DOI: 10.7554/eLife.101114

Resource: RRID:BDSC_5073

Curator: @maulamb

SciCrunch record: RRID:BDSC_5073

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DOI: 10.1016/j.devcel.2025.04.005

Resource: (Santa Cruz Biotechnology Cat# sc-32233, RRID:AB_627679)

Curator: @scibot

SciCrunch record: RRID:AB_627679

What is this?

Đã thử sử dụng Android Studio Cloud, nhưng sau phiên làm việc thì không thể Bookmark URL nên chưa biết lưu Project ở đâu 17/05/2025 : Sẽ thử nghiệm lại sau

DOI: 10.1016/j.celrep.2025.115603

Resource: (Agilent Cat# P0448, RRID:AB_2617138)

Curator: @scibot

SciCrunch record: RRID:AB_2617138

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DOI: 10.1016/j.cell.2025.04.006

Resource: (LI-COR Biosciences Cat# 926-32213, RRID:AB_621848)

Curator: @scibot

SciCrunch record: RRID:AB_621848

What is this?

This rationality feels almost contradictory to me, where despite how they feel sexuality isn't important, adults oftentimes enforce heteronormativity on children, such as calling a boy-girl pairing "boyfriend and girlfriend", or only presenting literature with heterosexual pairings. Regardless of what is "wanted", sexuality is already a part of day-to-day life.

The notion that sexuality is not an appropriate topic for young people is completely ridiculous. If anything, it's one of the most important topics for young people who are in the midst of discovering themselves. For it to be completely ignored in curriculum and school discussion is a failure on the part of the school.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

This is a very well-written paper presenting interesting findings related to the recovery following the end-Permian event in continental settings, from N China. The finding is timely as the topic is actively discussed in the scientific community. The data provides additional insights into the faunal, and partly, floral global recovery following the EPE, adding to the global picture.

Strengths:

The conclusions are supported by an impressive amount of sedimentological and paleontological data (mainly trace fossils) and illustrations.

We thank Reviewer #1 for the positive assessments.

Weaknesses:

The occurrence of MISS (Microbially Induced Sedimentary Structures) could be discussed more in detail as these provide interesting information directly linked to the delayed recovery of the biota.

We appreciate the reviewer for highlighting this important point. In the Phanerozoic, increase of microbial abundances generally occurred with rapid warming when documented and those hyperthermal events had causal links to mass extinction in continental realms, including the Permian–Triassic mass extinction (Mays et al., 2021). Accumulations of cyanobacteria and other microbes was favored by low dissolved oxygen concentrations (Pacton et al., 2011) and the produced secondary metabolites may also be toxic to animals (Paerl and Otten, 2013). Therefore, repeated algal and bacterial blooms in the post-extinction interval could disrupt ecological stability and inhibit the restoration of ecosystems.

So, the sentence from Lines 127–130 “The depauperate ichnofauna of the late Smithian were monospecific, representing initial recolonization of empty niches by opportunists, but the coeval thrived microbial mats indicated harsh environments, which might have inhibited the recovery of freshwater ecosystems (Tu et al., 2016; Chu et al., 2017; Mays et al., 2021).” is rephased by:

“The depauperate ichnofauna of the late Smithian were monospecific, representing initial recolonization of empty niches by opportunists. However, recurrent occurrences of microbial induced sedimentary structures (MISS) in the Liujiagou Formation imply that depressed ecosystems persisted until the Smithian (Tu et al., 2016; Chu et al., 2017). Studies revealed that the increase in microbial abundances were generally associated with hyperthermals, which would be the principal causes for mass extinction on land (Mays et al., 2021). Accumulations of microbes were favored by low dissolved oxygen concentration condition and their secondary metabolites could also be toxic to animals (Pacton et al., 2011; Paerl and Otten, 2013). Therefore, repeated thriving of MISS during the Dienerian–Smithian disrupted ecological stability in freshwater ecosystem and delayed biotic recovery in North China.”

References:

Mays, C., et al. 2021. Lethal microbial blooms delayed freshwater ecosystem recovery following the end-Permian extinction. Nat. Commun. 12, 5511. https://doi.org/10.1038/s41467-021-25711-3

Pacton, M., et al. 2011. Amorphous organic matter—Experimental data on formation and the role of microbes. Rev. Palaeobot. Palynol. 166, 253–267. https://doi.org/10.1016/j.revpalbo.2011.05.011

Paerl, H. W. & Otten, T. G. 2013. Harmful cyanobacterial blooms: causes, consequences, and controls. Microb. Ecol. 65, 995–1010. https://doi.org/10.1007/s00248-012-0159-y

Reviewer #2 (Public review):

Summary:

A rapid recovery of the ecosystems during the late Early Triassic, in the aftermath of the end-Permian mass extinction, is discussed based on different types of fossils.

Strengths:

The combined study of invertebrate trace fossils, tetrapod bones, and plant remains together with their stratigraphic distribution in different sections provides a convincing case to support a rapid recovery as the authors hypothesize.

We thank Reviewer #2 for the positive comments on our work.

Weaknesses:

The study is based on three regions with Triassic successions from the North China block. While a first-hand study of other localities of similar age would be ideal, this is of course a difficult task. Instead, the authors provide comparisons with other worldwide regions to build their case and support the initial hypothesis.

Globally, ichnoassemblages reported from the Lower Triassic are relatively impoverished (Guo et al., 2019). We have compiled ichnoassemblages from several continental basins before, including South Africa, Antarctica, North America, European Basin and North China (Fig. 14 in Guo et al., 2019). However, most of the Early Triassic strata lack bioturbation (e.g., Guo et al., 2019, Buatois et al., 2021). On the contrary, the coeval deposits in North China contain diverse trace fossils, making it an ideal place for ichnological investigations. Hence, this study mainly focuses on the ichnological records in North China, but we hope more work will be done in other basins. 

References:

Guo, W.W, et al. 2019. Secular variations of ichnofossils from the terrestrial Late Permian–Middle Triassic succession at the Shichuanhe section in Shaanxi Province, North China. Glob. Planet. Change 181, 102978. https://doi.org/10.1016/j.gloplacha.2019.102978

Buatois, L.A., et al. 2021. Impact of Permian mass extinctions on continental invertebrate infauna. Terra Nova 33, 455–464. https://doi.org/10.1111/ter.12530

Reviewer #3 (Public review):

Summary:

This manuscript by Guo and colleagues features the documentation and interpretation of three successions of continental to marginal marine deposits spanning the P/T transition and their respective ichnofaunas. Based on these new data inferences concerning end-Permian mass extinction and Triassic recovery in the tropical realm are discussed.

Strengths:

The manuscript is well-written and organized and includes a large amount of new lithological and ichnological data that illuminate ecosystem evolution in a time of large-scale transition. The lithological documentations, facies interpretations, and ichnotaxonomic assignments look okay (with a few exceptions).

We thank Reviewer #3 for the positive assessments.

Weaknesses:

Some interpretations in Table 1 could be questioned: For facies association FA2 the interpretation as „terrestrial facies with periodical flooding" should be put into the right column and, given the fossil content, other interpretations, such as "marine facies" or "lagoonal environment" with some plant debris and (terrestrial) animal remains washed in, could also be possible. For FA3 the statement "bioturbation is absent" is in conflict with the next statement "strata are moderately reworked". For FA5 the observation of a "monospecific ichnoassemblage" contradicts the listing of several ichnotaxa.

We thank the reviewer for this feedback. The “FA2: terrestrial facies with periodical flooding” has been moved to the right column. As for the interpretation of depositional environment of FA2, this interval was basically terrestrial accordingly to the well-developed paleosols (Yu et al., 2022). Meanwhile, regional geological surveys have shown a faunal transition in this interval among a series of successions, from typical marine fauna containing Lingula, Eumorphotis, etc. in the southwest to a marine bivalve-terrestrial conchostracan mixed fauna in the northeast (Yin and Lin, 1979; Chu et al., 2019). Therefore, occurrence of episodic transgressions is suggested.

The FA3: Costal mudplain facies distributed to both the upper Sunjiagou Formation and Lower Heshang Formation (Fig S1), where the former lack bioturbation and the latter were moderately disturbed. We have stated this clearly in the table S1.

Ichnofauna in FA5 are dominated by Skolithos, Lockeia and Gordia, with only one poorly preserved specimen of Palaeophycus, which are distributed at the Shichuanhe and Liulin sections. However, there ichnotaxa were distributed separately, characterized by low diversity (single ichnogenus) and high density. We have deleted the “monospecific ichnoassemblage” for clarity.

References:

Chu, D., et al. 2019, Mixed continental-marine biotas following the Permian-Triassic mass extinction in South and North China: Palaeogeography, Palaeoclimatology, Palaeoecology, v. 519, p. 95–107, doi:10.1016/j.palaeo.2017.10.028.

Yu, Y., et al. 2021, Latest Permian–Early Triassic paleoclimatic reconstruction by sedimentary and isotopic analyses of paleosols from the Shichuanhe section in central North China Basin: Palaeogeography, Palaeoclimatology, Palaeoecology, v. 585, p. 110726, doi:10.1016/j.palaeo.2021.110726.

Yin, H.F., Lin, H.M., 1979. Marine Triassic faunas and the geologic time from Shihchienfeng Group in the northern Weihe River Basin, Shaanxi Province. Acta Stratigr. Sin. 3, 233–241 (in Chinese).

Concerning the structure of the manuscript, certain hypotheses related to the end-Permian mass extinction and the process of the P/T extinction and recovery, namely the existence of a long-persisting "tropic dead zone" are introduced as a foregone conclusion to which the new data seemingly shall be fit as corroborating evidence. Some of the data - e.g. the presence of a supposedly Smithian-age ichnofauna are interpreted as a fast recovery shortening the duration of the "tropic dead zone" episode - but these interpretations could also be interpreted as contradicting the idea of a "dead zone" sensu stricto in favour of a "normal" post-extinction environment with low diversity and occurrence of typical disaster taxa. Due to their large error bars the early Triassic radiometric ages did not put much of a constraint on the age determination of the earliest post-extinction ichnofaunas discussed here.

In the first ~5 Myr of the Triassic, there is evidence for a broad equatorial belt (30°N-40°S) where marine and terrestrial animals were nearly absent (namely “equatorial tetrapod gap”; Sun et al., 2012). However, the nature, duration and range of the “equatorial tetrapod gap” remain debated. Allen et al. (2020) show poleward migrations of terrestrial tetrapods during the Late Permian to Middle Triassic, with marine reptile diversity peak still restricted to northern low latitudes. Romano et al. (2020) argued that the Early Triassic equatorial terrestrial tetrapod gap would be narrower and restricted the “death belt” between 15° N and about 31° S, while Liu et al. (2022) consider that the exact boundaries of this gap likely varied with climate change (hot phases). Moreover, duration of the gap is also questioned, it’s long-lasting (Late Permian to Middle Triassic), during Induan (Bernardi et al., 2018), or from Induan to the early Spathian (Liu et al., 2022). Regardless of these discrepancies, all the related studies show the existence of the “low latitudinal tetrapod gap”, which is mentioned as background information. On this basis, this study aims to reveal when and how terrestrial ecosystems recovered from the “tropic dead zone” from the ecological point of view, rather than tetrapods only.

The fast recovered terrestrial ecosystems are represented by diverse traces, and concurrent tetrapods and plants found in the Heshanggou Formation. We acknowledge that the chronostratigraphy of the Lower Triassic in North China (and most of continental basins globally) are not controlled by precise ages, this formation, however, could be constrained to Spathian (or even straddle to earliest Middle Triassic), based on integrated magnetostratigraphic correlation, fossil records and geochemical data (Liu, 2018; Guo et al., 2022). The Smithian-age ichnofaunas here are not interpreted as a rapidly recovering biota, but early occurring opportunist-dominated communities that explore the empty ecospace under inhospitable environments. Our study also constrains roughly the “tropical dead zone” from Induan to late Smithian in North China (Fig. 4).

References:

Allen, B.J., et al. 2020. The latitudinal diversity gradient of tetrapods across the Permo-Triassic mass extinction and recovery interval. Proc Biol Sci 287, 20201125. https://doi.org/10.1098/rspb.2020.1125

Bernardi, M., et al. 2018. Tetrapod distribution and temperature rise during the Permian-Triassic mass extinction. Proc Biol Sci 285, 20172331. https://doi.org/10.1098/rspb.2017.2331

Guo, W., et al. 2022. Late Permian–Middle Triassic magnetostratigraphy in North China and its implications for terrestrial-marine correlations. Earth Planet. Sci. Lett. 585, 117519. https://doi.org/10.1016/j.epsl.2022.117519

Liu, J. 2018. New progress on the correlation of Chinese terrestrial Permo-Triassic strata. Vertebrata Palasiatica, 56, 327-342. 10.19615/j.cnki.1000-3118.180709

Liu, J., et al. 2021. Permo-Triassic tetrapods and their climate implications. Glob. Planet. Change 103618. https://doi.org/10.1016/j.gloplacha.2021.103618

Romano, M., et al. 2020. Early Triassic terrestrial tetrapod fauna: a review. Earth-Sci. Rev. 210, 103331. https://doi.org/10.1016/j.earscirev.2020.103331

Sun, Y., er al. 2012. Lethally hot temperatures during the early triassic greenhouse. Science 338, 366–70. https://doi.org/10.1126/science.1224126

Considering the somewhat equivocal evidence and controversial ideas about the P/T transition, the introduction could be improved by describing how the idea of a "tropic dead zone" arose against the background of earlier ideas, alternative views, and conflicting data. In the discussion section, alternative interpretations of the extensive data presented here - e.g. proximal-distal shifts in lithofacies with respect to the sediment source, sea level changes, preservation bias, the local occurrence of hostile environments instead of a regional scale, etc. should be discussed, also to avoid the impression that the author's conclusion was driven by confirmation bias.

As mentioned above, it’s still controversial about the nature, duration and range of the “equatorial tetrapod gap”, which primarily derived from the database (body fossils only vs. both skeletal and footprint data) and analytical methods. However, detailed discussions about these differences are beyond the scope of our study. This paper provides new evidence for the "tropical dead zone" from the ecological perspective (invertebrate ichnology, paleobotany and newly found tetrapods). Our results show that the "tropical dead zone" in North China terminated in the Smithian, followed by the reappearance of many animals in the Spathian, shedding light on the more rapidly recovering terrestrial ecosystems than previously thought.

We have improved the Introduction section by providing a summary of the “equatorial tetrapod gap”. Lines 33-35: “A tropical “tetrapod gap”, spanning between 15°N and ~31°S, prevailed through the Early Triassic, or at least during particular intervals of intense global warming (Bernardi et al., 2018; Allen et al., 2020; Romano et al., 2020; Liu et al., 2022).” is revised to:

“A tropical “tetrapod gap”, spanning between 15°N and ~31°S, prevailed in the Early Triassic, or at particular interval of intense global warming, even though the nature, duration and range remain debated (Bernardi et al., 2018; Allen et al., 2020; Romano et al., 2020; Liu et al., 2022).”

In the Discussion section, Lines 180-181: “Although the specimens are not yet fully prepared for taxonomic description, they clearly show the existence of tetrapod at this level” is revised to:

“Although the specimens are not yet fully prepared for taxonomic description, they clearly show the existence of tetrapods at this level, narrowing the “tetrapod gap” to the Spathian.”

we also add a new paragraph from Line 208:

“Our results also shed light on the timing of the tropical dead zone. The late Smithian-age ichnofauna, although impoverished, represents early opportunist-dominated communities that explored empty ecospace under inhospitable environments, which constrains the equatorial death belt to the late Smithian in North China.”

Contrary to the authors' claim, Figures S7 and S8 suggest that burrow size does not vary much within the studied sections. Size decreases and increases in the Shichuanhe and Liulin sections do not contemporaneously, are usually within the error-bar range, and might be driven by ichnotaxa composition, i.e. the presence or absence of larger ichnotaxa, rather than by size changes in the same ichnotaxon (and producer group). Here the measurement data would be needed as well to check the basis of the authors' interpretations.

We thank the reviewer for highlighting this important point. We have checked the accuracy of our raw data. Both the average size of all ichnogenera and single ichnogenera do not change obviously, but increase slightly upwards in the Spathian (Figures S7c and S8). This tendency is congruent with other coeval studies in North China (e.g., Shu et al., 2018; Xing et al., 2020). The presence of larger ichnotaxa will indeed improve the average sizes of fossil-bearing horizons, however, burrows of single ichnogenera in the Spathian generally show wider size distributions than in the Smithian, which might be associated with enriched producer groups or different growth stages of the same biota.

The asynchronous burrow size changes in the Shichuanhe and Liulin sections could be attributed to sedimentary facies. Late Permian deposits at Shichuanhe are finer than those at Linlin, which is located at the basin margin. As a result, tiny traces, like Helminthoidichnites, which were widely distributed at Shichuanhe, are absent at Linlin section. Those traces significantly reduce the average sizes in this interval, leading to inconsistent size variation patterns.

References:

Shu, W., et al. 2018. Limuloid trackways from Permian-Triassic continental successions of North China. Palaeogeogr. Palaeoclimatol. Palaeoecol. 508, 71–90. https://doi.org/10.1016/j.palaeo.2018.07.022

Xing, Z.F., et al. 2020. Trace fossils from the Lower Triassic of North China—a potential signature of the gradual recovery of a terrestrial ecosystem. Palaeoworld 30, 95–105. https://doi.org/10.1016/j.palwor.2020.06.002

Some arthropod tracks assigned here to Kouphichnium might not represent limulid traces but other (non-marine) arthropod taxa in accordance with their occurrence in terrestrial facies/non-marine units of the succession. More generally, the ichnotaxonomy of arthropod trackways is not yet well reserved - beyond Kouphichnium and Diplichnites various similar-looking types may occur that can have a variety of distinct insect, crustacean, millipede, etc. producers (including larval stages).

Well, individual trace-makers can produce different traces, and different organisms can make morphologically similar traces. In consideration of this, it’s hard to give a one-on-one relationship between trace fossils and their producers in most cases, especially for the invertebrates. So, Kouphichnium could be made by arthropods other than limuloidss.

However, horseshoe crabs, originating in the early Ordovician, invaded freshwater environments twice in the Paleozoic and once in the Mesozoic (Lamsdell, 2016), and their body fossils have been found from the Early Triassic of Germany (e.g., Hauschke and Wilde, 2008) and North China (which occur with their traces; unpublished data). Accordingly, we tentatively speculate Kouphichnium found in this interval could be primarily produced by limuloids.

References:

Hauschke, N., Wilde, V. 2008. Limuliden aus dem Oberen Buntsandstein von Süddeutschland. Hallesches Jahrb. Für Geowiss. 30, 21–26.

Lamsdell, J.C. 2016. Horseshoe crab phylogeny and independent colonizations of fresh water: ecological invasion as a driver for morphological innovation. Palaeontology 59, 181–194. https://doi.org/10.1111/pala.12220

Recommendations for the authors:

Reviewer #1 (Recommendations for The Authors):

(1)  Line 112 - was identified during..; please change to ...was identified in successions of late Changsian-early Smithian age.

Revised as suggested.

(2)  Line 116 - change prolong to prolonged.

Revised as suggested.

(3) Line 121 - change ichnofaunal to ichnofauna (check the entire sentence).

We checked the manuscript thoroughly and revised as suggested.

(4) Figure 1 caption - check sentence starting with - Base map...(delete 'of is')

Revised as suggested.

(5) Line 471 - tiny instead of tinny.

Revised as suggested.

(6) Figure S9 - would it be possible to include this reconstruction in the main manuscript?

We have moved the artistic illustration to the main text as Figure 5.

(7) Add the illustrators name / or indicate if it is produced by AI.

We have added the sentence “The artistic illustration is credited to J. Sun” at the end.

Reviewer #2 (Recommendations for The Authors):

(1) Line 15 – change 252 million years ago to ca. 252 million years ago.

Revised as suggested.

(2) Line 18 – change low-latitude North China to low-latitude present-day North China.

Actually, the paleolatitude of North China during the Early Triassic is about 17-18°N according to paleomagnetic results (Huang et al., 2018; Guo et al., 2022,).

References:

Guo, W., et al. 2022. Late Permian–Middle Triassic magnetostratigraphy in North China and its implications for terrestrial-marine correlations. Earth Planet. Sci. Lett. 585, 117519. https://doi.org/10.1016/j.epsl.2022.117519

Huang, B., et al. 2018. Paleomagnetic constraints on the paleogeography of the east asian blocks during Late Paleozoic and Early Mesozoic times. Earth-Sci. Rev. 186, 8–36. https://doi.org/10.1016/j.earscirev.2018.02.004

(3) Line 25 - "possible" doesn't seem the appropriate term here for the structure of the sentence. Could it be "to make possible" that it meant? Or otherwise you could write "possibly". Please revise this.

Revised “possible” to “possibly”.

(4) Line 33 – change “are” to “were”.

Revised as suggested.

(5) Line 43 – There are other, more appropriate articles that should (also) be cited here, especially because Mujal et al. (2017) doesn't deal with the Central European Basin (so you could even remove this reference). For sure this one should be cited:

Scholze, F., Wang, Z., Kirscher, U., Kraft, J., Schneider, J.W., Götz, A.E., Joachimski, M.M., Bachtadse, V., 2017. A multistratigraphic approach to pinpoint the Permian-Triassic boundary in continental deposits: the Zechstein–Lower Buntsandstein transition in Germany. Glob. Planet. Chang. 152, 129–151. http://dx.doi.org/10.1016/j.gloplacha.2017.03.004.

We have replaced Mujal’s paper with Scholze et al., (2017) in the main text.

(6) Line 46 – change “Roopnarinev et al., 2019” to “Roopnarine et al., 2019”.

Revised as suggested.

(7) Line 53 – Here Mujal et al. (2017) would be more appropriate, since it deals with a basin from the western peri-Tethys, also, this other article by Mujal et al. (2017) discussed the recovery in the western peri-Tethys based on tetrapod footprints:

Mujal, E., Fortuny, J., Bolet, A., Oms, O., López, J.Á., 2017. An archosauromorph dominated ichnoassemblage in fluvial settings from the late Early Triassic of the Catalan Pyrenees (NE Iberian Peninsula). PLoS One 12 (4), e0174693. http://dx.doi.org/10.1371/journal.pone.0174693.

Revised as suggested.

(8) Line 58 – change “relatively diversified trace fossils have been found during the late Early Triassic” to “because relatively diversified trace fossils have been found in upper Lower Triassic deposits”.

Revised as suggested.

(9) Line 58 – change “recovered” to “ecosystems recovered”.

Revised as suggested.

(10) Line 81 – These two paragraphs could be under a section named Geological setting or similar.

Yes, these two paragraphs are brief introductions of the geological background of North China, so we change the section name to “Geological Settings and Methods”.

(11) Line 99 – change “behavioural” to “behavioral”.

Revised as suggested and check spelling throughout.

(12) Line 103 – add “is” before adopted.

The sentence “Tiering, referring to the life position of an animal vertically in the sediment, is divided into surficial, semi-infaunal (0–0.5 cm), shallow (0.5–6 cm), intermediate (6–12 cm) and deep infaunal tiers (> 12 cm), adopted from Minter et al. (2017).” is changed to “…, based on Minter et al. (2017).”

(13) Line 113 –change “mainly” to “were mainly”.

Revised as suggested

(14) Line 116 - change prolong to prolonged.

Revised as suggested.

(15) Line 121 – add “preserved” before in.

Revised as suggested.

(16) Line 123 - change “were” to “are”.

Revised as suggested.

(17) Line 127 – “Kouphichnium” instead of “Kouphichnim”.

Revised as suggested.

(18) Line 135 – change to “Occupied by”.

Revised as suggested.

(19) Line 140 – change “bioturbations” to “bioturbated deposits”.

Revised as suggested.

(20) Line 145 – “Spathian” rather than “Spthian”.

Revised as suggested.

(21) Line 140 – change “displayed” to “displays”.

Revised as suggested.

(22) Line 160 – change “continental” to “terrestrial”.

Revised as suggested.

(23) Line 165 – “Marchetti” rather than “Marchettti”.

Revised as suggested.

(24) Line 168 – change “relationships” to “relation”.

Revised as suggested.

(25) Line 177 – “including” instead of “includes”.

Revised as suggested.

(26) Line 181 and Line 214– change “tetrapod” to “tetrapods”.

Revised as suggested.

(27) Line 195 and Line 218 – change “cooccurred” to “co-occurring”.

Revised as suggested.

(28) Line 540 – delete “herein”.

Revised as suggested.

(28) Line 559 – “Helminthoidichnites tenuis”, it should be in italics.

Revised as suggested.

Ocurre cuando un óvulo o un espermatozoide carece del cromosoma sexual X. Si ese gameto se une con uno normal (que tiene un X), el embrión resultante tendrá solo un cromosoma X: 45,X (XO). Conteo anormal de cromosomas

Septima a octava semana: Comienza diferenciación genital aunque aun no se distinguen, en la semana 12 ya se cierra la hendidura urogenital y forman o pene o vagina

A Barcelona I l’AMB no em coincideix cap valor amb les dades del power point. Quines dades s’han fet servir per Barcelona i l’AMB. La Gemma m’ha dit que et pregunti: S’han fet servir desplaçaments residents a Barcelona o be son desplaçaments origen /destinació Barcelona de tots els residents del SIMMB?

Al SIMMB hi ha un valors que no coincideixen per molt poc. A Altres on posa 0,2% hauria de posar 0,3%. Potser aquesta diferencia sigui pels decimals?

A Barcelona I l’AMB no em coincideix cap valor amb les dades del power point. Quines dades s’han fet servir per Barcelona i l’AMB. La Gemma m’ha dit que et pregunti: S’han fet servir desplaçaments residents a Barcelona o be son desplaçaments origen /destinació Barcelona de tots els residents del SIMMB?

Al SIMMB hi ha alguns valors que no coincideixen per molt poc. Benzina on posa 46,4% hauria de posar 46,3%.A GNC GLP on posa 47.510 hauria de posar 47.509. Potser aquesta diferencia sigui pels decimals?

The contrast of beliefs is an important way of emphasizing the otherness of the Egyptians, who may be a stand-in for polytheists in general. The condemnation of other religions successfully creates a divide between groups. Religion is one of the greatest tools to forge a sense of identity with, seeing as how it builds community. It follows, then, that it would also be one of the greatest tools to other a foreign nation. CC BY-NC-SA

Joseph's brothers plot against him out of jealousy. He is innocent and suffers unjustly common in tragic and epic narratives. His endurance parallels heroes like Jesus or Sita from the Ramayana, who suffer betrayal by their own. I would say the hero trait would be its Innocent sufferer

SCM Electric Typewriters by [[Joe Van Cleave]]

Tips for cleaning the Smith-Corona 6 series electric typewriters and details about how they work.

S-C also made this series of typewriter for both Sears and Singer under their branding. (including the Singer Electric in this video.)

The belts on the electric motor and cams was originally a rubber 'V' belt which is no longer manufactured. Using 1/8" round cross-section o-rings of appropriate sizes (for water cannister applications) is the recommended replacement, however one may need to slightly move the drive motor down a bit so that the belt doesn't hit the frame of the typewriter and thereby destroying it over time.

Cleaning and lubricating the drive motors and moving pieces before using may help before trying out a typewriter which has been sitting for long periods of time.

Some later models had an electric return, which can tend to be violent. Electro 220 and Coronet Automatic 12 have an additional clutch and draw band (and lack of return lever on the carriage) for their electric returns.

because social > profit

one supervises and one manages

managers pay

rt. 330. A petição inicial será indeferida quando:

I - for inepta;

II - a parte for manifestamente ilegítima;

III - o autor carecer de interesse processual;

IV - não atendidas as prescrições dos arts. 106 e 321 .

§ 1º Considera-se inepta a petição inicial quando:

I - lhe faltar pedido ou causa de pedir;

II - o pedido for indeterminado, ressalvadas as hipóteses legais em que se permite o pedido genérico;

III - da narração dos fatos não decorrer logicamente a conclusão;

IV - contiver pedidos incompatíveis entre si.

§ 2º Nas ações que tenham por objeto a revisão de obrigação decorrente de empréstimo, de financiamento ou de alienação de bens, o autor terá de, sob pena de inépcia, discriminar na petição inicial, dentre as obrigações contratuais, aquelas que pretende controverter, além de quantificar o valor incontroverso do débito.

§ 3º Na hipótese do § 2º, o valor incontroverso deverá continuar a ser pago no tempo e modo contratados.

Acórdão 894/2025 Plenário (Pedido de Reexame, Relator Ministro Augusto Nardes)

Responsabilidade. Licitação. Revogação. Prejuízo. Multa. Pressupostos. Lei de Introdução às Normas do Direito Brasileiro. - A revogação do certame licitatório não obsta a aplicação de sanção ao agente público, uma vez que se pune a mera conduta, não se exigindo a consumação e a produção de todos os efeitos do ato administrativo. A natureza da multa aplicada pelo TCU se ampara no direito administrativo sancionador, cujo objetivo é prevenir e punir condutas de acordo com o seu grau de reprovabilidade, o que afasta a exigência da concretização de prejuízo, prevista no art. 22, § 2º, da Lindb.

Lei 8.212/91

Art. 31. A empresa contratante de serviços executados mediante cessão de mão de obra, inclusive em regime de trabalho temporário, deverá reter 11% (onze por cento) do valor bruto da nota fiscal ou fatura de prestação de serviços e recolher, em nome da empresa cedente da mão de obra, a importância retida até o dia 20 (vinte) do mês subsequente ao da emissão da respectiva nota fiscal ou fatura, ou até o dia útil imediatamente anterior se não houver expediente bancário naquele dia, observado o disposto no § 5º do art. 33 desta Lei.

No credenciamento, é possível atribuir o objeto da contratação a terceiro; no entanto, deverá haver expressa autorização da Administração.

Acórdão 886/2025 Plenário (Representação, Revisor Ministro Bruno Dantas)

Responsabilidade. Multa. Pressupostos. Conduta. Excludente de culpabilidade. Sanção. Exceção. - O TCU deve, diante de circunstâncias excepcionais devidamente demonstradas, afastar a responsabilização de agente público cuja conduta, embora em desconformidade com a norma, tenha se dado num contexto de inexigibilidade de conduta diversa (art. 22 da Lindb), removendo-se, assim, o pressuposto da culpabilidade, necessário à imposição de sanção.

Für einen Blogartikel gut, auch die Abbildung ist zum Verständnis hilfreich. Für eine wiss. Arbeit ist allerdings eine qualitätsgesicherte Quelle besser zu wählen

Wenn etwas so indirekt zitiert wird wie hier, ist eine Seiten- oder Absatzangabe nötig - oder eine bessere Quelle

Quelle unvollständig: Da weder Verlag noch Link angegeben sind, ist es nicht möglich, die Quelle aufzurufen oder in der Bib zu finden.

caracteristicas arquiteturais

Parece que fuera solo un concepto, por lo que quitaría la coma o lo aclararía

Author response:

The following is the authors’ response to the original reviews

Public Reviews:  

Reviewer #1 (Public Review):

Summary:

This paper reports an intracranial SEEG study of speech coordination, where participants synchronize their speech output with a virtual partner that is designed to vary its synchronization behavior. This allows the authors to identify electrodes throughout the left hemisphere of the brain that have activity (both power and phase) that correlates with the degree of synchronization behavior. They find that high-frequency activity in the secondary auditory cortex (superior temporal gyrus) is correlated to synchronization, in contrast to primary auditory regions. Furthermore, activity in the inferior frontal gyrus shows a significant phase-amplitude coupling relationship that is interpreted as compensation for deviation from synchronized behavior with the virtual partner.

Strengths:

(1) The development of a virtual partner model trained for each individual participant, which can dynamically vary its synchronization to the participant's behavior in real-time, is novel and exciting.

(2) Understanding real-time temporal coordination for behaviors like speech is a critical and understudied area.

(3) The use of SEEG provides the spatial and temporal resolution necessary to address the complex dynamics associated with the behavior.

(4) The paper provides some results that suggest a role for regions like IFG and STG in the dynamic temporal coordination of behavior both within an individual speaker and across speakers performing a coordination task.

We thank the Reviewer for their positive comments on our manuscript.

Weaknesses:

(1) The main weakness of the paper is that the results are presented in a largely descriptive and vague manner. For instance, while the interpretation of predictive coding and error correction is interesting, it is not clear how the experimental design or analyses specifically support such a model, or how they differentiate that model from the alternatives. It's possible that some greater specificity could be achieved by a more detailed examination of this rich dataset, for example by characterizing the specific phase relationships (e.g., positive vs negative lags) in areas that show correlations with synchronization behavior. However, as written, it is difficult to understand what these results tell us about how coordination behavior arises.

We understand the reviewer’s comment. It is true that this work, being the first in the field using real-time adapting synchronous speech and intracerebral neural data, is a descriptive work, that hopefully will pave the way for further studies. We have now added more statistical analyses (see point 2) to go beyond a descriptive approach and we have also rewritten the discussion to clarify how this work can possibly contribute to disentangle different models of language interaction. Most importantly we have also run new analyses taking into account the specific phase relationship, as suggested.

We already had an analysis using instantaneous phase difference in the phase-amplitude coupling approach, that bridges phase of behaviour to neural responses (amplitude in the high-frequency range). However, this analysis, as the reviewer noted, does not distinguish between positive and negative lags, but rather uses the continuous fluctuations of coordinative behaviour. Following the reviewer’s suggestion, we have now run a new analysis estimating the average delay (between virtual partner speech and patient speech) in each trial, using a cross-correlation approach. This gives a distribution of delays across trials that can then be “binned” as positive or negative. We have thus rerun the phase-amplitude coupling analyses on positive and negative trials separately, to assess whether the phase amplitude relationship depends upon the anticipatory (negative lags) or compensatory (positive lags) behaviour. Our new analysis (now in the supplementary, see figure below) does not reveal significant differences between positive and negative lags. This lack of difference, although not easy to interpret, is nonetheless interesting because it seems to show that the IFG does not have a stronger coupling for anticipatory trials. Rather the IFG seems to be strongly involved in adjusting behaviour, minimizing the error, independently of whether this is early or late.

We have updated the “Coupling behavioural and neurophysiological data” section in Materials and methods as follows:  

“In the third approach, we assessed whether the phase-amplitude relationship (or coupling) depends upon the anticipatory (negative delays) or compensatory (positive delays) behaviour between the VO and the patients’ speech. We computed the average delay in each trial using a cross-correlation approach on speech signals (between patient and VP) with the MATLAB function xcorr. A median split (patient-specific ; average median split = 0ms, average sd = 24ms) was applied to conserve a sufficient amount of data, classifying trials below the median as “anticipatory behaviour” and trials above the median as “compensatory behaviour”. Then we conducted the phase-amplitude coupling analyses on positive and negative trials separately.”

We also added a paragraph on this finding in the Discussion:

“Our results highlight the involvement of the inferior frontal gyrus (IFG) bilaterally, in particular the BA44 region, in speech coordination. First, trials with a weak verbal coordination (VCI) are accompanied by more prominent high frequency activity (HFa, Fig.4; Fig.S4). Second, when considering the within-trial time-resolved dynamics, the phase-amplitude coupling (PAC) reveals a tight relation between the low frequency behavioural dynamics (phase) and the modulation of high-frequency neural activity (amplitude, Fig.5B ; Fig.S5). This relation is strongest when considering the phase adjustments rather than the phase of speech of the VP per se : larger deviations in verbal coordination are accompanied by increase in HFa. Additionally, we also tested for potential effects of different asynchronies (i.e., temporal delay) between the participant's speech and that of the virtual partner but found no significant differences (Fig.S6). While lack of delay-effect does not permit to conclude about the sensitivity of BA44 to absolute timing of the partner’s speech, its neural dynamics are linked to the ongoing process of resolving phase deviations and maintaining synchrony.”

(2) In the results section, there's a general lack of quantification. While some of the statistics reported in the figures are helpful, there are also claims that are stated without any statistical test. For example, in the paragraph starting on line 342, it is claimed that there is an inverse relationship between rho-value and frequency band, "possibly due to the reversed desynchronization/synchronization process in low and high frequency bands". Based on Figure 3, the first part of this statement appears to be true qualitatively, but is not quantified, and is therefore impossible to assess in relation to the second part of the claim. Similarly, the next paragraph on line 348 describes optimal clustering, but statistics of the clustering algorithm and silhouette metric are not provided. More importantly, it's not entirely clear what is being clustered - is the point to identify activity patterns that are similar within/across brain regions? Or to interpret the meaning of the specific patterns? If the latter, this is not explained or explored in the paper.

The reviewer is right. We have now added statistical analyses showing that:

(1) the ratio between synchronization and desynchronization evolves across frequencies (as often reported in the literature).

(2) the sign of rho values also evolves across frequencies.

(3) the clustering does indeed differ when taking into account behaviour. We have also clarified the use of clustering and the reasoning behind it.

We have updated the Materials and methods section as follows:

“The statistical difference between spatial clustering in global effect and brain-behaviour correlation was estimated with linear model using the R function lm (stat package), post-hoc comparisons were corrected for multiple comparisons using the Tukey test (lsmeans R package ; Lenth, 2016). The statistical difference between clustering in global effect and behaviour correlation across the number of clusters was estimated using permutation tests (N=1000) by computing the silhouette score difference between the two conditions.” We have updated the Results section as follows:

(1) “This modulation between synchronization and desynchronization across frequencies was significant (F(5) = 6.42, p < .001 ; estimated with linear model using the R function lm).”

(2) “The first observation is a gradual transition in the direction of correlations as we move up frequency bands, from positive correlations at low frequencies to negative ones at high frequencies (F(5) = 2.68, p = .02). This effect, present in both hemispheres, mimics the reversed desynchronization/synchronization process in low and high frequency bands reported above.”

(3) “Importantly, compared to the global activity (task vs rest, Fig 3A), the neural spatial profile of the behaviour-related activity (Fig 3B) is more clustered, in the left hemisphere. Indeed, silhouette scores are systematically higher for behaviour-related activity compared to global activity, indicating greater clustering consistency across frequency bands (t(106) = 7.79, p < .001, see Figure S3). Moreover, silhouette scores are maximal, in particular for HFa, for five clusters (p < .001), located in the IFG BA44, the IPL BA 40 and the STG BA 41/42 and BA22 (see Figure S3).”

(3) Given the design of the stimuli, it would be useful to know more about how coordination relates to specific speech units. The authors focus on the syllabic level, which is understandable. But as far as the results relate to speech planning (an explicit point in the paper), the claims could be strengthened by determining whether the coordination signal (whether error correction or otherwise) is specifically timed to e.g., the consonant vs the vowel. If the mechanism is a phase reset, does it tend to occur on one part of the syllable?

Thank you for this thoughtful feedback. We agree that the relationship between speech coordination and specific speech units, such as consonants versus vowels, is an intriguing question. However, in our study, both interlocutors (the participant and the virtual partner) are adapting their speech production in real-time. This interactive coordination makes it difficult to isolate neural signatures corresponding to precise segments like consonants or vowels, as the adjustments occur in a continuous and dynamic context.

The VP's ability to adapt depends on its sensitivity to spectral cues, such as the transition from one phonetic element to another. This is likely influenced by the type of articulation, with certain transitions being more salient (e.g., between a stop consonant like "p" and a vowel like "a") and others being less distinct (e.g., between nasal consonants like "m" and a vowel). Thus, the VP’s spectral adaptation tends to occur at these transitions, which are more prominent in some cases than in others.

For the participants, previous studies have shown a greater sensitivity during the production of stressed vowels (Oschkinat & Hoole, 2022; Li & Lancia, 2024), which may reflect a heightened attentional or motor adjustment to stressed syllables.

Here, we did not specifically address the question of coordination at the level of individual linguistic units. Moreover, even if we attempted to focus on this level, it would be challenging to relate neural dynamics directly to specific speech segments. The question of how synchronization at the level of individual linguistic units might relate to neural data is complex. The lack of clear, unit-specific predictions makes it difficult to parse out distinct neural signatures tied to individual segments, particularly when both interlocutors are continuously adjusting their speech in relation to one another.

Therefore, while we recognize the potential importance of examining synchronization at the level of individual phonetic elements, the design of our task and the nature of the coordination in this interactive context (realtime bidirection adaptation) led us to focus more broadly on the overall dynamics of speech synchronization at the syllabic level, rather than on specific linguistic units.

We now state at the end of the Discussion section:

“It is worth noting that the influence of specific speech units, such as consonants versus vowels, on speech coordination remains to be explored. In non-interactive contexts, participants show greater sensitivity during the production of stressed vowels, possibly reflecting heightened attentional or motor adjustments (Oschkinat & Hoole, 2022; Li & Lancia, 2024). In this study, the VP’s adaptation relies on sensitivity to spectral cues, particularly phonetic transitions, with some (e.g., formant transitions) being more salient than others. However, how these effects manifest in an interactive setting remains an open question, as both interlocutors continuously adjust their speech in real time. Future studies could investigate whether coordination signals, such as phase resets, preferentially align with specific parts of the syllable.” References cited:

– Oschkinat, M., & Hoole, P. (2022). Reactive feedback control and adaptation to perturbed speech timing in stressed and unstressed syllables. Journal of Phonetics, 91, 101133.

– Li, J., & Lancia, L. (2024). A multimodal approach to study the nature of coordinative patterns underlying speech rhythm. In Proc. Interspeech, 397-401.

(4) In the discussion the results are related to a previously-described speech-induced suppression effect. However, it's not clear what the current results have to do with SIS, since the speaker's own voice is present and predictable from the forward model on every trial. Statements such as "Moreover, when the two speech signals come close enough in time, the patient possibly perceives them as its own voice" are highly speculative and apparently not supported by the data.

We thank the reviewer for raising thoughtful concerns about our interpretation of the observed neural suppression as related to speaker-induced suppression (SIS). We agree that our study lacks a passive listening condition, which limits direct comparisons to the original SIS effect, traditionally defined as the suppression of neural responses to self-produced speech compared to externally-generated speech (Meekings & Scott, 2021).

In response, we have reconsidered our terminology and interpretation. In the revised Discussion section, we refer to our findings as a "SIS-related phenomenon specific to the synchronous speech context". Unlike classic SIS paradigms, our interactive task involves simultaneous monitoring of self- and externally-generated speech, introducing additional attentional and coordinative demands.

The revised Discussion also incorporates findings by Ozker et al. (2022, 2024), which link SIS and speech monitoring, suggesting that suppressing responses to self-generated speech facilitates error detection. We propose that the decrease in high-frequency activity (HFa) as verbal coordination increases reflects reduced error signals due to closer alignment between perceived and produced speech. Conversely, HFa increases with reduced coordination may signify greater prediction error.

Additionally, we relate our findings to the "rubber voice" effect (Zheng et al., 2011; Lind et al., 2014; Franken et al., 2021), where temporally and phonetically congruent external speech can be perceived as self-generated. We speculate that this may occur in synchronous speech tasks when the participant's and VP's speech signals closely align. However, this interpretation remains speculative, as no subjective reports were collected to confirm this perception. Future studies could include participant questionnaires to validate this effect and relate subjective experience to neural measures of synchronization.

Overall, our findings extend the study of SIS to dynamic, interactive contexts and contribute to understanding internal forward models of speech production in more naturalistic scenarios.

We have now added these points to the discussion as follows:

“The observed negative correlation between verbal coordination and high-frequency activity (HFa) in STG BA22 suggests a suppression of neural responses as the degree of behavioural synchrony increases. This result is reminiscent of findings on speaker-induced suppression (SIS), where neural activity in auditory cortex decreases during self-generated speech compared to externally-generated speech (Meekings & Scott, 2021; Niziolek et al., 2013). However, our paradigm differs from traditional SIS studies in two critical ways: (1) the speaker's own voice is always present and predictable from the forward model, and (2) no passive listening condition was included. Therefore, our findings cannot be directly equated with the original SIS effect.

Instead, we propose that the suppression observed here reflects a SIS-related phenomenon specific to the synchronous speech context. Synchronous speech requires simultaneous monitoring of self- and externallygenerated speech, a task that is both attentionally demanding and coordinative. This aligns with evidence from Ozker et al. (2024, 2022), showing that the same neural populations in STG exhibit SIS and heightened responses to feedback perturbations. These findings suggest that SIS and speech monitoring are related processes, where suppressing responses to self-generated speech facilitates error detection. In our study, suppression of HFa as coordination increases may reflect reduced prediction errors due to closer alignment between perceived and produced speech signals. Conversely, increased HFa during poor coordination may signify greater mismatch, consistent with prediction error theories (Houde & Nagarajan, 2011; Friston et al., 2020). Furthermore, when self- and externally-generated speech signals are temporally and phonetically congruent, participants may perceive external speech as their own. This echoes the "rubber voice" effect, where external speech resembling self-produced feedback is perceived as self-generated (Zheng et al., 2011; Lind et al., 2014; Franken et al., 2021). While this interpretation remains speculative, future studies could incorporate subjective reports to investigate this phenomenon in more detail.” References cited:

– Franken, M. K., Hartsuiker, R. J., Johansson, P., Hall, L., & Lind, A. (2021). Speaking With an Alien Voice: Flexible Sense of Agency During Vocal Production. Journal of Experimental Psychology-Human perception and performance, 47(4), 479-494. https://doi.org/10.1037/xhp0000799

– Houde, J. F., & Nagarajan, S. S. (2011). Speech production as state feedback control. Frontiers in human neuroscience, 5, 82.

– Lind, A., Hall, L., Breidegard, B., Balkenius, C., & Johansson, P. (2014). Speakers' acceptance of real-time speech exchange indicates that we use auditory feedback to specify the meaning of what we say. Psychological Science, 25(6), 1198-1205. https://doi.org/10.1177/0956797614529797

– Meekings, S., & Scott, S. K. (2021). Error in the Superior Temporal Gyrus? A Systematic Review and Activation Likelihood Estimation Meta-Analysis of Speech Production Studies. Journal of Cognitive Neuroscience, 33(3), 422-444. https://doi.org/10.1162/jocn_a_01661

– Niziolek C. A., Nagarajan S. S., Houde J. F (2013) What does motor efference copy represent? Evidence from speech production Journal of Neuroscience 33:16110–16116Ozker M., Doyle W., Devinsky O., Flinker A (2022) A cortical network processes auditory error signals during human speech production to maintain fluency PLoS Biology 20.

– Ozker, M., Yu, L., Dugan, P., Doyle, W., Friedman, D., Devinsky, O., & Flinker, A. (2024). Speech-induced suppression and vocal feedback sensitivity in human cortex. eLife, 13, RP94198. https://doi.org/10.7554/eLife.94198

– Zheng, Z. Z., MacDonald, E. N., Munhall, K. G., & Johnsrude, I. S. (2011). Perceiving a Stranger's Voice as Being One's Own: A 'Rubber Voice' Illusion? PLOS ONE, 6(4), e18655.

(5) There are some seemingly arbitrary decisions made in the design and analysis that, while likely justified, need to be explained. For example, how were the cutoffs for moderate coupling vs phase-shifted coupling (k ~0.09) determined? This is noted as "rather weak" (line 212), but it's not clear where this comes from. Similarly, the ROI-based analyses are only done on regions "recorded in at least 7 patients" - how was this number chosen? How many electrodes total does this correspond to? Is there heterogeneity within each ROI?

The reviewer is correct, we apologize for this missing information. We now specify that the coupling values were empirically determined on the basis of a pilot experiment in order to induce more or less synchronization, but keeping the phase-shifted coupling at a rather implicit level.  

Concerning the definition of coupling as weak, one should consider that, in the Kuramoto model, the strength of coupling (k) is relative to the spread of the natural frequencies (Δω) in the system. In our study, the natural frequencies of syllables range approximately from 2 Hz to 10Hz, resulting in a frequency spread of Δω = 8 Hz. For coupling to strongly synchronize oscillators across such a wide range, k must be comparable to or exceed Δω. Thus, since k = 0.1 is far much smaller than Δω, it is therefore classified as weak coupling.

We have now modified the Materials and methods section as follows:

“More precisely, for a third of the trials the VP had a neutral behaviour (close to zero coupling: k = +/- 0.01). For a third it had a moderate coupling, meaning that the VP synchronised more to the participant speech (k = -0.09). And for the last third of the trials the VP had a moderate coupling but with a phase shift of pi/2, meaning that it moderately aimed to speak in between the participant syllables (k = + 0.09). The coupling values were empirically determined on the basis of a pilot experiment in order to induce more or less synchronization but keeping the phase-shifted coupling at a rather implicit level. In other terms, while participants knew that the VP would adapt, they did not necessarily know in which direction the coupling went.”

Regarding the criterion of including regions recorded in at least 7 patients, our goal was to balance data completeness with statistical power. Given our total sample of 16 patients, this threshold ensures that each included region is represented in at least ~44% of the cohort, reducing the likelihood of spurious findings due to extremely small sample sizes. This choice also aligns with common neurophysiological analysis practices, where a minimum number of subjects (at least 2 in extreme cases) is required to achieve meaningful interindividual comparisons while avoiding excessive data exclusion. Additionally, this threshold maintains a reasonable tradeoff between maximizing patient inclusion and ensuring that statistical tests remain robust.

We have now added more information in the Results section “Spectral profiles in the language network are nuanced by behaviour” on this point as follows:

“To balance data completeness and statistical power, we included only brain regions recorded in at least 7 patients (~44% of the cohort) for the left hemisphere and at least 5 patients for the right hemisphere (~31% of the cohort), ensuring sufficient representation while minimizing biases due to sparse data.”

Reviewer #2 (Public Review):

Summary:

This paper investigates the neural underpinnings of an interactive speech task requiring verbal coordination with another speaker. To achieve this, the authors recorded intracranial brain activity from the left hemisphere in a group of drug-resistant epilepsy patients while they synchronised their speech with a 'virtual partner'. Crucially, the authors were able to manipulate the degree of success of this synchronisation by programming the virtual partner to either actively synchronise or desynchronise their speech with the participant, or else to not vary its speech in response to the participant (making the synchronisation task purely one-way). Using such a paradigm, the authors identified different brain regions that were either more sensitive to the speech of the virtual partner (primary auditory cortex), or more sensitive to the degree of verbal coordination (i.e. synchronisation success) with the virtual partner (secondary auditory cortex and IFG). Such sensitivity was measured by (1) calculating the correlation between the index of verbal coordination and mean power within a range of frequency bands across trials, and (2) calculating the phase-amplitude coupling between the behavioural and brain signals within single trials (using the power of high-frequency neural activity only). Overall, the findings help to elucidate some of the left hemisphere brain areas involved in interactive speaking behaviours, particularly highlighting the highfrequency activity of the IFG as a potential candidate supporting verbal coordination.

Strengths:

This study provides the field with a convincing demonstration of how to investigate speaking behaviours in more complex situations that share many features with real-world speaking contexts e.g. simultaneous engagement of speech perception and production processes, the presence of an interlocutor, and the need for inter-speaker coordination. The findings thus go beyond previous work that has typically studied solo speech production in isolation, and represent a significant advance in our understanding of speech as a social and communicative behaviour. It is further an impressive feat to develop a paradigm in which the degree of cooperativity of the synchronisation partner can be so tightly controlled; in this way, this study combines the benefits of using prerecorded stimuli (namely, the high degree of experimental control) with the benefits of using a live synchronisation partner (allowing the task to be truly two-way interactive, an important criticism of other work using pre-recorded stimuli). A further key strength of the study lies in its employment of stereotactic EEG to measure brain responses with both high temporal and spatial resolution, an ideal method for studying the unfolding relationship between neural processing and this dynamic coordination behaviour.

We sincerely appreciate the Reviewer's thoughtful and positive feedback on our manuscript.

Weaknesses:

One major limitation of the current study is the lack of coverage of the right hemisphere by the implanted electrodes. Of course, electrode location is solely clinically motivated, and so the authors did not have control over this. However, this means that the current study neglects the potentially important role of the right hemisphere in this task. The right hemisphere has previously been proposed to support feedback control for speech (likely a core process engaged by synchronous speech), as opposed to the left hemisphere which has been argued to underlie feedforward control (Tourville & Guenther, 2011). Indeed, a previous fMRI study of synchronous speech reported the engagement of a network of right hemisphere regions, including STG, IPL, IFG, and the temporal pole (Jasmin et al., 2016). Further, the release from speech-induced suppression during a synchronous speech reported by Jasmin et al. was found in the right temporal pole, which may explain the discrepancy with the current finding of reduced leftward high-frequency activity with increasing verbal coordination (suggesting instead increased speech-induced suppression for successful synchronisation). The findings should therefore be interpreted with the caveat that they are limited to the left hemisphere, and are thus likely missing an important aspect of the neural processing underpinning verbal coordination behaviour.

We have now included, in the supplementary materials, data from the right hemisphere, although the coverage is a bit sparse (Figures S2, S4, S5, see our responses in the ‘Recommendation for the authors’ section, below). We have also revised the Discussion section to add the putative role of right temporal regions (see below as well).

A further limitation of this study is that its findings are purely correlational in nature; that is, the results tell us how neural activity correlates with behaviour, but not whether it is instrumental in that behaviour. Elucidating the latter would require some form of intervention such as electrode stimulation, to disrupt activity in a brain area and measure the resulting effect on behaviour. Any claims therefore as to the specific role of brain areas in verbal coordination (e.g. the role of the IFG in supporting online coordinative adjustments to achieve synchronisation) are therefore speculative.

We appreciate the reviewer’s observation regarding the correlational nature of our findings and agree that this is a common limitation of neuroimaging studies. While elucidating causal relationships would indeed require intervention techniques such as electrical stimulation, our study leverages the unique advantages of intracerebral recordings, offering the best available spatial and temporal resolution alongside a high signal-tonoise ratio. These attributes ensure that our data accurately reflect neural activity and its temporal dynamics, providing a robust foundation for understanding the relationship between neural processes and behaviour. Therefore, while causal claims are beyond the scope of this study, the precision of our methodology allows us to make well-supported observations about the neural correlates of synchronous speech tasks.

Recommendations for the authors:

Reviewing Editor Comment:

After joint consultation, we are seeing the potential for the report to be strengthened and the evidence here to be deemed ultimately at least 'solid': to us (editors and reviewers) it seems that this would require both (1) clarifying/acknowledging the limitations of not having right hemisphere data, and (2) running some of the additional analyses the reviewers suggest, which should allow for richer examination of the data e.g. phase relationships in areas that correlate with synchronisation.

We have now added data on the right hemisphere (RH) that we did not previously report due to a rather sparse sampling of the RH. These results are now reported in the Results section as well as in the Supplementary section, where we put all right hemisphere figures for all analyses (Figure S2, S4, S5). We have also run additional analyses digging into the phase relationship in areas that correlate with synchronisation (Figure S6). These additional analyses allowed us to improve the Discussion section as well.

Reviewer #1 (Recommendations For The Authors):

In some sections, the writing is a bit unclear, with both typos and vague statements that could be fixed with careful proofreading.

We thank the reviewer for pointing out areas where the writing could be improved. We carefully proofread the manuscript to address typos and clarify any vague statements. Specific sections identified as unclear have been rephrased for better precision and readability.

In Figure 1, the colors repeat, making it impossible to tell patients apart.

We have now updated Figure 1 colormap to avoid redundancy and added the right hemisphere.

Line 132: "16 unilateral implantations (9 left, 7 bilateral implantations)". Should this say 7 right hemisphere? If so, the following sentence stating that there was "insufficient cover [sic] of the right hemisphere" is unclear, since the number of patients between LH and RH is similar.

The confusion was due to the fact that the lateralization refers to the presence/absence of electrodes in the Heschl’s gyrus (left : H’ ; right : H) exclusively.

We have thus changed this section as follows:

“16 patients (7 women, mean age 29.8 y, range 17 - 50 y) with pharmacoresistant epilepsy took part in the study. They were included if their implantation map covered at least partially the Heschl's gyrus and had sufficiently intact diction to support relatively sustained language production.” The relevant part (previously line 132) now states:

“Sixteen patients with a total of 236 electrodes (145 in the left hemisphere) and 2395 contacts (1459 in the left hemisphere, see Figure 1). While this gives a rather sparse coverage of the right hemisphere, we decided, due to the rarity of this type of data, to report results for both hemispheres, with figures for the left hemisphere in the main text and figures for the right hemisphere in the supplementary section.”

Reviewer #2 (Recommendations For The Authors):

(1) To address the concern regarding the absence of data from the right hemisphere, I would advise the authors to directly acknowledge this limitation in their Discussion section, citing relevant work suggesting that the right hemisphere has an important role to play in this task (e.g. Jasmin et al., 2016). You should also make this clear in your abstract e.g. you could rewrite the sentence in line 40 to be: "Then, we recorded the intracranial brain activity of the left hemisphere in 16 patients with drug-resistant epilepsy...".

We are grateful to the reviewer for this comment that incited us to look into the right hemisphere data. We have now included results in the right hemisphere, although the coverage is a bit sparse. We have also revised the Discussion section to add the putative role of right temporal regions. Interestingly, our results show, as suggested by the reviewer, a clear involvement of the RH in this task.

First, the full brain analyses show a very similar implication of the RH as compared to the LH (see Figure below). We have now added in the Results section:

“As expected, the whole language network is strongly involved, including both dorsal and ventral pathways (Fig 3A). More precisely, in the left temporal lobe the superior, middle and inferior temporal gyri, in the left parietal lobe the inferior parietal lobule (IPL) and in the left frontal lobe the inferior frontal gyrus (IFG) and the middle frontal gyrus (MFG). Similar results are observed in the right hemisphere, neural responses being present across all six frequency bands with medium to large modulation in activity compared to baseline (Figure S2A) in the same regions. Desynchronizations are present in the theta, alpha and beta bands while the low gamma and HFa bands show power increases.”

As to compared to the left hemisphere, assessing brain-behaviour correlations in the right hemisphere does not provide the same statistical power, because some anatomical regions have very few electrodes. Nonetheless, we observe a strong correlation in the right IFG, similar to the one we previously reported in the left hemisphere, and we now report in the Results section:

“The decrease in HFa along the dorsal pathway is replicated in the right hemisphere (Figure S4). However, while both the right STG BA41/42 and STG BA22 present a power increase (compared to baseline) — with a stronger increase for the STG BA41/42 — neither shows a significant correlation with verbal coordination (t(45)=-1.65, p=.1 ; t(8)=-0.67, p=.5 ; Student’s T test, FDR correction). By contrast, results in the right IFG BA44 are similar to the one observed in the left hemisphere with a significant power increase associated with a negative brainbehaviour correlation (t(17) = -3.11, p = .01 ; Student’s T test, FDR correction).”

Interestingly, the phase-amplitude coupling analysis yields very similar results in both hemispheres (exception made for BA22). We have thus updated the Results section as follows:

“Notably, when comparing – within the regions of interest previously described – the PAC with the virtual partner speech and the PAC with the phase difference, the coupling relationship changes when moving along the dorsal pathway: a stronger coupling in the auditory regions with the speech input, no difference between speech and coordination dynamics in the IPL and a stronger coupling for the coordinative dynamics compared to speech signal in the IFG (Figure 5B ). When looking at the right hemisphere, we observe the same changes in the coupling relationship when moving along the dorsal pathway, except that no difference between speech and coordination dynamics is present in the right secondary auditory regions (STG BA22; Figure S5).”

We also included in the Discussion section the right hemisphere results also mentioning previous work of Guenther and the one of Jasmin. On the section “Left secondary auditory regions are more sensitive to coordinative behaviour” one can read:

“Furthermore, the absence of correlation in the right STG BA22 (Figure S4) seems in first stance to challenge influential speech production models (e.g. Guenther & Hickok, 2016) that propose that the right hemisphere is involved in feedback control. However, one needs to consider the the task at stake heavily relied upon temporal mismatches and adjustments. In this context, the left-lateralized sensitivity to verbal coordination reminds of the works of Floegel and colleagues (2020, 2023) suggesting that both hemispheres are involved depending on the type of error: the right auditory association cortex monitoring preferentially spectral speech features and the left auditory association cortex monitoring preferentially temporal speech features. Nonetheless, the right temporal pole seems to be sensitive to speech coordinative behaviour, confirming previous findings using fMRI (Jasmin et al., 2016) and thus showing that the right hemisphere has an important role to play in this type of tasks (e.g. Jasmin et al., 2016).”

References cited:

– Floegel, M., Fuchs, S., & Kell, C. A. (2020). Differential contributions of the two cerebral hemispheres to temporal and spectral speech feedback control. Nature Communications, 11(1), 2839.

– Floegel, M., Kasper, J., Perrier, P., & Kell, C. A. (2023). How the conception of control influences our understanding of actions. Nature Reviews Neuroscience, 24(5), 313-329.

– Guenther, F. H., & Hickok, G. (2016). Neural models of motor speech control. In Neurobiology of language (pp. 725-740). Academic Press.

(2) When discussing previous work on alignment during synchronous speech, you may wish to include a recently published paper by Bradshaw et al (2024); this manipulated the acoustics of the accompanist's voice during a synchronous speech task to show interactions between speech motor adaptation and phonetic convergence/alignment.

We thank the reviewer for pointing to this recent and interesting paper. We added the article as reference as follows

“Furthermore, synchronous speech favors the emergence of alignment phenomena, for instance of the fundamental frequency or the syllable onset (Assaneo et al., 2019 ; Bradshaw & McGettigan, 2021 ; Bradshaw et al., 2023; Bradshaw et al., 2024).”

(3) Line 80: "Synchronous speech resembles to a certain extent to delayed auditory feedback tasks"- I think you mean "altered auditory feedback tasks" here.

In the case of synchronous speech it is more about timing than altered speech signals, that is why the comparison is done with delayed and not altered auditory feedback. Nonetheless, we understand the Reviewer’s point and we have now changed the sentence as follows:

“Synchronous speech resembles to a certain extent to delayed/altered auditory feedback tasks”

(4) When discussing superior temporal responses during such altered feedback tasks, you may also want to cite a review paper by Meekings and Scott (2021).

We thank the reviewer for this suggestion, indeed this was a big oversight!

The paper is now quoted in the introduction as follows:

“Previous studies have revealed increased responses in the superior temporal regions compared to normal feedback conditions (Hirano et al., 1997 ; Hashimoto & Sakai, 2003 ; Takaso et al., 2010 ; Ozerk et al., 2022 ; Floegel et al., 2020 ; see Meekings & Scott, 2021 for a review of error-monitoring and feedback control in the STG during speech production).”

Furthermore, we updated the discussion part concerning the speaker-induced suppression phenomenon (see below our response to the point 10).

(5) Line 125: "The parameters and sound adjustment were set using an external low-latency sound card (RME Babyface Pro Fs)". Can you please report the total feedback loop latency in your set-up? Or at the least cite the following paper which reports low latencies with this audio device.

Kim, K. S., Wang, H., & Max, L. (2020). It's About Time: Minimizing Hardware and Software Latencies in Speech Research With Real-Time Auditory Feedback. Journal of Speech, Language, and Hearing Research, 63(8), 25222534. https://doi.org/10.1044/2020_JSLHR-19-00419

We now report the total feedback loop latency (~5ms) and also cite the relevant paper (Kim et al., 2020).

(6) Line 127 "A calibration was made to find a comfortable volume and an optimal balance for both the sound of the participant's own voice, which was fed back through the headphones, and the sound of the stimuli." What do you mean here by an 'optimal balance'? Was the participant's own voice always louder than the VP stimuli? Can you report roughly what you consider to be a comfortable volume in dB?

This point was indeed unlcear. We have now changed as follows:

“A calibration was made to find a comfortable volume and an optimal balance for both the sound of the participant's own voice, which was fed back through the headphones, and the sound of the stimuli. The aim of this procedure was that the patient would subjectively perceive their voice and the VP-voice in equal measure. VP voice was delivered at approximately 70dB.”

(7) Relatedly, did you use any noise masking to mask the air-conducted feedback from their own voice (which would have been slightly out of phase with the feedback through the headphones, depending on your latency)?

Considering the low-latency condition allowed with the sound card (RME Babyface Pro Fs), we did not use noise masking to mask the air-conducted feedback from the self-voice of the patients.

(8) Line 141: "four short sentences were pre-recorded by a woman and a man." Did all participants synchronise with both the man and woman or was the VP gender matched to that of the participant/patient?

We thank the reviewer for this important missing detail. We know changed the text as follows:

“Four stimuli corresponding to four short sentences were pre-recorded by both a female and a male speaker. This allowed to adapt to the natural gender differences in fundamental frequency (i.e. so that the VP gender matched that of the patients). All stimuli were normalised in amplitude.”

(9) Can you clarify what instructions participants were given regarding the VP? That is, were they told that this was a recording or a real live speaker? Were they naïve to the manipulation of the VP's coupling to the participant?

We have now added this information to the task description as follows:

“Participants, comfortably seated in a medical chair, were instructed that they would perform a real-time interactive synchronous speech task with an artificial agent (Virtual Partner, henceforth VP, see next section) that can modulate and adapt to the participant’s speech in real time.”

“The third step was the actual experiment. This was identical to the training but consisted of 24 trials (14s long, speech rate ~3Hz, yielding ~1000 syllables). Importantly, the VP varied its coupling behaviour to the participant. More precisely, for a third of the sequences the VP had a neutral behaviour (close to zero coupling : k = +/- 0.01). For a third it had a moderate coupling, meaning that the VP synchronised more to the participant speech (k = - 0.09). And for the last third of the sequences the VP had a moderate coupling but with a phase shift of pi/2, meaning that it moderately aimed to speak in between the participant syllables (k = + 0.09). The coupling values were empirically determined on the basis of a pilot experiment in order to induce more or less synchronization, but keeping the phase-shifted coupling at a rather implicit level. In other terms, while participants knew that the VP would adapt, they did not necessarily know in which direction the coupling went.”  

(10) The paragraph from line 438 entitled "Secondary auditory regions are more sensitive to coordinative behaviour" includes an interesting discussion of the relation of the current findings to the phenomenon of speech-induced suppression (SIS). However, the authors appear to equate the observed decrease in highfrequency activity as speech coordination increases with the phenomenon of SIS (in lines 456-457), which is quite a speculative leap. I would encourage the authors to temper this discussion by referring to SIS as a potentially related phenomenon, with a need for more experimental work to determine if this is indeed the same phenomenon as the decreases in high-frequency power observed here. I believe that the authors are arguing here for an interpretation of SIS as reflecting internal modelling of sensory input regardless of whether this is self-generated or other-generated; if this is indeed the case, I would ask the authors to be more explicit here that these ideas are not a standard part of the traditional account of SIS, which only includes internal modelling of self-produced sensory feedback.

As stated in the public review, we thank both reviewers for raising thoughtful concerns about our interpretation of the observed neural suppression as related to speaker-induced suppression (SIS). We agree that our study lacks a passive listening condition, which limits direct comparisons to the original SIS effect, traditionally defined as the suppression of neural responses to self-produced speech compared to externally-generated speech (Meekings & Scott, 2021).

In response, we have reconsidered our terminology and interpretation. In the revised discussion, we refer to our findings as a "SIS-related phenomenon specific to the synchronous speech context." Unlike classic SIS paradigms, our interactive task involves simultaneous monitoring of self- and externally-generated speech, introducing additional attentional and coordinative demands.

The revised discussion also incorporates findings by Ozker et al. (2024, 2022), which link SIS and speech monitoring, suggesting that suppressing responses to self-generated speech facilitates error detection. We propose that the decrease in high-frequency activity (HFa) as verbal coordination increases reflects reduced error signals due to closer alignment between perceived and produced speech. Conversely, HFa increases with reduced coordination may signify greater prediction error.

Additionally, we relate our findings to the "rubber voice" effect (Zheng et al., 2011; Lind et al., 2014; Franken et al., 2021), where temporally and phonetically congruent external speech can be perceived as self-generated. We speculate that this may occur in synchronous speech tasks when the participant's and VP's speech signals closely align. However, this interpretation remains speculative, as no subjective reports were collected to confirm this perception. Future studies could include participant questionnaires to validate this effect and relate subjective experience to neural measures of synchronization.

Overall, our findings extend the study of SIS to dynamic, interactive contexts and contribute to understanding internal forward models of speech production in more naturalistic scenarios.

We have now added these points to the discussion as follows:

“The observed negative correlation between verbal coordination and high-frequency activity (HFa) in STG BA22 suggests a suppression of neural responses as the degree of synchrony increases. This result aligns with findings on speaker-induced suppression (SIS), where neural activity in auditory cortex decreases during self-generated speech compared to externally-generated speech (Meekings & Scott, 2021; Niziolek et al., 2013). However, our paradigm differs from traditional SIS studies in two critical ways: (1) the speaker's own voice is always present and predictable from the forward model, and (2) no passive listening condition was included. Therefore, our findings cannot be directly equated with the original SIS effect.

Instead, we propose that the suppression observed here reflects a SIS-related phenomenon specific to the synchronous speech context. Synchronous speech requires simultaneous monitoring of self- and externally generated speech, a task that is both attentionally demanding and coordinative. This aligns with evidence from Ozker et al. (2024, 2022), showing that the same neural populations in STG exhibit SIS and heightened responses to feedback perturbations. These findings suggest that SIS and speech monitoring are related processes, where suppressing responses to self-generated speech facilitates error detection.

In our study, suppression of HFa as coordination increases may reflect reduced prediction errors due to closer alignment between perceived and produced speech signals. Conversely, increased HFa during poor coordination may signify greater mismatch, consistent with prediction error theories (Houde & Nagarajan, 2011; Friston et al., 2020).”

(11) Within this section, you also speculate in line 460 that "Moreover, when the two speech signals come close enough in time, the patient possibly perceives them as its own voice." I would recommend citing studies on the 'rubber voice' effect to back up this claim (e.g. Franken et al., 2021; Lind et al., 2014; Zheng et al., 2011).

We are grateful to the Reviewer for this interesting suggestion. Directly following the previous comment, the section now states:

“Furthermore, when self- and externally-generated speech signals are temporally and phonetically congruent, participants may perceive external speech as their own. This echoes the "rubber voice" effect, where external speech resembling self-produced feedback is perceived as self-generated (Zheng et al., 2011; Lind et al., 2014; Franken et al., 2021). While this interpretation remains speculative, future studies could incorporate subjective reports to investigate this phenomenon in more detail.”

(12) As noted in my public review, since your methods are correlational, you need to be careful about inferring the causal role of any brain areas in supporting a specific aspect of functioning e.g. line 501-504: "By contrast, in the inferior frontal gyrus, the coupling in the high-frequency activity is strongest with the input-output phase difference (input of the VP - output of the speaker), a metric that reflects the amount of error in the internal computation to reach optimal coordination, which indicates that this region optimises the predictive and coordinative behaviour required by the task." I would argue that the latter part of this sentence is a conclusion that, although consistent with, goes beyond the current data in this study, and thus needs tempering.

We agree with the Reviewer and changed the sentence as follows:

“By contrast, in the inferior frontal gyrus, the coupling in the high-frequency activity is strongest with the inputoutput phase difference (input of the VP - output of the speaker), a metric that could possibly reflect the amount of error in the internal computation to reach optimal coordination. This indicates that this region could have an implication in the optimisation of the predictive and coordinative behaviour required by the task.”

Me genera dudas la relación entre promedio de edad y lo que sigue

¿Es posible pensar que se mantienen los métodos tradicionales, solo que se median por lo tecnológico? En estudios sobre lectura y escritura se han visto que el objeto no necesariamente modifica las prácticas.

Te sugiero que puedas pensar en términos menos ambiguos ya que en la escritura del trabajo es vital que ubiques al lector lo menos posible en la duda. Busca estadísticas o estudios que te permitan desde el principio citar contextos específicos del contexto.

Reviewer #2 (Public review):

The study by Setogawa et al. aims to understand the role that different striatal subregions belonging to parallel brain circuits have in associative learning and discrimination learning (S-O-R and S-R tasks). Strengths of the study are the use of multiple methodologies to measure and manipulate brain activity in rats, from microPET imaging to excitotoxic lesions and multielectrode recordings across anterior dorsolateral (aDLS), posterior ventral lateral (pVLS)and dorsomedial (DMS) striatum.

The main conclusions are that the aDLS promotes stimulus-response association and suppresses response-outcome associations. The pVLS is engaged in the formation and maintenance of the stimulus-response association. There is a lot of work done and some interesting findings however, the manuscript can be improved by clarifying the presentation and reasoning. The inclusion of important controls will enhance the rigor of the data interpretation and conclusions.

Comments on revisions:

The authors have made important revisions to the manuscript and it has improved in clarity. They also added several figures in the rebuttal letter to answer questions by the reviewers. I would ask that these figures are also made public as part of the authors' response or if not, included in the manuscript.

Reviewer #2 (Public review):

This valuable paper studies the problem of learning from feedback given by sources of varying credibility. The solid combination of experiment and computational modeling helps to pin down properties of learning, although some ambiguity remains in the interpretation of results.

Summary:

This paper studies the problem of learning from feedback given by sources of varying credibility. Two bandit-style experiments are conducted in which feedback is provided with uncertainty, but from known sources. Bayesian benchmarks are provided to assess normative facets of learning, and alternative credit assignment models are fit for comparison. Some aspects of normativity appear, in addition to deviations such as asymmetric updating from positive and negative outcomes.

Strengths:

The paper tackles an important topic, with a relatively clean cognitive perspective. The construction of the experiment enables the use of computational modeling. This helps to pinpoint quantitatively the properties of learning and formally evaluate their impact and importance. The analyses are generally sensible, and parameter recovery analyses help to provide some confidence in the model estimation and comparison.

Weaknesses:

(1) The approach in the paper overlaps somewhat with various papers, such as Diaconescu et al. (2014) and Schulz et al. (forthcoming), which also consider the Bayesian problem of learning and applying source credibility, in terms of theory and experiment. The authors should discuss how these papers are complementary, to better provide an integrative picture for readers.

Diaconescu, A. O., Mathys, C., Weber, L. A., Daunizeau, J., Kasper, L., Lomakina, E. I., ... & Stephan, K. E. (2014). Inferring the intentions of others by hierarchical Bayesian learning. PLoS computational biology, 10(9), e1003810.<br /> Schulz, L., Schulz, E., Bhui, R., & Dayan, P. Mechanisms of Mistrust: A Bayesian Account of Misinformation Learning. https://doi.org/10.31234/osf.io/8egxh

(2) It isn't completely clear what the "cross-fitting" procedure accomplishes. Can this be discussed further?

(3) The Credibility-CA model seems to fit the same as the free-credibility Bayesian model in the first experiment and barely better in the second experiment. Why not use a more standard model comparison metric like the Bayesian Information Criterion (BIC)? Even if there are advantages to the bootstrap method (which should be described if so), the BIC would help for comparability between papers.

(4) As suggested in the discussion, the updating based on random feedback could be due to the interleaving of trials. If one is used to learning from the source on most trials, the occasional random trial may be hard to resist updating from. The exact interleaving structure should also be clarified (I assume different sources were shown for each bandit pair). This would also relate to work on RL and working memory: Collins, A. G., & Frank, M. J. (2012). How much of reinforcement learning is working memory, not reinforcement learning? A behavioral, computational, and neurogenetic analysis. European Journal of Neuroscience, 35(7), 1024-1035.

(5) Why does the choice-repetition regression include "only trials for which the last same-pair trial featured the 3-star agent and in which the context trial featured a different bandit pair"? This could be stated more plainly.

(6) Why apply the "Truth-CA" model and not the Bayesian variant that it was motivated by?

(7) "Overall, the results from this study support the exact same conclusions (See SI section 1.2) but with one difference. In the discovery study, we found no evidence for learning based on 50%-credibility feedback when examining either the feedback effect on choice repetition or CA in the credibility-CA model (SI 1.2.3)" - this seems like a very salient difference, when the paper reports the feedback effect as a primary finding of interest, though I understand there remains a valence-based difference.

(8) "Participants were instructed that this feedback would be "a lie 50% of the time but were not explicitly told that this meant it was random and should therefore be disregarded." - I agree that this is a possible explanation for updating from the random source. It is a meaningful caveat.

(9) "Future studies should investigate conditions that enhance an ability to discard disinformation, such as providing explicit instructions to ignore misleading feedback, manipulations that increase the time available for evaluating information, or interventions that strengthen source memory." - there is work on some of this in the misinformation literature that should be cited, such as the "continued influence effect". For example: Johnson, H. M., & Seifert, C. M. (1994). Sources of the continued influence effect: When misinformation in memory affects later inferences. Journal of experimental psychology: Learning, memory, and cognition, 20(6), 1420.

(10) Are the authors arguing that choice-confirmation bias may be at play? Work on choice-confirmation bias generally includes counterfactual feedback, which is not present here.

Reviewer #1 (Public review):

Summary:

Parise presents another instantiation of the Multisensory Correlation Detector model that can now accept stimulus-level inputs. This is a valuable development as it removes researcher involvement in the characterization/labeling of features and allows analysis of complex stimuli with a high degree of nuance that was previously unconsidered (i.e., spatial/spectral distributions across time). The author demonstrates the power of the model by fitting data from dozens of previous experiments, including multiple species, tasks, behavioral modalities, and pharmacological interventions.

Strengths:

One of the model's biggest strengths, in my opinion, is its ability to extract complex spatiotemporal co-relationships from multisensory stimuli. These relationships have typically been manually computed or assigned based on stimulus condition and often distilled to a single dimension or even a single number (e.g., "-50 ms asynchrony"). Thus, many models of multisensory integration depend heavily on human preprocessing of stimuli, and these models miss out on complex dynamics of stimuli; the lead modality distribution apparent in Figures 3b and c is provocative. I can imagine the model revealing interesting characteristics of the facial distribution of correlation during continuous audiovisual speech that have up to this point been largely described as "present" and almost solely focused on the lip area.

Another aspect that makes the MCD stand out among other models is the biological inspiration and generalizability across domains. The model was developed to describe a separate process - motion perception - and in a much simpler organism - Drosophila. It could then describe a very basic neural computation that has been conserved across phylogeny (which is further demonstrated in the ability to predict rat, primate, and human data) and brain area. This aspect makes the model likely able to account for much more than what has already been demonstrated with only a few tweaks akin to the modifications described in this and previous articles from Parise.

What allows this potential is that, as Parise and colleagues have demonstrated in those papers since our (re)introduction of the model in 2016, the MCD model is modular - both in its ability to interface with different inputs/outputs and its ability to chain MCD units in a way that can analyze spatial, spectral, or any other arbitrary dimension of a stimulus. This fact leaves wide open the possibilities for types of data, stimuli, and tasks a simplistic, neutrally inspired model can account for.

And so it's unsurprising (but impressive!) that Parise has demonstrated the model's ability here to account for such a wide range of empirical data from numerous tasks (synchrony/temporal order judgement, localization, detection, etc.) and behavior types (manual/saccade responses, gaze, etc.) using only the stimulus and a few free parameters. This ability is another of the model's main strengths that I think deserves some emphasis: it represents a kind of validation of those experiments, especially in the context of cross-experiment predictions (but see some criticism of that below).

Finally, what is perhaps most impressive to me is that the MCD (and the accompanying decision model) does all this with very few (sometimes zero) free parameters. This highlights the utility of the model and the plausibility of its underlying architecture, but also helps to prevent extreme overfitting if fit correctly (but see a related concern below).

Weaknesses:

There is an insufficient level of detail in the methods about model fitting. As a result, it's unclear what data the models were fitted and validated on. Were models fit individually or on average group data? Each condition separately? Is the model predictive of unseen data? Was the model cross-validated? Relatedly, the manuscript mentions a randomization test, but the shuffled data produces model responses that are still highly correlated to behavior despite shuffling. Could it be that any stimulus that varies in AV onset asynchrony can produce a psychometric curve that matches any other task with asynchrony judgements baked into the task? Does this mean all SJ or TOJ tasks produce correlated psychometric curves? Or more generally, is Pearson's correlation insensitive to subtle changes here, considering psychometric curves are typically sigmoidal? Curves can be non-overlapping and still highly correlated if one is, for example, scaled differently. Would an error term such as mean-squared or root mean-squared error be more sensitive to subtle changes in psychometric curves? Alternatively, perhaps if the models aren't cross-validated, the high correlation values are due to overfitting?

While the model boasts incredible versatility across tasks and stimulus configurations, fitting behavioral data well doesn't mean we've captured the underlying neural processes, and thus, we need to be careful when interpreting results. For example, the model produces temporal parameters fitting rat behavior that are 4x faster than when fitting human data. This difference in slope and a difference at the tails were interpreted as differences in perceptual sensitivity related to general processing speeds of the rat, presumably related to brain/body size differences. While rats no doubt have these differences in neural processing speed/integration windows, it seems reasonable that a lot of the differences in human and rat psychometric functions could be explained by the (over)training and motivation of rats to perform on every trial for a reward - increasing attention/sensitivity (slope) - and a tendency to make mistakes (compression evident at the tails). Was there an attempt to fit these data with a lapse parameter built into the decisional model as was done in Equation 21? Likewise, the fitted parameters for the pharmacological manipulations during the SJ task indicated differences in the decisional (but not the perceptual) process and the article makes the claim that "all pharmacologically-induced changes in audiovisual time perception" can be attributed to decisional processes "with no need to postulate changes in low-level temporal processing." However, those papers discuss actual sensory effects of pharmacological manipulation, with one specifically reporting changes to response timing. Moreover, and again contrary to the conclusions drawn from model fits to those data, both papers also report a change in psychometric slope/JND in the TOJ task after pharmacological manipulation, which would presumably be reflected in changes to the perceptual (but not the decisional) parameters.

The case for the utility of a stimulus-computable model is convincing (as I mentioned above), but its framing as mission-critical for understanding multisensory perception is overstated, I think. The line for what is "stimulus computable" is arbitrary and doesn't seem to be followed in the paper. A strict definition might realistically require inputs to be, e.g., the patterns of light and sound waves available to our eyes and ears, while an even more strict definition might (unrealistically) require those stimuli to be physically present and transduced by the model. A reasonable looser definition might allow an "abstract and low-dimensional representation of the stimulus, such as the stimulus envelope (which was used in the paper), to be an input. Ultimately, some preprocessing of a stimulus does not necessarily confound interpretations about (multi)sensory perception. And on the flip side, the stimulus-computable aspect doesn't necessarily give the model supreme insight into perception. For example, the MCD model was "confused" by the stimuli used in our 2018 paper (Nidiffer et al., 2018; Parise & Ernst, 2025). In each of our stimuli (including catch trials), the onset and offset drove strong AV temporal correlations across all stimulus conditions (including catch trials), but were irrelevant to participants performing an amplitude modulation detection task. The to-be-detected amplitude modulations, set at individual thresholds, were not a salient aspect of the physical stimulus, and thus only marginally affected stimulus correlations. The model was of course, able to fit our data by "ignoring" the on/offsets (i.e., requiring human intervention), again highlighting that the model is tapping into a very basic and ubiquitous computational principle of (multi)sensory perception. But it does reveal a limitation of such a stimulus-computable model: that it is (so far) strictly bottom-up.

The manuscript rightly chooses to focus a lot of the work on speech, fitting the MCD model to predict behavioral responses to speech. The range of findings from AV speech experiments that the MCD can account for is very convincing. Given the provided context that speech is "often claimed to be processed via dedicated mechanisms in the brain," a statement claiming a "first end-to-end account of multisensory perception," and findings that the MCD model can account for speech behaviors, it seems the reader is meant to infer that energetic correlation detection is a complete account of speech perception. I think this conclusion misses some facets of AV speech perception, such as integration of higher-order, non-redundant/correlated speech features (Campbell, 2008) and also the existence of top-down and predictive processing that aren't (yet!) explained by MCD. For example, one important benefit of AV speech is interactions on linguistic processes - how complementary sensitivity to articulatory features in the auditory and visual systems (Summerfield, 1987) allow constraint of linguistic processes (Peelle & Sommers, 2015; Tye-Murray et al., 2007).

References

Campbell, R. (2008). The processing of audio-visual speech: empirical and neural bases. Philosophical Transactions of the Royal Society B: Biological Sciences, 363(1493), 1001-1010. https://doi.org/10.1098/rstb.2007.2155<br /> Nidiffer, A. R., Diederich, A., Ramachandran, R., & Wallace, M. T. (2018). Multisensory perception reflects individual differences in processing temporal correlations. Scientific Reports 2018 8:1, 8(1), 1-15. https://doi.org/10.1038/s41598-018-32673-y<br /> Parise, C. V, & Ernst, M. O. (2025). Multisensory integration operates on correlated input from unimodal transient channels. ELife, 12. https://doi.org/10.7554/ELIFE.90841<br /> Peelle, J. E., & Sommers, M. S. (2015). Prediction and constraint in audiovisual speech perception. Cortex, 68, 169-181. https://doi.org/10.1016/j.cortex.2015.03.006<br /> Summerfield, Q. (1987). Some preliminaries to a comprehensive account of audio-visual speech perception. In B. Dodd & R. Campbell (Eds.), Hearing by Eye: The Psychology of Lip-Reading (pp. 3-51). Lawrence Erlbaum Associates.<br /> Tye-Murray, N., Sommers, M., & Spehar, B. (2007). Auditory and Visual Lexical Neighborhoods in Audiovisual Speech Perception: Trends in Amplification, 11(4), 233-241. https://doi.org/10.1177/1084713807307409

problemas de transito de líquidos o alimentos desde la boca a esófago

Proč se k tomuto přistoupilo?

Este capítulo da autora Gilles Salmon, descreve o modelo do processo ensino aprendizagem em contexto digital baseado num sistema em que o conhecimento é construído em 5 etapas em que o professor assume papeis de moderador e de suporte técnico ao longo deste processo. é da responsabilidade do professor construir e-atividades que permitam ao aluno construir o seu conhecimento ao mesmo tempo que o aluno deve dominar determinadas capacidades técnicas para que consiga ascendes nas diferentes etapas da construção de conhecimento. é referido também que é entre as etapas 3-5 que se dá desenvolvimento em termos de aquisição de competências e conhecimento, no entanto este processo é mais fluido se tiverem acesso as e-atividades das etapas 1 e 2.

two separate words

What have you already done to develop this competency?

Reviewer #1 (Public review):

Summary:

In this manuscript the Treisman and colleagues address the question of how protein phosphatase 1 (PP1) regulatory subunits (or PP1-interacting protein (PIPs)) confer specificity on the PP1 catalytic subunit which by itself possesses little substrate specificity. In prior work the authors showed that the PIP Phactrs confers specificity by remodelling a hydrophobic groove immediately adjacent to the PP1 catalytic site through residues within the RVxF- ø ø -R-W string of Phactrs. Specifically, the residues proximal and including the 'W' of the RVxF- ø ø -R-W string remodel the hydrophobic groove. Other residues the of the RVxF- ø ø -R-W string (i.e. the RVxF- ø ø -R) are not involved in this remodelling.

The authors suggest that the RVxF- ø ø -R-W string is a conserved feature of many PIPs including PNUTS, Neurabin/spinophilin and R15A. However from a sequence and structural perspective only the RVxF- ø ø -R- is conserved. The W is not conserved in most and in the R15A structure (PDB:7NZM) the Trp side chain points away from the hydrophobic channel - this could be a questionable interpretation due to model building into the low resolution cryo-EM map (4 A).

In this paper the authors convincingly show that Neurabin confers substrate specificity through interactions of its PDZ domain with the PDZ domain-binding motif (PBM) of 4E-BP. They show the PBM motif is required for Neurabin to increase PP1 activity towards 4E-BP and a synthetic peptide modelled on 4E-BP and also a synthetic peptide based on IRSp53 with a PBM added. The PBM of 4E-BP1 confers high affinity binding to the Neurabin PDZ domain. A crystal structure of a PP1-4E-BP1 fusion with Neurabin shows that the PBM of 4E-BP interacts with the PDZ domain of Neurabin. No interactions of 4E-BP and the catalytic site of PP1 are observed. Cell biology work showed that Neurabin-PP1 regulates the TOR signalling pathway by dephosphorylating 4E-BPs.

Strengths:

This work demonstrates convincingly using a variety of cell biology, proteomics, biophysics and structural biology that the PP1 interacting protein Neurabin confers specificity on PP1 through an interaction of its PDZ domain with a PDZ-binding motif of 4E-BP1 proteins. Remodelling of the hydrophobic groove of the PP1 catalytic subunit is not involved in Neurabin-dependent substrate specificity, in contrast to how Phactrs confers specificity on PP1. The active site of the Neurabin/PP1 complex does not recognise residues in the vicinity of the phospho-residue, thus allowing for multiple phospho-sites on 4E-BP to be dephosphorylated by Neurabin/PP1. This contrasts with substrate specificity conferred by the Phactrs PIP that confers specificity of Phactrs/PP1 towards its substrates in a sequence-specific context by remodelling the hydrophobic groove immediately adjacent to the catalytic. The structural and biochemical insights are used to explore the role of Neurabin/PP1 in dephosphorylation 4E-BPs in vivo, showing that Neurabin/PP1 regulates the TOR signalling pathway, specifically mTORC1-dependent translational control.

Weaknesses:

The only weakness is the suggestion that a conserved RVxF- ø ø -R-W string exists in PIPs. The 'W' is not conserved in sequence and 3-dimensions in most of the PIPs discussed in this manuscript. The lack of conservation of the W would be consistent with the finding based on multiple PP1-PIP structures that apart from Phactrs, no other PIP appears to remodel the PP1 hydrophobic channel.

Comments on revisions:

The authors have addressed my comments.

One aspect of the manuscript and response to reviewers is misleading regarding the statement: 'Like many PIPs, they interact with PP1 using the previously defined "RVxF", "ΦΦ", and "R" motifs (Choy et al, 2014).' This statement, and similar in the authors' response, implies that Choy et al discovered the "RVxF" and "ΦΦ" motifs. The Choy et al, 2014 paper reports the discovery of the "R" motif. The "RVxF" and "ΦΦ" motifs were discovered and reported in earlier papers not cited in the authors' manuscript. Perhaps the authors can correct this.

In Reconsidering Reparations, Philosopher Olufemu O. Taiwo lays out the history of the creation of the global racial empire.

“We are swimming on the aqueducts of history” - Olufemu O. Taiwo

grammar

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript assesses the utility of spatial image correlation spectroscopy (ICS) for measuring physiological responses to DNA damage. ICS is a long-established (~1993) method similar to fluorescence correlation spectroscopy, for deriving information about the fluorophore density that underlies the intensity distributions of images. The authors first provide a technical but fairly accessible background to the theory of ICS, then compare it with traditional spot-counting methods for its ability to analyze the characteristics of γH2AX staining. Based on the degree of aggregation (DA) value, the authors then survey other markers of DNA damage and uncover some novel findings, such as that RPA aggregation inversely tracks the sensitivity to PARP inhibitors of different cell lines.

The need for a more objective and standardized tool for analyzing DNA damage has long been felt in the field and the authors argue convincingly for this. The data in the manuscript are in general well-supported and of high quality, and show promise of being a robust alternative to traditional focus counting. However, there are a number of areas where I would suggest further controls and explanations to strengthen the authors' case for the robustness of their ICS method.

Strengths:

The spatial ICS method the authors describe and demonstrate is easy to perform and applicable to a wide variety of images. The DDR was well-chosen as an arena to showcase its utility due to its well-characterized dose-responsiveness and known variability between cell types. Their method should be readily useable by any cell biologist wanting to assess the degree of aggregation of fluorescent tags of interest.

Weaknesses:

The spatial ICS method, though of longstanding history, is not as intuitive or well-known as spot-based quantitation. While the Theory section gives a standard mathematical introduction, it is not as accessible as it could be. Additionally, the values of TNoP and DA shown in the Results are not discussed sufficiently with regard to their physical and physiological interpretation.

We agree that a major limitation in adaption of this approach is a deeper understanding of the theory and results. We have updated the theory section to include further discussion (Page 4 line 132)

The correlation of TNoP with γH2AX foci is high (Figure 2) and suggestive that the ICS method is suitable for measuring the strength of the DDR. The authors correctly mention that the number of spots found using traditional means can vary based on the parameters used for spot detection. They contrast this with their ICS detection method; however, the actual robustness of spatial ICS is not given equal consideration.

We found it difficult to give equal consideration of robustness to ICS. The major limitation of traditional approaches is proper selection of an intensity threshold that is necessary to define and separate foci from background intensity. However, ICS does not employ a threshold, therefore we could not test different thresholding applications in ICS as we did with traditional methods. In our view the absence of the need for a threshold is profoundly advantageous. The only inputs we employ in the ICS analysis are used to segment cell nuclei, yet these have no impact on the ICS calculation and are necessary for any analysis of the DDR.

Reviewer #2 (Public review):

Summary:

Immunostaining of chromatin-associated proteins and visualization of these factors through fluorescence microscopy is a powerful technique to study molecular processes such as DNA damage and repair, their timing, and their genetic dependencies. Nonetheless, it is well-established that this methodology (sometimes called "foci-ology") is subject to biases introduced during sample preparation, immunostaining, foci visualization, and scoring. This manuscript addresses several of the shortcomings associated with immunostaining by using image correlation spectroscopy (ICS) to quantify the recruitment of several DNA damage response-associated proteins following various types of DNA damage.

The study compares automated foci counting and fluorescence intensity to image correlation spectroscopy degree of aggregation study the recruitment of DNA repair proteins to chromatin following DNA damage. After validating image correlation spectroscopy as a reliable method to visualize the recruitment of γH2AX to chromatin following DNA damage in two separate cell lines, the study demonstrates that this new method can also be used to quantify RPA1 and Rad51 recruitment to chromatin following DNA damage. The study further shows that RPA1 signal as measured by this method correlates with cell sensitivity to Olaparib, a widely-used PARP inhibitor.

Strengths:

Multiple proof-of-concept experiments demonstrate that using image correlation spectroscopy degree of aggregation is typically more sensitive than foci counting or foci intensity as a measure of recruitment of a protein of interest to a site of DNA damage. The sensitivity of the SKOV3 and OVCA429 cell lines to MMS and the PARP inhibitors Olaparib and Veliparib as measured by cell viability in response to increasing amounts of each compound is a valuable correlate to the image correlation spectroscopy degree of aggregation measurements.

Weaknesses:

The subjectivity of foci counting has been well-recognized in the DNA repair field, and thus foci counts are usually interpreted relative to a set of technical and biological controls and across a meaningful time period. As such:

(1) A more detailed description of the numerous prior studies examining the immunostaining of proteins such as γH2AX, RAD51, and RPA is needed to give context to the findings presented herein.

We apologize for not providing enough detail. We have added further references and discussion. γH2AX foci counting, in particular, has been used in thousands of previous studies. (Pages 18 line 513 and 517)

(2) The benefits of adopting image correlation spectroscopy should be discussed in comparison to other methods, such as super-resolution microscopy, which may also offer enhanced sensitivity over traditional microscopy.

Thank you for raising this point. We have added this discussion (page 19 line 553). The limiting factor that ICS addresses is the partition coefficient of signal in a foci or cluster versus outside the cluster. Super-resolution will not necessarily improve this unless it is resolved down to single molecule counting. However, one would still need to evaluate how to define a cluster or foci in the background of non-cluster distribution.

(3) Additional controls demonstrating the specificity of their antibodies to detection of the proteins of interest should be added, or the appropriate citations validating these antibodies included.

We have added text stating that we only use validated antibodies (page 6 line 193). One thing to note is that we are measuring differences between treatment conditions, thus, if an antibody has non-specific labeling of proteins of cellular structures that do not change upon treatment, our approach would overcome this limitation.

Reviewer #3 (Public review):

Summary:

This paper described a new tool called "Image Correlation Spectroscopy; ICS) to detect clustering fluorescence signals such as foci in the nucleus (or any other cellular structures). The authors compared ICS DA (degree of aggregation) data with Imaris Spots data (and ImageJ Find Maxima data) and found a comparable result between the two analyses and that the ICS sometimes produced a better quantification than the Imaris. Moreover, the authors extended the application of ICS to detect cell-cycle stages by analyzing the DAPI image of cells. This is a useful tool without the subjective bias of researchers and provides novel quantitative values in cell biology.

Strengths:

The authors developed a new tool to detect and quantify the aggregates of immunofluorescent signals, which is a center of modern cell biology, such as the fields of DNA damage responses (DDR), including DNA repair. This new method could detect the "invisible" signal in cells without pre-extraction, which could prevent the effect of extracted materials on the pre-assembled ensembles, a target for the detection. This would be an alternative method for the quantification of fluorescent signals relative to conventional methods.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Major comments:

(1) The ICS theory section is essential and based on an excellent review from one of the authors. It would benefit greatly from a diagram showing where the quantities 𝒈(𝟎, 𝟎), 𝝎𝟎, and 𝒈inf come from in the 2D Gaussian fit, ideally for two cases where these quantities differ (i.e., how they correspond to different DA or TNoP values). In my opinion, this addition would greatly increase the manuscript's accessibility for DDR researchers. The citation of the review at the beginning would also be a plus.

We have added the review citation at the front of the theory section (page 3 line 87).We have highlighted where g(0,0), the most critical measurement for determination of TNoP and DA, derives from in Figure 2D. However, it is difficult to describe all the curve fit parameters in an image as they have some interdependency on each other and thus labeling one in a single image would not independently capture how they might be observed in a different curve fit.

(2) The TNoP measured in Figure 2 is a quantity about 2000-3000 times greater than the number of "traditionally detected" foci by both methods and the linear relations have very low Y intercepts. Can the authors comment explicitly on the physical interpretation of this number - are 2 to 3 thousand independent particles present within each "focus" detected by traditional means? If so, then what might one "particle" correspond to? (a single secondary antibody or fluorophore? a nucleosome?). In a similar vein, the X intercepts lie at around 25 foci, meaning that in images with fewer than that number of foci detected by ImageJ or Imaris, the ICS method should detect zero TNoP - is this in line with the authors' predictions? Is it possible that a first-order line fit is not the most appropriate relation between the two methods?

We apologize for our brevity here. Since DA proved to be a more useful metric we did not spend much effort discussing TNoP. TNoP correlates to the number of clustered particles, or non-diffuse fluorophores. TNoP is the inverse of the number of individual particles per nucleus, but the value is not a direct measure of foci. If a sample had no clustering at all, the number of individual particles would be at a maximum and the TNoP would be at a minimum. However, as fluorophores cluster, the number of individual particles (i.e. non-clustered fluorophores) decreases, which increases the TNoP value. Therefore, TNoP has a correlation to the number of foci detected through traditional measurements, as we found here. Yet, TNoP is a relative measurement and cannot be compared across different conditions. Similar to foci counting, TNoP is unable to factor the size or intensity of each cluster, thus DA is a more appropriate quantification of the DNA damage response.

The value of TNoP is dependent on the fitted point spread function and the area of the nucleus. The y=0 intercept of TNoP is defined by the optical setup and is not expected to necessarily go through x=0. Intriguingly, other groups have found that some foci identified through traditional measurements are actually clusters of multiple smaller foci, thus the concept of what a foci represents is difficult to interpret. Thus, here we aimed to show a general correlation of TNoP with foci count through traditional methods to reflect how ICS is similar to foci counting, then employed DA to overcome the limitations of defining a foci.

We have tried to clarify this in the text (page 8, line 266)

(3) Some suggestions to address the robustness of ICS:

For a given sample (i.e. one segmented nucleus), the calculation of DA and TNoP should be similar between different images of that same nucleus taken at different times, similar to how the number of traditionally detected foci would be fairly invariant. In particular, it should be shown that these values are not just scaling with the higher normalized intensity seen in stronger DDR responses. In the same vein, the linear relationship between TNoP and "foci" should not change even if the confocal settings are slightly different (i.e., higher/lower illumination intensity) as long as the condition stipulated by the authors in the Discussion holds ("ICS can be implemented on any fluorescence image as long as the square relative fluorescence intensity fluctuations are detectable above noise fluctuations."). To show, as the title states, that spatial ICS is a robust tool, it would be desirable to demonstrate this with a series of images of the same cell at the same or varying excitation intensities.

Thank you for your suggestions. Indeed, the calculation will be the same over sequential images of the same cell. Observations of dose dependent DA that does not correlate with intensity for RPA1 and RAD51 results (Fig. S5) directly demonstrates that DA does not just scale with intensity.

We would not expect the TNoP to change with confocal setting, however we show in Figure 1 that the number of foci does indeed change with intensity settings as captured by thresholds. Therefore, any interpretation of TNoP vs. foci count would be very difficult to make at different microscope settings. To ensure we are fairly comparing ICS to existing analysis we keep the settings the same and measure changes between conditions.

(4) More information is needed on how intensity normalization was performed. The Methods states "Measurements across experiments were normalized by the control in each dataset." The DMSO (0mM drug) plots all appear to have a mean of 1.0, so it appears the values for each set of control nuclei were divided by their own mean, and then the values for each set of experimental nuclei were divided by the mean value of all 3 controls as an aggregate; is this correct?

We apologize for not being more clear. Thank you for raising this point. We normalized data to a control from each experimental group. Thus, in figures 3,4 and 5 data were collected over multiple experiments with one control per experiment and each treatment condition included in each experiment. Therefore, we normalized each result to the corresponding control from that imaging session. However, in Figure 8 we ran experiments at much higher throughput with multiple controls per experiment, thus the data were normalized to the overall average of the controls, which is why the control averages are not all at a value of 1. We have clarified this in the text. (Page 7 line 218).

(5) Some more information about the ICS analysis should be given if the full code is not provided - in particular, how the nucleus mask was implemented on the "signal" channel (were the edges abruptly set to zero or was a window function introduced to avoid edge effects in the discrete FFT?

Thank you for raising this point. We have added the code to GitHub - github.com/ dubachLab/ics. The signal region was established by simply applying the nuclear mask from the DAPI channel to the IF channel. Each region is padded with average intensity value at the edges for 2x the dimensions of the ROI to remove edge effects in the FFT.

Minor comments:

(1) Figure 3, 4, 5: I think it would aid figure readability if channels were labeled in the images themselves, not just in the legend.

Thank you for the suggestion, we tried doing this and struggle to fit a label with the layout of the images. We were also concerned about interpretation of data in each column and the potential to assign data to each figure if they were so prominently labeled.

(2) Supplemental Figures are mislabeled; the order given in the legends is S1, S2, S3, S2, S3. S4 is called out in the main text where it should be S5.

Thank you for catching this error. We have made the necessary corrections. S4 contains data on cellular response to the drugs, while S5 contains intensity data in response to MMS.

(3) It should be stated for each Figure what kind of microscopy was performed - I assume that it is confocal for everything except when widefield is explicitly stated, but for clarity please add this information.

Indeed, this is correct, we have indicated which microscopy was used for each figure.

(4) The MATLAB code and full (uncropped) Western blots should be provided as supplemental data if possible.

We have included a GitHub link for the code and un-cropped western blots.

(5) The p values from significance tests should indicate whether multiple comparisons correction was necessary (if suggested by Prism) and performed.

Apologies for a lack of clarity but this was not necessary, significance was calculated vs. the next lower dose (e.g. 10 micromolar vs. 1 micromolar). We have clarified this in the methods (page 7 line 221).

Reviewer #2 (Recommendations for the authors):

Major points:

In addition to the weaknesses noted above, to encourage widespread adoption of this method, the authors should make the tools that they used for their analysis publicly available. In a few instances (e.g., compare Figures 3J and 3L), other methods outperform DA. It would be meaningful to discuss when especially DA may be a better measure than others (such as intensity or number of foci).

We have made code available on Github. We expect results, such as those in Figures 3J and 3L where intensity is significantly higher at the highest concentration but DA is not are reflective of the underlying biology and this may be interpreted differently under different experimental conditions. Imaris spots (Fig. 3K) also does not capture a significant increase at the highest dose of olaparib, suggesting that intensity may raise but it doesn’t not generate more foci. These results are likely highly dependent on the mechanism of olaparib at such a high concentration and the DDR response. We are hesitant to draw biological conclusions from these results and instead would like to highlight the capacity of ICS to evaluate the DDR, therefore we don’t want to make any broad comments about different applications.

Minor points:

(1) Pg. 12: "We used MMS to induce DNA damage in SKOV3 and OVCA429 cells. As expected, normalized intensity for RPA1 and RAD51 values (Figure S5) did not display a dose dependence on MMS concentration."

Please provide a citation for the claim that RPA1 and RAD51 normalized intensities do not display a dose dependence on MMS concentration.

These were data that we generated. We were not expecting an intensity change as that would presumably require increased protein generation in response to MMS, compared to gH2AX where the phospho-specific H2AX is generated in the DDR.

(2) Pg. 12: "Similar to RPA1, RAD51 does not form distinguishable foci in the nuclei in cells without preextraction (Fig. 5)." Please provide a citation for this claim.

We did not do pre-extraction and our results don’t produce changes in distinguishable foci. We provided citations discussing how, without pre extraction, foci formation for these proteins is not obvious (REF 38 and 39).

(3) I noted that the authors cite one paper [38] apparently showing that RPA and Rad51 do not always form foci, however, this is in the C. elegans germline in response to micro irradiation, therefore I am not sure that it is applicable to human cells.

We apologize for referencing a paper on C elegans. Most papers looking at RPA and RAD51 in the DDR use pre-extraction as it seems necessary to observe foci. Therefore, there are not as many papers, that we could find, that do not use pre-extraction. Reference 39 is in Hela cells.

Reviewer #3 (Recommendations for the authors):

Major points:

(1) Page 8, the second paragraph: In the Result section, it is better to describe how the authors carried out immuno-staining (without pre-extract subtraction) and ICS briefly, although the method is described in detail in the Method section.

Thank you for the suggestion, we have added this description (page 8, line 259)

(2) In Figure 5K-P: The authors analyzed "invisible" RAD51 foci on the image (Fig. 5L, M, O, and P) without pre-extraction. As a control experiment, it is useful to check whether pre-extraction would provide "visible" RAD51 foci and to examine the similar MMS concentration dependency shown in Figure 5R (or 5T). This would strengthen the power of the ICS analysis.

Thank you for the suggestion. In our hands, pre-extraction is extremely subjective. We have tried performing pre-extraction but find highly variable results depending on conditions. Therefore, we did not include any pre-extraction here. We expect that performing these experiments may or may not agree with results in Figure 5 largely because we are unable to achieve repeatable pre-extraction foci counting.

(3) Figure 6D (and 6C) looks very interesting. It would be important to show the interpretation of this correlation shown in the graph. Although the authors argued that ICS analysis results shown in the graph could provide new insight into the DDR (page 14, last line 5), as shown in another part, it is important to carry out the same analysis by using Imaris Spots. Moreover, it is interesting to apply the analysis to RAD51 foci (shown in Figure 5), given that the PARPi effect is enhanced in the absence of RAD51mediated recombination.

We completely agree that this analysis may generate interesting results to help interpret the DDR response to PARP inhibition. These experiments are part of an ongoing follow up study where we extend the use of ICS to other parts of the DDR and investigate protein clustering across several proteins with impact on PARPi response. Therefore, since the focus of this manuscript is introducing ICS as a tool to study the DDR, we believe that omitting those data here does not deter from the central points of the manuscript. We including results in Figure 6 because we wanted to show how ICS could impact DDR research. Furthermore, combined with our advances shown in Figures 7 and 8, we are currently working on adapting ICS to be high-throughput and much simpler than Imaris spots for handling large datasets needed to generate results like those in Figure 6.

Minor points:

(1) Figure 1I, blue arrows: These showed an area with a higher background. Because of a low magnification, it is very hard to see the difference from the other areas of the background. It is better to show a magnified image of the representative region with a higher background.

We hope that readers can see the higher intensity in the diffuse area. We attempted to construct a zoomed in area, but that either blocked a significant portion of the nonzoomed image or added complexity to the figure. We have noted that images in Figure S1 are larger and more obviously capture an increase in background intensity.

(2) Figure 2 legend, line 5, the same as "A)": This should be "B".

Here, the number of independent particle clusters is intended to be the same as A, the difference is that the independent particles are clusters in C and individual fluorophores in A.

(3) Page 9, the first paragraph, last line, foci formation, and foci composition: These should be "focus formation and focus composition".

We have changed this.

(4) Page 15, the first paragraph, line 5, palbociclib, camptothecin, or etoposide: please explain what kinds of the drugs are.

We have added that these drugs cause cells to stall at different cell cycle stages. Explaining the drugs would take considerable room in the text.

(5) Page 16, the first paragraph, line 1, bleomycin: Please explain what this drug is.

Similar to above, we have stated that this drug causes DNA damage, going into detail would take several sentences.

O wyraźnie nasyconych kolorach, drukowana na wodoodpornej tkaninie.

Namioty z serii Octa Pro zapewniają zadaszenie o powierzchni od 9 do 32 m². Model 4×8 m, dzięki swoim dużym rozmiarom, umożliwia jednoczesne zaparkowanie dwóch samochodów. To sprawia, że doskonale sprawdza się podczas rajdów – często pełni funkcję namiotu serwisowego.

Szukasz namiotu, który poradzi sobie nawet w najtrudniejszych warunkach? Octa Pro to nasz najmocniejszy model, sprawdzony w boju podczas Rajdu Dakar od 2018 roku. Dzięki wyjątkowo wytrzymałej konstrukcji i odporności na wiatr o prędkości do 100 km/h, doskonale sprawdza się podczas ekstremalnych wydarzeń – od rajdów, przez targi, po wymagające imprezy plenerowe.

Esto es un test

"Errors invite opportunities!" Absolutely agree with this as we don't master everything the first time and many practices need to happen. Making mistakes is essential in learning with positive feedback to help a student feel more comfortable to keep trying.

'Lord Kroak, why didn't you lift the siege four hundred years ago?' 'Eh, just didn't feel like it.' Literally what most Lizardmen lore sounds like.

Of course the elves.

That is actually hilarious. I'm imagining this Chaos moon also takes on the guise of the regular moon just to fuck with those astronomers even more. Maybe it has a leering gobbo face on one side that it constantly hides away until it's too late. Majora's Mask style.

What great policy, guys-- spend all your time obsessing over these artefacts, and once you get 'em, promptly stop thinking about them. These lizards are run by packrats.

example of lack of backing up the evidence

A Barcelona I l’AMB no em coincideix cap valor amb les dades del power point. Quines dades s’han fet servir per Barcelona i l’AMB. La Gemma m’ha dit que et pregunti: S’han fet servir desplaçaments residents a Barcelona o be son desplaçaments origen /destinació Barcelona de tots els residents del SIMMB?

Al SIMMB hi ha un valors que no coincideixen per molt poc. A Altres on posa 0,2% hauria de posar 0,3%. Potser aquesta diferencia sigui pels decimals?

A Barcelona I l’AMB no em coincideix cap valor amb les dades del power point. Quines dades s’han fet servir per Barcelona i l’AMB. La Gemma m’ha dit que et pregunti: S’han fet servir desplaçaments residents a Barcelona o be son desplaçaments origen /destinació Barcelona de tots els residents del SIMMB?

Al SIMMB hi ha alguns valors que no coincideixen per molt poc. Benzina on posa 46,4% hauria de posar 46,3%.A GNC GLP on posa 47.510 hauria de posar 47.509. Potser aquesta diferencia sigui pels decimals?

Mitjans de tranport

Aquí potser hauriem de posar gènere, entenc que explotem la variable gènere diria. Acabar-ho de confirmar amb la Gemma. Si ho fem així, s'hauria de canviar a tot arreu.

Haurem de decidir com posem els valors, si en milers i un decimal o bé com ho ho has fet aquí.

You already mention this later when you decribe the selection constraints

Taula Individus i desplaçaments segons tipus de població Barcelona – 2022

Desplaçaments

Poblacio general amb mobilitat columna absolutes on posa 5.512.674 hauria de posar 5.573.280

Poblacio general amb mobilitat columna % on posa 98,9% hauria de posar 96,9%

Total poblacio general columna absolutes on posa – hauria de posar 5.573.280

Total poblacio general columna % on posa – hauria de posar 96,9%

Poblacio amb 8, o més, desplaçaments laborals diaris* columna absolutes on posa 60.606 hauria de posar 178.893

Poblacio amb 8, o més, desplaçaments laborals diaris* columna % on posa 1,1% hauria de posar 3,1%

Total Barcelona columna absolutes on posa 5.573.280 hauria de posar 5.752.172

Taula Individus i desplaçaments segons tipus de població AMB – 2022

Desplaçaments

Poblacio general amb mobilitat columna absolutes on posa 11.001.252 hauria de posar 11.183.157

Poblacio general amb mobilitat columna % on posa 98,4% hauria de posar 94,3%

Total poblacio general columna absolutes on posa – hauria de posar 11.183.157

Total poblacio general columna % on posa – hauria de posar 94%

Poblacio amb 8, o més, desplaçaments laborals diaris* columna absolutes on posa 181.905 hauria de posar 674.143

Poblacio amb 8, o més, desplaçaments laborals diaris* columna % on posa 1,6% hauria de posar 5,7%

Total Barcelona columna absolutes on posa 11,183.158 hauria de posar 11.857.300

Taula Individus i desplaçaments segons tipus de població SIMMB – 2022

Desplaçaments

Poblacio general amb mobilitat columna absolutes on posa 18.839.157 hauria de posar 19.212.260

Poblacio general amb mobilitat columna % on posa 98,1% hauria de posar 94,3%

Total poblacio general columna absolutes on posa – hauria de posar 19.212.260

Total poblacio general columna % on posa – hauria de posar 94,3%

Poblacio amb 8, o més, desplaçaments laborals diaris* columna absolutes on posa 373.102 hauria de posar 1.152.993

Poblacio amb 8, o més, desplaçaments laborals diaris* columna % on posa 1,9% hauria de posar 5,7%

Total SIMMB columna absolutes on posa 19.212.260 hauria de posar 20.365.253

Taula Individus i desplaçaments segons tipus de població Barcelona – 2023

Desplaçaments

Poblacio general amb mobilitat columna absolutes on posa 5.872.759 hauria de posar 5.945.945

Poblacio general amb mobilitat columna % on posa 98,8% hauria de posar 96,3%

Total poblacio general columna absolutes on posa – hauria de posar 5.945.945

Total poblacio general columna % on posa – hauria de posar 96,3%

Poblacio amb 8, o més, desplaçaments laborals diaris* columna absolutes on posa 73.186 hauria de posar 231.191

Poblacio amb 8, o més, desplaçaments laborals diaris* columna % on posa 1,2% hauria de posar 3,7%

Total Barcelona columna absolutes on posa 5.945.945 hauria de posar 6.177.136

Taula Individus i desplaçaments segons tipus de població AMB – 2023

Desplaçaments

Poblacio general amb mobilitat columna absolutes on posa 11.693.378 hauria de posar 11.877.892

Poblacio general amb mobilitat columna % on posa 98,4% hauria de posar 93,7%

Total poblacio general columna absolutes on posa – hauria de posar 11.877.892

Total poblacio general columna % on posa – hauria de posar 94%

Poblacio amb 8, o més, desplaçaments laborals diaris* columna absolutes on posa 184.514 hauria de posar 802.111

Poblacio amb 8, o més, desplaçaments laborals diaris* columna % on posa 1,6% hauria de posar 6,3%

Total Barcelona columna absolutes on posa 11.877.892 hauria de posar 12.680.004

Taula Individus i desplaçaments segons tipus de població SIMMB – 2023

Individu

Poblacio general amb mobilitat columna absolutes on posa 4.565.451 hauria de posar 4.476.205

Poblacio general amb mobilitat columna % on posa 92,6% hauria de posar 90,8%

Total poblacio general columna absolutes on posa 4.927.771 hauria de posar 4.838.525

Desplaçaments

Poblacio general amb mobilitat columna absolutes on posa 19.887.259 hauria de posar 20.262.244

Poblacio general amb mobilitat columna % on posa 98,1% hauria de posar 93,3%

Total poblacio general columna absolutes on posa – hauria de posar 20.262.244

Total poblacio general columna % on posa – hauria de posar 93,3%

Poblacio amb 8, o més, desplaçaments laborals diaris* columna absolutes on posa 374.985 hauria de posar 1.463.620

Poblacio amb 8, o més, desplaçaments laborals diaris* columna % on posa 1,9% hauria de posar 6,7%

Total Barcelona columna absolutes on posa 20.262.244 hauria de posar 21.725.864

El grafic percentatge de població no mòbil 2023 Total SIMMB on posa 7,3% hauria de posar 7,4%

la dada de total AMB i total RMB no surt al power point i no tinc manera de saber si està bé o no.

Taula Individus i desplaçaments segons tipus de població Barcelona – 2022

Desplaçaments

Poblacio general amb mobilitat columna absolutes on posa 5.512.674 hauria de posar 5.573.280

Poblacio general amb mobilitat columna % on posa 98,9% hauria de posar 96,9%

Total poblacio general columna absolutes on posa – hauria de posar 5.573.280

Total poblacio general columna % on posa – hauria de posar 96,9%

Poblacio amb 8, o més, desplaçaments laborals diaris* columna absolutes on posa 60.606 hauria de posar 178.893

Poblacio amb 8, o més, desplaçaments laborals diaris* columna % on posa 1,1% hauria de posar 3,1%

Total Barcelona columna absolutes on posa 5.573.280 hauria de posar 5.752.172

Taula Individus i desplaçaments segons tipus de població AMB – 2022

Desplaçaments

Poblacio general amb mobilitat columna absolutes on posa 11.001.252 hauria de posar 11.183.157

Poblacio general amb mobilitat columna % on posa 98,4% hauria de posar 94,3%

Total poblacio general columna absolutes on posa – hauria de posar 11.183.157

Total poblacio general columna % on posa – hauria de posar 94%

Poblacio amb 8, o més, desplaçaments laborals diaris* columna absolutes on posa 181.905 hauria de posar 674.143

Poblacio amb 8, o més, desplaçaments laborals diaris* columna % on posa 1,6% hauria de posar 5,7%

Total Barcelona columna absolutes on posa 11,183.158 hauria de posar 11.857.300

Taula Individus i desplaçaments segons tipus de població SIMMB – 2022

Desplaçaments

Poblacio general amb mobilitat columna absolutes on posa 18.839.157 hauria de posar 19.212.260

Poblacio general amb mobilitat columna % on posa 98,1% hauria de posar 94,3%

Total poblacio general columna absolutes on posa – hauria de posar 19.212.260

Total poblacio general columna % on posa – hauria de posar 94,3%

Poblacio amb 8, o més, desplaçaments laborals diaris* columna absolutes on posa 373.102 hauria de posar 1.152.993

Poblacio amb 8, o més, desplaçaments laborals diaris* columna % on posa 1,9% hauria de posar 5,7%

Total SIMMB columna absolutes on posa 19.212.260 hauria de posar 20.365.253

Taula Individus i desplaçaments segons tipus de població Barcelona – 2023

Desplaçaments

Poblacio general amb mobilitat columna absolutes on posa 5.872.759 hauria de posar 5.945.945

Poblacio general amb mobilitat columna % on posa 98,8% hauria de posar 96,3%

Total poblacio general columna absolutes on posa – hauria de posar 5.945.945

Total poblacio general columna % on posa – hauria de posar 96,3%

Poblacio amb 8, o més, desplaçaments laborals diaris* columna absolutes on posa 73.186 hauria de posar 231.191

Poblacio amb 8, o més, desplaçaments laborals diaris* columna % on posa 1,2% hauria de posar 3,7%

Total Barcelona columna absolutes on posa 5.945.945 hauria de posar 6.177.136

Taula Individus i desplaçaments segons tipus de població AMB – 2023

Desplaçaments

Poblacio general amb mobilitat columna absolutes on posa 11.693.378 hauria de posar 11.877.892

Poblacio general amb mobilitat columna % on posa 98,4% hauria de posar 93,7%

Total poblacio general columna absolutes on posa – hauria de posar 11.877.892

Total poblacio general columna % on posa – hauria de posar 94%

Poblacio amb 8, o més, desplaçaments laborals diaris* columna absolutes on posa 184.514 hauria de posar 802.111

Poblacio amb 8, o més, desplaçaments laborals diaris* columna % on posa 1,6% hauria de posar 6,3%

Total Barcelona columna absolutes on posa 11.877.892 hauria de posar 12.680.004

Taula Individus i desplaçaments segons tipus de població SIMMB – 2023

Individu

Poblacio general amb mobilitat columna absolutes on posa 4.565.451 hauria de posar 4.476.205

Poblacio general amb mobilitat columna % on posa 92,6% hauria de posar 90,8%

Total poblacio general columna absolutes on posa 4.927.771 hauria de posar 4.838.525

Desplaçaments

Poblacio general amb mobilitat columna absolutes on posa 19.887.259 hauria de posar 20.262.244

Poblacio general amb mobilitat columna % on posa 98,1% hauria de posar 93,3%

Total poblacio general columna absolutes on posa – hauria de posar 20.262.244

Total poblacio general columna % on posa – hauria de posar 93,3%

Poblacio amb 8, o més, desplaçaments laborals diaris* columna absolutes on posa 374.985 hauria de posar 1.463.620

Poblacio amb 8, o més, desplaçaments laborals diaris* columna % on posa 1,9% hauria de posar 6,7%

Total Barcelona columna absolutes on posa 20.262.244 hauria de posar 21.725.864

El grafic percentatge de població no mòbil 2023 Total SIMMB on posa 7,3% hauria de posar 7,4%

la dada de total AMB i total RMB no surt al power point i no tinc manera de saber si està bé o no.

Mensaje booleano es falso O es verdadero

He condoms the holiday as fraud from the perspecitve of the enlaved, turing patriotic symbols into evidence of hypocrisy

CLT

Art. 884 - Garantida a execução ou penhorados os bens, terá o executado 5 (cinco) dias para apresentar embargos, cabendo igual prazo ao exeqüente para impugnação.

• § 1º - A matéria de defesa será restrita às alegações de cumprimento da decisão ou do acordo, quitação ou prescrição da divida.

§ 2º - Se na defesa tiverem sido arroladas testemunhas, poderá o Juiz ou o Presidente do Tribunal, caso julgue necessários seus depoimentos, marcar audiência para a produção das provas, a qual deverá realizar-se dentro de 5 (cinco) dias.

§ 3º - Somente nos embargos à penhora poderá o executado impugnar a sentença de liquidação, cabendo ao exeqüente igual direito e no mesmo prazo. - § 4º Julgar-se-ão na mesma sentença os embargos e as impugnações à liquidação apresentadas pelos credores trabalhista e previdenciário.

• ADI 5348
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. CÁRMEN LÚCIA
• Julgamento: 11/11/2019
• Publicação: 28/11/2019

Ementa

AÇÃO DIRETA DE INCONSTITUCIONALIDADE. ART. 1º-F DA LEI N. 9.494/1997, ALTERADO PELA LEI N. 11.960/2009. ÍNDICE DE REMUNERAÇÃO DA CADERNETA DE POUPANÇA COMO CRITÉRIO DE CORREÇÃO MONETÁRIA EM CONDENAÇÕES DA FAZENDA PÚBLICA. INCONSTITUCIONALIDADE. - 1. Este Supremo Tribunal declarou inconstitucional o índice de remuneração da caderneta de poupança como critério de correção monetária em condenações judiciais da Fazenda Pública ao decidir o Recurso Extraordinário n. 870.947, com repercussão geral (Tema 810). - 2. Assentou-se que a norma do art. 1º-F da Lei n. 9.494/1997, pela qual se estabelece a aplicação dos índices oficiais de remuneração da caderneta de poupança para atualização monetária nas condenações da Fazenda Pública, configura restrição desproporcional ao direito fundamental de propriedade. - 3. Ação direta de inconstitucionalidade julgada procedente.

DIREITO CONSTITUCIONAL. REGIME DE ATUALIZAÇÃO MONETÁRIA E JUROS MORATÓRIOS INCIDENTE SOBRE CONDENAÇÕES JUDICIAIS DA FAZENDA PÚBLICA. ART. 1º-F DA LEI Nº 9.494/97 COM A REDAÇÃO DADA PELA LEI Nº 11.960/09. IMPOSSIBILIDADE JURÍDICA DA UTILIZAÇÃO DO ÍNDICE DE REMUNERAÇÃO DA CADERNETA DE POUPANÇA COMO CRITÉRIO DE CORREÇÃO MONETÁRIA. VIOLAÇÃO AO DIREITO FUNDAMENTAL DE PROPRIEDADE (CRFB, ART. 5º, XXII). INADEQUAÇÃO MANIFESTA ENTRE MEIOS E FINS. INCONSTITUCIONALIDADE DA UTILIZAÇÃO DO RENDIMENTO DA CADERNETA DE POUPANÇA COMO ÍNDICE DEFINIDOR DOS JUROS MORATÓRIOS DE CONDENAÇÕES IMPOSTAS À FAZENDA PÚBLICA, QUANDO ORIUNDAS DE RELAÇÕES JURÍDICO-TRIBUTÁRIAS. DISCRIMINAÇÃO ARBITRÁRIA E VIOLAÇÃO À ISONOMIA ENTRE DEVEDOR PÚBLICO E DEVEDOR PRIVADO (CRFB, ART. 5º, CAPUT). RECURSO EXTRAORDINÁRIO PARCIALMENTE PROVIDO. - 1. O princípio constitucional da isonomia (CRFB, art. 5º, caput), no seu núcleo essencial, revela que o art. 1º-F da Lei nº 9.494/97, com a redação dada pela Lei nº 11.960/09, na parte em que disciplina os juros moratórios aplicáveis a condenações da Fazenda Pública, é inconstitucional ao incidir sobre débitos oriundos de relação jurídico-tributária, os quais devem observar os mesmos juros de mora pelos quais a Fazenda Pública remunera seu crédito; nas hipóteses de relação jurídica diversa da tributária, a fixação dos juros moratórios segundo o índice de remuneração da caderneta de poupança é constitucional, permanecendo hígido, nesta extensão, o disposto legal supramencionado. - 2. O direito fundamental de propriedade (CRFB, art. 5º, XXII) repugna o disposto no art. 1º-F da Lei nº 9.494/97, com a redação dada pela Lei nº 11.960/09, porquanto a atualização monetária das condenações impostas à Fazenda Pública segundo a remuneração oficial da caderneta de poupança não se qualifica como medida adequada a capturar a variação de preços da economia, sendo inidônea a promover os fins a que se destina. - 3. A correção monetária tem como escopo preservar o poder aquisitivo da moeda diante da sua desvalorização nominal provocada pela inflação. É que a moeda fiduciária, enquanto instrumento de troca, só tem valor na medida em que capaz de ser transformada em bens e serviços. A inflação, por representar o aumento persistente e generalizado do nível de preços, distorce, no tempo, a correspondência entre valores real e nominal (cf. MANKIW, N.G. Macroeconomia. Rio de Janeiro, LTC 2010, p. 94; DORNBUSH, R.; FISCHER, S. e STARTZ, R. Macroeconomia. São Paulo: McGraw-Hill do Brasil, 2009, p. 10; BLANCHARD, O. Macroeconomia. São Paulo: Prentice Hall, 2006, p. 29). - 4. A correção monetária e a inflação, posto fenômenos econômicos conexos, exigem, por imperativo de adequação lógica, que os instrumentos destinados a realizar a primeira sejam capazes de capturar a segunda, razão pela qual os índices de correção monetária devem consubstanciar autênticos índices de preços. - 5. Recurso extraordinário parcialmente provido.

(RE 870.947, Tema 810, Relator(a): LUIZ FUX, Tribunal Pleno, julgado em 20-09-2017, ACÓRDÃO ELETRÔNICO REPERCUSSÃO GERAL - MÉRITO DJe-262 DIVULG 17-11-2017 PUBLIC 20-11-2017)

EMENTA RECURSO EXTRAORDINÁRIO COM REPERCUSSÃO GERAL. TEMA N. 1.170. CONSTITUCIONAL E PROCESSUAL CIVIL. CONDENAÇÕES JUDICIAIS DA FAZENDA PÚBLICA. RELAÇÃO JURÍDICA NÃO TRIBUTÁRIA. TÍTULO EXECUTIVO. TRÂNSITO EM JULGADO. JUROS DE MORA. PARÂMETROS. ALTERAÇÃO. POSSIBILIDADE. ART. 1º-F DA LEI N. 9.494/1997, COM A REDAÇÃO DADA PELA DE N. 11.960/2009. OBSERVÂNCIA IMEDIATA. CONSTITUCIONALIDADE. RE 870.947. TEMA N. 810 DA REPERCUSSÃO GERAL. AUSÊNCIA DE OFENSA À COISA JULGADA. - 1. A Lei n. 11.960, de 29 de junho de 2009, alterou a de n. 9.494, de 10 de setembro de 1997, e deu nova redação ao art. 1º-F, o qual passou a prever que, nas condenações impostas à Fazenda Pública, para fins de atualização monetária, remuneração do capital e compensação da mora, incidirão, de uma só vez, até o efetivo pagamento, os índices oficiais de remuneração básica e de juros aplicados à caderneta de poupança. - 2. A respeito das condenações oriundas de relação jurídica não tributária, o Supremo Tribunal Federal, ao apreciar o RE 870.947 (Tema n. 810/RG), ministro Luiz Fux, declarou a constitucionalidade do art. 1º-F da Lei n. 9.494/1997, na redação dada pela de n. 11.960/2009, concernente à fixação de juros moratórios segundo o índice de remuneração da caderneta de poupança. - 3. O trânsito em julgado de sentença que tenha fixado percentual de juros moratórios não impede a observância de alteração legislativa futura, como no caso, em que se requer a aplicação da Lei n. 11.960/2009. - 4. Inexiste ofensa à coisa julgada, uma vez não desconstituído o título judicial exequendo, mas apenas aplicada legislação superveniente cujos efeitos imediatos alcançam situações jurídicas pendentes, em consonância com o princípio tempus regit actum. - 5. Recurso extraordinário provido, para reformar o acórdão recorrido, a fim de que seja aplicado o índice de juros moratórios estabelecido pelo art. 1º-F da Lei n. 9.494/1997, na redação dada pela de n. 11.960/2009. 6. Proposta de tese: “É aplicável às condenações da Fazenda Pública envolvendo relações jurídicas não tributárias o índice de juros moratórios estabelecido no art. 1º-F da Lei n. 9.494/1997, na redação dada pela Lei n. 11.960/2009, a partir da vigência da referida legislação, mesmo havendo previsão diversa em título executivo judicial transitado em julgado.”

(RE 1317982,Tema 1.170, Relator(a): NUNES MARQUES, Tribunal Pleno, julgado em 12-12-2023, PROCESSO ELETRÔNICO REPERCUSSÃO GERAL - MÉRITO DJe-s/n DIVULG 19-12-2023 PUBLIC 08-01-2024)

Reviewer #1 (Public review):

This is a very elegant and convincing study. Using systematic screening of actin tail formation in two bacterial strains and employing a panel of CRISPR-CAS ko cell lines, the authors identify a novel dynamin-related GTPase GVIN, which forms an oligomeric coat around an intracellular Burkholderia strain. The bacterial O-antigen LPS layer is required for the formation of the GVIN coat, which disturbs the polar localization of the bacterial actin-polymerizing BimA protein.

I am not an expert in infection studies, but the experiments appear to be of high quality, the figures are well prepared, and clean and statistically significant results are provided. I have no criticism of the presented approaches.

The identification of a novel GBP1-independent pathway targeting intracellular bacteria is not only of fundamental importance for the immunity field but also of high interest to researchers in other areas, for example, evolutionary or structural biologists.

Reviewer #2 (Public review):

Summary:

The authors wanted to investigate how cells defend against intracellular pathogens, such as Shigella and Burkholderia species, that co-opt the host actin machinery to spread from cell-to-cell. Previous work has identified IFNg-inducible GTPase of the Guanylate Binding Protein (GBP) family in cytosolic defence against Gram-negative bacteria. By forming a coat around Shigella, human GBP1 suppresses actin-based motility by displacing IcsA, which is the actin-polymerising virulence factor present at bacterial poles. In addition, GBP1 recruits the cytosolic LPS-sensor, caspase-4, to the bacterial surface, which results in the removal of bacterial replicative niches via pyroptotic cell death. Here, they followed up their finding that GBP1 can reduce actin-based motility of Shigella in HeLa cells and, surprisingly, fails to do so during Burkholderia infection. In contrast, in T24 bladder epithelial cells, GBP1 is competent in blocking Burkholderia actin-tails. They therefore wanted to identify the GBP1-independent factor that blocks actin-based motility in IFNg-treated cells that is absent in HeLa cells.

Major strengths and weaknesses of the methods and results:

The authors report a second IFNg-dependent pathway involving the protein product of the gene GVIN1, which was previously thought to be a pseudogene. GVIN1 (GTPase, very large interferon inducible 1) is thus the first human member of this family of ~250 kDa putative GTPases to be demonstrated to be functional and have potential antimicrobial roles. The discovery that GVIN1 is indeed functional, forms coats on Burkholderia in an LPS O-antigen-dependent manner, and limits actin-dependent motility are the main strengths of this paper. The authors use CRISPR/Cas9-based knockouts in HeLa and T24 cells, and complement them to demonstrate that GBP1 and GVIN1 are both required to inhibit actin-based motility.

An appraisal of whether the authors achieved their aims and whether the results support their conclusions:

The authors achieved their main goals through well-planned experiments and unbiased screens. They succeeded in finding the factor that blocks actin-based motility independently of GBP1. This is driven by GVIN1, which coats bacteria and limits actin-tail formation by reducing the expression of BimA through currently unknown mechanisms. Further, they found that an O-antigen mutant can escape coating by GVIN1, indicating the requirement for these polysaccharides in GVIN1-dependent bacterial sensing. However, the authors have not investigated whether GVIN1, which has two GTPase-domains, does indeed have GTPase activity and whether GVIN1 and GBP1 together completely block cell-to-cell spread by Burkholderia and thereby restrict bacterial numbers over the infection time course. They also do not show whether GBP1 and GVIN1 target the same bacterial cell or different populations of bacteria.

A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

This work uncovers the antimicrobial actions of a member of yet another family of IFNg-induced GTPases, which potentially acts against other intracellular pathogens. GVIN1 appears to operate independently and in parallel to GBP1, pointing to the breadth and complexity of the IFNg-inducible GTPase families.

Reviewer #1 (Public review):

Summary:

Gruskin and colleagues use twin data from a movie-watching fMRI paradigm to show how genetic control of cortical function intersects with the processing of naturalistic audiovisual stimuli. They use hyperalignment to dissect heritability into the components that can be explained by local differences in cortical-functional topography and those that cannot. They show that heritability is strongest at slower-evolving neural time scales and is more evident in functional connectivity estimates than in response time series.

Strengths:

This is a very thorough paper that tackles this question from several different angles. I very much appreciate the use of hyperalignment to factor out topographic differences, and I found the relationship between heritability and neural time scales very interesting. The writing is clear, and the results are compelling.

Weaknesses:

The only "weaknesses" I identified were some points where I think the methods, interpretation, or visualization could be clarified.

(1) On page 16, the authors compare heritability in functional connectivity (FC) and response time series, and find that the heritability effect is larger in FC. In general, I agree with your diagnosis that this is in large part due to the fact that FC captures the covariance structure across parcels, whereas response time series only diverge in terms of univariate time-point-by-time-point differences. Another important factor here is that (within-subject) FC can be driven by intrinsic fluctuations that occur with idiosyncratic timing across subjects and are unrelated to the stimulus (whereas time-locked metrics like ISC and time-series differences cannot, by definition). This makes me wonder how this connectivity result would change if the authors used intersubject functional connectivity (ISFC) analysis to specifically isolate the stimulus-driven components of functional connectivity (Simony et al., 2016). This, to me, would provide a closer comparison to the ISC and response time series results, and could allow the authors to quantify how much of the heritability in FC is intrinsic versus stimulus-driven. I'm not asking that the authors actually perform this analysis, as I don't think it's critical for the message of the manuscript, but it could be an interesting future direction. As the authors discuss on page 17, I also suspect there's something fundamentally shared between response time series and connectivity as they relate to functional topography (Busch et al., 2021) that drives part of the heritability effect.

(2) The observation that regions with intermediate ISC have the largest differences between MZ, DZ, and UR is very interesting, but it's kind of hard to see in Figure 1B. Is there any other way to plot this that might make the effect more obvious? For example, I could imagine three scatter plots where the x- and y-axes are, e.g., MZ ISC and UR ISC, and each data point is a parcel. In this kind of plot, I would expect to see the middle values lifted visibly off the diagonal/unity line toward MZ. The authors could even color the data points according to networks, like in Figure 3C. (They also might not need to scale the ISC axis all the way to r = 1, which would make the differences more visible.)

(3) On page 9, if I understand correctly, the authors regress the vector of ISC values across parcels out of the vector of heritability values across parcels, and then plot the residual heritability values. Do they center the heritability values (or include some kind of intercept) in the process? I'm trying to understand why the heritability values go from all positive (Figure 2A) to roughly balanced between positive and negative (Figure 2B). Important question for me: How should we interpret negative values in this plot? Can the authors explain this explicitly in the text? (I also wonder if there's a more intuitive way to control for ISC. For example, instead of regressing out ISC at the parcel/map level, could they go into a single parcel and then regress the subject-level pairwise ISC values out when computing the heritability score?).

(4) On page 4 (line 155), the authors say "we shuffled dyad labels"- is this equivalent to shuffling rows and columns of the pairwise subject-by-subject matrix combined across groups? I'm trying to make sure their approach here is consistent with recommendations by Chen et al., 2016. Is this the same kind of shuffling used for the kinship matrix mentioned in line 189?

(5) I found panel A in Figure 4 to be a little bit misleading because their parcel-wise approach to hyperalignment won't actually resolve topographic idiosyncrasies across a large cortical distance like what's depicted in the illustration (at the scale of the parcels they are performing hyperalignment within). Maybe just move the green and purple brain areas a bit closer to each other so they could feasibly be "aligned" within a large parcel. Worth keeping in mind when writing that hyperalignment is also not actually going to yield a one-to-one mapping of functionally homologous voxels across individuals: it's effectively going to model any given voxel time series as a linear combination of time series across other voxels in the parcel.

(6) I believe the subjects watched all different movies across the two days, however, for a moment I was wondering "are Day 1 and Day 2 repetitions of the same movies?" Given that Day 1 and Day 2 are an organizational feature of several figures, it might be worth making this very explicit in the Methods and reminding the reader in the Results section.

References:

Busch, E. L., Slipski, L., Feilong, M., Guntupalli, J. S., di Oleggio Castello, M. V., Huckins, J. F., Nastase, S. A., Gobbini, M. I., Wager, T. D., & Haxby, J. V. (2021). Hybrid hyperalignment: a single high-dimensional model of shared information embedded in cortical patterns of response and functional connectivity. NeuroImage, 233, 117975. https://doi.org/10.1016/j.neuroimage.2021.117975

Chen, G., Shin, Y. W., Taylor, P. A., Glen, D. R., Reynolds, R. C., Israel, R. B., & Cox, R. W. (2016). Untangling the relatedness among correlations, part I: nonparametric approaches to inter-subject correlation analysis at the group level. NeuroImage, 142, 248-259. https://doi.org/10.1016/j.neuroimage.2016.05.023

Simony, E., Honey, C. J., Chen, J., Lositsky, O., Yeshurun, Y., Wiesel, A., & Hasson, U. (2016). Dynamic reconfiguration of the default mode network during narrative comprehension. Nature Communications, 7, 12141. https://doi.org/10.1038/ncomms12141

Me quedo la duda de si este término es así "Aginación" o es "Asignación" porque sería el valor que se le asigna a x para ser igual a tres.

Estas partes resultan bastante familiares, teniend en cuenta la gama de herramientas que han sido trabajadas en el marco de la clase, lo cuál tiene sentido teniendo en cuenta que de alguna forma todas las herramientas hacen parte de un ecosistema más grande

Suspensão considionada

Stawiasz na bezpieczeństwo? Wybierz trudnopalny poliester o gramaturze 275 g/m². Nasze poszycie spełnia normy PN-EN ISO 6940, 6941 oraz DIN4102-B1, co potwierdza jego odporność na ogień. Dodatkowo materiał jest dwukrotnie impregnowany, co zapewnia jego wodoodporność.

O wyraźnie nasyconych kolorach, drukowana na wodoodpornej tkaninie.

Dwie dodatkowe warstwy tkaniny chronią najwrażliwsze miejsca dachu przed przetarciem.

Los efectos pronociceptivos son aquellos que favorecen o aumentan la percepción del dolor.

Acá está el problema principal.

En hypothes.is no subís los PDF a ningún lado, podes resaltar cualquier PDF, ya sea online o local.

Limitarte a solo 5 PDFs o pagar premium va en contra de los principios que proponen.

Es curioso como decidió irse por objetos en vez de mensajes, pero tiene muchísimo más sentido. Solo lo refuerza si lo pensamos con los operadores boléanos, que son conjuntos que se basan en el mensaje. Se resume en seleccionar que menajes quieres y/o excluyes, los objetos en este caso, como dice, quedan como idea menor.

Lo valioso de esto es que ya no limitamos el mensaje en si mismo, sino que lo expandimos y lo pones en un rol donde, por si mismo, al ser un objeto, se comunica e interactúa con otros y consigo mismo de forma autónoma; este saber sobre su propio estado es una herramienta que debemos entender y saber aprovechar, pues, siguiendo la metáfora que hemos seguido con nuestra moderadora, una célula está en ese habitad de relacionen entre otras y ella misma, pero no define la integridad de todo el organismo. Los objetos y los mensajes funcionan por sí mismos, pero se comunican con los demás, sin dejar de saber su propio estado.

Como vimos en las charlas de Anjana Vakil, se puso desde metáforas/marco biológico y matemático como entendemos la computación, o cómo puede entenderse.

Es curioso como decidió irse por objetos en vez de mensajes, pero tiene muchísimo más sentido. Solo lo refuerza si lo pensamos con los operadores boléanos, que son conjuntos que se basan en el mensaje. Se resume en seleccionar que menajes quieres y/o excluyes, los objetos en este caso, como dice, quedan como idea menor.

Lo valioso de esto es que ya no limitamos el mensaje en si mismo, sino que lo expandimos y lo pones en un rol donde, por si mismo, al ser un objeto, se comunica e interactúa con otros y consigo mismo de forma autónoma; este saber sobre su propio estado es una herramienta que debemos entender y saber aprovechar, pues, siguiendo la metáfora que hemos seguido con nuestra moderadora, una célula está en ese habitad de relacionen entre otras y ella misma, pero no define la integridad de todo el organismo. Los objetos y los mensajes funcionan por sí mismos, pero se comunican con los demás, sin dejar de saber su propio estado.

While traveling to Honolulu in 1988, the author had the opportunity to tour the USS Missouri, the battleship on which Japan surrendered during World War II. Walking on the very deck where that historic occasion took place recalled memories of playing with a model of the ship as a kid, and recollections of General MacArthur's hopeful post surrender speeches and the complex world that ultimately came to be.

This section covers the historical context of the Ghost Dance movement among Western American Indian tribes, who participated in the Battle of Little Bighorn. It covers the spiritual goal of the movement to regain their land and way of life, and the terrified response of the U.S. government resulting in atrocities such as the Wounded Knee massacre.

just a good quote in general about how resistance in queer films is an essential theme. we should use queer film to empower those identities. not just make them conform to everything else and perpetuate the stereotypes that people already have of queer people. this is reistnace because it keeps these stereotypes OUT of place

Example?

I wonder if this pulls the data out of reserv-o-matic using data federation, or a DLO.

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Reply to the reviewers

Manuscript number____: RC-2024-02806R

Corresponding author(s): Hamed Jafar-Nejad, Carmen Paradas

Point-by-point description of the revisions

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

The manuscript by Cho et al uses conditional and inducible conditional mouse models to characterize the function of protein O-glucosyltransferase 1 (POGLUT1), known to cause a type of Limb Girdle Muscular Dystrophy (LGMD-R21), in skeletal muscle satellite cells, differentiation and regeneration. The Authors find that conditional deletion of POGLUT1 in the myogenic progenitors leads to postnatal muscle defects and lethality by postnatal day 30 or so. Muscle progenitors lacking POGLUT1 undergo reduced proliferation and accelerated differentiation, possibly leading to impairment in muscle regeneration. This is supported by an inducible conditional deletion of POGLUT1 in adult satellite cells. Finally, in vitro experiments suggest that POGLUT1 is required for NOTCH pathway activation in myogenic cells and that POGLUT1 could potentially glycosylate specific residues in NOTCH3.

• We thank the reviewer for the concise summary of the major findings of our manuscript and for her/his comments, which have helped us improve the manuscript.

  Major comments

1. What is the control used in Figure 1B and other panels? The genotype should be clearly specified instead of saying controls, in all the figures. It will be easier to interpret the data if densitometry of the western blots is provided, normalized to GAPDH levels. Also, is there some POGLUT1 protein remaining in the satellite cells in the cKOs? Data should be shown for an adult time point also (P30 or later), in addition to P0 and P4, to see whether Poglut1 levels are reduced in adult stages in the muscle and satellite cells in the cKOs.

• As described in the Methods section of the original submission, for all experiments involving Poglut1-cKO, siblings without Poglut1 deletion (Poglut1+/+, Poglut1flox/flox, or Pax7Cre; Poglut1+/+) were used as controls. To address the reviewer's comment, we added this information to the legend of all figures showing Poglut1-cKO

• In the revised version, the densitometry is provided for all western blot images (Figures 1B and 7A). We added western blot with anti-POGLUT1 on whole muscle extract from P21 animals (Figure 1B), which is the latest stage at which we consistently obtain Poglut1-cKO PAX7 staining and injury studies indicate that at P21 there is a significant reduction in the number of satellite cells. Therefore, isolation of sufficient satellite cells from later stages is not feasible.

  1. In Figure 1C, can the tibia weight be normalized to total body weight instead of tibia length and analyzed? Similarly, can the grip strength measurements in Figure 1G be normalized to total body weight and represented? Grip strength measurements in neonates is tricky; the Authors should clearly explain how this was done. The NMJ defects can be characterized better, especially since the Authors discuss about Agrin in detail.

• Normalizing tibialis anterior muscle weight to tibia bone length is commonly used and is especially useful when assessing muscle-specific growth or atrophy, as it provides a standardized measure to compare between different mice. To address the reviewer's comment, in the revised manuscript we have provided the tibialis anterior weight (not normalized), tibia length, and then the normalized TA weight (which was also shown in the first submission). Before normalization, the TA weight in cKO showed ~ 56% reduction compared to control (on average). The tibia length did not show a statistically significant difference between cKO and sibling control. After normalization to tibia length, the TA muscle weight still showed a substantial (55%) reduction in cKO mice compared to controls. These data indicate that a reduction in muscle size is the major if not the sole reason for the reduction observed in cKO body size.

• We reanalyzed the grip strength based on the reviewer's comment and found that when normalized to body weight, the grip strength is not significantly different between cKO and control animals (please see the following figure for the reviewer). Accordingly, we have modified the Results section to avoid any direct suggestion that the muscle function itself is impaired in the cKO animals. Specifically, instead of writing "... suggesting that the muscle function is impaired in the mutant mice" we wrote " ... suggesting reduced muscle strength in the mutant mice". Moreover, at the end of this paragraph, instead of referring to "a severe muscle weakness" we just describe the phenotype as "muscle weakness". Our goal from this paragraph is to suggest that since the animals have much smaller muscles, they are weaker and cannot eat properly; that's why switching to soft food somewhat improves their survival. The observed NMJ defects suggest that there might be functional muscle defects as well. However, to draw conclusions about the muscle function we will need additional experiments, which we believe are beyond the scope of the current manuscript. If the reviewer believes the normalized grip test should be added to the manuscript, we will be happy to do so as a supplementary figure.
• As stated in the original manuscript, grip test was done starting at P12. Following the Boston University Guideline (https://www.bu.edu/research/ethics-compliance/animal-subjects/animal-care/anesthesia/anesthesia-and-analgesia-neonatal-mice-and-rats-iacuc/; recommended by Jacksons Laboratory when working with neonatal mice), we consider P10 to be the end of the neonatal period in mice. This is also supported by the majority of the papers in literature. For example, in a paper entitled "Quantitative analysis of neonatal skeletal muscle functional improvement in the mouse" (PMID 18310108), the authors show that by P28, most anatomical and gene expression parameters of mouse skeletal muscle are close to adults, and that by P21, these parameters are closer to P28 than the early postnatal mice. Also, they show that the expression of neonatal myosin heavy chain is dramatically reduced between P7 and P14, with another 3-4-fold reduction between P14 and P21. Therefore, for muscle development, similar to many other contexts studied in mice, neonatal period ends between P7 and P14 (most likely P8-P10). Therefore, the grip tests have not been performed on neonatal mice. In the revised manuscript, we have clarified this issue in the Methods section.

• The NMJ defects were not the main focus of the manuscript and were performed as a standard characterization of this new mouse model. We agree that additional experiments can be performed to better characterize the NMJ defects in this model, but we believe those experiments are beyond the scope of the current manuscript. Also, given the new data and text added to the revised manuscript, we removed the section related to NMJ and agrin from the Discussion to reduce the manuscript length.

  1. What are the small myofibers seen at the corners of the larger myofibers at P21 in the cKOs in Figure 2E? What is the point that the Authors want to conclude from Figure 2C and D? Clearly, if there are fewer satellite cells, Pax7 transcript levels will decrease. In Figure 2D, how are the satellite cell samples normalized between control and cKOs; did they start with equal number of satellite cells or equal amount of satellite cell RNA between control and cKOs? In Figure 2F, representative images for cKOs should be shown? The conclusion from Figure 2F-G is unclear. Since the Authors claim that the weak laminin staining is resolved in the cKOs by P21, why is α-dystroglycan hypoglycosylation seen in the P21 muscle?

• The structures to which the reviewer is referring are blood vessels. As shown in the following figure generated for the reviewer, staining the muscle sections from control and cKO mice using the endothelial marker CD31 confirms the vascular nature of those structures.

• The decrease in PAX7+ cells was shown based on antibody staining, but this does not necessarily mean that Pax7 mRNA is similarly reduced. Figure 2C and 2D show that loss of Poglut1 in these cells affects Pax7 expression at the mRNA level. We started with equal amounts of RNA for Figure 2D and have added additional text to describe RNA isolation and RT-PCR in the Methods section of the revised manuscript.

• Representative images for cKOs are added to the revised Figure 2, as suggested.
• The conclusion from 2F-G is that a statistically significant reduction is specifically observed in those PAX7+ cells which co-express M-cadherin, which is another well-established satellite cell marker. The data also indicate that some PAX7+ cells persist in the mutant muscle, highlighting the importance of examining the ability of mutant muscle to repair muscle injury, as shown in Figure 4.

• The glycans on α-dystroglycan mediate its binding to laminin and other extracellular matrix proteins, but laminin expression level is not thought to regulate the degree of α-dystroglycan glycosylation. In other words, laminin staining intensity and the degree of α-dystroglycan are not causally related to each other. What we observed in P21 animals mirrors what we have reported for adult patient biopsies: normal level of extracellular matrix protein collagen VI associated with α-dystroglycan hypoglycosylation. It is worth mentioning that based on our previous report using primary LGMD-R21 patient myoblasts and C2C12 cells treated with a Notch inhibitor, reducing Notch signaling leads to α-dystroglycan hypoglycosylation, likely by altering the differentiation dynamics of the myoblasts (PMID: 27807076).

  1. Representative images should be shown as examples for all time points for both genotypes in Figure 3A. Figure 3H should be represented with statistically significant differences marked clearly. Have the Authors checked whether cell death contributes to the decrease in cultured satellite cells and Pax7+ cells in the cKOs in Figure 3F, G? Are the differences between the control and cKOs in fusion index and Myogenin expression (Figure 3J, K) statistically significant?

• Representative images are added in a new supplementary figure (Figure S3A) as requested by the reviewer.

• Thanks for pointing out the lack of statistics in Figure 3H. To address this point and a comment by reviewer 2, we removed those quantifications from the manuscript. Instead, we performed PAX7/MYOD/Ki67 co-staining on cultured cells and quantified the percentage of each cell state based on the expression of these three markers as a more accurate measure of quiescent versus cycling satellite cells, as well as progenitors and precursor cells. The revised quantification and statistical analysis is presented in the revised Figure 3H. This quantification is represented similarly to the data shown in Gattazzo et al 2020.
• Thanks for bringing up the issue of cell death. To address this point, we performed two rounds of anti-Caspase 3 staining on a limited number of myogenic progenitors that we were able to isolate from cKO and control animals during the revisions. Unfortunately, the stainings did not work. To ensure that we do not rule out the possibility of apoptosis without experimental data, we added the following sentence to the Results section: "These data suggest reduced proliferation of myogenic cells upon loss of Poglut1, although we cannot exclude that cell death also contributes to the reduced cell number."

• Thanks for pointing out the lack of statistics for these data. We have added additional independent samples to the data presented in 3J, K and found that the differences between the control and cKOs in fusion index and Myogenin expression are indeed statistically significant.

  1. Figure 4 has multiple problems in my opinion. First, P21 is too early a time point to be talking about regeneration, since satellite cells are just transitioning to become quiescent cells at this time (described in Lepper et al, Nature 2009). It is difficult to distinguish the regenerative and neonatal developmental roles of satellite cells in the experiment that the Authors have carried out. Another issue is that the Authors show a decrease in satellite cells in the cKOs; satellite cell function is clearly known to be required for regeneration. Therefore, there is not much novelty that the cKOs exhibit poor regenerative capability. The data shown in Figure 4B-E is more or less a repetition of what has been shown in Figures 2 and 3.

• After carefully reviewing publications including the one suggested by the reviewer, we found that multiple studies have performed muscle injury experiments at P21 or even younger ages to study regeneration capabilities. Also, please consider that many of our mutant mice are lethal around weaning age which was another reason for selecting P21 as our injury timepoint. To better explain the logic for this choice, we have added the following sentences to the revised manuscript (new text is underlined: "To address this question, we induced muscle injury in mutant and control mice by injecting cardiotoxin (CTX) into TA muscles at P21 (Fig. 4A), an age at which we consistently obtain Poglut1-cKO animals without any dietary changes (Fig. 1E). Importantly, in WT C57BL/6 mice, 51% of PAX7+ cells are reported to be in the quiescent state at P21 (Gattazzo et al., 2020) and the TA muscles of P21 mice exhibit a robust regenerative response to cardiotoxin-induced injury (Lepper et al., 2009)."

• While the reduction in satellite cells (SCs) in cKOs suggests impaired regeneration, it was essential to confirm this experimentally, as remaining SCs could still compensate. Therefore, it was necessary to assess whether the residual satellite cells could proliferate and contribute to muscle repair. Our results demonstrate that our mutant mice fail to repair muscle, providing evidence that the reduction in PAX7+ cells in Poglut1-cKO mice is functionally significant. The goal of these experiments was not to increase novelty; it was to increase experimental rigor and reproducibility so that we don't draw functional conclusions merely based on tissue staining.

• While Figures 2 and 3 examine muscle sections and myoblast cultures, Figure 4B-E presents isolated fibers in an ex vivo culture system. This approach allows us to assess satellite cell activation and proliferation in a different injury model. Figure 4 lets us confirm that the proliferation defect observed in the cKO is consistent across multiple experimental conditions. Additionally, we utilized two forms of muscle injury-in vivo and ex vivo-to comprehensively evaluate the function of PAX7+ cells in WT versus mutant muscles. The ex vivo findings from EDL fibers align with our cell culture experiments in Figure 3, further supporting the observed defect in muscle repair. We believe this approach will increase the likelihood of obtaining reproducible data.

  1. In Figure 5C, D, have the Authors checked the Pax7+ cell numbers between the controls and the i-cKOs? Is the difference seen in 1X TAM due to preexisting reduction in satellite cells in the i-cKOs? What is the explanation by the Authors for the large number of Tomato+ fibers seen in the 2X TAM and 3X TAM uninjured muscle (Figure 3C, E)? Figure 5E is difficult to comprehend and should be represented in a clearer manner.

• New PAX7 staining shows that most of the TOM+ cells (93% in i-cKO, 97% in control) co-express PAX7 (revised Figure 6D and 6E). If preexisting meant "before tamoxifen injection", a preexisting reduction in satellite cells should be highly unlikely, as the Pax7-Cre-ERT2 line has been used by multiple groups to study adult satellite cells, and we have not observed any abnormalities in the Poglut1[flox/flox] strain. Moreover, our control animals harbor both Pax7-Cre-ERT2 and the tdTomato transgenes. The observed difference in 1xTAM most likely reflects spontaneous activation and fusion of these cells following loss of Poglut1, as evidenced by the enhanced appearance of Tomato+ fibers in i-cKO muscles compared to control muscles. At 1xTAM, we can observe a modest increase in Tomato⁺ The reduction becomes more pronounced with repeated tamoxifen, consistent with progressive differentiation of the satellite cell pool and their fusion with myofibers.

• For Figure 5E (now Figure 6C), we made some changes to make the graph clearer and added a sentence to the figure legend to make it easier to follow.

  1. Densitometry for Figure 6A should be included, since N1 ICD levels seem quite variable in the controls also. POGLUT1 expression and shRNA knockdown efficiency should be shown in Figure 6B. What is the correlation between the NOTCH3 glycosylation with reduced NOTCH pathway activation and satellite cell function? This should be clearly discussed. Why was the NOTCH3 glycosylation assay done in HEK293 cells?

• In the revised version, we have included densitometry for Figure 6A (now Figure 7A).

• In the revised version, we show qRT-PCR data indicating that C2C12 cells stably expressing Poglut1 shRNA show ~ 75% decrease in Poglut1 mRNA levels compared to control cells (now Figure 7B), in agreement with our original report of these cells (PMID 21490058). We also performed qRT-PCR for Pax7 and found a significant reduction in Pax7 mRNA levels in Poglut1-knockdown C2C12 cells, further supporting their usage in these signaling assays.
• As discussed in detail in the Results and Discussion sections of the original submission, reports from our lab and others have provided strong evidence that glycosylation of NOTCH1 and NOTCH2 by POGLUT1 promotes signaling mediated by these receptors. Since it was not clear whether this phenomenon is ligand-specific, in the current manuscript we showed that both DLL1-madiated and JAG1-mediated signaling by NOTCH1 and NOTCH2 require the expression of POGLUT1 in the signal-receiving cells. Since NOTCH3 also plays a key role in muscle stem cell biology (albeit a not so well characterized one), we showed in the current manuscript that reducing POGLUT1 in the NOTCH3-expressing cell also leads to a significant reduction in signaling mediated by this receptor. Moreover, we found that NOTCH3 is broadly glycosylated by POGLUT1. In fact, during the revision, we succeeded in confirming four additional POGLUT1 target sites on the mouse NOTCH3 protein (added to the revised Figure 7D and Supplementary Figure S4). These data suggest that similar to NOTCH1 and NOTCH2, POGLUT1-medaited O-glucosylation of NOTCH3 is required for its signaling. To address the reviewer's comment, in the revised manuscript, we have added the following phrase to the Discussion to address the reviewer's comment: "..., strongly suggesting that O-glucosylation of NOTCH3 by POGLUT1 promotes its ligand-mediated activation."
• HEK293 cells express robust levels of POGLUT1 and have been used in previous studies for the characterization of POGLUT1-mediated glycosylation of its target proteins. Analysis of overexpressed target proteins in this cell line is used to establish assay conditions and glycopeptide identification parameters and thereby sets the stage to analyze glycosylation of endogenous protein (in this case, mouse NOTCH3) in specific cell types. We note that in cases examined so far, the glycosylation data obtained from overexpressed proteins in HEK293 or other commonly used mammalian cell lines recapitulate the glycosylation of endogenously expressed POGLUT1 targets very well (both in cell culture and in vivo; for example see PMID 27268051).

  Reviewer #1 (Significance (Required)):

  Based on my expertise as a muscle and stem cell biologist, the manuscript is not clearly thought through, with not many novel inferences that one can draw from the data provided. While the manuscript could be informative to muscle biologists and stem cell investigators, several additional experiments are required to better characterize the phenotypes and provide meaningful conclusions from the study. The role of POGLUT1 in the muscle could be of great interest, especially in light of its role in LGMD-R21, as described by some of the Authors previously. Several pieces of data provided in the current manuscript are disjointed, with few connecting links, such as the NMJ characterization, the NOTCH glycosylation data and the regeneration experiments done on P21 neonates. Better quantitation of data is required as well, as detailed below. Overall, the manuscript may be revised to address the specific comments and reconsidered at a suitable journal.

Response: We hope that our explanations, additional experiments, and changes to the text have helped address the concerns raised by the reviewer.

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

Poglut1 mutations have been identified in an autosomal recessive form of muscular dystrophy. Poglut1 encodes an O-glucosyltransferase that modifies Notch receptors and ligands but also has other substrates. In mice, null mutations in Poglut1 are embryonic lethal, which has previously precluded the analysis of Poglut1's role in muscle development and regeneration using murine models. To address this limitation, the authors generated a conditional Poglut1 allele and introduced mutations using Pax7Cre or Pax7CreERT2. They characterize the phenotypic consequences of these mutations and further demonstrate that signaling through the Notch1, Notch2, and Notch3 receptors requires Poglut1 using the C2C12 cell culture model.

Some aspects of the manuscript's description of muscle stem cell behavior and regeneration are not fully up to date. These points should be addressed before publication.

Specific points

1) Evidence Supporting Reduced Notch Signaling as the Cause of the Phenotype The comparison of muscle phenotypes observed in other Notch signaling mutations strongly supports the hypothesis that the phenotype is due to reduced Notch signaling. Relevant studies, such as those addressing development (PMID: 17194759, PMID: 17360543) and those focusing on adult muscle and regeneration (PMID: 21989910, PMID: 22069237, PMID: 22045613, PMID: 30862660) should be cited and discussed in the manuscript. Including these references will strengthen the argument and provide a broader context for the findings.

• We thank the reviewer for this comment. To address this point and a similar comment made by reviewer 3, we have modified the Introduction and Discussion in the revised manuscript. In the Introduction, we have added the following sentences and have cited all 6 references listed by the reviewer: "Loss of function studies for key components of the Notch signaling pathway including Rbpj and Dll1 indicated that disruption of this pathway during muscle development leads to premature differentiation and depletion of muscle progenitor cells (Schuster-Gossler et al., 2007; Vasyutina et al., 2007). In addition, multiple studies have shown that Notch signaling is required in adult mice to prevent the spontaneous or premature differentiation of satellite cells and to maintain a muscle stem cell pool capable of repairing muscle damage (Bjornson et al., 2012; Fukada et al., 2011; Lahmann et al., 2019; Mourikis et al., 2012)." In the third paragraph of the Discussion, we have highlighted the similarities between Poglut1-cKO and i-cKO phenotypes and the phenotypes observed in "animals with germline or conditional loss of various Notch pathway components (Bjornson et al., 2012; Fukada et al., 2011; Lahmann et al., 2019; Mourikis et al., 2012; Schuster-Gossler et al., 2007; Vasyutina et al., 2007)".

  2) Figure 1: NMJ Deficits - Pre- or Postsynaptic? The authors describe the mutant synaptic vesicles as showing a significantly reduced evoked neurotransmitter release (quantal content) compared to controls. This phrasing raises the question: are motor neurons mutated in these animals? It should be clarified why the synaptic vesicles are referred to as "mutant." To my knowledge, Pax7Cre does not recombine in motor neurons, and this discrepancy needs to be addressed. The text should be rephrased to accurately reflect the origins of the observed deficits.

• We are not aware of any reports on recombination in motor neurons by the Pax-Cre line used in our study and had implied this in the initial submission in the following sentence in Discussion: "Therefore, even if agrin is indeed glycosylated by POGLUT1, loss of Poglut1 with Pax7-Cre is not expected to affect the glycosylation of agrin expressed by motoneurons." [please note that given the new data and text added to the revised manuscript, we removed the paragraph related to NMJ and agrin from the Discussion to reduce the manuscript length.] We agree that referring to synaptic vesicles as "mutant" can be misleading and have revised "mutant synaptic vesicles" to "in the NMJs of Poglut1-cKO LAL muscles". To address the discrepancy, we have added the following sentence to the Results section after describing a reduction in quantal content: "Since the Pax7-Cre strain used in our study is not reported to induce recombination in motor neurons (Murdoch et al., 2012), this presynaptic NMJ defect might be secondary to defects in postsynaptic NMJ abnormalities."

  3) Quantification of Myofibers with Internal Nuclei The statement, "We first quantified the ratio of myofibers with internal nuclei, which is an indication for recent fusion of myoblasts to myofibers," is not entirely accurate. Recent studies, such as PMID: 38569550, provide a more nuanced explanation of this phenomenon. The manuscript should reference this study and update the description to ensure it accurately reflects the current understanding of myofiber internal nuclei as markers of muscle pathology or regeneration.

• We thank the reviewer for bringing this paper to our attention. In the revised manuscript, we have changed the above sentence to make it aligned with the observations of the paper mentioned by the reviewer: "We first quantified the ratio of myofibers with internal nuclei, recently reported to specifically result from the fusion of embryonic myogenic cells during limb myogenesis and also driven by myocyte-myocyte fusion in the first phase of postnatal muscle regeneration (Collins et al., 2024)." We also added the following sentence, which we believe makes our interpretation more accurate in light of Collins et al, 2024: "These data suggest enhanced differentiation of Poglut1-deficient progenitors into myocytes followed by continued myocyte-myocyte fusion and/or a delay in the peripheral migration of internal nuclei which normally occurs in the perinatal period (Collins et al., 2024)." This way, in the revised manuscript we do not link the presence of internal nuclei with recent fusion of myoblasts anymore. Finally, we also referred to Collins et al 2024 when describing the appearance of internal nuclei in injured muscles 5 days after injury (Figure 4A).

  4) Figure 3H: Cell Identification in Culture

  The use of PAX7+ MYOD− to identify quiescent cells in culture is not accurate. Instead, PAX7+ Ki67− should be used for this purpose. Similarly, PAX7− MYOD+ does not reliably identify differentiating cells. Instead, staining for MYOG should be used to ensure accuracy. The figure and accompanying text should be adjusted accordingly to reflect these updates.

• To address this issue and a comment by reviewer 1, we performed PAX7/MYOD/Ki67 co-staining on cultured cells and quantified the percentage of each cell state based on the expression of these three markers as a more accurate measure of quiescent versus cycling satellite cells, as well as progenitors and precursor cells. The revised quantification and statistical analysis is presented in the revised Figure 3H. This quantification is represented similarly to the data shown in Gattazzo et al 2020. Based on the reviewer's comment and the results of these quantification, we have modified the Results section as follows (new text is underlined): "These observations indicate that loss of Poglut1 impairs the ability of muscle stem cells to remain in a quiescent state and suggest that the mutant myogenic progenitors might undergo premature differentiation." We then present the fusion index and myogenin staining data to conclude enhanced differentiation.

  5) Description of Satellite Cell Quiescence

  The statement, "About 2-3 weeks after birth, some of the PAX7+ cells generated by active proliferation of embryonic myogenic progenitors enter a quiescent state to generate adult satellite cells," is not entirely correct. The description should be updated based on the findings in PMID: 32763161, which provide a more accurate account of the transition of PAX7+ cells to quiescence and their role in generating adult satellite cells.

• We thank the reviewer for bringing this paper to our attention. To address this issue and one of the concerns raised by Reviewer 1, we have made the following changes to the corresponding paragraph in the revised version: (A) We removed the statement highlighted by the reviewer. (B) We provided better justification for using P21 mice to assess muscle regeneration by quiescent satellite cells by adding the underlined sentences to this section: "To address this question, we induced muscle injury in mutant and control mice by injecting cardiotoxin (CTX) into TA muscles at P21 (Fig. 4A), an age at which we consistently obtain Poglut1-cKO animals without any dietary changes (Fig. 1E). Importantly, 51% of PAX7+ cells are reported to be in the quiescent state at P21 (Gattazzo et al., 2020) and the TA muscles of P21 mice exhibit a robust regenerative response to cardiotoxin-induced injury (Lepper et al., 2009)." We note that Gattazzo et al 2020 is PMID: 32763161, the paper with a more accurate account of satellite cell quiescence mentioned by the reviewer.

  6) Figure 5: Loss of Quiescence in Satellite Cells

  A hallmark phenotype of mutations in Notch signaling genesis the loss of quiescence in satellite cells when the mutation is introduced in the adult,. The authors should include data on Ki67 and MyoD expression in PAX7+ cells of mice with the Poglut1 mutation introduced in the adult by an analysis of the uninjured muscle. This would provide insight into the maintenance of quiescence in the mutant satellite cells.

• Based on the reviewer's recommendation, we aimed to assess Ki67 and MyoD expression in PAX7⁺ cells of adult inducible-cKO (i-cKO) mice in the uninjured muscle. However, due to technical limitations including antibody species incompatibility and the number of available fluorescence channels, we were unable to perform simultaneous triple staining combined with tdTomato visualization on the same tissue sections. Instead, we utilized the Tomato reporter as a marker for PAX7⁺ cells, based on new data indicating that 93-97% of TOM⁺ cells co-express PAX7 (Figure 6E). We performed Ki67 and MyoD double staining and quantified the percentage of Tomato⁺ cells that expressed one or both of these markers. Our analysis showed that TOM⁺/Ki67⁻/MyoD⁻ (quiescent) satellite cells were significantly reduced in the i-cKO mice compared to controls, while the proportions of single-positive cells (Ki67⁺ or MyoD⁺) were increased (Figure 6F-G). These findings are consistent with loss of quiescence and increased activation of satellite cells upon loss of POGLUT1 in the adult mice.

• During the revision, we performed additional experiments (not suggested by the reviewers) to better assess the role of Poglut1 in self-renewal of satellite cells upon injury. First, in a new cohort of i-cKO and control animals, we performed two rounds of tamoxifen injections but skipped the first round of injury and only induced CTX injury after the second round of recombination. Interestingly, analysis of 14 dpi muscles from these animals showed a full repair in both control and i-cKO mice (shown in revised Figure 5B). Second, we inspected the repaired control and i-cKO muscle after one round of recombination + injury for the presence of single tdTomato+ cells next to the repaired myofibers, which would represent the satellite cells formed after the repair. While single tdTomato+ cells were readily seen in the repaired muscle in control mice, we did not see any such cells next to the repaired muscle from i-cKO animals (shown in the revised Fig 5C, n = 4 animals per condition). Together, these new data provide strong evidence that loss of Poglut1 in adult muscle stem cells impairs their ability to return to quiescence and form new satellite cells upon repair of injured muscle.

  7) Figure 6A: Assay of Cleaved NOTCH1 Intracellular Domain

  The text describes that satellite cells isolated from Poglut1-cKO muscles showed a strong reduction in the level of cleaved (active) NOTCH1 intracellular domain compared to control cells. It is unclear whether these satellite cells were freshly isolated and directly assayed or cultured before the assay. Please specify this in the result section.

• These satellite cells were freshly isolated from whole muscle, and the proteins were extracted directly from the isolated satellite cells without culturing them. This is now included in the Results section related to the revised Figure 7A.

  8) Figure 6: Notch Receptor Overexpression in C2C12 Cells The results section should explicitly explain that the Notch 1-3 receptors were overexpressed or transfected in the C2C12 cells. This detail is essential for understanding the experimental design.

• Thank you. The C2C12 cells were transfected with mouse NOTCH receptors 1, 2, or 3 to overexpress each receptor. This is now included in the Results section as suggested.

  Reviewer #2 (Significance (Required)):

  The manuscript by Cho et al. demonstrates that the muscular dystrophy phenotype associated with Poglut1 mutations is caused by a Notch signaling deficit in muscle stem cells. Although previous studies had suggested this connection, alternative mechanisms could not be excluded, as Poglut1 glycosylates a multitude of proteins. Overall, this is a thorough and careful analysis of Poglut1's role in muscle development and regeneration, providing valuable insights into the mechanisms underlying this rare muscle disease.

Response: We sincerely thank the reviewer for her/his positive assessment of our manuscript and for the constructive comments.

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

POGLUT1 is a key enzyme essential for the glycosylation and signaling activity of Notch receptors in C2C12 muscle cells. Elegant work from multiple groups has demonstrated the essential role of Notch signaling in maintaining the quiescence of muscle stem cells known as satellite cells. For instance, genetic deletion of Rbpj in tamoxifen-inducible conditional knockout mouse models causes ectopic expression of MyoD, diminished Pax7 expression, and precocious differentiation and fusion of quiescent satellite cells without entering the cell cycle. This is accompanied by the complete loss of tissue regeneration following repetitive muscle injuries. Hypomorphic mutations of POGLUT1 cause limb-girdle muscular dystrophy (type R21), accompanied by a significant reduction of Notch signaling and a decrease in the number of satellite cells in patient muscles.

Despite the substantial body of evidence establishing the key function of the POGLUT1-Notch-Pax7/MyoD axis in satellite cells, the in vivo function of POGLUT1 in mice has not been studied. In the present study, Cho et al. carefully examined muscle development and regeneration using the POGLUT1 conditional knockout mouse model. The results, at both phenotypic and cellular/molecular levels, align perfectly with the previously established working model of the POGLUT1-Notch-Pax7/MyoD axis. Overall, the experiment is well-designed and executed, and the data are generally of high quality supporting the conclusions. No specific experimental issues was identified.

Major suggestions include adding references in the discussion section: 1) studies of Rbpj cKO in mice (PMID: 22069237; 22045613), as genetic deletion of POGLUT1 using the same Pax7 driver showed almost identical phenotypes across all levels: phenotype, cellular fate changes, and gene expression changes; 2) studies of Notch-Rbpj targets (Fig. S1, PMID: 29795344), as this paper identified multiple genes encoding collagen V and VI as direct targets of Notch signaling in quiescent satellite cells. These findings are consistent with the authors' observations in Fig. 2e.

• We thank the reviewer for the suggestion to add these highly relevant papers to the manuscript. 1) To address this comment and the specific point #1 raised by reviewer 2, we have highlighted the similarities between Poglut1-cKO and i-cKO phenotypes and the phenotypes observed in "animals with germline or conditional loss of various Notch pathway components (Bjornson et al., 2012; Fukada et al., 2011; Lahmann et al., 2019; Mourikis et al., 2012; Schuster-Gossler et al., 2007; Vasyutina et al., 2007)" in the Discussion. Mourikis et al 2012 and Bjornson et al 2012 are the two papers that the reviewer has referred to. 2) To address this point, we have added the following sentences to the Discussion: "Notch signaling has been shown to directly activate the transcription of several genes encoding collagen V and VI in muscle stem/progenitor cells, and collagen V expressed by satellite cells plays a key role in the niche to maintain satellite cell quiescence (Baghdadi et al., 2018). Therefore, ECM abnormalities caused by reduced Notch signaling might contribute to the loss of satellite cell quiescence in i-cKO animals as well." Baghdadi et al, 2018 is PMID: 29795344, to which the reviewer has referred.

  Minor suggestions: 1B: "P0" is an awkward term. Does it refer to newborn day 1 or a near full-term embryo?

• By saying "P0", we are referring to the day the litters are born.

  Fig. 1C: The y-axis label includes an extra space.

• The extra space is removed in the revised figure.

  Fig. 1D: Provide the N number for each genotype/diet group.

• The N number has been added (now Figure 1E).

  Gene nomenclature: Use POGLUT1 exclusively for the human protein and Poglut1 for the mouse protein.

• According to the guidelines of the International Committee for Standardized Genetic Nomenclature for Mice, protein symbols for mice should use all uppercase letters. Therefore, while the gene symbols for mouse and human are different in terms of uppercase versus lowercase usage, the protein symbols for mouse and human are usually identical. Please see the following webpage: https://www.informatics.jax.org/mgihome/nomen/gene.shtml#ps (accessed January 18, 2025).

  Reviewer #3 (Significance (Required)):

  In the present study, Cho et al. carefully examined muscle development and regeneration using the POGLUT1 conditional knockout mouse model. The results, at both phenotypic and cellular/molecular levels, align perfectly with the known working model of the POGLUT1-Notch-Pax7/MyoD axis in satellite cells and muscle regeneration. Overall, the experiment is well-designed and executed, and the data are generally of high quality.

Response: We sincerely thank the reviewer for the positive evaluation of our manuscript as well as the helpful suggestions.

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

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Referee #2

Evidence, reproducibility and clarity

Poglut1 mutations have been identified in an autosomal recessive form of muscular dystrophy. Poglut1 encodes an O-glucosyltransferase that modifies Notch receptors and ligands but also has other substrates. In mice, null mutations in Poglut1 are embryonic lethal, which has previously precluded the analysis of Poglut1's role in muscle development and regeneration using murine models. To address this limitation, the authors generated a conditional Poglut1 allele and introduced mutations using Pax7Cre or Pax7CreERT2. They characterize the phenotypic consequences of these mutations and further demonstrate that signaling through the Notch1, Notch2, and Notch3 receptors requires Poglut1 using the C2C12 cell culture model.

Some aspects of the manuscript's description of muscle stem cell behavior and regeneration are not fully up to date. These points should be addressed before publication.

Specific points

1. Evidence Supporting Reduced Notch Signaling as the Cause of the Phenotype

The comparison of muscle phenotypes observed in other Notch signaling mutations strongly supports the hypothesis that the phenotype is due to reduced Notch signaling. Relevant studies, such as those addressing development (PMID: 17194759, PMID: 17360543) and those focusing on adult muscle and regeneration (PMID: 21989910, PMID: 22069237, PMID: 22045613, PMID: 30862660) should be cited and discussed in the manuscript. Including these references will strengthen the argument and provide a broader context for the findings. 2. Figure 1: NMJ Deficits - Pre- or Postsynaptic?

The authors describe the mutant synaptic vesicles as showing a significantly reduced evoked neurotransmitter release (quantal content) compared to controls. This phrasing raises the question: are motor neurons mutated in these animals? It should be clarified why the synaptic vesicles are referred to as "mutant." To my knowledge, Pax7Cre does not recombine in motor neurons, and this discrepancy needs to be addressed. The text should be rephrased to accurately reflect the origins of the observed deficits. 3. Quantification of Myofibers with Internal Nuclei

The statement, "We first quantified the ratio of myofibers with internal nuclei, which is an indication for recent fusion of myoblasts to myofibers," is not entirely accurate. Recent studies, such as PMID: 38569550, provide a more nuanced explanation of this phenomenon. The manuscript should reference this study and update the description to ensure it accurately reflects the current understanding of myofiber internal nuclei as markers of muscle pathology or regeneration. 4. Figure 3H: Cell Identification in Culture

The use of PAX7+ MYOD− to identify quiescent cells in culture is not accurate. Instead, PAX7+ Ki67− should be used for this purpose. Similarly, PAX7− MYOD+ does not reliably identify differentiating cells. Instead, staining for MYOG should be used to ensure accuracy. The figure and accompanying text should be adjusted accordingly to reflect these updates. 5. Description of Satellite Cell Quiescence

The statement, "About 2-3 weeks after birth, some of the PAX7+ cells generated by active proliferation of embryonic myogenic progenitors enter a quiescent state to generate adult satellite cells," is not entirely correct. The description should be updated based on the findings in PMID: 32763161, which provide a more accurate account of the transition of PAX7+ cells to quiescence and their role in generating adult satellite cells. 6. Figure 5: Loss of Quiescence in Satellite Cells

A hallmark phenotype of mutations in Notch signaling genesis the loss of quiescence in satellite cells when the mutation is introduced in the adult,. The authors should include data on Ki67 and MyoD expression in PAX7+ cells of mice with the Poglut1 mutation introduced in the adult by an analysis of the uninjured muscle. This would provide insight into the maintenance of quiescence in the mutant satellite cells. 7. Figure 6A: Assay of Cleaved NOTCH1 Intracellular Domain

The text describes that satellite cells isolated from Poglut1-cKO muscles showed a strong reduction in the level of cleaved (active) NOTCH1 intracellular domain compared to control cells. It is unclear whether these satellite cells were freshly isolated and directly assayed or cultured before the assay. Please specify this in the result section. 8. Figure 6: Notch Receptor Overexpression in C2C12 Cells

The results section should explicitly explain that the Notch 1-3 receptors were overexpressed or transfected in the C2C12 cells. This detail is essential for understanding the experimental design.

Significance

The manuscript by Cho et al. demonstrates that the muscular dystrophy phenotype associated with Poglut1 mutations is caused by a Notch signaling deficit in muscle stem cells. Although previous studies had suggested this connection, alternative mechanisms could not be excluded, as Poglut1 glycosylates a multitude of proteins. Overall, this is a thorough and careful analysis of Poglut1's role in muscle development and regeneration, providing valuable insights into the mechanisms underlying this rare muscle disease.

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

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

The manuscript by Cho et al uses conditional and inducible conditional mouse models to characterize the function of protein O-glucosyltransferase 1 (POGLUT1), known to cause a type of Limb Girdle Muscular Dystrophy (LGMD-R21), in skeletal muscle satellite cells, differentiation and regeneration. The Authors find that conditional deletion of POGLUT1 in the myogenic progenitors leads to postnatal muscle defects and lethality by postnatal day 30 or so. Muscle progenitors lacking POGLUT1 undergo reduced proliferation and accelerated differentiation, possibly leading to impairment in muscle regeneration. This is supported by an inducible conditional deletion of POGLUT1 in adult satellite cells. Finally, in vitro experiments suggest that POGLUT1 is required for NOTCH pathway activation in myogenic cells and that POGLUT1 could potentially glycosylate specific residues in NOTCH3.

Major comments

1. What is the control used in Figure 1B and other panels? The genotype should be clearly specified instead of saying controls, in all the figures. It will be easier to interpret the data if densitometry of the western blots is provided, normalized to GAPDH levels. Also, is there some POGLUT1 protein remaining in the satellite cells in the cKOs? Data should be shown for an adult time point also (P30 or later), in addition to P0 and P4, to see whether Poglut1 levels are reduced in adult stages in the muscle and satellite cells in the cKOs.
2. In Figure 1C, can the tibia weight be normalized to total body weight instead of tibia length and analyzed? Similarly, can the grip strength measurements in Figure 1G be normalized to total body weight and represented? Grip strength measurements in neonates is tricky; the Authors should clearly explain how this was done. The NMJ defects can be characterized better, especially since the Authors discuss about Agrin in detail.
3. What are the small myofibers seen at the corners of the larger myofibers at P21 in the cKOs in Figure 2E? What is the point that the Authors want to conclude from Figure 2C and D? Clearly, if there are fewer satellite cells, Pax7 transcript levels will decrease. In Figure 2D, how are the satellite cell samples normalized between control and cKOs; did they start with equal number of satellite cells or equal amount of satellite cell RNA between control and cKOs? In Figure 2F, representative images for cKOs should be shown? The conclusion from Figure 2F-G is unclear. Since the Authors claim that the weak laminin staining is resolved in the cKOs by P21, why is α-dystroglycan hypoglycosylation seen in the P21 muscle?
4. Representative images should be shown as examples for all time points for both genotypes in Figure 3A. Figure 3H should be represented with statistically significant differences marked clearly. Have the Authors checked whether cell death contributes to the decrease in cultured satellite cells and Pax7+ cells in the cKOs in Figure 3F, G? Are the differences between the control and cKOs in fusion index and Myogenin expression (Figure 3J, K) statistically significant?
5. Figure 4 has multiple problems in my opinion. First, P21 is too early a time point to be talking about regeneration, since satellite cells are just transitioning to become quiescent cells at this time (described in Lepper et al, Nature 2009). It is difficult to distinguish the regenerative and neonatal developmental roles of satellite cells in the experiment that the Authors have carried out. Another issue is that the Authors show a decrease in satellite cells in the cKOs; satellite cell function is clearly known to be required for regeneration. Therefore, there is not much novelty that the cKOs exhibit poor regenerative capability. The data shown in Figure 4B-E is more or less a repetition of what has been shown in Figures 2 and 3.
6. In Figure 5C, D, have the Authors checked the Pax7+ cell numbers between the controls and the i-cKOs? Is the difference seen in 1X TAM due to preexisting reduction in satellite cells in the i-cKOs? What is the explanation by the Authors for the large number of Tomato+ fibers seen in the 2X TAM and 3X TAM uninjured muscle (Figure 3C, E)? Figure 5E is difficult to comprehend and should be represented in a clearer manner.
7. Densitometry for Figure 6A should be included, since N1 ICD levels seem quite variable in the controls also. POGLUT1 expression and shRNA knockdown efficiency should be shown in Figure 6B. What is the correlation between the NOTCH3 glycosylation with reduced NOTCH pathway activation and satellite cell function? This should be clearly discussed. Why was the NOTCH3 glycosylation assay done in HEK293 cells?

Significance

Based on my expertise as a muscle and stem cell biologist, the manuscript is not clearly thought through, with not many novel inferences that one can draw from the data provided. While the manuscript could be informative to muscle biologists and stem cell investigators, several additional experiments are required to better characterize the phenotypes and provide meaningful conclusions from the study. The role of POGLUT1 in the muscle could be of great interest, especially in light of its role in LGMD-R21, as described by some of the Authors previously. Several pieces of data provided in the current manuscript are disjointed, with few connecting links, such as the NMJ characterization, the NOTCH glycosylation data and the regeneration experiments done on P21 neonates. Better quantitation of data is required as well, as detailed below. Overall, the manuscript may be revised to address the specific comments and reconsidered at a suitable journal.

Conforme destacam Cabero e Gisbert (2002), os ambientes digitais não podem ser a mera transposição da geografia física da sala de aula para uma geografia digital. Para evitar que os ambientes digitais se tornem espaços de ensino expositivos, é essencial incluir e-atividades que promovam a compreensão e aplicação prática do conhecimento. Isso pode ser alcançado através de atividades que desafiem os estudantes a aplicar o conhecimento em contextos do mundo real, tornando a aprendizagem mais relevante e significativa. Neste contexto, podem ser implementadas atividades como estudos de caso, pequenos projetos de investigação, simulações que replicam cenários do mundo real, debates online sobre temas relevantes, ou a criação de conteúdos educativos, como vídeos, podcasts ou blogs, sobre os tópicos estudados.

As e-atividades são muito importantes na promoção de uma aprendizagem ativa e participativa. No ensino digital, é essencial criar e-atividades que incentivem a colaboração e a construção de conhecimento entre os alunos. Isso pode ser feito através de projetos de grupo, discussões em fóruns e outras atividades interativas que envolvam os alunos de maneira significativa.

Um ponto importante, mas que merece ser expandido. Que indicadores de sustentabilidade estão em jogo aqui? Seria útil exemplificar como o design instrucional beneficia das e-atividades ao longo do tempo, tanto em termos de adaptação pedagógica como de escalabilidade.

A clareza, a acessibilidade e a intencionalidade pedagógica no design das e-atividades contribuem para ambientes digitais inclusivos e estimulantes. Daí a importância de se cumprirem com estes aspetos. Assim se verifica a intencionalidade das atividades pedagógicas para que se: promovam competências transversais como o pensamento crítico, a resolução de problemas e a comunicação digital. Cumprindo estes pressupostos, transforma-se o espaço virtual num ambiente de aprendizagem significativo e centrado no desenvolvimento integral do estudante.

A interação entre os sujeitos e os conteúdos promove a construção colaborativa do saber. Remete para a ideia de que recursos poderão ser adequados utilizar em função das aprendizagens que se pretende. Destaca o papel fundamental da e-moderação.<br /> Ainda assim, importa definir objetivos claros e metodologias participativas. O papel do docente, neste contexto, é essencial para orientar, motivar e garantir a coesão do processo formativo.

Seria interessante compreender esta visão de ambiente virtual e treino aplicado a cursos eminentemente práticos, como a enfermagem. Julgo que, provavelmente, serão recursos interessantes para o desenvolvimento do decisão clínica e do juízo crítico.

As e-atividades devem considerar a diversidade dos estudantes, promovendo personalização e autonomia. Devem ser claras, acessíveis e intencionais, desenvolvendo competências como pensamento crítico e comunicação digital. Dessa forma, os ambientes virtuais tornam-se espaços de aprendizagem significativa.

Contextualizar as e-atividades como estratégias pedagógicas significa integrá-las de forma intencional e alinhada com os objetivos de aprendizagem, respeitando as características dos conteúdos, dos estudantes e do ambiente digital. Estas atividades não devem ser encaradas como tarefas isoladas, mas como parte de um percurso formativo coeso, que favoreça o envolvimento ativo e reflexivo dos intervenientes. Ao serem bem desenhadas, permitem estimular competências cognitivas, colaborativas e digitais, fundamentais na educação contemporânea.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review):

Summary:

The paper addresses the knowledge gap between the representation of goal direction in the central complex and how motor systems stabilize movement toward that goal. The authors focused on two descending neurons, DNa01 and 02, and showed that they play different roles in steering the fly toward a goal. They also explored the connectome data to propose a model to explain how these DNs could mediate response to lateralized sensory inputs. They finally used lateralized optogenetic activation/inactivation experiments to test the roles of these neurons in mediating turnings in freely walking flies.

Strengths:

The experiments are well-designed and controlled. The experiment in Figure 4 is elegant, and the authors put a lot of effort into ensuring that ATP puffs do not accidentally activate the DNs. They also have explained complex experiments well. I only have minor comments for the authors.

We are grateful for this positive feedback.

Weaknesses:

(1) I do not fully understand how the authors extracted the correlation functions from the population data in Figure 1. Since the ipsilateral DNs are anti-correlated with the contralateral ones, I expected that the average will drop to zero when they are pooled together (e.g., 1E-G). Of course, this will not be the case if all the data in Figure 1 are collected from the same brain hemisphere. It would be helpful if the authors could explain this.

We regret that this information was not easy to find in our initial submission. As noted in the Figure 1D legend, Here and elsewhere, ipsi and contra are defined relative to the recorded DN(s). We have now added a sentence to the Results (right after we introduce Figure 1D) that also makes this point.

(2) What constitutes the goal directions in Figures 1-3 and 8, as the authors could not use EPG activity as a proxy for goal directions? If these experiments were done in the dark, without landmarks, one would expect the fly's heading to drift randomly at times, and they would not engage the DNa01/02 for turning. Do the walking trajectories in these experiments qualify as menotactic bouts?

Published work (Green et al., 2019) has shown that, even in the dark, flies will often walk for extended periods while holding the bump of EPG activity at a fixed location. During these epochs, the brain is essentially estimating that the fly is walking in a straight line in a fixed direction. (The fact that the fly is actually rotating a bit on the spherical treadmill is not something the fly can know, in the dark.) Thus, epochs where the EPG bump is held fixed are treated as menotactic bouts, even in darkness.

Our results provide additional support for this interpretation. We find that, when flies are walking in darkness and holding the bump of EPG activity at a fixed location, they will make a corrective behavioral turning maneuver in response to an imposed bump-jump. This result argues that the flies are actually engaging in goal-directed straight-line walking, i.e. menotaxis, and it reproduces the findings of Green et al. (2019).

To clarify this point, we have adjusted the wording of the Results pertaining to Figure 4.

(3) In Figure 2B, the authors mentioned that DNa02 overpredicts and 01 underpredicts rapid turning and provided single examples. It would be nice to see more population-level quantification to support this claim.

In this revision, we have reorganized Figures 1 and 2 (and associated text) to improve clarity. As part of this reorganization, we have removed this passage from the text, as it was a minor point in any event.

Reviewer #2 (Public review):

The data is largely electrophysiological recordings coupled with behavioral measurements (technically impressive) and some gain-of-function experiments in freely walking flies. Loss-of-function was tested but had minimal effect, which is not surprising in a system with partially redundant control mechanisms. The data is also consistent with/complementary to subsequent manuscripts (Yang 2023, Feng 2024, and Ros 2024) showing additional descending neurons with contributions to steering in walking and flying.

The experiments are well executed, the results interesting, and the description clear. Some hypotheses based on connectome anatomy are tested: the insights on the pre-synaptic side - how sensory and central complex heading circuits converge onto these DNs are stronger than the suggestions about biomechanical mechanisms for how turning happens on the motor side.

Of particular interest is the idea that different sensory cues can converge on a common motor program. The turn-toward or turn-away mechanism is initiated by valence rather than whether the stimulus was odor or temperature or memory of heading. The idea that animals choose a direction based on external sensory information and then maintain that direction as a heading through a more internal, goal-based memory mechanism, is interesting but it is hard to separate conclusively.

To clarify, we mention the role of memory in connection with two places in the manuscript. First, we note that the EPG/head direction system relies on learning and memory to construct a map of directional cues in the environment. These cues are, in principle, inherently neutral, i.e. without valence. Second, we note that specific mushroom body output neurons rely on learning and memory to store the valence associated with an odor. This information is not necessarily associated with an allocentric direction: it is simply the association of odor with value. Both of these ideas are well-attested by previous work.

The reviewer may be suggesting a sequential scheme whereby the brain initializes an allocentric goal direction based on valence, and then maintains that goal direction in memory, based on that initialization. In other words, memory is used to associate valence with some allocentric direction. This seems plausible, but it is not a claim we make in our manuscript.

The "see-saw", where left-right symmetry is broken to allow a turn, presumably by excitation on one side and inhibition of the other leg motor modules, is interesting but not well explained here. How hyperpolarization affects motor outputs is not clear.

We have added several sentences to the Discussion to clarify this point. According to this see-saw model, steering can emerge from right/left asymmetries in excitation, or inhibition, or both. It may be nonintuitive to think that inhibitory input to a DN can produce an action. However, this becomes more plausible given our finding that DNa02 has a relatively high basal firing rate (Fig. 1D), and DNa02 hyperpolarization is associated with contraversive turning (Fig. 5A). It is also relevant to note that there are many inhibitory cell types that form strong unilateral connections onto DNa02 (e.g., AOTU019).

The statement near Figure 5B that "DNa02 activity was higher on the side ipsilateral to the attractive stimulus, but contralateral to the aversive stimulus" is really important - and only possible to see because of the dual recordings.

We thank the reviewer for this positive feedback.

Reviewer #3 (Public review):

Summary:

Rayshubskiy et al. performed whole-cell recordings from descending neurons (DNs) of fruit flies to characterize their role in steering. Two DNs implicated in "walking control" and "steering control" by previous studies (Namiki et al., 2018, Cande et al., 2018, Chen et al., 2018) were chosen by the authors for further characterization. In-vivo whole-cell recordings from DNa01 and DNa02 showed that their activity predicts spontaneous ipsilateral turning events. The recordings also showed that while DNa02 predicts transient turns DNa01 predicts slow sustained turns. However, optogenetic activation or inactivation showed relatively subtle phenotypes for both neurons (consistent with data in other recent preprints, Yang et al 2023 and Feng et al 2024). The authors also further characterized DNa02 with respect to its inputs and showed a functional connection with olfactory and thermosensory inputs as well as with the head-direction system. DNa01 is not characterized to this extent.

Strengths:

(1) In-vivo recordings and especially dual recordings are extremely challenging in Drosophila and provide a much higher resolution DN characterization than other recent studies that have relied on behavior or calcium imaging. Especially impressive are the simultaneous recordings from bilateral DNs (Figure 3). These bilateral recordings show clearly that DNa02 cells not only fire more during ipsilateral turning events but that they get inhibited during contralateral turns. In line with this observation, the difference between left and right DNa02 neuronal activity is a much better predictor of turning events compared to individual DNa02 activity.

(2) Another technical feat in this work is driving local excitation in the head-direction neuronal ensemble

(PEN-1 neurons), while simultaneously imaging its activity and performing whole-cell recordings from DNa02

(Figure 4). This impressive approach provided a way to causally relate changes in the head-direction system to DNa02 activity. Indeed, DNa02 activity could predict the rate at which an artificially triggered bump in the PEN-1 ring attractor returns to its previous stable point.

(3) The authors also support the above observations with connectomics analysis and provide circuit motifs that can explain how the head direction system (as well as external olfactory/thermal stimuli) communicated with DNa02. All these results unequivocally put DNa02 as an essential DN in steering control, both during exploratory navigation as well as stimulus-directed turns.

We are grateful for this detailed positive feedback.

Weaknesses:

(1) I understand that the first version of this preprint was already on biorxiv in 2020, and some of the "weaknesses" I list are likely a reflection of the fact that I'm tasked to review this manuscript in late 2024 (more than 4 years later). But given this is a 2024 updated version it suffers from laying out the results in contemporary terms. For instance, the manuscript lacks any reference to the DNp09 circuit implicated in object-directed turning and upstream to DNa02 even though the authors cite one of the papers where this was analyzed (Braun et al, 2024). More importantly, these studies (both Braun et al 2024 and Sapkal et al 2024) along with recent work from the authors' lab (Yang et al 2023) and other labs (Feng et al 2024) provide a view that the entire suite of leg kinematics changes required for turning are orchestrated by populations of heterogeneous interconnected DNs. Moreover, these studies also show that this DN-DN network has some degree of hierarchy with some DNs being upstream to other DNs. In this contemporary view of steering control, DNa02 (like DNg13 from Yang et al 2023) is a downstream DN that is recruited by hierarchically upstream DNs like DNa03, DNp09, etc. In this view, DNa02 is likely to be involved in most turning events, but by itself unable to drive all the motor outputs required for the said events. This reasoning could be used while discussing the lack of major phenotypes with DNa02 activation or inactivation observed in the current study, which is in stark contrast to strong phenotypes observed in the case of hierarchically upstream DNs like DNp09 or DNa03. In the section, "Contributions of single descending neuron types to steering behavior": the authors start off by asking if individual DNs can make measurable contributions to steering behavior. Once more, any citations to DNp09 or DNa03 - two DNs that are clearly shown to drive strong turning-on activation (Bidaye et al, 2020, Feng et al 2024) - are lacking. Besides misleading the reader, such statements also digress the results away from contemporary knowledge in the field. I appreciate that the brief discussion in the section titled "Ensemble codes for steering" tries to cover these recent updates. However, I think this would serve a better purpose in the introduction and help guide the results.

We apologize for these omissions of relevant citations, which we have now fixed. Specifically, in our revised Discussion, we now point out that:

- Braun et al. (2024) reported that bilateral optogenetic activation of either DNa02 or DNa01 can drive turning (in either direction). 

- Braun et al. (2024) also identified DNb02 as a steering-related DN.

- Bidaye et al. (2020), Sapkal et al. (2024), and Braun et al. (2024) all contributed to the identification of DNp09 as a broadcaster DN with the capacity to promote ipsiversive turning.

We have also revised the beginning of the Results section titled “Contributions of single descending neuron types to steering behavior”, as suggested by the Reviewer.

Finally, we agree with the Reviewer’s overall point that steering is influenced by multiple DNs. We have not claimed that any DN is solely responsible for steering. As we note in the Discussion: “We found that optogenetically inhibiting DNa01 produced only small defects in steering, and inhibiting DNa02 did not produce statistically significant effects on steering; these results make sense if DNa02 is just one of many steering DNs.”

(2) The second major weakness is the lack of any immunohistochemistry (IHC) images quantifying the expression of the genetic tools used in these studies. Even though the main split-Gal4 tools for DNa01 and DNa02 were previously reported by Namiki et al, 2018, it is important to document the expression with the effectors used in this work and explicitly mention the expression in any ectopic neurons. Similarly, for any experiments where drivers were combined together (double recordings, functional connectivity) or modified for stochastic expression (Figure 8), IHC images are absolutely necessary. Without this evidence, it is difficult to trust many of the results (especially in the case of behavioral experiments in Figure 8). For example, the DNa01 genetic driver used by the authors is also expressed in some neurons in the nerve cord (as shown on the Flylight webpage of Janelia Research Campus). One wonders if all or part of the results described in Figure 8 are due to DNa01 manipulation or manipulation of the nerve cord neurons. The same applies for optic lobe neurons in the DNa02 driver.

This is a reasonable request. We used DN split-Gal4 lines to express three types of UAS-linked transgenes:

(1) GFP

In these flies, we know that expression in DNs is restricted to the DN types in question, based on published work (Namki et al., 2018), as well as the fact that we see one labeled DN soma per hemisphere. When we label both cells with GFP, we use the spike waveform to identify DNa02 and DNa01, as described in Figure S1

(2) ReaChR

In these flies, expression patterns were different in different flies because ReaChR expression was stochastically sparsened using hs-FLP. Expression was validated in each fly after the experiment, as described in the Methods (“Stochastic ReaChR expression”). hs-FLP-mediated sparsening will necessarily produce stochastic patterns of expression in both DNa02 and off-target cells, and this is true of all the flies in this experiment. What makes the “unilateral” flies distinct from the “bilateral” flies is that unilateral flies express ReaChR in one copy of DNa02, whereas bilateral flies express ReaChR in both copies of DNa02. On average, off-target expression will be the same in both groups.

(3) GtACR1

In these flies, we initially assumed that GtACR1 expression was the same as GFP expression under control of the same driver. However, we agree with the reviewer’s point that these two expression patterns are not necessarily identical. Therefore, to address the reviewer’s question, we performed immunofluorescence microscopy to characterize GtACR1 patterns in the brain and VNC of both genotypes. These expression patterns are now shown in a new supplemental figure (Figure S8). This figure shows that, as it happens, expression of GtACR1 is indeed indistinguishable from the GFP expression patterns for the same lines (archived on the FlyLight website). Both DN split-Gal4 lines are largely selective for the DNs in question, with limited off-target labeling. We have now drawn attention to this off-target labeling in the last paragraph of the Results, where the GtACR1 results are discussed.

(3) The paper starts off with a comparative analysis of the roles of DNa01 and DNa02 during steering. Unfortunately, after this initial analysis, DNa01 is largely ignored for further characterization (e.g. with respect to inputs, connectomics, etc.), only to return in the final figure for behavioral characterization where DNa01 seems to have a stronger silencing phenotype compared to DNa02. I couldn't find an explanation for this imbalance in the characterization of DNa01 versus DNa02. Is this due to technical reasons? Or was it an informed decision due to some results? In addition to being a biased characterization, this also results in the manuscript lacking a coherent thread, which in turn makes it a bit inaccessible to the non-specialist.

Yes, the first portion of the manuscript focuses on DNa01 and DNa02. The latter part of the manuscript transitions to focus mainly on DNa02. 

Our rationale is noted at the point in the manuscript where we make this transition, with the section titled “Steering toward internal goals”: “Having identified steering-related DNs, we proceeded to investigate the brain circuits that provide input to these DNs. Here we decided to focus on DNa02, as this cell’s activity is predictive of larger steering maneuvers.” When we say that DNa02 is predictive of larger steering maneuvers, we are referring to several specific results:

- We obtain larger filter amplitudes for DNa02 versus DNa01 (Fig. 2A-C). This means that, just after a unit change in DN firing rate, we see on average a larger change in steering velocity for DNa02 versus DNa01.

- The linear filter for DNa02 has a higher variance explained, as compared to DNa01 (Fig. 2D). This means that DNa02 is more predictive of steering.

- The relationship between firing rate and rotational velocity (150 ms later) is steeper for DNa02 than for DNa01 (Fig. 2G). This means that, if we ignore dynamics and we just regress firing rate against subsequent rotational velocity, we see a higher-gain relationship for DNa02.

Our focus on DNa02 was also driven by connectivity considerations. In the same paragraph (the first paragraph in the section titled “Steering toward internal goals”). We note that “there are strong anatomical pathways from the central complex to DNa02”; the same is not true of DNa01. This point has also been noted by other investigators (Hulse et al. 2021).

We don’t think this focus on DNa02 makes our work biased or inaccessible. Any study must balance breadth with depth. A useful general way to balance these constraints is to begin a study with a somewhat broader scope, and then narrow the study’s focus to obtain more in-depth information. Here, we began with comparative study of two cell types, and we progressed to the cell type that we found more compelling.

(4) There seems to be a discrepancy with regard to what is emphasized in the main text and what is shown in Figures S3/S4 in relation to the role of these DNs in backward walking. There are only two sentences in the main text where these figures are cited.

a) "DNa01 and DNa02 firing rate increases were not consistently followed by large changes in forward velocity

(Figs. 1G and S3)."

b) "We found that rotational velocity was consistently related to the difference in right-left firing rates (Fig. 3B). This relationship was essentially linear through its entire dynamic range, and was consistent across paired recordings (Fig. 3C). It was also consistent during backward walking, as well as forward walking (Fig. S4)." These main text sentences imply the role of the difference between left and right DNa02 in turning. However, the actual plots in the Figures S3 and S4 and their respective legends seem to imply a role in "backward walking". For instance, see this sentence from the legend of Figure S3 "When (ΔvoltageDNa02>>ΔvoltageDNa01), the fly is typically moving backward. When (firing rateDNa02>>firing rateDNa01), the fly is also often moving backward, but forward movement is still more common overall, and so the net effect is that forward velocity is small but still positive when (firing rateDNa02>>firing rateDNa01). Note that when we condition our analysis on behavior rather than neural activity, we do see that backward walking is associated with a large firing rate differential (Fig. S4)." This sort of discrepancy in what is emphasized in the text, versus what is emphasized in the figures, ends up confusing the reader. More importantly, I do not agree with any of these conclusions regarding the implication of backward walking. Both Figures S3 and S4 are riddled with caveats, misinterpretations, and small sample sizes. As a result, I actually support the authors' decision to not infer too much from these figures in the "main text". In fact, I would recommend going one step further and removing/modifying these figures to focus on the role of "rotational velocity". Please find my concerns about these two figures below:

a) In Figures S3 and S4, every heat map has a different scale for the same parameter: forward velocity. S3A is -10 to +10mm/s. S3B is -6 to +6 S4B (left) is -12 to +12 and S4B (right) is -4 to +4. Since the authors are trying to depict results based on the color-coding this is highly problematic.

b) Figure S3A legend "When (ΔvoltageDNa02>>ΔvoltageDNa01), the fly is typically moving backward." There are also several instances when ΔvoltageDNa02= ΔvoltageDNa01 and both are low (lower left quadrant) when the fly is typically moving backwards. So in my opinion, this figure in fact suggests DNa02 has no role in backward velocity control.

c) Based on the example traces in S4A, every time the fly walks backwards it is also turning. Based on this it is important to show absolute rotational velocity in Figure S4C. It could be that the fly is turning around the backward peak which would change the interpretation from Figure S4C. Also, it is important to note that the backward velocities in S4A are unprecedentedly high. No previous reports show flies walking backwards at such high velocities (for example see Chen et al 2018, Nat Comm. for backward walking velocities on a similar setup).

d) In my opinion, Figure S4D showing that right-left DNa02 correlates with rotational velocity, regardless of whether the fly is in a forward or backward walking state, is the only important and conclusive result in Figures S3/S4. These figures should be rearranged to only emphasize this panel.

We agree that it is difficult to interpret some of the correlations between DN activity and forward velocity, given that forward velocity and rotational velocity are themselves correlated to some degree. This is why we did not make claims based on these results in the main text. In response to these comments, we have taken the Reviewer’s suggestion to preserve Figure S4D (now Figure S3). The other components of these supplemental figures have been removed.

(5) Figure 3 shows a really nice analysis of the bilateral DNa02 recordings data. While Figure S5 [now Figure S4] shows that authors have a similar dataset for DNa01, a similar level analysis (Figures 3D, E) is not done for DNa01 data. Is there a reason why this is not done?

The reason we did not do the same analysis for DNa01 is that we only have two paired DNa01-DNa01 recordings. It turned out to be substantially more difficult to perform DNa01-DNa01 recordings, as compared to DNa02-DNa02 recordings. For this reason, we were not able to get more than two of these recordings.

(6) In Figure 4 since the authors have trials where bump-jump led to turning in the opposite direction to the DNa02 being recorded, I wonder if the authors could quantify hyperpolarization in DNa02 as is predicted from connectomics data in Figure 7.

We agree this is an interesting question. However, DNa02 firing rate and membrane potential are variable, and stimulus-evoked hyperpolarizations in these DNs tend to be relatively small (on the order of 1 mV, in the case of a contralateral fictive olfactory stimulus, Figure 5A). In the case of our fictive olfactory stimuli, we could look carefully for these hyperpolarizations because we had a very large number of trials, and we could align these trials precisely to stimulus onset. By contrast, for the bump-jump experiments, we have a more limited number of trials, and turning onset is not so tightly time-locked to the chemogenetic stimuli; for these reasons, we are hesitant to make claims about any bump-jump-related hyperpolarization in these trials.

(7) Figure 6 suggests that DNa02 contains information about latent steering drives. This is really interesting. However, in order to unequivocally claim this, a higher-resolution postural analysis might be needed. Especially given that DNa02 activation does not reliably evoke ipsilateral turning, these "latent" steering events could actually contain significant postural changes driven by DNa02 (making them "not latent"). Without this information, at least the authors need to explicitly mention this caveat.

This is a good point. We cannot exclude the possibility that DNa02 is driving postural changes when the fly is stopped, and these postural changes are so small we cannot detect them. In this case, however, there would still be an interesting mismatch between the stimulus-evoked change in DNa02 firing rate (which is large) and the stimulus-evoked postural response (which would be very small). We have added language to the relevant Results section in order to make this explicit.

(8) Figure 7 would really benefit from connectome data with synapse numbers (or weighted arrows) and a corresponding analysis of DNa01.

In response to this comment, we have added synapses number information (represented by weighted arrows) to Figures 7C, E, and F. We also added information to the Methods to explain how cells were chosen for inclusion in this diagram. (In brief: we thresholded these connections so as to discard connections with small numbers of synapses.)

We did perform an analogous connectome circuit analysis for DNa01, but if we use the same thresholds as we do for DNa02, we obtain a much sparser connectivity graph. We now show this in a new supplemental figure (Figure S9). MBON32 makes no monosynaptic connections onto DNa01, and it only forms one disynaptic connection, via LAL018, which is relatively weak. PFL3 and PFL2 make no mono- or disynaptic connections onto DNa01 comparable in strength to what we find for DNa02. 

The sparser connectivity graph for DNa01 is partly due to the fact that fewer cell types converge onto DNa01 as compared to DNa02 (110 cell types, versus 287 cell types). Also, it seems that DNa01 is simply less closely connected to the central complex and mushroom body, as compared to DNa02.

(9) In Figure 8E, the most obvious neuronal silencing phenotype is decreased sideways velocity in the case of DNa01 optogenetic silencing. In Figure S2, the inverse filter for sideways velocity for DNa01 had a higher amplitude than the rotational velocity filter. Taken together, does this point at some role for DNa01 in sideways velocity specifically?

No. The forward filters describe the average velocity impulse response, given a brief step change in firing rate.

Figure 1 and Figure S2 show that the sideways velocity forward filter is actually smaller for DNa01 than for DNa02. This means that a brief step change in DNa01 firing rate is followed by only a very small sideways velocity response. Conversely, the reverse filters describe the average firing rate impulse response, given a brief step change in sideways velocity. Figure S2 shows that the sideways velocity reverse filter is larger for DNa01 than for DNa02, but this means that the relationship between DNa01 activity and sideways velocity is so weak that we would need to see a very large neural response in order to get a brief step change in sideways velocity. In other words, the reverse filter says that DNa01 likely has very little role in determining sideways velocity.

(10) In Figure 8G, the effect on inner hind leg stance prolongation is very weak, and given the huge sample size, hard to interpret. Also, it is not clear how this fits with the role of DNa01 in slow sustained turning based on recordings.

Yes, this effect is small in magnitude, which is not too surprising, given that many DNs seem to be involved in the control of steering in walking. To clarify the interpretation of these phenotypes, we have added a paragraph to the end of the Results:

“All these effects are weak, and so they should be interpreted with caution. Also, both DN split-Gal4 lines drive expression in a few off-target cell types, which is another reason for caution (Fig. S8). However, they suggest that both DNs can lengthen the stance phase of the ipsilateral back leg, which would cause ipsiversive turning. These results are also compatible with a scenario where both DNs decrease the step length in the ipsilateral legs, which would also cause ipsiversive turning. Step frequency does not normally change asymmetrically during turning, so the observed decrease in step frequency during optogenetic inhibition may just be a by-product of increasing step length when these DNs are inhibited.” We have also added caveats and clarifications in a new Discussion paragraph:

“Our study does not fully answer the question of how these DNs affect leg kinematics, because we were not able to simultaneously measure DN activity and leg movement. However, our optogenetic experiments suggest that both DNs can lengthen the stance phase of the ipsilateral back leg (Fig. 8G), and/or  decrease the step length in the ipsilateral legs (Fig. 8H), either of which would cause ipsiversive turning. If these DNs have similar qualitative effects on leg kinematics, then why does DNa02 precede larger and more rapid steering events? This may be due to the fact that DNa02 receives stronger and more direct input from key steering circuits in the brain (Fig. S9). It may also relate to the fact that DNa02 has more direct connections onto motor neurons (Fig. 1B).”

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) I found the sign conventions for rotational velocity particularly confusing. Figure 3 represents clockwise rotations as +ve values, but Figure 4H represents anticlockwise rotations as positive values. But for EPG bumps, anticlockwise rotations are given negative values. Please make them consistent unless I am missing something obvious.

Different fields use different conventions for yaw velocity. In aeronautics, a clockwise turn is generally positive. In robotics and engineering of terrestrial vehicles, a counterclockwise turn is generally positive. Historically, most Drosophila studies that quantified rotational (yaw) velocity were focused on the behavior of flying flies, and these studies generally used the convention from aeronautics, where a clockwise turn is defined as a positive turn. When we began working in the field, we adopted this convention, in order to conform to previous literature. It might be argued that walking flies are more like robots than airplanes, but it seemed to us that it was confusing to have different conventions for different behaviors of the same animal. Thus, all of the published studies from our lab define clockwise rotation as having positive rotational velocity.

Figure 4 focuses on the role of the central complex in steering. As the fly turns clockwise (rightward), the bump of activity in EPG neurons normally moves counterclockwise around the ellipsoid body, as viewed from the posterior side (Turner-Evans et al., 2017). The posterior view is the conventional way to represent these dynamics, because (1) we and others typically image the brain from the posterior side, not the anterior side, and (2) in a posterior view, the animal’s left is on the left side of the image, and vice versa. We have added a sentence to the Figure 4A legend to clarify these points.

Previous work has shown that, when an experimenter artificially “jumps” the EPG bump, this causes the fly to make a compensatory turn that returns the bump to (approximately) its original location (Green et al., 2019). Our work supports this observation. Specifically, we find that clockwise bump jumps are generally followed by rightward turns (which drive the bump to return to its approximate original location via a counterclockwise path), and vice versa. This is noted in the Figure 4D legend. Note that Figure 4D plots the fly’s rotational velocity during the bump return, plotted against the initial bump jump. 

Figure 4H shows that clockwise (blue) bump returns were typically preceded by leftward turning, counter-clockwise (green) bump returns were preceded by rightward turning, as expected. This is detailed in the Figure 4H legend, and it is consistent with the coordinate frame described above.

(2) It would be helpful to have images of the DNa01 and DNa02 split lines used in this paper, considering this paper would most likely be used widely to describe the functions of these neurons. Similarly, images of their reconstructions would be a useful addition.

High-quality three-dimensional confocal stacks of all the driver lines used in our study are publicly available. We have added this information to the Methods (under “Fly husbandry and genotypes”). Confocal images of the full morphologies of DNa01 and DNa02 have been previously published (Namiki et al., 2018). Figure 1A is a schematic that is intended to provide a quick visual summary of this information.

EM reconstructions of DNa01 and DNa02 are publicly accessible in a whole-brain dataset (https://codex.flywire.ai/) and a whole-VNC dataset (https://neuprint.janelia.org/). Both datasets are referenced in our study. As these datasets are easy to search and browse via user-friendly web-based tools, we expect that interested readers will have no difficulty accessing the underlying datasets directly.

Reviewer #2 (Recommendations for the authors):

(1) The description of the activity of the DNs that they "PREDICT steering during walking". This is an interesting word choice. Not causes, not correlates with, not encodes... does that mean the activity always precedes the action? Does that mean when you see activity, you will get behavior? This is important for assessing whether the DN activity is a cause or an effect. It is good to be cautious but it might be worth expanding on exactly what kind of connection is implied to justify the use of the word 'predict'.

Conventionally, “predict” means “to indicate in advance”. We write that DNs “predict” certain features of behavior. We use this term because (1) these DNs correlate with certain features of behavior, and (2) changes in DN activity precede changes in behavior.

The notion that neurons can “predict” behavior is not original to our study. Whenever neuroscientists summarize the relationship between neural activity and behavior by fitting a mathematical model (which may be as simple as a linear regression), the fitted model can be said to represent a “prediction” of behavior. These models are evaluated by comparing their predictions with measured behaviors. A good model is predictive, but it also implies that the underlying neural signal is also predictive (Levenstein et al., 2023 Journal of Neuroscience 43: 1074-1088; DOI: 10.1523/JNEUROSCI.1179-22.2022). Here, prediction simply means correlation, without necessarily implying causation. We also use “prediction” to imply correlation.

We do not think the term “prediction” implies determinism. Meteorologists are said to predict the weather, but it is understood that their predictions are probabilistic, not deterministic. Certainly, we would not claim that there is a deterministic relationship between DN activity and behavior. Figure 2D shows that neither DN type can explain all the variance in the fly’s rotational or sideways velocity. At the same time, both DNs have significant predictive power.

We might equally say that these DNs “encode” behavior. We have chosen to use the word “predict” rather than “encode” because we do not think it is necessary to use the framework of symbolic communication in connection with these DNs.

We agree with the Reviewer that it is helpful to test whether any neuron that “predicts” a behavior might also “cause” this behavior. In Figure 8, we show that directly perturbing these DNs can indeed alter locomotor behavior, which suggests a causal role. Connectome analyses also suggest a causal role for these DNs in locomotor behavior (Figure 1B, see especially also Cheong et al., 2024).

At the same time, it is clear from our results that these DNs are not “command neurons” for turning: they do not deterministically cause turning. Therefore, to avoid misunderstanding, we have generally been careful to summarize the results of our perturbation experiments by avoiding the statement that “this DN causes this behavior”. Rather, we have generally tried to say that “this DN influences this behavior”, or “this DN promotes this behavior”.

(2) There is some concern about how the linear filter models were developed and then used to predict the relationship between firing rate and steering behavior: how exactly were the build and test data separated to avoid re-extracting the input? It reads like a self-fulfilling prophecy/tautology.

We used conventional cross-validation for model fitting and evaluation. We apologize that this was not made explicit in our original submission; this was due to an oversight on our part. To be clear: linear filters were computed using the data from the first 20% of a given experiment. We then convolved each cell’s firing rate estimate with the computed Neuron→Behavior filter (the “forward filter”) using the data from the final 80% of the experiment, in order to generate behavioral predictions. Thus, when a model has high variance explained, this is not attributable to overfitting: rather, it quantifies the bona fide predictive power of the model. We have added this information to the Methods (under “Data analysis - Linear filter analysis”).

(3) Type-O right above Figure 2 [now Figure 1E]: I assume spike rate fluctuations in DNa02 precede DNa01?

Fixed. Thank you for reading the manuscript carefully.

(4) The description of the other manuscripts about neural control of the steering as "follow-up" papers is a bit diminishing. They were likely independent works on a similar theme that happened afterwards, rather than deliberate extensions of this paper, so "subsequent" might be a more accurate description.

We apologize, as we did not intend this to be diminishing. Given this request, we have revised “follow-up” to “subsequent”.

(5) The idea that DNa02 is high-gain because it is more directly connected to motor neurons is a hypothesis and this should be made clear. We really don't know the functional consequences of the directness of a path or the number of synapses, and which circuits you compare to would change this. DNa02 may be a higher gain than DNa01, but what about relative to the other DNs that enter pre-motor regions? How do you handle a few synapses and several neurons in a common class? All of these connectivity-based deductions await functional tests - like yours! I think it is better to make this clear so readers don't assume a higher level of certainty than we have.

The Reviewer asks how we handled few-synapse connections, and how we combined neurons in the same class. We apologize for not making this explicit in our original submission. We have now added this information to the Methods. Briefly, to select cell types for inclusion in Figures 7C, we identified all individual cells postsynaptic to PFL3 and presynaptic to DNa02, discarding any unitary connections with <5 synapses. We then grouped unitary connections by cell type, and then summed all synapse numbers within each connection group (e.g., summing all synapses in all PFL3→LAL126 connections). We then discarded connection groups having <200 synapses or <1% of a cell type’s pre- or postsynaptic total. Reported connection weights are per hemisphere, i.e. half of the total within each connection group. For Figure 7F we did the same, but now discarding connection groups having <70 synapses or <0.4% of a cell type’s pre- or postsynaptic total. In Figure S9, we used the same procedures for analyzing connections onto DNa01. 

We agree that it is tricky to infer function from connectome data, and this applies to motor neuron connectivity. We bring up DN connectivity onto motor neurons in two places. First, in the Results, we note that “steering filters (i.e., rotational and sideways velocity filters) were larger for DNa02 (Fig. 2A,B). This means that an impulse change in firing rate predicts a larger change in steering for this neuron. In other words, this result suggests that DNa02 operates with higher gain. This may be related to the fact that DNa02 makes more direct output synapses onto motor neurons (Fig. 1B) [emphasis added].” We feel this is a relatively conservative statement.

Subsequently, in the Discussion, we ask, “why does DNa02 precede larger and more rapid steering events? This may be due to the fact that DNa02 receives stronger and more direct input from key steering circuits in the brain (Fig. S9). It may also relate to the fact that DNa02 has more direct connections onto motor neurons (Fig. 1B) [emphasis added].” Again, we feel this is a relatively conservative statement.

To be sure, none of the motor neurons postsynaptic to DNa02 actually receive most of their synaptic input from DNa02 (or indeed any DN), and this is typical of motor neurons controlling leg muscles. Rather, leg motor neurons tend to get most of their input from interneurons rather than motor neurons (Cheong et al. 2024). Available data suggests that the walking rhythm originates with intrinsic VNC central pattern generators, and the DNs that influence walking do so, in large part, by acting on VNC interneurons. These points have been detailed in recent connectome analyses (see especially Cheong et al. 2024).

We are reluctant to broaden the scope of our connectome analyses to include other DNs for comparison, because we think these analyses are most appropriate to full-central-nervous-system-(CNS)-connectomes (brain and VNC together), which are currently under construction. Without a full-CNS-connectome, many of the DN axons in the VNC cannot be identified. In the future, we expect that full-CNS-connectomes will allow a systematic comparison of the input and output connectivity of all DN types, and probably also the tentative identification of new steering DNs. Those future analyses should generate new hypotheses about the specializations of DNa02, DNa01, and other DNs. Our study aims to help lay a conceptual foundation for that future work.

(6) Given the emphasis on the DNa02 to Motor Neuron connectivity shown (Figure 1B) and multiple text mentions, could you include more analyses of which motor neurons are downstream and how these might be expected to affect leg movements? I would like to see the synapse numbers (Figure 1B) as well as the fraction of total output synapses. These additions would help understand the evidence for the "see-saw" model.

We agree this is interesting. In follow-up work from our lab (Yang et al., 2023), we describe the detailed VNC connectivity linking DNa02 to motor neurons. We refer the Reviewer specifically to Figure 7 of that study (https://www.cell.com/cell/fulltext/S0092-8674(24)00962-0).

We regret that the see-saw model was perhaps not clear in our original submission. Briefly, this model proposes that an increase in excitatory synaptic input to one DN (and/or a disinhibition of that DN) is often accompanied by an increase in inhibitory synaptic input to the contralateral DN. This model is motivated by connectome data on the brain inputs to DNa02 (Figure 7), along with our observation that excitation of one DN is often accompanied by inhibition of the contralateral DN (Figure 5). We have now added text to the Results in several places in order to clarify these points. 

This model specifically pertains to the brain inputs to DNs, comparing the downstream targets of these DNs in the VNC would not be a test of this hypothesis. The Reviewer may be asking to see whether there is any connectivity in the brain from one DN to its contralateral partner. We do not find connections of this sort, aside from multisynaptic connections that rely on very weak links (~10 synapses per connection). Figure 7 depicts a much stronger basis for this hypothesis, involving feedforward see-saw connections from PFL3 and MBON32. 

(7) The conclusions from the data in Figure 8 could be explained more clearly. These seem like small effect sizes on subtle differences in leg movements - maybe like what was seen in granular control by Moonwalker's circuits? Measuring joint angles or step parameters might help clarify, but a summary description would help the reader.

We agree that these results were not explained very well in our original submission. 

In our revised manuscript, we have added a new paragraph to the end of this Results section providing some summary and interpretation:

“All these effects are weak, and so they should be interpreted with caution. However, they suggest that both DNs can lengthen the stance phase of the ipsilateral back leg, which would promote ipsiversive turning. These results are also compatible with a scenario where both DNs decrease the step length in the ipsilateral legs, which would also promote ipsiversive turning. Step frequency does not normally change asymmetrically during turning, so the observed decrease in step frequency during optogenetic inhibition may just be a by-product of increasing step length when these DNs are inhibited.”

Moreover, in the Discussion, we have also added a new paragraph that synthesizes these results with other results in our study, while also noting the limitations of our study:

“Our study does not fully answer the question of how these DNs affect leg kinematics, because we were not able to simultaneously measure DN activity and leg movement. However, our optogenetic experiments suggest that both DNs can lengthen the stance phase of the ipsilateral back leg (Fig. 8G), and/or  decrease the step length in the ipsilateral legs (Fig. 8H), either of which would promote ipsiversive turning. If these DNs have similar qualitative effects on leg kinematics, then why does DNa02 precede larger and more rapid steering events? This may be due to the fact that DNa02 receives stronger and more direct input from key steering circuits in the brain (Fig. S9). It may also relate to the fact that DNa02 has more direct connections onto motor neurons (Fig. 1B).”

In Figure 8D-H, we measure step parameters in freely walking flies during acute optogenetic inhibition of DNa01 and DNa02. In experiments measuring neural activity in flies walking on a spherical treadmill, we did not have a way to measure step parameters. Subsequently, this methodology was developed by Yang et al. (2023) and results for DNa02 are described in that study. 

Reviewer #3 (Recommendations for the authors):

Minor Points:

(1) If space allows, actual membrane potential should be mentioned when raw recordings are shown (for example Figure 1D).

We have now added absolute membrane potential information to Figure 1d.

(2) Typo in the sentence "To address this issue directly, we looked closely at the timing of each cell's recruitment in our dual recordings, and found that spike rate fluctuations in DNa02 typically preceded the spike rate fluctuations in DNa02 (Fig. 2A)." The final word should be "DNa01".

Fixed. Thank you for reading the manuscript carefully.

(3) Figure 2A - although there aren't direct connections between a01 and a02 in the connectome, the authors never rule out functional connectivity between these two. Given a02 precedes a01, shouldn't this be addressed?

In the full brain FAFB data set, there are two disynaptic connections from DNa02 onto the ipsilateral copy of DNa01. One connection is via CB0556 (which is GABAergic), and the other is via LAL018 (which is cholinergic). The relevant DNa02 output connections are very weak: each DNa02→CB0556 connection consists of 11 synapses, whereas each DNa02→LAL018 connection consists of 10 synapses (on average). Conversely, each CB0556→DNa01 connection consists of 29 synapses, whereas  each LAL018→DNa01 connection consists of 64 synapses. In short, LAL018 is a nontrivial source of excitatory input to DNa01, but DNa02 is not positioned to exert much influence over LAL018, and the two disynaptic connections from DNa02 onto DNa01 also have the opposite sign. Thus, it seems unlikely that DNa02 is a major driver of DNa01 activity. At the same time, it is difficult to completely exclude this possibility, because we do not understand the logic of the very complicated premotor inputs to these DNs in the brain. Thus, we are hesitant to make a strong statement on this point.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Cognitive and brain development during the first two years of life is vast and determinant for later development. However, longitudinal infant studies are complicated and restricted to occidental high-income countries. This study uses fNIRS to investigate the developmental trajectories of functional connectivity networks in infants from a rural community in Gambia. In addition to resting-state data collected from 5 to 24 months, the authors collected growing measures from birth until 24 months and administrated an executive functioning task at 3 or 5 years old.

The results show left and right frontal-middle and right frontal-posterior negative connections at 5 months that increase with age (i.e., become less negative). Interestingly, contrary to previous findings in high-income countries, there was a decrease in frontal interhemispheric connectivity. Restricted growth during the first months of life was associated with stronger frontal interhemispheric connectivity and weaker right frontal-posterior connectivity at 24 months. Additionally, the study describes that some connectivity patterns related to better cognitive flexibility at pre-school age.

Strengths:

- The authors analyze data from 204 infants from a rural area of Gambia, already a big sample for most infant studies. The study might encourage more research on different underrepresented infant populations (i.e., infants not living in occidental high-income countries).

- The study shows that fNIRS is a feasible instrument to investigate cognitive development when access to fMRI is not possible or outside a lab setting.

- The fNIRS data preprocessing and analysis are well-planned, implemented, and carefully described. For example, the authors report how the choices in the parameters for the motion artifacts detection algorithm affect data rejection and show how connectivity stability varies with the length of the data segment to justify the threshold of at least 250 seconds free of artifacts for inclusion.

- The authors use proper statistical methods for analysis, considering the complexity of the dataset.

We thank the reviewer for highlighting the strengths of this work.

Weaknesses:

- No co-registration of the optodes is implemented. The authors checked for correct placement by looking at pictures taken during the testing session. However, head shape and size differences might affect the results, especially considering that the study involves infants from 5 months to 24 months and that the same fNIRS array was used at all ages.

The fNIRS array used in this work was co-registered onto age-appropriate MNI templates at every time point in a previous published work L. H. Collins-Jones, et al., Longitudinal infant fNIRS channel-space analyses are robust to variability parameters at the group-level: An image reconstruction investigation. Neuroimage 237, 118068 (2021). This is reference No. 68 in the manuscript.

As we mentioned in the section fNIRS preprocessing and data-analysis: ‘The sections were established via the 17 channels of each hemisphere which were grouped into front, middle and back (for a total of six regions) based on a previous co-registration of the BRIGHT fNIRS arrays onto age-appropriate templates’. The procedure mentioned by the reviewer, involving the examination of pictures showing the placement of headbands on participants, aimed to exclude infants with excessive cap displacement from further analysis.

- The authors regress the global signal to remove systemic physiological noise. While the authors also report the changes in connectivity without global signal regression, there are some critical differences. In particular, the apparent decrease in frontal inter-hemispheric connections is not present when global signal regression is omitted, even though it is present for deoxy-Hb. The authors use connectivity results obtained after applying global signal regression for further analysis. The choice of regressing the global signal is questionable since it has been shown to introduce anti-correlations in fMRI data (Murphy et al., 2009), and fNIRS in young infants does not seem to be highly affected by physiological noise (Emberson et al., 2016). Systemic physiological noise might change at different ages, which makes its remotion critical to investigate functional network development. However, global signal regression might also affect the data differently. The study would have benefited from having short separation channels to measure the systemic psychological component in the data.

The work of Emberson et. al (2016) mentioned by the reviewer highlights indeed the challenges of removing systemic changes from the infants’ haemodynamic signal with short-channel separation (SSC). In fact, even a SSC of 1 cm detected changes in the blood in the brain, therefore by regressing this signal from the recorded one, the authors removed both systemic changes AND haemodynamic signal. This paper from Emberson et. al (2016) is taken as a reference in the field to suggest that SSC might not be an ideal tool to remove systemic changes when collecting fNIRS data on young infants, as we did in this work.

We agree with the reviewer's observation that systemic physiological noise may vary with age and among infants. Therefore, for each infant at each age, we regressed the mean value calculated across all channels. This ensures that the regressed signal is not biased by averaged calculations at group levels.

We are aware of the criticisms directed towards global signal regression in the fMRI literature, although some other works showed anticorrelations in functional connectivity networks both with and without global signal regression (Chaia, 2012). Furthermore, Murphy himself revised his criticism on the use of global signal regression in functional connectivity analysis in one of his more recent works (Murphy et al, 2017). The fact that the decreased FC is significant in results from data pre-processed without global signal regression gives us confidence that this finding is statistically robust and not solely driven by this preprocessing choice in our pipeline.

An interesting study by Abdalmalak et al. (2022) demonstrated that failing to correct for systemic changes using any method is inappropriate when estimating FC with fNIRS, as it can lead to a high risk of elevated connectivity across the whole brain (see Figure 4 of the mentioned paper). Consequently, we strongly advocate for the implementation of global signal regression in our analysis pipeline as a fundamental step for accurate functional connectivity estimations.

References:

Emberson, L. L., Crosswhite, S. L., Goodwin, J. R., Berger, A. J., & Aslin, R. N. (2016). Isolating the effects of surface vasculature in infant neuroimaging using short-distance optical channels: a combination of local and global effects. Neurophotonics, 3(3), 031406-031406.

Chaia, X. J., Castañóna, A. N., Öngürb, D., & Whitfield-Gabrielia, S. (2012). Anticorrelations in resting state networks without global signal regression. NeuroImage, 59(2), 1420–1428. https://doi.org/10.1515/9783050076010-014

Murphy, K., & Fox, M. D. (2017). Towards a consensus regarding global signal regression for resting state functional connectivity MRI. NeuroImage, 154(November 2016), 169–173. https://doi.org/10.1016/j.neuroimage.2016.11.052

Abdalmalak, A., Novi, S. L., Kazazian, K., Norton, L., Benaglia, T., Slessarev, M., ... & Owen, A. M. (2022). Effects of systemic physiology on mapping resting-state networks using functional near-infrared spectroscopy. Frontiers in neuroscience, 16, 803297.

- I believe the authors bypass a fundamental point in their framing. When discussing the results, the authors compare the developmental trajectories of the infants tested in a rural area of Gambia with the trajectories reported in previous studies on infants growing in occidental high-income countries (likely in urban contexts) and attribute the differences to adverse effects (i.e., nutritional deficits). Differences in developmental trajectories might also derive from other environmental and cultural differences that do not necessarily lead to poor cognitive development.

We agree with the reviewer that other factors differing between low- and poor-resource settings might have an impact on FC trajectories. We therefore specified this in the discussion as follows: “We acknowledge that differences in FC could also be attributed to other environmental and cultural disparities between high-resource and low-resource settings, and future studies are needed to investigate this further” (line 238).

- While the study provides a solid description of the functional connectivity changes in the first two years of life at the group level, the evidence regarding the links between adverse situations, developmental trajectories, and later cognitive capacities is weaker. The authors find that early restricted growth predicts specific connectivity patterns at 24 months and that certain connectivity patterns at specific ages predict cognitive flexibility. However, the link between development trajectories (individual changes in connectivity) with growth and later cognitive capacities is missing. To address this question adequately, the study should have compared infants with different growing profiles or those who suffered or did not from undernutrition. However, as the authors discussed, they lacked statistical power.

We agree with the reviewer, and indeed we highlighted this as one of the main limitation of our work: “Even given the large sample in our study, we were underpowered to test for group comparisons between sets of infants with distinct undernutrition growth profiles, e.g., infants with early poor growth that later resolved and infants with standard growth early that had a poor growth later. We were also underpowered to test the associations between early growth and FC on clinically undernourished infants (defined as having DWLZ two standard deviations below the mean) (line 311, discussion section).

We believe this is an important point to consider for the field, as it addresses the sample size required for studies investigating brain development in clinically malnourished infants. We hope this will serve as a valuable reference for future studies in the field. For example, a new study led by Prof. Sophie Moore and other members of the BRIGHT team (INDiGO) is currently recruiting six-hundreds pregnant women with the aim of obtaining a broader distribution of infants’ growth measures (https://www.kcl.ac.uk/research/sophie-moore-research-group).

Reviewer #2 (Public Review):

Summary and strengths:

The article pertains to a topic of importance, specifically early life growth faltering, a marker of undernutrition, and how it influences brain functional connectivity and cognitive development. In addition, the data collection was laborious, and data preprocessing was quite rigorous to ensure data quality, utilizing cutting-edge preprocessing methods.

We thank the reviewer for highlighting the strengths of this work.

Weaknesses:

However, the subsequent analysis and explanations were not very thorough, which made some results and conclusions less convincing. For example, corrections for multiple tests need to be consistently maintained; if the results do not survive multiple corrections, they should not be discussed as significant results. Additionally, alternative plans for analysis strategies could be worth exploring, e.g., using ΔFC in addition to FC at a certain age. Lastly, some analysis plans lacked a strong theoretical foundation, such as the relationship between functional connectivity (FC) between certain ROIs and the development of cognitive flexibility.

Thus, as much as I admire the advanced analysis of connectivity that was conducted and the uniqueness of longitudinal fNIRS data from these samples (even the sheer effort to collect fNIRS longitudinally in a low-income country at such a scale!), I have reservations about the importance of this paper's contribution to the field in its present form. Major revisions are needed, in my opinion, to enhance the paper's quality. 

We acknowledge the reviewer’s concern regarding the reporting of results that do not survive multiple comparisons. However, considering the uniqueness of our dataset and the novelty of our work, we believe it is crucial to report all significant findings as well as hypothesis-generating findings that may not pass stringent significance thresholds. We have taken great care to transparently distinguish between results that survived multiple comparisons and those that did not in both the Results and Discussion sections, ensuring that readers are not misled. It is possible that future studies may replicate and further strengthen these associations. Therefore, by sharing these results with the research community, we provide valuable insights for future investigations.

The relationship between FC and cognitive flexibility (as well as the relationship between growth and FC) has been explored focusing on those FC that showed a significant change with age, as specified in the results sections: ‘To investigate the impact of early nutritional status on FC at 24 months, we used multiple regression with the infant growth trajectory [...] and FC at 24 months [...]. To maximise power, we considered only those FC that showed a statistically significant change with age’ (line 183) and ‘To investigate whether FC early in life predicted cognitive flexibility at preschool age, we used multiple regression of FC across the first two years of life against later cognitive flexibility in preschoolers at three and five years. As per the analysis above, we focused on only those FC that showed a statistically significant change with age’ (line 198).

We explored the possibility of investigating the relationship between changes in FC and changes in growth. However, the degrees of freedom in these analyses dropped dramatically (~25/30), thereby putting the significance and the meaning of the results at risk. We look forward to future longitudinal studies with less attrition across these time points to maintain the statistical power necessary to run such analyses.

Reviewer #3 (Public Review):

Summary:

This study aimed to investigate whether the development of functional connectivity (FC) is modulated by early physical growth and whether these might impact cognitive development in childhood. This question was investigated by studying a large group of infants (N=204) assessed in Gambia with fNIRS at 5 visits between 5 and 24 months of age. Given the complexity of data acquisition at these ages and following data processing, data could be analyzed for 53 to 97 infants per age group. FC was analyzed considering 6 ensembles of brain regions and thus 21 types of connections. Results suggested that: i) compared to previously studied groups, this group of Gambian infants have different FC trajectory, in particular with a change in frontal inter-hemispheric FC with age from positive to null values; ii) early physical growth, measured through weight-for-length z-scores from birth on, is associated with FC at 24 months. Some relationships were further observed between FC during the first two years and cognitive flexibility at 4-5 years of age, but results did not survive corrections for multiple comparisons.

Strengths:

The question investigated in this article is important for understanding the role of early growth and undernutrition on brain and behavioral development in infants and children. The longitudinal approach considered is highly relevant to investigate neurodevelopmental trajectories. Furthermore, this study targets a little-studied population from a low-/middle-income country, which was made possible by the use of fNIRS outside the lab environment. The collected dataset is thus impressive and it opens up a wide range of analytical possibilities.

We thank the reviewer for highlighting the strengths of this work.

Weaknesses:

- Analyzing such a huge amount of collected data at several ages is not an easy task to test developmental relationships between growth, FC, and behavioral capacities. In its present form, this study and the performed analyses lack clarity, unity and perhaps modeling, as it suggests that all possible associations were tested in an exploratory way without clear mechanistic hypotheses. Would it be possible to specify some hypotheses to reduce the number of tests performed? In particular, considering metrics at specific ages or changes in the metrics with age might allow us to test different hypotheses: the authors might clarify what they expect specifically for growth-FC-behaviour associations. Since some FC measures and changes might be related to one another, would it be reasonable to consider a dimensionality reduction approach (e.g., ICA) to select a few components for further correlation analyses?

We confirm that this work was motivated by a compelling theoretical question: whether neural mechanisms, specifically FC, can be influenced by early adversity, such as growth, and subsequently impact cognitive outcomes, such as cognitive flexibility. This aligns with the overarching goal of the BRIGHT project, established in 2015 (Lloyd-Fox, 2023). We believe this was evident throughout the manuscript in several instances, for example:

- “The goal of the study was to investigate early physical growth in infancy, developmental trajectories of brain FC across the first two years of life, and cognitive outcome at school age in a longitudinal cohort of infants and children from rural Gambia, an environment with high rates of maternal and child undernutrition. Specifically, we aimed to: (i) investigate whether differences in physical growth through the first two years of life are related to FC at 24 months, and (ii) investigate if trajectories of early FC have an impact on cognitive outcome at pre-school age in these children.” (page 4, introduction)

- “This study investigated how early adversity via undernutrition drives longitudinal changes in brain functional connectivity at five time points throughout the first two years of life and how these developmental trajectories are associated with cognitive flexibility at preschool age.” (page 6, discussion)

- We had a clear hypothesis regarding short-range connectivity decreasing with age and long-range connectivity increasing with age, as stated at the end of the introduction: We hypothesized that (i) long-range FC would increase and short-range FC would decrease throughout the first two years of life” (page 4, line 147). However, we were not able to formulate clear hypotheses about the localization of these connections due to the scarcity of previous studies conducted within this age range, particularly in low-resource settings. The ROI approach for analysis was chosen to mitigate this challenge by reducing the number of comparisons while still enabling us to estimate the developmental trajectories of all the connections from which we acquired data.

Regarding the use of dimensionality reduction approach, we have not considered the use of ICA in our analysis. These methods require selecting a fixed number of components to remove from all participants. However, due to the high variability of infant fNIRS data across the five timepoints, we considered it untenable to precisely determine the number of components to remove at the group level. Such a procedure carries the risk of over-cleaning the data for some participants while leaving noise in for others (Di Lorenzo, 2019). We also felt that using PCA in this initial study would be beyond the scope of the brain-region-specific hypotheses and would be more appropriate in a follow-up analysis of these important data.

References:

Lloyd-Fox, S., McCann, S., Milosavljevic, B., Katus, L., Blasi, A., Bulgarelli, C., Crespo-Llado, M., Ghillia, G., Fadera, T., Mbye, E., Mason, L., Njai, F., Njie, O., Perapoch-Amado, M., Rozhko, M., Sosseh, F., Saidykhan, M., Touray, E., Moore, S. E., … Team, and the B. S. (2023). The Brain Imaging for Global Health (BRIGHT) Study: Cohort Study Protocol. Gates Open Research, 7(126).

Di Lorenzo, R., Pirazzoli, L., Blasi, A., Bulgarelli, C., Hakuno, Y., Minagawa, Y., & Brigadoi, S. (2019). Recommendations for motion correction of infant fNIRS data applicable to multiple data sets and acquisition systems. NeuroImage, 200(April), 511–527.

- It seems that neurodevelopmental trajectories over the whole period (5-24 months) are little investigated, and considering more robust statistical analyses would be an important aspect to strengthen the results. The discussion mentions the potential use of structural equation modelling analyses, which would be a relevant way to better describe such complex data.

We appreciate the complexity of the dataset we are working with, which includes multiple measures and time points. Currently, our focus within the outputs from the BRIGHT project is on examining the relationship between selected measures. While this may not involve statistically advanced modelling at the moment, it is worth noting that most of the results presented in this work have survived correction for multiple comparisons, indicating their statistical robustness. We believe that more advanced statistical analyses are beyond the scope of this rich initial study. In the next phase of the project, known as BRIGHT IMPACT, our team is collaborating with statisticians and experts in statistical modelling to apply more sophisticated and advanced statistical techniques to the data.

- Given the number of analyses performed, only describing results that survive correction for multiple comparisons is required. Unifying the correction approach (FDR / Bonferroni) is also recommended. For the association between cognitive flexibility and FC, results are not significant, and one might wonder why FC at specific ages was considered rather than the change in FC with age. One of the relevant questions of such a study would be whether early growth and later cognitive flexibility are related through FC development, but testing this would require a mediation analysis that was not performed.

We acknowledge the reviewer’s concern regarding the reporting of results that do not survive multiple comparisons. However, considering the uniqueness of our dataset and the novelty of our work, we believe it is crucial to report all significant findings. We have taken great care to transparently distinguish between results that survived multiple comparisons and those that did not in both the Results and Discussion sections, ensuring that readers are not misled. It is possible that future studies may replicate and further strengthen these associations. Therefore, by sharing these results with the research community, we provide valuable insights for future investigations.

We did not perform a mediation analysis as i) ΔWLZ between birth and the subsequent time points positively predicted frontal interhemispheric FC at 24 months, ii) frontal interhemispheric FC at 18 months (and right fronto-posterior connectivity at 24 months) predicted cognitive flexibility at preschool age. Considering that the frontal interhemispheric FC at 24 months that was positively predicted by growth, did not significantly predicted cognitive outcome at preschool age, we did not perform mediation models.

The reviewer raised concerns about using different methods to correct for multiple comparisons throughout the work. Results showing changes in FC with age were Bonferroni corrected, while we used FDR correction for the regression analyses investigating the relationship between growth and FC, as well as FC and cognitive flexibility. Both methods have good control over Type I errors (false positives), but Bonferroni is very conservative, increasing the likelihood of Type II errors (false negatives). We considered Bonferroni an appropriate method for correcting results showing changes in FC with age, where we had a large sample with strong statistical power (i.e. linear mixed models with 132 participants who had at least 250 seconds of good data for 2 out of 5 visits). However, Bonferroni was too conservative for the regression analyses, with N between 57 and 78) (Acharya, 2014; Félix & Menezes, 2018; Narkevich et al., 2020; Narum, 2006; Olejnik et al., 1997).

References:

Acharya, A. (2014). A Complete Review of Controlling the FDR in a Multiple Comparison Problem Framework--The Benjamini-Hochberg Algorithm. ArXiv Preprint ArXiv:1406.7117.

Félix, V. B., & Menezes, A. F. B. (2018). Comparisons of ten corrections methods for t-test in multiple comparisons via Monte Carlo study. Electronic Journal of Applied Statistical Analysis, 11(1), 74–91.

Narkevich, A. N., Vinogradov, K. A., & Grjibovski, A. M. (2020). Multiple comparisons in biomedical research: the problem and its solutions. Ekologiya Cheloveka (Human Ecology), 27(10), 55–64.

Narum, S. R. (2006). Beyond Bonferroni: less conservative analyses for conservation genetics. Conservation Genetics, 7, 783–787.

Olejnik, S., Li, J., Supattathum, S., & Huberty, C. J. (1997). Multiple testing and statistical power with modified Bonferroni procedures. Journal of Educational and Behavioral Statistics, 22(4), 389–406.

- Growth is measured at different ages through different metrics. Justifying the use of weight-for-length z-scores would be welcome since weight-for-age z-scores might be a better marker of growth and possible undernutrition (this impacting potentially both weight and length). Showing the distributions of these z-scores at different ages would allow the reader to estimate the growth variability across infants.

We consistently used WLZ as the metric to measure growth throughout. Our analysis investigating the relationship between WLZ and growth included HCZ at 7/14 days to correct for head size at birth. When selecting the best growth measure for this paper, we opted for WLZ over WAZ, given extant evidence that infants in our sample are smaller and shorter compared to the reference WHO standard for the same age group (Nabwera et al., 2017). Therefore, using WLZ allows us to adjust each infant's weight for its own length.

References:

Nabwera, H. M., Fulford, A. J., Moore, S. E., & Prentice, A. M. (2017). Growth faltering in rural Gambian children after four decades of interventions: a retrospective cohort study. The Lancet Global Health, 5(2), e208–e216.

- Regarding FC, clarifications about the long-range vs short-range connections would be welcome, as well as drawing a summary of what is expected in terms of FC "typical" trajectory, for the different brain regions and connections, as a marker of typical development. For instance, the authors suggest that an increase in long-range connectivity vs a decrease in short-range is expected based on previous fNIRS studies. However anatomical studies of white matter growth and maturation would suggest the reverse pattern (short-range connections developing mostly after birth, contrarily to long-range connections prenatally).

We expected an increase in long-range functional connectivity with age, as discussed in the introduction:

- “Based on data from fMRI, current models hypothesize that FC patterns mature throughout early development (23–27), where in typically developing brains, adult-like networks emerge over the first years of life as long-range functional connections between pre-frontal, parietal, temporal, and occipital regions become stronger and more selective (28–31). This maturation in FC has been shown to be related to the cascading maturation of myelination and synaptogenesis (32, 33) - fundamental processes for healthy brain development (34)” (line 93, page 3, introduction);

- “Importantly, normative developmental patterns may be disrupted and even reversed in clinical conditions that impact development; e.g., increased short-range and reduced long-range FC have been observed in preterm infants (36) and in children with autism spectrum disorder (37, 38)” (line 103, page 3, introduction);

- “We hypothesized that (i) long-range FC would increase and short-range FC would decrease throughout the first two years of life” (line 147, page 4, introduction).

Since inferences about FC patterns recorded with fNIRS are highly limited by the number and locations of the optodes, it is challenging to make strong inferences about specific brain regions. Moreover, infant FC fNIRS studies are still limited, which is why we focused our inferences on long-range versus short-range connectivity, without specifically pinpointing particular brain regions.

Additionally, were unable to locate the works mentioned by the reviewer regarding an increase in short-range white matter connectivity immediately after birth. On the contrary, we found several studies documenting an increase in white-matter long-range connectivity after birth, which is consistent with the hypothesised increase in FC long-range connectivity, such as:

Yap, P. T., Fan, Y., Chen, Y., Gilmore, J. H., Lin, W., & Shen, D. (2011). Development trends of white matter connectivity in the first years of life. PloS one, 6(9), e24678.

Dubois, J., Dehaene-Lambertz, G., Kulikova, S., Poupon, C., Hüppi, P. S., & Hertz-Pannier, L. (2014). The early development of brain white matter: a review of imaging studies in fetuses, newborns and infants. Neuroscience, 276, 48-71.

Stephens, R. L., Langworthy, B. W., Short, S. J., Girault, J. B., Styner, M. A., & Gilmore, J. H. (2020). White matter development from birth to 6 years of age: a longitudinal study. Cerebral Cortex, 30(12), 6152-6168.

Hagmann, P., Sporns, O., Madan, N., Cammoun, L., Pienaar, R., Wedeen, V. J., ... & Grant, P. E. (2010). White matter maturation reshapes structural connectivity in the late developing human brain. Proceedings of the National Academy of Sciences, 107(44), 19067-19072.

Collin G, van den Heuvel MP. The ontogeny of the human connectome: development and dynamic changes of brain connectivity across the life span. Neuroscientist. 2013 Dec;19(6):616-28. doi: 10.1177/1073858413503712.

The authors test associations between FC and growth, but making sense of such modulation results is difficult without a clearer view of developmental changes per se (e.g., what does an early negative FC mean? Is it an increase in FC when the value gets close to 0? In particular, at 24m, it seems that most FC values are not significantly different from 0, Figure 2B). Observing positive vs negative association effects depending on age is quite puzzling. It is also questionable, for some correlation analyses with cognitive flexibility, to focus on FC that changes with age but to consider FC at a given age.

We thank the reviewer for bringing up this important point and understand that it requires some additional consideration. The negative FC values decreasing with age indicate that these regions go from being anti-correlated to becoming increasingly correlated. Hence, FC of these ROIs increased with age. The trajectory seems to suggest that this will keep increasing with age but of course further data need to be collected to assess this.

Unfortunately, when considering ΔFC to predict cognitive flexibility, the numbers of participants dropped significantly, with N=~15/20 infants per group of preschoolers, making it very challenging to interpret the results with meaningful statistical power.

- The manuscript uses inappropriate terms "to predict", "prediction" whereas the conducted analyses are not prediction analyses but correlational.

We thank the reviewer for giving us to opportunity to thoroughly revise the manuscript about this matter. In this work, we had clear hypotheses regarding which variables predicted which certain measures (such as growth predicting FC and FC predicting cognitive outcomes). Therefore, we performed regression analyses rather than correlational analyses to investigate these associations. Hence, we believe that using the term ‘predict and ‘prediction’ is appropriate

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) In the introduction and discussion, the authors talk about the link between developmental trajectories and cognitive capacities, and undernutrition. However, they did not compare developmental trajectories but connectivity patterns at different ages with ΔWLZ and cognitive flexibility. I recommend that the authors rephrase the introduction and discussion.

We thank the reviewer for pointing out places requiring better clarity in the text. We made edits through the introduction to better match our investigations. In particular we changed:

- ‘our understanding of the relationships between early undernutrition, developmental trajectories of brain connectivity, and later cognitive outcomes is still very limited,’ to, ‘our understanding of the relationships between early undernutrition, brain connectivity, and later cognitive outcomes is still very limited’ (line 89, introduction);

- ‘(ii) investigate if trajectories of early FC have an impact on cognitive outcome at pre-school age in these children,’ to, ‘(ii) investigate if early FC has an impact on cognitive outcome at pre-school age in these children’ (line 137, introduction);

- ‘This study investigated how early adversity via undernutrition drives longitudinal changes in brain functional connectivity at five time points throughout the first two years of life and how these developmental trajectories are associated with cognitive flexibility at preschool age,’ to, ‘This study investigated how early adversity via undernutrition drives brain functional connectivity throughout the first two years of life and how these early functional connections are associated with cognitive flexibility at preschool age’ (line 215, discussion).

(2) Considering most research is done in occidental high-income countries, and this work is one of the few presenting research in another context, I think the authors should discuss in the manuscript that differences with previous studies might also be due to environmental and cultural differences. Since the study lacks the statistical power to perform a statistical analysis that directly establishes a link between developmental trajectories and restricted growth and cognitive flexibility, the authors cannot disentangle which differences are related to undernutrition and which might result from growing up in a different environment. I recommend that the authors avoid phrases like (lines 57-58): "We observed that early physical growth before the fifth month of life drove optimal developmental trajectories of FC..." or (lines 223-224) "...our cohort of Gambian infants exhibit atypical developmental trajectories of functional connectivity...".

We thank the reviewer for this observation, and we agree with the reviewer that other factors differing between low- and poor-resource settings might have an impact on FC trajectories. We therefore specified this in the discussion as follows: “We acknowledge that differences in FC could also be attributed to other environmental and cultural disparities between high-resource and low-resource settings, and future studies are needed to explore this further” (line 238). We revised the whole manuscript to reflect similar statements.

(3) To better interpret the results, it would be interesting to know if poor early growth predicts late cognitive flexibility in the tested sample and if the ΔWLZ distributions differ compared to a population in a high-income country where undernutrition is less frequent.

We explored the relationship between changes in growth and cognitive flexibility in the two preschooler group, but there were no significant associations.

Mean and SD values of WLZ are reported in Table 3. The values at every age are negative, indicating that the infants' weight-for-length is below the expected norm at all ages. To our knowledge, no other studies have assessed changes in growth in an infant sample with similar closely spaced age time points in high-income countries, making comparisons on growth changes challenging.

(4) It is unclear why WLZ at birth and HCZ at 7-14 days are included in the models. I imagine this is to ensure that differences are not due to growing restrictions before birth. It would be nice if the authors could explain this.

As the reviewer pointed out, HCZ at 7-14 days was included to ensure associations between growth and FC are not due to physical differences at birth. This case be considered as a 'baseline' measure for cerebral development, in the same way that WLZ at birth was used as a baseline for physical development. Therefore, we can more confidently  assume that the associations between growth and FC were specific to the impact of change in WLZ postnatally and not confounded by the size or maturity of the infant at birth. We specified this in the manuscript as follows: “These analyses were adjusted by WLZ at birth and HCZ at 7/14 days, to more confidently assume that the associations between growth and FC were specific to the impact of change in WLZ postnatally and not confounded by the size or maturity of the infant at birth” (line 520, statistical analysis section in the method section).

(5) Right frontal-posterior connections at 24 months negatively correlate with ΔWLZ. Thus, restricted growth results in stronger frontal-posterior connections at 24 months. However, the same connections at 24 months positively correlate with cognitive flexibility (stronger connections predict better cognitive flexibility). Do the authors have any interpretation of this? How could this relate to previous findings of the authors (Bulgarelli et al. 2020), showing first an increase and then a decrease in functional connectivity between frontal and parietal regions?

We acknowledge that interpreting the negative relationship between changes in growth and fronto-posterior FC at 24 months, alongside the positive association between the same connection and later cognitive flexibility, is challenging. We refrain from relating these findings to those published by Bulgarelli in 2020 due to differences in optode locations and because in that work the decrease in fronto-posterior FC was observed after 24 months (up to 36 months), whereas the endpoint in this study is right at 24 months.

(6) With the growth of the head, the frontal channels move to more temporal areas, right? Could this determine the decrease in frontal inter-hemisphere connections?

As shown in Nabwera (2017) head size does not increase that much in Gambian infants, or at least as expected by the WHO standard measures. We have added HCZ mean and SD values per age in Table 3.

Minor points

- HCZ is used in line 184 but not defined.

We thank the reviewer for spotting this, we have now specified HCZ at line 184 as follows: ‘head-circumference z-score (HCZ)’.

- Table SI2: NIRS not undertaken = the participant was assessed but did want or could not perform... I imagine there is a missing "not".

We thank the reviewer for spotting this, we have now modified the legend of Table SI2 as follows: ‘the participant was assessed but did not want or could not perform the NIRS assessments.’

- The authors should explain what weight-for-length is for those who are not familiar with it.

We have added an explanation of weight-for-length in the experimental design section, line 339 as follows: ‘We then tested for relationships between brain FC at age 24 months with measures of early growth, as indexed by changes in weight-for-length z-scores (reflecting body weight in proportion to attained growth in length) at one month of age, and at each of the four subsequent visits (details provided below).’

Reviewer #2 (Recommendations For The Authors):

(1) I am confused about the authors' interpretation that left and right front-middle and right front-back FC increased with age. It appears in Figure 2 that the negative FC among these ROIs should actually decrease with age. This means that as individuals grow older, the FC values between these regions and zero diminished, albeit starting with negative FC (anticorrelation values) in younger age groups.

Yes, the reviewer is correct. The negative values of the left and right front-middle and right front-back FC decreasing with age indicate that these regions go from being anti-correlated to becoming increasingly correlated. Hence, FC of these ROIs increased with age.

(2) Are these negative values mentioned above at 24 months still negative? Have t-tests been run to examine the differences from zero?

As suggested, we performed t-tests against zero for the mentioned FC at 24 months, and only the left and right fronto-middle FC are significantly different than zero (left fronto-middle FC: t(94) = 1.8, p = 0.036; right fronto-middle FC t(94) = 2.7, p = 0.003).

(3) With so many correlation analyses, have multiple comparisons been consistently controlled for? While I assume this was done according to the Methods section, could the authors clarify whether FDR adjustment was applied to all the p-values at once or to a group of p-values each time? I found the following way of reporting FDR-adjusted p-values quite informative, such as PFDR, 24 pairs of ROIs < 0.05.

We thank the reviewer for this insightful comment. P-values of regression analyses were FDR corrected per connection investigated, i.e. 21 possible ΔWLZ values per connection. We have specified this in the method section as follows: “To ensure statistical reliability, results from the regression analyses on each FC were corrected for multiple comparisons using false discovery rate (FDR)(Benjamini & Hochberg, 1995) per each connection investigated, i.e. 21 possible ΔWLZ values per each connection,” (page 12, Statistical Analyses section).

(4) Can early growth trajectories predict changes in FC? Why not use ΔWLZ to predict ΔFC?

Unfortunately, when considering ΔWLZ to predict ΔFC, the numbers of participants dropped significantly, with N=~30 infants, making it very challenging to interpret the results. We believe this emphasizes the importance of recruiting large samples when conducting longitudinal studies involving infants and employing multiple measures.

(5) I might have missed the rationale, but why weren't the growth changes after 5 months studied?

ΔWLZ between all time points were assessed as predictors of FC at 24 months. We have specified this at line 183 as follows: ‘we used multiple regression with the infant growth trajectory (delta weight for length z-score between all time points, DWLZ) and FC at 24 months’. As indicated in Table 2 and 3 the associations between ΔWLZ at all time points and FC at 24 months were tested, but only those with DWLZ calculated between birth and 1 month and the subsequent time points were significant. DWLZ between 5 months and the subsequent time points, DWLZ between 8 months and the subsequent time points, DWLZ between 12 months and the subsequent time points, DWLZ between 18 months and the subsequent time points did not significantly predict FC at 24 months. These are highlighted in Table 2 and Figure 3 in blue and marked as NS (non-significant).

(6) Once more, the advantage of longitudinal data is that it allows us to tap into developmental changes. Analyzing and predicting cognitive development based solely on FC values at a single age stage (i.e., 24 months) would overlook the benefits of a longitudinal design, which is regrettable. I suggest that the authors attempt to use ΔFC for predictions and observe the outcomes.

As mentioned to point (4) raised by the reviewer, unfortunately, when considering ΔWLZ to predict ΔFC, the numbers of participants dropped significantly, with N=~30 infants, making it very challenging to interpret the results. We believe this emphasizes the importance of recruiting large samples when conducting longitudinal studies involving infants and employing various measures.

(7) In the section "Early FC predicts cognitive flexibility at preschool age", the authors pointed out that "...,none of these survived FDR correction for multiple comparisons." However, the paper discussed the association between FC at 24 months of age and cognitive flexibility, as it was supported by the statistical analysis in the following sections. If FDR correction cannot be satisfied, I would rephrase the implication/conclusion of the results to suggest that early FC does not predict cognitive flexibility at preschool age.

We acknowledge the reviewer’s concern regarding the reporting of results that do not survive multiple comparisons. However, considering the uniqueness of our dataset and the novelty of our work, we believe it is crucial to report all significant findings, even those not passing multiple comparisons corrections, as they may motivate hypothesis-generation for future studies. We have taken great care to transparently distinguish between results that survived multiple comparisons and those that did not in both the Results and Discussion sections, ensuring that readers are not misled. It is possible that future studies may replicate and further support these associations. Therefore, by sharing these results with the research community, we provide valuable insights for future investigations.

Following the reviewer’ suggestion, we specified that results from regression analysis are significant but they did not survive multiple comparisons in the discussion as follows: ‘While our results are consistent with previous studies, we acknowledge that the significant association between early FC and later cognitive flexibility does not withstand multiple comparisons. Therefore, we encourage future studies that may replicate these findings with a larger sample. (line 290, discussion section).

(8) Have the authors assessed the impact of growth trajectories on cognitive flexibility?

We explored the relationship between changes in growth and cognitive flexibility in the two preschooler groups, but there were no significant associations.

(9) Are there no other cognitive or behavioural measures available? Cognitive flexibility is just one domain of cognitive development, and would the impact of undernutrition on cognitive development be domain-specific? There is a lack of theoretical support here. Why choose cognitive flexibility, and should the impact of undernutrition be domain-specific or domain-general?

We agree with the reviewer that in this work, we chose to focus on one specific cognitive outcome. While this does not imply that the impact of undernutrition is domain-specific, cognitive flexibility, being a core executive function, has been extensively studied in terms of its neural underpinnings using other neuroimaging modalities, especially fMRI (for example see Dajani, 2015; Uddin, 2021).

Moreover, other studies looking at the effect of adversity on cognitive outcomes focus on specific cognitive skills, such as working memory (Roberts, 2017), reading and arithmetic skills (Soni, 2021).

We did assess infants also with Mullen Scales of Early Learning (MSEL), although the cognitive flexibility task within the Early Years Toolbox has been specifically designed for preschoolers (Howard, 2015), and this set of tasks has recently been validated in our team in The Gambia (Milosavljevic, 2023).Future works from the BRIGHT team will investigate performance at the MSEL in relation to other variable of the project.

References:

D. R. Dajani, L. Q. Uddin, Demystifying cognitive flexibility: Implications for clinical and developmental neuroscience. Trends Neurosci. 38, 571–578 (2015).

L. Q. Uddin, Cognitive and behavioural flexibility: neural mechanisms and clinical considerations. Nat. Rev. Neurosci. 22, 167–179 (2021).

Roberts, S. B., Franceschini, M. A., Krauss, A., Lin, P. Y., de Sa, A. B., Có, R., ... & Muentener, P. (2017). A pilot randomized controlled trial of a new supplementary food designed to enhance cognitive performance during prevention and treatment of malnutrition in childhood. Current developments in nutrition, 1(11), e000885.

Soni, A., Fahey, N., Bhutta, Z. A., Li, W., Frazier, J. A., Moore Simas, T., ... & Allison, J. J. (2021). Early childhood undernutrition, preadolescent physical growth, and cognitive achievement in India: A population-based cohort study. PLoS Medicine, 18(10), e1003838.

Howard, S. J., & Melhuish, E. (2015). An Early Years Toolbox (EYT) for assessing early executive function, language, self-regulation, and social development: Validity, reliability, and preliminary norms. Journal of Psychoeducational Assessment, 35(3), 255-275.

Milosavljevic, B., Cook, C. J., Fadera, T., Ghillia, G., Howard, S. J., Makaula, H., ... & Lloyd‐Fox, S. (2023). Executive functioning skills and their environmental predictors among pre‐school aged children in South Africa and The Gambia. Developmental Science, e13407.

(10) I would review more previous fNIRS studies on infants if they exist (e.g., the work by S Lloyd-Fox, L Emberson, and others). These studies can help identify brain ROIs likely linked to undernutrition and cognitive flexibility. The current analysis methods lean towards exploratory research. This makes the paper more of a proof-of-concept report rather than a strongly theoretically-driven study.

We thank the reviewer for this important point. While we have reviewed existing fNIRS infant studies, there are no extant works that showed whether specific brain regions are related undernutrition. However, several fMRI studies assessed regions that do support cognitive flexibility, and we mentioned these in the manuscript (for example see Dajani, 2015; Uddin, 2021).

Other than the BRIGHT project, we are aware of two other projects that assessed the effect of undernutrition on brain development, assessing cognitive outcomes in poor-resource settings:

- the BEAN project in Bangladesh in which fNIRS data were recorded from the bilateral temporal cortex (i.e. Pirazzoli, 2022);

- a project in India in which fNIRS data were recorded from frontal, temporal and parietal cortex bilaterally (i.e. Delgado Reyes, 2020)

The brain regions recorded in these studies largely overlap with the brain regions we recorded from in this study.

Another aspect to consider is that infants underwent several fNIRS tasks as part of the BRIGHT project, focusing on social processing, deferred imitation, and habituation responses. Therefore, brain regions for data acquisition were chosen to maximize the likelihood of recording meaningful data for all tasks (Lloyd-Fox, 2023). To clarify the text, we specified this information in the methods section (line 383).

References:

D. R. Dajani, L. Q. Uddin, Demystifying cognitive flexibility: Implications for clinical and developmental neuroscience. Trends Neurosci. 38, 571–578 (2015).

Pirazzoli, L., Sullivan, E., Xie, W., Richards, J. E., Bulgarelli, C., Lloyd-Fox, S., ... & Nelson III, C. A. (2022). Association of psychosocial adversity and social information processing in children raised in a low-resource setting: an fNIRS study. Developmental Cognitive Neuroscience, 56, 101125.

Delgado Reyes, L., Wijeakumar, S., Magnotta, V. A., Forbes, S. H., & Spencer, J. P. (2020). The functional brain networks that underlie visual working memory in the first two years of life. NeuroImage, 219, Article 116971.

Lloyd-Fox, S., McCann, S., Milosavljevic, B., Katus, L., Blasi, A., Bulgarelli, C., Crespo-Llado, M., Ghillia, G., Fadera, T., Mbye, E., Mason, L., Njai, F., Njie, O., Perapoch-Amado, M., Rozhko, M., Sosseh, F., Saidykhan, M., Touray, E., Moore, S. E., … Team, and the B. S. (2023). The Brain Imaging for Global Health (BRIGHT) Study: Cohort Study Protocol. Gates Open Research, 7(126).

(11) Last but not least, in the paper, the authors mentioned that fNIRS offers better spatial resolution and anatomical specificity compared to EEG, thereby providing more precise and reliable localization of brain networks. While I partially agree with this perspective, it remains to be explored whether the current fNIRS analysis strategies indeed yield higher spatial resolution. It is hoped that the authors will delve deeper into this discussion in the paper.

The brain regions of focus were selected based on coregistration work previously conducted at each time point on the array used in this project (Collins-Jones, 2019). We deliberately avoided making claims about small brain regions, considering that head size might increase slightly less with age in The Gambia compared to Western countries (Nabwera, 2017) . However, we maintain that the conclusions drawn in this study offer higher brain-region specificity than could have been  identified with current common EEG methods alone.

References:

L. H. Collins-Jones, et al., Longitudinal infant fNIRS channel-space analyses are robust to variability parameters at the group-level: An image reconstruction investigation. Neuroimage 237, 118068 (2021).

Nabwera, H. M., Fulford, A. J., Moore, S. E., & Prentice, A. M. (2017). Growth faltering in rural Gambian children after four decades of interventions: a retrospective cohort study. The Lancet Global Health, 5(2), e208–e216.

Reviewer #3 (Recommendations For The Authors):

Introduction

- Among important developmental mechanisms to mention are the development of exuberant connections and the further selection/stabilization of the relevant ones according to environmental stimulation, vs the pruning of others.

We agree with the reviewer that the development of exuberant connections and subsequent pruning is a universal process of paramount importance during the first years of life. However, after revising our introduction, given the word limit of the journal, we maintained focus on neurodevelopment and early adversity.

Results

- Adding a few more information on the 6 sections and 21 connections would be welcome. In particular for within-section FC: how was this computed?

The 6 sections were created based on the co-registration of the array used in this study at each age in a previous published work L. H. Collins-Jones, et al., Longitudinal infant fNIRS channel-space analyses are robust to variability parameters at the group-level: An image reconstruction investigation. Neuroimage 237, 118068 (2021). This is reference No. 68 in the manuscript.

As we mentioned in the section fNIRS preprocessing and data-analysis: ‘The sections were established via the 17 channels of each hemisphere which were grouped into front, middle and back (for a total of six regions) based on a previous co-registration of the BRIGHT fNIRS arrays onto age-appropriate templates’.

The 21 connections were defined as all the possible links between the 6 regions, specifically: the interhemispheric homotopic connections (in orange in Figure SI1), which connect the same regions between hemispheres (i.e., front left with front right); the intrahemispheric connections (in green in Figure SI1), which correlate channels belonging to the same region; the fronto-posterior connections (in blue in Figure SI1), which link front and middle, middle and back, and front and back regions of the same hemisphere; and the crossing interhemispheric connections (non-homotopic interhemispheric, in yellow in Figure SI1), which link the front, middle, and back areas between left and right hemispheres. We added these specifications also in the legend of Figure SI1 for clarity.

- The denomination intrahemispheric vs fronto-posterior vs crossed connections is not clear. Maybe prefer intra-hemispheric vs inter-hemispheric homotopic vs inter-hemispheric non-homotopic (also in Figure SI1).

We appreciate the reviewer's suggestion regarding terminology. However, we believe that the term 'inter-hemispheric non-homotopic' could potentially refer to both connections within the same brain hemisphere from front to back and connections crossing between hemispheres, leading to increased confusion. Therefore, we have chosen not to include the term 'non-homotopic' and instead added 'homotopic' to 'interhemispheric' throughout the manuscript to emphasize that these functional connections occur between corresponding regions of the two hemispheres.

- with time -> with age.

We replaced “with time” with “with age” as suggested through the manuscript.

- The description of both HbO2 and HHb results overloads the main text: would it be relevant to present one of the two in Supplementary Information if the results are coherent?

We understand the reviewer’s concern regarding overloading the results section with reporting both chromophores. However, reporting results for both HbO and HHb is considered a crucial step for publications in the fNIRS field, as emphasized in recent formal guidance (Yücel et al., 2020). One of the strengths of fNIRS compared to fMRI is its ability to record from both chromophores, enabling a more precise characterization of brain activations and oscillations. Moreover, in FC studies like this one, ensuring that HbO and HHb results overlap is an important check that increases confidence in interpreting the findings.

References:

Yücel, M. A., von Lühmann, A., Scholkmann, F., Gervain, J., Dan, I., Ayaz, H., Boas, D., Cooper, R. J., Culver, J., Elwell, C. E., Eggebrecht, A. ., Franceschini, M. A., Grova, C., Homae, F., Lesage, F., Obrig, H., Tachtsidis, I., Tak, S., Tong, Y., … Wolf, M. (2020). Best Practices for fNIRS publications. Neurophotonics, 1–34. https://doi.org/10.1117/1.NPh.8.1.012101

- HCZ is not defined when first used.

We thank the reviewer for spotting this, we have now specified HCZ at line 184 as follows: ‘head-circumference z-score (HCZ)’.

- Choosing the analyzed measures to "maximize power" could be criticised.

We appreciate the reviewer’s concern. However, correlating all the FC values with all changes in growth would have raised an important issue for multiple comparisons. We therefore we made a priori decision to focus on investigating the relationship between changes in growth and those FC that showed a significant change with age, considering these as the most interesting ones from a developmental perspective in our sample.

Discussion

- I would recommend using the same order to synthesize results and further discuss them.

We agree with the reviewer that the suggested structure is optimal for a clear discussion section. We have indeed followed it, with each paragraph covering specific aspects:

- Recap of the study aims

- Results summary and discussion of developmental changes

- Results summary and discussion of the relationship between changes in growth and FC

- Results summary and discussion of the relationship between FC and cognitive flexibility

- Limitations

- Conclusion

Given the numerous results presented in this paper, we believe that readers will better digest them by first reading a summary of the results followed by their interpretations, rather than condensing all the interpretations together.

- Highlighting how "atypical" developmental trajectories are in Gambian infants would be welcome in the Results section. Other interpretations can be found than "The observed decrease in frontal inter-hemispheric FC with increasing age may be due to the exposure to early life undernutrition adversity".

We agree with the reviewer that other factors that differ between low- and high-resource settings might have an impact on FC trajectories. We therefore specified this in the discussion as follows: “We acknowledge that differences in FC could also be attributed to other environmental and cultural disparities between high-resource and low-resource settings, and future studies are needed to further investigate cultural, environmental, and genetic effects on brain FC” (line 238).

- Focusing on FC at 24m for the relationship with growth is questionable.

Correlating the FC values at 5 time points with all changes in growth would have raised an important issue for multiple comparisons. We therefore we made a decision a priori to focus on investigating the relationship between changes in growth and FC at 24 months as our final time point of data collection. We added this information in the methods section as follows: “To investigate the impact of undernutrition on FC development, we used DWLZ as independent variables in regression analyses on HbO2 (as the chromophore with the highest signal-to-noise ratio) FC at 24 months, our final time point of data collection” (line 517, method section).

- There is too much emphasis on the correlation between FC and cognitive flexibility, whereas results are not significant after correction for multiple comparisons.

Following the reviewer’ suggestion, we specified that results from regression analysis are significant but they did not survive multiple comparisons in the discussion as follows: While our results are consistent with previous studies, we acknowledge that the significant association between early FC and later cognitive flexibility does not withstand multiple comparisons. Therefore, we encourage future studies that may replicate these findings with a larger sample. (line 290, discussion section).

Methods

- I would recommend detailing how z-scores were computed in the paragraph "Anthropometric measures".

We specified how z-scores were computed in the statistical analysis section as follows: “Anthropometric measures were converted to age and sex adjusted z‐scores that are based on World Health Organization Child Growth Standards (93). Weight‐for‐Length (WLZ) and Head Circumference (HCZ) z-scores were computed” (line 509, method section). As transforming data is the first step of statistical analysis and is not directly related to data collection, we believe it is more appropriate to retain this description in the statistical analysis section.

- FC computation: the mention of "correlating the first and the last 250s" is not clear.

We specified this more clearly in the text as follows: We found that correlating the first and the last 250 seconds of valid data after pre-processing provided the highest percentage of infants with strong correlation between the first and the last portion of data (line 467).

- The manuscript mentions "age 3 years" for the younger preschoolers but ~48months rather corresponds to 4 years.

We revised the entire manuscript and the supplementary materials, but we could not find any instance in which preschoolers are referred with age in months rather than in years.

- Specify the number of children evaluated at 4 and 5 years. Is the test of cognitive flexibility normalized for age? If not, how were the 2 groups considered in the analyses? (age as a confounding factor).

We have added the number of children in the two preschooler groups as follows: younger preschoolers (age mean ± SD=47.96 ± 2.77 months, N=77) and older preschoolers (age mean ± SD=57.58 ± 2.11 months, N=84). (line 484).

The cognitive flexibility test was not normalized for age, as this task was specifically developed for preschoolers (Howard, 2015). As mentioned in ‘Cognitive flexibility at preschool age’ of the methods section, “data were collected in two ranges of preschool ages”, which guided our decision to perform regression analysis on the impact of FC on cognitive flexibility separately within these two age groups, rather than treating them as a single group of preschoolers.

References:

Howard, S. J., & Melhuish, E. (2015). An Early Years Toolbox (EYT) for assessing early executive function, language, self-regulation, and social development: Validity, reliability, and preliminary norms. Journal of Psychoeducational Assessment, 35(3), 255-275.

Figures and Tables

- Table 1 could highlight the significant results. It is not clear what the "baseline" results correspond to.

We have marked in bold the results that are statistically significant in Table 1. In the linear mixed model we performed, the first time point (i.e. 5 months) is chosen as ‘baseline’, i.e. the reference against which the other time points are compared to, and its statistical values refer to its significance against 0 (as it has been performed in Bulgarelli 2020).

- Figures 2 B and C seem redundant? What is SE vs SD?

We believe that both figures 2B and 2C are useful for the readers. While the first one shows the mean FC values at the group level, the second one highlights the individual variability of FC values (typical of infant neuroimaging data), which also why it is interesting to relate these measures to other variables of our dataset (i.e. growth and cognitive flexibility). Figure 2C also reports mean FC values per age, but these might be less visible considering that also one dot per infant is also plotted.

SE stands for standard error, and in the legend of the figure we specified this as follows: ‘Mean and standard error of the mean (SE)’. SD stands for standard deviation, and we have now specified this as follows: ‘mean ± standard deviation (SD)’ .

- Table 2: I would recommend removing results that don't survive corrections for multiple comparisons.

We acknowledge the reviewer’s concern regarding the reporting of results that do not survive multiple comparisons. However, considering the uniqueness of our dataset and the novelty of our work, we believe it is crucial to report all significant findings. We have taken great care to transparently distinguish between results that survived multiple comparisons and those that did not in both the Results and Discussion sections, ensuring that readers are not misled. It is possible that future studies may replicate and further strengthen these associations. Therefore, by sharing these results with the research community, we provide valuable insights for future investigations.

- Figure 3: the top is redundant with Table 2: to be merged? B: the statistical results might be shown in a Table.

We agree with the reviewer that the top part of Figure 3 and Table 2 report the same results. However, given the richness of these findings, we believe that the top part of Figure 3 serves as a useful summary for readers. Additionally, examining both the top and bottom parts of Figure 3 provides a comprehensive overview of the regression analysis conducted in this study.

- Figure SI6: Is it really a % in x-axis?

We thank the reviewer for spotting this typo, the percentage is relevant for the y-axis only. We removed the % symbol from ticks of the x-axis.

- Table SI1: the presented p-values don't seem to survive Bonferroni correction, contrary to what is written.

We thank the reviewer for spotting this mistake, we removed the reference to the Bonferroni correction for the p-values.

- Table SI2: For the proportion of children included in the analysis, maybe be precise that the proportion was computed based on the ones with acquired data. Maybe also add the proportion according to all children, to better show the high drop-out rate at certain ages?

We thank the reviewer for these useful suggestions. We have specified in the legend of the table how we calculated the proportion of infants included as follows: ‘The proportion of children included in the analysis was computed based on the infants with FC data’. We have also added a column in the table called ‘Inclusion rate (from the 204 infants recruited)’, following the reviewer’s suggestion. This will be a useful reference for future studies.

- A few typos should be corrected throughout the manuscript.

We thoroughly revised the main manuscript and the supplementary materials for typos.

Tema 128

• O exercício concomitante da função de cobrador pelo motorista de ônibus urbano não gera direito à percepção de acréscimo salarial.

RR-0100221-76.2021.5.01.0074

O abono de férias somente poderá ser requisitado até 15 dias antes do encerramento do período aquisitivo.

Isso é, não são 15 dias de sair de férias, mas sim 15 dias antes da conclusão do período aquisitivo.

Licença remunerada por mais de 30 dias extingue o direito a férias.

OJ-SDI1-191

CONTRATO DE EMPREITADA. DONO DA OBRA DE CONSTRUÇÃO CIVIL. RESPONSABILIDADE (nova re-dação) - Res. 175/2011, DEJT divulgado em 27, 30 e 31.05.2011 - Diante da inexistência de previsão legal específica, o contrato de empreitada de construção civil entre o dono da obra e o empreiteiro não enseja responsabilidade solidária ou subsidiária nas obrigações trabalhistas contraídas pelo empreiteiro, salvo sendo o dono da obra uma empresa construtora ou incorporadora.

INCIDENTE DE RECURSO DE REVISTA REPETITIVO. TEMA Nº 0006. CONTRATO DE EMPREITADA. DONO DA OBRA. RESPONSABILIDADE. ORIENTAÇÃO JURISPRUDENCIAL Nº 191 DA SbDI-1 DO TST VERSUS SÚMULA Nº 42 DO TRIBUNAL REGIONAL DO TRABALHO DA TERCEIRA REGIÃO 1. A exclusão de responsabilidade solidária ou subsidiária por obrigação trabalhista, a que se refere a Orientação Jurisprudencial nº 191 da SbDI-1 do TST, não se restringe a pessoa física ou micro e pequenas empresas. Compreende igualmente empresas de médio e grande porte e entes públicos. 2. A excepcional responsabilidade por obrigações trabalhistas, prevista na parte final da Orientação Jurisprudencial nº 191 da SbDI-1 do TST, por aplicação analógica do artigo 455 da CLT, alcança os casos em que o dono da obra de construção civil é construtor ou incorporador e, portanto, desenvolve a mesma atividade econômica do empreiteiro. 3. Não é compatível com a diretriz sufragada na Orientação Jurisprudencial nº 191 da SbDI-1do TST jurisprudência de Tribunal Regional do Trabalho que amplia a responsabilidade trabalhista do dono da obra, excepcionando apenas “a pessoa física ou micro e pequenas empresas, na forma da lei, que não exerçam atividade econômica vinculada ao objeto contratado”. 4. Exceto ente público da Administração direta e indireta, se houver inadimplemento das obrigações trabalhistas contraídas por empreiteiro que contratar, sem idoneidade econômico-financeira, o dono da obra responderá subsidiariamente por tais obrigações, em face de aplicação analógica do art. 455 da CLT e de culpa in eligendo.

• O prazo para embargos à execução é o mesmo do embargos de declaração, isto é, são 5 dias o prazo para propor embargos à execução a contar da data da garantia ao juízo ou da penhora de bens, não se aplicando o prazo comum de 8 dias.

• O prazo de 8 dias é o definido para as partes se manifestarem a respeito da sentença de liquidação, sob pena de preclusão da oposição de embargos à execução.

A celebração de CCT ou ACT depende de deliberação da Assembleia Geral, a qual deve ser convocada especialmente para este fim.

O quórum de votação é: - Se CCT: Comparecimento e Votação de 2/3 dos associados em 1ª Convocação - 1/3 em 2ª Convocação; - Se ACT: Comparecimento e Votação de 2/3 dos interessados em 1ª Convocação - 1/3 em 2ª Convocação.

Se o sindicato contar com mais de 5.000 associados, a primeira convocação deverá contar com mais de 2/3 dos associados.

Porém, a segunda convocação poderá se limitar ao quórum de 1/8 dos associados.

Art. 616 - Os Sindicatos representativos de categorias econômicas ou profissionais e as emprêsas, inclusive as que não tenham representação sindical, quando provocados, não podem recusar-se à negociação coletiva.

• § 3º - Havendo convenção, acordo ou sentença normativa em vigor, o dissídio coletivo deverá ser instaurado dentro dos 60 (sessenta) dias anteriores ao respectivo termo final, para que o novo instrumento possa ter vigência no dia imediato a esse termo.

O quórum de aprovação da representação destinada à instauração de instância é o seguinte: - 1ª Convocação: 2/3 dos associados interessados; OU - 2ª Convocação: 2/3 dos presentes.

That book, [[Monopoly Capital]], eventually expresses the principle marxist idea of [[surplus value]], the one Jack London famously had advocated about, specifically that by distributing only 50% of their profits as wages, capitalists provoke the next Crisis, hence Capitalism is unstable (coupled with the diminithing returns of capital).

The Russian marxists knew since the 1920's that the answer to these capitalistic contradictions is Imperialism, the Highest Stage of Capitalism.

Capitalism needs ([[Graeber]]) Bullshit Jobs, as Baran & Sweezy claimed in their 1966 book "Monopoly Capital: An Essay on the American Economic and Social Order".

Binder Launch fails. Error during build: Microsoft killed MRAN, the source of R package snapshots before 2018-12-07. This repo has a snapshot date of 2018-04-01 specified in runtime.txt. Please use a newer snapshot dateError in event stream: Error

泊松盘采样（Poisson Disk Sampling）是一种在空间中生成点集的方法，其核心特性是保证任意两点之间的距离不小于一个预设的最小值 $ r $，从而避免点过于密集或稀疏。这种特性使其在高维点云处理、图形学、纹理生成等领域具有广泛应用。以下是对这一特性的深入解析：

1. 点间最小距离约束的核心思想

• 目标：在空间中生成一组点，使得：
• 所有点之间的距离 至少为 $ r $；
• 点的分布尽可能随机且均匀（蓝噪声特性）。
• 对比传统方法：
• 完全随机采样：可能导致点聚集或稀疏区域（图A）；
• 规则网格采样：分布均匀但缺乏随机性，容易产生伪影（图B）；
• 泊松盘采样：在随机性基础上加入最小距离约束，既避免聚集又保留自然感（图C）。

2. 为什么最小距离约束重要？

(1) 避免过度密集的局部结构

• 在完全随机采样中，点的密度服从泊松分布，可能出现局部点密度过高的区域（如“扎堆”现象）。最小距离约束通过强制排斥机制，确保每个新点不会与已有点过近。
• 示例：模拟森林中树木的位置时，树与树之间需要一定的生长间距，泊松盘采样能自然模拟这一特性。

(2) 提升视觉质量

• 在计算机图形学中，泊松盘采样的蓝噪声特性使渲染结果更接近人眼感知的自然分布，减少伪影和噪点。
• 应用场景：
• 毛发生成（毛孔位置）、粒子系统（烟花爆炸）、地形建模（植被分布）等。

(3) 高维空间的适应性

• 在高维空间（如3D点云、多维特征空间），传统采样方法（如蒙特卡洛）容易因“维度灾难”导致低效或分布不均。泊松盘采样的最小距离约束通过以下方式应对：
• 网格划分：将空间划分为大小为 $ r/\sqrt{d} $ 的超立方体（$ d $ 为维度），确保每个单元格最多包含一个点。
• 高效搜索：新点仅需检查相邻单元格内的点，而非所有已有点，降低时间复杂度。

3. 泊松盘采样的实现原理

以 Bridson 算法（2007）为例，关键步骤如下： 1. 初始化： - 将空间划分为网格，每个网格边长为 $ r/\sqrt{d} $（确保同网格内任意两点半径大于 $ r $）。 - 随机选择一个初始点，并将其加入活动列表（active list）。 2. 迭代生成新点： - 从活动列表中随机取出一个点（当前中心点）。 - 在该点周围生成一个环形区域（半径范围 $ [r, 2r] $），随机生成候选点。 - 检查候选点是否与邻近网格中的点满足最小距离约束： - 若满足，则接受该点，并将其加入活动列表； - 若不满足，则丢弃该点。 3. 终止条件： - 当活动列表为空时，所有可生成的点已被耗尽。

4. 高维点云中的适用性

泊松盘采样在高维点云处理中的优势体现在：

(1) 保持几何结构

• 在点云降采样或上采样时，最小距离约束确保保留原始点云的关键几何特征（如边缘、尖锐区域）。
• 示例：Open3D 中的 PoissonDiskSampling 方法利用此特性进行点云简化（见 [1]）。

(2) 平衡密度与效率

• 通过网格划分和活动列表管理，算法在高维空间中仍能高效运行（时间复杂度约为 $ O(n) $）。
• 对比：传统“飞镖投掷”（dart throwing）方法在高维空间中效率极低，因为冲突概率随维度增加指数级上升。

(3) 处理噪声与异常值

• 泊松盘采样通过排除过近的点，天然过滤掉噪声点或异常值（如点云中的离群点）。

5. 实际应用案例

(1) 图形学

• 纹理生成：使用灰度图像控制采样密度（亮区密集，暗区稀疏），生成自然纹理（见 [2]）。
• 光照采样：在路径追踪中，泊松盘采样减少方差，提升渲染质量。

(2) 点云处理

• 去噪与简化：Open3D 和 PCL 库提供现成实现，用于点云压缩和预处理（见 [1][5]）。
• 表面重建：泊松盘采样的均匀分布特性适合后续的 Delaunay 三角化或隐式曲面重建（见 [5]）。

(3) 游戏开发

• 程序化生成：玩家出生点、敌人分布、资源放置等场景中，确保分布合理且无重叠（见 [4]）。

6. 局限性

• 参数敏感性：最小距离 $ r $ 过大会导致采样不足，过小则增加计算量。
• 边界效应：靠近边界的点可能无法完全满足最小距离约束（可通过虚拟镜像扩展空间缓解）。
• 高维性能瓶颈：尽管优于随机采样，但随着维度增加，算法效率仍可能下降。

总结

泊松盘采样的最小距离约束通过排斥机制实现了点集的均匀随机分布，其核心在于平衡随机性与规则性。在高维点云处理中，这一特性不仅避免了过度密集的局部结构，还能保留关键几何信息，因此成为点云简化、表面重建、图形学渲染等领域的关键技术。

Author response:

The following is the authors’ response to the original reviews

Joint Public Review:

Idiopathic scoliosis (IS) is a common spinal deformity. Various studies have linked genes to IS, but underlying mechanisms are unclear such that we still lack understanding of the causes of IS. The current manuscript analyzes IS patient populations and identifies EPHA4 as a novel associated gene, finding three rare variants in EPHA4 from three patients (one disrupting splicing and two missense variants) as well as a large deletion (encompassing EPHA4) in a Waardenburg syndrome patient with scoliosis. EPHA4 is a member of the Eph receptor family. Drawing on data from zebrafish experiments, the authors argue that EPHA4 loss of function disrupts the central pattern generator (CPG) function necessary for motor coordination.

The main strength of this manuscript is the human genetic data, which provides convincing evidence linking EPHA4 variants to IS. The loss of function experiments in zebrafish strongly support the conclusion that EPHA4 variants that reduce function lead to IS.

The conclusion that disruption of CPG function causes spinal curves in the zebrafish model is not well supported. The authors' final model is that a disrupted CPG leads to asymmetric mechanical loading on the spine and, over time, the development of curves. This is a reasonable idea, but currently not strongly backed up by data in the manuscript. Potentially, the impaired larval movements simply coincide with, but do not cause, juvenile-onset scoliosis. Support for the authors' conclusion would require independent methods of disrupting CPG function and determining if this is accompanied by spine curvature. At a minimum, the language of the manuscript could be toned down, with the CPG defects put forward as a potential explanation for scoliosis in the discussion rather than as something this manuscript has "shown". An additional weakness of the manuscript is that the zebrafish genetic tools are not sufficiently validated to provide full confidence in the data and conclusions.

We highly appreciate the reviewer’s insightful comments and the acknowledgment of the main values of our study. We agree with the reviewer that further experiments are needed to fully establish the relationship between CPG and scoliosis. In response, we have revised the conclusion in the manuscript to better reflect this. Additionally, we conducted further analyses on the mutants to provide additional evidence supporting this concept.

Reviewer #1 (Recommendations for the authors):

Epha4a mutant zebrafish exhibited mild spinal curves, mostly laterally and in the tail. This was 75% of homozyous mutants but also, surprisingly, about 20% of heterozygotes. epha4b mutants also developed some mild scoliosis. If the two zebrafish paralogs can compensate for each other (partial redundancy), we might expect more severe scoliosis in double mutants. Did the authors generate and analyze double mutants? I believe it would be very useful for this study to report the zebrafish phenotype of loss of both paralogs together.

We appreciate the reviewer’s insightful comment regarding the potential value of reporting the phenotype of eph4a/eph4b double mutants. While we fully agree that this analysis would be valuable, our attempts to generate double mutants have been unsuccessful. These two genes are closely linked on the chromosome, with less than 100 kb separating them, which makes it challenging to generate double mutants through standard genetic crossing. Establishing a double mutant line would require more than a year due to the technical constraints of the process. Although we are unable to address this question directly at this time, we hypothesize that eph4a/eph4b double mutants may exhibit a higher likelihood of body axis abnormalities based on the phenotypes observed in single mutants and the known functions of these genes.

We hope this perspective will provide some useful context despite the limitations.

In Figure 1F, a pCDK5 western blot is performed as a readout of EPH4A signaling after either WT or C849Y mutant EPH4A is transfected into HEK 293T cells. It would be useful to mention in the text, or at least the figure legend, how this experiment was performed/where the protein samples came from. It is included in the methods, but in the main text, it simply says "we conducted western blotting" without mentioning whether the protein samples were from cell lines, patients, or another source.

Sorry for our ignorance. A detailed description of the western blotting conduction was supplemented at both “results” part (page 8, line 187-190) and the Figure 1 legend.

Was the relative turn angle biased to the left or right side of the fish? (i.e. is a positive angle a rightward or leftward turn?)

We are sorry for our unclear description. In Figure 3D, positive angle means turning left, while negative angle means turning right. In wild-type larvae, the average turning angle over a 4-minute period is approximately 0, whereas in mutants, this value deviates from 0, indicating a directional preference (positive for leftward and negative for rightward turns) in swimming behavior during the recording period. We have also made the necessary supplementation in the text and figure legend.

In Figure 4, morpholinos rather than mutants are used, but it is not clear why. Has it been established that the MO used disrupts gene function specifically? Can the effect of the MO be rescued by expressing a wild-type mRNA of Epha4a? Does MO knockdown induce spinal curves if fish are raised? Indeed, this could be a way to determine whether the spinal curves are caused by early events in development (when MOs are active).

Thanks for the comments. The efficacy of relevant MOs has been well-documented in numerous previous studies (Addison et al., 2018; Cavodeassi et al., 2013; Letelier et al., 2018; Royet et al., 2017). Following this reviewer’s suggestion, we have raised the epha4a morphants into adults, while no scoliosis were observed, suggesting that the spinal curvature formation may be induced by long-term defects in the absence of Epha4a. Additionally, we reconfirmed the abnormal motor neuron activation frequency phenotype in the mutants background. The corresponding data have replaced the original Figure 4 in the manuscript. 

References

(1) Addison, M., Xu, Q., Cayuso, J., and Wilkinson, D.G. (2018). Cell Identity Switching Regulated by Retinoic Acid Signaling Maintains Homogeneous Segments in the Hindbrain. Dev Cell 45, 606-620 e603.

(2) Cavodeassi, F., Ivanovitch, K., and Wilson, S.W. (2013). Eph/Ephrin signalling maintains eye field segregation from adjacent neural plate territories during forebrain morphogenesis. Development 140, 4193-4202.

(3) Letelier, J., Terriente, J., Belzunce, I., Voltes, A., Undurraga, C.A., Polvillo, R., Devos, L., Tena, J.J., Maeso, I., Retaux, S., et al. (2018). Evolutionary emergence of the rac3b/rfng/sgca regulatory cluster refined mechanisms for hindbrain boundaries formation. Proc Natl Acad Sci U S A 115, E3731-E3740.

(4) Royet, A., Broutier, L., Coissieux, M.M., Malleval, C., Gadot, N., Maillet, D., Gratadou-Hupon, L., Bernet, A., Nony, P., Treilleux, I., et al. (2017). Ephrin-B3 supports glioblastoma growth by inhibiting apoptosis induced by the dependence receptor EphA4. Oncotarget 8, 23750-23759.

Reviewer #2 (Recommendations for the authors):

Supplementary Table 3 is missing.

Sorry for any inconvenience caused to the reviewers. Due to the size of the supplementary Table 3, we have separately uploaded an Excel file as supplementary materials. We have also double-checked during the resubmission process of the revised manuscript. Thanks for your thorough review.

The authors report only a single mutant allele for zebrafish epha4a and epha4b. Additionally, they provide no information about how many generations each allele has been outcrossed. The authors should provide some type of validation that the phenotypes they describe result from loss of function of the targeted gene and not from an off-targeting event.

Thanks for the comments. For epha4a and epha4b mutants, each homozygous mutant was initially derived from the self-crossing of first filial generation heterozygotes, and subsequent homozygous generations were maintained for fewer than three rounds of in-crossing. Interestingly, we observed a reduction in the incidence of scoliosis across successive generations. This trend may be attributed to potential genetic compensation mechanisms, which could mitigate the phenotypic severity over time. To address concerns about possible off-target effects, we synthesized and injected epha4a mRNA to test for phenotypic rescue. Our data show that epha4a mRNA injection partially restored swimming coordination in the mutants (Fig. S5). Moreover, similar motor coordination defects have been reported in Epha4-deficient mice, as documented in previous studies (Kullander et al., 2003; Borgius et al., 2014). These findings collectively strengthen the hypothesis that Epha4a plays a critical role in regulating motor coordination.

References

(1) Borgius, L., Nishimaru, H., Caldeira, V., Kunugise, Y., Low, P., Reig, R., Itohara, S., Iwasato, T., and Kiehn, O. (2014). Spinal glutamatergic neurons defined by EphA4 signaling are essential components of normal locomotor circuits. J Neurosci 34, 3841-3853.

(2) Kullander, K., Butt, S.J., Lebret, J.M., Lundfald, L., Restrepo, C.E., Rydstrom, A., Klein, R., and Kiehn, O. (2003). Role of EphA4 and EphrinB3 in local neuronal circuits that control walking. Science 299, 1889-1892.

The authors need to provide allele designations for the mutant alleles following accepted nomenclature guidelines.

Thank you for your careful review! We have reviewed and made revisions to the genes and mutation symbols throughout the entire text.

The three antisense morpholino oligonucleotides need to be validated for efficacy and specificity.

Thanks for the comments. The morpholinos were extensively used and validated in previous studies, and the efficacy of these morpholinos has been thoroughly validated in multiple studies (Addison et al., 2018; Cavodeassi et al., 2013; Letelier et al., 2018; Royet et al., 2017). Furthermore, we also performed swimming behavior analysis in the mutant background, which showed similar results as the morphants. Moreover, we also performed rescue experiments to confirm the specificity of the mutants (Fig. S5). Finally, we reconfirmed the abnormal calcium signaling in the mutants (Fig. 4), which further support our previous knockdown results.

References

(1) Addison, M., Xu, Q., Cayuso, J., and Wilkinson, D.G. (2018). Cell Identity Switching Regulated by Retinoic Acid Signaling Maintains Homogeneous Segments in the Hindbrain. Dev Cell 45, 606-620 e603.

(2) Cavodeassi, F., Ivanovitch, K., and Wilson, S.W. (2013). Eph/Ephrin signalling maintains eye field segregation from adjacent neural plate territories during forebrain morphogenesis. Development 140, 4193-4202.

(3) Letelier, J., Terriente, J., Belzunce, I., Voltes, A., Undurraga, C.A., Polvillo, R., Devos, L., Tena, J.J., Maeso, I., Retaux, S., et al. (2018). Evolutionary emergence of the rac3b/rfng/sgca regulatory cluster refined mechanisms for hindbrain boundaries formation. Proc Natl Acad Sci U S A 115, E3731-E3740.

(4) Royet, A., Broutier, L., Coissieux, M.M., Malleval, C., Gadot, N., Maillet, D., Gratadou-Hupon, L., Bernet, A., Nony, P., Treilleux, I., et al. (2017). Ephrin-B3 supports glioblastoma growth by inhibiting apoptosis induced by the dependence receptor EphA4. Oncotarget 8, 23750-23759.

Line 229. "While in consistent with previous reports, the hindbrain rhombomeric boundaries were found to be defective....". This sentence is not clear. Please describe how it is "inconsistent".

Thanks for the comments and sorry for the unclear description, we have described this more clearly in our revised manuscript (page 9, line 229-230).

Animals frequently are described as "heterozygous mutants" or "mutants". Please make clear that the latter are homozygous mutant animals.

Thanks for the comments. In the manuscript, all references to mutants specifically indicate homozygous mutants. Heterozygous mutants are explicitly identified as such.

The chromatin interaction portion of the Methods does not include any information on how these experiments were conducted or where the data were obtained. This information needs to be provided.

Thanks for your advice. The detailed information of chromatin interaction mapping has been provided in “Methods and Materials” (page 18-19, line 450-455). Information about the interacting regions was derived from Hi-C datasets of 21 tissues and cell types provided by GSE87112. The significance of interactions for Hi-C datasets was computed by Fit-Hi-C, with an FDR ≤ 10-6 considered significant.

The authors present single-cell RNA-seq data in Supplementary Figure 5 for which they cite Cavone et al, 2021. This seems like an odd database to use. Can the authors provide an explanation for choosing it? In any case, the citation should also be made in the Supplementary Figure 5 legend.

Thank you for your rigorous comment, we have cited this literature in the proper place of the revised manuscript. Cavone et al. used the her4.3:GFP line to label ependymo-radial glia (ERG) progenitor cells and performed single-cell RNA-seq on FACS-isolated fluorescent cells. The isolated cells included not only ERG progenitors but also undifferentiated and differentiated neurons and oligodendrocytes. The authors attributed this to the relative stability of the GFP protein, which remained in the progeny of GFP-expressing her4.3+ ERG progenitor cells, thus effectively acting as a short-term cell lineage tracer. Indeed, clustering analysis of this data successfully identifies neural progenitors and other neural clusters. Therefore, we consider that this scRNA-seq data encompasses a comprehensive range of neural cell types and is suitable for analyzing the expression of genes of interest. Furthermore, we downloaded and analyzed the scRNA-seq data of the zebrafish nervous system reported by Scott et al. in 2021 (Fig. S7B) (Scott et al., 2021). Despite differences in the developmental stages of the larvae analyzed (Cavone et al. examined larvae at 4 dpf, whereas Scott et al. analyzed larvae at 24, 36, and 48 hpf), our findings are consistent. Specifically, epha4a and epha4b are expressed in interneurons, whereas efnb3a and efnb3b are enriched in floor plate cells.

References

(1) Scott, K., O'Rourke, R., Winkler, C.C., Kearns, C.A., and Appel, B. (2021). Temporal single-cell transcriptomes of zebrafish spinal cord pMN progenitors reveal distinct neuronal and glial progenitor populations. Dev Biol 479, 37-50.

In Figure Legend 1, "expressed from the EPHA4-mutant plasmid" is not an accurate description of the experiment.

Sorry for the previous inaccurate description. The description has been revised to accurately reflect the experiment. “Western blot analysis of EPHA4-c.2546G>A variant showing the protein expression levels of EPHA4 and CDK5 and the amount of phosphorylated CDK5 (pCDK5) in HEK293T cells transfected with EPHA4-mutant or EPHA4-WT plasmid”.

Figure 3 panels J and K need more explanation. I don't understand what the different colors represent nor do I understand what are wild type and what are mutant data.

Thank you for your valuable feedback. We apologize for the lack of clarity in the original figure legend. To address this, we have revised the legend of Figure 3 to provide a more detailed explanation. In panels J and K, each color-coded curve represents the response of an individual larva from an independent experimental trial to the stimulus. Specifically, panel J depicts the response data for the wild-type larvae, whereas panel K presents the response data for the homozygous epha4a mutants.

Please provide the genotypes for the images in Figure 5A.

Thanks for the comments and we are sorry for our unclear description, we have described this more clearly in the Figure 5.

Figure legend 6B should also note the heterozygote data with the wild type and homozygous mutant data.

Thanks for the comments, the data are now included in Figure 6B.

Epha4 and Efnb3 have well-established roles in axon guidance. Although this is noted in the Discussion, I think a more extensive description of prior findings would be helpful.

Thanks for your valuable feedback. A more detailed description of the roles of Epha4 and Efnb3 in axon guidance was provided in the “Discussion” (page 16, line 388-396).

The main conclusion of this manuscript is that EPHA4 variants cause IS by disrupting central pattern generator function. I think this is misleading. I think that the more valid conclusion is that EPHA4 loss of function causes axon pathfinding defects that impair locomotion by disrupting CPG activity, thereby leading to IS. I urge the authors to consider this more nuanced interpretation.

Thank you for your insightful comments. We appreciate your suggestion to refine our main conclusion. We agree that the proposed revision more accurately reflects our findings and will revise the manuscript accordingly to state that “EPHA4 loss of function causes axon pathfinding defects, which impair locomotion by disrupting central pattern generator activity, potentially leading to IS.”

I'm on MacOS x86_64 and the following error occurs:

```

=== ERROR while compiling ocaml-base-compiler.5.2.0 ==========================

context 2.3.0 | macos/x86_64 | | https://opam.ocaml.org#8106bcf57fc33218275bbf0d02e725f4e9fcd0f6

path ~/.opam/cs3110-2025sp/.opam-switch/build/ocaml-base-compiler.5.2.0

command ~/.opam/opam-init/hooks/sandbox.sh build make -j7

exit-code 2

env-file ~/.opam/log/ocaml-base-compiler-68106-48fec6.env

output-file ~/.opam/log/ocaml-base-compiler-68106-48fec6.out

output

/Library/Developer/CommandLineTools/usr/bin/make coldstart

[...]

CC runtime/fail_byt.b.o

CC runtime/fix_code.b.o

CC runtime/interp.b.o

CC runtime/startup_byt.b.o

CC runtime/zstd.b.o

MKLIB runtime/libcamlrun.a

MKEXE runtime/ocamlrun

ld: archive member '/' not a mach-o file in 'runtime/libcamlrun.a'

clang: error: linker command failed with exit code 1 (use -v to see invocation)

make[1]: *** [runtime/ocamlrun] Error 1

make: *** [world.opt] Error 2

```

I solved this by uninstalling GNU binutils with brew following this stackoverflow question.

Se trata de un valor binario Ojo no es un operador binario.

• Mientras que los operadores binarios corresponden a las operaciones de la lógica binaria y son múltiples (and, or, not, xor, implicación).
• Los valores binarios corresponde a aquellos que pueden usarse para dichas operaciones y son sólo dos: verdadero o falso (a veces representados como 1 y 0, respectivamente).

Es un bloque, porque está delimitado por paréntesis cuadrados. En este caso la barra vertica |, no es el operador lógico "ó", sino un separador entre la variables del bloque y las operaraciones del mismo.

Para mayor ampliación, ver la sección de bloques en Representando y Procesando datos en Pharo y la anotación de la sesión anterior que incluía una mención al respecto de bloques.

• Tipo mensaje: binario.
• Receptor: true
• Selector: |(ó lógica o conjunción)
• Argumento: false
• Resultado: true
• Interpretación: Estamos encontrando el resultado del operador lógico "ó", también llamado conjunción. Sirve para distintas operaciones, en particular la búsqueda de información. Ver más en lógica boolena

• Receptor: SmallInteger
• Selector: maxVal
• Argumento: no existe.

El receptor es una clase, pues

• Empieza en mayúsculas.
• Se refiere a un conjunto o todo.

Estamos encontrando el mayor valor del subconjunto o clase de todos los enteros pequeños.

• receptor: #(2 33-4 67)
• selector: collect:
• argumento: [ :each | each abs ]

Este es un caso de la combinación de tres cosas: las colecciones, los bloques y los iteraradores.

each es el iterador, que toma la forma de las llamadas variables mudas, pues puedo colocar su nombre de manera arbitraria (se define en la parte a la izquiera de la barra del bloque [ :each y a la derecha, lo que quiero hacer con dicha variable, una vez vaya tomando los valores de cada elemento en el arreglo, que, para este ejercicio, es sacar el valor absoluto (each abs ]). Podría haberle llamado number o de otra forma. Lo clave es que el nombres :each sea el mismo usado antes y después de la barra del bloque, que separa la definición de variables, de lo que hago con ellas.

El argumento en este caso es nulo, pues no existe ningún elemento extra o parámetro que le esté dando al mensaje.

Como vimos en las charlas de Anjana Vakil, se puso desde metáforas/marco biológico y matemático como entendemos la computación, o cómo puede entenderse.

El valor agregado de este tipo de documentos, más que los hipervínculos que extienden la información, pues esto ya es popularizado y utilizado en distintos lugares: principalmente Wikipedia, pero también Fandom, Wikcionario o parecidas; sino el hecho de poder comentar sobre el mismo texto. Hay un valor tanto comunitario como personal. Comunitario porque podemos ver comentarios, ideas, o anotaciones de la comunidad que lee los textos junto sus etiquetas, como también en le valor personal en apoyo de procesos de aprendizaje, ya que nos permite recuperar y catalogar textos que vamos leyendo en la web, como agregarlo nuestras propias anotaciones.

El valor agregado de este tipo de documentos, más que los hipervínculos que extienden la información, pues esto ya es popularizado y utilizado en distintos lugares: principalmente Wikipedia, pero también Fandom, Wikcionario o parecidas; sino el hecho de poder comentar sobre el mismo texto. Hay un valor tanto comunitario como personal. Comunitario porque podemos ver comentarios, ideas, o anotaciones de la comunidad que lee los textos junto sus etiquetas, como también en le valor personal en apoyo de procesos de aprendizaje, ya que nos permite recuperar y catalogar textos que vamos leyendo en la web, como agregarlo nuestras propias anotaciones.

“¡Bienvenido a América! Aquí nada es realmente real, pero todo es perfectamente vendible.”

Creio serem fundamentais as atividades de aprendizagem digital, com recursos recreativos, ao permitirem de forma lúdica e amplia, assumir diferentes formatos para cumprir o processo de aprendizagem dos alunos, abordando assim muitas maneiras de transferência do conhecimento.

Tendo sempre em conta os diferentes e variáveis caminhos, para promover o papel ativo do estudante, estimulando uma aprendizagem e construção autónoma do conhecimento, mediante os princípios para desenhar atividades assíncronas, num ambiente ‘online’. Permitindo a participação de todos os estudantes, a seu próprio tempo, permitindo também a partilha das reflexões e assim uma aprendizagem colaborativa.

Um dos recursos do “learnign”, com grande potencial, são os audiovisuais, sendo um elemento central importante nos contextos virtuais de aprendizagem, com todas as suas plataformas e redes de distribuição e acesso, que permitem novos recursos que propõem um novo paradigma da aquisição do conhecimento e da educação.

Estas atividades devem ser criadas com uma “boa estrutura de comunicação para gerar uma autêntica comunidade virtual de aprendizagem”, como propõe Moreira, José António Marques; Henriques, Susana; Barros, Daniela (2020, p. 353), estando claro que devem ter presente os elementos básicos de organização, seleção de recursos, preparação e avaliação de e-atividades de aprendizagem.

A reflexão sobre o papel do e-moderador é essencial, na medida em que é um agente fundamental para a promoção da reflexão e motivação dos estudantes. Sem uma boa orientação, atividades como a discussão em fóruns (e que foi uma prática comum desta microcredencial) podem ficar sem o foco e o objetivo pretendido. Cabe ao e-moderador manter a dinâmica da participação, incentivar a partilha de ideias e ajudar os estudantes a construir conhecimento de forma colaborativa. Além disso, é importante que saiba encerrar as discussões no momento certo, sintetizando os principais contributos e reforçando as aprendizagens alcançadas. Acredito que um e-moderador atento faz toda a diferença na qualidade da experiência de aprendizagem online.

a apresentação global e detalhada da estrutura de conteúdos é fundamental para que a motivação inicial se mantenha durante o decurso do tempo... e para que possamos usar esse momento inicial como memória construída.

Ao ler este trecho, refleti sobre como é importante reconhecer o conhecimento prévio dos participantes e criar atividades que partam desse ponto. Em vez de sobrecarregar com novas informações, o foco na exploração do que já é familiar favorece a construção de confiança e autonomia no processo de aprendizagem. Isso me faz perceber o valor de estratégias que respeitem o ritmo dos alunos e que promovam descobertas significativas a partir de suas próprias experiências e repertórios.

Este trecho destaca a relação entre habilidades técnicas e motivação inicial no ensino online. Como poderíamos estruturar microcredenciais para garantir que, além da aquisição técnica, haja também suporte emocional, especialmente nos primeiros contatos do estudante com o ambiente virtual? Thamires Fiuza