these are helpful and evidence that are good for writers.

explaining also help not everybody knows everything so it better to explain facts just readers can understand you're going and what you mean by saying that.

the body paragraphs is where all the datils go.

main points need to specific so that the audience get the idea.

selecting your thesis is the best part because what supports your points

best thing about introduction is to engage you reader let people engage in to it

some writer found idea of an introduction after writing a body others begin with introduction.

the feelings of desperation and desire are interconnected. Both are full of charge, suffering and passion.

It actually seems that the smile is more forced. This sentence is articulating vaguely that this love has become a performance, over a true and authentic feeling of love and affection.

This was an interesting parallel that I feel helps paint the picture of the situation to something in a context that feels more familiar to me. Phrasing veiling with this comparison models it as a choice to be culturally/socially respectable.

What a comment to really get a large group of people, most of which know next to nothing of Islamic culture and tradition, to use women's rights as a means to rally support for a series of military actions that were based on primarily other reasons. Convincing speech writing for sure, but I see how they're taking advantage of the fact that their audience does not know the complexity of womens' dignity in Afghanistan to rally support for a fight.

Online remixing can easily slip into cultural appropriation when dominant groups use language and styles without respecting the communities they come from. People can benefit from remixing while original communities lose credit while others benefit from it.

Memes can be seen as cultural units that spread and change through imitation and it can always evolve. Thinking about culture this way helps explain why some ideas catch on quickly while others fade out.

I wonder how social media have ways of tracking our online behavior and recommending content. I think they keep the algorithms secret because of privacy and not wanting other sites to copy.

To prevent illegal content and misinformation that can affect people's mental health. I think it should promote positivity in people's feed as well.

I think the positive parts about social media and my mental health is that some videos can really entertain me and just make my day chill to the point where I can just relax, watch videos, and dm my friends in the group chat. Negative aspects are that sometimes I can compare my life to others like celebrities.

This line is a reference to Isaiah 53, an Old Testament prophecy referring to the coming of the messiah:

"He was despised and rejected by men, a man of sorrows and acquainted with grief; and as one from whom men hide their faces he was despised, and we esteemed him not." (Isaiah 53:3 ESV)

I think they should receive strong mental health support with clear guidelines and safe working conditions. This can help them with the traumatic material they could possibly review and ensures moderator safety.

I feel like crowdsourcing can also be harmful when it comes to legal proceedings. It's easy for people to find evidence online and spread it publicly as a way to help the court findings, and I may be wrong, but I think that may only leads to the evidence being concluded as inadmissible because it was found externally.

This can be a major concern for influencers who post a lot about their daily life online. They can be victims of stalking and harassment.

This is extremely prominent in TikTok. I feel like people are always making something up and spreading it around, and because so many people are covering it, it seems believable. Even the TikTok searches start to cater toward the rumor by highlighting searches that other people are making about the rumor. This is why I don't rely on information I find out about on TikTok, and instead research it myself by reading a few articles I find on Google to generate my own conclusion.

The case of Justine Sacco shows how social media crowdsourcing can quickly turn into harassment, as users collaborated in real time to track her flight and monitor her return. Twitter enabled people to pool gather info, and even capture and circulate a photo of her the moment she turned her phone back on.

The topic I chose to speak about is Primary care, which can be found in multiple health care locations like family clinics, pediatric clinics, urgent care, public health, and primary health care offices. Primary care plays a vital role in healthcare as it is the first interactions the patients will have when it comes to their care, followed by secondary care. All of te different types of care are absolutely necessary to have proper diagnosis and care plans given to a patient. The overall goal of the primary care providers is to idenetify the potential acute issues in the patient, prevent any infections or illnesses, and overall maintain adequate health of the patient. Some of the healthcare members that are involved in primary care are doctors, MAs, NPs, RNs, LPNs. and more. These workers ensure to build trust with the patient and make them feel safe in the onset of their care by performing therapeutic communication. Overall, primary care can be considered the foundation of healthcare as its focus is management and prevention of possible health issues.

-Kourtney

Hey class, I will talk about primary care today. I chose it because primary care is the first interactions with the health system when a health problem arises. This level of care would be seen when attempting to promote health, disease prevention, as well as rehab and the treatment of acute diseases. The people handling this level of care would be anyone involved with the first steps of taking care of a patient, namely nurses, physicians, pharmacists, or any other health professional that the patient may come into contact with in the initial stages of care. Payment options for primary care may include copays from insurance, Medicare for 65+ with disabilities, Medicaid for low-income individuals, and my personal favorite, TRICARE. Thanks, Lucas

Hello Class, Tertiary care is a form of care that is highly specialized and occurs when a patient needs more advanced/specific care than what they can receive from primary or secondary care. This kind of care is usually referred by a secondary specialist, but occasionally a primary physician can refer the patient to the tertiary care facility directly. These facilities are often set up by region and are typically found in large teaching hospitals or stand alone centers specific to its service. Some common care services within tertiary care include neurosurgery, advanced neonatal care, plastic surgery, transplants, burn treatments, and dialysis. Due to the complexity of care involved in these kinds of treatments, patients can expect to interact with a large interdisciplinary team. This team may include different specialist, respiratory therapists, case managers, physical therapists, surgeons, nurses, advanced practice nurses, and many others. Because of the increased complexity of this form of healthcare, it typically ends up being a lot more expensive then primary and secondary care. Often times this treatment is funded by insurance, but it is unfortunately common to this not be covered in full. If this happens some patients pay out of pocket, utilize financial assistance programs, or sometimes even obtain funding from donations/GoFundMe's. Thank you, Emily.

Hello class, Today I would like to discuss with you about what secondary care is and why it is important in the healthcare setting. In just about every health care system, region, or nation; each system is made up of numerous amounts of different providers and healthcare members. Through this, each member has their own responsibilities of services and specialties they provide to their patient. With this, each member will typically represent different levels of care in which are often arranged into four main categories including: primary care, secondary care, tertiary care, and quaternary care. When focusing on secondary care, it is important to first understand what it includes. Following primary care, secondary care is the next stage of care provided to patients by specialists who do not typically have the first contact with their patients. In other words, this care is often implemented when the patients condition requires specialized treatment and monitoring that is outside of the scope primary care can provide to the patient. Through this, patients will often meet with multi-professional teams such as nurse professionals, professions related to medicine, and imaging specialists. In addition, patients will commonly meet with specialists such as surgeons, physicians, psychiatrists, or even pediatricians in order to treat their condition. Common examples of where this would be seen include areas such as inpatient and outpatient care, surgeries, conditions relating to cardiology, neurology, gastroenterology, and so much more. One big concern related to patients who receive secondary care is the payment, however many factors and services have been put into effect in order to aid financial takings. Most commonly, secondary care is paid for through insurances like government health insurance systems, private health insurance systems, and in some cases, paid out of pocket for by the patient. depending on the type of hospital, financial services may be funded as for government ran hospitals, this is provided through the development of budgets based on departmental line item costs or grants. This allows patients to be given adequate care without the stressors of financial costs discouraging them. Thank you, Wolfe

The most I really do is report accounts for impersonation because they're impersonating someone I know. It's either that a new account has been made impersonating a friend or acquaintance of mine and they've followed me, or the original account was hacked and I find out because they message me asking for "help." Other times that I've reported someone was for hateful behavior thats beyond regular hate that people get, such as hateful comments followed with racial slurs.

This passage explains a viewpoint that blames racial and ethnic inequality on the culture of people of color instead of social systems. It claims that poverty exists because of supposed cultural flaws, like not valuing hard work or strong families. This view ignores the impact of discrimination, inequality, and lack of opportunity, and places blame on individuals rather than society.

In a way, I feel like this doesn't hold up in most cases. Of course, social media platforms have their functionalities to prevent trolling and harassment, but there are thousands of cases (or more) where their users are genuinely in danger or their safety is compromised in some sort of way, and the companies don't do anything productive to help. If anything, their actions may be more beneficial toward the perpetrators rather than the victims. For example, in stalking cases for content creators (especially big content creators), little is done on the company's end to actually ensure safety and justice for the creators. Twitch has a handful of occasions (that I know of) where their creators were attacked by their stalkers during Twitchcon, and hardly anything was done to arrest the attacker or permanently ban the attacker from their platform.

This response explains that gender inequality is still common, even if it looks different in different societies. It points out that unequal pay, roles, and expectations still affect women today. It also explains that being financially supported by a man is not a problem if it is a personal choice. The main issue is lack of choice or opportunity. The key idea is that real equality means having freedom and options.

This passage explains that sociologists study social problems by looking for patterns. Fuller and Myers created a model called the natural history of a social problem, which explains how social problems start, grow, and are addressed over time.

This passage explains that after a policy is created, it still needs to be put into action. In the civil rights movement, this meant actually integrating schools, registering people to vote, and ending legal segregation. Even though laws changed, inequality still exists in everyday life.

This step is about pointing out a problem and getting others to take it seriously. People make a claim, meaning they argue that something is wrong and needs to be fixed. At this stage, people often disagree about whether the problem exists or who should act. In the example, civil rights activists argued that racial segregation was unfair and needed to end, especially because it harmed Black communities.

The idea of power users and lurkers makes me wonder how much online spaces are actually shaped by a very small group of people. If most content comes from just a tiny percentage of users, then the tone, norms, and even political direction of a platform might reflect that minority more than the silent majority. At the same time, lurkers still benefit from the content without contributing, which makes crowdsourcing feel less evenly distributed than it first appears.

When it comes to lurkers, I think the best way is not credit them if it's a project or something else that requires crediting the people who have worked on it. It may not be as effective, but I think, as of now, that is the best way to avoid lurkers.

This passage explains that different sociological perspectives look at crime in different ways. Using armed robbery as an example, it shows how each theory offers a different explanation. The idea is that no single perspective explains crime fully, but together they give a better understanding.

This passage explains how the Industrial Revolution caused major social changes. As people moved to cities for factory work, living conditions became crowded and unsafe, and crime increased. These problems made intellectuals believe society was breaking down, which led them to start closely studying social order and social problems.

This passage explains blaming-the-victim, which is when people blame individuals for problems instead of looking at larger systems. In this example, poor children are blamed for doing poorly in school by saying their parents do not care. Ryan argues this ignores the real problem, which is underfunded and overcrowded schools. He explains that fixing education means improving the schools, not blaming families.

There have been many such demonstrations in the past, and in recent history, and it reminds me of how many times it is college students who tend to lead public protests. It is very interesting how each time such protests take place, they are considered especially audacious, despite the fact that there is real historic precident of people doing this for years.

I find it interesting how crowdsourcing depends so much on the design of communication systems. Features like anonymity, archiving, and time delay can really shape how willing people are to participate. For example, anonymity might encourage more honest contributions, but it can also reduce accountability. It makes me think that crowdsourcing isn’t just about “using the crowd,” but about carefully designing the communication environment that enables the crowd to collaborate effectively.

The core argument lies in moving from "replication" to "transcendence." It argues that instead of fixating on making digital communication infinitely approximate offline interaction, we should leverage the inherent characteristics of computer systems to identify scenarios where they offer distinct advantages.

I never really thought about the difference between crowdsourcing and just regular outsourcing before reading this. It’s pretty interesting how the 'open call' part is what makes it unique, but it also makes me wonder how platforms actually filter out bad data when anyone can join in.

I never really thought about the difference between crowdsourcing and just regular outsourcing before reading this. It’s pretty interesting how the 'open call' part is what makes it unique, but it also makes me wonder how platforms actually filter out bad data when anyone can join in.

It's happening!!

How do people in the medical field manage this? With these chronic stressors and exposures, what are the effects that take toll in their personal lives. The human brain isn't meant to experience death or traumatic experiences at such high rates. What does this contribute to their life expectancy?

It is vital to consider the social location in regards to social determents in health. This was also mentioned in the documentary we watched in class as well as the one we were instructed to watch on our own. Why is it only recently we are studying this? What are we doing about it?

As mentioned in the documentary it is noted that underrepresentation in healthcare. One of the students even mentioned that we need more black doctors because of how disproportional health care is amongst the black population, and how their care differentiates from others.

When it is worded like this to read about it just sounds rude, however it makes sense for doctors to behave this way to ensure they hold that detached concern. If doctors did not have this detached concern then with every patient they would build a strong bond with, therefore making it harder for them if something bad were to happen to the patient.

Within their first year students are becoming aware that it is not possible to master all of the facts they are learning, they then become aware that the knowledge they are learning is incomplete, and lastly they start the find limitations when it comes to answering patient questions (whether that is lack of their own knowledge or just lack of knowledge within the field)

Make Bode Diagram and look at -3 dB

Read Breakpoint Frequency, w_b $$s = a \pm jb$$

In transfer function G(s), substitute $$s=jw_b=\frac{j}{\tau}$$

Solve the Transfer Function

Convert to Polar Form $$z = a + jb , $$ $$z = r \angle \theta , \quad r = \sqrt{a^2 + b^2}$$ $$ \quad \theta = \tan^{-1}!\left(\frac{b}{a}\right)$$

$$r = k_dc$$

No transfer function for returning pass (unity)

Input=Output for returning pass.

ex: cruise control. r=desired speed, y=actual speed

• $$r-y = e$$

• \(e\) represents the tracking error, the difference between the desired output \(r\) and the actual output \(y\).

$$ u(t) = K_p e(t) + K_i \int e(t)\,dt + K_d \frac{de}{dt} $$

• The control signal \(u\) to the plant is equal to the proportional gain \(K_p\) times the magnitude of the error plus the integral gain \(K_i\) times the integral of the error plus the derivative gain \(K_d\) times the derivative of the error.

A time I found myself in a new discourse community was when I started my job at the electric cooperative. I literally knew nothing about the electric industry, so it felt super overwhelming at first and like everyone was speaking a different language. I definitely felt pressure to conform and “act like I knew what I was doing” until I learned the terminology, processes, and expectations. Things like the industry jargon, the way coworkers communicated with members, and the standards for accuracy and professionalism made it clear what was expected, and over time I started to feel more comfortable being myself in that space.

I typically like to skim texts then reread, it helps me truly process what I'm reading.

I've never really thought about reading in this way. I'm excited to practice this!

To me, reading means slowing down and actually letting myself connect with a story or idea, not just scanning words on a page. As a reader, I’m someone who enjoys reading when I’m interested in the topic, but struggles when it feels forced or purely academic. My reading process is usually to skim first to get the general idea, then go back and read more closely, especially if I know I’ll need to write or discuss it.

When you route error into the control system.

Example gravel depositing on weighted belt * Weight too heavy, system controls flow rate to reduce weight * Weight is sufficient, system does no work to correct error = neutral zone

I think individual experience is important because everyone comes into a space with different backgrounds, emotions, and ways of seeing the world, and that shapes how we understand things. In academic settings, I think we can start connecting experiences by letting people share their perspectives, asking questions, and actually listening instead of assuming there’s only one “right” way to think about something. When learning connects to real life, it feels more meaningful and easier to care about.

It truly is so important to have a flexible mindset and experiment. You won't truly grow unless you step out of your comfort zone and try something new.

I am a visual learner. My education has been a mix of all the zones. My math and science classes tend to fall in the learning zone and panic zone. English was always in the comfort zone. Arts and history were typically a mix of comfort and learning zone.

While I absolutely love learning, a lot of my classes have led to me staying in the "Comfort Zone" and I would not truly learn anything.

I completely agree with this. A test doesn't determine someone's ability to think.

This is critical, especially for college students. If I'm not genuinely interested in what I'm learning, I won't give it the time of day.

This is how you get students genuinely excited to learn.

Overall, I have had a good educational experience. I have had a handful of teachers/professors that were passionate about what they taught and were passionate about helping their students learn. However, the majority of my teachers and professors just gave lectures, assigned busy work, and did not interact with students very much.

Questioning is definitely how I learn best. The old school learning model sounds way too familiar and while I got amazing grades, I did not truly learn much.

I think this is such an interesting and helpful way to truly get students to engage in class and learn instead of students not actively paying attention to lectures and not putting effort into assignments.

Wikipedia’s open, crowdsourced model makes knowledge accessible and constantly evolving, allowing anyone to contribute and improve content. However, this openness also requires strong community oversight to maintain neutrality and prevent conflicts of interest.

Wikipedia is an example of a crowdsourcing platform because its content is created and edited by volunteers rather than a centralized editorial staff. Anyone can click “edit” on most pages and contribute, and even powerful institutions do not receive special privileges, highlighting how authority on the platform is meant to come from collective oversight rather than formal status.

The part about Amazon Mechanical Turk is kind of eye-opening. It seems super efficient for companies to break down big tasks, but at the same time, it feels a bit weird that these workers are basically treated like human processing units without much protection.

I would like to add that Reddit can also be a platform for crowdsourcing, especially for younger people whoares asking for help or recommendations. Additionally, sometimes with crowdsourcing, there are people who might be there just to cause trouble, which can be a gray area in this topic.

DO: The homepage of GOV.UK is completely keyboard-friendly. Without ever using a mouse, users can tab through all menus, links, and forms. You never lose your position because a visible highlight moves with your cursor. Anyone who prefers keyboard navigation for speed will also benefit from this, especially those with motor impairments who rely on keyboards.

DO: The accessibility of this "Is this page useful?" question with a "Report a problem" link is good. If something on the page doesn't work for users, it provides them with an easy way to provide feedback. This enables users with disabilities to report problems they discover so that the website can be improved and made more user-friendly. It shows the website values each and every user.

DO: This is an excellent illustration of a descriptive link with clear supporting text. An explanation in simple terms follows the link "Child Trust Funds": "Verify whether you have a Child Trust Fund." This makes it clear to all users including those with cognitive impairments or those who are not familiar with government terminology.

DO: Everyone can easily use this search bar. Its "Search" label makes it obvious what it does. A highlight appears around it when you click or tab into it. This makes it easier for those who are unable to use a mouse to navigate the page.

When using sources, make sure you check the credibility of the source and its intended purpose

DON'T: The term "Benefits" by itself is too ambiguous. Screen reader users who navigate by clicking between links may only hear "Benefits" without the context below, even though it has descriptive text underneath ("Includes eligibility, appeals, tax credits and Universal Credit"). More information should be included in the link itself, such as "Benefits: eligibility, appeals and tax credits."

In this passage, Taney explains the historical disputes between the states during and after the Revolutionary War over who should control western lands and benefit from their sale. He uses this early political conflict to argue that these territories were treated as shared property and were never meant to be controlled by the federal government in the way Scott’s case suggests. This is originalist reasoning because Taney relies on historical practices and debates from the founding era to define the Constitution’s meaning. I disagree with this point because political compromises and disagreements at that time do not automatically create permanent constitutional limits, and Taney treats history like clear legal proof instead of recognizing how the government’s role evolved over time.

Taney is basically saying that Congress never had the constitutional power to pass the Missouri Compromise because the framers did not originally intend for Congress to regulate slavery in territories gained later, like Louisiana. This shows originalist thinking because he relies heavily on what he believes the framers meant back when the Constitution was written and refuses to go beyond that historical viewpoint. I disagree with this because it locks the Constitution into the 1700s, ignores how much the country grew and changed, and ends up protecting slavery instead of allowing the Constitution to adapt to new situations.

I agree with this since it demonstrates how they were not even treated with human respect, which is heartbreaking.

This just proves how citizenship and rights were historically denied based on race, reinforcing systems of slavery and exclusion. It highlights how the Court viewed citizenship as limited and tied to racial hierarchy at the time. which is very sad

I believe this language shows and example of originalism.

I'm a little confused about this portion are they stating that even without evidence, they opted to pursue the case? If that's the case, I view the entire situation to be extremely unfair and simply discriminatory.

test

heavy dependance on technology has caused rupture on the ability to connect and communicate

to what extent will the robots perform? What are these limitations that the companies will be ridding them off?

are the robots acting like humans, or humans like robots?

Technology is used for the replacement of intimacy and comfort. Lack of human connection between parent and kid

Technology being very heavily related to capitalism. Prioritising profit over the care of citizens

DO: This link makes use of concise, informative text that explains to users exactly what will happen when they click on it. The phrase "Sign in to your childcare account," which incorporates the context (childcare account) and the action (sign in), is used in place of ambiguous phrases like "Click here" or "Sign in." This facilitates jumping between links navigation for screen reader users.

Systems thinking involves understanding environmental problems as interconnected parts of a whole rather than isolated components. Transdisciplinary approaches integrate knowledge across scientific, social, and institutional boundaries, while resilience science focuses on a system’s ability to adapt to change and disturbances without losing core functions.

One major concept of social ecological systems (SES) science is the concept of resilience which adds investment to the capacity of the interconnected social and ecological systems to absorb disruptions, adapt to them and still survive.

If environmental management continues to operate along disciplinary boundaries, how realistic is it to expect meaningful integration of SES principles without institutional reform? What changes would be required within agencies to support this shift?

The authors argue that current environmental management approaches are insufficient because they operate within disciplinary silos. This sets up the need for a social-ecological systems (SES) framework that integrates social and ecological dimensions to address complex environmenal problems.

This passage explains that environmental problems result from the interaction between natural and social systems. However, environmental management still tends to treat these issues separately within individual disciplines. The authors argue that a more integrated approach is needed to effectively address complex environmental challenges.

Low quality land with little agricultural value. They also lacked water.

It is so sad to read about all these people who didn't have enough money for their families.

has autofluorescence from the glass coverslip and microscope slide been problematic, especially for low-mag objectives and at 785 nm excitation where glass is highly absoptive?

This idea particularly resonated with me. If a particular archive is meant to be a cultural space that is meant for community to come together in some way, then affect is the main way to make someone come back to a space. It brings warmth and welcoming energy.

It makes me curious to explore archives that are not so insistent on being a cultural space and more concerned with research. I've only spent time in community archives, which seems natural but an archive that has less concern with being an active space then has a different audiance and purpose and meaning in and of itself.

This is a cool way to "induce" more phenotypic variation from cells in response to drugs. Are there other pathways that could be stimulated and be orthogonal to PKC for even more possibility of finding phenotypes?

How physiologically relevant is this activation of cells? Is this perturbation just a way to stress the cells or does it actually place them in a more clinically-relevant state?

Is it possible to distinguish drug-induced cytotoxicity from a drug-induced phenotype?

Is there a specific reason why activation should happen after drug treatment versus before? Does the phenotype from activation start to reverse after some amount of time?

"Might be a politician"? He was a president of the United States in the 1920s, of course he was a politician.

Many times, both experiences and witnessed. One major once that came to mind is accounts in highschool to point out other people. For example, my school had an account for anyone with bad posture, which ended with people taking photos of people around them and submitting them to this account, who would then post them. I know I got on there once, and the angle from the photo was from a table with people I did not know. Similar accounts were made frequently, mainly confession accounts, where students could essentially talk bad about each other and post it openly.

Its actually better

Analytically (and in Drake), what would the exponential stability requirement look like in this case? Would it be

$$ V(x) \text{ is SOS, } -\dot{V} - \lambda \left( s^2 + c^2 - 1 \right) - \beta V \text{ is SOS } $$

for some small user-selected β > 0, to ensure that the time derivative of V is always proportional to V itself?

Within both of these stories Salinan and Cherokee they both have a view of animals creating the great discovery of the their new world but also have a lens that can tie this to religion and that God created the land. With the strong religious lens of both stories was the end goal for both groups the same but just founded differently through the path "God" gave them?

Similar to the Salinan Coyote the little Water-beetle is the experiment. The animal that is going to sink or swim. Offering to explore this possible new world in order to bring light into the already unknown. Taking the belief that is established about earth already and see what more earth has to offer the founders of the people. The unexpected animals being the mastermind of trial and error. More than likely being this experiment because they are commonly found on the land already. So one being in danger is okay. Thus coming back with great success allows for better idolization among the other animals. A complex discovery turning into a holy grail and working from the bottom up is true importance.

The Coyote isn't as sacred to the Bald Eagle but more of an experiment or useless animal. While Coyotes are hunters and prey the Bald Eagle uses that to advantage. In order to see if the Bald Eagle can execute a new world he has to see if this creation will work. So using his power he told the Coyote to try the women first because the Eagle knew the Coyote idolized him. In short threw the Coyote in the deep end and was like this will sink or swim. While the sinking occurred first that brought light to the Eagle and ultimately held more value to which he had to revive the Coyote back to life to swim.

The Bald Eagle being chief is represented of the creator for Salians. Without the Eagle then their world wouldn't be created because the Eagle can fly so high and be the eyes needed. Hinting to why he saw the world being incomplete and creating humans. A community needed to be built and a story needed to be made. Holding power to build a lifetime creation. Also connecting to why the United States holds great value to the Bald Eagle because of the foundation and representation it brings. Founding new worlds with endless possibilities and being this idolizer that morally brought freedoms and liberty for all because it is the creator.

like the corporate learning and development teams? This could sell well to L&D in HR. I have a lot of connections there, but we will need to get SAML login and integrate with Okta. This might be a whole can of worms that this stage. Maybe phase 2 or 3?

Like a content authoring platform?

I'd love to think through this a big more. The platform is both learning and certification focused, so wondering how we can offer multiple certificates around one role type that can be automatically generated.

I wonder if we could integrate with Notebook LM potentially. Also, I have a few friends working on Learn Your Way at Google. They are still dogfooding, but I see what the product roadmap is for it. Thoughts?

Many people don't know what career path they want. There are a lot of career assessments out there. This is the one on C-Net. Honestly, I am not a big fan of this, but it relates very well to the API data.

https://onetinterestprofiler.org/

LOVE!

This is a landing page before users sign up?

In terms of the tech build, are you thinking of building this natively? Or using an admin backend like GHL for any of the pieces?

Each of the audiences' meeds are quite different, so we'll need to think through where to start.

Curious about what a learner monthly subscription would look like.

While I like the sound of this, this will need to be fleshed out as the goal is to be open-ended to any role.

One concern is the coaches or companies unique role specific content that they want to include in their cert.

This is very accurate. I was war stories from working in companies where once we finished an exam we needed to revise or redo because industries and roles change so quickly!

This is so great. Really, this can be a B2B with other coaches as you mention, B2C with career transitioners, people ready to upskill their AI skills for their roles in the age of AI.

Additionally, many customer education programs would eat this up. My background is primarily in L&D and certifications. When I worked at Meta, they would spend 1.5M per exam that would take over a year to create. Once the product is built, there are so many ways to tailor towards different audiences.

People know when a poem is good when they can feel something when reading or relate to it a lot.

The truth is, people do not like boring consistency, they like one good thing and will stick to it for a long time.

why does he refer to himself in third person here? is there significance in that?

they were moving again, right when things were getting good for him

self-destroys

Why would he want to work with his father and brother in the heat instead of being in school?

He does not have good English

the work was hard and grueling, especially for a young boy.

I am in love with how the author describes the sun setting

they're supposed to be in school but they're not. that shows poverty, but it also shows the hardships of being an immigrant

some kind of berry crop, possibly grapes or blueberries

Its dilapidated and old, and its barely livable. I don't see how they're happy about this

she is excited about finding work

the older and more soul it acquired the more mama liked it

Roberto is the older brother

This seems significant to me but why did papa need to know who owned the car before?

they're moving

Why are they so quiet? are they just tired? or are they planning something?

why would it sadden them? shouldn't being off work make someone happy?

I love this description of the sun setting

interesting

the narrator is also a Mexican

slow days on the farm

climate change

Leviathan- 1. (in biblical use) a sea monster, identified in different passages with the whale and the crocodile 2. an autocratic monarch or state 3. a thing that is very large or powerful, especially an organization or vehicle:

Its basically a fattener

why would they not have allowed it if it wasn't a special occasion?

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We appreciate the time and effort the reviewers have invested in providing constructive feedback on our manuscript. Below, we’ve detailed additional work, corrections, and improvements that we will complete during the revision process.

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

Summary

Folding is a major morphogenetic process that shapes tissues and organs in three dimensions. The mechanisms underlying tissue folding have been extensively explored and are often driven by actomyosin-based apical constriction. Here, the authors describe changes in cell geometry and mechanics during mouse neural tube formation. They build on quantitative fixed imaging and live junction ablation to extract cell geometry and junctional tension. These analyses are performed at different developmental stages and in both male and female embryos to propose a mechanical mechanism for neural tube elevation in the brain.

Major comments

The authors report quantitative data on cell geometry and junctional tension inferred from laser ablation. Overall, there are numerous statements that require stronger support from the experimental data. To substantiate several of their claims, the authors need to provide a larger number of data points-or at least comparable numbers across experimental conditions-for the tension measurements. Additional statistical analyses are required throughout to support the conclusions.

Figure 1

1. Does the projection algorithm account for tissue curvature when computing cell geometrical parameters such as area and anisotropy? At present, our projection algorithm does not correct for tissue curvature. Curvature in the tissue can make larger cells appear smaller in projections, skew the angle of cell orientations, and change aspect ratios. The largest curvature in the midbrain neural tube samples that we analyze is found in the transition region from the midline and lateral regions (~10-30% of tissue width) of 5 ss and 8ss embryos. The regions at the midline and more laterally are relatively flat. Therefore, distortion from curvature will not dramatically alter our key conclusions. We will apply a curvature correction using existing tools (Herbert S., et al (2021) BMC Biology) to sample images and determine if there are substantial differences in curvature-sensitive cells shape metrics. These will be included in a supplement to Figure 1. If there is a significant difference, we will expand the correction to all images that we analyze and update our analysis.

The authors should provide information on the accuracy and reliability of the cell segmentation.

We can provide a supplement to Figure 1 to demonstrate the accuracy of the segmentation. We have used F-Actin to segment cells in our images, which is enriched along the cell junctions but can also form medial cables that cross the cell surface. Junctional actomyosin is notably brighter than medial cables, and segmentation with our trained CellPose model is consistently able to distinguish the junctions. We also checked segmentation and performed manual corrections to ensure accuracy. To demonstrate this for our readers, we will prepare samples stained with both F-actin and ZO-1, a tight junction component that is localized to cell junctions. We will then segment the image twice in CellPose, once using the F-actin signal and once using the ZO-1 signal. The resulting cell outlines will then be digitally superimposed to show how much the signals overlap, and we will plot out the cell frequency as a function of area to determine if F-actin segmentations can segment with the same fidelity as ZO-1. Recent work by a co-author has shown excellent corroboration of neuroepithelial apical cell areas segmented using F-Actin and ZO-1 (Ampartzidis I., et al. eLife 2026). We are confident that our data will show a similar result.

The authors indicate that the rate of apical constriction differs between male and female embryos. However, apical sizes differ only at specific positions along the ML axis (Fig. 2H, I).

In Figure 2H, we show that at 5 ss males have larger apical areas than females at the midline, adjacent lateral cells, and at the surface ectoderm-neural epithelium border. By 8ss (Figure 2I), cells at the midline are smaller in males than females, while cells in more lateral regions are now equivalent between sexes. This change in apical area over time suggests that males have faster rates of constriction than females at the midline and adjacent lateral region where male cells become smaller or equivalent in size to female cells, respectively. We will perform statistical analysis (see comment #4) to determine if there are regions with significant differences in rate and amend our language to clarify that these differences are region specific as appropriate.

The authors should provide statistical analyses for the rates shown in Fig. 2J. Are these rates significantly different between males and females, and between medial and lateral regions?

Currently we calculate our rates using the difference in population averages of apical area at each stage shown in Figure 2H and 2I for each sex, and dividing by the number of somite stages, 3. As a result, there is only one rate value at each midline-lateral bin for each sex which is not amenable to statistical analysis. To correct this, we will calculate rates by subtracting the average apical area of each embryo at 8 ss from the population average of embryos at 5 ss. This will create 5 rates for both females and males at each 10% midline-lateral bin. We plan to perform a two-way ANOVA to determine if there are statistical differences in rates between males and females at each bin position and between medial and lateral regions. We will also add a section describing these calculations to the “Statistical Analysis” portion of the methods.

Please clearly state the main novelty of this study relative to the work published by Brooks et al.

Our study builds on the work of Brooks ER, et al. (2020) eLife. Brooks demonstrates that cells in a region of the lateral neural folds undergo apical constriction (Figure 1) and that cells at the midline do not (Figure 2). We expand and improve upon this work in the following ways:

1. A) As required by our funding sources at the NIH (NOT-OD-15-102) we have collected, analyzed, and reported on sex as a biological variable of interest. In doing so, we have shown that there are clear sex differences in apical area in the neural tube that were not previously shown. We also show that there is apical constriction within the neural tube midline in a sex dependent manner. Brooks et al do not address sex in their work.
2. B) We have provided more complete and spatially precise information on midline-lateral patterns of apical area and apical constriction. To show changes in apical area of lateral cells, Brooks selects a 100 x 100 µm region of interest in the midbrain (Figure 1E-F, Figure 2A) but does not specify the midline-lateral or rostral-caudal location of this region of interest or standardize it between embryos of different ages and dimensions. In our study, we’ve standardized our measurements to a 100 µm wide band across the midbrain adjacent to the midbrain/hindbrain boundary (Figure 2A-C). We also standardize positions as a percent distance from midline to account for differences in width between embryos and ages. This allows us to consistently compare similar populations of cells along the midline-lateral axis and determine changes in apical area over time.
3. C) We connect patterns of apical area and constriction to F-actin and Myosin-IIB density. Though Brooks et al report some analysis of F-actin in lateral cells (Figure 6), they do not analyze the midline cells or explore the relationship between cell shape and actomyosin.
4. D) Finally, we tested the mechanical properties of the tissue through laser ablation in living mouse embryos. From these ablations we’ve found that tension at the midline is less than in more lateral regions. Work in the neural tubes of frog (Haigo S., et al. (2003) Current Biology, Baldwin AT., et al. (2022) eLife, Matsuda M., et al. (2023) Nature Communications) and chicken (Kinoshita N., (2008) * Cell, Nishimura T., et al. (2012) Cell) embryos has conclusively shown that enriched midline actomyosin promotes apical contractility and drives hinge formation. It was therefore largely believed that a similar contractile hinge was employed in mammals (Copp AJ. and Green NDE. (2010) J. Pathol, Nikolopoulo E., et al. (2017) Development). Collectively, our work is the first to demonstrate that such a contractile hinge is not present in the mammalian brain neural tube. Figure 3*

The authors need to provide statistical support for the claim that large midline cells exhibit reduced F-actin and Myosin IIB levels.

We will conduct a two-way ANOVA to determine if there are statistical differences in F-actin and Myosin IIB density at the midline and more lateral regions in both males and females. We will update our language in the text and plots as appropriate from these results.

F-actin and Myosin IIB intensities should be plotted as a function of cell area to support the proposed anticorrelation between apical area and actomyosin levels.

We will make plots of cell areas vs. F-actin or Myosin IIB density for cells in each embryo. We will then fit a line to determine the R value for each embryo to determine if there is a negative correlation between cell area and actomyosin intensity. We will also adjust our language in the text as appropriate based on the results of these tests.

Statistical analyses are missing to substantiate the increase in F-actin levels between stages ss5 and ss8.

We will perform an F-test to determine homogeneity of variance between F-actin at 5 ss and 8 ss followed by the appropriate t-test to determine if there is a statistical increase in F-actin over time. We will also amend our language in the text to reflect the results of this test.

Figure S3 should be supported by plots showing Myosin II and F-actin intensity as a function of position along the ML axis, together with appropriate statistics.

In Figure 3A-D, we show representative images of F-Actin and Myosin IIB density in female embryos. These are plotted as the purple lines in Figure 3 E-H. Figure 3 Supplement 1 shows representative images of F-actin and Myosin IIB density in male embryos. These are plotted as the green lines in Figure 3 E-H. We will add a line in the caption of Figure 3 Supplement 1 indicating that these samples are represented and plotted in Figure 3. We also noted a typo in the respective captions, incorrectly indicating male or females were shown in the figure. We will correct these typos as well. Additionally, we will perform the statistical tests indicated under comment #6.

Figure 4

The authors state that lateral tension in male embryos is not different from midline tension, yet the number of data points is much lower than in females. To support this claim, the number of ablations should be comparable across sexes.

As part of this study we performed 270 ablations in the neural tubes of 83 mouse embryos: an exceptional scale of ablations that is the first of its kind in early embryos. We conducted our initial recoil velocity analysis blinded to information on sex. Male embryos were statistically underrepresented in our data set because male embryos develop faster than their female littermates (Seller MJ. and Perkins-Cole KJ. (1987) J. Reprod. Fert.). As such, the neural folds of male embryos were too elevated to ablate. At present we do not have the resources or justification to perform laser ablations on additional animals to obtain the number of male embryos needed to supplement the already exceptionally large data set. We will instead perform a power analysis to determine if: 1) we have a sample size large enough to detect a biologically-meaningful difference with suitable power, 2) the sample size required to detect the observed difference is so large that the difference would not be biologically meaningful, or 3) we do not have a sample size large enough to detect a difference confidently. With the results of this analysis, we will amend our language in the text to reflect the most accurate claims that can be made.

Is lateral tension different between males and females?

In Figure 4G we show that females have statistically different tension between the lateral and midline regions, while males do not. However, we do not test if the lateral or midline tension is different between females and males. We will perform an F-test and t-test to determine if there are statistical differences between males and females in this region.

Similarly, the data in Fig. S4 used to claim no change in tension over time are not supported by sufficient data points.

As discussed in comment #10, the scale of ablations is already substantial, and the initial recoil velocities were analyzed blinded to information on embryo age. We will calculate a best fit line for these plots to demonstrate if there is a trend in recoil velocity over time. We will then adjust our language in the text as appropriate with this added information.

Would the medial and lateral tensions reported in Fig. 4G remain unchanged if the authors perform statistical analyses on 10-15 ablations per condition?

We do not have a justification for removal or exclusion of any of the laser ablations analyzed in this study. We will instead perform a power analysis, as indicated in comment # 10, and adjust the language in the text as appropriate given the results of that analysis.

Figure 5

The number of data points in Fig. 5J and L is insufficient to support claims of no difference. The only detectable difference arises in the comparison with much higher sample size (Fig. 5L, ML vs RC).

In Figure 5J we disaggregate ablations performed at the midline by directionality (midline-lateral or rostral-caudal). We were unable to detect a statistically significant difference based on the direction of initial recoil velocity in either sex, though N’s for all categories are comparable. As discussed in comments #10 and #12, the scale of ablations conducted in this study is uniquely substantial. We will perform a power analysis for our anisotropy measurements in the lateral region of the tissue to determine if we have a sample size large enough to have detect a biologically-relevant difference with high confidence or if the sample size required to detect the observed difference is so large that the difference would not be biologically meaningful. Given the results of this analysis, we will amend our language in the text to reflect the most accurate claims that can be made.

The authors conclude that males have higher ML tension than RC tension, but given the limited data this conclusion should be amended to "no detectable difference."

In Figure 5L, we disaggregate ablations performed in the lateral regions, by directionality (midline-lateral or rostral-caudal). We find a statistical difference in the directionality of initial recoil velocity in females. In males, though we can observe a difference in the initial recoil velocity means, we are unable to detect a statistical difference, likely due to the smaller male sample size. As discussed in comments #10 and #12, the scale of ablations conducted in this study is uniquely substantial and was conducted blinded to embryo sex. Given that males develop faster than their female littermates (Seller MJ. and Perkins-Cole KJ. (1987) J. Reprod. Fert.) we were unable to obtain more males in our data set. We will perform a power analysis for our anisotropy measurements in the lateral region of the tissue to determine if: 1) we have a sample size large enough to detect a biologically-meaningful difference with suitable power, 2) the sample size required to detect the observed difference is so large that the difference would not be biologically meaningful, or 3) we do not have a sample size large enough to detect a difference confidently. With the results of this analysis, we will amend our language in the text to reflect the most accurate claims that can be made.

Code availability

The authors should provide access to the code used to generate the projections.

We are committed to ensuring open access to all code used as part of this study, including components of the projection workflow, data analysis, and figure creation. We are in the process of assembling a GitHub repository containing these files as well as documentation to allow for use by other members of the research community and public. We will publicly publish this documentary upon completion of the repository or at time of publication, whichever comes first.

Reviewer #1 (Significance (Required)):

The authors propose a mechanical model for neural tube elevation based on analyses of cell geometry and tension at two developmental stages. The reported differences in cell geometry or actomyosin levels do not appear to explain the differences in geometry or tension suggested between male and female embryos. This raises questions about the relationship between these measurements and their relevance for understanding the mechanisms of neural tube elevation.

If the major concerns outlined above are rigorously addressed, the manuscript will offer a valuable descriptive characterization of neural tube cell geometry and mechanical stress during morphogenesis. Such datasets could form a foundation for future studies investigating the mechanisms driving neural tube elevation.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

The manuscript investigates the role of apical constriction and actomyosin organization in shaping the mouse brain neural epithelium during neural tube elevation, with particular emphasis on sex-specific differences. The authors develop an imaging and analysis pipeline to reconstruct the apical surface of the neural plate in three dimensions and perform quantitative measurements of apical cell area, actin, and myosin IIB distributions. Targeted laser ablation experiments are used to infer regional tissue tension.

The main findings can be summarized as follows. First, the authors identify a mediolateral gradient in apical cell area, with larger cells at the midline and smaller cells on the lateral neural folds, which inversely correlates with actomyosin density. Laser ablation experiments suggest that apical tension is lower and isotropic at the midline, whereas it is higher and anisotropic on the lateral folds, particularly in females. Second, sex-dependent differences in apical cell area, constriction rates, and actomyosin levels are reported at early somite stages, preceding previously described sex biases in neural tube defects.

The experimental work is technically solid, and the imaging and quantification pipeline represents a useful advance for analyzing large, curved epithelial surfaces. However, the study feels incomplete in its current form. Despite addressing neural tube elevation, the manuscript does not provide a comprehensive analysis of the folding process itself. Key aspects such as three-dimensional tissue morphology, curvature evolution, or global shape changes of the neural folds are not quantified. In addition, other potentially relevant cellular behaviors, such as proliferation, cell rearrangements, or contributions from neighboring tissues, are not examined, nor are they compared systematically between sexes.

Conceptually, the study focuses narrowly on correlations between apical cell area, actomyosin density, and inferred tension. While these measurements are carefully performed, the relationship between differential actomyosin contractility and three-dimensional tissue folding remains largely descriptive. No mechanical model or simulation framework is provided to link changes in actomyosin organization and cell shape to the emergence of neural folds and hinge formation. As a result, it is difficult to assess whether the measured differences in tension (on the order of ~40%) are sufficient to account for the proposed mechanical behavior of the tissue.

The central hypothesis advanced by the authors is that a relatively "soft" midline, flanked by stiffer, tension-bearing lateral folds, facilitates hinge formation during brain neurulation. However, this hypothesis is not directly tested by perturbation. For example, experimentally increasing contractility or stiffness at the midline (e.g., via optogenetic activation of apical constriction machinery) would provide a more direct test of causality. As it stands, the data demonstrate correlation rather than necessity or sufficiency.

Relatedly, alternative interpretations are not fully addressed. Large apical cell areas and low actomyosin levels at the midline could arise as a consequence of tissue geometry, contact with underlying structures such as the notochord, or extrinsic mechanical constraints, rather than being the primary cause of hinge formation. Similarly, anisotropic stresses generated at the tissue or embryo scale could align cells and actomyosin cables, producing the observed patterns without requiring locally specified apical tension differences as the initiating mechanism. The manuscript does not clearly distinguish whether apical tension asymmetries are a driver of folding or an emergent outcome of folding dynamics.

Finally, while the identification of sex differences is intriguing, it remains unclear what mechanistic insight is gained beyond establishing that such differences exist. The functional consequences of these differences for neural tube closure, robustness, or failure are not explored, nor is it clear how they integrate into the proposed lateral tension model.

In summary, this study provides high-quality measurements of apical cell geometry, actomyosin organization, and inferred tension in the mouse neural epithelium. However, the lack of direct perturbations, mechanical modeling, and quantitative analysis of three-dimensional tissue deformation limits the strength of the mechanistic conclusions. Addressing these gaps would substantially strengthen the manuscript and clarify the causal role of apical tension patterns in neural fold formation.

__ __The reviewer makes an excellent point, that direct perturbation of the system would enable us to test our hypothesis and inform whether the reduced contractility at the midline is essential for neural tube elevation. However, at present the technology needed to conduct an optogenetic experiment like that described by the reviewer does not exist. As with the laser ablations, an optogenetic experiment requires access to live and healthy embryos. Currently, mouse embryos can be cultured for several days in roller culture, where they are continuously rotated, or for several hours in static culture (Aguilera-Castrejon A. and Hanna JH. (2021) J. Vis. Exp.). Both techniques require that the yolk and amniotic sacs remain intact around the embryo. To access the apical surface of the brain neural tube for imaging, both sacs must be breached, after which the embryo has about 30 minutes before it begins to exhibit altered cellular morphology and tissue integrity and ultimate embryo death.

The neural tube elevates over several hours and closes fully after more than a day (Jacobson AG. and Tam PPL. (1982) The Anatomical Record). Even if we did acquire mice expressing photoactivatable constructs, the support membranes of the embryos would need to be breached to activate protein interactions. The embryos would die before any meaningful progress in neural tube elevation could be evaluated. Conducting an experiment like this would greatly advance our understanding of the system, and we hope that the needed technologies are developed to enable future work of this nature. The Galea lab previously purchased a photo-activatable Cre line, but was unable to induce deletion of a protein of interest using this allele before closure of the neural tube was completed (and the blue light needed to activate the cre was photo-toxic).

At present, there is some experimental evidence to suggest that lack of apical constriction at the midline if important for proper neural tube closure. Brooks ER, et al. (2020) eLife shows that a truncated Ift122 mutant, leads to abnormal constriction of the midline cells but does not disrupt lateral cell apical constriction, leading to a failure in brain neural tube closure in these embryos. Ift122 regulates trafficking and signaling proteins in cilia, which in turn regulates Sonic hedgehog signaling which Brooks ER, et al. also demonstrates regulates apical constriction. While this disruption is clearly multifaceted and nuanced, it provides some genetic support for the lack of apical constriction at the midline being important for neural tube closure.

Major Comments

Figure quality. Figure 1 contains very low-resolution images, which makes it difficult to evaluate the segmentation quality and tissue morphology. Higher-resolution versions should be provided.

In Figure 1, we outline the conceptual strategy and approach used to create and analyze shell projections of the curved neural tube. As much of our analysis builds from segmentation of cells in the projections, being able to assess segmentation quality from high resolution images is critical to evaluating the quality of the data shown. As discussed in comment #2, we will create a supplement to Figure 1 to demonstrate the accuracy of the segmentation. This will include high resolution images of both the label used to segment and the resulting segmentation, with corresponding overlays.

Cell segmentation strategy and validation. The authors segment cell areas using Myosin II and F-actin signals. This approach may introduce inaccuracies, as actomyosin cables can traverse the apical surface of individual cells and do not always coincide with cell boundaries. Segmentation based on junctional markers such as ZO-1 may be more appropriate. At minimum, the authors should provide a quantitative validation of segmentation accuracy, for example by overlaying segmentation results on raw images together with a nuclear marker (e.g., DAPI or H2B-GFP), to demonstrate that the number of segmented cells corresponds to the number of nuclei.

We will provide a supplement to Figure 1 to demonstrate the accuracy of the segmentation. We have used F-Actin to segment cells in our images. F-actin is enriched along junctions but cells can also have medial pools and F-actin cables, which might lead to errors. Though we understand the reviewer’s logic in asking to align segmentations with marked nuclei, the morphology of the neural epithelium makes this approach infeasible. The neural epithelium is pseudostratified, and nuclear position varies along the apical-basal axis depending on the cell cycle phase of each cell. As a result, an apical shell projection of nuclei would not capture all nuclei and a maximum intensity projection in Z of all nuclei would be uninterpretable as there would be substantial XY overlap between nuclei. Instead, we will create a supplement to Figure 1 to demonstrate the accuracy of the segmentation as discussed in comment #2. We will segment samples stained for both F-Actin and junctional markers like ZO-1. We will then create overlays of the resulting cell outlines and a cell area frequency plot for both segmentations to evaluate if F-actin based segmentation deviates from tight junction-based segmentation.

Lack of cross-sectional views of neural tube morphology. The manuscript would benefit from the inclusion of cross-sectional images of the neural tissue at different developmental stages. This would serve two purposes: (i) to demonstrate that the authors have a comprehensive understanding of the full three-dimensional folding process during neural tube closure, including medial and lateral hinge formation, and (ii) to allow readers to visualize the tissue geometry corresponding to the analyzed projection datasets (e.g., at 5 ss and 8 ss).

A key component of our model states that the changes in cell-level morphology and features correspond to changes in tissue level morphology (Figure 6). Specifically, that lateral apical constriction coincides with the flattening and elevation of the dorsal bulges on the lateral neural folds. We agree that it is beneficial to include additional visuals of tissue morphology. We plan to add an additional figure at the start of manuscript that details both the dorsal and relevant cross-sectional views of the somite stages analyzed. These visuals will take the form of graphical illustrations along with 3D confocal microscopy images and optical reconstructions of samples.

Sex-specific differences in overall neural plate morphology. The authors report that at 5 ss, males consistently have larger apical cell areas than females. It is unclear whether this difference reflects a global difference in neural plate morphology. Showing representative images of female and male neural plates would help readers directly assess whether there are overt morphological differences beyond those revealed by quantitative analysis.

If one sex has larger cells than the other, it would be reasonable to expect that the neural folds may be wider as well. In Figure 2B-C, we show representative images of male embryos at 5 and 8 ss. As part of the additions we indicated in comment #19, we will also include dorsal and cross-sectional views of both male and female embryos at the stages analyzed. If there is a difference in tissue morphology between sexes, we will also quantify these differences in tissue size, curvature, etc.

Cell number analysis. The authors state, based on prior literature, that cell numbers do not change between 5 and 8 ss. Given that the tissue is already segmented in the current study, this claim should be directly verified using the authors' own data. This analysis should be straightforward and would strengthen the conclusions.

We agree and will determine the number of cells analyzed for each embryo to test if there are changes in cell numbers at different stages and between sexes, along with appropriate statistical tests.

Relation between tissue curvature and cellular properties. It would be highly informative to extract the three-dimensional morphology of the neural plate, in particular its curvature, and examine how curvature correlates with two-dimensional cell anisotropy, apical area, and F-actin/Myosin intensity. For example, at 8 ss the authors report a U-shaped dependence of cell area along the mediolateral axis. How does this pattern relate to local tissue curvature?

We agree with this assessment and will create optical reslices in the midbrain adjacent to but excluding the midbrain hindbrain boundary. We will then divide the apical surface into 10% bins and fit a circle to the apical surface of the neural epithelium in each to calculate the local radius of curvature, which is the reciprocal of curvature for the surface. We can then correlate these values with two-dimensional cell shape and actomyosin density metrics.

Visualization of sex differences in medial actin levels (Figure 3). In Figure 3, the reported female-male difference in medial actin levels would benefit from visualization of the raw data. A zoomed-in inset of the midline region, shown separately for females and males, would help substantiate this claim.

In Figure 3, we demonstrate patterns of the whole-cell apical F-actin (Fig. 3A, B) and Myosin IIB (Fig. 3C, D) density. We find that there is no difference in F-Actin density between males and females (Fig. 3E, F), but a significant difference in midline Myosin IIB density at 5 ss that is mostly absent by 8 ss (Fig. 3G, H). We currently provide representative images for female and male myosin IIB expression across the midline-lateral axis in Figure 3C, D, and Figure 3-Supplement 1C and D. We can provide a close-up image of Myosin IIB in the midline region for both sexes as part of Figure 3, with additional annotations on existing representative image to indicate their origin.

Typographical error. Line 143: please correct "cell are" to "cell area".

We thank the reviewer for pointing out this error and will correct this typo and perform additional editing to correct any other typos present in the manuscript.

Quantitative correlation analysis between cell area and actomyosin. The authors qualitatively discuss the relationship between cell area dynamics and actomyosin levels. It would strengthen the analysis to directly compute and report correlations between these variables, and to explicitly test whether actin and myosin levels are anti-correlated with apical cell area.

As discussed in comment #6, we will plot cell area vs. F-actin or Myosin IIB density for each embryo and fit a line to calculate their correlation coefficient. From there, we will determine if there is a negative correlation between cell area and actomyosin intensity.

Interpretation of anti-correlation and contractile hinge mechanism. In lines 143-157, the authors state that the observed anti-correlation between actomyosin and cell area argues against a contractile hinge mechanism. However, this anti-correlation could also suggest that apical cell area is determined by local mechanical or geometric constraints rather than by local actomyosin contractility. The authors should clarify and discuss this alternative interpretation.

Within the neural epithelium of mice and other vertebrates, F-actin and myosin-IIB are enriched on the apical surface relative to other regions of the cell (Sadler TW, et al. (1982) Science, Matsuda M., et al. (2023), Nat. Communication, Röper, K. (2013) BioArchitecture). This poises the actomyosin network to be able to selectively constrict the apical surface relative to the basal side of the cells. Apical constriction is observed to actively facilitate the formation of hinges in folding tissues (Chanet S, et al. (2017), Nature Communications, Nishimura T., et al. (2012) Cell, Chistrodoulou N., et al. (2015) Cell Reports) in what we term the contractile hinge model of tissue folding. Tissues that employ this model of folding are expected to have small apical areas and apical enrichment of contractile actomyosin at the hinge point during folding. We observe large apical areas, low apical actomyosin density, and low apical tension at the midline hinge of the mouse midbrain neural tube, which are all inconsistent with a contractile hinge mechanism being employed in this tissue folding process. We agree with the reviewer that “cell shape does not always match [acto]myosin contractility levels, because cell shape depends on extrinsic, as well as intrinsic forces” (Line 147-149). We also agree that anticorrelation of actomyosin density and apical cell area does not per se argue against the contractile hinge model and will amend our language to be clearer. We will also further elaborate on potential extrinsic factors that may lead to the observed cell behaviors at the midline in the discussion.

Statistical robustness of laser ablation results (Figure 4). The differences in recoil velocity between regions appear small, with substantial overlap between the distributions. In addition, the sample sizes for lateral versus midline ablations appear unequal (with visibly more data points in the lateral condition). These factors raise concerns about the robustness and statistical significance of the reported differences, which should be addressed more carefully.

In Figure 4E, we show initial recoil velocities binned only by region: lateral vs. midline and report a 3.03 μm/s vs. 2.40 μm/s, or 26% difference between the two regions. We then show in Figure 4G that by considering another relevant variable, sex, we find initial recoil to be 3.15 μm/s vs. 2.30 μm/s, or 37% difference in females and 2.68 μm/s vs. 2.57 μm/s, or 4% difference in males. We go on to show In Figure 5L that within the lateral region that recoils also vary by direction, with a 38% difference. Ultimately the final conclusions that we draw regarding tissue tension that we present in our model are derived from the most finely disaggregated data in Figure 5. Our goal in presenting a stepwise disaggregation of the data was to demonstrate which variables had the greatest impact on the variance within our data set. We agree with the reviewer that a more precise statistical analysis of this data set is warranted that accounts for the complexity and multitude of variables that can influence our conclusions. In addition to the power analysis described in comment #10, we plan to conduct a mixed-effect model analysis of our data that considers factors including sex, age, cut direction, cut region, cut number, and embryos to determine which factors explain the most variance in the population. We will add this analysis as a supplement to Figure 4 alongside a description of the tests performed in the Statistical Analysis section of the methods. We will also adjust our language in the text to clearly state the limitations of the data as presented and qualify conclusions as appropriate.

Speculative statement regarding anisotropic tension in males. Line 278: "We believe that both sexes demonstrate anisotropic tension, given that males have cell aspect ratios and orientations in the lateral neural folds similar to females." This statement is speculative. Either anisotropic tension in males should be directly measured and reported, or this statement should be removed.

As discussed in comment # 15, in Figure 5L, though we can observe a difference in the initial recoil velocity means, we are unable to detect a statistical difference. Ablations were conducted blinded to embryo sex, but fewer male embryos were suitable for ablation because males develop faster than their female littermates (Seller MJ. and Perkins-Cole KJ. (1987) J. Reprod. Fert.). We were therefore unable to obtain more males in our data set. At present we do not have the resources to perform additional laser ablations to supplement the existing data set. We will instead perform a power analysis for our anisotropy measurements in the lateral region of the tissue to determine if: 1) we have a sample size large enough to detect a biologically-meaningful difference with suitable power, 2) the sample size required to detect the observed difference is so large that the difference would not be biologically meaningful, or 3) we do not have a sample size large enough to detect a difference confidently. With the results of this analysis, we will amend our language in the text to reflect the most accurate claims that can be made.

Reviewer #2 (Significance (Required)):

This study provides high-quality measurements of apical cell geometry, actomyosin organization, and inferred tension in the mouse neural epithelium. However, the lack of direct perturbations, mechanical modeling, and quantitative analysis of three-dimensional tissue deformation limits the strength of the mechanistic conclusions. Addressing these gaps would substantially strengthen the manuscript and clarify the causal role of apical tension patterns in neural fold formation. At the end of the day, the authors suggest an hypothesis that is not well support by their data, which is of high quality.

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

Summary

This manuscript by De La O et al addresses a long-standing question of how actomyosin contributes mechanically to cranial neural tube elevation in the mouse, a system in which classical midline contractile hinge models appear insufficient. The authors develop an image-processing and analysis pipeline that enables reconstruction and quantitative analysis of the apical actomyosin network across the large, curved dorsal surface of the mouse brain neuroepithelium. Using this approach, combined with laser ablation-based tension measurements in live embryos, they report a medio-lateral gradient of apical cell area and an inverse gradient of actomyosin density. Contrary to contractile hinge models described in frog, chick and invertebrate systems, they find that the midline exhibits low, isotropic tension, while the lateral neural folds show higher anisotropic apical tension, consistent with their proposal of a "lateral tension" mechanism for neural tube elevation.

The work provides an important reframing of actomyosin function in mammalian cranial neurulation, supported by extensive quantitative imaging and mechanical measurements. The finding that lateral, rather than midline, actomyosin networks dominate tissue tension is compelling and helps reconcile previous observations that midline hinge formation in mouse can proceed despite actomyosin perturbation. The study is technically sophisticated and addresses a biologically important process with clear relevance to neural tube defect etiology. However, several aspects of the statistical treatment, interpretation of laser ablation data, and mechanistic framing require clarification or tempering to fully support the authors' conclusions.

Major comments

Statistical unit and pseudo-replication in cell-based analyses (Figures 2-3)

In Figures 2 and 3, it is unclear whether statistical comparisons were performed at the level of individual cells or embryos. Because cells are nested within embryos, treating cells as independent observations raises concerns about pseudo-replication and inflated statistical significance, particularly for sex-dependent effects. While the color-coded maps are visually compelling, they may overstate confidence in differences between conditions if embryo-to-embryo variability is not explicitly accounted for.

Clarification is needed as to whether statistical testing was performed on embryo-level summary values (e.g., one value per embryo per positional bin), or whether hierarchical or mixed-effects models were used with embryo treated as a random effect. Providing embryo-level summary plots would also help readers assess inter-embryo variability. Addressing this point is important for confidence in both the reported medio-lateral gradients and the sex differences.

We agree with the reviewer that it is inappropriate to calculate statistics based on measurements of individual cells. As indicated under the ‘Statistical Analysis and Figure Assembly’ section of our methods “For fixed images, cell shape and protein intensity analysis (Figure 2H-J, Figure 3E-H, Figure 5E-H), N = 5 embryos for all conditions and n, or the number of cells in each 10% bin, is ≥ 150 cells for each embryo” (Line 556 – 558). The average and SD between embryos are shown in these plots and is calculated at the embryo level, not the cell level. We chose to consolidate this information in the methods section as the same data set is used across the three figures. We will add a line to the figure captions that N values for all experiments can be found in this section of the methods. We will also provide supplementary plots showing the bin averages for each individual embryo, color coded by embryo to show the distribution of the data set.

Interpretation of actomyosin density as a proxy for contractility (Figure 3)

The descriptive correlation between apical cell area and actomyosin density is clear and consistent. However, actomyosin abundance alone does not necessarily equate to force generation, particularly in the absence of measurements of myosin activation state (e.g., pMLC), actomyosin dynamics, or direct perturbations linking actomyosin levels to mechanical output. Although the authors appropriately note that cell shape does not always reflect intrinsic contractility, actomyosin density is nevertheless used to argue against a contractile hinge mechanism.

While the subsequent laser ablation experiments address tissue tension more directly, the mechanistic conclusions drawn from actomyosin density measurements alone would benefit from more careful qualification. Tempering language that equates actomyosin enrichment with contractile output, or explicitly acknowledging these limitations, would strengthen the interpretation.

It is largely believed that apical pools of actomyosin are active and that apical localization of actomyosin is dependent on activation. Shroom3, an actin-binding protein, is localized to the apical adherens junctions in the neural tube (Haigo SL., et al. (2003) Curr. Biol., Hildebrand JD. and Soriano P. (1999) Cell), where it can recruit Rho kinases (ROCKs) that in turn phosphorylate and activate Myosin IIB (Nishimura T. and Takeichi M. (2008) Dev.). Mutations in Shroom3 lead to neural tube close defects and its overexpression in the neural tube can induce apical constriction and increased apical accumulation of Myosin II tube (Haigo SL., et al. (2003) Curr. Biol., Hildebrand JD. (2005) J. Cell Sci.). In the mouse neural tube, Myosin IIB intensity is greater in cells that can apically constrict than in those that cannot constrict (Galea GL., et al. (2021) Nat Commun). Additionally, inhibition of ROCK reduces apical tension, presumably by reduction of activated Myosin II (Butler MB., et al. (2019) J. Cell Sci.). We agree with the reviewer’s assessment that to definitively state that the apical pools of Myosin IIB and F-actin are promoting apical contractility, a demonstration of the phosphorylation state of the Myosin II regulatory light chain (pMLC) or observations/perturbations in live embryos is necessary. We will adjust our language to reflect this limitation. We will also provide information on the relationship between apically localized actomyosin and contractility.

Statistical and biological independence of laser ablation measurements (Figures 4-5)

The Methods indicate that 155 laser ablations were analyzed across 71 embryos, implying that multiple ablations were performed per embryo. It would be helpful to clarify how this hierarchical data structure was handled statistically. Specifically, were recoil velocities averaged per embryo, paired with embryos for ML vs. RC comparisons, or analyzed using hierarchical/mixed-effects models?

Our laser ablation data set captures variables including embryo sex, age, cut location, cut direction, and cut number. Therefore, we did not feel it appropriate to average recoils within the same embryo as these cuts were intentionally in different regions (lateral vs midline) or in different orientations (i.e. a rostral-caudal cut and midline-lateral cut on opposite lateral folds), which our analysis has shown would lead to averaging out potential differences. Ablations were far apart from each other, and we had checked that ablation order did not predict changes in recoil. However, we agree with the reviewer that a more precise statistical analysis of this data set is warranted that accounts for the complexity of variables potentially influencing initial recoil velocities. As discussed in comment #27, we plan to conduct a mixed-effect model analysis of our data that considers the above and add this analysis as a supplement to figure 4. We will include a description of this in the methods and our language in the text to clearly state the limitations of the data as presented and qualify conclusions as appropriate.

In addition, embryos were subjected up to 5 ablations within a short time window. Because laser ablation disrupts tissue integrity and can induce rapid cytoskeletal remodeling, it is unclear whether later ablations represent independent measurements of the native tension state. Clarification is needed regarding whether the authors tested for effects of ablation order (e.g., first vs. later cuts), ensured sufficient spatial separation between ablation sites, or verified that repeated ablations did not systematically alter recoil measurements. Demonstrating that initial recoil velocity is independent of cut number would substantially strengthen confidence in the mechanical conclusions.

We agree with the reviewer that laser ablations cause disruptions to tissue, and these disruptions can impact the results of additional ablations performed near the site of prior ablations. The average embryo in our data set has three ablations: one on either neural fold and one at the midline, with hundreds of µm distances from each other. In embryos that had more than 3 ablations made far away from each other (additional ablations were performed in the hindbrain rhombomeres, rhombomere boundaries, at the neuroepithelium and surface ectoderm boundary, or at the zipper point, but n numbers of these are insufficient for analysis). We will supplement the methods text describing the laser ablations to clarify this for readers. Additionally, after an ablation, displacement is not detectable further than 3-5 cell lengths away from the cut even after several seconds post ablation. We will provide visual examples of these cuts after ablation to demonstrate this phenomenon. As discussed in comment #27 and #32, we will also perform mixed-effect modeling to determine if cut number impacts observed initial recoil velocities. We will also provide plots demonstrating relevant examples of these comparisons (e.g. sequential lateral cuts made in the same direction).

Interpretation of sex-dependent tension differences (Figures 4-5)

Figure 4 shows a clear lateral-greater-than-midline tension difference in females, whereas this pattern is not detected in males under initial analysis. Later, Figure 5 reveals directional anisotropy in the lateral neural folds of both sexes. As currently framed, this creates some ambiguity regarding whether the proposed lateral tension mechanism is sex-specific, sex-biased in magnitude, or sex-general but masked by directional averaging in males.

Clarifying this distinction, both in figure presentation and in the text, would strengthen the mechanistic interpretation and prevent confusion. In particular, it would be helpful to more clearly explain how directional anisotropy reconciles the apparent absence of regional tension differences in males in Figure 4.

We appreciate the reviewer taking the time to indicate this point of confusion. We ultimately conclude that the lateral tension model of neural tube elevation is agnostic of sex. Though there are nuanced differences in some of the details regarding Myosin IIB density, midline apical constriction and tension anisotropy, we do not believe these differences would fundamentally change the mechanical model used between sexes. With specific regards to masking of the lateral neural fold tension in males, we briefly address this in the discussion: “The averaging of [Rostra-Caudal] and [Midline-Lateral] [Initial Recoil Velocities] likely masked tension differences between the midline hinge and lateral neural folds, creating the false impression that males did not have high tension on the lateral neural folds” (Line 280-282). We will adjust the text in the results and discussion section to clearly indicate that are lateral tension model applies to both sexes, though some differences in specific details exist, and that averaging may have led to the result in Figure 4G.

Causal overreach in mechanistic interpretation of anisotropic tension

While the laser ablation data convincingly demonstrates spatial and directional differences in recoil consistent with patterned mechanical anisotropy, the manuscript frequently treats anisotropic apical tension as a mechanistic explanation for neural tube elevation. The presented experiments do not directly test whether anisotropic apical tension is necessary or sufficient for tissue bending, nor whether isotropic tension at the midline plays a causal role. Initial recoil velocity reflects not only pre-existing tension but also tissue geometry and viscoelastic properties, which may differ between midline and lateral regions.

As such, statements suggesting that anisotropic lateral tension "explains" neural fold elevation should be tempered or reframed. The data strongly support spatial patterning of mechanical properties but do not yet establish causal primacy. Recasting the model as a mechanically consistent framework rather than a definitive mechanism would better align conclusions with the data.

Our lateral tension model proposes that a regionalized difference in tension, with high tension in the lateral neural folds and low tension at the midline, is needed to enable neural tube elevation and ultimate closure. We agree with the reviewer, our work demonstrates that the results of our laser ablation experiments, along with measurements of cell shapes and protein density, are consistent with the lateral tension model that we propose. Our model is also supported by past work that shows that perturbations that disrupt actomyosin contractility leads to defects in brain neural tube elevation and closure but not midline hinge formation. For example, chemical perturbation of actin polymerization with Cytochalasin D (Ybot-Gonzalez P. and Copp AJ. (1999) Dev. Dyn), and genetic perturbations of Shroom 3, which apically localizes actomyosin (Hildebrand JD. And Soriano P. (1999) Cell) or Fhod3, which promotes actin polymerization (Sulistomo HW, et al. (2019) J. Biol. Chem.) all have brain neural tube closure defects but form a midline hinge. However, since we do not directly perturb tension, we have only demonstrated consistency rather than causality or sufficiency. We will adjust and temper our language accordingly in the relevant sections of the results and discussion.

Minor comments

Manuscript length and clarity

The manuscript is longer and more complex than necessary for its central message. Several sections of the Results, particularly methodological validation and somite-stage stratification, could be streamlined.

We agree with the reviewer and will continue editing the manuscript, prioritizing clarity, brevity, and precision of language so that readers are able to quickly understand the key points of the manuscript.

Sex differences section

The section on sex differences is interesting but somewhat tangential. Clarifying whether these findings are intended as mechanistic insight or observational motivation for future work could improve focus.

  We intended this section to offer up perspectives that inform and motivate future work to continue to track, analyze, and report on sex difference during development. We will edit this section of the discussion to improve clarity and brevity so that the reader can easily acquire this takeaway. Sex differences in the penetrance of exencephaly is an active area of research and our manuscript provides the first cell-level measurements which will guide the field in disaggregating future analyses by embryo sex.

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Referee #3

Evidence, reproducibility and clarity

Summary

This manuscript by De La O et al addresses a long-standing question of how actomyosin contributes mechanically to cranial neural tube elevation in the mouse, a system in which classical midline contractile hinge models appear insufficient. The authors develop an image-processing and analysis pipeline that enables reconstruction and quantitative analysis of the apical actomyosin network across the large, curved dorsal surface of the mouse brain neuroepithelium. Using this approach, combined with laser ablation-based tension measurements in live embryos, they report a medio-lateral gradient of apical cell area and an inverse gradient of actomyosin density. Contrary to contractile hinge models described in frog, chick and invertebrate systems, they find that the midline exhibits low, isotropic tension, while the lateral neural folds show higher anisotropic apical tension, consistent with their proposal of a "lateral tension" mechanism for neural tube elevation.

The work provides an important reframing of actomyosin function in mammalian cranial neurulation, supported by extensive quantitative imaging and mechanical measurements. The finding that lateral, rather than midline, actomyosin networks dominate tissue tension is compelling and helps reconcile previous observations that midline hinge formation in mouse can proceed despite actomyosin perturbation. The study is technically sophisticated and addresses a biologically important process with clear relevance to neural tube defect etiology. However, several aspects of the statistical treatment, interpretation of laser ablation data, and mechanistic framing require clarification or tempering to fully support the authors' conclusions.

Major comments

1. Statistical unit and pseudo-replication in cell-based analyses (Figures 2-3) In Figures 2 and 3, it is unclear whether statistical comparisons were performed at the level of individual cells or embryos. Because cells are nested within embryos, treating cells as independent observations raises concerns about pseudo-replication and inflated statistical significance, particularly for sex-dependent effects. While the color-coded maps are visually compelling, they may overstate confidence in differences between conditions if embryo-to-embryo variability is not explicitly accounted for.

Clarification is needed as to whether statistical testing was performed on embryo-level summary values (e.g., one value per embryo per positional bin), or whether hierarchical or mixed-effects models were used with embryo treated as a random effect. Providing embryo-level summary plots would also help readers assess inter-embryo variability. Addressing this point is important for confidence in both the reported medio-lateral gradients and the sex differences. 2. Interpretation of actomyosin density as a proxy for contractility (Figure 3) The descriptive correlation between apical cell area and actomyosin density is clear and consistent. However, actomyosin abundance alone does not necessarily equate to force generation, particularly in the absence of measurements of myosin activation state (e.g., pMLC), actomyosin dynamics, or direct perturbations linking actomyosin levels to mechanical output. Although the authors appropriately note that cell shape does not always reflect intrinsic contractility, actomyosin density is nevertheless used to argue against a contractile hinge mechanism.

While the subsequent laser ablation experiments address tissue tension more directly, the mechanistic conclusions drawn from actomyosin density measurements alone would benefit from more careful qualification. Tempering language that equates actomyosin enrichment with contractile output, or explicitly acknowledging these limitations, would strengthen the interpretation. 3. Statistical and biological independence of laser ablation measurements (Figures 4-5) The Methods indicate that 155 laser ablations were analyzed across 71 embryos, implying that multiple ablations were performed per embryo. It would be helpful to clarify how this hierarchical data structure was handled statistically. Specifically, were recoil velocities averaged per embryo, paired with embryos for ML vs. RC comparisons, or analyzed using hierarchical/mixed-effects models?

In addition, embryos were subjected up to 5 ablations within a short time window. Because laser ablation disrupts tissue integrity and can induce rapid cytoskeletal remodeling, it is unclear whether later ablations represent independent measurements of the native tension state. Clarification is needed regarding whether the authors tested for effects of ablation order (e.g., first vs. later cuts), ensured sufficient spatial separation between ablation sites, or verified that repeated ablations did not systematically alter recoil measurements. Demonstrating that initial recoil velocity is independent of cut number would substantially strengthen confidence in the mechanical conclusions. 4. Interpretation of sex-dependent tension differences (Figures 4-5) Figure 4 shows a clear lateral-greater-than-midline tension difference in females, whereas this pattern is not detected in males under initial analysis. Later, Figure 5 reveals directional anisotropy in the lateral neural folds of both sexes. As currently framed, this creates some ambiguity regarding whether the proposed lateral tension mechanism is sex-specific, sex-biased in magnitude, or sex-general but masked by directional averaging in males.

Clarifying this distinction, both in figure presentation and in the text, would strengthen the mechanistic interpretation and prevent confusion. In particular, it would be helpful to more clearly explain how directional anisotropy reconciles the apparent absence of regional tension differences in males in Figure 4. 5. Causal overreach in mechanistic interpretation of anisotropic tension While the laser ablation data convincingly demonstrates spatial and directional differences in recoil consistent with patterned mechanical anisotropy, the manuscript frequently treats anisotropic apical tension as a mechanistic explanation for neural tube elevation. The presented experiments do not directly test whether anisotropic apical tension is necessary or sufficient for tissue bending, nor whether isotropic tension at the midline plays a causal role. Initial recoil velocity reflects not only pre-existing tension but also tissue geometry and viscoelastic properties, which may differ between midline and lateral regions.

As such, statements suggesting that anisotropic lateral tension "explains" neural fold elevation should be tempered or reframed. The data strongly support spatial patterning of mechanical properties but do not yet establish causal primacy. Recasting the model as a mechanically consistent framework rather than a definitive mechanism would better align conclusions with the data.

Minor comments

1. Manuscript length and clarity The manuscript is longer and more complex than necessary for its central message. Several sections of the Results, particularly methodological validation and somite-stage stratification, could be streamlined.
2. Sex differences section The section on sex differences is interesting but somewhat tangential. Clarifying whether these findings are intended as mechanistic insight or observational motivation for future work could improve focus.

Significance

General assessment.

This study provides a technically sophisticated and carefully executed analysis of the mechanical organization of the mouse cranial neural epithelium during neural tube elevation. Its principal strengths lie in the development of a large-scale apical imaging and reconstruction pipeline, the quantitative mapping of medio-lateral gradients in cell shape and actomyosin organization, and the use of laser ablation to directly probe regional and directional tissue tension in live embryos. Together, these approaches allow the authors to address a long-standing discrepancy between classical contractile hinge models and prior observations in mouse neurulation. The main limitations of the study relate not to data quality, but to interpretation: several conclusions rely on correlational relationships between actomyosin enrichment, cell shape anisotropy, and tissue tension, and the mechanistic language at times exceeds what is directly tested. Clarification of statistical structure and tempering of causal claims would substantially strengthen the work.

Advance.

Relative to prior studies of neural tube closure in frog, chick, and invertebrate systems, this work advances the field by providing direct, spatially resolved measurements of tissue tension in the mouse cranial neural tube. The identification of low, largely isotropic tension at the midline and higher, anisotropic tension in the lateral neural folds represents a conceptual advance that reframes how actomyosin contributes to mammalian neurulation. While the study does not establish causality between anisotropic apical tension and tissue bending, it offers a mechanically consistent alternative to contractile hinge models and provides a valuable framework for interpreting species-specific differences in neural tube morphogenesis. The advance is therefore primarily conceptual and technical, rather than mechanistic in the strict causal sense.

Audience.

This work will be of strong interest to a specialized but broad audience spanning developmental biology, epithelial mechanics, morphogenesis, and neural tube defect research. The imaging and analytical approaches are likely to be useful beyond neurulation, particularly for investigators studying force patterning in large, curved epithelial tissues. With appropriate framing, the study should also be of interest to researchers investigating the biomechanical basis of congenital defects, even if its immediate implications are primarily basic rather than translational.

Field of expertise.

My expertise lies in epithelial morphogenesis, tissue mechanics, actomyosin-based force generation, and quantitative imaging in developmental systems. I do not have specific expertise in clinical aspects of neural tube defect diagnosis or treatment.

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Referee #2

Evidence, reproducibility and clarity

The manuscript investigates the role of apical constriction and actomyosin organization in shaping the mouse brain neural epithelium during neural tube elevation, with particular emphasis on sex-specific differences. The authors develop an imaging and analysis pipeline to reconstruct the apical surface of the neural plate in three dimensions and perform quantitative measurements of apical cell area, actin, and myosin IIB distributions. Targeted laser ablation experiments are used to infer regional tissue tension.

The main findings can be summarized as follows. First, the authors identify a mediolateral gradient in apical cell area, with larger cells at the midline and smaller cells on the lateral neural folds, which inversely correlates with actomyosin density. Laser ablation experiments suggest that apical tension is lower and isotropic at the midline, whereas it is higher and anisotropic on the lateral folds, particularly in females. Second, sex-dependent differences in apical cell area, constriction rates, and actomyosin levels are reported at early somite stages, preceding previously described sex biases in neural tube defects.

The experimental work is technically solid, and the imaging and quantification pipeline represents a useful advance for analyzing large, curved epithelial surfaces. However, the study feels incomplete in its current form. Despite addressing neural tube elevation, the manuscript does not provide a comprehensive analysis of the folding process itself. Key aspects such as three-dimensional tissue morphology, curvature evolution, or global shape changes of the neural folds are not quantified. In addition, other potentially relevant cellular behaviors, such as proliferation, cell rearrangements, or contributions from neighboring tissues, are not examined, nor are they compared systematically between sexes.

Conceptually, the study focuses narrowly on correlations between apical cell area, actomyosin density, and inferred tension. While these measurements are carefully performed, the relationship between differential actomyosin contractility and three-dimensional tissue folding remains largely descriptive. No mechanical model or simulation framework is provided to link changes in actomyosin organization and cell shape to the emergence of neural folds and hinge formation. As a result, it is difficult to assess whether the measured differences in tension (on the order of ~40%) are sufficient to account for the proposed mechanical behavior of the tissue.

The central hypothesis advanced by the authors is that a relatively "soft" midline, flanked by stiffer, tension-bearing lateral folds, facilitates hinge formation during brain neurulation. However, this hypothesis is not directly tested by perturbation. For example, experimentally increasing contractility or stiffness at the midline (e.g., via optogenetic activation of apical constriction machinery) would provide a more direct test of causality. As it stands, the data demonstrate correlation rather than necessity or sufficiency.

Relatedly, alternative interpretations are not fully addressed. Large apical cell areas and low actomyosin levels at the midline could arise as a consequence of tissue geometry, contact with underlying structures such as the notochord, or extrinsic mechanical constraints, rather than being the primary cause of hinge formation. Similarly, anisotropic stresses generated at the tissue or embryo scale could align cells and actomyosin cables, producing the observed patterns without requiring locally specified apical tension differences as the initiating mechanism. The manuscript does not clearly distinguish whether apical tension asymmetries are a driver of folding or an emergent outcome of folding dynamics.

Finally, while the identification of sex differences is intriguing, it remains unclear what mechanistic insight is gained beyond establishing that such differences exist. The functional consequences of these differences for neural tube closure, robustness, or failure are not explored, nor is it clear how they integrate into the proposed lateral tension model.

In summary, this study provides high-quality measurements of apical cell geometry, actomyosin organization, and inferred tension in the mouse neural epithelium. However, the lack of direct perturbations, mechanical modeling, and quantitative analysis of three-dimensional tissue deformation limits the strength of the mechanistic conclusions. Addressing these gaps would substantially strengthen the manuscript and clarify the causal role of apical tension patterns in neural fold formation.

Major Comments

1. Figure quality. Figure 1 contains very low-resolution images, which makes it difficult to evaluate the segmentation quality and tissue morphology. Higher-resolution versions should be provided.
2. Cell segmentation strategy and validation. The authors segment cell areas using Myosin II and F-actin signals. This approach may introduce inaccuracies, as actomyosin cables can traverse the apical surface of individual cells and do not always coincide with cell boundaries. Segmentation based on junctional markers such as ZO-1 may be more appropriate. At minimum, the authors should provide a quantitative validation of segmentation accuracy, for example by overlaying segmentation results on raw images together with a nuclear marker (e.g., DAPI or H2B-GFP), to demonstrate that the number of segmented cells corresponds to the number of nuclei.
3. Lack of cross-sectional views of neural tube morphology. The manuscript would benefit from the inclusion of cross-sectional images of the neural tissue at different developmental stages. This would serve two purposes: (i) to demonstrate that the authors have a comprehensive understanding of the full three-dimensional folding process during neural tube closure, including medial and lateral hinge formation, and (ii) to allow readers to visualize the tissue geometry corresponding to the analyzed projection datasets (e.g., at 5 ss and 8 ss).
4. Sex-specific differences in overall neural plate morphology. The authors report that at 5 ss, males consistently have larger apical cell areas than females. It is unclear whether this difference reflects a global difference in neural plate morphology. Showing representative images of female and male neural plates would help readers directly assess whether there are overt morphological differences beyond those revealed by quantitative analysis.
5. Cell number analysis. The authors state, based on prior literature, that cell numbers do not change between 5 and 8 ss. Given that the tissue is already segmented in the current study, this claim should be directly verified using the authors' own data. This analysis should be straightforward and would strengthen the conclusions.
6. Relation between tissue curvature and cellular properties. It would be highly informative to extract the three-dimensional morphology of the neural plate, in particular its curvature, and examine how curvature correlates with two-dimensional cell anisotropy, apical area, and F-actin/Myosin intensity. For example, at 8 ss the authors report a U-shaped dependence of cell area along the mediolateral axis. How does this pattern relate to local tissue curvature?
7. Visualization of sex differences in medial actin levels (Figure 3). In Figure 3, the reported female-male difference in medial actin levels would benefit from visualization of the raw data. A zoomed-in inset of the midline region, shown separately for females and males, would help substantiate this claim.
8. Typographical error. Line 143: please correct "cell are" to "cell area".
9. Quantitative correlation analysis between cell area and actomyosin. The authors qualitatively discuss the relationship between cell area dynamics and actomyosin levels. It would strengthen the analysis to directly compute and report correlations between these variables, and to explicitly test whether actin and myosin levels are anti-correlated with apical cell area.
10. Interpretation of anti-correlation and contractile hinge mechanism. In lines 143-157, the authors state that the observed anti-correlation between actomyosin and cell area argues against a contractile hinge mechanism. However, this anti-correlation could also suggest that apical cell area is determined by local mechanical or geometric constraints rather than by local actomyosin contractility. The authors should clarify and discuss this alternative interpretation.
11. Statistical robustness of laser ablation results (Figure 4). The differences in recoil velocity between regions appear small, with substantial overlap between the distributions. In addition, the sample sizes for lateral versus midline ablations appear unequal (with visibly more data points in the lateral condition). These factors raise concerns about the robustness and statistical significance of the reported differences, which should be addressed more carefully.
12. Speculative statement regarding anisotropic tension in males. Line 278: "We believe that both sexes demonstrate anisotropic tension, given that males have cell aspect ratios and orientations in the lateral neural folds similar to females." This statement is speculative. Either anisotropic tension in males should be directly measured and reported, or this statement should be removed.

Significance

This study provides high-quality measurements of apical cell geometry, actomyosin organization, and inferred tension in the mouse neural epithelium. However, the lack of direct perturbations, mechanical modeling, and quantitative analysis of three-dimensional tissue deformation limits the strength of the mechanistic conclusions. Addressing these gaps would substantially strengthen the manuscript and clarify the causal role of apical tension patterns in neural fold formation. At the end of the day, the authors suggest an hypothesis that is not well support by their data, which is of high quality.

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Referee #1

Evidence, reproducibility and clarity

Summary

Folding is a major morphogenetic process that shapes tissues and organs in three dimensions. The mechanisms underlying tissue folding have been extensively explored and are often driven by actomyosin-based apical constriction. Here, the authors describe changes in cell geometry and mechanics during mouse neural tube formation. They build on quantitative fixed imaging and live junction ablation to extract cell geometry and junctional tension. These analyses are performed at different developmental stages and in both male and female embryos to propose a mechanical mechanism for neural tube elevation in the brain.

Major comments

The authors report quantitative data on cell geometry and junctional tension inferred from laser ablation. Overall, there are numerous statements that require stronger support from the experimental data. To substantiate several of their claims, the authors need to provide a larger number of data points-or at least comparable numbers across experimental conditions-for the tension measurements. Additional statistical analyses are required throughout to support the conclusions.

Figure 1

• Does the projection algorithm account for tissue curvature when computing cell geometrical parameters such as area and anisotropy?
• The authors should provide information on the accuracy and reliability of the cell segmentation.

Figure 2

• The authors indicate that the rate of apical constriction differs between male and female embryos. However, apical sizes differ only at specific positions along the ML axis (Fig. 2H, I). The authors should provide statistical analyses for the rates shown in Fig. 2J. Are these rates significantly different between males and females, and between medial and lateral regions?
• Please clearly state the main novelty of this study relative to the work published by Brooks et al.

Figure 3

• The authors need to provide statistical support for the claim that large midline cells exhibit reduced F-actin and Myosin IIB levels.
• F-actin and Myosin IIB intensities should be plotted as a function of cell area to support the proposed anticorrelation between apical area and actomyosin levels.
• Statistical analyses are missing to substantiate the increase in F-actin levels between stages ss5 and ss8.
• Figure S3 should be supported by plots showing Myosin II and F-actin intensity as a function of position along the ML axis, together with appropriate statistics.

Figure 4

• The authors state that lateral tension in male embryos is not different from midline tension, yet the number of data points is much lower than in females. To support this claim, the number of ablations should be comparable across sexes. Is lateral tension different between males and females?
• Similarly, the data in Fig. S4 used to claim no change in tension over time are not supported by sufficient data points. Would the medial and lateral tensions reported in Fig. 4G remain unchanged if the authors perform statistical analyses on 10-15 ablations per condition?

Figure 5

• The number of data points in Fig. 5J and L is insufficient to support claims of no difference. The only detectable difference arises in the comparison with much higher sample size (Fig. 5L, ML vs RC). The authors conclude that males have higher ML tension than RC tension, but given the limited data this conclusion should be amended to "no detectable difference."

Code availability

The authors should provide access to the code used to generate the projections.

Significance

The authors propose a mechanical model for neural tube elevation based on analyses of cell geometry and tension at two developmental stages. The reported differences in cell geometry or actomyosin levels do not appear to explain the differences in geometry or tension suggested between male and female embryos. This raises questions about the relationship between these measurements and their relevance for understanding the mechanisms of neural tube elevation.

If the major concerns outlined above are rigorously addressed, the manuscript will offer a valuable descriptive characterization of neural tube cell geometry and mechanical stress during morphogenesis. Such datasets could form a foundation for future studies investigating the mechanisms driving neural tube elevation.

Very important to understand how the regulations put onto the labor unions are put into effect in their structuring

I'm happy to learn about clean-ups like this! I'd definitely want to connect and perhaps bring them into my scope of work and/or academic services as a student leader. This is definitely what I'm bringing with me from this read!

I don't fully agree with this quote. Surely house pricing skyrocketing is a factor, but I think it's a generalization. There are many reasons why people are unhoused these days -- and yes, dealing with addiction is another factor that is massively important when discussing the issue. The problem is that more often than not people think about it as "oh, you got into drugs because you wanted to, people always have a choice" etc, when they do not consider facts such as the painkiller and fentanyl crisis, the systemic poverty inflicted by the healthcare system, and the rigged food system which traps certain pay brackets within an unhealthy cycle, which later on condemns them to crippling poverty, debt, and unaffordable medical expenses -- often without them having a support system or a safety net.

Well I didn't even know about the Great Raleigh Cleanup, which is definitely something new I have learned about today and I'm excited for. I wish we had more organizations like this who take care of our communities. In a way, I wish we didn't need it, but who knows, maybe one day...

I found this really funny, in a way. I've met people like this in my life, people who sort of developed a sense of excitement for being a "green fiend", turning into "garbage goblins" as the thought of cleaning up and "doing good" by fixing other people's mess gives them a feeling of purpose. In this case, Preston profits from it - which helps. But it's sorta bittersweet to see how something so tragic can be someone's livelihood.

sometimes when people have the want or at times the need to write, their psyche doesn't let them at times especially when they see professionally published and written pieces of text. even I get that intimidation of looking at those other texts and books and I end up thinking to myself "i'll never reach that level of professional writing"

i feel a resonance to this part of the text since my little brother at 15 years old is still pretty illiterate and has to ask someone else what something says, this is most partly due to the fact that the education system doesn't give a rat's ass about it's future workforce, politicians, and service members. and the fact that during the pandemic in 2020 my little brother did nothing but break MY xbox controllers while raging on fortnite.

sources showing the effectiveness and welfare enhancing properties of targeted ads

more evidence for consumer welfare enhancements of ad targeting

proposed solution from this paper

this is probably the most problematic issue on targeted ads. It exacerbates political polarization

This site is such a fascinating object of modern labor, and how the processes of the internet abstract humanity from code. So much of the algorithms of Amazon, Google, and YouTube work partly on the basis of human labor, yet because the internet allows asynchronous, atomized labor, we forget this fact and think of the algorithm as a magical bit of technology. I wonder if there is a way to organize the internet which presents the labor that goes into its creation in a way that is unobtrusive to the user.

The text takes the form of an scientific article on a science blog website.

It would be good to add a background, the easiest would be to use the gif that your already have in the page? The experience has greatly improved with the addition of the sound

This makes me think about how forms of the internet focused on geographical vicinity have pretty much been entirely relegated to dating apps at this point. It is unlikely that a source of content (i.e. YouTube videos, TikTok algorithm) is surrounding a sense of place, rather than an account or movement which stands in for the place. I wonder if there is a place for an internet that is based in vicinity outside of dating, such as YikYak back in the day.

Grene, Marjorie. “Authenticity: An Existential Virtue.” Ethics, vol. 62, no. 4, 1952, pp. 266–74. JSTOR, http://www.jstor.org/stable/2378633. Accessed 25 Feb. 2026.

Absolutely, they have a duty to protect their users in case things get overboard. I am thinking about that woman with the racist tweet before going on her plane, although she was messed up for tweeting it, the situation could've turned really ugly really quickly because of the amount of people ready to attack her and find where she is. Yes she brought it upon herself, but there are situations like that that can happen without racist tweets, people can gan up on a person for any reason at any time and it can be unsafe.

I have probably participated in crowdsourcing at some point. Maybe like fundraising on the internet, or commenting something along with others to achieve some goal. I think that collective input into something can be a fun use of online communities and bring people together despite distance, however I do see drawbacks on how it can overload the system and overwhelm people.

Multiple authors

With the founding of a 'romantic' middle ages, the () of gothic archetecture was revived in england, while artisticatributes of medieval religious art were adopted by the Pre-raphaelites. But how much of an impact did this make on Wales. Unlike England, Wales had a () of barbarism, and as such, it is possible that gothic architecture was not recieved as warmly by the Welsh. However, this did not mean that the country was bereft of contributions to this movement. Perhaps the most oppulent and well-preservediscastle coch by (blah). but was it Welsh in character?

Alternatively, Wales boasted many gothic revival churches, often restorations of authentic medieval churches, although many were built too. While the style itself was largely English, the movement saw some of the first Welsh speaking architects involved, with john jones first building a chapel in blah, and ten going on to aid the building of crystal palace in blah. While an image of liberal nonconformity has come to present the Welsh of the victorian period, not all who were proud to be Welsh were nonconformists, the scathing () of the 'blue books' actually written by an anglican. As such, could we argue that such building came from the Welsh people also, inspired, like many across Europe, to return to medieval architecture in a time of great change.

Interestingly

Interesting, what is the link to wales? Was he innfluenced by lady guest?

babelicious babes xoxo

was it common in Wales

I think the blending of old and new is definitely a really important part of revolution and innovation. Past trial and error is an important part of revolution and creating new ideas for the future of a society.

It's interesting that even then it was recognized as a "century of revolutions"

This is very important for ethos because you’re open to the fact that one will not agree with you and that shows maturity and leads to being trustworthy. Coates isn’t trying to push done with opinion onto everyone and making it enforced. For example, let's say people on the left will make a statement that they claim is to be true, like guns are completely wrong. People on the right will be upset and not even consider their argument because of them claiming it was true. I would never want to listen to someone that isn't considering or open to what I believe in. The writer wouldn’t be able to gain my trust this way.

eLife Assessment

This fundamental work advances our understanding of the role of human hippocampal theta oscillations in memory encoding and retrieval. The evidence supporting the conclusions is convincing, using both scopolamine administration and intracranial EEG recordings. This work will be of broad interest to neuroscientists and has translational implications.

Reviewer #1 (Public review):

Summary:

The authors report intracranial EEG findings from 12 epilepsy patients performing an associative recognition memory task under the influence of scopolamine. They show that scopolamine administered before encoding disrupts hippocampal theta phenomena and reduces memory performance, and that scopolamine administered after encoding but before retrieval impairs hippocampal theta phenomena (theta power, theta phase reset) and neural reinstatement but does not impair memory performance. This is an important study with exciting, novel results and translational implications. The manuscript is well written, the analyses are thorough and comprehensive, and the results seem robust.

Strengths:

- Very rare experimental design (intracranial neural recordings in humans coupled with pharmacological intervention);

- Extensive analysis of different theta phenomena;

- Well-established task with different conditions for familarity versus recollection;

- Clear presentation of findings;

- Translational implications for diseases with cholinergic dysfuction (e.g., AD);

- Findings challenge existing memory models and the discussion presents interesting novel ideas.

Reviewer #2 (Public review):

Summary:

In this study, performed in human patients, the authors aimed at dissecting out the role of cholinergic modulation in different types of memory (recollection-based vs familiarity and novelty-based) and during different memory phases (encoding and retrieval). Moreover, their goal was to obtain the electrophysiological signature of cholinergic modulation on network activity of the hippocampus and the entorhinal cortex.

Strengths:

Authors combined cognitive tasks and intracranial EEG recordings in neurosurgical epilepsy patients. The study confirms previous evidence regarding the deleterious effects of scopolamine, a muscarinic acetylcholine receptor antagonist, on memory performance when administered prior the encoding phase of the task. During both encoding and retrieval phases scopolamine disrupts the power of theta oscillations in terms of amplitude and phase synchronization. These results raise the question on the role of theta oscillations during retrieval and the meaning of scopolamine effect on retrieval-associated theta rhythm without cognitive changes. The authors clearly discussed this issue in the discussion session.

A major point is the finding that scopolamine-mediated effect is selective for recollection-based memory and not for familiarity- and novelty-based memory.

The methodology used is powerful and the data underwent a detailed and rigorous analysis.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors report intracranial EEG findings from 12 epilepsy patients performing an associative recognition memory task under the influence of scopolamine. They show that scopolamine administered before encoding disrupts hippocampal theta phenomena and reduces memory performance, and that scopolamine administered after encoding but before retrieval impairs hippocampal theta phenomena (theta power, theta phase reset) and neural reinstatement but does not impair memory performance. This is an important study with exciting, novel results and translational implications. The manuscript is well-written, the analyses are thorough and comprehensive, and the results seem robust.

Strengths:

(1) Very rare experimental design (intracranial neural recordings in humans coupled with pharmacological intervention).

(2) Extensive analysis of different theta phenomena.

(3) Well-established task with different conditions for familiarity versus recollection.

(4) Clear presentation of findings and excellent figures.

(5) Translational implications for diseases with cholinergic dysfunction (e.g., AD).

(6) Findings challenge existing memory models, and the discussion presents interesting novel ideas.

Weaknesses:

(1) One of the most important results is the lack of memory impairment when scopolamine is administered after encoding but before retrieval (scopolamine block 2). The effect goes in the same direction as for scopolamine during encoding (p = 0.15). Could it be that this null effect is simply due to reduced statistical power (12 subjects with only one block per subject, while there are two blocks per subject for the condition with scopolamine during encoding), which may become significant with more patients? Is there actually an interaction effect indicating that memory impairment is significantly stronger when scopolamine is applied before encoding (Figure 1d)? Similar questions apply to familiarity versus recollection (lines 78-80). This is a very critical point that could alter major conclusions from this study, so more discussion/analysis of these aspects is needed. If there are no interaction effects, then the statements in lines 84-86 (and elsewhere) should be toned down.

The reviewer highlights important concerns regarding the statistical power of the behavioral effects. We address these concerns in the revised manuscript in two ways: (1) we provide a supplemental analysis using a matched number of blocks between the placebo and scopolamine conditions to avoid statistical bias related to differing trial counts, and (2) we include a supplemental figure illustrating paired comparisons between blocks.

(2) Further, could it simply be that scopolamine hadn't reached its major impact during retrieval after administration in block 2? Figure 2e speaks in favor of this possibility. I believe this is a critical limitation of the experimental design that should be discussed.

The reviewer raises an important methodological concern regarding the time required for scopolamine's effect to manifest and the subsequent impact on the study outcomes. Previous studies report that the average time to maximum serum concentration after intravenous (IV) scopolamine administration is approximately 5 minutes (Renner et al., 2005), with the corresponding clinical onset estimated at 10 minutes. In our study, the retrieval period in Block 2 commenced at 15 ± 0.2 post-injection across all subjects. Given this timing, there is sufficient reason to conclude that scopolamine had reached its major impact during the Block 2 retrieval phase. Furthermore, the observation of significant disruptions to theta oscillations during this same retrieval phase provides strong evidence that the drug was in full effect at that time.

(3) It is not totally clear to me why slow theta was excluded from the reinstatement analysis. For example, despite an overall reduction in theta power, relative patterns may have been retained between encoding and recall. What are the results when using 1-128 Hz as input frequencies?

Slow theta (2–4 Hz) was excluded from the reinstatement analysis to avoid potential confounding effects. Given the observed disruption to slow theta power following scopolamine administration, any subsequent changes in slow theta reinstatement would be causally ambiguous, potentially arising directly from the power effects. Therefore, we would be unable to determine whether changes in slow theta reinstatement were genuinely independent of changes in power.

(4) In what way are the results affected by epileptic artifacts occurring during the task (in particular, IEDs)?

To exclude abnormal events and interictal activity, a kurtosis threshold of 4 was applied to each trial, effectively filtering out segments exhibiting significant epileptic artifacts.

Reviewer #2 (Public review):

Summary:

In this study, performed in human patients, the authors aimed at dissecting out the role of cholinergic modulation in different types of memory (recollection-based vs familiarity and novelty-based) and during different memory phases (encoding and retrieval). Moreover, their goal was to obtain the electrophysiological signature of cholinergic modulation on network activity of the hippocampus and the entorhinal cortex.

Strengths:

The authors combined cognitive tasks and intracranial EEG recordings in neurosurgical epilepsy patients. The study confirms previous evidence regarding the deleterious effects of scopolamine, a muscarinic acetylcholine receptor antagonist, on memory performance when administered prior to the encoding phase of the task. During both encoding and retrieval phases, scopolamine disrupts the power of theta oscillations in terms of amplitude and phase synchronization. These results raise the question of the role of theta oscillations during retrieval and the meaning of scopolamine's effect on retrieval-associated theta rhythm without cognitive changes. The authors clearly discussed this issue in the discussion session. A major point is the finding that the scopolamine-mediated effect is selective for recollection-based memory and not for familiarity- and novelty-based memory.

The methodology used is powerful, and the data underwent a detailed and rigorous analysis.

Weaknesses:

A limited cohort of patients; the age of the patients is not specified in the table.

To comply with human subject privacy protection policies, age was not reported; however, we did not find any significant effects of age on the behavioral or neural measures.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) Regarding dosage, did you take the patients' body weight into account? Do the effects hold when controlling for it?

We controlled for participant weight, yet the observed effects were more strongly correlated with the absolute scopolamine dosage, irrespective of weight. This outcome indicates that scopolamine likely rapidly crosses the blood-brain barrier, producing swift effects that are not initially influenced by metabolic variability.

(2) Line 96: Corrected for what kind of multiple comparisons?

We apologize for this confusion. The statistical analysis presented in this line does not require multiple-comparison correction, and we will therefore remove the annotation.

(3) Line 165: These are very interesting results. How do they relate to Rizzuto et al., NeuroImage, 2006?

Our findings show that successful retrieval is tied to an encoding-retrieval phase match, which is a refinement and application of the Rizzuto et al. (2006) work. Rizzuto et al. showed that memory events are phase-locked; we show that maintaining a specific, matched phase relationship between encoding and retrieval events is critical for memory success, and that this process is dependent on the cholinergic system.

Reviewer #2 (Recommendations for the authors):

Figure 1b: It would be useful for clarity to have the cartoon of the treatment paradigm for the encoding phase (blocks 3 and 4).

The treatment paradigm only involved a single intravenous (IV) injection of scopolamine (or saline, for the placebo condition). The injections were administered by the participant's attending nurse, with a board-certified anesthesiologist present at the time of injection and available throughout the experiment. These details are fully documented in the Methods section.

So, your secrets /are/ stored on your filesystem. They are encrypted by Bitwarden, though.

Or you could not. Put a badge in the UI that indicates, "Hey, this thing was deleted. We still have a copy, though, and that's what you're seeing here. Pinafore is working for you."

This is the prime example I use to illustrate implicit step zero.

How can we do this more effectively though? Is there a place where such a concern was able to actualize into developed institutions representing fisher's interests?

How can we properly manage economic and environmental impacts of developing a properly sustainable fishery industry in the developing world?

What is the best suggestion to the development of strong governmental institutions to support SSF in developing nations, who primarily rely on this for their nutritional portfolio?

eLife Assessment

This valuable manuscript investigates the localisation of nutrient receptors in bloodstream stage trypanosomes, with implications for both nutrient uptake and immune evasion. Results after direct fixation of the cells in culture medium (as opposed to fixation after centrifugation) provide compelling evidence that the amounts of receptors on the surface of the cell, as opposed to the flagellar pocket, have previously been severely underestimated.

Reviewer #1 (Public review):

Summary:

An interesting manuscript from the Carrington lab is presented investigating the behavior of single vs double GPI-anchored nutrient receptors in bloodstream form (BSF) T. brucei. These include the transferrin receptor (TfR), the HpHb receptor (HpHbR), and the factor H receptor (FHR). The central question is why these critical proteins are not targeted by host acquired immunity. It has generally been thought that they are sequestered in the flagellar pocket (FP), where they are subject to rapid endocytosis - any Ab:receptor complexes would be rapidly removed from the cell surface. This manuscript challenges that assumption by showing that these receptors can be found all over the outer cell body and flagella surfaces - if one looks in an appropriate manner (rapid direct fixation in culture media).

Strengths and weaknesses:

(1) The presence of a second ESAG6 gene in the BES7 expression site was noted in the previous review. This is now noted and discussed appropriately in the current version.

(2) Surface binding studies: The ability of cells to bind tagged-Tf while in complete media was challenged and it was suggested that classic competition studies be performed to validate saturable ligand binding. This has been done now and the results confirm that this is so. A reasonable discussion of the results is presented.

(3) Variable TfR expression in different BESs: The claim that specific ES environment is the dominant factor controlling TfR expression levels was challenged in that the presented results could be due to technical issues. RNA seq has now been performed confirming that the differences in TfR abundance is indeed directly related to mRNA levels

(4) Surface immuno-localization of receptors: In regard to the novel immunofluorescence (direct fixation) methodology used to demonstrate TfR on the cell surface the authors were asked of they had attempted more traditional methods that involve centrifugation/washing. These data are now provided (Fig S5) and do indicate that centrifugation does reduce signal, likely due to shedding and/or internalization during the procedure. Nevertheless, significant signal is present after centrifugation leaving the issue of why others have never detected significant surface TfR.

These responses address all the major concerns with the original submission and a greatly improved manuscript is now submitted.

Reviewer #2 (Public review):

The revised data support the conclusion that methodological differences can influence apparent receptor localization. However, key claims regarding functional surface engagement of TfR and hydrodynamic clearance remain based largely on indirect evidence and model-based interpretation. These conclusions should therefore be phrased more cautiously.

I thank the authors for their careful rebuttal and the additional experiments included in the revised manuscript. The new fixation comparisons and transferrin competition assays substantially strengthen the technical basis of the study and address several of the original concerns.

However, some conclusions remain more inferential than directly supported by the data. While the fixation and washing controls demonstrate that methodology influences apparent TfR localisation, they do not directly establish that previous protocols quantitatively redistribute surface TfR into the flagellar pocket. Statements implying such redistribution should therefore be phrased more cautiously.

Similarly, the added transferrin binding controls argue against non-specific interactions, but functional engagement of surface-exposed TfR in intact bloodstream-form parasites remains supported mainly by indirect evidence. The proposed explanation involving rapid on/off rates and newly arriving receptors is plausible but should be more clearly identified as an inference.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

An interesting manuscript from the Carrington lab is presented investigating the behavior of single vs double GPI-anchored nutrient receptors in bloodstream form (BSF) T. brucei. These include the transferrin receptor (TfR), the HpHb receptor (HpHbR), and the factor H receptor (FHR). The central question is why these critical proteins are not targeted by host-acquired immunity. It has generally been thought that they are sequestered in the flagellar pocket (FP), where they are subject to rapid endocytosis - any Ab:receptor complexes would be rapidly removed from the cell surface. This manuscript challenges that assumption by showing that these receptors can be found all over the outer cell body and flagella surfaces, if one looks in an appropriate manner (rapid direct fixation in culture media).

The main part of the manuscript focuses on TfR, typically a GPI1 heterodimer of very similar E6 (GPI anchored) and E7 (truncated, no GPI) subunits. These are expressed coordinately from 15 telomeric expression sites (BES), of which only one can be transcribed at a time. The authors identify a native E6:E7 pair in BES7 in which E7 is not truncated and therefore forms a GPI2 heterodimer. By in situ genetic manipulation, they generate two different sets of GPI1:GPI2 TfR combinations expressed from two different BESs (BES1 and BES7). Comparative analyses of these receptors form the bulk of the data.

The main findings are:

(1) Both GPI1 and GPI2 TfR can be found on the cell body/flagellar surface.

(2) Both are functional for Tf binding and uptake.

(3) GPI2 TfR is expressed at ~1.5x relative to GPI1 TfR

(4) Ultimate TfR expression level (protein) is dependent on the BES from which it is expressed.

Most of these results are quite reasonably explained in light of the hydrodynamic flow model of the Engstler lab and the GPI valence model of the Bangs lab. Additional experiments, again by rapid fixation, with HpHbR and FHR, show that these GPI1 receptors can also be seen on the cell surface, in contrast to published localizations.

It is quite interesting that the authors have identified a native GPI2 TfR. However, essentially all of the data with GPI2 TfR are confirmatory for the prior, more detailed studies of Tiengwe et al. (2017). That said, the suggestion that GPI2 was the ancestral state makes good evolutionary sense, and begs the question of why trypanosomes prefer GPI1 TfR in 14 of 15 ESs (i.e., what is the selection pressure?)

Strengths and weaknesses:

(1) BES7 TfR subunit genes (BES7_Tb427v10): There are actually three (in order 5'3'): E7gpi, E6.1 and E6.2. E6.1 and E6.2 have a single nucleotide difference. This raises the issue of coordinate expression. If overall levels of E6 (2 genes) are not down-regulated to match E7 (1 gene), this will result in a 2x excess of E6 subunits. The most likely fate of these is the formation of non-functional GPI2 homodimers on the cell surface, as shown in Tiengwe et al. (2017), which will contribute to the elevated TfR expression seen in BES7.

We would like to thank the reviewer for pointing out that there are two ESAG6 genes in BES7, we had relied on the publicly available annotation and should have known better.

For transferrin expression levels, see the discussion in response to reviewer 1 point 3 below

(2) Surface binding studies: This is the most puzzling aspect of the entire manuscript. That surface GPI2 TfR should be functional for Tf binding and uptake is not surprising, as this has already been shown by Tiengwe et al. (2017), but the methodology for this assay raises important questions. First, labeled Tf is added at 500 nM to live cells in complete media containing 2.5 uM unlabeled Tf - a 5x excess. It is difficult to see how significant binding of labeled TfR could occur in as little as 15 seconds under these conditions.

The k_on for transferrin is very rapid (BES1 TfR / bovine transferrin at pH7.4 = 4.5 x 10⁵ M^-1s^-1 (Trevor et al., 2019) and binding would occur to unoccupied receptors within 15 sec. The k_off is also fast (BES1 TfR / bovine transferrin at pH7.4 = 3.6 x 10^-2 s^-1 (Trevor et al., 2019) and there would be exchange of transferrin within the time taken for endocytosis. These values are in vitro with purified proteins, the in vivo values may be affected by the VSG coat.

The failure to bind canine transferrin (Supp. Figure 4B) acts as a control for specificity of the interaction.

We have now performed a competition experiment as an additional control; cells in culture were supplemented with: A, 0.5 µM labelled transferrin; B, 0.5 µM labelled and 2.5 µM unlabelled transferrin; C, 0.5 µM labelled and 5 µM unlabelled transferrin, fixed after 60 s and visualised by fluorescence microscopy (Figure S4C). There was effective competition and greatly reduced binding of transferrin was seen in the presence of a 10-fold excess of unlabelled. We would like to thank the reviewer for suggesting this experiment.

Second, Tiengwe et al. (2017) found that trypanosomes taken directly from culture could not bind labeled Tf in direct surface labelling experiments. To achieve binding, it was necessary to first culture cells in serum-free media for a sufficient time to allow new unligated TfR to be synthesized and transported to the surface. This result suggests that essentially all surface TfR is normally ligated and unavailable to the added probe.

As part of the preliminary experiments for this paper we found that centrifugation followed by resuspension in either complete or serum free (but 1% BSA) medium resulted in a reduction is total cellular TfR and determined by western blotting. We have now included this experiment (Figure S4D). The inference from this experiment is that centrifugation and subsequently incubation will have an effect on receptor detection and endocytosis rates for a discreet time period.

The amount of binding of labelled transferrin to cells in culture will depend on the specific activity of the labelled transferrin. This reasoning was behind the use of 0.5 µM labelled transferrin when roughly 1 in 6 molecules in the culture medium are labelled and there was only a small effect on the overall concentration of transferrin.

Third, the authors have themselves argued previously, based on binding affinities, that all surface-exposed TfR is likely ligated in a natural setting (DOI:10.1002/bies.202400053). Could the observed binding actually be non-specific due to the high levels of fixative used?

The absence of binding/uptake of canine transferrin argues against a non-specific interaction. In our previous publication, we did not pay enough attention to the on and off rates which allow for a degree of exchange and, here, TfR newly appearing on the cell surface has a 1 in 6 chance of binding a labelled transferrin.

(3) Variable TfR expression in different BESs: It appears that native TfR is expressed at higher levels from BES7 compared to BES1, and even more so when compared to BES3. This raises the possibility that the anti-TfR used in these experiments has differential reactivity with the three sets of TfRs. The authors discount this possibility due to the overall high sequence similarities of E6s and E7s from the various ESs. However, their own analyses show that the BES1, BES3, and BES7 TfRs are relatively distal to each other in the phylogenetic trees, and this Reviewer strongly suspects that the apparent difference in expression is due to differential reactivity with the anti-TfR used in this work. In the grand scheme, this is a minor issue that does not impact the other major conclusions concerning TfR localization and function, nor the behavior of HpHbR and FHR. However, the authors make very strong conclusions about the role of BESs in TfR expression levels, even claiming that it is the 'dominant determinant' (line 189).

This point is valid but exceptionally difficult to address at the protein level. As an orthogonal approach, we performed RNAseq analysis of the ‘wild type’ BES1, BES3, and BES7 cell lines to determine whether differences in receptor mRNA levels were consistent with the proposed difference in protein levels (Table S1). The analysis showed total ESAG6/7 mRNA levels to vary in a similar manner to the protein estimates with BES3 < BES1 < BES7 providing support for the differences in protein levels.

The strongest evidence for the expression site determining the TfR level is the comparison of the cell lines in which the VSG were exchanged. This had no effect on TfR levels and so there is no evidence that the identity of the VSG alters TfR expression.

(4) Surface immuno-localization of receptors: These experiments are compelling and useful to the field. To explain the difference with essentially all prior studies, the authors suggest that typical fixation procedures allow for clearance of receptor:ligand complexes by hydrodynamic flow due to extended manipulation prior to fixation (washing steps). Despite the fact that these protocols typically involve ice-cold physiological buffers that minimize membrane mobility, this is a reasonable possibility. Have the authors challenged their hypothesis by testing more typical protocols themselves? Other contributing factors that could play a role are the use of deconvolution, which tends to minimize weak signals, and also the fact that investigators tend to discount weak surface signals as background relative to stronger internal signals.

We have added preliminary experiments that compared fixation protocols in two parts. First the effect on TfR levels of washing and resuspending cells discussed above (Figure S4D), and second how different fixation protocols alter apparent TfR immunolocalisation (Supp Figure S5A-B). The comparison shows that both the absence of glutaraldeyde and the use of washing alters the outcome.

(5) Shedding: A central aspect of the GPI valence model (Schwartz et al., 2005, Tiengwe et al., 2017) is that GPI1 reporters that reach the cell body surface are shed into the media because a single dimyristoylglycerol-containing GPI anchor does not stably associate with biological membranes. As the authors point out, this is a major factor contributing to higher steady-state levels of cell-associated GPI2 TfR relative to GPI1 TfR. Those studies also found that the size/complexity of the attached protein correlated inversely with shedding, suggesting exit from the flagellar pocket as a restricting factor in cell body surface localization. The amount of newly synthesized TfR shed into the media was ~5%, indicating that very little actually exits the FP to the outer surface. In this regard, is it possible to know the overall ratio of cell surface:FP:endosomal localized receptors? Could these data not be 'harvested' from the 3D structural illumination imaging?

A ratio could be determined but we did not do this as it would only be valid if the antibody has equal access to the internal TfR in a diluted VSG environment and the external VSG embedded in a densely packed and cross-linked VSG layer As such, we would have no confidence in the accuracy of any estimate.

Reviewer #2 (Public review):

The work has significant implications for understanding immune evasion and nutrient uptake mechanisms in trypanosomes.

While the experimental rigor is commendable, revisions are needed to clarify methodological limitations and to broaden the discussion of functional consequences.

The authors argue that prior studies missed surface-localized TfR due to harsh washing/fixation (e.g., methanol). While this is plausible, additional evidence would strengthen the claim.

Preliminary experiments that compared fixation protocols are now included to show that method affects outcome.

It remains unclear how centrifugation steps of various lengths (as in previous publications) can equally and quantitatively redistribute TfR into the flagellar pocket. If this were the case, it should be straightforward for the authors to test this experimentally.

Not aware of previous studies that demonstrate equal and quantitative redistribution to the flagellar pocket. In previous reports, there is variation in cell surface/flagellar pocket localisation depending on expression levels, for example (Mussmann et al., 2003) (Mussmann et al., 2004), it’s worth noting that the increase in TfR expression in these papers is similar to the difference in the cell lines used here. In addition, most report the presence of TfR in endosomal compartments. In the experiments here, there are cells where the majority of signal from labelled transferrin is present in the flagellar pocket and the argument is that this is a stage of a continuous process in which the receptor picks up a transferrin on the cell surface and is swept towards the pocket.

If TfR is distributed over the cell surface, live-cell imaging with fluorescent transferrin should be performed as a control. Modern detection limits now reach the singlemolecule level, and transient immobilization of live trypanosomes has been established, which would exclude hydrodynamic surface clearance as a confounding factor.

This is non-trivial and is a longer-term aim. The immobilisation involves significant manipulation of the cells prior to restraining.

In most images, TfR is not evenly distributed on the surface but rather appears punctate. Could this reflect localization to membrane domains? Immuno-EM with high-pressure frozen parasites could resolve this question and is relatively straightforward.

There is a non-uniform appearance in the super-resolution images for both TfR and FHR. We cannot distinguish whether this represents random variation in receptor density over the cell surface or results from a biological phenomenon. Whatever the cause, the experiments showed unambiguous cell surface localisation.

The authors might consider discussing whether differences in parasite life cycle stages (procyclic versus bloodstream forms) or culture conditions (e.g., cell density) affect localization. The developmentally regulated retention of GPI-anchored procyclin in the flagellar pocket might be worth mentioning.

The aim of this paper was to determine the localisation of receptors in proliferating bloodstream form trypanosomes in culture. TfR and HpHbR are not expressed in insect stages in culture. FHR is expressed in insect stages and is present all over the cell surface (Macleod et al., 2020). A procyclin-based reporter was distributed over the whole cell surface in one report (Schwartz et al. 2005). In other reports, the retention of procyclin in the flagellar pocket of proliferating bloodstream forms is probably dependent on structure/sequence as other single GPI-anchored proteins, such as FHR (Macleod et al., 2020) and GPI-anchored sfGFP (Martos-Esteban et al., 2022) can access the surface.

References:

MacGregor, P., Gonzalez-Munoz, A. L., Jobe, F., Taylor, M. C., Rust, S., Sandercock, A. M., Macleod, O. J. S., Van Bocxlaer, K., Francisco, A. F., D’Hooge, F., Tiberghien, A., Barry, C. S., Howard, P., Higgins, M. K., Vaughan, T. J., Minter, R., & Carrington, M. (2019). A single dose of antibody-drug conjugate cures a stage 1 model of African trypanosomiasis. PLoS Neglected Tropical Diseases, 13(5), e0007373. https://doi.org/10.1371/journal.pntd.0007373

Macleod, O. J. S., Bart, J.-M., MacGregor, P., Peacock, L., Savill, N. J., Hester, S., Ravel, S., Sunter, J. D., Trevor, C., Rust, S., Vaughan, T. J., Minter, R., Mohammed, S., Gibson, W., Taylor, M. C., Higgins, M. K., & Carrington, M. (2020). A receptor for the complement regulator factor H increases transmission of trypanosomes to tsetse flies. Nature Communications, 11(1), 1326. https://doi.org/10.1038/s41467-020-15125-y

Martos-Esteban, A., Macleod, O. J. S., Maudlin, I., Kalogeropoulos, K., Jürgensen, J. A., Carrington, M., & Laustsen, A. H. (2022). Black-necked spitting cobra (Naja nigricollis) phospholipases A2 may cause Trypanosoma brucei death by blocking endocytosis through the flagellar pocket. Scientific Reports, 12(1), 6394. https://doi.org/10.1038/s41598-02210091-5

Mussmann, R., Engstler, M., Gerrits, H., Kieft, R., Toaldo, C. B., Onderwater, J., Koerten, H., van Luenen, H. G. A. M., & Borst, P. (2004). Factors affecting the level and localization of the transferrin receptor in Trypanosoma brucei. The Journal of Biological Chemistry, 279(39), 40690–40698. https://doi.org/10.1074/jbc.M404697200

Mussmann, R., Janssen, H., Calafat, J., Engstler, M., Ansorge, I., Clayton, C., & Borst, P. (2003). The expression level determines the surface distribution of the transferrin receptor in Trypanosoma brucei. Molecular Microbiology, 47(1), 23–35. https://doi.org/10.1046/j.13652958.2003.03245.x

Schwartz, K. J., Peck, R. F., Tazeh, N. N., & Bangs, J. D. (2005). GPI valence and the fate of secretory membrane proteins in African trypanosomes. Journal of Cell Science, 118(Pt 23), 5499–5511. https://doi.org/10.1242/jcs.02667

Trevor, C. E., Gonzalez-Munoz, A. L., Macleod, O. J. S., Woodcock, P. G., Rust, S., Vaughan, T. J., Garman, E. F., Minter, R., Carrington, M., & Higgins, M. K. (2019). Structure of the trypanosome transferrin receptor reveals mechanisms of ligand recognition and immune evasion. Nature Microbiology, 4(12), 2074–2081. https://doi.org/10.1038/s41564-019-0589-0

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Major Recommendations:

(1) 2 E6 gene in BES7s: This does not affect the overall conclusions, but the text should be modified to reflect the existence of the second gene, and to discuss the ramifications.

This has been corrected

(2) Surface binding studies: To clarify this issue, two experimental approaches are strongly recommended. First: additional excess unlabelled Tf should be added. If binding is truly receptor-mediated, it must by definition be saturable at some experimentally achievable level. Second: TfR expression should be abrogated by RNAi silencing to show that binding is TfR-dependent. Without some validation of specific binding by one or both of these approaches, these counter-intuitive results must be questioned.

The excess unlabelled transferrin experiment is now included (we would like to thank the reviewer for this suggestion). The absence of binding of canine transferrin provides strong evidence for the specificity.

(3) Variable TfR expression in different BESs: To make such claims, quantitative RTPCR should be performed with conserved primers to assess the actual relative expression at the transcriptional level. Absent this, the claims should be eliminated, or at the very least greatly tempered.

This has been done using an RNAseq analysis.

(4) Surface immuno-localization of receptors: An example of discounting weak signals as background can be seen in Figure 8 of Duncan et al. (2024). It has also been shown that at least one other GPI1 reporter (procyclin) is readily detected on the outer cell surface under ectopic expression in BSF trypanosomes (Schwartz et al., 2005) using typical fixation procedures. This could be cited, and the authors could discuss the fact that procyclin is not a receptor and may not be susceptible to hydrodynamic drag.

Yes

Minor issues:

(1) Fully appreciating the data presented requires an understanding of the hydrodynamic flow and GPI valence models of the Engstler and Bangs labs, respectively. For the uninitiated,d it might perhaps be useful to include brief summaries of each in the Introduction.

Added to the introduction

(2) Lines 110-112: ISG65 and ISG75 both have strong localizations in endosomal compartments. This should be noted with citation of any of the work from the Field lab.

Added

(3) Lines 121-132: This passage presents the role of GPI anchors (1 vs 2) in a rather digital manner (in or out). Schwartz et al (2005) present a much more nuanced view of what is likely taking place. This is one reason summaries of hydrodynamic flow and GPI valence would be helpful.

Modified

(4) Lines 182-184: The increased size of GPI-anchored E7 is in part due to the presence of the GPI itself, as the authors state, but there are also 24 additional amino acid residues in this protein that contribute.

Modified

(5) Lines 212-214: Do p>0.95 and p>0.99 indicate statistical significance? This must be a typo.

Thank you, corrected

(6) Lines 218-219: The better references documenting GPI number in regard to turnover/shedding are Schwartz et al. 2005 and Tiengwe et al. 2017.

Changed

(7) Line 241 and Figures 3, 4, and 6: The transverse sections add little to the presentation. That there is signal variation in all dimensions is readily apparent from the images themselves, and similar profiles would be obtained regardless of the transect. Was there some process/rationale in the selection of the individual transects intended to make a broader point? If so, a description of the process should be provided.

The point was to show that the signal had a pattern consistent with plasma membrane (two distal peaks) as opposed to cytoplasm (single central peak). As such, we think it is important.

(8) Lines 582-596: Methodology for quantitation of cellular fluorescent signals should be provided.

Has been expanded

Reviewer #2 (Recommendations for the authors):

(1) As a less critical but still useful control, antibody accessibility assays on live versus fixed parasites could test whether VSG coats limit detection.

This could only be quantified by using a range of monoclonal antibodies which are not available.

(2) The rapid transferrin uptake (15-60 seconds) could reflect fast endocytic recycling rather than stable surface residency. A pulse-chase experiment tracking receptor movement would clarify this (though I acknowledge that this is technically challenging).

We agree that endocytic recycling is probably the main source of unoccupied TfR on the cell surface. It is hard to see how the pulse chase experiment could be performed without centrifugation which will affect the outcome – see above.

(3) Statistical and quantitative reporting

Added as Table S2- S4

(4) Report confidence intervals (e.g., for fluorescence intensity comparisons in Figure 3B) to contextualize claims of "no significant difference."

We do not claim ‘no significant difference’ and the SD overlap due to a high level of variation in the population

(5) Specify the number of biological replicates and cells analyzed per condition in the figure legends.

Added

(6) The study notes that surface-exposed receptors avoid antibody detection, but does not explore how.

We don’t claim that receptors avoid detection and have published evidence to the contrary. The cell has evolved mechanisms to reduce/minimise the effect of antibody binding.

(7) Comparing antibody binding to TfR in VSG221 versus VSG224 coats.

This is already present in Figure 3D

(8) Testing whether receptor shedding or conformational masking contributes to immune evasion.

A lifetime’s work

(9) Evolutionary trade-offs: Discuss why T. brucei maintains ~15 TfR variants if the GPI-anchor number has minimal impact on function (Figure 3).

The possible reason for the evolution of ~15 TfR variants was discussed in a previous publication.

(10) How do their findings align with recent studies on ISG75 surface exposure?

If this refers to the finding that ISG75 is an Ig Fc receptor, this has been included

(11) Add scale bars to 3D reconstructions (Figure 5).

Added

(12) Include a schematic summarizing key findings in the main text.

Chosen not to do

(13) Explicitly state where raw microscopy images, flow cytometry data, and analysis scripts are deposited.

Microscope Images have deposited in Bioimage Archive repository at EMBL/EBI No flow cytometry used

(14) Correct inconsistent GPI-anchor terminology (e.g., "glycosylphosphoinositol" to "glycosylphosphatidylinositol").

Our typo, corrected

(15) Clarify ambiguous phrases (e.g., "subtle mechanisms" in the Discussion).

Corrected

policy implication, restates central metaphor being bridge.

challeneges common belief

reframes controversial concept

supports integration htesis

This article is part of the special JITC series: SITC 40th Anniversary: I-O Progress and Potential

This article is part of the special JITC series: Myeloid Cells in Immuno-Oncology

Coming soon to this special series: Leading experts provide their perspectives and answer provocative questions surrounding myeloid cells. Stay tuned!