Below is not the molecule for caffeine. Below is just a rectangle.

Gimana kalau KPI yang memastikan ketersediaan basis data dari lembaga penyiaran baik yang bersifat terbuka atau diatur melalui perjanjian kerja sesuai peraturan perundang-undangan?

Sementara kegiatan analisis, monitoring/evaluasi dilakukan oleh pihak ketiga misalnya Perguruan Tinggi, Lembaga Penelitian, Ormas dsb.

🤭🤭🤭

Atas dasar apa, bagaimana, dan siapa yang berwenang memutuskan terjadinya monopoli informasi dan atau monopoli pembentukan opini oleh lembaga penyiaran?

👍👍👍 PR berikutnya bagaimana mekanisme pencabutan hak siar dan keberanian KPI menerapkan

Jika republik ini masih mempercayai asas desentralisasi dalam kerangka otonomi daerah, Semestinya UU ini mengikutsertakan Pemerintah Daerah baik sebagai Wakil Pemerintah Pusat (Provinsi) maupun sebagai unsur penyelenggara pemerintahan daerah (Provinsi/ Kabupaten/Kota) untuk ikut bertanggung jawab.

Hal ini sejalan dengan ketentuan sebagaimana tercantum dalam UU 24/2019 tentang Ekonomi Kreatif di mana televisi dan radio merupakan bagiannya. Lihat catatan sebelumnya

Pasal ini mengerdilkan fungsi KPI hanya sebatas mengatur dan mengawasi isi dan konten siaran. Sementara problem penyiaran di Indonesia cukup kompleks dan sarat kepentingan. Sudah banyak kajian yang membahas 'mandul'nya KPI sebagai Independent Regulatory Agency. Lihat

Mengapa hanya urusan pemeringkatan isi siaran? Selain berat sebelah pada sisi ekonomi, ada potensi bias algoritma pemeringkatan.

Perlu ditambahkan poin penegasan tentang kedudukan penyiaran sebagai produk ekonomi sekaligus produk budaya serta kejelasan tanggung jawab pemerintah maupun pemerintah daerah. Hal ini senada dengan poin b dan c pada UU 24/2019 tentang Ekonomi Kreatif, sebagaimana tertera dalam pertimbangan berikut

b. bahwa Pemerintah dan/atau Pemerintah Daerah bertanggung jawab dalam menciptakan dan mengembangkan ekosistem ekonomi kreatif sehingga mampu memberikan kontribusi bagi perekonomian nasional dan meningkatkan daya saing global guna tercapainya tujuan pembangunan berkelanjutan;

c. bahwa untuk memberikan dasar kepastian hukum kepada Pemerintah dan/atau Pemerintah Daerah dalam menciptakan dan mengembangkan ekosistem ekonomi kreatif, perlu pengaturan tentang ekonomi kreatif;

RUU ini belum secara jelas memberikan gambaran bagaimana tata kelola penyiaran menuju arah yang diharapkan. Perlu ada kebijakan afirmatif terhadap keberadaan lembaga pendidikan bidang komunikasi, penyiaran, maupun komunitas sebagai bagian yang tak terpisahkan dari ekosistem penyelenggaraan penyiaran di Indonesia.

Deonde Express is the leading multi-restaurant delivery software solution, meticulously designed to streamline multi-vendor food ordering. Try our multi-restaurant online ordering system now!

https://web.archive.org/web/20240503124032/https://karl-voit.at/2022/01/29/How-to-Use-Tags/

Long post on 'how to' tag with a set of rules. Not a word on why to tag as a personal practice. Retrieval is key, and not just retrieval but retrieval in contexts. Not merely descriptive but mostly associative. That there is e.g. a #longtail of tags only used once is also a piece of information itself. E.g. when finding the starting point for a new branch of exploration. I find that [[Tags are valuable as pivots 20070815104800]]. The mostly used tags (unavoidable if you use a limited set as suggested in rule 2) become useless because they no longer demarcate a manageble chunck of material.

kai nustatinėjam 12ps

Paspausti ant jo įjungimo mygtuką! taip pat srove stogo padaryt i 98proc!!!

-4000

Try out new versions of your self, your creative self even if they make you uncomfortable.

Explore the truths inherent in your craft, speak them out loud, share them with others in and out of your discipline.

What are the dynamics that make your craft work, that makes it hard to do or easy to do, that explains why you are so passionate about it. Always be observing and reflecting.

Observe what makes your discipline worth caring about.

Understand the psychology of your craft and what draws you to its mastery.

Keep an attitude of support not competition as part of your approach/stance to your craft.

Gather with your community in the real world: conventions, performances, etc.

Find your community, preferably a small one.

Observe the amazing part of your craft that drew you to it in the first place and continues to do so.

Every skill has both a personal history and a historical context, big history that you can learn as way of bolstering your own passion for the craft.

Relate your work to other parts of your life.

Share your wishes out loud.

Fly under the radar with yourself as an audience of one. Lots of these types of performances. For example if you need to act out a Moth story night use an app like Oasis to be your audience of one. Or to share with audiences of one elsewhere.

Observe your learning self.

Perform/do the thing in a low stakes public place.

find a practitioner who can introduce you to the skill.

Say yes to stuff that fits your protopassion.

Look for entry trick: Twigger's Micromastery.

I am trying to tease out how to go from zero to skilled, a worthwhile checklist for anyone contemplating any future.

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

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Reply to the reviewers

1. Point-by-point description of the revisions

__Response to Reviewers’ comments and suggestions __

We are thankful to the reviewers for their time and effort to review our study and for their constructive suggestions. We address below their comments to further improve the manuscript.

__Reviewer #1 __

Major revision points:

The authors should consider using CENH3 as a marker instead of NDC80 to claim NEK1's role in chromosome segregation. Using a direct marker like CENH3 would strengthen their conclusion. However, if the authors choose not to generate the cell line and new set of data, it would be advisable to tone down their conclusion regarding chromosome segregation. I acknowledge the extensive work and data present in the paper.

Response: Thanks to the reviewer for the suggestion. We have tried previously to generate a CENH3-GFP marker line but were not successful. We also requested a CenH3 antibody from the group who published it, but without success. These are the reasons why we generated the NDC80 line, which is another kinetochore marker for chromosome segregation. We have characterised the NDC80-GFP parasite line extensively in a previous study using live cell imaging and super-resolution microscopy to follow its spatiotemporal dynamics at different stages of the Plasmodium life cycle including its correlation with the kinetochore (Zeeshan et al, 2020). We showed its binding to the centromeric region of chromosomes by ChIP seq analysis (See the figure below-Zeeshan et al, 2020). In our recent studies we also showed its dynamic location with other spindle markers like EB1 and ARK2 (Zeeshan et al, 2023). Based on these data, we believe that both CENH3 and NDC80 are appropriate markers for chromosome segregation in Plasmodium, and we hope that the reviewer appreciates this interpretation.

We are pleased to read that the reviewer recognises the extensive amount of work and data present in the paper.

Minor revision points: The figures are well-designed and presented to a high standard. I especially appreciate the guide schematics associated with the IFAs. However, one area that could be improved is the presentation of the expanded parasites. Firstly, the insets cover a major section of the cell, concealing data from the figure. Secondly, the NHS-ester signal is currently saturated and could be dimmed to more accurately represent the MTOC.

Response: We thank the reviewer for their appreciation and suggestions to further improve the figures. We have shifted the insets on the figures to avoid concealing the data. We have tried to improve the NHS-ester signal and provided more Z-stacks to show the MTOC more accurately. Please see the new supplementary figure S7.

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

Major points (please note that both points do not necessarily need further experiments):

1) there is a scarcity for n numbers for many parts of the manuscript. Please give some indications how many cells were inspected, how often a phenotype was observed and how often an experiment was independently carried out (see also minor points).

Response: We have now provided better quantification for all the observations, with the number of cells observed and how many times each experiment was performed reported in the figure legends and in the method section.

2) While the -omics data is interesting, it is somewhat disconnected from the remainder of the manuscript in that it does not lead to any experimental work on the cell level to bring it in connection with NEK1 nor is there any validation. For instance, while the interactome analysis includes a good proportion of very plausible hits, not a single one is validated. I do see that it may be beyond this manuscript to do extensive validations but as this data has limits what can be concluded from it, this should at least be stated in the text. For instance in the paragraph in the discussion (line 613etc). Some experimental validations, if only to show location at the expected site, would be even better.

Response: We thank the reviewer for this insight. We have reanalysed the proteomics data and identified some other MTOC and spindle proteins, including kinesin-8B and kinesin-13 (see new Figure 4). We have already validated kinesin-8B as an MTOC/basal body marker in a previous study, with a similar phenotype during male gametogenesis resulting from its ablation (Zeeshan et al, 2019). Here we show the relative location of NEK1 with respect to that of kinesin-8B, which has a similar MTOC location in early-stage male gametogenesis. In another study we showed that kinesin-13 is localised at the MTOC and spindle and has a similar role during male gametogenesis (Zeeshan et al, 2022). Other proteins, including PF16 and kinesin15, have also been shown to be important for male gametogenesis (Straschil et al, 2010, Zeeshan et al, 2022).

Minor points: 1) Plasmodium is a species name, better not use it on its own (Anopheles would also not be used on its own if Anopheles mosquitoes are meant).

Response: We now mentioned the particular species of the Plasmodium genus or in general, we use Plasmodium spp.

2) While the imaging is very nice, I do have some issues with backgrounds containing mostly pixels of zero intensity (which seems to be the case for some of the images). In western blots this is not permitted anymore (because it is unclear what was clipped and whether this made weaker bands disappear). The same should apply to microscopy, unless very specific and defined analyses were used that caused this. If this was the case here (e.g. deconvolution, back ground subtraction etc), this should be stated for each type of imaging (including parameters). If this was just due to adjusting levels in Photoshp to clip low intensity, I would recommend to reduce that to a degree where no pixel has 0 intensity anymore to ensure no information in the image was lost.

Response: We thank the reviewer for pointing out this issue. Here, we have used several different types of imaging, including live cell, structural illumination microscopy (SIM), expansion microscopy and fixed immunofluorescence. In general, we adjusted the backgrounds for most of these images as necessary but did not use photoshop for any of them. For live cell imaging, the GFP/RFP fluorescent cell signals are well captured at different time points with an auto-exposure time and then processed simply using Axiovision/Zeiss zen software. SIM images were captured using different parameters based on fluorescence signal intensity and processed to remove background at a threshold level. We provide the processing parameters in the SIM processing method section). For expansion microscopy (ExM), the Z stacks were collected for different channels and the brightness adjusted to remove background. The SIM and ExM resolution images are presented as maximum intensity projections, as explained in the methods section; please see pages 23, 25 and 27. An example of SIM image processing is shown below:

3) Line 158: The role of NEK1 in centrosome splitting in Toxoplasma. Given that Plasmodium NEKs are not analogous to mammalian NEKs, a quick word on the relation of Plasmodium and Toxoplasma NEKs may be beneficial. Does Toxo also have 4 NEKs and is TgNEK1 an orthologoue of Plasmodium NEK1?

Response: In fact, Toxoplasma gondii encodes 4 NEKs (Miranda-Saavedra et al., 2012; PMID: 22587893), with TgNEK1 the orthologue of Plasmodium spp NEK1.

4) Fig. S1A: is hDHFR really fused to NEK1? Maybe the scheme can be updated to clarify this without readers having to consult the cited publication (Guttery et al., 2014a)

Response: We apologise for the confusion; we have updated and clarified the schematic for readers.

5) Line 135: those other model eukaryotes -> insert "of" before other

Response: We thank the reviewer for spotting this error and have now inserted “of”.

6) Line 149: is substituted by an SMASH -> by a SMASH

Response: We thank the reviewer for spotting this error and have now substituted by “a SMASH”.

7) Line 151: in the modulation of MAPK pathway -> an article seems to be missing here

Response: We thank the reviewer for pointing out the missing article and have now added Dorin-Semblat et al., 2007 article on MAP kinases to the text.

8) Line 153: remove first bracket

Response: We thank the reviewer for noticing the surplus bracket which has now been removed.

9) Line 189; insert „the" before parasite life cycle

Response: We thank the reviewer for noticing this and have now inserted “the”.

10) Line 211: We observed an overlap of NEK1 and centrin signals but in this case NEK1 was closer than centrin to the DNA (Fig 1C). In contrast to the NDC80 statement ("NDC80-mCherry was always closer to the DNA"), there is no quantitative information. Looking at the images this also seems a bit less clear cut. Can the authors put a number to his in some way?

Response: We thank the reviewer for highlighting this issue. To clarify, we have added some images showing the locations of Centrin-4/NDC80/DNA (New Fig 1B). We also calculated the overlap of DNA with centrin and DNA with NEK1 in the images showing the signals of these proteins (new Fig 1E). Similarly, the overlap of DNA with Ndc80 and DNA with NEK1 was calculated in the images showing the signals of these proteins (new Fig 1F). This analysis describes the order of signals for the different markers showing centrin is further than NDC80 from the DNA.

11) Line 216: The live cell images of the proliferative liver schizogony (Fig S1D) and sporogony (Fig S1E) stages showed similar patterns of NEK1-GFP foci formation during proliferative stages. Can the authors specify what they mean with similar? I do not see any change from cytoplasmic to one focus per nucleus in the liver schizont and also in sporogony there only seems one focus per nucleus in the sporozoite.

Response: The focal points are not very clear in these stages. Plasmodium liver and oocyst stages contain thousands of progenies and accurate study of the temporal dynamics of GFP expression is difficult. We highlight here that NEK1-GFP is localised at focal points in the nucleus, together with a diffused cytoplasmic location similar to that in asexual blood and male gametocyte stages. These foci are only observed during proliferative stages in cells undergoing active endomitosis in both oocyst and liver stages. No signal is observed in later stages when nuclear division is finished.

12) Not all videos seem to have been treated the same way. Video 1 shows strong increase in the DAPI signal, suggesting post-acquisition boosting of the signal in later time points to compensate for GFP bleaching. In contrast the DAPI in Video S2 stays in a reasonable dynamic range. Can the type of processing used be indicated in the materials or legends?

Response: We agree with the reviewer. Our main aim here was to observe the dynamic location of GFP- and RFP-tagged proteins at various stages during male gametogenesis, and not focus on quantifying the signal. We adjusted different channels according to fluorescence intensity only to show the protein location. We could collect a series of timelapse images for only two to three minutes because the GFP/RFP signals are bleached quickly.

13) Particularly for Fig. 2F and Fig. S2, Fig. 3I-L and Fig. 6A but also for others, please indicate how many cells and independent experiments this is based on and give the number in the legend (image representative of X inspected cells or something along these lines). Fig. 6B has some of that information in the main text but also their total number of cells inspected should be added to the figure legend.

Response: We thank the reviewer for this important suggestion. We now include the total number of cells analysed for each representative image, and the number of experiments performed, in both the figure legends and methods section.

14) Line318/Figure 3: "Live cell imaging showed that both NEK1 and kinesin-8B were located in the cytoplasm (Fig 3G, S3D)". And also later in this paragraph: Fig. S3D should be S3G and S3H. Also, the recruitment of Kinesin-8B within the first 30 seconds that is mentioned is not shown, a pre-induction image would be generally good to show. At the start of video 6 Kinesin-8B is also already recruited.

Response: We thank the reviewer for this suggestion. We have now included pre-induction gametocytes images showing expression of these proteins in new Fig 3A.

15) Line 339: "the" before nucleus might make this long sentence clearer.

Response: We thank the reviewer for pointing this out. “the” has now been added.

16) Line 341: Fig. 3I, beaded NDC80 signal. This signal does not seem that much different to some of the other markers in the SIM images in this figure part and the signal looks quite processed. How sure are the authors that the beads are real? This would be a very fascinating data point, so maybe worth providing some more image data. Does the number of NDC80 foci make sense? See also point 13.

Response: We thank the reviewer for pointing out this observation. We agree that that the NDC80 signal in this image does not look disimilar from some others, but this beaded structure is present in many images we have captured. Our focus was to locate the NEK1 signal relative to the signal of NDC80, and it was very challenging to capture both focal points, especially using two markers. We have replaced the image in Fig 3I with another with better signals for NDC80 and included more images in supplementary figures (S3I and J) to validate the observation. In previous studies we have observed similar NDC80 foci (about 28 NDC80 foci in a diploid gametocyte). (Zeeshan et al 2022 and Zeeshan et al 2023); please see the following figure. The NDC80 focal points represent the number of unclustered kinetochores; for example, in a diploid gametocyte during spindle formation, this would be expected for the Plasmodium haploid genome consisting of 14 chromosomes and the centromeric region of each chromosome associated with the kinetochore multi-protein complex that facilitates spindle attachment.

17) Is S4A a replicate of Fig. 5A, it looks like the identical gel? Was this done more than once? Maybe also add that it was a 1 h induction time into the figure. Three replicates were done for the qPCR for the clag promoter strategy, but again this graph is in both, Fig. 5 and Fig. S4. Can the authors weed out the redundancies in these two figures and provide all n numbers?

Response: We thank the reviewer for highlighting this duplication of the image to show depletion/downregulation of NEK1. The image that was originally part of main Figure 5 has now been deleted, leaving it in supplementary Figure S4.

18) Line 419: please help the reader here and modify to start of this paragraph with something along the lines of "To generate PTD lines..."

Response: We have modified the start of the sentence to make clear to readers the importance of the PTD lines.

19) Line 432: Is this how this is typically phrased (In mosquitoes fed NEK1clag parasites)? I would remove "s" from mosquitoes or insert "with" after "fed" (or maybe fed on NEK1clag infected mice?).

Response: We thank the reviewer for this suggestion. We have added “with” after “fed”.

20) Line 436: on the naïve mice, remove "the"

Response: We thank the reviewer for noticing this. We have removed “the” before naïve mice.

21) Line 446: log2fold -1.5

Response: We thank the reviewer for pointing this out and have corrected this.

22) Fig. S5: It might be beneficial to give in the figure the information to at a glance see what kind of data the figure parts show because it is a mix of RNASeq, phosphoproteomics, NEK1clag and NEK1-AID/HA gametocytes.

Response: We thank the reviewer for this suggestion and have now described the data/plots showing RNA-seq and phosphoproteomic analyses in Figure S5.

23) Line 500: consider replacing imaging with information.

Response: We thank the reviewer for this suggestion and have now replaced “imaging” with “information”.

24) Line 585: remove "be"

Response: We thank the reviewer for spotting this and have now removed “be”.

25) Not all symbol fonts did survive PDF conversion, see e.g. line 1050 or 1101 or 1197.

Response: We are not quite sure why the symbols did not survive conversion to pdf; we have tested conversion of the Word file to pdf format and all the symbols survived.

Reviewer #2 (Significance (Required)):

The strength of this work is the very comprehensive imaging data that combines several high-end techniques and provides a coherent picture. It has all the necessary markers to firstly localize NEK1 in the context of mitosis and then understand the phenotype when it is inactivated. The weakness of this work lies in the limited quantitative information on some of the observed phenotypes.

Response: We appreciate the concern of the reviewer and have revised the manuscript by adding further quantitative information in figure legends and in the method section.

and in the limited pursuit of the findings of the -omics data although, in favour of the paper - these data do add meaningfully to the overall picture even if they were not further validated.

Response: We have discussed more about the omics data. Please line number 656-58 in discussion

__Reviewer #3 __

Major comments:

1. NEK1 mutant parasites show a defect in male gametogenesis. Did authors observe any defect on female gamete formation? This data can be included in the main manuscript. An IFA with female gametocyte marker such as Pbg337 can be included to demonstrate sex-specific expression of NEK1. Response: We have investigated sex-specificity by using P28 antibody (13.1), a reagent which is generally used in P. berghei to identify female gametes and zygotes (we do not have a Pbg337-specific reagent and assume that the reviewer is referring to Pf g377). We see no defect in the number of female gametes formed and identified by surface expression of P28 (Fig S4I and J). This observation was supported by the lack of NEK1-GFP expression in female gametocytes at any time point (Fig. S1F). Furthermore, the rescue experiment (now added in Fig 5F) proves that the defect is only in the male and not the female lineage.

Authors should include genetic crosses experiments to demonstrate female gametes of NEK1 mutant are fertile. Authors appear to have sex-sterile lines available in their lab and have performed these experiments in their previous studies (PMID: 37704606).

Response: We thank the reviewer for this suggestion. We have now performed the genetic rescue experiment by crossing NEK1Clag parasites with two female deficient lines (Δnek4 and Δdozi) and one male deficient line (Δhap2). In three independent set of experiments, we could rescue the defect in NEK1Clag parasites by crossing with female deficient lines, but not by crossing with the male deficient line, by observing ookinete formation. These data are presented in Fig 5F.

1. Proteomic and phosphoproteomic data using mutant NEK1 parasites showed differential phosphorylation of several proteins but authors did not observe same proteins to be differentially phosphorylated in different replicates. Using in vitro experiments involving peptides or recombinant protein fragments, authors should validate and demonstrate some of the substrates to be direct parasite substrates for NEK1. Kinesin-15 can be one good candidate substrate as kinesin-15 is less phosphorylated in NEK1-AID/HA and is enriched in NEK1-GFP Immunoprecipitates. This is very relevant especially since authors observe perturbation of levels for several other kinases in NEK1 knock down parasites.

Response: We appreciate the reviewer’s suggestion to do in vitro biochemical experimentation to validate NEK1 kinase substrates. With respect, this suggestion is well beyond the focus of this study since our aim was to characterise the cell biology of NEK1 function in vivo, with a focus on male gametogenesis. Kinesin-15 together with NEK1 was one of the few proteins for which we detected a significant reduction of phosphorylated peptides across all replicates, despite the high sample variability (likely linked to the pre-treatment involving ethanol and/or auxin to degrade the auxin-induced degron). It is therefore highly likely that phosphorylation of Kinesin-15-S454 is dependent on NEK1. We observed no differential phosphorylation of other kinases in the dataset, but we cannot exclude that other kinases or phosphatases are involved in this signalling pathway.

NEK1 is known to phosphorylate MAP2 kinase in vitro in P. falciparum. Did authors find MAP2 to be differentially phosphorylated in their dataset? It should be discussed accordingly in the current manuscript.

Response: We observed phosphorylation of serine 301 in MAPK2. At 6 min post-activation we detected a two-fold reduction of the corresponding peptide with a Q-value of 0.0528, which is just above the selected threshold. Given the high replicate variability, it is possible that this serine is phosphorylated by NEK1 upon gametocyte activation, but more targeted analyses would be necessary to test this hypothesis. As this observation is still rather speculative, we preferred to refer to it in the discussion.

1. Authors show that NEK1 is expressed in pre-erythrocytic stages. Since authors have multiple tools/ transgenic parasites to study relative expression of NEK1, authors can test relative expression of NEK1 in pre-erythrocytic stages and discuss possible function of NEK1 during liver stages.

Response: We appreciate this suggestion of the reviewer, but we consider the proposed work to be outside the scope of this manuscript, where our focus is on the role of NEK1 in sexual cells. We agree that it will be interesting to examine the possible function of NEK1 in liver stages and believe that several groups are now embarking upon such work.

Minor comments: Authors should cite relevant literature on role of kinases in male gametogenesis by adding a paragraph. e.g. PMID: 18532880, PMID: 29042501, PMID: 29311293, PMID: 32568069, PMID: 34724830, PMID: 32681115, PMID: 36154191 and other kinases.

Response: These references have been added in the discussion section, line numbers 632-635.

Reviewer #3 (Significance (Required)):

Expansion microscopy and live cell imaging are cutting edge and provide the detail and dynamic picture of NEK1 expression in the context of components associated with rapid mitosis, spindle formation, and Kinetochore attachment. This study adds new information to our understanding of the process of male gametogenesis in apicomplexan parasites.

Response: We appreciate these encouraging comments.

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

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Referee #3

Evidence, reproducibility and clarity

The manuscript by Zeeshan et al. investigates the function of Never in mitosis (NIMA)-like kinase (NEK 1) during Plasmodium gametogenesis with a focus on NEK1 in the rodent malaria parasites. The current study shows NEK1 as an important component present near MTOC, kinetochore complex and assisting in spindle formation during rapid mitosis during gametogenesis. Using NEK1-GFP parasites, authors show general association of NEK1 with axoneme/ciliary proteins as well as subunits of the replication machinery. By conditional knock down approaches, authors showed that NEK1 is required during male gametogenesis and parasite transmission to mosquitoes suggesting it to be a significant target for developing transmission blocking interventions.

Major comments:

1. NEK1 mutant parasites show a defect in male gametogenesis. Did authors observe any defect on female gamete formation? This data can be included in the main manuscript. . An IFA with female gametocyte marker such as Pbg337 can be included to demonstrate sex-specific expression of NEK1.
2. Authors should include genetic crosses experiments to demonstrate female gametes of NEK1 mutant are fertile. Authors appear to have sex-sterile lines available in their lab and have performed these experiments in their previous studies (PMID: 37704606).
3. Proteomic and phosphoproteomic data using mutant NEK1 parasites showed differential phosphorylation of several proteins but authors did not observe same proteins to be differentially phosphorylated in different replicates. Using in vitro experiments involving peptides or recombinant protein fragments, authors should validate and demonstrate some of the substrates to be direct parasite substrates for NEK1. Kinesin-15 can be one good candidate substrate as kinesin-15 is less phosphorylated in NEK1-AID/HA and is enriched in NEK1-GFP Immunoprecipitates. This is very relevant especially since authors observe perturbation of levels for several other kinases in NEK1 knock down parasites.
4. NEK1 is known to phosphorylate MAP2 kinase in vitro in P. falciparum. Did authors find MAP2 to be differentially phosphorylated in their dataset? It should be discussed accordingly in the current manuscript.
5. Authors show that NEK1 is expressed in pre-erythrocytic stages. Since authors have multiple tools/ transgenic parasites to study relative expression of NEK1, authors can test relative expression of NEK1 in pre-erythrocytic stages and discuss possible function of NEK1 during liver stages.

Minor comments:

Authors should cite relevant literature on role of kinases in male gametogenesis by adding a paragraph. e.g. PMID: 18532880, PMID: 29042501, PMID: 29311293, PMID: 32568069, PMID: 34724830, PMID: 32681115, PMID: 36154191 and other kinases.

Significance

Expansion microscopy and live cell imaging are cutting edge and provide the detail and dynamic picture of NEK1 expression in the context of components associated with rapid mitosis, spindle formation, and Kinetochore attachment. This study adds new information to our understanding of the process of male gametogenesis in apicomplexan parasites.

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

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Referee #2

Evidence, reproducibility and clarity

A fascinating aspect of malaria parasite biology is the incredibly fast generation of 8 nuclei in microgamete formation. In this work the authors identify NEK1, one of the 4 NEKs of the parasite, as an essential protein in that process. They use impressive imaging (but see small comment on that below) including live cell time lapse, superresolution, expansion and EM with markers to describe the location of NEK1 in relation to mitosis relevant structures and the impact of its loss. This is bolstered by some IP, RNASeq and phosphoproteomic data, providing a very thorough characterization of NEK1 although these data are less thorough than the imaging. The sound conclusion from the authors is that Nek1 is needed for MTOC organization, spindle assembly and kinetochore attachment in mitosis during male gamete formation.

Major points(please note that both points do not necessarily need further experiments):

1. there is a scarcity for n numbers for many parts of the manuscript. Please give some indications how many cells were inspected, how often a phenotype was observed and how often an experiment was independently carried out (see also minor points).
2. while the -omics data is interesting, it is somewhat disconnected from the remainder of the manuscript in that it does not lead to any experimental work on the cell level to bring it in connection with NEK1 nor is there any validation. For instance, while the interactome analysis includes a good proportion of very plausible hits, not a single one is validated. I do see that it may be beyond this manuscript to do extensive validations but as this data has limits what can be concluded from it, this should at least be stated in the text. For instance in the paragraph in the discussion (line 613etc). Some experimental validations, if only to show location at the expected site, would be even better.

Minor points:

1. Plasmodium is a species name, better not use it on its own (Anopheles would also not be used on its own if Anopheles mosquitoes are meant).
2. While the imaging is very nice, I do have some issues with backgrounds containing mostly pixels of zero intensity (which seems to be the case for some of the images). In western blots this is not permitted anymore (because it is unclear what was clipped and whether this made weaker bands disappear). The same should apply to microscopy, unless very specific and defined analyses were used that caused this. If this was the case here (e.g. deconvolution, back ground subtraction etc), this should be stated for each type of imaging (including parameters). If this was just due to adjusting levels in Photoshp to clip low intensity, I would recommend to reduce that to a degree where no pixel has 0 intensity anymore to ensure no information in the image was lost.
3. Line 158: The role of NEK1 in centrosome splitting in Toxoplasma. Given that Plasmodium NEKs are not analogous to mammalian NEKs, a quick word on the relation of Plasmodium and Toxoplasma NEKs may be beneficial. Does Toxo also have 4 NEKs and is TgNEK1 an orthologoue of Plasmodium NEK1?
4. Fig. S1A: is hDHFR really fused to NEK1? Maybe the scheme can be updated to clarify this without readers having to consult the cited publication (Guttery et al., 2014a)
5. Line 135: those other model eukaryotes -> insert "of" before other
6. Line 149: is substituted by an SMASH -> by a SMASH
7. Line 151: in the modulation of MAPK pathway -> an article seems to be missing here
8. Line 153: remove first bracket
9. Line 189; insert „the" before parasite life cycle
10. Line 211: We observed an overlap of NEK1 and centrin signals but in this case NEK1 was closer than centrin to the DNA (Fig 1C). In contrast to the NDC80 statement ("NDC80-mCherry was always closer to the DNA"), there is no quantitative information. Looking at the images this also seems a bit less clear cut. Can the authors put a number to his in some way?
11. Line 216: The live cell images of the proliferative liver schizogony (Fig S1D) and sporogony (Fig S1E) stages showed similar patterns of NEK1-GFP foci formation during proliferative stages. Can the authors specify what they mean with similar? I do not see any change from cytoplasmic to one focus per nucleus in the liver schizont and also in sporogony there only seems one focus per nucleus in the sporozoite.
12. Not all videos seem to have been treated the same way. Video 1 shows strong increase in the DAPI signal, suggesting post acquisition boosting of the signal in later time points to compensate for GFP bleaching. In contrast the DAPI in Video S2 stays in a reasonable dynamic range. Can the type of processing used be indicated in the materials or legends?
13. Particularly for Fig. 2F and Fig. S2, Fig. 3I-L and Fig. 6A but also for others, please indicate how many cells and independent experiments this is based on and give the number in the legend (image representative of X inspected cells or something along these lines). Fig. 6B has some of that information in the main text but also there total number of cells inspected should be added to the figure legend.
14. Line318/Figure 3: "Live cell imaging showed that both NEK1 and kinesin-8B were located in the cytoplasm (Fig 3G, S3D)". And also later in this paragraph: Fig. S3D should be S3G and S3H. Also, the recruitment of Kinesin-8B within the first 30 seconds that is mentioned is not shown, a pre-induction image would be generally good to show. At the start of video 6 Kinesin-8B is also already recruited.
15. Line 339: "the" before nucleus might make this long sentence clearer.
16. Line 341: Fig. 3I, beaded NDC80 signal. This signal does not seem that much different to some of the other markers in the SIM images in this figure part and the signal looks quite processed. How sure are the authors that the beads are real? This would be a very fascinating data point, so maybe worth providing some more image data. Does the number of NDC80 foci make sense? See also point 13.
17. Is S4A a replicate of Fig. 5A, it looks like the identical gel? Was this done more than once? Maybe also add that it was a 1 h induction time into the figure. Three replicates were done for the qPCR for the clag promoter strategy, but again this graph is in both, Fig. 5 and Fig. S4. Can the authors weed out the redundancies in these two figures and provide all n numbers?
18. Line 419: please help the reader here and modify to start of this paragraph with something along the lines of "To generate PTD lines..."
19. Line 432: Is this how this is typically phrased (In mosquitoes fed NEK1clag parasites)? I would remove "s" from mosquitoes or insert "with" after "fed" (or maybe fed on NEK1clag infected mice?).
20. Line 436: on the naïve mice, remove "the"
21. Line 446: log2fold 1.5 likely should be log2fold>-1.5
22. Fig. S5: It might be beneficial to give in the figure the information to at a glance see what kind of data the figure parts show because it is a mix of RNASeq, phosphoproteomics, NEK1clag and NEK1-AID/HA gametocytes.
23. Line 500: consider replacing imaging with information.
24. Line 585: remove "be"
25. Not all symbol fonts did survive PDF conversion, see e.g. line 1050 or 1101 or 1197.

Significance

The strength of this work is the very comprehensive imaging data that combines several high-end techniques and provides a coherent picture. It has all the necessary markers to firstly localize NEK1 in the context of mitosis and then understand the phenotype when it is inactivated. The weakness of this work lies in the limited quantitative information on some of the observed phenotypes and in the limited pursuit of the findings of the -omics data although, in favour of the paper - these data do add meaningfully to the overall picture even if they were not further validated.

The study provides very interesting information to a field that currently is gaining momentum in malaria research. It will be of interest to researchers working on

• mitosis in malaria parasites and other apicomplexans
• kinases in these parasites and likely also for researchers working on mitosis in model organisms.

Expertise: I am a cell biologist working with P. falciparum blood stages

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Referee #1

Evidence, reproducibility and clarity

Summary:

This article by Zeeshan et al. investigates the role of NEK1 kinase in Plasmodium, the malaria parasite, focusing on its essential functions in microtubule organizing center (MTOC) organization and kinetochore attachment during the rapid mitosis involved in male gamete formation. Utilizing a combination of live cell imaging, ultrastructure expansion microscopy (U-ExM), and various molecular biology techniques, the authors elucidate the spatiotemporal dynamics of NEK1 in relation to MTOC dynamics across different stages of the Plasmodium life cycle. Their findings reveal that NEK1 is crucial for coordinating spindle formation and chromosome segregation, highlighting its potential as a target for malaria intervention strategies.

Strengths:

Comprehensive Methodological Approach: The combination of cutting-edge imaging techniques with conditional gene knockdown and proteomics provides a robust framework for investigating NEK1's role in Plasmodium mitosis, ensuring the reliability and depth of the findings. Novel Insights into Plasmodium Biology: The study offers groundbreaking insights into the mitotic mechanisms of Plasmodium, particularly the atypical processes involved in male gametogenesis, thereby filling a significant knowledge gap. Implications for Malaria Control: By identifying a potential new drug target, this research directly contributes to the ongoing malaria control and eradication efforts, highlighting the translational potential of basic biological research. Major revision points: The authors should consider using CENH3 as a marker instead of NDC80 to claim NEK1's role in chromosome segregation. Using a direct marker like CENH3 would strengthen their conclusion. However, if the authors choose not to generate the cell line and new set of data, it would be advisable to tone down their conclusion regarding chromosome segregation. I acknowledge the extensive work and data present in the paper. Minor revision points: The figures are well-designed and presented to a high standard. I especially appreciate the guide schematics associated with the IFAs. However, one area that could be improved is the presentation of the expanded parasites. Firstly, the insets cover a major section of the cell, concealing data from the figure. Secondly, the NHS-ester signal is currently saturated and could be dimmed to more accurately represent the MTOC.

Significance

This study significantly advances our understanding of the cell cycle mechanisms in Plasmodium, particularly the unique mitotic processes involved in male gametogenesis. By elucidating the role of NEK1 kinase, the research addresses a critical gap in malaria biology, offering insights into the parasite's ability to proliferate and transmit between hosts. The identification of NEK1 as a key regulator of MTOC organization and kinetochore attachment during Plasmodium mitosis not only broadens our fundamental knowledge of cellular division in divergent eukaryotes. The study lays a solid foundation for future research to disrupt malaria transmission through targeted intervention strategies. Further exploration of NEK1's interactions and the development of specific inhibitors could pave the way for novel antimalarial therapies, highlighting the importance of continued research in this area.

The article by Zeeshan et al. contributes significantly to our understanding of Plasmodium biology, particularly the role of NEK1 kinase in the parasite's cell cycle. Despite some limitations, such as the scope of kinase investigation and the direct translation to therapeutic applications, the study lays a solid foundation for future research to disrupt malaria transmission through targeted intervention strategies. Further exploration of NEK1's interactions and the development of specific inhibitors could pave the way for novel antimalarial therapies, highlighting the importance of continued research in this area.

.

https://www.theguardian.com/environment/article/2024/may/03/britain-climate-action-plan-unlawful-high-court

The Git's way. Snapshots as the source of truth, derive deltas to merge.

Résumé de la vidéo [00:00:00][^1^][1] - [00:30:16][^2^][2] : La vidéo présente une conférence sur la décentralisation des organisations, explorant la relation entre autonomie et contrôle. Les intervenants, Carolyn Mc Lain Pirard et Eric Della Valle, partagent leurs perspectives sur l'innovation managériale et l'entreprise libérée, en mettant l'accent sur l'importance de la cohérence des pratiques organisationnelles et du maintien d'un équilibre entre autonomie et contrôle social.

Points forts : + [00:00:00][^3^][3] Introduction à la conférence * Présentation du thème de la décentralisation * Discussion sur l'autonomie versus le contrôle * Introduction des intervenants et de leurs parcours + [00:02:15][^4^][4] Carolyn Mc Lain Pirard sur l'entreprise libérée * Définition et historique de l'entreprise libérée * Analyse des pratiques managériales innovantes * Importance de la vision et de l'orientation client + [00:05:07][^5^][5] Eric Della Valle sur les formes organisationnelles * Son expérience en tant que consultant et dirigeant * Intérêt pour l'évolution des structures organisationnelles * Présentation du modèle organisationnel cellulaire + [00:09:31][^6^][6] Articulation entre autonomie et contrôle * Méthodes de décentralisation des organisations * Impact de la structure, des processus et des pratiques sur l'autonomie * Rôle du leadership et de la philosophie humaniste dans l'innovation managériale Résumé de la vidéo [00:30:20][^1^][1] - [00:57:45][^2^][2]:

La vidéo traite de la décentralisation des organisations et de l'importance de l'autonomie dans les processus de transformation. Elle souligne les défis liés au contrôle social et à la performance sociale des organisations qui cherchent à s'émanciper des structures hiérarchiques traditionnelles.

Points forts: + [00:30:20][^3^][3] Défis de la décentralisation * Risques de dérives dans le contrôle social * Impact sur l'investissement des employés dans les projets + [00:31:22][^4^][4] Pérenniser la libération * Adoption des pratiques en grappes pour une transformation efficace * Importance de l'ordre d'adoption des pratiques + [00:33:27][^5^][5] Contrôle dans les organisations transformées * Transition du contrôle hiérarchique vers l'autocontrôle * Différentes approches de transformation et leur impact sur le contrôle + [00:37:11][^6^][6] Progressivité dans l'adoption des pratiques * Nécessité d'une transformation progressive pour une adoption durable * Importance de l'accompagnement des employés vers l'autonomie Résumé de la vidéo [00:57:49][^1^][1] - [01:23:22][^2^][2]:

La vidéo aborde la décentralisation des organisations en présentant un modèle d'organisation cellulaire, où les équipes sont autonomes mais alignées sur des objectifs communs. Elle discute de la hiérarchie, de la subsidiarité et de l'importance de l'alignement des équipes pour atteindre des objectifs supérieurs.

Points forts: + [00:57:49][^3^][3] Autonomie et contrôle * Introduction du management par objectifs clairs * Concept de mini-entreprises autonomes * Nécessité de reporter quotidiennement les activités + [01:00:12][^4^][4] Hiérarchie organisationnelle * Distinction entre hiérarchie humaine et organisationnelle * Utilisation de la subsidiarité plutôt que de la délégation * Les niveaux supérieurs servent les niveaux inférieurs + [01:02:21][^5^][5] Fonctions support dans les organisations cellulaires * Importance des fonctions support pour l'autonomie des équipes * Responsabilité des entités opérationnelles pour l'efficacité et l'efficience * Les fonctions support agissent en tant que fournisseurs de compétences + [01:04:41][^6^][6] Dynamique organisationnelle * Préférence pour les petites équipes et le principe de Dunbar * Importance des valeurs et règles internes * Adoption de l'amélioration continue et du changement permanent Résumé de la vidéo [01:23:24][^1^][1] - [01:31:30][^2^][2]:

Cette partie de la vidéo aborde la décentralisation des organisations, en mettant l'accent sur l'évaluation des performances et des compétences au sein des équipes auto-organisées. Elle explore les différences entre les rôles et les fonctions dans ces équipes, ainsi que les méthodes d'évaluation adaptées à ce type de structure organisationnelle.

Points forts: + [01:24:04][^3^][3] Pratiques d'évaluation * Différenciation entre performance et compétences * Évaluation des compétences individuelles * Co-responsabilité de la performance en équipe + [01:25:28][^4^][4] Évaluation chez Spotify * Utilisation de la méthode Scrum * Évaluation des compétences par les chapters * Évaluation à 360° par les coéquipiers + [01:27:15][^5^][5] Organisations cellulaires dans des structures pyramidales * Possibilité d'intégrer des équipes auto-organisées * Approche empirique et accompagnement des managers * Infusion progressive de l'auto-organisation

Chaque point fort est accompagné de trois sous-points qui offrent un aperçu rapide du contenu abordé dans la vidéo.

Le lien ne marche pas quand je le met sur VSC

https://web.archive.org/web/20240503092551/https://protos.com/creator-of-rabbit-ai-assistant-has-hidden-nft-past/

r1 founder's (scrubbed) past as another nail in Rabbit's coffin. I guess the 'large action model' is vaporware, back to Automator :D

これどうなの？

Matplotlibで作った図

DataFrameで作った表

SymPyで作った数式

てすと

Résumé de la vidéo [00:00:01][^1^][1] - [00:22:54][^2^][2] : La vidéo aborde le problème de la violence chez les jeunes en France, en se concentrant sur les aspects sociaux et les réponses pénales. Elle examine les causes profondes, telles que l'influence des bandes et la culture de la rue, et discute de l'efficacité des interventions judiciaires et éducatives.

Points forts : + [00:00:01][^3^][3] La violence juvénile comme problème social * Augmentation de la violence dans les quartiers prioritaires * Influence des bandes et de la culture de la rue sur les jeunes * Rôle des réseaux sociaux dans l'amplification de la violence + [00:12:00][^4^][4] La réponse pénale à la violence des jeunes * Différences entre la justice pour mineurs et pour majeurs * Importance de l'éducation et de l'accompagnement des adolescents * Défis rencontrés par les institutions judiciaires dans certains quartiers + [00:18:01][^5^][5] Évolution de la violence et nécessité d'adaptation des réponses * Difficultés des institutions à travailler dans certains quartiers * Transmission intergénérationnelle de la culture de la rue * Besoin de solutions d'accueil et de formation pour les jeunes vulnérables Résumé de la vidéo [00:22:57][^1^][1] - [00:34:11][^2^][2]:

La vidéo aborde le problème de la violence chez les jeunes dans les quartiers, l'influence des réseaux sociaux, et les réponses pénales et éducatives. Elle met en lumière l'adaptation des jeunes à la vie de ghetto, l'impact des réseaux sociaux sur la mobilisation et la diffusion de la violence, et l'importance de l'éducation et de la prévention.

Points forts: + [00:23:10][^3^][3] Rôle des réseaux sociaux * Valorisation d'actes violents * Point de ralliement pour les jeunes * Diffusion rapide de la violence + [00:25:01][^4^][4] Culture de rue et résonance * Influence des voyous sur les jeunes * Production de clips de violence * Conformisme des jeunes dans les écoles + [00:27:00][^5^][5] Réforme de la justice des mineurs * Proposition de modèles alternatifs de réussite * Importance de l'éducation au système juridique * Réponses pénales et réparations pour les dommages + [00:31:00][^6^][6] Débat sur la culture de l'excuse * Fausse perception de l'impunité des mineurs * Atténuation de la responsabilité des mineurs * Importance des principes constitutionnels et internationaux

Résumé de la vidéo [00:00:00][^1^][1] - [00:23:09][^2^][2]:

Cette vidéo présente une conférence de Pierre-Michel Menger sur l'évaluation de la pénibilité au travail dans les sciences, les arts et les organisations. Il aborde les liens entre les préoccupations sociales et scientifiques, l'importance de la pénibilité au travail dans l'agenda social, et les défis de l'évaluation de la pénibilité.

Points forts: + [00:00:22][^3^][3] Contexte et introduction * Présentation du sujet et de son importance sociale et scientifique * Discussion sur la pénibilité au travail et son impact + [00:01:37][^4^][4] La pénibilité comme épreuve * Exploration de la polysémie du terme "épreuve" * Structure du plan de la conférence + [00:06:00][^5^][5] Effets du travail sur la santé * Inégalités sociales d'espérance de vie liées au travail * Impact des conditions de travail sur la santé à long terme + [00:14:08][^6^][6] Évaluation de la pénibilité * Difficultés et méthodes d'évaluation de la pénibilité * Distinction entre différents types de pénibilité + [00:20:31][^7^][7] Quantification de la pénibilité * Exemples de quantification des effets du travail sur la santé * Importance de l'intégration des connaissances scientifiques dans le débat social Résumé de la vidéo [00:23:11][^1^][1] - [00:45:33][^2^][2]:

La vidéo traite de l'évaluation de la pénibilité dans les métiers, en particulier chez les éboueurs. Elle explore les impacts de l'âge, les conditions de travail, et les changements organisationnels sur la perception de la pénibilité et la santé des travailleurs.

Points forts: + [00:23:11][^3^][3] Impact de l'âge sur la pénibilité * La consommation de somnifères varie avec l'âge * Les différences significatives apparaissent après 40 ans * Discussion sur la tolérance au travail urgent avec l'âge + [00:25:56][^4^][4] Étude démographique sur les éboueurs * Comparaison de la longévité des éboueurs avec d'autres personnels * Écart d'espérance de vie de trois ans à 60 ans * Importance de considérer les caractéristiques pénalisantes du travail + [00:27:02][^5^][5] Entretiens rétrospectifs avec les éboueurs * Analyse des conditions de travail et de la fin de carrière * Impact de l'évolution des équipements et des interactions sociales * Perception de la dégradation du comportement des usagers + [00:32:05][^6^][6] Évaluation ergonomique et analyse d'activité * Réduction des contraintes physiques avec l'utilisation de conteneurs * Observation des courses et des efforts physiques nécessaires * Importance de la transmission des savoirs professionnels Résumé de la vidéo [00:45:37][^1^][1] - [01:00:03][^2^][2]:

La vidéo présente une conférence sur l'évaluation de la pénibilité dans les sciences, les arts et les organisations. Elle aborde la reconnaissance de l'expérience des employés, les stratégies de travail individuelles et collectives, et la responsabilité des employeurs de créer des conditions de travail favorables.

Points forts: + [00:45:37][^3^][3] Reconnaissance de l'expérience * Importance de valoriser l'ancienneté et l'expérience * Nécessité de respecter les employés expérimentés * Impact des stratégies de travail construites sur l'expérience + [00:46:31][^4^][4] Amélioration des conditions de travail * Effets sur la santé à long terme et l'accueil des personnes ayant une santé déficiente * Importance des actions ciblées et de la cessation d'activité * Valorisation de l'expérience pour une fin de vie professionnelle moins pénible + [00:49:01][^5^][5] Expérimentations et impact à long terme * Discussion sur les expérimentations grandeur nature * Intégration du changement immédiat dans la réalisation à long terme * Questions sur la causalité complexe et la position de l'expertise dans le débat public + [00:50:01][^6^][6] Mécanismes de compensation et de prévention * Différents mécanismes pour gérer la pénibilité * Rôle de la réflexivité individuelle et collective * Importance de la transmission d'expérience et de la prise de conscience

Résumé de la vidéo [00:00:00][^1^][1] - [00:27:04][^2^][2]:

Cette vidéo présente une conférence de Pierre-Michel Menger sur l'évaluation dans les sciences, les arts et les organisations. Il aborde les raisons pour lesquelles nous avons besoin d'évaluations, les critères utilisés pour les établir et l'efficacité de ces évaluations.

Points saillants: + [00:00:22][^3^][3] Introduction de Victor Ginsburgh * Économiste de renom * Spécialiste en économie de l'art et de la culture * A publié sur la thématique des langues + [00:02:52][^4^][4] Pourquoi les évaluations sont nécessaires * Aident à choisir et à distinguer la qualité * Les concours et les prix fournissent des incitations + [00:03:25][^5^][5] Histoire des concours et évaluations * Origines dans l'antiquité grecque * Exemples de concours dans divers domaines + [00:07:32][^6^][6] Critères d'évaluation * Différence entre critères objectifs et subjectifs * Complexité de l'agrégation des jugements + [00:15:57][^7^][7] Perspectives des sociologues, économistes et philosophes * Bourdieu : évaluation basée sur des conventions sociales * Économistes : les prix reflètent la valeur * Philosophes : jugement subjectif et propriétés esthétiques + [00:25:01][^8^][8] Propriétés esthétiques et évaluation * Expérience de pensée sur l'évaluation de l'art * Influence des titres sur la perception des œuvres Résumé de la vidéo [00:27:08][^1^][1] - [00:54:02][^2^][2]:

La vidéo traite de l'évaluation dans les arts et les organisations, en se concentrant sur l'histoire de la critique d'art et l'importance des propriétés dans l'évaluation des œuvres d'art. Elle explore comment les propriétés artistiques ont été évaluées et pondérées au fil du temps, en utilisant des exemples de la peinture et du cinéma.

Points forts: + [00:27:08][^3^][3] L'histoire de la critique d'art * Discussion sur la querelle entre les défenseurs du dessin et du coloris * Analyse de la "balance des peintres" de Roger de Piles + [00:35:11][^4^][4] Évaluation des propriétés artistiques * Importance de pondérer les propriétés pour une évaluation globale * Utilisation de la régression économétrique pour inférer les pondérations + [00:37:00][^5^][5] Les Oscars comme étude de cas * Corrélation entre les Oscars secondaires et l'Oscar du meilleur film * Prédiction des gagnants basée sur les propriétés attribuées aux films + [00:39:07][^6^][6] Conclusion sur les propriétés * Les propriétés existent et peuvent être quantifiées * L'impact des propriétés sur l'évaluation des œuvres d'art à travers le temps Résumé de la vidéo [00:54:05][^1^][1] - [01:00:54][^2^][2]:

La partie 3 de la vidéo aborde l'évaluation des performances dans les concours, en se concentrant sur un cas d'étude de musiciens évalués lors d'un concours. L'analyse révèle une corrélation entre l'ordre de passage des participants et leur classement final, suggérant que le succès peut être influencé par des facteurs aléatoires plutôt que par le talent seul.

Points forts: + [00:54:05][^3^][3] Analyse des résultats d'un concours * Présentation des données de classement des participants * Observation d'une corrélation entre l'ordre de passage et le classement * Discussion sur l'impact de l'ordre de passage sur le succès + [00:57:00][^4^][4] Questionnement sur la validité du classement * Interrogation sur le rôle du talent dans le succès des participants * Évaluation de la réussite basée sur les enregistrements et les critiques * Remise en question de l'équité du processus d'évaluation + [00:57:50][^5^][5] Critique de la nécessité de choisir un vainqueur * Réflexion sur la culture de la compétition et ses conséquences * Exemples de concours où le premier prix n'a pas été attribué * Proposition de repenser les concours basés sur des jugements subjectifs

Résumé de la vidéo [00:00:00][^1^][1] - [00:26:34][^2^][2]:

Cette vidéo présente une conférence sur l'évaluation dans les sciences, les arts et les organisations, donnée par P.-M. Menger. Elle explore l'évolution de l'évaluation académique, l'impact des évaluations sur les carrières universitaires et la gestion des universités.

Points forts: + [00:00:00][^3^][3] Introduction à l'évaluation académique * Présentation du sujet et du contexte historique de l'évaluation * Évolution de l'évaluation depuis le XVIIe siècle * Changements survenus autour des années 1980 + [00:03:10][^4^][4] L'évaluation et les carrières universitaires * Lien entre évaluation et carrières * Influence de l'évaluation sur les décisions de carrière * Disparité entre évaluation et progression de carrière + [00:05:03][^5^][5] Managerialisation des universités * Transformation des universités dans les années 1980 * Impact de la gestion sur l'évaluation académique * Évaluation encadrée par des outils organisationnels + [00:07:38][^6^][6] Caractérisation du travail universitaire * Hétérogénéité et multifonctionnalité du travail universitaire * Importance de l'évaluation par les pairs dans la reproduction académique * Difficulté de mesurer la valeur relative des différentes tâches académiques + [00:12:01][^7^][7] Le rôle des comités dans l'évaluation * Fonctionnement et délibérations des comités d'évaluation * Évaluation de divers aspects du travail universitaire * Incommensurabilité des activités évaluées + [00:20:23][^8^][8] La fabrique organisationnelle de l'évaluation * Évolution du rôle des départements et des directeurs * Introduction de la gestion des ressources humaines dans les universités * Coexistence de l'auto-régulation professionnelle et des méthodes de gestion des ressources humaines Résumé de la vidéo [00:26:36][^1^][1] - [00:54:25][^2^][2]:

Cette partie de la vidéo aborde l'évaluation dans les universités du Royaume-Uni, en se concentrant sur les changements dans les carrières universitaires et l'impact des évaluations sur celles-ci. L'orateur discute de l'évolution des systèmes d'évaluation et de la manière dont ils influencent la différenciation et la stratification des postes universitaires.

Points forts: + [00:26:36][^3^][3] Évaluation des universités britanniques * Introduction des évaluations continues * Importance accrue du directeur de département * Impact sur les carrières universitaires + [00:32:02][^4^][4] Travail d'organisation et production normative * Redéfinition des postes et des fonctions * Établissement de critères d'évaluation * Prescription des opérations d'évaluation + [00:37:20][^5^][5] Effets sur les carrières universitaires * Différenciation fonctionnelle des postes * Stratification institutionnelle croissante * Division du travail d'évaluation + [00:44:31][^6^][6] Discussion et questions * Comparaison avec d'autres pays * Mécanismes de différenciation endogène * Justice dans les systèmes d'évaluation * Évaluation de l'enseignement et comparaison internationale Résumé de la vidéo [00:54:28][^1^][1] - [01:01:45][^2^][2]:

La partie 3 de la vidéo aborde l'évaluation dans les universités, la mobilité académique, la production scientifique, et l'évaluation de l'enseignement au Royaume-Uni. L'orateur discute de l'hybridation des rôles universitaires et managériaux, de l'augmentation de la mobilité interinstitutionnelle depuis les années 80, et de l'inflation des publications scientifiques. Il souligne également les défis posés par les programmes de formation des enseignants et les effets de la diffusion en ligne des contenus d'enseignement.

Points saillants: + [00:54:28][^3^][3] Hybridation des rôles dans l'évaluation * Évaluation effectuée par des bureaucrates et des universitaires * Difficulté à opposer universitaires et managers * Profession universitaire différenciée par rapport à l'action managériale + [00:55:46][^4^][4] Mobilité académique et production scientifique * Augmentation de la mobilité académique interinstitutionnelle * Inflation des publications scientifiques depuis les années 80 * Nécessité d'examiner la qualité des travaux et le nombre de revues + [00:57:00][^5^][5] Évaluation de l'enseignement * Programmes de formation pour les enseignants depuis 1997 * Débats sur l'efficacité de la formation 'Learning to Teach' * Certification des universitaires presque automatique après la formation + [01:00:45][^6^][6] Diffusion en ligne et impact sur l'enseignement * Formatage des contenus d'enseignement par la diffusion en ligne * Compétition et standardisation des enseignements diffusés * Ouverture des contenus d'enseignement via la technologie

Author response:

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

We thank the reviewers for their constructive comments and suggestions. We have prepared a revised manuscript with updated quantification of theta cycle skipping, new statistical comparisons of the difference between the two behavioral tasks, and general improvements to the text and figures.

Reviewer #1 (Public Review):

Summary

The authors provide very compelling evidence that the lateral septum (LS) engages in theta cycle skipping.

Strengths

The data and analysis are highly compelling regarding the existence of cycle skipping.

Weaknesses

The manuscript falls short on in describing the behavioral or physiological importance of the witnessed theta cycle skipping, and there is a lack of attention to detail with some of the findings and figures:

More/any description is needed in the article text to explain the switching task and the behavioral paradigm generally. This should be moved from only being in methods as it is essential for understanding the study.

Following this suggestion, we have expanded the description of the behavioral tasks in the Results section.

An explanation is needed as to how a cell can be theta skipping if it is not theta rhythmic.

A cell that is purely theta skipping (i.e., always fires on alternating theta cycles and never on adjacent theta cycles) will only have enhanced power at half theta frequency and not at theta frequency. Such a cell will therefore not be considered theta rhythmic in our analysis. Note, however, that there is a large overlap between theta rhythmic and theta skipping cell populations in our data (Figure 3 - figure supplement 2), indicating that most cells are not purely theta skipping.

The most interesting result, in my opinion, is the last paragraph of the entire results section, where there is more switching in the alternation task, but the reader is kind of left hanging as to how this relates to other findings. How does this relate to differences in decoding of relative arms (the correct or incorrect arm) during those theta cycles or to the animal's actual choice? Similarly, how does it relate to the animal's actual choice? Is this phenomenon actually behaviorally or physiologically meaningful at all? Does it contribute at all to any sort of planning or decision-making?

We agree that the difference between the two behavioral tasks is very interesting. It may provide clues about the mechanisms that control the cycle-by-cycle expression of possible future paths and the potential impact of goal-directed planning and (recent) experience. In the revised manuscript, we have expanded the analysis of the differences in theta-cycle dynamics between the two behavioral tasks. First, we confirm the difference through a new quantification and statistical comparison. Second, we performed additional analyses to explore the idea that the alternation of non-local representations reflects the number of relevant paths available to the animal (Figure 11 – figure supplements 2 and 3), but this did not appear to be the case. However, these results provide a starting point for future studies to clarify the task dependence of the theta- cycle dynamics of spatial representations and to address the important question of behavioral/physiological relevance.

The authors state that there is more cycle skipping in the alternation task than in the switching task, and that this switching occurs in the lead-up to the choice point. Then they say there is a higher peak at ~125 in the alternation task, which is consistent. However, in the final sentence, the authors note that "This result indicates that the representations of the goal arms alternate more strongly ahead of the choice point when animals performed a task in which either goal arm potentially leads to reward." Doesn't either arm potentially lead to a reward (but different amounts) in the switching task, not the alternation task? Yet switching is stronger in the alternation task, which is not constant and contradicts this last sentence.

The reviewer is correct that both choices lead to (different amounts of) reward in the switching task. As written, the sentence that the reviewer refers to is indeed not accurate and we have rephrased it to: “This result indicates that the representations of the goal arms alternate more strongly ahead of the choice point when animals performed a task in which either goal arm potentially leads to a desirable high-value reward.”.

Additionally, regarding the same sentence - "representations of the goal arms alternate more strongly ahead of the choice point when the animals performed a task in which either goal arm potentially leads to reward." - is this actually what is going on? Is there any reason at all to think this has anything to do with reward versus just a navigational choice?

We appreciate the reviewer’s feedback and acknowledge that our statement needs clarification. At the choice point in the Y-maze there are two physical future paths available to the animal (disregarding the path that the animal took to reach the choice point) – we assume this is what the reviewer refers to as “a navigational choice”. One hypothesis could be that alternation of goal arm representations is present whenever there are multiple future paths available, irrespective of the animal’s (learned) preference to visit one or the other goal arm. However, the reduced alternation of goal arm representations in the switching task that we report, suggests that the animal’s recent history of goal arm visits and reward expectations likely do influence the theta-cycle representations ahead of the choice point. We have expanded our analysis to test if theta cycle dynamics differ for trials before and after a switch in reward contingency in the switching task, but there was no statistical difference in our data. We have rewritten and expanded this part of the results to make our point more clearly.

Similarly, the authors mention several times that the LS links the HPC to 'reward' regions in the brain, and it has been found that the LS represents rewarded locations comparatively more than the hippocampus. How does this relate to their finding?

Indeed, Wirtshafter and Wilson (2020) reported that lateral septum cells are more likely to have a place field close to a reward site than elsewhere in their double-sided T-maze. It is possible that this indicates a shift towards reward or value representations in the lateral septum. In our study we did not look at reward-biased cells and whether they are more or less likely to engage in theta cycle skipping. This could be a topic for future analyses. It should be noted that the study by Wirtshafter and Wilson (2020) reports that a reward bias was predominantly present for place fields in the direction of travel away from the reward site. These reward-proximate LS cells may thus contribute to theta-cycle skipping in the inbound direction, but it is not clear if these cells would be active during theta sweeps when approaching the choice point in the outbound direction.

Reviewer #2 (Public Review)

Summary

Recent evidence indicates that cells of the navigation system representing different directions and whole spatial routes fire in a rhythmic alternation during 5-10 Hz (theta) network oscillation (Brandon et al., 2013, Kay et al., 2020). This phenomenon of theta cycle skipping was also reported in broader circuitry connecting the navigation system with the cognitive control regions (Jankowski et al., 2014, Tang et al., 2021). Yet nothing was known about the translation of these temporally separate representations to midbrain regions involved in reward processing as well as the hypothalamic regions, which integrate metabolic, visceral, and sensory signals with the descending signals from the forebrain to ensure adaptive control of innate behaviors (Carus-Cadavieco et al., 2017). The present work aimed to investigate theta cycle skipping and alternating representations of trajectories in the lateral septum, neurons of which receive inputs from a large number of CA1 and nearly all CA3 pyramidal cells (Risold and Swanson, 1995). While spatial firing has been reported in the lateral septum before (Leutgeb and Mizumori, 2002, Wirtshafter and Wilson, 2019), its dynamic aspects have remained elusive. The present study replicates the previous findings of theta-rhythmic neuronal activity in the lateral septum and reports a temporal alternation of spatial representations in this region, thus filling an important knowledge gap and significantly extending the understanding of the processing of spatial information in the brain. The lateral septum thus propagates the representations of alternative spatial behaviors to its efferent regions. The results can instruct further research of neural mechanisms supporting learning during goal-oriented navigation and decision-making in the behaviourally crucial circuits entailing the lateral septum.

Strengths

To this end, cutting-edge approaches for high-density monitoring of neuronal activity in freely behaving rodents and neural decoding were applied. Strengths of this work include comparisons of different anatomically and probably functionally distinct compartments of the lateral septum, innervated by different hippocampal domains and projecting to different parts of the hypothalamus; large neuronal datasets including many sessions with simultaneously recorded neurons; consequently, the rhythmic aspects of the spatial code could be directly revealed from the analysis of multiple spike trains, which were also used for decoding of spatial trajectories; and comparisons of the spatial coding between the two differently reinforced tasks.

Weaknesses

Possible in principle, with the present data across sessions, longitudinal analysis of the spatial coding during learning the task was not performed. Without using perturbation techniques, the present approach could not identify the aspects of the spatial code actually influencing the generation of behaviors by downstream regions.

Reviewer #3 (Public Review)

Summary

Bzymek and Kloosterman carried out a complex experiment to determine the temporal spike dynamics of cells in the dorsal and intermediate lateral septum during the performance of a Y-maze spatial task. In this descriptive study, the authors aim to determine if inputting spatial and temporal dynamics of hippocampal cells carry over to the lateral septum, thereby presenting the possibility that this information could then be conveyed to other interconnected subcortical circuits. The authors are successful in these aims, demonstrating that the phenomenon of theta cycle skipping is present in cells of the lateral septum. This finding is a significant contribution to the field as it indicates the phenomenon is present in neocortex, hippocampus, and the subcortical hub of the lateral septal circuit. In effect, this discovery closes the circuit loop on theta cycle skipping between the interconnected regions of the entorhinal cortex, hippocampus, and lateral septum. Moreover, the authors make 2 additional findings: 1) There are differences in the degree of theta modulation and theta cycle skipping as a function of depth, between the dorsal and intermediate lateral septum; and 2) The significant proportion of lateral septum cells that exhibit theta cycle skipping, predominantly do so during 'non-local' spatial processing.

Strengths

The major strength of the study lies in its design, with 2 behavioral tasks within the Y-maze and a battery of established analyses drawn from prior studies that have established spatial and temporal firing patterns of entorhinal and hippocampal cells during these tasks. Primary among these analyses, is the ability to decode the animal's position relative to locations of increased spatial cognitive demand, such as the choice point before the goal arms. The presence of theta cycle skipping cells in the lateral septum is robust and has significant implications for the ability to dissect the generation and transfer of spatial routes to goals within and between the neocortex and subcortical neural circuits.

Weaknesses

There are no major discernable weaknesses in the study, yet the scope and mechanism of the theta cycle phenomenon remain to be placed in the context of other phenomena indicative of spatial processing independent of the animal's current position. An example of this would be the ensemble-level 'scan ahead' activity of hippocampal place cells (Gupta et al., 2012; Johnson & Redish, 2007). Given the extensive analytical demands of the study, it is understandable that the authors chose to limit the analyses to the spatial and burst firing dynamics of the septal cells rather than the phasic firing of septal action potentials relative to local theta oscillations or CA1 theta oscillations. Yet, one would ideally be able to link, rather than parse the phenomena of temporal dynamics. For example, Tingley et al recently showed that there was significant phase coding of action potentials in lateral septum cells relative to spatial location (Tingley & Buzsaki, 2018). This begs the question as to whether the non-uniform distribution of septal cell activity within the Y-maze may have a phasic firing component, as well as a theta cycle skipping component. If so, these phenomena could represent another means of information transfer within the spatial circuit during cognitive demands. Alternatively, these phenomena could be part of the same process, ultimately representing the coherent input of information from one region to another. Future experiments will therefore have to sort out whether theta cycle skipping, is a feature of either rate or phase coding, or perhaps both, depending on circuit and cognitive demands.

The authors have achieved their aims of describing the temporal dynamics of the lateral septum, at both the dorsal extreme and the intermediate region. All conclusions are warranted.

Reviewer #1 (Recommendations For The Authors)

The text states: "We found that 39.7% of cells in the LSD and 32.4% of cells in LSI had significantly higher CSI values than expected by chance on at least one of the trajectories." The text in the supplemental figure indicates a p-value of 0.05 was used to determine significance. However, four trajectory categories are being examined so a Bonferroni correction should be used (significance at p<0.0125).

Indeed, a p-value correction for multiple tests should be performed when determining theta cycle skipping behavior for each of the four trajectories. We thank the reviewer for pointing out this oversight. We have implemented a Holm-Sidak p-value correction for the number of tested trajectories per cell (excluding trajectories with insufficient spikes). As a consequence, the number of cells with significant cycle-skipping activity decreased, but overall the results have not changed.

Figure 4 is very confusing as raster plots are displayed for multiple animals but it is unclear which animal the LFP refers to? The bottom of the plot is also referenced twice in the figure caption.

We apologize for the confusion. We have removed this figure in the revised manuscript, as it was not necessary to make the point about the spatial distribution of theta cycle skipping. Instead, we show examples of spatially-resolved cycle skipping in Figure 4 (formerly Figure 5 - supplementary figures 1 and 2) and we have added a plot with the spatially-resolved cycle skipping index for all analyzed cells in Figure 5A.

Figure 6 has, I think, an incorrect caption or figure. Only A and B are marked in the figure but A-G are mentioned in the caption but do not appear to correspond to anything in the figure.

Indeed, the caption was outdated. This has now been corrected.

Figure 8 is also confusing for several reasons: how is the probability scale on the right related to multiple semi-separate (top and middle) figures? In the top and bottom figures, it is not clear what the right and left sides refer to. It is also unclear why a probability of 0.25 is used for position (seems potentially low). The caption also mentions Figure A but there are no lettered "sub" figures in Figure 8.

The color bar on the right applies to both the top plot (directional decoding) and the middle plot (positional decoding). However, the maximum probability that is represented by black differs between the top and middle plots. We acknowledge that a shared color bar may lead to confusion and we have given each of the plots a separate color bar.

As for the maximum probability of 0.25 for position: this was a typo in the legend. The correct maximum value is 0.5. In general, the posterior probability will be distributed over multiple (often neighboring) spatial bins, and the distribution of maximum probabilities will depend on the number of spatial bins, the level of spatial smoothing in the decoding algorithm, and the amount of decodable information in the data. It would be more appropriate to consider the integrated probability over a small section of the maze, rather than the peak probability that is assigned to a single 5 cm bin. Also, note that a posterior probability of 0.5 is many times higher than the probability associated with a uniform distribution, which is in our case.

The left and right sides of the plots represent two different journeys that the animal ran. On the left an outbound journey is shown, and on the right an inbound journey. We have improved the figure and the description in the legend to make this clearer.

The reviewer is correct that there are no panels in Figure 8 and we have corrected the legend.

Some minor concerns

The introduction states that "a few studies have reported place cell-like activity in the lateral septum (Tingley and Buzsaki, 2018; Wirtshafter and Wilson, 2020, 2019)." However, notably and controversially, the Tingley study is one of the few studies to find NO place cell activity in the lateral septum. This is sort of mentioned later but the citation in this location should be removed.

The reviewer is correct, Tingley and Buzsaki reported a spatial phase code but no spatial rate code. We have removed the citation.

Stronger position/direction coding in the dLS consistent with prior studies and they should be cited in text (not a novel finding).

Thank you for pointing out this omission. Indeed, a stronger spatial coding in the dorsal lateral septum has been reported before, for example by Van der Veldt et al. (2021). We now cite this paper when discussing these findings.

Why is the alternation task administered for 30m but the switching task for 45m?

The reason is that rats received a larger reward in the switching task (in the high-reward goal arm) and took longer to complete trials on average. To obtain a more-or-less similar number of trials per session in both tasks, we extended the duration of switching task sessions to 45 minutes. We have added this explanation to the text.

Regarding the percentage of spatially modulated cells in the discussion, it is also worth pointing out that bits/sec information is consistent with previous studies.

Thank you for the suggestion. We now point out that the spatial information in our data is consistent with previous studies.

Reviewer #2 (Recommendations For The Authors)

While the results of the study are robust and timely, further details of behavioural training, additional quantitative comparisons, and improvements in the data presentation would make the study more comprehensible and complete.

Major comments

(1) I could not fully comprehend the behavioural protocols. They require a clearer explanation of both the specific rationale of the two tasks as well as a more detailed presentation of the protocols. Specifically:

(1.1) In the alternation task, were the arms baited in a random succession? How many trials were applied per session? Fig 1D: how could animals reach high choice accuracy if the baiting was random?

We used a continuous version of the alternation task, in which the animals were rewarded for left→home→right and right→home→left visit sequences. In addition, animals were always rewarded on inbound journeys. There was no random baiting of goal arms. Perhaps the confusion stems from our use of the word “trial” to refer to a completed lap (i.e., a pair of outbound/inbound journeys). On average, animals performed 54 of such trials per 30-minute session in the alternation task. We have expanded the description of the behavioral tasks in the Results and further clarified these points in the Methods section.

(1.2) Were they rewarded for correct inbound trials? If there was no reward, why were they considered correct?

Yes, rats received a reward at the home platform for correct inbound trials. We have now explicitly stated this in the text.

(1.3) In the switch alternation protocol, for how many trials was one arm kept more rewarding than the other, and how many trials followed after the rewarding value switch?

A switch was triggered when rats (of their own volition) visited the high-reward goal arm eight times in a row. Following a switch, the animals could complete as many trials as necessary until they visited the new high- reward goal arm in eight consecutive trials, which triggered another switch. As can be seen in Figure 1D, at the population level, animals needed ~13 trials to fully commit to the high-reward goal arm following a switch. We have further clarified the switching task protocol in the Results and Methods sections.

(1.4) What does the phrase "the opposite arm (as 8 consecutive visits)" exactly mean? Sounds like 8 consecutive visits signalled that the arm was rewarded (as if were not predefined in the protocol).

The task is self-paced and the animals initially visit both goal arms, before developing a bias for the high- reward goal arm. A switch of reward size was triggered as soon as the animal visited the high-reward goal arm for eight consecutive trials. We have rewritten the description of the switching task protocol, including this sentence, which hopefully clarifies the procedure.

(1.5) P. 15, 1st paragraph, Theta cycle skipping and alternation of spatial representations is more prominent in the alternation task. Why in the switching task, did rats visit the left and right arms approximately equally often if one was more rewarding than the other? How many switches were applied per recording session, and how many trials were there in total?

Both the left and right goal arms were sampled more or less equally by the animals because both goal arms at various times were associated with a large reward following switches in reward values during sessions. The number of switches per session varied from 1 to 3. Sampling of both goal arms was also evident at the beginning of each session and following each reward value switch, before animals switched their behavior to the (new) highly rewarded goal arm. In Table 1, we have now listed the number of trials and the number of reward-value switches for all sessions.

(1.6) Is the goal arm in figures the rewarded/highly rewarded arm only or are non-baited arms also considered here?

Both left and right arms are considered goal arms and were included in the analyses, irrespective of the reward that was received (or not received).

(2) The spatial navigation-centred behavioural study design and the interpretation of results highlight the importance of the dorsal hippocampal input to the LS. Yet, the recorded LSI cells are innervated by intermediate and ventral aspects of the hippocampus, and LS receives inputs from the amygdala and the prefrontal cortex, which together may together bring about - crucial for the adaptive behaviours regulated by the LS - reward, and reward-prediction-related aspects in the firing of LS cells during spatial navigation. Does success or failure to acquire reward in a trial modify spatial coding and cycle skipping of LSD vs. LSI cells in ensuing inbound and outbound trials?

This is an excellent question and given the length of the current manuscript, we think that exploration of this question is best left for a future extension of our study.

A related question: in Figure 10, it is interesting that cycle skipping is prominent in the goal arm for outbound switching trials and inbound trials of both tasks. Could it be analytically explained by task contingencies and behaviour (e.g. correct/incorrect trial, learning dynamics, running speed, or acceleration)?

Our observation of cycle skipping at the single-cell level in the goal arms is somewhat surprising and, we agree with the reviewer, potentially interesting. However, it was not accompanied by alternation of representations at the population level. Given the current focus and length of the manuscript, we think further investigation of cycle skipping in the goal arm is better left for future analyses.

(3) Regarding possible cellular and circuit mechanisms of cycle skipping and their relation to the alternating representations in the LS. Recent history of spiking influences the discharge probability; e.g. complex spike bursts in the hippocampus are associated with a post-burst delay of spiking. In LS, cycle skipping was characteristic for LS cells with high firing rates and was not uniformly present in all trajectories and arms. The authors propose that cycle skipping can be more pronounced in epochs of reduced firing, yet the opposite seems also possible - this phenomenon can be due to an intermittently increased drive onto some LS cells. Was there a systematic relationship between cycle skipping in a given cell and the concurrent firing rate or a recent discharge with short interspike intervals?

In our discussion, we tried to explain the presence of theta cycle skipping in the goal arms at the single-cell level without corresponding alternation dynamics at the population level. We mentioned the possibility of a decrease in excitatory drive. As the reviewer suggests, an increase in excitatory drive combined with post- burst suppression or delay of spiking is an alternative explanation. We analyzed the spatial tuning of cells with theta cycle skipping and found that, on average, these cells have a higher firing rate in the goal arm than the stem of the maze in both outbound and inbound run directions (Figure 5 – figure supplement 1). In contrast, cells that do not display theta cycle skipping do not show increased firing in the goal arm. These results are more consistent with the reviewer’s suggested mechanism and we have updated the discussion accordingly.

(4) Were the differences between the theta modulation (cycle skipping) of local vs. non-local representations (P.14, line 10-12, "In contrast...", Figure 9A) and between alternation vs. switching tasks (Figure 10 C,D) significantly different?

We have added quantification and statistical comparisons for the auto- and cross-correlations of the local/non-local representations. The results indeed show significantly stronger theta cycle skipping of the non-local representations as compared to the local representations (Figure 10 - figure supplement 1A), a stronger alternation of non-local representations in the outbound direction (Figure 10 - figure supplement 1B), and significant differences between the two tasks (Figure 11E,F).

(5) Regarding the possibility of prospective coding in LS, is the accurate coding of run direction not consistent with prospective coding? Can the direction be decoded from the neural activity in the start arm? Are the cycling representations of the upcoming arms near the choice point equally likely or preferential for the then- selected arm?

The coding of run direction (outbound or inbound) is distinct from the prospective/retrospective coding of the goal arm. As implemented, the directional decoding model does not differentiate between the two goal arms and accurate decoding of direction with this model can not inform us whether or not there is prospective (or retrospective) coding. To address the reviewer’s comments, we performed two additional analyses. First, we analyzed the directional (outbound/inbound) decoding performance as a function of location in the maze (Figure 6 - figure supplement 3E). The results show that directional decoding performance is high in both stem and goal arms. Second, we analyzed how well we can predict the trajectory type (i.e., to/from the left or right goal arm) as a function of location in the maze, and separately for outbound and inbound trajectories (Figure 6 - figure supplement 3C,D). The results show that on outbound journeys, decoding the future goal arm is close to chance when the animals are running along the stem. The decoding performance goes up around the choice point and reaches the highest level when animals are in the goal arm.

(6) Figure 10 seems to show the same or similar data as Figures 5 (A,B) and 9 (C,D).

Figure 10 (figure 11 in revised manuscript) re-analyzes the same data as presented in Figures 5 and 9, but separates the experimental sessions according to the behavioral task. We now explicitly state this.

Minor comments

(1) If cycle skipping in the periodicity of non-local representations was more prominent in alternation than in the switching task, one might expect them to be also prominent in early trials of the switching task, when the preference of a more rewarding arm is not yet established. Was this the case?

The reviewer makes an interesting suggestion. Indeed, if theta cycle skipping and the alternation of non-local representations reflect that there are multiple paths that the animal is considering, one may predict that the theta skipping dynamics are similar between the two tasks in early trials (as the reviewer suggests). Similarly, one may predict that in the switching task, the alternation of non-local representations is weaker immediately before a reward contingency switch (when the animal has developed a bias towards the goal arm with a large reward) as compared to after the switch.

We have now quantified the theta cycle dynamics of spatial representations in the early trials in each session of both tasks (Figure 11 - figure supplement 2) and in the trials before and after each switch in the switching task (Figure 11 - figure supplement 3).

The results of the early trial analysis indicate stronger alternation of non-local representations in the alternation task than in the switching task (consistent with the whole session analysis), which is contrary to the prediction.

The pre-/post-switch analysis did not reveal a significant difference between the trials before and after a reward contingency switch. If anything, there was a trend towards stronger theta cycle skipping/alternation in the trials before a switch, which would be opposite to the prediction.

These results do not appear to support the idea that the alternation of non-local representations reflects the number of relevant paths available to the animal. We have updated the text to incorporate these new data and discuss the implications.

(2) Summary: sounds like the encoding of spatial information and its readout in the efferent regions are equally well established.

Thank you for pointing this out.

(3) Summary: "motivation and reward processing centers such as the ventral tegmental area." How about also mentioning here the hypothalamus, which is a more prominent output of the lateral septum than the VTA?

We have now also mentioned the hypothalamus.

(4) "lateral septum may contribute to the hippocampal theta" - readers not familiar with details of the medial vs. lateral septum research may misinterpret the modest role of LS in theta compared to MS.

We have added “in addition to the strong theta drive originating from the medial septum” to make clear that the lateral septum has a modest role in hippocampal theta generation.

(5) "(Tingley and Buzsáki, 2018) found a lack of spatial rate coding in the lateral septum and instead reported a place coding by specific phases of the hippocampal theta rhythm (Rizzi-Wise and Wang, 2021) " needs rephrasing.

Thank you, we have rephrased the sentence.

(6) Figure 4 is a bit hard to generalize. The authors may additionally consider a sorted raster presentation of the dataset in this main figure.

We have removed this figure in the revised manuscript, as it was not necessary to make the point about the location of theta cycle skipping. Instead, we show examples of spatially-resolved cycle skipping in Figure 4 (formerly Figure 5 - supplementary figures 1 and 2), and, following the reviewer’s suggestion, we have added a plot with the spatially-resolved cycle skipping index for all analyzed cells (Figure 5A).

(7) It would help if legends of Figure 5 (and related supplementary figures) state in which of the two tasks the data was acquired, as it is done for Figure 10.

Thank you for the suggestion. The legends of Figure 4A,B (formerly Figure 5 – supplemental figures 1 and 2) and Figure 5 now include in which behavioral task the data was acquired.

(8) Page 10, "Spatial coding...", 1st Citing the initial report by Leugeb and Mizumori would be appropriate here too.

The reviewer is correct. We have added the citation.

(9) The legend in Figure 6 (panels A-G) does not match the figure (only panels A,B). What is shown in Fig. 6B, the legend does not seem to fully match.

Indeed, the legend was outdated. This has now been corrected.

(10) 7 suppl., if extended to enable comparisons, could be a main figure. Presently, Figure 7C does not account for the confounding effect of population size and is therefore difficult to interpret without complex comparisons with the Supplementary Figure which is revealing per se.

We thank the reviewer for their suggestion. We have changed Figure 7 such that it only shows the analysis of decoding performed with all LSD and LSI cells. Figure 7 – supplemental figure 1 has been transformed into main Figure 8, with the addition of a panel to show a statistical comparison between decoding performance in LSD and LSI with a fixed number of cells.

(11) 14, line 10 there is no Figure 8A

This has been corrected.

(12) 15 paragraph 1, is the discussed here model the one from Kay et al?

From Kay et al. (2020) and also Wang et al. (2020). We have added the citations.

(13) Figure 5 - Figure Supplement 1 presents a nice analysis that, in my view, can merit a main figure. I could not find the description of the colour code in CSI panels, does grey/red refer to non/significant points?

Indeed, grey/red refers to non-significant points and significant points respectively. We have clarified the color code in the figure legend. Following the reviewer’s suggestion, we have made Figure 5 Supplement 1 and 2 a main figure (Figure 4).

(14) Figure 5 -Figure Supplement 2. Half of the cells (255 and 549) seems not to be representative of the typically high SCI in the goal arm in left and right inbound trials combined (Figure 5 A). Were the changes in CSI in the right and left inbound trials similar enough to be combined in Fig 5A? Otherwise, considering left and right inbound runs separately and trying to explain where the differences come from would seem to make sense.

Figure 5 – figure supplement 2 is now part of the new main Figure 4. Originally, the examples were from a single session and the same cells as shown in the old Figure 4. However, since the old Figure 4 has been removed, we have selected examples from different sessions and both left/right trajectories that are more representative of the overall distribution. We have further added a plot with the spatially-resolved cycle skipping for all analyzed cells in Figure 5A.

(15) In the second paragraph of the Discussion, dorso-ventral topography of hippocampal projections to the LS (Risold and Swanson, Science, 90s) could be more explicitly stated here.

Thank you for the suggestion. We have now explicitly mentioned the dorsal-ventral topography of hippocampal-lateral septum projections and cite Risold & Swanson (1997).

(16) Discussion point: why do the differences in spatial information of cells in the ventral/intermediate vs. dorsal hippocampus not translate into similarly prominent differences in LSI vs. LSD?

In our data, we do observe clear differences in spatial coding between LSD and LSI. Specifically, cell activity in the LSD is more directional, has higher goal arm selectivity, and higher spatial information (we have now added statistical comparisons to Figure 6 – figure supplement 1). As a result, spatial decoding performance is much better for LSD cell populations than LSI cell populations (see updated Figure 8, with statistical comparison of decoding performance). Spatial coding in the LS is not as strong as in the hippocampus, likely because of the convergence of hippocampal inputs, which may give the impression of a less prominent difference between the two subregions.

(17) Discussion, last paragraph: citation of the few original anatomical and neurophysiological studies would be fitting here, in addition to the recent review article.

Thank you for the suggestion. We have added selected citations of the original literature.

(18) Methods, what was the reference electrode?

We used an external reference electrode that was soldered to a skull screw, which was positioned above the cerebellum. We have added this to the Methods section.

(19) Methods, Theta cycle skipping: bandwidth = gaussian kerner parameter?

The bandwidth is indeed a parameter of the Gaussian smoothing kernel and is equal to the standard deviation.

Reviewer #3 (Recommendations For The Authors)

Below I offer a short list of minor comments and suggestions that may benefit the manuscript.

(A) I was not able to access the Open Science Framework Repository. Can this be rectified?

Thank you for checking the OSF repository. The data and analysis code are now publicly available.

(B) In the discussion the authors should attempt to flesh out whether they can place theta cycle skipping into context with left/right sweeps or scan ahead phenomena, as shown in the Redish lab.

Thank you for the excellent suggestion. We have now added a discussion of the possible link between theta cycle skipping and the previously reported scan-ahead theta sweeps.

(C) What is the mechanism of cycle skipping? This could be relevant to intrinsic vs network oscillator models. Reference should also be made to the Deshmukh model of interference between theta and delta (Deshmukh, Yoganarasimha, Voicu, & Knierim, 2010).

We had discussed a potential mechanism in the discussion (2nd to last paragraph in the revised manuscript), which now includes a citation of a recent computational study (Chu et al., 2023). We have now also added a reference to the interference model in Deshmukh et al, 2010.

(D) Little background was given for the motivation and expectation for potential differences between the comparison of the dorsal and intermediate lateral septum. I don't believe that this is the same as the dorsal/ventral axis of the hippocampus, but if there's a physiological justification, the authors need to make it.

We have added a paragraph to the introduction to explain the anatomical and physiological differences across the lateral septum subregions that provide our rationale for comparing dorsal and intermediate lateral septum (we excluded the ventral lateral septum because the number of cells recorded in this region was too low).

(E) It would help to label "outbound" and "inbound" on several of the figures. All axes need to be labeled, with appropriate units indicated.

We have carefully checked the figures and added inbound/outbound labels and axes labels where appropriate.

(F) In Figure 6, the legend doesn't match the figure.

Indeed, the legend was outdated. This has now been corrected.

(G) The firing rate was non-uniform across the Y-maze. Does this mean that the cells tended to fire more in specific positions of the maze? If so, how would this affect the result? Would increased theta cycle skipping at the choice point translate to a lower firing rate at the choice point? Perhaps less overdispersion of the firing rate (Fenton et al., 2010)?

Individual cells indeed show a non-uniform firing rate across the maze. To address the reviewer’s comment and test if theta cycle skipping cells were active preferentially near the choice point or other locations, we computed the mean-corrected spatial tuning curves for cell-trajectory pairs with and without significant theta cycle skipping. This additional analysis indicates that, on average, the population of theta cycle skipping cells showed a higher firing rate in the goal arms than in the stem of the maze as compared to non-skipping cells for outbound and inbound directions (shown in Figure 5 - figure supplement 1).

(H) As mentioned above, it could be helpful to look at phase preference. Was there an increased phase preference at the choice point? Would half-cycle firing correlate with an increased or decreased phase preference? Based on prior work, one would expect increased phase preference, at least in CA1, at the choice point (Schomburg et al., 2014). In contrast, other work might predict phasic preference according to spatial location (Tingley & Buzsaki, 2018). Including phase analyses is a suggestion, of course. The manuscript is already sufficiently novel and informative. Yet, the authors should state why phase was not analyzed and that these questions remain for follow-up analyses. If the authors did analyze this and found negative results, it should be included in this manuscript.

We thank the reviewer for their suggestion. We have not yet analyzed the theta phase preference of lateral septum cells or other relations to the theta phase. We agree that this would be a valuable extension of our work, but prefer to leave it for future analyses.

(I) One of the most important aspects of the manuscript, is that there is now evidence of theta cycle skipping in the circuit loop between the EC, CA1, and LS. This now creates a foundation for circuit-based studies that could dissect the origin of route planning. Perhaps the authors should state this? In the same line of thinking, how would one determine whether theta cycle skipping is necessary for route planning as opposed to a byproduct of route planning? While this question is extremely complex, other studies have shown that spatial navigation and memory are still possible during the optogenetic manipulation of septal oscillations (Mouchati, Kloc, Holmes, White, & Barry, 2020; Quirk et al., 2021). However, pharmacological perturbation or lesioning of septal activity can have a more profound effect on spatial navigation (Bolding, Ferbinteanu, Fox, & Muller, 2019; Winson, 1978). As a descriptive study, I think it would be helpful to remind the readers of these basic concepts.

We thank the reviewer for their comment and for pointing out possible future directions for linking theta cycle skipping to route planning. Experimental manipulations to directly test this link would be very challenging, but worthwhile to pursue. We now mention how circuit-based studies may help to test if theta cycle skipping in the broader subcortical-cortical network is necessary for route planning. Given that the discussion is already quite long, we decided to omit a more detailed discussion of the possible role of the medial septum (which is the focus of the papers cited by the reviewer).

Very minor points

(A) In the introduction, "one study" begins the sentence but there is a second reference.

Thank you, we have rephrased the sentence.

(B) Also in the introduction, it could be helpful to have an operational definition of theta cycle skipping (i.e., 'enhanced rhythmicity at half theta frequency').

We followed the reviewer’s suggestion.

(C) The others should be more explicit in the introduction about their main question. Theta cycle skipping exists in CA1, and then import some of the explanations mentioned in the discussion to the introduction (i.e., attractors states of multiple routes). The main question is then whether this phenomenon, and others from CA1, translate to the output in LS.

We have edited the introduction to more clearly state the main question of our study, following the suggestion from the reviewer.

(D) There are a few instances of extra closing parentheses.

We checked the text but did not find instances of erroneous extra closing parentheses. There are instances of nested parentheses, which may have given the impression that closing parentheses were duplicated.

(E) The first paragraph of the Discussion lacks sufficient references.

We have now added references to the first paragraph of the discussion.

(F) At the end of the 2nd paragraph in the Discussion, the comparison is missing. More than what? It's not until the next reference that one can assume that the authors are referring to a dorsal/ventral axis. However, the physiological motivation for this comparison is lacking. Why would one expect a dorsal/intermediate continuum for theta modulation as there is along the dorsal/ventral axis of the hippocampus?

Thank you for spotting this omission. We have rewritten the paragraph to more clearly make the parallel between dorsal-ventral gradients in the lateral septum and hippocampus and how this relates to the topographical connections between the two structures.

eLife assessment

In this study, the authors present convincing evidence to demonstrate theta cycle skipping by individual neurons of the lateral septum, which they then relate to population coding of future trajectories encapsulated by theta cycles. This valuable finding furthers our understanding of how the septum conveys navigational information downstream.

Reviewer #1 (Public Review):

Summary:

The authors provide very compelling evidence that the lateral septum (LS) engages in theta cycle skipping.

Strengths:

The data and analysis is highly compelling regarding the existence of cycle skipping.

Comments on the revised version:

All previous recommendations were addressed in this revision.

Reviewer #2 (Public Review):

Summary

Recent evidence indicates that cells of the navigation system representing different directions and whole spatial routes fire in a rhythmic alternation during 5-10 Hz (theta) network oscillation (Brandon et al., 2013, Kay et al., 2020). This phenomenon of theta cycle skipping was also reported in broader circuitry connecting the navigation system with the cognitive control regions (Jankowski et al., 2014, Tang et al., 2021). Yet nothing was known about the translation of these temporally separate representations to midbrain regions involved in reward processing as well as the hypothalamic regions, which integrate metabolic, visceral, and sensory signals with the descending signals from the forebrain to ensure adaptive control of innate behaviors (Carus-Cadavieco et al., 2017). The present work aimed to investigate theta cycle skipping and alternating representations of trajectories in the lateral septum, neurons of which receive inputs from large number of CA1 and nearly all CA3 pyramidal cells (Risold and Swanson, 1995). While spatial firing has been reported in the lateral septum before (Leutgeb and Mizumori, 2002, Wirtshafter and Wilson, 2019), its dynamic aspects have remained elusive. The present study replicates the previous findings of theta-rhythmic neuronal activity in the lateral septum and reports a temporal alternation of spatial representations in this region, thus filling an important knowledge gap and significantly extending the understanding of the processing of spatial information in the brain. The lateral septum thus propagates the representations of alternative spatial behaviors to its efferent regions. The results can instruct further research of neural mechanisms supporting learning during goal-oriented navigation and decision-making in the behaviourally crucial circuits entailing the lateral septum.

Strengths

To this end, cutting-edge approaches for high-density monitoring of neuronal activity in freely behaving rodents and neural decoding were applied. Strengths of this work include comparisons of different anatomically and probably functionally distinct compartments of the lateral septum, innervated by different hippocampal domains and projecting to different parts of the hypothalamus; large neuronal datasets including many sessions with simultaneously recorded neurons; consequently, the rhythmic aspects of the spatial code could be directly revealed from the analysis of multiple spike trains, which were also used for decoding of spatial trajectories; and comparisons of the spatial coding between the two differently reinforced tasks.

Weaknesses

Without using perturbation techniques, the present approach could not identify the aspects of the spatial code actually influencing the generation of behaviors by downstream regions.

Résumé de la vidéo [00:00:00][^1^][1] - [00:24:00][^2^][2]:

La vidéo présente une conférence de Yves Gingras, sociologue et historien des sciences, sur l'évaluation dans les sciences, les arts et les organisations. Il aborde l'histoire de l'évaluation scientifique, l'importance de l'expertise et la montée de la bibliométrie dans l'évaluation de la recherche.

Points forts: + [00:00:22][^3^][3] Introduction d'Yves Gingras * Présentation de son parcours et de ses spécialités * Discussion sur la biométrie et l'histoire des sciences + [00:01:51][^4^][4] L'évaluation de la recherche * Importance croissante de l'évaluation dans les dernières décennies * Lien entre l'évaluation et la valeur du travail scientifique + [00:03:03][^5^][5] Histoire de l'évaluation scientifique * Évaluation comme pratique ancienne remontant au XVIIe siècle * Expertise et évaluation intrinsèques à l'institutionnalisation de la science + [00:09:01][^6^][6] Évaluation par les pairs et exemples historiques * Exemples de Newton et Einstein pour illustrer l'évaluation par les pairs * Impact de l'évaluation sur la qualité et la validation des travaux scientifiques + [00:16:04][^7^][7] Évaluation institutionnelle et financement de la recherche * Évolution des mécanismes d'évaluation et leur importance pour le financement * Rôle des organismes de recherche et des comités de sélection + [00:19:00][^8^][8] Évaluation des structures et programmes universitaires * Évaluation des départements, centres de recherche et programmes de doctorat * Reconnaissance de la priorité et citations comme mécanisme institutionnel Résumé de la vidéo [00:24:01][^1^][1] - [00:46:48][^2^][2]:

Cette partie de la vidéo discute de l'évaluation dans les sciences, les arts et les organisations, en se concentrant sur l'importance des citations et des indicateurs bibliométriques dans la recherche scientifique. Elle explique comment ces outils ont évolué pour devenir des instruments de politique scientifique et d'évaluation institutionnelle.

Points forts: + [00:24:01][^3^][3] L'origine des citations bibliométriques * L'utilisation des citations pour trouver des publications pertinentes * La création d'une base de données par Eugene Garfield + [00:26:04][^4^][4] L'impact des indicateurs bibliométriques * L'influence sur les politiques scientifiques et les décisions de financement * L'introduction du facteur d'impact et son utilisation dans la gestion des bibliothèques + [00:30:03][^5^][5] La massification de la science * La croissance du nombre de chercheurs et la nécessité de nouveaux mécanismes d'évaluation * L'utilisation des données quantitatives pour contester les pratiques subjectives + [00:34:05][^6^][6] La transition de l'évaluation par les pairs aux experts * La quantification des évaluations permet un contrôle plus large de l'expertise * Les chiffres remplacent le jugement des pairs dans les comités d'évaluation + [00:36:13][^7^][7] Les conséquences des indicateurs quantitatifs * Modification des comportements des chercheurs en réponse aux métriques * L'effet sur le choix des objets de recherche et les pratiques institutionnelles + [00:41:53][^8^][8] Le passage de l'évaluation au classement * La prolifération des classements universitaires et leurs effets pervers * La critique des méthodologies de classement et l'appel à des indicateurs valides Résumé de la vidéo [00:46:51][^1^][1] - [01:06:53][^2^][2]:

Cette vidéo présente une conférence de Pierre-Michel Menger sur l'évaluation dans les sciences, les arts et les organisations. Il discute des indicateurs utilisés pour évaluer les performances des universités et des institutions, soulignant les défis et les pièges potentiels de ces méthodes d'évaluation.

Points forts: + [00:46:51][^3^][3] Les indicateurs d'évaluation * Définition et utilisation des indicateurs * Exemple de la température comme indicateur physique * Critique des classements universitaires fluctuants + [00:51:01][^4^][4] Les classements universitaires * L'absurdité de certains indicateurs * L'importance de la relation entre le concept mesuré et l'indicateur * Les effets pervers des classements sur les politiques universitaires + [00:53:00][^5^][5] Évaluer les évaluations * Nécessité de critiquer et d'améliorer les méthodes d'évaluation * Application des principes du management public à l'évaluation * Contrôle des dérives potentielles de l'évaluation + [00:55:01][^6^][6] L'usage des données chiffrées * Limites et manipulation des données chiffrées * Les données comme aides au jugement, pas comme décisions * La politique indépendante des chiffres et l'importance de la critique + [00:59:00][^7^][7] Les conséquences des évaluations * Impact sur les politiques et les missions académiques * Dérives liées à l'accent mis sur la recherche au détriment de l'enseignement * Nécessité d'une prise de conscience et d'une évaluation équilibrée

Résumé de la vidéo [00:00:00][^1^][1] - [00:22:42][^2^][2]:

Cette vidéo présente une conférence de Pierre Merle, sociologue spécialisé dans l'évaluation scolaire, qui explore la notation des élèves et ses implications. Il discute des différents types d'évaluation, de l'expérience subjective des élèves face à la notation, et des résultats des recherches en docimologie qui remettent en question la fiabilité des notes.

Points forts: + [00:00:25][^3^][3] Introduction de Pierre Merle * Sociologue spécialisé dans les questions scolaires * Professeur de sociologie à l'école supérieure du professorat et de l'éducation * Auteur d'une biographie sur son père, Robert Merle + [00:01:50][^4^][4] La notation des élèves * Distinction entre évaluation et notation * Types d'évaluation: sommative, certificative, diagnostique, formative * L'institution scolaire privilégie les évaluations basées sur les notes + [00:06:17][^5^][5] Expérience subjective des élèves * Enquêtes statistiques révèlent différents profils d'élèves face à la notation * Impact de la notation sur la motivation et le rapport à la discipline * Les élèves sereins, insatisfaits et critiques envers la notation + [00:15:00][^6^][6] Conception institutionnelle et recherches en docimologie * La croyance en la note juste et ses hypothèses sous-jacentes * Études de multi-correction démontrent la variabilité des notes * L'impact de l'aléa dans la notation et la pertinence des examens Résumé de la vidéo [00:22:45][^1^][1] - [00:45:34][^2^][2]:

La vidéo aborde l'évaluation dans l'éducation, en se concentrant sur les biais sociaux et les stéréotypes qui peuvent influencer les notes des élèves. Elle explore les expériences psychologiques et sociologiques qui démontrent comment l'origine sociale, le genre, et d'autres facteurs externes peuvent affecter les évaluations des enseignants.

Points forts: + [00:22:45][^3^][3] Biais dans l'évaluation * Expérience montrant l'influence du statut social sur les notes * Les copies attribuées à des fils de journalistes reçoivent de meilleures notes * Impact de l'origine sociale et du genre sur l'évaluation + [00:31:00][^4^][4] La notation comme arrangement * La notation peut être vue comme un arrangement entre élèves et enseignants * Les pratiques de notation varient selon les dynamiques de classe et les politiques d'établissement * Les enseignants adaptent leur notation en fonction des contraintes et des négociations + [00:36:00][^5^][5] Négociations et relations personnelles * Les élèves négocient parfois leurs notes avec les enseignants * Les relations personnelles et la séduction peuvent influencer les notes * Exemples de négociations et d'interactions en classe affectant l'évaluation + [00:42:50][^6^][6] Principes pour une notation équitable * Importance de préserver l'anonymat social et scolaire des élèves * Mutualisation des épreuves et barèmes pour une évaluation plus juste * Adoption d'une notation encourageante pour favoriser les progrès des élèves Résumé de la vidéo [00:45:36][^1^][1] - [01:03:10][^2^][2]:

Cette partie de la vidéo aborde l'évaluation dans le contexte éducatif, en se concentrant sur les systèmes de notation en Suède, en Allemagne et en France. L'orateur discute des effets des différentes échelles de notation sur les élèves et propose des méthodes d'évaluation plus encourageantes et équitables.

Points forts: + [00:45:36][^3^][3] Systèmes de notation en Suède et en Allemagne * En Suède, une seule note pour indiquer l'échec * En Allemagne, une échelle traditionnelle avec peu de notes pour l'échec + [00:46:15][^4^][4] Impact des notes basses sur les élèves * Les notes basses peuvent décourager les élèves * Importance d'une notation qui évite de décourager et qui contractualise les pratiques d'évaluation + [00:47:01][^5^][5] L'évaluation par contrat de confiance * Éviter les évaluations pièges * Préparer les élèves en difficulté avec des exercices accessibles + [00:48:11][^6^][6] Fondement de l'évaluation sur des compétences standardisées * S'inspirer des pratiques de notation étrangères * Varier les modalités d'évaluation pour mieux noter différentes compétences + [00:49:16][^7^][7] Évaluation des établissements en Finlande * Les établissements sont évalués et aidés sans afficher de palmarès * Insérer l'évaluation des élèves dans une pédagogie explicite + [00:50:06][^8^][8] Effets des comparaisons sociales sur les élèves * Comparaisons ascendantes et descendantes affectent l'auto-évaluation et la motivation * Importance de construire une image positive pour améliorer l'attention et la performance

Résumé de la vidéo [00:00:00][^1^][1] - [00:24:45][^2^][2]:

Cette vidéo présente une conférence de P.-M. Menger sur l'évaluation dans les sciences, les arts et les organisations, mettant l'accent sur l'expérimentation sociale et son rôle dans la compréhension des politiques publiques et des comportements sociaux.

Points forts: + [00:00:22][^3^][3] Introduction à l'évaluation * Importance de l'évaluation des politiques publiques * Rôle de l'expérimentation sociale dans la compréhension des comportements * Citation de Robert Solow sur les politiques sociales + [00:03:27][^4^][4] Définition de l'expérimentation sociale * Mise en œuvre à petite échelle d'un dispositif * Comparaison des bénéficiaires et non bénéficiaires * Pertinence de l'expérimentation sociale pour l'évaluation + [00:04:37][^5^][5] Progression de l'expérimentation sociale * Utilisation croissante aux États-Unis depuis les années 1970 * Impact dans l'économie du développement et en France * Importance politique et scientifique de l'expérimentation sociale + [00:07:49][^6^][6] Posture épistémologique de l'expérimentation sociale * Obsession pour la mesure de relation causale * Importance de l'empirisme et de la réalité concrète * Influence sur la pratique de la recherche empirique + [00:10:33][^7^][7] Problème de la validité externe * Applicabilité des résultats empiriques à d'autres circonstances * Distinction entre expérimentation sociale et de laboratoire * Enjeux spécifiques aux sciences sociales + [00:16:18][^8^][8] Impact causal et théorie contrefactuelle * Définition de l'impact causal par rapport à une situation de référence * Importance de la manipulation expérimentale pour établir la causalité * Difficultés liées à la sélection des individus dans les études empiriques Résumé de la vidéo [00:24:47][^1^][1] - [00:45:25][^2^][2]:

La vidéo présente une conférence sur l'évaluation dans les expérimentations sociales, en se concentrant sur une étude de cas concernant la taille des classes et leur impact sur la performance scolaire. L'orateur, P.-M. Menger, explique l'importance de l'expérimentation contrôlée et randomisée pour établir la causalité et la validité des résultats. Il discute également des défis liés à la généralisation des résultats à d'autres contextes et populations.

Points forts: + [00:24:47][^3^][3] L'importance de l'expérimentation * Observation des individus dans différentes situations * Création de groupes de test et de contrôle comparables + [00:27:15][^4^][4] Étude de cas sur la taille des classes * Coût et faisabilité des politiques publiques * Expérience randomisée dans le Tennessee + [00:35:58][^5^][5] Résultats et implications politiques * Impact significatif sur la performance scolaire * Effets hétérogènes selon les populations + [00:39:01][^6^][6] Comparaison avec les opérateurs privés de placement * Réfutation de la théorie de l'efficacité du secteur privé * Importance de l'évaluation empirique pour les politiques publiques Résumé de la vidéo [00:45:28][^1^][1] - [01:04:26][^2^][2] : La vidéo aborde l'évaluation des interventions dans les domaines des sciences, des arts et des organisations, en mettant l'accent sur les problèmes de causalité et le changement d'échelle. L'orateur, Pierre-Michel Menger, discute des effets de l'accompagnement des chômeurs et de la théorie économique sous-jacente, en utilisant des expériences pour illustrer les concepts de validité interne et externe dans la recherche sociale.

Points forts : + [00:45:28][^3^][3] Problèmes d'interprétation causale * Difficultés liées à l'interprétation des effets des interventions * Importance de considérer le changement d'échelle * Impact des mécanismes du marché et des interactions sociales + [00:46:35][^4^][4] Effets d'éviction sur le marché du travail * Théories opposées sur les effets agrégés de l'accompagnement des chômeurs * Explication des théories du marché du travail fixe et de la courbe de Beveridge * Importance de l'expérimentation pour comprendre les effets réels + [00:48:55][^5^][5] Protocole expérimental pour traiter la généralisation * Utilisation d'un protocole complexe pour évaluer les effets d'éviction * Suivi de dix mille demandeurs d'emploi sur 20 mois * Comparaison des taux d'emploi entre différents groupes + [00:52:17][^6^][6] Résultats de l'expérimentation et implications politiques * Observation d'effets d'éviction importants * Considération de l'accompagnement des chômeurs comme une politique redistributive * Réflexion sur la validité externe et l'impact des contextes économiques

eLife assessment

This important work identifies a previously uncharacterized capacity for songbird to recover vocal targets even without sensory experience. The evidence supporting this claim is convincing, with technically difficult and innovative experiments exploring goal-directed vocal plasticity in deafened birds. This work has broad relevance to the fields of vocal and motor learning.

Reviewer #1 (Public Review):

Summary:

Zai et al test if songbirds can recover the capacity to sing auditory targets without singing experience or sensory feedback. Past work showed that after the pitch of targeted song syllables are driven outside of birds' preferred target range with external reinforcement, birds revert to baseline (i.e. restore their song to their target). Here the authors tested the extent to which this restoration occurs in muted or deafened birds. If these birds can restore, this would suggest an internal model that allows for sensory-to-motor mapping. If they cannot, this would suggest that learning relies entirely on feedback dependent mechanisms, e.g. reinforcement learning (RL). The authors find that deafened birds exhibit moderate but significant restoration, consistent with the existence of a previously under-appreciated internal model in songbirds.

Strengths:

The experimental approach of studying vocal plasticity in deafened or muted birds is innovative, technically difficult and perfectly suited for the question of feedback-independent learning. The finding in Figure 4 that deafened birds exhibit subtle but significant plasticity toward restoration of their pre-deafening target is surprising and important for the songbird and vocal learning fields, in general.

In this revision, the authors suitably addressed confusion about some statistical methods related to Fig. 4, where the main finding of vocal plasticity in deafened birds was presented.

There remain minor issues in the presentation early in the results section and in Fig. 4 that should be straightforward to clarify in the revision.

Reviewer #3 (Public Review):

Summary:

Zai et al. test whether birds can modify their vocal behavior in a manner consistent with planning. They point out that while some animals are known to be capable of volitional control of vocalizations, it has been unclear if animals are capable of planning vocalizations-that is, modifying vocalizations towards a desired target without the need to learn this modification by practising and comparing sensory feedback of practised behavior to the behavioral target. They study zebra finches that have been trained to shift the pitch of song syllables away from their baseline values. It is known that once this training ends, zebra finches have a drive to modify pitch so that it is restored back to its baseline value. They take advantage of this drive to ask whether birds can implement this targeted pitch modification in a manner that looks like planning, by comparing the time course and magnitude of pitch modification in separate groups of birds who have undergone different manipulations of sensory and motor capabilities. A key finding is that birds who are deafened immediately before the onset of this pitch restoration paradigm, but after they have been shifted away from baseline, are able to shift pitch partially back towards their baseline target. In other words, this targeted pitch shift occurs even when birds don't have access to auditory feedback, which argues that this shift is not due to reinforcement-learning-guided practice, but is instead planned based on the difference between an internal representation of the target (baseline pitch) and current behavior (pitch the bird was singing immediately before deafening).

The authors present additional behavioral studies arguing that this pitch shift requires auditory experience of song in its state after it has been shifted away from baseline (birds deafened early on, before the initial pitch shift away from baseline, do not exhibit any shift back towards baseline), and that a full shift back to baseline requires auditory feedback. The authors synthesize these results to argue that different mechanisms operate for small shifts (planning, which does not need auditory feedback) and large shifts (through a mechanism that requires auditory feedback).

The authors also make a distinction between two kinds of planning: covert-not requiring any motor practice and overt-requiring motor practice but without access to auditory experience from which target mismatch could be computed. They argue that birds plan overtly, based on these deafening experiments as well as an analogous experiment involving temporary muting, which suggests that indeed motor practice is required for pitch shifts.

Strengths:

The primary finding (that partially restorative pitch shift occurs even after deafening) rests on strong behavioral evidence. It is less clear to what extent this shift requires practice, since their analysis of pitch after deafening takes the average over within the first two hours of singing. If this shift is already evident in the first few renditions then this would be evidence for covert planning. Technical hurdles, such as limited sample sizes and unstable song after surgical deafening, make this difficult to test. (Similarly, the authors could test whether the first few renditions after recovery from muting already exhibit a shift back towards baseline.)

This work will be a valuable addition to others studying birdsong learning and its neural mechanisms. It documents features of birdsong plasticity that are unexpected in standard models of birdsong learning based on reinforcement and are consistent with an additional, perhaps more cognitive, mechanism involving planning. As the authors point out, perhaps this framework offers a reinterpretation of the neural mechanisms underlying a prior finding of covert pitch learning in songbirds (Charlesworth et al., 2012).

A strength of this work is the variety and detail in its behavioral studies, combined with sensory and motor manipulations, which on their own form a rich set of observations that are useful behavioral constraints on future studies.

Weaknesses:

The argument that pitch modification in deafened birds requires some experience hearing their song in its shifted state prior to deafening (Fig. 4) is solid but has an important caveat. Their argument rests on comparing two experimental conditions: one with and one without auditory experience of shifted pitch. However, these conditions also differ in the pitch training paradigm: the "with experience" condition was performed using white noise training, while the "without experience" condition used "lights off" training (Fig. 4A). It is possible that the differences in ability for these two groups to restore pitch to baseline reflects the training paradigm, not whether subjects had auditory experience of the pitch shift. Ideally, a control study would use one of the training paradigms for both conditions, which would be "lights off" or electrical stimulation (McGregor et al. 2022), since WN training cannot be performed in deafened birds. In the Discussion, in response to this point, the authors point out that birds are known to recover their pitch shift if those shifts are driven using electrical stimulation as reinforcement (McGregor et al. 2022); however, it is arguably still relevant to know whether a similar recovery occurs for the "lights off" paradigm used here.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Recommendations For The Authors):

In this revision the authors address some of the key concerns, including clarification of the balanced nature of the RL driven pitch changes and conducting analyses to control for the possible effects of singing quantity on their results. The paper is much improved but still has some sources of confusion, especially around Fig. 4, that should be fixed. The authors also start the paper with a statistically underpowered minor claim that seems unnecessary in the context of the major finding. I recommend the authors may want to restructure their results section to focus on the major points backed by sufficient n and stats.

Major issues.

(1) The results section begins very weak - a negative result based on n=2 birds and then a technical mistake of tube clogging re-spun as an opportunity to peak at intermittent song in the otherwise muted birds. The logic may be sound but these issues detract from the main experiment, result, analysis, and interpretation. I recommend re-writing this section to home in on, from the outset, the well-powered results. How much is really gained from the n=2 birds that were muted before ANY experience? These negative results may not provide enough data to make a claim. Nor is this claim necessary to motivate what was done in the next 6 birds. I recommend dropping the claim?

We thank the reviewer for the recommendation. We moved the information to the Methods.

(2) Fig. 4 is very important yet remains very confusing, as detailed below.

Fig. 4a. Can the authors clarify if the cohort of WNd birds that give rise to the positive result in Fig 4 ever experienced the mismatch in the absence of ongoing DAF reinforcement pre-deafening? Fig4a does nor the next clearly specifies this. This is important because we know that there are day timescale delays in LMAN-dependent bias away from DAF and consolidation into the HVC-RA pathway (Andalman and Fee, 2009). Thus, if birds experienced mismatch pre-deafening in the absence of DAF, then an earnly learning phase in Area X could be set in place. Then deafening occurs, but these weight changes in X could result in LMAN bias that expresses only days later -independent of auditory feedback. Such a process would not require an internal model as the authors are arguing for here. It would simply arise from delays in implementing reinforcement-driven feedback. If the birds in Fig 4 always had DAF on before deafening, then this is not an issue. But if the birds had hours of singing with DAF off before deafening, and therefore had the opportunity to associate DA error signals with the targeted time in the song (e.g. pauses on the far-from-target renditions (Duffy et al, 2022), then the return-to-baseline would be expected to be set in place independent of auditory feedback. Please clarify exactly if the pitch-contingent DAF was on or off in the WNd cohort in the hours before deafening. In Fig. 3b it looks like the answer is yes but I cannot find this clearly stated in the text.

We did not provide DAF-free singing experience to the birds in Fig. 4 before deafening. Thus, according to the reviewer, the concern does not apply.

Note that we disagree with the reviewer’s premise that there is ‘day timescale delay in LMAN-dependent bias away from DAF and consolidation into the HVC-RA pathway’. More recent data reveals immediate consolidation of the anterior forebrain bias without a night-time effect (Kollmorgen, Hahnloser, Mante 2020; Tachibana, Lee, Kai, Kojima 2022). Thus, the single bird in (Andalman and Fee 2009) seems to be somewhat of an outlier.

Hearing birds can experience the mismatch regardless of whether they experience DAF-free singing (provided their song was sufficiently shifted): even the renditions followed by white noise can be assessed with regards to their pitch mismatch, so that DAF imposes no limitation on mismatch assessment.

We disagree with their claim that no internal model would be needed in case consolidation was delayed in Area X. If indeed, Area X stores the needed change and it takes time to implement this change in LMAN, then we would interpret the change in Area X as the plan that birds would be able to implement without auditory feedback. Because pitch can either revert (after DAF stops) or shift further away (when DAF is still present), there is no rigid delay that is involved in recovering the target, but a flexible decision making of implementing the plan, which in our view amounts to using a model.

Fig 4b. Early and Late colored dots in legend are both red; late should be yellow? Perhaps use colors that are more distinct - this may be an issue of my screen but the two colors are difficult to discern.

We used colors yellow to red to distinguish different birds and not early and late. We modified the markers to improve visual clarity: Early is indicated with round markers and late with crosses.

Fig 4b. R, E, and L phases are only plotted for 4c; not in 4b. But the figure legend says that R, E and L are on both panels.

In Fig. 4b E and L are marked with markers because they are different for different birds. In Fig. 4c the phases are the same for all birds and thus we labeled them on top. We additionally marked R in Fig. 4b as in Fig. 4c.

Fig 4e. Did the color code switch? In the rest of Fig 4, DLO is red and WND is blue. Then in 4e it swaps. Is this a typo in the caption? Or are the colors switch? Please fix this it's very confusing.

Thank you for pointing out the typo in the caption. We corrected it.

The y axes in Fig 4d-e are both in std of pitch change - yet they have different ylim which make it visually difficult to compare by eye. Is there a reason for this? Can the authors make the ylim the same for fig 4d-e?.

We added dashed lines to clarify the difference in ylim.

Fig 4d-3 is really the main positive finding of the paper. Can the others show an example bird that showcases this positive result, plotted as in Fig 3b? This will help the audience clearly visualize the raw data that go into the d' analyses and get a more intuitive sense of the magnitude of the positive result.

We added example birds to figure 4, one for WNd and one for dLO.

Please define 'late' in Fig.4 legend.

Done

Minor

Define NRP In the text with an example. Is an NRP of 100 where the birds was before the withdrawal of reinforcement?

We added the sentence to the results:

"We quantified recovery in terms of 𝑵𝑹𝑷 to discount for differences in the amount of initial pitch shift where 𝑵𝑹𝑷 = 𝟎% corresponds to complete recovery and 𝑵𝑹𝑷 = 𝟏𝟎𝟎% corresponds pitch values before withdrawal of reinforcement (R) and thus no recovery."

Reviewer #3 (Recommendations For The Authors):

The use of "hierarchically lower" to refer to the flexible process is confusing to me, and possibly to many readers. Some people think of flexible, top-down processes as being _higher_ in a hierarchy. Regardless, it doesn't seem important, in this paper, to label the processes in a hierarchy, so perhaps avoid using that terminology.

We reformulated the paragraph using ‘nested processes’ instead of hierarchical processes.

In the statement "a seeming analogous task to re-pitching of zebra finch song, in humans, is to modify developmentally learned speech patterns", a few suggestions: it is not clear whether "re-pitching" refers to planning or feedback-dependent learning (I didn't see it introduced anywhere else). And if this means planning, then it is not clear why this would be analogous to "humans modifying developmentally learned speech patterns". As you mentioned, humans are more flexible at planning, so it seems re-pitching would _not_ be analogous (or is this referring to the less flexible modification of accents?).

We changed the sentence to:

"Thus, a seeming analogous task to feedback-dependent learning of zebra finch song, in humans, is to modify developmentally learned speech patterns."

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

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Reply to the reviewers

Response to reviewer comments

R: We really appreciate the reviewer positive comments and consideration, and we believe that the review process has significantly strengthened our manuscript.

We have responded to all the reviewer comments, as follows:

Response (R)

FROM REVIEWER #1

Major comments:

The manuscript is mostly well written (it could use a few minor grammatical corrections), the significance of the problem is well described, and the results are clearly presented with adequate controls. The movies, provided as supplementary material, are of the highest quality and are essential additions to the stills provided in the figures. The data convincingly support the key conclusions of the manuscript.

R: We sincerely appreciate the positive comments provided by the reviewer. In response, we have thoroughly revised the manuscript to address any grammatical issue.

Does the MO knockdown both S and L homeologs of X. laevis? Since the level of GAPDH in Figure 1H also looks reduced in Gai2 MO lane, it should be made clear that the apparent knockdown of Gai2 was normalized to GAPDH, rather than being the results of unequal loading of the gel. Yes, I recognize that Figure 1I says normalized, but this is not stated in the results or the methods. Also, was this experiment done with X. laevis or X. tropicalis? I could imagine that if done in X. laevis, the lack of complete knockdown might be due to only one homeolog being affected.

R: We appreciate the reviewer comment, and we described in Material and Methods section the region targeted by the morpholino, in both Xenopus species. We added the next paragraph in the Material and Methods section, see page 24, paragraph 2, lines: 4-11:

"MO against Xenopus Gαi2 was designed by GeneTools to target the 5' UTR site of X. tropicalis (X.t) and X. laevis (X.l) transcripts (Gαi2MO: 5'-CGACACAGCCCCAGATAGTGCGT-3'). Specifically, it hybridizes with the 5' UTR of X. t Gαi2 (NM_203919), 17 nucleotides upstream of the ATG start codon. For X. l Gαi2, the morpholino hybridizes with both isoforms described in Xenbase. It specifically targets the 5' UTR of the Gαi2.L isoform (XM_018258962), located 17 nucleotides upstream of the ATG start codon, and the 5' UTR of the Gαi2.S isoform (NM_001097056), situated 275 nucleotides upstream of the ATG."

With respect to Figure 1H and 1I, we have specified in the Fig. 1 legend that we normalized the data to GAPDH to quantifying the decrease in Gαi2 expression induced by the morpholino.

See page 40, Figure 1H-I, Legends section. Finally, the result showed in Fig. 1A-I was done in X.t., that was now stated at the legend from the figure. We added at the Supplementary material Fig.1S, the result done in X.l. experiment.

The knowledge of the efficacy of knockdown in each Xenopus species provided by the information requested in the previous point, would allow the reader to assess the level of knockdown in the remaining assays. To do this, the authors should tell us which assays were done in which species. I am not suggesting that each experiment needs to be done in each species, only that the information should be provided. If the MO is more effective in X. tropicalis - which assays used this species? If the knock down is partial, as shown in Figure 1H-I, which species this represents in the remaining assays would be useful knowledge.

R: We greatly appreciate the reviewer's valuable comments and suggestions, and as a response, we have incorporated a new supplementary figure (Figure S1). This figure includes a western blot and an in situ hybridization assay illustrating the efficiency of the knockdown in Xenopus laevis. The results presented in Figure S1 demonstrate that the knockdown efficiency is similar in both Xenopus species, allowing for a comparison between Figure 1A-I (X. tropicalis) and Figure 1S (X. laevis).

To complement this information, we have also improved the section of Material and Methods regarding the experiments in both Xenopus species (Xenopus tropicalis and Xenopus laevis). As detailed in the Materials and Methods section, we employed 20 ng of Gai2MO for Xenopus tropicalis embryos and 35 ng of Gai2MO for Xenopus laevis embryos to deplete cell migration. In both species, in vivo migration was analyzed, resulting in a substantial inhibition of cranial neural crest (NC) migration, ranging from 60% to 80%. Additionally, we conducted dispersion assays in both species. In X. laevis, in vitro migration was monitored for 10 hours, while in X. tropicalis, it was tracked for 4 hours, both yielding the same phenotype. We also studied cell morphology and microtubule dynamics in both Xenopus models. However, we used different tracer concentrations for each, with 200 pg for X. laevis and 100 pg for X. tropicalis, as specified in the Materials and Methods section. Our Rac1 and RhoA timelapse experiments were conducted in both species as well, employing pGBD-GFP and rGBD-mCherry probes, respectively, and different probe concentrations as outlined in the Materials and Methods section. These experiments revealed polarity impairment and consistent Rac1 behavior in both Xenopus species. The study of focal adhesion in vivo dynamics using the FAK-GFP tracer was carried out also in both species, resulting in the same phenotype. It is worth noting that the only experiment conducted exclusively in X. tropicalis was the focal adhesion disassembly assay with nocodazole.

Regarding the improvements of the Materials and Method section see page 24, paragraph 1.

We want to highlight that at the beginning of the Materials and Methods section, we incorporated a paragraph to clarify that "All experiments were conducted in both Xenopus species (X.t and X.l) using distinct concentrations of the morpholino (MO) and mRNA, as specified in each respective methodology description". This approach consistently yielded similar results. It is important to note that for the figures, we selected the most representative images.

We have also specified in each figure legend which Xenopus species is depicted.

Minor comments:

While prior studies are referenced appropriately, and the text and figures are mostly clear and accurately presented, the following are a few suggestions that would help the authors improve the presentation of their data and conclusions:

The cell biological experiments convincingly demonstrate that knockdown of Gai2 causes cells to move more slowly. It would be a nice addition to bring the explant experimental data back to the embryo by showing whether the slower moving NC cells in morphants eventually populate the BA. DO they cease to migrate or are they just slower getting to their destination? This could be done by performing snail2 ISH at a later stage (34-35?).

R: We appreciate the reviewer's insightful point, and in response, we conducted the in situ hybridization assay at stages 32-36 to address this question. The result has been included in Figure S1F-H, revealing a delayed migration of cranial neural crest cells. Consequently, we have updated the text in the results section, page 6, paragraph 1, line 18:

"In later developmental stages, such as stage 32, WISH revealed alterations in migration as well, albeit to a lesser extent compared to the early stages (22-23). This suggests a phenotype characterized by delayed migration (supplementary material Fig S1F-H)."

There are places in the manuscript where the authors use the terms "silencing" or "suppression" of Gai2, when they really mean reduced translation - their system is not a genetic knockout, as clearly demonstrated in Figure 1H-I. I suggest that more accurate wording be used.

R: We appreciate the reviewer's comment, and we agree that the Gαi2 morpholino impedes Gαi2 translation, leading to a reduction in Gαi2 protein expression. Consequently, we have revised the entire manuscript, replacing the terms "silencing" and "suppression" with "knockdown".

In Figures 1-5 there are scale of bars on the cell images, but these are not defined in any of the figure legends.

R: We value the reviewer's comment, and we have revised all the figure legends by including the scale information. Each image has been scaled to 10 µm with varying magnifications.

The abstract is the weakest section of the manuscript, and would have greater impact if it were more clearly written.

R: We appreciate the reviewer's comment on the abstract, and we have revised and edited it to enhance its quality.

Abstract:

"Cell migration is a complex and essential process in various biological contexts, from embryonic development to tissue repair and cancer metastasis. Central to this process are the actin and tubulin cytoskeletons, which control cell morphology, polarity, focal adhesion dynamics, and overall motility in response to diverse chemical and mechanical cues. Despite the well- established involvement of heterotrimeric G proteins in cell migration, the precise underlying mechanism remains elusive, particularly in the context of development. This study explores the involvement of Gαi2, a subunit of heterotrimeric G proteins, in cranial neural crest cell migration, a critical event in embryonic development. Our research uncovers the intricate mechanisms underlying Gαi2 influence, revealing a direct interaction with the microtubule-associated protein EB1, and through this with tubulin, suggesting a regulatory function in microtubule dynamics modulation. Here, we show that Gαi2 knockdown leads to microtubule stabilization, alterations in cell polarity and morphology with an increased Rac1-GTP concentration at the leading edge and cell-cell contacts, impaired cortical actin localization and focal adhesion disassembly. Interestingly, in Gai2 depleted cells RhoA-GTP was found to be reduced at cell-cell contacts and concentrated at the leading edge, providing evidence of Gαi2 significant role in polarity. Remarkably, treatment with nocodazole, a microtubule-depolymerizing agent, effectively reduces Rac1 activity, restoring cranial NC cell morphology, actin distribution, and overall migration. Collectively, our findings shed light on the intricate molecular mechanisms underlying cranial neural crest cell migration and highlight the pivotal role of Gαi2 in orchestrating microtubule dynamics through EB1 and EB3 interaction, modulating Rac1 activity during this crucial developmental process."

Reviewer #1 (Significance (Required)):

The molecular regulation of cell movement is a key feature of a number of developmental and homeostatic processes. While many of the proteins involved have been identified, how they interact to provide motility has not been elucidated in any great detail, particularly in embryo-derived cells (as opposed to cell lines). The results obtained from the presented experiments are novel, in-depth and provide a novel paradigm for how G proteins regulate microtubule dynamics which in turn regulate other components of the cytoskeleton required for cell movement. The results will be applicable to many migrating cell types, not just neural crest cells.

Because of the application of the data to many types of cells that migrate, the audience is expected to include a broad array of developmental biologists, basic cell biologists and those interested in clinically relevant aberrant cell migrations.

R: We really appreciate the reviewer positive comments and consideration

FROM REVIEWER #2

Reviewer: Major comments:

The authors aim to address two issues in this manuscript: a) the role of Gai2 in neural crest development; and b) the mechanism of Gai2 function. While they have done a good job demonstrating a role of Gai2 in NC migration both in vivo and in vitro as well as the effects of Gai2 knockdown on cytoskeleton dynamics, protein distribution of selected polarity and focal adhesion molecules, and Rac1 activation, the link between Gai2 and the downstream effectors is largely correlative. Because of this, the model suggesting the sequential events flowing from Gai2 to microtubule to Rac1 to focal adhesion/actin should be modified to allow room for direct and indirect regulation at potentially multiple entry points.

R: We appreciate the valuable comments provided by the reviewer. To further elucidate the mechanism underlying Gαi2 regulation of cranial neural crest cell migration, we have incorporated new data from interaction analysis conducted by PLA (proximity ligand assay). This analysis supports our proposed model, indicating Gαi2 interacts with EB proteins to form a complex with tubulin, thereby regulating microtubules dynamics and subsequently influencing Rac1 and RhoA activity, cell morphology (actin cytoskeleton) and cell-matrix adhesion, ultimately affecting migration. However, we cannot exclude that this regulation may also involve other intermediary proteins, such as GEFs, GAPs, GDIs, and others. Finally, as a result, we have revised our model and its description to provide a more detailed explanation of the potential mechanism in line with the reviewer suggestion. Specifically, we have edited the discussion/conclusion, model and the legend for Figure 6. Please refer to page 16 (paragraph 1, 2 and 3), 22 (paragraph 1), 23 (paragraph 1), 44 (Legend Fig. 6).

__Reviewer: __Specific major comments are as the following:

Strengths:

-Determination of a role of Gai2 in neural crest migration is novel.

-The effect of Gai2 knockdown on membrane protrusion morphology and microtubule stability and dynamics are demonstrated nicely.

-Quantification of experimental perimeters has been performed throughout the manuscript in all the figures, and statistical analysis is included in the figures.

R: We appreciate the reviewer positive comments

Weaknesses: -The heavy focus of the study on microtubule is due to the previous publication on the function of Gai2 in regulation of microtubule during asymmetrical cell division. However, the activity of Gai2 is likely cell type-specific, as it has not been shown to control microtubule during cytokinesis in general. It is equally likely that Gai2 primarily regulates Rac1 or actin regulators to influence both microtubule and actin dynamics. The tone of the discussion should therefore be softened.

R: We greatly appreciate and agree with the comment from the reviewer, highlighting the possibility that Gαi2 primarily regulates Rac1 or actin regulators to influence both microtubule and actin dynamics. In this regard, we have revised our manuscript to include a discussion of this point. We added the next paragraph in the Discussion/Conclusion section, page 22-23.

"It is well established that the activity from the Rho family of small GTPases is controlling cytoskeletal organization during migration (Ridley et al., 2015). Contrariwise, it has been described in many cell types, that microtubules dynamic polymerization plays a crucial role in establishing the structural foundation for cell polarization, consequently influencing the direction of cell motility (Watanabe et al., 2005). Our results appear to align with this latter view. While it is reasonable to postulate the possibility that Gαi2 regulates Rac1 activity, subsequently influencing actin and microtubule dynamics, our findings in the context of cranial NC cells, lend support to an alternative sequence of events. Initially, Gαi2 knockdown leads to a decrease in microtubule dynamics, which in turn increase Rac-GTP towards the leading edge. This shift is accompanied by reduced levels of cortical actin and impaired focal adhesion disassembly, culminating in compromised cell migration. Notably, nocodazole, a microtubule-depolymerizing agent, not only diminishes Rac-GTP localization at the leading edge but also rescues cell morphology, restores normal cortical actin localization, and promotes focal adhesion disassembly, thereby facilitating cell migration. If Rac1 activity were indeed upstream of microtubules, it would be expected that nocodazole would not reduce Rac-GTP levels at the cell leading edge. These results suggest that the regulation of Rac1 activity may follow, rather than precede, alterations in microtubule dynamics, in the context of NC cells. Furthermore, in support of our model, our protein interaction analysis demonstrates Gαi2 interacting with microtubule components such as EB proteins and tubulin. As we already mention above, earlier studies have reported that microtubule dynamics promote Rac1 signaling at the leading edge and by releasing RhoGEFs promote RhoA signaling as well (Best et al., 1996; Garcin and Straube, 2019; Moore et al., 2013; Waterman-Storer et al., 1999). In addition, it is well-documented that RhoGEFs interact with microtubules, including bPix, a GEF for Rac1 and Cdc42, which, in turn, promotes tubulin acetylation (Kwon et al., 2020). Interestingly, in ovarian cancer cells, Gαi2 has been shown to activate Rac1 through an interaction with bPix, thereby jointly regulating migration in response to LPA (Ward et al., 2015). Taken together, these findings further support our proposed model (refer to Fig. 6)."

The effect of rescue of NC migration with Rac1 inhibitor is marginal and the result is hard to interpret considering the inhibitor also blocks control NC migration. Either lower doses of Rac1 inhibitor can be used or the experiment can be removed from the manuscript, as Rac1 is required for membrane protrusions and the inhibitor doses can be hard to titrate.

R: We appreciate and agree with the reviewer's comments. To address this concern and enhance clarity, we have incorporated the following paragraph into the manuscript within the Discussion section. Additionally, we have included information on the range of NSC23766 concentrations used for this analysis in the Materials and Methods section. Page 25, Explants and microdissection.

In the results section see page 11 and 12, paragraph 2.

"It is worth noting that we conducted Rac inhibitor NSC23766 trials at concentrations ranging from 20 nM to 50 nM for X. laevis and between 10 nM to 30 nM for X. tropicalis. In both cases, higher concentrations of the Rac inhibitor proved to be lethal (data not shown), underscoring the essential role of Rac1 in both cell migration and cell survival. Remarkably, our results show partial restoration in cranial NC cells dispersion following a 5-minute treatment with a low concentration of the Rac1 inhibitor (20 nM of NSC23766 X. laevis and 10 nM for X. tropicalis) (Fig. 3L-P, supplementary material movie S5). This suggests that these concentrations are sufficient to demonstrate that the increase in Rac1-GTP resulting from Gαi2 morpholino knockdown impairs cell migration."

The partial rescue can be attributed to the crucial role of microtubule dynamics in cell migration, which acts upstream of Rac activity. Additionally, Rac is pivotal for the modulation of cell polarity at the leading edge of migration. It is worth emphasizing that Rac1 levels are critical for cell migration, as demonstrated by other researchers. Lower concentrations of Rac1-GTP have been shown to hinder cell migration in cells deficient in Rac1, leading to a significant reduction in wound closure and random cell migration (Steffen et al., 2013).

"Therefore, we believe that the lower concentration of NSC23766 used in our assay was adequate to reduce the abnormal Rac1-GTP activity in the morphant NC cells. However, it is important to note that for normal NC cell, this level of reduction in Rac1-GTP activity is critical and sufficient to impair normal migration".

See page 11 and 12, paragraph 2.

Steffen A, Ladwein M, Dimchev GA, Hein A, Schwenkmezger L, Arens S, Ladwein KI, Margit Holleboom J, Schur F, Victor Small J, Schwarz J, Gerhard R, Faix J, Stradal TE, Brakebusch C, Rottner K. Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation. J Cell Sci. 2013 Oct 15;126(Pt 20):4572-88. doi: 10.1242/jcs.118232. Epub 2013 Jul 31. PMID: 23902686; PMCID: PMC3817791.

Since the defects seem to result partially from the inability of the NC cells to retract and move away, it may help to either include some data on Rho activation patterns in knockdown cells or simply add some discussion about the issue.

R: We acknowledge and sincerely appreciate the reviewer's valuable comments on this pivotal aspect, which significantly enhances our capacity to elucidate the impact of Gαi2 knockdown on cell polarity. To address this crucial point, we have introduced an experiment that examines RhoA-GTP localization under Gαi2 knockdown conditions, and we have incorporated a supplementary figure S3 into our manuscript. This newly added figure clearly demonstrates that, under Gαi2 knockdown conditions, and in contrast to control cells, RhoA-GTP localization is substantially disrupted at cell-cell contacts and now detected at the leading edge of the cell, providing compelling evidence of cell polarity defects (refer to Figure S3A-C). In response to these results, we have included a description of these findings in the Results section (please see page 10) and a dedicated paragraph in the Discussion section (please see page 19, paragraph 2, last line, page 19-21).

Results section 1 (page 10, paragraph 1 line 6-12): "To achieve this, we explored whether Gαi2 regulates the subcellular distribution of active Rac1 and RhoA in cranial NC explants under Gαi2 loss-of-function conditions, considering their pivotal roles in cranial NC migration and contact inhibition of locomotion (CIL) (Carmona-Fontaine et al., 2011; Moore et al., 2013; Leal et al., 2018). Hence, we employed mRNA encoding the small GTPase-based probe, enabling specific visualization of the GTP-bound states of these proteins."

Results section 2 (page 10, paragraph 1 line 14-27): "Consistent with earlier observations by Carmona-Fontaine et al. (2011), in control cranial NC cells, active Rac1 displayed prominent localization at the leading edge of migrating cells, whereas its presence was reduced at cell-cell contacts, coincident with an increase in RhoA-GTP levels (white arrows in Fig. 3A, supplementary material Figure S3A,C). On the contrary, in comparison to the control cells, Gαi2 morphants exhibit a pronounced accumulation of active Rac1 protein in the protrusions at cell-cell contacts, where active RhoA localization is conventionally expected (white arrow in Fig. 4B, supplementary material Figure S3A,C and movie S4). In contrast to control cells, a notable shift in the localization of active RhoA protein was observed, with its predominant accumulation now detected at the leading edge of the cell, instead of the typical localization towards the trailing edge or cell-cell contacts (__supplementary material Figure S3B,C). __These findings suggest a dysregulation of contractile forces that align with the observed distribution of active RhoA, cortical actin disruption, and diminished retraction in cell treated with Gαi2MO."

*Discussion section: (page 19 last line, page 20, paragraph 1, line 1-20) *

"Other studies have reported that microtubule assembly promotes Rac1 signaling at the leading edge, while microtubule depolymerization stimulates RhoA signaling through guanine nucleotide exchange factors associated with microtubule-binding proteins controlling cell contractility, via Rho-ROCK and focal adhesion formation (Krendel et al., 2002; Ren et al., 1999; Best et al., 1996; Garcin and Straube, 2019; Waterman-Storer et al., 1999; Bershadsky et al., 1996; Moore et al., 2013). This mechanism would contribute to establishing the antero-posterior polarity of cells, crucial for maintaining migration directionality, underscoring the significance of regulating microtubule dynamics in directed cell migration. These findings closely align with the results obtained in this investigation, demonstrating that Gαi2 loss of function reduces microtubule catastrophes and promotes tubulin stabilization, resulting in increased localization of active Rac1 at the leading edge and cell-cell contacts, while decreasing active RhoA at the cell-cell contact but increasing it at the leading edge. This possibly reinforces focal adhesion, which is consistent with the presence of large and highly stable focal adhesions under Gαi2 knockdown conditions. This finding also suggests a dysregulation of contractile forces in comparison to control cells, a result that aligns with the observed distribution of active RhoA, cortical actin distribution and diminished retraction in cells treated with Gαi2MO. This strikingly contrasts with the normal cranial NC migration phenotype, where Rac1 is suppressed while active RhoA is increased at cell-cell contacts during CIL, leading to a shift in polarity towards the cell-free edge to sustain directed migration (Theveneau et al., 2010; Shoval and Kalcheim, 2012; Leal et al., 2018)."

To consider focal adhesion dynamics, live imaging should be used in the analysis. The fixed samples are different from each other, and natural variations of focal adhesion may exist among the samples. This can obscure data collection and quantification.

R: We agree with the reviewer that focal adhesion (FA) dynamics need to be analysed using live imaging. Indeed, Fig 5E-H shows an extensive analysis of FA using live imaging of neural crest expressing FAK-GFP. As complement to this live imaging analysis, and in order to analyse the effect on the endogenous levels of FA proteins, we performed immunostaining against FA. Both experiments using live imaging or fixed cells produce similar results, and they are consistent with our model on the role of Gαi2 on FA dynamics.

Reviewer: minor comments

Fig. 2, the centrosomes in control cells are not always obvious. The microtubules simply seem to be more networked and more fluid in control cells. This should be clarified with either marking the centrosomes in the figure or modifying the wording in the manuscript.

R: We appreciate and concur with the reviewer's comment on this matter. As pointed out by the reviewer, the precise localization of the centrosome is not consistently clear in all cells. In response to this observation, we have revised the manuscript to emphasize this aspect solely as "microtubule morphology". Please refer to the Results section description Figure 2.

In Fig. 3, a better negative control for co-IP should be using anti-V5 antibody to IP against tubulin/EB1/EB3 in the absence of Gai2-V5.

R: We appreciate the reviewer's comment, and we agree with the suggested control. Accordingly, we have included this control in Supplementary material Figure S4A. Additionally, we conducted all Co-IPP in triplicate, and these data have been incorporated into Supplementary material Figure S4B. Furthermore, as mentioned earlier, we have reorganized some of the sections of the results to improve the logical flow of the manuscript's description. As a result, the Figure presenting the interaction analysis by Co-IPP now corresponds to Figure 5.

The data for cell polarity proteins Par3 and PKC-zeta seem to be out of place. It is unclear whether mis-localization of these proteins has anything to do with NC migration defects induced by Gai2 knockdown. The conclusion does not seem to be affected if the data are taken out of the manuscript.

R: We appreciate the reviewer's concern, and we would like to highlight two points in this regard. Firstly, we have included these results as additional data to support the impact of Gai2 knockdown on cell polarity, given that these two proteins are commonly used as polarity markers. Secondly, we have discussed this aspect extensively in the Discussion section of the manuscript. (See page 20, paragraph 1, lines 21-31).

In that section, we delve into the relationship between aPKC, Par3, and Gαi2 in controlling cell polarity during asymmetric cell division, as described in Hao et al., 2010. Par3 is known to play a role in regulating microtubule dynamics and Rac1 activation through its interaction with Rac-GEF Tiam1 (Chen et al., 2005). Additionally, it has been shown to promote microtubule catastrophes and inhibit Rac1/Trio signaling, regulating Contact Inhibition of Locomotion (CIL) as demonstrated in Moore et al., 2013. Thus, we believe that the data we present support the relationship between Par3 and aPKC localization changes and the neural crest migration defects induced by Gαi2 knockdown, probably by controlling microtubule dynamics. However, we have moved these results as part of the supplementary Figure S3D-G.

In Suppl. Fig. 1, protrusion versus retraction should be defined more clearly. The retraction shown in this figure seems to be just membrane between protrusions instead of actively retracting membrane.

R: We appreciate the reviewer's comments, and here we aim to provide a clearer description of our approach to this analysis. For the measurement of protrusion extension/retraction, we conducted two distinct experiments. The first, as described in Figure 1, involved measuring membrane extension and retraction in live cell using membrane-GFP by utilizing the image subtraction tool in ImageJ, which highlights changes in the membrane in red. Secondly, we employed ADAPT software to quantify cell perimeter based on fluorescence intensity in live cell using lifeactin-GFP, distinguishing membrane extension in green and retraction in red (as has been shown similarly in Barry et al., 2015). In both approaches, we observed a substantial increase in membrane protrusion (both in area and extension) and protrusion stability in Gαi2 morphants. Hence, we have revised the Materials and Methods section of the manuscript and included this clarification.

See Materials and Methods section, Cell dispersion and morphology, page 28.

In addition we inform hat this images now are included in Supplementary material Fig S2G,H.

Barry DJ, Durkin CH, Abella JV, Way M. Open source software for quantification of cell migration, protrusions and fluorescence intensities. J Cell Biol. 2015. Doi: 10.1083/jcb.201501081

Discussion can be improved by better incorporating all the components to make a cohesive story on how Gai2 works to regulate migration in the context of the neural crest cells.

R: We appreciate the reviewer's comment and agree. To enhance the manuscript, we have included a new paragraph at the end of the Discussion/Conclusion section specifically addressing this point. For more details, please refer to page 23.

"Therefore, in the context of collective cranial NC cells migration, our findings reveal the pivotal role played by Gαi2 in orchestrating the intricate interplay of microtubule dynamics and cellular polarity. When Gαi2 levels are diminished, we observe significant impediments in the ability of cells to efficiently navigate through their environment, resulting in a range of distinct effects. First and foremost, Gαi2 deficiency leads to the diminished ability of cells to adjust and reorient new protrusions effectively. Primary protrusions exhibit higher stability and heightened levels of active Rac1/RhoA when compared to control conditions in the leading edge. In addition, we observe a notable increase in protrusion area, a decrease in retraction velocity, and an enhanced level of cell-matrix adhesion in Gαi2 knockdown cells. These findings underscore the pivotal role that Gαi2 plays in the modulation of various cellular dynamics essential for collective cranial NC cells migration. Notably, the application of nocodazole, a microtubule-depolymerizing agent, and NSC73266, a Rac1 inhibitor, to Gαi2 knockdown cells leads to the rescue of the observed effects, thus facilitating migration. This observed response closely mirrors the outcomes associated with Par3, a known regulator of microtubule catastrophe during contact inhibition of locomotion (CIL) in NC cells (Moore et al., 2013). This parallel implies that there exists a delicate equilibrium between microtubule dynamics and Rac1-GTP levels, crucial for the establishment of proper cell polarity during collective migration. Our findings collectively position Gαi2 as a central master regulator within the intricate framework of collective cranial NC migration. This master regulator role is pivotal in orchestrating the dynamics of polarity, morphology, and cell-matrix adhesion by modulating microtubule dynamics through interactions with EB1 and EB3 proteins, described here for the first time, possible in a protein complex involving other intermediary proteins such as other microtubules accessory proteins like CLIP170, actin intermediaries, like mDia1-2, and signaling proteins such as GDIs, GAPs and GEFs, thus fostering crosstalk between the actin and tubulin cytoskeletons. This orchestration ultimately ensures the effective collective migration of cranial NC cells (Fig. 6)."

Review____er #2 (Significance (Required)):

The authors demonstrate a role of Gai2 in regulation of neural crest migration in Xenopus by modulating microtubule dynamics. In addition, they show an effect of Gai2 knockdown on Rac1 spatial activation and focal adhesion stability. These are novel discoveries of the study. Some limitations exist in linking Gai2 with downstream effectors that directly or indirectly impact on cytoskeleton and Rac1 small GTPase.

R: We really appreciate the reviewer positive comments and consideration. We believe that the review process has significantly strengthened our manuscript in this regard.

FROM REVIEWER #3

__ ____Reviewer: mayor comments:__

The authors focus exclusively on the analysis of the subcellular levels of Rac1, which is probably related to the fact that they observe large extended protrusions with high Rac1 activity. However, as the authors note, a global fine-tuning of Rho GTPase activity is required for neural crest migration. One of the observed phenotypes of Gαi2-morphant neural crest cells is a decrease in cell dispersion, which may be caused by defects in contact inhibition of locomotion (CIL). This process involves a local activation of RhoA at cell-cell contact sites (Carmona-Fontaine et al., 2008). Furthermore, in fibroblast, RhoA/ROCK activity is required for the front-rear polarity switch during CIL (Kadir et al., 2011). Interestingly, similar to the Gαi2 loss of function phenotype, ROCK inhibition leads to microtubule stabilization, which can be rescued by nocodazole treatment, restoring microtubule dynamics and CIL. Therefore, it would also be interesting to know how RhoA activity is affected in Gαi2-morphant NC cells. At a minimum, this point should be be included in the discussion.

R: We acknowledge and sincerely appreciate the reviewer's valuable comments on this pivotal aspect, which significantly enhances our capacity to elucidate the impact of Gαi2 knockdown on cell polarity. To address this crucial point, we have introduced an experiment that examines RhoA-GTP localization under Gαi2 knockdown conditions, and we have incorporated a supplementary figure S3A-C into our manuscript. This newly added figure clearly demonstrates that, under Gαi2 knockdown conditions and in contrast to control cells, RhoA-GTP localization is substantially disrupted at cell-cell contacts and now detected at the leading edge of the cell, providing compelling evidence of cell polarity defects (refer to Figure S3). In response to these results, we have included a description of these findings in the Results section (please see page 10) and a dedicated paragraph in the Discussion section (please see page 19-20).

Results section 1 (page 10, paragraph 1 line 6-12): "To achieve this, we explored whether Gαi2 regulates the subcellular distribution of active Rac1 and RhoA in cranial NC explants under Gαi2 loss-of-function conditions, considering their pivotal roles in cranial NC migration and contact inhibition of locomotion (CIL) (Carmona-Fontaine et al., 2011; Moore et al., 2013; Leal et al., 2018). Hence, we employed mRNA encoding the small GTPase-based probe, enabling specific visualization of the GTP-bound states of these proteins."

Results section 2 (page 10, paragraph 1 line 14-27): "Consistent with earlier observations by Carmona-Fontaine et al. (2011), in control cranial NC cells, active Rac1 displayed prominent localization at the leading edge of migrating cells, whereas its presence was reduced at cell-cell contacts, coincident with an increase in RhoA-GTP levels (white arrows in Fig. 3A, supplementary material Figure S3A,C). On the contrary, in comparison to the control cells, Gαi2 morphants exhibit a pronounced accumulation of active Rac1 protein in the protrusions at cell-cell contacts, where active RhoA localization is conventionally expected (white arrow in Fig. 4B, supplementary material Figure S3A,C and movie S4). In contrast to control cells, a notable shift in the localization of active RhoA protein was observed, with its predominant accumulation now detected at the leading edge of the cell, instead of the typical localization towards the trailing edge or cell-cell contacts (__supplementary material Figure S3B,C). __These findings suggest a dysregulation of contractile forces that align with the observed distribution of active RhoA, cortical actin disruption, and diminished retraction in cell treated with Gαi2MO."

*Discussion section: (page 19, second paragraph, line 12 and page 20, paragraph 1, line 1-18) *

"Other studies have reported that microtubule assembly promotes Rac1 signaling at the leading edge, while microtubule depolymerization stimulates RhoA signaling through guanine nucleotide exchange factors associated with microtubule-binding proteins controlling cell contractility, via Rho-ROCK and focal adhesion formation (Krendel et al., 2002; Ren et al., 1999; Best et al., 1996; Garcin and Straube, 2019; Waterman-Storer et al., 1999; Bershadsky et al., 1996; Moore et al., 2013). This mechanism would contribute to establishing the antero-posterior polarity of cells, crucial for maintaining migration directionality, underscoring the significance of regulating microtubule dynamics in directed cell migration. These findings closely align with the results obtained in this investigation, demonstrating that Gαi2 loss of function reduces microtubule catastrophes and promotes tubulin stabilization, resulting in increased localization of active Rac1 at the leading edge and cell-cell contacts, while decreasing active RhoA at the cell-cell contact but increasing it at the leading edge. This possibly reinforces focal adhesion, which is consistent with the presence of large and highly stable focal adhesions under Gαi2 knockdown conditions. This finding also suggests a dysregulation of contractile forces in comparison to control cells, a result that aligns with the observed distribution of active RhoA, cortical actin distribution and diminished retraction in cells treated with Gαi2MO. This strikingly contrasts with the normal cranial NC migration phenotype, where Rac1 is suppressed while active RhoA is increased at cell-cell contacts during CIL, leading to a shift in polarity towards the cell-free edge to sustain directed migration (Theveneau et al., 2010; Shoval and Kalcheim, 2012; Leal et al., 2018)."

The co-Immunoprecipitation data lack marker bands (larger images/sections of the blots would be preferable) and the labelling is not clear. What do the white arrows in Fig. 3H,I mean? What does "elu" and "non eluted" mean?. ? Did the reverse IP work as well?

R: We appreciate the reviewer's comments, and here we intend to provide a more detailed explanation of our approach to this analysis. Since we do not possess a secondary antibody specific to the heavy chain, our method involves eluting the co-immunoprecipitated proteins to visualize those with weights close to that of the light chain (such as EB1). We have outlined this elution step in the "Cell lysates and co-immunoprecipitation" protocol in the Materials and Methods section. To ensure proper control, we load both fractions - the eluted (or supernatant) and non-eluted (or resin) fractions - to monitor the amount of protein extracted from the resin using a 1% SDS solution. It's important to note that the elution step, as indicated by the V5 signal, is not entirely efficient, and a significant portion of the protein remains bound to the resin. This issue may also apply to the EB1 protein; however, it is still possible to visualize both bands (Gαi2V5 and EB1).

As we mentioned earlier the Co-IPP analysis now are in Figure 5. We have revised the legend for Figure 5 to include an explanation of the terms 'elu' (eluted fraction) and 'non-eluted' (non-eluted fraction). We have also included the explanation of the white arrows' significance in the legends for Figure 5H and 5I. These arrows indicate the bands corresponding to the immunoprecipitated proteins.

We also agree with the reviewer's suggestion to conduct the reverse IP. To address this point, and in favour of the lack of this control, accordingly, we have included a negative control for co-IP using anti-V5 antibody to IP, this control was included in Supplementary material Figure S4A. Additionally, we conducted all Co-IPP in triplicate, and these data have been incorporated into Supplementary material Figure S4B.

The presentation of the Delaunay triangulations varies in quality. In Fig. 1 J/K the cells are clearly visible in the images, while this is not the case in Fig. 3 J-M and Fig. 4K-N. Conversely, the Delaunay triangulations in Fig. 1L are mainly black, while they are clear in Fig. 3 and 4. Perhaps the authors could find a more consistent way to present the data. Were the explants all approximately the same size at the beginning of the experiment? The Gαi2-morphant explant in Fig. 3K appears to be unusually small.

R: We appreciate the reviewer's concerns and have taken steps to address them. To improve the quality of our data, we have made enhancements to the presentation of Figures 3 (panels L-O) and Figure 5 (panels P-S). Specifically, we have standardized the Delaunay triangulation representations.

Regarding the size of the explants at the beginning of the experiments, they were indeed approximately similar in size. We confirmed this by including a reference point (point 0) for each condition in the figures 5. However, in the panels presented, we show the results after 10 hours (Figure 5, X. laevis, in the actual Figure organization) and 4 hours (Figure 3, X. tropicalis, in the actual Figure organization) to assess cell dispersion, as indicated in the respective figure legends. This uniformity in size was further ensured by the calculation used to quantify dispersion. For the dispersion assay, we normalized each initial size of the explant upon the control, and we have added another representative explant of Gαi2 morpholino with its Delaunay triangulation to facilitate the experiment interpretation. Every Delaunay triangulation calculates the area generated between three adjacent cells and it grows depending on how much disperse are the cells between each other in the final point. (See Material and Methods section, Cell dispersion and morphology). As we can see in the manuscript, in every dispersion experiment that we have performed with Gαi2 morpholino, the cells cannot disperse at all. Furthermore, to analyze the dispersion rate in this experiment we use Control n= 21 explants, Gαi2MO n= 24 explants, Gαi2MO + 65 nM Nocodazole n= 36 explants, Control + 65 nM Nocodazole n= 30 explants (as we mentioned in the manuscript legend).

Why was the tubulin distribution in Fig. 2F measured from the nucleus to the cell cortex? Would it not make more sense to include cell protrusions? This does not seem to be the case in the example shown in Fig. 2F.

R: We appreciate the reviewer's concern. We would like to clarify that for the tubulin distribution measurements, we indeed measured from the nucleus to the cell protrusion. As a result, we have made an edit to Figure 2 (panel 2F) to provide further clarity on this matter.

The immunostaining for acetylated tubulin (Fig. 3A,B) looks potentially unspecific and seems to co-localize with actin (for comparison see Bance et al., 2019). For imaging and quantification, it may be better to use tubulin co-staining to calculate the percentage of acetylated tubulin. Please also add marker bands to the Western blot in Fig. 3C. If this issue cannot be resolved it may be better to only include the Western blot data.

R: We appreciate the reviewer's concern about the potential unspecific nature of acetylated-tubulin and its co-localization with actin. Regarding the co-localization with actin, it is predominantly observed in panel B, and we attribute this phenomenon to the Gαi2 morphant phenotype, where cortical actin is notably reduced, creating the appearance of co-localization. In response to the reviewer comment, we have retained the acetylated tubulin western blot analysis in the main Figure 5A,B, while relocating the immunofluorescence analysis to Supplementary material Figure S4C-H. Additionally, we have included the measurements of the acetylated tubulin fluorescence intensity for both conditions Gαi2MO and control, as depicted Supplementary material Figure S4I.

We have also included marker weight indications on the western blot panel in now Figure 5A.

The model in Fig.6 indicates that Gαi2 inhibits EB1/3. What is the experimental evidence and the proposed mechanism for this? In the discussion, the authors cite evidence that Gαi activates the intrinsic GTPase activity of tubulin, which would destabilize microtubules by removing the GTP cap. However, this mechanism would not directly affect EB1 and EB3 stability as the Fig. 6A seems to suggest. The authors also mention that EB3 appears to be permanently associated with microtubules in Gαi2-morphant cells. How would this work, given that end-binding proteins bind to the cap region? Are the authors suggesting that there is an extended cap region in Gαi2 morphants?

R: We appreciate the reviewer's valuable comments. We have revised our model accordingly to our data and new data that we have incorporated regarding interaction analysis conducted by PLA (proximity ligand assay), in order to further elucidate the mechanism underlying Gαi2 regulation of cranial neural crest cell migration. This analysis supports our actual proposed model, indicating Gαi2 interacts with EB proteins to form a complex with tubulin, thereby regulating microtubules dynamics and subsequently influencing Rac1 and RhoA activity, cell morphology (actin cytoskeleton) and cell-matrix adhesion, ultimately affecting migration. Therefore, we have revised our model and its description to provide a more detailed explanation of the potential mechanism in line with the reviewer suggestion. Specifically, we have edited the discussion/conclusion, model and the legend for Figure 6. Please refer to page 16 (paragraph 1, 2 and 3), 22 (paragraph 1), 23 (paragraph 1), 45 (Legend Fig. 6). In addition, in Gαi2 knockdown conditions we have found a strong reduction in microtubules dynamics following EB3-GFP comets. Regarding the observation that EB3 seems to be persistently associated with microtubules in Gαi2-morphant cells, we wish to clarify that this is a speculation based on the microtubule phenotype observed during our dynamic analysis, where they appear more like lines rather than comets. It is important to note that none of the experiments conducted in this study conclusively demonstrate this, and thus, it remains a suggestion. As a result, we have revised our model in accordance with the reviewer suggestion.

Reviewer 3: minor comments:

The citation of Wang et al. 2018 in the introduction does not seem to fit.

R: We appreciate the correction provided by the reviewer. We have carefully reviewed the Introduction and Reference sections and have corrected this error.

Does the graph in Fig. 4S show average values for the three conditions? If so, what is the standard deviation?

R: We appreciate the reviewer's concern and we have added the standard deviation to now Figure 4J.

From the images in Fig. 2G and H, it is difficult to understand what the difference is between the four groups shown.

R: We appreciate the reviewer's comment, and to clarify this point, we would like to explain that the comparison has been made between each type of comet. The PlusTipTracker software separates comets based on their speed and lifetime, classifying them as fast long-lived, fast short-lived, slow long-lived, or slow short-lived. In both conditions (control and morphant cells), we compared the percentage of each type of comet, as previously described in Moore et al., 2013. The results demonstrate that morphant cells exhibit an increase in slow comets compared to control cells. The same explanation is described in the Material and Methods section on page 28, Microtubule dynamics analysis.

Review____er #3: (Significance (Required)):

Overall, the study is well executed and significantly advances our understanding of the control and role of microtubule dynamics in cell migration, which is much less understood compared to the function of the actin cytoskeleton in this process. The strength of the study is the use of state-of-the-art (live imaging) techniques to characterize the role of Gαi in neural crest migration at the cellular/subcellular level. This article will be of interest to a broad readership, including researchers interested in basic embryonic morphogenesis, cell migration, and cytoskeletal dynamics, as well as translational/clinical researchers interested in cancer progression or wound healing.

R: We really appreciate the reviewer positive comments and consideration. We believe that the review process has significantly strengthened our manuscript.

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Referee #3

Evidence, reproducibility and clarity

Summary: The manuscript by Villaseca et al. analyzes the role of Gαi2 in cranial neural crest migration and reveals a novel mechanistic link to microtubule dynamics. The authors nicely demonstrate that Gαi2 is required for Xenopus neural crest migration and affects cell dispersion, cell polarity, focal adhesion turnover, and microtubule dynamics. They find that Gαi2-morphant neural crest cells are elongated, have larger, more stable protrusions, higher active Rac1 levels, and a concentration of microtubules at the leading edge. Using co-immunoprecipitation, the authors show that Gαi2 forms a complex with α-tubulin and the microtubule plus-end binding proteins EB1 and EB3, which are known regulators of microtubule dynamics. Time-lapse imaging shows that Gαi2 loss of function increases microtubule stability, which is further supported by an increase in acetylated tubulin levels. Consistently, treatment with nocodazole, which inhibits microtubule polymerization, as well as treatment with a Rac1 inhibitor, is able to rescue cell dispersion and morphology of Gαi2-morphant neural crest cells. The authors propose a model, whereby Gαi2 interacts with components of the plus-tip microtubule-binding complex to control microtubule dynamics and Rac1 activity to establish cell polarity, disassemble focal adhesion, and thereby facilitate neural crest migration.

Major comments:

1. The authors focus exclusively on the analysis of the subcellular levels of Rac1, which is probably related to the fact that they observe large extended protrusions with high Rac1 activity. However, as the authors note, a global fine-tuning of Rho GTPase activity is required for neural crest migration. One of the observed phenotypes of Gαi2-morphant neural crest cells is a decrease in cell dispersion, which may be caused by defects in contact inhibition of locomotion (CIL). This process involves a local activation of RhoA at cell-cell contact sites (Carmona-Fontaine et al., 2008). Furthermore, in fibroblast, RhoA/ROCK activity is required for the front-rear polarity switch during CIL (Kadir et al., 2011). Interestingly, similar to the Gαi2 loss of function phenotype, ROCK inhibition leads to microtubule stabilization, which can be rescued by nocodazole treatment, restoring microtubule dynamics and CIL. Therefore, it would also be interesting to know how RhoA activity is affected in Gαi2-morphant NC cells. At a minimum, this point should be be included in the discussion.
2. The co-Immunoprecipitation data lack marker bands (larger images/sections of the blots would be preferable) and the labelling is not clear. What do the white arrows in Fig. 3H,I mean? What does "elu" and "non eluted" mean? Did the reverse IP work as well?
3. The presentation of the Delaunay triangulations varies in quality. In Fig. 1 J/K the cells are clearly visible in the images, while this is not the case in Fig. 3 J-M and Fig. 4K-N. Conversely, the Delaunay triangulations in Fig. 1L are mainly black, while they are clear in Fig. 3 and 4. Perhaps the authors could find a more consistent way to present the data. Were the explants all approximately the same size at the beginning of the experiment? The Gαi2-morphant explant in Fig. 3K appears to be unusually small.
4. Why was the tubulin distribution in Fig. 2F measured from the nucleus to the cell cortex? Would it not make more sense to include cell protrusions? This does not seem to be the case in the example shown in Fig. 2F.
5. The immunostaining for acetylated tubulin (Fig. 3A,B) looks potentially unspecific and seems to co-localize with actin (for comparison see Bance et al., 2019). For imaging and quantification, it may be better to use tubulin co-staining to calculate the percentage of acetylated tubulin. Please also add marker bands to the Western blot in Fig. 3C. If this issue cannot be resolved it may be better to only include the Western blot data.
6. The model in Fig.6 indicates that Gαi2 inhibits EB1/3. What is the experimental evidence and the proposed mechanism for this? In the discussion, the authors cite evidence that Gαi activates the intrinsic GTPase activity of tubulin, which would destabilize microtubules by removing the GTP cap. However, this mechanism would not directly affect EB1 and EB3 stability as the Fig. 6A seems to suggest. The authors also mention that EB3 appears to be permanently associated with microtubules in Gαi2-morphant cells. How would this work, given that end-binding proteins bind to the cap region? Are the authors suggesting that there is an extended cap region in Gαi2 morphants?

Minor comments

1. The citation of Wang et al. 2018 in the introduction does not seem to fit.
2. Does the graph in Fig. 4S show average values for the three conditions? If so, what is the standard deviation?
3. From the images in Fig. 2G and H, it is difficult to understand what the difference is between the four groups shown.

Referees cross-commenting The concerns raised by my colleagues are entirely valid.

Significance

Overall, the study is well executed and significantly advances our understanding of the control and role of microtubule dynamics in cell migration, which is much less understood compared to the function of the actin cytoskeleton in this process. The strength of the study is the use of state-of-the-art (live imaging) techniques to characterize the role of Gαi in neural crest migration at the cellular/subcellular level. This article will be of interest to a broad readership, including researchers interested in basic embryonic morphogenesis, cell migration, and cytoskeletal dynamics, as well as translational/clinical researchers interested in cancer progression or wound healing.

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

Summary

The manuscript by Villaseca et al. describes functional analysis of Gai2 in cranial neural crest (CNC) migration using the frog Xenopus as their model system. The authors performed the loss-of-function assay to knock down expression of endogenous of Gai2 and discovered that CNC migration was impaired in the absence of changes of CNC fate specification. Based on the literature on Gai2 activities in other cellular contexts, the authors speculated that Gai2 might regulate microtubule dynamics and Rac1 function. Their studies using immunofluorescence (IF) and live-cell imaging indeed showed that microtubules were stabilized in membrane protrusions with concurrent activation of Rac1 in Gai2 knockdown cells. In addition, focal adhesion turnover was reduced. They further demonstrated that the CNC migration defects could be partially rescued by destabilization of microtubules with chemical treatment. The authors conclude from the studies that Gai2 orchestrates microtubule dynamics and modulates Rac1 activation during neural crest migration.

Major comments

The authors aim to address two issues in this manuscript: a) the role of Gai2 in neural crest development; and b) the mechanism of Gai2 function. While they have done a good job demonstrating a role of Gai2 in NC migration both in vivo and in vitro as well as the effects of Gai2 knockdown on cytoskeleton dynamics, protein distribution of selected polarity and focal adhesion molecules, and Rac1 activation, the link between Gai2 and the downstream effectors is largely correlative. Because of this, the model suggesting the sequential events flowing from Gai2 to microtubule to Rac1 to focal adhesion/actin should be modified to allow room for direct and indirect regulation at potentially multiple entry points.

Specific major comments are as the following:

Strengths:

-Determination of a role of Gai2 in neural crest migration is novel. -The effect of Gai2 knockdown on membrane protrusion morphology and microtubule stability and dynamics are demonstrated nicely. -Quantification of experimental perimeters has been performed throughout the manuscript in all the figures, and statistical analysis is included in the figures.

Weaknesses:

• The heavy focus of the study on microtubule is due to the previous publication on the function of Gai2 in regulation of microtubule during asymmetrical cell division. However, the activity of Gai2 is likely cell type-specific, as it has not been shown to control microtubule during cytokinesis in general. It is equally likely that Gai2 primarily regulates Rac1 or actin regulators to influence both microtubule and actin dynamics. The tone of the discussion should therefore be softened.
• The effect of rescue of NC migration with Rac1 inhibitor is marginal and the result is hard to interpret considering the inhibitor also blocks control NC migration. Either lower doses of Rac1 inhibitor can be used or the experiment can be removed from the manuscript, as Rac1 is required for membrane protrusions and the inhibitor doses can be hard to titrate.
• Since the defects seem to result partially from the inability of the NC cells to retract and move away, it may help to either include some data on Rho activation patterns in knockdown cells or simply add some discussion about the issue.
• To consider focal adhesion dynamics, live imaging should be used in the analysis. The fixed samples are different from each other, and natural variations of focal adhesion may exist among the samples. This can obscure data collection and quantification.

Minor comments

• Fig. 2, the centrosomes in control cells are not always obvious. The microtubules simply seem to be more networked and more fluid in control cells. This should be clarified with either marking the centrosomes in the figure or modifying the wording in the manuscript.
• In Fig. 3, a better negative control for co-IP should be using anti-V5 antibody to IP against tubulin/EB1/EB3 in the absence of Gai2-V5.
• The data for cell polarity proteins Par3 and PKC-zeta seem to be out of place. It is unclear whether mis-localization of these proteins has anything to do with NC migration defects induced by Gai2 knockdown. The conclusion does not seem to be affected if the data are taken out of the manuscript.
• In Suppl. Fig. 1, protrusion versus retraction should be defined more clearly. The retraction shown in this figure seems to be just membrane between protrusions instead of actively retracting membrane.
• Discussion can be improved by better incorporating all the components to make a cohesive story on how Gai2 works to regulate migration in the context of the neural crest cells.

Referees cross-commenting I agree with other reviewers' comments.

Significance

The authors demonstrate a role of Gai2 in regulation of neural crest migration in Xenopus by modulating microtubule dynamics. In addition, they show an effect of Gai2 knockdown on Rac1 spatial activation and focal adhesion stability. These are novel discoveries of the study. Some limitations exist in linking Gai2 with downstream effectors that directly or indirectly impact on cytoskeleton and Rac1 small GTPase.

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Referee #1

Evidence, reproducibility and clarity

Summary:

This manuscript examines the role of a G protein, Gai2, in regulating the migration of cranial neural crest cells. Although previous literature has established that heterotrimeric G proteins are involved in cell migration, a central process during embryogenesis and adult homeostasis, the underlying cell biological mechanisms of their activities have not been elucidated. This manuscript rigorously examines the various aspects of Gai2 protein interactions to generate an exciting new paradigm in which Gai2 maintains normal microtubule dynamics by binding to tubulin and EB proteins. This normally dynamic microtubular intracellular environment then promotes cortical actin formation in the leading edge of the migrating cell as well as rapid focal adhesion disassembly by controlling Rac1 activity. Under conditions in which the levels of Gai2 are reduced by MO-mediated knockdown, cells display reduced microtubule dynamics and a decreased catastrophe rate, resulting in slower and more stable microtubules to which EB3 is more persistently associated. A stable microtubule environment leads to enhanced Rac1 activation at the leading edge and stable and larger focal adhesions, resulting in reduced migration. The authors utilize cutting edge approaches to examine the interactions between Gai2 and these other cellular components, taking advantage of the well characterized cell migration model - the cranial neural crest - both in embryos and in cultured explants of these cells.

Major comments:

The manuscript is mostly well written (it could use a few minor grammatical corrections), the significance of the problem is well described, and the results are clearly presented with adequate controls. The movies, provided as supplementary material, are of the highest quality and are essential additions to the stills provided in the figures. The data convincingly support the key conclusions of the manuscript.

Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? No

Would additional experiments be essential to support the claims of the paper? No additional experiments are essential.

Are the experiments adequately replicated and statistical analysis adequate? The number of embryos/ explants per assay and the number of explant replicates for each assay and the statistical assessments are rigorous.

Are the data and the methods presented in such a way that they can be reproduced? Mostly, however, the description of the MO used for Gai2 knockdown needs more detail:

1. Does the MO knockdown both S and L homoeologs of X. laevis? Since the level of GAPDH in Figure 1H also looks reduced in Gai2 MO lane, it should be made clear that the apparent knockdown of Gai2 was normalized to GAPDH, rather than being the results of unequal loading of the gel. Yes, I recognize that Figure 1I says normalized, but this is not stated in the results or the methods. Also, was this experiment done with X. laevis or X. tropicalis? I could imagine that if done in X. laevis, the lack of complete knockdown might be due to only one homoeolog being affected.
2. The knowledge of the efficacy of knockdown in each Xenopus species provided by the information requested in the previous point, would allow the reader to assess the level of knockdown in the remaining assays. To do this, the authors should tell us which assays were done in which species. I am not suggesting that each experiment needs to be done in each species, only that the information should be provided. If the MO is more effective in X. tropicalis - which assays used this species? If the knock down is partial, as shown in Figure 1H-I, which species this represents in the remaining assays would be useful knowledge.

Minor comments:

While prior studies are referenced appropriately, and the text and figures are mostly clear and accurately presented, the following are a few suggestions that would help the authors improve the presentation of their data and conclusions:

1. The cell biological experiments convincingly demonstrate that knockdown of Gai2 causes cells to move more slowly. It would be a nice addition to bring the explant experimental data back to the embryo by showing whether the slower moving NC cells in morphants eventually populate the BA. DO they cease to migrate or are they just slower getting to their destination? This could be done by performing snail2 ISH at a later stage (34-35?)
2. There are places in the manuscript where the authors use the terms "silencing" or "suppression" of Gai2, when they really mean reduced translation - their system is not a genetic knockout, as clearly demonstrated in Figure 1H-I. I suggest that more accurate wording be used.
3. In Figures 1-5 there are scale of bars on the cell images, but these are not defined in any of the figure legends.
4. The abstract is the weakest section of the manuscript, and would have greater impact if it were more clearly written.

Referees cross-commenting

The concerns are fair assessments. However, most can be addressed in the text and by clearer presentation of existing data rather than more experimentation.

Significance

The molecular regulation of cell movement is a key feature of a number of developmental and homeostatic processes. While many of the proteins involved have been identified, how they interact to provide motility has not been elucidated in any great detail, particularly in embryo-derived cells (as opposed to cell lines). The results obtained from the presented experiments are novel, in-depth and provide a novel paradigm for how G proteins regulate microtubule dynamics which in turn regulate other components of the cytoskeleton required for cell movement. The results will be applicable to many migrating cell types, not just neural crest cells.

Because of the application of the data to many types of cells that migrate, the audience is expected to include a broad array of developmental biologists, basic cell biologists and those interested in clinically relevant aberrant cell migrations.

Reviewer keywords: Xenopus embryology; neural crest gene expression; use of MO-mediated knockdown of gene expression. Not an expert in microtubule dynamics.

Author response:

We would first like to thank the editor for considering our findings for publication in eLife. Furthermore, we thank the reviewers and editors for their encouraging reviews and for providing helpful and insightful comments.

Reviewer #1 (Public Review):

Summary:

The pituitary gonadotropins, FSH and LH, are critical regulators of reproduction. In mammals, synthesis and secretion of FSH and LH by gonadotrope cells are controlled by the hypothalamic peptide, GnRH. As FSH and LH are made in the same cells in mammals, variation in the nature of GnRH secretion is thought to contribute to the differential regulation of the two hormones. In contrast, in fish, FSH and LH are produced in distinct gonadotrope populations and may be less (or differently) dependent on GnRH than in mammals. In the present manuscript, the authors endeavored to determine whether FSH may be independently controlled by a distinct peptide, cholecystokinin (CCK), in zebrafish.

Strengths:

The authors demonstrated that the CCK receptor is enriched in FSH-producing relative to LH-producing gonadotropes, and that genetic deletion of the receptor leads to dramatic decreases in gonadotropin production and gonadal development in zebrafish. Also, using innovative in vivo and ex vivo calcium imaging approaches, they show that LH- and FSH-producing gonadotropes preferentially respond to GnRH and CCK, respectively. Exogenous CCK also preferentially stimulated FSH secretion ex vivo and in vivo.

Weaknesses:

The concept that there may be a distinct FSH-releasing hormone (FSHRH) has been debated for decades. As the authors suggest that CCK is the long-sought FSHRH (at least in fish), they must provide data that convincingly leads to such a conclusion. In my estimation, they have not yet met this burden. In particular, they show that CCK is sufficient to activate FSH-producing cells, but have not yet demonstrated its necessity. Their one attempt to do so was using fish in which they inactivated the CCK receptor using CRISPR-Cas9. While this manipulation led to a reduction in FSH, LH was affected to a similar extent. As a result, they have not shown that CCK is a selective regulator of FSH.

Our conclusion regarding the necessity of CCK signaling for FSH secretion is based on the following evidence:

(1) CCK-like receptors are expressed in the pituitary gland predominantly on FSH cells.

(2) Application of CCK to pituitaries elicits FSH cell activation and FSH release, and, to a lesser degree, activation of LH cells.

(3) Mutating the CCK-like receptor causes a decrease in fsh and lh mRNA synthesis.

(4) Mutating the CCK-like receptor gives rise to a phenotype which is identical to that caused by mutation of both lh and fsh genes in zebrafish.

(5) Mutating the FSH-specific CCK receptor in a different species of fish (medaka) also causes a complete shutdown of FSH production and phenocopies a fsh-mutant phenotype (Uehara et al, BioRxiv, DOI: 10.1101/2023.05.26.542428).

Taken together, we believe that this data strongly supports the conclusion that CCK is necessary for FSH production and release from the fish pituitary. Admittedly, the overlapping effects of CCK on both FSH and LH cells in zebrafish (evident in both our calcium imaging experiments and the KO phenotype) complicates the interpretation of the phenotype. We speculate that the effect of CCK on LH cells in zebrafish can be caused either by paracrine signaling within the gland or by the effects of CCK on higher levels of the axis. In our revised manuscript we will make sure to highlight the overlapping effects of CCK on LH cells rather than portray it as a selective activator of FSH cells.

Moreover, they do not yet demonstrate that the effects observed reflect the loss of the receptor's function in gonadotropes, as opposed to other cell types.

Although there is evidence for the expression of CCK receptor in other tissues, we do show a direct decrease of FSH and LH expression in the gonadotrophs of the pituitary of the mutant fish; taken together with its significant expression in FSH cells, it is the most reasonable and forward explanation for the mutant phenotype. Unfortunately, unlike in mice, technologies for conditional knockout of genes in specific cell types are not yet available for our model and cell types. However, in the revised manuscript we will add a supplementary figure describing the distribution of this receptor in other tissues.

It also is not clear whether the phenotypes of the fish reflect perturbations in pituitary development vs. a loss of CCK receptor function in the pituitary later in life. Ideally, the authors would attempt to block CCK signaling in adult fish that develop normally. For example, if CCK receptor antagonists are available, they could be used to treat fish and see whether and how this affects FSH vs. LH secretion.

While the observed gonadal phenotype of the KO (sex inversion) should have a developmental origin since it requires a long time to manifest, the effect of the KO on FSH and LH cells is probably more acute.

In the Discussion, the authors suggest that CCK, as a satiety factor, may provide a link between metabolism and reproduction. This is an interesting idea, but it is not supported by the data presented. That is, none of the results shown link metabolic state to CCK regulation of FSH and fertility. Absent such data, the lengthy Discussion of the link is speculative and not fully merited.

In the revised manuscript, we will address this comment by either providing data to link cck with metabolic status or tuning down the Discussion of this topic.

Also in the Discussion, the authors argue that "CCK directly controls FSH cells by innervating the pituitary gland and binding to specific receptors that are particularly abundant in FSH gonadotrophs." However, their imaging does not demonstrate innervation of FSH cells by CCK terminals (e.g., at the EM level).

Innervation of the fish pituitary does not imply a synaptic-like connection between axon terminals and endocrine cells. In fact, such connections are extremely rare, and their functionality is unclear. Instead, the mode of regulation between hypothalamic terminals and endocrine cells in the fish pituitary is more similar to "volume transmission" in the CNS, i.e. peptides are released into the tissue and carried to their endocrine cell targets by the circulation or via diffusion.

Moreover, they have not demonstrated the binding of CCK to these cells. Indeed, no CCK receptor protein data are shown.

Our revised manuscript will include detailed experiments showing the activation of the receptor by its ligand. Unfortunately, no antibody is available against this fish- specific receptor (one of the caveats of working with fish models); therefore, we cannot present receptor protein data.

The calcium responses of FSH cells to exogenous CCK certainly suggest the presence of functional CCK receptors therein; but, the nature of the preparations (with all pituitary cell types present) does not demonstrate that CCK is acting directly in these cells.

We agree with the reviewer that there are some disadvantages in choosing to work with a whole-tissue preparation. However, we believe that the advantages of working in a more physiological context far outweigh the drawbacks as it reflects the natural dynamics more precisely. Since our transcriptome data as well as our ISH staining, show that the CCK receptor is exclusively expressed on FSH cells, it is improbable that the observed calcium response is mediated via a different pituitary cell type.

Indeed, the asynchrony in responses of individual FSH cells to CCK (Figure 4) suggests that not all cells may be activated in the same way. Contrast the response of LH cells to GnRH, where the onset of calcium signaling is similar across cells (Figure 3).

The difference between the synchronization levels of LH and FSH cells activity stems from the gap-junction mediated coupling between LH cells that does not exist between FSH cells (Golan et al 2016, DOI: 10.1038/srep23777). Therefore, the onset of calcium response in FSH cells is dependent on the irregular diffusion rate of the peptide within the preparation, whereas the tight homotypic coupling between LH cells generates a strong and synchronized calcium rise that propagates quickly throughout the entire population; we will make sure this is clear in the final revision.

Finally, as the authors note in the Discussion, the data presented do not enable them to conclude that the endogenous CCK regulating FSH (assuming it does) is from the brain as opposed to other sources (e.g., the gut).

We agree with the reviewer that, for now, we are unable to determine whether hypothalamic or peripheral CCK are the main drivers of FSH cells. While the strong innervation of the gland by CCK-secreting hypothalamic neurons strengthens the notion of a hypothalamic-releasing hormone and also fits with the dogma of the neural control of the pituitary gland in fish (Ball, 1981; doi: 10.1016/0016-6480(81)90243-4.), more experiments are required to resolve this question.

Reviewer #2 (Public Review):

Summary:

This manuscript builds on previous work suggesting that the CCK peptide is the releasing hormone for FSH in fishes, which is different than that observed in mammals where both LH and FSH release are under the control of GnRH. Based on data using calcium imaging as a readout for stimulation of the gonadotrophs, the researchers present data supporting the hypothesis that CCK stimulates FSH- containing cells in the pituitary. In contrast, LH-containing cells show a weak and variable response to CCK but are highly responsive to GnRH. Data are presented that support the role of CCK in the release of FSH. Researchers also state that functional overlap exists in the potency of GnRH to activate FSH cells, thus the two signalling pathways are not separate.

The results are of interest to the field because for many years the assumption has been that fishes use the same signalling mechanism. These data present an intriguing variation where a hormone involved in satiation acts in the control of reproduction.

Strengths:

The strengths of the manuscript are that researchers have shed light on different pathways controlling reproduction in fishes.

Weaknesses:

Weaknesses are that it is not clear if multiple ligand/receptors are involved (more than one CCK and more than one receptor?). The imaging of the CCK terminals and CCK receptors needs to be reinforced.

Reviewer consultation summary:

• The data presented establish sufficiency, but not necessity of CCK in FSH regulation. The paper did not show that CCK endogenously regulates FSH in fish. This has not been established yet.

This is a very important comment, also raised by reviewer 1. To avoid repetition, please see our detailed response to the comment above.

• The paper presents the pharmacological effects of CCK on ex vivo preparations but does not establish the in vivo physiological function of the peptide. The current evidence for a novel physiological regulatory mechanism is incomplete and would require further physiological experiments. These could include the use of a CCK receptor antagonist in adult fish to see the effects on FSH and LH release, the generation of a CCK knockout, or cell-specific genetic manipulations.

As detailed in the responses to the first reviewer,we cannot conduct conditional, cell- specific gene knockout in our model.

• Zebrafish have two CCK ligands: ccka, cckb and also multiple receptors: cckar, cckbra and cckbrb. There is ambiguity about which CCK receptor and ligand are expressed and which gene was knocked out.

In the revised manuscript, we will clarify which of the receptors are expressed and which receptor is targeted. We will also provide data showing the specificity of the receptors (both WT and mutant) to the ligands.

• Blocking CCK action in fish (with receptor KO) affects FSH and LH. Therefore, the work did not demonstrate a selective role for CCK in FSH regulation in vivo and any claims to have discovered FSHRH need to be more conservative.

We agree with the reviewer that the overlap in the effect of CCK measured in the calcium activation of cells and in the KO model does not allow us to conclude selectivity. In this context, it is crucial to highlight that CCK-R exhibits high expression on FSH cells but not on LH cells. Therefore, the effect of CCK on LH cells is likely paracrine rather than solely endocrine. We will tone down our claims of selectivity in the revised manuscript.

• The labelling of the terminals with anti-CCK looks a lot like the background and the authors did not show a specificity control (e.g. anti-CCK antibody pre-absorbed with the peptide or anti-CCK in morphant/KO animals).

We will update the colors of the image for better clarity. Also, The same antibody had been previously used to mark CCK-positive cells in the gut of the red drum fish (K.A. Webb, Jr. 2010; DOI: https://doi.org/10.1016/j.ygcen.2009.10.010), where a control (pre-absorbed with the peptide) experiment had been conducted.

eLife assessment

This study presents valuable findings on the potential role of a peptide typically associated with feeding in the control of a pituitary hormone, FSH, which is a critical regulator of reproductive physiology. The evidence supporting the main claims of the authors is thought-provoking but incomplete. In particular, the authors demonstrate that the peptide is sufficient to regulate FSH, but they have not established its necessity. The work will be of interest to reproductive biologists, especially those with an interest in the endocrine control of fertility.

Reviewer #1 (Public Review):

Summary:

The pituitary gonadotropins, FSH and LH, are critical regulators of reproduction. In mammals, synthesis and secretion of FSH and LH by gonadotrope cells are controlled by the hypothalamic peptide, GnRH. As FSH and LH are made in the same cells in mammals, variation in the nature of GnRH secretion is thought to contribute to the differential regulation of the two hormones. In contrast, in fish, FSH and LH are produced in distinct gonadotrope populations and may be less (or differently) dependent on GnRH than in mammals. In the present manuscript, the authors endeavored to determine whether FSH may be independently controlled by a distinct peptide, cholecystokinin (CCK), in zebrafish.

Strengths:

The authors demonstrated that the CCK receptor is enriched in FSH-producing relative to LH-producing gonadotropes, and that genetic deletion of the receptor leads to dramatic decreases in gonadotropin production and gonadal development in zebrafish. Also, using innovative in vivo and ex vivo calcium imaging approaches, they show that LH- and FSH-producing gonadotropes preferentially respond to GnRH and CCK, respectively. Exogenous CCK also preferentially stimulated FSH secretion ex vivo and in vivo.

Weaknesses:

The concept that there may be a distinct FSH-releasing hormone (FSHRH) has been debated for decades. As the authors suggest that CCK is the long-sought FSHRH (at least in fish), they must provide data that convincingly leads to such a conclusion. In my estimation, they have not yet met this burden. In particular, they show that CCK is sufficient to activate FSH-producing cells, but have not yet demonstrated its necessity. Their one attempt to do so was using fish in which they inactivated the CCK receptor using CRISPR-Cas9. While this manipulation led to a reduction in FSH, LH was affected to a similar extent. As a result, they have not shown that CCK is a selective regulator of FSH. Moreover, they do not yet demonstrate that the effects observed reflect the loss of the receptor's function in gonadotropes, as opposed to other cell types. It also is not clear whether the phenotypes of the fish reflect perturbations in pituitary development vs. a loss of CCK receptor function in the pituitary later in life. Ideally, the authors would attempt to block CCK signaling in adult fish that develop normally. For example, if CCK receptor antagonists are available, they could be used to treat fish and see whether and how this affects FSH vs. LH secretion.

In the Discussion, the authors suggest that CCK, as a satiety factor, may provide a link between metabolism and reproduction. This is an interesting idea, but it is not supported by the data presented. That is, none of the results shown link metabolic state to CCK regulation of FSH and fertility. Absent such data, the lengthy discussion of the link is speculative and not fully merited.

Also in the Discussion, the authors argue that "CCK directly controls FSH cells by innervating the pituitary gland and binding to specific receptors that are particularly abundant in FSH gonadotrophs." However, their imaging does not demonstrate innervation of FSH cells by CCK terminals (e.g., at the EM level). Moreover, they have not demonstrated the binding of CCK to these cells. Indeed, no CCK receptor protein data are shown. The calcium responses of FSH cells to exogenous CCK certainly suggest the presence of functional CCK receptors therein; but, the nature of the preparations (with all pituitary cell types present) does not demonstrate that CCK is acting directly in these cells. Indeed, the asynchrony in responses of individual FSH cells to CCK (Figure 4) suggests that not all cells may be activated in the same way. Contrast the response of LH cells to GnRH, where the onset of calcium signaling is similar across cells (Figure 3). Finally, as the authors note in the Discussion, the data presented do not enable them to conclude that the endogenous CCK regulating FSH (assuming it does) is from the brain as opposed to other sources (e.g., the gut).

Reviewer #2 (Public Review):

Summary:

This manuscript builds on previous work suggesting that the CCK peptide is the releasing hormone for FSH in fishes, which is different than that observed in mammals where both LH and FSH release are under the control of GnRH. Based on data using calcium imaging as a readout for stimulation of the gonadotrophs, the researchers present data supporting the hypothesis that CCK stimulates FSH-containing cells in the pituitary. In contrast, LH-containing cells show a weak and variable response to CCK but are highly responsive to GnRH. Data are presented that support the role of CCK in the release of FSH. Researchers also state that functional overlap exists in the potency of GnRH to activate FSH cells, thus the two signalling pathways are not separate.

The results are of interest to the field because for many years the assumption has been that fishes use the same signalling mechanism. These data present an intriguing variation where a hormone involved in satiation acts in the control of reproduction.

Strengths:

The strengths of the manuscript are that researchers have shed light on different pathways controlling reproduction in fishes.

Weaknesses:

Weaknesses are that it is not clear if multiple ligand/receptors are involved (more than one CCK and more than one receptor?). The imaging of the CCK terminals and CCK receptors needs to be reinforced.

Reviewer consultation summary:

- The data presented establish sufficiency, but not necessity of CCK in FSH regulation. The paper did not show that CCK endogenously regulates FSH in fish. This has not been established yet.

- The paper presents the pharmacological effects of CCK on ex vivo preparations but does not establish the in vivo physiological function of the peptide. The current evidence for a novel physiological regulatory mechanism is incomplete and would require further physiological experiments. These could include the use of a CCK receptor antagonist in adult fish to see the effects on FSH and LH release, the generation of a CCK knockout, or cell-specific genetic manipulations.

- Zebrafish have two CCK ligands: ccka, cckb and also multiple receptors: cckar, cckbra and cckbrb. There is ambiguity about which CCK receptor and ligand are expressed and which gene was knocked out.

- Blocking CCK action in fish (with receptor KO) affects FSH and LH. Therefore, the work did not demonstrate a selective role for CCK in FSH regulation in vivo and any claims to have discovered FSHRH need to be more conservative.

- The labelling of the terminals with anti-CCK looks a lot like the background and the authors did not show a specificity control (e.g. anti-CCK antibody pre-absorbed with the peptide or anti-CCK in morphant/KO animals).

Résumé de la vidéo [00:00:00][^1^][1] - [00:23:54][^2^][2]:

La vidéo présente une conférence de Jean-Emmanuel Ray, professeur de droit privé à Paris 1 Sorbonne, sur l'évaluation dans le travail. Il discute des défis de l'évaluation des employés et des étudiants, l'impact des technologies de l'information, et l'évolution du droit du travail depuis l'ère industrielle jusqu'à l'ère de la connaissance.

Points forts: + [00:00:22][^3^][3] Introduction de Jean-Emmanuel Ray * Présentation de son expertise en droit du travail * Discussion sur l'évaluation dans les universités et les entreprises + [00:07:01][^4^][4] La vraie vie au travail * L'importance de comprendre le travail au-delà des chiffres * La nécessité d'évaluer le travail réel plutôt que les résultats visibles + [00:13:09][^5^][5] L'individualisation de l'évaluation * Transition de l'évaluation collective vers l'évaluation individuelle * Impact de la technologie et de la crise économique sur l'évaluation + [00:19:00][^6^][6] Les défis de l'évaluation moderne * La recherche de leaders plutôt que de supérieurs hiérarchiques * L'importance de l'expertise et de l'exemplarité dans le management moderne Résumé de la vidéo [00:23:57][^1^][1] - [00:47:40][^2^][2]:

La vidéo présente une conférence sur l'évaluation dans les sciences, les arts et les organisations, en se concentrant sur les différences culturelles et les défis juridiques associés aux systèmes d'évaluation, notamment dans le contexte de la mondialisation et des entreprises internationales.

Points forts: + [00:23:57][^3^][3] Les défis de l'évaluation * Difficultés d'évaluer les compétences et performances * Importance de l'adaptation culturelle dans les évaluations * Impact des évaluations sur les carrières et le bien-être des employés + [00:30:25][^4^][4] L'évaluation dans un contexte juridique * Droits et devoirs des employeurs dans l'évaluation des employés * Conséquences juridiques des évaluations inappropriées * Importance de la transparence et de l'objectivité dans les évaluations + [00:37:39][^5^][5] L'évaluation des délégués syndicaux * Complexité de l'évaluation des représentants syndicaux * Discrimination potentielle et exigences légales * Approches pour évaluer de manière équitable et légale + [00:42:31][^6^][6] Les systèmes d'évaluation et la santé mentale * Influence des évaluations sur la santé mentale des employés * Rôle du CHSCT dans la surveillance des pratiques d'évaluation * Légalité des évaluations et leur conformité avec la loi sur la santé et la sécurité Résumé de la vidéo [00:47:43][^1^][1] - [01:02:24][^2^][2]:

La vidéo présente une conférence sur l'évaluation dans les sciences, les arts et les organisations, mettant en lumière les défis de l'évaluation des compétences techniques et personnelles dans divers domaines professionnels.

Points forts: + [00:47:43][^3^][3] L'évaluation des compétences * Différenciation entre compétences techniques et qualités personnelles * Importance de l'évaluation holistique dans la sélection des juges et des hauts fonctionnaires * Exemple de l'épreuve du grand oral dans les concours de la haute fonction publique + [00:49:00][^4^][4] Cas de l'affaire d'Outreau * Discussion sur la suffisance de la technicité juridique pour être un bon juge * Mention de l'affaire d'Outreau comme exemple de l'importance de l'expérience personnelle + [00:50:10][^5^][5] Évaluation des évaluateurs * Questionnement sur la validité et l'équité des processus d'évaluation * Exemple d'une étude américaine sur les différences de gestion entre managers hommes et femmes + [00:56:02][^6^][6] Impact de l'évaluation sur la rémunération * Analyse de l'évolution des pratiques d'évaluation et leur effet sur les rémunérations variables * Discussion sur l'individualisation des bonus et la collaboration dans le contexte du web 2.0 + [01:00:00][^7^][7] Pratiques d'évaluation et harcèlement moral * Exploration des implications légales des méthodes d'évaluation sur la santé mentale des employés * Influence de la réputation de l'entreprise sur les pratiques d'évaluation + [01:01:00][^8^][8] Technologie d'évaluation et relations horizontales * Réflexion sur la mesure de la performance des outils d'évaluation * Discussion sur le manque de relations horizontales et la rigidité des systèmes d'évaluation

eLife assessment

This study provides evidence that the quality of research in female-dominated fields of research is systematically undervalued by the research community. The authors' findings are based on analyses of data from a research assessment exercise in New Zealand and data on funding success rates in Australia, Canada, the European Union and the United Kingdom. This work is an important contribution to the discourse on gender biases in academia, underlining the pervasive influence of gender on whole fields of research, as well as on individual researchers. The evidence supporting the conclusions is solid, but the work would benefit from further explorations into the nuances of specific fields of fields of research.

Reviewer #2 (Public Review):

Summary:<br /> The authors used four datasets spanning 30 countries to examine funding success and research quality score for various disciplines. They examined whether funding or research quality score were influenced by majority gender of the discipline and whether these affected men, women, or both within each discipline. They found that disciplines dominated by women have lower funding success and research quality score than disciplines dominated by men. These findings, are surprising because even the men in women-dominated fields experienced lower funding success and research quality score.

Strengths:<br /> - The authors utilized a comprehensive dataset covering 30 countries to explore the influence of the majority gender in academic disciplines on funding success and research quality scores.<br /> - Findings suggest a systemic issue where disciplines with a higher proportion of women have lower evaluations and funding success for all researchers, regardless of gender.<br /> - The manuscript is notable for its large sample size and the diverse international scope, enhancing the generalizability of the results.<br /> - The work accounts for various factors including age, number of research outputs, and bibliometric measures, strengthening the validity of the findings.<br /> - The manuscript raises important questions about unconscious bias in research evaluation and funding decisions, as evidenced by lower scores in women-dominated fields even for researchers that are men.<br /> - The study provides a nuanced view of gender bias, showing that it is not limited to individuals but extends to entire disciplines, impacting the perception and funding and quality or worth of research.<br /> - This work underscores the need to explore motivations behind gender distribution across fields, hinting at deep-rooted societal and institutional barriers.<br /> - The authors have opened a discussion on potential solutions to counter bias, like adjusting funding paylines or anonymizing applications, or other practical solutions.<br /> - While pointing out limitations such as the absence of data from major research-producing countries, the manuscript paves the way for future studies to examine whether its findings are universally applicable.

Weaknesses:<br /> - The study does not provide data on the gender of grant reviewers or stakeholders, which could be critical for understanding potential unconscious bias in funding decisions. These data are likely not available; however, this could be discussed. Are grant reviewers in fields dominated by women more likely to be women?<br /> - There could be more exploration into whether the research quality score is influenced by inherent biases towards disciplines themselves, rather than only being gender bias.<br /> - The manuscript should discuss how non-binary gender identities were addressed in the research. There is an opportunity to understand the impact on this group.<br /> - A significant limitation is absence of data from other major research-producing countries like China and the United States, raising questions about the generalizability of the findings. How comparable are the findings observed to these other countries?<br /> - The motivations and barriers that drive gender distribution in various fields could be expanded on. Are fields striving to reach gender parity through hiring or other mechanisms?<br /> - The authors could consider if the size of funding awards correlates with research scores, potentially overlooking a significant factor in the evaluation of research quality. Presumably there is less data on smaller 'pilot' funds and startup funds for disciplines where these are more common. Would funding success follow the same trend for these types of funds?<br /> - The language used in the manuscript at times may perpetuate bias, particularly when discussing "lower quality disciplines," which could influence the reader's perception of certain fields.<br /> - The manuscript does not clarify how many gender identities were represented in the datasets or how gender identity was determined, potentially conflating gender identity with biological sex.

Reviewer #3 (Public Review):

This study seeks to investigate one aspect of disparity in academia: how gender balance in a discipline is valued in terms of evaluated research quality score and funding success. This is important in understanding disparities within academia.<br /> This study uses publicly available data to investigate covariation between gender balance in an academic discipline and:<br /> i) Individual research quality scores of New Zealand academics as evaluated by one of 14 broader subject panels.<br /> ii) Funding success in Australia, Canada, Europe, UK.

The study would benefit from further discussion of it limitations, and from the clarification of some technical points (as described in the recommendations for the authors).

Reviewer #3 (Public Review):

Summary:

The goal of this paper is to characterize an anti-diuretic signaling system in insects using Drosophila melanogaster as a model. Specifically, the authors wished to characterize a role of ion transport peptide (ITP) and its isoforms in regulating diverse aspects of physiology and metabolism. The authors combined genetic and comparative genomic approaches with classical physiological techniques and biochemical assays to provide a comprehensive analysis of ITP and its role in regulating fluid balance and metabolic homeostasis in Drosophila. The authors further characterized a previously unrecognized role for Gyc76C as a receptor for ITPa, an amidated isoform of ITP, and in mediating the effects of ITPa on fluid balance and metabolism. The evidence presented in favor of this model is very strong as it combines multiple approaches and employs ideal controls. Taken together, these findings represent an important contribution to the field of insect neuropeptides and neurohormones and have strong relevance for other animals.

Strengths:

Many approaches are used to support their model. Experiments were well-controlled, used appropriate statistical analyses, and were interpreted properly and without exaggeration.

Weaknesses:

No major weaknesses were identified by this reviewer. More evidence to support their model would be gained by using a loss-of-function approach with ITPa, and by providing more direct evidence that Gyc76C is the receptor that mediates the effects of ITPa on fat metabolism. However, these weaknesses do not detract from the overall quality of the evidence presented in this manuscript, which is very strong.

eLife assessment

This important study provides a comprehensive analysis of ITP and its role as an anti-diuretic and metabolic hormone in Drosophila. The evidence supporting the conclusion is solid in general with combined genetic, comparative genomic approaches, classical physiological techniques, and biochemical assays. However, the evidence of direct binding between ITPa and Gyc76C and their physiological functions is incomplete. This work represents a contribution to the field of neuropeptides and neurohormones in insects and other animals.

Reviewer #1 (Public Review):

Summary:

In Drosophila melanogaster, ITP has functions on feeding, drinking, metabolism, excretion, and circadian rhythm. In the current study, the authors characterized and compared the expression of all three ITP isoforms (ITPa and ITPL1&2) in the CNS and peripheral tissues of Drosophila. An important finding is that they functionally characterized and identified Gyc76C as an ITPa receptor in Drosophila using both in vitro and in vivo approaches. In vitro, the authors nicely confirmed that the inhibitory function of recombinant Drosophila ITPa on MT secretion is Gyc76C-dependent (knockdown Gyc76C specifically in two types of cells abolished the anti-diuretic action of Drosophila ITPa on renal tubules). They also used a combination of multiple approaches to investigate the roles of ITPa and Gyc76C on osmotic and metabolic homeostasis modulation in vivo. They revealed that ITPa signaling to renal tubules and fat body modulates osmotic and metabolic homeostasis via Gyc76C.

Furthermore, they tried to identify the upstream and downstream of ITP neurons in the nervous system by using connectomics and single-cell transcriptomic analysis. I found this interesting manuscript to be well-written and described. The findings in this study are valuable to help understand how ITP signals work on systemic homeostasis regulation. Both anatomical and single-cell transcriptome analysis here should be useful to many in the field.

Strengths:

- The question (what receptors of ITPa in Drosophila) that this study tries to address is important. The authors ruled out the Bombyx ITPa receptor orthologs as potential candidates. They identified a novel ITP receptor by using phylogenetic, anatomical analysis, and both in vitro and in vivo approaches.

- The authors exhibited detailed anatomical data of both ITP isoforms and Gyc76C (in the main and supplementary figures), which helped audiences understand the expression of the neurons studied in the manuscript.

- They also performed connectomes and single-cell transcriptomics analysis to study the synaptic and peptidergic connectivity of ITP-expressing neurons. This provided more information for better understanding and further study on systemic homeostasis modulation.

Weaknesses:

In the discussion section, the authors raised the limitations of the current study, which I mostly agree with, such as the lack of verification of direct binding between ITPa and Gyc76C, even though they provided different data to support that ITPa-Gyc76C signaling pathway regulates systemic homeostasis in adult flies.

Reviewer #2 (Public Review):

Summary:

The physiology and behaviour of animals are regulated by a huge variety of neuropeptide signalling systems. In this paper, the authors focus on the neuropeptide ion transport peptide (ITP), which was first identified and named on account of its effects on the locust hindgut (Audsley et al. 1992). Using Drosophila as an experimental model, the authors have mapped the expression of three different isoforms of ITP (Figures 1, S1, and S2), all of which are encoded by the same gene.

The authors then investigated candidate receptors for isoforms of ITP. Firstly, Drosophila orthologs of G-protein coupled receptors (GPCRs) that have been reported to act as receptors for ITPa or ITPL in the insect Bombyx mori were investigated. Importantly, the authors report that ITPa does not act as a ligand for the GPCRs TkR99D and PK2-R1 (Figure S3). Therefore, the authors investigated other putative receptors for ITPs. Informed by a previously reported finding that ITP-type peptides cause an increase in cGMP levels in cells/tissues (Dircksen, 2009, Nagai et al., 2014), the authors investigated guanylyl cyclases as candidate receptors for ITPs. In particular, the authors suggest that Gyc76C may act as an ITP receptor in Drosophila.

Evidence that Gyc76C may be involved in mediating effects of ITP in Bombyx was first reported by Nagai et al. (2014) and here the authors present further evidence, based on a proposed concordance in the phylogenetic distribution ITP-type neuropeptides and Gyc76C (Figure 2). Having performed detailed mapping of the expression of Gyc76C in Drosophila (Figures 3, S4, S5, S6), the authors then investigated if Gyc76C knockdown affects the bioactivity of ITPa in Drosophila. The inhibitory effect of ITPa on leucokinin- and diuretic hormone-31-stimulated fluid secretion from Malpighian tubules was found to be abolished when expression of Gyc76C was knocked down in stellate cells and principal cells, respectively (Figure 4). However, as discussed below, this does not provide proof that Gyc76C directly mediates the effect of ITPa by acting as its receptor. The effect of Gyc76C knockdown on the action of ITPa could be an indirect consequence of an alteration in cGMP signalling.

Having investigated the proposed mechanism of ITPa in Drosophila, the authors then investigated its physiological roles at a systemic level. In Figure 5 the authors present evidence that ITPa is released during desiccation and accordingly, overexpression of ITPa increases survival when animals are subjected to desiccation. Furthermore, knockdown of Gyc76C in stellate or principal cells of Malphigian tubules decreases survival when animals are subject to desiccation. However, whilst this is correlative, it does not prove that Gyc76C mediates the effects of ITPa. The authors investigated the effects of knockdown of Gyc76C in stellate or principal cells of Malphigian tubules on i). survival when animals are subject to salt stress and ii). time taken to recover from of chill coma. It is not clear, however, why animals over-expressing ITPa were also not tested for its effect on i). survival when animals are subject to salt stress and ii). time taken to recover from of chill coma. In Figures 6 and S8, the authors show the effects of Gyc76C knockdown in the female fat body on metabolism, feeding-associated behaviours and locomotor activity, which are interesting. Furthermore, the relevance of the phenotypes observed to potential in vivo actions of ITPa is explored in Figure 7. The authors conclude that "increased ITPa signaling results in phenotypes that largely mirror those seen following Gyc76C knockdown in the fat body, providing further support that ITPa mediates its effects via Gyc76C." Use of the term "largely mirror" seems inappropriate here because there are opposing effects- e.g. decreased starvation resistance in Figure 6A versus increased starvation resistance in Figure 7A. Furthermore, as discussed above, the results of these experiments do not prove that the effects of ITPa are mediated by Gyc76C because the effects reported here could be correlative, rather than causative.

Lastly, in Figures 8, S9, and S10 the authors analyse publicly available connectomic data and single-cell transcriptomic data to identify putative inputs and outputs of ITPa-expressing neurons. These data are a valuable addition to our knowledge ITPa expressing neurons; but they do not address the core hypothesis of this paper - namely that Gyc76C acts as an ITPa receptor.

Strengths:

(1) The main strengths of this paper are i) the detailed analysis of the expression and actions of ITP and the phenotypic consequences of over-expression of ITPa in Drosophila. ii). the detailed analysis of the expression of Gyc76C and the phenotypic consequences of knockdown of Gyc76C expression in Drosophila.

(2) Furthermore, the paper is generally well-written and the figures are of good quality.

Weaknesses:

(1) The main weakness of this paper is that the data obtained do not prove that Gyc76C acts as a receptor for ITPa. Therefore, the following statement in the abstract is premature: "Using a phylogenetic-driven approach and the ex vivo secretion assay, we identified and functionally characterized Gyc76C, a membrane guanylate cyclase, as an elusive Drosophila ITPa receptor." Further experimental studies are needed to determine if Gyc76C acts as a receptor for ITPa. In the section of the paper headed "Limitations of the study", the authors recognise this weakness. They state "While our phylogenetic analysis, anatomical mapping, and ex vivo and in vivo functional studies all indicate that Gyc76C functions as an ITPa receptor in Drosophila, we were unable to verify that ITPa directly binds to Gyc76C. This was largely due to the lack of a robust and sensitive reporter system to monitor mGC activation." It is not clear what the authors mean by "the lack of a robust and sensitive reporter system to monitor mGC activation". The discovery of mGCs as receptors for ANP in mammals was dependent on the use of assays that measure GC activity in cells (e.g. by measuring cGMP levels in cells). Furthermore, more recently cGMP reporters have been developed. The use of such assays is needed here to investigate directly whether Gyc76C acts as a receptor for ITPa. In summary, insufficient evidence has been obtained to conclude that Gyc76C acts as a receptor for ITPa. Therefore, I think there are two ways forward, either:<br /> (a) The authors obtain additional biochemical evidence that ITPa is a ligand for Gyc76C.<br /> or<br /> (b) The authors substantially revise the conclusions of the paper (in the title, abstract, and throughout the paper) to state that Gyc76C MAY act as a receptor for ITPa, but that additional experiments are needed to prove this.

(2) The authors state in the abstract that a phylogenetic-driven approach led to their identification of Gyc76C as a candidate receptor for ITPa. However, there are weaknesses in this claim. Firstly, because the hypothesis that Gyc76C may be involved in mediating effects of ITPa was first proposed ten years ago by Nagai et al. 2014, so this surely was the primary basis for investigating this protein. Nevertheless, investigating if there is correspondence in the phylogenetic distribution of ITP-type and Gyc76C-type genes/proteins is a valuable approach to addressing this issue. Unfortunately, the evidence presented is rather limited in scope. Essentially, the authors report that they only found ITP-type and Gyc76C-type genes/proteins in protostomes, but not in deuterostomes. What is needed is a more fine-grained analysis at the species level within the protostomes. Thus, are there protostome species in which both ITP-type and Gyc76C-type genes/proteins have been lost? Furthermore, are there any protostome species in which an ITP-type gene is present but an Gyc76C-type gene is absent, or vice versa? If there are protostome species in which an ITP-type gene is present but a Gyc76C-type gene is absent or vice versa, this would argue against Gyc76C being a receptor for ITPa. In this regard, it is noteworthy that in Figure 2A there are two ITP-type precursors in C. elegans, but there are no Gyc76C-type proteins shown in the tree in Figure 2B. Thus, what is needed is a more detailed analysis of protostomes to investigate if there really is correspondence in the phylogenetic distribution of Gyc76C-type and ITP-type genes at the species level.

(3) The manuscript would benefit from a more comprehensive overview and discussion of published literature on Gyc76C in Drosophila, both as a basis for this study and for interpretation of the findings of this study.

Reviewer #1 (Public Review):

Summary:

In the work: "Endosomal sorting protein SNX4 limits synaptic vesicle docking and release" Josse Poppinga and collaborators addressed the synaptic function of Sortin-Nexin 4 (SNX4). Employing a newly developed in vitro KO model, with live imaging experiments, electrophysiological recordings, and ultrastructural analysis, the authors evaluate modifications in synaptic morphology and function upon loss of SNX4. The data demonstrate increased neurotransmitter release and alteration in synapse ultrastructure with a higher number of docked vesicles and shorter AZ. The evaluation of the presynaptic function of SNX4 is of relevance and tackles an open and yet unresolved question in the field of presynaptic physiology.

Strengths:

The sequential characterization of the cellular model is nicely conducted and the different techniques employed are appropriate for the morpho-functional analysis of the synaptic phenotype and the derived conclusions on SNX4 function at presynaptic site. The authors succeeded in presenting a novel in vitro model that resulted in chronical deletion of SNX4 in neurons. A convincing sequence of experimental techniques is applied to the model to unravel the role of SNX4, whose functions in neuronal cells and at synapses are largely unknown. The understanding of the role of endosomal sorting at the presynaptic site is relevant and of high interest in the field of synaptic physiology and in the pathophysiology of the many described synaptopathies that broadly result in loss of synaptic fidelity and quality control at release sites.

Weaknesses:

The flow of the data presentation is mostly descriptive with several consistent morphological and functional modifications upon SNX loss. The paper would benefit from a wider characterization that would allow us to address the physiological roles of SNX4 at the synaptic site and speculate on the underlying molecular mechanisms. In addition, due to the described role of SNX4 in autophagy and the high interest in the regulation of synaptic autophagy in the field of synaptic physiology, an initial evaluation of the autophagy phenotype in the neuronal SNX4KO model is important, and not to be only restricted to the discussion section.

Reviewer #3 (Public Review):

Summary:

The study aims to determine whether the endosomal protein SNX4 performs a role in neurotransmitter release and synaptic vesicle recycling. The authors exploited a newly generated conditional knockout mouse to allow them to interrogate the SNX4 function. A series of basic parameters were assessed, with an observed impact on neurotransmitter release and active zone morphology. The work is interesting, however as things currently stand, the work is descriptive with little mechanistic insight. There are a number of places where the data appear to be a little preliminary, and some of the conclusions require further validation.

Strengths:

The strengths of the work are the state-of-the-art methods to monitor presynaptic function.

Weaknesses:

The weaknesses are the fact that the work is largely descriptive, with no mechanistic insight into the role of SNX4. Further weaknesses are the absence of controls in some experiments and the design of specific experiments.

eLife assessment

This valuable study presents a series of results aimed at uncovering the involvement of the endosomal sorting protein SNX4 in neurotransmitter release. While the evidence supporting the conclusions is solid, the molecular mechanisms remain unclear, and the study would significantly benefit from additional experiments to strengthen its findings. This paper will be of interest to cell biologists and neurobiologists.

Reviewer #2 (Public Review):

Summary:

SNX4 is thought to mediate recycling from endosomes back to the plasma membrane in cells. In this study, the authors demonstrate the increases in the amounts of transmitter release and the number of docked vesicles by combining genetics, electrophysiology, and EM. They failed to find evidence for its role in synaptic vesicle cycling and endocytosis, which may be intuitively closer to the endosome function.

Strengths:

The electrophysiological data and EM data are in principle, convincing, though there are several issues in the study.

Weaknesses:

It is unclear why the increase in the amounts of transmitter release and docked vesicles happened in the SNX4 KO mice. In other words, it is unclear how the endosomal sorting proteins in the end regulate or are connected to presynaptic, particularly the active zone function.

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Résumé de la vidéo [00:00:00][^1^][1] - [00:28:40][^2^][2]:

Cette vidéo présente une conférence de Pierre-Michel Menger sur l'évaluation dans les sciences, les arts et les organisations, donnée en 2013-2014. Menger, directeur de recherche au CNRS et co-directeur de la Revue française de sociologie, aborde la sociologie des valeurs et leur impact sur la société.

Points forts: + [00:00:22][^3^][3] Introduction de Pierre-Michel Menger * Présentation de son parcours académique * Discussion sur ses thèmes de recherche antérieurs * Annonce du sujet de la conférence + [00:02:08][^4^][4] La sociologie des valeurs * Explication des différentes approches en sociologie * Présentation des enquêtes sur les valeurs en France et en Europe * Analyse de l'évolution des valeurs depuis les années 80 + [00:07:25][^5^][5] Travaux influents sur les valeurs * Référence aux recherches de Lazarsfeld, Rokeach et Inglehart * Discussion sur les méthodes empiriques et théories associées * Importance de l'universalisme dans l'étude des valeurs + [00:12:01][^6^][6] Méthodologie des enquêtes sur les valeurs * Détails sur la conception et l'objectif des enquêtes * Explication des techniques d'analyse factorielle * Construction d'échelles de valeurs à partir des données recueillies + [00:18:15][^7^][7] Limites des approches quantitatives * Discussion sur les défis de mesurer les valeurs de manière quantitative * Importance de l'interprétation des données collectées * Nécessité de compléter les enquêtes quantitatives par des méthodes qualitatives + [00:24:30][^8^][8] Étude systématique des valeurs * Présentation d'une étude approfondie sur la structure et l'évolution des valeurs * Utilisation d'analyses multivariées pour synthétiser les données * Examen des scores construits pour mesurer l'adhésion aux différentes valeurs Résumé de la vidéo [00:28:42][^1^][1] - [00:55:49][^2^][2] : La vidéo présente une conférence sur l'évaluation des valeurs traditionnelles et modernes dans les sociétés européennes, l'impact de l'affiliation religieuse sur ces valeurs, et la convergence des valeurs entre les générations. L'analyse factorielle révèle deux axes principaux : traditionalisme et intégration sociale, montrant des différences significatives entre les pays européens.

Points forts : + [00:28:42][^3^][3] Rôles sexuels et travail * Débat sur les rôles sexués dans la société française * Construction d'une échelle de valeurs sur le travail et l'économie de marché * Introduction de nouvelles questions sur l'immigration et l'environnement + [00:31:58][^4^][4] Maintien des valeurs traditionnelles * Permanence des différences de valeurs entre les sociétés nationales * Europe des valeurs : cohérence des valeurs européennes par rapport aux américaines * Impact fort de l'affiliation religieuse sur les valeurs + [00:37:02][^5^][5] Opposition entre traditionalisme et autonomie * Contredit les théories de la modernisation des années 50 et 60 * Évolution non linéaire des sociétés traditionnelles vers des valeurs modernes * Maintien d'un clivage des valeurs au sein des sociétés occidentales + [00:44:01][^6^][6] Convergence générationnelle autour des valeurs * Mouvement de convergence des valeurs entre les jeunes et les âgés * Retour à l'étiage des valeurs traditionnelles chez les jeunes * Diminution des valeurs traditionnelles chez les classes d'âge plus âgées + [00:47:29][^7^][7] Variabilité des valeurs traditionnelles selon les pays * Quatre groupes de pays avec des positions différentes dans l'univers des valeurs * Importance de l'appartenance nationale et religieuse dans la différenciation des valeurs * Convergence des valeurs malgré des positions nationales différentes + [00:52:19][^8^][8] Effet de l'affiliation religieuse sur l'intégration * L'appartenance religieuse comme facteur d'intégration ou de désintégration * Influence positive de l'appartenance musulmane et protestante, négative de l'orthodoxie * Effet intégrateur de la religion selon Durkheim et son maintien en Europe moderne Résumé de la vidéo [00:55:50][^1^][1] - [01:09:03][^2^][2]:

La partie 3 de la vidéo se concentre sur l'évaluation de l'intégration dans les sociétés à travers divers facteurs tels que la religion, la confiance dans les institutions, et l'individualisme. L'orateur, Pierre-Michel Menger, analyse les différences entre les affiliés religieux et les non-affiliés, ainsi que l'impact des différentes affiliations religieuses sur l'intégration sociale et les valeurs du travail.

Points forts: + [00:55:50][^3^][3] L'indicateur d'intégration * Un indicateur composite basé sur plusieurs variables * Examine comment l'intégration est liée à ces variables + [00:56:48][^4^][4] Affiliation religieuse et intégration * Les affiliés religieux adhèrent plus aux normes et sont moins individualistes * Les non-affiliés sont plus ouverts et participatifs + [00:58:34][^5^][5] Impact du protestantisme * Forte participation sociale et confiance élevée * Influence significative sur tous les aspects de l'intégration + [01:00:39][^6^][6] Valeurs du travail et économie de marché * Les protestants adhèrent fortement à l'économie de marché mais s'éloignent des valeurs du travail normatif * Les musulmans adhèrent à l'éthique du travail mais restent éloignés de l'universalisme économique

Résumé de la vidéo [00:00:00][^1^][1] - [00:23:42][^2^][2] : La vidéo présente une conférence de François Vatin, professeur de sociologie, sur l'évaluation du travail, ses valeurs et ses mesures. Il s'inspire de ses ouvrages sur la sociologie du travail et économique, et aborde la notion de travail à travers différentes perspectives historiques, philosophiques et techniques.

Points forts : + [00:01:15][^3^][3] Introduction de la conférence * Présentation de François Vatin et de ses travaux * Annonce du sujet de la conférence : le travail, ses valeurs et ses mesures + [00:06:09][^4^][4] Le travail comme valeur * Discussion sur la notion de travail et son évolution historique * Questionnement sur le travail en tant que valeur universelle + [00:14:01][^5^][5] Le paradigme énergétique * Analyse critique du paradigme énergétique dans la représentation du travail * Exploration de la connexion entre le travail en physique et en sociologie + [00:20:43][^6^][6] Le travail automatisé * Réflexion sur l'organisation du travail dans les industries automatisées * Distinction entre le travail prescrit et le travail réel Résumé de la vidéo [00:23:43][^1^][1] - [00:47:16][^2^][2]:

La vidéo présente une conférence sur l'évaluation de l'efficacité dans les sciences, les arts et les organisations, en se concentrant sur l'histoire et l'évolution des concepts de travail et d'énergie. Elle aborde la norme taylorienne, les théories économiques du travail, et la distinction entre le travail comme dépense d'énergie et comme création de valeur.

Points forts: + [00:23:43][^3^][3] La norme taylorienne et l'efficacité industrielle * Critique de la norme taylorienne comme obsolète * Distinction entre le coût d'immobilisation du capital et l'intensité du travail + [00:25:09][^4^][4] Concepts de travail et d'énergie * Évolution des concepts depuis la mécanique vers la thermodynamique * Le travail comme produit d'une force par une distance + [00:32:00][^5^][5] Le travail humain et la thermodynamique * Application des principes thermodynamiques à l'étude du travail humain * La machine humaine comme système thermodynamique + [00:38:00][^6^][6] Repenser le travail productif * La nécessité de comprendre le travail au-delà des contraintes sociales * Distinction entre le travail artistique et le travail salarié Résumé de la vidéo [00:47:19][^1^][1] - [01:02:33][^2^][2]:

Cette partie de la vidéo explore l'évaluation dans les sciences, les arts et les organisations, en se concentrant sur les tentatives historiques et contemporaines de mesurer le travail et la fatigue. Elle discute des limites des modèles universels et de l'importance de considérer les aspects sociaux et économiques dans l'évaluation.

Points forts: + [00:47:19][^3^][3] Les tentatives historiques de mesure * Échec des modèles universels de la fatigue au début du 20e siècle * Transition de la psychophysiologie à la psychosociologie du travail * Développement de l'ergonomie avec une approche prophylactique + [00:52:15][^4^][4] Les débats contemporains * Critique des modèles économiques simplistes * Succès des modèles épidémiologiques malgré leur régression intellectuelle * Importance de la valeur monétaire dans l'évaluation du travail + [00:57:04][^5^][5] La valorisation de la carcasse d'agneau * Exemple complexe de valorisation au-delà des modèles économiques standards * Interaction entre la reconnaissance de la qualité et la valorisation marchande * Nécessité d'une analyse fine pour comprendre le processus de valorisation

Test de note

We lose trust in messages. They don't prove authenticity. So peers talk "off the record". And we can't trust anything said.

Version Vectors does not represent equivocations / forks.

E.g., it conveys "Alice's 3rd" (is X remotely). Where Alice could have created equivocation, and locally Alice's 3rd event is Y.

Namely, less metadata.

Perhaps it could be mitigated via metadata compaction on sync and snapshots to garbage-collect history.

Résumé de la vidéo [00:00:20][^1^][1] - [00:22:58][^2^][2]:

Cette vidéo présente une conférence de Luc PELLISSIER sur la complexité des textes juridiques et leur traitement calculatoire. Il explore les notions de complexité en informatique théorique, la simplification et la codification du droit, et comment ces processus affectent l'accessibilité et l'intelligibilité de la loi. Il discute également des différentes époques de la codification en France et de l'impact de l'informatisation sur la législation.

Points forts: + [00:00:20][^3^][3] Introduction et contexte * Présentation du sujet et de l'importance de la complexité en informatique * Lien entre l'enseignement du droit et la complexité + [00:01:15][^4^][4] Complexité et droit * Exploration de la complexité en informatique théorique * Absence de la notion de complexité dans la littérature juridique + [00:04:27][^5^][5] Simplification et codification * Discussion sur la simplification du droit comme objectif constitutionnel * Relation entre simplification et codification dans l'histoire juridique française + [00:07:04][^6^][6] Les trois âges de la codification en France * Analyse des différentes méthodes de codification depuis le Consulat jusqu'à aujourd'hui * Impact de l'informatisation sur la codification et la législation + [00:17:19][^7^][7] Le droit comme système calculatoire * Proposition d'une nouvelle perspective sur le droit comme objet calculatoire * Exemple de la rémunération des heures complémentaires dans les universités françaises Résumé de la vidéo [00:23:00][^1^][1] - [00:44:29][^2^][2] : La vidéo présente une analyse détaillée des textes juridiques en tant qu'objets calculatoires, en explorant la complexité des modifications législatives et réglementaires, ainsi que leur impact sur la consolidation des textes de loi.

Points saillants : + [00:23:00][^3^][3] Classification des dispositions législatives * Différenciation entre les dispositions substantielles et celles qui appellent à l'action d'autres autorités. * Exemples concrets de modifications législatives et leur effet direct ou indirect sur le monde réel. * Discussion sur la complexité des textes qui modifient d'autres textes. + [00:25:02][^4^][4] Modifications précises vs. générales * Comparaison entre les modifications qui indiquent exactement quel texte changer et celles qui sont plus larges et moins spécifiques. * Impact des modifications générales sur la clarté et l'interprétation des textes. * Exemple de la réforme du Conseil national des universités et ses implications. + [00:31:02][^5^][5] Citations et références dans les textes législatifs * Utilisation de citations textuelles pour lier différents codes, comme le Code de l'éducation et le Code de la santé publique. * Problèmes posés par les modifications qui ne sont pas consolidées et laissent l'utilisateur final interpréter le texte. + [00:37:00][^6^][6] Conséquences des modifications non consolidées * Difficultés rencontrées par les utilisateurs du droit en raison de modifications automatiques non reflétées dans les textes consolidés. * Questions soulevées sur la simplification du droit et l'accessibilité des informations légales actuelles. Résumé de la vidéo [00:44:31][^1^][1] - [01:03:14][^2^][2]:

La partie 3 de la vidéo aborde la complexité du texte juridique en tant qu'objet calculatoire, en se concentrant sur les processus d'amendement, de consolidation et de codification. Luc Pellissier explore les défis théoriques et pratiques liés à la gestion des versions d'un texte de loi et la nécessité d'une approche formelle pour comprendre les modifications et leur impact sur la structure du texte.

Points forts: + [00:44:31][^3^][3] Théorie du versionnement * Discussion sur la gestion des versions d'un fichier ou d'une loi * Importance des modifications explicites pour une théorie correcte + [00:46:11][^4^][4] Analogie avec le logiciel libre * Comparaison entre le texte juridique et le code source d'un logiciel * Le rôle de la compilation dans la compréhension du logiciel + [00:49:00][^5^][5] Questions épistémologiques * Débat sur la neutralité du droit et l'impact des hypothèses de recherche * Lien entre la simplification du droit et la qualité démocratique + [00:55:01][^6^][6] Développement d'un logiciel de versionnage * Spécification du logiciel pour la gestion des versions du droit * Défis liés à la création d'une théorie propre pour un "code spaghetti" juridique + [01:01:03][^7^][7] Structure formelle du texte juridique * Visualisation du texte de loi comme un arbre avec des branches et des modifications * Impact des outils informatiques sur la précision des modifications législatives

https://www.youtube.com/watch?v=iTFM54VKKc4<br /> How To: Replace a Ribbon on Remington Quiet-Riter Typewriter by Typewriter Minutes

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eLife assessment

In this study, the authors developed a cell-based screening assay for the identification of small molecule inhibitors of nonsense-mediated decay (NMD). They used it to validate a novel small molecule SMG1 kinase inhibitor that inhibits NMD in cultured cells leading to the expression of neoantigens from NMD-targeted genes, and in vivo slows tumor growth of cells with a significant number of out-of-frame indel mutations. The conclusions are supported by convincing evidence, and the significance of this work consists in the development of a novel and very promising NMD inhibitor drug that acts as an inhibitor of the SMG1 NMD kinase and is suitable for use in animals. This is an important advance for the field, as previous NMD inhibitors were not specific, lacked efficacy, or were very toxic and hence not suitable for animal application.

Reviewer #1 (Public Review):

Summary:

This work identified new NMD inhibitors and tested them for cancer treatment, based on the hypothesis that inhibiting NMD could lead to the production of cancer neoantigens from the stabilized mutant mRNAs, thereby enhancing the immune system's ability to recognize and kill cancer cells. Key points of the study include:

• Development of an RNA-seq based method for NMD analysis using mixed isogenic cells that express WT or mutant transcripts of STAG2 and TP53 with engineered truncation mutations.

• Application of this method for a drug screen and identified several potential NMD inhibitors.

• Demonstration that one of the identified compounds, LY3023414, inhibits NMD by targeting the SMG1 protein kinase in the NMD pathway in cultured cells and mouse xenografts.

• Due to the in vivo toxicity observed for LY3023414, the authors developed 11 new SMG1 inhibitors (KVS0001-KVS0011) based on the structures of the known SMG1 inhibitor SMG1i-11 and the SMG1 protein itself.

• Among these, KVS0001 stood out for its high potency, excellent bioavailability, and low toxicity in mice. Treatment with KVS0001 caused NMD inhibition and increased presentation of neoantigens on MHC-I molecules, resulting in the clearance of cancer cells in vitro by co-cultured T cells and cancer xenografts in mice by the immune system.

These findings support the strategy of targeting the NMD pathway for cancer treatment and provide new research tools and potential lead compounds for further exploration.

Strengths:

The RNA-seq-based NMD analysis, using isogenic cell lines with specific NMD-inducing mutations, represents a novel approach for the high-throughput identification of potential NMD modulators or genetic regulators. The effectiveness of this method is exemplified by the identification of a new activity of AKT1/mTOR inhibitor LY3023414 in inhibiting NMD.

The properties of KVS0001 described in the manuscript as a novel SMG1 inhibitor suggest its potential as a lead compound for further testing the NMD-targeting strategies in cancer treatment. Additionally, this compound may serve as a useful research tool.

The results of the in vitro cell killing assay and in vivo xenograft experiments in both immuno-proficient and immune-deficient mice indicate that inhibiting NMD could be a viable therapeutic strategy for certain cancers.

Weaknesses:

The authors did not address the potential effects of NMD/SMG1 inhibitors on RNA splicing. Given that the transcripts of many RNA-binding proteins are natural targets of NMD, inhibiting NMD could significantly alter splicing patterns. This, in turn, might influence the outcomes of the RNA-seq-based method for NMD analysis and result interpretation.

While the RNA-seq-based approach offers several advantages for analyzing NMD, the effects of NMD/SMG1 inhibitors observed through this method should be confirmed using established NMD reporters. This step is crucial to rule out the possibility that mutations in STAG2 or TP53 affect NMD in cells, as well as to address potential clonal variations between different engineered cell lines.

The results from the SMG1/UPF1 knockdown and SMG1i-11 experiments presented in Figure 3 correlate with the effects seen for LY3023414, but they do not conclusively establish SMG1 as the direct target of LY3023414 in NMD inhibition. An epistatic analysis with LY3023414 and SMG1-knockdown is needed.

Reviewer #2 (Public Review):

Summary:

Several publications during the past years provided evidence that NMD protects tumor cells from being recognized by the immune system by suppressing the display of neoantigens, and hence NMD inhibition is emerging as a promising anti-cancer approach. However, the lack of an efficacious and specific small-molecule NMD inhibitor with suitable pharmacological properties is currently a major bottleneck in the development of therapies that rely on NMD inhibition. In this manuscript, the authors describe their screen for identifying NMD inhibitors, which is based on isogenic cell lines that either express wild-type or NMD-sensitive transcript isoforms of p53 and STAG2. Using this setup, they screened a library of 2658 FDA-approved or late-phase clinical trial drugs and had 8 hits. Among them they further characterized LY3023414, showing that it inhibits NMD in cultured cells and in a mouse xenograft model, where it, however, was very toxic. Because LY3023414 was originally developed as a PI3K inhibitor, the authors claim that it inhibits NMD by inhibiting SMG1. While this is most likely true, the authors do not provide experimental evidence for this claim. Instead, they use this statement to switch their attention to another previously developed SMG1 inhibitor (SMG1i-11), of which they design and test several derivatives. Of these derivatives, KVS0001 showed the best pharmacological behavior. It upregulated NMD-sensitive transcripts in cultured cells and the xenograft mouse model and two predicted neoantigens could indeed be detected by mass spectrometry when the respective cells were treated with KVS0001. A bispecific antibody targeting T cells to a specific antigen-HLA complex led to increased IFN-gamma release and killing of cancer cells expressing this antigen-HLA complex when they were treated with KVS0001. Finally, the authors show that renal (RENCA) or lung cancer cells (LLC) were significantly inhibited in tumor growth in immunocompetent mice treated with KVS0001. Overall, this establishes KVS0001 as a novel and promising ant-cancer drug that by inhibiting SMG1 (and therewith NMD) increases the neoantigen production in the cancer cells and reveals them to the body's immune system as "foreign".

Strengths:

The novelty and significance of this work consists in the development of a novel and - judging from the presented data - very promising NMD inhibiting drug that is suitable for applications in animals. This is an important advance for the field, as previous NMD inhibitors were not specific, lacked efficacy, or were very toxic and hence not suitable for animal application. It will be still a long way with many challenges ahead towards an efficacious NMD inhibitor that is safe for use in humans, but KVS0001 appears to be a molecule that bears promise for follow-up studies. In addition, while the idea of inhibiting NMD to trigger neoantigen production in cancer cells and so reveal them to the immune system has been around for quite some time, this work provides ample and compelling support for the feasibility of this approach, at least for tumors with a high mutational burden.

Main weaknesses:

There is a disconnect between the screen and the KVS0001 compound, that they describe and test in the second part of the manuscript since KVS0001 is a derivative of the SMG1 inhibitors developed by Gopalsamy et al. in 2012 and not of the lead compound identified in the screen (LY3023414). Because of high toxicity in the mouse xenograft experiments, the authors did not follow up LY3023414 but instead switched to the published SMG1i-11 drug of Gopalsamy and colleagues, a molecule that is widely used among NMD researchers for NMD inhibition in cultured cells. Therefore, in my view, the description of the screen is obsolete, and the paper could just start with the optimization of the pharmacological properties of SMG1i-11 and the characterization of KVS0001. Even though the screen is based on an elegant setup and was executed successfully, it was ultimately a failure as it didn't reveal a useful lead compound that could be further optimized.

Additional points:

- Compared to SMG1i-11, KVS0001 seems less potent in inhibiting SMG1 (higher IC50). It would therefore be important to also compare the specificity of both drugs for SMG1 over other kinases at the applied concentrations (1 uM for SMG1i-11, 5 uM for KVS0001). The Kinativ Assay (Fig. S13) was performed with 100 nM KVS0001, which is 50-fold less than the concentration used for functional assays and hence not really meaningful. In addition, more information on the pharmacokinetic properties and toxicology of KVS0001 would allow a better judgment of the potential of this molecule as a future therapeutic agent.

- On many figures, the concentrations of the used drugs are missing. Please ensure that for every experiment that includes drugs, the drug concentration is indicated.

- Do the authors have an explanation for why LY3023414 has a much stronger effect on the p53 than on the STAG2 nonsense allele (Figure 1B, S8), whereas emetine upregulates the STAG2 nonsense alleles more than the p53 nonsense allele (Figure S5). I find this curious, but the authors do not comment on it.

- While it is a strength of the study that the NMD inhibitors were validated on many different truncation mutations in different cell lines, it would help readers if a table or graphic illustration was included that gives an overview of all mutant alleles tested in this study (which gene, type of mutation, in which cell type). In the current version, this information is scattered throughout the manuscript.

- Lines 194 and 302: That SMG1i-11 was highly insoluble in the hands of the authors is surprising. It is unclear why they used variant 11j, since variant 11e of this inhibitor is widely used among NMD researchers and readily dissolves in DMSO.

- Line 296: The authors claim that they were able to show that LY3023414 inhibited the SMG1 kinase, which is not true. To show this, they would have for example to show that LY3023414 prevents SMG1-mediated UPF1 phosphorylation, as they did for KVS0001 and SMG1i-11 in Fig. 3F. Unless the authors provide this data, the statement should be deleted or modified.

Author response:

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

The authors have addressed my comments. As a final minor point, regarding comment 2, these condensates are likely viscoelastic rather than purely viscous. It is prudent to indicate that the data may refer to an apparent viscosity.

We added the following text to the manuscript to highlight the viscoelastic nature of ELP condensates, and the relationship of reported values with the steady state viscosity. “It is worth noting that the reported values, although related, may not quantitatively represent the steady-state viscosity. This discrepancy arises from the slow relaxation timescale inherent in ELP condensates with viscoelastic properties.”

eLife assessment

This important study investigates the structural organization of a series of diblock elastin-like polypeptide condensates. The methodology is highly compelling, as it combines multiscale simulations and fluorescence lifetime imaging microscopy experiments. The results increase our understanding of model biomolecular condensates.

Reviewer #1 (Public Review):

This is an interesting, informative, and well-designed study that combines theoretical and experimental methodologies to tackle the phenomenon of higher-resolution structures/substructures in model biomolecular condensates.

The authors have adequately addressed my previous concerns.

Reviewer #2 (Public Review):

Summary:

Latham A.P. et al. apply simulations and FLIM to analyse several di-block elastin-like polypetides and connect their sequence to the micro-structure of coacervates resulting from their phase-separation.

Strengths:

Understanding the molecular grammar of phase separating proteins and the connection with mesoscale properties of the coacervates is highly relevant. This work provides insights into micro-structures of coacervates resulting from di-block polypetides.

Weaknesses:

The results apply to a very specific architecture (di-block polypetides) with specific sequences.

the tension between Jewish thought and Enlightenment thought is the intertwined influence of Christianity; rationalism is considered consistent with Christianity but finds a tension with the basic teachings in Jewish sources; thus the connection between rationalism and natural religion becomes important; further, it seems that Enlightenment religion arrived at the natural conclusion that a rational person would become Christian

eLife assessment:

This work describes an easily implemented method for measuring solid food intake in Drosophila, which is necessary for studying the consumption of experimentally challenging diets, such as high-fat foods, as well as their nutritional impacts on the organism. It is a valuable technical contribution with solid evidence supporting the conclusions, filling a significant gap in the field.

Reviewer #1 (Public Review):

Summary:

Thakare et al propose a gravimetric method to evaluate feeding from solid food in Drosophila adults that can be used to evaluate the nutritional impact of high-fat food.

Strengths:

This method is new and fills a gap in the methods used in Drosophila research.

Weaknesses:

The data presented address a number of questions that are mainly interesting for people needing to reproduce such experiments. The work could be improved by being presented within a broader scope.

Reviewer #2 (Public Review):

Summary:

Thakare et al. present the DIETS assay for quantifying food consumption in adult Drosophila. DIETS measures food intake by weighing fly food before and after feeding. Technically, this is a well-designed, executed, and analyzed study. The interpretations are generally conservative and justified by the results. Although the results aren't always consistent with other published studies, which might reflect some of the unique conditions of the DIETS assay, the technique can clearly distinguish between some expected differences in food intake. Although lifespan is shortened in the DIETS chamber, the method seems robust for various time scales up to a week. DIETS adds another useful and versatile tool for fly researchers interested in studying feeding behavior.

Strengths:

The authors test various conditions, including food presentation, surface area, and humidity (by changing the food cup distance to an agar base) to demonstrate an optimized technique for quantifying consumption. Under these conditions, evaporation is generally limited to <10%.

The authors use DIETS to validate diverse feeding paradigms, including the published effects of temperature, food dilution, and intermittent fasting on food intake.

Weaknesses:

The studies to optimize and test the DIETS assay are technically rigorous and well-designed. However, the results reveal some weaknesses or potential caveats of the assay. As highlighted below, how much nutrition flies are actually obtaining may be misestimated due to vapor diffusion, and crowding/competition for food. This appears largely acceptable though, since the 'group' measurement can still clearly distinguish between expected feeding differences under different conditions, but it likely reduces accuracy, which may be important in some studies, and probably nullifies the effectiveness of using DIETS to restrict caloric intake.

It is my understanding that flies suck out nutrients from the medium, leaving behind the agar/cornmeal matrix. This seems consistent with the images in Figure S2B, where the spheroidal medium in the food cup maintains its shape as it shrinks, but there don't seem to be any pits or holes from fly consumption. Given that flies in DIETS consume a significant portion of the available food, it seems that the food concentration at the medium surface may be changing throughout the experiment. This may also make it challenging to use other common fly food ingredients, such as cornmeal, much of which is indigestible.

Similarly, vapor diffusion is expected between the agar bed and food cup (which the authors observed; in line 385), which may further affect assay accuracy, especially in comparisons between foods with different osmolarity.

In DIETS, increased feeding is observed with increased flies per chamber, but this is not observed in other techniques, such as EX-Q (Wu et al. 2020). It is unclear whether sensitivity to adult density is a DIETS-specific feature, or if adult density instead directly affects food intake estimates using DIETS (e.g., by affecting chamber humidity).

In another example, there is a ~300% difference in absolute feeding when the DIETS food cup is presented in different formats (Figure 3C). Again, it is unclear whether food presentation has an inherently greater effect in DIETS, or if the measurements themselves are highly sensitive to the environment.

Although the control of total food mass given to the animals is a novel feature of the assay, the likely differences in nutrient intake between individuals (and shortened lifespan) in a DIETS chamber makes this a challenging method to use to study caloric restriction. The shortened lifespan likely stems from the high adult density per vial, which has been explored in previous publications (e.g., Pearl in the 1920s; Mueller in the 1990s).

eLife assessment

This study reports some useful information on the mechanisms by which a high-fat diet induces arrhythmias in the model organism Drosophila. Specifically, the authors propose that adipokinetic hormone (Akh) secretion is increased with this diet, and through binding of Akh to its receptor on cardiac neurons, arrhythmia is induced. The presented data, however, incompletely support the conclusions, with a number of concerns identified, such as the need for editorial clarifications, issues with experimental design (including additional control experiments), and over or misinterpretation of some of the experimental data. Nonetheless, some of the data will be helpful to those who wish to extend the research to a more complex model system, such as the mouse.

Reviewer #1 (Public Review):

Summary:

In the manuscript submission by Zhao et al. entitled, "Cardiac neurons expressing a glucagon-like receptor mediate cardiac arrhythmia induced by high-fat diet in Drosophila" the authors assert that cardiac arrhythmias in Drosophila on a high-fat diet are due in part to adipokinetic hormone (Akh) signaling activation. High-fat diet induces Akh secretion from activated endocrine neurons, which activate AkhR in posterior cardiac neurons. Silencing or deletion of Akh or AkhR blocks arrhythmia in Drosophila on a high-fat diet. Elimination of one of two AkhR-expressing cardiac neurons results in arrhythmia similar to a high-fat diet.

Strengths:

The authors propose a novel mechanism for high-fat diet-induced arrhythmia utilizing the Akh signaling pathway that signals to cardiac neurons.

Weaknesses:

Major comments:

(1) The authors state, "Arrhythmic pathology is rooted in the cardiac conduction system." This assertion is incorrect as a blanket statement on arrhythmias. There are certain arrhythmias that have been attributable to the conduction system, such as bradycardic rhythms, heart block, sinus node reentry, inappropriate sinus tachycardia, AV nodal reentrant tachycardia, bundle branch reentry, fascicular ventricular tachycardia, or idiopathic ventricular fibrillation to name a few. However the etiological mechanism of many atrial and ventricular arrhythmias, such as atrial fibrillation or substrate-based ventricular tachycardia, are not rooted in the conduction system. The introduction should be revised to reflect a clear focus on atrial fibrillation (AF). In addition, AF susceptibility is known to be modulated by autonomic tone, which is topically relevant to this manuscript.

(2) The authors state that "HFD led to increased heartbeat and an irregular rhythm." In representative examples shown, HFD resulted in pauses, slower heart rate, and increased irregularity in rhythm but not consistently increased heart rate (Figures 1B, 3A, and 4C). Based on the cited work by Ocorr et al (https://doi.org/10.1073/pnas.0609278104), Drosophila heart rate is highly variable with periods of fast and slow rates, which the authors attributed to neuronal and hormonal inputs. Ocorr et al then describe the use of "semi-intact" flies to remove autonomic input to normalize heart rate. Were semi-intact flies used? If not, how was heart rate variability controlled? And how was heart rate "increase" quantified in high-fat diet compared to normal-fat diet? Lastly, how does one measure "arrhythmia" when there is so much heart rate variability in normal intact flies?

(3) The authors state, "to test whether the HFD-induced increase in Akh in the APC affects APC neuron activity, we used CaLexA (https://doi.org/10.3109/01677063.2011.642910)." According to the reference, CaLexA is a tool to map active neurons and would not indicate, as the authors state, whether Akh affects APC neuron activity specifically. It is equally possible that APC neurons may be activated by HFD and produce more Akh. Please clarify this language.

(4) Are the AkhR+ neurons parasympathetic or sympathetic? Please provide additional experimentation that characterizes these neurons. The AkhR+ neurons appear to be anti-arrhythmic. Please expand the discussion to include a working hypothesis of the overall findings on Akh, AkhR, and AkhR+ neurons.

(5) The authors state, "Heart function is dependent on glucose as an energy source." However, the heart's main energy source is fatty acids with minimal use of glucose (doi: 10.1016/j.cbpa.2006.09.014). Glucose becomes more utilized by cardiomyocytes under heart failure conditions. Please amend/revise this statement.

Reviewer #2 (Public Review):

This manuscript explores mechanisms underlying heart contractility problems in metabolic disease using Drosophila as a model. They confirm, as others have demonstrated, that a high-fat diet (HFD) induces cardiac problems in flies. They showed that a high-fat diet increased Akh mRNA levels and calcium levels in the Akh-producing cells (APC), suggesting there is increased production and release of this hormone in a HFD context. When they knock down Akh production in the APCs using RNAi they see that cardiac contractility problems are abolished. They similarly show that levels of the Akh receptor (Akhr) are increased on a HFD and that loss of Akhr also rescues contractility problems on a HFD.

One highlight of the paper was the identification of a pair of neurons that express a receptor for the metabolic hormone Akh, and showing initial data that these neurons innervate the cardiac muscle. They then overexpress cell death gene reaper (rpr) in all Akhr-positive cells with Akhr-GAL4 and see that cardiac contractility becomes abnormal.

However, this paper contains several findings that have been reported elsewhere and it contains key flaws in both experimental design and data interpretation. There is some rationale for doing the experiments, and the data and images are of good quality. However, others have shown that HFD induces cardiac contractility problems (Birse 2010), that Akh mRNA levels are changed with HFD (Liao 2021) that Akh modulates cardiac rhythms (Noyes 1995), so Figures 1-4 are largely a confirmation of what is already known. This limits the overall magnitude of the advances presented in these figures. Overall, the stated concerns limit the impact of the manuscript in advancing our understanding of heart contractility.

Reviewer #3 (Public Review):

Zhao et al. provide new insights into the mechanism by which a high-fat diet (HFD) induces cardiac arrhythmia employing Drosophila as a model. HFD induces cardiac arrhythmia in both mammals and Drosophila. Both glucagon and its functional equivalent in Drosophila Akh are known to induce arrhythmia. The study demonstrates that Akh mRNA levels are increased by HFD and both Akh and its receptor are necessary for high-fat diet-induced cardiac arrhythmia, elucidating a novel link. Notably, Zhao et al. identify a pair of AKH receptor-expressing neurons located at the posterior of the heart tube. Interestingly, these neurons innervate the heart muscle and form synaptic connections, implying their roles in controlling the heart muscle. The study presented by Zhao et al. is intriguing, and the rigorous characterization of the AKH receptor-expressing neurons would significantly enhance our understanding of the molecular mechanism underlying HFD-induced cardiac arrhythmia.

Many experiments presented in the manuscript are appropriate for supporting the conclusions while additional controls and precise quantifications should help strengthen the authors' augments. The key results obtained by loss of Akh (or AkhR) and genetic elimination of the identified AkhR-expressing cardiac neurons do not reconcile, complicating the overall interpretation.

It is intriguing to see an increase in Akh mRNA levels in HFD-fed animals. This is a key result for linking HFD-induced arrhythmia to Akh. Thus, demonstrating that HFD also increases the Akh protein levels and Akh is secreted more should significantly strengthen the manuscript.

The experiments employing an AkhR null allele nicely demonstrate its requirement for HFD-induced cardiac arrhythmia. Depletion of Akh in Akh-expressing cells recapitulates the consequence of AkhR knockout, supporting that both Akh and its receptor are required for HFD-induced cardiac arrhythmia. Given that RNAi is associated with off-target effects and some RNAi reagents do not work, testing multiple independent RNAi lines is the standard procedure. It is also important to show the on-target effect of the RNAi reagents used in the study.

The most exciting result is the identification of AkhR-expressing neurons located at the posterior part of the heart tube (ACNs). The authors attempted to determine the function of ACNs by expressing rpr with AkhR-GAL4, which would induce cell death in all AkhR-expressing cells, including ACNs. The experiments presented in Figure 6 are not straightforward to interpret. Moreover, the conclusion contradicts the main hypothesis that elevated Akh is the basis of HFD-induced arrhythmia. The results suggest the importance of AkhR-expressing cells for normal heartbeat. However, elimination of Akh or AkhR restores normal rhythm in HFD-fed animals, suggesting that Akh and AkhR are not important for maintaining normal rhythms. If Akh signaling in ACNs is key for HFD-induced arrhythmia, genetic elimination of ACNs should unalter rhythm and rescue the HFD-induced arrhythmia. An important caveat is that the experiments do not test the specific role of ACNs. ACNs should be just a small part of the cells expressing AkhR. The experiments presented in Figure 6 cannot justify the authors' conclusion. Specific manipulation of ACNs will significantly improve the study. Moreover, the main hypothesis suggests that HFD may alter the activity of ACNs in a manner dependent on Akh and AkhR. Testing how HFD changes calcium, possibly by CaLexA (Figure 2) and/or GCaMP, in wild-type and AkhR mutants could be a way to connect ACNs to HFD-induced arrhythmia. Moreover, optogenetic manipulation of ACNs will allow for specific manipulation of ACNs, which is crucial for studying the specific role of ACNs in controlling cardiac rhythms.

Interestingly, expressing rpr with AkhR-GAL4 was insufficient to eliminate both ACNs. It is not clear why it didn't eliminate both ACNs. Given the incomplete penetrance, appropriate quantifications should be helpful. Additionally, the impact on other AhkR-expressing cells should be assessed. Adding more copies of UAS-rpr, AkhR-GAL4, or both may eliminate all ACNs and other AkhR-expressing cells. The authors could also try UAS-hid instead of UAS-rpr.

Résumé de la vidéo [00:00:00][^1^][1] - [00:17:29][^2^][2] :

Cette vidéo présente une analyse approfondie de la méritocratie, abordant les controverses et les implications sociologiques. Pierre-Michel Menger discute des enjeux liés à l'éducation, l'inégalité, et la mobilité sociale, tout en examinant les défis posés par les hiérarchies et les stratifications dans la société moderne.

Points forts : + [00:00:26][^3^][3] Clôture du colloque * Importance de l'analyse empirique des inégalités * Rôle de l'éducation dans la méritocratie * Impact de la massification scolaire + [00:02:55][^4^][4] Critiques de l'enseignement supérieur * Valeur et conditions d'admission dans les filières sélectives * Effet des diplômes sur la réussite sociale * Débat sur la justice proportionnelle + [00:06:49][^5^][5] Inégalités et mécanismes de 'winner takes all' * Composition complexe des 1% les plus riches * Influence du capitalisme financier et des hautes technologies * Rôle des professions libérales et de l'entertainment + [00:10:21][^6^][6] Défis de l'ascension sociale * Importance de l'égalisation des chances * Interdépendance des sociétés et impact de la migration * Enjeux de la compétition mondiale et de l'innovation technologique

Résumé de la vidéo [00:00:00][^1^][1] - [00:31:31][^2^][2]:

La vidéo présente une conférence de Pierre-Michel Menger sur la méritocratie, ses analyses et controverses. Menger explore les théories libérales de la justice sociale, la non-méritocratie et l'antiméritocratie dans ces théories, et les différences entre les représentations spontanées de la justice sociale et les théories influentes.

Points forts: + [00:00:26][^3^][3] Introduction et contexte * Menger revisite ses recherches sur la justice sociale * Il aborde les théories de Friedrich Hayek et Robert Nozick + [00:02:33][^4^][4] Théories non méritocratiques * Menger explique pourquoi les théories influentes sur la justice sociale ne sont pas méritocratiques * Il discute de l'ouvrage "Le sacrifice et l'envie" et de son analyse du libéralisme + [00:03:18][^5^][5] Études de cas culturelles * Menger présente trois études de cas du Brésil, de la France du 17e siècle et de la société contemporaine * Il examine les attitudes envers le mérite et la chance dans différentes cultures + [00:15:15][^6^][6] Le mérite dans la société égalitariste * Menger discute des composants du mérite : les talents innés et les efforts personnels * Il souligne la contradiction dans les sociétés égalitaristes concernant la reconnaissance du mérite + [00:25:52][^7^][7] La théorie de la justice de John Rawls * Menger analyse la théorie de la justice de Rawls comme une théorie anti-méritocratique * Il détaille les principes de justice de Rawls et leur application dans la société Résumé de la vidéo [00:31:34][^1^][1] - [00:47:43][^2^][2]:

Cette partie de la vidéo discute de la méritocratie, analysant les idées de Pierre-Michel Menger sur les circonstances sociales de la naissance et les talents naturels. Il explore la notion de mérite moral et les efforts individuels, en se référant aux théories de John Rawls sur la justice et l'égalité des chances.

Points saillants: + [00:31:34][^3^][3] Circonstances de la naissance et mérite * Les circonstances sociales et les gènes sont arbitraires moralement * Aucun mérite ne s'attache aux talents et capacités naturels + [00:32:08][^4^][4] Les efforts et les talents selon Rawls * Même les efforts pour cultiver les dons ne sont pas méritoires * Les talents et les efforts doivent être neutralisés dans la sphère morale + [00:36:04][^5^][5] Principe de différence de Rawls * Les inégalités justes maximisent la part du moins avantagé * La corrélation entre talents et richesse sociale n'est pas méritocratique + [00:42:02][^6^][6] Menaces des passions et justice sociale * Les passions négatives menacent la théorie de la justice * La société juste nécessite l'égalité des chances et la reconnaissance publique

Résumé de la vidéo [00:00:00][^1^][1] - [00:24:39][^2^][2] : La vidéo présente une conférence sur la méritocratie dans le domaine des mathématiques, analysant les autobiographies de mathématiciens célèbres et leur perception du mérite. Elle explore la hiérarchie interne et les controverses liées à la reconnaissance du talent et du travail dans la communauté mathématique.

Points forts : + [00:00:26][^3^][3] Introduction à la méritocratie * Présentation du sujet et de l'approche méthodologique * Discussion sur la méritocratie à travers des autobiographies de mathématiciens + [00:01:15][^4^][4] Citations et contexte historique * Citation de Sylvia Nazar sur la culture mathématique à Princeton après-guerre * Analyse de la hiérarchie et du prestige dans la communauté mathématique + [00:02:35][^5^][5] La rigueur des mathématiques * Les mathématiques comme discipline scolaire, universitaire et domaine de recherche * L'importance de la rigueur, de l'abstraction et de la sélectivité + [00:10:37][^6^][6] Utilisation du terme 'mérite' * Examen de la polysémie du terme 'mérite' dans les textes mathématiques * Discussion sur la valeur et la reconnaissance dans la communauté mathématique + [00:11:09][^7^][7] Influence familiale et éducative * Rôle des antécédents familiaux et des trajectoires éducatives des mathématiciens * Impact de l'origine familiale sur l'orientation vers les mathématiques + [00:19:29][^8^][8] La communauté mathématique et la méritocratie * Représentation de la communauté mathématique comme méritocratique * Critique des conséquences négatives d'une vision stricte de la méritocratie Résumé de la vidéo [00:24:41][^1^][1] - [00:36:00][^2^][2]:

La vidéo explore les analyses et controverses autour de la notion de méritocratie dans le domaine mathématique. Pierre-Michel Menger discute des effets de la hiérarchisation basée sur le mérite, notamment la fatuité et le mépris envers les autres, et comment cela peut conduire à une compétition contre-productive et à l'ignorance de certains collègues ou étudiants.

Points forts: + [00:24:41][^3^][3] La méritocratie et ses conséquences * Discussion sur la hiérarchisation par le mérite * Effets d'identification et de connivence entre mathématiciens * Fatuité et mépris comme résultats de la hiérarchisation + [00:27:33][^4^][4] L'idéologie du mérite * Le mérite comme leurre cachant le mépris * Effets contre-productifs sur le développement des mathématiques * Rejet de la compétition par Grothendieck + [00:32:01][^5^][5] La confiance en soi et la modestie * Comparaison des thèmes de la fatuité et de la confiance en soi * Schwartz sur l'importance de la compétition pour révéler les talents * Grothendieck sur la réflexion existentielle et le rejet de la compétition + [00:33:06][^6^][6] Les antidotes à la méritocratie * La présence physique comme rappel des réalités essentielles * L'amitié indépendante du mérite et le respect de la personne * La règle de publication scientifique comme correctif à l'attitude méritocratique

Résumé de la vidéo [00:00:00][^1^][1] - [00:23:43][^2^][2]:

Cette vidéo présente une analyse approfondie de la méritocratie dans le football, en se concentrant sur la concurrence que subissent les jeunes footballeurs, l'effet de l'âge relatif, et les aspects financiers de la carrière des footballeurs.

Points forts: + [00:00:26][^3^][3] La concurrence chez les jeunes footballeurs * La sélection rigoureuse dès le jeune âge * L'importance du mois de naissance sur les chances de devenir footballeur * La durée de carrière et l'effet super star + [00:15:01][^4^][4] L'effet d'âge relatif * L'avantage des individus nés en début d'année * Les disparités de développement physique et les prophéties auto-réalisatrices * L'impact sur la probabilité de devenir footballeur professionnel + [00:19:50][^5^][5] Les aspects financiers de la carrière des footballeurs * La segmentation du marché du travail et l'effet super star * L'impact de l'arrêt Bosman sur la mobilité et les salaires des joueurs * La corrélation entre les salaires et les performances sportives Résumé de la vidéo [00:23:45][^1^][1] - [00:43:04][^2^][2]:

Cette partie de la vidéo aborde la notion de méritocratie dans le contexte du football professionnel, en analysant les salaires des joueurs et les dynamiques du marché. Pierre-Michel Menger discute des inégalités salariales entre les joueurs, l'impact des budgets des ligues sur les salaires moyens, et la distinction entre les joueurs moyens, les bons joueurs, et les superstars.

Points forts: + [00:23:45][^3^][3] Dynamiques du marché du football * Discussion sur le fonctionnement du marché * Explication des contrats courts et des salaires variables * Mention de la dualité du marché avec des exemples concrets + [00:26:28][^4^][4] Salaires moyens dans différents championnats * Comparaison des salaires moyens en fonction des budgets des ligues * Analyse de la distribution des salaires et de l'inégalité * Exemple de la Ligue 1 et de la Major League Soccer (MLS) + [00:29:14][^5^][5] Croissance des inégalités salariales * Évolution des salaires depuis les années 90 * Augmentation des inégalités entre les joueurs * Discussion sur la part des salaires des joueurs les mieux payés + [00:33:29][^6^][6] Théorie économique des superstars * Présentation de l'économie des superstars et de l'effet Pavarotti * Analyse de l'impact du talent et de la popularité sur les salaires * Exemples historiques et contemporains pour illustrer les concepts

where the programmers are fungible - not the software -

they produce

If te software could be fungible capable of organic

re - usable - mixable - sumable

digital executable artifats

auto-poietic co-evolving body of organic growth

we would not need big teams

and the realm of software itself will become - an Open Sea of - effective articulation of capabilities

that are tinkerable capable of self-organization and autopoietic synthetic symmathetic growth living everrgreen and permanent

a systems that are lifelike co-evol

at scale in the long tail of the internet

The highest possible praise

Let's overcome the "Curse of LISP"

Amazon care about money; they don't care about humans.

https://www.repubblica.it/economia/2024/05/02/news/gazprom_bilancio_in_rosso_vendite_gas_gnl_europa-422805952/?ref=RHLF-BG-P2-S1-T1

https://taz.de/Gesundheitliche-Folgen-der-Klimakrise/!6003297/

https://taz.de/Petersberger-Klimadialog/!6007095/

Max, Kim. “Seoul Has Less Homelessness than L.A. but a Huge Housing Crisis - Los Angeles Times.” Los Angeles Times, April 11, 2023.

https://www.theguardian.com/us-news/article/2024/may/02/youth-climate-lawsuit-juliana-appeals-court

Google intellectual firepower

boldy go where no programmer has gone before

and transcend programming itself

intellectual firepower CERN

Join Deborah Halpern's sculpture workshop to unlock your creativity. Under her guidance, explore sculpting techniques and unleash your imagination to craft vibrant, dynamic forms. Whether you're a novice or an experienced artist, Halpern's workshop offers a welcoming environment for learning and expression, fostering artistic growth and inspiration.

This is an interesting new entry -- keep an eye on Tracy!

虚拟币 wp 收款渠道

Karya rekam baik cetak maupun digital, termasuk di dalamnya film, multimedia, sinetron adalah produk budaya yang perlu diarsipkan.

Poin ini relevan dengan RUU Penyiaran terkait penyedia konten maupun penyelenggara platform (masukkan pasal-pasal terkait)

Dari keseluruhan dokumen Rencana Induk Ekonomi Kreatif, "televisi dan radio" hanya disebut sekali. Ini menunjukkan adanya 'policy blind spot' dalam tata kelola ekosistem penyiaran. Ini sekaligus menjadi hub antara UU Ekonomi Kreatif dengan RUU Penyiaran yang sedang dibahas DPR.

well

Girl not the lack of a square or something to indicate the proof is over like be real...

So what was all the hoopla about RecA-coating about exactly...

Maybe look at this idk

can probably give examples / draw diagrams here

OK so we're finally getting to the stuff from the first paper...

Expose them

Yet more examples!

Might just have to say like ok usually the DNA forms rational tangles

Seems like it would just be called a tangle idk

This seems to be like...the worst way to describe this idk

so the unit circle...be serious

Ok I would give more clear pictures of how these are braided together

Mahdi, Balali-Mood, Kobra Naseri, Zoya Tahergorabi, Mohammad Reza Khazdair, and Mahmood Sadeghi. “Toxic Mechanisms of Five Heavy Metals: Mercury, Lead, Chromium, Cadmium, and Arsenic.” Frontiers in Pharmacology 12 (April 13, 2021): 643972. https://doi.org/10.3389/fphar.2021.643972.

S. W., Jeong et al.

S. W., Jeong, Y. -H. Wu, Y. C. Cho, and S. W. Ji. “Flow Behavior and Mobility of Contaminated Waste Rock Materials in the Abandoned Imgi Mine in Korea.” Geomorphology 301 (January 15, 2018): 79–91. https://doi.org/10.1016/j.geomorph.2017.10.021.

cause

Résumé de la vidéo [00:00:00][^1^][1] - [00:27:48][^2^][2] : La vidéo présente une conférence de Pierre-Michel Menger sur la méritocratie, analysant les sociétés islamiques, indiennes et chinoises du 18ème et 19ème siècle. Il explore les systèmes de classe et de gouvernance, contrastant les sociétés d'ordre européennes avec les structures non héréditaires de l'Orient et la méritocratie apparente de la Chine.

Points saillants : + [00:00:27][^3^][3] Introduction de la conférence * Présentation par Pierre-Michel Menger * Annonce du sujet : Orientalisme et méritocratie * Contextualisation historique et géographique + [00:01:37][^4^][4] L'orientalisme et son émergence * Origines européennes au 17ème siècle * Influence des érudits et des voyageurs * Impact des jésuites sur la pensée européenne + [00:08:04][^5^][5] Le système mamelouk * Structure inverse de la noblesse européenne * Mamelouks comme esclaves et soldats * Interdiction de succession pour les enfants de mamelouks + [00:18:59][^6^][6] Les jésuites et le système d'examen chinois * Découverte du système méritocratique chinois * Comparaison avec l'éducation européenne * Influence sur la pensée des Lumières + [00:27:22][^7^][7] Influence sur l'administration moderne * Création de l'école des ponts et chaussées en France * Évolution des concours et examens * Parallèles avec le système des mandarins chinois Résumé de la vidéo [00:27:51][^1^][1] - [00:44:31][^2^][2]:

La vidéo présente une analyse approfondie du concept de méritocratie à travers le prisme du système des castes en Inde. Elle explore l'histoire et l'évolution des castes, leur signification sociale et religieuse, et la manière dont elles ont été interprétées par les colonisateurs européens et les penseurs occidentaux.

Points forts: + [00:27:51][^3^][3] Origines du terme 'caste' * Introduction du mot 'casta' par les Portugais * Distinction entre les quatre castes principales + [00:31:37][^4^][4] Interprétation des castes * La caste vue comme un phénomène religieux et social * Influence des missionnaires et des penseurs comme Max Müller et Max Weber + [00:35:02][^5^][5] Impact des textes anciens * Les lois de Manou et leur influence sur la perception des castes * La collaboration des Brahmanes avec les Britanniques + [00:40:44][^6^][6] Comparaisons contemporaines * Parallèles entre les castes et les structures sociales occidentales * Utilisation du terme 'Brahmane' pour décrire des élites dans d'autres contextes