teor - Cinge-se a controvérsia em saber se a realização de protestos <u>sem comunicação prévia às autoridades</u> e com <u>paralisação de diversas vias de acesso à capital do Estado</u> configura dano moral coletivo, justificando a condenação ao pagamento de indenização.

• A configuração do dano moral coletivo requer que a conduta antijurídica afete intoleravelmente os valores e interesses coletivos fundamentais, mediante conduta de grave lesão.

• A jurisprudência desta Corte Superior firmou-se no sentido de que o dano moral coletivo se configura in re ipsa, ou seja, <u>independentemente</u> da comprovação de dor, sofrimento ou abalo psicológico.

• No caso, ficou demonstrado o abuso no exercício do direito de reunião, configurando ofensa intolerável aos interesses coletivos, capaz de ensejar a condenação por dano moral coletivo. Isso porque, a pretexto de defender seus associados, o sindicato olvidou-se de que <u>o exercício da cidadania pressupõe o respeito ao direito dos demais indivíduos</u>, tendo obstruído importantes vias públicas de acesso à capital do Estado por lapso temporal considerável, até mesmo com a interrupção total em uma delas, com o uso de material inflamável e a queima de pneus na via, colocando em risco não só a população em geral, mas os próprios manifestantes.

Conforme LC nº 141/2012, as aplicações mínimas em saúde por Estados e Municípios não deverão ser inferiores a: - 15% da RCL do Município; - 12% da RCL do Estado.

contribute your own thinking

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This work aims to elucidate the molecular mechanisms affected in hypoxic conditions, causing reduced cortical interneuron migration. They use human assembloids as a migratory assay of subpallial interneurons into cortical organoids and show substantially reduced migration upon 24 hours of hypoxia. Bulk and scRNA-seq show adrenomedullin (ADM) up-regulation, as well as its receptor RAMP2, confirmed atthe protein level. Adding ADM to the culture medium after hypoxic conditions rescues the migration deficits, even though the subtype of interneurons affected is not examined. However, the authors demonstrate very clearly that ineffective ADM does not rescue the phenotype, and blocking RAMP2 also interferes with the rescue. The authors are also applauded for using 4 different cell lines and using human fetal cortex slices as an independent method to explore the DLXi1/2GFP-labelled iPSC-derived interneuron migration in this substrate with and without ADM addition (after confirming that also in this system ADM is up-regulated). Finally, the authors demonstrate PKA-CREB signalling mediating the effect of ADM addition, which also leads to up-regulation of GABAreceptors. Taken together, this is a very carefully done study on an important subject - how hypoxia affects cortical interneuron migration. In my view, the study is of great interest.

Strengths:

The strengths of the study are the novelty and the thorough work using several culture methods and 4 independent lines.

Weaknesses:

The main weakness is that other genes regulated upon hypoxia are not confirmed, such that readers will not know until which fold change/stats cut-off data are reliable.

Reviewer #2 (Public review):

Summary

The manuscript by Puno and colleagues investigates the impact of hypoxia on cortical interneuron migration and downstream signaling pathways. They establish two models to test hypoxia, cortical forebrain assembloids, and primary human fetal brain tissue. Both of these models provide a robust assay for interneuron migration. In addition, they find that ADM signaling mediates the migration deficits and rescue using exogenous ADM.

Strengths:

The findings are novel and very interesting to the neurodevelopmental field, revealing new insights into how cortical interneurons migrate and as well, establishing exciting models for future studies. The authors use sufficient iPSC lines including both XX and XY, so the analysis is robust. In addition, the RNAseq data with re-oxygenation is a nice control to see what genes are changed specifically due to hypoxia. Further, the overall level of validation of the sequencing data and involvement of ADM signaling is convincing, including the validation of ADM at the protein level. Overall, this is a very nice manuscript.

Weaknesses:

I have a few comments and suggestions for the authors. See below.

Reviewer #3 (Public review):

Summary:

The authors aimed to test whether hypoxia disrupts the migration of human cortical interneurons, a process long suspected to underlie brain injury in preterm infants but previously inaccessible for direct study. Using human forebrain assembloids and ex vivo developing brain tissue, they visualized and quantified interneuron migration under hypoxic conditions, identified molecular components of the response, and explored the effect of pharmacological intervention (specifically ADM) on restoring the migration deficits.

Strengths:

The major strength of this study lies in its use of human forebrain assembloids and ex vivo prenatal brain tissue, which provide a direct system to study interneuron migration under hypoxic conditions. The authors combine multiple approaches: long-term live imaging to directly visualize interneuron migration, bulk and single-cell transcriptomics to identify hypoxia-induced molecular responses, pharmacological rescue experiments with ADM to establish therapeutic potential, and mechanistic assays implicating the cAMP/PKA/pCREB pathway and GABA receptor expression in mediating the effect. Together, this rigorous and multifaceted strategy convincingly demonstrates that hypoxia disrupts interneuron migration and that ADM can restore this defect through defined molecular mechanisms.

Overall, the authors achieve their stated aims, and the results strongly support their  conclusions. The work has a significant impact by providing the first direct evidence of hypoxia-induced interneuron migration deficits in the human context, while also nominating a candidate therapeutic avenue. Beyond the specific findings, the methodological platform - particularly the combination of assembloids and live imaging - will be broadly useful to the community for probing neurodevelopmental processes in health and disease.

Weaknesses:

The main weakness of the study lies in the extent to which forebrain assembloids

recapitulate in vivo conditions, as the migration of interneurons from hSO to hCO does not fully reflect the native environment or migratory context of these cells. Nevertheless, this limitation is tempered by the fact that the work provides the first direct observation of human interneuron migration under hypoxia, representing a major advance for the field. In addition, while the transcriptomic analyses are valuable and highlight promising candidates, more in-depth exploration will be needed to fully elucidate the molecular mechanisms governing neuronal migration and maturation under hypoxic conditions.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) The authors should examine if all cortical interneurons are affected by ADM or only subtypes (Parvalbumin/Somatostatin).

We thank the reviewer for raising this important question. In our study, we utilized the Dlx1/2b::eGFP reporter to broadly label cortical interneurons; however, this system does not distinguish specific interneuron subtypes. To address this, in the revised version of the manuscript we will use the single-cell RNA sequencing data and immunostainings to provide this information. Based on previous analyses from Birey et al (Cell Stem Cell, 2022), we expect interneurons within assembloids to express mostly calbindin (CALB2) and somatostatin (SST) at this in vitro stage of development; parvalbumin subtype appears later based on data from Birey et al (Nature, 2017) and more recently from Varela et al, (bioRxiv, 2025).

In parallel, we will analyze available scRNA-seq data from developing human primary brain tissue a similar age as the one used in the manuscript, and check whether these subtypes of interneurons are similar to the ones within assembloids.

(2) The authors should test more candidates from their bulk RNA-seq data with different fold changes for regulation after hypoxia, to allow the reader to judge at which cut-off the DEGs may be reproducible. This would make this database much more valuable for the field of hypoxia research.

We appreciate the reviewers’ thoughtful suggestion. In addition to the bulk RNA-seq analysis, we did validate several upregulated hypoxia-responsive genes with varying fold changes by qPCR; these include PDK1, PFKP, VEGFA (Figure S1). 

We go agree that in-depth investigation of specific cut-offs would be interesting, however, this could be the focus of a different manuscript.

Reviewer #2 (Recommendations for the authors):

(1) Can the authors comment on the possibility of inflammatory response pathways being activated by hypoxia? Has this been shown before? While not the focus of the manuscript, it could be discussed in the Discussion as an interesting finding and potential involvement of other cells in the Hypoxic response.

We thank the reviewer this important comment about inflammation. Indeed, hypoxia has been shown to activate the inflammatory response pathways. In various studies, it was found that HIF-1a can interact with NF-κB signaling, leading to the upregulation of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α (Rius et al., Cell, 2008; Hagberg et al., Nat Rev Neurol, 2015).

In our transcriptomics data (Figure 2D), and to the reviewers’ point, we identified enrichment of inflammatory signaling response following the hypoxic exposure. Since hSO at the time of analyses do contain astrocytes, we think these glia contribute to the observed pro-inflammatory changes. Based on these results and because ADM is known to have strong anti-inflammatory properties, the effects of ADM on hypoxic astrocytes should be investigated in future studies focused on hypoxia-induced inflammation. In the revision, we will address this comment in the discussion section and cite the appropriate papers.

(2) Could the authors comment on the mechanism at play here with respect to ADM and binding to RAMP2 receptors - is this a potential autocrine loop, or is the source of ADM from other cell types besides inhibitory neurons? Given the scRNA-seq data, what cell-to-cell mechanisms can be at play? Since different cells express ADM, there could be different mechanisms in place in ventral vs dorsal areas.

Based on our scRNA-seq data in hSOs showing significant upregulation of ADM expression in astrocytes and progenitors, we speculate that the primary mechanism is likely to involve paracrine interactions. However, we cannot exclude autocrine mechanisms with the included experiments. Dissecting these interactions in a cell-type specific manner could be an important focus for future ADM-related studies.

To address the question about the possible different mechanisms in ventral versus dorsal areas, in the revision we will plot and include in the figures the data about the cell-type expression of ADM and its receptors in hCOs.

(3) For data from Figure 6 - while the ELISA assays are informative to determine which pathways (PKA, AKT, ERK) are active, there is no positive control to indicate these assays are "working" - therefore, if possible, western blot analysis from assembloid tissue could be used (perhaps using the same lysates from Figure 3) as an alternative to validate changes at the protein level (however, this might prove difficult); further to this, is P-CREB activated at the protein level using WB?

We thank the reviewer for this comment and the observation. Although we did not include a traditional positive control in these ELISA assays, several lines of evidence indicate that the measurements are reliable. First, the standard curves behaved as expected, and all sample values fell within the assay’s dynamic range. Second, technical replicates showed low variability, and the observed changes across experimental conditions (e.g., hypoxia vs. control) were consistent with the expected biological responses based on previous literature. We agree that including western blot validation would strengthen the findings, and we will note this for our future studies focused on CREB and ADM.

(4) Could the authors comment further on the mechanism and what biological pathways and potential events are downstream of ADM binding to RAMP2 in inhibitory neurons? What functional impact would this have linked to the CREB pathway proposed? While the link to GABA receptors is proposed, CREB has many targets beyond this.

We appreciate the reviewers’ insightful question. Currently, not much is known about the molecular pathways and downstream cellular events triggered by ADM binding to RAMP2 in inhibitory neurons, and in general in brain cells. The data from our study brings the first information about the cell-type specific expression of ADM in baseline and hypoxic conditions and is one of the key novelties of our study.

While the signaling landscape of ADM in interneurons is largely unexplored, several studies in other (non-brain) cell types have demonstrated that ADM binding to RAMP2 can activate downstream cascades such as the cAMP/PKA/CREB pathway, PI3K/AKT, and ERK/MAPK, all of which are also known to be critical regulators of neuronal development and survival. These previously published data along with our CREB-targeted findings in hypoxic interneurons, suggest ADM–RAMP2 signaling could influence multiple aspects of interneuron biology, but these remain to be evaluated in future studies.

We agree with the reviewer that CREB has a wide range of transcriptional targets. We decided to focus on GABA as a target of CREB for two main reasons, including: (i) GABA signaling has been previously shown to play an important role in the migration of cortical interneurons, and (ii) a previous study by Birey et al. (Cell Stem Cell, 2022) demonstrated that CREB pathway activity is essential for regulating interneuron migration in assembloid models of Timothy Syndrom, thus further providing evidence that dysregulation of CREB activity disrupts migration dynamics.

While our study provides a first step toward uncovering the mechanisms of interneuron migration protection by ADM, we fully acknowledge that future work will be needed to delineate the full spectrum of ADM–RAMP2 downstream signaling events in inhibitory neurons and other brain cells.

(5) Does hypoxia cause any changes to inhibitory neurogenesis (earlier stages than migration?) - this might always be known, but was not discussed.

We appreciate this question from the reviewer; however, this was not something that we focused on in this manuscript due to the already large amount of data included. A separate study focusing on neurogenesis defects and the molecular mechanisms of injury for that specific developmental process would be an important next step.

(6) In the Discussion section, it might be worth detailing to the readers what the functional impact of delayed/reduced migration of inhibitory neurons into the cortex might result in, in terms of functional consequences for neural circuit development.

We thank the Reviewer for the suggestion of detailing the functional impact of reduced inhibitory neuron migration. We will revise the manuscript by incorporating a paragraph about this in the Discussion section.

Reviewer #3 (Recommendations for the authors):

Most of the evidence presented is convincing in supporting the conclusions, and I have only minor suggestions for improvement:

(1) The bulk RNA-seq was performed in hSOs only, which may not fully capture the phenotypes of migrating or migrated interneurons. It would be valuable, if feasible, to sort migrated cells from hSO-hCO assembloids and specifically examine their molecular mediators.

We thank the reviewer for this suggestion. While it is likely that the cellular environment will have some influence on a subset of the molecular changes, based on all the data from the manuscript and our specific target, the RNA-sequencing on hCOs was sufficient to capture essential changes like ADM upregulation. The in-depth exploration on differential responses of migrated versus non-migrated interneurons to hypoxia could be the focus of a different project.

(2) In Figure 3, it is striking that cell-type heterogeneity dominates over hypoxia vs. control conditions. A joint embedding of hSO and hCO cells could provide further insight into molecular differences between migrated and non-migrated interneurons.

We thank the reviewer for this observation and opportunity to clarify. Since we manually separated the assembloids before the analyses, we processed these samples separately. That is why they separate like this. In the revision, we will add data about ADM expression and its receptors’ expression in the hCOs.

(3) It would be helpful to expand the discussion on how closely the migration observed in hSO-hCO assembloids reflects in vivo conditions, and what environmental aspects are absent from this model. This would better frame the interpretation and translational relevance of the findings.

We thank the Reviewer for bringing up this important point. Although the assembloid model offers the unique advantage of allowing the direct investigation of migration patterns of hypoxic interneurons, we fully agree it does not fully recapitulate the in vivo environment. While there are multiple aspects that cannot be recapitulated in vitro at this time (e.g. cellular complexity, vasculature, immune response, etc), we are encouraged by the validation of our main findings in ex vivo developing human brain tissue, which strongly supports the validity of our findings for in vivo conditions.

We will expand our discussion to include more details and the need to validate these findings using in vivo models, while also acknowledging that different species (e.g. rodents versus non-human primates versus humans) might have different responses to hypoxia.

(4) The authors suggest that hypoxia is also associated with delayed interneuron maturation, yet the bulk RNA-seq data primarily reveal stress and hypoxia-related genes. A more detailed discussion of why genes linked to interneuron maturation and function were not strongly affected would clarify this point.

We thank the Reviewer for the opportunity to clarify.

The RNAseq data was performed during the acute stages of hypoxia/reoxygenation and we think a maturation phenotype might be difficult to capture at this point and would require analysis at later in vitro assembloid maturation stages.

Our speculation about a possible maturation defect is based on data from previous studies from developmental biology that showed failure of interneurons to reach their final cortical location within a specified developmental window will impair their integration within the neuronal network, and thus lead to maturation defects and possible elimination by apoptosis.

Since preterm infants suffer from countless hypoxic events over multiple months, we suggest these repetitive events are likely to induce cumulative delays in migration, inability of interneurons to reach their target in time, followed by abnormal integration within the excitatory network, and eventual elimination of some of these interneurons through apoptosis. However, the direct demonstration of this effect following a hypoxic insult would require prolonged in vivo experiments in rodents to follow the migration, network integration and apoptosis of interneurons; to our knowledge this experimental design is not technically feasible at this time.

(5) Relatedly, while the focus on interneuron migration is well justified, acknowledging how hypoxia might also impact other aspects of cortical development (e.g., progenitor proliferation, neuronal maturation, or circuit integration) would place the findings in a broader developmental framework and strengthen their relevance.

We appreciate the Reviewer’s suggestion to discuss the role of hypoxia on other processes during cortical development. In the revised manuscript, we will include citations about the effects of hypoxia on interneuron proliferation, maturation and circuit integration as available, and also expand to other cell types known to be affected.

(6) Very minor: in Figure S3C and D, it was not stated what the colors mean (grey: control, yellow: hypoxia)

Thank you for pointing out this error and we will correct it in our revision.

In "Researchers Are Willing to Trade Their Results for Journal Prestige: Results from a Discrete Choice Experiment", the authors investigate researchers’ publication preferences using a discrete choice experiment in a cross-sectional survey of international health and medical researchers. The study investigates publishing decisions in relation to negotiation of trade-offs amongst various factors like journal impact factor, review helpfulness, formatting requirements, and usefulness for promotion in their decisions on where to publish. The research is timely; as the authors point out, reform of research assessment is currently a very active topic. The design and methods of the study are suitable and robust. The use of focus groups and interviews in developing the attributes for study shows care in the design. The survey instrument itself is generally very well-designed, with important tests of survey fatigue, understanding (dominant choice task) and respondent choice consistency (repeat choice task) included. Respondent performance was good or excellent across all these checks. Analysis methods (pMMNL and latent class analysis) are well-suited to the task. Pre-registration and sharing of data and code show commitment to transparency. Limitations are generally well-described.

In the below, I give suggestions for clarification/improvement. Except for some clarifications on limitations and one narrower point (reporting of qualitative data analysis methods), my suggestions are only that – the preprint could otherwise stand, as is, as a very robust and interesting piece of scientific work.

1. Respondents come from a broad range of countries (63), with 47 of those countries represented by fewer than 10 respondents. Institutional cultures of evaluation can differ greatly across nations. And we can expect variability in exposure to the messages of DORA (seen, for example, in level of permeation of DORA as measured by signatories in each country, https://sfdora.org/signers/)..%3B!!NVzLfOphnbDXSw!HdeyeHHei6yWQHFjhN3deSSfp82ur9i9JNOLEVOYZN0BvyslUO2S8DlvjBbautmafJEvlUsxQZbT0JLQX7lO8EcOYtZsJkA%24&data=05%7C02%7Ca.l.brasil.varandas.pinto%40cwts.leidenuniv.nl%7C9f47a111adec49d04bb608dd0614ae94%7Cca2a7f76dbd74ec091086b3d524fb7c8%7C0%7C0%7C638673408085242099%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=by5mhPfSM0MFFG9LE2iiYjdtSs5IhvpuukqVv%2FLak2s%3D&reserved=0 "https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.com%2Fv3%2F__https%3A%2F%2Fsfdora.org%2Fsigners%2F).%3B!!NVzLfOphnbDXSw!HdeyeHHei6yWQHFjhN3deSSfp82ur9i9JNOLEVOYZN0BvyslUO2S8DlvjBbautmafJEvlUsxQZbT0JLQX7lO8EcOYtZsJkA%24&data=05%7C02%7Ca.l.brasil.varandas.pinto%40cwts.leidenuniv.nl%7C9f47a111adec49d04bb608dd0614ae94%7Cca2a7f76dbd74ec091086b3d524fb7c8%7C0%7C0%7C638673408085242099%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=by5mhPfSM0MFFG9LE2iiYjdtSs5IhvpuukqVv%2FLak2s%3D&reserved=0") In addition, some contexts may mandate or incentivise publication in some venues using measures including IF, but also requiring journals to be in certain databases like WoS or Scopus, or having preferred journal lists). I would suggest the authors should include in the Sampling section a rationale for taking this international approach, including any potentially confounding factors it may introduce, and then adding the latter also in the limitations.

2. Reporting of qualitative results: In the introduction and methods, the role of the focus groups and interviews seems to have been just to inform the design of the experiment. But then, results from that qualitative work then appear as direct quotes within the discussion to contextualise or explain results. In this sense though, the qualitative results are being used as new data. Given this, I feel that the methods section should include description of the methods and tools used for qualitative data analysis (currently it does not). But in addition, to my understanding (and this may be a question of disciplinary norms – I’m not a health/medicine researcher), generally new data should not be introduced in the discussion section of a research paper. Rather the discussion is meant to interpret, analyse, and provide context for the results that have already been presented. I personally hence feel that the paper would benefit from the qualitative results being reported separately within the results section.

3. Impact factors – Discussion section: While there is interesting new information on the relative trade-offs amongst other factors, the most emphasised finding, that impact factors still play a prominent role in publication venue decisions, is hardly surprising. More could perhaps be done to compare how the levels of importance reported here differ with previous results from other disciplines or over time (I know a like-for-like comparison is difficult but other studies have investigated these themes, e.g., https://doi.org/10.1177/01655515209585). In addition, beyond the question of whether impact factors are important, a more interesting question in my view is why they still persist. What are they used for and why are they still such important “driver[s] of researchers’ behaviour”? This was not the authors’ question, and they do provide some contextualisation by quoting their participants, but still I think they could do more to contextualise what is known from the literature on that to draw out the implications here. The attribute label in the methods for IF is “ranking”, but ranking according of what and for what? Not just average per-article citations in a journal over a given time frame. Rather, impact factors are used as a proxy indicators of less-tangible desirable qualities – certainly prestige (as the title of this article suggests), but also quality, trust (as reported by one quoted focus group member “I would never select a journal without an impact factor as I always publish in journals that I know and can trust that are not predatory”, p.6), journal visibility, importance to the field, or improved chances of downstream citations or uptake in news media/policy/industry etc. Picking apart the interactions of these various factors in researchers’ choices to make use of IFs (which is not in all cases bogus or unjustified) could add valuable context. I’d especially recommend engaging at least briefly with more work from Science and Technology Studies - especially Müller and de Rijcke’s excellent Thinking with Indicators study (doi: 10.1093/reseval/rvx023), but also those authors other work, as well as work from Ulrike Felt, Alex Rushforth (esp https://doi.org/10.1007/s11024-015-9274-5), Björn Hammerfelt and others.

4. Disciplinary coverage: (1) A lot of the STS work I talk about above emphasises epistemic diversity and the ways cultures of indicator use differ across disciplinary traditions. For this reason, I think it should be pointed out in the limitations that this is research in Health/Med only, with questions on generalisability to other fields. (2) Also, although the abstract and body of the article do make clear the disciplinary focus, the title does not. Hence, I believe the title should be slightly amended (e.g., “Health and Medical Researchers Are Willing to Trade …”)

Decreto nº 9.830/2019

Responsabilização na hipótese de dolo ou erro grosseiro

Art. 12. O agente público somente poderá ser responsabilizado por suas decisões ou opiniões técnicas se agir ou se omitir com dolo, direto ou eventual, ou cometer erro grosseiro, no desempenho de suas funções.

• § 1º Considera-se erro grosseiro aquele manifesto, evidente e inescusável praticado com culpa grave, caracterizado por ação ou omissão com elevado grau de negligência, imprudência ou imperícia.

• § 2º Não será configurado dolo ou erro grosseiro do agente público se não restar comprovada, nos autos do processo de responsabilização, situação ou circunstância fática capaz de caracterizar o dolo ou o erro grosseiro.

• § 3º O mero nexo de causalidade entre a conduta e o resultado danoso não implica responsabilização, exceto se comprovado o dolo ou o erro grosseiro do agente público.

• § 4º A complexidade da matéria e das atribuições exercidas pelo agente público serão consideradas em eventual responsabilização do agente público.

• § 5º O montante do dano ao erário, ainda que expressivo, não poderá, por si só, ser elemento para caracterizar o erro grosseiro ou o dolo.

• § 6º A responsabilização pela opinião técnica não se estende de forma automática ao decisor que a adotou como fundamento de decidir e somente se configurará se estiverem presentes elementos suficientes para o decisor aferir o dolo ou o erro grosseiro da opinião técnica ou se houver conluio entre os agentes.

• § 7º No exercício do poder hierárquico, só responderá por culpa in vigilando aquele cuja omissão caracterizar erro grosseiro ou dolo.

• § 8º O disposto neste artigo não exime o agente público de atuar de forma diligente e eficiente no cumprimento dos seus deveres constitucionais e legais.

Acórdão 1525/2025 Primeira Câmara (Tomada de Contas Especial, Relator Ministro Jhonatan de Jesus)

Responsabilidade. Culpa. Erro grosseiro. Omissão no dever de prestar contas. Débito. Multa.

• A omissão no dever de prestar contas constitui grave inobservância do dever de cuidado no trato com a coisa pública, revelando a existência de culpa grave, uma vez que se distancia do que seria esperado de um administrador médio, o que caracteriza erro grosseiro a que alude o art. 28 do Decreto-lei 4.657/1942 (Lindb), incluído pela Lei 13.655/2018, legitimando a condenação em débito do responsável e a aplicação da sanção pecuniária prevista no art. 57 da Lei 8.443/1992.

Acórdão 755/2025 Plenário (Tomada de Contas Especial, Relator Ministro Jhonatan de Jesus)

Responsabilidade. Culpa. Erro grosseiro. Conduta. Avaliação. Sanção.

• Para fins do exercício do poder sancionatório do TCU, o erro grosseiro a que alude o art. 28 do Decreto-lei 4.657/1942 (Lindb) fica configurado quando a conduta do agente público se distancia acentuadamente daquela que seria esperada do administrador médio, parâmetro que retrata o dever de cuidado objetivo esperado de um gestor comum, capaz e prudente.

Acórdão 1089/2025 - Plenário (Tomada de Contas Especial, Relator Ministro Benjamin Zymler)

Responsabilidade. Culpa. Parecerista. Fundamentação. Parecer jurídico.

• Os pareceres jurídicos desprovidos de fundamentação adequada, favoráveis a contratações manifestamente ilegais ou que deixem de considerar jurisprudência pacificada do TCU podem ensejar a responsabilização do seu autor, se o ato concorrer para eventual irregularidade praticada pela autoridade que nele se embasou.

Acórdão 2467/2025 - Segunda Câmara (Tomada de Contas Especial, Relator Ministro-Substituto Marcos Bemquerer)

Responsabilidade. Culpa. Erro grosseiro. Convênio. Execução física. Plano de trabalho.

• Para fins do exercício do poder sancionatório do TCU, considera-se erro grosseiro (art. 28 do Decreto-lei 4.657/1942 - Lindb) a execução do objeto conveniado em desacordo com o plano de trabalho aprovado pelo concedente, sem qualquer justificativa.

Acórdão 5284/2025 - Segunda Câmara (Recurso de Reconsideração, Relator Ministro Augusto Nardes)

Responsabilidade. Débito. Culpa. Dolo. Lei de Introdução às Normas do Direito Brasileiro. Erro grosseiro.

• A regra prevista no art. 28 do Decreto-lei 4.657/1942 (Lindb), que estabelece que o agente público só responderá pessoalmente por suas decisões ou opiniões técnicas em caso de dolo ou erro grosseiro, <u>não se aplica à responsabilidade financeira por dano ao erário</u>. O dever de indenizar prejuízos aos cofres públicos permanece sujeito à comprovação de dolo ou culpa, sem qualquer gradação, tendo em vista o tratamento constitucional dado à matéria (art. 37, § 6º, da Constituição Federal).

Reviewer #1 (Public review):

Summary:

This manuscript assesses the differences between young and aged chondrocytes. Through transcriptomic analysis and further assessments in chondrocytes, GATA4 was found to be increased in aged chondrocyte donors compared to young. Subsequent mechanistic analysis with lentiviral vectors, siRNAs, and a small molecule were used to study the role of GATA4 in young and old chondrocytes. Lastly, an in vivo study was used to assess the effect of GATA4 expression on osteoarthritis progression in a DMM mouse model.

Strengths:

This work linked the over expression of GATA4 to NF-kB signaling pathway activation, alterations to the TGF-b signaling pathway, and found that GATA4 increased the progression of OA compared to the DMM control group. Indicating that GATA4 contributes to the onset and progression of OA in aged individuals.

Comments on revised version:

Great work! All my concerns have been well addressed.

Reviewer #3 (Public review):

Summary:

This is an exciting, comprehensive paper that demonstrates the role of GATA4 on OA-like changes in chondrocytes. The authors present elegant reverse translational experiments that justify this mechanism and demonstrate the sufficiency of GATA4 in a mouse model of osteoarthritis (DMM), where GATA4 drove cartilage degeneration and pain in a manner that was significantly worse than DMM alone. This could pave the way for new therapies for OA that account for both structural changes and pain.

Strengths:

(1) GATA4 was identified from human chondrocytes.

(2) IHC and sequencing confirmed GATA4 presence.

(3) Activation of SMADs is clearly shown in vitro with GATA4 overexpression.

(4) The role of GATA4 was functionally assessed in vivo using the mouse DMM model, where the authors uncovered that GATA4 worsens OA structure and hyperalgesia in male mice.

(5) It is interesting that GATA4 is largely known to be found in cardiac cells and to have a role in cardiac repair, metabolism, and inflammation, among other things listed by the authors in the discussion (in liver, lung, pancreas). What could this new knowledge of GATA4 mean for OA as a potentially systemically mediated disease, where cardiac disease and metabolic syndrome are often co-morbid?

Weaknesses:

I do not have further comments. Thank you for addressing the previously mentioned concerns.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #2 (Public review):

The only aspect that would benefit from further clarification is a more detailed discussion of aging-associated ECM changes in the context of prior literature. 

Thank you. Please refer to the new section (Lines 604-617)

Reviewer #3 (Public review):

(1) It would be useful to explain why GATA4 was chosen over HIF1a, which was the most differentially expressed. 

Thank you. Please refer to Lines 530-537.  

“Of note, Hypoxia-Inducible Factor 1α (HIF1 α) was the most differentially expressed gene predicted to regulate chondrocyte aging. The connection between HIF1 α and aging has been previously reported.[32] Furthermore, additional studies have investigated HIF1 in association with OA and assessed its use as a therapeutic target.[33,34] Therefore, we decided to focus on GATA4, which was less studied in chondrocytes but highly associated with cellular senescence, an aging hallmark. However, our selection did not dampen the importance of HIF1α and other molecules listed in Figure 1D in chondrocyte aging. They can be further studied in the future using the same strategy employed in the current work.”

(2) In Figure 5, it would be useful to demonstrate the non-surgical or naive limbs to help contextualize OARSI scores and knee hyperalgesia changes. 

In the current study, we focused on the DMM control and DMM Gata4 virus groups so we did not include a sham control group. We recognized this was a limitation of this study.  

(3) While there appear to be GATA4 small-molecule inhibitors in various stages of development that could be used to assess the effects in age-related OA, those experiments are out of scope for the current study.  

We agree with this comment that the results are still preliminary, which was the reason that we put it in the supplementary materials. However, we felt like the result is informative, which will support the potential of GATA4 as a therapeutic target and inspire the development of more specific inhibitors. Therefore, we would still keep the results in the current study.

Reviewer #2 (Public review):

In this work Thapliyal and Glauser tried to provide mechanistic understanding by which animals modulate their neural circuit responses to control nociceptive behavior on the basis of the dynamic internal feeding state. It is an important study that adds to growing body of evidences coming from multiple model systems. They have used elegant genetics, behavioral and Ca-imaging experiments to demonstrate how the auxiliary thermosensory neuron pair, AWC and one of the internal state sensing interneuron pair, ASI, respond to dynamic internal starvation-state to modulate behavioral response to noxious heat. Interestingly, these neuron pairs use distinct molecular mechanisms along with some other unidentified neurons to suppress heat-indued reversal response under short-term and prolonged starvations. The experiments are well performed that support most of the claims and provide important framework for future studies.

I have some queries that if answered, will certainly enhance the study,

(1) The results suggests that ASI is one of the primary drivers for the starvation-evoked behavioral plasticity, which regulates AWC activity under prolonged starvation. It raises many important questions including, a) how starvation modulates ASI response to heat? b) under prolonged starvation, whether ASI also promotes other, non-AWC, glutamatergic inhibitory neurons to suppress heat-induced reversal and how?

(2) How does ASI regulate AWC activity? In the proposed model (figure 8) authors suggested an independent, unknown signal, other than INS-32 and NLP-18, from ASI to regulate AWC activity. However, from the results the existence of another signal is not very clear.

(3) Previously, Takeishi et. al., showed that ins-1 dynamically modulates AWC-AIA mediated thermotaxis behavior based on the feeding state of the animal. It raises questions whether ins-1 also contributes to noxious heat-induced reversal behavior.

(4) Experiments with AWC fate conversion mutants (nsy-1 and nsy-7) were very good ideas, however the results obtained were confusing. flp-6 mutant data suggests AWCoff would be essential for heat induced reversal, especially at the low intensity stimulus level. However, nsy-1 mutant forming two AWCon neurons showed complete rescue at the low heat level, which is quite opposite. Similarly, although less prominent, eat-4 rescue experiments suggested both nsy-1 and nsy-7 should behave normally at high heat condition, which was not the result observed.

• 1. O eventual acolhimento do pedido na ação de usucapião acarreta perda patrimonial imediata, ou seja, perda da propriedade do imóvel, gerando enorme prejuízo para os credores da massa falida. Assim, deve-se reconhecer a competência do juízo universal da falência para apreciar demandas dessa natureza.
• 2. "A arrecadação é ato de apreensão judicial executiva que visa à guarda e conservação dos bens do falido para futura alienação, em benefício dos credores. Sendo assim, nada mais coerente que todas as questões relacionadas aos bens arrecadados sejam decididas pelo juízo falimentar." (CC 84.752/RN, Rel. Ministra NANCY ANDRIGHI, SEGUNDA SEÇÃO, julgado em 27/06/2007, DJ 01/08/2007, p. 433)
• 3. Conflito de competência não conhecido em relação aos Juízos da 16ª e 17ª Varas Cíveis de Brasília/DF e, quanto ao incidente suscitado em face do Juízo da 11ª Vara Cível de Brasília/DF e do Tribunal de Justiça do Distrito Federal e dos Territórios, conflito conhecido para declarar a competência do Juízo da 11ª Vara Cível de Goiânia/GO. (CC n. 114.842/GO, relator Ministro Luis Felipe Salomão, Segunda Seção, julgado em 25/2/2015, DJe de 3/3/2015.)

1. Nos termos da jurisprudência consolidada pela Segunda Seção do STJ, no julgamento do CC 114.842/GO, em 25/02/2015, eventual acolhimento do pedido na ação de usucapião acarreta perda patrimonial imediata, ou seja, perda da propriedade do imóvel, gerando enorme prejuízo para os credores da massa falida, devendo ser reconhecida a competência do juízo universal da falência para apreciar demandas dessa natureza. (AgInt no REsp n. 2.004.910/CE, relatora Ministra Nancy Andrighi, Terceira Turma, julgado em 28/11/2022, DJe de 30/11/2022.)

Reviewer #1 (Public review):

Summary:

The authors state the study's goal clearly: "The goal of our study was to understand to what extent animal individuality is influenced by situational changes in the environment, i.e., how much of an animal's individuality remains after one or more environmental features change." They use visually guided behavioral features to examine the extent of correlation over time and in a variety of contexts. They develop new behavioral instrumentation and software to measure behavior in Buridan's paradigm (and variations thereof), the Y-maze, and a flight simulator. Using these assays, they examine the correlations between conditions for a panel of locomotion parameters. They propose that inter-assay correlations will determine the persistence of locomotion individuality.

Strengths:

The OED defines individuality as "the sum of the attributes which distinguish a person or thing from others of the same kind," a definition mirrored by other dictionaries and the scientific literature on the topic. The concept of behavioral individuality can be characterized as: (1) a large set of behavioral attributes, (2) with inter-individual variability, that are (3) stable over time. A previous study examined walking parameters in Buridan's paradigm, finding that several parameters were variable between individuals, and that these showed stability over separate days and up to 4 weeks (DOI: 10.1126/science.aaw718). The present study replicates some of those findings, and extends the experiments from temporal stability to examining correlation of locomotion features between different contexts.

The major strength of the study is using a range of different behavioral assays to examine the correlations of several different behavior parameters. It shows clearly that the inter-individual variability of some parameters is at least partially preserved between some contexts, and not preserved between others. The development of high-throughput behavior assays and sharing the information on how to make the assays is a commendable contribution.

Weaknesses:

The definition of individuality considers a comprehensive or large set of attributes, but the authors consider only a handful. In Supplemental Fig. S8, the authors show a large correlation matrix of many behavioral parameters, but these are illegible and are only mentioned briefly in Results. Why were five or so parameters selected from the full set? How were these selected? Do the correlation trends hold true across all parameters? For assays in which only a subset of parameters can be directly compared, were all of these included in the analysis, or only a subset?

The correlation analysis is used to establish stability between assays. For temporal re-testing, "stability" is certainly the appropriate word, but between contexts it implies that there could be 'instability'. Rather, instead of the 'instability' of a single brain process, a different behavior in a different context could arise from engaging largely (or entirely?) distinct context-dependent internal processes, and have nothing to do with process stability per se. For inter-context similarities, perhaps a better word would be "consistency".

The parameters are considered one-by-one, not in aggregate. This focuses on the stability/consistency of the variability of a single parameter at a time, rather than holistic individuality. It would appear that an appropriate measure of individuality stability (or individuality consistency) that accounts for the high-dimensional nature of individuality would somehow summarize correlations across all parameters. Why was a multivariate approach (e.g. multiple regression/correlation) not used? Treating the data with a multivariate or averaged approach would allow the authors to directly address 'individuality stability', along with the analyses of single-parameter variability stability.

The correlation coefficients are sometimes quite low, though highly significant, and are deemed to indicate stability. For example, in Figure 4C top left, the % of time walked at 23{degree sign}C and 32{degree sign}C are correlated by 0.263, which corresponds to an R2 of 0.069 i.e. just 7% of the 32{degree sign}C variance is predictable by the 23{degree sign}C variance. Is it fair to say that 7% determination indicates parameter stability? Another example: "Vector strength was the most correlated attention parameter... correlations ranged... to -0.197," which implies that 96% (1 - R2) of Y-maze variance is not predicted by Buridan variance. At what level does an r value not represent stability?

The authors describe a dissociation between inter-group differences and inter-individual variation stability, i.e. sometimes large mean differences between contexts, but significant correlation between individual test and retest data. Given that correlation is sensitive to slope, this might be expected to underestimate the variability stability (or consistency). Is there a way to adjust for the group differences before examining correlation? For example, would it be possible to transform the values to in-group ranks prior to correlation analysis?

What is gained by classifying the five parameters into exploration, attention, and anxiety? To what extent have these classifications been validated, both in general, and with regard to these specific parameters? Is increased walking speed at higher temperature necessarily due to increased 'explorative' nature, or could it be attributed to increased metabolism, dehydration stress, or a heat-pain response? To what extent are these categories subjective?

The legends are quite brief and do not link to descriptions of specific experiments. For example, Figure 4a depicts a graphical overview of the procedure, but I could not find a detailed description of this experiment's protocol.

Using the current single-correlation analysis approach, the aims would benefit from re-wording to appropriately address single-parameter variability stability/consistency (as distinct from holistic individuality). Alternatively, the analysis could be adjusted to address the multivariate nature of individuality, so that the claims and the analysis are in concordance with each other.

The study presents a bounty of new technology to study visually guided behaviors. The Github link to the software was not available. To verify successful transfer or open-hardware and open-software, a report would demonstrate transfer by collaboration with one or more other laboratories, which the present manuscript does not appear to do. Nevertheless, making the technology available to readers is commendable.

The study discusses a number of interesting, stimulating ideas about inter-individual variability, and presents intriguing data that speaks to those ideas, albeit with the issues outlined above.

While the current work does not present any mechanistic analysis of inter-individual variability, the implementation of high-throughput assays sets up the field to more systematically investigate fly visual behaviors, their variability, and their underlying mechanisms.

Comments on revisions:

While the incorporation of a hierarchical mixed model (HMM) appears to represent an improvement over their prior single-parameter correlation approach, it's not clear to me that this is a multivariate analysis. They write that "For each trait, we fitted a hierarchical linear mixed-effects model in Matlab (using the fit lme function) with environmental context as a fixed effect and fly identity (ID) as a random intercept... We computed the intraclass correlation coefficient (ICC) from each model as the between-fly variance divided by total variance. ICC, therefore, quantified repeatability across environmental contexts."

Does this indicate that HMM was used in a univariate approach? Can an analysis of only five metrics of several dozen total metrics be characterized as 'holistic'?

Within Figure 10a, some of the metrics show high ICC scores, but others do not. This suggests that the authors are overstating the overall persistence and/or consistency of behavioral individuality. It is clear from Figure S8 that a large number of metrics were calculated for each fly, but it remains unclear, at least to me, why the five metrics in Figure 10a are justified for selection. One is left wondering how rare or common is the 0.6 repeatability of % time walked among all the other behavioral metrics. It appears that a holistic analysis of this large data set remains impossible.

The authors write: "...fly individuality persists across different contexts, and individual differences shape behavior across variable environments, thereby making the underlying developmental and functional mechanisms amenable to genetic dissection." However, presumably the various behavioral features (and their variability) are governed by different brain regions, so some metrics (high ICC) would be amenable to the genetic dissection of individuality/variability, while others (low ICC) would not. It would be useful to know which are which, to define which behavioral domains express individuality, and could be targets for genetic analysis, and which do not. At the very least, the Abstract might like to acknowledge that inter-context consistency is not a major property of all or most behavioral metrics.

I hold that inter-trial repeatability should rightly be called "stability" while inter-context repeatability should be called "consistency". In the current manuscript, "consistency" is used throughout the manuscript, except for the new edits, which use "stability". If the authors are going to use both terms, it would be preferable if they could explain precisely how they define and use these terms.

Author response:

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

Reviewer #1 (Public review):  

Summary:  

The authors state the study's goal clearly: "The goal of our study was to understand to what extent animal individuality is influenced by situational changes in the environment, i.e., how much of an animal's individuality remains after one or more environmental features change." They use visually guided behavioral features to examine the extent of correlation over time and in a variety of contexts. They develop new behavioral instrumentation and software to measure behavior in Buridan's paradigm (and variations thereof), the Y-maze, and a flight simulator. Using these assays, they examine the correlations between conditions for a panel of locomotion parameters. They propose that inter-assay correlations will determine the persistence of locomotion individuality.

Strengths:  

The OED defines individuality as "the sum of the attributes which distinguish a person or thing from others of the same kind," a definition mirrored by other dictionaries and the scientific literature on the topic. The concept of behavioral individuality can be characterized as: (1) a large set of behavioral attributes, (2) with inter-individual variability, that are (3) stable over time. A previous study examined walking parameters in Buridan's paradigm, finding that several parameters were variable between individuals, and that these showed stability over separate days and up to 4 weeks (DOI: 10.1126/science.aaw718). The present study replicates some of those findings and extends the experiments from temporal stability to examining correlation of locomotion features between different contexts.  

The major strength of the study is using a range of different behavioral assays to examine the correlations of several different behavior parameters. It shows clearly that the inter-individual variability of some parameters is at least partially preserved between some contexts, and not preserved between others. The development of high-throughput behavior assays and sharing the information on how to make the assays is a commendable contribution.

Weaknesses:  

The definition of individuality considers a comprehensive or large set of attributes, but the authors consider only a handful. In Supplemental Fig. S8, the authors show a large correlation matrix of many behavioral parameters, but these are illegible and are only mentioned briefly in Results. Why were five or so parameters selected from the full set? How were these selected? Do the correlation trends hold true across all parameters? For assays in which only a subset of parameters can be directly compared, were all of these included in the analysis, or only a subset?  

The correlation analysis is used to establish stability between assays. For temporal re-testing, "stability" is certainly the appropriate word, but between contexts it implies that there could be 'instability'. Rather, instead of the 'instability' of a single brain process, a different behavior in a different context could arise from engaging largely (or entirely?) distinct context-dependent internal processes, and have nothing to do with process stability per se. For inter-context similarities, perhaps a better word would be "consistency".  

The parameters are considered one-by-one, not in aggregate. This focuses on the stability/consistency of the variability of a single parameter at a time, rather than holistic individuality. It would appear that an appropriate measure of individuality stability (or individuality consistency) that accounts for the high-dimensional nature of individuality would somehow summarize correlations across all parameters. Why was a multivariate approach (e.g. multiple regression/correlation) not used? Treating the data with a multivariate or averaged approach would allow the authors to directly address 'individuality stability', along with the analyses of single-parameter variability stability.

The correlation coefficients are sometimes quite low, though highly significant, and are deemed to indicate stability. For example, in Figure 4C top left, the % of time walked at 23{degree sign}C and 32{degree sign}C are correlated by 0.263, which corresponds to an R2 of 0.069 i.e. just 7% of the 32{degree sign}C variance is predictable by the 23{degree sign}C variance. Is it fair to say that 7% determination indicates parameter stability? Another example: "Vector strength was the most correlated attention parameter... correlations ranged... to -0.197," which implies that 96% (1 - R2) of Y-maze variance is not predicted by Buridan variance. At what level does an r value not represent stability?

The authors describe a dissociation between inter-group differences and inter-individual variation stability, i.e. sometimes large mean differences between contexts, but significant correlation between individual test and retest data. Given that correlation is sensitive to slope, this might be expected to underestimate the variability stability (or consistency). Is there a way to adjust for the group differences before examining correlation? For example, would it be possible to transform the values to in-group ranks prior to correlation analysis?

What is gained by classifying the five parameters into exploration, attention, and anxiety? To what extent have these classifications been validated, both in general, and with regard to these specific parameters? Is increased walking speed at higher temperature necessarily due to increased 'explorative' nature, or could it be attributed to increased metabolism, dehydration stress, or a heat-pain response? To what extent are these categories subjective?

The legends are quite brief and do not link to descriptions of specific experiments. For example, Figure 4a depicts a graphical overview of the procedure, but I could not find a detailed description of this experiment's protocol.

Using the current single-correlation analysis approach, the aims would benefit from re-wording to appropriately address single-parameter variability stability/consistency (as distinct from holistic individuality). Alternatively, the analysis could be adjusted to address the multivariate nature of individuality, so that the claims and the analysis are in concordance with each other.

The study presents a bounty of new technology to study visually guided behaviors. The Github link to the software was not available. To verify successful transfer or open-hardware and open-software, a report would demonstrate transfer by collaboration with one or more other laboratories, which the present manuscript does not appear to do. Nevertheless, making the technology available to readers is commendable.

The study discusses a number of interesting, stimulating ideas about interindividual variability and presents intriguing data that speaks to those ideas, albeit with the issues outlined above.

While the current work does not present any mechanistic analysis of interindividual variability, the implementation of high-throughput assays sets up the field to more systematically investigate fly visual behaviors, their variability, and their underlying mechanisms.  

Comments on revisions:  

I want to express my appreciation for the authors' responsiveness to the reviewer feedback. They appear to have addressed my previous concerns through various modifications including GLM analysis, however, some areas still require clarification for the benefit of an audience that includes geneticists.  

(1) GLM Analysis Explanation (Figure 9)  

While the authors state that their new GLM results support their original conclusions, the explanation of these results in the text is insufficient. Specifically:

The interpretation of coefficients and their statistical significance needs more detailed explanation. The audience includes geneticists and other nonstatistical people, so the GLM should be explained in terms of the criteria or quantities used to assess how well the results conform with the hypothesis, and to what extent they diverge.

The criteria used to judge how well the GLM results support their hypothesis are not clearly stated.

The relationship between the GLM findings and their original correlationbased conclusions needs better integration and connection, leading the reader through your reasoning.

We thank the reviewer for highlighting this important point. We have revised the Results section in the reviseed manuscript to include a more detailed explanation of the GLM analysis. Specifically, we now clarify the interpretation of the model coefficients, including the direction and statistical significance, in relation to the hypothesized effects. We also outline the criteria we used to assess how well the GLM supports our original correlation-based conclusions—namely, whether the sign and significance of the coefficients align with the expected relationships derived from our prior analysis. Finally, we explicitly describe how the GLM results confirm or extend the patterns observed in the correlation-based analysis, to guide readers through our reasoning and the integration of both approaches.

(2) Documentation of Changes  

One struggle with the revised manuscript is that no "tracked changes" version was included, so it is hard to know exactly what was done. Without access to the previous version of the manuscript, it is difficult to fully assess the extent of revisions made. The authors should provide a more comprehensive summary of the specific changes implemented, particularly regarding:

We thank the reviewer for bringing this to our attention. We were equally confused to learn that the tracked-changes version was not visible, despite having submitted one to eLife as part of our revision. 

Upon contacting the editorial office, they confirmed that we did submit a trackedchanges version, but clarified that it did not contain embedded figures (as they were added manually to the clean version).  The editorial response said in detail: “Regarding the tracked-changes file: it appears the version with markup lacked figures, while the figure-complete PDF had markup removed, which likely caused the confusion mentioned by the reviewers.” We hope this answer from eLife clarifies the reviewers’ concern.

(2)  Statistical Method Selection  

The authors mention using "ridge regression to mitigate collinearity among predictors" but do not adequately justify this choice over other approaches. They should explain:

Why ridge regression was selected as the optimal method  

How the regularization parameter (λ) was determined  

How this choice affects the interpretation of environmental parameters' influence on individuality

We appreciate the reviewer’s thoughtful question regarding our choice of statistical method. In response, we have expanded the Methods section in the revised manuscript to provide a more detailed justification for the use of a GLM, including ridge regression. Specifically, we explain that ridge regression was selected to address collinearity and to control for overfitting.

We now also describe how the regularization parameter (λ) was selected: we used 5-fold cross-validation over a log-spaced grid (10^⁻⁶ - 10<sup>⁶</sup) to identify the optimal value that minimized the mean squared error (MSE).

Finally, we clarify in both the Methods and Results sections how this modeling choice affects the interpretation of our findings. 

Reviewer #2 (Public review):  

Summary:  

The authors repeatedly measured the behavior of individual flies across several environmental situations in custom-made behavioral phenotyping rigs.

Strengths:  

The study uses several different behavioral phenotyping devices to quantify individual behavior in a number of different situations and over time. It seems to be a very impressive amount of data. The authors also make all their behavioral phenotyping rig design and tracking software available, which I think is great, and I'm sure other folks will be interested in using and adapting to their own needs.

Weaknesses/Limitations:  

I think an important limitation is that while the authors measured the flies under different environmental scenarios (i.e. with different lighting, temperature) they didn't really alter the "context" of the environment. At least within behavioral ecology, context would refer to the potential functionality of the expressed behaviors so for example, an anti-predator context, or a mating context, or foraging. Here, the authors seem to really just be measuring aspects of locomotion under benign (relatively low risk perception) contexts. This is not a flaw of the study, but rather a limitation to how strongly the authors can really say that this demonstrates that individuality is generalized across many different contexts. It's quite possible that rank-order of locomotor (or other) behaviors may shift when the flies are in a mating or risky context.  

I think the authors are missing an opportunity to use much more robust statistical methods It appears as though the authors used pearson correlations across time/situations to estimate individual variation; however far more sophisticated and elegant methods exist. The problem is that pearson correlation coefficients can be anti-conservative and additionally, the authors have thus had to perform many many tests to correlate behaviors across the different trials/scenarios. I don't see any evidence that the authors are controlling for multiple testing which I think would also help. Alternatively, though, the paper would be a lot stronger, and my guess is, much more streamlined if the authors employ hierarchical mixed models to analyse these data, which are the standard analytical tools in the study of individual behavioral variation. In this way, the authors could partition the behavioral variance into its among- and within-individual components and quantify repeatability of different behaviors across trials/scenarios simultaneously. This would remove the need to estimate 3 different correlations for day 1 & day 2, day 1 & 3, day 2 & 3 (or stripe 0 & stripe 1, etc) and instead just report a single repeatability for e.g. the time spent walking among the different strip patterns (eg. figure 3). Additionally, the authors could then use multivariate models where the response variables are all the behaviors combined and the authors could estimate the among-individual covariance in these behaviors. I see that the authors state they include generalized linear mixed models in their updated MS, but I struggled a bit to understand exactly how these models were fit? What exactly was the response? what exactly were the predictors (I just don't understand what Line404 means "a GLM was trained using the environmental parameters as predictors (0 when the parameter was not changed, 1 if it was) and the resulting individual rank differences as the response"). So were different models run for each scenario? for different behaviors? Across scenarios? What exactly? I just harp on this because I'm actually really interested in these data and think that updating these methods can really help clarify the results and make the main messages much clearer!

I appreciate that the authors now included their sample sizes in the main body of text (as opposed to the supplement) but I think that it would still help if the authors included a brief overview of their design at the start of the methods. It is still unclear to me how many rigs each individual fly was run through? Were the same individuals measured in multiple different rigs/scenarios? Or just one?

I really think a variance partitioning modeling framework could certainly improve their statistical inference and likely highlight some other cool patterns as these methods could better estimate stability and covariance in individual intercepts (and potentially slopes) across time and situation. I also genuinely think that this will improve the impact and reach of this paper as they'll be using methods that are standard in the study of individual behavioral variation

Reviewer #3 (Public review):  

This manuscript is a continuation of past work by the last author where they looked at stochasticity in developmental processes leading to inter-individual behavioural differences. In that work, the focus was on a specific behaviour under specific conditions while probing the neural basis of the variability. In this work, the authors set out to describe in detail how stable individuality of animal behaviours is in the context of various external and internal influences. They identify a few behaviours to monitor (read outs of attention, exploration, and 'anxiety'); some external stimuli (temperature, contrast, nature of visual cues, and spatial environment); and two internal states (walking and flying).

They then use high-throughput behavioural arenas - most of which they have built and made plans available for others to replicate - to quantify and compare combinations of these behaviours, stimuli, and internal states. This detailed analysis reveals that:

(1) Many individualistic behaviours remain stable over the course of many days.  

(2) That some of these (walking speed) remain stable over changing visual cues. Others (walking speed and centrophobicity) remain stable at different temperatures.

(3) All the behaviours they tested fail to remain stable over spatially varying environment (arena shape).

(4) and only angular velocity (a read out of attention) remains stable across varying internal states (walking and flying)

Thus, the authors conclude that there is a hierarchy in the influence of external stimuli and internal states on the stability of individual behaviours.

The manuscript is a technical feat with the authors having built many new high-throughput assays. The number of animals are large and many variables have been tested - different types of behavioural paradigms, flying vs walking, varying visual stimuli, different temperature among others.  

Comments on revisions:'  

The authors have addressed my previous concerns.  

We thank the reviewer for the positive feedback and are glad our revisions have satisfactorily addressed the previous concerns. We appreciate the thoughtful input that helped us improve the clarity and rigor of the manuscript.

Reviewer #1 (Recommendations for the authors):  

Comment on Revised Manuscript  

Recommendations for Improvement  

(1) Expand the Results section for Figure 9 with a more detailed interpretation of the GLM coefficients and their biological significance

(2) Provide explicit criteria (or at least explain in detail) for how the GLM results confirm or undermine their original hypothesis about environmental context hierarchy

While the claims are interesting, the additional statistical analysis appears promising. However, clearer explanation of these new results would strengthen the paper and ensure that readers from diverse backgrounds can fully understand how the evidence supports the authors' conclusions about individuality across environmental contexts. 

We thank the reviewer for these constructive suggestions. In response to these suggestions, we have expanded both the Methods and Results sections to provide a more detailed explanation of the GLM coefficients, including their interpretation and how they relate to our original correlation-based findings.

We now clarify how the direction, magnitude, and statistical significance of specific coefficients reflect the influence of different environmental factors on the persistence of individual behavioral traits. To make this accessible to readers from diverse backgrounds, we explicitly outline the criteria we used to evaluate whether the GLM results support our hypothesis about the hierarchical influence of environmental context, namely, whether the structure and strength of effects align with the patterns predicted from our prior correlation analysis.

These additions improve clarity and help readers understand how the new statistical results reinforce our conclusions about the context-dependence of behavioral individuality.

Reviewer #2 (Recommendations for the authors):  

Thanks for the revision of the paper! I updated my review to try and provide a little more guidance by what I mean about updating your analyses. I really think this is a super cool data set and I genuinely wish this were MY dataset so that way I could really dig into it to partition the variance. These variance partitioning methods are standard in my particular subfield (study of individual behavioral variation in ecology and evolution) and so I think employing them is 1) going to offer a MUCH more elegant and holistic view of the behavioral variation (e.g. you can report a single repeatability estimate for each behavior rather than 3 different correlations) and 2) improve the impact and readership for your paper as now you'll be using methods that a whole community of researchers are very familiar with. It's just a suggestion, but I hope you consider it!

We sincerely thank the reviewer for the insightful and encouraging feedback and for introducing us to this modeling approach. In response to this suggestion, we have incorporated a hierarchical linear mixed-effects model into our analysis (now presented in Figure 10), accompanied by a new supplementary table (Table T3). We also updated the Methods, Results, and Discussion sections to describe the rationale, implementation, and implications of the mixed-model analysis.

We agree with the reviewer that this approach provides a more elegant way to quantify behavioral variation and individual consistency across contexts. In particular, the ability to estimate repeatability directly aligns well with the core questions of our study. It facilitates improved communication of our findings to ecology, evolution, and behavior researchers. We greatly appreciate the suggestion; it has significantly strengthened both the analytical framework and the interpretability of the manuscript.

Reviewer #1 (Public review):

Summary:

In this manuscript, Mahajan et.al introduce two innovative macroscopic measures-intrachromosomal gene correlation length (𝓁∗) and transition energy barrier-to investigate chromatin structural dynamics associated with aging and age-related syndromes such as Hutchinson-Gilford Progeria Syndrome (HGPS) and Werner Syndrome (WRN). The authors propose a compelling systems-level approach that complements traditional biomarker-driven analyses, offering a more holistic and quantitative framework to assess genome-wide dysregulation. The concept of 𝓁∗ as a spatial correlation metric to capture chromatin disorganization is novel and well-motivated. The use of autocorrelation on distance-binned gene expression adds depth to the interpretation of chromatin state shifts. The energy landscape framework for gene state transitions is an elegant abstraction, with the notion of "irreversibility" providing a thermodynamic interpretation of transcriptional dysregulation. The application to multiple datasets (Fleischer, Line-1) and pathological states adds robustness to the analysis. The consistency of chromosome 6 (and to some extent chromosomes 16 and X) emerging as hotspots aligns well with known histone cluster localization and disease-relevant pathways. The manuscript does an excellent job of integrating transcriptomic trends with known epigenetic hallmarks of aging, and the proposed metrics can be used in place of traditional techniques like PCA in capturing structural transcriptome features. However, a direct correlation with ATACseq/ HiC data with the present analysis will be more informative.

Strengths:

Novel inclusion of statistical metrics that can help in systems-level studies in aging and chromatin biology.

Weaknesses:

(1) In the manuscript, the authors mention "While it may be intuitive to assume that highly expressed genes originate from euchromatin, this cannot be conclusively stated as a complete representation of euchromatin genes, nor can LAT be definitively linked to heterochromatin". What percentage of LAT can be linked to heterochromatin? What is the distribution of LAT and HAT in the euchromatin?

(2) In Figure 2, the authors observe "that the signal from the HAT class is the stronger between two and the signal from the LAT class, being mostly uniform, can be constituted as background noise." Is this biologically relevant? Are low-abundance transcripts constitutively expressed? The authors should discuss this in the Results section.

(3) The authors make a very interesting observation from Figure 3: that ASO-treated LINE-1 appears to be more effective in restoring HGPS cell lines closer to wild-type compared to WRN.. This can be explained by the difference in the basal activity of L1 elements in the HGPS vs WRN cell types. The authors should comment on this.

(4) The authors report that "from the results on Fleicher dataset is the magnitude of the difference in similarity distance is more pronounced in 𝓁∗ than in gene expression." Does this mean that the alterations in gene distance and chromatin organization do not result in gene expression change during aging?

(5) "In Fleischer dataset, as evident in Figure 4a, although changes in the heterochromatin are not identical for all chromosomes shown by the different degrees of variation of 𝓁∗ in each age group." The authors should present a comprehensive map of each chromosome change in gene distance to better explain the above statement.

(6) While trends in 𝓁∗ are discussed at both global and chromosome-specific levels, stronger statistical testing (e.g., permutation tests, bootstrapping) would lend greater confidence, especially when differences between age groups or treatment states are modest.

(7) While the transition energy barrier is an insightful conceptual addition, further clarification on the mathematical formulation and its physical assumptions (e.g., energy normalization, symmetry conditions) would improve interpretability. Also, in between Figures 7 and 8, the authors first compare the energy barrier of Chromosome 1 and then for all other chromosomes. What is the rationale for only analyzing chromosome 1? How many HAT or LAT are present there?

Reviewer #2 (Public review):

The authors report that intra-chromosomal gene correlation length (spatial correlations in gene expressions along the chromosome) serves as a proxy of chromatin structure and hence gene expression. They further explore changes in these metrics with aging. These are interesting and important findings. However, there are fundamental problems at this time.

(1) The basic method lacks validation. There is no validation of the method by approaches that directly measure chromatin structure, for example ATAC-seq, ChIP-seq, or CUT n RUN.

(2) There is no validation by interventions that directly probe chromatin structure, such as HDAC inhibitors. The authors employ datasets with knockdown of LINE-1 for validation. However, this is not a specific chromatin intervention.

(3) There is no statistical analysis, e.g., in Figures 4 and 5.

(4) The authors state, "in Figure 4a changes in the heterochromatin are not identical for all chromosomes shown...." I do not see the data for individual chromosomes.

(5) In comparisons of WT vs HGPS NT or HGPS SCR (Figure S6), is this a fair comparison? The WT and HGPS are presumably from different human donors, so they have genetic and epigenetic differences unrelated to HGPS.

Author response:

Reviewer #1 (Public review):

Summary:

In this manuscript, Mahajan et. al. introduce two innovative macroscopic measures-intrachromosomal gene correlation length (𝓁∗) and transition energy barrier-to investigate chromatin structural dynamics associated with aging and age-related syndromes such as Hutchinson-Gilford Progeria Syndrome (HGPS) and Werner Syndrome (WRN). The authors propose a compelling systems-level approach that complements traditional biomarker-driven analyses, offering a more holistic and quantitative framework to assess genome-wide dysregulation. The concept of 𝓁∗ as a spatial correlation metric to capture chromatin disorganization is novel and well-motivated. The use of autocorrelation on distance-binned gene expression adds depth to the interpretation of chromatin state shifts. The energy landscape framework for gene state transitions is an elegant abstraction, with the notion of "irreversibility" providing a thermodynamic interpretation of transcriptional dysregulation. The application to multiple datasets (Fleischer, Line-1) and pathological states adds robustness to the analysis. The consistency of chromosome 6 (and to some extent chromosomes 16 and X) emerging as hotspots aligns well with known histone cluster localization and disease-relevant pathways. The manuscript does an excellent job of integrating transcriptomic trends with known epigenetic hallmarks of aging, and the proposed metrics can be used in place of traditional techniques like PCA in capturing structural transcriptome features. However, a direct correlation with ATACseq/HiC data with the present analysis will be more informative.

(1) In the manuscript, the authors mention "While it may be intuitive to assume that highly expressed genes originate from euchromatin, this cannot be conclusively stated as a complete representation of euchromatin genes, nor can LAT be definitively linked to heterochromatin". What percentage of LAT can be linked to heterochromatin? What is the distribution of LAT and HAT in the euchromatin?

Thank you for this insightful question. In the revision we will add chromatin state annotations using ChromHMM to identify overlap between HAT/LAT and corresponding chromatin state. This should provide the specific percentages and distributions you requested.

We would like to take this opportunity to clarify that based on the plots Fig S1, and differential gene expressions, HAT is most likely a subset of euchromatin and LAT may contain both euchromatin and heterochromatin. The HAT/LAT cutoff occurs around the knee point in the log-log plot (Figure S1), where the linear portion indicates scale-invariant behavior with similar relative changes across expression ranks. The non-linear portion represents departure from power-law scaling, where low-expression genes exhibit sharper decline than expected. This suggests potential biological mechanisms such as chromatin silencing, detection limits, or technical artifacts related to sequencing depth.

We will provide detailed chromatin state analysis in the revision. For reference, HAT gene lists per chromosome are available in our GitHub repository at: https://github.com/altoslabs/papers-2025-rnaseq-chrom-aging/tree/main/data/Preprocessed_dat a under /<dataset>/chromosome_{}/data_hi.

(2) In Figure 2, the authors observe "that the signal from the HAT class is the stronger between two and the signal from the LAT class, being mostly uniform, can be constituted as background noise." Is this biologically relevant? Are low-abundance transcripts constitutively expressed? The authors should discuss this in the Results section.

We apologize for the confusion arising from the usage of the term “background noise”. We agree that the distinction between high-abundance transcripts (HATs) and low-abundance transcripts (LATs) deserves more explicit discussion in the Results.

Our intention is to say that HAT has a higher signal-to-noise ratio (SNR) compared to LAT. This is coming from the power law graph of FigS1.  Our intention is to state that the HAT class provides a strong, robust signal, consistent across chromosomes and the LAT class exhibits lower SNR and a more uniform background-like distribution in the context of the problem we are solving and not rather a generic biological statement. The experiment result that led to this statement is presented in FigS3. This does not imply that low-abundance transcripts lack biological relevance, but rather that they contribute less to the spatial organization patterns we measure.

(3) The authors make a very interesting observation from Figure 3: that ASO-treated LINE-1 appears to be more effective in restoring HGPS cell lines closer to wild-type compared to WRN.. This can be explained by the difference in the basal activity of L1 elements in the HGPS vs WRN cell types. The authors should comment on this.

We thank the reviewer for this incisive biological observation. While the differential effectiveness of ASO-treated LINE-1 in HGPS versus WRN cell lines is indeed an interesting phenomenon that may relate to basal L1 activity differences, this biological mechanism falls outside the scope of our current study.

Our paper focuses on demonstrating that the 𝓁∗ metric can sensitively detect chromatin structural changes that have been independently validated. We utilize the Della Valle et al. (2022) dataset specifically because it provides experimentally confirmed chromatin structural differences (Progeroid vs wild-type vs ASO-treated Progeriod), allowing us to validate that 𝓁∗ correlates with these established changes.

For detailed discussion of the biological mechanisms underlying differential LINE-1 ASO effectiveness between progeroid syndromes, we would direct readers to Della Valle et al. (2022) and related LINE-1 biology literature. Our contribution lies in demonstrating that 𝓁∗ can capture these chromatin organizational changes with enhanced sensitivity compared to traditional expression-based approaches. We are reluctant, without further experimentation, to venture into over-interpreting these results from a biology perspective.  

(4) The authors report that "from the results on Fleischer dataset is the magnitude of the difference in similarity distance is more pronounced in 𝓁∗ than in gene expression." Does this mean that the alterations in gene distance and chromatin organization do not result in gene expression change during aging?

Thank you for this important clarification request. This observation, illustrated in Figure 3, highlights two key points: (1) 𝓁∗ shows similar trends to PCA analysis, and (2) 𝓁∗ demonstrates higher sensitivity than traditional gene expression analysis.

This enhanced sensitivity enables better discrimination between aging states, particularly in the Fleischer dataset representing natural aging where changes are more gradual. The higher sensitivity stems from 𝓁∗'s ability to capture transcriptional spatial organization through spatial autocorrelation, which can detect subtle organizational changes that may precede or accompany expression changes rather than replacing them.

We will clarify in the revision that chromatin organizational changes and gene expression changes are complementary rather than mutually exclusive phenomena during aging.

(5) "In Fleischer dataset, as evident in Figure 4a, although changes in the heterochromatin are not identical for all chromosomes shown by the different degrees of variation of 𝓁∗ in each age group." The authors should present a comprehensive map of each chromosome change in gene distance to better explain the above statement.

Thank you for the feedback. If we understand your comment correctly, we need to provide a chromosome-wise distribution for Fig3c. We will update the paper and the supplementary.

(6) While trends in 𝓁∗ are discussed at both global and chromosome-specific levels, stronger statistical testing (e.g., permutation tests, bootstrapping) would lend greater confidence, especially when differences between age groups or treatment states are modest.

Thank you for the helpful suggestion. In the revision, we will incorporate permutation-based significance testing by shuffling the gene annotation and count table to generate a null distribution for our 𝓁∗ calculation. This will allow us to more rigorously assess whether the observed differences across age groups or treatment states deviate from chance expectations and thereby lend greater statistical confidence to our findings.

(7) While the transition energy barrier is an insightful conceptual addition, further clarification on the mathematical formulation and its physical assumptions (e.g., energy normalization, symmetry conditions) would improve interpretability. Also, in between Figures 7 and 8, the authors first compare the energy barrier of Chromosome 1 and then for all other chromosomes.

What is the rationale for only analyzing chromosome 1? How many HAT or LAT are present there?

Regarding chromosome 1 focus: we initially presented chromosome 1 as a representative example, but we will include energy landscape analysis for all chromosomes in the supplementary materials

We use the same HATs that were extracted during 𝓁∗ for the energy landscape as well. The HAT details are present in the github repo, the link provided in response to 1st feedback.

The normalization of the energy barrier ensures comparability across chromosomes of different sizes and across samples with different absolute expression scales. Specifically, we normalize with respect to the total area under the two-dimensional energy landscape while using the thermal energy (k_B T) as a scaling factor to place transition energy barriers on the scale of thermal fluctuations. This is formally expressed as in Eq. (1). 

The physical consequences of symmetry in the energy landscape are discussed in lines 472-491 of the manuscript, where we also introduce the concept of irreversibility. In brief, the chromatin energy landscape (Figure 8) is constructed by quantifying the energy contributions of genes that are upregulated (lower triangular matrix) and downregulated (upper triangular matrix) between two states. If the integrated energy contributions of upregulated and downregulated genes are equal, the landscape is symmetric, representing a thermodynamically reversible process, for example, nucleosome repositioning between euchromatic and heterochromatic regions without net gain or loss of nucleosomes. However, in cases where epigenetic modifications alter nucleosome density (e.g., disease states that reduce nucleosome numbers), the integrated energies are unequal, reflecting an irreversible energy cost. In this case, restoring chromatin requires additional energy input (e.g., to replace “missing” nucleosomes), which manifests as asymmetry in the landscape.

Reviewer #2 (Public review):

The authors report that intra-chromosomal gene correlation length (spatial correlations in gene expressions along the chromosome) serves as a proxy of chromatin structure and hence gene expression. They further explore changes in these metrics with aging. These are interesting and important findings. However, there are fundamental problems at this time.

(1) The basic method lacks validation. There is no validation of the method by approaches that directly measure chromatin structure, for example ATAC-seq, ChIP-seq, or CUT n RUN.

We appreciate the reviewer’s point that direct measurements such as ATAC-seq and ChIP-seq remain the gold standard for characterizing chromatin structure. Our method is designed to complement, not replace, these approaches by leveraging RNA-seq data to detect large-scale transcriptional patterns that correlate with chromatin dynamics.

We agree that integrating datasets with paired RNA-seq and chromatin accessibility assays would strengthen the manuscript and plan to include one such dataset in the revision.

Based on this feedback, we will also take the opportunity during revision to clarify and soften certain statements. Specifically, we will reposition ℓ∗ as a sensitive, computational proxy for detecting transcriptional signatures that are suggestive of chromatin structural changes. In other words, ℓ∗ provides an indirect window into chromatin dynamics through transcriptional spatial organization, allowing detection of patterns that may precede or accompany structural changes. Direct assays such as ATAC-seq or ChIP-seq remain essential for confirming the underlying physical modifications. To make this scope clear, we will revise the title to: “Macroscopic RNA-seq Analysis to Detect Transcriptional Patterns Associated with Chromatin State Changes,” and adjust the main text.  

We would like to take this opportunity to clarify why our initial version focused on the Della Valle and Fleischer datasets rather than including new paired datasets with direct chromatin measurements. The primary objective of our paper is to introduce two macroscopic RNA-seq–based measures, ℓ∗ and the energy landscape, that are designed to detect transcriptional signatures suggestive of chromatin structural changes in the context of aging and age-related diseases. These measures explicitly model transcriptional spatial organization and provide a sensitive, scalable way to analyze RNA-seq data in domains where direct chromatin assays may not be readily available.

The datasets we used (Della Valle et al., Fleischer et al.) have been rigorously validated and independently demonstrated differences in chromatin structure between conditions. Our goal was to show that ℓ∗ and the energy landscape align with and extend these established findings, offering a more sensitive measure of transcriptional spatial organization. Specifically, in the Della Valle dataset, chromatin structural differences between progeroid and healthy donors — and their partial rescue by LINE-1 ASO treatment — were experimentally confirmed, providing a strong foundation for testing whether our metrics reflect these known changes. Similarly, the Fleischer dataset captures natural, in vivo aging, which has also been linked to chromatin alterations in prior studies.

Thus, our approach builds on this well-established biological context rather than attempting to re-demonstrate these chromatin differences from scratch. Finally, we emphasize that our current focus is aging and age-related diseases. While the framework could potentially be applied to other chromatin modification contexts, we have not tested it outside this domain and do not claim general applicability at this stage.

(2) There is no validation by interventions that directly probe chromatin structure, such as HDAC inhibitors. The authors employ datasets with knockdown of LINE-1 for validation. However, this is not a specific chromatin intervention.

We request the reviewer to refer to our response to (1) as it includes the rationale behind the selection of LINE-1 and Fleischer dataset. We would also like to state that while the focus of Della Valle et al. was LINE-1 treated ASO to show rescue of progeroid samples, it also contains data for non-treated as well as healthy samples. Importantly, untreated progeroid samples show distinctly different chromatin structure compared to healthy samples, with substantial differences detectable by both PCA and our 𝓁∗ metric.

Our 𝓁∗ method provides additional interpretability by capturing transcriptional spatial organization, resulting in shorter correlation lengths for healthy patients and longer lengths for progeroid patients.

But as mentioned in our response to (1) we will try to add an additional dataset with paired rna-seq and one of ATAC, ChIP-seq or CUT n RUN in the revision

(3) There is no statistical analysis, e.g., in Figures 4 and 5.

We have provided statistical analysis for Fig 4 (lines 237-241). We will do a similar analysis for Fig. 5. 

(4) The authors state, "in Figure 4a changes in the heterochromatin are not identical for all chromosomes shown...." I do not see the data for individual chromosomes.

The data for individual chromosomes is available in supplementary Fig. S11 – references at line 425. We will make this cross-reference clearer in the main text and consider whether some of this chromosome-specific information should be elevated to the main figures for better accessibility.

(5) In comparisons of WT vs HGPS NT or HGPS SCR (Figure S6), is this a fair comparison? The WT and HGPS are presumably from different human donors, so they have genetic and epigenetic differences unrelated to HGPS.

Figure S6 demonstrates that 𝓁∗ analysis identifies chromosome 6 as most affected, consistent with differential gene expression patterns.

Regarding donor differences in WT vs HGPS comparisons, we defer to the experimental design of Della Valle et al., which follows standard practices in progeroid research. Our review of the literature indicates that progeroid studies typically use either parent/child samples or different donor comparisons (as individuals cannot simultaneously represent both WT and HGPS states).

Importantly, the LINE-1 ASO treatment comparisons use the same cell lines, eliminating donor variability concerns. This experimental design allows us to validate that 𝓁∗ can detect rescue effects within genetically identical samples, supporting the method's sensitivity to chromatin structural changes  

Reviewing Editor Comments:

You'll note that both reviewers were very thoughtful in their comments, and in principle are supportive and excited by the work. However, their evaluation of the strength of evidence diverged substantially. I'm inclined to suggest that finding a way to support the novel method with an alternative approach would greatly improve the impact of this work. I encourage you to consider a revision that provides such data, in the context of technology currently available to the field.

We sincerely thank the editor for their thoughtful and encouraging assessment of our work. We are grateful for their recognition of the novelty of our macroscopic measures (ℓ∗ and the transition energy barrier) and their potential to provide a systems-level understanding of chromatin structural dynamics in aging and age-related syndromes. In response to the editor’s suggestion for direct validation with chromatin accessibility data, we plan to integrate an additional dataset containing paired RNA-seq and ATAC-seq or related measurements in our revision. This will help strengthen the link between our RNA-seq–based metrics and direct chromatin assays. We have also clarified and softened the manuscript text to ensure it is clear that ℓ∗ serves as a complementary, computational proxy, not a replacement, for direct experimental approaches. Very specifically, to make this scope clear, we will revise the title to: “Macroscopic RNA-seq Analysis to Detect Transcriptional Patterns Associated with Chromatin State Changes,” and adjust the main text. We thank the editor for the feedback. We have provided additional details in response to specific comments made by the reviewers.

Reviewer #1 (Public review):

This is an interesting and timely computational study using molecular dynamics simulation as well as quantum mechanical calculation to address why tyrosine (Y), as part of an intrinsically disordered protein (IDP) sequence, has been observed experimentally to be stronger than phenylalanine (F) as a promoter for biomolecular phase separation. Notably, the authors identified the aqueous nature of the condensate environment and the corresponding dielectric and hydrogen bonding effects as a key to understand the experimentally observed difference. This principle is illustrated by the difference in computed transfer free energy of Y- and F-containing pentapeptides into solvent with various degrees of polarity. The elucidation offered by this work is important. The computation appears to be carefully executed, the results are valuable, and the discussion is generally insightful. However, there is room for improvement in some parts of the presentation in terms of accuracy and clarity, including, e.g., the logic of the narrative should be clarified with additional information (and possibly additional computation), and the current effort should be better placed in the context of prior relevant theoretical and experimental works on cation-π interactions in biomolecules and dielectric properties of biomolecular condensates. Accordingly, this manuscript should be revised to address the following, with added discussion as well as inclusion of references mentioned below.

(1) Page 2, line 61: "Coarse-grained simulation models have failed to account for the greater propensity of arginine to promote phase separation in Ddx4 variants with Arg to Lys mutations (Das et al., 2020)". As it stands, this statement is not accurate, because the cited reference to Das et al. showed that although some coarse-grained model, namely the HPS model of Dignon et al., 2018 PLoS Comput did not capture the Arg to Lys trend, the KH model described in the same Dignon et al. paper was demonstrated by Das et al. (2020) to be capable of mimicking the greater propensity of Arg to promote phase separation than Lys. Accordingly, a possible minimal change that would correct the inaccuracy of this statement in the manuscript would be to add the word "Some" in front of "coarse-grained simulation models ...", i.e., it should read "Some coarse-grained simulation models have failed ...". In fact, a subsequent work [Wessén et al., J Phys Chem B 126: 9222-9245 (2022)] that applied the Mpipi interaction parameters (Joseph et al., 2021, already cited in the manuscript) showed that Mpipi is capable of capturing the rank ordering of phase separation propensity of Ddx4 variants, including a charge scrambled variant as well as both the Arg to Lys and the Phe to Ala variants (see Fig.11a of the above-cited Wessén et al. 2022 reference). The authors may wish to qualify their statements in the introduction to take note of these prior results. For example, they may consider adding a note immediately after the next sentence in the manuscript "However, by replacing the hydrophobicity scales ... (Das et al., 2020)" to refer to these subsequent findings in 2021-2022.

(2) Page 8, lines 285-290 (as well as the preceding discussion under the same subheading & Fig.4): "These findings suggest that ... is not primarily driven by differences in protein-protein interaction patterns ..." The authors' logic in terms of physical explanation is somewhat problematic here. In this regard, "Protein-protein interaction patterns" appears to be a straw man, so to speak. Indeed, who (reference?) has argued that the difference in the capability of Y and F in promoting phase separation should be reflected in the pairwise amino acid interaction pattern in a condensate that contains either only Y (and G, S) and only F (and G, S) but not both Y and F? Also, this paragraph in the manuscript seems to suggest that the authors' observation of similar contact patterns in the GSY and GSF condensates is "counterintuitive" given the difference in Y-Y and F-F potentials of mean force (Joseph et al., 2021); but there is nothing particularly counterintuitive about that. The two sets of observations are not mutually exclusive. For instance, consider two different homopolymers, one with a significantly stronger monomer-monomer attraction than the other. The condensates for the two different homopolymers will have essentially the same contact pattern but very different stabilities (different critical temperatures), and there is nothing surprising about it. In other words, phase separation propensity is not "driven" by contact pattern in general, it's driven by interaction (free) energy. The relevant issue here is total interaction energy or critical point of the phase separation. If it is computationally feasible, the authors should attempt to determine the critical temperatures for the GSY condensate versus the GSF condensate to verify that the GSY condensate has a higher critical temperature than the GSF condensate. That would be the most relevant piece of information for the question at hand.

(3) Page 9, lines 315-316: "...Our ε [relative permittivity] values ... are surprisingly close to that derived from experiment on Ddx4 condensates (45{plus minus}13) (Nott et al., 2015)". For accuracy, it should be noted here that the relative permittivity provided in the supplementary information of Nott et al. was not a direct experimental measurement but based on a fit using Flory-Huggins (FH), but FH is not the most appropriate theory for polymer with long-spatial-range Coulomb interactions. To this reviewer's knowledge, no direct measurement of relative permittivity in biomolecular condensates has been made to date. Explicit-water simulation suggests that relative permittivity of Ddx4 condensate with protein volume fraction ≈ 0.4 can have relative permittivity ≈ 35-50 (Das et al., PNAS 2020, Fig.7A), which happens to agree with the ε = 45{plus minus}13 estimate. This information should be useful to include in the authors' manuscript.

(4) As for the dielectric environment within biomolecular condensates, coarse-grained simulation has suggested that whereas condensates formed by essentially electric neutral polymers (as in the authors' model systems) have relative permittivities intermediate between that of bulk water and that of pure protein (ε = 2-4, or at most 15), condensates formed by highly charge polymers can have relative permittivity higher than that of bulk water [Wessén et al., J Phys Chem B 125:4337-4358 (2021), Fig.14 of this reference]. In view of the role of aromatic residues (mainly Y and F) in the phase separation of IDPs such as A1-LCD and LAF-1 that contain positively and negatively charged residues (Martin et al., 2020; Schuster et al., 2020, already cited in the manuscript), it should be useful to address briefly how the relationship between the relative phase-separation promotion strength of Y vs F and dielectric environment of the condensate may or may not be change with higher relative permittivities.

(5) The authors applied the dipole moment fluctuation formula (Eq.2 in the manuscript) to calculate relative permittivity in their model condensates. Does this formula apply only to an isotropic environment? The authors' model condensates were obtained from a "slab" approach (p.4) and thus the simulation box has a rectangular geometry. Did the authors apply their Eq.2 to the entire simulation box or only to the central part of the box with the condensate (see, e.g., Fig.3C in the manuscript). If the latter is the case, is it necessary to use a different dipole moment formula that distinguishes between the "parallel" and "perpendicular" components of the dipole moment (see, e.g., Eq.16 in the above-cited Wessén et al. 2021 paper). A brief added comments will be useful.

(6) With regard to the general role of Y and F in the phase separation of biomolecules containing positively charged Arg and Lys residues, the relative strength of cation-π interactions (cation-Y vs cation-F) should be addressed (in view of the generality implied by the title of the manuscript), or at least discussed briefly in the authors' manuscript if a detailed study is beyond the scope of their current effort. It has long been known that in the biomolecular context, cation-Y is slightly stronger than cation-F, whereas cation-tryptophan (W) is significantly stronger than either cation-Y and cation-F [Wu & McMahon, JACS 130:12554-12555 (2008)]. Experimental data from a study of EWS (Ewing sarcoma) transactivation domains indicated that Y is a slightly stronger promoter than F for transcription, whereas W is significantly stronger than either Y or F [Song et al., PLoS Comput Biol 9:e1003239 (2013)]. In view of the subsequent general recognition that "transcription factors activate genes through the phase-separation capacity of their activation domain" [Boija et al., Cell 175:1842-1855.e16 (2018)] which is applicable to EWS in particular [Johnson et al., JACS 146:8071-8085 (2024)], the experimental data in Song et al. 2013 (see Fig.3A of this reference) suggests that cation-Y interactions are stronger than cation-F interactions in promoting phase separation, thus generalizing the authors' observations (which focus primarily on Y-Y, Y-F and F-F interactions) to most situations in which cation-Y and cation-F interactions are relevant to biomolecular condensation.

(7) Page 9: The observation of a weaker effective F-F (and a few other nonpolar-nonpolar) interaction in a largely aqueous environment (as in an IDP condensate) than in a nonpolar environment (as in the core of a folded protein) is intimately related to (and expected from) the long-recognized distinction between "bulk" and "pair" as well as size dependence of hydrophobic effects that have been addressed in the context of protein folding [Wood & Thompson, PNAS 87:8921-8927 (1990); Shimizu & Chan, JACS 123:2083-2084 (2001); Proteins 49:560-566 (2002)]. It will be useful to add a brief pointer in the current manuscript to this body of relevant resource in protein science.

Comments on revisions:

The authors have largely addressed my previous concerns and the manuscript has been substantially improved. Nonetheless, it will benefit the readers more if the authors had included more of the relevant references provided in my previous review so as to afford a broader and more accurate context to the authors' effort. This deficiency is particularly pertinent for point number 6 in my previous report about cation-pi interactions. The authors have now added a brief discussion but with no references on the rank ordering of Y, F, and W interactions. I cannot see how providing additional information about a few related works could hurt. Quite the contrary, having the references will help readers establish scientific connections and contribute to conceptual advance.

Author response:

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

Reviewer #1 (Public review):

This is an interesting and timely computational study using molecular dynamics simulation as well as quantum mechanical calculation to address why tyrosine (Y), as part of an intrinsically disordered protein (IDP) sequence, has been observed experimentally to be stronger than phenylalanine (F) as a promoter for biomolecular phase separation. Notably, the authors identified the aqueous nature of the condensate environment and the corresponding dielectric and hydrogen bonding effects as a key to understanding the experimentally observed difference. This principle is illustrated by the difference in computed transfer free energy of Y- and F-containing pentapeptides into a solvent with various degrees of polarity. The elucidation offered by this work is important. The computation appears to be carefully executed, the results are valuable, and the discussion is generally insightful. However, there is room for improvement in some parts of the presentation in terms of accuracy and clarity, including, e.g., the logic of the narrative should be clarified with additional information (and possibly additional computation), and the current effort should be better placed in the context of prior relevant theoretical and experimental works on cation-π interactions in biomolecules and dielectric properties of biomolecular condensates. Accordingly, this manuscript should be revised to address the following, with added discussion as well as inclusion of references mentioned below.

We are grateful for the referee’s assessment of our work and insightful suggestions, which we address point by point below.

(1) Page 2, line 61: "Coarse-grained simulation models have failed to account for the greater propensity of arginine to promote phase separation in Ddx4 variants with Arg to Lys mutations (Das et al., 2020)". As it stands, this statement is not accurate, because the cited reference to Das et al. showed that although some coarse-grained models, namely the HPS model of Dignon et al., 2018 PLoS Comput did not capture the Arg to Lys trend, the KH model described in the same Dignon et al. paper was demonstrated by Das et al. (2020) to be capable of mimicking the greater propensity of Arg to promote phase separation than Lys. Accordingly, a possible minimal change that would correct the inaccuracy of this statement in the manuscript would be to add the word "Some" in front of "coarse-grained simulation models ...", i.e., it should read "Some coarse-grained simulation models have failed ...". In fact, a subsequent work [Wessén et al., J Phys Chem B 126: 9222-9245 (2022)] that applied the Mpipi interaction parameters (Joseph et al., 2021, already cited in the manuscript) showed that Mpipi is capable of capturing the rank ordering of phase separation propensity of Ddx4 variants, including a charge scrambled variant as well as both the Arg to Lys and the Phe to Ala variants (see Figure 11a of the above-cited Wessén et al. 2022 reference). The authors may wish to qualify their statements in the introduction to take note of these prior results. For example, they may consider adding a note immediately after the next sentence in the manuscript "However, by replacing the hydrophobicity scales ... (Das et al., 2020)" to refer to these subsequent findings in 2021-2022.

We agree with the referee that the wording used in the original version was inaccurate. We did not want to expand too much on the previous results on Lys/Arg, to avoid overwhelming our readers with background information that was not directly relevant to the aromatic residues Phe and Tyr. We have now introduced some of the missing details in the hope that this will provide a more accurate account of what has been achieved with different versions of coarse-grained models. In the revised version, we say the following:

Das and co-workers attempted to explain arginine’s greater propensity to phase separate in Ddx4 variants using coarse-grained simulations with two different energy functions (Das et al., 2020). The model was first parametrized using a hydrophobicity scale, aimed to capture the “stickiness” of different amino acids (Dignon et al., 2018), but this did not recapitulate the correct rank order in the stability of the simulated condensates (Das et al., 2020). By replacing the hydrophobicity scale with interaction energies from amino acid contact matrices —derived from a statistical analysis of the PDB (Dignon et al., 2018; Miyazawa and Jernigan, 1996; Kim and Hummer, 2008)— they recovered the correct trends (Das et al., 2020). A key to the greater propensity for LLPS in the case of Arg may derive from the pseudo-aromaticity of this residue, which results in a greater stabilization relative to the more purely cationic character of Lys (Gobbi and Frenking, 1993; Wang et al., 2018; Hong et al., 2022).

(2) Page 8, lines 285-290 (as well as the preceding discussion under the same subheading & Figure 4): "These findings suggest that ... is not primarily driven by differences in protein-protein interaction patterns ..." The authors' logic in terms of physical explanation is somewhat problematic here. In this regard, "Protein-protein interaction patterns" appear to be a straw man, so to speak. Indeed, who (reference?) has argued that the difference in the capability of Y and F in promoting phase separation should be reflected in the pairwise amino acid interaction pattern in a condensate that contains either only Y (and G, S) and only F (and G, S) but not both Y and F? Also, this paragraph in the manuscript seems to suggest that the authors' observation of similar contact patterns in the GSY and GSF condensates is "counterintuitive" given the difference in Y-Y and F-F potentials of mean force (Joseph et al., 2021); but there is nothing particularly counterintuitive about that. The two sets of observations are not mutually exclusive. For instance, consider two different homopolymers, one with a significantly stronger monomer-monomer attraction than the other. The condensates for the two different homopolymers will have essentially the same contact pattern but very different stabilities (different critical temperatures), and there is nothing surprising about it. In other words, phase separation propensity is not "driven" by contact pattern in general, it's driven by interaction (free) energy. The relevant issue here is total interaction energy or the critical point of the phase separation. If it is computationally feasible, the authors should attempt to determine the critical temperatures for the GSY condensate versus the GSF condensate to verify that the GSY condensate has a higher critical temperature than the GSF condensate. That would be the most relevant piece of information for the question at hand.

We are grateful for this very insightful comment by the referee. We have followed this suggestion to address whether, despite similar interaction patterns in GSY and GSF condensates, their stabilities are different. As in our previous work (De Sancho, 2022), we have run replica exchange MD simulations for both condensates and derived their phase diagrams. Our results, shown in the new Figure 5 and supplementary Figs. S6-S7, clearly indicate that the GSY condensate has a lower saturation density than the GSF condensate. This result is consistent with the trends observed in experiments on mutants of the low-complexity domain of hnRNPA1, where the relative amounts of F and Y determine the saturation concentration (Bremer et al., 2022).

(3) Page 9, lines 315-316: "...Our ε [relative permittivity] values ... are surprisingly close to that derived from experiment on Ddx4 condensates (45{plus minus}13) (Nott et al., 2015)".  For accuracy, it should be noted here that the relative permittivity provided in the supplementary information of Nott et al. was not a direct experimental measurement but based on a fit using Flory-Huggins (FH), but FH is not the most appropriate theory for a polymer with long-spatial-range Coulomb interactions. To this reviewer's knowledge, no direct measurement of relative permittivity in biomolecular condensates has been made to date. Explicit-water simulation suggests that the relative permittivity of Ddx4 condensate with protein volume fraction ≈ 0.4 can have a relative permittivity ≈ 35-50 (Das et al., PNAS 2020, Fig.7A), which happens to agree with the ε = 45{plus minus}13 estimate. This information should be useful to include in the authors' manuscript.

We thank the referee for this useful comment. We are aware that the estimate we mentioned is not direct. We have now clarified this point and added the additional estimate from Das et al. In the new version of the manuscript, we say:

Our 𝜀 values for the condensates (39 ± 5 for GSY and 47 ± 3 for GSF) are surprisingly close to that derived from experiments on Ddx condensates using Flory-Huggins theory (45±13) (Nott et al., 2015) and from atomistic simulations of Ddx4 (∼35−50 at a volume fraction of 𝜙 = 0.4) (Das et al., 2020).

(4) As for the dielectric environment within biomolecular condensates, coarse-grained simulation has suggested that whereas condensates formed by essentially electric neutral polymers (as in the authors' model systems) have relative permittivities intermediate between that of bulk water and that of pure protein (ε=2-4, or at most 15), condensates formed by highly charged polymers can have relative permittivity higher than that of bulk water [Wessén et al., J Phys Chem B 125:4337-4358 (2021), Fig.14 of this reference]. In view of the role of aromatic residues (mainly Y and F) in the phase separation of IDPs such as A1-LCD and LAF-1 that contain positively and negatively charged residues (Martin et al., 2020; Schuster et al., 2020, already cited in the manuscript), it should be useful to address briefly how the relationship between the relative phase-separation promotion strength of Y vs F and dielectric environment of the condensate may or may not be change with higher relative permittivities.

We thank the referee for their comment regarding highly charged polymers. However, we have chosen not to address these systems in our manuscript, as they are significantly different from the GSY/GSF peptide condensates under investigation. In polyelectrolyte systems, condensate formation is primarily driven by electrostatic interactions and counterion release, while we highlight the role of transfer free energies. At high dielectric constants (and dielectrics even higher than that of water), the strength of electrostatic interactions will be greatly reduced. In our approach to estimate differences between Y and F, the transfer free energy should plateau at a value of ΔΔG=0 in water. At greater values of ε>80, it becomes difficult to predict whether additional effects might become relevant. As this lies beyond the scope of our current study, we prefer not to speculate further.

(5) The authors applied the dipole moment fluctuation formula (Eq.2 in the manuscript) to calculate relative permittivity in their model condensates. Does this formula apply only to an isotropic environment? The authors' model condensates were obtained from a "slab" approach (page 4 and thus the simulation box has a rectangular geometry. Did the authors apply Equation 2 to the entire simulation box or only to the central part of the box with the condensate (see, e.g., Figure 3C in the manuscript). If the latter is the case, is it necessary to use a different dipole moment formula that distinguishes between the "parallel" and "perpendicular" components of the dipole moment (see, e.g., Equation 16 in the above-cited Wessén et al. 2021 paper). A brief added comment will be useful.

We have calculated the relative permittivity from dense phases only. These dense phases were sliced from the slab geometry and then re-equilibrated. Long simulations were then run to converge the calculation of the dielectric constant. We have clarified this in the Methods section of the paper. We say:

For the calculation of the dielectric constant of condensates, we used the simulations of isolated dense phases mentioned above.

(6) Concerning the general role of Y and F in the phase separation of biomolecules containing positively charged Arg and Lys residues, the relative strength of cation-π interactions (cation-Y vs cation-F) should be addressed (in view of the generality implied by the title of the manuscript), or at least discussed briefly in the authors' manuscript if a detailed study is beyond the scope of their current effort. It has long been known that in the biomolecular context, cation-Y is slightly stronger than cation-F, whereas cation-tryptophan (W) is significantly stronger than either cation-Y and cation-F [Wu & McMahon, JACS 130:12554-12555 (2008)]. Experimental data from a study of EWS (Ewing sarcoma) transactivation domains indicated that Y is a slightly stronger promoter than F for transcription, whereas W is significantly stronger than either Y or F [Song et al., PLoS Comput Biol 9:e1003239 (2013)]. In view of the subsequent general recognition that "transcription factors activate genes through the phase-separation capacity of their activation domain" [Boija et al., Cell 175:1842-1855.e16 (2018)] which is applicable to EWS in particular [Johnson et al., JACS 146:8071-8085 (2024)], the experimental data in Song et al. 2013 (see Figure 3A of this reference) suggests that cation-Y interactions are stronger than cation-F interactions in promoting phase separation, thus generalizing the authors' observations (which focus primarily on Y-Y, Y-F and F-F interactions) to most situations in which cation-Y and cation-F interactions are relevant to biomolecular condensation.

We thank our referee for this insightful comment. While we restrict our analysis to aromatic pairs in this work, the observed crossover will certainly affect other pairs where tyrosine or phenylalanine are involved. We now comment on this point in the discussions section of the revised manuscript. This topic will be explored in detail in a follow-up manuscript we are currently completing. We say:

We note that, although we have not included in our analysis positively charged residues that form cation-π interactions with aromatics, the observed crossover will also be relevant to Arg/Lys contacts with Phe and Tyr. Following the rationale of our findings, within condensates, cation-Tyr interactions are expected to promote phase separation more strongly than cation-Phe pairs.

(7) Page 9: The observation of weaker effective F-F (and a few other nonpolar-nonpolar) interactions in a largely aqueous environment (as in an IDP condensate) than in a nonpolar environment (as in the core of a folded protein) is intimately related to (and expected from) the long-recognized distinction between "bulk" and "pair" as well as size dependence of hydrophobic effects that have been addressed in the context of protein folding [Wood & Thompson, PNAS 87:8921-8927 (1990); Shimizu & Chan, JACS 123:2083-2084 (2001); Proteins 49:560-566 (2002)]. It will be useful to add a brief pointer in the current manuscript to this body of relevant resources in protein science.

We thank the referee for bringing this body of work to our attention. In the revised version of our work, we briefly mention how it relates to our results. We also note that the suggested references have pointed to another of the limitations of our study, that of chain connectivity, addressed in the work by Shimizu and Chan. While we were well aware of these limitations, we had not mentioned them in our manuscript. Concerning the distinction between pair and bulk hydrophobicities, we include the following in the concluding lines of our work:

The observed context dependence has deep roots in the concepts of “pair” and “bulk” hydrophobicity (Wood and Thompson, 1990; Shimizu and Chan, 2002). While pair hydrophobicity is connected to dimerisation equilibria (i.e. the second step in Figure 2B), bulk hydrophobicity is related to transfer processes (the first step). Our work stresses the importance of considering both the pair contribution that dominates at high solvation, and the transfer free energy contribution, which overwhelms the interaction strength at low dielectrics.

Reviewer #2 (Public review):

Summary:

In this preprint, De Sancho and López use alchemical molecular dynamics simulations and quantum mechanical calculations to elucidate the origin of the observed preference of Tyr over Phe in phase separation. The paper is well written, and the simulations conducted are rigorous and provide good insight into the origin of the differences between the two aromatic amino acids considered.

We thank the referee for his/her positive assessment of our work. Below, we address all the questions raised one by one.

Strengths:

The study addresses a fundamental discrepancy in the field of phase separation where the predicted ranking of aromatic amino acids observed experimentally is different from their anticipated rankings when considering contact statistics of folded proteins. While the hypothesis that the difference in the microenvironment of the condensed phase and hydrophobic core of folded proteins underlies the different observations, this study provides a quantification of this effect. Further, the demonstration of the crossover between Phe and Tyr as a function of the dielectric is interesting and provides further support for the hypothesis that the differing microenvironments within the condensed phase and the core of folded proteins is the origin of the difference between contact statistics and experimental observations in phase separation literature. The simulations performed in this work systematically investigate several possible explanations and therefore provide depth to the paper.

Weaknesses:

While the study is quite comprehensive and the paper well written, there are a few instances that would benefit from additional details. In the methods section, it is unclear as to whether the GGXGG peptides upon which the alchemical transforms are conducted are positioned restrained within the condensed/dilute phase or not. If they are not, how would the position of the peptides within the condensate alter the calculated free energies reported? 

The peptides are not restrained in our simulations and can therefore diffuse out of the condensate given sufficient time. Although the GGXGG peptide can, given sufficient time, leave the peptide condensate, we did not observe any escape event in the trajectories we used to generate starting points for switching. Hence, the peptide environment captured in our calculations reflects, on average, the protein-protein and protein-solvent interactions inside the model condensate. We believe this is the right way of performing the calculation of transfer free energy differences into the condensate. We have clarified this point when we describe the equilibrium simulation results in the revised manuscript. We say:

Also, the peptide that experiences the transformation, which is not restrained, must remain buried within the condensate for all the snapshots that we use as initial frames, to avoid averaging the work in the dilute and dense phases.

On the referee’s second point of whether there would be differences if the peptide visited the dilute phase, the answer is that, indeed, we would. We expect that the behaviour of the peptide would approach ΔΔG=0, considering the low protein concentration in the dilute phase. For mixed trajectories with sampling in both dilute and dense phases, our expectation would be a bimodal distribution in the free energy estimates from switching (see e.g. Fig. 8 in DOI:10.1021/acs.jpcb.0c10263). Because we are exclusively interested in the transfer free energies into the condensate, we do not pursue such calculations in this work.

It would also be interesting to see what the variation in the transfer of free energy is across multiple independent replicates of the transform to assess the convergence of the simulations. 

Upon submission of our manuscript, we were confident that the results we had obtained would pass the test of statistical significance. We had, after all, done many more simulations than those reported, plus the comparable values of ΔΔG_Transfer for both GSY and GSF pointed in the right direction. However, we acknowledge that the more thorough test of running replicates recommended by the referee is important, considering the slow diffusion within the Tyr peptide condensates due to its stickiness. Also, the non-equilibrium switching method had not been tested before for dense phases like the ones considered here.

We have hence followed our referee's suggestion and done three different replicates, 1 μs each, of the equilibrium runs starting from independent slab configurations, for both the GSY and GSF condensates (see the new supporting figures Fig. S1, S2 and S5). We now report the errors from the three replicates as the standard error of the mean (bootstrapping errors remain for the rest of the solvents). Our results are entirely consistent with the values reported originally, confirming the validity of our estimates.

Additionally, since the authors use a slab for the calculation of these free energies, are the transfer free energies from the dilute phase to the interface significantly different from those calculated from the dilute phase to the interior of the condensate? 

We thank the referee for this valuable comment, as it has pointed us in the direction of a rapidly increasing body of work on condensate interfaces, for example, as mediators of aggregation, that we may consider for future study with the same methodology. However, as discussed above, we have not considered this possibility in our work, as we decided to focus on the condensate environment, rather than its interface.

The authors mention that the contact statistics of Phe and Tyr do not show significant difference and thereby conclude that the more favorable transfer of Tyr primarily originates from the dielectric of the condensate. However, the calculation of contacts neglects the differences in the strength of interactions involving Phe vs. Tyr. Though the authors consider the calculation of energy contact formation later in the manuscript, the scope of these interactions are quite limited (Phe-Phe, Tyr-Tyr, Tyr-Amide, Phe-Amide) which is not sufficient to make a universal conclusion regarding the underlying driving forces. A more appropriate statement would be that in the context of the minimal peptide investigated the driving force seems to be the difference in dielectric. However, it is worth mentioning that the authors do a good job of mentioning some of these caveats in the discussion section.

We thank the referee for this important comment. Indeed, the similar contact statistics and interaction patterns that we reported originally do not necessarily imply identical interaction energies. In other words, similar statistics and patterns can still result in different stabilities for the Phe and Tyr condensates if the energetics are different. Hence, we cannot conclude that the GSF and GSY condensate environments are equivalent.

To address this point, we have run new simulations for the revised version of our paper, using the temperature-replica exchange method, as before. From the new datasets, we derive the phase diagrams for both the GSF and GSY condensates (see the new Fig. 5). We find that the tyrosine-containing condensate is more stable than that of phenylalanine, as can be inferred from the lower saturation density in the low-density branch of the phase diagram. In consequence, despite the similar contact statistics, the energetics differ, making the saturation density of the GSY slightly lower than that of GSF. This result is consistent with experimental data by Bremer et al (Nat. Chem. 2022). 

Reviewer #3 (Public review):

Summary:

In this study, the authors address the paradox of how tyrosine can act as a stronger sticker for phase separation than phenylalanine, despite phenylalanine being higher on the hydrophobicity scale and exhibiting more prominent pairwise contact statistics in folded protein structures compared to tyrosine.

We are grateful for the referee’s favourable opinion on the paper. Below, we address all of the issues raised.

Strengths:

This is a fascinating problem for the protein science community with special relevance for the biophysical condensate community. Using atomistic simulations of simple model peptides and condensates as well as quantum calculations, the authors provide an explanation that relies on the dielectric constant of the medium and the hydration level that either tyrosine or phenylalanine can achieve in highly hydrophobic vs. hydrophilic media. The authors find that as the dielectric constant decreases, phenylalanine becomes a stronger sticker than tyrosine. The conclusions of the paper seem to be solid, it is well-written and it also recognises the limitations of the study. Overall, the paper represents an important contribution to the field.

Weaknesses:

How can the authors ensure that a condensate of GSY or GSF peptides is a representative environment of a protein condensate? First, the composition in terms of amino acids is highly limited, second the effect of peptide/protein length compared to real protein sequences is also an issue, and third, the water concentration within these condensates is really low as compared to real experimental condensates. Hence, how can we rely on the extracted conclusions from these condensates to be representative for real protein sequences with a much more complex composition and structural behaviour?

We agree with the main weakness identified by the referee. In fact, all these limitations had already been stated in our original submission. Our ternary peptide condensates are just a minimal model system that bears reasonable analogies with condensates, but definitely is not identical to true LCR condensates. The analogies between peptide and protein condensates are, however, worth restating: 

(1) The limited composition of the peptide condensates is inspired by LCR sequences (see Fig. 4 in Martin & Mittag, 2018).

(2) The equilibrium phase diagram, showing a UCST, is consistent with that of LCRs from Ddx4 or hnRNPA1.

(3) The dynamical behaviour is intermediate between liquid and solid (De Sancho, 2022). 

(4) The contact patterns are comparable to those observed for FUS and LAF1 (Zheng et al, 2020).

The third issue pointed out by the referee requires particular attention. Indeed, the water content in the model condensates is low (~200 mg/mL for GSY) relative to the experiment (e.g. ~600 mg/mL for FUS and LAF-1 from simulations). Considering that both interaction patterns and solvation contribute to the favorability of Tyr relative to Phe, we speculate that a greater degree of solvation in the true protein condensates will further reinforce the trends we observe.

In any case, in the revised version of the manuscript, we have made an effort to insist on the limitations of our results, some of which we plan to address in future work.

Reviewer #3 (Recommendations for the authors):

(1) The fact that protein density is so high within GSY or GSF peptide condensates may significantly alter the conclusions of the paper. Can the authors show that for condensates in which the protein density is ~0.2-0.3 g/cm3, the same conclusions hold? Could the authors use a different peptide sequence that establishes a more realistic protein concentration/density inside the condensate?

Unfortunately, recent work with a variety of peptide sequences suggests that finding peptides in the density range proposed by the referee may be very challenging. For example, Pettit and his co-workers have extensively studied the behaviour of GGXGG peptides. In a recent work, using the CHARMM36m force field and TIP3P water, they report densities of ~1.2-1.3 g/mL for capped pentapeptide condensates (Workman et al, Biophys. J. 2024; DOI: 10.1016/j.bpj.2024.05.009). Brown and Potoyan have recently run simulations of zwitterionic GXG tripeptides with the Amber99sb-ILDNQ force field and TIP3P water, starting with a homogenous distribution in cubic simulation boxes (Biophys. J. 2024, DOI: 10.1016/j.bpj.2023.12.027). In a box with an initial concentration of 0.25 g/mL, upon phase separation, the peptide ends up occupying what would seem to be ~1/3 of the box, although we could not find exact numbers. This would imply densities of ~0.75 g/mL in the dense phase, with the additional problem of many charges. Finally, Joseph and her co-workers have recently simulated a set of hexapeptide condensates with varied compositions using a combination of atomistic and coarse-grained simulations. For the atomistic simulations, the Amber03ws force field and TIP4P water were used (see BioRxiv reference 10.1101/2025.03.04.641530). They have found values of the protein density in the dense phase ranging between 0.8 and 1.2 g/mL.  The consistency in the range of densities reported in these studies suggests that short peptides, at least up to 7-residues long, tend to form quite dense condensates, akin to those investigated in our work. While the examples mentioned do not comprehensively span the full range of peptide lengths, sequences, and force fields, they nonetheless support the general behaviour we observe. A systematic exploration of all these variables would require an extensive search in parameter space, which we believe falls outside the scope of the present study.

(2) Do the conclusions hold for phase-separating systems that mostly rely on electrostatic interactions to undergo LLPS, like protein-RNA complex coacervates? In other words, could the authors try the same calculations for a binary mixture composed of polyR-polyE, or polyK-polyE?

This is an excellent idea that we may attempt in future work, but the remit of the current work is aromatic amino acids Phe and Tyr only. Hence, we do not include calculations or discussion on polyR-polyE systems in our revised manuscript.

(3) One of the major approximations made by the authors is the length of the peptides within the condensates, which is not realistic, or their density. Specifically, could they double or triple the length of these peptides while maintaining their composition so it can be quantified the impact of sequence length in the transfer of free energies?

We thank the referee for this comment and agree with the main point, which was stated as a limitation in our original submission. The suggested calculations anticipate research that we are planning but will not include in the current work. One of the advantages of our model systems is that the small size of the peptides allows for small simulation boxes and relatively rapid sampling. Longer peptide sequences would require conformational sampling beyond our current capabilities, if done systematically. An example of these limitations is the amount of data that we had to discard from the new simulations we report, which amounts to up to 200 ns of our replica exchange runs in smaller simulation boxes (i.e. >19 μs in total for the 48 replicas of the two condensates!). As stated in the answer to point 1, we have found in the literature work on peptides in the range of 1-7 residues with consistent densities. Additionally, a recent report using alchemical transformations using equilibrium techniques with tetrapeptide condensates, pointing to the role of transfer free energy as driving force for condensate formation, further supports the observations from our work.

Minor issues:

(1) The caption of Figure 3B is not clear. It can only be understood what is depicted there once you read the main text a couple of times. I encourage the authors to clarify the caption.

We have rewritten the caption for greater clarity. Now it reads as follows:

Time evolution of the density profiles calculated across the longest dimension of the simulation box (L) in the coexistence simulations. In blue we show the density of all the peptides, and in dark red that of the F/Y residue in the GGXGG peptide.

(2) Why was the RDF from Figure 5A cut at such a short distance? Can the authors expand the figure to clearly show that it has converged?

In the updated Figure 5 (now Fig. 6), we have extended the g(r) up to r=1.75 nm so that it clearly plateaus at a value of 1.

Author response:

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

Reviewer #1 (Public review):

Thank you very much for your recognition of our work and for pointing out the shortcomings. We have made revisions one by one and provided corresponding explanations regarding the issues you raised.

Weaknesses:

One of the main EEG results is based on the weighted phase lag index (wPLI) between oscillations in the alpha and theta bands. In my opinion, this is problematic, as wPLI measures the locking of oscillations at the same frequency. It quantifies how reliably the phase difference stays the same over time. If these oscillations have different frequencies, the phase difference cannot remain consistent. Even worse, modeling data show that even very small fluctuations in frequency between signals make wPLI artificially small (Cohen, 2015).

thank you for raising the question regarding the application of wPLI between the alpha and theta bands, which indeed deserves further explanation. In our study, we referred to some relevant previous literatures and adopted their approach of using wPLI to measure cross-frequency coupling strength, as this index itself can reflect the stability of phase differences. We have also considered the point you mentioned that the phase differences of oscillations with different frequencies are difficult to remain consistent. However, in this study, the presentation times of the two memory items are the same, which is fair to both from this perspective. Moreover, the study observed that the wPLI values of these two items alternately dominate over time, and this changing pattern is consistent with the regularity of behavioral data. It seems hard to explain this as a mere coincidence. 

The corresponding discussion has been added to the revised part of the paper：“the present study referenced previous research by using the wPLI index as a measure of cross-frequency coupling strength31,64-66 (this index quantifies the stability of phase differences), yet the phases of different oscillations inherently change over time. However, this is fair to the two memory items in the present study, as their presentation times were balanced. The study found that the wPLI values of the two items alternately dominated over time, consistent with the pattern of behavioral data, which is hardly explicable by coincidence”

Another result from the electrophysiology data shows that the attentional capture effect is positively correlated with the mean amplitude of alpha power. In the presented scatter plot, it seems that this result is driven by one outlier. Unfortunately, Pearson correlation is very sensitive to outliers, and the entire analysis can be driven by an extreme case. I extracted data from the plot and obtained a Pearson correlation of 0.4, similar to what the authors report. However, the Spearman correlation, which is robust against outliers, was only 0.13 (p = 0.57), indicating a non-significant relationship.

you mentioned that the correlation between the attentional capture effect and the mean amplitude of alpha power in the electrophysiological data might be influenced by an outlier, and you also compared the results of Pearson and Spearman correlation coefficients, which we fully agree with.

It is true that the small sample size of the current study makes the results vulnerable to interference from extreme data. Regarding this point, I have already explained it in the limitations section of the discussion in the revised manuscript:“the sample size of the current study is small, which may render the results vulnerable to interference from extreme cases”

The behavioral data are interesting, but in my opinion, they closely replicate Peters and colleagues (2020) using a different paradigm. In that study, participants memorized four spatial positions that formed the endpoints of two objects, and one object was cued. Similarly, reaction times fluctuated at theta frequency, and there was an anti-phase relationship between the two objects. The main novelty of the present study is that this bias can be transferred to an unrelated task. While the current study extends Peters and colleagues' findings to a different task context, the lack of a thorough, direct comparison with Peters et al. limits the clarity of the novel insights provided.

thank you very much for your attention to the behavioral data and its relevance to the study by Peters et al. (2020). We have noticed that there are similarities in some results between the two studies, which also indicates the stability of the relevant phenomena from one aspect.

However, we would also like to further explain the differences between this study and the study by Peters et al. In the study by Peters et al., participants memorized four spatial positions that formed the endpoints of two objects (one of which was cued), and their results showed that after the two objects disappeared, attention fluctuated at the theta rhythm between their original positions with an inverse correlation. In contrast, the present study explores the manner of memory maintenance indirectly by leveraging the guiding effect of working memory on attention, effectively avoiding the influence of spatial positions.

The study by Peters et al. directly examined differences in probe positions, clearly demonstrating that attention undergoes rhythmic changes at the two spatial locations and persists after the objects vanish, but it hardly clarifies the rhythmicity of working memory performance. Whereas the present study directly investigates such performance using the attention-capture effect of working memory, revealing that when maintaining multiple memory items, their attention-capturing capabilities alternate in dominance, i.e., multiple working memory items alternately become priority templates in a rhythmic manner. This is also some new attempts in the research perspective and method of this study.

The corresponding discussion has been added to the revised part of the paper

“Similar to the present study, Peters et al. had participants memorize four spatial positions forming the endpoints of two objects (one cued), and their results showed that after the two objects disappeared, attention fluctuated at the theta rhythm between their original positions with an inverse correlation; in contrast, the present study explores the manner of memory maintenance indirectly by leveraging the guiding effect of working memory on attention, effectively avoiding the influence of spatial positions—while Peters et al.’s study, which directly examined differences in probe positions, clearly demonstrates that attention undergoes rhythmic changes at the two spatial locations and persists after the objects vanish, it hardly clarifies the rhythmicity of working memory performance, whereas the present study directly investigates such performance using the attention-capture effect of working memory, revealing that when maintaining multiple memory items, their attention-capturing capabilities alternate in dominance, i.e., multiple working memory items alternately become priority templates in a rhythmic manner.”

Reviewer #2 (Public review):

The information provided in the current version of the manuscript is not sufficient to assess the scientific significance of the study.

thank you very much for pointing out the multiple issues in our manuscript. Due to several revisions of this work, including experimental adjustments, there have been some inconsistencies in details. We appreciate you identifying them one by one.  We have made corresponding revisions based on your comments:

(1) In many cases, the details of the experiments or behavioral tasks described in the main text are not consistent with those provided in the Materials and Methods section. Below, I list only a few of these discrepancies as examples:

a) For Experiment 1, the Methods section states that the detection stimulus was presented for 2000 ms (lines 494 and 498), but Figure 1 in the main text indicates a duration of 1500 ms.

we greatly appreciate you catching this inconsistency. We have made unified revisions by referring to the final implemented experimental procedures.  Corresponding revisions have been made in the paper：

b) For Experiment 2, not only is the range of SOAs mentioned in the Methods section inconsistent with that shown in the main text and the corresponding figure, but the task design also differs between sections.

Thank you for bringing this discrepancy to our attention. We have made unified revisions by referring to the final implemented experimental procedures. The correct SOAs are 233：33：867 ms.

Corresponding revisions have been made in the paper：

c) For Experiment 3, the main text indicates that EEG recordings were conducted, but in the Methods section, the EEG recording appears to have been part of Experiment 2 (lines 538-540).

we’re grateful for you noticing this mix-up. In fact, only Experiment 3 is an EEG experiment, and we have made corresponding corrections in the "Methods" section. Corresponding revisions have been made in the paper: “The remaining components after this process were then projected back into the channel space. We extracted data from -500 ms to 2000 ms relative to cue stimulus presentation in Experiment 3.”  

(2) The results described in the text often do not match what is shown in the corresponding figure. For example:

a) In lines 171-178, the SOAs at which a significant difference was found between the two conditions do not appear to match those shown in Figure 2A.

Many thanks for spotting this error. The previous results missed one SOA time, namely 33 ms, leading to a 33 ms difference in time. We have corrected it in the revised manuscript.

Corresponding revisions have been made in the paper：“Specifically, the capture effect of cued items was significantly greater than that of uncued items at SOAs of 267ms (t(24) = 2.72, p = 0.03, Cohen's d = 1.11), 667ms (t(24) = 2.37, p = 0.03, Cohen's d= 0.97) and 833ms (t(24) = 3.53, p = 0.002, Cohen's d = 1.44), while the capture effect of uncued items was significantly greater than that of cued items at SOAs of 333ms (t(24) = 2.97, p = 0.007, Cohen's d = 1.21), 367ms (t(24) = 2.14, p = 0.04, Cohen's d = 0.87), 433ms (t(24 )= 2.49, p = 0.02, Cohen's d = 1.02), 467ms (t(24)=2.37, p = 0.03, Cohen's d = 0.97) and 567ms (t(24)=2.72, p = 0.02, Cohen's d = 1.11). ”

(b) In Figure 4, the figure legend (lines 225-228) does not correspond to the content shown in the figure.

we appreciate you pointing out this oversight. When adjusting the color scheme during the revision of the manuscript, we neglected to revise the legend, which has now been corrected in the revised manuscript.

Corresponding revisions have been made in the paper：“Figure 4. The red line represents the average across all participants of the Fourier transforms of the differences in capture effects between left and right memory items at the individual level. The gray area represents values below the group average of medians derived from 1000 permutations, with each permutation involving Fourier transforms for each participant. *: p < 0.05.”

(c) In Figure 9, not sufficient information is provided within the figure or in the text, making it difficult to understand. Consequently, the results described in the text cannot be clearly linked to the figure.

Thank you for drawing our attention to this issue. We have revised Figure 9 and its legend in the revised manuscript to make them clearer and easier to understand.

Corresponding revisions have been made in the paper

(3) Insufficient information is provided regarding the data analysis procedures, particularly the permutation tests used for the data presented in Figures 2B, 4, and 10. The results shown in these figures are critical for the main conclusions drawn in the manuscript.

we’re thankful for you highlighting this gap. In the revised manuscript, we have provided a more detailed explanation in the "Methods" section, especially regarding the content related to frequency analysis, to make the expression clearer.

Corresponding revisions have been made in the paper：“As shown in Figure 8, the alpha power (8-14 Hz) induced by cued and uncued items alternated in dominance during the memory retention phase. To quantify this rhythmic alternation, we conducted a spectral analysis following these steps: First, we computed the power difference between cued and uncued items within the 8-14 Hz range during the retention phase. These differences were then downsampled to 100 Hz using a 10 ms window for averaging, generating a one-dimensional time series spanning the 0-2000 ms retention period. This time series was subsequently subjected to amplitude spectrum analysis across frequencies from 1 Hz to 50 Hz using Fourier transformation.

To assess the statistical significance of the observed spectral features, we employed a permutation test. Specifically, we randomly shuffled the temporal order of the time series of power differences between cued and uncued items—thereby preserving the amplitude distribution of the data while eliminating temporal correlations in the original sequence—and repeated the Fourier transform and spectral analysis for each shuffled time series. This permutation process was replicated 1000 times to generate a null distribution of spectral power values. A frequency component in the original data was considered statistically significant if its power ranked within the top 5% of the corresponding null distribution (p < 0.05).

We applied the same analytical pipeline to investigate differences in the weighted phase-lag index (wPLI) between the contralateral regions of the two items and the prefrontal cortex during the retention phase. Specifically, wPLI differences (i.e., the difference between the two conditions) were computed, downsampled to 100 Hz using a 10 ms window for averaging to generate a time series spanning 0-2000 ms, and then subjected to amplitude spectrum analysis (1-50 Hz) using Fourier transformation. Significance was assessed via the identical permutation test procedure described above (randomly shuffling the temporal order of the difference time series).”

DOI: 10.1101/2025.09.15.676412

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DOI: 10.1016/j.xcrm.2025.102365

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DOI: 10.1016/j.scr.2025.103841

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DOI: 10.1016/j.cels.2025.101402

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What is this?

Catalogue: http://hdl.handle.net/1887.1/item:2669083 Visor: https://iiifviewer.universiteitleiden.nl/?manifest=https%3A//digitalcollections.universiteitleiden.nl/iiif_manifest/item%253A2669083/manifest

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Cobertura temporal de la consulta

Nota: revisar

Si entiendo bien ,estas testeando el modelo publicado en tu paper anterior con estos datos no? entiendo que el formato de esta revista tiene la metodologia al final, es por eso que empiezas con un modo resumen de la metodologia en tus resultados? (este primer parrafo?

It depends on the rule of the college as to whether IA can not be used. If it is prohibited by the school policy , then it is a clear cheating .IA is a guardian jump start your idea , but can not be fully use.

with their son in law

Los nuevos significados de la familia le han permitido a la mujer trabajar, sin descuidar sus responsabilidades familiares.

Me llama la atencion estas oraciones, porque resaltan que la familia no solo consiste en una casa y las personas que viven en ella.

Los diferentes tipos de conceptos sobre la familia siempre sera un tema para debatir, con el pasar de el tiempo el significado de que es una familia a cambio mucho alredor de el mundo. Lo cual me hace reflexionar en que debemos estar dispuestos a escuchar diferentes opiniones.

Yo estoy de acuerdo con esto, ya que el cuidado entre generaciones es muy importante para el desarrollo.

Creo que los adultos mayores están empezando a ser más independientes más pronto que tarde debido a la época en la que vivimos. Con esto me refiero a cómo las redes sociales han persuadido a la gente a ser más abierta (explorar), mientras que antes solo contábamos con nuestra familia de sangre y nos creíamos la mayoría de lo que decían. No sé si me explico, es un poco confuso. Y sí, puede haber desafíos al volverse más independiente, ¡pero esto es bueno! Nos ayuda a crecer y aprender de nuestros errores, y eso es lo que nos prepara para la edad adulta.

Para mí, la familia no son solo aquellos con la misma sangre, sino aquellos a quienes amas, aquellos que han estado contigo cuando más los has necesitado. Digo esto porque a veces ciertos miembros de la "familia" (misma sangre) son de las peores personas posibles. Desafortunadamente, eso sucede.

No me identifico personalmente con esto, pero tengo un hermano que se fue a tener hijos y siguió viviendo en casa de mis padres con su conyuge. Les llevó un tiempo conseguir su propio lugar, pero al final lo consiguieron. Dicho esto, entiendo que esto sea cierto en cierto modo.

El texto muestra que los padres que no viven con sus hijos y la ayuda entre generaciones son importantes para el desarrollo infantil. Tambien senala que los programas sociales para ninos vulnerables ayudan a mejorar su bienestar.

El texto senala que, a diferencia de lo que ocurre en Estados Unidos, en America Latina y el Caribe el aumento del nivel educativo de las mujeres no disminuye la probabilidad de que sean madres solteras.

El informe Pulso Social 2016 muestra que en America Latina y el Caribe casa vez mas adultos mayores viven solos so solo con su conyuge an lugar de con la familia extendida. Aunque este cambio puede favorecer la independencia, talmbien tiene retos que podrian poner los cuidados de la vejez en riesgo.

unidad singular e irrepetible que forma parte de un sistema más grande, ya sea biológico, social o estadístico.

Confirmo esto, la familia moderna busca mas el bienestar de todos que el propio

Personas que no son de tu familia tambien puede llegar a sentirse como una si el sentimiento es lo bastante fuerte.

Dans le corrigé, vous mettez cette balise dans le "header", mais dans le cours, on voit qu'elle vient après le "header". Du coup, je n'ai pas le même rendu que vous. De plus, vous ajoutez un lien "à propos" dans la page "à propos". Pour moi, cela est inutile : on ne va pas cliquer sur le lien d'une page où on y est déjà !

Another artist I have never heard of.

How big a difference would be introduced? It seems like there isn't a huge drop off if they were combined, if both of them?

Personally, I don't think the MVP should be a sychronized file as it doesn't align with WCAG 2.1 AA requirements. I think this should be something much similar like:

A machine generated transcript delivered in plain text.

Mucho de la población de Los Estados Unions son hispanohablantes. Entonces, cuando los etudiantes aprenden inglés necesita se esfuerza como preservar español tambien y mezcla los culturas.

Dominicano, Cubano, Puertorriqueño, Mexicano, Salvadoreño

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OJO

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IMPORTANTE

MUY IMPORTANTE

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Author response:

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

Reviewer #1 (Public review):

“Can the authors offer a hypothesis as to how decreased coactivity promotes increased movement velocity.” 

In our revision we have added an additional metric measuring how spatial coactivity changes during movement onset, the spatial correlation index, which replicates a previous finding that co-activity among proximal neurons is statistically greater surrounding movement onset. We did not find, as outlined in the revision, that mGluR5 manipulations significantly altered this relationship. Our data therefore shows, consistent with that shown previously, that ensembles of dSPNs that are co-active during movement onset, in particular ambulatory movement, are more likely to contain neurons that are closer together and the neurons are highly active. In contrast, rest ensembles contain neurons that are less active but have more highly correlated activity, across all pairwise distances. Additionally, mGluR5 inhibition, genetic or pharmacological, promotes the activation of rest ensembles but does not affect the properties of movement ensembles. Previous studies (e.g. Klaus A. et al., 2017) have shown that neurons in rest ensembles are, in general, unlikely to also be members of movement ensembles, We therefore hypothesize that corticostriatal synapses onto SPNs of rest ensembles are more likely, during spontaneous behavior, to have reduced synaptic weight due to mGluR5 signaling, potentially due to eCB mediated inhibition of neurotransmitter release. Therefore, when we inhibit mGluR5 at these synapses, we increase synaptic weight and increase the probability of activation of this coordinated rest ensemble, which suppresses movement. If, on the other hand, the synapses that govern activation of neurons in movement ensembles have a higher weight, they may be unaffected by mGluR5 inhibition. 

The use of the Jaccard similarity index in this study is not intuitive and not fully explained by the methods or the diagram in Figure 1. 

We have added more detail to the paper to explain the methodology of the jaccard similarity measure. The advantage of this method is that is specifically captures cells that are jointly active, as opposed to jointly inactive and is therefore useful for capturing co-activity in our sparsely active Ca²⁺ imaging data. 

The analysis of a possible 2-AG role in the mGlu5 mediated processes is incomplete. 

We agree that, as an experiment to outline which endocannabinoids are involved in modulating synaptic strength through mGluR5, this experiment alone is not sufficient.

However, our main focus in this paper is how manipulations of mGluR5 affect the spatiotemporal dynamics of dSPNs and we chose not to focus on specific mechanisms of endocannabinoid signaling, though these would certainly be interesting to investigate further in vivo.

It would seem to be a simple experiment to examine effects of the mGlu5 NAM in the dSPN mGlu5 cKO mice. If effects of the two manipulations occluded one another this would certainly support the hypothesis that the drug effects are mediated by receptors expressed in dSPNs. A similar argument can be made for examining effects of the JNJ PAM in the cKO mice. 

We agree that this experiment would be valuable and extend our findings presented in the paper, however, it has practically been outside the scope of the current work. 

Reviewer #2 (Public review):

Pharmacological and genetic manipulations of mGluR5 do not differentially/preferentially modulate the activity of proximal vs distal dSPNs, therefore, it could also be interpreted that mGluR5 is blanketly boosting/suppressing all dSPN activity as opposed to differential proximal/distal spatial relationships. 

As in the response to reviewer 1 above, we have added additional clarification to the text explaining that our manipulations do not differentially affect the co-activity of proximal vs distal dSPNs, this is also quantified throughout the text using the spatial coordination index. However, we disagree that “it could also be interpreted that mGluR5 is blanketly boosting/suppressing all dSPN activity” as we do not observe statistically significant changes in the event rate following either pharmacological or genetic manipulations of mGluR5. Rather, we consistently observe statistically significant changes in co-activity among neurons, the extent to which activity of active neurons during either rest or movement are correlated with each other. This is the central finding of our manuscript, inhibiting or potentiating mGluR5 signaling alters behavior, not by blanket suppression or enhancement of the activity as measured using the event rate, of dSPNs, but by affecting their ensemble dynamic properties.  Co-activity during rest versus ambulatory movement is statistically greater in both proximal and distal cells and inhibiting mGluR5 increases this co-activity and decreases movement. 

For these analyses of prox vs distal and all others, please include the detail of how many proximal vs distal cells were involved and per subject. 

We have added a supplemental table that details the number of cells included per subject in all analyses

Ln. 151-152: Please provide data concerning how volumes of infectivity differ between injecting AAV vs. coating the lens? If these numbers are very different, this could impact the number of Jaccard pairings and bias results. 

While viral injection may lead to a larger volume of expression, with this one photon imaging method only those cells within ~200 microns of the edge of the lens will be able to be resolved, therefore practically, if there is an additional volume of infected tissue outside of the field of view of the lens, it would not affect the results as these neurons will not be resolved by the endoscope camera. Accordingly, the average number of cells detected per session is very similar following each approach (mean # of cells per session with coating 90.93 ± 23.69 cells, with viral injection 90.03 ± 29.29 cells)

Is mGluR5 affecting dSPN activity in other measures beyond co-activity and rate? Does the amplitude of events change?

We have added supplemental data for figures 2, 3, and 5 demonstrating that manipulations of mGluR5 do not affect the amplitude or length of Ca²⁺ events included in the analysis. 

What is the model of mGluR5 signaling in a resting state vs. movement? What other behaviors are occurring when the mouse is in a low velocity "resting state" (0-0.5 cm/s). If this includes other forms of movement (i.e. rearing, grooming) then the animal really isn't in a resting state. This is not mentioned in the open field behavior section of the methods and should be described (Ln. 486) in addition to greater explanation of what behavior measures were obtained from the video tracking software (only locomotion?)

It would be very interesting to determine if during “rest,” when the animals is not engaged in ambulatory behavior, it may be engaged in some fine motor behavior. However, the resolution of the cameras used to measure locomotor activity in this dataset does not allow us to do this. 

There is large variability in co-activity in proximal dSPNs when animals are "resting" (2j). Could this be explained by different behavior states within your definition of "rest"?

We agree that if the animal is engaging in fine motor behavior that we cannot resolve with our behavior setup, this could produce some variability in coactivity. However, as shown previously (e.g. Klaus A. et al., 2017), ensembles active when the animal is not moving (our definition of “resting”), regardless of additional fine motor behaviors the animal may be engaged in when not moving, are substantially different that those ensembles that are active when the animal is moving. We therefore expect that this may limit, although potentially not eliminate, variability due to different behavioral states we may have grouped into our “resting” category. Unfortunately, as mentioned above, we are not able resolve variations in fine motor output in this behavioral data. 

Have you performed IHC, ISH or another measure to validate D1 cell specific cKO?

The mGluR5^loxP/loxP mice used in this study were characterized previously by our lab (Xu et al., 2009), we used the same mice here with a different, but also published and characterized Cre-driver line, Drd1a-Cre Ey262 (Gerfen et al., 2013).

Why are the "Mean Norm Co-activity" values in 5e so high in this experiment relative to figures 2-4?  

In experiments where we treated the same animal with vehicle and a drug (i.e., experiments in Figure 2 and 3), we normalized the values for each animal in the drug treatment group to the distal bin of that animal following vehicle treatment. This allowed us to more clearly resolve the changes within each animal due to drug treatment. As comparisons in the data in figure 5 d–f are between different animals (rather than different treatments of the same animal) we could not perform this normalization procedure.  

Reviewer #3 (Public review):

Some D1 Cre lines have expression in the cortex. Which specific Cre line was used in this study? 

We used, Drd1a-Cre Ey262. This is included in methods. 

The text says JNJ treatment .... increased locomotor speed (Figure 3b) and increased the duration but not frequency of movement bouts (Figure 3c, d). However, the statistics of the figure legends say: however the change in mean velocity (3b) is not significant (p=0.060, U=3, Mann-Whitney U test), nor is the mean bout length during vehicle and JNJ (p=0.060, U=3, Mann-Whitney U test) (3d) Comparison of mean number of bouts of each animal during vehicle and JNJ (p=0.403, U=8, Mann-Whitney U test). 

This has been corrected to indicate only the change in time spend at rest is statistically significant.

This effect was most pronounced during periods of rest (Figure 3i, j). The decrease was only in rest? Are the colors in Figure 3J inverted? Therefore, JNJ treatment had effects that were qualitatively the inverse to the effects of fenobam on locomotion and dSPN activity. 

We have corrected the text to state that, overall, and during periods of rest but not movement, JNJ had effects that were qualitatively the opposite of fenobam.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

This work addresses an important question in the field of Drosophila aggression and mating. Prior social isolation is known to increase aggression in males, manifesting as increased lunging, which is suppressed by group housing (GH). However, it is also known that single housed (SH) males, despite their higher attempts to court females, are less successful. Here, Gao et al., develop a modified aggression assay to address this issue by recording aggression in Drosophila males for 2 hours, with a virgin female immobilized by burying its head in the food. They found that while SH males frequently lunge in this assay, GH males switch to higher intensity but very low frequency tussling. Constitutive neuronal silencing and activation experiments implicate cVA sensing Or67d neurons in promoting high frequency lunging, similar to earlier studies, whereas Or47b neurons promote low frequency but higher intensity tussling. Optogenetic activation revealed that three pairs of pC1SS2 neurons increase tussling. Cell-type-specific DsxM manipulations combined with morphological analysis of pC1SS2 neurons and side-by-side tussling quantification link the developmental role of DsxM to the functional output of these aggression-promoting cells. In contrast, although optogenetic activation of P1a neurons in the dark did not increase tussling, thermogenetic activation under visible light drove aggressive tussling. Using a further modified aggression assay, GH males exhibit increased tussling and maintain territorial control, which could contribute to a mating advantage over SH males, although direct measures of reproductive success are still needed.

Strengths:

Through a series of clever neurogenetic and behavioral approaches, the authors implicate specific subsets of ORNs and pC1 neurons in promoting distinct forms of aggressive behavior, particularly tussling. They have devised a refined territorial control paradigm, which appears more robust than earlier assays using a food cup (Chen et al., 2002). This new setup is relatively clutter-free and could be amenable to future automation using computer vision approaches. The updated Figure 5, which combines cell-type-specific developmental manipulation of pC1SS2 neurons with behavioral output, provides a link between developmental mechanisms and functional aggression circuits. The manuscript is generally well written, and the claims are largely supported by the data.

Thank you for the precise summary of the manuscript and acknowledgment of the novelty and significance of the study.

Weakness:

Although most concerns have been addressed, the manuscript still lacks a rigorous, objective method for quantifying lunging and tussling. Because scoring appears to have been done manually and a single lunge in a 30 fps video spans only 2-3 frames, the 0.2 s cutoff seems arbitrary, and there are no objective criteria distinguishing reciprocal lunging from tussling. Despite this, the study offers valuable insights into the neural and behavioral mechanisms of Drosophila aggression.

Thank you for this comment. The duration of each lunge was measured by analyzing the videos frame by frame—from the frame before the initiation of the lunge to the frame after its completion—resulting in an average span of 3–5 frames. Given a frame rate of 30 fps, this corresponds to approximately 0.1–0.17 seconds. We acknowledge that there are certain limitations for manually quantifying the two types of aggressive behaviors, which has now been stated in the newly added “Limitations of the Study” section in the revised manuscript.

Reviewer #2 (Public review):

Summary:

Gao et al. investigated the change of aggression strategies by the social experience and its biological significance by using Drosophila. Two modes of inter-male aggression in Drosophila are known: lunging, high-frequency but weak mode, and tussling, low-frequency but more vigorous mode. Previous studies have mainly focused on the lunging. In this paper, the authors developed a new behavioral experiment system for observing tussling behavior and found that tussling is enhanced by group rearing, while lunging is suppressed. They then searched for neurons involved in the generation of tussling. Although olfactory receptors named Or67d and Or65a have previously been reported to function in the control of lunging, the authors found that these neurons do not function in the execution of tussling and another olfactory receptor, Or47b, is required for tussling, as shown by the inhibition of neuronal activity and the gene knockdown experiments. Further optogenetic experiments identified a small number of central neurons pC1[SS2] that induce the tussling specifically. These neurons express doublesex (dsx), a sex-determination factor, and knockdown of dsx strongly suppresses the induction of tussling. In order to further explore the ecological significance of the aggression mode change in group-rearing, a new behavioral experiment was performed to examine the territorial control and the mating competition. And finally, the authors found that differences in the social experience (group vs. solitary rearing) and the associated change in aggression strategy are important in these biologically significant competitions. These results add a new perspective to the study of aggression behavior in Drosophila. Furthermore, this study proposes an interesting general model in which the social experience modified behavioral changes play a role in reproductive success.

Strengths:

A behavioral experiment system that allows stable observation of tussling, which could not be easily analyzed due to its low-frequency, would be very useful. The experimental setup itself is relatively simple, just the addition of a female to the platform, so it should be applicable to future research. The finding about the relationship between the social experience and the aggression mode change is quite novel. Although the intensity of aggression changes with the social experience was already reported in several papers (Liu et al., 2011 etc), the fact that the behavioral mode itself changes significantly has rarely been addressed, and is extremely interesting. The identification of sensory and central neurons required for the tussling makes appropriate use of the genetic tools and the results are clear. A major strength of this study in neurobiology is the finding that another group of neurons (Or47b-expressing olfactory neurons and pC1[SS2] neurons), distinct from the group of neurons previously thought to be involved in low-intensity aggression (i.e. lunging), function in the tussling behavior. Furthermore, the results showing that the regulation of aggression by pC1[SS2] neurons is based on the function of the dsx gene will bring a new perspective to the field. Further investigation of the detailed circuit analysis is expected to elucidate the neural substrate of the conflict between the two aggression modes. The experimental systems examining the territory control and the reproductive competition in Fig. 6 are novel and have advantages in exploring their biological significance. It is important to note that in addition to showing the effects of age and social experience on territorial and mating behaviors, the authors experimentally demonstrated that altered fighting strategy has effects with respect to these behaviors.

Thank you for your precise summary of our study and being very positive on the novelty and significance of the study.

Reviewer #3 (Public review):

In this revised manuscript, Gao et al. presented a series of well-controlled behavioral data showing that tussling, a form of high-intensity fighting among male fruit flies (Drosophila melanogaster) is enhanced specifically among socially experienced and relatively old males. Moreover, results of behavioral assays led authors to suggest that increased tussling among socially experienced males may increase mating success. They also concluded that tussling is controlled by a class of olfactory sensory neurons and sexually dimorphic central neurons that are distinct from pathways known to control lunges, a common male-type attack behavior.

A major strength of this work is that it is the first attempt to characterize behavioral function and neural circuit associated with Drosophila tussling. Many animal species use both low-intensity and high-intensity tactics to resolve conflicts. High-intensity tactics are mostly reserved for escalated fights, which are relatively rare. Because of this, tussling in the flies, like high-intensity fights in other animal species, have not been systematically investigated. Previous studies on fly aggressive behavior have often used socially isolated, relatively young flies within a short observation duration. Their discovery that 1) older (14-days old) flies tend to tussle more often than younger (2 to 7-days-old) flies, 2) group-reared flies tend to tussle more often than socially isolated flies, and 3) flies tend to tussle at later stage (mostly ~15 minutes after the onset of fighting), are the result of their creativity to look outside of conventional experimental settings. These new findings are key for quantitatively characterizing this interesting yet under-studied behavior.

Newly presented data have made several conclusions convincing. Detailed descriptions of methods to quantify behaviors help understand the basis of their claims by improving transparency. However, I remain concerned about authors' persistent attempt to link the high intensity aggression to reproductive success. The authors' effort to "tone down" the link between the two phenomena remains insufficient. There are purely correlational. I reiterate this issue because the overall value of the manuscript would not change with or without this claim.

Thank you for acknowledging the novelty and significance of the study. Regarding the relationship you mentioned between high-intensity aggression and reproductive success, we further toned down the statement between them throughout the manuscript in the revised manuscript. We also modified the title to “Social Experience Shapes Fighting Strategies in Drosophila”. In addition, we now added a ‘Limitations of the Study’ section to clearly state the correlation between tussling and reproductive success.

Reviewer #1 (Recommendations for the authors):

If possible, mention the EM-connectome data showing the minimal interneuronal path from Or47b ORNs to pC1SS2 neurons (even if derived from the female connectome), which can strengthen the model of parallel sensory-central pathways.

Thank you for this comment. According to data from the EM connectome, connecting Or47b ORNs to pC1d neurons requires at least two intermediate neurons. An example minimal pathway is: ORN_VA1v (L) → AL-AST1 (L) → PLP245 (L) → pC1d (R). We have added this point in the Discussion section of the revised manuscript.

I'm not convinced that labeling lunges as "gentle" combat behavior works, either in the abstract or elsewhere. While lunging is indeed a lower-intensity form of aggression compared to tussling, applying anthropomorphic descriptors risks misleading readers.

Thank you for this comment. We now use “low-intensity” instead of “gentle” to describe lunging.

In Materials & Methods, please cross-check all figure-panel references after the recent re-numbering (e.g. "Figure 5A6A" etc.).

Thank you for this comment. We have thoroughly verified the figure panel references in the Materials & Methods section.

Ensure that Table S1 is clearly cited in the main text where you first describe fly genotypes.

Thank you for this comment. We have now cited Table S1 in the main text.

There are multiple grammatical errors and typos throughout the manuscript. Please correct them. Some examples are below, but this is not an exhaustive list:

Line 98-102 requires rephrasing as the results are already published and not being observed by the authors.

Thank you for this comment. We have revised the manuscript to “we occasionally observed the high-intensity boxing and tussling behavior in male flies as previously reported (Chen et al., 2002; Nilsen et al., 2004), which….”

line 116- lower not 'lowed'.

Corrected.

line 942 & 945- knock-down males not 'knocking down males'.

Corrected. Thank you very much for these comments.

Reviewer #2 (Recommendations for the authors):

The authors have almost completely answered the major comments I have noted on the ver.1 manuscript: (1) They clearly show changes in fighting strategy in the territory control behavior experiment in Fig. 6-figure supplements. (2) A detailed description of how aggressive behavior is measured. Thus, I am convinced by this revision.

Thank you for these comments that make the manuscript a better version.

Furthermore, in Fig. 5, which examined the relationship of pC1[SS2] characteristics with the function of dsx, is a novel data and very interesting. I look forward to further developments.

Thank you. We will continue to explore this part in our future study.

However, one point still concerns me.

Line 192: Although the authors describe it as "usage-dependent," the trans-Tango technique is essentially a postsynaptic cell-labeling technique. It is possible that the labeling intensity in postsynaptic cells increases from the change in expression levels of the Or47b gene due to GH. However, there is no difference in the expression level of the Or47b gene labeled by GFP between SH and GH. Therefore, we cannot conclude that the expression of the Or47b gene is increased by rearing conditions.

The original paper on trans-TANGO (Talay et al., 2017) does not discuss the usage-dependency. A review of trans-synaptic labeling techniques (Ni, Front Neural Circuits. 2021) discusses that the increase in trans-TANGO signaling with aging may be related to synaptic strength, but there is no experimental evidence for this. In my opinion, the results in Figure 3-figure supplement 2 only weakly suggest that the increase in trans-TANGO signaling may be explained by an increase in synaptic strength due to group rearing.

We appreciate the reviewer’s insightful comment regarding the interpretation of the trans-Tango signal. Indeed, the original trans-Tango study (Talay et al., 2017) does not claim that the method is usage-dependent. The observed increase in trans-Tango labeling with age, as reported in their supplemental figures, may reflect accumulation over time, potentially influenced by synaptic maturation or increased component expression. To avoid overstating our results, we have revised the relevant statement in the manuscript to remove the term "usage-dependent" and now describe the change in trans-Tango signal more cautiously.  

Reviewer #3 (Recommendations for the authors):

Below are the cases where their professed attempts to "tone down the statement" appear ignored:

Lines 27-29:

"Our findings... suggest how social experience shapes fighting strategies to optimize reproductive success".

We have now revised the manuscript to “Our findings… suggest that social experience may shape fighting strategies to optimize reproductive success.”

Lines 85-86:

"... discover that this infrequent yet intense form of combat is... crucial for territory dominance and mating competition".

We have now revised the manuscript to “…discover that this infrequent yet intense form of combat is enhanced by social enrichment, while the low-intensity lunging is suppressed by social enrichment.” 

Lines 335-339:

"Here, we found that... GH males tend to... increase the high-intensity tussling, which enhances their territorial and mating competition."

We have removed “which enhances their territorial and mating competition” in the revised manuscript.

Lines 343-344:

"... presenting a paradox between social experience, aggression and reproductive success. Our result resolved this paradox..."

We have now revised the manuscript to “...Our results provide an explanation for this paradox…”

Lines 355-358:

"Interestingly, we found that the mating advantage gained through social enrichment can even offset the mating disadvantage associated with aging, further supporting the vital role of shifting fighting strategies in experienced, aged males."

We have removed “further supporting the vital role of shifting fighting strategies in experienced, aged males” in the revised manuscript.

Lines 361-362:

"These results separate the function of the two fighting forms and rectify out understanding of how social experiences regulate aggression and reproductive success."

We have removed this sentence in the revised manuscript.

Some may say that a speculative statement is harmless, but I think it indeed is harmful unless it is clearly indicated as a speculation. It is regrettable that authors remain reluctant to change their claim without providing any new supporting evidence. All three reviewers raised the same concern in the first round of review.

We apologize for not making the speculative nature of the statement clearer in the previous version. In the revised manuscript, we have now explicitly rephrased sentences to only suggest a correlation but not a causal link between tussling and reproductive success.

I have no choice but to keep my evaluation of the manuscript as "Incomplete" unless the authors thoroughly eliminate any attempt to link these two. This must go beyond changing a few words in the lines listed above.

Thank you for this comment. In addition to the lines listed above, we carefully checked all statements regarding the correlation between fighting strategies and reproductive success throughout the full text. Furthermore, we have also added a “Limitations of the Study” section to address the shortcomings of this study in the revised manuscript.

I do not have the same level of concern over the interpretation of Fig. 6A-C, because this is directly linked to aggressive interactions. Even if the socially isolated males do not engage in tussling, it is not a leap to assume that a different fighting tactic of socially experienced males can give them an advantage in defending a territory. To me, this is a sufficient ethological link with the observed behavioral change.

Thank you for this insightful comment.

The following are relatively minor, although important, concerns.

I beg to differ over the authors' definition of "tussling". Supplemental movies S1 and S2 appear to include "tussling" bouts in which 2 flies lunging at each other in rapid succession, and supplemental movie S3 appears to include bouts of "holding", in which one fly holds the opponent's wings and shakes vigorously. These cases suggest that the definition of "tussling" as opposed to "lunging" has a subjective element. However, I would not delve on this matter further because it is impossible to be completely objective over behavioral classification, even by using a computational method. An important point is that the definition is applied consistently within the publication. I have no reason to doubt that this was not the case.

Thank you for this comment. Since the analysis of tussling behavior was conducted manually, it is challenging to achieve complete objectivity. However, we made every effort to apply consistent criteria throughout the analysis. We have added a “Limitations of the Study” section in the revised manuscript to clearly state this caveat. We appreciate your understanding.

Authors now state that "all tester flies were loaded by cold anesthesia" (lines 432-433). I would like to draw attention to the well-known fact that anesthesia, whether by ice or by CO2, are long known to affect fly's subsequent behaviors (for aggression, see Trannoy S. et al., Learn. Mem. 2015. 22: 64-68). It will be prudent to acknowledge the possibility that this handling method could have contributed to unusually high levels of spontaneous tussling, which has not been reported elsewhere before.

Thank you for this comment. The increased tussling behavior observed in our study is unlikely due to cold anesthesia, as noted by Trannoy S. et al. (2015), cold anesthesia profoundly reduces locomotion and general aggressiveness in flies. We acknowledge that the use of cold anesthesia in behavioral experiments may have potential effects on aggression. To minimize this influence, we allowed the flies to recover and adapt for at least 30 minutes before behavioral recording. Moreover, both control and experimental groups were treated in exactly the same manner to ensure consistency.

It is intriguing that pC1SS2 neurons are dsx+ but fru-. Authors convincingly demonstrated that these neurons are clearly distinct from the P1a neurons, a well-characterized hub for male social behaviors. It is possible that pC1SS2 neurons overlap with previously characterized dsx+ neurons that are important for male aggressions (measured by lunges), such as in Koganezawa et al., Curr. Biol. 2016 and Chiu et al., Cell 2020, a point authors could have explicitly raised.

Thank you for this comment. We have added this point into the Discussion section of the revised manuscript, as follows: “That tussling-promoting… aggression (Koganezawa et al., 2016). Moreover, the anatomical features of pC1^SS2 neurons are highly similar to the male-specific aggression-promoting (MAP) neurons identified by another previous study (Chiu et al., 2021).

I acknowledge the authors' courage to initiate an investigation to a less characterized, high intensity fighting behavior. Tussling requires the simultaneous engagement of two flies. Even if there are confusion over the distinction between lunges and tussling, authors' conclusion that socially experienced flies and socially isolated flies employ distinct fighting strategy is convincing. The concern I raised above is about the interpretation of the data, not about the quality of data.

Thank you for your constructive comments to make this manuscript better.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review):

Summary: 

This study provides compelling evidence suggesting that ghrelin, a molecule released in the surroundings of the major adult brain neurogenic niche (V-SVZ) by blood vessels with high blood flow, controls the migration of newborn interneurons towards the olfactory bulbs. 

Strengths:

This study is a tour de force as it provides a solid set of data obtained by time-lapse recordings in vivo. The data demonstrate that the migration and guidance of newborn neurons rely on factors released by selective types of blood vessels. 

Weaknesses:

Some intermediate conclusions are weak and may be reinforced by additional experiments. 

We thank the reviewer for the thoughtful evaluation and constructive comments outlined in the “Recommendations for The Authors”. In response, we have incorporated additional data, revised relevant figures, and clarified explanations in the revised manuscript.

Reviewer #2 (Public review)

Summary: 

The authors establish a close spatial relationship between RMS neurons and blood vessels. They demonstrated that high blood flow was correlated with migratory speed. In vitro, they demonstrate that Ghrelin functions as a motogen that increases migratory speed through augmentation of actin cup formation. The authors proceed to demonstrate through the knockdown of the Ghrelin receptor that fewer RMS neurons reach the OB.

They show the opposite is true when the animal is fasted. 

Strengths: 

Compelling evidence of close association of RMS neurons with blood vessels (tissue clearing 3D), preferentially arterioles. Good use of 2-photon imaging to demonstrate migratory speed and its correlation with blood flow. In vitro analysis of Ghrelin administration to cultured RMS neurons, actin visualization, Ghsr1KD, is solid and compelling. 

We sincerely thank the reviewer for the encouraging comments and helpful suggestions. As noted, our original manuscript lacked sufficient in vivo evidence connecting blood flow with ghrelin signaling. To address this, we have added new data and revised the explanations throughout the manuscript as described below.

Weaknesses: 

(1) Novelty of findings attenuated due to prior work, especially Li et al., Experimental Neurology 2014. Here, the authors demonstrated that Ghrelin enhances migration in adultborn neurons in the SVZ and RMS. 

We agree with the reviewer that the idea that ghrelin enhances migration of new neurons is not entirely novel. The study by Li et al. (2014) provided critical insights that guided our investigation into ghrelin as a blood-derived factor promoting neuronal migration. However, our study expands on this by demonstrating that ghrelin directly stimulates migration via GHSR1a in cultured new neurons, and we further identified the cellular and cytoskeletal mechanisms involved. Specifically, we showed that ghrelin enhances somal translocation by activating actin dynamics at the rear of the cell soma. We have revised the Results and Discussion sections accordingly to emphasize these novel aspects as follows:

“A previous study demonstrated that the migration of V-SVZ-derived new neurons was attenuated in ghrelin knockout mice (Li et al., 2014). In our study, we found that the migration of cultured new neurons was enhanced by the application of ghrelin to the culture medium, and this effect was abolished by Ghsr1a knockdown (KD). These findings suggest that ghrelin directly stimulates neuronal migration through its receptor, GHSR1a, on new neurons. A previous study showed that GHSR1a is expressed in various regions of the brain (Zigman et al., 2006). In our experiments, new neuron-specific KD of Ghsr1a indicated that ghrelin signaling acts in a cell-autonomous manner to regulate neuronal migration.” (Discussion, page 13, lines 10–18)

“Furthermore, we identified the cellular and cytoskeletal mechanisms underlying this effect on migration. The results indicate that ghrelin enhances somal translocation during migration by activating actin cytoskeletal dynamics at the rear of the neuronal soma.” (Discussion, page 13, lines 24–26)

(2) The evidence for blood delivery of Ghrelin is not very convincing. Fluorescently-labeled Ghrelin appears to be found throughout the brain parenchyma, irrespective of the distance from vessels. It is also not clear from the data whether there is a link between increased blood flow and Ghrelin delivery. 

We agree that the correlation between blood flow and ghrelin transcytosis is not very convincing in our study. As the reviewer pointed out, Figure 3A gives the impression that fluorescent-labeled ghrelin is uniformly distributed throughout the brain parenchyma. However, high-magnification images newly added in Figure 3 show that some, but not all, vessels have particularly strong fluorescent signals in the parenchymal area adjacent to the abluminal side of vascular endothelial cells, visualized by CD31 immunostaining (Feng et al., 2004) (Figure 3A′, A′′). To quantify these observations, we defined two regions: Area I (perivascular area), within 10 μm of the abluminal surface of CD31-positive endothelium; and Area II (distant area), located 10–20 μm away (Figure 3E). Of note, Area I corresponds to the perivascular region where new neurons are frequently observed (Figure 1).

Importantly, we found strong ghrelin signals in vascular endothelial cells of endomucin-negative high-flow vessels (Figure 3C, D). This suggests that transcytosis of blood-derived ghrelin may occur more frequently in high-flow vessels due to increased endocytosis at the endothelium. To test this, we quantified signal gradients in the extra-vessel regions as fold changes (Area I / Area II), as illustrated in Figure 3E. The proportion of vessel segments with >1.5-fold increases was significantly higher in endomucin-negative vessels than in endomucin-positive ones (Figure 3F). Furthermore, vessels with >2-fold increases were observed exclusively in the endomucinnegative group (6.48% ± 1.18%). 

These data suggest that, in high-flow vessels, blood-derived ghrelin accumulates more in the immediate perivascular region than in areas further away. This supports the possibility that elevated blood flow delivers a larger amount of ghrelin to the vascular endothelium, enhancing its transcytosis into adjacent brain parenchyma. This mechanism may underlie the preferential migration of new neurons along perivascular regions with high blood flow, as shown in Figure 1.  We have incorporated this new data in Figure 3 and corresponding explanations into the Results, Figure legend and Methods

(3) The in vivo link between Ghsr1KD and migratory speed is not established. Given the strong work to open the study on blood flow and migratory speed and the in vitro evidence that migratory speed is augmented by Ghrelin, the paper would be much stronger with direct measurement of migration speed upon Ghsr1KD. Indeed, blood flow should also be measured in this experiment since it would address concerns in 2. If blood flow and ghrelin delivery are linked, one would expect that Ghsr1KD neurons would not exhibit increased migratory speed when associated with slow or fast blood flow vessels. 

In Figure 3, we showed that ghrelin transcytosis occurs preferentially in high-flow vessels, suggesting a role for ghrelin in mediating the effects of blood flow on neuronal migration. However, whether this dependence is solely attributable to ghrelin signaling remains unclear. 

To address this, we tested whether Ghsr1a-KD modifies the impact of reduced blood  flow on neuronal migration by combining Ghsr1a-KD with bilateral common carotid artery stenosis (BCAS), a chronic cerebral hypoperfusion model (Figure S9A). We found that BCAS decreased the percentage of Ghsr1a-KD new neurons reaching the OB, similar to the effect seen in control neurons (Figure S9B, see also Figure 2A–C). This suggests that blood flow influences neuronal migration even under Ghsr1a-KD conditions. 

Furthermore, we analyzed the distribution of Ghsr1a-KD neurons with respect to vessel flow characteristics. Even under Ghsr1a-KD, a higher proportion of new neurons were located in the area of endomucin-negative (high-flow) vessels compared with endomucin-positive (low-flow) vessels (Figure S9C), indicating that Ghsr1a-KD does not abolish the preferential association of migrating neurons with high flow vessels. These findings suggest that although ghrelin signaling contributes to blood flow-dependent migration, it is not the sole factor. Other blood-derived signals may also mediate this effect. We have included these new data in Figure S9 and updated the corresponding sections in the Results

Reviewer #1 (Recommendations for the authors) :

Major 

Page 6, Line 13. Please provide in the result section some explanation about how photothrombic clot is induced.  

We added the following explanation to the Results section to clarify the method used to induce photothrombotic clot formation.

“For clot formation, a restricted area of selected vessels was irradiated by a two-photon laser immediately after intravenous injection of rose bengal.” (Results, Page 7, lines 27–28)

Page 6, Line 18. The authors use the marmoset as an additional experimental model. Here, V-SVZ-derived newborn neurons migrate in other brain regions as compared to rodents. Please provide a clear rationale for moving from rodents to "common marmosets" as an experiment model. And why use marmosets only for this set of experiments? 

We clarified the rationale for using common marmosets in addition to mice as follows:

“Because blood vessel-guided neuronal migration in the adult brain is a conserved phenomenon across species (Kishimoto et al., 2011; Akter et al., 2021; Shvedov et al., 2024), we hypothesized that blood flow may also influence neuronal migration in other brain regions of primates. The neocortex, which supports higher-order brain functions and has undergone evolutionary expansion in primates, was selected as a target region. In common marmosets, but not in mice, V-SVZ-derived new neurons migrate toward the neocortex and ventral striatum (Akter et al., 2021) (Supplemental Movies S4 and S5).” (Results, Page 6, lines 19–25)

Figure 2B. The experimental setup is possibly problematic as the lentiviral tracing measurement does not take into consideration the rate of neurogenesis or newborn neuron survival. Can authors assess the rate of proliferation and survival in the VSVZ/RMS upon BCAS to decipher whether the reduced number of cells observed in the OB only results from migration changes? (comparable remark stands for Figure 5) 

To evaluate whether the reduction in the number of new neurons observed in the OB after BCAS (Figure 2B, C) is due solely to impaired migration, we assessed cell proliferation and survival in the V-SVZ and RMS. Specifically, we quantified the density of Ki67+ proliferating cells and cleaved caspase-3+ apoptotic cells in the sham and BCAS groups. BCAS significantly decreased cell proliferation and increased cell death in both the V-SVZ and RMS (Figure S4), suggesting that reduced neurogenesis and/or survival may contribute to the decreased neuronal distribution in the OB. 

Although we cannot exclude the possibility that changes in cell proliferation or survival contributed to this effect, our photothrombotic clot formation experiments are better suited to directly examine how acute reduction in blood flow affects neuronal migration. These experiments allowed us to measure the migration speed of new neurons shortly after inducing localized blood flow inhibition. We found that clot formation significantly reduced the migration speed of new neurons (Figure 2E, H), indicating that blood flow changes directly impair neuronal migration in the adult brain. 

We have included these new data in Figure S4 and updated the corresponding text in the Results, Discussion, Figure legend, and Methods as follows:

Figure 3. About ghrelin signaling. It is unclear whether its transcytosis occurs in endomucin-negative because of the high bloodstream flow. How can this be explained? What happens upon BCAS, is there still a close relation between ghrelin transcytosis, blood flow, and neuron migration? 

As correctly noted, our initial explanation and data did not provide sufficient evidence that higher blood flow delivers a larger amount of ghrelin into the brain parenchyma. We found that some vessels had particularly strong fluorescent signals in the parenchymal area adjacent to the abluminal surface of vascular endothelial cells, as visualized by CD31 immunostaining (Feng et al., 2004) (Figure 3A′, A′′). On the basis of our observation that strong fluorescent signals were detected in vascular endothelial cells of endomucin-negative (high-flow) vessels (Figure 3C, D), we hypothesized that ghrelin transcytosis may occur more frequently in high-flow vessels due to increased endocytosis at the vessel endothelium. 

To test this hypothesis, we quantified signal gradients in the extra-vessel regions by calculating fold changes in fluorescent intensity between two zones: Area I (0–10 μm from the abluminal surface of the endothelium) and Area II (10–20 μm away), as illustrated in Figure 3E. Area I corresponds to the perivascular region where new neurons are frequently found (Figure 1). We found that the proportion of vessel segments with >1.5-fold signal increase in Area I relative to Area II was significantly higher in endomucin-negative vessels than endomucin-positive ones (Figure 3F). Furthermore, vessel segments with >2-fold increases were observed exclusively in the endomucin-negative group (6.48% ± 1.18%). These results support the idea that higher blood flow increases the amount of ghrelin that reaches the luminal surface of vascular endothelial cells, thereby increasing the possibility of ghrelin transcytosis into the brain parenchyma.

We also examined whether blood flow inhibition–induced by BCAS or photothrombotic clot formation–affects the relationship between ghrelin transcytosis, blood flow, and neuronal migration. The above results suggest that blood flow reduction may decrease ghrelin transcytosis, thereby contributing to impaired neuronal migration. To further explore this, we analyzed the distribution of new neurons around high- versus low-flow vessels under BCAS conditions. In the BCAS group, we still observed a higher density of new neurons in the region of high-flow (endomucin-negative) vessels compared with in low-flow (endomucin-positive) ones (Figure S9C). This suggests that even under reduced blood flow, neuronal migration preferentially occurs near high-flow vessels. Taken together, these results suggest that ghrelin transcytosis, blood flow and neuronal migration are connected, and that this relationship persists under conditions of blood flow reduction.

Figure 4. Is ghrelin controlling both individual Dcx+ neuron migration as well as chain migration (cells moving more together)? This should be assessed and clarified. 

How is ghrelin controlling actin dynamics in newborn migrating neurons? Since somal translocation speed and somal stride length are both modulated by ghrelin, this factor may also control MT remodeling, could that be checked? 

We have revised the manuscript to better explain the role of ghrelin in both modes of neuronal migration–chain and individual. Initially, we demonstrated that ghrelin enhances the migration of new neurons in V-SVZ culture (Figure 4A, B), where these neurons migrate outward as chains, indicating that ghrelin facilitates chain migration. In subsequent in vitro experiments (Figure 4C–M), we showed that ghrelin also enhances the migration of individual neurons. To examine this in vivo, we injected Ghsr1a-KD and control lentiviruses into two different anatomical regions: the V-SVZ, where chain migration originates, and the OB core, where new neurons migrate individually. These experiments enabled us to assess the role of ghrelin signaling in each mode of migration independently. We found that ghrelin enhanced both chain migration in the RMS and individual migration in the OB. These results indicate that ghrelin signaling facilitates both forms of neuronal migration. We added the following text in the Results section:

“To assess the direct effect of ghrelin on neuronal migration, we applied recombinant ghrelin to V-SVZ cultures, in which new neurons emerge and migrate as chains (Figure 4A). Ghrelin significantly increased the migration distance of these neurons (Figure 4B), indicating enhanced chain migration. We then used super-resolution time-lapse imaging to examine individually migrating neurons with or without knockdown (KD) of growth hormone secretagogue receptor 1a (GHSR1a), a ghrelin receptor expressed in V-SVZ-derived new neurons (Li et al., 2014) (Figure 4C). Ghrelin enhanced the migration speed of control cells (lacZ-KD) cells, indicating that it also facilitates individual migration (Figure 4D).” (Results, Page 9, lines 5–12)

“Of the total labeled Dcx+ cells, the percentage of Dcx+ cells reaching the GL was significantly lower in the Ghsr1a-KD group than in the control group (Figure 5B, C), suggesting that ghrelin enhances individual radial migration of new neurons in the OB.” (Results, Page 10, lines 5–8) “These data indicate that ghrelin signaling facilitates both individual migration in the OB and chain migration in the RMS.” (Results, Page 10, lines 17–18)

We also added discussion on how ghrelin may regulate cytoskeletal dynamics in migrating neurons. Ghrelin signaling has been reported to control actin cytoskeletal remodeling in astrocytoma cells (Dixit et al., 2006), which led us to investigate similar effects in migrating neurons. Rac, a member of the Rho GTPase family, was shown to mediate this actin remodeling in astrocytoma migration, suggesting it may also be involved in ghrelin-induced actin cup formation in new neurons. Furthermore, because somal translocation depends not only on actin but also on microtubule dynamics (Kaneko et al., 2017), it is possible that ghrelin influences both systems. Supporting this idea, ghrelin signaling was shown to modulate microtubule behavior via SFK-dependent phosphorylation of α-tubulin (Slomiany and Slomiany, 2017). These findings suggest that ghrelin may enhance somal translocation through coordinated regulation of both the actin and microtubule systems. We added following text in the Results and Discussion sections:

“Ghrelin signaling has been reported to regulate actin cytoskeletal dynamics in astrocytoma cells (Dixit et al., 2006), which led us to examine whether a similar mechanism operates in migrating neurons.”(Results, Page 9, lines 23–25)

“Further studies are needed to elucidate how ghrelin promotes actin cup formation in migrating neurons. Given that Rac, a Rho family GTPase, mediates actin remodeling downstream of ghrelin in astrocytoma cells (Dixit et al., 2006), it is possible that Rac may also be involved in ghrelininduced cytoskeletal regulation in new neurons.” (Discussion, Page 13, lines 28–31)

“In addition to actin remodeling, ghrelin may regulate microtubule dynamics. Ghrelin signaling was shown to modulate microtubules via SFK-dependent phosphorylation of α-tubulin (Slomiany and Slomiany, 2017), raising the possibility that ghrelin promotes somal translocation of new neurons through coordinated regulation of both actin and microtubule networks (Kaneko et al., 2017).” (Discussion, Page 13, line 31–Page 14, line 2)

It would also be informative to provide immunolabeling of Ghsr1 in the V-SVZ / RMS/ OB to have a clear picture of the expression pattern of this receptor. Newborn neurons migrate along blood vessels, which are surrounded by astrocytes that have also been reported to express Ghsr1, thus could newborn neuron migration change may also arise from activation of Ghsr1 in their surrounding astrocytes? 

A previous study reported that GHSR1a is expressed in DCX+ new neurons in the RMS and OB, and in V-SVZ neural progenitor cells (Li et al., 2014). To visualize the spatial expression pattern of Ghsr1a, we performed RNAscope in situ hybridization because specific anti-GHSR1a antibodies suitable for immunohistochemistry were not available. Consistent with the previous report, we detected Ghsr1a mRNA in DCX+ new neurons in the VSVZ, RMS, and OB (Figure S5A), indicating that new neurons directly receive ghrelin signaling. 

Moreover, our KD experiments demonstrated that ghrelin enhanced the migration of new neurons in a cell-autonomous manner via GHSR1a (Figure 4, 5). Nevertheless, a recent study (Stark et al., 2024) showed that GHSR1a was expressed in various cell types, including glutamatergic and GABAergic neurons, suggesting that ghrelin may also exert non-cellautonomous effects on neuronal migration. Given the presence of diverse cell types, including neurons, microglia, pericytes, and astrocytes, along the migratory route, it remains possible that GHSR1a activation in these neighboring cells contributes to the overall regulation of neuronal migration. 

Figure 5. About the in vivo knockdown of Ghsr1a. The results section (page 9, line 3) mentioned that mice were either injected with one or the other construct but Figure 5 shows coincidence of GFP and dsRed positive cells. Were control and Ghsr1a shRNAs injected together into the same mouse? Could you quantify the number of cells in green (control), red (Ghsr1a KD), and yellow (both)? Won't they mostly be yellow? Have you tried injecting control and Ghsr1a separately? If yes, do you get the same result? Such analysis would be important to separate cell autonomous from noncell autonomous effects. 

To minimize variability in injection conditions, we initially coinjected control and Ghsr1a-KD lentiviruses into the same mice and analyzed their migration using a paired design. As the reviewer correctly noted, some cells were coinfected and expressed both EmGFP and DsRed (18.7% ± 2.86% of EmGFP+ cells and 10.8% ± 0.533% of DsRed+ cells). To ensure that this overlap did not affect our analysis, we excluded EmGFP+/DsRed+ double-positive cells and focused solely on EmGFP+/DsRed− (control) and EmGFP−/DsRed+ (Ghsr1a-KD) single-positive cells. 

We agree with the reviewer that coinjection could lead to reciprocal interactions between control and Ghsr1a-KD cells, potentially masking cell-autonomous effects. To address this, we performed an independent experiment in which control and Ghsr1a-KD lentiviruses were injected separately into different mice (Figure S7A), as suggested. Consistent with the results of the coinjection experiment, we found that the Ghsr1a-KD cells showed significantly reduced distribution in the GL compared with that in control cells (Figure S7B). Although we cannot exclude the possibility of a non-cell-autonomous effect of ghrelin, this result supports the conclusion that ghrelin signaling enhances neuronal migration in a cell-autonomous manner. 

Who is expressing Ghsr1a, newborn neurons, and or their progenitors? The production and survival of newborn V-ZVS cells should be assessed upon knockdown of the ghrelin receptor too. 

To determine whether the altered distribution of new neurons observed upon Ghsr1aKD is due to impaired migration rather than decreased cell production or survival, we examined the effects of Ghsr1a-KD on the proliferation and survival of new neurons and their progenitors, which express GHSR1a (Li et al., 2014). 

We compared the proportion of cleaved caspase-3+ cells and Ki67+ cells from the total labeled cells in the V-SVZ and RMS between the control and Ghsr1a-KD groups. There was no significant difference in the proportion of cleaved caspase-3+ cells between the groups (Control: 874 cells from 5 mice; Ghsr1a-KD: 678 cells from 7 mice), suggesting that ghrelin signaling does not affect the survival of new neurons and their progenitors. 

Similarly, the proportion of Ki67+ cells in the RMS did not differ significantly between the two groups (Figure S8), indicating that Ghsr1a-KD does not impair cell proliferation in the RMS. However, it remains technically difficult to evaluate whether Ghsr1a-KD affects proliferation in the VSVZ, because lentivirus injection into the VSVZ may interfere with GHSR1a expression not only in new neurons and neural progenitors, but also in other cell types known to express GHSR1a (Zigman et al., 2006). A previous study reported that ghrelin signaling promoted cell proliferation in the V-SVZ (Li et al., 2014), thus we cannot exclude the possibility that Ghsr1a-KD may affect V-SVZ proliferation.

To overcome this limitation, we assessed the effects of Ghsr1a-KD on neuronal migration using in vitro KD experiments (Figure 4C–J) and in vivo OB-core lentivirus injections (Figure 5A–C), both of which did not interfere with proliferation in the V-SVZ. These complementary approaches consistently demonstrated that Ghsr1a-KD reduces the migration speed of new neurons. 

“To determine whether the altered distribution of new neurons after Ghsr1a-KD is due to impaired migration rather than changes in cell production or survival, we assessed the effects of Ghsr1aKD on the proliferation and survival of new neurons and their progenitors, which express GHSR1a (Li et al., 2014). We quantified the proportion of cleaved caspase-3+ cells and Ki67+ cells from the total labeled cells in the V-SVZ and RMS in both control and Ghsr1a-KD groups. We found no significant difference in cleaved caspase-3+ cell proportions between the groups (Control: 874 cells from 5 mice; Ghsr1a-KD: 678 cells from 7 mice), suggesting that ghrelin signaling does not influence the survival of new neurons and their progenitors. Similarly, the percentage of Ki67+ cells in the RMS was similar between the two groups (Figure S8), indicating that Ghsr1a-KD does not impair cell proliferation in the RMS. However, technical limitations prevented a reliable evaluation of proliferation in the V-SVZ, as lentivirus injection into this region may interfere with GHSR1a expression in not only neural progenitors and new neurons, but also other GHSR1aexpressing cell types (Zigman et al., 2006). Although ghrelin signaling has been reported to promote cell proliferation in the V-SVZ (Li et al., 2014), our complementary in vitro KD experiments (Figure 4C–J) and in vivo OB-core lentivirus injections (Figure 5A–C), which did not affect the V-SVZ, consistently demonstrated that Ghsr1a-KD reduces neuronal migration. Taken together, our results suggest that blood-derived ghrelin enhances neuronal migration in the RMS and OB by stimulating actin cytoskeleton contraction in the cell soma, rather than by altering cell proliferation or survival.” (Results, Page 10, line 19–Page 11, line 4)

“rat anti-Ki67 (1:500, #14-5698-82, eBioscience); and rabbit anti-cleaved caspase-3 (1:200, #9661, Cell Signaling Technology)” (Methods, Page 48, lines 14–16)

How much is ghrelin/Ghsr1 signaling conserved in marmosets? 

How ghrelin signaling is conserved between mice and common marmosets is important to clarify. A previous study reported the existence of a ghrelin homolog in common marmoset, which shares high sequence similarity with that in mice (Takemi et al., 2016). Moreover, the GHSR1a homolog in the common marmoset (https://www.ncbi.nlm.nih.gov/protein/380748978) shares 95.36% amino acid identity with its mouse counterpart. These findings suggest that blood-derived ghrelin may similarly promote neuronal migration in the marmoset brain, as observed in mice. 

We have added the following text in the Discussion section:

“Our data showed that new neurons preferentially migrate along arteriole-side vessels rather than venule-side vessels in both mouse and common marmoset brains, suggesting that the mechanism of blood flow-dependent neuronal migration is conserved across rodent and primate species, as well as across brain regions. A previous study identified a ghrelin homolog in the common marmoset with high sequence similarity to the murine version (Takemi et al., 2016). In addition, the marmoset GHSR1a homolog shares 95.36% amino acid identity with that of the mouse (https://www.ncbi.nlm.nih.gov/protein/380748978). These findings suggest that bloodderived ghrelin promotes neuronal migration in the common marmoset brain in a manner similar to that in mice.” (Discussion, Page 15, lines 8–16)

Page 9. Starvation has been shown to boost ghrelin blood levels. What is the exact protocol used in this experiment and is this indeed increasing Ghrelin release from blood vessels in the V-SVZ? What about Ghsr1 expression level in newborn neurons? 

We have clarified the calorie restriction (CR) protocol used in our experiments. We adopted a 70% CR protocol, which was previously shown to enhance hippocampal neurogenesis when administered for 14 days (Hornsby et al., 2016). In our study, the daily food intake under ad libitum (AL) conditions was first measured, and CR mice were then fed 70% of that amount for 5 consecutive days (see Figure 5I and Figure S10A). 

To assess whether CR enhances ghrelin transcytosis into the brain parenchyma, we performed ELISA to quantify ghrelin levels in the OB and RMS. However, ghrelin concentrations were below the detection limit in both groups, precluding a direct comparison.

We also considered whether CR modulates the expression level of the ghrelin receptor GHSR1a. A recent study reported that fasting increased GHSR1a expression in the OB (Stark et al., 2024), raising the possibility that CR may exert a similar effect. To test this, we performed in situ hybridization and quantified Ghsr1a mRNA puncta in Dcx+ cells in the OB. No significant difference was found between the AL and CR groups (Figure S5B), suggesting that CR does not alter GHSR1a expression levels in new neurons. 

Although we cannot exclude the possibility that CR increases GHSR1a expression in other OB cell types, our combined CR and Ghsr1a-KD experiments strongly support a cellautonomous contribution of ghrelin signaling to the enhanced neuronal migration observed under CR conditions. Corresponding data and text have been added to Figure S5 and the Results, Discussion, and the Figure legend sections as follows:

Minor 

Page 4 

Line 19 In Supplemental movies 1 and 2, it is unclear where to see the GFP+ new neurons interact with BV. Can you add arrows as an indication for the readers? It will be better to add the anatomy term for orientation, caudal, or rostral in the video. (The same for Supplemental movies 3, 4, and 5).  

To clarify the regions of interest in Supplemental Movies 1 and 2, where neuron–vessel interactions in the RMS are highlighted, we added dotted lines indicating the RMS boundaries. In addition, we created a new movie (Supplemental Movie S1′) showing a high-magnification view of Supplemental Movie S1, in which arrows mark EGFP+ new neurons interacting with blood vessels. We also added orientation indicators (e.g., caudal and rostral) and arrows to highlight new neuron–vessel interactions in Supplemental Movies S1–S5. 

The following descriptions have been added to the Figure legends:

“Supplemental Movie S1′ 

High-magnification view extracted from Supplemental Movie S1. Arrows indicate EGFP+ cells interacting with blood vessels.” (Figure legend, Page 46, lines 6–8)

“Arrows indicate EGFP+ cells interacting with blood vessels.” (Figure legend, Supplemental Movie S3, Page 46, lines 16–17)

“Arrows indicate Dcx+ cells interacting with blood vessels.” (Figure legend, Supplemental Movies S4 and S5, Page 46, lines 21–22, 26–27)

Blood vessels are labeled in the Supplemental movies 2 and 3 by employing Flt1DsRed transgenic mice instead of RITC-Dex-GMA. However, Flt1-DsRed transgenic mice are not mentioned in the results section. 

We have now included an explanation regarding the use of Flt1-DsRed mice, in which vascular endothelial cells were labeled with DsRed.

“To visualize blood vessels, we also used Flt1-DsRed transgenic mice, in which vascular endothelial cells were specifically labeled with DsRed (Matsumoto et al., 2012). Using DcxEGFP/Flt1-DsRed double transgenic mice, we observed close spatial relationships between new neurons and blood vessels (Supplemental Movies S2 and S3).” (Results, Page 4, lines 22– 26)

Figure 5. Can you indicate (in the figure legend and the result section) the stage of the adult brain used for this experiment? 

We used 6- to 12-week-old adult male mice in all experiments in this study. To specify this, we have added the age of animals to both the Results and the relevant Figure legends as follows:

“Therefore, we first studied blood vessel-guided neuronal migration in the RMS and OB using three-dimensional imaging in 6- to 12-week-old adult mice, which enabled analysis of the in vivo spatial relationship between new neurons and blood vessels.” (Results, Page 4, lines 14–16)

“Figure 1 New neurons migrate along blood vessels with abundant flow in the adult brain.” (Figure legend, Page 25, line 4)

“(B, C) Three-dimensional reconstructed images of a new neuron (green) and blood vessels (red) in the rostral migratory stream (RMS) (B) and glomerular layer (GL) (C) of 6- to 12-weekold adult mice.” (Figure legend, Page 25, lines 6–8)

“(E) Transmission electron microscopy image of a new neuron (green) in close contact with a blood vessel (red) in the GL of a 6- to 12-week-old adult mouse.” (Figure legend, Page 26, lines 4–5)

“(F) Time-lapse images of a migrating neuron (indicated by asterisks) in the GL of a 6- to 12week-old Dcx-EGFP mouse.” (Figure legend, Page 26, lines 6–7)

“Figure 3 Ghrelin is delivered from the bloodstream to the RMS and OB in the adult brain (A) Representative images of the OB and cortex of a fluorescent ghrelin-infused mouse (6 to 12 weeks old).” (Figure legend, Page 30, lines 1–3)

“Lentivirus injection into the OB core (A) and the VSVZ (D) was performed in 6- to 12-week-old adult mice.” (Figure legend, Page 33, lines 3–4)

Reviewer #2 (Recommendations for author):

Major:

Ghsr1KD and blood flow 2-photon experiments to directly measure migratory speed. Could also do the same with fasting with or without Ghsr1KD.  

We thank the reviewer for the valuable suggestion to strengthen our study. As pointed out in the Public Review, we agree that direct in vivo measurement of neuronal migration speed under Ghsr1a-KD conditions is important to clarify the link between ghrelin signaling and blood flow. 

Two-photon imaging is the most suitable method for this purpose. Although we attempted two-photon imaging of Ghsr1a-KD new neurons, the number of virus-infected cells observed in vivo was too low to yield reliable data. Therefore, we chose an alternative strategy, combining Ghsr1a-KD with blood flow reduction using the BCAS model (Figure S9A), in which migration speed can be quantified based on the percentage of labeled cells reaching the OB. As stated in the Public Review response, BCAS significantly decreased the migration speed of Ghsr1a-KD new neurons (Figure S9B), indicating that Ghsr1a-KD does not abolish the influence of blood flow reduction. These findings suggest that ghrelin signaling is involved, but is not essential, for blood flow-dependent neuronal migration. 

As suggested by the reviewer, direct observation of migration dynamics (e.g., somal translocation, leading process extension, stationary and migratory phases) is needed, especially in calorie restriction experiments. Although our data indicate that ghrelin signaling is required for fasting-induced increases in migration speed of new neurons, calorie restriction could also change concentrations of other factors in blood (Bonnet et al., 2020; Wu et al., 2024; Alogaiel et al., 2025), which may independently affect behavior of migrating neurons. Given that ghrelin is not the sole factor contributing to blood flow-dependent neuronal migration, other circulating factors could affect behavior of migrating neurons in a different manner during fasting. In vivo twophoton imaging would be a powerful approach to determine whether fasting-induced neuronal migration is caused by upregulated somal translocation speed, which would further support a role for ghrelin in this process.

We have added the following text in the Discussion:

“Although our data indicate that ghrelin signaling is essential for fasting-induced acceleration of neuronal migration, calorie restriction may also alter the concentrations of other circulating factors (Bonnet et al., 2020; Wu et al., 2024; Alogaiel et al., 2025), which could independently influence the behavior of migrating neurons.” (Discussion, Page 14, lines 25–29)

Minor: 

(1) Show fluorescent Ghreliin in Figure 3 for all brain areas measured in Figure 1 (GL, EPL, GCL, and RMS) for direct comparison.  

To allow for direct comparison across brain regions, we added a new Supplemental figure showing the distribution of fluorescently labeled ghrelin in the OB, including the GL, EPL, GCL and RMS. This comprehensive view highlights ghrelin localization relative to vasculature and migrating neurons in the regions analyzed in Figure 1.

(1) Figure 1, panel I is presented in a confusing manner. High blood flow points to 0 degrees, low blood flow to 180 degrees. It implies (unintentionally, I am sure) that low blood flow results in migration away from OB. Maybe plot separately?

We agree that the original presentation of Figure 1I could be misinterpreted as referring to anatomical orientation (i.e., toward or away from the OB). To avoid confusion, we revised the figure to categorize new neuron–vessel interactions into four groups according to (1) the angle between the migration direction and vessel axis (small or large), and (2) whether the new neuron is migrating toward or away from the direction of higher blood flow. This new presentation avoids implying a fixed anatomical direction and better reflects the relationship between local blood flow and neuronal migration behavior. The revised figure is presented as Supplemental Figure S1.

Reviewer #1 (Public review):

The manuscript by Zhang et al describes the use of a protein language model (pLM) to analyse disordered regions in proteins, with a focus on those that may be important in biological phase separation. While the paper is relatively easy to read overall, my main comment is that the authors could perhaps make it clearer which observations are new, and which support previous work using related approaches. Further, while the link to phase separation is interesting, it is not completely clear which data supports the statements made, and this could also be made clearer.

Major comments:

(1) With respect to putting the work in a better context of what has previously been done before, this is not to say that there is not new information in it, but what the authors do is somewhat closely related to work by others. I think it would be useful to make those links more directly. Some examples:

(1a) Alderson et al (reference 71) analysed in detail the conservation of IDRs (via pLDDT, which is itself related to conservation) to show, for example, that conserved residues fold upon binding. This analysis is very similar to the analysis used in the current study (using ESM2 as a different measure of conservation). Thus, the approach (pages 7-8) described as "This distinction allows us to classify disordered regions into two types: "flexible disordered" regions, which show high ESM2 scores and greater mutational tolerance, and "conserved disordered" regions, which display low ESM2 scores, indicating varying levels of mutational constraint despite a lack of stable folding." is fundamentally very similar to that used by Alderson et al. Thus, the result that "Given that low ESM2 scores generally reflect mutational constraint in folded proteins, the presence of region a among disordered residues suggests that certain disordered amino acids are evolutionarily conserved and likely functionally significant" is in some ways very similar to the results of that paper.

(1b) Dasmeh et al (https://doi.org/10.1093/genetics/iyab184), Lu et al (https://doi.org/10.1371/journal.pcbi.1010238) and Ho & Huang (https://doi.org/10.1002/pro.4317) analysed conservation in IDRs, including aromatic residues and their role in phase separation

(1c) A number of groups have performed proteomewide saturation scans using pLMs, including variants of the ESM family, including Meier (reference 89, but cited about something else) and Cagiada et al (https://doi.org/10.1101/2024.05.21.595203) that analysed variant effects in IDRs using a pLM. Thus, I think statements such as "their applicability to studying the fitness and evolutionary pressures on IDRs has yet to be established" should possibly be qualified.

(2) On page 4, the authors write, "The conserved residues are primarily located in regions associated with phase separation." These results are presented as a central part of the work, but it is not completely clear what the evidence is.

(3) It would be useful with an assessment of what controls the authors used to assess whether there are folded domains within their set of IDRs.

Reviewer #1 (Public review):

This study presents an exploration of PPGL tumour bulk transcriptomics and identifies three clusters of samples (labeled as subtypes C1-C3). Each subtype is then investigated for the presence of somatic mutations, metabolism-associated pathways and inflammation correlates, and disease progression.

The proposed subtype descriptions are presented as an exploratory study. The proposed potential biomarkers from this subtype are suitably caveated, and will require further validation in PPGL cohorts together with a mechanistic study.

The first section uses WGCNA (a method to identify clusters of samples based on gene expression correlations) to discover three transcriptome-based clusters of PPGL tumours.

The second section inspects a previously published snRNAseq dataset, and labels some of the published cells as subtypes C1, C2, C3 (Methods could be clarified here), among other cells labelled as immune cell types. Further details about how the previously reported single-nuclei were assigned to the newly described subtypes C1-C3 require clarification.

The tumour samples are obtained from multiple locations in the body (Figure 1A). It will be important to see further investigation of how the sample origin is distributed among the C1-C3 clusters, and whether there is a sample-origin association with mutational drivers and disease progression.

Author response:

Reviewer #1 (Public Review):

This study presents an exploration of PPGL tumour bulk transcriptomics and identifies three clusters of samples (labeled as subtypes C1-C3). Each subtype is then investigated for the presence of somatic mutations, metabolism-associated pathways and inflammation correlates, and disease progression. The proposed subtype descriptions are presented as an exploratory study. The proposed potential biomarkers from this subtype are suitably caveated and will require further validation in PPGL cohorts together with a mechanistic study.

The first section uses WGCNA (a method to identify clusters of samples based on gene expression correlations) to discover three transcriptome-based clusters of PPGL tumours. The second section inspects a previously published snRNAseq dataset, and labels some of the published cells as subtypes C1, C2, C3 (Methods could be clarified here), among other cells labelled as immune cell types. Further details about how the previously reported single-nuclei were assigned to the newly described subtypes C1-C3 require clarification.

Thank you for your valuable suggestion. In response to the reviewer’s request for further clarification on “how previously published single-nuclei data were assigned to the newly defined C1-C3 subtypes,” we have provided additional methodological details in the revised manuscript (lines 103-109). Specifically, we aggregated the single-nucleus RNA-seq data to the sample level by summing gene counts across nuclei to generate pseudo-bulk expression profiles. These profiles were then normalized for library size, log-transformed (log1p), and z-scaled across samples. Using genesets scores derived from our earlier WGCNA analysis of PPGLs, we defined transcriptional subtypes within the Magnus cohort (Supplementary Figure. 1C). We further analyzed the single-nucleus data by classifying malignant (chromaffin) nuclei as C1, C2, or C3 based on their subtype scores, while non-malignant nuclei (including immune, stromal, endothelial, and others) were annotated using canonical cell-type markers (Figure. 4A).

The tumour samples are obtained from multiple locations in the body (Figure 1A). It will be important to see further investigation of how the sample origin is distributed among the C1-C3 clusters, and whether there is a sample-origin association with mutational drivers and disease progression.

Thank you for your valuable suggestion. In the revised manuscript (lines 74-79), Figure. 1A, Table S1 and Supplementary Figure. 1A, we harmonized anatomic site annotations from our PPGL cohort and the TCGA cohort and analyzed the distribution of tumor origin (adrenal vs extra-adrenal) across subtypes. The site composition is essentially uniform across C1-C3—approximately 75% pheochromocytoma (PC) and 25% paraganglioma (PG)—with only minimal variation. Notably, the proportion of extra-adrenal origin (paraganglioma origin) is slightly higher in the C1 subtype (see Supplementary Figure 1A), which aligns with the biological characteristics of tumors from this anatomical site, which typically exhibit more aggressive behavior.

Reviewer #2 (Public Review):

A study that furthers the molecular definition of PPGL (where prognosis is variable) and provides a wide range of sub-experiments to back up the findings. One of the key premises of the study is that identification of driver mutations in PPGL is incomplete and that compromises characterisation for prognostic purposes. This is a reasonable starting point on which to base some characterisation based on different methods. The cohort is a reasonable size, and a useful validation cohort in the form of TCGA is used. Whilst it would be resource-intensive (though plausible given the rarity of the tumour type) to perform RNA-seq on all PPGL samples in clinical practice, some potential proxies are proposed.

We sincerely thank the reviewer for their positive assessment of our study’s rationale. We fully agree that RNA sequencing for all PPGL samples remains resource-intensive in current clinical practice, and its widespread application still faces feasibility challenges. It is precisely for this reason that, after defining transcriptional subtypes, we further focused on identifying and validating practical molecular markers and exploring their detectability at the protein level.

In this study, we validated key markers such as ANGPT2, PCSK1N, and GPX3 using immunohistochemistry (IHC), demonstrating their ability to effectively distinguish among molecular subtypes (see Figure. 5). This provides a potential tool for the clinical translation of transcriptional subtyping, similar to the transcription factor-based subtyping in small cell lung cancer where IHC enables low-cost and rapid molecular classification.

It should be noted that the subtyping performance of these markers has so far been preliminarily validated only in our internal cohort of 87 PPGL samples. We agree with the reviewer that larger-scale, multi-center prospective studies are needed in the future to further establish the reliability and prognostic value of these markers in clinical practice.

The performance of some of the proxy markers for transcriptional subtype is not presented.

We agree with your comment regarding the need to further evaluate the performance of proxy markers for transcriptional subtyping. In our study, we have in fact taken this point into full consideration. To translate the transcriptional subtypes into a clinically applicable classification tool, we employed a linear regression model to compare the effect values (β values) of candidate marker genes across subtypes (Supplementary Figure. 1D-F). Genes with the most significant β values and statistical differences were selected as representative markers for each subtype.

Ultimately, we identified ANGPT2, PCSK1N, and GPX3—each significantly overexpressed in subtypes C1, C2, and C3, respectively, and exhibiting the most pronounced β values—as robust marker genes for these subtypes (Figure. 5A and Supplementary Figure. 1D-F). These results support the utility of these markers in subtype classification and have been thoroughly validated in our analysis. 

There is limited prognostic information available.

Thank you for your valuable suggestion. In this exploratory revision, we present the available prognostic signal in Figure. 5C. Given the current event numbers and follow-up time, we intentionally limited inference. We are continuing longitudinal follow-up of the PPGL cohort and will periodically update and report mature time-to-event analyses in subsequent work.

DOI: 10.1161/JAHA.124.039220

Resource: RRID:Addgene_49437

Curator: @olekpark

SciCrunch record: RRID:Addgene_49437

What is this?

jhfhjghj

I didn't find that Arnold's friend was even older than him.

A nivel general, considero que es una propuesta interesante, pero como bien señala la autora, quizá no sea para todos, no obstante no está demás intentarla para empezar a ser más conscientes de cómo empleamos nuestro tiempo, de como fluctúan nuestros pensamiento y emociones, e ir finalmente encontrando estrategias que nos ayuden a enfocarnos en este mundo en el que todo el tiempo nos vemos tentados a desviarnos por caminos que nos roban nuestro tiempo y atención.

Esto también me resulta muy interesante ya que por lo general no ponemos atención a nuestro estado de ánimo al momento de desarrollar actividades o en el día a día en general, y esto impacta directamente nuestro desempeño y capacidad. Muchas veces solo actuamos en automático y esperamos dar resultados similares día a día sin considerar cómo nos estamos sintiendo.

De nuevo, creo que esta puede ser una de las partes más útiles de la herramienta, pero la veo más como algo que puedo aplicar a mi vida personal como estratégica de autoconocimiento o reconocimiento de mis fortalezas, debilidades, intereses, hábitos, etc., en vez de algo que realice en medio de una tarea que he de desarrollar durante un periodo de tiempo específico.

Creo que con las pausas de procrastinación podría ocurrir lo mismo que con las pausas proactivas. Si nos damos cuenta de los desvíos de atención que nos hemos permitido, con base en mi experiencia, nos puede costar más retomar la tarea inicial al desanimarnos y sentir culpa de nuestra falta de atención.

Veo que las pausas proactivas son una herramienta que invita a la reflexión, a mirar el avance que se ha logrado y prepararse para continuar planeando anticipadamente. También veo que su fin es liberar la mente de aquellas distracciones que van surgiendo con el fin de enfocarnos en el desarrollo de la tarea en que nos hemos embarcado. Personalmente lo encuentro útil y lógico pero un poco complicado por la tendencia que tengo a sobre pensar y a abrumarme fácilmente. Planear nos supone la necesidad de cumplir el paso a paso que hemos diseñado y puede ser fácil desanimarse y bloquearse mentalmente cuando nos damos cuenta de que no hemos podido seguir lo que nos planteamos.

A pesar de las ventajas que podría tener el interstitial journaling para la productividad y el aprendizaje autónomo, a nivel personal me parece necesario reconocer que esta estrategia quizá no se ajusta a las necesidades y a las formas de todas y todos, y que en algunos casos podría resultar siendo un obstáculo o una carga adicional para llevar a cabo las tareas que se espera desarrollar.

Creo que este es un buen punto de partida para reflexionar sobre el interstitial journaling, porque más que su utilidad en relación con la productividad a la que tanto estamos llamados hoy en día, me interesa la idea de poder hacernos más consciente del tiempo y cómo hacemos uso de el, sobretodo cuando estamos buscando desarrollar una tarea en específico, pero también como herramienta para entendernos y conocernos mejor cuando tenemos dificultades para manejar nuestro tiempo y para enfocarnos en desarrollar tareas que nos proponemos o que debemos realizar.

* what a foolish reason se traduce "que motivo mas estúpido". La a que se traduciría como un se omite pero en el ingles no por que expresa sustantivo singular contable.