The website highlights how important it is, for us to understand accessibility. This lets us understand the various disabilities and what is the best and needed approach to help ensure they have the best experience.

Users can easily navigate between sections of the website thanks to its well structured and understandable links. Users can easily recognize clickable parts because the links are easily distinguished from regular text by their distinct color and underlining. This design decision improves accessibility by guaranteeing that visitors can easily and clearly discern between links and ordinary content.

This summary gives information that is valuable, along with different content that is available. The video also provides accessibiltiy to various users from all backgrounds who are deaf or have difficulty hearing. It provides very clear and needed information of the principles of W3C as well as the services provided. Not to mention the captions that allow for better engagment with those whos first language may not be english.

It seems difficult to give annotation a specific definition. It should be adapted to each, and adapted depending on the results / the way they were received.

Annatation are worthless if they are made by obligation and not to give interactive and worthful feedbacks.

Annotation may not work depending on the profile of the one receiving it.

Feedback are only working when the one receiving it actually cares and try to improve.

Feedback purpose

Annotation gives specific feedback when the general feedback doesn't help that much the student to improve.

Feedback is here to engage not just to say if its true or wrong. It is a conversation, an interaction.

Writing some annotation can should allow the reader to interact and understand more the reading.

How an annotation should be in order to give the best feedbacks and help.

feedbacks and annotations are powerful tools for growth, development and understandings.

learning is by trying then getting feedbacks to understand what is the next step. Figuring out yourself the conclusion of an action can be really difficult and take long. That is why feedbacks are important for students.

Annotation is a powerful feedback for students to improve. It allows them to have a feedback exactly where it's needed and on the context for better understandings.

Annotating always existed since medieval writing but at some points depending on the technologies it was less or more used. Nowadays with the online technology, it is really easy to annotate and moreover to share them instantly.

Annotating is more common than we think it is, especially in the area of school especially between students and teachers.

"Audioeye.com", implies that the website titled "Scope.org.uk" is digitally accessible as the colour contrast is an aid for the visually impaired. Although less bright without the yellow that the "Scope" website features, "Audioeye.com" features a bright purple colour, which could aid with visual accessibility. Citation : Rank, Sojin. "10 Accessible and ADA-Compliant Website Examples". Web. 7 August 2024. Accessed 10 Sept. 2025. https://www.audioeye.com/post/accessible-website-design-examples/ Scope. Home | Disability charity Scope UK, Scope, 2025. Web. Accessed 10 Sept. 2025. https://www.scope.org.uk/

To navigate "Audioeye.com"'s lengthy article, there is a directory that sits under the article title that clearly states the three main points of the article, which, when clicked, brings you directly to the sectioned paragraphs. This quick directory function is a wide range accessibility aid. Citation : Rank, Sojin. "10 Accessible and ADA-Compliant Website Examples". Web. 7 August 2024. Accessed 10 Sept. 2025. https://www.audioeye.com/post/accessible-website-design-examples/

Full statistics can be cited separately if called upon.

Your process is going to develop from your own experiences, good and bad, and your work will reflect if your process still needs work.

Evaluation paragraphs are likely to be the conclusion to summary, analysis, and synthesis paragraphs.

Many children do not know how to interact or "play" anymore. Imagination seems to be diminishing, and even the simplest things, such as a lunch conversation, are too much now.

A very important point to the article.

Yes! I do not want my children on social media for as long as I possibly can. However, many coaches and extra curriculars use apps and social media to communicate. There is then minimal choices in keeping them away from it.

Many were filled with anxiety and struggled to adapt back to the "new" normal.

To this day, it is still crazy to see the increased problems within schools following COVID.

Although Apple has innovative visuals for their visitors to get a real time experience while using their webpage. Their lack of audible features may hinder some of their users who may need access to listen to them. There are no integrated text-to-speech or read aloud options, so users with difficulty reading or visual/hearing impairments would struggle to access content effectively.

Apple incorporates large headings providing a smooth reading experience for visitors on their webpage to identify the products they are looking for. This practice ensures that those with visual impairments, or are not familiar with technology have a similar experience of those who are more tech savvy.

Group 1 has no summary posted

Group 2: Gorbachev’s Non-Violent Revolution. Key points: Perestroika was about empowering the people and breaking away from totalitarian power. Gorbachev had a very humanistic approach that was against violence His attitude towards women was of true respect There was a lack of a clear plan from Gorbachev in the beginning

Conclusions: How and why was he able to gain power under communism? Gorbachev’s goal was to reform the system in which the people had continuously been oppressed by, and his reform was called Perestroika. His upbringing was full of hardships and famine, making him more understanding towards the people in his policies and shaping the way he took on his non-violent revolution and New Thinking.

Group 4 has no summary posted

Group 5: Linking Gorbachev’s Domestic and Foreign Policies Key points: The Soviet Union has a neutral position regarding the conflict between ex-Union countries. Gorbachev’s policies are seen as “ambitious” by the author of the article There was an anti-alcohol campaign that failed Gorbachev’s international goals are focused on improving relations with countries like the US, UK and Israel, which was unusual in Russian policies.

Conclusions: Gorbachev’s domestic reforms are linked to his foreign policy actions during this crucial time in Soviet history. The ongoing tension between Armenia and Azerbaijan, both of which used to be part of the Union, bring the Soviet Union at a neutral state, and shifting its attention towards foreign relations, driven by the realization that China was no longer a dependable ally, so there was a need to form new allays and have stability.

A good cognitive practice Apple follows design principles that make information easier to understand for people with diverse cognitive needs. For example, it uses a top navigation bar, a search tab and a cart tab in consistent locations. Apple also utilizes white space so it's easier for users to focus on one idea at a time. The text is usually short and direct, avoiding long, wordy sentences that users struggle to understand. For example, “Thinnest Iphone ever.”

A good cognitive practice Apple follows design principles that make information easier to understand for people with diverse cognitive needs. For example, it uses a top navigation bar, a search tab and a cart tab in consistent locations. Apple also utilizes white space so it's easier for users to focus on one idea at a time. The text is usually short and direct, avoiding long, wordy sentences that users struggle to understand. For example, “Thinnest Iphone ever.”

I feel that dismissing a teacher for striking is unfair because the majority of these strikes are for improving their pay, working conditions, or benefits. There have been cases where the teachers are striking for the rights or interests of their students.

this text highlighted that the main purpose of education is not just academics but also preparing our students to be the best versions of them they can be to want to make a respectful and compassionate society. Teachers can allow children to gain curiosity, as well as allow them to know that knowledge alone isn't enough but understanding what theyre taking in is important too. Educators can guide students in many ways like in getting meaningful goals not just academics ones.

I agree with this statement because all children should have equal opportunities and feel included regardless of their background, race or socioeconomic status.

I appreciate this because no students should have to feel left out or change their appearances due to their beliefs, unless it's something inappropriate/offensive. Some schools are more diverse than other's and it's nice when the different cultures are noticed and appreciated by the school.

Although I had always experienced this situation, I had never thought about this question and if we look closely, in reality my teacher wasn't equal to all of us because when she called us in-front of the class to answer the question we no longer had the option if whether we wanted to o not.

I think that it is so important that teachers do everyday these four things because they play a big role in the success of all students.

I know here at CSUMB, as a Liberal Studies major, they have us take a government class about how they play a role in public education and how they influence public education.

This is super important to recognize. The education system should be fair and equal for all students, however sometimes the system tends to fail us. As educators we should push for equinity.

I appreciate how this educator takes what he's assigning to the students into consideration. It's important for educators to put things out that are beneficial for their students or that students may have a take away.

Literally a lightbulb came to me when I read this paragraph. In Middle School and High School we would have our student handbooks/planners which we always had to carry and annotate to see what we are learning through out the year. Before we started the class we would read the objective of the course that we were learning. Also, why it was important.

I do remember that my teachers would always have the state guidelines that we need to follow and we would always talk about any new changes or continuing with the old guidelines.

I feel that teachers should be able to wear what they want to wear as long as they're covered or aren't wearing super-tight attire. If students are allowed to express themselves, then they should have the same right. I believe the only time this could be challenged is if you're working at a private school where they do have a dress code that you need to follow.

I believe this is the core essentials of reporting. It's more true when it comes to data journalism.

important

important

Bill's talking points revolved around highlighting government spending and he spent a majority of his time speaking on it, but his tone almost seemed mocking, while Jon's speech highlights were towards more common societal issues, but a majority of his speech was catered towards getting laughs and reactions from the audience.

The tone you convey to your audience should serve to create a connection between the audience and you.

Your tone must reflect your own interest in your topic and keep the audience engaged.

Know what type of audience you are writing for and what the best technique to dispense that writing to them would be.

Emotions through texts.

Haraway wants us to look beyond the traditional view of the Anthropocene as "humans changed the planet" in "Anthropocene, Capitalocene, Plantationocene, Chthulucene: Making Kin." The larger problems of capitalism, imperialism, and plantation systems that first drove environmental damage can be overlooked by this perspective, she says. Haraway proposes new concepts, such as Capitalocene and Plantationocene, to tell us that climate change and environmental damage are related to specific histories of exploitation. Additionally, she presents "Chthulucene" as a means of visualizing a future based on care, connection, and "making kin" with other species. She challenges us to consider accountability and creating better livable environments together rather than giving up.

This quote shows how Haraway frames the Anthropocene as a turning point rather than just a time period. The idea that we can work to minimize the impact of the time period on the climate to create a future full of refuge is very hopeful. It shows how there is a possibility of repairing the world even after the damage has been done.

I like how Haraway uses “The Dithering” to describe humanity’s indecision in responding to the climate crisis. It shows the confusion many people face when they know they must do something to fix the issue, but dont know what to do. This in-turn causes people to feel powerless to make a different to the climate crisis. He contacts this with Sam-chthonics who take action to make change by participating in acts like recycling. This shows that truly caring for the climate means actively participating in the change.

This quote resonates because it connects the climate collapse to the loss of safe places of refuge for both humans and nonhumans. In this quote, Haraway shows how mourning irreversible climate destruction can cause the rebuilding of areas where life can grow and change. It shows that loss is unavoidable, but also shows how the mourning can be a starting point for creating new refuges.

Haraway introduces a slogan she came up with “Make Kin Not Babies!” to establish her vision in terms of Chthulucene. She emphasizes the urgency of reimagining human relations beyond reproduction and genealogical ties. More so the part of “Make Kin” is to deliver the idea of taking responsibility for the life that we affect around us and to form a deeper connection with species other than ourselves. Haraway's purpose for this slogan is to project more awareness of the Anthropocene we currently live in and Chthulucene.

In this singular sentence, Haraway highlights the destruction of refugia as the “Inflection point” marking the Anthropocene. Building on Anna Tsing’s argument, she emphasizes that without places of refuge for recovery, all life on earth including us humans are living in a displacement and uncertainty of survival in the future. This point broadens the scope of the systemic crisis outlined by Haraway earlier, demonstrating that the Anthropocene is not only characterized by the deterioration of our ecosystem but also by the eradication of the very spaces that would prevent the mass extinction of life.

Haraway begins with a strong compelling opening that frames the argument she is trying to make. She proposes the question of the "inflection point” that changes the “game of life on earth”; she immediately emphasizes the gravity of the ecological and systemic crisis. She expands beyond the understanding of climate change to include toxic chemistry, mining, water depletion, ecosystem simplification, and mass extinctions to establish the interconnect and recursive nature of the collapse of our planet.

Ties to confucianism?

what is the punishment for illegal reproduction?

when did this law become an actuality?

That's interesting, her parents, unlike most (or the expectations) are worried more for her future than about what society will have to say

Might be a great Segway into marriage market!

when a mother would give birth to a daughter and they would end that infants life because it was a girl seems so cruel. Part of me wonders if families would do this in fear of what would become of their daughters and their daughters future, but also, I feel as if families, especially women were shamed if they gave birth to girls. The women I'm sure felt so much shame not birthing males. And the mothers who did choose to keep their daughters, the things they did to hopefully ensure that their daughters were not a shame to their families is a lot. If only it wasn't all men that ruled the Chinese society, then maybe women would have a little value to the Chinese society.

I believe that this is why the Chinese became so successful and powerful in time. They have maintained their power for so long and by doing so they in the way that they have done this is so smart. People don't so much come into power just because they were born with it, it has to be earned and to earn it is to prove that they belong. They are well educated people who possibly came from nothing at one point but because they are educated and put in the work to their education they moved up in class and in position.

Smith's vision on government.

Smith saw collusion as the main market failure.

Smith's vision on self-interest.

Productivity and the scale of the market.

The invisible hand metaphor.

I think that use of VR in this way would be very interesting in a game format but I think that bringing things like VR into such serious topics as politics could get messy. Politics are already such a controversial topic that adding a "what could've been" scenario could be harmful.

Its so crazy to me how much VR can and will change the world. I think that its really cool to use as a fun game or activity but I do not think that it should be incorporated into everyday life. I feel as though its going to make the world into such a fake environment and ruin true socialness and connection.

I have a hard time agreeing with this statement. I feel that the idea of calling "Virtual realities genuine realities" almost disproves itself. If what we see and do in VR is real, then how real is what we do outside of VR, in real life? You can die in VR a million times and nothing will happen to the person playing. I feel like the word "virtual reality" perfectly describes what it is, as there are huge advancements making the games feel very real, but in all they are just virtual.

I think the question of if we are in a simulation is pretty interesting to think about. I believe the question if we will ever know is also interesting. My mind always comes back to the question of how would we know? What if simulations have become so advanced that it would be impossible to detect, or ever figure out. My mind also normally goes to the thought that if we are in fact inside of a virtual simulated world, how ever we got here probably also made sure that we wouldn't be able to ever figure it out.

I think this is a really nice thought; while he was Sushila, no time passed in the reality of Narada. However, I’m inclined to believe that all objective realities are running at the same time, like when you spend time in a virtual reality, the time still passes in real life. On the other hand, I thought that maybe different realities can have different speeds, like the time passes much more slowly in Narada’s reality than in Sushila’s.

This concept is easier to grasp for video games, like Animal Crossing, where it’s synced to real time. The “villagers” have thoughts, feelings, and words to say while I’m playing, but do they still have thoughts while I’m gone? I say yes, because they exist in a reality, even though it’s created by humans and is simple compared to our reality. For movies, the characters are actors and are only replicating emotions, but to me, it has its own reality because I’m observing it. It’s a reality apart from mine that exists because I interpreted it.

I disagree with this thesis. Some people might use virtual reality to escape from what is happening in their real world life, but that doesn't mean it all just goes away. At some point when you stop using VR, you have to come back to the reality of your life. You may lead a fully meaningful life in the virtual world but it all comes back to that being fake and used to deceive your mind into thinking its real. In virtual reality, you aren't fully taking care of yourself mentally and physically so there isn't really an satisfaction you could get from that. Yes, depending on your choices in the reality world, you could get everything you wanted and still have connections emotionally, but you can't touch, smell or feel anything which is partly something I believe makes your life have meaning because it is catered to you as a human.

I remember when Pokemon Go first came out, it became a part of everyone's routine to find them and they became sort of to immersid in the game because it had a tie in through the real world and through a simulation. It had some backlash because players were to focused on their when walking to find these pokemon, they would be lured into dangerous areas, be injured from vehicles, falling, etc. Much like VR, it was fun because our in your own reality while playing because it feels real to the players.

I think it is interesting how much VR has developed through the years and how realistic it is becoming to the point you have to wonder if you can experience everything the world has to offer through the simulation of VR what then is the point of going outside and doing the stuff yourself vs staying at home with you VR and not having to face any of world consequences (finical loss, prison, loss of life, emotional distress, etc.). For comfort reason many people might choice VR compared to real world experience but then how does that effect ones mental and physical health if you aren't going outside to go view the real world?

I don't completely agree with this thesis. Sure, when the goggles are put on you're tricking your brain into thinking what you are seeing is real. This is not necessarily true. What happens in the virtual world does not coincide with what happens in the world outside of the goggles. If we started to adopt this idea that the VR world is the real world then we would begin to neglect aspects of life and to an extent even ourselves and our health.

remove "FYI" as this was covered previously in the FMR block.

Bart (the apostrophe in the older Bart's is being dropped)

HbS (this is the standard naming convention)

Streptococcus (to be consistent with naming of all the pathogens listed; also the names should be italicized)

Combination Hemoglobinopathies (remove the word Thalassemia from this title)

Means taking pleasure not just in the story, but in how it’s put together.

TV used to be about flow = continuous broadcast schedule. With DVDs, TV became more like publishing

Shows how TV has shifted from broadcast schedules to collectible box sets. TV series are now organized like books: alphabetically, for study and easy access.

I appreciate the clear policy when it comes to attendance. Some professors aren't straight forward when it comes to leaving class early.

I think that responding in a helpful manner is crucial for success. Getting feedback from several perspectives can only help in the long run. I like how the word helpful in used here because we are all here to do our best not just for ourselves but for others too.

This is a very good point. The issue is not AI itself, but rather the lack of laws and regulations surrounding its use. Currently, there are few protections in place to safeguard workers or hold tech companies accountable for how they implement AI. Without clear policies and oversight, the concerns about job displacement, unfair labor practices, and the misuse of AI are very real and justified. Establishing legal frameworks and ethical guidelines will be essential to ensuring that AI develops in a way that benefits workers rather than harms them.

This is a fair point. It’s not that AI is necessarily causing consultants to lose their jobs consultants are still needed because they bring a level of creativity that AI currently lacks. However, since AI is such a new and rapidly developing field, consultants don’t necessarily have more experience implementing it than the employees already working within companies.

Clear and bold font is used which is beneficial to those with vision problems or for those who's first language doesn't use the English alphabet. There is a high contrast between the white and blue used throughout the page, making it easier on the eyes. When looking at Google's inspect feature, we can see that alt text is provided for all pictures. I feel like this homepage does suffer from a lack of organization. The page could benefit from having a drop down menu, instead of having some subheadings on the main page and others on a bar at the top of the screen.

I remember last class talking about the mending and translation of the outside view of dance versus the internal view of dance? How much responsibility does a person have to educate another person? How does intersectionality play a role in the discussion of dance?

Helps determine the deeper meanings of maps and their intents.

This half of the paragraphs helps me understand the complexities of visions and versions of lands.

This sentence makes me feel connected a bit; I agree and understand what the author may feel here

I never knew the history of Ethnic Studies or what motivated the people to develop it. Now after reading it I am inspired.

This is a good indication that as an individual ages these focused areas pop up the most. As a nurse we know that receiving older clients we will most likely see one of these 9 conditions, and must know interventions for correct care.

the strike was a success!!

This passage is very important because it shows how no matter what ethnicity, background, cultural, etc. when we come together, we can make huge movements with a positive effect.

1/6 Capital attack

By this Pickard highlights the human labor support of AI as it keeps low wages hidden, this hides the workers that power the system, they go without credit.

this is something we touched on in class

the urgency of ai development also reminds me of the space race

I've witnessed this being done in the classroom. It really does work more than you would think. Students get nervous if you move closer towards them and if you linger, they may redirect their focus back towards you.

I'm not sure that I agree with that statement. Even as an adult, I enjoy hearing that my efforts are recognized and it does help push me to keep being successful. Granted, it may not work for everyone, but I have witnessed it working in several cases and to varying degrees (including teenagers and adults in the workforce). I can't tell you how many times I've heard the saying "It's nice to just feel appreciated".

Sometimes there were teachers that I had growing up, you could just tell they came to school in a bad mood. This is obviously something we should avoid. I read an article once about exercising before school to get in a better mood. However, I don't want to come to school all gross. Does anyone have any ideas how to maintain a good work/life balance so as not to let the stresses of the day affect the home life, or vice versa?

I've learned that this is crucial for teenagers. I coach a competitive softball team and the girls on the team tend to shut down if you are correcting them too much. It's a balance, truly. I feel like highlighting what someone does right helps to soften the correction. Regardless of the correct answer or way to do something, people want their efforts to be acknowledged.

Another aspect of the site that is done well (in terms of accessibility) is that the website is robust and supported on multiple different screen sizes and devices, from a phone to a different size monitor (vertical or horizontal) the website adapts and ends up being an example of robust web design looking through an accessibility lens.

The homepage is mostly keyboard accessible, although tabbing through all the options works until you get to the textbox below this text which then starts to scroll through all the different groups in their system. This is an example of bad web design taking accessibility in mind.

The image next to this textbox has alt text for the image which is a positive use of web accessibility. It reads "A confident woman with curly black hair wearing a pink blazer stands and gestures toward a floating text box. The text box reads bundle and save and features a positive Trustpilot rating."

On the other hand

eLife Assessment

This important study significantly advances our understanding of the skeleton of cartilaginous fishes by using a range of state of the art and complementary approaches to compare the skeleton amongst three cartilagenous fishes (catshark, little skate and ratfish). The evidence presented is compelling and likely to impact several fields of study.

Reviewer #2 (Public review):

General comment:

This is a very valuable and unique comparative study. An excellent combination of scanning and histological data from three different species is presented. Obtaining the material for such a comparative study is never trivial. The study presents new data and thus provides the basis for an in-depth discussion about chondrichthyan mineralised skeletal tissues.

Comments on previous revisions:

The manuscript has been revised and improved and can be published. A very nice manuscript, indeed. My only recommendation (point of discussion, not a requirement) would still be to think about the claim of paedomorphosis in a holocephalan.

Within the chondrichthyes, how distant holocephali are in relation to elasmobranchii remains uncertain, holocephali are quite a specialised group. Holocephali are also older than Batoidea and Selachii. As paedomorphosis is a derived character, I imagine it is difficult to establish that development in an extant holocephalan is derived compared to development in elasmobranchii. If this type of development would have been typical for the "older" holocephali it would not be paedomorphic. Also, the uncertainty how distant holocephali are from elasmobranchii makes it difficult to identify paedomorphosis with reference to chondrichthyes.

[Editors note: the authors have made further revisions in response to the previous reviews.]

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public Review):

Summary:

It seems as if the main point of the paper is about the new data related to rat fish although your title is describing it as extant cartilaginous fishes and you bounce around between the little skate and ratfish. So here's an opportunity for you to adjust the title to emphasize ratfish is given the fact that leader you describe how this is your significant new data contribution. Either way, the organization of the paper can be adjusted so that the reader can follow along the same order for all sections so that it's very clear for comparative purposes of new data and what they mean. My opinion is that I want to read, for each subheading in the results, about the the ratfish first because this is your most interesting novel data. Then I want to know any confirmation about morphology in little skate. And then I want to know about any gaps you fill with the cat shark. (It is ok if you keep the order of "skate, ratfish, then shark, but I think it undersells the new data).

The main points of the paper are 1) to define terms for chondrichthyan skeletal features in order to unify research questions in the field, and 2) add novel data on how these features might be distributed among chondrichthyan clades. However, we agree with the reviewer that many readers might be more interested in the ratfish data, so we have adjusted the order of presentation to emphasize ratfish throughout the manuscript.

Strengths:

The imagery and new data availability for ratfish are valuable and may help to determine new phylogenetically informative characters for understanding the evolution of cartilaginous fishes. You also allude to the fossil record.

Thank you for the nice feedback.

Opportunities:

I am concerned about the statement of ratfish paedomorphism because stage 32 and 33 were not statistically significantly different from one another (figure and prior sentences). So, these ratfish TMDs overlap the range of both 32 and 33. I think you need more specimens and stages to state this definitely based on TMD. What else leads you to think these are paedomorphic? Right now they are different, but it's unclear why. You need more outgroups.

Sorry, but we had reported that the TMD of centra from little skate did significantly increase between stage 32 and 33. Supporting our argument that ratfish had features of little skate embryos, TMD of adult ratfish centra was significantly lower than TMD of adult skate centra (Fig1).  Also, it was significantly higher than stage 33 skate centra, but it was statistically indistinguishable from that of stage 33 and juvenile stages of skate centra.  While we do agree that more samples from these and additional groups would bolster these data, we feel they are sufficiently powered to support our conclusions for this current paper.

Your headings for the results subsection and figures are nice snapshots of your interpretations of the results and I think they would be better repurposed in your abstract, which needs more depth.

We have included more data summarized in results sub-heading in the abstract as suggested (lines 32-37).

Historical literature is more abundant than what you've listed. Your first sentence describes a long fascination and only goes back to 1990. But there are authors that have had this fascination for centuries and so I think you'll benefit from looking back. Especially because several of them have looked into histology and development of these fishes.

I agree that in the past 15 years or so a lot more work has been done because it can be done using newer technologies and I don't think your list is exhaustive. You need to expand this list and history which will help with your ultimate comparative analysis without you needed to sample too many new data yourself.

We have added additional recent and older references: Kölliker, 1860; Daniel, 1934; Wurmbach, 1932; Liem, 2001; Arratia et al., 2001.

I'd like to see modifications to figure 7 so that you can add more continuity between the characters, illustrated in figure 7 and the body of the text.

We address a similar comment from this reviewer in more detail below, hoping that any concerns about continuity have been addressed with inclusion of a summary of proposed characters in a new Table 1, re-writing of the Discussion, and modified Fig7 and re-written Fig7 legend.

Generally Holocephalans are the outgroup to elasmobranchs - right now they are presented as sister taxa with no ability to indicate derivation. Why isn't the catshark included in this diagram?

While a little unclear exactly what was requested, we restructured the branches to indicate that holocephalans diverged earlier from the ancestors that led to elasmobranchs. Also in response to this comment, we added catshark (S. canicula) and little skate (L. erinacea) specifically to the character matrix.

In the last paragraph of the introduction, you say that "the data argue" and I admit, I am confused. Whose data? Is this a prediction or results or summary of other people's work? Either way, could be clarified to emphasize the contribution you are about to present.

Sorry for this lack of clarity, and we have changed the wording in this revision to hopefully avoid this misunderstanding.

Reviewer #2 (Public Review):

General comment:

This is a very valuable and unique comparative study. An excellent combination of scanning and histological data from three different species is presented. Obtaining the material for such a comparative study is never trivial. The study presents new data and thus provides the basis for an in-depth discussion about chondrichthyan mineralised skeletal tissues.

many thanks for the kind words

I have, however, some comments. Some information is lacking and should be added to the manuscript text. I also suggest changes in the result and the discussion section of the manuscript.

Introduction:

The reader gets the impression almost no research on chondrichthyan skeletal tissues was done before the 2010 ("last 15 years", L45). I suggest to correct that and to cite also previous studies on chondrichthyan skeletal tissues, this includes studies from before 1900.

We have added additional older references, as detailed above.

Material and Methods:

Please complete L473-492: Three different Micro-CT scanners were used for three different species? ScyScan 117 for the skate samples. Catshark different scanner, please provide full details. Chimera Scncrotron Scan? Please provide full details for all scanning protocols.

We clarified exact scanners and settings for each micro-CT experiment in the Methods (lines 476-497).

TMD is established in the same way in all three scanners? Actually not possible. Or, all specimens were scanned with the same scanner to establish TMD? If so please provide the protocol.

Indeed, the same scanner was used for TMD comparisons, and we included exact details on how TMD was established and compared with internal controls in the Methods. (lines 486-488)

Please complete L494 ff: Tissue embedding medium and embedding protocol is missing. Specimens have been decalcified, if yes how? Have specimens been sectioned non-decalcified or decalcified?

Please complete L506 ff: Tissue embedding medium and embedding protocol is missing. Description of controls are missing.

Methods were updated to include these details (lines 500-503).

Results:

L147: It is valuable and interesting to compare the degree of mineralisation in individuals from the three different species. It appears, however, not possible to provide numerical data for Tissue Mineral Density (TMD). First requirement, all specimens must be scanned with the same scanner and the same calibration values. This in not stated in the M&M section. But even if this was the case, all specimens derive from different sample locations and have, been preserved differently. Type of fixation, extension of fixation time in formalin, frozen, unfrozen, conditions of sample storage, age of the samples, and many more parameters, all influence TMD values. Likewise the relative age of the animals (adult is not the same as adult) influences TMD. One must assume different sampling and storage conditions and different types of progression into adulthood. Thus, the observation of different degrees of mineralisation is very interesting but I suggest not to link this observation to numerical values.

These are very good points, but for the following reasons we feel that they were not sufficiently relevant to our study, so the quantitative data for TMD remain scientifically valid and critical for the field moving forward.  Critically, 1) all of the samples used for TMD calculations underwent the same fixation protocols, and 2) most importantly, all samples for TMD were scanned on the same micro-CT scanner using the same calibration phantoms for each scanning session.  Finally, while the exact age of each adult was not specified, we note for Fig1 that clear statistically significant differences in TMD were observed among various skeletal elements from ratfish, shark, and skate.  Indeed, ratfish TMD was considerably lower than TMD reported for a variety of fishes and tetrapods (summarized in our paper about icefish skeletons, who actually have similar TMD to ratfish: https://doi.org/10.1111/joa.13537).

In  , however, we added a caveat to the paper’s Methods (lines 466-469), stating that adult ratfish were frozen within 1 or 2 hours of collection from the wild, staying frozen for several years prior to thawing and immediate fixation.

Parts of the results are mixed with discussion. Sometimes, a result chapter also needs a few references but this result chapter is full of references.

As mentioned above, we reduced background-style writing and citations in each Results section.

Based on different protocols, the staining characteristics of the tissue are analysed. This is very good and provides valuable additional data. The authors should inform the not only about the staining (positive of negative) abut also about the histochemical characters of the staining. L218: "fast green positive" means what? L234: "marked by Trichrome acid fuchsin" means what? And so on, see also L237, L289, L291

We included more details throughout the Results upon each dye’s first description on what is generally reflected by the specific dyes of the staining protocols. (lines 178, 180, 184, 223, 227, and 243-244)

Discussion

Please completely remove figure 7, please adjust and severely downsize the discussion related to figure 7. It is very interesting and valuable to compare three species from three different groups of elasmobranchs. Results of this comparison also validate an interesting discussion about possible phylogenetic aspects. This is, however, not the basis for claims about the skeletal tissue organisation of all extinct and extant members of the groups to which the three species belong. The discussion refers to "selected representatives" (L364), but how representative are the selected species? Can there be a extant species that represents the entire large group, all sharks, rays or chimeras? Are the three selected species basal representatives with a generalist life style?

These are good points, and yes, we certainly appreciate that the limited sampling in our data might lead to faulty general conclusions about these clades.  In fact, we stated this limitation clearly in the Introduction (lines 126-128), and we removed “representative” from this revision.  We also replaced general reference to chondrichthyans in the Title by listing the specific species sampled.  However, in the Discussion, we also compare our data with previously published additional species evaluated with similar assays, which confirms the trend that we are concluding.  We look forward to future papers specifically testing the hypotheses generated by our conclusions in this paper, which serves as a benchmark for identifying shared and derived features of the chondrichthyan endoskeleton.

Please completely remove the discussion about paedomorphosis in chimeras (already in the result section). This discussion is based on a wrong idea about the definition of paedomorphosis. Paedomorphosis can occur in members of the same group. Humans have paedormorphic characters within the primates, Ambystoma mexicanum is paedormorphic within the urodeals. Paedomorphosis does not extend to members of different vertebrate branches. That elasmobranchs have a developmental stage that resembles chimera vertebra mineralisation does not define chimera vertebra centra as paedomorphic. Teleost have a herocercal caudal fin anlage during development, that does not mean the heterocercal fins in sturgeons or elasmobranchs are paedomorphic characters.

We agree with the reviewer that discussion of paedomorphosis should apply to members of the same group.  In our paper, we are examining paedomorphosis in a holocephalan, relative to elasmobranch fishes in the same group (Chrondrichthyes), so this is an appropriate application of paedomorphosis.  In response to this comment, we clarified that our statement of paedomorphosis in ratfish was made with respect to elasmobranchs (lines 37-39; 418-420).

L432-435: In times of Gadow & Abott (1895) science had completely wrong ideas bout the phylogenic position of chondrichthyans within the gnathostomes. It is curious that Gadow & Abott (1895) are being cited in support of the paedomorphosis claim.

If paedomorphosis is being examined within Chondrichthyes, such as in our paper and in the Gadow and Abbott paper, then it is an appropriate reference, even if Gadow and Abbott (and many others) got the relative position of Chondrichthyes among other vertebrates incorrect.

The SCPP part of the discussion is unrelated to the data obtained by this study. Kawaki & WEISS (2003) describe a gene family (called SCPP) that control Ca-binding extracellular phosphoproteins in enamel, in bone and dentine, in saliva and in milk. It evolved by gene duplication and differentiation. They date it back to a first enamel matrix protein in conodonts (Reif 2006). Conodonts, a group of enigmatic invertebrates have mineralised structures but these structure are neither bone nor mineralised cartilage. Cat fish (6 % of all vertebrate species) on the other hand, have bone but do not have SCPP genes (Lui et al. 206). Other calcium binding proteins, such as osteocalcin, were initially believed to be required for mineralisation. It turned out that osteocalcin is rather a mineralisation inhibitor, at best it regulates the arrangement collagen fiber bundles. The osteocalcin -/- mouse has fully mineralised bone. As the function of the SCPP gene product for bone formation is unknown, there is no need to discuss SCPP genes. It would perhaps be better to finish the manuscript with summery that focuses on the subject and the methodology of this nice study.

We completely agree with the reviewer that many papers claim to associate the functions of SCPP genes with bone formation, or even mineralization generally.  The Science paper with the elephant shark genome made it very popular to associate SCPP genes with bone formation, but we feel that this was a false comparison (for many reasons)!  In response to the reviewer’s comments, however, we removed the SCPP discussion points, moving the previous general sentence about the genetic basis for reduced skeletal mineralization to the end of the previous paragraph (lines 435-439).  We also added another brief Discussion paragraph afterwards, ending as suggested with a summary of our proposed shared and derived chondrichthyan endoskeletal traits (lines 440-453).

Reviewer #1 (Recommendations For The Authors):

Further Strengths and Opportunities:

Your headings for the results subsection and figures are nice snapshots of your interpretations of the results and I think they would be better repurposed in your abstract, which needs more depth. It's a little unusual to try and state an interpretation of results as the heading title in a results section and the figures so it feels out of place. You could also use the headings as the last statement of each section, after you've presented the results. In order I would change these results subheadings to:

Tissue Mineral Density (TMD)

Tissue Properties of Neural Arches

Trabecular mineralization

Cap zone and Body zone Mineralization Patterns

Areolar mineralization

Developmental Variation

Sorry, but we feel that summary Results sub-headings are the best way to effectively communicate to readers the story that the data tell, and this style has been consistently used in our previous publications.  No changes were made.

You allude to the fossil record and that is great. That said historical literature is more abundant than what you've listed. Your first sentence describes a long fascination and only goes back to 1990. But there are authors that have had this fascination for centuries and so I think you'll benefit from looking back. Especially because several of them have looked into histology of these fishes. You even have one sentence citing Coates et al. 2018, Frey et al., 2019 and ørvig 1951 to talk about the potential that fossils displayed trabecular mineralization. That feels like you are burying the lead and may have actually been part of the story for where you came up with your hypothesis in the beginning... or the next step in future research. I feel like this is really worth spending some more time on in the intro and/or the discussion.

We’ve added older REFs as pointed out above.  Regarding fossil evidence for trabecular mineralization, no, those studies did not lead to our research question.  But after we discovered how widespread trabecular mineralization was in extant samples, we consulted these papers, which did not focus on the mineralization patterns per se, but certainly led us to emphasize how those patterns fit in the context of chondrichthyan evolution, which is how we discussed them.

I agree that in the past 15 years or so a lot more work has been done because it can be done using newer technologies. That said there's a lot more work by Mason Dean's lab starting in 2010 that you should take a look at related to tesserae structure... they're looking at additional taxa than what you did as well. It will be valuable for than you to be able to make any sort of phylogenetic inference as part of your discussion and enhance the info your present in figure 7. Go further back in time... For example:

de Beer, G. R. 1932. On the skeleton of the hyoid arch in rays and skates. Quarterly

Journal of Microscopical Science. 75: 307-319, pls. 19-21.

de Beer, G. R. 1937. The Development of the Vertebrate Skull. The University Press,Oxford.

Indeed, we have read all of Mason’s work, citing 9 of his papers, and where possible, we have incorporated their data on different species into our Discussion and Fig7.  Thanks for the de Beer REFs.  While they contain histology of developing chondrichthyan elements, they appear to refer principally to gross anatomical features, so were not included in our Intro/Discussion.

Most sections with in the results, read more like a discussion than a presentation of the new data and you jump directly into using an argument of those data too early. Go back in and remove the references or save those paragraphs for the discussion section. Particularly because this journal has you skip the method section until the end, I think it's important to set up this section with a little bit more brevity and conciseness.  For instance, in the first section about tissue mineral density, change that subheading to just say tissue mineral density. Then you can go into the presentation of what you see in the ratfish, and then what you see in the little skate, and then that's it. You save the discussion about what other elasmobranch's or mineralizing their neural arches, etc. for another section.

We dramatically reduced background-style writing and citations in each Results section (other than the first section of minor points about general features of the ratfish, compared to catshark and little skate), keeping only a few to briefly remind the general reader of the context of these skeletal features.

I like that your first sentence in the paragraph is describing why you are doing. a particular method and comparison because it shows me (the reader) where you're sampling from. Something else is that maybe as part of the first figure rather than having just each with the graph have a small sketch for little skate and catch shark to show where you sampled from for comparative purposes. That would relate back, then to clarifying other figures as well.

done (also adding a phylogenetic tree).

Second instance is your section on trabecular mineralization. This has so many references in it. It does not read like results at all. It looks like a discussion. However, the trabecular mineralization is one of the most interesting aspect of this paper, and how you are describing it as a unique feature. I really just want a very clear description of what the definition of this trabecular mineralization is going to be.

In addition to adding Table 1 to define each proposed endoskeletal character state, we have changed the structure of this section and hope it better communicates our novel trabecular mineralization results.  We also moved the topic of trabecular mineralization to the first detailed Discussion point (lines 347-363) to better emphasize this specific topic.

Carry this reformatting through for all subsections of the results.

As mentioned above, we significantly reduced background-style writing and citations in each Results section.

I'd like to see modifications to figure 7 so that you can add more continuity between the characters, illustrated in figure 7 and the body of the text. I think you can give the characters a number so that you can actually refer to them in each subsection of the results. They can even be numbered sequentially so that they are presented in a standard character matrix format, that future researchers can add directly to their own character matrices. You could actually turn it into a separate table so it doesn't taking up that entire space of the figure, because there need to be additional taxa referred to on the diagram. Namely, you don't have any out groups in figure 7 so it's hard to describe any state specifically as ancestral and wor derived. Generally Holocephalans are the outgroup to elasmobranchs - right now they are presented as sister taxa with no ability to indicate derivation. Why isn't the catshark included in this diagram?

The character matrix is a fantastic idea, and we should have included it in the first place!  We created Table 1 summarizing the traits and terminology at the end of the Introduction, also adding the character matrix in Fig7 as suggested, including specific fossil and extant species.  For the Fig7 branching and catshark inclusion, please see above. 

You can repurpose the figure captions as narrative body text. Use less narrative in the figure captions. These are your results actually, so move that text to the results section as a way to truncate and get to the point faster.

By figure captions, we assume the reviewer refers to figure legends.  We like to explain figures to some degree of sufficiency in the legends, since some people do not read the main text and simply skim a manuscript’s abstract, figures, and figure legends.  That said, we did reduce the wording, as requested.

More specific comments about semantics are listed here:

The abstract starts negative and doesn't state a question although one is referenced. Potential revision - "Comprehensive examination of mineralized endoskeletal tissues warranted further exploration to understand the diversity of chondrichthyans... Evidence suggests for instance that trabecular structures are not common, however, this may be due to sampling (bring up fossil record.) We expand our understanding by characterizing the skate, cat shark, and ratfish... (Then add your current headings of the results section to the abstract, because those are the relevant takeaways.)"

We re-wrote much of the abstract, hoping that the points come across more effectively.  For example, we started with “Specific character traits of mineralized endoskeletal tissues need to be clearly defined and comprehensively examined among extant chondrichthyans (elasmobranchs, such as sharks and skates, and holocephalans, such as chimaeras) to understand their evolution”.  We also stated an objective for the experiments presented in the paper: “To clarify the distribution of specific endoskeletal features among extant chondrichthyans”. 

In the last paragraph of the introduction, you say that "the data argue" and I admit, I am confused. Whose data? Is this a prediction or results or summary of other people's work? Either way, could be clarified to emphasize the contribution you are about to present.

Sorry for this lack of clarity, and we have changed the wording in this revision to hopefully avoid this misunderstanding.

In the second paragraph of the TMD section, you mention the synarcual comparison. I'm not sure I follow. These are results, not methods. Tell me what you are comparing directly. The non-centrum part of the synarcual separate from the centrum? They both have both parts... did you mean the comparison of those both to the cat shark? Just be specific about which taxon, which region, and which density. No need to go into reasons why you chose those regions here.. Put into methods and discussion for interpretation.

We hope that we have now clarified wording of that section.

Label the spokes somehow either in caption or on figure direction. I think I see it as part of figure 4E, I, and J, but maybe I'm misinterpreting.

Based upon histological features (e.g., regions of very low cellularity with Trichrome unstained matrix) and hypermineralization, spokes in Fig4 are labelled with * and segmented in blue.  We detailed how spokes were identified in main text (lines 241-243; 252-254) and figure legend (lines 597-603). 

Reviewer #2 (Recommendations For The Authors):

Other comments

L40: remove paedomorphism

no change; see above

L53: down tune languish, remove "severely" and "major"

done (lines 57-59)

L86: provide species and endoskeletal elements that are mineralized

no change; this paragraph was written generally, because the papers cited looked at cap zones of many different skeletal elements and neural arches in many different species

L130: remove TMD, replace by relative, descriptive, values

no change; see above

L135: What are "segmented vertebral neural arches and centra" ?

changed to “neural arches and centra of segmented vertebrae” (lines 140-141)

L166: L168 "compact" vs. "irregular". Partial mineralisation is not necessarily irregular.

thanks for pointing out this issue; we changed wording, instead contrasting “non-continuous” and “continuous” mineralization patterns (lines 171-174)

L192: "several endoskeletal regions". Provide all regions

all regions provided (lines 198-199)

L269: "has never been carefully characterized in chimeras". Carefully means what? Here, also only one chimera is analyses, not several species.

sentence removed

302: Can't believe there is no better citation for elasmobranch vertebral centra development than Gadow and Abott (1895)

added Arriata and Kolliker REFs here (lines 293-295)

L318 ff: remove discussion from result chapter

references to paedomorphism were removed from this Results section

L342: refer to the species studied, not to the entire group.

sorry, the line numbering for the reviewer and our original manuscript have been a little off for some reason, and we were unclear exactly to which line of text this comment referred.  Generally in this revision, however, we have tried to restrict our direct analyses to the species analyzed, but in the Discussion we do extrapolate a bit from our data when considering relevant published papers of other species.

346: "selected representative". Selection criteria are missing

“selected representative” removed

L348: down tune, remove "critical"

Done

L351: down tune, remove "critical"

done

L 364: "Since stem chondrichthyans did not typically mineralize their centra". Means there are fossil stem chondrichthyans with full mineralised centra?

Re-worded to “Stem chondrichthyans did not appear to mineralize their centra” (lines 379)

L379: down tune and change to: "we propose the term "non-tesseral trabecular mineralization. Possibly a plesiomorphic (ancestral) character of chondrichthyans"

no change; sorry, but we feel this character state needs to be emphasized as we wrote in this paper, so that its evolutionary relationship to other chondrichthyan endoskeletal features, such as tesserae, can be clarified.

L407: suggests so far palaeontologist have not been "careful" enough?

apologies; sentence re-worded, emphasizing that synchrotron imaging might increase details of these descriptions (lines 406-408)

414: down tune, remove "we propose". Replace by "possibly" or "it can be discussed if"

sentence re-worded and “we propose” removed (lines 412-415)

L420: remove paragraph

no action; see above

L436: remove paragraph

no action; see above

L450: perhaps add summery of the discussion. A summery that focuses on the subject and the methodology of this nice study.

yes, in response to the reviewer’s comment, we finished the discussion with a summary of the current study.  (lines 440-453)

I struggle with why he thinks this question must be asked, when it doesn't have to be asked in the other scenarios he brings up. Why does it matter what the school is if the check is to blanketly support children's transportation to education? (just food for thought)

I find the tension between not using laws to aid the practice of religion and making sure laws don't prevent or discourage this practice to be very interesting. It is like walking on a tight rope, where on either side there is a disregard to the 1st amendment in one way or the other.

I think it is interesting to think about how this "indignation" is connected to the feelings of indignation around the taxation without representation which led directly to the revolutionary war. When considering the historical context of the constitution it is important to keep in mind not just the thoughts and concerns of the framers, but also the ways that the historical context was effecting public opinion.

Text Accessibility

The page allows to increase or decreasing the text size ensuring is readable for all users

Contrast

The page contains good contrast making accessible for all users

Long Alternative Text

The image has a very long alternative text, lengthy alternative text often indicates content is not available to sighted users

Classic "don't judge a book by its cover" argument. What makes the perivous lines more interesting is how advanced technology and social media have made it easier than ever for people to be deceived. The anecdote about the man in the gray truck highlights how easily accessible information can be used to mislead others.

The idea of having a completely unbiased opinion is interesting because it's almost impossible to have one. Even if the author's identity or the purpose behind the piece is unknown, it's difficult to form an opinion that isnt influenced in some shape or form by personal bias. It speaks to how our experiences and beliefs inevitably shape how we process information.

This is an important skill for law enforcement officers to have, which connects back to the anecdote on the first page about the importance of attention to detail. In high-stakes situations like the ones police officers might face, being highly observant can be beneficial, as it can reduce misjudgments and unnecessary violence.

I think this is a really interesting way to express the idea that our actions do, in fact, impact the lives of those around us. Especially through our perceptions and how closely we observe the world and things around us

People only see things from their own eyes.

I really like how this story encapsulates how different people view the world. The religious man sees the Eskimos helping him as a sign of God. The atheist only sees it as the Eskimos helping.

fuck off pretentious

visuals present images hat are to appeal to the audience's emotions

Sometimes I get distracted and do not wanna do my work all the time. If I am in an environment that is talkative and very loud I get distracted easier than when im with a small amount of people and we help each other out.

Sometimes when I have to study an assignment I do not find interesting I just struggle to have the energy to get it done. But, I use music to motivate me and keep me energized.

I think it is very important for me to do my assignments right when I am told about them and right after the class ends because im already in the working mood and it will help me focus.

connects back to first article

i feel like this ties back to the act now, handle consequences later idea from the last article

My preferred method of active reading has always been to write down what I think is important as I read. I have terrible memory retention when just reading, but I was always told that writing down what you learn as you read uses two different parts of your brain, and thus can help you memorize things better. It also never hurts to have notes to look back on if you find yourself struggling.

listening and understanding others view points Is key

can't miss more than one class

Homework is not given to students for credit but It's to access Important Information

I think this is a really important concept especially as a college student. I love the idea that I am not just sitting here but a "creator of knowledge."

It surprised me because it seemed innocent at first, but later it's revealed to be for violence. It shows how normalized cruelty is in the community.

Co-existing in the same age or time period

A genre which requires nontrivial effort from the reader to navigate the text in order to find some type of answer or solution.

Narrative structure or sequence pertaining to that point of the narrative in which hypertext poems become the opposite of that

A notable feature of the BBC website is that it is operable with the use of only a keyboard. Meaning users can get the experience they wish by just clicking the 'tab' key. This is useful for those with restricted physical abilities.

Most of the text on this page is paired with imagery placed next to important navigation tools, such as save, share, and search functions.

The text beneath each photo is formatted consistently, making it cohesive and easy to read, especially for people with difficulties. (Understandable)

“How to Read Like a Writer” is an essay by Mike Bunn. It’s to focus on looking deeper into the text for example the writing style, the layout, the wordings, etc. Just reading would be reading and enjoying a book rather than analyzing it.

There have been many times where I saw certain things in black and white. There have been many times where I then saw another persons opinions and their take on a book and it helped me see a bigger picture. Reading can be super broad and up to how the reader takes it. These times are when I really love seeing people’s annotation.

I heavily agree with this. Learning could be just behavior or perspective even from annotations. It helps me understand how other people could have read a certain event and how they felt about it. I love reading annotations because it gives off personality and perspective. I love when people are open minded to see other perspectives and willing to listen. Learned behaviors are a really big thing everyone goes through and being able to broaden your minds really opens you to understand others.

I have seen many annotations specifically in books. I love to read and many of my friends like to read as well. Most of the readers I’ve met have annotated their books. This means adding their reaction to a specific text with words. I like to use thin sticky notes and associate a certain emotion or theme via color and add them to the books to mark parts that really stood out to me. Recently, I bought a book that comes with annotations. It leaves a star after certain words and gives some context to the reader like “remember this name”. I really like that this book came with interactive like annotation on the bottom of the page. I like how annotating books gives readers a way to express themselves into the books they read.

i visited a university in china and in order to access the campus it used facial recognition- one of the american students who i went with is asian and the facial recognition would sometimes work on her

Angolban: "Additionally, the deposit breakage can be initiated before the maturity date here." helyett

Additionally, early withdrawal of the deposit can be initiated before the maturity date here.

Since sleep is necessary and dreams can almost be considered a privilege, the line is saying that it won't be needed to do things out of the genuine need to, but out of the want to do so.

Since this person is going to the underworld they are saying goodbye to all of their possessions as they cannot take their property with them. They are also stating how they no longer have to deal with any burdens they may have had along with their debts.

Atlas was the Titan charged with holding up the sky to keep it from crushing the world. This addition of him makes it seem as if Eurydice is saying that there is no point of him keeping up the sky, because sooner or later everything will end.

This is meaningful because it show how the burdens have been let go of and the line after No further debts to be paid shows how he can rest now and he doesn't have to hold the weight of the world anymore

All of the responsibilities and hard duties go away and it becomes easier.

The writer isn't necessarily longing for extreme joy or happiness, instead she is looking to just have a life of peace and calm.

Running short on that at the moment

Not surpising, at least the migration piece

Easily manipulated voter base

Obviously important but doesn't scream to me the way the others do

Just as an immigrant community can help receive new migrants, they also soften the landing of society around them to be less discriminatory

huh

But I think this will somewhat depend on the place and like mindset of where they are being integrated

Yeah the right is likely to deny why and how they left

Countries can get benefits from bolstering dying cities

This is smart, take advantage of the human disposition already at play

Who gets to allocate then? Just a lottery system, and how does one verify that it is for climate reasons (although maybe that isn't important)

requires so much individual generosity on the part of the country

We need a more strict definition of climate migrant b/c climate change so often works in tandem with other push factors, the scope needs to be narrower

Strategic migration is more economically or individually motivated while managed retreat will happen as that place slowly becomes unlivable (as opposed to rapidly in the case of disaster)

Avoiding the most random and devastating of climate change effects

TLDR: It makes everything worse bozos

This migration would have been internal, will it scale to international

What are the good outcomes for receiving communities

But you are actively fighting against the dominant american thought

Good fucking luck bro

angolban on the day of booking helyett deposit start date

Angolul: "In the case of FIRST, the period of an interest coupon comprises the following interval" helyett The period of an interest coupon comprises the following interval

Angol verzióban "After selecting the break date, by checking "Unique Interest," a manual break interest rate can be specified; otherwise, the system will use the accrued interest associated with the break date." helyett: After selecting the date of early withdrawal, by checking the box "Unique Interest," the interest rate of early withdrawal can be inserted manually; otherwise, the system will use the accrued interest associated with the early withdrawal date.

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Reply to the reviewers

Manuscript number: RC-2025-03094

Corresponding author(s): Saurabh S. Kulkarni

1. General Statements

We thank the reviewers for their strong praise of the manuscript, highlighting its rigor, depth, and conceptual importance. They consistently described the study as a beautiful, fascinating, and conceptually strong piece of work that addresses a timely question in multiciliated cells. They also noted the high quality of the data, careful quantification, and the use of multiple genetic and pharmacological approaches, all of which improve the reproducibility and credibility of the findings. Importantly, they emphasized the novelty of discovering a direct mechanistic link between Piezo1-mediated mechanotransduction and Foxj1-driven transcriptional control of multiciliation, representing a significant breakthrough for both the cilia field and mechanobiology more broadly. Collectively, these strengths highlight the manuscript’s wide impact and make it highly suitable for publication in a high-impact journal.

2. Description of the planned revisions

Reviewer #1:

There are two experiments that would significantly strengthen these claims.

• First if their model is correct then even short term treatment with Yoda1 should induce the pathway and effect centriole numbers. While I appreciate the challenge of long term Yoda1 treatment its not clear to me why it would be needed if short term treatment is setting off the transcriptional cascade. Yoda is used throughout the paper to induce all the pathways but we don't know if it actually induces the phenotype. I think this should be addressed with either short term treatments or a dose response to find a dose that does not lead to skin pealing. It is hard to ignore this obvious deficiency.
• Second, the model predicts that all of this is to regulate Foxj1 levels to regulate the subtle balance between cell size and centriole number. If this is correct, then the overexpression of Foxj1 should have a profound effect on centriole number in multiciliated cells. This is such an easy experiment that would validate many of the claims. RESPONSE:

We recognize that the reviewer is asking us to test the sufficiency of the pathway with these comments: “If their model is correct, then they should be able to activate the pathway in one way or another to stimulate centriole number. This is a significant limitation to their overall model.” And “If this is correct, then the overexpression of Foxj1 should have a profound effect on centriole number in multiciliated cells.”

To address reviewers’ suggestions, we will perform the following experiments.

1. A brief exposure (15 and 30 mins) to Yoda1 and wait for 3 hours to examine changes in centriole amplification. This will avoid skin peeling from long-term exposure.
2. A brief exposure to Yoda1 (15 mins) followed by a 30-minute wait period, and the cycle repeats a total of 4 times for a total of 3 hours to examine centriole amplification.
3. The above two experiments will also be done in a constitutively active-Yap background to increase the probability that synergistic activation can lead to centriole amplification.
4. Although Foxj1 is essential for multiciliogenesis, it is not sufficient to induce multiciliogenesis, as shown by multiple previous studies. Therefore, we do not expect overexpression of Foxj1 to have a profound effect on centriole number. While we will conduct the experiments because we truly want to address the suggestions and gain insight into the answers ourselves, we respectfully ask the Reviewer to consider the following responses to their concern.

Yoda1 sufficiency: We agree that testing whether acute Yoda1 treatment can induce centriole amplification is an important question. We will conduct experiments with short-pulse and cyclic Yoda1 exposure, including in a constitutively active-YAP background (listed above), to address this possibility. However, several challenges complicate interpretation: (i) PIEZO1 adapts and desensitizes upon activation, (ii) transient signaling may be sufficient to cause secondary signaling but insufficient to drive stable transcriptional programs required for amplification, and (iii) centriole number is inherently variable, making modest effects difficult to resolve. However, we must recognize that failure to observe sufficiency under these conditions would not invalidate the model for two reasons: 1) absence of evidence is not evidence of absence, and thus, we may not have found the right experimental design. 2) PIEZO1–YAP is a necessary input but not sufficient on its own, as elaborated below. For both reasons, we are very careful about the interpretation of results in the manuscript, which shows that this pathway is necessary for centriole amplification using loss-of-function approaches.

Foxj1 overexpression: Foxj1 is a well-established regulator essential for motile and multiciliogenesis across species (Xenopus, zebrafish, mouse). Loss of Foxj1 reduces cilia number in MCCs, but its activation alone does not have a profound effect on ciliogenesis/cilia number in MCCs. This is because Foxj1 is a part of a larger network essential for multiciliogenesis. This parallels the behavior of other transcriptional regulators, such as Myb, where loss of function impairs centriole amplification, but overexpression does not drive the formation of supernumerary centrioles. Both studies are seminal discoveries in the field of ciliogenesis, but they did not demonstrate the sufficiency of these molecules/pathways. Thus, our results, demonstrating that Foxj1 is necessary to induce tension-dependent centriole amplification, are significant, as the reviewer mentioned. The lack of Foxj1 sufficiency to induce centriole amplification is not a deficiency of the study, but rather evidence that Foxj1 is a part of a larger network essential for tension-dependent centriole amplification.

Necessity versus sufficiency: We respectfully emphasize that sufficiency is not a prerequisite for demonstrating the significance of a pathway. Mechanochemical signaling is inherently complex, involving many mechanosensitive proteins and pathways. In our case, mechanical stretch increases centriole amplification, with PIEZO1–YAP signaling identified as a key mediator. However, we do not claim that PIEZO1–YAP alone is sufficient. Other pathways, including cadherin-mediated junctions, F-actin–myosin contractility, integrin–focal adhesion signaling, and nuclear mechanotransduction, likely contribute and may regulate unique downstream effectors that collectively promote centriole amplification. Therefore, PIEZO1–YAP should be regarded as one essential component within a larger network.

__TIMELINE: __We will perform these additional proposed experiments. Since the first author, a postdoctoral researcher on this manuscript, has started a new job and will be coming in on weekends to complete the experiments, we estimate it will take approximately 2-3 months to finish them.

Reviewer #2:

1. Considering the Yap-piezo mechanism of action, the authors' logic for the selection of myb, foxj, plk4 and ccno as transcriptional targets is clear, but the HCR-derived signal and the differences seen in the yap morphants are not very strong, notwithstanding the statistical significance. There appear to be distinct subgroups within the treated populations (in Figure S6B, although these data seem quite different in Fig. 7H, so a comment on the technical differences might be helpful), so that the extent to which Yap1 regulates (Myb-)Foxj1 expression in MCCs is not clearly demonstrated by this experiment. Related to this point, it is unclear why 20-25% of the yap1/ piezo1 MO-treated embryos do not show a decline in FOXj1 in Fig. 6, given the qualitative nature of the scoring. Assuming the KD penetrance would vary on a cell-to-cell basis, rather than an embryo-to-embryo basis, this may suggest that there are additional relevant targets (some of which are discussed by the authors). Single-cell analysis might be a way to address this; however, this is not a trivial experiment, it might be sufficient to include a caveat in the text. Furthermore, the conclusion that Foxj1 regulates centriole amplification in a tension-dependent manner is well-supported by the data.

RESPONSE: We appreciate the reviewer’s thoughtful observation. Differences in the expression of Foxj1 from experiment to experiment are possible due to a combination of factors, including heterogeneity in MCC development across embryos, slightly different embryonic stages, differences in embryo quality between fertilizations, and variability in morpholino delivery and knockdown penetrance, which can occur both across embryos and on a cell-to-cell basis within an embryo. We also note that technical aspects of HCR RNA-FISH, such as proteinase K treatment and washing steps, can affect signal intensity, potentially contributing to the appearance of distinct subgroups within treated populations.

We agree that single-cell analysis would be a powerful way to dissect these differences, but as the reviewer notes, this is not a trivial experiment and is beyond the scope of the present study. We have therefore added clarifications in the text and discussion to acknowledge these sources of variability and to highlight the possibility of parallel pathways regulating foxj1 expression.

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Controls for the knockdowns by the various MOs should be provided.

RESPONSE: We appreciate the reviewer’s comment. The piezo1 MO has been previously established in Kulkarni et al. (2021). Additionally, the current manuscript includes MO control experiments for both erk2 and yap1, through KD at the 1-cell stage using the MO oligonucleotide, followed by mosaic-rescue with the respective WT RNA constructs (mCherry-ERK2 and yap1-GFP) and a nuclear tracer molecule such as H2B-RFP (Fig. 5, E-H, Fig. S5, C&D, Fig. 3, D-F). The mosaic-rescue is a robust experiment that provides an internal control within the same embryo, thereby avoiding differences that may arise due to embryo-to-embryo variability, embryo quality, or differences in fertilization batches. This approach also serves as a valuable tool for detecting cell-autonomous effects, providing a clear readout against uninjected neighboring cells, as the injected cells are labeled with a tracer. We will perform a similar mosaic-rescue experiment for the foxj1 MO.

TIMELINE: We will conduct mosaic-rescue experiments for the foxj1 MO. We will need 1 month to complete the experiment.

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__Minor comments:

__

Autocorrection of ERK1/2 or MEK1/2 pathways to 1/2 should be avoided. – We are unclear on this comment. Can reviewer please clarify what they mean.

Reviewer # 3

Major concerns

1- The presented data do not yet establish a specific, direct pathway linking mechanotransduction to centriole number, because the molecular players tested (PIEZO1, Ca²⁺, PKC, ERK, YAP, Foxj1) are highly pleiotropic. As such, the observed centriole number phenotypes, and some of the major conclusions, could be indirect. It is therefore critical to test the specificity and causality of the proposed pathway. This could be done with the authors' own strategies and/or with the following potential approaches:

• Genetic dependency and sufficiency tests: It could be shown that Yoda1 has no effect in PIEZO1 loss-of-function MCCs, and that wild-type PIEZO1, but not conductance-ad PIEZO1 pore mutants restores Yoda1 responsiveness across centriole number, pERK, and YAP readouts. For example, PIEZO1 C terminus was shown to govern Ca²⁺ influx and ERK1/2 activation. Comparing full length PIEZO1 with a C terminal deletion in MCC restricted rescue; loss of rescue of centriole amplification and ERK/YAP activation with the C terminal deletion can provide a genetics anchored specificity test beyond broad inhibitors.

RESPONSE:

• To address the reviewer’s concern, we will test whether Yoda1 affects ERK and Yap activation when Piezo1 is depleted. We appreciate the reviewer’s thoughtful suggestion to employ genetic rescue experiments with Piezo1 mutants. Unfortunately, these are not technically feasible in Xenopus, as the Piezo1 coding sequence is exceptionally large (~7.5 kb)____, and repeated attempts by our group to generate and express stable, translatable transcripts have been unsuccessful. To address genetic dependency and specificity despite these technical barriers, we have employed a combination of orthogonal strategies that together provide strong genetic and mechanistic evidence:

• Mosaic loss-of-function experiments (Fig. 1) demonstrate that Piezo1 regulates centriole number in a cell-autonomous manner, ruling out global epithelial or indirect tissue-wide effects.

• Pharmacological activation/inhibition with Piezo1-specific agonist (Yoda1) and inhibitors (GSMTx4, gadolinium) produced consistent phenotypes, including activation of downstream ERK and YAP readouts. Notably, Yoda1 is a Piezo-specific agonist, not a broad pharmacological agent.
• Downstream pathway dissection (calcium chelation, PKC inhibition, ERK2 depletion, and YAP1 knockdown/rescue) consistently converges on the same phenotypes, reduced centriole amplification and altered Foxj1 expression, providing multiple independent lines of evidence that the Piezo1–Ca²⁺–PKC–ERK–YAP axis specifically controls centriole number.

• Positive feedback regulation of Piezo1 expression by YAP/Foxj1 (Fig. 7) further strengthens the argument for a pathway-specific role rather than pleiotropic, indirect effects. Taken together, while full-length Piezo1 rescue experiments are technically not possible in Xenopus due to gene size constraints, our data employ state-of-the-art genetic, pharmacological, and orthogonal functional assays to rigorously test pathway specificity. These complementary approaches provide compelling evidence for the causal role of Piezo1-mediated mechanotransduction in centriole number control in MCCs.

• Downstream bypass/rescue experiments: In PIEZO1 loss-of-function or BAPTA conditions, can enforcing MEK/ERK activation or YAP rescue centriole number defect? Conversely, can MEK inhibitors block Yoda1-induced effects.

RESPONSE: We appreciate the reviewer’s insightful questions.

• We will express CA Yap in the Piezo1 KD background to assess if we can rescue centriole number. We also note that the converse experiment has already been performed in our study: 1) PKC inhibition abolishes Yoda1-induced ERK phosphorylation and nuclear localization (Fig. 2), 2) both MEK inhibition and ERK2 depletion block Yoda1-induced Yap activation and nuclear entry (Figs. 4, S2). Thus, we have directly demonstrated that MEK inhibition prevents Yoda1-induced effects, satisfying this aspect of the reviewer’s concern.

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2- Image quantification and analysis must be described in greater detail in the Methods section, as they are central to the major conclusions of the manuscript. For example, the authors should explain how nuclear, cytoplasmic, and centriole segmentation were performed, and how relative protein levels in the nucleus versus the cytoplasm (e.g., YAP, volume- or area-based) were quantified. Specifically, the thresholds and segmentation criteria applied to different cellular structures under various conditions, as well as the use of Imaris and other software, should be clearly detailed.

RESPONSE: We will describe the methods in greater detail.

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3- PIEZO1 mRNA was shown to incrase in a Foxj1 linked feedback loop. Does this increase translate into an increase in total protein levels?

RESPONSE: If the reviewer is referring to Figure 7B, that is the Piezo1 antibody, so yes, the Piezo1 protein levels have increased.

If the reviewer is referring to Figure 7C and D, we show that loss of Foxj1 leads to a reduction in Piezo1 mRNA expression.

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4- Is the proposed signaling cascade active in mammalian multiciliated cells (e.g., airway epithelium). If possible, testing this by using one of the major players of the pathway as a readout such as as ERK phosphorylation, YAP nuclear localization in mammalian MCCs will reveal whether regulation of centriole number through this pathway is conserved and would strengthen the generality.

RESPONSE: We agree with the reviewer that testing conservation of this pathway in mammalian MCCs is of great interest. Indeed, another group is currently investigating the role of Yap in the mammalian airway epithelium; in their temporally controlled Yap knockout model (the global Yap KO being embryonic lethal), they observed that Yap loss led to a reduction in centriole number. To avoid overlap and direct competition with this ongoing work, we chose to focus our efforts on Xenopus.

Importantly, Xenopus has become a widely recognized and powerful system for MCC biology, enabling mechanistic dissection of centriole amplification and ciliogenesis. Several key discoveries in the field, including the identification of MCIDAS as a master regulator of MCC fate, were first made in Xenopus before being validated in mammals. Similarly, our study provides a mechanistic framework in Xenopus that can inform and guide ongoing studies in the mammalian airway.

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5- Throughout the results section, there are multiple times where authors raised specific hypothesis about their data (e.g. foxj1 regulation of number control, apical actin/YAP). However, they have not tested them. These hypothesis are very exciting and if possible, testing experimentally, would strengthen the conclusions associated with them.

RESPONSE: We are not sure what the reviewer means here by “authors raised specific hypothesis about their data (e.g., foxj1 regulation of number control, apical actin/YAP). However, they have not tested them”,

BECAUSE:

• Foxj1 regulation of centriole number: We demonstrate a clear reduction in centriole number upon Foxj1 depletion, and importantly, we extend this finding by showing that the reduction is tension-dependent (Fig. 6). We will perform a rescue assay to demonstrate the specificity.
• Foxj1 and YAP: We never claimed that Foxj1 regulates YAP expression, and this is not part of our proposed model. Instead, our data show that Piezo1–ERK–YAP signaling regulates Foxj1
• Foxj1 and apical actin: Foxj1 regulation of apical F-actin has already been established in prior work, and in our study, we clearly observe reduced apical actin intensity in Foxj1-depleted MCCs (Fig. 6). To further strengthen this conclusion, we will provide a quantitative analysis of apical actin intensity in Foxj1 morphants. ********************************************

__TIMELINE: __We will perform these additional proposed experiments. Since the first author, a postdoc on this manuscript, has started a new job and will be coming in on weekends to finish the experiments, we estimate it will take approximately 2-3 months to complete them.

Minor comments

MCC vs non MCC identification (Fig. 1): Clarify how non MCCs were distinguished from MCCs (e.g. markers/criteria). – Can the reviewer please clarify which panel or panels? Or provide more specific text that needs to be changed.

Add the Kintner group reference linking motile cilia number and centriole number in Xenopus MCCs.– Can the reviewer clarify where and which reference? Thank you.

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

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

Reviewer 2

Major comments:

1. It should be clarified whether the immunoblots and the related quantitations in Figs. 2 and S2 are all from separate blots/ exposures. If so, they are not useful as controls, and these blots should be repeated with the relevant samples analyzed in parallel. Size markers and labels should be included (2B, 2G; S2B and S2G). An increase in total ERK would alter the interpretation of the increase in nuclear pERK in the IF experiments. RESPONSE: We thank the reviewer for raising this important point regarding clarification of the immunoblots. All experimental groups were analyzed in parallel with their corresponding controls. Because the primary antibodies for pERK and ERK were both raised in rabbit, we optimized our workflow to prevent protein loss during stripping and to ensure accurate visualization. Specifically, lysates from each experimental group were loaded in duplicate on the same gel, separated by a molecular weight ladder that served as a reference point. After transfer, the blot was cut along the ladder, and the two halves were processed in parallel: one probed with anti-pERK and the other with anti-ERK. This strategy ensured that all samples from a single experiment (e.g., Control and Yoda1-treated groups) were analyzed under identical conditions, with staining and imaging performed together at the same exposure. To enhance clarity, we have provided this data as __uncut, full-length __as Supplemental Figure 7 (Figure S7) in the revised revision.

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Minor comments:

1. Reference list should be checked for completeness; some citations lack journal/ volume/ page/ year details. – We have corrected the references.
2. An 'overexposed' version of the image selected for centrioles in Figure 5F might be included with the Chibby-BFP at the same level as in the other figures. At present, the Yap KD cell in the image appears to have normal centrioles; this is potentially confusing, even though the authors clearly explain the matter in the text. – __We have added a new panel to Fig. 5F to avoid confusion.

__ 3. It might be clearer to present injected/ uninjected in the same orientation in Fig. 6A and B. – __Unfortunately, that is not possible because the injected and uninjected sides are left and right, and they cannot be in the same orientation.

__ 4. Figure 7B lacks the schematic described in the figure legend. – We have removed the Schematic sentence from the figure legend. That was an error on our side. Thank you for catching it.

Reviewer 3

1. Abstract: "how MCCs regulate centriole/cilia numbers remains a major knowledge gap" overstates the field; please soften to reflect recent advances (mechanics/apical area scaling; PIEZO1 implication). – We changed the text to “incompletely understood”.
2. GsMTx4 rationale: State that GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (including PIEZO1) and justify its use alongside Yoda1.– GsMTx4 was used in the previous manuscript, and its use was justified there. Here, we are only comparing the results. However, we have added a sentence describing what GSMTx4 is. We have also included a sentence explaining the use of Yoda1. “GsMTx4, a spider venom peptide used in our previous study, inhibits cationic mechanosensitive channels, including Piezo1.”

“For this experiment, we used the Piezo1 channel-specific chemical agonist, Yoda1, to increase the sensitivity of Piezo1 and upregulate calcium entry into cells”

Timeline statement: "Centriole amplification to migration and apical docking takes ~4-5 h (personal observation)" is not appropriate; either cite time lapse literature or include your own time lapse data.– We have added a reference that showed imaging for 2 hours, but it was not enough to capture the entire process from intercalation to maturation, so we also kept “personal observation” still in the manuscript. We are unaware of any study that has done time-lapse imaging for 4 hours to capture the entire process of centriole amplification.

Redundancy: The description of Yoda1 as a channel specific agonist is repeated; keep only once.- Removed

"WT yap1 GFP construct previously used by Dr. Lance Davidson ..." should move construct description to Methods and keep only the citation in Results.– We moved it to Methods.

"(Unpublished data; Dr. Mahjoub)" should be removed unless data are shown.- Removed

Replace "as shown previously in our eLife paper" with "as we previously showed or shown previously (Kulkarni et al., 2021)".– We have made the change.

The two hypotheses for how Foxj1 could regulate number under tension (actin remodeling vs. transcriptional control of amplification genes) belong in the Discussion unless tested. Moreover, the part on the discussion on yap sequestration by apical actin and the two possibilities presented also should go do discussion. – We have moved both to the discussion section.

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

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

Reviewer 3

1- The hypothesis about the centriole pool of Piezo as the mechnosensor for centriole number regulation is very exciting and novel. Can localization controlled variants be used to test whether a centriole associated pool directly senses tension for number control (for example, centrosome targeted PIEZO1 via a PACT tag). Alternatively, broad cellular Ca sensors (GcaMP) or centrosome proximal Ca sensors (e.g., PACT GCaMP) can be used detect local calcium microdomains during tethering or Yoda1 treatment.

RESPONSE: We appreciate the reviewer's curiosity and excitement; however, these experiments will not alter the conclusion of this paper and will be part of the next study, which aims to delve deeper into how different pools of Piezo1 at centrioles versus cell junctions function in MCCs. To that point, we had thought about these experiments. As mentioned earlier, the Piezo1 coding sequence is exceptionally large (~7.5 kb)____, and repeated attempts by our group to generate and express stable, translatable transcripts have been unsuccessful. Thus, the idea of centrosome-targeted PIEZO1 via a PACT is very exciting; however, it is not technically feasible. Beyond size, PIEZO1 is a trimeric, large plasma-membrane mechanosensitive channel that requires proper ER processing and bilayer incorporation. PACT localizes cargo to the centriole/pericentriolar material, not a membrane compartment; thus, a PACT-anchored PIEZO1 would be membrane-mismatched and almost certainly nonfunctional even if expressed/

Second, Centrosome-proximal GCaMP (PACT-GCaMP) would show correlation, not causation. This experiment does not address the question “centriole pool of Piezo as the mechanosensor for centriole number regulation”. It will only show if the Ca2+ influx is happening at the basal bodies, but not whether and how that Ca2+ is essential for centriole amplification. For this purpose, we will need to find a way to block Ca2+ influx specifically at basal bodies, rather than junctions, which will require extensive controls.

We do not claim that any specific Piezo1 or Ca2+ pool is critical for controlling centriole number and thus the suggested experiment would not alter the manuscript's conclusions. We therefore view the above as exciting future directions rather than prerequisites.

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2- Because the proposed pathway is tension-sensing and YAP pathway is tightly linked to the actin cytoskeleton, the role of actin cysoskeleton in the proposed pathway should be tested directly. The authors mention different hypothesis around actin but has not tested them in the manuscript. For example, actin-depedent sequestration of Yap at the apical surface is intriguing. Does actin polymerization induced by drugs release Yap from the apical surface?

RESPONSE: We would like to thank the reviewer for their suggestion. As per the reviewers' suggestion, we have moved this section to discussion, stating that “In the future, we plan to address this question by examining how Yap is sequestered by apical actin.”.

However, we appreciate the reviewer’s enthusiasm and would like to share some experiments we are thinking/planning of to test the hypothesis.

We plan to examine if the actin polymerization or contractility is responsible for Yap sequestration/release from the apical surface with the following experiments: 1) if the Yap is displaced by Jasplakinolide treatment, which stabilizes filamentous actin, 2) use of ROCK inhibitor to decrease contractility in the absence or presence of Yoda1, 3) Use genetic constructs such as Shroom3 to increase ROCK-mediated contractility to observe changes in Yap localization and dynamics.

Although these experiments are interesting, they do not alter the conclusion of the current manuscript, and they represent future directions for our research.

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Referee #3

Evidence, reproducibility and clarity

This manuscript investigates how mechanical tension is transduced into centriole amplification in Xenopus multiciliated cells (MCCs). Building on prior work that centriole number scales with MCC apical area and that this scaling depends on PIEZO1, the study proposes that MCCs repurpose a canonical mechanochemical axis-PIEZO1 → Ca²⁺/PKC → ERK1/2 → YAP → Foxj1-to regulate centriole number rather than mitosis. The authors use tethered vs. untetheredanimal cap explants to modulate tissue tension, combine pharmacologic perturbations with genetic loss of function and rescue, quantititative image analysis and present a model in which tension gated PIEZO1 activates ERK/YAP, influences Foxj1, and tunes centriole number in MCCs.

The manuscript tackles an important and timely problem with clear disease relevance. It major advance is their presented model that posits that post mitotic MCCs repurpose a canonical mechanotransduction module to regulate organelle number rather than proliferation. It is a conceptually strong study addressing an important problem with a clean mechanical paradigm. However, to support the central claim that centriole number control is a specific, direct consequence of the PIEZO1-Ca²⁺-ERK/YAP pathway within MCCs, the revision should establish specificity and causality and provide experimental data for some of the major conclusions as detailed below. Addressing these points are critical to support the mechanistic conclusions and impact.

Major concerns:

1) The presented data do not yet establish a specific, direct pathway linking mechanotransduction to centriole number, because the molecular players tested (PIEZO1, Ca²⁺, PKC, ERK, YAP, Foxj1) are highly pleiotropic. As such, the observed centriole number phenotypes, and some of the major conclusions, could be indirect. It is therefore critical to test the specificity and causality of the proposed pathway. This could be done with the authors' own strategies and/or with the following potential approaches:

• Genetic dependency and sufficiency tests: It could be shown that Yoda1 has no effect in PIEZO1 loss-of-function MCCs, and that wild-type PIEZO1, but not conductance-dead PIEZO1 pore mutants restores Yoda1 responsiveness across centriole number, pERK, and YAP readouts. For example, PIEZO1 C terminus was shown to govern Ca²⁺ influx and ERK1/2 activation. Comparing full length PIEZO1 with a C terminal deletion in MCC restricted rescue; loss of rescue of centriole amplification and ERK/YAP activation with the C terminal deletion can provide a genetics anchored specificity test beyond broad inhibitors.

• Downstream bypass/rescue experiments: In PIEZO1 loss-of-function or BAPTA conditions, can enforcing MEK/ERK activation or YAP rescue centriole number defect? Conversely, can MEK inhibitors block Yoda1-induced effects.

2) The hypothesis about the centriole pool of Piezo as the mechnosensor for centriole number regulation is very exciting and novel. Can localization controlled variants be used to test whether a centriole associated pool directly senses tension for number control (for example, centrosome targeted PIEZO1 via a PACT tag). Alternatively, broad cellular Ca sensors (GcaMP) or centrosome proximal Ca sensors (e.g., PACT GCaMP) can be used detect local calcium microdomains during tethering or Yoda1 treatment.

3) Because the proposed pathway is tension-sensing and YAP pathway is tightly linked to the actin cytoskeleton, the role of actin cysoskeleton in the proposed pathway should be tested directly. The authors mention different hypothesis around actin but has not tested them in the manuscript. For example, actin-depedent sequestration of Yap at the apical surface is intriguing. Does actin polymerization induced by drugs release Yap from the apical surface?

4) Image quantification and analysis must be described in greater detail in the Methods section, as they are central to the major conclusions of the manuscript. For example, the authors should explain how nuclear, cytoplasmic, and centriole segmentation were performed, and how relative protein levels in the nucleus versus the cytoplasm (e.g., YAP, volume- or area-based) were quantified. Specifically, the thresholds and segmentation criteria applied to different cellular structures under various conditions, as well as the use of Imaris and other software, should be clearly detailed.

5) PIEZO1 mRNA was shown to incrase in a Foxj1 linked feedback loop. Does this increase translate into an increase in total protein levels?

6) Is the proposed signaling cascade active in mammalian multiciliated cells (e.g., airway epithelium). If possible, testing this by using one of the major players of the pathway as a readout such as as ERK phosphorylation, YAP nuclear localization in mammalian MCCs will reveal whether regulation of centriole number through this pathway is conserved and would strengthen the generality.

7) Throughout the results section, there are multiple times where authors raised specific hypothesis about their data (e.g. foxj1 regulation of number control, apical actin/YAP). However, they have not tested them. These hypothesis are very exciting and if possible, testing experimentally, would strengthen the conclusions associated with them.

Minor concerns:

1) Abstract: "how MCCs regulate centriole/cilia numbers remains a major knowledge gap" overstates the field; please soften to reflect recent advances (mechanics/apical area scaling; PIEZO1 implication).

2) MCC vs non MCC identification (Fig. 1): Clarify how non MCCs were distinguished from MCCs (e.g. markers/criteria).

3) GsMTx4 rationale: State that GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (including PIEZO1) and justify its use alongside Yoda1.

4) Timeline statement: "Centriole amplification to migration and apical docking takes ~4-5 h (personal observation)" is not appropriate; either cite time lapse literature or include your own time lapse data.

5) Redundancy: The description of Yoda1 as a channel specific agonist is repeated; keep only once.

6) "WT yap1 GFP construct previously used by Dr. Lance Davidson ..." should move construct description to Methods and keep only the citation in Results.

7) "(Unpublished data; Dr. Mahjoub)" should be removed unless data are shown.

8) Add the Kintner group reference linking motile cilia number and centriole number in Xenopus MCCs.

9) Replace "as shown previously in our eLife paper" with "as we previously showed or shown previously (Kulkarni et al., 2021)".

10) The two hypotheses for how Foxj1 could regulate number under tension (actin remodeling vs. transcriptional control of amplification genes) belong in the Discussion unless tested. Moreover, the part on the discussion on yap sequestration by apical actin and the two possibilities presented also should go do discussion.

Significance

This manuscirpt dissects Piezo1-mediated mechanotransduction to regulation of centriole number in Xenopus multiciliated cells (MCCs) via Ca²⁺, ERK/YAP, and Foxj1. While Piezo1 and its downstream effectors have been implicated broadly in mechanosensation, cellular tension responses, and transcriptional regulation, their specific role in centriole nubmer control in MCCs has been unknown By integrating pharmacological manipulation, genetic perturbation, and functional readouts, the authors demonstrate that this pathway directly influences centriole number.

The findings extend published knowledge in two main ways:

(1) they connect a mechanosensitive ion channel to the transcriptional program governing Foxj1 expression and multiciliation, a mechanistic link not previously defined, and

(2) they highlight the pleiotropic yet coordinated nature of Piezo1 signaling in organelle biogenesis. This work will be of broad interest to cell and developmental biologists studying ciliogenesis, epithelial differentiation, and mechanotransduction, as well as to biomedical researchers interested in multicilaited cells and ciliopathies. By situating a well-studied mechanosensor within the context of MCC biology, the study opens new directions for understanding how tissue-level forces shape organelle number control and function.

At the same time, the impact of the study is weakened by concerns regarding the causability and specificity of the pathway, since the signaling components examined are highly pleiotropic and it remains challenging to separate direct effects on centriole number from broader cellular consequences. The causal relationships among Piezo1 activity, downstream signaling, and Foxj1 expression require stronger substantiation, and the extent to which this pathway operates in mammalian multiciliated cells remains an open question. Addressing these limitations would strengthen the robustness, generality, and translational relevance of the conclusions.

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Referee #2

Evidence, reproducibility and clarity

Narayanan, Kulkami and colleagues here examine how the Piezo-Erk-Yap pathway is involved in centriole numerical control in multiciliated cells (MCCs). Using reverse genetic and pharmacological methods in Xenopus embryos, they show that Piezo-mediated ERK signalling through to Yap regulates tension-sensitive centriole number, through a mechanism that involves Foxj1, very likely acting as a transcription factor. The data are carefully controlled, robustly analysed and well presented. Statistical analyses are notably thorough.

Main points:

1. It should be clarified whether the immunoblots and the related quantitations in Figs. 2 and S2 are all from separate blots/ exposures. If so, they are not useful as controls, and these blots should be repeated with the relevant samples analysed in parallel. Size markers and labels should be included (2B, 2G; S2B and S2G). An increase in total ERK would alter the interpretation of the increase in nuclear pERK in the IF experiments.

2. Considering the Yap-piezo mechanism of action, the authors' logic for the selection of myb, foxj, plk4 and ccno as transcriptional targets is clear, but the HCR-derived signal and the differences seen in the yap morphants are not very strong, notwithstanding the statistical significance. There appear to be distinct subgroups within the treated populations (in Figure S6B, although these data seem quite different in Fig. 7H, so a comment on the technical differences might be helpful), so that the extent to which Yap1 regulates (Myb-)Foxj1 expression in MCCs is not clearly demonstrated by this experiment. Related to this point, it is unclear why 20-25% of the yap1/ piezo1 MO -treated embryos do not show a decline in FOXj1 in Fig. 6, given the qualitative nature of the scoring. Assuming the KD penetrance would vary on a cell-to-cell basis, rather than an embryo-to-embryo basis, this may suggest that there are additional relevant targets (some of which are discussed by the authors). Single-cell analysis might be a way to address this; however, this is not a trivial experiment, it might be sufficient to include a caveat in the text. Furthermore, the conclusion that Foxj1 regulates centriole amplification in a tension-dependent manner is well-supported by the data.

3. Controls for the knockdowns by the various MOs should be provided.

Minor points:

1. Autocorrection of ERK1/2 or MEK1/2 pathways to 1/2 should be avoided.

2. Reference list should be checked for completeness; some citations lack journal/ volume/ page/ year details.

3. An 'overexposed' version of the image selected for centrioles in Figure 5F might be included with the Chibby-BFP at the same level as in the other figures. At present, the Yap KD cell in the image appears to have the normal centrioles; this is potentially confusing, even though the authors clearly explain matters in the text.

4. It might be clearer to present injected/ uninjected in the same orientation in Fig. 6A and B.

5. Figure 7B lacks the schematic described in the figure legend.

Significance

This study presents novel insight into the developmentally important process of ciliogenesis in multiciliated cells that will be of specific interest to the fields of cilium biology and mechanobiology, with additional general interest in calcium signalling and cell biology.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #1

Evidence, reproducibility and clarity

The manuscript from Narayanan addresses the fascinating question of how Multiciliated cells regulate centriole number to scale with cell size. They have generated a tremendous amount of high quality data that supports a model in which mechanosensitive signaling via piezo1 leads to an increase in intracellular Ca++ that leads to an activation of the Erk pathway which in turn activates the Yap pathway that in turn regulates FoxJ1 levels which they propose regulates centriole number. This is complicated but they have strong quantifiable data that supports most of the claims. I think this is a beautiful study that adds significantly to the field. There is a lot of evidence that disrupting these pathways has a negative consequence on centriole number. What is lacking is a positive connection showing a role of these processes in fine tuning the centriole number as the title suggests. Several key experiments would significantly strengthen their claims.

• The data is presented in a way that proposes that the ultimate role of these pathways is to regulate Foxj1 levels to fine tune centriole number based on the level of tension. There are two experiments that would significantly strengthen these claims. First if their model is correct then even short term treatment with Yoda1 should induce the pathway and effect centriole numbers. While I appreciate the challenge of long term Yoda1 treatment its not clear to me why it would be needed if short term treatment is setting off the transcriptional cascade. Yoda is used throughout the paper to induce all the pathways but we don't know if it actually induces the phenotype. I think this should be addressed with either short term treatments or a dose response to find a dose that does not lead to skin pealing. It is hard to ignore this obvious deficiency. Second, the model predicts that all of this is to regulate Foxj1 levels to regulate the subtle balance between cell size and centriole number. If this is correct, then the overexpression of Foxj1 should have a profound effect on centriole number in multiciliated cells. This is such an easy experiment that would validate many of the claims.

Minor issues:

• The authors attempt to measure an effect of plk4 and ccno in the Yap MO experiment. However, the fact that they could not be scored means the experiment wasn't really performed. I think it is more appropriate to leave out rather than risk giving the impression that these genes were unaffected.

• The authors indicate that the foxj1 result suggests two alternatives, one that foxj1 regulates actin (pan 2007) and the other that it is a transcription factor. I think the evidence for foxj1 being a transcription factor is extremely well established and while it is possible for it to have an additional unrelated role my interpretation of the Pan paper is that the failed apical docking leads to disrupted actin which is also well established. I don't think there is a lot of evidence for foxj1 being anything other than a TF.

Significance

• This is a really beautiful paper that will be well appreciated by the cilia community but also should be appreciated by the broader cell biology community.

• The strengths of this paper are a high level of rigor in which they perform detailed quantification of a wide range of processes. For many experiments they have multiple methods for disrupting function which again adds to the rigor. They have successfully linked Piezo1, Erk, Yap and FoxJ1 function to proper centriole biogenesis, which is a significant advance.

• The limitation is that all their perturbations negatively effect centriole number which could be indirect. If their model is correct then they should be able to activate the pathway in one way or another to stimulate centriole number. This is a significant limitation to their overall model.

Often, hospital imagery becomes almost cliché. Every story talks about the mechanical and sanitized atmosphere, the cold harsh lights. However, the author managed to inject a new visualization. Hospitals can be described as "gruesome" with all the disease and wounds inside, but to mix the robotic with the gruesome synthesizes a new perspective. I also like how he describes his brother's body both in an active and passive sense, emphasizing his liminal state. His lips "were parted" in passive voice but chose to "admit" the breathing tube. These two lines of figurative language very much elevate the entire story.

RR\ID Summary of Reviews: This preprint evaluates the diagnostic performance of Quantum-enhanced nanodiamond rapid test for early SARS-CoV-2 antigen detection. Peer reviewers praised the rigor of the study noting it as the first large-scale blinded clinical evaluation of spin-enhanced nanodiamond LFTs, moving beyond proof-of-concept to a performance assessment with real clinical samples. They urged the need for further work on membrane chemistry and blocking strategies to bring performance closer to its theoretical potential. They also suggested discussion of the drop in sensitivity at lower viral loads (<10⁴ copies/mL) in terms of balancing infectiousness detection with over diagnosis risk and suggest the authors to briefly comment on the potential utility of this assay for public health applications such as surveillance or contact tracing.  

Read directly in RR\ID: https://rrid.mitpress.mit.edu/pub/vi2iegeo/release/1

Reviewer #1: Evidentiary Rating: Potentially Informative

Written Review: The authors claim that a spin-enhanced fluorescent nanodiamond (FND) lateral flow test for SARS-CoV-2 antigen detection achieves up to 1,100-fold greater sensitivity than conventional gold nanoparticle LFTs using identical antibodies. In a large, blinded clinical evaluation, the assay demonstrated 95.1% sensitivity (Ct ≤ 30) and 100% specificity, enabling SARS-CoV-2 detection on average two days earlier than conventional LFTs and within 0.6 days of RT-qPCR. They assert that this quantum-enhanced diagnostic platform could be adapted to other infectious and non-infectious diseases. 1. The study represents the first large-scale blinded clinical evaluation of spin-enhanced nanodiamond LFTs, moving beyond proof-of-concept to a performance assessment with real clinical samples. 2. The antibody screening process using biolayer interferometry is well executed, but screening on a single recombinant antigen source introduces potential epitope bias; inclusion of diverse antigen sources could further validate pair robustness. 3. The direct head-to-head comparison with in-house AuNP LFTs using identical reagents is a strong methodological choice, eliminating confounding variables common in cross-platform sensitivity claims. 4. While the assay achieves sub-pg/mL LoDs, residual non-specific binding limits ultimate sensitivity exploitation; more work on membrane chemistry or blocking strategies could push performance closer to the theoretical limit. 5. The sample size is adequate for preliminary evaluation, but a larger, more demographically and geographically diverse cohort is necessary to confirm real-world performance, especially in asymptomatic and early infection cases. 6. The assay’s 95.1% sensitivity at Ct ≤ 30 exceeds WHO “desirable” criteria, but the drop in sensitivity at lower viral loads (<10⁴ copies/mL) should be discussed in terms of balancing infectiousness detection with overdiagnosis risk. 7. The ROC, Bayesian regression, and infection dynamics modelling are sophisticated and well described, but providing raw Ct distribution histograms for positive samples would help readers assess viral load representativeness.

Reviewer #2: Evidentiary Rating: Reliable

Written Review: The authors present the development of a lateral flow assay prototype for the detection of SARS-CoV-2 nucleocapsid antigen, utilizing spin-enhanced nanodiamonds as fluorescent sensors. The manuscript provides sufficient detail regarding the evaluation of fluorescent nanodiamonds, optimization of anti-SARS-CoV-2 nucleocapsid capture and detection antibody pairs, assessment of assay sensitivity, and assay specificity for SARS-CoV-2 in comparison to other coronaviruses. Additionally, the authors include data demonstrating detection of SARS-CoV-2 nucleocapsid antigen in clinically relevant specimens, including nasal and nasopharyngeal swabs. The supplementary material includes a comparative analysis of the assay’s sensitivity relative to published data on various lateral flow tests, highlighting superior sensitivity achieved through the use of fluorescent nanodiamonds. Increased assay sensitivity may lead to earlier detection of SARS-CoV-2 infection thus providing opportunity for early intervention. Overall, the manuscript is well-organized, and the data are scientifically robust. I have a few minor comments for the authors’ consideration: 1. The authors state that the sensitivity of their assay is comparable to that of the Quanterix Simoa assay. Please provide a reference for the Simoa SARS-CoV-2 assay. 2. Could the authors briefly comment on the potential utility of this assay for public health applications such as surveillance or contact tracing?

When I was working in school board, all employees where put into groups to tackle specific issue more precisely. With the huge team broken up into small groups, more focused what put into each committee and its respective workloads.

the society identity comes from peoples character, its environment, and political system, working together to form how that civilization grow and behaves.

The speaker is Launfal’s mysterious lover and she is addressing King Arthur. She acknowledges her love for Launfal and defends him. She explains that Launfal was unfairly treated in the court. Her goal is to plead his case and persuade the King to understand Launfal’s actions in a more compassionate way.

The mysterious lady intervenes directly in Launfal’s trial, defending him. The effect on the reader is sympathy and admiration. I really liked these lines because it feels like a really big turning point in the story.

The things I noticed was tone and repetition. The tone of these lines is respectful and obviously thought out. She says "Sire" and "vassals" these both have an elevated tone of respect. There is also a repetive theme of misjudgment in these lines. She says "misprized," "turned to blame," and "over hasty." All of those words lead the reader to the idea that something morally wrong is going on.

Overall, these lines were my really good because Marie de France elevates the voice of a woman. She does this not just to defend Launfal, but to critique a system where justice can be corrupted by jealousy!!

It's not an accident that workers get locked out of knowledge. This hits me hard as a teacher—the gatekeeping of knowledge feels designed. Keep workers (or students) ignorant, and you keep them compliant. How am I supposed to help this be a better world for everyone if I never have the vision of its potential?

high-tech isn't required to enforce domination. Sometimes the simplest tools are the most insidious. We underestimate the genius of simplicity in control—pacing is everything in schools too, from bell schedules to testing windows. Behaviorism teaches us how to condition the masses, and setting forth routines and expectations helps administrators assert a culture of control.

Tech is never neutral. This is exactly why I'm skeptical of ed-tech "solutions". The tool might be brilliant, but who owns it, and how do they wield it? That's the real story. AI as a tutor becomes a uniform process of indoctrination as it guides you to think like the programmers want you to think, and refuses to answer questions you have based on an 'ethical' pretense. Who's ethics matter to me? Someone else's or my own?

The machine isn't just replacing labor, it's erasing identities built on skill. I get why strikes weren't just about wages—they were about dignity and purpose being stripped away.

An example would be helpful and appreciated.

Is the relative frequency of different alleles the best way to measure evolution in action?