diverse perspectives

open up as 1 adopts an omni optional integral perpective with universal maximalist perspective

Yes boiling the ocean is required to make the impossible inevitable

Count your blessings to find what you look for (Woah)

We must give credit to the authors

When using someone else's work word for word you must include the source for your reader.

Why plagiarize when you’re intelligent!

When you write your paper, make sure your own ideas stand out the most, and use sources only to support what you’re saying.

Accurate citations that match your Works Cited and show where each detail came from.

Citations are placed in parentheses so they stand out to the reader. It gives credit to the original author and lets the reader find the source if they want to learn more.

The tone is so serious in this section, there were no comments.

any time you use someone else's words, ideas, or research, you must give credit to the source

This made me realize that media literacy covers so much more than just the social media or news that we see on our phones every day. Older forms of media, like books, newspapers, and magazines, also shape the way that we think and understand the world. Media surrounds us in countless ways, and noticing across all formats shows how important it is to develop the skills to analyze and critically engage with everything we consume.

The interpretation of media isn't universal. What I might find funny, obvious, or harmless in a video might be confusing, unfunny, or even offensive to someone else. I regularly see this in my daily life. Sometimes I show my parents a TikTok that I found really funny, and they give me a confused expression. They don't share the same feeling. However, understanding perspectives and recognizing that people's backgrounds, experiences, and viewpoints shape how they see the world is a key aspect of media literacy. It's not all about spotting bias and errors.

I like how this frames media literacy as a conversation instead of just a lecture. It emphasizes collaboration and the value of multiple perspectives. Even though De Abreu is speaking to teachers, I can imagine how powerful this would feel if students heard it directly. I believe they might feel more confident sharing their own ideas and questioning what they see. It reminds me that learning media literacy is as much about dialogue and understanding others' viewpoints as it is about facts.

This line effectively highlights the significant influence the media has over our daily lives, often without our even being aware of it. De Abreu's call to action pushes teachers to take responsibility. However, it makes me wonder how students themselves could take a more active role in questioning what they see. Media literacy is truly about building the skills to navigate, challenge, and understand the messages that surround us every day.

This part hit hard because it shows how deep-rooted bias in media shapes public perception. By constantly under-representing or stereotyping certain groups, the media reinforces inequality. Discussing this in class would help students recognize bias, not just in obvious hate speech but in everyday portrayals. De Abreu is essentially saying: to think critically, we must see whose stories are missing—not just who is shown.

De Abreu’s argument here is powerful—schools can’t ignore how much media affects learning, identity, and truth. Media education shouldn’t be optional; it’s as essential as reading or math because students form opinions and make decisions based on what they see online. Teaching media literacy gives students the tools to tell the difference between reliable information and manipulation. It also shifts teachers’ roles from content deliverers to guides in critical reasoning.

This line really captures the heart of media literacy—it’s not about memorizing facts about media, but about questioning what we see and hear. De Abreu emphasizes that without critical thinking, media literacy becomes empty. It reminds me that consuming news or social posts passively makes us more likely to be influenced by bias or misinformation. True literacy means asking who made this, why, and how it’s shaping what I believe.

I think she is optimistic that she would find happiness as her father had in the colonies!!

https://pelicram.github.io/The-Typewriter-Blog-You-Will-Eventually-Read/blog/2023/continental-identification/

Following the correct format is best to avoid confusion and irritation.

is means MLA was made for book and print sources, so sometimes its hard to fit online sources into the same format

you're a genius

i'm sure!!!!!!

Yes, Mingi isn't the type to give up on you. He's Mingi after all. What does time mean in the face of... love? Well, too much. But also: nothing at all

i love the extended water metaphors, how water flows naturally and to work against the tide is to set yourself up for struggle. taoism and how if you have to force something too much it's the universe's way of telling you it isn't for you. oh yunho. you are fighting so much against your own want and feelings and love, the universe is trying to tell you something.

because like this he becomes attached to this being deemed bigger than him. he doesn't have to be catalyst of his own life, he can be absorbed into something else entirely, his purpose someone else's, his want something else's. small in his own life isn't small enough.

you're lying to yourself, yunho. you're lying!

He believes so much in him, I'm crying. Because to Yunho, that's all real.What he feels is so real to him... What he believes dictates everything about his life. He believes Mingi will go far and he believes he won't and so he think, he knows, it'll never work out. But he's wrong.

He wants to be Mingi's equal in every way, be the man he deserves, but he's Too Much. Too hungry. Too... Yunho.

oh my god. the word ruin here. because they'd be left unrecognizable, destroyed landscapes of people, without the other.

i feel violently ill. don't leave me behind. DONT LEAVE ME BEHIND. it's like standing still while watching everyone run ahead. don't forget me here, in your memory. i think of all the people you meet for the last time without realizing, condemning them to this thing called the past. lucky for yunho, mingi's been knocking on his door all his life, just open the door for him.

Are you perhaps fond of Song Mingi?

You know, Yunho dehumanizes himself, seeing himself so lowly, but at the same time he dehumanizes Mingi, seeing him so highly, putting him up on that pedestal. Let him be hurt, wounded, loved by you, Yunho. He's just human, too. Doesn't he deserve to be human too?

So he can Be an asshole because he sees himself that way already, he believes. I see. But also because being an asshole requires nothing of him, no true expectation, more, it's a lacking. A void of where something else should be (obedience? kindness? consideration?) What do we call that failure? And why is it we are always trying to rise above it? Why are we born way down here, so far away

WE KNOW! FUCK! i love how you implement so much of reality into your writing it is truly a touchstone of... of their relationship, wading between fiction and reality. All fiction is a true story, somewhere, sometime, for someone

the voices are so loud...

This isn't very holy of you Yunho. You're supposed to only see God... Oh silly how could I forget, he's made a mantle of this man he loves. He gets down on his knees for him. He prays. He fears. He wants more...

you romantic sap, mingudding, you romantic sap!

What is the world without judgment? What would we do? And does he perform in fear of judgment? Is that what it is? But from who? Mingi? Or himself. Because he hate himself for not being able to control this one thing... his want. It's proof of his undoing, his animal nature, his straying from God. God and religion being related to obedience, and Yunho not being able to tame his emotions.... making him just like the rest, meaning just like every other person. Human.

The dirt isn't such a terrible place to be...

my precious yunho ;-;

The no choice here, the fate of it all, he's doomed because he wants, he's saved because he wants I'd go as far as to say that his want is what makes him a human, it's what sets him off the path of fate. Because yes there is fate, it's that we're all going to die, but some of us still Want anyway. And I'm beginning to think that's the whole point.

But he does say them. They're engraved on the roof of his mouth

The performance

I love them.

This part makes me lose my mind, imagining younger Yunho not able to hold back any longer and kissing Mingi and it's Mingi's first kiss and Yunho is so far from being able to call it love. It's heartbreaking

He wants so badly for that to be enough, to be able to take from Mingi what he wants and have that be what he wants, but it isn't. It isn't enough because it's all mental and not physical. Sex is so in your head, such a mental experience, while also being so purely physical. The mind makes up for what the body can't comprehend and in that gap... it's almost as if Yunho feels so much that he cannot process it as anything but something too much for him to deserve. He feels so much his mind has to call it something else entirely. Well, baby, it's love.

so interesting to see hm fluctuate between being the thing to burn and damage mingi but then mingi to be the one to melt him... these two fire signs... an aries and a leo :,)

feels good, sounds good, tastes good - when does sense become being? And do the senses, does how we perceive anything, matter when we don't have faith? When we don't also Believe?

Fool, fall for it, the leap of faith

Did you know "satisfy" comes from the Latin noun "satis" meaning "enough."

'Be good," oh mingi you don't even know the half of it...

This is the greatest sentence of all time. My jaw dropped. The prayer engraved in the roof of his mouth, spoken over and over again like it's the only thing that could save him. Saying it out loud.

He holds God so highly here. That's fascinating. it's almost like yunho can't be the main character here, in his own life, he needs to be part of something bigger because he's afraid of just having to exist with himself and his want. so it becomes about God, it becomes a test, it becomes a test of faith, in himself and the universe.

the panopticon...

In a panel I went to once, someone said "in the pursuit of happiness, there is no justice" in regards to love and relationships. because you may Want and Want, but simply wanting isn't enough for someone to want you back or to want you in the way you want to be wanted and that isn't anyone's fault... and so there is no "fair" and there is no "justice" (i need to go dig through my notes and read more about that)

even this sentence alludes to how yunho thinks they should've ended by now.... oh! the anguish!

you've mad the yearning so palpable i am huffing in his longing fumes, his wanting fumes, and i'm so sad for him. all he ever did was want... and even that feels like too much to him.

Yunho acts as if... by doing enough good he can somehow qualify to love Mingi. Oh Yunho, you can love anyway. The concept of "good enough" is so belittling and freakish. Self worth being something you have to accomplish, not something you intrinsically have, it really feeds into this concept of life as a performance and Yunho just has to perform Well Enough to be worth anything at all. Sad shit!

What a puppy! Dog mention... Yunho loving with the devotion of the ground, face in the dirt for his hunger...

Thank you for writing these smaller details out, they make Yunho feels so real and alive to me. Also this is just so endearing I had to stop and scream into my hands. He's so careful, so freaking careful around the boy he loves.

like the north star, or a tall building in London :)

I really love how you play around with language and use words that denote something more, something filthier here. Usually it's "churning in his stomach" but the word gut here is so... animal. Like an animal that's already dead, guts out on the floor. Churning in his gut, oh it makes me shiver. Churning where he's most animal, the messy intestines that are of no use anymore...

I really get that sense of determination here, fate, that the way it is is how it will always be. Yunho thinking he's always disappointing Mingi, it reminds me a lot of how in religious contextual stories the protagonist is meant to fear God, and in that fear is love. That quote:

Love, for you, is larger than the usual romantic love. It’s like a religion. It’s terrifying. No one will ever want to sleep with you.

I like that the segment emphasizes the importance of encouraging participation regardless of current ability. This reminds me so much of the work done within the behavior classroom that I work in. We help children complete a task until they can master it themselves. Encouragement, inclusion, and engagement can go a long way.

This segment truly deepened my understanding of the concept of LOPI and its role in my life and the lives of those around me. My grandmother, a German immigrant, practiced LOPI. My great-grandmother, an African American woman, did the same. This was at sharp contrast to many of my peers at my PWI. This segment also reminds me of some of the work that is done in the enclosed classroom that I work in. When teaching children life. skills, we hand-over-hand the aspects that they are having difficulty with until they themselves develop the ability to complete the action. One program that is very successful in this is washing hands.

This seems like a key concept to remember when referring back to or even practicing LOPI. To effectively participate in it there must be an active component. This sets it apart from other methods.

I am completely new to education and these concepts. This is a great explanation of a term that I have heard before, but have not necessarily had an adequate breakdown of. After reading the description I have come to realize that this is one thing my family practiced when teaching new life skills. I like having this segment to refer back to.

I see my own students more engaged when they are in groups and we are working together. I have put two big tables together in my classroom and started getting away from the solitary exercises because my special education students are already behind in social skills and understanding roles in group activities. I do this because their peers are so far ahead of them academically that they can not engage or contribute to their group the same and then the groups accuse the students of cheating when really they just cannot comprehend what the class is doing. I am trying to help the students learn social cues and academics with the activities that are centered around group work.

This really hit home for me because at home during my childhood everything was done in solitary, from getting ready for the day to chores and even meals. We as a family never ate together except on special occasions. If I got all of my chores, homework or whatever it might need to be taken care of done as soon as possible so I could go to my friends’ homes where we had dinner together and family engagement. I did not mind if I needed to help the family with more work.

This shared task and collaboration ethic is my favorite way to work and learn. As a young adult somewhere in the multiple homes I visited I came to the conclusion that if you could not work in the kitchen together with a potential mate then that was not the person you should be dating or considering for marriage. It may sound silly however the theory has proven itself more times than I would like to count in my life, my children's lives and many friends and family. There is a bond that cannot be explained when you can work together for common goals.

I had never experienced the segregation mentioned here growing up and as an adult at a family gathering for Thanksgiving at my in-law's dinner I was told my children would have to be seated at a different table. Children were not included at the adult table or later in activities because their belief was that children are seen not heard. This was and still is horrifying to me as a parent because I wanted the children to be with us and never had that kind of separation with my children or my family growing up. That was the only dinner I ever ate without my children unless my kids did not want to have me at the table with them and preferred someone else like the favorite Aunt and Uncle.

The Assembly-Line Instruction has been horrible for students and teachers alike. I have always learned more in the "non-traditional" ways of learning and encourage “non-traditional” learning in my classrooms. As a special education teacher and foods teacher I want my students to be successful in life not just school so I teach mostly by using activity based learning and I always remind kids it is ok for our project or food not to turn out ok because we will learn from it as long as we are putting forth an earnest effort.

an idiom referring to the internal workings, hidden mechanisms, or complex details of something, similar to how a mechanic looks at a car's engine

not adjusting adequately or appropriately to the environment or situation.

For this explanation would there be core skills that every person in childhood possesses and any specific skill learned would be a branch off of this main skill? For example, motor skill is a core skill and a specific skill learned later in life if dribbling for soccer. In order to be able to dribble you need to first develop your motor skills, meaning dribbling is a more advanced motor skill.

I know that infants tend to feel things with their feet rather than their hands. I wonder if our sensitivity in our feet get worse as we age. Especially because we use shoes nowadays, so we don't need them to have the same capabilities as our hands to interact with the world.

Alt text for images as described before is often essential for certain people to use the internet. It is not always helpful however, since there are no rules or regulations for making alt text, often it just says "image" or "jpeg" or the like. Other than that, the main issue is that no one includes alt text with their images, either because its too inconvenient or because it seems unnecessary. Recently, companies have tried using AI to generated alt text for images with mixed results.

This link shows us the concepts of disabled vary across eras and cultures. For example, in the middle ages, the physical differences could be considered religious punishment. But later on, the disability turn into a more biological differences definition way. However, here is a detail, that disability is not simply a physical or cognitive difference, but more about the society defines and responds to those difference. I think it is some how correct since people are social animals, we need to be recognized by society and the collective to a great extent. Disability may initially mean that one is physically unable to participate in social activities, but when a person is completely excluded from social activities, the consequences are similar to those of disability to some extent.

Another good example of this I like are curb buts, those dips on sidewalks that go down to the street. It's said that they were originally designed just to help those with wheelchairs navigate off and on the street and sidewalk. But, as it turns out, it helped not only people in wheelchairs, but most people in general, like parents rolling their kids in strollers, people with wheeled carts trying to transport stuff, skateboarders and roller-skates. Whether this story is true or not, it has inspired the term the curb-cut effect, where something designed to aid a disabled person also aids everyone.

What does this barter-based system say about how people adapted to having limited cash and relied on social trust in trade?

What does Sarah Knight's attitude toward the Indigenous people reveal about the colony's beliefs, and how could that shape our understanding of the relationships between the English and the Natives?

Sarah Knight's comment shows slavery's presence in the northern colonies. Although slavery is often associated with the South, enslaved labor was common in New England, Massachusetts, and Connecticut households and farms during the early 1700s. Sarah Knight criticizes how farmers and their slaves would eat at the same table and eat the same food. Her disapproval of farmers being "too indulgent" with the enslaved people reflects her beliefs in racial hierarchies. It also shows how normalized slavery was in everyday colonial life.

The “consumer revolution” was a period of time during the late 1600s to 1700s. During this period, there was an increase in buying and selling imported goods such as textiles, household items, and other goods from British colonists in Europe and North America. This revolution started in Britain before later spreading to other parts of the world. Sarah Knight's account of trading practices shows the variety of goods that were available during this period, and how everyday people participated in bargaining. Her account shows us how colonial life became connected to global markets and consumer habits.

The phrase "Lex Mercatoria" translates to "the law of merchants and it refers to the trade rules that merchants developed in Europe to ensure fair trade and settle disputes.

The phrase "aromatic tincture" refers to a strongly scented liquid mixture. The word tincture means a concentrated liquid, and aromatic describes a fragrant or pungent smell. Sarah Knight's use of aromatic tincture in the sentence describes someone chewing tobacco and spitting out the juice.

spread

how about autonomous social mutual learning networks Weaving the Indy Learning Commons

Typically the beginning of a journal contains enough info to evaluate if its pertinent to your research

I really agree with the idea in this passage about affordances — it makes so much sense when thinking about how we interact with interfaces every day. The point that affordances are not just about what something can do, but whether the user recognizes what can be done, feels super relevant. It’s one thing for a button to be clickable, but it’s another for users to know it’s clickable. I also like how the passage connects affordances to signifiers, like visual or sensory cues that guide users. It reminds me of how modern apps use animations, color changes, or shadows to make buttons feel “touchable.” It’s a small detail, but it really changes how intuitive something feels.

I used to think prototyping was something you had to do in every design process, but this made me realize it’s more about thinking strategically than just building for the sake of it. The idea that prototyping helps you decide what to learn instead of just testing something random really stood out to me. I also find it super useful that it points out how sometimes it’s better to just go ahead and build the final version if it saves time or effort. Not every project needs multiple rounds of prototypes — it really depends on your goals, skills, and the level of uncertainty. Overall, I think this perspective helps designers (especially students like us) use their time more wisely and make smarter decisions about when and why to prototype.

This really changed my perspective on what prototyping actually means. I have never created a prototype for something before and I honestly just thought of it as a crude version of the final product. This use of prototypes makes much more sense for the design process and the idea of prototypes teaching you about the effectiveness of your solution seems incredibly useful.

Locke’s treatise is relevant to the purpose and need for the Declaration of Independence and the Constitution because of the historical context the authors (Locke, Jefferson and other American Revolutionaries) were writing in. When Locke wrote this treatise he was identifying what rulers (specifically a king) should be allowed (or not allowed) to do with their power and the purpose of that power. He was rebutting an argument that Kings should have absolute power because of patriarchy and his treatise defended a movement that limited a monarch’s power while increasing a parliament’s authority over the power of one individual. Thomas Jefferson and the other American revolutionaries are writing the Declaration to limit and abolish an oppressive king's power and the Constitutin to protect Americans from an oppressive government where one individual wields all power. Since the writer of the treatise and the authors of the American documents share similar purposes, it is natural that Locke’s treatise hugely inspired the Declaration and the Constitution.

I used to think that the job of college advisors was only to help with class schedules, but now I see that they also give emotional support and guidance when students face problems. It’s interesting to know that building a relationship with an advisor can really make a big difference in college life.

This situation shows how easily anxiety can keep someone from connecting with others. It’s sad that John feels the need to hide behind his phone instead of giving himself a chance to meet new people. It reminds me how difficult social situations can be when fear takes over, and how important it is to slowly build confidence to open up.

When we take care of ourselves, we have more energy and positivity to share with others. It made me realize that loving yourself is the first step to loving others. I used to think taking care of myself was less important than caring for others, but now I see how closely they are connected.

In this page, it shows us in the facebook's privacy setting, it allows users to find their facebook accounts by using their phone number, but even if user want to close that feature other people can still use their phone number to find their facebook accounts. One detail is facebook said they did not find evidence of abuse for the find feature. So I think for the large platform of such bid companies, they have to protect user's privacy, since their consumer base is really large. They have the responsibility to do that.

I really understand of today's recommendation algorithm. For example, when me and my friends are chatting on a specific topic, later the posts will on my phone, also the posts sent to my friends will later send on my homepage. If you usually click and comments on a specific topic, it will send you more similar posts for you and even for the search bar will jump out the related words for you and guess you might want to search that topic.

Hi, I learned EDSPE last winter quarter, and that course talked about disability people and education. Let me realized the real world problem of disability people faced, for example, their works, social identity and so on. Also disability not just refer to physical disabilities, but also includes learning disabilities, cognitive disabilities, emotional disorders, etc. We also discussed appearance anxiety. Therefore, there may be many cases of disability around us, which require us to observe carefully and then be tolerant and understanding.

This creates a problem worth investigating.

because the collision is assumed to be perfectly "Elastic", which is not real but only assumed for the Ideal Gases In Real life the molecules loses very small amount of energy.

How mental disorders are classified.

a single class in the grade had treatment

im not sure how good of a result this is

lol what if they think they know sm ab finance that they can take on these decisions. but borrowing money from friends is not an outcome that should come out of this. also if goal is to introduce saving and budgeting practice then outcome shouldnt bhi ki ye nai cheez ke bare me pata chala so chalo ab kartein hein

but that means ur study failed if its main goal was savings and budgeting

all they had to do was not spend what they dont have as a clear guideline if these were the goals. must be issues w study material that introduce these fun payment methods as new and innovative and ingenius

is this even significant

but these results are a smaller section of the sample anyway. plus recheck if 1/2 of treatment (finance) is compared to whole control (health)

good so that teachers can discuss what the others said/discussed, but again teachers may be lying

even within the treatment for parent workshops the attendance rates were half

so treatment didnt work half the case

this means each teacher did this once at the end of the program?

less 5 + 7 %? Also 2 and/or 3 means diff textbooks required for sem 2 and sem 3 so dk how many got in sem 3 and no dropped from sem 1 to sem 2

so this means we are assuming that some people's starting points are replaced with no financial knowledge? which might increase the positive effect shown by the program??

dummy pairs are matched to each other based on similar characteristics under a certain criteria

better administration can be loosely correlated with better care for students and better faculty and therefore better outcomes

most developed states, so maybe it is a certain type of students. maybe they have more opp to experiment w monetary investments and decisions, maybe they have seen recently started earning good amounts so they are curious and have the means to try out things. maybe students in poorer areas would be more cautious and disciplined due to diff financial goals?

I believe saying this to students can lead to a fixed mindset. Instead, I usually say, "Thanks for working hard on this problem. Do you have another strategy?" I would think that the other students will notice that this comes from efforts, not innate ability, and they will also try to do the same as the other students and work hard because success doesn't come from fixed talent. In the future, I would have the students who worked hard to lead the other students and give them advice on what to do and how to solve their problems.

What I usually do during morning work is advise my students that learning from mistakes helps our brains process more ideas and grow. And I always have students to avoid saying, "I can't do this. "Try to ask for help. In the future, I will show my students a short video about how neurons make connections in the brain that they can learn instantly and learn on time.

I wonder how this contrasts, historically, with vegetarians in India…

circuit breaker a guard in front of your call. when a downstream keeps failing beyond a set threshold (say 5 fails in a row), the circuit “opens.” once open, all future calls immediately fail instead of wasting resources on requests that’ll fail anyway. after a cooldown (timeout), it switches to half-open, allowing a few test calls through. if they succeed, it closes back to normal; if they fail, it stays open longer.

allowing all other operations to occur - within the user's browser,

• with data fetched either peer-to-peer or
• from remote HTTP trustless gateways.

both peer-to-peer and HTTP)

verifying that block hashes match the expected CID, and

re-assembling blocks into deserialized bytes that - can be rendered by the browser,

all happens within the end user's machine.

gateway in the browser

DOI: 10.1016/j.isci.2025.113564

Resource: Virtual Cell at the National Resource for Cell Analysis and Modeling (RRID:SCR_007421)

Curator: @scibot

SciCrunch record: RRID:SCR_007421

What is this?

for - US Republican governance failure - blue states provide welfare to red states - youtube - Dave Pakman - blue states vs red states - The US survives Trump's mismanagement because the US is a welfare state in which the blue states, with far better social policies is forced to bail out the tax-friendly red states - The red states keep choosing the same dysfunctional policies, and keep having to get bailed out by the blue states - In this sense, the federal government is being exploited to keep red states doing the same thing

Sometimes friendships can also deteriorate due to internal conflict on one side, or personal issues, I've had reasons to end relationships, while I also have been the one who has been theoretically "dumped" in a relationship.

I think this also is affected by the situation you're in as well, I currently feel incredibly lonely because I struggle to get to the point where I feel comfortable or ready to hang out with someone or be 'real' friends as I say, instead of just acquaintances.

I feel like this is a situational friend, like if you go to class with someone and you go to study group for that class, but after the class ends we part ways.

A lot of the time, or maybe just with me, I can see myself rushing into this stage to get to the integrating stage, but usually it's because i'm so excited to be around the person and to have a 'someone.' which reminds me of how a happy dog is so excited for you to be homme they run up to you and jump on you at the door, and you're so overwhelmed you push them away.

I just learned about The Ba'ba. It is called "The Holiest of Holies." It was built during the Old Testament times when God told them exactly how to build it. They have had to rebuild it, but it has real gold, and the doors (covered in the cloth) are pure gold.

Random thought but I feel like there are many emperors in China that rule for very long periods of time.

This is so soon after Theodoric died. Many times, in history when there is a new ruler(s) they get weakened down and invaded.

I wonder what factors weakened the Sasanian Empire before outside attacks?

I find it impressive that Wen was able to unite the north and then launch such a massive army to capture Nanjing.

I think it’s interesting that the Gothic War came right after the victories over the vandals.

On the Similarities Between the SGI Doctrinal Text and Professor Miyata’s Paper

Issue 1.

Lately, on social media, there have been discussions suggesting that The Book on the Doctrinal Foundation of the SGI, published immediately after President Daisaku Ikeda’s passing, closely resembles several papers written by Professor Koichi Miyata. I also share this impression.

Mr. Suda, former Vice Leader of the SGI Study Department, makes the following claim in his Letter to President Harada regarding the “Teaching Outline”: “I have heard that the main individuals behind the creation of the Teaching Outline were Mr. Miyata and Mr. Kanno, both professors emeritus at Soka University. From their standpoint as researchers, however, they have shown a marked tendency to defer to the Minobu sect of Nichiren Buddhism, which represents the mainstream of academic Nichiren studies, seemingly out of fear of criticism from that sect. As a result, the entire Teaching Outline can be seen as having been assimilated to the doctrines of the Minobu sect.” On the other hand, the SGI Men’s Division Doctrinal Office officially denied any involvement of Professors Miyata and Kanno, stating on the Seikyo Shimbun website that “this claim is factually incorrect, and neither Professor Miyata nor Professor Kanno were members of the publication committee.” (Reference: Seikyo Online) In response, I conducted a careful comparative analysis of The Book on the Doctrinal Foundation of the SGI and Professor Miyata’s papers, employing not only a close textual reading but also the analytical functions of a natural language processing AI (ChatGPT) to ensure objectivity. The results revealed a level of similarity far beyond my expectations—one that can hardly be overlooked.

SGI is a large religious organization with approximately twelve million members in Japan and around the world, and The Book on the Doctrinal Foundation of the SGI appears to have become the organization’s core doctrinal text following President Ikeda’s passing. In this regard, Suda, in his Letter to President Harada regarding the “Teaching Outline”, points out that “Even now, it seems that the ‘Teaching Outline’ is being followed in articles in the Soka Shimpo and Seikyo Shimbun newspapers, as well as in the commentary on The Object of Devotion for Observing the Mind, and I think that this is a dangerous situation.” Therefore, this issue is of considerable importance for the future of the SGI’s doctrinal study.

At the same time, it should be emphasized that the purpose of this paper is not to criticize any individual personally, but rather to ensure the accuracy and scholarly transparency of doctrinal materials.To maintain the credibility of SGI’s doctrinal studies, it is increasingly necessary to clarify bibliographical sources and secure philosophical consistency in future publications.

Professor Koichi Miyata, “The Structure and Issues of Udana Nichiko’s Honzon Ryakuben” (2017–2018, pp. 38–39)

“The passage ‘The doctrine of three thousand realms in a single moment of life is found in only one place, hidden in the depths of the “Life Span” chapter of the essential teaching of the Lotus Sutra’ (WND I: 30) can be interpreted, from the overall context, to mean that the doctrine of the true “three thousand realms in a single moment of life” is hidden in the text of the “Life Span” chapter—that is, in the passage revealing enlightenment countless kalpas ago.” The Book on the Doctrinal Foundation of the SGI (Kyōgaku Yōkō, 2023, p. 70) “The great sage Nichiren states in The Opening of the Eyes, ‘The doctrine of three thousand realms in a single moment of life is found in only one place, hidden in the depths of the “Life Span” chapter of the essential teaching of the Lotus Sutra’ (WND I: 30). He perceived that, at the very depths of the chapter The Life Span of the Thus Come One, which expounds enlightenment countless kalpas ago, the essential doctrine of three thousand realms in a single moment of life in the Lotus Sutra is revealed.” Commentary The Book on the Doctrinal Foundation of the SGI makes no reference to Miyata’s paper. Yet both the book and Miyata’s interpretation share a distinctive view: that the passage describing “enlightenment countless kalpas ago” conceals the doctrine of three thousand realms in a single moment of life. In the Book on the Doctrinal Foundation of the SGI, this interpretation is presented as if it were Nichiren’s own insight, raising serious concerns regarding the proper attribution of scholarly authority. In this respect, the book may be seen as raising a significant issue of academic integrity. Incidentally, the traditional SGI interpretation—found in the third President Daisaku Ikeda’s works such as The Lecture on the Orally Transmitted Teachings II (pp. 32–33)—is that this doctrine is concealed in the passage “Originally I practiced the bodhisattva way” (Burton Watson, The Lotus Sutra, p. 268) in the Life Span chapter. From this perspective, The Book on the Doctrinal Foundation represents a problematic departure from that established tradition.

In this respect, Suda, former Vice Leader of the SGI Study Department (2024, p. 53), criticizes this book, stating that “Although The Book on the Doctrinal Foundation was published immediately after Ikeda’s passing and claims to have been supervised by him, its content significantly deviates from Ikeda’s ideological framework during his lifetime. Thus, the assertion that the book was under Ikeda’s supervision can be seen as an attempt to misuse his name rather than a genuine reflection of his doctrinal stance.”

Furthermore, in his paper, Professor Miyata rejects the traditional interpretation that locates the doctrine in the passage “Originally I practiced the bodhisattva way” (Burton Watson, The Lotus Sutra, p. 268) and offers his own alternative view. The following section examines the possibility that this reinterpretation found its way into The Book on the Doctrinal Foundation of the SGI.

Reference

Ikeda, D. (1968) Gogikuden Kōgi [Lecture on the Oral Transmission of the Teachings, vol. 2]. Tokyo: Soka Gakkai.

Miyata, K. (2017–2018). The Structure and Issues of Udana Nichiko’s Honzon Ryakuben. Available at: http://hw001.spaaqs.ne.jp/miya33x/honzonryakuben.pdf [Accessed 24 October 2025].

Nakamura, M. (2025) Kyôgaku Yōkō to Moto Sōka Daigaku Kyōju no Ronbun no Hikaku Kensō [A Comparative Examination of the Book on the Doctrinal Foundation of SGI and Papers by Former Soka University Professors]. Available at: https://jikatsu.net/wp-content/uploads/2025/10/4f92b9e0e1e7f83a456ee2c6d262748b.pdf [Accessed 24 October 2025].

Soka Gakkai. (1999). The Writing of Nichiren Daishonin. Tokyo: Soka Gakkai.

Soka Gakkai (2023) Kyōgaku Yōkō [The Book on the Doctrinal Foundation of SGI]. Tokyo: Soka Gakkai. Available at: https://www.amazon.co.jp/dp/4412017028 (Accessed: 24 October 2025).

SGI Study Department (2024). “The Teaching Outline” is the Culmination of the Soka Renaissance. Available at: https://www.seikyoonline.com/article/603E8EF7E9B96D20AF2920005F5C1C6B [Accessed 10 October 2025].

Suda, H. (2024). Letter to President Harada regarding the “Teaching Outline”, 12 September 2024. file_20240930-185744.pdf [Accessed 24 October 2025].

Suda, H. (2024). A Critique of the Book on Doctrinal Foundation of Soka Gakkai from the Perspective of Buddhist History: In Light of Nichiren Buddhism as the Global Religion Taught by SGI President Daisaku Ikeda. Available at: https://jikatsu.net/download/1st-english-edition-a-critique-of-the-book-on-doctrinal-foundation-of-soka-gakkai-from-the-perspective-of-buddhist-history/

Watson, B. (1993). The Lotus Sutra. New York: Columbia University Press.

Figure 1.2.3 does not have the "dog" on the left side, and the "cat" on the right side. Which feels like an ironic typo given the subject matter of the text.

• Running can counteract some negative effects of an unhealthy, Western-style diet, according to researchers from University College Cork.
• The study found that exercise restores key metabolites linked to mental well-being and balances crucial hormones disrupted by a diet high in sugar, fat, and processed foods.
• Rats fed high-calorie “cafeteria diet” foods showed major disturbances in gut metabolism, with 100 out of 175 metabolites significantly altered; running partially restored balance, especially for anserine, indole-3-carboxylate, and deoxyinosine.
• Physically active rats on the same poor diet had normalized levels of insulin and leptin compared to sedentary ones, showing exercise helps regulate hormonal balance.
• The body activated compensatory hormonal mechanisms, such as changes in GLP-1 and PYY levels, to stabilize metabolism under poor dietary conditions.
• The study also linked exercise to improved neurogenesis (formation of new neurons) in the hippocampus, but only when paired with a healthy diet—junk food appeared to suppress this regenerative effect.
• Despite running’s benefits, researchers emphasized that good nutrition remains essential for full brain health and mental performance.
• The findings offer insight into how exercise may protect mental well-being even amid widespread consumption of processed foods.

Artists can decide how many tools and which tools they wish to use, depending on their skills, priorities, and amount of time available.

While AI tools are being widely integrated in industries such as music, it is very important not to overuse them, otherwise the human element and critical thinking skills required for these complex tasks are completely lost.

AI makes promotion of music through analytics and social media easier than ever.

The age of balanced AI use like Diana's is already coming to an end, as AI tools continually get implemented everywhere.

These AI tools are difficult to learn with a steep learning curve, but the efficiency boost outweighs this.

Diana's use of AI does not undermine the human aspect of it; rather, it makes the process more efficient and keeps the balance between technological assistance and human input.

This chapter does not mention energy use or environmental impact at all.

The music industry is very fast-paced, so AI tools are able to help artists like Diana adapt fast to new market trends.

AI tools also have access to global analytics that weren't previously easily accessible or centralized in one place.

The way that Diana uses AI for music production is very ethical, simply making complex processes more efficient, and ensuring fair royalty splits.

AI financial tools also ensure maximum revenue by analyzing market trends.

An AI tool called Boomplay reveals analytics of popular songs, with the ability to provide a playbook of what release strategies made it successful, so that it can be used for releasing your own music.

Additionally, AI tools track music plays across all platforms, helping to distribute royalties fairly. AI analytics also help guide decisions and help musicians like Dians remain successful in today's industry.

Heaven 11 is an AI platform that includes tools for music distribution and copyright management, a pivotal part to solving the problem of copyright management with music generated by AI.

Adoption of AI tools is becoming widespread in all industries, for people like Diana, these tools free up time and allow her to focus on her music more.

AI has allowed Diana to work more efficiently and better understand the business metrics of her music.

She continued her education, getting a master's degree decades later into her career. Her dedication has allowed her to grow and adapt through the decades.

She is a very influential figure in the music industry.

She is a well-established artist who has been in the industry for several decades.

How do we identify the folks who are just faking it until the make it with synthetic knowledge.

Pre-emptive note: it seems logical that the very next paragraph references Paul Graham directly; my very next move was going to be to connect the writer's self-reported experiences here with Graham on writing, had it not been the case that that job was already done.

As I've said before: I'm not in the same boat with Graham on the writing-is-thinking stance. The difficulty with seeing my own thoughts fixed in words after an initial pass is not in their inadequacy or their being a source of illusory and fleeting comfort with said illusion now made stark for everyone to see, but a mixture of (a) a lack of "punch", and/or (b) the places where a dishonest broker could exploit the yet-to-be shored up wording to suggest/assert the presence of some weakness in thought regarding the thing being argued for, where such purported weakness would be the real illusion.

The lack of satisfaction I feel when trying to capture my thoughts in English (my first language) isn't too far off from the lack of satisfaction at being able to comprehensively express a simple declarative in another language only because of the fact that don't know, say, the right word for the noun in that language. It doesn't lead me to agonize about how well-supported my observations about a backhoe are just because I've never been introduced to the word for backhoe in that language.

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

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Reply to the reviewers

Manuscript number: RC-2025-03130

Corresponding author(s): Ellie S. Heckscher

[The "revision plan" should delineate the revisions that authors intend to carry out in response to the points raised by the referees. It also provides the authors with the opportunity to explain their view of the paper and of the referee reports.

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The document is important for the editors of affiliate journals when they make a first decision on the transferred manuscript. It will also be useful to readers of the reprint and help them to obtain a balanced view of the paper.

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1. General Statements [optional]

We thank all three reviewers for their feedback on the paper. Reviewers stated that the paper was of broad interest to developmental biologists and neurobiologists. However, we want to ensure that our two key conceptual contributions are clear. We clarify in the following paragraph and include a revised abstract. We will update the introduction and paper to better reflect these advances. We also attach a supplemental table 1, which was inadvertently omitted from the previous submission due to our error.

The first advance is that serially homologous neuroblasts follow a multimodal production model: In principle, stem cells can divide any number of times, from once to throughout the entire lifetime of the animal. And, on each division, a stem cell can generate either a proliferative daughter cell or a post-mitotic neuron. Together, therefore, there is a vast potential number of neurons any given stem cell could produce. From the literature on the vertebrate neocortex, we had the following models: (1) "random production" model, in which any number of neurons could be made by a stem cell; or (2) "unitary production" model, in which the same number of neurons (~eight) is produced by a stem cell regardless of context. Our data revealed an entirely new "multi-modal production" model, which could not have been predicted by prior literature. In the context of serially homologous neuroblasts arrayed along the Drosophila larval body axis, sets of five to seven neurons are produced in increments of one, two, or four. These increments correspond to units called temporal cohorts. Temporal cohorts are lineage fragments, or small set of neurons that share synaptic partners, making them lineage-based units of circuit assembly. Thus, in a multimodal production model, serially homologous stem cells produce different numbers of temporal cohorts depending on location. Our data advance the field by showing that stem cells produce circuit-relevant sets of neurons by adding or omitting temporal cohorts from a region, to meet regional needs.

Key to understanding the second advance is that there are multiple types of temporal cohorts: early-born Notch OFF, early-born Notch ON, late-born Notch OFF, and late-born Notch ON. One temporal cohort type, the early-born Notch OFF, is found in every segment, which we term the "ubiquitous" temporal cohort. The other temporal cohort types can be produced in various combinations depending on the stem cell division pattern and segmental location. In a result that could not have been predicted, we found that the ubiquitous temporal cohorts are refined both in terms of the number of neurons and their connectivity, depending on body region. In contrast, when other temporal cohort types are produced, they are not refined to the same degree.

The impact of this work is to advance how we think about stem cell-based circuit assembly.

2. Description of the planned revisions

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

*Summary: The study by Vasudevan et al intends to address how serially homologous neural progenitors generate different numbers and types of neurons depending on their location along the body axis. *

Investigation of full repertoire of neurogenesis for these progenitors necessitates a precise ability to track the fates of both progenitors and their neuronal progeny making it extremely difficult in vertebrate paradigm. The authors used NB3-3 in the developing fly embryo as a model to investigate the full extent of the flexibility in neurogenesis from a single type of serially homologous stem cell. Previous work showed NB3-3 generates neurons including lateral interneurons that can be positively labeled by Even-skipped, but detailed characterization of the NB3-3 lineage mainly focused on 3 segments during embryogenesis. The authors defined the number of EL neurons in all segments of the central nervous system in early larvae after the completion of circuit formation and carried out clonal analyses to determine the proliferation pattern of NB3-3. They described the failure to express Eve in Notch OFF/B neurons as a new mechanism for controlling the number of EL neurons and PCD limits EL neurons in terminal segments.

• *Thank you! In addition to the contributions highlighted by the reviewer, we also showed that all segments have ELs with early-born molecular identities, but only a subset have ELs with late-born identities (Figure 5). And we showed that early-born temporal cohorts can be mapped into different circuits depending on the axial region (Figure 6).

*Major comments: The authors performed careful analyses of the NB3-3 lineage using EL neurons. My main concerns are limited applicability of their findings and lack of mechanisms as how NB3-3 generate various numbers of EL neurons. Their findings are exclusively relevant to the NB3-3 lineage despite their effort in highlighting that other NB lineages also generate temporal cohorts of EL neurons. *

  Thank you for raising these points. First, to clarify, as Reviewer 4 also mentioned, NB3-3 is the only lineage to produce EL neurons. We will ensure that this is clearly stated in the revised text.

We agree that our findings might not apply beyond the NB3-3 lineage. However, as this is the first study of its kind, it is impossible to know a priori to what extent the concepts surfaced here are generalizable. In our opinion, this speaks to the novelty and impact of the study. A contribution is to motivate a need for future studies. We will make this explicit in our updated manuscript in the Discussion section.

  Our manuscript provides cell biological mechanisms that explain how stem cells give rise to different numbers of EL neurons in different regions, including stem cell division duration and type, neural cell death, identity gene expression, and differentiation state. If the reviewer is interested in genetic or molecular mechanisms, this is an interesting point. Several prior studies using NB3-3 as a model (e.g., Tsuji et al., 2008, Birkholz et al., 2013, Baumgardt et al., 2014) have elucidated the genetic regulation of specific cell biological processes. However, these studies provided fragmentary insight with regard to serially homologous stem cell development along the body axis. A comprehensive understanding of how the NB3-3 lineage, or any other serially homologous lineage, develops was missing. This is what makes our study both novel and needed. Without an analysis that both examines every segment and assays multiple cell biological processes, we would have missed key insights: that there is a ubiquitous type of temporal cohort, and that neurons within the ubiquitous temporal cohort are selectively refined post-mitotically (See General Statements for more details).

*I disagreed with their conclusion that failure to express Eve as a mechanism for controlling EL neuron numbers when Eve serves as the marker for these neurons. Are there any other strategy to assess the fates and functions of these cells beside relying solely on Eve expression? I am not familiar with the significance of Eve expression on the functions of these neurons. Is it possible to perform clonal analyses of NB3-3 mutant for Eve and see if these neurons adopt different functionalities/identities? *

• We agree that if Eve were only a marker, our logic would be circular. The Eve homolog, Evx1/2 is crucial for vertebrate interneuron cell fate (Moran-Rivard et al., 2001). Eve is essential for motor neuron morphology in Drosophila *(Fujioka et al., 2003). Eve is critical in Even-skipped for both the morphology and function of Even-skipped interneurons (Marshall et al., 2022). Hence, ELs cannot fully differentiate or incorporate into circuits without Eve. Thus, we use the failure to express Eve as a mechanism for controlling EL number. Furthermore, our prior study (Wang et al., 2022) showed that NB3-3 Notch OFF neurons in A1 that fail to express Eve have small soma and "stick-like" neurite projections that are typical of undifferentiated neurons. We will be sure to add this context to the revised manuscript.

*If NB3-3 in the SEZ continually generate GMCs based on the interpretation of clonal analyses and depicted in Fig. 2A, why is the percent of clones that are 1:0 virtually at or near 100% from division 6-11 shown in 2G? *

Admittedly, the ts-MARCM heat-shock-based lineage tracing experiments are inherently messy. This is part of the reason why we included the G-TRACE lineage tracing experiments in Figure 3. In Figure 3E, one can see that the number of Notch ON/A neurons in SEZ3 is equal to the number of ELs in that segment (Figure 1E). This is a second independent method that supports the assertion that in SEZ, NB3-3 stem cells continually generate GMCs. Given this independent observation, it leads us to believe that this question is most likely explained by technical issues inherent in ts-MARCM. These issues include but are not limited to: cell-type specific accessibility/success of heat-shock induced recombination; variably effective RNAi; and idiosyncrasies of the EL-GAL4 line used to detect recombination events. If the question is why the data is only reported for division 6-11, the answer is that the ts-MARCM dataset, which included SEZ clones only used later heat-shock time points (line from the paper "for the SEZ-containing dataset, inductions started at NB3-3's 5th division"). Along with this revision plan, we will include Supplemental Table 1, which was inadvertently omitted from the previous submission due to our error. This table shows all of the clonal data. We will include a section in the discussion to describe limitations in ts-MARCM.

The authors also indicate that NB3-3 in the abdomen directly generate Notch OFF/B cells that assume EL neuronal identity. In this scenario, shouldn't the percent of 1:0 clones be 100% in later divisions in Fig. 2G? Based on the number of clones in abdomen shown in Fig. 2E, I cannot seem to understand how the authors come to the percent of 1:0 clones shown in Fig. 2G

  We agree that one might expect the 12th division to be 100% 1:0 clones in the abdomen. Unfortunately, we didn't sample that late in our dataset, and even when we sampled the inferred 11th division, we had a small sample size (Figure 2E). Other studies suggest that NB3-3 in the abdomen directly generates Notch OFF/B neurons (Baumgardt et al., 2014), which served as our starting point. We will revise the text to make this clearer. As you can see from Figure 3E, there is only one NB3-3 Notch ON/ A neuron produced in each abdominal segment in comparison to the number of NB3-3 Notch OFF/B/EL neurons (Figure 1E). According to two independent assessments, Figure 3 and Baumgardt et al., 2014, the data support the conclusion that NB3-3 in the abdomen directly generates Notch OFF/B cells that assume EL identity for all but one of their divisions. Again, we believe technical issues make the ts-MARCM dataset messy. We will include a section in the discussion to describe limitations in ts-MARCM.

*There are many potentially interesting questions related to this study that can significantly broaden the impact of this study. For example, are other NB lineages that also generate distinct temporal cohorts of EL neurons display similar proliferation patterns (type 1 division in SEZ, early termination of cell division in thoracic segments and type 0 division in abdomen)? *

• *NB3-3 is the only lineage that makes ELs; Many lineages switch proliferation fates along the body axis. Previous studies have described how this switch in division patterns produces the wedge-shaped CNS: Cobeta et al., 2017. In the revision, we will be sure to clarify both points.

*Why does NB3-3 in the thoracic segment become quiescence so much sooner than SEZ and abdominal segments? *

• *NB3-3 in the thorax enters quiescence due to Hox genes and temporal transcription factors (Tsuji et al., 2008). In the revision, we will be sure to clarify this point.

The authors' observations suggest that NB3-3 in SEZ and abdomen generate a similar number of EL neurons despite the difference in their division patterns (type 1 vs type 0). Are the mechanisms that promote EL neuron generate in NB3-3 in SEZ and abdomen the same? Anything else is known beside Notch OFF?

• We agree this is an interesting point. Previous work has detailed NB3-3 division patterns, showing Type 1 divisions in the thorax, and Type 1 to Type 0 switch in the abdomen (Baumgardt et al., 2014). However, the proliferation pattern of NB3-3 in the SEZ had not been addressed until our study. Figures 2 and 3 suggest the following (1) SEZ proliferates for the duration of embryonic neurogenesis; (2) It produces a GMC on each division; (3) the GMC divides to produce one EL Notch OFF neuron and one Notch ON neuron. In our revision, we will manipulate the Notch pathway using two mutants, sanpodo, which produces two Notch OFF cells, and numb*, which produces two Notch ON cells (Skeath et al., 1998), to specifically test how ELs in the SEZ are regulated by Notch signaling. The other difference we know of between the SEZ, and abdomen is Hox gene expression. In Figure S2, we show that a subset of ELs in the SEZ express the anterior Hox genes, Sex combs reduced (Scr). The role of Hox genes in this lineage is an interesting question, as addressed in the discussion. This is an important future direction that merits in-depth study and is beyond the scope of what of this study is trying to accomplish.

Minor commentsThe authors' writing style is highly unusual especially in the result section. There is an overwhelming large amount of background information in the result section but very thin description on their observations. The background information portion also includes previously published observations. Since the nature of this study is not hypothesis-driven, it is very confusing to read in many places and difficult to distinguish their original observations from previously published results and making. One easily achievable improvement is to insert relevant figure numbers into the text more often.

Thank you for this comment. It is invaluable. In the revision, we will expand the background into a more comprehensive introduction and present the results more clearly. We will certainly insert relevant figure numbers. In responding to the reviewer's comments above, we can see where our writing lacked clarity and will improve these areas. Thank you again.

Reviewer #1 (Significance (Required)):

The study by Vasudevan et al intends to address how serially homologous neural progenitors generate different numbers and types of neurons depending on their location along the body axis. Investigation of full repertoire of neurogenesis for these progenitors necessitates a precise ability to track the fates of both progenitors and their neuronal progeny making it extremely difficult in vertebrate paradigm. The authors used NB3-3 in the developing fly embryo as a model to investigate the full extent of the flexibility in neurogenesis from a single type of serially homologous stem cell. Previous work showed NB3-3 generates neurons including lateral interneurons that can be positively labeled by Even-skipped, but detailed characterization of the NB3-3 lineage mainly focused on 3 segments during embryogenesis. The authors defined the number of EL neurons in all segments of the central nervous system in early larvae after the completion of circuit formation and carried out clonal analyses to determine the proliferation pattern of NB3-3. They described the failure to express Eve in Notch OFF/B neurons as a new mechanism for controlling the number of EL neurons and PCD limits EL neurons in terminal segments.

Because this text is the same as the summary, please see our response to that section.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

In this manuscript, Vasudevan et al provide a detailed characterisation of the different numbers and temporal birthdates of Even-skipped Lateral (EL) neurons produced at in different segments from the same neuroblast, NB3-3. The work highlights the differences in EL neuronal generation across segments is achieved through a combination of different division patterns, failure to upregulate EL marker Eve and segment-specific program cell death. For neurons born within the same window and segment, the authors describe additional heterogeneity in their circuit formation. The work underscores the large diversity that the same neuroblast can generate across segments.

Thank you!

Major comments:

- Based on the ts-MARCM 1:0 clones representing 100% of the SEZ clones at any given inferred cell division, the authors conclude "NB3-3 neuroblasts generate proliferative daughter GMCs in the SEZ and thorax on most divisions". Figure 2G does not have any data for SEZ before inferred division 5, whereas there is data in other regions. The authors also state "In the SEZ and abdomen, ELs were labelled regardless of induction time." In reference to Fig 2F, which seems inaccurate given there are no SEZ clones before inferred division 5. There is no comment on this fact, which is surprising give their focus on temporal cohorts. The authors should explain this discrepancy, if known, or modify their statements to reflect the data.

• *Thank you for raising this point. The reason is because we produced two ts-MARCM datasets. One had SEZ clones, the other did not. The dataset with SEZ clones used heat shock protocols only for later time points, because those were most informative. The text from the paper is "We combined a published ts-MARCM (Wang et al., 2022) dataset with a new one (Table S1). The differences between the datasets are (1) CNSs were imaged either at low resolution for all regions (SEZ to terminus) or higher resolution for nerve cords (thorax to terminus); (2) for the SEZ-containing dataset, inductions started at NB3-3's 5th division. The combined data includes ~12 different heat shock protocols, 80 CNS, and 234 clones (Table S2)". In response to this comment, however, we will further clarify this point. In addition, we are submitting Supplemental table 1, which contains all the clonal data, as you can see experiments a-h lack SEZ data and experiments i-k contain SEZ data.

- The temporal cohort (early-born vs late-born) identity is exclusively examined based on markers. Given the absence of SEZ clones from early NB3-3 divisions, a time course showing that the SEZ generate early-born Els or some other complementary method would be desirable.

Thank you for raising this point. We show early-born versus late-born identity using markers in Figure 5. We conducted the time-course experiment as suggested and can confirm that there are early-born ELs in the SEZ at stage 13. We will include a new Supplemental Figure that includes a time course of EL number at stages 11, 13, 15, and 17 for segments SEZ3 to Te2 in the revision. See figure below.

- The authors repeatedly refer to their work as showing how a stem cell type can have "flexibility". Flexibility would imply that NB3-3 from one segment could adopt a different behaviour (different division pattern, or cell death or connectivity) if it were placed in a different segment. This is not what is being shown. In my opinion, "heterogeneity" of the same neuroblast across different segments would be more appropriate.

• *Thank you for this comment. We will change the wording to heterogeneity in the revision.

Minor comments:

- Figure 2A depicts a combination of known data and conclusions from their own (mainly SEZ). The authors might consider editing the figure to highlight what is new. A possibility would be for figure A to be a diagram of the experimental design and their summary division pattern to be shown after the new data instead of being panel A.

Thank you for this suggestion. We will make the suggested change.

- The authors state that they combined published ts-MARCM with their new one, which differed in a number ways that they list, but they don't specify which limitations are associated with the published vs new dataset. Could the authors please clarify?

  We now include Supplemental Table 1, which shows the complete combined datasets. In the first dataset, experiments a-h, the CNS was imaged at high resolution, but in a smaller region. The limitation is that the SEZ is missing. In the second dataset, i-k, inductions started at NB3-3's 5th division. The limitation is that we fail to sample early time points. This was a strategic decision. There were two possible scenarios: (1) in the SEZ, NB3-3 divided early, made GMCs, but both daughters expressed Eve. (2) in the SEZ, NB3-3 divided for the entirety of the embryonic neurogenesis, making GMCs, with only the Notch OFF daughters expressing Eve-our data support (2). Only late heat shocks were needed to distinguish between these possibilities. As these experiments are labor-intensive, we focused our efforts on the later time points. We will make this clearer in our revised text.

- The title refers exclusively to "temporal cohorts", which in the manuscript are defined quite narrowly and do not seem to apply to all segments.

• *Thank you! This, in our opinion, is a central, not a minor point to raise, because the impact of this study involves temporal cohort biology. We outlined the essential concepts in Part 1 "general statements" section of this revision plan. We did not mean to use "temporal cohort" in a limited sense, and we can see how the writing of our results section led to this comment. We will revise to make this clear.

- Several cited references are missing from the Reference list at the end. Could the authors please double check this? (e.g. Matsushita, 1997; Sweeney et al., 2018)

• *Thank you, we will remedy this!

- Legend for figure 2 is a bit confusing, there is a "(A)" within the legend for (D), which indicates that segments A1-A7 are shown (this seems inaccurate, as it only goes to A6).

Thank you, we will remedy this!

Reviewer #3 (Significance (Required)):

This study provides a comprehensive analysis of different cell biological scenarios for a neuroblast to generate distinct progeny across repeating axial units. The strength is the detailed and systematic approach across segments and possible scenarios: different division patterns, cell death, molecular marker expression. While it focuses on one specific neuroblast of the ventral nerve cord of Drosophila, the authors have done extensive work to place their findings and interpretation in the context of other cell types and across model organisms both in the introduction and discussion. This makes the work of interest for developmental biologists in general, neurodevelopment research in particular and those interested in circuit assembly, beyond their specialised community. This point of view comes from someone working in vertebrate CNS development.

Thank you!

Reviewer #4 (Evidence, reproducibility and clarity (Required)):

Summary

This manuscript addresses the question of how the number of neurons produced by each progenitor in the nervous system is determined. To address this question the authors use the Drosophila embryo model. They focus on a single type of neural stem cell (neuroblast), with homologues in each hemisegment along the anterior-posterior axis.

Using a combination of clonal labelling, antibody stainings, and blockade of programmed cell death, they provide a detailed description of segment-specific differences in the proliferation patterns of these neuroblasts, as well as in the fate and survival of their neuronal progeny.

Furthermore, by employing trans-synaptic labelling, they demonstrate that neurons derived from the same progenitor type receive distinct patterns of synaptic input depending on their segmental origin, in part due to their temporal window origin.

Overall this work shows that different mechanisms contribute to the final number and identity of the neuronal progeny arising from a single progenitor, even within homologous progenitors along the anterior posterior body axis.

Thank you!

Major Comments

I would suggest adding line numbers to the text for future submissions, this massively helps providing comments.

  Thank you for this comment. We will definitely add line numbers to the revised manuscript. We also thank you for providing comments despite this oversight on our part. We appreciate your time, and did not mean to make extra work.

*The authors propose that all neuroblasts produce the same type of temporal cohort (early born) and that, by changing the pattern of cell division, different temporal cohorts can be added. The way this this presented in the abstract sounds like an obvious thing, what would be the alternative scenario/s? *

  Thank you for raising the point that the abstract should be updated. We have included a revised abstract. The things that are obvious are: (1) changing a neuroblast's division pattern will change the number of neurons produced, and (2) if you have late-born neurons, the stem cell must at some point, have made early-born neurons. However, within those bounds is an extremely large parameter space. Each stem cell can choose to divide or not, and it can also choose to produce a proliferative daughter or not. The stem cell must navigate these choices at every division. The field had two models for what a stem cell might do - a "random production" model and a "unitary production" model. Our data support a third "multimodal production" model, which could not have been predicted based on prior literature or data.

We had raised these points in the discussion as follows-

"Under a null model, the durations and types of proliferation would vary stochastically across segments, resulting in a continuous and unstructured distribution of neuron numbers (Llorca et al., 2019). In a unitary production model, based on the vertebrate neocortex, there is a fixed neurogenic output of ~8-9 neurons per progenitor (Gao et al., 2014). However, our data support a third model, a multimodal production model. In a multimodal model, serially homologous neuroblasts generate different numbers of neurons depending on the segment."

We will now update the text to address this concern.

Here it's the late born neurons that lack in thoracic segments because of early NB quiescence, but it cannot be excluded that different neuroblast types adopt a different strategy.

• *True. Neural development is complex. Other lineages could easily employ alternative strategies. Our study presents a new conceptual framework that should inspire future research.

I found the ts-MARCM results confusing for 2 reasons:

1- It's not clear to me why there are so many single cell clones in div 3 and 4 in abdominal segments. This is not compatible with the division model depicted for abdominal segments, unless GMCs are produced in those division window and the MARCM hits the GMC, as also mentioned in the legend for G. This aspect is important because, either the previous model by Baumgardt et al. - please correct cit. currently Gunnar et al. 2026 - is wrong, or something strange happens in this experiment, or the relative temporal order is incorrect.

Thank you for raising this point. Having multiple single-cell (i.e., 1:0) clones in divisions 3 and 4 is not precisely what would be predicted by the model in Figure 2C. In part because heat-shock-based recombination methods in fly are stochastic and inherently "messy", we also conducted a second set of lineage tracing experiments, as shown in Figure 3, using G-TRACE. Figure 3E shows one Notch ON/A neuron in each abdominal segment, suggesting there is only one GMC present during lineage progression. But Figure 3E's result does not localize the GMC to any particular division. One possibility is that the GMC is generated once, but randomly throughout lineage progression. This possibility is consistent with the idea that the relative temporal order is incorrect and suggests that Baumgardt is erroneous. However, the Baumgardt data are strong, so we do not favor this idea. A second possibility, which we favor, is that something strange happened in this experiment. Here is how we envision the strange occurrence: heterogeneity in the EL driver. Ts-MARCM's recombination timing dictates the upper limit for the number of cells within a clone. However, recombination is detected by GAL4. So, if the GAL4 driver for some reason detects fewer cells than one expects, then one would see unusually small clones as is the case in question. To detect Ts-MARCM recombination in Figure 2, we used the EL-GAL4 driver. The EL-GAL4 driver is an enhancer fragment, ~400KB, meaning that it does not capture the full regulatory context of the eve locus. In our experience (e.g., Manning et al., 2012), drivers using small enhancers tend to give highly-specific, but somewhat variable expression, and this is the case for EL-GAL4 in our experience. We will update the discussion to discuss the ts-MARCM dataset and its limitations. And, we will correct the citation to Baumgardt et al., 2014, not Gunnar. Thank you!

2- In segments other than abdomen, it is quite rare to hit proper clones, it appears that only GMCs are hit by recombination, with very few exceptions. Could the author please provide an explanation for this or at least mention this aspect?

• *This is true. We cannot explain it. It could have something to do with the RNAi cassettes that are used in ts-MARCM, because in the original paper they mention that RNAi can be differently regulated in GMCs versus neuroblasts (Yu et al., 2009). We will mention it in the revised discussion about ts-MARCM limitations.

It is also unclear whether in F the graph includes all types of clones (including 1:0 clones). This is important, because the timing of division for NBs and GMCs is different, and inclusion of 1:0 might lead to a wrong estimate of the NB proliferation window (longer than it actually is because GMCs divide for longer). This is particularly important for the SEZ, where most clones in normalised division 10 and 11 are with ratio 1:0, thus compatible with both terminal division as well as GMC division.

• *The graph in F does include all types of clones. We provide Supplemental Table 1, which shows the full dataset. Unfortunately, we do not have enough data to analyze only NB clones. We agree that the estimate of the NB proliferation window is coarse using this analysis method and could overrepresent the division time by one cell division. We will mention this in the discussion and make sure that our results text is free from any overreaching claims about the precision of these measurements.

To obtain an estimate of the timing of division, the authors normalise clone size to the size of the bigger clone in the abdomen. What happened to those samples where no abdominal clones were hit? Were they simply excluded from the analysis?

  From the analysis in Figure 2, we excluded the clones that were SEZ, thorax, or terminus only. They were rare. They are shown in Supplemental Table 1, which will now be added in our revision plan.

It is proposed that in the thorax late temporal cohort neurons are not produced, yet the ts-MARCM experiment detects some 1:0 clones. What is the fate of these cells? Are they all derived from GMC division and therefore decoupled from the temporal identity window? Or is this a re-activation of division?

Figure 2F shows at the inferred 11th NB3-3 division, 100% of thoracic clones are of the 1:0 type. This is an n=1 observation (Supplemental Table 1, row f-Jan20-2). When we look at the morphology of this thoracic EL, we can see that it is a fully differentiated neuron that crosses the midline and ascends to the CNS, which is similar to EL morphologies in A1, so we don't think it's a whole new cell type. We have no way of determining whether this neuron was derived from a GMC division. It is also possible that this is an infrequent event or a technical anomaly. To address the question of reactivation of the thoracic NB3-3 division, we plan to include a Supplemental Figure of EL number over developmental time (stages 11, 13, 15, 17) for segments SEZ3 to Te2. This is the same data that we mentioned to Reviewer 3. This will reveal the extent to which the thorax produces late-born ELs.

*"in A1, a majority of segments had one Notch OFF/B neuron that failed to label with Eve" does "the majority" in this sentence mean that there were cases where all B neurons were labelled with Eve? If yes, where would this stochasticity come from? *

• • Yes, "the majority" in this sentence means that there were cases where all B neurons were labeled by Eve. In Figure 3F, for segment A1, that number is four. In contrast, there are 6 cases where B neurons failed to label with Eve. We can only speculate about the origin of the stochasticity. It could be biological (e.g., low level of Eve expression) or technical (e.g., poor antibody penetration). We plan to mention this in the discussion.

Additionally, there is no evidence that it's the first born NotchOFF neuron in A1 that does not express Eve. The authors should clarify where this speculation comes from.

• *The evidence that the first-born Notch OFF neuron in A1 does not express Eve comes from our ts-MARCM data: "So far, our ts-MARCM analyses grouped segments into regions (Figure 2A-C), however, EL number varies on a segment-by-segment basis (Figure 1). Therefore, we looked for segment-by-segment differences in ts-MARCM data (Table S1). The only detectable difference was between A1 and the other abdominal segments: When both A1 and another abdominal segment were labeled in a single CNS, a majority had smaller A1 clones. These data suggest that the production of ELs by NB3-3 neuroblasts lags in A1 compared to A2-A7." We will add a representation of these data to the ts-MARCM figure. As we stated above, we will add a Supplemental Figure of EL number over developmental time (stages 11, 13, 15, 17) for segments SEZ3 to Te2, which could strengthen this point.

When discussing trends shared with other phyla:

A- "In the mammalian spinal cord, more neurons are present in regions that control limbs (Francius et al., 2013). Analogously, EL numbers do not smoothly taper from anterior to posterior; instead, the largest number of ELs is found in two non-adjacent regions, SEZ and the abdomen." It's unclear what is the link between the figure in the mammalian spinal cord and the Drosophila embryo. The embryo doesn't even have limbs and the number of neurons measured here refer only to a single lineage, while there could be (and in fact there are) lineage-to-lineage differences that could depict a different scenario.

Thank you for this comment. We will rewrite this sentence, "in the mammalian spinal cord, more neurons are present in regions that control limbs (Francius et al., 2013)" to more accurately reflect the data in the Francius paper, and make the parallel more explicit. We will say "the size of columns of V3, V1, V2a, V2b, and V0v neurons differ at brachial compared to lumbar levels in the developing spinal cord." This removes the confusion about limbs and somewhat mitigates the concern about lineage-to-lineage differences, at least from the perspective of the spinal cord.

B- The parallelism between V1 mouse neurons and EL Drosophila neurons is also unclear to me. The similarity in fold change across segments could be a pure coincidence and, from what I understand, the two cell types are not functionally linked.

  Thank you for this comment. We believe this is the sentence in question (sorry about no line numbers). "(3) In the mouse spinal cord, ~10-fold differences in molecular subtypes for V1 neurons (Sweeney et al., 2018). In *Drosophila*, NB3-3 neuroblasts show differences in EL number, depending on region, with similar fold changes, suggesting this trait is shared across phyla."  The emphasis was intended to be on the fold-changes, not cell types. Coincidence or not, it is parallel. We will update the sentence to say "(3) In the mouse spinal cord, ~10-fold differences in molecular subtypes for V1 neurons (Sweeney et al., 2018). Although V1 neurons are not direct homologs of EL neurons, the number also varies ~10-fold depending on the region. One possibility is that this trait is shared across phyla." And, we will remove the final part of the paragraph, which distracts from the point "Thus, for this study and future research, NB3-3 development now offers a uniquely tractable, detailed, and comprehensive model for studying how stem cells flexibly produce neurons."

Minor comments:

I found the manuscript somewhat difficult to follow, even though I am familiar with both the model and the topic. For non-specialist readers, I expect it will be even more challenging. The presentation of the results often feels fragmented, at times resembling a sequence of brief statements rather than a continuous narrative. I would encourage the authors to provide more synthesis and interpretation, for example by summarising key findings, rather than listing in detail the number of neurons labelled in each segment for every experiment. This would make the results more accessible and easier to digest.

• *Thank you for this comment. We will provide more synthesis and interpretation in results by summarizing key findings.

From the way the MS is written it's not clear from the beginning that the work focuses exclusively on embryonic-born neurons. Since in Drosophila neuronal stem cells undergo two rounds of neurogenesis, one in the embryo and one in the larva, this omission could lead to confusion.

  Thank you for this comment. We will mention this in the abstract, introduction and discussion.

In the abstract, what would be the other temporal cohorts generated in specific regions? (ref to: "In specific regions, NB3-3 neuroblasts produce additional types of temporal cohorts, including but not limited to the late-born EL temporal cohort.")

  In this manuscript, we use lineage tracing to identify four types of temporal cohorts- early-born Notch ON, early-born Notch OFF, late-born Notch ON, and late-born Notch OFF. This is now reflected in the revised abstract. ELs are early-born Notch OFF and/or late-born Notch OFF.

This sentence in the introduction is inaccurate: "The Drosophila CNS is

organized into an anterior hindbrain-like subesophageal zone (SEZ) and a posterior spinal cord-like nerve cord". The anterior hindbrain-like portion of the CNS is in fact the supraesophageal ganglion (or cerebrum), while the SEZ is a posterior-like region.

  Thank you. We will change this sentence to: "The *Drosophila* CNS is

organized into a hindbrain-like subesophageal zone (SEZ) and a spinal cord-like nerve cord".

Fig 1E: the encoding of the significance is not immediately clear. In the legend the 4 stars could also be arranged in the same way for clarity.

• *Thank you. We will change it for clarity.

Fig 2E legend: it is mentioned that B corresponds to a 1:4 clone, however the MARCM example is shown for C and it's a 1:5.

Thank you. We will fix this.

The occurrence of "undifferentiated" neurons in Th segments is in less than 10% of the clones, I wonder if this a stochastic or deterministic event and to what extent small cell bodies could just be the consequence of local differences in tissue architecture.

• Because we are using a stochastic technique, it is difficult for us to determine whether the occurrence of neurons with small somas is a stochastic or deterministic event. Several papers suggest neurons with small axons are found across insect species (Pearson and Fourtner, 1975; Burrows, 1996). Neurons with a small soma and short axons/ axonless are found in the Drosophila embryonic abdominal nerve cord (Lacin et al., 2009). In our unpublished work from the Drosophila* nerve cord at a first instar larval stage, we found small somas with short axons in segment A1 (see Figure 4.6 below). This leads us to believe it is not a consequence of local tissue architecture.

Fig 2I: it's unclear what the purple means (I suppose it might be Eve expression) and why in J there should be one purple cell not labelled by the ts-MARCM when this is not present in H and I.

Purple is Eve. We will add labels for stains used in H and I, and remove the extra purple cell from the illustration in J.

"When synapses do occur, they are numerically similar from segment to segment". It's unclear where the evidence for this statement comes from, please clarify or remove the sentence.

We calibrated our trans-Tango data against available connectomic data using segment A1 as a reference. We learned that the trans-tango method only identifies strongly (>15 synapses) connected neurons.

"First, we calibrated trans-Tango for use in larval Drosophila, focusing on segment A1, where connectome data are available (Wang et al., 2022). In the connectome, of the five early-born ELs in A1, three are strongly connected to CHOs (>15 synapses), two are weakly connected (15 synapses) connected to somatosensory neurons."

  We will modify this sentence to say "when synapses do occur they are of similar strengths from segment to segment"

"In SEZ2, NB3-3 divides 10 times (Figure 2F)". Figure 2F does not support this statement and Figure 7 shows 12 divisions. Possibly SEZ2 and 3 have been inverted in this statement, please clarify.

Thank you for pointing this out. We will correct it!

**Referees cross-commenting**

I agree with most of the comments/suggestions provided by the other two reviewers.

In particular:

I agree with reviewer #1's comment about failure to express Eve being a mechanism for controlling neurons number, as this is a circular argument.

• *We address this earlier and direct you to that text. Briefly, Eve is not just a marker, but a key differentiation gene for ELs.

I agree with reviewer #2's concern about the use of the word "flexibility"; "heterogeneity" would be a more appropriate term, as I would associate the word "flexibility" to the ability of a single neuroblast in a single segment to produce neurons with different fates under, for example, unusual growth conditions. Here no genetic/epigenetic manipulations were performed to address flexibility and the observed (stereotypical) differences result from axial patterning.

• *We will change this, thank you.

*As a note, Reviewer #1 asks about other temporal cohorts of EL neurons produced by other lineages, but these neurons are specifically generated from NB3-3. *

• *Thank you for adding this clarification.

To generalise the observations reported in this study, the authors would need to focus on other molecularly defined temporal cohorts or, more generally, on other lineages, which, however, are likely to adopt different combinations of mecahnisms to tune progeny number across segments.

• *We agree that further studies are needed to assess the generalizability of our findings.

Reviewer #4 (Significance (Required)):

In Drosophila melanogaster, the relationship between neural progenitors and their neuronal progeny has been studied in great detail. This work has provided a comprehensive description of the number of progenitors present in each embryonic segment, their molecular identities, the number of neurons they produce, and the temporal transcriptional cascades that couple progenitor temporal identity to neuronal fate.

This work adds to the existing knowledge a detailed characterisation of intersegmental differences in the pattern of proliferation of a single type of neuronal progenitor as well as in post-divisional fate depending on anterior-posterior position in the body axis (i.e. programmed cell death and Notch signalling activation). This is a first step towards understanding the cellular and molecular mechanisms underlying such differences, but it's not disclosing them.

We have disclosed the cellular mechanisms- stem cell division duration and type, neural cell death, identity gene expression, and differentiation state -unless something else is envisaged by this comment. The molecular mechanisms are beyond the scope of this paper.

That homologous neuroblasts can generate variable numbers of progeny neurons depending on their segmental position has been established previously. What this manuscript adds is the demonstration that these differences arise through a combination of altered division patterns and differential programmed cell death, thereby revealing a more complex and less predictable scenario than could have been anticipated from existing knowledge in other contexts. The advance provided by this study is therefore incremental, refining rather than overturning our understanding of how segmental diversity in neuroblast lineages is achieved.

The key conceptual advances provided by this study are described in the General Statements section above. We don't overturn, but we advance the field.

By touching on the general question of how progenitors generate diversity, this work could be of broad interest to developmental neuroscientists beyond the fly field. However, the way it is currently written does not make it very accessible to non-specialists.

Thank you for this comment. We will endeavor to make it more accessible in the revised manuscript. Reviewer 3, an expert in vertebrate neurobiology, agreed that our work was of broad interest.

My expertise: Drosophila neurodevelopment, nerve cord, cell types specification

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

Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

With this Revision Plan, we submit a revised abstract, and a supplemental table 1. We plan to address every point raised by the reviewers.

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

Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

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Referee #3

Evidence, reproducibility and clarity

Summary

This manuscript addresses the question of how the number of neurons produced by each progenitor in the nervous system is determined. To address this question the authors use the Drosophila embryo model. They focus on a single type of neural stem cell (neuroblast), with homologues in each hemisegment along the anterior-posterior axis.

Using a combination of clonal labelling, antibody stainings, and blockade of programmed cell death, they provide a detailed description of segment-specific differences in the proliferation patterns of these neuroblasts, as well as in the fate and survival of their neuronal progeny. Furthermore, by employing trans-synaptic labelling, they demonstrate that neurons derived from the same progenitor type receive distinct patterns of synaptic input depending on their segmental origin, in part due to their temporal window origin. Overall this work shows that different mechanisms contribute to the final number and identity of the neuronal progeny arising from a single progenitor, even within homologous progenitors along the anterior posterior body axis.

Major Comments

I would suggest adding line numbers to the text for future submissions, this massively helps providing comments.

The authors propose that all neuroblasts produce the same type of temporal cohort (early born) and that, by changing the pattern of cell division, different temporal cohorts can be added. The way this this presented in the abstract sounds like an obvious thing, what would be the alternative scenario/s? Here it's the late born neurons that lack in thoracic segments because of early NB quiescence, but it cannot be excluded that different neuroblast types adopt a different strategy.

I found the ts-MARCM results confusing for 2 reasons:

1. It's not clear to me why there are so many single cell clones in div 3 and 4 in abdominal segments. This is not compatible with the division model depicted for abdominal segments, unless GMCs are produced in those division window and the MARCM hits the GMC, as also mentioned in the legend for G. This aspect is important because, either the previous model by Baumgardt et al. - please correct cit. currently Gunnar et al. 2026 - is wrong, or something strange happens in this experiment, or the relative temporal order is incorrect.
2. In segments other than abdomen, it is quite rare to hit proper clones, it appears that only GMCs are hit by recombination, with very few exceptions. Could the author please provide an explanation for this or at least mention this aspect? It is also unclear whether in F the graph includes all types of clones (including 1:0 clones). This is important, because the timing of division for NBs and GMCs is different, and inclusion of 1:0 might lead to a wrong estimate of the NB proliferation window (longer than it actually is because GMCs divide for longer). This is particularly important for the SEZ, where most clones in normalised division 10 and 11 are with ratio 1:0, thus compatible with both terminal division as well as GMC division.

To obtain an estimate of the timing of division, the authors normalise clone size to the size of the bigger clone in the abdomen. What happened to those samples where no abdominal clones were hit? Were they simply excluded from the analysis?

It is proposed that in the thorax late temporal cohort neurons are not produced, yet the ts-MARCM experiment detects some 1:0 clones. What is the fate of these cells? Are they all derived from GMC division and therefore decoupled from the temporal identity window? Or is this a re-activation of division?

"in A1, a majority of segments had one Notch OFF/B neuron that failed to label with Eve" does "the majority" in this sentence mean that there were cases where all B neurons were labelled with Eve? If yes, where would this stochasticity come from? Additionally, there is no evidence that it's the first born NotchOFF neuron in A1 that does not express Eve. The authors should clarify where this speculation comes from. When discussing trends shared with other phyla:

A- "In the mammalian spinal cord, more neurons are present in regions that control limbs (Francius et al., 2013). Analogously, EL numbers do not smoothly taper from anterior to posterior; instead, the largest number of ELs is found in two non-adjacent regions, SEZ and the abdomen." It's unclear what is the link between the figure in the mammalian spinal cord and the Drosophila embryo. The embryo doesn't even have limbs and the number of neurons measured here refer only to a single lineage, while there could be (and in fact there are) lineage-to-lineage differences that could depict a different scenario.

B- The parallelism between V1 mouse neurons and EL Drosophila neurons is also unclear to me. The similarity in fold change across segments could be a pure coincidence and, from what I understand, the two cell types are not functionally linked.

Minor comments:

I found the manuscript somewhat difficult to follow, even though I am familiar with both the model and the topic. For non-specialist readers, I expect it will be even more challenging. The presentation of the results often feels fragmented, at times resembling a sequence of brief statements rather than a continuous narrative. I would encourage the authors to provide more synthesis and interpretation, for example by summarising key findings, rather than listing in detail the number of neurons labelled in each segment for every experiment. This would make the results more accessible and easier to digest.

From the way the MS is written it's not clear from the beginning that the work focuses exclusively on embryonic-born neurons. Since in Drosophila neuronal stem cells undergo two rounds of neurogenesis, one in the embryo and one in the larva, this omission could lead to confusion.

In the abstract, what would be the other temporal cohorts generated in specific regions? (ref to: "In specific regions, NB3-3 neuroblasts produce additional types of temporal cohorts, including but not limited to the late-born EL temporal cohort.")

This sentence in the introduction is inaccurate: "The Drosophila CNS is organized into an anterior hindbrain-like subesophageal zone (SEZ) and a posterior spinal cord-like nerve cord". The anterior hindbrain-like portion of the CNS is in fact the supraesophageal ganglion (or cerebrum), while the SEZ is a posterior-like region.

Fig 1E: the encoding of the significance is not immediately clear. In the legend the 4 stars could also be arranged in the same way for clarity.

Fig 2E legend: it is mentioned that B corresponds to a 1:4 clone, however the MARCM example is shown for C and it's a 1:5.

The occurrence of "undifferentiated" neurons in Th segments is in less than 10% of the clones, I wonder if this a stochastic or deterministic event and to what extent small cell bodies could just be the consequence of local differences in tissue architecture.

Fig 2I: it's unclear what the purple means (I suppose it might be Eve expression) and why in J there should be one purple cell not labelled by the ts-MARCM when this is not present in H and I.

"When synapses do occur, they are numerically similar from segment to segment". It's unclear where the evidence for this statement comes from, please clarify or remove the sentence.

"In SEZ2, NB3-3 divides 10 times (Figure 2F)". Figure 2F does not support this statement and Figure 7 shows 12 divisions. Possibly SEZ2 and 3 have been inverted in this statement, please clarify.

Referees cross-commenting

I agree with most of the comments/suggestions provided by the other two reviewers. In particular: I agree with reviewer #1's comment about failure to express Eve being a mechanism for controlling neurons number, as this is a circular argument. I agree with reviewer #2's concern about the use of the word "flexibility"; "heterogeneity" would be a more appropriate term, as I would associate the word "flexibility" to the ability of a single neuroblast in a single segment to produce neurons with different fates under, for example, unusual growth conditions. Here no genetic/epigenetic manipulations were performed to address flexibility and the observed (stereotypical) differences result from axial patterning. As a note, Reviewer #1 asks about other temporal cohorts of EL neurons produced by other lineages, but these neurons are specifically generated from NB3-3. To generalise the observations reported in this study, the authors would need to focus on other molecularly defined temporal cohorts or, more generally, on other lineages, which, however, are likely to adopt different combinations of mecahnisms to tune progeny number across segments.

Significance

In Drosophila melanogaster, the relationship between neural progenitors and their neuronal progeny has been studied in great detail. This work has provided a comprehensive description of the number of progenitors present in each embryonic segment, their molecular identities, the number of neurons they produce, and the temporal transcriptional cascades that couple progenitor temporal identity to neuronal fate. This work adds to the existing knowledge a detailed characterisation of intersegmental differences in the pattern of proliferation of a single type of neuronal progenitor as well as in post-divisional fate depending on anterior-posterior position in the body axis (i.e. programmed cell death and Notch signalling activation). This is a first step towards understanding the cellular and molecular mechanisms underlying such differences, but it's not disclosing them.

That homologous neuroblasts can generate variable numbers of progeny neurons depending on their segmental position has been established previously. What this manuscript adds is the demonstration that these differences arise through a combination of altered division patterns and differential programmed cell death, thereby revealing a more complex and less predictable scenario than could have been anticipated from existing knowledge in other contexts. The advance provided by this study is therefore incremental, refining rather than overturning our understanding of how segmental diversity in neuroblast lineages is achieved. By touching on the general question of how progenitors generate diversity, this work could be of broad interest to developmental neuroscientists beyond the fly field. However, the way it is currently written does not make it very accessible to non-specialists.

My expertise: Drosophila neurodevelopment, nerve cord, cell types specification

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Referee #2

Evidence, reproducibility and clarity

In this manuscript, Vasudevan et al provide a detailed characterisation of the different numbers and temporal birthdates of Even-skipped Lateral (EL) neurons produced at in different segments from the same neuroblast, NB3-3. The work highlights the differences in EL neuronal generation across segments is achieved through a combination of different division patterns, failure to upregulate EL marker Eve and segment-specific program cell death. For neurons born within the same window and segment, the authors describe additional heterogeneity in their circuit formation. The work underscores the large diversity that the same neuroblast can generate across segments.

Major comments:

• Based on the ts-MARCM 1:0 clones representing 100% of the SEZ clones at any given inferred cell division, the authors conclude "NB3-3 neuroblasts generate proliferative daughter GMCs in the SEZ and thorax on most divisions". Figure 2G does not have any data for SEZ before inferred division 5, whereas there is data in other regions. The authors also state "In the SEZ and abdomen, ELs were labelled regardless of induction time." In reference to Fig 2F, which seems inaccurate given there are no SEZ clones before inferred division 5. There is no comment on this fact, which is surprising give their focus on temporal cohorts. The authors should explain this discrepancy, if known, or modify their statements to reflect the data.
• The temporal cohort (early-born vs late-born) identity is exclusively examined based on markers. Given the absence of SEZ clones from early NB3-3 divisions, a time course showing that the SEZ generate early-born Els or some other complementary method would be desirable.
• The authors repeatedly refer to their work as showing how a stem cell type can have "flexibility". Flexibility would imply that NB3-3 from one segment could adopt a different behaviour (different division pattern, or cell death or connectivity) if it were placed in a different segment. This is not what is being shown. In my opinion, "heterogeneity" of the same neuroblast across different segments would be more appropriate.

Minor comments:

• Figure 2A depicts a combination of known data and conclusions from their own (mainly SEZ). The authors might consider editing the figure to highlight what is new. A possibility would be for figure A to be a diagram of the experimental design and their summary division pattern to be shown after the new data instead of being panel A.
• The authors state that they combined published ts-MARCM with their new one, which differed in a number ways that they list, but they don't specify which limitations are associated with the published vs new dataset. Could the authors please clarify?
• The title refers exclusively to "temporal cohorts", which in the manuscript are defined quite narrowly and do not seem to apply to all segments.
• Several cited references are missing from the Reference list at the end. Could the authors please double check this? (e.g. Matsushita, 1997; Sweeney et al., 2018)
• Legend for figure 2 is a bit confusing, there is a "(A)" within the legend for (D), which indicates that segments A1-A7 are shown (this seems inaccurate, as it only goes to A6).

Significance

This study provides a comprehensive analysis of different cell biological scenarios for a neuroblast to generate distinct progeny across repeating axial units. The strength is the detailed and systematic approach across segments and possible scenarios: different division patterns, cell death, molecular marker expression. While it focuses on one specific neuroblast of the ventral nerve cord of Drosophila, the authors have done extensive work to place their findings and interpretation in the context of other cell types and across model organisms both in the introduction and discussion. This makes the work of interest for developmental biologists in general, neurodevelopment research in particular and those interested in circuit assembly, beyond their specialised community. This point of view comes from someone working in vertebrate CNS development.

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Referee #1

Evidence, reproducibility and clarity

Summary: The study by Vasudevan et al intends to address how serially homologous neural progenitors generate different numbers and types of neurons depending on their location along the body axis. Investigation of full repertoire of neurogenesis for these progenitors necessitates a precise ability to track the fates of both progenitors and their neuronal progeny making it extremely difficult in vertebrate paradigm. The authors used NB3-3 in the developing fly embryo as a model to investigate the full extent of the flexibility in neurogenesis from a single type of serially homologous stem cell. Previous work showed NB3-3 generates neurons including lateral interneurons that can be positively labeled by Even-skipped, but detailed characterization of the NB3-3 lineage mainly focused on 3 segments during embryogenesis. The authors defined the number of EL neurons in all segments of the central nervous system in early larvae after the completion of circuit formation and carried out clonal analyses to determine the proliferation pattern of NB3-3. They described the failure to express Eve in Notch OFF/B neurons as a new mechanism for controlling the number of EL neurons and PCD limits EL neurons in terminal segments.

Major comments: The authors performed careful analyses of the NB3-3 lineage using EL neurons. My main concerns are limited applicability of their findings and lack of mechanisms as how NB3-3 generate various numbers of EL neurons. Their findings are exclusively relevant to the NB3-3 lineage despite their effort in highlighting that other NB lineages also generate temporal cohorts of EL neurons. I disagreed with their conclusion that failure to express Eve as a mechanism for controlling EL neuron numbers when Eve serves as the marker for these neurons. Are there any other strategy to assess the fates and functions of these cells beside relying solely on Eve expression? I am not familiar with the significance of Eve expression on the functions of these neurons. Is it possible to perform clonal analyses of NB3-3 mutant for Eve and see if these neurons adopt different functionalities/identities? If NB3-3 in the SEZ continually generate GMCs based on the interpretation of clonal analyses and depicted in Fig. 2A, why is the percent of clones that are 1:0 virtually at or near 100% from division 6-11 shown in 2G? The authors also indicate that NB3-3 in the abdomen directly generate Notch OFF/B cells that assume EL neuronal identity. In this scenario, shouldn't the percent of 1:0 clones be 100% in later divisions in Fig. 2G? Based on the number of clones in abdomen shown in Fig. 2E, I cannot seem to understand how the authors come to the percent of 1:0 clones shown in Fig. 2G

There are many potentially interesting questions related to this study that can significantly broaden the impact of this study. For example, are other NB lineages that also generate distinct temporal cohorts of EL neurons display similar proliferation patterns (type 1 division in SEZ, early termination of cell division in thoracic segments and type 0 division in abdomen)? Why does NB3-3 in the thoracic segment become quiescence so much sooner than SEZ and abdominal segments? The authors' observations suggest that NB3-3 in SEZ and abdomen generate a similar number of EL neurons despite the difference in their division patterns (type 1 vs type 0). Are the mechanisms that promote EL neuron generate in NB3-3 in SEZ and abdomen the same? Anything else is known beside Notch OFF?

Minor comments:

The authors' writing style is highly unusual especially in the result section. There is an overwhelming large amount of background information in the result section but very thin description on their observations. The background information portion also includes previously published observations. Since the nature of this study is not hypothesis-driven, it is very confusing to read in many places and difficult to distinguish their original observations from previously published results and making. One easily achievable improvement is to insert relevant figure numbers into the text more often.

Significance

The study by Vasudevan et al intends to address how serially homologous neural progenitors generate different numbers and types of neurons depending on their location along the body axis. Investigation of full repertoire of neurogenesis for these progenitors necessitates a precise ability to track the fates of both progenitors and their neuronal progeny making it extremely difficult in vertebrate paradigm. The authors used NB3-3 in the developing fly embryo as a model to investigate the full extent of the flexibility in neurogenesis from a single type of serially homologous stem cell. Previous work showed NB3-3 generates neurons including lateral interneurons that can be positively labeled by Even-skipped, but detailed characterization of the NB3-3 lineage mainly focused on 3 segments during embryogenesis. The authors defined the number of EL neurons in all segments of the central nervous system in early larvae after the completion of circuit formation and carried out clonal analyses to determine the proliferation pattern of NB3-3. They described the failure to express Eve in Notch OFF/B neurons as a new mechanism for controlling the number of EL neurons and PCD limits EL neurons in terminal segments.

What is the source for this? The word "way" forms the word "exodus"?

Oh, I found it. Exodus is from the Greek word ἔξοδος (éxodos), which means "a going out" or "departure". It is a compound word from ἐξ (ex), meaning "out of," and ὁδός (hodos)

Interesting point about paying attention to the "the". Normally we skip over "the". But here, "the" has sigficiant meaning.

You can insert a TOC using the Reference Tab in MS Word.

I am a bit confused on how to interpret this. From looking at the link, the "Philosopher" he refers to is Plato, and the quote I am guessing, from the citations, is from Plato's ethics. Plato lists several virtues: courage, moderation, piety, and justice (Scavone 2023). I'd like to read the original by Plato before making real conclusions, but I believe the author's argument is appealing to both the virtues of moderation and piety. We've established already that among the differing medieval attitudes around games, one of the conclusions was moderation was key, such as this passage selected from the textbook, which was originally by John Salisbury, "'There are, however, times when, viewed from a certain aspect, games of chance are permissible. For example, if without evil consequences they alleviate the strain of heavy responsibilities and if without harming character they introduce an agreeable period of relaxation. Liberty to do as one pleases is justified if moderation controls the act'" (Milliman, 587), and so it's easy to see how, say, dice games would contradict Plato's virtue of moderation from a more severe perspective, since they can become addictive and make a gambler of a person. There is also a contradiction to the virtue of piety that the author may be appealing to, that since a person should "direct his action to something else" he means, potentially, that games distract a person from both their other responsibilities (both religion and justice if you were to look at it under the lens of Plato's virtues) and from the worship of God, not because a person should be spending every second of their time on these things, but because they have the capacity to steer a person the wrong way and tempt them away from keeping to the virtues.

I've left a link to the article I looked at here, it is by Daniel C. Scavone.

In this article Carnival seems to be a day for boys to bring their roosters to fight each other. Previously, we read this was a celebration before Lent, " a rejoicing period of time" (Milliman, 597). Lent from my understanding is where you give something up in order to develop or strengthen your relationship to God. This seems to be an interesting game especially since this would be a game to relax the students before a time of ceremonial divinity. This game also fits in with ceremonial combat mention by Milliman (591). I wonder why it was deemed morally acceptable to have animals fight but not have tournaments. This seems like it would feed a love of violence. However, this book does not mention details of how far the cock-fight would go, so maybe not. There seems to be a respect for knowing how to fight as a discipline and actually fighting as a sport.

Too much distortion harms performance. Not too sure why the contrastive noise can't be too much. I mean if we take an image of completely random noise, doesn't this expose the hallucination the most? Maybe not, because the model will at some point, when there is too much noise, refuse to provide an answer?

In the bipolar world order, the two global superpowers greatly benefited from competing from the other and there not being any countries competing against them. With a new world order there is less security from great powers and emerging powers without two clear hegemonic powers. And so, there is less security felt from other countries who do not have a power to assist them leading to a higher possibility of conflicts especially in unstable regions

The author's perspective is that Russia seeks to become a global power but has a number of obstacles. At the end of the cold war left Russia with not the same status as a world power like the Soviet Union was. To gain the status as a global superpower once again countries have to rely on their own resources and advantages for hegemony. Being naive with other global powers can bring down the status that they are building up. But the author questions its overall power such as military capabilities and how it can counter threats from the middle east, central and south Asia how they can compete with other great powers to gain influence near its borders.

via u/schematical at https://reddit.com/user/schematical/comments/1oe41bx/what_is_a_database_index_as_explained_to_a_1930s/

Perhaps it's a question of the "long search" versus the "short search"? Long searches with proper connecting tissue are more often the thing that produces innovation out of serendipity and this is the thing of greatest value versus "What time does the Superbowl start?". How do you build a database index to improve the "long search"?

See, for example Keith Thomas' problem: https://hyp.is/DFLyZljJEe2dD-t046xWvQ/www.lrb.co.uk/the-paper/v32/n11/keith-thomas/diary

nftables firewall configuration

This is an interesting perspective, and I completely resonate with Nick's character being very lackluster, especially with the grand personalities that he associates himself with. I wonder if Nick would appear less bland if he was less ignorant of his own bias? We discussed while reading the novel how Nick may have some biases towards Gatsby because of his involvement in World War I and seems to type the women as frailer and more elegant in a way, as if they are not capable of understanding the things he does. Many times, throughout the novel we see that Nick is rather ignorant, and there are many things that he simply does not pick up on that are happening right in front of him. I kind of enjoy Nick's narration style and the idea that he is the one "normal" character in a world full of very curious people, yet unfortunately that is tainted with his unreliability as a narrator that makes him much less appealing.

Is this a warning not to be overwhelmed? or assume that nothing is possible?

eLife Assessment

This important study resolves the structure of one missing piece of the eukaryotic DNA replication fork, the leading strand clamp loader. Overall, the data are convincing, with electron microscopy data providing a strong basis for analyzing differences and similarities with other RFC complexes. A minor point is that the evidence supporting the proposed role of the β-hairpin is incomplete.

Reviewer #1 (Public review):

Summary:

The authors report the structure of the human CTF18-RFC complex bound to PCNA. Similar structures (and more) have been reported by the O'Donnell and Li labs. This study should add to our understanding of CTF18-RFC in DNA replication and clamp loaders in general. However, there are numerous major issues that I recommend the authors fix.

Strengths:

The structures reported are strong and useful for comparison with other clamp loader structures that have been reported lately.

Comments on revisions:

The revised manuscript is greatly improved. The comparison with hRFC and the addition of direct PCNA loading data from the Hedglin group are particular highlights. I think this is a strong addition to the literature.

I only have minor comments on the revised manuscript.

(1) The clamp loading kinetic data in Figure 6 would be more easily interpreted if the three graphs all had the same x axes, and if addition of RFC was t=0 rather than t=60 sec.

(2) The author's statement that "CTF18-RFC displayed a slightly faster rate than RFC" seems to me a bit misleading, even though this is technically correct. The two loaders have indistinguishable rate constants for the fast phase, and RFC is a bit slower than CTF18-RFC in the slow phase. However, the data also show that RFC is overall more efficient than CTF18-RFC at loading PCNA because much more flux through the fast phase (rel amplitudes 0.73 vs 0.36). Because the slow phase represents such a reduced fraction of loading events, the slight reduction in rate constant for the slow phase doesn't impact RFC's overall loading. And because the majority of loading events are in the fast phase, RFC has a faster halftime than CTF18-RFC. (Is it known what the different phases correspond to? If it is known, it might be interesting to discuss.)

(3) AAA+ is an acronym for "ATPases Associated with diverse cellular Activities" rather than "Adenosine Triphosphatase Associated".

Reviewer #2 (Public review):

Summary

Briola and co-authors have performed a structural analysis of the human CTF18 clamp loader bound to PCNA. The authors purified the complexes and formed a complex in solution. They used cryo-EM to determine the structure to high resolution. The complex assumed an auto-inhibited conformation, where DNA binding is blocked, which is of regulatory importance and suggests that additional factors could be required to support PCNA loading on DNA. The authors carefully analysed the structure and compared it to RFC and related structures.

Strength & Weakness

Their overall analysis is of high quality, and they identified, among other things, a human-specific beta-hairpin in Ctf18 that flexible tethers Ctf18 to Rfc2-5. Indeed, deletion of the beta-hairpin resulted in reduced complex stability and a reduction in a primer extension assay with Pol ε. Moreover, the authors identify that the Ctf18 ATP-binding domain assumes a more flexible organisation.

The data are discussed accurately and relevantly, which provides an important framework for rationalising the results.

All in all, this is a high-quality manuscript that identifies a key intermediate in CTF18-dependent clamp loading.

Comments on revisions:

The authors have done a nice job with the revision.

Reviewer #3 (Public review):

Summary:

CTF18-RFC is an alternative eukaryotic PCNA sliding clamp loader which is thought to specialize in loading PCNA on the leading strand. Eukaryotic clamp loaders (RFC complexes) have an interchangeable large subunit which is responsible for their specialized functions. The authors show that the CTF18 large subunit has several features responsible for its weaker PCNA loading activity, and that the resulting weakened stability of the complex is compensated by a novel beta hairpin backside hook. The authors show this hook is required for the optimal stability and activity of the complex.

Relevance:

The structural findings are important for understanding RFC enzymology and novel ways that the widespread class of AAA ATPases can be adapted to specialized functions. A better understanding of CTF18-RFC function will also provide clarity into aspects of DNA replication, cohesion establishment and the DNA damage response.

Strengths:

The cryo-EM structures are of high quality enabling accurate modelling of the complex and providing a strong basis for analyzing differences and similarities with other RFC complexes.

Weaknesses:

The manuscript would have benefited from a more detailed biochemical analysis using mutagenesis and assays to tease apart the differences with the canonical RFC complex. Analysis of the FRET assay could be improved.

Overall appraisal:

Overall, the work presented here is solid and important. The data is mostly sufficient to support the stated conclusions.

Comments on revisions:

While the authors addressed my previous specific concerns, they have now added a new experiment which raises new concerns.

The FRET clamp loading experiments (Fig. 6) appear to be overfitted so that the fitted values are unlikely to be robust and it is difficult to know what they mean, and this is not explained in this manuscript. Specifically, the contribution of two exponentials is floated in each experiment. By eye, CTF18-RFC looks much slower than RFC1-RFC (as also shown previously in the literature) but the kinetic constants and text suggest it is faster. This is because the contribution of the fast exponential is substantially decreased, and the rate constants then compensate for this. There is a similar change in contribution of the slow and fast rates between WT CTF18 and the variant (where the data curves look the same) and this has been balanced out by a change in the rate constants, which is then interpreted as a defect. I doubt the data are strong enough to confidently fit all these co-dependent parameters, especially for CTF18, where a fast initial phase is not visible. I would recommend either removing this figure or doing a more careful and thorough analysis.

Author response:

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

Reviewer #1 (Public review): 

Summary: 

The authors report the structure of the human CTF18-RFC complex bound to PCNA. Similar structures (and more) have been reported by the O'Donnell and Li labs. This study should add to our understanding of CTF18-RFC in DNA replication and clamp loaders in general. However, there are numerous major issues that I recommend the authors fix. 

Strengths: 

The structures reported are strong and useful for comparison with other clamp loader structures that have been reported lately. 

Weaknesses: 

The structures don't show how CTF18-RFC opens or loads PCNA. There are recent structures from other groups that do examine these steps in more detail, although this does not really dampen this reviewer's enthusiasm. It does mean that the authors should spend their time investigating aspects of CTF18-RFC function that were overlooked or not explored in detail in the competing papers. The paper poorly describes the interactions of CTF18-RFC with PCNA and the ATPase active sites, which are the main interest points. The nomenclature choices made by the authors make the manuscript very difficult to read. 

Reviewer #2 (Public review): 

Summary 

Briola and co-authors have performed a structural analysis of the human CTF18 clamp loader bound to PCNA. The authors purified the complexes and formed a complex in solution. They used cryo-EM to determine the structure to high resolution. The complex assumed an auto-inhibited conformation, where DNA binding is blocked, which is of regulatory importance and suggests that additional factors could be required to support PCNA loading on DNA. The authors carefully analysed the structure and compared it to RFC and related structures. 

Strength & Weakness 

Their overall analysis is of high quality, and they identified, among other things, a human-specific beta-hairpin in Ctf18 that flexibly tethers Ctf18 to Rfc2-5. Indeed, deletion of the beta-hairpin resulted in reduced complex stability and a reduction in a primer extension assay with Pol ε. This is potentially very interesting, although some more work is needed on the quantification. Moreover, the authors argue that the Ctf18 ATP-binding domain assumes a more flexible organisation, but their visual representation could be improved. 

The data are discussed accurately and relevantly, which provides an important framework for rationalising the results. 

All in all, this is a high-quality manuscript that identifies a key intermediate in CTF18dependent clamp loading. 

Reviewer #3 (Public review): 

Summary: 

CTF18-RFC is an alternative eukaryotic PCNA sliding clamp loader that is thought to specialize in loading PCNA on the leading strand. Eukaryotic clamp loaders (RFC complexes) have an interchangeable large subunit that is responsible for their specialized functions. The authors show that the CTF18 large subunit has several features responsible for its weaker PCNA loading activity and that the resulting weakened stability of the complex is compensated by a novel beta hairpin backside hook. The authors show this hook is required for the optimal stability and activity of the complex. 

Relevance: 

The structural findings are important for understanding RFC enzymology and novel ways that the widespread class of AAA ATPases can be adapted to specialized functions. A better understanding of CTF18-RFC function will also provide clarity into aspects of DNA replication, cohesion establishment, and the DNA damage response. 

Strengths: 

The cryo-EM structures are of high quality enabling accurate modelling of the complex and providing a strong basis for analyzing differences and similarities with other RFC complexes. 

Weaknesses: 

The manuscript would have benefitted from more detailed biochemical analysis to tease apart the differences with the canonical RFC complex. 

I'm not aware of using Mg depletion to trap active states of AAA ATPases. Perhaps the authors could provide a reference to successful examples of this and explain why they chose not to use the more standard practice in the field of using ATP analogues to increase the lifespan of reaction intermediates. 

Overall appraisal: 

Overall the work presented here is solid and important. The data is sufficient to support the stated conclusions and so I do not suggest any additional experiments. 

Reviewer #1 (Recommendations for the authors): 

We thank the reviewer for their positive comments and for their thorough review. All raised points have been addressed below.

Major points 

(1) The nomenclature used in the paper is very confusing and sometimes incorrect. The authors refer to CTF18 protein as "Ctf18", and the entire CTF18-RFC complex as "CTF18". This results in massive confusion because it is hard to ascertain whether the authors are discussing the individual subunits or the entire complex. Because these are human proteins, each protein name should be fully capitalized (i.e. CTF18, RFC4 etc). The full complex should be referred to more clearly with the designation CTF18-RFC or CTF18-RLC (RFC-like complex). Also, because the yeast and human clamp loader complexes use the same nomenclature for different subunits, it would be best for the authors to use the "A, B, C, D, E subunit" nomenclature that has been standard in the field for the past 20 years. Finally, the authors try to distinguish PCNA subunits by labeling them "PCNA2" or "PCNA1" (see Page 8 lines 180,181 for an example). This is confusing because the names of the RFC subunits have similar formats (RFC2, RFC3, RFC4, etc). In the case of RFC this denotes unique genes, whereas PCNA is a homotrimer. Could the authors think of another way to denote the different subunits, such as super/subscript? PCNA-I, PCNA-II, PCNA-III? 

We thank the reviewer for pointing out the confusing nomenclature. Following the referee suggestion, we now refer to the CTF18 full complex as “CTF18-RFC”. We prefer keeping the nomenclature used for CTFC18 subunits as RFC2, RFC3 etc., as recently used in Yuan et al, Science, 2024. However, we followed the referee’s suggestion for PCNA subunits, now referred to as PCNA-I, PCNA-II and PCNA-III.

(2) I believe that the authors are over-interpreting their data in Figure 1. The claim that "less sharp definition" of the map corresponding to the AAA+ domain of Ctf18 supports a relatively high mobility of this subunit is largely unsubstantiated. There are several reasons why one could get varying resolution in a cryo-EM reconstruction, such as compositional heterogeneity, preferred orientation artifacts, or how the complex interacts with the air-water interface. If other data were presented that showed this subunit is flexible, this evidence would support that data but cannot alone as justification for subunit mobility. Along these lines, how was the buried surface area (2300 vs 1400 A2) calculated? Is this the total surface area or only the buried surface area involving the AAA+ domains? It is surprising that these numbers are so different considering that the subunits and complexes look so similar (Figures 1c and 2b). 

We respectfully disagree with the suggestion that our interpretation of local flexibility in the AAA+ domain of Ctf18 is overreaching. Several lines of evidence support this interpretation. First, compositional heterogeneity is unlikely, as the A′ domain of Ctf18 is well-resolved and forms stable interactions with RFC3, indicating that Ctf18 is consistently incorporated into the complex. Second, preferred orientation artifacts are excluded, as the particle distribution shows excellent angular coverage (Fig. S9a). Third, we now include a 3D variability analysis (3DVA; Supplementary Video 1), which reveals local conformational heterogeneity centered around the AAA+ domain of Ctf18, consistent with intrinsic flexibility.

Regarding the buried surface area values, the reported numbers refer specifically to the interfaces between the AAA+ domain of Ctf18 and RFC2, and are derived from buried surface area calculations performed with PISA. The smaller interface (~1400 Ų) compared to RFC1–RFC2 (~2300 Ų) reflects low sequence identity (~26%) and divergent structural features, including the absence of conserved elements such as the canonical PIP-box in Ctf18. We have clarified and expanded this explanation in the revised manuscript (Page 7).

(3) The authors very briefly discuss interactions with PCNA and how the CTF18-RFC complex differs from the RFC complex. This is amongst the most interesting results from their work, but also not well-developed. Moreover, Figure 3D describing these interactions is extremely unclear. I feel like this observation had potential to be interesting, but is largely ignored by the authors. 

We thank the referee for pointing this out. We have expanded the section describing the interactions of CTF18-RFC and PCNA (Page 9 in the new manuscript), and made a new panel figure with further details (Fig. 3D).  

(4) The authors make the observation that key ATP-binding residues in RFC4 are displaced and incompatible with nucleotide binding in their CTF18-RFC structure compared to the hRFC structure. This should be a main-text figure showing these displacements and how it is incompatible with ATP binding. Again, this is likely an interesting finding that is largely glossed over by the authors. 

We now discuss this feature in detail (Pag 11 in the new manuscript), and added two figure insets (Fig. 4c) describing the incompatibility of RFC4 with nucleotide binding.

(5) The authors claim that the work of another group (citation 50) "validate(s) our predictions regarding the significant similarities between CTF18-RFC and canonical RFC in loading PCNA onto a ss/dsDNA junction." However, as far as this reviewer can tell the work in citation 50 was posted online before the first draft of this manuscript appeared on biorxiv, so it is dubious to claim that these were "predictions." 

We agree with the referee about this claim. We have now revised the text as follows:

“While our work was being finalized, several cryo-EM structures of human CTF18-RFC bound to PCNA and primer/template DNA were reported by another group (He et al, PNAS, 2024). These findings are consistent with the distinct features of CTF18-RFC observed in our structures and independently support the notion of significant mechanistic similarity between CTF18-RFC and canonical RFC in loading PCNA onto a ss/dsDNA junction”.

(6) The authors use a primer extension assay to test the effects of truncating the Nterminal beta hairpin of CTF18. However, this assay is only a proxy for loading efficiency and the observed effects of the mutation are rather subtle. The authors could test their hypothesis more clearly if they performed an ATPase assay or even better a clamp loading assay. 

We thank the referee for this valuable suggestion. In response, we have performed clamp loading assays comparing the activities of human RFC, wild-type CTF18-RFC, and the β-hairpin–truncated CTF18-RFC mutant. The results, now presented in Fig. 6 and Table 1 of the revised manuscript, clearly show that truncation of the N-terminal βhairpin results in a slower rate of PCNA loading. We propose that this reduced loading rate likely contributes to the diminished Pol ε–mediated DNA synthesis observed in the primer extension assays.

Minor points 

(1) Page 3 line 53 the introduction suggests that ATP hydrolysis prompts clamp closure. While this may be the case, to my knowledge all recent structural work shows that closure can occur without ATP hydrolysis. It may be better to rephrase it to highlight that under normal loading conditions, ATP hydrolysis occurs before clamp closure. 

The text now reads (Page 3): 

“DNA binding prompts the closure of the clamp and hydrolysis of ATP induces the concurrent disassembly of the closed clamp loader from the sliding clamp-DNA complex, completing the cycle necessary for the engagement of the replicative polymerases to start DNA synthesis.”

(2) Page 3 line 60, I do not see how the employment of alternative loaders highlights the specificity of the loading mechanism - would it not be possible for multiple loaders to have promiscuous clamp loading? 

We thank the referee for this comment. The text now reads (Page 3):

“However, eukaryotes also employ alternative loaders (20), including CTF18-RFC (6, 21-24), which likely use a conserved loading mechanism but are functionally specialized through specific protein interactions and context-dependent roles in DNA replication.”

(3) Page 4 line 75 could you please cite a study that shows Ctf8 and Dcc1 bind to the Ctf18 C-terminus and that a long linker is predicted to be flexible? 

Two references have been added (Stokes et al, NAR, 2020 and Grabarczyk et al, Structure, 2018)

(4) Figure 2A has the N-terminal region of Ctf18 as bound to RFC3 but should likely be labeled as bound to RFC5. This caused significant confusion while trying to parse this figure. Further, the inclusion of "X" as a sequence - does this refer to a sequence that was not buildable in the cryo-EM map? I would be surprised that density immediately after the conserved DEXX box motif is unbuildable. If this is the case, it should be clearly stated in the figure legend that "X" denotes an unbuildable sequence. For the conserved beta-hairpin in the sequence, could the authors superimpose the AlphaFold prediction onto their structure? It would be more informative than just looking at the sequence. 

We apologize for this confusion. The error in Figure 2A has been corrected. The figure caption now explicitely says that “X” refers to amino acid residues in the sequence which were not modelled. A superposition of the cryo-EM model of the N-terminal Beta hairpin in human Ctf18 and AlphaFold predictions for this feature in drosophila and yeast Ctf18 is now presented in Figure 2A.

(5) Page 8 line 168, the use of the term "RFC5" here feels improper, since the "C" subunit is not RFC5 in all lower eukaryotes (see comment above about nomenclature). For instance, in S cerevisiae, the C subunit is RFC3. I would expect this interaction to be maintained in all C subunits, not all RFC5 subunits. 

The text now reads (Page 8):

“Therefore, lower eukaryotes may use a similar b-hairpin motif to bind the corresponding subunit of the RFC-module complex (RFC5 in human, Rfc3 in S. cerevisiae), emphasizing its importance.”  

(6) Page 10 line 228, the authors claim that hydrolysis is dispensable at the Ctf18/RFC2 interface based on evidence from RFC1/RFC2 interface, by analogy that this is the "A/B" interface in both loaders. However, the wording makes it sound as if the cited data were collected while studying Ctf18 loaders. The authors should clarify this point. 

The text has been modified as follows (Pag 11): 

“Prior research has indicated that hydrolysis at the large subunit/RFC2 interface is not essential for clamp loading by various loaders (48-51), while the others are critical for the clamp-loading activity of eukaryotic RFCs. “

(7) Page 11 line 243/244 the authors introduce the separation pin. Could they clarify whether Ctf18 contains any aromatic residues in this structural motif that would suggest it serves the same functional purpose? Also, the authors highlight this is similar to yeast RFC, which makes it sound like this is not conserved in human RFC, but the structural motif is also conserved in human RFC. 

We thank the reviewer for this helpful comment. We have clarified in the revised text (Page 12) that the separation pin is conserved not only in yeast RFC but also in human RFC, and now note that human Ctf18 also harbors aromatic residues at the corresponding positions. This observation is supported by the new panel in Figure 4e.

Minutia 

(1) Page 2 line 37 please remove the word "and" before PCNA. 

This has been corrected.

(2) Please define AAA+ and update the language to clarify that not all pentameric AAA+ ATPases are clamp loaders. 

AAA+ has been now defined (Page 3).

(3) Page 4 line 86 Given the relatively weak interaction of Pol ε. 

This has been corrected.

(4) Page 8 line 204 the authors likely mean "leucine" and not "lysine". 

We thank the reviewer for catching this. The error has been corrected.

(5) Page 14 line 300, the authors claim that CTF18 utilizes three subunits but then list four. 

We have corrected this.

Reviewer #2 (Recommendations for the authors): 

We thank the reviewer for their positive comments and valuable suggestions. The points raised by the referee have been addressed below.

Major point: 

(1) Please quantify Figure 6 and S9 from 3 independent repeats and determine the standard deviation to show the variability of the Ctf18 beta hairpin deletion.  The authors suggest that a suboptimal Ctf18 complex interaction with PCNA impacts the stability of the complex, but do not test this hypothesis. Could the suboptimal PIP motif in Ctf18 be changed to an improved motif and the impact tested in the primer extension assay? Although not essential, it would be a nice way to explore the mechanism. 

We thank the reviewer for the suggestion. However, we note that Figure 6b (now 7b) already presents the quantification of the primer extension assay from three independent replicates, with error bars showing standard deviations, and includes the calculated rate of product accumulation. These data clearly indicate a 42% reduction in primer synthesis rate upon deletion of the Ctf18 β-hairpin.

We agree that we do not provide direct evidence of impaired complex stability upon deletion of the Ctf18 β-hairpin. However, the 2D classification of the cryo-EM dataset (Figure S9) shows a marked reduction in the number of particles corresponding to intact CTF18-RFC–PCNA complexes in the β-hairpin deletion sample, with the majority of particles corresponding to free PCNA. This contrasts with the wild-type dataset, where complex particles are predominant. These findings indirectly suggest that deletion of the β-hairpin compromises the stability or assembly of the clamp-loader–clamp complex.

We thank the reviewer for the valuable suggestion to mutate the weak PIP-box of Ctf18. While an interesting direction, we instead sought to directly test the mechanism by performing quantitative clamp loading assays. These assays revealed a significant reduction in the rate of PCNA loading by the CTF18^Δ165–194-RFCmutant (Figure 6), supporting the conclusion that the β-hairpin contributes to productive PCNA loading. This loading delay likely underlies the reduced rate of primer extension observed in the Pol ε assay (Figure 7), consistent with impaired formation of processive polymerase– clamp complexes.

(2) I did not see the method describing how the 2D classes were quantified to evaluate the impact of the Ctf18 beta hairpin deletion on complex formation. Please add the relevant information. 

The relevant information has been added to the Method section:

“For quantification of complex stability, the number of particles contributing to each 2D class was extracted from the classification metadata (Datasets 1 and 3). All classes showing isolated PCNA rings were summed and compared to the total number of particles in classes representing intact CTF18-RFC–PCNA complexes. This analysis was performed for both wild-type and β-hairpin deletion mutant datasets. Notably, no 2D classes corresponding to free PCNA were observed in the wild-type dataset, whereas in the mutant dataset, a substantial fraction of particles corresponded to isolated PCNA, suggesting reduced stability of the mutant complex.”

Minor point: 

(1) Page 2, line 25. Detail what type of mobility is referred to. Do you mean flexibility in the EM-map? 

We have clarified this. The text now reads:

“The unique RFC1 (Ctf18) large subunit of CTF18-RFC, which based on the cryo-EM map shows high relative flexibility, is anchored to PCNA through an atypical low-affinity PIP box”

(2) Page 4, line 82. Please introduce CMGE, or at least state what the abbreviation stands for. 

This has been addressed.

(3) Page 4, line 89. Specify that the architecture of the HUMAN CTF18-RFC module is not known, as the yeast one has been published. 

At the time our study was initiated, the architecture of the human CTF18-RFC module was unknown. A structure of the human complex was published by another group during the final stages of our work and is now properly acknowledged in the Discussion.

(4) Page 6. Is it possible to illustrate why the autoinhibited state cannot bind to DNA? A visual representation would be nice. 

We thank the reviewer for this suggestion. Figure 4b in the original manuscript already illustrates why the autoinhibited, overtwisted conformation of the CTF18-RFC pentamer cannot accommodate DNA. In this state, the inner chamber of the loader is sterically occluded, precluding the binding of duplex DNA.

Reviewer #3 (Recommendations for the authors): 

We thank Reviewer #3 for their constructive feedback and positive overall assessment of our work.

We also thank the reviewer for their remarks on the use of Mg depletion to halt hydrolysis. Magnesium is an essential cofactor for ATP hydrolysis, and its depletion is expected to effectively prevent catalysis by destabilizing the transition state, possibly more completely than the use of slowly hydrolysable analogues such as ATPγS. We have recently employed Mg^²+ depletion to successfully trap a pre-hydrolytic intermediate in a replicative AAA+ helicase engaged in DNA unwinding (Shahid et al., Nature, 2025). This precedent supports the rationale for our choice, and the reference has now been included in the revised manuscript.

I think the authors deposited the FSC curve for the +Mg structure in the -Mg structure PDB/EMDB entry according to the validation report. 

We thank the reviewer for their careful inspection of the deposition materials. The discrepancy in the deposited FSC curve has now been corrected, and the appropriate FSC curves have been assigned to the correct PDB/EMDB entries.

This commentary begins here and awkwardly re-engaged in the Registration Periods section. Let's avoid this redundancy and strive for more paragraph-level coherence and claim progression

Remove adjective 'temporal' and revise sentence structure with theoretical support from Jonathan Crary's 24/7, particularly his commentary on the ends of sleep amid the late capitalist state of constant socioeconomic hardship and compounding austerity conditions.

Add commentary from Crary's 24/7 here to close out the paragraph

This is a vague generalization and overuse of austerity as an abstract driver. Let's get more specific with theoretical applications such as these: "As history has shown," writes Crary," war-related innovations are inevitably assimilated into a broader social sphere, and the sleepless soldier would be the forerunner of the sleepless worker or consumer" (3).

Update this section to follow the new outline structure and organization as of Oct 24.

This section should effectively chart the seasonal patterns of engagement and be placed above the daily and hourly patterns of engagement, subsequently followed by commentary on space and mobility, transit and navigation across the boroughs.

Scale back this expanded edit to only one longer paragraph and make sure to recalibrate the section with an awareness of the Section header's organizing principle being temporal, seasonal patterns of activity. Revise the prose to help frame this from the start and move some the discourse analysis work being done here to the linguistic analysis section, effectively coordinate the transition to highlight CUNY discourse conventions as part of the local public sphere of these subreddits

SBU Logo - add The State University of New York. Change alt text to the name + logo instead of just "logo"

why do you guys keep on ADDING FORSAKEN STUFF IN THE TAGS IT UPSETS ME

A hidden concept here is Occam's razor. In many cases, we somehow come up with parsimonious theories of the generative rule behind the data. This will (I think) be discussed in the following chapters.

This claim needs context. Consider Figure 1.1: it illustrates what machine learning typically calls few‑shot learning—inferring a task from a small number of labeled examples and then generalizing to new instances of the same task. The crucial question is whether such generalization truly happens without relying on previous experience.

Impedance Summary

Impedance is the combination of Resistance (\(R\)) and Reactance (\(X\)).

Resistance is the real part of impedance; when a circuit is driven with DC, there is no distinction between impedance and resistance.

Reactance is the imaginary part of impedance. A capacitor has purely reactive impedance that is inversely proportional to signal frequency. \(X_C = \frac{-1}{2\pi f C}\). Inductive reactance is proportional to signal frequency \(f \) and the inductance \(L\). \(X_L = 2 \pi f L\).

The total reactance is given by \(X = X_L + X_C\). Note \(X_C\) is negative.

The total impedance is \(Z = R + jX\)

Traduce en tu mente el siguiente extracto al español sin mirar el texto y luego comprueba si lo has hecho correctamente o si existen otras alternativas de vocabulario:

The advantages of sharing housing are extensive: the most prominent is the savings you have by paying only for a room and not for the whole house. By sharing expenses, there is the possibility of having a larger space for a lower price. As if that were not enough, following the roommate guide that you can see in the video, it is most likely that no misunderstandings will be generated between the cohabitants and you can even make plans together.

Although there are also disadvantages, the list of advantages can be longer than the list of problems. It is up to the individual to decide which lifestyle he or she prefers to lead.

Sin mirar el texto intenta traducir en tu mente esta parte al español y luego, mirando el texto, comprueba si lo has hecho correctamente:

Most apartment sharers are students or young people who have just started working. The fact that several people of the same age live together can sometimes lead to clashes of character.

this represents the conscious effort to go beyond the surface stories 'myths' and confront the deeper, often hidden realities 'the wreck'

eLife Assessment

This valuable study uses single-molecule imaging to characterize factors controlling the localization, mobility, and function of RNase E in E. coli, a key bacterial ribonuclease central to mRNA catabolism. The supporting evidence for the differential roles of RNAse E's membrane targeting sequence (MTS) and the C-terminal domain (CTD) to RNAse E's diffusion and membrane association is convincing. It provides insight into how RNAse E shapes the spatiotemporal organization of RNA processing in bacterial cells. This interdisciplinary work will be of interest to cell biologists, microbiologists, biochemists, and biophysicists.

Reviewer #1 (Public review):

This paper by Troyer et al. measures the positioning and diffusivity of RNaseE-mEos3.2 proteins in E. coli as a function of rifampicin treatment, compares RNaseE to other E. coli proteins, and measures the effect of changes in domain composition on this localization and motion. The straightforward study is thoroughly presented, including very good descriptions of the imaging parameters and the image analysis/modeling involved, which is good because the key impact of the work lies in presenting this clear methodology for determining the position and mobility of a series of proteins in living bacteria cells.

Most of my concerns in the original review were addressed in this round of revisions based on new text, experiments, and analysis, including most notably:

-A revision of the abstract to focus on the actual topic of the manuscript.<br /> -New experiments (Fig. S1) to confirm that there is no significant undercounting of the fast-moving cytoplasmic population<br /> -Removing the experiments discussion related to degradosome proteins rather than overstating results.<br /> -Improving the logical flow and writing.

One minor concern still remains:

-Though the discussion of the rifampicin-treated cells is improved, this experiment is motivated (line 196) as "To test the effect of mRNA substrates on RNE diffusion", but the conclusion of the paragraph (based on similarities with the effect on LacY) is that the observed changes are due to factors other than the concentration of mRNA substrates, such that the effect of mRNA has not been tested.

Reviewer #2 (Public review):

Summary:

Troyer and colleagues have studied the in vivo localisation and mobility of the E.coli RNaseE (a protein key for mRNA degradation in all bacteria) as well as the impact of two key protein segments (MTS and CTD) on RNase E cellular localisation and mobility. Such sequences are important to study since there is significant sequence diversity within bacteria, as well as lack of clarity about their functional effects. Using single-molecule tracking in living bacteria, the authors confirmed that >90% of RNaseE localised on the membrane, and measured its diffusion coefficient. Via a series of mutants, they also showed that MTS leads to stronger membrane association and slower diffusion compared to a transmembrane motif (despite the latter being more embedded in the membrane), and that the CTD weakens membrane binding. The study also rationalised how the interplay of MTS and CTD modulate mRNA metabolism (and hence gene expression) in different cellular contexts.

The authors have also done an excellent job addressing reviewer's concerns and improving the manuscript during revision.