Children were treated as badly as adults.

Slaves had unlivable conditions; the bare minimum was not provided.

Colonel Lloyd's home is at the top of the slave hierarchy, hence the usage of the term "home plantation".

The masters would sell their own children to appease their wives. White women expressed jealousy toward these enslaved children because of sexual envy and the fear of favoritism upon the children.

The separation of a mother and her child happens before the child turns one. This prevents them from bonding and emotional connections.

This portion of his excerpt expresses how the poor should be grateful towards the rich for giving them a better life. Almost as if comparing them to gods whoms purpose is to become as rich as possible so that giving to the poor has no negative impact on them. For example Andrews quote "A man who dies rich dies disgraced".

algorithms are instructions that will solve the problem, and it may solve as a general solution

Tin Tin, Brains, Alan and I always like to play in the mud and get caked from head to toe in the stuff, always laughing like crazy and having mud fights - that is, if the buffalo don't mind!

Tin Tin always wanders unclothed as nature intended through the jungle. She snuggles down to sleep beside her animal friends, and she always loves to play with them.

If Tin Tin were living and breathing under the sea, she'd befriend a dolphin and go on many sensual and wonder-filled adventures deep under the water.

I always snuggle near Lady Penelope's body whenever we are treated to a nice pampering session at the spa

The jungle is so beautiful to Tin Tin, she likes to swim in rivers and lakes and play with her animal friends.

A gaggle of friendly characters: Kipper, Franklin, Little Bear, Llama Llama, Bear, Winnie The Pooh & Piglet, Oswald, Blue (Blue's Room), Percy The Park Keeper, Noddy, Postman Pat, Fireman Sam, Pingu, Joshua Jones, Oakie Doke

Bob The Builder: Bob's Big Plan

Playing with the kids as part of a tribal community

Veggietales: The Ballad Of Little Joe

important

for - progress trap - psychedelics

for - quote - the worst kind of pseudoscience is this kind of dogmatic scientism

for - what's it like to be a bat? - what's it like to be a plant? - it begs the most general question: - what's it like to be the other?

for - adjacency - reality is incredibly rich and powerful - poverty mentality - What he says here is in line with the Buddhist concept of poverty mentality, in which we cannot believe we are the very happiness and richness we have been searching for - we've been on a life goal of searching for enlightenment our whole lives, - not realizing that we are it

for - citation - book - Patrick Harpur - Daimonic Reality: A field guide to the otherworld - to - book Daimonic Reality: A field guide to the otherworld - Patrick Harpur - adjacency - realm between fact and fiction - Donald Hoffman interview - Deep Humanity - self / other gestalt - the Indyweb - physiosphere - symbolosphere - this is exactly the intetwingledness of - the subject and the object - consciousness and phenomenal reality - Deep Humanity - the individual / collective gestalt - the self / other gestalt - symbolosphere / physiosphere - to - Youtube - The Diary of a CEO - Donald Hoffman interview - https://hyp.is/go?url=https%3A%2F%2Fwww.youtube.com%2Fwatch%3Fv%3DW0vTZrZny6A&group=world - internet Archive - https://hyp.is/egkk-IvhEfCpxyM0mIOqLA/archive.org/details/daimonicrealityf0000harp - Patrick Harpur - book webpage - https://hyp.is/1iPUDovhEfC4PStyYJoYnQ/www.harpur.org/x1Daimonic.htm

for - citation - film - Werner Hertzog - Ecstatic Truth vs documentary

for - compare - technocratic superhuman - vs - innate superhuman

for - adjacency - alternative - transhumanism - Deep Humanity

for - citation - book - More Everything Forever - Adam Becker - to - book - More Everything Forever - Adam Becker - https://hyp.is/rGo8uovJEfCovHv7HL0Rgg/freelanceastrophysicist.com/

for - key inisght - book - Surveillance Capitalism - Once capitalism conquers the physical, it has to expand inwards to conquer (our inner word)

for - citation - book - The Age of Surveillance Capitalism - Shoshana Zuboff - to book - The Age of Surveillance Capitalism - Shoshana Zuboff - https://hyp.is/W4il7ovIEfCh30P8h49-Hg/www.hbs.edu/faculty/Pages/item.aspx?num=56791

for - progress trap - transhumanism -quote - transhumanism - mandating change on the human species

for - adjacency- transhumanism - consciousness - quote - dark force of transhumanism - The transhumanist agenda to me is a very dark force. - It's a force that wants to extinguish humankind while telling us it's going to be great. - Consciousness is going to be key here

for - metaphor - hard problem of consciousness - trick interview question

for - quote - We shouldn't have called it the hard problem of consciousness - By calling it a hard problem, - Yeah. Hard problems you can still solve and we shouldn't have called it a hard problem. - We should have said okay materialism just died.

Comment : insightful observation!

for - quote - materialism crime scene - Alex Gomez- Marin - the hard problem was I would say a covering up of the crime scene where materialism had died

what are some practical ways to incorporate cultural understanding into the planning of service or treatment?

amazing how the adaptation approach can help clinicians focus on cultural awareness in their training.

does it lead to better connections with clients from diverse bachgrounds?

this is true, CBT is soo defferent for each person, especially for asians. the therapy can really change in its relevance and effectiveness for them.

its really interesting how clinicians trained in the coordinated strategic action models use more cbt techniques with families.

supporting each other in these types of fields helps everyone, supporting one another is very crucial its not only enriches the field but also ensures that care is more effective for diverse communities.

i think its crucial to recognize that traditional cbt often emphasizws individual independec, whihc might not resonate with cultures that prioritize community and togetherness.

what specific cultural adaptation or training might enhance the efffectivenss of cbt for enthic minorities?

its effectiveness in treating various mental health issues.

Carlton's 'Bring The Magic Home' - Thunderbirds, Annabelle's Wish, A Monkey's Tale, Rudolph The Red Nosed Reindeer, Jellikins, Bananas In Pyjamas, Tots TV, The World Of Beatrix Potter, Casper's Haunted Christmas

History of big data and also DBMS

The process of discovering meaningful insights is Data analysis.

for - from - Youtube - Essentia Foundation - interview - Alex Gomez-Marin - Neuroscientist speaks out on the hidden war on consciousness - https://hyp.is/ile8TIvJEfCl35MW3f5B8Q/www.youtube.com/watch?v=K7NIicE_h9w

for - from - Youtube - Essentia Foundation - interview - Alex Gomez-Marin - Neuroscientist speaks out on the hidden war on consciousness - https://hyp.is/ile8TIvJEfCl35MW3f5B8Q/www.youtube.com/watch?v=K7NIicE_h9w

for - adjacency - synchronicity - intense retreat experience - Mingyur Rinpoche - I'm listening to Mingyur Rinpoche and there's some synchronicity that in the live talk, he is talking about the same thing as the monk in this interview - They both went into a multiyear retreat and suffered huge panic attacks - to - Youtube - Mingyur Rinpoche - Anytime Anywhere meditation - South Africa - https://hyp.is/coluBIvcEfCRpD_roJ5NsQ/www.youtube.com/watch?v=P_GmQMZqtGU

بتعي بناء الواجهة

Testing git issue

داخل أي مكوّن Livewire، عندك كائن سحري اسمه $wire متاح في Alpine.

$wire هو “نسخة جافاسكربت” من المكوّن: فيه نفس الخصائص والميثودز، لكن القراءة منه لا تُرسل طلبًا للسيرفر.

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

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Reply to the reviewers

Manuscript number: RC-2025-02922

Corresponding author(s): Christian Specht

[Please use this template only if the submitted manuscript should be considered by the affiliate journal as a full revision in response to the points raised by the reviewers.

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If you wish to submit a preliminary revision with a revision plan, please use our "Revision Plan" template. It is important to use the appropriate template to clearly inform the editors of your intentions.]

1. General Statements [optional]

This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

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We thank the reviewers for their thorough and constructive evaluation of our work. We have revised the manuscript carefully and addressed all the criticisms raised, in particular the issues mentioned by several of the reviewers (see point-by-point response below). We have also added a number of explanations in the text for the sake of clarity, while trying to keep the manuscript as concise as possible.

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In our view, the novelty of our research is two-fold. From a neurobiological point of view, we provide conclusive evidence for the existence of glycine receptors (GlyRs) at inhibitory synapses in various brain regions including the hippocampus, dentate gyrus and sub-regions of the striatum. This solves several open questions and has fundamental implications for our understanding of the organisation and function of inhibitory synapses in the telencephalon. Secondly, our study makes use of the unique sensitivity of single molecule localisation microscopy (SMLM) to identify low protein copy numbers. This is a new way to think about SMLM as it goes beyond a mere structural characterisation and towards a quantitative assessment of synaptic protein assemblies.

2. Point-by-point description of the revisions

This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

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__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __

In this manuscript, the authors investigate the nanoscopic distribution of glycine receptor subunits in the hippocampus, dorsal striatum, and ventral striatum of the mouse brain using single-molecule localization microscopy (SMLM). They demonstrate that only a small number of glycine receptors are localized at hippocampal inhibitory synapses. Using dual-color SMLM, they further show that clusters of glycine receptors are predominantly localized within gephyrin-positive synapses. A comparison between the dorsal and ventral striatum reveals that the ventral striatum contains approximately eight times more glycine receptors and this finding is consistent with electrophysiological data on postsynaptic inhibitory currents. Finally, using cultured hippocampal neurons, they examine the differential synaptic localization of glycine receptor subunits (α1, α2, and β). This study is significant as it provides insights into the nanoscopic localization patterns of glycine receptors in brain regions where this protein is expressed at low levels. Additionally, the study demonstrates the different localization patterns of GlyR in distinct striatal regions and its physiological relevance using SMLM and electrophysiological experiments. However, several concerns should be addressed.

The following are specific comments:

1. Colocalization analysis in Figure 1A. The colocalization between Sylite and mEos-GlyRβ appears to be quite low. It is essential to assess whether the observed colocalization is not due to random overlap. The authors should consider quantifying colocalization using statistical methods, such as a pixel shift analysis, to determine whether colocalization frequencies remain similar after artificially displacing one of the channels. *Following the suggestion of reviewer 1, we re-analysed CA3 images of Glrbeos/eos hippocampal slices by applying a pixel-shift type of control, in which the Sylite channel (in far red) was horizontally flipped relative to the mEos4b-GlyRβ channel (in green, see Methods). As expected, the number of mEos4b-GlyRβ detections per gephyrin cluster was markedly reduced compared to the original analysis (revised__ Fig. 1B__), confirming that the synaptic mEos4b detections exceed chance levels (see page 5). *

Inconsistency between Figure 3A and 3B. While Figure 3B indicates an ~8-fold difference in the number of mEos4b-GlyRβ detections per synapse between the dorsal and ventral striatum, Figure 3A does not appear to show a pronounced difference in the localization of mEos4b-GlyRβ on Sylite puncta between these two regions. If the images presented in Figure 3A are not representative, the authors should consider replacing them with more representative examples or providing an expanded images with multiple representative examples. Alternatively, if this inconsistency can be explained by differences in spot density within clusters, the authors should explain that.

*The pointillist images in Fig. 3A are essentially binary (red-black). Therefore, the density of detections at synapses cannot be easily judged by eye. For clarity, the original images in Fig. 3A have been replaced with two other examples that better reflect the different detection numbers in the dorsal and ventral striatum. *

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Quantification in Figure 5. It is recommended that the authors provide quantitative data on cluster formation and colocalization with Sylite puncta in Figure 5 to support their qualitative observations.

*This is an important point that was also raised by the other reviewers. We have performed additional experiments to increase the data volume for analysis. For quantification, we used two approaches. First, we counted the percentage of infected cells in which synaptic localisation of the recombinant receptor subunit was observed (Fig. 5C). We found that mEos4b-GlyRa1 consistently localises at synapses, indicating that all cells express endogenous GlyRb. When neurons were infected with mEos4b-GlyRb, fewer cells had synaptic clusters, meaning that indeed, GlyR alpha subunits are the limiting factor for synaptic targeting. In cultures infected with mEos4b-GlyRa2, only very few neurons displayed synaptic localisation (as judged by epifluorescence imaging). We think this shows that GlyRa2 is less capable of forming heteromeric complexes than GlyRa1, in line with our previous interpretation (see pp. 9-10, 13). *

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Secondly, we quantified the total intensity of each subunit at gephyrin-positive domains, both in infected neurons as well as non-infected control cultures (Fig. 5D). We observed that mEos4b-GlyRa1 intensity at gephyrin puncta was higher than that of the other subunits, again pointing to efficient synaptic targeting of GlyRa1. Gephyrin cluster intensities (Sylite labelling) were not significantly different in GlyRb and GlyRa2 expressing neurons compared to the uninfected control, indicating that the lentiviral expression of recombinant subunits does not fundamentally alter the size of mixed inhibitory synapses in hippocampal neurons. Interestingly, gephyrin levels were slightly higher in hippocampal neurons expressing mEos4b-GlyRa1. In our view, this comes from an enhanced expression and synaptic targeting of mEos4b-GlyRa1 heteromers with endogenous GlyRb, pointing to a structural role of GlyRa1/b in hippocampal synapses (pp. 10, 13).

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The new data and analyses have been described and illustrated in the relevant sections of the manuscript.

Potential for pseudo replication. It's not clear whether they're performing stats tests across biological replica, images, or even synapses. They often quote mean +/- SEM with n = 1000s, and so does that mean they're doing tests on those 1000s? Need to clarify.

All experiments were repeated at least twice to ensure reproducibility (N independent experiments). Statistical tests were performed on pooled data across the biological replicates; n denotes the number of data points used for testing (e.g., number of synaptic clusters, detections, cells, as specified in each case). We have systematically given these numbers in the revised manuscript (n, N, and other experimental parameters such as the number of animals used, coverslips, images or cells). Data are generally given as mean +/- SEM or as mean +/- SD as indicated.

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Does mEoS effect expression levels or function of the protein? Can't see any experiments done to confirm this. Could suggest WB on homogenate, or mass spec?

The Glrbeos/eos knock-in mouse line has been characterised previously and does not to display any ultrastructural or functional deficits at inhibitory synapses (Maynard et al. 2021 eLife). GlyRβ expression and glycine-evoked responses were not significantly different to those of the wild-type. The synaptic localisation of mEos4b-GlyRb in KI animals demonstrates correct assembly of heteromeric GlyRs and synaptic targeting. Accordingly, the animals do not display any obvious phenotype. We have clarified this in the manuscript (p. 4). In the case of cultured neurons, long-term expression of fluorescent receptor subunits with lentivirus has proven ideal to achieve efficient synaptic targeting. The low and continuous supply of recombinant receptors ensures assembly with endogenous subunits to form heteropentameric receptor complexes (e.g. [Patrizio et al. 2017 Sci Rep]). In the present study, lentivirus infection did not induce any obvious differences in the number or size of inhibitory synapses compared to control neurons, as judged by Sylite labelling of synaptic gephyrin puncta (new__ Fig. 5D__).

Quantification of protein numbers is challenging with SMLM. Issues include i) some of FP not correctly folded/mature, and ii) dependence of localisation rate on instrument, excitation/illumination intensities, and also the thresholds used in analysis. Can the authors compare with another protein that has known expression levels- e.g. PSD95? This is quite an ask, but if they could show copy number of something known to compare with, it would be useful.

We agree that absolute quantification with SMLM is challenging, since the number of detections depends on fluorophore maturation, photophysics, imaging conditions, and analysis thresholds (discussed in Patrizio & Specht 2016, Neurophotonics). For this reason, only very few datasets provide reliable copy numbers, even for well-studied proteins such as PSD-95. One notable exception is the study by Maynard et al. (eLife 2021) that quantified endogenous GlyRb-containing receptors in spinal cord synapses using SMLM combined with correlative electron microscopy. The strength of this work was the use of a KI mouse strain, which ensures that mEos4b-GlyRb expression follows intrinsic regional and temporal profiles. The authors reported a stereotypic density of ~2,000 GlyRs/µm² at synapses, corresponding to ~120 receptors per synapse in the dorsal horn and ~240 in the ventral horn, taking into account various parameters including receptor stoichiometry and the functionality of the fluorophore. These values are very close to our own calculations of GlyR numbers at spinal cord synapses that were obtained slightly differently in terms of sample preparation, microscope setup, imaging conditions, and data analysis, lending support to our experimental approach. Nevertheless, the obtained GlyR copy numbers at hippocampal synapses clearly have to be taken as estimates rather than precise figures, because the number of detections from a single mEos4b fluorophore can vary substantially, meaning that the fluorophores are not represented equally in pointillist images. This can affect the copy number calculation for a specific synapse, in particular when the numbers are low (e.g. in hippocampus), however, it should not alter the average number of detections (Fig. 1B) or the (median) molecule numbers of the entire population of synapses (Fig. 1C). We have discussed the limitations of our approach (p. 11).

Rationale for doing nanobody dSTORM not clear at all. They don't explain the reason for doing the dSTORM experiments. Why not just rely on PALM for coincidence measurements, rather than tagging mEoS with a nanobody, and then doing dSTORM with that? Can they explain? Is it to get extra localisations- i.e. multiple per nanobody? If so, localising same FP multiple times wouldn't improve resolution. Also, no controls for nanobody dSTORM experiments- what about non-spec nb, or use on WT sections?

*As discussed above (point 6), the detection of fluorophores with SMLM is influenced by many parameters, not least the noise produced by emitting molecules other than the fluorophore used for labelling. Our study is exceptional in that it attempts to identify extremely low molecule numbers (down to 1). To verify that the detections obtained with PALM correspond to mEos4b, we conducted robust control experiments (including pixel-shift as suggested by the reviewer, see point 1, revised__ Fig. 1B__). The rationale for the nanobody-based dSTORM experiments was twofold: (1) to have an independent readout of the presence of low-copy GlyRs at inhibitory synapses and (2) to analyse the nanoscale organisation of GlyRs relative to the synaptic gephyrin scaffold using dual-colour dSTORM with spectral demixing (see p. 6). The organic fluorophores used in dSTORM (AF647, CF680) ensure high photon counts, essential for reliable co-localisation and distance analysis. PALM and dSTORM cannot be combined in dual-colour mode, as they require different buffers and imaging conditions. *

The specificity of the anti-Eos nanobody was demonstrated by immunohistochemistry in spinal cord cultures expressing mEos4b-GlyRb and wildtype control tissue (Fig. S3). In response to the reviewer's remarks, we also performed a negative control experiment in Glrbeos/eos slices (dSTORM), in which the nanobody was omitted (new__ Fig. S4F,G__). Under these conditions, spectral demixing produced a single peak corresponding to CF680 (gephyrin) without any AF647 contribution (Fig. S4F). The background detection of "false" AF647 detections at synapses was significantly lower than in the slices labelled with the nanobody. We conclude that the fluorescence signal observed in our dual-colour dSTORM experiments arises from the specific detection of mEos4b-GlyRb by the nanobody, rather than from background, cross-reactivity or wrong attribution of colour during spectral demixing. We have added these data and explanations in the results (p. 7) and in the figure legend of Fig. S4F,G.

What resolutions/precisions were obtained in SMLM experiments? Should perform Fourier Ring Correlation (FRC) on SR images to state resolutions obtained (particularly useful for when they're presenting distance histograms, as this will be dependent on resolution). Likewise for precision, what was mean precision? Can they show histograms of localisation precision.

This is an interesting question in the context of our experiments with low-copy GlyRs, since the spatial resolution of SMLM is limited also by the density of molecules, i.e. the sampling of the structure in question (Nyquist-Shannon criterion). Accordingly, the priority of the PALM experiments was to improve the sensibility of SMLM for the identification of mEos4b-GlyRb subunits, rather than to maximize the spatial resolution. The mean localisation precision in PALM was 33 +/- 12 nm, as calculated from the fitting parameters of each detection (Zeiss, ZEN software), which ultimately result from their signal-to-noise ratio. This is a relatively low precision for SMLM, which can be explained by the low brightness of mEos4b compared to organic fluorophores together with the elevated fluorescence background in tissue slices.

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In the case of dSTORM, the aim was to study the relative distribution of GlyRs within the synaptic scaffold, for which a higher localisation precision was required (p. 6). Therefore, detections with a precision ≥ 25 nm were filtered during analysis with NEO software (Abbelight). The retained detections had a mean localisation precision of 12 +/- 5 for CF680 (Sylite) and 11 +/- 4 for AF647 (nanobody). These values are given in the revised manuscript (pp. 18, 22).

Why were DBSCAN parameters selected? How can they rule out multiple localisations per fluor? If low copy numbers (

Multiple detections of the same fluorophore are intrinsic to dSTORM imaging and have not been eliminated from the analysis. Small clusters of detections likely represent individual molecules (e.g. single receptors in the extrasynaptic regions, Fig. 2A). DBSCAN is a robust clustering method that is quite insensitive to minor changes in the choice of parameters. For dSTORM of synaptic gephyrin clusters (CF680), a relatively low length (80 nm radius) together with a high number of detections (≥ 50 neighbours) were chosen to reconstruct the postsynaptic domain with high spatial resolution (see point 8). In the case of the GlyR (nanobody-AF647), the clustering was done mostly for practical reasons, as it provided the coordinates of the centre of mass of the detections. The low stringency of this clustering (200 nm radius, ≥ 5 neighbours) effectively filters single detections that can result from background noise or incorrect demixing. An additional reference explaining the use of DBSCAN including the choice of parameters is given on p. 22 (see also R2 point 4).

For microscopy experiment methods, state power densities, not % or "nominal power".

*Done. We now report the irradiance (laser power density) instead of nominal power (pp. 18, 21). *

In general, not much data presented. Any SI file with extra images etc.?

*The original submission included four supplementary figures with additional data and representative images that should have been available to the reviewer (Figs. S1-S4). The SI file has been updated during revision (new Fig. S4E-G). *

Clarification of the discussion on GlyR expression and synaptic localization: The discussion on GlyR expression, complex formation, and synaptic localization is sometimes unclear, and needs terminological distinctions between "expression level", "complex formation" and "synaptic localization". For example, the authors state:"What then is the reason for the low protein expression of GlyRβ? One possibility is that the assembly of mature heteropentameric GlyR complexes depends critically on the expression of endogenous GlyR α subunits." Does this mean that GlyRβ proteins that fail to form complexes with GlyRα subunits are unstable and subject to rapid degradation? If so, the authors should clarify this point. The statement "This raises the interesting possibility that synaptic GlyRs may depend specifically on the concomitant expression of both α1 and β transcripts." suggests a dependency on α1 and β transcripts. However, is the authors' focus on synaptic localization or overall protein expression levels? If this means synaptic localization, it would be beneficial to state this explicitly to avoid confusion. To improve clarity, the authors should carefully distinguish between these different aspects of GlyR biology throughout the discussion. Additionally, a schematic diagram illustrating these processes would be highly beneficial for readers.

We thank the reviewer to point this out. We are dealing with several processes; protein expression that determines subunit availability and the assembly of pentameric GlyRs complexes, surface expression, membrane diffusion and accumulation of GlyRb-containing receptor complexes at inhibitory synapses. We have edited the manuscript, particularly the discussion and tried to be as clear as possible in our wording.

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We chose not to add a schematic illustration for the time being, because any graphical representation is necessarily a simplification. Instead, we preferred to summarise the main numbers in tabular form (Table 1). We are of course open to any other suggestions.

Interpretation of GlyR localization in the context of nanodomains. The distribution of GlyR molecules on inhibitory synapses appears to be non-homogeneous, instead forming nanoclusters or nanodomains, similar to many other synaptic proteins. It is important to interpret GlyR localization in the context of nanodomain organization.

The dSTORM images in Fig. 2 are pointillist representations that show individual detections rather than molecules. Small clusters of detections are likely to originate from a single AF647 fluorophore (in the case of nanobody labelling) and therefore represent single GlyRb subunits. Since GlyR copy numbers are so low at hippocampal synapses (≤ 5), the notion of nanodomain is not directly applicable. Our analysis therefore focused on the integration of GlyRs within the postsynaptic scaffold, rather than attempting to define nanodomain structures (see also response to point 8 of R1). A clarification has been added in the revised manuscript (p. 6).

__Reviewer #1 (Significance (Required)): __

The paper presents biological and technical advances. The biological insights revolve mostly on the documentation of Glycine receptors in particular synapses in forebrain, where they are typically expressed at very low levels. The authors provide compelling data indicating that the expression is of physiological significance. The authors have done a nice job of combining genetically-tagged mice with advanced microscopy methods to tackle the question of distributions of synaptic proteins. Overall these advances are more incremental than groundbreaking.

We thank the reviewer for acknowledging both the technical and biological advances of our study. While we recognize that our work builds upon established models, we consider that it also addresses important unresolved questions, namely that GlyRs are present and specifically anchored at inhibitory synapses in telencephalic regions, such as the hippocampus and striatum. From a methodological point of view, our study demonstrates that SMLM can be applied not only for structural analysis of highly abundant proteins, but also to reliably detect proteins present at very low copy numbers. This ability to identify and quantify sparse molecule populations adds a new dimension to SMLM applications, which we believe increases the overall impact of our study beyond the field of synaptic neuroscience.

__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

In their manuscript "Single molecule counting detects low-copy glycine receptors in hippocampal and striatal synapses" Camuso and colleagues apply single molecule localization microscopy (SMLM) methods to visualize low copy numbers of GlyRs at inhibitory synapses in the hippocampal formation and the striatum. SMLM analysis revealed higher copy numbers in striatum compared to hippocampal inhibitory synapses. They further provide evidence that these low copy numbers are tightly linked to post-synaptic scaffolding protein gephyrin at inhibitory synapses. Their approach profits from the high sensitivity and resolution of SMLM and challenges the controversial view on the presence of GlyRs in these formations although there are reports (electrophysiology) on the presence of GlyRs in these particular brain regions. These new datasets in the current manuscript may certainly assist in understanding the complexity of fundamental building blocks of inhibitory synapses.

However I have some minor points that the authors may address for clarification:

1) In Figure 1 the authors apply PALM imaging of mEos4b-GlyRß (knockin) and here the corresponding Sylite label seems to be recorded in widefield, it is not clearly stated in the figure legend if it is widefield or super-resolved. In Fig 1 A - is the scale bar 5 µm? Some Sylite spots appear to be sized around 1 µm, especially the brighter spots, but maybe this is due to the lower resolution of widefield imaging? Regarding the statistical comparison: what method was chosen to test for normality distribution, I think this point is missing in the methods section.

*This is correct; the apparent size of the Sylite spots does not reflect the real size of the synaptic gephyrin domain due to the limited resolution of widefield imaging including the detection of out-of-focus light. We have clarified in the legend of Fig. 1A that Sylite labelling was with classic epifluorescence microscopy. The scale bar in Fig. 1A corresponds to 5 µm. Since the data were not normally distributed, nonparametric tests (Kruskal- Wallis one-way ANOVA with Dunn’s multiple comparison test or Mann-Whitney U-test for pairwise comparisons) were used (p. 23). *

Moreover I would appreciate a clarification and/or citation that the knockin model results in no structural and physiological changes at inhibitory synapses, I believe this model has been applied in previous studies and corresponding clarification can be provided.

The Glrbeos/eos mouse model has been described previously and does not exhibit any structural or physiological phenotypes (Maynard et al. 2021 eLife). The issue was also raised by reviewer R1 (point 5) and has been clarified in the revised manuscript (p. 4).

2) In the next set of experiments the authors switch to demixing dSTORM experiments - an explanation why this is performed is missing in the text - I guess better resolution to perform more detailed distance measurements? For these experiments: which region of the hippocampus did the authors select, I cannot find this information in legend or main text.

Yes, the dSTORM experiments enable dual-colour structural analysis at high spatial resolution (see response to R1 point 7). An explanation has been added (p. 6).

3) Regarding parameters of demixing experiments: the number of frames (10.000) seems quite low and the exposure time higher than expected for Alexa 647. Can the authors explain the reason for chosing these particular parameters (low expression profile of the target - so better separation?, less fluorophores on label and shorter collection time?) or is there a reference that can be cited? The laser power is given in the methods in percentage of maximal output power, but for better comparison and reproducibility I recommend to provide the values of a power meter (kW/cm2) as lasers may change their maximum output power during their lifetime.

Acquisition parameters (laser power, exposure time) for dSTORM were chosen to obtain a good localisation precision (~12 nm; see R1 point 8). The number of frames is adequate to obtain well sampled gephyrin scaffolds in the CF680 channel. In the case of the GlyR (nanobody-AF647), the concept of spatial resolution does not really apply due to the low number of targets (see R1, point 13). Power density (irradiance) values have now been given (pp. 18, 21).

4) For analysis of subsynaptic distribution: how did the authors decide to choose the parameters in the NEO software for DBSCAN clustering - was a series of parameters tested to find optimal conditions and did the analysis start with an initial test if data is indeed clustered (K-ripley) or is there a reference in literature that can be provided?

DBSCAN parameters were optimised manually, by testing different values. Identification of dense and well-delimited gephyrin clusters (CF680) was achieved with a small radius and a high number of detections (80 nm, ≥ 50 neighbours), whereas filtering of low-density background in the AF647 channel (GlyRs) required less stringent parameters (200 nm, ≥ 5) due to the low number of target molecules. Similar parameters were used in a previous publication (Khayenko et al. 2022, Angewandte Chemie). The reference has been provided on p. 22 (see also R1 point 9).

5) A conclusion/discussion of the results presented in Figure 5 is missing in the text/discussion.

*This part of the manuscript has been completely overhauled. It includes new experimental data, quantification of the data (new Fig.5), as well as the discussion and interpretation of our findings (see also R1, point 3). In agreement with our earlier interpretation, the data confirm that low availability of GlyRa1 subunits limits the expression and synaptic targeting of GlyRa1/b heteropentamers. The observation that GlyRa1 overexpression with lentivirus increases the size of the postsynaptic gephyrin domain further points to a structural role, whereby GlyRs can enhance the stability (and size) of inhibitory synapses in hippocampal neurons, even at low copy numbers (pp. 13-14). *

6) in line 552 "suspension" is misleading, better use "solution"

Done.

__Reviewer #2 (Significance (Required)): __

Significance: The manuscript provides new insights to presence of low-copy numbers by visualizing them via SMLM. This is the first report that visualizes GlyR optically in the brain applying the knock-in model of mEOS4b tagged GlyRß and quantifies their copy number comparing distribution and amount of GlyRs from hippocampus and striatum. Imaging data correspond well to electrophysiological measurements in the manuscript.

Field of expertise: Super-Resolution Imaging and corresponding analysis

__Reviewer #4 (Evidence, reproducibility and clarity (Required)): __

In this study, Camuso et al., make use of a knock-in mouse model expressing endogenously mEos4b-tagged GlyRβ to detect endogenous glycine receptors using single-molecule localization microscopy. The main conclusion from this study is that in the hippocampus GlyRβ molecules are barely detected, while inhibitory synapses in the ventral striatum seem to express functionally relevant GlyR numbers.

I have a few points that I hope help to improve the strength of this study.

• In the hippocampus, this study finds that the numbers of detections are very low. The authors perform adequate controls to indicate that these localizations are above noise level. Nevertheless, it remains questionable that these reflect proper GlyRs. The suggestion that in hippocampal synapses the low numbers of GlyRβ molecules "are important in assembly or maintenance of inhibitory synaptic structures in the brain" is on itself interesting, but is not at all supported. It is also difficult to envision how such low numbers could support the structure of a synapse. A functional experiment showing that knockdown of GlyRs affects inhibitory synapse structure in hippocampal neurons would be a minimal test of this.

*It is not clear what the reviewer means by “it remains questionable that these reflect proper GlyRs”. The PALM experiments include a series of stringent controls (see R1, point 1) demonstrating the existence of low-copy GlyRs at inhibitory synapses in the hippocampus (Fig. 1) and in the striatum (Fig. 3), and are backed up by dSTORM experiments (Fig. 2). We have no reason to doubt that these receptors are fully functional (as demonstrated for the ventral striatum (Fig. 4). However, due to their low number, a role in inhibitory synaptic transmission is clearly limited, at least in the hippocampus and dorsal striatum. *

• *

We therefore propose a structural role, where the GlyRs could be required to stabilise the postsynaptic gephyrin domain in hippocampal neurons. This is based on the idea that the GlyR-gephyrin affinity is much higher than that of the GABAAR-gephyrin interaction (reviewed in Kasaragod & Schindelin 2018 Front Mol Neurosci). Accordingly, there is a close relationship between GlyRs and gephyrin numbers, sub-synaptic distribution, and dynamics in spinal cord synapses that are mostly glycinergic (Specht et al. 2013 Neuron; Maynard et al. 2021 eLife; Chapdelaine et al. 2021 Biophys J). It is reasonable to assume that low-copy GlyRs could play a similar structural role at hippocampal synapses. A knockdown experiment targeting these few receptors is technically very challenging and beyond the scope of this study. However, in response to the reviewer's question we have conducted new experiments in cultured hippocampal neurons (new__ Fig. 5__). They demonstrate that overexpression of GlyRa1/b heteropentamers increases the size of the postsynaptic domain in these neurons, supporting our interpretation of a structural role of low-copy GlyRs (p. 14).

• The endogenous tagging strategy is a very strong aspect of this study and provides confidence in the labeling of GlyRβ molecules. One caveat however, is that this labeling strategy does not discriminate whether GlyRβ molecules are on the cell membrane or in internal compartments. Can the authors provide an estimate of the ratio of surface to internal GlyRβ molecules?

Gephyrin is known to form a two-dimensional scaffold below the synaptic membrane to which inhibitory GlyRs and GABAARs attach (reviewed in Alvarez 2017 Brain Res). The majority of the synaptic receptors are therefore thought to be located in the synaptic membrane, which is supported by the close relationship between the sub-synaptic distribution of GlyRs and gephyrin in spinal cord neurons (e.g. Maynard et al. 2021 eLife). To demonstrate the surface expression of GlyRs at hippocampal synapses we labelled cultured hippocampal neurons expressing mEos4b-GlyRa1 with anti-Eos nanobody in non-permeabilised neurons (see Figure below for the reviewer only). The close correspondence between the nanobody (AF647) and the mEos4b signal confirms that the majority of the GlyRs are indeed located in the synaptic membrane.

• *

Figure (for the reviewer only).* Left: Lentivirus expression of mEos4b-GlyRa1 in fixed and non-permeabilised hippocampal neurons (mEos4b signal). Right: Surface labelling of the recombinant subunit with anti-Eos nanoboby (AF647). *

• 'We also estimated the absolute number of GlyRs per synapse in the hippocampus. The number of mEos4b detections was converted into copy numbers by dividing the detections at synapses by the average number of detections of individual mEos4b-GlyRβ containing receptor complexes'. In essence this is a correct method to estimate copy numbers, and the authors discuss some of the pitfalls associated with this approach (i.e., maturation of fluorophore and detection limit). Nevertheless, the authors did not subtract the number of background localizations determined in the two negative control groups. This is critical, particularly at these low-number estimations.

We fully agree that background subtraction can be useful with low detection numbers. In the revised manuscript, copy numbers are now reported as background-corrected values. Specifically, the mean number of detections measured in wildtype slices was used to calculate an equivalent receptor number, which was then subtracted from the copy number estimates across hippocampus, spinal cord and striatum. This procedure is described in the methods (p. 20) and results (p. 5, 8), and mentioned in the figure legends of Fig. 1C, 3C. The background corrected values are given in the text and Table 1.

Furthermore, the authors state that "The advantage of this estimation is that it is independent of the stoichiometry of heteropentameric GlyRs". However, if the stoichometry is unknown, the number of counted GlyRβ subunits cannot simply be reported as the number of GlyRs. This should be discussed in more detail, and more carefully reported throughout the manuscript.

*The reviewer is right to point this out. There is still some debate about the stoichiometry of heteropentameric GlyRs. Configurations with 2a:3b, 3a:2b and 4a:1b subunits have been advanced (e.g. Grudzinska et al. 2005 Neuron; Durisic et al. 2012 J Neurosci; Patrizio et al. 2017 Sci Rep; Zhu & Gouaux 2021 Nature). We have therefore chosen a quantification that is independent of the underlying stoichiometry. Since our quantification is based on very sparse clusters of mEos4b detections that likely originate from a single receptor complex (irrespective of its stoichiometry), the reported values actually reflect the number of GlyRs (and not GlyRb subunits). We have clarified this in the results (p. 5) and throughout the manuscript (Table 1). *

• The dual-color imaging provides insights in the subsynaptic distribution of GlyRβ molecules in hippocampal synapses. Why are similar studies not performed on synapses in the ventral striatum where functionally relevant numbers of GlyRβ molecules are found? Here insights in the subsynaptic receptor distribution would be of much more interest as it can be tight to the function.

This is an interesting suggestion. However, the primary aim of our study was to identify the existence of GlyRs in hippocampal regions. At low copy numbers, the concept of sub-synaptic domains (SSDs, e.g. Yang et al. 2021 EMBO Rep) becomes irrelevant (see R1 point 13). It should be pointed out that the dSTORM pointillist images (Fig. 2A) represent individual GlyR detections rather than clusters of molecules. In the striatum, our specific purpose was to solve an open question about the presence of GlyRs in different subregions (putamen, nucleus accumbens).

• It is unclear how the experiments in Figure 5 add to this study. These results are valid, but do not seem to directly test the hypothesis that "the expression of α subunits may be limiting factor controlling the number of synaptic GlyRs". These experiments simply test if overexpressed α subunits can be detected. If the α subunits are limiting, measuring the effect of α subunit overexpression on GlyRβ surface expression would be a more direct test.

Both R1 and R2 have also commented on the data in Fig. 5 and their interpretation. We have substantially revised this section as described before (see R1 point 3) including additional experiments and quantification of the data (new Fig. 5). The findings lend support to our earlier hypothesis that GlyR alpha subunits (in particular GlyRa1) are the limiting factor for the expression of heteropentameric GlyRa/b in hippocampal neurons (pp. 13-14). Since the GlyRa1 subunit itself does not bind to gephyrin (Patrizio et al. 2017 Sci Rep), the synaptic localisation of the recombinant mEos4b-GlyRa1 subunits is proof that they have formed heteropentamers with endogenous GlyRb subunits and driven their membrane trafficking, which the GlyRb subunits are incapable of doing on their own.

__Reviewer #4 (Significance (Required)): __

These results are based on carefully performed single-molecule localization experiments, and are well-presented and described. The knockin mouse with endogenously tagged GlyRβ molecules is a very strong aspect of this study and provides confidence in the labeling, the combination with single-molecule localization microscopy is very strong as it provides high sensitivity and spatial resolution.

The conceptual innovation however seems relatively modest, these results confirm previous studies but do not seem to add novel insights. This study is entirely descriptive and does not bring new mechanistic insights.

This study could be of interest to a specialized audience interested in glycine receptor biology, inhibitory synapse biology and super-resolution microscopy.

my expertise is in super-resolution microscopy, synaptic transmission and plasticity

As we have stated before, the novelty of our study lies in the use of SMLM for the identification of very small numbers of molecules, which requires careful control experiments. This is something that has not been done before and that can be of interest to a wider readership, as it opens up SMLM for ultrasensitive detection of rare molecular events. Using this approach, we solve two open scientific questions: (1) the demonstration that low-copy GlyRs are present at inhibitory synapses in the hippocampus, (2) the sub-region specific expression and functional role of GlyRs in the ventral versus dorsal striatum.

• *

• *

The following review was provided later under the name “Reviewer #4”. To avoid confusion with the last reviewer from above we will refer to this review as R4-2.

__Reviewer #4-2 (Evidence, reproducibility and clarity (Required)): __

Summary:

Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

The authors investigate the presence of synaptic glycine receptors in the telencephalon, whose presence and function is poorly understood.

Using a transgenically labeled glycine receptor beta subunit (Glrb-mEos4b) mouse model together with super-resolution microscopy (SLMM, dSTORM), they demonstrate the presence of a low but detectable amount of synaptically localized GLRB in the hippocampus. While they do not perform a functional analysis of these receptors, they do demonstrate that these subunits are integrated into the inhibitory postsynaptic density (iPSD) as labeled by the scaffold protein gephyrin. These findings demonstrate that a low level of synaptically localized glycerine receptor subunits exist in the hippocampal formation, although whether or not they have a functional relevance remains unknown.

They then proceed to quantify synaptic glycine receptors in the striatum, demonstrating that the ventral striatum has a significantly higher amount of GLRB co-localized with gephyrin than the dorsal striatum or the hippocampus. They then recorded pharmacologically isolated glycinergic miniature inhibitory postsynaptic currents (mIPSCs) from striatal neurons. In line with their structural observations, these recordings confirmed the presence of synaptic glycinergic signaling in the ventral striatum, and an almost complete absence in the dorsal striatum. Together, these findings demonstrate that synaptic glycine receptors in the ventral striatum are present and functional, while an important contribution to dorsal striatal activity is less likely.

Lastly, the authors use existing mRNA and protein datasets to show that the expression level of GLRA1 across the brain positively correlates with the presence of synaptic GLRB.

The authors use lentiviral expression of mEos4b-tagged glycine receptor alpha1, alpha2, and beta subunits (GLRA1, GLRA1, GLRB) in cultured hippocampal neurons to investigate the ability of these subunits to cause the synaptic localization of glycine receptors. They suggest that the alpha1 subunit has a higher propensity to localize at the inhibitory postsynapse (labeled via gephyrin) than the alpha2 or beta subunits, and may therefore contribute to the distribution of functional synaptic glycine receptors across the brain.

Major comments:

• Are the key conclusions convincing?

The authors are generally precise in the formulation of their conclusions.

• They demonstrate a very low, but detectable, amount of a synaptically localized glycine receptor subunit in a transgenic (GlrB-mEos4b) mouse model. They demonstrate that the GLRB-mEos4b fusion protein is integrated into the iPSD as determined by gephyrin labelling. The authors do not perform functional tests of these receptors and do not state any such conclusions.
• The authors show that GLRB-mEos4b is clearly detectable in the striatum and integrated into gephyrin clusters at a significantly higher rate in the ventral striatum compared to the dorsal striatum, which is in line with previous studies.
• Adding to their quantification of GLRB-mEos4b in the striatum, the authors demonstrate the presence of glycinergic miniature IPSCs in the ventral striatum, and an almost complete absence of mIPSCs in the dorsal striatum. These currents support the observation that GLRB-mEos4b is more synaptically integrated in the ventral striatum compared to the dorsal striatum.

• The authors show that lentiviral expression of GLRA1-mEos4b leads to a visually higher number of GLR clusters in cultured hippocampal neurons, and a co-localization of some clusters with gephyrin. The authors claim that this supports the idea that GLRA1 may be an important driver of synaptic glycine receptor localization. However, no quantification or statistical analysis of the number of puncta or their colocalization with gephyrin is provided for any of the expressed subunits. Such a claim should be supported by quantification and statistics A thorough analysis and quantification of the data in Fig.5 has been carried out as requested by all the other reviewers (e.g. R1, point 3). The new data and results have been described in the revised manuscript (pp. 9-10, 13-14).

• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

One unaddressed caveat is the fact that a GLRB-mEos4b fusion protein may behave differently in terms of localization and synaptic integration than wild-type GLRB. While unlikely, it is possible that mEos4b interacts either with itself or synaptic proteins in a way that changes the fused GLRB subunit’s localization. Such an effect would be unlikely to affect synaptic function in a measurable way, but might be detected at a structural level by highly sensitive methods such as SMLM and STORM in regions with very low molecule numbers (such as the hippocampus). Since reliable antibodies against GLRB in brain tissue sections are not available, this would be difficult to test. Considering that no functional measures of the hippocampal detections exist, we would suggest that this possible caveat be mentioned for this particular experiment.

*This question has also been raised before (R1, point 5). According to an earlier study the mEos4b-GlyRb knock-in does not cause any obvious phenotypes, with the possible exception of minor loss of glycine potency (Maynard et al. 2021 eLife). The fact that the synaptic levels in the spinal cord in heterozygous animals are precisely half of those of homozygous animals argues against differences in receptor expression, heteropentameric assembly, forward trafficking to the plasma membrane and integration into the synaptic membrane as confirmed using quantitative super-resolution CLEM (Maynard et al. 2021 eLife). Accordingly, we did not observe any behavioural deficits in these animals, making it a powerful experimental model. We have added this information in the revised manuscript (p. 4). *

In addition, without any quantification or statistical analysis, the author’s claims regarding the necessity of GLRA1 expression for the synaptic localization of glycine receptors in cultured hippocampal neurons should probably be described as preliminary (Fig. 5).

As mentioned before, we have substantially revised this part (R1, point 3). The quantification and analysis in the new Fig. 5 support our earlier interpretation.

• Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

The authors show that there is colocalization of gephyrin with the mEos4b-GlyRβ subunit using the Dual-colour SMLM. This is a powerful approach that allows for a claim to be made on the synaptic location of the glycine receptors. The images presented in Figure 1, together with the distance analysis in Figure 2, display the co-localization of the fluorophores. The co-localization images in all the selected regions, hippocampus and striatum, also show detections outside of the gephyrin clusters, which the authors refer to as extrasynaptic. These punctated small clusters seem to have the same size as the ones detected and assigned as part of the synapse. It would be informative if the authors analysed the distribution, density and size of these non-synaptic clusters and presented the data in the manuscript and also compared it against the synaptic ones. Validating this extrasynaptic signal by staining for a dendritic marker, such as MAP-2 or maybe a somatic marker and assessing the co-localization with the non-synaptic clusters would also add even more credibility to them being extrasynaptic.

The existence of extrasynaptic GlyRs is well attested in spinal cord neurons (e.g. Specht et al. 2013 Neuron; this study see Fig. S2). The fact that these appear as small clusters of detections in SMLM recordings results from the fact that a single fluorophore can be detected several times in consecutive image frames and because of blinking. Therefore, small clusters of detections likely represent single GlyRs (that can be counted), and not assemblies of several receptor complexes. Due to their diffusion in the neuronal membrane, they are seen as diffuse signals throughout the somatodendritic compartment in epifluorescence images (e.g. Fig. 5A). SMLM recordings of the same cells resolves this diffuse signal into discrete nanoclusters representing individual receptors (Fig. 5B). It is not clear what information co-localisation experiments with specific markers could provide, especially in hippocampal neurons, in which the copy numbers (and density) of GlyRs is next to zero.

In addition we would encourage the authors to quantify the clustering and co-localization of virally expressed GLRA1, GLRA2, and GLRB with gephyrin in order to support the associated claims (Fig. 5). Preferably, the density of GLR and gephyrin clusters (at least on the somatic surface, the proximal dendrites, or both) as well as their co-localization probability should be quantified if a causal claim about subunit-specific requirements for synaptic localization is to be made.

Quantification of the data have been carried out (new Fig.5C,D). The results have been described before (R1, point 3) and support our earlier interpretation of the data (pp. 13-14).

Lastly, even though it may be outside of the scope of such a study analysing other parts of the hippocampal area could provide additional important information. If one looks at the Allen Institute’s ISH of the beta subunit the strongest signal comes from the stratum oriens in the CA1 for example, suggesting that interneurons residing there would more likely have a higher expression of the glycine receptors. This could also be assessed by looking more carefully at the single cell transcriptomics, to see which cell types in the hippocampus show the highest mRNA levels. If the authors think that this is too much additional work, then perhaps a mention of this in the discussion would be good.

We have added the requested information from the ISH database of the Allen Institute in the discussion as suggested by the reviewer (p. 12). However, in combination with the transcriptomic data (Fig. S1) our finding strongly suggest that the expression of synaptic GlyRs depends on the availability of alpha subunits rather than on the presence of the GlyRb transcript. This is obvious when one compares the mRNA levels in the hippocampus with those in the basal ganglia (striatum) and medulla. While the transcript concentrations of GlyRb are elevated in all three regions and essentially the same, our data show that the GlyRb copy numbers *at synapses differ over more than 2 orders of magnitude (Fig. 1B, Table 1). *

• Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

Since the labeling and some imaging has been performed already, the requested experiment would be a matter of deploying a method of quantification. In principle, it should not require any additional wet-lab experiments, although it may require additional imaging of existing samples.

• Are the data and the methods presented in such a way that they can be reproduced?

Yes, for the most part.

• Are the experiments adequately replicated and statistical analysis adequate?

Yes

Minor comments:

• Specific experimental issues that are easily addressable.

N/A

• Are prior studies referenced appropriately?

Yes

• Are the text and figures clear and accurate?

Yes, although quantification in figure 5 is currently not present.

A quantification has been added (see R1, point 3).

• Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

This paper presents a method that could be used to localize receptors and perhaps other proteins that are in low abundance or for which a detailed quantification is necessary. I would therefore suggest that Figure S4 is included into Figure 2 as the first panel, showcasing the demixing, followed by the results.

We agree in principle with this suggestion. However, the revised Fig. S4 is more complex and we think that it would distract from the data shown in Fig. 2. Given that Fig. S4 is mostly methodological and not essential to understand the text, we have kept it in the supplement for the time being. We leave the final decision on this point to the editor.

__Reviewer #4-2 (Significance (Required)): __

[This review was supplied later]

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

Using a novel and high resolution method, the authors have provided strong evidence for the presence of glycine receptors in the murine hippocampus and in the dorsal striatum. The number of receptors calculated is small compared to the numbers found in the ventral striatum. This is the first study to quantify receptor numbers in these region. In addition it also lays a roadmap for future studies addressing similar questions.

• Place the work in the context of the existing literature (provide references, where appropriate).

This is done well by the authors in the curation of the literature. As stated above, the authors have filled a gap in the presence of glycine receptors in different brain regions, a subject of importance in understanding the role they play in brain activity and function.

• State what audience might be interested in and influenced by the reported findings.

Neuroscientists working at the synaptic level, on inhibitory neurotransmission and on fundamental mechanisms of expression of genes at low levels and their relationship to the presence of the protein would be interested. Furthermore, researchers in neuroscience and cell biology may benefit from and be inspired by the approach used in this manuscript, to potentially apply it to address their own aims.

*We thank the reviewer for the positive assessment of the technical and biological implications of our work, as well as the interest of our findings to a wide readership of neuroscientists and cell biologists. *

• Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

Synaptic transmission, inhibitory cells and GABAergic synapses functionally and structurally, cortex and cortical circuits. No strong expertise in super-resolution imaging methods.

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

Learn more at Review Commons

Referee #4

Evidence, reproducibility and clarity

Summary: Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

The authors investigate the presence of synaptic glycine receptors in the telencephalon, whose presence and function is poorly understood.

Using a transgenically labeled glycine receptor beta subunit (Glrb-mEos4b) mouse model together with super-resolution microscopy (SLMM, dSTORM), they demonstrate the presence of a low but detectable amount of synaptically localized GLRB in the hippocampus. While they do not perform a functional analysis of these receptors, they do demonstrate that these subunits are integrated into the inhibitory postsynaptic density (iPSD) as labeled by the scaffold protein gephyrin. These findings demonstrate that a low level of synaptically localized glycerine receptor subunits exist in the hippocampal formation, although whether or not they have a functional relevance remains unknown.

They then proceed to quantify synaptic glycine receptors in the striatum, demonstrating that the ventral striatum has a significantly higher amount of GLRB co-localized with gephyrin than the dorsal striatum or the hippocampus. They then recorded pharmacologically isolated glycinergic miniature inhibitory postsynaptic currents (mIPSCs) from striatal neurons. In line with their structural observations, these recordings confirmed the presence of synaptic glycinergic signaling in the ventral striatum, and an almost complete absence in the dorsal striatum. Together, these findings demonstrate that synaptic glycine receptors in the ventral striatum are present and functional, while an important contribution to dorsal striatal activity is less likely.

Lastly, the authors use existing mRNA and protein datasets to show that the expression level of GLRA1 across the brain positively correlates with the presence of synaptic GLRB. The authors use lentiviral expression of mEos4b-tagged glycine receptor alpha1, alpha2, and beta subunits (GLRA1, GLRA1, GLRB) in cultured hippocampal neurons to investigate the ability of these subunits to cause the synaptic localization of glycine receptors. They suggest that the alpha1 subunit has a higher propensity to localize at the inhibitory postsynapse (labeled via gephyrin) than the alpha2 or beta subunits, and may therefore contribute to the distribution of functional synaptic glycine receptors across the brain.

Major comments: - Are the key conclusions convincing?

The authors are generally precise in the formulation of their conclusions.

1) They demonstrate a very low, but detectable, amount of a synaptically localized glycine receptor subunit in a transgenic (GlrB-mEos4b) mouse model. They demonstrate that the GLRB-mEos4b fusion protein is integrated into the iPSD as determined by gephyrin labelling. The authors do not perform functional tests of these receptors and do not state any such conclusions. 2) The authors show that GLRB-mEos4b is clearly detectable in the striatum and integrated into gephyrin clusters at a significantly higher rate in the ventral striatum compared to the dorsal striatum, which is in line with previous studies. 3) Adding to their quantification of GLRB-mEos4b in the striatum, the authors demonstrate the presence of glycinergic miniature IPSCs in the ventral striatum, and an almost complete absence of mIPSCs in the dorsal striatum. These currents support the observation that GLRB-mEos4b is more synaptically integrated in the ventral striatum compared to the dorsal striatum. 4) The authors show that lentiviral expression of GLRA1-mEos4b leads to a visually higher number of GLR clusters in cultured hippocampal neurons, and a co-localization of some clusters with gephyrin. The authors claim that this supports the idea that GLRA1 may be an important driver of synaptic glycine receptor localization. However, no quantification or statistical analysis of the number of puncta or their colocalization with gephyrin is provided for any of the expressed subunits. Such a claim should be supported by quantification and statistics

• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

One unaddressed caveat is the fact that a GLRB-mEos4b fusion protein may behave differently in terms of localization and synaptic integration than wild-type GLRB. While unlikely, it is possible that mEos4b interacts either with itself or synaptic proteins in a way that changes the fused GLRB subunit's localization. Such an effect would be unlikely to affect synaptic function in a measurable way, but might be detected at a structural level by highly sensitive methods such as SMLM and STORM in regions with very low molecule numbers (such as the hippocampus). Since reliable antibodies against GLRB in brain tissue sections are not available, this would be difficult to test. Considering that no functional measures of the hippocampal detections exist, we would suggest that this possible caveat be mentioned for this particular experiment.

In addition, without any quantification or statistical analysis, the author's claims regarding the necessity of GLRA1 expression for the synaptic localization of glycine receptors in cultured hippocampal neurons should probably be described as preliminary (Fig. 5).

• Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

The authors show that there is colocalization of gephyrin with the mEos4b-GlyRβ subunit using the Dual-colour SMLM. This is a powerful approach that allows for a claim to be made on the synaptic location of the glycine receptors. The images presented in Figure 1, together with the distance analysis in Figure 2, display the co-localization of the fluorophores. The co-localization images in all the selected regions, hippocampus and striatum, also show detections outside of the gephyrin clusters, which the authors refer to as extrasynaptic. These punctated small clusters seem to have the same size as the ones detected and assigned as part of the synapse. It would be informative if the authors analysed the distribution, density and size of these non-synaptic clusters and presented the data in the manuscript and also compared it against the synaptic ones. Validating this extrasynaptic signal by staining for a dendritic marker, such as MAP-2 or maybe a somatic marker and assessing the co-localization with the non-synaptic clusters would also add even more credibility to them being extrasynaptic.

In addition we would encourage the authors to quantify the clustering and co-localization of virally expressed GLRA1, GLRA2, and GLRB with gephyrin in order to support the associated claims (Fig. 5). Preferably, the density of GLR and gephyrin clusters (at least on the somatic surface, the proximal dendrites, or both) as well as their co-localization probability should be quantified if a causal claim about subunit-specific requirements for synaptic localization is to be made.

Lastly, even though it may be outside of the scope of such a study analysing other parts of the hippocampal area could provide additional important information. If one looks at the Allen Institute's ISH of the beta subunit the strongest signal comes from the stratum oriens in the CA1 for example, suggesting that interneurons residing there would more likely have a higher expression of the glycine receptors. This could also be assessed by looking more carefully at the single cell transcriptomics, to see which cell types in the hippocampus show the highest mRNA levels. If the authors think that this is too much additional work, then perhaps a mention of this in the discussion would be good.

• Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

Since the labeling and some imaging has been performed already, the requested experiment would be a matter of deploying a method of quantification. In principle, it should not require any additional wet-lab experiments, although it may require additional imaging of existing samples.

• Are the data and the methods presented in such a way that they can be reproduced?

Yes, for the most part.

• Are the experiments adequately replicated and statistical analysis adequate?

Yes

Minor comments: - Specific experimental issues that are easily addressable.

N/A

• Are prior studies referenced appropriately?

Yes

• Are the text and figures clear and accurate?

Yes, although quantification in figure 5 is currently not present.

• Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

This paper presents a method that could be used to localize receptors and perhaps other proteins that are in low abundance or for which a detailed quantification is necessary. I would therefore suggest that Figure S4 is included into Figure 2 as the first panel, showcasing the demixing, followed by the results.

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

Using a novel and high resolution method, the authors have provided strong evidence for the presence of glycine receptors in the murine hippocampus and in the dorsal striatum. The number of receptors calculated is small compared to the numbers found in the ventral striatum. This is the first study to quantify receptor numbers in these region. In addition it also lays a roadmap for future studies addressing similar questions.

• Place the work in the context of the existing literature (provide references, where appropriate).

This is done well by the authors in the curation of the literature. As stated above, the authors have filled a gap in the presence of glycine receptors in different brain regions, a subject of importance in understanding the role they play in brain activity and function.

• State what audience might be interested in and influenced by the reported findings.

Neuroscientists working at the synaptic level, on inhibitory neurotransmission and on fundamental mechanisms of expression of genes at low levels and their relationship to the presence of the protein would be interested. Furthermore, researchers in neuroscience and cell biology may benefit from and be inspired by the approach used in this manuscript, to potentially apply it to address their own aims.

• Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

Synaptic transmission, inhibitory cells and GABAergic synapses functionally and structurally, cortex and cortical circuits. No strong expertise in super-resolution imaging methods.

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

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

In this study, Camuso et al., make use of a knock-in mouse model expressing endogenously mEos4b-tagged GlyRβ to detect endogenous glycine receptors using single-molecule localization microscopy. The main conclusion from this study is that in the hippocampus GlyRβ molecules are barely detected, while inhibitory synapses in the ventral striatum seem to express functionally relevant GlyR numbers.

I have a few points that I hope help to improve the strength of this study.

• In the hippocampus, this study finds that the numbers of detections are very low. The authors perform adequate controls to indicate that these localizations are above noise level. Nevertheless, it remains questionable that these reflect proper GlyRs. The suggestion that in hippocampal synapses the low numbers of GlyRβ molecules "are important in assembly or maintenance of inhibitory synaptic structures in the brain" is on itself interesting, but is not at all supported. It is also difficult to envision how such low numbers could support the structure of a synapse. A functional experiment showing that knockdown of GlyRs affects inhibitory synapse structure in hippocampal neurons would be a minimal test of this.
• The endogenous tagging strategy is a very strong aspect of this study and provides confidence in the labeling of GlyRβ molecules. One caveat however, is that this labeling strategy does not discriminate whether GlyRβ molecules are on the cell membrane or in internal compartments. Can the authors provide an estimate of the ratio of surface to internal GlyRβ molecules?
• 'We also estimated the absolute number of GlyRs per synapse in the hippocampus. The number of mEos4b detections was converted into copy numbers by dividing the detections at synapses by the average number of detections of individual mEos4b-GlyRβ containing receptor complexes'. In essence this is a correct method to estimate copy numbers, and the authors discuss some of the pitfalls associated with this approach (i.e., maturation of fluorophore and detection limit). Nevertheless, the authors did not subtract the number of background localizations determined in the two negative control groups. This is critical, particularly at these low-number estimations. Furthermore, the authors state that "The advantage of this estimation is that it is independent of the stoichiometry of heteropentameric GlyRs". However, if the stoichometry is unknown, the number of counted GlyRβ subunits cannot simply be reported as the number of GlyRs. This should be discussed in more detail, and more carefully reported throughout the manuscript.
• The dual-color imaging provides insights in the subsynaptic distribution of GlyRβ molecules in hippocampal synapses. Why are similar studies not performed on synapses in the ventral striatum where functionally relevant numbers of GlyRβ molecules are found? Here insights in the subsynaptic receptor distribution would be of much more interest as it can be tight to the function.
• It is unclear how the experiments in Figure 5 add to this study. These results are valid, but do not seem to directly test the hypothesis that "the expression of α subunits may be limiting factor controlling the number of synaptic GlyRs". These experiments simply test if overexpressed α subunits can be detected. If the α subunits are limiting, measuring the effect of α subunit overexpression on GlyRβ surface expression would be a more direct test.

Significance

These results are based on carefully performed single-molecule localization experiments, and are well-presented and described. The knockin mouse with endogenously tagged GlyRβ molecules is a very strong aspect of this study and provides confidence in the labeling, the combination with single-molecule localization microscopy is very strong as it provides high sensitivity and spatial resolution.

The conceptual innovation however seems relatively modest, these results confirm previous studies but do not seem to add novel insights. This study is entirely descriptive and does not bring new mechanistic insights.

This study could be of interest to a specialized audience interested in glycine receptor biology, inhibitory synapse biology and super-resolution microscopy.

my expertise is in super-resolution microscopy, synaptic transmission and plasticity

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Referee #2

Evidence, reproducibility and clarity

In their manuscript "Single molecule counting detects low-copy glycine receptors in hippocampal and striatal synapses" Camuso and colleagues apply single molecule localization microscopy (SMLM) methods to visualize low copy numbers of GlyRs at inhibitory synapses in the hippocampal formation and the striatum. SMLM analysis revealed higher copy numbers in striatum compared to hippocampal inhibitory synapses. They further provide evidence that these low copy numbers are tightly linked to post-synaptic scaffolding protein gephyrin at inhibitory synapses. Their approach profits from the high sensitivity and resolution of SMLM and challenges the controversial view on the presence of GlyRs in these formations although there are reports (electrophysiology) on the presence of GlyRs in these particular brain regions. These new datasets in the current manuscript may certainly assist in understanding the complexity of fundamental building blocks of inhibitory synapses.

However I have some minor points that the authors may address for clarification:

1. In Figure 1 the authors apply PALM imaging of mEos4b-GlyRß (knockin) and here the corresponding Sylite label seems to be recorded in widefield, it is not clearly stated in the figure legend if it is widefield or super-resolved. In Fig 1 A - is the scale bar 5 µm? Some Sylite spots appear to be sized around 1 µm, especially the brighter spots, but maybe this is due to the lower resolution of widefield imaging? Regarding the statistical comparison: what method was chosen to test for normality distribution, I think this point is missing in the methods section. Moreover I would appreciate a clarification and/or citation that the knockin model results in no structural and physiological changes at inhibitory synapses, I believe this model has been applied in previous studies and corresponding clarification can be provided.
2. In the next set of experiments the authors switch to demixing dSTORM experiments - an explanation why this is performed is missing in the text - I guess better resolution to perform more detailed distance measurements? For these experiments: which region of the hippocampus did the authors select, I cannot find this information in legend or main text.
3. Regarding parameters of demixing experiments: the number of frames (10.000) seems quite low and the exposure time higher than expected for Alexa 647. Can the authors explain the reason for chosing these particular parameters (low expression profile of the target - so better separation?, less fluorophores on label and shorter collection time?) or is there a reference that can be cited? The laser power is given in the methods in percentage of maximal output power, but for better comparison and reproducibility I recommend to provide the values of a power meter (kW/cm2) as lasers may change their maximum output power during their lifetime.
4. For analysis of subsynaptic distribution: how did the authors decide to choose the parameters in the NEO software for DBSCAN clustering - was a series of parameters tested to find optimal conditions and did the analysis start with an initial test if data is indeed clustered (K-ripley) or is there a reference in literature that can be provided?
5. A conclusion/discussion of the results presented in Figure 5 is missing in the text/discussion.
6. in line 552 "suspension" is misleading, better use "solution"

Significance

Significance: The manuscript provides new insights to presence of low-copy numbers by visualizing them via SMLM. This is the first report that visualizes GlyR optically in the brain applying the knock-in model of mEOS4b tagged GlyRß and quantifies their copy number comparing distribution and amount of GlyRs from hippocampus and striatum. Imaging data correspond well to electrophysiological measurements in the manuscript.

Field of expertise: Super-Resolution Imaging and corresponding analysis

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Referee #1

Evidence, reproducibility and clarity

In this manuscript, the authors investigate the nanoscopic distribution of glycine receptor subunits in the hippocampus, dorsal striatum, and ventral striatum of the mouse brain using single-molecule localization microscopy (SMLM). They demonstrate that only a small number of glycine receptors are localized at hippocampal inhibitory synapses. Using dual-color SMLM, they further show that clusters of glycine receptors are predominantly localized within gephyrin-positive synapses. A comparison between the dorsal and ventral striatum reveals that the ventral striatum contains approximately eight times more glycine receptors and this finding is consistent with electrophysiological data on postsynaptic inhibitory currents. Finally, using cultured hippocampal neurons, they examine the differential synaptic localization of glycine receptor subunits (α1, α2, and β). This study is significant as it provides insights into the nanoscopic localization patterns of glycine receptors in brain regions where this protein is expressed at low levels. Additionally, the study demonstrates the different localization patterns of GlyR in distinct striatal regions and its physiological relevance using SMLM and electrophysiological experiments. However, several concerns should be addressed.

The following are specific comments:

1. Colocalization analysis in Figure 1A. The colocalization between Sylite and mEos-GlyRβ appears to be quite low. It is essential to assess whether the observed colocalization is not due to random overlap. The authors should consider quantifying colocalization using statistical methods, such as a pixel shift analysis, to determine whether colocalization frequencies remain similar after artificially displacing one of the channels.
2. Inconsistency between Figure 3A and 3B. While Figure 3B indicates an ~8-fold difference in the number of mEos4b-GlyRβ detections per synapse between the dorsal and ventral striatum, Figure 3A does not appear to show a pronounced difference in the localization of mEos4b-GlyRβ on Sylite puncta between these two regions. If the images presented in Figure 3A are not representative, the authors should consider replacing them with more representative examples or providing an expanded images with multiple representative examples. Alternatively, if this inconsistency can be explained by differences in spot density within clusters, the authors should explain that.
3. Quantification in Figure 5. It is recommended that the authors provide quantitative data on cluster formation and colocalization with Sylite puncta in Figure 5 to support their qualitative observations.
4. Potential for pseudo replication. It's not clear whether they're performing stats tests across biological replica, images, or even synapses. They often quote mean +/- SEM with n = 1000s, and so does that mean they're doing tests on those 1000s? Need to clarify.
5. Does mEoS effect expression levels or function of the protein? Can't see any experiments done to confirm this. Could suggest WB on homogenate, or mass spec?
6. Quantification of protein numbers is challenging with SMLM. Issues include i) some of FP not correctly folded/mature, and ii) dependence of localisation rate on instrument, excitation/illumination intensities, and also the thresholds used in analysis. Can the authors compare with another protein that has known expression levels- e.g. PSD95? This is quite an ask, but if they could show copy number of something known to compare with, it would be useful.
7. Rationale for doing nanobody dSTORM not clear at all. They don't explain the reason for doing the dSTORM experiments. Why not just rely on PALM for coincidence measurements, rather than tagging mEoS with a nanobody, and then doing dSTORM with that? Can they explain? Is it to get extra localisations- i.e. multiple per nanobody? If so, localising same FP multiple times wouldn't improve resolution. Also, no controls for nanobody dSTORM experiments- what about non-spec nb, or use on WT sections?
8. What resolutions/precisions were obtained in SMLM experiments? Should perform Fourier Ring Correlation (FRC) on SR images to state resolutions obtained (particularly useful for when they're presenting distance histograms, as this will be dependent on resolution). Likewise for precision, what was mean precision? Can they show histograms of localisation precision.
9. Why were DBSCAN parameters selected? How can they rule out multiple localisations per fluor? If low copy numbers (<10), then why bother with DBSCAN? Could just measure distance to each one.
10. For microscopy experiment methods, state power densities, not % or "nominal power".
11. In general, not much data presented. Any SI file with extra images etc.?
12. Clarification of the discussion on GlyR expression and synaptic localization: The discussion on GlyR expression, complex formation, and synaptic localization is sometimes unclear, and needs terminological distinctions between "expression level", "complex formation" and "synaptic localization". For example, the authors state:"What then is the reason for the low protein expression of GlyRβ? One possibility is that the assembly of mature heteropentameric GlyR complexes depends critically on the expression of endogenous GlyR α subunits." Does this mean that GlyRβ proteins that fail to form complexes with GlyRα subunits are unstable and subject to rapid degradation? If so, the authors should clarify this point. The statement "This raises the interesting possibility that synaptic GlyRs may depend specifically on the concomitant expression of both α1 and β transcripts." suggests a dependency on α1 and β transcripts. However, is the authors' focus on synaptic localization or overall protein expression levels? If this means synaptic localization, it would be beneficial to state this explicitly to avoid confusion. To improve clarity, the authors should carefully distinguish between these different aspects of GlyR biology throughout the discussion. Additionally, a schematic diagram illustrating these processes would be highly beneficial for readers.
13. Interpretation of GlyR localization in the context of nanodomains. The distribution of GlyR molecules on inhibitory synapses appears to be non-homogeneous, instead forming nanoclusters or nanodomains, similar to many other synaptic proteins. It is important to interpret GlyR localization in the context of nanodomain organization.

Significance

The paper presents biological and technical advances. The biological insights revolve mostly on the documentation of Glycine receptors in particular synapses in forebrain, where they are typically expressed at very low levels. The authors provide compelling data indicating that the expression is of physiological significance. The authors have done a nice job of combining genetically-tagged mice with advanced microscopy methods to tackle the question of distributions of synaptic proteins. Overall these advances are more incremental than groundbreaking.

for - book - More Everything Forever - Adam Becker - from - Youtube - Essentia Foundation - interview - Alex Gomez-Marin - Neuroscientist speaks out on the hidden war on consciousness - https://hyp.is/ile8TIvJEfCl35MW3f5B8Q/www.youtube.com/watch?v=K7NIicE_h9w

Summary - Interesting adjacency with another video I've been watching, that focused on a Western monk's practice of Tibetan Buddhism, who after 12 years, entered a 4 year retreat and panicked - His demons emerged in the first 2 years of the retreat and he left but returned - This monk emphasized accepting the relationship with his demons instead of averting them and how craving and desire emphasized by Western civilllization is the cause of modernity's meaning crisis - to - Youtube - Diary of a CEO - Your brain is lying to you - Interview - Gerong Tupton - https://hyp.is/go?url=https%3A%2F%2Fwww.youtube.com%2Fwatch%3Fv%3DvIbLQQ1i56Y&group=world

for - book - More Everything Forever - Adam Becker - from - Youtube - Essentia Foundation - interview - Alex Gomez-Marin - Neuroscientist speaks out on the hidden war on consciousness - https://hyp.is/ile8TIvJEfCl35MW3f5B8Q/www.youtube.com/watch?v=K7NIicE_h9w

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Reply to the reviewers

Manuscript number: RC-2025-03111

Corresponding author(s): Qingyin Qian and Ryusuke Niwa

1. General Statements [optional]

We would like to thank reviewers for their feedback on our initial submission. Changes in figures were noted in the point-to-point reply. For submission of our current revised manuscript, we provide two Word files, which are the “clean” and “Track-and-Change” files. Page and line numbers described below correspond to those of the “clean” file. The “Track-and-Change” file might be helpful for Reviewers to find what we have changed for the current revision.

In the revised manuscript, major changes in the text were tracked, while minor edits in figure numbers and legends were not tracked. In the Discussion, the section “Xrp1-mediated EE plasticity…” was moved before “Xrp1, a transcription factor …”, to follow the order of the Results, and was split into two: “EE plasticity …” and “Xrp1-mediated EE plasticity …”.

2. Description of the planned revisions

- The authors should investigate the regenerative growth of the adult midgut after irradiation. Is there an impact on ISCs proliferation or cell turn over. Is Xrp1 in EEs required in this adaptive response. It would be elegant to use the recently generated tracing method by Tobias Reiff lab to observe overall impact on tissue renewal (rapport-tracing esglexReDDM esg-lexA, 13xLexAop2-CD8::GFP, 13xLexAop2-H2B::mCherry::HA, tub-Gal80ts on the second chromosome. It can be combined with any EEs Gal4-driver (see Nat Commun 2025, https://doi.org/10.1038/s41467-024-55664-2, the stock is already existing, see table1). This reviewer thinks that it is a key experiment to support the proposed model.

2.1. Author response:

We will conduct the following experiments to answer these criticisms.

(1) We will investigate the ISC behavior, proliferation and differentiation, after 100 Gy of radiation by examining changes in the number of progenitor cells and their progenies, using esgtsF/O (esg-Gal4, UAS-GFP, tub-Gal80ts; Act>Cd2>Gal4, UAS-Flp) generated in the study (Jiang et al. Cell 2009 DOI: 10.1016/j.cell.2009.05.014) or esgReDDM (esg-Gal4, UAS-CD8::GFP; UAS-H2B::RFP, tubGal80ts) generated in the study (Antonello et al. EMBO J. 2015 DOI: 10.15252/embj.201591517). Flies will have progenitor cell lineages traced for 7 days, irradiated on day 6, and examined at different time points after radiation, following the design shown in Fig. 2A. Based on the previous findings (Sharma et al. Sci. Rep. 2020 DOI: 10.1038/s41598-020-75867-z; Pyo et al. Radiat. Res. 2014 DOI: 10.1667/RR13545.1), we anticipate that radiation compromises ISCs’ proliferation and differentiation. Should this be the case, our results can be interpreted in relation to those earlier studies.

(2) In parallel, we will examine whether Xrp1 expression in EEs affects radiation-induced ISC behaviors. As suggested, we will use “EE Rapport” (esg-lexA, 13xLexAop2-CD8::GFP, 13xLexAop2-H2B::mCherry::HA, tub-Gal80ts; Rab3-Gal4) generated in the study (Zipper et al. Nat. Commun. 2025 DOI: 10.1038/s41467-024-55664-2) and compare control flies to flies with Xrp1 knocked down in EEs to assess the impact on ISC behaviors.

- Is p53 required for Xrp1 induction in the gut after irradiation?

2.2. Author response:

To answer this point, we will perform immunostaining of anti-Xrp1 antibody to examine whether p53 is required for Xrp1 induction in irradiated flies with p53 knocked down in EEs.

- Xrp1 over expression has been shown to induce upd3 ligand and nutrient-driven dedifferentiation of enteroendocrine cells is occuring by activation of the JAK-STAT pathway (DOI: 10.1016/j.devcel.2023.08.022). Could the authors test the function of this signaling pathway during irradiation (upd3-lacZ and Stat-GFP can be used in parallel of upd3 RNAi and UAS Dome-DN.

2.3. Author response:

We will conduct the following experiments to answer these points.

(1) We will examine the cell type in which upd3 ligand induction occurs after radiation by using the upd3.1-LacZ reporter generated in the study (Jiang et al. Cell Stem Cell 2011 DOI: doi.org/10.1016/j.stem.2010.11.026).

(2) One possibility is that upd3.1-LacZ is detected in EEs. In this case, we will examine the requirement of upd3 in EEs for radiation-induced EE plasticity by knocking down upd3. Another possibility is that upd3.1-LacZ is detected in non-EE cells. If so, we will examine the requirement of the JAK-STAT pathway in EEs by overexpressing dome[△cyt] generated in the study (Brown et al. Curr. Biol. 2001 DOI: 10.1016/s0960-9822(01)00524-3) or knocking down Stat92E in EEs. Because these conditions are not mutually exclusive, both approaches may be pursued, with the latter relating our results to nutrient-driven EE dedifferentiation.

- Xrp1 is known for its role in cell competition and elimination of looser cells by induction of apoptosis. It would be interesting to check for induction of cell death and/or caspase activation in the fly gut after irradiation and verify a non apoptotic role of DRONC activation in this context using a Dronc RNAi (as proposed by Bergmann lab (https://doi.org/10.1038/s41598-021-81261-0) or Baena-Lopez lab (DOI: 10.15252/embr.201948892)). Overexpression of Xrp1 could be combined with UAS-p35.

2.4. Author response:

To address these points, we will investigate apoptosis induction following radiation with anti-cleaved Dcp-1 immunostaining. Based on the previous finding (Sharma et al. Sci. Rep. 2020 DOI: 10.1038/s41598-020-75867-z), we anticipate seeing increased cleaved Dcp-1 signals in all cell types after radiation. We intend to clarify whether radiation increases the ratio of apoptotic EEs among EEs; however, we cannot yet be certain whether it will be feasible.

Regarding Dronc activation, we previously requested the antibody used in the study (Wilson et al. Nat. Cell Biol. 2002 DOI: 10.1038/ncb799; Lindblad et al. Sci. Rep. 2021 DOI: 10.1038/s41598-021-81261-0) and tested it in our context, after radiation and by Xrp1-S O/E in EEs. We present our data below. In the anterior midgut, anti-Dronc signals were not observed under both control conditions. After radiation and by Xrp1-S O/E in EEs, anti-Dronc signals were seen in part of past EEs (#2 past) and progenitor cells (#3 prgn), implying their EB identity. However, anti-Dronc signals were never observed in current EEs (#1 current), suggesting Dronc does not act directly downstream to Xrp1.

We will address UAS-p35 in 3.3. Author response and Dronc-RNAi in 4.2. Author response.

- The authors do not justify or explain why they used 100 Gy of radiation. This is higher than doses used in comparable regeneration studies in adult Drosophila (e.g., PMID25959206, PMID: 28925355). The authors should clarify why this dose was chosen.

2.5. Author response:

Our initial rationale was based on the paper (Sharma et al. Sci. Rep. 2020 DOI: 10.1038/s41598-020-75867-z), where the authors claimed that ISC proliferation was inhibited and the ISC number was decreased by 100 Gy of radiation.

Nevertheless, we understand the reviewer’s concern and will examine 50 Gy of radiation as used in the papers the reviewer listed. We will examine radiation-induced changes in EE lineages and ISC behaviors. Depending on the results, we will evaluate whether and how they should be incorporated into the manuscript.

- Fig. 2C, the number of past EE’s increased transiently so that baseline number is restored at 18 hr after IR. The authors conclude that fate plasticity is a transient event. Can they rule out loss due to cell death?

2.6. Author response:

In our system, past EEs were detected transiently but did not persist. We agree that we cannot distinguish whether the transient appearance of past EEs reflects transient adoption of another identity that ends in cell death or reversible plasticity.

To partially address this criticism, as noted in 2.4. Author response, we will examine the apoptosis marker cleaved Dcp-1, which also tests whether cleaved Dcp-1-positive cells can be past EEs. However, regardless of detecting apoptosis markers in past EEs, we have changed “transient” into “temporary” to describe a short-lived cell state (see Page 8, Line 178; Page 15, Line 338).

- They authors interpret fate-conversion as beneficial for tissue repair but never test whether blocking this process impairs recovery or organismal survival or whether promoting it improves outcomes.

2.7. Author response:

We have removed this potentially misleading interpretation (see Page 4, removed the last part of the previous introduction, “and propose the possibility that such plasticity contributes to tissue repair”). We present below the data showing a severe reduction of the ISC number in 7-day post-radiation guts, suggesting the inability of tissue repair. We will add this to the manuscript together with results from the following experiments.

(1) We will examine if the blockage of radiation-induced EE plasticity, via knocking down Xrp1 in EEs, alters the epithelial cell number and cell junction protein localization.

(2) To complement the result of plasticity inhibition, we attempt to promote plasticity by overexpressing Xrp1 in EEs, to test whether this rescues ISC loss or restores junctions.

Should knockdown worsen ISC loss and junction integrity, or overexpression rescue them, we will describe EE plasticity as beneficial; otherwise, we will present it as a radiation-induced response without inferring benefits, while noting our limitations.

We will address organismal survival in 4.3. Author response.

- Related to the above, it would be helpful to know if fate-converted cells function as true ISCs or ECs (e.g., through proliferation or absorption assays).

2.8. Author response:

To partially answer this criticism, we will examine whether EE-derived ISCs are proliferative by examining whether they can be positive for the mitotic marker phospho-histone 3.

We will address absorption assays in 4.4. Author response.

3. Description of the revisions that have already been incorporated in the transferred manuscript

- It is surprising to observe EEs dedifferentiation at a steady state during homeostasis, a condition in which Xrp1 is not detected in the gut. Can the authors comment this point in the discussion?

3.1. Author response:

We have added our thoughts in terms of Xrp1 being not detectable in homeostatic EE lineages (see Page 15, Line 350 - 356). We have also added our thoughts regarding observation of EE plasticity in homeostatic guts (see Page 14, Line 322 - 332).

- Xrp1 is existing as a short of long isoforms. The short form has been recently proposed to be required for cell competition (https://doi.org/10.1101/2025.06.15.659587) whereas Xrp1 long isoform may be responsible for reduced cell growth. Could the authors test which isoform is induced in the gut after irradiation? Is the overexpression of Xrp1 long isoform having the same effect that the short isoform used by the authors.

3.2. Author response:

We have added data on the effect of Xrp1 long isoform overexpression on EE plasticity (see Fig. 5A - 5B, Page 12, Line 276 - 278), showing that overexpression of the Xrp1 long isoform caused a similar increase in past EEs. In addition, we have changed Xrp1 O/E to Xrp1-S O/E in the contents related to Figs 4, 5, S4, and S5.

We will address radiation-induced Xrp1 isoforms in 4.1. Author response.

- Xrp1 is known for its role in cell competition and elimination of looser cells by induction of apoptosis. It would be interesting to check for induction of cell death and/or caspase activation in the fly gut after irradiation and verify a non apoptotic role of DRONC activation in this context using a Dronc RNAi (as proposed by Bergmann lab (https://doi.org/10.1038/s41598-021-81261-0) or Baena-Lopez lab (DOI: 10.15252/embr.201948892)). Overexpression of Xrp1 could be combined with UAS-p35.

3.3. Author response:

We have added data regarding p35 O/E combined with Xrp1 O/E, showing that p35 O/E did not further increase the number of past EEs, thereby suggesting that Xrp1-driven EE plasticity has a non-apoptotic nature (see Fig. 5C - 5D, Page 13, Line 293 - 297).

- Line 221: fig S3E should be S3F

- Line 230: fig S3F-G should be S3G-H

• • Line 230, Fig S3F-G should be Fig S3G-H.*

3.4. Author response:

We have fixed this error.

- The posterior gut region R4 is more proliferative than the anterior part and is usually used for testing regenerative growth. What is happening there after irradiation?

3.5. Author response:

We present below radiation-induced changes in EE lineages and ISC number in the R4bc gut region. Radiation did not alter the proportion of past EEs among EE lineages but reduced the ISC number. We acknowledge differences between anterior and posterior gut regions, but we do not plan to further analyze regional differences or underlying mechanisms.

- The authors’ explanation for cells with weak GFP in Figure 1 is not convincing. Induction of GFP is an all or nothing event as it results from Pros-driven FLPase and a recombination that removes the transcription stop signals to express GFP from a Ubi promotor. Once that happens, it should not matter how strong or weak Pros is, GFP should be the same. So, another explanation is needed. Nuclear staining of cell #2 in Fig 1B resembles a metaphase chromosome arrangement. Nuclear GFP may appear ‘weak’ in mitosis as the nuclear envelope breaks down. It is positive for the purple Pros/Dl stain, which makes it hard to tell if it is Pros+ or Pros- even though the authors state that cells with weak GFP are Pros- in line 104 (see the point above regarding confusing same-color stain for ISC and EE markers). Could cell #2 be a pre-EE that is undergoing mitosis since the lineage tracer marks both EE and pre-EE cells (line 119)? Or do the authors mean recombination on one or both homologs? This should not be possible since the cells are heterozygotes for the Ubi-GFP locus.

3.6. Author response:

For cell #5, RFP- GFPweak may result from the leakiness of the G-TRACE system. We have added our observations of the G-TRACE strains and changed our previous explanation (see Fig. S1B - S1C, Page 5, Line 94 - 97, 103 - 106).

For cell #2, we agree that RFP+ GFPweak cells may either be a cell turning on pros expression just before sample preparation or a pre-EE undergoing mitosis. Nevertheless, it is not a past EE that has lost the EE marker Pros, so it is considered a current EE. We have removed our previous interpretation of cell #2 (see Page 5, removed “which likely had not yet fully activated recombination”), and changed the image to avoid confusion (see Fig. 1C).

- Fig. 2C, if past-EE’s increased in number while current EE’s stayed the same, where are new past-EE’s coming from? There cannot be compensatory proliferations since EE’s are post-mitotic. For fate conversion, one would expect the generation of each past-EE to accompany loss of one current EE.

3.7. Author response:

We agree that the generation of one past EE should be accompanied by the loss of one current EE. We do not have a clear answer to this question. Our data showed cell numbers per ROI rather than the total cell number across the whole gut. To address this, we have changed the number to the proportion, calculated from [past EE] / ([past EE] + [current EE]), in experiments examining damage-induced EE plasticity, which provides a more informative measure for EE fate conversion (see Fig. 2C, also Fig. S2B and 3E).

- Fig. 2E. Dl+ past-EE cell number declined at 14 and 18 h after IR and because cell sized increased, the authors conclude that EE cells that de-differentiated into ISCs subsequently re-differentiated into EC’s. To reach this conclusion, the authors should count past-EEs that are positive for EC markers. Cell size alone is insufficient evidence.

3.8. Author response:

We have added data quantifying the proportion of past EEs that are positive for the EC marker Pdm1, showing that past EEs were more likely to be ECs in guts examined 14 h after radiation (see Fig. 2F - 2G, Page 9, Line 189).

- Fig. 6. Where are the % numbers for ISC, EB and EE’s coming from? And wouldn’t these change with time after IR, etc?

3.9. Author response:

The numbers came from the calculation of the percentage of the absolute values of control and 14 h post-IR conditions from Fig. 2E. These numbers changed with time after radiation. We realized that the precise numbers were misleading. We therefore have removed such illustration and instead added phrases “more current EEs → past EEs, more past EEs being ISCs → past EEs being ECs” to describe the increase in past EE cell number and the shift in the composition of past EEs (see Fig. 6).

- Improve Figure 1B: Pros and Dl are shown in the same color, creating confusion. If both are stained together, different colors or clearer labeling should be used. Clarify how cells are identified as Pros+ vs Dl+.

3.10. Author response:

Anti-Pros and anti-Dl antibodies were produced from the same host species and were detected with the same secondary antibody, so they were in the same color. We have stated that solid nuclear staining indicates Pros, whereas punctate cytoplasmic staining indicates Dl (see Page 5, Line 100, 102, and 103). Such staining has been reported in previous studies (for example, Fig. 2A - 2B, Veneti et al. Nat. Commun. 2024 DOI: 10.1038/s41467-024-46119-9).

- Why is Dl (supposed to be cytoplasmic) overlapping with nuclear GFP in cells #3 and 4 in Fig. 1B?

3.11. Author response:

Because Dl signals were located apically to DAPI/GFP signals, the overlap was likely due to Z-projection from stacked slices. We present below orthogonal slices along the z-axis, from top to bottom by row, and composite and individual color channels, from left to right by columns, for cell #3 (left) and cell #4 (right).

For cell #3, Dl signals were present in slices 1/8 and 2/8 and disappeared in slice 3/8, whereas DAPI signals appeared from slice 2/8. For cell #4, Dl signals surrounded DAPI signals when viewed separately. In addition, we realized that nuclear GFP signals slightly outgrew DAPI signals, despite our confirmation that the GFP channel was not saturated.

We have included separate color channels for DAPI signals and Pros, Dl and DAPI merged channels, showing that Dl signals were absent from the nucleus. For cell #3, in which the nuclear DAPI and cytoplasmic Dl cannot be distinguished in the stacked view, we show the images from a single orthogonal slice in the main panel, and the image from stacked slices as insets (see Fig. 1C).

- Fig. S1E and F. Very hard to see what the authors describe about Arm and Cora. One problem is that cell boundaries are not visible, just the nuclei, so it is hard to know whether cell-cell interactions the authors describe as normal are really normal. Another problem is the overlap of Arm (supposed to be cytoplasmic) with the nuclear GFP signal. What is that?

3.12. Author response:

Regarding the invisibility of cell boundaries, we have improved the image of anti-Cora staining and added anti-Mesh staining and a separate color channel for DAPI signals to reinforce junction integrity (see Fig. S1H - S1I).

Regarding the overlap of Arm signals with nuclear GFP signals, we realized similar problems as those noted in 3.11. Author response. We present below orthogonal slices along the z-axis and combined and individual color channels, for cell #2 (left) and cell #3 (right). For both cells, Arm signals did not overlap with DAPI signals. We have adjusted the maximum intensity projection to include slices 1-4 instead of 1-8 and added a separate color channel for DAPI signals to avoid the signals appearing to overlap (see Fig. S1G).

- Include a simple schematic of ISC to EE/EC lineages for readers unfamiliar with Drosophila gut biology.

3.13. Author response:

We have included a schematic (see Fig. 1A). Although not requested, we have also improved Fig. 1B to enhance clarity.

- Discuss the regional difference in Xrp1 efficacy (R2a vs R2b). Is there something known about gene expression differences in different gut regions that can explain the results?

3.14. Author response:

At present, we do not have an explanation for these results. We have refined our discussion regarding such regional differences (see Page 16 - 17, Line 381 - 390).

- Consider moving scRNAseq (Fig. S1G) into main paper: this is a central part of the conclusion.

3.15. Author response:

We have moved Fig. S1G, as well as Fig. S1H and S1I, into the main figure (see Fig. 1G - 1I).

4. Description of analyses that authors prefer not to carry out

- Xrp1 is existing as a short of long isoforms. The short form has been recently proposed to be required for cell competition (https://doi.org/10.1101/2025.06.15.659587) whereas Xrp1 long isoform may be responsible for reduced cell growth. Could the authors test which isoform is induced in the gut after irradiation? Is the overexpression of Xrp1 long isoform having the same effect that the short isoform used by the authors.

4.1. Author response:

We prefer not to distinguish whether the long or short Xrp1 isoform is induced in the gut after radiation. This presents technical challenges and falls outside the scope of the present study. As noted in 3.2. Author response, we instead report in the revised manuscript that both isoforms similarly promote EE plasticity.

- Xrp1 is known for its role in cell competition and elimination of looser cells by induction of apoptosis. It would be interesting to check for induction of cell death and/or caspase activation in the fly gut after irradiation and verify a non apoptotic role of DRONC activation in this context using a Dronc RNAi (as proposed by Bergmann lab (https://doi.org/10.1038/s41598-021-81261-0) or Baena-Lopez lab (DOI: 10.15252/embr.201948892)). Overexpression of Xrp1 could be combined with UAS-p35.

4.2. Author response:

We prefer not to perform Dronc-RNAi, because we did not observe Dronc activation downstream to Xrp1, as shown in 2.4. Author response.

- They authors interpret fate-conversion as beneficial for tissue repair but never test whether blocking this process impairs recovery or organismal survival or whether promoting it improves outcomes.

4.3. Author response:

We prefer not to examine organismal survival. We agree that organismal survival would be informative, but our study focuses on epithelial cell number, which will be tested as noted in 2.7. Author response. We will not mention broad claims at the organismal level.

- Related to the above, it would be helpful to know if fate-converted cells function as true ISCs or ECs (e.g., through proliferation or absorption assays).

4.4. Author response:

We prefer not to perform absorptive assays due to technical challenges. We will instead test proliferation, as noted in 2.8. Author response, and note our limitations.

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Referee #2

Evidence, reproducibility and clarity

Summary

Qian and colleagues report a study on radiation induced cell fate plasticity in the intestine of Drosophila. Using lineage tracing to mark pre-EE and EE cells, the authors how that these cells can lose EE/pre-EE marker Pros and express ISC or EC markers, indicating fate conversion. Single cell RNAseq analysis showed that even under basal conditions, ISC/EB cell population includes those with EE/pre-EE lineage tracer, confirming fate conversion. The same analysis showed that fate converted ISC/EB cells express transcription factor Ets21C, which is associated with regeneration but not normal development. Exposure to ionizing radiation (IR) increases the frequency of fate conversion and accompanies the induction of Xrp1 (which is not expressed normally in the EE's). Xrp1 knock down reduced IR-induced fate conversion, demonstrating necessity. Xrp1 is also sufficient because overexpression of it resulted in increased fate conversion without IR. scRNAseq analysis showed that overexpression of Xrp1 in pre-EE/EE cells (without IR) resulted in the induction of ISC/progenitor state genes such as esg and Sox homologs. Functional testing of the latter group of genes demonstrated their essential role in cell fate plasticity induced by Xrp1.

Major comments

• The authors do not justify or explain why they used 100 Gy of radiation. This is higher than doses used in comparable regeneration studies in adult Drosophila (e.g., PMID25959206, PMID: 28925355). The authors should clarify why this dose was chosen.
• The authors' explanation for cells with weak GFP in Figure 1 is not convincing. Induction of GFP is an all or nothing event as it results from Pros-driven FLPase and a recombination that removes the transcription stop signals to express GFP from a Ubi promotor. Once that happens, it should not matter how strong or weak Pros is, GFP should be the same. So, another explanation is needed. Nuclear staining of cell #2 in Fig 1B resembles a metaphase chromosome arrangement. Nuclear GFP may appear 'weak' in mitosis as the nuclear envelope breaks down. It is positive for the purple Pros/Dl stain, which makes it hard to tell if it is Pros+ or Pros- even though the authors state that cells with weak GFP are Pros- in line 104 (see the point above regarding confusing same-color stain for ISC and EE markers). Could cell #2 be a pre-EE that is undergoing mitosis since the lineage tracer marks both EE and pre-EE cells (line 119)? Or do the authors mean recombination on one or both homologs? This should not be possible since the cells are heterozygotes for the Ubi-GFP locus.
• Fig. 2C, if past-EE's increased in number while current EE's stayed the same, where are new past-EE's coming from? There cannot be compensatory proliferations since EE's are post-mitotic. For fate conversion, one would expect the generation of each past-EE to accompany loss of one current EE.
• Fig. 2C, the number of past EE's increased transiently so that baseline number is restored at 18 hr after IR. The authors conclude that fate plasticity is a transient event. Can they rule out loss due to cell death?
• Fig. 2E. Dl+ past-EE cell number declined at 14 and 18 h after IR and because cell sized increased, the authors conclude that EE cells that de-differentiated into ISCs subsequently re-differentiated into EC's. To reach this conclusion, the authors should count past-EEs that are positive for EC markers. Cell size alone is insufficient evidence.
• Fig. 6. Where are the % numbers for ISC, EB and EE's coming from? And wouldn't these change with time after IR, etc?
• They authors interpret fate-conversion as beneficial for tissue repair but never test whether blocking this process impairs recovery or organismal survival or whether promoting it improves outcomes.
• Related to the above, it would be helpful to know if fate-converted cells function as true ISCs or ECs (e.g., through proliferation or absorption assays).

Minor comments

• Improve Figure 1B: Pros and Dl are shown in the same color, creating confusion. If both are stained together, different colors or clearer labeling should be used. Clarify how cells are identified as Pros+ vs Dl+.
• Why is Dl (supposed to be cytoplasmic) overlapping with nuclear GFP in cells #3 and 4 in Fig. 1B?
• Fig. S1E and F. Very hard to see what the authors describe about Arm and Cora. One problem is that cell boundaries are not visible, just the nuclei, so it is hard to know whether cell-cell interactions the authors describe as normal are really normal. Another problem is the overlap of Arm (supposed to be cytoplasmic) with the nuclear GFP signal. What is that?
• Include a simple schematic of ISC to EE/EC lineages for readers unfamiliar with Drosophila gut biology.
• Discuss the regional difference in Xrp1 efficacy (R2a vs R2b). Is there something known about gene expression differences in different gut regions that can explain the results?
• Consider moving scRNAseq (Fig. S1G) into main paper: this is a central part of the conclusion.
• Line 230, Fig S3F-G should be Fig S3G-H.

Significance

Xrp1 is known to have a role in DNA Damage Responses and in cell competition and to function in the context of the p53 network, but this is the first time its role in fate conversion has been demonstrated. For the most part, the data are convincing and include strong genetic evidence from loss- and gain-of-function approaches that demonstrate a role for Xrp1 in activating progenitor gene expression and fate conversion. However, there are several experimental and presentation issues that need to be addressed first as outlined in the previous sections.

The work highlights how mature cells may revert to stem-like states in response to injury, a theme with broad relevance in regenerative medicine.

My field of expertise lies in DNA damage responses in Drosophila and human cancer models.

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Referee #1

Evidence, reproducibility and clarity

Adult tissue homeostasis refers to the process by which tissues maintain a stable and functional state over time. This usually depends on stem cell activity and the balance between cell proliferation and differentiation to ensure that tissues can repair damage, replace old or dead cells, and maintain their structure and function.

Damage-induced plasticity plays an important role in restoring tissue homeostasis. Cellular plasticity is the ability of differentiated cells to acquire alternative phenotypic identities. It is typically constrained under homeostatic conditions but can be activated in response to tissue damage to support regeneration. In this study entitled "Xrp1 drives damage-induced cellular plasticity of enteroendocrine cells in the adult Drosophila midgut", Qian Q. et al., describe damage-induced plasticity of secretory enteroendocrine cells (EEs) in the adult Drosophila midgut. They found that ionizing radiation enhances EE plasticity, enabling EEs to dedifferentiate into intestinal stem cells (ISCs), which subsequently re-differentiate into absorptive enterocytes (ECs). Mechanistically, radiation triggers the expression of Xrp1, a stress-responsive transcription factor, within EE lineages. Xrp1 upregulation is necessary for initiating EE plasticity by expressing progenitor specific genes (like escargot for example), as verified by single-cell RNA sequencing of midguts with EE-specific Xrp1 overexpression. This is suggesting that Xrp1 reprograms EEs by promoting progenitor-like transcriptional states.

The authors nicely describe the dedifferentiation of EEs using the G-TRACE system in response to irradiation and the role of Xrp1 in this process. Yet, the authors need to show the requirement of the EEs dedifferenciation during regenerative growth.

Major comments:

• The authors should investigate the regenerative growth of the adult midgut after irradiation. Is there an impact on ISCs proliferation or cell turn over. Is Xrp1 in EEs required in this adaptive response. It would be elegant to use the recently generated tracing method by Tobias Reiff lab to observe overall impact on tissue renewal (rapport-tracing esglexReDDM esg-lexA, 13xLexAop2-CD8::GFP, 13xLexAop2-H2B::mCherry::HA, tub-Gal80ts on the second chromosome. It can be combined with any EEs Gal4-driver (see Nat Commun 2025, https://doi.org/10.1038/s41467-024-55664-2, the stock is already existing, see table1). This reviewer thinks that it is a key experiment to support the proposed model.
• It is surprising to observe EEs dedifferentiation at a steady state during homeostasis, a condition in which Xrp1 is not detected in the gut. Can the authors comment this point in the discussion?

Minor comments:

• Is p53 required for Xrp1 induction in the gut after irradiation?
• Xrp1 is existing as a short of long isoforms. The short form has been recently proposed to be required for cell competition (https://doi.org/10.1101/2025.06.15.659587) whereas Xrp1 long isoform may be responsible for reduced cell growth. Could the authors test which isoform is induced in the gut after irradiation? Is the overexpression of Xrp1 long isoform having the same effect that the short isoform used by the authors.
• Xrp1 over expression has been shown to induce upd3 ligand and nutrient-driven dedifferentiation of enteroendocrine cells is occuring by activation of the JAK-STAT pathway (DOI: 10.1016/j.devcel.2023.08.022). Could the authors test the function of this signaling pathway during irradiation (upd3-lacZ and Stat-GFP can be used in parallel of upd3 RNAi and UAS Dome-DN.
• Xrp1 is known for its role in cell competition and elimination of looser cells by induction of apoptosis. It would be interesting to check for induction of cell death and/or caspase activation in the fly gut after irradiation and verify a non apoptotic role of DRONC activation in this context using a Dronc RNAi (as proposed by Bergmann lab (https://doi.org/10.1038/s41598-021-81261-0) or Baena-Lopez lab (DOI: 10.15252/embr.201948892)). Overexpression of Xrp1 could be combined with UAS-p35.
• Line 221: fig S3E should be S3F
• Line 230: fig S3F-G should be S3G-H
• The posterior gut region R4 is more proliferative than the anterior part and is usually used for testing regenerative growth. What is happening there after irradiation?

Significance

Altogether, the paper present compiling lines of evidence supporting the proposed model. The experiments are well designed and are convincing. The papers is interesting and relevant for a broad audience.

for - from - Youtube - Essentia Foundation - interview - Alex Gomez-Marin - neuroscientist speaks out on the hidden war on consciousness - https://hyp.is/GEWtIovIEfCHTlOdPqfE7A/www.youtube.com/watch?v=K7NIicE_h9w

DOI: 10.1371/journal.pgen.1011791

Resource: The Cancer Genome Atlas (RRID:SCR_003193)

Curator: @scibot

SciCrunch record: RRID:SCR_003193

What is this?

DOI: 10.1158/2159-8290.CD-24-1788

Resource: The European Genome-phenome Archive (EGA) (RRID:SCR_004944)

Curator: @scibot

SciCrunch record: RRID:SCR_004944

What is this?

DOI: 10.1158/1078-0432.CCR-25-1667

Resource: The Cancer Genome Atlas (RRID:SCR_003193)

Curator: @scibot

SciCrunch record: RRID:SCR_003193

What is this?

DOI: 10.1158/0008-5472.CAN-24-3904

Resource: The Cancer Genome Atlas (RRID:SCR_003193)

Curator: @scibot

SciCrunch record: RRID:SCR_003193

What is this?

DOI: 10.1016/j.xgen.2025.100927

Resource: None

Curator: @olekpark

SciCrunch record: RRID:IMSR_JAX:007850

What is this?

This is the key word to grade distortion

I’m definitely aiming to improve my writing and reading skills. Perfection isn't the goal, but by continuously striving for progress, we can develop our own unique approach to writing a paper.

This statement emphasizes the importance of writing, reading, and critical thinking is; no matter the specific area of study. Writing is crucial because it helps you clearly communicate ideas, while reading deepens your understanding of various topics. Critical thinking allows you to analyze, evaluate, and form reasoned conclusions.

I find this relatable because there are gonna be people better at something than you for sure. I'm not the best when it comes to reading & writing I don't do it as much as I think I should but alas I am not Shakespeare.

Taking advantage of resources availability is only going to help you succeed in the colloge atmosphere

The textbook will help build my discipline. When writing, I will be more attentive with my grammar choices and rhetorical situations. After I complete this course, I should be excellent in my writing in every form..

This is an interesting principle I have never heard of. Of course the durability of a building must be strongly considered by an architect in its design.

I really like how the Backward Design begins with the Academic Standard, then the learning goal, then the assessment, then the learning activity.

It is based "Bayes Theoram" and it works on the assumption of the independence between every pair of featues

Visual just as important as what is written, the "SLAP"s are placed in a way that makes it seem like the speaker is chasing the mosquito around. Uses pattern poem method

Stanza 2 has a rhythm to it, which is then interrupted by the SLAP

The Bean Eaters -A lyric? Doesn't follow a linear story, sounds like it talks of an old couple -Assonance of the a sound in stanza 1 -Enjambment "...their rented back room that / is full..." -Old objects to represent the aging subjects

The same can be asked about Indigenous film, specifically Pacific films. What makes a film Indigenous? Does it have to follow particular themes, ideas, or structures?

It's incredible how Dr. King's words are still so relevant, even today. It's as if society has not learned. As a student of history, I am reminded that change is slow but it does happen.

time is needed to fully understand the text.

It establishes the importance of having clear objectives before reading. Clear goals help in choosing the right reading strategies.

I am a person who takes notes especially if I have not mastered the content.

I did not know "pre-reading" was a term used but I have caught myself doing this before actually reading the book and in most cases it has helped me understand my reading.

I have a hard time reading during the day since I do work a full time job. So the only time i am able to read is at night.

Around 1970, sitcoms like All in the Family replaced single-conflict formats with deeper family dynamics and social issues, showing the shift toward richer domestic comedy.

If cultural shifts and audience aging influenced sitcoms, why didn’t they directly cause the genre to change?

Just as Aristotle classified living beings by essential traits, genre theory, developed by Frye and applied by Feuer, uses taxonomy to organize scripted TV shows by shared narrative features, helping audiences and producers make sense of meaning and structure.

How does television genre act like biological taxonomy—sorting scripted shows into categories based on shared traits like setting, tone, and character types, just as animals are grouped by species and phyla?

The Office is a modern example of the sitcom genre, using mockumentary style and workplace humor to meet audience expectations.

This is interesting to bring up because I have also thought about this when it came to indigenous movements like the protest for TMT. I also sometimes question the motives of some indigenous "spokespeople" who claim authority on knowledge. I wonder what others think about this.

This is a poignant argument to make. I cannot imagine the horror of having to be forcibly removed from your homeland, taken somewhere you've never been before, and stripped of your cultural identity. It's incredible to see how African slaves tried to hold onto their identity and use it to persevere, but also how it evolved due to their horrific experiences.

I am glad that Mays points out the hypocrisy of American democracy. As a teacher who teaches both US Government and Hawaiian History, I cannot help but always recognize the dichotomy between the two histories. I try to remind my students that, despite the ideals that the US nation is founded on, it has fallen short of what it promises.

Page 259 : “ I suppose it was a inevitable that as my Wordbase rotten in. I could for the first time, pick up a book and read and now begin to Understand. What The tampoco se anyone who has read a great deal. Can imagine that the new world that opened.” - This was significant to me, because once you can learn how to read and can advance to bigger dialogue books. You really do open up a new world of fiction and nonfiction.

With a sequence of instructions, the code will be able to run the code and create an output, because it's using conditional execution and repetition, since some of these sequences may be repeated

It's interesting because I have never heard this before and I would love to research more about it.

.py is important for the program to be identified as a code.

So it is CollcectiveSenseCommons.org Mattermost is shutting down. Should do a project /task to extend the life of CSC Mattermost held conversations via OpenLearningcommons/IndyLearningCommons what an opportunity to demonstrate the value of hyperpost! and engage with people not just as Users but co-creators

make it lifelong and autonomous andmutualy owned by its participants as they join the Indy/web

or even without that!

on the hypothesis.margins

No signup to spaces but the opportunity to own their own spaces. Control their own(ed) information spaces, control and shape their own destiny

This content is snarfed from

chat.collectivesensecommons.org/

by one of its participants

for sharing with other participants and interested trusted individuals

without requiring sing in or sing up

This is important as the site itself is slated to be discontinued

It is a conversation following the anouncement for OGM's MatterMost forum, that is, as announced here, slated to be shutdown net month

Would like to use hyperpost.peergos.me to keep it alive and thriving on the IndyWeb by lead envisioneer and first developer of the IndyWeb and Hyperpost itself Warning This is alll new development/test in progress

HyperPost is dedicated to

Content Liberation from ephemeral silos, for evergreen conversations

Snarf key conversations by the participants and share over the web for keeping the conversation autonomous evergreen continuous without being synchronous hosted by the participant's owned autonomous named networks of Indy.Web.Spaces

Use annotations on the margins via hypothes.is and eventually other tools to provide links to evergreen Individual and/or community owned evergreen conversational spaces that provide full verifiable provenance and recapitulate-able history of their co-evolutions and emergent mutual learning

This passage shows that the Bell Beaker culture was largely descended from the Yamnaya people, with about 70% of their ancestry coming from them. It also notes that ancestry percentages differed by region, suggesting random variation in how native and immigrant groups mixed.

This passage explains that while some ancient people lived in harsh, remote areas, most likely chose richer, more comfortable environments. It contrasts survival in barren regions with the preference for fertile river valleys.

How did farming and herding move into Europe, and how did it change the way people lived?

This explains how well how farming has spread. It also explains how it's methods were developed and shared. However, it's yet to address the origin of farming. Is the origin of farming really this mystery that we are unable to find a unanimous answer to?

This is interesting because it shows how people changed their way of life over time to survive in different environments.

It's remarkable how religiosity played an enormous role in the prehistoric world. I thought it would be assumed that they were too busy surviving and providing for themselves and their family to put any significance on religion. It's truly revealing of their complex social and spiritual life.

I remember learning this in class. I was so fascinated by this concept of species.

I find it really interesting that people actually found this as the normal way to live. It makes me very grateful to live how I live right now. I would want to learn more about this.

I found this very informative. I did not hear about this before. I found the map very helpful and it helped me visualize it better.

This is interesting it shows how farming spread both by people learning from neighbors and migration. This also shows how connected and mobile people were even in ancient times.

I find this interesting because this shows that even though Chinese society had so many advancements, they still had a lot of flaws.

China invented all this stuff way before Europe. These inventions eventually shaped the world today, even though Europe gets more historical credit for using them.

I think this is really interesting and most people would just read right over this and not actually understand how many ships and people this is. Hutch has about 15,000 people and there were almost twice that many that left on the first voyage.

I find this interesting because the Chinese were based on someones own education getting them power and no one could get them there but themselves, it didn't matter how much money you had or who you knew.

This was a very dissapointing thing to read for me. You'd think that after hearing about some of the successes of Zheng He under the Imperial Power, that the Chinese navy wouldn't have gone under so easily.

I liked this section a lot. Giving us more of a takeaway with the ships being used by Zheng He. There is a picture above this section displaying the very large size of the ships being used by Zheng He, and they are exceptionally large! Finding out more information about how they are able to operate and their strategies of efficiency across the water our quite intriguing, in my opinion.

I think that this is an interesting passage because of how Hongwu is willing to give up the throne just because he is unable to give it to his eldest son. In my last post, I had mentioned that Hongwu was a great leader. Perhaps in motivation from the terrible and possibly impoverished childhood. However, I wouldn't expect him to make such a harsh decision. He'd rather give up his title, his life, to someone else rather than one of his own sons!

I think this is a very good section of the chapter due to the actions Hongwu takes in memory of the dysfunction of his youth. The way society wasn't very beneficial for the greater good. Hongwu is able to work with irrigation and agriculture. Creating better environments of his land.

Having all of my work given to me at one time for the entire week.

All of the above

it's interesting because it shows how the same germ can cause different kinds of disease, changing how it spreads and how deadly it is.

this is like trying to explain to someone there where never plums in Sugar plums.

Shared values and opinions and how to communicate a groups end result. For example, as a Patient Care Tech in the NBICU we might discuss where something is the inventory but someone from another unit, i.e. Neuro ICU would understand what or where a specific item is.

Reading different articles and identifying what common goals, question they have on the topic.

Discourse communities share common goals, genres, expertise.

Humans in different parts of the world could not have communicated with each other and taught each other farming... is this the same case with domestication? If they did not teach other domestication, did a more simple circumstance incite this discovery?

Dithyrambic- described a powerful, emotional song performed by a chorus in ancient Greece to celebrate Dionysus( the god of wine and ecstasy).

O: Oates seems to be trying to enforce the idea that prose fiction is a space for readers to take in the 'art' of the novel/story/etc., and grow an interpretation. I think the earlier statement, "Where in prose fiction the writer is accustomed to shaping subtleties of meaning by way of carefully composed language," explains this best. While the play on the other hand is given directly to the viewer, with little space to fill in imaginative blanks (although the depth behind acting should not be disregarded.) I find this comparison impactful because it demonstrates the depth of prose fiction that may not be initially realized. A play becomes a predominately collective experience and interpretation, while prose fiction becomes a living document. I can think back to times when I was so immersed in a piece of prose fiction that I began to imagine it in my free time, the characters coming alive in my head, my imagination becoming incredibly involved in the story.

is culture an emerging capability under the survival pressure?

important

Philosophy helps educators analyze and shape their position on the goal of education, and make decisions based on values that shape their beliefs and behaviors. This will shape their choices of teaching strategies and guide them in developing relationships with students and embracing their career.

At the end of a long and vital process intended to reach the student, we find the teachers developing unit and lesson plans and activities. They are at the stage of learning experience, and their role is crucial to the success of the students. Whether they are seen as facilitators, guides, or coaches, so much depends on the way they approach their work, their values, and professionalism.

Another misconception is that instructional design is the same as curriculum development. They are not.

why?

Celecoxib is safer for the stomach than standard NSAIDs, but if the patient is also taking aspirin (even baby aspirin), the GI safety advantage is lost.

TLDR: technological advancement and globalization means we need to do more work keeping track of where things are coming from and going to

different algorithms are better for doing different things

i.e., take the old strategies n use them to make the AI better at its job/ make sure the AI gives us the backstory on where we got the info

I am Testing this out

Everyone has there different way of writing up a esay or even a noval so when it comes to having your own style its common it what makes you differnt and possibly even a great writer

The individual nature of writing and the importance of discovering a personalized writing process through exploration.

I have been out of high school for so long. When i do help my nephews who are in high school I do not understand what they are being taught.

I deeply admire FDR’s humanity in this statement. He openly acknowledges that he is not perfect and that mistakes are inevitable along the way. Yet, he reassures the people of his unwavering determination to do everything in his power to ensure success. To me, this reflects not only his humility but also his strength as a practical and transparent leader.

I think this is a very interesting quote that not only shows how the author gets his perspective across, but also how extreme the changes used to be back then.

The author gets his perspective across in a mostly objective and evidence-based way. The use of this quote (and many others throughout) shows that well. Instead of subjectively saying what his own opinion is that the reforms became more radical, he gives the primary source of Gorbachev's own words. This allows him to build a credible argument by not only pointing out that he believed the changes to be more and more extreme, but also by showing that Gorbachev himself believed so. He shows Gorbachev's personal and subjective viewpoint that simple reconstruction wasn't enough anymore.

That’s also why this quote itself reveals how extreme the changes got. Gorbachev himself changes wording from restructuring to revolution, which is something many people connect to things like the French Revolution, which were inherently violent. This really shows how he later on wasn't just trying to fix the economy by fighting the deeper issues, he wanted a genuine revolution of the entire system and society. The goal shifted into a complete psychological and also cultural transformation. This, among other things, also explains why the reforms caused opposition, as extreme changes almost always do.

This caught my attention because it highlights an important lesson in political reform. This is the point where Gorbachev and his allies realized that the issue is a deeper lying one, and not just one that can be fixed with a more simple economic focus. Gorbachev's team started to realize that countries’ economic struggles were actually just the symptom of the underlying "disease," which is a social and cultural one. It also makes apparent that Gorbachev's steps weren't one whole master plan; his strategy evolved over time. He realized that earlier measures were either unsuccessful or constrained by a too rigid system and the elites, and that they needed a deeper lying change and more support within the country. The move towards glasnost can be seen as the turn around from trying to treat the symptoms (the economy) to attempting to treat the underlying social issues by getting the public’s support. This reminder and also takeaway stays relevant today. Economic problems, maybe even in modern European countries, are often just the surface of, in reality, much deeper lying and more complicated societal issues.

Esta foto me resulta muy interesante. Todas las demás fotos de esta lista me parecen muy deprimentes, tristes y sucias. Sin embargo, en esta, todos están bien vestidos, el hombre está feliz y todo parece estar limpio. ¿Por qué esta es diferente y significativa para la lista? Solo sé que esta foto fue tomada durante la Segunda Guerra Mundial y están haciendo fila para ver una película.

Me gusta que esta foto muestre un poco de alegría en esa época. Ver a tanta gente haciendo fila para ver una película sin duda despierta la felicidad en muchos. Y esta foto lo demuestra a la perfección.

10 roles common to the work of all managers, the ten roles are divided into three groups . 1. - Interpersonal Figurehead Leader Liaison 2. -Informational Monitor Disseminator spokesperson 3. - Decisional Entrepreneur Disturbance Handler Resource Allocator Negotiator

Note that aldosterone, the end product of the mineralocorticoid pathway, does not cause negative feedback inhibition on ACTH or CRH production.

spell out Adrenal Insufficiency in the table legend.

high

It is interesting that until now as a coach I did not have to include student data as part of my evaulation. This year I do and I am struggling with how to write my goal. It is hard to do when my goal is buidling capacity in the teacher and there is nothing in the rubric about students.

Our district redid the induction process this year and it is much more clear which is really helpful!

This year I created a new to the document with lots of important links and went over it with all that were new to the building. I got a lot of positive feedback on it. I spoke with people who were relatively new and asked them what they wish they knew when they started to help me.

I am an Instructional Coach and in our district we define a mentor as teacher in a similiar job that offers basic support. New teachers have an Instructional coach and a mentor. Mentors in our district Job-alike/ job-adjacent Side-by-side observations Expert in the field Conduit to understanding grade level/ content/ team More informal Nuts & bolts “What I might do ” and Instructional coaches Coaching cycles Trained expert in coaching, facilitating and teaching adults including PD Conduit of district vision Student data-driven Goal-oriented “What might you do _”

I find it kind of confusing.

I feel like a lot of my STEM friends and colleagues would disagree with me if I said this to them.

This is a new concept to me, but I can recognize that I have done it in my own writing. This is interesting.

Reflects the goal of true inclusion: every student is valued, supported, and encouraged to grow academically and socially. How can teachers create a classroom culture that values each student’s unique contributions while supporting social and academic growth?

Shows that inclusion is more than behavior management—it’s about social belonging.

Highlights the importance of balancing academics with social/emotional learning for all students, including those with disabilities.

Shows fairness, respect, and individualized support in practice; could reflect on how these strategies might be applied in your own classroom experiences.

How can teachers create a classroom culture where all students feel valued and supported, not just treated equally?

Evidence that the Responsive Classroom approach effectively promotes inclusion

Emphasizes the focus on inclusion and community, not just academic skills.

Which principle would be hardest to implement in a real classroom? I think knowing each child individually can be challenging in large classrooms but is essential for supporting inclusion and social growth.

Emphasizes understanding students needs to support inclusion.

Morning Meeting is important because it gives all students a predictable routine to participate, build friendships, and practice social skills. I notice in classrooms that activities like Morning Meeting help students with disabilities feel part of the group and learn from peers. I also see them eager to participate in the classroom.

IDEA requires that all students have access to general education, but I see firsthand that placement alone isn’t enough for true inclusion. Planned strategies and support are essential to help students with disabilities develop social skills and participate fully in the classroom community.

What other strategies could help students like Matthew besides peer support?

Rachel’s modeling of the song’s motions for Matthew is an example of peer tutoring, showing how classmates can support students with disabilities and help them feel included in classroom activities. This is so important, I see firsthand in classrooms how peer support helps students with disabilities feel included and participate.

The person I'm describing has wavey raven hair, brown eyes, no taller than 5ft 5inches tall with a hue that is a deep tan in color. We are the same ethnically, speaking the same language with identical religious backgrounds.

1. Clear Text - This text and description is clear, concise, and uses simple wording. It communicates this section of the sites purpose, which benefits users with cognitive disabilities and those just scanning the page for information.

1. Clear Link In Footer - This link text is very clear and describes exactly where the link takes you to. It doesn't just say "click here", which is helpful for everyone particularly screen reader users.

1. Link Label Clarity - Several links on the website such as "Learn more", "TRY FOR FREE", etc, have no context to them and are pretty generic if taken out of surrounding texts. Descriptive label links like "Learn more about VSCO Workspace" or 'Try VSCO for free' would inform users particularly those using screen readers where each link leads.

Seems like everyone is ordered to leave the fight scene or they will be punished badly(death)

Good point to be noted he these two family been fighting and their war destroyes the city, that’s why they called it a civil brawl

Montague family head is also on the scene now

Fight to settle the quarrel

This character represents group of older people that were fed up both families of Montague and Capulet for disrupting the peace. They attacked both families as a result.

Through careful research, This act of biting of the thumb is equivalent to giving someone the middle finger. Sampson seems to be antagonizing the men of the house of Montague. This act clearly shows that he essentially itching for a fight.

This statement highlight Sampson's disdain for the women of the house of Montague. This extreme violent act of stating cutting their heads went so far that it made Gregory question Sampson "the heads of maids"?, he literally meant are you being serious right now? Are aware of what you are saying?

Disapproval from Juliet to be wed?

Questioning the quarrel between the two houses

Shows the conflict from the two houses

I wondered what this term meant, it means the provoking of the beings of the house, comparing them to feeble dogs.

She is the surviving child

Symbolizes the fact that no one is perfect but Obtala thought they were beautiful

Egg, palm nuts, maize and seeds from baobab tree seem like they are symbols for things.

He has powers that are unlike others. Sounds like he might be the "hero" in the story.

The use of imagery of where the story is set.

Gives us a lot of context to the upcoming story.

But they will have to write and speak (I think a lot of writing skills translate over into speaking) for the rest of their lives and careers. A friend of mine that just started teaching recently talked to me about how she had to emphasize to her students that no matter their field they will need to write.

Infinite

I'm confused with what this means.

He is over hearing words he wants actions.

Seeing this as one of the requirements for a passing grade is refreshing to see listed. We sometimes forget, as students, when we are navigating new courses, job opportunities, and everyday life, that it is vital to feed our minds with ideas that don't initially come to mind. Thinking deeply and forming connections with past readings and courses has helped me tremendously as a student when it comes to passing a class. I believe it's excellent that you brought this to students' attention in hopes of them succeeding within your course.

Lee seems like a villain, I wonder if that is based on my reactions to/perception of him, bias, or the way he has been portrayed by the author.

no words, just -0-

It's interesting that the author thought to include this.

Interesting and something I have recognized, but I dont think it is just Gen Z. I think this is a multi-generational problem, especially when it comes to recognizing how truthful AI content is.

This is so interesting!