set up a similarities and differences between your sources.

I think that GA culture is in favor of a quiet individualistic pietism that doesn't place demands on others

I think that CA culture is more accomodating to that, but still is dubious about Christian beliefs on sexuality, etc.

What does it look like to be truly a diverted insider in an environment with a lot of phony diverted insiders?

I think the social cost here comes from actually living differently — that then gives the lie to the profession of faith of others who are happy to compromise with the world

This strikes me of AD's experience; living with one foot in the church and one foot in the world — obedience looks like embracing the social cost that comes with being all in for Christ

It's weird to consider what it looks like to live as estranged for the sake of Christ in an environment where it seems like Christ is much beloved

go back into Evangelical Theology textbook and see what Bird has to say about these categories

the sect is able to expect stricter adherence to its instruction than the church is

I definitely think that the world of GA is the world of churches, in that, it is very hard to expect much out of anyone, since it is extremely easy for people to defect to other churches (or nonchurches)

it seems like each has its own elements that commend it; the church wants to be all embracing and draw the world to itself, the sect wants purity and builds high walls

Police have the power to shape public opinion

Made them an institution against their will

I am interested in this

We are learning the buildup to the jail lecture

Too much of the second face of gov combined with not enough of the first

Not paying attention to the right things

First face is the bad/good job they are supposed to be doing, second face is what they do with their power

second face is the implied power that comes with being in the government

Corruption still

Cryptogyny, term coined in 2020 https://www.eldiario.es/comunitat-valenciana/criptoginia-paraula-nova-fenomen-antic_132_1003396.html

a phrase to sugarcoat as well as make the goverment looke bettter for what they have done. The understanding is more worth it then the individuals who may get upset by it.

these are some changes that are necessary. If it may not fit the moment dont use it. But if they do and dont understand anger is unneccessary. Coffin, Corpse, Dead/Died. all can hurt but in a moment of disspair. they are words that help understand better. some of these words are totally rediculous. Like sure coffin may upset some people, but me and a lot of others would not be upset at someone saying my loved one was laying in their coffin. its the same thing with deathbed. Its person to person and you dont have to change for one person, but you may better understand what words shouldn't be used in situations.

It can cause harm, but thats the point it can. Not everyone will be hurt by a word, You have to be able to use every word you can. Not against people, but for your own needs. It needs to be an understanding of if you dont like it say something. But you cannot expect it to change for everyone. there will be always someone who doesnt understand that it may be hurtful and what then? You tell them the truth that you dont like it. but a word is a word and you have every right to ask for it not to be said as they do to say and write it. ITS EQUALITY. not a reason to get upset. Understand comes from talking not rules.

more words used to downplay the significance, dont sugar coat the truth. It will always be better to understand the full picture then have my job downplay my loss of position.

the violent phrases used to sway the audience more using emotional tone. slash prices, beat the competition, stranglehold of the market. these are just words but tied to an emotional feeling that are used again and again to manipulate the audience with feelings.

The changing of one word to flatter or irratate people. it affects confidences and personal belief. For someone learning the language they understand it as the same thing, but it has 2 different emotional meanings in which they only understand the literal and are unnatched to the words.

the understanding that words change the point of view, Doublespeaking is to using obscure words to twist the truth, this isnt just politians its manipulation by words done by everyone. Even a fluent speaker of english will have trouble understanding the true meaning

The Literal meaning vs the emotional, cultural, or associated meanings. understanding that if you are learning a language having a cultural and literal meaning for all words, it will stray you away from the language as well as if you already know the language the understanding will be skewed and possibly continue generation to generation

People misunderstanding the differences. Changing something for everyone is going to affect a lot of speakers, and if you dont follow you are looked down upon

Words Matter just as much as the argument for it. It is necessary to have people understanding the whole picture not a sugar coated phrase. War isnt always about defense.

A big thing today, One group wanting it one way another a differerent way. Say what you wanna say, without forcing yourself to change. Personal views matter but with some words it can be a necessary change.

Political Bias, but the words changing in urgency as we find it necessary to sway people

I wonder if you have considered wither blind source separation or machine learning methods to try to identify specific spectral signatures for your differing cell types? Non-negative matrix factorization is possibly particularly interesting here as it can separate out the spectral signals that vary across the cell types.

Context matters and changes the meaning of words uttered - circumstances are also important - potentially has even more impact than what we are tempted to think as the "mental activity" defining it

We want to say that "Slab!" and "Bring me a slab!" are in our language, saying the same thing - why are they the same? because they can be used in the same way - not that they relate to the same thing - use of words depends on the circumstance

Meaning is use and that's all there is to it

no, you just do it without thought (as long as you're a master of the language)

To think this way is not the right track - these are tempting explanations but this is not what happens when someone says "Slab!"

we have the ability to contrast it with other similar sentences

if they mean the same thing, why do I feel the need to "translate it" to the longer version?

The builders and Wittgenstein have different languages - the builders cannot say anything more than "Slab!" - different forms of life - conceptions are available to us that is not accessible to them This is why we are tempted to "translate" it to have more context, and this is where the problems come out - we ought to avoid this temptation - accounting for what they mean turns into a problem - can they mean something that goes beyond their language? - mind stuff that is not "contaminated" by language

when the builder says "Slab!", he has in mind "bring me a slab!" --> this is the target of the investigations - meaning something is not a state of mind

Why would "bring me a slab" be the more perfect version of "slab!", where "slab!" is a shortened version, and not that "bring me a slab!" is a lengthened version of "slab!"

the tourist mentality is when you strive to keep readers engaged by briefly covering a subject and later explains it in more detail with the hopes to keep the reader in suspense. being fascinated by differences in language is natural and appropriate for those who are seeing them for the first time.

to bring translingual writing effectively into a classroom the teacher must have an extensive all those languages.

teachers strive to find ways to incorporate translingual writing into their work. Another possibility for the growing popularity of translingual writing could be a result of curiosity.

some perspectives on translingual have been "silenced" due to the fear of being on the wrong side of the argument

You mention that longevity is negatively correlated with effective population size, which would create correlated shifts in selection intensity across many sites simultaneously in real data. I'm curious whether the H0 simulations, which use constant fitness profiles I believe, might underestimate the false positive rate in the empirical setting, since that correlated background signal isn't captured in the benchmark. Do you have a sense of how much this might affect the transfer of the FPR estimates to real data?

If the goal is species species classification, it makes complete sense to average multiple spectra per cell, but, given cells are dynamical systems, it seems totally reasonable that (even for isogenic lines in identical environments) much of this variability might reflect real biology. Furthermore, it might be true that averaging across samples (as I think you are doing here?) is difficult and multiple spectra from a given cell in a given part of its dynamical process might be very similar to one another. Have you thought about modeling frameworks that would characterize the possible spectra from a given isogenic line in a fixed environment? Perhaps generating a probability distribution of spectra for a given species or cell type? This could lead to more robust classification by taking into account the true variability of the spectra for a given cell type.

This is a good list of methods, but, given the data efficiency benefits and the ability to learn non-linear relationships, did you try random forrest? and, possibly SVM with an RBF kernel instead of linear? It seems possible there might be informative, non-linear relationships among parts of the spectra and while a NN could find these, they usually require large amounts of data to do so.

slay?

not everyone agreed with the tractariansn, nor were gothic churches the standard form of architecture this, however, does not make it by any means less relevant

but we still get slay churches in the south??

very interesting for later tractarianism!!! Left a legacy of a desire to renew faith!!

SLAYYY - movement expressed itself through gothic architecture girly poops - but was this solely to do with industrialisation. yes people were worried, but it would be innacurate to solely place the emergence of gothic architecture on it - yes it provided the funding and impetus, but the religious background pre-dating significant industrialisation in Wales blah blah

As such, the gateway to heaven needed to be in a suitable form, reconstruction and rebuilding increasingly gothic from (blah)

Hope in the ancient ways also translated to gothic architecture

Interesting link to nationalism and general medievalism through estedfodu

It sounds like the motivating concern is that ML rescoring could exploit construction artifacts, but Oktoberfest is primarily spectrum-driven, and rescoring compresses rather than amplifies the gap between generators. It might be useful to consider testing with a rescoring model that takes sequence-derived features (PLM embeddings) as direct input, where exploitation of construction fingerprints would be more mechanistically plausible?

Terence Eden on human.json and how he added it to WP. and compares it with FOAF and in person key vouching

I have mentioned something like mutually vouching for someone that they're human, on my Reverse Turing page.

Still not sure if having a machine readable file makes the right point though

Version 3 of this preprint has been peer-reviewed and recommended by Peer Community in Genomics.<br /> See the peer reviews and the recommendation.

Interesting

I didn’t know this

It is certainly true that technological advancement is happening at a much greater speed than society and the law can adapt. Technologies such as deepfakes, bots, and recommendation algorithms are capable of affecting millions of individuals prior to regulatory bodies creating guidelines to govern their use. This has led me to believe that companies should be developing new technology with an eye toward the ethical implications it may have on its users, rather than relying upon subsequent laws or regulations to govern how they are used.

This is something that sounds incredibly familiar to what I had been taught since I began writing papers.

When I first started writing papers, I found this to be a major problem, where my sources would contradict themselves and I would not analyze them correctly.

When you put it in these categories, it makes it easier to digest and understand.

while hyperlinks are convenient, they also take away from adding to what you are writing about and your own personal perspective.

I found digital sources to be easier to manage and use when working on an essay.

It is interesting to see how the internet has evolved over time and continues to evolve.

Utilitarianism is another theory associated with Consequentialism; a version of Consequentialism focused upon maximizing overall happiness or general well-being. Social Media and technology are common areas in which utilitarianism is applied due to the fact that many companies will defend their choices using claims such as "my platform supports/benefits millions." The problem with this form of reasoning, however, is that it may support causing harm to a small group of people while benefiting a large one. For example, features designed to create user engagement could lead to an increase in harassment and misinformation.

Claude, I think we can prove it!

theoretical_g_factor ∈[experimental_value - 12th_digit_error, experimental_value + 12th_digit_error]

Also: Let users choose their own weights with sliders as well

potentially we should ask the raters to give category ratings as well, to help refine this

not necessarily, it could also be published in a journal and still on NBER ... Sometimes you can see that in NBER, and sometimes not.

**(①)They are the largest papillae of the tongue with 8–12 in front of the sulcus terminalis.

Sulcus terminalis’in önünde yer alan, dilin en büyük papillalarıdır ve sayıları 8–12 arasındadır.

**(②)It has a bitter taste sensory body.

Acı (bitter) tat duyusunu algılayan tat tomurcuklarına sahiptir.

Bu bezin ağız tabanındaki uzantısını tarif ediyor. Arkada 3. azı dişinden (yirmi yaş dişi hizasından) başlayıp, önde dil bağına (dilin altındaki o ince dikey perdeye) kadar uzandığını anlatıyor.

"Extends from the 3rd molar tooth to the tongue frenulum": Bu bezin ağız tabanındaki uzantısını tarif ediyor. Arkada 3. azı dişinden (yirmi yaş dişi hizasından) başlayıp, önde dil bağına (dilin altındaki o ince dikey perdeye) kadar uzandığını anlatıyor.

Lobular sublingual salivary gland": Dil altı tükürük bezinin lobüllü (yani tek bir parça değil, küçük tanecikli/boğumlu bir yapıda) olduğunu belirtiyor.

*(①)They are round-shaped, red-colored formations on the dorsal surface of the tongue.

Dil sırtında (dorsal yüzeyinde) bulunan yuvarlak şekilli, kırmızı renkli oluşumlardır.

**(②)These papillary contain mechanical thermal receptors as well as taste buds.

Bu papillalar, tat tomurcuklarının yanı sıra mekanik ve termal reseptörler de içerir.

**(③)These can sometimes appear brown due to the intense melanin pigment in brunettes.

Esmer bireylerde yoğun melanin pigmenti nedeniyle bazen kahverengi görünebilirler.

Submandibular tükürük bezinin (Wharton kanalı) ve sublingual tükürük bezinin (Bartholin kanalı) boşaltım kanallarının ağız içine birlikte açıldığı yerdir.

Stenon kanalı (parotis kanalı), ağız boşluğuna üst ikinci molar dişler seviyesinde açılır. Mukozal yüzeyde hafif kabarık bir yapı oluşturur ve enfeksiyon durumunda hiperemik (kızarık) bir görünüm alır.

Ağız mukozasında hemen her yerde çok sayıda küçük tükürük bezi bulunur. Bunlar toplam tükürük miktarının yaklaşık %10’unu üretir.

Sublingual bölge venöz damar yapısı açısından zengindir. Bu durum, yaşla birlikte damar duvarlarının genişlemesi nedeniyle ortaya çıkabilir. Genellikle dilin ventral (alt) yüzeyinde görülür, ancak ağız tabanı mukozasında, dudaklarda ve ağız köşesi (angular) bölgesinde de görülebilir.

Bazen bu lezyonlar hiçbir belirti vermeden, hasta veya hekim tarafından tesadüfen fark edilir.

Ağrı, ülseratif lezyonlarda bıçak saplar gibi keskin, iltihaplı lezyonlarda ise künt olarak tanımlanabilir.

Ülser olup olmadığı Alt dokulara infiltrasyonu (yayılımı) Kenar sınırının belirginliği Sertliği Ve hacim artışı gösterip göstermediği

Bu bağlamda, premalign (kanser öncülü) lezyonların tanınması ve erken tedavisi hastalar için büyük bir öneme sahiptir.

Tüm oral patolojiler arasında, premalign (kanser öncülü) lezyonların ve ağız boşluğu kanserlerinin görülme sıklığı yüksektir.

Bu lezyonlar, enfeksiyöz, otoimmün, premalign ve malign lezyonların öncülleri olabilir veya sistemik bir hastalığın ağız tutulumuna sekonder olarak ortaya çıkabilir.

Bu durum konuşma bozukluğu, yutma güçlüğü ve çiğneme zorluğu gibi birçok probleme neden olur ve hastanın yaşam kalitesi üzerinde doğrudan etki yapar.

The final, peer-reviewed Abstract can be found on pages 7-8 of the Accepted Manuscript PDF.

The final, peer-reviewed Abstract can be found on pages 7-8 of the Accepted Manuscript PDF.

The final, peer-reviewed abstract can be found on pages 7-8 of the Accepted Manuscript PDF.

Let's also incorporate GFI reports on other components, especially the growth factors.

quick tooltip and also link to an explainer (in the 'learn') section detailing these differences between these definitions of output ... Also, which papers do what here? If it's complicated, do that in a tooltip.

Column widths are off. Make the columns with more text wider than the columns with little text. Make that a skill or a sort of general instruction. It comes up a lot whenever you generate these HTML documents. !

eLife Assessment

This article presents valuable findings on how the timing of cooling affects the timing of autumn bud set in European beech saplings. The study leverages extensive experimental data and provides an interesting conceptual framework for the various ways in which warming can affect but set timing. The statistical analysis is compelling, but indicates some factors that may temper the authors' claims, while the designs of experiments offer incomplete support for the current claims as they rely on one population under extreme conditions for only one year each while a confounding effect (time in a chamber) sometimes lacks a control.

Reviewer #1 (Public review):

Summary:

This study provided key experimental evidence for the "Solstice-as-Phenology-Switch Hypothesis" through two temperature manipulation experiments.

Strengths:

The research is data-rich, particularly in exploring the effects of pre- and post-solstice cooling, as well as daytime versus nighttime cooling, on bud set timing, showcasing significant innovation. The article is well-written, logically clear, and is likely to attract a wide readership.

Comments on revisions:

This is the second round of review, and I am generally very satisfied with the authors' revisions. However, a few detailed issues still require attention:

The authors identified the summer solstice (June 21) as a phenological "switch point", but the flexibility of this switch point remains poorly understood. A more precise explanation of what "flexibility" means in this context is needed, along with a description of the specific experimental results that would demonstrate this flexibility.

The experiment did not directly measure the specific date of the phenological switch point. Instead, it was inferred by comparing temperature effects before and after the solstice. The manuscript should clearly state that this switch point remains an inferred conceptual node rather than a directly measured variable.

In Experiment 1, the effect of bud type (terminal vs. lateral) was inconsistent across the overall model and the different leafing groups. The authors should provide a more thorough discussion of potential reasons for this inconsistency. In addition, the statistical model for Experiment 1 indicates that the measured variables (summer cooling and leaf emergence date) explain only 23.4% of the variation in bud formation timing. This leaves over 76% of the variation unexplained, suggesting that other important factors are involved. The discussion should address this limitation in greater depth, moving beyond a focus on the measured variables.

Reviewer #2 (Public review):

In 'Developmental constraints mediate the summer solstice reversal of climate effects on European beech bud set [their original title]' Rebindaine and co-authors report on two experiments on Fagus sylvatica where they manipulated temperatures of saplings between day and night and at different times of year. I think the experiments are interesting, but I found the exact methods of them somewhat extreme compared to how the authors present them. Further, given that much of the experiment happened outside, I am not sure how much we can generalize from one year for each experiment, especially when conducted on one population of one species. I was also very concerned by the revisions.

I expand briefly on these concerns and a few others for readers of the paper (see `The below comments relate to my original review'). Subsequent edits to the paper addressed some of these by providing a new figure and moving around the methods. Further, I am at a loss about their hypothesis, when they write in their letter: "Importantly, the Solstice-as-Phenology-Switch hypothesis does not assume that the reversal is fixed to June 21." Why on earth reference the solstice if the authors do not mean to exactly reference the solstice?

The comments below relate to my original review with many of them still applying.

Methods: As I read the Results I was surprised the authors did not give more info on the methods here. For example, they refer to the 'effect of July cooling' but never say what the cooling was. Once I read the methods I feared they were burying this as the methods feel quite extreme given the framing of the paper. The paper is framed as explaining observational results of natural systems, but the treatments are not natural for any system in Europe of which I have worked in. For example a low of 2 deg C at night and 7 deg C during the day through end of May and then 7/13 deg C in July is extreme. I think these methods need to be clearly laid out for the reader so they can judge what to make of the experiment before they see the results.

I also think the control is confounded with growth chamber experience in Experiment 1. That is, the control plants never experience any time in a chamber, but all the treatments include significant time in a chamber. The authors mention how detrimental chamber time can be to saplings (indeed, they mention an aphid problem in experiment 2) so I think they need to be more upfront about this. The study is still very valuable, but -- again -- we may need to be more cautious in how much we infer from the results.

Also, I suggest the authors add a figure to explain their experiments as they are very hard to follow. Perhaps this could be added to Figure 1?

Finally, given how much the authors extrapolate to carbon and forests, I would have liked to see some metrics related to carbon assimilation, versus just information on timing.

Fagus sylvatica: Fagus sylvatica is an extremely important tree to European forests, but it also has outlier responses to photoperiod and other cues (and leafs out very late) so using just this species to then state 'our results likely are generalisable across temperate tree species' seems questionable at best.

Measuring end of season (EOS): It's well known that different parts of plants shut down at different times and each metric of end of season -- budset, end of radial expansion, leaf coloring etc. -- relate to different things. Thus I was surprised that the authors ignore all this complexity and seem to equate leaf coloring with budset (which can happen MONTHS before leaf coloring often) and with other metrics. The paper needs a much better connection to the physiology of end of season and a better explanation for the focus on budset. Relatedly, I was surprised the authors cite almost none of the literature on budset, which generally suggests is it is heavily controlled by photoperiod and population-level differences in photoperiod cues, meaning results may different with a different population of plants.

Somewhat minor comments:<br /> (1) How can a bud type -- which is apical or lateral -- be a random effect? The model needs to try to estimate a variance for each random effect so doing this for n=2 is quite odd to me. I think the authors should also report the results with bud type as fixed, or report the bud types separately.<br /> (2) I didn't fully see how the authors results support the Solstice as Switch hypothesis, since what timing mattered seemed to depend on the timing of treatment and was not clearly related to solstice. Could it be that these results suggest the Solstice as Switch hypothesis is actually not well supported (e.g., line 135) and instead suggest that the pattern of climate in the summer months affects end of season timing?

Author Response:

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

eLife Assessment

This article presents valuable findings on how the timing of cooling affects the timing of autumn bud set in European beech saplings. The study leverages extensive experimental data and provides an interesting conceptual framework of the various ways in which warming can affect bud set timing. The support for the findings is incomplete, though extra justifications of the experimental settings, clarifications of the interpretation of the results, and alternative statistical analyses can make the conclusions more robust.

We thank the editors and reviewers for their expert assessment of our findings and their interest in our conceptual framework. Below we respond to the specific reviewer and editor comments.

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study provided key experimental evidence for the "Solstice-as-PhenologySwitch Hypothesis" through two temperature manipulation experiments.

Strengths:

The research is data-rich, particularly in exploring the effects of pre- and postsolstice cooling, as well as daytime versus nighttime cooling, on bud set timing, showcasing significant innovation. The article is well-written, logically clear, and is likely to attract a wide readership.

Thank you for your generous description of our study and the manuscript.

Weaknesses:

However, there are several issues that need to be addressed.

(1) In Experiment 1, significant differences were observed in the impact of cooling in July versus August. July cooling induced a delay in bud set dates that was 3.5 times greater in late-leafing trees compared to early-leafing ones, while August cooling induced comparable advances in bud set timing in both early- and late-leafing trees.

The study did not explain why the timing (July vs. August) resulted in different mechanisms. Can a link be established between phenology and photosynthetic product accumulation? Additionally, can the study differentiate between the direct warming effect and the developmental effect, and quantify their relative contributions?

We thank the reviewer for pointing out that we could improve our explanation of the different responses to July and August cooling in experiment 1. Whilst we incorporated this in the conceptual model and the figure caption (Fig. 1b), we now also address this topic in more depth in the discussion section, focussing on daylength and photosynthetic assimilation as the possible mediators of this change in responses (L350-371).

For the early-season development effect vs the late-season temperature effect we can use the leaf-out day-of-year (as a proxy for development), and the summer cooling treatments (direct temperature effect) to assess the relative importance of these two components of our model. We have now included a variance partitioning analysis following this logic, see L246-252 for methods, L278-281 for results.

(2) The two experimental setups differed in photoperiod: one used a 13-hour photoperiod at approximately 4,300 lux, while the other used an ambient day length of 16 hours with a light intensity of around 6,900 lux. What criteria were used to select these conditions, and do they accurately represent real-world scenarios? Furthermore, as shown in Figure S1, significant differences in soil moisture content existed between treatments - could this have influenced the conclusions?

This question may reflect a misunderstanding regarding the light availability that we hope to address with improved clarification. The duration and intensity of the lighting in these experiments was always set to reflect the average conditions experienced in Zurich for those respective times of the year. Day length in spring is shorter than it is in summer, so the durations were simply adjusted to reflect this reality. The 13-hour, 4,300 lux conditions in experiment 1 were only for the April-May period, when we reduced developmental rates for the late-leafing trees (L125-129). In July, the photoperiod was set to 16 hours and light intensity was approximately 7,300 lux (L150-154). This is equitable to experiment 2–when treatments were applied in June and July–where photoperiod was 16 hours and light intensity approximately 6,900 lux (L206-207). These conditions reflect the average daylengths in Zurich, and the maximum light intensity output by the chambers.

As mentioned in our initial author response, we do not think small differences in soil moisture levels should influence our conclusions. All pots were watered sufficiently to avoid water deficit, and all efforts were made to minimise differences in water availability. A Tukey honest significant difference test showed that only one treatment pair (6 - Late_July_Extreme vs. 7 - Early_August_Moderate, difference = 6%, p < 0.05) had significantly different soil water content, a pair whose responses are not compared. We have added words to this effect in the figure legend of Fig. S1.

(3) The authors investigated how changes in air temperature around the summer solstice affected primary growth cessation, but the summer solstice also marks an important transition in photoperiod. How can the influence of photoperiod be distinguished from the temperature effect in this context?

We agree that photoperiod likely plays a central role. Our conceptual model (Fig. 1) explicitly incorporates photoperiod as the framework within which temperature responses are regulated (L72-75, L627-629 & L638-641). The Solstice-as-Phenology-Switch hypothesis assumes that the annual progression of daylength sets the physiological “window” for trees’ responsiveness to temperature. Our experiments therefore focused on how temperature responses differ before versus after the solstice, while recognising that this reversal is likely enabled by the photoperiod signal. In other words, photoperiod provides the regulatory backdrop, and our results identify how diel and seasonal temperature cues are interpreted within that photoperiodic framework.

(4) The study utilized potted trees in a controlled environment, which limits the generalization of the results to natural forests. Wild trees are subject to additional variables, such as competition and precipitation. Moreover, climate differences between years (2022 vs. 2023) were not controlled. As such, the conclusions may be overgeneralized to "all temperate tree species", as the experiment only involved potted European beech seedlings. The discussion would benefit from addressing species-specific differences.

We agree that extrapolation from our experiments on Fagus sylvatica to other species and natural forests requires caution. However, it is precisely the controlled nature of our design that allowed us to isolate the precise mechanisms that appear to underpin the solstice switch, highlighting the role of diel and seasonal temperature variation. In natural systems, additional variables such as competition, precipitation, and soil heterogeneity can strongly influence phenology, but they also make it difficult to disentangle causal mechanisms. By minimising these confounding factors, our experiment provided a clear test of how temperature before and after the solstice regulates growth cessation.

To acknowledge the limitation, we have toned down statements about generalisation (e.g. “likely generalisable” to “other temperate tree species may display similarities”; L409-411) and explicitly call for follow-up studies across species and forest contexts (L413–414). At the same time, we highlight that our findings align with independent evidence from manipulative experiments, satellite observations, flux measurements, and ground-based phenology, which suggests the mechanisms we report may extend beyond the specific populations studied here.

Reviewer #2 (Public review):

In 'Developmental constraints mediate the summer solstice reversal of climate effects on European beech bud set', Rebindaine and co-authors report on two experiments on Fagus sylvatica where they manipulated temperatures of saplings between day and night and at different times of year. I enjoyed reading this paper and found it well written. I think the experiments are interesting, but I found the exact methods somewhat extreme compared to how the authors present them. Further, given that much of the experiment happened outside, I am not sure how much we can generalize from one year for each experiment, especially when conducted on one population of one species. I next expand briefly on these concerns and a few others.

Thank you for the kind comments. We appreciate your concerns regarding the severity of our treatments and the generalisability of our results, and you can find our detailed responses below.

Concerns:

(1) As I read the Results, I was surprised the authors did not give more information on the methods here. For example, they refer to the 'effect of July cooling' but never say what the cooling was. Once I read the methods, I feared they were burying this as the methods feel quite extreme given the framing of the paper. The paper is framed as explaining observational results of natural systems, but the treatments are not natural for any system in Europe that I have worked in. For example, a low of 2 {degree sign}C at night and 7 {degree sign}C during the day through the end of May and then 7/13 {degree sign}C in July is extreme. I think these methods need to be clearly laid out for the reader so they can judge what to make of the experiment before they see the results.

We understand the concern regarding the structure of the manuscript and note that the methods section was moved to the end of the paper in accordance with eLife’s recommended formatting. We have now moved the methods section before the results to ensure that readers are familiar with the treatments before encountering the outcomes.

We recognise that our temperature treatments were severe and do not mimic real world scenarios. They were deliberately designed to create large contrasts in developmental rates, thereby maximising our ability to detect the mechanisms underpinning the solstice switch. For example, the severe cooling between 4 April and 24 May was specifically designed to slow spring development as much as possible without damaging the plants (L129-L133). We have added text in the Methods to clarify this aim (L129-131 & L156-161).

Regarding presentation, treatment details are now described in both the Methods and the relevant figure legends. Given this structure, we have chosen not to restate the full treatment conditions in the main Results text to avoid repetition.

(2) I also think the control is confounded with the growth chamber experience in Experiment 1. That is, the control plants never experience any time in a chamber, but all the treatments include significant time in a chamber. The authors mention how detrimental chamber time can be to saplings (indeed, they mention an aphid problem in experiment 2), so I think they need to be more upfront about this. The study is still very valuable, but again, we may need to be more cautious in how much we infer from the results.

We appreciate the reviewer’s concern about the potential confounding effect of chamber exposure in experiment 1. We have now discussed this limitation more explicitly, adding further explanation to the Methods (L146-148) and Discussion (L345-346).

Note that chamber-related problems (e.g. aphid infestations) primarily occurred under warm chamber conditions, whereas our experiment 1 cooling treatments maintained low temperatures that suppressed such issues. This means that an equivalent “warm chamber control” could have been associated with its own artefacts, as trees kept under warm chamber conditions would have been exposed to additional stressors that were not present under natural growing conditions. To address this point, we included a chamber control in experiment 2. While aphid abundance was indeed higher in the warm chamber controls, chamber exposure itself had no detectable effect on autumn phenology. This suggests that the main findings of experiment 1 are unlikely to be artefacts of chamber conditions (L141145).

Nevertheless, we agree that chamber exposure remains a potential limitation of experiment 1, which requires clear acknowledgement. We now state this more explicitly in the manuscript while also emphasising that our results are supported by experiment 2 and by converging lines of external evidence.

(3) I suggest the authors add a figure to explain their experiments, as they are very hard to follow. Perhaps this could be added to Figure 1?

We have now added figures to the methods section to depict the experimental timelines and settings more clearly (Figs. 2 and 3).

(4) Given how much the authors extrapolate to carbon and forests, I would have liked to see some metrics related to carbon assimilation, versus just information on timing.

We agree that including more data on photosynthetic assimilation would be valuable for interpreting phenological responses. Indeed, it was our intention to collect this information. However, unfortunately, we experienced technical challenges with the equipment available to us during the experimental period, which prevented us from collecting a full dataset. Nevertheless, we were able to obtain measurements during pre-solstice cooling (now presented as Fig. S12, including data for all treatments), which show that cooling treatments strongly reduced assimilation rates compared to controls. Importantly, these strong reductions occurred across all cooling treatments, yet their phenological outcomes differed markedly, demonstrating that assimilation alone cannot explain the observed responses. As we discuss, our findings are consistent with previous manipulative and observational studies reporting a weak role of late-season assimilation in controlling autumn phenology.

(5) Fagus sylvatica is an extremely important tree to European forests, but it also has outlier responses to photoperiod and other cues (and leafs out very late), so using just this species to then state 'our results likely are generalisable across temperate tree species' seems questionable at best.

We agree that Fagus sylvatica has a stronger photoperiod dependence than many other European tree species. As we note in our response to Reviewer 1 (comment 4), our findings align with previous research across temperate northern forests. Within our framework, interspecific variation in leaf-out timing would not alter the overall response pattern, though it could shift the specific timing of effect reversals. For example, earlier-leafing species may approach completion of development sooner and thus show sensitivity to late-season cooling earlier than F. sylvatica. Nevertheless, we acknowledge the importance of not overstating generality. We have therefore revised the manuscript to phrase conclusions more cautiously (L409411) and highlight the need for further research across species (L413–414).

(6) Another concern relates to measuring the end of season (EOS). It is well known that different parts of plants shut down at different times, and each metric of end of season - budset, end of radial expansion, leaf coloring, etc - relates to different things. Thus, I was surprised that the authors ignore all this complexity and seem to equate leaf coloring with budset (which can happen MONTHS before leaf coloring often) and with other metrics. The paper needs a much better connection to the physiology of end of season and a better explanation for the focus on budset. Relatedly, I was surprised that the authors cite almost none of the literature on budset, which generally suggests it is heavily controlled by photoperiod and population-level differences in photoperiod cues, meaning results may be different with a different population of plants.

We thank the reviewer for pointing out that our discussion of the responses of different EOS metrics needs more clarity. We agree with much of this perspective, and we have added an additional analysis of leaf chlorophyll content data to use leaf discolouration as an alternative EOS marker (L179-195 for methods, L296-311 for results). On this we would like to make two important points:

Firstly, we agree that bud set often occurs before leaf discolouration, although this can depend on which definition of leaf discolouration is used. In experiment 1, bud set occurred on average on day-of-year (DOY) 262 and leaf senescence (50% loss of leaf chlorophyll) occurred on DOY 320. However, we do not necessarily agree that this excludes the combined discussion of bud set and leaf senescence timing. Whilst environmental drivers can affect parts of plants differently, often responses from different end-of-season indicators (e.g. bud set and loss of leaf chlorophyll) are similar, even if only directionally. Figure S11 shows how, across both experiments, treatment effects were tightly conserved (R² = 0.49) amongst the two phenometrics. In accordance with these revisions, we have updated the manuscript title to “Developmental constraints mediate the summer solstice reversal of climate effects on the autumn phenology of European beech” (L1-2).

Secondly, shifts in bud set timing remain the primary focus of the manuscript as these shifts are of direct physiological relevance to plant development and dormancy induction, whereas leaf discolouration may simply follow bud set as a symptom of developmental completion. This is supported by our results, which show stronger responses of bud set than leaf senescence (Figs. 4 & 5 vs. Figs. S9 & S10).

Following the reviewer’s suggestion, we have included more references on the topic of bud set and its environmental controls. The reviewer rightly stresses that photoperiod is considered the most important factor. As mentioned above (see Reviewer 1 comment 3), photoperiod is therefore key in our conceptual model. However, the responses we observed in F. sylvatica cannot be explained by photoperiod alone. For example, in experiment 1, July cooling delayed the autumn phenology of late-leafing trees but had negligible impact on early-leafing trees, even though both experienced the exact same photoperiod. Moreover, in experiment 2, day, night and full-day cooling showed substantial variations in their effects despite equal photoperiod across the climate regimes. This is why we suggest that the annual progression of photoperiod modulates the responses to temperature variations instead of eliciting complete control.

(7) I didn't fully see how the authors' results support the Solstice as Switch hypothesis, since what timing mattered seemed to depend on the timing of treatment and was not clearly related to the solstice. Could it be that these results suggest the Solstice as Switch hypothesis is actually not well supported (e.g., line 135) and instead suggest that the pattern of climate in the summer months affects end-of season timing?

We interpret this concern as relating to the flexibility in reversal timing that we observed. Importantly, the Solstice-as-Phenology-Switch hypothesis does not assume that the reversal is fixed to June 21. Rather the hypothesis implies that reversal occurs around the solstice, when photoperiod cues cause tree individuals to shift from accelerating to decelerating their seasonal development. Our conceptual model (Fig. 1) explicitly incorporates this flexibility by showing how the timing of the reversal depends on developmental speed: Individuals that develop more slowly (or leaf out later) cross the compensatory point later in the summer, whereas fast developing individuals reach it earlier.

Our experiments support this framework: pre-solstice full-day cooling delayed bud set, whereas post-solstice full-day cooling advanced it, with differences between early- and late-developing individuals consistent with the model. Moreover, the contrasting impacts of daytime vs. night time cooling demonstrate how diel conditions can further shape when the reversal is expressed. Thus, rather than contradicting the Solstice-as-Phenology-Switch hypothesis, our findings reinforce it and extend it by showing how flexibility arises from interactions between developmental progression, diel temperature responses, and photoperiod.

We have added an additional section in the Discussion that elaborates on how our results support the Solstice-as-Phenology-Switch hypothesis (L416-432).

Recommendations for the authors:

Reviewing Editor (Recommendations for the authors):

(1) The current strength of evidence is incomplete. Extra justifications of the experimental settings, clarifications of the interpretation of the results, and alternative statistical analyses could make the conclusions more solid.

We agree with the vast majority of the reviewer comments and have made the relevant edits. We believe that these have dramatically improved the clarity of the manuscript. The revised analyses have not changed our conclusions, though we have toned down generalisations.

(2) The Solstice as Switch hypothesis is about the effect of temperature warming. However, the two experiments did not simulate warming but rather cooling. Although a temperature difference can be obtained compared to the control in both cases, the impacts on plant physiology and phenology should still be different between the two scenarios.

Thank you for raising this point, which requires clearer communication in our manuscript. The Solstice-as-Phenology-Switch hypothesis posits that changes in temperature before and after the summer solstice have opposite effects on the autumn phenology of northern forest trees. While the hypothesis has most often been framed in terms of warming, the underlying mechanism concerns whether development is accelerated or slowed relative to ambient conditions. In essence, we are exploring the effect of changes in temperature – not warming per se. In warmer springs, development begins earlier and/or proceeds faster, while in colder springs the opposite occurs; the same logic applies to post-solstice conditions. We have extended our explanation in the Introduction (L69-71).

In our experiments, we applied cooling to create strong contrasts in developmental rates without damaging the trees. These treatments allow us to test the direction of phenological responses relative to ambient conditions. Thus, although we used cooling rather than warming, the results are directly informative for the Solstice-as Switch framework, which concerns the relative effect of temperature changes rather than the absolute direction of manipulation.

(3) The number of groups for bud type and summer temperature treatment is too small to be used as a random effect; it would be more appropriate to treat them as fixed-effect terms.

We have revised the analysis to include bud type as a fixed effect. There are only very minor numerical adjustments (e.g. rounding to 4.8 days instead of 4.9, see L271) and inferences are not altered. We also report the bud type effects for experiment 1 (L262-266) and experiment 2 (L292-293)

(4) Please add more clarifications for Figure 4 about what this figure is for and how you derived this figure, whether the data were from your experiments or others.

We have rewritten the caption for Figure 6 (Fig. 4 in the previous manuscript) to clarify where the data came from and how the figure was generated (L687-693). This figure serves as a visual guide to aid the understanding of the processes that may govern the patterns we have observed. Figure 6a uses data from previous studies on diel patterns in F. sylvatica, specifically growth (Zweifel et al., 2021) and photosynthetic assimilation rates (Urban et al., 2014). To aid visualisation, we linearly interpolated between measurements points, converted the values to a relative percentage (compared to observed maximum), and then smoothed the resulting curves. Based on the evidence from experiment 2, we suggest there may be a temperature threshold below which overwintering responses (e.g. bud set) are induced in F. sylvatica. Figure 6b depicts a theoretical diel pattern of this potential threshold. In simple terms, the threshold must be lower at night because nights are typically colder than days.

Reviewer #2 (Recommendations for the authors):

(1) How can a bud type -- which is apical or lateral -- be a random effect? The model needs to try to estimate a variance for each random effect, so doing this for n=2 is quite odd to me. I think the authors should also report the results with bud type as fixed, or report the bud types separately.

See point (3) in reviewing editor’s recommendations for the authors.

(2) Could the authors move the methods earlier and remind readers of them in the results?

We have addressed this issue, please see detailed response under reviewer 2’s concerns.

Urban O, Klem K, Holišová P, Šigut L, Šprtová M, Teslová-Navrátilová P, Zitová M, Špunda V, Marek MV, Grace J. 2014. Impact of elevated CO2 concentration on dynamics of leaf photosynthesis in Fagus sylvatica is modulated by sky conditions. Environmental Pollution 185: 271–280.

Zweifel R, Sterck F, Braun S, Buchmann N, Eugster W, Gessler A, Häni M, Peters RL, Walthert L, Wilhelm M, et al. 2021. Why trees grow at night. New Phytologist 231: 2174–2185.

I wanted to expand on the term "intrinsically quite different." This means that the difference was presented at -Natural (built into who people are) -Permanent (not changeable) -Fundamental (affecting values, behavior, and identity)

I wanted to highlight this comparison. Communism (influenced by Karl Marx) focuses on struggle between social classes (rich vs. poor) while fascism focuses on struggle between nations (us vs. them).

I was wondering how did the Great Depression challenge people’s confidence in capitalism, and why did some see it as evidence supporting Karl Marx’s predictions?

I find it interesting that some ruling governments, especially traditional, monarchy-based, or elite-controlled ones, saw advantages in going to war, even if they didn’t directly cause it.

every other line should be indented.

Every other line should be indented.

Supposed to have "- - -" after the word.

Every other line should be indented.

Strange Title for the FAQ

Remove

One way to assess platform evolution is by reviewing update frequency and recently released features. More frequent updates may indicate ongoing product development, while less frequent updates may suggest a more stable but slower release cycle.

Based on publicly available release information, ChMeetings publishes updates more frequently, while Breeze appears to follow a less frequent release cycle. The exact frequency may vary over time and should be verified through official product updates or release notes.

When evaluating a church management system, it may be useful to consider how actively the platform evolves over time. This includes factors such as feature updates, product releases, and overall development activity.

ChMeetings supports multi-site and diocesan structures, allowing centralized management across multiple locations while maintaining local autonomy. Breeze does not indicate equivalent functionality for multi-level organizational structures within its standard offering.

This may be relevant for organizations managing multiple churches or planning to scale across locations.

ChMeetings includes a built-in accounting module that supports managing funds, tracking expenses, and generating financial reports within the same system. Breeze focuses on contribution tracking and typically relies on integrations with external accounting tools for full financial management.

This difference may impact whether churches prefer an integrated financial workflow or a combination of specialized tools.

ChMeetings does not apply additional platform fees on top of payment provider charges, and transactions are processed through external providers such as Stripe or PayPal. Breeze may apply additional fees depending on the payment method and region, particularly when using internally managed payment processing.

These differences in fee structures may affect overall donation costs depending on usage volume and chosen payment setup.

Payment Processing Approach

Key Differences That May Influence Your Choice

Based on the comparisons above, the following differences may influence how each platform fits specific church requirements.

Allows members to give via SMS by linking their payment method to their phone number. After initial setup, donations can be completed by sending a text with the desired amount.

The following features are available in Breeze and are not included in ChMeetings:

Provides centralized management capabilities for multi-church organizations, including shared administration and optional dedicated infrastructure.

remove as breeze have it now

Elections: Supports election workflows, including candidate management, voting, and result tracking. Available in higher-tier plans.

Blog: Provides a built-in blogging feature accessible within the application.

Accounting: Includes a built-in accounting module for managing accounts, funds, transactions, and reports. Integrations with external tools such as QuickBooks are available via Zapier.

Mobile Push Notifications: Allows sending push notifications to users logged into the mobile app. This feature is included in paid plans.

The following features are available in ChMeetings and are not included in Breeze:

Breeze manages event registration and payments through forms. Registration data is collected via forms and can be associated with events through configuration, rather than through a dedicated event registration module.

ChMeetings includes a dedicated event module for managing registrations and payments. This includes features such as ticketing, payment handling, and registration management within the event context.

Both platforms support user accounts and member access. In Breeze, member access through the mobile app depends on additional bundled services.

Both platforms offer data import services. With ChMeetings, this service is included with paid plans, while Breeze provides data import as part of onboarding.

Both platforms offer mobile applications available on iOS and Android. ChMeetings provides mobile access for both administrators and members. Breeze includes a mobile app for administrators in its standard plan, while member-facing app access is available through a separate bundle.

Both ChMeetings and Breeze provide a range of church management features covering administration, communication, events, and giving. While they overlap in core functionality, there are differences in how certain features are implemented and accessed. This section outlines shared capabilities and highlights key differences between the two platforms.

Breeze offers website functionality through Tithely Sites, which is available as part of a bundled plan priced at approximately $119 per month. This allows churches to build a website integrated with their church management system.

ChMeetings includes a member portal builder within its paid plans. This can be used to create a simple church page for sharing updates and providing access to member-related content.

Both platforms support established payment processing providers and offer similar capabilities for handling online donations. Differences in fee structures and provider options may influence the overall cost depending on usage and regional availability.

Online giving fees can accumulate depending on donation volume. Both ChMeetings and Breeze allow donors to optionally cover transaction fees, although this may vary in practice.

Website and Member Portal Options

Samir: this need to be same layout like the above card about online giving and messaging

ChMeetings does not process payments directly and does not apply additional platform fees. Payments are processed through providers such as Stripe or PayPal, and standard transaction fees apply.

Breeze processes payments internally in some regions (such as the USA and Canada) and applies transaction fees. In other regions, it integrates with providers such as Stripe, PayPal, Authorize.net, and Tithely Giving.

Both platforms allow donors to optionally cover transaction fees, although this may vary depending on user behavior.

ChMeetings does not include free text messages by default. Messages can be sent using an external phone plan or through purchased messaging packages. For example, in the USA, a package may cost around $15 for 1000 messages (approximately $0.015 per message).

Breeze includes 250 messages and charges additional messages at approximately $0.019 per message. Sending 1000 messages would cost around $19. Breeze does not support sending messages through an external phone plan.

Some cost factors depend on usage and configuration. The following sections outline key differences to consider when evaluating overall cost.

For churches that require managing an unlimited number of members, both platforms offer higher-tier plans. Breeze’s standard plan is priced at $72 per month, while ChMeetings offers a plan at $60 per month for unlimited people.

ChMeetings also provides an annual billing option, where the total cost is equivalent to paying for 10 months instead of 12. Breeze’s pricing is presented as a monthly subscription, with no annual discount indicated.

The differences between these plans reflect variations in pricing structure, feature availability, and usage limits, which may impact suitability depending on church requirements.

A direct comparison between ChMeetings Small and Breeze Basic is relevant, as both plans are similarly priced at $25/month (excluding annual discounts). The table below outlines the differences in included features.

For churches managing between 500 and 1000 people, ChMeetings pricing ranges from $40/month ($400/year) for 500 people to $50/month ($500/year) for 1000 people. Breeze’s standard plan is priced at $72/month, with no annual discount currently indicated.

These differences in pricing and features may influence which plan is more suitable depending on budget constraints and required functionality.

The feature coverage between these plans includes differences such as:

At similar price points, ChMeetings Small and Breeze Basic are both priced at $25 per month. When billed annually, ChMeetings costs $250 per year, while Breeze Basic costs $300 per year. The included features and usage limits differ between the two plans.

For smaller churches, ChMeetings offers a free plan and lower-tier paid plans that scale based on the number of people managed. Breeze offers a basic paid plan with limits on the number of people and administrators. As needs grow, both platforms provide higher-tier plans with expanded features and capacity.

ChMeetings uses a pricing model based on the number of people managed in the system. Breeze follows a flat-rate pricing structure, with different tiers depending on feature access and usage limits.

The free plan includes core functionality such as member management, groups, events, forms, basic communication, and limited online giving.

ChMeetings offers a free plan for churches managing up to 50 people, with a limited set of features. Breeze does not provide a free tier and instead operates on paid plans.

ChMeetings vs Breeze: Pricing Structure and Cost Comparison

When Each Platform May Be a Better Fit

Breeze Church Management is a cloud-based church management system that was launched in 2015 and later acquired by Tithely in 2021. It focuses on providing core church administration features, including member management, event tracking, giving, forms, and basic automation tools.

ChMeetings is a cloud-based church management system (ChMS) developed by Jios Apps Inc. It was launched in 2018 to support churches in managing member data and administrative processes digitally. The platform includes features such as member management, event scheduling and check-in, communication tools, giving, and reporting.

As above: it isn't itself an estimator (of anything useful), but minimizing it tends to identify the nearest model to the truth.

The criteria themselves don't estimate p. They are just penalised log-likelihoods. I think the text should say "can help us choose the optimal lag length p".

shows how in professional public speaking classes, mother tongues/dialects are refused

Enforcing the SAE for someone who speaks in their mother tongue

BIOPIC: Black, Indigenous and People of Color

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Sınırları belirgin olan, genişliği yüksekliğinden fazla olan deri kabarıklığıdır.

Eritema multiforme’de dudakta hemorajik (kanlı) kabuk oluşumu.

Keratin tabakasının kalınlığında artış veya normalde keratinize olmayan bölgelerde keratin oluşmasıdır.

Yumuşak dokularda oluşan derin oluk veya çatlak.

Epitel yüzeyinin tamamen kaybı ve alttaki bağ dokusunun açığa çıkmasıdır. Nekrotik doku, granülasyon dokusu veya fibrin ile örtülebilir.

Bağ dokusunu açığa çıkarmadan epitel yüzeyinin kısmi kaybıdır.

Hücre katmanlarının sayısında azalma, epitel genişliğinde daralma ve rete peg’lerin kaybı veya azalması.

Çapı 5 mm’den küçük olan, epitel yüzeyinde veya epitel altında sıvı içeren ve sınırları belirgin lezyonlardır.

pulls the loosened mucosa towards the muscle durıng chewıng protectıng ıt form beıng bıtten-gevsemıs mukozayı kasa dogru cekerek ısırılmaya karsı korur

① Connects the epithelium to the underlying connective tissue. Epitel ile alttaki bağ dokusunu birbirine bağlar.

② It consists of lamina densa and lamina lucida Lamina densa ve lamina lucida’dan oluşur.

③ Lamina densa is a fibrillar layer (type IV collagen + heparan sulfate) Lamina densa fibriller bir tabakadır (Tip IV kollajen + heparan sülfat içerir).

④ Lamina lucida electron permeable layer (Laminin) Lamina lucida elektron geçirgen bir tabakadır (laminin içerir).

⑤ Approximately 400 Å thick under the basal layer of the epithelium Epitelin bazal tabakası altında yaklaşık 400 Å kalınlığındadır.

① THE RATE OF SHEDDING AND MITOSIS MUST BE THE SAME TO STABILIZE THE EPITHELIUM. Epitelin stabil kalabilmesi için dökülme hızı ile mitoz hızı aynı olmalıdır.

② OTHERWISE PATHOLOGIES OCCUR Aksi takdirde patolojiler ortaya çıkar.

① IN THE BASAL LAYER, TISSUE REGENERATES ITSELF BY MITOSIS. Basal tabakada doku, mitoz ile kendini yeniler.

② MITOTIC CELLS MOVE UPWARDS Mitoz geçiren hücreler yukarı doğru hareket eder.

③ WHEN THEY REACH THE STRATUM CORNEUM, THEY ARE SHED INTO THE MOUTH Stratum corneum’a ulaştıklarında ağız içine dökülürler.

eLife Assessment

The authors previously identified SLAP as a key suppressor of the Src tyrosine kinase and a tumor suppressor. In this important study, the authors show SLAP functions in a cell-autonomous fashion in colon stem cells and propose solid evidence that SLAP reduces tumorigenesis by inhibiting an EphB2-SRC axis.

Reviewer #1 (Public review):

Naim et al. use genetically engineered mouse models and tissue culture cell lines to investigate the role of the SLAP adaptor protein in colonic epithelium and colon tumour formation. The SLAP adaptor protein is known to be a negative regulator of tyrosine kinase signaling in hematopoietic cells, but its role outside the immune system is less well defined. Here, the authors use genetically engineered SLAP-deficient mice, tissue-specific SLAP KO, and colonic organoids to demonstrate that SLAP is expressed in cells of the colonic epithelium, where it acts as a cell-autonomous regulator of proliferation and differentiation. In addition, they provide biochemical evidence that loss of SLAP expression in cultured colonic organoids results in increased Src family kinase activity and global tyrosine phosphorylation, consistent with its known role as a suppressor of tyrosine kinase activity in immune cells. Consistently, treatment with an SRC kinase inhibitor inhibited the growth of SLAP-deficient organoids. These data provide solid evidence of a cell-autonomous role of SLAP in the colonic epithelium.

This work would be improved by further description and interpretation of the SLAP expression pattern shown in the constitutive and tissue-specific KO to further support the conclusions made. In Supplementary Figure 1, magnification of the colon epithelium areas with SLAP expression shown by b-gal and anti-SLAP staining, highlighting regions of interest, would better support the conclusions regarding SLAP expression in specific regions of the colon epithelium. In Supplementary Figure 1B, the authors should indicate that the SLAP staining referred to is epithelial and in resident immune cells, as is mentioned in the text. Also, magnification of the boxed area of LRG5 staining in Figure 1 would improve this figure.

Using a chemically induced model of colitis-associated cancer, the authors demonstrate that inactivation of SLAP shows a trend toward increased tumor formation (though this did not reach significance) as well as increased Src family kinase activity within tumors. Tumor spheres from SLAP-deficient animals showed enhanced growth that was suppressed by treatment with a Src family kinase inhibitor. Of note, the latter effect was specific to SLAP-deficient tumor spheres. These observations are convincing and support the authors' conclusion that SLAP has a tumor suppressor role in CRC through inhibition of SFK signaling.

Mechanistically, elevated expression of the RTK, EphB2, was detected in immunoblots of SLAP KO colonic crypts, while overexpression of SLAP in CRC cell lines downregulated EphB2 protein levels. Using an EPHB2 inhibitor, the role of EPHB2 in the growth of SLAP-deficient colonic organoids was demonstrated. While these data generally support the authors' conclusion that SLAP limits colonic organoid growth by downregulating RTKS such as EphB2 and downstream Src family kinase activity, they do not show which cell types/regions in the colonic epithelium have increased EPHB2 protein and how this relates to SLAP and phospho-SRC expression, as shown in Figure 1 and Figure S1 immunocytochemistry. The expression of EphB2 and its role in colonic tumorsphere growth were not investigated.

Overall, this work provides evidence of SLAP adaptor function in restricting tyrosine kinase signaling in the colonic epithelium, and suggests that loss of SLAP expression could promote tumorigenesis in this context.

Reviewer #2 (Public review):

Summary:

Protein tyrosine kinases are subject to diverse regulatory mechanisms controlling their activity in normal situations. The authors previously identified SLAP (Src-like adaptor protein), a negative regulator of receptor tyrosine kinase (RTK) signaling, as a key suppressor of the cytoplasmic tyrosine kinase SRC in the normal colon and demonstrated that SLAP is downregulated in a majority of colorectal cancers (CRCs).

In this study, the authors further explored SLAP functions in mouse models using constitutive and inducible epithelial-specific Slap deletion (villin-CreERT2 model). They found that loss of SLAP augments colonic epithelial cell proliferation and that induction of tumorigenesis by the AOM/DSS protocol mimicking CRC leads to more aggressive tumors in the absence of SLAP. This effect is apparently cell-autonomous as growth of normal and tumoral colonic organoids is SLAP-dependent in in vitro settings. Finally, the authors define that, in colon, SLAP represses EphB2, an RTK lying upstream of SRC, and show that inhibitors of EphB2 can partially limit tumorigenic development in vitro.

Strengths:

The manuscript is clearly and concisely written, making it easy to follow. The data obtained in the mouse models are very convincing.

Weaknesses:

Direct evidence that EphB2 is activated/phosphorylated in the absence of SLAP is lacking, as conclusions are only based on results obtained with inhibitors. Some other issues have to be addressed before acceptance, in particular, the relevance of the findings in CRC patients.

Author Response:

Public Reviews:

Reviewer #1 (Public review):

Naim et al. use genetically engineered mouse models and tissue culture cell lines to investigate the role of the SLAP adaptor protein in colonic epithelium and colon tumour formation. The SLAP adaptor protein is known to be a negative regulator of tyrosine kinase signaling in hematopoietic cells, but its role outside the immune system is less well defined. Here, the authors use genetically engineered SLAP-deficient mice, tissue-specific SLAP KO, and colonic organoids to demonstrate that SLAP is expressed in cells of the colonic epithelium, where it acts as a cell-autonomous regulator of proliferation and differentiation. In addition, they provide biochemical evidence that loss of SLAP expression in cultured colonic organoids results in increased Src family kinase activity and global tyrosine phosphorylation, consistent with its known role as a suppressor of tyrosine kinase activity in immune cells. Consistently, treatment with an SRC kinase inhibitor inhibited the growth of SLAP-deficient organoids. These data provide solid evidence of a cell-autonomous role of SLAP in the colonic epithelium.

This work would be improved by further description and interpretation of the SLAP expression pattern shown in the constitutive and tissue-specific KO to further support the conclusions made. In Supplementary Figure 1, magnification of the colon epithelium areas with SLAP expression shown by b-gal and anti-SLAP staining, highlighting regions of interest, would better support the conclusions regarding SLAP expression in specific regions of the colon epithelium. In Supplementary Figure 1B, the authors should indicate that the SLAP staining referred to is epithelial and in resident immune cells, as is mentioned in the text. Also, magnification of the boxed area of LRG5 staining in Figure 1 would improve this figure.

We thank the reviewer for their positive and constructive evaluation of our work.

We agree that a more detailed description and visualization of SLAP expression in the colonic epithelium would strengthen our conclusions. In response, we will revise Fig 1 and S1 to better highlight SLAP expression patterns. Specifically, we will include higher-magnification images of the colonic epithelial regions in Suppl Fig 1, with clearly indicated regions of interest. We will also clarify in the legend of Suppl Figure 1B that SLAP staining is observed in both epithelial and resident immune cells, as described in the text. Additionally, we will provide a magnified view of the boxed area showing LGR5 staining in Figure 1 to improve clarity.

Using a chemically induced model of colitis-associated cancer, the authors demonstrate that inactivation of SLAP shows a trend toward increased tumor formation (though this did not reach significance) as well as increased Src family kinase activity within tumors. Tumor spheres from SLAP-deficient animals showed enhanced growth that was suppressed by treatment with a Src family kinase inhibitor. Of note, the latter effect was specific to SLAP-deficient tumor spheres. These observations are convincing and support the authors' conclusion that SLAP has a tumor suppressor role in CRC through inhibition of SFK signaling.

Mechanistically, elevated expression of the RTK, EphB2, was detected in immunoblots of SLAP KO colonic crypts, while overexpression of SLAP in CRC cell lines downregulated EphB2 protein levels. Using an EPHB2 inhibitor, the role of EPHB2 in the growth of SLAP-deficient colonic organoids was demonstrated. While these data generally support the authors' conclusion that SLAP limits colonic organoid growth by downregulating RTKS such as EphB2 and downstream Src family kinase activity, they do not show which cell types/regions in the colonic epithelium have increased EPHB2 protein and how this relates to SLAP and phospho-SRC expression, as shown in Figure 1 and Figure S1 immunocytochemistry. The expression of EphB2 and its role in colonic tumorsphere growth were not investigated.

Overall, this work provides evidence of SLAP adaptor function in restricting tyrosine kinase signaling in the colonic epithelium, and suggests that loss of SLAP expression could promote tumorigenesis in this context.

We also thank the reviewer for their positive comments regarding our tumor studies and the role of SLAP in regulating SFK signaling.

Regarding the mechanistic insights involving EphB2, we appreciate the reviewer’s suggestion to further define its spatial expression and relationship with SLAP and phospho-SRC. To address this, we plan to extend our analysis to assess the effect of Slap depletion on EphB2 protein levels throughout the intestinal epithelium.

We recognize that directly testing EphB2’s role in murine colonic tumorsphere formation would require a new cohort of SLAP knockout mice treated with AOM/DSS for 90 days, which is not feasible in the short term. To address this, we will instead use human colorectal cancer models to assess how SLAP modulation affects the response of tumoroids derived from cell lines to EphB2 inhibition, providing complementary mechanistic insights.

Overall, we believe these additions will strengthen the manuscript and more fully address the reviewer’s concerns.

Reviewer #2 (Public review):

Summary:

Protein tyrosine kinases are subject to diverse regulatory mechanisms controlling their activity in normal situations. The authors previously identified SLAP (Src-like adaptor protein), a negative regulator of receptor tyrosine kinase (RTK) signaling, as a key suppressor of the cytoplasmic tyrosine kinase SRC in the normal colon and demonstrated that SLAP is downregulated in a majority of colorectal cancers (CRCs).

In this study, the authors further explored SLAP functions in mouse models using constitutive and inducible epithelial-specific Slap deletion (villin-CreERT2 model). They found that loss of SLAP augments colonic epithelial cell proliferation and that induction of tumorigenesis by the AOM/DSS protocol mimicking CRC leads to more aggressive tumors in the absence of SLAP. This effect is apparently cell-autonomous as growth of normal and tumoral colonic organoids is SLAP-dependent in in vitro settings. Finally, the authors define that, in colon, SLAP represses EphB2, an RTK lying upstream of SRC, and show that inhibitors of EphB2 can partially limit tumorigenic development in vitro.

Strengths:

The manuscript is clearly and concisely written, making it easy to follow. The data obtained in the mouse models are very convincing.

Weaknesses:

Direct evidence that EphB2 is activated/phosphorylated in the absence of SLAP is lacking, as conclusions are only based on results obtained with inhibitors. Some other issues have to be addressed before acceptance, in particular, the relevance of the findings in CRC patients.

We thank the reviewer for their positive and constructive evaluation of our work.

We agree that our conclusions regarding the SLAP–EphB2–SRC signaling axis rely in part on pharmacological inhibition. As outlined in the manuscript, EphB2 was selected primarily as a proof-of-concept receptor to illustrate how SLAP may indirectly regulate SRC activity through modulation of upstream receptor tyrosine kinases. We note that the use of two distinct classes of EphB inhibitors supports the robustness of our observations.

To further strengthen this aspect of the study, we will assess EphB2 phosphorylation status in SLAP-deficient conditions, which will provide more direct evidence of its activation state and its contribution to SRC signaling.

eLife Assessment

This study presents an important study of the relationship between morphogen signaling and cell fate choices in the forming zebrafish neural tube, addressing a topical question in developmental biology. The authors provide a solid characterization of the precision limit for gene regulatory networks interpreting Shh, with single-cell resolution and state-of-the-art in vivo approaches. While the depth of analysis is restricted, particularly by the number of cell traces, the study will be of interest to developmental biologists interested in cellular decision-making.

Reviewer #1 (Public Review):

[Editors' note: This version has been assessed by the Reviewing Editor without further input from the original reviewers. Given the time elapsed since the original data collection, the authors have addressed the previous concerns by providing a more nuanced discussion of their results and acknowledging the limitations of the study to ensure the conclusions are supported by the existing data.]

Throughout the paper, the authors do a fantastic job of highlighting caveats in their approach, from image acquisition to analysis. Despite this, some conclusions and viewpoints portrayed in this study do not appear well-supported by the provided data. Furthermore, there are a few technical points regarding the analysis that should be addressed.

(1) Analysis of signaling traces

- Relevance of "modeled signaling level": It is not clear whether this added complexity and potential for error (below) provides benefits over a more simple analysis such as taking the derivative (shown in Figure 3C). Could the authors provide evidence for the benefits? For example, does the "maximal response" given a simpler metric correlate less well with cell fate than that calculated from the fitted response?

- Assumptions for "modeled signaling level": According to equation (1) Kaede levels are monotonically increasing. This is assumed given the stability of the fluorescent protein. However, this only holds for the "totally produced Kaede/fluorescence". Other metrics such as mean fluorescence can very well decrease over time due to growth and division. Does "intensity" mean total fluorescence? Visual inspection of the traces shown in Figure 2 suggests that "fluorescence intensity" can decrease. What does this mean for the inferred traces?

- Estimation of Kaede reporter half-live: It is not clear how the mRNA stability of Kaede is estimated. It sounds like it was just assessed visually, which seems not entirely appropriate given the quantitative aspects of the rest of the study. Also, given that Shh signaling was inhibited on the level of Smoothened, it is not obvious how the dynamics of signaling shutdown affect the estimate. Most results in Figure 7 seem to be quite robust to the estimate of the half-live. That they are, might suggest that the whole analysis is unnecessary in the first place. However, not all are. Thus, it would be important to make this estimate more quantitative.

(2) Assignment of fates and correlations

- Error estimate for cell-type assignment: Trying to correlate signaling traces to cell fate decisions requires accurate cell fate assignment post-tracking. The provided protocol suggests a rather manual, expert-directed process of making those decisions. Can the authors provide any error-bound on those decisions, for example comparing the results obtained by two experts or something comparable? I am particularly concerned about the results regarding the higher degree of variability in the correlation between signaling dynamics and cell fate in the posterior neural tube. Here, the expression of Olig2 does not seem to segregate between different assigned fates, while it does so nicely in the anterior neural tube. This would suggest to me that cells in the posterior neural tube might not yet be fully committed to a fate or that there could be a relatively high error rate in assigning fates. Thus, the results could emerge from technical errors or differences in pure timing. Could the authors please comment on these possibilities?

- Clustering and fates: One approach the authors use to analyze the correlation between signaling and fate is clustering of cell traces and comparison of the fate distributions in those clusters. There is a large number of clusters with only single traces, suggesting that the data (number of traces) might not be sufficient for this analysis. Furthermore, I am skeptical about clustering cells of different anterior-posterior identities together, given potential differences in the timing of signal reception and signaling. I am not convinced that this analysis reveals enough about how signaling maps to fate given the heterogeneity in traces in large clusters and the prevalence of extremely small clusters.

- Signaling vector and hand-picked metrics: As an alternative approach, that might be better suited for their data, the authors then pick three metrics (based on their model-predicted signaling dynamics) and show that the maximal response is a very good predictor of fate for different anterior-posterior identities. Previous information-theoretic analysis of signaling dynamics has found that a whole time-vector of signaling can carry much more information than individual metrics (Selimkhanov et al, 2014, PMID: 25504722). Have the authors tried to use approaches that make use of the whole trace (such as simple classifiers (Granados et al, 2018, PMID: 29784812), or can comment on why this is not feasible for their data? The authors should at least make clear that their results present a lower bound to how accurately cells can make cell-fate decisions based on signaling dynamics.

(3) Consequences of signaling heterogeneity

The authors focus heavily on portraying that signaling dynamics are highly variable, which seems visually true at first glance. However, there is no metric used or a description given of what this actually means. Mainly, the variability seems to relate to the correlation between signaling and fate. However, given the data and analysis, I would argue that the decoding of signaling dynamics into fate is surprisingly accurate. So signaling dynamics that seem quite noisy and variable by visual inspection can actually be very well discriminated by cells, which to me appears very exciting.

Indeed, simple features of signaling traces can predict cell fate as well as position (for anterior progenitors). Given that signaling should be a function of position, it naively seems as if signaling read-out could be almost perfect. It might be interesting to plot dorsal-ventral position vs the signaling metrics, to also investigate how Shh concentration/position maps to signaling dynamics, this would give an even more comprehensive view of signal transmission.

There remains the discrepancy between signaling traces and fate in the posterior neural tube. The authors point towards differences in tissue architecture and difficulties in interpreting a "small" Shh gradient. However, the data seems consistent with differences in timing of cell-fate decisions between anterior and posterior cells. The authors show that fate does initially not correlate well with position in the posterior neural tube. So, signaling dynamics should likely also not, as they should rather be a function of position, given they are downstream of the Shh gradient. As mentioned above, not even Olig2 expression does segregate the assigned fates well. All this points towards a difference in the time of fate assignment between the anterior and posterior. Given likely delays in reporter protein production and maturation, it can thus not be expected that signaling dynamics correlate better with cell fate than the reporter "83%". Can the authors please discuss this possibility in the paper?

Thus, while this paper represents an example of what the community needs to do to gain a better understanding of robust patterning under variability, the provided data is not always sufficient to make clear conclusions regarding the functional consequences of signaling dynamics.

Reviewer #2 (Public Review):

Summary:

In this work, Xiong and colleagues examine the relationship between the profile of the morphogen Shh and the resulting cell fate decisions in the zebrafish neural tube. For this, the authors combine high-resolution live imaging of an established Shh reporter with reporter lines for the different progenitor types arising in the forming neural tube. One of the key observations in this manuscript is that, while, on average, cells respond to differences in Shh activity to adopt distinct progenitor fates, at the single cell level there is strong heterogeneity between Shh response and fate choices. Further, the authors showed that this heterogeneity was particularly prominent for the pMN fate, with similar Shh response dynamics to those observed in neighboring LFP progenitors.

Strengths:

It is important to directly correlate Shh activity with the downstream TFs marking distinct progenitor types in vivo and with single cell resolution. This additional analysis is in line with previous observations from these authors, namely in Xiong, 2013. Further, the authors show that cells in different anterior-posterior positions within the neural tube show distinct levels of heterogeneity in their response to Shh, which is a very interesting observation and merits further investigation.

Weaknesses:

This is a convincing work, however, adding a few more analyses and clarifications would, in my view, strengthen the key finding of heterogeneity between Shh response and the resulting cell fate choices.

Author Response:

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

Public Reviews:

Reviewer #1 (Public Review):

Throughout the paper, the authors do a fantastic job of highlighting caveats in their approach, from image acquisition to analysis. Despite this, some conclusions and viewpoints portrayed in this study do not appear well-supported by the provided data. Furthermore, there are a few technical points regarding the analysis that should be addressed.

We thank the reviewer for the comments, due to the age of the work and logistic constraints, we are unable to perform further experiments and analysis to address some of the concerns. We revised conclusions and viewpoints accordingly to reflect reviewer concerns.

(1) Analysis of signaling traces

Relevance of "modeled signaling level": It is not clear whether this added complexity and potential for error (below) provides benefits over a more simple analysis such as taking the derivative (shown in Figure 3C). Could the authors provide evidence for the benefits? For example, does the "maximal response" given a simpler metric correlate less well with cell fate than that calculated from the fitted response?

We think the benefits of modeled signaling level are the conceptual accuracy to the extent possible with the data. It’s true that the assumptions brought-in may cause certain biases. We perform this and the simplest (raw data averaging, Fig.2). Intermediate results in between (such as the first derivative in Fig.3C) may correlate well or less well, but cannot be interpreted biologically.

Assumptions for "modeled signaling level": According to equation (1) Kaede levels are monotonically increasing. This is assumed given the stability of the fluorescent protein. However, this only holds for the "totally produced Kaede/fluorescence." Other metrics such as mean fluorescence can very well decrease over time due to growth and division. Does "intensity" mean total fluorescence? Visual inspection of the traces shown in Figure 2 suggests that "fluorescence intensity" can decrease. What does this mean for the inferred traces?

Yes the segmentations measure intensity in a fixed volume inside a cell, therefore it’s a spatial average (concentration) and is susceptible to cell volume changes. This has been noted in the revision. The raw measurement does fluctuate and can decrease, we think the short-time-scale fluctuations are likely measurement variations/errors rather than underlying big changes in concentration.

Estimation of Kaede reporter half-live: It is not clear how the mRNA stability of Kaede is estimated. It sounds like it was just assessed visually, which seems not entirely appropriate given the quantitative aspects of the rest of the study. Also, given that Shh signaling was inhibited on the level of Smoothened, it is not obvious how the dynamics of signaling shutdown affect the estimate. Most results in Figure 7 seem to be quite robust to the estimate of the half-live. That they are, might suggest that the whole analysis is unnecessary in the first place. However, not all are. Thus, it would be important to make this estimate more quantitative.

Yes we agree. Unfortunately we don’t have the quantitative data required to better estimate Kaede mRNA stability. The timing of Cyc inhibition to the ceasing of ptch mRNA production is roughly estimated but not necessarily precise in this context.

(2) Assignment of fates and correlations

Error estimate for cell-type assignment: Trying to correlate signaling traces to cell fate decisions requires accurate cell fate assignment post-tracking. The provided protocol suggests a rather manual, expert-directed process of making those decisions. Can the authors provide any error-bound on those decisions, for example comparing the results obtained by two experts or something comparable? I am particularly concerned about the results regarding the higher degree of variability in the correlation between signaling dynamics and cell fate in the posterior neural tube. Here, the expression of Olig2 does not seem to segregate between different assigned fates, while it does so nicely in the anterior neural tube. This would suggest to me that cells in the posterior neural tube might not yet be fully committed to a fate or that there could be a relatively high error rate in assigning fates. Thus, the results could emerge from technical errors or differences in pure timing. Could the authors please comment on these possibilities?

This is a very insightful point. We did examine the posterior data again (cross-checked by 2 co-authors) to make sure the mixed situation has correct cell fate assignment. As established by others’ and our previous studies (See also Fig.1A), the identification of MFPs and LFPs in zebrafish spinal cord is very robust. The MFPs are the apical constricted single column of cells along the midline on top of the notochord, and the LFPs are the 2 columns of cells next to MFP on both sides. LFPs’ expression of olig2:gfp did vary more in the posterior (timing of response/commitment could be a factor as the reviewer pointed out), but eventually the cells at those positions will be V3 interneurons or floor plates and have not been observed to make motoneurons. There are 3 low Olig2:GFP pMNs in the anterior dataset (Fig.2B’) and 3 high Olig2:GFP LFPs in the posterior dataset (Fig.2D’) that we checked carefully. The heterogeneity argument is based on the verified tracking and final positioning of these cells.

Clustering and fates: One approach the authors use to analyze the correlation between signaling and fate is clustering of cell traces and comparison of the fate distributions in those clusters. There is a large number of clusters with only single traces, suggesting that the data (number of traces) might not be sufficient for this analysis. Furthermore, I am skeptical about clustering cells of different anterior-posterior identities together, given potential differences in the timing of signal reception and signaling. I am not convinced that this analysis reveals enough about how signaling maps to fate given the heterogeneity in traces in large clusters and the prevalence of extremely small clusters.

We agree. Due to the age of the work and logistic constraints, we are unable to perform further experiments and analysis to enrich the tracks for this revision. We are aware of upcoming, independent studies with many more systematic tracks and analysis which will address these concerns. We have added the caveats the reviewer raised.

Signaling vector and hand-picked metrics: As an alternative approach, that might be better suited for their data, the authors then pick three metrics (based on their model-predicted signaling dynamics) and show that the maximal response is a very good predictor of fate for different anterior-posterior identities. Previous information-theoretic analysis of signaling dynamics has found that a whole time-vector of signaling can carry much more information than individual metrics (Selimkhanov et al, 2014, PMID: 25504722). Have the authors tried to use approaches that make use of the whole trace (such as simple classifiers (Granados et al, 2018, PMID: 29784812), or can comment on why this is not feasible for their data? The authors should at least make clear that their results present a lower bound to how accurately cells can make cell-fate decisions based on signaling dynamics.

Thanks for these suggestions. We are limited by the measurement noise, coverage window of the traces and the number of tracks to make use of the full dynamics in a more informative manner.

(3) Consequences of signaling heterogeneity

The authors focus heavily on portraying that signaling dynamics are highly variable, which seems visually true at first glance. However, there is no metric used or a description given of what this actually means. Mainly, the variability seems to relate to the correlation between signaling and fate. However, given the data and analysis, I would argue that the decoding of signaling dynamics into fate is surprisingly accurate. So signaling dynamics that seem quite noisy and variable by visual inspection can actually be very well discriminated by cells, which to me appears very exciting.

Yes – we agree that most cells are actually accurate in such a highly dynamic tissue. In the literature, the view has been more focused on how the GRN enables this accuracy. We therefore highlighted the heterogeneity and limit of accuracy of the GRN here. We added this point to make our presentation more balanced.

Indeed, simple features of signaling traces can predict cell fate as well as position (for anterior progenitors). Given that signaling should be a function of position, it naively seems as if signaling read-out could be almost perfect. It might be interesting to plot dorsal-ventral position vs the signaling metrics, to also investigate how Shh concentration/position maps to signaling dynamics, this would give an even more comprehensive view of signal transmission.

We’d refer readers to our earlier study Xiong et al., 2013 where ptch2:kaede, nkx2:gfp and olig2:gfp were plotted against position over time in single cell tracks. It was found that position was not a good predictor of signaling levels or cell fates at early stages when the cell fates were specified.

There remains the discrepancy between signaling traces and fate in the posterior neural tube. The authors point towards differences in tissue architecture and difficulties in interpreting a "small" Shh gradient. However, the data seems consistent with differences in timing of cell-fate decisions between anterior and posterior cells. The authors show that fate does initially not correlate well with position in the posterior neural tube. So, signaling dynamics should likely also not, as they should rather be a function of position, given they are downstream of the Shh gradient. As mentioned above, not even Olig2 expression does segregate the assigned fates well. All this points towards a difference in the time of fate assignment between the anterior and posterior. Given likely delays in reporter protein production and maturation, it can thus not be expected that signaling dynamics correlate better with cell fate than the reporter "83%". Can the authors please discuss this possibility in the paper?

Yes this is an important point/caveat of live signaling and fate tracking. As discussed in the manuscript, due to the sensitivity limit of fluorescent imaging, it’s difficult to determine the time when cells start to respond to the signal, and how variable that is from cell to cell. The posterior cells may be more variable in either spatial or temporal responses compared to the anterior and we are not able to distinguish that. However, signaling dynamics is not necessarily a good function of position or time either, there is no evidence for that in our results here. The 83% correlation is thus striking for the posterior progenitors indicating a certain robust logic in the GRN to capture a strong (even short-lived) response to Shh, regardless of position or time. This is an interest possibility (we do not claim it a mechanism as we have not tested it with perturbations) that challenges the prevailing view in the field that these progenitors integrate Shh exposure over time, or that they acquire positional information by reading a gradient.

The discussion has been modified to be more nuanced about these points.

Thus, while this paper represents an example of what the community needs to do to gain a better understanding of robust patterning under variability, the provided data is not always sufficient to make clear conclusions regarding the functional consequences of signaling dynamics.

We quite agree. Together with the reviewer, we look forward to seeing the publication of some recent, independent progresses overcoming the challenges in our work by other colleagues.

Reviewer #2 (Public Review):

Summary:

In this work, Xiong and colleagues examine the relationship between the profile of the morphogen Shh and the resulting cell fate decisions in the zebrafish neural tube. For this, the authors combine high-resolution live imaging of an established Shh reporter with reporter lines for the different progenitor types arising in the forming neural tube. One of the key observations in this manuscript is that, while, on average, cells respond to differences in Shh activity to adopt distinct progenitor fates, at the single cell level there is strong heterogeneity between Shh response and fate choices. Further, the authors showed that this heterogeneity was particularly prominent for the pMN fate, with similar Shh response dynamics to those observed in neighboring LFP progenitors.

Strengths:

It is important to directly correlate Shh activity with the downstream TFs marking distinct progenitor types in vivo and with single cell resolution. This additional analysis is in line with previous observations from these authors, namely in Xiong, 2013. Further, the authors show that cells in different anterior-posterior positions within the neural tube show distinct levels of heterogeneity in their response to Shh, which is a very interesting observation and merits further investigation.

Weaknesses:

This is a convincing work, however, adding a few more analyses and clarifications would, in my view, strengthen the key finding of heterogeneity between Shh response and the resulting cell fate choices.

We thank the reviewer for the comments, due to the age of the work and logistic constraints, we are unable to perform further experiments and analysis to address some of the concerns. We revised conclusions and viewpoints accordingly to reflect reviewer concerns.

Recommendations for the authors:

Reviewer #1 (Recommendations for The Authors):

Minor comments:

y-axis label suddenly changes to Ptch2-reporter level in Figure 5. Is what is plotted different from what is seen as examples in Figure 3?

Thanks! Figure 5 tracks are as Figure 3B, this has been annotated in the figure legends.

There are random bounding boxes in some of the figures.

Sometimes the m in "More dorsal" is stylized with a capital M and sometimes not. It is somewhat confusing as a name for cell types but it is fine if no alternative can be found.

This study unfortunately does not include markers that distinguish the interneurons dorsal to pMNs. We categorized them collectively as “more dorsal”.

Response-time is defined as "the amount of time with an above-basal Shh response". This seems to me as the definition of response duration. I would assume that response-time, means the time it takes until a response is first observed. Please consider changing this.

We did not use “duration” because a response time course recorded in these tracks may include multiple durations (on and off). The duration of exposure/response has been specifically used in the field as a single period of response. So it’s a sum of active responding time here. Clarified in the text.

Reviewer #2 (Recommendations for The Authors):

(1) The authors address several possible setbacks of transforming the measured fluorescence intensity of the patched reporter into a readout of the Shh signaling activity over time, however, one aspect that isn't directly addressed is the potential effect of differences in the z position of analyzed cells. These could, at least in principle, be sufficient to introduce significant noise in the fluorescence measurements. Can the authors subset their datasets by initial, as well as average, z position and then re-examine the measured trends for both Shh activity and the intensity of the cell fate reporters used in the study?

The zebrafish early neural plate/tube has a small thickness in z in dorsal-ventral imaging and the tissue is transparent. The depth-associated scattering contributes very little, if at all to the fluorescent signals in the imaged time window. This can be seen in the nuclear/membrane signal of the movies, which is largely uniform across the tissue in z in the neural tissue. It can also be seen that the notochord cells, further ventral, appears to be dimmer.

(2) It is critical for the validity of this study that the intensity of the patched reporter introduced by the authors in 2012, and used again in this study, faithfully represents the signaling activity of Shh. In this study, the authors provide measurements of the transcriptional rate of Kaede and additional modeling for this purpose. However, an important point is to determine how sensitive is the reporter to changes in Shh signaling of different magnitudes?

We consider this BAC reporter line a good (probably still the best live reporter) one as it resolves the endogenous gradient up to the dorsal interneuron domains (Huang et al., 2012, Xiong et al., 2013) and responds well to perturbations (Notch, Cyclopamine, etc). But it’s true that we don’t have information of how sensitive it responds to changes of different magnitude. As far as we know, there is no in vivo, single cell information of how Shh targets respond to signaling of different magnitudes.

(3) To strengthen the previous point, it would be nice to extend the analysis in Figure 2, at least partially, using other readouts for Shh activity (e.g. GBS-GFP)?

We have used a GBS-RFP line previously and found it to be lower resolution in terms of showing the DV gradient, compared to ptch2:kaede.

(4) It is unclear to me what is the relevant time window during which cells respond to Shh in the anterior versus posterior domains to determine progenitor specification. This is a concern to me, since: i) the average heterogeneity of Shh activity seems to increase strongly in time (Figure 2A/C); and ii) it is important to exclude that the finding of heterogeneous relationship between Shh activity and fate choices is largely driven by later timepoints, where potentially its activity is no longer relevant for cell fate specification. Can this point be clarified when this data is introduced in the manuscript and further discussed?

Yes this is an important point/caveat of live signaling and fate tracking. As discussed in the manuscript, due to the sensitivity limit of fluorescent imaging, it’s difficult to determine the time when cells start to respond to the signal, and how variable that is from cell to cell. The posterior cells may be more variable in either spatial or temporal responses compared to the anterior and we are not able to distinguish that.

(i) The ptch2:kaede reporter variability is higher in terms of magnitude (the signal gets brighter) in later times but the heterogeneity (overlap between difference cell fate groups) is lower in later times

(ii) Similarly, the heterogenous relationship is more pronounced in early time points. Since we do not know exactly when the activity becomes no longer relevant (from our earlier studies we do think that the cells become specified early, when Shh signaling is noisy), we modelled the response profile and searched for a good predictor. The maximum response stands out, particularly as a good indicator for the posterior cells, suggests an early window/time of specification.

Discussion has been modified to clarify these points.

(5) Is the response of the patched reporter, as well as cell fate reporters, to defined concentrations of exogenously provided Shh heterogeneous, for instance, in in vitro experiments?

Well-controlled (e.g., microfluidics and labeled Shh molecules) in vitro experiments will be fantastic future directions. Existing tissue explant + Shh dose approaches do not resolve the heterogeneity of exposure at single cell level but may be helpful in testing the limits and variabilities at different magnitudes.

(6) The source of noise in this system is not entirely clear to me. The authors seem to attribute the heterogeneity they observe to the way cells respond to Shh, but can it be excluded that the morphogen profile is itself noisy to start with? It is currently difficult to distinguish between these two possibilities, given that the Shh activity reporter used in this study is itself a transcriptional output of the pathway. Can the distribution of Shh itself be analyzed (even if in immunostainings) during neural tube formation?

Yes we fully agree. More quantitative analysis may help dissecting the sources of noise. The morphogen profile (particularly through time) will be great. Currently no reagent is available to achieve that. Studies using an engineered morphogen or tagged morphogen suggest that the pattern through tissue reasonably captures simple diffusion dynamics. However, at single cell level considerable randomness may still remain and difficult to quantitatively compare with still staining.

(7) It is unclear to me how the authors define the ultimate cell fate of cells in their analysis in Figure 6. The brief description in the methods and in the manuscript seems to suggest that, in combination with marker expression, the cell position is used as a criteria to assign the fate to the progenitors - if this is the case, I guess the observed relationship in Figure 6 with LMDV distance is almost a control? This could be clarified for the readers.

Yes indeed Figure 6 is a control as LMDV distances lead to final positions which form part of our determination of cell fates.

As established by others’ and our previous studies (See also Fig.1A), the identification of MFPs and LFPs in zebrafish spinal cord is very robust. The MFPs are the apical constricted single column of cells along the midline on top of the notochord, and the LFPs are the 2 columns of cells next to MFP on both sides. LFPs’ expression of olig2:gfp did vary more in the posterior (timing of response/commitment could be a factor as the reviewer pointed out), but eventually the cells at those positions will be V3 interneurons or floor plates and have not been observed to make motoneurons. There are 3 low Olig2:GFP pMNs in the anterior dataset (Fig.2B’) and 3 high Olig2:GFP LFPs in the posterior dataset (Fig.2D’) that we checked carefully.

The methods of fate determination are described in detail in methods.

(8) The graphs in Figures 6 and 7 are difficult to interpret. What proportion, and absolute number, of cells are "mis specified" when the authors show the distinct colored lines in the pMN, LFP or more dorsal domains? How do the authors determine where each cell fate domain begins and ends to access for "mis-specified" cells? Can the authors also provide the corresponding experimental images in the figure?

We apologize for the difficulties to interpret these figures. The graphs are a ranked list of all cells using the specified metric. The visual is to help generate an intuition of how mixed vs clear-cut the pattern is given the tested metric. They are not to be interpreted as the actual pattern in the tissue and there are no data images that show these patterns.

(9) Given the experimental limitations/technical challenges discussed by the authors during the paper, the score of around 90% of predictability of cell fate choices is rather high in the anterior domain, suggesting a minor functional role for heterogeneity in this region. Even for the posterior domain, the score of 83% predictability based on the maximum response to Shh is still relatively high. In my view, this author's conclusions should be adjusted to make this difference clearer in the abstract and discussion, highlighting that the heterogeneity between Shh response and cell fate choices, particularly in the pMN fate, are stronger in the posterior domain affecting the precision of cell fate decisions particularly in this region. Can the authors further comment on potential mechanisms driving this difference?

Yes – we agree that most cells are actually accurate in such a highly dynamic tissue. In the literature, the view has been more focused on how the GRN enables this accuracy. We therefore highlighted the heterogeneity and limit of accuracy of the GRN here.

We have added the fact that the Shh response is still the main determinant of the pattern despite the heterogeneity in the Discussion. We also further discussed possibilities of the anterior posterior differences.

(10) Following up from the previous point, the data in Figure 7 suggests that there might be different underlying mechanisms in how anterior and posterior cells interpret the Shh profile, with anterior cells potentially responding to the integrated concentration of Shh (since response time, average response, or maximum response to Shh all provide similar predictability scores for cell fate choices). In contrast, only the maximum response to Shh can provide a good prediction of posterior cell fate, consistent with a more instantaneous response to morphogen concentration (and thus potentially more error-prone measurement of the Shh profile?). This is a very interesting observation in my view. Could this be further tested?

Thank you. Yes we found this very interesting too. We discussed the possibilities, including the reviewer’s suggestion that these cells may have different contexts or strategy to interpret the signal. It is also possible that the anterior cells use the same strategy (maximum response at an early time) and the subsequent response/duration do not matter to their fate commitment. A precise approach to shut down Shh response dynamics in single cells (e.g., optogenetics) will enable the test of these ideas. We hope following up studies will take such approaches.

This one uses Peergos Secret link to file and opens it in a view

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eLife Assessment

In this important study, DNA and RNA are co-imaged in single cells to show that the proximity of topologically associated domain (TAD) boundaries is uncoupled from the transcriptional activity of nearby genes. The evidence supporting these conclusions is convincing for the regions examined, with high-throughput imaging providing robust statistics. This work will be of interest to researchers studying genome architecture and its relationship to gene regulation.

Reviewer #2 (Public review):

Summary:

Almansour et al., investigate whether the proximity of TAD boundaries is directly linked to gene activity. The authors use high-throughput imaging to simultaneously measure the gene activity and physical distances between boundary regions in an allele-specific manner. Using transcriptional inhibitors, expression induction, and acute depletion of CTCF and cohesin, they test whether proximity of boundaries affects, or is affected by, gene activity.

Strengths:

The combined use of DNA and RNA imaging enabled simultaneous measurement of boundary proximity and transcriptional status at individual alleles. This allows single-allele correlation between boundary proximity and gene activity at multiple loci across thousands of alleles.

The use of both transcription inhibitors and transcription stimulation provides compelling and consistent evidence that boundary proximity can be disconnected from a gene's activity. The data convincingly support the conclusion that stable proximity between boundary regions is not required for ongoing transcription at the loci and timescales examined.

This work strengthens the emerging view that genome organization at the level of domain boundaries does not impose a deterministic control over transcription.

Strong disruption of boundary distances is only observed upon depletion of cohesin. Notably, this corresponds with the largest changes in gene activity. In contrast, depletion of CTCF actually had minimal impact on boundary distances and also had minimal impact on gene activity. This makes sense in light of previous work, where live cell imaging demonstrated that cohesin is more important for domain-structure, whereas CTCF is only important for blocking cohesin from continuing on, such that the fully formed loop occurs in a very small percentage of cells. Therefore, the fact that disruption of cohesin (more important for internal domain structure) affects gene activity while disruption of CTCF does not is exceptionally interesting.

Weaknesses:

In untreated cells, the distribution of distance measurements between boundary probes is exceptionally narrow. While depletion of RAD21 clearly demonstrates an ability to detect changes in this distribution, this tight baseline distribution may limit sensitivity to more subtle changes (like those one might expect from transcriptional influences).

This approach primarily tests the role of boundary interactions rather than domain organization as a whole.

Reviewer #3 (Public review):

Summary:

This study addresses a central question in genome organization: whether the positions of chromosomal domain boundaries are functionally coupled to gene activity. The authors use high-throughput imaging to simultaneously measure distances between boundary markers and nascent RNA production in thousands of individual cells, enabling direct comparison of boundary positions and transcriptional status at single chromosomal copies. This approach is applied across multiple loci, genes, and cell types, and is combined with acute transcriptional perturbations and depletion of architectural proteins to test the relationship between chromosome structure and gene activity in both directions.<br /> This work makes a meaningful contribution by providing direct, single-cell evidence that domain boundary positions and gene activity are largely uncoupled in this system.

Strengths:

A major strength of the work is its single-cell, single-allele resolution, which overcomes the averaging inherent to population-based assays. The authors consistently find that boundary proximity is largely independent of transcriptional status: active and inactive alleles have similar boundary distances, transcriptional perturbations do not shift boundary distributions, and depletion of the boundary factor CTCF does not alter gene expression, whereas cohesin depletion affects both boundary organization and transcription. These conclusions are supported by large numbers of alleles, multiple loci and cell types, and internal controls that distinguish boundary-specific effects from broader chromatin influences. The study offers a robust, scalable imaging pipeline that will be valuable for future studies linking genome organization and transcription at single-cell resolution.

Weaknesses:

The study has important limitations that are acknowledged by the authors. Measurements are restricted to distances between flanking boundaries and do not capture internal domain architecture, sub-domain structure, or finer-scale regulatory contacts. Resolution is limited by probe size and imaging, potentially masking subtle positional changes, and only a small set of loci is examined, leaving open how broadly the uncoupling generalizes. Some perturbation effects, particularly for RAD21, may involve mechanisms beyond boundary disruption.

Author Response:

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

Public Reviews:

Reviewer #1 (Public review):

(1) Conceptual framing and interpretation:

The central conclusion may require more precise framing to avoid potential overreach. The authors' interpretation equating "physical distance between TAD boundaries" with overall "TAD boundary architecture," and "transcriptional bursting events" with broader "gene activity," could benefit from clarification. This framing may not fully capture the temporal dynamics of transcription or the regulatory complexity within TADs. Furthermore, the broad conclusion of an uncoupled relationship appears to challenge extensive prior evidence from perturbation studies showing that disrupting TAD boundaries can alter gene expression. The authors' own observation of reduced gene activity upon RAD21 degradation suggests that global TAD disruption can affect transcription. A more precise and limited conclusion, acknowledging that their data demonstrate a lack of detectable correlation between boundary distance and bursting activity in their system, would be more accurate and help reconcile these findings with the existing literature.

We have modified statements throughout the manuscript, including in the title, to enhance the precision of our conclusions to avoid overreach. We have also added on p. 16 of our Discussion, a separate section on the limitations of the study, noting that our conclusions are limited to TAD boundary distances and do not reflect the structure of TAD boundaries or of TADs themselves. We have also expanded our Discussion of possible TAD functions on p. 14/15.

(2) Technical methods and data presentation:

(2.1) Accuracy and dimensionality of distance measurements: The manuscript does not clearly state whether distances are measured in 2D or 3D, nor does it sufficiently address precision limits. The stated Z-step size (1 µm) may be inadequate for accurately measuring sub-micron chromatin distances in 3D.

We state in both the Results and Methods that our data represent 2D distances derived from maximal-intensity projections of 3D image stacks. We previously published a detailed analysis of the precision of this measurement approach applied to chromatin interactions and documented the effect of 2D vs 3D analysis on these types of measurements. This study by Finn et al., 2022 is cited in the text. We also show in Figure S3 and mention on p. 6 and 10 that we observe similar results using either 2D or 3D analysis.

(2.2) Probe design and systematic error: The genomic coverage size of the BAC probes used for DNA FISH is not explicitly stated. Large probe coverage could inherently blur the precise spatial location of adjacent DNA loci. The reported average distance (~300 nm) may be influenced by the physical size of the probes, as well as systematic expansion or distortion introduced by sample fixation and FISH processing. Although such technical limitations are currently unavoidable, the authors should clarify how these factors might affect their ability to detect subtle distance changes.

The genomic location and size of all probes are provided in Supplementary Table 1. We deliberately use relatively large BAC probes both to generate robust, highly reproducible signals and to eliminate effects arising from local chromatin behavior. In line with earlier characterization of BAC probes (Finn et al., Cell, 2019; Finn et al., Methods, 2022), we find a strong correlation between micro-C/Hi_C interaction frequency and distance measurements. Systematic errors such as sample fixation and FISH processing have previously been evaluated by comparison to live cell data (see Finn et al., 2019) and found to be negligible, especially as all our analyses involve pairwise comparisons, which would both be similarly affected by systematic errors. We discuss resolution limits due to probe size in our new section on study limitations on p. 16.

(2.3) Data Visualization: The manuscript would benefit from including representative, zoomed-in regions of interest from the raw imaging data. This would allow readers to visually assess measured distance differences against background noise.

Raw images for inspection at any magnification are available at https://figshare.com/projects/_b_TAD_boundaries_and_gene_activity_are_uncoupled_b_/271078.

(2.4) Potential impact of resolution limits: In Figure 5, the micro-C data reveal a clear difference in interaction patterns inside versus outside the VARS2 locus TAD, yet the imaging data show no corresponding distance difference. This strongly suggests that the current imaging system, limited by optical resolution, probe size, and localisation accuracy, may be unable to resolve finer-scale spatial reorganizations associated with specific chromatin conformations (e.g., enhancer-promoter loops). The authors should explicitly discuss that their conclusion of "no coupling observed" may be constrained by the resolution and sensitivity of their method and does not preclude the possibility of detecting such associations with higher-precision measurements or in live-cell dynamics.

We generally see good agreement between micro-C/Hi-C data and distance measurements. Specifically, we consistently find closer proximity of boundaries than non-boundaries and larger boundary distances for larger TADs than for smaller ones, as presented throughout the study. Contrary to the reviewer’s statement, this is also true for the VARS2 TAD, where we find statistically significant shorter boundary distances for boundary probes (350 nm) vs the outside control region (390 nm), which correlates with the difference in micro-C interaction score of 5847 vs 2308. These data are shown in Figure 3. Regardless, we mention the issue of resolution due to probe size in the study limitation section on p. 16.

Reviewer #2 (Public review):

In untreated cells, the distribution of distance measurements between boundary probes is exceptionally narrow. While depletion of RAD21 clearly demonstrates an ability to detect changes in this distribution, this tight baseline distribution may limit sensitivity to more subtle changes (like those one might expect from transcriptional influences). In addition, the correlation analysis is asymmetric, primarily stratifying by transcriptional status and then comparing boundary distances. Given the central claim that boundary architecture does not influence gene activity, the analysis should be done from the opposite perspective (stratifying by boundary distance).

We mention the limitations on resolution of our approach in our discussion of study limitations on p. 16. An example of an analysis of stratifying by boundary distance is presented in Figure S3C. The conclusion is the same as stratifying by activity status.

Strong disruption of boundary distances is only observed upon depletion of cohesin. Notably, this corresponds with the largest changes in gene activity. In contrast, depletion of CTCF actually had minimal impact on boundary distances and also had minimal impact on gene activity. This makes sense in light of previous work, where live cell imaging demonstrated that cohesin is more important for domain-structure, whereas CTCF is only important for blocking cohesin from continuing on, such that the fully formed loop occurs in a very small percentage of cells. Therefore, the fact that disruption of cohesin (more important for internal domain structure) affects gene activity while disruption of CTCF does not is exceptionally interesting but is lacking from the discussion.

We mention the stronger effect of cohesion depletion compared to CTCF loss on gene expression in multiple locations in the Results and Discussion.

On a related note, this approach primarily tests the role of boundary interactions rather than domain organization as a whole, and it should be acknowledged that internal domain structures are not directly assessed.

We have modified statements throughout the manuscript to clearly indicate that our conclusions relate to boundary interactions rather than domain organization as a whole. We also discuss this in our section on study limitations.

The comparison to work in other organisms (particularly the comparisons made to Drosophila) should be handled with care. The mechanisms underlying domain formation differ substantially across these systems, particularly regarding the differences in CTCF's role.

We have modified our discussion of the data on Drosophila TADs, particularly as it relates to CTCF.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

I couldn't locate the image data from figshare with the information provided (DOI: 10.6084/m9.figshare.30728354)

The link has been updated

https://figshare.com/projects/_b_TAD_boundaries_and_gene_activity_are_uncoupled_b_/271078.

Reviewer #2 (Recommendations for the authors):

Some of the conclusions overreach. I recommend revising the claims and discussion to focus solely on the proximity of boundaries, instead of TADs themselves. This would match better with your experiments.

We have modified statements throughout the manuscript, including in the title, to enhance the precision of our conclusions to avoid overreach. We have also added on p. 16, a separate section on limitations of our study, noting that our conclusions are limited to TAD boundary distances and do not reflect on the structure of the TADs themselves. We have also expanded our Discussion of possible TAD functions on p. 14/15.

I do disagree with the interpretation of the data in some parts, particularly at the end, where you state that disruption of TADs does not impact gene activity. For example, "Altogether, these results demonstrate that disruption of TAD boundary architecture is insufficient to alter gene expression" doesn't seem to match the results. Sure, depletion of CTCF minimally impacted gene expression, but it also minimally impacted the boundary distances. I think it is interesting that depletion of RAD21 had a bigger impact on both gene expression and boundary distances, and this should be discussed.

We have deleted this statement and now mention on p. 13 that RAD21 depletion affected gene expression, whereas loss of CTCF did not, and on p. 15 that loss of RAD21 had a greater impact on boundary distances than loss of CTCF. We have also expanded our Discussion of possible TAD functions on p. 14/15.

Related to this, I also recommend expanding the discussion of prior live-cell imaging work (ref 32) that showed that the fully formed CTCF loop is a rare event.

We have expanded the discussion of prior live-cell imaging work in several locations.

All the analysis is done from the perspective of the gene expression (e.g. group by expression and then measure distances). It would help to show that the inverse analysis is consistent (e.g. group by distances and measure gene expression).

Analysis of data stratified by distance measurements is shown in Figure S3C.

The discussion of the Drosophila work is strange, given that CTCF in Drosophila has a very different N-terminus, explaining why it doesn't really form loops. Sure, maybe it contributes to domains in some way, but probably no more than the dozens of other architectural proteins that have been found in that system. This work clearly focuses on CTCF-loop domains, so I would be specific about that. In the introduction, you do a good job of saying "in human cells, TADs are.... marked by binding sites for the CTCF protein". However, then you overgeneralize and state that TADs form via a process of loop extrusion. I think a simple statement before this to say that TADs in human cells have become somewhat synonymous with CTCF loop domains, and that is how you will use the term here. However, other organisms have TADs despite the lack of conservation of the CTCF protein.

We have modified the text accordingly.

On a related note, in the discussion, you cite two papers in Drosophila to state that "TADs form prior to the establishment of cell-type-specific gene expression programs", but that's not entirely accurate for those papers. They actually show that TADs occur coincident with ZGA, but loops form before that (ref 23: Espinola et al), or that there are indeed a few boundaries that show up before ZGA, but these correspond to RNA Polymerase (ref 24: Ing-Simmons et al.).

We have corrected this statement.

This setence is important because it uses official statistics to make the argument seem objective, while actually reinforcing fear about the future impact of immigration.

Put me in front of a computer, day after day, week after week, and the will to live will slowly leave my body. Put me in charge of a live production or event and I really shine, juggling all the variables in play.

eLife Assessment

The manuscript presents important findings on how C. elegans can utilize distinct molecular mechanisms and circuit engagements to regulate tactile-dependent locomotory behaviours through the AFD thermosensory neuron. The authors use multiple techniques including microfluidics, genetic manipulations and single-copy rescue experiments, to provide compelling evidence for the role of AFD/AIB electrical synaptic connections in this behaviour. The reviewers are satisfied with the comprehensive revisions made by the authors.

Reviewer #1 (Public review):

Summary:

In this manuscript, Rosero and Bai examined how the well-known thermosensory neuron in C. elegans, AFD, regulates context-dependent locomotory behavior based on the tactile experience. Here they show that AFD uses discrete cGMP signalling molecules and independent of its dendritic sensory endings regulates this locomotory behavior. The authors also show here that AFD's connection to one of the hub interneurons, AIB, through gap junction/electrical synapses, is necessary and sufficient for the regulation of this context-dependent locomotion modulation.

Strengths:

This is an interesting paper showcasing how a sensory neuron in C. elegans can employ a distinct set of molecular strategies and different physical parts to regulate a completely distinct set of behaviors, which were not been shown to be regulated by AFD before. The experiments were well performed and the results are clear. However, there are some questions about the mechanism of this regulation. This reviewer thinks that the authors should address these concerns before the final published version of this manuscript.

Comments on revisions:

In this revised manuscript, Rosero and Bai satisfactorily addressed all the concerns raised by this reviewer regarding their original manuscript. This reviewer appreciates the authors' effort. This revised and improved manuscript demonstrates that a sensory neuron in C. elegans can utilize distinct molecular strategies and circuit engagements to regulate distinct sets of behaviors. This reviewer believes that the manuscript is suitable for final acceptance in eLife.

Reviewer #2 (Public review):

The goal of the study was to uncover the mechanisms mediating tactile-context-dependent locomotion modulation in C. elegans, which represents an interesting model of behavioral plasticity. Starting from a candidate genetic screen focusing on guanylate cyclase (GCY) mutants, the authors identified the AFD-specific gcy-18 gene as essential for tactile-context-dependent locomotion modulation. AFD has been primarily characterized as a thermosensory neuron. However, key thermosensory transduction genes and the sensory ending structure of AFD were shown here to be dispensable for tactile-context locomotion modulation. AFD actuates tactile-context locomotion modulation via the cell-autonomous actions of GCY-18 and the CNG-3 cyclic nucleotide-gated channel, and via AFD's connection with AIB interneurons through electrical synapses. At the circuit level, AIB also receive inputs from the mechanosensory neuron FLP, which was also shown to be relevant for tactile-context-dependent locomotion modulation.

For this study, the authors combined a very clever microfluidic-based behavioral assay with a large set of genetic manipulations to dissect the molecular and cellular pathways involved. Rescue experiments with single-copy transgenes are particularly convincing. The study is very clearly written, and the figures are nicely illustrated with diagrams that effectively convey the authors' interpretation. Overall, the convergence of behavioral assays, genetics, and circuit analysis provides convincing support for the proposed role of the AFD-AIB connection, potentially downstream of FLP via synapic and of other mechanosensory neurons via extra-synaptic communication.

The facts that AFD mediates tactile-context locomotion modulation, that this role relies on GCY-18, and on electrical synapses linking AFD to AIB are new, somewhat unexpected, and interesting. The study raises intriguing and addressable questions about the role of innexin-based cellular communication in a multimodal sensory-behavior microcircuit, including the direction and nature of the signal(s) transmitted through these electrical synapses. These questions remain difficult to address in most experimental systems. The compact and genetically tractable nervous system of C. elegans provides a powerful entry point for addressing them in the context of an intact in vivo circuit.

Reviewer #3 (Public review):

Summary:

Rosero and Bai report an unconventional role of AFD neurons in mediating tactile-dependent locomotion modulation, independent of their well-established thermosensory function. They partially elucidate the signaling mechanisms underlying this AFD-dependent behavioral modulation. The regulation does not require the sensory dendritic endings of AFD but rather the AFD neurons themselves. This process involves a distinct set of cGMP signaling proteins and CNG channel subunits separate from those involved in thermosensation or thermotaxis. Furthermore, the authors demonstrate that AIB interneurons connect AFD to mechanosensory circuits through electrical synapses. They conclude that, beyond its primary function in thermosensation, AFD contributes to context-dependent neuroplasticity and behavioral modulation via broader circuit connectivity.

While the discovery of multifunctionality in AFD is not entirely unexpected, given the limited number of neurons in C. elegans (302 in total), the molecular and cellular mechanisms underlying this AFD-dependent behavioral modulation, as revealed in this study, provide valuable insights into the field.

Strengths:

(1) The authors uncover a novel role of AFD neurons in mediating tactile-dependent locomotion modulation, distinct from their well-established thermosensory function, providing an important conceptual contribution to our understanding of how individual neurons can support multiple, mechanistically separable behavioral functions.

(2) They provide meaningful mechanistic insight into how AFD, GCY-18-dependent cGMP signaling, and AFD-AIB electrical coupling contribute to this AFD-dependent behavioral modulation.

(3) The neural behavior assays utilizing two types of microfluidic chambers (uniform and binary chambers) are innovative and well-designed. In the revised manuscript the authors introduce a removable-barrier assay that physically separates exploration and assay phases. This independent behavioral approach addresses prior concerns about ongoing sensory input and confirms that tactile experience alone is sufficient to modulate locomotion.

(4) By comparing AFD's role in locomotion modulation to its thermosensory function throughout the study, the authors present strong evidence supporting these as two independent functions of AFD.

(5) The finding that AFD contributes to context-dependent behavioral modulation is significant, further reinforcing the growing evidence that individual neurons can serve multiple functions through broader circuit connectivity.

Weaknesses:

While the requirement for AFD, GCY-18, and AFD-AIB electrical coupling is well supported, the directionality of information flow and the precise mode of interaction between mechanosensory neurons, AIB, and AFD remain unclear and an area of future studies.

Overall, the authors successfully achieve their primary aim of identifying and characterizing a novel role for AFD in tactile experience-dependent locomotion modulation. This work contributes meaningfully to the growing body of literature demonstrating multifunctionality and context-dependent reconfiguration of individual neurons within compact nervous systems.

Author Response:

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

Public Reviews:

Although the reviewers agree on the potential importance of this study, they have brought out multiple pertinent queries with respect to the interpretation of some of the results presented in the manuscript, that the authors should consider addressing. The reviewers have also suggested modifications that would increase the clarity of the manuscript.

We appreciate the thoughtful evaluation of our manuscript by the reviewers and the editor. We are encouraged by their recognition of the importance of our study and have carefully considered all the points raised. In response, we have added new data and revised the text to address the concerns and improve the clarity of the manuscript. Our detailed responses to the reviewers’ comments are provided below.

Reviewer #1 (Public review):

Summary:

In this manuscript, Rosero and Bai examined how the well-known thermosensory neuron in C. elegans, AFD, regulates context-dependent locomotory behavior based on the tactile experience. Here they show that AFD uses discrete cGMP signaling molecules and independent of its dendritic sensory endings regulates this locomotory behavior. The authors also show here that AFD's connection to one of the hub interneurons, AIB, through gap junction/electrical synapses, is necessary and sufficient for the regulation of this context-dependent locomotion modulation.

Strengths:

This is an interesting paper showcasing how a sensory neuron in C. elegans can employ a distinct set of molecular strategies and different physical parts to regulate a completely distinct set of behaviors, which were not been shown to be regulated by AFD before. The experiments were well performed and the results are clear. However, there are some questions about the mechanism of this regulation. This reviewer thinks that the authors should address these concerns before the final published version of this manuscript.

Weaknesses:

(1) The authors argued about the role of prior exposure to different physical contexts which might be responsible for the difference in their locomotory behavior. However, the worms in the binary chamber (with both non-uniformly sized and spaced pillars) experienced both sets of pillars for one hour prior to the assay and they were also free to move between two sets of environments during the assay. So, this is not completely a switch between two different types of tactile barriers (or not completely restricted to prior experience), but rather a difference between experiencing a more complex environment vs a simple uniform environment. They should rephrase their findings. To strictly argue about the prior experience, the authors need to somehow restrict the worms from entering the uniform assay zone during the 1hr training period.

We agree that, in the original design, worms in the binary chamber experience a more complex physical environment while retaining access to both exploration and assay zones. We have therefore revised the manuscript to more clearly distinguish between behavioral differences due to exposure to a complex environment and modulation driven by prior experience.

To directly test whether locomotion modulation can be sustained by prior physical experience in the absence of continued access to the exploration zone, we introduced a barrier-based assay that prevents worms from re-entering the exploration zone before locomotion is measured. The results section has been revised accordingly to explicitly address this point.

Revisions to the manuscript:

Lines 122-139: Added two paragraphs describing the new assay and summarizing the corresponding results.

“Because worms in the binary chamber are exposed to both pillar types and remain free to move between exploration and assay zones, the behavioral differences described above could reflect exposure to a more complex physical environment rather than prior experience alone. To directly test whether locomotion is modulated by prior physical experience independently of continued access to the exploration zone, we designed microfluidic chambers in which the assay zone could be separated from the exploration zone by a removable barrier (Fig. 1–Supplement 1A). In these chambers, worms were initially allowed to explore the entire device, including exploration zones that either matched or differed from the assay zone. A barrier was then inserted to prevent worms in the assay zone from re-entering the exploration zones.

Under these conditions, locomotion immediately after barrier insertion was higher in worms that had previously explored physical settings matching the assay zone (205 ± 8 µm/s) than in worms that had explored non-matching settings (151 ± 7 µm/s; p = 0.006; Fig. 1–Supplement 1B). This difference persisted when worms were recorded 40 minutes after barrier insertion, with animals in matching chamber retaining their higher locomotion rates (218 ± 11 µm/s) compared to those in non-matching chambers (185 ± 8 µm/s; p = 0.02; Fig. 1–Supplement 1B). These findings demonstrate that prior exploration of distinct physical environments can modulate locomotion even when worms are prevented from returning to those environments, supporting a role for prior physical experience independent of ongoing sensory input.”

Figure 1–Supplement 1: New figure showing the experimental design and behavioral results.

(2) The authors here argued that the sensory endings of AFD are not required for this novel role of AFD in context-dependent locomotion modulation. However, gcy-18 has been shown to be exclusively localized to the ciliated sensory endings of AFD and even misexpression of GCY-18 in other sensory neurons also leads to localizations in sensory endings (Nguyen et. al., 2014 and Takeishi et. al., 2016). They should check whether gcy-18 or tax-2 gets mislocalized in kcc-3 or tax-1 mutants.

As the reviewer suggested, we examined GCY-18 localization in wild type animals and in mutants with defective sensory microvilli using a split-GFP strategy (He et al., 2019). We generated a gcy18::gfp11×7 knock-in strain using CRISPR–Cas9 to visualize endogenous GCY-18 localization. Consistent with prior studies, GCY-18 localized strongly to the AFD dendritic ending in wild-type animals (Figure 4– Supplement 1A, A′, A′′), with an additional weaker signal detectable near the soma and axon (Figure 4– Supplement 1A′′′).

In kcc-3 mutants, GCY-18 remained localized to the distal dendrite despite disruption of sensory microvillar morphology (Figure 4–Supplement 1B–B′′). Similarly, in ttx-1 mutants, which completely lack AFD sensory microvilli, GCY-18 still localized to the distal dendrite (Figure 4–Supplement 1C–C′′) and remained detectable near the soma and axon (Figure 4–Supplement 1C′′′).

In the revised manuscript, we clarify both the implications and the limitations of these imaging experiments, noting that “although these experiments do not identify the precise subcellular site at which GCY-18 acts, they show that disruption of sensory microvilli does not substantially alter GCY-18 localization within AFD.” The exact site at which GCY-18 functions to support locomotion modulation therefore remains an important open question for future investigation.

Revisions to the manuscript:

Figure 4-Supplement 1: Added a new figure reporting GCY-18 localization in wild type and mutant worms.

Lines 268-280: Added a new paragraph reporting GCY-18 localization in wild type, kcc-3, and ttx-1 mutants and clarifying its relevance to the reviewer’s concern.

“Given that gcy-18 is required for context-dependent locomotion modulation and that GCY-18 localizes to the distal dendrite of AFD, we next examined how disruption of sensory microvilli affects its localization in AFD. We used a split-GFP strategy to visualize endogenous GCY-18 [73]. A tandem array of seven GFP11 β-strands (GFP11x7) was inserted at the C-terminus of GCY-18 using CRISPR-Cas9. When complemented with GFP1-10, GCY-18::GFP11x7 fluorescence was strongly enriched at the AFD sensory microvilli near the nose (Fig. 4–Supplement 1A-A′′), consistent with previous reports [42,74,75]. In addition, weaker but reproducible GCY-18 signal was detected near the AFD soma and axon (Fig. 4–Supplement 1A′′′). Importantly, in kcc-3, which exhibit disrupted sensory microvilli, and ttx-1 mutants, which lack sensory microvilli, GCY-18 remained localized to the distal dendrite and was still detectable near the soma and axon (Fig. 4–Supplement 1B-B′′’ and 1C-C′′′). Although these experiments do not identify the precise subcellular site at which GCY-18 acts, they show that disruption or loss of sensory microvilli does not substantially alter GCY-18 localization within AFD.”

(3) MEC-10 was shown to be required for physical space preference through its action in FLP and not the TRNs (PMID: 28349862). Since FLP is involved in harsh touch sensation while TRNs are involved in gentle touch sensation, which are the neuron types responsible for tactile sensation in the assay arena? Does mec-10 rescue in TRNs rescue the phenotype in the current paper?

We performed cell-specific rescue experiments of mec-10. Single-copy expression of mec-10 cDNA in either FLP neurons alone (egl-44p) or TRNs alone (mec-18p) did not restore context-dependent locomotion modulation (Fig. 5A). In contrast, co-expression in both FLP and TRNs (egl-44p::mec-10 + mec18p::mec-10), as well as expression from the mec-10 promoter, rescued the phenotype.

These results indicate that input from multiple mec-10-expressing neurons, including both FLP and TRNs, is required for context-dependent locomotion adjustment. This requirement differs from spatial preference behavior, where mec-10 acts specifically in FLP (Han et al., 2017), suggesting distinct mechanosensory circuits are engaged by different tactile-driven behaviors.

Revisions to the manuscript:

Fig. 5A: Updated to include the cell-specific rescue data.

Lines 317-331: Added a new paragraph describing these findings.

“The mec-10 gene is expressed in several mechanosensory neurons, including the six touch receptor neurons (TRNs) and the polymodal nociceptors FLP and PVD [77,79]. To determine which neurons are required for tactile-dependent locomotion modulation, we expressed mec-10 cDNA under cell-specific promoters: mec-18p (TRNs) [80], egl-44p (FLP) [81], or mec-10p (TRNs, FLP, and PVD) [79]. Expression in either FLP or TRNs alone did not restore modulation, as worms carrying egl-44p::mec-10 (Δspeed: -11± 4%) or mec-18p::mec-10 (Δspeed: -13 ± 4%) transgenes showed significantly reduced Δspeed compared to wild type (Δ speed: N2: 33 ± 6%; p < 0.0001 for both; Fig. 5A). By contrast, mec-10 co-expression in both FLP and TRNs (Δspeed: 16 ± 4%), or expression from the mec-10 promoter (Δspeed: 23 ± 4%), restored Δ speed to wild type levels (p = 0.20 and p = 0.57, respectively; Fig. 5A). These findings indicate that mec10 expression across multiple mechanosensory neuron types is required for context-dependent locomotion modulation. It is also worth noting that, while both tactile-dependent locomotion modulation and previously reported spatial preference require FLP, only the former depends on TRNs. Together, these findings suggest that distinct subsets of mechanosensory neurons differentially contribute to behaviors shaped by tactile experience.”

(4) The authors mention that the most direct link between TRNs and AFD is through AIB, but as far as I understand, there are no reports to suggest synapses between TRNs and AIB. However, FLP and AIB are connected through both chemical and electrical synapses, which would make more sense as per their mec10 data. (the authors mentioned about the FLP-AIB-AFD circuit in their discussion but talked about TRNs as the sensory modality). mec-10 rescue experiment in TRNs would clarify this ambiguity.

We agree with the reviewer that there are no reported synapses between TRNs and AIB, and we have revised Fig. 5 and the corresponding text to clarify this point. In the revised manuscript, we removed any implication of a direct TRN-AIB connection and instead focus on the established FLP-AIB-AFD pathway, while considering potential indirect contributions from TRNs.

As the reviewer suggested, we performed cell-specific mec-10 rescue experiments. Expression of mec-10 in either FLP alone or TRNs alone was insufficient to restore tactile-dependent locomotion modulation, whereas co-expression in both cell types rescued the phenotype (revised Fig. 5A). These results indicate that FLP is essential for this behavior, consistent with the known FLP-AIB-AFD connectivity, and that TRNs are also required.

Given that TRNs lack direct synapses with AIB, TRN requirement suggests the involvement of indirect communication, likely mediated through modulatory mechanisms such as neuropeptide signaling. Accordingly, we have revised the model (revised Fig. 5C) and the corresponding text to clarify that tactiledependent locomotion modulation integrates inputs from multiple mec-10-expressing neurons and does not rely on a direct TRN-AIB synaptic connection.

Revisions to the manuscript:

Lines 334–345: Revised paragraph to clarify circuit logic and remove implication of direct TRN-AIB synapses.

“Touch-sensitive neurons that express mec-10, including TRNs, FLP, and PVD, do not form direct synapses with AFD, suggesting that tactile information is relayed through intermediary neurons. Because the interneuron AIB receives synaptic input from FLP and forms electrical synapses with AFD, we hypothesized that AIB could serve as a conduit for mechanosensory signals to reach AFD. To test whether AIB is required for tactile-dependent modulation, we examined locomotion in worms with genetically ablated AIB neurons using npr-9p::caspase expression [82]. AIB-ablated worms failed to adjust locomotion speed, showing a near-complete loss of modulation (∆speed: -1 ± 5%) compared to wild type (30 ± 8%, p = 0.001, Fig. 5B). These results demonstrate that AIB is required for AFD-mediated tactile-dependent locomotion modulation. However, because mec-10-expressing TRNs are also required, additional pathways beyond AIB likely contribute to transmitting tactile information to AFD, potentially involving indirect synaptic connections through other interneurons or long-distance signaling via neuropeptides or other modulators (Fig. 5C).”

Fig. 5: Updated to include new cell-specific mec-10 rescue data and revised model.

(5) Do inx-7 or inx-10 rescue in AFD and AIB using cell-specific promoters rescue the behavior?

Yes. We tested this during revision. Using the AFD-specific srtx-1b promoter, we expressed inx10 cDNA selectively in AFD neurons of inx-10 mutant worms. This manipulation significantly restored tactile-dependent locomotion modulation compared to non-transgenic inx-10 mutants (Fig. 6D), demonstrating that inx-10 expression in AFD alone is sufficient to rescue the behavioral defect.

Revisions to the manuscript:

Line 366-370: Added a description of the AFD-specific inx-10 rescue results.

“We next tested whether restoring inx-10 specifically in AFD would be sufficient to rescue the behavioral defect. Using the AFD-specific srtx-1b promoter, we expressed inx-10 cDNA in inx-10 mutant worms. These transgenic animals displayed significantly improved locomotion modulation (∆speed: 42 ± 5%) compared to non-transgenic inx-10 mutants (15 ± 4%; p = 0.018; Fig. 6D), indicating that inx-10 expression in AFD alone is sufficient to restore function.”

Fig. 6D: Updated to include new cell-specific inx-10 rescue data.

(6) How Guanylyl cyclase gcy-18 function is related to the electrical synapse activity between AFD and AIB? Is AFD downstream or upstream of AIB in this context?

At present, the precise relationship between GCY-18 signaling and the AFD-AIB electrical synapse is not fully resolved. Given that AIB receives mechanosensory input from FLP, it is likely that AIB acts upstream of AFD during tactile-dependent locomotion modulation. However, because the AIB-AFD connection is mediated by gap junctions, communication could also be bi-directional, especially since small signaling molecules such as cGMP and Ca²⁺ are known to diffuse through electrical synapses.

We have therefore revised the manuscript to state explicitly that the directionality of information flow between AFD and AIB remains open, and that this will be an important question for future investigation (Line 455-458).

“Together, these findings support a model in which AIB functions as a hub neuron that relays mechanosensory input from FLP to AFD to modulate locomotion (Fig. 5C). However, because electrical synapses are often bidirectional, information flow may also occur in the opposite direction, from AFD to AIB.”

Reviewer #2 (Public review):

Summary:

The goal of the study was to uncover the mechanisms mediating tactile-context-dependent locomotion modulation in C. elegans, which represents an interesting model of behavioral plasticity. Starting from a candidate genetic screen focusing on guanylate cyclase (GCY) mutants, the authors identified the AFDspecific gcy-18 gene as essential for tactile-context-dependent locomotion modulation. AFD is primarily characterized as a thermo-sensory neuron. However, key thermosensory transduction genes and the sensory ending structure of AFD were shown here to be dispensable for tactile-context locomotion modulation. AFD actuates tactile-context locomotion modulation via the cell-autonomous actions of GCY-18 and the CNG-3 cyclic nucleotide-gated channel, and via AFD's connection with AIB interneurons through electrical synapses. This represents a potentially relevant synaptic connection linking AFD to the mechanosensory-behavior circuit.

Strengths:

(1) The fact that AFD mediates tactile-context locomotion modulation is new, rather surprising, and interesting.

(2) The authors have combined a very clever microfluidic-based behavioral assay with a large set of genetic manipulations to dissect the molecular and cellular pathways involved. Rescue experiments with singlecopy transgenes are very convincing.

(3) The study is very clearly written, and figures are nicely illustrated with diagrams that effectively convey the authors' interpretation.

Weaknesses:

(1) Whereas GCY-18 in AFD and the AFD-AIB synaptic connection clearly play a role in tactile-context locomotion modulation, whether and how they actually modulate the mechanosensory circuit and/or locomotion circuit remains unclear. The possibility of non-synaptic communication linking mechanosensory neurons and AFD (in either direction) was not explored. Thus, in the end, we have not learned much about what GCY-18 and the AFD-AIB module are doing to actuate tactile context-dependent locomotion modulation.

We agree with the reviewer that although GCY-18 in AFD and the AFD-AIB connection are clearly required for tactile context-dependent locomotion modulation, the precise mechanisms by which they influence mechanosensory and locomotor circuits remain unresolved. In particular, the possibility of nonsynaptic communication or bidirectional signaling between mechanosensory neurons and AFD cannot be addressed by the current experiments and warrants future investigation.

At the same time, we believe this study reveals several previously unrecognized aspects of tactiledependent locomotion modulation that provide a foundation for future mechanistic investigation.

Specifically, we show that (i) GCY-18 functions in AFD to support tactile-dependent locomotion modulation; (ii) the cGMP-gated channel TAX-4, required for thermosensation, is dispensable for this process, whereas CNG-3 is required, revealing functional specialization within AFD; (iii) the interneuron AIB is necessary for this modulation; and (iv) restoring a single electrical connection between AFD and AIB using mammalian Cx36 is sufficient to rescue tactile-dependent modulation in innexin mutants.

Accordingly, we now explicitly state in the revised Discussion that “a limitation of this study is that the directionality and mode of information flow between AFD and AIB remain unresolved, and defining this relationship will be an important goal for future investigation” (Line 472-475).

(2) The authors only focused on speed readout, and we don't know if the many behavioral parameters that are modulated by tactile context are also under the control of AFD-mediated modulation.

We used locomotion speed as the primary behavioral readout because it provides a robust measure for detecting whether behavior is modified by prior tactile experience, rather than to capture the full spectrum of motor outputs. This strategy is often used to assess experience-dependent behavioral plasticity across sensory modalities and enabled us to uncover the unexpected role of AFD in tactile-dependent plasticity.

In the revised manuscript, we expanded our analysis to include additional behavioral parameters. As described in the Results, AFD-ablated worms showed a complete loss of context-dependent modulation not only in speed, but also in idle time and turning frequency, with no detectable differences between uniform and binary chambers (Fig. 4E). These data strengthen the conclusion that AFD broadly supports tactiledependent behavioral modulation rather than selectively affecting a single locomotor parameter.

Revisions to the manuscript:

Fig. 4E: Revised panel to include additional locomotion parameters, including idle time and turning frequency, in wild type and AFD-ablated worms.

Lines 283–285: Expanded the results to describe changes in locomotion speed, idle time, or turning frequency of AFD-ablated mutant worms. “These animals showed no detectable differences between uniform and binary chambers in locomotion speed, idle time, or turning frequency (Fig. 4E).”

(3) The AFD-AIB gap junction reconstruction experiment was conducted in an innexin double mutant background, in which the whole nervous system's functioning might be severely impaired, and its results should be interpreted with this limitation in mind.

We appreciate the reviewer’s concern that the innexin double-mutant background may broadly affect nervous system function, and we agree that loss of innexins is not restricted to the AFD-AIB synapse and could introduce global circuit perturbations.

Importantly, however, the specificity of the rescue is informative. In an innexin double-mutant background, where electrical coupling is broadly disrupted, re-establishing a single electrical synapse between AFD and AIB using Cx36 was sufficient to restore tactile-dependent locomotion modulation (Fig. 6D). The ability of a targeted AFD-AIB connection to rescue behavior despite the absence of many other electrical synapses argues against a purely global network defect and instead identifies the AFD-AIB electrical synapse as a critical locus for this modulation.

To further address this concern, we performed an additional rescue experiment in a less perturbed genetic background. In the revised manuscript, we show that AFD-specific expression of inx-10 rescues locomotion modulation in inx-10 single mutants (Fig. 6D). Together, these complementary rescue approaches, one restoring endogenous innexin function in AFD and the other reconstituting an electrical synapse using Cx36, support the conclusion that AFD-AIB electrical coupling is sufficient to enable tactile-dependent locomotion modulation, rather than reflecting nonspecific recovery of global circuit function.

Revision to the manuscript:

Fig. 6D and Lines 366-370: Added new data and revised text showing that AFD-specific inx-10 expression restores tactile-dependent locomotion modulation.

“We next tested whether restoring inx-10 specifically in AFD would be sufficient to rescue the behavioral defect. Using the AFD-specific srtx-1b promoter, we expressed inx-10 cDNA in inx-10 mutant worms. These transgenic animals displayed significantly improved locomotion modulation (∆speed: 42 ± 5%) compared to non-transgenic inx-10 mutants (15 ± 4%; p = 0.018; Fig. 6D), indicating that inx-10 expression in AFD alone is sufficient to restore function.”

Reviewer #3 (Public review):

Summary:

Rosero and Bai report an unconventional role of AFD neurons in mediating tactile-dependent locomotion modulation, independent of their well-established thermosensory function. They partially elucidate the signaling mechanisms underlying this AFD-dependent behavioral modulation. The regulation does not require the sensory dendritic endings of AFD but rather the AFD neurons themselves. This process involves a distinct set of cGMP signaling proteins and CNG channel subunits separate from those involved in thermosensation or thermotaxis. Furthermore, the authors demonstrate that AIB interneurons connect AFD to mechanosensory circuits through electrical synapses. They conclude that, beyond its primary function in thermosensation, AFD contributes to context-dependent neuroplasticity and behavioral modulation via broader circuit connectivity.

While the discovery of multifunctionality in AFD is not entirely unexpected, given the limited number of neurons in C. elegans (302 in total), the molecular and cellular mechanisms underlying this AFD-dependent behavioral modulation, as revealed in this study, provide valuable insights into the field.

Strengths:

(1) The authors uncover a novel role of AFD neurons in mediating tactile-dependent locomotion modulation, distinct from their well-established thermosensory function.

(2) They provide partial insights into the signaling mechanisms underlying this AFD-dependent behavioral modulation.

(3) The neural behavior assays utilizing two types of microfluidic chambers (uniform and binary chambers) are innovative and well-designed.

(4) By comparing AFD's role in locomotion modulation to its thermosensory function throughout the study, the authors present strong evidence supporting these as two independent functions of AFD.

(5) The finding that AFD contributes to context-dependent behavioral modulation is significant, further reinforcing the growing evidence that individual neurons can serve multiple functions through broader circuit connectivity.

Weaknesses:

(1) Limited Behavioral Assays: The study relies solely on neural behavior assays conducted using two types of microfluidic chambers (uniform and binary chambers) to assess context-dependent locomotion modulation. No additional behavioral assays were performed. To strengthen the conclusions, the authors should validate their findings using an independent method, at the very least by testing AFD-ablated animals and gcy-18 mutants with a second behavioral approach.

The reviewer points out that the original study relied on locomotion assays in two microfluidic environments (uniform and binary chambers) and suggests validation using an independent behavioral approach, particularly for AFD-ablated animals and gcy-18 mutants.

To address this concern, we developed an independent behavioral assay in which the exploration and assay environments are physically separated by a removable barrier (Figure 1–Supplement 1A). In this design, worms first explored distinct physical settings, after which a barrier was inserted to confine them to an identical assay zone. This approach allowed us to directly test whether context-dependent locomotion modulation can be maintained when worms are prevented from re-entering the exploration environment and must rely solely on prior experience.

Using this assay, we found that wild-type worms that had previously explored environments matching the assay zone moved significantly faster than those that had explored non-matching environments (Figure 1– Supplement 1B-C). These results demonstrate that context-dependent locomotion modulation is retained even when ongoing sensory input from the exploration zone is eliminated, independently validating our original findings using a distinct behavioral paradigm.

Further, using this same assay, we found that locomotion modulation was significantly impaired in both gcy-18 mutants and AFD-ablated worms (Figure 4–Supplement 2A). Together, these results provide independent behavioral evidence supporting the conclusion that AFD and gcy-18 are required for contextdependent locomotion modulation.

Revision to the manuscript:

Figure 1–Supplement 1A: Added schematic and results from the removable-barrier assay in wild type animals.

Lines 120-137: Added corresponding Results text describing the new assay and wild-type behavior.

“Because worms in the binary chamber are exposed to both pillar types and remain free to move between exploration and assay zones, the behavioral differences described above could reflect exposure to a more complex physical environment rather than prior experience alone. To directly test whether locomotion is modulated by prior physical experience independently of continued access to the exploration zone, we designed microfluidic chambers in which the assay zone could be separated from the exploration zone by a removable barrier (Fig. 1–Supplement 1A). In these chambers, worms were initially allowed to explore the entire device, including exploration zones that either matched or differed from the assay zone. A barrier was then inserted to prevent worms in the assay zone from re-entering the exploration zones.

Under these conditions, locomotion immediately after barrier insertion was higher in worms that had previously explored physical settings matching the assay zone (205 ± 8 µm/s) than in worms that had explored non-matching settings (151 ± 7 µm/s; p = 0.006; Fig. 1–Supplement 1B). This difference persisted when worms were recorded 40 minutes after barrier insertion, with animals in matching chamber retaining their higher locomotion rates (218 ± 11 µm/s) compared to those in non-matching chambers (185 ± 8 µm/s; p = 0.02; Fig. 1–Supplement 1B). These findings demonstrate that prior exploration of distinct physical environments can modulate locomotion even when worms are prevented from returning to those environments, supporting a role for prior physical experience independent of ongoing sensory input.” Figure 4–Supplement 2A: Added data for gcy-18 mutants and AFD-ablated worms in the removable barrier assay.

Lines 288-296: Added text describing behavioral defects in gcy-18 mutants and AFD-ablated worms using the new assay.

“Building on our finding that locomotion modulation can be driven by prior physical experience even after worms are prevented from re-entering the exploration zones, we next tested whether AFD is required for this modulation using chambers in which the exploration and assay zones were separated by a removable barrier (Fig. 1–Supplement 1A). Under these conditions, locomotion modulation was significantly reduced in AFD-ablated worms (∆speed: -AFD = 1 ± 6% vs. N2 = 23 ± 7%; p = 0.036; Fig. 4–Supplement 2A). Similarly, gcy-18 mutants showed defective locomotion modulation (∆speed: gcy-18 = -1 ± 8% vs. N2 = 23 ± 7%; p = 0.034; Fig. 4–Supplement 2A). These results indicate that AFD and gcy-18 are required to generate locomotion modulation in response to recent physical experience, even when continued access to surrounding environments is restricted.”

(2) Clarity in Behavioral Assay Methodology: The methodology for conducting the behavioral assays is unclear. It appears that worms were free to move between the exploration and assay zones, with no control over the duration each worm spent in either zone. This lack of regulation may introduce variability in tactile experience across individuals, potentially affecting the reproducibility and quantitativeness of the method. The authors should clarify whether and how they accounted for this variability.

In the primary assay, worms were allowed to move freely between the exploration and assay zones for one hour, and each animal’s tactile experience depended on its exploratory trajectory. To address the resulting variability, we performed an a priori power analysis, which determined that approximately 160 worms distributed across more than 20 chambers per condition were sufficient to obtain reliable populationlevel measurements. This sampling strategy was applied consistently across all experiments. Accordingly, analyses emphasize well-powered population means rather than individual trajectories, ensuring robust and reproducible comparisons despite variability in individual experience.

In addition, as described above, we developed a removable-barrier assay that eliminates variability from ongoing exploration by confining worms to the assay zone after a defined exploration period. The consistency of behavioral effects across both assays further supports the robustness and reproducibility of the approach.

(3) Potential Developmental and Behavioral Confounds in Mutant Analysis: Several neuronal mutant strains were used in this study, yet the effects of these mutations on development and general behavior (e.g., movement ability) were not discussed. Although young adult worms were used for behavioral assays, were they at similar biological ages? To rule out confounding factors, locomotion assays assessing movement ability should be conducted (see reference PMID 25561524).

To address the possibility that behavioral phenotypes in mutant strains arise from developmental defects or impaired general locomotion, we directly measured locomotion speed on agar plates and body length in gcy-18 mutant and AFD-ablated worms. Neither genotype showed defects in basal locomotion speed or body length compared to wild type animals (Figure 4–Supplement 2B-C), indicating that the observed modulation defects are not explained by impaired development or gross motor ability.

To further control for developmental variability, all behavioral assays were performed using agesynchronized populations. Animals were selected at a defined gravid adult stage, identified by the presence of 5-10 eggs arranged in a single row within the gonad. All mutant strains reached this developmental stage approximately three days after egg laying, comparable to wild type animals.

Revision to the manuscript:

Figure 4–Supplement 2B-C: Added quantification of locomotion speed on agar plates and body length for gcy-18 mutants and AFD-ablated worms.

Lines 297-304: Added text describing the data presented in Figure 4–Supplement 2B-C.

“Finally, to determine whether the modulation defects observed in gcy-18 mutants and AFD-ablated worms could be attributed to developmental abnormalities or gross motor impairments, we measured locomotion speed and body length on standard NGM plates. Both day-1 adult AFD-ablated worms (speed: 281 ± 10 µm/s; p = 0.33; body length: 1.12 ± 0.01 mm; p = 0.76) and gcy-18 mutants (speed: 291 ± 13 µm/s; p = 0.22; body length: 1.15 ± 0.02 mm; p = 0.86) showed locomotion speeds and body lengths comparable to wild type controls (speed: 252 ± 30 µm/s; body length: 1.14 ± 0.02 mm; Fig. 4–Supplement 2B, C). These results indicate that the loss of context-dependent locomotion modulation is not due to developmental defects or gross impairments in locomotion.”

(4) Definition and Baseline Measurements for Locomotion Categories: The finding that tax-4 and kcc-3 contribute to basal locomotion but not to context-dependent locomotion modulation is intriguing. The authors argue that distinct mechanisms regulate these two processes; however, the study does not clearly define the concepts of "basal locomotion" and "context-dependent locomotion," nor does it provide baseline measurements. A clear definition and baseline data are needed to support this conclusion.

We define basal locomotion as the locomotion speed of worms measured in the binary chamber, where wild-type animals consistently exhibit lower locomotion rates. Measurements from the binary chamber therefore serve as the baseline reference for locomotion speed in our microfluidic assays. Context-dependent locomotion modulation is defined as the quantified difference in locomotion speed between worms in uniform chambers and those in binary chambers. These definitions are now stated in:

Lines 199-201: “We examined the locomotion speed of mutant worms in the binary chambers, which we refer to as the basal speed because wild type worms consistently move slowest in this environment.”

Lines 645-46: “Asterisks above horizontal black lines indicate statistically significant differences in basal speed, defined as speed of worms in the binary chamber”

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

The availability of strains has not been mentioned. This should be addressed.

The revised Methods section now includes a complete list of strains used in this study, and we have added a statement indicating that all strains are available upon request.

Minor comment:

Figure 1C - it should be Idle, not Idel.

We have corrected the y-axis label in Figure 1C to ‘Idle.’

Reviewer #2 (Recommendations for the authors):

This is an interesting and well-written article, which I greatly appreciated reading. There are a few concerns that the authors should address, in my opinion, to provide a more complete and convincing story.

Major points:

(1) Maybe the material transmitted to me was incomplete, but I did not find the gcy gene screen results. It seems important to present the screen results in full, together with the description of the alleles tested for the 24 gcy genes.

The revised manuscript now includes the complete results of the gcy mutant screen in Figure 2– Supplement 1, with the alleles tested for all 24 gcy genes listed in Table S1.

(2) I did not find the actual p-values, sample sizes for each condition, or raw data; nor a data availability statement indicating where to retrieve these.

Statistical significance is indicated by asterisks in all figures, with definitions provided in each figure legend (n.s., p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Sample sizes are shown as individual data points in the plots, and we have now added explicit n values to each figure legend for clarity. A Data Availability Statement has also been added to indicate where the raw data can be accessed. Where possible, we have included exact p-values. For analyses using Tukey-Kramer post hoc tests, p-values are reported to four decimal places, reflecting the output limits of the statistical software used.

(3) It is not clear why the authors only quantified animal speed for most of the study. What about idle time, turns, and reversals? This choice limits the reach of the study, as we only partly understand what AFD is doing, notably to explain the phenotype in the preference assay.

Data on idle time, turning frequency, and reversal frequency for wild-type worms are now included in Figure 1F. In addition, we present new data showing that AFD ablation disrupts context-dependent modulation of locomotion speed, idle time, and turning frequency (Figure 4E).

(4) Figure 2D and related text: these conclusions are based on a single mutant analysis. Were the millionmutation project lines outcrossed? It would be much more convincing if more gcy alleles were tested (this should be relatively easy since classical alleles are available at the CGC for gcy-8 and gcy-18).

The million-mutation project lines used in this study were outcrossed prior to analysis. In addition, we confirmed that the observed defects were specifically due to loss of gcy-18 function by rescuing the phenotype through expression of gcy-18 cDNA under AFD-specific promoters. This cell-specific rescue shows that the behavioral defects arise from disruption of gcy-18 rather than from background mutations.

(5) It is hard to interpret the speed phenotype when the authors switch between Delta speed and absolute speed display from one figure to another, or even from one panel to another. If only tax-4 and kcc-3 display a constitutive speed phenotype, then there should be no problem showing the absolute speed data in every panel. This is important to convince the reader that major speed changes in mutants are not biasing the interpretation based on Deltas. Indeed, if some mutants move very fast, there might be a ceiling effect. Conversely, if they move very slowly, there might be a 'sickness' effect. Both effects could prevent seeing a tactile-context-dependent modulation, and the results would need to be interpreted much more carefully. Providing the full view on absolute speed levels would also really help support the whole discussion paragraph about the differential regulation of constitutive versus context-dependent locomotion (from L339 onward).

We focus on ∆speed because it directly quantifies experience-dependent locomotion modulation relative to each strain’s own baseline, making it an appropriate metric for comparing tactile plasticity across genotypes. This approach avoids confounding effects from strain-specific differences in overall locomotion levels.

At the same time, we agree that absolute locomotion speed is important to consider when interpreting behavioral phenotypes. To address this, we added plate-based locomotion speed and body length measurements for two key genotypes that lack modulation, gcy-18 mutants and AFD-ablated worms (Figure 4–Supplement 2B–C). Both exhibit normal locomotion on agar plates, indicating that their defects in tactiledependent modulation are not due to impaired motor ability or general sickness.

In addition, among the mutants tested in microfluidic chambers, tax-4 mutants display elevated basal speed yet retain robust context-dependent modulation, indicating that ceiling effects do not limit detection of modulation.

(6) The gap junction expression is a nice experiment. But there is a major limitation that should be stated: the electrical synapse re-construction is made in a double mutant background in which the whole animal circuitry might be severely affected. It might well be that the restoration of behavioral plasticity represents something totally irrelevant to wild-type nervous system functioning. A cell-specific innexin knockout is needed to fully support the relevance of the AFD-AIB connection.

We agree that reconstruction of an electrical synapse in an innexin double-mutant background carries the limitation that global circuit function may be broadly affected. To address this concern, we performed an additional rescue experiment in a less perturbed genetic background.

As described above, we show that AFD-specific expression of inx-10 is sufficient to restore tactiledependent locomotion modulation in inx-10 single mutants (Fig. 6D). This cell-specific rescue does not rely on a double-mutant background and converges on the same outcome as the Cx36-based electrical synapse reconstruction. Together, these complementary approaches support the conclusion that restoring AFD-AIB coupling is sufficient to enable tactile-dependent locomotion modulation, rather than reflecting nonspecific recovery from global circuit disruption.

(7) How was developmental age controlled? It seems that all genotypes were grown for a fixed duration (72h). Some mutants, like gcy-8, might grow slower. It would be useful to at least provide control data in wildtype animals showing that behavioral performance is similar even in slightly younger animals (covering the developmental age of the youngest mutant).

Developmental age was controlled by strict age synchronization and staging criteria rather than growth duration alone. Worms were synchronized by allowing 40-50 young adults to lay eggs on OP50-seeded NGM plates for two hours, after which adults were removed. Developmental stage was further assessed by gonadal morphology, and only young adult animals with 5-10 eggs arranged in a single row were selected for behavioral assays. Using these criteria, all strains, including mutants, consistently reached the assayed stage approximately three days after egg laying, comparable to wild type animals.

To further address the possibility that subtle developmental differences could influence behavior, we measured locomotion speed on agar plates and body length for genotypes that show defects in contextdependent modulation. gcy-18 mutants and AFD-ablated worms exhibited normal locomotion rates and body size, indicating that their behavioral phenotypes are unlikely to arise from developmental delay or impaired general motor ability. These control data are now included in the revised manuscript (Figure 4– Supplement 2B–C).

(8) Plasmid construction description is entirely lacking.

Description of plasmid construction has been added to the revised Methods.

Minor points:

(1) 'Context-dependent locomotion' should be replaced by 'tactile context-dependent locomotion' or something similar throughout the manuscript when referring to the impact of the pillar environment.

Presently, this phrasing shortcut makes the communication too vague throughout, and even confusing when presenting the result of supplementary Figure 2 (where both thermal and tactile contexts are manipulated).

We appreciate this suggestion and have revised the terminology for clarity where appropriate. Prior to introducing the mechanosensory origin of the modulation (that is, before presenting the mec-10 data), we retain the broader term “context-dependent modulation” to avoid presupposing a tactile mechanism before it is experimentally established.

(2) L97: Suggested change along the same lines: "prior experience" -> "prior tactile experience".

We have made this change as suggested.

(3) Figure 1A: Would the author consider swapping the order of conditions displayed in this diagram? It would make more sense to have the same left-to-right order in the whole figure with the binary chamber on the left, particularly since the author describes the results considering the binary chamber as the 'reference point'.

The order of chambers in Figure 1A has been revised as suggested, with the binary chamber now shown on the left.

(4) Figure 1C: 'idel' typo in the axis label.

The y-axis label has been updated from “idel” to “idle.”

(5) Without AFD-specific manipulations, the data with tax-4 and tax-2 mutants provide limited information regarding TAX-4 and TAX-2 role in AFD. It should be explicitly mentioned in the Results section that they might work in other neurons.]

The revised manuscript now explicitly states that the tax-2(p694) allele affects multiple neurons, including BAG, ASE, ADE, and AFD (Lines 421–422).

(6) L220-222: The strict meaning of this sentence implies that one attributes a role to AFD in controlling constitutive locomotion, but none of the presented data directly shows this (both kcc-3 and tax-4 mutant phenotypes could arise from additional neurons, regardless of any perturbation in AFD). This should be corrected.

To avoid implying that AFD directly controls constitutive locomotion, we have removed the sentence in question, “Together, these findings suggest that the role of AFD neurons in modulating context-dependent locomotion is distinct from their thermosensory functions and differs from the mechanisms controlling basal locomotion”, from the revised manuscript.

(7) L328-329: Overstatement. Without AFD-specific manipulation of TAX-2 and TAX-4, the different mutant phenotypes could be due to different cell types, rather than different protein pairs in the channel heteromers. I would recommend addressing this alternative possibility directly in the discussion, rather than focusing only on one (I agree, very cool) possibility.

We have clarified this point in the revised text. We now explicitly note that the tax-2(p694) mutation affects tax-2 expression in multiple neurons (AFD, BAG, ASE, and ADE) (Lines 421–422).

Reviewer #3 (Recommendations for the authors):

(1) Clarification of inx Gene Expression Analysis (Lines 270-271): The authors should specify how the expression of inx genes in individual neurons was identified.

The revised manuscript now specifies that innexin expression patterns were identified using the CeNGEN single-cell transcriptomic database (Lines 352–354).

(2) Cx36 Expression in AFD and AIB (Lines 287-288): Further clarification is needed on how Cx36 expression was achieved in AFD and AIB.

We have clarified that Cx36 was expressed specifically in AFD using the srtx-1b promoter and in AIB using the inx-1 promoter, as stated in the main text (Lines 372–373) and the Fig. 6 legend.

The author argues that "human-induced species extinctions" are not just a result of modern fossil fuel and environmental practices, but from hunting ever since humans became a dominant species. Humans have greatly effected the environment for much longer than many give credit for. Environmental damage doesn't necessarily come from burning fossil fuels, it seems to increase when humans make social and technological advancements.

I'm trying to find this on the Nuke Repo and I can't find this section. Is this on the doc side and if so, could we remove this?

Remove "Windows 10 (64-bit) or"

for:

17.1 17.0 16.1

Please and thank you