ok... but the zen-like peace gets displaced by the entrepreneurial ethos that says you always have to be working (in order to produce your own happiness):

meta-cognition!

context...

mindfulness as emancipating...

ends off with a strangely positive-sounding note...

...

how do you think this accords with entrepreneurial thinking? Clearly, one ought not be wrapped up in the past (either failures or successes), but what about a future-focus? A fixed mindset is clearly bad, but is a growth mindset also (especially if the pressure to "grow" and "develop" takes one away from a present focus)?

to limn = to depict/describe (from the root word "illuminate")

this isn't the original "zen"

**

linking the first week to grit etc.

Wow - this sounds, at first, like it's antithetical to entrepreneurial undertakings. Shouldn't one want to achieve something and invest oneself wholly in it?

indeed - this is a summation of the relevance of the last number of weeks, connected under the rubric of "mindset."

FYI - cognitive relates to thinking, affective relates to emotions, and conative relates to behaviour, specifically the inclination to act 'on purpose'. It's an effort or a drive to do something, but this isn't entirely rational...

what a link - liquid selves in an entrepreneurial context = zen-like...

...

zen is awfully implied here - not at all explicit.

whew!

**

!!

now we're getting somewhere!

interesting - how do you relate this to the entrepreneurial quest that motivates entrepreneurial personalities -- to turn the impossible (dream) into the real? To not be held by the bounds of the ordinary and others' expectations but to transcend them and become extraordinary...

thus, we get things like this:

does this sound wise (or good/meaningful) to you?

Your response will locate you relative to spirituality/mindfulness/mediation as a lens for self-actualization ...

...

background...

...

...

aha

*

*

this seems like a critique rather than something to be celebrated...

!

part of their overall argument

(fancy) theoretical framing...

Yowza!!

even if we're not always working (at our jobs), we're always working on ourselves ...

...

interesting

context

(see my last comment)

...

ok

ok!

connection to other article!

interesting

!

!

ok!

*

Interestingly [NSFW language alert!], as Tim Desmond argued in his 2019 book “How to stay human in a fucked-up world: Mindfulness practices for real life,” one can be “too” compassionate: “The challenge of staying human in a fucked-up world comes down to how we respond to the immensity of suffering that confronts us from every direction. [one has] to find a way to take care of the compassion in me so that I don’t end up overwhelmed. If I can’t I’ll find myself in despair.” One must be mindful in order to not drown in anger or bitterness.

food for thought - seeing one's own emotions and detaching from them is posited as good (but doesn't this assume that those emotions get one into trouble?) -- The idea is that over-identifying with feelings is bad. But does this apply to "good emotions" (like compassion)?

good examples, but maybe a little too manichean -- compassion = a good emotion and greed/aggresiveness = a bad emotion. Can you have a well-functioning mix of the two?

...

*

in other words...

possible route #1

(as another article put it, this is an attempt to offer ‘the wisdom and the heart of Buddhist meditation without the Buddhism’)

the basis for the aforementioned hope

bringing the different parts of the literature review together...

the hope...

implications...

nice term.

to review...

one of the reasons we've been focusing on affect just as much as effect, recently...

hah! easier said than done

widening the aperture!

interesting. What do you think of this (and how this idea connects to this course in general, or this week in particular)?

that's a tall order!

interesting. akin to re-framing

(link to experiencing failure too)

! with great power comes great responsibility...

to recap...

...

and as the very first reading of the year stated, we're all living in an environment of perpetual uncertainty now, having to all be entrepreneurs and assess risk and make our own opportunities...

quite frankly, this has been one of the "hidden agendas" of this course - how do we think about thinking (often by thinking about ourselves first)

!

mindfulness ought to help us eliminate cognitive biases and see things the way they are (not the way we want them to be, or to be trapped in vicious circles of judgement)

attitude

(with the idea that mindfulness can help us with this task)

connecting the axioms to the definition

"flow"

good - starting out with a definition (kind of ... more of a prefatory comment)

attention

intention (including the entrepreneurial type...)

entrepreneurial traits/actions can be bad (for the entrepreneurial agent, and for others) ... but mindfulness can avert that (or re-center one while on an entrepreneurial journey)

an attitude of non-judgement

more background, linking it to the need of all entrepreneurs to be creative ... creating opportunities, creating change...

link to EQ!

I appreciate Tim Desmond's observation that mindfulness is a very conscious exercise of relating to the world (in a way that many think meditation is not): Mindfulness does not mean taking deep breaths, sitting on a cushion, watching your thoughts and feelings with disinterest (like you’re watching a boring TV show). RATHER, mindfulness = a way of relating to the world (and specifically to suffering) that contains compassion, joy, equanimity, and wisdom. It is precisely the quality that allows us to stay human in fucked-up situations – to stay open, caring, and able to relate.

meditation...

d'uh

implications of mindfulness - connecting the internal to the external

a better definition

The purpose - bringing mindfulness into the entrepreneurial equation (how does it help with discovering opportunities...)

the point is that one ought to aspire to ...

d'uh

!

whoa! Big question. And something to consider for yourselves...

Theoretically, your highest aspiration should (hopefully) coincide with what we would imagine others also seek to fulfill ...

It basically boils down to your version of the "good life" (not necessarily good for others, or even only good for you, but what makes a life good or what makes one's life filled with well-being? What does it take to "be well"? Being good doesn't necessarily mean "doing" good...

I couldn't agree more with this quote! The more and more people begin to understand this the better the World Wide Web will be.

This is a really important statistic to understand. When making apps or softwares, creators need to be aware of disabilities and how they affect a lot of people. Having this understanding will help them to make sure their creations are accessible.

super important

Crow, Belknap, and Cheyenne Reservations - wyola

badlands

1.) Reparations, romanticized, loyal evangelists, exploitative, colonized, history, slavery, emancipation, property, psychological violence, obedience, narrative, industry. 2.) Short history of rum and sugar industry, slavery and exploitation in the Caribbean on the basis of skin color and production of rum. Modern impact of colonizer influence follows, which focuses on its effects in the corporate aspects, being that people of color are not allowed into the higher levels of rum companies, financial inequity, and lack of Caribbean representation in positions of power in outsider-owned spirit companies operating in the Caribbean.

This is representative of the argument, as it lists the acts committed against the people, the reason why current "reparation" actions being taken are not sufficient, and what the people want.

FINAL THOUGHTS: This article touched a lot on India's relationship with the US, China, and Russia, specially after the US had withdrawn from Afghanistan. India has developed a growing connection with the US, and there are lots of concerns about China's influence in the region. Some questions I had throughout the reading include, how can India leverage its economic strength to navigate its relationships with other powers? Additionally, are there potential risks of increased US and India cooperation?

Historical support from Russia is crucial for India.

India’s being quiet about it's concern.

Afghanistan’s situation could heavily increase tensions between India and China.

So this pushed India closer to the US.

Russia has a growing rivalry with India and its allies.

Hurts India's diplomatic efforts.

India's commitment to Kabul is very different than Russia's approach.

Russia's shifting alliances complicate India's plan.

Cautiously increasing its influence in Afghanistan.

Does this mean India’s actions are warranted given the stakes and concern they had?

Pakistan’s role could boost China’s strategy.

China’s increased involvement could destabilize the states.

US's withdrawal creates new challenges for India.

Shifting dynamics.

India's strategic outlook.

Shift in focus for India.

A more strategic evolution in India’s approach to its neighbors.

Increased terrorism.

Historical irritants in US and India relations.

A weakened US and Pakistan relationship could shift the power dynamics more favorably for India.

A need for India to recalibrate its expectations of US support.

What specific dangers are they anticipating?

How will it change relations with China?

Sense of ownership for artifacts of Troy

Thesis

n the U.S., freedom of expression is highly protected under the First Amendment, with only limited exceptions. Two key cases illustrate this broad interpretation:

Texas v. Johnson (1989): Gregory Lee Johnson burned an American flag in protest during a political demonstration, violating a Texas law that prohibited flag desecration. The U.S. Supreme Court ruled in Johnson's favor, holding that his act of flag burning was a form of "symbolic speech" protected by the First Amendment. The Court emphasized that the government cannot prohibit expression simply because society finds it offensive. United States v. Eichman (1990): After Texas v. Johnson, Congress passed the Flag Protection Act to criminalize flag desecration on a federal level, attempting to override the Court's decision. In U.S. v. Eichman, the Supreme Court again ruled in favor of free speech, declaring the federal law unconstitutional. The Court reaffirmed that flag desecration, as a form of protest, is protected by the First Amendment, regardless of its symbolism. These cases underscore the Court's strong stance on protecting freedom of expression, even when it involves controversial or offensive acts like flag burning.

explicitly states his position

reasons and evidence for how Honore came to see work as a blessing

Honore takes a position in response to another position: his father's.

search: "sd-ndn" ipfs

to: https://hyp.is/xCrXKoyREe-zZCOJFZ39mg/www.journaltocs.ac.uk/index.php?action=tocs&journalID=18837

searched in vein for the acroym IPFS

or Inter Planetary File System

which is an SD-NDN

a transformative approach in network architectures poised to deliver substantial benefits. By addressing the limitations inherent in traditional host-centric network architectures, SD-NDN offers improvements in network performance, scalability, and efficiency. The paper commences with an overview of named data networking (NDN) and software-defined networking (SDN), the two fundamental building blocks of SD-NDN. It then explores the specifics of integrating NDN with SDN, illustrating examples of various SD-NDN models. These models are designed to leverage SDN for NDN routing, caching, and forwarding. The paper concludes by proposing potential strategies for further integration of SDN and NDN and some open research questions. These proposed strategies aim to stimulate further exploration and innovation in the field of SD-NDN.

subjunctive time

this section discusses how we can inject communication errors into the game. The form used creates a tradeoff between more signals (expressivity) and accuracy (expected no of correct messages)

if stems have a hierarchy via phonemic similarity and there is a rich morphology, learning words may be greatly simplified by a learning a few systematic rules. Idealy we need to learn a lexicon with just one base form and apply a group action to get all the forms. In this case we only need to memorize a small lexicon.

Here we may be considering a form of compositionality, where semantics are established by preferential selection of languages with related patterns of phonemes. What is missing is that these patterns are not grounded in the structural features if the state being expressed..

decoding the state is mandated on decoding the each signal in the sequence

this q is a re-normalised transposed of p<br /> this is what receivers learn in lewis games.

having this transpose solution means that for each agent there is randomly chosen a lewis equilibrium in place at the start of equilibrium and selection is acting the fitness function which is highest for a signaling system. As n increases the number of partial pooling equilibria grows much faster then the number of separating equilibria

I agree with this statement because if you know there's something you don't want anybody to take advantage of or see, the best thing you should do is think about what you post on the internet . If you accidentally post something that wasn't meant to be seen by other users, the chances of people knowing and probably sharing it with numerous people is very high. The internet is not always trust worthy.

The very essens

all you need is naming mutbl a What's in a name?

everything is possible if you know how to name things

nd immutable data

all in th way you name things

Its both interesting and a great source of ethos to be able to admit and address this problem to the general public.

Is this because of the tenth amendment?

Were people generally excited about a way out of this/this crucial change?

Bradley states that it is likely that we “share our general morality with a common man.” Eliot, however, suggests that it is not simply these values that we share, but rather a confinement within them.

This notion is reflected in the title: “What the thunder said” becomes ironic. We don’t care what the thunder had to say; instead, we interpret it according to our own perspectives. The presumable thunder echoes its teaching—“da”— to gods, humans, and demons, each of whom constrains it to their singular interpretation: to give (“datta”), to control oneself (“dayahadvam”), and to sympathize (damyata). Thus, it appears that the teaching itself is less significant than its interpretation. What if “da” were intended to encompass all three meanings? Yet, different creatures choose to confine it within just one.

Eliot goes further than that and crafts a world in which “da” means none of these interpretations. The first pronoun following each of the “da” gives insight into this irony of the values we established. The first “datta” – to give – is dominated by the pronoun “we.” By definition, “give” is “to freely transfer the possession to.” The collective pronoun “we” evokes a sense of shared responsibility, suggesting that giving is a communal activity rather than a purely selfless act. With a simple use of “we” Eliot hints at how noble intentions – this “freely” – turn into transactional relationships and the concept of giving becomes confined within the bounds of reciprocity.

As we switch to “dayahadvam,” however, Eliot introduces a pronoun “I.” I represents a personal agency, where individual confinement leaves no room for compassion to others. Eliot reinforces this idea with a double repetition of “each.” Without compassion and connection with others we are isolated – “each” in their own prison, where thinking of escape we further confine us within this trap. The use of “I” reveals the limitations of self-centered thinking, suggesting that without genuine connection and compassion, we are ultimately confined, entrapped, and isolated from one another.

Finally, as we transition to “damyata,” the pronoun shifts again, this time to the third-person “the boat.” This transition signifies a move from internal self-control, which damyata is supposed to signify, to a search for external control. The boat, an object, thus, becomes a metaphor for the external forces we seek to exert control over our lives. In this way, the use of the third person reflects a desire to externalize control rather than cultivating it from within. This search, however, never culminates with a discovery; instead, Eliot uses a hypothetical tense, where “the heart would have responded gaily” if it was “invited” into these “controlling hands.” The hypothetical tense suggests an absence of such an external force that could have provided stability from the outside.

Through these pronouns, thus, it appears that Eliot suggests that it is not the values that connect us; instead it is the entrapment within the interpretations of these values, obscuring their true significance, that we share. The act of giving becomes transactional, compassion is separated by barriers of isolation, and self-control turns into an unfulfilled quest for external authority. Consequently, the teachings of connection only serve to highlight the constraints we ourselves have imposed, trapping us in this prison of distorted interpretations.

for - definition - physiocrats - Steve Keen - economy - history - economic flow as biomimicry of body's circulation system

definition - physiocrat - During the 18th and 19th century, a group of mostly French "economists" led by Francois Quesnay, physician to the King of France at the time, performed some of the first autopsies of the time. - Autopsies were banned for the longest time for religious reasons - When Quesnay performed autopsies, he discovered networks of tubes in the circulation system and this led him to surmise a network of circulation in another field, economics - Quesnay advised the king, hence the name physiocrat - So modern economics has its roots in biology - it was a case of biomimicry!

for - from - Fair Share Commons discussion thread - Marie - discussing the use of the word "capital" in "spiritual capital" - webcast - Great Simplification - On the Origins of Energy Blindness - Steve Keen - Energy Blindness

It's impossible to write an algorithm that can determine, given an arbitrary program and input, whether the program will halt (stop running) or run forever.

dus bij functioneel level gaat het om valure chains die bestaan uit; 1. kwaliteit, 2. innovatie 3. klant responsiveness 4. efficientie

The nature of the "tech-solutionism" is not so much about the reasoning as the (pro-)activity

Previously it was stated that the doctrine and confession can't be changed, but here it is stated that 3/4 majority can change technical aspects of the confession.

I think this part should be fortified against heresies such as female pastors, universalism etc.

When there is conflict between the pastor and the congregation, without public sins or heresy, the bishop is the middleman in trying to resolve these issues.

Also, only the consistory can (or it requires their permission) sack a pastor from his office, not the congregation alone. Here again the power of the congregation has been shifted to the upper echelons of the church.

This part just talks about how the thing (investigation) must be kept a secret/hidden while it's going on, how there have to usually be multiple witnesses and that the pastor is innocent until proven guilty.

A lot of very good, protective legal text, which for a pastor is good. IF a single congregant tries to smear you, it very like won't succeed.

The bishop and the consistory have the means to deal with problematic situations, the MD has to intervene if the sin of the pastor is: 1. exceptionally grave act or of neglect. 2. habitual 3. public or known in the congregation 4. directly related to the congregation or his office.

What does this mean exactly? I'm not sure. Adultery, alcoholism come to mind, but the bishop and consistory have the final say in the matter.

This may differ from the US context, but here it is explicitly stated that pastors can't, according to the 2 regiments, in congregation endorse a party or political members. Talking about questions that are political (abortion for an example) is allowed, but you can't preach that "Christians can only vote for party X".

This is a good thing and the MD seeks to remain faithful in the questions of two kingdoms by keeping them clearly separated.

The interesting part is that the congregation does call the pastor, and has the final say in the call, but it is the bishop who suggests candidates and potential pastors for the congregation.

The bishop and the congregation of course discuss these things together, but it's remarkable that the bishop can veto a congregation's choice if he sees fit.

This works for as long as the bishop isn't heretical, but problems may arise if the church becomes corrupted from the top end. It is odd that the congregation lacks freedom to call here whoever they wish to call, but this again shows the rigid nature of order in the MD.

It has to be noted though that in normal situations the communication is healthy and both parties are satisfied with results

Interesting that the pastor is responsible for ALL preaching and spiritual activity in the congregation, not the board or a collection of members.

I think this means that laypeople can teach, but it requires the permission of the pastor, which is a good thing.

Of course here it is stated that the sacrament is not to be given to heretics or unrepented sinners (public sins especially), so if a bishop or a pastor of the state church came to our service, they would not be served the sacrament.

However, why do we use this kind of strictness on the pastors of the state church, but not the laity who can be members of it?

This is what I dislike quite a bit about our communion practices, it says that everyone who have been baptised and confirmed (though children who haven't been confirmed yet but understand the meaning can partake per 13 §) and adhere to the Lutheran confessions can partake of the sacrament.

This means that a person can come and be a member of the state church and partake according to these rules, everyone who claims to be Lutheran can come to the table.

I know that this is an intermediate model, as we move towards registeration in the coming years (hopefully), but I would reserve the sacrament only for the members of the MD, because the current rules are way too open about this.

Here it is clearly stated that the eucharist is to be administrated by a/the pastor, not a layman. Layperson can be assisting, as they hold the paten, but they can't administer it.

This is a very strict, and good, decision for the sake of order and clarity, who are the representatives of Christ, who holds the keys in the sense that who is let to the table and who is not. I know that in some conservative revivalist movements in the state church lay women can be administering the sacrament, which destroys the whole "picture" of Christ feeding His flock.

I again encourage the reader to translate everything what is said here if he has time, but this is the point I have been talking about earlier.

"Divine services are to be held according to the Liturgical tradition, obeying the instructions of the consistory." This seems to put a very firm nail into the coffin of CoWo and soloing the liturgy. Pointing this out again, individual freedom of a congregation is very much limited here for the sake of order and unity.

The idea being that you could go to any MD divine service and they would be 90-95% liturgically same

This part is excellent, "If the person (whom is to be baptized) isn't going to become a member of the MD or he isn't going to become a member by the consent of his/her parents (infants), then the baptism in ordinary circumstances must not be administrated".

I love how clearly this outlines that baptism just for cultural reasons or to appease your grandparents isn't a good enough reason for it.

It is said that the congregations are free to "do what they want" as long as it doesn't go against this document and the confessions. I just have to ask how much freedom this actually entails, since this document is fairly strict regarding questions of doctrine and liturgy, which I believe is a good thing.

Though one has to ask, could a congregation have a contemporary worship service? I'm not advocating for it, but to me it would seem that this document doesn't allow it.

The Mission Diocese is split into regions (canons?), these regions consisting of congregations are led by a 'Rovasti' (dean, canon, not sure what is the English equivalent here, maybe DP for LCMS). This again helps with administrational and spiritual problems, a pastor facing trouble doesn't have to talk to the bishop directly, but can contact his closer superior first.

This is what I like about "strict" order in the church, yes, individual congregations aren't as free as in other models regarding their policies, economy, decisions (they aren't slaved of course) but they have unity and very clear structures to help with different kinds of problems.

Congregations cover the costs of the church body (of all congregations) together, like mentioned before, there is no flying solo. For rich, well off, big congregations this may seem unfair, but it does benefit the mission of the church all-together as long as the funds are being used wisely (the good stewardship aspect applies here to the whole church body, not just individual congregations).

The bishop must regularly (usually once a year) visit a congregation, when this happens there are specific instructions on how to handle that service and what happens after that, as the bishop is interacting with the congregation.

These visitations can be had to discuss policies or if the pastor isn't behaving well, meaning in church planting you as a pastor can call the bishop to visit if you are having issues you can't resolve.

I suggest the dear reader to translate the whole page with google translate about the consistory, because it's interesting, but I want to point out this last bit. The Bishop holds the authority (as a chairman) to call the consistory into session. This once again shows how much power the bishop (rightly) holds, I think the question remains then that can the dean or a pastor call up a consistory meeting if the bishop is being heretical?

Even though the consistory has a single layman with it, which is fine, it's good that they have outlined it to be male. Women should not be in positions to make theological decisions concerning the church body.

There is a lot of stuff I have chosen not to annotate due to the sheer amount of text alone, but this one is interesting. Before consistory can send a document to the gathering of representatives, it must hear the college of pastors.

All of this can seem a bit complex when trying to understand how all of these different decision-making bodies relate to each other, especially when a person can (and usually is) be a member of these different bodies simultaneously.

I would question here that why can laypeople vote when electing a bishop?

As the bishop is the overseer of other pastors, it would seem fitting to me that only pastors can vote on the issue.

Bishop is the oversee of all congregations in the MD; but as our church body is growing, it's becoming increasingly difficult to overseer about 50 different congregations for one single bishop. I think we should soon have two bishops or change the overseer system.

If we got two bishops, then which would be the chairman in various organizational tasks? I'm not sure.

Chapter 7 is about the office of the Bishop, here it says that the bishop is to ordain new pastors. We don't hold that ONLY a bishop could ordain pastors, but again, for the sake of order and unity, and continuation of the Finnish-Lutheran tradition, the bishop is the one who ordains.

"The doctrine and confession mentioned in the first part can't be changed."

More assuring that this whole church body would need to be torn apart before things like "female pastors" could come forth. But because history shows that things can be "reinterpreted" I would use even stronger, clearer language in some parts for the sake of the future generations.

In doctrinal and theological questions the consistory HAS TO BE heard! This all shows that the administration is very tightly tied together, anchored in many different places to avoid heresies from coming forth.

This helps church planting and pastors, because the unity of the church is upheld by a very high order. You don't have to again fly solo, be afraid if problems you can't fix come up in a congregation etc.

Though I would question whether the consistory or the gathering of representatives should be the highest authority, wouldn't it be better for the best, trained theologians to have the ulimate final say?

I mean, I can understand how easy it is to say this, but it's way more difficult than that because we have students who haven't had the same education as others because these students don't have enough money to get tutoring, get books, or maybe even go to school so I think we must consider other things instead of just making them take a class and basing their education on this class.

for - article - Why Human (Contributive) Labor remains the creative principle of human society - Michael Bauwens - PhD thesis - From Modes of Production to the Resurrection of the Body: A Labor Theory of Revolutionary Subjectivity & Religious Ideas (2016) - Benjamin Suriano - to - P2P Foundation - more detailed presentation of Benjamin Suriano's PhD paper

Summary - This is a review and high recommendation of the PhD dissertation of Benjamin Suriano by Michael Bauwens - The subject is the historical analysis of labour in medieval times, and - how Christian monasticism provided a third perspective on labour that was an important alternative to the false dichotomy of - cleric - warrior - that was inclusive of the alienated within class majority - a proposal for revival the spirit of this spiritual view of labour - as a means to mitigate modernity's meaning crisis as it relates to the lack of purpose usually associated with work in contemporary society

to - P2P Foundation - more detailed presentation of Benjamin Suriano's PhD paper - https://hyp.is/7PeMMIxtEe-NOmuU08T3jg/wiki.p2pfoundation.net/From_Modes_of_Production_to_the_Resurrection_of_the_Body

for - from - recommendation - from - Michel Bauwens - on Fair Share Commons chat thread, 2024 Oct 17 - context Karl Marx liberation of the individual - to - substack article - Why Human (Contributive) Labor remains the creative principle of human society - Michel Bauwens article details - title: From Modes of Production to the Resurrection of the Body: A Labor Theory of Revolutionary Subjectivity & Religious Ideas" (2016) - author: Benjamin Suriano

to - Substack article - Why Human (Contributive) Labor remains the creative principle of human society - Michel Bauwens - https://hyp.is/go?url=https%3A%2F%2F4thgenerationcivilization.substack.com%2Fp%2Fwhy-human-contributive-labor-remains&group=world

eLife Assessment

This valuable study focuses on the regulation of Notch signaling during the immune response in Drosophila. The authors provide solid evidence in support of roles for Su(H) and Pkc53E-induced phosphorylation in Drosophila immunity. The work will be of interest to colleagues in immunity and receptor signaling.

Reviewer #1 (Public review):

The authors previously showed in cell culture that Su(H), the transcription factor mediating Notch pathway activity in Drosophila, was phosphorylated on S269 and they found that a phospho-deficient Su(H) allele behaves as a moderate gain of Notch activity in flies, notably during blood cell development. Since downregulation of Notch signaling is important for the production of specialized blood cell types (lamellocytes) in response to wasp parasitism, the authors hypothesized that Su(H) phosphorylation might be involved in this cellular immune response.<br /> Consistent with their hypothesis, the authors now show that Su(H)S269A knock-in flies display a reduced response to wasp parasitism and that Su(H) is phosphorylated upon infestation. Using in vitro kinase assays and a genetic screen, they identify the PKCa family member Pkc53E as the putative kinase involved in Su(H) phosphorylation and they show that Pkc53E can bind Su(H). They further show that Pkc53E deficit or its knock-down in larval blood cells results in similar blood cell phenotypes as Su(H)S269A and their epistatic analyses indicate that Pkc53E acts upstream of Su(H). Finally, they show that Pkc53E mutants aslo display a compromised immune response to wasp parasitism.

Strengths

The manuscript is well presented and the experiments are sound, with a good combination of genetic and biochemical approaches and several clear phenotypes backing the main conclusions. Notably Su(H)S269A mutation strongly reduces lamellocyte production. Moreover, the epistatic data are convincing, notably concerning the relationship between Notch/Su(H) and Pkc53E for crystal cell production.<br /> Even though it is not fully established, the overall model is credible and interesting. In addition, it opens further avenues of research to study the activation of Pkc in response to an immune challenge.

Weaknesses

Apparently, the hypothesis that Pkc53E is required for Su(H) phosphorylation in vivo could not be directly tested due to the lack of an appropriate tool (the specificity and sensitivity of the current anti-pS269 antibody was insufficient).<br /> Also, the poor immune response of Pkc53E mutant might rather be linked to their constitutively reduced circulating blood cell number than to a deficit in Notch/Su(H) down-regulation following wasp infestation.

Reviewer #2 (Public review):

The current draft by Deischel et.al., describes the role of Pkc53E in the phosphorylation of Su(H) to down regulate its transcriptional activity to mount a successful immune response upon parasitic wasp-infection. Overall, I find the study interesting and relevant especially the identification of Pkc53E in phosphorylation of Su(H) is very nice. The authors have proved the central idea linking phosphorylation of Su(H) via Pkc53E to implying its modulation of Notch activity to mount a robust immune response is now well addressed in its entirety and I find the paper indeed very interesting.

Comments on revised version:

The authors have addressed all pending concerns and I have no further comments. I indeed complement the authors for their wonderful piece of work.

Reviewer #3 (Public review):

Diechsel et al. provide important and valuable insights into how Notch signaling is shut down in response to parasitic wasp infestation in order to suppress crystal cell fate and favor lamellocyte production. The study shows that CSL transcription factor Su(H) is phosphorylated at S269A in response to parasitic wasp infestation and this inhibitory phosphorylation is critical for shutting down Notch. The authors go on to perform a screen for kinases responsible for this phosphorylation and have identified Pkc53E as the specific kinase acting on Su(H) at S269A. Using analysis of mutants, RNAi and biochemistry-based approaches the authors convincingly show how Pkc53E-Su(H) interaction is critical for remodeling hematopoiesis upon wasp challenge. I find the study interesting, and the data presented supports the overall conclusions made by the authors. The authors have addressed all my comments satisfactorily in the revised submission.

Strengths:

The manuscript is well presented, and the conclusions made are backed by genetic, biochemical and molecular biology-based approaches. Overall, the authors convincingly demonstrate how Pkc53E mediated phosphorylated of Su(H) shuts down Notch signaling during wasp infestation in Drosophila.

Weaknesses:

The exact molecular trigger for activation of Pkc53E is still uncharacterized and it would be interesting to know how Pkc53E gets activated during wasp infestation and whether Pkc53E gets activated turning down Notch in other stress induced scenarios.

The authors have addressed comments satisfactorily. Overall, I think the findings are interesting and would be useful to the field of developmental biology and immunology and address an important gap in the field. The most significant conclusion from the work is how Notch acts as a molecular switch during parasitic wasp infestation.

Author response:

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

Reviewer #1 (Public Review):

The authors previously showed in cell culture that Su(H), the transcription factor mediating Notch pathway activity, was phosphorylated on S269 and they found that a phospho-deficient Su(H) allele behaves as a moderate gain of Notch activity in flies, notably during blood cell development. Since a downregulation of Notch signaling was proposed to be important for the production of a specialized blood cell types (lamellocytes) in response to wasp parasitism, the authors hypothesized that Su(H) phosphorylation might be involved in this cellular immune response.

Consistent with their hypothesis, the authors show that Su(H)S269A knock-in flies display a reduced response to wasp parasitism and that Su(H) is phosphorylated upon infestation. Using in vitro kinase assays and a genetic screen, they identify the PKCa family member Pkc53E as the putative kinase involved in Su(H) phosphorylation and they show that Pkc53E can bind Su(H). They further show that Pkc53E deficit or its knock-down in larval blood cells results in similar blood cell phenotypes as Su(H)S269A, including a reduced response to wasp parasitism, and their epistatic analyses indicate that Pkc53E acts upstream of Su(H).

Strengths

The manuscript is well presented and the experiments are sound, with a good combination of genetic and biochemical approaches and several clear phenotypes which back the main conclusions. Notably Su(H)S269A mutation or Pkc53E deficiency strongly reduces lamellocyte production and the epistatic data are convincing.

Weaknesses

The phenotypic analysis of larval blood cells remains rather superficial. Looking at melanized cells is a crude surrogate to quantify crystal cell numbers as it is biased toward sessile cells (with specific location) and does not bring information concerning the percentage of blood cells differentiated along this lineage.

In Su(H)S269A knock-in or Pkc53E zygotic mutants, the increase in crystal cells in uninfected conditions and the decreased capacity to induce lamellocytes following infection could have many origins which are not investigated. For instance, premature blood cell differentiation could promote crystal cell differentiation and reduce the pool of lamellocytes progenitors. These mutations could also affect the development and function of the posterior signaling center in the lymph gland, which plays a key role in lamellocyte induction.

Similarly, the mild decrease on resistance to wasp infestation (Fig. 2A) could reflect a constitutive reduction in blood cell numbers in Su(H)S269A larvae rather than a defective down-regulation of Notch activity.

We fully agree with the reviewer that sessile crystal cells counts are a coarse approach to capture hemocytes. However, they allowed the screening of numerous genotypes in the course of our kinase candidate screen. We recorded the hemocyte numbers in the various genetic backgrounds and with regard to wasp infestation. There was no significant difference between Su(H)S269A and Su(H)gwt control, independent of infection. This is in agreement with earlier observations of unchanged plasmatocyte numbers in N or Su(H) mutants compared to the wild type (Duvic et al., 2002). We noted, however, a small drop in hemocyte numbers in Su(H)S269D and a strong one in Pkc53ED28 mutants in both conditions relative to control. Presumably, Pkc53E has a more general role in blood cell development, which we have not further analysed. The results were included in new Figure 1_S1 and Figure 9_S1 supplements. Based on the link between hemocyte numbers and wasp resistance (e.g. McGonigle et al., 2017), we cannot exclude that the lowered resistance of Pkc53ED28 mutants regarding wasp attacks is partly due to reduced hemocyte numbers, albeit we did not see significant differences between either Su(H)S269A, nor Pkc53ED28 nor the double mutant. We have included this notion in the text.

Lamellocytes arise in response to external challenges like parasitoid wasp infestation by trans-differentiation from larval plasmatocytes, and by maturation of lamellocyte precursors in the lymph gland, yet barely in the Su(H)S269A and Pkc53ED28 mutants.

We find it hard to envisage, however, that a premature differentiation of plasmatocytes into crystal cells in our case could deplete the pool of lamellocyte progenitors in the hemolymph. (Is there a precedent?). Crystal cells make up about 5% of the hemocyte pool; they are increased max. 2 fold in the Su(H)S269A and Pkc53E mutants. Even if these extra crystal cells (now  ̴10%) had arisen by premature differentiation, there should be still enough plasmatocytes (̴ 80%) remaining with a potential to further divide and transdifferentiate into lamellocytes.

Indeed, we cannot exclude an effect of the Su(H)S269A mutant on the development and function of the posterior signaling center of the lymph gland. We noted, however, a slight but significant enlargement of the PS in the Su(H)S269A mutant, that to our understanding cannot explain the reduced lamellocyte numbers.

Whereas the authors also present targeted-knock down/inhibition of Pkc53E suggesting that this enzyme is required in blood cells to control crystal cell fate (Fig. 6), it is somehow misleading to use lz-GAL4 as a driver in the lymph gland and hml-GAL4 in circulating hemocytes as these two drivers do not target the same blood cell populations/steps in the crystal cell development process.

We fully agree with the reviewer that the two driver lines target different blood cell populations/ steps in hematopoiesis. The hml-Gal4 driver is regarded pan-hemocyte, common to both plasmatocytes and pre-crystal cells (e.g. Tattikota et al., 2020). It has been reported to drive specifically within differentiated hemocytes prior to or at the stage of crystal cells commitment (Mukherjee et al., 2011). Hence, hml-Gal4 appeared suitable to hit sessile and circulating hemocytes prior to final differentiation into crystal cells or lamellocytes, respectively.

In the lymph gland, however, hml is expressed within the cortical zone, where it appears specific to the plasmatocytes lineage, and not present in the crystal cell precursors (Blanco-Obregon et al., 2020). In contrast, lz-Gal4 is specific to the differentiating crystal cells in both lineages, i.e. in circulating and sessile hemocytes and in the lymph gland. Hence, we choose lz-Gal4 instead of hml-Gal4 at the risk of driving markedly later in the course of crystal cell differentiation. We included the reasoning in the text. Overall, we feel that this choice does not limit our conclusions.

In addition, the authors do not present evidence that Pkc55E function (and Su(H) phosphorylation) is required specifically in blood cells to promote lamellocyte production in response to infestation.

We have tried to address this interesting question by several means. Firstly, we show that Pkc53E is indeed expressed in the various cell types of larval hemocytes, shown in a new Figure 8 and Figure 8_S1 supplement. I.e., there is the potential of Pkc53E to promote lamellocyte formation. Moreover, RNAi-mediated downregulation of Pkc53E within hemocytes affected crystal cell formation similar to the Pkc53ED28 mutant, in agreement with a specific requirement within blood cells (Figure 6). Finally, we show a major drop in Notch target gene transcription (NRE-GFP) in response to wasp infestation within isolated hemocytes from Su(H)gwt in contrast to Su(H)S269A larvae (see new Figure 1 G). These data show that Su(H)-mediated Notch activity must be downregulated in hemocytes prior to lamellocyte formation in agreement with our hypothesis.

Finally, the conclusion that Pkc53E is (directly) responsible for Su(H) phosophorylation needs to be strengthened. Most importantly, the authors do not demonstrate that Pkc53E is required for Su(H) phosphorylation in vivo (i.e. that Su(H) is not phosphorylated in the absence of Pkc53E following infestation).

We would very much like to show respective results. Unfortunately, the low affinity of our pS269 antibody does not allow any in situ or in vivo experiments. We very much hope to obtain a more specific phosphoS269-Su(H) antibody allowing us further in situ studies, and show, for example co-localization with Pkc53E.

In addition, the in vitro kinase assays with bacterially purified Pkc53E (in the presence of PMA or using an activated variant of Pkc53E) only reveal a weak activity on a Su(H) peptide encompassing S269 (Fig. 4).

The reviewer correctly notes the poor activity of our purified Pkc53EEDDD kinase. This low activity also holds true for the standard peptide (PS), which in fact is even less well accepted than the Swt substrate. Indeed, the commercially available PKCα is a magnitude more active. Whether this reflects the poor quality of our isolated protein compared to the commercial PKCα, or whether it reflects a true biochemical property of Pkc53E remains to be shown in the future. We noted this observation in the manuscript.

Moreover, while the authors show a coIP between an overexpressed Pkc53E and endogenous Su(H) (Fig. 7) (in the absence of infestation), it has recently been reported that Pkc53E is a cytoplasmic protein in the eye (Shieh et al. 2023), calling for a direct assessment of Pkc53E expression and localization in larval blood cells under normal conditions and upon infestation.

Indeed, it is interesting that a Pkc53E-GFP fusion protein is cytoplasmic in the eye. The construct reported by Shieh et al. however, i.e. the B-isoform, is preferentially expressed in photoreceptors, where it regulates the de-polymerization of the actin cytoskeleton.

Due to the eye-specific expression, we unfortunately cannot use the Pkc53E-B-GFP construct to test for Pkc53E’s distribution in other tissues.

As this construct is of little use for studying hematopoiesis, we have instead used Pck53E-GFP (BL59413) derived from a protein trap: again, GFP is primarily seen in the cytoplasm of hemocytes, including lamellocytes of infected larvae. However, in a small number of hemocytes, GFP appears to be also nuclear (Fig. 8A), leaving the possibility that activated Pkc53E may localize to the nucleus, eventually phosphorylating Su(H) and downregulating Notch activity. As Su(H) enters the nucleus piggy-back with NICD, however, phosphorylation may as well occur at the membrane or within the cytoplasm. We note, however, that these hypotheses require a much more detailed analysis.

Furthermore, the effect of the PKCa agonist PMA on Su(H)-induced reporter gene expression in cell culture and crystal cell number in vivo is somehow consistent with the authors hypothesis, but some controls are missing (notably western blots to show that PMA/Staurosporine treatment does not affect Su(H)-VP16 level) and it is unclear why STAU treatment alone promotes Su(H)-VP16 activity (in their previous reports, the authors found no difference between Su(H)S269A-VP16 and Su(H)-VP16) or why PMA treatment still has a strong impact on crystal cell number in Su(H)S269A larvae.

We have added a Western blot showing that the treatment does not affect Su(H)-VP16 expression levels (Figure 5_supplement 1). As STAU is a general kinase inhibitor, it may obviate any inhibitory phosphorylation of Su(H)-VP16 in the HeLa cells, e.g. that by Akt1, CAMK2D or S6K which pilot T271, phosphorylation of which is expected to affect the DNA-binding of Su(H) as well (Figure 3_supplement 2). Moreover, in the previous report, we used different constructs with regard to the promoter, and we used RBPJ instead of Su(H), which may explain some of the discrepancies. As PMA is not specific to just Pkc53E, the altered crystal cell numbers may result from the influence on other kinases involved in blood cell homeostasis, as predicted by our genetic screen (Figure 3_supplement 1).

Reviewer #1 (Recommendations For The Authors):

(1) The authors should provide a more elaborate examination of larval blood cell types and blood cell counts under normal conditions and following infestation in the different zygotic mutants as well as upon Pkc53 knock-down. A thorough examination of PSC integrity should be performed and the maintenance of core blood cell progenitors examined. The authors should also clarify when after infestation the LG and larval bleeds are analyzed.

- a more elaborate examination of larval blood cell types:

- examination of larval blood cell counts under normal conditions: hemocyte # in gwt, SA, SD, & Pkc

- examination of larval blood cell counts after infestation: hemocyte # in gwt, SA, SD, & Pkc

- thorough examination  of PSC integrity: in gwt, SA, SD, & Pkc

- thorough examination of blood cell progenitors: in gwt, SA, SD, & Pkc

- clarify timing

Hemocyte numbers of the various genotypes and conditions were recorded and are presented in Figure 1_S1 and Figure 9_S1. Timing was elaborated in the text and the Methods section.

(2) The authors should clarify why they use lz-GAL4 or hml-GAL4 and what we can infer from using these different drivers.

See above. The reasoning was included in the text.

(3) The percentage of hatching of Su(H)S269A and Su(H)gwt flies in the absence of infestation should also be scored; a small decrease in Su(H)S269A viability might explain the observed differences in survival to wasp infestation. Absolute blood cell numbers (in the absence of infestation) have also been correlated with survival to infection and should be checked.

Percentage of the emerging flies and hemocyte numbers in the absence of infestation were recorded and included in Figure 2, Figure 1_S1, Figure 9_S1.

(4) Whereas the impact of Su(H)S269A or Pkc53E mutation on lamellocytes production is clear, there is still a substantial reduction in crystal cell production following infestation. So I wouldn't conclude that the Su(H) larvae are "unable" to detect this immune challenge or respond to it (line 116).

Thank you for the hint, we corrected the text.

(5) The expression and localization of Pkc53E in larval blood cells should be investigated, for instance using the Pkc53E-GFP line recently published by Shieh et al. (or at least at the RNA level).

Firstly, we confirmed expression of Pkc53E in hemocytes by RT-PCR (Figure 8_S1 supplement). Secondly, expression of Pkc53E-GFP was monitored in hemocytes (Figure 8). To this end, we used the protein trap (BL59413), since the one published by Shieh et al., 2023 is restricted to photoreceptors.

(6) It would be interesting to test the anti-pS269 antibody in immunostaining (using Su(H)S269A as negative control).

Unfortunately, the pS269 antiserum does not work in situ at all.

(7) The authors must perform a western blot with anti-pS269 in Pkc53e mutant to show that Su(H) is not phosphorylated anymore after wasp infestation.

The blot gives a negative result.

(8) It is surprising that no signal is seen in the absence of infestation with anti-pS269: the fact that Su(H)S269A have more crystal cells suggest that there is a constitutive level of phosphorylation of Su(H).

We fully agree: In the ideal world, we would expect a low level of S269 phosphorylation in the wild type as well. However, given the lousy specificity of our antibody, we were happy to see phospho-Su(H) in infected larvae. We are currently working hard to get a better antibody. 

(9) The authors should check Su(H)-VP16 levels and phosphorylation status after PMA and/or staurosporine treatment. Some clarifications are also needed to explain the impact of PMA in Su(H)S269 larvae (this clearly suggests that PKC has other substrates implicated in crystal cell development).

Su(H)-VP16 expression levels were monitored by Western blot and were not altered conspicuously (Figure 5_1 supplement). Presumably, Pkc53E is not the only kinase involved in Su(H) phosphorylation or the transduction of stress signals. Moreover, PMA may have a more general effect on larval development and hematopoiesis affecting both genotypes. We included this reasoning in the text.

(10) Concerning the redaction, the authors forgot to mention and discuss the work of Cattenoz et al. (EMBO J 2020). The presentation of the screen for kinase candidates could be streamlined and better illustrated (notably supplement table 4, which would be easier to grasp as a figure/graph). The discussion could be shortened (notably the part on T cells), and I don't really understand lines 374-376 (why is it consistent?).

We are sorry for omitting Cattenoz et al. 2020, which we have now included. We fully agree that this paper is of utmost importance to our work. We streamlined the screen and included a new figure in addition to table 4 summarizing the results graphically (Figure 3_S1 supplement). We cut on the T cell part and omitted the strange lines.

Reviewer #2 (Public Review):

Summary:

The current draft by Deischel et.al., entitled "Inhibition of Notch activity by phosphorylation of CSL in response to parasitization in Drosophila" decribes the role of Pkc53E in the phosphorylation of Su(H) to downregulate its transcriptional activity to mount a successful immune response upon parasitic wasp-infection. Overall, I find the study interesting and relevant especially the identification of Pkc53E in phosphorylation of Su(H) is very nice. However, I have a number of concerns with the manuscript which are central to the idea that link the phosphorylation of Su(H) via Pkc53E to implying its modulation of Notch activity. I enlist them one by one subsequently.

Strengths:

I find the study interesting and relevant especially because of the following:

(1) The identification of Pkc53E in phosphorylation of Su(H) is very interesting.

(2) The role of this interaction in modulating Notch signaling and thereafter its requirement in mounting a strong immune response to wasp infection is also another strong highlight of this study.

Weaknesses:

(1) Epistatic interaction with Notch is needed: In the entire draft, the authors claim Pkc53E role in the phosphorylation of Su(H) is down-stream of notch activity. Given the paper title also invokes Notch, I would suggest authors show this in a direct epistatic interaction using a Notch condition. If loss of Notch function makes many more lamellocytes and GOF makes less, then would modulating Pkc53E (and SuH)) in this manifest any change? In homeostasis as well, given gain of Notch function leads to increased crystal cells the same genetic combinations in homeostasis will be nice to see.

While I understand that Su(H) functions downstream of Notch, but it is now increasingly evident that Su(H) also functions independent of Notch. An epistatic relationship between Notch and Pkc will clarify if this phosphorylation event of Su(H) via Pkc is part of the canonical interaction being proposed in the manuscript and not a non-canoncial/Notch pathway independent role of Su(H).

This is important, as I worry that in the current state, while the data are all discussed inlight of Notch activity, any direct data to show this affirmatively is missing. In our hands we do find Notch independent Su(H) function in immune cells, hence this is a suggestion that stems from our own personal experience.

The role of Notch in Drosophila hematopoiesis, notably during crystal cell development in both hematopoietic compartments is well established; likewise the role of Su(H) as integral signal transducer in this context (e.g. Duvic et al., 2002). Not only promotes Notch activity crystal cell fate by upregulating target genes, at the same time it prevents adopting the alternative plasmatocyte fate (e.g. Terriente-Felix et al., 2013). We could confirm the downregulation of Notch target gene expression in response to wasp infestation by qRT-PCR, which was discovered earlier by Small et al. (2014). This is clearly in favor of a repression of Notch activity rather than a relief of inhibition by Su(H). A ligand-independent activation of Notch signaling has been uncovered in the context of crystal cell maintenance in the lymph gland involving Sima/Hif-α, including Su(H) as transcriptional mediator (Mukherjee et al., 2011). However, we are unaware of a respective Su(H) activity independent of Notch.

Certainly, Su(H) acts independently of Notch in terms of gene repression. Here, Su(H) forms a repressor complex together with H and co-repressors Groucho and CtBP to silence Notch target genes. Accordingly, loss of Su(H) or H may induce the upregulation of respective gene expression independent of Notch activity. This has been demonstrated, for example, during wing and heart development (Klein et al., 2000; Kölzer, Klein, 2006; Panta et al., 2020). Moreover, during axis formation of the early embryo, global repression is brought about by Su(H) and relieved by activated Notch (Koromila, Stathopolous, 2019). In all these instances, Su(H) is thought to act as a molecular switch, and the activation of Notch causes a strong expression of the respective genes. Likewise, the loss of DNA-binding resulting from the phosphorylation of Su(H) allows the upregulation of repressed Notch target genes in wing imaginal discs, e.g. dpn, as we have demonstrated before with overexpression and clonal analyses (Nagel et al. 2017; Frankenreiter et al., 2021). However, H does not contribute to crystal cell homeostasis, i.e. de-repression of Notch target genes does not appear to be a major driver in this context, asking for additional mechanisms to downregulate Notch activity. Our work provides evidence that these inhibitory mechanisms involves the phosphorylation of Su(H) by Pkc53E. Formally, we cannot exclude alternative mechanisms. Hence, we have tried to avoid the direct link between Su(H) phosphorylation and the inhibition of Notch activity throughout the text, including the title. Moreover, we have discussed the possible consequences of Su(H) lack of DNA binding, interfering either with the activation of Notch target genes or abrogating their repression.

In addition, we have performed new experiments addressing the epistasis between Notch and Su(H) during crystal cell formation (Figure 1_supplement 1). To this end, we knocked down Notch activity in hemocytes by RNAi (hml::N-RNAi) in the Su(H)gwt and Su(H)S269A background, respectively. Indeed, Notch downregulation strongly impairs crystal cell development independent of the genetic background as expected if Notch were epistatic to Su(H). We attribute the slightly elevated crystal cell numbers observed in the Su(H)S269A background to the increase in the embryonic precursors (see Fig. 4; Frankenreiter et al. 2021). Of note, the Notch gain of function allele Ncos479 also displayed a likewise increase in embryonic crystal cell precursors as well as in crystal cells within the lymph gland (Frankenreiter et al. 2021).

(2) Temporal regulation of Notch activity in response to wasp-infection and its overlapping dynamics of Su(H) phosphorylation via Pkc is needed:

First, I suggest the authors to show how Notch activity post infection in a time course dependent manner is altered. A RT-PCR profile of Notch target genes in hemocytes from infected animals at 6, 12, 24, 48 HPI, to gauge an understanding of dynamics in Notch activity will set the tone for when and how it is being modulated. In parallel, this response in phospho mutant of Su(H) will be good to see and will support the requirement for phosphorylation of Su(H) to manifest a strong immune response.

Indeed, it would be extremely nice to follow the entire processes in every detail, ideally at the cellular level. The challenge, however, is quantities. The mRNA isolated from hemocytes could be barely quantified, although the subsequent ct-values were ok. We quantified NRE-GFP expression, introduced into Su(H)gwt and Su(H)S269A, as well as atilla expression. We were able to generate data for two time slots, 0-6 h and 24-30 h post infection. The data are provided in the extended Figure 1G, and show a strong drop of NRE-GFP in the infected Su(H)gwt control compared to the uninfected animals, whereas expression in Su(H)S269A plateaus at around 60%-70% of the infected Su(H)gwt control. Atilla expression jumps up in the control, but stays low in Su(H)S269A hemocytes.

Second, is the dynamics of phosphorylation in a time course experiment is missing. While the increased phosphorylation of Su(H) in response to wasp-infestation shown in Fig.2B is using whole animal, this implies a global down-regulation of Su(H)/Notch activity. The authors need to show this response specifically in immune cells. The reader is left to the assumption that this is also true in immune cells. Given the authors have a good antibody, characterizing this same in circulating immune cells in response to infection will be needed. A time course of the phosphorylation state at 6, 12, 24, 48 HPI, to guage an understanding of this dynamics is needed.

We really would love to do these experiments. Unfortunately, our pS269 antibody is rather lousy. It does not allow to detect Su(H) protein in tissue or cells, nor does it work on protein extracts in Westerns or for IP. Hence, we have no way so far to demonstrate cell or tissue specificity of Su(H) phosphorylation. So far, we were lucky to detect mCherry-tagged Su(H) proteins pulled down in rather large amounts with the highly specific nano-bodies. We have tried very hard to repeat the experiment with hemolymph and lymph glands only, but we have failed so far. Hence, we have to state that our antibody is neither suitable for in vivo analyses, nor for a detection of phospho-Su(H) at lower levels.

The authors suggest, this mechanism may be a quick way to down-regulate Notch, hence a side by side comparison of the dynamics of Notch down-regulation (such as by doing RT-PCR of Notch target genes following different time point post infection) alongside the levels of pS269 will strengthen the central point being proposed.

We fully agree and hope to address these issues in the future by improving our tools.

Last, in Fig7. the authors show Co-immuno-precipitation of Pkc53EHA with Su(H)gwt-mCh 994 protein from Hml-gal4 hemocytes. I understand this is in homeostasis but since this interaction is proposed to be sensitive to infection, then a Co-IP of the two in immune cells, upon infection should be incorporated to strengthen their point.

We do not fully agree with the reviewer. Although we also think that the interaction between Pkc53E and Su(H) might occur more frequently upon infection, we propose that this is a transient process occurring in several but not all hemocytes at a given time. Moreover, in the described experiment, Pkc53E-HA was expressed in hemocytes via the UAS/Gal4 system. We cannot exclude that this approach causes an overexpression. Hence, we would not expect considerable differences between unchallenged and infested animals.

(3) In Fig 5B, the authors show the change in crystal cell numbers as read out of PMA induced activation of Pkc53E and subsequent inhibition of Su(H) transcriptional activity, I would suggest the authors use more direct measures of this read out. RT-PCR of Su(H) target genes, in circulating immune cells, will strengthen this point. Formation of crystal cells is not just limited to Notch, I am not convinced that this treatment or the conditions have other affect on immune cells, such as any impact on Hif expression may also lead to lowering of CC numbers. Hence, the authors need to strengthen this point by showing that effects are direct to Notch and Su(H) and not non-specific to any other pathway also shown to be important for CC development.

We agree with the Reviewer that the rather general influence of PMA on PKCs might present a systemic stress to the animal. For example, we observed a slight drop of crystal cell numbers also in Su(H)S269A, suggesting other kinases apart from Pkc53E were affected that are involved in crystal cell homeostasis. We have included this notion in the text. To provide more conclusive evidence we also fed Staurosporine to the larvae which reversed the PMA effect. In addition, we assayed the expression of NRE-GFP in hemocytes of infected animals by qRT-PCR, and observed a strong drop in the infected versus uninfected control but less so in Su(H)S269A. The new data are provided in extended Figures 1G and 5B.

(4) In addition to the above mentioned points, the data needs to be strengthened to further support the main conclusions of the manuscript. I would suggest the authors present the infection response with details on the timing of the immune response. Characterization of the immune responses at respective time points (as above or at least 24 and 48 HPI, as norms in the field) will be important. Also, any change in overall cell numbers, other immune cells, plasmatocytes or CC post infection is missing and is needed to present the specificity of the impact. The addition of these will present the data with more rigor in their analysis.

Total hemocyte numbers of the various genotypes, i.e. control, Su(H)S269A, Su(H)S269D, and Pkc53ED28 were included before and after wasp infestation in supplemental Figures 1_S1 and 9_S1. 

(5) Finally, what is the view of the authors on what leads to activation of Pkc53E, any upstream input is not presented. It will be good to see if wasp infection leads to increased Pkc53 kinase activity.

The analysis of the full process is an ongoing project. We propose that ROS is produced upon the wasps’ sting, which is to trigger the subsequent cascade of events. These have to end with activation of Pkc53E in the presumptive pre-lamellocyte pool of both lineages, i.e. in plasmatocyte of the hemolymph, presumably in the sessile compartment (Tattikotta et al., 2021) and at the same time in the lymph gland cortex harboring the LM precursors (Blanco-Obregon et al., 2020). One of the known upstream kinases, Pdk1 has a similar impact on crystal cell development as Pkc53E, making its involvement likely. Moreover, we think that other PKCs influence the process as well.

Without a good read out, e.g. a functional pSu(H) antiserum working in situ or a Pkc-activity reporter, it will be quite difficult to follow up this question. However, we already know that Pkc53E is expressed in hemocytes of all types independent of wasp infestation, in agreement with a role during lamellocyte differentiation. We hope to unravel the process in more of it in the future.

Overall, I think the findings in the current state are interesting and fill an important gap, but the authors will need to strengthen the point with more detailed analysis that includes generating new data and also presenting the current data with more rigor in their approach. The data have to showcase the relationship with Notch pathway modulation upon phosphorylation of CSL in a much more comprehensive way, both in homeostasis and in response to infection which is entirely missing in the current draft.

Reviewer #3 (Public Review):

Diechsel et al. provide important and valuable insights into how Notch signalling is shut down in response to parasitic wasp infestation in order to suppress crystal cell fate and favour lamellocyte production. The study shows that CSL transcription factor Su(H) is phosphorylated at S269A in response to parasitic wasp infestation and this inhibitory phosphorylation is critical for shutting down Notch. The authors go on to perform a screen for kinases responsible for this phosphorylation and have identified Pkc53E as the specific kinase acting on Su(H) at S269A. Using analysis of mutants, RNAi and biochemistry-based approaches the authors convincingly show how Pkc53E-Su(H) interaction is critical for remodelling hematopoiesis upon wasp challenge. The data presented supports the overall conclusions made by the authors. There are a few points below that need to be addressed by the authors to strengthen the conclusions:

(1) The authors should check melanized crystal cells in Su(H)gwt and Su(H)S269A in presence of PMA and Staurosporine?

Thank you for the suggestion. We included the results of PMA + Staurosporine feeding into an extended Fig. 5B; they match those from the HeLa cells. Unfortunately, Staurosporine alone was lethal for the larvae at various concentrations, presumably owing to the overarching inhibition of kinase activity. This global effect also explains the high crystal cell numbers in the control fed with PMA + STAU compared to the untreated animals, as the downregulation of many kinases results in higher crystal cell numbers, a fact uncovered in our genetic screen.

(2) Data for number of dead pupae, flies eclosed, wasps emerged post infestation should be monitored for the following genotypes and should be included:

Pkc53EΔ28_, Su(H)S269A,_ Pkc53EΔ28 Su(H)S269A, Su(H)S269D, Su(H)S269D Pkc53EΔ28

We extended the data with and without infection. The respective data are shown in a new Fig. 9 and an extended Fig. 2,  except for the Su(H)S269D allele. Su(H)S269D is larval lethal, i.e. dies too early for wasp development, and hence could not be included in the assay. Overall, Pkc53EΔ28 matched Su(H)S269A_._

(3) The exact molecular trigger for activation of Pkc53E upon wasp infestation is not clear.

Indeed, and we would love to know! Perhaps, the generation of Ca2+ by the wasp’s breach of the larval cuticle results in Pkc53E activation. The generation of ROS could be involved as well. At this point, we can only speculate. We hope to be able in the future to obtain direct experimental evidence for the one or the other hypothesis.

(4) The authors should check if activating ROS alone or induction of Calcium pulses/DUOX activation can mimic this condition and can trigger activation of Pkc53E and thereby cause phosphorylation of Su(H) at S269

The reviewer’s suggestions open up a new field of investigations, and are hence beyond of the scope of this article. However, we want to pursue the research in this direction, albeit we realize that counting crystal cells is too coarse but to give a first impression, and that lamellocytes may form already by breaching the larval cuticle. A major challenge shall be direct measurements of Pkc53E activation. To date, we have no tools for this, but ideally, we would like to have a direct, biochemical read out. Although we have been unsuccessful in the past, we want to develop a strong and specific phospho-S269 antibody that is also working in situ. Alternatively, we think of developing a PS-phosphorylation reporter, to allow reasonably addressing these questions.

(5) Does Pkc53E get activated during sterile inflammation?

We are in the process of addressing this issue, however, feel that his topic is beyond the scope of this paper. Our preliminary experiments, however, support the notion of a phospho-dependent regulation of Su(H) also in this context.

Reviewer #3 (Recommendations For The Authors):

The authors provide a graphical representation of major phenotypes that form the basis of their investigation and conclusions but have not supplemented the quantitation with images that represent these phenotypes. The authors need to include the following data to strengthen their conclusions:

(1) The authors should include representative images for each of the genotypes/conditions (in presence and absence of wasp infestation) based on which corresponding plots have been made in Figure 1. Please include this for both circulating lamellocytes in the hemolymph and in the lymph glands since this is one of the main figures presenting the key findings.

The data have been included in Figure 1-S2 supplement.

(2) Please include representative images of LG with Hnt staining and corresponding images for melanization for each of the genotypes used in the plots in Figure 6A and B.

The data have been included in Figure 6-S2 supplement.

(3) Representative images for each of the genotypes in Figure 7A & B should be included (circulating crystal cells and lymph gland crystal cell numbers).

Representative images for each of the genotypes for Fig. 7A have been included in Figure 7-S1 and for the old Fig. 7B in Figure 9-S2 supplement, respectively.

docker容器间访问ollama localhost要变 ubuntu获取主机ip命令： hostname -I

eLife Assessment

This important study provides a new perspective on how human immunity shapes the antigenic evolution of pathogens. By combining theory and simulation the authors make a compelling case for the importance of eco-evolutionary interactions in population-level virus-host dynamics, which arise due to coupling between the dynamics of immune memories and viral variants. Although the work does not propose improved data-driven viral forecasting methods, it makes a conceptual contribution that advances the field's understanding of this problem's intrinsic difficulty.

Reviewer #1 (Public review):

In this work, the authors study the dynamics of fast-adapting pathogens under immune pressure in a host population with prior immunity. In an immunologically diverse population, an antigenically escaping variant can perform a partial sweep, as opposed to a sweep in a homogeneous population. In a certain parameter regime, the frequency dynamics can be mapped onto a random walk with zero mean, which is reminiscent of neutral dynamics, albeit with differences in higher order moments. Next, they develop a simplified effective model of time dependent selection with expiring fitness advantage, and posit that the resulting partial sweep dynamics could explain the behaviour of influenza trajectories empirically found in earlier work (Barrat-Charlaix et al. Molecular Biology and Evolution, 2021). Finally, the authors put forward an interesting hypothesis: the mode of evolution is connected to the age of a lineage since ingression into the human population. A mode of meandering frequency trajectories and delayed fixation has indeed been observed in one of the long-established subtypes of human influenza, albeit so far only over a limited period from 2013 to 2020. The paper is overall interesting and well-written.

In the revised version, the authors have addressed questions on the role of clonal interference by new simulations in the SI, clarified the connection between the SIR model and vanishing-fitness models, and placed their analysis into the broader context of consumer resource dynamics.

However, the general conclusion, as stated in the abstract, that variant trajectories become unpredictable as a consequence of the SIR dynamics remains somewhat misleading. Two aspects contribute to this problem. (1) The empirical observation of ``quasi-neutrality', i.e. the absence of a net frequency increase inferred as an average of many trajectories at intermediate frequencies, does not imply that individual trajectories are neutral (i.e., fully stochastic and unpredictable) over the time span of observation. Rather, it just says that some have a positive and some have a negative selection coefficient over that time span. (2) As stated by the authors, the observation of average quasi-neutrality is indeed incompatible with the travelling wave model, where initially successful new variants are assumed to retain a fixed, positive selection coefficient from origination to fixation. This observation also limits predictions by extrapolation, where a positive selection coefficient inferred at small frequency is assumed to remain the same at later times and higher frequencies. However, predictions derived from Gog and Grenfell's multi-strain SIR model, as used by several authors, do not make the assumption of fixed selection coefficients and incorporate trajectory-specific, time-dependent expiration effects into their model predictions. This distinction remains blurred throughout the text of the paper.

Reviewer #3 (Public review):

In this work the authors present a multi-strain SIR model in which viruses circulate in a heterogeneous population with different groups characterized by different cross-immunity structures. They reformulate the qualitative features of these SIR dynamics as a random walk characterized by new variants saturating at intermediate frequencies. Then they recast their microscopic description to an effective formalism in which viral strains lose fitness independently from one another. They study several features of this process numerically and analytically, such as the average variants frequency, the probability of fixation, and the coalescent time. They compare qualitatively the dynamics of this model to variants dynamics in RNA viruses such as flu and SARS-CoV-2

The idea that vanishing fitness mechanisms that produce partial sweeps may explain important features of flu evolution is very interesting. Its simplicity and potential generality make it a powerful framework. As noted by the authors, this may have important implications for predictability of virus evolution and such a framework may be beneficial when trying to build predictive models for vaccine design. The vanishing fitness model is well analyzed and produces interesting structures in the strains coalescent. Even though the comparison with data is largely qualitative, this formalism would be helpful when developing more accurate microscopic ingredients that could reproduce viral dynamics quantitatively.<br /> This general framework has the potential to be more universal than human RNA viruses, in situations where invading mutants would saturate at intermediate frequencies.

The qualitative connection between the coarse-grained features of these vanishing fitness dynamics and structured SIR processes offers additional intuition relevant to host-pathogens interactions, although as noted by the authors other ecological processes could drive similar evolutionary patterns. The additions in the revised manuscript, substantiating more thoroughly the connection between the SIR and the vanishing fitness description, are important to better appreciate the scope of the work.

Author response:

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

Response to reviewers

We thank the Editor and the Reviewers for their constructure review. In the light of this feedback, we have made a number of changes and additions to the manuscript, that we think improved the presentation and hopefully address the majority of the concerns by the reviewers.

Main changes:

•   We added a new SI section (B1) with a population dynamics simulation in the high clonal interference regime and without expiring fitness (see R1: (1)).

•   We added a new SI section (A9) with the derivation of the equilibrium state of our SIR model in the case of 𝑀 immune groups and in the limit 𝜀 → 0 (see R1: (5)).

•   The text of the section Abstraction as “expiring” fitness advantage has been modified.

•   We added a new SI section (A4) describing the links between parameters of the “expiring fitness” and SIR models.

All three reviewers had concerns about the relation between our SIR model and the “expiring fitness” model, that we hope will be addressed by the last two items listed above. In particular, we would like to underline the following points:

•   The goal of our SIR model is to give a mechanistic explanation of partial sweeps using traditional epidemiological models. While ecological models (e.g. consumer resource) can give rise to the same phenomenology, we believe that in the context of host-pathogen interaction it is relevant to explicitely show that SIR models can result in partial sweeps.

•   The expiring fitness model is mainly an effective model: it reproduces some qualitative features of the SIR but does not quantitatively match all aspects of the frequency dynamics in SIR models.

•   It is possible to link the parameters of the SIR (𝛼,𝛾,𝑏,𝑓) and expiring fitness (𝑠,𝑥,𝜈) models at the beginning of the invasion of the variant (new SI section A4). However, the two models also differ in significant ways (the SIR model can for example oscillate, while the effective model can not). The correspondence of quantities like the initial invasion rate and the ‘expiration rate’ of fitness effects is thus only expected to hold for some time after the emergence of a novel variant.

Public reviews:

Reviewer 1:

Summary In this work, the authors study the dynamics of fast-adapting pathogens under immune pressure in a host population with prior immunity. In an immunologically diverse population, an antigenically escaping variant can perform a partial sweep, as opposed to a sweep in a homogeneous population. In a certain parameter regime, the frequency dynamics can be mapped onto a random walk with zero mean, which is reminiscent of neutral dynamics, albeit with differences in higher order moments. Next, they develop a simplified effective model of time dependent selection with expiring fitness advantage, and posit that the resulting partial sweep dynamics could explain the behaviour of influenza trajectories empirically found in earlier work (Barrat-Charlaix et al. Molecular Biology and Evolution, 2021). Finally, the authors put forward an interesting hypothesis: the mode of evolution is connected to the age of a lineage since ingression into the human population. A mode of meandering frequency trajectories and delayed fixation has indeed been observed in one of the long-established subtypes of human influenza, albeit so far only over a limited period from 2013 to 2020. The paper is overall interesting and well-written. Some aspects, detailed below, are not yet fully convincing and should be treated in a substantial revision.

We thank the reviewer for their constructive criticism. The deep split in the A/H3N2 HA segment from 2013 to 2020 is indeed the one of the more striking examples of such meandering frequency dynamics in otherwise rapidly adapting populations. But the up and down of H1N1pdm clade 5a.2a.1 in recent years might be a more recent example. We argue that such meandering dynamics might be a common contributor to seasonal influenza dynamics, even if it only spans 3-6 years.

(1) The quasi-neutral behaviour of amino acid changes above a certain frequency (reported in Fig, 3), which is the main overlap between influenza data and the authors’ model, is not a specific property of that model. Rather, it is a generic property of travelling wave models and more broadly, of evolution under clonal interference (Rice et al. Genetics 2015, Schiffels et al. Genetics 2011). The authors should discuss in more detail the relation to this broader class of models with emergent neutrality. Moreover, the authors’ simulations of the model dynamics are performed up to the onset of clonal interference 𝜌/ 𝑠0 \= 1 (see Fig. 4). Additional simulations more deeply in the regime of clonal interference (e.g. 𝜌/ 𝑠0 \= 5) show more clearly the behaviour in this regime.

We agree with the reviewer that we did not discuss in detail the effects of clonal interference on quasi-neutrality and predictability. As suggested, we conducted additional simulations of our population model in the regime of high clonal interference (𝜌/ 𝑠0 ≫ 1) and without expiring fitness effects. The results are shown in a new section of the supplementary information. These simulations show, as expected, that increasing clonal interference tends to decrease predictability: the fixation probability of an adaptive mutation found at frequency 𝑥 moves closer to 𝑥 as 𝜌 increases. However, even in a case of strong interference 𝜌/ 𝑠0 \= 32, 𝑝fix remains significantly different from the neutral expectation. We conclude from this that while it is true that dynamics tend to quasi-neutrality in the case of strong interference, this effect alone is unlikely to explain observations of H3N2 influenza dynamics. In our previous publication (BarratCharlaix et al, MBE, 2021) we have also investigated the effect of epistatic interactions between mutations, along side strong clonal interference. We concluded that, while most of these processes make evolution less predictable and push 𝑝fix towards the diagonal, it is hard to reproduce the empirical observations with realistic parameters. The “expiring fitness” model, however, produces this quite readily.

But there are qualitative differences between quasi-neutrality in traveling wave models and the expiring fitness model. In the traveling wave, a genotype carrying an adaptive mutation is always fitter than if it didn’t carry the mutation. Quasi-neutrality emerges from the accumulation of fitness variation at other loci and the fact that the coalescence time is not much bigger than the inverse selection coefficient of the mutation. In the expiring fitness model, the selective effect of the mutation itself goes away with time. We now discuss the literature on quasi-neutrality and cite Rice et al. 2015 and Schiffels et al. 2011.

In this context, I also note that the modelling results of this paper, in particular the stalling of frequency increase and the decrease in the number of fixations, are very similar to established results obtained from similar dynamical assumptions in the broader context of consumer resource models; see, e.g., Good et al. PNAS 2018. The authors should place their model in this broader context.

We thank the reviewer for pointing out the link between consumer resource models and our work. We further strengthened our discussion of the similarity of the phenomenology to models typically used in ecology and made an effort to highlight the link between consumer-resource models and ours in the introduction and in the part on the SIR model.

(2) The main conceptual problem of this paper is the inference of generic non-predictability from the quasi-neutral behaviour of influenza changes. There is no question that new mutations limit the range of predictions, this problem being most important in lineages with diverse immune groups such as influenza A(H3N2). However, inferring generic non-predictability from quasi-neutrality is logically problematic because predictability refers to individual trajectories, while quasi-neutrality is a property obtained by averaging over many trajectories (Fig. 3). Given an SIR dynamical model for trajectories, as employed here and elsewhere in the literature, the up and down of individual trajectories may be predictable for a while even though allele frequencies do not increase on average. The authors should discuss this point more carefully.

We agree with the reviewer that the deterministic SIR model is of course predictable. Similarly, a partial sweep is predictable. But we argue that expiring fitness makes evolution less predictable in two ways: (i) When a new adaptive mutation emerges and rises in frequency, we typically don’t know how rapidly its fitness effect is ‘expiring’. Thus even if we can measure its instantaneous growth rate accurately, we can’t predict its fate far into the future. (ii) Compared to the situation where fitness effects are not expiring, time to fixation is longer and there are more opportunities for novel mutations to emergence and change the course of the trajectory. We have tried to make this point clearer in the manuscript.

(3) To analyze predictability and population dynamics (section 5), the authors use a Wright-Fisher model with expiring fitness dynamics. While here the two sources of the emerging neutrality are easily tuneable (expiring fitness and clonal interference), the connection of this model to the SIR model needs to be substantiated: what is the starting selection 𝑠0 as a function of the SIR parameters (𝑓,𝑏,𝑀,𝜀), the selection decay 𝜈 = 𝜈(𝑓,𝑏,𝑀,𝜀,𝛾)? This would enable the comparison of the partial sweep timing in both models and corroborate the mapping of the SIR onto the simplified W-F model. In addition, the authors’ point would be strengthened if the SIR partial sweeps in Fig.1 and Fig.2 were obtained for a combination of parameters that results in a realistic timescale of partial sweeps.

We added a new section to the SI (A4) that relates the parameters of the SIR and expiring fitness models. In particular, we compute the initial growth rate 𝑠0 and a proxy for the fitness expiry rate 𝜈 as a function of the SIR parameters 𝛼,𝛾,𝑓,𝑏,𝑀, at the instant where the variant is introduced. The initial growth rate depends primarily on the degree of immune escape 𝑓, while the expiration rate 𝜈 is related to incidence 𝐼wt + 𝐼𝑚. However, as both models have fundamentally different dynamics, these relations are only valid on time scales shorter than potential oscillations of the SIR model. Beyond that, the connection between the models is mostly qualitative: both rely on the fact that growth rate of a strain diminishes when the strain becomes more frequent, and give rise to partial sweeps.

In Figure 1, the time it takes a partial sweep to finish is roughly 100− 200 generations (bottom right panel). If we consider H3N2 influenza and take one generation to be one week, this corresponds to a sweep time of 2 to 4 years, which is slightly slower but roughly in line with observations for selective sweeps. This time is harder to define if oscillatory dynamics takes place (middle right panel), but the time from the introduction of the mutant to the peak frequency is again of about 4 years. The other parameters of the model correspond to a waning time of 200 weeks and immune escape on the order of 20-30% change in susceptibility.

Reviewer 2:

Summary

This work addresses a puzzling finding in the viral forecasting literature: high-frequency viral variants evince signatures of neutral dynamics, despite strong evidence for adaptive antigenic evolution. The authors explicitly model interactions between the dynamics of viral adaptations and of the environment of host immune memory, making a solid theoretical and simulation-based case for the essential role of host-pathogen eco-evolutionary dynamics. While the work does not directly address improved data-driven viral forecasting, it makes a valuable conceptual contribution to the key dynamical ingredients (and perhaps intrinsic limitations) of such efforts.

Strengths

This paper follows up on previous work from these authors and others concerning the problem of predicting future viral variant frequency from variant trajectory (or phylogenetic tree) data, and a model of evolving fitness. This is a problem of high impact: if such predictions are reliable, they empower vaccine design and immunization strategies. A key feature of this previous work is a “traveling fitness wave” picture, in which absolute fitnesses of genotypes degrade at a fixed rate due to an advancing external field, or “degradation of the environment”. The authors have contributed to these modeling efforts, as well as to work that critically evaluates fitness prediction (references 11 and 12). A key point of that prior work was the finding that fitness metrics performed no better than a baseline neutral model estimate (Hamming distance to a consensus nucleotide sequence). Indeed, the apparent good performance of their well-adopted “local branching index” (LBI) was found to be an artifact of its tendency to function as a proxy for the neutral predictor. A commendable strength of this line of work is the scrutiny and critique the authors apply to their own previous projects. The current manuscript follows with a theory and simulation treatment of model elaborations that may explain previous difficulties, as well as point to the intrinsic hardness of the viral forecasting inference problem.

This work abandons the mathematical expedience of traveling fitness waves in favor of explicitly coupled eco-evolutionary dynamics. The authors develop a multi-compartment susceptible/infected model of the host population, with variant cross-immunity parameters, immune waning, and infectious contact among compartments, alongside the viral growth dynamics. Studying the invasion of adaptive variants in this setting, they discover dynamics that differ qualitatively from the fitness wave setting: instead of a succession of adaptive fixations, invading variants have a characteristic “expiring fitness”: as the immune memories of the host population reconfigure in response to an adaptive variant, the fitness advantage transitions to quasi-neutral behavior. Although their minimal model is not designed for inference, the authors have shown how an elaboration of host immunity dynamics can reproduce a transition to neutral dynamics. This is a valuable contribution that clarifies previously puzzling findings and may facilitate future elaborations for fitness inference methods.

The authors provide open access to their modeling and simulation code, facilitating future applications of their ideas or critiques of their conclusions.

We thank the reviewer for their summary, assessement, and constructive critique.

(1) The current modeling work does not make direct contact with data. I was hoping to see a more direct application of the model to a data-driven prediction problem. In the end, although the results are compelling as is, this disconnect leaves me wondering if the proposed model captures the phenomena in detail, beyond the qualitative phenomenology of expiring fitness. I would imagine that some data is available about cross-immunity between strains of influenza and sarscov2, so hopefully some validation of these mechanisms would be possible.

We agree with the reviewer that quantitatively confronting our model with data would be very interesting. Unfortunately, most available serological data for influenza and SARS-CoV-2 is obtained using post-infection sera from previoulsy naive animal models. To test our model, we would require human serology data, ideally demographically resolved, and a way to link serology to transmission dynamics. Furthermore, our model is mostly an explanation for qualitative features of variant dynamics and their apparent lack of predictability. We therefore considered that quantitative validation using data is out of scope of this work.

(2) After developing the SIR model, the authors introduce an effective “expiring fitness” model that avoids the oscillatory behavior of the SIR model. I hoped this could be motivated more directly, perhaps as a limit of the SIR model with many immune groups. As is, the expiring fitness model seems to lose the eco-evolutionary interpretability of the SIR model, retreating to a more phenomenological approach. In particular, it’s not clear how the fitness decay parameter 𝜈 and the initial fitness advantage 𝑠0 relate to the key ecological parameters: the strain cross-immunity and immune group interaction matrices.

The expiring fitness model emerges as a limiting case, at least qualitatively, of the SIR model when growth rate of the new variant is small compared to the waning rate and the SIR model does not oscillate. This can be readily achieved by many immune groups, which reconciles the large effect of many escape mutations and the lack of oscillation by confining the escape to some fraction of the population. Beyond that, the expiring fitness model is mainly an effective model that allows us to study the consequences of partial sweeps on predictability on long timescales. As stated in the “Main changes” section at the start of this reply, we added an SI section which links parameters of the two models. However, we underline the fact that beyond the phenomenon of partial sweeps, the dynamics of the two are different.

Reviewer 3:

Summary

In this work the authors start presenting a multi-strain SIR model in which viruses circulate in an heterogeneous population with different groups characterized by different cross-immunity structures. They argue that this model can be reformulated as a random walk characterized by new variants saturating at intermediate frequencies. Then they recast their microscopic description to an effective formalism in which viral strains lose fitness independently from one another. They study several features of this process numerically and analytically, such as the average variants frequency, the probability of fixation, and the coalescent time. They compare qualitatively the dynamics of this model to variants dynamics in RNA viruses such as flu and SARS-CoV-2.

Strengths

The idea that a vanishing fitness mechanisms that produce partial sweeps may explain important features of flu evolution is very interesting. Its simplicity and potential generality make it a powerful framework. As noted by the authors, this may have important implications for predictability of virus evolution and such a framework may be beneficial when trying to build predictive models for vaccine design. The vanishing fitness model is well analyzed and produces interesting structures in the strains coalescent. Even though the comparison with data is largely qualitative, this formalism would be helpful when developing more accurate microscopic ingredients that could reproduce viral dynamics quantitatively. This general framework has a potential to be more universal than human RNA viruses, in situations where invading mutants would saturate at intermediate frequencies.

We thank the reviewer for their positive remarks and constructive criticism below.

Weaknesses

The authors build the narrative around a multi-strain SIR model in which viruses circulate in an heterogeneous population, but the connection of this model to the rest of the paper is not well supported by the analysis. When presenting the random walk coarse-grained description in section 3 of the Results, there is no quantitative relation between the random walk ingredients importantly 𝑃(𝛽) - and the SIR model, just a qualitative reasoning that strains would initially grow exponentially and saturate at intermediate frequencies. So essentially any other microscopic description with these two features would give rise to the same random walk.

As also highlighted in the response to other reviewers, we now discuss how the parameter of the SIR model are related to the initial growth rate and the ‘expiration’ rate of the effective model. While the phenomenology of the SIR model is of course richer, this correspondence describes its overdamped limit qualitatively well.

Currently it’s unclear whether the specific choices for population heterogeneity and cross-immunity structure in the SIR model matter for the main results of the paper. In section 2, it seems that the main effect of these ingredients are reduced oscillations in variants frequencies and a rescaled initial growth rate. But ultimately a homogeneous population would also produce steady state coexistence between strains, and oscillation amplitude likely depends on parameters choices. Thus a homogeneous population may lead to a similar coarse-grained random walk.

The reviewer is correct that the primary effects of using many immune groups is to slow down the increase of novel variant, which in turn dampens the oscillations. Having multiple immune groups widens the parameter space in which partial sweeps without dramatic oscillations are observed. For slow sweeps, similar dymamics are observed in a homogeneous population.

Similarly, it’s unclear how the SIR model relates to the vanishing fitness framework, other than on a qualitative level given by the fact that both descriptions produce variants saturating at intermediate frequencies. Other microscopic ingredients may lead to a similar description, yet with quantitative differences.

Both of these points were also raised by other reviewers and we agree that it is worth discussing them at greater length. We now discuss how the parameters of the ‘expiring fitness’ model relate to those of the SIR. We also discuss how other models such as ecological models give rise to similar coarse grained models.

At the same time, from the current analysis the reader cannot appreciate the impact of such a mean field approximation where strains lose fitness independently from one another, and under what conditions such assumption may be valid.

In the SIR model, the rate at which strains lose fitness does depend on the precise state of the host population through the quantities 𝑆𝑚 and 𝑆wt , which is apparent in equation (A27) of the new SI section. The fact that a new variant shifts the equilibrium frequencies of previous strains in a proportional way is valid if the “antigenic space” is of very high dimensions, as explained in section Change in frequency when adding subsequent strains of the SI. It would indeed be interesting to explore relaxations of this assumption by considering a larger class of cross immunity matrices 𝐾. However, in the expiring fitness model, the fact that strains lose fitness independently from each ohter is a necessary simplification.

In summary, the central and most thoroughly supported results in this paper refer to a vanishing fitness model for human RNA viruses. The current narrative, built around the SIR model as a general work on host-pathogen eco-evolution in the abstract, introduction, discussion and even title, does not seem to match the key results and may mislead readers. The SIR description rather seems one of the several possible models, featuring a negative frequency dependent selection, that would produce coarse-grained dynamics qualitatively similar to the vanishing fitness description analyzed here.

We have revised the text throughout to make the connections between the different parts of the manuscript, in particular the SIR model and the expiring fitness model, clearer. We agree that the phenomenology of the expiring fitness model is more general than the case of human RNA viruses described by the SIR model, but we think this generality is an attractive feature of the coarse-graining, not a shortcoming. Indeed, other settings with negative frequency dependent selection or eco-systems that adapt on appropriate time scale generate similar dynamics.

Recommendations for the authors:

Reviewer 1:

(4) Line 74: what does fitness mean?

Many population dynamics models, including ones used for viral forecasting, attach a scalar fitness to each strain. The growth rate of each strain is then computed by substracting the average population fitness to the strain’s fitness. In this sentence, fitness is intended in this way.

(5) Fig. 1: The equilibrium frequency in the middle and bottom rows is hardly smaller than the equilibrium frequency in the top row for one immune group. This is surprising since for M=10, the variant escapes in only 1/10th of the population, which naively should impact the equilibrium frequency more strongly. Could the authors comment on this?

This is indeed non-trivial, and a hand-waving argument can be made by considering the extreme case 𝜀 = 0. The variant is then completely neutral for the immune groups 𝑖 > 1, and would be at equilibrium at any frequency in these immune groups. Its equilibrium frequency is then only determined by group 1, which is the only one breaking degeneracy. For 𝜀 > 0 but small, we naturally expect a small deviation from the 𝜀 = 0 case and thus 𝛽 should only change slightly.

A more rigorous argument with a mathematical proof in the case 𝜀 = 0 is now given in section A4 of the supplementary information.

(6) Fig. 1: In the caption, it is stated that the simulations are performed with 𝜀 = 0.99. Is this a typo? It seems that it should be 𝜀 = 0.01, as in and just below equation (7).

This was indeed a typo. It is now fixed.

(7) Fig. 3: The data analysis should be improved. In order to link the average frequency trajectories to standard population genetics of conditional fixation probabilities, the focal time should always be the time where the trajectory crosses the threshold frequency for the first time. Plotting some trajectories from a later time onwards, on their downward path destined to loss, introduces a systematic bias towards negative clonal interference (for these trajectories, the time between the first and the second crossing of the threshold frequency is simply omitted). The focal time of first crossing of the threshold frequency can easily be obtained, e.g., by linear interpolation of the trajectory between subsequent time points of frequency evalution. In light of the modified procedure, the statements on the on the inertia of the trajectories after crossing 𝑥⋆ (line 356) should be re-examined.

The way we process the data is already in line with the suggestions of the reviewer. In particular, we use as focal time the first time at which a trajectory is found in the threshold frequency bin. Trajectories that are never seen in the bin because of limited time-resolution are simply ignored.

In Fig. 3, there are no trajectories that are on their downward path at the focal time and when crossing the threshold frequency. Our other work on predictability of flu Barrat-Charlaix et. al. (2021) has a similar figure, which maybe created confusion.

(8) Fig. 4: authors write 𝛼/ 𝑠0 in the figure, but should be 𝜈/ 𝑠0.

Fixed.

(9) Line 420: authors refer to the blue curve in panel B as the case with strong interference. However, strong interference is for higher 𝜌/ 𝑠0, that is panel D (see point 1).

Fixed.

(10) Line 477: typo “there will a variety of mutations”.

Fixed.

Reviewer 2:

Should 𝛼 be 𝜈 in Figure 4 legends?

Thank you very much for spotting this error. We fixed it.

Equations 4-5 could be further simplified.

We factorised the 𝐼 term in equation 4. In equation 5, we prefered to keep the 1− 𝛿/ 𝛼 term as this quantity appears in different calculations concerning the model. For instance, 𝑆 = 𝛿/ 𝛼 at equilibrium.

The sentence before equation 8 references 𝑃𝛽(𝛽), but this wasn’t previously introduced.

We now introduce 𝑃𝑏𝜂 at the beginning of the section Ultimate fate of the variant.

In the last paragraph of page 12, “monotonously” maybe should be “monotonically”.

Fixed.

For the supplement section B, you might want a more descriptive title than “other”.

We renamed this section to Expiring fitness model and random walk.

Reviewer 3:

To expand on my previous comments, my main concerns regard the connection of section 2 and the SIR model with the rest of the paper.

In the first paragraph of page 9 the authors argue that a stochastic version of the SIR model would lead to different fixation dynamics in homogeneous vs heterogeneous populations due to the oscillations. This paragraph is quite speculative, some numerical simulations would be necessary to quantitatively address to what extent these two scenarios actually differ in a stochastic setting, and how that depends on parameters.

Likewise, the connection between the SIR model, the random walk coarse-grained description and the vanishing fitness model can be investigated through numerical simulations of a stochastic SIR given the chosen population and cross-immunity structures with i.e. 10-20 strains. This would allow for a direct comparison of individual strain dynamics rather than the frequency averages, as well as other scalar properties such as higher moments, coalescent, and fixation probability once reaching a given frequency. It would also be possible to characterize numerically the SIR P(beta) bridging the gap with the random walk description. It’s not obvious to me that the SIR P(beta) would not depend on the population size in the presence of birth-death stochasticity, potentially changing the moments scalings. I appreciate that such simulations may be computationally expensive, but similar numerical studies have been performed in previous phylodynamics works so it shouldn’t be out of reach.

An alternative, the authors should consider re-centering the narrative directly on the random walk of the vanishing fitness model, mentioning the SIR more briefly as a possible qualitative way to get there. Either way the authors should comment on other ways in which this coarse-grained dynamics could arise.

In the vanishing fitness model, where variants fitnesses are independent, is an infinite dimensional antigenic space implicitly assumed? If that’s the case, it should be explained in the main text.

A long simulation of the SIR model would indeed be interesting, but is numerically demanding and our current simulation framework doesn’t scale well for many strains and susceptibilities. We thus refrained from adding extensive simulations.

In Figure 2B of the main text, the simulation with 7 strains illustrates the qualitative match between the expiring fitness and the SIR model. However, it is clearly not long enough to discuss statistical properties of the corresponding random walk. Furthermore, we do not expect the individual strain dynamics of the SIR and expiring fitness models to match. The latter depends on few parameters (𝛼, 𝑠0), while the former depends on the full state of the host population and of the previous variants.

In the sectin linking the parameters of the two models, we now discuss the distribution 𝑃(𝛽) of the SIR model for two strains and a specific choice of distribution for the cross immunity 𝑏 and 𝑓.

Minor comments:

There is some back and forth in the writing. For instance, when introducing the model, 𝐶𝑖𝑗 is first defined as 1/ 𝑀, then a few paragraphs later the authors introduce that in another limit 𝐶𝑖𝑖 is just much higher than any 𝐶𝑖𝑗, and finally they specify that the former is the fast mixing scenario.

Another example is in section 2, in the first paragraph they put forward that heterogeneity and crossimmunity have different impacts on the dynamics, but the meaning attributed to these different ingredients becomes clear only a while later after the homogeneous population analysis. Uniforming the writing would make it easier for the reader to follow the authors’ train of thought.

We removed the paragraph below Equation (1) mentioning the 𝐶𝑖𝑗 \= 1/ 𝑀 case, which we hope will linearize the writing.

When mentioning geographical structure, why would geography affect how immunity sees pairs of viral strains (differences in 𝐾)?

Geographic structure could influence cross-immunity because of exposure histories of hosts. For instance in the case of influenza, different geographical regions do not have the same dominating strains in each season, and hosts from different regions may thus build up different immunity.

In the current narrative there are some speculations about non-scalar fitness, especially in section 2. The heterogeneity in this section does not seem so strong to produce a disordered landscape that defies the notion of scalar fitness in the same way some complex ecological systems do. A more parsimonious explanation for the coexistence dynamics observed here may be a negative frequency dependent selection.

Our language here was not very precise and we agree that the phenomenology we describe is related to that of frequency dependent selection (mediated by via immunity of the host population that integrates past frequencies). Traveling wave models typically use fitness function that are independent of the population distribution and only account for the evolution via an increasing average fitness. We have made discussion more accurate by stating that we consider a case where fitness depends explicitly on present and past population composition, which includes the case of negative frequency dependent selection.

I don’t understand the comparison with genetic drift (typo here, draft) in the last paragraph of section 3 given that there is no stochasticity in growth death dynamics.

We compare the random walk to genetic drift because of the expression of the second moment of the step size. The genetic draft has the same functional form. If one defines the effective population size as in the text, the drift due to random sampling of alleles (neutral drift) and the changes in strain frequency in our model have the same first and second moments. The stochasticity here does not come from the dynamics, which are indeed deterministic, but from the appearance of new mutations (variants) on backgrounds that are randomly sampled in the population. This latter property is shared with genetic draft.

In the vanishing fitness model, I think the reader would benefit from having 𝑃(𝑠) in the main text, and it should be made more clear what simulations assume what different choice of 𝑃(𝑠).

We added the expression of 𝑃(𝑠) in the main text. Simulations use the value 𝑠0 \= 0.03, which we added in the caption of Figure 4.

When comparing the model and data, is the point that COVID is not reproduced due to clonal interference? It seems from the plot that flu has clonal interference as well though. Why is that negligible?

A similar point has been raised by the first reviewer (see R1-(1)). Clonal interference is not negligible, but we find it to be insufficient to explain the observations made for H3N2 influenza, namely the lack of inertia of frequency trajectories or the probability of fixation. This is shown in the new section (B1) of the SI. Both SARS-CoV-2 and H3N2 influenza experience clonal interference, but the former is more predictable than the latter. Our point is that expiring fitness effects should be stronger in influenza because of the higher immune heterogeneity of the host population, making it less predictable than SARS-CoV-2.

Does the fixation probability as a function of frequency threshold match the flu data for some parameters sets?

For H3N2 influenza, the fixation probability is found to be equal to the threshold frequency (see Barrat-Charlaix MBE 2021, also indirectly visible from Fig. 3). In Figure 4, we obtain that either a high expiry rate or intermediate expiry rates and clonal interference regimes match this observation.

It would be instructive to see examples of the individual variant dynamics of the vanishing fitness model compared to the presented data.

We added an extra SI figure (S7) showing 10 randomly selected trajectories of individual variants in the case of H3N2/HA influenza and for the expiring fitness model with different parameter choices.

Figure 4E has no colorbar label. The reader shouldn’t have to look for what that means in the bottom of the SIs. In panels A and B the label should be 𝜈, not 𝛼. Same thing in most equations of page 42.

We added the colorbar label to the figure and also updated the caption: a darker color corresponds to a higher probability of sweeps to overlap. We fixed the 𝜈 – 𝛼 confusion in the SI and in the caption of the figure.

Author response:

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

Recommendations For The Authors:

Reviewer #1:

●      It might help the reader if you make it explicit that mDES allows you to create an approximate amalgam of different kinds of experiences by assuming that, across individuals, there is a general consensus of experiences at particular points in the movie. Whether this assumption is an accurate reflection of the way in which each individual's brain is an important, testable prediction that could be discussed/examined in different projects. For instance, in other projects there are clear idiosyncratic responses to the same naturalistic stimuli: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8064646/.

Thank you, this is an excellent point. We have included this article in our revision and expanded on the introduction to emphasize how this study relates to our work. Additionally, we have included an additional figure that helps illustrate how mDES can be used to evaluate the idiosyncrasy for each respective thought component to visually display the variance across moments in the film:

Page 6-7 [137-148] In our study, we used multi-dimensional experience sampling (mDES) to describe ongoing thought patterns during the movie-watching experience [8]. mDES is an experience sampling method that identifies different features of thought by probing participants about multiple dimensions of their experiences. mDES can provide a description of a person’s thoughts, generating reliable thought patterns across laboratory cognitive tasks [22, 32, 33] and in daily life [34, 35], and is sensitive to accompanying changes in brain activity [24, 36]. Studies that use mDES to describe experience ask participants to provide experiential reports by answering a set of questions about different features of their thought on a continuous scale from 1 (Not at all) to 10 (Completely) [24, 32-41]. Each question describes a different feature of experience such as if their thoughts are oriented in the future or the past, about oneself or other people, deliberate or intrusive in nature, and more (See methods for a full list of questions used in the current study).

●      A cartoon describing the mDES technique could be helpful for uninitiated readers.

Thank you for your suggestion, we have added an additional figure (Figure 3) that illustrates the process of mDES in the laboratory during this experiment, clarifying that participants answer mDES items using a slider to indicate their score (rather than expressing it verbally).

●      Did the authors check for any measures of reliability across mDES estimates other than split-half reliability? For instance, the authors could demonstrate construct validity by showing that engagement with certain features of the thought-sampling space aligned with specific points in the movies. If so, the start of the Results section would be a great place to demonstrate the reliability of the approach. For instance, did any two participants sample the same 15-second window of time in a particular stimulus? If so, you could compare their experience samples to determine whether the method was extensible across subjects.

This is a great point, thank you very much for highlighting this. We have eight individuals at each time point in our analysis, which is probably not enough to calculate meaningful reliability measures. However, we have added a time series analysis of experience in each clip to our revision (Figure 3). In these time plots, it is possible to see clear moments in the film in which scores do not straddle 0 (using 95% CI), and often, these persist across successive moments (Figure 3; see time-series plot four for the clearest example).  When the confidence intervals of a sampling epoch do not overlap with zero, this suggests a high degree of agreement in thought content across participants. At the same time, our analysis shows that individual differences do exist since the relative presence of each component for each participant was linked to objective measures of movie watching (in this case, comprehension). In this revision we have specifically addressed this question by conducting ANOVAs to determine how scores on each component across the clip (See also supplementary table 11). This additional analysis shows that mDES effectively captures shared aspects of movie-watching and is also sensitive to individual variation (since it can describe individual differences).

Page 15 [304-323]: Next, we examined how each pattern of thought changes across each movie clip. For this analysis, we conducted separate ANOVA for each film clip for the four components (see Table 1 and Figure 3). Clear dynamic changes were observed in several components for different films. We analyzed these data using an Analysis of Variance (ANOVA) in which the time in each clip were explanatory variables of interest. This identified significant change in “Episodic Social Cognition” scores across Little Miss Sunshine, F(1, 712) = 10.80, p = .001, , η2 = .03, and Citizenfour, F(1, 712) = 5.23, p = .023, , η2 = .02. There were also significant change in “Verbal Detail” scores across Little Miss Sunshine, F(1, 712) = 31.79, p <.001, η2 = .09. Lastly, there were significant changes in “Sensory Engagement” scores for both Citizenfour, F(1, 712) = 6.22, p = .013, η2 = .02, and 500 Days of Summer, F(1, 706) = 80.41, p <.001, η2 = .18. These time series are plotted in Figure 3 and highlight how mDES can capture the dynamics of different types of experience across the three movie clips. Moreover, in several of these time series plots, it is clear that thought patterns reported extend beyond adjacent time periods (e.g. scores above zero between time periods 150 to 400 for Sensory Engagement in 500 days of Summer and for time periods between 175 and 225 for Verbal Detail in Little Miss Sunshine). It is important to note that no participant completed experience sampling reports during adjacent sampling points (see Supplementary Figure 7), so the length of these intervals indicates agreement in how specific scenes within a film were experienced and conserved across different individuals. Notably, the component with the least evidence for temporal dynamics was “Intrusive Distraction.”

●      P10: "Generation of the thought-space" - how stable are these word clouds to individual subjects? If there are subject-specific differences, are there ways to account for this with some form of normalization?

Thank you for bringing up this point. Our current goal was to show how the average experience of one group of participants relates to the brain activity of a second group. In this regard it is important to seek the patterns of similarity across individuals in how they experience the film. However, as is normal in our studies using mDES, we can also use the variation from the mean to predict other cognitive measures and, in this way, account for the variability that individuals have in their movie-watching experience. In other words, the word clouds reflect the mean of a particular dimension, so when an individual score is close to 0, their thought content does not align with this dimension -- however, deviating scores, positive or negative, indicating that this dimension provides meaningful information about the individual's experience. Evidence of the meaningful nature of this variation can be seen in the links between the reported thoughts and the individuals’ comprehension (e.g. individuals whose thoughts do not contain strong evidence of “Intrusive Distraction”, or in other words, a negative score, tended to do better on comprehension tests of information in the movies they watched).

●      P11: "Variation in thought patterns" - can the authors use a null model here to demonstrate that the associations they've observed would occur above chance levels (e.g., for a comparison of time series with similar temporal autocorrelation but non-preserved semantic structure)? Further, were there any pre-defined hypotheses over whether any of the three different movies would engage any of the 4 observed dimensions?

This is a great point. We chose to sample from three distinctly different films to help us understand if mDES was sensitive to different semantic and affective features of films. Our analysis, therefore, shows that at a broad level, mDES is able to discriminate between films, highlighting its broad sensitivity to variation in semantic or affective content. Armed with this knowledge, researchers in the future could derive mechanistic insights into how the semantic features may influence the mDES data. For example, future studies could ask participants to watch movies in a scrambled order to understand how varying the structure of semantics or information breaks the mapping between brains and ongoing experience. In this revision we have amended the text to reflect this possibility:

Page 34 [674-679]. Our analysis shows that mDES is able to discriminate between films, highlighting its broad sensitivity to variation in semantic or affective content. Armed with this knowledge, we propose that in the future, researchers could derive mechanistic insights into how the semantic features may influence the mDES data. For example, it may be possible to ask participants to watch movies in a scrambled order to understand how the structure of semantic or information influences the mapping between brains and ongoing experience as measured by mDES.

●      P14: "Brain - Thought Mappings: Voxel-space Analysis" - this is a cool analysis, and a nice validation of the authors' approach. I would personally love to see some form of reliability analysis on these approaches - e.g., do the same locations in the cerebral cortex align with the four features in all three movies? Across subjects?

This is another great point, and we thank you for your enthusiasm. The data we have has only sampled mDES during a relatively short period of brain activity which we suspect would make an individual-by-individual analysis underpowered. In the future, however, it may be possible to adopt a precision mapping approach in which we sample mDES during longer periods of movie watching and identify how group-level mappings of experience relate to brain activity within a single subject. To reflect this possibility, we have amended the text in this revision in the following way:

Page 34-35 [672-687]: In addition, our study is correlational in nature, and in the future, it could be useful to generate a more mechanistic understanding of how brain activity maps onto the participants' experience. Our analysis shows that mDES is able to discriminate between films, highlighting its broad sensitivity to variation in semantic or affective content. Armed with this knowledge, we propose that in the future, researchers could derive mechanistic insights into how the semantic features may influence the mDES data. For example, it may be possible to ask participants to watch movies in a scrambled order to understand how the structure of semantic or information influences the mapping between brains and ongoing experience as measured by mDES. Finally, our study focused on mapping group-level patterns of experience onto group-level descriptions of brain activity. In the future, it may be possible to adopt a “precision-mapping” approach by measuring longer periods of experience using mDES and determining how the neural correlates of experience vary across individuals who watched the same movies while brain activity was collected [1]. In the future, we anticipate that the ease with which our method can be applied to different groups of individuals and different types of media will make it possible to build a more comprehensive and culturally inclusive understanding of the links between brain activity and movie-watching experience

Reviewer #2:

(1) The three-dimensional scatter plot in Figure 2 does not represent "Intrusive Distraction." Would it make sense to color-code dots by this important dimension?

Thank you for this suggestion. Although it could be possible to indicate the location of each film in all four dimensions, we were worried that this would make the already complex 3-D space confusing to a naive reader. In this case, we prefer to provide this information in the form of bar graphs, as we did in the previous submission.

(2) The coloring of neural activation patterns in Figure 3 is not distinct enough between the different dimensions of thought. Please reconsider color intensities or coding. The same applies to the left panel in Figure 4.

Thanks for this comment; we found it quite difficult to find a colour mapping that allows us to show the distinction between four states in a simple manner, yet we believe it is valuable to show all of the results on a similar brain. Nonetheless, to provide a more fine-grained viewing of our results in this revision we have provided a supplementary figure (Supplementary Figure 6) that shows each of the observed patterns of activity in isolation.

(3) The new method (mDES) is mentioned too often without explanation, making it hard to follow without referring to the methods section. It would be helpful to state prominently that participants rated their thoughts on different dimensions instead of verbalizing them.

Thank you for this point, we have adjusted the Introduction to clarify and expand on the mDES method. We have also included an example of the mDES method in an additional figure that we have now included to visually express how participants respond to mDES probes (Figure 3).

Page 6-7 [136-148]: In our study, we used multi-dimensional experience sampling (mDES) to describe ongoing thought patterns during the movie-watching experience [2]. mDES is an experience sampling method that identifies different features of thought by probing participants about multiple dimensions of their experiences. mDES can provide a description of a person’s thoughts, generating reliable thought patterns across laboratory cognitive tasks [3-5] and in daily life [6, 7], and is sensitive to accompanying changes in brain activity when reports are gained during scanning [8, 9]. Studies that use mDES to describe experience ask participants to provide experiential reports by answering a set of questions about different features of their thought on a continuous scale from 1 (Not at all) to 10 (Completely) [3, 5-14]. Each question describes a different feature of experience, such as if their thoughts are oriented in the future or the past, about oneself or other people, deliberate or intrusive in nature, and more (See Methods for a full list of questions used in the current study).

Author response image 1.

(4) Reporting of single-movie thought patterns seems quite extensive. Could this be condensed in the main text?

Thank you for this point, upon re-visiting the manuscript, we have adjusted the text to be more concise.

Reviewer #3:

●      This is a very elegant experiment and seems like a very promising approach. The text is currently hard to read.

Thank you for this point, we have since revisited the text and adjusted the manuscript to be more concise and add more clarity.

●      The introduction (+ analysis goals) fails to explain the basic aspects of the analysis and dataset. It is not clear how many participants and datapoints were used to establish the group-level thought patterns, nor is it entirely clear that the fMRI data is a separate existing dataset. Some terms are introduced and highlighted and never revisited (e.g decoupled states and the role of the DMN).

Thank you for this critique, we have since adjusted the introduction to clearly explain the difference between Sample 1 and Sample 2 and further clarify that the fMRI data is an entirely separate, independent sample compared to the laboratory mDES sample:

Page 7-8 [158-174]: Thus, to overcome this obstacle, we developed a novel methodological approach using two independent sample participants. In the current study, one set of 120 participants was probed with mDES five times across the three ten-minute movie clips (11 minutes total, no sampling in the first minute). We used a jittered sampling technique where probes were delivered at different intervals across the film for different people depending on the condition they were assigned. Probe orders were also counterbalanced to minimize the systematic impact of prior and later probes at any given sampling moment. We used these data to construct a precise description of the dynamics of experience for every 15 seconds of three ten-minute movie clips. These data were then combined with fMRI data from a different sample of 44 participants who had already watched these clips without experience sampling [15]. By combining data from two different groups of participants, our method allows us to describe the time series of different experiential states (as defined by mDES) and relate these to the time series of brain activity in another set of participants who watched the same films with no interruptions. In this way, our study set out to explicitly understand how the patterns of thoughts that dominate different moments in a film in one group of participants relate to the brain activity at these time points in a second set of participants and, therefore, better understand the contribution of different neural systems to the movie-watching experience.

Page 8-9 [177-188] The goal of our study, therefore, was to understand the association between patterns of brain activity over time during movie clips in one group of participants and the patterns of thought that participants reported at the corresponding moment in a different set of participants (see Figure 1). This can be conceptualized as identifying the mapping between two multi-dimensional spaces, one reflecting the time series of brain activity and the other describing the time series of ongoing experience (see Figure 1 right-hand panel). In our study, we selected three 11-minute clips from movies (Citizenfour, Little Miss Sunshine and 500 Days of Summer) for which recordings of brain data in fMRI already existed (n = 44) [15] (Figure 1, Sample 1). A second set of participants (n = 120) viewed the same movie clips, providing intermittent reports on their thought patterns using mDES (Figure 1, Sample 2). Our goal was to understand the mapping between the patterns of brain activity at each moment of the film and the reports of ongoing thought recorded at the same point in the movies.

●      It is unclear what the utility of the method is - is it meant to be done in fMRI studies on the same participants? Or is the idea to use one sample to model another?

Great point, thank you for highlighting this important question. This paper aimed to interrogate the relationship between experience and neural states while preserving the novelty of movie-watching. Although it could be done in the same sample, it may be difficult to collect frequent reports of experience without interrupting the dynamics of the brain. However, in the future it could be possible to collect mDES and brain activity in the same individuals while they watched movies. For example, our prior studies (e.g. [9]) where we combined mDES with openly-available brain data activity during tasks. In the future, this online method could also be applied during movie watching to identify direct mapping between brain activity and films. However, this online approach would make it very expensive to produce the time series of experience across each clip given that it would require a large number of participants (e.g. 200 as we used in our current study). The following has been included in our manuscript:

Page 7 [149-159] One challenge that arises when attempting to map the dynamics of thought onto brain activity during movie watching is accounting for the inherently disruptive nature of experience sampling: to measure experience with sufficient frequency to map the dynamics of thoughts during movies would disrupt the natural dynamics of the brain and would also alter the viewer’s experience (for example, by pausing the film at a moment of suspense). Therefore, if we periodically interrupt viewers to acquire a description of their thoughts while recording brain activity, this could impact capturing important dynamic features of the brain. On the other hand, if we measured fMRI activity continuously over movie-watching (as is usually the case), we would lack the capacity to directly relate brain signals to the corresponding experiential states. Thus, to overcome this obstacle, we developed a novel methodological approach using two independent sample participants

●      The conclusions currently read as somewhat trivial (e.g "Our study, therefore, establishes both sensory and association cortex as core features of the movie-watching experience", "Our study supports the hypothesis that perceptual coupling between the brain and external input is a core feature of how we make sense of events in movies").

Thank you for this comment. In this revision we have attempted to extend the theoretical significance of our work in the discussion (for example, in contrasting the links between Intrusive distraction and the other components). To this end we have amended the text in this revision by including the following sections:

Page 33-35 [654-687]: Importantly, our study provides a novel method for answering these questions and others regarding the brain basis of experiences during films that can be applied simply and cost-effectively. As we have shown mDES can be combined with existing brain activity allowing information about both brain activity and experience to be determined at a relatively low cost.  For example, the cost-effective nature of our paradigm makes it an ideal way to explore the relationship between cognition and neural activity during movie-watching during different genres of film. In neuroimaging, conclusions are often made using one film in naturalistic paradigm studies [16]. Although the current study only used three movie clips, restraining our ability to form strong conclusions regarding how different patterns of thought relate to specific genres of film, in the future, it will be possible to map cognition across a more extensive set of movies and discern whether there are specific types of experience that different genres of films engage. One of the major strengths of our approach, therefore, is the ability to map thoughts across groups of participants across a wide range of movies at a relatively low cost.

Nonetheless, this paradigm is not without limitations. This is the first study, as far as we know, that attempts to compare experiential reports in one sample of participants with brain activity in a second set of participants, and while the utility of this method enables us to understand the relationship between thought and brain activity during movies, it will be important to extend our analysis to mDES data during movie watching while brain activity is recorded. In addition, our study is correlational in nature, and in the future, it could be useful to generate a more mechanistic understanding of how brain activity maps onto the participants experience. Our analysis shows that mDES is able to discriminate between films, highlighting its broad sensitivity to variation in semantic or affective content. Armed with this knowledge, we propose that in the future, researchers could derive mechanistic insights into how the semantic features may influence the mDES data. For example, it may be possible to ask participants to watch movies in a scrambled order to understand how the structure of semantic or information influences the mapping between brains and ongoing experience as measured by mDES. Finally, our study focused on mapping group-level patterns of experience onto group-level descriptions of brain activity. In the future it may be possible to adopt a “precision-mapping” approach by measuring longer periods of experience using mDES and determining how the neural correlates of experience vary across individuals who watched the same movies while brain activity was collected [1]. In the future, we anticipate that the ease with which our method can be applied to different groups of individuals and different types of media will make it possible to build a more comprehensive and culturally inclusive understanding of the links between brain activity and movie-watching experience

●      The beginning of the discussion is very clear and explains the study very well. Some of it could be brought up in the intro/analysis goal sections.

Thank you for this comment, this is an excellent idea. We have revisited the introduction and analysis goals section to mirror this clarity across the manuscript.

●      The different components are very interesting, and not entirely clear. Some examples in the text could help. Especially regarding your thought that verbal components would refer to a "decoupled" mental verbal analysis participants might be performing in their thoughts.

Thank you for this point. We would prefer not to elaborate on this point since, at present, it would simply be conjecture based on our correlational design. However, we have included a section in the discussion which explains how, in principle, we would draw more mechanistic conclusions (for example, by shuffling the order of scenes in a movie as suggested by another reviewer). In the current revision, we have amended the text in the following way:

Page 34 [674-679]: Our analysis shows that mDES is able to discriminate between films, highlighting its broad sensitivity to variation in semantic or affective content. Armed with this knowledge, we propose that in the future, researchers could derive mechanistic insights into how the semantic features may influence the mDES data. For example, it may be possible to ask participants to watch movies in a scrambled order to understand how the structure of semantic or information influences the mapping between brains and ongoing experience as measured by mDES

●      The reference to using neurosynth as performing a meta-analysis seems a little stretched.

We have adjusted the manuscript to remove ‘meta-analysis’ when referring to the analysis computed with neurosynth. Thank you for bringing this to our attention.

●      State-space is defined as brain-space in the methods.

Thank you, we have since updated this.

●      It could be useful to remind the reader what thought and brain spaces are at the top of the state-space results section.

This is an excellent point, and it has since been updated to remind the reader of thought- and brain-space. Thank you for this comment.

Page 24 [458-467]: Our next analysis used a “state-space” approach to determine how brain activity at each moment in the film predicted the patterns of thoughts reported at these moments (for prior examples in the domain of tasks, see [12, 17], See Methods). In this analysis, we used the coordinates of the group average of each TR in the “brain-space” and the coordinates of each experience sampling moment in the “thought-space.”. To clarify, the location of a moment in a film in “brain-space” is calculated by projecting the grand mean of brain activity for each volume of each film against the first five dimensions of brain activity from a decomposition of the Human Connectome Project (HCP) resting state data, referred to as Gradients 1-5. “Thought-space” is the decomposition of mDES items to create thought pattern components, referred to as “Episodic Knowledge”, “Intrusive Distraction”, “Verbal Detail” and “Sensory Engagement.”

●      DF missing from the t-test for episodic knowledge/grad 4.

Thank you for catching this, the degrees of freedom has since been included in this revision.

Page 24 [474-476]: First, we found a significant main effect of Gradient 4 (DAN to Visual), which predicted the similarity of answers to the “Episodic Knowledge” component, t(2046) = 2.17, p = .013, η2 = .01.

Public Reviews:

Reviewer #1:

●      The lack of direct interrogation of individual differences/reliability of the mDES scores warrants some pause.

Our study's goal was to understand how group-level patterns of thought in one group of participants relate to brain activity in a different group of participants. To this end, we decomposed trial-level mDES data to show dimensions that are common across individuals, which demonstrated excellent split-half reliability. Then we used these data in two complementary ways. First, we established that these ratings reliably distinguished between the different films (showing that our approach is sensitive to manipulations of semantic and affective features in a film) and that these group-level patterns were also able to predict patterns of brain activity in a different group of participants (suggesting that mDES dimensions are also sensitive to broad differences in how brain activity emerges during movie watching). Second, we established that variation across individuals in their mDES scores predicted their comprehension of information from the films. This establishes that when applied to movie-watching, mDES is sensitive to individual differences in the movie-watching experience (as determined by an individual's comprehension). Given the success of this study and the relative ease with which mDES can be performed, it will be possible in the future to conduct mDES studies that hone in on the common and distinct features of the movie-watching experience.

Reviewer #2:

(1) The dimensions of thought seem to distinguish between sensory and executive processing states. However, it is unclear if this effect primarily pertains to thinking. I could imagine highly intrusive distractions in movie segments to correlate with stagnating plot development, little change in scenery, or incomprehensible events. Put differently, it may primarily be the properties of the movies that evoke different processing modes, but these properties are not accounted for. For example, I'm wondering whether a simple measure of engagement with stimulus materials could explain the effects just as much. How can the effects of thinking be distinguished from the perceptual and semantic properties of the movie, as well as attentional effects? Is the measure used here capturing thought processes beyond what other factors could explain?

Our study used mDES to identify four distinct components of experience, each of which had distinct behavioural and neural correlates and relationships to comprehension. Together this makes it unlikely that a single measure of engagement would be able to capture the range of effects we observed in our study. For example, “Intrusive Distraction” was associated with regions of association cortex, while the other three components highlighted regions of sensory cortex. Behaviorally, we found that some components had a common effect on comprehension (e.g. “Intrusive distraction” was related to worse comprehension across all films), while others were linked to clear benefits to comprehension in specific films (e.g. “Episodic Knowledge” was associated with better comprehension in only one of the films). Given the complex nature of these effects, it would be difficult for a single metric of engagement to explain this pattern of results, and even if it did, this could be misleading because our analysis implies that they are better explained by a model of movie-watching experience in which there are several relatively orthogonal dimensions upon which our experience can vary.

At the same time, we also found that films vary in the general types of experience they can engender. For example, Citizenfour was high on “Intrusive Distraction” and participants performed relatively low on comprehension. This shows that manipulations of the semantic and affective content of films also have implications for the movie-watching experience. This pattern is consistent with laboratory studies that applied mDES during tasks and found that different tasks evoke different types of experience (for example, patterns of ‘intrusive’ thoughts were common in movie clips that were suspenseful, [18]). At the same time, in the same study, patterns of intrusive thought across the tasks were also associated with trait levels of dysphoria reported by participants. Other studies using mDES in daily life have shown that the data can be described by multiple dimensions and that each of these types of thought is more prevalent in certain activities than others ([19]). For example, in daily life, patterns of ‘intrusive distraction’ thoughts were more prevalent when individuals were engaged in activities that were relatively unengaging (such as resting). Collectively, therefore, studies using mDES suggest that is likely that human thought is multidimensional in nature and that these dimensions vary in a complex way in terms of (a) the contexts that promote them, and (b) how they are impacted by features of the individual (whether they be traits like anxiety or depression or memory for information in a film).

(2) I'm skeptical about taking human thought ratings at face value. Intrusive distraction might imply disengagement from stimulus materials, but it could also be an intended effect of the movie to trigger higher-level, abstract thinking. Can a label like intrusive distraction be misleading without considering the actual thought and movie content?

Our method uses a data-driven approach to identify the dimensions that best describe the range of answers that our participants provided to describe their experience. We use these dimensions to understand how these patterns of thought emerge in different contexts and how they vary across individuals (in this case, in different movies, but in other studies, laboratory tasks [3, 8, 9, 12, 20-22] or activities in daily life[6, 7]). These context relationships help constrain interpretations of what the components mean. For example, “Intrusive Distraction” scores were highest in the film with the most real-world significance for the participants (Citizenfour) and were associated with worse comprehension. In daily life, however, patterns of “Intrusive Distraction” thoughts tend to occur when activities engage in non-demanding activities, like resting. Psychological perspectives on thoughts that arise spontaneously occur in this manner since there is evidence that they occur in non-demanding tasks with no semantic content (when there is almost no external stimulus to explain the occurrence of the experience, see [23]), however, other studies have shown that specific cues in the environment can also cue the experience (see [23]). Consistent with this perspective, and our current data, patterns of ‘Intrusive Distraction’ thought are likely to arise for multiple reasons, some of which are more intrinsic in nature (the general association with poor comprehension across all films) and others which are extrinsic in nature (the elevation of intrusive distraction in Citizenfour).

It is also important to note that our data-driven approach also found patterns of experience that provide more information about the content of their experience, for example, the dimension of “Episodic Knowledge” is characterized by thoughts based on prior knowledge, involving the past, and concerning oneself, and was most prevalent in the romance film (500 Days of Summer). Likewise, “Sensory Engagement” was associated with experiences related to sensory input and positive emotionality and occurred more during the romance movie (500 Days of Summer) than in the documentary (Citizenfour) and was linked to increased brain activity across the sensory systems. This shows that mDES can also provide information about the content of that experience, and discriminate between different sources of experience. In the future, it will be possible to improve the level of detail regarding the content of experiences by changing the questions used to interrogate experience.     

(3) A jittered sampling approach is used to acquire thought ratings every 15 seconds. Are ratings for the same time point averaged across participants? If so, how consistent are ratings among participants? High consistency would suggest thoughts are mainly stimulus-evoked. Low consistency would question the validity of applying ratings from one (group of) participant(s) to brain-related analyses of another participant.

In this experiment, we sampled experience every 15 seconds in each clip, and in each sampling epoch, we gained mDES responses from eight participants. Furthermore, no participant was sampled at an adjacent time point, as our approach jittered probes approximately 2 minutes apart (See Supplementary Figure 7). To illustrate the consistency of mDES data, we have included an additional figure (Figure 3) highlighting how experience varies over time in each clip. It is evident from these plots that there are distinct moments in which group-averaged reported thoughts across participants are stable and that these can extend across adjacent sampling points (i.e. when the confidence intervals of the score at a timepoint do not overlap with zero). Therefore, in some cases, adjacent sampling points, consisting of different sets of eight participants, describe their experiences as having similar positions on the same mDES dimension. This suggests that there is agreement among individuals regarding how they experienced a specific moment in a film, and in some cases, this agreement was apparent in successive sets of eight participants. Together, our findings indicate a conservation of agreement across participants that spans multiple moments in a film. A clear example of agreement on experience across multiple sets of 10 participants can be seen between 150-400 seconds in the clip from 500 Days of Summer for the dimension of “Sensory Engagement” (time series plot 4 in Figure 3).

(4) Using three different movies to conclude that different genres evoke different thought patterns (e.g., line 277) seems like an overinterpretation with only one instance per genre.

We found that mDES was able to distinguish between each film on at least one dimension of experience. In other words, information encoded in the mDES dimensions was sensitive to variation in semantic and affective experiences in the different movie clips. This provides evidence that is necessary but not sufficient to conclude that we can distinguish different genres of films (i.e. if we could not distinguish between films, then we would not be able to distinguish genres). However, it is correct that to begin answering the broader question about experiences in different genres then it would be necessary to map cognition across a larger set of movies, ideally with multiple examples of each genre.

(5) I see no indication that results were cross-validated, and no effect sizes are reported, leaving the robustness and strength of effects unknown.

Thank you for drawing this to our attention. We have re-run the LMMs and ANOVA models to include partial eta-squared values to clarify the strength of the effects in each of our reported outcomes.

Reviewer #3:

●      What are the considerations for treating high-order thought patterns that occur during film viewing as stable enough to be used across participants? What would be the limitations of this method? (Do all people reading this paper think comparable thoughts reading through the sections?)

It is likely, based on our study, that films can evoke both stereotyped thought patterns (i.e. thoughts that many people will share) and others that are individualistic. It is clear that, in principle, mDES is capable of capturing empirical information on both stereotypical thoughts and idiosyncratic thoughts. For example, clear differences in experiences across films and, in particular, during specific periods within a film, show that movie-watching can evoke broadly similar thought patterns in different groups of participants (see Figure 3 right-hand panel). On the other hand, the association between comprehension and the different mDES components indicate that certain individuals respond to the same film clip in different ways and that these differences are rooted in objective information (i.e. their memory of an event in a film clip). A clear example of these more idiosyncratic features of movie watching experience can be seen in the association between “Episodic Knowledge” and comprehension. We found that “Episodic Knowledge” was generally high in the romance clip from 500 Days of Summer but was especially high for individuals who performed the best, indicating they remembered the most information. Thus good comprehends responded to the 500 Days of Summer clip with responses that had more evidence of “Episodic Knowledge” In the future, since the mDES approach can account for both stereotyped and idiosyncratic features of experience, it will be an important tool in understanding the common and distinct features that movie watching experiences can have, especially given the cost effective manner with which these studies can be run.   

●      How does this approach differ from collaborative filtering, (for example as presented in Chang et al., 2021)?

Our study is very similar to the notion of collaborative filtering since we can use an approach that is similar to crowd-sourcing as a tool for understanding brain activity. One of its strengths is its generalizability since it is also a method that can be used to understand cognition because it is not limited to movie-watching. We can use the same mDES method to sample cognition in multiple situations in daily life ([6, 19]), while performing tasks in the behavioural lab [18, 24], and while brain activity is being acquired [8, 25, 26]. In principle, therefore, we can use mDES to understand cognition in different contexts in a common analytic space (see [27] for an example of how this could work)

Page 5 [106-110]: In our study, we acquired experiential data in one group of participants while watching a movie clip and used these data to understand brain activity recorded in a second set of participants who watched the same clip and for whom no experiential data was recorded. This approach is similar to what is known as “collaborative filtering” [28].

●      In conclusion, this study tackles a highly interesting subject and does it creatively and expertly. It fails to discuss and establish the utility and appropriateness of its proposed method.

Thank you very much for your feedback and critique. In our revision and our responses to these questions, we provided more information about the method's robustness utility and application to understanding cognition.

References

(1) Gordon, E.M., et al., Precision Functional Mapping of Individual Human Brains. Neuron, 2017. 95(4): p. 791-807.e7.

(2) Smallwood, J., et al., The neural correlates of ongoing conscious thought. Iscience, 2021. 24(3).

(3) Konu, D., et al., Exploring patterns of ongoing thought under naturalistic and conventional task-based conditions. Consciousness and Cognition, 2021. 93.

(4) Smallwood, J., et al., The default mode network in cognition: a topographical perspective. Nature Reviews Neuroscience, 2021. 22(8): p. 503-513.

(5) Turnbull, A., et al., Age-related changes in ongoing thought relate to external context and individual cognition. Consciousness and Cognition, 2021. 96: p. 103226.

(6) McKeown, B., et al., The impact of social isolation and changes in work patterns on ongoing thought during the first COVID-19 lockdown in the United Kingdom. Proceedings of the National Academy of Sciences, 2021. 118(40): p. e2102565118.

(7) Mulholland, B., et al., Patterns of ongoing thought in the real world. Consciousness and Cognition, 2023. 114: p. 103530.

(8) Konu, D., et al., A role for the ventromedial prefrontal cortex in self-generated episodic social cognition. NeuroImage, 2020. 218: p. 116977.

(9) Turnbull, A., et al., Left dorsolateral prefrontal cortex supports context-dependent prioritisation of off-task thought. Nature Communications, 2019. 10.

(10) Ho, N.S.P., et al., Facing up to the wandering mind: Patterns of off-task laboratory thought are associated with stronger neural recruitment of right fusiform cortex while processing facial stimuli. NeuroImage, 2020. 214: p. 116765.

(11) Karapanagiotidis, T., et al., Tracking thoughts: Exploring the neural architecture of mental time travel during mind-wandering. NeuroImage, 2017. 147: p. 272-281.

(12) McKeown, B., et al., Experience sampling reveals the role that covert goal states play in task-relevant behavior. Scientific Reports, 2023. 13(1): p. 21710.

(13) Vatansever, D., et al., Distinct patterns of thought mediate the link between brain functional connectomes and well-being. Network Neuroscience, 2020. 4(3): p. 637-657.

(14) Wang, H.-T., et al., Dimensions of Experience: Exploring the Heterogeneity of the Wandering Mind. Psychological Science, 2017. 29(1): p. 56-71.

(15) Aliko, S., et al., A naturalistic neuroimaging database for understanding the brain using ecological stimuli. Scientific Data, 2020. 7(1).

(16) Yang, E., et al., The default network dominates neural responses to evolving movie stories. Nature Communications, 2023. 14(1): p. 4197.

(17) Turnbull, A., et al., Reductions in task positive neural systems occur with the passage of time and are associated with changes in ongoing thought. Scientific Reports, 2020. 10(1): p. 9912.

(18) Konu, D., et al., Exploring patterns of ongoing thought under naturalistic and conventional task-based conditions. Consciousness and cognition, 2021. 93: p. 103139.

(19) Mulholland, B., et al., Patterns of ongoing thought in the real world. Consciousness and cognition, 2023. 114: p. 103530.

(20) Christoff, K., et al., Experience sampling during fMRI reveals default network and executive system contributions to mind wandering. Proc Natl Acad Sci U S A, 2009. 106(21): p. 8719-24.

(21) Zhang, M., et al., Perceptual coupling and decoupling of the default mode network during mind-wandering and reading. eLife, 2022. 11: p. e74011.

(22) Zhang, M.C., et al., Distinct individual differences in default mode network connectivity relate to off-task thought and text memory during reading. Scientific Reports, 2019. 9.

(23) Smallwood, J. and J.W. Schooler, The science of mind wandering: Empirically navigating the stream of consciousness. Annual review of psychology, 2015. 66(1): p. 487-518.

(24) Turnbull, A., et al., The ebb and flow of attention: Between-subject variation in intrinsic connectivity and cognition associated with the dynamics of ongoing experience. Neuroimage, 2019. 185: p. 286-299.

(25) Turnbull, A., et al., Left dorsolateral prefrontal cortex supports context-dependent prioritisation of off-task thought. Nature communications, 2019. 10(1): p. 3816.

(26) Mckeown, B., et al., Experience sampling reveals the role that covert goal states play in task-relevant behavior. Scientific reports, 2023. 13(1): p. 21710.

(27) Chitiz, L., et al., Mapping cognition across lab and daily life using experience-sampling. 2023.

(28) Chang, L.J., et al., Endogenous variation in ventromedial prefrontal cortex state dynamics during naturalistic viewing reflects affective experience. Science Advances, 2021. 7(17): p. eabf7129.

eLife Assessment

This study presents a valuable methodological advancement in quantifying thoughts over time. A novel multi-dimensional experience-sampling approach is presented, identifying data-driven patterns that the authors use to interrogate fMRI data collected during naturalistic movie-watching. The experimentation is inventive and the analyses carried out are convincing.

Reviewer #1 (Public review):

Summary:

The authors used a novel multi-dimensional experience sampling (mDES) approach to identify data-driven patterns of experience samples that they use to interrogate fMRI data collected during naturalistic movie-watching data. They identify a set of multi-sensory features of a set of movies that delineate low-dimensional gradients of BOLD fMRI signal patterns that have previously been linked to fundamental axes of cortical organization.

Strengths:

* The novel solution to challenges associated with experience sampling offer potential access to aspects of experience that have been challenging to assess.

Weaknesses:

* The lack of direct interrogation of individual differences/reliability of the mDES scores warrants some pause.

Reviewer #2 (Public review):

Summary:

The present study explores how thoughts map onto brain activity, a notoriously challenging question because of the dynamic, subjective, and abstract nature of thoughts. To tackle this question, the authors collected continuous thought ratings from participants watching a movie, and additionally made use of an open-source fMRI dataset recorded during movie watching as well as five established gradients of brain variation as identified in resting state data. Using a voxel-space approach, the results show that episodic knowledge, verbal detail, and sensory engagement of thoughts commonly modulate visual and auditory cortex, while intrusive distraction modulates the frontoparietal network. Additionally, sensory engagement mapped onto a gradient from primary to association cortex, while episodic knowledge mapped onto a gradient from the dorsal attention network to visual cortex. Building on the association between behavioral performance and neural activation, the authors conclude that sensory coupling to external input and frontoparietal executive control are key to comprehension in naturalistic settings.

The manuscript stands out for its methodological advancements in quantifying thoughts over time and its aim to study the implementation of thoughts in the brain during naturalistic movie watching. However, the conceptualization of thoughts remains vague, limiting the study's insights into brain function.

Strengths:

(1) The study raises a question that has been difficult to study in naturalistic settings so far but is key to understanding human cognition, namely how thoughts map onto brain activation.<br /> (2) The thought ratings introduce a novel method for continuously tracking thoughts, promising utility beyond this study.<br /> (3) The authors used diverse data types, metrics, and analyses to substantiate the effects of thinking from multiple perspectives.

Weaknesses:

(1) The distinction between thinking and stimulus processing (in the sense of detecting and assigning meaning to features, modulated by factors such as attention) remains unclear. Is "thinking" a form of conscious access or a reportable read-out from sensory and higher-level stimulus processing? Or does it simply refer to the method used here to identify different processing states?<br /> (2) The dimensions of thought appear to be directly linked to brain areas traditionally associated with core faculties of perception and cognition. For example, superior temporal cortex codes for speech information, which is also where thought reports on verbal detail localize in this study. This raises the question of whether the present study truly captures mechanisms specific to thinking and distinct from processing, especially given that individual variations in reports were not considered and movie-specific features were not controlled for.

Reviewer #3 (Public review):

This study attempted to investigate the relations between processing in the human brain during movie watching and corresponding thought processes. This is a highly interesting question, as movie watching presents a semi-constrained task, combining naturally occurring thoughts and common processing of sensory inputs across participants. This task is inherently difficult because in order to know what participants are thinking at any given moment, one has to interrupt the same thought process which is the object of study.

This study attempts to deal with this issue by aggregating staggered experience sampling data across participants in one behavioral study and using the population level thought patterns to model brain activity in different participants in an open access fMRI dataset.

The behavioral data consist of 120 participants who watched 3 11-minute movie clips. Participants responded to the mDES questionnaire: 16 visual scales characterizing ongoing thought 5 times, two minutes apart, in each clip. The 16 items are first reduced to 4 factors using PCA, and their levels are compared across the different movies. The factors are "episodic knowledge", "intrusive distraction", "verbal detail", and "sensory engagement". The factors differ between the clips, and distraction is negatively correlated with movie comprehension and sensory engagement is positively correlated with comprehension.

The components are aggregated across participants (transforming single subject mDES answers into PCA space and concatenating responses of different participants) and are used as regressors in a GLM analysis. This analysis identifies brain regions corresponding to the components. The resulting brain maps reveal activations that are consistent with the proposed mental processes (e.g. negative loading for intrusion in frontoparietal network, positive loadings for visual and auditory cortices for sensory engagement).

Then, the coordinates for brain regions which were significant for more than one component are entered into a paper search in neurosynth. It is not clear what this analysis demonstrates beyond the fact that sensory engagement contained both visual and auditory components.

The next analysis projected group-averaged brain activation onto gradients (based on previous work) and used gradient timecourses to predict the behavioral report timecourses. This revealed that high activations in gradient 1 (sensory→association) predicted high sensory engagement, and that "episodic knowledge" thought patterns were predicted by increased visual cortex activations. Then, permutation tests were performed to see whether these thought pattern related activations corresponded to well defined regions on a given cluster.

This paper is framed as presenting a new paradigm but it does little to discuss what this paradigm serves, what are its limitations and how it should have been tested. The novelty appears to be in using experience sampling from 1 sample to model the responses of a second sample.

What are the considerations for treating high-order thought patterns that occur during film viewing as stable enough to use across participants? What would be the limitations of this method? (Do all people reading this paper think comparable thoughts reading through the sections?) This is briefly discussed in the revised manuscript and generally treated as an opportunity rather than as a limitation.

In conclusion, this study tackles a highly interesting subject and does it creatively and expertly. It fails to discuss and establish the utility and appropriateness of its proposed method.

Differences between the skills that you learn starting vs finishing

eLife Assessment

This study presents an important finding on the involvement of a Caspase 3-dependent pathway in the elimination of synapses for retinogeniculate circuit refinement and eye-specific territory segregation. This work fits well with the concept of "synaptosis" which has been proposed in the past but lacked in vivo support. Despite its elegant design and many strengths, the evidence supporting the claims of the authors is incomplete, particularly regarding whether Caspase-3 expression can really be isolated to synapses vs locally dying cells, whether microglia direct or instruct synapse elimination, and whether astrocytes are also involved. The work will be of interest to investigators studying cell death pathways, neurodevelopment, and neurodegenerative disease.

Reviewer #1 (Public Review):

In this manuscript, the authors study the effects of synaptic activity on the process of eye-specific segregation, focusing on the role of caspase 3, classically associated with apoptosis. The method for synaptic silencing is elegant and requires intrauterine injection of a tetanus toxin light chain into the eye. The authors report that this silencing leads to increased caspase 3 in the contralateral eye (Figure 1) and demonstrate evidence of punctate caspase 3 that does not overlap neuronal markers like map2. However, the quantifications showing increased caspase 3 in the silenced eye (done at P5) are complicated by overlap with the signal from entire dying cells in the thalamus. The authors also show that global caspase 3 deficiency impairs the process of eye-specific segregation and circuit refinement (Figures 3-4).

The authors also report that "synapse weakening-induced caspase-3 activation determines the specificity of synapse elimination mediated by microglia but not astrocytes" (abstract). They report that microglia engulf fewer RGC axon terminals in caspase 3 deficient animals (Figure 5), and that this preferentially occurs in silenced terminals, but this preferential effect is lost in caspase 3 knockouts. Based on this, the authors conclude that caspase 3 directs microglia to eliminate weaker synapses. However, a much simpler and critical experiment that the authors did not perform is to eliminate microglia and show that the caspase 3 dependent effects go away. Without this experiment, there is no reason to assume that microglia are directing synaptic elimination.

Finally, the authors also report that caspase 3 deficiency alters synapse loss in 6-month-old female APP/PS1 mice, but this is not really related to the rest of the paper.

Reviewer #2 (Public Review):

Summary:

This manuscript by Yu et al. demonstrates that activation of caspase-3 is essential for synapse elimination by microglia, but not by astrocytes. This study also reveals that caspase 3 activation-mediated synapse elimination is required for retinogeniculate circuit refinement and eye-specific territories segregation in dLGN in an activity-dependent manner. Inhibition of synaptic activity increases caspase-3 activation and microglial phagocytosis, while caspase-3 deficiency blocks microglia-mediated synapse elimination and circuit refinement in the dLGN. The authors further demonstrate that caspase-3 activation mediates synapse loss in AD, loss of caspase-3 prevented synapse loss in AD mice. Overall, this study reveals that caspase-3 activation is an important mechanism underlying the selectivity of microglia-mediated synapse elimination during brain development and in neurodegenerative diseases.

Strengths:

A previous study (Gyorffy B. et al., PNSA 2018) has shown that caspase-3 signal correlates with C1q tagging of synapses (mostly using in vitro approaches), which suggests that caspase-3 would be an underlying mechanism of microglial selection of synapses for removal. The current study provides direct in vivo evidence demonstrating that caspase-3 activation is essential for microglial elimination of synapses in both brain development and neurodegeneration.

The paper is well-organized and easy to read. The schematic drawings are helpful for understanding the experimental designs and purposes.

Weaknesses:

It seems that astrocytes contain large amounts of engulfed materials from ipsilateral and contralateral axon terminals (Figure S11B) and that caspase-3 deficiency also decreased the volume of engulfed materials by astrocytes (Figures S11C, D). So the possibility that astrocyte-mediated synapse elimination contributes to circuit refinement in dLGN cannot be excluded.

Does blocking single or dual inactivation of synapse activity (using TeTxLC) increase microglial or astrocytic engulfment of synaptic materials (of one or both sides) in dLGN?

Author response:

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

eLife Assessment 

This study presents an important finding on the involvement of a Caspase 3-dependent pathway in the elimination of synapses for retinogeniculate circuit refinement and eye-specific territory segregation. This work fits well with the concept of "synaptosis" which has been proposed in the past but lacked in vivo support. Despite its elegant design and many strengths, the evidence supporting the claims of the authors is incomplete, particularly regarding whether Caspase-3 expression can really be isolated to synapses vs locally dying cells, whether microglia direct or instruct synapse elimination, and whether astrocytes are also involved. The work will be of interest to investigators studying cell death pathways, neurodevelopment, and neurodegenerative disease.

Regarding significance:

This study provides in vivo evidence that caspase-3 is important for synapse elimination in the visual pathway (Figure 3 and 4) and corroborates the previously proposed but not yet validated “synaptosis” hypothesis. But more significantly, we show that caspase-3 is activated in dLGN relay neurons in response to synapse inactivation (Figure 1) when synaptic competition is present (Figure 2), and that caspase-3 is important for efficient elimination of weakened synapses by microglia (Figure 5 and 6). We consider the causal link between synapse weakening/inactivation and caspase-3 activation to be the most important finding of this study and believe it is an error to not include this aspect of the study in the assessment. The mechanism by which neuronal activity influences synapse elimination is a fundamental question in neuroscience, and our study presents a significant advancement in understanding this problem.

Regarding strength of evidence:

We do not agree with the assessment that our evidence should be broadly labeled as “incomplete”. In fact, we argue that many concerns raised by the reviewers are not focused on the main claims made in this study.

(1) Regarding whether caspase-3 activation (not “expression”, which is the term used in the assessment) is isolated to synapses or occurs in entire cells, we show in Figure 1 that both types of signals can be present. The main concern of the reviewers seems to be that activated caspase-3 signals in apoptotic dLGN relay neurons are irrelevant to our analysis and confound interpretation. We argue that this is not the case.

In Figure 1, we have two sets of controls demonstrating that the observed apoptosis of dLGN relay neurons occurs specifically in response to synapse inactivation. For each animal that received TeTxLC injection in the right eye, activated caspase-3 signal is compared between the left dLGN, where most of the inactivated synapses are located, and the right dLGN, where the minority of the inactivated synapses are located (between Figure 1B and 1C, also between the first and second group of Figure 1E). We observed apoptotic neurons in the right dLGN with more inactivated synapses but not in the left dLGN with fewer inactivated synapses. The second control is between TeTxLC-injected animals (Figure 1B) and mock-injected animals (Figure 1D). We observed apoptotic relay neurons in the dLGN of TeTxLC-injected animals (Figure 1B) but not mock-injected animals (Figure 1D). Both these controls show that the observed apoptosis of dLGN relay neurons is caused by synapse inactivation.

In addition, in our synapse inactivation experiment (Figure 1), AAV-hSyn-TeTxLC is injected into the right eye and expressed only in RGCs, not in dLGN relay neurons. Since dLGN relay neurons in this experiment do not receive a perturbation that is independent of synaptic transmission, we conclude that their apoptosis occurs through synapse-dependent mechanisms.

Furthermore, if the apoptotic neurons are confounding the analysis (as implied by reviewers and editors) and do not occur through synapse-dependent mechanisms, then inhibiting both eyes with TeTxLC (Figure 2C, rightmost group) should cause high levels of caspase-3 activation, like that in the single-inhibition condition. Instead, we observe the opposite (Figure 2C, middle group) – overall caspase-3 activity goes down significantly in the dual-inhibition condition and is closer to the unperturbed condition, which can be explained by a loss of interaction between “strong” and “weak” synapses. Taken together, our data demonstrate that apoptosis of relay neurons in Figure 1 occurs specifically in response to synapse inactivation through synapse-dependent mechanisms, and the activated caspase-3 signal in the neurons should be included in our analysis.

Why does synaptic caspase-3 activation manifest in different forms: puncta, “blobs”, and cells?  This is not surprising when considering the mechanisms that neurons must utilize to spatially confine caspase-3 activation and the nature of the apoptotic signaling cascade. On one hand, it has been proposed that caspase-3 activity in dendrites can be locally confined by proteasomal degradation of cleaved caspase-3 (Erturk et al., DOI: 10.1523/JNEUROSCI.3121-13.2014 ). On the other hand, caspase-3 activation is known to trigger explosive feedback amplification of apoptotic signaling events (McComb et al., DOI: 10.1126/sciadv.aau9433 ). For caspase-3 activation to remain localized to dendrites, the negative regulation must outweigh the positive feedback amplification. By expressing TeTxLC in RGCs of one eye, we create a strong perturbation that silences a large fraction of the synapses in the retinogeniculate pathway, which likely shifts the balance between positive and negative regulation of caspase-3 activity in some relay neurons. To be more specific, if a given dLGN relay neuron receives too many inactivated synapses, which is likely the case in our perturbation, caspase-3 activity that is initially localized can overwhelm the physiological negative regulation mechanisms that act to spatially confine it, resulting in whole cell apoptosis. In fact, previous in vitro evidence (Enturk et al., DOI: 10.1523/JNEUROSCI.3121-13.2014 ) demonstrated that, while caspase-3 activation in a single distal dendrite can be locally contained, activating apoptosis signaling in dendrites proximal to the cell body can result in whole-cell apoptosis. Similarly, a few inactivated retinogeniculate synapses can elicit locally contained caspase-3 activity in dLGN relay neurons, but a large number of inactivated synapses on a single relay neuron may trigger sufficient caspase-3 activity that can lead to whole-cell apoptosis. We discussed how to interpret synapse inactivation-induced apoptosis in dLGN relay neurons both in the main text and in the discussion (line 123-132, and line 411-421).

(2) Regarding microglia, we did not claim that “microglia direct or instruct synapse elimination”. Our main claim is that caspase-3 activation is important for efficient elimination of weakened synapses by microglia. This claim emphasizes a regulatory role for caspase-3 activation in microglia-mediated synapse elimination, but not a regulatory role of microglia in synapse elimination. To be more specific, our data suggest that lack of synaptic activity induces caspase-3 activity, and caspase-3 activity in turn influences which synapses are preferentially eliminated by microglia. Therefore, the elimination specificity is fundamentally determined (i.e. instructed) by neuronal activity, not by microglia. We also did not presume the manner in which microglia engage in synapse elimination. We specifically address this point in the discussion at line 458 through 465 where we acknowledge that microglia may indirectly mediate synapse elimination by engulfing shed neuronal material. In our title and text, we use the phrase “microglia-mediated synapse elimination”, which is not the same as microglia-instructed synapse elimination and does not presume any instructive/directive role of microglia.

(3) Regarding whether astrocytes are involved, we did not challenge the notion that astrocytes play important roles in synapse elimination. Rather, our claim is that, unlike what we observed with microglia, the amount of synaptic material engulfed by astrocytes does not robustly depend on whether caspase-3 is present. We acknowledge that there might be a caspase-3 dependent phenotype that we were unable to detect (line 309-310), and that it is plausible that astrocytes mediate activity-dependent synapse elimination through other caspase-3-independent mechanisms. This claim is not central to our study, and we would like to qualify the statements in the manuscript. We will remove the phrase “but not astrocytes” in line 18 of the abstract.

In summary, using a state-of-the-art method to inactivate retinogeniculate synapses, we discovered a causal link between synapse weakening/inactivation and caspase-3 activation. Coupled with well-established in vivo assays (e.g., segregation analysis, electrophysiology, and engulfment analysis) that are used in many landmark studies we cite, we provide solid evidence supporting our claim that “caspase-3 is essential for synapse elimination driven by both spontaneous and experience-dependent neural activity”, and that “synapse weakening-induced caspase-3 activation determines the specificity of synapse elimination mediated by microglia”.

Public Reviews: 

Reviewer #1 (Public Review): 

In this manuscript, the authors study the effects of synaptic activity on the process of eye-specific segregation, focusing on the role of caspase 3, classically associated with apoptosis. The method for synaptic silencing is elegant and requires intrauterine injection of a tetanus toxin light chain into the eye. The authors report that this silencing leads to increased caspase 3 in the contralateral eye (Figure 1) and demonstrate evidence of punctate caspase 3 that does not overlap neuronal markers like map2. However, the quantifications showing increased caspase 3 in the silenced eye (done at P5) are complicated by overlap with the signal from entire dying cells in the thalamus. The authors also show that global caspase 3 deficiency impairs the process of eye-specific segregation and circuit refinement (Figures 3-4). 

The reviewer states: “this silencing leads to increased caspase 3 in the contralateral eye”. We observed increased caspase-3 activity, not protein levels, in the contralateral dLGN, not eye.

The reviewer states: “and demonstrate evidence of punctate caspase 3 that does not overlap neuronal markers like map2”. This is not accurate. We show that the punctate active caspase-3 signals overlap with the dendritic marker MAP2 (Figure S4A).

The reviewer states: “, the quantifications showing increased caspase 3 [activity] in the silenced [dLGN] (done at P5) are complicated by overlap with the signal from entire dying cells in the thalamus”. This is not accurate. The apoptotic neurons we observed are relay neurons located in the dLGN (confirmed by their morphology and positive staining of NeuN – Figure S4B-C), not “cells” of unknown lineage (as suggested by the reviewer) in the general “thalamus” area (as suggested by the reviewer). If the dying cells were non-neuronal cells, that would indeed confound our quantification and conclusions, but that is not the case.

We argue that the active caspase-3 signals in apoptotic dLGN relay neurons are not a confounding factor but a bona fide response to synaptic silencing and therefore should be included in the quantification. We have two sets of controls (please also see the general response above), one is between the strongly inactivated dLGN and the weakly inactivated dLGN in each TeTxLC-injected animal, second is between dLGN of TeTxLC-injected animals and mock-injected animals. In both controls, only the dLGN receiving strong synapse inactivation has these apoptotic dLGN relay neurons, demonstrating that these cells occur as a consequence of synapse inactivation. It is also unlikely that our perturbation is causing cell death through a non-synaptic mechanism. As mock injections do not cause apoptosis in dLGN neurons, this phenomenon is not related to surgical damage. TeTxLC is injected into the eyes and only expressed in presynaptic RGCs, not in postsynaptic relay neurons, so this phenomenon is also unlikely to be caused by TeTxLC-related toxicity. Furthermore, if apoptosis of dLGN relay neurons is not related to synapse inactivation, then when TeTxLC is injected into both eyes, one would expect to see either the same amount or more apoptotic relay neurons, but we instead observed a reduction in dLGN neuron apoptosis, suggesting a synapse-related mechanism must be responsible. Considering the above, apoptosis of relay neurons in TeTxLC-inactivated dLGN is causally linked to synapse inactivation, and active caspase-3 signals in these neurons are true signals that should be included in the quantification.

The authors also report that "synapse weakening-induced caspase-3 activation determines the specificity of synapse elimination mediated by microglia but not astrocytes" (abstract). They report that microglia engulf fewer RGC axon terminals in caspase 3 deficient animals (Figure 5), and that this preferentially occurs in silenced terminals, but this preferential effect is lost in caspase 3 knockouts. Based on this, the authors conclude that caspase 3 directs microglia to eliminate weaker synapses. However, a much simpler and critical experiment that the authors did not perform is to eliminate microglia and show that the caspase 3 dependent effects go away. Without this experiment, there is no reason to assume that microglia are directing synaptic elimination. 

The reviewer states: “microglia engulf fewer RGC axon terminals in caspase 3 deficient animals (Figure 5), and that this preferentially occurs in silenced terminals, but this preferential effect is lost in caspase 3 knockouts”. We are not sure what the reviewer means by “this preferentially occurs in silenced terminals”. Our results show that microglia preferentially engulf silenced terminals, and such preference is lost in caspase-3 deficient mice (Figure 6).

We do not understand the experiment where the reviewer suggested to: “eliminate microglia and show that the caspase 3 dependent effects go away”. To quantify caspase-3 dependent engulfment of synaptic material by microglia or preferential engulfment of silenced terminals by microglia, microglia must be present in the tissue sample. If we eliminate microglia, neither of these measurements can be made. What could be measured if microglia are eliminated is the refinement of retinogeniculate pathway. This experiment would test whether microglia are required for caspase-3 dependent phenotypes. This is not a claim made in the manuscript. Instead, we claimed caspase-3 is required for microglia to preferentially eliminate weak synapses.

We did not claim that “microglia are directing synaptic elimination”. Our claim is that synapse inactivation induces caspase-3 activity, and this caspase-3 activity in turn determines the substrate preference of microglia-mediated synapse elimination. Based on this model, it is the neuronal activity that fundamentally directs synapse elimination. Throughout the manuscript, we used the term “microglia-mediated synapse elimination”. This terminology does not assume a directive/instructive role of microglia in synapse elimination and only describes the observed engulfment of synaptic material by microglia. We also did not assume how microglia engage in synapse elimination. We acknowledge in the discussion (line 458 through 465) that microglia may mediate synapse elimination in an indirect, passive way by engulfing shed neuronal material. This topic is a matter of debate in the field (Eyo et al., DOI: 10.1126/science.adh7906 ).

Finally, the authors also report that caspase 3 deficiency alters synapse loss in 6-month-old female APP/PS1 mice, but this is not really related to the rest of the paper. 

We respectfully disagree that Figure 7 is not related to the rest of the paper. Many genes involved in postnatal synapse elimination, such as C1q and C3, have been implicated in neurodegeneration. It is therefore natural and important to ask whether the function of caspase-3 in regulating synaptic homeostasis extends to neurodegenerative diseases in adult animals. The answer to this question may have broad therapeutic impacts.

Reviewer #2 (Public Review): 

Summary: 

This manuscript by Yu et al. demonstrates that activation of caspase-3 is essential for synapse elimination by microglia, but not by astrocytes. This study also reveals that caspase 3 activation-mediated synapse elimination is required for retinogeniculate circuit refinement and eye-specific territories segregation in dLGN in an activity-dependent manner. Inhibition of synaptic activity increases caspase-3 activation and microglial phagocytosis, while caspase-3 deficiency blocks microglia-mediated synapse elimination and circuit refinement in the dLGN. The authors further demonstrate that caspase-3 activation mediates synapse loss in AD, loss of caspase-3 prevented synapse loss in AD mice. Overall, this study reveals that caspase-3 activation is an important mechanism underlying the selectivity of microglia-mediated synapse elimination during brain development and in neurodegenerative diseases. 

Strengths: 

A previous study (Gyorffy B. et al., PNSA 2018) has shown that caspase-3 signal correlates with C1q tagging of synapses (mostly using in vitro approaches), which suggests that caspase-3 would be an underlying mechanism of microglial selection of synapses for removal. The current study provides direct in vivo evidence demonstrating that caspase-3 activation is essential for microglial elimination of synapses in both brain development and neurodegeneration. 

The paper is well-organized and easy to read. The schematic drawings are helpful for understanding the experimental designs and purposes. 

Weaknesses: 

It seems that astrocytes contain large amounts of engulfed materials from ipsilateral and contralateral axon terminals (Figure S11B) and that caspase-3 deficiency also decreased the volume of engulfed materials by astrocytes (Figures S11C, D). So the possibility that astrocyte-mediated synapse elimination contributes to circuit refinement in dLGN cannot be excluded.

The experiments presented in Figure S11 aim to determine whether astrocyte-mediated synapse elimination depends on caspase- 3 signaling.  We do not claim that astrocytes are unimportant for synapse elimination or circuit refinement. We did observe a small decrease in synaptic material engulfed by astrocytes when caspase-3 is deficient, and we acknowledged that there could be defects that we were not able to detect (line 309-310). The claim that caspase-3 does not regulate astrocyte-mediated synapse elimination is not a central claim of the manuscript and we will qualify our statements in the text. We will remove the phrase “but not astrocytes” in the abstract (line 18).

Does blocking single or dual inactivation of synapse activity (using TeTxLC) increase microglial or astrocytic engulfment of synaptic materials (of one or both sides) in dLGN? 

We assume that by “blocking single or dual inactivation of synapse activity”, the reviewer refers to inactivating retinogeniculate synapses from one or both eyes.

We showed that inactivating retinogeniculate synapses from one eye (single inactivation) increases microglia-mediated engulfment of presynaptic terminals of inactivated synapses (Figure 6). We did not measure microglia-mediated engulfment of synaptic material while inactivating retinogeniculate synapses from both eyes (dual inactivation). However, based on the total active caspase-3 signal (Figure 2) in the dual inactivation scenario, we do not expect to see an increase in engulfment of synaptic material.

We did not measure astrocyte-mediated engulfment with single or dual inactivation, as we did not see a robust caspase-3 dependent phenotype in astrocyte-mediated engulfment.