the presence of two or more cell lines with different genetic makeup within a single organism, resulting from an error in cell division after fertilization

"They’re like us.” Yes, they are, think about politics, data centers, crime that happens every day.

"Ignorance is not bliss. Well, in some special cases, it's better not to know available information," because it's scary to know all, life is frightening, so sometimes peace of mind is acting as if nothing "exists".

Latin: "at the point of death"

A delusion of the truth?

Ignorance is not bliss. Well, in some special cases, it's better not to know information that is available. However, knowledge is power and can contribute to status and financial gain.

Freedom is not just physical, it's also mental, emotional, and spiritual.

I agree with this statement, without knowledge, humans are just followers of the most ignorant of them all.

Humans who dwell in the caves only "know" what others tell them. Of the fears and judgments of others.

Other than the abundance argument, wouldn't it be better to pick one of the ARGs itself as an indicator rather than this proxy?

Aspects of language transcend time and linger on todays vocabularies.

A quote that really highlights what this article is about.

Diving into the history of this English is so helpful in understanding the way it is today, and the why/how of it all.

The comparison here is intriguing to me to see how similar it is across time.

I am always curious about language and such before we were able to record things. The sounds of our past are so recent, and to really know what something sounded like before we had the ability to record is so unknown. Fascinating to me.

This is an interesting bit of information to me, I think that it's quite fascinating that some of these 'improper uses of English' have a British/white source.

This has been a common theme throughout many of the course readings in Section 2 of the English 1110.

This is what they are being fed by the gov.

So not only will there be cultural divides but also a fallout from whatever they were fleeing

I suspect this will hit on similair notes from the book

again any outcomes here you can mention? how many people used this? what problem did it solve?

Here Taylor and Hunt-Hendrix argue against the idea that identities must be thrown off to promote solidarity and that ,on the contrary, inclusion of various identities is the only way to build transformative solidarity and to overcome entrenched divisions.

It seems that transformative solidarity is centered not on necessarily changing the "Us" but reframing it. In order to bring change we must take the solidarity we feel from friends and family and expand those feelings to broader societal categories. In doing so we practice transformative solidarity and create more uniformed collective identities.

Speed dating!! I'd love to know more about how well this worked

Stronger Alternatives: A stronger argument for Strauss would be to emphasize Aquinas's theological framework, since every natural human good depends on its relation to the supernatural end. Without knowledge of the supernatural, Aquinas' argument lacks the standard against which it is measured, making his view of the perfect and imperfect views of human good incomplete. It could strengthen Strauss's point by showing that Aquinas's logic renders political science entirely theological, even when it seems reasonable.

Ambiguity/Misleading Language: The phrase "Supreme Authority" is unclear whether it refers to political or supernatural authority. Whereas the use of "political goods" could be misleading in the argument about the paranormal. It is unclear whether it concerns the natural good of humans or if it serves a supernatural end.

Consistency: It's consistent in linking physical, moral, and supernatural needs. But some phrases blur the lines between the natural political order and Aquinas' supernatural theology. The argument maintains the same spiritual justification but fails to distinguish between theology and politics.

Soundness: The argument is sound and supported by Aquinas' own writing, as Strauss claims that Aquinas is establishing superiority through natural virtue rather than appealing to supernatural order. Where Aqunia treats virtue as the highest reasonable attainable by reason, the text relies on Aquinas's philosophical beliefs, even if the debate is from a theological standpoint.

Some flaws in this argument are an appeal to consequences, as Bourke argues that the supernatural end has brought "rational order" to some people's lives, so it is reasonable to accept that Aquinas' belief is true. But it also falls into the hasty generalization, assuming that everyone shares Aquinas's religious beliefs, even though this does not represent all philosophical and religious beliefs. Lastly, there's a categorical mistake because we cannot logically confirm the existence of a supernatural theoretical end.

Validity: The premise is valid because, in the conclusion, Strauss argues that if Aquinas believes that no good can be perfect from human nature, then human fulfillment has to lie in the supernatural end. Since political science is dependent on an understanding of the ultimate end, he relies on theology rather than philosophy. However, the argument's validity doesn't imply it's true, as Strauss's reasoning is sound and free of contradictions, unlike Aquinas's.

Seeing a formatted page matters because format equals communication. The page tells everyone what kind of day, where we are, and how to prep

Cinema’s gotten super complex image, sound, VR, all that, but the heartbeat is still story. If the fireworks don’t serve a clean narrative, we leave cold.

I have never seen a noir film. I always thought they were interesting, but I still have been reluctant.

Composition is like the choreography of a film, where things are placed and how they move both inside and outside the frame. Those choices shape how we feel the scene, turning simple shots into something dynamic and alive.

Lighting is more than just turning things on; it’s an art form that shapes the mood and meaning of a scene. Every choice with key, fill, or back light changes how we see characters and space, making light itself part of the story.

I have always liked this perspective for actors. A time where you get to immerse yourself in a whole new person and time.

Whether in the background or directly in a character’s hands, each object adds layers of meaning and helps shape how we read the scene.

The use of CGI has grown throughout the years, but so has its quality. Still giving us something to real as possible.

It’s not just about building sets, but about locking down light, sound, and even time of day so the director’s vision can play out without outside interruptions.

The architect of the film’s look. They’re the ones making sure every detail in the environment lines up with the director’s vision, so the world on screen actually feels intentional and consistent.

It’s part of the story itself. The way a space is built or chosen shapes how we feel about the scene, almost like another character working under the director’s vision.

The director feels more like a glue role than a boss role. They’re less about controlling every move and more about making sure all the parts flow into the same bigger picture.

I agree, because a director really does leave their fingerprints all over a film. Their vision ties every piece together, so it makes sense to see them as the true author of the story on screen.

mise-en-scène is really just the full package, every single detail we see on screen, shaping the vibe. Nothing is random; everything adds to the story being told.

important proposal research

how does the increase of technology affect how students perform, and does it affect our district?

You can see how the population was reduced and what had all happened.

It's insane to see how an outbreak can wreck so many things and effect all these people.

It's really cool to see how he died without a male heir and this is how it was passed to his young grandson

eLife Assessment

This study presents a valuable finding on a new role of glia in activity-dependent synaptic remodeling using the Drosophila NMJ as a model system. The evidence supporting the claims of the authors is convincing. The authors have addressed most of the reviewers' concerns and help to further clarify the claims. The work will be of interest to neuroscientists working on glia-neuron interaction and synaptic remodeling.

Reviewer #2 (Public review):

In this paper Chang et al follow up on their lab's previous findings about the secreted protein Shv and its role in activity-induced synaptic remodeling at the fly NMJ. Previously they reported that shv mutants have impaired synaptic plasticity. Normally a high stimulation paradigm should increase bouton size and GluR expression at synapses but this does not happen in shv mutants. The phenotypes relating to activity-dependent plasticity were completely recapitulated when Shv was knocked down only in neurons and could be completely rescued by incubation in exogenously applied Shv protein. The authors also showed that Shv activation of integrin signaling on both the pre- and post-synapse was the molecular mechanism underlying its function in plasticity. Here they extend their study to consider a role of Shv derived from glia in modulating synaptic features at baseline and remodeling conditions. The authors show evidence that Shv is expressed in both neurons and glia. Despite the fact that neuron-specific RNAi knockdown of Shv recapitulated the plasticity phenotypes seen in whole animal mutants, the authors asked whether glial-specific knockdown would have any effects. Surprisingly, knockdown of Shv only in glia also blocked plasticity, just like neuron-specific knockdown, and supporting an important role for glial-derived Shv in plasticity. Unlike neuronal knockdown, though, glial knockdown also caused abnormally high baseline GluR expression. Restoring Shv in ONLY glia in mutant animals is sufficient to completely rescue the plasticity phenotypes and baseline GluR expression, but glial-Shv does not appear to activate integrin signaling which was shown to be the mechanism for neuronally derived Shv to control plasticity. This suggests a different or indirect mechanism of action for glial-derived Shv. This led the authors to hypothesize that glial Shv might work via controlling the levels of neuronal Shv and/or extracellular glutamate. To test these hypotheses, they provide evidence that in the absence of glial Shv, synaptic levels of Shv go up overall, suggesting that glial Shv could somehow have a suppressive effect on release of neuronal Shv. This would indirectly modulate integrin signaling to control plasticity. Using an extracelluar glutamate sensor in presynaptic boutons, they also observe decreased signal (extracellular glutamate) from the sensor in glial Shv KD animals, and increased signal in glial Shv overexpression animals, supporting the hypothesis that glial Shv can regulate glutamate levels somehow. These results establish glia as an important source of Shv in these processes and identify some mechanisms for how this might be accomplished. Several outstanding questions remain-most importantly: how/why do glial-derived and neuronal-derived Shv have different effects when in the same space? No obvious isoform or size differences were found, and the same rescue construct expressed either in neurons or glia could have different effects on integrin activation or glutamate levels. Answering these questions using modified rescue constructs will be an important future direction to understand Shv function specifically and how neurons and glia work together in this context--and potentially many other contexts.

Comments on revisions:

The authors addressed my and the other reviewers' concerns from the original review adequately and this has strengthened the paper substantially.

One small omission to correct: In Figures 4 and 6, the graphs in the figures do not have a legend for the colored bars.

Reviewer #3 (Public review):

Summary:

The manuscript by Chang and colleagues provides compelling evidence that glia-derived Shriveled (Shv) modulates activity-dependent synaptic plasticity at the Drosophila neuromuscular junction (NMJ). This mechanism differs from the previously reported function of neuronally released Shv, which activates integrin signaling. They further show that this requirement of Shv is acute and that glial Shv supports synaptic plasticity by modulating neuronal Shv release and the ambient glutamate levels. However, there are a number of conceptual and technical issues that need to be addressed.

Major comments

(1) From the images provided for Fig 2B +RU486, the bouton size appears to be bigger in shv RNAi + stimulation, especially judging from the outline of GluR clusters.

(2) The shv result needs to be replicated with a separate RNAi.

(3) The phenotype of shv mutant resembles that of neuronal shv RNAi - no increased GluR baseline. Any insights why that is the case?

(4) In Fig 3B, SPG shv RNAi has elevated GluR baseline, while PG shv RNAi has a lower baseline. In both cases, there is no activity induced GluR increase. What could explain the different phenotypes?

(5) In Fig 4C, the rescue of PTP is only partial. Does that suggest neuronal shv is also needed to fully rescue the deficit of PTP in shv mutants?

(6) The observation in Fig 5D is interesting. While there is a reduction in Shv release from glia after stimulation, it is unclear what the mechanism could be. Is there a change in glial shv transcription, translation or the releasing machinery? It will be helpful to look at the full shv pool vs the released ones.

(7) In Fig 5E, what will happen after stimulation? Will the elevated glial Shv after neuronal shv RNAi be retained in the glia?

(8) It would be interesting to see if the localization of shv differs based on if it is released by neuron or glia, which might be able to explain the difference in GluR baseline. For example, by using glia-Gal4>UAS-shv-HA and neuronal-QF>QUAS-shv-FLAG. It seems important to determine if they mix together after release? It is unclear if the two shv pools are processed differently.

(9) Alternatively, do neurons and glia express and release different Shv isoforms, which would bind different receptors?

(10) It is claimed that Sup Fig 2 shows no observable change in gross glial morphology, further bolstering support that glial Shv does not activate integrin. This seems quite an overinterpretation. There is only one image for each condition without quantification. It is hard to judge if glia, which is labeled by GFP (presumably by UAS-eGFP?), is altered or not.

(11) The hypothesis that glutamate regulates GluR level as a homeostatic mechanism makes sense. What is the explanation of the increased bouton size in the control after glutamate application in Fig 6?

(12) What could be a mechanism that prevents elevated glial released Shv to activate integrin signaling after neuronal shv RNAi, as seen in Fig 5E?

(13) Any speculation on how the released Shv pool is sensed?

Comments on revisions:

The authors have addressed most of my previous comments and questions in their revision.

Author response:

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

Reviewer #1 (Public review):

In this manuscript, Chang et al. investigated the cell type-specific role of the integrin activator Shv in activity-dependent synaptic remodeling. Using the Drosophila larval neuromuscular junction as a model, they show that glial-secreted Shv modulates synaptic plasticity by maintaining the extracellular balance of neuronal Shv proteins and regulating ambient extracellular glutamate concentrations, which in turn affects postsynaptic glutamate receptor abundance. Furthermore, they report that genetic perturbation of glial morphogenesis phenocopies the defects observed with the loss of glial Shv. Altogether, their findings propose a role for glia in activity-induced synaptic remodeling through Shv secretion. While the conclusions are intriguing, several issues related to experimental design and data interpretation merit further discussion.

We appreciate the insightful and constructive comments. We have added new data and modified the text to address your concerns.  In doing so, the manuscript has been substantially strengthened.  Please see our detailed point-by-point response below. 

Reviewer #2 (Public review):

In this paper Chang et al follow up on their lab's previous findings about the secreted protein Shv and its role in activity-induced synaptic remodeling at the fly NMJ. Previously they reported that shv mutants have impaired synaptic plasticity. Normally a high stimulation paradigm should increase bouton size and GluR expression at synapses but this does not happen in shv mutants. The phenotypes relating to activity dependent plasticity were completely recapitulated when Shv was knocked down only in neurons and could be completely rescued by incubation in exogenously applied Shv protein. The authors also showed that Shv activation of integrin signaling on both the pre- and post- synapse was the molecular mechanism underlying its function. Here they extend their study to consider the role of Shv derived from glia in modulating synaptic features at baseline and remodeling conditions. This study is important to understand if and how glia contribute to these processes. Using cell-type specific knockdown of Shv only in glia causes abnormally high baseline GluR expression and prevents activity-dependent increases in bouton size or GluR expression post-stimulation. This does not appear to be a developmental defect as the authors show that knocking down Shv in glia after basic development has the same effects as lifelong knockdown, so Shv is acting in real time. Restoring Shv in ONLY glia in mutant animals is sufficient to completely rescue the plasticity phenotypes and baseline GluR expression, but glial-Shv does not appear to activate integrin signaling which was shown to be the mechanism for neuronally derived Shv to control plasticity. This led the authors to hypothesize that glial Shv works by controlling the levels of neuronal Shv and extracellular glutamate. They provide evidence that in the absence of glial Shv, synaptic levels of Shv go up overall, presumably indicating that neurons secrete more Shv. In this context which could then work via integrin signaling as described to control plasticity. They use a glutamate sensor and observe decreased signal (extracellular glutamate) from the sensor in glial Shv KD animals, however, this background has extremely high GluR levels at the synapse which may account for some or all of the decreases in sensor signal in this background. Additional controls to test if increased GluR density alone affects sensor readouts and/or independently modulating GluR levels in the glial KD background would help strengthen this data. In fact, glialspecific shv KD animals have baseline levels of GluR that are potentially high enough to have hit a ceiling of expression or detection that accounts for the inability for these levels to modulate any higher after strong stimulation and such a ceiling effect should be considered when interpreting the data and conclusions of this paper. Several outstanding questions remain-why can't glial derived Shv activate integrin pathways but exogenously applied recombinant Shv protein can? The effects of neuronal specific rescue of shv in a shv mutant are not provided vis-à-vis GluR levels and bouton size to compare to the glial only rescue. Inclusion of this data might provide more insight to outstanding questions of how and why the source of Shv seems to matter for some aspects of the phenotypes but not others despite the fact that exogenous Shv can rescue and in some experimental paradigms but not others.

We appreciate your insightful comments. We have added new data and modified the text to address your concerns.  In doing so, the manuscript has been substantially strengthened.  Please also see the enclosed point-by-point response.

To address the question of whether altered GluR density alone affects sensor readouts, we expressed GluR using a mhc promoter-driven GluRIIA fusion line, which increases total GluRIIA expression in muscle independently of the Gal4/UAS system. As shown in Figure 6 – figure supplement 1, mhc-GluRIIA animals exhibited elevated levels of not only GluRIIA but also the obligatory GluRIIC subunit. Despite this increase in GluR expression, we did not observe any change in extracellular glutamate levels, as measured by live imaging using the neuronal iGluSnFR sensor (updated Figure 6A). These results suggest that elevated GluR density alone does not alter iGluSnFR sensors dynamics and further support our conclusions.

In regard to the question about ceiling effect, we do not think that the lack of GluR enhancement in repo>shv-RNAi is due to a saturated postsynaptic state. This is based on results in Figure 6, which shows that GluR levels can increase up to fourfold upon stimulation in the presence of glutamate, whereas repo>shv-RNAi results in only a ~2-fold increase in baseline GluR concentration. These results suggest that the synapse retains the capacity for further upregulation. 

To address the question of why exogenously applied Shv activates integrin while glial derived Shv does not, we tested whether glia and neurons could differentially modify Shv. Based on Western blot analyses of adult heads and larval brains showing that Shv is present as a single band (Fig. 1A and Figure 2 – figure supplement 1B), the functional differences in neuronal or glial Shv is not likely due to the presence of different isoforms. Consistent with this, FlyBase also suggests that shv encodes a single isoform. However, while we did not detect obvious posttranslational modifications when Shv protein was expressed in neurons or glia (Figure 5 – figure supplement 1A), we cannot exclude the possibility that different cell types process Shv differently through post-transcriptional or post-translational mechanisms. Notably, shv is predicted to undergo A-to-I RNA editing, including an editing site in the coding region, which will result in a single amino acid change (St Laurent et al., 2013). Given that ADAR, the editing enzyme, is enriched in neurons and absent from glia (Jepson et al., 2011), such cell-specific editing could contribute to functional differences. It will be interesting to investigate this in the future. We have now included this in the Discussion section.

Additionally, we have now included new data on neuronal Shv rescue of shv¹ mutants as suggested in the updated Figure 4. Consistent with previous findings that neuronal Shv rescues integrin signaling and electrophysiological phenotypes (Lee et al., 2017), we found that it also restores bouton size, GluR levels, and activity-induced synaptic remodeling. These results support the functional contribution of neuronal Shv. 

Reviewer #3 (Public review):

Summary:

The manuscript by Chang and colleagues provides compelling evidence that glia-derived Shriveled (Shv) modulates activity-dependent synaptic plasticity at the Drosophila neuromuscular junction (NMJ). This mechanism differs from the previously reported function of neuronally released Shv, which activates integrin signaling. They further show that this requirement of Shv is acute and that glial Shv supports synaptic plasticity by modulating neuronal Shv release and the ambient glutamate levels. However, there are a number of conceptual and technical issues that need to be addressed.

We appreciate the insightful and constructive comments. We have added new data and modified the text to address your concerns.  In doing so, the manuscript has been substantially strengthened.  Please see our detailed point-by-point response below.

Major comments:

(1) From the images provided for Fig 2B +RU486, the bouton size appears to be bigger in shv RNAi + stimulation, especially judging from the outline of GluR clusters.

Thank you for pointing this out. We have selected another image to better represent the data.

(2) The shv result needs to be replicated with a separate RNAi.

We have used another independent RNAi line targeting shv to confirm our findings (BDSC 37507). This shv-RNAi³⁷⁵⁰⁷ line also showed the same phenotype, including increased GluR levels and impaired activity-induced synaptic remodeling line (new Figure 2 – figure supplement 1A).

(3) The phenotype of shv mutant resembles that of neuronal shv RNAi - no increased GluR baseline. Any insights why that is the case?

This is an interesting question. We speculate that neuronal Shv normally has a dominant role in maintaining GluR levels during development, mainly through its ability to activate integrin signaling. Consistent with this, we have shown that mutations in integrin leads to a drastic reduction in GluR levels at the NMJ (Lee et al., 2017). While we have shown that neuronal knockdown of shv elevates Shv from glia (Fig. 5E), glial Shv cannot activate integrin signaling (Fig. 5B, 5C). Additionally, high levels of glial Shv will elevate ambient glutamate concentrations (Figure 6A), which will likely reduce GluR abundance and impair synaptic remodeling (Augustin et al.  2007, Chen et al., 2009, and Figure 6B). Therefore, neuronal knockdown of Shv resulted in the same phenotype as shv¹ mutant. 

(4) In Fig 3B, SPG shv RNAi has elevated GluR baseline, while PG shv RNAi has a lower baseline. In both cases, there is no activity induced GluR increase. What could explain the different phenotypes?

SPG is the middle glial cell layer in the fly peripheral nervous system and may also influence the PG layer through signaling mechanisms (Lavery et al., 2007), therefore having a stronger effect. We have now mentioned this in the text. 

(5) In Fig 4C, the rescue of PTP is only partial. Does that suggest neuronal shv is also needed to fully rescue the deficit of PTP in shv mutants?

This is indeed a possibility. We have shown that neuronal and glial Shv each contribute to activity-induced synaptic remodeling through different mechanisms. It will be interesting test this in the future.

(6) The observation in Fig 5D is interesting. While there is a reduction in Shv release from glia after stimulation, it is unclear what the mechanism could be. Is there a change in glial shv transcription, translation or the releasing machinery? It will be helpful to look at the full shv pool vs the released ones. 

Thank you for the suggestion. To address this, we monitored the levels of intracellular Shv using a permeabilized preparation (we found that the addition of detergent to permeabilize the sample strips away extracellular Shv). Combined with the extracellular staining results, we can get an idea about the total amount of Shv. As shown in the updated Figure 5D, intracellular Shv levels (permeabilized) remained unchanged following stimulation, indicating that there is no intracellular accumulation and that the observed decrease in extracellular Shv is unlikely due to impaired release machinery.

(7) In Fig 5E, what will happen after stimulation? Will the elevated glial Shv after neuronal shv RNAi be retained in the glia? 

Thank you for the interesting question. We agree that examining Shv distribution following neuronal activity would be highly informative. While we plan to perform time-lapse experiments in future studies to address this, we feel that such analyses are beyond the scope of the current manuscript.

(8) It would be interesting to see if the localization of shv differs based on if it is released by neuron or glia, which might be able to explain the difference in GluR baseline. For example, by using glia-Gal4>UAS-shv-HA and neuronal-QF>QUAS-shv-FLAG. It seems important to determine if they mix together after release? It is unclear if the two shv pools are processed differently.

We agree that investigating whether neuronal and glial shv pools colocalize or are differentially processed is an important future direction. We hope to examine how each pool responds to stimulation in the shv¹ mutant background using LexA and Gal4 systems in the future

(9) Alternatively, do neurons and glia express and release different Shv isoforms, which would bind different receptors?

Thank you for the questions. We have now addressed this in the discussion and also enclosed below:

Based on Western blot analyses of adult heads and larval brains showing that Shv is present as a single band (Fig. 1A and Figure 2 – figure supplement 1B), the functional differences in neuronal or glial Shv is not likely due to the presence of different isoforms. Consistent with this, FlyBase also suggests that shv encodes a single isoform (Ozturk-Colak et al., 2024). However, while we did not detect obvious post-translational modifications when Shv protein was expressed in neurons or glia (Figure 5 – figure supplement 1A), we cannot exclude the possibility that different cell types process Shv differently through posttranscriptional or post-translational mechanisms. Notably, shv is predicted to undergo A-to-I RNA editing, including an editing site in the coding region, which could result in a single amino acid change (St Laurent et al., 2013). Given that ADAR, the editing enzyme, is enriched in neurons and absent from glia (Jepson et al., 2011), such cell-specific editing could contribute to functional differences. It will be interesting to investigate this in the future.

(10) It is claimed that Sup Fig 2 shows no observable change in gross glial morphology, further bolstering support that glial Shv does not activate integrin. This seems quite an overinterpretation. There is only one image for each condition without quantification. It is hard to judge if glia, which is labeled by GFP (presumably by UAS-eGFP?), is altered or not.

Thank you for raising this concern. To strengthen our claim, we now include additional images (Figure 5, figure supplement 2). No obvious change in overall glial morphology was observed, with glia continuing to wrap the segmental nerves and extend processes that closely associate with proximal synaptic boutons (Figure 5, figure supplement 2). These observations suggest that glial  Shv is not essential for maintaining normal glial structure or survival, and is consistent with the idea that glial Shv does not activate integrin, as integrin signaling is required to maintain the integrity of peripheral glial layers. 

(11) The hypothesis that glutamate regulates GluR level as a homeostatic mechanism makes sense. What is the explanation of the increased bouton size in the control after glutamate application in Fig 6?

We speculate that it could be due to a retrograde signaling mechanism activated by elevated extracellular glutamate, allowing neurons to modulate bouton morphology in response to synaptic demand. It will be interesting to investigate this possibility in the future.  

(12) What could be a mechanism that prevents elevated glial released Shv to activate integrin signaling after neuronal shv RNAi, as seen in Fig 5E?

One potential mechanism is post-translational or post-transcriptional processing of Shv. Although our Western blots did not reveal differences in the molecular weight of glial vs. neuronal Shv, we cannot exclude the possibility that modifications not readily detectable by this method are responsible. Additionally, as mentioned in the Discussion section, post-transcriptional processing such as A-to-I RNA editing could introduce changes in the Shv protein, potentially altering its ability to interact with or activate integrin. 

(13) Any speculation on how the released Shv pool is sensed?

The same RNA editing modification mentioned earlier or post-translational modifications in Shv may also influence how it is sensed by target cells. 

Reviewer #1 (Recommendations for the authors):

Issues Regarding Cell Type-Specific Secretion and the Role of Shv:

Extracellular Secretion of Shv:

(1) The data in Figure 1 suggest that Shv is not secreted under resting conditions, challenging the proposed extracellular role of Shv. It remains unclear whether Shv secretion can be confirmed using Shv-eGFP (knock-in) following high K+ stimulation.

We apologize for not being clear. In Figure 1, Shv signals we’ve shown are from permeabilized preparation, which preferentially labels intracellular Shv. We do observe secreted Shv-eGFP following stimulation (Figure 5E), consistent with our hypothesis. However, endogenous extracellular Shv-eGFP signal is very weak, and was therefore detected using the GFP antibody and amplified with a  fluorescent secondary antibody. We have now also included additional controls in Figure 5E to demonstrate the specificity of the staining.

(2) In Figure 5D, total Shv staining should be included to evaluate potential presynaptic accumulation of intracellular Shv, which may lead to extracellular secretion upon stimulation. Additionally, the representative images of glial rescue do not seem to align with the quantification data; more extracellular Shv signals were observed after stimulation.

Thank you for the comments. We monitored the levels of intracellular Shv using a permeabilized preparation (detergent treatment stripped away extracellular Shv signal). When combined with non-permeabilized extracellular staining, this approach provides insights into total Shv levels. We found no intracellular accumulation of Shv and the intracellular levels remained unchanged following stimulation (updated Figure 5D), suggesting that reduced extracellular Shv is not likely due to impaired release. Additionally, we have selected another image for glial rescue by avoiding the trachea region, which better represent the quantification data.

(3) In Figure 5E, "extracellular" Shv staining in repo>shv-RNAi samples appears localized within synaptic boutons. This raises concerns about the staining protocol potentially labeling intracellular proteins. Control experiments using presynaptic cytosolic markers are needed to confirm staining specificity.

Thank you for the thoughtful suggestion. To validate that our staining protocol is selective for extracellular proteins, we also stained for cysteine string protein (CSP), an intracellular synaptic vesicle protein predominantly located in the presynaptic terminals (Zinsmaier et al., 1990; Umbach et al., 1994), under the same conditions. CSP was detected only in the permeabilized condition (updated Figure 5E), suggesting that the non-permeabilizing protocol is selective for extracellular proteins. 

(4) The study does not clarify why Shv knockdown in either perineurial glia or subperineurial glia abolishes stimulus-dependent synaptic remodeling. Does Shv secretion occur from PG, SPG, or both toward the synaptic bouton?

Thank you for raising this point. SPG is the middle glial cell layer in the fly peripheral nervous system and may also influence the PG layer through signaling mechanisms (Lavery et al., 2007). Consistent with this, we observed a stronger effect on GluR levels when SPG was disrupted compared to PG. It will be interesting to distinguish whether Shv is released by PG or SPG in the future.

(5) The possibility of an inter-glial role for Shv via integrin signaling in regulating glial morphogenesis is underexplored. The rough morphological characterization in Supplemental Figure 2 requires more detailed quantification and the use of sub-glial typespecific GAL4 drivers.

We now include additional images (Figure 5, figure supplement 2) to examine the overall glial morphology. There was no obvious change in gross glial morphology, with glia continuing to wrap the segmental nerves and extend processes that closely associate with proximal synaptic boutons when shv is knocked down in glia (Figure 5, figure supplement 2). These observations suggest that glial  Shv is not essential for maintaining normal glial structure or survival, and is consistent with the idea that glial Shv does not activate integrin, as integrin signaling is required to maintain the integrity of peripheral glial layers (Xie and Auld, 2011; Hunter et al., 2020).

(6) While repo>shv rescues stimulus-dependent bouton size and GluR increases in the shv mutant (Figure 5), the interaction between neuronal and glial Shv remains unclear. Does neuronal Shv influence the expression or distribution of glial Shv?

We agree that investigating whether neuronal and glial shv pools influence each other’s expression or distribution is an important future direction. We hope to investigate this in more detail in the future using LexA-LexOp and GAL4/UAS dual expression systems.

Issues Regarding the Regulation of GluR and Perisynaptic Glutamate by Glial Shv:

(7) The methodology for iGluSnFR measurement (Figure 6A) is inadequately described. If anti-HRP staining was used to normalize signals, it suggests the experiment may have involved fixed tissue. However, iGluSnFR typically measures glutamate levels in live cells, raising concerns about the validity of this approach in fixed samples.

We apologize for not being clear about the method used to measure iGluSnFR. The original figure was generated from imaging iGluSnFR signals immediately following fixation. To address the reviewer’s concern and validate these results, we have now performed live imaging experiments using a water dipping objective to measure iGluSnFR intensity in unfixed preparations (new Figure 6A). To label synaptic boutons, we co-expressed mtdTomato using the neuronal driver, nSybGAL4. The results from the live imaging experiments confirmed our original observations that glial Shv required to control ambient extracellular glutamate levels (see updated Fig. 6A and text). Additionally, to ascertain that the decrease in iGluSnFR signal reflects a decrease in ambient extracellular glutamate levels rather than glutamate depletion caused by high levels of GluR, we upregulated GluR levels using mhc-GluRIIA, which drives GluRIIA expression in muscles (Petersen et al., 1997). We found mhc-GluRIIA animals exhibited elevated levels of not only GluRIIA but also the obligatory GluRIIC subunit. However, iGluSnFR signals at the synapse remained unchanged (Figure 6A), suggesting that elevated GluR density alone does not reduce signals. Taken together, these results suggest that glial Shv plays a critical role in controlling ambient extracellular glutamate levels. 

(8) As shown in Figure 2, repo>shv-RNAi increases GluR levels before high K+ stimulation, potentially saturating postsynaptic GluR expression and precluding further increases upon stimulation.

Our data in Figure 6 show that GluR levels can increase up to four-fold upon stimulation in the presence of glutamate, whereas repo>shv-RNAi results in only a ~2-fold increase in baseline GluR concentration. These results suggest that the synapse retains the capacity for further upregulation. Thus, we do not think that the lack of GluR enhancement in repo>shv-RNAi is due to a saturated postsynaptic state, but rather reflects a requirement for glial Shv in activity-dependent modulation.

(9) Despite glial shv knockdown lowering extracellular glutamate levels, GluR levels unexpectedly increase (Figure 6B). This contradicts the known requirement for high ambient glutamate concentrations to promote GluR clustering and membrane expression (Chen et al., 2009). Furthermore, adding 2 mM glutamate reverses these increases, suggesting additional complexity in the regulation of Shv synaptic remodeling.

Thank you for the comment and the opportunity to clarify this point. While it may seem counterintuitive at first glance, our observations are in line with previous reports that showed low ambient glutamate levels significantly elevated GluR intensity at the Drosophila NMJ (Chen et al., 2009), but such increase can be reversed by glutamate supplementation (Augustin et al., 2007; Chen et al., 2009). We have revised the text to more clearly reflect this connection.

(10) If glial Shv promotes GluR expression, why does the increased extracellular Shv from neuronal shv knockdown (elav>shv-RNAi, Figure 5E) fail to elicit stimulus-dependent GluR elevation?

We speculate that this is because glial Shv does not activate integrin signaling (Figure 5B, C), and elevated glial Shv increases ambient glutamate concentration (Figure 6A), thereby reducing GluR expression (Augustin et al., 2007; Chen et al., 2009). This is indeed what we observed when shv is knocked down in neurons. 

Additional Issues:

(11) The type of bouton used for quantification (e.g., Ib or Is boutons) is not specified, which is critical for interpreting the results.

We apologize for not being clear. We analyzed type Ib boutons as done previously (Lee et al., 2017 and Chang et al., 2024), and have now included this information in the Methods section.  

(12) The extent of Shv protein depletion in the repo-GeneSwitch system needs validation to confirm the efficacy of the knockdown.

Thank you for the suggestion. We confirmed the efficiency of acute shv knockdown by the repo-GeneSwitch system by performing Western blot analysis of dissected larval brains (Figure 2 – figure supplement 1B). Acute glial knockdown using the repo-GeneSwitch driver resulted in a 30% reduction in Shv levels, similar to the decrease observed with the repo-GAL4 driver, suggesting that the GeneSwitch driver is functional. Furthermore, knockdown of shv by the ubiquitous tubulin-GAL4 driver completely eliminated Shv protein, indicating that the RNAi construct is effective.  

Reviewer #2 (Recommendations for the authors):

(1) General comment on statistics/data presentation: The authors employ an unusual method of using both one-way ANOVA and multiple t-test stats for the same data. Would a 2-way ANOVA be the more appropriate solution to this problem (to analyze across genotype and stimulation condition)? Also a chart in the supplementals showing all comparisons rather than just the fraction explicitly reported in the graphs would be helpful (it is not clear if no indication on significance indicates no difference or just not reported between some of the baseline levels, especially since everything is presented as ratios and in some cases this could help with data interpretation of which baseline levels are different and how they compare to other baselines and other post-stim levels). Further, there are no sample sizes given for any experiment, nor are any values of means, SD, etc ever explicitly given.

We appreciate the thoughtful suggestion. While a two-way ANOVA could be used to examine interaction effects between genotype and stimulation condition, our analysis was designed to address a specific biological question: whether each genotype, independent of baseline levels, is capable of undergoing activitydependent synaptic remodeling. To this end, we used t-tests to directly compare unstimulated vs. stimulated conditions within each genotype, allowing us to determine whether stimulation produces a significant effect in an all-or-none manner. In parallel, we applied one-way ANOVA with post hoc tests to analyze differences among baseline (unstimulated) conditions across genotypes. This approach is justified by the fact that stimulation was applied acutely and separately, and therefore the baseline values should not be influenced by the stimulated condition. Because we were not aiming to compare the extent of synaptic remodeling between genotypes, we did not use a two-way ANOVA to analyze interaction effects across all conditions.

In response to the reviewer’s suggestion, we have now added the sample number in the graphs. Additionally, in the Methods section, we include information that each sample represents biological repeats, and that data are presented as fold-change relative to unstimulated controls from the same experimental batch. This normalization is necessary, as absolute GluR intensities can vary depending on microscope settings and staining conditions.

(2) To clarify distinct roles of Shv coming from neurons vs glia it would help if the authors could include more data on the rescue of shv mutants with UAS-Shv in neurons alone. This data is never shown in the manuscript and data on what effect this rescue has on the pertinent phenotypes in this paper (bouton size and GluR staining) is not reported in the referred to 2017 paper. What this does and does not do for these phenotypes has important implications for how to interpret the glia-only rescue findings.

Thank you for the suggestion. We have now included new data on neuronal Shv rescue in shv¹ mutants as suggested (updated Figure 4A). Consistent with previous findings that neuronal Shv rescues integrin signaling and electrophysiological phenotypes (Lee et al., 2017), we found that it also restores bouton size, GluR levels, and activity-induced synaptic remodeling. These results support the functional contribution of neuronal Shv. 

(3) Figure 1C: Where are the images in the periphery taken? The morphology of the glia is odd in that "blobs" of glial membrane seemingly unattached to anything else are floating about? Perhaps these are a thin stack projection and so the connection to the main glia "stalks" are just cut off? Could a specific individual synapse be shown? Also consider HRP shown on its own so that where the actual boutons are could be more clear. It seems like both the Tomato and HRP channels are really overexposed making visualizing the morphology quite confusing. Also why not use the antibody against Shv to directly visualize expression which is more direct than a knock-in tagged version?

Figure 1C shows a single optical slice of the NMJ at muscle segment 2, selected to clearly highlight Shv-eGFP localization at a branch in close contact with the glial membrane. The glial stalk is not visible in this image because it lies in a different focal plane from the branch of interest. We have now specified this information in the figure legend. In the original figure, the HRP signal (405 channel) was oversaturated, which interfered with visual clarity. In the updated Figure 1C, we reduced the intensity of overexposed channels to better reveal the weak ShveGFP signal and fine glial processes. While we have generated an antibody against Shv, the amount is extremely limited, and hence the Shv-eGFP fusion serves as a valuable tool for visualizing subcellular localization.

(4) Do glutamate levels really rise in glia Shv KD? Although iGluSnFR signal changes could it be the high level of GluR at the synapse acting as sponges to sequester glutamate so that it can't stimulate the sensor as well? One way to test this would be to overexpress or KD GluRs in muscle in wildtype (or in the repo>Shv RNAi background) to see if that alone can modulate iGluSnfR signals?

Thank you for suggesting this important control. To address the question of whether high level GluR density alone could influence neuronal iGluSnFR sensor readouts, we expressed GluR using a mhc promoter-driven GluRIIA fusion line, which increases total GluRIIA expression in muscle independently of the Gal4/UAS system. As shown in Figure 6 – figure supplement 1, mhc-GluRIIA animals exhibited elevated levels of not only GluRIIA but also the obligatory GluRIIC subunit. Despite this increase in GluR expression, we did not observe any change in extracellular glutamate levels, as measured by live imaging using the neuronal iGluSnFR sensor (updated Figure 6A). These results suggest that elevated GluR density alone does not alter iGluSnFR sensors  dynamics and further support our conclusions.

(5) The authors have some Shv constructs that can't be secreted or can't bind to integrins. Performing cell type specific rescues with these constructs might also help distinguish how source matters for each proposed sub-function of Shv though this may be outside the scope of this study. 

Thank you for noticing the Shv constructs we have. We hope to further test subfunctions of Shv in the future.

(6) At one point the authors discuss experiments that measure how much Shv is released by glia during neuronal stimulation. Then state that "These data indicate that glial Shv does not directly inhibit integrin signaling." But how this experiment relates to integrin signaling is not explained and unclear.

We apologize for the confusion. We have now updated the text to better explain our logic: “This activity-induced decrease in glial Shv levels, along with reduced integrin activation (Fig. 5B), suggest that glial Shv does not act by directly inhibiting integrin signaling.”

Reviewer #3 (Recommendations for the authors):

Minor comments

(1) Readers are left wondering what causes the increased baseline of GluR after glial shv RNAi at Fig 1, which is addressed much later. It would be helpful to preemptively mention this.

Thank you for the suggestion. To maintain a logical flow, we chose to first present the phenotypic data in Figures 1 and 2 and then return to the mechanistic explanation once we introduced ambient glutamate measurements. 

(2) Be consistent with eGFP vs EGFP.

Thank you, we have corrected the inconsistencies.  

(3) Scale bar for Fig 1B is missing in the low-magnification panel.

Thank you for pointing out. We’ve put in the scale bar for Figure 1B.   

(4) Fig 1C, it would be helpful to elaborate on the anatomy. For example, what NMJ/abdominal segment is this? Why only some axons are surrounded by glia?

Figure 1C presents a single optical slice of the NMJ at muscle segment 2, chosen to highlight Shv-eGFP localization at a branch closely juxtaposed to the glial membrane. The glial stalk is not shown in this image because it resides in a different focal plane than the branch being visualized. We have now included this information in the figure legend.

(5) For Fig 3B, while it is stated that "we observed normal synaptic remodeling using alrmGAL4," the effect size is smaller. There seems to be a decrease in the amount of synaptic remodeling occurring?

Thank you for pointing this out. Our primary goal was to determine whether each genotype, regardless of baseline GluR levels, is capable of undergoing activitydependent synaptic remodeling in response to stimulation. For this reason, we focused on detecting the presence or absence of remodeling rather than comparing the extent of remodeling across genotypes. While a smaller effect on activity-induced bouton size was observed with alrm-GAL4, the change was still statistically significant, indicating that remodeling does occur in this genotype. Currently, we do not have a clear biological interpretation for differences in the magnitude of remodeling, and therefore chose not to emphasize cross-genotype comparisons.

In early childhood this is so important. the students have all had limited and very differnt exposure to learning and experiences. We work hard to deliver developmentally appropraite and interesting lessons

Success of a chinatown isndependent on how well the de facto leaders get along

What explains tge immigrant integration

are you guys even doing it for the love of the game or are you just doing it to beat the french

shady bastard

The tool for controlling what’s inside tells the story, and what’s left out is just as loud. That choice decides how we read the whole scene.

Movies ain’t just about the way they’re shot, but also what they’re actually saying about us as people. Like every film ends up being a mirror, showing back the values and beliefs we carry around, whether we notice it or not.

I have yet to hear this saying before, but it's a funny saying nonetheless.

Just thinking about all the iconic one-liners like "You're killing me, smalls", or just iconic music like Michael Myers chasing someone.

It is very rare, in my opinion, that I see a bad movie. I have not experienced a movie where there was not a theme in any way.

I have always heard that literture or books ar most of the time differ in slight details when in movie adaptation.

It's wild how he doesn't take his own wife seriously. It sounds like he's just saying "add it to the list of problems" or "now the women are complaining?"

It seems that this whole part just disregards anything Abigail had written about. He says something along the lines of "men know their role, and they'll go easy on women, but they need to go wo war and fight". In the second part of the paragraph, he just restates what he's saying in the beginning, that women are just the next subgroup of people to rebel against the patriots.

This as well as the rest of the paragraph shows that she is in touch with the town, as well as all she is doing for their family. Just goes to show it's ridiculous that women can fill this important role but cant have any rights outside of their home.

I can feel her sense of longing, as well as impatience for his replies. I wonder how long it took to send mail back then, it's been a few weeks since she's heard from him, is that a long time for their postal service?

It just doesn't sound right even coming from her words, but the harsh reality was women seems like they were just there to satisfy men, which is absolutely terrible, I don't blame her for speaking in this tone.

Very Powerful! I even take a liking to the threat of rebellion, and although at this point women have a long way to go, it is impressive to see this much fire from a woman in this time period; she speaks for those who want to represent.

Abigail is saying "How can men be passionate for liberty if they won't allow it for women?"

Dunmore Passed "Lord Dunmore's Proclamation" which freed slaves of the south so they could join the British army.

This was the mentality of so many people i knew in middle school and highschool they chose to drop out of education in order to pursue work in trade or rap or whatever they could get their hands on because the education system failed them and denied them their ability to pursue something higher

when i was a child i faced this exact situation where no one would be home but me the oldest to care for all the children while my parents went to work to make enough to pay for rent and the kids wants

From the perspective of immigrant students, this passage reveals how uncertainty about the future can deeply affect motivation and self-worth. Many undocumented students grow up believing in the same dreams as their peers—going to college, finding a good job, contributing to society—but later realize that their immigration status limits those possibilities. Seeing friends move forward while they remain stuck creates a painful sense of immobility and isolation. The confusion about what’s even possible makes it hard for them to plan or stay engaged in the present. For these students, education becomes a source of both hope and frustration—it represents opportunity but also reminds them of the barriers they face just to belong.

As an international student, I feel Flor perfectly captures the reality of our situation. We find ourselves in a phase completely devoid of social connections, family, or status. We must muster the courage to make our first “local friend” during our studies. We need to master the use of a “second language.” Every seemingly simple task becomes a challenge for us, and we rely entirely on ourselves.

This shows how family responsibility can pull students away from school, especially when emotional or academic support is missing. It’s sad because even though the student cares deeply about their family, the lack of encouragement from teachers makes it harder to stay motivated in education.

This is interesting because I have never really linearly placed my ideas. This is also something I would try in the future and apply it to my creative thinking skills.

what i was thinking

personality disorder

the way i see it being able to maintain that original piece of you is important to the way you portray yourself as to others. schools equating english proficiency with intelligence is unfair and easily hurtful to one’s self identity

having immigrant parents and grandparents my grandparents saw it as extremely important that my parents and their siblings learned English in order to maintain better opportunities. its amazing the lengths parents will go to support their children no matter the cost

the fact that the author pointed oht the fact that first gen and second gen students face different challenges is extremely important. especially since grouping them together can hide real differences in experience especially when creating policies or studies meant to support them

This made me curious because I have never tried this method before. I always only jot down good ideas and try to avoid writing down those I deem as bad. This will be something I try in the future.

This statement is useful because it's the first step on how you guide your creative thinking and ideation to produce solutions and results. Without a creative objective, you wouldn't know how to source your thoughts and facilitate the direction they are going in.

eLife Assessment

This useful study describes a mechanism of microbial modulation of anti-tumor immunity, which is of considerable interest in the field. However, the experimental supports for the key mechanistic claim, the interaction between RadD and NKp46, are not robust. Multiple experimental inconsistencies, especially in vivo, weaken the conclusions, making the strength of evidence incomplete. Additional controls, direct binding assays, and clarification of in vivo mechanistic relevance would strengthen the work.

Reviewer #1 (Public review):

In this manuscript, Rishiq et al. investigate whether natural killer (NK) cells can interact with Fusobacterium nucleatum and identify the molecular mediators involved in this interaction. The authors propose that the bacterial adhesin RadD may bind to the activating NK cell receptor NKp46 (NCR1 in mice), leading to NK cell activation and tumor control. While the topic is of significant interest and the hypothesis intriguing, the manuscript lacks critical experimental evidence, contains several technical concerns, and requires substantial revisions.

Major Concerns:

(1) Lack of Direct Evidence for RadD-NKp46 Interaction

The central claim that RadD interacts with NKp46 is not formally demonstrated. A direct binding assay (e.g., Biacore, ELISA, or pull-down with purified proteins) is essential to support this assertion. The absence of this fundamental experiment weakens the mechanistic conclusions of the study.

(2) Figure 2: Binding Specificity and Bacterial Strains

A CEACAM1-Ig control should be included in all binding experiments to distinguish between specific and non-specific Ig interactions. There is differential Ig binding between strains ATCC 23726 and 10953. The authors should quantify RadD expression in each strain to determine if the difference in binding is due to variation in RadD levels.

(3) Figure 3: Flow Cytometry Inconsistencies and Missing Controls

What do the FITC-negative, Ig-negative events represent? The authors should clarify whether these are background signals, bacterial aggregates, or debris.

Panel B, CEACAM1-Ig binding appears markedly increased compared to WT bacteria. The reason for this enhancement should be discussed-does it reflect upregulation of the bacterial ligand or an artifact of overexpression? Fluorescence compensation should be carefully reviewed for the NKp46/NCR1-Ig binding assays to ensure that the signals are not due to spectral overlap or nonspecific binding. Importantly, binding experiments using the FadI/RadD double knockout strain are missing and should be included. This control is essential.

In Panel E, the basis for calculating fold-change in MFI is unclear. Please indicate the reference condition to which the change is normalized.

(4) Figure 4: Binding Inhibition and Receptor Sensitivity

Panel A lacks representative FACS plots and is currently difficult to interpret. Differences in the sensitivity of human vs. mouse NKp46 to arginine inhibition should be discussed, given species differences in receptor-ligand interactions. What are the inhibition results using F. nucleatum strains deficient in FadI?

In Panel B, CEACAM1-Ig and RadD-deficient bacteria must be included as negative controls for binding specificity upon anti-NKp46 blocking.

(5) Figure 5: Functional NK Activation and Tumor Killing

In Panels B and C, the key control condition (NK cells + anti-NKp46, without bacteria) is missing. This is needed to evaluate if NKp46 recognition is involved in tumor killing. The authors should explicitly test whether pre-incubation of NK cells with bacteria enhances their anti-tumor activity. Alternatively, could bacteria induce stress signals in tumor cells that sensitize them to NK killing? This distinction is critical.

(6) Figure 5D: Mechanism of Peripheral Activation

It is suggested that contact between bacteria and NK cells in the periphery leads to their activation. Can the authors confirm whether this pre-activation leads to enhanced killing of tumor targets, or if bacteria-tumor co-localization is required? The literature indicates that F. nucleatum localizes intracellularly within tumor cells. If so, how is RadD accessible to NKp46 on infiltrating NK cells?

(8) Figure 5E and In Vivo Relevance

Surprisingly, F. nucleatum infection is associated with increased tumor burden. Does this reflect an immunosuppressive effect? Are NK cells inhibited or exhausted in infected mice (TGIT, SIGLEC7...)? If NK cell activation leads to reduced tumor control in the infected context, the role of RadD-induced activation needs further explanation. RadD-deficient bacteria, which do not activate NK cells, result in even poorer tumor control. This paradox needs to be addressed: how can NK activation impair tumor control while its absence also reduces tumor control?

(9) NKp46-Deficient Mice: Inconsistencies

In Ncr1⁻/⁻ mice, infection with WT or RadD-deficient F. nucleatum has no impact on tumor burden. This suggests that NKp46 is dispensable in this context and casts doubt on the physiological relevance of the proposed mechanism. This contradiction should be discussed more thoroughly.

Reviewer #2 (Public review):

Summary:

In the present study, Rishiq et al. investigated whether the RadD protein expressed by Fusobacterium nucleatum subsp. Nucleatum serves as a natural ligand for the NK-activating receptor NKp46, and whether RadD-NKp46 interaction enhances NK cell cytotoxicity against tumor cells. To address this, the authors first performed an association analysis of F. nucleatum abundance and NKp46 expression in head and neck squamous cell carcinoma (HNSC) and colorectal cancer (CRC) using the TCMA and TCGA databases, respectively. While a positive association between NKp46⁺ and F. nucleatum⁺ status with improved overall survival was observed in HNSC patients, no such correlation was found in CRC.

Next, they examined the binding of NKp46-Ig to various F. nucleatum strains. To confirm that this interaction was mediated specifically by RadD, they employed a RadD-deficient mutant strain. Finally, to establish the functional relevance of the RadD-NKp46 interaction in promoting NK cell cytotoxicity and anti-tumor responses, they utilized a syngeneic mouse breast cancer model. In this setup, AT3 cells were orthotopically implanted into the mammary fat pad of C57BL/6 wild-type (WT) or Ncr1-deficient (NCR1⁻/⁻; murine orthologue of human NKp46) mice, followed by intravenous inoculation with either WT F. nucleatum or the ∆RadD mutant strain.

Strengths:

A notable strength of the work is that it identifies a previously unrecognized activating interaction between F. nucleatum RadD and the NK cell receptor NKp46, demonstrating that the same bacterial protein can engage distinct NK cell receptors (activating or inhibitory) to exert context-dependent effects on anti-tumor immunity. This dual-receptor insight adds depth to our understanding of F. nucleatum-immune interactions and highlights the complexity of microbial modulation of the tumor microenvironment.

Weaknesses:

(1) A previous study by this group (PMID: 38952680) demonstrated that RadD of F. nucleatum binds to NK cells via Siglec-7, thereby diminishing their cytotoxic potential. They further proposed that the RadD-Siglec-7 interaction could act as an immune evasion mechanism exploited by tumor cells. In contrast, the present study reports that RadD of F. nucleatum can also bind to the activating receptor NKp46 on NK cells, thereby enhancing their cytotoxic function.

While F. nucleatum-mediated tumor progression has been documented in breast and colon cancers, the current study proposes an NK-activating role for F. nucleatum in HNSC. However, it remains unclear whether tumor-infiltrating NK cells in HNSC exhibit differential expression of NKp46 compared to Siglec-7. Furthermore, heterogeneity within the NK cell compartment, particularly in the relative abundance of NKp46⁺ versus Siglec-7⁺ subsets, may differ substantially among breast, colon, and HNSC tumors. Such differences could have been readily investigated using publicly available single-cell datasets. A deeper understanding of this subset heterogeneity in NK cells would better explain why F. nucleatum is passively associated with a favorable prognosis in HNSC but correlates with poor outcomes in breast and colon cancers.

(2) The in vivo tumor data (Figure 5D-F) appear to contradict the authors' claims. Specifically, Figure 5E suggests that WT mice engrafted with AT3 breast tumors and inoculated with WT F. nucleatum exhibited an even greater tumor burden compared to mice not inoculated with F. nucleatum, indicating a tumor-promoting effect. This finding conflicts with the interpretation presented in both the results and discussion sections.

(3) Although the authors acknowledge that F. nucleatum may have tumor context-specific roles in regulating NK cell responses, it is unclear why they chose a breast cancer model in which F. nucleatum has been reported to promote tumor growth. A more appropriate choice would have been the well-established preclinical oral cancer model, such as the 4-nitroquinoline 1-oxide (4NQO)-induced oral cancer model in C57BL/6 mice, which would more directly relate to HNSC biology.

(4) Since RadD of F. nucleatum can bind to both Siglec-7 and NKp46 on NK cells, exerting opposing functional effects, the expression profiles of both receptors on intratumoral NK cells should be evaluated. This would clarify the balance between activating and inhibitory signals in the tumor microenvironment and provide a more mechanistic explanation for the observed tumor context-dependent outcomes.

Author response:

Reviewer #1 (Public review):

Major Concerns:

(1) Lack of Direct Evidence for RadD-NKp46 Interaction

The central claim that RadD interacts with NKp46 is not formally demonstrated. A direct binding assay (e.g., Biacore, ELISA, or pull-down with purified proteins) is essential to support this assertion. The absence of this fundamental experiment weakens the mechanistic conclusions of the study.

The reviewer is correct. Direct assays are currently quite impossible because RadD is huge protein and it will take years to purify it. Instead, we used immunoprecipitation assays using NKp46-Ig (Author response images 1 and 2). Fusobacteria were lysed using RIPA buffer, and the lysates were centrifuged twice to separate the supernatant from the pellet (which contains the bacterial membranes). The resulting lysates were incubated overnight with 2.5 µg of purified NKp46 and protein G-beads. After thorough washing, the bound proteins were placed in sample buffer and heated at 95 °C for 8 minutes. The eluates were run on a 10% acrylamide gel and visualized by Coomassie blue staining. As can be seen the NKp46-Ig was able to precipitate protein band around 350Kd in both F. polymorphum ATCC10953 (Author response image 1) and in F. nucleatum ATCC23726 (Author response image 2).

Author response image 1. NKp46 immunoprecipitation with Fusobacterium polymorphum (ATCC 10953) lysates. The resulting lysates of supernatant and pellet of Fusobacterium were immunoprecipitated (IP) with 2.5 μg of control fusion protein (RBD-Ig) or with NKp46-Ig. A 2.5 μg of purified fusion proteins were also run on gel.

Author response image 2. NKp46 immunoprecipitation with Fusobacterium nucleatum (ATCC 23726) lysates. The resulting lysates of supernatant and pellet of Fusobacterium were immunoprecipitated (IP) with 2.5 μg of Control fusion protein (RBD-Ig) or with NKp46-Ig. 2.5 μg of purified fusion proteins were also run on gel.

(2) Figure 2: Binding Specificity and Bacterial Strains

A CEACAM1-Ig control should be included in all binding experiments to distinguish between specific and non-specific Ig interactions. There is differential Ig binding between strains ATCC 23726 and 10953. The authors should quantify RadD expression in each strain to determine if the difference in binding is due to variation in RadD levels.

No significant difference in mCEACAM-1-Ig binding was observed across multiple independent experiments. Author response image 3 shows a representative histogram showing mCEACAM-1-Ig binding to F. nucleatum ATCC 23726 and F. polymorphum ATCC 10953. Comparable binding levels were detected in both bacterial species (upper histogram). Similarly, NKp46-Ig and Ncr1-Ig fusion proteins exhibited comparable binding patterns (lower histogram). It is currently not possible to quantify RadD expression directly, as no anti-RadD antibody is available.

Author response image 3. CEACAM-1 Ig binding to Fusobacterium ATCC 23726 and ATCC 10953. Upper histograms show staining with secondary antibody alone (gray) compared to CEACAM-1 Ig (black line). Lower histograms show binding of NKp46 and Ncr1 fusion proteins to the two Fusobacterium strains. Gray represent secondary antibody controls.

(3) Figure 3: Flow Cytometry Inconsistencies and Missing Controls

What do the FITC-negative, Ig-negative events represent? The authors should clarify whether these are background signals, bacterial aggregates, or debris.

We now present the gating strategy used in these experiments (Author response image 4). Fusion negative Ig samples were the bacterial samples stained only with the secondary antibody APC (anti-human AF647). The TITC-negative represent unlabeled bacteria.

Author response image 4. Gating strategy for FITC-labeled Fusobacterium stained with fusion proteins. Bacteria were first gated as shown in the left panel. The gated population was then further analyzed in the right plot: the lower-left quadrant represents bacterial debris, the upper-left quadrant corresponds to FITC-stained bacteria only, and the upper-right quadrant shows bacteria double-positive for FITC and APC, indicating binding of the fusion proteins.

Panel B, CEACAM1-Ig binding appears markedly increased compared to WT bacteria. The reason for this enhancement should be discussed-does it reflect upregulation of the bacterial ligand or an artifact of overexpression? Fluorescence compensation should be carefully reviewed for the NKp46/NCR1-Ig binding assays to ensure that the signals are not due to spectral overlap or nonspecific binding. Importantly, binding experiments using the FadI/RadD double knockout strain are missing and should be included. This control is essential.

We don’t know why expression of CEACAM1-Ig binding is increased. Indeed, it will be nice to have the FadI/RadD double knockout strain which we currently don’t have.

In Panel E, the basis for calculating fold-change in MFI is unclear. Please indicate the reference condition to which the change is normalized.

The mean fluorescence intensity (MFI) fold change was calculated by dividing the MFI obtained from staining with the fusion proteins by the MFI of the corresponding secondary antibody control (bacteria incubated without fusion proteins).

(4) Figure 4: Binding Inhibition and Receptor Sensitivity

Panel A lacks representative FACS plots and is currently difficult to interpret.

Fusobacteria binding to CEACAM-1, NKp46, and NCR1 fusion proteins was tested in the presence of 5 and 10 mM L-arginine (Author response image 5). L-arginine inhibited the binding of NKp46-Ig and NCR1-Ig, whereas no effect was observed on CEACAM-1-Ig binding.

Author response image 5. Fusobacterium binding inhibition by L-Arginine. The figure shows the binding of CEACAM1-Ig (left panel), NKp46-Ig (middle panel), and Ncr1-Ig (right panel) in the presence of 0 mM (black), 5 mM (red), and 10 mM (blue) L-arginine.

Differences in the sensitivity of human vs. mouse NKp46 to arginine inhibition should be discussed, given species differences in receptor-ligand interactions.

Ncr1, the murine orthologue of human NKp46, shares approximately 58% sequence identity with its human counterpart (1). The observed differences in arginine-mediated inhibition of bacterial binding between mouse and human NKp46 might stem from structural differences or distinct posttranslational modifications, such as glycosylation. Indeed, prediction algorithms combined with high-performance liquid chromatography analysis revealed that Ncr1 possesses two putative novel O-glycosylation sites, of which only one is conserved in humans (2).

References

(1) Biassoni R., Pessino A., Bottino C., Pende D., Moretta L., Moretta A. The murine homologue of the human NKp46, a triggering receptor involved in the induction of natural cytotoxicity. Eur J Immunol. 1999 Mar; 29(3).

(2) Glasner A., Roth Z., Varvak A., Miletic A., Isaacson B., Bar-On Y., Jonjić S., Khalaila I., Mandelboim O. Identification of putative novel O-glycosylations in the NK killer receptor Ncr1 essential for its activity. Cell Discov. 2015 Dec 22; 1:15036.

What are the inhibition results using F. nucleatum strains deficient in FadI?

The inhibition pattern observed in the F. nucleatum ΔFadI mutant was comparable to that of the wild-type strain (Author response image 6). When cultured under identical conditions and exposed to increasing concentrations of arginine (0, 5, and 10 mM), the F. nucleatum ΔFadI strain also demonstrated a dose-dependent reduction in binding to NKp46 and Ncr1.

Author response image 6. Arginine inhibition of NKp46-Ig and Ncr1-Ig binding in F. nucleatum ΔFadI. Histograms show NKp46-Ig (A, C) and Ncr1-Ig (B, D) binding to F. nucleatum ATCC10953 ΔFadI (A and B) and to F. nucleatum ATCC23726 ΔFadI (A and B) following exposure to 5 mM and 10 mM L-Arginine. Panels (E) and (F) display the mean fluorescence intensity (MFI) quantification corresponding to (A and B) and (C and D), respectively.

In Panel B, CEACAM1-Ig and RadD-deficient bacteria must be included as negative controls for binding specificity upon anti-NKp46 blocking.

We appreciate the request to include CEACAM1-Ig and RadD-deficient bacteria as negative controls for specificity under anti-NKp46 blocking. We don’t not think it is necessary since the 02 antibody is specific for NKp46, we used other anti0NKp46 antibodies that did not block the interaction and an irrelevant antibofy, we showed that arginine produced a dose-dependent reduction in NKp46/Ncr1 binding, consistent with an arginine-inhibitable RadD interaction already shown in our manuscript (Fig. 4A). The ΔRadD strains we used already demonstrate loss of NKp46/Ncr1 binding and loss of NK-boosting activity (Figs. 3, 5). Collectively, these data establish that NKp46/Ncr1 recognition of a high-molecular-weight ligand consistent with RadD is specific and functionally relevant.

Figure 5: Functional NK Activation and Tumor Killing

In Panels B and C, the key control condition (NK cells + anti-NKp46, without bacteria) is missing. This is needed to evaluate if NKp46 recognition is involved in tumor killing. The authors should explicitly test whether pre-incubation of NK cells with bacteria enhances their anti-tumor activity.

No significant difference in NK cell cytotoxicity was observed between untreated NK cells and NK cells incubated with anti-NKp46 antibody in the absence of bacteria. Therefore, the NK + anti-NKp46 (O2) group was included as an additional control alongside the other experimental conditions shown in Figures 5b and 5c, and is presented in Author response image 7 below.

Author response image 7. NK cytotoxicity against breast cancer cell lines. NK cell cytotoxicity against T47D (left) and MCF7 (right) breast cancer cell lines. This experiment follows the format of Figure 5b and 5c, with the addition of the NK cells + O2 antibody group. No significant differences were observed when values were normalized to NK cells alone.

Could bacteria induce stress signals in tumor cells that sensitize them to NK killing? This distinction is critical.

It remains unclear whether the bacteria induce stress-related signals in tumor cells that render them more susceptible to NK cell–mediated cytotoxicity.

(6) Figure 5D: Mechanism of Peripheral Activation

It is suggested that contact between bacteria and NK cells in the periphery leads to their activation. Can the authors confirm whether this pre-activation leads to enhanced killing of tumor targets, or if bacteria-tumor co-localization is required? The literature indicates that F. nucleatum localizes intracellularly within tumor cells. If so, how is RadD accessible to NKp46 on infiltrating NK cells?

We do not expect that pre-activation of NK cells with bacteria would enhance their tumor-killing capacity. In fact, when NK cells were co-incubated with bacteria, we occasionally observed NK cell death. Although F. nucleatum can reside intracellularly, bacterial entry requires prior adhesion to tumor cells. At this stage—before internalization—the bacteria are accessible for recognition and binding by NK cells.

(8) Figure 5E and In Vivo Relevance

Surprisingly, F. nucleatum infection is associated with increased tumor burden. Does this reflect an immunosuppressive effect? Are NK cells inhibited or exhausted in infected mice (TGIT, SIGLEC7...)? If NK cell activation leads to reduced tumor control in the infected context, the role of RadD-induced activation needs further explanation. RadD-deficient bacteria, which do not activate NK cells, result in even poorer tumor control. This paradox needs to be addressed: how can NK activation impair tumor control while its absence also reduces tumor control?

Siglec-7 lacks a direct orthologue in mice, and neither mouse TIGIT nor CEACAM1 bind F. nucleatum. The increased tumor burden observed in infected mice may therefore result from bacterial interference with immune cell infiltration and accumulation within the tumor microenvironment (Parhi, L., Alon-Maimon, T., Sol, A. et al. Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression. Nat Commun 11, 3259 (2020)). Consequently, the NK cells that do reach the tumor site can recognize and kill F. nucleatum–bearing tumor cells through RadD–NKp46 interactions. In the absence of RadD, this recognition is impaired, leading to reduced NK-mediated cytotoxicity and increased tumor growth.

(9) NKp46-Deficient Mice: Inconsistencies

In Ncr1⁻/⁻ mice, infection with WT or RadD-deficient F. nucleatum has no impact on tumor burden. This suggests that NKp46 is dispensable in this context and casts doubt on the physiological relevance of the proposed mechanism. This contradiction should be discussed more thoroughly.

Ncr1 is also directly involved in mediating NK cell–dependent killing of tumor cells, even in the absence of bacterial infection. Therefore, in Ncr1-deficient mice, F. nucleatum has no additional effect on tumor progression (Glasner, A., Ghadially, H., Gur, C., Stanietsky, N., Tsukerman, P., Enk, J., Mandelboim, O. Recognition and prevention of tumor metastasis by the NK receptor NKp46/NCR1. J Immunol. 2012).

Reviewer #2 (Public review):

Weaknesses:

(1) A previous study by this group (PMID: 38952680) demonstrated that RadD of F. nucleatum binds to NK cells via Siglec-7, thereby diminishing their cytotoxic potential. They further proposed that the RadD-Siglec-7 interaction could act as an immune evasion mechanism exploited by tumor cells. In contrast, the present study reports that RadD of F. nucleatum can also bind to the activating receptor NKp46 on NK cells, thereby enhancing their cytotoxic function.

Siglec-7 lacks a direct orthologue in mice, and neither mouse TIGIT nor CEACAM1 bind F. nucleatum. In contrast, NKp46 and its murine homologue, Ncr1, both recognize and bind the bacterium.

While F. nucleatum-mediated tumor progression has been documented in breast and colon cancers, the current study proposes an NK-activating role for F. nucleatum in HNSC. However, it remains unclear whether tumor-infiltrating NK cells in HNSC exhibit differential expression of NKp46 compared to Siglec-7. Furthermore, heterogeneity within the NK cell compartment, particularly in the relative abundance of NKp46⁺ versus Siglec-7⁺ subsets, may differ substantially among breast, colon, and HNSC tumors. Such differences could have been readily investigated using publicly available single-cell datasets. A deeper understanding of this subset heterogeneity in NK cells would better explain why F. nucleatum is passively associated with a favorable prognosis in HNSC but correlates with poor outcomes in breast and colon cancers.

Currently, there are no publicly available single-cell datasets suitable for characterizing NK cell heterogeneity in the context of F. nucleatum infection—particularly regarding the expression of Siglec-7, NKp46, or CEACAM1 and their potential association with poor clinical outcomes in breast, head and neck squamous cell carcinoma (HNSC), or colorectal cancer (CRC). Furthermore, no RNA-seq datasets are available for breast cancer cases specifically associated with F. nucleatum infection and poor prognosis. Therefore, we analyzed bulk RNA expression datasets for Siglec-7 and CEACAM1 and evaluated their associations with HNSC and CRC using the same patient databases utilized in our manuscript (Author response image 8). No significant differences in Siglec-7 expression were detected between HNSC and CRC samples (Author response image 8A). Although CEACAM1 mRNA levels did not differ between F. nucleatum–positive and –negative cases within either cancer type, its overall expression was higher in CRC compared to HNSC (Author response image 8B).

Author response image 8. Siglec7 and Ceacam1 expression and the prognostic effect of F. nucleatum in a tumor-type-specific manner. Comparison of Siglec7 (A) and Ceacam1 (B) expression across HNSC and CRC tumors. Log₂ expression levels of NKp46 mRNA were compared across HNSC and CRC cohorts, stratified by F. nucleatum positive and negative. Results were analyzed by one-way ANOVA with Bonferroni post hoc correction.

(2) The in vivo tumor data (Figure 5D-F) appear to contradict the authors' claims. Specifically, Figure 5E suggests that WT mice engrafted with AT3 breast tumors and inoculated with WT F. nucleatum exhibited an even greater tumor burden compared to mice not inoculated with F. nucleatum, indicating a tumor-promoting effect. This finding conflicts with the interpretation presented in both the results and discussion sections.

Siglec-7 lacks a direct orthologue in mice, and neither mouse TIGIT nor CEACAM1 bind F. nucleatum. The increased tumor burden observed in infected mice may therefore result from bacterial interference with immune cell infiltration and accumulation within the tumor microenvironment (Parhi, L., Alon-Maimon, T., Sol, A. et al. Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression. Nat Commun 11, 3259 (2020)). Consequently, the NK cells that do reach the tumor site can recognize and kill F. nucleatum–bearing tumor cells through RadD–NKp46 interactions. In the absence of RadD, this recognition is impaired, leading to reduced NK-mediated cytotoxicity and increased tumor growth.

(3) Although the authors acknowledge that F. nucleatum may have tumor context-specific roles in regulating NK cell responses, it is unclear why they chose a breast cancer model in which F. nucleatum has been reported to promote tumor growth. A more appropriate choice would have been the well-established preclinical oral cancer model, such as the 4-nitroquinoline 1-oxide (4NQO)-induced oral cancer model in C57BL/6 mice, which would more directly relate to HNSC biology.

The tumor model we employed is, to date, the only model in which F. nucleatum has been shown to exert a measurable effect, which is why we selected it for our study (Parhi, L., Alon-Maimon, T., Sol, A. et al. Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression. Nat Commun. 2020; 11: 3259). We have not tested the 4-nitroquinoline-1-oxide (4NQO)–induced oral cancer model, and we are uncertain whether its use would be ethically justified.

(4) Since RadD of F. nucleatum can bind to both Siglec-7 and NKp46 on NK cells, exerting opposing functional effects, the expression profiles of both receptors on intratumoral NK cells should be evaluated. This would clarify the balance between activating and inhibitory signals in the tumor microenvironment and provide a more mechanistic explanation for the observed tumor context-dependent outcomes.

This question was answered in Author response image 8 above.

With my stats class, the only way of knowing materials/big assignments are coming is looking at the modules as the professor has not and will not express any reminders or talk about it at all in class. I don’t even know the reason why I’m going to class. If most of us have to look online, he doesn’t talk about anything.

I never had this issue as most of my teachers were student teachers training for a degree to teach in English one exception is the professor. I have now which is the reason why I am making this annotation He makes it easy to understand at least for me.

Most of my teachers did not express the expectations to the students and the course/Learning goals isn't clear instead very confusing not efficient as way of communicating. Mostly through Pages of work but not explaining what this actually means.

I strongly disagree with this claim. Regulating your emotions makes you dull, and apathetic. While in arguments, yes, you should stay away from emotional arguments and claims because they are not factual, however, arguing itself is the emotion. It is how you feel about the piece you are writing and defending, the passion, that there itself is emotion that shouldn't be regulated. Critical thinking doesn't require such emotional intelligence or control, but rather being able to fluctuate it and allow it to improve your writing and your voice.

Bouncing off my other annotation, while we should use the rubric to help us out on writings and assignments, we should rely too much on it, because that also can make it harder to write a successful paper. It's a "It's not the destination, but the journey," of a saying. As a writer, you shouldn't be tunneled into just hitting all the checkboxes to have a bare minimum piece, but to also explore and take your own paths, while still following the correct way.

A lot of the times when I come across a rubric, I skim over the basics and write what the main question is asking, without really thinking the little things through, and this in often times, has shown holes of errors through my work. Reading and using the rubric actively is one of the best ways to guide someone through there paper, especially if they are not as experienced or if they are struggling. Rubrics arent just strict outlines that you have to follow but rather are "the road map" to success.

The common saying "if a million monkeys are typing randomly on a keyboard eventually one will write Shakespeare" comes to mind. I often hate free writing, but what the author says here is something I need to keep in mind. I do not need to analyze the words I am putting down in this time. If I end up writing something that sparks an idea then that will happen without me thinking about it, it will be more of an emotional reaction, which will then trigger the intellectual side of my brain that was blocked off.

Your writing is a cumulation of hours of reading your topic, translating the language to something you can understand, then taking your words and configuring them to fit that same language.

Do not write like you are explaining your topic to someone who does not understand. Write like you are proving you understand it to someone who understands it better than you.

The professors are grading your paper on the weekend or in the evening because they need to work during the day and have other things. You should put yourself in your professor's shoes and ask yourself. How would your professor feel if you were in their shoes? To see how they feel. The professor has their own life and needs a break from grading and teaching. Sometimes you need to remind them that they have a life outside of teaching and grading your homework. If they forgot to grade your homework, you can politely email them to ask them to grade it or remind them, since they are so busy.

Some assignments may be an argumentative paper, an action paper, a reflective paper, a lab report, etc. Other assignments, such as papers, involve a learning process. Some of the students understand the essay and the prompt. The teacher or the professor is looking for grammar, whether the students are following the prompt or not. Most of the essays are different. Some of it is research needed for the sites

The professor does not have to assign the writing lightly, and grading the student's writing is the hardest. It is the most intensive work for the instructor with every assignment, even though they give you and the professor, and try to demonstrate that it can be completed while they are also preparing for the class meeting, advancing their scholarly and creative work, advising students, and serving on the student body.

Telling/showing different ways students stayed in tune with using different technologies

Talking about COVID back in 2020

Informing me about the different technologies I can use to learn more about civic engagements.

As far as I can see, the Toh number of the Tantra section version is not given anywhere- neither on the title page nor here. It should be given somewhere - depending on what our convention is - either on the title page or here in brackets ie: Action Tantra (Skt. kriyātantra) section (Toh 721).

the glossary should pick both parts of the name together as a single glossary entry for 'jam dpal gzhon nur gyur pa (which could also link to just Mañjuśrī, but the point is that both parts of the name should highlight, not just the first part). The same issue is found for other instances of the name that occur later in the sūtra.

change to: the divine knowledge of the thus-gone ones

make all lower case (samādhi name).

also make the corresponding glossary entry all lower case.

this samādhi name should all be lower case (like the other samādhi names)

Like for example this class the standard of writing is much higher degree as in high school when you wrote there was usually no clear goal to the writing but in a college you have a goal which makes it easier to write as you have an angle of looking and what you need to do.

Like stated in the article, becoming an excellent communicator will make anything you do more efficient and smooth as you can deliver information to a better degree but don’t agree with honing my writing because the limited time I have can be spent on better things than writing.

With such high level of writing in high school has made most students have a harsh reality check when they have English writing in college as the writing in college is different from high school in so many different other ways.

In collage teaching is the technical term for what professors do, but in reality they more give out the information they have accumulated to people who also wish to go into the fields they have spent, sometimes, decades studying. It is not so much teaching as it is the sharing.

I feel like most people go into college with a fixed mindset, and professors are trying to rewire that with their students. They are trying to force them out of the comfort zone of a script that high school has fabricated for them. By the time a student has finished college or at least a class that has a message to be shared, they should leave with a growth mindset. Professors now want to see how you've grown from high school and how much "time and effort" you've put into your writing.

This line is a metaphor, and to me, it's saying that writing is an immersive experience. It requires academic writing to have some sort of depth, curiosity emotion, and persistence. It shouldn't be the same every time, and also, some specifics are just not for every other person. Students should try to adapt to their writing skills and become “travelers learning new cultures," or learning how to write differently to become better and more vast.

I find this paragraph, and this specific line, most interesting. Most of high school, you focus on organization and writing a paper properly; however, in college, writing demands intellectual independence, rather than just summarizing and following a guide. The main goal of a writer isn't supposed to be being perfect at what you write and checking off a list of what you're supposed to write, but instead a discovery and an exploration of your own personal thoughts and ideas.

We communicate more through text than with our voice. We send emojis to show our faces. We have digitized ourselves completely, so what is the point in meeting with someone? To talk and form a connection, to create individuality with one another, you remember one person's face as opposed to another, but when everyone is just black text, or a single image in the corner of your screen, who is anyone anymore?

woefully inadequate conception of meaning

Like Knowledge without the knowing subject

There is evidence that many hunter and gatherer society's resisted the transformation into permanent society's, often associating them with disease and state controlled society's.

The author argues that much of history and archaeology is influenced by modern states. They argue that societies that leave ruins to be found dominate our historical views compared to societies that built with perishable materials.

The author argues against the traditional view of the progression of society. They say that it is thought that once people learn about agriculture they would never continue to be nomads. The author says people view it this way because they think that each step is a leap in mankind's well being which can be proven to not be the case.

The author says that the first stratified, walled states pop up more than four millenia after the first crop domestications. They explain that this lag in time is a problem for the theory that states/empires arose immediately after the domestication of crops.

Always keep those units checked and transform back to the real length.

once its been ntroduce with the little omega, its complex number now. So use complex calculation rules ,and especially in EXCEL. Make the form in complex and easy to do the finnal magnitude operation

once its been ntroduce with the little omega, its complex number now. So use complex calculation rules ,and especially in EXCEL. Make the form in complex and easy to do the finnal magnitude operation

eLife Assessment

This work is an important contribution to understanding the role of FGF signaling in the induction of primitive-like cells in a 2D system of human gastrulation. The authors provide compelling evidence showing that endogenous FGF ligands, acting through FGF receptors localized basolaterally, are determinant in the acquisition of specific cell fates. These observations will be of broad relevance to the FGF field.

Reviewer #1 (Public review):

Summary:

This is an interesting study on the role of FGF signaling in the induction of primitive streak like-cells (PS-LC) in human 2D-gastruloids. The authors use a previously characterized standard culture that generates a ring of PS-LCs (TBXT+) and correlate this with pERK staining. A requirement for FGF signaling in TBXT induction is demonstrated via pharmacological inhibition of MEK and FGFR activity. A second set of culture conditions (with no exogenous FGFs) suggests that endogenous FGFs are required for pERK and TBXT induction. The authors then characterize, via scRNA-seq, various components of the FGF pathway (genes for ligand, receptors, ERK regulators, HSPG regulation). They go on to characterize the pFGFR1, receptor isoforms and polarized localization of this receptor. Finally, they perform FGF4 inhibition and use a cell line with a limited FGF17 inactivation (heterozygous null) and show that loss of these FGFs reduce PS-LC and derivative cell types.

Strengths:

(1) As the authors point out, the role of FGF signaling in gastrulation is less well understood than other signaling pathways. Hence this is a valuable contribution to that field.

(2) The FGF4 and FGF17 loss-of-function experiments in Figure 5 are very intriguing. This is especially so given the intriguing observation that these FGFs appear to be dominating in this model of human gastrulation, in contrast to what FGFs dominate in mice, chick and frogs.

(3) In general this paper is valuable as a further development of the Human gastruloid system and the role of FGF signaling in the induction of PS-CLs. The wide net that the authors cast in characterizing FGF ligand gene, receptor isoforms, and downstream components provides a foundation for future work. As the authors write near the beginning of the Discussion "Many questions remain."

Weaknesses:

(1) FGFs are cell survival factors in various aspects of development. The authors fail to address cell death due to loss of FGF signaling in any of their experiments. For example, in Figure 1E (which requires statistical analysis) and 1G (the bottom FGFRi row), there appears to be a significant amount of cell loss. Is this due to cell death? The authors should address the question of whether the role of FGF/ERK signaling is to keep the cells alive.

(2) Regarding the sparse cells in 1G, is there a reduction in cell number only with FGFRi and not MEKi? Is this reproducible? Gattiglio et al (Development, 2023, PMID: 37530863) present data supporting a "community effect" in the FGF-induced mesoderm differentiation of mouse embryonic stem cells. Could a community effect be at play in this human system (especially given the images in the bottom row of 1G). If the authors don't address this experimentally they should at least address the ideas in Gattoglio et al.

(3) Do the FGF4 and FGF17 LOF experiments in Figure 5 affect cell number like FGFRi in Figure 1? Why examine PS-LC induction only in FGF17 heterozygous cells and not homozygous FGF17 nulls?

(4) The idea that FGF8 plays a dominant role during gastrulation of other species but not humans is so intriguing it warrants deeper testing. The authors dismiss FGF8 because its mRNA "...levels always remained low." (line 363) as well as the data published in Zhai et al (PMID: 36517595) and Tyser et al (PMID: 34789876). But there are cases in mouse development where a gene was expressed at levels so low, it might be dismissed, and yet LOF experiments revealed it played a role or even was required in a developmental process. The authors should consider FGF8 inhibition or inactivation to explore its potential role, despite its low levels of expression.

(5) Redundancy is a common feature in FGF genetics. What is the effect of inhibiting FGF4 in FGF17 LOF cells?

(6) I suggest stating that the authors take more caution describing FGF gradients. For example, in one Results heading they write "Endogenous FGF4 and FGF17 gradients underly the ERK activity pattern.", implying an FGF protein gradient. However, they only present data for FGF mRNA , not protein. This issue would be clarified if they used proper nomenclature for gene, mRNA (italics) and protein (no italics) throughout the paper.

Comments on revisions:

The authors have addressed my concerns.

Reviewer #2 (Public review):

Summary:

The role of FGFs in embryonic development and stem cell differentiation has remained unclear due to its complexity. In this study, the authors utilized a 2D human stem cell-based gastrulation model to investigate the functions of FGFs. They discovered that FGF-dependent ERK activity is closely linked to the emergence of primitive streak cells. Importantly, this 2D model effectively illustrates the spatial distribution of key signaling effectors and receptors by correlating these markers with cell fate markers, such as T and ISL1. Through inhibition and loss-of-function studies, they further corroborated the needs of FGF ligands. Their data shows that FGFR1 is the primary receptor, and FGF2/4/17 are the key ligands for primitive streak development, which aligns with observations in primate embryos. Additional experiments revealed that the reduction of FGF4 and FGF17 decreases ERK activity.

Strengths:

This study provides comprehensive data and improves our understanding of the role of FGF signaling in primate primitive streak formation. The authors provide new insights related to the spatial localization of the key components of FGF signaling and attempt to reveal the temporal dynamics of the signal propagation and cell fate decision, which has been challenging.

Reviewer #3 (Public review):

Jo and colleagues set out to investigate the origins and functions of localized FGF/ERK signaling for the differentiation and spatial patterning of primitive streak fates of human embryonic stem cells in a well-established micropattern system. They demonstrate that endogenous FGF signaling is required for ERK activation in a ring-domain in the micropatterns, and that this localized signaling is directly required for differentiation and spatial patterning of specific cell types. Through high-resolution microscopy and transwell assays, they show that cells receive FGF signals through basally localized receptors. Finally, the authors find that there is a requirement for exogenous FGF2 to initiate primitive streak-like differentiation, but endogenous FGFs, especially FGF4 and FGF17, fully take over at later stages.

Even though some of the authors' findings - such as the localized expression of FGF ligands during gastrulation and the importance of FGF/ERK signaling for cell differentiation in the primitive streak - have been reported in model organisms before, this is one of the first studies to investigate the role of FGF signaling during primitive streak-like differentiation of human cells. In doing so, the paper reports a number of interesting and valuable observations, namely the basal localization of FGF receptors which mirrors that of BMP and Nodal receptors, as well as the existence of a positive feedback loop centered on FGF signaling that drives primitive-streak differentiation. In the revised version of their work, the authors have furthermore dissected the role of different FGFs through knockdown approaches. These experiments reveal discrete functions for different FGF genes in their system, as well as interesting differences between the role of specific FGFs in human compared to model systems.

Comments on revisions:

The authors have appropriately addressed all comments and suggestions from the previous round of review. The only textual change that I would still like to suggest is to write explicitly in the main text corresponding to Fig. 1 that the mTESR1 medium used for these initial experiments already contains FGF. This is something that is probably known to experts in the field, but not necessarily to a broader readership.

Author response:

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

Reviewer #1 (Public review): 

Summary: 

This is an interesting study on the role of FGF signaling in the induction of primitive streak-like cells (PS-LC) in human 2D-gastruloids. The authors use a previously characterized standard culture that generates a ring of PSLCs (TBXT+) and correlate this with pERK staining. A requirement for FGF signaling in TBXT induction is demonstrated via pharmacological inhibition of MEK and FGFR activity. A second set of culture conditions (with no exogenous FGFs) suggests that endogenous FGFs are required for pERK and TBXT induction. The authors then characterize, via scRNA-seq, various components of the FGF pathway (genes for ligands, receptors, ERK regulators, and HSPG regulation). They go on to characterize the pFGFR1, receptor isoforms, and polarized localization of this receptor. Finally, they perform FGF4 inhibition and use a cell line with a limited FGF17 inactivation (heterozygous null) and show that loss of these FGFs reduces PS-LC and derivative cell types. 

Strengths: 

(1) As the authors point out, the role of FGF signaling in gastrulation is less well understood than other signaling pathways. Hence this is a valuable contribution to that field. 

(2) The FGF4 and FGF17 loss-of-function experiments in Figure 5 are very intriguing. This is especially so given the intriguing observation that these FGFs appear to be dominating in this model of human gastrulation, in contrast to what FGFs dominate in mice, chicks, and frogs. 

(3) In general this paper is valuable as a further development of the Human gastruloid system and the role of FGF signaling in the induction of PS-CLs. The wide net that the authors cast in characterizing the FGF ligand gene, receptor isoforms, and downstream components provides a foundation for future work. As the authors write near the beginning of the Discussion "Many questions remain." 

We thank the reviewer for these positive comments.

Weaknesses: 

(1) FGFs are cell survival factors in various aspects of development. The authors fail to address cell death due to loss of FGF signaling in their experiments. For example, in Figure 1E (which requires statistical analysis) and 1G (the bottom FGFRi row), there appears to be a significant amount of cell loss. Is this due to cell death? The authors should address the question of whether the role of FGF/ERK signaling is to keep the cells alive. 

Indeed, FGF also strongly affects cell survival and it is an interesting question to what extent this depends on ERK. Our manuscript focuses instead on the role of FGF/ERK signaling in cell fate patterning. As mentioned in our discussion, figure 1de show that doxycycline induced pERK leads to more TBXT+ cells than the control without restoring cell number, suggesting the role of FGF in controlling cell number is independent of the requirement for FGF/ERK in PS-LC differrentiation. To further support this, we have added data showing low doses of MEKi are sufficient to inhibit differentiation without affecting cell number (Supp. Fig. 1i).

To address the reviewers question regarding the cause of cell loss, we now stained for BrdU and cleaved Cas3 to assess proliferation and apoptosis in the presence and absence of MEK and FGFR inhibition (new Supp. Fig.

1ef). This shows that the effect of these inhibitors on cell number is primarily due to a reduction in proliferation. We have also included statistical analysis in Fig.1e. 

(2) Regarding the sparse cells in 1G, is there a reduction in cell number only with FGFRi and not MEKi? Is this reproducible? Gattiglio et al (Development, 2023, PMID: 37530863) present data supporting a "community effect" in the FGF-induced mesoderm differentiation of mouse embryonic stem cells. Could a community effect be at play in this human system (especially given the images in the bottom row of 1G)? If the authors don't address this experimentally they should at least address the ideas in Gattoglio et al. 

Indeed, FGFRi reproducibly affects cell number more than MEKi, in line with the fact that pathways other than MAPK/ERK downstream of FGF (e.g. PI3K) play important roles in cell survival and growth. However, we think the lack of differentiation in MEKi and FGFRi in Fig.1g cannot be attributed to a loss of cells combined with a community effect. This is because without FGFRi or MEKi cells efficiently differentiate to primitive streak at much lower densities than those originally shown, consistent with the data we discuss in response to (1) arguing against a primarily indirect effect of FGF on PS-LC differentiation through cell density. In the context of directed differentiation (rather than 2D gastruloids), we have now shown in a controlled manner that the effect of MEKi and FGFRi does not depend on a community effect by repeating the experiment in Fig.1g while adjusting cell seeding densities to obtain similar final cell densities in all three conditions (new Fig.1g, new Supp Fig.1g). Furthermore we have included new data showing extremely sparse cells without MEKi or FGFRi still differentiate without problems (new Supp Fig 1h). We have also include Gattoglio et al in our revised discussion.

(3) Do the FGF4 and FGF17 LOF experiments in Figure 5 affect cell numbers like FGFRi in Figure 1? 

We did not observe major changes in cell number in the FGF4 and FGF17 loss of function experiments. This is in line with our observation that low levels of ERK signaling are sufficient to maintain proliferation (new Supp. Fig. 1i), and the fact that low levels of ERK signaling are maintained in the absence of FGF4 and FGF17 (Fig.5), likely by FGF2 (Fig. 2). In contrast, FGFRi treatment in Fig.1 leads to a nearly complete loss of FGF signaling (ERK and other pathways) that has a dramatic effect on cell number.

Why examine PS-LC induction only in FGF17 heterozygous cells and not homozygous FGF17 nulls? 

We were unable to obtain homozygous FGF17 nulls, it is not clear if there is a reason for this. In the absence of homozygous nulls, we have now further corroborated our findings with additional knockdown data (described in response to other comments below).

(4) The idea that FGF8 plays a dominant role during gastrulation of other species but not humans is so intriguing it warrants deeper testing. The authors dismiss FGF8 because its mRNA "...levels always remained low." (line 363) as well as the data published in Zhai et al (PMID: 36517595) and Tyser et al (PMID: 34789876). But there are cases in mouse development where a gene was expressed at levels so low, that it might be dismissed, and yet LOF experiments revealed it played a role or even was required in a developmental process. The authors should consider FGF8 inhibition or inactivation to explore its potential role, despite its low levels of expression. 

We thank the reviewer for this suggestion. We have now analyzed the role of FGF8 using FISH to visualize its expression and siRNA to understand its function (Fig.5d,f,h; Supp.Fig.5e,g,6e). We found that FGF8 expression is higher earlier in differentiation, preceding most expression of TBXT. Our scRNA-seq only analyzed samples at 42h so did not capture this. Furthermore, FGF8 expression localized inside the PS-like ring rather than coinciding with it like FGF4. Surprisingly, FGF8 knockdown led to an increase in primitive streak-like differentiation, suggesting it may counteract FGF4. The results are shown in the revised Fig. 5 and Supplemental Fig. 5. While this certainly merits further investigation, understanding the role of FGF8 in more detail is beyond the scope of the current work. 

(5) Redundancy is a common feature in FGF genetics. What is the effect of inhibiting FGF4 in FGF17 LOF cells? 

Further siRNA and shRNA experiments showed that FGF17 knockdown had a much smaller effect than FGF4 knockdown on expression of primitive streak markers (Fig.5i, Supp.Fig.6f-i) but that FGF17 knockdown did lead to a complete loss of the mesoderm marker TBX6 (Fig.5j, Supp.Fig.6j). A double knockdown of FGF4+FGF17 looked similar to FGF4 alone (Supp.Fig.6k). Thus, we now think the more likely scenario is that FGF17 is downstream of FGF4-dependent PS-differentiation and although this may have a positive feedback effect whereby this FGF17 can then enhance further PS-differentiation, which we previously interpreted as partial redundancy, the primary role of FGF17 may be later, in mesoderm differentiation.

(6) I suggest stating that the authors take more caution in describing FGF gradients. For example, in one Results heading they write "Endogenous FGF4 and FGF17 gradients underly the ERK activity pattern.", implying an FGF protein gradient. However, they only present data for FGF mRNA , not protein. This issue would be clarified if they used proper nomenclature for gene, mRNA (italics), and protein (no italics) throughout the paper. 

Thank you for the suggestion. We have edited the paper to more clearly distinguish protein and mRNA. We do think our data provide substantial indirect evidence for a protein gradient which is what the results heading is meant to convey. Receptor activation is high where ERK activity is high (Fig.3), and receptor activation is limited by ligands, since creating a scratch to let exogenous FGF reach the basal side of cells in the center leads to receptor activation (Fig.4). This strongly suggests ERK activity reflects an FGF protein gradient. 

Reviewer #2 (Public review): 

Summary: 

The role of FGFs in embryonic development and stem cell differentiation has remained unclear due to its complexity. In this study, the authors utilized a 2D human stem cell-based gastrulation model to investigate the functions of FGFs. They discovered that FGF-dependent ERK activity is closely linked to the emergence of primitive streak cells. Importantly, this 2D model effectively illustrates the spatial distribution of key signaling effectors and receptors by correlating these markers with cell fate markers, such as T and ISL1. Through inhibition and loss-of-function studies, they further corroborated the needs of FGF ligands. Their data shows that FGFR1 is the primary receptor, and FGF2/4/17 are the key ligands for primitive streak development, which aligns with observations in primate embryos. Additional experiments revealed that the reduction of FGF4 and FGF17 decreases ERK activity. 

Strengths: 

This study provides comprehensive data and improves our understanding of the role of FGF signaling in primate

primitive streak formation. The authors provide new insights related to the spatial localization of the key components of FGF signaling and attempt to reveal the temporal dynamics of the signal propagation and cell fate decision, which has been challenging. 

Weaknesses: 

Given the solid data, the work only partially clarifies the complex picture of FGF signaling, so details remain somewhat elusive. The findings lack a strong punchline, which may limit their broader impact. 

We thank this reviewer for their valuable feedback and compliment on the solidity of our data. The punchline of our work is that FGF4 and FGF17-dependent ERK signaling plays a key role in differentiation of human PS-like cells and mesoderm, and that these are different FGFs than those thought to drive mouse gastrulation. A second key point is that like BMP and TGFβ signaling, FGF signaling is restricted to the basolateral sides of pluripotent stem cell colonies due to polarized receptor expression, which is crucial for understanding the response to exogenous ligands added to the cell medium. Indeed, many facets of FGF signaling remain to be investigated in the future, such as how FGF regulates and is regulated by other signals, which we will dedicate a different manuscript to. 

Reviewer #3 (Public review): 

Jo and colleagues set out to investigate the origins and functions of localized FGF/ERK signaling for the differentiation and spatial patterning of primitive streak fates of human embryonic stem cells in a well-established micropattern system. They demonstrate that endogenous FGF signaling is required for ERK activation in a ringdomain in the micropatterns, and that this localized signaling is directly required for differentiation and spatial patterning of specific cell types. Through high-resolution microscopy and transwell assays, they show that cells receive FGF signals through basally localized receptors. Finally, the authors find that there is a requirement for exogenous FGF2 to initiate primitive streak-like differentiation, but endogenous FGFs, especially FGF4 and FGF17, fully take over at later stages. 

Even though some of the authors' findings - such as the localized expression of FGF ligands during gastrulation and the importance of FGF/ERK signaling for cell differentiation in the primitive streak - have been reported in model organisms before, this is one of the first studies to investigate the role of FGF signaling during primitive streak-like differentiation of human cells. In doing so, the paper reports a number of interesting and valuable observations, namely the basal localization of FGF receptors which mirrors that of BMP and Nodal receptors, as well as the existence of a positive feedback loop centered on FGF signaling that drives primitive-streak differentiation. The authors also perform a comparison of the role of different FGFs across species and try to assign specific functions to individual FGFs. In the absence of clean genetic loss-of-function cell lines, this part of the work remains less strong. 

We thank the reviewer for emphasizing the value of our findings in a human model for gastrulation. We agree more loss-of-function experiments would provide further insight into the role of different FGFs. While we did not manage to create knockout cell lines, we have now performed both siRNA and shRNA knock-down of all FGF4, and FGF17 in two different hPSC lines, performed siRNA knockdown of FGF8, and also made a FGF4+FGF17 shRNA double knockdown cell lines to more completely test the functions of the individual FGFs (Fig.5, Supp.Fig.5,6). Our data suggest FGF17 may be downstream of FGF4 and primarily required for mesoderm differentiation while FGF8 appears to counteract FGF4. In doing this we have added a large amount of new data to the manuscript and we have removed the heterozygous knockout data in the first version of the manuscript which we felt added little to the new data. Further experiments are still needed to solidify our interpretation but those are beyond the scope of the current work.   

Reviewer #1 (Recommendations for the authors): 

(1) FGF2 is added to culture experiments (e.g. Figure 4), but the commercial source is not mentioned in Methods. For example, it could be added to "Supplementary Table 1: Cell signaling reagents." 

We apologize for this oversight and have now added the information to Supplementary Table 1.

(2) Line 117-118: "For example, by controlling the expression of Wnt or Nodal which are both required for PS-like differentiation". It is clear what the authors mean, but this is not a complete sentence. 

We edited this for clarity, it now reads: “First, is FGF/ERK signaling required directly for PS-like differentiation, or does it act indirectly? These possibilities are not mutually exclusive. For example, FGF/ERK could be required directly but also act indirectly by controlling Wnt or Nodal expression, as both Wnt and Nodal signaling are required for PS-like differentiation.”

(3) Line 246 "...found its spatial pattern to strongly resembles that of pERK..." either remove "to" or change "resembles" to "resemble" 

Thank you for catching this. We removed “to”.

(4) Lines 391- 393 seem to be missing a word in the last phrase: "...with FGF17 more important continued differentiation to mesoderm and endoderm." Maybe "during" after the word "important"? 

Thank you for catching this, indeed the word “during” was missing and we have now added it.

(5) Please define acronyms in Figure 3D (PS-LC was defined previously, but not others). 

We apologize for the oversight, we have now defined the acronyms.

(6) The three blue lines in Figure 5B (right) are hard to discern (and I'm not colorblind). I suggest also using a variety of dotted lines in a subset of these FGFs. 

Thanks you for the suggestion. We have now given all the FGFs colors that are more clearly distinct and made the TBXT and TBX6 lines dashed.  

Reviewer #2 (Recommendations for the authors): 

(1) The reviewer acknowledges that FGF signaling is complex, particularly when dynamics and its correlation with cell fates are considered. To improve the clarity of the findings, the authors are encouraged to provide an additional schematic figure that clearly delineates the main findings of this study.  

Thank you for the suggestion. We have now added a summary figure (Fig.6) to our discussion, which we hope helps present our findings more clearly.

(2) The data suggest that FGF signaling may function differently in mice compared to primates, and their stem cell model aligns more closely with the latter. While the authors discuss this in the contents only based on sequencing data, it would be valuable to conduct some experiments with mouse embryos to validate the key differences. 

It is unclear to us which experiments the reviewer has in mind. There is ample data on FGF expression in the mouse literature, as are many knockout phenotypes. Furthermore, verifying loss of function phenotypes (e.g. FGF17 knockout) in mouse is beyond our expertise.

(3) Heparan sulfate proteoglycan (HSPG) is mentioned as an important component of FGF signaling; however, the only data related to HSPG is single-cell sequencing results. The authors should consider performing immunostaining or other assays to validate HSPG expression and spatial distribution, similar to the approach they used for other signaling components. 

Our scratch experiments in Fig. 4 strongly argue against HSPGs as being responsible for the spatial pattern of FGF receptor activation: after a scratch across the colony the response is strong all along the scratch as expected if presence of FGF (an FGF gradient) controls the level of activity. If HSPGs were limiting, FGF flowing in from the media show not be able to uniformly activate receptors around the scratch.

In addtion, we have now included an immunostain for HS in a newly added Supp. Fig. 4 which does not explain the observed pattern of ERK signaling.

(4) In the scratch experiment, particularly high PERK expression is observed at the edge of the scratch. The authors should provide an explanation for why this expression is significantly higher compared to the edges of the colony. Additionally, it would be interesting to investigate the fate of the cells with super high PERK expression.  

We have now determined that adaptive response to FGF is the reason that the response around the scratch is initially much higher than in the ERK activity ring that overlaps with the primitive streak-like cells. We have added figures showing that although the intial response to FGF exposure after scratching is very high, the response around the scratch adapts to levels similar in those in the ERK ring over the course of 6 hours (Fig.4ij). 

(5) For some of the key experiments, multiple cell lines should be used to ensure that the findings are reproducible and applicable across different human stem cell lines.

We have now checked FISH stainings and knockdown phenotypes for different FGFs in two different cell lines: ESI17 (hESC, XX) and PGP1 (hiPSC, XY). These results are shown in Supplementary Figures 6. We found all results to be consistent.

(6) Where applicable, the meaning of error bars needs to be more clearly presented, including details on the number of independent experiments or samples used. 

Thank you for pointing this out. Where error bar definitions were missing we have now added them to the figure captions.

Reviewer #3 (Recommendations for the authors): 

(1) The authors only analyze the ppERK ring in micropatterns of a single size. What was the motivation for the choice of this size? Can the authors how the ppERK ring is expected to depend on colony size? 

Much smaller patterns lose the interior pluripotent regions while much larger patters have a much larger pluripotent region, which requires larger tilings to image without providing additional insight. The colony sizedependence of cell fate patterning was described in the paper that established the 2D gastruloids model (Warmflash Nat Methods 2014) and we later showed this due to a fixed length scale of the BMP and Nodal signaling gradients from the colony edge (Jo et al Elife 2022). We have now included data showing that the ERK patterns behaves similarly, with a fixed length scale of the pattern implying that in smaller colonies the ERK ring becomes a disc and the entire center of the colony has high ERK signaling (Supp Fig 1a).

(2) The scRNAseq is somewhat confusing - why do the two datasets not overlap in the PHATE representation? This is unexpected, because the two samples have been treated similarly, and the authors have integrated their data to iron out possible batch effects. This discrepancy should be discussed. The authors should also specify from which reference exactly the first dataset comes from.  

The two datasets do overlap nicely, the same fates are well mixed in the same place and the gene expresison profiles for the integrated data (e.g., Fig.2e) look smooth, so we believe the integration is good, but different cell fates are represented to different degrees. In particular, sample 2 shows much more mesoderm differentiation making the mesoderm branch mostly orange. Occassionally samples differentiate faster or slower than average which we see here, and these samples were collected far apart in time. We do not believe this affects our conclusions, if anything, we think performing the analysis on two samples that differ this much should make the conclusions more robust.  

(3) If find it intriguing that exogenous FGF2 is important early on for primitive streak-like differentiation, although the authors show that it does not reach the center of the colony. The authors may want to discuss this conundrum. Does the FGF2 effect propagate from the outside to the inside, or does it act at an early stage when the cells have not yet formed a tight epithelium on the micropattern? 

The cells in the experiment in Fig. 5a were given 24h to epithelialize, so we we do believe it acts from the edge. We believe this may be due to FGF2 modulating the early BMP response on the edge and are working on a manuscript that further explores this pathway crosstalk.

(4) The authors' statement that FGF4 and FGF17 have partially redundant functions is not very strong, mainly because the study lacks a full FGF17 loss-of-function cell line. If the authors wanted to improve on this point, they could knock down FGF4 in the FGF17 heterozygous line, or produce a homozygous FGF17 KO line. If there are specific reasons why FGF17 homozygous lines cannot be produced, this could be interesting to discuss, too. Finally, I noticed that the methods list experiments with an FGF17 siRNA, but these are not shown in the manuscript. 

We agree our evidence was previously not as strong as it could be. While there is no reason we know of why homozygous knockout lines cannot be produced, we failed to produce on. To strengthen our evidence we have therefore included substantial new knockdown data.  We have now performed both siRNA and shRNA knockdown of all FGF4, and FGF17 in two different hPSC lines, performed siRNA knockdown of FGF8, and also made a FGF4+FGF17 shRNA double knockdown cell lines to more completely test the functions of the individual FGFs (Fig.5, Supp.Fig.5,6). These experiments showed that FGF17 knockdown had a much smaller effect than FGF4 knockdown on expression of primitive streak markers (Fig.5i, Supp.Fig.6f-i) but that FGF17 knockdown did lead to a complete loss of the mesoderm marker TBX6 (Fig.5j, Supp.Fig.6j). A double knockdown of FGF4+FGF17 looked similar to FGF4 alone (Supp.Fig.6k). Thus, we now think the more likely scenario is that FGF17 is downstream of FGF4-dependent PS-differentiation and although this may have a positive feedback effect whereby this FGF17 can then enhance further PS-differentiation, which we previously interpreted as partial redundancy, the primary role of FGF17 may be later, in mesoderm differentiation. Furthermore, our new data suggests FGF8 may counteract FGF4 and limit PS-like differentiation. 

Minor 

(5) Line 63: Reference(s) appear to be missing. 

This whole paragraph summarizes the results of the references given on line 55, we have now repeated the relevant references where the reviewer indicated.

(6) Supplementary Figure 1a,b does not show ppERK, unlike stated in lines 102 - 104. 

Indeed, the data described in lines 102-104 is shown in Fig.1a and we have removed the original Supplementary Figure 1ab since it did not provide relevant information.

(7) Line 201: It is not clear whether this is a new sequencing dataset, or if existing datasets have been reanalyzed. 

We agree our description was unclear. We have edited the text, which now explicitly states that our analysis is based on one dataset we collected previously and a replicate that was newly collected and deposited on GEO for this manuscript.

(8) Figure 2f; Supplementary Figure 2b, c: The colors need to be explained in scale bars. How has this data been normalized to allow for comparison between very different sample types? 

We have now added color bars indicating the scale for each of these figure panels. As the caption stated, the interspecies comparison was normalized within each species, so the highest FGF level for any FGF at any time within each species is normalized to one. We are thus comparing between species the relative expression of different FGFs within each species. Indeed there is no good way to compare absolute expression between species. For extra clarity we have expanded our description of the interspecies comparison analysis and normalization in the methods section.

(9) Line 232: Where is the expression of SEF shown? 

It is shown in Fig. 2i, under the official gene name IL17RD.

(10) Supplementary Figure 4 seems to be missing. 

Thank you for pointing this out. We have now added a supplementary Fig.4.

(11) Line 437: Citation needed. 

We have included citations now.

(12) Line 439: A similar feedback loop has been proposed to operate during mesoderm differentiation in mouse ESC (pmid: 37530863 ). The authors may consider citing this work. 

Thank you for the suggestion, we have now included this work in the discussion. The feedback loop proposed in that work involves FGF8, while we were trying to explain why FGF4 and not FGF8 appears to be conserved across species by invoking an FGF4 feedback loop. Thus, it becomes even harder to explain differences in FGF4 and FGF8 expression between human and mouse gastrulation.

(13) Supplementary Figure 6 is not described in the main text. 

We have removed the original Supplementary Figure 6 and corresponding heterozygous knockout data in the main figure which we felt added little to the extensive knockdown data we now present. We did create a new Supplementary Figure 6 showing additional knockdown data which is described in the main tekst.

(14) Submission of sequencing data to GEO needs to be updated. 

We have now made the GEO data public.

given that use of the word I came up with the word

prenitel

as a special value in the context of an algorithm which uses its presence as a condition of a block

It may need some additonal logic to actually then trigger a special interpretative process

This proposition sounds crazy, but not when you consider this is what humans have been doing since the beginning of time. When you go so far into history, the patterns of civilization and society become very apparent, yet maybe not much easier to avoid.

This frames the archaeological narrative around tribes, nomads and others not traditionally thought of as being part of a rigid state as not only important for historical understanding, but also as a heavily politicized topic that still has implications in our understanding of freedom today.

The author writes that tribal societies were not only written and reconfirmed as 'barbaric' for millenia, but also were thought of largely as not contributing to a larger world order or production despite their often large numbers. He goes into further the effect of ideas of population on this narrative later in the article.

Wengrow is saying that we need more push-back on the idea that Empires are the best, or only, thing to study when trying to understand human progress. We need to recognize other forms of society as well.

Wengrow stresses the importance of using up to date resources when discussing historical topics. This is because our knowledge is ever-growing and the only way to improve historical conjecture is to use up to date information.

eLife Assessment

This fundamental study substantially advances our current understanding of mechanotransduction within endothelial cells. The evidence provided by the authors in the revised manuscript is compelling, which taken together, provides strong support for the authors' major findings. The work will be of broad interest to cell biologists and vascular biologists.

Reviewer #1 (Public review):

This manuscript puts forward the concept that there is a specific time window during which YAP/TAZ driven transcription provides feedback for optimal endothelial cell adhesion, cytoskeletal organization and migration. The study follows up on previous elegant findings from this group and others which established the importance of YAP/TAZ-mediated transcription for persistent endothelial cell migration. The data presented here extends the concept at two levels: first, the data may explain why there are differences between experimental setups where YAP/TAZ activity are inhibited for prolonged times (e.g. cultures of YAP knockdown cells), versus experiments in which the transient inhibition of YAP/TAZ and (global) transcription affects endothelial cell dynamics prior to their equilibrium.

All experiments are convincing, clearly visualized and quantified.

The strength of the paper is that it clearly indicates that there are temporal controlled feedback systems, which is important knowledge for understanding the mechanisms that drive endothelial collective cell behavior.

A potential limitation of the in vivo experiments is that the inhibitors may include off-target effects as well. To solve this caveat in future research endeavours, which is beyond the scope of the current study, it would be interesting to study this process in knockout models, combined with optogenetics and transgenic zebrafish lines that visualize endothelial cell functional properties such as proliferation and migration.

Reviewer #2 (Public review):

Summary:

Here the effect of overall transcription blockade, and then specifically depletion of YAP/TAZ transcription factors was tested on cytoskeletal responses, starting from a previous paper showing YAP/TAZ-mediated effects on the cytoskeleton and cell behaviors. Here, primary endothelial cells were assessed on substrates of different stiffness and parameters such as migration, cell spreading, and focal adhesion number/length were tested upon transcriptional manipulation. Zebrafish subjected to similar manipulations were also assessed during the phase of intersegmental vessel elongation. The conclusion was that there is a feedback loop of 4 hours that is important for the effects of mechanical changes to be translated into transcriptional changes that then permanently affect the cytoskeleton.

The idea is intriguing and a previous paper contains data supporting the overall model. The fish washout data is quite interesting and supports the kinetics conclusions. New transcriptional profiling in this version supports that cytoskeletal genes are differentially regulated with YAP/TAZ manipulations.

Major strengths:

The combination of in vitro and in vivo assessment provides evidence for timing in physiologically relevant contexts, and rigorous quantification of outputs is provided. The idea of defining temporal aspects of the system is quite interesting. New RNA profiling supports the model.

Weaknesses:

Actinomycin D blocks most transcription so exposure for hours likely leads to secondary and tertiary effects and perhaps effects on viability.

Comments on latest version:

I read the author response to previous reviews, and it seems they agree with the weaknesses stated in the reviews but did not provide any text or data revisions.

Reviewer #4 (Public review):

Summary:

Mason DE et al. have extended their previous study on continuous migration of cells regulated by a feedback loop that controls gene expression by YAP and TAZ. Time scale of the negative feedback loop is derived from the authors' adhesion-spreading-polarization-migration (ASPM) assay. Involvement of transcription-translation in the negative feedback loop is evidenced by the experiments using Actinomycin D. The time scale of mechanotransduction-dependent feedback demonstrated by cytoskeletal alteration in the actinomycin D-treated endothelial colony forming cells (ECFCs) and that shown in the ECFCs depleted of YAP/TAZ by siRNA. The authors examine the time scale when ECFCs are attached to MeHA matrics (soft, moderate, and stiff substrate) and show the conserved time scale among the conditions they use, although instantaneous migration, cell area, and circularity vary. Finally, they tried to confirm that the time scale of the feedback loop-dependent endothelial migration by the effect of washout of Actinomycin D (inhibition of gene transcription), Puromycin (translational inhibition), and Verteporfin (YAP/TAZ inhibitor) on ISV extension during sprouting angiogenesis. They conclude that endothelial motility required for vascular morphogenesis is regulated by a mechanotransduction-mediated feedback loop that is dependent on YAP/TAZ-dependent transcriptional regulation.

Strengths:

The authors conduct ASPM assay to find the time scale of feedback when ECFCs attach to three different matrics. They observe the common time scale of feedback. Thus, under very specific conditions they use, the reproducibility is validated by their ASPM assay. The feedback loop mediated by inhibition of gene expression by Actinomycin D is similar to that obtained from YAP/TAZ-depleted cells, suggesting the mechanotranduction might be involved in the feedback loop. The time scale representing infection point might be interesting when considering the continuous motility in cultured endothelial cells, although it might not account for the migration of endothelial cells that is controlled by a wide variety of extracellular cues. In vivo, stiffness of extracellular matrix is merely one of the cues.

Weaknesses:

ASPM assay is based on attachment-dependent phenomenon. The time scale, including the inflection point determined by ASPM experiments using cultured cells and the mechanotransduction-based theory, do not seem to fit in vivo ISV elongation. Although it is challenging to find the conserved theory of continuous cell motility of endothelial cells, the data is preliminary and does not support the authors' claim. There is no evidence that mechanotransduction solely determines the feedback loop during elongation of ISVs.

Comments on revisions:

The authors' methods using ASPM assay might suggest the feedback loop by their in vitro culture assay. They still need to confirm the loop in vivo using zebrafish intersegmental vessels. The time course of the feedback loop is supported by the ASPM assay. However, the feedback loop is not confirmed in vivo, although it might be suggested by the phenotypes of the ISV treated with drugs. Thus, in the abstract and in the results section, they had better rewrite the interpretation. They have not yet confirmed the feedback loop in vivo.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

All experiments are convincing, clearly visualized and quantified. 

The strength of the paper is that it clearly indicates that there are temporal controlled feedback systems which is important for endothelial collective cell behavior. 

A limitation of the study is that the inhibitory studies in vivo may include off-target effects as well. Future endeavors, including specific knockout models, optogenetics and/or transgenic zebrafish lines that visualize endothelial cell properties (proliferation and migration) will be informative to track individual endothelial cell responses upon feedback signals.

We agree with the reviewer and are currently conducting experiments with optogenetic tools, knockout models, and transgenic zebrafish lines to dissect the feedback loop dynamics at the cellular scale.    

Reviewer #2 (Public review):

Major strengths: The combination of in vitro and in vivo assessment provides evidence for timing in physiologically relevant contexts, and rigorous quantification of outputs is provided. The idea of defining temporal aspects of the system is quite interesting. New RNA profiling supports the model. 

Weaknesses: Actinomycin D blocks most transcription so exposure for hours likely leads to secondary and tertiary effects and perhaps effects on viability.

We agree with the reviewer that “off-target” effects are a limitation of the pharmacologic approach. We have also previously shown that long-term treatment with actinomycin D reduces ECFC survival (Mason et al., 2019). 

Reviewer #3 (Public review):

Strengths: The authors conduct ASPM assay to find the time scale of feedback when ECFCs attach to three different matrics. They observe the common time scale of feedback. Thus, under very specific conditions they use, the reproducibility is validated by their ASPM assay. The feedback loop mediated by inhibition of gene expression by Actinomycin D is similar to that obtained from YAP/TAZ-depleted cells, suggesting the mechanotranduction might be involved in the feedback loop. The time scale representing infection point might be interesting when considering the continuous motility in cultured endothelial cells, although it might not account for the migration of endothelial cells that is controlled by a wide variety of extracellular cues. In vivo, stiffness of extracellular matrix is merely one of the cues. 

Weaknesses: ASPM assay is based on attachment-dependent phenomenon. The time scale including the inflection point determined by ASPM experiments using cultured cells and the mechanotransduction-based theory do not seem to fit in vivo ISV elongation. Although it is challenging to find the conserved theory of continuous cell motility of endothelial cells, the data is preliminary and does not support the authors' claim. There is no evidence that mechanotransduction solely determines the feedback loop during elongation of ISVs. The points to be addressed are listed in recommendations for the authors.

The ASPM assay enabled us to define temporal dynamics of YAP/TAZ mechanotransduction. We then used those insights to design ISV washout experiments that tested if the characteristic time scales were conserved in vivo. However, we agree with the limitations identified by the reviewer. Cells behave and respond to mechanical cues differently in 2D vs 3D environments, and the microenvironment in vivo is much more complex. Future work with optogenetic tools will be useful to dissect the temporal kinetics in vivo during ISV elongation.

eLife Assessment

These valuable studies explore the consequences of exposure to the toxin hydrogen sulfide (H2S) on the behavior and physiology of C. elegans. The work finds that behavioral changes evoked by H2S exposure are modulated by several regulatory pathways known to influence chemosensory-evoked locomotor behavior, but there is incomplete data to support the authors' claim of comprehensive mechanistic insight into the consequences of H2S exposure. Nevertheless, the findings may be informative for those studying organismal stress responses and the effects of mitochondrial ROS on behavior and physiology.

Reviewer #3 (Public review):

Summary:

The manuscript explores behavioral responses of C. elegans to hydrogen sulfide, which is known to exert remarkable effects on animal physiology in a range of contexts. The possibility of genetic and precise neuronal dissection of responses to H2S motivates the study of responses in C. elegans.

The authors have followed up observations in the initial version of the manuscript, and their data do not support the direct sensing of H2S by the ASJ neurons or other sensory neurons. Genetic and parallel analysis of O2 and CO2 responsive pathways do not reveal further insights regarding potential mechanisms underlying H2S sensing. Gene expression analysis extends prior work. Finally, the authors have examined how H2S-evoked locomotory behavioral responses are affected in mutants with altered stress and detoxification response to H2S, most notably hif-1 and egl-9. These data, while examining locomotion, are more suggestive that observed effects on animal locomotion are secondary to altered organismal toxicity as opposed to specific behavioral responedse

Overall, the manuscript provides a wide range of preliminary observations of genetic interactions that may influence locomotory responses to H2S, but mechanistic insight or a synthesis of disparate data is lacking.

Reviewer #4 (Public review):

Summary:

The authors establish a behavioral paradigm for avoidance of H2S and conduct a large candidate screen to identify genetic requirements. They follow up by genetically dissecting a large number of implicated pathways - insulin, TGF-beta, oxygen/HIF-1, and mitochondrial ROS, which have varied effects on H2S avoidance. They additionally assay whole-animal gene expression changes induced by varying concentrations and durations of H2S exposure.

Strengths:

The implicated pathways are tested extensively through mutants of multiple pathway molecules. The authors address previous reviewer concerns by directly testing the ability of ASJ to respond to H2S via calcium imaging. This allows the authors to revise their previous conclusion and determine that ASJ does not directly respond to H2S and likely does not initiate the behavioral response. Extensive experiments manipulating the mitochondrial ETC and ROS support the authors' revised model that mitochondrial toxicity is the major driver of H2S avoidance.

It seems possible that HIF-1 and SKN-1 signaling directly modulate ROS toxicity while ASJ neurons and the oxygen sensing circuit could modulate the avoidance behavior. How this neuronal interaction happens remains unknown.

Author response:

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

Reviewer #3 (Public review): 

Summary: 

The manuscript explores behavioral responses of C. elegans to hydrogen sulfide, which is known to exert remarkable effects on animal physiology in a range of contexts. The possibility of genetic and precise neuronal dissection of responses to H2S motivates the study of responses in C. elegans. The revised manuscript does not seem to have significantly addressed what was lacking in the initial version. 

The authors have added further characterization of possible ASJ sensing of H2S by calcium imaging but ASJ does not appear to be directly involved. Genetic and parallel analysis of O2 and CO2 responsive pathways do not reveal further insights regarding potential mechanisms underlying H2S sensing. Gene expression analysis extends prior work. Finally, the authors have examined how H2S-evoked locomotory behavioral responses are affected in mutants with altered stress and detoxification response to H2S, most notably hif-1 and egl-9. These data, while examining locomotion, are more suggestive that observed effects on animal locomotion are secondary to altered organismal toxicity as opposed to specific behavioral responedse 

Overall, the manuscript provides a wide range of intriguing observations, but mechanistic insight or a synthesis of disparate data is lacking. 

We thank the reviewer for the valuable feedback. We agree that while our investigation provides broad coverage, it does not fully resolve the mechanisms of H₂S perception. As both reviewers noted, the avoidance response to high levels of H₂S is most likely driven by its toxicity, particularly at the level of mitochondria, rather than by direct perception of H₂S. We also favor this model and have revised the results and discussion to highlight this interpretation, while acknowledging that other mechanisms cannot be excluded (main changes lines 387-402 and 535-547).

Building on this view, our observations point toward mitochondrial ROS transients as the trigger for H₂S avoidance. First, toxic levels of H₂S are known to promote ROS production (1). Second, similar to acute H₂S, brief exposure to rotenone, an ETC complex I inhibitor that rapidly generates mitochondrial ROS, triggers locomotory responses (Figure 7E) (Lines 393-396). Third, regardless of duration, rotenone exposure inhibits H₂S-evoked avoidance (Figure 7E) (Lines 389-391), likely by preventing or dampening H₂S-evoked mitochondrial ROS bursts when ETC function is impaired and ROS is already high. Notably, animals subjected to prolonged rotenone exposure, ETC mutants, and quintuple sod mutants, each experiencing chronically high ROS levels, fail to respond to H₂S and display reduced locomotory activity, presumably due to ROS toxicity and/or activation of stress-adaptive mechanisms (Figure 7).

Consistent with the activation of stress-responsive pathways, H₂S exposure alters expression of genes controlled by SKN-1 and HIF-1 signaling. Both pathways are ROS-sensitive and promote adaptation to chronic ROS production (2-4). Their activation, as in egl-9, render these animals insensitive to H₂S-evoked ROS transients (Figure 5B) (Lines 303-305). Conversely, mutants defective in these adaptive pathways, such as hif-1, still show initial locomotory responses to H₂S, but rapidly lose activity during prolonged H₂S exposure (Figure 5D) (Lines 318-319). These observations suggest that HIF-1 pathway is dispensable for initiating the response to H₂S evoked ROS transients, but essential for protecting against ROS toxicity.

In this context, the neural circuit we examined, such as ASJ neurons, is not directly involved in H₂S perception (Line 165-169 and 448-457). Instead, it likely modulates a circuit that is responsive to ROS toxicity. This circuit is also influenced by ambient O₂ levels, the state of O₂ sensing circuit, and nutrient status, in a manner reminiscent of the CO₂ responses (5, 6).

Reviewer #4 (Public review): 

Summary: 

The authors establish a behavioral paradigm for avoidance of H2S and conduct a large candidate screen to identify genetic requirements. They follow up by genetically dissecting a large number of implicated pathways - insulin, TGF-beta, oxygen/HIF-1, and mitochondrial ROS, which have varied effects on H2S avoidance. They additionally assay whole-animal gene expression changes induced by varying concentrations and durations of H2S exposure. 

Strengths: 

The implicated pathways are tested extensively through mutants of multiple pathway molecules. The authors address previous reviewer concerns by directly testing the ability of ASJ to respond to H2S via calcium imaging. This allows the authors to revise their previous conclusion and determine that ASJ does not directly respond to H2S and likely does not initiate the behavioral response. 

We thank the reviewer for the supportive comments.

Weaknesses: 

Despite the authors focus on acute perception of H2S, I don't think the experiments tell us much about perception. I think they indicate pathways that modulate the behavior when disrupted, especially because most manipulations used broadly affect physiology on long timescales. For instance, genetic manipulation of ASJ signaling, oxygen sensing, HIF-1 signaling, mitochondrial function, as well as starvation are all expected to constitutively alter animal physiology, which could indirectly modulate responses to H2S. The authors rule out effects on general locomotion in some cases, but other physiological changes could relatively specifically modulate the H2S response without being involved in its perception. 

I am actually not convinced that H2S is directly perceived by the C. elegans nervous system at all. As far as I can tell, the avoidance behavior could be a response to H2S-induced tissue damage rather than the gas itself. 

We thank the reviewer for the valuable insights, and fully agree that the H₂S may not be directly perceived by C. elegans. Please see detailed responses below.

Reviewer #4 (Recommendations for the authors): 

The clarity of the paper is improved in this version. My main issue has to do with "perception" of H2S. At times the authors suggest that hydrogen sulfide should be perceived by a neural circuit ("we did not specifically identify the neural circuit mediating H2S signaling"), while at other times they discuss the possibility that it is not directly perceived neuronally ("Supporting the idea that acute mitochondrial ROS generation initiates avoidance of high H2S levels,"). The authors should clearly state their model for H2S perception. Do they think there is a receptor and sensory neuron for H2S (not identified in this paper)? If not, what does it mean for there to be a neural circuit mediating the response? To me, it looks more like what is being "perceived" by a neural circuit is ROS-induced toxicity, not H2S itself. 

To drill down on direct modulation of acute perception, are any of the pathway manipulations used in this paper performed on the timescale of perception? Rotenone for 10 mins is close to that timescale, and in fact it increases speed independently of H2S, consistent with ROSinduced toxicity, not H2S being the signal that induces the behavior. Optogenetic activation of RMG could also be on the acute timescale. Can the authors clarify for how long blue light was on the worms before the start of the assay? Or was it turned on at the same time as video acquisition commenced? This could be evidence that RMG acutely modulates this behavioral response. 

I feel that the ASJ calcium imaging data should be in the main figure given its importance in revising the original model. 

We thank the reviewer for the valuable advice.

As suggested, ASJ calcium imaging data are displayed in the main figure (Figure 2I) (Line 167).

As both reviewers noted, our initial presentation was not sufficiently clear regarding the mechanism underlying H₂S avoidance. We agree with the reviewer that H₂S avoidance is unlikely mediated by direct perception via a H₂S-specific receptor, but likely arises from acute mitochondrial dysfunction and ROS generation. 

ROS

In line with the reviewer’s perspective, our observations point toward mitochondrial ROS transients as the trigger for H₂S avoidance. First, toxic levels of H₂S are known to promote ROS production (1). Second, similar to acute H₂S, brief exposure to rotenone, an ETC complex I inhibitor that rapidly generates mitochondrial ROS, triggers locomotory responses (Figure 7E) (Lines 393-396). Third, regardless of duration, rotenone exposure inhibits H₂S-evoked avoidance (Figure 7E) (Lines 389-391), likely by preventing or dampening H₂S-evoked mitochondrial ROS bursts when ETC function is impaired and ROS is already high. Notably, animals subjected to prolonged rotenone exposure, ETC mutants, and quintuple sod mutants, each experiencing chronically high ROS levels, fail to respond to H₂S and display reduced locomotory activity, presumably due to ROS toxicity and/or activation of stress-adaptive mechanisms (Figure 7). We revised the Results and Discussion to present the model more consistently (main changes lines 387-402 and 535-547).

Consistent with the activation of stress-responsive pathways, H₂S exposure alters expression of genes controlled by SKN-1 and HIF-1 signaling. Both pathways are ROS-sensitive and promote adaptation to chronic ROS production (2-4). Their activation, as in egl-9, render these animals insensitive to H₂S-evoked ROS transients (Figure 5B) (Lines 303-305). Conversely, mutants defective in these adaptive pathways, such as hif-1, still show initial locomotory responses to H₂S, but rapidly lose activity during prolonged H₂S exposure (Figure 5D) (Lines 318-319). These observations suggest that HIF-1 pathway is dispensable for initiating the response to H₂ Sevoked ROS transients, but essential for protecting against ROS toxicity.

ASJ neurons

ASJ neurons and DAF-11 signaling are required for H₂S-evoked behavioral responses. However, ASJ does not exhibit an H₂S-evoked calcium transient. It suggests that ASJ neurons do not directly detect H₂S (Line 165-169 and 448-457), but likely modulate the circuit responsive to ROS toxicity. This circuit can also be modulated by ambient O₂ levels, the state of O₂ sensing circuit, and nutrient status, in a manner reminiscent of the CO₂ responses (5, 6). 

O₂ sensing circuit

Consistent with the reviewer’s view, we favor the model that H₂S avoidance is likely induced by ROS transients. We believe that the state of O₂ sensing circuit, similar to ASJ neurons, modulates the neural circuit that is responsive to H₂S-evoked ROS toxicity. This circuit is inhibited as long as O₂ sensing circuit is active. In the RMG optogenetic experiment, channelrhodopsin was photo-stimulated as soon as the assay was initiated at 7% O₂ (Methods Lines 633-634 and Figure legend Lines 1177-1178), therefore RMG remained active throughout the assay including at 7% O₂. Our interpretation is that RMG activation inhibits this ROSresponsive circuit and H₂S avoidance. However, these observations do not resolve if H₂S is acutely and directly perceived. The modulation of H₂S response by O₂ circuit was discussed between Lines 437-447.

References

(1) J. Jia et al., SQR mediates therapeutic effects of H(2)S by targeting mitochondrial electron transport to induce mitochondrial uncoupling. Sci Adv 6, eaaz5752 (2020).

(2) S. J. Lee, A. B. Hwang, C. Kenyon, Inhibition of Respiration Extends C. elegans Life Span via Reactive Oxygen Species that Increase HIF-1 Activity. Current Biology 20, 2131-2136 (2010).

(3) C. Lennicke, H. M. Cocheme, Redox metabolism: ROS as specific molecular regulators of cell signaling and function. Mol Cell 81, 3691-3707 (2021).

(4) D. A. Patten, M. Germain, M. A. Kelly, R. S. Slack, Reactive oxygen species: stuck in the middle of neurodegeneration. J Alzheimers Dis 20 Suppl 2, S357-367 (2010).

(5) A. J. Bretscher, K. E. Busch, M. de Bono, A carbon dioxide avoidance behavior is integrated with responses to ambient oxygen and food in Caenorhabditis elegans. Proc Natl Acad Sci U S A 105, 8044-8049 (2008).

(6) E. A. Hallem, P. W. Sternberg, Acute carbon dioxide avoidance in Caenorhabditis elegans. Proc Natl Acad Sci U S A 105, 8038-8043 (2008).

So basically the constant to evaluate the same maps was that they has the same administrative geography or vertical relationship between places: Provineces / states / departments, counties, cities, villages.

eLife Assessment

This valuable study uses EEG and computational modeling to investigate hemispheric oscillatory asymmetries in unilateral spatial neglect. The work benefits from rare patient data and a careful multimethod approach. However, the evidence is incomplete because key assumptions about alpha‑band entrainment and methodological confounds such as lesion variability and eye‑movement artifacts remain insufficiently addressed.

Reviewer #1 (Public review):

Summary:

Okazaki et al. showed flickering stimuli to patients with unilateral spatial neglect (USN) and measured EEG responses. They compared this with another patient group (post-stroke, but no USN) and healthy controls. The author's rationale was to entrain intrinsic brain rhythms using the flicker of different frequencies (3-30 Hz). Effects found unique to the 9-Hz stimulation condition differentiate USN patients from the other groups, leading them to conclude that USN can be characterized by increased hemispheric alpha asymmetry, driven by a relatively increased response in the intact hemisphere.

Strengths:

This study is principled empirical work that benefits from access to special patient groups of considerable size (about 60 stroke patients in total, and 20 USN). The authors use state-of-the-art established methods to (1) deliver and (2) quantify the responses to the flicker stimulation in the EEG recordings. In addition, they use phase-coupling measures to investigate cross-frequency coupling (here: alpha-gamma) and a measure of directed connectivity between brain areas, transfer entropy. The results are supported by means of simulations using a coupled-oscillators model.

Weaknesses:

In my eyes, the major conceptual weakness of the study is that the authors make the a priori assumption that the flicker stimulation entrains intrinsic brain rhythms, especially alpha (9 Hz). To date, there is no direct (and only equivocal indirect) evidence that alpha rhythms can be entrained with periodic visual stimulation. In the present study, the assumption of alpha entrainment permeates some analytical decisions - where it would be possible to separate stimulus-driven from intrinsic rhythms more strongly than is currently the case, potentially yielding deeper insights into the oscillopathy of USN - and, ultimately, the interpretation of the results. Another potential issue to consider here is the analysis of gamma rhythms in EEG data, absent a control of miniature eye movements, a known problem (Yuval-Greenberg et al., 2008, https://doi.org/10.1016/j.neuron.2008.03.027) that may be exacerbated here, given that USN patients could show different auxiliary gaze behaviour.

Reviewer #2 (Public review):

This study investigates how altered neural oscillations may contribute to unilateral spatial neglect (USN) following right-hemisphere stroke. By combining steady-state visual evoked potentials (SSVEPs), phase-amplitude coupling (PAC), transfer entropy (TE), and computational modeling, the authors aim to show that USN arises from disrupted hemispheric synchronization dynamics rather than simply from lesion extent. The integration of empirical EEG data with a mechanistic model is a major strength and offers a valuable new perspective on how frequency-specific neural dynamics relate to clinical symptoms.

The work has several notable strengths. The combination of experimental and modeling approaches is innovative and powerful, and the findings provide a coherent mechanistic framework linking abnormal neural entrainment to attentional deficits. The study also provides concrete evidence to support the potential for frequency-specific neuromodulatory interventions, which could have translational relevance.

At the same time, there are areas where the evidence could be clarified or contextualized further. The manuscript would benefit from more detailed characterization of lesions, since differences in lesion topography (white vs. gray matter, occipital vs. parietal areas) could greatly improve our understanding of the physiopathology causing unilateral spatial neglect and the altered neural oscillations reported. Methodological choices, such as focusing analyses on occipital electrodes rather than parietal sites, and the potential influence of volume conduction in transfer entropy analyses, also need clearer justification/elaboration. In addition, while the authors report several neural metrics, it is not always clear why SSVEP power was chosen as the primary correlate of clinical severity over other measures. More broadly, the manuscript would be strengthened by clearer definitions of dependent variables and reporting of software and toolboxes used.

Overall, the study makes a significant contribution by demonstrating that USN can be conceptualized as a disorder of disrupted oscillatory dynamics. With some clarifications and expansions, the paper will provide readers with a clearer understanding of both the strengths and the limitations of the evidence, and it will stand as a valuable reference for future work on oscillatory mechanisms in stroke and attention.

Author response:

Reviewer #1 (Public review):

Summary:

Okazaki et al. showed flickering stimuli to patients with unilateral spatial neglect (USN) and measured EEG responses. They compared this with another patient group (post-stroke, but no USN) and healthy controls. The author's rationale was to entrain intrinsic brain rhythms using the flicker of different frequencies (3-30 Hz). Effects found unique to the 9-Hz stimulation condition differentiate USN patients from the other groups, leading them to conclude that USN can be characterized by increased hemispheric alpha asymmetry, driven by a relatively increased response in the intact hemisphere.

Strengths:

This study is principled empirical work that benefits from access to special patient groups of considerable size (about 60 stroke patients in total, and 20 USN). The authors use state-of-the-art established methods to (1) deliver and (2) quantify the responses to the flicker stimulation in the EEG recordings. In addition, they use phase-coupling measures to investigate cross-frequency coupling (here: alpha-gamma) and a measure of directed connectivity between brain areas, transfer entropy. The results are supported by means of simulations using a coupled-oscillators model.

Weaknesses:

In my eyes, the major conceptual weakness of the study is that the authors make the a priori assumption that the flicker stimulation entrains intrinsic brain rhythms, especially alpha (9 Hz). To date, there is no direct (and only equivocal indirect) evidence that alpha rhythms can be entrained with periodic visual stimulation. In the present study, the assumption of alpha entrainment permeates some analytical decisions - where it would be possible to separate stimulus-driven from intrinsic rhythms more strongly than is currently the case, potentially yielding deeper insights into the oscillopathy of USN - and, ultimately, the interpretation of the results. Another potential issue to consider here is the analysis of gamma rhythms in EEG data, absent a control of miniature eye movements, a known problem (Yuval-Greenberg et al., 2008, https://doi.org/10.1016/j.neuron.2008.03.027) that may be exacerbated here, given that USN patients could show different auxiliary gaze behaviour.

Reviewer #1 expressed concern that alpha entrainment is assumed a priori; however, our interpretation is based on the empirical observation of frequency-specific (9 Hz) hemispheric asymmetry, not on a prior assumption. This 9 Hz specificity is difficult to explain by a simple summation of stimulus-evoked responses and is more appropriately interpreted as a resonance phenomenon in the alpha band, which is close to the intrinsic resonance frequency of the visual system [1, 2]. In the revision, we will strengthen the conceptual distinction between stimulus-driven and intrinsic components and clarify that entrainment is a conclusion supported by our data and modeling.

Gamma contamination by eye movements is a valid theoretical concern. However, it is unlikely that saccadic spike potentials explain our α-γ coupling findings, due to several factors including timing constraints and spectral properties. In the revision, we will add explicit discussion of this limitation while explaining why our coupling patterns are more consistent with physiological neural coupling than with artifacts.

Reviewer #2 (Public review):

This study investigates how altered neural oscillations may contribute to unilateral spatial neglect (USN) following right-hemisphere stroke. By combining steady-state visual evoked potentials (SSVEPs), phase-amplitude coupling (PAC), transfer entropy (TE), and computational modeling, the authors aim to show that USN arises from disrupted hemispheric synchronization dynamics rather than simply from lesion extent. The integration of empirical EEG data with a mechanistic model is a major strength and offers a valuable new perspective on how frequency-specific neural dynamics relate to clinical symptoms.

The work has several notable strengths. The combination of experimental and modeling approaches is innovative and powerful, and the findings provide a coherent mechanistic framework linking abnormal neural entrainment to attentional deficits. The study also provides concrete evidence to support the potential for frequency-specific neuromodulatory interventions, which could have translational relevance At the same time, there are areas where the evidence could be clarified or contextualized further. The manuscript would benefit from more detailed characterization of lesions, since differences in lesion topography (white vs. gray matter, occipital vs. parietal areas) could greatly improve our understanding of the physiopathology causing unilateral spatial neglect and the altered neural oscillations reported. Methodological choices, such as focusing analyses on occipital electrodes rather than parietal sites, and the potential influence of volume conduction in transfer entropy analyses, also need clearer justification/elaboration. In addition, while the authors report several neural metrics, it is not always clear why SSVEP power was chosen as the primary correlate of clinical severity over other measures. More broadly, the manuscript would be strengthened by clearer definitions of dependent variables and reporting of software and toolboxes used.

Overall, the study makes a significant contribution by demonstrating that USN can be conceptualized as a disorder of disrupted oscillatory dynamics. With some clarifications and expansions, the paper will provide readers with a clearer understanding of both the strengths and the limitations of the evidence, and it will stand as a valuable reference for future work on oscillatory mechanisms in stroke and attention.

We agree that further lesion characterization would be generally useful. However, as shown in Supplementary Figure 1, lesions in our USN cohort involved both cortical and subcortical regions, and cortical damage often extended into adjacent white matter. Therefore, a strict gray-versus-white-matter classification was not feasible. This anatomical diversity suggests that the frequency-specific hemispheric asymmetry observed here cannot be fully explained by lesion location or size alone, but rather may reflect altered network dynamics following right-hemisphere damage. We will clarify this point in the revised Discussion.

Regarding transfer entropy (TE) and volume conduction, TE is theoretically insensitive to zero-lag correlations and quantifies temporally directed information transfer. Furthermore, we used amplitude envelopes rather than raw oscillations as input, which should greatly reduce the risk of spurious causal estimation due to sinusoidal autocorrelation structure. Moreover, if such spurious connectivity due to autocorrelation had occurred, it would have been expected to appear equally in both feedforward and feedback directions. Therefore, the feedforward-limited (visual→frontal) asymmetry observed in our study cannot be explained by volume conduction or autocorrelation effects. We will maintain this position clearly in the revision.

Regarding other methodological points: we focused on occipital electrodes (O1/O2) because visual stimuli primarily drive the visual system (we also analyzed parietal sites but found no significant hemispheric differences; Figure 4). We chose SSVEP power for clinical correlation because it was the primary phenomenon distinguishing USN from non-USN patients. In the revision, we will clarify these points and include software and toolbox information.

We believe these revisions will substantially strengthen the manuscript and clarify the conceptual and methodological contributions of our study.

References

(1) Rosanova, M., Casali, A., Bellina, V., Resta, F., Mariotti, M., and Massimini, M. (2009). Natural frequencies of human corticothalamic circuits. J Neurosci 29, 7679-7685.

(2) Okazaki, Y.O., Nakagawa, Y., Mizuno, Y., Hanakawa, T., and Kitajo, K. (2021). Frequency- and Area-Specific Phase Entrainment of Intrinsic Cortical Oscillations by Repetitive Transcranial Magnetic Stimulation. Front Hum Neurosci 15, 608947.

No - it begins when he is told by the drunk that he is not his father's son (plastos)? I.e. that opens up a space of questioning, some interval in certainty about parentage, that requires answers...

eLife Assessment

This study presents an important toolkit for visualising the endogenous expression of four classes of neurotransmitter vesicular transporters. Using their toolkit, the authors find that there is co-transmission of neurotransmitters in over 10% of neurons tested. Although the evidence presented in the manuscript is solid, one weakness of this study is the failure of the authors to compare and contrast their results with available single-cell sequencing datasets and with well-established synaptic reporter lines (i.e., co-localization experiments). This toolkit will be of great use to multiple labs, and the authors should indicate their plan to disseminate the reagents and the associated information that is part of this kit.

Reviewer #1 (Public review):

Summary:

This study presents a novel toolkit for visualizing and manipulating neurotransmitter-specific vesicles in C. elegans neurons, addressing the challenge of tracking neurotransmitter dynamics at the level of individual synapses. The authors engineered endogenously tagged vesicular transporters for glutamate, GABA, acetylcholine, and monoamines, enabling cell-specific labeling while maintaining physiological function. Additionally, they developed conditional knockout strains to disrupt neurotransmitter synthesis in single neurons. The study reveals that over 10% of neurons in C. elegans exhibit co-transmission, with a detailed case study on the ADF sensory neuron, where serotonin and acetylcholine are trafficked in distinct vesicle pools. The approach provides a powerful platform for studying neurotransmitter identity, synaptic architecture, and co-transmission.

Strengths:

(1) This toolkit offers a generalizable framework that can be applied to other model organisms, advancing the ability to investigate synaptic plasticity and neural circuit logic with molecular precision.

(2) Through the use of this toolkit, the authors uncover molecular heterogeneity at individual synapses, revealing co-transmission in over 10% of neurons, and offer new insights into neurotransmitter trafficking and synaptic plasticity, advancing our understanding of synaptic organization.

Weaknesses:

(1) While the article introduces valuable tools for visualizing neurotransmitter vesicles in vivo, the core techniques are based on previously established methods. The study does not present significant technological breakthroughs, limiting the novelty of the methodological advancements.

(2) The article does not fully explore the potential implications or the underlying mechanisms governing this process, while the discovery of co-transmission in over 10% of neurons is an intriguing finding. A deeper investigation into the functional uniqueness and interactions of neurotransmitters released from individual co-transmitting neurons - perhaps through case study examples - would strengthen the study's impact.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors developed fluorescent reporters to visualize the subcellular localization of vesicular transporters for glutamate, GABA, acetylcholine, and monoamines in vivo. They also developed cell-specific knockout methods for these vesicular transporters. To my knowledge, this is the first comprehensive toolkit to label and ablate vesicular transporters in C. elegans. They carefully and strategically designed the reporters and clearly explained the rationale behind their construct designs. Meanwhile, they used previously established functional assays to confirm that the reporters are functional. They also tested and confirmed the effect of cell-specific and pan-neuronal knockout of several of these transporters.

Strengths:

The tools developed are versatile: they generated both green and red fluorescent reporters for easy combination with other reporters; they established the method for cell-type-specific KO to analyze the function of the neurotransmitter in different cell types. The reagents allow visualization of specific synapses among other processes and cell bodies. In addition, they also developed a binary expression method to detect co-transmission "We reasoned that if two neurotransmitters were co-expressed in the same neuron, driving Flippase under the promoter of one transmitter would activate the conditional reporter - resulting in fluorescence - only in cells also expressing a second neurotransmitter identity". Overall, this is a versatile and valuable toolkit with well-designed and carefully validated reagents. This toolkit will likely be widely used by the C. elegans community.

Weaknesses:

The authors evaluated the positions of fluorescent puncta by visually comparing their positions with the positions of synapses indicated by EM reconstruction. It would provide stronger supportive evidence if the authors also examined co-localization of these reporters with well-established synaptic reporters previously published by their lab, such as reporters that label presynaptic sites of AIY interneurons.

This toolkit will likely be widely used by the C. elegans community. To facilitate the adoption of the approach and method by worm labs, the authors should include their plan for the dissemination of all of the reagents included in the kit, along with all of the associated information, including construct sequences and the protocols for their use.

Reviewer #3 (Public review):

Summary:

Cuentas-Condori et al. generate cell-specific tools for visualizing the endogenous expression of, as well as knocking out, four different classes of neurotransmitter vesicular transporters (glutamatergic, cholinergic, GABAergic, and monoaminergic) in C. elegans. They then use these tools in an intersectional strategy to provide evidence for the co-expression of these transporters in individual neurons, suggesting co-transmission of the associated neurotransmitters.

Strengths:

A major strength of the work is the generation of several endogenous tools that will be of use to the community. Additionally, this adds to accumulating evidence of co-transmission of different classes of neurotransmitters in the nervous system.

Weaknesses:

A weakness of the study is a lack of comparison to previously published single-cell sequencing data. These tools are alternatively described in the manuscript as superior to the sequencing data and as validation of the sequencing data, but neither claim can be assessed without knowing how they compare and contrast to that data. It is thus not clear to what extent the conclusions of this paper are an advance over what could be determined from the sequencing data on its own. Finally, some technical considerations should be discussed as potential caveats to the robustness of their intersectional strategy for concluding that certain genes are indeed co-expressed. Overall, claims about co-transmission should be tempered by the caveats presented in the discussion, suggesting that co-expression of these transporters is not in and of itself sufficient for neurotransmitter release.

eLife Assessment

This study introduces Megabouts, a transformer-based classifier for larval zebrafish movement bouts. This useful tool is thoughtfully implemented and has clear potential to unify analyses across labs. However, the evidence supporting its robustness is incomplete. How the method generalizes across datasets, how sensitive it is to noise, and the specific sources of misclassification are unclear. The method would also be strengthened by providing options for users to fine-tune the clusters under different experimental conditions, which would further enhance reliability and flexibility.

Reviewer #1 (Public review):

Jouary et al. present Megabouts, a Transformer-based classifier and Python toolbox for automated categorization of zebrafish movement bouts into 13 bout types. This is potentially a very useful tool for the zebrafish community. It is broadly applicable to a wide variety of behavioral paradigms and could help to unify behavioral quantification across labs. The overall implementation is technically sound and thoughtfully engineered. The choice of standard Transformer architecture is well-justified (e.g., it can handle long-term tracking data and process missing data, integrates posture and trajectory information over time, and shows robustness to variable frame rates and partial occlusion). The data augmentation strategies (e.g., downsampling, tail masking, and temporal jitter) are well designed to enhance cross-condition generalization. Thus, I very much support this work.

For the benefit of the end users of this tool, several clarifications and additional analyses would be helpful:

(1) What is the source and nature of the classification errors? The reported accuracy is <80% with trajectory data and still <90% with trajectory + tail data.

(1a) Is this due to model failure (is overfitting a concern? How unbiased were the test sets?), imperfections of the preprocessing step (how sensitive is this to noise in the input data?), or underlying ambiguity in the biological data (e.g., do some "errors" reflect intermediate patterns that don't map neatly onto the 13 discrete classes)?

(1b) A systematic error analysis would be helpful. Which classes are most often confused? Are errors systematic (e.g., slow swims vs. routine turns) or random?

(1c) Can confidence of classification be provided for each bout in the data? How would the authors recommend that the end user deal with misclassifications (e.g., by manual correction)?<br /> Overall, the end user would benefit greatly from more information on potential failure modes and their root causes.

(2) How well does the trained network generalize across labs and setups? To what extent have the authors tested this on datasets from other labs to determine how well the pretrained model transfers across datasets? Having tested the code provided by the authors on a short stretch of x-y zebrafish trajectory data obtained independently, the pipeline generates phantom movement annotations. The underlying cause is unclear.

(2a) One possibility is that preprocessing steps may be highly sensitive to slight noise in the x-y positional data, which leads to noise in the speed data. The neural net, in turn, classifies noise into movement annotations. It would be helpful if the authors could add Gaussian noise to the x-y trajectory data and then determine the extent to which the computational pipeline is robust to noise.

(2b) When testing the pipeline, some stationary periods are classified as movements. Which step of the pipeline gave rise to the issue is unclear. Thus, explicit cross-lab validation and robustness tests (e.g., adding Gaussian noise to trajectories) would strengthen the claims of this paper.

(2c) Lastly, given the potential issue of generalization across labs, it would be helpful to provide/outline the steps for users in different labs to retrain and fine-tune the model.

Reviewer #2 (Public review):

Summary:

Overall, the manuscript is well organized and clearly written. However, in this reviewer's opinion, the manuscript suffers from multiple major weaknesses.

Strengths:

The strengths of the paper are unclear; they have not been articulated well by the authors.

Weaknesses:

The pipeline is designed to analyze larval zebrafish behaviors, which by definition is considered a highly specialized, if not niche, application. Hence, the scope of this manuscript is extremely narrow, and consequently, the overall significance and the broader impact on the field of behavioral neuroscience are rather low. Broadening the scope would significantly improve the manuscript's impact. Second, it was noted that the authors neglect to present an unbiased discussion of how their pipeline compares to well-established and time-proven pipelines used to track larval zebrafish behaviors. This reviewer also failed to detect any new biological insights presented or improvements compared to existing methods, further questioning the overall significance and impact of this manuscript. Finally, the core claim of the manuscript lacks meaningful experimental data that would allow an unbiased and more definitive evaluation of the claims made regarding the Megabouts pipeline. The critical experiment to achieve this would be to run an identical set of behavioral assays (e.g., PPI, social behaviors) on different platforms (e.g., a commercial and a non-commercial one) and then determine if Megabouts correctly analyzes and integrates the results. While this might sound to the authors like an 'outside the scope' experiment, this reviewer would argue that it is the only meaningful experiment to validate the central claim put forward in this manuscript.

Reviewer #3 (Public review):

In this manuscript, the authors introduce Megabouts, a software package designed to standardize the analysis of larval zebrafish locomotion, through clustering the 2D posture time series into canonical behavioral categories. Beyond a first, straightforward segmentation that separates glides from powered movements, Megabouts uses a Transformer neural network to classify the powered movements (bouts). This Transformer network is trained with supervised examples. The authors apply their approach to improve the quantification of sensorimotor transformations and enhance the sensitivity of drug-induced phenotype screening. Megabouts also includes a separate pipeline that employs convolutional sparse coding to analyze the less predictable tail movements in head-restrained fish.

I presume that the software works as the authors intend, and I appreciate the focus on quantitative behavior. My primary concerns reflect an implicit oversimplification of animal behavior. Megabouts is ultimately a clustering technique, categorizing powered locomotion into distinct, labelled states which, while effective for analysis, may confuse the continuous and fluid nature of animal behavior. Certainly, Megabouts could potentially miss or misclassify complex, non-stereotypical movements that do not fit the defined categories. In fact, it appears that exactly this situation led the authors to design a new clustering for head-restrained fish. Can we anticipate even more designs for other behavioral conditions?

Ultimately, I am not yet convinced that Megabouts provides a justifiable picture of behavioral control. And if there was a continuous "control knob", which seems very likely, wouldn't that confuse the clustering process, as many distinct clusters would correspond to, say, different amplitudes of the same control knob?

There has been tremendous recent progress in the measurement and analysis of animal behavior, including both continuous and discrete perspectives. However, the supervised clustering approach described here feels like a throwback to an earlier era. Yes, it's more automatic and quantifiable, and the amount of data is fantastic. But ultimately, the method is conceptually bound to the human eye in conditions where we are already familiar.

eLife Assessment

This valuable work potentially advances our understanding of melody extraction in polyphonic music listening by identifying spontaneous attentional focus in uninstructed listening contexts. However, the evidence supporting the main conclusions is incomplete. The work will be of interest to psychologists and neuroscientists working on music listening, attention, and perception in ecological settings.

Reviewer #1 (Public review):

Summary:

This manuscript investigates the interplay between spontaneous attention and melody formation during polyphonic music listening. The authors use EEG recordings during uninstructed listening to examine how attention bias influences melody processing, employing both behavioural measures and computational modelling with music transformers. The study introduces a very clever pitch-inversion manipulation design to dissociate high-voice superiority from melodic salience, and proposes a "weighted integration" model where attention dynamically modulates how multiple voices are combined into perceived melody.

Strengths:

(1) The attention bias findings (Figure 2) are compelling and methodologically sound, with convergent evidence from both behavioral and neural measures.

(2) The pitch-inversion manipulation appears to super elegantly dissociate two competing factors (high-voice superiority vs melodic salience), moreover, the authors claim that the chosen music lends itself perfectly to his PolyInv condition. A claim I cannot really evaluate, but which would make it even more neat.

(3) Nice bridge between hypotheses and operationalisations.

Weaknesses:



The results in Figure 3 are very striking, but I have a number of questions before I can consider myself convinced. 


(1) Conceptual questions about surprisal analysis:


The pattern of results seems backwards to me. Since the music is inherently polyphonic in PolyOrig, I'd expect the polyphonic model to fit the brain data better - after all, that's what the music actually is. These voices were composed to interact harmonically, so modeling them as independent monophonic streams seems like a misspecification. Why would the brain match this misspecified model better?
<br /> Conversely, it would seem to me the pitch inversion in PolyInv disrupts (at least to some extent) the harmonic coherence, so if anywhere, I'd a priori expect that in this condition, listeners would rather be processing streams separately - making the monophonic model fit better there (or less bad), not in PolyOrig. The current pattern is exactly opposite to what seems logical to me.


(2) Missing computational analyses:


If the transformer is properly trained, it should "understand" (i.e., predict/compress) the polyphonic music better, right? Can the authors demonstrate this via perplexity scores, bits-per-byte, or other prediction metrics, comparing how well each model (polyphonic vs monophonic) handles the music in both conditions? Similarly, if PolyInv truly maintains musical integrity as claimed, the polyphonic model should handle it as well as PolyOrig. But if the inversion does disrupt the music, we should see this reflected in degraded prediction scores. These metrics would validate whether the experimental manipulation works as intended. Also, how strongly are the surprisal streams correlated? There are many non-trivial modelling steps that should be reported in more detail.


(3) Methodological inconsistencies:

Why are the two main questions (Figures 2 and 3) answered with completely different analytical approaches? The switch from TRF to CCA with match-vs-mismatch classification seems unmotivated. I think it's very important to provide a simpler model comparison - just TRF with acoustic features plus either polyphonic or monophonic surprisal - evaluated on relevant electrodes or the full scalp. This would make the results more comparable and interpretable.

(4) Presentation and methods:

a) Coming from outside music/music theory, I found the paper somewhat abstract and hard to parse initially. The experimental logic becomes clearer with reflection, but you're doing yourselves a disservice with the jargon-heavy presentation. It would be useful to include example stimuli.

b) The methods section is extremely brief - no details whatsoever are provided regarding the modelling: What specific music transformer architecture? Which implementation of this "anticipatory music transformer"? Pre-trained on what corpus - monophonic, polyphonic, Western classical only? What constituted "technical issues" for the 9 excluded participants? What were the channel rejection criteria?

Reviewer #2 (Public review):

Summary:

The authors sought to understand the drivers of spontaneous attentional bias and melodic expectation generation during listening to short two-part classical pieces. They measured scalp EEG data in a monophonic condition and trained a model to reconstruct the audio envelope from the EEG. They then used this model to probe which of the two voices was best reflected in the neural signal during two polyphonic conditions. In one condition, the original piece was presented, in the other, the voices were switched in an attempt to distinguish between effects of (a) the pitch range of one voice compared to the other and (b) intrinsic melodic features. They also collected a behavioural measure of attentional bias for a subset of the stimuli in a separate study. Further modelling assessed whether expectations of how the melody would unfold were formed based on an integrated percept of melody across the two voices, or based on a single voice. The authors sought to relate the findings to different theories of how musical/auditory scene analysis occurs, based on divided attention, figure-ground perception, and stream integration.

Strengths:

(1) A clever but simple manipulation - transposing the voices such that the higher one became the lower one - allowed an assessment of different factors that might affect the allocation of attention.

(2) State-of-the-art analytic techniques were applied to (a) build a music attention decoder (these are more commonly encountered for speech) and (b) relate the neural data to features of the stimulus at the level of acoustics and expectation.

(3) The effects appeared robust across the group, not driven by a handful of participants.

Weaknesses:

(1) A key goal of the work is to establish the relative importance for the listener's attention of a voice's (a) mean pitch in the context of the two voices (high-voice superiority) and (b) intrinsic melodic statistics/motif attractiveness. The rationale of the experimental manipulation is that switching the relative height of the lines allows these to be dissociated by imparting the same high-voice benefit to the new high-voice and the same preferred intrinsic melodic statistics to the new low voice. However, previous work suggests that the high-voice superiority effect is not all-or-nothing. Electrophysiology supported by auditory nerve modelling found it to depend on the degree of voice separation in a non-monotonic way (see https://doi.org/10.1016/j.heares.2013.07.014 at p. 68). Although the authors keep the overall pitch of the lower (and upper) line fixed across conditions, systematically different contour patterns across the voices could give rise to a sub-optimal distribution of separations in the PolyInv versus PolyOrig condition. This could weaken the high-voice superiority effect in PolyInv and explain the pattern of results. One could argue that such contour differences are examples of the "intrinsic melodic statistics" put forward as the effect working in opposition to high-voice superiority, but it is their interaction across voices that matters here.

(2) Although melody statistics are mentioned throughout, none have been calculated. It would be helpful to see the features that presumably lead to "motif attractiveness" quantified, as well as how they differ across lines. The work of David Huron, such as at https://dl.acm.org/doi/abs/10.1145/3469013.3469016, provides examples that could be calculated with ease and compared across the two lines: "the tendency for small over large pitch movements, for large leaps to ascend, for musical phrases to fall in pitch, and for phrases to begin with an initial pitch rise". The authors also mention differences in ornamentation. Such comparisons would make it more tangible for the reader as to what differs across the original "melody" and "support" line. In particular, as the authors themselves note, lines in double-counterpoint pieces can, to a degree, operate interchangeably. Bach's inventions in particular use a lot of direct repetition (up to octave invariance), which one would expect to minimise differences in the statistics mentioned. The references purporting to relate to melodic statistics (11-14 in original numbering) seem rather to relate to high-voice superiority.

(3) The exact nature of the transposition manipulation is obscured by a confusing Figure 1B, which shows an example in which the transposed line does not keep the same note-to-note interval structure as the original line.

(4) The transformer model is barely described in the main text. Even readers who are familiar with the Hidden Markov Models (e.g., in IDyOM) previously used by some of the authors to model melodic surprise and entropy would benefit from a brief description in the main text at least of how transformer models are different. The Methods section goes a little further but does not mention what the training set was, nor the relative weight given to long- and short-term memory models.

(5) The match-mismatch procedure should be explained in enough detail for readers to at least understand what value represents chance performance and why performance would be measured as an average over participants. Relatedly, there is no description at all of CCA or the match-mismatch procedure in the Methods.

(6) Details of how the integration model was implemented will be critical to interpreting the results relating to melodic expectations. It is not clear how "a single melody combining the two streams" was modelled, given that at least some notes presumably overlapped in time.

(7) The authors propose a weighted integration model, referring in the Discussion to dynamics and an integration rate. They do show that in the PolyOrig case, the top stream bias is highest and the monophonic model gives the best prediction, while in the PolyInv case, the top stream bias is weaker and the polyphonic model provides the best prediction. However, that doesn't seem to say anything about the temporal rate of integration, just the degree, which could be fixed over the whole stimulus. Relatedly, the terms "strong attention bias" and "weak attention bias" in Highlight 4 might give the impression of different attention modes for a given listener, or perhaps different types of listeners, but this seems to be shorthand for how attention is allocated for different types of stimuli (namely those that have or have not had their voices reversed).

(8) Another aspect of the presentation relating to temporal dynamics is that in places (e.g., Highlight 1), the authors suggest they are tracking attention dynamically. However, as acknowledged in the Discussion, neither the behavioural nor neural measure of attentional bias are temporally resolved. The measures indicate that on average participants attend more to the higher line (less so when it formed the lower line in the original composition).

(9) It is not clear whether the sung-back data were analysed (and if not why participants were asked to sing the melody back rather than just listen to the two components and report which they thought was the melody). It is also not stated whether the order in which the high and low voices were played back was randomised. If not, response biases or memory capacity might have affected the behavioural attention data.

Reviewer #3 (Public review):

Summary:

In this paper, Winchester and colleagues investigated melodic perception in natural music listening. They highlight the central role of attentional processes in identifying one particular stream in polyphonic material, and propose to compare several theoretical accounts, namely (1) divided attention, (2) figure-ground separation, and (3) stream integration. In parallel, the authors compare the relative strength of exogenous attentional effects (i.e., salience) produced by two common traits of melodies: high-pitch (compared to other voices), and attractive statistics. To ensure the generalisability of their results to real-life listening contexts, they developed a new uninstructed listening paradigm in which participants can freely attend to any part of a musical stimulus.

Major strengths and weaknesses of the methods and results:

(1) Winchester and colleagues capitalized on previous attention decoding techniques and proposed an uninstructed listening paradigm. This is an important innovation for the study of music perception in ecological settings, and it is used here to investigate the spontaneous attentional focus during listening. The EEG decoding results obtained are coherent with the behavioral data, suggesting that the paradigm is robust and relevant.

(2) The authors first evaluate the relative importance of high-pitch and statistics in producing an attentional bias (Figure 2). Behavioral results show a clear pattern, in which both effects are present, with a dominance of the high-pitch one. The only weakness inherent to this protocol is that behavioral responses are measured based on a second presentation of short samples, which may induce a different attentional focus than in the first uninstructed listening.

(3) Then, the analyses of EEG data compare the decoding results of each melody (the high or low voice, and with "richer" or "poorer" statistics), and show a similar pattern of results. However, this report leaves open the possibility of a confounding factor. In this analysis, a TRF decoding model is first trained based on the presentation of monophonic samples, and it is later used to decode the envelope of the corresponding melodies in the polyphonic scenario. The fitting scores of the training phase are not reported. If the high-pitch or richer melodies were to produce higher decoding scores during monophonic listening (due to properties of the physiological response, or to perceptual processes), a similar difference could be expected during polyphonic listening. To capture attentional biases specifically, the decoding scores in the polyphonic conditions should be compared to the scores in the monophonic conditions, and attention could be expected to increase the decoding of the attended stream or decrease the unattended one.

(4) Then, Winchester and colleagues investigate the processing of melodic information by evaluating the encoding of melodic surprise and uncertainty (Figure 3). They compare the surprise and uncertainty estimated from a monophonic or a polyphonic model (Anticipatory Music Transformer), and analyse the data with a CCA analysis. The results show a double dissociation, where the processing of melodies with a strong attentional bias (high-pitch, rich statistics) is better approximated with a monophonic model, while a polyphonic model better classifies the other melodies. While this global result is compelling, it remains a preliminary and intriguing finding, and the manuscript does not further investigate it. As it stands, the result appears more like a starting point for further exploration than a definitive finding that can support strong theoretical claims. First, it could be complemented by a comparison of the encoding of individual melodies (e.g., AMmono high-voice vs AMmono low-voice, in PolyOrig and PolyInv conditions) to highlight a more direct correspondence with the previous results (Figure 2) and allow a more precise interpretation. Second, additional analyses or experiments would be needed to unpack this result and provide greater explanatory power. Additionally, the CCA analysis is not described in the method. The statistical testing conducted on this analysis seems to be performed across the 250 repetitions of the evaluation rather than across the 40 participants, which may bias the resulting p-values. Moreover, the choice and working principle of the Anticipatory Music Transformer are not described in the method. Overall, these results seem at first glance solid, but the missing parts of the method do not allow for full evaluation or replication of them.

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

(1) Winchester and colleagues aimed at identifying the melodic stream that attracts attention during the listening of natural polyphonic music, and the underlying attentional processes. Their behavioral results confirm that high-pitched and attractive statistics increase melodic salience with a greater effect size of the former, as stated in the discussion. The TRF analyses of EEG data seem to show a similar pattern, but could also be explained by confounding factors. Next, the authors interpret the CCA results as the results of stream segregation when there is a high melodic salience, and stream integration when there are weaker attentional biases. These interpretations seem to be supported by the data, but unfortunately, no additional analyses or experiments have been conducted to further evaluate this hypothesis. The authors also acknowledge that their results do not show whether stream segregation occurs via divided attention or figure-ground separation. However, the lack of information about the music model used (Anticipatory Music Model) and the way it was set up raises some questions about its relevance and limits as a model of cognition (e.g. Is this transformer a "better" model of the listeners' expectations than the well-established IDyOM model, and why ?), and about the validity of those results.

(2) Overall, the authors achieved most of the aims presented in the introduction, although they couldn't give a more precise account of the attentional processes at stake. The interpretations are sound and not overstated, with the exception of potential confounding factors that could compromise the conclusions on the neural tracking of salient melodies (EEG results, Figure 2).

Impact of the work on the field, and the utility of the methods and data to the community:

The new uninstructed listening paradigm introduced in this paper will likely have an important impact on psychologists and neuroscientists working on music perception and auditory attention, enabling them to conduct experiments in more ecological settings. While the attentional biases towards melodies with high-pitch and attractive statistics are already known, showing their relative effect is an important step in building precise models of auditory attention, and allows future paradigms to explore more fine-grained effects. Finally, the stream segregation and integration shown with this paradigm could be important for researchers working on music perception. Future work may be necessary to identify the models (Markov chains, deep learning) and setup (data analysis, stimuli, control variables) that do or do not replicate these results.

eLife Assessment

This study provides an important contribution by showing that whiteflies and planthoppers use salivary effectors to suppress plant immunity through the receptor-like protein RLP4, suggesting convergent evolution in these insect lineages. The topic is of clear interest for understanding plant-insect interactions and offers ideas that could stimulate further research in the field. However, the strength of evidence is incomplete, as some aspects of the data and experimental design limit the extent to which the main claims are fully supported.

Reviewer #1 (Public review):

Summary:

This is a well-structured and interesting manuscript that investigates how herbivorous insects, specifically whiteflies and planthoppers, utilize salivary effectors to overcome plant immunity by targeting the RLP4 receptor.

Strengths:

The authors present a strong case for the independent evolution of these effectors and provide compelling evidence for their functional roles.

Weaknesses:

Western blot evidence for effector secretion is weak. The possibility of contamination from insect tissues during the sample preparation should be avoided.

Below are some specific comments and suggestions to strengthen the manuscript.

(1) Western blot evidence for effector secretion:

The western blot evidence in Figure 1, which aims to show that the insect protein is secreted into plants, is not fully convincing. The band of the expected size (~30 kDa) in the infested tissues is very weak. Furthermore, the high and low molecular weight bands that appear in the infested tissues do not match the size of the protein in the insects themselves, and a high molecular weight band also appears in the uninfested control tissues. It is difficult to draw a definitive conclusion that this protein is secreted into the plants based on this evidence. The authors should also address the possibility of contamination from insect tissues during the sample preparation and explain how they have excluded this possibility.

(2) Inconsistent conclusion (Line 156 and Figure 3c): T

The statement in line 156 is inconsistent with the data presented in Figure 3c. The figure clearly shows that the LRR domain of the protein is the one responsible for the interaction with BtRDP, not the region mentioned in the text. This is a critical misrepresentation of the experimental findings and must be corrected. The conclusion in the text should accurately reflect the data from the figure.

(3) Role of SOBIR1 in the RLP4/SOBIR1 Complex:

The authors demonstrate that the salivary effectors destabilize the RLP4 receptor, leading to a decrease in its protein levels and a reduction in the RLP4/SOBIR1 complex. A key question remains regarding the fate of SOBIR1 within this complex. The authors should clarify what happens to the SOBIR1 protein after the destabilization of RLP4. Does SOBIR1 become unbound, targeted for degradation itself, or does it simply lose its function without RLP4? This would provide further insight into the mechanism of action of the effectors.

(4) Clarification on specificity and evolutionary claims:

The paper's most significant claim is that the effectors from both whiteflies and planthoppers "independently evolved" to target RLP4. While the functional data is compelling, this evolutionary claim would be more convincing with stronger evidence. Showing that two different effector proteins target the same host protein is a fascinating finding but without a robust phylogenetic analysis, the claim of independent evolution is not fully supported. It would be valuable to provide a more detailed evolutionary analysis, such as a phylogenetic tree of the effector proteins, showing their relationship to other known insect proteins, to definitively rule out a shared, but highly divergent, common ancestor.

(5) Role of SOBIR1 in the interaction:

The results suggest that the effectors disrupt the RLP4/SOBIR1 complex. It is not entirely clear if the effectors are specifically targeting RLP4, SOBIR1, or both. Further experiments, such as a co-immunoprecipitation assay with just RLP4 and the effector, could clarify if the effector can bind to RLP4 in the absence of SOBIR1. This would help to definitively place RLP4 as the primary target.

(6) Transcriptome analysis (Lines 130-143):

The transcriptome analysis section feels disconnected from the rest of the manuscript. The findings, or lack thereof, from this analysis do not seem to be directly linked to the other major conclusions of the paper. This section could be removed to improve the manuscript's overall focus and flow. If the authors believe this data is critical, they should more clearly and explicitly connect the conclusions of the transcriptome analysis to the core findings about the effector-RLP4 interaction.

(7) Signal peptide experiments (Lines 145 and beyond):

The experiments conducted with the signal peptide (SP) are questionable. The SP is typically cleaved before the protein reaches its final destination. As such, conducting experiments with the SP attached to the protein may have produced biased observations and could lead to unjustified conclusions about the protein's function within the plant cell. We suggest the authors remove the experiments that include the signal peptide.

(8) Overly strong conclusion and unclear evidence (Line 176):

The use of the word "must" on line 176 is very strong and presents a definitive conclusion without sufficient evidence. The authors state that the proteins must interact with SOBIR1, but they do not provide a clear justification for this claim. Is SOBIR1 the only interaction partner for NtRLP4? The authors should provide a specific reason for focusing on SOBIR1 instead of demonstrating an interaction with NtRLP4 first. Additionally, do BtRDP or NlSP694 also interact with SOBIR1 directly? The authors should either tone down their language to reflect the evidence or provide a clearer justification for this strong claim.

Reviewer #2 (Public review):

Summary:

The authors tested an interesting hypothesis that white flies and planthoppers independently evolved salivary proteins to dampen plant immunity by targeting a receptor-like protein.

Strengths:

The authors used a wide range of methods to dissect the function of the white fly protein BtRDP and identify its host target NtRLP4.

Weaknesses:

(1) Serious concerns about protein work.

I did not find the indicated protein bands for anti-BtRDP in Figures 1a and 1b in the original blot pictures shown in Figure S30. In Figure 1a, I can't get the point of showing an unspecific protein band with a size of ~190 kD as a loading control for a protein of ~ 30 kD.

The data discrepancy led me to check other Western blot pictures. Similarly, Figures 2d, 3b, 3d, and S15b (anti-Myc) do not correspond to the original blots shown. In addition, the anti-Myc blot in Figure 4i, all blot pictures in Figures 5b, 5h, and S19a appeared to be compressed vertically. These data raised concerns about the quality of the manuscript.

Blots shown in Figure 3d, 4f, 4g, and 4h appeared to be done at a different exposure rate compared to the complete blot shown in Figure S30. The undesirable connection between Western blot pictures shown in the figures and the original data might be due to the reduced quality of compressed figures during submission. Nevertheless, clarification will be necessary to support the strength of the data provided.

(2) Misinterpretation of data.

I am afraid the authors misunderstood pattern-triggered immunity through receptor-like proteins. It is true that several LRR-type RLPs constitutively associate with SOBIR1, and further recruit BAK1 or other SERKs upon ligand binding. One should not take it for granted that every RLP works this way. To test the hypothesis that NtRLP4 confers resistance to B.tabaci infestation, the author compared transcriptional profiles between an EV plant line and an RLP4 overexpression line. If I understood the methods and figure legends correctly, this was done without B. tabaci treatment. This experimental design is seriously flawed. To provide convincing genetic evidence, independent mutant lines (optionally independent overexpression lines) in combination with different treatments will be necessary. Otherwise, one can only conclude that overexpressing the RLP4 protein generated a nervous plant. In addition, ROS burst, but not H2O2 accumulation, is a common immune response in pattern-triggered immunity.

(3) Lack of logic coherence.

The written language needs substantial improvement. This impeded the readability of the work. More importantly, the logic throughout the manuscript appeared scattered. The choice of testing protein domains for protein-protein interactions, using plants overexpressing an insect protein to study its subcellular localization, switching back and forth between using proteins with signal peptides and without signal peptides, among others, lacks a clear explanation.

Reviewer #3 (Public review):

Summary:

In this study, Wang et al. investigate how herbivorous insects overcome plant receptor-mediated immunity by targeting plant receptor-like proteins. The authors identify two independently evolved salivary effectors, BtRDP in whiteflies and NlSP694 in brown planthoppers, that promote the degradation of plant RLP4 through the ubiquitin-dependent proteasome pathway. NtRLP4 from tobacco and OsRLP4 from rice are shown to confer resistance against herbivores by activating defense signaling, while BtRDP and NlSP694 suppress these defenses by destabilizing RLP4 proteins.

Strengths:

This work highlights a convergent evolutionary strategy in distinct insect lineages and advances our understanding of insect-plant coevolution at the molecular level.

Weaknesses:

(1) I found the naming of BtRDP and NlSP694 somewhat confusing. The authors defined BtRDP as "B. tabaci RLP-degrading protein," whereas NlSP694 appears to have been named after the last three digits of its GenBank accession number (MF278694, presumably). Is there a standard convention for naming newly identified proteins, for example, based on functional motifs or sequence characteristics? As it stands, the inconsistency makes it difficult for readers to clearly distinguish these proteins from those reported in other studies.

(2) Figure 2 and other figures. Transgenic experiments require at least two independent lines, because results from a single line may be confounded by position effects or unintended genomic alterations, and multiple lines provide stronger evidence for reproducibility and reliability.

(3) Figure 3e. Quantitative analysis of NtRLP4 was required. Additionally, since only one band was observed in oeRLP, were any tags included in the construct?

(4) Figure 4a. The RNAi effect appears to be well rescued in Line 1 but poorly in Line 2. Could the authors clarify the reason for this difference?

(5) ROS accumulation is shown for only a single leaf. A quantitative analysis of ROS accumulation across multiple samples would be necessary to support the conclusion. The same applies to Figure 16f.

(6) Figure 4f: NtRLP4 abundance was significantly reduced in oeBtRDP plants but not in oeBtRDP-SP. Although coexpression analysis suggests that BtRDP promotes NtRLP4 degradation in an ubiquitin-dependent manner, the reduced NtRLP4 levels may not result from a direct interaction between BtRDP and NtRLP4. It is possible that BtRDP influences other factors that indirectly affect NtRLP4 abundance. The authors should discuss this possibility.

(7) The statement in lines 335-336 that 'Overexpression of NtRLP4 or NtSOBIR1 enhances insect feeding, while silencing of either gene exerts the opposite effect' is not supported by the results shown in Figures S16-S19. The authors should revise this description to accurately reflect the data.

(8) BtRDP is reported to attach to the salivary sheath. Does the planthopper NlSP694 exhibit a similar secretion localization (e.g., attachment to the salivary sheath)? The authors should supplement this information or discuss the potential implications of any differences in secretion localization between BtRDP and NlSP694 for their respective modes of action.

eLife Assessment

This manuscript provides a valuable contribution by identifying stress-responsive neurons in the supramammillary nucleus and their ventral subiculum inputs and assessing the regulation of anxiety-related behaviors. The evidence is convincing that the supramammillary nucleus contains stress-responsive neurons, and activation of these neurons increases anxiety-like behaviors. However, evidence that the ventral subiculum input to the supramammillary nucleus encodes and regulates anxiety and that the supramammillary nucleus generates an anxiety engram is incomplete. This work has the potential to offer new insights into how distinct circuits encode different emotional states and will be of interest to those interested in brain systems of aversive emotional and behavioral states.

Reviewer #1 (Public review):

A summary of what the authors were trying to achieve:

Zhang et al. examine connections between supramammillary (SuM) neurons and the subiculum in the context of stress-induced anxiety-like behaviors. They identify stress-activated neurons (SANs) in the SuM using Fos2A-iCreERT2 TRAP mice and show that reactivation of SANs increases anxiety-like behavior and corticosterone levels. Circuit mapping reveals inputs from glutamatergic neurons in both ventral and dorsal subiculum (Sub) to SANs. vSub neurons showing calcium dynamics correlated with open-arm exploration in the elevated zero maze (EZM), which is interpreted to indicate a link to e. Finally, chronic inhibition of vSub→SuM neurons during chronic social defeat stress (CSDS) reduces anxiety-like behaviors.

An account of the major strengths and weaknesses of the methods and results:

Strengths:

The manuscript provides compelling evidence for monosynaptic connections from the subiculum to SuM neurons activated by stress. Demonstrating that SuM neuronal activity is altered after CSDS is of particular interest, potentially linking SuM circuits to stress-related psychiatric disorders. The TRAP approach highlights a stress-responsive population of neurons, and reactivation studies suggest behavioral relevance. Together, these data contribute to an emerging literature implicating SuM in stress and anxiety regulation.

Weaknesses

As presented, the manuscript has limitations that weaken support for the central conclusions drawn by the authors. Many of the findings align with prior work on this topic, but do not extend those findings substantially.<br /> An overarching limitation is the lack of temporal resolution in the manipulations relative to the behavioral assays. This is particularly important for anxiety-like behaviors, as antecedent exposures can alter performance. In the open field and elevated zero maze assays, testing occurred 30 minutes after CNO injection. During much of this interval, the targeted neurons were likely active, making it difficult to determine whether observed behavioral changes were primary - resulting directly from SuM neuronal activity - or secondary, reflecting a stress-like state induced by prolonged activation of SuM and related circuits. This concern also applies to the chronic inhibition of ventral subiculum (vSub) neurons during 10 days of CSDS.

The combination of stressors (foot shock and CSDS) and behavioral assays further complicates interpretation. The precise role of SuM neurons, including SANs, remains unclear. Both vSub and dSub neurons responded to foot shock, but only vSub neurons showed activity differences associated with open-arm transitions in the EZM.

In light of prior studies linking SuM to locomotion (Farrell et al., Science 2021; Escobedo et al., eLife 2024), the absence of analyses connecting subpopulations to locomotor changes weakens the claim that vSub neurons selectively encode anxiety. Because open- and closed-arm transitions are inherently tied to locomotor activity, locomotion must be carefully controlled to avoid confounding interpretations.

Another limitation is the narrow behavioral scope. Beyond open field and EZM, no additional assays were used to assess how SAN reactivation affects other behaviors. Without richer behavioral analyses, interpretations about fear engrams, freezing, or broader stress-related functions of SuM remain incomplete.

In addition, small n values across several datasets reduce confidence in the strength of the conclusions.

Figure level concerns:

(1) Figure 1: In Figure 1, the acute recruitment of SuM neurons by for shock is paired with changes in neural activity induced by social defeat stress. Although interesting, the connections of changes induced by a chronic stressor to Fos induction following acute foot shock are unclear and do not establish a baseline for the studies in Figure 3 on activation of SANs by social stressors.

(2) Figure 2: The chemogenetic experiments using AAV-hSyn-Gq-DREADDs lack data or images, or hit maps showing viral spread across animals. This omission is critical given the small size of SuM, where viral spread directly determines which neurons are manipulated. Without this, it is difficult to interpret findings in the context of prior studies on SuM circuits involved in threats and rewards.

(3) Figure 3: The TRAP experiments show that the number of labeled neurons following foot shock (Figure 3F) is approximately double that of baseline home-cage animals, though y-axis scaling complicates interpretation. It is unclear whether this reflects true Fos induction, low TRAP efficiency, or baseline recombination. Overlap analyses are also limited. For example, it is not shown what proportion of foot shock SANs are reactivated by subsequent foot shock. Comparisons of Fos induction after sucrose reward are also weakened by the very low Fos signal observed. If sucrose reward does not robustly induce Fos in SuM, its utility in distinguishing reward- versus stress-activated neurons is questionable. Thus, conclusions about overlap between SANs and socially stressed neurons remain uncertain due to the missing quantification of Fos+ populations.

(4) Supplemental Figure 3: The claim that "SANs in the SuM encode anxiety but not fear memory" is not well supported. Inhibition of SANs (Gi-DREADDs) did not alter freezing behavior, but the absence of change could reflect technical issues (e.g., insufficient TRAP efficiency, low expression of Gi-DREADDs). Moreover, the manuscript does not provide a positive control showing that SuM SANs inhibition alters anxiety-like behavior, making it difficult to interpret the negative result. Prior work (Escobedo et al., eLife 2024) suggests SuM neurons drive active responses, not freezing, raising further interpretive questions.

(5) Figure 4: The statement that corticosterone concentration is "usually used to estimate whether an individual is anxious" (line 236) is an overstatement. Corticosterone fluctuates dynamically across the day and responds to a broad range of stimuli beyond anxiety.

(6) Figures 5-6: The conclusion that vSub neurons encode anxiety-like behavior is not firmly supported. Data from photo-activating terminals in SuM is shown for ex vivo recording, but not in vivo behavior, which would strengthen support for this conclusion. Both vSub and dSub neurons responded to foot shock. The key evidence comes from apparent differential recruitment during open-arm exploration. However, the timing appears to lag arm entry, no data are provided for closed-arm entry, and there is heterogeneity across animals. These limitations reduce confidence in the authors' central claim regarding vSub-specific encoding of anxiety.

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

(1) From the data presented, the authors conclude that "the SuM is the critical brain region that regulates anxiety" (line 190). This interpretation appears overstated, as it downplays well-established contributions of other brain regions and does not place SuM's role within a broader network context. The data support that SuM neurons are recruited by foot shock and, to a lesser extent, by acute social stress. However, the alterations in activity of SuM subpopulations following chronic stress reported in Figure 1 remain largely unexplored, limiting insight into their functional relevance.

(2) The limited temporal resolution of DREADD-based manipulations leaves alternative explanations untested. For example, if SANs encode signals of threat, generalized stress, or nociception, then prolonged activation could indirectly alter behavior in the open field and EZM assays, rather than reflecting direct anxiety regulation.

(3) The conclusion that "SuM store information about stress but not memory" (line 240) is not fully supported, particularly with respect to possible roles in memory. The lack of a role in memory of events, as opposed to the output of threat or stress memory, may be true, but is functionally untested in presented experiments. The data do indicate activation of the SuM neuron by foot shock, which has been previously reported(Escobedo et al eLife 2024). The changes in SuM activity following chronic stress (Figure 1) are intriguing, but their relationship to "stress information storage" is not clearly established.

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

The reported results align with prior studies on SuM and Sub areas' roles in stress in anxiety. There are limitations due to narrowly focused behavioral assays and the limited temporal resolution of the tools used. Overall, the study further supports a role for SuM in threat and stress responses. The reported changes in SuM neuron activity following chronic stress may offer new insights into stress-induced disorders and behavioral changes.

Reviewer #2 (Public review):

This manuscript investigates the neural mechanisms of anxiety and identifies the supramammillary nucleus (SuM) as a critical hub in mediating anxiety-related behaviors. The authors describe a population of neurons in the SuM that are activated by acute and chronic stress. While their activity is not required for fear memory recall, reactivation of these neurons after chronic stress robustly increases anxiety-like behaviors as well as physiological stress markers. Circuit analysis further shows that these stress-activated neurons are driven by inputs from the ventral, but not dorsal, subiculum, and inhibition of this pathway exerts an anxiolytic effect.

The study provides an elegant integration of techniques to link stress, neuronal ensembles, and circuit function, thereby advancing our understanding of the neural substrates of anxiety. A particularly notable point is the selective role of these stress-activated neurons in anxiety, but not in associative fear memory, which highlights functional distinctions between neural circuits underlying anxiety and fear.

Some aspects would benefit from clarification. For example, how selective is the recruitment of this population to stress compared with other aversive states, and how should one best interpret their definition as "stress-activated neurons" given the relatively modest overlap across stress exposures? In addition, the use of the term "engram" in this context raises conceptual questions. Is it appropriate to describe a neuronal ensemble encoding an emotional state as an engram, a term usually tied to specific memory recall?

Overall, this work makes a valuable contribution by identifying SuM stress-activated neurons and their ventral subiculum inputs as central elements of the circuitry underlying anxiety. These findings provide a valuable framework for future studies investigating anxiety circuitry and may inform the development of targeted interventions for stress-related disorders.

Reviewer #3 (Public review):

Summary:

The authors aim to investigate the mechanisms of anxiety. The paper focuses on the supramammillary nucleus (SuM) based on a fos screen and recordings showing that footshock and social defeat stress increase activity in this region. Using activity-dependent tagging, they show that reactivation of stress-activated neurons in SuM has an anxiety-like effect, reducing open-arm exploration in the elevated zero task. They then investigate the ventral subiculum as a potential source of anxiety-related information for SuM. They show that ventral subiculum (vSub) inputs to SuM are more strongly activated than dSub when mice explore the open arms of the elevated zero. Finally, they show that DREADD-mediated inhibition of vSub-SuM projections alleviates stress-enhanced anxiety. Overall, the results provide good evidence that SuM contains a stress-activated neuronal population whose later activity increases anxiety-like behavior. It further provides evidence that vSub projects to SuM are activated by stress, and their inhibition alleviates some effects of stress.

Strengths:

Strengths of this paper include the use of convergent methods (e.g., fos plus electrode recordings, footshock, and social defeat) to demonstrate that the SuM is activated by different forms of stress. The activity-dependent tagging experiment shows that footshock-activated SuM neurons are reactivated by social defeat but not by sucrose is also compelling because it provides evidence that SuM neurons are driven by some integrative aspect of stress rather than by a simple sensory stimulus.

Weaknesses:

The strength of some of the evidence is judged to be incomplete. The paper provides good evidence that SuM contains stress-responsive neurons, and the activity of these neurons increases some measure of anxiety-like behavior. However, the evidence that the vSub-SuM projection "encodes anxiety" and that the SuM is a key regulator of anxiety is judged to be incomplete. The claim that SuM generates an "anxiety engram" is also judged to be incompletely supported by the evidence. Namely, what is unclear is whether these cells/regions encode anxiety per se versus modulate behaviors (like exploration) that tend to correlate with anxiety. Since many brain regions respond to footshock and other stressors, the response of SuM to these stimuli is not strong evidence for a role in anxiety. I am not convinced that the identified SuM cells have a specific anxiety function. As the authors mention in the introduction, SuM regulates exploration and theta activity. Since theta potently regulates hippocampal function, there is the concern that SuM manipulations could have broad effects. As shown in Supplementary Figure 2, stimulating stress-responsive cells in SuM potently reduces general locomotor exploration. This raises concerns that the manipulation could have broader effects that go beyond just changes in anxiety-like behavior. Furthermore, the meaning of an "anxiety engram" is unclear. Would this engram encode stress, the sense of a potential threat, or the behavioral response? A more developed analysis of the behavioral correlates of SuM activity and the behavioral effects of SuM manipulations could give insight into these questions.

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eLife Assessment

This valuable study characterises receptors for calcitonin-related peptides from a deuterostomian animal, the echinoderm Apostichopus japonicus, by a combination of heterologous expression, pharmacological experiments, and the quantification of gene-expression levels. The authors provide convincing evidence for a functional calcitonin-related peptide system in the sea cucumber, but further work will be needed to confirm the proposed physiological functions of PDF receptor system in this species. This work should be of interest to scientists studying the signaling pathways, functions, and evolution of neuropeptides, and could be of relevance to improving the culture conditions of this economically key species.

Reviewer #1 (Public review):

Summary:

The manuscript characterizes a functional peptidergic system in the echinoderm Apostichopus japonicus that is related to the widely conserved family of calcitonin/diuretic hormone 31 (CT/DH31) peptides in bilaterian animals. In vitro analysis of receptor-ligand interactions, using multiple receptor activation assays, identifies three cognate receptors for two CT-like peptides in the sea cucumber, which stimulate cAMP, calcium, and ERK signaling. Only one of these receptors clusters within the family of calcitonin and calcitonin-like receptors (CTR/CLR) in bilaterian animals, whereas two other receptors cluster with invertebrate pigment dispersing factor receptors (PDFRs). In addition, this study sheds light on the expression and in vivo functions of CT-like peptides in A. japonicus, by quantitative real-time PCR, immunohistochemistry, pharmacological experiments on body wall muscle and intestine preparations, and peptide injection and RNAi knockdown experiments. This reveals a conserved function of CT-like peptides as muscle relaxants and growth regulators in A. japonicus.

Strengths:

This work combines both in vitro and in vivo functional assays to identify a CT-like peptidergic system in an economically relevant echinoderm species, the sea cucumber A. japonicus. A major strength of the study is that it identifies three G protein-coupled receptors for AjCT-like peptides, one related to the CTR/CLR family and two related to the PDFR family. A similar finding was previously reported for the CT-related peptide DH31 in Drosophila melanogaster that activates both CT-type and PDF-type receptors. Here, the authors expand this observation to a deuterostomian animal, which suggests that receptor promiscuity is a more general feature of the CT/DH31 peptide family and that CT/DH31-like peptides may activate both CT-type and PDF-type receptors in other animals as well.

Besides the identification of receptor-ligand pairs, the downstream signaling pathways of AjCT receptors have been characterized, revealing broad and in some cases receptor-specific effects on cAMP, calcium, and ERK signaling.

Functional characterization of the CT-related peptide system in heterologous cells is complemented with ex vivo and in vivo experiments. First, peptide injection and RNAi knockdown experiments establish transcriptional regulation of all three identified receptors in response to changing AjCT peptide levels. Second, ex vivo experiments reveal a conserved role for the two CT-like peptides as muscle relaxants, which have differential effects on body wall muscle and intestine preparations. Finally, peptide injection and knockdown experiments uncover a growth-promoting role for one CT-like peptide (AjCT2). Injection of AjCT2 at high concentration, or long-term knockdown of the AjCT precursor, affects diverse growth-related parameters including weight gain rate, specific growth rate, and transcript levels of growth-regulating transcription factors. The authors also reveal a growth-promoting function for the PDFR-like receptor AjPDFR2, suggesting that this receptor mediates the effects of AjCT2 on growth.

Weaknesses:

Expression of CT-like peptides was investigated both at transcript and protein level, but insight into the expression of the three peptide receptors is limited. This makes it difficult to understand the mechanism underlying the (different) functions of the two CT-like peptides in vivo. The authors identify differences in signal transduction cascades activated by each peptide, which might underpin distinct functions, but these differences were established only in heterologous cells.

The authors show overlapping phenotypes for a long-term knockdown of the AjCT precursor and the AjPDFR2 receptor, suggesting that the growth-regulating functions of AjCT2 are mediated by this receptor pathway. However, it remains unclear whether this mechanism underpins the growth-regulating function of AjCT2, until further in vivo evidence for this ligand-receptor interaction is presented. For example, the authors could investigate whether knockdown of AjPDFR2 attenuates the effects of AjCT2 peptide injection. In addition, a functional PDF system in this species remains uncharacterized, and a potential role of PDF-like peptides in growth regulation has not yet been investigated in A. japonicus. Therefore, it also remains unclear whether the ability of CT-like peptides to activate PDFRs is an evolutionary ancient property of this peptide family or whether this is an example of convergent evolution in some protostomian (Drosophila) and deuterostomian (sea cucumber) species.

Reviewer #2 (Public review):

Summary:

The authors show that A. japonicus calcitonins (AjCT1 and AjCT2) activate not only the calcitonin/calcitonin-like receptor, but they also activate the two "PDF receptors", ex vivo. They also explore secondary messenger pathways that are recruited following receptor activation. They determine the source of CT1 and CT2 using qPCR and in situ hybridization and finally test the effects of these peptides on tissue contractions, feeding and growth. This study provides solid evidence that CT1 and CT2 act as ligands for calcitonin receptors; however, evidence supporting cross-talk between CT peptides and "PDF receptors" is weak.

Strengths:

This is the first study to report pharmacological characterization of CT receptors in an echinoderm. Multiple lines of evidence in cell culture (receptor internalization and secondary messenger pathways) support this conclusion.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

Summary:

The manuscript characterizes a functional peptidergic system in the echinoderm Apostichopus japonicus that is related to the widely conserved family of calcitonin/diuretic hormone 31 (CT/DH31) peptides in bilaterian animals. In vitro analysis of receptor-ligand interactions, using multiple receptor activation assays, identifies three cognate receptors for two CT-like peptides in the sea cucumber, which stimulate cAMP, calcium, and ERK signaling. Only one of these receptors clusters within the family of calcitonin and calcitonin-like receptors (CTR/CLR) in bilaterian animals, whereas two other receptors cluster with invertebrate pigment dispersing factor receptors (PDFRs). In addition, this study sheds light on the expression and in vivo functions of CT-like peptides in A. japonicus, by quantitative real-time PCR, immunohistochemistry, pharmacological experiments on body wall muscle and intestine preparations, and peptide injection and RNAi knockdown experiments. This reveals a conserved function of CT-like peptides as muscle relaxants and growth regulators in A. japonicus.

Strengths:

This work combines both in vitro and in vivo functional assays to identify a CT-like peptidergic system in an economically relevant echinoderm species, the sea cucumber A. japonicus. A major strength of the study is that it identifies three G protein-coupled receptors for AjCT-like peptides, one related to the CTR/CLR family and two related to the PDFR family. A similar finding was previously reported for the CT-related peptide DH31 in Drosophila melanogaster that activates both CT-type and PDF-type receptors. Here, the authors expand this observation to a deuterostomian animal, which suggests that receptor promiscuity is a more general feature of the CT/DH31 peptide family and that CT/DH31-like peptides may activate both CT-type and PDF-type receptors in other animals as well.

Besides the identification of receptor-ligand pairs, the downstream signaling pathways of AjCT receptors have been characterized, revealing broad and in some cases receptor-specific effects on cAMP, calcium, and ERK signaling.

Functional characterization of the CT-related peptide system in heterologous cells is complemented with ex vivo and in vivo experiments. First, peptide injection and RNAi knockdown experiments establish transcriptional regulation of all three identified receptors in response to changing AjCT peptide levels. Second, ex vivo experiments reveal a conserved role for the two CT-like peptides as muscle relaxants, which have differential effects on body wall muscle and intestine preparations. Finally, peptide injection and knockdown experiments uncover a growth-promoting role for one CT-like peptide (AjCT2). Injection of AjCT2 at high concentration, or long-term knockdown of the AjCT precursor, affects diverse growth-related parameters including weight gain rate, specific growth rate, and transcript levels of growth-regulating transcription factors. The authors also reveal a growth-promoting function for the PDFR-like receptor AjPDFR2, suggesting that this receptor mediates the effects of AjCT2 on growth.

Weaknesses:

The authors present a more detailed phylogenetic analysis in the revised version, including a larger number of species. But some clusters in the analysis are not well supported because they have only low bootstrap values. This makes it difficult to interpret the clustering in some parts of the tree.

Thank you for the reviewer’s comments. In response, we have produced a new phylogenetic analysis using the maximum likelihood method. This was done by Nayeli Escudero Castelán and Kite Jones in the Elphick group at QMUL and therefore they have been added as co-authors of this paper. The new phylogenetic tree (Figure 2, line 206) includes broad taxonomic sampling of CT-type receptors and PDF-type receptors. CRH-type receptors, which are also members of the secretin-type GPCR sub-family, have been included as an outgroup to root the tree. In the previous version the much more distantly related vasopressin/oxytocin-type receptors, which are rhodopsin-type GPCRs, were included as an outgroup. Furthermore, VIP-type receptors were also included in the previous tree but these have been omitted from the new tree because VIP receptor orthologs only occur in vertebrates and therefore they are not representative of a bilaterian GPCR family. The new tree shows high bootstrap support for key clades, notably achieving a bootstrap value of 100 for a clade comprising both deuterostomian and protostomian PDF receptors. This provides important evidence that the A. japonicus PDF-type receptors characterised in this study (AjPDFR1, AjPDFR2) are co-orthologs of the PDF-type receptor that has been characterised previously in Drosophila. Similarly, there is strong bootstrap support (100) for a clade comprising CT/DH31-type receptors and, importantly, the CT-type receptor characterised in this study (AjCTR) is positioned in a branch of this clade that comprises deuterostomian CT-type receptors (with bootstrap support of 100). Details of methods employed to produce the new receptor tree are included in lines 727-739. The new phylogenetic tree is shown below and has been incorporated into the revised manuscript (Figure 2, line 206). The description of new phylogenetic tree has also been modified accordingly in the revised manuscript (line 169-183).

References:

Bauknecht P, Jékely G. Large-Scale Combinatorial Deorphanization of Platynereis Neuropeptide GPCRs. Cell reports, 2015, 12(4), 684–693. doi:  10.1016/j.celrep.2015.06.052.

Beets I, Zels S, Vandewyer E, Demeulemeester J, et al. System-wide mapping of peptide-GPCR interactions in C. elegans. Cell reports, 2023, 42(9), 113058. doi: 10.1016/j.celrep.2023.113058.

Cardoso J C, Mc Shane J C, Li Z, et al. Revisiting the evolution of Family B1 GPCRs and ligands: Insights from mollusca. Molecular and cellular endocrinology, 2024, 586, 112192. doi: 10.1016/j.mce.2024.112192.

Gorn A H, Lin H Y, Yamin M, et al. Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line. The Journal of clinical investigation, 1992, 90(5), 1726–1735. doi: 10.1172/JCI116046.

Huang T, Su J, Wang X, et al. Functional Analysis and Tissue-Specific Expression of Calcitonin and CGRP with RAMP-Modulated Receptors CTR and CLR in Chickens. Animals: an open access journal from MDPI, 2024, 14(7), 1058. doi: 10.3390/ani14071058.

Johnson E C, Shafer O T, Trigg J S, et al. A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling. Journal of Experimental Biology, 2005, 208(7): 1239-1246. doi: 10.1242/jeb.01529.

McLatchie L M, Fraser N J, Main M J, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature, 1998, 393(6683): 333-339. doi: 10.1038/30666.

Schwartz J, Réalis-Doyelle E, Dubos M P, et al. Characterization of an evolutionarily conserved calcitonin signaling system in a lophotrochozoan, the Pacific oyster (Crassostrea gigas). Journal of Experimental Biology, 2019, 222(13): jeb201319. doi: 10.1242/jeb.201319.

Sekiguchi T, Kuwasako K, Ogasawara M, et al. Evidence for conservation of the calcitonin superfamily and activity-regulating mechanisms in the basal chordate Branchiostoma floridae: insights into the molecular and functional evolution in chordates. Journal of Biological Chemistry, 2016, 291(5): 2345-2356. doi: 10.1074/jbc.M115.664003.

Expression of CT-like peptides was investigated both at transcript and protein level, but insight into the expression of the three peptide receptors is limited. This makes it difficult to understand the mechanism underlying the (different) functions of the two CT-like peptides in vivo. The authors identify differences in signal transduction cascades activated by each peptide, which might underpin distinct functions, but these differences were established only in heterologous cells.

We appreciate the reviewer's insightful comments. Regarding expression of CT-like peptide receptors, we have quantitatively analyzed the mRNA expression levels of the three receptors in key tissues using qRT-PCR (Figure 6, line 319) and receptor expression exhibits significant tissue-specific differences. Combined with the heterologous expression assays and In vivo functional validation, we believe our findings have provided clear mechanistic insights into the functional divergence of the two CT-like peptides. Investigation of the expression of the three receptor proteins in A. japonicus would require generation of specific antibodies, which was beyond the scope of this study. Furthermore, immunohistochemical visualization of neuropeptide receptor expression in other invertebrates has not been reported widely, which likely reflects technical difficulties in generation of antibodies that can be used to specifically detect receptor proteins that are typically expressed a low level in comparison to the neuropeptides that act as their ligands. 

We acknowledge that investigating signal transduction cascades in heterologous cells (rather than native A. japonicus cells) is a limitation. However, as a non-model organism, A. japonicus currently lacks established cell lines for such research. Therefore, using heterologous cells was the most feasible approach to examine the differential signaling cascades activated by the peptides through the three receptors. Importantly, our in vivo experiments demonstrated that long-term knockdown of either the AjCT precursor or AjPDFR2 resulted in similar and significant growth defects. The phenotypic consistency strongly suggests that AjCT2 and AjPDFR2 function within the same signaling pathway, with AjPDFR2 serving as the key receptor functionally activated by AjCT2.

The authors show overlapping phenotypes for a long-term knockdown of the AjCT precursor and the AjPDFR2 receptor, suggesting that the growth-regulating functions of AjCT2 are mediated by this receptor pathway. However, it remains unclear whether this mechanism underpins the growth-regulating function of AjCT2, until further in vivo evidence for this ligand-receptor interaction is presented. For example, the authors could investigate whether knockdown of AjPDFR2 attenuates the effects of AjCT2 peptide injection. In addition, a functional PDF system in this species remains uncharacterized, and a potential role of PDF-like peptides in growth regulation has not yet been investigated in A. japonicus. Therefore, it also remains unclear whether the ability of CT-like peptides to activate PDFRs is an evolutionary ancient property of this peptide family or whether this is an example of convergent evolution in some protostomian (Drosophila) and deuterostomian (sea cucumber) species.

Thank you for the reviewer’s insightful comments and constructive questions. We acknowledge the request for more direct evidence to demonstrate how AjCT2 functions in vivo through AjPDFR2. However, long-term knockdown of the AjCT precursor and AjPDFR2 both resulted in identical and significant growth defect phenotypes. The high phenotypic consistency, combined with the activation effect of AjCT2 on AjPDFR2 in heterologous cells, strongly suggests that they function within the same signaling pathway, with AjPDFR2 serving as the key receptor functionally activated by AjCT2. While exogenous peptide injection combined with receptor knockdown is a classic method for verifying receptor activation, phenotypic overlap itself is widely accepted in genetic research as robust evidence for pathway association (Shafer and Taghert, 2009; Van Sinay et al., 2017). A. japonicus is a non-model organism with a 3-month aestivation period in summer followed shortly by winter hibernation. During these periods, we are unable to conduct in vivo experiments. Any single experimental suggestion from reviewers could potentially require one more year of research and we have already conducted an additional year of research, in response to reviewer feedback, since submitting the original manuscript. We hope therefore that these challenges associated with working with aquatic invertebrate non-model organisms is recognized by the reviewers.

We fully agree that the functional PDF/PDFR system in A. japonicus and its potential role in growth regulation remain uncharacterized. Currently, the precursors of the PDF-type neuropeptide in echinoderms remain unidentified, which precludes clear pharmacological characterization of the two receptors. While further exploration of echinoderm PDF-type neuropeptides is still needed, our phylogenetic analysis-conducted using the maximum likelihood method with optimized parameters and rigorous sequence curation-demonstrates that the deuterostomian PDFRs (including AjPDFR1 and AjPDFR2) are positioned in a clade with the well-characterized protostomian PDFR clades with extremely high bootstrap support (value=100). Therefore, these two receptors in A. japonicus clearly belong to the PDF receptor family and our findings clearly indicate that the ability of CT-like peptides to activate PDFRs is either an evolutionarily ancient and conserved property or has arisen independently in different lineages. Details of methods employed to produce the new receptor tree are included in line 727-739. The new phylogenetic tree is shown below and has been incorporated into the revised manuscript (Figure 2, line 206). The description of new phylogenetic tree has also been modified accordingly in the revised manuscript (line 169-183).

References:

Bauknecht P, Jékely G. Large-Scale Combinatorial Deorphanization of Platynereis Neuropeptide GPCRs. Cell reports, 2015, 12(4), 684–693. doi:  10.1016/j.celrep.2015.06.052.

Beets I, Zels S, Vandewyer E, Demeulemeester J, et al. System-wide mapping of peptide-GPCR interactions in C. elegans. Cell reports, 2023, 42(9), 113058. doi: 10.1016/j.celrep.2023.113058.

Cardoso J C, Mc Shane J C, Li Z, et al. Revisiting the evolution of Family B1 GPCRs and ligands: Insights from mollusca. Molecular and cellular endocrinology, 2024, 586, 112192. doi: 10.1016/j.mce.2024.112192.

Gorn A H, Lin H Y, Yamin M, et al. Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line. The Journal of clinical investigation, 1992, 90(5), 1726–1735. doi: 10.1172/JCI116046.

Huang T, Su J, Wang X, et al. Functional Analysis and Tissue-Specific Expression of Calcitonin and CGRP with RAMP-Modulated Receptors CTR and CLR in Chickens. Animals: an open access journal from MDPI, 2024, 14(7), 1058. doi: 10.3390/ani14071058.

Johnson E C, Shafer O T, Trigg J S, et al. A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling. Journal of Experimental Biology, 2005, 208(7): 1239-1246. doi: 10.1242/jeb.01529.

McLatchie L M, Fraser N J, Main M J, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature, 1998, 393(6683): 333-339. doi: 10.1038/30666.

Schwartz J, Réalis-Doyelle E, Dubos M P, et al. Characterization of an evolutionarily conserved calcitonin signaling system in a lophotrochozoan, the Pacific oyster (Crassostrea gigas). Journal of Experimental Biology, 2019, 222(13): jeb201319. doi: 10.1242/jeb.201319.

Sekiguchi T, Kuwasako K, Ogasawara M, et al. Evidence for conservation of the calcitonin superfamily and activity-regulating mechanisms in the basal chordate Branchiostoma floridae: insights into the molecular and functional evolution in chordates. Journal of Biological Chemistry, 2016, 291(5): 2345-2356. doi: 10.1074/jbc.M115.664003.

Shafer, O. T., & Taghert, P. H. (2009). RNA-interference knockdown of Drosophila pigment dispersing factor in neuronal subsets: the anatomical basis of a neuropeptide's circadian functions. PloS one, 4(12), e8298. doi: 10.1371/journal.pone.0008298.

Van Sinay, E., Mirabeau, O., Depuydt, G., Van Hiel, M. B., Peymen, K., Watteyne, J., Zels, S., Schoofs, L., & Beets, I. (2017). Evolutionarily conserved TRH neuropeptide pathway regulates growth in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America, 114(20), E4065–E4074. doi: 10.1073/pnas.1617392114.

Reviewer #2 (Public review):

Summary:

The authors show that A. japonicus calcitonins (AjCT1 and AjCT2) activate not only the calcitonin/calcitonin-like receptor, but they also activate the two "PDF receptors", ex vivo. They also explore secondary messenger pathways that are recruited following receptor activation. They determine the source of CT1 and CT2 using qPCR and in situ hybridization and finally test the effects of these peptides on tissue contractions, feeding and growth. This study provides solid evidence that CT1 and CT2 act as ligands for calcitonin receptors; however, evidence supporting cross-talk between CT peptides and "PDF receptors" is weak.

Strengths:

This is the first study to report pharmacological characterization of CT receptors in an echinoderm. Multiple lines of evidence in cell culture (receptor internalization and secondary messenger pathways) support this conclusion.

Weaknesses:

The authors claim that A. japonicus CTs activate "PDF" receptors and suggest that this cross-talk is evolutionary ancient since similar phenomenon also exists in the fly Drosophila melanogaster. These conclusions are not fully supported. The authors perform phylogenetic analysis to show that the two "PDF" receptors form an independent clade. The bootstrap support is quite low in a lot of instances, especially for the deuterostomian and protostomian PDFR clades which is below 30. With such low support, it is unclear if the clade comprising deuterostomian "PDFR" is in fact PDFRs and not another receptor type whose endogenous ligand (besides CT) remains to be discovered.

Thank you for the reviewer’s comments. In response, we have produced a new phylogenetic analysis using the maximum likelihood method. This was done by Nayeli Escudero Castelán and Kite Jones in the Elphick group at QMUL and therefore they have been added as co-authors of this paper. The new phylogenetic tree (Figure 2, line 206) includes broad taxonomic sampling of CT-type receptors and PDF-type receptors. CRH-type receptors, which are also members of the secretin-type GPCR sub-family, have been included as an outgroup to root the tree. In the previous version the much more distantly related vasopressin/oxytocin-type receptors, which are rhodopsin-type GPCRs, were included as an outgroup. Furthermore, VIP-type receptors were also included in the previous tree but these have been omitted from the new tree because VIP receptor orthologs only occur in vertebrates and therefore they are not representative of a bilaterian GPCR family. The new tree shows high bootstrap support for key clades, notably achieving a bootstrap value of 100 for a clade comprising both deuterostomian and protostomian PDF receptors. This provides important evidence that the A. japonicus PDF-type receptors characterized in this study (AjPDFR1, AjPDFR2) are co-orthologs of the PDF-type receptor that has been characterized previously in Drosophila. Similarly, there is strong bootstrap support (100) for a clade comprising CT/DH31-type receptors and, importantly, the CT-type receptor characterized in this study (AjCTR) is positioned in a branch of this clade that comprises deuterostomian CT-type receptors (with bootstrap support of 100). Details of methods employed to produce the new receptor tree are included in lines 727-739. The new phylogenetic tree is shown below and has been incorporated into the revised manuscript (Figure 2, line 206). The description of new phylogenetic tree has also been modified accordingly in the revised manuscript (line 169-183).

References:

Bauknecht P, Jékely G. Large-Scale Combinatorial Deorphanization of Platynereis Neuropeptide GPCRs. Cell reports, 2015, 12(4), 684–693. doi:  10.1016/j.celrep.2015.06.052.

Beets I, Zels S, Vandewyer E, Demeulemeester J, et al. System-wide mapping of peptide-GPCR interactions in C. elegans. Cell reports, 2023, 42(9), 113058. doi: 10.1016/j.celrep.2023.113058.

Cardoso J C, Mc Shane J C, Li Z, et al. Revisiting the evolution of Family B1 GPCRs and ligands: Insights from mollusca. Molecular and cellular endocrinology, 2024, 586, 112192. doi: 10.1016/j.mce.2024.112192.

Gorn A H, Lin H Y, Yamin M, et al. Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line. The Journal of clinical investigation, 1992, 90(5), 1726–1735. doi: 10.1172/JCI116046.

Huang T, Su J, Wang X, et al. Functional Analysis and Tissue-Specific Expression of Calcitonin and CGRP with RAMP-Modulated Receptors CTR and CLR in Chickens. Animals: an open access journal from MDPI, 2024, 14(7), 1058. doi: 10.3390/ani14071058.

Johnson E C, Shafer O T, Trigg J S, et al. A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling. Journal of Experimental Biology, 2005, 208(7): 1239-1246. doi: 10.1242/jeb.01529.

McLatchie L M, Fraser N J, Main M J, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature, 1998, 393(6683): 333-339. doi: 10.1038/30666.

Schwartz J, Réalis-Doyelle E, Dubos M P, et al. Characterization of an evolutionarily conserved calcitonin signaling system in a lophotrochozoan, the Pacific oyster (Crassostrea gigas). Journal of Experimental Biology, 2019, 222(13): jeb201319. doi: 10.1242/jeb.201319.

Sekiguchi T, Kuwasako K, Ogasawara M, et al. Evidence for conservation of the calcitonin superfamily and activity-regulating mechanisms in the basal chordate Branchiostoma floridae: insights into the molecular and functional evolution in chordates. Journal of Biological Chemistry, 2016, 291(5): 2345-2356. doi: 10.1074/jbc.M115.664003.

Reviewer #2 (Recommendations for the authors):

Figure 1C: The bootstrap support is quite low in a lot of instances, especially for the deuterostomian and protostomian PDFR clades which is below 30. With such support, I would be hesitant to label the blue clade as deuterostomian PDFR for two reasons: 1) no members of this clade have been shown to be activated by a PDF-like substance and 2) the current study shows that these receptors are activated by CT-type peptides. Therefore, the phylogenetic analyses do not support the conclusions of this paper. What is the basis for calling these receptors PDFR and not CTR in light of weak phylogenetic support?

Thank you for the reviewer’s comments. In response, we have produced a new phylogenetic analysis using the maximum likelihood method. This was done by Nayeli Escudero Castelán and Kite Jones in the Elphick group at QMUL and therefore they have been added as co-authors of this paper. The new phylogenetic tree (Figure 2, line 206) includes broad taxonomic sampling of CT-type receptors and PDF-type receptors. CRH-type receptors, which are also members of the secretin-type GPCR sub-family, have been included as an outgroup to root the tree. In the previous version the much more distantly related vasopressin/oxytocin-type receptors, which are rhodopsin-type GPCRs, were included as an outgroup. Furthermore, VIP-type receptors were also included in the previous tree but these have been omitted from the new tree because VIP receptor orthologs only occur in vertebrates and therefore they are not representative of a bilaterian GPCR family. The new tree shows high bootstrap support for key clades, notably achieving a bootstrap value of 100 for a clade comprising both deuterostomian and protostomian PDF receptors. This provides important evidence that the A. japonicus PDF-type receptors characterized in this study (AjPDFR1, AjPDFR2) are co-orthologs of the PDF-type receptor that has been characterized previously in Drosophila. Similarly, there is strong bootstrap support (100) for a clade comprising CT/DH31-type receptors and, importantly, the CT-type receptor characterized in this study (AjCTR) is positioned in a branch of this clade that comprises deuterostomian CT-type receptors (with bootstrap support of 100). Details of methods employed to produce the new receptor tree are included in lines 727-739 The new phylogenetic tree is shown below and has been incorporated into the revised manuscript (Figure 2, line 206). The description of new phylogenetic tree has also been modified accordingly in the revised manuscript (line 169-183).

We agree with the reviewer that no members of the PDF-type receptor clade in deuterostomes have yet been shown to be activated by a PDF-like substance. That is because the precursors of the PDF-type neuropeptides in echinoderms remain unidentified so far, which precludes clear pharmacological characterization of these receptors within the deuterostomian PDFR clade. However, the new phylogenetic tree now provides strong support (bootstrap value = 100) for the clade comprising deuterostomian and protostomian PDFRs, confirming the classification of AjPDFR1 and AjPDFR2 as PDF-type receptors. 

References:

Bauknecht P, Jékely G. Large-Scale Combinatorial Deorphanization of Platynereis Neuropeptide GPCRs. Cell reports, 2015, 12(4), 684–693. doi:  10.1016/j.celrep.2015.06.052.

Beets I, Zels S, Vandewyer E, Demeulemeester J, et al. System-wide mapping of peptide-GPCR interactions in C. elegans. Cell reports, 2023, 42(9), 113058. doi: 10.1016/j.celrep.2023.113058.

Cardoso J C, Mc Shane J C, Li Z, et al. Revisiting the evolution of Family B1 GPCRs and ligands: Insights from mollusca. Molecular and cellular endocrinology, 2024, 586, 112192. doi: 10.1016/j.mce.2024.112192.

Gorn A H, Lin H Y, Yamin M, et al. Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line. The Journal of clinical investigation, 1992, 90(5), 1726–1735. doi: 10.1172/JCI116046.

Huang T, Su J, Wang X, et al. Functional Analysis and Tissue-Specific Expression of Calcitonin and CGRP with RAMP-Modulated Receptors CTR and CLR in Chickens. Animals: an open access journal from MDPI, 2024, 14(7), 1058. doi: 10.3390/ani14071058.

Johnson E C, Shafer O T, Trigg J S, et al. A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling. Journal of Experimental Biology, 2005, 208(7): 1239-1246. doi: 10.1242/jeb.01529.

McLatchie L M, Fraser N J, Main M J, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature, 1998, 393(6683): 333-339. doi: 10.1038/30666.

Schwartz J, Réalis-Doyelle E, Dubos M P, et al. Characterization of an evolutionarily conserved calcitonin signaling system in a lophotrochozoan, the Pacific oyster (Crassostrea gigas). Journal of Experimental Biology, 2019, 222(13): jeb201319. doi: 10.1242/jeb.201319.

Sekiguchi T, Kuwasako K, Ogasawara M, et al. Evidence for conservation of the calcitonin superfamily and activity-regulating mechanisms in the basal chordate Branchiostoma floridae: insights into the molecular and functional evolution in chordates. Journal of Biological Chemistry, 2016, 291(5): 2345-2356. doi: 10.1074/jbc.M115.664003.

The new results following AjCT and AjPDFR2 knockdown are a welcome addition. While this additional evidence supports the claim that AjCT could mediate its effects via AjPDFR2, this evidence does not show that AjCT acts as an endogenous ligand for PDFR in vivo. In combination with the weak phylogenetic analyses, I would recommend the authors to key down their claims that they have functionally characterized a PDFR (in the title and text).

Thank you for your insightful comments and we do understand the reviewer’s concern. 

Regarding “the weak phylogenetic analyses”, as highlighted above, we have produced a new phylogenetic tree (Fig 2, line 206) that provides strong bootstrap support for the clade comprising deuterostome and protostome PDF-type receptors. For this reason, it is our opinion that inclusion of “pigment-dispersing factor-type receptors” in the title of the paper is appropriate. The details of phylogenetic analysis method were added in line 727-739, and the updated phylogenetic tree has been incorporated into the revised manuscript (Figure 2, line 206). The description of new phylogenetic tree has also been modified accordingly in the revised manuscript (line 169-183). Besides, long-term knockdown of the AjCT precursor and AjPDFR2 both resulted in identical and significant growth defect phenotypes. And the observation of phenotypic overlap is widely accepted in genetic research as strong evidence for pathway association (Shafer and Taghert, 2009; Van Sinay et al., 2017). This high degree of phenotypic consistency, coupled with our in vitro finding that AjCT2 specifically activates AjPDFR2, strongly supports the conclusion that AjCT2 and AjPDFR2 function within the same signaling pathway in vivo, with AjPDFR2 serving as the key receptor functionally activated by AjCT2.

References:

Shafer, O. T., & Taghert, P. H. (2009). RNA-interference knockdown of Drosophila pigment dispersing factor in neuronal subsets: the anatomical basis of a neuropeptide's circadian functions. PloS one, 4(12), e8298. doi: 10.1371/journal.pone.0008298.

Van Sinay, E., Mirabeau, O., Depuydt, G., Van Hiel, M. B., Peymen, K., Watteyne, J., Zels, S., Schoofs, L., & Beets, I. (2017). Evolutionarily conserved TRH neuropeptide pathway regulates growth in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America, 114(20), E4065–E4074. doi: 10.1073/pnas.1617392114.

Since there is no formal logic defining the use of "type" vs "like" vs "related", I would encourage the authors to use one term (of their choice) to avoid unnecessary confusion. Or another possibility is that these relationships are defined at some point in the manuscript so that it becomes clear to the reader.

Thank you for the reviewer’s comments. The “CT-related peptides” has defined in the Introduction (line 54-58). As per your suggestion, we have now defined both “CT-type peptides” and “CT-like peptides” in the Introduction (line 76-79). “CT-type peptides” are characterized by an N-terminal disulphide bridge, whereas “CT-like peptides” (diuretic hormone 31 (DH31)-type peptides) lack this feature. Additionally, in accordance with the definitions, we have corrected these three descriptions in the revised manuscript (line 80, 83, 88 for “CT-type peptides”) to ensure consistent and accurate usage of these terms.

"To provide in vivo evidence supporting CT-mediated activation of "PDF" receptors, we conducted the following experiments: Firstly, we confirmed that AjPDFR1 and AjPDFR2were the functional receptors of AjCT1and AjCT2 (Figure 2, 3 and 4). Secondly, injection of AjCT2 and siAjCTP1/2-1 in vivo induced corresponding changes in AjPDFR1and AjPDFR2expression levels in the intestine (Figure 8C, 9A, 9B and 9C)."

None of these experiments provide direct evidence that CT activates PDFR in vivo. The functional studies are indeed a welcome addition but they cannot discriminate between correlation and causation.

Thank you for the reviewer’s insightful comments. We agree that the functional studies do not constitute direct proof that CT’s activation of PDFR in vivo. However, we observed identical and significant growth defect phenotypes following long-term knockdown of the AjCT precursor and the AjPDFR2. This high degree of phenotypic congruence, combined with the established in vitro activation of AjPDFR2 by AjCT2, provides strong support for the conclusion that AjCT2 acts as the key endogenous ligand activating the AjPDFR2 signaling pathway in vivo. Importantly, such phenotypic overlap has been widely accepted in genetic research as strong evidence for functional pathway association (Shafer and Taghert, 2009; Van Sinay et al., 2017).

References:

Shafer, O. T., & Taghert, P. H. (2009). RNA-interference knockdown of Drosophila pigment dispersing factor in neuronal subsets: the anatomical basis of a neuropeptide's circadian functions. PloS one, 4(12), e8298. doi: 10.1371/journal.pone.0008298.

Van Sinay, E., Mirabeau, O., Depuydt, G., Van Hiel, M. B., Peymen, K., Watteyne, J., Zels, S., Schoofs, L., & Beets, I. (2017). Evolutionarily conserved TRH neuropeptide pathway regulates growth in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America, 114(20), E4065–E4074. doi: 10.1073/pnas.1617392114.

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DOI: 10.1101/2025.03.24.644980

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DOI: 10.1016/j.physbeh.2025.114958

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DOI: 10.1016/j.isci.2025.113675

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DOI: 10.1016/j.canep.2025.102803

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What is this?

I agree with Ko that the primary goal of a user interface designer is to define inputs, outputs, and event handlers to modify state. This really connects to my experience building my AI note-taking startup, where the interface had to translate messy, real-time lecture notes into structured, AI-generated summaries. I realized how much of UI design is about managing that flow of interaction — every click, edit, or pause changes the state of the system. Thinking about design in this way has helped me approach my product less as a static layout and more as a dynamic conversation between the user and the AI.

It makes sense that you must determine the functionality and architecture of something before you can deem what it looks like. This is a good reminder that design and software development work hand-in-hand, as it's easy to classify them as different fields entirely. I think, as a designer, being aware of how the backend of an interface works can be a leg up!

Wow, bad news for iron air

This article made me realize how apps “remember” more than we want them to — and don’t know when to stop. The author’s experience shows how technology treats emotional pain like just another data point.

Doesn't this seem a tad dangerous? I mean, Lavery's pathology has lead to some damaging behavior (like getting wasted beyond belief in order to write). Also, couldn't the pathology just be the compulsion to write?

This excerpt is pretty eye-catching because it gave me new knowledge on what prototyping is. It's interesting to learn that prototyping isn't meant to reflect the final product, rather its meant to help your ideate further. As someone who struggles with perfectionism and minor details, knowing that prototypes are meant to be disregarded (after they serve their purpose) helps with the pressure of perfecting my ideas.

I thought I knew what prototype means before but upon reading this chapter I realized my idea was somewhat wrong or at least incomplete. I had in mind that prototype would be a synonym for 'test'. By condensing it in something that help us make decisions and to acquire knowledge Ko gave me the insight that it would not be only testing if X works or not, but the knowledge that it works or not.

I think it’s kind of scary how recommendation algorithms know so much about us, yet we barely know how they actually work. The fact that platforms keep their algorithms secret makes me feel uneasy — it’s like we’re being studied without knowing what the experiment is. I’ve definitely noticed times when I talked about something and it suddenly showed up on my feed

It’s interesting that either her husband or her brother are all physician and had same idea about her. It shows that how stressful the narrator would be if none of them believed she was sick.

AI did some hallucinating on this page.

Any ongoing support beyond that would be on a separate, fixed hourly consultancy basis as needed at your discretion. Paul's hourly rate is $200/USD. We are happy to discuss during or after the 2-week sprint has been completed to your satisfaction.

Let's keep the part about flexibility at the end of the page.

Remove this, these are all something that we are interested in. We can bring this up after delivery on our own. No need to include these in this doc.

The following is from Sachin's original email to us with what he wants: What We Need from You Your role is Technical Consultant and Project Manager - providing strategic guidance, methodology, and oversight from project start through first client deployment. Based on your background (CTO experience, enterprise app development, TDD expertise, cloud architecture),