YEs Kauffman will fund a company that is NOT a not for profit if its terms of assocaition say it has a charitable function. 501c3 for kauffman is all about TAX ANd yes, we can set up an LLC with an FSC bye laws just as easily

Paul will createa 501c3 as a RESEARCH instrument to apply for Kauffman;s research grant My hope is that IAN's 501c3 will apply for the PROJECT Grant that holds the eco-system of 501c3 fair shares commons

So nto everyone has a telegram account so that is even a splinter group even though it was asked for by some members of the team So we still have an email list and a telegram grop and they are not synchrinised

The meeting with Gyuri was a request from him to meet PAUL so that we could ground on the threads for the RESEARCH application I asked Donan to attend but I did not not ask others to attend as I do not want to place demands on people's time and space This is why I recorded it and sent it around If people then ANNOTATE the call the way I am doing here, Gyuri has data that he can use to develop technology based on what he is himself involved in and not just as a service provider

This is where SROI (Social Return on Investment) DATA will help us bridge the gap In a recent call in Ireland a football team scored a 17:1 SROI score for their impact on community

Kauffman is just our sandbox. We may NOT get their funding. But as soon as we have clarity, structure, documents, templates, we will find the right funder

Tapping to the MMT theory, if a 501c3 is not taxed, then why does it need dollars??? We are close to some understanding some fundamental incoherencies

We welcome the idea of HYBRID SOCIAL ENTERPRISE which can have profit making processes that are professional and not for profit processes which are vocational

Not for Profit is a way to infiltrate the parasite of PROFIT that has come to symbolise incorporation

The reason we are setting up a 501c3 is that the donors have made it easier to receive funding beacse they get a tax write off

Why isnt Evolutesix then the most profitable FSC that it can be?

It is unsafe to assuem that we all knwo hwo a company is incorporated Even senior executives do nto know how their companies are incorporated Most SME founders do NOT know the meaning of how their company is incorporated

Thank you Marie. You embody the SPIRIT of a 555 FSC

Can we all know and understand this graphic. It is quite deep in the powerpoint.

Could we begin to use organism rather than organisation?

if we speak into the incoherence, we might creaet more of it?

We also need to fund a way to create a currency which also has at least the same level of systemic trust as money

The plan is to create a pool of learning and documents so that any one of us can apply for funding to create an FSC with a 501c3 as the legal entity with FSC bye laws that can be adapted

The emergenrt natur eis that we are holding spoace for the creation of an eco system of 501c3's with FSC bye laws

This is why we are raising the money - to offer this benign parenting support of helping us all to pay for what we need so that we do what we love For now, I am using thew term Universal Learning Income

I have realised why COLLABORATION is limited. The word only relates to LABOUR. It is only that we share work. We do not share heart, love, prayer, food, shadow, time, space, etc etc

CHECK AND TWO SECONDS PAUSE

As a Dialogue group, we have the chance to practice allowing the other to end their sentance, for which they can say CHECK Then TWO SECONDS Then the next person can join from the eternal silence

We are hoping to create a COLLECTIVE COMMONS for our annotations The ancient model is the Celtic SPirtual ANNAL such as the book of kells or Leabhar Gabhala (Book of INvasions) where multipel wisodm keepers over generations add their wisdom IN BETWEEN THE LINES

For now, Gyuri and Gien call it Hyperpost

We are using hypothesis (as in this annotation) to play with what tools we have now

Gyuir and Gien intend to make it CROSS PLATFORM

They are using the term INDYWEB and INDRAS NETS

Thank you so much for creating the meeting and for holding space It is not intended that any one person ought to be a central point of contact We are to become autocatalytic - energies arise and fall and all that is is and all that is not is not Some people will not show up Some people will show up all the time We are all equally included We are all One

Similar to Jeffrey Epstein. That case was so important for all the cases following after that, it encouraged victims to speak up and let their voices be heard.

These NDAs signifies the use of intimidation and sparking fear within people.

I feel like these sorts of cases continue to come out more and more. The Hollywood industry, which I would assume these men were apart of, does a terrible job at protecting vulnerable people who are looking to make social connections. Rather, people in high power continually take advantage of innocent people.

Were these victims also people who worked under him? The party setting also reminds me of the huge case revolving around P. Diddy at the moment.

This seems very similar to the Harvey Weinstein and #MeToo Movement. People see an incredibly wealthy and influential man who does not use his power for good, rather he abuses it.

Purpose: This part of the podcast as well as the whole podcast is mainly entertainment with sprinkles of information when it comes to the success of Bad bunny. Though they are providing facts and data the nature of the podcast is for the viewers to find entertainment in talking about music artists and their journey.

Audio: They used background music to set the atmosphere for the views for the rest of the podcast. The music gives the viewers excited to listed to the conversation of this topic which is music. At the end of the podcast they play a Bad Bunny song which gives the fans of this artist some enjoyment in listening to the song and shows others who don't know who he is a taste of his music.

call to action for players

players should be able to disconnect after a game the same way employees disconnect from work. These messages don't allow them to do so

direct evidence of how these messages impacts a player's thinking

same idea that was expressed in the Blame Game article I read

proof that this is a relevant issue and should not be overlooked

another direct experience with the issue

the city centers of large urban centers

the study shows different family types including their traditition

The institute

All products made in the USA need this USDA approval, so it is interesting that this wasn't caught earlier.

If true they need to fix that immediately, but it might not be the end of the company. Other companies have had similar issues when starting up, so it is only normal.

This could be the issue. Real cheese molds much quicker if not chilled correctly in shipping.

This is an important part to mention, since the product is new, having the cheese mold is not a good sign. either they are lying about how old the cheese is, or the specific blend they use is much less artificial and rots quicker.

I find this hard to believe even with the pictures, as the product was just launched mere weeks ago. If true this could be a quick end to the company.

Why and how are they bestfriends? - would have been a nice inclusion

I feel as if the author could've had alot more detail in this article if it was not posted during the 2nd quarter rather the end of the game.

It would've been nice for the author to incorporate a year that the previous record was set in.

This could be looked at as bias since Charean Williams says, 'The Ravens needed it" which comes out to be an opinion when in reality they really did need it to keep up in the game.

While the article was not focused specifically on how hurtful social media backlash is on athletes, I think it does showcase the extremity of some of these fans. Fans like this take abuse to the next level and the article highlights that while providing some solutions that could be helpful in mitigating the issue

yet another solution. But can you apply this to everyone who posts negative things?

another way to respond to hate. Similar to a solution from last week's article

seems like the article is focused on hate but not towards athletes in specific

This is interesting, I didn't actually know that there was a change in views on race in this time period, I thought that was a much more modern concept.

Ironic, given that slavery was still widespread in some places post-Revolution, meaning that all did not, in fact, lay claim to that freedom.

Interesting, because regardless of the actual activism, wasn't Haiti forced to pay a ridiculous sum by the French that left the country economically crippled? I wonder if some people were discouraged by that, and what the response to those arguments was.

Thi sis a annotation on this line

who cares what this says ... the point?

the daughters of "men" are aliens; and all united against God himself, the cutest little puppy that was ever created; and forced to listen to dave sing ...

coud i have been?

your little sister? anyone ... other than me?

https://www.youtube.com/watch?v=B9Ol1Rd9ABI

could i have been?

is this about or at gabriel, or taylor; or the source of Manhattan, or Hatteras; or the world that didn't even know what a "tt" was before there was a Sloan? Remember the days I refused to call Tay "tee" ... before "tee tea" had something to do with ... "keys"

little boy blue and the man in the moon ... when you cummings, georgia tech?

# you know we'll have all the tech giants then.

https://biogarabatos.blogspot.com/2024/10/the-rogue-scholar.html

Is he now scared of all black people?

a reasonable response?

Negative stereotyping that re-enforces the belief that black individuals are not being treated equally as previously stated

This seems unlikely given past descriptions of their actions

Overall getting the message that Melville is trying to depict old Europeans as monsters and Americans as more "humane"

So do most other mothers thus showing how stupid the concept of racism is.

The fact the spainards take women as slaves shows their cruelty and how contradictory they are to being "civilized"

A detailed potryal of the awfulness of both sides of the atlantic slave trade where tribal leaders sold slaves to white europeans

Could be a double meaning for converting the slaves to christianity?

Does the setting also make this american literature then (takes place in south america)

Then does this not kind of invalidate the whole concept of slavery and racism the spainards participate in?

The identity of the american is a lot less descriptive then the spainard, possible eluding to the concept of the melting pot

Same ethnicity as past writers like De la Casas

could this mean the captian or the actual ship itself?

who?

outing the foreshadowing?

Gradient of the text needs to be changed, see figma

Awesome to allow readers to learn more about E. Coli bacteria. The author gives readers a short summary of her own, and provides us with a direct link to the CDC.

This paragraph gives readers a possible answer for what is causing the outbreak. Although earlier sentences state there is no official cause, there are possibilities.

This is impact. It is affecting a lot of people. Ranging from those who already ate a Quarter Pounder to those who were interested in purchasing one.

Author Jonel Aleccia lets us readers know where she got her information to write this article, and provides us with the link to easily access it.

PMID: 8651275

Gene: GAMT

Disease: GAMT deficiency

Inheritance: Autosomal Recessive

From this point of view, I think the disadvantages of data mining outweigh the advantages. Although data mining can improve the user experience, when the platform uses this data to make profits, it will harm the users. It puts the cart before the horse and turns something that should serve the users into something that hurts the users.

I find it interesting that the goal of mining data is to collect information about users to make them stay longer on social media. Some may consider this collection of information an invasion of privacy.

Social media platforms use data to increase user engagement and profits through targeted advertising. While this can be useful for businesses and consumers, it raises ethical concerns when ads are directed at vulnerable groups, like children or addicts, exploiting their weaknesses for financial gain.

Word shading supposed to look more pink at the word 'Moment!'

There is supposed to be an arrow at the end of this -> See figma

Delete, also delete all XX on the other cards here

Font color = dark blue Font size = same as the segment below, "Download our Free STEM Activities..." currently the text here is larger than what's below

Agrandir font

Line spacing to be wider between lines

Missing - 3 boxes. see figma

Supposed to have multiple banner images here. See figma.

Also the words 'imagination' and 'flight' are supposed to be in yellow

Reviewer #1 (Public review):

Summary:

Juvenile Hormone (JH) plays a key role in insect development and physiology. Although the intracellular receptor for JH was identified long ago, a number of studies have shown that part of JH functions should be fulfilled through binding to an unknown membrane receptor, which was proposed to belong to the RTK family. In this study, the authors screened all RTKs from the H. armigera genome for their ability to mediate responses to JH III treatment both in cultured cells and in developping animals. They also present convincing evidence that CAD96CA and FGFR1 directly bind JH III, and that their role might be conserved in other insect species.

Strengths:

Altogether, the experimental approach is very complete and elegant, providing evidence for the role of CAD96CA and FGFR1 in JH signalling using different techniques and in different contexts. I believe that this work will open new perspectives to study the role of JH and better understand what is the contribution of signalling through membrane receptors for JH-dependent developmental processes.

Weaknesses:

Unfortunately, the revised manuscript does not show significant improvement. While the identification of the receptors is highly convincing, important issues about the biological relevance remain unaddressed.

First, the main point I raised about the first version of this article is that the redundancy and/or specificity of the two receptors should be clarified, even though I understand that it cannot be deeply investigated here. I believe that this point, shared by all reviewers, is highly relevant for the scope of this work. In this revised version, it is still unclear how to reconcile gain and loss-of-function experiments and the different expression profiles of the receptors.

Second, the newly added explanations and pieces of discussion provided about the mild in vivo phenotypes of early pupation upon Cad96ca or Fgfr1 knock-out do not clarify the issue but instead put emphasis on methodological issues. Indeed, it is not clear whether the mild phenotypes reflect the biological role of Cad96ca and Fgfr1, or the redundancy of these two RTKs (and/or others), or some issue with the knock-out strategy (partial efficiency, mosaicism...).

Finally, parts of the updated discussion and the modifications to the figures are confusing.

Reviewer #2 (Public review):

Summary:

Juvenile hormone (JH) is a pleiotropic terpenoid hormone in insects that mainly regulates their development and reproduction. In particular, its developmental functions are described as the "status quo" action, as its presence in the hemolymph (the insect blood) prevents metamorphosis-initiating effects of ecdysone, another important hormone in insect development, and maintains the juvenile status of insects.

While such canonical functions of JH are known to be mediated by its intracellular receptor complex composed of Met and Tai, there have been multiple reports suggesting the presence of cell membrane receptor(s) for JH, which mediate non-genomic effects of this terpenoid hormone. In particular, the presence of receptor tyrosine kinase(s) that phosphorylate Met/Tai in response to JH and thus indirectly affect the canonical JH signaling pathway has been strongly suggested. Given the importance of JH in insect physiology and the fact that the JH signaling pathway is a major target of insect growth regulators, elucidating the identify and functions of putative JH membrane receptors is of great significance from both basic and applied perspectives.

In the present study, the authors identified candidate receptors for such cell membrane JH receptors, CAD96CA and FGFR1, in the cotton bollworm Helicoverpa armigera.

Strengths:

Their in vitro analyses are conducted thoroughly using multiple methods, which overall supports their claim that these receptors can bind to JH and mediate their non-genomic effects.

Weaknesses:

Results of their in vivo experiments, particularly those of their loss-of-function analyses using CRISPR mutants are still preliminary, and the results rather indicate that these membrane receptors do not have any physiologically significant roles in vivo. More specifically, previous studies in lepidopteran species have clearly and repeatedly shown that precocious metamorphosis is the hallmark phenotype for all JH signaling-deficient larvae. In contrast, the present study showed that Cad96ca and Fgfr1 G0 mutants only showed slight acceleration in their pupation timing, which is not a typical phenotype one would expect from JH signaling deficiency. This is inconsistent with their working model provided in Figure 6, which indicates that these cell membrane JH receptors promote the canonical JH signaling by phosphorylating Met/Tai.

If the authors argue that this slight acceleration of pupation is indeed a major JH signaling-deficient phenotype in Helicoverpa, they need to provide more data to support their claim by analyzing CRISPR mutants of other genes involved in JH signaling, such as Jhamt and Met. An alternative explanation is that there is functional redundancy between CAD96CA and FGFR1 in mediating phosphorylation of Met/Tai. This possibility can be tested by analyzing double knockouts of these two receptors.

Currently, the validity of their calcium imaging analysis in Figure 5 is also questionable. When performing calcium imaging in cultured cells, it is critically important to treat all the cells at the end of each experiment with a hormone or other chemical reagents that universally induce calcium increase in each particular cell line. Without such positive control, the validity of calcium imaging data remains unknown, and readers cannot properly evaluate their results.

Reviewer #3 (Public review):

Summary:

In this study, Li et al. identified CAD96CA and FGF1 among 20 receptor tyrosine kinase receptors as mediators of JH signaling. By performing a screen in HaEpi cells with overactivated JH signaling, the authors pinpointed two main RTKs that contribute to the transduction of JH. Using the CRISPR/Cas9 system to generate mutants, the authors confirmed that these RTKs are required for normal JH activation, as precocious pupariation was observed in their absence. Additionally, the authors demonstrated that both CAD96CA and FGF1 exhibit a high affinity for JH, and their activation is necessary for the proper phosphorylation of Tai and Met, transcription factors that promote the transcriptional response. Finally, the authors provided evidence suggesting that the function of CAD96CA and FGF1 as JH receptors is conserved across insects.

Strengths:

The data provided by the authors are convincing and support the main conclusions of the study, providing ample evidence to demonstrate that phosphorylation of the transducers Met and Tai mainly depends on the activity of two RTKs. Additionally, the binding assays conducted by the authors support the function of CAD96CA and FGF1 as membrane receptors of JH. The study's results validate, at least in H. amigera, the predicted existence of membrane receptors for JH.

Weaknesses:

The authors have provided evidences that the Cad96Ca and FGF1 RTK receptors contribute to JH signaling through CRISPR/Cas9, inducing precocious metamorphosis, although not to the same extent as absence of JH. Therefore, it still remains unclear whether these RTKs are completely required for pathway activation or only necessary for high activation levels during the last larval stage.

While the authors have included some additional data, the mechanism by which different RTKs function in transducing JH signaling in a tissue specific manner is still unclear. As the authors note in the discussion, it is possible that other RTKs may also play a role in facilitating the transduction of JH signaling.

Lastly, the study does not yet explain how RTKs with known ligands could also bind JH and contribute to JH signaling activation. Although receptor promiscuity has been suggested as a possible mechanism, future studies could explore whether activation of RTK pathways by their known ligands induces certain levels of JH transducer phosphorylation, which, in the presence of JH, could contribute to full pathway activation without the need for direct JH-RTK binding.

Author response:

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

Reviewer #1 (Public Review):

Summary:

Juvenile Hormone (JH) plays a key role in insect development and physiology. Although the intracellular receptor for JH was identified long ago, a number of studies have shown that part of JH functions should be fulfilled through binding to an unknown membrane receptor, which was proposed to belong to the RTK family. In this study, the authors screened all RTKs from the H. armigera genome for their ability to mediate responses to JH III treatment both in cultured cells and in developing animals. They also present convincing evidence that CAD96CA and FGFR1 directly bind JH III, and that their role might be conserved in other insect species.

Strengths:

Altogether, the experimental approach is very complete and elegant, providing evidence for the role of CAD96CA and FGFR1 in JH signalling using different techniques and in different contexts. I believe that this work will open new perspectives to study the role of JH and better understand what is the contribution of signalling through membrane receptors for JH-dependent developmental processes.

Weaknesses:

I don't see major weaknesses in this study. However, I think that the manuscript would benefit from further information or discussion regarding the relationship between the two newly identified receptors. Experiments (especially in HEK-293T cells) suggest that CAD96CA and FGFR1 are sufficient on their own to transduce JH signalling. However, they are also necessary since loss-of-function conditions for each of them are sufficient to trigger strong effects (while the other is supposed to be still present).

Thank you for the suggestion. We have added the discussion in the text: "CAD96CA and FGFR1 have similar functions in JH signaling, including transmitting JH signal for Kr-h1 expression, larval status maintaining, rapid intracellular calcium increase, phosphorylation of transcription factors MET1 and TAI, and high affinity to JH III. CAD96CA and FGFR1 are essential in the JH signal pathway, and loss-of-function for each is sufficient to trigger strong effects on pupation. The difference is that CAD96CA expression has no tissue specificity, and the Fgfr1 gene is highly expressed in the midgut; possibly, it plays a significant role in the midgut. Other possibility is that they play roles by forming heterodimer with each other or other RTKs, which needs to be addressed in future study. CAD96CA and FGFR1 transmit JH III signals in three different insect cell lines, suggesting their conserved roles in other insects.".

In addition, despite showing different expression patterns, the two receptors seem to display similar developmental functions according to loss-of-function phenotypes. It is therefore unclear how to draw a model for membrane receptor-mediated JH signalling that includes both CAD96CA and FGFR1.

Thank you for your question. We have modified the figure and the legends to make the conception clear.

Reviewer #2 (Public Review):

Summary:

Juvenile hormone (JH) is a pleiotropic terpenoid hormone in insects that mainly regulates their development and reproduction. In particular, its developmental functions are described as the "status quo" action, as its presence in the hemolymph (the insect blood) prevents metamorphosis-initiating effects of ecdysone, another important hormone in insect development, and maintains the juvenile status of insects. While such canonical functions of JH are known to be mediated by its intracellular receptor complex composed of Met and Tai, there have been multiple reports suggesting the presence of cell membrane receptor(s) for JH, which mediate non-genomic effects of this terpenoid hormone. In particular, the presence of receptor tyrosine kinase(s) that phosphorylate Met/Tai in response to JH and thus indirectly affect the canonical JH signaling pathway has been strongly suggested. Given the importance of JH in insect physiology and the fact that the JH signaling pathway is a major target of insect growth regulators, elucidating the identification and functions of putative JH membrane receptors is of great significance from both basic and applied perspectives. In the present study, the authors identified candidate receptors for such cell membrane JH receptors, CAD96CA and FGFR1, in the cotton bollworm Helicoverpa armigera.

Strengths:

Their in vitro analyses are conducted thoroughly using multiple methods, which overall supports their claim that these receptors can bind to JH and mediate their non-genomic effects.

Weaknesses:

Results of their in vivo experiments, particularly those of their loss-of-function analyses using CRISPR mutants are still preliminary, and the results rather indicate that these membrane receptors do not have any physiologically significant roles in vivo. More specifically, previous studies in lepidopteran species have clearly and repeatedly shown that precocious metamorphosis is the hallmark phenotype for all JH signaling-deficient larvae. In contrast, the present study showed that Cad96ca and Fgfr1 G0 mutants only showed a slight acceleration in their pupation timing, which is not a typical phenotype one would expect from JH signaling deficiency. This is inconsistent with their working model provided in Figure 6, which indicates that these cell membrane JH receptors promote the canonical JH signaling by phosphorylating Met/Tai.

If the authors argue that this slight acceleration of pupation is indeed a major JH signaling-deficient phenotype in Helicoverpa, they need to provide more data to support their claim by analyzing CRISPR mutants of other genes involved in JH signaling, such as Jhamt and Met. An alternative explanation is that there is functional redundancy between CAD96CA and FGFR1 in mediating phosphorylation of Met/Tai. This possibility can be tested by analyzing double knockouts of these two receptors.

Thank you for your question and suggestion. The cadherin 96ca (CAD96CA) and fibroblast growth factor receptor 1 (FGFR1) were finally determined as JH cell membrane receptors by their roles in JH regulated-gene expression, maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and their JH-binding affinity. Their roles as JH cell membrane receptors were further determined by knockdown and knockout of them in vivo and in cell lines, and overexpression of them in mammal HEK-293T heterogeneously. Figure 6 is drafted by these solidate evidences.

Cad96ca and Fgfr1 G0 mutants caused slight acceleration of pupation is one of the types of evidence of JH signaling-deficient. Othe evidences include a set of gene expression and the block of JH induced-rapid intracellular calcium increase.

Kr-h1 is a typical indicator gene at the downstream of Jhamt and in JH signaling, so we used it as an indicator to examine JH signaling. Jhamt and Met or other genes might be affected in Cad96ca and Fgfr1 G0 mutants, which can be examined in future study.

We have discussed the question that Cad96ca and Fgfr1 G0 mutants only showed a slight acceleration in their pupation timing: "Homozygous Cad96ca null Drosophila die at late pupal stages (Wang et al., 2009). However, we found that 86% of the larvae of the Cad96ca mutant successfully pupated in G0 generation, although earlier than the control. Similarly, null mutation of Fgfr1 or Fgfr2 in mouse is embryonic lethal (Arman et al., 1998; Deng et al., 1994; Yamaguchi et al., 1994). In D. melanogaster, homozygous Htl (Fgfr) mutant embryos die during late embryogenesis, too (Beati et al., 2020; Beiman et al., 1996; Gisselbrecht et al., 1996). However, in H. armigera, 91% of larvae successfully pupated in G0 generation after Fgfr1 knockout. The low death rate after Cad96ca and Fgfr1 knockout might be because of following reasons, including the editing efficiency (67% and 61% for Cad96ca mutant and Fgfr1 mutant, respectively), the chimera of the gene knockout at the G0 generation, and the redundant RTKs that play similar roles in JH signaling, similar to the redundant roles of MET and Germ-cell expressed bHLH-PAS (GCE) in JH signaling (Liu et al., 2009), which needs to obtain alive G1 homozygote mutants and double knockout of these two receptors in future study. We indeed observed that the eggs did not hatch successfully after mixed-mating of G0 Cad96ca mutant or Fgfr1 mutant, respectively, but the reason was not addressed further due to the embryonic death. By the similar reasons, most of the Cad96ca and Fgfr1 mutants showed a slight acceleration of pupation (about one day) without the typical precocious metamorphosis (at least one instar earlier) phenotype caused by JH signaling defects (Daimon et al., 2012; Fukuda, 1944; Riddiford et al., 2010) and JH pathway gene deletions (Abdou et al., 2011; Liu et al., 2009). On other side, JH can regulate gene transcription by diffusing into cells and binding to the intracellular receptor MET to conduct JH signal, which might affect the results of gene knockdown and knockout.".

Currently, the validity of their calcium imaging analysis in Figure 5 is also questionable. When performing calcium imaging in cultured cells, it is critically important to treat all the cells at the end of each experiment with a hormone or other chemical reagents that universally induce calcium increase in each particular cell line. Without such positive control, the validity of calcium imaging data remains unknown, and readers cannot properly evaluate their results.

Thank you for your question. For Figure 5, our goal was to demonstrate that JH can induce calcium mobilization through CAD96CA and FGFR1. Controls have been established between different experimental groups within the same cell, as well as between different cells. Increasing the positive experimental group would make the results more complex.

Reviewer #3 (Public Review):

Summary:

In this study, Li et al. identified CAD96CA and FGF1 among 20 receptor tyrosine kinase receptors as mediators of JH signaling. By performing a screen in HaEpi cells with overactivated JH signaling, the authors pinpointed two main RTKs that contribute to the transduction of JH. Using the CRISPR/Cas9 system to generate mutants, the authors confirmed that these RTKs are required for normal JH activation, as precocious pupariation was observed in their absence. Additionally, the authors demonstrated that both CAD96CA and FGF1 exhibit a high affinity for JH, and their activation is necessary for the proper phosphorylation of Tai and Met, transcription factors that promote the transcriptional response. Finally, the authors provided evidence suggesting that the function of CAD96CA and FGF1 as JH receptors is conserved across insects.

Strengths:

The data provided by the authors are convincing and support the main conclusions of the study, providing ample evidence to demonstrate that phosphorylation of the transducers Met and Tai mainly depends on the activity of two RTKs. Additionally, the binding assays conducted by the authors support the function of CAD96CA and FGF1 as membrane receptors of JH. The study's results validate, at least in H. amigera, the predicted existence of membrane receptors for JH.

Weaknesses:

The study has several weaknesses that need to be addressed. Firstly, it is not clear what criteria were used by the authors to discard several other RTKs that were identified as repressors of JH signaling. For example, while NRK and Wsck may not fulfill all the requirements to become JH receptors, other evidence, such as depletion analysis and target gene expression, suggests they are involved in proper JH signaling activation.

Thank you for your question. We screened the RTKs sequentially, including examining the roles of 20 RTKs identified in the H. armigera genome in JH regulated-gene expression to obtain primary candidates, followed by screening of the candidates by their roles in maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and affinity to JH. WSCK was not involved in the phosphorylation of MET and TAI and was discarded during subsequent screening. NRK did not bind to JH III, did not meet the screening strategy, and was discarded.

We increased the information in the Introduction: "We screened the RTKs sequentially, including examining the roles of 20 RTKs identified in the H. armigera genome in JH regulated-gene expression to obtain primary candidates, followed by screening of the candidates by their roles in maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and affinity to JH. The cadherin 96ca (CAD96CA) and fibroblast growth factor receptor 1 (FGFR1) were finally determined as JH cell membrane receptors by their roles in JH regulated-gene expression, maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and their JH-binding affinity. Their roles as JH cell membrane receptors were further determined by knockdown and knockout of them in vivo and cell lines, and overexpression of them in mammal HEK-293T heterogeneously.".

We increased discussion: "This study found six RTKs that respond to JH induction by participating in JH induced-gene expression and intracellular calcium increase, however; they exert different functions in JH signaling, and finally CAD96CA and FGFR1 are determined as JH cell membrane receptors by their roles in JH induced-phosphorylation of MET and TAI and binding to JH III. We screen the RTKs transmitting JH signal primarily by examining some of JH induced-gene expression. By examining other genes or by other strategies to screen the RTKs might find new RTKs functioning as JH cell membrane receptors; however, the key evaluation indicators, such as the binding affinity of the RTKs to JH and the function in transmitting JH signal to maintain larval status are essential.".

Secondly, the expression of the six RTKs, which, when knocked down, were able to revert JH signaling activation, was mainly detected in the last larval stage of H. amigera. However, since JH signaling is active throughout larval development, it is unclear whether these RTKs are completely required for pathway activation or only needed for high activation levels at the last larval stage.

Thank you for the question. We knocked down the genes at last larval stage to observe pupation, which is a relatively simple and easily to be observed target to examine the role of the gene in JH-maintained larval status. The results from CRISPR/Cas9 experiments showed: "Most wild-type larvae showed a phenotype of pupation on time. However, in the Cad96ca mutant, 86% of the larvae (an editing efficiency of 67% by TA clone analysis) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 24 h earlier. In the Fgfr1 mutant, 91% of the larvae (an editing efficiency of 61%) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 23 h earlier (Figure 4D and E). The data suggested that CAD96CA and FGFR1 support larval growth and prevent pupation in vivo.".

Additionally, the mechanism by which different RTKs exert their functions in a specific manner is not clear. According to the expression profile of the different RTKs, one might expect some redundant role of those receptors. In fact the no reversion of phosphorilation of tai and met upon depletion of Wsck in cells with overactivated JH signalling seems to support this idea.

Nevertheless, and despite the overlapping expression of the different receptors, all RTKs seem to be required for proper pathway activation, even in the case of FGF1 which seems to be only expressed in the midgut. This is an intriguing point unresolved in the study.

Thank you for your comments. Yes, from our study, different RTKs exert their functions in a specific manner. We have increased discussion: "This study found six RTKs that respond to JH induction by participating in JH induced-gene expression and intracellular calcium increase, however; they exert different functions in JH signaling, and finally CAD96CA and FGFR1 are determined as JH cell membrane receptors by their roles in JH induced-phosphorylation of MET and TAI and binding to JH III. We screen the RTKs transmitting JH signal primarily by examining some of JH induced-gene expression. By examining other genes or by other strategies to screen the RTKs might find new RTKs functioning as JH cell membrane receptors; however, the key evaluation indicators, such as the binding affinity of the RTKs to JH and the function in transmitting JH signal to maintain larval status are essential.".

Finally, the study does not explain how RTKs with known ligands could also bind JH and contribute to JH signaling activation. in Drosophila, FGF1 is activated by pyramus and thisbe for mesoderm development, while CAD96CA is activated by collagen during wound healing. Now the authors claim that in addition to these ligands, the receptors also bind to JH. However, it is unclear whether these RTKs are activated by JH independently of their known ligands, suggesting a specific binding site for JH, or if they are only induced by JH activation when those ligands are present in a synergistic manner. Alternatively, another explanation could be that the RTK pathways by their known ligands activation may induce certain levels of JH transducer phosphorylation, which, in the presence of JH, contributes to the full pathway activation without JH-RTK binding being necessary.

Thank you for your professional questions. It is an exciting and challenging to explore the molecular mechanism by which multiple ligands transmit signals through the same receptor. It requires a long-term research plan and in-depth studies. We added discussion in the text: "CAD96CA (also known as Stitcher, Ret-like receptor tyrosine kinase) activates upon epidermal wounding in Drosophila embryos (Tsarouhas et al., 2014) and promotes growth and suppresses autophagy in the Drosophila epithelial imaginal wing discs (O'Farrell et al., 2013). There is a CAD96CA in the genome of the H. armigera, which is without function study. Here, we reported that CAD96CA prevents pupation by transmitting JH signal as a JH cell membrane receptor. We also showed that CAD96CA of other insects has a universal function of transmitting JH signal to trigger Ca2+ mobilization, as demonstrated by the study in Sf9 cell lines of S. frugiperda and S2 cell lines of D. melanogaster.

FGFRs control cell migration and differentiation in the developing embryo of D. melanogaster (Muha and Muller, 2013). The ligand of FGFR is FGF in D. melanogaste_r (Du et al., 2018_). FGF binds FGFR and triggers cell proliferation, differentiation, migration, and survival (Beenken and Mohammadi, 2009; Lemmon and Schlessinger, 2010). Three FGF ligands and two FGF receptors (FGFRs) are identified in Drosophila (Huang and Stern, 2005). The Drosophila FGF-FGFR interaction is specific. Different ligands have different functions. The activation of FGFRs by specific ligands can affect specific biological processes (Kadam et al., 2009). The FGFR in the membrane of Sf9 cells can bind to Vip3Aa (Jiang et al., 2018). One FGF and one FGFR are in the H. armigera genome, which has yet to be studied functionally. The study found that FGFR prevents insect pupation by transmitting JH signal as a JH cell membrane receptor. Exploring the molecular mechanism and output by which multiple ligands transmit signals through the same receptor is exciting and challenging.".

Reviewer #1 (Recommendations For The Authors):

As an experimental suggestion, I will only propose that authors test the double knock-down/knock-out or overexpression of CAD96CA and FGFR1 to give some hints into how redundant/independent the two receptors are.

Thank you very much for your professional advice. We agree with your point of view that double knockout of CAD96CA and FGFR1 is very important to resolve the redundant/independent of the two receptors, which can make our research more complete. Unfortunately, due to experimental difficulty and time constraints, we did not provide supplementary experiments. In this study, we aim to screen the cell membrane receptors of JH. Therefore, we focused on which RTKs can function as receptors. This article is a preliminary study to identify the cell membrane receptors of JH. To further understand the relationship between the two membrane receptors, we will conduct in-depth research in future work.

Apart from that, here are some minor points about the manuscript:

Figure 2A: changing the scale on the y-axis would help to better see the different genotypes (similar to the way it is presented in Figure 5).

Thanks for your reminding, we have changed the scale in Figure 2A.

Figure 4J: image settings could be improved to better highlight the green fluorescence.

Thank you for your advice, we have improved the imaged in Figure 4J.

In general, the manuscript would benefit from some proofreading since a number of sentences are incorrect.

Thanks for your reminding, we have carefully revised the manuscript.

Reviewer #2 (Recommendations For The Authors):

(1) Although the authors note that there are 21 RTK genes in Drosophila (line 55), I can only see 16 Drosophila RTKs in Figure 1 - Figure Supplement 1. Some important Drosophila RTKs such as breathless are missing. The authors need to redraw the phylogenetic tree.

Thanks for your reminding, we have presented the new phylogenetic tree in Figure 1-figure supplement 1.

(2) The accelerated pupation phenotype in Cad96ca and Fgfr1 G0 mutants needs to be better described. In particular, it is critical to examine which developmental stage(s) are shortened in these mutant larvae. Refer to a similar study on a JH biosynthetic enzyme in Bombyx (PMID: 22412378) regarding how to describe the developmental timing phenotype.

Thank you for your advice. We have re-shown Figure 4E and added the explanation in the text: "In 61 survivors of Cas9 protein plus Cad96ca-gRNA injection, 30 mutants were sequenced, and a mutation efficiency was 49.2%. Similarly, in the 65 survivors of Cas9 protein plus Fgfr1-gRNA injection, 35 mutants were sequenced, and a mutation efficiency was 53.8% (Figure 4C). The DNA sequences, deduced amino acids and off–target were analyzed (Figure 4—figure supplement 1). Most wild-type larvae showed a phenotype of pupation on time. However, in the Cad96ca mutant, 86% of the larvae (an editing efficiency of 67% by TA clone analysis) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 24 h earlier. In the Fgfr1 mutant, 91% of the larvae (an editing efficiency of 61%) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 23 h earlier (Figure 4D and E). The data suggested that CAD96CA and FGFR1 support larval growth and prevent pupation in vivo.".

(3) The editing efficiency described in lines 211-213 is obscure. Does this indicate the percentage of animals with noisy sequencing spectra or the percentage of mutation rates analyzed by TA cloning?

Thanks for your reminder. We have revised the description in the text: "In 61 survivors of Cas9 protein plus Cad96ca-gRNA injection, 30 mutants were sequenced, and a mutation efficiency was 49.2%. Similarly, in the 65 survivors of Cas9 protein plus Fgfr1-gRNA injection, 35 mutants were sequenced, and a mutation efficiency was 53.8% (Figure 4C). The DNA sequences, deduced amino acids and off–target were analyzed (Figure 4—figure supplement 1). Most wild-type larvae showed a phenotype of pupation on time. However, in the Cad96ca mutant, 86% of the larvae (an editing efficiency of 67% by TA clone analysis) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 24 h earlier. In the Fgfr1 mutant, 91% of the larvae (an editing efficiency of 61%) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 23 h earlier (Figure 4D and E). The data suggested that CAD96CA and FGFR1 support larval growth and prevent pupation in vivo.".

(4) In Figures 4F and G, the authors examined expression levels of some JH/ecdysone responsive genes only at 0 hr-old 6th instar larvae. This single developmental stage is not enough for this analysis. In particular, the expression level of Fgfr1 only goes up in the mid-6th instar according to their own data (Figure 1-Figure Supplement 4), so it is critical to examine expression levels of these genes at least throughout the 6th larval instar.

Thank you for your advice. Indeed, it is essential to detect the expression levels of JH/ecdysone response genes in the whole sixth instar larvae. Because we observed that the mutation has a shorter feeding stage at the sixth instar, we examined the expression level of the JH/ecdysone response gene at the early sixth instar. Due to the number of mutants obtained in the experiment was small and non-destructive sampling could not be performed in sixth instar period, there were no enough samples to test. In the future, we will generate Cad96ca Fgfr1 double mutations to carry out studies and detect the expression level of JH/ecdysone response genes in the whole sixth instar.

(5) As mentioned above, some important Drosophila RTKs such as breathless are missing in their analyses. As breathless is a close paralog of heartless (Htl), I am sure that Drosophila breathless is also orthologous to Helicoverpa FGFR1. The authors therefore need to analyze breathless in Figure 5B in addition to Htl.

Thank you for your advice. We added experiments and the results are shown in Figure 5B and Figure 5—figure supplement 1.

(6) More discussion about the reason why dsNrk and dsWsck can provide resistance to JHIII in Figure 1 is required.

Thank you for your advice. We added explanation in the discussion: "It is generally believed that the primary role of JH is to antagonize 20E during larval molting (Riddiford, 2008). The knockdown of Cad96ca, Nrk, Fgfr1, and Wsck showed phenotypes resistant to JH III induction and the decrease of Kr-h1 and increase of Br-z7 expression, but knockdown of Vegfr and Drl only decrease Kr-h1, without increase of Br-z7. Br-z7 is involved in 20E-induced metamorphosis in H. armigera (Cai et al., 2014), whereas, Kr-h1 is a JH early response gene that mediates JH action (Minakuchi et al., 2009) and represses Br expression (Riddiford et al., 2010). The high expression of Br-z7 is possible due to the down-regulation of Kr-h1 in Cad96ca, Nrk, Fgfr1 and Wsck knockdown larvae. The different expression profiles of Br-z7 in Vegfr and Drl knockdown larvae suggest other roles of Vegfr and Drl in JH signaling, which need further study."

Reviewer #3 (Recommendations For The Authors):

(1) The authors should consider optimizing their experimental approach by depleting the six candidate RTKs in an early larval stage rather than using a sensitized background with JH application in the last larval stage.

Thank you for your precious suggestion. We knocked down the genes at last larval stage to observe pupation, which is a relatively simple and easily to be observed target to examine the role of the gene in JH-maintained larval status. The results from CRISPR/Cas9 experiments showed: "Most wild-type larvae showed a phenotype of pupation on time. However, in the Cad96ca mutant, 86% of the larvae (an editing efficiency of 67% by TA clone analysis) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 24 h earlier. In the Fgfr1 mutant, 91% of the larvae (an editing efficiency of 61%) had a shortened feeding stage in the sixth instar and entered the metamorphic molting stage earlier, showing early pupation, with the pupation time being 23 h earlier (Figure 4D and E). The data suggested that CAD96CA and FGFR1 support larval growth and prevent pupation in vivo.". To know the roles of other RTKs in the whole larval development needs future work since a lot of experiments are needed.

(2) Including a positive control for JH signaling, such as met or tai, would strengthen the assays and provide a benchmark for evaluating the downregulation of target genes and phenotype reversion upon JH application. This addition, especially in Figure 1, would enhance the interpretability of the results.

Thank you for your suggestion. We agree with your point of view that adding the detection of Met or Tai as a positive control. Our laboratory has reported in previous studies that knockdown of Met leads to decreased expression of genes in the JH signaling pathway and precocious pupation (PMID: 24872508), so we did not repeat this related experiment in this study. In the future, when performg Cad96ca and Fgfr1 double mutant experiments, Met mutant can be generated as a control to provide more references for the interpretation of the results.

(3) I recommend revising the manuscript to improve readability, particularly in the Results section, where descriptions of the binding part are particularly dense.

Thank you for your advice. We have carefully revised the manuscript.

(4) In line 122, please add the reference Wang et al., 2016.

Thank you for your reminding, we have added the reference in line 125 of the new manuscript.

(5) The authors should clarify why they chose to test the possible binding to JH of only Cad96CA, FGFR1, and NRK after conducting various assays while including OTK in the study as a negative control. This explanation should be included in the text.

Thank you for the suggestion. We added the explanation, as described in the text: "We screened the RTKs sequentially, including examining the roles of 20 RTKs identified in the H. armigera genome in JH regulated-gene expression to obtain primary candidates, followed by screening of the candidates by their roles in maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and affinity to JH. The cadherin 96ca (CAD96CA) and fibroblast growth factor receptor 1 (FGFR1) were finally determined as JH cell membrane receptors by their roles in JH regulated-gene expression, maintaining larval status, JH induced-rapid increase of intracellular calcium levels, JH induced-phosphorylation of MET and TAI, and their JH-binding affinity. Their roles as JH cell membrane receptors were further determined by knockdown and knockout of them in vivo and cell lines, and overexpression of them in mammal HEK-293T heterogeneously.".

"Since Cad96CA, FGFR1, and NRK were not only involved in JH-regulated Kr-h1 expression, JH III-induced delayed pupation, and calcium levels increase, but also involved in MET and TAI phosphorylation, we further analyzed their binding affinity to JH III. OTK did not respond to JH III, so we used it as a control protein on the cell membrane to exclude the possibility of nonspecific binding.".

(6) The observed embryonic lethality of cad96ca and FGF1 mutants in Drosophila contrasts with the ability of the respective mutants in H. armigera to reach the pupal stage. The authors should discuss this significant difference.

Thank you for the suggestion. We added the explanation in the discussion, as described in the text: "Homozygous Cad96ca null Drosophila die at late pupal stages (Wang et al., 2009). However, we found that 86% of the larvae of the Cad96ca mutant successfully pupated in G0 generation, although earlier than the control. Similarly, null mutation of Fgfr1 or Fgfr2 in mouse is embryonic lethal (Arman et al., 1998; Deng et al., 1994; Yamaguchi et al., 1994). In D. melanogaster, homozygous Htl (Fgfr) mutant embryos die during late embryogenesis, too (Beati et al., 2020; Beiman et al., 1996; Gisselbrecht et al., 1996). However, in H. armigera, 91% of larvae successfully pupated in G0 generation after Fgfr1 knockout. The low death rate after Cad96ca and Fgfr1 knockout might be because of following reasons, including the editing efficiency (67% and 61% for Cad96ca mutant and Fgfr1 mutant, respectively), the chimera of the gene knockout at the G0 generation, and the redundant RTKs that play similar roles in JH signaling, similar to the redundant roles of MET and Germ-cell expressed bHLH-PAS (GCE) in JH signaling (Liu et al., 2009), which needs to obtain alive G1 homozygote mutants and double knockout of these two receptors in future study. We indeed observed that the eggs did not hatch successfully after mixed-mating of G0 Cad96ca mutant or Fgfr1 mutant, respectively, but the reason was not addressed further due to the embryonic death. By the similar reasons, most of the Cad96ca and Fgfr1 mutants showed a slight acceleration of pupation (about one day) without the typical precocious metamorphosis (at least one instar earlier) phenotype caused by JH signaling defects (Daimon et al., 2012; Fukuda, 1944; Riddiford et al., 2010) and JH pathway gene deletions (Abdou et al., 2011; Liu et al., 2009). On other side, JH can regulate gene transcription by diffusing into cells and binding to the intracellular receptor MET to conduct JH signal, which might affect the results of gene knockdown and knockout.".

(7) Building upon the previous point, it is noteworthy that the cad96ca and FGF1 mutants exhibit only a 24-hour early pupation phenotype, contrasting with the 48-hour early pupation induced by Kr-h1 depletion. This discrepancy suggests that while the function of these RTKs is necessary, it may not be sufficient to fully activate JH signaling. The expression profile of these receptors, primarily observed in the last larval stage, supports this hypothesis.

Thank you for your suggestion. We added the explanation in the discussion, as described in the text: "Homozygous Cad96ca null Drosophila die at late pupal stages (Wang et al., 2009). However, we found that 86% of the larvae of the Cad96ca mutant successfully pupated in G0 generation, although earlier than the control. Similarly, null mutation of Fgfr1 or Fgfr2 in mouse is embryonic lethal (Arman et al., 1998; Deng et al., 1994; Yamaguchi et al., 1994). In D. melanogaster, homozygous Htl (Fgfr) mutant embryos die during late embryogenesis, too (Beati et al., 2020; Beiman et al., 1996; Gisselbrecht et al., 1996). However, in H. armigera, 91% of larvae successfully pupated in G0 generation after Fgfr1 knockout. The low death rate after Cad96ca and Fgfr1 knockout might be because of following reasons, including the editing efficiency (67% and 61% for Cad96ca mutant and Fgfr1 mutant, respectively), the chimera of the gene knockout at the G0 generation, and the redundant RTKs that play similar roles in JH signaling, similar to the redundant roles of MET and Germ-cell expressed bHLH-PAS (GCE) in JH signaling (Liu et al., 2009), which needs to obtain alive G1 homozygote mutants and double knockout of these two receptors in future study. We indeed observed that the eggs did not hatch successfully after mixed-mating of G0 Cad96ca mutant or Fgfr1 mutant, respectively, but the reason was not addressed further due to the embryonic death. By the similar reasons, most of the Cad96ca and Fgfr1 mutants showed a slight acceleration of pupation (about one day) without the typical precocious metamorphosis (at least one instar earlier) phenotype caused by JH signaling defects (Daimon et al., 2012; Fukuda, 1944; Riddiford et al., 2010) and JH pathway gene deletions (Abdou et al., 2011; Liu et al., 2009). On other side, JH can regulate gene transcription by diffusing into cells and binding to the intracellular receptor MET to conduct JH signal, which might affect the results of gene knockdown and knockout.".

(8) The expression profile of the RTK hits described in Supplementary Figure 4A appears to be limited to the last larval stage until pupation. The authors should clarify whether these receptors are expressed earlier, and the meaning of the letters in the plot should be described in the figure legend.

Thank you for the suggestion. We added the explanation in the Figure 1—figure supplement 4 legend, as described in the text: "The expression profiles of Vegfr1, Drl, Cad96ca, Nrk, Fgfr1, and Wsck during development. 5F: fifth instar feeding larvae; 5M: fifth instar molting larvae; 6th-6 h to 6th-120 h: sixth instar at 6 h to sixth instar 120 h larvae; P0 d to P8 d: pupal stage at 0-day to pupal stage at 8-day F: feeding stage; M: molting stage; MM: metamorphic molting stage; P: pupae.".

We are very sorry, but due to time limitations, we will investigate the expression profile of RTK throughout the larval stage in future work.

(9) In Figure 4, panels F and G, the levels of Kr-h1 are shown in cad96ca and FGF1 mutants in the last larval stage. The authors should indicate whether Kr-h1 levels are also low in earlier larval stages or only detected in the last larval stage, as this would imply that these RTKs are only required at this stage.

Thank you for your suggestion. In this study, the Cad96ca and Fgfr1 mutants' feeding stage was shortened in the sixth instar, and they entered the metamorphic molting stage earlier. So, we detected the expression of Kr-h1 in the sixth instar. It is an excellent idea to detect the expression of Kr-h1 at various larvae stages to analyze the stages in which CAD96CA and FGFR1 play a role and to study the relationship between CAD96CA and FGFR1 in future.

(10) While Figure 5 demonstrates JH-triggered calcium ion mobilization in Sf9 cells and S2 cells, the authors should also include data on JH signaling target genes, such as Kr-h1, for a more comprehensive analysis.

Thank you for your advice. We added experiments, as described in the text: "To demonstrate the universality of CAD96CA and FGFR1 in JH signaling in different insect cells, we investigated JH-triggered calcium ion mobilization and Kr-h1 expression in Sf9 cells developed from S. frugiperda and S2 cells developed from D. melanogaster. Knockdown of Cad96ca and Fgfr1 (named Htl or Btl in D. melanogaster), respectively, significantly decreased JH III-induced intracellular Ca2+ release and extracellular Ca2+ influx, and Kr-h1 expression (Figure 5A, B, Figure 5—figure supplement 1A and B). The efficacy of RNAi of Cad96ca and Fgfr1 was confirmed in the cells (Figure 5—figure supplement 1C and D), suggesting that CAD96CA and FGFR1 had a general function to transmit JH signal in S. frugiperda and D. melanogaster.".

(11) The authors should consider improving the quality of images and some plots, particularly enlarging panels showing larval and pupal phenotypes, such as Figure 1B and Supplementary Figure C. Additionally, adding a plot showing the statistical analysis of the phenotype in Supplementary Figure C would enhance clarity. Some plots are overly busy and difficult to read due to small size, such as Figure 1C, Figure 2A, and all the plots in Figure 3. Figure 4E also requires improvement for better readability.

Thank you for your suggestion. We have adjusted Figure 1B, Figure 1C, Figure 1—figure supplement 1C, Figure 2A and Figure 4E. However, for Figure 3, we have not found a better way to arrange and adapt them, considering the overall arrangement of the results and the page space, so we keep them in their original state.

I think this is wrong. Although the platform can recommend better content to us after getting our information, such behavior will cause trouble to users. When people do not want their information to be discovered by others or their interests change and they are tired of their previous interests, seeing the content pushed by the platform based on data mining will make users irritated.

Not social media, but Google is egregious at using dystopian tactics to determine your profile. There is a famous study (that has sense been replicated in a livestream) where typing the number of letters required to autofill for "dog toys" in google is highly dependent on how frequently your device's microphone has picked up dog related words. Anecdotally, I know it's not just microphone data, but keystroke data as well.

This shows how social media data can reveal personal traits, such as political views or susceptibility to scams, and societal trends, like misinformation. While unconventional data can provide insights, such as linking COVID-19 to bad candle reviews, concerns about privacy, bias, and the use of flawed methods remain significant in the world of social media and content

This part is interesting about raising socil media, as it potentially let's us know abput the rough definitiion and meaning of data mining which is using data to present it in a way

I find it fascinating that data mining specifically regarding social media data can be used to determine public opinion by obtaining emotions, ideas and motives behind posts. In which case this application of data mining can be applied to the social media aspect of brands when they want to gauge their audience's interests.

This is really creative in leveraging unusual data to find a trend. Who would have imagined that COVID-19 cases are correlated with negative reviews of Yankee Candle-a proxy for not being able to smell, a known symptom-from product reviews? Indeed, an interesting case of how sometimes unrelated streams of data can point to a pattern or predict a trend in public health. It also underlines that any data correlations have to be subject to critical examination in order not to jump to misleading conclusions, as in the case of negative reviews, the reasons could be altogether different. This is a good example of how strong and/or limited data interpretation can be in a real-life setting.

It’s alarming that even interests or vulnerabilities, such as a susceptibility to financial scams or addiction, can be guessed from online behavior. What’s even more disturbing is the use of AI to repackage debunked, racist theories about facial features to make judgments about people. Social media also plays a role in tracking broader trends, like how misinformation spreads, which affects public opinion on a massive scale. It's a reminder of how our online data is used in ways we might not even realize, sometimes with harmful consequences.

many sections feel like they stand on their own, large contrast between these two stanzas

I personally have to work with data a lot and line graph are one of the chart I use the most to show how value of items can change over time.

While this report highlights of drugs that have shown up in the toxicology reports, it is important to note that he was human and he had many friends, family, and fans who cared about him. It's important to end such a heavy article like this to make it more sensitive rather than insensitive.

Could this death have been prevented if authorities or someone had taken action quicker?

This 911 call is important to add to news stories because there were things that people noticed before the star fell to his death. This call serves as a witness to this tragic death and is beneficial to reporting as well as determining what actually happened.

The headline is straight and to the point, but we should emphasize the word "reportedly." This implies that this is not 100% percent confirmed, but it is in speculation and possibility.

The toxicology and autopsy report of this star's tragic death has just been released which is very recent. There were many speculations in the media about how Liam Payne died because it wasn't completely apparent at the scene of his death. News like this can give readers the opportunity to creative new feelings for this situation as well.

This is important to note when analyzing content. NBC News admits that have yet to completely confirm the sources they used so readers must take "facts" with caution.

This is very similar to what the NFL does with Redzone where they show all the action from all the games

I think this is very human interest focused because they obviously brought it back because all the NHL fans across the world enjoyed it very much

I think this is a good way to open their article because it makes people actually think "Why are the games starting so early"

So the benefit of the responsibility approach is that it justifies why someone should be liable for a harm caused, or why they should owe nothing to the victim by having established a mechanism allowing an analysis to be undertaken as to whether or not someone is 'morally' liable

So the proposal of the 'responsibility approach' to tort is that under tort someone will be liable if they are (A) responsible for the outcome, and (B) morally responsible for causing that outcome.

The banner/general proposition of the responsibility approach to tort law

An outline/summary of the content of the responsibility approach to tort law

Key summary of modern tort law.

STILL so user-write oriented

very user-write oriented—people still have to do roughly the same amount of work

possible MM use case: ask your teammate who left a question

not enough

Hmm. I can't find anything about this! It would be nice if we had a scan.

(Since philsp.com links are unstable, this is cited as "Best Stories of All Time [v1 #6, November 1925]")

z

See I may not have the full story of Congo, the virus, and the people, but they told me pretty much everything I want to know in that one paragraph.

Lots of signs here, we're being offered a very sad image here, which sets the scene for dealing with the problem we're talking about.

I picked this because it has a non-politicized topic about something that still very strongly affects the lives of a lot of people. The values being offered are timeliness and human interest.

shifted topic from the headline.

Doesn't dive much deeper into this. Probably because it isn't related to Trump working at Mcdonald's but still an interesting addition.

Factual comment. Although this statement might sound like it has some sort of bias,I don't believe it does. Trump has disputed Kamala's claims of being a former Mcdonald's employee and hasn't offered evidence for that claim. In regard to the uncertainty of whether she has or hasn't worked for Mcdonald's before, there is no evidence that she has or hasn't.

I find it interesting that the Mcdonald's is making it clear to the public that they have no political affiliation in the race. Again, I do think it is necessary, but it leads me to ask further questions such as were the crew working with Trump happy with his attendance? Were employees given the option to work with him, or was it randomly mandatory regardless of the employee political opinions.

Clearly, the Mcdonald's that hosted Trump made it clear that they are not biased or in favor of one party over the other. They may have had to make this clear for the sake of their business. I wouldn't find it hard to believe that some people may boycott or even damage, or threaten the restaurant just for having Trump.

This clearly defines to the reader the circumstances of Trump's "shift". We now know that the people in the drive through weren't just random but "pre-selected". We can now infer that he really was handing food out to real people, but they weren't just anybody.

The first thing I noticed about this article was obviously the title. I like how it doesn't feel like there is an underlying agenda or bias. The title is very straightforward and factual. Clearly and simply states that Trump worked a drive-thru before town hall.

bootstrap peer list

I believe that API's are very important as they like Reddit’s PRAW library, enable structured access to data. While the PRAW documentation offers deeper insights into its capabilities, it may be challenging to navigate, requiring patience and exploration to master its use to ensure that its full potential and purpose is being utilized.

I think data poisoning can have severe consequences which are often overlooked including the idea that misleading information and drastically reduced accuracy can lead to big companies or brands especially on social media making big decisions leading to losses and failures.

This part raises an important point; If the purpose of data mining is usually to use data and present it in a way that gies insights and understandings, what would the point of having online surveys that use fake data and poisoning be as it will generate an inaccurate data representation?

I wonder how people working with data sets that may be poisoned go about fixing them or determining how detrimental the poison really is in the data set. It makes sense how one person with a specfic demographic fan base would be able to mess up the data in a survey but its kind of obvious thinking about it now. I wonder why i never heard of this story or ones similar.

This poisoning of the dataset is going to connect with the Deepfake issue in Korea, as data manipulation and biased inputs have severe consequences in terms of outcomes. Applications in deepfakes rely on enormous datasets of images and videos that feed into algorithms that generate realistic fake content. If these datasets are contaminated with biased or skewed data-intentional or unintentional-such as overrepresentation within certain demographics, then the outcomes will be very problematic. On one hand, it is similar that viral survey participation undermines data reliability, while deepfake datasets poisoned with biased material lead to applications considered very unethical or harmful, such as creating non-consensual videos or misinformation. In fact, these two examples drive the message home regarding the risk involved with unregulated data inputs in critical systems.

It made me realize how unpredictable, even unexpected, the influence of social media can be. A seemingly innocuous video can have such a profound impact on scientific research that the data can no longer be used. It also made me think about the importance of data quality and diversity, and how to prevent this kind of “data pollution” from happening again.

Unintentional and intentional data poisoning can severely compromise the usefulness of datasets. Whether caused by viral social media trends or deliberate sabotage, such as spamming job applications, these incidents highlight the vulnerability of data collection processes and the potential for disruption, often undermining research or organizational operations.

I did not use the google account that used UW email too much. However, surprisingly, google knows a lot of my private information. And a lot of them are pretty accurate. This also make me to think whether I need to improve my awareness on data security.

I did not use the google account that used UW email too much. However, surprisingly, google knows a lot of my private information. And a lot of them are pretty accurate. This also make me to think whether I need to improve my awareness on data security.

Yes, for example health data (especially mental health ) is very risky for users. Anxiety, depression, or suicidal ideation language on net can be identified using data mining. Social media exploit this to push targeted content or manipulate users during vulnerable times.

Whoa, are the 1920s issues really not that widely available?

Continuing to scroll down, "Our Core Team" [Minor edit: Capitalization]....

What is the "Resume Review" tab [at the top of your page] does not, however, appear sequentially, as everything else does, on this page.

Again, from the perspective of "job seeker" who might be unclear as to the breadth of services that you provide, not seeing the "Resume Review" as I scroll down the page - despite everything else appearing there - might leave me a bit confused.

On this "Home" tab....

I'd suggest inverting the order of the "Work: What We Do" and "Success: How We Help You Get the Right Team" sections.

As I read [through the eyes of "Job Seeker"], I was initially thrown off given that I don't need help "find[ing] the right people for the job", rather, to BE that "right person" who is found for a job!!

I feel like the information that is given to these social media sights is very private. They should make sure to ask for the proper permission when sharing sensitive data like search history or medical history.

I feel like i knew a lot about how different companies collect data on users but im always shocked to learn different ones that i didnt think about. I never would of thought that knowing when users are logged on and off is something that these companies tracked. Im curious to what other methods they use that people might not know about,

Personally, I do not like such targeted advertisements, because it is a violation of privacy to let advertisers know too much about users' privacy. Advertisers are using social media to collect our information and make profits for themselves. I think the advertising push on social media can only refer to the age and gender information in each user's profile.

I think this needs its own section for how to add students to a Cycle or how to add additional Forms.

GUARANTEES??

Are there any that you're ready to stand behind? Any that you'd be willing to offer your job seeker for choosing "MJL Projects LLC" over another résumé service?

Perhaps some type of "Post-Review Support Guarantee" that provides some type of ongoing support for a specified period (e.g., 30, 60, or 90 days) after the review is completed?

Again, with all the services and promises flung at desperate candidates, offering this type of limited/ongoing support might help to ease concerns about who to entrust with this #RésuméRevamp

"LINE BY LINE REVIEW":

But what, exactly, does that look like??

For job seekers that have sooooo many people pitching sooooo many different services, the one thing that I want to see is what this "line by line review" and the "detailed feedback on how...[to] improve" your résumé ACTUALLY looks like.

Is there a way that you can provide a couple of samples??

Just some thoughts:

• "Take your résumé bulletpoints from this [insert poorly (over)worded job description] to this [provide your polished version of the bullet" with our "Line by Line Review"

• "Let us help you market yourself most effectively to give you an edge in this highly competitive market"

'she felt sick' has the same impact

tone is too chatty

if she's laughing so hard her eyes water, how is her face barely moving?

tone is off

italics - also we know that she can control this, so the sentence is redundant

'in the frenzy to escape'

'the smell of eggs now mixing with the stench of tobacco...'

'- which came from the trash, definitely - '

what do the italics symbolise here

they're on the wall? how?

i think this word offsets the tone

this is too long

this is too long and we lose track of the sentence

interesting name but runs the risk of sounding too 2016 YA

i'm not sure about this paragraph - i don't think the repetition is effective here, and 'not even actually' is clunky. i also don't like the italics

Below the laptop, there's supposed to be a line, "Plus, don't miss out on our exclusive resources and tools -- available now on Eddy!"

Text is supposed to be dark blue. "Get in touch with our team" is supposed to be underlined and bolded

Color should be dark blue (same as the "Grants Available" line

Color of the text here is supposed to be a lighter blue. See figma

(same comment for all the body text in this section)

There's supposed to be subheader text below this, but it doesn't show up here. See the figma file

Author response:

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

Reviewer #1 (Public Review):

Summary:

The extra macrochaetae (emc) gene encodes the only Inhibitor of DNA binding protein (Id protein) in Drosophila. Its best-known function is to inhibit proneural genes during development. However, the emc mutants also display nonproneural phenotypes. In this manuscript, the authors examined four non-proneural phenotypes of the emc mutants and reported that they are all caused by inappropriate non-apoptotic caspase activity. These non-neuronal phenotypes are: reduced growth of imaginal discs, increased speed of the morphogenetic furrow, and failure to specify R7 photoreceptor neurons and cone cells during eye development. Double mutants between emc and either H99 (which deletes the three pro-apoptotic genes reaper, grim, and hid) or the initiator caspase dronc suppress these mutant phenotypes of emc suggesting that the cell death pathway and caspase activity are mediating these emc phenotypes. In previous work, the authors have shown that emc mutations elevate the expression of ex which activates the SHW pathway (aka the Hippo pathway). One known function of the SHW pathway is to inhibit Yorkie which controls the transcription of the inhibitor of apoptosis, Diap1. Consistently, in emc clones the levels of Diap1 protein are reduced which might explain why caspase activity is increased in emc clones giving rise to the four non-neural phenotypes of emc mutants.

However, this increased caspase activity is not causing ectopic apoptosis, hence the authors propose that this is nonapoptotic caspase activity. In the last part of the manuscript, the authors ruled out that Wg, Dpp, and Hh signaling are the target of caspases, but instead identified Notch signaling as the target of caspases, specifically the Notch ligand Delta. Protein levels of Delta are increased in emc clones in an H99- and dronc-dependent manner. The authors conclude that caspase-dependent non-apoptotic signaling underlies multiple roles of emc that are independent of proneural bHLH proteins.

Strengths:

Overall, this is an interesting manuscript and the findings are intriguing. It adds to the growing number of non-apoptotic functions of apoptotic proteins and caspases in particular. The manuscript is well written and the data are usually convincingly presented.

Weaknesses:

(1)  One major concern I have is the observation by the authors in Figure 3C in which protein levels of Diap1 are still reduced in emc H99 double mutant clones. If Diap1 is still reduced in these clones, shouldn't caspases still be derepressed? Given that emc H99 double mutants rescue all emc phenotypes examined, the observation that Diap1 levels are still reduced in emc H99 clones is inconsistent with the authors' model. The authors need to address this inconsistency.

The effect of H99 emc clones on Diap1 protein levels is consistent with our conclusions.  The reviewer’s concern probably relates to previous work that shows that RHG proteins act by antagonizing DIAP1, so that Diap1 is epistatic to RHG (PMID:10481910), and that RHG proteins affect DIAP1 protein levels, and in particular that HID promotes DIAP1 ubiquitylation leading to its destruction (PMID:12021767).  First, epistasis means that in the absence of DIAP1, RHG levels do not affect cell survival.  DIAP1 protein is not absent in emc/emc eye clones, however, it is reduced.  It is not only possible but expected that RHG levels would affect survival when DIAP1 levels are only reduced.  Secondly, we did not see a difference in DIAP1 levels between H99/H99 clones and H99/+ cells within the same specimen, suggesting that rpr, grim and hid might not affect DIAP1 levels. It is possible that Hid protein only affects DIAP1 levels when overexpressed, as in the aforementioned paper (PMID:12021767), and that physiological RHG levels affect DIAP1 activity.  The H99 deficiency also eliminates Rpr and Grim, which may affect DIAP1 without ubiquitylating it. In our experiments, however, there are no cells completely wild type for the H99 region for comparison in the same specimen, so our results do not rule out the H99 deletion having a dominant effect on DIAP1 levels both inside and outside the clones.  What our data clearly showed is that emc affected DIAP1 levels independently of any potential RHG effect, and we hypothesized this was through diap1 transcription, because we showed previously that emc affects yki, a transcriptional regulator of the diap1 gene, but we have not demonstrated transcriptional regulation of diap1 directly in emc clones.  We modified the manuscript to better delineate these issues (lines 275-284).    

(2) Are Diap1 protein levels reduced in all emc clones, including clones anterior to the furrow? This is difficult to see in Figure 3B. it is also recommended to look in emc mosaic wing discs.

We now mention that DIAP1 levels were only reduced in  emc clones posterior to the morphogenetic furrow, not anterior to the morphogenetic furrow or in emc clones in wing imaginal discs (lines 284-5) and Figure 3 supplement 1.  

(3) The authors speculate that Delta may be a direct target of caspase cleavage (Figure 9B), but then rule it out for a good reason. However, I assume that the increased protein levels of Delta in emc clones (Figure 7) are the results of increased transcription. In that case, shouldn't caspases control the transcriptional machinery leading to Delta expression?

Thank you for suggesting that caspases control the transcription of Dl.  We added this possibility to the manuscript (lines 499-500).  At one time there was a Dl-LacZ transcriptional reporter, which would have made it straightforward to assess Dl transcription in emc clones, but this strain does not seem to exist now.  We have not attempted in situ hybridization to Dl transcripts in mosaic discs.  

(4) How does caspase activity in emc clones cause reduced growth? Is this also mediated through Delta signaling?

We do not know what is the caspase target responsible for reduced growth in wing discs.

(5) Figure 1M: Is there a similar result with emc dronc mosaics?

The emc dronc clones do not show as dramatic a growth advantage in a Minute background.  This is consistent with the smaller effect of emc dronc in the non-Minute background also (Figure 1N).  We mention this in the revised paper (lines 232-3).     

Reviewer #2 (Public Review):

Id proteins are thought to function by binding and antagonizing basic helix-loop-helix (bHLH) transcription factors but new findings demonstrate roles for emc including in tissues where no proneural (Drosophila bHLH) genes are known to function. The authors propose a new mechanism for developmental regulation that entails restraining new/novel non-apoptotic functions of apoptotic caspases.

Specifically, the data suggest that loss of emc leads to reduced expression of diap1 and increased apoptotic caspase activity, which does not induce apoptosis but elevates Delta expression to increase N activity and cause developmental defects. Indeed, many of the phenotypes of emc mutant clones can be rescued by a chromosomal deficiency that reduces caspase activation or by mutations in the initiator caspase Dronc. A related manuscript that shows that loss of emc results in increased da, linked previously to diap1 expression, provides supporting data. There is increasing appreciation that apoptotic caspases have non-apoptotic roles. This study adds to the emerging field and should be of interest to readers.

The data, for the most part, support the conclusions but I do have concerns about some of the data and the interpretations that should be addressed.

Reviewer #3 (Public Review):

The work extends earlier studies on the Drosophila Id protein EMC to uncover a potential pathway that explains several tissue-scale developmental abnormalities in emc mutants. It also describes a non-apoptotic role for caspases in cell biology.

Strengths:

The work adds to an emerging new set of functions for caspases beyond their canonical roles as cell death mediators. This novelty is a major strength as well as its reliance on genetic-based in vivo study. The study will be of interest to those who are curious about caspases in general.

Weaknesses:

The manuscript relies on imaging experiments using genetic mosaic imaginal discs. It is for the most part a qualitative analysis, showing representative samples with a small number of mutant clones in each. Although the senior author has a long track record of using experiments like this to rigorously discover regulatory mechanisms in this system, it is straightforward in 2023 to use Fiji and other image analysis tools to measure fluorescence. Such measurements could be done for all replicate clones of a given genotype as well as genetic control sampling. These could be presented in plots that would not only provide quantitative and statistical measurements, but will be more reader- friendly to those who are not fly people.

We added quantification of anti-Delta and anti-Diap1 levels to the manuscript (Figures 3E and 7E).  We agree that this facilitates statistical confirmation of the results and may be more accessible to non-experts.  We do have concerns that these quantifications might be given too much weight.  For example, we cannot measure the background level of anti-DIAP1 labeling by labeling diap1 null mutant cells, because such cells do not survive.  Although we measure ~20% reduction in emc clones in the eye disc, and none in the wing disc, both measures could be underestimates if some of the labeling is non-specific, as is very possible.  We discuss this in the Methods (lines 166-9).

Likewise, more details are needed to describe how clone areas were measured in Figure 1. Did they measure each clone and its twin spot, and then calculate the area ratio for each clone and its paired twin spot? This would be the correct way to analyze the data, yielding many independent measurements of the ratio. And doing so would obviate the need to log transform the data which is inexplicable unless they were averaging clones and twins within a disc and making replicates. More explanation is needed and if they indeed averaged, then they need to calculate the ratios pairwise for each clone and twin.

We added details of clone size measurements and analysis to the methods (lines 141-6).  Although it might be useful to compare individual clones and corresponding twin spots, the only rigorous way to associate individual clones with individual twin spots, or even to determine what is one clone and what is one twin spot, is to use recombination rates low enough that significantly less than one recombination occurs per disc.  This would require many more dissections and we did not do this.  We now clarify in the manuscript that the analysis is indeed based on the ratio of total area of clones and twin spots with replicates, and that Log-transformation is to improve the normality of the ratio data suitable for parametric significance testing, not because clones and twin spots were summed from each sample.  We consulted with a statistician over this approach.  

Reviewer #1 (Recommendations For The Authors):

Lines 319/320: "Frizzled-3 RFP expression was not changed in in emc clones (Figure 4A)". This was actually not shown in Fig 4A (in fact this result was not shown at all). Fig 4A shows the result for emc nkd3 which the authors incorrectly assigned to Figure 4B (line 324).

We apologize for labeling Figure 4A and 4B incorrectly.

The title of Figure 6 is inaccurate. The title does not indicate what is shown in this figure. A more accurate title would be: Notch activity and function in emc mutant clones.

We provided a new title for Figure 6. 

Reviewer #2 (Recommendations For The Authors):

There is no information on how reproducible the data is. How many discs were examined in each experiment and in how many technical or biological replicates? Can fluorescence signals be quantified within and outside the clones and presented to illustrate reproducibility and significance? This is especially needed for Fig 7, which shows key data that N ligand Delta is elevated in emc clones but dronc and H99 mutations rescue this phenotype. I can see that the Dl signal is brighter in the GFP- emc clone in Fig 7B but I can also see a brighter Dl signal in the small clone and perhaps also in the large clone in C. The difference between B and C could be simply disc-to-disc variation, which should be addressed with quantification and presentation of all data points.

We added the number of samples to each figure legend.  We quantified the fluorescence signals for Figures 3 and 7.  Quantification shows that the difference between 7B and 7C is highly significant, not disc to disc variation.

Fig 2B does not support the conclusion. It is supposed to show premature Sens expression and therefore abnormal morphogenetic furrow progression in emc clones. But the yellow arrow is pointing to GFP+ (wild type) cells and it is within this GFP+ region that most premature Sens expression is seen.

We relocated the arrows in Figure 2B to point precisely to the premature differentiation.  When the morphogenetic furrow is accelerated in emc mutant, GFP – tissue, it does not stop when wild type, GFP+ tissue is encountered again, it continues at a normal pace.  Accordingly, emc+ regions that are anterior to emc- regions can also experience accelerated differentiation (please see lines 594-8).

Fig 1 shows that while H99 deficiency restores the growth of emc clones to wild type level (Fig 1N), placing these in the Minute background made emc clones grow better than emc wild type but Minute neighbors (Fig 1M). The latter cells were nearly absent, suggesting elimination through cell competition. For the rest of the figures, some experiments are done in the Minute background (e.g., emc H99 clones in Fig 2D) while others are not in the Minute background (e.g., emc H99 clones in Fig 7D). Why the switch between backgrounds from experiment to experiment?

Figure 2D shows emc H99 clones in a Minute background so that it can be compared with panels 2A-C, which show clones of other genotypes in a Minute background.  These clones almost take over the eye disc.  In Figure 7D, it was important to show the Dl expression pattern in a substantial wild type region, which could only be shown using the non-Minute background.  We have no indication that a Minute background changes the properties of the nonMinute clone, other than allowing its greater growth.  

The first 3 paragraphs of the Introduction are overly detailed and read more like a review article. These could be made more concise to focus on the founding data for this manuscript, which are the published findings that emc mutations elevate ex expression (line 129) and that ex mutants show elevated diap1 expression (line 125). These do not show up until the very end of the Introduction.

We shortened the Introduction to focus more rapidly on the topics relevant to these experiments.

In several places, the space between the end of the sentence and the citation is missing (e.g., lines 57, 68, and 75).

The spacing of citations was fixed.

Line 247. 'morphogenetic furrow that found each ommatidia...' should use a word besides 'found.'

We corrected line 247.

Reviewer #3 (Recommendations For The Authors):

(1) The authors show that inhibiting caspases rescues the growth defect of emc clones. However, they did not find excessive TUNEL staining in emc clones that would explain why the clones would be so small - excessive cell death. How reliable was their tunel staining in being able to detect excessive apoptosis (only negative data was shown). Could they induce excessive cell death using radiation or some other means to ensure the assay is robust? If death is not occurring in emc clones, a deficiency worth addressing is that they do not discuss or explore how the caspases then inhibit clone growth. Is it expanded cell cycle times, or smaller cells?? And that phenotype does not fit with their end model of Delta being the only moderator of emc since it is not playing a significant role in tissue growth anterior to the furrow.One would assume using the commercial antibody against activated caspase would be another readout for emc clones and this would bolster their claim that excessive caspase activation occurs in the emc cells.

We have added Dcp1 staining in Figure 2 supplement 3 to show that TUNEL staining is reliable.

(2) Figure 3D has really large emc clones when GMR-Diap is present. But the large clones are anterior to the furrow where Diap would not be overexpressed. Is this just an unusual sample with a coincidentally big emc M+ clone? It speaks to my concerns about the qualitative nature of the data.

We replaced Figure 3D with an example of smaller clones.  Nowhere have we suggested that  GMR-DIAP1 affects clone size.

(3) Figure 9B is very speculative and not appropriate since the authors have zero data to support that cleavage mechanism. It is fit for the next paper if the idea is correct. The panel should be removed.

We did not intend Figure 9B to imply that we think Dl itself is the relevant target of non-apoptotic caspases.  Since apparently we gave that impression, we removed this to a supplemental figure.  We still think it is worth showing that Dl does not contain predicted caspase sites expected to activate signaling. 

(4) Figure 9A could be made more clear. Their pathway represents the mutant cells in the mosaic disc. Why not also outline what you think is happening in the emc+ cells as well?

It is difficult to make a comparable diagram for normal cells, because none of this pathway happens in normal cells.  We modified the figure legend to indicate this (lines 677-8).

(5) The one emc ci clone they show spanning the furrow has a very non-continuous furrow advance phenotype. This is unlike the emc clones where the furrow advance is graded about the clone. And it resembles the SuH clones they show. This result and the synergistic effect on clone sizes they mention need more discussion and thought put into it. It argues ci is doing something with respect to emc action. loss of ci might not rescue size and furrow advance but actually, it makes it worse! This is interesting and might suggest an inhibitory role for ci in emc or a parallel role for ci in mediating growth and progression that is redundant with emc.

We agree that aspects of the emc ci phenotype are not clear.  We discuss this in the revised manuscript (lines 373-5).  

(6) Related to point 7, it is a weak argument for non-autonomy that graded furrow advance in emc clones is evidence for emc acting nonautonomously through Delta. Its weakness is combined with its lack of significance relative to the other findings. It should be deleted as should the SuH data.

We agree that the evidence that emc affects morphogenetic furrow progression non-autonomously is not compelling and have revised the manuscript to soften this conclusion (lines 426-7).  We do not want to remove this idea, because it does in fact have significance for other findings.  Specifically, it supports the idea that the emc effect in the morphogenetic furrow is due to trans-activation by Delta, whereas  the effect on R7 and cone cell differentiation is due to autonomous cis-inhibition.  We think this is important to keep in the paper.

Reviewer #1 (Public review):

Summary:

The extra macrochaetae (emc) gene encodes the only Inhibitor of DNA binding protein (Id protein) in Drosophila. Its best-known function is to inhibit proneural genes during development. However, the emc mutants also display non-proneural phenotypes. In this manuscript, the authors examined four non-proneural phenotypes of the emc mutants and reported that they are all caused by inappropriate non-apoptotic caspase activity. These non-neuronal phenotypes are: reduced growth of imaginal discs, increased speed of the morphogenetic furrow, and failure to specify R7 photoreceptor neurons and cone cells during eye development. Double mutants between emc and either H99 (which deletes the three pro-apoptotic genes reaper, grim, and hid) or the initiator caspase dronc suppress these mutant phenotypes of emc suggesting that the cell death pathway and caspase activity are mediating these emc phenotypes. In previous work, the authors have shown that emc mutations elevate the expression of ex which activates the SHW pathway (aka the Hippo pathway). One known function of the SHW pathway is to inhibit Yorkie which controls the transcription of the inhibitor of apoptosis, Diap1. Consistently, in emc clones the levels of Diap1 protein are reduced which might explain why caspase activity is increased in emc clones giving rise to the four non-neural phenotypes of emc mutants. However, this increased caspase activity is not causing ectopic apoptosis, hence the authors propose that this is non-apoptotic caspase activity. In the last part of the manuscript, the authors ruled out that Wg, Dpp, and Hh signaling are the target of caspases, but instead identified Notch signaling as the target of caspases, specifically the Notch ligand Delta. Protein levels of Delta are increased in emc clones in an H99- and dronc-dependent manner. The authors conclude that caspase-dependent non-apoptotic signaling underlies multiple roles of emc that are independent of proneural bHLH proteins.

Strengths:

Overall, this is an interesting manuscript and the findings are intriguing. It adds to the growing number of non-apoptotic functions of apoptotic proteins and caspases in particular. The manuscript is well written and the data are usually convincingly presented.

Weaknesses:

The authors have addressed all my concerns and questions.

Reviewer #2 (Public review):

Id proteins are thought to function by binding and antagonizing basic helix-loop-helix (bHLH) transcription factors but new findings demonstrate roles for emc including in tissues where no proneural (Drosophila bHLH) genes are known to function. The authors propose a new mechanism for developmental regulation that entails restraining new/novel non-apoptotic functions of apoptotic caspases.

Specifically, the data suggest that loss of emc leads to reduced expression of diap1 and increased apoptotic caspase activity, which does not induce apoptosis but elevates Delta expression to increase N activity and cause developmental defects. Indeed, many of the phenotypes of emc mutant clones can be rescued by a chromosomal deficiency that reduces caspase activation or by mutations in the initiator caspase Dronc. A related manuscript that shows that loss of emc results in increased da, linked previously to diap1 expression, provides supporting data. There is increasing appreciation that apoptotic caspases have non-apoptotic roles. This study adds to the emerging field and should be of interest to the readers.

The revised manuscript addresses my concerns from the first round of review.

Reviewer #3 (Public review):

The work extends earlier studies on the Drosophila Id protein EMC to uncover a potential pathway that explains several tissue-scale developmental abnormalities in emc mutants. It also describes a non-apoptotic role for caspases in cell biology.

Strengths:

The work adds to an emerging new set of functions for caspases beyond their canonical roles as cell death mediators. This novelty is a major strength as well as its reliance on genetic-based in vivo study. The study will be of interest to those who are curious about caspases in general.

Weaknesses:

The authors did an adequate job in dealing with the limitations of the reviewed preprint. Although they could have done more, they chose not to for reasons they adequately defended.

Author response:

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

Reviewer #1 (Public Review):

(1) This experiment sought to determine what effect congenital/early-onset hearing loss (and associated delay in language onset) has on the degree of inter-individual variability in functional connectivity to the auditory cortex. Looking at differences in variability rather than group differences in mean connectivity itself represents an interesting addition to the existing literature. The sample of deaf individuals was large, and quite homogeneous in terms of age of hearing loss onset, which are considerable strengths of the work. The experiment appears well conducted and the results are certainly of interest. I do have some concerns with the way that the project has been conceptualized, which I share below.

Thank you for acknowledging the strengths and novelty of our study. We have now addressed the conceptual issues raised; please see below in the specific comments.

(2) The authors should provide careful working definitions of what exactly they think is occurring in the brain following sensory deprivation. Characterizing these changes as 'largescale neural reorganization' and 'compensatory adaptation' gives the impression that the authors believe that there is good evidence in support of significant structural changes in the pathways between brain areas - a viewpoint that is not broadly supported (see Makin and Krakauer, 2023). The authors report changes in connectivity that amount to differences in coordinated patterns of BOLD signal across voxels in the brain; accordingly, their data could just as easily (and more parsimoniously) be explained by the unmasking of connections to the auditory cortex that are present in typically hearing individuals, but which are more obvious via MR in the absence of auditory inputs.

We thank the Reviewer for the suggestion to clarify and better support our stance regarding reorganization. We indeed believe that the adaptive changes in the auditory cortex in deafness represent real functional recruitment for non-auditory functions, even in the relatively limited large-scale anatomical connectivity changes. This is supported by animal works showing causal evidence for the involvement of deprived auditory cortices in non-auditory tasks, in a way that is not found in hearing controls (e.g., Lomber et al., 2010, Meredith et al., 2011, reviewed in Alencar et al., 2019; Lomber et al., 2020). Whether the word “reorganization” should be used is indeed debated recently (Makin and Krakauer, 2023). Beyond terminology, we do agree that the basis for the changes in recruitment seen in the brains of people with deafness or blindness is largely based on the typical anatomical connectivity at birth. We also agree that at the group level, there is poor evidence of large-scale anatomical connectivity differences in deprivation. However, we think there is more than ample evidence that the unmasking and more importantly re-weighting of non-dominant inputs gives rise to functional changes. This is supported by the relatively weaker reorganization found in late-onset deprivation as compared to early-onset deprivation. If unmasking of existing connectivity without any functional additional changes were sufficient to elicit the functional responses to atypical stimuli (e.g., non-visual in blindness and non-auditory in deafness), one would expect there to be no difference between early- and late-onset deprivation in response patterns. Therefore, we believe that the fact that these are based on functions with some innate pre-existing inputs and integration is the mechanism of reorganization, not a reason not to treat it as reorganization. Specifically, in the case of this manuscript, we report the change in variability of FC from the auditory cortex, which is greater in deafness than in typically hearing controls. This is not an increase in response per se, but rather more divergent values of FC from the auditory cortex, which are harder to explain in terms of ‘unmasking’ alone, unless one assumes unmasking is particularly variable. The mechanistic explanation for our findings is that in the absence of auditory input’s fine-tuning and pruning of the connectivity of the auditory cortex, more divergent connectivity strength remains among the deaf. Thus, auditory input not only masks non-dominant inputs but also prunes/deactivates exuberant connectivity, in a way that generates a more consistently connected auditory system. We have added a shortened version of these clarifications to the discussion (lines 351-372).

(3) I found the argument that the deaf use a single modality to compensate for hearing loss, and that this might predict a more confined pattern of differential connectivity than had been previously observed in the blind to be poorly grounded. The authors themselves suggest throughout that hearing loss, per se, is likely to be driving the differences observed between deaf and typically-hearing individuals; accordingly, the suggestion that the modality in which intentional behavioral compensation takes place would have such a large-scale effect on observed patterns of connectivity seems out of line.

Thank you for your critical insight regarding our rationale on modality use and its impact on connectivity patterns in the deaf compared to the blind. After some thought, we agree that the argument presented may not be sufficiently strong and could distract from the main findings of our study. Therefore, we have decided to remove this claim from our revised manuscript.

(4) The analyses highlighting the areas observed to be differentially connected to the auditory cortex and areas observed to be more variable in their connectivity to the auditory cortex seem somewhat circular. If the authors propose hearing loss as a mechanism that drives this variability in connectivity, then it is reasonable to propose hypotheses about the directionality of these changes. One would anticipate this directionality to be common across participants and thus, these areas would emerge as the ones that are differently connected when compared to typically hearing folks.

We are a little uncertain how to interpret this concern.  If the question was about the logic leading to our statement that variability is driven by hearing loss, then yes, we indeed were proposing hearing loss as a mechanism that drives this variability in connectivity to the auditory cortex; we regret this was unclear in the original manuscript. This logic parallels the proposal made with regard to the increased variability in FC in blindness; deprivation leads to more variable outcomes, due to the lack of developmental environmental constraints (Sen et al., 2022). Specifically, we first analyzed the differences in within-group variability between deaf and hearing individuals (Fig. 1A), followed by examining the variability ratio (Fig. 1B) in the same regions that demonstrated differences. The first analysis does not specify which group shows higher variability; therefore, the second analysis is essential to clarify the direction of the effect and identify which group, and in which regions, exhibits greater variability. We have clarified this in the revised manuscript (lines 125-127): “To determine which group has larger individual differences in these regions (Figure 1B), we computed the ratio of variability between the two groups (deaf/hearing) in the areas that showed a significant difference in variability (Figure 1A)”. Nevertheless, this comment can also be interpreted as predicting that any change in FC due to deafness would lead to greater variability. In this case, it is also important to mention that while we would expect regions with higher variability to also show group differences between the deaf and the hearing (Figure 2), our analysis demonstrates that variability is present even in regions without significant group mean differences. Similarly, many areas that show a difference between the groups in their FC do not show a change in variability (for example, the bilateral anterior insula and sensorimotor cortex). In fact, the correlation between the regions with higher FC variability (Figure 1A) and those showing FC group differences (Figure 2B) is significant but rather modest, as we now acknowledge in our revised manuscript (lines 324-328). Therefore, increased FC and increased variability of FC are not necessarily linked. 

(5) While the authors describe collecting data on the etiology of hearing loss, hearing thresholds, device use, and rehabilitative strategies, these data do not appear in the manuscript, nor do they appear to have been included in models during data analysis. Since many of these factors might reasonably explain differences in connectivity to the auditory cortex, this seems like an omission.

We thank the Reviewer for their comment regarding the inclusion of these variables in our manuscript. We have now included additional information in the main text and a supplementary table in the revised manuscript that elaborates further on the etiology of hearing loss and all individual information that characterizes our deaf sample. Although we initially intended to include individual factors (e.g., hearing threshold, duration of hearing aid use, and age of first use) in our models, this was not feasible for the following reasons: 1) for some subjects, we only have a level  of hearing loss rather than specific values, which we could not use quantitatively as a nuisance variable (it was typical in such testing to ascertain the threshold of loss as belonging to a deafness level, such as “profound” and not necessarily go into more elaborate testing to identify the specific threshold), and 2) this information was either not collected for the hearing participants (e.g., hearing threshold) or does not apply to them (e.g., age of hearing aid use), which made it impossible to use the complete model with all these variables. Modeling the groups separately with different variables would also be inappropriate. Last, the distribution of the values and the need for a large sample to rigorously assess a difference in variability also precluded sub-dividing the group to subgroup based on these values. 

Therefore, we opted for a different way to control for the potential influence of these variables on FC variability in the deaf. We tested the correlation between the FC from the auditory cortex and each of these parameters in the areas that showed increased FC in deafness (Figures 1A, B), to see if it could account for the increased variability. This ROI analysis did not reveal any significant correlations (all p > .05, prior to correction for multiple comparisons; see Figures S4, S5, and S6 for scatter plots). The maximal variability explained in these ROIs by the hearing factors was r2\=0.096, whereas the FC variability (Figure 1B) was increased by at least 2 in the deaf. Therefore, it does not seem like these parameters underlie the increased variability in deafness. To test if these variables had a direct effect on FC variability in other areas in the brain, we also directly computed the correlation between FC and each factor individually. At the whole-brain level, the results indicate a significant correlation between AC-FC and hearing threshold, as well as a correlation between AC-FC and the age of hearing aid use onset, but not for the duration of hearing aid use (Figure S3). While these may be interesting on their own, and are added to the revised manuscript, the regions that show significant correlations with hearing threshold and age of hearing aid use are not the same regions that exhibit FC variability in the deaf (Figures 1A, B).

Overall, these findings suggest that although some of these factors may influence FC, they do not appear to be the driving factors behind FC variability. Finally, in terms of rehabilitative strategies, only one deaf subject reported having received long-term oral training from teachers. This participant started this training at age 2, as now described in the participants’ section. We thank the reviewer for raising this concern and allowing us to show that our findings do not stem from simple differences ascribed to auditory experience in our participants. 

Reviewer #2 (Public Review):

(1) The paper has two main merits. Firstly, it documents a new and important characteristic of the re-organization of the brains of the deaf, namely its variability. The search for a welldefined set of functions for the deprived auditory cortex of the deaf has been largely unsuccessful, with several task-based approaches failing to deliver unanimous results. Now, one can understand why this was the case: most likely there isn't a fixed one well-defined set of functions supported by an identical set of areas in every subject, but rather a variety of functions supported by various regions. In addition, the paper extends the authors' previous findings from blind subjects to the deaf population. It demonstrates that the heightened variability of connectivity in the deprived brain is not exclusive to blindness, but rather a general principle that applies to other forms of deprivation. On a more general level, this paper shows how sensory input is a driver of the brain's reproducible organization.

We thank the Reviewer for their observations regarding the merits of our study. We appreciate the recognition of the novelty in documenting the variability of brain reorganization in deaf individuals. 

(2) The method and the statistics are sound, the figures are clear, and the paper is well-written. The sample size is impressively large for this kind of study.

We thank the Reviewer for their positive feedback on the methodology, statistical analysis, clarity of figures, and the overall composition of our paper. We are also grateful for the acknowledgment of our large sample size, which we believe significantly strengthens the statistical power and the generalizability of our findings.

(3) The main weakness of the paper is not a weakness, but rather a suggestion on how to provide a stronger basis for the authors' claims and conclusions. I believe this paper could be strengthened by including in the analysis at least one of the already published deaf/hearing resting-state fMRI datasets (e.g. Andin and Holmer, Bonna et al., Ding et al.) to see if the effects hold across different deaf populations. The addition of a second dataset could strengthen the evidence and convincingly resolve the issue of whether delayed sign language acquisition causes an increase in individual differences in functional connectivity to/from Broca's area. Currently, the authors may not have enough statistical power to support their findings.

We thank the Reviewer for their constructive suggestion to reinforce the robustness of our findings. While we acknowledge the potential value of incorporating additional datasets to strengthen our conclusions, the datasets mentioned (Andin and Holmer, Bonna et al., Ding et al.) are not publicly available, which limits our ability to include them in our analysis. Additionally, datasets that contain comparable groups of delayed and native deaf signers are exceptionally rare, further complicating the possibility of their inclusion. Furthermore, to discern individual differences within these groups effectively, a substantially larger sample size is necessary. As such, we were unfortunately unable to perform this additional analysis. This is a challenge we acknowledge in the revised manuscript (lines 442-445), especially when the group is divided into subcategories based on the level of language acquisition, which indeed reduces our statistical power. We have however, now integrated the individual task accuracy and reaction time parameters as nuisance variables in calculating the variability analyses; all the results are fully replicated when accounting for task difficulty. We also report that there was no group difference in activation for this task between the groups which could affect our findings. 

We would like to note that while we would like to replicate these findings in an additional cohort using resting-state, we do not anticipate the state in which the participants are scanned to greatly affect the findings. FC patterns of hearing individuals have been shown to be primarily shaped by common system and stable individual features, and not by time, state, or task (Finn et al., 2015; Gratton et al., 2018; Tavor et al., 2016). While the task may impact FC variability, we have recently shown that individual FC patterns are stable across time and state even in the context of plasticity due to visual deprivation (Amaral et al., 2024). Therefore, we expect that in deafness as well there should not be meaningful differences between resting-state and task FC networks, in terms of FC individual differences. That said, we are exploring collaborations and other avenues to access comparable datasets that might enable a more powerful analysis in future work. This feedback is very important for guiding our ongoing efforts to verify and extend our conclusions.

(4) Secondly, the authors could more explicitly discuss the broad implications of what their results mean for our understanding of how the architecture of the brain is determined by the genetic blueprint vs. how it is determined by learning (page 9). There is currently a wave of strong evidence favoring a more "nativist" view of brain architecture, for example, face- and object-sensitive regions seem to be in place practically from birth (see e.g. Kosakowski et al., Current Biology, 2022). The current results show what is the role played by experience.

We thank the Reviewer for highlighting the need to elaborate on the broader implications of our findings in relation to the ongoing debate of nature vs. nurture. We agree that this discussion is crucial and have expanded our manuscript to address this point more explicitly. We now incorporate a more detailed discussion of how our results contribute to understanding the significant role of experience in shaping individual neural connectivity patterns, particularly in sensory-deprived populations (lines 360-372).

Reviewer #3 (Public Review):

Summary:

(1) This study focuses on changes in brain organization associated with congenital deafness. The authors investigate differences in functional connectivity (FC) and differences in the variability of FC. By comparing congenitally deaf individuals to individuals with normal hearing, and by further separating congenitally deaf individuals into groups of early and late signers, the authors can distinguish between changes in FC due to auditory deprivation and changes in FC due to late language acquisition. They find larger FC variability in deaf than normal-hearing individuals in temporal, frontal, parietal, and midline brain structures, and that FC variability is largely driven by auditory deprivation. They suggest that the regions that show a greater FC difference between groups also show greater FC variability.

Strengths:

-  The manuscript is well written.

-  The methods are clearly described and appropriate.

-  Including the three different groups enables the critical contrasts distinguishing between different causes of FC variability changes.

-  The results are interesting and novel.

We thank the Reviewer for their positive and detailed feedback. Their acknowledgment of the clarity of our methods and the novelty of our results is greatly appreciated.

Weaknesses:

(2) Analyses were conducted for task-based data rather than resting-state data. It was unclear whether groups differed in task performance. If congenitally deaf individuals found the task more difficult this could lead to changes in FC.

We thank the Reviewer for their observation regarding possible task performance differences between deaf and hearing participants and their potential effect on the results. Indeed, there was a difference in task accuracy between these groups. To account for this variation and ensure that our findings on functional connectivity were not confounded by task performance, we now included individual task accuracy and reaction time as nuisance variables in our analyses. This approach allowed us to control for any performance differences. The results now presented in the revised manuscript account for the inclusion of these two nuisance variables (accuracy and reaction time) and completely align with our original conclusions, highlighting increased variability in deafness, which is found in both the entire deaf group at large, as well as when equating language experience and comparing the hearing and native signers. The correlation between variability and group differences also remains significant, but its significance is slightly decreased, a moderate effect we acknowledge in the revised manuscript (see comment #4). The differences between the delayed signers and native signers are also retained (Figure 3), now aligning better with language-sensitive regions, as previously predicted. The inclusion of the task difficulty predictors also introduced an additional finding in this analysis, a significant cluster in the right aIFG. Therefore, the inclusion of these predictors reaffirms the robustness of the conclusions drawn about FC variability in the deaf population.

We would like to note that while we would like to replicate these findings in an additional cohort using resting-state if we had access to such data, we do not anticipate the state in which the participants are scanned to greatly affect the findings. FC patterns of hearing individuals have been shown to be primarily shaped by common system and stable individual features, and not by time, state, or task (Finn et al., 2015; Gratton et al., 2018; Tavor et al., 2016). While the task may impact FC variability, we have recently shown that individual FC patterns are stable across time and state even in the context of plasticity due to visual deprivation (Amaral et al., 2024). Therefore, we expect that in deafness as well there should not be meaningful differences between resting-state and task FC networks, in terms of FC individual differences. We have also addressed this point in our manuscript (lines 442-451).

(3) No differences in overall activation between groups were reported. Activation differences between groups could lead to differences in FC. For example, lower activation may be associated with more noise in the data, which could translate to reduced FC.

We thank the reviewer for noting the potential implications of overall activation differences on FC. In our analysis of the activation for words, we found no significant clusters showing a group difference between the deaf and hearing participants (p < .05, cluster-corrected for multiple comparisons) - we also added this information to the revised manuscript (lines 542-544). This suggests that the differences in FC observed are not confounded by variations in overall brain activation between the groups under these conditions.

(4) Figure 2B shows higher FC for congenitally deaf individuals than normal-hearing individuals in the insula, supplementary motor area, and cingulate. These regions are all associated with task effort. If congenitally deaf individuals found the task harder (lower performance), then activation in these regions could be higher, in turn, leading to FC. A study using resting-state data could possibly have provided a clearer picture.

We thank the Reviewer for pointing out the potential impact of task difficulty on FC differences observed in our study. As addressed in our response to comment #2, task accuracy and reaction times were incorporated as nuisance variables in our analysis. Further, these areas showed no difference in activation between the groups (see response to comment #3 above). Notably, the referred regions still showed higher FC in congenitally deaf individuals even when controlling for these performance differences. Additionally, these findings are consistent with results from studies using resting-state data in deaf populations, further validating our observations. Specifically, using resting-state data, Andin & Holmer (2022), have shown higher FC for deaf (compared to hearing individuals) from auditory regions to the cingulate cortex, insular cortex, cuneus and precuneus, supramarginal gyrus, supplementary motor area, and cerebellum. Moreover, Ding et al. (2016) have shown higher FC for the deaf between the STG and anterior insula and dorsal anterior cingulated cortex. This suggests that the observed FC differences are likely reflective of genuine neuroplastic adaptations rather than mere artifacts of task difficulty. Although we wish we could augment our study with resting-state data analyzed similarly, we could not at present acquire or access such a dataset. We acknowledge this limitation of our study (lines 442-451) in the revised manuscript and intend to confirm that similar results will be found with resting state data in the future.

(5) The correlation between the FC map and the FC variability map is 0.3. While significant using permutation testing, the correlation is low, and it is not clear how great the overlap is.

We acknowledge that the correlation coefficient of 0.3, while statistically significant, indicates a moderate overlap. It's also worth noting that, using our new models that include task performance as a nuisance variable, this value has decreased somewhat, to 0.24 (which is still highly significant). It is important to note that the visual overlap between the maps is not a good estimate of the correlation, which was performed on the unthresholded maps, to estimate the link not only between the most significant peaks of the effects, but across the whole brain patterns. This correlation is meant to suggest a trend rather than a strong link, but especially due to its consistency with the findings in blindness, we believe this observation merits further investigation and discussion. As such, we kept it in the revised manuscript while moderating our claims about its strength.

Reviewer #1 (Recommendations For The Authors):

(1) Page 4: Does auditory cortex FC variability..." FC is not yet defined.

Corrected, thanks.

(2) Page 4: "It showed lower variability..." What showed this?

Clarified, thanks.

(3) Page 11: "highlining the importance" should read "highlighting the importance".

Corrected, thanks.

(4) Page 11: Do you really mean to suggest functional connectivity does not vary as a function of task? This would not seem well supported.

We do not suggest that FC doesn’t vary as a function of task, and have revised this section (lines 447-451). 

(5) Page 12: "there should not to be" should read "there should not be".

Corrected, thanks.

(6) Page 12: "and their majority" should read "and the majority".

Corrected, thanks.

Reviewer #2 (Recommendations For The Authors):

Major

(1) Although this is a lot of work, I nonetheless have another suggestion on how to test if your results are strong and robust. Perhaps you could analyze your data using an ROI/graph-theory approach. I am not an expert in graph theory analysis, but for sure there is a simple and elegant statistic that captures the variability of edge strength variability within a population. This approach could not only validate your results with an independent analysis and give the audience more confidence in their robustness, but it could also provide an estimate of the size of the effect size you found. That is, it could express in hard numbers how much more variable the connections from auditory cortex ROI's are, in comparison to the rest of the brain in the deaf population, relative to the hearing population.

We thank the Reviewer for suggesting the use of graph theory as a method to further validate our findings. While we see the potential value in this approach, we believe it may be beyond the scope of the current paper, and merits a full exploration of its own, which we hope to do in the future.  However, we understand the importance of showing the uniqueness of the connectivity of the auditory cortex ROI as compared to the rest of the brain. So, in order to bolster our results, we conducted an additional analysis using control regions of interest (ROIs). Specifically, we calculated the inter-individual variability using all ROIs from the CONN Atlas (except auditory and language regions) as the control seed regions for the FC. We showed that the variability of connectivity from the auditory cortex is uniquely more increased on deafness, as compared to these control ROIs (Figure S1). This additional analysis supports the specificity of our findings to the auditory cortex in the deaf population. We aim to integrate more analytic approaches, including graph theory methods, in our future work.

Minor

(1) Some citations display the initial of the author in addition to the last name, unless there is something I don't know about the citation system, the initial shouldn't be there.

This is due to the citation style we're using (APA 7th edition, as suggested by eLife), which requires including the first author's initials in all in-text citations when citing multiple authors with the same last name.  

Reviewer #3 (Recommendations For The Authors):

(1) I recommend that the authors provide behavioral data and results for overall neural activation.

Thanks. We have added these to the revised manuscript. Specifically, we report that there was no difference in the activation for words (p < .05, cluster-corrected for multiple comparisons) between the deaf and hearing participants. Further, we report the behavioral averages for accuracy and reaction time for each group, and have now used these individual values explicitly as nuisance variables in the revised analyses.

(2) For the correlation between FC and FC variability, it seemed a bit odd that the permuted data were treated additionally (through Gaussian smoothing). I understand the general logic (i.e., to reintroduce smoothness), but this approach provides more smoothing to the permutation than the original data. It is hard to know what this does to the statistical distribution. I recommend using a different approach or at least also reporting the p-value for non-smoothed permutation data.

In response to this suggestion and to ensure transparency in our results, we have now included also the p-value for the non-smoothed permutation data in our revised manuscript (still highly significant; p < .0001). Thanks for this proposal.

(3) For the map comparison, a plot with different colors, showing the FC map, the FC variability map, and one map for the overlap on the same brain may be helpful.

We thank the Reviewer for their suggestion to visualize the overlap between the maps. However, we performed the correlation analysis using the unthresholded maps, as mentioned in the methods section of our manuscript, specifically to estimate the link not only between the most significant peaks of the effects, but across the whole brain patterns. This is why the maps displayed in the figures, which are thresholded for significance, may not appear to match perfectly, and may actually obscure the correlation across the brain. This methodological detail is crucial for interpreting the relationship and overlap between these maps accurately but also explains why the visualization of the overlap is, unfortunately, not very informative.

eLife Assessment

This study presents valuable data on the increase in individual differences in functional connectivity with the auditory cortex in individuals with congenital/early-onset hearing loss compared to individuals with normal hearing. The evidence supporting the study's claims is convincing, although additional work using resting-state functional connectivity and further links to how the results align with the underlying biology could have further strengthened the study. The work will be of interest to neuroscientists working on brain plasticity and may have implications for the design of interventions and compensatory strategies.

Reviewer #1 (Public review):

This experiment sought to determine what effect congenital/early-onset hearing loss (and associated delay in language onset) has on the degree of inter-individual variability in functional connectivity to the auditory cortex. Looking at differences in variability rather than group differences in mean connectivity itself represents an interesting addition to the existing literature. The sample of deaf individuals was large, and quite homogeneous in terms of age of hearing loss onset, which are considerable strengths of the work. The experiment appears well conducted and the results are certainly of interest.

Reviewer #2 (Public review):

Summary:

This study focuses on changes in brain organization associated with congenital deafness. The authors investigate differences in functional connectivity (FC) and differences in the variability of FC. By comparing congenitally deaf individuals to individuals with normal hearing, and by further separating congenitally deaf individuals into groups of early and late signers, the authors can distinguish between changes in FC due to auditory deprivation and changes in FC due to late language acquisition. They find larger FC variability in deaf than normal-hearing individuals in temporal, frontal, parietal, and midline brain structures, and that FC variability is largely driven by auditory deprivation. They suggest that the regions that show a greater FC difference between groups also show greater FC variability.

Strengths:

The manuscript is well-written, and the methods are clearly described and appropriate. Including the three different groups enables the critical contrasts distinguishing between different causes of FC variability changes. The results are interesting and novel.

Weaknesses:

Analyses were conducted for task-based data rather than resting-state data. The authors report behavioral differences between groups and include behavioral performance as a nuisance regressor in their analysis. This is a good approach to account for behavioral task differences, given the data. Nevertheless, additional work using resting-state functional connectivity could remove the potential confound fully.

The authors have addressed my concerns well.

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a lot of good business people seem to reject the church- is it in this rejection from Church and tradition that they are successful?

capitalist spirit- re-organization and re-orientation of every part of business to increase profit beyond sustainability or expectation

capitalist behavior with traditionalist forms of business- non challenging nature

don't need to be capitalist entrepreneur to put forth "capitalist spirit"

Economic enterprise doesn't equal capitalism

Human caregivers are more reliable than robots caregivers because robots might malfunction. Then, the patient would have a hard time doing tasks temporarily without the robot. This would also lead to the costs of fixing the robot.

Test

Hippocampus atrophy → physiological inability to dampen the disease

The queerness of color is important because it can explain the difference between our worlds.

UTF-8

RPC and security. Kerberos V5 will get used.

At long last she has maybe reached her finally hope of getting out of that bird suit. This conveys that sometimes you have to accept the fate whether it turns out good or bad.

forcces them to kill yourself

blind to the danger, almost as if a sense of fear eludes them.

Margaret Atwood is referencing the song of a siren and how it is so irresistible it led sailors to their death.