Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review):

Strengths:

The genetic approaches here for visualizing the recombination status of an endogenous allele are very clever, and by comparing the turnover of wildtype and mutant cells in the same animal the authors can make very convincing arguments about the effect of chronic loss of pu.1. Likely this phenotype would be either very subtle or nonexistent without the point of comparison and competition with the wildtype cells.

Using multiple species allows for more generalizable results, and shows conservation of the phenomena at play.

The demonstration of changes to proliferation and cell death in concert with higher expression of tp53 is compelling evidence for the authors' argument.

Weaknesses:

This paper is very strong. It would benefit from further investigating the specific relationship between pu.1 and tp53 specifically. Does pu.1 interact with the tp53 locus? Specific molecular analysis of this interaction would strengthen the mechanistic findings.

We agree with the reviewer’s assessment regarding the significance of the relationship between PU.1 and TP53. To investigate the potential interaction between Pu.1 and Tp53 in zebrafish, we analyzed the promoter region of zebrafish tp53. Indeed, we found three PU.1 binding sites (GAGGAA) on tp53 promoter, which locate on the antisense strand from position -1047 to -1042, -1098 to -1093 and -1423 to -1418 relative to the transcriptional start site (Fig. S10). These potential Pu.1 binding sites indicate a direct interaction between Pu.1 and tp53 locus. Furthermore, a previous study by Tschan et al. (2008) elucidated the mechanism by which PU.1 attenuates the transcriptional activity of the P53 tumor suppressor family through direct binding to the DNA-binding and/or oligomerization domains of p53/p73 proteins. We have also cited this study (Line 399-401) and included all above information in the discussion of the revised manuscript (Line 399-405).

Reviewer #2 (Public review):

Strengths:

Generation of an elegantly designed conditional pu.1 allele in zebrafish that allows for the visual detection of expression of the knockout allele.

The combination of analysis of pu.1 function in two model systems, zebrafish and mouse, strengthens the conclusions of the paper.

Confirmation of the functional significance of the observed upregulation of tp53 in mutant microglia through double mutant analysis provides some mechanistic insight.

Weaknesses:

(1) The presented RNA-Seq analysis of mutant microglia is underpowered and details on how the data was analyzed are missing. Only 9-15 cells were analyzed in total (3 pools of 3-5 cells each). Further, the variability in relative gene expression of ccl35b.1, which was used as a quality control and inclusion criterion to define pools consisting of microglia, is extremely high (between ~4 and ~1600, Figure S7A).

We feel sorry for the unclearness of RNAseq procedures and have accordingly added the details about RNA-seq data analysis in the “Material and methods” section (Line 491-501). Briefly, reads were aligned to the zebrafish genome using the STAR package. Original counts were calculated with featureCounts package. Differential expression genes (DEGs) were identified with the DESeq2 package. Owing to the technical challenge of unambiguously distinguishing microglia from dendritic cells (DCs) in brain cell suspensions, we employed a strategy of isolating 3-5 cells per pool and quantifying the relative expression of the microglia-specific marker ccl34b.1 normalized to the DC-specific marker ccl19a.1. This approach aimed to reduce DC contamination in downstream analyses. Across all experimental groups subjected to RNA-seq analysis, the ccl34b.1/ccl19a.1 expression ratios exceeded 5, confirming microglia as the dominant cell population. Nonetheless, residual DC contamination in the RNA-seq data cannot be entirely ruled out. We have discussed this technical constraint in the revised manuscript to ensure methodological transparency (Line 498-501).

(2) The authors conclude that the reduction of microglia observed in the adult brain after cKO of pu.1 in the spi-b mutant background is due to apoptosis (Lines 213-215). However, they only provide evidence of apoptosis in 3-5 dpf embryos, a stage at which loss of pu.1 alone does lead to a complete loss of microglia (Figure 2E). A control of pu.1 KI/d839 mutants treated with 4-OHT should be added to show that this effect is indeed dependent on the loss of spi-b. In addition, experiments should be performed to show apoptosis in the adult brain after cKO of pu.1 in spi-b mutants as there seems to be a difference in the requirement of pu.1 in embryonic and adult stages.

We apologize for the omission of data regarding conditional pu.1 knockout alone in the embryos in our manuscript, which may have led to ambiguity. We would like to clarify that conditional pu.1 knockout alone at the embryonic stage does not induce microglial death (Fig S2). Microglial death occurs only in both embryonic and adult brains when Pu.1 is disrupted in the spi-b mutant background. The blebbing morphology of some microglia after pu.1 conditional knockout in adult spi-b mutant indicated microglia undergo apoptosis at both embryonic and adult stages (Figure S4 and Fig. S5). The reviewer’s concern likely arises from the distinct outcomes of global pu.1 knockout (Fig. 2) versus conditional pu.1 ablation (Fig. S2). Global knockout eliminates microglia during early development due to Pu.1’s essential role in myeloid lineage specification. We have included this clarification in the revised manuscript (Line 208-211).

(3) The number of microglia after pu.1 knockout in zebrafish did only show a significant decrease 3 months after 4-OHT injection, whereas microglia were almost completely depleted already 7 days after injection in mice. This major difference is not discussed in the paper.

We propose that zebrafish Pu.1 and Spi-b function cooperatively to regulate microglial maintenance, analogous to the role of PU.1 alone in mice. This cooperative mechanism likely explains the observed difference in microglial depletion kinetics between zebrafish and mice following pu.1 conditional knockout. Specifically, the compensatory activity of Spi-b in zebrafish may buffer the immediate loss of Pu.1, whereas in mice, the absence of Spi-b expression in microglia eliminates this redundancy, resulting in rapid microglial depletion. Furthermore, during evolution, SPI-B appears to have acquired lineage-specific roles, becoming absent in microglia. We have included the clarification in the revised manuscript (Line 302-305).

(4) Data is represented as mean +/-.SEM. Instead of SEM, standard deviation should be shown in all graphs to show the variability of the data. This is especially important for all graphs where individual data points are not shown. It should also be stated in the figure legend if SEM or SD is shown

We have represented our data as mean ± SD in the revised manuscript.

Recommendations for the authors:

Reviewing Editor:

To further strengthen the manuscript, we ask the authors to address the reviewers' comments through additional experiments where necessary. In cases where certain experiments may be challenging, we encourage the authors to address these concerns within the text, such as by referencing any prior evidence of pu.1 and tp53 interactions or incorporating in silico analyses that support such interaction.

As suggested, we have performed in-silico analysis of Pu.1 binding sites in zebrafish tp53 promoter and also cited previous paper showing how PU.1 attenuates the transcriptional activity of the P53 tumor suppressor family (Line 399-405).

Reviewer #1 (Recommendations for the authors):

It would be useful to investigate the relationship between pu.1 and tp53. The data presented here show that pu.1 deficient cells have higher expression of tp53, but this could be an indirect effect. However, since pu.1 has known DNA binding motifs, it would be worthwhile to investigate if there are any direct interactions between pu.1 and the tp53 locus -- does pu.1 directly bind and repress tp53 expression? This could be directly investigated with Cut & Run or an EMSA.

The interaction between Pu.1 and Tp53 has been discussed in the public review section.

The paper would likely also benefit from a more in-depth discussion of the relationship of the zebrafish alleles and their relationship to mammalian Pu.1 -- as presented here, the authors are implicitly arguing that zebrafish pu.1 and spi-b are both more closely related to mammalian Pu.1 than to mammalian Spi-b. A clear argument, perhaps backed up by sequence alignment and homology matching, would help readers, especially those less familiar with zebrafish genome duplications.

We have conducted detailed sequence alignment in our previous work (Yu et al., 2017, Blood) and found zebrafish Spi-b shares the highest similarity with the mammalian SPI-B among Ets family transcription factors in zebrafish. A unique P/S/T-rich region known to be essential for mammalian SPI-B transactivation activity is present in zebrafish Spi-b. Our data do not support the interpretation that Spi-b is more closely related to mammalian Pu.1 than to Spi-b. Instead, functional compensation between pu.1 and spi-b in microglia maintenance likely reflects their shared role as Ets-family transcriptional regulators, rather than ortholog-driven redundancy.

Reviewer #2 (Recommendations for the authors):

(1) The nomenclature of the genes in the SPI family in zebrafish is somewhat confusing as genes were renamed several times. It would make it easier for the reader to understand if in the abstract and the main text, spi-b would be referred to as the zebrafish orthologue of mouse SPI-B (as determined by the authors in previous work) rather than the paralogue of zebrafish pu.1. To clarify which genes were analyzed in both zebrafish and mouse, Gene accession numbers should be added.

Thanks for the recommendations. We have changed “the paralogue of zebrafish pu.1” to “the orthologue of mouse Spi-b” in the abstract (Line 22) and added gene accession numbers for both zebrafish and mouse gene (Line 105-106 and 301-302).

(2) Methods RNA-seq: Details on how the aligned reads were analyzed to detect differentially expressed genes are missing and should be added. In addition, a table with read counts, fold changes and adjusted p values should be added.

We have added details of RNA-seq analysis in the Material and Methods part (Line 491-501). A table generated by Deseq2 has been included as a supplemental file to show read counts, fold changes and adjusted p values (Supplemental file 2).

(3) Figure 2H: It would be helpful to the reader if the KO splicing would be shown in comparison to WT splicing.

Thank you for your suggestion. We have added the sequence result between exon 3 and exon 4 of pu.1 from wildtype cDNA to show WT splicing in Figure 2H.

(4) Legend Figure 5C. Relative expression should be replaced with transcripts per million (TPM).

We have corrected it in the figure legend of Figure 5C (Line 786-787).

(5) In Figure S3. the label on the y-axis in panel B is not visible.

We apologize for the mistake during figures assembling. We have corrected it and now the y-axis is visible.

(6) In Figure S7B an explanation for the colors in the heat map is missing and should be added.

Colors represent scaled TPM values. The red color represents high expression while the blue color represents low expression. We have added the information in the figure legend.

(7) A justification for the use of male mice only should be added or additional experiments in female mice should be performed.

Female mice were excluded to avoid variability associated with estrous cycle-dependent hormonal changes, which are known to influence microglial behavior (Habib P et al., 2015). We have added a justification in the revised manuscript (Line 547-548).

(8) The manuscript would benefit from some language editing. A few examples are listed below:

a) line 97: the rostral blood (RBI) should read the rostral blood island.

b) line 373 typo: nucleus translocation should read nuclear translocation.

c) line 393 typo: pu.1-dificent should read pu.1-deficient.

We apologize for the typos or grammar mistakes in the manuscript. We have checked the manuscript thoroughly and revised those typos or grammar mistakes.

Reference:

Tschan MP, Reddy VA, Ress A, Arvidsson G, Fey MF, Torbett BE (2008) PU.1 binding to the p53 family of tumor suppressors impairs their transcriptional activity. Oncogene 27: 3489-93

Yu T, Guo W, Tian Y, Xu J, Chen J, Li L, Wen Z (2017) Distinct regulatory networks control the development of macrophages of different origins in zebrafish. Blood 129: 509-519

Habib P, Beyer C (2015) Regulation of brain microglia by female gonadal steroids. J Steroid Biochem Mol Biol 146: 3-14

Document de Synthèse : Les Représentations de la Violence

Ce document de synthèse explore les multiples facettes de la représentation de la violence, en s'appuyant principalement sur l'analyse de la violence coloniale et de la violence guerrière.

Il met en lumière la complexité des perspectives, des intentions et des mediums à travers lesquels la violence est perçue et restituée.

I. La Représentation de la Violence : Une Dualité Fondamentale

Écrire sur la violence implique deux formes de "représentation" :

• La représentation mentale (pour les philosophes et psychologues) : Il s'agit de l'image que l'esprit perçoit d'un objet ou d'un événement, influencée par l'histoire collective, l'expérience personnelle, la vision du monde et les préjugés. Un même fait peut ainsi donner lieu à une "variété de représentations mentales possibles."
• La représentation sémantique/artistique (pour les écrivains et anthropologues) : Cela correspond à ce que les créateurs et chercheurs choisissent de "donner des faits dont il est question," impliquant un double rapport au fait : réalisme et esthétique. Un acte donné peut ainsi engendrer des "récits éloignés."
• En somme, représenter la violence met en jeu "à la fois la façon dont cela représente à soi-même et dont elle la représente pour les autres."

Le texte n'est qu'une des formes de représentation, d'autres étant le cinéma, la vidéo, le chant, le son, la peinture et la sculpture, souvent "plus expressives."

Une pensée critique dans les sciences sociales s'est développée, remettant en question l'idée que la représentation serait une "simple reproduction fidèle de la réalité," rappelant que toute représentation "impliqu[e] toujours des perspectives."

II. La Violence Coloniale : Fondatrice et Multiforme

La violence coloniale est présentée comme une expérience "fondatrice en ce qu'elle institue une relation radicale à l'autre," se traduisant par des "pratiques de cruauté et de déshumanisation qui ont permis des massacres et d'expoliation, l'esclavage et l'aliénation."

Elle est considérée comme un "principe nécessaire par où commencer" pour saisir l'histoire de la violence contemporaine.

A. Deux Modes de Représentation de la Violence Coloniale : Fiction vs. Rapport

L'analyse se concentre sur deux textes majeurs concernant le Congo belge :

• "Heart of Darkness" (1899) de Joseph Conrad (Fiction) :
• Contexte : Basé sur l'expérience de Conrad au Congo, propriété personnelle du roi Léopold II, marquée par une "répression féroce des populations."
• Description de la violence : Le roman dépeint une "odyssée de plus en plus sombre, inquiétante, hallucinatoire même où la violence est omniprésente."

Il illustre trois figures de l'altérité – "les ennemis, les criminels et les travailleurs" – qui correspondent à "trois formes majeures de la violence coloniale : la guerre forcenée, le châtiment inhumain et l'exploitation implacable."

• Racisme et déshumanisation : La violence est exercée dans le cadre d'un "racisme ouvertement affirmé" par les administrateurs coloniaux. La folie meurtrière de Kurt, culminant dans sa note manuscrite "exterminer toutes ses brutes," et sa dernière vision murmurée "the horror, the horror," révèlent l'horreur des massacres et des tortures derrière le commerce lucratif.

• Intention de l'auteur : Conrad affirme être parti de son expérience, "poussée un petit peu juste un petit peu au-delà des faits réels de l'affaire dans l'intention parfaitement légitime [...] de la rendre sensible à l'esprit et au cœur des lecteurs." Il visait à donner "à ce thème sinistre une sombre résonance, une tonalité spécifique, une vibration continue." Il s'agit d'une composition visant à "affecter" le lecteur ("pathos").

• Réception et influence : Initialement modeste, "Heart of Darkness" a connu un succès croissant au XXe siècle, devenant un ouvrage académique clé et étant adapté au cinéma (ex. "Apocalypse Now"), symbolisant le passage du colonialisme à l'impérialisme.

Hannah Arendt s'est inspirée de ce roman pour critiquer le "capitalisme colonial comme prémisse du totalitarisme européen," notant la "bureaucratie coloniale" comme "arrogante qu'incompétente, aussi raciste qu'inculte, aussi cruelle que lâche."

L'éloignement de la métropole et l'absence de contrôle "favorise la violence sans borde aux colonies."

Le "Casement Report" (1904) de Roger Casement (Rapport) :

• Contexte : Roger Casement, diplomate britannique, a mené une enquête au Congo belge, recueillant "de nombreuses preuves des violences" suite aux témoignages de missionnaires et militaires.

• Description des atrocités : Le rapport détaille des villages désertés, la privation de subsistance, l'emprisonnement et la mort d'hommes pour des quotas insuffisants. Il révèle les "meurtres" pour le caoutchouc et l'ivoire, avec une "vérification rigoureuse par l'administration" via la présentation des "mains droites des cadavres ainsi coupées." Il mentionne des enfants tués et amputés, et des parents obligés de vendre leurs enfants.

• Intention de l'auteur : Casement se veut "strictement factuel," se déplaçant de village en village, recueillant des témoignages, constatant des blessures, croisant les sources et examinant des documents. Son but est de "convaincre" par les faits ("logos").

• Réception et influence : Le rapport a eu une "reconnaissance immédiate," jouant un "rôle essentiel dans la découverte de la violence coloniale et dans l'émergence d'une mobilisation pour les droits des peuples colonisés." Bien qu'il ait connu une période d'oubli (en partie due au discrédit de son auteur condamné à mort pour trahison), il a finalement trouvé sa place dans l'histoire du colonialisme.

Michael Taussig, anthropologue australien, s'est appuyé sur le "Putumayo Report" de Casement (décrivant des atrocités similaires au Pérou) pour analyser la violence coloniale, y voyant une "subversion mythique du mythe de l'impérialisme."

Il met en lumière l'"inscription d'une mythologie sur le corps indien" pour justifier la cruauté, et la dimension de "jouissance" dans la torture et le meurtre des Indiens.

B. Pluralité des Représentations et Enjeux Éthiques :

La distinction entre fiction et rapport ne doit pas conduire à remettre en question ces représentations, mais à "admettre leur pluralité en terme à la fois d'imagibilité et d'interprétation possible et à porter sur elle un regard critique interrogeant sans cesse la frontière entre les faits et ce qu'on en donne à voir et à comprendre."

La vérité du roman peut l'emporter sur la réalité du témoignage (comme Arendt privilégiant Conrad), ou inversement (comme Taussig privilégiant Casement).

III. La Représentation de la Violence Guerrière :

Omniprésence et Brutalisation

La guerre est un "fait incontournable de la vie de chaque pays, chaque nation, chaque personne," et l'Europe est aujourd'hui "directement confrontée à deux guerres."

A. L'Iliade ou le Poème de la Force (Simone Weil) :

• Le thème central : Pour Simone Weil, le "vrai héros, le vrai sujet, le centre de l'Iliade, c'est la force." Cette force (grec ancien bia) inclut la "force vitale," la "force du corps" et son emploi, c'est-à-dire la "violence."

• La déshumanisation : La force "fait de quiconque lui est soumis une chose," allant jusqu'à transformer les individus en "cadavre," ou pire, en "êtres plus malheureux qui sans mourir sont devenus des choses pour toute leur vie," un "compromis entre l'homme et le cadavre." Cette description préfigure les "premiers camps de concentration."

• La jouissance des vainqueurs et l'arrogance : La force "enivre quiconque la possède ou croit la posséder." L'arrogance ("l'hybris") conduit les guerriers à leur perte, car "le fort n'est jamais absolument fort ni le faible absolument faible mais l'un et l'autre l'ignore."

• La leçon ultime : La violence "pétrifie différemment mais également les âmes de ceux qui la subissent et de ceux qui la manient."

Les batailles se déroulent entre des "hommes dépouillés de toutes ces facultés, transformés, tombés au rang de forces aveugles."

Les peuples d'Europe ne retrouveront leur esprit que lorsqu'ils sauront "ne jamais admirer la force, ne pas haïr les ennemis et ne pas mépriser les malheureux."

B. La Brutalisation des Sociétés (George Mosse) :

• "Fallen Soldiers" (1990) : L'historien George Mosse analyse la perception de la Première Guerre mondiale en Allemagne, soulignant le rôle de la "technologie" et des "nouvelles et plus efficaces modalités de communication" pour diffuser l'image de la guerre et stimuler l'imagination.

• Le mythe et le culte du soldat tombé : La guerre prend une "double dimension mythique et religieuse," culminant dans le "culte du soldat tombé au champ d'honneur," qui projette la croyance dans le martyre et la résurrection sur la nation comme une "religion civique."

• Banalisation et brutalisation :Banalisation (pendant la guerre) : Vise à "domestiquer les horreurs du conflit," à "éviter les évocations douloureuses et à rendre la représentation familière" à travers des objets du quotidien, des jeux pour enfants, et des films qui "distraire le public loin du front" sans montrer la réalité des tranchées. L'objectif est de donner aux gens le "sentiment de dominer les événements."

• Brutalisation (après la guerre) : Une "continuation des attitudes guerrières en temps de paix au sein du peuple des faits." La politique est conçue comme une bataille, l'idée nationaliste se cristallise autour de la "haine de l'ennemi souvent déshumanisée." La vie perd de sa valeur.

• Facteurs de brutalisation : La "camaraderie masculine" et le "racisme" (notamment contre les Juifs) sont des éléments cruciaux. Mosse suggère que la banalisation de la violence pendant la guerre a facilité l'adoption de discours et de pratiques violents par la suite, "nourri[ssant] l'expression du ressentiment sur lequel s'est édifié le régime nazi."

• Le paradoxe de Mosse : Steven Hashaim note une "certaine sympathie pour plusieurs des mythes et des symboles qu'il étudie," et une "identification aux mythes et symboles nationalistes qu'il a pourtant tant fait pour démystifier."

Mosse lui-même reconnaît la "défaite de sa raison critique et de son savoir historique" face à la "démonstration de force d'une communauté juive d'un guerre décimée."

Cela illustre comment "les représentations de la guerre mêlent toujours la rationalité et l'émotion, l'imaginaire et l'effacement."

C. La Guerre à Gaza (Depuis le 7 octobre 2023) : Une "Atrocity Propaganda" Double

Les événements récents au Moyen-Orient offrent une "démonstration saisissante autant que tragique" de cette transformation des représentations :

• Victimisation (côté israélien) : L'attaque du Hamas et du djihad islamique a produit un "traumatisme sans précédent." Elle a été comparée à la Shoah, les assaillants assimilés à des nazis, et a nourri un discours de "menace existentielle." Des récits d'exactions commises par l'ennemi (démontées par la suite) ont été diffusés, et la "représentation politiquement maîtrisée des victimes" a prévalu.

• Brutalisation (côté israélien) : Dès le début de la guerre à Gaza, une "autre représentation" s'est imposée, "documentée dans la requête déposée par l'Afrique du Sud devant la Cour internationale de justice." Elle s'est manifestée par un "discours officiel d'humiliation de la population palestinienne," dénonçant la "nation entière comme responsable" et utilisant des termes déshumanisants ("animaux humains").

Cette brutalisation s'est exprimée "ouvertement" dans les médias nationaux (montrant des prisonniers palestiniens humiliés) et surtout sur les réseaux sociaux, où les soldats postaient "avec fierté des images de brimade, d'humiliation, de destruction de quartier, de pillage d'habitation" et des "sortes de trophées de guerre."

Ces vidéos et photos, révélant des crimes relevant de la justice internationale, ont été diffusées "sans restriction" par les autorités israéliennes.

• "Atrocity Propaganda" : La publicisation des violences perpétrées depuis le 7 octobre 2023 relève de l'"atrocity propaganda," pratique ancienne visant à transformer l'ennemi en monstre pour justifier des moyens extrêmes.

La "singularité du cas israélien" réside dans une "double publicisation : d'abord des sévices exercés par l'ennemi [...] et des exactions ensuite perpétrées par l'armée."

• La première visait à "susciter un sentiment d'horreur" pour légitimer les représailles.

• La seconde, essentiellement militaire, visait à "permettre au public israélien de constater la campagne punitive menée par l'armée et d'assouvir ainsi le désir de vengeance."

• Cette nouvelle représentation inclut le "culte de la virilité" et l'"ironie d'une parodie érotique violant son intimité."

• Les médias internationaux ont souvent reflété cette distinction, utilisant des mots comme "horrific, slaughter, massacre" plus souvent pour les victimes israéliennes que palestiniennes.

D. La Représentation des Victimes Palestiniennes :

• Après une brève diffusion de vidéos exaltantes du 7 octobre, les images suivantes ont été "généralement celles recueillies par des reporters palestiniens," montrant la destruction, les souffrances, les privations.

Le film "From Ground Zero" de Rachid Masharaoui, composé de courts métrages réalisés par des Palestiniens, offre un rare aperçu du "quotidien de la vie à Gaza pendant la guerre."

Il dépeint le "deuil, la désolation, la tristesse, souvent la tendresse, parfois l'ironie, mais jamais la haine, jamais le ressentiment, jamais l'évocation même de l'ennemi, seulement l'espoir que cesse enfin la guerre et que la vie redevienne possible."

Il vise à rappeler que "les gens sont des humains," donnant "des visages, des noms, des histoires, des métiers, des rêves" à des "chiffres" macabres.

Le refus de projeter ce film à Cannes pour ne pas faire de "politique" est souligné comme paradoxal, la destruction de Gaza étant comparée à Gernika et inspirant des artistes palestiniens.

IV. Conclusion

Représenter la violence est un "défi éthique, politique, esthétique" auquel sont confrontés les écrivains, cinéastes, chercheurs, et de plus en plus de personnes via les nouvelles technologies.

L'analyse de la violence coloniale et guerrière illustre la "multiplication des modes de représentation," de la fiction au rapport factuel, de la propagande à la documentation journalistique, des célébrations des acteurs de la violence aux récits des victimes.

Il est crucial de considérer ce qui n'est pas représenté, comme le "traitement sélectif de l'information dans les médias" et la "non-représentation de certaines violences" (ex. la conquête française de l'Algérie, la Nakba), éléments essentiels à toute enquête sur la représentation de la violence.

DOI: 10.1016/j.xgen.2025.100894

Resource: None

Curator: @scibot

SciCrunch record: RRID:DGGR_124054

What is this?

Su equivalente en frase en español de estos códigos es que, primero, tomamos el link donde se encuentran todos los nombres de los Pokemon y los pasamos a información estructurada en JSON. Todo esto dentro de 'json'. Luego, esta lista de pokemon lo que hará es, dentro de todo la información que trae 'json', y de la misma forma que con el ejercicio de la altura, identificando los resultados por el nombre de cada uno. De está manera, PokemonList solo contendrá un largo listado de todos los nombres de los Pokemones.

Los modelos vectoriales de lenguaje nos ayudan a trabajar con texto de forma más eficiente, permitiendo comparar, buscar o generar información automáticamente. Son útiles para analizar y procesar grandes cantidades de datos de manera precisa.

Un torneo Pokémon es un evento competitivo donde los entrenadores demuestran sus habilidades en combate, compiten por premios y tienen la oportunidad de clasificar a eventos mayores, como los Campeonatos Mundiales. Además de ser una experiencia divertida y social, estos torneos ayudan a mejorar estrategias y habilidades. Existen distintos niveles de torneos, desde desafíos locales hasta competiciones internacionales de alto nivel.

Me es curioso como en varias ocasiones el mismo código ya ejecutado al guardar, cerrar y volver a entrar ya no ejecuta y aparecen errores

El resultado me es diferente, y vuelve al principio, es decir sin orden de diccionario ya prestablecido

Utilice diferentes a Charmander y Squirtle Aunque luego al ejecutar y en secuencia no leía el código

Los repositorios de código pueden ser de gran ayuda para nosotros, ya que nos permiten acceder, organizar y trabajar con el código de manera eficiente. Gracias a ellos, podemos colaborar con otros compañeros, seguir el historial de cambios y acceder a nuestros proyectos desde cualquier lugar. Aprender a extraer información de estos repositorios nos ayuda a desarrollar habilidades prácticas valiosas, como lo vimos al trabajar con PokeAPI. Además, este conocimiento puede aplicarse en otras asignaturas donde necesitemos analizar datos, gestionar proyectos o trabajar en equipo. Aun así, es importante seguir profundizando en su valor teórico para entender mejor su utilidad en el entorno académico.

La importancia de ir más allá de la presencia física o la participación física, y claramente las formas que afectan la falta de comunicación

Al realizar la ejecutar el combate en el torneo pokemon entre Pikachu y Pidgey, siguiendo la narrativa de datos se logro adicionar las filas correspondientes a los tres round en la tabla

Me presenta un error y no estoy seguro si se debe a que coloqué varios Fighter en el código. Posiblemente estoy repitiendo nombres o variables sin control, lo que podría estar generando conflicto entre lo que quiero ejecutar.

Esta instrucción permite al sistema obtener y sincronizar automáticamente una serie de documentos numerados del 1 al 29 desde una URL específica.

HedgeDoc es una herramienta colaborativa en línea que permite crear y editar documentos de manera simultánea entre varios usuarios, facilitando la sincronización de contenidos entre diferentes clases. Además, puede integrarse con Glamorous Toolkit, lo que permite sincronizar y gestionar esos documentos dentro de un entorno de desarrollo avanzado basado en Pharo Smalltalk.

Un DataFrame es una tabla de datos estructurada en filas y columnas que permite organizar, manipular y analizar información de forma sencilla y ordenada en programación y ciencia de datos.

No me está funcionando la tabla, creo que me falta definir alguna variable o estoy olvidando algún paso.

Estoy ejecutando Fighter 1 hasta Fighter 6 para organizar la lista de los Pokémon que tengo en sus Pokébolas, de esta manera puedo mantener un orden claro y definido sobre su respectiva posición o turno.

Pero no se si estoy ejecuntado mal al tener varios Fighter.

La primera generación de Pokémon, que inició con los juegos de Game Boy (Rojo, Verde y Azul en Japón), presenta 151 especies diferentes. Estos 151 Pokémon son todos originarios de la región de Kanto. El último Pokémon de esta generación es Mew, el número 151.

Se puede utilizar JSON para almacenar la información de un Pokémon, incluyendo su nombre, tipo, habilidades y estadísticas, organizados en una estructura de datos clara y accesible.

Es importante tener conocimientos básicos de programación, ya que al momento de ejecutar código pueden presentarse errores en algunos casos. Contar con esas bases permite identificar, corregir y entender mejor los problemas que surgen durante el desarrollo.

Grafoscopio es una plataforma de código abierto basada en Pharo Smalltalk, una versión actualizada que facilita la creación, lectura y distribución de cuadernos computacionales interactivos.

Ideal para dejar documentado no solo los resultados sino también el proceso que se lleva, permitiendo a otros replicarlo o adaptarlo.

El enlace que compartiste lleva a la línea de tiempo del repositorio “Valentina-P” en ChiselApp, gestionado por Valentina Penagos Ariza.

Este repositorio documenta sus actividades académicas y de programación, especialmente en temas de computación y lecturas hipertextuales.

En la línea de tiempo se pueden ver los distintos cambios y publicaciones que ha hecho en sus proyectos.

3 de junio de 2025: Eliminó un archivo con un nombre incorrecto.

2 de junio de 2025: Publicó el proyecto Batalla Pokémon.

1 de junio de 2025: Subió su proyecto final.

21 de mayo de 2025: Publicó una actividad autónoma usando JSON.

7 de mayo de 2025: Actualizó y publicó el ejercicio Challenge Yourself.

26 y 28 de abril de 2025: Publicó lecturas hipertextuales y ejercicios de clase.

siguiendo las instrucciones no agrega la fila porque devuelve un valor nulo

Cuando ejecuto el código y dejo en vista de impresión un resultado pero no como un arreglo como se ve en el ejemplo

Respecto a este apartado tengo problemas para poder importar esta misma narrativa. Puesto que cuando coloco el enlace como aprendimos a hacerlo en clase del 31 de marzo se me congela por completo el programa de Glamorous Toolkit. No sé si haya otra estrategia para poder importar la narrativa o si deba presentar una nueva narrativa completamente hecha por mí.

En este apartado, realice el ejercicio con las partes de código del torneo Pokémon, pero no pude organizar los datos en la tabla, porque se me genera solo con los daos vacíos. Entonces seguí los ejemplos del libro de los DataFrame, pero me sigue generando el mismo error, se crea la tabla, pero no se puede organizar dentro de la estructura y en algunos lados genera errores que desconozco si son de la ejecución del código, o es que falta adicionar algo al Software Grafoscopio

Aqui no se coloca el nombre, pero se entiende que el fighter1 es pikachu y el me va a traer los datos de él lo mismo si le digo fighter 2 el entiende que es Ditto...

Para el proyecto final he creado la CLASE: pokemon con sus objetos " fighter" y a la vez cada uno de estos con sus atributos para mi proyecto he creado 6 luchadores:

Sobre los códigos de las rondas tengo un problema. Precisamente cuando ejecuto el código tal cual como lo vimos en clase me aparece "value was sent to nil" Estuve revisando las grabaciones de las clases pero no encuentro ningún error anterior, de hecho, me había funcionado hasta la parte donde debemos utilizar los dataframes. Pero a partir de ahí cuando quiero poner una ronda no me lo permite. Este es el código que he estado utilizando. round1 := { fighter1 name. fighter2 name. (randomMove value: fighter1) at: 'move' at: 'name'. (randomMove value: fighter2) at: 'move' at: 'name'. { fighter1 . fighter2 } atRandom } Y esto es lo que aparece cuando lo ejecuto. De saber alguna solución posible lo agradecería

Funciona para obtener los datos de diferentes pokemones a la vez, y almacenarlos en una variable.

realicé el cambio de Pikachu por ''Snorlax'' y el resultado fue el mismo, únicamente con la edición del Pokémon que indiqué

Ha sido una forma diferente de entender la programación y la cantidad de posibilidades que existen una vez que aprendes a entender la lógica de los lenguajes. Pokémon en este caso es el conjunto de datos con los que estamos trabajando (Criaturas con diferentes poderes).

Atrandom es el comando que he estado implementando para elegir un Pokémon al azar, y hará parte da la construcción de los round de mis pokemones.

Si lo interpreto bien, se supone que lo que le estamos pidiendo es: en bloque tomando la variable 'item' que será cada valor de nuestro arreglo anterior, entrá a cada uno y tre el valor de los metadatos 'name' y 'height'. Así el resultado será un listado del número de valores de nuestro arreglo, con su respectivo nombre y altura.

Lo único que me molesta es que el orden que presenta es justamente como lo pedimos, es decir, los resultados están en el mismo orden que los valores de nuestro arreglo. No sé si haya una forma de volver la información de 'height' en un dato al que luego podamos ponerle una condición de mayor a menor número.

Al final esto es justamente lo que intenté hacer en la última clase, tener un listado con los nombres de los Pokemones y un bloque que me permita visualizar la información estructurada de cada uno. No pensé que este sería el resultado de ello, pero me voy satisfecha.

Esta representación es muy útil si lo queremos adaptar a otros ejercicios. Si bien el reto es, precisamente, como adaptamos este código para una nueva necesidad que surja, me parece interesante como los nombres son las llaves y el link personal de cada repositorio. Lo interpretó como una especie de BUSCARV de excel interesante para analizar a profundidad.

Figure énumérative.

(le recours à etc. laisse entendre qu’il y a d’autres facteurs – faiblesse?)

A version of this preprint has been published in the Open Access journal GigaScience (see paper https://doi.org/10.1093/gigascience/giaf048), where the paper and peer reviews are published openly under a CC-BY 4.0 license.

Revision 2 version

Reviewer 2: Benjamin D Rosen

The first near-complete genome assembly of pig: enabling more accurate genetic research.

General comments:

The authors have clarified how their HiC manual curation efforts were able to remove gaps from the assembly. This was my only remaining major issue. I only have a few minor comments remaining.

Minor comments:

Line 1 - Title: "A Near Telomere-to-Telomere Genome Assembly of the Jinhua Pig"

Line 369 - replace "only 6 gaps left in our final JH assembly" with "only 6 gaps remain in our final JH assembly"

Line 370 - Figure S5 needs a more detailed legend

Line 405 - I just noticed this, but are the authors proposing that chr9 has 2 centromeres? Given the know pig karyotype (metacentric chr9), it seems more likely that they have identified some other form of tandem repeat at the beginning of chr9.

A version of this preprint has been published in the Open Access journal GigaScience (see paper https://doi.org/10.1093/gigascience/giaf048), where the paper and peer reviews are published openly under a CC-BY 4.0 license.

Revision 1 version

Reviewer 1: Martien Groenen

In their revised version of the manuscript, the authors have addressed all my major concerns raised in my earlier review and have made the many editorial edits as suggested. I only have a few (mostly editorial) comments for the revised version. The most important one is the title of the manuscript. I realize I did not mention this in my earlier review, but I think the title is not very appropriate and could be more informative. I suggest something like "A telomere-to-telomere genome assembly of the Jinhua pig"

Minor editorial comments: Line 40: Replace "provides" by "provide"; "genome" to "genomes" and "JH" to "Jinhua" Lines 50-51: "This study produced a gapless and near-gapless assembly of the pig genome, and provides a set of diploid JH reference genome." Should be changes to something like "This study produced a near-gapless assembly of the pig genome and provides a set of haploid Jinhua reference genomes." Line 177: Change "with with" to "with" Line 194: Replace "population" by "populations" Lines 232-233: Referring to human as a "closely related species" is rather awkward and not correct. I suggest replacing this with "eleven other mammals" Lines 299, 301 and 303: Insert "of" after "consisting" Line 317: Insert "and" before "2.33 Gb" Line 319: Insert "and" before "2.17 Gb" Line 320-321: Change to "The more continuous contigs of the two assemblies were selected to construct the final haploid assemblies". Line 323: Replace "assembly" by "assembler "Line 354: Delete "ranging" Lines 358359: Change "The average properly mapped rate" to "The average rate of properly mapped reads" Line 379: Insert "respectively" after "60.07"Line 380: "suggested" (remove space)Line 385: Change "indicate a gapless and near-gapless" to "indicate a near-gapless" Line 455: Change "were overlapped with" to "were overlapping with" Lines 557-559" The sentence "The insertion found in the SLA-DOB gene, which serves to enhance the immune system's response and is relevant to transplant rejection" seems incomplete and sound awkward. Perhaps you mean something like "The insertion found in SLA-DOB, a gene involved in enhancing the immune system's response to infection, might be relevant in relation to transplant rejection"

Reviewer 2: Benjamin D Rosen

The first near-complete genome assembly of pig: enabling more accurate genetic research

General comments: I thank the authors for addressing most of my points and providing more details on the parameters they have used. Unfortunately, I still have some unanswered questions regarding the methodology. My current understanding from the authors responses to my previous comments leads me to believe that the assembly has been scaffolded incorrectly. If the authors did indeed use HiC data to place 8 contigs into gaps and then joined those contigs without placing gaps at the joins or doing any further gap filling, that calls into question the validity of the assembly. Finally, the language needs further improvement for readability.

Specific comments: Line 85 - *will contribute to. Lines 187-191 - HiC interaction maps do not provide information for gap filling. Either this has been explained insufficiently, or it has been done incorrectly. Placing assembled sequences in the correct order does not mean that it is okay to join them without a gap. It is necessary to return to the gap filling procedure now that the contigs are in the correct order and attempt to fill them as done previously. Line 191 - Figure S3 - These HiC contact maps are not very informative they need to be labeled and have a scale bar. Additionally, contact maps can have a lack of signal due to a gap in the sequence or due to multimapping reads in repetitive regions being filtered so it's not clear what they are trying to show in A-C. The authors reply to my previous concern regarding the labeling of this figure does not help, furthermore, the figure legend in the supplemental materials is still insufficient. I think I understand that panels D and E are chr3 before and after misassembly correction, it would be helpful if the two panels were at the same scale. I still don't know why panel F is shown, how is this related to panel C and I don't see any red ellipses indicated by the legend. Line 275 - "ensemble from Duroc pigs" is incorrect. It is an "assembly of a Duroc pig". Lines 299, 301, 303 - "containing" not "consisting" Lines 306-308 - Again, HiC data orders and orients contigs, but it does not fill gaps. Please clarify how the assembly was reduced from 14 gaps to 6 gaps with HiC data. Was an additional round of gap filling performed? Lines 313-314 - How is the contig N50 larger than the scaffold N50 above? Lines 335-336 - Does this refer to the Merqury analysis? I don't think "using mapped K-mers" is correct here, please reword. Lines 367-368 - what does it mean that "8 out of 63 gaps were corrected" is this from the HiC ordering of contigs? Line 369 - what does the mapping between Sscrofa11.1 and JH-T2T shown in figure S6 have to do with the JH-T2T gap filling being described here? Line 369 - I previously asked about this supplemental table only containing 55 entries. The authors response "The other filled 8 gaps were resolved through adjustments made to the Hi-C map to correct misassembles. As a result, these gaps cannot be precisely located within the existing order of the assembly." indicates that contigs must have been incorrectly joined solely based on the HiC signal between contigs. The authors must know what contigs were added or joined to form the final assembly. It would be trivial to align the two assembly versions and identify the positions of the old contigs in the new assembly. I believe that these incorrectly joined contigs should be broken and put through the same gap filling procedure as performed earlier. Lines 375-378 - Dramatic coverage changes in read mappings as found in these figures are usually indicative of assembly errors. I do not agree that "These findings confirmed the accuracy and reliability" of the assembly. I suggest replacing the last sentence with something more measured such as "Although supported by some read data, the inconsistency of coverage across these gap filled regions suggests that caution should be used when interpreting findings in these regions, cross-referencing results with the gap positions (Supplementary Table S9) is advised." Line 375 - "evidenced by fully coverage" remove "fully", it isn't proper usage of the word and I wouldn't interpret the low coverage in many of these regions as "full coverage". Line 385 - should read "Overall, our assembly quality metrics indicate a near-gapless assembly of the pig genome" Line 390 - should read "a gapless T2T sequence for 16 out of 20" Line 396 - Supplemental table 10 not 9.Lines 398399 - according to supplemental table S4 and figure 3A, chromosome 2 also has a single telomere. Line 402 - the centromeres are not marked in Figure 3A.Line 402 - Figure S8 - please rename chr19 and chr20, chrX and chrY. Line 406 - "at early research" unclear what is meant by this. please reword. Line 423 - as indicated on line 397, 33 telomeres were identified, not 35.Line 426 - "The JH-T2T assembly IDENTIFIED 17 centromeres" Line 450 - "are located in" Line 453 - "these SVs are located in" Line 455 - Moreover, 12,129 genes overlap these SVs" Line 502 - "which contained 544 gaps" Line 841 - Figure 2 legend description is still incorrect. Only A is mapping rates, B and C are PM rates and base error rates.

A version of this preprint has been published in the Open Access journal GigaScience (see paper https://doi.org/10.1093/gigascience/giaf048), where the paper and peer reviews are published openly under a CC-BY 4.0 license.

Original version

Reviewer 1: Martien Groenen

The manuscript describes the T2T genome assembly for the Chinese pig breed Jinhua, which presents a vast improvement compared to the current reference genome of the Duroc pig TJTabasco (build11.1). The results and methodology use for the assembly are described clearly and the authors show the improvement of this assembly by a detailed comparison with the current reference 11.1. While clearly of interest to be published, several aspects of the manuscript should be improved. Most of these changes are minor modifications or inaccuracies in the presentation of the results.

However, there are two major aspects that need further attention:

1. The T2T assembly presented, represents a combination of the two haplotypes of the pig sequenced. I am surprised why the authors did not also develop two haplotype resolved assemblies of this genome. Haplotype resolved assemblies will be the assemblies of choice for future developments of a reference pan-genome for pigs. The authors describe that they have sequenced the two parents of the sequenced F1 individual, so why did they not use the trio-binning approach to also develop haplotype resolved assemblies. I, think adding these to the manuscript would be a vast improvement for this important resource.

2. The results described for the identification of selective sweep regions is not very convincing. This analysis shows differences in the genomes of two breeds: Duroc and Jinhua. However, these breeds have a very different origin of domestication of wild boars that diverged 1 million years ago, followed by the development of a wide range of different breeds selected for different traits. Therefore, the comparison made by the authors cannot distinguish between differences in evolution of Chinese and European Wild Boar, more recent selection after breed formation and even drift. To be able to do so, these analyses would need the inclusion of additional breeds and wild boars from China and Europe. Alternatively, the authors can decide to tone down this part of the manuscript or even delete it altogether, as it does not add to the major message of the manuscript.Minor comments Line 34: Change the sentence to: "with thousands of segments and centromeres and telomeres missing" Line 37: Insert "and Hi-C" after "long reads "Line 46: Delete " such as GPAM, CYP2C18, LY9, ITLN2, and CHIA" Line 54: Insert "potential" before "xenotransplantation" Line 82: Delete "in response to the gap of a T2T-level pig genome" as this does not add anything and the use of "gap" in this context is confusing. Line 93: Change "The fresh blood" to "Fresh blood" Line 100: The authors need to provide a reference for the SDS method. Lines 152-153, line 444, and table S6: This is confusing. The authors mention Genotypes from 939 individuals, but in the table it is shown that they have used WGS data. You need to describe how the WGS data was used to call the genotypes for these individuals. Furthermore, in line 444 you mention 289 JH pigs and 616 DU pigs which together is 905. What about the other 34 individuals shown in table S6?Line 244: Replace "were" by "was" and delete "the" before "fastp" Lines 287292: Here you use several times "length of xx Gb and yy contigs". This is not correct as the value for the contigs refers to a number and not a length. Rephase e.g. like "length of xx Gb and consisting of yy contigs" Line 294: The use of "bone" sems strange. Either use "backbone" or "core"Line 306: Replace "chromosome" by "genome" Lines 308-309: For the comment "Second, 16 of the 20 chromosomes were each represented by a single contig" you refer to figure 1D however from this figure it cannot be seen if the different chromosomes consist of a single or multiple contigs. Line 346: Do you mean build 11.1 with "historical genome version". If so, please use that instead. Line 349: "post-gap filled" Line 353: The largest gap is 35 kb not 36 kb. Figures 2F-I should be better explained in the legends and the main text (lines 353-358). Lines 378: For the 23,924 genes you refer to supp table S13. However, that table shows a list of SV enriched QTL not these genes. Furthermore, I checked all tables but a table with all the protein coding genes is missing. Line 380: For the 799 newly anchored genes, refer to table S10. Now you refer to table S17 which shows genes enriched KEGG pathways. Lines 383-386: For the higher gene density in GC rich regions, you refer to figure 1D, but it is impossible to see this correlation from figure 1D. For the density of genes and telomeres, you refer to figure 1G. However, that figure does not show gene densities only repeat densities. Line 406-407. This should be table S11.Lines 409412: For this result you refer to table S11. However, that table only shows data for the gained genes, not the lost genes. Lines 419-420: You refer to table S12 and figure 3B, but the information is only shown in figure 3B and not in table S12.Line 420: Replace "were" by "is" Line 422: Better to use "repeats" instead of "they" Line 425: "Moreover, 12,129 genes located in these SVs". Unclear to what "these" refers to and I assume that you mean genes that (partially) overlap with SVs? Also, this is an incomplete sentence (verb missing). Likewise, this number is not very meaningful as many of these SVs are within introns. It is much more informative to mention for how many genes SVs affect the CDS. Line 433 and table S14: This validation is not clear at all. What exactly are these numbers that are shown? You also mention "greater than 1.00" but the table does not contain any number that is greater than 1.00. Line 435: "Table" not "Tables" Line 436: Change to " SVs with a length larger than 500 bp "The term "invalidate" in figure 3D is rather awkward. Better to use "not-validated" and "validated" in this figure. Line 449: This should be Table S16. Line 452: There is not Table S18Lines 484-486: Change to "Similarly, in human, the use of the T2T-CHM13 genome assembly yields a more comprehensive view of SVs genome-wide, with a greatly improved balance of insertions and deletions [61]." Lines 500-501: Change to "For example, in human, the T2T-CHM13 assembly was shown to improve the analysis of global" Lines 517-528: This paragraph should be deleted as these genes have already been annotated and described in previous genome builds including 11.1. Why discuss these genes here? Following that line of thinking, almost every gene of the 20,000 can be discussed. Line 532: "%" instead of "%%" and insert "which" after "SVs" Lines 537-542: These sentences should be deleted. It is common knowledge that second generation sequencing is not very sensitive to identify SVs. The authors also do not provide any results about dPCR. Line 544: "affect" rather than "harbor" Lines 544-547: This is repetitive and has been stated multiple times so better to delete. Line 561: "which is serve to immune system's response and relevant to transplant rejection" This is an incorrect sentence and should rephrased. Lines 562-568: I don't agree with is statement and suggest to remove it from the discussion.

Reviewer 2: Benjamin D Rosen

The first near-complete genome assembly of pig: enabling more accurate genetic research. The authors describe the telomere-to-telomere assembly of a Jinhua breed pig. They sequenced genomic DNA from whole blood with PacBio HiFi and Oxford Nanopore (ONT) long-read technologies as well as Illumina for short reads. They generated HiC data for scaffolding from blood and extracted RNA from 19 tissues for short read RNAseq for gene annotation. A hifiasm assembly was generated with the HiFi data and scaffolded with HiC to chromosome level with 63 gaps. The scaffolded assembly was gap filled with contigs from a NextDenovo assembly of the ONT data bringing the gaps down to 14. Finally, the assembly was manually curated with juicebox somehow closing a further 8 gaps. This needs to be clarified. Standard assembly assessments were performed as well as genome annotation. The authors compared their assembly to the current reference, Sscrofa11.1, and called SVs between the assemblies. The SVs were validated with additional Jinhua and Duroc animals. They then identified signatures of selection present in some of the largest SVs.

General comments: The manuscript is mostly easy to read but would benefit from further editing for language throughout. The described assembly appears to be high quality and quite contiguous. Although the authors do mention obtaining parental samples and claim the assembly is fully phased, there is no mention of how this was done. There are many additional places where the methods could be described more fully including the addition of parameters used.

Specific comments: Line 39 - Figure 1 only displays 34 telomeres, not 35. Additionally, I was only able to detect 33 telomeres using seqtk telo. Seqtk only reports telomeres at the beginning and end of sequences, digging further, the telomere on chr2 is ~59kb from the end of the chromosome, perhaps indicating a misassembly. Lines 79-81 - there are not hundreds of species with gap free genome assemblies and reference 19 does not claim that there are. Line 82 - the assembly is not gap-free, replace with "nearly gap-free" Line 95 - were these parental tissue samples ever used? Lines 151-156 - this section would be better located below the assembly methods. Please number supplementary tables in order of their appearance in the text. Line 171 - please provide parameters used here and for all analyses. Lines 187-188 - how did rearranging contigs decrease the gaps? Was the same gap filling procedure used after HiC manual adjustments? Line 188 - Figure S3 - I don't understand the relationship between the panels nor what the authors are attempting to show. If panels A-C display chromosomes 2, 8, and 13, Why does D display chr3? Both panels C and E are labeled chr13 but they look nothing alike. Are D-E whole chromosomes or zoomed in views? Missing description of panel F. Lines 222-224 - why weren't pig proteins used? Ensembl rapid release has annotated protein datasets for 9 pig assemblies. Line 264 - although most will know this, make it clear that Sscrofa11.1 is an assembly of a Duroc pig. Line 292 - how was polishing performed? This is missing from the methods. Line 294 - should this read "selected it for the backbone of the genome assembly."? Lines 298-299 - methods? Line 314 - what is meant by "using mapped K-mers from trio Illumina PCR-free reads data"? Line 331 - accession numbers for assemblies would be useful. Line 333 - what is "properly mapped rate"? Do you mean properly paired mapping rate? Line 346 - what is the historical genome version? Line 349 - Supplemental Table S8 only has 55 entries including the 6 remaining gaps. Where are the other filled 8 gaps located? Lines 350-358 - read depth displays wouldn't show the presence of clipped reads which would indicate an improperly closed gap. It would be more convincing to display IGV windows containing these alignments showing that there are no clipped reads. Line 354 - Figure S5 needs a better legend. What is ref and what is own? Line 359 - the assembly is near-gapless. Line 359 - where is the data regarding assembly phasing? How was this determined to be fully phased? Line 363 - 16 of 20 chromosomes are gapless. Line 370 - only 33 telomeres were found at the expected location (end of the chromosome), if you count the telomere on chr2 59kb from the end, then 34 telomeres were identified. Line 372 - chr13 also only has a single telomere. It does not have a telomere at the beginning. Line 372 - chr19 is chrX correct? Line 374 - Figure 1G - It would be nice to have the centromeres marked on this plot (or in Figure 3A). Are the long blocks of telomeric repeats internal to the chromosomes expected? Line 423 - Figure 3A - there is no telomeric repeat at the beginning of chr4 or chrXLine 431 - why were only 5 pigs of each breed used to validate SVs when 100's of WGS datasets from the two breeds had been aligned? How were these 5 selected? Line 481 - Sscrofa11.1 only has 544 gaps.Line 492 - ONT data was used to fill more than 6 gaps. Gaps in the assembly were reduced from 63 to 14 using ONT contigs. Lines 588-589 - please make your code publicly available through zenodo, github, figshare, or something similar. Line 815-824 - Figure 2 - legend description needs to be improved. Only A is mapping rates, B and C are PM rates and base error rates. The color switch from A-C having European pigs in blue to D having JH-T2T in blue might confuse readers.

A version of this preprint has been published in the Open Access journal GigaScience (see paper (https://doi.org/10.1093/gigascience/giaf035)), where the paper and peer reviews are published openly under a CC-BY 4.0 license.

Original Submission Reviewer 1: Hua Zhang

Wess et al reports a Python framework, MIIT (Multi-Omics Imaging Integration Toolset), for integrating spatially resolved multi-omics data. Multi-omics imaging represents a pivotal approach for systems molecular biology and biomarker discovery. This method introduces a timely and valuable tool to advance the field. However, in my opinion, this paper still has some issues that need to be addressed before consideration for publication. Cancer tissue exhibits significant heterogeneity effects, in this study, different molecular information obtaining from different tissue sections, this means from different cells as the tissue section is 10 um thickness, almost the diameter of the cells. Please height the meaningful of co-registration information if they are obtained from different cell layers. In particular, for the datasets of spatial transcriptomics and MSI, the experiments were conducted on serial sections with an axial sectioning distance of 40 to 100 μm. This means that the mRNA and metabolites originate from different cells, raising questions about how integrating these two datasets can provide meaningful insights. The multi-omics imaging integration toolset is based on the GreedyFHist, a non-rigid registration algorithm, it suggests including more details about this algorithm and highlight the difference comparing to previously reported non-rigid image co-registration algorithm. The author should demonstrate the accuracy of background segmentation, it concerns certain low signal sample area would be removed in the denoising step. What is criterion to define the background region and sample region in the background segmentation.

In the Method section, more details need to be included in the spatial transcriptomics part, what the spatial resolution of the 10x Genomics was used. As the MALDI resolution is 30 um, how the pixel alignment of the ST and MSI data if their spatial resolution is different. In the MALDI-MSI of prostate tissue, on tissue MS/MS data is missing to confirm the identification of target analytes of citrate, ZnCl3-, and spermine.

**Reviewer 2: Santhoshi Krishnan **

Overview: In this paper, the authors present the Multi-Omics Imaging Integration Toolset, which is a python framework for integration multiple spatial omics datatypes. To facilitate this, they also development a registration method (GreedyFHist) for jointly analyzing sequential tissue layers that have undergone different types of staining/phenotyping regimens. The method validation was done on a 244 fresh-frozen prostrate tissue sections. The highly detailed methods and results section is well appreciated and helps fully contextualize the significance of the study. The definitions of study-specific terms mentioned throughout the paper at the beginning are also appreciated. Data and Code Availability: Detailed code, tutorials and associated instructions have been made available for use by the public, which is appreciated. All systems requirements have also been explicitly laid out for ease of installation and use. The workflow examples provided are quite detailed; however, a more extensive codebase with stepwise explanations within the code will be appreciated. Data has not been made available publicly, except for the raw and processed spatial transcriptomics data; however, detailed and explicit instructions have been provided on data access, keeping in mind local regulations. Revisions: Major Revisions: 1. In recent years, a lot of other platforms, both free and paid, tend to support registration across multiple slides. For example, HALO has a registration feature available as well, along with a host of other open-source datatypes. In that regard, how is your platform different? 2. It is mentioned that downscaling occurs during the registration process in order to reduce runtime - how are nuances in features selected as registration landmarks preserved in such a case? 3. How is the fixed image determined in this case? The assumption would be that a standard H&E image is selected for this purpose- is that assumption, correct? 4. The authors have stated and justified their rationale for using the mentioned evaluation metrics in the paper. However, in the general image registration space, metrics such as the dice coefficient and jaccard index are commonly used and accepted. Is there a particular reason why these were not used as well? It would offer a more complete picture for the general user if these metrics were provided as well. 5. The validation of registering distance neighboring sections is quite a valuable contribution, as the authors rightly stated that in many multi-omics experiments, this might be a necessity. However, when looking at tissue sections that are 80-100 microns apart, it is quite likely that the set of cells that one may be looking at on the x-y coordinate system may not be the same at all; in fact, for a highly heterogeneous/flexible piece of tissue, they might be completely different. In such a circumstance, how much value is there in registering these two sections together instead of, say, separately analyzing them and using alternative methods to combine the results downstream? 6. In the proof of concept presented in the paper, the authors mention using ST and MSI data for validating their framework. Have they also investigated ST integration with more commonly available datatypes such as IHC/mIF? 7. The work that the authors have put in to validate the registration and MIIT framework using different approaches (selecting spatially distant slides, integration using augmented/artificial data) is thorough. However, different tissue types bring in their own challenges, and thus validation of this framework on an external dataset would lend more credence to this much needed framework, especially in the era of increased multiomics analyses.

Minor Revisions: 1. Please ensure all typos/grammatical mistakes are corrected. 2. In the 'preprocessing of stained histology images', can more details be given on the thresholding process? It is also stated that the threshold is manually adjusted for each image if necessary - how is this determination done? 3. The headings/subheadings organizations within sections can be done in a more organized manner, in some parts it was challenging to determine the organization of sections/subsections. 4. Can some more details be given on the landmarks that were identified per image? Could some examples be provided on what these landmarks are, and how they remain consistence across tissue layers? 5. Currently, the way various samples are used for validating the GreedyFHist and MIIT frameworks are listed out in the paper is quite confusing. It would be appreciated if the authors can distinctly mention the number of samples out of the set of samples, and the associated stained slides are used for each. 6. How were the annotations from the 3 annotators cross validated?

A lot of bright minds have been silenced by established authorities, particularly in medicine, where people's life are at stake, hence professor acquire semi-god powers.

y, the

This should be changed so that "you" is the subject, for example "you can find the code to create Figure 7.13 tibbles x and y below"

DOI: 10.1016/j.celrep.2025.115775

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_3097648

What is this?

Después de colocar los nombres a las columnas, así se ve en la vista DATA y en la vista TREE.

Aquí le asigno el nombre de las columnas a las filas no por que sale un errror y no deja ejecutar.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public Review):

(…) In my view, the part about NF-YA1 is less strong - although I realize this is a compelling candidate to be a regulator of cell cycle progression, the experimental approaches used to address this question falls a bit short, in particular, compared to the very detailed approaches shown in the rest of the manuscript. The authors show that the transcription factor NF-YA1 regulates cell division in tobacco leaves; however, there is no experimental validation in the experimental system (nodules). All conclusions are based on a heterologous cell division system in tobacco leaves. The authors state that NF-YA1 has a nodule-specific role as a regulator of cell differentiation. I am concerned the tobacco system may not allow for adequate testing of this hypothesis.

Reviewer #1 makes a valid point by asking to focus the manuscript more explicitly on the role of NF-YA1 as a differentiation factor in a symbiotic context. We have now addressed this formally and experimentally.

The involvement of A-type NF-Y subunits in the transition to the early differentiation of nodule cells has been documented in model legumes through several publications that we refer to in the revised version of the discussion (lines 617/623). We fully agree that the CDEL system, because it is heterologous, does not allow us more than to propose a parallel explanation for these observations - i.e_., that the Medicago NF-YA1 subunit presumably acts in post-replicative cell-cycle regulation at the G2/M transition. Considering your recommendations and those of reviewer #2, we sought to support this conclusion by testing the impact of localized over-expression of _NF-YA1 on cortical cell division and infection competence at an early stage of root colonization. The results of these experiments are now presented in the new Figure 9 and Figure 9-figure supplement 1-5 and described from line 435 to 495.

With the fluorescent tools the authors have at hand (in particular tools to detect G2/M transition, which the authors suggest is regulated by NF-YA1), it would be interesting to test what happens to cell division if NF-YA1 is over-expressed in Medicago roots?

To limit pleiotropic effects of an ectopic over-expression, we used the symbiosis-induced, ENOD11 promoter to increase NF-YA1 expression levels more specifically along the trajectory of infected cells. We chose to remain in continuity with the experiments performed in the CDEL system by opting for a destabilized version of the KNOLLE transcriptional reporter to detect the G2/M transition. The results obtained are presented in Figure 9B (quantification of split infected cells), in Figure 9-figure supplement 1B (ENOD11 expression profile), in Figure 9-figure supplement 3B (representative confocal images) and Figure 9-figure supplement 4D (quantification of pKNOLLE reporter signal). There, we show that mitosis remains inhibited in cells accommodating infection threads, but is completed in a higher proportion of outer cortical cells positioned on the infection trajectory, where ENOD11 gene transcription is active before their physical colonization.

Based on NF-YA1 expression data published previously and their results in tobacco epidermal cells, the authors hypothesize that NF-YA regulates the mitotic entry of nodule primordial cells. Given that much of the manuscript deals with earlier stages of the infection, I wonder if NF-YA1 could also have a role in regulating mitotic entry in cells adjacent to the infection thread?

The expression profile of NF-YA1 at early stages of cortical infection (Laporte et al., 2014) is indeed similar to the one of ENOD11 (as shown in Figure 9-figure supplement 1C) in wild-type Medicago roots, with corresponding transcriptional reporters being both activated in cells adjacent to the infection thread. Under our experimental conditions, additional expression of NF-YA1 (driven by the ENOD11 promoter) in these neighbouring cells did not impact their propensity to enter mitosis and to complete cell division. These results are presented in Figure 9-figure supplement 4D (quantification of pKNOLLE reporter signal) and Figure 9-figure supplement 5 (quantification of split neighbouring cells).

Reviewer #1 (Recommendations For The Authors):

- In the first part, images show the qualitative presence/absence of H3.1 or H3.3 histones.

Upon closer inspection, many cells seem to have both histones. In Fig1-S1 for example (root meristem), it is evident that there are many cells with low but clearly present H3.1 content in the green channel; however, in the overlay, the green is lost and H3.3 (pink) is mainly visible. What does this mean in terms of the cell cycle? 

We fully agree with reviewer #1 on these points. Independent of whether they have low or high proliferation potential, most cells retain histone H3.1 particularly in silent regions of the genome, while H3.3 is constitutively produced and enriched at transcriptionally active regions. When channels are overlaid, cells in an active proliferation or endoreduplication state (in G1, S or G2, depending on the size of their nuclei) will appear mainly "green" (H3.1-eGFP positive). Cells with a low proliferation potential (e.g., in the QC), G2-arrested (e.g., IT-traversed) or terminally differentiating (e.g., containing symbiosomes or arbuscules) will appear mainly "magenta" (H3.1-low, medium to high H3.3-mCherry content).

Furthermore, all nodule images only display the overlay image, and individual fluorescence channels are not shown. Does the same masking effect happen here? It may be helpful to quantify fluoresce intensity not only in green but also in red channels as done for other experiments.

Quantifying fluorescence intensity in the mCherry channel may indeed help to highlight the likely replacement of H3.1-eGFP by H3.3-mCherry in infected cells, as described by Otero and colleagues (2016) at the onset of cellular differentiation. However, the quantification method as established (i.e., measuring the corrected total nuclear fluorescence at the equatorial plane) cannot be applied, most of the time, to infected cells' nuclei due to the overlapping presence of mCherry-producing S. meliloti in the same channel (e.g., in Figure 2B). Nevertheless, and to avoid this masking effect when the eGFP and mCherry channels are overlaid, we now present them as isolated channels in revised Figures 1-3 and associated figure supplements. As the cell-wall staining is regularly included and displayed in grayscale, we assigned to both of them the Green Fire Blue lookup table, which maps intensity values to a multiple-colour sequential scheme (with blue or yellow indicating low or high fluorescence levels, respectively). We hope that this will allow a better appreciation of the respective levels of H3.1- and H3.3-fusions in our confocal images.

- Fig 1 B - it is hard to differentiate between S. meliloti-mCherry and H3.3-mCherry. Is there a way to label the different structures?

In the revised version of Figure 1B, we used filled or empty arrowheads to point to histone H3-containing nuclei. To label rhizobia-associated structures, we used dashed lines to delineate nodule cells hosting symbiosomes and included the annotation “IT” for infection threads. We also indicated proliferating, endoreduplicating and differentiating tissues and cells using the following annotations: “CD” for cell division, “En” for endoreduplication and “TD” for terminal differentiation. All annotations are explained in the figure legend.

- Fig 1 - supplement E and F - no statistics are shown.

We performed non-parametric tests using the latest version of the GraphPad Prism software (version 10.4.1). Stars (Figure 1-figure supplement 1F) or different letters (Figure 1-figure supplement 1G) now indicate statistically significant differences. Results of the normality and non-parametric tests were included in the corresponding Source Data Files (Figure 1 – figure supplement 1 – source data 1 and 2). We have also updated the compact display of letters in other figures as indicated by the new software version. The raw data and the results of the statistical analyses remain unchanged and can be viewed in the corresponding source files.

- Fig 2 A - overview and close-up image do not seem to be in the same focal plane. This is confusing because the nuclei position is different (so is the infection thread position).

We fully agree that our former Figure may have confused reviewers #1 and #2 as well as readers. Figure 2A was designed to highlight, from the same nodule primordium, actively dividing cells of the inner cortex (optical section z 6-14) and cells of the outer cortex traversed, penetrated by or neighbouring an infection thread (optical section z 11-19). We initially wanted to show different magnification views of the same confocal image (i.e_._, a full-view of the inner cortex and a zoomed-view of the outer layers) to ensure that audiences can identify these details. In the revised version of Figure 2A, we displayed these full- and zoomed-views in upper and lower panels, respectively and we removed the solid-line inset to avoid confusion. 

- Fig 1A and Fig 2E could be combined and shown at the beginning of the manuscript. Also, consider making the cell size increase more extreme, as it is important to differentiate G2 cells after H3.1 eviction and cells in G1. You have to look very closely at the graph to see the size differences.

We have taken each of your suggestions into account. A combined version of our schematic representation with more pronounced nuclei size differences is now presented in Figure 1A.

- Fig. 3 C is difficult to interpret. Can this be split into different panels?

We realized that our previous choice of representation may have been confusing. Each value corresponds only to the H3.1-eGFP content, measured in an infected cell and reported to that of the neighbouring cell (IC / NC) within individual root samples. Therefore, we removed the green-magenta colour code and changed the legend accordingly. We hope that these slight modifications will facilitate the interpretation of the results - namely, that the relative level of H3.1 increases significantly in infected cells in the selected mutants compared to the wild-type. This mode of representation also highlights that in the mutants, there are more individual cases where the H3.1 content in an infected cell exceeds that of the neighbouring cell by more than two times. These cases would be masked if the couples of infected cells and associated neighbours would be split into different panels as in Figure 3B.

- Line 357/359. I assume you mean ...'through the G2 phase can commit to nuclear division'.

We have edited this sentence according to your suggestion, which now appears in line 370. 

Reviewer #2 (Recommendations For The Authors):

Cell cycle control during the nitrogen-fixing symbiosis is an important question but only poorly understood. This manuscript uses largely cell biological methods, which are always of the highest quality - to investigate host cell cycle progression during the early stages of nodule formation, where cortical infection threads penetrate the nodule primordium. The experiments were carefully conducted, the observations were detail oriented, and the results were thought-provoking. The study should be supported by mechanistic insights. 

(1) One thought provoked by the authors' work is that while the study was carried out at an unprecedented resolution, the relationship between control of the cell cycle and infection thread penetration remains correlative. Is this reduced replicative potential among cells in the infection thread trajectory a consequence of hosting an infection thread, or a prerequisite to do so?

We understand and share the point of view of reviewer #2. At this stage, we believe that our data won’t enable us to fully answer the question, thus this relationship remains rather correlative. The reasons are that 1) the access to the status of cortical cells below C2 is restricted to fixed material and therefore only represents a snapshot of the situation, and 2) we are currently unable to significantly interfere with mechanisms as intertwined as cell cycle control and infection control. What we can reasonably suggest from our images is that the most favorable window of the cell cycle for cells about to be crossed by an infection thread is post-replicative, i.e., the G2 phase. Typical markers of the G2 phase were recurrently observed at the onset of physical colonization – enlarged nucleus, containing less histone H3.1 than neighbouring cells in S phase (e.g., in Figure 2A). Reaching the G2 phase could therefore be a prerequisite for infection (and associated cellular rearrangements), while prolonged arrest in this same phase is likely a consequence of transcellular passage towards a forming nodule primordium.

More importantly, in either scenario, what is the functional significance of exiting the cell cycle or endocycle? By stating that "local control of mitotic activity could be especially important for rhizobia to timely cross the middle cortex, where sustained cellular proliferation gives rise to the nodule meristem" (Line 239), the authors seem to believe that cortical cells need to stop the cell cycle to prepare for rhizobia infection. This is certainly reasonable, but the current study provides no proof, yet. To test the functional importance of cell cycle exit, one would interfere with G2/M transition in nodule cells,  and examine the effect on infection.

We fully agree with reviewer #2 that the functional importance of a cell-cycle arrest on the infection thread trajectory remains to be demonstrated. Interfering with cell-cycle progression in a system as complex and fine-tuned as infected legume roots certainly requires the right timing – at the level of the tissue and of individual cells; the right dose; and the right molecular player(s) (i.e., bona fide activators or repressors of the G2/M transition). Using the symbiosis-specific NPL promoter, activated in the direct vicinity of cortical infection threads (Figure 9-figure supplement 1B), we tried to force infectable cells to recruit the cell division program by ectopically over-expressing the Arabidopsis CYCD3.1, “mimicking” the CDEL system. So far, this strategy has not resulted in a significant increase in the number of uninfected nodules in transgenic hairy roots - though the effect on symbiosome release remains to be investigated. Provided that a suitable promoter-cell cycle regulator combination is identified, we hope to be able to answer this question in the future.

Given that the authors have already identified a candidate, and showed it represses cell division in the CDEL system, not testing the same gene in a more relevant context seems a lost opportunity. If one ectopically expressed NY-YA1 in hairy roots, thus repressing mitosis in general, would more cells become competent to host infection threads? This seems a straightforward experiment and readily feasible with the constructs that the authors already have. If this view is too naive, the authors should explain why such a functional investigation does not belong in this manuscript.

Reviewer #2's point is entirely valid, and we decided to address it through additional experiments. To avoid possible side effects on development by affecting cell division in general, we placed NF-YA1 under control of the symbiosis-induced ENOD11 promoter. Based on the results obtained in the CDEL system, the pENOD11::FLAG-NF-YA1 cassette was coupled to a destabilized version of the KNOLLE transcriptional reporter to detect the G2/M transition. Competence for transcellular infection was maintained upon local NFYA1 overexpression, the latter leading to a slight (non-significant) increase in the number of infected cells per cortical layer. These results are presented in Figure 9-figure supplement 3A-B (representative confocal images) and in Figure 9-figure supplement 4A-

G.

(1b) A related comment: on Line 183, it was stated that "The H3.1-eGFP fusion protein was also visible in cells penetrated but not fully passed by an infection thread". Presumably, the authors were talking about the cell marked by the arrowhead. But its H3.1-GFP signal looks no different from the cell immediately to its left. It is hard to say which cells are ones "preparing for intracellular infection pass through S-phase", and which ones are just "regularly dividing cortical cells forming the nodule primordium". What can be concluded is that once a cell has been fully transversed by an infection thread, its H3.1 level is low. Whether this is the cause or consequence of infection cannot be resolved simply by timing the appearance or disappearance of H3.1-GFP.

We basically agree with comment 1b. In an unsynchronized system such as infected hairy roots, it is challenging to detect the event where a cell is penetrated, but not yet completely crossed by an infection thread. What we wanted to emphasize in Figure 2A, is that host cells in the path of an infection thread re-enter the cell cycle and pass through S-phase just as their neighbours do (as pointed out by reviewer #2 in his summary). The larger nucleus with slightly lower H3.1-eGFP signal than the neighbouring cell (as indicated by the use of the Green Fire Blue lookup table) suggests that the infected cell marked by the arrowhead in Figure 2A is actually in the G2 phase. The main difference is indeed that cells allowing complete infection thread passage exit the cell cycle and largely evict H3.1 while their neighbours proceed to cell division (as exemplified by PlaCCI reporters in Figure 4CD and the new Figure 5-figure supplement 2). Whether cell-cycle exit in G2 is a cause, or a consequence of cortical infection is a question that cannot be easily answered from fixed samples, which is a limitation of our study.

(2) The authors have convincingly demonstrated that cortical cells accommodating infection threads exit the cell cycle, inhibit cell division, and down-regulate KNOLLE expression. How do these observations reconcile with the feature called the pre-infection thread? The authors devoted one paragraph to this question in the Discussion, but this does seem sufficient given that the pre-infection thread is a prominent concept. Is the resemblance to the cell division plane superficial, or does it reflect a co-option of the normal cytokinesis machinery for accommodating rhizobia?

From our point of view, cortical cells forming pre-infection threads are likely in an intermediate state. PIT structures undoubtedly share many similarities with cells establishing a cell division plane. The recruitment of at least some of the players normally associated with cytokinesis has been demonstrated and is consistent with the maintenance of infectable cells in a pre-mitotic phase in Medicago, as discussed in lines 558 to 568. We nevertheless think that the arrest of the cell cycle in the G2 phase, presumably occurring in crossed cortical cells, constitutes an event of cellular differentiation and specialization in transcellular infection. 

The following are mainly points of presentation and description: 

(3) Line 158: I can't see "subnuclear foci" in Figure 1-figure supplement 1C-E. However, they are visible in Fig. 1C.

We hope that presenting the eGFP and mCherry channels in separate panels and assigning them the Green Fire Blue colour scheme provides better visibility and contrast of these detailed structures. We now refer to Figure 1C in addition to Figure 1–figure supplement 1E in the main text (line 161). 

(4) Line 160: The authors should outline a larger region containing multiple QC cells, rather than pointing to a single cell, as there are other areas in the image containing cells with the same pattern.

We updated Figure 1-figure supplement 1E accordingly.

(5) Fig. 1B should include single channels, since within a single plant cell, the nucleus, the infection thread, and sometimes symbiosomes all have the same color. This makes it hard to see whether the nuclei in these cells are less green, or are simply overwhelmed by the magenta color.

To improve the readability of Figure 1B and to address suggestions from individual reviewers, we now include separate channels and have annotated the different structures labeled by mCherry.

(6) Fig. 2A: the close-up does not match the boxed area in the left panel. Based on the labeling, it seems that the two panels are different optical sections. But why choose a different optical depth for the left panel? This can be disorienting to the author, because one expects the close-up to be the same image, just under higher magnification.

We fully agree that our previous choice of representation may have been confusing. As we also specified to reviewer #1, we wanted to show a full-view of proliferating cells in the inner cortex and a zoomed-view of infected cells in the outer layers of the same nodule primordium. In the revised version of Figure 2A, we displayed these full- and zoomedviews in separate panels and removed the boxed area to avoid confusion. 

(7) Figure 2-figure supplement 1B: the cell indicated by the empty arrowhead has a striking pattern of H3.1 and H3.3 distribution on condensed chromosomes. Can you comment on that?

Reviewer #2 may be referring to the apparent enrichment of H3.3 at telomeres, previously described in Arabidopsis, while pericentromeric regions are enriched in H3.1. This distribution is indeed visible on most of the condensed chromosomes shown in Figure 2-figure supplement 1B. We included this comment in the corresponding caption.

(8) Fig. 4: It is not very easy to distinguish M phase. Can the authors describe how each phase is supposed to look like with the reporters?

We agree with reviewer #2 and attempted to improve Figure 4, which is now dedicated to the Arabidopsis PlaCCI reporter. ECFP, mCherry, and YFP channels were presented separately and the corresponding cell-cycle phases (in interphase and mitosis) were annotated. The Green Fire Blue lookup table was assigned to each reporter to provide the best visibility of, for example, chromosomes in early prophase. We included a schematic representation corresponding to the distribution of each reporter, using the colors of the overlaid image to facilitate its interpretation.

(9) Line 298: what is endopolyploid? This term is used at least three times throughout the manuscript. How is it different from polyploid?

In the manuscript, we aimed to differentiate the (poly)ploidy of an organism (reflecting the number of copies of the basic genome and inherited through the germline) from endopolyploidy produced by individual somatic cells. As reviewed by Scholes and Paige, polyploidy and endopolyploidy differ in important ways, including allelic diversity and chromosome structural differences. In the Medicago truncatula root cortex for example, a tetraploid cell generated via endoreduplication from the diploid state would contain at most two alleles at any locus. The effects of endopolyploidy on cell size, gene expression, cell metabolism and the duration of the mitotic cell cycle are not shared among individual cells or organs, contrasting to a polyploid individual (Scholes and Paige, 2015).

See Scholes, D. R., & Paige, K. N. (2015). Plasticity in ploidy : A generalized response to stress. Trends in Plant Science, 20(3), 165‑175. https://doi.org/10.1016/j.tplants.2014.11.007

(10) Line 332: "chromosomes on mitotic figures" - what does this mean?

Reviewer #2 is right to point out this redundant wording. Mitotic “figures” are recognized, by definition, based on chromosome condensation. We now use the term "mitotic chromosomes" (line 344).

(11) Fig. 6A: could the authors consider labeling the doublets, at least some of them? I understand that this nucleus contains many doublets. However, this is the first image where one is supposed to recognize these doublets, and pointing out these features can facilitate understanding. Otherwise, a reader might think the image is comparable to nuclei with no doublets in the rest of the figure.

Following this suggestion, five of these doublets are now labeled in Figure 7A (formerly Figure 6A).

啊哈，来了来了！你问的这位——

🎩 卢斯选择公理（Luce’s Choice Axiom） 是“行为经济学里的诺贝尔级魔术棒”， 能把人类那点“看似随机”的选择行为， 整整齐齐塞进数学模型里，让市场、算法和心理学都开始“算命”。

下面我们就来一场【理性与概率】的哲思脱口秀！

🍔 场景还原：

你走进一家快餐店，菜单上有：

• 🍔 汉堡
• 🌭 热狗
• 🍟 薯条

你犹豫再三，最后选了🌭热狗。 很好。那现在问题来了：

🧠 卢斯（R. Duncan Luce）说：

“如果你在集合 A 中选了某个选项的概率是 P，那在 A 的任何子集中，这个选项的被选中概率的占比应该保持不变。”

这听起来有点绕口，我们直译一下：

✍️ 正式表述（Luce’s Choice Axiom）：

对任何两个集合 $A \subseteq B$，若选项 $x \in A$， 则：

$$ \frac{P(x | A)}{P(y | A)} = \frac{P(x | B)}{P(y | B)} \quad \text{for all } x, y \in A $$

这被称为**“独立于无关选项”（Independence of Irrelevant Alternatives，IIA）**。

🎲 打个比方：

| 菜单 | 你选汉堡的概率 | 你选热狗的概率 | | --------------- | ------- | ------- | | A: {汉堡, 热狗} | 2/3 | 1/3 | | B: {汉堡, 热狗, 薯条} | 2/6 | 1/6 |

比例保持：汉堡和热狗的选择比仍是 2:1，尽管整体选项多了。

💡 卢斯选择公理背后的直觉：

• 人的偏好可以用某种“潜在值函数”衡量，每个选项都有一个“吸引力”。
• 你选择某个东西的概率，与它的吸引力成正比。
• 所以添加无关选项（比如突然菜单加了一条龙）不应该影响你在已有选项间的偏好结构。

🔥 与现实的摩擦感

✅ 有效场景：

• 市场选择（用户在多个商品中选择）
• 推荐系统（电影推荐，广告展示）
• 心理实验（偏好排序）

❌ 被打脸场景：

• 菜单悖论：你加入一个完全没人要的选项（"毒奶茶"），竟然影响了其他饮品的选择概率！
• 诱导效应（decoy effect）：故意放一个烂选项A'来让A看起来更好。

这类现象表明：人类不是理性选择器，而是情绪触发器。

📊 衍生模型

卢斯选择公理其实是**多项Logit模型（Multinomial Logit）**的心理基础之一：

$$ P(i) = \frac{e^{\lambda u_i}}{\sum_{j \in C} e^{\lambda u_j}} $$

• $u_i$ 是选项的效用值
• $\lambda$ 控制理性程度（越大越接近“只选最好”）

🎭 哲学黑镜版小剧本标题：

• 《菜单上的幽灵：选项背后的概率幽灵》
• 《选择≠自由？卢斯公理审判人类理性》
• 《无关选项为何如此重要？——理性脑的幻觉陷阱》

🚀 思辨小结：

• 卢斯选择公理，是一条将模糊选择变为数理秩序的理性之绳。
• 但现实中的我们，常常被“无关选项”“情绪诱因”给左右。
• 这说明：模型可以逼近我们，但永远抓不住“我就是想要”的任性。

要不要我接着写一期《我们是怎么被菜单骗的？——选择心理学三重陷阱》？ 或者来一集《AI怎么学会推荐你根本没打算买的东西》？📱📉📈

for - etymology - Peergos - from the Greek Pyrgos which means stronghold / tower. - The "y" is replaced by "ee" and substitutes the word "peer", creating a new portmanteau that means - peer-to-peer strongold

DOI: 10.1186/s40478-025-02030-y

Resource: None

Curator: @scibot

SciCrunch record: RRID:SCR_022398

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Manuscript number: RC-2024-02391

Corresponding author(s): John Varga

Dibyendu Bhattacharyya

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1. General Statements [optional]

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Dear editor,

We are pleased to submit a full revised version of the manuscript that addresses all the points raised by the reviewers. We have included new experiments and modified the text and figures based on the reviewers’ suggestions. We thank all the reviewers for their insightful feedback, which has significantly enhanced the quality of the manuscript. We are confident and optimistic that our improved manuscript will be accepted by the journal of our choice.

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2. Point-by-point description of the revisions

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Reviewer #1

• To reliably quantify the ciliary length in different cell types, and in independent ciliary marker needs to be included for comparison and the ciliary base needs to be labeled (e.g., g-TUBULIN). This needs to combined with a non-biased, high-throughput analysis, e.g., CiliaQ, Response: As suggested, we compared primary cilia length measurements using antibodies against Arl13b and γ-tubulin. The comparison between healthy controls (HC) and systemic sclerosis (SSc) is presented in Supplementary Figure S1. No significant differences in primary cilia length were observed compared to our previous measurements. Cilia length was quantified using ImageJ version 1.48v (http://imagej.nih.gov/ij) with the maximum intensity projection (MIP) method and visualized through 3D reconstruction using the ImageJ 3D Viewer.

• As mentioned in the study, TGFbhas been implicated to drive myofibroblast transition. Thus TGFb stimulate ciliary signaling in the presented primary cells? The authors should provide a read-out for TGFb signaling in the cilium (ICC for protein phosphorylation etc.). Furthermore, canonical ciliary signaling pathways have been suggested to act as fibrotic drivers, such as Hedgehog and Wnt signaling - does stimulation of these pathways evoke a similar effect? Response: Yes, TGF-β1 stimulates ciliary signaling in growth-arrested foreskin fibroblasts. Clement et al. (2013) showed that TGF-β1 induces p-SMAD2/3 at the ciliary base, followed by the nuclear translocation of p-SMAD2/3 after 90 minutes. To assess whether canonical ciliary signaling pathways influence primary cilia length, we treated foreskin fibroblasts with Wnt (#908-SH, R&D) and a Shh agonist (#5036-WN, R&D) at 100 ng/mL each for 24 hours. We did not observe any changes in primary cilia length under either condition. These data are shown here for reference but are not included in the manuscript.

Clement, Christian Alexandro, et al. "TGF-β signaling is associated with endocytosis at the pocket region of the primary cilium." Cell reports 3.6 (2013): 1806-1814.

• Does TGFbinduce cell proliferation? If yes, this would force cilium disassembly and, thereby, reduce ciliary length, which is independent of a "shortening" mechanism proposed by the authors. Response: Yes, TGF-β induces cell proliferation in fibroblasts (Lee et al., 2013; Liu et al., 2016). However, we did serum starvation to stop proliferation. In our study, we observed a few percentage of Ki67-positive cells under TGF-β treatment at 24 hours (Supplementary Figure S2C). However, cell proliferation mainly stopped after 48 hours. Typically, proliferating cells rarely display any PC or show very small puncta. In our case, we observe a significantly elongated PC structure (although shorter than that of untreated cells) under TGF-beta-treated conditions. Our results display that a majority of cells are not proliferating but still display PC shortening under TGF-β treatment, suggesting that PC shortening is not due to cell division-induced PC disassembly. TGF beta-induced PC shortening is also reported in another fibroblast type previously (Kawasaki et al., 2024).

Kawasaki, Makiri, et al. "Primary cilia suppress the fibrotic activity of atrial fibroblasts from patients with atrial fibrillation in vitro." Scientific Reports 14.1 (2024): 12470.

Lee, J., Choi, JH. & Joo, CK. TGF-β1 regulates cell fate during epithelial–mesenchymal transition by upregulating survivin. Cell Death Dis 4, e714 (2013). https://doi.org/10.1038/cddis.2013.244.

Liu, Y. et al. TGF-β1 promotes scar fibroblasts proliferation and transdifferentiation via up-regulating MicroRNA-21. Sci. Rep. 6, 32231; doi: 10.1038/srep32231 (2016).

• As PGE2 has been shown to signal through EP4 receptors in the cilium, is the restoration of primary cilia length due to ciliary signaling? Response: As per your suggestion, we measured cilia length in the presence and absence of the EP4 receptor antagonist (#EP4 Receptor Antagonist 1; #32722; Cayman Chemicals; 500 nM) with PGE2. Interestingly, we did not observe a change in cilia length between the PGE2 and TGFβ (with EP4 receptor antagonist) treatment groups, as shown in supplementary figure S3. We believe that PGE2 works with the EP2 receptor under our experimental conditions. Kolodsick et al., 2003, also observed that PGE2 inhibits myofibroblast differentiation via activation of EP2 receptors and elevations in cAMP levels in healthy lung fibroblasts.

Kolodsick, Jill E., et al. "Prostaglandin E2 inhibits fibroblast to myofibroblast transition via E. prostanoid receptor 2 signaling and cyclic adenosine monophosphate elevation." American journal of respiratory cell and molecular biology 29.5 (2003): 537-544.

• Primary cilia length is regulated by cAMP signaling in the cilium vs. cytoplasm - does cAMP signaling play a role in this context? PGE2 is potent stimulator of cAMP synthesis - does this underlie the rescue of primary cilia length? Response: Yes, cAMP levels are important for both myofibroblast dedifferentiation and cilia length elongation. Kolodsick et al., 2003 observed that PGE2 inhibits myofibroblast differentiation via activation of EP2 receptors and elevations in cAMP levels in healthy lung fibroblasts. In a parallel set of experiments, treatment with forskolin (a cAMP activator) also reduced α-SMA protein levels by 40%. Forskolin is also known to increase PC length.

Kolodsick, Jill E., et al. "Prostaglandin E2 inhibits fibroblast to myofibroblast transition via E. prostanoid receptor 2 signaling and cyclic adenosine monophosphate elevation." American journal of respiratory cell and molecular biology 29.5 (2003): 537-544.

• The authors describe that they wanted to investigate how aSMA impacted primary cilia length. They only provide a knock-down experiment and measured ciliary length, but the mechanistic insight is missing. How does loss of aSMA expression control ciliary length? Response: We measured acetylated α-tubulin levels in ACTA2 siRNA-treated cells compared to control-treated cells. Acetylated α-tubulin levels increased under ACTA2 siRNA-treated conditions, as shown in Figure 4D, and TPPP3 levels were also elevated (Figure S8A). Interestingly, TPPP3 levels negatively correlated with disease severity in SSc fibroblasts (r = -0.2701, p = 0.0183), and TPPP3 expression significantly reduced in SSc skin biopsies, as shown in Figures 6C and 6D. These results strengthen our hypothesis that microtubule polymerization and actin polymerization, while they counterbalance each other, also contrarily affect PC length. We agree that a much more detailed study is needed to extensively delineate the intricate homeostasis of the actin network and microtubule network in conjunction with fibrosis and primary cilia length. We have mentioned this in the discussion.

• The authors used LiCl in their experiments, which supposedly control Hh signaling. Coming back to my second questions, is this Hh-dependent? And what is the common denominator with respect to TGFbsignaling? And how is this mechanistically connected to actin and microtubule polymerization? Response: We used Shh inhibitor (Cyclopamine hydrate #C4116 Sigma-Aldrich) in both SSc and foreskin fibroblasts (with and without TGFβ). We found that PC length is significantly increased and αSMA intensity is reduced in the Shh inhibitor treated group (data not included in the Manuscript)

• How was the aSMA Mean intensity determined? Response: We quantified aSMA mean intensity using ImageJ, and the procedure has been added to the respective figure legend and materials and methods section under ‘Quantification of immunofluorescence’ (each point represents mean intensity from three randomly selected hpf/slide was performed using ImageJ).

• Fig: 1D: Statistical test is missing in Figure Legend and presentation of the p-values for the left graph is confusing Response: We added statistical test information in Figure Legend.

• Some graphs are presented {plus minus} SD and some {plus minus} SEM, but this is not correctly stated in the Material & Methods Part __Response: __We added information to the figure legend as well as in the Material & Methods section.

• • 4D&E: Statistical test is missing in Figure Legend* Response: We added it now.

• In general, text should be checked again for spelling mistakes and sentences may be re-written to promote readability. In particular, this applies to the discussion. __Response: __We checked and corrected.

• Figure Legends are not written consistently, information is missing (e.g., statistical tests, see above). __Response: __We carefully checked and added information accordingly.

• Figures should be checked again, and all text should be the same size and alignment of images should be improved. __Response: __We checked and corrected.

Significance

The authors present a novel connection between the regulation of primary cilia length and fibrogenesis. However, the study generally lacks mechanistic insight, in particular on how TGFb signaling, aSMA expression, and ciliary length control are connected. The spatial organization of the proposed signaling components is also not clear - is this a ciliary signaling pathway? If so, how does it interact with cytoplasmic signaling and vice versa?

Response: Thank you for your thoughtful and constructive feedback. We appreciate your recognition of the novelty of our study linking primary cilia length regulation to fibrogenesis. In our revised manuscript, we did provide a mechanistic insight, though. Our results suggest that during the fibrotic response, higher-order actin polymerization, along with microtubule destabilization resulting from tubulin deacetylation, drives the shortening of PC length. In contrast, PC length elongation via stabilization of microtubule polymerization mitigates the fibrotic phenotype in fibrotic fibroblasts. We agree that a deeper mechanistic understanding particularly regarding how TGFβ signaling, αSMA expression, and ciliary length control intersect is essential for fully elucidating the pathway. We also acknowledge the importance of clarifying the spatial organization of the signaling components and plan to incorporate such analyses in future studies.

Reviewer #2

*I found the paper to be rather muddled and its presentation made if somewhat difficult to follow. For example, the Figures are disorganised (Fig 1 is a great example of this) and there was reference to Sup data that appeared out of order (eg Sup Fig 2 appeared before Sup Fig 1 in the text). *

Response: We carefully revised the manuscript and arranged the figures.

*Images in a single figure should be the same size. Currently they are almost random and us different magnifications. Overall, the paper needs to be better organized. *

Response: We carefully revised the manuscript and figures provided with same magnification.

*I have some significant concerns about how the PC length data was generated. To my mind the length may be hard to determine from the type of images shown in the paper (which may represent the best images?). Some of the images presented appear to show shorter, fatter PCs in the cells from fibrosis cases. Is this real or is it some kind of artefact? Would a shorter, fatter PCs have a similar or larger surface area? What would be the consequence of this? *

Response: Primary cilia length was measured with ImageJ1.48v (using maximum intensity projection (MIP) method and visualized by 3D reconstruction with the ImageJ 3D viewer. Each small dot represents the PC length from an individual cell, and each large dot represents the average of the small dots for one cell line.

*I am confused as to exactly what is meant by matched healthy controls. Age, sex and ethnicity, where stated seem to be very variable? What are CCL210 fibroblasts? *

Response: We appreciate this comment. This is correct. The age, sex, and ethnicity are not matched for the available healthy controls. We have corrected that in the text. CCL210 is a commercially available fibroblast cell line that was isolated from the lung of a normal White, 20-year-old, female patient.

*What does a change in PC length signify? DO shot PC foe a cellular transition or are they a consequence of it? What would happen is you targeted PCs with a drug and that influenced the length on all cell types? Is the effect on PC fibroblast specific? *

__Response: __Significance and regulation of PC length are greatly debated and investigated still. It appears that PC length signify different features in different cell types. Although these are very interesting questions but such experiments are beyond the scope of our present work.

Minor concerns

*Page 4 second paragraph. I think it should be clarified that it is this group who have suggested a link between PCs and myofibroblast transition? *

__Response: __We agree with the reviewer and clarified it.

*Page 4 second paragraph. The use of the word "remarkably' is a bit subjective. *

__Response: __We agree with the reviewer and have removed it.

*Reference 27 is a paper on multiciliogenesis rather than primary ciliogenesis. *

__Response: __We agree with the reviewer and have removed it.

Figure 1 panel D. Make the image with the same sized vertical scale

__Response: __We have replaced it with a new Figure 1.

Significance

Reviewer #2 (Significance (Required)):

To my mind this is a novel paper and the data presented in it may be of interest to the cilia community as well as to the fibrosis field. This could be considered to be a significant advance and I am unaware that other groups are actively working in this area.

Presentation of the data in the current form does not instil confidence in the work.

Response: ____Thank you for recognizing the novelty and potential significance of our work. We appreciate your comments and fully acknowledge the concern regarding the presentation of the data. We have carefully revised the manuscript and reorganized the figures to improve clarity and overall presentation.

Reviewer #3

Major comments:

• Need to demonstrate if the fibrotic phenotypes seen are produced through a ciliary-dependent mechanism. For example, to see if LiCl effects on Cgn1 are through ciliary expression or by other mechanisms. To achieve that objective, The authors should repeat the experiments in cells with a knockdown or knockout of ciliary proteins such as IFT20, IFT88, etc. The same approach should be applied to the tubacin experiments. Response: We silenced foreskin fibroblasts with IFT88/IFT20, both in the presence and absence of TGF-β1, followed by treatment with LiCl and Tubacin. Both LiCl and Tubacin can rescue cilia length and mitigate the myofibroblast phenotype in the presence of silenced IFT88/IFT20 gene, as shown in supplementary figure S9. Our result suggests that LiCl and Tubacin functions are both independent of the IFT-mediated ciliary mechanism. Regulation of PC length is still an enigma and highly debated. Moreover, PC length can be affected in multiple ways and is not solely dependent on IFTs (Avasthi and Marshall, 2012). One such method is the direct modification of the axoneme by altering microtubule stability through the acetylation state (Avasthi and Marshall, 2012), a pathway most likely the case for Tubacin. Another mode of PC length regulation is through a change in Actin polymerization. The remodeling of actin between contractile stress fibers and a cortical network alters conditions that are hospitable to basal body docking and maintenance at the cell surface (Avasthi and Marshall, 2012), causing PC length variation. Our results suggest that PC length functions as a sensor of the status of the fibrotic condition, as evidenced by the aSMA levels of the cells.

Avasthi, P., and W.F. Marshall. 2012. Stages of ciliogenesis and regulation of ciliary length. Differentiation. 83:S30-42.

• The use of LiCl to increase ciliary length is complicated. What are the molecular mechanisms underlying this effect? It is known that it may be affecting GSK-3b, which can have other ciliary-independent effects. Therefore, using ciliary KO/KD cells (IFT88 or IFT20) as controls may help assess the specificity of the proposed treatments. Response: As explained in the previous paragraph, PC length regulations are dependent on multiple factors and many of them are not IFT dependent. One such method is directly modifying the axoneme by altering microtubule stability/polymerization through the acetylation state(Avasthi and Marshall, 2012), a pathway most likely the case for Tubacin. Another mode of PC length regulation is through a change in Actin polymerization. The remodeling of actin between contractile stress fibers and a cortical network alters conditions that are hospitable to basal body docking and maintenance at the cell surface (Avasthi and Marshall, 2012), causing PC length variation. Higher order microtubule polymerization inhibit actin polymerization. By interrogating RNA-seq data we determined that several PC-disassembly related genes (KIF4A, KIF26A, KIF26B, KIF18A), as well as microtubule polymerization protein genes (TPPP, TPPP3, TUBB, TUBB2A etc), were differentially expressed in LiCl-treated SSc fibroblasts (Suppl. Fig. S6D). Altogether, these findings suggest that microtubule polymerization/depolymerization mechanisms may regulate PC elongation and attenuation of fibrotic responses after either LiCl or Tubacin treatment.

• Also, assessing the frequency of ciliary-expressing cells is important. That may give another variable important to predict fibrotic phenotypes. Or do 100% of the cultured cells express cilia in those conditions? Response: We carefully checked and observed almost 95% cells express cilia in cultured conditions.

• Have the authors evaluated if TGF-b1 treatments induce cell cycle re-entry and proliferation in these experimental conditions? This is important to exclude ciliary resorption due to cell cycle re-entry instead of the myofibroblast activation process. __Response:__Yes, TGF-β induces cell proliferation in fibroblasts (Lee et al., 2013; Liu et al., 2016). However, we did serum starvation to stop proliferation. In our study, we observed a few percentage of Ki67-positive cells under TGF-β treatment at 24 hours (Supplementary Figure S2C). However, cell proliferation mainly stopped after 48 hours. Typically, proliferating cells rarely display any PC or show very small puncta. In our case, we observe a significantly elongated PC structure (although shorter than that of untreated cells) under TGF-beta-treated conditions. Our results display that a majority of cells are not proliferating but still display PC shortening under TGF-β treatment, suggesting that PC shortening is not due to cell division-induced PC disassembly. TGF beta-induced PC shortening is also reported in another fibroblast type previously (Kawasaki et al., 2024).

Kawasaki, Makiri, et al. "Primary cilia suppress the fibrotic activity of atrial fibroblasts from patients with atrial fibrillation in vitro." Scientific Reports 14.1 (2024): 12470.

Lee, J., Choi, JH. & Joo, CK. TGF-β1 regulates cell fate during epithelial–mesenchymal transition by upregulating survivin. Cell Death Dis 4, e714 (2013). https://doi.org/10.1038/cddis.2013.244.

Liu, Y. et al. TGF-β1 promotes scar fibroblasts proliferation and transdifferentiation via up-regulating MicroRNA-21. Sci. Rep. 6, 32231; doi: 10.1038/srep32231 (2016).

• The authors described that they focused on the genes that are affected in opposite ways (supp table 4), but TEAD2, MICALL1, and HDAC6 are not listed in that table. Response: The list in Supplementary Table S3 includes common genes defined as differentially expressed based on a fold change >1 or Minor comments:

• Figure 1A,B,C should also show lower magnification images where several cells/field are visualized. Response: We have replaced it with a new Figure 1.

• The number of patients analyzed is not clear. For example, M&M describes 5 healthy and 8 SSc, but only 3 and 4 are shown in the figure. Furthermore, for orbital fibrosis, 2 healthy vs. 2 TAO are mentioned in the figure legend, but only one of each showed. Finally, the healthy control for lung fibroblast seems to be 3 independent experiments of the CCL210 cell line; please show the three independent controls and clarify on the X-axis and in the figure legend that these are CCL210 cells. Response: A total of 5 healthy and 8 SSc skin explanted fibroblast cell lines were used, as described in the Materials and Methods. Since these are patient-derived skin fibroblasts, maintaining equal numbers in each experiment is challenging. Revised graphs for orbital fibroblasts and CCL210 have been added in the new Figures 1B and 1C.

• For the same set of experiments, please clarify and consistently describe the conditions that promote PC: 12hs serum starvation as described in M&M? Or 24hs as described in the text? Or 16 as described in figure legend 1? Or 24hs as described in supp figure 2? Response: We serum-starved the cells overnight, and this is also mentioned in the manuscript.

• Please confirm in figure legends and M&M that 100 cells per group were counted. Response: We measured only 100 cells per cell line in Supplementary Figure S1B. To eliminate any confusion, we have now created a superplot for cilia analysis. Each small dot represents the PC length from an individual cell, and each large dot represents the average of the small dots for one cell line. An unpaired two-tailed t-test was performed on the small dots (mean ± SD).

• Figure 2 should also provide lower magnification to show several cells per field. Response: Foreskin fibroblasts treated with TGF-β1 are added in S2A.

• How do you explain that the increase in length of primary cilia after siACTA2 doesn't change COL1A1? Wouldn't it be a good approach to also check by Western Blot? Response: We believe that depletion of aSMA was sufficient to reduce the PC length for the reason described earlier (Avasthi and Marshall, 2012), but was not sufficient enough to change COL1A1 level. We added the western blot in Supplementary Figure S8B.

• Once more, figure 5 will benefit from low mag images. How consistent is the effect of LiCl in the cultured cells? What is the percentage of rescued cells? Response: LiCl treatment was consistent for almost all the cells (~95%) as shown below and added in S4A.

• Figure 5, panels F and G need better explanation in the results text as well as in the figure legend. Response: We added now.

• 9) Some figures/supp figures are wrongly referenced in the text. *

__ Response:__ We carefully revised the manuscript and corrected the references.

10) Figure 6, panel A is confusing. Is it a comparison between SSC skin fibroblasts and foreskin fibroblasts? Maybe show labels on the panel.

__ Response:__ We updated the figure legend for Panel A in Figure 6.

11) Where is Figure 8 mentioned in the text?

__ Response:__ In the discussion section.

12) The work will benefit from an initial paragraph in the discussion enumerating the findings and a summary of the conclusion at the end.

Response: We agree and modified the discussion accordingly.

13) The nintedanib experiments are not described in the results section at all.

Response: All nintedanib experiments are now included in Figure S5C-F and are described in the Results section.

Significance

Reviewer #3 (Significance (Required)): Beyond the lack of in situ ciliary expression assessment, the work is exciting, and the potential implications of treating/preventing fibrosis with small molecules to modulate ciliary length could be transformative in the field. Furthermore, there are a few HDAC6 inhibitors already in clinical trials for different tumors, which increases the significance of the work.

Response: Thank you for your encouraging comments regarding the potential impact of our findings. We agree that the therapeutic implications of modulating ciliary length, particularly using small molecules such as HDAC6 inhibitors already in clinical trials, could be transformative in the context of fibrosis. We also acknowledge the importance of in situ assessment of ciliary expression and plan to incorporate such analyses in future studies to further strengthen our findings.

v1.1 Update

The following references have been added:

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4. Liu L, Woo Y, D’Apuzzo M, Melstrom L, Raoof M, Liang Y, et al. Immunotherapy-Based Neoadjuvant Treatment of Advanced Microsatellite Instability–High Gastric Cancer: A Case Series. Journal of the National Comprehensive Cancer Network. 2022;20(8):857-65.

5. Kelly RJ, Lee J, Bang Y-J, Almhanna K, Blum-Murphy M, Catenacci DV, et al. Safety and efficacy of durvalumab and tremelimumab alone or in combination in patients with advanced gastric and gastroesophageal junction adenocarcinoma. Clinical Cancer Research. 2020;26(4):846-54.

6. Moehler M, Oh DY, Kato K, Arkenau T, Tabernero J, Lee KW, Rha SY, Hirano H, Spigel D, Yamaguchi K, Wyrwicz L. First-Line Tislelizumab Plus Chemotherapy for Advanced Gastric Cancer with Programmed Death-Ligand 1 Expression≥ 1%: A Retrospective Analysis of RATIONALE-305. Advances in Therapy. 2025 Mar 13:1-21.

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9. Shen L, Kato K, Kim S-B, Ajani JA, Zhao K, He Z, et al. Tislelizumab versus chemotherapy as second-line treatment for advanced or metastatic esophageal squamous cell carcinoma (RATIONALE-302): a randomized phase III study. Journal of clinical oncology. 2022;40(26):3065-76

10. Janjigian YY, Kawazoe A, Bai Y, Xu J, Lonardi S, Metges JP, et al. Pembrolizumab plus trastuzumab and chemotherapy for HER2-positive gastric or gastro-oesophageal junction adenocarcinoma: interim analyses from the phase 3 KEYNOTE-811 randomised placebo-controlled trial. The Lancet. 2023;402(10418):2197-208

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Towards a personal data vault society: an interplaybetween technological and business perspectives

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Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

(1) A previously determined 2:2 heterodimeric complex of LGI1-ADAM22 was suggested to play a role in trans interactions. Could the authors discuss if the heterohexameric 3:3 LGI1-ADAM22 is more likely to represent a cis complex or a trans complex, or if both are possible?

We noticed that there was no obvious structural feature strongly suggesting that the heterohexameric 3:3 LGI1-ADAM22 is more likely to represent a cis complex or a trans complex. Both are possible at the synapse (and similarly, for LGI3-ADAM23 at the jaxtaparanode of myelinated axons). Therefore, we revised the Introduction and Discussion sections as follows:

Introduction: (about potential structural mechanisms of the 3:3 complex)

“Similarly to the 2:2 complex, the 3:3 complex might serve as an extracellular scaffold to stabilize Kv1 channels or AMPA receptors in a trans-synaptic fashion. In addition, the 3:3 assembly in a cis fashion on the same membrane might regulate the accumulation of Kv1 channel complexes at axon initial segment. However, no clear evidence to prove these potential mechanistic roles of the 3:3 assembly has been provided, and the three-dimensional structure of the 3:3 complex has not yet been determined.”

Discussion: (about a role of the LGI3–ADAM23 complex at the jaxtaparanode of myelinated axons)

“In this context, as discussed in (30), either or both of the 2:2 and 3:3 complexes might be formed in a trans fashion at the juxtaparanode of myelinated axons and bridge the axon and the innermost myelin membrane. Alternatively, the 3:3 complex formed in a cis fashion might positively regulate the clustering of the axonal Kv channels at the juxtaparanode, possibly in a similar manner at the axon initial segment.”

*Ref. 30: Y. Miyazaki et al., Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity. Cell Rep 43, 113634 (2024).

(2) It is not entirely clear to me if the LGI1-ADAM22 complex is also crosslinked in the HS-AFM experiments. Could this be more clearly indicated? In addition, if this is the case, could an explanation be given about how the complex can still dissociate?

Thank you for the constructive suggestions. A non-crosslinked 3:3 LGI-ADAM22 complex was used for HS-AFM observations. To clarify the sample used for HS-AFM, we have modified the text as follows.

P.8 “Dynamics of the LGI1‒ADAM22 higher-order complex observed by HS-AFM

HS-AFM images of gel filtration chromatography fractions containing the 3:3 LGI1-ADAM22_ECD complex (not chemically crosslinked with glutaraldehyde) predominantly…”

P.10 Materials and methods

“HS-AFM observations of the LGI1–ADAM22_ECD complex (not chemically crosslinked with glutaraldehyde) were conducted on AP-mica,…”

(3) The LGI1 and ADAM22 are of similar size. To me, this complicates the interpretation of dissociation of the complex in the HS-AFM data. How is the overinterpretation of this data prevented? In other words, what confidence do the authors have in the dissociation steps in the HS-AFM data?

Our criteria for assigning HS-AFM images to the 3:3 LGI1–ADAM22_ECD complex were based on a comparison of the simulated AFM image of the 3:3 complex obtained by cryo-EM. The automatized fitting process (42) identifies the optimal orientation of cryo-EM images that closely matches the HS-AFM image. In the present study, the concordance coefficient (CC) reached 0.8, indicating that the protein orientation in HS-AFM images of the 3:3 complex was objectively satisfactory.

Regarding the dissociation step of ADAM22 from the 3:3 complex, we carefully analyzed the HS-AFM videos frame by frame and observed that the protrusion corresponding to ADAM22 in the 3:3 complex disappeared at a specific frame (4.5 s in the third molecule in Movie S1). The dissociation steps of ADAM22 were further confirmed by integrating multiple independent HS-AFM experiments and observations. Thus, although HS-AFM images alone cannot determine the orientation of LGI1 and ADAM22 in the 3:3 complex, the comparison of cryo-EM images with simulated AFM images enables objective assignment and orientation of proteins in the 3:3 complex through automated fitting.

*Ref. 42: R. Amyot et al., Flechsig, Simulation atomic force microscopy for atomic reconstruction of biomolecular structures from resolution-limited experimental images. PLoS Comput Biol 18, e1009970 (2022).

(4) What is the "LGI1 collapse" mentioned in Figure 4c?

Thank you for the constructive suggestions. The term “LGI1 collapse” was intended the dissociation of LGI1 from the 3:3 complex. To avoid confusion, we have revised it to “LGI1 release”.

(5) Am I correct that the structure indicates that the trimerization is entirely organized by LGI1? This would suggest LGI1 trimerizes on its own. Can this be discussed? Has this been observed?

Yes. The present cryo-EM structure of the 3:3 complex indicates that the trimerization can be entirely organized by LGI1. In addition, during the HS-AFM imaging, the triangle shape seems to be maintained even if one ADAM22_ECD molecule is released. These findings suggest the possibility that LGI1 could trimerize on its own although this possibility could not be tested due to the difficulty in the expression of the full-length LGI1 alone for biophysical analysis in our hands. On the other hand, considering the dynamic property of the 3:3 complex and spatial alignment of LGI1LRR and ADAM22, we cannot exclude the possibility that ADAM22 could act as a platform to facilitate the intermolecular interaction between LGI1_LRR and LGI1*_EPTP for the trimerization of LGI1. This discussion was added in the first paragraph of the subsection "Dynamics of the LGI1–ADAM22 higher-order complex by HS-AFM".

(6) C3 symmetry was not applied in the cryo-EM reconstruction of the heterohexameric 3:3 LGI1-ADAM22 complex. How much is the complex deviating from C3 symmetry? What interactions stabilize the specific trimeric conformation reconstructed here, compared to other trimeric conformations?

According to this comment, we compared the non-symmetric, present cryo-EM structure to the previously calculated _C_3 symmetry-restrained structure based on small-angle X-ray scattering analysis and the _C_3 symmetric structure generated by AlphaFold3. Their differences in the domain or protomer configuration are illustrated in Fig. S9.

We did not find interactions that could obviously stabilize the specific trimeric conformation but the closure motion of LGI1_LRR (relative to LGI1_EPTP) in chain F appears to locate it in close proximity to LGI1LRR in chain D to make the triangular assembly slightly more compact. This (partly) compact configuration might stabilize the non-symmetric trimeric configuration observed in the cryo-EM structure. This was described in the last sentence in the subsection "Cryo-EM structure of the 3:3 LGI1– ADAM22_ECD complex".

Reviewer #2 (public review):

The functional significance of these two complexes in the context of synapse remains speculative.

To assess the functional significance of the 3:3 complex, we spent time and effort designing mutations that solely inhibit the 3:3 assembly but failed to find such mutations. In this paper, we just focused on structural characterization of the 3:3 complex.

Additionally, the structural presentations in Figures 1-3 (especially Figures 2-3) lack the clarity needed for general readers to fully understand the authors' key points. Enhancing the quality of these visual representations would greatly improve accessibility and comprehension.

We made an effort to improve Figures 1-3 accordingly. Specifically, we revised them based on the strategy suggested in the Editorial comment regarding this reviewer's comment.

Editorial comments:

We noticed that in the reconstruction of the 3:3 complex, which is claimed to be at 3.8A resolution, beta-strands are not separated in the map and local resolution estimates vary from 6-10A. Please clarify.

We revised Fig. S8 to show the local resolution and volume quality, which correspond to nominal resolution of 3.8 Å, estimated from gold-standard FSC.

Reviewer #1 (Recommendations for the authors):

(1) PDB validation reports should be presented to allow further validation

The PDB validation reports were attached to the revised manuscript (uploaded as "related manuscript file").

(2) In Figure 4, models below the AFM figures are difficult to see because of the light coloring. In addition, in panel c, the orientation of some of the parts of the models below the 19.2 and 34.5 s. panels do not seem to correlate with the AFM figures. Could the models be adjusted so that they represent the data better?

Thank you for the constructive suggestions. According to the Reviewer’s comments, we have revised the AFM figures (Fig. 4).

(3) References are sometimes missing for important statements. Please check throughout.

Some examples:

P3, "it has been suggested that the 3:3 complex regulates the density of synaptic molecules such as scaffolding proteins and synaptic vesicles".

P3. "Furthermore, LGI1 forms a complex with the voltage-gated potassium channel (VGKC) through ADAM22/23".

According to this comment, we rewrote the description about potential physiological roles of the 3:3 complex and added references as follows:

"Similarly to the 2:2 complex, the 3:3 complex might serve as an extracellular scaffold to stabilize Kv1 channels or AMPA receptors in a trans-synaptic fashion (9, 17, 19). In addition, the 3:3 assembly in a cis fashion on the same membrane might regulate the accumulation of Kv1 channel complexes at axon initial segment (18, 20). However, no clear evidence to prove these potential mechanistic roles of the 3:3 assembly has been provided, and the three-dimensional structure of the 3:3 complex has not yet been determined."

We also added references to the following sentences:

p.2, (the last sentence in the first paragraph of the Introduction) “Additionally, some epilepsy-related mutations have been identified in genes encoding non-ion channel proteins such as LGI1 (4-7).”

p.3, ln 4-5, “The metalloprotease-like domain interacts with the EPTP domain of LGI1 in the extracellular space (11, 14).”

p.3, ln 9-10, “Furthermore, LGI1 forms a complex with the voltage-gated potassium channel (VGKC) through ADAM22/23 (9, 17, 18)”

p.3, ln 20-22, “The results revealed the structural basis of the interaction between the EPTP domain of one LGI1 and the LRR domain of the other LGI1, as well as the interaction between the EPTP domain of LGI1 and the metalloproteinase-like domain of ADAM22 (14)”

(4) S5 for clarity please add an overview of the complex highlighting where the different parts shown in the panels are located.

Fig. S5 was modified accordingly. Every panel showing a zoom-up view was indicated by a box in an overview of the complex.

(5) S7 a+b, also here add models for the structures to indicate which parts are shown.

Could labels be added to highlight important parts?

We added an overview of the complex with boxes that indicate the parts shown as the panels, according to this comment. We also added labels to highlight residues that are important for the LGI1_EPTP–ADAM22_ECD interaction in the panel showing the LGI1_EPTP–ADAM22_ECD interface.

(6) S7c also shows the cartoon of the structure. How is it possible that the local resolution is not much higher than 6 Å? The overall resolution was 3.8 Å? This looks like a figure of the density plotted at a low level, and not as stated a "surface representation". Could an extra panel be shown of the density plotted at a higher level? Also, please add Å to the legend in this figure.

Local resolution maps of the 3:3 LGI1-ADAM22_ECD complex were shown as Fig. S8 in the revised manuscript. According to this comment, the distribution of the resolution was plotted onto the density at high (0.06) and low (0.03) levels. "Å" was added to the legend in the figure.

Reviewer #2 (Recommendations for the authors):

(1) The study was conducted using the ectodomain (ECD) of ADAM22. It remains unclear whether the 3:3 complex could form if the transmembrane domain (TMD) of ADAM22 were included. In other words, it is difficult to assess whether the observed 3:3 complex represents plausible cis interactions.

As mentioned in our reply to the first comment from Reviewer #1, we noticed that there was no obvious structural feature strongly suggesting that the heterohexameric 3:3 LGI1–ADAM22 is more likely to represent a cis complex or a trans complex. Both are possible at the synapse (and similarly, for LGI3–ADAM23 at the jaxtaparanode of myelinated axons). Therefore, we revised the Introduction and Discussion sections as follows:

Introduction: (about potential structural mechanisms of the 3:3 complex)

“Similarly to the 2:2 complex, the 3:3 complex might serve as an extracellular scaffold to stabilize Kv1 channels or AMPA receptors in a trans-synaptic fashion. In addition, the 3:3 assembly in a cis fashion on the same membrane might regulate the accumulation of Kv1 channel complexes at axon initial segment. However, no clear evidence to prove these potential mechanistic roles of the 3:3 assembly has been provided, and the three-dimensional structure of the 3:3 complex has not yet been determined.”

Discussion: (about a role of the LGI3–ADAM23 complex at the jaxtaparanode of myelinated axons)

“In this context, as discussed in (30), either or both of the 2:2 and 3:3 complexes might be formed in a trans fashion at the juxtaparanode of myelinated axons and bridge the axon and the innermost myelin membrane. Alternatively, the 3:3 complex formed in a cis fashion might positively regulate the clustering of the axonal Kv channels at the juxtaparanode, possibly in a similar manner at the axon initial segment.”

*Ref. 30: Y. Miyazaki et al., Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity. Cell Rep 43, 113634 (2024).

(2) Page 2, line 1: "...caused by genetic mutations." - Specify the mutations involved. Which genes are mutated? Providing this information would enhance clarity and context.

According to this comment, we rephrased the sentence as follows:

"LGI1 is linked to epilepsy, a neurological disorder that can be caused by genetic mutations of genes regulating neuronal excitability (e.g., voltage- or ligand-gated ion channels)."

(3) The experimental strategy and data for both cryo-EM and HS-AFM are of high quality. However, improvements are needed in the cryo-EM/structural figures to enhance clarity. Structural components should be labeled, and the protein interfaces should be identified within the overall complex figures in Figures 2 and 3, as the current presentation is challenging for general readers to follow. For example, in Figure 2, panel a would benefit from clear labeling to indicate the locations of ADAM22 and LGI1. Panels b and c lack context unless the authors specify which interface corresponds to panel a. Additionally, panels e and f are unlabelled, making it difficult to interpret the figures. Improved annotations and descriptions would significantly enhance figure accessibility and comprehension.

Thank you for the constructive suggestion for enhancing accessibility and comprehension of cryo-EM/structural figures. According to this comment, we labeled structural components and indicated the protein interfaces as boxes in the overall complex figures in Figures 2 and 3. Further, in Figure 2, the locations that panels b and c show were indicated as two boxes in the close-up view in panel a.

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Reply to the reviewers

1. Response to reviewers

We would like to thank the reviewers for carefully reading our manuscript and for their valuable comments in support for the publication of our investigation of rapid promoter evolution of accessory gland genes between Drosophila species and hybrids. We are glad to read that the reviewers find our work interesting and that it provides valuable insights into the regulation and divergence of genes through their promoters. We are encouraged by their acknowledgement of the overall quality of the work and the importance of our analyses in advancing the understanding of cis-regulatory changes in species divergence.

2. Point-by-point description of the revisions

Reviewer #

Reviewer Comment

Author Response/Revision

Reviewer 1

The authors test the hypothesis that promoters of genes involved in insect accessory glands evolved more rapidly than other genes in the genome. They test this using a number of computational and experimental approaches, looking at different species within the Drosophila melanogaster complex. The authors find an increased amount of sequence divergence in promoters of accessory gland proteins. They show that the expression levels of these proteins are more variable among species than randomly selected proteins. Finally, they show that within interspecific hybrids, each copy of the gene maintains its species-specific expression level.

We thank Reviewer 1 for their detailed review and positive feedback on our manuscript, and for their helpful suggestions. We have now fully addressed the points raised by Reviewer 1 and have provided the suggested clarifications and revisions to improve the flow, readability, and presentation of the data, which we believe have improved the manuscript significantly.

The work is done with expected standards of controls and analyses. The claims are supported by the analysis. My main criticism of the manuscript has to do not with the experiments or conclusion themselves but with the presentation. The manuscript is just not very well written, and following the logic of the arguments and results is challenging.

The problem begins with the Abstract, which is representative of the general problems with the manuscript. The Abstract begins with general statements about the evolution of seminal fluid proteins, but then jumps to accessory glands and hybrids, without clarifying what taxon is being studied, and what hybrids they are talking about. Then, the acronym Acp is introduced without explanation. The last two sentences of the Abstract are very cumbersome and one has to reread them to understand how they link to the beginning of the Abstract.

More generally, if this reviewer is to be seen as an "average reader" of the paper, I really struggled through reading it, and did not understand many of the arguments or rationale until the second read-through, after I had already read the bottom line. The paragraph spanning lines 71-83 is another case in point. It is composed of a series of very strongly worded sentences, almost all starting with a modifier (unexpectedly, interestingly, moreover), and supported by citations, but the logical flow doesn't work. Again, reading the paragraph after I knew where the paper was going was clearer, but on a first read, it was just a list of disjointed statements.

Since most of the citations are from the authors' own work, I suspect they are assuming too much prior understanding on the part of the reader. I am sure that if the authors read through the manuscript again, trying to look through the eyes of an external reader, they will easily be able to improve the flow and readability of the text.

We thank the reviewer for their detailed feedback and are glad that they acknowledge our work fully supports the claims of our manuscript. We also appreciate their helpful suggestions for improving the readability of the manuscript and have done our best to re-write the abstract and main text where indicated. In particular, the paragraph between lines 71-83 have been rewritten and we have taken care to write to non-expert readers.

1) In the analysis of expression level differences, it is not clear what specific stage / tissue the levels taken from the literature refer to. Could it be that the source of the data is from a stage or tissue where seminar fluid proteins will be expressed with higher variability in general (not just inter-specifically) and this could be skewing the results? Please add more information on the original source of the data and provide support for their validity for this type of comparison.

These were taken from publicly available adult male Drosophila datasets, listed in the data availability statement and throughout the manuscript. We have provided more detail on the tissue used for analysis of Acp gene expression levels.

2) The sentence spanning lines 155-157 needs more context.

We have added more context to lines 155-157.

3) Line 203-204: What are multi-choice enhancers?

We replaced the sentence with "... such as rapidly evolving enhancers or nested epistasis enhancer networks"

4) Figure 1: The terminology the authors use, comparing the gene of interest to "Genome" is very confusing. They are not comparing to the entire genome but to all genes in the genome, which is not the same.

We have changed the word "genome" to "all genes in the genome" on the reviewer's suggestion.

5) Figure 2: Changes between X vs. Y is redundant (either changes between X and Y or changes in X vs. Y).

We assume that the reviewer is referring to Fig. 2B, which does not measure changes between X and Y, but changes in distribution between Acps and the control group. We have explained this in the figure legend.

The manuscript addresses a general question in evolutionary biology - do control regions diverge more quickly protein coding regions. The answer is that yes, they do, but this is actually not very surprising. The work is probably thus of more interest to people interested in the copulatory proteins or in the evolution of mating systems, than to people interested in broader evolutionary questions.

We appreciate this reviewer's recognition of the significance of our work and would like to point out that there are very few studies looking at promoter evolution as detailed in the introduction. Of particular relevance, our study using Acp genes allows us to directly test the impact of promoter mutations on the expression by comparing two alleles in male accessory glands of Drosophila hybrids. Male accessory glands consist of only two secretory cell types allowing us to study evolution of gene expression in a single cell type (Acps are either expressed in main cells or secondary cells). Amid this unique experimental set up we can conclude that promoter mutations can act dominant, in contrast to mutations in protein coding regions, which are generally recessive. Thus, our study is unique in pointing out a largely overseen aspect of gene evolution.

Reviewer 2

This manuscript explores promoter evolution of genes encoding seminal fluid proteins expressed in the male accessory gland of Drosophila and finds cis-regulatory changes underlie expression differences between species. Although these genes evolve rapidly it appears that the coding regions rarely show signs of positive selection inferring that changes in their expression and hence promoter sequences can underlie the evolution of their roles within and among species.

We thank Reviewer 2 for their thorough review, positive feedback on the importance of our work, and suggestions for improving the manuscript. We have addressed all points raised by the reviewer, including analysis of Acp coding region evolution, additional analyses of hybrid expression data, and improved the clarity of the text.

Figure 1 illustrates evidence that the promoter regions of these gene have accumulated more changes than other sampled genes from the Drosophila genome. While this convinces that the region upstream of the transcription start site has diverged considerably in sequence (grey line compared to black line), Figure 1A also suggests the "Genespan" region which includes the 5'UTR but presumably also part of the coding region is also highly diverged. It would be useful to see how the pattern extends into the coding region further to compare further to the promoter region (although Fig 1H does illustrate this more convincingly).

The reviewer raises an interesting point, and certainly all parts of genes evolve. Fig. 1A shows the evolutionary rates of Acps compared to the genome average from phyloP27way scores calculated from 27 insect species. Since these species are quite distant it is unsurprising that they show divergence in coding regions as well as promoter regions. In fact, we addressed whether promoter regions evolve fast in closely related Drosophila species in Fig. 1H compared to coding regions. We have included an additional analysis of coding region evolution in Figure 1B.

Figure 2 presents evidence for significant changes in (presumably levels of) expression of male accessory gland protein (AcP) genes and ribosomal proteins genes between pairs of species, which is reflected in the skew of expression compared to randomly selected genes.

Correct, we have rephrased the statement for clarity.

Figure 3 shows detailed analysis for 3 selected AcP genes with significantly diverged expression. The authors claim this shows 'substitution' hotspots in the promoter regions of all 3 genes but this could be better illustrated by extending the plots in B-D further upstream and downstream to compare to these regions.

We picked the 300-nucleotide promoter region for this analysis as it accumulated significant changes as shown in Fig. 1E-H, and extending the G plots (Fig. 3B-D) to regions with lower numbers of sequence changes would not substantially change the conclusion. Specifically, this analysis identifies sequence change hotspots within fast-evolving promoter regions, rather than comparing promoter regions to other genomic regions, as we previously addressed. The plot is based on a cumulative distribution function and the significant positive slope in the upstream region where promoters are located identifies a hotspot for accumulation of substitutions. There could be other hotspots, but the point being made is that significant hotspots consistently appear in the promoter region of these three genes.

Figure 4 shows the results of expression analysis in parental lines of each pair of species and F1 hybrids. However the results are very difficult to follow in the figure and in the relevant text. While the schemes in A, C. E and G are helpful, the gel images are not the best quality and interpretations confusing. An additional scheme is needed to illustrate hypothetical outcomes of trans change, cis change and transvection to help interpret the gels. On line 169 (presumably referring to panels D and F although C and D are cited on the next line) the authors claim that Obp56f and CG11598 'were more expressed in D. melanogaster compared to D. simulans' but in the gel image the D. sim band is stronger for both genes (like D. sechellia) compared to the D. mel band. The authors also claim that the patterns of expression seen in the F1s are dominant for one allele and that this must be because of transvection. I agree this experiment is evidence for cis-regulatory change. However the interpretation that it is caused by transvection needs more explanation/justification and how do the authors rule out that it is not a cis X trans interaction between the species promoter differences and differences in the transcription factors of each species in the F1? Also my understanding is that transvection is relatively rare and yet the authors claim this is the explanation for 2/4 genes tested.

We appreciate the reviewer's comments on Figure 4 and the opportunity to improve its clarity. To address these concerns, we have carefully checked the figure citations and corrected any inconsistencies.

The reviewer raises an important point about our interpretation of transvection. We have expanded our discussion of this result to consider why transvection is a plausible explanation for the observed dominance patterns and also consider cis x trans interactions between species-specific promoters and transcription factor binding. While rare, transvection likely has more relevance in hybrid regulatory contexts involving homologous chromosome pairing which we discuss this in the revised text.

Line 112 states that the melanogaster subgroup contains 5 species - this is incorrect - while this study looked at 5 species there are more species in this subgroup such as mauritiana and santomea.

We have corrected the statement about the number of species in the melanogaster subgroup.

Lines 131-134 could explain better what the conservation scores and their groupings mean and the rationale for this approach.

We have clarified what the conservation scores and their groupings mean and the rationale for this approach.

Line 162 - the meaning of the sentence starting on this line is unclear - it sounds very circular.

We have rephrased the statement for more clarity.

Line 168 should cite Fig 4 H instead of F.

We have amended citation of Fig 4F to H.

Reviewer 3

In this study, McQuarrie et al. investigate the evolution of promoters of genes encoding accessory gland proteins (Acps) in species within the D. melanogaster subgroup. Using computational analyses and available genomic and transcriptomic datasets, they demonstrate that promoter regions of Acp genes are highly diverse compared to the promoters of other genes in the genome. They further show that this diversification correlates with changes in gene expression levels between closely related species. Complementing these computational analyses, the authors conduct experiments to test whether differences in expression levels of four Acp genes with highly diverged promoter regions are maintained in hybrids of closely related species. They find that while two Acp genes maintain their expression level differences in hybrids, the other two exhibit dominance of one allele. The authors attribute these findings to transvection. Based on their data, they conclude that rapid evolution of Acp gene promoters, rather than changes in trans, drives changes in Acp gene expression that contribute to speciation.

We thank Reviewer 3 for their thorough review and suggestions. We further thank the reviewer for acknowledging the importance of our findings and for pointing out that it contributes to our understanding of speciation. We have thoroughly addressed all comments from the reviewer and significantly revised the manuscript. We believe that this has greatly improved the manuscript.

Unfortunately, the presented data are not sufficient to fully support the conclusions. While many of the concerns can be addressed by revising the text to moderate the claims and acknowledge the methodological limitations, some key experiments require repetition with more controls, biological replicates, and statistical analyses to validate the findings.

Specifically, some of the main conclusions heavily rely on the RT-PCR experiments presented in Figure 4, which analyze the expression of four Acp genes in hybrid flies. The authors use PCR and RFLP to distinguish species-specific alleles but draw quantitative conclusions from what is essentially a qualitative experiment. There are several issues with this approach. First, the experiment includes only two biological replicates per sample, which is inadequate for robust statistical analysis. Second, the authors did not measure the intensity of the gel fragments, making it impossible to quantify allele-specific expression accurately. Third, no control genes were used as standards to ensure the comparability of samples.

The gold standard for quantifying allele-specific expression is using real-time PCR methods such as TaqMan assays, which allow precise SNP genotyping. To address this major limitation, the authors should ideally repeat the experiments using allele-specific real-time PCR assays. This would provide a reliable and quantitative measurement of allele-specific expression.

If the authors cannot implement real-time PCR, an alternative (though less rigorous) approach would be to continue using their current method with the following adjustments:

• Include a housekeeping gene in the analysis as an internal control (this would require identifying a region distinguishable by RFLP in the control).

• Quantify the intensity of the PCR products on the gel relative to the internal standard, ensuring proper normalization.

• Increase the sample size to allow for robust statistical analysis.

These experiments could be conducted relatively quickly and would significantly enhance the validity of the study's conclusions.

We thank the reviewer for their detailed suggestions for improving the conclusions in Fig. 4. Indeed, incorporating a housekeeping gene as a control supports our results for qualitative analysis of gene expression in hybrids assessing each allele individually (Fig 4), and improves interpretation for non-experts. We have also included an additional analysis in the new Fig. 5 which analyses RNA-seq expression changes in D. melanogaster x D. simulans hybrid male accessory glands. We believe these additions have significantly improved the manuscript and its conclusions.

While the following comments are not necessarily minor, they can be addressed through revisions to the text without requiring additional experimental work. Some comments are more conceptual in nature, while others concern the interpretation and presentation of the experimental results. They are provided in no particular order.

1. A key limitation of this study is the use of RNA-seq datasets from whole adult flies for interspecies gene expression comparisons. Whole-body RNA-seq inherently averages gene expression across all tissues, potentially masking tissue-specific expression differences. While Acp genes are likely restricted to accessory glands, the non-Acp genes and the random gene sets used in the analysis may have broader expression profiles. As a result, their expression might be conserved in certain tissues while diverging in others- an aspect that whole-body RNA-seq cannot capture. The authors should acknowledge that tissue-specific RNA-seq analyses could provide a more precise understanding of expression divergence and potentially reveal reduced conservation when considering specific tissues independently.

We have added a section discussing the limitations in gene expression analysis in the discussion. In addition, we have included an additional Figure analysing gene expression in hybrid male accessory glands (Fig. 5).

1. The statement in line 128, "Consistent with this model," does not accurately reflect the findings presented in Figures 2A and B. Specifically, the data in Figure 2A show that Acp gene expression divergence is significantly different from the divergence of non-Acp genes or a random sample only in the comparison between D. melanogaster and D. simulans. However, when these species are compared to D. yakuba, Acp gene expression divergence aligns with the divergence patterns of non-Acp genes or random samples. In contrast, Figure 2B shows that the distribution of expression changes is skewed for Acp genes compared to random control samples when D. melanogaster or D. simulans are compared to D. yakuba. However, this skew is absent when the two D. melanogaster and D. simulans are compared. Therefore, the statement in line 128 should be revised to accurately reflect these nuanced results and the trends shown in Figure 2A and B.

We have updated the statement for clarity. Here, the percentage of Acps showing significant gene expression changes is greater between more closely related species, but the distribution of expression changes increases between more distantly related species.

1. The statement in lines 136-138, "Acps were enriched for significant expression changes in the faster evolving group across all species," while accurate, overlooks a key observation. This trend was also observed in other groups, including those with slower evolving promoters, in some of the species' comparisons. Therefore, the enrichment is not unique to Acps with rapidly evolving promoters, and this should be explicitly acknowledged in the text.

This is a valid point, and we have updated this statement as suggested.

1. It would be helpful for the authors to explain the meaning of the d score at the beginning of the paragraph starting in line 131, to ensure clarity for readers unfamiliar with this metric.

This scoring method is described in the methods sections, and we have now included reference to thorough explanation of how d was calculated at the indicated section.

1. In Figure 2C-E - the title of the Y-axis does not match the text. If it represents the percentage of genes with significant expression changes, as in Figure 2A, the discrepancies between the percentages in this figure and those in Figure 2A need to be addressed.

We have updated the method used to categorise significant changes in gene expression in the text and the figure legend for clarity.

1. The experiment in Figure 3 needs a better explanation in the text. What is the analysis presented in Figure 3B-D. How many species were compared?

We have added additional details in the results section and an explanation of how sequence change hotspots were calculated in the results section is available.

1. The concept of transvection should be omitted from this manuscript. First, the definition provided by the authors is inaccurate. Second, even if additional experiments were to convincingly show that one allele in hybrid animals is dominant over the other, there are alternative explanations for this phenomenon that do not involve transvection. The authors may propose transvection as a potential model in the discussion, but they should do so cautiously and explicitly acknowledge the possibility of other mechanisms.

We have updated the text to more conservatively discuss transvection, moving this to the discussion section with additional possibilities discussed.

1. The statement at the end of the introduction is overly strong and would benefit from more cautious phrasing. For instance, it could be reworded as: "These findings suggest that promoter changes, rather than genomic background, play a significant role in driving expression changes, indicating that promoter evolution may contribute to the rise of new species."

We have reworded this line following the reviewer's suggestion.

1. Line 32 of the abstract: The term "Acp" is introduced without explaining what it stands for. Please define it as "Accessory gland proteins (Acp)" when it first appears.

We have updated the manuscript to define Acp where it is first mentioned.

1. Line 61: The phrase "...through relaxed,..." is unclear. Specify what is relaxed (e.g., "relaxed selective pressures").

We have included description of relaxed selective pressures.

1. The sentence in lines 74-76, starting in "Interestingly,...." Needs revision for clarity.

We have removed the word interestingly.

1. Line 112: Revise "we focused on the melanogaster subgroup which is made up of five species" to: "we focused on the melanogaster subgroup, which includes five species."

We have made this change in the text.

1. In line 144 use the phrase "promoter conservation" instead of "promoter evolution"

We have updated the phrasing.

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Referee #3

Evidence, reproducibility and clarity

Summary:

In this study, McQuarrie et al. investigate the evolution of promoters of genes encoding accessory gland proteins (Acps) in species within the D. melanogaster subgroup. Using computational analyses and available genomic and transcriptomic datasets, they demonstrate that promoter regions of Acp genes are highly diverse compared to the promoters of other genes in the genome. They further show that this diversification correlates with changes in gene expression levels between closely related species. Complementing these computational analyses, the authors conduct experiments to test whether differences in expression levels of four Acp genes with highly diverged promoter regions are maintained in hybrids of closely related species. They find that while two Acp genes maintain their expression level differences in hybrids, the other two exhibit dominance of one allele. The authors attribute these findings to transvection. Based on their data, they conclude that rapid evolution of Acp gene promoters, rather than changes in trans, drives changes in Acp gene expression that contribute to speciation.

Major comments:

Unfortunately, the presented data are not sufficient to fully support the conclusions. While many of the concerns can be addressed by revising the text to moderate the claims and acknowledge the methodological limitations, some key experiments require repetition with more controls, biological replicates, and statistical analyses to validate the findings.

Specifically, some of the main conclusions heavily rely on the RT-PCR experiments presented in Figure 4, which analyze the expression of four Acp genes in hybrid flies. The authors use PCR and RFLP to distinguish species-specific alleles but draw quantitative conclusions from what is essentially a qualitative experiment. There are several issues with this approach. First, the experiment includes only two biological replicates per sample, which is inadequate for robust statistical analysis. Second, the authors did not measure the intensity of the gel fragments, making it impossible to quantify allele-specific expression accurately. Third, no control genes were used as standards to ensure the comparability of samples.

The gold standard for quantifying allele-specific expression is using real-time PCR methods such as TaqMan assays, which allow precise SNP genotyping. To address this major limitation, the authors should ideally repeat the experiments using allele-specific real-time PCR assays. This would provide a reliable and quantitative measurement of allele-specific expression.

If the authors cannot implement real-time PCR, an alternative (though less rigorous) approach would be to continue using their current method with the following adjustments:

• Include a housekeeping gene in the analysis as an internal control (this would require identifying a region distinguishable by RFLP in the control).
• Quantify the intensity of the PCR products on the gel relative to the internal standard, ensuring proper normalization.
• Increase the sample size to allow for robust statistical analysis. These experiments could be conducted relatively quickly and would significantly enhance the validity of the study's conclusions.

Minor comments

While the following comments are not necessarily minor, they can be addressed through revisions to the text without requiring additional experimental work. Some comments are more conceptual in nature, while others concern the interpretation and presentation of the experimental results. They are provided in no particular order. 1. A key limitation of this study is the use of RNA-seq datasets from whole adult flies for interspecies gene expression comparisons. Whole-body RNA-seq inherently averages gene expression across all tissues, potentially masking tissue-specific expression differences. While Acp genes are likely restricted to accessory glands, the non-Acp genes and the random gene sets used in the analysis may have broader expression profiles. As a result, their expression might be conserved in certain tissues while diverging in others- an aspect that whole-body RNA-seq cannot capture. The authors should acknowledge that tissue-specific RNA-seq analyses could provide a more precise understanding of expression divergence and potentially reveal reduced conservation when considering specific tissues independently. 2. The statement in line 128, "Consistent with this model," does not accurately reflect the findings presented in Figures 2A and B. Specifically, the data in Figure 2A show that Acp gene expression divergence is significantly different from the divergence of non-Acp genes or a random sample only in the comparison between D. melanogaster and D. simulans. However, when these species are compared to D. yakuba, Acp gene expression divergence aligns with the divergence patterns of non-Acp genes or random samples. In contrast, Figure 2B shows that the distribution of expression changes is skewed for Acp genes compared to random control samples when D. melanogaster or D. simulans are compared to D. yakuba. However, this skew is absent when the two D. melanogaster and D. simulans are compared. Therefore, the statement in line 128 should be revised to accurately reflect these nuanced results and the trends shown in Figure 2A and B. 3. The statement in lines 136-138, "Acps were enriched for significant expression changes in the faster evolving group across all species," while accurate, overlooks a key observation. This trend was also observed in other groups, including those with slower evolving promoters, in some of the species' comparisons. Therefore, the enrichment is not unique to Acps with rapidly evolving promoters, and this should be explicitly acknowledged in the text. 4. It would be helpful for the authors to explain the meaning of the d score at the beginning of the paragraph starting in line 131, to ensure clarity for readers unfamiliar with this metric. 5. In Figure 2C-E - the title of the Y-axis does not match the text. If it represents the percentage of genes with significant expression changes, as in Figure 2A, the discrepancies between the percentages in this figure and those in Figure 2A need to be addressed. 6. The experiment in Figure 3 needs a better explanation in the text. What is the analysis presented in Figure 3B-D. How many species were compared? 7. The concept of transvection should be omitted from this manuscript. First, the definition provided by the authors is inaccurate. Second, even if additional experiments were to convincingly show that one allele in hybrid animals is dominant over the other, there are alternative explanations for this phenomenon that do not involve transvection. The authors may propose transvection as a potential model in the discussion, but they should do so cautiously and explicitly acknowledge the possibility of other mechanisms. 8. The statement at the end of the introduction is overly strong and would benefit from more cautious phrasing. For instance, it could be reworded as: "These findings suggest that promoter changes, rather than genomic background, play a significant role in driving expression changes, indicating that promoter evolution may contribute to the rise of new species."

Text edits:

Throughout the manuscripts there are incomplete sentences and sentences that are not clear. Below is a list of corrections:

1. Line 32 of the abstract: The term "Acp" is introduced without explaining what it stands for. Please define it as "Accessory gland proteins (Acp)" when it first appears.
2. Line 61: The phrase "...through relaxed,..." is unclear. Specify what is relaxed (e.g., "relaxed selective pressures").
3. The sentence in lines 74-76, starting in "Interestingly,...." Needs revision for clarity.
4. Line 112: Revise "we focused on the melanogaster subgroup which is made up of five species" to: "we focused on the melanogaster subgroup, which includes five species."
5. In line 144 use the phrase "promoter conservation" instead of "promoter evolution"

Significance

This study addresses an important question in evolutionary biology: how seminal fluid proteins achieve rapid evolution despite showing limited adaptive changes in their coding regions. By focusing on accessory gland proteins (Acps) and examining their promoter regions, the authors suggest promoter-driven evolution as a potential mechanism for rapid seminal fluid protein diversification. While this hypothesis is intriguing and can contribute to our understanding of speciation, more rigorous analysis and experimental validation would be needed to support the conclusions. The revised manuscript can be of interest to fly geneticists and to scientists in the fields of gene regulation and evolution.

Keywords for my expertise: Enhancers, transcriptional regulation, development, evolution, Drosophila.

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

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Referee #1

Evidence, reproducibility and clarity

The authors test the hypothesis that promoters of genes involved in insect accessory glands evolved more rapidly than other genes in the genome. They test this using a number of computational and experimental approaches, looking at different species within the Drosophila melanogaster complex. The authors find an increased amount of sequence divergence in promoters of accessory gland proteins. They show that the expression levels of these proteins are more variable among species than randomly selected proteins. Finally, they show that within interspecific hybrids, each copy of the gene maintains its species-specific expression level.

The work is done with expected standards of controls and analyses. The claims are supported by the analysis. My main criticism of the manuscript has to do not with the experiments or conclusion themselves but with the presentation. The manuscript is just not very well written, and following the logic of the arguments and results is challenging. The problem begins with the Abstract, which is representative of the general problems with the manuscript. The Abstract begins with general statements about the evolution of seminal fluid proteins, but then jumps to accessory glands and hybrids, without clarifying what taxon is being studied, and what hybrids they are talking about. Then, the acronym Acp is introduced without explanation. The last two sentences of the Abstract are very cumbersome and one has to reread them to understand how they link to the beginning of the Abstract.

More generally, if this reviewer is to be seen as an "average reader" of the paper, I really struggled through reading it, and did not understand many of the arguments or rationale until the second read-through, after I had already read the bottom line. The paragraph spanning lines 71-83 is another case in point. It is composed of a series of very strongly worded sentences, almost all starting with a modifier (unexpectedly, interestingly, moreover), and supported by citations, but the logical flow doesn't work. Again, reading the paragraph after I knew where the paper was going was clearer, but on a first read, it was just a list of disjointed statements.

Since most of the citations are from the authors' own work, I suspect they are assuming too much prior understanding on the part of the reader. I am sure that if the authors read through the manuscript again, trying to look through the eyes of an external reader, they will easily be able to improve the flow and readability of the text.

More specific comments:

1. In the analysis of expression level differences, it is not clear what specific stage / tissue the levels taken from the literature refer to. Could it be that the source of the data is from a stage or tissue where seminar fluid proteins will be expressed with higher variability in general (not just inter-specifically) and this could be skewing the results? Please add more information on the original source of the data and provide support for their validity for this type of comparison.
2. The sentence spanning lines 155-157 needs more context.
3. Line 203-204: What are multi-choice enhancers?
4. Figure 1: The terminology the authors use, comparing the gene of interest to "Genome" is very confusing. They are not comparing to the entire genome but to all genes in the genome, which is not the same.
5. Figure 2: Changes between X vs. Y is redundant (either changes between X and Y or changes in X vs. Y).

Significance

The manuscript addresses a general question in evolutionary biology - do control regions diverge more quickly protein coding regions. The answer is that yes, they do, but this is actually not very surprising. The work is probably thus of more interest to people interested in the copulatory proteins or in the evolution of mating systems, than to people interested in broader evolutionary questions.

question: where did the depth come from initially?

L'autonomisation n'est vue que sous la forme du statut du travailleur (salarié, entrepreneur, indépendant, travailleur ubérisé...). Oui il y a plus de travailleur.euses indépendant.es, mais est ce un choix ? Le recours à la sous-traitance et à l'externalisation des activités s'est accompagné, dans de nombreuses entreprises et administrations, par des suppressions de postes (ménage, courrier, livraison, fonctions transverses...).

Gain de place et donc de productivité, oui puisque tout es monétisé dans une entreprise (y compris les m² occupés). Cela dit les veilles juridiques continuent d'exister, et celles-ci doivent être compilées, classées et archivées pour en faire ressortir facilement les jurisprudences intéressantes. De ce point de vue le lien de cause à effet que fait l'auteur est un peu rapide. Il ne tient pas compte du savoir-faire et des nomenclatures propres à chaque individu/collectif.

Dans ce chapitre l'auteur met en avant les facilités dans le travail permises par les nouvelles technologies, ainsi que le gain de temps grâce à celles-ci. En revanche s'il pose bien la question de partage et de transit de données, il ne se positionne pas sur la question de la confidentialité et de l'utilisation qui en sont faites (traitement des datas, reventes des données à des sociétés privées à des fins commerciales par exemple). Par ailleurs l'affirmation selon laquelle l'information est gratuite est erronée. En effet l'accès à certaines informations est soumis à abonnement (payant) ou au paiement d'un droit d'accès temporaire.

Una vez hecho el ejercicio en GT y agregado el DataFrame, y cliquear en la solapa Tree, me salen los datos vacíos, no se si es que falta agregar una parte de código para ejecutar algo y organizarlos.

Docente @Offray, sobre el siguiente bloque tengo las siguientes inquietudes: 1. técnicamente que significa := ? ya que esta siempre antecede un bloque. 2. con base en el documento Representando y procesando datos en Pharo no veo ejemplos como :name donde los dos puntos anteceden el valor, técnicamente que significa dicho mensaje dentro del boque, es lo referente al elemento que presentará los cambios dentro de un mensaje?

En el presente caso podemos ver un ARREGLO, el cual está representado por #( ) y que son maneras de guardar colecciones de información diversa como en este caso se agrupan los repositorios de la clase Unisemánticas. Para mayor información ver el apartado 4.1. del documento [Representando y procesando datos en Pharo] (https://mutabit.com/repos.fossil/labci/doc/25A1/wiki/representando-y-procesando-datos-en-pharo--cek90.md.html)

La siguiente operación implica un Receptor(pokemonExampleLink), un Selector (asUrl) y un Argumento (retrieveContents) donde básicamente se interpreta que la operación pokemonRawData indique que la cadena pokemonExampleLink como URL permita la sustracción de los contenidos de https://pokeapi.co/api/v2/pokemon/pikachu

Pokémon es una de las franquicias más populares del mundo del entretenimiento, originada en Japón. El nombre viene de la contracción de "Pocket Monsters" (Monstruos de Bolsillo) y fueron creados en 1996.

En este caso, para la selección de otros peleadores, por ejemplo, no me queda claro, si debo, en este código cambiar el fighter1 y 2 por 3 y 4.

Author response:

The following is the authors’ response to the previous reviews

General Response to Reviewers:

We thank the Reviewers for their comments, which continue to substantially improve the quality and clarity of the manuscript, and therefore help us to strengthen its message while acknowledging alternative explanations.

All three reviewers raised the concern that we have not proven that Rab3A is acting on a presynaptic mechanism to increase mEPSC amplitude after TTX treatment of mouse cortical cultures.  The reviewers’ main point is that we have not shown a lack of upregulation of postsynaptic receptors in mouse cortical cultures. We want to stress that we agree that postsynaptic receptors are upregulated after activity block in neuronal cultures.  However, the reviewers are not acknowledging that we have previously presented strong evidence at the mammalian NMJ that there is no increase in AChR after activity blockade, and therefore the requirement for Rab3A in the homeostatic increase in quantal amplitude points to a presynaptic contribution. We agree that we should restrict our firmest conclusions to the data in the current study, but in the Discussion we are proposing interpretations. We have added the following new text:

“The impetus for our current study was two previous studies in which we examined homeostatic regulation of quantal amplitude at the NMJ.  An advantage of studying the NMJ is that synaptic ACh receptors are easily identified with fluorescently labeled alpha-bungarotoxin, which allows for very accurate quantification of postsynaptic receptor density. We were able to detect a known change due to mixing 2 colors of alpha-BTX to within 1% (Wang et al., 2005).  Using this model synapse, we showed that there was no increase in synaptic AChRs after TTX treatment, whereas miniature endplate current increased 35% (Wang et al., 2005). We further showed that the presynaptic protein Rab3A was necessary for full upregulation of mEPC amplitude (Wang et al., 2011). These data strongly suggested Rab3A contributed to homeostatic upregulation of quantal amplitude via a presynaptic mechanism.  With the current study showing that Rab3A is required for the homeostatic increase in mEPSC amplitude in cortical cultures, one interpretation is that in both situations, Rab3A is required for an increase in the presynaptic quantum.”

The point we are making is that the current manuscript is an extension of that work and interpretation of our findings regarding the variability of upregulation of postsynaptic receptors in our mouse cortical cultures further supports the idea that there is a Rab3Adependent presynaptic contribution to homeostatic increases in quantal amplitude.

Public Reviews:

Reviewer #1 (Public review):

Koesters and colleagues investigated the role of the small GTPase Rab3A in homeostatic scaling of miniature synaptic transmission in primary mouse cortical cultures using electrophysiology and immunohistochemistry. The major finding is that TTX incubation for 48 hours does not induce an increase in the amplitude of excitatory synaptic miniature events in neuronal cortical cultures derived from Rab3A KO and Rab3A Earlybird mutant mice. NASPM application had comparable effects on mEPSC amplitude in control and after TTX, implying that Ca2+-permeable glutamate receptors are unlikely modulated during synaptic scaling. Immunohistochemical analysis revealed no significant changes in GluA2 puncta size, intensity, and integral after TTX treatment in control and Rab3A KO cultures. Finally, they provide evidence that loss of Rab3A in neurons, but not astrocytes, blocks homeostatic scaling. Based on these data, the authors propose a model in which neuronal Rab3A is required for homeostatic scaling of synaptic transmission, potentially through GluA2-independent mechanisms.

The major finding - impaired homeostatic up-scaling after TTX treatment in Rab3A KO and Rab3 earlybird mutant neurons - is supported by data of high quality. However, the paper falls short of providing any evidence or direction regarding potential mechanisms. The data on GluA2 modulation after TTX incubation are likely statistically underpowered, and do not allow drawing solid conclusions, such as GluA2-independent mechanisms of up-scaling.

The study should be of interest to the field because it implicates a presynaptic molecule in homeostatic scaling, which is generally thought to involve postsynaptic neurotransmitter receptor modulation. However, it remains unclear how Rab3A participates in homeostatic plasticity.

Major (remaining) point:

(1) Direct quantitative comparison between electrophysiology and GluA2 imaging data is complicated by many factors, such as different signal-to-noise ratios. Hence, comparing the variability of the increase in mini amplitude vs. GluA2 fluorescence area is not valid. Thus, I recommend removing the sentence "We found that the increase in postsynaptic AMPAR levels was more variable than that of mEPSC amplitudes, suggesting other factors may contribute to the homeostatic increase in synaptic strength." from the abstract.

We have not removed the statement, but altered it to soften the conclusion. It now reads, “We found that the increase in postsynaptic AMPAR levels in wild type cultures was more variable than that of mEPSC amplitudes, which might be explained by a presynaptic contribution, but we cannot rule out variability in the measurement.”.

Similarly, the data do not directly support the conclusion of GluA2-independent mechanisms of homeostatic scaling. Statements like "We conclude that these data support the idea that there is another contributor to the TTX- induced increase in quantal size." should be thus revised or removed.

This particular statement is in the previous response to reviewers only, we deleted the sentence that starts, “The simplest explanation Rab3A regulates a presynaptic contributor….”. and “Imaging of immunofluorescence more variable…”. We deleted “ our data suggest….consistently leads to an increase in mEPSC amplitude and sometimes leads to….” We added “…the lack of a robust increase in receptor levels leaves open the possibility that there is a presynaptic contributor to quantal size in mouse cortical cultures. However, the variability could arise from technical factors associated with the immunofluorescence method, and the mechanism of Rab3A-dependent plasticity could be presynaptic for the NMJ and postsynaptic for cortical neurons.”

Reviewer #2 (Public review):

I thank the authors for their efforts in the revision. In general, I believe the main conclusion that Rab3A is required for TTX-induced homeostatic synaptic plasticity is wellsupported by the data presented, and this is an important addition to the repertoire of molecular players involved in homeostatic compensations. I also acknowledge that the authors are more cautious in making conclusions based on the current evidence, and the structure and logic have been much improved.

The only major concern I have still falls on the interpretation of the mismatch between GluA2 cluster size and mEPSC amplitude. The authors argue that they are only trying to say that changes in the cluster size are more variable than those in the mEPSC amplitude, and they provide multiple explanations for this mismatch. It seems incongruous to state that the simplest explanation is a presynaptic factor when you have all these alternative factors that very likely have contributed to the results. Further, the authors speculate in the discussion that Rab3A does not regulate postsynaptic GluA2 but instead regulates a presynaptic contributor. Do the authors mean that, in their model, the mEPSC amplitude increases can be attributed to two factors- postsynaptic GluA2 regulation and a presynaptic contribution (which is regulated by Rab3A)? If so, and Rab3A does not affect GluA2 whatsoever, shouldn't we see GluA2 increase even in the absence of Rab3A? The data in Table 1 seems to indicate otherwise.

The main body of this comment is addressed in the General Response to Reviewers. In addition, we deleted text “current data, coupled with our previous findings at the mouse neuromuscular junction, support the idea that there are additional sources contributing to the homeostatic increase in quantal size.” We added new text, so the sentence now reads: “Increased receptors likely contribute to increases in mESPC amplitudes in mouse cortical cultures, but because we do not have a significant increase in GluA2 receptors in our experiments, it is impossible to conclude that the increase is lacking in cultures from Rab3A^-/- neurons.”

I also question the way the data are presented in Figure 5. The authors first compare 3 cultures and then 5 cultures altogether, if these experiments are all aimed to answer the same research question, then they should be pooled together. Interestingly, the additional two cultures both show increases in GluA2 clusters, which makes the decrease in culture #3 even more perplexing, for which the authors comment in line 261 that this is due to other factors. Shouldn't this be an indicator that something unusual has happened in this culture?

Data in this figure is sufficient to support that GluA2 increases are variable across cultures, which hardly adds anything new to the paper or to the field. 

A major goal of performing the immunofluorescence measurements in the same cultures for which we had electrophysiological results was to address the common impression that the homeostatic effect itself is highly variable, as the reviewer notes in the comment “…GluA2 increases are variable across cultures…” Presumably, if GluA2 increases are the mechanism of the mEPSC amplitude increases, then variable GluA2 increases should correlate with variable mEPSC amplitude increases, but that is not what we observed. We are left with the explanation that the immunofluorescence method itself is very variable. We have added the point to the Discussion, which reads, “the variability could arise from technical factors associated with the immunofluorescence method, and the mechanism of Rab3A-dependent homeostatic plasticity could be presynaptic for the NMJ and postsynaptic for cortical neurons.”

Finally, the implication of “Shouldn’t this be an indicator that something unusual has happened in this culture?” if it is not due to culture to culture variability in the homeostatic response itself, is that there was a technical problem with accurately measuring receptor levels. We have no reason to suspect anything was amiss in this set of coverslips (the values for controls and for TTX-treated were not outside the range of values in other experiments). In any of the coverslips, there may be variability in the amount of primary anti-GluA2 antibody, as this was added directly to the culture rather than prepared as a diluted solution and added to all the coverslips. But to remove this one experiment because it did not give the expected result is to allow bias to direct our data selection.

The authors further cite a study with comparable sample sizes, which shows a similar mismatch based on p values (Xu and Pozzo-Miller 2007), yet the effect sizes in this study actually match quite well (both ~160%). P values cannot be used to show whether two effects match, but effect sizes can. Therefore, the statement in lines 411-413 "... consistently leads to an increase in mEPSC amplitudes, and sometimes leads to an increase in synaptic GluA2 receptor cluster size" is not very convincing, and can hardly be used to support "the idea that there are additional sources contributing to the homeostatic increase in quantal size.”

We have the same situation; our effect sizes match (19.7% increase for mEPSC amplitude; 18.1% increase for GluA2 receptor cluster size, see Table 1), but in our case, the p value for receptors does not reach statistical significance. Our point here is that there is published evidence that the variability in receptor measurements is greater than the variability in electrophysiological measurements. But we have softened this point, removing the sentences containing “…consistently leads and sometimes...” and “……additional sources contributing…”.

I would suggest simply showing mEPSC and immunostaining data from all cultures in this experiment as additional evidence for homeostatic synaptic plasticity in WT cultures, and leave out the argument for "mismatch". The presynaptic location of Rab3A is sufficient to speculate a presynaptic regulation of this form of homeostatic compensation.

We have removed all uses of the word “mismatch,” but feel the presentation of the 3 matched experiments, 23-24 cells (Figure 5A, D), and the additional 2 experiments for a total of 5 cultures, 48-49 cells (Figure 5C, F), is important in order to demonstrate that the lack of statistically significant receptor response is due neither to a variable homeostatic response in the mEPSC amplitudes, nor to a small number of cultures.

Minor concerns:

(1) Line 214, I see the authors cite literature to argue that GluA2 can form homomers and can conduct currents. While GluA2 subunits edited at the Q/R site (they are in nature) can form homomers with very low efficiency in exogenous systems such as HEK293 cells (as done in the cited studies), it's unlikely for this to happen in neurons (they can hardly traffic to synapses if possible at all).

We were unable to identify a key reference that characterized GluA2 homomers vs. heteromers in native cortical neurons, but we have rewritten the section in the manuscript to acknowledge the low conductance of homomers:

“…to assess whether GluA2 receptor expression, which will identify GluA2 homomers and GluA2 heteromers (the former unlikely to contribute to mEPSCs given their low conductance relative to heteromers (Swanson et al., 1997; Mansour et al., 2001)…”

(2) Lines 221-222, the authors may have misinterpreted the results in Turrigiano 1998. This study does not show that the increase in receptors is most dramatic in the apical dendrite, in fact, this is the only region they have tested. The results in Figures 3b-c show that the effect size is independent of the distance from soma.

Figure 3 in Turrigiano et al., shows that the increase in glutamate responsiveness is higher at the cell body than along the primary dendrite. We have revised our description to indicate that an increase in responsiveness on the primary dendrite has been demonstrated in Turrigiano et al. 1998.

“We focused on the primary dendrite of pyramidal neurons as a way to reduce variability that might arise from being at widely ranging distances from the cell body, or, from inadvertently sampling dendritic regions arising from inhibitory neurons. In addition, it has been shown that there is a clear increase in response to glutamate in this region (Turrigiano et al., 1998).”

“…synaptic receptors on the primary dendrite, where a clear increase in sensitivity to exogenously applied glutamate was demonstrated (see Figure 3 in (Turrigiano et al., 1998)).

(3) Lines 309-310 (and other places mentioning TNFa), the addition of TNFa to this experiment seems out of place. The authors have not performed any experiment to validate the presence/absence of TNFa in their system (citing only 1 study from another lab is insufficient). Although it's convincing that glia Rab3A is not required for homeostatic plasticity here, the data does not suggest Rab3A's role (or the lack of) for TNFa in this process.

We have modified the paragraph in the Discussion that addresses the glial results, to describe more clearly the data that supported an astrocytic TNF-alpha mechanism: “TNF-alpha accumulates after activity blockade, and directly applied to neuronal cultures, can cause an increase in GluA1 receptors, providing a potential mechanism by which activity blockade leads to the homeostatic upregulation of postsynaptic receptors (Beattie et al., 2002; Stellwagen et al., 2005; Stellwagen and Malenka, 2006).”

We have also acknowledged that we cannot rule out TNF-alpha coming from neurons in the cortical cultures: “…suggesting the possibility that neuronal Rab3A can act via a non-TNF-alpha mechanism to contribute to homeostatic regulation of quantal amplitude, although we have not ruled out a neuronal Rab3A-mediated TNF-alpha pathway in cortical cultures.”

Reviewer #3 (Public review):

This manuscript presents a number of interesting findings that have the potential to increase our understanding of the mechanism underlying homeostatic synaptic plasticity (HSP). The data broadly support that Rab3A plays a role in HSP, although the site and mechanism of action remain uncertain.

The authors clearly demonstrate that Rab3A plays a role in HSP at excitatory synapses, with substantially less plasticity occurring in the Rab3A KO neurons. There is also no apparent HSP in the Earlybird Rab3A mutation, although baseline synaptic strength is already elevated. In this context, it is unclear if the plasticity is absent, already induced by this mutation, or just occluded by a ceiling effect due to the synapses already being strengthened. Occlusion may also occur in the mixed cultures when Rab3A is missing from neurons but not astrocytes. The authors do appropriately discuss these options. The authors have solid data showing that Rab3A is unlikely to be active in astrocytes, Finally, they attempt to study the linkage between changes in synaptic strength and AMPA receptor trafficking during HSP, and conclude that trafficking may not be solely responsible for the changes in synaptic strength during HSP.

Strengths:

This work adds another player into the mechanisms underlying an important form of synaptic plasticity. The plasticity is likely only reduced, suggesting Rab3A is only partially required and perhaps multiple mechanisms contribute. The authors speculate about some possible novel mechanisms, including whether Rab3A is active pre-synaptically to regulate quantal amplitude.

As Rab3A is primarily known as a pre-synaptic molecule, this possibility is intriguing. However, it is based on the partial dissociation of AMPAR trafficking and synaptic response and lacks strong support. On average, they saw a similar magnitude of change in mEPSC amplitude and GluA2 cluster area and integral, but the GluA2 data was not significant due to higher variability. It is difficult to determine if this is due to biology or methodology - the imaging method involves assessing puncta pairs (GluA2/VGlut1) clearly associated with a MAP2 labeled dendrite. This is a small subset of synapses, with usually less than 20 synapses per neuron analyzed, which would be expected to be more variable than mEPSC recordings averaged across several hundred events. However, when they reduce the mEPSC number of events to similar numbers as the imaging, the mESPC amplitudes are still less variable than the imaging data. The reason for this remains unclear. The pool of sampled synapses is still different between the methods and recent data has shown that synapses have variable responses during HSP. Further, there could be variability in the subunit composition of newly inserted AMPARs, and only assessing GluA2 could mask this (see below). It is intriguing that pre-synaptic changes might contribute to HSP, especially given the likely localization of Rab3A. But it remains difficult to distinguish if the apparent difference in imaging and electrophysiology is a methodological issue rather than a biological one. Stronger data, especially positive data on changes in release, will be necessary to conclude that pre-synaptic factors are required for HSP, beyond the established changes in post-synaptic receptor trafficking.

Regarding the concern that the lack of increase in receptors is due to a technical issue, please see General Response to Reviewers, above. We have also softened our conclusions throughout, acknowledging we cannot rule out a technical issue.

Other questions arise from the NASPM experiments, used to justify looking at GluA2 (and not GluA1) in the immunostaining. First, there is a strong frequency effect that is unclear in origin. One would expect NASPM to merely block some fraction of the post-synaptic current, and not affect pre-synaptic release or block whole synapses. But the change in frequency seems to argue (as the authors do) that some synapses only have CP-AMPARs, while the rest of the synapses have few or none. Another possibility is that there are pre-synaptic NASPM-sensitive receptors that influence release probability. Further, the amplitude data show a strong trend towards smaller amplitude following NASPM treatment (Fig 3B). The p value for both control and TTX neurons was 0.08 - it is very difficult to argue that there is no effect. The decrease on average is larger in the TTX neurons, and some cells show a strong effect. It is possible there is some heterogeneity between neurons on whether GluA1/A2 heteromers or GluA1 homomers are added during HSP. This would impact the conclusions about the GluA2 imaging as compared to the mEPSC amplitude data.

The key finding in Figure 3 is that NASPM did not eliminate the statistically significant increase in mEPSC amplitude after TTX treatment (Fig 3A).  Whether or not NASPM sensitive receptors contribute to mESPC amplitude is a separate question (Fig 3B). We are open to the possibility that NASPM reduces mEPSC amplitude in both control and TTX treated cells (p = 0.08 for both), but that does not change our conclusion that NASPM has no effect on the TTX-induced increase in mEPSC amplitude. The mechanism underlying the decrease in mEPSC frequency following NASPM is interesting, but does not alter our conclusions regarding the role of Rab3A in homeostatic synaptic plasticity of mEPSC amplitude. In addition, the Reviewer does not acknowledge the Supplemental Figure #1, which shows a similar lack of correspondence between homeostatic increases in mEPSC amplitude and GluA1 receptors in two cultures where matched data were obtained. Therefore, we do not think our lack of a robust increase in receptors can be explained by our failing to look at the relevant receptor.

To understand the role of Rab3A in HSP will require addressing two main issues:

(1) Is Rab3A acting pre-synaptically, post-synaptically or both? The authors provide good evidence that Rab3A is acting within neurons and not astrocytes. But where it is acting (pre or post) would aid substantially in understanding its role. The general view in the field has been that HSP is regulated post-synaptically via regulation of AMPAR trafficking, and considerable evidence supports this view. More concrete support for the authors' suggestion of a pre-synaptic site of control would be helpful.

We agree that definitive evidence for a presynaptic role of Rab3A in homeostatic plasticity of mEPSC amplitudes in mouse cortical cultures requires demonstrating that loss of Rab3A in postsynaptic neurons does not disrupt the plasticity, whereas loss in presynaptic neurons does. Without these data, we can only speculate that the Rab3A-dependence of homeostatic plasticity of quantal size in cortical neurons may be similar to that of the neuromuscular junction, where it cannot be receptors. We have added to the Discussion that the mechanism of Rab3A regulation of homeostatic plasticity of quantal amplitude could different between cortical neurons and the neuromuscular junction (lines 448-450 in markup,). Establishing a way to co-culture Rab3A-/- and Rab3A+/+ neurons in ratios that would allow us to record from a Rab3A-/- neuron that has mainly Rab3A+/+ inputs (or vice versa) is not impossible, but requires either transfection or transgenic expression with markers that identify the relevant genotype, and will be the subject of future experiments.

(2): Rab3A is also found at inhibitory synapses. It would be very informative to know if HSP at inhibitory synapses is similarly affected. This is particularly relevant as at inhibitory synapses, one expects a removal of GABARs or a decrease in GABA release (ie the opposite of whatever is happening at excitatory synapses). If both processes are regulated by Rab3A, this might suggest a role for this protein more upstream in the signaling; an effect only at excitatory synapses would argue for a more specific role just at those synapses.

We agree with the Reviewer, that it is important to determine the generality of Rab3A function in homeostatic plasticity. Establishing the homeostatic effect on mIPSCs and then examining them in Rab3A-/- cultures is a large undertaking and will be the subject of future experiments.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Minor (remaining) points:

(1) The figure referenced in the first response to the reviewers (Figure 5G) does not exist.

We meant Figure 5F, which has been corrected in the current response.

(2) I recommend showing the data without binning (despite some overlap).

The box plot in Origin will not allow not binning, but we can make the bin size so small that for all intents and purposes, there is close to 1 sample in each bin. When we do this, the majority of data are overlapped in a straight vertical line. Previously described concerns were regarding the gaps in the data, but it should be noted that these are cell means and we are not depicting the distributions of mEPSC amplitudes within a recording or across multiple recordings.

(3) Please auto-scale all axes from 0 (e.g., Fig 1E, F).

We have rescaled all mEPSC amplitude axes in box plots to go from 0 (Figures 1, 2 and 6).

(4) Typo in Figure legend 3: "NASPM (20 um)" => uM

Fixed.

Reviewer #2 (Recommendations for the authors):

(1) Line 140, frequencies are reported in Hz while other places are in sec-1, while these are essentially the same, they should be kept consistent in writing.

All mEPSC frequencies have been changed to sec^-1, except we have left “Hz” for repetitive stimulation and filtering.

(2) Paragraph starting from line 163 (as well as other places where multiple groups are compared, such as the occlusion discussion), the authors assessed whether there was a change in baseline between WT and mutant group by doing pairwise tests, this is not the right test. A two-way ANOVA, or at least a multivariant test would be more appropriate.

We have performed a two-way ANOVA, with genotype as one factor, and treatment as the other factor. The p values in Figures 1 and 2 have been revised to reflect p values from the post-hoc Tukey test on the specific interactions (for each particular genotype, TTX vs CON effects). The difference in the two WT strains, untreated, was not significant in the Post-Hoc Tukey test, and we have revised the text. The difference between the untreated WT from the Rab3A+/Ebd colony and the untreated Rab3AEbd/Ebd mutant was still significant in the Post-Hoc Tukey test, and this has replaced the Kruskal-Wallis test. The two-way ANOVA was also applied to the neuron-glia experiments and p values in Figure 6 adjusted accordingly.

(3) Relevant to the second point under minor concerns, I suggest this sentence be removed, as reducing variability and avoiding inhibitory projects are reasons good enough to restrict the analysis to the apical dendrites.

We have revised the description of the Turrigiano et al., 1998 finding from their Figure 3 and feel it still strengthens the justification for choosing to analyze only synapses on the apical dendrite.

Reviewer #3 (Recommendations for the authors):

Minor points:

The comments on lines 256-7 could seem misleading - the NASPM results wouldn't rule out contribution of those other subunits, only non-GluA2 containing combinations of those subunits. I would suggest revising this statement. Also, NASPM does likely have an effect, just not one that changes much with TTX treatment.

At new line 213 (markup) we have added the modifier “homomeric” to clarify our point that the lack of NASPM effect on the increase in mEPSC amplitude after TTX indicates that the increase is not due to more homomeric Ca²⁺-permeable receptors. We have always stated that NASPM reduces mEPSC amplitude, but it is in both control and treated cultures.

Strong conclusions based on a single culture (lines 314-5) seem unwarranted.

We have softened this statement with a “suggesting that” substituted for the previous “Therefore,” but stand by our point that the mEPSC amplitude data support a homeostatic effect of TTX in Culture #3, so the lack of increase in GluA2 cluster size needs an explanation other than variability in the homeostatic effect itself.

Saying (line 554) something is 'the only remaining possibility' also seems unwarranted.

We have softened this statement to read, “A remaining possibility…”.

Beattie EC, Stellwagen D, Morishita W, Bresnahan JC, Ha BK, Von Zastrow M, Beattie MS, Malenka RC (2002) Control of synaptic strength by glial TNFalpha. Science 295:2282-2285.

Mansour M, Nagarajan N, Nehring RB, Clements JD, Rosenmund C (2001) Heteromeric AMPA receptors assemble with a preferred subunit stoichiometry and spatial arrangement. Neuron 32:841-853. Stellwagen D, Malenka RC (2006) Synaptic scaling mediated by glial TNF-alpha. Nature 440:1054-1059.

Stellwagen D, Beattie EC, Seo JY, Malenka RC (2005) Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha. J Neurosci 25:3219-3228.

Swanson GT, Kamboj SK, Cull-Candy SG (1997) Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition. J Neurosci 17:5869.

Turrigiano GG, Leslie KR, Desai NS, Rutherford LC, Nelson SB (1998) Activity-dependent scaling of quantal amplitude in neocortical neurons. Nature 391:892-896.

Wang X, Wang Q, Yang S, Bucan M, Rich MM, Engisch KL (2011) Impaired activity-dependent plasticity of quantal amplitude at the neuromuscular junction of Rab3A deletion and Rab3A earlybird mutant mice. J Neurosci 31:3580-3588.

Wang X, Li Y, Engisch KL, Nakanishi ST, Dodson SE, Miller GW, Cope TC, Pinter MJ, Rich MM (2005) Activity-dependent presynaptic regulation of quantal size at the mammalian neuromuscular junction in vivo. J Neurosci 25:343-351.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study uncovers a protective role of the ubiquitin-conjugating enzyme variant Uev1A in mitigating cell death caused by over-expressed oncogenic Ras in polyploid Drosophila nurse cells and by RasK12 in diploid human tumor cell lines. The authors previously showed that overexpression of oncogenic Ras induces death in nurse cells, and now they perform a deficiency screen for modifiers. They identified Uev1A as a suppressor of this Ras-induced cell death. Using genetics and biochemistry, the authors found that Uev1A collaborates with the APC/C E3 ubiquitin ligase complex to promote proteasomal degradation of Cyclin A. This function of Uev1A appears to extend to diploid cells, where its human homologs UBE2V1 and UBE2V2 suppress oncogenic Ras-dependent phenotypes in human colorectal cancer cells in vitro and in xenografts in mice.

Strengths:

(1) Most of the data is supported by a sufficient sample size and appropriate statistics.

(2) Good mix of genetics and biochemistry.

(3) Generation of new transgenes and Drosophila alleles that will be beneficial for the community.

We greatly appreciate these comments.

Weaknesses:

(1) Phenotypes are based on artificial overexpression. It is not clear whether these results are relevant to normal physiology.

Downregulation of Uev1A, Ben, and Cdc27 together significantly increased the incidence of dying nurse cells in normal ovaries (Figure 2-figure supplement 4), indicating that the mechanism we uncovered also protects nurse cells from death during normal oogenesis.

(2) The phenotype of "degenerating ovaries" is very broad, and the study is not focused on phenotypes at the cellular level. Furthermore, no information is provided in the Materials and Methods on how degenerating ovaries are scored, despite this being the most important assay in the study.

Thanks for pointing out this issue. We quantified the phenotype of nurse cell death using “degrading/total egg chambers per ovary”, not “degenerating ovaries” (see all quantification data in our manuscript). Notably, this phenotype ranges from mild to severe. In normal nurse cells, nuclei exhibit a large, round morphology in DAPI staining (see the first panel in Figure 1D). During early death, nurse cell nuclei become disorganized and begin to condense and fragment (see the third panel in Figure 2-figure supplement 2E). In late-stage death, the nuclei are completely fragmented into small, condensed spherical structures (see the second panel in Figure 1D), making cellular-level phenotypic quantification impossible. Since all nurse cells within the same egg chamber are interconnected, their death process is synchronous. Thus, quantifying the phenotype at the egg-chamber level is more practical than at the cellular level. To improve clarity, we will provide a detailed description of the phenotype and integrate this explanation into the main text of the revised manuscript.

(3) In Figure 5, the authors want to conclude that uev1a is a tumor-suppressor, and so they over-express ubev1/2 in human cancer cell lines that have RasK12 and find reduced proliferation, colony formation, and xenograft size. However, genes that act as tumor suppressors have loss-of-function phenotypes that allow for increased cell division. The Drosophila uev1a mutant is viable and fertile, suggesting that it is not a tumor suppressor in flies. Additionally, they do not deplete human ubev1/2 from human cancer cell lines and assess whether this increases cell division, colony formation, and xenograph growth.

We apologize for our misleading description. In Figure 5, we aimed to demonstrate that UBE2V1/2, like Uev1A in Drosophila “nos>Ras^G12V+bam-RNAi” germline tumors (Figure 4), suppress oncogenic KRAS-driven overgrowth in diploid human cancer cells. Importantly, this function of Uev1A and UBE2V1/2 is dependent on Ras-driven tumors; there is no evidence that they act as broad tumor suppressors in the absence of oncogenic Ras. Drosophila uev1a mutants were lethal, not viable (see Lines 131-133), and germline-specific knockdown of uev1a (nos>uev1a-RNAi) caused female sterility without inducing tumors. These findings suggest that Uev1A lacks tumor-suppressive activity in the Drosophila female germline in the absence of Ras-driven tumors. We will revise the manuscript to prevent misinterpretation. Furthermore, we will investigate whether depletion of UBE2V1, UBE2V2, or both promotes oncogenic KRAS-driven overgrowth in human cancer cells.

(4) A critical part of the model does not make sense. CycA is a key part of their model, but they do not show CycA protein expression in WT egg chambers or in their over-expression models (nos.RasV12 or bam>RasV12). Based on Lilly and Spradling 1996, Cyclin A is not expressed in germ cells in region 2-3 of the germarium; whether CycA is expressed in nurse cells in later egg chambers is not shown but is critical to document comprehensively.

We appreciate this critical comment. CycA is a key cyclin that partners with Cdk1 to promote cell division (Edgar and Lehner, 1996). Notably, nurse cells are post-mitotic endocycling cells (Hammond and Laird, 1985) and typically do not express CycA (Lilly and Spradling, 1996) (see the last sentence, page 2518, paragraph 3). However, their death induced by oncogenic Ras^G12V is significantly suppressed by monoallelic deletion of either cycA or cdk1 (Zhang et al., 2024). Conversely, ectopic CycA expression in nurse cells triggers their death (Figure 2C, 2D). These findings suggest that polyploid nurse cells exhibit high sensitivity to aberrant division-promoting stress, which may represent a distinct form of cellular stress unique to polyploid cells. To further test our model, we will compare CycA expression levels in normal nurse cells versus those undergoing oncogenic Ras^G12V-induced cell death.

(5) The authors should provide more information about the knowledge base of uev1a and its homologs in the introduction.

Thanks for this suggestion. We will include this information in the introduction of the revised manuscript.

Reviewer #2 (Public review):

Summary:

The authors performed a genetic screen using deficiency lines and identified Uev1a as a factor that protects nurse cells from Ras^G12V-induced cell death. According to a previous study from the same lab, this cell death is caused by aberrant mitotic stress due to CycA upregulation (Zhang et al.). This paper further reveals that Uev1a forms a complex with APC/C to promote proteasome-mediated degradation of CycA.

In addition to polyploid nurse cells, the authors also examined the effect of Ras^G12V-overexpression in diploid germline cells, where Ras^G12V-overexpression triggers active proliferation, not cell death. Uev1a was found to suppress its overgrowth as well.

Finally, the authors show that the overexpression of the human homologs, UBE2V1 and UBE2V2, suppresses tumor growth in human colorectal cancer xenografts and cell lines. Notably, the expression of these genes correlates with the survival of colorectal cancer patients carrying the Ras mutation.

Strength:

This paper presents a significant finding that UBE2V1/2 may serve as a potential therapy for cancers harboring Ras mutations. The authors propose a fascinating mechanism in which Uev1a forms a complex with APC/C to inhibit aberrant cell cycle progression.

We greatly appreciate these comments.

Weakness:

The quantification of some crucial experiments lacks sufficient clarity.

Thanks for highlighting this issue. We will provide requested details regarding these quantification data in the revised manuscript.

References

Edgar, B.A., and Lehner, C.F. (1996). Developmental control of cell cycle regulators: a fly's perspective. Science 274, 1646-1652.

Hammond, M.P., and Laird, C.D. (1985). Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster. Chromosoma 91, 267-278.

Lilly, M.A., and Spradling, A.C. (1996). The Drosophila endocycle is controlled by Cyclin E and lacks a checkpoint ensuring S-phase completion. Genes Dev 10, 2514-2526.

Zhang, Q., Wang, Y., Bu, Z., Zhang, Y., Zhang, Q., Li, L., Yan, L., Wang, Y., and Zhao, S. (2024). Ras promotes germline stem cell division in Drosophila ovaries. Stem Cell Reports 19, 1205-1216.

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What is this?

Reviewer #2 (Public review):

This is an excellent paper. The ability to measure the immune response to multiple viruses in parallel is a major advancement for the field, which will be relevant across pathogens (assuming the assay can be appropriately adapted). I only have a few comments, focused on maximising the information provided by the sera.

Firstly, one of the major findings is that there is wide heterogeneity in responses across individuals. However, we could expect that individuals' responses should be at least correlated across the viruses considered, especially when individuals are of a similar age. It would be interesting to quantify the correlation in responses as a function of the difference in ages between pairs of individuals. I am also left wondering what the potential drivers of the differences in responses are, with age being presumably key. It would be interesting to explore individual factors associated with responses to specific viruses (beyond simply comparing adults versus children).

Relatedly, is the phylogenetic distance between pairs of viruses associated with similarity in responses?

Figure 5C is also a really interesting result. To be able to predict growth rates based on titers in the sera is fascinating. As touched upon in the discussion, I suspect it is really dependent on the representativeness of the sera of the population (so, e.g., if only elderly individuals provided sera, it would be a different result than if only children provided samples). It may be interesting to compare different hypotheses - so e.g., see if a population-weighted titer is even better correlated with fitness - so the contribution from each individual's titer is linked to a number of individuals of that age in the population. Alternatively, maybe only the titers in younger individuals are most relevant to fitness, etc.

In Figure 6, the authors lump together individuals within 10-year age categories - however, this is potentially throwing away the nuances of what is happening at individual ages, especially for the children, where the measured viruses cross different groups. I realise the numbers are small and the viruses only come from a small numbers of years, however, it may be preferable to order all the individuals by age (y-axis) and the viral responses in ascending order (x-axis) and plot the response as a heatmap. As currently plotted, it is difficult to compare across panels

sub-500ms can be either really slow or really fast all depending on context. For example, league of legends ping versus downloading a video. I would probably choose a different metric here so it's less confusing and more "wow"y

Boost: Aumentar, mejorar o fortalecer. • Rigorous instruction: Se refiere a una enseñanza desafiante, profunda y de alta calidad que promueve el pensamiento crítico y la comprensión profunda, no solo la memorización. • Student feedback: Retroalimentación que reciben los estudiantes sobre su desempeño, útil para mejorar su aprendizaje. • Assessment: Evaluación del aprendizaje para medir el progreso y las necesidades. • All content areas: Todas las asignaturas o áreas del currículo (como matemáticas, lengua, ciencias, etc.).

Ici, l’auteur généralise un profil type en se basant sur une figure caricaturale. Cette typologie souffre d’un manque de nuance.

Reviewer #1 (Public review):

In this study the authors aim to understand why decision formation during behavioural tasks is distributed across multiple brain areas. They hypothesize that multiple areas are used in order to implement an information bottleneck (IB). Using neural activity recorded from monkey DLPFC and PMd performing a 2-AFC task, they show that DLPFC represents various task variables (decision, color, target configuration), while downstream PMd primarily represents decision information. Since decision information is the only information needed to make a decision, the authors suggest that PMd has a minimal sufficient representation (as expected from an IB). They then train 3-area RNNs on the same task, and show that activity in the first and third areas resemble the neural representations of DLPFC and PMd, respectively. In order to propose a mechanism, they analyse the RNN and find that area 3 ends up with primarily decision information because feedforward connections between areas primarily propagate decision information.

Overall, the paper reads well and the data analysis and RNN modeling are well done and mostly correct. I agree with the authors that PMd has less information than DLPFC, meaning that some of the target and color information is attenuated. I also agree that this also happens in their multi-area RNN.

However, I find the use of the IB principle here muddles the water rather than clarifying anything. The key problem is that the authors evoke the information bottleneck in a mostly intuitive sense, but they do not actually use it (say, in their modelling). Rather, the IB is simply used to motivate why information will be or should be lost. Since the IB is a generic compressor, however, it does not make any statements about how a particular compression should be distributed or computed across brain areas.

If I ignore the reference to the information bottleneck, I still see a more mechanistic study that proposes a neural mechanism of how decisions are formed, in the tradition of RNN-modelling of neural activity as in Mante et al 2013. Seen through this more limited sense, the present study succeeds at pointing out a good model-data match.

Major points

(1) The IB is a formal, information-theoretic method to identify relevant information. However, in the paper, reference to the information bottleneck method (IB) is only used to motivate why (task-irrelevant) information should be lost in higher areas. The IB principle itself is actually never used. The RNNs are fitted using standard techniques, without reference to the IB. Without a formal link, I think the authors should describe their findings using words (e.g., task-irrelevant information is lost), rather than stating this as evidence for an information-theoretic principle.

(2) The advantage of employing a formal theory is that all assumptions have to be clarified. Since the authors only evoke the IB, but never employ it, they refrain from clarifying some of their assumptions. That is what creates unnecessary confusion.

For instance, the authors cite the following predictions of the IB principle: "(1) There exists a downstream area of cortex that has a minimal and sufficient representation to perform a task ... (2) there exists an upstream area of cortex that has more task information than the minimal sufficient area" - However, since the information bottleneck method is a generic compressor, it does not make any predictions about areas (or neurons). For a given sensory input p(x), a given task output p(y|x), and a given information loss, the IB generates exactly one optimal representation. In other words, the predictions made by the authors relie on other assumptions (e.g. feedforward processing, hierarchy, etc.) and these are not clearly stated.

(3) A corrollary to this problem is that the authors do not formally define task-irrelevant information. It seems the authors simply use the choice or decision as the thing that needs to be computed, and identify all other information as task-irrelevant. That's at least what I glean from the RNN model. However, I find that highly confusing because it suggests the conclusion that color information or target information are task-irrelevant. Surely, that cannot be true, since the decision is based on these quantities!

(4) If we define the output as the only task-relevant information, then any representation that is a pure motor representation would qualify as a minimal sufficient representation to carry out the correct actions. However, it is well-known that sensory information is lost in motor areas. It is not clear to me what exactly we gain by calling motor representations "minimal sufficient representations."

In summary, I think the authors should refrain from evoking the IB - which is a formal, mathematical principle - unless they actually use it formally as well.

Author response:

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

Reviewer #1 (Public review):

Summary:

In this manuscript, Pakula et al. explore the impact of reactive oxygen species (ROS) on neonatal cerebellar regeneration, providing evidence that ROS activates regeneration through Nestin-expressing progenitors (NEPs). Using scRNA-seq analysis of FACS-isolated NEPs, the authors characterize injury-induced changes, including an enrichment in ROS metabolic processes within the cerebellar microenvironment. Biochemical analyses confirm a rapid increase in ROS levels following irradiation and forced catalase expression, which reduces ROS levels, and impairs external granule layer (EGL) replenishment post-injury.

Strengths:

Overall, the study robustly supports its main conclusion and provides valuable insights into ROS as a regenerative signal in the neonatal cerebellum.

Comments on revisions:

The authors have addressed most of the previous comments. However, they should clarify the following response:

*"For reasons we have not explored, the phenotype is most prominent in these lobules, that is why they were originally chosen. We edited the following sentence (lines 578-579):

First, we analyzed the replenishment of the EGL by BgL-NEPs in vermis lobules 3-5, since our previous work showed that these lobules have a prominent defect."*

It has been reported that the anterior part of the cerebellum may have a lower regenerative capacity compared to the posterior lobe. To avoid potential ambiguity, the authors should clarify that "the phenotype" and "prominent defect" refer to more severe EGL depletion at an earlier stage after IR rather than a poorer regenerative outcome. Additionally, they should provide a reference to support their statement or indicate if it is based on unpublished observations.

Our comment does not refer to a more severe EGL depletion at an earlier stage. There is instead poorer regeneration of the anterior region. The irradiation approach used provides consistent cell killing of GCPs across the cerebellum. This can be seen in Fig. 1c, e, g, i in our previous publication: Wojcinski, et al. (2017) Cerebellar granule cell replenishment post-injury by adaptive reprogramming of Nestin+ progenitors. Nature Neuroscience, 20:1361-1370). Also, Fig 2e, g, k, m in the paper shows that by P5 and P8, posterior lobule 8 recovers better than anterior lobules 1-5.

Reviewer #2 (Public review):

Summary:

The authors have previously shown that the mouse neonatal cerebellum can regenerate damage to granule cell progenitors in the external granular layer, through reprogramming of gliogenic nestin-expressing progenitors (NEPs). The mechanisms of this reprogramming remain largely unknown. Here the authors used scRNAseq and ATACseq of purified neonatal NEPs from P1-P5 and showed that ROS signatures were transiently upregulated in gliogenic NEPs ve neurogenic NEPs 24 hours post injury (P2). To assess the role of ROS, mice transgenic for global catalase activity were assessed to reduce ROS. Inhibition of ROS significantly decreased gliogenic NEP reprogramming and diminished cerebellar growth post-injury. Further, inhibition of microglia across this same time period prevented one of the first steps of repair - the migration of NEPs into the external granule layer. This work is the first demonstration that the tissue microenvironment of the damaged neonatal cerebellum is a major regulator of neonatal cerebellar regeneration. Increased ROS is seen in other CNS damage models, including adults, thus there may be some shared mechanisms across age and regions, although interestingly neonatal cerebellar astrocytes do not upregulate GFAP as seen in adult CNS damage models. Another intriguing finding is that global inhibition of ROS did not alter normal cerebellar development.

Strengths:

This paper presents a beautiful example of using single cell data to generate biologically relevant, testable hypotheses of mechanisms driving important biological processes. The scRNAseq and ATACseq analyses are rigorously conducted and conclusive. Data is very clearly presented and easily interpreted supporting the hypothesis next tested by reduce ROS in irradiated brains.

Analysis of whole tissue and FAC sorted NEPS in transgenic mice where human catalase was globally expressed in mitochondria were rigorously controlled and conclusively show that ROS upregulation was indeed decreased post injury and very clearly the regenerative response was inhibited. The authors are to be commended on the very careful analyses which are very well presented and again, easy to follow with all appropriate data shown to support their conclusions.

Weaknesses:

The authors also present data to show that microglia are required for an early step of mobilizing gliogenic NEPs into the damaged EGL. While the data that PLX5622 administration from P0-P5 or even P0-P8 clearly shows that there is an immediate reduction of NEPs mobilized to the damaged EGL, there is no subsequent reduction of cerebellar growth such that by P30, the treated and untreated irradiated cerebella are equivalent in size. There is speculation in the discussion about why this might be the case. Additional experiments and tools are required to assess mechanisms. Regardless, the data still implicate microglia in the neonatal regenerative response, and this finding remains an important advance.

As stated previously, the suggested follow up experiments while relevant are extensive and considered beyond the scope of the current paper.

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this manuscript, Pakula et al. explore the impact of reactive oxygen species (ROS) on neonatal cerebellar regeneration, providing evidence that ROS activates regeneration through Nestin-expressing progenitors (NEPs). Using scRNA-seq analysis of FACS-isolated NEPs, the authors characterize injury-induced changes, including an enrichment in ROS metabolic processes within the cerebellar microenvironment. Biochemical analyses confirm a rapid increase in ROS levels following irradiation, and forced catalase expression, which reduces ROS levels, and impairs external granule layer (EGL) replenishment post-injury.

Strengths:

Overall, the study robustly supports its main conclusion and provides valuable insights into ROS as a regenerative signal in the neonatal cerebellum.

Weaknesses:

(1) The diversity of cell types recovered from scRNA-seq libraries of sorted Nes-CFP cells is unexpected, especially the inclusion of minor types such as microglia, meninges, and ependymal cells. The authors should validate whether Nes and CFP mRNAs are enriched in the sorted cells; if not, they should discuss the potential pitfalls in sampling bias or artifacts that may have affected the dataset, impacting interpretation.

In our previous work, we thoroughly assessed the transgene using RNA in situ hybridization for Cfp, immunofluorescent analysis for CFP and scRNA-seq analysis for Cfp transcripts (Bayin et al., Science Adv. 2021, Fig. S1-2)(1), and characterized the diversity within the NEP populations of the cerebellum. Our present scRNA-seq data also confirms that Nes transcripts are expressed in all the NEP subtypes. A feature plot for Nes expression has been added to the revised manuscript (Fig 1E), as well as a sentence explaining the results. Of note, since this data was generated from FACS-isolated CFP+ cells, the perdurance of the protein allows for the detection of immediate progeny of Nes-expressing cells, even in cells where Nes is not expressed once cells are differentiated. Finally, oligodendrocyte progenitors, perivascular cells, some rare microglia and ependymal cells have been demonstrated to express Nes in the central nervous system; therefore, detecting small groups of these cells is expected (2-4). We have added the following sentence (lines 391-394):

“Detection of Nes mRNA confirmed that the transgene reflects endogenous Nes expression in progenitors of many lineages, and also that the perdurance of CFP protein in immediate progeny of Nes-expressing cells allowed the isolation of these cells by FACS (Figure 1E)”.

(2) The authors should de-emphasize that ROS signaling and related gene upregulation exclusively in gliogenic NEPs. Genes such as Cdkn1a, Phlda3, Ass1, and Bax are identified as differentially expressed in neurogenic NEPs and granule cell progenitors (GCPs), with Ass1 absent in GCPs. According to Table S4, gene ontology (GO) terms related to ROS metabolic processes are also enriched in gliogenic NEPs, neurogenic NEPs, and GCPs.

As the reviewer requested, we have de-emphasized that ROS signaling is preferentially upregulated in gliogenic NEPs, since we agree with the reviewer that there is some evidence for similar transcriptional signatures in neurogenic NEPs and GCPs. We added the following (lines 429-531):

“Some of the DNA damage and apoptosis related genes that were upregulated in IR gliogenic-NEPs (Cdkn1a, Phlda3, Bax) were also upregulated in the IR neurogenic-NEPs and GCPs at P2 (Supplementary Figure 2B-E).”

And we edited the last few sentences of the section to state (lines 453-459):

“Interestingly, we did not observe significant enrichment for GO terms associated with cellular stress response in the GCPs that survived the irradiation compared to controls, despite significant enrichment for ROS signaling related GO-terms (Table S4). Collectively, these results indicate that injury induces significant and overlapping transcriptional changes in NEPs and GCPs. The gliogenic- and neurogenic-NEP subtypes transiently upregulate stress response genes upon GCP death, and an overall increase in ROS signaling is observed in the injured cerebella.”

(3) The authors need to justify the selection of only the anterior lobe for EGL replenishment and microglia quantification.

We thank the reviewers for asking for this clarification. Our previous publications on regeneration of the EGL by NEPs have all involved quantification of these lobules, thus we think it is important to stay with the same lobules. For reasons we have not explored, the phenotype is most prominent in these lobules, that is why they were originally chosen. We edited the following sentence (lines 578-579):

“First, we analyzed the replenishment of the EGL by BgL-NEPs in vermis lobules 3-5, since our previous work showed that these lobules have a prominent defect.”

(4) Figure 1K: The figure presents linkages between genes and GO terms as a network but does not depict a gene network. The terminology should be corrected accordingly.

We have corrected the terminology and added the following (lines 487-489):

“Finally, linkages between the genes in differentially open regions identified by ATAC-seq and the associated GO-terms revealed an active transcriptional network involved in regulating cell death and apoptosis (Figure 1K).”

(5) Figure 1H and S2: The x-axis appears to display raw p-values rather than log10(p.value) as indicated. The x-axis should ideally show -log10(p.adjust), beginning at zero. The current format may misleadingly suggest that the ROS GO term has the lowest p-values.

Apologies for the mistake. The data represents raw p-values and the x-axis has been corrected.

(6) Genes such as Ppara, Egln3, Foxo3, Jun, and Nos1ap were identified by bulk ATAC-seq based on proximity to peaks, not by scRNA-seq. Without additional expression data, caution is needed when presenting these genes as direct evidence of ROS involvement in NEPs.

We modified the text to discuss the discrepancies between the analyses. While some of this could be due to the lower detection limits in the scRNA-seq, it also highlights that chromatin accessibility is not a direct readout for expression levels and further analysis is needed. Nevertheless, both scRNA-seq and ATAC-seq have identified similar mechanisms, and our mutant analysis confirmed our hypothesis that an increase in ROS levels underlies repair, further increasing the confidence in our analyses. Further investigation is needed to understand the downstream mechanisms. We added the following sentence (lines 478-481):

“However, not all genes in the accessible areas were differentially expressed in the scRNA-seq data. While some of this could be due to the detection limits of scRNA-seq, further analysis is required to assess the mechanisms of how the differentially accessible chromatin affects transcription.”

(7) The authors should annotate cell identities for the different clusters in Table S2.

All cell types have been annotated in Table S2.

(8) Reiterative clustering analysis reveals distinct subpopulations among gliogenic and neurogenic NEPs. Could the authors clarify the identities of these subclusters? Can we distinguish the gliogenic NEPs in the Bergmann glia layer from those in the white matter?

Thank you for this clarification. As shown in our previous studies, we can not distinguish between the gliogenic NEPs in the Bergmann glia layer and the white matter based on scRNA-seq, but expression of the Bergmann glia marker Gdf10 suggests that a large proportion of the cells in the Hopx+ clusters are in the Bergmann glia layer. The distinction within the major subpopulations that we characterized (Hopx-, Ascl1-expressing NEPs and GCPs) are driven by their proliferative/maturation status as we previously observed. We have included a detailed annotation of all the clusters in Table S2, as requested and a UMAP for mKi57 expression in Fig 1E. We have clarified this in the following sentence (lines 383-385):

“These groups of cells were further subdivided into molecularly distinct clusters based on marker genes and their cell cycle profiles or developmental stages (Figure 1D, Table S2).”

(9) In the Methods section, the authors mention filtering out genes with fewer than 10 counts. They should specify if these genes were used as background for enrichment analysis. Background gene selection is critical, as it influences the functional enrichment of gene sets in the list.

As requested, the approach used has been added to the Methods section of the revised paper. Briefly, the background genes used by the goseq function are the same genes used for the probability weight function (nullp). The mm8 genome annotation was used in the nullp function, and all annotated genes were used as background genes to compute GO term enrichment. The following was added (lines 307-308):

“The background genes used to compute the GO term enrichment includes all genes with gene symbol annotations within mm8.”

(10) Figure S1C: The authors could consider using bar plots to better illustrate cell composition differences across conditions and replicates.

As suggested, we have included bar plots in Fig. S1D-F.

(11) Figures 4-6: It remains unclear how the white matter microglia contribute to the recruitment of BgL-NEPs to the EGL, as the mCAT-mediated microglia loss data are all confined to the white matter.

We have thought about the question and had initially quantified the microglia in the white matter and the rest of the lobules (excluding the EGL) separately. However, there are very few microglia outside the white matter in each section, thus it is not possible to obtain reliable statistical data on such a small population. We therefore did not include the cells in the analysis. We have added this point in the main text (line 548).

“As a possible explanation for how white matter microglia could influence NEP behaviors, given the small size of the lobules and how the cytoarchitecture is disrupted after injury, we think it is possible that secreted factors from the white matter microglia could reach the BgL NEPs. Alternatively, there could be a relay system through an intermediate cell type closer to the microglia.” We have added these ideas to the Discussion of the revised paper (lines 735-738).

Reviewer #2 (Public review):

Summary:

The authors have previously shown that the mouse neonatal cerebellum can regenerate damage to granule cell progenitors in the external granular layer, through reprogramming of gliogenic nestin-expressing progenitors (NEPs). The mechanisms of this reprogramming remain largely unknown. Here the authors used scRNAseq and ATACseq of purified neonatal NEPs from P1-P5 and showed that ROS signatures were transiently upregulated in gliogenic NEPs ve neurogenic NEPs 24 hours post injury (P2). To assess the role of ROS, mice transgenic for global catalase activity were assessed to reduce ROS. Inhibition of ROS significantly decreased gliogenic NEP reprogramming and diminished cerebellar growth post-injury. Further, inhibition of microglia across this same time period prevented one of the first steps of repair - the migration of NEPs into the external granule layer. This work is the first demonstration that the tissue microenvironment of the damaged neonatal cerebellum is a major regulator of neonatal cerebellar regeneration. Increased ROS is seen in other CNS damage models including adults, thus there may be some shared mechanisms across age and regions, although interestingly neonatal cerebellar astrocytes do not upregulate GFAP as seen in adult CNS damage models. Another intriguing finding is that global inhibition of ROS did not alter normal cerebellar development.

Strengths:

This paper presents a beautiful example of using single cell data to generate biologically relevant, testable hypotheses of mechanisms driving important biological processes. The scRNAseq and ATACseq analyses are rigorously conducted and conclusive. Data is very clearly presented and easily interpreted supporting the hypothesis next tested by reduce ROS in irradiated brains.

Analysis of whole tissue and FAC sorted NEPS in transgenic mice where human catalase was globally expressed in mitochondria were rigorously controlled and conclusively show that ROS upregulation was indeed decreased post injury and very clearly the regenerative response was inhibited. The authors are to be commended on the very careful analyses which are very well presented and again, easy to follow with all appropriate data shown to support their conclusions.

Weaknesses:

The authors also present data to show that microglia are required for an early step of mobilizing gliogenic NEPs into the damaged EGL. While the data that PLX5622 administration from P0-P5 or even P0-P8 clearly shows that there is an immediate reduction of NEPs mobilized to the damaged EGL, there is no subsequent reduction of cerebellar growth such that by P30, the treated and untreated irradiated cerebella are equivalent in size. There is speculation in the discussion about why this might be the case, but there is no explanation for why further, longer treatment was not attempted nor was there any additional analyses of other regenerative steps in the treated animals. The data still implicate microglia in the neonatal regenerative response, but how remains uncertain.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

This is an exemplary manuscript.

The methods and data are very well described and presented.

I actually have very little to ask the authors except for an explanation of why PLX treatment was discontinued after P5 or P8 and what other steps of NEP reprogramming were assessed in these animals? Was NEP expansion still decreased at P8 even in the presence of PLX at this stage? Also - was there any analysis attempted combining mCAT and PLX?

We agree with the reviewer that a follow up study that goes into a deeper analysis of the role of microglia in GCP regeneration and any interaction with ROS signaling would interesting. However, it would require a set of tools that we do not currently have. We did not have enough PLX5622 to perform addition experiments or extend the length of treatment. Plexxikon informed us in 2021 that they were no longer manufacturing PLX5622 because they were focusing on new analogs for in vivo use, and thus we had to use what we had left over from a completed preclinical cancer study. We nevertheless think it is important to publish our preliminary results to spark further experiments by other groups.

References

(1) Bayin N. S. Mizrak D., Stephen N. D., Lao Z., Sims P. A., Joyner A. L. Injury induced ASCL1 expression orchestrates a transitory cell state required for repair of the neonatal cerebellum. Sci Adv. 2021;7(50):eabj1598.

(2) Cawsey T, Duflou J, Weickert CS, Gorrie CA. Nestin-Positive Ependymal Cells Are Increased in the Human Spinal Cord after Traumatic Central Nervous System Injury. J Neurotrauma. 2015;32(18):1393-402.

(3) Gallo V, Armstrong RC. Developmental and growth factor-induced regulation of nestin in oligodendrocyte lineage cells. The Journal of neuroscience : the official journal of the Society for Neuroscience. 1995;15(1 Pt 1):394-406.

(4) Huang Y, Xu Z, Xiong S, Sun F, Qin G, Hu G, et al. Repopulated microglia are solely derived from the proliferation of residual microglia after acute depletion. Nat Neurosci. 2018;21(4):530-40.

DOI: 10.1038/s42003-025-08148-y

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DOI: 10.1038/s42003-025-08148-y

Resource: (GeneTex Cat# GTX135357, RRID:AB_2868464)

Curator: @scibot

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DOI: 10.1007/s10875-025-01886-y

Resource: Johns Hopkins University School of Medicine Genetic Resources Core Facility (RRID:SCR_018669)

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Note de Synthèse : Où commence la folie ?

Ce document de synthèse examine les principales thématiques et idées importantes présentées dans les extraits de "Où commence la folie ? | 42 - La réponse à presque tout | ARTE".

Thèmes Principaux :

• La Normalité comme Construction Sociale : Le concept de "norme" est présenté non pas comme une réalité intrinsèque mais comme une construction sociale nécessaire à l'interaction. En conséquence, la folie, définie par opposition à la norme, devient également relative.

• La Folie au Sens Psychiatrique : La psychiatrie tend à éviter le terme "folie", préférant parler de troubles variés, principalement la psychose. La psychose est caractérisée par une altération du lien avec la réalité et affecte le raisonnement et la perception.

• Mécanismes de la Psychose (notamment la Schizophrénie) : Les extraits décrivent comment la psychose, souvent associée à la schizophrénie, peut impliquer l'interprétation de coïncidences comme des signes ou des messages codés. Un sentiment d'insécurité identitaire et environnementale peut conduire à des interprétations délirantes.

• La "Bouffée Délirante" : Un épisode intense d'angoisse et d'interprétation délirante, qui paradoxalement peut apporter un soulagement en trouvant une explication (même absurde) à l'insécurité ressentie.

• La Folie comme Hyper-sensibilité ou Perception Décalée : L'idée est explorée que les troubles psychiatriques, y compris la psychose, pourraient être des formes amplifiées de caractéristiques présentes chez les personnes dites "normales". Notre perception de la réalité est toujours une interprétation influencée par notre histoire personnelle.

• Absence de Frontière Claire entre Folie et Normalité : Il n'existe pas de critères strictement définis pour séparer clairement la folie de la normalité. Le critère principal d'une conviction erronée et inébranlable peut aussi s'appliquer à d'autres formes de croyances non pathologiques.

• Le Diagnostic et la Souffrance : Le but du diagnostic en psychiatrie est de soigner les personnes en souffrance. Cependant, la souffrance n'est pas toujours perçue par la personne concernée (ex: épisode maniaque), mais peut être ressentie par l'entourage ou apparaître après l'épisode (souffrance des conséquences). La pertinence d'un diagnostic se mesure plutôt par la souffrance qu'il vise à soulager.

• Symptômes Psychotiques chez les Personnes "en Bonne Santé" : Un pourcentage significatif de personnes sans diagnostic formel peuvent présenter des symptômes psychotiques (perceptions différentes, voix, idées bizarres) sans être considérées comme malades.

• L'Approche centrée sur le Patient : L'importance de déterminer les objectifs d'un traitement en fonction du patient, et non uniquement du médecin. Un symptôme (comme entendre une voix) peut ne pas être source de souffrance pour le patient et ne pas nécessiter d'être supprimé.

• La Neurodiversité : Ce concept remet en question l'idée que certaines particularités, notamment neurodéveloppementales (comme l'autisme), soient des maladies. Elles sont vues comme des expressions naturelles de la diversité humaine. La distinction est faite entre troubles neurodéveloppementaux (avec lesquels on naît et qui ne guérissent pas) et maladies psychiatriques (acquises et potentiellement curables).

• L'Évolution du Diagnostic et la Stigmatisation Historique : Les critères diagnostiques ont évolué, menant à une augmentation des diagnostics pour certaines conditions (comme l'autisme). L'histoire montre une persécution et une stigmatisation des personnes jugées différentes, culminant avec les atrocités du régime nazi. La stigmatisation et la discrimination persistent aujourd'hui, notamment dans la sphère professionnelle.

• La Folie et la Créativité : Le lien potentiel entre une prédisposition aux troubles psychotiques et une tendance accrue à la créativité est évoqué, citant des études génétiques. Cependant, l'idéalisation de ce lien est tempérée par le fait que les phases aiguës d'une psychose entravent généralement la création.

• Le Diagnostic et les Particularités : Certaines particularités (comme le haut potentiel intellectuel) peuvent être confondues avec des troubles (comme le TDAH), soulignant la nécessité d'une évaluation précise pour distinguer une aptitude d'un handicap. Le diagnostic est évolutif et peut enfermer l'individu.

• L'Acceptation de la Différence et de sa Propre "Folie" : L'importance pour la société d'accepter les écarts à la norme et pour les individus d'accepter leurs propres singularités et vulnérabilités.

Idées ou Faits Importants :

• La norme est une invention sociale et non une réalité absolue.

• La "folie" dans le langage courant correspond souvent à ce que la psychiatrie appelle "psychose", caractérisée par une perte du lien avec la réalité.

• Un critère clé de la folie est une conviction inébranlable jugée erronée par les autres, mais ce critère peut s'appliquer au-delà des pathologies.

• Il n'y a pas de frontière nette entre folie et normalité.

• La souffrance est un critère important pour le diagnostic psychiatrique, même si elle n'est pas toujours perçue par le patient lui-même durant l'épisode.

• Des symptômes psychotiques peuvent exister chez des personnes sans diagnostic formel.

• L'objectif d'un traitement devrait être défini par le patient.

• Le concept de neurodiversité voit les particularités (notamment neurodéveloppementales) comme des expressions de la diversité humaine, et non nécessairement comme des maladies.

• Les troubles neurodéveloppementaux sont innés, tandis que les maladies psychiatriques sont acquises.

• L'histoire a montré une stigmatisation et une persécution des personnes jugées différentes, et cette stigmatisation persiste aujourd'hui.

• Il pourrait exister un lien entre la prédisposition aux troubles psychotiques et la créativité.

• Le monde peut parfois nous rendre "fous" face aux expériences traumatisantes, soulignant la vulnérabilité humaine.

• Accepter la diversité humaine et même sa propre "folie" est crucial.

Citations Clés :

• "je pense que la norme en tant que telle elle n'existe pas c'est un concept qu'on a qu'on a inventé qu'on a construit parce qu'on en a besoin pour interagir socialement tous ensemble"

• "ce que l'on désigne par folie correspond en général à ce que la psychiatrie appelle une psychose à savoir un état dans lequel tout lien avec la réalité ou du moins ce qu'on perçoit comme tel se brouille"

• "lorsque vous vous dites "Je suis en train de donner une interview" vous savez pertinemment qui est le jeu en question pour une personne schizophrène en revanche c'est loin d'être aussi évident"

• "il n'existe pas de critères rigoureusement définis qui permettent de tracer une frontière claire entre la folie et la normalité"

• "ce qui compte dans le diagnostic des maladies psychiatriques c'est plutôt de savoir si une personne souffre et non pas si elle est atteinte de folie ou si elle a un comportement normal"

• "le but d'un traitement doit être déterminé par le patient pas par le médecin"

• "en définitive la folie n'est qu'une question de point de vue tout ce qui semble décalé n'a pas forcément besoin d'être soigné"

• "Le concept de neurodiversité remet en question l'idée même que certaines particularités soient considérées comme des maladies ou des troubles il les entrevoi plutôt comme une expression naturelle de la diversité humaine"

• "la différence entre un trouble neurodéveloppemental et une maladie psychiatrique c'est que la maladie psychiatrique on on l'acquiert comme on se casse une jambe finalement... alors qu'un trouble neuréveloppemental on vient au monde avec et on le garde toute sa vie"

• "l'idée reste profondément ancrée dans notre société [la stigmatisation] et que la seule manière d'y faire face ce qui est fondamental notamment pour les personnes atteintes de troubles psychiatriques c'est de sensibiliser le grand public"

• "il pourrait exister un lien entre une prédisposition au trouble psychotique et une tendance accrue à la créativité"

• "le monde peut parfois nous rendre fou des expériences traumatisantes peuvent conduire certaines personnes à sombrer dans la folie au fond je crois que cela fait partie de la condition humaine"

• "peut-être devrions-nous aussi apprendre à mieux accepter notre propre folie"

En résumé, les extraits remettent en question les notions rigides de folie et de normalité, les présentant comme des constructions sociales ou des spectres de perception et de fonctionnement humain.

Ils soulignent la complexité des troubles psychiatriques, l'importance de l'approche centrée sur le patient, et appellent à une plus grande acceptation de la diversité humaine et à la déstigmatisation.

Compte rendu détaillé des thèmes et idées clés du podcast "Les enfants rendent-ils heureux ? | Unhappy | ARTE"

Ce podcast explore la question complexe de l'impact des enfants sur le bonheur parental, en interrogeant différentes perspectives et expériences.

Il s'appuie sur des témoignages personnels, des opinions d'experts et des études socio-économiques.

Thèmes principaux et idées clés :

• La parentalité : Un défi enrichissant mais exigeant ("Une vie d'extrémiste")

• Le podcast commence par souligner que, bien que les parents puissent parfois préférer des activités solitaires comme regarder la télévision, le temps partagé avec les enfants est souvent source d'enrichissement et de sens.

• Le bonheur parental ne réside pas toujours dans des moments de joie continue, mais aussi dans la confrontation et le dépassement des défis inhérents à l'éducation.

• Un parallèle est établi entre la parentalité et des défis extrêmes comme l'escalade du Kilimanjaro, mais avec la différence fondamentale qu'on ne peut pas "revenir en arrière". La philosophe d'iter Tomy est citée, affirmant que la parentalité est "réservée aux extrémistes".

• Louise, une mère célibataire, témoigne du sentiment d'être constamment dépassée et de la peur de "couler" au début, un mélange de surmenage, d'insomnie, d'angoisse existentielle et de crainte face à la vulnérabilité d'un si petit enfant.

• La parentalité est décrite comme une "activité extrêmement intense" qui demande énormément de temps et d'énergie, conduisant à reléguer les propres besoins au second plan.

L'instinct versus la raison dans la décision d'avoir des enfants :

• La question de savoir si l'on doit suivre son instinct ou la raison pour décider d'avoir un enfant est soulevée.

• Établir une liste de "pour" et de "contre" pour avoir un enfant est jugé "incongru", notamment en considérant l'impact potentiel sur l'enfant qui grandit et pourrait découvrir qu'il ne "coche pas tous les éléments de la liste".

• La parentalité est présentée comme un "mythe redoutable très fortement chargé émotionnellement par la société". Il est conseillé de se demander sincèrement si le désir d'avoir un enfant vient de soi ou est influencé par les "désirs des autres".

• Louise, ayant découvert sa grossesse de manière inattendue, témoigne de s'être fiée à son instinct et de n'avoir jamais douté de vouloir garder son enfant malgré les difficultés.

Les aspects du bonheur parental :

• Au-delà des défis, les enfants sont sources de bonheur. Louise explique que le bonheur vient en partie de l'acceptation que son fils peut aussi lui apprendre beaucoup de choses et lui a permis de découvrir de nouvelles facettes d'elle-même. Cela aide aussi à "être plus tolérant envers soi-même".

• L'attirance innée pour les caractéristiques physiques des bébés (grands yeux, front haut, joues rebondies) est mentionnée comme un facteur initial de sentiment positif.

• Tilman, père de quatre filles, affirme que pour lui, le bonheur est indissociable : celui qu'il partage en couple, en famille avec les enfants, et celui qu'il cultive de son côté. C'est un bonheur "avec les enfants", et non "malgré" ou "à cause" d'eux.

Les réalités socio-économiques de la parentalité :

• Une étude longitudinale allemande (panel socio-économique) révèle que la satisfaction de vie augmente avant la naissance, atteint un pic la première année, puis diminue et revient au niveau initial. La satisfaction de vie ne diffère pas significativement entre les personnes avec ou sans enfants sur le long terme.

• Le coût financier de l'éducation d'un enfant est souligné (environ 150 000 € pour le premier enfant en Allemagne) ainsi que l'impact sur la carrière professionnelle de nombreux parents.

• Une augmentation du niveau de colère quotidienne chez les parents 5 ans après la naissance est notée, probablement liée à l'augmentation du stress.

La dynamique du couple et le partage des tâches :

• La parentalité transforme la relation de couple en une "sorte de relation de travail", nécessitant une communication constante et une répartition des tâches.

• Il est observé que les hommes ont tendance à surestimer leur part de travail ménager par rapport à la réalité.

• Le manque de congé parental pris par les pères est souligné (seulement 18% en Allemagne, prenant en moyenne 2 mois contre 13 pour les femmes), indiquant un déséquilibre persistant dans la charge parentale.

• Tilman et Ilana, avec quatre enfants, insistent sur l'importance de l'improvisation et de la communication permanente pour concilier famille nombreuse et vies professionnelles.

Les regrets et le tabou de la non-maternité :

• La publication de l'étude "Regretting Motherhood" par Orna Donat, interrogeant des femmes qui aiment leurs enfants mais regretteraient de devenir mères si elles pouvaient revenir en arrière, est mentionnée comme ayant brisé un tabou important.

• La recherche suggère qu'environ 10% des parents, s'ils pouvaient revenir en arrière, choisiraient de ne pas avoir d'enfants.

• Il est souligné que les femmes devraient avoir la liberté de dire que les enfants ne sont pas toujours une bénédiction, une liberté que les hommes possèdent depuis longtemps.

• Certains parents peuvent regretter de ne pas avoir consacré autant d'énergie à leur développement personnel après être devenus parents.

Le concept de "village" et la parentalité partagée :

• Louise, en tant que mère célibataire, a construit un "village" autour de son fils, un réseau d'amis, de baby-sitters et de connaissances qui la soutiennent. Ce réseau est crucial mais doit être entretenu.

• Le podcast suggère d'élargir la définition de la parentalité et de revenir à l'idée qu'il faut "tout un village pour élever un enfant". Il n'est pas nécessaire d'avoir enfanté pour être un "parent" dans ce sens.

• L'expérience en Namibie, où les enfants sont confiés à la garde plus tôt, est citée comme une source d'inspiration pour Louise, lui montrant qu'il est possible de concilier parentalité et autres engagements.

La transformation de soi et la responsabilité parentale :

• Devenir parent est décrit comme une "seconde naissance" et une "expérience transformatrice", au point de devenir "quelqu'un d'autre". Il est difficile de savoir à l'avance si l'on appréciera ce changement.

• Être parent implique d'être "sous surveillance" et de "servir d'exemple", ce qui peut être difficile. Louise confie échouer souvent dans ce rôle, notamment par manque de patience.

• La capacité à rire de soi et des autres est présentée comme un outil essentiel pour faire face aux imperfections de la vie de famille.

• L'être humain est vu comme un être en devenir, avec un potentiel de transformation, y compris sur le plan émotionnel.

Le détachement et la projection dans l'avenir :

• La relation parent-enfant est réciproque en termes d'attachement, et les deux parties doivent éventuellement parvenir à se détacher l'une de l'autre.

• Malgré les défis, les enfants permettent aux parents de "voir le monde à travers leurs yeux", de les regarder grandir et devenir autonomes, offrant une "projection dans la société à venir".

Le rôle de l'instinct et du plaisir des parents :

• Les parents ne doivent pas se forcer à faire des choses avec leurs enfants s'ils n'en ont pas envie. Le plaisir des parents dans certaines activités est un "acquis précieux pour la vie" des enfants.

• Les enfants ont besoin de parents "vraiment là", présents et impliqués, plutôt que de directives données de loin.

• Tilman et Ilana soulignent que la vie avec des enfants est "plus riche", "plus imprévisible", les entraîne sur des chemins inattendus, et surtout, qu'ils rient beaucoup en famille, ce qui serait moins le cas sans leurs enfants.

Citations importantes :

• "...le bonheur vient notamment du fait de se confronter à des défis que ce soit en élevant un enfant en courant un marathon ou en escaladant une montagne Surmonter ces difficultés enrichit nos vies"

• "Je suis d'accord avec le philosophe d'iter Tomy qui a dit un jour que la parentalité était réservée aux extrémistes car contrairement à l'ascension du Kilimanjaro par exemple on ne peut pas revenir en arrière On ne peut pas faire demi-tour On est parent Vitam et Ternam On reste toute sa vie sur le Kilimanjaro quoi qu'il arrive Une vie d'extrémiste"

• "...la parentalité est un mythe redoutable très fortement chargé émotionnellement par la société Je conseillerai donc de se poser la question est-ce que je le veux vraiment ou est-ce plutôt le désir des autres que j'essaie de satisfaire"

• "J'étais perdu Je n'étais pas sûr de savoir m'y prendre Mais il a 6 ans maintenant Tu as surmonté pas mal de choses déjà Étonnamment Oui Qu'est-ce qui rend heureux dans le fait d'avoir un enfant pour moi c'est d'admettre qu'il puisse lui aussi m'apprendre beaucoup de choses Grâce à lui j'ai découvert de nombreuses facettes de moi que je n'aurais peut-être pas remarqué sans lui On apprend à être plus tolérant envers soi-même Aujourd'hui je me rends compte que cela me rend vraiment heureuse et que c'est un cadeau"

• "Une vie d'extrémiste Ce n'est pas vraiment comme ça que j'imaginais la parentalité mais à la réflexion les jeunes parents ont parfois un petit côté fanatique"

• "...nos résultats montrent que la satisfaction de vie et le sentiment de bonheur augmente au cours des années précédents la naissance Atteignent un pic au cours de la première année après la naissance puis diminuent et reviennent à leur niveau initial La satisfaction de vie ne diffère pas énormément entre les personnes avec ou sans enfant Celles avec enfant ne sont pas plus heureuse ou plus contente de leur vie que les autres"

• "...lorsque en tant qu'être humain nous sommes face à un être avec de grands yeux un front haut et des joues rebondis nous ressentons un sentiment positif Nous nous sentons bien et nous voulons nous en occuper Et ce sont bien sûr exactement les caractéristiques des bébés"

• "Quand on devient parent on s'engage dans un travail d'équipe au sein du couple On est dans une sorte de relation de travail et on se critique mutuellement"

• "Cette étude a brisé un tabou Les femmes devraient être autorisées à dire que les enfants ne sont pas toujours une bénédiction Cela fait longtemps qu'on accorde aux hommes cette liberté de parole"

• "Peut-être devrions-nous élargir la définition de la parentalité et revenir à l'idée qu'il faut tout un village pour élever un enfant Il n'est pas nécessaire d'avoir enfanté pour être parent"

• "En devenant parents on devient quelqu'un d'autre et il est difficile de savoir à l'avance si on va apprécier ce changement"

• "Pour moi le bonheur ne fait qu'un Celui que nous partageons tous les deux celui que nous vivons en famille avec les enfants et celui que chacun cultive de son côté Je trouve que c'est difficile de les séparer C'est un bonheur qui n'est pas malgré à cause d'eux mais avec les enfants"

• "Ce dont les enfants ont besoin ce sont des parents qui sont vraiment là qui ne crient pas de loin 'Rangent ta chambre' mais qui sont présents mettent la main à la pâte et disent 'Vien on va faire ça ensemble.'"

• "À une époque où l'on cherche à se prémunir contre tous les risques où l'on cherche à tout sécuriser je trouve tout à fait formidable d'oser se lancer Se jeter ou pas dans le grand bain de la parentalité c'est peut-être au bout du compte aussi une question de confiance"

• "la vie devient plus riche elle devient aussi plus imprévisible Cela nous entraîne sur des chemins que l'on aurait pas emprunté sinon Et puis c'est très amusant On rit beaucoup en famille On ne rirait pas autant je pense si on avait pas tous ces enfants autour de nous"

• En résumé, le podcast offre une perspective nuancée sur la parentalité, reconnaissant à la fois ses difficultés et ses récompenses.

Il remet en question les mythes sociaux, souligne l'importance du soutien et de l'adaptation, et explore les multiples facettes du bonheur que les enfants peuvent apporter à la vie des parents, malgré les défis inévitables.

pf = np.polyfit(x, y, 1) 和 p = np.poly1d(pf)  对 (x, y)（x 为当前特征的所有样本值，y 为房价中位数数组）做一次（degree=1）多项式拟合，得到拟合系数 pf，再用 np.poly1d 封装成函数 p(x)，以便后面直接计算拟合直线上的 y 值。

• 1 表示“拟合多项式的最高次数”，也就是一次多项式（直线）。
• poly1d 专门用于一维多项式（\(p(x) = a_n x^n + \dots + a_1 x + a_0\)）的封装和运算。

如果你想处理双变量或更高维度的多项式拟合／评估，不是去找 poly2d，而是用 numpy.polynomial 里的相应函数（如 polyfit + polyval2d）或自己基于输出系数矩阵构造计算逻辑。

ax.plot(x, y, 'o')  在子图上绘制散点——'o' 指用圆点表示。

ax.plot(x, p(x), "r--")  在同一个子图上画出红色虚线 ("r--") 代表的拟合直线。

ax.set_title(col + ' vs Median House Value')、ax.set_xlabel(col)、ax.set_ylabel('Median House Value')  分别设置每个子图的标题和坐标轴标签，横轴是当前特征名，纵轴都是“Median House Value”。

Ici l'auteure évoque une communication sur la sphère publique que le sociologue Dominique Cardon qualifie de "clair-obscur", à savoir des propos assumés, clairement affichés mais peu lisibles par les lecteurs "hors cercle" puisqu'on y trouve seulement des sous-entendus. Pour autant, même sans être clairs pour les autres lecteurs, de tels propos, sous couverts de faire réagir pourraient avoir un impact ou une interprétation négative et participer à cette spirale du "bad buzz", de la polémique, à échelle du public évoquée plus haut.

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

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Reply to the reviewers

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

In this manuscript the authors have done cryo-electron tomography of the manchette, a microtubule-based structure important for proper sperm head formation during spermatogenesis. They also did mass-spectrometry of the isolated structures. Vesicles, actin and their linkers to microtubules within the structure are shown.

__We thank the reviewer for the critical reading of our manuscript; we have implemented the suggestions as detailed below, which we believe indeed improved the manuscript. __

Major:

The data the conclusions are based on seem very limited and sometimes overinterpreted. For example, only one connection between actin and microtubules was observed, and this is thought to be MACF1 simply based on its presence in the MS.

__We regret giving the impression that the data is limited. We in fact collected >100 tilt series from 3 biological replicas for the isolated manchette. __

__In the revised version, we added data from in-situ studies showing vesicles interacting with the manchette (as requested below, new Fig. 1). __

Specifically, for the interaction of actin with microtubule we added more examples (Revised Fig. 6) and we toned down the discussion related to the relevance of this interaction (lines 193-194, 253-255). MACF1 is mentioned only as a possible candidate in the discussion (line 254).

Another, and larger concern, is that the authors do a structural study on something that has been purified out of the cell, a process which is extremely disruptive. Vesicles, actin and other cellular components could easily be trapped in this cytoskeletal sieve during the purification process and as such, not be bona fide manchette components. This could create both misleading proteomics and imaging. Therefore, an approach not requiring extraction such as high-pressure freezing, sectioning and room-temperature electron tomography and/or immunoEM on sections to set aside this concern is strongly recommended. As an additional bonus, it would show if the vesicles containing ATP synthase are deformed mitochondria.

__We recognise the concern raised by the reviewer. __

__To alleviate this concern, we added imaging data of manchettes in-situ that show vesicles, mitochondria and filaments interacting with the manchette (new Fig. 1), essentially confirming the observations that were made on the isolated manchette. __

__The benefits of imaging the isolated manchette were better throughput (being able to collect more data) and reaching higher resolution allowing to resolve unequivocally the dynein/dynactin and actin filaments. __

Minor: Line 99: "to study IMT with cryo-ET, manchettes were isolated ...(insert from which organism)..."

__Added in line 102 in the revised version. __

Line 102 "...demonstrating that they can be used to study IMT".. can the authors please clarify?

This paragraph was revised (lines 131-137), we hope it is now more clear.

Line 111 "densities face towards the MT plus-end" How can a density "face" anywhere? For this, it needs to have a defined front and back.

Microtubule motor proteins (kinesin and dynein) are often attached to the microtubules with an angle and dynactin and cargo on one side (plus end). We rephrased this part and removed the word “face” in the revised version to make it more clear (lines 161-162).

Line 137: is the "perinuclear ring" the same as the manchette?

The perinuclear ring is the apical part of the manchette that connects it to the nucleus. We added to the revised version imaging of the perinuclear ring with observations on how it changes when the manchette elongates (new Fig. 2).

Figure 2B: How did the authors decide not to model the electron density found between the vesicle and the MT at 3 O'clock? Is there no other proteins with a similar lollipop structure as ATP synthase, so that this can be said to be this protein with such certainty?

__The densities connecting the vesicles to the microtubules shown in (now) Fig. 4D are not consistent enough to be averaged. __

__The densities resembling ATP synthase are inside the vesicles. Nevertheless, we have decided to remove the averaging of the ATP synthases from the revised manuscipt as they are not of great importance for this manuscript. Instead, the new in-situ data clearly show mitochondria (with their characteristic double membrane and cristae) interacting with manchette microtubule (new Fig 1C). __

Line 189: "F-actin formed organized bundles running parallel to mMTs" - this observation needs confirming in a less disrupted sample.

__Phalloidin (actin marker) was shown before to stain the manchette (PMID: 36734600). As actin filaments are very thin (7 nm) they are very hard to observe in plastic embedded EM. __

In the in-situ data we added to the revised manuscipt (new Fig 1D), we observe filaments with a diameter corresponding to actin. In addition, we added more examples of microtubules interacting with actin in isolated manchette (new Fig. 6 E-K).

Line 242 remove first comma sign.

Removed.

Line 363 "a total of 2 datasets" - is this manuscript based on only two tilt-series? Or two datasets from each of the 4 grids? In any case, this is very limited data.

We apologise for not clearly providing the information about the data size in the original manuscipt. The data is based on three biological replicas (3 animals). We collected more than 100 tomograms of different regions of the manchettes. As such, we would argue that the data is not limited per se.

Reviewer #1 (Significance (Required)):

The article is very interesting, and if presented together with the suggested controls, would be informative to both microtubule/motorprotein researchers as well as those trying studying spermatogenesis.

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

The manchette appears as a shield-like structure surrounding the flagellar basal body upon spermiogenesis. It consists of a number of microtubules like a comb, but actin (Mochida et al. 1998 Dev. Biol. 200, 46) and myosin (Hayasaka et al. 2008 Asian J. Androl. 10, 561) were found, suggesting transportation inside the manchette. Detailed structural information and functional insight into the manchette was still awaited. There is a hypothesis called IMT (intra-machette transport) based on the fact that machette and IFT (intraflagellar transport) share common components (or homologues) and on their transition along the stages of spermiogenesis. While IMT is considered as a potential hypothesis to explain delivery of centrosomal and flagellar components, no one has witnessed IMT at the same level as IFT. IMT has never been purified, visualized in motion or at high resolution. This study for the first time visualized manchette using high-end cryo-electron tomography of isolated manchettes, addressing structural characterization of IMT. The authors successfully microtubular bundles, vesicles located between microtubules and a linker-like structure connecting the vesicle and the microtubule. On multilamellar membranes in the vesicles they found particles and assigned them to ATPase complexes, based on intermediate (~60A) resolution structure. They further identified interesting structures, such as (1) particles on microtubules, which resemble dynein and (2) filaments which shows symmetry of F-actin. All the molecular assignments are consistent with their proteomics of manchettes.

__We thank the reviewer for highlighting the novelty of our study.____ __

Their assignment of ATPase will be strengthened by MS data, if it proves absence of other possible proteins forming such a membrane protein complex.

All the ATPase components were indeed found in our proteomics data. Nevertheless, we have decided to remove the averaging of the ATPase as it does not directly relate to IMT, the focus of this manuscript.

They discussed possible role of various motor proteins based on their abundance (Line 134-151, Line 200). This makes sense only with a control. Absolute abundance of proteins would not necessarily present their local importance or roles. This reviewer would suggest quantitative proteomics of other organelles, or whole cells, or other fractions obtained during manchette isolation, to demonstrate unique abundance of KIF27 and other proteins of their interest.

We agree with the reviewer that absolute abundance does not necessarily indicate importance or a role. As such, we removed this part of the discussion from the revised manuscript.

A single image from a tomogram, Fig.6B, is not enough to prove actin-MT interaction. A gallery and a number (how many such junctions were found from how many MTs) will be necessary.

We agree that one example is not enough. In the new Fig. 6E-K, we provide a gallery of more examples. We have revised the text to reflect the point that these observations are still rare and more data will be needed to quantify this interaction (Lines 253-254).

Minor points: Their manchette purification is based on Mochida et al., which showed (their Fig.2) similarity to the in vivo structure (for example, Fig.1 of Kierszenbaum 2001 Mol. Reproduc. Dev. 59, 347). Nevertheless, since this is not a very common prep, it is helpful to show the isolated manchette’s wide view (low mag cryo-EM or ET) to prove its intactness.

We thank the reviewer for this suggestion, in the revised version, new Fig. 2 provides a cryo-EM overview of purified manchette from different developmental stages.

Line 81: Myosin -> myosin (to be consistent with other protein names)

Corrected.

This work is a significant step toward the understanding of manchettes. While the molecular assignment of dynein and ATPase is not fully decisive, due to limitation of resolution (this reviewer thinks the assignment of actin filament is convincing, based on its helical symmetry), their speculative model still deserves publication.

Reviewer #2 (Significance (Required)):

This work is a significant step toward the understanding of manchettes. While the molecular assignment of dynein and ATPase is not fully decisive, due to limitation of resolution (this reviewer thinks the assignment of actin filament is convincing, based on its helical symmetry), their speculative model still deserves publication.

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

->Summary:

The manchette is a temporary microtubule (MT)-based structure essential for the development of the highly polarised sperm cell. In this study, the authors employed cryo-electron tomography (cryo-ET) and proteomics to investigate the intra-manchette transport system. Cryo-EM analysis of purified rat manchette revealed a high density of MTs interspersed with actin filaments, which appeared either bundled or as single filaments. Vesicles were observed among the MTs, connected by stick-like densities that, based on their orientation relative to MT polarity, were inferred to be kinesins. Subtomogram averaging (STA) confirmed the presence of dynein motor proteins. Proteomic analysis further validated the presence of dynein and kinesins and showed the presence of actin crosslinkers that could bundle actin filaments. Proteomics data also indicated the involvement of actin-based transport mediated by myosin. Importantly, the data indicated that the intraflagellar transport (IFT) system is not part of the intra-manchette transport mechanism. The visualisation of motor proteins directly from a biological sample represents a notable technical advancement, providing new insights into the organisation of the intra-manchette transport system in developing sperm.

We thank the reviewer for summarising the novelty of our observations.

-> Are the key conclusions convincing? Below we comment on three main conclusions. MT and F-actin bundles are both constituents of the manchette While the data convincingly shows that MT and F-actin are part of the manchette, one cannot conclude from it that F-actin is an integral part of the manchette. The authors would need to rephrase so that it is clear that they are speculating.

We have rephrased our statements and replaced “integral” with ‘actin filaments are associated’. Of note previous studies suggested actin are part of the manchette including staining with phalloidin (PMID: 36734600, PMID: 9698455, PMID: 18478159) and we here visualised the actin in high resolution.

The transport system employs different transport machinery on these MTs Proteomics data indicates the presence of multiple motor proteins in the manchette, while cryo-EM data corroborates this by revealing morphologically distinct densities associated with the MTs. However, the nature of only one of these MT-associated densities has been confirmed-specifically, dynein, as identified through STA. The presence of kinesin or myosin in the EM data remains unconfirmed based on just the cryo-ET density, and therefore it is unclear whether these proteins are actively involved in cargo transport, as this cannot be supported by just the proteomics data. In summary, we recommend that the authors rephrase this conclusion and avoid using the term "employ".

We agree that our cryo-ET only confirmed the motor protein dynein. As such, we removed the term employ and rephrased our claims regarding the active transport and accordingly changed the title.

Dynein mediated transport (Line 225-227) The data shows that dynein is present in the manchette; however, whether it plays and active role in transport cannot be determined from the cryo-ET data provided in the manuscript, as it does not clearly display a dynein-dynactin complex attached to cargo. The attachment to cargo is also not revealed via proteomics as no adaptor proteins that link dynein-dynactin to its cargo have been shown.

A list of cargo adaptor proteins were found in our proteomics data but we agree that cryo-ET and proteomics alone cannot prove active transport. As such we toned down the discussion about active transport (lines 212-220).

-> Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

F-actin • In the abstract, the authors state that F-actin provides tracks for transport as well as having structural and mechanical roles. However, the manuscript does not include experiments demonstrating a mechanical role. The authors appear to base this statement on literature where actin bundles have been shown to play a mechanical role in other model systems. We suggest they clarify that the mechanical role the authors suggest is speculative and add references if appropriate.

__ ____We removed the claim about the mechanical role of the actin from the abstract and rephrased this in the discussion to suggest this role for the F-actin (lines 242-243).__

• Lines 15,92, 180 and 255: The statement "Filamentous actin is an integral part of the manchette" is misleading. While the authors show that F-actin is present in their purified manchette structures, whether it is integral has not been tested. Authors should rephrase the sentence.

We removed the word integral.

• To support the claim that F-actin plays a role in transport within the manchette, the authors present only one instance where an unidentified density is attached to an actin filament. This is insufficient evidence to claim that it is myosin actively transporting cargo. Although the proteomics data show the presence of myosin, we suggest the authors exercise more caution with this claim.

We agree that our data do not demonstrate active transport as such we removed that claim. We mention the possibility of cargo transport in the discussion (lines 250-255).

• The authors mention the presence of F-actin bundles but do not show direct crosslinking between the F-actin filaments. They could in principle just be closely packed F-actin filaments that are not necessarily linked, so the term "bundle" should be used more cautiously.

We do not assume that a bundle means that the F-actin filaments are crosslinked. A bundle simply indicates the presence of multiple F-actin filaments together. We rephrased it to call them actin clusters.

Observations of dynein • Relating to Figure 2B: From the provided image it is not clear whether the density corresponds to a dynein complex, as it does not exhibit the characteristic morphological features of dynein or dynactin molecules.

We indeed do not claim that the densities in this figure are dynein or dynactin. __We revised this paragraph and hope that it is now more clear (lines 135-137). __

• Lines 171-172 and Figure 4: It is well established that dynein is a dimer and should always possess two motor domains. The authors have incorrectly assumed they observed single motor heads, except possibly in Figure 4A (marked by an arrow). In all other instances, the dynein complexes show two motor domains in proximity, but these have not been segmented accurately. Furthermore, the "cargos" shown in grey are more likely to represent dynein tails or the dynactin molecule, based on comparisons with in vitro structures of these complexes (see references 1-3).

We thank the reviewer for this correction. We improved the annotations in the figure and revised the text to clarify that we identified dimers of dynein motor heads (lines 140-144). We further added a projection of a dynein dynactin complex to compare to the observation on the manchette (new Fig. 5E). We further changed claims on the presence of protein cargo to the presence of dynein/dynactin that allows cargo tethering based on the presence of cargo adaptors in the proteomics data.

• Lines 21, 173, and 233 mention cargos, but as noted above, it seems to be parts of the dynein complex the authors are referring to.

This was corrected as mentioned above.

• Panel 4B appears to show a dynein-dynactin complex, but whether there is a cargo is unclear and if there is it should be labelled accordingly. To assessment of whether there is any cargo bound to the dynein-dynactin complex a larger crop of the panel would be helpful In summary, we recommend that the authors revisit their segmentations in Figures 2B and 4, revise their text based on these observations, and perform quantification of the data (as suggested in the next section).

We thank the reviewers for sharing their expertise on dynein-dynactin complexes. We have revised the text as detailed above and excluded the assignment of any cargo, as we cannot (even from larger panels) see a clear association of cargo. We have made clear that we only refer to dynein dynactin with the capability of linking cargo based on the presence of proteomics data. We have removed claims on active transport with dynein.

Dynein versus kinesin-based transport The calculation presented in lines 147-151 does not account for the fact that both the dynein-dynactin complex and kinesin proteins require cargo adaptors to transport cargo. Additionally, the authors overlook the possibility that multiple motors could be attached to a single cargo. If the authors did not observe this, they should explicitly mention it to support their argument. In short, the calculations are based on an incorrect premise, rendering the comparison inaccurate. Unless the authors have identified any dynein-dynactin or kinesin cargo adaptors in their proteomics data which could be used for such a comparison, we believe the authors lack sufficient data to accurately estimate the "active transport ratio" between dynein and kinesin.

Even though we detect cargo adaptors in our proteomics, we agree that calculating relative transport based only on the proteomics can be inaccurate as such we removed absolute quantification and comparison between dynein and kinesin-based IMT.

• Would additional experiments be essential to support the claims of the paper?

F-actin distance and length distribution • To support the claim that F-actin is bundled (line 189), could the authors provide the distance between each F-actin filament and its neighbours? Additionally, could they compare the average distance to the length of actin crosslinkers found in their proteomics data, or compare it to the distances between crosslinked F-actin observed in other research studies?

We measured distances between the actin filaments and added a plot to new Fig 6.

• While showing that F-actin is important for the manchette would require cellular experiments, authors could provide quantification of how frequently these actin structures are observed in comparison to MTs to support their claims that these actin filaments could be important for the manchette structure.

We agree that claims on the role and function of actin in the manchette require cellular experiments that are beyond the scope of this study. Absolute quantification of the ratio between MTs and actin from cryoET is very hard and will be inaccurate as the manchette cannot be imaged as a whole due to its size and thickness. The ratio we have is based on the relative abundance provided by the proteomics (Fig. 5F).

• In line 193, the authors claim that the F-actin in bundles appears too short for transport. Could they provide length distributions for these filaments? This might provide further support to their claim that individual F-actin filaments can serve as transport tracks (line 266).

__In addition to the limitation mentioned in the previous point, quantification of length from high magnification imaging will likely be inaccurate as the length of the actin in most cases is bigger than the field of view that is captured. Nevertheless, we removed the claim about the actin being too short for transport. __

• Could the authors also quantify the abundance of individual F-actin filaments observed, compared to MTs and F-actin bundles, to support the idea that they could play a role in transport?

As explained for the above points absolute quantification of the ratio between MTs and actin is not feasible from cryoET data that cannot capture all of the manchette in high enough resolution to resolve the actin.

• In the discussion, the authors mention "interactions between F-actin singlets and mMTs" (line 269), yet they report observing only one instance of this interaction (lines 210 and 211). Given the limited data, they should refer to this as a single interaction in the discussion. The scarcity of data raises questions about how representative this event truly is.

We agree that one example is not enough. In the new Fig. 6E-K, we provide a gallery of more examples as also requested by reviewers 1 and 2. We have also revised the text to reflect the point that these observations are still rare (Lines 190-194).

Quantifications for judgement of representativity The authors should quantify how often they observed vesicles with a stick-like connection to MTs (lines 106-107); this would strengthen the interpretation of the density, as currently only one example is shown in the manuscript (Figure 4A). If possible, they could show how many of them are facing towards the MT plus end.

__As mentioned in the text (lines 135-137), the linkers connecting vesicles to MTs were irregular and so we could not interpret them further this is in contrast to dynein that were easily recognisable but were not associated with vesicles. __

Dynein quantifications • The authors are recommended to quantify how many dynein molecules per micron of MT they observe and how often they are angled with their MT binding domain towards the minus-end.

As the manchette is large and highly dense any quantification will likely be biased towards parts of the manchette that are easier to image, for example the periphery. As such we do not think quantifying the dynein density will yield meaningful insight.

• Could the authors quantify how many dynein densities they found to be attached to a (vesicle) cargo, if any (line 175)? They could show these observations in a supplementary figure.

We did not observe any case of a connection between a vesicle and dynein motors, we edited this sentence to be more clear on that.

• For densities that match the size and location of dynein but lack clear dynein morphology (as seen in Figure 2B), could the authors quantify how many are oriented towards the MT minus end?

We had many cases where the connection did not have a clear dynein morphology, and as the morphology is not clear, it is impossible to make a claim about whether they are oriented towards the minus end.

Artefacts due to purification: Authors should discuss if the purification could have effects on visualizing components of the manchette. For example, if it has effect on the MTs and actin structure or the abundance/structure of the motor protein complexes (bound to cargo or isolated).

We have followed a protocol that was published before and showed the overall integrity of the manchette. Nevertheless, losing connections between manchette and other cellular organelles are expected. To address this point, we added in-situ data (new Fig 1) showing manchette in intact spermatids interacting with vesicles and mitochondria, as well as overviews of manchettes (new Fig 2), the text was revised accordingly.

• Are the experiments adequately replicated and statistical analysis adequate? The cryo-ET data presented in the manuscript is collected using two separate sample preparations. Along with the quantifications of the different observations suggested above which will help the reader assess how abundant and representative these observations are, the authors could further strengthen their claims by acquiring data from a third sample preparation and then analysing how consistent their observations are between different purifications. This however could be time consuming so it is not a major requirement but recommended if possible within a short time frame.

We regret not explicitly mentioning our data set size, it was added now to the revised version. In essence, the data is based on three biological replicas (3 animals). We collected more than 100 tomograms of different regions of the manchettes. We provided in the revised version more observations (new Fig 1, 2, 4B-C and 6E-K).

• Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

Most of the comments deal with either modifying the text or analysing the data already presented, so the revision could be done with 1-3 months.

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Minor comments: - Specific experimental issues that are easily addressable. 1) Could the authors state how many tilt series were collected for each dataset/independent sample preparation? We recommend that they upload their raw data or tomograms to EMPAIR.

We added this information in the material and methods.

2) It is not clear to me if the same sample was used for cryo-ET and proteomics. Could the authors clarify how comparable the sample preparation for the cryo-ET and proteomics data is or if the same sample was used for both. If there is a discrepancy between these preparations, they would need to discuss how this can affect comparing observations from cryo-ET and mass spectrometry. Ideally both samples should be the same.

After sample preparation the manchettes were directly frozen on grids. The rest of the samples was used for proteomics. Consequently, EM and MS data were acquired on the same samples. We clarified this in the text (lines 327-328).

• Are prior studies referenced appropriately? We recommend including additional references to support the claim that F-actin has a mechanical role (line 242). Could the authors compare their proteomics data to other mass spectrometry studies conducted on the Manchette (for example, see reference 4)?

We added the comparison but it is important to point out that in reference 4 the manchettes were isolated from mice testes.

• Are the text and figures clear and accurate? Text: We do not see the necessity of specifying the microtubules (MTs) in the data as "manchette MTs" or "mMTs" rather than simply "MTs". However, we recommend that the authors use either "MT" or "mMT" consistently throughout the manuscript.

We changed to only MTs.

The authors appear to refer to both dynein-1 (cytoplasmic dynein) and dynein-2 (axonemal dynein or IFT dynein). To avoid confusion, it is important that the authors clearly specify which dynein they are referring to throughout the text. This is particularly relevant as the study aims to demonstrate that IFT is not part of the manchette transport system.

• Introduction: In the third paragraph (lines 59-75), the authors should specify that they are referring to dynein-2, which is distinct from cytoplasmic dynein discussed in the previous paragraph (lines 44-58).

We specify the respective dyneins in the text (line 66,140-141,145).

• Figure 4D: The authors could fit a dynein-1 motor domain instead of a dynein-2 into the density to stay consistent with the fact that the density belongs to cytoplasmic dynein-1.

__We changed the figure and fitted a cytosolic dynein-1 structure (5nvu) instead. __

Figures: • Figure 2B: The legend mentions a large linker complex; however, this may correspond to two or three separate densities.

We have addressed this and changed the wording.

• Figure 4: please revisit the segmentation of this whole figure based on previous comments.

__We revised as suggested. __

• Figures 1, 2, 4, 5, and 6: It would be helpful to state in the legends that the tomograms are denoised. There are stripe-like densities visible in the images (e.g., in the vesicle in Figure 2B). Do these artefacts also appear in the raw data?

As stated in the Methods section, tomograms were generally denoised with CryoCare for visualisation purposes. The “stripe-like densities” are artefacts of the gold fiducials used for tomogram alignment and appear in the raw data (before denoising).

• Do you have suggestions that would help the authors improve the presentation of their data and conclusions? We suggest revising the paragraph title "Dynein-mediated cargo along the manchette" (line 165) to "Dynein-mediated cargo transport along the manchette".

__We have changed this in the revised version. __

We recommend that the authors provide additional evidence to support the interpretation that the observed EM densities correspond to motor proteins. Specifically: • Include scale bars or reference lines indicating the known dimensions of motor proteins, based on previous data, to demonstrate that the observed densities match the expected size.

The dynein structure is provided for reference. We also added the cytosolic dynein–dynactin as a reference (Fig 5E).

• Make direct comparisons to existing EM data and highlight morphological similarities.

We have added a comparison to existing data (Fig 5E).

In the discussion (lines 249-254), the authors could speculate on alternative roles for the IFT components in the manchette, particularly if they are not part of the IFT trains. We also suggest rephrasing the claim in line 266 to make it more speculative in tone.

__We have addressed this in the revised version (lines 221-230). __

Finally, a schematic overview of the manchette ultrastructure in a spermatid would greatly aid the reader in understanding the material presented.

We now include a graphical abstract and overviews of isolated manchettes on cryo-EM grids.

References: 1. Chowdhury, S., Ketcham, S., Schroer, T. et al. Structural organization of the dynein-dynactin complex bound to microtubules. Nat Struct Mol Biol 22, 345-347 (2015). https://doi.org/10.1038/nsmb.2996

1. Grotjahn, D.A., Chowdhury, S., Xu, Y. et al. Cryo-electron tomography reveals that dynactin recruits a team of dyneins for processive motility. Nat Struct Mol Biol 25, 203-207 (2018). https://doi.org/10.1038/s41594-018-0027-7

2. Chaaban, S., Carter, A.P. Structure of dynein-dynactin on microtubules shows tandem adaptor binding. Nature 610, 212-216 (2022).https://doi.org/10.1038/s41586-022-05186-y

3. W. Hu, R. Zhang, H. Xu, Y. Li, X. Yang, Z. Zhou, X. Huang, Y. Wang, W. Ji, F. Gao, W. Meng, CAMSAP1 role in orchestrating structure and dynamics of manchette microtubule minus-ends impacts male fertility during spermiogenesis, Proc. Natl. Acad. Sci. U.S.A. 120 (45) e2313787120, https://doi.org/10.1073/pnas.2313787120 (2023).

Reviewer #3 (Significance (Required)):

This study employs cryo-electron tomography (cryo-ET) and proteomics to elucidate the architecture of the manchette. It advances our understanding of the components involved in intracellular transport within the manchette and introduces the following technical and conceptual innovations:

a) Technical Advances: The authors have visualized the manchette at high resolution using cryo-ET. They optimized a purification pipeline capable of retaining, at least partially, the transport machinery of the manchette. Notably, they observed dynein and putative kinesin motors attached to microtubules-a significant achievement that, to our knowledge, has not been reported previously.

b) Conceptual Advances: This study provides novel insights into spermatogenesis. The findings suggest that intraflagellar transport (IFT) is unlikely to play a role at this stage of sperm development while shedding light on alternative transport systems. Importantly, the authors demonstrate that actin filaments organize in two distinct ways: clustering parallel to microtubules or forming single filaments.

This work is likely to be of considerable interest to researchers in sperm development and structural biology. Additionally, it may appeal to scientists studying motor proteins and the cytoskeleton.

We thank the reviewers for appreciating the significance and novelty of our study.

The reviewers possess extensive expertise in in situ cryo-electron tomography and single-particle microscopy, including work on dynein-based complexes. Collectively, they have significant experience in the field of cytoskeleton-based transport.

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

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Referee #3

Evidence, reproducibility and clarity

Summary:

The manchette is a temporary microtubule (MT)-based structure essential for the development of the highly polarised sperm cell. In this study, the authors employed cryo-electron tomography (cryo-ET) and proteomics to investigate the intra-manchette transport system. Cryo-EM analysis of purified rat manchette revealed a high density of MTs interspersed with actin filaments, which appeared either bundled or as single filaments. Vesicles were observed among the MTs, connected by stick-like densities that, based on their orientation relative to MT polarity, were inferred to be kinesins. Subtomogram averaging (STA) confirmed the presence of dynein motor proteins. Proteomic analysis further validated the presence of dynein and kinesins and showed the presence of actin crosslinkers that could bundle actin filaments. Proteomics data also indicated the involvement of actin-based transport mediated by myosin. Importantly, the data indicated that the intraflagellar transport (IFT) system is not part of the intra-manchette transport mechanism. The visualisation of motor proteins directly from a biological sample represents a notable technical advancement, providing new insights into the organisation of the intra-manchette transport system in developing sperm.

Are the key conclusions convincing?

Below we comment on three main conclusions.

MT and F-actin bundles are both constituents of the manchette While the data convincingly shows that MT and F-actin are part of the manchette, one cannot conclude from it that F-actin is an integral part of the manchette. The authors would need to rephrase so that it is clear that they are speculating.

The transport system employs different transport machinery on these MTs Proteomics data indicates the presence of multiple motor proteins in the manchette, while cryo-EM data corroborates this by revealing morphologically distinct densities associated with the MTs. However, the nature of only one of these MT-associated densities has been confirmed-specifically, dynein, as identified through STA. The presence of kinesin or myosin in the EM data remains unconfirmed based on just the cryo-ET density, and therefore it is unclear whether these proteins are actively involved in cargo transport, as this cannot be supported by just the proteomics data. In summary, we recommend that the authors rephrase this conclusion and avoid using the term "employ".

Dynein mediated transport (Line 225-227) The data shows that dynein is present in the manchette; however, whether it plays and active role in transport cannot be determined from the cryo-ET data provided in the manuscript, as it does not clearly display a dynein-dynactin complex attached to cargo. The attachment to cargo is also not revealed via proteomics as no adaptor proteins that link dynein-dynactin to its cargo have been shown.

Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

F-actin

• In the abstract, the authors state that F-actin provides tracks for transport as well as having structural and mechanical roles. However, the manuscript does not include experiments demonstrating a mechanical role. The authors appear to base this statement on literature where actin bundles have been shown to play a mechanical role in other model systems. We suggest they clarify that the mechanical role the authors suggest is speculative and add references if appropriate.
• Lines 15,92, 180 and 255: The statement "Filamentous actin is an integral part of the manchette" is misleading. While the authors show that F-actin is present in their purified manchette structures, whether it is integral has not been tested. Authors should rephrase the sentence.
• To support the claim that F-actin plays a role in transport within the manchette, the authors present only one instance where an unidentified density is attached to an actin filament. This is insufficient evidence to claim that it is myosin actively transporting cargo. Although the proteomics data show the presence of myosin, we suggest the authors exercise more caution with this claim.
• The authors mention the presence of F-actin bundles but do not show direct crosslinking between the F-actin filaments. They could in principle just be closely packed F-actin filaments that are not necessarily linked, so the term "bundle" should be used more cautiously.

Observations of dynein

• Relating to Figure 2B: From the provided image it is not clear whether the density corresponds to a dynein complex, as it does not exhibit the characteristic morphological features of dynein or dynactin molecules.
• Lines 171-172 and Figure 4: It is well established that dynein is a dimer and should always possess two motor domains. The authors have incorrectly assumed they observed single motor heads, except possibly in Figure 4A (marked by an arrow). In all other instances, the dynein complexes show two motor domains in proximity, but these have not been segmented accurately. Furthermore, the "cargos" shown in grey are more likely to represent dynein tails or the dynactin molecule, based on comparisons with in vitro structures of these complexes (see references 1-3).
• Lines 21, 173, and 233 mention cargos, but as noted above, it seems to be parts of the dynein complex the authors are referring to.
• Panel 4B appears to show a dynein-dynactin complex, but whether there is a cargo is unclear and if there is it should be labelled accordingly. To assessment of whether there is any cargo bound to the dynein-dynactin complex a larger crop of the panel would be helpful In summary, we recommend that the authors revisit their segmentations in Figures 2B and 4, revise their text based on these observations, and perform quantification of the data (as suggested in the next section).

Dynein versus kinesin-based transport

The calculation presented in lines 147-151 does not account for the fact that both the dynein-dynactin complex and kinesin proteins require cargo adaptors to transport cargo. Additionally, the authors overlook the possibility that multiple motors could be attached to a single cargo. If the authors did not observe this, they should explicitly mention it to support their argument. In short, the calculations are based on an incorrect premise, rendering the comparison inaccurate. Unless the authors have identified any dynein-dynactin or kinesin cargo adaptors in their proteomics data which could be used for such a comparison, we believe the authors lack sufficient data to accurately estimate the "active transport ratio" between dynein and kinesin.

Would additional experiments be essential to support the claims of the paper?

F-actin distance and length distribution

• To support the claim that F-actin is bundled (line 189), could the authors provide the distance between each F-actin filament and its neighbours? Additionally, could they compare the average distance to the length of actin crosslinkers found in their proteomics data, or compare it to the distances between crosslinked F-actin observed in other research studies?
• While showing that F-actin is important for the manchette would require cellular experiments, authors could provide quantification of how frequently these actin structures are observed in comparison to MTs to support their claims that these actin filaments could be important for the manchette structure.
• In line 193, the authors claim that the F-actin in bundles appears too short for transport. Could they provide length distributions for these filaments? This might provide further support to their claim that individual F-actin filaments can serve as transport tracks (line 266).
• Could the authors also quantify the abundance of individual F-actin filaments observed, compared to MTs and F-actin bundles, to support the idea that they could play a role in transport?
• In the discussion, the authors mention "interactions between F-actin singlets and mMTs" (line 269), yet they report observing only one instance of this interaction (lines 210 and 211). Given the limited data, they should refer to this as a single interaction in the discussion. The scarcity of data raises questions about how representative this event truly is.

Quantifications for judgement of representativity

The authors should quantify how often they observed vesicles with a stick-like connection to MTs (lines 106-107); this would strengthen the interpretation of the density, as currently only one example is shown in the manuscript (Figure 4A). If possible, they could show how many of them are facing towards the MT plus end.

Dynein quantifications

• The authors are recommended to quantify how many dynein molecules per micron of MT they observe and how often they are angled with their MT binding domain towards the minus-end.
• Could the authors quantify how many dynein densities they found to be attached to a (vesicle) cargo, if any (line 175)? They could show these observations in a supplementary figure.
• For densities that match the size and location of dynein but lack clear dynein morphology (as seen in Figure 2B), could the authors quantify how many are oriented towards the MT minus end?

Artefacts due to purification: Authors should discuss if the purification could have effects on visualizing components of the manchette. For example, if it has effect on the MTs and actin structure or the abundance/structure of the motor protein complexes (bound to cargo or isolated).

Are the experiments adequately replicated and statistical analysis adequate?

The cryo-ET data presented in the manuscript is collected using two separate sample preparations. Along with the quantifications of the different observations suggested above which will help the reader assess how abundant and representative these observations are, the authors could further strengthen their claims by acquiring data from a third sample preparation and then analysing how consistent their observations are between different purifications. This however could be time consuming so it is not a major requirement but recommended if possible within a short time frame.

Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

Most of the comments deal with either modifying the text or analysing the data already presented, so the revision could be done with 1-3 months.

Minor comments:

Specific experimental issues that are easily addressable.

1. Could the authors state how many tilt series were collected for each dataset/independent sample preparation? We recommend that they upload their raw data or tomograms to EMPAIR.
2. It is not clear to me if the same sample was used for cryo-ET and proteomics. Could the authors clarify how comparable the sample preparation for the cryo-ET and proteomics data is or if the same sample was used for both. If there is a discrepancy between these preparations, they would need to discuss how this can affect comparing observations from cryo-ET and mass spectrometry. Ideally both samples should be the same.

Are prior studies referenced appropriately?

We recommend including additional references to support the claim that F-actin has a mechanical role (line 242). Could the authors compare their proteomics data to other mass spectrometry studies conducted on the Manchette (for example see reference 4)?

Are the text and figures clear and accurate?

Text: We do not see the necessity of specifying the microtubules (MTs) in the data as "manchette MTs" or "mMTs" rather than simply "MTs". However, we recommend that the authors use either "MT" or "mMT" consistently throughout the manuscript.

The authors appear to refer to both dynein-1 (cytoplasmic dynein) and dynein-2 (axonemal dynein or IFT dynein). To avoid confusion, it is important that the authors clearly specify which dynein they are referring to throughout the text. This is particularly relevant as the study aims to demonstrate that IFT is not part of the manchette transport system.

• Introduction: In the third paragraph (lines 59-75), the authors should specify that they are referring to dynein-2, which is distinct from cytoplasmic dynein discussed in the previous paragraph (lines 44-58).
• Figure 4D: The authors could fit a dynein-1 motor domain instead of a dynein-2 into the density to stay consistent with the fact that the density belongs to cytoplasmic dynein-1. Figures:
• Figure 2B: The legend mentions a large linker complex; however, this may correspond to two or three separate densities.
• Figure 4: please revisit the segmentation of this whole figure based on previous comments.
• Figures 1, 2, 4, 5, and 6: It would be helpful to state in the legends that the tomograms are denoised. There are stripe-like densities visible in the images (e.g., in the vesicle in Figure 2B). Do these artefacts also appear in the raw data?

Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

We suggest revising the paragraph title "Dynein-mediated cargo along the manchette" (line 165) to "Dynein-mediated cargo transport along the manchette".

We recommend that the authors provide additional evidence to support the interpretation that the observed EM densities correspond to motor proteins. Specifically:

• Include scale bars or reference lines indicating the known dimensions of motor proteins, based on previous data, to demonstrate that the observed densities match the expected size.
• Make direct comparisons to existing EM data and highlight morphological similarities. In the discussion (lines 249-254), the authors could speculate on alternative roles for the IFT components in the manchette, particularly if they are not part of the IFT trains. We also suggest rephrasing the claim in line 266 to make it more speculative in tone. Finally, a schematic overview of the manchette ultrastructure in a spermatid would greatly aid the reader in understanding the material presented.

References:

1. Chowdhury, S., Ketcham, S., Schroer, T. et al. Structural organization of the dynein-dynactin complex bound to microtubules. Nat Struct Mol Biol 22, 345-347 (2015). https://doi.org/10.1038/nsmb.2996
2. Grotjahn, D.A., Chowdhury, S., Xu, Y. et al. Cryo-electron tomography reveals that dynactin recruits a team of dyneins for processive motility. Nat Struct Mol Biol 25, 203-207 (2018). https://doi.org/10.1038/s41594-018-0027-7
3. Chaaban, S., Carter, A.P. Structure of dynein-dynactin on microtubules shows tandem adaptor binding. Nature 610, 212-216 (2022). https://doi.org/10.1038/s41586-022-05186-y
4. W. Hu, R. Zhang, H. Xu, Y. Li, X. Yang, Z. Zhou, X. Huang, Y. Wang, W. Ji, F. Gao, W. Meng, CAMSAP1 role in orchestrating structure and dynamics of manchette microtubule minus-ends impacts male fertility during spermiogenesis, Proc. Natl. Acad. Sci. U.S.A. 120 (45) e2313787120, https://doi.org/10.1073/pnas.2313787120 (2023).

Significance

This study employs cryo-electron tomography (cryo-ET) and proteomics to elucidate the architecture of the manchette. It advances our understanding of the components involved in intracellular transport within the manchette and introduces the following technical and conceptual innovations:

a) Technical Advances:

The authors have visualized the manchette at high resolution using cryo-ET. They optimized a purification pipeline capable of retaining, at least partially, the transport machinery of the manchette. Notably, they observed dynein and putative kinesin motors attached to microtubules-a significant achievement that, to our knowledge, has not been reported previously.

b) Conceptual Advances:

This study provides novel insights into spermatogenesis. The findings suggest that intraflagellar transport (IFT) is unlikely to play a role at this stage of sperm development while shedding light on alternative transport systems. Importantly, the authors demonstrate that actin filaments organize in two distinct ways: clustering parallel to microtubules or forming single filaments.

This work is likely to be of considerable interest to researchers in sperm development and structural biology. Additionally, it may appeal to scientists studying motor proteins and the cytoskeleton.

The reviewers possess extensive expertise in in situ cryo-electron tomography and single-particle microscopy, including work on dynein-based complexes. Collectively, they have significant experience in the field of cytoskeleton-based transport.

Author response:

Reviewer #1:

Lipid transfer proteins (LTPs) play a crucial role in the intramembrane lipid exchange within cells. However, the molecular mechanisms that govern this activity remain largely unclear. Specifically, the way in which LTPs surmount the energy barrier to extract a single lipid molecule from a lipid bilayer is not yet fully understood. This manuscript investigates the influence of membrane properties on the binding of Ups1 to the membrane and the transfer of phosphatidic acid (PA) by the LTP. The findings reveal that Ups1 shows a preference for binding to membranes with positive curvature. Moreover, coarse-grained molecular dynamics simulations indicate that positive curvature decreases the energy barrier associated with PA extraction from the membrane. Additionally, lipid transfer assays conducted with purified proteins and liposomes in vitro demonstrate that the size of the donor membrane significantly impacts lipid transfer efficiency by Ups1-Mdm35 complexes, with smaller liposomes (characterized by high positive curvature) promoting rapid lipid transfer.

This study offers significant new insights into the reaction cycle of phosphatidic acid (PA) transfer by Ups1 in mitochondria. Notably, the authors present compelling evidence that, alongside negatively charged phospholipids, positive membrane curvature enhances lipid transfer - an effect that is particularly relevant at the mitochondrial outer membrane. The experiments are technically robust, and my primary feedback pertains to the interpretation of specific results.

(1) The authors conclude from the lipid transfer assays (Figure 5) that lipid extraction is the rate-limiting step in the transfer cycle. While this conclusion seems plausible, it should be noted that the authors employed high concentrations of Ups1-Mdm35 along with less negatively charged phospholipids in these reactions. This combination may lead to binding becoming the rate-limiting factor. The authors should take this point into consideration. In this type of assay, it is challenging to clearly distinguish between binding, lipid extraction, and membrane dissociation as separate processes.

We thank the reviewer for the constructive and positive evaluation of our manuscript. We agree that, while our data support the interpretation that the rate-limiting step occurs at the donor membrane, it is difficult to dissect in our assay which of the individual steps at the donor membrane - such as binding of Ups1, lipid extraction into the binding pocket, or dissociation of Ups1 - is rate-limiting. Nevertheless, although we cannot exclude contributions from membrane binding or dissociation, several observations suggest that lipid extraction is a rate-limiting step under our experimental conditions.

The acceptor membrane has a similar lipid composition to the donor membrane (in tendency, the donor membrane is even a bit richer in binding-promoting lipids). If binding was ratelimiting, similar constraints would be expected at the acceptor membrane during lipid insertion. However, this is not observed.

Regarding dissociation, if this step were rate-limiting, one would expect similar constraints to be evident at the acceptor vesicles as well. Nevertheless, membrane dissociation might be mechanistically coupled to lipid extraction and thus difficult to evaluate as an independent step.

Based on our data and the considerations described above, we suggest that lipid extraction is the dominant rate-limiting step at the donor membrane under our conditions. However, we agree that a clear separation of these individual steps is not possible with the current experimental design. We will revise the corresponding passage to clarify that the rate-limiting step occurs at the donor membrane and, based on our observations, likely involves lipid extraction. Future studies aiming on dissecting these steps, will be important for elucidating the mechanism and regulation of Ups1-mediated lipid transfer both in vitro and in vivo.

(2) The authors should discuss that variations in the size of liposomes will also affect the distance between them at a constant concentration, which may affect the rate of lipid transfer. Therefore, the authors should determine the average size and size distribution of liposomes after sonication (by DLS or nanoparticle analyzer, etc.)

We agree that variations in liposome size will influence the average distance between vesicles at a given lipid concentration, which may in turn affect the rate of lipid transfer. As suggested, we will include DLS measurements to characterize the size distribution of our different liposome preparations.

Our setup was designed to keep the total membrane surface area comparable across conditions. This approach ensures a comparable overall binding capacity for Ups1 and enables the comparison of membrane binding and lipid extraction from different membranes. However, we agree that vesicle spacing, which is affected by liposome size at constant lipid concentration, could potentially influence certain steps in the transfer process, such as the time required for Ups1 to travel between donor and acceptor membranes. Whether this intermembrane travel time contributes to rate limitation is indeed an interesting question, and we will address this point through further discussion in the revised manuscript.

Investigating such effects in our current experimental system would require altering the vesicle concentration, which would in turn change the total membrane surface area and introduce additional variables. Nevertheless, exploring the influence of vesicle spacing and intermembrane distance on lipid transfer represents a promising direction for future studies aimed at dissecting the rate-limiting steps of the transfer cycle.

(3) The authors use NBD-PA in the lipid transfer assays. Does the size of the donor liposomes affect the transfer of NBD-PA and DOPA similarly? Since NBD-labeled lipids are somewhat unstable within lipid bilayers (as shown by spontaneous desorption in Figure 5B), monitoring the transfer of unlabeled PA in at least one setting would strengthen the conclusion of the swap experiments.

Ups1-mediated transfer of PA has been demonstrated both by mass spectrometry analysis of donor and acceptor vesicles (Connerth et al., 2012) and by NBD-fluorescence-based lipid transfer assays (Lu et al., 2020; Miliara et al., 2015; Miliara et al., 2019; Miliara et al., 2023; Potting et al., 2013; Watanabe et al., 2015). The fluorescence-based approach has been the most widely applied across multiple studies and has enabled detailed analysis of various aspects of lipid transfer by Ups1. It has been used to investigate mutants of key structural elements—such as the lipid-binding pocket and the α2–loop region. It has also been used to analyze fusion constructs between Ups1 and Mdm35, the influence of Mdm35 variants, and competition with excess Mdm35. Additionally, by comparing the transfer of NBD-labeled PA and NBD-labeled PS, this assay has provided insights into the determinants of the lipid specificity of Ups1. Hence, our experiments are based on the standard assay used to analyse lipid transfer in the field and thus can be corralated with the majority of published data.

Nevertheless, we agree that it is important to keep in mind that NBD labeling may alter the biophysical properties of lipids and, consequently, affect their transfer efficiency. Moreover, NBD-labeled lipids are not suitable for comparing the transfer efficiency of different PA species, as the label itself may mask differences in acyl chain composition. Therefore, it will be valuable to establish complementary methods that do not rely on NBD-labeled PA. We aim to develop these non-standard methods for possible inclusion in the present study, but even if not fully implemented at this stage, they will certainly form an important part of future investigations.

(4) The present study suggests that membrane domains with positive curvature at the outer membrane may serve as starting points for lipid transport by Ups1-Mdm35. Is anything known about the mechanisms that form such structures? This should be discussed in the text.

The origin of positively curved membrane domains is indeed highly relevant in the context of our findings, and while not the primary focus of this work, we will place more emphasis on discussing how such curvature may arise. Mechanisms include the action of curvature-generating proteins, asymmetric lipid composition and curvature induced at membrane contact sites. We have so far included examples of proteins in the outer mitochondrial membrane that are expected to influence curvature in their vicinity, and we will expand on this aspect and other contributing factors more thoroughly in the revised text.

Reviewer #2:

Summary:

Lipid transfer between membranes is essential for lipid biosynthesis across different organelle membranes. Ups1-Mdm35 is one of the best-characterized lipid transfer proteins, responsible for transferring phosphatidic acid (PA) between the mitochondrial outer membrane (OM) and inner membrane (IM), a process critical for cardiolipin (CL) synthesis in the IM. Upon dissociation from Mdm35, Ups1 binds to the intermembrane space (IMS) surface of the OM, extracts a PA molecule, re-associates with Mdm35, and moves through the aqueous IMS to deliver PA to the IM. Here, the authors analyzed the early steps of this PA transfer - membrane binding and PA extraction - using a combination of in vitro biochemical assays with lipid liposomes and purified Ups1-Mdm35 to measure liposome binding, lipid transfer between liposomes, and lipid extraction from liposomes. The authors found that membrane curvature, a previously overlooked property of the membrane, significantly affects PA extraction but not PA insertion into liposomes. These findings were further supported by MD simulations.

Strengths:

The experiments are well-designed, and the data are logically interpreted. The present study provides an important basis for understanding the mechanism of lipid transfer between membranes.  

Weaknesses:

The physiological relevance of membrane curvature in lipid extraction and transfer still remains open.

We thank the reviewer for the constructive feedback on our work. We agree that the physiological relevance of membrane curvature in lipid extraction and transfer remains an open question. Our data show that Ups1 binding to native-like OM membranes under physiological pH conditions is curvature-dependent, supporting the idea that this mechanism may optimize lipid transfer in vivo. While the intricate biophysical basis of this behaviour can only be dissected in vitro, these findings offer valuable insight into how curvature may functionally regulate Ups1 activity in the cellular context. To directly test this, it will be important in future studies to identify Ups1 mutants that lack curvature sensitivity and assess their performance in vivo, which will help clarify the physiological importance of this mechanism.

Reviewer #3:

The manuscript by Sadeqi et al. studies the interactions between the mitochondrial protein Ups1 and reconstituted membranes. The authors apply synthetic liposomal vesicles to investigate the role of pH, curvature, and charge on the binding of Ups1 to membranes and its ability to extract PA from them. The manuscript is well wrifen and structured. With minor exceptions, the authors provide all relevant information (see minor points below) and reference the appropriate literature in their introduction. The underlying question of how the energy barrier for lipid extraction from membranes is overcome by Ups1 is interesting, and the data presented by the authors could offer a valuable new perspective on this process. It is also certainly a challenging in vitro reconstitution experiment, as the authors aim to disentangle individual membrane properties (e.g., curvature, charge, and packing density) to study protein adsorption and lipid transfer. I have one major suggestion and a few minor ones that the authors might want to consider to improve their manuscript and data interpretation:

Major Comments:

The experiments are performed with reconstituted vesicles, which are incubated with recombinant protein variants and quantitatively assessed in flotation and pelleting assays. According to the Materials and Methods section, the lipid concentration in these assays is kept constant at 5 µM. However, the authors change the size of the vesicles to tune their curvature. Using the same lipid concentration but varying vesicle sizes results in different total vesicle concentrations. Moreover, larger vesicles (produced by freeze-thawing and extrusion) tend to form a higher proportion of multilamellar vesicles, thus also altering the total membrane area available for binding. Could these differences in the experimental system account for the variation in binding? To address this, the authors would need to perform the experiments either under saturation (excess protein) conditions or find an experimental approach to normalize for these differences.

We thank the reviewer for the constructive and positive comments. We agree that, since the total number of lipids was kept constant, the number of vesicles varied with vesicle size in our experiments. However, the setup was specifically designed to maintain a comparable total membrane surface area across conditions, ensuring a comparable number of available binding sites for Ups1. Because membrane surface area decreases with the square of the vesicle radius, keeping vesicle number constant would have led to a marked reduction in binding surface. Our approach was therefore aimed at preserving comparable binding capacity while varying membrane curvature.

With respect to multilamellarity, we thank the reviewer for addressing this important point. As described above, we aimed to maintain a constant total membrane surface area across all conditions to ensure an equal number of potential binding sites. We agree that multilamellarity in large liposomes could restrict accessibility to part of the membrane surface. However, we see in our experiments that even when the total membrane surface area of the small liposomes is reduced to one quarter of the standard amount, binding to the small liposomes remained stronger than to the larger liposomes at the higher concentration. This strongly indicates that restricted accessibility cannot account for the curvature-specific effect observed. Nonetheless, we will further address this aspect experimentally and in the discussion of the revised manuscript.

References

Connerth, M., Tatsuta, T., Haag, M., Klecker, T., Westermann, B., & Langer, T. (2012). Intramitochondrial transport of phosphatidic acid in yeast by a lipid transfer protein. Science, 338(6108), 815-818. https://doi.org/10.1126/science.1225625 

Lu, J., Chan, C., Yu, L., Fan, J., Sun, F., & Zhai, Y. (2020). Molecular mechanism of mitochondrial phosphatidate transfer by Ups1. Commun Biol, 3(1), 468. https://doi.org/10.1038/s42003-020-01121-x 

Miliara, X., Garnef, J. A., Tatsuta, T., Abid Ali, F., Baldie, H., Perez-Dorado, I., Simpson, P., Yague, E., Langer, T., & Mafhews, S. (2015). Structural insight into the TRIAP1/PRELI-like domain family of mitochondrial phospholipid transfer complexes. EMBO Rep, 16(7), 824-835. https://doi.org/10.15252/embr.201540229 

Miliara, X., Tatsuta, T., Berry, J. L., Rouse, S. L., Solak, K., Chorev, D. S., Wu, D., Robinson, C. V., Mafhews, S., & Langer, T. (2019). Structural determinants of lipid specificity within Ups/PRELI lipid transfer proteins. Nat Commun, 10(1), 1130. https://doi.org/10.1038/s41467-019-09089-x 

Miliara, X., Tatsuta, T., Eiyama, A., Langer, T., Rouse, S. L., & Mafhews, S. (2023). An intermolecular hydrogen bonded network in the PRELID-TRIAP protein family plays a role in lipid sensing. Biochim Biophys Acta Proteins Proteom, 1871(1), 140867. https://doi.org/10.1016/j.bbapap.2022.140867 

Posng, C., Tatsuta, T., Konig, T., Haag, M., Wai, T., Aaltonen, M. J., & Langer, T. (2013). TRIAP1/PRELI complexes prevent apoptosis by mediating intramitochondrial transport of phosphatidic acid. Cell Metab, 18(2), 287-295. https://doi.org/10.1016/j.cmet.2013.07.008 

Watanabe, Y., Tamura, Y., Kawano, S., & Endo, T. (2015). Structural and mechanistic insights into phospholipid transfer by Ups1-Mdm35 in mitochondria. Nat Commun, 6, 7922. https://doi.org/10.1038/ncomms8922

Author response:

The following is the authors’ response to the original reviews

Reviewer #1:

(1) 8 molar urea not only denatures proteins but also denatures DNA. Obviously, this does not affect the ChIP, since antibodies often recognize small linear epitopes and the proteins are crosslinked. However, under high urea conditions the BUR elements should be rendered single-stranded, and one wonders whether this has any effect on the procedure. The authors should alert the reader of these circumstances.

Thank you for raising this important question about the effects of 8M urea. We have added a brief paragraph explaining this point in the revised manuscript. Despite common misconceptions, 8M urea by itself does not actively convert double-stranded DNA to single-stranded DNA. For this conversion to occur, a heat denaturation step is required. Once DNA is heat-denatured to become single-stranded, urea can maintain this configuration. This is why the addition of 8M urea to acrylamide gel electrophoresis is a standard method for analyzing single-stranded oligonucleotides, but the DNA must first be denatured by heat (Summer et al., J. Vis. Exp. (32), e1485, DOI : 10.3791/1485). This is clearly described in published work comparing the status of DNA with and without heat treatment in an 8M urea-containing buffer (Hegedus et al., Nucl.Acids Res. 2009 (doi:10.1093/nar/gkp539).

We have additional evidence supporting this conclusion in the context of our urea ultracentrifugation experiment. Both crosslinked and un-crosslinked genomic DNA purified by 8M urea centrifugation can be digested with restriction enzymes, which indicates that the DNA remains double-stranded. For instance, we previously published SATB1 ChIP-3C results using Sau3A-digested DNA after urea purification. In the current paper, we used HindIII to digest urea-purified DNA for urea4C-seq. The BUR reference map can also be generated after restriction digestion of urea-purified DNA and isolating and sequencing SATB1-bound restriction fragments in vitro. If genomic DNA were denatured by 8M urea ultracentrifugation, we would not have been able to digest it with restriction enzymes to obtain these results.

We have now added a sentence noting that SATB1 is a double-stranded DNA-binding protein that does not bind to single-stranded DNA, as we have previously shown (Dickinson et al., 1992, Ref 32).

(2) An important conclusion is that urea-ChIP reveals direct DNA binding events, whereas standard ChIP shows indirect binding (which is stripped off by urea). I do not see any evidence for direct binding. At low resolution, predicted BUR elements are enriched in domains where SATB-1 is mapped by urea-ChIP. A statement like 'In a zoomed-in view, covering a 430 kb region, SATB1 sites identified from urea ChIP-seq precisely coincided with BUR peaks' is certainly not correct: most BUR peaks do not show significant SATB-1 binding. The randomly chosen regions shown in Figure 4 – Supplement 1 show how poor the overlap of SATB-1 and BURs is; indeed, they show that SATB-1 binds DNA mostly at non-BUR sites. I see Figure 2D, but such cumulative plots can be highly biased by very few cases. I suggest showing these data in heat maps instead.

We believe there may be some confusion regarding the interpretation of our figures. Looking at Track 3 (BUR reference map, RED peaks) and urea SATB1 Tracks 4 and 5 (replicas from two independent experiments) in Fig. 2B, the SATB1 peaks detected by urea ChIP-seq do indeed coincide with BUR peaks. In the revised manuscript, we have provided a further ‘zoomed-in’ view to better illustrate this point and also provided the underlying BUR sequence from one of these SATB1-bound regions (Figure 2—supplement figure 1).

It is true that many more BURs exist than SATB1-bound BURs, especially in gene-poor regions where BURs are clustered. However, from the perspective of SATB1-bound peaks, the majority of these coincide with BURs, as shown by both deepTools analyses and new heatmap, as suggested (Figure 2E, and Figure 7—supplement figure 3).

The results from our genome-wide quantitative analyses using deepTools to compare peaks from urea SATB1 ChIP-seq data and the BUR reference map shown in Supplementary Tables 1 and 2 are consistent with the heatmap analyses.

We must apologize for an error in the scaling of the y-axis in Figure 4-supplement figure 1 that likely contributed to some confusion. We have corrected our mistake in the revised manuscript. As we were preparing our figures, when placed in the figure and axes relabeled for legibility, the BUR reference peaks were mislabeled on their y-axis. In the figure the peaks were erroneously labeled on a scale of 0.1-1 read counts/million reads, but the data shown is actually scaled at 0.1 to 2 read counts per million reads. Unfortunately, we did not realize this error and, using the figure as a guide for scaling, provided urea SATB1 ChIP-seq peaks at a scale of 0.1-1 read counts/million reads to match the mislabeled BUR reference track. This had the effect of reducing the signal/noise in the SATB1 ChIP-seq data (Figure 1). We have now standardized the y-axis for fair comparison using a scaling of the y-axis at 0.1-2 for all tracks.  This will more clearly show that there are indeed more BUR peaks than SATB1-bound sites, consistent with our quantitative analysis.

We hope that these clarifications as well as the added heatmaps and binding site example allay the concerns about the specificity and overlap of SATB1 binding on BURS.

(3) In Figure 6C 'peaks' are compared. However, looking at Figure 4 - Supplement 1 again it is clear that peak calling can yield a misleading impression. Figure 6D suggests that there are more BURs than SATB-1 peaks but this is not true from looking at the browser.

We thank the reviewer for this observation. As noted in our response to point 2 above, the inconsistent y-axis scaling in Figure 4-supplement figure 1 created a misleading impression, which we have corrected in the revised manuscript. When properly displayed with consistent y-axis scaling, the browser view aligns with our quantitative data showing that there are indeed many more BURs than SATB1-bound sites. As mentioned under 2 above, we have performed genome-wide quantitative analysis by deepTools (Supplementary Tables 1 and 2) to confirm the results shown by bar graphs in Fig. 6C, 6D and Fig. 2D. 

In Figure 6C, the bars show the percentage of SATB1-bound peaks in each cell type (denominator) that overlap with confirmed BUR sites in the BUR reference map (numerator). In Figure 6D, we show the percentage of total BUR sites in the BUR reference map (denominator) that are bound by SATB1 from urea ChIP-seq (numerator). To avoid any confusion, we have added brief subtitles to Figures 6C and 6D in the revised manuscript.

(4) An important conclusion is that urea-ChIP reveals direct DNA binding events, whereas standard ChIP shows indirect binding (which is stripped off by urea). I do not yet see any evidence for direct binding. It cannot be excluded that the binding is RNA-mediated. The authors mention in passing that urea-ChIP material still contains (specific!) RNA. Given that this is a new procedure, the authors should document the RNA content of urea-ChIP and RNase-treat their samples prior to ChIP to monitor an RNA contribution.

Thank you for raising this important point. The direct binding of SATB1 to BURs is well-established in our previous work. Indeed, this was the main motivation to explore the reason for the lack of evidence for genome-wide SATB1 binding to BURs in the DNA-binding profile by standard ChIP-seq. This has been a major point of confusion for us for many years.

SATB1 was originally identified through a search for mammalian proteins that could recognize BURs specifically and not just any A+T-rich sequence. The Satb1 gene was originally cloned by an expression cDNA library and encoded SATB1 protein bound the BUR probe but not a mutated AT-rich BUR (control) probe.  Subsequent experiments confirmed that SATB1 specifically binds to many BURs without requiring additional factors. Furthermore, SATB1 recognizes BURs by binding in the minor groove of double-stranded DNA, presumably recognizing the altered phosphate backbone structure of BUR DNA, rather than accessing nucleotide bases (Dickinson et al, 1992).

We do agree with the reviewer, however, that there is a possibility that RNA can redirect SATB1 to different subsets of BURs and/or to interact indirectly with different regulatory regions depending on cell type or developmental stage. Although urea ultracentrifugation clearly separates most RNA (found in the middle region of the tube) from genomic DNA (pelleted at the bottom) (de Belle et al., 1998), upon crosslinking cells, a small quantity of RNA is found co-pelleted with DNA (our recent unpublished results). This RNA, tightly associated with crosslinked chromatin, may have some impact on SATB1 function.

Based on our preliminary data, we are currently planning to study the impact of RNA using RNase A as well as by targeting specific RNAs employing an anti-sense approach. We believe that thoroughly addressing the impact of RNA warrants a full paper, including the potential roles of specific non-coding RNAs in SATB1 function, and thus is beyond the scope of the current paper. However, we have now added discussion of this important point in the manuscript.

(5) An important aspect of the model is that SATB1 tethers active genes to inactive LADs. However, in the 4C experiment the BUR elements used to anchor the looping are both in the accessible, active chromatin domain. If the authors want to maintain their statement, they must show a 4C result that connects the 2 distinct domains and transverses A/B domain boundaries. Currently, the data only show a looping within accessible chromatin.

We appreciate REVIEWER 1 for bringing up the important point that our model could potentially be interpreted as “SATB1 tethers active genes to inactive LADs.” Since we describe that BURs are enriched in LADs and that SATB1 binds a subset of BURs, readers may assume that we aim to demonstrate, through urea 4C-seq, that SATB1 tethers active genes to transcriptionally-inactive LADs (via BURs). However, this is not our intention in the model (Figure 8). In the experiment we designed for our present study,  we selected BUR-1 and BUR-2 as viewpoints from a non-LAD gene-rich region (inter-LAD). Because these BURs are bound by SATB1, it indicates that these BURs are part of the “hard-to-access” SATB1-rich subnuclear structure, which resists extraction, in contrast to accessible chromatin. Thus, we illustrate in the model that BURs anchored to the SATB1-rich nuclear substructure make contact with accessible chromatin over long distances in a SATB1-dependent manner. Therefore, we do not intend to conclude that SATB1 mediates interactions between LADs and inter-LADs (accessible chromatin) from our current study: this would be a topic for future research. In the original model in the submitted manuscript, we used the terms “inaccessible” and “accessible.” In the revised version, we clarified this in the model by changing “inaccessible” to “SATB1-rich subnuclear structure” and carefully revised  the text in the Figure 8 legend to clarify the model. 

At this time, we do not know exactly how LADs and SATB1 nuclear architecture are related spatially and functionally. While LADs are mapped as genomic domains in proximity to Lamin B1 by LaminB1-DamID, BURs are mapped at ~300-500 bp resolution by urea ChIP-seq. To gain further insight into this important question, a large body of DNA-FISH and immunoDNA-FISH experiments will be required, comparing different cell types to see whether and how specific BURs move between LADs and SATB1 nuclear architecture. Such experiments may benefit from testing the Gabrg1 and Gabra2 loci, where many BURs are anchored to SATB1 in neurons but not in thymocytes, for instance.  This is included in Discussion in the revised manuscript.

Regarding the reviewer's second point about showing more extended domains for 4C interactions, we would like to highlight that Figure 5—supplement figure 3 in our submitted manuscript addresses this concern. This figure shows that BUR-interactions extend to multiple gene-rich regions across intervening gene-poor regions. Interestingly, BUR-1 and BUR-2 interactions skip a transcriptionally silent gene-rich region containing olfactory receptor genes but interact with subsequent gene-rich regions containing active genes. These data demonstrate that BUR-interactions do indeed traverse A- and B-compartment boundaries.  In the revised manuscript (in Figure 5—supplement figure 3), we newly added a Lamin B1-DamID (thymocyte) track.  Comparing with LADs, BUR-1 interactions occur mostly in non-LAD regions. Some minor overlap with LADs was detected in high resolution views (not shown). Future experiments testing BUR viewpoints that reside within LADs are required to assess whether SATB1 mediates interactions between B and A compartments.

(6) The description of the urea-co-immunoprecipitation experiment (Figure 3C) could be improved to make it unequivocally clear that co-binding to chromatin is tested, not protein-protein interaction (which is destroyed by urea).

Thank you for this helpful suggestion. We have revised the text in the manuscript by stating “Distinct from protein-protein co-immunoprecipitation (co-IP) using whole cell or nuclear extracts, we examined the direct co-binding status on chromatin in vivo of SATB1 and CTCF or cohesin by urea ChIP-Western”.

Reviewer #2:

(1) Since SATB1 has been described to interact with beta-catenin, I wonder if the authors have looked at TCF4/TCF7l2 binding patterns and their potential overlap with SATB1 binding patterns. This might appear a trivial request. However, uncontrolled WNT signalling is a major feature of cancer undergoing metastasis - a process that the authors have earlier associated with unscheduled SATB1 expression in triple-negative breast cancer.

We thank the reviewer for highlighting this important point about the potential relationship between SATB1 and TCF4/TCF7l2 binding patterns. Based on published observations with other factors (Rad21, CTCF, BRG1, RUNX) that show substantial overlap with SATB1 in standard ChIP-seq peaks(Kakugawa et al., Cell Rep 19, 1176-1188 (2017). DOI: 10.1016/j.celrep.2017.04.038. Poterlowicz et al., PLoS Genet, 2017 DOI: 10.1371/journal.pgen.1006966), we would anticipate that TCF4 might also show significant overlap with SATB1. An important question is whether the DNA binding profile of TCF4 depends on SATB1.

We have not yet generated ChIP-seq data for TCF4 in the presence and absence of SATB1, but we agree that such experiments could provide important insights into cancer progression as well as brain function. This represents an interesting direction for future work. We have added this point in our discussion based on your kind suggestion.

(2) The CTCF sizes indicated in the western blot analyses of Figures 3C and Figure 3 - supplement figure 2 do not display the normal size, which is around 130 kDa. Either the issue is erroneous marking or a so-called salt effect to slow the migration in the gel. Alternatively, it reflects a slower migrating form of CTCF generated by for example PARylation (by PARP1) that is known to approach 180 kDa. It would be useful if the authors could clarify this minor issue.

We appreciate the reviewer pointing out this discrepancy. As the reviewer correctly noted, CTCF can appear at a higher molecular weight due to post-translational modifications such as PARylation and O-GlcNAcylation, which alter its migration during electrophoresis.

Upon re-examination of our raw data for Figure 3—supplement figure 2A, we discovered that the marker lane for the CTCF panel was broken, and the 150kDa band was erroneously assigned. This led to the 150kDa marker being placed below the CTCF migration position, which is clearly an error. We thank the reviewer for bringing this to our attention.

We have checked our other data and consistently observe CTCF migrating below the 150kDa band, similar to the pattern shown on the Abcam website for the antibody we used (ab128873) (Figure 2). For Figure 3-supplement figure 2, we will use a marker lane from a parallel gel with identical composition and run time to correctly indicate the molecular weight. We havealso corrected the marker position in Figure 3C.

Reviewing Editor (Recommendations for the authors):

(1) The introduction states that urea ChIP-seq is "unbiased", which is difficult to unambiguously determine and therefore might be an overstatement. Maybe the authors could consider rephrasing.

We agree with the reviewer's assessment and have rephrased our description of the urea ChIP-seq method to avoid using the term "unbiased."

(2) The authors propose that in standard ChIP, most SATB1 is in the insoluble fraction. This seems easy to test and demonstrating it may help to further clarify the differences between the protocols.

We appreciate this suggestion and would like to clarify our description. What we stated in the manuscript was:

"We envision that SATB1 bound to inaccessible nuclear regions may be lost in the insoluble fraction."

This refers specifically to a subpopulation of SATB1 that is bound to the high-salt extraction-resistant nuclear substructure, not to the total SATB1 protein. We also noted elsewhere in the manuscript that:

"SATB1 proteins are found in high salt-resistant fraction as well as salt-extracted fraction (40). Thus, it is possible that soluble SATB1 may associate with open chromatin."

Our unpublished results show that SATB1 proteins exist in at least two distinct forms based on protein mobility: SATB1 with high mobility and another with very low or no mobility. While we have identified the SATB1 domain responsible for each of these distinct mobility patterns, we have not yet identified biochemical differences that would allow us to distinguish them conclusively. Therefore, an experiment to test the distribution of SATB1 in soluble versus insoluble fractions would show SATB1 in both fractions but would not necessarily provide information about the functional significance of these different populations. We believe this is an important area for future research and are working to develop tools to specifically distinguish and characterize SATB1 in the soluble versus insoluble fractions.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This work studies representations in a network with one recurrent layer and one output layer that needs to path-integrate so that its position can be accurately decoded from its output. To formalise this problem, the authors define a cost function consisting of the decoding error and a regularisation term. They specify a decoding procedure that at a given time averages the output unit center locations, weighted by the activity of the unit at that time. The network is initialised without position information, and only receives a velocity signal (and a context signal to index the environment) at each timestep, so to achieve low decoding error it needs to infer its position and keep it updated with respect to its velocity by path integration.

The authors take the trained network and let it explore a series of environments with different geometries while collecting unit activities to probe learned representations. They find localised responses in the output units (resembling place fields) and border responses in the recurrent units. Across environments, the output units show global remapping and the recurrent units show rate remapping. Stretching the environment generally produces stretched responses in output and recurrent units. Ratemaps remain stable within environments and stabilise after noise injection. Low-dimensional projections of the recurrent population activity forms environment-specific clusters that reflect the environment's geometry, which suggests independent rather than generalised representations. Finally, the authors discover that the centers of the output unit ratemaps cluster together on a triangular lattice (like the receptive fields of a single grid cell), and find significant clustering of place cell centers in empirical data as well.

The model setup and simulations are clearly described, and are an interesting exploration of the consequences of a particular set of training requirements - here: path integration and decodability. But it is not obvious to what extent the modelling choices are a realistic reflection of how the brain solves navigation. Therefore it is not clear whether the results generalize beyond the specifics of the setup here.

Strengths:

The authors introduce a very minimal set of model requirements, assumptions, and constraints. In that sense, the model can function as a useful 'baseline', that shows how spatial representations and remapping properties can emerge from the requirement of path integration and decodability alone. Moreover, the authors use the same formalism to relate their setup to existing spatial navigation models, which is informative.

The global remapping that the authors show is convincing and well-supported by their analyses. The geometric manipulations and the resulting stretching of place responses, without additional training, are interesting. They seem to suggest that the recurrent network may scale the velocity input by the environment dimensions so that the exact same path integrator-output mappings remain valid (but maybe there are other mechanisms too that achieve the same).

The clustering of place cell peaks on a triangular lattice is intriguing, given there is no grid cell input. It could have something to do with the fact that a triangular lattice provides optimal coverage of 2d space? The included comparison with empirical data is valuable, although the authors only show significant clustering - there is no analysis of its grid-like regularity.

First of all, we would like to thank the reviewer for their comprehensive feedback, and their insightful comments. Importantly, as you point out, our goal with this model was to build a minimal model of place cell representations, where representations were encouraged to be place-like, but free to vary in tuning and firing locations. By doing so, we could explore what upstream representations facilitate place-like representations, and even remapping (as it turned out) with minimal assumptions. However, we agree that our task does not capture some of the nuances of real-world navigation, such as sensory observations, which could be useful extensions in future work. Then again, the simplicity of our setup makes it easier to interpret the model, and makes it all the more surprising that it learns many behaviors exhibited by real world place cells.

As to the distribution of phases - we also agree that a hexagonal arrangement likely reflects some optimal configuration for decoding of location.

And we agree that the symmetry within the experimental data is important; we have revised analyses on experimental phase distributions, and included an analysis of ensemble grid score, to quantify any hexagonal symmetries within the data.

Weaknesses:

The navigation problem that needs to be solved by the model is a bit of an odd one. Without any initial position information, the network needs to figure out where it is, and then path-integrate with respect to a velocity signal. As the authors remark in Methods 4.2, without additional input, the only way to infer location is from border interactions. It is like navigating in absolute darkness. Therefore, it seems likely that the salient wall representations found in the recurrent units are just a consequence of the specific navigation task here; it is unclear if the same would apply in natural navigation. In natural navigation, there are many more sensory cues that help inferring location, most importantly vision, but also smell and whiskers/touch (which provides a more direct wall interaction; here, wall interactions are indirect by constraining velocity vectors). There is a similar but weaker concern about whether the (place cell like) localised firing fields of the output units are a direct consequence of the decoding procedure that only considers activity center locations.

Thank you for raising this point; we absolutely agree that the navigation task is somewhat niche. However, this was a conscious decision, to minimize any possible confounding from alternate input sources, such as observations. In part, this experimental design was inspired by the suggestion that grid cells support navigation/path integration in open-field environments with minimal sensory input (as they could, conceivably do so with no external input). This also pertains to your other point, that boundary interactions are necessary for navigation. In our model, using boundaries is one solution, but there is another way around this problem, which is conceivably better: to path integrate in an egocentric frame, starting from your initial position. Since the locations of place fields are inferred only after a trajectory has been traversed, the network is free to create a new or shifted representation every time, independently of the arena. In this case, one might have expected generalized solutions, such as grid cells to emerge. That this is not the case, seems to suggest that grid cells may somehow not be optimal for pure path integration, or at the very least, hard to learn (but may still play a part, as alluded to by place field locations). We have tried to make these points more evident in the revised manuscript.

As for the point that the decoding may lead to place-like representations, this is a fair point. Indeed, we did choose this form of decoding, inspired by the localized firing of place cells, in the hope that it would encourage minimally constrained, place-like solutions. However, compared to other works (Sorscher and Xu) hand tuning the functional form of their place cells, our (although biased towards centralized tuning curves) allows for flexible functional forms such as the position of the place cell centers, their tuning width, whether or not it is center-surround activity, and how they should tune to different environments/rooms. This allows us to study several features of the place cell system, such as remapping and field formation. We have revised to make this more clear in the model description.

The conclusion that 'contexts are attractive' (heading of section 2) is not well-supported. The authors show 'attractor-like behaviour' within a single context, but there could be alternative explanations for the recovery of stable ratemaps after noise injection. For example, the noise injection could scramble the network's currently inferred position, so that it would need to re-infer its position from boundary interactions along the trajectory. In that case the stabilisation would be driven by the input, not just internal attractor dynamics. Moreover, the authors show that different contexts occupy different regions in the space of low-dimensional projections of recurrent activity, but not that these regions are attractive.

We agree that boundary interactions could facilitate the convergence of representations after noise injection. We did try to moderate this claim by the wording “attractor-like”, but we agree that boundaries could confound this result. We have therefore performed a modified noise injection experiment, where we let the network run for an extended period of time, before noise injection (and no velocity signal), see Appendix Velocity Ablation in the revised text. Notably, representations converge to their pre-scrambled state after noise injection, even without a velocity signal. However, place-like representations do not converge for all noise levels in this case, possibly indicating that boundary interactions do serve an error-correcting function, also. Thank you for pointing this out.

As for the attractiveness of contexts, we agree that more analyses were required to demonstrate this. We have therefore conducted a supplementary analysis where we run the trained network with a mismatch in context/geometry, and demonstrate that the context signal fixes the representation, up to geometric distortions.

The authors report empirical data that shows clustering of place cell centers like they find for their output units. They report that 'there appears to be a tendency for the clusters to arrange in hexagonal fashion, similar to our computational findings'. They only quantify the clustering, but not the arrangement. Moreover, in Figure 7e they only plot data from a single animal, then plot all other animals in the supplementary. Does the analysis of Fig 7f include all animals, or just the one for which the data is plotted in 7e? If so, why that animal? As Appendix C mentions that the ratemap for the plotted animal 'has a hexagonal resemblance' whereas other have 'no clear pattern in their center arrangements', it feels like cherrypicking to only analyse one animal without further justification.

Thank you for pointing this out; we agree that this is not sufficiently explained and explored in the current version. We have therefore conducted a grid score analysis of the experimental place center distributions, to uncover possible hexagonal symmetries. The reason for choosing this particular animal was in part because it featured the largest number of included cells, while also demonstrating the most striking phase distribution, while including all distributions in the supplementary. Originally, this was only intended as a preliminary analysis, suggesting non-uniformity in experimental place field distributions, but we realize that these may all provide interesting insight into the distributional properties of place cells.

We have explained these choices in the revised text, and expanded analyses on all animals to showcase these results more clearly.

Reviewer #2 (Public Review):

Summary:

The authors proposed a neural network model to explore the spatial representations of the hippocampal CA1 and entorhinal cortex (EC) and the remapping of these representations when multiple environments are learned. The model consists of a recurrent network and output units (a decoder) mimicking the EC and CA1, respectively. The major results of this study are: the EC network generates cells with their receptive fields tuned to a border of the arena; decoder develops neuron clusters arranged in a hexagonal lattice. Thus, the model accounts for entorhinal border cells and CA1 place cells. The authors also suggested the remapping of place cells occurs between different environments through state transitions corresponding to unstable dynamical modes in the recurrent network.

Strengths:

The authors found a spatial arrangement of receptive fields similar to their model's prediction in experimental data recorded from CA1. Thus, the model proposes a plausible mechanisms to generate hippocampal spatial representations without relying on grid cells. This result is consistent with the observation that grid cells are unnecessary to generate CA1 place cells.

The suggestion about the remapping mechanism shows an interesting theoretical possibility.

We thank the reviewer for their kind feedback.

Weaknesses:

The explicit mechanisms of generating border cells and place cells and those underlying remapping were not clarified at a satisfactory level.

The model cannot generate entorhinal grid cells. Therefore, how the proposed model is integrated into the entire picture of the hippocampal mechanism of meｍory processing remains elusive.

We appreciate this point, and hope to clarify: From a purely architectural perspective, place-like representations are generated by linear combinations of recurrent unit representations, which, after training, appear border-like. During remapping, the network is simply evaluated/run in different geometries/contexts, which, it turns out, causes the network to exhibit different representations, likely as solutions to optimally encoding position in the different environments. We have attempted to revise the text to make some of these interpretations more clear. We have also conducted a supplementary analysis to demonstrate how representations are determined by the context signal directly, which helps to explain how recurrent and output units form their representations.

We also agree that our model does not capture the full complexity of the Hippocampal formation. However, we would argue that its simplicity (focusing on a single cell type and a pure path integration task), acts as a useful baseline for studying the role of place cells during spatial navigation. The fact that our model captures a range of place cell behaviors (field formation, remapping and geometric deformation) without grid cells also point to several interesting possibilities, such that grid cells may not be strictly necessary for place cell formation and remapping, or that border cells may account for many of the peculiar behaviors of place cells. However, we wholeheartedly agree that including e.g. sensory information and memory storage/retrieval tasks would prove a very interesting extension of our model to more naturalistic tasks and settings. In fact, our framework could easily accommodate this, e.g. by decoding contexts/observations/memories from the network state, alongside location.

Reviewer #3 (Public Review):

Summary:

The authors used recurrent neural network modelling of spatial navigation tasks to investigate border and place cell behaviour during remapping phenomena.

Strengths:

The neural network training seemed for the most part (see comments later) well-performed, and the analyses used to make the points were thorough.

The paper and ideas were well explained.

Figure 4 contained some interesting and strong evidence for map-like generalisation as environmental geometry was warped.

Figure 7 was striking, and potentially very interesting.

It was impressive that the RNN path-integration error stayed low for so long (Fig A1), given that normally networks that only work with dead-reckoning have errors that compound. I would have loved to know how the network was doing this, given that borders did not provide sensory input to the network. I could not think of many other plausible explanations... It would be even more impressive if it was preserved when the network was slightly noisy.

Thank you for your insightful comments! Regarding the low path integration error, there is a slight statistical signal from the boundaries, as trajectories tend to turn away from arena boundaries. However, we agree, that studying path integration performance in the face of noise would make for a very interesting future development.

Weaknesses:

I felt that the stated neuroscience interpretations were not well supported by the presented evidence, for a few reasons I'll now detail.

First, I was unconvinced by the interpretation of the reported recurrent cells as border cells. An equally likely hypothesis seemed to be that they were positions cells that are linearly encoding the x and y position, which when your environment only contains external linear boundaries, look the same. As in figure 4, in environments with internal boundaries the cells do not encode them, they encode (x,y) position. Further, if I'm not misunderstanding, there is, throughout, a confusing case of broken symmetry. The cells appear to code not for any random linear direction, but for either the x or y axis (i.e. there are x cells and y cells). These look like border cells in environments in which the boundaries are external only, and align with the axes (like square and rectangular ones), but the same also appears to be true in the rotationally symmetric circular environment, which strikes me as very odd. I can't think of a good reason why the cells in circular environments should care about the particular choice of (x,y) axes... unless the choice of position encoding scheme is leaking influence throughout. A good test of these would be differently oriented (45 degree rotated square) or more geometrically complicated (two diamonds connected) environments in which the difference between a pure (x,y) code and a border code are more obvious.

Thank you for pointing this out. This is an excellent point, that we agree could be addressed more rigorously. Note that there is no position encoding in our model; the initial state of the network is a vector of zeros, and the network must infer its location from boundary interactions and context information alone. So there is no way for positional information to leak through to the recurrent layer directly. However, one possible reason for the observed symmetry breaking, is the fact that the velocity input signal is aligned with the cardinal directions. To investigate this, we trained a new model, wherein input velocities are rotated 45 degrees relative to the horizontal, as you suggest. The results, shown and discussed in appendix E (Learned recurrent representations align with environment boundaries), do indicate that representations are tuned to environment boundaries, and not the cardinal directions, which hopefully improves upon this point.

Next, the decoding mechanism used seems to have forced the representation to learn place cells (no other cell type is going to be usefully decodable?). That is, in itself, not a problem. It just changes the interpretation of the results. To be a normative interpretation for place cells you need to show some evidence that this decoding mechanism is relevant for the brain, since this seems to be where they are coming from in this model. Instead, this is a model with place cells built into it, which can then be used for studying things like remapping, which is a reasonable stance.

This is a great point, and we agree. We do write that we perform this encoding to encourage minimally constrained place-like representations (to study their properties), but we have revised to make this more evident.

However, the remapping results were also puzzling. The authors present convincing evidence that the recurrent units effectively form 6 different maps of the 6 different environments (e.g. the sparsity of the code, or fig 6a), with the place cells remapping between environments. Yet, as the authors point out, in neural data the finding is that some cells generalise their co-firing patterns across environments (e.g. grid cells, border cells), while place cells remap, making it unclear what correspondence to make between the authors network and the brain. There are existing normative models that capture both entorhinal's consistent and hippocampus' less consistent neural remapping behaviour (Whittington et al. and probably others), what have we then learnt from this exercise?

Thanks for raising this point! We agree that this finding is surprising, but we hold that it actually shows something quite important: that border-type units are sufficient to create place-like representations, and learns several of the behaviors associated with place cells and remapping (including global remapping and field stretching). In other words, a single cell type known to exist upstream of place cells is sufficient to explain a surprising range of phenomena, demonstrating that other cell types are not strictly necessary. However, we agree that understanding why the boundary type units sometimes rate remap, and whether that can be true for some border type cells in the brain (either directly, or through gating mechanisms) would be important future developments. Related to this point, we also expanded upon the influence of the context signal for representation selection (appendix F)

Concerning the relationship to other models, we would argue that the simplicity of our model is one of its core strengths, making it possible to disentangle what different cell types are doing. While other models, including TEM, are highly important for understanding how different cell types and brain regions interact to solve complex problems, we believe there is a need for minimal, understandable models that allows us to investigate what each cell type is doing, and this is where we believe our work is important. As an example, our model not only highlights the sufficiency of boundary-type cells as generators of place cells, its lack of e.g. grid cells also suggest that grid cells may not be strictly necessary for e.g. open-field/sensory-deprived navigation, as is often claimed.

One striking result was figure 7, the hexagonal arrangement of place cell centres. I had one question that I couldn't find the answer to in the paper, which would change my interpretation. Are place cell centres within a single clusters of points in figure 7a, for example, from one cell across the 100 trajectories, or from many? If each cluster belongs to a different place cell then the interpretation seems like some kind of optimal packing/coding of 2D space by a set of place cells, an interesting prediction. If multiple place cells fall within a single cluster then that's a very puzzling suggestion about the grouping of place cells into these discrete clusters. From figure 7c I guess that the former is the likely interpretation, from the fact that clusters appear to maintain the same colour, and are unlikely to be co-remapping place cells, but I would like to know for sure!

This is a good point, and you are correct: one cluster tends to correspond to one unit. To make this more clear, we have revised Fig. 7, so that each decoded center is shaded by unit identity, which makes this more evident. And yes, this is, seemingly in line with some form of optimal packing/encoding of space, yes!

I felt that the neural data analysis was unconvincing. Most notably, the statistical effect was found in only one of seven animals. Random noise is likely to pass statistical tests 1 in 20 times (at 0.05 p value), this seems like it could have been something similar? Further, the data was compared to a null model in which place cell fields were randomly distributed. The authors claim place cell fields have two properties that the random model doesn't (1) clustering to edges (as experimentally reported) and (2) much more provocatively, a hexagonal lattice arrangement. The test seems to collude the two; I think that nearby ball radii could be overrepresented, as in figure 7f, due to either effect. I would have liked to see a computation of the statistic for a null model in which place cells were random but with a bias towards to boundaries of the environment that matches the observed changing density, to distinguish these two hypotheses.

Thanks for raising this point. We agree that we were not clear enough in our original manuscript. We included additional analyses in one animal, to showcase one preliminary case of non-uniform phases. To mitigate this, we have performed the same analyses for all animals, and included a longer discussion of these results (included in the supplementary material). We have also moderated the discussion on Ripley’s H to encompass only non-uniformity, and added a grid score analysis to showcase possible rotational symmetries in the data. We hope this gets our findings across more clearly

Some smaller weaknesses:

- Had the models trained to convergence? From the loss plot it seemed like not, and when including regularisors recent work (grokking phenomena, e.g. Nanda et al. 2023) has shown the importance of letting the regularisor minimise completely to see the resulting effect. Else you are interpreting representations that are likely still being learnt, a dangerous business.

Longer training time did not seem to affect representations. However, due to the long trajectories and statefulness involved, training was time-intensive and could become unstable for very long training. We therefore stopped training at the indicated time.

- Since RNNs are nonlinear it seems that eigenvalues larger than 1 doesn't necessarily mean unstable?

This is a good point; stability is not guaranteed. We have updated the text to reflect this.

- Why do you not include a bias in the networks? ReLU networks without bias are not universal function approximators, so it is a real change in architecture that doesn't seem to have any positives?

We found that bias tended to have a detrimental effect on training, possibly related to the identity initialization used (see e.g. Le et al. 2015), and found that training improved when biases were fixed to zero.

- The claim that this work provided a mathematical formalism of the intuitive idea of a cognitive map seems strange, given that upwards of 10 of the works this paper cite also mathematically formalise a cognitive map into a similar integration loss for a neural network.

We agree that other works also provide ways of formalizing this concepts. However, our goal by doing so was to elucidate common features across these seemingly disparate models. We also found that the concept of a learned and target map made it easier to come up with novel models, such as one wherein place cells are constructed to match a grid cell label.

Aim Achieved? Impact/Utility/Context of Work

Given the listed weaknesses, I think this was a thorough exploration of how this network with these losses is able to path-integrate its position and remap. This is useful, it is good to know how another neural network with slightly different constraints learns to perform these behaviours. That said, I do not think the link to neuroscience was convincing, and as such, it has not achieved its stated aim of explaining these phenomena in biology. The mechanism for remapping in the entorhinal module seemed fundamentally different to the brain's, instead using completely disjoint maps; the recurrent cell types described seemed to match no described cell type (no bad thing in itself, but it does limit the permissible neuroscience claims) either in tuning or remapping properties, with a potentially worrying link between an arbitrary encoding choice and the responses; and the striking place cell prediction was unconvincingly matched by neural data. Further, this is a busy field in which many remapping results have been shown before by similar models, limiting the impact of this work. For example, George et al. and Whittington et al. show remapping of place cells across environments; Whittington et al. study remapping of entorhinal codes; and Rajkumar Vasudeva et al. 2022 show similar place cell stretching results under environmental shifts. As such, this papers contribution is muddied significantly.

Thank you for this perspective; we agree that all of these are important works that arrive at complementary findings. We hold that the importance of our paper lies in its minimal nature, and its focus on place cells, via a purpose-built decoding that enables place-like representations. In doing so, we can point to possibly under explored relationships between cell types, in particular place cells and border cells, while challenging the necessity of other cell types for open-field navigation (i.e. grid cells). In addition, our work points to a novel connection between grid cells, place cells and even border cells, by way of the hexagonal arrangement of place unit centers. However, we agree that expanding our model to include more biologically plausible architectures and constraints would make for a very interesting extension in the future.

Thank you again for your time, as well as insightful comments.  

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Even after reading Methods 5.3, I found it hard to understand how the ratemap population vectors that produce Fig 3e and Fig 5 are calculated. It's unclear to me how there can be a ratemap at a single timestep, because calculating a ratemap involves averaging the activity in each location, which would take a whole trajectory and not a single timestep. But I think I've understood from Methods 5.1 that instead the ratemap is calculated by running multiple 'simultaneous' trajectories, so that there are many visited locations at each timestep. That's a bit confusing because as far as I know it's not a common way to calculate ratemaps in rodent experiments (probably because it would be hard to repeat the same task 500 times, while the representations remain the same), so it might be worth explaining more in Methods 5.3.

We understand the confusion, and have attempted to make this more clear in the revised manuscript. We did indeed create ratemaps over many trajectories for time-dependent plots, for the reasons you mentioned. We also agree that this would be difficult to do experimentally, but found it an interesting way to observe convergence of representations in our simulated scenario.

Fig 3b-d shows multiple analyses to support output unit global remapping, but no analysis to support the claim that recurrent units remap by rate changes. The examples in Fig 3ai look pretty convincing, but it would be useful to also have a more quantitative result.

We agree, and only showed that units turn off/become silent using ratemaps. We have therefore added an explicit analysis, showcasing rate remapping in recurrent units (see appendix G; Recurrent units rate remap)

Reviewer #2 (Recommendations For The Authors):

Some parts of the current manuscript are hard to follow. Particularly, the model description is not transparent enough. See below for the details.

Major comments:

(1) Mathematical models should be explained more explicitly and carefully. I had to guess or desperately search for the definitions of parameters. For instance, define the loss function L in eq.(1). Though I can assume L represents the least square error (in A.8), I could not find the definition in Model & Objective. N should also be defined explicitly in equation (3). Is this the number of output cells?

Thank you for pointing this out, we have revised to make it more clear.

(2) In Fig. 1d, how were the velocity and context inputs given to individual neurons in the network? The information may be described in the Methods, but I could not identify it.

This was described in the methods section (Neural Network Architecture and Training), but we realize that we used confusing notation, when comparing with Fig. 1d. We have therefore changed the notation, and it should hopefully be clearer now. Thanks for pointing out this discrepancy.

(3) I took a while to understand equations (3) and (4) (for instance, t is not defined here). The manuscript would be easier to read if equations (5) and (6) are explained in the main text but not on page 18 (indeed, these equations are just copies of equations 3 and 4). Otherwise, the authors may replace equations (3) and (4) with verbal explanations similar to figure legend for Fig. 1b.

(4) Is there any experimental evidence for uniformly strong EC-to-CA1 projections assumed in the non-trainable decoder? This point should be briefly mentioned.

Thank you for raising this point. The decoding from EC (the RNN) to CA1 (the output layer) consists of a trainable weight matrix, and may thus be non-uniform in magnitude. The non-trainable decoding acts on the resulting “CA1” representation only. We hope that improvements to the model description also makes this more evident.  

(5) The explanation of Fig. 3 in the main text is difficult to follow because subpanels are explained in separate paragraphs, some of which are very short, as short as just a few lines.

This presentation style makes it difficult to follow the logical relationships between the subpanels. This writing style is obeyed throughout the manuscript but is not popular in neuroscience.

Thanks for pointing this out, we have revised to accommodate this.

(6) Why do field centers cluster near boundaries? No underlying mechanisms are discussed in the manuscript.

This is a good point; we have added a note on this; it likely reflects the border tuning of upstream units.

(7) In Fig. 4, the authors presented how cognitive maps may vary when the shape and size of open arenas are modified. The results would be more interesting if the authors explained the remapping mechanism. For instance, on page 8, the authors mentioned that output units exhibit global remapping between contexts, whereas recurrent units mainly rate remapping.

Why do such representational differences emerge?

We agree! Thanks for raising this point. We have therefore expanded upon this discussion in section 2.4.

(8) In the first paragraph of page 10, the authors stated ".. some output units display distinct field doubling (see both Fig. 4c), bottom right, and Fig. 4d), middle row)". I could not understand how Fig. 4d, middle row supports the argument. Similarly, they stated "..some output units reflect their main boundary input (with greater activity near one boundary)." I can neither understand what the authors mean to say nor which figures support the statement. Please clarify.

This is a good point, there was an identifier missing; we have updated to refer to the correct “magnification”. Thanks!

(9) The underlying mechanism of generating the hexagonal representation of output cells remains unclear. The decoder network uses a non-trainable decoding scheme based on localized firing patterns of output units. To what extent does the hexagonal representation depend on the particular decoding scheme? Similarly, how does the emergence of the hexagonal representation rely on the border representation in the upstream recurrent network? Showing several snapshots of the two place representations during learning may answer these questions.

This is an interesting point, and we have added some discussion on this matter. In particular, we speculate whether it’s an optimal configuration for position reconstruction, which is demanded by the task and thus highly likely dependent on the decoding scheme. We have not reached a conclusive method to determine the explicit dependence of the hexagonal arrangement on the choice of decoding scheme. Still, it seems this would require comparison with other schemes. In our framework, this would require changing the fundamental operation of the model, which we leave as inspiration for future work. We have also added additional discussion concerning the relationship between place units, border units, and remapping in our model. As for exploring different training snapshots, the model is randomly initialized, which suggests that earlier training steps should tend to reveal unorganized/uninformative phase arrangements, as phases are learned as a way of optimizing position reconstruction. However, we do call for more analysis of experimental data to determine whether this is true in animals, which would strongly support this observation. We also hope that our work inspires other models studying the formation and remapping of place cells, which could serve as a starting point for answering this question in the future.

(10) Figure 7 requires a title including the word "hexagonal" to make it easier to find the results demonstrating the hexagonal representations. In addition, please clarify which networks, p or g, gave the results shown here.

We agree, and have added it!

Minor comments:

(11) In many paragraphs, conclusions appear near their ends. Stating the conclusion at the beginning of each paragraph whenever possible will improve the readability.

We have made several rewrites to the manuscript, and hope this improves readability.

(12) Figure A4 is important as it shows evidence of the CA1 spatial representation predicted by the model. However, I could not find where the figure is cited in the manuscript. The authors can consider showing this figure in the main text.

We agree, and we have added more references to the experimental data analyses in the main text, as well as expanded this analysis.

(13) The main text cites figures in the following format: "... rate mapping of Fig. 3a), i), boundary ...." The parentheses make reading difficult.

We have removed the overly stringent use of double parentheses, thanks for letting us know.

(14) It would be nice if the authors briefly explained the concept of Ripley's H function on page 14.

Yes, we have added a brief descriptor.

Author response

eLife Assessment

The authors investigated KLF Transcription Factor 16 (KLF16) as an inhibitor of osteogenic differentiation, which plays a critical role in bone development, metabolism and repair. The results of the study are valuable as they could help to facilitate future research on the regulation of osteogenesis in vitro and in vivo. However, the evidence overall is incomplete, as validation by knockout mouse models would help to strengthen the conclusions.

We appreciate the editors’ evaluation and recognition of the importance of our research. The primary goal and value of our study is to provide robust bioinformatics analyses of 20 independent iPSC lines, which can lead to the identification of novel genes involved in osteogenic differentiation. The identification of KLF16 serves to illustrate this goal. A thorough analysis of the function of any single gene both in vitro and in vivo is beyond the initial scope of this study. To validate KLF16’s inhibitory role in osteogenic differentiation, we provided evidence showing overexpression of Klf16 suppressed osteogenic differentiation in vitro, and Klf16^+/- mice exhibited enhanced bone mineral content and density in vivo.

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this manuscript, Ru and colleagues investigated regulatory gene interactions during osteogenic differentiation. By profiling transcriptomic changes during mesenchymal stem cell differentiation, they identified KLF16 as a key transcription factor that inhibits osteogenic differentiation and mineralization. It was found that overexpression of KLF16 suppressed osteogenesis in vitro, while Klf16^+/- mice exhibited enhanced bone density, underscoring its regulatory role in bone formation.

Strengths:

(1) Bioinformatics is strong and comprehensive.

(2) Identification of KLF16 in osteoblast differentiation is exciting and innovative.

We appreciate the reviewer’s comments on our bioinformatic analyses of MSC osteogenic differentiation and the identification of KLF16 as a new osteogenesis regulator. The differentiation of iPSC-derived MSCs to OBs serves as a valuable model for investigating gene expression and regulatory networks in osteogenic differentiation. This study provides insights into the complex and dynamic regulation of the transcriptomic landscape in osteogenic differentiation and supplies a foundational resource for additional investigation into normal bone formation and the mechanisms underlying pathological conditions.

Weaknesses:

(1) The mechanism of KLF16 function is not studied.

(2) Studies of KLF16 in bone development, from both in vitro and in vivo perspectives, are descriptive.

Our study aims to apply rigorous bioinformatic analyses of 20 iPSC lines to identify novel genes involved in osteogenic differentiation. With this strategy, we successfully identified KLF16 as a regulator of osteogenic differentiation. We validated this with both in vitro and in vivo models even though we had limited availability of Klf16 knockout mice when the study was conducted. We demonstrated that overexpression of Klf16 suppressed osteogenesis in vitro, while Klf16^+/- mice exhibited increased bone mineral density, trabecular number, and cortical bone area, highlighting its role in bone formation. With these mice now available, further investigation into the mechanism of KLF16's function is possible.

(3) Findings in bioinformatics analysis are mostly redundant with previous studies in the field, and can be simplified.

We compared our bulk RNA-seq data with our previously published single-cell RNA-seq (scRNA-seq) data generated from iPSC-induced cells during osteogenic differentiation (Housman et al., 2022). The purpose is to corroborate the expression patterns of the genes we focused on during osteogenic differentiation. We found similar differential expression patterns in a pseudobulk analysis of the scRNA-seq data, even though there are significant differences between these two studies, including: cell culture conditions, sequencing approaches (bulk vs. single cell), goals of the studies (key TF drivers of osteoblast differentiation vs. mapping differentiation stages and inter-species gene programs in human and chimp), and findings (identification of TFs vs. identification of interspecific regulatory differences) .

Importantly, we performed network analyses to identify key transcription factors, which were not redundant with previous studies. We constructed a transcription factor regulatory network analysis during human osteogenic differentiation, and identified a network organized into five interactive modules. The most exciting finding was the identification of KLF16 as one of the strongest regulators in Module 5 (Figure 3), which previously was not demonstrated to be involved in bone formation. We also demonstrated known TF genes regulating osteogenic differentiation in these modules, and performed gene ontology (GO) and reactome pathway (RP) analyses for regulatory functions and pathways specific to each module. To clarify that our findings do not overlap with previous studies, we will revise the manuscript focusing on Module 5 and simplify the description of the bioinformatics analysis as the reviewer suggested.

Reviewer #2 (Public review):

In their manuscript with the title "Integrated transcriptomic analysis of human induced pluripotent stem cell (iPSC)-derived osteogenic differentiation reveals a regulatory role of KLF16", Ru et al. have analyzed the gene expression changes during the osteogenic differentiation of iPSC-derived mesenchymal stem/stromal cells into preosteoblasts and osteoblasts. As part of the computational analyses, they have investigated the transcription factor regulatory network mediating this differentiation process, which has also led to the identification of the transcription factor KLF16. Overexpression experiments in vitro and the analysis of heterozygous KLF16 knockout mice in vivo indicate that KLF16 is an inhibitor of osteogenic differentiation.

The integrated analysis of iPSC bulk transcriptomic data is a major strength of the study, and it is also great that the authors provide deeper functional characterization of the transcription factor KLF16, one of the newly identified candidate regulators of osteogenic differentiation.

We appreciate the reviewer’s summary and comments on the strength of our bioinformatic analyses of iPSC/MSC osteogenic differentiation and the deep functional characterization of the KLF16, as well as the novelty of our findings.

However, characterization of KLF16 expression in the mouse and validation of the knockout model are currently lacking. Alternative explanations for the mutant phenotype should be considered to improve the strength of the conclusions.

If all issues can be addressed, the study would provide an important resource for the field that would facilitate future research on the regulation of osteogenesis in vitro and in vivo, with potential implications for preclinical and clinical research as well as bioengineering.

We appreciate the reviewer’s valuable suggestions. Klf16 is highly expressed in mandibular, maxillary and tail mesenchyme at embryonic Day 12 (D'Souza et al., 2002), indicating its role in early bone development. We will further characterize the expression of Klf16 in mice, especially in the developing bones.

We identified Klf16 as a potential regulator of osteogenic differentiation, and then validated this with both in vitro and in vivo models. Overexpression of Klf16 suppressed osteogenesis in vitro, and Klf16^+/- mice showed increased bone mineral content and density, indicating its regulatory role in bone formation. We agree with the reviewer that the bone phenotypes of Klf16 knockout mice potentially can be affected by other factors in addition to osteogenic differentiation. As both bone formation and resorption are critical for bone development, we evaluated osteoclastogenesis in the Klf16^+/- mice by analyzing the expression of osteoclast marker CALCR and regulator RANKL in the femurs of the Klf16^+/- mice. Neither CALCR nor RANKL decreased in the bone of Klf16^+/- mice, indicating that osteoclastogenesis is not decreased; therefore, increased bone mineral content and density in the mutant mice is more likely attributed to enhanced bone formation rather than reduced resorption by osteoclasts. Additionally, we will discuss other alternative explanations for the bone phenotypes of Klf16 knockout mice as suggested by the reviewer.

References

D'Souza, U. M., Lammers, C.-H., Hwang, C. K., Yajima, S. and Mouradian, M. M. (2002). Developmental expression of the zinc finger transcription factor DRRF (dopamine receptor regulating factor). Mechanisms of Development 110, 197-201.

Housman, G., Briscoe, E. and Gilad, Y. (2022). Evolutionary insights into primate skeletal gene regulation using a comparative cell culture model. PLOS Genetics 18, e1010073-e1010073.

Note de synthèse : L'écriture de la violence dans les sciences sociales et la littérature

Source : Excerpts from "Les formes de la violence (6) - Didier Fassin (2024-2025)"

**Contexte : Ce texte est une exploration des défis et des méthodes liés à l'écriture et à la représentation de la violence, en particulier du point de vue des sciences sociales, en la comparant à la littérature.

L'auteur, Didier Fassin, aborde la violence non pas sous l'angle de son absence ou de sa définition contestée dans les sciences sociales, mais comme un "objet d'une recherche portant sur les manières d'écrire de l'écrire de la représenter de la tester de la qualifier et finalement peut-être de la comprendr**e voire même de la refuser".

Thèmes principaux :

• Les défis éthiques et méthodologiques de l'écriture de la violence : Comment représenter la violence sans sensationnalisme ni obscénité ? Faut-il privilégier la voix des victimes ou inclure celle des auteurs ? Comment concilier le devoir de témoigner avec la protection des personnes impliquées ?

• La distinction entre "vérité" (littérature) et "réalité" (sciences sociales) dans la représentation de la violence : La fiction offre une liberté créative pour atteindre une "vérité" humaine et psychologique, tandis que les sciences sociales sont contraintes par la "factualité" des faits.

• La "pauvreté des mots" pour saisir l'expérience de la violence extrême : Certaines violences, en particulier les violences historiques et déshumanisantes, laissent des traces difficiles à exprimer directement, se manifestant dans les interstices du langage ordinaire et les violences plus banales.

• La pluralité des perspectives et des récits de la violence : L'importance de présenter différentes versions des événements violents, notamment celles des victimes, des auteurs et des témoins, pour saisir la complexité et les ambiguïtés.

• La violence institutionnelle et structurelle : La violence ne se limite pas aux actes interpersonnels, mais s'incarne aussi dans les systèmes, les politiques et les structures sociales, comme l'apartheid ou le système carcéral.

Idées ou faits les plus importants :

• L'écriture de la violence comme objet de recherche : Fassin propose d'étudier comment la violence est écrite, représentée et comprise, en s'intéressant à "ce qu'elle est et ce qu'on en fait".

• Les cinq écueils identifiés par Valentine Daniel (anthropologue srilankais) face à l'écriture de la violence :Produire du sensationnalisme (potentiellement obscène ou pornographique).

• Éloigner la théorie de l'expérience de la souffrance.

• Trahir les personnes qui se sont confiées en se taisant.

• Mettre en danger les personnes impliquées en les rendant reconnaissables.

• Se mettre soi-même en danger (interdiction de séjour, répression).

• Le dilemme de l'anthropologue face au témoignage de la violence : L'exemple de la fille dont le père a été lynché montre le conflit entre le devoir de témoigner ("dites à tous et tous ce qu'ils ont fait à mon père") et la volonté de protection de la dignité de la victime ("ne racontez pas cette histoire").

• La distinction entre littérature et sciences sociales :Littérature (poètes, romanciers) : Privilège de l'imagination, création de langages, de personnages, d'histoires pour accéder à une forme de "vérité" (ex: Emily Dickinson, Mahmoud Darwish, Marcel Proust, Tony Morrison, J.M. Coetzee, David Simon). La fiction permet une liberté créative et une efficacité narrative pour explorer les "profondeurs humaines" et la "complexité psychologique".

• Sciences sociales (anthropologues, sociologues) : Tenues par le "principe de réalité", la "factualité des faits".

Leur écriture est contrainte par les sources (archives, entretiens, observations). L'ethnographie est limitée par ce que l'ethnographe saisit, sans pouvoir "combler les trous de son ignorance et les lacunes de son incertitude par son imagination". Elles visent la "réalité".

• La violence comme trace "invisible et indiscible" (Veena Das) : L'étude de la violence de la partition de l'Inde révèle comment les violences extrêmes ("historique, extrême") peuvent être "trop profonde trop humiliante trop dévalorisante" pour être dites directement, se manifestant dans les violences "domestiques tristement banale" (disputes, vexations) et les "interstices du discours". Das parle de la "pauvreté des mots" face à ces violences.

• L'expérience des auteurs de violence : L'enquête de Martha Huggins sur les policiers tortionnaires brésiliens montre qu'ils étaient souvent des "hommes ordinaires", endoctrinés, formés à une culture du secret, développant des représentations déshumanisantes des victimes et des justifications morales de leurs actes, avec des pratiques "normalisées".

Cela rappelle la "banalité du mal" d'Hannah Arendt. Les méthodes sociologiques (entretiens circonscrits) peuvent tendre vers une lecture objectivante, tandis que les méthodes anthropologiques (observation prolongée) permettent une analyse plus réflexive.

• La violence institutionnelle et l'effacement des distinctions auteurs/victimes : L'exemple du détenu éborgné et violent dans une prison française illustre comment les positions d'auteurs et de victimes peuvent s'alterner ou s'estomper dans le contexte d'un système carcéral qui "l'a fait".

• La multiperspective comme stratégie narrative : Carl Jacobi, dans "Shadows at Dawn", présente quatre versions distinctes du massacre de Camp Grant en Arizona, basées sur les perspectives des différents groupes impliqués (Odham, Mexicains, États-Unis, Apaches), pour saisir la complexité historique.

• La juxtaposition des récits face à l'incompatibilité des versions : Dans son propre travail sur l'homicide par le GIGN ("Mort d'un voyageur"), Fassin choisit de juxtaposer les différentes versions des faits (gendarmes, famille) sans "préjuger de qui dit la vérité", à la manière de Kurosawa dans "Rashômon".

Cette approche permet de montrer comment chacun dit que la scène "s'est déroulé".

• Le rôle de la contre-enquête : Face aux "incohérences du dossier" et aux "altérations par la justice", une contre-enquête basée sur l'analyse des preuves peut permettre de reconstituer la scène "telle qu'elle s'est vraisemblablement déroulée", même si elle contredit la version officielle.

• Les "plusieurs vies" des écrits de sciences sociales sur la violence : Les travaux peuvent avoir un impact au-delà de leur publication académique, par exemple en étant utilisés dans des recours juridiques (Cour européenne des droits de l'homme) ou en inspirant des œuvres artistiques (film).

Citations clés :

• "...il s'agit de s'intéresser à ce qu'elle est et ce qu'on en fait je vais donc la traiter comme un objet de d'une recherche portant sur les manières d'écrire de l'écrire de la représenter de la tester de la qualifier et finalement peut-être de la comprendre voire même de la refuser"

• "y a-t-il des faits qu'il vaut mieux taire et d'autres dont au contraire il faut parler s'il importe de faire entendre la voix des victimes doit-on pour autant se priver de celle des auteurs de la violence"

• "d'épeindre la violence de manière brute sans l'intention d'y rendre son lectorat sensible expose cependant à produire un sensationnalisme potentiellement obsène qu'il qualifie d'ailleurs de pornographique"

• "il me semble qu'à cette analyse il faudrait toutefois ajouter deux éléments premièrement l'écriture des poètes et des romanciers procède de leur imagination ils invent langages ils créent des personnages ils conçoivent des histoires qui peuvent leur donner accès à une forme de vérité que l'on pense pour la poésie à Emily Dickinson ou à Marmou Darwish ou pour le roman à Marcel Prou et Tony Morrison anthropologue et sociologues eux sont en principe tenus par une sorte de principe de réalité... Leur écriture est circonscrite par ce qu'on peut appeler la factualité des faites"

• "en somme la fiction détient les clés de la vérité l'ethnographie celle de la réalité"

• "la pauvreté des mots Vinadas se dit je cite frappé par le silence autour de la violence subie ou exercée dans le contexte de la partition trop profonde trop humiliante trop dévalorisante pour les victimes la trace laissée ne se donne à lire que dans les interstices du discours dans des allusions blessantes dans des attitudes méprisantes"

• "c'est le système carcéral qui l'a fait"

• "La violence peut être énoncée par plusieurs voix"

• "...fallait-il en rester là en laissant à la justice le dernier mot il m'a semblé qu'au regard des incohérences du dossier et des éléments mis à ma disposition une contre-enquête était possible"

Conclusion :

Didier Fassin conclut en soulignant que l'écriture de la violence, qu'elle soit littéraire ou relevant des sciences sociales, implique de nombreuses questions et précautions (rhétoriques, méthodologiques, politiques, éthiques).

Si la littérature et les sciences sociales abordent la violence différemment, elles partagent une préoccupation commune quant à ce qui peut et doit être écrit, aux voix à rendre audibles et aux différentes versions des faits à considérer.

Le texte se concentre sur la violence physique, laissant entendre que d'autres formes de violence pourraient poser des défis d'écriture distincts.

Document de Synthèse : Les Formes de la Violence et les Mythes d'Origine

Ce briefing document analyse les thèmes et idées principales tirées des extraits de la conférence "Les formes de la violence (4) - Didier Fassin (2024-2025)".

L'objectif est d'examiner comment la violence, longtemps négligée par les sciences sociales, est appréhendée à travers les mythes d'origine, les rituels associés, et leur instrumentalisation politique dans les sociétés modernes et contemporaines.

1. La Violence dans les Sciences Sociales : Reconnaissance Tardive et Complexité

Le conférencier souligne que la violence a été tardivement étudiée par les sciences sociales.

Les anthropologues ont rencontré des difficultés à la définir précisément en raison d'une relativisation ("ce que certains voyaient comme tel n'étaient pas considéré de la même manière par d'autres") et les sociologues ont élargi la notion, menant à une dissolution de la violence dans d'autres concepts de domination.

• Idée Clé : La difficulté à cerner la violence, perçue différemment selon les perspectives et étendue à des formes de domination non physiques, a initialement entravé son étude.

• Correction Épistémologique : Fassin propose de parler de "perspectivisme" plutôt que de relativisation, pour comprendre les différentes logiques et raisons des points de vue divergents sur la violence. Il suggère également de parler d'"approfondissement" plutôt que de dissolution, pour ne pas réduire la violence aux seules agressions physiques, tout en les incluant.

• Citation Clé : "cette double précaution épistémologique loin d'être un obstacle à la production d'un savoir sur la violence en est la condition... ne pas avoir une définition précise de la violence rend possible de la saisir dans toute sa complexité et sa diversité".

• Contraste avec le Sens Commun : Cette approche va à l'encontre du sens commun où chacun pense comprendre la violence.

2. La Violence et les Mythes des Origines : Une Association Profonde

Bien que l'étude scientifique de la violence soit récente, elle a toujours été présente dans la pensée humaine, notamment à travers les mythes des origines. Ces mythes lient souvent l'origine de la violence à celle du monde.

• Idée Clé : Les mythes des origines, qu'ils soient religieux, théâtraux ou philosophiques, ont servi de discours sur la violence et ses débuts.

• Exemple Grec : La Théogonie d'Hésiode : Le mythe de Chronos est présenté comme un exemple emblématique. L'origine du monde et des dieux est marquée par une violence extrême : parricide (Chronos castre Uranos), infanticide (Chronos dévore ses enfants), viol (Chronos viole Rhéa).

• Nature de la Violence Mythologique : Ces violences vont au-delà de l'usage excessif de la force. Elles manifestent un "excès de la violence elle-même", un registre de monstruosité et d'atrocité qui rompt avec les valeurs élémentaires.

• Fonction de la Violence Mythologique : Elle n'est pas seulement instrumentale (se débarrasser de rivaux, se venger), elle semble aussi "expressive", voire source de jouissance pour elle-même et pour le public.

• Cadre Familial : Fait remarquable, ces scènes de violence extrême se déroulent souvent dans un cadre familial (famille des divinités, famille des Atrides).

• Mythes et Rituels : La relation entre mythe et rituel est débattue par les anthropologues. Le mythe de Chronos a été associé à des pratiques rituelles ambivalentes : sacrifices sanglants et anthropophages côtoyant des offrandes pacifiques et des festivités de tolérance sociale (Cronia).

• Ambivalence de Chronos : Le mythe présente une image ambivalente de Chronos : le meurtrier anthropophage et le souverain juste d'un âge d'or.

3. La Croyance dans les Mythes : Une Question Complexe et Contemporaine

La question de savoir si les Grecs croyaient littéralement à leurs mythes est posée. Fassin établit un parallèle avec les croyances contemporaines pour éclairer cette question.

• Idée Clé : La croyance dans les mythes, même contradictoires, n'est pas l'apanage d'un passé lointain. Des phénomènes contemporains comme le créationnisme ou l'Intelligent Design démontrent une croyance persistante en des récits originels, parfois habillés de justifications scientifiques.

• Parallèle Contemporain : Le taux élevé de croyance au créationnisme aux États-Unis, y compris parmi les évolutionnistes qui pensent que Dieu guide l'évolution, suggère que la crédulité n'a pas disparu.

4. Instrumentalisation Politique des Mythes des Origines : De l'Antiquité aux Temps Modernes

Les mythes des origines ont une pertinence sociale en ce qu'ils révèlent les tensions et la nécessité d'un ordre social, tout en ouvrant la possibilité d'un autre monde. Cependant, leur mobilisation est souvent politique.

• Pertinence Sociale du Mythe Grec : Le mythe de Chronos peut être interprété comme révélant une période de chaos nécessitant l'ordre social, tout en offrant une soupape de relâchement des contraintes à travers les rituels des Cronia.

• Mythes Modernes et Invention de la Tradition : À l'inverse des mythes antiques issus de traditions culturelles, les mythes modernes sont souvent des "inventions de tradition" (Hobsbawm & Ranger), créées à des fins politiques.

• Fonction Politique des Mythes Nationaux : Les mythes nationaux servent un projet nationaliste, renforçant la légitimité de l'État, créant une histoire unifiée, ou justifiant des revendications indépendantistes.

• Exemple Français : Le Baptême de Clovis et la Bataille de Poitiers : Fassin analyse comment l'historiographie nationaliste française a instrumentalisé le baptême de Clovis pour fonder l'identité nationale catholique et la bataille de Poitiers pour construire une altérité arabe et musulmane rejetée, nourrissant le racisme et l'islamophobie.

• Citation Clé (Patrick Geary) : "L'histoire moderne est née au 19e siècle, conçue et développé comme un instrument du nationalisme européen, ce qui a eu pour effet de transformer notre compréhension du passé en une décharge de déchets toxiques remplie du poison du nationalisme ethnique."

• Héritage Symbolique : Des symboles issus de ces mythes (la Francisque, le casque franc) sont repris par l'extrême droite.

• Généalogie Distinguée : L'islamophobie (forgée par des mythes nationaux) et l'antisémitisme (lié à des légendes urbaines) pourraient avoir des généalogies distinctes en France.

5. Le Mythe Sioniste des Origines et la Violence Contemporaine en Israël/Palestine

Le conférencier consacre une analyse détaillée à l'instrumentalisation des récits bibliques dans la construction du mythe national israélien et sa connexion avec les violences actuelles.

• Idée Clé : Le mythe fondateur de l'exil, basé sur une interprétation spécifique des textes bibliques, a servi à légitimer l'immigration juive en Palestine et le droit au retour, tout en niant l'existence et l'histoire du peuple palestinien.

• Citation Clé (Schlomo Sand) : "Si toute écriture de l'histoire est porteur de mythes, ceux de l'historiographie nationale sont particulièrement flagrants. Le cas d'Israël est selon lui exemplaire même s'il est loin d'être unique."

• La Terre sans Peuple pour un Peuple sans Terre : Le conférencier retrace l'origine et la réécriture de cette phrase de Israel Zangwill, souvent utilisée pour justifier l'immigration juive, en omettant que Zangwill lui-même a révisé son jugement face à la population arabe existante.

• La Violence dans les Récits Bibliques : Les textes sacrés décrivent l'origine de l'histoire d'Israël comme une série de guerres et de conquêtes (conquête de Canaan, combat contre les Amalécites).

• Instrumentalisation Contemporaine : Des références à la guerre contre les Amalécites sont mobilisées pour nommer l'ennemi archétypal (les Palestiniens) et justifier leur élimination. La figure de Josué, conquérant et destructeur, est invoquée pour légitimer l'occupation et les colonies.

• Citation Clé (Benyamin Netanyahou) : "se souvenir de ce qu'Amalek leur a fait".

• Lien entre Mythes et Violence Réelle : L'appel au génocide dans les textes bibliques, bien que certains exégètes en donnent une interprétation non littérale, est réactivé dans le discours politique et populaire, notamment après le 7 octobre 2023.

• Fusion des Mémoires : Une fusion s'opère entre la mémoire héroïque (les conquêtes bibliques) et la mémoire douloureuse (la Shoah), assimilant le Hamas à Amalek et aux nazis.

• Dénégation de la Nakba : La violence de l'expulsion des Palestiniens en 1948 (la Nakba) est largement absente de ce complexe mémoriel officiel.

6. La Violence Sacrificielle : Une Perspective Contestée

Fassin critique la thèse de René Girard sur la violence sacrificielle comme origine de la violence dans les sociétés.

• Thèse de René Girard : Le sacrifice rituel permet de détourner la violence intrinsèque au groupe vers une victime émissaire (animale ou humaine considérée comme de moindre valeur sociale), rétablissant ainsi l'unité du groupe. Le désir mimétique (désirer ce qu'un autre désire) est à l'origine de la rivalité et de la violence fondatrice.

• Critiques de la Thèse de Girard : La thèse de Girard est jugée trop simple et universelle, réduisant la pluralité des formes de violence à une cause unique (le désir mimétique) et appliquant mécaniquement une même logique à toutes les sociétés.

• Alternative Anthropologique : Hubert et Mauss : Leurs travaux sur le sacrifice définissent le sacrifice comme un acte religieux qui, par la consécration d'une victime, modifie l'état de la personne ou de l'objet. La violence est absente de leur définition.

• Clé de Compréhension : Le Sacré : Pour Hubert et Mauss, le cœur du sacrifice n'est pas la violence, mais le double travail rituel de sacralisation et de désacralisation.

• Substituion et Dédramatisation : La possibilité de substituer une victime (un concombre pour un bœuf chez les Nuer, selon Lévi-Strauss) dans le sacrifice dédramatise l'acte et contredit l'idée d'une violence primordiale nécessitant impérativement un sacrifice sanglant.

Conclusion sur Girard : L'anthropologie sociale ne valide pas la thèse de Girard, jugée trop spéculative et possiblement influencée par sa conversion catholique.

7. Conclusion : L'Importance de la Violence Originaire comme Imaginaire

Malgré les limites de la théorie girardienne, la violence fondatrice présente dans les récits mythiques et les textes sacrés reste essentielle, non pas tant par sa réalité historique que par les usages politiques qui en sont faits.

• Idée Clé : L'intérêt de la violence originaire réside dans son instrumentalisation politique par divers acteurs et à différentes époques, notamment par les extrêmes droites contemporaines.

• Fonction de l'Imaginaire de la Violence Originaire : La mobilisation de cet imaginaire sert à légitimer stigmatisations, discriminations, exclusions et exactions contre des ennemis perçus (intérieurs ou extérieurs).

• Citation Clé : "En ce sens, la violence originaire dont j'ai parlé ici éclaire bien les violences contemporaines."

• Cycle Nationaliste : La construction des récits nationalistes suit souvent un cycle d'âge d'or, de déclin et de régénération, se nourrissant d'une double genèse : héroïque et victimaire.

• Dimension Sacrificielle Réappropriée : La mémoire douloureuse de la Shoah (appelée "Holocauste", c'est-à-dire sacrifice) confère une obligation morale aux générations suivantes, se mêlant désormais à la mémoire héroïque des conquêtes.

• En résumé, le conférencier met en évidence la difficulté intrinsèque à appréhender la violence dans sa complexité.

Il montre ensuite comment les mythes des origines ont servi de cadre pour penser la violence, et surtout, comment ces récits, qu'ils soient antiques ou "inventés" à l'époque moderne, sont politiquement mobilisés pour construire des identités nationales, justifier la violence et exclure des groupes.

L'analyse du cas israélien illustre de manière particulièrement frappante la façon dont les récits scripturaires peuvent être réactivés pour légitimer des violences contemporaines.

Enfin, il réfute la thèse d'une violence fondatrice universelle nécessitant un sacrifice, tout en reconnaissant la pertinence de l'idée de victime émissaire dans l'analyse des persécutions et des brutalités contre les minorités.

Briefing sur les Formes de la Violence selon Didier Fassin (Extraits 2024-2025)

Ce texte de Didier Fassin examine l'évolution de l'étude de la violence dans les sciences sociales, notamment l'anthropologie et la sociologie.

Il met en évidence le silence initial entourant le sujet, les raisons de cette absence, les défis méthodologiques et moraux liés à son étude, et l'expansion progressive de sa conceptualisation.

Thèmes Principaux:

• Le Silence Initial autour de la Violence dans les Sciences Sociales: L'auteur souligne l'ignorance ou le manque d'étude explicite de la violence durant le premier siècle de l'anthropologie et de la sociologie, malgré un XXe siècle marqué par des conflits et des atrocités majeures.

• Les Raisons du Silence: Fassin identifie des raisons morales, méthodologiques et théoriques pour cette absence d'étude. La violence est perçue comme impliquant un jugement normatif qui rend difficile l'objectivité scientifique, elle échappe à la construction d'un objet homogène, et elle est difficile à définir de manière univoque.

• Les Défis Moraux et Méthodologiques de l'Étude de la Violence: L'étude de la violence est confrontée à la dimension normative et subjective du terme, ce qui soulève la question de l'impartialité de l'observateur, en particulier pour les anthropologues confrontés à des pratiques violentes qui ne sont pas perçues comme telles localement. Les défis méthodologiques incluent la difficulté à circonscrire la violence comme un objet d'étude homogène et observable, ainsi que les obstacles pratiques de l'observation sur le terrain.

• L'Expansion Conceptuelle de la Violence: Le texte trace un cheminement de la compréhension de la violence, passant d'une focalisation sur la violence physique interpersonnelle à l'inclusion des animaux, des objets inanimés, et enfin des formes de violence non directement attribuables à un acteur intentionnel (violence structurelle et symbolique).

• La Violence Structurelle (Johan Galtung et Paul Farmer): Introduction du concept de violence structurelle, définie par l'écart entre le potentiel et l'effectif de la réalisation humaine dû à l'influence négative de structures sociales.

L'exemple de l'épidémie de sida et de tuberculose en Haïti est utilisé pour illustrer comment l'histoire sociale et l'économie politique peuvent constituer des formes de violence.

• La Violence Symbolique (Pierre Bourdieu): Présentation du concept de violence symbolique, qui explique comment les dominants obtiennent l'adhésion des dominés à leur domination sans recourir à la force physique, mais en imposant une légitimité par le langage et les significations partagées, conduisant à des souffrances psychiques et à l'incorporation de l'ordre social.

• Lien entre les Différentes Formes de Violence: Malgré leurs différences, les formes de violence (physique, structurelle, symbolique) se manifestent dans le corps et provoquent de la souffrance.

• L'Apport de l'Étymologie et de la Philologie:

L'analyse étymologique (grec et latin) et philologique (français, allemand) montre la richesse et la complexité historique du terme "violence", révélant un passage d'un sens initial lié à la force physique à des significations incluant l'excès, l'infraction à la norme, la contravention à la morale, et la contrainte psychologique.

L'étude comparée des termes allemands (Gevalt, Macht) et de leurs traductions souligne les difficultés à saisir les différentes dimensions du pouvoir et de la violence.

• La Problématisation plutôt que la Définition: En conclusion, l'auteur suggère que la valeur de l'étude de la violence réside moins dans la recherche d'une définition univoque que dans la problématisation constante de la notion, ouvrant ainsi la voie à de nouvelles questions.

Idées et Faits Importants:

• Le XXe siècle a été marqué par une violence omniprésente, pourtant l'anthropologie et la sociologie l'ont longtemps ignorée en tant qu'objet d'étude spécifique.

• La nature normative et subjective du terme "violence" a été un obstacle majeur à son étude objective.

• La difficulté à définir la violence de manière univoque et à la circonscrire comme un objet d'étude cohérent a également contribué à son ignorance initiale.

• Les anthropologues sont confrontés au défi de la relativité culturelle de la violence, où ce qui est considéré violent par l'observateur ne l'est pas nécessairement par la société étudiée.

• La définition de Françoise Héritier élargit considérablement le champ de la violence au-delà du corps humain, incluant le psychique, les animaux et les objets inanimés.

• La violence structurelle (Galtung) met l'accent sur les inégalités sociales et les structures qui limitent le potentiel humain, indépendamment de l'intention d'un acteur.

• Paul Farmer a appliqué le concept de violence structurelle à la santé, montrant comment les épidémies peuvent être comprises dans un contexte historique et économique de domination.

• La violence symbolique (Bourdieu) explique comment la domination est reproduite par l'intériorisation et l'acceptation des rapports de pouvoir par les dominés, entraînant des souffrances psychiques et des manifestations corporelles.

• La notion d'hégémonie chez Gramsci offre une perspective alternative ou complémentaire à la violence symbolique, en soulignant la possibilité de visions du monde alternatives émanant des classes dominées.

• L'étymologie et la philologie révèlent la polysémie et l'évolution historique du terme "violence", soulignant que son sens contemporain, centré sur les relations humaines et la morale, est relativement récent.

• Les difficultés de traduction du terme allemand "Gevalt" (force, violence, pouvoir) mettent en lumière les nuances conceptuelles et les défis de la reconnaissance de la violence dans les institutions et derrière les revendications de légitimité.

• La problématisation de la notion de violence est plus féconde pour la recherche que la recherche d'une définition rigide.

Citations Pertinentes:

• Sur l'absence initiale de l'étude de la violence : "la violence a donc été ignorée nous l'avons vu pendant le 1er siècle de l'existence de l'anthropologie et de la sociologie ou plus exactement elle n'a pas été traité en tant que telle n'a pas fait l'objet d'entrée spécifique dans les encyclopédies et les dictionnaires de ces disciplines n'a pas donné lieu à des travaux empiriques ou théoriques se réclamant explicitement de son étude"

• Cit. Anna Arendt sur le rôle de la violence dans les affaires humaines : "il n'est personne qui s'intéressant à l'histoire et à la politique puisse ne pas être conscient du rôle majeur joué par la violence dans les affaires humaines il est donc à première vue surprenant que la violence a été si rarement reconnue comme méritant une considération particulière"

• Cit. David Riches sur la difficulté du terme "violence" : "En raison notamment des émotions qu'il provoquent le mot violence se situe au sommet de la liste des termes qui présentent un danger de ce double point de vue en effet poursuit-il violence que note fortement un comportement illégitime et inacceptable."

• Cit. Jean-Luc Nancy sur la question interne de la violence : "il y a aujourd'hui une question de la violence qui n'est pas simplement une question externe ouvrant sur la résistance éthique et politique aux violences effectives... mais aussi une question interne à savoir quelque chose de la violence ou de ce qui est trop rapidement classé sous l'unique terme de violence reste à penser au par-delà son évidente condamnation l'essence de la violence n'est sans doute pas épuisé par sa réprobation"

• Cit. Gérard Lanclude sur la possibilité d'une ethnographie de la violence : "si l'on fait l'hypothèse qui ne paraît pas déraisonnable qu'aucune société ne l'ignore que chacune s'attache à désigner comme violent certains comportements à l'exclusion d'autres leur confèrent ou non un sens et une fonction et les sanctionne selon des procédures qui lui appartiennent en propre"

• Cit. Gérard Lanclude sur la nature de la violence : "la violence ne se donne pas d'emblé pour tel sa spécification résulte d'une procédure toujours ouverte de qualification et tout semble se passer comme si la notion de violence chargée de représentation composite référait à des phénomènes de nature très différente"

• Cit. Élisabeth Claveri sur le regard de l'ethnographe : "chacun sait bien ce que la violence ce que violence veut dire et est à même de la reconnaître sous des formes extrêmement diverses dans différentes sociétés étudiées peut-être parce qu'il en a l'expérience dans l'univers dont il est lui-même indigène"

• Cit. Françoise Héritier pour une définition large de la violence : "appelons violence toute contrainte de nature physique ou psychique susceptible d'entraîner la terreur le déplacement le malheur la souffrance ou la mort d'un être animé tout acte d'intrusion qui a pour effet volontaire ou involontaire la dépossession d'autruie le dommage ou la destruction d'objets inanimés"

• Cit. Michel Napels interrogeant l'extension de la définition de la violence : "est-il légitime écrit-il de rassembler les objets inanimés les animaux et les humains dans un même registre d'expérience ?"

• Cit. Johan Galtung sur la violence structurelle : "La violence s'exerce lorsque des êtres humains ne se réalisent pas physiquement et mentalement comme il devrait le pouvoir du fait de facteurs qui ont sur eux une influence négative"

• Cit. Paul Farmer sur la distribution du sida et de la tuberculose en Haïti : "que la distribution du sida et de la tuberculose comme celle de l'esclavage autrefois est historiquement et économiquement déterminé et qu'elle est en relation avec le racisme l'inégalité genrée et la pauvreté massive"

• Cit. Bourdieu et Passeron sur la violence symbolique : "tout pouvoir de violence symbolique c'est-à-dire tout pouvoir qui parvient à imposer des significations et à à les imposer comme légitime en dissimulant les rapports de force qui sont au fondement de sa force ajoute sa force propre c'est-à-dire proprement symbolique à ses rapports de force"

• Cit. Michel Perrot sur la "pensée claustrale" dans La domination masculine : "une pensée claustrale à force d'être implacable dont la rigueur consiste en la mise en ordre de matériaux soumis à la question d'une grille contraignante"

• Cit. Robert Bernasconi sur la reconnaissance de la violence : "quel moyen avons-nous de reconnaître la violence lorsqu'elle est dissimulée dans les institutions et occulté par les revendications de légitimité ?"

• Cit. Anna Arendt sur la distinction entre pouvoir et violence : "power correspond à la possibilité non seulement d'agir mais d'agir de concert car il relève pas de l'individu mais du groupe... Violence enfin se distingue par son caractère instrumental"

En résumé, ces extraits offrent une exploration approfondie des défis rencontrés par les sciences sociales dans l'étude de la violence, tout en soulignant l'importance cruciale d'élargir sa compréhension au-delà des manifestations physiques pour inclure les dimensions structurelles et symboliques qui affectent profondément les corps et les vies.

La difficulté de définir la violence devient alors moins un obstacle qu'une invitation à une analyse critique constante.

Note de Synthèse : Démocratie Participative et Outils

Source : Extraits de "Démocratie participative : quels outils ?" (Conférence/Table Ronde)

• Introduction : La source analyse la démocratie participative en se concentrant sur la notion d'« ingénierie de la participation ».

Elle compare notamment le Grand Débat National (GDN) et la Convention Citoyenne pour le Climat (CCC) pour illustrer l'impact du design et de la conception des dispositifs sur leur qualité, leur portée et leur légitimité.

Les intervenants soulignent l'importance de l'ingénierie non pas comme une simple neutralité instrumentale, mais comme une manifestation de théories politiques en acte, porteuses de valeurs et produisant des effets concrets.

La discussion aborde également le rôle des acteurs locaux et des outils numériques (civic tech) dans le paysage de la participation.

Thèmes Principaux et Idées Clés :

• La Démocratie comme Égalité des Intelligences et Aptitude Universelle à la Réflexion Politique :
• La source commence par rappeler le principe fondamental de la démocratie, "qui consiste à acquiescer au principe de l'égalité des intelligences".
• S'appuyant sur Jacques Rancière, il est souligné que "n'importe qui est apte à réfléchir aux enjeux qui nous sont communs et que la politique relève de réflexion de considérations qui ne concerne aucune compétence spécifique". Cette idée pose la base théorique de la participation citoyenne large.
• L'Ingénierie de la Participation : Une Dimension Essentielle et Non Neutre :
• Le terme "ingénierie" est central, défini comme "la personne qui élabore dresse les plans d'ouvrages d'art de machines et parfois on dirige on surveille l'exécution". Appliqué à la participation, il s'agit de concevoir et piloter des dispositifs.
• L'ingénierie n'est pas neutre : "les choix de procédure de participation la manière dont on va articuler les outils ne sont pas neutres".
• Elle "porte bien l'intention politique et elle embarque elle embarque un projet".
• Une critique de l'idée d'une démocratie "sauvage" ou "radicale" venant uniquement d'en bas est formulée. L'institutionnalisation et l'organisation de la participation sont jugées nécessaires et non nécessairement dénaturantes, contrairement à une vision "risquée et limitative" qui craint toute mise en forme.
• Inversement, l'absence de structure peut mener à la "tyrannie de l'absence de structure", où des rapports de domination invisibles se développent.
• Les outils ne sont pas de simples instruments ; "ils sont aussi des théories politiques en actes", porteurs de valeurs et produisant des effets.
• Comparaison entre le Grand Débat National (GDN) et la Convention Citoyenne pour le Climat (CCC) :
• Ces deux dispositifs sont présentés comme "deux exercices participatifs" aux dynamiques contradictoires.
• Grand Débat National :Conçu comme une "réponse politique en forme de conte politique de coups" au mouvement des Gilets Jaunes.
• Il a été une "concession procédurale", offrant la possibilité de s'exprimer sans pour autant garantir des concessions substantielles sur le fond.
• Il a "contribué aussi à disqualifier les gilet jaune" en opposant les "bons citoyens" (débattant calmement) aux "mauvais citoyens" (manifestant violemment).
• La plateforme numérique était un outil de "pure et simple consultation d'agrégation de d'opinion individuelles", s'apparentant à un "vaste opération de sondages" plutôt qu'à de la délibération.
• Il a souffert d'un manque d'impartialité dans son pilotage, ce qui a généré de la suspicion.
• La "masse absolument gigantesque" de contributions n'a pas été correctement analysée ni synthétisée de manière accessible au grand public.
• Il a donné le "sentiment finalement d'un exercice présidentielle de communication politique au risque de discréditer et de disqualifier cette fois ci la démocratie participative au regard des français".
• Malgré ses limites, il a été une "première période de purge" et un "premier pas" vers la démocratie numérique, ayant potentiellement contribué à "reconnecter de la confiance interpersonnelle entre les citoyens" via les réunions locales.
• Convention Citoyenne pour le Climat :Présentée comme "l'anti grand débat", issue d'une "coproduction" entre l'exécutif et une volonté de répondre à la demande écologique et aux Gilets Jaunes.
• Caractérisée par un "design" et une "technologie" (au sens de conception du dispositif) visant l'impartialité et la véritable délibération.
• Pilotage impartial par un "comité de gouvernance" pluripartite et indépendant.
• Accueillie par le Conseil Économique Social et Environnemental (CESE), garantissant une absence de tutelle de l'exécutif.
• Organisation du temps (six rencontres sur quatre mois) pour permettre aux citoyens de "s'approprier l'information" et les enjeux.
• Question posée aux citoyens : "comment diminuer d'au moins 40% les émissions de gaz à effet de serre de d'ici 2030... et dans un esprit de justice sociale", avec maîtrise des réponses sur les mesures législatives.
• Représentativité de la société française par tirage au sort, incluant des publics généralement absents des arènes participatives habituelles (jeunes, précaires, sans diplôme).
• L'exécutif s'est engagé à transmettre les propositions "au parlement ou au référendum".
• Enjeux de la Démocratie Participative :
• Compenser les défaillances de la démocratie représentative : Particulièrement face à l'"urgence climatique" qui pourrait rapidement empêcher de "nous gouverner démocratiquement". L'enjeu est de "sauver la planète le climat mais la démocratie en même temps", d'une manière "qui ne soit pas autoritaire".
• Articuler démocratie institutionnelle et contestataire : La "démocratie contestataires qui s'exprime dans la rue" doit trouver des "débouchés". Il doit y avoir une "synergie entre ces mouvements".
• Vérifier les consensus : Tester ce que la société est "prêt à accepter" en termes de mesures.
• Lien à la décision : C'est le "nerf de la guerre". Il faut "faire reconnaître essayer de mettre au jour un lien entre ce qui se dit dans ces dispositifs et les décisions qui sont prises". L'importance de la justification par les élus des décisions prises, même si elles ne suivent pas totalement les préconisations citoyennes.
• Déclinaison territoriale des politiques publiques : La participation est un "véritable laboratoire" dans les territoires, permettant de "décliner les politiques publiques beaucoup plus finement" en tenant compte des singularités locales. Exemple du projet Garonne Amont face au changement climatique.
• Défis de l'Analyse et de la Représentativité dans la Participation de Masse :
• La "notion de synthèse" est jugée "l'enfer" face à une masse de contributions "incalculables" et intéressantes. L'idée n'est pas de produire un "top 5 des grandes propositions" mais un "référentiel qui est l'exact expression de l'expression citoyenne".
• L'application de théories comme les "arbres de connaissances" pour représenter les consensus (le tronc) et les oppositions ou idées spécifiques (les branches et les feuilles) est préférée à une simple agrégation.
• La question de la légitimité de la masse de données se pose : "on ne sait pas qui la parole". L'anonymat (sauf pour les codes postaux) dans le GDN a empêché de "construire la légitimité au niveau de la représentativité".
• Un mini-public comme la CCC (150 citoyens) peut produire une "richesse une qualité de délibération" supérieure à une masse de 500 000 contributions non délibératives.
• Le Rôle des Outils Numériques (Civic Tech) et la Pédagogie Civique :
• Des outils comme les comparateurs de programmes politiques (Wax) visent à rendre l'information "claire et efficace", en particulier pour les jeunes.
• La civic tech cherche à "faire de la sensibilisation" et dire : "toi aussi tu as droit d'avoir une idée".
• L'idée est de rendre l'information politique "plus accessible plus interactive" (via des chatbots par exemple) pour amener progressivement les citoyens à se positionner et à s'engager ("le niveau zéro de l'engagement").
• Il s'agit d'"éduquer notre audience" pour leur montrer qu'ils sont "légitime" et que "la démocratie c'est à elle".
• Il faut accepter qu'il n'y ait pas que la démocratie délibérative "dans les règles de l'art". La "première marche" (comme répondre à un questionnaire simple) est déjà "pas mal" et peut être un point de départ vers une participation plus profonde.
• Il faut "prendre le citoyen par la main" et l'accompagner dans son cheminement.
• La Délibération comme Pierre Angulaire de la Légitimité Démocratique :
• Une forte conviction dans l'idée que "pour qu'une décision soit légitime il faut que ceux qui sont affectés par cette décision aient pu en débattre auparavant".
• La nécessité de "multiplier les occasions de s'exposer au point de vue de l'autre" pour lutter contre la "défiance généralisée à la fois horizontal et vertical".
• La délibération permet de "progresser dans la réflexion" et de réaliser qu'on ne dispose pas de toutes les informations, comme l'illustre l'exemple du citoyen face au fiscaliste dans une réunion locale.
• Faire le "pari de l'intelligence de tous" implique le "corollaire" de la "responsabilité de tous". La question est : "jusqu'où je suis prêt à m'engager", "de passer de la posture de spectateurs à la posture de d'auteur du monde qui m'entoure ensuite à celle d'acteur".
• Défis et Risques :
• L'Instrumentalisation : Risque que la participation soit perçue et utilisée uniquement comme un outil d'acceptabilité ou de communication politique.
• L'Enfumage : La crainte des citoyens d'être manipulés ou que leur participation ne mène à rien.
• Le Lien à la Décision Effectif : Malgré de bonnes procédures, les recommandations ne sont pas toujours reprises ou justifiées par le pouvoir politique.
• L'Influence et les Biais : Les dispositifs de masse sont sensibles aux biais (salariés d'une industrie dominante dans une consultation par exemple) et aux jeux d'influence. Il est crucial de "savoir qui parle" et de prendre en compte ces dynamiques.
• Le Risque de Discrédit : Utiliser des outils comme la CCC sur des sujets majeurs et clivants comporte le risque, en cas d'échec (faible impact sur la décision, résultats jugés tièdes, ou rejet par référendum), de discréditer l'outil lui-même.

Conclusion :

La source met en évidence la complexité de la démocratie participative, loin d'une simple opposition entre "bons" et "mauvais" outils.

L'efficacité et la légitimité des dispositifs dépendent crucialement de leur "ingénierie" (conception, pilotage, règles) et de leur capacité à favoriser une véritable délibération, à garantir l'impartialité, à s'articuler avec le processus décisionnel et à prendre en compte la diversité des formes de participation (de la simple consultation à la délibération profonde, du niveau national au local).

Les outils numériques peuvent jouer un rôle important de porte d'entrée et de pédagogie civique, mais ne remplacent pas la nécessité de rencontres et d'échanges pour construire la confiance et la compréhension mutuelle.

Le principal obstacle reste souvent la "volonté politique de faire en sorte qu'elles servent effectivement à à démocratiser la démocratie", en utilisant le plein potentiel des technologies et méthodes disponibles et en les articulant de manière significative avec les lieux de pouvoir.