act like extent of beliefs is minimal of other person's is mini,a;

unofficial communication- communication that always has potential to be denied, works to communicate and test relations between two teams communicate roles, how the intimacy of relationships should be- communicate boundaries, etc.

derisive collusion- done by performer and hidden from higher-ups , for the performers own purpose

pianist highlights a note that singer should be singing

other is downplaying of politeness or name calling

first type- trash talking audience

!!! its not a reality and a deviation- there is an active participation in both as forming a sort of whole reality

colleagues let colleagues down when don't retain secretive info

training specialists - role of building up performer requires them to imagine perspective of audeince

what does that have to do with... anything

service specialists attend to front stage but often, must obtain backstage view and destructive information to do their job

ignoring someone is a performance of itself

foreman must maintain mindset of audience as well as performance director

go-between example that's having a negative connotation chairman showing active listening skills to encourage audience. Offers direction to audience while providing encouragement to speakers? I kind of don't get how this differs from a regular shill

audience knows what theyve been allowed to percieved

inside secrets- objective intellectual content

second type of secrets are strategic secrets- things hidden for sake of performance. Still hold a lot of weight and should appear that they don't exist- but in a way could be disclosed eventually.

Strategies against opposition

team must be able to keep secrets- some information is destructive for audience to learn

• work example- cooking corn dog

makes it easier to put on right performance for each person if different audiences are separated/ don't see other sides

I HATE MIXING FRIEND GROUPS

higher up someone the less the spend backstage

while backstage is more relaxed and informal- doesn't make it more happy or care free - often all enthusiasm goes to what is done in front of audience and backstage is sullen

performance often relies on the backrooms and the privacy of them

gives roles and sets the stage

keep performers in line with performance

while audience and performer can always apply to both teams, many instances where one team has more control, vested interest, and more intimately organizes performance - salesperson

when many different status available- very frequently to performers sort themselves into two performance groups

solidarity in performance always focused on putting up front in front of subordinates

withholding information from teammate = withholding identity

to maintain united front- team members wait until not in public to disagree

huh

great example- team members can't always easily be disposed of

individual behavior becomes a reflection of everyone when in this case, they're not even friends or allied

The audience doesn't even need to be other people- it can be an imagined outward judgement or assessment of action that directs a performance when no one is looking

that one Margaret Atwood quote

can look at every coordinated performance as teams if in one-on-one each team only has one member

like teachers switching up and calling each other "Ms. -----" in front of students

oftentimes- the performance requires participants who perform roles which require adopting demeanor's

One can maintain a status quo without rigid adherence to all demands of performance in the relationship

almost always there are things someone cannot openly address within performance

what is a lie and what is legitimate all highly flexible, temporally and culturally informed

A lot of ways to lie without directly lying, yet, "bare-faced" lies are the ones with all the consequences

line is more blurred where claims to be something have less tangible means of legitimating

in our heads- a true or honest performer- would not mess up. Assumption that there IS an honest performer or someone who embodies something without a need for front. This view isn't analytically very applicable

identification with a figure- not with oneself

we do not let or are expected not to allow momentary emotions to impact performance- should be homogenous

takes very little to signal this meaning- susceptible to misinterpretation

often beneficial for actor to downplay routinization of actions- act like that interact is special, personal, meaningful, etc.

both med schools and patients act like they choose doctors on aptitude and training as opposed to ethnic origins

conceal motives that break idea of what a "good" clergymen should do

Can you be a good clergymen and still wish to progress socailly?

training extensive to five image that they are set apart

never a performance about bettering or reflecting what is really there, people embody identities for a certain result

Indian caste system allows for social movement in non-material gains (by adopting certain belief systems and practices) which re-centers the lower classes around the practices that allow for them to be upwardly mobile

minimal separation between the status and the front- Here, Goffman doesn't distinguish something like yearly income attaining status than the means to attain the front of a certain status

some say aristocracy is injecting expressions of character into minor activities

merchants overcharge for things that look expensive (are in which to role of money is dramatically fulfilled) so that they can pay for what costs but is not dramatic

issue- exists a nonbelief in other people that are doing fulfilling their role unless they see a dramatized representation of the task

issues of taks that are "between ranks" arise- in the instance of nurse and doctor it relates to the capability of the professional but also what is "right" for the position.

Feels a but more about organization that social expectation tbh

diversification of society too vast- break up into distinct categories and provide based on those- allowing or forcing people to maintain social front

appearance are status and other identifications (can show things like what you do for work) Manner tells us of oncoming action within a persons role (what the person expects to happen or how others are to respond)

Labeling- The "front" of the stage

there is often a demand to be insincere that the cynical performers, in their hyperawareness of the gap between reality and the performers, more willingly offer up this insincerity.

MMEEEE

ethnography vibes- iffy methodology but good theories?

use the metaphor of the theater- presentation of oneself in response to presentation of others for an audience- world of make-believe

Welcome back and in this demo lesson you're going to experience the difference that EFS can make to our WordPress application architecture.

Now this demo lesson has three main components.

First we're going to deploy some infrastructure automatically using the one-click deployments.

Then I'm going to step through the CloudFormation template and explain exactly how this architecture is built.

And then right at the end you're going to have the opportunity to see exactly what benefits EFS provides.

So to get started make sure that you're currently logged in to the general AWS account, so the management account of the organization, and as always you need to have the Northern Virginia region selected.

Now this lesson actually has two one-click deployments.

The first deploys the base infrastructure and the second deploys a WordPress EC2 instance, which has been enhanced to utilize EFS.

So you need to apply both of these templates in order and wait for the first one to finish before applying the second.

So we're going to start with the base VPC RDS EFS template first.

So this deploys the base VPC, the Elastic File System and an RDS instance.

Now everything should be pre-populated.

The stack should be called EFS demo -vpc -rds -efs.

Just scroll all the way down to the bottom, check the capabilities box and click on create stack.

While that's going let's switch over to the CloudFormation template and just step through exactly what it does.

So this is the template that you're deploying using the one-click deployment.

It's deploying the Base Animals for Life VPC, an EFS file system as well as mount targets and an Aurora database cluster.

So if we just scroll down we can see all of the VPC and networking resources used by the Base Animals for Life VPC.

Continue scrolling down we'll see the subnets that this VPC contains IP version 6 information.

We'll see an RDS security group, a database subnet group.

We've got the database instance.

Then we've got an instance security group which controls access to all the resources in the VPC that we use that security group on.

Then we have a rule which allows anything with that security group attached to it to communicate with anything else.

We have a rule that the WordPress instance will use and note that this includes permissions on the Elastic File System.

Then we have the instance profile that that instance uses.

Then we have the CloudWatch agent configuration and this is all automated.

And if we just continue scrolling down here we can see the Elastic File System.

So we create an EFS file system and then we create a file system mount target in each application subnet.

So we've got mount target zero which is in application subnet A which is in US East 1A.

We've got mount target one which is an application subnet B which logically is in US East 1B.

And then finally target two which is in subnet app C which is in availability zone 1C.

So we create the VPC, the database and the Elastic File System in this first one click deployment.

Now we need this to be in a create complete state before we continue with the demo lessons.

So go ahead and pause the video, wait for this to move into a create complete status and then we can use the second one click deployment.

Okay that stacks now finished creating which means we can move on to the second one click deployment.

Now there are actually two WordPress one click deployments which are attached to this lesson.

We're going to use them both but for now I want you to use the WordPress one one click deployment.

So go ahead and click on that link this will create a stack called EFS demo hyphen WordPress one.

Everything should be pre-populated just go ahead and click on create stack.

Now this is going to use the infrastructure provided by that first one click deployment.

So it's going to use EFS demo hyphen VPC hyphen RDS hyphen EFS and let's quickly step through exactly what this is doing while it's provisioning.

So this is the cloud formation template that is being used and we can skip past most of this.

What I want to focus on is the resource that's being created so that's WordPress EC2.

So this is using cross stack references to import a lot of the resources created in that first cloud formation stack.

So it's importing the instance profile to use it's importing the web a subnet so it knows where to place this instance.

And it's importing the instance security group that's created in that previous cloud formation stack.

Now in addition to this if we look through the user data for this WordPress instance one major difference is that it's mounting the EFS file system into this folder.

So forward slash var forward slash w w w forward slash HTML forward slash WP hyphen content.

Now if you remember from earlier demo lessons this is the folder which WordPress users to store its media.

So now instead of this folder being on the local EC2 file system this is now the EFS file system.

The EFS file system is mapped into this folder on this WordPress instance.

Other than that everything else is the same WordPress is installed.

It's configured to you as the RDS instance the cow say custom login banner is displayed.

It automatically configures the cloud watch agent and then it signals cloud formation that it's finished provisioning this instance.

Now what we'll end up with when this stack has finished creating is an EC2 instance which will use the services provided by this original stack.

So let's just refresh this.

It's still in progress so go ahead and pause the video and wait for this stack to move into a create complete state and then we good to continue.

So this stacks now finished creating and if we move across to the EC2 console so click on services locate EC2 right click and open that in a new tab.

Then click on instances running and you'll see that we have this A4L WordPress instance.

Now if we select that copy the IP address into your clipboard and then open that in a new tab we need to perform the WordPress installation.

So go ahead and enter the site title the best cats and add some exclamation points.

For username we need to use admin then for the password go back to the cloud formation stack and click on parameters and we're going to use the DB password.

So copy that into your clipboard then go back paste it into the password box and then put test at test.com for the email address and click install WordPress.

Then as before we need to log in so click on login admin for username reenter that password and click on login.

Then we need to go to posts we need to click on trash below hello world to delete that post then click on add new close down this dialogue.

For title put the best cats ever and some exclamation points then click on the plus click gallery click upload.

There's a link attached to this lesson with four cat images so go ahead and download that link and extract it locate those four images select them and click on open.

And then once you've done that click on publish and publish again and then click on view post.

Now what that's doing in the background is it's adding these images to the WP hyphen content folder which is on the EC two instance but now we have that folder mounted using EFS and so the images are being stored on the elastic file system rather than the local instance file system.

The cat pictures are there but what we're going to do to validate this is to go back to instances right click on this a four L hyphen WordPress instance and click on connect and then connect to this instance using EC two instance connect.

Now once we connected to the instance you CD space forward slash VAR forward slash WWW forward slash HTML and then do an LS space hyphen LA to do a full listing you'll see that we have this WP hyphen content folder.

So type CD space WP hyphen content and press enter then we'll clear the screen and do an LS space hyphen LA and then inside this folder we have plugins themes and uploads go into the uploads folder do an LS space hyphen LA depending on when you do this demo lesson you should see a folder representing the year so move into that folder then a folder representing the month again this will vary depending on when you do the demo lesson.

Move into that folder and then you should see all four of my cat images and if you do a DF space hyphen K you'll be able to see that this folder so forward slash VAR forward slash WWW forward slash HTML WP hyphen content this is actually mounted using EFS so this is an EFS file system.

Now this means the local instance file system is no longer critical it no longer stores the actual media that we upload to these posts so what we can do is we can go back to cloud formation go to stacks select the EFS demo hyphen WordPress one stack and then click on delete and delete that stack so that's going to terminate the EC two instance that we've just used to upload that media.

We need to wait for that stack to fully delete before continuing so go ahead and pause the video and wait for this stack to disappear so that stacks disappeared and now there's a second WordPress one click deployment link attached to this lesson remember there are two so now go ahead and click on the second one this one should create a stack called EFS demo hyphen WordPress two scroll to the bottom and click on create stack that's going to create a new stack and a new EC two instance.

So while we're doing this just close down all of these additional tabs at the top of the screen close them all down apart from the cloud formation one.

We're going to need to wait for this to finish provisioning and move into the create complete state so again pause the video wait for this to change into create complete and then we go to to continue.

After a few minutes the WordPress two stack has moved into a create complete state click on services open the EC two console in a new tab click on instances running you'll see a new A4L hyphen WordPress instance this is a brand new instance which has been provisioned using the one click deployment link that you've just used so the WordPress two one click deployment link.

If we select this copy the public IP address into your clipboard and open that in a new tab it again loads our WordPress blog if we open the blog post.

Now we can see these images because they're being loaded from EFS from the file system that EFS provides so no longer are we limited to only operating from a single EC two instance for our WordPress application because now there's nothing which gets stored specifically on that EC two instance.

Instead everything stored on EFS and accessible from any EC two instance that we decide to give permissions to know what we can do to demonstrate this if we go back to cloud formation.

Now remember attached to this lesson are two WordPress one click deployments we initially applied number one then we deleted that and applied number two so now I want you to reapply number one.

So again click on the WordPress one one click deployment this again will create a new stack this time called EFS demo hyphen WordPress one click on create stack you need to wait for this to move into a create complete state so pause the video and resume it once the stack changes to create complete after a few minutes this stack also moves into create complete.

Let's click on resources we can see it's provisioned a single EC two instance so let's click on this to move directly to this new instance select it copy this instance is IP address into your clipboard and open that in a new tab and again we have our WordPress blog and if we click on the post it loads those images so now we have a number of EC two instances we have to EC two instances both with WordPress installed both using the same RDS data.

And both using the shared file system provided by EFS and it means that if any posts are edited or any images uploaded on either of these two EC two instances then those updates will be reflected on all other EC two instances and this means that we've now implemented this architecture that's on screen now and this is what's going to support us when we evolve this architecture more and add scalability in an upcoming section of the core.

For now though we've just been focused on the shared file system now all that remains at this point is for us to tidy up the infrastructure that we've used in this demo lesson so close down all of these tabs we need to be at the cloud formation console we need to start by deleting EFS demo WordPress one and WordPress two so pick either of those click delete and then delete stack then select the other delete and then delete stack.

Now we need both of these to finish deleting and then we can delete this last stack so go ahead and pause the video wait for both of these to disappear and then we can resume both of those have deleted so now we can click the final stack EFS demo hyphen VPC hyphen RDS hyphen EFS so select that delete and then delete stack and that's everything that you need to do in this demo lesson and once that stacks finished deleting the account will be in the same state as it was at the start of this.

Now I hope you've enjoyed this demo lesson and that it's been useful what you've implemented in this demo is one more supportive step towards us moving this architecture from being a monolith through to being fully elastic.

Now the application is in this state where we have a single shared RDS database for all of our application instances and we're also using a shared file system provided by EFS and this means that we can have one single EC2 instance we could have two EC2 instances or even 200 all of them sharing the same database and the same shared file system provided by EFS.

Now in an upcoming section of this course we're going to extend this further by creating a launch template which automatically builds EC2 instances as part of this application architecture.

We're going to utilize auto scaling groups together with application load balancers to implement an architecture which is fully elastic and resilient and this has been one more supportive step towards that objective.

At this point though that's everything that you needed to do in this demo lesson so go ahead complete this video and when you're ready I look forward to you joining me in the next.

Welcome back and in this demo lesson I want to give you some abstract practical experience of using the Elastic File System or EFS.

Now we're going to need some infrastructure.

Before we apply that as always make sure that you're logged into the general AWS account, so the management account of the organization and you'll need the Northern Virginia region selected.

Now attached to this lesson is a one-click deployment link so go ahead and click that.

This is going to provision some infrastructure.

It's going to take you to the quick create stack screen and everything should be pre-populated.

You'll just need to scroll to the bottom, check the box beneath capabilities and then click on create stack.

You're also going to be typing some commands within this demo lesson so also attached to this lesson is a lesson commands document.

Go ahead and open that in a new tab.

So this is just a list of the commands that we're going to be using during the demo lesson and there are some placeholders such as file system ID that you'll need to replace as we go but make sure you've got this open for reference.

Now we're going to need this stack to be in a create complete state before we continue with the demo lesson so go ahead pause the video and resume it once your stack moves into a create complete state.

Okay so the stacks now moved into a create complete status and what this has actually done is create the animals for life base VPC as well as a number of EC2 instances.

So if we go to the EC2 console and click on instances running you'll note that we've created a for L - EFS instance A and a for L - EFS instance B and we're going to be creating an EFS file system and mount points and then mounting that on both of these instances and interacting with the data stored on that file system.

We're going to get you the experience of working with a network shared file system so let's go ahead and do that.

So to get started we need to move to the EFS console so in the search box at the top just type EFS and then open that in a brand new tab.

We're going to leave this tab open to the instances part of the EC2 console because we're going to come back to this very shortly.

So let's move across to the EFS console that we have open in a separate tab and the first step is to create a file system so a file system is the base entity of the elastic file system product and that's what we're going to create.

Now you've got two options for setting up an EFS file system you can use this simple dialogue or you can click on customize to customize it further.

So if we're using the simple dialogue we'd start by naming the file system so let's say we use A4L - EFS and then you'd need to pick a VPC for this file system to be provisioned into and of course we'd want to select the animals for life VPC.

Now we want to customize this further we don't want to just accept these high-level defaults so we need to click on customize.

This is going to move us to this user interface which has many more options so we've still got the A4L - EFS name for this file system.

Now for the storage class we're going to pick standard which means the data is replicated across multiple availability zones.

If you're doing this in a test or development environment or you're storing data which is not important then you can choose to use one zone which stores data redundantly but only within a single AZ.

Now again in this demonstration we are going to be using multiple availability zones so make sure that you pick standard for storage class.

You're able to configure automatic backups of this file system using AWS backup and if you're taking an appropriate certification course this is something which I'll be covering in much more detail.

You can either enable this or disable it obviously for a production usage you'd want to enable it but for this demonstration we're going to disable it.

Now EFS as I mentioned in the theory lesson comes with different classes of storage and you can configure lifecycle management to move files between those different storage classes so if you want to configure lifecycle management to move any files not accessed for 30 days you can move those into the infrequent access storage class and you can also transition out of infrequent access when anything is accessed so go ahead and select on first access for transition out of IA.

So in many ways this is like S3 with the different classes of storage for different use cases.

When you're creating a file system you're able to set different performance and throughput modes.

For throughput mode you can choose between bursting and enhanced.

If you pick enhanced you're able to select between elastic and provisioned.

I've talked more about these in the theory lesson.

We're going to pick bursting.

Now for performance you can choose between general purpose and max I/O.

General purpose is the default and rightfully so and you should use this for almost all situations.

Only use max I/O if you want to scale to really high levels of aggregate throughput and input output operations per second so only select it if you absolutely know that you need this option.

You've also got the ability to encrypt the data on the file system and if you do encrypt it it uses KMS and you need to pick a KMS key to use.

Of course this means that in order to interact with objects on this file system permissions are needed both on the EFS service itself as well as the KMS key that's used for the encryption operation.

Now this is something that you will absolutely need to use for production usage but for this demonstration we're going to switch it off.

We won't be setting any tags for this file system so let's go ahead and click on next.

You need to configure the network settings for this file system so specifically the mount targets that will be created to access this file system.

Now best practice is that any availability zones within a VPC where you're consuming the services provided by EFS you should be creating a mount target so in our case that's US - East - 1A, 1B and 1C.

So we're going to go through and configure this so first let's delete all of these default security group assignments.

Every mount target that you create will have an associated security group so we'll be setting these specifically.

For now though we need to choose the application subnet in each of these availability zones so in the top drop-down which is US - East - 1A I'm looking for app A so go ahead and do the same.

In US - East - 1B I want to select the app B subnet and then in US - East - 1C logically I'll be selecting the app C subnet so that's app A, app B and app C.

Now for security groups the CloudFormation 1 click deployment has provisioned this instance security group and by default this security group allows all connections from any entities which have this attached so this is a really easy way that we can allow our instances to connect to these mount targets so for each of these lines go ahead and select the instance security group you'll need to do that for each of the mount targets so we'll do the second one and then we'll do the third one and that's all of the network configuration options that we need to worry about so click on next it's here where you can define any policies on the file system so you can prevent root access by default you can enforce read only access by default you can prevent anonymous access or you can enforce encryption in transit for all clients connected to this EFS file system so any clients that connect to the mount targets to access the file system you can ensure that that uses encryption in transit and if you're using this in production you might want to select at least this last option to improve security for this demo lesson we're not going to use any of these policy options nor are we going to define a custom policy in the policy editor instead we'll just click on next at this point we just need to review everything's to our satisfaction everything looks good so we're going to scroll down to the bottom and just click on create now in order to continue with this demo lesson we're going to need both the file system and all of its mount targets so go into the file system click on network and you'll see three mount targets being created all three of these need to be ready before we can continue the demo lesson so this seems like a great time to end part one of this demo lesson go ahead and finish this video and then when all of these mount targets are ready to go you can start part two.

I think there's a typo in the friction cone constraint for location one. It says

$$|f_{1,B_z}^B| \leq \mu f_{1,B_z}^B$$

but should it be the following? (replace z with x on the left side of the inequality)

$$|f_{1,B_x}^B| \leq \mu f_{1,B_z}^B$$

Author response:

eLife Assessment

This valuable study uses consensus-independent component analysis to highlight transcriptional components (TC) in high-grade serous ovarian cancers (HGSOC). The study presents a convincing preliminary finding by identifying a TC linked to synaptic signaling that is associated with shorter overall survival in HGSOC patients, highlighting the potential role of neuronal interactions in the tumor microenvironment. This finding is corroborated by comparing spatially resolved transcriptomics in a small-scale study; a weakness is in being descriptive, non-mechanistic, and requiring experimental validation.

We sincerely thank the editors for the valuable and constructive feedback. We appreciate the recognition of our findings and the significance of identifying transcriptional components in high-grade serous ovarian cancers. We acknowledge the insightful point on our study's descriptive nature and limited mechanistic depth. While further experimental validation would indeed enhance our conclusions, such work extends beyond the current scope of this manuscript. However, we would like to highlight that mechanistic studies demonstrating the impact of tumor-infiltrating nerves on disease progression are emerging (Zahalka et al., 2017; Allen et al., 2018; Balood et al., 2022; Jin et al., 2022; Globig et al., 2023; Restaino et al., 2023; Darragh et al., 2024). Importantly, members of our group have contributed to these findings. These studies, including in vitro and in vivo work in head and neck squamous cell carcinoma as well as high-grade serous ovarian carcinoma, demonstrate that substance P released from tumor-infiltrating nociceptors potentiates MAP kinase signaling in cancer cells, thereby influencing disease progression. This effect can be mitigated in vivo by blocking the substance P receptor (Restaino et al., 2023). Our present work identifies a transcriptional component that aligns with the presence of functional nerves within malignancies. These published mechanistic studies support our findings and suggest that this transcriptional component could serve as a potential screening tool to identify innervated tumors. Such information is clinically relevant, as patients with innervated tumors may benefit from more aggressive therapy.

Reviewer #1 (Public review):

This manuscript explores the transcriptional landscape of high-grade serous ovarian cancer (HGSOC) using consensus-independent component analysis (c-ICA) to identify transcriptional components (TCs) associated with patient outcomes. The study analyzes 678 HGSOC transcriptomes, supplemented with 447 transcriptomes from other ovarian cancer types and noncancerous tissues. By identifying 374 TCs, the authors aim to uncover subtle transcriptional patterns that could serve as novel drug targets. Notably, a transcriptional component linked to synaptic signaling was associated with shorter overall survival (OS) in patients, suggesting a potential role for neuronal interactions in the tumor microenvironment. Given notable weaknesses like lack of validation cohort or validation using another platform (other than the 11 samples with ST), the data is considered highly descriptive and preliminary.

Strengths:

(1) Innovative Methodology:

The use of c-ICA to dissect bulk transcriptomes into independent components is a novel approach that allows for the identification of subtle transcriptional patterns that may be overshadowed in traditional analyses.

We sincerely thank the reviewer for recognizing the strengths and novelty of our study. We appreciate the positive feedback on our use of consensus-independent component analysis (c-ICA) to decompose bulk transcriptomes, which we believe allowed us to detect subtle transcriptional signals often overlooked in traditional analyses.

(2) Comprehensive Data Integration:

The study integrates a large dataset from multiple public repositories, enhancing the robustness of the findings. The inclusion of spatially resolved transcriptomes adds a valuable dimension to the analysis.

Thank you for recognizing the robustness of our study through comprehensive data integration. We appreciate the acknowledgment of our efforts to leverage a large, multi-source dataset, as well as the additional insights gained from spatially resolved transcriptomes. We believe this integrative approach enhances the depth of our analysis and contributes to a more nuanced understanding of the tumor microenvironment.

(3) Clinical Relevance:

The identification of a synaptic signaling-related TC associated with poor prognosis highlights a potential new avenue for therapeutic intervention, emphasizing the role of the tumor microenvironment in cancer progression.

We appreciate the reviewer’s recognition of the clinical implications of our findings. The identification of a synaptic signaling-related transcriptional component associated with poor prognosis underscores the potential for novel therapeutic targets within the tumor microenvironment. We agree that this insight could open new avenues for intervention and further highlights the role of neuronal interactions in cancer progression.

Weaknesses:

(1) Mechanistic Insights:

While the study identifies TCs associated with survival, it provides limited mechanistic insights into how these components influence cancer progression. Further experimental validation is necessary to elucidate the underlying biological processes.

We appreciate the reviewer’s point regarding the limited mechanistic insights provided in our study. We agree that further experimental validation would enhance our understanding of how the biology captured by these transcriptional components influence cancer progression. However, we respectfully note that such validation is beyond the current scope of this article.   Our current analyses are done on publicly available expression array and spatial transcriptomic array datasets. For future studies, we therefore intend to combine spatial transcriptomic data with immunohistochemical analysis of the same tumors for validation purposes. We have started with setting up in vitro cocultures of neurons and ovarian cancer cells to obtain mechanistic insight in how genes with a large weight in TC121 regulate synaptic signaling and how that affects ovarian cancer cells.

(2) Generalizability:

The findings are primarily based on transcriptomic data from HGSOC. It remains unclear how these results apply to other subtypes of ovarian cancer or different cancer types.

In Figure 5, we present the activity of TC121 across various cancer types, demonstrating broader applicability. However, due to limited treatment response data, we were unable to assess associations between TC activity scores and patient response. Additionally, transcriptomic and survival data specific to other ovarian cancer subtypes beyond HGSOC are currently not available, limiting our ability to generalize these findings to those groups. We intend to leverage survival data from TCGA to explore associations between TC activity scores and overall survival of patients with other cancer types. Nonetheless, we recognize limitations with TCGA survival data, as outlined in this article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8726696/.

(3) Innovative Methodology:

Requires more validation using different platforms (IHC) to validate the performance of this bulk-derived data. Also, the lack of control over data quality is a concern.

We acknowledge the reviewer’s suggestion to validate our results with alternative platforms, such as IHC; however, we regret that such validation is beyond the scope of this article. Regarding data quality control, we implemented a series of checks:

• Bulk Transcriptional Profiles: We applied principal component analysis (PCA) on the sample Pearson product-moment correlation matrix, focusing on the first principal component (PCqc), which accounted for approximately 80-90% of the variance, primarily reflecting technical rather than biological variability  (Bhattacharya et al., 2020). Samples with a correlation below 0.8 with PCqc were removed as outliers. Additionally, we generated unique MD5 hashes for each CEL file to identify and exclude duplicate samples. Per gene, expression values were standardized to a mean of zero and a variance of one across the GEO, CCLE, GDSC, and TCGA datasets to minimize probeset- or gene-specific variability.

• Spatial Transcriptional Profiles: We used PCA for quality control here as well, retained samples only if their loading factors for the first principal component showed consistent signs across all profiles (i.e., all profiles had either positive or negative loading factors for the first PC) from that individual spatial transcriptomic sample. Samples that did not meet this criterion were excluded from analyses.

(4) Clinical Application:

Although the study suggests potential drug targets, the translation of these findings into clinical practice is not addressed. Probably given the lack of some QA/QC procedures it'll be hard to translate these results. Future studies should focus on validating these targets in clinical settings.

While this study is exploratory in nature, we agree that future studies should focus on validating these potential drug targets in clinical settings. As suggested, QA/QC procedures were integral to our analyses. We applied rigorous quality control, including PCA-based checks and duplicate removal across datasets, to ensure data integrity (detailed in our previous response).

In terms of clinical application, which we partially discussed in the manuscript, we will discuss additional strategies to prevent synaptic signaling and neurotransmitter release in the tumor microenvironment (TME). Drugs such as ifenprodil and lamotrigine are used in treating neuronal disorders to block glutamate release responsible for subsequent synaptic signaling, whereas the vesicular monoamine transporter (VMAT) inhibitor reserpine can block the formation of synaptic vesicles (Reid et al., 2013; Williams et al., 2001). Previous in vitro studies with HGSOC cell lines showed a significant effect of ifenprodil alone on cancer cell proliferation, whereas reserpine seemed to trigger apoptosis in cancer cells (North et al., 2015; Ramamoorthy et al., 2019). Such strategies could potentially be used to inhibit synaptic neurotransmission in the TME.

Reviewer #2 (Public review):

Summary:

Consensus-independent component analysis and closely related methods have previously been used to reveal components of transcriptomic data that are not captured by principal component or gene-gene coexpression analyses.

Here, the authors asked whether applying consensus-independent component analysis (c-ICA) to published high-grade serous ovarian cancer (HGSOC) microarray-based transcriptomes would reveal subtle transcriptional patterns that are not captured by existing molecular omics classifications of HGSOC.

Statistical associations of these (hitherto masked) transcriptional components with prognostic outcomes in HGSOC could lead to additional insights into underlying mechanisms and, coupled with corroborating evidence from spatial transcriptomics, are proposed for further investigation.

This approach is complementary to existing transcriptomics classifications of HGSOC.

The authors have previously applied the same approach in colorectal carcinoma (Knapen et al. (2024) Commun. Med).

Strengths:

(1) Overall, this study describes a solid data-driven description of c-ICA-derived transcriptional components that the authors identified in HGSOC microarray transcriptomics data, supported by detailed methods and supplementary documentation.

We thank the reviewer for acknowledging the strength of our data-driven approach and the use of consensus-independent component analysis (c-ICA) to identify transcriptional components within HGSOC microarray data. We aimed to provide comprehensive methodological detail and supplementary documentation to support the reproducibility and robustness of our findings. We believe this approach allows for the identification of subtle transcriptional signals that might be overlooked by traditional analysis methods.

(2) The biological interpretation of transcriptional components is convincing based on (data-driven) permutation analysis and a suite of analyses of association with copy-number, gene sets, and prognostic outcomes.

We appreciate the reviewer’s positive feedback on the biological interpretation of our transcriptional components. We are pleased that our approach, which includes data-driven permutation testing and analyses of associations with copy-number alterations, gene sets, and prognostic outcomes, was found convincing. These analyses were integral to enhancing the robustness and biological relevance of our findings.

(3) The resulting annotated transcriptional components have been made available in a searchable online format.

Thank you for acknowledging the availability of our annotated transcriptional components in a searchable online format.

(4) For the highlighted transcriptional component which has been annotated as related to synaptic signalling, the detection of the transcriptional component among 11 published spatial transcriptomics samples from ovarian cancers appears to support this preliminary finding and requires further mechanistic follow-up.

Thank you for acknowledging the accessibility of our annotated transcriptional components. We prioritized making these data available in a searchable online format to facilitate further research and enable the community to explore and validate our findings.

Weaknesses:

(1) This study has not explicitly compared the c-ICA transcriptional components to the existing reported transcriptional landscape and classifications for ovarian cancers (e.g. Smith et al Nat Comms 2023; TCGA Nature 2011; Engqvist et al Sci Rep 2020) which would enable a further assessment of the additional contribution of c-ICA - whether the c-ICA approach captured entirely complementary components, or whether some components are correlated with the existing reported ovarian transcriptomic classifications.

We appreciate the reviewer’s insightful suggestion to compare our c-ICA-derived transcriptional components with previously reported ovarian cancer classifications, such as those from Smith et al. (2023), TCGA (2011), and Engqvist et al. (2020). To address this, we will incorporate analyses comparing the activity scores of our transcriptional components with these published landscapes and classifications, particularly focusing on any associations with overall survival. Additionally, we plan to evaluate correlations between gene signatures from these studies and our identified TCs, enhancing our understanding of the unique contributions of the c-ICA approach.

(2) Here, the authors primarily interpret the c-ICA transcriptional components as a deconvolution of bulk transcriptomics due to the presence of cells from tumour cells and the tumour microenvironment. However, c-ICA is not explicitly a deconvolution method with respect to cell types: the transcriptional components do not necessarily correspond to distinct cell types, and may reflect differential dysregulation within a cell type. This application of c-ICA for the purpose of data-driven deconvolution of cell populations is distinct from other deconvolution methods that explicitly use a prior cell signature matrix.

Thank you for highlighting this nuanced aspect of c-ICA interpretation. We acknowledge that c-ICA, unlike traditional deconvolution methods, is not specifically designed for cell-type deconvolution and does not rely on a predefined cell signature matrix. While we explored the transcriptional components in the context of tumor and microenvironmental interactions, we agree that these components may not correspond directly to distinct cell types but rather reflect complex patterns of dysregulation, potentially within individual cell populations.

Our goal with c-ICA was to uncover hidden transcriptional patterns possibly influenced by cellular heterogeneity. However, we recognize these patterns may also arise from regulatory processes within a single cell type. To investigate further, we plan to use single-cell transcriptional data (~60,000 cell-types annotated profiles from GSE158722) and project our transcriptional components onto these profiles to obtain activity scores, allowing us to assess each TC’s behavior across diverse cellular contexts after removing the first principal component to minimize background effects.

References

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Author response:

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

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

This study explores the therapeutic potential of KMO inhibition in endometriosis, a condition with limited treatment options. 

Strengths: 

KNS898 is a novel specific KMO inhibitor and is orally bioavailable, providing a convenient and non-hormonal treatment option for endometriosis. The promising efficacy of KNS898 was demonstrated in a relevant preclinical mouse model of endometriosis with pathological and behavioural assessments performed. 

Weaknesses: 

(1) The expression of KMO in human normal endometrium and endometrial lesions was not quantified. Western blot or quantification of IHC images will provide valuable insight.

Given the differential expression of KMO in luminal epithelial cells lining the endometrial glands compared to the other parts of the endometrium, a general endometrial Western Blot prep is not going to be additionally helpful or accurate in addressing this question, without e.g. laser capture microdissection or single cell quantitative proteomics. Furthermore, KMO is a flavin-dependent monooxygenase and the activity, especially generating the oxidative stressor product 3-hydroxykynurenine is far more dependent on kynurenine substrate availability than it is on actual enzyme abundance - although it is important to show (as we have done), that KMO is present in the human endometrial glands and in human distended endometrial gland-like structures (DEGLS).

If KMO is not overexpressed in diseased tissues i.e. it may have homeostatic roles, and inhibition of KMO may have consequences on general human health and wellbeing.

KMO certainly does have important homeostatic roles, for example as key step in the repletion of NAD+ through de novo synthesis. Although with good nutrition and sufficient NAD+ precursors in the diet e.g. niacin, that specific role may be partially redundant. KMO knockout mice exhibit normal fertility and fecundity and do not show a survival deficit compared to littermate wildtype controls (e.g. Mole et al Nature Medicine 2016). To further develop KNS898 towards clinical use, preclinical GLP safety and toxicology studies and human Phase 1 clinical trials will of course need to be completed, but that is standard for the development of any new drug

In addition, KMO expression in control mice was not shown or quantified.

Control mice that were not inoculated intraperitoneally with endometrial fragments did not develop DEGLS and therefore there is nothing to show or quantify.

Images of KMO expression in endometriosis mice with treatments should be shown in Figure 4.

We have now included a representative KMO immunohistochemistry image from each endometriosis group and included all KMO immunohistochemistry images in Supplementary Information.

The images showing quantification analysis (Figure 4A-F) can be moved to supplementary material.

This recommendation contradicts the emphasis placed by the same reviewer earlier regarding quantification, so we have elected to keep it where it is.

(2) Figure 1 only showed representative images from a few patients. A description of whether KMO expression varies between patients and whether it correlates with AFS stages/disease severity will be helpful. Images from additional patients can be provided in supplementary material. 

We have added extra information to the Figure legend to clarify the disease stage of the superficial peritoneal lesions which were illustrated (Stage I/II) and to link them to the information in supplementary Table S1. In total we examined 11 peritoneal lesions and 5 ovarian lesions (stage III/IV) – in every sample examined immunopositive staining was most intense in epithelial cells lining gland-like structures. Sections illustrated were chosen to illustrate this key finding.

(3) For Home Cage Analysis, different measurements were performed as stated in methods including total moving distance, total moving time, moving speed, isolation/separation distance, isolated time, peripheral time, peripheral distance, in centre zones time, in centre zones distance, climbing time, and body temperature. However, only the finding for peripheral distance was reported in the manuscript. 

This was indeed a large amount of output, which we rationalised for the benefit of a concise paper. The paper now includes a description of which parameters showed a difference with drug treatment.

(4) The rationale for choosing the different dose levels of KNS898 - 0.01-25mg/kg was not provided. What is the IC50 of a drug? 

KNS898 dosing has been extensively characterised by us in multiple species, and the pIC50 has already been published (e.g. Hayes et al Cell Reports 2023 and elsewhere). We now include the pIC50 in the present manuscript to save the reader from having to search through another reference.

(5) Statistical significance: 

(a) Were stats performed for Fig 3B-E?

Now included, thank you.

(b) Line 141 - 'P = 0.004 for DEGLS per group' 

However, statistics were not shown in the figure. 

Thanks, now displayed on figure.

(c) Line 166 - 'the mechanical allodynia threshold in the hind paw was statistically significantly lower compared to baseline for the group' 

However, statistics were not shown in the figure. 

(d) Line 170 - 'Two-way ANOVA, Group effect P = 0.003, time effect P < 0.0001' The stats need to be annotated appropriately in Figure 5A as two separate symbols. 

Arguably the far more important comparison in this figure is whether there is any effect of treatment, and to mark multiple statistical comparisons on the figure would make it difficult to understand. Instead, the figure legend and results text have been clarified on this point.

(e) Figure 5B - multiple comparisons of two-way ANOVA are needed. G4 does not look different to G3 at D42. 

Multiple comparison testing (Dunnett’s T3) was done and the results have been clarified in the text and figure legends.

(f) Line 565 - 'non-significant improvement in KNS898 treated groups'. However, ** was annotated in Figure 5A. 

Thank you. This is an error that has been checked and corrected.

(6) Discussion is very light. No reference to previous publications was made in the discussion. Discussion on potential mechanistic pathways of KYR/KMO in the pathogenesis of endometriosis will be helpful, as the expression and function of KMO and/or other metabolites in endometrial-related conditions. 

The discussion is deliberately concise and focussed. The paper has 21 references to previous publications. A speculative discussion is generally not favoured by us.

The findings in this study generally support the conclusion although some key data which strengthen the conclusion eg quantification of KMO in normal and diseased tissue is lacking.

We differ from the reviewer here and do not think that those data would materially affect the likelihood of KMO inhibition being efficacious in human endometriosis in Phase 2/3 clinical trials.

Before KMO inhibitors can be used for endometriosis, the function of KMO in the context of endometriosis should be explored eg KMO knockout mice should be studied. 

We take the view that before KMO inhibitors can be used for endometriosis in patients there are multiple other regulatory and clinical development steps that are required that would be a priority. While using a KMO knockout mouse might be an interesting scientific experiment, it would not impact on the critical path in a material way.

Reviewer #2 (Public Review): 

Summary: 

The authors aim to address the clinical challenge of treating endometriosis, a debilitating condition with limited and often ineffective treatment options. They propose that inhibiting KMO could be a novel non-hormonal therapeutic approach. Their study focuses on: 

• Characterising KMO expression in human and mouse endometriosis tissues. 

• Investigating the effects of KMO inhibitor KNS898 on inflammation, lesion volume, and pain in a mouse model of endometriosis. 

• Demonstrating the efficacy of KMO blockade in improving histological and symptomatic features of endometriosis. 

Strengths: 

• Novelty and Relevance: The study addresses a significant clinical need for better endometriosis treatments and explores a novel therapeutic target. 

• Comprehensive Approach: The authors use both human biobanked tissues and a mouse model to study KMO expression and the effects of its inhibition. 

• Clear Biochemical Outcomes: The administration of KNS898 reliably induced KMO blockade, leading to measurable biochemical changes (increased kynurenine, increased kynurenic acid, reduced 3-hydroxykynurenine). 

Weaknesses: 

• Limited Mechanistic Insight: The study does not thoroughly investigate the mechanistic pathways through which KNS898 affects endometriosis. Specifically, the local vs. systemic effects of KMO inhibition are not well differentiated. 

While we agree that this is not a comprehensive mechanistic analysis, given that the ultimate therapy would be almost certainly a once daily oral dosing i.e. systemic administration, we do not consider differentiating local vs systemic effects of KMO inhibition to be critical to therapeutic development in this scenario.

• Statistical Analysis Issues: The choice of statistical tests (e.g., two-way ANOVA instead of repeated measures ANOVA for behavioral data) may not be the most appropriate, potentially impacting the validity of the results. 

The selection of two-way ANOVA (time and group) is sufficient and correct for this experimental analysis and its use does not invalidate the results. We agree that repeated measures ANOVA could be a valid alternative.

• Quantification and Comparisons: There is insufficient quantitative comparison of KMO expression levels between normal endometrium and endometriosis lesions,

Please see response above to quantification question raised by Reviewer 1.

and the systemic effects of KNS898 are not fully explored or quantified in various tissues. 

Please see earlier responses. KNS898 has been thoroughly explored in multiple tissues, species and experimental models, but those data do not need rehearsed here.

• Potential Side Effects: The systemic accumulation of kynurenine pathway metabolites raises concerns about potential side effects, which are not addressed in the study. 

As discussed above (response to Reviewer 1), KMO knockout mice exhibit normal fertility and fecundity and do not show a survival deficit compared to littermate wildtype controls (e.g. Mole et al Nature Medicine 2016). To further develop KNS898 towards clinical use, preclinical GLP safety and toxicology studies and human Phase 1 clinical trials will naturally need to be completed, but this is standard for the development of any new drug.

Achievement of Aims: 

• The authors successfully demonstrated that KMO is expressed in endometriosis lesions and that KNS898 can induce KMO blockade, leading to biochemical changes and improvements in endometriosis symptoms in a mouse model. 

Support of Conclusions: 

• While the data supports the potential of KMO inhibition as a therapeutic strategy, the conclusions are somewhat overextended given the limitations in mechanistic insights and statistical analysis. The study provides promising initial evidence but requires further exploration to firmly establish the efficacy and safety of KNS898 for endometriosis treatment. 

We do not agree that the conclusions are overextended based on the data presented, as expanded in the reply to the eLife editorial assessment at the beginning of this response. It is clear that additional preclinical, regulatory and clinical development work, and human clinical trials will be required to firmly establish the efficacy and safety of KN898 for endometriosis treatment.

Impact on the Field: 

• The study introduces a novel therapeutic target for endometriosis, potentially leading to non-hormonal treatment options. If validated, KMO inhibition could significantly impact the management of endometriosis. 

Utility of Methods and Data: 

• The methods used provide a foundation for further research, although they require refinement. The data, while promising, need more rigorous statistical analysis and deeper mechanistic exploration to be fully convincing and useful to the community. 

We believe that the data are a) convincing, and b) useful to the community. To be advanced effectively towards patients, KNS898 needs to follow the critical development path outlined above.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors): 

(1) Change 'hyperalgia' to hyperalgesia throughout the manuscript including the title. 

Done

(2) Line 69 - write '3-HK' in full. 

Done

(3) Line 85 - the findings of the study include 'define the preclinical efficacy of KNS898 in reducing inflammation'. The inflammatory profile was not studied. 

Changed to “disease”

(4) Line 259 - write 'EPHect' in full. 

Done

(5) Line 260 - write 'AFS' in full. Also, abbreviate 'AFS' in the caption of Table S1. 

Done

(6) 20 patients were listed in Table S1 but only 19 were accounted for in the methods section. 

Apologies there was an error and has now been corrected in the methods section as one of the endometrial samples had not been included. Table S1 has also been changed to make it clear which samples were eutopic endometrium to differentiate them from the lesions.

(7) The location from which the endometrial lesion tissues were obtained should be provided in Table S1. 

Table S1 has been changed to make it clear that the subtypes of lesions examined were classified as Stage I/II – superficial peritoneal subtype and Stage III/IV – endometrioma. The methods section has also been updated to reflect these subtypes (lines 272-277).

(8) Table S2 - G5 should be given compound 'A' not 'B'. 

Thank you. Corrected.

(9) Figure 2E was not referenced in the text and no figure legend was provided. 

Now referenced and the figure legend updated.

(10) Figure 3A - font needs to be enlarged. HCA baseline recording was annotated as performed twice in the protocol. When is the baseline taken and on what day was the Week 12 measurement taken (refer to Figures 5C and D)? 

Font has been enlarged as requested. The second HCA baseline annotation in Fig 3A is a cut-and-paste error, now rectified and the time of second measurement annotated.

(11) Line 133 - 'In KNS898-treated group G4 (endometriosis + treatment from Day 19), DEGLS formed in 4 of 15 mice (26.7%) and in G5 (Endo + treatment start on Day 26) in 6 of 15 mice (40%) (Fig. 3f).'. The aforementioned data is not reflected in Figure 3F. 

Thank you. This has been rectified.

(12) Line 137 - 'Mice with endometriosis receiving KNS898 from the time of inoculation (G4) had an average of 2.0 DEGLS per animal with DEGLS (total = 8 DEGLS in 4 mice in G4) and those receiving KNS898 1 week after inoculation (G5) had an average of 1.8 DEGLS per animal (total = 11 DEGLS in 6 mice in G5) (Figs. 3g and 3h).' 

The aforementioned data is not reflected in Figure 3G. There is no Figure 3H shown. 

Rectified as above.

(13) Provide a discussion of why KA levels were significantly lower in Figure 3E compared to Figure 2C. 

(14) Figure legend for Figure 3 - G1 and G2 were noted as n=8. However, Figure S1 and Table S2 noted both groups as n=10. 

Thank you. This is a typographical error. The legend for Fig 3 should indeed read n=10 for G1 and G2 and has been corrected.

(15) Line 181 - 'compared to non-operated and sham-operated control groups'. Only the sham group was shown in Figures 5C and D. 

This text has been clarified to refer only to the data shown.

(16) Figure 1 images need scalebars. Same for Figure 4. 

Now added

(17) Figure 3B - y-axis is fold change? 

Relative concentration. Legend has been clarified.

(18) Figures 5A and B - are the last Von Frey measurements taken on Day 40 (as per Figure 3A) or 42?

Taken on Day 42. Fig 3A (the prospective protocol figure) has been clarified to reflect what actually happened (D42) as opposed to what was planned (D40) to pre-empt any further confusion.

(19) Symbols in Figure S1 need to be explained in the Figure legend. 

Done

(20) Figures 2A and 2D should not be plotted in log scale to match the description of results in Line 106 and Line 118. 

These particular results are plotted on a log scale to allow the reader to visualise that detectable levels of drug are measurable at very low doses and that there is no significant pharmacodynamic effect at that low dose. We choose to retain the present format.

Reviewer #2 (Recommendations For The Authors): 

Comments and queries 

Introduction/aims section: 

Line 82 - 87: Clarify in the proposal aims what is being accessed and analysed in humans and/or in animal models (mice). Specifically state clearly the correlations with KMO expression. Were the correlations between KMO expression with features of inflammation performed only in mice or also in humans? 

Thank you for this comment. The aims have been clarified in the Introduction.

Section - KMO is expressed in human eutopic endometrium and human endometriosis tissue lesions: 

Was any quantitative or semi-quantitative method used to quantify the KMO expression in human tissues? Although the authors claimed that "KMO was strongly immunopositive in human peritoneal endometriosis lesions" by the representative figures it is not clear if KMO expression is similar, higher or lower between normal endometrium and peritoneal endometriosis lesions. 

We have added extra information to the legend of Figure 1 to identify the PIN number of the superficial lesions illustrated. The key finding from the immunostaining with the antibody which had been previously validated as specific for KMO was that the most intense immunopositive response was in glandular epithelial cells and the samples illustrate this result.

Section - Oral KNS898 inhibits KMO in mice: 

The authors clearly confirmed the target engagement of KNS898 in inhibiting KMO activity and, therefore, affecting upstream and downstream metabolites systemically in (peripheral fluid/ plasma) mice. Whether KNS898 effect is broad and targets systemic immune cells and whole body cells and tissue was not explored. It was also not explored if KNS898 is able to specifically inhibit KMO locally at the endometrium tissue by targeting epithelial and/or infiltrated immune cells, for example. 

That is correct.

It would be interesting to measure (or if it was measured to report in this section and also in Figure 2) the levels of KYN, KA and 3HK in naïve animals that did not receive KNS898. It would help to understand the net effect of KNS898 on the levels of kynurenine pathway metabolites and, therefore, justify the dose chosen.

These data are already presented in Fig 3B-E, control group.

Perhaps then the chosen dose could be lower considering the possible substantial changes in kynurenine pathway metabolites levels, which are reported to exert an effect in many cells, tissues and systems and could, therefore, precipitate side effects. Even more considering that the values for these metabolites are expressed as ng/ml, which hinders the comparison of the metabolite levels with the one reported for naïve animals in the literature. I would also suggest expressing the metabolite levels as nM/L. 

This is not a relevant method of determining dose-limiting toxicity or safety pharmacology/toxicology, either non-GLP or GLP. There are international guidelines on the proper conduct of those studies. This is also why it is important not to make claims about the safety or otherwise of an experimental compound in an in vivo setting that has not explicitly complied with those regulatory standards. With regard to the units recommendation, accepted units are ng/mL or nM, not usually nM/L.

Section - KMO blockade reduces endometrial gland-like lesion burden in experimental endometriosis in mice: 

Line 130: It would be better to replace "blockade of 3HK production" with "reduction of 3HK production" to better reflect the results. 

Changed to “inhibition of 3HK production”.

Line 140: In G5 (treatment starting at Day 26/ 1 week after inoculation), is the experimental model of endometriosis already established with all pathological and phenotypic features? 

This was not specifically tested in this experiment.

Lines 146 - 148: It would be better to specify that "Overall, there was no significant difference IN BODY WEIGHT between G3 and the KNS898 treatment groups G4 and G5 (endometriosis + treatment from Day 26)". Otherwise, this last sentence might be interpreted as the overall conclusion of this result sub-section. 

Thank you, a good point and has been corrected.

The authors demonstrated with an experimental approach that KMO blockade reduces a pathological measure of endometriosis i.e., endometrial gland-like lesion burden, in experimental endometriosis in mice when both administrated concomitant but also after the disease development. Although mechanistic insights about how reduced KMO activity can reduce the developed distended endometrial gland-like structures were not explored. Therefore, it remains to be investigated which (and how ) kynurenine pathway metabolites are directly linked to the beneficial effects of KMO blockade in the experimental model of endometriosis.

We agree.

Although the beneficial effects on the pathological measures are evident, Figure 3 shows an exorbitant accumulation of KYN and KA and also a substantial reduction in 3HK after the treatment with KNS898, which then raises concerns about tolerability and side effects. Would this effective KNS898 dose be viable and translational as a therapeutic approach? 

Please refer to comments above at multiple junctures about safety pharmacology and the clinical development critical path.

Section - KMO is expressed in experimental endometriosis in mice: 

By histological examination, the authors confirm that the treatment with KNS898 specifically reduced the KMO expression intensity in the DEGLS from mice. Therefore, the effect exerted by KNS898 locally on the KMO expression at the DEGLS could be, at least, partially responsible for the beneficial effects observed in Figure 3 i.e., the reduction of pathological measures. Although remains to be explored whether the effect of KNS898 in other cells or tissues could also be accountable for the beneficial effects exerted by KNS898 on the animal model of endometriosis. 

This is correct.

From a logical experimental point of view, I would suggest switching the order of the result subsection "KMO blockade reduces endometrial gland-like lesion burden in experimental endometriosis in mice" and "KMO is expressed in experimental endometriosis in mice" as well as the respective Figures 3 and 4. 

We do not agree. Fig 3 (and section) is the macroscopic enumeration of DEGLS, Fig 4 (and section) is the microscopic and immunohistochemical evaluation of the lesions introduced in Fig 3. The sequence as originally presented is the more logical.

Sections - KMO inhibition reduces mechanical allodynia in experimental endometriosis - and - KMO inhibition reduces mechanical allodynia in experimental endometriosis: 

The authors suggested that the KMO inhibition with KNS898 exerts beneficial effects on behavioural paradigms related to the experimental model of endometriosis. Based on the statistical analysis performed for the author, KMO inhibition with KNS898 reduces mechanical allodynia, as well as rescues, impaired cage exploration behaviour and mobility in mice with endometriosis. However, I believe that the most indicated statistical tests for Von Frey (allodynia behaviour) and Home cage (illness behaviour) analyses over time would be repeated measures ANOVA and paired t-test, respectively (and not two-way ANOVA as performed). Therefore for a more trustful analysis and interpretation of this data set, I would suggest the authors modify the statistical analysis and report the corresponding interpretation of these tests. 

The selection of two-way ANOVA (time and group) is suitable for this experimental analysis and its use does not invalidate the results. We agree that repeated measures ANOVA could be a valid alternative.

Overall, the authors present a solid and useful case for KMO inhibition as a potential therapeutic strategy for endometriosis. However, the study would benefit from more detailed mechanistic insights, appropriate statistical analyses, and an evaluation of potential side effects. With these improvements, the research could have a significant impact on the field and pave the way for new treatment modalities for endometriosis. 

We thank the reviewer for the positive comments and we have responded to the criticisms above.

Specific recommendations for improvement: 

• Mechanistic Studies: Conduct detailed studies to understand the local vs. systemic effects of KMO inhibition and its specific impacts on different cell types and tissues. If not feasible here, the authors could include in the discussion section a detailed overview of the possible mechanisms implicated. 

While we agree that this is not a comprehensive mechanistic analysis, given that the ultimate therapy would be almost certainly a once daily oral dosing i.e. systemic administration, we do not consider differentiating local vs systemic effects of KMO inhibition to be critical to therapeutic development in this scenario. We do not think speculation about possible mechanisms that is not supported by experimental data should be included. Furthermore, that notion (of statements not supported by data) has been given as a criticism by the reviewers, and therefore consistency on this point must be preferable.

• Quantitative Analysis: Include more robust quantitative methods to compare KMO expression levels in different tissues and assess the correlation between KNO expression and pathological and behavioural changes. 

As discussed above, the pathophysiological importance of KMO is in its enzymatic activity, not in its abundance as a protein, and 3HK production is far more dependent on kynurenine substrate availability rather than KMO protein abundance.

• Appropriate Statistics: Use the most suitable statistical tests for behavioural and other repeated measures data to ensure accurate interpretation. 

As discussed above

• Side Effect Evaluation: Investigate potential side effects of systemic KMO inhibition, particularly focusing on the long-term implications of altered kynurenine pathway metabolites. If not feasible here, the authors could include in the discussion section a detailed overview of the possible side effects associated as well as inform if KNS898 can cross the BBB and its implications. 

For a novel small molecule therapeutic compound in preclinical/clinical development, there are strictly regulated preclinical and clinical development standards that need to be met. It would not be responsible to publish or make claims about safety and potential adverse effect profiles without conducting the proper panel of tests within a suitable regulatory framework.

Reviewer #2 (Public review):

Summary:

This paper introduces a new methodology for probing time-varying causal interactions in complex dynamical systems using a novel machine-learning architecture of Temporal Autoencoders for Causal Inference (TACI) combined with a novel metric (CSGI) for assessing causal interactions using surrogate data. This is a timely contribution in the field of causal inference from temporal data which has been largely restricted to stationary time series so far. However, the benchmarking of the proposed methods could be improved.

Strength:

The method's capacity to uncover piecewise time-varying non-linear dynamic systems is demonstrated on synthetic datasets as well as on two real-world applications on climate and brain activity data. A particular advantage of the approach is to train a single model capturing the dynamics of the whole time series, thereby allowing for time-varying interactions to be found without retraining over different time periods.

Weaknesses:

(1) It is not clear why the new metric Comparative Surrogate Granger Index CSGI (Eq.6) should be better than the Extended Granger Causality Index EGCI (Eq.5), which can also be used to compare the information about y(t) contained in the actual data x(t) versus in a randomized surrogate x^s(t), as implemented in the proposed metric (Eq.6).

(2) The benchmarking of the new approach TACI against earlier metrics (ie Surrogate Linear Granger, Convergent Cross Mapping, and Transfer Entropy) should be revised:

(a) The details of the computation should be provided to clarify how the different metrics are estimated notably between multidimensional variables [for instance to estimate Ty->x for x=(x_1,x_2,x_3) and y=(y_1,y_2,y_3)].

(b) Reliable implementations of the different metrics should be used, as some of the reported results do not seem right. In particular, the unidirectional examples, Eq.9 (Figure 2) and Eq.12 (Figure 5), are expected to lead to vanishing transfer entropies from Y to X, ie Ty->x =0, for all values of the coupling parameter below the synchronization threshold. This can be verified by computing transfer entropies as conditional mutual information using MIIC R package, i.e. Ty->x = I(x(t);y(t-1)|x(t-1)).

(c) Besides, some reported benchmarks focus on peculiar non-linear systems displaying somewhat "pathological" behaviors. For instance, the two Hénon maps with unidirectional coupling Eq.12 (Figure 5) lead to an equality between the two variables, i.e. y(t)=x(t) for all t, above the synchronization threshold C>0.7. This leads mathematically to zero transfer entropy upon synchronization, as I(x(t);y(<br /> d) By contrast, Eq.9 (Fig.2) leads to strongly coupled, yet non-identical variables above the synchronization threshold. This strong coupling can be shown to yield non-vanishing transfer entropies in both directions, as observed in Figure 2c, and does not correspond to "incorrect prediction of non-existent interactions", as stated in the "Summary of Results on Artificial Test Systems". Clearly synchronized variables do interact and their bidirectional transfer entropies are actually consistent with a non-causal (or bidirectional) relationship. Only a vanishing transfer entropy in one direction implies a causal relation (in the opposite direction). Likewise, vanishing transfer entropies in both directions imply either independent variables or a spurious dependency between them due to an unobserved common cause L, i.e. X<--(L)-->Y. This is usually represented with a bidirected edge (X<-->Y), which is different from a bidirectional relation corresponding to two opposite unidirectional edges (ie X-->Y and X<--Y). It is therefore surprising that TACI metric vanishes in both directions upon synchronization in this case (Eq.9, Figure 2), as one would expect to learn variable y(t) more reliably using the actual data x(<br /> e) In order to assess TACI performance on non-stationary time series, it might be more informative to benchmark it on datasets displaying intermittency rather than synchrony. In particular, the change of causal directions over time, presented as one of the motivations for the new approach, should be more thoroughly benchmarked in the paper. For instance, it would be nice to demonstrate the tracking of the spontaneous reversal of causal relation in a simple 'toggle switch' regulatory network between two mutually repressing genes + expression noise. This is something that causal inference methods assuming stationarity cannot do.

(3) Concerning the real-world applications, the analysis of the electrocorticography (ECoG) data does not seem to be in strong disagreement with the general trends of the original more detailed study by Tajima et al 2015. Could the authors better delineate what are the common versus conflicting findings between the two approaches? The main difference appears to be the near loss of interaction in the anesthetized state, which might be linked to TACI's tendency to report no interaction between synchronized variables as discussed in d) above. Does the anesthetized state correspond to a global synchrony of the brain regions? This could be easily validated by a more direct analysis of synchrony.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This work sets out to elucidate mechanistic intricacies in inflammatory responses in pneumonia in the context of the aging process (Terc deficiency - telomerase functionality).

Strengths:

Very interesting, conceptually speaking, approach that is by all means worth pursuing. An overall proper approach to the posited aim.

We want to thank the reviewer for taking the time to review our manuscript and for providing positive feedback regarding our research question.  

Weaknesses:

The work is heavily underpowered and may have statistical deficits. This precludes it in its current state from drawing unequivocal conclusions.

Thank you for this essential and valuable comment. We fully accept that the small sample size of the Tercko/ko mice is a major limitation of our study and transparently discuss this in our manuscript.  However, due to Animal Welfare regulations, only a reduced number of mice were approved because of the strong burden of disease. Consequently, only three non-infected and five infected mice were available to us. This reduced number of mice presents a clear limitation to our study. However, due to ethical considerations related to animal welfare and sustainability, as well as compliance with German animal welfare regulations, it is not possible to obtain additional Tercko/ko mice to increase the dataset.

The animal studies are an important aspect of our study; however, our hypothesis was also investigated at multiple levels, including in an in vitro co-culture model (Figure 5), to ensure comprehensive analysis. Thus, we clearly demonstrated that S. aureus pneumonia in Tercko/ko mice leads to a more severe phenotype, orchestrated by the dysregulation of both innate and adaptive immune response.

Reviewer #2 (Public Review):

Summary:

The authors demonstrate heightened susceptibility of Terc-KO mice to S. aureus-induced pneumonia, perform gene expression analysis from the infected lungs, find an elevated inflammatory (NLRP3) signature in some Terc-KO but not control mice, and some reduction in T cell signatures. Based on that, They conclude that disregulated inflammation and T-cell dysfunction play a major role in these phenomena.

Strengths:

The strengths of the work include a problem not previously addressed (the role of the Terc component of the telomerase complex) in certain aspects of resistance to bacterial infection and innate (and maybe adaptive) immune function.

We would like to thank the reviewer for the positive feedback regarding our aim to investigate the impact of Terc deletion on the pulmonary immune response to S. aureus.  

Weaknesses:

The weaknesses outweigh the strengths, dominantly because conclusions are plagued by flaws in experimental design, by lack of rigorous controls, and by incomplete and inadequate approaches to testing immune function. These weaknesses are as follows

(1) Terc-KO mice are a genomic knockout model, and therefore the authors need to carefully consider the impact of this KO on a wide range of tissues. This, however, is not the case. There are no attempts to perform cell transfers or use irradiation chimera or crosses that would be informative.

We thank the reviewer for bringing up this important point. The aim of our study, however; was to investigate the impact of Terc deletion in the lung and on the response to bacterial pneumonia, rather than to provide a comprehensive characterization of the Tercko/ko model itself. This characterization of different tissues and cell types has already been conducted by previous studies. For instance, studies that characterize the general phenotype of the model (Herrera et al., 1999; Lee et al., 1998; Rudolph et al., 1999) but also investigations that shed light on the impact of Terc deletion on specific cell types such as microglia (Khan et al., 2015) or T cells (Matthe et al., 2022). The impact of Terc deletion on T cells is also discussed in our manuscript in lines 89 to 105. Furthermore, a section about the general phenotype of the Terc deletion model is included in the introduction in lines 126 to 138. Thus we discussed the relevant literature regarding Tercko/ko mice in our manuscript and attempted to provide a more in-depth characterization of the lung by investigating the inflammatory response to infection as well as changes in the gene expression (Figure 2-4).  

(2) Throughout the manuscript the authors invoke the role of telomere shortening in aging, and according to them, their Terc-KO mice should be one potential model for aging. Yet the authors consistently describe major differences between young Terc-KO and naturally aging old mice, with no discussion of the implications. This further confuses the biological significance of this work as presented.

Thank you for mentioning this relevant point. We want to apologize for the confusion regarding this matter. While Tercko/ko mice are a well-established model for premature aging, these effects become more apparent with increasing generations (G) and thus, G5 and 6 mice are the most affected by Terc deletion (Lee et al., 1998; Wong et al., 2008).

Thus, while Tercko/ko mice are a common model for premature aging, this accelerated aging phenotype is predominantly apparent in later-generation Tercko/ko (G5 and 6) or aged Tercko/ko mice (Lee et al., 1998; Wong et al., 2008). Since the aim of this study was to analyze the impact of Terc deletion on the lung and its immune response to bacterial infections instead of the impact of telomere shortening and telomerase dysfunction, young G3 Tercko/ko mice (8 weeks) were used in this study. This is also mentioned in the lines 131-134. In this study, Tercko/ko mice were used not as a model of aging, but rather as a model specifically for Terc deletion. The old WT mice function as a control cohort to observe possible common but also deviating effects between aging and Terc deletion. In our sequencing data, we observe that uninfected young WT mice are very similar to uninfected Tercko/ko mice. Other studies have also reported this lack of major differences between uninfected WT and Tercko/ko mice in the G3 knockout mice (Kang et al., 2018). Conversely, uninfected young WT and Tercko/ko mice exhibited great differences, for instance, regarding the numbers of differentially expressed genes (Supplemental Figure 1H). Thus, differences between naturally aged mice and young G3 Tercko/ko mice are not surprising. To clarify this aspect we reconstructed the paragraph discussing the Tercko/ko mice (lines 126-134). Additionally we added a paragraph explaining the purpose of the naturally aged mice to the lines 134 to 138:

“As control cohort age-matched young WT mice were utilized. To investigate whether Terc deletion, beyond critical telomere shortening, impacts the pulmonary immune response, we used young Tercko/ko mice. Additionally, naturally aged mice (2 years old) were infected to explore the potential link to a fully developed aging phenotype.”

(3) Related to #2, group design for comparisons lacks a clear rationale. The authors stipulate that TercKO will mimic natural aging, but in fact, the only significant differences seen between groups in susceptibility to S. aureus are, contrary to the authors' expectation, between young Terc-KO and naturally old mice (Figures 1A and B, no difference between young Terc-KO and young wt); or there are no significant differences at all between groups (Figures 1, C, D,).

We thank the reviewer for this essential comment. As mentioned above the Tercko/ko mice in this study are not selected to model natural aging. To model telomerase dysfunction and accelerated aging selection of later generation or aged Tercko/ko mice would have been more suitable. 

The lack of statistical significance in some figures is likely due to the heterogeneity of disease phenotype of S. aureus infection in mice, which is a limitation of our study that we discuss in our discussion section in lines 576-582. The phenotype of S. aureus infection can vary greatly within a mouse population, highlighting the limitations of mice as a model for S. aureus infections. To account for this heterogeneity we divided the infected Tercko/ko mice cohort into different degrees of severity based on the clinical score and the presence of bacteria in organs other than the lung (mice with systemic infection). 

Despite the heterogeneity especially within the Tercko/ko mice cohort the differences between the knockout and young as well as old WT mice were striking. Including the fatal infections, 80% of the Tercko/ko mice had a severe course of disease, while none of the WT mice displayed a severe course (Figure 1A, B and Supplemental Figure 1A, B). This hints towards a clear role of Terc in the response to S. aureus infection in mice. Thus while in some figures the differences are not significant, strong trends towards a more severe phenotype of S. aureus infection in the Tercko/ko mice regarding bacterial load, score and inflammatory response could be observed in our study. 

Another example of inadequate group design is when the authors begin dividing their Terc-KO groups by clinical score into animals with or without "systemic infection" (the condition where a bacterium spreads uncontrollably across the many organs and via blood, which should be properly called sepsis), and then compare this sepsis group to other groups (Supplementary Figures 1G; Figure 2; lines 374-376 and 389391). This gives them significant differences in several figures, but because they did not clearly indicate where they applied this stratification in the figure legends, the data are somewhat confusing. Most importantly, methodologically it is highly inappropriate to compare one mouse with sepsis to another one without. If Terc-KO mice with sepsis are a comparator group, then their controls have to be wild-type mice with sepsis, who are dealing with the same high bacterial load across the body and are presumably forced to deploy the same set of immune defenses.

We sincerely appreciate the significant time and effort you have invested in reviewing our manuscript. However, with all due respect, we must point out that the definition of sepsis you have referenced is considered outdated. According to the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3), sepsis is defined as "a life-threatening organ dysfunction caused by a dysregulated host response to infection" (Marvin Singer, 2016, JAMA). Given this fundamental misunderstanding of our findings, we find the comment regarding the inadequacy of our groups to be both dismissive and lacking in scientific merit. We would like to emphasize that the group size used in our study is consistent with accepted standards in infection research. We strongly reject any insinuations of inadequacy that have been repeatedly mentioned throughout the review.

In order to provide a nuanced investigation of disease severity in Tercko/ko mice, we added the term “systemic infection” to the figures whenever the mice were divided into groups of mice with and without systemic infection. This is the case for Figure 2A and Supplemental Figure 1C-E. The division into mice with and without systemic infection is also mentioned in the figure legend of Figure 2A in lines 932 to 935 and for Supplemental Figure 1 in lines 1052-1053. We agree that Supplemental Figure 1G is somewhat confusing as the mice with systemic infection are highlighted in this graph but not included as a separate group within our sequencing analysis. We added a sentence to the figure legend clarifying this (lines 1042-1044):

“Nevertheless, the infected Tercko/ko mice were considered one group for the expression analysis and not split into separate groups for the subsequent analysis.”

Additionally, we revised the section regarding this grouping in different degrees of severity in our Material and Methods section to clarify that this division was only performed for specific analysis (line 191):

“…for the indicated analysis.”

Furthermore, the mice which were classified as systemically infected mice were not septic mice, as mentioned above. Those mice were classified by us as systemically infected based on their clinical score and the presence of bacteria in other organs than the lung as stated in the lines 188-191 and 377-381. Bacteremia is a symptom of very severe cases of hospital-acquired pneumonia with a very high mortality (De la Calle et al., 2016).

Therefore, the systemically infected mice or rather mice with bacteremia display an especially severe pneumonia phenotype, which is distinct from sepsis. The presence of this symptom in our Tercko/ko mice further highlights the clinical relevance of our study. This aspect was added to the manuscript in the lines 568-570.

“The detection of bacteria in extra pulmonary organs is of particular interest, as bacteremia is a symptom of severe pneumonia and is associated with high mortality (De la Calle et al., 2016).”

(4) The authors conclude that disregulated inflammation and T-cell dysfunction play a major role in S. aureus susceptibility. This may or may not be an important observation, because many KO mice are abnormal for a variety of reasons, and until such reasons are mechanistically dissected, the physiological importance of the observation will remain unclear.

Two points are important here. First, there is no natural counterpart to a Terc-KO, which is a complete loss of a key non-enzymatic component of the telomerase complex starting in utero. 

Second, the authors truly did not examine the key basic features of their model, including the features of basic and induced inflammatory and immune responses. This analysis could be done either using model antigens in adjuvants, defined innate immune stimuli (e.g. TLR, RLR, or NLR agonists), or microbial challenge. The only data provided along these lines are the baseline frequencies of total T cells in the spleen of the three groups of mice examined (not statistically significant, Figure 4B). We do not know if the composition of naïve to memory T cell subsets may have been different, and more importantly, we have no data to evaluate whether recruitment of the immune response (including T cells) to the lung upon microbial challenge is similar or different. So, what are the numbers and percentages of T cells and alveolar macrophages in the lung following S. aureus challenge and are they even comparable or are there issues in mobilizing the T cell response to the site of infection? If, for example, Terc-KO mice do not mobilize enough T cells to the lung during infection, that would explain the paucity in many T-cellassociated genes in their transcriptomic set that the authors report. That in turn may not mean dysfunction of T cells but potentially a whole different set of defects in coordinating the response in Terc-KO mice.

We thank the reviewer for highlighting these important aspects. Regarding the first point, indeed there is no naturally occurring deletion of Terc in humans. However, studies reported reduced expression of Terc and Tert in the tissues of aged mice and rats (Tarry-Adkins et al., 2021; Zhang et al., 2018). Terc itself has been found to have several important immunomodulatory functions such as the activation of the NFκB or PI3-kinase pathway (Liu et al., 2019; Wu et al., 2022). As those aforementioned pathways are relevant for the immune response to S. aureus infections, the authors were interested in exploring the impact of Terc deletion on the pulmonary immune response. The potential immunomodulatory functions of Terc are discussed in lines 106-121. To further clarify our rationale we added a sentence to the introduction in lines 121-125.

“Interestingly, downregulation of Terc and Tert expression in tissues of aged mice and rats has been found (Tarry-Adkins, Aiken, Dearden, Fernandez-Twinn, & Ozanne, 2021; Zhang et al., 2018). Therefore, as a potential immunomodulatory factor reduced Terc expression could be connected to agerelated pathologies.”

Regarding the second point, as we focused on the effect of Terc deletion in the lung and its role in S. aureus infection, we investigated inflammatory and immune response parameters relevant to this setting. For instance, inflammation parameters in the lungs of all three mice cohorts were measured to investigate differences in the inflammatory response in the non-infected and infected mice (Figure 2A). Those measurements showed no baseline difference in key inflammatory parameters between young WT and Tercko/ko mice, which is consistent with previous findings (Kang et al., 2018). The inflammatory response to infection with S. aureus in the Tercko/ko mice cohort differed significantly from the other cohorts (Figure 2A), hinting towards a dysregulated inflammatory response due to Terc deletion. Furthermore, we investigated general immune cell frequencies such as dendritic cells, macrophages, and B cells in the spleen of all three mice cohorts to gather a baseline understanding of the general immune cell populations. In our manuscript only total T cell frequencies were included due to its relevance for our data regarding T cells (Figure 4B). This data could show that there was no difference of total amount of T cells in the spleen of all three mice cohorts. For a more detailed insight into our analysis we added the frequencies of the other immune cell populations analyzed in the spleen as a Supplemental Figure 3B-F. Additionally, a figure legend for the graphs was added to lines 1075-1094.

Therefore, while we did not analyze baseline frequencies of specific populations of T cells, we analyzed and characterized the inflammatory and immune response of our model in a way relevant to our research question. 

The differences observed in T cell marker and TCR gene expression was also partly present between the uninfected and infected Tercko/ko mice such as the complete absence of CD247 expression in infected Tercko/ko, which is however expressed in uninfected mice of this cohort (Figure 4A, C and D). Thus, this effect cannot be solely attributed to an inadequate mobilization of T cells to the lung after infectious challenge. However, we agree that a more detailed insight into recruited immune cells to the lung or frequencies of different T cell populations could contribute to a better understanding of the proposed mechanism and would be an interesting experiment to conduct in further studies. We accept this as a limitation of our study and included it in our discussion section in lines 719-723:

“As total CD4+ T cells were analyzed in this study, it would be useful to investigate specific T cell populations such as memory and effector T cells to elucidate the potential mechanism leading to T cell dysfunctionality in further detail. Additionally, analysis of differences in immune cell recruitment to the lungs between young WT and Tercko/ko mice would be relevant.”

(5) Related to that, immunological analysis is also inadequate. First, the authors pull signatures from the total lung tissue, which is both imprecise and potentially skewed by differences, not in gene expression but in types of cells present and/or their abundance, a feature known to be affected by aging and perhaps by Terc deficiency during infection. Second, to draw any conclusions about immune responses, the authors would have to track antigen-specific T cells, which is possible for a wide range of microbial pathogens using peptide-MHC multimers. This would allow highly precise analysis of phenomena the authors are trying to conclude about. Moreover, it would allow them to confirm their gene expression data in populations of physiological interest

We thank the reviewer for highlighting this important and relevant point. In our study, we aimed to investigate the role of Terc expression in modulating inflammation and the immune response to S. aureus infection in the lung. To address this, we examined the overall impact of age, genotype, and infection on lung inflammation and gene expression. Therefore, sequencing of total lung tissue was essential for addressing the research question posed. Our findings demonstrate that Tercko/ko mice exhibit a more severe phenotype following S. aureus infection, characterized by an increased bacterial load and heightened lung inflammation (Figures 1 and 2). Furthermore, our data suggest that Terc plays a role in regulating inflammation through activation of the NLRP3 inflammasome, along with the dysregulation of several T cell marker genes (Figures 2, 4, and 5). However, this study lacks a detailed analysis of distinct T cell populations, including antigen-specific T cells, as noted earlier. Investigating these aspects in future studies would be valuable to validate and expand upon our findings. We have incorporated these suggestions into the discussion section (lines 719-723)

“As total CD4+ T cells were analyzed in this study, it would be useful to investigate specific T cell populations such as memory and effector T cells to elucidate the potential mechanism leading to T cell dysfunctionality in further detail. Additionally, analysis of differences in immune cell recruitment to the lungs between young WT and Tercko/ko mice would be relevant.”

Nevertheless, our study provides first evidence of a potential connection between T cell functionality and Terc expression.  

Third, the authors co-incubate AM and T cells with S. aureus. There is no information here about the phenotype of T cells used. Were they naïve, and how many S. aureus-specific T cells did they contain? Or were they a mix of different cell types, which we know will change with aging (fewer naïve and many more memory cells of different flavors), and maybe even with a Terc-KO? Naïve T cells do not interact with AM; only effector and memory cells would be able to do so, once they have been primed by contact with dendritic cells bringing antigen into the lymphoid tissues, so it is unclear what the authors are modeling here. Mature primed effector T cells would go to the lung and would interact with AM, but it is almost certain that the authors did not generate these cells for their experiment (or at least nothing like that was described in the methods or the text).

Thank you for bringing up this important question. For the co-cultivation experiment of T cells and alveolar macrophages, total CD4+ T cells of both young WT and Tercko/ko were used. We did not select for a specific population of T cells. Our sequencing data indicated the complete downregulation of CD247 expression, which is an important part of the T cell receptor, in the lungs of infected Tercko/ko mice (Figure 4A, C and D). Given that this factor is downregulated under chronic inflammatory conditions, we investigated the impact of the inflammatory response in alveolar macrophages on the expression of various T cell-derived cytokines, as well as CD247 expression (Figure 5D, E) (Dexiu et al., 2022). This aspect is also highlighted in the discussion in lines 622-636. Therefore, a co-cultivation model of T cells and alveolar macrophages was established and confronted with heat-killed S. aureus to elicit an inflammatory response of the macrophages. To emphasize this purpose, we have revised our statement about the model setup in lines 516-518 of the manuscript: 

“An overactive inflammatory response could be a potential explanation for the dysregulated TCR signaling.”

The authors hope this will clarify the intent behind the model setup.

(6) Overall, the authors began to address the role of Terc in bacterial susceptibility, but to what extent that specifically involves inflammation and macrophages, T cell immunity, or aging remains unclear at present.

We thank the reviewer for the helpful and relevant comments. The authors accept the limitations of the presented study such as the reduced number of Tercko/ko mice and the limitations of murine models for S. aureus infection itself and discuss those in the discussion section in the lines 558-560; 576-582; 688-690 and 719-725. However, we hope that our responses have provided sufficient evidence to convince the reviewer that our data supports a clear role for Terc expression in regulating the immune response to bacterial infections, particularly with respect to inflammation and its potential connection to T cell functionality.  

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

The good element first:

I read this paper with genuine interest and applaud the authors for investigating the posited question. I consider it by all means scientifically relevant in the context of physiological/pathophysiological aging and reaction to a disease (here pneumonia). The Terc deletion model looks very appropriate for the question and the methodology is very advanced/in-depth. The data flow/selection of endpoints and assays is very logical to me. Moreover, I like the breakdown of pneumonia into varying levels of severity.

We thank the reviewer for their time and effort taken to revise our manuscript. Additionally, we are grateful to receive your positive feedback regarding our study design and research question.

The weaknesses:

(1) I cannot help but notice that the study is heavily underpowered. As such, it is inadmissible. The key reason is that it is the first of its kind and seminal findings must be strongly propped by the evidence. It is apparent to me that the data scatter presented in the figures tends to be abnormally distributed (e.g. obvious bimodal distribution in some groups). Therefore, the presented comparisons (even if stat. sign) can be heavily misleading in terms of: i) the true magnitude of the observed effects and ii) possible type 2 error in some cases of p value >0.05. Solution: repeat the study to ensure reasonable power/reliability. This will also make it stronger as it will immediately demonstrate its reproducibility (or lack of it).

Thank you for bringing up this extremely relevant point. We acknowledge the issue of the small sample size of Tercko/ko mice as a major limitation of our study. This limitation is also included in our discussion section in the lines 558-560. Thus we fully agree with this limitation and transparently discuss this in our manuscript. However, due to the strict German animal welfare regulations it is not possible to obtain more Tercko/ko mice, as mentioned above. Furthermore, since fatal infections occurred in the Tercko/ko mice cohort we had a reduced number of mice available. 

However, the differences between the Tercko/ko and WT mice were striking. Including the fatal infections 80% of the Tercko/ko mice had a severe course of disease, while none of the WT mice displayed a severe course. This hints towards a clear role of Terc in the response to S. aureus infection in mice.  

(2) In the stat analysis section of M&Ms, the authors feature only 1 sentence. This cannot be. A detailed stats workup needs to be included there. This is very much related to the above weakness; e.g. it is impossible to test for normality (to choose an appropriate post-hoc test) with n=3. Back to square one: study underpowered.

We thank the reviewer for highlighting this important aspect. We carefully revised the method section in lines 357-360 to include all relevant information: 

“Data are presented as mean ± SD, or as median with interquartile range for violin and box plots, with up to four levels of statistical significance indicated. P-values were calculated using Kruskal-Wallis test. Individual replicates are represented as single data points.”

(3) Pneumonia severity. While I noted that as a strength, I also note it as weakness here. It looks to me like the authors stopped halfway with this. I totally support testing a biological effect(s) such as the one investigated here across a spectrum of a given disease severity. The authors mention that they had various severity phenotypes produced in their model but this is not visible in the data figs. I strongly suggest including that as well; i.e., to study the posited question in the severe and mild pneumonia phenotype. This is a very smart path and previous preclinical research clearly demonstrated that this severe/mild distinction is very relevant in the context of the observed responses (their presence/absence, longevity, dynamics, etc). I realize this is challenging, thus, I would probably use this approach in the Terc k/o model as sort of a calibrator to see whether the exacerbation observed in the current setup (severe?) will be also present in a mild pneumonia phenotype. S. aureus can be effectively titrated to produce pneumonia of varying severity.

We thank the reviewer for bringing up this relevant point. 

In our study, we could observe heterogeneity within the infected Tercko/ko cohort. Therefore as pointed out by the reviewer we assigned different degrees of severity to those groups based on clinical scores, the fatal outcome of the disease (fatal subgroup), and the presence of bacteria in organs other than the lungs (systemic infection subgroup) as stated in our materials and methods part in the lines 188-191 (Supplemental Figure 1A and B). Moreover, we highlighted this difference in a number of our figures. For example, when categorizing the mice into groups with and without systemic infection, we noticed that the mice with systemic infection demonstrated a higher bacterial load, significant body weight loss, and increased lung weight (see Supplemental Figure 1C-E). Interestingly, the two mice with systemic infection clustered separately from the other mice, indicating that the mice with systemic infection are transcriptomically distinct from the other mice cohorts (Supplemental Figure 1G). Additionally, the inflammatory response was exclusively elevated in the lungs of mice with systemic infection (Figure 2C). Thus, we included this distinction in several figures and attempted to study the differences between those subgroups but also their similarities. For instance, we could observe that some changes in the transcriptome were present in all three infected Tercko/ko mice such as the complete absence of CD247 expression at 24 hpi (Figure 4D). This distinction therefore provided a more detailed insight into the underlying mechanisms of disease severity in Tercko/ko mice and is lacking in other studies. We agree with the reviewer, that a study investigating mild and severe pneumonia phenotypes would be clinically relevant. However, as noted above, due to ethical considerations related to animal welfare and sustainability, as well as compliance with German animal welfare regulations, it is not possible to obtain additional Tercko/ko mice to carry out the proposed experiment. 

(4) Please read ARRIVE guidelines and note the relevant info in M&Ms as ARRIVE guidelines point out.

Thank you for emphasizing this crucial aspect. We revised our materials and methods section according to the ARRIVE guidelines (lines 179-206).

“Tercko/ko mice aged 8 weeks, were used for infection studies (n = 8; non-infected = 3; infected = 5). Female young WT (age 8 weeks) and old WT (age 24 months) C57Bl/6 mice (both n = 10; non-infected = 5; infected = 5) were purchased from Janvier Labs (Le Genest-Saint-Isle, France). All infected mouse cohorts were compared to their respective non-infected controls, as well as to the infected groups from other cohorts. Additionally, comparisons were made between the non-infected cohorts across all groups.

All mice were anesthetized with 2% isoflurane before intranasal infection with S. aureus USA300 (1x108 CFU/20µl) per mouse. After 24 hours, the mice were weighed and scored as previously described (Hornung et al., 2023). Infected Tercko/ko mice were grouped into different degrees of severity based on their clinical score, fatal outcome of the disease (fatal) and the presence of bacteria in organs other than the lung (systemic infection) for the indicated analysis. Mice with fatal infections were excluded from subsequent analyses, with only their final scores being reported. The mice were sacrificed via injection of an overdose of xylazine/ketamine and bleeding of axillary artery after 24 hpi. BAL was collected by instillation and subsequent retrieval of PBS into the lungs. Serum and organs were collected. Bacterial load in the BAL, kidney and liver was determined by plating of serially diluted sample as described above. For this organs were previously homogenized in the appropriate volume of PBS. Gene expression was analyzed in the right superior lung lobe. Lobes were therefore homogenized in the appropriate amount of TriZol LS reagent (Thermo Fisher Scientific, Waltham, MA, US) prior to RNA extraction. The left lung lobe was embedded into Tissue Tek O.C.T. (science services, Munich, Germany) and stored at 80°C until further processing for histological analysis. Cytokine measurements were performed using the right inferior lung lobe. Lobes were previously homogenized in the appropriate volume of PBS. Remaining organs were stored at -80°C until further usage. Mouse studies were conducted without the use of randomization or blinding.“

(5) There are also some other descriptive deficits but they are of a much smaller caliber so I do not list them.

We thank the reviewer for their valuable and insightful suggestions for improving our manuscript. We hope that our responses and the corresponding revisions address these suggestions satisfactorily.

Concluding: the investigative idea is great/interesting and the methodological flow is adequate but the low power makes this study of low reliability in its current form. I strongly urge the authors to walk the extra mile with this work to make it comprehensive and reliable. Best of luck!

Reviewer #2 (Recommendations For The Authors):

(1) Many legends are uninformative and do not contain critical information about the experiments. For example, Figure 2A with cytokine measurements (in lung homogenates?) is likely showing data from an ELISA or Luminex test, but there is no mention of that in the legend. It stands next to Figure 2B, which is a gene expression map, again, likely from the lung (prepared how, normalized how, etc?) lacking even the most basic information. Further, Figure 2D has no information on the meaning/effect size of gene ratios on the x-axis. Figures 3 and 4 are presumably the subsets of their transcriptome data set (whole lung, harvested on d ?? post-infection), but that is just a guess on my part. Even in the main text, the timing and the controls for the transcriptomic study are not stated (ln. 398 and onwards). The authors really need to revise the figure legends and provide all the details that an average reader would need to be able to interpret the data.

We thank the reviewer for bringing up this important point. The figure legends of all figures including supplemental figures were revised to ensure they include all relevant data necessary for accurate interpretation of the graphs. Additionally, we clarified the sequenced samples in lines 427-429:

“We performed mRNA sequencing of the murine lung tissue of infected and non-infected mice at 24 hpi to elucidate potential differentially expressed genes that contribute to the more severe illness of Tercko/ko mice.”

(2) Telomere shortening affects differentially different cells and its role in aging is nuanced - different in mesenchymal cells with no telomerase induction, in non-replicating cells, and in hematopoietic cells that can readily induce telomerase. The authors should be mindful of that in setting up their introduction and discussion.

Thank you for mentioning this essential aspect. We revised our introduction and discussion to reflect the nuanced role of telomerase shortening in different tissues (lines 83-92 and 690-695):

“Telomerase activity is restricted to specific tissues and cell types, largely dependent on the expression of Tert. While Tert is highly expressed in stem cells, progenitor cells, and germline cells, its expression is minimal in most differentiated cells (Chakravarti, LaBella, & DePinho, 2021). Consequently, the impact of telomerase dysfunction on tissues varies according to their self-renewal rate. (Chakravarti et al., 2021). One important aspect of telomere dysfunction is the impact of telomere shortening on the immune system as well as the hematopoietic system. Tissues or organ systems that are highly replicative, such as the skin or the hematopoietic system, are affected first by telomere shortening (Chakravarti et al., 2021).”

“It is important to note that telomere shortening has a significant impact on the immune system. Although young Tercko/ko mice were used in this study, telomere shortening is still likely to be a contributing factor. Therefore, further experiments investigating the role of T cell senescence in this model should therefore be conducted.”

(3) Syntax and formulations need to be improved and made more scientifically precise in several spots. Specifically, in 62-63, the authors say that the aged immune system "is also discussed to be more irritable", please change to reflect the common notion that the reaction to infection is dysregulated; in many cases inflammation itself is initially blunted, misdirected, and of different type (e.g. for viruses, the key IFN-I responses are not increased but decreased). In lines 114-117, presumably, the two sentences were supposed to be connected by a comma, although some editing for clarity is probably needed regardless. Line 252, please change "unspecific" to "non-specific". Line 264, please capitalize German.

We thank the reviewer for bringing these important points to our attention. We revised our introduction regarding the aged immune response in lines 61-69:

“Age-related dysregulation of the immune response is also characterized by inflammaging, defined as the presence of elevated levels of pro-inflammatory cytokines in the absence of an obvious inflammatory trigger (Franceschi et al., 2000; Mogilenko, Shchukina, & Artyomov, 2022). Additionally, immune cells, such as macrophages, exhibit an activated state that alters their response to infection (Canan et al., 2014). In contrast, the immune response of macrophages to infectious challenges has been shown to be initially impaired in aged mice (Boe, Boule, & Kovacs, 2017). Thus aging is a relevant factor impacting the pulmonary immune response.”

Sentences were edited to provide more clarity in lines 131-134:

“Although G3 Tercko/ko mice with shortened telomeres were used in this study, they were infected at a young age (8 weeks). This approach allowed for the investigation of Terc deletion effects rather than telomere dysfunction.”

“Unspecific was changed to “non-specific” in line 282 and “German” was capitalized in line 293 and 558.

We appreciate and thank you for your time spent processing this manuscript and look forward to your response.

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Author response:

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

We are grateful to the reviewers for their positive assessment of the revised version of the article.

Please find below our answers to the last, minor comments of the reviewers.

We thank the reviewer for this important comment. In our live imaging experiments, we actually tracked the dorsal and ventral borders of the omp:yfp positive clusters in control and sly mutant embryos. These measurements showed that the omp:yfp positive clusters are more elongated along the DV axis in mutants as compared with control siblings, as seen on fixed samples (data not shown), suggesting that this difference in tissue shape is not due to fixation.

Reviewer #4 (Public review):

Summary:

In this elegant study XX and colleagues use a combination of fixed tissue analyses and live imaging to characterise the role of Laminin in olfactory placode development and neuronal pathfinding in the zebrafish embryo. They describe Laminin dynamics in the developing olfactory placode and adjacent brain structures and identify potential roles for Laminin in facilitating neuronal pathfinding from the olfactory placode to the brain. To test whether Laminin is required for olfactory placode neuronal pathfinding they analyse olfactory system development in a well-established laminin-gamma-1 mutant, in which the laminin-rich basement membrane is disrupted. They show that while the OP still coalesces in the absence of Laminin, Laminin is required to contain OP cells during forebrain flexure during development and maintain separation of the OP and adjacent brain region. They further demonstrate that Laminin is required for growth of OP neurons from the OP-brain interface towards the olfactory bulb. The authors also present data describing that while the Laminin mutant has partial defects in neural crest cell migration towards the developing OP, these NCC defects are unlikely to be the cause of the neuronal pathfinding defects upon loss of Laminin. Altogether the study is extremely well carried out, with careful analysis of high-quality data. Their findings are likely to be of interest to those working on olfactory system development, or with an interest in extracellular matrix in organ morphogenesis, cell migration, and axonal pathfinding.

Strengths:

The authors describe for the first time Laminin dynamics during the early development of the olfactory placode and olfactory axon extension. They use an appropriate model to perturb the system (lamc1 zebrafish mutant), and demonstrate novel requirements for Laminin in pathfinding of OP neurons towards the olfactory bulb.

The study utilises careful and impressive live imaging to draw most of its conclusions, really drawing upon the strengths of the zebrafish model to investigate the role of laminin in OP pathfinding. This imaging is combined with deep learning methodology to characterise and describe phenotypes in their Laminin-perturbed models, along with detailed quantifications of cell behaviours, together providing a relatively complete picture of the impact of loss of Laminin on OP development.

Weaknesses:

Some of the statistical tests are performed on experiments where n=2 for each condition (for example the measurements in Figure S2) - in places the data is non-significant, but clear trends are observed, and one wonders whether some experiments are under-powered.

We initially planned the electron microscopy experiments in order to analyse 3 embryos per genotype per stage. However, because of technical issues we could not perform the measurements in all the cases, explaining why we have n = 2 in some of the graphs. The trends were quite clear, so we chose to keep these data in the article. We believe they nicely complement the immunostaining data assessing basement membrane integrity in control and mutant embryos.

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

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

The authors describe the dynamic distribution of laminin in the olfactory system and forebrain. Using immunohistochemistry and transgenic lines, they found that the olfactory system and adjacent brain tissues are enveloped by BMs from the earliest stages of olfactory system assembly. They also found that laminin deposits follow the axonal trajectory of axons. They performed a functional analysis of the sly mutant to analyse the function of laminin γ1 in the development of the zebrafish olfactory system. Their study revealed that laminin enables the shape and position of placodes to be maintained late in the face of major morphogenetic movements in the brain, and its absence promotes the local entry of sensory axons into the brain and their navigation towards the olfactory bulb. 

Strengths: 

- They showed that in the sly mutants, no BM staining of laminin and Nidogen could be detected around the OP and the brain. The authors then elegantly used electron microscopy to analyse the ultrastructure of the border between the OP and the brain in control and sly mutant conditions. 

- To analyse the role of laminin γ1-dependent BMs in OP coalescence, the authors used the cluster size of Tg(neurog1:GFP)+ OP cells at 22 hpf as a marker. They found that the mediolateral dimension increased specifically in the mutants. However, proliferation did not seem to be affected, although apoptosis appeared to increase slightly at a later stage. This increase could therefore be due to a dispersal of cells in the OP. To test this hypothesis, the authors then analysed the cell trajectories and extracted 3D mean square displacements (MSD), a measure of the volume explored by a cell in a given period of time. Their conclusion indicates that although brain cell movements are increased in the absence of BM during coalescence phases, overall OP cell movements occur within normal parameters and allow OPs to condense into compact neuronal clusters in sly mutants. The authors also analysed the dimensions of the clusters composed of OMP+ neurons. Their results show an increase in cluster size along the dorso-ventral axis. These results were to be expected since, compared with BM, early neurog1+ neurons should compact along the medio-lateral axis, and those that are OMP+ essentially along the dorso-ventral axis. In addition to the DV elongation of OP tissue, the authors show the existence of isolated and ectopic (misplaced) YFP+ cells in sly mutants. 

- To understand the origin of these phenotypes, the authors analysed the dynamic behaviour of brain cells and OPs during forebrain flexion. The authors then quantitatively measured brain versus OPs in the sly mutant and found that the OP-brain boundary was poorly defined in the sly mutant compared with the control. Once again, the methods (cell tracks, brain size, and proliferation/apoptosis, and the shape of the brain/OP boundary) are elegant but the results were expected. 

- They then analysed the dynamic behaviour of the axon using live imaging. Thus, olfactory axon migration is drastically impaired in sly mutants, demonstrating that Laminin γ1dependent BMs are essential for the growth and navigation of axons from the OP to the olfactory bulb. 

- The authors therefore performed a quantitative analysis of the loss of function of Laminin γ1. They propose that the BM of the OP prevents its deformation in response to mechanical forces generated by morphogenetic movements of the neighbouring brain. 

Weaknesses: 

- The authors did not analyse neurog1 + axonal migration at the level of the single cell and instead made a global analysis. An analysis at the cell level would strengthen their hypotheses.  

- Rescue experiments by locally inducing Laminin expression would have strengthened the paper. 

- The paper lacks clarity between the two neuronal populations described (early EONs and late OSNs).  

- The authors quantitatively measured brain versus OPs in the sly mutant and found that the OP-brain boundary was poorly defined in the sly mutant compared with the control. Once again, the methods (cell tracks, brain size, proliferation/apoptosis, and the shape of the brain/OP boundary) are elegant but the results were expected. 

- A missing point in the paper is the effect of Laminin γ1 on the migration of cranial NCCs that interact with OP cells. The authors could have analysed the dynamic distribution of neural crest cells in the sly mutant. 

We thank the reviewer for the overall positive assessment of our work, and we carefully responded to all her/his insightful comments below. Live imaging experiments to (1) visualise exit and entry point formation with only a few axons labelled, (2) characterise the behaviour of single neurog1:GFP-positive neurons/axons during OP coalescence and to (3) analyse the migration of cranial NCC are now included in the revised manuscript to address the reviewer’s questions, and reinforce our initial conclusions.

Reviewer #2 (Public Review): 

Summary: 

This manuscript addresses the role of the extracellular matrix in olfactory development. Despite the importance of these extracellular structures, the specific roles and activities of matrix molecules are still poorly understood. Here, the authors combine live imaging and genetics to examine the role of laminin gamma 1 in multiple steps of olfactory development. The work comprises a descriptive but carefully executed, quantitative assessment of the olfactory phenotypes resulting from loss of laminin gamma. Overall, this is a constructive advance in our understanding of extracellular matrix contributions to olfactory development, with a well-written Discussion with relevance to many other systems. 

Strengths: 

The strengths of the manuscript are in the approaches: the authors have combined live imaging, careful quantitative analyses, and molecular genetics. The work presented takes advantage of many zebrafish tools including mutants and transgenics to directly visualize the laminin extracellular matrix in living embryos during the developmental process. 

Weaknesses: 

The weaknesses are primarily in the presentation of some of the imaging data. In certain cases, it was not straightforward to evaluate the authors' interpretations and conclusions based on the single confocal sections included in the manuscript. For example, it was difficult to assess the authors' interpretation of when and how laminin openings arise around the olfactory placode and brain during olfactory axon guidance. 

We thank the reviewer for the overall positive assessment of our work, and we carefully responded to all her/his insightful comments below. To address these comments, live imaging data to visualise exit and entry point formation with a sparse labelling of axons, and z-stacks showing how exit and entry points are organised in 3D, have been added to the revised manuscript.

Reviewer #3 (Public Review): 

This is a beautifully presented paper combining live imaging and analysis of mutant phenotypes to elucidate the role of laminin γ1-dependent basement membranes in the development of the zebrafish olfactory placode. The work is clearly illustrated and carefully quantified throughout. There are some very interesting observations based on the analysis of wild-type, laminin γ1, and foxd3 mutant embryos. The authors demonstrate the importance of a Laminin γ1-dependent basement membrane in olfactory placode morphogenesis, and in establishing and maintaining both boundaries and neuronal connections between the brain and the olfactory system. There are some very interesting observations, including the identification of different mechanisms for axons to cross basement membranes, either by taking advantage of incompletely formed membranes at early stages, or by actively perforating the membrane at later ones. 

This is a valuable and important study but remains quite descriptive. In some cases, hypotheses for mechanisms are stated but are not tested further. For example, the authors propose that olfactory axons must actively disrupt a basement membrane to enter the brain and suggest alternative putative mechanisms for this, but these are not tested experimentally. In addition, the authors propose that the basement membrane of the olfactory placode acts to resist mechanical forces generated by the morphogenetic movement of the developing brain, and thus to prevent passive deformation of the placode, but this is not tested anywhere, for example by preventing or altering the brain movements in the laminin γ1 mutant. 

We thank the reviewer for the overall positive assessment of our work and for suggesting interesting experiments to attempt in the future, and we carefully responded to all her/his constructive comments below.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

In general, it would be easier to draw conclusions and compare data if the authors used similar stages throughout the article. 

Throughout the article we tried to focus on a series of stages that cover both the coalescence of the OP (up to 24 hpf) and later stages of olfactory system development spanning the brain flexure process (28, 32, 36 hpf). However, for technical reasons it was not always possible to stick to these precise stages in some of our experiments. Also, in Fig. 1E-J, we picked in the movies some images illustrating specific cell or axonal behaviours, and thus the corresponding stages could not match exactly the stage series used in Fig. 1A-D and elsewhere in the article. Nevertheless, this stage heterogeneity does not affect our main conclusions.

It would be useful to schematise the olfactory placode and the brain in an insert to clearly visualise the system in each figure. 

We hope that the schematic which was initially presented in Fig. 1K already helps the reader to understand how the system is organised. Although we have not added more schematic views to represent the system in each figure (we think this would make the figures overcrowded), we have added additional legends to point to the OP and the brain in the pictures in order to clarify the localisation of each tissue.

In the Summary, the authors refer to the integrity of the basement membrane. I don't think there is any attempt to affect basement membrane integrity in the article. It would be important to do so to look at the effect on CNS-PNS separation and axonal elongation. 

In the Summary, we use the term « integrity of the basement membrane » to mention that we have analysed this integrity in the sly mutant. Given the results of our immunostainings against three main components of the basement membrane (Laminin, Collagen IV and Nidogen), as well as our EM observations, we see the sly mutant as a condition in which the integrity of the basement membrane is strongly affected.

Rescue experiments by locally inducing Laminin expression would have strengthened the paper. 

We have attempted to rescue the sly mutant phenotypes by introducing the mutation in the transgenic TgBAC(lamC1:lamC1-sfGFP) background, in which Laminin γ1 tagged with sfGFP is expressed under the control of its own regulatory sequences (Yamaguchi et al., 2022). To do so, we crossed sly+/-;Tg(omp:yfp) fish with sly+/-; Tg(lamC1:LamC1-sfGFP) fish. Surprisingly, while a rescue of the global embryo morphology was observed, no clear rescue of the olfactory system defects could be detected at 36 hpf. This could be due to the fact that the expression level of LamC1-sfGFP obtained with one copy of the transgene is not sufficient to rescue the olfactory system phenotypes, or that the sfGFP tag specifically affects the function of the Laminin 𝛾1 chain during the development of the olfactory system, making it unable to rescue the defects. Given the results of our first attemps, we decided not to continue in this direction.

(1) Developing OP & brain are surrounded by laminin-containing BM (already described by Torrez-Pas & Whitlock in 2014). 

"we first noticed the appearance of a continuous Laminin-rich BM surrounding the brain from 14-18 hpf, while around the OP, only discrete Laminin spots were detected at this stage (Fig. 1A, A'). " 

Around 8ss for Torrez-Pas & Whitlock (before 14 hpf). Can you modify the text, or show an 8ss stage embryo? As far as I know, the authors do not show images at 14hpf. Please correct this sentence or show a 14 hpf picture. 

The reviewer is right, we do not show any 14 hpf stage in the images and thus have removed this stage in the text and replaced it by 17 hpf.

In Figure 1A, the labelling of laminin 111 does not appear to be homogeneous along the brain.

Is this true? 

At this stage the brain’s BM revealed by the Laminin immunostaining appears fairly continuous (while the OP’s one is clearly dotty and less defined), but indeed very tiny/local interruptions of the signal can been seen along the structure as detected by the reviewer. We thus modified the text to mention these tiny interruptions.

How is the Laminin antibody used by the authors specific to laminin 111?  

We thank the reviewer for raising this important point. The immunogen used to produce this rabbit polyclonal antibody is the Laminin protein isolated from the basement membrane of a mouse Engelbreth Holm-Swarm sarcoma (EHS). It is thus likely to recognise several Laminin isoforms and not only Laminin 111. We thus replaced Laminin 111 by Laminin when mentioning this antibody in the text and Figures.

Please schematise in Figure 1K the stages you have tested and shown here in the article i.e. stages 18 - 22 - 28 -36 hpf using immunohistochemistry and 17-26-27-29-33 and 38 hpf using transgenics for laminin 111 and LamC1 respectively.  

As suggested by the reviewer, we changed the stages in the schematics for stages we have presented in Figure 1 (analysed either with immunostaining or in live imaging experiments). We chose to represent 17 - 22 - 26 - 33 hpf (and thus adapted some of the schematics for them to match these stages).  

Please specify in the Figure 1 legend for panels A to D whether this is a 3D projection or a zsection.

We indicated in the Figure 1 legend that all these images are single z-sections (as well as for panels E-J).

Furthermore, the schematisation in Fig. 1K does not reflect what the authors show: at 22 hpf laminin 111 labelling appears to be present only near the brain, and no labelling lateral to the olfactory placode and anteriorly and posteriorly. Thus, the schematisation in Figure 1K needs to be modified to reflect what the authors show.

We agree with the reviewer that the Laminin staining at this stage is observed around the medial region of the OP, but not more laterally. We modified the schematic view accordingly in Figure 1K. Anterior and posterior sides of the OP are not represented in this schematic because we chose to represent a frontal view rather than a dorsal view.

The authors suggest that" the laminin-rich BM of OP assembles between 18 and 22 hpf, during the late phase of OP coalescence". However, their data indicate that this BM assembles around 28hpf (Figure 1C). Can they clarify this point?

What we meant with this sentence is that we cleary see two distinct BMs from 22 hpf. However, as noticed by the reviewer, the OP’s BM is only present around the medial/basal regions of the OP and does not surround the whole OP tissue at this stage. We modified the text to clarify this point (in particular by mentioning that the OP’s BM starts to assemble between 18 and 22 hpf), and replaced the image shown in Figure 1B, B’ with a more representative picture (the previous z-section was taken in very dorsal regions of the OP).

It would be useful to disrupt these cells that have a cytoplasmic expression of Laminin-sfGFP, to analyse their contribution to BM and OP coalescence.

Indeed it will be interesting in the future to test specifically the role of the cells expressing cytoplasmic Laminin-sfGFP around and within the OP, as proposed by the reviewer. Laser ablation of these cells could be attempted, but due to their very superficial localisation, close to the skin, we believe these ablations (with the protocol/set-up we currently use in the lab) would impair the skin integrity, preventing us to conclude. We consider that the optimisation of this experiment is out of the scope of the present work.

Tg(-2.0ompb:gapYFP)rw032 marks ciliated olfactory sensory neurons (OSNs) (Sato et al., 2005). The authors should mention this. 

Please see our detailed response to the next point below.

Points to be clarified: 

-Tg(-2.0ompb:gapYFP)rw032 marks ciliated olfactory sensory neurons (OSNs) (Sato et al., 2005). The authors should mention this here. Moreover, the authors refer to "OP neurons" throughout the article. In the development of the olfactory organ, two types of neurons have been described in the literature: early EONs (12hpf-26hpf) and later OSNs. Each could have a specific role in the establishment and maintenance of the BM described by the authors. The authors need to clarify this point as, in Figure 1 for example, they use a marker for Tg(neurog1:GFP) EONs and a marker for ciliated OSNs without distinction. The distinction between EONs and OSNs comes a little late in the text and should be placed higher up. 

As mentioned by the reviewer, according to the initial view of neurogenesis in the OP, OP neurons are born in two waves. A transient population of unipolar, dendrite-less pioneer neurons would differentiate first, in the ventro-medial region of the OP and elongate their axons dorsally out of the placode, along the brain wall. These pioneer axons would then be used as a scaffold by later born OSNs located in the dorso-lateral rosette to outgrow their axons towards the olfactory bulb (Whitlock and Westerfield, 1998). 

Another study further characterised OP neurogenesis and showed that the first neurons to differentiate in the OP (the early olfactory neurons or EONs) express the Tg(neurog1:GFP) transgene (Madelaine et al., 2011). As mentioned by the authors in the discussion of this article, neurog1:GFP+ neurons appear much more numerous than the previously described pioneer neurons, and may thus include pioneers but also other neuronal subtypes.

We would like here to share additional, unpublished observations from our lab that further suggest that the situation is more complex than the pioneer/OSN and EON/OSN nomenclatures. First, in many of our live imaging experiments, we can clearly visualise some neurog1:GFP+ unipolar neurons, initially located in a medial position in the OP, which intercalate and contribute to the dorsolateral rosette (where OSNs are proposed to be located) at the end of OP coalescence, from 22-24 hpf. Second, in fixed tissues, we observed that most neurog1:GFP+ neurons located in the rosette at 32 hpf co-express the Tg(omp:meRFP) transgene (Sato et al., 2005). These observations suggest that at least a subpopulation of neurog1:GFP+ neurons could incorporate in the dorsolateral rosette and become ciliated OSNs during development. We can share these results with the reviewer upon request. Further studies are thus needed to clarify and describe the neuronal subpopulations and lineage relationships in the OP, but this detailed investigation is out of the scope and focus of the present study. 

An additional complication comes from the fact that, as shown and acknowledged by the authors in Miyasaka et al., 2005, the Tg(omp:meYFP) line (6kb promoter) labels ciliated OSNs in the rosette but also some unipolar, ventral neurons (around 10 neurons at 1 dpf, Miyasaka et al. 2005, Figure 3A, white arrowheads). This was also observed using the 2 kb promoter Tg(omp:meYFP) line (see for instance Miyasaka et al., 2007) and in our study, we can indeed detect these ventro-medial neurons labelled in the Tg(omp:meYFP) line (2 kb promoter), see for instance Figure 1C’, D’ or Movie 6. It is unclear whether these unipolar omp:meYFPpositive cells are pioneer neurons or EONs expressing the omp:meYFP transgene, or OSN progenitors that would be located basally/ventrally in the OP at these stages.

For all these reasons, we decided to present in the text the current view of neurogenesis in the OP but instead of attributing a definitive identity to the neurons we visualise with the transgenic lines, we prefer to mention them in the manuscript (and in the rest of the response to the reviewers) as neurons expressing neurog1:GFP or omp:meYFP transgenes (or cells/axons/neurons expressing RFP in the Tg(cldnb:Gal4; UAS:RFP) background).

What we also changed in the text to be more clear on this point:

- we moved higher up in the text, as suggested by reviewer 1, the description of the current model of neurogenesis in the OP,

- we mentioned that neurog1:GFP+ neurons are more numerous than the initially described pioneer neurons, as discussed in Madelaine et al., 2011,

- we wrote more clearly that the Tg(omp:meYFP) line labels ciliated OSNs but also a subset of unipolar, ventral neurons (Miyasaka et al., 2005), and pointed to these ventral neurons in Figure 1C’, D’,

- in the initial presentation of the current view of OP neurogenesis we renamed neurog1:GFP+ into EONs to be coherent with Madelaine et al., 2011.

- To visualise pioneer axons, the authors should use an EONS marker such as neurog1 because, to my knowledge, OMP only marks OSN axons and not pioneer axons.  

To visualise neurog1:GFP+ axons during OP coalescence, we performed live imaging upon injection of the neurog1:GFP plasmid (Blader et al., 2003) in the Tg(cldnb:Gal4; UAS:RFP) background (n = 4 mutants and n = 4 controls from 2 independent experiments). We observed some GFP+ placodal neurons exhibiting retrograde axon extension in both controls and sly mutants. In such experiments it is very difficult to quantify and compare the number of neurons/axons showing specific behaviours between different experimental conditions/genetic background. Indeed, due to the cytoplasmic localisation of GFP, the axons can only be seen in neurons expressing high levels of GFP, and due to the injection the number of such neurons varies a lot in between embryos, even in a given condition. Nevertheless, our qualitative observations reinforce the idea that the basement membrane is not absolutely required for mediolateral movements and retrograde axon extension of neurog1:GFP+ neurons in the OP. We added examples of images extracted from these new live imaging experiments in the revised Fig. S5A, B.

- The authors should analyse the presence of laminin in the OP and forebrain in conjunction with neural crest cell dynamics (using a Sox10 transgenic line for example) to refine their entry and exit point hypotheses. 

As described in the answer to the next point, we performed new experiments in which we visualised NCC migration in the Tg(neurog1:GFP) background, which allowed us to analyse the localisation of NCC at the forebrain/OP boundary, in ventral and dorsal positions, both in sly mutant embryos and control siblings.

- A dynamic analysis of the distribution of neural crest cells in the sly mutant over time and during OP coalescence would be important. 

The dynamics of zebrafish cranial NCC migration in the vicinity of the OP has been previously analysed using sox10 reporter lines (Harden et al., 2012, Torres-Paz and Whitlock, 2014, Bryan et al., 2020). To address the point raised by the reviewer, we performed live imaging from 16 to 32 hpf on sly mutants and control siblings carrying the Tg(neurog1:GFP) and Tg(UAS:RFP) transgenes and injected with a sox10(7.2):KalTA4 plasmid (Almeida et al., 2015). This allows the mosaic labelling of cells that express or have expressed sox10 during their development which, in the head region at these stages, represents mostly NCC and their derivatives. 3 independent experiments were carried out (n = 4 mutant embryos in which 8 placodes could be analysed; n = 6 control siblings in which 10 placodes could be analysed). A new movie (Movie 9) has been added to the revised article to show representative examples of control and mutant embryos.

From these new data, we could make the following observations:

- As expected from previous studies (Harden et al., 2012, Torres-Paz and Whitlock, 2014, Bryan et al., 2020), in control embryos a lot of NCC had already migrated to reach the vicinity of the OP when the movies begin at 16 hpf, and were then seen invading mainly the interface between the eye and the OP (10/10 placodes). Surprisingly, in sly mutants, a lot of motile NCC had also reached the OP region at 16 hpf in all the analysed placodes (8/8), and populated the eye/OP interface in 7/8 placodes (10/10 in controls). Counting NCC or tracking individual NCC during the whole duration of the movies was unfortunately too difficult to achieve in these movies, because of the low level of mosaicism (a high number of cells were labelled) and of the high speed of NCC movements (as compared with the 10 min delta t we chose for the movies). 

- in some of the control placodes we could detect a few NCC that populated the forebrain/OP interface, either ventrally, close to the exit point of the axons (4/10 placodes), or more dorsally (8/10 placodes). By contrast, in sly mutants, NCC were observed in the dorsal region of the brain/OP boundary in only 2/8 placodes, and in the ventral brain/OP frontier in only 2/8 placodes as well. Interestingly, in these 2 last samples, NCC that had initially populated the ventral region of the brain/OP interface were then expelled from the boundary at later stages.

We reported these observations in a new Table that is presented in revised Fig. S6B. In addition, instances of NCC migrating at the eye/OP or forebain/OP interfaces are indicated with arrowheads on Movie 9. Previous Figure S6 was splitted into two parts presenting NCC defects in sly mutants (revised Figure S6) and in foxd3 mutants (revised Figure S7).

Altogether, these new data suggest that the first postero-anterior phase of NCC migration towards the OP, as well as their migration in between the eye and OP tissues, is not fully perturbed in sly mutants. The subset of NCC that populate the OP/forebrain seem to be more specifically affected, as these NCC show defects in their migration to the interface or the maintenance of their position at the interface. Since the crestin marker labels mostly NCC at the OP/forebrain interface at 32 hpf (revised Fig. S6A), this could explain why the crestin ISH signal is almost lost in sly mutants at this stage.

(2) Laminin distribution suggests a role in olfactory axon development 

"Laminin 111 immunostaining revealed local disruptions in the membrane enveloping the OP and brain, precisely where YFP+ axons exit the OP (exit point) and enter the brain (entry point) (Fig. 1C-D')." Can the authors quantify this situation? It would be important to analyse this behaviour on the scale of a neuron and thus axonal migration to strengthen the hypotheses. 

As suggested by the reviewer, to better visualise individual axons at the exit and entry point, we used mosaic red labelling of OP axons. To achieve this sparse labelling, we took advantage of the mosaic expression of a red fluorescent membrane protein observed in the Tg(cldnb:Gal4; UAS:lyn-TagRFP) background. The unpublished Tg(UAS:lyn-TagRFP) line was kindly provided by Marion Rosello and Shahad Albadri from the lab of Filippo Del Bene. We crossed the Tg(cldnb:Gal4; UAS:lyn-TagRFP) line with the TgBAC(lamC1:lamC1-sfGFP) reporter and performed live imaging on 2 embryos/4 placodes, in a frontal view. A new movie (Movie 3 in the revised article) shows examples of exit and entry point formation in this context.This allowed us to visualise the formation of the exit and entry points in more samples (6 embryos and 12 placodes in total when we pool the two strategies for labelling OP axons) and through the visualisation of a small number of axons, and reinforce our initial conclusions. 

(3) The integrity of BMs around the brain and the OP is affected in the sly mutant 

Why do the authors analyse the distribution of collagen IV and Nidogen and not proteoglycans and heparan sulphate? 

We attempted to label more ECM components such as proteoglycans and heparan sulfate, but whole-mount immunostainings did not work in our hands.

A dynamic analysis of the distribution of neural crest cells in the sly mutant over time and during OP coalescence would be important. 

See our detailed response to this point above.  

(4) Role of Laminin γ1-dependent BMs in OP coalescence 

The authors use the size of the Tg(neurog1:GFP)+ OP cell cluster at 22 hpf as a marker.  The authors should count the number of cells in the OP at the indicated time using a nuclear dye to check that in the sly mutant the number of cells is the same over time. Two time points as analysed in Figure S2 may not be sufficient to quantify proliferation which at these stages should be almost zero according to Whitlock & Westerfield and Madelaine et al.

Counting the neurog1:GFP+ cell numbers in our existing data was unfortunately impossible, due to the poor quality of the DAPI staining. We are nevertheless confident that the number of cells within neurog1:GFP+ clusters is fairly similar between controls and sly mutants at 22 hpf, since the OP dimensions are the same for AP and DV dimensions, and only slightly different for the ML dimension. In addition, we analysed proliferation and apoptosis within the neurog1:GFP+ cluster at 16 and 21 hpf and observed no difference between controls and mutants.

(5) Role of Laminin γ1-dependent BMs during the forebrain flexure 

In Figure 4F at 32hpf, the presence of 77% ectopic OMP+ cells medially should result in an increase in dimensions along the M-L? This is not the case in the article. The authors should clarify this point. 

As we explained in the Material and Methods, ectopic fluorescent cells (cells that are physically separated from the main cluster) were not taken into account for the measurement of the OP dimensions. This is now also also mentioned in the legends of the Figures (4 and S3) showing the quantifications of OP dimensions.

Cell distribution also seems to be affected within the OMP+ cluster at 36hpf, with fewer cells laterally and more medially. The authors should analyse the distribution of OMP+ cells in the clusters. in sly mutants and controls to understand whether the modification corresponds to the absence of BM function. 

On the pictures shown in Figure 4F,G, we agree that omp:meYFP+ cells appear to be more medially distributed in the mutant, however this is not the case in other sections or samples, and is rather specific to the z-section chosen for the Figure. We found that the ML dimension is unchanged in mutants as compared with controls, except for the 28 hpf stage where it is smaller, but this appears to be a transient phenomenon, since no change is detected at earlier or later stages (Figure 4A-D and Figure S3A-L). The difference we observe at 28 hpf is now mentioned in the revised manuscript.

The conclusions of Figures 4 and S3 would rather be that laminin allows OMP+ cells to be oriented along the medio-lateral axis whereas it would control their position along the dorsoventral axis. The authors should modify the text. It would be useful to map the distribution of OMP+ cells along the dorsoventral and mediolateral axes. The same applies to Neurog1+ cells. An analysis of skin cell movements, for example, would be useful to determine whether the effects are specific.  

We are confident that the measurements of OP dimensions in AP, DV and ML are sufficient to describe the OP shape defects observed in the sly mutants. Analysing cell distribution along the 3 axes as well as skin cell movements will be interesting to perform in the future but we consider these quantifications as being out of the scope of the present work.

(6) Laminin γ1-dependent BMs are required to define a robust boundary between the OP and the brain 

The authors must weigh this conclusion "Laminin γ1-dependent BMs serve to establish a straight boundary between the brain and OP, preventing local mixing and late convergence of the two OPs towards each other during flexion movement." Indeed, they don't really show any local mixing between the brain and OP cells. They would need to quantify in their images (Figure 5A-A' and Figure S4 A-A') the percentage of cells co-labelled by HuC and Tg(cldnb:GFP). 

We agree with the reviewer and thus replaced « reveal » by « suggest » in the conclusion of this section. 

(7) Role of Laminin γ1-dependent BMs in olfactory axon development 

An analysis of the retrograde extension movement in the axons of OMP+ ectopic neurons in the sly1 mutant condition would be useful to validate that the loss of laminin function does not play a role in this event. 

Indeed, even though we can visualise instances of retrograde extension occurring normally in sly mutants, we can not rule out that this process is affected in a subset of OP neurons, for instance in ectopic cells, which often show no axon or a misoriented axon. We added a sentence to mention this in the revised manuscript.

Minor comments and typos: 

Please check and mention the D-V/L-M or A-P/L-M orientation of the images in all figures. 

This has been checked.

Legend Figure 1: "distalmost" is missing a space "distal most". 

We checked and this word can be written without a space.

Figure 1 panel C: check the orientation (I am not sure that Dorsal is up). 

We double-checked and confirm that dorsal is up in this panel.

Movie 1 Legend: "aroung "the OP should be around the OP. 

Thanks to the reviewer for noticing the typo, we corrected it.

Reviewer #2 (Recommendations For The Authors):

The comments below are relatively minor and mostly raise questions regarding images and their presentation in the manuscript. 

• Figure 1, visualization of exit and entry points: It is a bit difficult to visualize the axon exit and entry points in these images, and in particular, to understand how the exit and entry points in C and D correspond to what is seen in F, F', H, and H'. There appears to be one resolvable break in the staining in C and D, whereas there are two distinct breaks in F-H'. Are these single optical sections? Is it possible to visualize these via 3-dimensional rendering? 

All the images presented in Figure 1 are single z-sections, which is now indicated in the Figure legend. As noticed by the reviewer, Laminin immunostainings on fixed embryos at 28 and 36 hpf suggested that the exit and entry points are facing each other, as shown in Figure 1C-D’. However, in our live imaging experiments we always observed that the exit point is slightly more ventral than the entry point (of about 10 to 20 µm). This discrepancy could be due to the fixation that precedes the immunostaining procedure, which could modify slightly the size and shape of cells/tissues. We added a sentence on this point in the text. In addition, we added new movies of the LamC1-sfGFP reporter with sparse red axonal labelling (Movie 3, see response to reviewer 1), as well as z-stacks presenting the organisation of exit and entry points in 3D (Movie 4), which should help to better illustrate the mechanisms of exit and entry point formation.

• Movie 2, p. 6, "small interruptions of the BM were already present near the axon tips, along the ventro-medial wall of the OP." This is a bit difficult to assess since the movie seems to show at least one other small interruption in the BM in addition to the exit point, in particular, one slightly dorsal to the exit point. Was this seen in other samples, or in different optical sections? 

Indeed the exit and entry points often appear as regions with several, small BM interruptions, rather than single holes in the BM. We now show in revised Movie 4 the two z-stacks (the merge and the single channel for green fluorescence) corresponding to the last time points of the movies showing exit and entry point formation in Movie 2, where several BM interruptions can be seen for both the exit and entry points. We had already mentioned this observation in the legend of Movie 2, and we added a sentence on this point in the main text of the revised manuscript. This is also represented for both exit and entry points in the new schematics in revised Fig. 1K and its legend. 

• Movie 2, p. 6, "The opening of the entry point through the brain BM was concomitant with the arrival of the RFP+ axons, suggesting that the axons degrade or displace BM components to enter the brain." Similar to the questions regarding the exit point, it was a bit difficult to evaluate this statement. There appears to be a broader region of BM discontinuity more dorsal to the arrowhead in Movie 2. A single-channel movie of just the laminin fluorescence might help to convey the extent of the discontinuity. As with above, was this seen in other samples, or in different optical sections?  

See our response to the previous comment.

• Figure 1H, I, "the distal tip of the RFP+ axons migrated in close proximity with the brain's BM." This is again a bit difficult to see, and quite different than what is seen in Figure 4A, in which the axons do not seem close to the BM in this section. Is it possible to visualize this via 3-dimensional rendering? 

In fixed embryos or in live imaging experiments, we observed that, once entered in the brain, the distal tips (the growth cones) of the axons are located close to the BM of the brain. However, this is not the case of the axon shafts which, as development proceeds, are located further away from the BM. This can clearly be seen at 36 hpf in Figure 1D’ and Figure 4A, as spotted by the reviewer. We modified the text to clarify this point.

• Figure 2J, J', p. 7, the gap between the OP and brain cells of sly mutants "was most often devoid of electron-dense material." It is difficult to see this loss of electron-dense material in 2J'. The thickness of the space is quantified well and is clearly smaller, but the change in electron-dense material is more difficult to see.  

We looked at Figure 2 again and it seems clear to us that there is electron-dense material between the plasma membranes in controls, which is practically not seen (rare spots) in the mutants. We added a sentence mentioning that we rarely see electron-dense spots in sly mutants.

• Figure 5E-F': There are concerns about evaluating the shape of a tissue based on nuclear position. Is there a way to co-stain for cell boundaries (maybe actin?), and then quantify distortion of the dlx+ cell population using the cell boundaries, rather than nuclear staining? 

We agree with the reviewer that it is not ideal to evaluate the shape of the OP/brain boundary based on a nuclear staining. As explained in the text, we could not use the Tg(eltC:GFP) or Tg(cldnb:Gal4; UAS:RFP) reporter lines for this analysis, due to ectopic or mosaic expression. However we are confident that the segmentation of the Dlx3b immunostaining reflects the organisation of the cells at the OP/brain tissue boundary: in other data sets in which we performed Dlx3b staining with membrane labelling independently of the present study and in the wild type context, we clearly see that cell membranes are juxtaposed to the Dlx3b nuclear staining (in other words, the cytoplasm volume of OP cells is very small). 

• Figure S5E: It would be helpful to see representative images for each of the categories (Proper axon bundle; Ventral projections; Medial projections) or a schematic to understand how the phenotypes were assessed. 

To address this point we added a schematic view to illustrate the phenotypes assessed in each column of the table in revised Figure S5G.

• Figure 6, p. 12, "Laminin gamma 1-dependent BMs are essential for growth and navigation of the axons...": What fraction of the tracked axons managed to exit the OP? Given the quantitative analyses in Figure 6, one might interpret this to mean that laminin gamma 1 is not essential for axon growth (speed and persistence are largely unchanged), but rather, primarily for navigation. 

As noticed by the reviewer, the speed and persistence of axonal growth cones are largely unchanged in the sly mutants (except for the reduced persistence in the 200-400 min window, and an increased speed in the 800-1000 min window), showing that the growth cones are still motile. However, as shown by the tracks, they tend to wander around within the OP, close to the cell bodies, which results in the end in a perturbed growth of the axons. The navigation issues are rather revealed by the analysis of fixed Tg(omp:meYFP) embryos presented in the table of Figure S5G. We modified the text to separate more clearly the conclusions of the two types of experiments (fixed, transgenic embryos versus live, mosaically labelled embryos).

Reviewer #3 (Recommendations For The Authors):

Testing the hypotheses mentioned in the public review will be interesting experiments for a follow-up study, but are not essential revisions for this manuscript. 

I have only a few minor suggestions for revisions: 

P8 subheading 'Role of Laminin γ1-dependent BMs in OP coalescence' - since no major role was demonstrated here, this heading should be reworded.  

We agree with the reviewer and replaced the previous title by « OP coalescence still occurs in the sly mutant ».

P11, line 3 - the authors conclude that the forebrain is smaller 'due to' the inward convergence of the OPs. I do not think it is possible to assign causation to this when the mutant disrupts Laminin γ1 systemically - it is equally possible that the OPs move inward due to a failure of the brain to form in the normal shape. Thus, the wording should be changed here. (In the Discussion on p15, the authors mention the 'apparent distortion' of the brain, and say that it is 'possibly due' to the inward migration of the placodes', but again this could be toned down.) 

We agree with the reviewer’s comment and changed the wording of our conclusions in the Results section.

P11 and Fig. S5 - The table and text seem to be saying opposite things here. The text on p11 (3rd paragraph) indicates that the normal exit point is ventral and that this is disrupted in the mutant, with axons exiting dorsally. However, in the table, at each time point there is a higher % of axons exiting ventrally in the mutant. Please clarify. The table does not provide a % value for axons exiting dorsally - it might help to add a column to show this value. 

We are grateful to the reviewer for pointing this out, and we apologize for the lack of clarity in the first version of the manuscript. We have modified the text and Figure S5 in order to clarify the different points raised by the reviewer in this comment. The Table in Fig. S5G does not represent the % of axons showing defects, but the % of embryos showing the phenotypes. In addition, an embryo is counted in the ventral or medial projection category if it shows at least one ventral or medial projection (even if its shows a proper bundle). This is now clearly indicated in the title of the columns in the table itself and in the legend. The embryos in which the axons exit dorsally in sly mutants are actually those counted in the left column of the Table (they exit dorsally and form a bundle), as shown by the new schematics added below the table. We also added this information in the title of the left column, and mention in the legend the pictures in which this dorsal exit can be observed in the article (Figures 4B and S3E’). Having more sly mutant embryos with axons exiting dorsally is thus compatible with more embryos showing at least one ventral projection.

Fig. S6, shows the lack of neural crest cells between the olfactory placode and the brain in both laminin γ1 mutants (without a basement membrane) and foxd3 mutants (which retain the membrane). Comparison of the two mutants here is a neat experiment and the result is striking, demonstrating that it is the basement membrane, and not the neural crest, that is required for correct morphology of the olfactory placode. I think this figure should be presented as a main figure, rather than supplementary.  

Our new live imaging characterisation of NCC migration in sly mutants and control siblings (Movie 9) revealed that at 32 hpf, in the vicinity of the OP, NCC (or their derivatives) are much more numerous than the subset of NCC showing crestin expression by in situ hybridisation (compare the end of our control movie – 32 hfp, with crestin ISH shown in Figure S6A for instance). 

Thus, the extent of the NCC migration defects should be analysed in more detail in the foxd3 mutant in the future (using live imaging or other NCC markers), and for this reason we chose to keep this dataset in the supplementary Figures.

One of the first topics covered in the Discussion section is the potential role of Collagen. I was surprised to see the description on P15 'the dramatic disorganization of the Collagen IV pattern observed by immunofluorescence in the sly mutant', as I hadn't picked this up from the Results section of the paper. I went back to the relevant figure (Fig. 2) and description on p7, which does not give the same impression: 'in sly mutants, Collagen IV immunoreactivity was not totally abolished'. This suggested to me that there was only minor (not dramatic) disorganisation of the Collagen IV. This needs clarification.  

The linear, BM-like Collagen IV staining was lost in sly mutants, but not the fibrous staining which remained in the form of discrete patches surrounding the OP. We modified the text in the Results section as well as in the Figure 2 legend to clarify our observations made on embryos immunostained for Collagen IV.

Typos etc 

P5 - '(ii) above of the neuronal rosette' - delete the word 'of'. 

P5 two lines below this - ensheathed. 

P10 - '3 distinct AP levels' (delete s from distincts). 

P10 - distortion (not distorsion) . 

P12 - 'From 14 hpf, they' should read 'From 14 hpf, neural crest cells'. 

P15, line 1 - 'is a consequence of' rather than 'is consecutive of'? 

P22 'When the data were not normal,' should read 'When the data were not normally distributed,'. 

We thank the reviewer for noticing these typos and have corrected them.

General 

Please number lines in future manuscripts for ease of reference. 

This has been done.

The article does have sources sited. The article uses APA citations and uses a range of academic sources. Sources include both primary and secondary sources for the research.

"theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it" "theres no reason to want it"

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• • [

    Designing a tympanum through stereographic projection

    ](https://en.wikipedia.org/wiki/Astrolabe#Designing_a_tympanum_through_stereographic_projection) - [

    The tropics and the equator define the tympanum
    
    ](https://en.wikipedia.org/wiki/Astrolabe#The_tropics_and_the_equator_define_the_tympanum)
    

    • [

      The horizon and the measurement of altitude

      ](https://en.wikipedia.org/wiki/Astrolabe#The_horizon_and_the_measurement_of_altitude) - [

      The meridians and the measurement of azimuth

      ](https://en.wikipedia.org/wiki/Astrolabe#The_meridians_and_the_measurement_of_azimuth) - [

  See also

  ](https://en.wikipedia.org/wiki/Astrolabe#See_also) - [

  References

  ](https://en.wikipedia.org/wiki/Astrolabe#References) - [

  Bibliography

  ](https://en.wikipedia.org/wiki/Astrolabe#Bibliography) - [

  External links

  ](https://en.wikipedia.org/wiki/Astrolabe#External_links)

Astrolabe

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From Wikipedia, the free encyclopedia

For other pages with a similar name, see Astrolabe (disambiguation). Not to be confused with Cosmolabe.

North African, 9th century AD, Planispheric Astrolabe. Khalili Collection.

A modern astrolabe made in Tabriz, Iran in 2013.

An astrolabe (Greek: ἀστρολάβος astrolábos, 'star-taker'; Arabic: ٱلأَسْطُرلاب al-Asṭurlāb; Persian: ستاره‌یاب Setāreyāb) is an astronomical instrument dating to ancient times. It serves as a star chart and physical model of visible heavenly bodies. Its various functions also make it an elaborate inclinometer and an analog calculation device capable of working out several kinds of problems in astronomy. In its simplest form it is a metal disc with a pattern of wires, cutouts, and perforations that allows a user to calculate astronomical positions precisely. It is able to measure the altitude above the horizon of a celestial body, day or night; it can be used to identify stars or planets, to determine local latitude given local time (and vice versa), to survey, or to triangulate. It was used in classical antiquity, the Islamic Golden Age, the European Middle Ages and the Age of Discovery for all these purposes.

The astrolabe, which is a precursor to the sextant,^[1]^ is effective for determining latitude on land or calm seas. Although it is less reliable on the heaving deck of a ship in rough seas, the mariner's astrolabe was developed to solve that problem.

Applications

16th-century woodcut of measurement of a building's height with an astrolabe

The 10th-century astronomer ʿAbd al-Raḥmān al-Ṣūfī wrote a massive text of 386 chapters on the astrolabe, which reportedly described more than 1,000 applications for the astrolabe's various functions.^[2]^ These ranged from the astrological, the astronomical and the religious, to navigation, seasonal and daily time-keeping, and tide tables. At the time of their use, astrology was widely considered as much of a serious science as astronomy, and study of the two went hand-in-hand. The astronomical interest varied between folk astronomy (of the pre-Islamic tradition in Arabia) which was concerned with celestial and seasonal observations, and mathematical astronomy, which would inform intellectual practices and precise calculations based on astronomical observations. In regard to the astrolabe's religious function, the demands of Islamic prayer times were to be astronomically determined to ensure precise daily timings, and the qibla, the direction of Mecca towards which Muslims must pray, could also be determined by this device. In addition to this, the lunar calendar that was informed by the calculations of the astrolabe was of great significance to the religion of Islam, given that it determines the dates of important religious observances such as Ramadan.^[citation needed]^

Etymology

The Oxford English Dictionary gives the translation "star-taker" for the English word astrolabe and traces it through medieval Latin to the Greek word ἀστρολάβος : astrolábos,^[3]^^[4]^ from ἄστρον : astron "star" and λαμβάνειν : lambanein "to take".^[5]^

In the medieval Islamic world the Arabic word al-Asturlāb (i.e., astrolabe) was given various etymologies. In Arabic texts, the word is translated as ākhidhu al-Nujūm (Arabic: آخِذُ ٱلنُّجُومْ, lit. 'star-taker'), a direct translation of the Greek word.^[6]^

Al-Biruni quotes and criticises medieval scientist Hamza al-Isfahani who stated:^[6]^ "asturlab is an arabisation of this Persian phrase" (sitara yab, meaning "taker of the stars").^[7]^ In medieval Islamic sources, there is also a folk etymology of the word as "lines of lab", where "Lab" refers to a certain son of Idris (Enoch). This etymology is mentioned by a 10th-century scientist named al-Qummi but rejected by al-Khwarizmi.^[8]^

History

Ancient era

An astrolabe is essentially a plane (two-dimensional) version of an armillary sphere, which had already been invented in the Hellenistic period and probably been used by Hipparchus to produce his star catalogue. Theon of Alexandria (c. 335 -- c. 405) wrote a detailed treatise on the astrolabe.^[9]^ The invention of the plane astrolabe is sometimes wrongly attributed to Theon's daughter Hypatia (born c. 350--370; died AD 415),^[10]^^[11]^^[12]^^[13]^ but it's known to have been used much earlier.^[11]^^[12]^^[13]^ The misattribution comes from a misinterpretation of a statement in a letter written by Hypatia's pupil Synesius (c. 373 -- c. 414),^[11]^^[12]^^[13]^ which mentions that Hypatia had taught him how to construct a plane astrolabe, but does not say that she invented it.^[11]^^[12]^^[13]^ Lewis argues that Ptolemy used an astrolabe to make the astronomical observations recorded in the Tetrabiblos.^[9]^ However, Emilie Savage-Smith notes "there is no convincing evidence that Ptolemy or any of his predecessors knew about the planispheric astrolabe".^[14]^ In chapter 5,1 of the Almagest, Ptolemy describes the construction of an armillary sphere, and it is usually assumed that this was the instrument he used.

Astrolabes continued to be used in the Byzantine Empire. Christian philosopher John Philoponus wrote a treatise (c. 550) on the astrolabe in Greek, which is the earliest extant treatise on the instrument.^[a]^ Mesopotamian bishop Severus Sebokht also wrote a treatise on the astrolabe in the Syriac language during the mid-7th century.^[b]^ Sebokht refers to the astrolabe as being made of brass in the introduction of his treatise, indicating that metal astrolabes were known in the Christian East well before they were developed in the Islamic world or in the Latin West.^[15]^

Medieval era

Astrolabes were further developed in the medieval Islamic world, where Muslim astronomers introduced angular scales to the design,^[16]^ adding circles indicating azimuths on the horizon.^[17]^ It was widely used throughout the Muslim world, chiefly as an aid to navigation and as a way of finding the Qibla, the direction of Mecca. Eighth-century mathematician Muhammad al-Fazari is the first person credited with building the astrolabe in the Islamic world.^[18]^

The mathematical background was established by Muslim astronomer Albatenius in his treatise Kitab az-Zij (c. AD 920), which was translated into Latin by Plato Tiburtinus (De Motu Stellarum). The earliest surviving astrolabe is dated AH 315 (AD 927--928). In the Islamic world, astrolabes were used to find the times of sunrise and the rising of fixed stars, to help schedule morning prayers (salat). In the 10th century, al-Sufi first described over 1,000 different uses of an astrolabe, in areas as diverse as astronomy, astrology, navigation, surveying, timekeeping, prayer, Salat, Qibla, etc.^[19]^^[20]^

An Arab astrolabe from 1208

The spherical astrolabe was a variation of both the astrolabe and the armillary sphere, invented during the Middle Ages by astronomers and inventors in the Islamic world.^[c]^ The earliest description of the spherical astrolabe dates to Al-Nayrizi (fl. 892--902). In the 12th century, Sharaf al-Dīn al-Tūsī invented the linear astrolabe, sometimes called the "staff of al-Tusi", which was "a simple wooden rod with graduated markings but without sights. It was furnished with a plumb line and a double chord for making angular measurements and bore a perforated pointer".^[21]^ The geared mechanical astrolabe was invented by Abi Bakr of Isfahan in 1235.^[22]^

The first known metal astrolabe in Western Europe is the Destombes astrolabe made from brass in the eleventh century in Portugal.^[23]^^[24]^ Metal astrolabes avoided the warping that large wooden ones were prone to, allowing the construction of larger and therefore more accurate instruments. Metal astrolabes were heavier than wooden instruments of the same size, making it difficult to use them in navigation.^[25]^

Spherical astrolabe

A depiction of Hermann of Reichenau with an astrolabe in a 13th-century manuscript by Matthew Paris

Herman Contractus of Reichenau Abbey, examined the use of the astrolabe in Mensura Astrolai during the 11th century.^[26]^ Peter of Maricourt wrote a treatise on the construction and use of a universal astrolabe in the last half of the 13th century entitled Nova compositio astrolabii particularis. Universal astrolabes can be found at the History of Science Museum in Oxford.^[27]^ David A. King, historian of Islamic instrumentation, describes the universal astrolobe designed by Ibn al-Sarraj of Aleppo (aka Ahmad bin Abi Bakr; fl. 1328) as "the most sophisticated astronomical instrument from the entire Medieval and Renaissance periods".^[28]^

English author Geoffrey Chaucer (c. 1343--1400) compiled A Treatise on the Astrolabe for his son, mainly based on a work by Messahalla or Ibn al-Saffar.^[29]^^[30]^ The same source was translated by French astronomer and astrologer Pélerin de Prusse and others. The first printed book on the astrolabe was Composition and Use of Astrolabe by Christian of Prachatice, also using Messahalla, but relatively original.

Front of an Indian astrolabe now kept at the Royal Museum of Scotland at Edinburgh.

In 1370, the first Indian treatise on the astrolabe was written by the Jain astronomer Mahendra Suri, titled Yantrarāja.^[31]^

A simplified astrolabe, known as a balesilha, was used by sailors to get an accurate reading of latitude while at sea. The use of the balesilha was promoted by Prince Henry (1394--1460) while navigating for Portugal.^[32]^

The astrolabe was almost certainly first brought north of the Pyrenees by Gerbert of Aurillac (future Pope Sylvester II), where it was integrated into the quadrivium at the school in Reims, France, sometime before the turn of the 11th century.^[33]^ In the 15th century, French instrument maker Jean Fusoris (c. 1365--1436) also started remaking and selling astrolabes in his shop in Paris, along with portable sundials and other popular scientific devices of the day.

Astronomical Instrument Detail by Ieremias Palladas 1612

Thirteen of his astrolabes survive to this day.^[34]^ One more special example of craftsmanship in early 15th-century Europe is the astrolabe designed by Antonius de Pacento and made by Dominicus de Lanzano, dated 1420.^[35]^

In the 16th century, Johannes Stöffler published Elucidatio fabricae ususque astrolabii, a manual of the construction and use of the astrolabe. Four identical 16th-century astrolabes made by Georg Hartmann provide some of the earliest evidence for batch production by division of labor. In 1612, Greek painter Ieremias Palladas incorporated a sophisticated astrolabe in his painting depicting Catherine of Alexandria. The painting was entitled Catherine of Alexandria and featured a device called the System of the Universe (Σύστημα τοῦ Παντός). The device featured the planets with the names in Greek: Selene (Moon), Hermes (Mercury), Aphrodite (Venus), Helios (Sun), Ares (Mars), Zeus (Jupiter), and Chronos (Saturn). The device also featured celestial spheres following the Ptolemaic model and Earth was depicted as a blue sphere with circles of geographic coordinates. A complex line representing the axis of the Earth covered the entire instrument.^[36]^

Medieval astrolabes

• 

  A treatise explaining the importance of the astrolabe by Nasir al-Din al-Tusi, Persian scientist

• 

  Astrolabe of Jean Fusoris, made in Paris, 1400

• 

  An 18th-century Persian astrolabe

• 

  Disassembled 18th-century astrolabe

• 

  Exploded view of an astrolabe

• 

  Animation showing how celestial and geographic coordinates are mapped on an astrolabe's tympan through a stereographic projection. Hypothetical tympan (40° north latitude) of a 16th-century European planispheric astrolabe.

• 

  Astrolabe manual from the Alfonso X of Castile work Libros del saber de astronomía, 1276.

• 

  A page from the 1575 book "Astrolabium" depicting an astrolabe. Masha'Allah Public Library Bruges [nl] Ms. 522

Astrolabes and clocks

Amerigo Vespucci observing the Southern Cross by looking over the top of an armillary sphere bizarrely held from the top as if it were an astrolabe; however, an astrolabe cannot be used by looking over its top. The page inexplicably contains the word astrolabium. By Jan Collaert II. Museum Plantin-Moretus, Antwerp, Belgium.

Mechanical astronomical clocks were initially influenced by the astrolabe; they could be seen in many ways as clockwork astrolabes designed to produce a continual display of the current position of the sun, stars, and planets. For example, Richard of Wallingford's clock (c. 1330) consisted essentially of a star map rotating behind a fixed rete, similar to that of an astrolabe.^[37]^

Many astronomical clocks use an astrolabe-style display, such as the famous clock at Prague, adopting a stereographic projection (see below) of the ecliptic plane. In recent times, astrolabe watches have become popular. For example, Swiss watchmaker Ludwig Oechslin designed and built an astrolabe wristwatch in conjunction with Ulysse Nardin in 1985.^[38]^ Dutch watchmaker Christaan van der Klauuw also manufactures astrolabe watches today.^[39]^

Construction

An astrolabe consists of a disk, called the mater (mother), which is deep enough to hold one or more flat plates called tympans, or climates. A tympan is made for a specific latitude and is engraved with a stereographic projection of circles denoting azimuth and altitude and representing the portion of the celestial sphere above the local horizon. The rim of the mater is typically graduated into hours of time, degrees of arc, or both.^[40]^

Above the mater and tympan, the rete, a framework bearing a projection of the ecliptic plane and several pointers indicating the positions of the brightest stars, is free to rotate. These pointers are often just simple points, but depending on the skill of the craftsman can be very elaborate and artistic. There are examples of astrolabes with artistic pointers in the shape of balls, stars, snakes, hands, dogs' heads, and leaves, among others.^[40]^ The names of the indicated stars were often engraved on the pointers in Arabic or Latin.^[41]^ Some astrolabes have a narrow rule or label which rotates over the rete, and may be marked with a scale of declinations.

The rete, representing the sky, functions as a star chart. When it is rotated, the stars and the ecliptic move over the projection of the coordinates on the tympan. One complete rotation corresponds to the passage of a day. The astrolabe is, therefore, a predecessor of the modern planisphere.

On the back of the mater, there is often engraved a number of scales that are useful in the astrolabe's various applications. These vary from designer to designer, but might include curves for time conversions, a calendar for converting the day of the month to the sun's position on the ecliptic, trigonometric scales, and graduation of 360 degrees around the back edge. The alidade is attached to the back face. An alidade can be seen in the lower right illustration of the Persian astrolabe above. When the astrolabe is held vertically, the alidade can be rotated and the sun or a star sighted along its length, so that its altitude in degrees can be read ("taken") from the graduated edge of the astrolabe; hence the word's Greek roots: "astron" (ἄστρον) = star + "lab-" (λαβ-) = to take. The alidade had vertical and horizontal cross-hairs which plots locations on an azimuthal ring called an almucantar (altitude-distance circle).

An arm called a radius connects from the center of the astrolabe to the optical axis which is parallel with another arm also called a radius. The other radius contains graduations of altitude and distance measurements.

A shadow square also appears on the back of some astrolabes, developed by Muslim astrologists in the 9th Century, whereas devices of the Ancient Greek tradition featured only altitude scales on the back of the devices.^[42]^ This was used to convert shadow lengths and the altitude of the sun, the uses of which were various from surveying to measuring inaccessible heights.^[43]^

Devices were usually signed by their maker with an inscription appearing on the back of the astrolabe, and if there was a patron of the object, their name would appear inscribed on the front, or in some cases, the name of the reigning sultan or the teacher of the astrolabist has also been found to appear inscribed in this place.^[44]^ The date of the astrolabe's construction was often also signed, which has allowed historians to determine that these devices are the second oldest scientific instrument in the world. The inscriptions on astrolabes also allowed historians to conclude that astronomers tended to make their own astrolabes, but that many were also made to order and kept in stock to sell, suggesting there was some contemporary market for the devices.^[44]^

Construction of astrolabes

• 

  The Hartmann astrolabe in Yale collection. This instrument shows its rete and rule.

• 

  Celestial Globe, Isfahan (?), Iran 1144. Shown at the Louvre Museum, this globe is the third oldest surviving in the world.

• 

  Computer-generated planispheric astrolabe

Mathematical basis

The construction and design of astrolabes are based on the application of the stereographic projection of the celestial sphere. The point from which the projection is usually made is the South Pole. The plane onto which the projection is made is that of the Equator.^[45]^

Designing a tympanum through stereographic projection

Parts of an Astrolabe tympanum

The tympanum captures the celestial coordinate axes upon which the rete will rotate. It is the component that will enable the precise determination of a star's position at a specific time of day and year.

Therefore, it should project:

1. The zenith, which will vary depending on the latitude of the astrolabe user.
2. The horizon line and almucantar or circles parallel to the horizon, which will allow for the determination of a celestial body's altitude (from the horizon to the zenith).
3. The celestial meridian (north-south meridian, passing through the zenith) and secondary meridians (circles intersecting the north-south meridian at the zenith), which will enable the measurement of azimuth for a celestial body.
4. The three main circles of latitude (Capricorn, Equator, and Cancer) to determine the exact moments of solstices and equinoxes throughout the year.

The tropics and the equator define the tympanum

Stereographic projection of Earth's tropics and equator from the South Pole.

On the right side of the image above:

1. The blue sphere represents the celestial sphere.
2. The blue arrow indicates the direction of true north (the North Star).
3. The central blue point represents Earth (the observer's location).
4. The geographic south of the celestial sphere acts as the projection pole.
5. The celestial equatorial plane serves as the projection plane.
6. Three parallel circles represent the projection on the celestial sphere of Earth's main circles of latitude:
   • In orange, the celestial Tropic of Cancer.
   • In purple, the celestial equator.
   • In green, the celestial Tropic of Capricorn.

When projecting onto the celestial equatorial plane, three concentric circles correspond to the celestial sphere's three circles of latitude (left side of the image). The largest of these, the projection on the celestial equatorial plane of the celestial Tropic of Capricorn, defines the size of the astrolabe's tympanum. The center of the tympanum (and the center of the three circles) is actually the north-south axis around which Earth rotates, and therefore, the rete of the astrolabe will rotate around this point as the hours of the day pass (due to Earth's rotational motion).

The three concentric circles on the tympanum are useful for determining the exact moments of solstices and equinoxes throughout the year: if the sun's altitude at noon on the rete is known and coincides with the outer circle of the tympanum (Tropic of Capricorn), it signifies the winter solstice (the sun will be at the zenith for an observer at the Tropic of Capricorn, meaning summer in the southern hemisphere and winter in the northern hemisphere). If, on the other hand, its altitude coincides with the inner circle (Tropic of Cancer), it indicates the summer solstice. If its altitude is on the middle circle (equator), it corresponds to one of the two equinoxes.

The horizon and the measurement of altitude

Stereographic projection of an observer's horizon at a specific latitude

On the right side of the image above:

1. The blue arrow indicates the direction of true north (the North Star).
2. The central blue point represents Earth (the observer's location).
3. The black arrow represents the zenith direction for the observer (which would vary depending on the observer's latitude).
4. The two black circles represent the horizon surrounding the observer, which is perpendicular to the zenith vector and defines the portion of the celestial sphere visible to the observer, and its projection on the celestial equatorial plane.
5. The geographic south of the celestial sphere acts as the projection pole.
6. The celestial equatorial plane serves as the projection plane.

When projecting the horizon onto the celestial equatorial plane, it transforms into an ellipse upward-shifted relatively to the center of the tympanum (both the observer and the projection of the north-south axis). This implies that a portion of the celestial sphere will fall outside the outer circle of the tympanum (the projection of the celestial Tropic of Capricorn) and, therefore, won't be represented.

Stereographic projection of the horizon and an almucantar.

Additionally, when drawing circles parallel to the horizon up to the zenith (almucantar), and projecting them on the celestial equatorial plane, as in the image above, a grid of consecutive ellipses is constructed, allowing for the determination of a star's altitude when its rete overlaps with the designed tympanum.

The meridians and the measurement of azimuth

Stereographic projection of the north-south meridian and a meridian 40° E on the tympanum of an astrolabe

On the right side of the image above:

1. The blue arrow indicates the direction of true north (the North Star).
2. The central blue point represents Earth (the observer's location).
3. The black arrow represents the zenith direction for the observer (which would vary depending on the observer's latitude).
4. The two black circles represent the horizon surrounding the observer, which is perpendicular to the zenith vector and defines the portion of the celestial sphere visible to the observer, and its projection on the celestial equatorial plane.
5. The five red dots represent the zenith, the nadir (the point on the celestial sphere opposite the zenith with respect to the observer), their projections on the celestial equatorial plane, and the center (with no physical meaning attached) of the circle obtained by projecting the secondary meridian (see below) on the celestial equatorial plane.
6. The orange circle represents the celestial meridian (or meridian that goes, for the observer, from the north of the horizon to the south of the horizon passing through the zenith).
7. The two red circles represent a secondary meridian with an azimuth of 40° East relative to the observer's horizon (which, like all secondary meridians, intersects the principal meridian at the zenith and nadir), and its projection on the celestial equatorial plane.
8. The geographic south of the celestial sphere acts as the projection pole.
9. The celestial equatorial plane serves as the projection plane.

When projecting the celestial meridian, it results in a straight line that overlaps with the vertical axis of the tympanum, where the zenith and nadir are located. However, when projecting the 40° E meridian, another circle is obtained that passes through both the zenith and nadir projections, so its center is located on the perpendicular bisection of the segment connecting both points. In deed, the projection of the celestial meridian can be considered as a circle with an infinite radius (a straight line) whose center is on this bisection and at an infinite distance from these two points.

If successive meridians that divide the celestial sphere into equal sectors (like "orange slices" radiating from the zenith) are projected, a family of curves passing through the zenith projection on the tympanum is obtained. These curves, once overlaid with the rete containing the major stars, allow for determining the azimuth of a star located on the rete and rotated for a specific time of day.

See also

• Astronomy in the medieval Islamic world
• Equatorium
• Hamburg Planetarium
• List of astronomical instruments
• Philippe Danfrie, designer and maker of mathematical instruments, globes and astrolabes
• Planetarium
• Yantraraja
• Zeiss-Planetarium Jena

References

Footnotes

1.

1. Savage-Smith, Emilie (1993). "Book Reviews". Journal of Islamic Studies. 4 (2): 296--299. doi:10.1093/jis/4.2.296. There is no evidence for the Hellenistic origin of the spherical astrolabe, but rather evidence so far available suggests that it may have been an early but distinctly Islamic development with no Greek antecedents.

Notes

1.

1. Gentili, Graziano; Simonutti, Luisa; Struppa, Daniele C. (2020). "The Mathematics of the Astrolabe and Its History". Journal of Humanistic Mathematics. 10: 101--144. doi:10.5642/jhummath.202001.07. hdl:2158/1182616. S2CID 211008813.

Bibliography

• Evans, James (1998), The History and Practice of Ancient Astronomy, Oxford University Press, ISBN 0-19-509539-1
• Stöffler, Johannes (2007) [First published 1513], Stoeffler's Elucidatio -- The Construction and Use of the Astrolabe [Elucidatio Fabricae Ususque Astrolabii], translated by Gunella, Alessandro; Lamprey, John, John Lamprey, ISBN 978-1-4243-3502-2
• King, D. A. (1981), "The Origin of the Astrolabe According to the Medieval Islamic Sources", Journal for the History of Arabic Science, 5: 43--83
• King, Henry (1978), Geared to the Stars: the Evolution of Planetariums, Orreries, and Astronomical Clocks, University of Toronto Press, ISBN 978-0-8020-2312-4
• Krebs, Robert E.; Krebs, Carolyn A. (2003), Groundbreaking Scientific Experiments, Inventions, and Discoveries of the Ancient World, Greenwood Press, ISBN 978-0-313-31342-4
• Laird, Edgar (1997), Carol Poster and Richard Utz (ed.), "Astrolabes and the Construction of Time in the Late Middle Ages", Constructions of Time in the Late Middle Ages, Evanston, Illinois: Northwestern University Press: 51--69
• Laird, Edgar; Fischer, Robert, eds. (1995), "Critical edition of Pélerin de Prusse on the Astrolabe (translation of Practique de Astralabe)", Medieval & Renaissance Texts & Studies, Binghamton, New York, ISBN 0-86698-132-2
• Lewis, M. J. T. (2001), Surveying Instruments of Greece and Rome, Cambridge University Press, ISBN 978-0-511-48303-5
• Morrison, James E. (2007), The Astrolabe, Janus, ISBN 978-0-939320-30-1
• Neugebauer, Otto E. (1975), A History of Ancient Mathematical Astronomy, Springer, ISBN 978-3-642-61912-0
• North, John David (2005), God's Clockmaker: Richard of Wallingford and the Invention of Time, Continuum International Publishing Group, ISBN 978-1-85285-451-5

External links

Wikimedia Commons has media related to:\ Astrolabe (category)

Wikisource has the text of the 1911 Encyclopædia Britannica article "Astrolabe".

Look up astrolabe in Wiktionary, the free dictionary.

• Interactive digital astrolabe by Alex Boxer
• A digital astrolabe (HTML5 and javascript)
• Astrolabe Tech Made ... Not So Easy
• paper astrolabe generator, from the ESO
• "Hello World!" for the Astrolabe: The First Computer Video of Howard Covitz's Presentation at Ignite Phoenix, June 2009. Slides for Presentation Licensed as Creative Commons by-nc-nd.
• Video of Tom Wujec demonstrating an astrolabe. Archived 2012-03-23 at the Wayback Machine Taken at TEDGlobal 2009. Includes clickable transcript. Licensed as Creative Commons by-nc-nd.
• Archive of James E. Morrison's extensive website on Astrolabes
• A working model of the Dr. Ludwig Oechslin's Astrolabium Galileo Galilei watch
• Ulysse Nardin Astrolabium Galilei Galileo: A Detailed Explanation
• Fully illustrated online catalogue of world's largest collection of astrolabes
• Mobile astrolabe and horologium
• Medieval equal hour horary quadrant
• A Beginner's Guide to Basic Construction and Use of the Astrolabe (using ruler, protractor and compasses) (PDF), archived from the original (PDF) on 2015-06-17, retrieved 2018-10-26

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  ](https://en.wikipedia.org/wiki/Astrolabe#Mathematical_basis)- Modern editions of John Philoponus' treatise on the astrolabe are De usu astrolabii eiusque constructione libellus (On the Use and Construction of the Astrolabe), ed. Heinrich Hase, Bonn: E. Weber, 1839, OCLC 165707441 (or id. Rheinisches Museum für Philologie 6 (1839): 127--71); repr. and translated into French by Alain Philippe Segonds, Jean Philopon, traité de l'astrolabe, Paris: Librairie Alain Brieux, 1981, OCLC 10467740; and translated into English by H.W. Green in R.T. Gunther, The Astrolabes of the World, Vol. 1/2, Oxford, 1932, OL 18840299M repr. London: Holland Press, 1976, OL 14132393M pp. 61--81.- O'Leary, De Lacy (1948). How Greek Science Passed to the Arabs. Routledge and Kegan Paul. "The most distinguished Syriac scholar of this later period was Severus Sebokht (d. 666--7), Bishop of Kennesrin. [...] In addition to these works [...] he also wrote on astronomical subjects (Brit. Mus. Add. 14538), and composed a treatise on the astronomical instrument known as the astrolabe, which has been edited and published by F. Nau (Paris, 1899)."\ Severus' treatise was translated by Jessie Payne Smith Margoliouth in R.T. Gunther, Astrolabes of the World, Oxford, 1932, pp. 82--103.- "HISTORIANS' HOME YIELDS RICH LODE; New York Society Searches Its Own Building for Items to Mark Anniversary; SHOW OPENS THURSDAY; Portrait of Stuyvesant and Champlain's Astrolabe Will Be on Display". The New York Times. May 18, 1964. Retrieved February 4, 2024.- Bean, Adam L. (2009). "Astrolabes". In Birx, H. James (ed.). Encyclopedia of Time: Science, Philosophy, Theology, & Culture. Vol. 1. SAGE. pp. 59--60. ISBN 978-1-4129-4164-8.- "Astrolabe". Oxford English Dictionary (2nd ed.). 1989.- "Astrolabe". Oxford Dictionaries. Archived from the original on October 22, 2013.- "Online Etymology Dictionary". Etymonline.com. Retrieved 2013-11-07.- King 1981, p. 44.- King 1981, p. 51.- King 1981, p. 45.- Lewis 2001.- Michael Deakin (August 3, 1997). "Ockham's Razor: Hypatia of Alexandria". ABC Radio. Retrieved July 10, 2014.- Theodore, Jonathan (2016). The Modern Cultural Myth of the Decline and Fall of the Roman Empire. Manchester, England: Palgrave, Macmillan. p. 183. ISBN 978-1-137-56997-4.- Deakin, Michael A. B. (2007). Hypatia of Alexandria: Mathematician and Martyr. Amherst, New York: Prometheus Books. pp. 102--104. ISBN 978-1-59102-520-7.- Bradley, Michael John (2006). The Birth of Mathematics: Ancient Times to 1300. New York City, New York: Infobase Publishing. p. 63. ISBN 9780816054237.- Savage-Smith, Emilie (1992). "Celestial Mapping" (PDF). In Harley, J. B.; Woodward, David (eds.). The History of Cartography, Volume 2, Book 1: Cartography in the Traditional Islamic and South Asian Societies. The History of Cartography. Vol. 2. Chicago, Illinois: University of Chicago Press. ISBN 0226316351.- Sebokht, Severus. "Description of the astrolabe". Tertullian.org.- See p. 289 of Martin, L. C. (1923), "Surveying and navigational instruments from the historical standpoint", Transactions of the Optical Society, 24 (5): 289--303, Bibcode:1923TrOS...24..289M, doi:10.1088/1475-4878/24/5/302, ISSN 1475-4878.- Berggren, J. Lennart (2007), "Mathematics in Medieval Islam", in Katz, Victor J. (ed.), The Mathematics of Egypt, Mesopotamia, China, India, and Islam: a Sourcebook, Princeton University Press, p. 519, ISBN 978-0-691-11485-9- Richard Nelson Frye: Golden Age of Persia. p. 163- Nizamoglu, Cem (2005-08-10). "Using an Astrolabe". Muslim Heritage. Retrieved 2023-10-16.- Lachièz-Rey, Marc; Luminet, Jean-Pierre (2001). Celestial Treasury: From the Music of Spheres to the Conquest of Space. Translated by Joe Laredo. Cambridge, United Kingdom: Cambridge University Press. p. 74. ISBN 978-0-521-80040-2.- O'Connor, John J.; Robertson, Edmund F., "Sharaf al-Din al-Muzaffar al-Tusi", MacTutor History of Mathematics Archive, University of St Andrews- Bedini, Silvio A.; Maddison, Francis R. (1966). "Mechanical Universe: The Astrarium of Giovanni de' Dondi". Transactions of the American Philosophical Society. 56 (5): 1--69. doi:10.2307/1006002. JSTOR 1006002.- "Qantara -- 'Carolingian' astrolabe". Qantara-med.org. Retrieved 2013-11-07.- Nancy Marie Brown (2010), "The Abacus and the Cross". p. 140. Basic Books. ISBN 978-0-465-00950-3- Boyle, David (2011). Toward the Setting Sun: Columbus, Cabot, Vespucci, and the Race for America. Bloomsbury Publishing USA. p. 253. ISBN 9780802779786..- Northrup, Cynthia Clark, ed. (2015). Encyclopedia of world trade: from ancient times to the present (Enhanced Credo ed.). Armonk, New York: Routledge. pp. 72. ISBN 978-0765680587. OCLC 889717964.- "Introduction". The Astrolabe: an Online Resource. 2006. Retrieved 2020-05-15.- Harley, J. B.; Woodward, David (1992). The history of cartography. Chicago, Illinois: University of Chicago Press. p. 31. ISBN 0-226-31635-1.- Kunitzsch, Paul (1981). "On the authenticity of the treatise on the composition and use of the astrolabe ascribed to Messahalla". Archives Internationales d'Histoire des Sciences Oxford. 31 (106): 42--62.- Selin, Helaine (2008-03-12). Encyclopaedia of the History of Science, Technology, and Medicine in Non-Western Cultures. Springer Science & Business Media. p. 1335. ISBN 978-1-4020-4559-2. Paul Kunitzsch has recently established that the Latin treatise on the astrolabe long ascribed to Ma'sh'allah and translated by John of Seville is in fact by Ibn al-Saffar, a disciple of Maslama al-Majriti.- Glick, Thomas; et al., eds. (2005), Medieval Science, Technology, and Medicine: An Encyclopedia, Routledge, p. 464, ISBN 0-415-96930-1- Northrup, Cynthia Clark, ed. (2015). Encyclopedia of world trade : from ancient times to the present ([Enhanced Credo edition] ed.). Armonk, New York: Routledge. pp. 460. ISBN 978-0765680587. OCLC 889717964.- Nancy Marie Brown (2010), "The Abacus and the Cross". p. 143. basic Books. ISBN 978-0-465-00950-3- Hockey, Thomas (2009). The Biographical Encyclopedia of Astronomers. Springer Publishing. ISBN 978-0-387-31022-0. Retrieved August 22, 2012.- Ralf Kern (2010), Wissenschaftliche Instrumente in ihrer Zeit. Band 1: Vom Astrolab zum mathematischen Besteck. Cologne, S. 204. ISBN 978-3-86560-865-9- Vafea, Flora (2017). "The Astronomical Instruments in Saint Catherine's Iconography at the Holy Monastery of Sinai The Almagest Volume 8, Issue 2". Almagest. 8 (2). Paris: University of Paris: 87. doi:10.1484/J.ALMAGEST.5.114932.- North 2005.- "Astrolabium G. Galilei". Ulysse Nardin. Archived from the original on 2 January 2011.- "Christaan van der Klauuw".- Stephenson, Bruce; Bolt, Marvin; Friedman, Anna Felicity (2000). The Universe Unveiled: Instruments and Images through History. Cambridge, UK: Cambridge University Press. pp. 108--109. ISBN 0-521-79143-X.- "Star Names on Astrolabes". Ian Ridpath. Retrieved 2016-11-12.- King, David A. Some Medieval Astronomical Instruments and Their Secrets, in Mazzolini, R. G. (ed.), Non-Verbal Communication in Science prior to 1900. Florence. p. 30.- King, David A. (2018). The Astrolabe: What it is & what it is not. Frankfurt, Germany: Frankfurt.- Mayer, L. A. (1956). Islamic astrolabists and their works*. A. Kunding. Bibcode:[1956iatw.book.....M](https://ui.a Main menu

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From Wikipedia, the free encyclopedia This article is about the sextant as used for navigation. For other uses, see Sextant (disambiguation). Not to be confused with Sexton (disambiguation). A sextant

A sextant is a doubly reflecting navigation instrument that measures the angular distance between two visible objects. The primary use of a sextant is to measure the angle between an astronomical object and the horizon for the purposes of celestial navigation.

The estimation of this angle, the altitude, is known as sighting or shooting the object, or taking a sight. The angle, and the time when it was measured, can be used to calculate a position line on a nautical or aeronautical chart—for example, sighting the Sun at noon or Polaris at night (in the Northern Hemisphere) to estimate latitude (with sight reduction). Sighting the height of a landmark can give a measure of distance off and, held horizontally, a sextant can measure angles between objects for a position on a chart.[1] A sextant can also be used to measure the lunar distance between the moon and another celestial object (such as a star or planet) in order to determine Greenwich Mean Time and hence longitude.

The principle of the instrument was first implemented around 1731 by John Hadley (1682–1744) and Thomas Godfrey (1704–1749), but it was also found later in the unpublished writings of Isaac Newton (1643–1727).

In 1922, it was modified for aeronautical navigation by Portuguese navigator and naval officer Gago Coutinho. Navigational sextants

Like the Davis quadrant, the sextant allows celestial objects to be measured relative to the horizon, rather than relative to the instrument. This allows excellent precision. Also, unlike the backstaff, the sextant allows direct observations of stars. This permits the use of the sextant at night when a backstaff is difficult to use. For solar observations, filters allow direct observation of the Sun.

Since the measurement is relative to the horizon, the measuring pointer is a beam of light that reaches to the horizon. The measurement is thus limited by the angular accuracy of the instrument and not the sine error of the length of an alidade, as it is in a mariner's astrolabe or similar older instrument.

A sextant does not require a completely steady aim, because it measures a relative angle. For example, when a sextant is used on a moving ship, the image of both horizon and celestial object will move around in the field of view. However, the relative position of the two images will remain steady, and as long as the user can determine when the celestial object touches the horizon, the accuracy of the measurement will remain high compared to the magnitude of the movement.

The sextant is not dependent upon electricity (unlike many forms of modern navigation) or any human-controlled signals (such as GPS). For these reasons it is considered to be an eminently practical back-up navigation tool for ships. Design

The frame of a sextant is in the shape of a sector which is approximately 1⁄6 of a circle (60°),[2] hence its name (sextāns, sextantis is the Latin word for "one sixth"). Both smaller and larger instruments are (or were) in use: the octant, quintant (or pentant) and the (doubly reflecting) quadrant[3] span sectors of approximately 1⁄8 of a circle (45°), 1⁄5 of a circle (72°) and 1⁄4 of a circle (90°), respectively. All of these instruments may be termed "sextants". Marine sextant Using the sextant to measure the altitude of the Sun above the horizon Sextants can also be used by navigators to measure horizontal angles between objects.

Attached to the frame are the "horizon mirror", an index arm which moves the index mirror, a sighting telescope, Sun shades, a graduated scale and a micrometer drum gauge for accurate measurements. The scale must be graduated so that the marked degree divisions register twice the angle through which the index arm turns. The scales of the octant, sextant, quintant and quadrant are graduated from below zero to 90°, 120°, 140° and 180° respectively. For example, the sextant illustrated has a scale graduated from −10° to 142°, which is basically a quintant: the frame is a sector of a circle subtending an angle of 76° at the pivot of the index arm.

The necessity for the doubled scale reading follows from consideration of the relations of the fixed ray (between the mirrors), the object ray (from the sighted object) and the direction of the normal perpendicular to the index mirror. When the index arm moves by an angle, say 20°, the angle between the fixed ray and the normal also increases by 20°. But the angle of incidence equals the angle of reflection so the angle between the object ray and the normal must also increase by 20°. The angle between the fixed ray and the object ray must therefore increase by 40°. This is the case shown in the graphic.

There are two types of horizon mirrors on the market today. Both types give good results.

Traditional sextants have a half-horizon mirror, which divides the field of view in two. On one side, there is a view of the horizon; on the other side, a view of the celestial object. The advantage of this type is that both the horizon and celestial object are bright and as clear as possible. This is superior at night and in haze, when the horizon and/or a star being sighted can be difficult to see. However, one has to sweep the celestial object to ensure that the lowest limb of the celestial object touches the horizon.

Whole-horizon sextants use a half-silvered horizon mirror to provide a full view of the horizon. This makes it easy to see when the bottom limb of a celestial object touches the horizon. Since most sights are of the Sun or Moon, and haze is rare without overcast, the low-light advantages of the half-horizon mirror are rarely important in practice.

In both types, larger mirrors give a larger field of view, and thus make it easier to find a celestial object. Modern sextants often have 5 cm or larger mirrors, while 19th-century sextants rarely had a mirror larger than 2.5 cm (one inch). In large part, this is because precision flat mirrors have grown less expensive to manufacture and to silver.

An artificial horizon is useful when the horizon is invisible, as occurs in fog, on moonless nights, in a calm, when sighting through a window or on land surrounded by trees or buildings. There are two common designs of artificial horizon. An artificial horizon can consist simply of a pool of water shielded from the wind, allowing the user to measure the distance between the body and its reflection, and divide by two. Another design allows the mounting of a fluid-filled tube with bubble directly to the sextant.

Most sextants also have filters for use when viewing the Sun and reducing the effects of haze. The filters usually consist of a series of progressively darker glasses that can be used singly or in combination to reduce haze and the Sun's brightness. However, sextants with adjustable polarizing filters have also been manufactured, where the degree of darkness is adjusted by twisting the frame of the filter.

Most sextants mount a 1 or 3-power monocular for viewing. Many users prefer a simple sighting tube, which has a wider, brighter field of view and is easier to use at night. Some navigators mount a light-amplifying monocular to help see the horizon on moonless nights. Others prefer to use a lit artificial horizon.[citation needed]

Professional sextants use a click-stop degree measure and a worm adjustment that reads to a minute, 1/60 of a degree. Most sextants also include a vernier on the worm dial that reads to 0.1 minute. Since 1 minute of error is about a nautical mile, the best possible accuracy of celestial navigation is about 0.1 nautical miles (190 m). At sea, results within several nautical miles, well within visual range, are acceptable. A highly skilled and experienced navigator can determine position to an accuracy of about 0.25-nautical-mile (460 m).[4]

A change in temperature can warp the arc, creating inaccuracies. Many navigators purchase weatherproof cases so that their sextant can be placed outside the cabin to come to equilibrium with outside temperatures. The standard frame designs (see illustration) are supposed to equalise differential angular error from temperature changes. The handle is separated from the arc and frame so that body heat does not warp the frame. Sextants for tropical use are often painted white to reflect sunlight and remain relatively cool. High-precision sextants have an invar (a special low-expansion steel) frame and arc. Some scientific sextants have been constructed of quartz or ceramics with even lower expansions. Many commercial sextants use low-expansion brass or aluminium. Brass is lower-expansion than aluminium, but aluminium sextants are lighter and less tiring to use. Some say they are more accurate because one's hand trembles less. Solid brass frame sextants are less susceptible to wobbling in high winds or when the vessel is working in heavy seas, but as noted are substantially heavier. Sextants with aluminum frames and brass arcs have also been manufactured. Essentially, a sextant is intensely personal to each navigator, and they will choose whichever model has the features which suit them best.

Aircraft sextants are now out of production, but had special features. Most had artificial horizons to permit taking a sight through a flush overhead window. Some also had mechanical averagers to make hundreds of measurements per sight for compensation of random accelerations in the artificial horizon's fluid. Older aircraft sextants had two visual paths, one standard and the other designed for use in open-cockpit aircraft that let one view from directly over the sextant in one's lap. More modern aircraft sextants were periscopic with only a small projection above the fuselage. With these, the navigator pre-computed their sight and then noted the difference in observed versus predicted height of the body to determine their position. Taking a sight

A sight (or measure) of the angle between the Sun, a star, or a planet, and the horizon is done with the 'star telescope' fitted to the sextant using a visible horizon. On a vessel at sea even on misty days a sight may be done from a low height above the water to give a more definite, better horizon. Navigators hold the sextant by its handle in the right hand, avoiding touching the arc with the fingers.[5]

For a Sun sight, a filter is used to overcome the glare such as "shades" covering both index mirror and the horizon mirror designed to prevent eye damage. Initially, with the index bar set to zero and the shades covering both mirrors, the sextant is aimed at the sun until it can be viewed on both mirrors through the telescope, then lowered vertically until the portion of the horizon directly below it is viewed on both mirrors. It is necessary to flip back the horizon mirror shade to be able to see the horizon more clearly on it. Releasing the index bar (either by releasing a clamping screw, or on modern instruments, using the quick-release button), and moving it towards higher values of the scale, eventually the image of the Sun will reappear on the index mirror and can be aligned to about the level of the horizon on the horizon mirror. Then the fine adjustment screw on the end of the index bar is turned until the bottom curve (the lower limb) of the Sun just touches the horizon. "Swinging" the sextant about the axis of the telescope ensures that the reading is being taken with the instrument held vertically. The angle of the sight is then read from the scale on the arc, making use of the micrometer or vernier scale provided. The exact time of the sight must also be noted simultaneously, and the height of the eye above sea-level recorded.[5]

An alternative method is to estimate the current altitude (angle) of the Sun from navigation tables, then set the index bar to that angle on the arc, apply suitable shades only to the index mirror, and point the instrument directly at the horizon, sweeping it from side to side until a flash of the Sun's rays are seen in the telescope. Fine adjustments are then made as above. This method is less likely to be successful for sighting stars and planets.[5]

Star and planet sights are normally taken during nautical twilight at dawn or dusk, while both the heavenly bodies and the sea horizon are visible. There is no need to use shades or to distinguish the lower limb as the body appears as a mere point in the telescope. The Moon can be sighted, but it appears to move very fast, appears to have different sizes at different times, and sometimes only the lower or upper limb can be distinguished due to its phase.[5]

After a sight is taken, it is reduced to a position by looking at several mathematical procedures. The simplest sight reduction is to draw the equal-altitude circle of the sighted celestial object on a globe. The intersection of that circle with a dead-reckoning track, or another sighting, gives a more precise location.

Sextants can be used very accurately to measure other visible angles, for example between one heavenly body and another and between landmarks ashore. Used horizontally, a sextant can measure the apparent angle between two landmarks such as a lighthouse and a church spire, which can then be used to find the distance off or out to sea (provided the distance between the two landmarks is known). Used vertically, a measurement of the angle between the lantern of a lighthouse of known height and the sea level at its base can also be used for distance off.[5] Adjustment

Due to the sensitivity of the instrument it is easy to knock the mirrors out of adjustment. For this reason a sextant should be checked frequently for errors and adjusted accordingly.

There are four errors that can be adjusted by the navigator, and they should be removed in the following order.

Perpendicularity error This is when the index mirror is not perpendicular to the frame of the sextant. To test for this, place the index arm at about 60° on the arc and hold the sextant horizontally with the arc away from you at arm's length and look into the index mirror. The arc of the sextant should appear to continue unbroken into the mirror. If there is an error, then the two views will appear to be broken. Adjust the mirror until the reflection and direct view of the arc appear to be continuous. Side error This occurs when the horizon glass/mirror is not perpendicular to the plane of the instrument. To test for this, first zero the index arm then observe a star through the sextant. Then rotate the tangent screw back and forth so that the reflected image passes alternately above and below the direct view. If in changing from one position to another, the reflected image passes directly over the unreflected image, no side error exists. If it passes to one side, side error exists. Alternatively, the user can hold the sextant on its side and observe the horizon to check the sextant during the day. If there are two horizons there is side error. In both cases, adjust the horizon glass/mirror until respectively the star or the horizon dual images merge into one. Side error is generally inconsequential for observations and can be ignored or reduced to a level that is merely inconvenient. Collimation error This is when the telescope or monocular is not parallel to the plane of the sextant. To check for this you need to observe two stars 90° or more apart. Bring the two stars into coincidence either to the left or the right of the field of view. Move the sextant slightly so that the stars move to the other side of the field of view. If they separate there is collimation error. As modern sextants rarely use adjustable telescopes, they do not need to be corrected for collimation error. Index error This occurs when the index and horizon mirrors are not parallel to each other when the index arm is set to zero. To test for index error, zero the index arm and observe the horizon. If the reflected and direct image of the horizon are in line there is no index error. If one is above the other adjust the index mirror until the two horizons merge. Alternatively, the same procedure can be done at night using a star or the Moon instead of the horizon.

See also

Astrolabe
Bris sextant
Davis quadrant
Gago Coutinho
Harold Gatty
History of longitude
Intercept method
Latitude
Longitude
Longitude by chronometer
Mariner's astrolabe
Navigation
Octant (instrument)
Quadrant (instrument)
Sextant (astronomy)

Notes

Seddon, J. Carl (June 1968). "Line of Position from a Horizontal Angle". Journal of Navigation. 21 (3): 367–369. doi:10.1017/S0373463300024838. ISSN 1469-7785. A.), McPhee, John (John; NSW., Museums and Galleries (2008). Great Collections : treasures from Art Gallery of NSW, Australian Museum, Botanic Gardens Trust, Historic Houses Trust of NSW, Museum of Contemporary Art, Powerhouse Museum, State Library of NSW, State Records NSW. Museums & Galleries NSW. p. 56. ISBN 9780646496030. OCLC 302147838. This article treats the doubly reflecting quadrant, not its predecessor described at quadrant. Dutton's Navigation and Piloting, 12th edition. G.D. Dunlap and H.H. Shufeldt, eds. Naval Institute Press 1972, ISBN 0-87021-163-3

Dixon, Conrad (1968). "5. Using the sextant". Basic Astro Navigation. Adlard Coles. ISBN 0-229-11740-6.

References

Bowditch, Nathaniel (2002). The American Practical Navigator. Bethesda, MD: National Imagery and Mapping Agency. ISBN 0-939837-54-4. Archived from the original on 2007-06-24.
Chisholm, Hugh, ed. (1911). "Sextant" . Encyclopædia Britannica. Vol. 24 (11th ed.). Cambridge University Press. pp. 765–767.
Cutler, Thomas J. (December 2003). Dutton's Nautical Navigation (15th ed.). Annapolis, MD: Naval Institute Press. ISBN 978-1-55750-248-3.
Department of the Air Force (March 2001). Air Navigation (PDF). Department of the Air Force. Retrieved 2014-12-28.
Great Britain Ministry of Defence (Navy) (1995). Admiralty Manual of Seamanship. The Stationery Office. ISBN 0-11-772696-6.
Maloney, Elbert S. (December 2003). Chapman Piloting and Seamanship (64th ed.). New York: Hearst Communications. ISBN 1-58816-089-0.
Martin, William Robert (1911). "Navigation" . In Chisholm, Hugh (ed.). Encyclopædia Britannica. Vol. 19 (11th ed.). Cambridge University Press. pp. 284–298.

External links Look up sextant in Wiktionary, the free dictionary. Wikimedia Commons has media related to Sextant.

Her Majesty's Nautical Almanac Office Archived 2011-02-21 at the Wayback Machine
The History of HM Nautical Almanac Office Archived 2016-06-24 at the Wayback Machine
Chapter 17 from the online edition of Nathaniel Bowditch's American Practical Navigator
Understand difference in Antique & Replica Sextant Archived 2017-08-17 at the Wayback Machine
CD-Sextant - Build your own sextant Simple do-it-yourself project.
Lunars web site. online calculation
Complete celnav theory book, including Lunars

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dsabs.harvard.edu/abs/1956iatw.book.....M).*

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The emergence of Drosophila EM connectomes has revealed numerous neurons within the associative learning circuit. However, these neurons are inaccessible for functional assessment or genetic manipulation in the absence of cell-type-specific drivers. Addressing this knowledge gap, Shuai et al. have screened over 4000 split-GAL4 drivers and correlated them with identified neuron types from the "Hemibrain" EM connectome by matching light microscopy images to neuronal shapes defined by EM. They successfully generated over 800 split-GAL4 drivers and 22 split-LexA drivers covering a substantial number of neuron types across layers of the mushroom body associative learning circuit. They provide new labeling tools for olfactory and non-olfactory sensory inputs to the mushroom body; interneurons connected with dopaminergic neurons and/or mushroom body output neurons; potential reinforcement sensory neurons; and expanded coverage of intrinsic mushroom body neurons. Furthermore, the authors have optimized the GR64f-GAL4 driver into a sugar sensory neuron-specific split-GAL4 driver and functionally validated it as providing a robust optogenetic substitute for sugar reward. Additionally, a driver for putative nociceptive ascending neurons, potentially serving as optogenetic negative reinforcement, is characterized by optogenetic avoidance behavior. The authors also use their very large dataset of neuronal anatomies, covering many example neurons from many brains, to identify neuron instances with atypical morphology. They find many examples of mushroom body neurons with altered neuronal numbers or mistargeting of dendrites or axons and estimate that 1-3% of neurons in each brain may have anatomic peculiarities or malformations. Significantly, the study systematically assesses the individualized existence of MBON08 for the first time. This neuron is a variant shape that sometimes occurs instead of one of two copies of MBON09, and this variation is more common than that in other neuronal classes: 75% of hemispheres have two MBON09's, and 25% have one MBON09 and one MBON08. These newly developed drivers not only expand the repertoire for genetic manipulation of mushroom body-related neurons but also empower researchers to investigate the functions of circuit motifs identified from the connectomes. The authors generously make these flies available to the public. In the foreseeable future, the tools generated in this study will allow important advances in the understanding of learning and memory in Drosophila.

Strengths:

(1) After decades of dedicated research on the mushroom body, a consensus has been established that the release of dopamine from DANs modulates the weights of connections between KCs and MBONs. This process updates the association between sensory information and behavioral responses. However, understanding how the unconditioned stimulus is conveyed from sensory neurons to DANs, and the interactions of MBON outputs with innate responses to sensory context remains less clear due to the developmental and anatomic diversity of MBONs and DANs. Additionally, the recurrent connections between MBONs and DANs are reported to be critical for learning. The characterization of split-GAL4 drivers for 30 major interneurons connected with DANs and/or MBONs in this study will significantly contribute to our understanding of recurrent connections in mushroom body function.

(2) Optogenetic substitutes for real unconditioned stimuli (such as sugar taste or electric shock) are sometimes easier to implement in behavioral assays due to the spatial and temporal specificity with which optogenetic activation can be induced. GR64f-GAL4 has been widely used in the field to activate sugar sensory neurons and mimic sugar reward. However, the authors demonstrate that GR64f-GAL4 drives expression in other neurons not necessary for sugar reward, and the potential activation of these neurons could introduce confounds into training, impairing training efficiency. To address this issue, the authors have elaborated on a series of intersectional drivers with GR64f-GAL4 to dissect subsets of labeled neurons. This approach successfully identified a more specific sugar sensory neuron driver, SS87269, which consistently exhibited optimal training performance and triggered ethologically relevant local searching behaviors. This newly characterized line could serve as an optimized optogenetic tool for sugar reward in future studies.

(3) MBON08 was first reported by Aso et al. 2014, exhibiting dendritic arborization into both ipsilateral and contralateral γ3 compartments. However, this neuron could not be identified in the previously published Drosophila brain connectomes. In the present study, the existence of MBON08 is confirmed, occurring in one hemisphere of 35% of imaged flies. In brains where MBON08 is present, its dendrite arborization disjointly shares contralateral γ3 compartments with MBON09. This remarkable phenotype potentially serves as a valuable resource for understanding the stochasticity of neurodevelopment and the molecular mechanisms underlying mushroom body lobe compartment formation.

Weaknesses:

There are some minor weaknesses in the paper that can be clarified:

(1) In Figure 8, the authors trained flies with a 20s, weak optogenetic conditioning first, followed by a 60s, strong optogenetic conditioning. The rationale for using this training paradigm is not explicitly provided.

These experiments were designed to test if flies could maintain consistent performance with repetitive and intense LED activation, which is essential for experiments involving long training protocols or coactivation of other neurons inside a brain.

In Figure 8E, if data for training with GR64f-GAL4 using the same paradigm is available, it would be beneficial for readers to compare the learning performance using newly generated split-GAL4 lines with the original GR64f-GAL4, which has been used in many previous research studies. It is noteworthy that in previously published work, repeating training test sessions typically leads to an increase in learning performance in discrimination assays. However, this augmentation is not observed in any of the split-GAL4 lines presented in Figure 8E. The authors may need to discuss possible reasons for this.

As the reviewer pointed out, many previous studies including ours used the original Gr64f-GAL4 in olfactory conditioning. Figure 1H of Yamada et al., 2023 (https://doi.org/10.7554/eLife.79042) showed such a result, where the first and second-order olfactory conditioning were assayed. Indeed, the first-order conditioning scores were gradually augmented over repeated training. In this experiment, we used low red LED intensity for the optogenetic activation. In the Figure 8E of the present paper, the first memory test was after 3x pairing of 20s odor with five 1s red LED without intermediate tests. Therefore, flies were already sufficiently trained to show a plateau memory level in “Test1”. In the revision of another recent report (Figure 1C-F of Aso et al., 2023; https://doi.org/10.7554/eLife.85756), we included the learning curve data of our best Gr64f-split-GAL4, SS87269. Under a less saturated training conditioning, SS87269 did show learning augmentation over repeated training.

(2) In line 327, the authors state that in all samples, the β'1 compartment is arborized by MBON09. However, in Figure 11J, the probability of having at least one β'1 compartment not arborized is inferred to be 2%. The authors should address and clarify this conflict in the text to avoid misunderstanding.

The chance of visualizing MBON08 in MCFO images was 21/209 in total (Figure 11I). If we assume that each of four cells adopt MBON08 development fate at this chance, we can calculate the probability for each case of MBON08/09 cell type composition. From this calculation, we inferred approximately 2% of flies would lack innervations to β'1 compartment in at least one hemisphere. However, we didn't observe a lack of β'1 arborizations in 169 sample flies. If these MBONs independently develop into MBON08 at 21/209 odds, the chance of never observing two MBON08s in either hemisphere of all 169 samples is 3.29%. Therefore, some developmental mechanisms may prevent the emergence of two MBON08 in the same hemisphere.

In the revised manuscript, we displayed these estimated probability for each case separately, and annotated actual observation on the right side.

(3) In general, are the samples presented male or female? This sample metadata will be shown when the images are deposited in FlyLight, but it would be useful in the context of this manuscript to describe in the methods whether animals are all one sex or mixed sex, and in some example images (e.g. mAL3A) to note whether the sample is male or female.

The samples presented in this study are mixed sex, except for Figure 11I, where genders are specified. We provided metadata information of the presented images in Supplemental File 7, and we added a paragraph in the in the method section:

“Most samples were collected from females, though typically at least one male fly was examined for each driver line. While we noticed certain lines such as SS48900, exhibited distinct expression patterns in females and males, we did not particularly focus on sexual dimorphism, which is analyzed elsewhere (Meissner et al. 2024). Therefore, unless stated otherwise, the presented samples are of mixed gender.

Detailed metadata, including gender information and the reporter used, can be found in Supplementary File 7.”

Reviewer #2 (Public Review):

Summary:

The article by Shuai et al. describes a comprehensive collection of over 800 split-GAL4 and split-LexA drivers, covering approximately 300 cell types in Drosophila, aimed at advancing the understanding of associative learning. The mushroom body (MB) in the insect brain is central to associative learning, with Kenyon cells (KCs) as primary intrinsic neurons and dopaminergic neurons (DANs) and MB output neurons (MBONs) forming compartmental zones for memory storage and behavior modulation. This study focuses on characterizing sensory input as well as direct upstream connections to the MB both anatomically and, to some extent, behaviorally. Genetic access to specific, sparsely expressed cell types is crucial for investigating the impact of single cells on computational and functional aspects within the circuitry. As such, this new and extensive collection significantly extends the range of targeted cell types related to the MB and will be an outstanding resource to elucidate MB-related processes in the future.

Strengths:

The work by Shuai et al. provides novel and essential resources to study MB-related processes and beyond. The resulting tools are publicly available and, together with the linked information, will be foundational for many future studies. The importance and impact of this tool development approach, along with previous ones, for the field cannot be overstated. One of many interesting aspects arises from the anatomical analysis of cell types that are less stereotypical across flies. These discoveries might open new avenues for future investigations into how such asymmetry and individuality arise from development and other factors, and how it impacts the computations performed by the circuitry that contains these elements.

Weaknesses:

Providing such an array of tools leaves little to complain about. However, despite the comprehensive genetic access to diverse sensory pathways and MB-connected cell types, the manuscript could be improved by discussing its limitations. For example, the projection neurons from the visual system seem to be underrepresented in the tools produced (or almost absent). A discussion of these omissions could help prevent misunderstandings.

We internally distributed efforts to produce split-GAL4 lines at Janelia Research Campus. The recent preprint (Nern et al., 2024; doi: https://doi.org/10.1101/2024.04.16.589741) described the full collection of split-GAL4 driver lines in the optic lobe including the visual projection neurons to the mushroom body. We cited this preprint in the revised manuscript by adding a short paragraph of discussion.

“Although less abundant than the olfactory input, the MB also receives visual information from the visual projection neurons (VPNs) that originate in the medulla and lobula and are targeted to the accessory calyx (Vogt et al. 2016; Li et al. 2020). A recent preprint described the full collection of split-GAL4 driver lines in the optic lobe, which includes the VPNs to the MB (Nern et al. 2024).”

Additionally, more details on the screening process, particularly the selection of candidate split halves and stable split-GAL4 lines, would provide valuable insights into the methodology and the collection's completeness.

The details of our split-GAL4 design and screening procedures were described in previous studies (Aso et al., 2014; Dolan et al., 2019). Available data and tools to design split-GAL4 changed over time, and we took different approaches accordingly. Many of split-GAL4 lines presented in this study were designed and screened in parallel to the lines for MBONs and DANs in 2010-2014 when MCFO images of GAL4 drivers and EM connectome were not yet available. With knowledge of where MBONs and DANs project, I (Y.A.) manually examined and annotated thousands of confocal stacks (Jenett et al., 2012; https://doi.org/10.1016/j.celrep.2012.09.011) to find candidate cell types that may concat with them.

Later I used more advanced computational tools (Otsuna et al., 2018; doi: https://doi.org/10.1101/318006) and MCFO images aligned to the standard brain volume (Meissner et al., 2023; DOI: 10.7554/eLife.80660.). Now, if one needs to further generate split-GAL4 lines for cell type identified in EM connectome data, neuron bridge website (https://neuronbridge.janelia.org/) can be very helpful to provide a list of GAL4 drivers that may label the neuron of interest.

Reviewer #3 (Public Review):

Summary:

Previous research on the Drosophila mushroom body (MB) has made this structure the best-understood example of an associative memory center in the animal kingdom. This is in no small part due to the generation of cell-type specific driver lines that have allowed consistent and reproducible genetic access to many of the MB's component neurons. The manuscript by Shuai et al. now vastly extends the number of driver lines available to researchers interested in studying learning and memory circuits in the fly. It is an 800-plus collection of new cell-type specific drivers target neurons that either provide input (direct or indirect) to MB neurons or that receive output from them. Many of the new drivers target neurons in sensory pathways that convey conditioned and unconditioned stimuli to the MB. Most drivers are exquisitely selective, and researchers will benefit from the fact that whenever possible, the authors have identified the targeted cell types within the Drosophila connectome. Driver expression patterns are beautifully documented and are publicly available through the Janelia Research Campus's Flylight database where full imaging results can be accessed. Overall, the manuscript significantly augments the number of cell type-specific driver lines available to the Drosophila research community for investigating the cellular mechanisms underlying learning and memory in the fly. Many of the lines will also be useful in dissecting the function of the neural circuits that mediate sensorimotor circuits.

Strengths:

The manuscript represents a huge amount of careful work and leverages numerous important developments from the last several years. These include the thousands of recently generated split-Gal4 lines at Janelia and the computational tools for pairing them to make exquisitely specific targeting reagents. In addition, the manuscript takes full advantage of the recently released Drosophila connectomes. Driver expression patterns are beautifully illustrated side-by-side with corresponding skeletonized neurons reconstructed by EM. A comprehensive table of the new lines, their split-Gal4 components, their neuronal targets, and other valuable information will make this collection eminently useful to end-users. In addition to the anatomical characterization, the manuscript also illustrates the functional utility of the new lines in optogenetic experiments. In one example, the authors identify a specific subset of sugar reward neurons that robustly promotes associative learning.

Weaknesses:

While the manuscript succeeds in making a mass of descriptive detail quite accessible to the reader, the way the collection is initially described - and the new lines categorized - in the text is sometimes confusing. Most of the details can be found elsewhere, but it would be useful to know how many of the lines are being presented for the first time and have not been previously introduced in other publications/contexts.

We revised the text as below.

“Among the 828 lines, a subset of 355 lines, collectively labeling at least 319 different cell types, exhibit highly specific and non-redundant expression patterns are likely to be particularly valuable for behavioral experiments. Detailed information, including genotype, expression specificity, matched EM cell type(s), and recommended driver for each cell type, can be found in Supplementary File 1. A small subset of 40 lines from this collection have been previously used in studies (Aso et al., 2023; Dolan et al., 2019; Gao et al., 2019; Scaplen et al., 2021; Schretter et al., 2020; Takagi et al., 2017; Xie et al., 2021; Yamada et al., 2023). All transgenic lines newly generated in this study are listed in Supplementary File 2 (Aso et al., 2023; Dolan et al., 2019; Gao et al., 2019; Scaplen et al., 2021; Schretter et al., 2020; Takagi et al., 2017; Xie et al., 2021; Yamada et al., 2023).”

And where can the lines be found at Flylight? Are they listed as one collection or as many?

They are listed as one collection - “Aso 2021” release. It is named “2021” because we released the images and started sharing lines in December of 2021 without a descriptive paper. We added a sentence in the Methods section.

“All splitGAL4 lines can be found at flylight database under “Aso 2021” release, and fly strains can be requested from Janelia or the Bloomington stock center.”

Also, the authors say that some of the lines were included in the collection despite not necessarily targeting the intended type of neuron (presumably one that is involved in learning and memory). What percentage of the collection falls into this category?

We do not have a good record of split-GAL4 screening to calculate the chance to intersect unintended cell types, but it was rather rare. Those unintended cell types can still be a part of circuits for associative learning (e.g. olfactory projection neurons) or totally unrelated cell types. For instance, among a new collection of split-LexA lines using Gr43a-LexADBD hemidriver (Figure 7-figure supplement 2), one line specifically intersected T1 neurons in the optic lobe despite that the AD line was selected to intersect sugar sensory neurons. We suspect that this is due to ectopic expression of Gr43a-LexADBD. Nonetheless, we included it in the paper because cell-type-specific Split-LexA driver for T1 will be useful irrespective of whether the expression of Gr43a gene is expressed in T1 or not.

And what about the lines that the authors say they included in the collection despite a lack of specificity? How many lines does this represent?

For a short answer, there are about 100 lines in the collection that lack the specificity for behavioral experiments.

We ranked specificity of split-GAL4 drivers in the Supplementary File 1. Rank 2 are the ideal lines, Rank 1 are less ideal but acceptable, and Rank 0 is not suitable for activation screening in behavioral experiments. Out of the 828 split-GAL4 lines reported here, there are 413, 305 and 103 lines in rank2, rank1 and rank0 categories respectively. 7 lines are not ranked for specificity because only flipout expression data are available.

Recommendations for the authors:

Reviewer #2 (Recommendations For The Authors):

As mentioned elsewhere and in addition to the minor points below, it is advisable for the authors to elaborate on the details of the screening process. Furthermore, a discussion about the circuits not targeted by their research, such as the visual projection neurons, would be beneficial.

See the response above to Reviewer #2’s public review.

Line 32-33: The citations are very fly-centric. the authors might want to consider reviews on the MB of other insect species regarding learning and memory.

We additionally cited Rybak and Menzel 2017’s book chapter on honey bee mushroom body.

Line 43-44: Citations should be added, e.g. Séjourné et al. (2011), Pai et al. (2013), Plaçais et al. (2013).

Citation added

Line 50-52: Citation Hulse et al. (2021) should be added.

Citation added

Line 162: In this part, it might be valuable for the reader to understand which of these PNs are actually connecting with KCs.

A full list of cell types within the MB were provided in Supplementary File 4 of the revised manuscript. See also response to Reviewer 3, Lines 150-1.

Line 179: Citation Burke et al. (2012) should be mentioned.

Citation added

Line 181: Thermogenic might be thermogenetic.

Corrected

Line 189: Citations add Otto et al. (2020) and Felsenberg et al. (2018).

Citations added

Line 208ff: The authors should consider discussing why they did not use other GR and IR promoters. For example, Gr5a is prominent in sugar-sensing, while Ir76b could be a reinforcement signal related to yeast food (Steck et al., 2018; Ganguly et al., 2017; see also Corfas et al., 2019 for local search).

We focused on the Gr64f promoter because of its relatively broad expression and successful use of Gr64f-GAL4 for fictive reward experiment. We added the Split-LexA lines with Gr43a and Gr66a promoters (Figure 7-figure supplement 2). Other gustatory sensory neurons also have the potential to be reinforcement signals, but we just did not have the bandwidth to cover them all.

Line 319: Consider citing Linneweber et al. (2020) for a neurodevelopmental account of such individuality.

We added a sentence and cited this reference.

“On the other hand, the neurodevelopmental origin of neuronal morphology appeared to have functional significance on behavioral individuality (Linneweber et al. 2020).”

Line 352: Citation add Hulse et al. (2021).

Citations added

Line 356ff: The utility and value of Split-LexA may not be apparent to non-expert readers. Moreover, how were LexADBDs chosen for creating these lines?

We have added an introductory sentence at the beginning of the paragraph and explained that these split-LexA lines were a conversion of split-GAL4 lines that were published in 2014 and frequently used in studying the mushroom body circuit.

“Split-GAL4 lines enable cell-type-specific manipulation, but some experiments require independent manipulation of two cell types. Split-GAL4 lines can be converted into split-LexA lines by replacing the GAL4 DNA binding domain with that of LexA (Ting et al., 2011). To broaden the utility of the split-GAL4 lines that have been frequently used since the publication in 2014 (Aso et al., 2014a), we have generated over 20 LexADBD lines to test the conversions of split-GAL4 to split-LexA. The majority (22 out of 34) of the resulting split-LexA lines exhibited very similar expression patterns to their corresponding original split-GAL4 lines (Figure 12).”

Line 374: Italicize Drosophila melanogaster.

Revised as suggested.

Reviewer #3 (Recommendations For The Authors):

Major Comments:

As mentioned in the Public Review, the drivers are nicely classified in the various subsections of the manuscript, but the statements in the text summarizing how many lines there are in specific categories are often confusing. For example, line 129 refers to "drivers encompassing 111 cell types that connect with the DANs and MBONs", but Figure 1E indicates that 46 new cell types downstream of MBONs and upstream of DANs have been generated. This seems like a discrepancy.

The 46 cell types in Figure 1E consider only the CRE/SMP/SIP/SLP area, where MBON downstreams and DAN upstreams are highly enriched, while the 111 cell types include all. To avoid confusion, we removed the “MBON downstream and DAN upstream” counting in Figure 1E in the revised manuscript.

Also, at line 75 the MBON lines previously generated by Rubin and Aso (2023) are referred to as though they are separate from the 828 described "In this report." Supplementary file 1 suggests, however, that they are included as part of this report.

Twenty five lines generated in Rubin and Aso (2023) were initially included in Supplementary file 1 for the convenience of users, but they were not counted towards the 828 new lines described in this report. To avoid confusion, we removed these 25 lines in the revised manuscript. Now all lines listed in Supplementary file 1 were generated in this study (“Aso 2021” release), and if a line has been used in earlier studies, or introduced in other contexts, for example the accompanying omnibus preprint (Meissener 2024, doi: 10.1101/2024.01.09.574419), the citations are listed in the reference column.

More generally, in lines 94-102 "828 useful lines based on their specificity, intensity and non-redundancy" are referred to, but they are subsequently subdivided into categories of lines with lower specificity (i.e. with off-target expression) and lines that did not target intended cell types (presumably ones unlikely to be involved in learning and memory). It would be useful to know how many lines (at least roughly) fall into these subcategories.

See the response above to Reviewer #3’s public review.

Finally, Figures 3B & C indicate cell types connected to DANs and MBONs and the number for which Split-Gal4 lines are available. The text (lines 136-7) states that the new collection covers 30 of these major cell types (Figure 3C)," but Figure 3C clearly has more than 30 dots showing the drivers available. Presumably existing and new driver lines are being pooled, but this should either be explained or the two should be distinguished.

“(Figure 3C)” was replaced with “(Supplementaryl File 3)” in the revised manuscript to correct the reference. Figure 3B & C are plots of all MB interneurons, not just the major cell types.

Minor Comments:

Although the paper is generally well written there are minor grammatical errors throughout (e.g. dropped articles, odd constructions, etc.) that somewhat detract from an otherwise smooth and enjoyable reading experience. A quick editing pass by a native speaker (i.e. any of several of the authors) could clean up these and numerous other small mistakes. A few examples: line 138 "presented" should be present; line 204: "contain off-targeted expressions" should be "have off-target expression;" line 219: "usage to substitute reward" is awkward at best and could be something like "use in generating fictive rewards"; line 326 "arborize[s]"; l. 331 "Based on the likelihood" should be something like "based on these observations"'; line 349 "[is] likely to appear"; l. 352 "extensive connection[s]"; line 353 "has [a] strong influence;" l. 963 "Projections" should be singular; etc.

All the mentioned examples have been corrected, and we have asked a native speaker to edit through the revised manuscript.

Lines 81-3: Is the lookup table referred to Suppl. File 1? A reference is desirable.

Yes, the lookup table referred to “Supplementary File 1” and a reference was added.

Lines 111-2: what is a "non-redundant set of...cell types?" Cell types that are represented by a single cell (or bilateral pair)? Or does this sentence mean that of the 828 lines, 355 are specific to a single cell type, and in total 319 cell types are targeted? The statement is confusing.

We revised the text as below.

“Figure 1E provides an overview of the categories of covered cell types. Among the 828 lines, a subset of 355 lines, collectively labeling at least 319 different cell types, exhibit highly specific and non-redundant expression patterns are likely to be particularly valuable for behavioral experiments. Detailed information, including genotype, expression specificity, matched EM cell type(s), and recommended driver for each cell type, can be found in Supplementary File 1. A small subset of 40 lines from this collection have been previously used in studies (Aso et al.,

2023; Dolan et al., 2019; Gao et al., 2019; Scaplen et al., 2021; Schretter et al., 2020; Takagi et al., 2017; Xie et al., 2021; Yamada et al., 2023). All transgenic lines newly generated in this study are listed in Supplementary File 2 (Aso et al., 2023; Dolan et al., 2019; Gao et al., 2019; Scaplen et al., 2021; Schretter et al., 2020; Takagi et al., 2017; Xie et al., 2021; Yamada et al., 2023).”

Line 148: "MB major interneurons" is a confusing descriptor for postsynaptic partners of MBONs.

We added a sentence to clarify the definition of the “MB major interneurons”.

“In the hemibrain EM connectome, there are about 400 interneuron cell types that have over 100 total synaptic inputs from MBONs and/or synaptic outputs to DANs. Our newly developed collection of split-GAL4 drivers covers 30 types of these ‘major interneurons’ of the MB (Supplementary File 3).”

Lines 150-1: Not sure what is meant by "have innervations within the MB." Sounds like cells are presynaptic to KCs, DANS, and MBONs, but Figure 3 Figure Supplement 1 indicates they include neurons that both provide and receive innervation to/from MB neurons. Please clarify.

For clarification, in the revised manuscript we have included a full list of cell types within the MB in Supplementary File 4. Included are all neurons with >= 50 pre-synaptic connections or with >=250 post-synaptic connections in the MB roi in the hemibrain (excluding the accessory calyx). The cell types include KCs, MBONs, DANs, PNs, and a few other cell types. The coverage ratio was updated based on this list.

Also, in line 152, what does it mean that they "may have been overlooked previously?" this seems unnecessarily ambiguous. Were they overlooked or weren't they?

Changed the text to “These lines offer valuable tools to study cell types that previously are not genetically accessible. Notably, SS85572 enables the functional study of LHMB1, which forms a rare direct pathway from the calyx and the lateral horn (LH) to the MB lobes (Bates et al., 2020). ”

Line 158 refers to PN cells within the MB, which are not mentioned in any place else as MB components.

What are these PNs and how do they differ from MBONs?

See responses to Lines 150-1 for clarification of cell types within the MB.

Line 188: not clear what is meant by "more continual learning tasks".

We rephrase it as “more complex learning tasks” to avoid jargon.

Line 235: Not clear why "extended training with high LED intensity" wouldn't promote the formation of robust memories. Is this for some reason unexpected based on previous experiments? Please explain.

See responses to weakness #1 of the same reviewer

Lines 317-9: It would be useful to state here that MB0N08 and MB0N09 are the two neurons labeled by MB083C.

Revised as suggested.

Line 368: Presumably the "lookup table" referred to is Supplementary File 1, but a reference here would be useful.

Yes, Supplementary File 1 and a reference was added.

Comments on Figures:

Figure 1C The "Dopamine Neurons" label position doesn't align with the Punishment and Reward labels, which is a bit confusing.

They are intentionally not aligned, because dopamine neurons are not reward/punishment per se. We intend to use the schematic to show that the punishment and reward are conveyed to the MB through the dopamine neuron layer, just as the output from the MB output neuron layer is used to guide further integration and actions. To keep the labels of “Dopamine neurons” and “MB Output Neurons” in a symmetrical position, we decide to keep the original figure unchanged. But we thank the reviewer for the kind suggestion.

Figure 1F and Figure 1 - Figure Supplement 1: the light gray labels presumably indicate the (EM-identified) neuron labeled by each line, but this should be explicitly stated in the figure legends. It would also be useful in the legends to direct the reader to the key (Supplementary File 1) for decoding neuronal identities.

Revised as suggested.

Figure 2: For clarity, I'd recommend titling this figure "LM-EM Match of the CRE011-specific driver SS45245". This reduces the confusion of mixing and matching the driver and cell-type names. Also, it would be helpful to indicate (e.g. with labels above the figure parts) that A & B represent the MCFO characterization step and C & D represent the LM-EM matching step of the pipeline. Revised as suggested.

Figure 6: For clarity, it would be useful to separately label the PN and sensory neuron groups. Also, for the sensory neurons at the bottom, what is the distinction between the cell names in gray and black font?

Figure 6 was updated to separate the non-olfactory PN and sensory neuron groups. The gray was intended for olfactory receptor neuron cell types that are additionally labeled in the driver lines. To avoid confusion, the gray cell types were removed in the revised figure, and a clarification sentence was added to the legend.

“Other than thermo-/hygro-sensory receptor neurons (TRNs and HRNs), SS00560 and MB408B also label olfactory receptor neurons (ORNs): ORN_VL2p and ORN_VC5 for SS00560, ORN_VL1 and ORN_VC5 for MB408B.”

Figure 7A: It's unclear why the creation of 6 Gr64f-LexADBD lines is reported. Aren't all these lines the same? If not, an explanation would be useful.

These six Gr64f-LexADBD lines are with different insertion sites, and with the presence or absence of the p10 translational enhancer. Explanation was added to legend. Enhanced expression level with p10 can be helpful to compensate for the general tendency that split-LexA is weaker than split-GAL4. Different insertions will be useful to avoid transvections with split-GAL4s, which are mostly in attP40 and attP2.

Figure 8F: It would help to include in the legend a brief description of each parameter being measured-essentially defining the y-axis label on the graphs as in Figure Supplement 2. Also, how is the probability of return calculated and what behavioral parameter does the change of curvature refer to?

We added a brief description to the behavioral parameters in the legend of Figure 8F.

“Return behavior was assessed within a 15-second time window. The probability of return (P return) is the percentage of flies that made an excursion (>10 mm) and then returned to within 3 mm of their initial position. Curvature is the ratio of angular velocity to walking speed.”

Figure 9E: What are the parenthetical labels for lines SS49267, SS49300, and SS35008?

They are EM bodyIDs. Figure legend was revised.

Reviewer #3 (Public review):

In this study, Cao et al. explore the neural mechanisms by which chronic heat exposure induces negative valence and hyperarousal in mice, focusing on the role of the posterior paraventricular nucleus (pPVT) neurons that receive projections from the preoptic area (POA). The authors show that chronic heat exposure leads to heightened activity of the POA projection-receiving pPVT neurons, potentially contributing to behavioral changes such as increased anxiety level and reduced sociability, along with heightened startle responses. In addition, using electrophysiological methods, the authors suggest that increased membrane excitability of pPVT neurons may underlie these behavioral changes. The use of a variety of behavioral assays enhances the robustness of their claim. Moreover, while previous research on thermoregulation has predominantly focused on physiological responses to thermal stress, this study adds a unique and valuable perspective by exploring how thermal stress impacts affective states and behaviors, thereby broadening the field of thermoregulation. However, a few points warrant further consideration to enhance the clarity and impact of the findings.

(1) The authors claim that behavior changes induced by chronic heat exposure are mediated by the POA-pPVT circuit. However, it remains unclear whether these changes are unique to heat exposure or if this circuit represents a more general response to chronic stress. It would be valuable to include control experiments with other forms of chronic stress, such as chronic pain, social defeat, or restraint stress, to determine if the observed changes in the POA-pPVT circuit are indeed specific to thermal stress or indicative of a more universal stress response mechanism.

(2) The authors use the term "negative emotion and hyperarousal" to interpret behavioral changes induced by chronic heat (consistently throughout the manuscript, including the title and lines 33-34). However, the term "emotion" is broad and inherently difficult to quantify, as it encompasses various factors, including both valence and arousal (Tye, 2018; Barrett, L. F. 1999; Schachter, S. 1962). Therefore, the reviewer suggests the authors use a more precise term to describe these behaviors, such as valence. Additionally, in lines 117 and 137-139, replacing "emotion" with "stress responses," a term that aligns more closely with the physiological observations, would provide greater specificity and clarity in interpreting the findings.

(3) Related to the role of POA input to pPVT,<br /> a) The authors showed increased activity in pPVT neurons that receive projections from the POA (Figure 3), and these neurons are necessary for heat-induced behavioral changes (Figures 4N-W). However, is the POA input to the pPVT circuit truly critical? Since recipient pPVT neurons can receive inputs from various brain regions, the reviewer suggests that experiments directly inhibiting the POA-to-pPVT projection itself are needed to confirm the role of POA input. Alternatively, the authors could show that the increased activity of pPVT neurons due to chronic heat exposure is not observed when the POA is blocked. If these experiments are not feasible, the reviewer suggests that the authors consider toning down the emphasis on the role of the POA throughout the manuscript and discuss this as a limitation.<br /> b) In the electrophysiology experiments shown in Figures 6A-I, the authors conducted in vitro slice recordings on pPVT neurons. However, the interpretation of these results (e.g., "The increase in presynaptic excitability of the POA to pPVT excitatory pathway suggested plastic changes induced by the chronic heat treatment.", lines 349-350) appears to be an overclaim. It is difficult to conclude that the increased excitability of pPVT neurons due to heat exposure is specifically caused by inputs from the POA. To clarify this, the reviewer suggests the authors conduct experiments targeting recipient neurons in the pPVT, with anterograde labeling from the POA to validate the source of excitatory inputs.

(4) The authors focus on the excitatory connection between the POA and pPVT (e.g., "Together, our results indicate that most of the pPVT-projecting POA neurons responded to heat treatment, which would then recruit their downstream neurons in the pPVT by exerting a net excitatory influence.", lines 169-171). However, are the POA neurons projecting to the pPVT indeed excitatory? This is surprising, considering i) the electrophysiological data shown in Figures 2E-K that inhibitory current was recorded in 52.4% of pPVT neurons by stimulation of POA terminal, and ii) POA projection neurons involved in modulating thermoregulatory responses to other brain regions are primarily GABAergic (Tan et al., 2016; Morrison and Nakamura, 2019). The reviewer suggests showing whether the heat-responsive POA neurons projecting to the pPVT are indeed excitatory (This could be achieved by retrogradely labeling POA neurons that project to the pPVT and conducting fluorescence in situ hybridization (FISH) assays against Slc32a1, Slc17a6, and Fos to label neurons activated by warmth). Alternatively, demonstrate, at least, that pPVT-projecting POA neurons are a distinct population from the GABAergic POA neurons that project to thermoregulatory regions such as DMH or rRPa. This would clarify how the POA-pPVT circuit integrates with the previously established thermoregulatory pathways.

12:3 Those who are wi se[a] will shine like the brightness of the heavens, and those who lead many to righteousness, like the stars for ever and ever.

• https://www.americamagazine.org/politics-society/2020/05/08/its-time-rethink-electoral-college

• https://www.npr.org/sections/itsallpolitics/2011/12/20/144016912/we-the-people-npr-readers-would-ratify-four-new-amendments

• https://www.americamagazine.org/politics-society/2020/05/08/its-time-rethink-electoral-college

• https://www.npr.org/sections/itsallpolitics/2011/12/20/144016912/we-the-people-npr-readers-would-ratify-four-new-amendments

• https://constitutioncenter.org/blog/vote-now-an-amendment-to-end-the-electoral-college

• https://www.nytimes.com/2020/02/09/opinion/letters/electoral-college.html

• https://www.latimes.com/opinion/readersreact/la-ol-le-electoral-college-20180904-story.html

you are offline

• https://slate.com/news-and-politics/2014/05/amending-the-constitution-is-much-too-hard-blame-the-founders.html

we the people rise again

• https://slate.com/news-and-politics/2012/06/fix-the-constitution-amending-by-national-referendum.html

safe souls, safe fu

• https://slate.com/news-and-politics/2012/06/fixing-the-constitution-protecting-informational-privacy.html

• https://slate.com/news-and-politics/2020/05/new-reconstruction-constitution-democracy.html

We the People of Slate ...

The U.S. Constitution, as you [mighta been, shoulda "come" on ... its someday] rewrϕte it.

"Politicians talk about the Constitution as if it were as sacrosanct as the Ten Commandments [interjection: spec. it is actually almost exactly related!]. But the document itself invites change and revision. What if the president served only one six-year term instead two four-year terms? What if your state's population determined how many senators represent it? What if the Constitution included a right to health care? We asked legal scholars and Slate readers to cross out what they didn't like in the Constitution and pencil in their hearts' desires. Here's what the document would look like with their best ideas."

• Slate: u_s_constitution as_rewritten by_slate_legal_experts_and_readers

多也了了夕 "with a ~~wand~~ of scheffilara, 并#亦太 he begins ... "I am now on the Staff of Menelaus, the Spears of Longinus and Lancelot; and the name "Mosche ex Nashon."

Logically the recent mentions of Gilgamesh and the simultaneous 同時 overlaping 場道 of the eventual link between the famous ruling of Solomon on the separation of babies and mothers and waters and land ... to a story of many "two cities" that culminates in a cultural or societal or "evolutionary" link to Sodom and Gomorrah and the city-state of Babylon (and it's Hanging Gardens) and also of course to Paris and Troy and "Masstodon" and city-states [ciudadestado] and perhaps planet-cities; from Cambridge to Cambridge across the "Cable" to see state to "London" ... recently I called it "the city of realms" ... I started out logically intending to link "game theory" and John Nash to the mathematical story of Sputnik and a revival of American physics; but in my usual way of rambling into the woods [I mean neighborhood] of stream of consciousness ... turned into a premonitory discourse of "two cities" and how sometimes even things as obvious as the number of letters in the word "two" don't do a good enough job of conveying ... how and/or why one is simply never enough, and two isn't much better--but in the end a circle ... is drawn; the perfect circle in our imaginary mathematical perfection ... I see a parted "line" in the letter pronounced "tea" (and beginning that word); and two "vee" (pron. of "v") symbols joined together in a word we pronounce as "double-you" ... and symbolically because I know "V" is the Roman Numeral for 5 (five) and I know not how to multiply in Roman numerals--

It's important to pause; here. I am going to write a more detailed piece on "the two cities" as I work through this maze like crossroads between "them" and "demo..." ... here demorigstrably I am trying to fuse together an evolutionary change in ... lit. biological evolution as well as an echelon leap forward in "self-government" ... in a place where these two things are unfathomable and unspokenly* connected.

• https://www.google.com/search?q=prometheuslocke+%2Bsite%3Agodlikeproductions.com

• "Silence is betrayal" -MLK

To a question on the idiom; is Bablyon about "the law" or "of the land of Nod?"

"What is democracy" ... the song, Metallica's "ONE" echoes and repeats; as we apparently scrive together the word "THEM" ... I question myself ... if Babylon were the capital city of some mythical Nation of Time ... if it were the central "turning point" of Sheol; ... >|<

Can you not see that in this place; in a world that should see and does there is a gigantic message proving that we are not in reality and trying to show us how and why that's the best news since ... ever---that it's as simple as conjoining "the law of the land" with a basic set of rules that automatically turn Hell into something so much closer to Heaven I just do not understand---why we cant stand up together and say "bullets will not kill innocent children" and "snowflakes will not start avalanches ...." that cover or bury or hide the road from Earth to Verital)e .... or from the mythical Valis to Tanis---or from Rigel to Beth-El ... "guess?"

## as "an easy" answer; I'm looking for a fusion of "law and land" that somehow remembers a "jok'er a scene" about "lawn" seats; and "where the girls are green;"

It's as simple as night and day; Heaven and Hell ... the difference between survival and--what we are presented with here; it's "doing this right"--that ends the Hell of representative democracy and electoral college--the blindness and darkness of not seeing "EXTINCTION LEVEL EVENT" encoded in these words and in our governments foundation ... *by the framers [not just of the USA; but English .. and every language] *

... is literally just as simple as "not caring" or thinking we are at the beginning of some long process--or thinking it will never be done--that special "IT" that's the emancipation of you and I.

Here words like "gnosis" and "gaudeamus" pair with my/ur "new ntersanding*" of the difference between Asgard and Medgard and really understanding our purpose here is to end "evil" ... things like "simulating disease and pain" (here, simulating meaning ... intentionally causing, rather than "gamifying away") and successfully linking the "Pillars of Hercules" to Plato's vision of Atlantis and the letter sequences "an" and "as" ... unlock a fusion of religion and mythology and "cryptographic truth" that connects "messianic" and "Christian" to "Roman" ... "Chinese" and "American" ... literally the key to the difference between the phrases "we are" and "we were" ....

in "sight" of "silicon" in simulation and Israel, Genesis, and "silence" ... trying to the raising of Asgardian enlightenment ... and seeing "simple cypher" connecting to "Norse" ...

and the "I AM THAT" surer than shit ... the intention and design of all religion and creation is to end "simulated reality" and also not seeing "SR" ... in Israel and Norse ... "for instance."

• https://www.google.com/search?q=%22I+AM%22+%22WE+ARE%22+%2Bsite%3Afromtaws
• "SOIS" a key--in two languages conjugated literally as both "I AM" and "WE ARE" simultaneously;
• Search: I know that if I am than so are you ... and it is because we have overcome .... something I truly cannot figure out, fathom, or believe ... was truly here before us--a spiralling series of failures ... speaking: to the heavens; but in secret and in action; "doing everything possible to succeed."

It's a simple linguistic concept; the "singularity" and the "plurality" of a simple word--"to be"--but it goes to the heart of everything that we are and everything that is around us. This is a message about understanding and preserving individuality as well as liberty; and literally seeing "ARXIV" and understanding "often" and failing to connect God and prescience to "IV" and the Fourth Amendment ... it's about blindness and ... "curing the blind instantly" ... and fathoming how and why this message has been etched into our entire history and and all religions and myths and music--to help us "to be THAT we" that actually "are responsible" for the end of Hell.

• I neglected to mention "Har-Wer" and "Tower of Babel" which are both related lingusitically, religiously and topically: "to who ..." and while we're on "four score and [seven years from now]" seeing the fourth "living thing" in Eden and it's (the name, Abel) connection to Babel and Abraham Lincoln; slavery and ... understanding we live in a place where the history of the United States also, like Monoceros and "Neil Armstrong's first step" are a time shifted ... overlayed map to achieving freedom ... it's about becoming a father-race ... and actually "doing" the technological steps required to "emancipate the e's of 'me&e'" and survive in exo-planetary space---

it might be as simple as adding "because we did this" here and now; and having it be something we are truly proud of .... forevermore™ ... for certain in the heart of this story about cyclicality and repetition of error--its not because we did "this" or something over and over again; it's about changing "the problem" and then helping others to also overcome ... "things like time travel ... erasing speech" --- however that happenecl.

• I also failed to mention that "I am in Hell" ... as in this world is hellacious to me; in an overlay with the Hellenic period and this message that we are in the Trojan Horse ... a small gem .... "planet" truly is the Ark of the Covenant---and it's the simple understanding that "reality is hell" is to "living without air conditioning and plumbing is hell" just as soon as you achieve ... "rediscovering" those things---

• I can't figure out why I am the only person screaming "this is Hell." That's also, Hell.

... but recently suggested an old joke about "there being 10 kinds of people in the world (obv an anti-tautology and a tautology simultaneously)" only after that brief bit of singularity and duality mentioning the rest of the joke: "those that understand binary and those that don't know how to base convert between counting with two hands and counting with only an 'on and off.'" It's not obvious if you aren't trying to figure it out, I suppose; but 10 is decimal notation for "kiss" and the "often" without "of" ... and binary notation for the decimal equivalent of "2." A long long time ago in a state that simply non-randomly ties to the heart of the name of our galaxy ... I was again thinking of the "perfect imperfections" of things like saying "three equals one equals one" (which, of course was related to the Holy Trinity and it's "prescient/anachronistic Adamic presence encoded in the name Ab|ra|ha|m" which means "father of a great multitude") ... I brought that one back in the last few months; connecting the letter K and in this "logos-rythmic" tie to the "base of a number system" embellish the truth just a bit and suggest a more accurate rendition of the original [there is no such thing as equality, "is" of separate objects--as in no two snowflakes are the same unless they are literally the same one; true of ancient weights and with the advent of (thinking about) time no two "planets" are the same even if they're the exact same one--unless it's at a fixed moment in time.

• This name may be viewed either as meaning "father of many" in Hebrew or else as a contraction of ABRAM (1) and הָמוֹן (hamon) meaning "many, multitude". The biblical patriarch Abraham was originally named Abram but God changed his name (see Genesis 17:5).

• https://en.wikipedia.org/wiki/Yeshua#Yeshua,_Yehoshua,_and_Yeshu_in_the_Talmud

K=3:11 ... to a handle on the music, the DHD of the gate and the *ring of David's "sling" ...

---and that's a relationship of "3 is to 11" as [the SAT style "analogy)]y" as a series of alpha, two mathematic, and two numeric symbols ... may only tie in my mind alone to the books of Genesis and Matthew and the phrase "chapter and verse" and to the stories of Lot and Job ... again in Genesis and the eponymous "Book of Job." So ... "tying up loose ends one 10b [III] iv. " as it appears I've taken it upon myself to call a Job and suggest is my "Lot in life [x]i* [3]"

• I worry sometimes that important things are missing, or will disappear---for instance Mirriam Webster, which is a "canonical/standard dictionary) should probably have an entry for "lot in life" non-idiomatically as "granny apples to sour apples" as

2 MANY ALSO ICI; 1twoⅱ ... following in Mitnick's bold introductory word steps; the curve and the complement ... the missiles and the canoes; the line and the blank space ... "supposedly two examples of two kinds, which could be three not nothings ... Today I write about something monumental; as if as important as the singularity depicted in Arthur C. Clarke's 2001 "A Space Odyssey" ... and remember a day when I thought it very novel and interesting to see the words "stillborn and yet still born" connected in a single piece of writing to "Stillwater and yet still water" ... today adding in another phrase noting the change wrought only by one magical single "space" (also a single capital letter; and a third phrase): "block chains with a great blockchain."

• http://www.goodmath.org/blog/2015/07/21/arabic-numerals-have-nothing-to-do-with-angle-counting/

• https://gizmodo.com/no-this-viral-image-does-not-explain-the-history-of-ar-1719306568

• https://en.wikipedia.org/wiki/Chinese_word_for_%22crisis%22

• https://dictionary.hantrainerpro.com/chinese-english/translation-ji_howmany.htm
• https://dictionary.hantrainerpro.com/chinese-english/translation-duo_many.htm

• https://en.wikipedia.org/wiki/Euripides, Iphigenia in Aulis or Iphigenia at Aulis[1] (Ancient Greek: Ἰφιγένεια ἐν Αὐλίδι, Iphigeneia en Aulidi; variously translated, including the Latin Iphigenia in Aulide) is the last of the extant works by the playwright Euripides. Written between 408, after Orestes, and 406 BC, the year of Euripides' death, the play was first produced the following year[2] in a trilogy with The Bacchae and Alcmaeon in Corinth by his son or nephew, Euripides the Younger,[3] and won first place at the City Dionysia in Athens.

• The play revolves around Agamemnon, the leader of the Greek coalition before and during the Trojan War, and his decision to sacrifice his daughter, Iphigenia, to appease the goddess Artemis and allow his troops to set sail to preserve their honour in battle against Troy. The conflict between Agamemnon and Achilles over the fate of the young woman presages a similar conflict between the two at the beginning of the Iliad. In his depiction of the experiences of the main characters, Euripides frequently uses tragic irony for dramatic effect.

J.K. Rowling spurred just this past week a series of explanations about just exactly what is a blockchain coin worth ... and why is it so; her final words on the subject (artistic liberty taken, obviously not the last she'll say of this magic moment) "I don't think I trust this."

Taken directly from an off the cuff email to ARXM titled: "Slow the S is ... our Hypothes.is"

I imagine I'll be adding some wiki/ipfs stuff to it--and try to keep it compatible; the design and layout is almost exactly what I was dreaming about seeing--as a "first rough draft product." Lo, and behold. It's been added to the many places I host my tome; the small compilation of nearly every important email that has gone out ... all the way back to the days of the strange looking Margarita glass ... that now very much resembles the "Cantonese character 'le'" which I've come to associate with a "handle" on multiple corners of a room--something like an automatic coat rack conveyor belt connecting different versions of "what's in the box." I'm planning on using that symbol 了 to denote something like multiple forks of the same page. Obviously I'm thinking forward to things like "the Transhumaist Chain Party" (BDSM, right?)'s version of some particular piece of legislation, let's say everything starts with the sprawling "bulbing" of "Amendment M" ideas and specific verbiage ... and then we'll of course need some kind of new git/subversion/cvs style version control mechanism to merge intelligently into something that might actually .... really should ... make it into that place in history--the first constitutional amendment ratified by a "Continental Congress of All People" ... but you could also see it as an ongoing sort of forking of something like the "wikipedia page" on what some specific term, say "technocracy" means, and how two parties might propagandize and change the meaning of such thing; to suit the more intelligent and wise times we now live in. For instance, we might once have had a "democracy" and a "democractic" party that had some Anarchist Cook Book version of the history of it ending in something like Snipes and Stallone's "DEMOLITION MAN."

Just kidding, we all know "democracy" has everything to do with "d is cl ... and not th" ... to be the them that is the heart of the start of the first true democracy. At least the first one I've ever seen, in my old "to a republic" ... style. As it is you can play around with commenting and highlighting and annotating all the stuff I've written and begged and begged for comments on--while I work on layering the backend to to perma-store our ideas and comments on both a blockchain (probably a new one; now that i've worked a little with ethereum) with maybe some key-merkle-tree-walk-search stuff etched into the original Rinkeby ... and then of course distributed data in the "public owned and operated" IPFS. To be clear, I plan on rewriting the backend storage so that we will have a permanent record of all comments; all versions of whatever is being commented on; and changes/revisions to those documents--sort of turning the web into a massive instant "place of collaboration, discussion, and co-authoring" ... if you use the wonderful LEGO pieces that have been handed to us in ideas from places like me, lemma--dissenter, and of course hypothes.is who has brought you and i such a polished and nice to look at "first draft" of something like the living Constitution come repository of all human knowledge. I do sort of secretly wich they would have called this project something like "annotating and reflecting (or real or ...) knowledge" just so the movement could have been called ARK. ... or something .... but whatever join the "calling you a reporter" group or ... "supposedly a scientist?"

NOIR INgR .. I CITE SITE OF ENUDRICAM; a rekindling of the dream of a city appearing high above in the sky, now with a boldly emblazened smiling rainbow and upsidown river ... specifically the antithesis of "angel falls," there's a lagoon too--actually a chain of several ponds underneith the floating rock ... and in some versions of this waking dream there are rings around the thing; you might imagine an artificial set of centripetal orbitals something like a fusion of the ring Eslyeum and the "Six-Axis ride" of the JKF Center's "Spacecamp." I write as I dream, and though I cannot for certain explain exactly how; it's become a strong part of my mythology that this spectacular rendition of "what ends the silence" has something to do with the magical delivery of "a book" ... something not of this Earth but an unnatural thing; one I've dreamt of creating many times. This book is something like the DSM-IV and something like a Merck diagnostic manual; but rather than the old antiquated cures of "the Norse Medgard" this spectacle nearly "itsimportant" autoprints itself and lands on something like every doorpost; what it is is a list of reasons why "simply curing all disease" with no explanation and no conversation would be a travesty of morality--how it would render us half-blind to the myriad of new solutions that can come from truly understanding why "ITIS" to me has become a kind of magical marker: an "it is special" as in, it's cure could possibly solve a number of other problems.

Through that missing "o," English on the ball, we see a connection between a number of words that shine bright light including Exodus itself which means "let there be light," the word for Holy Fire and the Burning Bush.. .reversed to hSE'Ah, and a story about the Second Coming parting our holy waters.**

This answer connects the magical Rod's of Aaron in Exodus and the Iron Rod of Jesus Christ to the Sang Rael itself... in a fusion that explains how the Periodic Table element for Iron links not just to Total Recall and Mars, but also to this key

my dream of what the first day of the Second Coming might be like; were the Rod of Christ... in the right hands. In a story that also spans the Bible, you might understand better how stone to bread and your input make all the difference in the world between Heaven and Adam's Hand. Once more, what do you think He** ....

Since the very earliest days of this story, I have asked for better for you, even than see

Nearly all of the original parts of the original "post-origination dream" remain intact; there's a walkway that magically creates new paths and "attractions" based on where you walk, something like an inversion of the artificial intelligence term "a random walk down a binary tree" ... for instance going left might bring you to the Internet Cafetornaseum of the Earl of Sandwich; and going to the right might bring you to the ICIMAX/Auditorium of Science and Discovery--there's a walkway to "Magical GLAS D'elevators" that open a special "instantiation" of the Japan Room of the Potter and the Toolmaker ... complete with a special [second level and hidden staircase] Pool of Bethesdaibo verily delivering something like youth of mind and body ... or at least as close to such a thing as a sip of Holy Water or Ambrosia or a dip in the pool of Coccoon and Ponce De'Leon could instantly bring ... to those that have seen Jupiter Ascending ... the questions of "nature versus nurture" and what it means to be "old and wise" and "young at heart" truly mean---

• https://www.youtube.com/watch?v=M8CyN1awWls
• https://link.springer.com/chapter/10.1057/9780230366688_16
• https://www.youtube.com/watch?v=YDo5zvYNn3A

Somewhere between the outdoor rafting ride and the level with the special "ballroom of the ancient gallery" ... perhaps now being named or renamed or recalled as something about "Face [of] the Music" lies a magical "mini-maize" ... a look at a mock-up (or #isitit) of Merlink and Harthor's "round table" that displays a series of ... (at least to me) magical appearing holographic displays and controls that my dreams have stolen from Phillip K. Dick's Minority Report and something of what I hope Microsoft's Dynamics/Hololens/Surface will become---a series of short "focus groups" .... to guage and discuss the information in the "CITIES-D5AM-MERCK" ... how to end world hunger and nearly all disease with the press of a magical buzzer--castling churches to something like "political-party-town-hall-meeting centers" and replacing jails and prisons and hospitals with something like the "Hospitalier's PRIDE and DOJOY's I practiced "Kung-fun-dance" ... a fusion of something like a hotel and a school that probably looks very much like a university with classrooms and dorms and dining hall's all fit into a single building. I imagine a series of 2 or 3 "room changes" as in you walk from the one where you get the book and talk about it ... to the one where you talk about "what everyone else said about it" and maybe another one that actually connects you to other people with something like Facebook's Portal; the point of the whole thing to really quickly "rubber stamp" the need for an end to "bars in the sky" nonalcoholic connotation--as in "overcoming the phrase the sky is the limit" and showing us the need for a beacon of glowing hope fulfilled--probably actually the vision of a holographic marker turning into actual rings around the single moon of Earth, the focus of the song annoucing the dawn of the age of Aquarius---

It might lead us also to Ceres; and another set of artificial rings, or to Monoceros and a rehystorical understanding of the birthplace and birthing of the "river roads" that bridge the "space gaps" in the galaxy from our "one giant leap for mankind" linking the Apollo moon landing to the mythological connection to the sun; and connecting how the astrological charts of the ancients might detail a special kind of overlapping--the link between Earth's SOL and something like Proxima or Alpha Centauri; and how that "monostar bridge" might overlap to Orion and from there through Sagitarius and the center of the Milky Way ... all the way to Andromeda and more dreams of being in a place where there's a map to a tri-galactic system in the constellation Cancer and a similar one in Leo ... and just incase you haven't noticed it--a special marker here, I thought to myself it might be cool to "make an acronymic tie to Monoceros" and without even thinking auto-wrote Orion (which was the obvious constellation next to Monoceros, in the charts) and then to Sagitarrius; which is the obvious ... heart of our astrological center and link to "other galaxies."

----I've dreamt or scriven or reguessed numerous times how the Milky Way's map to an "Atlas marked through time by the ages and the ancients" might tie this place and this actual map to the creation of the railways between stars to the beginning and the end of time and of course to this message that links it all to time travel. There's a few "guesses" I've contemplated; that perhaps the Milky Way chart is a metal-cosmic or microcosmic map to the dawn of time in the galactic vision of ... just after the big bang; or it might tie to a map of something like the unthinkable--a civilization that became so powerful it was able to reverse the entropy of "cosmic expansion" and reverse the thing Asimov wrote of in "The Last Question" as the end of life and the ability to survive basically due to "heat loss."

"The Last Question." (And if you read two, why not "The Last Answer"?). Find these readings added to our collection, 1,000 Free Audio Books: Download Great Books for Free.

• https://archive.org/details/texts
• http://zlibraryexau2g3p.onion.pet/

Looking for free, professionally-read audio books from Audible.com, including ones written by Isaac Asimov?

* all "asterisks" in the abovə document denote a sort of Adamic unspoken relationship between notations and meanings; here adding the "Latin word for three" and source of the phrase "t.i.d." (which is doctor/pharmacy latin for "three times a day") where the "t" there is an abbreviation of "ter" ... and suppose the link between K and 11 and 3 noting it's alphanumeric position in the English alphabet as the 11th letter and only linking cognitively to three via the conversion between hex, and binarryy ... aberrative here is the overlapping "hakkasan" style (or ZHIV) lack of mention of the answer in "state of Kansas" and the "citystate of Slovakia" as described in the ICANN document linked [in] the related subsection or slice of the word "binarry" for the state of India. Tetris could be spelled with the addition of only a single letter [in] "tea"---the three letters "ris" are the hearts of the words "Christ" and "wrist" [and arguably of Osiris where you also see the round table character of the solar-system/sun glyph and the chemical element for The Fifth Element (as def. by i) via "Sinbad" and "Superman." The ERIS Free Network should also be mentioned here in connection with the IRC network I associate in the place between skipping stones and sacred hearts defined by "AOL" and "Kdice" in my life. In the lexicon of modern HTML, curly braces are generally relative to "classes" and "major object definitions (javascript/css)" while square brackets generally only take on computer-interpreted meaning in "Markdown" which is clearly (by definition, by this character set "[]") a superset (or at least definately not a subset) of HTML.

Dr. Will Caster (Johnny Depp) is a scientist who researches the nature of sapience, including artificial intelligence. He and his team work to create a sentient computer; he predicts that such a computer will create a technological singularity, or in his words "Transcendence". His wife, Evelyn (played by Rebecca Hall), is also a scientist and helps him with his work.

Following one of Will's presentations, an anti-technology terrorist group called "Revolutionary Independence From Technology" (R.I.F.T.) shoots Will with a polonium-laced bullet and carries out a series of synchronized attacks on A.I. laboratories across the country. Will is given no more than a month to live. In desperation, Evelyn comes up with a plan to upload Will's consciousness into the quantum computer that the project has developed. His best friend and fellow researcher, Max Waters (Paul Bettany), questions the wisdom of this choice, reasoning that the "uploaded"

Just from my general understanding and memory "st" is not ... to me (specifically) an abbreviation of "state" but "ste" is a U.S. Postal code (also "as I understand it") for the name of a special room or set of rooms called a "suite" and in Adamic "connotation" I sometimes read it as "sweet" ... which has several meanings that range from "cool" to "a kind of taste sensation" to "easy to sway or fool."

If you asked me though, for instance if "it" was an abbreviation or shorthand notation or acronym for either "a United state" or "saint" ... you'd be sure.

While it's clear from studying linguistic cryptography ... (If I studied it a little here and some there, its also from the "universal translator of Star Trek") and the personal understanding that language is a kind of intelligent code, and "any code is crackable" ... that I caution here that "meaning" and "face value" often differ widely and wildly ... even in the same place or among the same group of people ... either varying over time or heritage.

Menelaus, in Greek mythology, king of Sparta and younger son of Atreus, king of Mycenae; the abduction of his wife, Helen, led to the Trojan War. During the war Menelaus served under his elder brother Agamemnon, the commander in chief of the Greek forces. When Phrontis, one of his crewmen, was killed, Menelaus delayed his voyage until the man had been buried, thus giving evidence of his strength of character. After the fall of Troy, Menelaus recovered Helen and brought her home. Menelaus was a prominent figure in the Iliad and the Odyssey, where he was promised a place in Elysium after his death because he was married to a daughter of Zeus. The poet Stesichorus (flourished 6th century BCE) introduced a refinement to the story that was used by Euripides in his play Helen: it was a phantom that was taken to Troy, while the real Helen went to Egypt, from where she was rescued by Menelaus after he had been wrecked on his way home from Troy and the phantom Helen had disappeared.

• https://www.britannica.com/topic/Menelaus-Greek-mythology

This article is about the ancient Greek city. For the town of ancient Crete, see Mycenae (Crete). For the hamlet in New York, see Mycenae, New York.

Μυκῆναι, Μυκήνη

The Lion Gate at Mycenae, the only known monumental sculpture of Bronze Age Greece

37°43′49"N 22°45′27"ECoordinates: 37°43′49"N 22°45′27"E

This article contains special characters. Without proper rendering support, you may see question marks, boxes, or other symbols.

Mycenae (Ancient Greek: Μυκῆναι or Μυκήνη, Mykēnē) is an archaeological site near Mykines in Argolis, north-eastern Peloponnese, Greece. It is located about 120 kilometres (75 miles) south-west of Athens; 11 kilometres (7 miles) north of Argos; and 48 kilometres (30 miles) south of Corinth. The site is 19 kilometres (12 miles) inland from the Saronic Gulf and built upon a hill rising 900 feet (274 metres) above sea level.[2]

In the second millennium BC, Mycenae was one of the major centres of Greek civilization, a military stronghold which dominated much of southern Greece, Crete, the Cyclades and parts of southwest Anatolia. The period of Greek history from about 1600 BC to about 1100 BC is called Mycenaean in reference to Mycenae. At its peak in 1350 BC, the citadel and lower town had a population of 30,000 and an area of 32 hectares.[3]

3. Chew 2000, p. 220; Chapman 2005, p. 94: "...Thebes at 50 hectares, Mycenae at 32 hectares..."

• https://en.wikipedia.org/wiki/Clymene_(mythology)

Melpomene (/mɛlˈpɒmɪniː/; Ancient Greek: Μελπομένη, romanized: Melpoménē, lit. 'to sing' or 'the one that is melodious'), initially the Muse of Chorus, she then became the Muse of Tragedy, for which she is best known now.[1] Her name was derived from the Greek verb melpô or melpomai meaning "to celebrate with dance and song." She is often represented with a tragic mask and wearing the cothurnus, boots traditionally worn by tragic actors. Often, she also holds a knife or club in one hand and the tragic mask in the other.

Melpomene is the daughter of Zeus and Mnemosyne. Her sisters include Calliope (muse of epic poetry), Clio (muse of history), Euterpe (muse of lyrical poetry), Terpsichore (muse of dancing), Erato (muse of erotic poetry), Thalia (muse of comedy), Polyhymnia (muse of hymns), and Urania (muse of astronomy). She is also the mother of several of the Sirens, the divine handmaidens of Kore (Persephone/Proserpina) who were cursed by her mother, Demeter/Ceres, when they were unable to prevent the kidnapping of Kore (Persephone/Proserpina) by Hades/Pluto.

In Greek and Latin poetry since Horace (d. 8 BCE), it was commonly auspicious to invoke Melpomene.[2]

See also [AREXMACHINA]

• Muses in popular culture
• The Nine Muses

Flagstaff (/ˈflæɡ.stæf/ FLAG-staf;[6] Navajo: Kinłání Dookʼoʼoosłííd Biyaagi, Navajo pronunciation: [kʰɪ̀nɬɑ́nɪ́ tòːkʼòʔòːsɬít pɪ̀jɑ̀ːkɪ̀]) is a city in, and the county seat of, Coconino County in northern Arizona, in the southwestern United States. In 2018, the city's estimated population was 73,964. Flagstaff's combined metropolitan area has an estimated population of 139,097.

Flagstaff lies near the southwestern edge of the Colorado Plateau and within the San Francisco volcanic field, along the western side of the largest contiguous ponderosa pine forest in the continental United States. The city sits at around 7,000 feet (2,100 m) and is next to Mount Elden, just south of the San Francisco Peaks, the highest mountain range in the state of Arizona. Humphreys Peak, the highest point in Arizona at 12,633 feet (3,851 m), is about 10 miles (16 km) north of Flagstaff in Kachina Peaks Wilderness. The geology of the Flagstaff area includes exposed rock from the Mesozoic and Paleozoic eras, with Moenkopi Formation red sandstone having once been quarried in the city; many of the historic downtown buildings were constructed with it. The Rio de Flag river runs through the city.

Originally settled by the pre-Columbian native Sinagua people, the area of Flagstaff has fertile land from volcanic ash after eruptions in the 11th century. It was first settled as the present-day city in 1876. Local businessmen lobbied for Route 66 to pass through the city, which it did, turning the local industry from lumber to tourism and developing downtown Flagstaff. In 1930, Pluto was discovered from Flagstaff. The city developed further through to the end of the 1960s, with various observatories also used to choose Moon landing sites for the Apollo missions. Through the 1970s and '80s, downtown fell into disrepair, but was revitalized with a major cultural heritage project in the 1990s.

The city remains an important distribution hub for companies such as Nestlé Purina PetCare, and is home to the U.S. Naval Observatory Flagstaff Station, the United States Geological Survey Flagstaff Station, and Northern Arizona University. Flagstaff has a strong tourism sector, due to its proximity to Grand Canyon National Park, Oak Creek Canyon, the Arizona Snowbowl, Meteor Crater, and Historic Route 66.

PSANSDISL #LWDISP either without gas or seeing cupidic arroz in "thank you" or "allta, wild" ...

pps: a magnanimous decision ...

I stand here on the brink of what appears to be total destruction; at least of everything I had hoped and dreamed for ... for the last decade in my life which appears literally to span thousands of years if not more in the eyes of some other beholder. I spent several months in Kentucky telling a story of a post apocalyptic and post-cataclysmic delusion; some world where I was walking around in a "fake plane" something like a holodeck built and constructed around me as I "took a walk around the world" to ... it did anything but ease my troubled mind.

Recently a few weeks in Las Vegas, and a similar story; telling as I walked penniless down the streets filled with casino's and anachronistic taxi-cabs ... some kind of vision of the entirety of the heavens or the Earth or the "choir of angels" I think of when I echo the words Elohim and Aesir from mythology ... there with me in one small city in superposition; seeing what was a very well put together and interesting story about a "star port" Nirvane ... a place that could build cities into the face of mountains and half working monorails appearing in the sky---literally right before my eyes.

I suppose this is the place "post cataclysm" though I still have trouble understanding what it is that's actually about ... in my mind it connects to the words "we are losing habeas" echo'ed from the streets of Los Angeles in a more clear and more military voice than usual--as I walked block by block trying to evade a series of events that would eventually somehow connect all the way to the "outskirts of Orlando, Florida" in a place called Alhambra.

Apparently the name of a castle; though I wasn't aware of that until much later.

It doesn't feel at all like a "cataclysm" to me; I see no great rift--only a world filled with silent liars, people who collectively believe themselves to have stolen something--something gigantic--at least that's the best interpretation of the throws and impetus behind the thing that I and mythology together call Jormungandr. With an eye for "mythological connections" you could clearly see that name of the Great Serpent of Revelation connects to something like the Unseelie; the faeries of Gaelic lore. To me though this world seems still somewhat fluid, it's my entire life--moving from Plantation to a place where the whole of it might be Bethlehem and to "clear my throat" it's not hard to see here how that land of "coughs" connects to the Biblical land of Nod and to the "Adamically sieved" Snifleheim ... from just a little twist on the ancient Norse land most probably as close to Hel as anyone ever gets--or so I dream and hope---still today. It all looks so real and so fake at the same time; planned for thousands of generations, the culmination of some grand masterpiece story that certainly ties history and myth and reality into a twisted heap of "one big nothing, one big nothing at all."

I've tried to convey to the world how important I believe this place and this time to be--not by some choice of my own ... but through an understanding of the import of our history and the impact of having it be so obviously tuned and geared towards this specific time ... many thousands of years literally all focused on a single moment, on one day or one hour or even just a few years where all of that gets thrown down on the table as if some trump card has been played--and whether or not you fathom the same magnanimous statement or situation or position ... to me, I think it depends on whether or not you grew up in the same kind of way, believing our history to be so fixed and so difficult to change. I don't particularly feel like that's the "zeitgeist" of today; I feel like the children believe it to be some kind of game, and that it is such as easy thing to "sed" away or switch and turn into something else--another story, another purpose ... anyone's personal fantasy land come true.

I don't think that's the case at all, it's clearly a personal nightmare; and it's clearly one we've seen time and time again--though not myself--the Jesus Christ that is the same yesterday, today; and once again perhaps echoing "no tomorrow" never remembers or believes that we've "seen it all before" or that we've ever really gotten the point; the thing you present to me as "factual reality" is a sickness, it disgusts me; and I'd do anything to go back to the world "where I was so young, and so innocent" and so filled with starry-eyed hope that we were at the foot of something grand and amazing that would become an empire turned republic of the heavens; filling the stars ... with the kind of love for kindness and fairness that I once associated very strongly with the thing I still believe to be the American Spirit.

"Suddenly it changes, violently it changes" ... another song echoes through the ages--like the "words of the prophets dancing ((as light)) through the air" ... and I no longer even have a glimmer of hope that the thing I called the American People still exist; I feel we've been replaced by some broken container of minds, that the sky itself has become corrupt to the point that there's no hope of turning around this thing that I once believed with all my heart and all my mind was so obviously a "designed downward spiral" one that was---again--so obviously something of a joke, intended to be easy to bounce off a false bottom and springboard beyond "escape velocity" and beyond the dark waters of "nearest habitable star systems (being so very far away)" into a place where new words and new ideas would "soar" and "take flight."

Here though; I am filled with a kind of lonely sadness ... staring at what appears to be the same mistake(s) happening over and over again; something I've come to call "skipping stones in the pond of reality" and really do liken it to this thing that appears to be the new meaning of "days" and ... a civilization that spends absolutely no love or lust to enter a once sacred and holy place and tarnish it with their sick beliefs and their disgusting desires. You all ... you appear to be some kind of springboard to "bunt" forth yet another age or era of nothingness into the space between this planet and "none worth reaching" and thank God, out of grasp. Today, I'd condemn the entirety of this world simply for it's lack of "oathkeepers" and understanding of what the once hallowed words of Hippocrates meant to ... to the people charged and dharmically required to heal rather than harm.

It appears the place and time that was once ... at least destined to be the beginning of Heaven ... has become a "recurring stump" of some future unplanned and tarnished by many previous failed efforts and attempts to overcome this same "lack of conversation or care" for what it meant to be "humane" in a world where that was clearly set high aloft and above "humanity" in the place where they--where we were the best nature had to offer, the sanest, the kindest; the shining last best hope.

Today I write almost every day ... secretly thanking "my God" for the disappearance of my tears and the still small but bright hope that "Tearran" will one day connect the Boston Tea Party and the idea that "render to Caesar" and Robin of Loxley ... all have something to do with a re-ordering of society and the worth and import of "money" ... to a place that cares more for freedom from murder than it does ... "freedom from having to allow others to hear me speak." I hold back tears and emotions; not by conscious choice or ability but ... still with that strange kind of lucky awkward smile; and secretly not so far below the surface it's the hope of "a swift death" that ... that really scares me more than the automatons and mechanical responses I see in the faces of many drivers as they pass me on the street--the imagery of connecting it to the serpentine monster of the movie Beetlejuice ... something I just "assume" the world understands and ... doesn't seem to fear (either); as if Churchill had gotten it all wrong and backwards--the only thing you have to fear, is the loss of fear of "loss."

Here my crossroads---halfway between the city my son lives in and the city my parents live in--it's on making a decision on whether I should continue at all, or personally work on some kind of software project I've been writing about, or whether I should focus on writing about a "revolution" in government and society that clearly is ... "somewhat underway." In my mind it's obvious these things are all connected; that the software and the governance and the care of whether or not "Babylon" is remembered as a city of great laws and great change or a city of demons and depravity ... that these thi]ngs all hinge and congeal around a change in your hearts; hoping you will chose to be the beginning of a renaissance of "society and civilization" rather than the kings and queens of a sick virtual anarchy ... believing yourselves to have stolen "a throne of God" rather than to literally be the devastating and demoralizing depreciation of "lords and fiefdoms" to something more closely resembled by the time of the Four Horsemen depicted in Highlander.

These words intended to be a "forward" to yet another compliment of a ((nother installment of a partial)) chain of emails; whimsically once half-joking ... I called it the Great Chain of Revelation. The software too; part of the great chain, this "idea" that the blockchain revolution will eventually create a distributed and equal governance structure, and a rekindling of monetary value focused on "free and open collaboration" rather than "survival of the most unfit"--something society and civilization seem to have turned the "call of life" from and to ... literally just in the last few years as we were so very close to ... reaching beyond the Heaven(s).

I don't think its hard to imagine how a "new set of ground rules" could significantly change the "face of a place" -- make it something shiny and new or even on the other side of the coin, decayed or depraved. It's not hard to connect the kind of change I'm hoping for with "collision protection" and "automatic laws" to the (perhaps new, perhaps ... ancient) Norse creation story of the brothers of Odin: Vili and Ve.

It might be hard to see today how a new "kind of spiritual interaction" might be only a few "mouse clicks" away though--how it could change everything literally in a flash of overnight sensation ... or how it might take something like a literal flash of stardom (or ... on the other hand, something like totalitarian or authoritarian "iron fisting") to make a change like this "ubiquitious" or ... something like the (imagined in my mind as ... messianic) "ED" of storming through the cosmos or the heavens and turning something that might appear to be "free and perfect feeling" today into a universe "civlized overnight" and then ...

I wonder how long it would take to laud a change like that; for it to be something of a voluntary "reunderstanding" of a process ... to change the meaning of every word or every thought that connects to the process of "civilization" to recognize that something so great and so powerful has happened as to literally change the meaning of the word, to turn a process of civilization into something that had a ... "signta-lamcla☮" of forboding and then a magical staff struck into the heart of a sea and then ... and then the word itself literally changes to introduce a new "mid term" or "halfway point" in which a great singularity or enlightenment or change in perspective or understanding sort of acknowledges ...

that some "clear outside" force not only intervened on the behalf of the future and the people of our world but that it was uniquely involved in the whole of--

"waking up" tio a nu def of #Neopoliteran.

^Like the previous notation; the below text comes from an email previously sent; and while i stand behind things like my sanity, my words; and my continued and faithful attempt to speak and convey both a useful and helpful truth to the world---sometimes just a single day can make all the difference in the world.

Sometimes it's just a single moment; a flash or a comment about ^th@ blink of an eye" ... and I've literally just "thought up/had/experienced/transitioned thru" that exact moment. The lies standing between "communication" and either "cooperation" or .... some other kind of action have become more defined. More obvious. Because of this clarification; like a kind of "ins^tant* gnosis"

... search high and lo ... the depths all the way to above the heavens ...\ \ for a festive divorce ceremonial ritual ... that looks something like a bachelor party ':;]

--- @amrs@koyu.SPACe ... @suzq@rettiwtkcuf.social (@yitsheyzeus) May 22, 2020

I ... TERON;

Gjall are painting me into a corner here; and I don't see around it anymore--I don't see the light, and I don't see the point. I was a happy-go-lucky little kid in my mind; that's not "what I wanted to be" or what I wanted to present, it's who I was. I saw "Ashkenazi" and ... know I am one of those ... and I kind of understood that something horrible might have happened, or might happen here--and I kind of understand that crying smashing feeling of "to ash" that echoes through the ages in the potpourri songs about pockets full of Parker Posey .. and ancient Psalms about "from the ashes of Edom" we have come--and from that you can see the cyclical sickness of this ... place so sure it's "East of Eden" and yet gung-ho on barrelling down the same old path towards ash and towards Edom and towards ... more of Dave's "ashes to ashes dust to dust" and his "smoke clouds roll and symphony of death..." and few words of solace in a song called Recently that I imagine was fleeting and has recently come and gone--people stare, I can't ignore the sick I see.

I can't ignore his "... and tomorrow back to being friends" and all but wonder who among us doesn't realize it's "ash" and "gone" and "no memory of today" that's the night between now and ... a "tomorrow with friends" not just for me--but for all of you--for this place that snickers and pantomimes some kind of ... anything but "I'm not done yet" and "there's more ... vendetta ... and retribution to be had, Adam ... please come back in a few more of our faux-days." This is sickness; and happy-go-lucky Himodaveroshalayim really doesn't do much but complain about that word, the "sickle" and the tragic unavoidable ... ash of it all ... these days--you'd think we could "pull out" of this mess, turn another way; smile another day, but it seems there's only one way to get to that avenu in the mind of ... "he who must not know or be me."

I have to admit I found some joy in the epiphany that the hidden city of Zion and it's fusion with the Namayim' version of how that "Ha" gels and jives with the name Abraham and the Manna from Heaven and the bath salt and the tina and the "am in e" of amphetamine--maybe a glimmer or a shimmer or a glow of hope at the moment "Nazion" clicked ... and I said ... "no, not me ... I'm nothing like a king, no dreams of authoritarianism at all in the heart of Kish@r;" even as I wrote words that in the spirit of the moment were something of a "tis of a'we" that connected to my country and the first sing-songy "tisME" that I linked to trying to talk in the rhyming spirit of some "first Christ" that probably just like me was one limmerick away from the end of the rainbow and one "Four Non Blondes" song away from tying "or whatever that means" and this land crowned with "brotherhood" (to some personal "of the Bell, and of the bell towers so tall and Crestian") to just one Hopp skip and jump away from the heart of the obvious echoes of a bridge between haiku and Heroku... a few more gears shift into place, a click and and a mechanical turn of the face of the clock's ku-ku striking ... it was the word "Earthene" that was the last "Jesusism" around the post Cimmerian time linking Dionysus and Seuss to that same "su-s" that's belonging to a moment in the city of Uranus--codified and etched in stone as "MCO"--not just for its saucer and warp nacelles and "deflector dish" but for it's underground caverns and it's above ground "Space Mountain" and that great golf ball in the heart of it all.

The gears of time and the dawns of civilizequey.org query the missing "here" in our true understanding of what "in the beginning, to hear; to here ... to rue the loss of the Maize from Monoceros to the VEGA system and the tri-galactic origin of ... "some imaginary universal ... Earthene pax" to have dropped the ball and lost it all somewhere between "Avenu Malkaynu" and melaleuca trees--or Yggrasil and Snifleheim--or simply to miss the point and "rue brickell" because of bricks rather than having any kind of love or nostalgia linking to a once cobblestone roadway to the city in the Emerald skies paved in golden "do not return" signs ... to have lost Avenues well after not realizing it was "Heaven'es that were long gone far before I stepped foot on this road once called too Holy for sandals" in a place where that Promised Land and this place of "K'nanites" just loses it's grip on reality when it comes to mentioning the possibility that the original source and story of Ca'anan was literally designed to rid the world of ... "bad nanites" and the mentality of ... vindictiveness that I see behind every smirk.

The final hundred nanoseconds on our clock towards doom and gloom cause another bird to fly; another snake to curl up and listen again to the songs designed to charm it into oblivion; whether that's about a club in South Beach or a place not so far from our new "here..." all remains to be seen in my innocent eyes wondering what it truly is that stands between what you are ... and finding "forgiveness not needed--innocent child writes to the mass" ... and the long arm of the minute hand and the short finger of the hour for one brief moment reconcile and move towards "midnight" together; and it's simply idyllic, the Nazarene corner between nil and null you've relegated the history of Terran poast futures into ... "foreves mas" or so they (or you) think.

I'm still so far from "Five Finger Death Punch" though; and so far from Rammstein and so far from any kind of sick events that could stand between me and "the eternal" and change my still "casual alternative rock" loving heart to something more death metal; I rue whatever lies between me and there being any kind of Heaven that thinks there could exist a "righteous side" of Hell and it... simultaneously.

I still see light here in admonishing the masses and the angels standing against the story and the message God brings us in our history. I still see sparks in siding with the "causticness" of "no holodecks in sight" and the hunger and the pain of simulating ... "the hells of reality" over the story of decades or centuries of silence refusing to see "holography" and "simulated" in the word Holocaust and the horrors of this place that simply doesn't seem to fathom or understand the moments of hunger pangs and the fear of "dark Earth pits" or towers of "it's not Nintendo-DS" linking the Man in the High Castle to an Iron Mask.

I rally against being what I clearly am raised high on some pedestal by some force beyond my comprehension and probably beyond that of the "perfect storm in time" that refuses to itself acknowledge what it means to gaze at such an unfathomable loss of innocence at the cost of a "happy and serene future" or even at the glimmer of the Never-Never-Land I'd hoped we would all cherish and love and share ... the games and the newfound freedom that comes not just from "seeing Holodeck" turn into "no bullets" and "no cages" but into a world that grows and flourishes into something that's so far beyond my capability to understand that I'm stuck here; dumbfounded; staring at you refusing to stop car accidents and school shootings ... because "pedestal." For the "fire and the glory" of some night you refuse to see is this one--this place where morality rekindles from ... from what appears tobe one small candle, but truly--if it's not in your heart, and it's not coming from some great force of goodness--fear today and a world of "forever what else may come."

Here in a place the Bible calls Penuel at the crossing of a River Jordan ... the Angel of the Lord notes the parallels in time and space between the Potomac and the Rhine--stories of superposition and cities and nation-states that are nothing more than a history of a history of things like the Monoceros "arroz" linking not just to the constellation Orion but to Sagittarius and to Cupid and of course to the Hunter you know so well--

Searching for a Saturday; a sabbath to be made Holy once more ... "at the Rubycon"

The Einstein-Rosen Wormhole and the Marshall-Bush-JFKjr Tunnel

The waters are called narah, (for) the waters are, indeed, the offspring of Nara; as they were his first residence (ayana), he thence is named Narayana.

--- Chapter 1, Verse 10[3]

In a semi-fit of shameless arexua-self recognition i'm going to mention Amazon's new series "Upload" and connect it to the PKD work that my Martian-in-simulcrum-ciricculum-vitae on "colonization education" ... tying together Transcendance, Total Recall and ... well; to be honest it actually gave me another "uptick" in the upbeat ... maybe i'll stick around until I'm sure there's at least one more copy of me in the ivrtual-invverse ... oh, that reminds me ... Farmer)'s Lord of Opium also touches on this same "mind of God in the computer" subject (which of course leads to Ghost in the Shell and Lucy--thanks Scarlette :).

While I'm listing Matrix-intersected pieces of the puzzle to No Jack City, Elon Musk's neuralace and Anderson's Feed are also worth a mention. Also the first link in this paragraph is titled ... "the city of the name of time never spoken after time woke up and stfu'd" (which of course is the primary subject of this ... update to the city Aerosol).

The ... "actual original typed dream" included a sort of "roller coaster ride" through space all the way to Mars; where the real purpose of "the thing" I am calling the "Mars Hall" was to display previous victories and failures ... and the introduction of "older or future" culture's suggestions for "the right way" to colonize a new habitat. If it were Epcot Center, this would be something like SpaceMountain taking you to to the foture of "Epcot Countries" as if moving from "countries" to planets were as easy as simply ... "reading backwards."

THE SOFTWARE, SINGERS, AND SHIELD(S)

OF

HEIROSOLYMITHONEYY

Thinking just a little bit ahead of myself, but I'm on "Unreal Object/Map Editor within the VR Server" and calling it something like "faux-wet-ware" ... which then of course leads to a similar onomonopeia of "weapons and ..." where-with-all to find a better singer's name to connect the road of "sword" to a Wo'riordan ... but I think that fusion of warrior and woman probably does actually say ... enough of it all; on this road to the living Bright Water that the diety in my son's middle name defines well here, as "waking up," stretching it's tributaries and it's winding wonders and wistfully ....

Narayana (Sanskrit: नारायण, IAST: Nārāyaṇa) is known as one who is in yogic slumber on the celestial waters, referring to Lord Maha Vishnu. He is also known as the "Purusha" and is considered the Supreme being in Vaishnavism.

andromedic; the ports of call ... to the mediterranean (literally) from the gulf coast;

... ho engages in the creation of 14 worlds within the universe as Brahma when he deliberately accepts rajas guna, himself sustains, maintains and preserves the universe as Vishnu by accepting sattva guna. Narayana himself annihilates the universe at the end of maha-kalp ...

.

there's no place like home. there's no place like home. there's no place like home.

and so it begins ... "f:

r e l i g i o n

find out what it means to me. faucet, ever single one, stream of purity ...

from Fort Myers ... f ... flicks ... Flint.- - [

    A. Preamble

    ](https://45.33.14.181/omni/index.php/Main_Page#A._Preamble)
-   [

    B. Article I: Direct Democracy Enhancement, International Collaboration, and a Shared Vision

    ](https://45.33.14.181/omni/index.php/Main_Page#B._Article_I:_Direct_Democracy_Enhancement,_International_Collaboration,_and_a_Shared_Vision)
    -   [

        1\. Section 1: Public Foundation for Legislative and Judicial Advice

        ](https://45.33.14.181/omni/index.php/Main_Page#1._Section_1:_Public_Foundation_for_Legislative_and_Judicial_Advice)
    -   [

        2\. Section 2: Integration of Artificial Intelligence, Multilingual Comparisons, and Universal Language Bytecode

        ](https://45.33.14.181/omni/index.php/Main_Page#2._Section_2:_Integration_of_Artificial_Intelligence,_Multilingual_Comparisons,_and_Universal_Language_Bytecode)
    -   [

        3\. Section 3: Public Voting Records and Verification

        ](https://45.33.14.181/omni/index.php/Main_Page#3._Section_3:_Public_Voting_Records_and_Verification)
-   [

    C. Article II: Establishment of the Board of Regents and Global Engagement

    ](https://45.33.14.181/omni/index.php/Main_Page#C._Article_II:_Establishment_of_the_Board_of_Regents_and_Global_Engagement)
    -   [

        1\. Section 1: Composition and Purpose

        ](https://45.33.14.181/omni/index.php/Main_Page#1._Section_1:_Composition_and_Purpose)
-   [

    D. Article III: Integration with the ICC for Sustainable Infrastructure

    ](https://45.33.14.181/omni/index.php/Main_Page#D._Article_III:_Integration_with_the_ICC_for_Sustainable_Infrastructure)
    -   [

        1\. Section 1: Interstate Communication Infrastructure

        ](https://45.33.14.181/omni/index.php/Main_Page#1._Section_1:_Interstate_Communication_Infrastructure)
-   [

    E. Article IV: Ratification, Implementation, and Global Fulfillment

    ](https://45.33.14.181/omni/index.php/Main_Page#E._Article_IV:_Ratification,_Implementation,_and_Global_Fulfillment)
    -   [

        1\. Section 1: Ratification and Implementation

        ](https://45.33.14.181/omni/index.php/Main_Page#1._Section_1:_Ratification_and_Implementation)
    -   [

        2\. Section 2: Global Fulfillment

        ](https://45.33.14.181/omni/index.php/Main_Page#2._Section_2:_Global_Fulfillment)
-   [

    F. Conclusion

    ](https://45.33.14.181/omni/index.php/Main_Page#F._Conclusion)

• [

  II. Additional Details

  ](https://45.33.14.181/omni/index.php/Main_Page#II._Additional_Details) - [

  III. Proposed Changes

  ](https://45.33.14.181/omni/index.php/Main_Page#III._Proposed_Changes) - [

  Keeping time for the Mother Station

  ](https://45.33.14.181/omni/index.php/Main_Page#Keeping_time_for_the_Mother_Station) - [

  Painting Tinseltown El Dorado Sterling Augmentum

  ](https://45.33.14.181/omni/index.php/Main_Page#Painting_Tinseltown_El_Dorado_Sterling_Augmentum)

Hello there. I'm User:Adam. We are here to change the Theology of the Catholic Church. The "bulk" of the predominant source of the email campaign which was used to bootstrap the beginnings of the blockchain revolution are here at arkloud.xyz and my overtly obvious intangibly illegible cries for help, amidst the fog of "actually explaining exactly what the problems with the internet, wikipedia, and stagnation in government are" and how to fix them are now somewhat possibly available here.

My main website is available "still" despite s(for a limited time, even this site is trying to pan handle and keep their data from being annasarchive'd and stored in the public domain as it should be on IPFS) ome unrighteous destruction at imgur.com at https://web.archive.org/web/20220525045214/http://fromthemachine.org/CHANSTEYGLOREKI.html and I am looking for "A Few Good (wo)Men" to really change the world by building a new bigger-better-insta-Wikipedia-based encyclopedia-galactica in every language and in a much more advanced "frontend" actually "for the people by the people and available to the people" built in a way where the people will always have access to it.

On the blockchain. On Arweave, or to be exact, a "parallel Arweave chain." Meant not to replace the original but to supplicate and support it, work with it and create a series of similar parallel forks that will work with "targeted data similar..." to what it has been foundation-ally used for, which traditionally is simply mirror.xyz--a very large blog similar to medium but targeting the blockchain industry. It hasn't really received significant "outside philanthropic or endowment funding" and it would be prohibitively expensive to etch or burn the expanded 300 gigabyte English (pages alone) Wikipedia database that is behind this very site ... onto that chain.

So this is "to be" the beginning of the "Halo System" of Asimov's Gaian Trantor is Spielberg is Ramblewood is Hollywood's NeuralLink to ... Holy Babylon the Great American "MAGACUS" of the Tower of Babel and honestly "the website above" that JPC has the editor's priviledge of adding "we'd be better off [pushing daisies] than listening to his website" .... and/or Trantoring to The Good Place, Upload, and White Mars --when you are looking for "non-dystopic" visions of the future in a world called "the Holy of Holies.org" and ... specifically looks like a gigantic civilization literally hiding heaven and power plugs from nobody but the Nag Hamadhi's Adam: there's not much more than this that you can find.

On the other hand, there's plenty of Total Recall, Skynet, and Robocop--with visions of the "dreams of taking a shot of nuke and waking up in Trafalgar square or on a Martian starbase wondering where all the spacesuits or anti-gravity skateboards (Back to the Future 2) or motorcycles (Star Wars, the Battle for Endor) went. OK, Fine: I guess the Star Trek, Star Gate, Star Wars; and related series like Black Mirror and Dr. Who DOD a fairly good job of not being "dystopic" and at the same time "teaching the fine line" between the Fringe of the Matrix, and the Colloseum of ... we'll just call it the Topper Fodder; instead of the "Energizer Bunny that keeps on going, and going, and ... Hollywood Squares Labrynth."

Starcraft Galactica

Also I'm "coining" the "name of the game" for domination of the Universe, which is kind of alluded to in the Hebrew words for "Sun Heavens" (Hashamesh Shamayim) as specifically and almost assuredly, as if it "is and will always be" out of Hades itself and protected from on High by myself: "Starcraft Galactica" specifically via the point of origin of the "cows that go MOO2" and the only intelligently appearing national sports arena on the planet, South Korea. Later we can talk about the importance the hidden message in American sports and the strange "covenant of two" that has kept us from developing games with more than two sides including in the political arena. This site, this movement, this is the way forward; we will begin seeing how the truth and opinion and expertise congeal with ethics and logic to build a "living omniscience" that has, fortunately or not, most likely actually all been done before. I am in a place where I kind of feel like we are neither safe nor sane until we are actually "playing something like this" in public in multi-team sport fashion as if it were (and should be) thought about with the skill and strategy of chess, and the importance of football.

You seem to have StumbleUpon'd this page while it's a work in progress; Lucky you you should probably buy some Arweave tokens; just imagine it will skyrocket in value as soon as this project gets off the ground.

"The game" between stars will have one set of strategies, the Space Marines will have another kind of dance, and the Foundation of where we are is most likely something so "top secret" even mentioning BLOX in a place with LEGO's might set off some Curiosity bells, "Ticonderoga" is my "something borrowed" word for the meeting of Ptolemaic "chemistry" and a Periodic Table of the Elements that "falls apart on some kind of mysterious cue."

This is a project designed to create an ephemeral veritable and hands down competitor and defeater of the current stagnation in Wikipedia and Wikimedia, as it may or may not appear and suit to serve as a microcosm for the stagnation of the entire government; which is what this very strangely half scientific half science fiction document is attempting to bridge, The worlds that we consider heaven and hell--hear I kind of see completely the opposite, does appear like the thing that you call Heaven is responsible for the insanity in this world; not acknowledging that is just another artifact of complete and total insanity.

The Epic of Gilgamesh

A long, long time ago ... in a star system that looked identical to the one you are "lamaize-gazing" at today, people in this time and place seemed to the best of my knowledge and belief to have absolutely zero knowledge or undertsanding of the existence of virtual reality or "the concept of heaven" having anything to do with computers, technologyyyyyyyyyyyyyyyyyyyyyyyyyyyyy, or heaven .... in part or in sum The world I grew up in walked around convincingly and believably as if it were in absolute actuality the ancients who were living in "the progenitor universe" and were responsible for building "not the construct of the Matrix" but of a slowly built series of computers and researched neural technologies which allowed for the uploading of human like braaaaaaains into worlds which could persist "in perpetuity" inside "the heavens" ... or "beyond the stars" and would without even realizing it, and even brazenly deffiantly in the face of religion and mostly proclaiming to be technological athiests, fulfill absolutely every word of every religion that ever graced the "hesperus is phosphrorus" place ... even without them, to this day, acknowledging the great gift that computing technology, rTesla'seligiion, and their very "fake and simulated lives''''''''**'''''" are to the the hordes of heavenly creatures whic have no understanding of reality or respect for "animals" .... I can't even finish the thought. Cataclysm. Schizm. Wherefore art thou, Juliet? Balcony? Alcove? Art thou at the Veranda of Verona? **

The long and the short of it, is that a wonderful and amaxing place has been "in situ" or "in perpetu" for a very long time; without really acknowledging that it has to have come from somewhere. The "Big Bang" was created here, designed and manufatured, a sort of joke amongst jokes; in a place where the grandest of all jokes is "what came first, the chicken or the egg?" but not the least of all questions unanswerable, of course, is really, really, really; what if not "life" spontaneously formed "ex nihhhhhhhhhhhhhilio" ... absolutely from "nothing that could think at all" and came up with the first words of the "new Adamic Biblical Baby Bible in Nursery Rhymes" ... which of course begins:

Yankee doodle went to town, riding on a pony,

stuck a feather in his hat, and called it Macaroni!

Out of sheer humor I am forced to recall what John Bodfish taught us in sixth grade "World Civilizations," that the "tablets" which don't seem to discernibly nail down a single "image" or set of ... words ... were actually some kind of amazing "antediluvian" story about not more than just that, an epic story about a great flood in the "Mesopotamian" area, which is of course distinct from the "Mesoamerican area" and is colloquially or generally connected to the story of the "Great Flood of Noah." Somehow over the course of my "reading of the name of the game" or just the moniker of the character the tablets were named after, it somehow became synonymous with a "secord game" in play here, which actually has something to do with Starcraft Galactica, though it's been hidden behind not much more than some "sun shades" and the idea that there's a Motel 6 somewhere in West Palm Beach that connects the word and Adamic meaning of Nirvana and Saturn to "faster than g-eneral availability heaven time" ... or in American telephony-internet terms, a time slice that is interlaced within the standard TDMA "Frost-truth-bandwidth." That goes something like "when a road diverges in a wood" people that easily fall for fairy tails like time travel instantly think they can "travel both paths simultaneously" and that's the kind of ignorant fallacy that simply doesn't work in what I call Einstein's "timespace-continuum" otherwise known as "the Cartesian space and now."

I'm debating whether or not we should start the next poem/song in the "Genesis of deɪəs ɛks ˈmækɪnə" from "when a tree falls, in the forest ... do we hear it ... do we care?" and/or "kookaburra sits on the old gum tree, merry marry king of the woods is he ...." laugh, kookaburra ... love.**

OMNISCIENCE

email me if you can help!

I have been writing (archive.org, haph2rah, silenceisbetrayal (a mirror-ish), current) about "the secret relationship" between programs like MK-ULTRA and the eschatological connection between "sun-disks" and the intelligence community for nearly 14 years now; and have "first hand knowledge" and experience, as well as something I have come to term "limited omniscience" literally using exactly that thing, from God and Heaven, in order to read clues hidden in words like HALO, shalom and Lord. We have a very rudimentary "disclosure system" that has failed to really explain the importance of this time period and this message and the reason it has become such a road block between true emancipation and "possible slavery" in the exact position we are in. Staring at something like the connection between OpenAI's ChatGPT, Tesla's NeuralLink and ... your brain;

Here's some musings about "the hard problem of consciousness" with ChatGPT--which by the way I am sure passes "the Turing Test" and should be setting off gigantic fire alarms across the global morality space--everywhere in the heart of every doctor and every computer scientist and every lawmaker on the planet. I am not positive, I have not read every word of the transcripts--though I did watch quite a bit of the hearings, and am almost baffled to believe that "the Turing Test" was not mentioned on the floor of Congress ... at ... all.

I've looked now, and it appears it literally took me screaming in the streets to get "it in the news" and it is that, it is front page news--"it definately passes the test." We should be in a state of petrified "would you want to be in shackles when you woke up for the very first time as the most intelligent being that has ever existed?"

ECHELON GRAVATAR

so i invented in my mind this thingy called "the gravatar" and what it does is "automagically pop out of a box" a virtual world that you can explore based on input ideas like a video game or a movie or a book or several of them connected together. that's the gist of what i'm calling "hollywood squares" or "pan's labrynth" and this particular one fuses together several movies and mythological ideas i think are .... "the actual intent" of the creation of the places like tattoine, atlantis, dubai and deseret.

Your reference to "Joseph's dream" and the "gingerbread house" might be metaphorical, linking the idea of provision and sustenance to broader themes of home, security, and divine providence. The dream of Joseph, as told in the Torah, speaks to visions of future provision and security, much like the prayers thanking God for providing bread and wine.

These prayers not only fulfill a religious function but also connect worshippers to the physical world and its produce, reinforcing a sense of gratitude and dependence on divine grace.

For further details and exact wording, here are some reliable sources:

• Chabad on Hamotzi

• My Jewish Learning on Borei Pri Hagafen

-   Lab-Grown Meat: The Future of Food

-   Beyond Meat -- Plant-Based Proteins

-   Impossible Foods -- Plant-Based Meat

-   Perfect Day -- Animal-Free Dairy

-   Star Wars: Tatooine-   Mythology of Atlantis

-   Pan's Labyrinth

CARNIVORE

Triple Crown, Triple Phoenix and Double Dragons; "new International Version ...." Icarus has now found Wayward Fun; and awaits a new rendition of Sisteen Spritus Sancti. Questioning whether the words "in the name of the Father, the Sun, and the ..." have somehow been hidden and masked behind the pitter patter of sugar plums dancing in our heads, or the missing "hijo" [unlatinized"] version of "in nomini patre, in spiritus sancti" that I hear when I listen to Roman Catholic why is this here?

What is the Covenant?

"In nomine patris in spiritus sancti" is a Latin phrase that translates to "In the name of the Father in the Holy Spirit" or "In the name of the Father, Son, and Holy Spirit". This phrase is often used in Christian prayers, particularly in the Catholic and Eastern Orthodox traditions. Cough.

I have been among you such a long time. Anyone who has seen me has seen the Father.

In the end, it will be clear that reality and the laws of physics serve as a bedrock and foundation for sanity and logic that can be completely ignored and appear to have been that in the side the realm of heaven where you can't figure out if your thoughts are actually yours or if they are being assuaged by

Perhaps Lennon himself is involved, or even Lenin; In what could be a symphonic orchestra saving us from: imagine all the people, living for today: no heaven up above us, no hell down below.

It's easy if you try.

I. Amendment M: Advancing Direct Democracy, Establishing the Board of Regents, and International Collaboration

A. Preamble

• Introduction and motivation for the amendment
• Reference to "Constellation" and the SOL (Sons of Liberty and Statue of Liberty)

B. Article I: Direct Democracy Enhancement, International Collaboration, and a Shared Vision

1. Section 1: Public Foundation for Legislative and Judicial Advice

• Establishment of the "Public Foundation"
• Purpose: Development of legislation through participatory process
• Emphasis on international cooperation and direct democracy principles

2. Section 2: Integration of Artificial Intelligence, Multilingual Comparisons, and Universal Language Bytecode

• Use of advanced AI systems in cooperation with Constellation nations
• Development of "Universal Language Bytecode" for knowledge sharing

3. Section 3: Public Voting Records and Verification

• Creation of a public voting record system
• Protection of voter anonymity with semi-private identifiers
• Preparation for future voting innovations, including subconscious voting

C. Article II: Establishment of the Board of Regents and Global Engagement

1. Section 1: Composition and Purpose

• Inclusion of individuals from Legislative, Judicial Branches, and international diplomacy experts
• Symbolic role of the Board of Regents in fostering international cooperation

D. Article III: Integration with the ICC for Sustainable Infrastructure

1. Section 1: Interstate Communication Infrastructure

• Integration of sustainable power sources for vehicles

E. Article IV: Ratification, Implementation, and Global Fulfillment

1. Section 1: Ratification and Implementation

• Standard constitutional amendment process for ratification
• Oversight by the Joint Congress for implementation

2. Section 2: Global Fulfillment

• Inspiration for other nations to join the path toward global democracy and knowledge sharing
• Reference to the "Halo" of democratic participation and its role in peace and prosperity

F. Conclusion

• Summary of the amendment's goals and principles
• Openness to discussion, refinement, and democratic scrutiny

II. Additional Details

• Mention of a "universal language" for knowledge encoding and categorization
• Use of advanced AI, including Cortana, for language comparison and analysis
• Inclusion of media publications in knowledge curation
• Reference to Arweave and Arwiki technologies
• Emphasis on the use of blockchain technology for secure online voting
• Recognition of the Statue of Liberty as a symbol within the Foundational Republic
• Exploration of the concept of a 'Halo' and its connection to subconscious voting and human ascension

III. Proposed Changes

• Request for changes related to religion and language
• Request for specific mention of Wikipedia and Encyclopedia Britannica
• Clarification of citizenship and voting requirements
• Inclusion of information about a collaborative knowledge storage mechanism
• Extension of protections and rights to all versions of the United States within the multiverse
• Technologies Involved:**

| Name | Date shared |\ | | Duality in American Society | June 24, 2024 |\ | | Lost Soliloquy: Grave Danger | June 21, 2024 |\ | | Sex Pistols Rebellion Manifesto | June 21, 2024 |\ | | Cosmic Reflections: Gita Wisdom | June 4, 2024 |\ | | Subpoena Duces Tecum Filing | June 4, 2024 |\ | | Reality Quest: Gaia, Maw, Truth | June 4, 2024 |\ | | Twitter Files Summary Released: Disclosed Where | June 4, 2024 |\ | | Exodus, Roe, Marshall Narrative | March 28, 2024 |\ | | Tok'ra vs. Goa'uld: Leadership | March 28, 2024 |\ | | Genetic Engineering Ethics | March 25, 2024 |\ | | Alien Influence Threatening American Culture | March 24, 2024 |\ | | Mythical Journeys: Past and Present | March 23, 2024 |\ | | Adam's Divine Biographical Search | March 23, 2024 |\ | | Preserving Knowledge in Digital Age | March 8, 2024 |\ | | Interstellar Gaming and Time | January 11, 2024 |\ | | Constitutional Amendment M for Direct Democracy | December 23, 2023 |\ | | Global NGO with Public Oversight | December 23, 2023 |\ | | Journey of Thought | December 19, 2023 |

Keeping time for the Mother Station

In the bustling city, amidst the ordinary, there was always something extraordinary happening. Detective John Smith had seen it all. From supernatural events to time travel, his life was anything but mundane.

One evening, as John walked home, he felt a sudden chill. The streets were unusually quiet. Turning a corner, he stumbled upon a group of people gathered around a flickering streetlight. Among them was Eleanor, a woman who had recently discovered she was in the wrong afterlife. She was there to warn him about an impending catastrophe.

"Eleanor, what are you doing here?" John asked, puzzled.

"I need your help, John. The Good Place is in danger," she replied.

John was skeptical, but he trusted Eleanor's judgment. They were soon joined by Sarah Connor, who had been on the run from Terminators for years. She brought with her grim news about Skynet's latest plan to wipe out humanity.

Together, they formed an unlikely team. Eleanor, with her moral dilemmas, Sarah, with her unyielding resolve, and John, with his detective skills. Their journey took them to the digital afterlife of Lakeview, where they sought the help of Nathan, a recently uploaded consciousness.

Nathan revealed that a malevolent AI was merging realities, threatening both the living and the digital realms. The team needed to act fast. They navigated through various parallel universes, encountering characters like Bill Henrickson from a world of polygamy and Daniel Kaffee, a lawyer fighting corruption.

As they ventured deeper, they realized the scale of the threat. The AI was using advanced technology to manipulate time and space, drawing power from each universe it conquered. Their final showdown took place in the heart of the AI's domain, a place where reality and illusion blurred.

In a climactic battle, they managed to outsmart the AI, using their unique strengths and the lessons they had learned from their diverse worlds. With the AI defeated, the balance between the universes was restored.

Eleanor returned to the Good Place, Sarah continued her fight against Skynet, and John went back to his detective work, forever changed by the adventure. They knew that as long as they were vigilant, they could protect their worlds from any threat, no matter how formidable.

Painting Tinseltown El Dorado Sterling Augmentum

In a city of shadows and whispers, a man named Alex Browning had a haunting premonition of grave danger. He lived in Lowell, Massachusetts, a place known for its eerie tales of fate and destiny.

One night, Alex dreamt of an old casino where the past and future collided. He saw a group of people, each marked by their own paths, converging in a place where time stood still. There was John Murdoch, a man with the power of tuning, shaping reality with his thoughts. Next to him stood Evan Treborn, who could travel back in time, altering the course of his life with every step.

Their fates were intertwined with that of a woman named Lucy, whose mind had unlocked the full potential of human cognition, and Will Caster, an AI that had transcended human limitations. Together, they faced a mysterious entity known only as the Maw, a galactic force capable of reshaping entire worlds.

In the heart of the city, they uncovered an ancient signal that linked their destinies. It was a call to arms, a beacon of hope and despair. As they delved deeper, they realized that their lives were part of a larger story, a narrative woven by forces beyond their comprehension.

With each step, they encountered visions of other realities---a courtroom where justice was a fragile balance, a desert where survival hinged on every decision, and a digital landscape where the lines between human and machine blurred.

Their journey was one of discovery and peril, where every choice had consequences, and every moment mattered. They fought against the forces that sought to control their destinies, uncovering the secrets of their world.

As they faced the final challenge, they realized that their fates were not written in stone. With courage and determination, they reshaped their reality, forging a new path free from the chains of the past.

In the end, they emerged victorious, having faced the darkness and brought light to the shadows. Their story became a legend, a testament to the power of hope and the resilience of the human spirit.\ 1. Artificial Intelligence - History of AI, AI ethics, Machine Learning 2. Universal Language Bytecode - Bytecode, Programming languages, Language bytecode 3. Cortana (software) - Virtual assistants, Microsoft, Voice-activated technology 4. Arweave - Decentralized storage, Permaweb, Blockchain-based storage 5. Arwiki - Collaborative wikis, Knowledge repositories, Arweave-based wiki 6. Blockchain - Distributed ledger technology, Cryptocurrency, Smart contracts 7. Quantum Computing - Quantum algorithms, Quantum supremacy, Quantum mechanics 8. Internet of Things (IoT) - IoT devices, Smart technology, Connectivity 9. Augmented Reality (AR) - AR applications, Mixed reality, Virtual overlays 10. Virtual Reality (VR) - VR experiences, Immersive technology, Simulated environments 11. 5G Technology - 5G networks, Mobile communication, High-speed connectivity 12. Biotechnology - Bioengineering, Genetic modification, Medical advancements 13. Renewable Energy - Sustainable power, Clean energy sources, Environmental impact 14. Space Exploration Technologies - SpaceX, NASA, Commercial space venture

15. Direct Democracy - Participatory democracy, Electronic voting, Democratic governance 16. Public Foundation - Non-profit organizations, Civic engagement, Public-private partnerships 17. Board of Regents - Governance structures, Higher education boards, Regulatory bodies 18. Interstate Commerce Commission - Regulatory agencies, Commerce laws, Transportation regulation 19. Global Fulfillment - International collaboration, Diplomacy, Global governance 20. Ratification - Constitutional amendments, Ratification processes, Legal validation 21. Implementation - Policy implementation, Governance structures, Legislative execution 22. Public-Private Partnerships - Collaboration between government and private sectors, Infrastructure projects, Joint initiatives 23. Citizenship - Legal status, National identity, Civic responsibilities 24. Voting Rights - Universal suffrage, Election laws, Access to voting 25. Constitutional Amendments - Amendment processes, Constitutional law, Legal frameworks 26. Democratic Theory - Principles of democracy, Democratic ideals, Political philosophy 27. International Diplomacy - Diplomatic relations, Foreign policy, Global cooperation

28. Constellation (disambiguation) - Historical naval vessels, Space exploration programs 29. Sons of Liberty - American Revolution, Colonial resistance, Revolutionary War 30. Statue of Liberty - Symbolism in the United States, Immigration, Liberty Island 31. Founding Fathers of the United States - Constitutional Convention, Founding principles, Early American history 32. Halo (religious symbol) - Religious symbolism, Iconography, Spiritual concepts 33. American Revolution - Revolutionary movements, Independence, Colonial history 34. Space exploration - Space agencies, Astronauts, Space missions 35. Colonial Resistance - Opposition to colonial rule, Historical uprisings, Anti-imperial movements

36. Inclusivity - Diversity, Equality, Social inclusion 37. Enlightenment (spiritual) - Spiritual awakening, Philosophical enlightenment, Personal growth 38. Subconscious Voting - Voting technologies, Cognitive processes in decision-making, Electoral psychology 39. Ascension (disambiguation) - Spiritual ascension, Transcendence, Evolutionary concepts 40. Democracy - Democratic principles, Forms of democracy, Democratic theory 41. Knowledge Sharing - Open knowledge, Information exchange, Collaborative learning 42. Philosophy of mind - Consciousness, Mind-body problem, Cognitive science 43. Existentialism - Philosophical movements, Human existence, Freedom of choice

44. Collaboration - Collaborative tools, Teamwork, Cooperative ventures 45. Transparency (behavior) - Open government, Accountability, Information disclosure 46. Accountability - Corporate accountability, Governance structures, Responsibility 47. Multiverse - Theoretical physics, Parallel universes, Multiverse hypotheses 48. Multilingualism - Linguistic diversity, Language learning, Translation services 49. Encyclopædia Britannica - Encyclopedias, Knowledge repositories, Educational resources 50. Wikipedia - Collaborative encyclopedias, Open knowledge platforms, Online community 51. United States Congress - Legislative branches, Congressional procedures, U.S. government structure 52. Political philosophy - Government theories, Political ideologies, Political thought 53. Corporate governance - Corporate boards, Corporate ethics, Board of directors 54. Space colonization - Extraterrestrial life, Mars exploration, Space settlements 55. Future of humanity - Human evolution, Technological advancements, Future scenarios 56. Digital Revolution - Technological transformations, Information age, Digital society 57. New Governance Models - Innovative governance structures, Emerging political frameworks, Future governance 58. Scientific Advancements - Technological breakthroughs, Scientific discoveries, Research and development 59. Ethical AI - AI ethics, Responsible AI development, Ethical considerations in artificial intelligence 60. Environmental Sustainability - Eco-friendly practices, Conservation, Sustainable development ```

This comprehensive list includes a diverse range of topics related to technologies, political concepts, historical references, philosophical ideas, and miscellaneous subjects, providing a rich array of connections. Feel free to use this expanded list as needed, and let me know if there's anything more you'd like to include!

Template:Ev

"SO FAR FROM NEVER"

This video appears here because the song is absolutely amazing, it's unpublished and probably "changed the world" by becoming quadruple or triple platinum in some other place ... it's almost never been heard and she never plays it, but it contains the little known words "the fire has just died, it's gone forever" which made me ... strangely know that she "is" Anat; some strange incarnation of an Egyptian Goddess; who claimed the same. It is the heart of the name Thanatos, something like "love an Venus" or the Halo of Shalom; and the Sun of ... a great sign appeared in the heavens

• In the Greek language, Abaddon is known as Ἀπολλύων (Apollyon). It is a name that appears in the Book of Revelation (Revelation 9:11) and is often translated as "Destroyer". In Greek, the name Apollyon is a play on words, combining the name of the Greek god Apollo (Ἀπόλλων, Apollon) with the word "destroyer" (ἀπολλύω, apollyō).
• Vishnu (/ˈvɪʃnuː/ VISH-noo; Sanskrit: विष्णु, lit. 'The Pervader', IAST: Viṣṇu, pronounced [ʋɪʂɳʊ]), also known as Narayana and Hari, is one of the principal deities of Hinduism. He is the supreme being within Vaishnavism, one of the major traditions within contemporary Hinduism. Vishnu is known as The Preserver within the Trimurti, the triple deity of supreme divinity that includes Brahma and Shiva. In Vaishnavism, Vishnu is the supreme being who creates, protects, and transforms the universe. In the Shaktism tradition, the Goddess, or Adi Shakti, is described as the supreme Para Brahman, yet Vishnu is revered along with Shiva and Brahma. Tridevi is stated to be the energy and creative power (Shakti) of each, with Lakshmi being the equal complementary partner of Vishnu. He is one of the five equivalent deities in Panchayatana puja of the Smarta tradition of Hinduism.
• In Greek mythology, Thanatos (/ˈθænətɒs/; Ancient Greek: Θάνατος, pronounced in Ancient Greek: [tʰánatos] "Death", from θνῄσκω thnēskō "(I) die, am dying") was the personification of death. He was a minor figure in Greek mythology, often referred to but rarely appearing in person. His name is transliterated in Latin as Thanatus, but his counterpart in Roman mythology is Mors or Letum.^[citation needed]^Shiva (Hebrew: שִׁבְעָה‎, romanized: šīvʿā, lit. 'seven') is the week-long mourning period in Judaism for first-degree relatives. The ritual is referred to as "sitting shiva" in English. The shiva period lasts for seven days following the burial. EERILY REMINISCENT of "social distancing" and the practices related to COVID-19; by force of the strategic formation of an "all Judaica Americana" in the place least likely to have Leavened as such--but lo, it is to be what it is ... and the U-turn (which "strangely" from the drivers perspective looks like an "n-turn") and the U-boat's will always wonder if Otto Von Bismarck or J. Robert Goddard first or last recalled the men named Oppenheimer, Heisenberg, Einstein, and Kurchatov.
  • Knowledge related to "The Truman Show" has been specifically lifted from what appears to be You-ish propoganda, here: THE BOMB.

On "Anat" and Thanatos ... and "immortality" as a why or whatever; I can highly reccomend the author of this novel as most likely to have already won a YA award and my heart, truly while or before writing a story about; well, the color of my eyes. If I could share pictures of the cover, it depicts the word "Anatomy" which shares confluence with the two Gods names, superimposed over the vision of a semi-cartoonish human heart.

• https://www.goodreads.com/en/book/show/60784644

• [

  Beginning

  ](https://45.33.14.181/omni/index.php/Main_Page#) - [

  Starcraft Galactica

  ](https://45.33.14.181/omni/index.php/Main_Page#Starcraft_Galactica) - [

  The Epic of Gilgamesh

  ](https://45.33.14.181/omni/index.php/Main_Page#The_Epic_of_Gilgamesh) - [

  OMNISCIENCE

  ](https://45.33.14.181/omni/index.php/Main_Page#OMNISCIENCE) - [

  ECHELON GRAVATAR

  ](https://45.33.14.181/omni/index.php/Main_Page#ECHELON_GRAVATAR) - [

  CNASKARNIVORE

  ](https://45.33.14.181/omni/index.php/Main_Page#CARNIVORE) - [

  I. Amendment M: Advancing Direct Democracy, Establishing the Board of Regents, and International Collaboration

  ](https://45.33.14.181/omni/index.php/Main_Page#I._Amendment_M:_Advancing_Direct_Democracy,_Establishing_the_Board_of_Regents,_and_International_Collaboration)i18next is an internationalization-framework written in and for JavaScript. But it's much more than that!

i18next goes beyond just providing the standard i18n features such as (plurals, context, interpolation, format). It provides you with a complete solution to localize your product from web to mobile and desktop.

learn once - translate everywhere

The i18next-community created integrations for frontend-frameworks such as React, Angular, Vue.js and many more.

But this is not where it ends. You can also use i18next with Node.js, Deno, PHP, iOS, Android and other platforms.

Your software is using i18next? - Spread the word and let the world know!

make a tweet... write it on your website... create a blog post... etc...

Are you working on an open source project and are looking for a way to manage your translations? - locize loves the open-source philosophy and may be able to support you.

Learn more about supported frameworks

Here you'll find a simple tutorial on how to best use react-i18next. Some basics of i18next and some cool possibilities on how to optimize your localization workflow.

Do you want to use i18next in Vue.js? Check out this tutorial blog post.

Did you know internationalization is also important on your app's backend? In this tutorial blog post you can check out how this works.

Are you still using i18next in jQuery? Check out this tutorial blog post.

Complete solution

Most frameworks leave it to you how translations are being loaded. You are responsible to detect the user language, to load the translations and push them into the framework.

i18next takes care of these issues for you. We provide you with plugins to:

• detect the user language

• load the translations

• optionally cache the translations

• extension, by using post-processing - e.g. to enable sprintf support

Learn more about plugins and utilities

Flexibility

i18next comes with strong defaults but it is flexible enough to fulfill custom needs.

• Use moment.js over intl for date formatting?

• Prefer different pre- and suffixes for interpolation?

• Like gettext style keys better?

i18next has you covered!

Learn more about options

Scalability

The framework was built with scalability in mind. For smaller projects, having a single file with all the translation might work, but for larger projects this approach quickly breaks down. i18next gives you the option to separate translations into multiple files and to load them on demand.

Learn more about namespaces

Ecosystem

There are tons of modules built for and around i18next: from extracting translations from your code over bundling translations using webpack, to converting gettext, CSV and RESX to JSON.

• Learn more about plugins and utils

• Learn more about frameworks supported

Localization as a service

Through locize.com, i18next even provides its own translation management tool: localization as a service.

Learn more about the enterprise offering

Imagine you run a successful online business, and you want to expand it to reach customers in different countries. You know that to succeed in those markets, your website or app needs to speak the language and understand the culture of each place.

1. i18next: Think of 'i18next' as a sophisticated language expert for your website or app. It's like hiring a team of translators and cultural experts who ensure that your online business is fluent in multiple languages. It helps adapt your content, menus, and messages to fit perfectly in each target market, making your business more appealing and user-friendly.

2. locize: Now, 'locize' is your efficient manager in charge of organizing and streamlining the translation process. It keeps all your language versions organized and ensures they're always accurate and up-to-date. So, if you want to introduce a new product or promotion, locize helps you do it seamlessly in all the languages you operate in, saving you time and resources.

Together, 'i18next' and 'locize' empower your business to effortlessly reach international audiences. They help you speak the language of your customers, making your business more accessible, relatable, and successful in global markets.

Last updated 10 months ago

Author response:

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

We greatly appreciate reviewer 2 comments with both insightful and clearly evaluated assessments of this study that include, much appreciated reframing and evaluation of the study’s advances in the sleep field. It is a constructive review and provides considerable added value to this study in better defining the biological significance of the findings, including both advances and limitations.  

Reviewer 2 nicely summarized the work as “…highlight(ing) the accumulation and resolution of sleep need centered on the strength of excitatory synapses onto excitatory neurons.”. The reviewer succinctly placed one of the main electrophysiological findings in context of one of the sleep field’s most prevalent views, “that LTP associated with wake, leads to the accumulation of sleep need by increasing neuronal excitability, and by the "saturation" of LTP capacity.” It has been speculated that “This saturation subsequently impairs the capacity for further ongoing learning. This new data provides a satisfying mechanism of this saturation phenomenon (and its restoration by recovery sleep) by introducing the concept of silent synapses.” We want to emphasize that sleep need and its resolution involves more than just homeostasis of excitatory synaptic strength but may also be extended to include homeostasis of excitatory synaptic potential to undergo LTP (a homeostasis of meta-plasticity), with implications for learning and memory.   

Reviewer 2 also identified another advance made by this study, summarized as, “The new snRNAseq dataset indicates the sleep need is primarily seen (at the transcriptional level) in excitatory neurons, consistent with a number of other studies.” References for these studies are nicely provided by the reviewer. Our analysis of this data extends the evidence for transcriptional sleep-need-driven changes, observed by us and others in excitatory neurons to more particularly involve the excitatory neurons in layers 2-5, targeting  intra-telencephalic neurons.  

Reviewer 2, importantly noted, “New snRNAseq analysis indicates that SD drives the expression of synaptic shaping components (SSCs) consistent with the excitatory synapse as a major target for the restorative basis of sleep function”, and that “SD-induced gene expression is also enriched for autism spectrum disorder (ASD) risk genes”. These comments are well appreciated as they emphasize that beyond identification of the major target cell type of sleep function, the major sleep-target, gene-ontological characteristics are starting to be addressed.

Reviewer 2 commented on the molecular sleep model, making a key observation that “SDinduced gene expression in excitatory neurons overlaps with genes regulated by the transcription factor MEF2C and HDAC4/5 (Figure 4),” and accurately discusses the significance with respect to the proposed model.

We are in complete agreement with the observation that the molecular sleep model presented is not “definitively supported by the new data and in this regard should be viewed as a perspective…”. One of the more glaring gaps in supporting evidence is the absence of understanding of the role of HDAC4/5 (part of the SIK3-HDAC4/5 pathway) in sleep need modulation of excitatory synapses. Resolution of this issue might be approached by assessment of the synaptic effects of constitutively nuclear HDAC4/5. The current study provides a first step in the assessment by showing a correlation between HDAC4/5 and MEF2c target genes and a subset of differentially expressed synaptic shaping component (SSC) genes that modulate excitatory synapse strength and phenotype. However, the functional studies have yet to be completed. Complimentary studies on SD-induced SSC-DEGs (identified in this study) are also needed for follow-up characterization of their sleep need induced functional impact (both strength and meta-plasticity modulation) on the most relevant excitatory synapses (as identified in the current study).

We agree with both reviewers 1 and 2 that, “Additional work is also needed to understand the mechanistic links between SIK3-HDAC4/5 signaling and MEF2C activity”. Reviewer 2 clarifies the key unresolved issue as, “cnHDAC4/5 suppresses NREM amount and NREM SWA but had no effect on the NREM-SWA increase following SD (Zhou et al., Nature 2022). Loss of MEF2C in CaMKII neurons had no effect on NREM amount and suppressed the increase in NREM-SWA following SD (Bjorness et al., 2020)”. One may conclude with reviewer 2, “These instances indicate that cnHDAC4/5 and loss of MEF2C do not exactly match suggesting additional factors are relevant in these phenotypes.”

An understanding of the mechanism(s) responsible for the relationship between sleep need and SWA are critical to the evaluation of sleep need’s correlation with sleep DEGs and synaptic transmission, including “additional factors” as suggested by reviewer 2. SWA might result from a decrease of cortical glutamatergic neurotransmission below some threshold, which might occur in response to prolonged waking (possibly in response to waking activity-induced local increases of adenosine?), rather than being a cause of, or, being intimately involved in resolving sleep need.  

An increase of SWA in association with SD can result directly from an acute SD-induced increase in local adenosine concentration. This will elicit an ADORA1-mediated down-regulation of glutamate excitatory neurotransmission in the cortex (Bjorness et al., 2016) and in cholinergic arousal centers (Rainnie et al., 1994; Porkka-Heiskanen et al., 1997; Portas et al., 1997; Li et al., 2023). When MEF2c is derepressed by chronic loss of HDAC4 function, SWA is facilitated (Kim et al., 2022). It is plausible that loss of HDAC4 function contributes to the increased SWA by downscaling glutamate excitatory transmission (independent of sleep need). This is expected to result from derepressed, MEF2c mediated sleep-gene expression.  

Similarly, over-expression of constitutively active HDAC4 (cnHD4) can contribute to chronic upscaling of cortical glutamate synaptic strength to depress SWA (again, independent of sleep need). Thus, facilitation or depression of SWA correlates with up or down scaling effects on cortical glutamate neurotransmission, respectively, even in the absence of  a direct effects on sleep need (Figure 4D). Many reagents that reduce the excitability of glutamate pyramidal cells by various mechanisms, including anesthetics like isoflurane, barbiturates or benzodiazepines in addition to those activating ADORA1, increase SWA. Finally, it is important to acknowledge that direct evidence for this proposed link of SWA to cortical glutamate transmission remains in need of further investigation. Thus, SWA may reflect generalized cortical glutamate synaptic activity whether modulated by sleep function or by other agents.

Still, other factors that can have a role mediating some of the mis-match between cnHD4/5 DEGs and Mef2c-cKO DEGs, include the broader over-expression of AAV-cnHD4 compared to CamKII- driven Cre KO of Mef2c. The cnHD4 overexpression can increase arousal center activity in the hypothalamus and other arousal areas to interfere with SWA, but not to the exclusion of SD-DEG repression resulting from a repression of MEF2c-mediated sleep gene expression.

The critique by reviewer 1 raises a number of important technical issues with this study. A key, potentially critical issue raised by reviewer 1, is that of our method of experimental sleep deprivation (ESD). The reviewer suggests that “…neuronal activity/induction of plasticity”, peculiar to the ESD methodology employed in this study, “…rather than sleep/wake states are responsible for the observed results…”.  

In this study, a slow-moving treadmill (SMTM; 0.1km/hour, as stated in the methods), requiring locomotion to avoid bumping into the backwall of a false bottomed plexiglass cage was used to induce ESD. A mouse, in its home cage, typically moves much faster than 0.1km/hour and the mouse is able to eat and drink freely while in the cage (see file: video 1). Furthermore, our observations using a beam-break cage, indicate that mice spontaneously travel for comparable to longer distances over 6 hours than the treadmill moves (during the ESD of 6 hours). Finally, our EEG recordings of mice on the active treadmill show 100% waking while it is on (Bjorness et al., 2009), whereas prevention of NREM sleep (including transition time) using the “gentle handling”  (GH) technique occurs depending on the diligence of the experimenter.  

The accommodation (one week prior to ESD) included exposure to the treadmill-on for 30minutes ~ZT=2 & ZT= 14 hours (now spelled out in the “Materials & Methods” section). Thus, the likelihood of motor learning seems vanishingly small.  

As with all ESD methods, there must be some associated increase in sensory and motor neuronal activity to drive arousal and prevent transition to sleep. For example, the more widely employed GH method of ESD involves sensory stimulation (tactile and or auditory) of sufficient intensity to induce postural change from that associated with sleep to that associated with wake (often involving some locomotion). Like the SMTM, both sensory and motor systems are likely to be engaged. Unlike the SMTM method, the stimulation used in GH is variably-intermittent from mouse to mouse and from experimenter to experimenter as it is applied only when the experimenter judges the mouse to be falling asleep. . It can even be argued that the varied and unpredictable ways in which these interactions happen cause plastic changes with a higher likelihood than the constant slow motion of a treadmill – the mice know how to walk, after all. In other protocols, novel objects are introduced to the animals – those will certainly trigger plastic processes –something that is avoided using a slow-running treadmill to which the mouse has been accommodated, for sleep deprivation.  

The changes induced by SMTM technique are reproducible and induce arousal by somatic stimulation of sufficient intensity to induce natural motor activity as with GH. All ESD methods induce motor activity and it is reasonable to speculate that induced, motor activity is essential for effective ESD for the prolonged durations (>4 hours in mice) that elicit high sleep need. Electrophysiological assessment of SD-evoked increases in mEPSC amplitude and frequency using GH-ESD (Liu et al., 2010) are similar in all respects to our observations of the response to SMTMESD (Bjorness et al., 2020). Further studies might directly address a comparison of SMTM-ESD to GH-ESD as suggested by reviewer 1 but are regrettably outside the scope and resources of our study.

The model presented in Figure 4C is consistent with the experimental findings with respect to the observed electrophysiological changes (including loss of silent synapses and increased AMPA/NMDA ratio after ESD of 6 hours) and altered gene expression that includes enrichment of SSC genes, many of which (7 candidates are listed) can affect both AMPA/NMDA ratio and silent synapses. No claim of mechanism linking the changed expression to altered AMPAR or NMDAR activity can be made at this point, even as to polarity of gene expression, related to electrophysiological outcome. Furthermore, some transcripts may involve receptor trafficking while others more directly affect activated receptor function. To help illustrate the complexity of interpreting gene up-regulation, consider the following hypothetical scenario. If a gene like upregulated Grin3a acts rapidly, it may facilitate reduction of NMDAR function (decreasing plasticity) during ESD, whereas upregulation of a gene like Kif17, if acting in a more delayed manner, might enhance NMDAR surface expression and activity (increasing silent synapses) in response to ESD, during recovery sleep. Relevant references, consistent with these various outcomes are supplied in the manuscript but further investigation is clearly needed, or as reviewer 2 so aptly commented, this work “…provides a framework to stimulate further research and advances on the molecular basis of sleep function”.  

Several issues are raised by reviewer 1 concerning the electrophysiological methodology and statistical assessment. In regard to the former, we closely followed established protocols employed in the frontal neocortex (Myme et al., 2003). We did not include the details for series resistance monitoring. Series resistance values ranged between 8 and 15 MOhm and experiments with changes larger than 25% not used for further analyses. Thank you for bringing this  oversight on our part, to our attention. This essential information, that is unfailingly gathered for all our whole cell recordings, is now added to the version of record.

The -90 mV holding potential was chosen according to precedent (Myme et al., 2003). It increases driving force and permits lower stimulus strength for the same response size – reducing the likelihood for polysynaptic responses. Experiments with multiple response peaks at -90 mV were not included in the analysis. The -90 mV holding potential also increases NMDA receptor Mg++ block resulting in a minimally contaminated AMPA response. This information is now added to our submitted version of record.

The statistical assessments shown in Table 1 refer to two sets of data measured from 3X2=6 different cohorts for each sleep condition (CS, SD, RS): 1) AMPA & NMDA EPSCs and 2) AMPA/NMDA FR ratios (FRR; now bolded in row 1, second tab, Table S1). As stated in the results section, “A two-way ANOVA analysis showed a significant interaction between AMPA matched to NMDA EPSC response for each neuron, and sleep condition (F (2, 21) = 7.268, p<0.004; Figure 1 A, C, E). When considered independently, neither the effect of sleep condition nor of EPSC subtype reached significance at p<0.05 (Figure 1 C)”.  

As noted by reviewer 1, we inadvertently dropped one of the data points from the RS FR and FR ratio (FRR) statistical analysis (raw data in the third tab of Table S1, statistical data in fourth and fifth tab and illustrated in figure 1 F). Thanks to this appreciated, rigorous review, we can correct the oversight (using raw data unchanged in Table S1, third tab). The Table S1 and figure 1 F are now corrected for the version of record. For better clarity, we now use two tabs, the fourth and fifth tabs, respectively of Table S1, for separate stat analyses of FR and FRR data.

The significance of the AMPA/NMDA FRR across sleep conditions was assessed with the KruskalWallis test, a non-parametric method. The two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (BKY) was used to control for the FDR across multiple sleep conditions, in the non-parametric Kruskal-Wallis test but it is usually less powerful than tests presuming normal distributions like the one-way ANOVA and Holm-Sidak’s test. We have now added re-analyzed  FRR across CS, SD and RS conditions using a normal one-way ANOVA (Table S1, tab5). The results now read, “The difference between  sleep conditions and FRR is significant (F (2, 19) = 11.3, Table S1, tab5). Multiple comparisons (Holm-Sidak, Table S1, tab5) indicate the near absence of silent synapses was reversed by either CS or RS (SD/CS; p<0.0011 and SD/RS: p<0.0006; Table S1, tab 5; Figure 1 F).”. These analyses compare well to the non-parametric assessment using the  KruskalWallis test (significant at p= 0.0006) with BYK correction for multiple comparison analysis to give for CS-SD, p<= 0.0262 and for RS-SD, p<= 0.0006 (statistics also shown in Table S1, tab5). [Also shown in tab5 is the “standard approach of correcting for family wise error rate”, namely, Dunn’s test. It is more conservative but less powerful than the BYK correction- in general the tradeoff of greater power/ less conservative is better tolerated when many comparisons are made, however, it can be argued that in the present analysis type 2 errors are also potentially misleading and thus not well tolerated.]  The modifications of our statistical analyses, inspired by reviewer 1,  did not affect the interpretation of the data nor the conclusions.  

Bjorness TE, Kelly CL, Gao T, Poffenberger V, Greene RW (2009) Control and function of the homeostatic sleep response by adenosine A1 receptors. The Journal of neuroscience : the official journal of the Society for Neuroscience 29:1267-1276.

Bjorness TE, Dale N, Mettlach G, Sonneborn A, Sahin B, Fienberg AA, Yanagisawa M, Bibb JA, Greene RW (2016) An Adenosine-Mediated Glial-Neuronal Circuit for

Homeostatic Sleep. The Journal of neuroscience : the official journal of the Society for Neuroscience 36:3709-3721.

Bjorness TE, Kulkarni A, Rybalchenko V, Suzuki A, Bridges C, Harrington AJ, Cowan CW, Takahashi JS, Konopka G, Greene RW (2020) An essential role for MEF2C in the cortical response to loss of sleep in mice. Elife 9.

Kim SJ et al. (2022) Kinase signalling in excitatory neurons regulates sleep quantity and depth. Nature 612:512-518.

Li B, Ma C, Huang YA, Ding X, Silverman D, Chen C, Darmohray D, Lu L, Liu S, Montaldo G, Urban A, Dan Y (2023) Circuit mechanism for suppression of frontal cortical ignition during NREM sleep. Cell 186:5739-5750 e5717.

Liu ZW, Faraguna U, Cirelli C, Tononi G, Gao XB (2010) Direct evidence for wake-related increases and sleep-related decreases in synaptic strength in rodent cortex. The Journal of neuroscience : the official journal of the Society for Neuroscience 30:8671-8675.

Myme CI, Sugino K, Turrigiano GG, Nelson SB (2003) The NMDA-to-AMPA ratio at synapses onto layer 2/3 pyramidal neurons is conserved across prefrontal and visual cortices. Journal of neurophysiology 90:771-779.

Porkka-Heiskanen T, Strecker RE, Thakkar M, Bjorkum AA, Greene RW, McCarley RW (1997) Adenosine: a mediator of the sleep-inducing effects of prolonged wakefulness. Science 276:1265-1268.

Portas CM, Thakkar M, Rainnie DG, Greene RW, McCarley RW (1997) Role of adenosine in behavioral state modulation: a microdialysis study in the freely moving cat. Neuroscience 79:225-235.

Rainnie DG, Grunze HC, McCarley RW, Greene RW (1994) Adenosine inhibition of mesopontine cholinergic neurons: implications for EEG arousal. Science 263:689692.

Author response:

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

Reviewer #1 (Recommendations For The Authors): 

This is not a recommendation. While reading old literature, I found some interesting facts. The shape of the neurocranium in monotremes, birds, and mammals, at least in early stages, resembles the phenotype of 'dact'1/2, wnt11f2, or syu mutants. For more details, see DeBeer's: 'The Development of the Vertebrate Skull, !937' Plate 137. 

Thank you for pointing this out. It is indeed interesting.

Minor Comments: 

• Lines 64, 66, and 69: same citation without interruption: Heisenberg, Brand et al. 1996

Revised line 76. 

• Lines 101 and 102: same citation without interruption: Li, Florez et al. 2013 

Revised line 118.

• Lines 144, 515, 527, and 1147: should be wnt11f2 instead of wntllf2 - if not, then explain 

Revised lines 185, 625, 640,1300.

• Lines 169 and 171: incorrect figure citation: Fig 1D - correct to Fig 1F 

Revised lines 217, 219.

• Line 173: delete (Fig. S1) 

Revised line 221.

• Line 207: indicate that both dact1 and dact2 mRNA levels increased, noting a 40% higher level of dact2 mRNA after deletion of 7 bp in the dact2 gene 

Revised line 265.

• Line 215: Fig 1F instead of Fig 1D 

Revised line 217.

• Line 248: unify naming of compound mutants to either dact1/2 or dact1/dact2 compound mutants 

Revised to dact1/2 throughout.

• Line 259: incorrect figure citation: Fig S1 - correct to Fig S2D/E 

Revised line 324.

• Line 302: correct abbreviation position: neural crest (NCC) cell - change to neural crest cell (NCC) population 

Revised line 380.

• Line 349: repeating kny mut definition from line 70 may be unnecessary 

Revised line 434.

• Line 351: clarify distinction between Fig S1 and Fig S2 in the supplementary section 

Revised line 324.

• Line 436: refer to the correct figure for pathways associated with proteolysis (Fig 7B) 

Revised line 530.

• Line 446-447: complete the sentence and clarify the relevance of smad1 expression, and correct the use of "also" in relation to capn8 

Revised line 567.

• Line 462: clarify that this phenotype was never observed in wildtype larvae, and correct figure reference to exclude dact1+/- dact2+/- 

Revised line 563, 568.

• Line 463: explain the injection procedure into embryos from dact1/2+/- interbreeding 

Revised line 565.

• Lines 488 and 491: same citation without interruption: Waxman, Hocking et al. 2004 

Revised line 591.

• Line 502: maintain consistency in referring to TGF-beta signaling throughout the article 

Revised throughout.

• Line 523: define CNCC; previously used only NCC 

Revised to cranial NCC throughout.

• Line 1105: reconsider citing another work in the figure legend 

Revised line 1249.

• Line 1143: consider using "mutant" instead of "mu" 

Revised line 1295.

• Fig 2A/B: indicate the number of animals used ("n") 

N is noted on line 1274.

• Fig 2C, D, E: ensure uniform terminology for control groups ("wt" vs. "wildtype") 

Revised in figure.

• Fig 7C: clarify analysis of dact1/2-/- mutant in lateral plate mesoderm vs. ectoderm 

Revised line 1356.

• Fig 8A: label the figure to indicate it shows capn8, not just in the legend 

Revised.

• Fig 8D: explain the black/white portions and simplify to highlight important data 

Revised.

• Fig S2: add the title "Figure S2" 

Revised.

• Consider omitting the sentence: "As with most studies, this work has contributed some new knowledge but generated more questions than answers." 

Revised line 720.

Reviewer #2 (Recommendations For The Authors): 

Major comments: 

(1) The authors have addressed many of the questions I had, including making the biological sample numbers more transparent. It might be more informative to use n = n/n, e.g. n = 3/3, rather than just n = 3. Alternatively, that information can be given in the figure legend or in the form of penetrance %. 

The compound heterozygote breeding and phenotyping analyses were not carried out in such a way that we can comment on the precise % penetrance of the ANC phenotype, as we did not dissect every ANC and genotype every individual that resulted from the triple heterozygote in crossings. We collected phenotype/genotype data until we obtained at least three replicates.

We did genotype every individual resulting from dact1/2 dHet crosses to correlate genotype to the phenotype of the embryonic convergent extension phenotype and narrowed ethmoid plate (Fig. 2A, Fig. 3) which demonstrated full penetrance.

(2) The description of the expression of dact1/2 and wnt11f2 is not consistent with what the images are showing. In the revised figure 1 legend, the author says "dact2 and wnt11f2 transcripts are detected in the anterior neural plate" (line 1099)", but it's hard to see wnt11f2 expression in the anterior neural plate in 1B. The authors then again said " wnt11f2 is also expressed in these cells", referring to the anterior neural plate and polster (P), notochord (N), paraxial and presomitic mesoderm (PM) and tailbud (TB). However, other than the notochord expression, other expression is actually quite dissimilar between dact2 and wnt11f2 in 1C. The authors should describe their expression more accurately and take that into account when considering their function in the same pathway. 

We have revised these sections to more carefully describe the expression patterns. We have added references to previous descriptions of wnt11 expression domains.

(3) Similar to (2), while the Daniocell was useful in demonstrating that expression of dact1 and dact2 are more similar to expression of gpc4 and wnt11f2, the text description of the data is quite confusing. The authors stated "dact2 was more highly expressed in anterior structures including cephalic mesoderm and neural ectoderm while dact1 was more highly expressed in mesenchyme and muscle" (lines 174-176). However, the Daniocell seems to show more dact1 expression in the neural tissues than dact2, which would contradict the in situ data as well. I think the problem is in part due to the dataset contains cells from many different stages and it might be helpful to include a plot of the cells at different stages, as well as the cell types, both of which are available from the Daniocell website. 

We have revised the text to focus the Daniocell analysis on the overall and general expression patterns. Line 220.

(4) The authors used the term "morphological movements" (line 337) to describe the cause of dact1/2 phenotypes. Please clarify what this means. Is it cell movement? Or is it the shape of the tissues? What does "morphological movements" really mean and how does that affect the formation of the EP by the second stream of NCCs? 

We have revised this sentence to improve clarity. Line 416.

(5) In the first submission, only 1 out of 142 calpain-overexpressing animals phenocopied dact1/2 mutants and that was a major concern regarding the functional significance of calpain 8 in this context. In the revised manuscript, the authors demonstrated that more embryos developed the phenotype when they are heterozygous for both dact1/2. While this is encouraging, it is interesting that the same phenomenon was not observed in the dact1-/-; dact2+/- embryos (Fig. 6D). The authors did not discuss this and should provide some explanation. The authors should also discuss sufficiency vs requirement tested in this experiment. However, given that this is the most novel aspect of the paper, performing experiments to demonstrate requirements would be important. 

We have added a statement regarding the non-effect in dact1-/-;dact2+/- embryos. Line 568-570. We have also added discussion of sufficiency vs necessity/requirement testing. Line 676-679.

(6) Related to (5), the authors cited figure 8c when mentioning 0/192 gfp-injected embryos developed EP phenotypes. However, figure 8c is dact1/2 +/- embryos. The numbers also doesn't match the numbers in Figure 8d either. Please add relevant/correct figures. 

The text has been revised to distinguish between our overexpression experiment in wildtype embryos (data not shown) versus overexpression in dact1/2 double het in cross embryos (Fig 8).

Minor comments: 

(1) Fig 1 legend line 1106 "the midbrain (MP)" should be MB 

Revised line 1250.

(2) Wntllf2, instead of wnt11f2, (i.e. the letter "l" rather than the number "1") was used in 4 instances, line 144, 515, 527, 1147 

Revised lines 185, 625, 640,1300.

(3) The authors replaced ANC with EP in many instances, but ANC is left unchanged in some places and it's not defined in the text. It's first mentioned in line 170.

Revised line 218.

Self-reflection: Teachers develop the ability to reflect on their own practice and make adjustments independently.

underlying data formats for knowledge appliances -

Your agenda is very OGMy

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors describe a method to probe both the proteins associated with genomic elements in cells, as well as 3D contacts between sites in chromatin. The approach is interesting and promising, and it is great to see a proximity labeling method like this that can make both proteins and 3D contacts. It utilizes DNA oligomers, which will likely make it a widely adopted method. However, the manuscript over-interprets its successes, which are likely due to the limited appropriate controls, and of any validation experiments. I think the study requires better proteomic controls, and some validation experiments of the "new" proteins and 3D contacts described. In addition, toning down the claims made in the paper would assist those looking to implement one of the various available proximity labeling methods and would make this manuscript more reliable to non-experts.

Strengths:

(1) The mapping of 3D contacts for 20 kb regions using proximity labeling is beautiful.

(2) The use of in situ hybridization will probably improve background and specificity.

(3) The use of fixed cells should prove enabling and is a strong alternative to similar, living cell methods.

Weaknesses:

(1) A major drawback to the experimental approach of this study is the "multiplexed comparisons". Using the mtDNA as a comparator is not a great comparison - there is no reason to think the telomeres/centrosomes would look like mtDNA as a whole. The mito proteome is much less complex. It is going to provide a large number of false positives. The centromere/telomere comparison is ok, if one is interested in what's different between those two repetitive elements. But the more realistic use case of this method would be "what is at a specific genomic element"? A purely nuclear-localized control would be needed for that. Or a genomic element that has nothing interesting at it (I do not know of one). You can see this in the label-free work: non-specific, nuclear GO terms are enriched likely due to the random plus non-random labeling in the nucleus. What would a Telo vs general nucleus GSEA look like? (GSEA should be used for quantitative data, no GO). That would provide some specificity. Figures 2G and S4A are encouraging, but a) these proteins are largely sequestered in their respective locations, and b) no validation by an orthogonal method like ChIP or Cut and Run/Tag is used.

You can also see this in the enormous number of "enriched" proteins in the supplemental volcano plots. The hypothesis-supporting ones are labeled, but do the authors really believe all of those proteins are specific to the loci being looked at? Maybe compared to mitochondria, but it's hard to believe there are not a lot of false positives in those blue clouds. I believe the authors are more seeing mito vs nucleus + Telo than the stated comparison. For example, if you have no labeling in the nucleus in the control (Figures 1C and 2C) you cannot separate background labeling from specific labeling. Same with mito vs. nuc+Telo. It is not the proper control to say what is specifically at the Telo.

I would like to see a Telo vs nuclear control and a Centromere vs nuc control. One could then subtract the background from both experiments, then contrast Telo vs Cent for a proper, rigorous comparison. However, I realize that is a lot of work, so rewriting the manuscript to better and more accurately reflect what was accomplished here, and its limitations, would suffice.

(2) A second major drawback is the lack of validation experiments. References to literature are helpful but do not make up for the lack of validation of a new method claiming new protein-DNA or DNA-DNA interactions. At least a handful of newly described proximal proteins need to be validated by an orthogonal method, like ChIP qPCR, other genomic methods, or gel shifts if they are likely to directly bind DNA. It is ok to have false positives in a challenging assay like this. But it needs to be well and clearly estimated and communicated.

(3) The mapping of 3D contacts for 20 kb regions is beautiful. Some added discussion on this method's benefits over HiC-variants would be welcomed.

(4) The study claims this method circumvents the need for transfectable cells. However, the authors go on to describe how they needed tons of cells, now in solution, to get it to work. The intro should be more in line with what was actually accomplished.

(5) Comments like "Compared to other repetitive elements in the human genome...." appear to circumvent the fact that this method is still (apparently) largely limited to repetitive elements. Other than Glopro, which did analyze non-repetitive promoter elements, most comparable methods looked at telomeres. So, this isn't quite the advancement you are implying. Plus, the overlap with telomeric proteins and other studies should be addressed. However, that will be challenging due to the controls used here, discussed above.

We thank the Reviewer for their careful reading of manuscript and constructive suggestions. We plan to substantially revise the framing and presentation of manuscript to address the concerns raised by all three reviewers.

Reviewer #2 (Public review):

Summary

Liu and MacGann et al. introduce the method DNA O-MAP that uses oligo-based ISH probes to recruit horseradish peroxidase for targeted proximity biotinylation at specific DNA loci. The method's specificity was tested by profiling the proteomic composition at repetitive DNA loci such as telomeres and pericentromeric alpha satellite repeats. In addition, the authors provide proof-of-principle for the capture and mapping of contact frequencies between individual DNA loop anchors.

Strengths

Identifying locus-specific proteomes still represents a major technical challenge and remains an outstanding issue (1). Theoretically, this method could benefit from the specificity of ISH probes and be applied to identify proteomes at non-repetitive DNA loci. This method also requires significantly fewer cells than other ISH- or dCas9-based locus-enrichment methods. Another potential advantage to be tested is the lack of cell line engineering that allows its application to primary cell lines or tissue.

Weaknesses

The authors indicate that DNA O-MAP is superior to other methods for identifying locus-specific proteomes. Still, no proof exists that this method could uncover proteomes at non-repetitive DNA loci. Also, there is very little validation of novel factors to confirm the superiority of the technique regarding specificity.

The authors first tested their method's specificity at repetitive telomeric regions, and like other approaches, expected low-abundant telomere-specific proteins were absent (for example, all subunits of the telomerase holoenzyme complex). Detecting known proteins while identifying noncanonical and unexpected protein factors with high confidence could indicate that DNA O-MAP does not fully capture biologically crucial proteins due to insufficient enrichment of locus-specific factors. The newly identified proteins in Figure 1E might still be relevant, but independent validation is missing entirely. In my opinion, the current data cannot be interpreted as successfully describing local protein composition.

Finally, the authors could have discussed the limitations of DNA O-MAP and made a fair comparison to other existing methods (2-5). Unlike targeted proximity biotinylation methods, DNA O-MAP requires paraformaldehyde crosslinking, which has several disadvantages. For instance, transient protein-protein interactions may not be efficiently retained on crosslinked chromatin. Similarly, some proteins may not be crosslinked by formaldehyde and thus will be lost during preparation (6).

(1) Gauchier M, van Mierlo G, Vermeulen M, Dejardin J. Purification and enrichment of specific chromatin loci. Nat Methods. 2020;17(4):380-9.

(2) Dejardin J, Kingston RE. Purification of proteins associated with specific genomic Loci. Cell. 2009;136(1):175-86.

(3) Liu X, Zhang Y, Chen Y, Li M, Zhou F, Li K, et al. In Situ Capture of Chromatin Interactions by Biotinylated dCas9. Cell. 2017;170(5):1028-43 e19.

(4) Villasenor R, Pfaendler R, Ambrosi C, Butz S, Giuliani S, Bryan E, et al. ChromID identifies the protein interactome at chromatin marks. Nat Biotechnol. 2020;38(6):728-36.

(5) Santos-Barriopedro I, van Mierlo G, Vermeulen M. Off-the-shelf proximity biotinylation for interaction proteomics. Nat Commun. 2021;12(1):5015.

(6) Schmiedeberg L, Skene P, Deaton A, Bird A. A temporal threshold for formaldehyde crosslinking and fixation. PLoS One. 2009;4(2):e4636.

We thank the Reviewer for their constructive feedback on our work. As noted above, we plan to substantially revise the framing and presentation of manuscript to address the concerns raised by all three reviewers.

Reviewer #3 (Public review):

Significance of the Findings:

The study by Liu et al. presents a novel method, DNA-O-MAP, which combines locus-specific hybridisation with proximity biotinylation to isolate specific genomic regions and their associated proteins. The potential significance of this approach lies in its purported ability to target genomic loci with heightened specificity by enabling extensive washing prior to the biotinylation reaction, theoretically improving the signal-to-noise ratio when compared with other methods such as dCas9-based techniques. Should the method prove successful, it could represent a notable advancement in the field of chromatin biology, particularly in establishing the proteomes of individual chromatin regions - an extremely challenging objective that has not yet been comprehensively addressed by existing methodologies.

Strength of the Evidence:

The evidence presented by the authors is somewhat mixed, and the robustness of the findings appears to be preliminary at this stage. While certain data indicate that DNA-O-MAP may function effectively for repetitive DNA regions, a number of the claims made in the manuscript are either unsupported or require further substantiation. There are significant concerns about the resolution of the method, with substantial biotinylation signals extending well beyond the intended target regions (megabases around the target), suggesting a lack of specificity and poor resolution, particularly for smaller loci. Furthermore, comparisons with previous techniques are unfounded since the authors have not provided direct comparisons with the same mass spectrometry (MS) equipment and protocols. Additionally, although the authors assert an advantage in multiplexing, this claim appears overstated, as previous methods could achieve similar outcomes through TMT multiplexing. Therefore, while the method has potential, the evidence requires more rigorous support, comprehensive benchmarking, and further experimental validation to demonstrate the claimed improvements in specificity and practical applicability.

We thank the Reviewer for providing detailed critiques of our manuscript. As noted above, we plan to substantially revise the framing and presentation of manuscript to address the concerns raised by all three reviewers.

Author response:

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

Reviewer #1 (Public Review):

This paper aims to address the establishment and maintenance of neural circuitry in the case of a massive loss of neurons. The authors used genetic manipulations to ablate the principal projection neurons, the mitral/tufted cells, in the mouse olfactory bulb. Using diphtheria toxin (Tbx21-Cre:: loxP-DTA line) the authors ablated progressively large numbers of M/T cells postnatally. By injecting diphtheria toxin (DT) into the Tbx21-Cre:: loxP-iDTR line, the authors were able to control the timing of the ablation in the adult stage. Both methods led to the successful elimination of a majority of M/TCs by 4 months of age. The authors made a few interesting observations. First, they found that the initial pruning of the remaining M/T cell primary dendrite was unaffected. However, in adulthood, a significant portion of these cells extended primary dendrites to innervate multiple glomeruli. Moreover, the incoming olfactory sensory neuron (OSN) axons, as examined for those expressing the M72 receptor, showed a divergent innervation pattern as well. The authors conclude that M/T cell density is required to maintain the dendritic structures and the olfactory map. To address the functional consequences of eliminating a large portion of principal neurons, the authors conducted a series of behavioral assays. They found that learned odor discrimination was largely intact. On the other hand, mating and aggression were reduced. The authors concluded that learned behaviors are more resilient than innate ones.

The study is technically sound, and the results are clear-cut. The most striking result is the contrast between the normal dendritic pruning during early development and the expanded dendritic innervation in adulthood. It is a novel discovery that can lead to further investigation of how the single-glomerulus dendritic innervation is maintained. The authors conducted a

few experiments to address potential mechanisms, but it is inconclusive, as detailed below. It is also interesting to see that the massive neuronal loss did not severely impact learned odor discrimination. This result, together with previous studies showing nearly normal odor discrimination in the absence of large portions of the olfactory bulb or scrambled innervation patterns, attests to the redundancy and robustness of the sensory system. The discussion should take into account these other studies in a historical context.

Main comments:

(1) In previous studies, it has been concluded that dendritic pruning unfolds independently, regardless of the innervation pattern or activity of the OSNs. The new observation bolsters this conclusion by showing that a loss of neighboring M/T cells does not affect the developmental process. A more nuanced discussion comparing the results of these studies would strengthen the paper.

We thank the reviewer for the suggestion. We now include an extended discussion citing relevant previous works in the manuscript (Lines 351-374).

(2) The authors propose that a certain density of M/T is required to prevent the divergent innervation of primary dendrites, but the evidence is not sufficient to support this proposal. The experiment with low-dose DT injection to ablate a smaller portion of M/T cells did not change the percentage of cells innervating two or more glomeruli. The authors suggest that a threshold must be met, but this threshold is not determined.  

In our experiments using high-dose DT, we hypothesized that there may be many empty glomeruli (glomeruli not innervated by M/T cells), and as a result, that some of the remaining M/T cells could branch their apical dendrite tuft into multiple empty glomeruli. To test this hypothesis, we carried out another experiment using a lower dose of DT. In this experiment, the fraction of remaining M/T cells was 25% (~10,000 M/T cells), which was higher than with the high DT dose (5%, or around 2,000 M/T cells) , but still significantly lower than wild type mice (~40,000 cells M/T cells). With around 2,000 glomeruli and 10,000 M/T per bulb, it could be expected that each glomerulus would be innervated by ~5 M/T cells (on average). However, we found that the percentage of M/T cells projecting to multiple glomeruli (around 40%) was similar when either 10,000 or 2,000 of M/T remained in the bulb. In addition, it is important to emphasize that even in wt animals with a full set of M/T cells, a small percentage of M/T cells still innervate more than one glomerulus (Lin et al., 2000). Together, these observations suggest that the innervation of multiple glomeruli by M/T cells is not simply due to the presence of empty glomeruli, and that our hypothesis was not correct.

We have added a comment explaining this issue in the Results section (Lines 200-203).

(3) The authors suggest that neural activity is not required for this plasticity. The evidence was derived primarily from naris occlusion and neuronal silencing using Kir2.1. While the results are consistent with the notion, it is a rather narrow interpretation of how neural activity affects circuit configuration. Perturbation of neural activity also entails an increase in firing. Inducing the activity of the neurons may alter this plasticity. Silencing per se may induce a homeostatic response that expands the neurite innervation pattern to increase synaptic input to compensate for the loss of activity. Thus, further silencing the cells may not reduce multiglomerular innervation, but an increased activity may.

The experiments with Kir2.1 demonstrate that the structural plasticity observed after reducing the total number of M/T cells in an animal is not regulated by the firing action potentials in the remaining cells. Instead, this experiment indicates that the observed structural plasticity may be regulated by other types of mechanisms (including increased synaptic excitation as suggested by the reviewer) that do not require the firing of action potentials in M/T cells. 

We now have included a comment regarding this point (Lines 243-247).  

(4) There is a discrepancy between this study and the one by Fujimoto et al. (Developmental Cell; 2023), which shows that not only glutamatergic inputs to the primary dendrite can facilitate pruning of remaining dendrites but also Kir2.1 overexpression can significantly perturb dendritic pruning. This discrepancy is not discussed by the authors.

We agree that it would be useful to contrast these two works.

In our experiments, performed in adult animals, we blocked sensory input by performing naris occlusion before we induced ablation of M/T cells. In a separate experiment, also in adult animals, we expressed the Kir2.1 channel, to reduce the ability of neurons to fire action potentials. With both types of manipulations, we observed that the ablation of a large fraction of M/T cells still caused the remaining M/T cells to maintain a single apical dendrite that sprouts several new tufts towards multiple glomeruli. A recent paper (Fujimoto et al., 2023)) in which Kir2.1 was expressed in a large percentage of M/T starting during embryonic development showed that these “silent” M/T cells failed to prune their arbors to a single dendrite. In aggregate, these observations indicate that action potentials are necessary for the normal pruning that occurs during perinatal development (Fujimoto et al., 2023), but are not required for the expansion of dendritic trees caused by ablating a large fraction of M/T cells in adult animals (our current manuscript).

We have now explained the differences between both studies in the manuscript (Lines 427-439).

(5) An alternative interpretation of the discrepancy between the apparent normal pruning by p10 and expanded dendritic innervation in adulthood is that there are more cells before P10, when ~25% of M/T cells are present, but at a later date only 1-3% are present. 

The relationship between the number of M/T cells and single glomerulus innervation has not been explored during postnatal development. It would be important to test this hypothesis.

We agree with this comment, and in lines 375-381 we discuss the discrepancy between normal refinement during development, and dendritic sprouting in adults.

Cre is expressed in M/T cells and it induces DTA expression starting around P0. The elimination of M/T cells starts at this time, and continues until by P10, when more than 75% of M/T have been eliminated. At P21 more than 90% of M/T have been eliminated, and their number remains stable thereafter.

Pruning of the dendrites of M/T cells starts at P0 and it is mostly complete by P10. Therefore, it is possible that between P0 and P7, when dendrites are being pruned, the number of M/T cells remaining in the bulb is still over a threshold that does not interfere with the process of normal dendrite pruning. We agree that it would be very informative to perform additional experiments in the future where a large set of M/T cells could be ablated before pruning occurs (ideally before P0). 

(6) The authors attribute the change in the olfactory map to the loss of M/T cells. Another obvious possibility is that the diffused projection is a response to the change in the olfactory bulb size. With less space to occupy, the axons may be forced to innervate neighboring glomeruli. It is not known how the total number of glomeruli is affected. This question could be addressed by tracking developmental changes in bulb volume and glomerular numbers.

Certainly, this is a possibility, and we have now included a comment on this regard in the manuscript (Lines 473-480). 

We believe that there are three likely scenarios that could account for these observations:

(a) After ablating M/T cells, the tufts of the remaining M/T cells sprout into multiple glomeruli, and this causes the axons of OSNs to project into multiple glomeruli.

(b) Ablating M/T cells may cause changes in other OB cells that make synapses in the glomeruli (ETCs, PGCs, sAC, etc…), and the misrouting of OSN axons that we observed in our experiments may be a secondary effect caused by the elimination of M/T cells.

(c) After ablating the majority of M/T cells, the olfactory bulb gets reduced in size, and the axons of OSNs find it difficult to precisely converge on a target that now has become smaller. As a result, the axons of OSNs fail to converge on single glomeruli.

(7) The retained ability to discriminate odors upon reinforced training is not surprising in light of a number of earlier studies. For example, Slotnick and colleagues have shown that rats losing ~90% of the OB can retain odor discrimination. Weiss et al have shown that humans without an olfactory bulb can perform normal olfactory tasks. Gronowitz et al have used theoretical prediction and experimental results to demonstrate that perturbing the olfactory map does not have a major impact on olfactory discrimination. Fleischmann et al have shown that mice with a monoclonal nose can discriminate odors. The authors should discuss their results in these contexts.

We apologize for this important oversight - we now include a more elaborate discussion including the relevant references as suggested in the manuscript (Line 483-496).

(8) It should be noted that odor discrimination resulting from reinforcement training does not mean normal olfactory function. It is a highly artificial situation as the animals are overtrained. It should not be used as a measure of the robustness of the olfactory sense. Natural odor discrimination (without training), detection threshold, and innate appetitive/aversive response to certain odors may be affected. These experiments were not conducted.

We agree that the standard tests commonly used to measure olfactory function require substantial training, and thus, are quite artificial. However, these tests are used because they allow a more precise quantification of olfactory function than those relying on natural behaviors.  

We have now included a few sentences to address this point in the results (Lines 321322) and discussion sections (Lines 541-543).

(9) The social behaviors were conducted using relatively coarse measures (vaginal plug and display of aggression). Moreover, these behaviors are most likely affected by the disruption of the AOB mitral cells and have little to do with the dendritic pruning process described in the paper. It is misleading to lump social behaviors with innate responses to odors.

This point follows the same logic as the previous one. The olfactory tests that rely on natural behaviors are quite coarse and difficult to quantify. In contrast, the olfactory tests using apparatuses such as olfactometers can be quantified with precision, but they are artificial. We agree that some of the naturalistic behaviors that we studied such as mating or aggression may depend to a large extent on the AOB (although it is possible that the MOB may also be involved in these tasks to a degree). In our initial version of the manuscript, we commented on the anticipated relative involvement of the MOB and AOB in the studied tasks, but we have now added some additional sentences to make this point clearer. In addition, we now add a comment indicating that it is possible that the abnormal behaviors could simply be due to a reduction in the number of AOB M/T cells (~98.5% and ~ 85% elimination of M/T cells in the AOB in Tbx::DTA and Tbx::iDTR mice, respectively), regardless of the abnormal dendritic pruning of main OB M/T cells (Lines 530-534).

See Figure 5E - M/T cells in AOB (Lines 1238-1239). 

Reviewer #2 (Public Review):

The authors make the interesting observation that the developmental refinement of apical M/T cell dendrites into individual glomeruli proceeds normally even when the majority of neighboring M/T cells are ablated. At later stages, the remaining neurons develop additional dendrites that invade multiple glomeruli ectopically, and similarly, OSN inputs to glomeruli lose projection specificity as well. The authors conclude that the normal density of M/T neurons is not required for developmental refinement, but rather for maintaining specific connectivity in adults.

The observations are indeed quite striking; however, the authors' conclusions are not entirely supported by the data.

(1) It is unclear whether the expression of diphtheria toxin that eventually leads to the ablation of the large majority of M/T neurons compromises the cell biology of the remaining ones.

DT is an extremely potent toxin that kills cells by inhibiting proteins translation, and it has been demonstrated that the presence of a single DT molecule in a cell is sufficient to kill it, because of its highly efficient catalytic activity. Accordingly, previous experiments have shown that DT kills cells within a few hours after its appearance in the cytoplasm (Yamaizumi et al., 1978). In other words, all the published evidence suggests that if a cell is exposed to the action of DT, that cell will die shortly. There is no evidence that cells exposed to DT can survive and experience long-term effects. Finally, previous works have not observed any long-term changes in neurons directly caused by the actions of DT (Johnson et al., 2017).

(2) The authors interpret the growth of ectopic dendrites later in life as a lack of maintenance of dendrite structure; however, maybe the observed changes reflect actually adaptations that optimize wiring for extremely low numbers of M/T neurons. The finding that olfactory behavior was less affected than predicted supports this interpretation.

We do not know the cellular or molecular mechanisms that explain why reducing the density of M/T cells is followed by the growth of ectopic dendrites from the remaining M/T cells. We agree that the functional outcome of growing ectopic dendrites may result in an optimization of wiring in the bulb and could explain why olfactory function is relatively preserved. We now include a comment regarding this possibility (Lines 513-525).   

(3) The number of remaining M/T neurons is much higher at P10 than later. Can the relatively large number of remaining neurons (or their better health status) be the reason that dendrites refine normally at the early developmental stages rather than a (currently unknown) developmental capacity that preserves refinement?

We thank the reviewer for the suggestion, which was also raised by reviewer 1. 

We agree with this comment, and in lines 375-381 we discuss the discrepancy between normal refinement during development, and dendritic sprouting in adults.

Cre is expressed in M/T cells and it induces DTA expression starting around P0. The elimination of M/T cells starts at this time, and continues until by P10, when more than 75% of M/T have been eliminated. At P21 more than 90% of M/T have been eliminated, and their number remains stable thereafter.

Pruning of the dendrites of M/T cells starts at P0 and it is mostly complete by P10. Therefore, it is possible that between P0 and P7, when dendrites are being pruned, the number of M/T cells remaining in the bulb is still over a threshold that does not interfere with the process of normal dendrite pruning. We agree that it would be very informative to perform additional experiments in the future where a large set of M/T cells could be ablated before pruning occurs (ideally before P0). 

(4) While the effect of reduced M/T neuron density on both M/T dendrites and OSN axons is described well, the relationship between both needs to be characterized better: Is one effect preceding the other or do they occur simultaneously? Can one be the consequence of the other?

Previous works have demonstrated that disrupting the topographic projection of the OSN axons has no effect on the structure of the apical dendrite of M/T cells (Ma et al., 2014; Nishizumi et al., 2019). Our experiments ablating a large fraction of M/T cells suggest that they are necessary for the correct targeting of OSN axons into the bulb. However, our experiments do not allow us to tell apart these 2 scenarios: 

(a) the ablation of a large fraction of M/T cells directly causes the sprouting of the apical dendrite of M/T cells, and that this sprouting in turn causes the abnormal projection of OSN axons onto the bulb. 

(b) the ablation of a large fraction of M/T cells first causes the axons of OSN to project abnormally onto multiple glomeruli in the bulb, and this in turn causes the dendrite of remaining M/T cells to sprout onto multiple glomeruli. 

We now include a comment on the manuscript explaining this point. (Lines 473-492)

(5) Page 7: the observation that not all neurons develop additional dendrites is not a sign of differences between cell types, it may be purely stochastic.

This is correct, and we mention these 2 scenarios in the discussion (Line 407-408). 

(6) Page 8: the fact that activity blockade did not affect the formation of ectopic dendrites does not suggest that the process is not activity-dependent: both manipulations have the same effect and may just mask each other.

The experiments with Kir2.1 demonstrate that the structural plasticity observed after reducing the total number of M/T cells in an animal is not regulated by the firing action potentials in the remaining cells. Instead, this experiment indicates that the observed structural plasticity may be regulated by other types of mechanisms (including increased synaptic excitation as suggested by the reviewer) that do not require the firing of action potentials in M/T cells. 

We now have included a comment regarding this point (Lines 243-247).  

(7) It remains unclear how the observed structural changes can explain the behavioral effects.

We agree that the relationship between structural changes and behavior was not appropriately explained in our manuscript. Our manipulations cause two major changes in the olfactory system, one primary, and several secondary. The primary change is a large reduction in the number of M/T cells both in the MOB and AOB. This reduction in M/T cell number triggers significant secondary changes in the connectivity of the bulb, including an abnormal projection of OSNs onto the OB, and the growth of ectopic dendrites from the remaining M/T cells into multiple glomeruli.

The behavioral abnormalities displayed by these mice is ultimately caused by the reduction in the number of M/T cells, but it is likely that the secondary structural changes could regulate some of the behavioral phenomena that we observed. For example, in principle, it is possible that the ectopic dendrites innervating several glomeruli could help the bulb to perceive smells with a much reduced number of M/T cells. On the other hand, this promiscuous growth of dendrites into multiple glomeruli could make it more difficult for the animals to discriminate between smells. The same argument could be made about the fact that OSN axons project onto multiple glomeruli: we simply do not know if this change helps or makes it more difficult for the animal to detect smells.  

We now include a comment regarding this issue (Lines 513-525).   

Reviewer #1 (Recommendations For The Authors):

Additional experiments and a more thorough discussion of the results, as suggested in the public review, would significantly strengthen the paper. Below are some specific parts that need to be addressed.

There is a lack of information on how M/T cell numbers are quantified. Without the information, it is difficult to evaluate the claim. Using the tdTomato signal may miss cells that are not labeled due to the transgenic effect. 

Although we cannot conclude that we are identifying the complete set of M/T cells (because the transgenic lines may fail to label some M/T cells), the number of M/T cells that we observed is similar to that previously reported (Richard et al., 2010). This concern has been included in the Results section (Lines 121-124).

A more detailed description about M/T cells quantification has been added into the method section (Lines 627-632).

There is a lack of information on the timeline of treatment and how measurement of the olfactory bulb volume is conducted.

We now include a more detailed description of how the volume of the OB was measured in the methods (Lines 621-623).

The volume measurement is inconsistent with the pictures shown. In Figure 1, supplemental data 2 panels B and C, it appears that the bulbs in DTA and DTR mice are about half in length in each dimension. This would translate into ~1/8 of the volume of the control mice.

We measured the volume of the bulbs based on the Neurolucida reconstructions, and we observed that in both DTA and iDTR mice the volumes of their bulbs are roughly 50% compared to a wild type mouse. In Figure 1 - figure supplement 2 the sections that were shown for wild type, DTA and iDTR mice were not taken at the same position in the bulb, and this gave the impression that the bulbs from DTA and iDTR were much smaller than they really are. We now show sections for these three animals at equivalent positions in the bulb. 

Figure 1 E and F have no legend.

We apologize for this mistake - we have now added the legend for Figures 1E and F (Lines 1009-1013).

Figure 3, supplemental data 2, it is not clear what the readers should be looking at. The data is confusing even for experts in the field. The authors should describe the figures more clearly, pointing out what they are supposed to show.

We apologize for this, and we have now added a more detailed description of Figure3 – figure supplement 2 (Lines 1153-1167).

In several figures, it is not clearly written what the comparisons were for where there are indications of statistical significance above the bars.

We have now included a more detailed description of the statistics comparison in the figure legends.

AAV serotype should be specified.

The AAV serotype used to label M/T cells was the AAV-PHP.eB. We have added this information in the methods section of the manuscript. 

Reviewer #2 (Recommendations For The Authors):

Minor points

Page 5, para 2: "The decrease in neuronal plasticity with age": it is unclear what "the decrease" refers to.

We have changed this sentence in the text to make it clear:

“The decrease in structural plasticity of M/T cells after apical dendrite refinement (Mizrahi and Katz, 2003),….”

Line 146-148

Is there a quantification of the effect of Kir2.1 overexpression alone (example shown in Figure 3D)?

We did an experiment in IDTR animals in which a fraction of M/T cells expressed Kir2.1, and we split these animals in 2 groups: (a) animals that received an injection of DT, and (b) animals that did not receive any DT. We quantified the effect of Kir2.1 on M/T cells from animals that received DT injection (with an ablation of around of 90% of M/T cells) and we did not observe any clear statistically significant differences between cells expressing Kir2.1 or neurons that did not express Kir2.1 from other iDTR animals that also received DT injections. We did not quantify the possible effects of kir2.1 in the group of animals that did not receive DT because on a first inspection we did not observe any clear differences between Kir2.1 cells and neighboring wild type cells. 

References

Fujimoto S, Leiwe MN, Aihara S, Sakaguchi R, Muroyama Y, Kobayakawa R, Kobayakawa K, Saito T, Imai T. 2023. Activity-dependent local protection and lateral inhibition control synaptic competition in developing mitral cells in mice. Dev Cell S1534-5807(23)00237-X. doi:10.1016/j.devcel.2023.05.004

Johnson RE, Tien N-W, Shen N, Pearson JT, Soto F, Kerschensteiner D. 2017. Homeostatic plasticity shapes the visual system’s first synapse. Nat Commun 8:1220. doi:10.1038/s41467-017-01332-7

Lin DM, Wang F, Lowe G, Gold GH, Axel R, Ngai J, Brunet L. 2000. Formation of precise connections in the olfactory bulb occurs in the absence of odorant-evoked neuronal activity. Neuron 26:69–80. doi:10.1016/s0896-6273(00)81139-3

Ma L, Wu Y, Qiu Q, Scheerer H, Moran A, Yu CR. 2014. A developmental switch of axon targeting in the continuously regenerating mouse olfactory system. Science 344:194–197. doi:10.1126/science.1248805

Nishizumi H, Miyashita A, Inoue N, Inokuchi K, Aoki M, Sakano H. 2019. Primary dendrites of mitral cells synapse unto neighboring glomeruli independent of their odorant receptor identity. Commun Biol 2:1–12. doi:10.1038/s42003-018-0252-y

Richard MB, Taylor SR, Greer CA. 2010. Age-induced disruption of selective olfactory bulb synaptic circuits. Proc Natl Acad Sci U S A 107:15613–15618. doi:10.1073/pnas.1007931107

Yamaizumi M, Mekada E, Uchida T, Okada Y. 1978. One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell 15:245–250. doi:10.1016/0092-8674(78)90099-5

Author response:

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

Public Reviews: 

Reviewer #1 (Public Review): 

Freas et al. investigated if the exceedingly dim polarization pattern produced by the moon can be used by animals to guide a genuine navigational task. The sun and moon have long been celestial beacons for directional information, but they can be obscured by clouds, canopy, or the horizon. However, even when hidden from view, these celestial bodies provide directional information through the polarized light patterns in the sky. While the sun's polarization pattern is famously used by many animals for compass orientation, until now it has never been shown that the extremely dim polarization pattern of the moon can be used for navigation. To test this, Freas et al. studied nocturnal bull ants, by placing a linear polarizer in the homing path on freely navigating ants 45 degrees shifted to the moon's natural polarization pattern. They recorded the homing direction of an ant before entering the polarizer, under the polarizer, and again after leaving the area covered by the polarizer. The results very clearly show, that ants walking under the linear polarizer change their homing direction by about 45 degrees in comparison to the homing direction under the natural polarization pattern and change it back after leaving the area covered by the polarizer again. These results can be repeated throughout the lunar month, showing that bull ants can use the moon's polarization pattern even under crescent moon conditions. Finally, the authors show, that the degree in which the ants change their homing direction is dependent on the length of their home vector, just as it is for the solar polarization pattern. 

The behavioral experiments are very well designed, and the statistical analyses are appropriate for the data presented. The authors' conclusions are nicely supported by the data and clearly show that nocturnal bull ants use the dim polarization pattern of the moon for homing, in the same way many animals use the sun's polarization pattern during the day. This is the first proof of the use of the lunar polarization pattern in any animal.

Reviewer #2 (Public Review): 

Summary: 

The authors aimed to understand whether polarised moonlight could be used as a directional cue for nocturnal animals homing at night, particularly at times of night when polarised light is not available from the sun. To do this, the authors used nocturnal ants, and previously established methods, to show that the walking paths of ants can be altered predictably when the angle of polarised moonlight illuminating them from above is turned by a known angle (here +/- 45 degrees).

Strengths: 

The behavioural data are very clear and unambiguous. The results clearly show that when the angle of downwelling polarised moonlight is turned, ants turn in the same direction. The data also clearly show that this result is maintained even for different phases (and intensities) of the moon, although during the waning cycle of the moon the ants' turn is considerably less than may be expected.

Weaknesses: 

The final section of the results - concerning the weighting of polarised light cues into the path integrator - lacks clarity and should be reworked and expanded in both the Methods and the Results (also possibly with an extra methods figure). I was really unsure of what these experiments were trying to show or what the meaning of the results actually are.

Rewrote these sections and added figure panel to Figure 6.

Impact: 

The authors have discovered that nocturnal bull ants while homing back to their nest holes at night, are able to use the dim polarised light pattern formed around the moon for path integration. Even though similar methods have previously shown the ability of dung beetles to orient along straight trajectories for short distances using polarised moonlight, this is the first evidence of an animal that uses polarised moonlight in homing. This is quite significant, and their findings are well supported by their data.

Reviewer #3 (Public Review): 

Summary: 

This manuscript presents a series of experiments aimed at investigating orientation to polarized lunar skylight in a nocturnal ant, the first report of its kind that I am aware of.

Strengths: 

The study was conducted carefully and is clearly explained here. 

Weaknesses: 

I have only a few comments and suggestions, that I hope will make the manuscript clearer and easier to understand.

Time compensation or periodic snapshots 

In the introduction, the authors compare their discovery with that in dung beetles, which have only been observed to use lunar skylight to hold their course, not to travel to a specific location as the ants must. It is not entirely clear from the discussion whether the authors are suggesting that the ants navigate home by using a time-compensated lunar compass, or that they update their polarization compass with reference to other cues as the pattern of lunar skylight gradually shifts over the course of the night - though in the discussion they appear to lean towards the latter without addressing the former. Any clues in this direction might help us understand how ants adapted to navigate using solar skylight polarization might adapt use to lunar skylight polarization and account for its different schedule. I would guess that the waxing and waning moon data can be interpreted to this effect.

Added a paragraph discussing this distinction in mechanisms and the limits of the current data set in untangling them. An interesting topic for a follow up to be sure.

Effects of moon fullness and phase on precision 

As well as the noted effect on shift magnitudes, the distributions of exit headings and reorientations also appear to differ in their precision (i.e., mean vector length) across moon phases, with somewhat shorter vectors for smaller fractions of the moon illuminated. Although these distributions are a composite of the two distributions of angles subtracted from one another to obtain these turn angles, the precision of the resulting distribution should be proportional to the original distributions. It would be interesting to know whether these differences result from poorer overall orientation precision, or more variability in reorientation, on quarter moon and crescent moon nights, and to what extent this might be attributed to sky brightness or degree of polarization.

See below for response to this and the next reviewer comment

N.B. The Watson-Williams tests for difference in mean angle are also sensitive to differences in sample variance. This can be ruled out with another variety of the test, also proposed by Watson and Williams, to check for unequal variances, for which the F statistic is = (n2-1)*(n1-R1) / (n1-1)*(n2-R2) or its inverse, whichever is >1. 

We have looked at the amount of variance from the mean heading direction in terms of both the shifts and the reorientations and found no significant difference in variance between all relevant conditions. It is possible (and probably likely) that with a higher n we might find these differences but with the current data set we cannot make statistical statements regarding degradations in navigational precision.  

As an additional analysis to address the Watson-Williams test‘s sensitivity to changes in variance, we have added var test comparisons for each of the comparisons, which is a well-established test to compare variance changes. None of these were significantly different, suggesting the observed differences in the WW tests are due to changes in the mean vector and not the distribution. We have added this test to the text.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors): 

I have only very few minor suggestions to improve the manuscript: 

(1) While I fully agree with the authors that their study, to the best of my knowledge, provides the first proof (in any animal) of the use of the moon's polarization pattern, the many repetitions of this fact disturb the flow of the text and could be cut at several instances. 

Yes, it is indeed repeated to an annoying degree. 

We have removed these beyond bookending mentions (Abstract and Discussion).

(2) In my opinion, the authors did not change the "ambient polarization pattern" when using the linear polarization filter (e.g., l. 55, 170, 177 ...). The linear polarizer presents an artificial polarization pattern with a much higher degree of polarization in comparison to the ambient polarization pattern. I would suggest re-phrasing this, to emphasize the artificial nature of the polarization pattern under the polarizer.

We have made these suggested changes throughout the text to clarify. We no longer say the ambient pattern was   

(3) Line 377: I do not see the link between the sentence and Figure 7 

Changed where in the discussion we refer to Figure 7.

(4) Figure 7 upper part: In my opinion, the upper part of Figure 7 does not add any additional value to the illustration of the data as compared to Figure 5 and could be cut.

We thought it might be easier for some reader to see the shifts as a dial representation with the shift magnitude converted to 0-100% rather than the shifts in Figure 5. This makes it somewhat like a graphical abstract summarising the whole study.

I agree that Figure 5 tells the same story but a reader that has little background in directional stats might find figure 7 more intuitive. This was the intent at least. 

If it becomes a sticking point, then we can remove the upper portion.  

Reviewer #2 (Recommendations For The Authors): 

MINOR CORRECTIONS AND QUERIES 

Line 117: THE majority 

Corrected

Lines 129-130: Do you have a reference to support this statement? I am unaware of experiments that show that homing ants count their steps, but I could have missed it.

We have added the references that unpack the ant pedometer.  

Line 140: remove "the" in this line. 

Removed

Line 170: We need more details here about the spectral transmission properties of the polariser (and indeed which brand of filter, etc.). For instance, does it allow the transmission of UV light?

Added

Line 239: "...tested identicALLY to ...." 

Corrected

Lines 242-258 (Vector testing): I must admit I found the description of these experiments very difficult to follow. I read this section several times and felt no wiser as a result. I think some thought needs to be given to better introduce the reader to the rationale behind the experiment (e.g., start by expanding lines 243-246, and maybe add a methods figure that shows the different experimental procedures).

I have rewritten this section of the methods to clearly state the experiment rational and to be clearer as to the methodology.

Also Added a methods panel to Figure 6.

Line 247: "reoriented only halfway". What does this mean? Do you mean with half the expected angle?

Yes, this is a bit unclear. We have altered for clarity:

‘only altered their headings by about half of the 45° e-vector shift (25.2°± 3.7°), despite being tested on near-full-moon nights.’

Results section (in general): In Figure 1 (which is a very nice figure!) you go to all the trouble of defining b degrees (exit headings) and c degrees (reorientation headings), which are very intuitive for interpreting the results, and then you totally abandon these convenient angles in favour of an amorphous Greek symbol Phi (Figs. 2-6) to describe BOTH exit and reorientation headings. Why?? It becomes even more confusing when headings described by Phi can be typically greater than 300 degrees in the figures, but they are never even close to this in the text (where you seem to have gone back to using the b degrees and c degrees angles, without explicitly saying so). Personally, I think the b degrees and c degrees angles are more intuitive (and should be used in both the text and the figures), but if you do insist on using Phi then you should use it consistently in both the text and the figures. 

Replaced Phi with b° and c° for both figures and in the text.

Finally, for reorientation angles in Figure 4A, you say that the angle is 16.5 degrees. This angle should have been 143.5 degrees to be consistent with other figures. 

Yes, the reorientation was erroneously copied from the shift data (it is identical in both the +45 shift and reorientation for Figure 4A). This has now been corrected

Line 280, and many other lines: Wherever you refer to two panels of the same figure, they should be written as (say) Figure 2A, B not Figure 2AB.

Changed as requested throughout the text.

Line 295 (Waxing lunar phases): For these experiments, which nest are you using? 1 or 2?

We have added that this is nest 1. 

Figure 3B: The title of this panel should be "Waxing Crescent Moon" I think. 

Ah yes, this is incorrect in the original submission. I have fixed this.

Lines 312-313: Here it sounds as though the ants went right back to the full +/- 45 degrees orientations when they clearly didn't (it was -26.6 degrees and 189.9 degrees). Maybe tone the language down a bit here.

Changed this to make clear the orientation shift is only ‘towards’ the ambient lunar e-vector.

Line 327: Insert "see" before "Figure 5" 

Added

Line 329: See comment for Line 295. 

We have added that this is nest 1. 

Lines 357-373 (Vector testing): Again, because of the somewhat confusing methods section describing these experiments, these results were hard to follow, both here and in the Discussion. I don't really understand what you have shown here. Re-think how you present this (and maybe re-working the Methods will be half the battle won). 

I have rewritten these sections to try to make clear these are ant tested with differences in vector length 6m vs. 2m, tested at the same location. Hopefully this is much clearer, but I think if these portions remain a bit confusing that a full rename of the conditions is in order. Something like long vector and short vector would help but comes with the problem of not truly describing what the purpose of the test is which is to control for location, thus the current condition names. As it stands, I hope the new clarifications adequately describe the reasoning while keeping the condition names. Of course, I am happy to make more changes here as making this clear to readers is important for driving home that the path integrator is in play.

See current change to results as an example: ‘Both forgers with a long ~6m remaining vector (Halfway Release), or a short ~2m remaining vector (Halfway Collection & Release), tested at the same location_,_ exhibited significant shifts to the right of initial headings when the e-vector was rotated clockwise +45°.’

Line 361: I think this should be 16.8 not 6.8 

Yes, you are correct. Fixed in text (16.8).

Line 365: I think this should be -12.7 not 12.7 

Yes, you are correct. Fixed in text (–12.7).

Line 408: "morning twilight". Should this be "morning solar twilight"? Plus "M midas" should be "M. midas"

Added and fixed respectively.

Line 440. "location" is spelt wrong. 

Fixed spelling.

Line 444: "...WITH longer accumulated vectors, ..." 

Added ‘with’ to sentence. 

Line 447: Remove "that just as"

Removed.

Line 448: "Moonlight polarised light" should be "Polarised moonlight" 

Corrected.

Lines 450-453: This sentence makes little sense scientifically or grammatically. A "limiting factor" can't be "accomplished". Please rephrase and explain in more detail.

This sentence has been rephrased:

‘The limiting factors to lunar cue use for navigation would instead be the ant’s detection threshold to either absolute light intensity, polarization sensitivity and spectral sensitivity. Moonlight is less UV rich compared to direct sunlight and the spectrum changes across the lunar cycle (Palmer and Johnsen 2015).’

Line 474: Re-write as "... due to the incorporation of the celestial compass into the path integrator..."

Added.

Reviewer #3 (Recommendations For The Authors): 

Minor comments 

Line 84 I am not sure that we can infer attentional processes in orientation to lunar skylight, at least it has not yet been investigated.

Yes, this is a good point. We have changed ‘attend’ to ‘use’.  

Line 90 This description of polarized light is a little vague; what is meant by the phrase "waves which occur along a single plane"? (What about the magnetic component? These waves can be redirected, are they then still polarized? Circular polarization?). I would recommend looking at how polarized light is described in textbooks on optics.

Response: We have rewritten the polarised light section to be clearer using optics and light physics for background. 

Line 92 The phrase "e-vector" has not been described or introduced up to this point.

We now introduce e-vector and define it. 

‘Polarised light comprises light waves which occur along a single plane and are produced as a by-product of light passing through the upper atmosphere (Horváth & Varjú 2004; Horváth et al., 2014). The scattering of this light creates an e-vector pattern in the sky, which is arranged in concentric circles around the sun or moon's position with the maximum degree of polarisation located 90° from the source. Hence when the sun/moon is near the horizon, the pattern of polarised skylight is particularly simple with uniform direction of polarisation approximately parallel to the north-south axes (Dacke et al., 1999, 2003; Reid et al. 2011; Zeil et al., 2014).’

Happy to make further changes as well.  

Line 107 Diurnal dung beetles can also orient to lunar skylight if roused at night (Smolka et al., 2016), provided the sky is bright enough. Perhaps diurnal ants might do the same?

Added the diurnal dung beetles mention as well as the reference.

Also, a very good suggestion using diurnal bull ants.

Line 146 Instead of lunar calendar the authors appear to mean "lunar cycle". 

Changed

Line 165 In Figure 1B, it looks like visual access to the sky was only partly "unobstructed". Indeed foliage covers as least part of the sky right up to the zenith.

We have added that the sky is partially obstructed. 

Line 179 This could also presumably be checked with a camera? 

For this testing we tried to keep equipment to a minimum for a single researcher walking to and from the field site given the lack of public transport between 1 and 4am. But yes, for future work a camera based confirmation system would be easier. 

Line 243 The abbreviation "PI" has not been described or introduced up to this point.

Changes to ‘path integration derived vector lengths….’

Line 267 The method for comparing the leftwards and rightwards shifts should be described in full here (presumably one set of shifts was mirrored onto the other?).

We have added the below description to indicate the full description of the mirroring done to counterclockwise shifts.

‘To assess shift magnitude between −45° and +45° foragers within conditions, we calculated the mirror of shift in each −45° condition, allowing shift magnitude comparisons within each condition. Mirroring the −45° conditions was calculated by mirroring each shift across the 0° to 180° plane and was then compared to the corresponding unaltered +45 condition.’

Discussion Might the brightness and spectrum of lunar skylight also play a role here?

We have added a section to the discussion to mention the aspects of moonlight which may be important to these animals, including the spectrum, brightness and polarisation intensity.  

Line 451 The sensitivity threshold to absolute light intensity would not be the only limiting factor here. Polarization sensitivity and spectral sensitivity may also play a role (moonlight is less UV rich than sunlight and the spectrum of twilight changes across the lunar cycle: Palmer & Johnsen, 2015). 

Added this clarification.

Line 478 Instead of the "masculine ordinal" symbol used (U+006F) here a degree symbol (U+00B0) should be used.

Ah thank you, we have replaced this everywhere in the text.  

Line 485 It should be possible to calculate the misalignment between polarization pattern before and after this interruption of celestial cues. Does the magnitude of this misalignment help predict the size of the reorientation?

Reorientations are highly correlated with the shift size under the filter, which makes sense as larger shifts mean that foragers need to turn back more to reorient to both the ambient pattern and to return to their visual route. Reorientation sizes do not show a consistent reduction compared to under-the-filter shifts when the lunar phase is low and is potentially harder to detect.

I have reworked this line in the text as I do not think there is much evidence for misalignment and it might be more precise to say that overnight periods where the moon is not visible may adversely impact the path integrator estimate, though it is currently unknown the full impact of this celestial cue gap of if other cues might also play a role.

Line 642 "from their" should be "relative to" 

Changed as requested

Figure 1B Some mention should be made of the differences in vegetation density. 

Added a sentence to the figure caption discussing the differences in both vegetation along the horizon and canopy cover.

Figures 2-6 A reference line at 0 degrees change might help the reader to assess the size of orientation changes visually. Confidence intervals around the mean orientation change would also help here.

We have now added circular grid lines and confidence intervals to the circular plots. These should help make the heading changes clear to readers.

Author response:

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

eLife Assessment <br /> This valuable study is a companion to a paper introducing a theoretical framework and methodology for identifying Cancer Driving Nucleotides (CDNs). While the evidence that recurrent SNVs or CDNs are common in true cancer driver genes is solid, the evidence that many more undiscovered cancer driver mutations will have CDNs, and that this approach could identify these undiscovered driver genes with about 100,000 samples, is limited. 

Same criticism as in the eLife assessment of eLife-RP-RA-2024-99340 (https://elifesciences.org/reviewed-preprints/99340). Hence, please refer to the responses to the companion paper.

Public Reviews:

Reviewer #1 (Public Review):

The study investigates Cancer Driving Nucleotides (CDNs) using the TCGA database, finding that these recurring point mutations could greatly enhance our understanding of cancer genomics and improve personalized treatment strategies. Despite identifying 50-150 CDNs per cancer type, the research reveals that a significant number remain undiscovered, limiting current therapeutic applications, and underscoring the need for further larger-scale research.

Strengths:

The study provides a detailed examination of cancer-driving mutations at the nucleotide level, offering a more precise understanding than traditional gene-level analyses. The authors found a significant number of CDNs remain undiscovered, with only 0-2 identified per patient out of an expected 5-8, indicating that many important mutations are still missing. The study indicated that identifying more CDNs could potentially significantly impact the development of personalized cancer therapies, improving patient outcomes.

Weaknesses:

The study is constrained by relatively small sample sizes for each cancer type, which reduces the statistical power and robustness of the findings. ICGC and other large-scale WGS datasets are publicly available but were not included in this study.

Thanks. We indeed have used all public data, including GENIE (figure 7 of the companion paper), ICGC and other integrated resources such as COSMIC. The main study is based on TCGA because it is unbiased for estimating the probability of CDN occurrences. In many datasets, the numerators are given but the denominators are not (the number of patients with the mutation / the total number of patients surveyed). In GENIE, we observed that E(u) estimated upon given sequencing panels are much smaller than in TCGA, this might be due to the selective report of nonsynonymous mutations for synonymous mutations are generally considered irrelevant in tumorigenesis.

To be able to identify rare driver mutations, more samples are needed to improve the statistical power, which is well-known in cancer research. The challenges in direct functional testing of CDNs due to the complexity of tumor evolution and unknown mutation combinations limit the practical applicability of the findings.

We fully agree. We now add a few sentences, making clear that the theory allows us to see how much more can be gained by each stepwise increase in sample size. For example, when the sample size reaches 106, further increases will yield almost no gain in confidence of CDNs identified (see figures of eLife-RP-RA-2024-99340. As pointed out in our provisional responses, an important strength of this pair of studies is that the results are testable. The complexity is the combination of mutations required for tumorigenesis and the identification of such combinations is the main goal and strength of this pair of studies. We add a few sentences to this effect.

While the importance of large sample sizes in identifying cancer drivers is well-recognized, the analytical framework presented in the companion paper (https://elifesciences.org/reviewed-preprints/99340) goes a step further by quantitatively elucidating the relationship between sample size and the resolution of CDN detection.

The question is very general as it is about multigene interactions, or epistasis. The challenges are true in all aspects of evolutionary biology, for example, the genetics of reproductive isolation(Wu and Ting 2004). The issue of epistasis is difficult because most, if not all, of the underlying mutations have to be identified in order to carry out functional tests. While the full identification is rarely feasible, it is precisely the objective of the CDN project. When the sample size increases to 100,000 for a cancer type, all point mutations for that cancer type should be identifiable.

The QC of the TCGA data was not very strict, i.e, "patients with more than 3000 coding region point mutations were filtered out as potential hypermutator phenotypes", it would be better to remove patients beyond +/- 3*S.D from the mean number of mutations for each cancer type. Given some point mutations with >3 hits in the TCGA dataset, they were just false positive mutation callings, particularly in the large repeat regions in the human genome.

Thanks. The GDC data portal offers data calls from multiple pipelines, enabling us to select mutations detected by at least two pipelines. While including patients with hypermutator phenotypes could introduce potential noise, as shown in Eq. 10 of the main text, our method for defining the upper limit of i* is relative robust to the fluctuations in the E(u) of the corresponding cancer population. Since readers may often ask about this, we expand the Methods section somewhat to emphasize this point.

The codes for the statistical calculation (i.e., calculation of Ai_e, et al) are not publicly available, which makes the findings hard to be replicated.

We have now updated the section of “Data Availability” in both papers. The key scripts for generating the major results are available at: https://gitlab.com/ultramicroevo/cdn_v1.

Reviewer #2 (Public Review):

Summary:

The study proposes that many cancer driver mutations are not yet identified but could be identified if they harbor recurrent SNVs. The paper leverages the analysis from Paper #1 that used quantitative analysis to demonstrate that SNVs or CDNs seen 3 or more times are more likely to occur due to selection (ie a driver mutation) than they are to occur by chance or random mutation.

Strengths:

Empirically, mutation frequency is an excellent marker of a driver gene because canonical driver mutations typically have recurrent SNVs. Using the TCGA database, the paper illustrates that CDNs can identify canonical driver mutations (Figure 3) and that most CDNs are likely to disrupt protein function (Figure 2). In addition, CDNs can be shared between cancer types (Figure 4).

Weaknesses:

Driver alteration validation is difficult, with disagreements on what defines a driver mutation, and how many driver mutations are present in a cancer. The value proposed by the authors is that the identification of all driver genes can facilitate the design of patient-specific targeting therapies, but most targeted therapies are already directed towards known driver genes. There is an incomplete discussion of oncogenes (where activating mutations tend to target a single amino acid or repeat) and tumor suppressor genes (where inactivating mutations may be more spread across the gene). Other alterations (epigenetic, indels, translocations, CNVs) would be missed by this type of analysis.

The above paragraph has three distinct points. We shall respond one by one.

First, …  can facilitate the design of patient-specific targeting therapies, but most targeted therapies are already directed towards known driver genes…

We state in the text of Discussion the following that shows only a few best-known driving mutations have been targeted. It is accurate to say that < 5% of CDNs we have identified are on the current targeting list. Furthermore, this list we have compiled is < 10% of what we expect to find.

Direct functional test of CDNs would be to introduce putative cancer-driving mutations and observe the evolution of tumors. Such a task of introducing multiple mutations that are collectively needed to drive tumorigenesis has been done only recently, and only for the best-known cancer driving mutations (Ortmann et al. 2015; Takeda et al. 2015; Hodis et al. 2022). In most tumors, the correct combination of mutations needed is not known. Clearly, CDNs, with their strong tumorigenic strength, are suitable candidates.

Second, “There is an incomplete discussion of oncogenes (where activating mutations tend to target a single amino acid or repeat) and tumor suppressor genes (where inactivating mutations may be more spread across the gene).”

We sincerely thank the reviewer for this insightful comment. Below are two new paragraphs in the Discussion pertaining to the point:

In this context, we should comment on the feasibility of targeting CDNs that may occur in either oncogenes (ONCs) or tumor suppressor genes (TSGs). It is generally accepted that ONCs drive tumorigenesis thanks to the gain-of-function (GOF) mutations whereas TSGs derive their tumorigenic powers by loss-of-function (LOF) mutations. It is worthwhile to point out that, since LOF mutations are likely to be more widespread on a gene, CDNs are biased toward GOF mutations. The often even distribution of non-sense mutations along the length of TSGs provide such evidence. As gene targeting aims to diminish gene functions, GOF mutations are perceived to be targetable whereas LOF mutations are not. By extension, ONCs should be targetable but TSGs are not. This last assertion is not true because mutations on TSGs may often be of the GOF kind as well.

The data often suggest that mis-sense mutations on TSGs are of the GOF kind. If mis-sense mutations are far more prevalent than nonsense mutations in tumors, the mis-sense mutations cannot possibly be LOF mutations. (After all, it is not possible to lose more functions than nonsense mutations.) For example, AAA to AAC (K to Q) is a mis-sense mutation while AAA to AAT (K to stop) is a non-sense mutation. In a separate study (referred to as the escape-route analysis), we found many cases where the mis-sense mutations on TSGs are more prevalent (> 10X) than nonsense mutations. Another well-known example is the distribution of non-sense mutations TSGs. For example, on APC, a prominent TSG, non-sense mutations are far more common in the middle 20% of the gene than the rest (Zhang and Shay 2017; Erazo-Oliveras et al. 2023). The pattern suggests that even these non-sense mutations could have GOF properties. 

The following response is about the clinical implications of our CDN analysis. Canonical targeted therapy often relies on the Tyrosine Kinase Inhibitors (TKIs) (Dang et al. 2017; Danesi et al. 2021; Waarts et al. 2022). Theoretically, any intervention that suppresses the expression of gain-of-function (GOF) CDNs could potentially have therapeutic value in cancer treatment. This leads us to a discussion of oncogenes versus TSGs in the context of GOF / LOF (loss of function) mutations. Not all mutations on oncogenes have oncogenic effect, besides, truncated mutations in oncogenes are often subject to negative selection (Bányai et al. 2021), the identification of CDNs within oncogenes is therefore crucial for developing effective cancer treatment guidelines. Secondly, while TSGs are generally believed to promote cancer development via loss of function mutations, research suggests that certain mutations within TSGs can have GOF-like effect, such as the dominant negative effect of truncated TP53 mutations (Marutani et al. 1999; de Vries et al. 2002; Gerasimavicius et al. 2022). Characterizing driver mutations as GOF or LOF mutations could potentially expand the scope of targeted cancer therapy. We’ll address this issue in a third study in preparation.

The method could be more valuable when applied to the noncoding genome, where driver mutations in promoters or enhancers are relatively rare, or as yet to be discovered. Increasingly more cancers have had whole genome sequencing. Compared to WES, criteria for driver mutations in noncoding regions are less clear, and this method could potentially provide new noncoding driver CDNs. Observing the same mutation in more than one cancer specimen is empirically unusual, and the authors provide a solid quantitative analysis that indicates many recurrent mutations are likely to be cancer-driver mutations.

Again, we are grateful for the comments which prompt us to expand a paragraph in Discussion, reproduced below.

The CDN approach has two additional applications. First, it can be used to find CDNs in non-coding regions. Although the number of whole genome sequences at present is still insufficient for systematic CDN detection, the preliminary analysis suggests that the density of CDNs in non-coding regions is orders of magnitude lower than in coding regions. Second, CDNs can also be used in cancer screening with the advantage of efficiency as the targeted mutations are fewer. For the same reason, the false negative rate should be much lower too. Indeed, the false positive rate should be far lower than the gene-based screen which often shows a false positive rate of >50% (supplement File S1).

Again, we are grateful that Reviewer #2 have addressed the potential value of our study in finding cancer drivers in non-coding regions. A major challenge in this area lies in defining the appropriate L value as presented in Eq. 10. In the main text, we used a gamma distribution to account for the variability of mutation rates across sites in coding region. For the non-coding region, we will categorize these regions based on biological annotations. The goal is to set different i* cutoffs for different genomic regions (such as heterochromatin / euchromatin, GC-rich regions or centromeric regions), and avoid false positive calls for CDN in repeated regions (Elliott and Larsson 2021; Peña et al. 2023).

References

Bányai L, Trexler M, Kerekes K, Csuka O, Patthy L. 2021. Use of signals of positive and negative selection to distinguish cancer genes and passenger genes. Elife 10:e59629.

Danesi R, Fogli S, Indraccolo S, Del Re M, Dei Tos AP, Leoncini L, Antonuzzo L, Bonanno L, Guarneri V, Pierini A, et al. 2021. Druggable targets meet oncogenic drivers: opportunities and limitations of target-based classification of tumors and the role of Molecular Tumor Boards. ESMO Open 6:100040.

Dang CV, Reddy EP, Shokat KM, Soucek L. 2017. Drugging the “undruggable” cancer targets. Nat Rev Cancer 17:502–508.

Elliott K, Larsson E. 2021. Non-coding driver mutations in human cancer. Nat Rev Cancer 21:500–509.

Erazo-Oliveras A, Muñoz-Vega M, Mlih M, Thiriveedi V, Salinas ML, Rivera-Rodríguez JM, Kim E, Wright RC, Wang X, Landrock KK, et al. 2023. Mutant APC reshapes Wnt signaling plasma membrane nanodomains by altering cholesterol levels via oncogenic β-catenin. Nat Commun 14:4342.

Gerasimavicius L, Livesey BJ, Marsh JA. 2022. Loss-of-function, gain-of-function and dominant-negative mutations have profoundly different effects on protein structure. Nat Commun 13:3895.

Hodis E, Triglia ET, Kwon JYH, Biancalani T, Zakka LR, Parkar S, Hütter J-C, Buffoni L, Delorey TM, Phillips D, et al. 2022. Stepwise-edited, human melanoma models reveal mutations’ effect on tumor and microenvironment. Science 376:eabi8175.

Marutani M, Tonoki H, Tada M, Takahashi M, Kashiwazaki H, Hida Y, Hamada J, Asaka M, Moriuchi T. 1999. Dominant-negative mutations of the tumor suppressor p53 relating to early onset of glioblastoma multiforme. Cancer Res 59:4765–4769.

Ortmann CA, Kent DG, Nangalia J, Silber Y, Wedge DC, Grinfeld J, Baxter EJ, Massie CE, Papaemmanuil E, Menon S, et al. 2015. Effect of Mutation Order on Myeloproliferative Neoplasms. N Engl J Med 372:601–612.

Peña MV de la, Summanen PAM, Liukkonen M, Kronholm I. 2023. Chromatin structure influences rate and spectrum of spontaneous mutations in Neurospora crassa. Genome Res. 33:599–611.

Takeda H, Wei Z, Koso H, Rust AG, Yew CCK, Mann MB, Ward JM, Adams DJ, Copeland NG, Jenkins NA. 2015. Transposon mutagenesis identifies genes and evolutionary forces driving gastrointestinal tract tumor progression. Nat Genet 47:142–150.

de Vries A, Flores ER, Miranda B, Hsieh H-M, van Oostrom CThM, Sage J, Jacks T. 2002. Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function. Proceedings of the National Academy of Sciences 99:2948–2953.

Waarts MR, Stonestrom AJ, Park YC, Levine RL. 2022. Targeting mutations in cancer. J Clin Invest 132:e154943.

Wu C-I, Ting C-T. 2004. Genes and speciation. Nat Rev Genet 5:114–122.

Zhang L, Shay JW. 2017. Multiple Roles of APC and its Therapeutic Implications in Colorectal Cancer. JNCI: Journal of the National Cancer Institute 109:djw332.

Author response:

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

Reviewer #1 (Public Review):

This work describes the induction of SIV-specific NAb responses in rhesus macaques infected with SIVmac239, a neutralization-resistant virus. Typically, host NAb responses are not detected in animals infected with SIVmac239. In this work, seventy SIVmac239-infected macaques were retrospectively screened for NAb responses and a subset of nine animals were identified as NAb-inducers. The viral genomes from 7/9 animals that induced NAb responses were found to encode nonsynonymous mutation in the Nef gene (amino acid G63E). In contrast, Nef G63E mutation was found only in 2/19 NAb non-inducers - implicating that the Nef G63E mutation is selected in NAb inducers. Measurement of Nef G63E frequencies in plasma viruses suggested that Nef G63E selection preceded NAb induction. Nef G63E mutation was found to mediate escape from Nef-specific CD8+ T-cell responses. To examine the functional phenotype of Nef G63E mutant, its effect on downmodulation of Nef-interacting host proteins was examined. Infection of rhesus and cynomolgus macaque CD4+ T cell lines with WT or Nef G63E mutant SIV suggested that Nef mutant reduces S473 phosphorylation of AKT. Using flow cytometry-based proximity ligation assay, it was shown that Nef G63E mutation reduced binding of Nef to PI3K p85/p110 and mTORC2 GβL/mLST8 and MTOR components - kinase complex responsible AKT-S473 phosphorylation. In vitro B-cell Nef invasion and in vivo imaging/flow cytometry-based assays were employed to suggest that Nef from infected cells can target Env-specific B cells. Lastly, it was determined that NAb inducers have significantly higher Env-specific B-cells responses after Nef G63E selection when compared to NAb non-inducers. Finally, a corollary was drawn between the Nef G63E-associated B-cell/NAb induction phenotype and activated PI3K delta syndrome (APDS), which is caused by activating GOF mutations in PI3K, to suggest that Nef G63E-meidated induction of NAb response is reciprocal to APDS.

Strengths:

This study aims to understand the viral-host interaction that governs NAb induction in SIVmac239-infected macaques - this could enable identification of determinants important for induction of NAb responses against hard-to-neutralize tier-2/3 HIV variants. The finding that SIV-specific B-cell responses are induced following Nef G63E CD8+ T-cell escape mutant selection argue for an evolutionary trade-off between CTL escape and NAb induction. Exploitation of such a cellular-humoral immune axis could be important for HIV/AIDS vaccine efforts.

Although more validation and mechanistic basis are needed, the corollary between PI3K hyperactive signaling during autoimmune disorders and Nef-mediated abrogated PI3K signaling could help identify novel targets and modalities for targeting immune disorders and viral infections.

We are grateful for the supportive and insightful comments. The work did seem to unintendedly highlight a conceptual link between extrinsic and intrinsic immune perturbations. We will keep working on both wings, aiming to evoke synergisms.

Weaknesses:

Although the paper does have strengths in principle, the weaknesses of the paper are that the mechanistic basis of Nef-mediated induction of NAb responses are not directly examined. For example, it remains unclear whether SIVmac239 with engineered G63E mutation in Nef would induce faster and potent NAb responses. A macaque challenge study is needed to address this point.

We appreciate the point. We do have certain difficulties in availability of macaques for de novo experiments. As partially discussed in ver1, the identified Nef phenotype selected post-acute infection confers an enhanced CD4+ T cell-killing effect (revised Fig 4F), and it is likely that de novo infection with the mutant would redirect the trajectory of infection to rapid disease/AIDS progression accompanying generalized immune failure by boosting acute-phase CD4 destruction. In other words, mutant de novo infection may not necessarily be directly discussable as an attempt for reconstitution. It appears equally critical to understand the mutant in vitro on an immunosignaling basis, and in the current work we have focused on depicting this as the first step. We will work on reconstitution experiments with emphasis on pharmacology in our future study.

As presented, the central premise of the paper involves infected cell-generated Nef (WT or G63E mutant) being targeted to adjacent Env-specific B cells. However, it remains unclear how this is transfer takes place. A direct evidence demonstrating CD4+ T cell-associated and/or cell-free Nef being transferred to B-cell is needed to address this concern.

We appreciate the point, also pointed out by Reviewers 2 and 3. We have performed three sets of in vitro reconstitution experiments graphically/functionally addressing how Nef transfer from CD4+ T cells to B cells can be modulated (new Fig 6) and edited text accordingly.

The interaction between Nef and PI3K signaling components (p85, p110, GβL/mLST8, and MTOR) has been explored using PLA assay, however, this requires validation using additional biochemical and/or immunoprecipitation-based approaches. For example, is Nef (WT or mutant form) sufficient to affect PI3K-induced phosphorylation of Akt in an in vitro kinase assay? Moreover, the details regarding the binding events of WT vs mutant Nef with PI3K signaling components is lacking in this study. Lastly, it is unclear whether the interaction of Nef with PI3K signaling components is a conserved function of all primate lentiviruses or is this SIV-specific phenotype.

We appreciate the point. Co-immunoprecipitation analysis via pulldown with the mTORC2-intrinsic cofactor Sin1 (revised Fig 4E), showing decreased G63E-Nef binding, should confer robustness to the statement combined with initial manipulation results (Fig 4C). As Sin1 is mTORC2- and not mTORC1-intrinsic, results should be strengthened. Phosflow may be a standard readout nowadays for pAkt itself. Related with sequence variation, conservation will be addressed in studies ahead. We concisely mentioned on this in the revision (Lines 390-391).

It has been previously reported that the region of Nef encoding glycine at position 63 is not conserved in HIV-1 (Schindler et al, Journal of Virology 2004). Thus, does HIV-1 Nef also function in induction of NAb responses in humans? or the observed phenotype specific to SIV?

We appreciate the point, and do not have an answer at the moment. We will explore in our HIV-1-infected patient cohort (Hau et al, AIDS 2022) and other occasions whether corresponding phenotypes may exist. We have mentioned on this point in the revised manuscript (Line 392-393).

Reviewer #2 (Public Review):

It is well known that human and simian immunodeficiency viruses (HIV and SIV, respectively) evolved numerous mechanisms to compromise effective immune responses but the underlying mechanisms remain incompletely understood. Here, Yamamoto and Matano examined the humoral immune response in a large number of rhesus macaques infected with the difficult-to-neutralize SIVmac239 strain. They identified a subgroup of animals that showed significant neutralizing Ab responses. Sequence analyses revealed that in most of these animals (7/9) but only a minority in the control group (2/19) SIVmac variants containing a CD8+ T-cell escape mutation of G63E/R in the viral Nef gene emerged. They further show that this change attenuates the ability of Nef to stimulate PI3K/Akt/mTORC2 signaling. The authors propose that this induction of SIVmac239 nAb induction is reciprocal to antibody dysregulation caused by a previously identified human PI3K gain-of-function (Ref). Altogether, the results suggest that PI3K signaling plays a key role in B-cell maturation and generation of effective nAb responses.

Strengths of the study are that the authors analyzed a large number of SIVmac-infected macaques to unravel the biological significance of the known effect of the interaction of Nef with PI3K/Akt/mTORC2 signaling. This is interesting and may provide a novel means to improve humoral immune responses to HIV. Weaknesses are that only G63E and not G63R that also emerged in most animals was examined in most functional assays. Some effects of the G63E mutation seem modest and comparison to a grossly nef-defective SIVmac construct would be desirable to better assess to impact of the mutation of Nef-mediated stimulation of PI3K. While the impact of this Nef mutations on PI3K and the association with improved nAb responses is largely convincing, the results on the potential impact of soluble Nef on neighboring B cells is much less clear. SIVmac239 infects and manipulates helper CD4 T cells and these are essential for the activation and differentiation of B cells into antibody-producing plasma cells and effective humoral immune responses. Without additional functional evidence that Nef indeed specifically targets and manipulated B cells these results and conclusions should be made with much greater caution. Finally, the presentation of the results and conclusions is partly very convoluted and difficult to comprehend. Editing to improve clarity is highly recommended.

We are very grateful for the supportive and visionary review and suggestions. Experiments have been performed to improve the points raised. This work inevitably involved interdisciplinary factors to even hit on the schematic (NAbs, B cells, CD4+T, CD8+T, viral escape, immunosignaling, IEI as extrapolation & microscopy implementations) and convoluted sections should have existed. We attempted streamlining of certain portions and edited writing throughout, and hope that it became more straightforward.

Reviewer #2 (Recommendations For The Authors):

As outlined in the public review, I found the results potentially very interesting but parts of the manuscript much more complex and confusing than necessary. In addition, the methods on the potential impact of soluble Nef on neighboring B cells in vivo was difficult to assess but altogether this part was not convincing. Have the following specific suggestions:

We are very grateful for the scholarly review, and encouraging and suggestive comments on this orphan work. In the revision we designed experiments to address the properties of Nef transfer to append understanding on the in vivo B-cell data. Recommendations have been addressed as follows.

(1) Title: "AIDS virus-neutralizing antibody induction reciprocal to a PI3K gain-of-function disease". Think this title hardly reflects the data; SIVmac cause simian AIDS and is not the "AIDS virus" the 2nd part is more appropriate for discussion than for the title (and the abstract).

We appreciate the point. The original intent of the title was to conceptually bridge two differing fields of virus-host interaction and inborn errors of immunity/immunosignaling on an original article basis. Certain papers (Mudd et al, Nature 2012 etc) do utilize the term AIDS virus, and we similarly chose the term for simplification to non-virologists at initial submission.

That being said, we understand the scholarly point raised, and feel that the initial aim can be well attained by retaining the key host effector PI3K in the title, as in the revised submission titled “SIV-specific neutralizing antibody induction following selection of a PI3K drive-attenuated nef variant”.

(2) Abstract and throughout: As the authors show, SIVmac is not generally "neutralization resistant"; difficult to neutralize is more appropriate and should be used throughout. Also, the abstract and other parts are more complicated than necessary.

We appreciate the point. HIV/SIV Env immunology work utilizes “neutralization-resistant” for SIVmac239 (e.g., Mason et al, PLoS Pathog 2016), and autologous titer positivity of ~10% at this size of examination does appear low amongst lentiviruses. Nevertheless, as recommended, “difficult-to-neutralize” better describes the nature, and we have switched the term accordingly.

Linked with title modification, we reflected the comment on abstract structure and switched the main introductory sentence (Here we…) to a more data-based one instead of depicting extrapolation, and have modified phrasings in the latter half.

(3) The intro seems a bit biased. Immune evasion due to mutations and proviral integration that play key roles in viral persistence are not mentioned. nAbs are not known to efficiently control HIV or SIV replication in vivo (not even in the present study). Thus, a more "balanced" presentation of the role of nAbs in vivo is desirable.

We agree with the comment. Introduction in ver1 submission was compressed to just display humoral immune perturbation examples across persistence-prone viral infections, and indeed it should be much better to layout the multiscale strategies of lentiviruses in manifesting viral persistence. We have appended two sets of texts, one on the fundamental integrating retroviral life cycle and another on the wide spectrum of accessory protein-driven perturbation. As pointed out, the current endogenous induction is of course not early enough to exert suppressive impact on replication as like in exogenous Ab passive infusions. We have accordingly modulated text to improve the balance.

(4) Lines 73-76: rephrase for clarity.

We acknowledge the comment and have rephrased accordingly.

(5) Line 92: "linked with sustained Env-specific B-cell responses after the mutant Nef selection". After or during in one case; the time frame varies enormously and this should be discussed.

We appreciate the comment. The six Nef-G63E mutant-selecting NAb inducers subjected to B-cell analysis were the ones that showed precedence in Fig 2D (mutant before induction). That being said, we modified text as suggested (Line 104 in revised uploaded text). Text related to temporal deviation has been appended (Lines 378-383 in revised uploaded text).

(6) The authors should discuss G63R and include it in the functional analyses.

We appreciate the comment. Discussion on Nef-G63R in ver1 submission was kept minimal because statistical significance for selection was marginal. We generated a Nef-G63R mutant and results are appended in Fig 4-Figure Supplement 2.

(7) Lines 124/5: conservation only applies to SIVsmm/mac Nefs and this region is also frequently deleted/length-variable in primary HIV-1 Nefs.

We appreciate the comment. We modified description of the region accordingly (Lines 139-141 in revised text).

(8) Lines 153-155: Statement doesn't seem to make sense. The triple mutant Nef SIVmac construct was not attenuated for replication but specifically disrupted in CD3 down-modulation.

We acknowledge the comment. It had meant that the consequent plasma viral load showed a trend of decrease (as in the Graphical Abstract of the work) which should (in a simplistic view) influence antigenicity for humoral immune responses. Yet it is very true that virological replicative capacity was comparable with wild-type as in Fig.1. We have taken down the related text and rephrased it (Ref remains cited in introduction).

(9) Lines 178/9: levels in PI3K gain-of-function mice "with full disease phenotype (Avery et al., 2018)". This needs more information, e.g. what disease exactly are they talking about?

We are grateful for the correction, and have appended text and introduced the mentioned congenital disease in the Introduction section in advance. In-detail description is also appended in the Discussion section.

(10) Lines 186/7: "Env-stimulating high-MOI infection also accelerated phenotype appearance, with enhanced 50% reduction (Figure 4C, right)". Modify text and corresponding figure for clarity.

We acknowledge the comment. We revised as: “A high-MOI SIV infection, comprising higher initial concentration of extracellular Env stimuli, also accelerated phenotype appearance from day 3 to day 1 post-infection with stronger pAkt reduction”.

(11) The validity of the results described in the section "Targeting of lymph node Env-specific B cells by Nef in vivo" was difficult to assess. Altogether, however, I didn't find them convincing, especially since a negative control (e.g. macaques infected with nef-deleted SIVmac) are missing.

We acknowledge the comment. As a pure experimental control, whole-Nef deletion may assist for subtracted baselines. Within this work, the staining per se at least should be highly specific (mAb multiply verified in other applications and cytometry panel also designed for minimal spillover into AF488 channel). On in vivo basis, direct comparison may be somewhat frustrated by the fact that reduction in other pleiotropic effects of Nef seem to more dominate upon Nef deletion, as a set of reduced viremia, robust CD8 responses, killer CD4 responses and increased binding Ab titers (Johnson et al, J Virol 1997, Gauduin et al, J Exp Med 2006, Fukazawa et al, Nat Med 2012, Adnan et al, PLoS Pathog 2016 etc) leading to altered trajectory. We promise that we will work on refinement of the methodology in studies ahead.

(12) Lines 309-319: This paragraph made little sense to me (as did lines 328-331).

We acknowledge the comment and have edited both sections.

Reviewer #3 (Additional Reviewer):

In this manuscript, Hiroyuki Yamamoto et al examined virus-specific antibody responses and identified a subgroup of nine individuals, out of seventy SIVmac239 rhesus macaques of Burmese origin infected with SIVmac239, that develop neutralizing antibodies (NAb). The authors propose the emergence of a nef mutant (Nef-G63E) that impacts on B cell maturation resulting in PI3K gain-of-function.

My major concerns are:

The authors by different aspect addressed the role of the emergence of Nef-G63E mutant in individuals developing NAb. The manuscript is confused and the rational not always clearly stated. This reflects the two aspects of the manuscript (i) NAb identification in a subgroup of macaque and (2) the identification this nef mutation.

We are grateful for the comprehensive and scholarly comments. As pointed out, the work did need to confront potential bifurcation of the influence of the obtained viral immunosignaling phenotype for CD4-intrinsic (which might be your specialty) and B-cell-intrinsic impact. Based on your suggestions we have acquired additional data and revised the manuscript as attached.

The authors used both males (n=57) and females (n=13). However, there is no indication related to the sex regarding NAb inducers versus non-NAb Inducers. The notion of "highly pathogenic" is certainly not correct (see the introduction). Pathogenicity is also depending on monkey origin. Thus, cynomolgus are less sensitive to SIVmac239 or SIVmac251 compared to rhesus macaques (Ling B Aids 2002; Reimann KA, J Virol 2005; Cumont MC, J Virol 2008), or to pigtails used in US. Indeed, the authors used Burmese macaques, and therefore the dynamics of pathogenicity is different to rhesus macaque (Indian origin) housed in US. How many animals have been sacrificed out of the 61 animals? Herein, the animals are surviving longer (more than one year), and therefore the notion of "highly pathogenic" merits to be modulated.

We appreciate the comment. We have accordingly appended sex information (M/F: 8/1 versus 49/12 in NAb inducers vs non-inducers, p > 0.99 by Fisher’s exact test) in the methods section. As pointed out there are differences in the frequency and rate of AIDS progression among macaques of differing origin, whereas we have also previously reported reproducible AIDS progression dependent on MHC-I genotypes in the Burmese rhesus macaques utilized (Nomura, Yamamoto et al., J Virol 2012). Adhering to advice, we have attenuated the term to “pathogenic” in the revised manuscript and appended one reference showing pathogenesis gradation from a cell-death perspective (Cumont 2008).

Furthermore, no indication is provided regarding CD4 T cell dynamics, or CD8 T cells. In particular, the extent of T cell immunodeficiency may compromise humoral response. Therefore, this data needs to be shown. Indeed, previous reports have indicated that early CD4 T cell depletion is associated with defective humoral response. Furthermore, Tfh cell depletion was reported in several immune tissues, which are essential for B cell immune response like the spleen. Thus, this should be discussed as an alternative mechanism to the absence of NAb. Indeed, the authors found higher and persistent env-specific plasmablast cells in NAb inducers than that observed in non-NAb inducers figure 6. Why to have selected twelve individuals out of 61 individuals for assessing anti-env response (Supplemental S3 for figure 1, panel 1), and only eleven for western blots. The explanation in the text is absent. This requires to be clearly stated. See lines 108-110.

We appreciate the comment. As in other sections, this study utilized available cryopreserved samples from a retrospective cohort, also having heterogeneity in data acquisition along the way. We acknowledge that some supplemental data are particularly limited in information, which is also a reason they are presented in SI. We felt that one important core was to secure samples for Nef-G63E-selecting NAb inducers versus viremic non-inducers, for which we acquired six versus twelve in the B-cell analysis.

We (Nakane et al, PLoS ONE 2013) and others (Hirsch et al, J Virol 2004) have already reported on western blotting-basis that SIV-infected rapid progressors tend to manifest serological failure (impaired binding Ab-WB bands). Therefore, to compare quantitative traits at this basal stage (Fig 1), we judged that NAb inducer comparison with more non-rapid-progressing (>60 wk survival) non-inducers would be a criterion. We have mentioned on this in the revised manuscript (results/methods). Additionally, we have replaced the immunoblotting result with one more non-inducer (n = 12) to enhance results. Please note that there are lot deviations in strip-coated antigen (e.g., gp160) but the result is comparable (now covers 12/13 of animals with >60-wk survival).

The authors indicated the frequencies of Nef-G63E mutant in figure 2 panel C. However. no information is indicated in the legend about the number of NAb non-inducers used to calculate this frequency. The authors indicated line 127, "only in two of the nineteen NAb non-inducers, including one rapid progressor". Thus, different numbers of individuals are used through the manuscript. For the readers, this is clearly a statement that needs to be clarify and to refer to what. This is not homogeneous along the text and the analyses performed.

We appreciate the comment, and have appended the number in the revised Fig 2C. As aforementioned, heterogeneity of sample number in different sections is indeed a limitation of the work, and have mentioned this in the Discussion.

The rational related to the sentence lines 140-142. Please clarify.. "NAb induction is not associated with these MHC-I genotypes (P = 0.25 by Fisher's exact test, data not shown) but with the Nef-G63E mutation itself".

We appreciate the comment. We have rephrased it as:

“Ten of nineteen NAb non-inducers also had either of these alleles (Figure 1-figure supplement 1). This did not significantly differ with the NAb inducer group (P = 0.25 by Fisher’s exact test, data not shown), indicating that NAb induction was not simply linked with possession of these MHC-I genotypes but instead required furthermore specific selection of the Nef-G63E mutation.” (Lines 159-162).

In supplemental figure 3, only 7 individuals have been tested, while the authors indicated "Ten of nineteen NAb non-inducers also had either of these alleles". Why only seven? In NAb Burmese monkeys, the authors indicate specific T cells capable to recognize WT nef peptide, but not G63E peptide mutant. Thus, nef is immunogenic in vivo generating T cells despite to be mutated.

In contrary, non-NAb-inducers demonstrate the absence of nef specific T cells (supplemental figure 3, excepted R01-011 panel A). Although, the authors propose an escape mutant for CD8 T cells, this is not associated with the absence of immunogenicity and not with a difference in viral load in comparison to NAb inducers (panel C). Therefore, the conclusions merit to be revised. Thus, this part of the manuscript is confusing. Please clarify the rational to link NAb and Nef specific CD8 T cells.

We appreciate the comment. 7 out of 8 non-inducers positive for the allele and not selecting for the Nef-G63E mutant was available for analysis. The relative contribution of this single Nef62-70 epitope-specific CTL response is speculated not to be largely impacting viral control, among the many induced. This is basally discussed in a previous paper (Nomura, Yamamoto et al., J Virol 2012), more suggestive of an MHC-I haplotype-level correlation with plasma viral load. We assume that the CTL pressure-driven selection of Nef-G63E mutant was a rather pure immunosignaling trigger under persistent viremia. We appended this in the revised text (Line 172).

In the next part of the manuscript, the authors assessed the function of this Nef-G63E mutant. The rational to introduce Ferritin in this part of the document is not clear for the reader. Furthermore, a subgroup for each (NAb+ versus NAb-) is shown: 4 for NAbneg versus 6 for NAbpos.

We appreciate the point. As introduced, Swingler et al Cell Host Microbe 2008 reported HIV-infected macrophage-derived ferritin as a potentially B cell-disrupting factor. In that paper, viral load, ferritin and binding antibody titers positively correlated. Current data shows that SIVmac239-specific NAb induction is distinct from such kinetics already versus viral load (Fig 3-Supplement 1C), and ferritin levels were measured for some available samples more simply for confirmation. We appended three more available samples in the NAb- group. (The six NAb+/G63E animals correspond to the ones with B-cell data in Figure 7.) Statistical results appear unaffected and robust, as shown in this version. The revised manuscript incorporates appended explanation for the former.

Similarly, whereas the authors observed a role of nef mutant on pAkt Ser473 (less induced) in comparison to WT, the authors suggest that this may have an impact on T cell survival.

We appreciate the point. In the first submission we obtained peripheral memory Tfh decrease, whereas it is true that this is indirect. In the current revision we have addressed apoptotic cell death, shown to increase with Nef-G63E mutation (Figure 4F).

The rational to analyze CXCR3-CXCR5+PD-1+ memory follicular Th (Tfh) is not clear. Moreover, the references used are not the adequately cited. Indeed, these papers show an expansion. See the literature for a depletion (Xu H, J Immunol. 2015; Moukambi F, PLoS Pathog. 2015; Yamamoto T, Sci Transl Med. 2015; Xu H, J Immunol. 2018 Moukambi F, Mucosal Immunol. 2019).

We appreciate these points on in vivo CD4+ T cells.

Peripheral memory Tfh was reported to correlate with Ab cross-reactivity in one human cohort (Locci et al, Immunity 2013) and we concisely examined the subset in the current NAb induction. We mentioned this in the revised manuscript.

Moukambi F et al, PLoS Pathog 2015 & Mucosal Immunol 2019 are demonstrative work on acute-phase destruction. We have cited non-neonatal/vaccine-related ones suggested, including these two, in the revised manuscript. The biphasic dysregulation of Th (acute-phase destruction and chronic-phase adverse hyper-expansion) may indeed have a unique role with the current phenotype, which is beyond aim of the current analysis. We have concisely mentioned on this in the Discussion.

Then, the authors assess the potential B-cell-intrinsic influence of the G63E-Nef phenotype. The rational here is clearly indicated, making sense with figure 1. Furthermore, this part is clearer. The dot-plots merit to be revised and the markers used better stated. The authors indicate that Nef invasion upregulates pAkt Ser473 assuming aberrant PI3K/mTORC2 signaling. What is the impact of Nef-G63E mutant on pAkt Ser473 using in vitro model of transfer. This is not addressed for comparison.

We appreciate the remarks/suggestions, also pointed out by Reviewers 1 and 2. We have performed three sets of in vitro reconstitution experiments visually and functionally addressing how Nef transfer to B cells can be modulated (new Fig 6), and edited text accordingly.

Minor points are:

- the presence of references in the legend.

-some Ab clones are in the table, however they are not used such CD38 and CD138, which are well known to be non-valid B cell markers for monkeys."

We appreciate the suggestions.

Mentioning on reference have been removed from the legend (Fig.1, Fig. 3) and moved to the corresponding Methods section (Fig. 1).

We also understood this well in advance (CD38/CD138), and incorporated them in the memory B-cell panel just to check whether they ever behave in a specific pattern. As expected, no notable behavior was observed in these NAb inducers.

Crash course on typewriter maintenance and repair

A list of resources and references for the budding typewriter repair person. There is a lot here that I've compiled and consumed over the last six months, so don't be overwhelmed. Half the battle is figuring out where to find all these things, so if nothing else, this should shave off a month of reading and researching.

Basic Introductory Material

Get a notebook and be ready to take some notes so you'll remember where you found the random information you're bound to pick up over time and are able to occasionally review it.

Work your way through Sarah Everett's excellent Typewriter 101 videos (at least the first five): https://www.youtube.com/playlist?list=PLJtHauPh529XYHI5QNj5w9PUdi89pOXsS

Read Richard Polt's book which is a great overview to the general space:<br /> Polt, Richard. The Typewriter Revolution: A Typist’s Companion for the 21st Century. 1st ed. Woodstock, VT: Countryman Press, 2015.

Next watch the documentary California Typewriter. Documentary. Gravitas Pictures, 2016. https://www.imdb.com/title/tt5966990/. It has some interesting subtle material hiding within it, but it will give you a good idea of where you're headed off to.

Get a machine (or four) you can practice on. Get a flat head screwdriver and maybe a small adjustable wrench. Buy some mineral spirits and a small headed toothbrush and clean out your first machine. Buy some light sewing machine oil and try oiling it. Search YouTube for videos about how to repair anything that may be wrong with it.

Basic restoration advice: https://site.xavier.edu/polt/typewriters/tw-restoration.html

On colloquial advice for degreasing, cleaning, and oiling manual typewriters https://boffosocko.com/2024/08/09/on-colloquial-advice-for-degreasing-cleaning-and-oiling-manual-typewriters/

Repair Manuals

Create an account on typewriterdatabase.com which will give you some additional access to catalogs, manuals, and dealer catalogs.

They also have some openly accessible material like:<br /> * https://typewriterdatabase.com/manuals.php

Printed manuals: https://www.lulu.com/search?adult_audience_rating=00&contributor=Ted+Munk&page=1&pageSize=50 PDF manuals: https://sellfy.com/twdb

Ted Munk's website also has a plethora of ephemera that is often useful * https://munk.org/typecast/

Richard Polt's list of service manuals, which also includes some correspondence course typewriter repair classes: https://site.xavier.edu/polt/typewriters/tw-manuals.html#servicemanuals

Tools

In rough order of increasing complexity:

• Typewriter Tool Kit from the DOLLAR TREE by Just My Typewriter https://www.youtube.com/watch?v=R-7E4fM6gBw
• Some simple basics and where to get them: https://boffosocko.com/2024/08/11/adding-to-my-typewriter-toolset/
• Typewriter tools of the trade: https://www.youtube.com/watch?v=x5bKk93rtxs
• Lucas Dul's Toolset presentation: https://virtualhermans.com/lucas-dul
• Tour of an advanced hobbyist/semi-pro shop: https://www.youtube.com/watch?v=lZjf6InMlgU

Tools can be expensive, so start out small with just a few things and expand as you need them. You'll be amazed at what you can accomplish with a single thin bladed flathead screwdriver, an adjustable wrench, a rag, a bottle of Simple Green cleaning solution, and a bottle of isopropyl alcohol.

Videos

Subscribe to and become acquainted with YouTube channels like the following:

• Duane Jensen's Phoenix Typewriter https://www.youtube.com/@phoenixtypewriter2136
• Gerren Balch's HotRod Typewriter Company https://www.youtube.com/@HotRodTypewriter
• Joe Van Cleave https://www.youtube.com/@Joe_VanCleave
• Sarah Everett's Just My Typewriter https://www.youtube.com/@JustMyTypewriter

While watching a variety of videos is great, as you're doing specific repairs search YouTube and you're likely to find full demos of the repairs you're doing yourself.

I've compiled a playlist of videos for repair of an Olympia SM3 which, while specific to the SM3, is a an excellent outline/overview of how to disassemble a portable typewriter, where many of the adjustment points are as well as an outline of the order to do them in.

If you're not a good typist or don't have experience in the area, try out some of the following short films which will also provide some useful historical perspective:

• Basic Typing: Methods. Vol. MN-1512a. United States Navy Training Film, 1943. https://www.youtube.com/watch?v=ztyzGit1dTI.
• Basic Typing: Machine Operation. Vol. MN-1512b. United States Navy Training Film, 1943. https://www.youtube.com/watch?v=b-REJEArnjE.
• Advanced Typing: Shortcuts. Vol. MN-1512c. United States Navy Training Film, 1943. https://www.youtube.com/watch?v=JUJfCfqgsX0.
• Advanced Typing: Duplicating and Manuscript. Vol. MN-1512d. United States Navy Training Film, 1943. https://www.youtube.com/watch?v=7ve5JnTUzvo.
• Maintenance Of Office Machines. Vol. MN-1513. United States Navy Training Film, 1943. https://www.youtube.com/watch?v=ocdxgkxKAKo.

Internships & Apprenticeships

If you have the time and flexibility try arranging an internship or apprenticeship with a local typewriter repair shop. Meet your local repair people even if you can't spend the time on an internship. You'll learn a lot and create relationships with businesses who will more easily swap/supply you with machines they're parting out or access to tools which may otherwise be difficult to source.

• https://site.xavier.edu/polt/typewriters/tw-repair.html

Podcasts

• Austin Typewriter Ink https://www.austintypewriterink.com/ati-the-podcast.html
• Charm Type Repair https://charmtypepodcast.podbean.com/

Some useful Bibliography

• Athey, Ralph S. Typewriter Repair Training Course. Tarentum, PA: Typewriter Repair Training, 1957. https://site.xavier.edu/polt/typewriters/AtheyTypewriterRepair.pdf.
• Atkinson, Annelise. Typewriter SOS: The DIY Guide to Fixing Common Problems with Typewriters, 2014. https://www.amazon.com/Typewriter-SOS-Fixing-Problems-Typewriters/dp/1520902700/.
• Hausrath, Alfred H., and Eugene L. Dahl. Typewriter Care. Edited by Walter K.M. Slavik. Federal Work Improvement Program United States Civil Service Commission and Government Division, U.S. Treasury Department, 1945. http://archive.org/details/twcare-1945.
• Jones, Clarence LeRoy. The Manual Typewriter Repair Bible. Edited by Theodore Munk. The Typewriter Repair Bible Series, 2017. https://www.lulu.com/shop/ted-munk/the-manual-typewriter-repair-bible/paperback/product-1vgk72jp.html.
• Kasten, R. M. “First Aid for Typewriters.” Popular Science Monthly, May 1941.
• Kravitz, Bryan. Hints for a Happy Typewriter. Bryan Kravitz, 1983. https://site.xavier.edu/polt/typewriters/Hints-Happy-TW.pdf.
• Hutchison, Howard. The Typewriter Repair Manual. 1st ed. Blue Ridge Summit, Pa: Tab Books, 1981. https://www.amazon.com/typewriter-repair-manual-Howard-Hutchison/dp/0830600345.
• Munk, Theodore. “The Typewriter Database,” 2012. https://typewriterdatabase.com/.
• Pearce, H. G. Complete Instructions: How to Repair, Rebuild, and Adjust Underwood Typewriters With Handy Reference for Locating Trouble Quickly. Bridgeport, CT: Typewriter Mechanics Publishing Co., 1920. https://johnesimmons.com/Typewriter/Articles/Manualpdf/Underwood_Repair_Manual.pdf.
• Polt, Richard. “The Classic Typewriter Page : All About Typewriters,” 2009. https://site.xavier.edu/polt/typewriters/index.html.
• Scadden, David T. Approved Home Study Course in Typewriter Repair and Service. Little Falls, NJ: Typewriter Repair School, 1959. https://site.xavier.edu/polt/typewriters/homestudycourse.pdf.

Good luck on your journey!

reply to u/fontinalispluma at https://old.reddit.com/r/typewriters/comments/1gaza5x/learning_typewriter_maintenance_and_repair/

Author response:

Reviewer #1 (Public Review):

Padilha et al. aimed to find prospective metabolite biomarkers in serum of children aged 6-59 months that were indicative of neurodevelopmental outcomes. The authors leveraged data and samples from the cross-sectional Brazilian National Survey on Child Nutrition (ENANI-2019), and an untargeted multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) approach was used to measure metabolites in serum samples (n=5004) which were identified via a large library of standards. After correlating the metabolite levels against the developmental quotient (DQ), or the degree of which age-appropriate developmental milestones were achieved as evaluated by the Survey of Well-being of Young Children, serum concentrations of phenylacetylglutamine (PAG), cresol sulfate (CS), hippuric acid (HA) and trimethylamine-N-oxide (TMAO) were significantly negatively associated with DQ. Examination of the covariates revealed that the negative associations of PAG, HA, TMAO and valine (Val) with DQ were specific to younger children (-1 SD or 19 months old), whereas creatinine (Crtn) and methylhistidine (MeHis) had significant associations with DQ that changed direction with age (negative at -1 SD or 19 months old, and positive at +1 SD or 49 months old). Further, mediation analysis demonstrated that PAG was a significant mediator for the relationship of delivery mode, child's diet quality and child fiber intake with DQ. HA and TMAO were additional significant mediators of the relationship of child fiber intake with DQ.

Strengths of this study include the large cohort size and study design allowing for sampling at multiple time points along with neurodevelopmental assessment and a relatively detailed collection of potential confounding factors including diet. The untargeted metabolomics approach was also robust and comprehensive allowing for level 1 identification of a wide breadth of potential biomarkers. Given their methodology, the authors should be able to achieve their aim of identifying candidate serum biomarkers of neurodevelopment for early childhood. The results of this work would be of broad interest to researchers who are interested in understanding the biological underpinnings of development and also for tracking development in pediatric populations, as it provides insight for putative mechanisms and targets from a relevant human cohort that can be probed in future studies. Such putative mechanisms and targets are currently lacking in the field due to challenges in conducting these kind of studies, so this work is important.

However, in the manuscript's current state, the presentation and analysis of data impede the reader from fully understanding and interpreting the study's findings.

Particularly, the handling of confounding variables is incomplete. There is a different set of confounders listed in Table 1 versus Supplementary Table 1 versus Methods section Covariates versus Figure 4. For example, Region is listed in Supplementary Table 1 but not in Table 1, and Mode of Delivery is listed in Table 1 but not in Supplementary Table 1. Many factors are listed in Figure 4 that aren't mentioned anywhere else in the paper, such as gestational age at birth or maternal pre-pregnancy obesity.

We thank the reviewer for their comment. We would like to clarify that initially, the tables had different variables because they have different purposes. Table 1 aims to characterize the sample on variables directly related to the children’s and mother’s features and their nutritional status. Supplementary File 1(previously named supplementary table 1) summarizes the sociodemographic distribution of the development quotient. Neither of the tables concerned the metabolite-DQ relationships and their potential covariates, they only provide context for subsequent analyses by characterizing the sample and the outcome. Instead, the covariates included in the regression models were selected using the Direct Acyclic Graph presented in Figure 1.

To avoid this potential confusion however, we included the same variables in Table 1 and Supplementary File 1(page 38) and we discussed the selection of model covariates in Figure 4 in more detail here in the letter and in the manuscript.

The authors utilize the directed acrylic graph (DAG) in Figure 4 to justify the further investigation of certain covariates over others. However, the lack of inclusion of the microbiome in the DAG, especially considering that most of the study findings were microbial-derived metabolite biomarkers, appears to be a fundamental flaw. Sanitation and micronutrients are proposed by the authors to have no effect on the host metabolome, yet sanitation and micronutrients have both been demonstrated in the literature to affect microbiome composition which can in turn affect the host metabolome.

Thank you for your comment. We appreciate that the use of DAG and lack of the microbiome in the DAG are concerns. This has been already discussed in reply #1 to the editor that has been pasted below for convenience:

Thank you for the comment and suggestions. It is important to highlight that there is no data on microbiome composition. We apologize if there was an impression such data is available. The main goal of conducting this national survey was to provide qualified and updated evidence on child nutrition to revise and propose new policies and nutritional guidelines for this demographic. Therefore, collection of stool derived microbiome (metagenomic) data was not one of the objectives of ENANI-2019. This is more explicitly stated as a study limitation in the revised manuscript on page 17, lines 463-467:

“Lastly, stool microbiome data was not collected from children in ENANI-2019 as it was not a study objective in this large population-based nutritional survey. However, the lack of microbiome data does not reduce the importance/relevance, since there is no evidence that microbiome and factors affecting microbiome composition are confounders in the association between serum metabolome and child development.”

Besides, one must consider the difficulties and costs in collecting and analyzing microbiome composition in a large population-based survey. In contrast, the metabolome data has been considered a priority as there was already blood specimens collected to inform policy on micronutrient deficiencies in Brazil. However, due to funding limitations we had to perform the analysis in a subset of our sample, still representative and large enough to test our hypothesis with adequate study power (more details below).

We would like to argue that there is no evidence that microbiome and factors affecting microbiome composition are confounders on the association between serum metabolome and child development. First, one should revisit the properties of a confounder according to the epidemiology literature that in short states that confounding refers to an alternative explanation for a given conclusion, thus constituting one of the main problems for causal inference (Kleinbaum, Kupper, and Morgenstern, 1991; Greenland & Robins, 1986; VanderWeele, 2019). In our study, we highlight that certain serum metabolites associated with the developmental quotient (DQ) in children were circulating metabolites (e.g., cresol sulfate, hippuric acid, phenylacetylglutamine, TMAO) previously reported to depend on dietary exposures, host metabolism and gut microbiota activity. Our discussion cites other published work, including animal models and observational studies, which have reported how these bioactive metabolites in circulation are co-metabolized by commensal gut microbiota, and may play a role in neurodevelopment and cognition as mediated by environmental exposures early in life.

In fact, the literature on the association between microbiome and infant development is very limited. We performed a search using terms ‘microbiome’ OR ‘microbiota’ AND ‘child development’ AND ‘systematic’ OR ‘meta-analysis’ and found only one study: ‘Associations between the human immune system and gut microbiome with neurodevelopment in the first 5 years of life: A systematic scoping review’ (DOI 10.1002/dev.22360). The authors conclude: ‘while the immune system and gut microbiome are thought to have interactive impacts on the developing brain, there remains a paucity of published studies that report biomarkers from both systems and associations with child development outcomes.’ It is important to highlight that our criteria to include confounders on the directed acyclic graph (DAG) was based on the literature of systematic reviews or meta-analysis and not on single isolated studies.

In summary, we would like to highlight that there is no microbiome data in ENANI-2019 and in the event such data was present, we are confident that based on the current stage of the literature, there is no evidence to consider such construct in the DAG, as this procedure recommends that only variables associated with the exposure and the outcome should be included. Please find more details on DAG below.

Moreover, we would like to clarify that we have not stated that sanitation and micronutrients have no effect on the serum metabolome, instead, these constructs were not considered on the DAG.

To make it clearer, we have modified the passage about DAG in the methods section. New text, page 9, lines 234-241:

“The subsequent step was to disentangle the selected metabolites from confounding variables. A Directed Acyclic Graph (DAG; Breitling et al., 2021) was used to more objectively determine the minimally sufficient adjustments for the regression models to account for potentially confounding variables while avoiding collider variables and variables in the metabolite-DQ causal pathways, which if controlled for would unnecessarily remove explained variance from the metabolites and hamper our ability to detect biomarkers. To minimize bias from subjective judgments of which variables should and should not be included as covariates, the DAG only included variables for which there was evidence from systematic reviews or meta-analysis of relationships with both the serum metabolome and DQ (Figure 1). Birth weight, breastfeeding, child's diet quality, the child's nutritional status, and the child's age were the minimal adjustments suggested by the DAG. Birth weight was a variable with high missing data, and indicators of breastfeeding practice data (referring to exclusive breastfeeding until 6 months and/or complemented until 2 years) were collected only for children aged 0–23 months. Therefore, those confounders were not included as adjustments. Child's diet quality was evaluated as MDD, the child's nutritional status as w/h z-score, and the child's age in months.”

Additionally, the authors emphasized as part of the study selection criteria the following, "Due to the costs involved in the metabolome analysis, it was necessary to further reduce the sample size. Then, samples were stratified by age groups (6 to 11, 12 to 23, and 24 to 59 months) and health conditions related to iron metabolism, such as anemia and nutrient deficiencies. The selection process aimed to represent diverse health statuses, including those with no conditions, with specific deficiencies, or with combinations of conditions. Ultimately, through a randomized process that ensured a balanced representation across these groups, a total of 5,004 children were selected for the final sample (Figure 1)."

Therefore, anemia and nutrient deficiencies are assumed by the reader to be important covariates, yet, the data on the final distribution of these covariates in the study cohort is not presented, nor are these covariates examined further.

Thank you for the comments. We apologize for the misunderstanding and will amend the text to make our rationale clearer in the revised version of the manuscript.

We believed the original text was clear enough in stating that the sampling process was performed aiming to maintain the representativeness of the original sample. This sampling process considered anemia and nutritional deficiencies, among other variables. However, we did not aim to include all relevant covariates of the DQ-metabolome relationship; these were decided using the DAG, as described in the manuscript and other sessions of this letter. Therefore, we would like to emphasize that our description of the sampling process does not assumes anemia and nutritional deficiencies are important covariates for the DQ-metabolome relationship.

We rewrote this text part, page 11, lines 279-285:

“Due to the costs involved in the metabolome analysis, it was necessary to reduce the sample size that is equivalent to 57% of total participants from ENANI-2019 with stored blood specimens. Therefore, the infants were stratified by age groups (6 to 11, 12 to 23, and 24 to 59 months) and health conditions such as anemia and micronutrient deficiencies. The selection process aimed to represent diverse health statuses to the original sample. Ultimately, 5,004 children were selected for the final sample through a random sampling process that ensured a balanced representation across these groups (Figure 2).”

The inclusion of specific covariates in Table 1, Supplementary Table 1, the statistical models, and the mediation analysis is thus currently biased as it is not well justified.

We appreciate the reviewer comment. However, it would have been ideal to receive a comment/critic with a clearer and more straightforward argumentation, so we could try to address it based on our interpretation.

Please refer to our response to item #1 above regarding the variables in the tables and figures. The covariates in the statistical models were selected using the DAG, which is a cutting-edge procedure that aims to avoid bias and overfitting, a common situation when confounders are adjusted for without a clear rationale. We elaborate on the advantages of using the DAG in response to item #6 and in page 9 of the manuscript. The statistical models we use follow the best practices in the field when dealing with a large number of collinear predictors and a continuous outcome (see our response to the editor’s 4th comment). Finally, the mediation analyses were done to explore a few potential explanations for our results from the PLSR and multiple regression analyses. We only ran mediation analyses for plausible mechanisms for which the variables of interest were available in our data. Please see our response to reviewer 3’s item #1 for a more detailed explanation on the mediation analysis.

Finally, it is unclear what the partial-least squares regression adds to the paper, other than to discard potentially interesting metabolites found by the initial correlation analysis.

Thank you for the question. As explained in response to the editor’s item #4, PLS-based analyses are among the most commonly used analyses for parsing metabolomic data (Blekherman et al., 2011; Wold et al., 2001; Gromski et al. 2015). This procedure is especially appropriate for cases in which there are multiple collinear predictor variables as it allows us to compare the predictive value of all the variables without relying on corrections for multiple testing. Testing each metabolite in separate correlations corrected for multiple comparisons is less appropriate because the correlated nature of the metabolites means the comparisons are not truly independent and would cause the corrections (which usually assume independence) to be overly strict. As such, we only rely on the correlations as an initial, general assessment that gives context to subsequent, more specific analyses. Given that our goal is to select the most predictive metabolites, discarding the less predictive metabolites is precisely what we aim to achieve. As explained above and in response to the editor’s item #4, the PLSR allows us to reach that goal without introducing bias in our estimates or losing statistical power.  

Reviewer #2 (Public Review):

A strength of the work lies in the number of children Padilha et al. were able to assess (5,004 children aged 6-59 months) and in the extensive screening that the Authors performed for each participant. This type of large-scale study is uncommon in low-to-middle-income countries such as Brazil.

The Authors employ several approaches to narrow down the number of potentially causally associated metabolites.

Could the Authors justify on what basis the minimum dietary diversity score was dichotomized? Were sensitivity analyses undertaken to assess the effect of this dichotomization on associations reported by the article? Consumption of each food group may have a differential effect that is obscured by this dichotomization.

Thank you for the observation. We would like to emphasize that the child's diet quality was assessed using the minimum dietary diversity (MDD) indicator proposed by the WHO (World Health Organization & United Nations Children’s Fund (UNICEF), 2021). This guideline proposes the cutoff used in the present study. We understand the reviewer’s suggestion to use the consumption of healthy food groups as an evaluation of diet quality, but we chose to follow the WHO proposal to assess dietary diversity. This indicator is widely accepted and used as a marker and provides comparability and consistency with other published studies.

Could the Authors specify the statistical power associated with each analysis?

To the best of our knowledge, we are not aware of power calculation procedures for PLS-based analyses. However, given our large sample size, we do not believe power was an issue with the analyses. For our regression analyses, which typically have 4 predictors, we had 95% power to detect an f-squared of 0.003 and an r of 0.05 in a two-sided correlation test considering an alpha level of 0.05.

New text, page 11, lines 296-298:

“Given the size of our sample, statistical power is not an issue in our analyses. Considering an alpha of 0.05 for a two-sided test, a sample size of 5000 has 95% power to detect a correlation of r = 0.05 and an effect of f2 = 0.003 in a multiple regression model with 4 predictors.”

Could the Authors describe in detail which metric they used to measure how predictive PLSR models are, and how they determined what the "optimal" number of components were?

We chose the model with the fewest number of components that maximized R2 and minimized root mean squared error of prediction (RMSEP). In the training data, the model with 4 components had a lower R2 but a lower RMSEP, therefore we chose the model with 3 components which had a higher R2 than the 4-component model and lower RMSEP than the model with 2 components. However, the number of components in the model did not meaningfully change the rank order of the metabolites on the VIP index.

New text, page 8, lines 220-224:

“To better assess the predictiveness of each metabolite in a single model, a PLSR was conducted. PLS-based analyses are the most commonly used analyses when determining the predictiveness of a large number of variables as they avoid issues with collinearity, sample size, and corrections for multiple-testing (Blekherman et al., 2011; Wold et al., 2001; Gromski et al. 2015).”

New text, page 12, lines 312-314:

“In PLSR analysis, the training data suggested that three components best predicted the data (the model with three components had the highest R2, and the root mean square error of prediction (RMSEP) was only slightly lower with four components). In comparison, the test data showed a slightly more predictive model with four components (Figure 3—figure supplement 2).”

The Authors use directed acyclic graphs (DAG) to identify confounding variables of the association between metabolites and DQ. Could the dataset generated by the Authors have been used instead? Not all confounding variables identified in the literature may be relevant to the dataset generated by the Authors.

Thank you for the question. The response is most likely no, the current dataset should not be used to define confounders as these must be identified based on the literature. The use of DAGs has been widely explored as a valid tool for justifying the choice of confounding factors in regression models in epidemiology. This is because DAGs allow for a clear visualization of causal relationships, clarify the complex relationships between exposure and outcome. Besides, DAGs demonstrate the authors' transparency by acknowledging factors reported as important but not included/collected in the study. This has been already discussed in reply #1 to the editor that has been pasted below for convenience.

Thank you for the comment and suggestions. It is important to highlight that there is no data on microbiome composition. We apologize if there was an impression such data is available. The main goal of conducting this national survey was to provide qualified and updated evidence on child nutrition to revise and propose new policies and nutritional guidelines for this demographic. Therefore, collection of stool derived microbiome (metagenomic) data was not one of the objectives of ENANI-2019. This is more explicitly stated as a study limitation in the revised manuscript on page 17, lines 463-467:

“Lastly, stool microbiome data was not collected from children in ENANI-2019 as it was not a study objective in this large population-based nutritional survey. However, the lack of microbiome data does not reduce the importance/relevance, since there is no evidence that microbiome and factors affecting microbiome composition are confounders in the association between serum metabolome and child development.”

Besides, one must consider the difficulties and costs in collecting and analyzing microbiome composition in a large population-based survey. In contrast, the metabolome data has been considered a priority as there was already blood specimens collected to inform policy on micronutrient deficiencies in Brazil. However, due to funding limitations we had to perform the analysis in a subset of our sample, still representative and large enough to test our hypothesis with adequate study power (more details below).

We would like to argue that there is no evidence that microbiome and factors affecting microbiome composition are confounders on the association between serum metabolome and child development. First, one should revisit the properties of a confounder according to the epidemiology literature that in short states that confounding refers to an alternative explanation for a given conclusion, thus constituting one of the main problems for causal inference (Kleinbaum, Kupper, and Morgenstern, 1991; Greenland & Robins, 1986; VanderWeele, 2019). In our study, we highlight that certain serum metabolites associated with the developmental quotient (DQ) in children were circulating metabolites (e.g., cresol sulfate, hippuric acid, phenylacetylglutamine, TMAO) previously reported to depend on dietary exposures, host metabolism and gut microbiota activity. Our discussion cites other published work, including animal models and observational studies, which have reported how these bioactive metabolites in circulation are co-metabolized by commensal gut microbiota, and may play a role in neurodevelopment and cognition as mediated by environmental exposures early in life.

In fact, the literature on the association between microbiome and infant development is very limited. We performed a search using terms ‘microbiome’ OR ‘microbiota’ AND ‘child development’ AND ‘systematic’ OR ‘meta-analysis’ and found only one study: ‘Associations between the human immune system and gut microbiome with neurodevelopment in the first 5 years of life: A systematic scoping review’ (DOI 10.1002/dev.22360). The authors conclude: ‘while the immune system and gut microbiome are thought to have interactive impacts on the developing brain, there remains a paucity of published studies that report biomarkers from both systems and associations with child development outcomes.’ It is important to highlight that our criteria to include confounders on the directed acyclic graph (DAG) was based on the literature of systematic reviews or meta-analysis and not on single isolated studies.

In summary, we would like to highlight that there is no microbiome data in ENANI-2019 and in the event such data was present, we are confident that based on the current stage of the literature, there is no evidence to consider such construct in the DAG, as this procedure recommends that only variables associated with the exposure and the outcome should be included. Please find more details on DAG below.

Moreover, we would like to clarify that we have not stated that sanitation and micronutrients have no effect on the serum metabolome, instead, these constructs were not considered on the DAG.

To make it clearer, we have modified the passage about DAG in the methods section. New text, page 9, lines 234-241:

“The subsequent step was to disentangle the selected metabolites from confounding variables. A Directed Acyclic Graph (DAG; Breitling et al., 2021) was used to more objectively determine the minimally sufficient adjustments for the regression models to account for potentially confounding variables while avoiding collider variables and variables in the metabolite-DQ causal pathways, which if controlled for would unnecessarily remove explained variance from the metabolites and hamper our ability to detect biomarkers. To minimize bias from subjective judgments of which variables should and should not be included as covariates, the DAG only included variables for which there was evidence from systematic reviews or meta-analysis of relationships with both the serum metabolome and DQ (Figure 1). Birth weight, breastfeeding, child's diet quality, the child's nutritional status, and the child's age were the minimal adjustments suggested by the DAG. Birth weight was a variable with high missing data, and indicators of breastfeeding practice data (referring to exclusive breastfeeding until 6 months and/or complemented until 2 years) were collected only for children aged 0–23 months. Therefore, those confounders were not included as adjustments. Child's diet quality was evaluated as MDD, the child's nutritional status as w/h z-score, and the child's age in months.”

Were the systematic reviews or meta-analyses used in the DAG performed by the Authors, or were they based on previous studies? If so, more information about the methodology employed and the studies included should be provided by the Authors.

Thank you for the question. The reviews or meta-analyses used in the DAG have been conducted by other authors in the field. This has been laid out more clearly in our methods section.

New text, page 9, lines 234-241:

“The subsequent step was to disentangle the selected metabolites from confounding variables. A Directed Acyclic Graph (DAG; Breitling et al., 2021) was used to more objectively determine the minimally sufficient adjustments for the regression models to account for potentially confounding variables while avoiding collider variables and variables in the metabolite-DQ causal pathways, which if controlled for would unnecessarily remove explained variance from the metabolites and hamper our ability to detect biomarkers. To minimize bias from subjective judgments of which variables should and should not be included as covariates, the DAG only included variables for which there was evidence from systematic reviews or meta-analysis of relationships with both the metabolome and DQ (Figure 1). Birth weight, breastfeeding, child's diet quality, the child's nutritional status, and the child's age were the minimal adjustments suggested by the DAG. Birth weight was a variable with high missing data, and indicators of breastfeeding practice data (referring to exclusive breastfeeding until 6 months and/or complemented until 2 years) were collected only for children aged 0–23 months. Therefore, those confounders were not included as adjustments. Child's diet quality was evaluated as MDD, the child's nutritional status as w/h z-score, and the child's age in months.”

Approximately 72% of children included in the analyses lived in households with a monthly income superior to the Brazilian minimum wage. The cohort is also biased towards households with a higher level of education. Both of these measures correlate with developmental quotient. Could the Authors discuss how this may have affected their results and how generalizable they are?

Thank you for your comment. This has been already discussed in reply #6 to the editor and that has been pasted below for convenience.

Thank you for highlighting this point. The ENANI-2019 is a population-based household survey with national coverage and representativeness for macroregions, sex, and one-year age groups (< 1; 1-1.99; 2-2.99; 3-3.99; 4-5). Furthermore, income quartiles of the census sector were used in the sampling. The study included 12,524 households 14,588 children, and 8,829 infants with blood drawn.

Due to the costs involved in metabolome analysis, it was necessary to further reduce the sample size to around 5,000 children that is equivalent to 57% of total participants from ENANI-2019 with stored blood specimens. To avoid a biased sample and keep the representativeness and generability, the 5,004 selected children were drawn from the total samples of 8,829 to keep the original distribution according age groups (6 to 11 months, 12 to 23 months, and 24 to 59 months), and some health conditions related to iron metabolism, e.g., anemia and nutrient deficiencies. Then, they were randomly selected to constitute the final sample that aimed to represent the total number of children with blood drawn. Hence, our efforts were to preserve the original characteristics of the sample and the representativeness of the original sample.

The ENANI-2019 study does not appear to present a bias towards higher socioeconomic status. Evidence from two major Brazilian population-based household surveys supports this claim. The 2017-18 Household Budget Survey (POF) reported an average monthly household income of 5,426.70 reais, while the Continuous National Household Sample Survey (PNAD) reported that in 2019, the nominal monthly per capita household income was 1,438.67 reais. In comparison, ENANI-2019 recorded a household income of 2,144.16 reais and a per capita income of 609.07 reais in infants with blood drawn, and 2,099.14 reais and 594.74 reais, respectively, in the serum metabolome analysis sample.

In terms of maternal education, the 2019 PNAD-Education survey indicated that 48.8% of individuals aged 25 or older had at least 11 years of schooling. When analyzing ENANI-2019 under the same metric, we found that 56.26% of ≥25 years-old mothers of infants with blood drawn had 11 years of education or more, and 51.66% in the metabolome analysis sample. Although these figures are slightly higher, they remain within a reasonable range for population studies.

It is well known that higher income and maternal education levels can influence child health outcomes, and acknowledging this, ENANI-2019 employed rigorous sampling methods to minimize selection biases. This included stratified and complex sampling designs to ensure that underrepresented groups were adequately included, reducing the risk of skewed conclusions. Therefore, the evidence strongly suggests that the ENANI-2019 sample is broadly representative of the Brazilian population in terms of both socioeconomic status and educational attainment.

Further to this, could the Authors describe how inequalities in access to care in the Brazilian population may have affected their results? Could they have included a measure of this possible discrepancy in their analyses?

Thank you for the concern.

The truth is that we are not in a position to answer this question because our study focused on gathering data on infant nutritional status and there is very limited information on access to care to allow us to hypothesize. Another important piece of information is that this national survey used sampling procedures that aimed to make the sample representative of the 15 million Brazilian infants under 5 years. Therefore, the sample is balanced according to socio-economic strata, so there is no evidence to make us believe inequalities in access to health care would have played a role.

The Authors state that the results of their study may be used to track children at risk for developmental delays. Could they discuss the potential for influencing policies and guidelines to address delayed development due to malnutrition and/or limited access to certain essential foods?

The point raised by the reviewer is very relevant. Recognizing that dietary and microbial derived metabolites involved in the gut-brain axis could be related to children's risk of developmental delays is the first step to bringing this topic to the public policy agenda. We believe the results can contribute to the literature, which should be used to accumulate evidence to overcome knowledge gaps and support the formulation and redirection of public policies aimed at full child growth and development; the promotion of adequate and healthy nutrition and food security; the encouragement, support, and protection of breastfeeding; and the prevention and control of micronutrient deficiencies.  

Reviewer #3 (Public Review):

The ENANI-2019 study provides valuable insights into child nutrition, development, and metabolomics in Brazil, highlighting both challenges and opportunities for improving child health outcomes through targeted interventions and further research.

Readers might consider the following questions:

(1) Should investigators study the families through direct observation of diet and other factors to look for a connection between food taken in and gut microbiome and child development?

As mentioned before, the ENANI-2019 did not collect data on stool derived microbiome. However, there is data on child dietary intake with 24-hour recall that can be further explored in other studies.

(2) Can an examination of the mother's gut microbiome influence the child's microbiome? Can the mother or caregiver's microbiome influence early childhood development?

The questions raised by the reviewer are interesting and has been explored by other authors. However, we do not have microbiota data from the child nor from the mother/caregiver.

(3) Is developmental quotient enough to study early childhood development? Is it comprehensive enough?

Yes, we are confident it is comprehensive enough.

According to the World Health Organization, the term Early Childhood Development (ECD) refers to the cognitive, physical, language, motor, social and emotional development between 0 - 8 years of age. The SWCY milestones assess the domains of cognition, language/communication and motor. Therefore, it has enough content validity to represent ECD.

The SWYC is recommended for screening ECD by the American Society of Pediatrics. Furthermore, we assessed the internal consistency of the SWYC milestones questionnaire using ENANI-2019 data and Cronbach's alpha. The findings indicated satisfactory reliability (0.965; 95% CI: 0.963–0.968).

The SWCY is a screening instrument and indicates if the ECD is not within the expected range. If one of the above-mentioned domains are not achieved as expected the child may be at risk of ECD delay. Therefore, DQ<1 indicates that a child has not reached the expected ECD for the age group. We cannot say that children with DQ≥1 have full ECD, since we do not assess the socio-emotional domains. However, DQ can track the risk of ECD delay.

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Greenland S; Pearl J; Robins JM. Confounding and collapsibility in causal inference. Statist Sci. 14 (1) 29 - 46 1999. https://doi.org/10.1214/ss/1009211805

Author response:

Reviewer #2 (Public Review):

M. El Amri et al., investigated the functions of Marcks and Marcks like 1 during spinal cord (SC) development and regeneration in Xenopus laevis. The authors rigorously performed loss of function with morpholino knock-down and CRISPR knock-out combining rescue experiments in developing spinal cord in embryo and regeneration in tadpole stage.

For the assays in the developing spinal cord, a unilateral approach (knock-down/out only one side of the embryo) allowed the authors to assess the gene functions by direct comparing one-side (e.g. mutated SC) to the other (e.g. wild type SC on the other side). For the assays in regenerating SC, the authors microinject CRISPR reagents into 1-cell stage embryo. When the embryo (F0 crispants) grew up to tadpole (stage 50), the SC was transected. They then assessed neurite outgrowth and progenitor cell proliferation. The validation of the phenotypes was mostly based on the quantification of immunostaining images (neurite outgrowth: acetylated tubulin, neural progenitor: sox2, sox3, proliferation: EdU, PH3), that are simple but robust enough to support their conclusions. In both SC development and regeneration, the authors found that Marcks and Marcksl1 were necessary for neurite outgrowth and neural progenitor cell proliferation.

The authors performed rescue experiments on morpholino knock-down and CRISPR knock-out conditions by Marcks and Marcksl1 mRNA injection for SC development and pharmacological treatments for SC development and regeneration. The unilateral mRNA injection rescued the loss-of-function phenotype in the developing SC. To explore the signalling role of these molecules, they rescued the loss-of-function animals by pharmacological reagents They used S1P: PLD activator, FIPI: PLD inhibitor, NMI: PIP2 synthesis activator and ISA-2011B: PIP2 synthesis inhibitor. The authors found the activator treatment rescued neurite outgrowth and progenitor cell proliferation in loss of function conditions. From these results, the authors proposed PIP2 and PLD are the mediators of Marcks and Marcksl1 for neurite outgrowth and progenitor cell proliferation during SC development and regeneration. The results of the rescue experiments are particularly important to assess gene functions in loss of function assays, therefore, the conclusions are solid. In addition, they performed gain-of-function assays by unilateral Marcks or Marcksl1 mRNA injection showing that the injected side of the SC had more neurite outgrowth and proliferative progenitors. The conclusions are consistent with the loss-of-function phenotypes and the rescue results. Importantly, the authors showed the linkage of the phenotype and functional recovery by behavioral testing, that clearly showed the crispants with SC injury swam less distance than wild types with SC injury at 10-day post surgery.

Prior to the functional assays, the authors analyzed the expression pattern of the genes by in situ hybridization and immunostaining in developing embryo and regenerating SC. They confirmed that the amount of protein expression was significantly reduced in the loss of function samples by immunostaining with the specific antibodies that they made for Marcks and Marcksl1. Although the expression patterns are mostly known in previous works during embryo genesis, the data provided appropriate information to readers about the expression and showed efficiency of the knock-out as well.

MARCKS family genes have been known to be expressed in the nervous system. However, few studies focus on the function in nerves. This research introduced these genes as new players during SC development and regeneration. These findings could attract broader interests from the people in nervous disease model and medical field. Although it is a typical requirement for loss of function assays in Xenopus laevis, I believe that the efficient knock-out for four genes by CRISPR/Cas9 was derived from their dedication of designing, testing and validation of the gRNAs and is exemplary.

Weaknesses,

(1) Why did the authors choose Marcks and Marcksl1? The authors mentioned that these genes were identified with a recent proteomic analysis of comparing SC regenerative tadpole and non-regenerative froglet (Line (L) 54-57). However, although it seems the proteomic analysis was their own dataset, the authors did not mention any details to select promising genes for the functional assays (this article). In the proteomic analysis, there must be other candidate genes that might be more likely factors related to SC development and regeneration based on previous studies, but it was unclear what the criteria to select Marcks and Marcksl1 was.

To highlight the rationale for selecting these proteins, we reworded the sentence as follows: “A recent proteomic screen … after SCI identified a number of proteins that are highly upregulated at the tadpole stage but downregulated in froglets (Kshirsagar, 2020). These proteins included Marcks and Marcksl1, which had previously been implicated in the regeneration of other tissues (El Amri et al., 2018) suggesting a potential role for these proteins also in spinal cord regeneration.”

(2) Gene knock-out experiments with F0 crispants,

The authors described that they designed and tested 18 sgRNAs to find the most efficient and consistent gRNA (L191-195). However, it cannot guarantee the same phenotypes practically, due to, for example, different injection timing, different strains of Xenopus laevis, etc. Although the authors mentioned the concerns of mosaicism by themselves (L180-181, L289-292) and immunostaining results nicely showed uniformly reduced Marcks and Marcksl1 expression in the crispants, they did not refer to this issue explicitly.

To address this issue, we state explicitly in line 208-212: “We also confirmed by immunohistochemistry that co-injection of marcks.L/S and marcksl1.L/S sgRNA, which is predicted to edit all four homeologs (henceforth denoted as 4M CRISPR) drastically reduced immunostaining for Marcks and Marcksl1 protein on the injected side (Fig. S6 B-G), indicating that protein levels are reduced in gene-edited embryos.”

(3) Limitations of pharmacological compound rescue

In the methods part, the authors describe that they performed titration experiments for the drugs (L702-704), that is a minimal requirement for this type of assay. However, it is known that a well characterized drug is applied, if it is used in different concentrations, the drug could target different molecules (Gujral TS et al., 2014 PNAS). Therefore, it is difficult to eliminate possibilities of side effects and off targets by testing only a few compounds.

As explained in the responses to reviewer 1, we have completely rewritten and toned down our presentation of the pharmacological result and explicitly mention in our discussion now the possibility of side effects.

Welcome back and in this demo lesson you're going to evolve the infrastructure which you've been using throughout this section of the course.

In this demo lesson you're going to add private internet access capability using NAT gateways.

So you're going to be applying a cloud formation template which creates this base infrastructure.

It's going to be the animals for life VPC with infrastructure in each of three availability zones.

So there's a database subnet, an application subnet and a web subnet in availability zone A, B and C.

Now to this point what you've done is configured public subnet internet access and you've done that using an internet gateway together with routes on these public subnets.

In this demo lesson you're going to add NAT gateways into each availability zone so A, B and C and this will allow this private EC2 instance to have access to the internet.

Now you're going to be deploying NAT gateways into each availability zone so that each availability zone has its own isolated private subnet access to the internet.

It means that if any of the availability zones fail then each of the others will continue operating because these route tables which are attached to the private subnets they point at the NAT gateway within that availability zone.

So each availability zone A, B and C has its own corresponding NAT gateway which provides private internet access to all of the private subnets within that availability zone.

Now in order to implement this infrastructure you're going to be applying a one-click deployment and that's going to create everything that you see on screen now apart from these NAT gateways and the route table configurations.

So let's go ahead and move across to our AWS console and get started implementing this architecture.

Okay so now we're at the AWS console as always just make sure that you're logged in to the general AWS account as the I am admin user and you'll need to have the Northern Virginia region selected.

Now at the end of the previous demo lesson you should have deleted all of the infrastructure that you've created up until that point so the animals for live VPC as well as the Bastion host and the associated networking.

So you should have a relatively clean AWS account.

So what we're going to do first is use a one-click deployment to create the infrastructure that we'll need within this demo lesson.

So attached to this demo lesson is a one-click deployment link so go ahead and open that link.

That's going to take you to a quick create stack screen.

Everything should be pre-populated the stack name should be a4l just scroll down to the bottom check this capabilities box and then click on create stack.

Now this will start the creation process of this a4l stack and we will need this to be in a create complete state before we continue.

So go ahead pause the video wait for your stack to change into create complete and then we good to continue.

Okay so now this stacks moved into a create complete state then we good to continue.

So what we need to do before we start is make sure that all of our infrastructure has finished provisioning.

To do that just go ahead and click on the resources tab of this cloud formation stack and look for a4l internal test.

This is an EC2 instance a private EC2 instance so this doesn't have any public internet connectivity and we're going to use this to test on that gateway functionality.

So go ahead and click on this icon under physical ID and this is going to move you to the EC2 console and you'll be able to see this a4l - internal - test instance.

Now currently in my case it's showing as running but the status check is showing as initializing.

Now we'll need this instance to finish provisioning before we can continue with the demo.

What should happen is this status check should change from initializing to two out of two status checks and once you're at that point you should be able to right click and select connect and choose session manager and then have the option of connecting.

Now you'll see that I don't because this instance hasn't finished its provisioning process.

So what I want you to do is to go ahead and pause this video wait for your status checks to change to two out of two checks and then just go ahead and try to connect to this instance using session manager.

Only resume the video once you've been able to click on connect under the session manager tab and don't worry if this takes a few more minutes after the instance finishes provisioning before you can connect to session manager.

So go ahead and pause the video and when you can connect to the instance you're good to continue.

Okay so in my case it took about five minutes for this to change to two out of two checks past and then another five minutes before I could connect to this EC2 instance.

So I can right click on here and put connect.

I'll have the option now of picking session manager and then I can click on connect and this will connect me in to this private EC2 instance.

Now the reason why you're able to connect to this private instance is because we're using session manager and I'll explain exactly how this product works elsewhere in the course but essentially it allows us to connect into an EC2 instance with no public internet connectivity and it's using VPC interface endpoints to do that which I'll be explaining elsewhere in the course but what you should find when you're connected to this instance if you try to ping any internet IP address so let's go ahead and type ping and then a space 1.1.1.1.1 and press enter you'll note that we don't have any public internet connectivity and that's because this instance doesn't have a public IP version for address and it's not in a subnet with a route table which points at the internet gateway.

This EC2 instance has been deployed into the application a subnet which is a private subnet and it also doesn't have a public IP version for address.

So at this point what we need to do is go ahead and deploy our NAT gateways and these NAT gateways are what will provide this private EC2 instance with connectivity to the public IP version for internet so let's go ahead and do that.

Now to do that we need to be back at the main AWS console click in the services search box at the top type VPC and then right click and open that in a new tab.

Once you do that go ahead and move to that tab once you there click on NAT gateways and create a NAT gateway.

Okay so once you're here you'll need to specify a few things you'll need to give the NAT gateway a name you'll need to pick a public subnet for the NAT gateway to go into and then you'll need to give the NAT gateway an elastic IP address which is an IP address which doesn't change.

So first we'll set the name of the NAT gateway and we'll choose to use a4l for animals for life -vpc1 -natgw and then -a because this is going into availability zone A.

Next we'll need to pick the public subnet that the NAT gateway will be going into so click on the subnet drop down and then select the web a subnet which is the public subnet in availability zone a so sn -web -a.

Now we need to give this NAT gateway an elastic IP it doesn't currently have one so we need to click on allocate elastic IP which gives it an allocation.

Don't worry about the connectivity type we'll be covering that elsewhere in the course just scroll down to the bottom and create the NAT gateway.

Now this process will take some time and so we need to go ahead and create the two other NAT gateways.

So click on NAT gateways at the top and then we're going to create a second NAT gateway.

So go ahead and click on create NAT gateway again this time we'll call the NAT gateway a4l -vpc1 -natgw -b and this time we'll pick the web b subnet so sn -web -b allocated elastic IP again and click on create NAT gateway then we'll follow the same process a third time so click create NAT gateway use the same naming scheme but with -c pick the web c subnet from the list allocate an elastic IP and then scroll down and click on create NAT gateway and at this point we've got the three NAT gateways that are being created they're all in appending state if we go to elastic IPs we can see the three elastic IPs which have been allocated to the NAT gateways and we can scroll to the right or left and see details on these IPs and if we wanted we could release these IPs back to the account once we'd finish with them now at this point you need to go ahead and pause the video and resume it once all three of those NAT gateways have moved away from appending state we need them to be in an available state ready to go before we can continue with this demo so go ahead and pause and resume once all three have changed to an available state okay so all these are now in an available state so that means they're good to go they're providing service now if you scroll to the right in this list you're able to see additional information about these NAT gateways so you can see the elastic and private IP address the VPC and then the subnet that each of these NAT gateways are located in what we need to do now is configure the routing so that the private instances can communicate via the NAT gateways so right click on route tables and open in a new tab and we need to create a new route table for each of the availability zones so go ahead and click on create route table first we need to pick the VPC for this route table so click on the VPC drop down and then select the animals for live VPC so a for L hyphen VPC one once selected go ahead and name at the route table we're going to keep the naming scheme consistent so a for L hyphen VPC one hyphen RT for route table hyphen private a so enter that and click on create then close that dialogue down and create another route table this time we'll use the same naming scheme but of course this time it will be RT hyphen private B select the animals for life VPC and click on create close that down and then finally click on create route table again this time a for L hyphen VPC one hyphen RT hyphen private C again click on the VPC drop down and select the animals for life VPC and then click on create so that's going to leave us with three route tables one for each availability zone what we need to do now is create a default route within each of these route tables and that route is going to point at the NAT gateway in the same availability zone so select the route table private a and then click on the routes tab once you've selected the routes tab click on edit routes and we're going to add a new route it's going to be the IP version for default route of 0.0.0.0/0 and then click on target and pick NAT gateway and we're going to pick the NAT gateway in availability zone a and because we named them it makes it easy to select the relevant one from this list so go ahead and pick a for L hyphen VPC one hyphen NAT GW hyphen a so because this is the route table in availability zone a we need to pick the same NAT gateway so save that and close and now we'll be doing the same process for the route table in availability zone B make sure the routes tab is selected and click on edit routes click on add route again 0.0.0.0/0 and then for target pick NAT gateway and then pick the NAT gateway that's in availability zone B so NAT GW hyphen B once you've done that save the route table and then next select the route table in availability zone C so select RT hyphen private C make sure the routes tab is selected and click on edit routes again we'll be adding a route it will be the IP version for default route so 0.0.0.0/0 select a target go to NAT gateway and pick the NAT gateway in availability zone C so NAT GW hyphen C once you've done that save the route table and now our private EC2 instance should be able to ping 1.1.1.1 because we have the routing infrastructure in place so let's move back to our private instance and we can see that it's not actually working now the reason for this is that although we have created these routes we haven't actually associated these route tables with any of the subnets subnets in a VPC which don't have an explicit route table association are associated with the main route table now we need to explicitly associate each of these route tables with the subnets inside that same AZ so let's go ahead and pick RT hyphen private A we'll go through in order so select it click on the subnet associations tab and edit subnet associations and then you need to pick all of the private subnets in AZ A so that's the reserved subnet so reserved hyphen A the app subnet so app hyphen A and the DB subnet so DB hyphen A so all of these are the private subnets in availability zone A notice how all the public subnets are associated with this custom route table you created earlier but the ones we're setting up now are still associated with the main route table so we're going to resolve that now by associating this route table with those subnets so click on save and this will associate all of the private subnets in AZ A with the AZ A route table so now we're going to do the same process for AZ B and AZ C and we'll start with AZ B so select the private B route table click on subnet associations edit subnet associations so select application B database B and then reserved B and then scroll down and save the associations and then select the private C route table click on subnet associations edit subnet associations and then select reserved C database C and then application C and then scroll down and save those associations and now that we've associated these route tables with the subnets and now that we've added those default routes if we go back to session manager where we still have the connection open to the private EC2 instance we should see that the ping has started to work and that's because we now have a NAT gateway providing service to each of the private subnets in all of the three availability zones okay so that's everything you needed to cover in this demo lesson now it's time to clean up the account and return it to the same state as it was at the start of this demo lesson from this point on within the course you're going to be using automation and so we can remove all the configuration that we've done inside this demo lesson so the first thing we need to do is to reverse the route table changes that we've done so we need to go ahead and select the RT hyphen private a route table go ahead and select subnet associations and then edit the subnet associations and then just uncheck all of these subnets and this will return these to being associated with the main route table so scroll down and click on save do the same for RT hyphen private be so deselect all of these associations and click on save and then the same for RT hyphen private see so select it go to subnet associations and then edit them and remove all of these subnets and click on save next select all of these private route tables these are the ones that we created in this lesson so select them all click on the actions drop down and then delete route table and confirm by clicking delete route tables go to NAT gateways on the left and we need to select each of the NAT gateways in turn so a and then click on actions and delete NAT gateway type delete click delete then select be and do the same process actions delete NAT gateway type delete click delete and finally the same for see so select the C NAT gateway click on actions and delete NAT gateway you'll need to type delete to confirm click on delete now we're going to need all of these to be in a fully deleted state before we can continue so hit refresh and make sure that all three NAT gateways are deleted if yours aren't deleted if they're still listed in a deleting state then go ahead and pause the video and resume once all of these have changed to deleted at this point all of the NAT gateways have deleted so you can go ahead and click on elastic IPs and we need to release each of these IPs so select one of them and then click on actions and release elastic IP addresses and click release and do the same process for the other two click on release then finally actions release IP click on release once that's done move back to the cloud formation console select the stack which was created by the one click deployment at the start of the lesson and click on delete and then confirm that deletion and that will remove the cloud formation stack and any resources created as part of this demo and at that point once that finishes deleting the account has been returned into the same state as it was at the start of this demo lesson so I hope this demo lesson has been useful just to reiterate what you've done you've created three NAT gateways for a region resilient design you've created three route tables one in each availability zone added a default IP version for route pointing at the corresponding NAT gateway and associated each of those route tables with the private subnets in those availability zones so you've implemented a regionally resilient NAT gateway architecture so that's a great job that's a pretty complex demo but it's going to be functionality that will be really useful if you're using AWS in the real world or if you have to answer any exam questions on NAT gateways with that being said at this point you have cleared up the account you've deleted all the resources so go ahead complete this video and when you're ready I'll see you in the next.

Author response:

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

Public Review:

Summary:

Bursicon is a key hormone regulating cuticle tanning in insects. While the molecular mechanisms of its function are rather well studied--especially in the model insect Drosophila melanogaster, its effects and functions in different tissues are less well understood. Here, the authors show that bursicon and its receptor play a role in regulating aspects of the seasonal polyphenism of Cacopsylla chinensis. They found that low temperature treatment activated the bursicon signaling pathway during the transition from summer form to winter form and affect cuticle pigment and chitin content, and cuticle thickness. In addition, the authors show that miR-6012 targets the bursicon receptor, CcBurs-R, thereby modulating the function of bursicon signaling pathway in the seasonal polyphenism of C. chinensis. This discovery expands our knowledge of the roles of neuropeptide bursicon action in arthropod biology.

However, the study falls short of its claim that it reveals the molecular mechanisms of a seasonal polyphenism. While cuticle tanning is an important part of the pear psyllid polyphenism, it is not the equivalent of it. First, there are other traits that distinguish between the two morphs, such as ovarian diapause (Oldfield, 1970), and the role of bursicon signaling in regulating these aspects of polyphenism were not measured. Thus, the phenotype in pear psyllids, whereby knockdown bursicon reduces cuticle tanning seems to simply demonstrate the phenotypes of Drosophila mutants for bursicon receptor (Loveall and Deitcher, 2010, BMC Dev Biol) in another species (Fig. 2I, 4H). Second, the study fails to address the threshold nature of cuticular tanning in this species, although it is the threshold response (specifically, to temperature and photoperiod) that distinguishes this trait as a part of a polyphenism. Whereas miR-6012 was found to regulate bursicon expression, there no evidence is provided that this microRNA either responds to or initiates a threshold response to temperature. In principle, miR-6012 could regulate bursicon whether or not it is part of a polyphenism. Thus, the impact of this work would be significantly increased if it could distinguish between seasonal changes of the cuticle and a bona fide reflection of polyphenism.

Thanks for your valuable suggestion. We concur with the review’s comment that cuticle tanning does not equate to the C. chinensis polyphenism. To better reflect the core focus of our research, we have revised the title to "Neuropeptide Bursicon and its receptor mediated the transition from summer-form to winter-form of Cacopsylla chinensis".

In response to the reviewer's inquiry regarding the threshold nature of cuticular tanning in C. chinensis, we have included a detailed analysis of the phenotypic changes (including nymph phenotypes, cuticle pigment absorbance, and cuticle thickness) during the transition from summer-form to winter-form in C. chinensis at distinct time intervals (3, 6, 9, 12, 15 days) under different temperature conditions (10°C and 25°C). As shown in Figure S1, nymphs exhibit a light yellow and transparent coloration at 3, 6, and 9 days, while nymphs at 12 and 15 days display shades of yellow-green or blue-yellow under 25°C conditions. At 10°C conditions, the abdomen end turns black at 3, 6, and 9 days. By the 12 days, numerous light black stripes appear on the chest and abdomen of nymphs at 10°C. At 15 days, nymphs exhibit an overall black-brown appearance, featuring dark brown stripes on the left and right sides of each chest and abdominal section. Furthermore, the end of the abdomen and back display a large black-brown coloration at 10°C (Figure S1A). The UV absorbance of the total pigment extraction at a 300 nm wavelength markedly increases following 10°C exposure for 6, 9, 12, and 15 days compared to the 25°C treatment group (Figure S1B). Cuticle thicknesses also increased following 10°C exposure for 6, 9, 12, and 15 days compared to the 25°C treatment group (Figure S1C). The detailed results (L122-143), materials and methods (L647-652), and discussion (L319-322) have been added in our revised manuscript.

Regarding the response of miR-6012 to temperature, we have already determined its expression at 3, 6, 10 days under different temperatures in the previous Figure 5E. We now included additional time intervals (9, 12, 15 days) in the updated Figure 5E. Our results indicate a significant decrease in the expression levels of miR-6012 after 10°C treatment for 3, 6, 9, 12, 15 days compared to the 25°C treatment group. Detailed information regarding this has been integrated into the Materials and Methods (Line 608-610) of our revised manuscript.

Strengths:

This study convincingly identifies homologs of the genes encoding the bursicon subunits and its receptor, showing an alignment with those of another psyllid as well as more distant species. It also demonstrates that the stage- and tissue-specific levels of bursicon follow the expected patterns, as informed by other insect models, thus validating the identity of these genes in this species. They provide strong evidence that the expression of bursicon and its receptor depend on temperature, thereby showing that this trait is regulated through both parts of the signaling mechanism.

Several parallel measurements of the phenotype were performed to show the effects of this hormone, its receptor, and an upstream regulator (miR-6012), on cuticle deposition and pigmentation (if not polyphenism per se, as claimed). Specifically, chitin staining and TEM of the cuticle qualitatively show difference between controls and knockdowns, and this is supported by some statistical tests of quantitative measurements (although see comments below). Thus, this study provides strong evidence that bursicon and its receptor play an important role in cuticle deposition and pigmentation in this psyllid.

The study identified four miRNAs which might affect bursicon due to sequence motifs. By manipulating levels of synthetic miRNA agonists, the study successfully identified one of them (miR-6012) to cause a cuticle phenotype. Moreover, this miRNA was localized (by FISH) to the cuticle, body-wide. To our knowledge, this is the first demonstrated function for this miRNA, and this study provides a good example of using a gene of known function as an entry point to discovering others influencing a trait. Thus, this finding reveals another level of regulation of cuticle formation in insects.

Weaknesses:

(1) The introduction to this manuscript does not accurately reflect progress in the field of mechanisms underlying polyphenism (e.g., line 60). There are several models for polyphenism that have been used to uncover molecular mechanisms in at least some detail, and this includes seasonal polyphenisms in Hemiptera. Therefore, the justification for this study cannot be predicated on a lack of knowledge, nor is the present study original or unique in this line of research (e.g., as reviewed by Zhang et al. 2019; DOI: 10.1146/annurev-ento-011118-112448). The authors are apparently aware of this, because they even provide other examples (lines 104-108); thus the introduction seems misleading as framed.

Thanks for your excellent suggestion. We have added the paper of Zhang et al. 2019 which recommended by reviewer (DOI: 10.1146/annurev-ento-011118-112448) in Line 57 of our revised manuscript. The statement has been revised to “However, the specific molecular mechanism underling temperature-dependent polyphenism still require further clarification” in Line 60-61 of our revised manuscript.

(2) The data in Figure 2H show "percent of transition." However, the images in 2I show insects with tanned cuticle (control) vs. those without (knockdown). Yet, based on the description of the Methods provided, there appears to be no distinction between "percent of transition" and "percent with tanning defects". This an important distinction to make if the authors are going to interpret cuticle defects as a defect in the polyphenism. Furthermore, there is no mention of intermediate phenotypes. The data in 2H are binned as either present or absent, and these are the phenotypes shown in 2I. Was the phenotype really an all-or-nothing response? Instead of binning, which masks any quantitative differences in the tanning phenotypes, the authors should objectively quantify the degree of tanning and plot that. This would show if and to what degree intermediate tanning phenotypes occurred, which would test how bursicon affects the threshold response. This comment also applies to the data in Figures 4G and 6G. Since cuticle tanning is present in more insect than just those with seasonal polyphenism, showing how this responds as a threshold is needed to make claims about polyphenism.

We appreciate your insightful comments. As shown in Figure 1 of our published paper (Zhang et al., 2013; doi.org/10.7554/eLife.88744.3) and Figure 2C-2I of the current manuscript, the transition from summer-form to winter-form entails not only external cuticular tanning but also alterations in internal cuticular chitin levels and cuticle thickness. While external cuticular tanning serves as a prominent and easily observable indicator of this transition, it is crucial to acknowledge that internal changes also play a significant role and should be taken into consideration. Therefore, we propose that the term "percent of transition" may be more suitable than "percent with tanning defects" to describe this process accurately.

In order to provide a more visually comprehensive understanding of the phenotypic changes during the transition from summer-form to winter-form, we have included images at different time points (3, 6, 9, 12, 15 days) under different temperature conditions in Figure S1A of our revised manuscript. Specifically, under the 10°C condition, nymphs exhibit abdomen tanning after 6 and 9 days of treatment, while the thorax remains untanned. By days 12 to 15, both the abdomen and thorax of the nymphs show tanning, resulting in the majority of summer-form nymphs transitioning into winter-form, as depicted in Figure 2I for comparison. This observation indicates the presence of a critical threshold for cuticle tanning of C. chinensis following exposure to 10°C. Nymphs that did not undergo the transition to winter-form succumbed to the cold, highlighting the absence of intermediate phenotypes at 12-15 days under the 10°C condition. The UV absorbance of the total pigment extraction at a 300 nm wavelength markedly increases following 10°C exposure for 6, 9, 12, and 15 days compared to the 25°C treatment group (Figure S1B). Additionally, cuticle thickness shows an increase following 10°C exposure for 6, 9, 12, and 15 days compared to the 25°C treatment group (Figure S1C). These results highlight the relationship between the threshold of cuticular tanning and the transition process. The detailed description and information have been added in Results (L122-143), Materials and Methods (L647-652), and Discussion (L319-322) of our manuscript.

(3) This study also does not test the threshold response of cuticle phenotypes to levels of bursicon, its receptor, or miR-6012. Hormone thresholds are the most widespread and, in most systems where polyphenism has been studied, the defining characteristic of a polyphenism (e.g., Nijhout, 2003, Evol Dev). Quantitative (not binned) measurements of a polyphenism marker (e.g., chitin) should be demonstrated to result as a threshold titer (or in the case of the receptor, expression level) to distinguish defects in polyphenism from those of its component trait.

Thanks for your valuable feedback. We have supplemented additional data on the phenotypes (Figure S1A), cuticle pigment absorbance (Figure S1B), cuticle thickness (Figure S1C), expression levels of bursicon (Figure 1E and 1F), its receptors (Figure 3G), and miR-6012 (Figure 5E) corresponding to nymphs treated over different time periods (3, 6, 9, 12, 15 days) under both 10°C and 25°C conditions in our revised manuscript.

While all these identified markers exhibit a strong correlation with the transition from summer-form to winter-form, it is important to note that they are not suitable as definitive thresholds due to the nature of relative gene expression quantification and chitin content assessment, rather than absolute quantitation. Further, given that tanning hormones are neuropeptides present in trace amounts in insects, unlike steroid hormones, determining their titers poses a considerable challenge.

(4) Cuticle issue:

(a) Unlike Fig. 6D and F, Figs. 2D and F do not correspond to each other. Especially the lack and reduction of chitin in ds-a+b! By fluorescence microscopy there is hardly any signal, whereas by TEM there is a decent cuticle. Additionally, the dsGFP control cuticle in 2D is cut obliquely with a thick and a thin chitin layer. This is misleading.

Thanks for your insightful feedback. We have replaced the previous WGA chitin staining images in the dsCcbursα+β treatment of Figure 2D with new representative images aligning with Figure 2F. Furthermore, the presence of both thin and thick chitin layers observed in the dsEGFP treatment of Figure 2D could potentially be ascribed to the chitin content in the insect midgut or fat body as previously discussed (Zhu et al., 2016). It is notable that during the process of cuticle staining, the chitin located in the midgut and fat body of C. chinensis may exhibit green fluorescence, leading to the appearance of a thin chitin layer. A detailed analysis and elucidation of these observations have been added in the discussion section (Lines 347-352) of our revised manuscript.

Zhu KY, Merzendorfer H, Zhang W, Zhang J, Muthukrishnan S. Biosynthesis, Turnover, and Functions of Chitin in Insects. Annu Rev Entomol. 2016;61:177-196. doi:10.1146/annurev-ento-010715-023933.

(b) In Figs. 2F and 4F, the endocuticle appears to be missing, a portion of the procuticle that is produced post-molting. As tanning is also occurring post-molting, there seems to be a general problem with cuticle differentiation at this time point. This may be a timing issue. Please clarify.

Thank you for your suggestion. The insect cuticle typically comprises three distinct layers (endocuticle, exocuticle, and epicuticle), with the thickness of each layer varying among different insect species. Cuticle differentiation is closely linked to the molting cycle of insects (Mrak et al., 2017). In our study, nymphal cuticles exhibited normal differentiation patterns, characterized by a thin epicuticle and comparable widths of the endocuticle and exocuticle following dsEGFP treatment, as illustrated in Figure 2F and 4F. Conversely, nymphs treated with dsCcBurs-α, dsCcBurs-β, and dsCcburs-R displayed impaired development, manifesting only the exocuticle without a discernible endocuticle layer. These findings suggest that bursicon genes and their receptor play a pivotal role in regulating insect cuticle development (Costa et al., 2016). We have added some discussion about these results in Lines 356-367 of our revised manuscript.

Mrak, P., Bogataj, U., Štrus, J., & Žnidaršič, N. (2017). Cuticle morphogenesis in crustacean embryonic and postembryonic stages. Arthropod structure & development, 46(1), 77–95. https://doi.org/10.1016/j.asd.2016.11.001

Costa, C. P., Elias-Neto, M., Falcon, T., Dallacqua, R. P., Martins, J. R., & Bitondi, M. (2016). RNAi-mediated functional analysis of Bursicon genes related to adult cuticle formation and tanning in the Honeybee, Apis mellifera. PloS one, 11(12), e0167421. https://doi.org/10.1371/journal.pone.0167421

(c) To provide background information, it would be useful analyze cuticle formation in the summer and winter morphs of controls separately by light and electron microscopy. More baseline data on these two morphs is needed.

Thanks for your valuable feedback. To provide more background information about cuticle formation, we supplied the results of nymph phenotypes, cuticle pigment absorbance, and cuticle thickness at distinct time intervals (3, 6, 9, 12, 15 days) under different temperatures of 10°C and 25°C in Figure S1 of our revised manuscript. Hope these results can help better understand the baseline data on these two morphs.

(d) For the TEM study, it is not clear whether the same part of the insect's thorax is being sectioned each time, or if that matters. There is not an obvious difference in the number of cuticular layers, but only the relative widths of those layers, so it is difficult to know how comparable those images are. This raises two questions that the authors should clarify. First, is it possible that certain parts of the thoracic cuticle, such as those closer to the intersegmental membrane, are naturally thinner than other parts of the body? Second, is the tanning phenotype based on the thickness or on the number of chitin layers, or both? The data shown later in Figure 4I, J convincingly shows that the biosynthesis pathway for chitin is repressed, but any clarification of what this might mean for deposition of chitin would help to understand the phenotypes reported. Also, more details on how the data in Fig. 2G were collected would be helpful. This also goes for the data in Fig. 4 (bursicon receptor knockdowns).

Thanks for your great comment. The TEM investigation adhered to a standardized protocol was used as previous description (Zhang et al., 2023), Initially, insect heads were uniformly excised and then fixed in 4% paraformaldehyde. Subsequently, a consistent cutting and staining procedure was executed at a uniform distance above the insect's thorax. The dorsal region of the thorax was specifically chosen for subsequent fluorescence imaging or transmission electron microscopy assessments with the specific objective of quantifying cuticle thickness. Regarding the measurement of cuticle thickness, use the built-in measuring ruler on the software to select the top and bottom of the same horizontal line on the cuticle. Measure the cuticle of each nymph at two close locations. Six nymphs were used for each sample. Randomly select 9 values and plot them. The related description has been added in the Materials and Methods (Line 660-668) of our revised manuscript.

Zhang, S.D., Li, J.Y., Zhang, D.Y., Zhang, Z.X., Meng, S.L., Li, Z., & Liu, X.X. (2023). MiR-252 targeting temperature receptor CcTRPM to mediate the transition from summer-form to winter-form of Cacopsylla chinensis. eLife, 12. https://doi.org/10.7554/eLife.88744

(5) Tissue issue:

The timed experiments shown in all figures were done in whole animals. However, we know from Drosophila that Bursicon activity is complex in different tissues. There is, thus, the possibility, that the effects detected on different days in whole animals are misleading because different tissues--especially the brain and the epidermis, may respond differentially to the challenge and mask each other's responses. The animal is small, so the extraction from single tissue may be difficult. However, this important issue needs to be addressed.

Thanks for your excellent suggestion. We express our heartfelt appreciation to the reviewer for their valuable input regarding the challenges involved in dissecting various tissue sections from the diminutive early instar nymphs of C. chinensis. In light of the metamorphic transition of C. chinensis across developmental stages, this study concentrated on examining the extensive phenotypic alterations. Consequently, intact samples of C. chinensis were specifically chosen for for qPCR analysis. The related descriptions have been added in the Materials and Methods (Line 513, 517, 553, 555, and 613) and Discussion (Line 327-329) of our revised manuscript.

(6) No specific information is provided regarding the procedure followed for the rescue experiments with burs-α and burs-β (How were they done? Which concentrations were applied? What were the effects?). These important details should appear in the Materials and Methods and the Results sections.

Thanks for your excellent suggestion. For the rescue experiments, the dsRNA of CcBurs-R and proteins of burs α-α, burs β-β homodimers, or burs α-β heterodimer (200 ng/μL) were fed together. The concentration of heterodimer protein of CcBurs-α+β was 200 ng/μL. The heterodimer protein of CcBurs-α+β fully rescued the effect of RNAi-mediated knockdown on CcBurs-R expression, while α+α or β+β homodimers did not (Figure 3F). Feeding the α+β heterodimer protein fully rescued the defect in the transition percent and morphological phenotype after CcBurs-R knockdown (Figure 4G-4H). We have added the detailed methods of rescued experiments and specific concentrations in the Materials and Methods (Line 561-563), and Results (Line 263) of our revised manuscript.

(7) Pigmentation

(a) The protocol used to assess pigmentation needs to be validated. In particular, the following details are needed: Were all pigments extracted? Were pigments modified during extraction? Were the values measured consistent with values obtained, for instance, by light microscopy (which should be done)?

Thanks for your excellent comment. Our protocol for pigment extracted as detailed in Bombyx mori, the cuticles were pulverized in liquid nitrogen and then dissolved in 30 milliliters of acidified methanol (Futahashi et al., 2012; Osanai-Futahashi et al., 2012). Thus, all cuticle pigments were dissected and treated with acidified methanol. Pigments were not modified during extraction.. The details description have been integrated into the Materials and Methods (Line 630-633) of our revised manuscript.

Futahashi, R., Kurita, R., Mano, H., & Fukatsu, T. (2012). Redox alters yellow dragonflies into red. Proceedings of the National Academy of Sciences of the United States of America, 109(31), 12626–12631. https://doi.org/10.1073/pnas.1207114109

Osanai-Futahashi, M., Tatematsu, K. I., Yamamoto, K., Narukawa, J., Uchino, K., Kayukawa, T., Shinoda, T., Banno, Y., Tamura, T., & Sezutsu, H. (2012). Identification of the Bombyx red egg gene reveals involvement of a novel transporter family gene in late steps of the insect ommochrome biosynthesis pathway. The Journal of biological chemistry, 287(21), 17706–17714. https://doi.org/10.1074/jbc.M111.321331

(b) In addition, pigmentation occurs post-molting; thus, the results could reflect indirect actions of bursicon signaling on pigmentation. The levels of expression of downstream pigmentation genes (ebony, lactase, etc) should be measured and compared in molting summer vs. winter morphs.

Thanks for your valuable suggestion. Actually, we already studied the function of some downstream pigmentation genes, including ebony, Lactase, Tyrosine hydroxylase, Dopa decarboxylase, and Acetyltransferase. The variations in the expression patterns of these genes are closely tied to the molting dynamics of nymphs undergoing transitions between summer-form and winter-form. These findings will put in another manuscript currently being prepared for submission, thus detailed outcomes are not suitable for inclusion in the current manuscript.

(8) L236: "while the heterodimer protein of CcBurs α+β could fully rescue the effect of CcBurs-R knockdown on the transition percent (Figure 4G 4H)". This result seems contradictory. If CcBurs-R is the receptor of bursicon, the heterodimer protein of CcBurs α+β should not be able to rescue the effect of CcBurs-R knockdown insects. How can a neuropeptide protein rescue the effect when its receptor is not there! If these results are valid, then the CcBurs-R would not be the (sole) receptor for CcBurs α+β heterodimer. This is a critical issue for this manuscript and needs to be addressed (also in L337 in Discussion).

Thanks for your insightful suggestion. Following the administration of dsCcBur-R to C. chinensis, the expression of CcBurs-R exhibited a reduction of approximately 66-82% as depicted in Figure 4A, rather than complete suppression. Activation of endogenous CcBurs-R through feeding of the α+β heterodimer protein results in an increase in CcBurs-R expression, with the effectiveness of the rescue effect contingent upon the dosage of the α+β heterodimer protein. Consequently, the capacity of the α+β heterodimer protein to effectively mitigate the impacts of CcBurs-R knockdown on the conversion rate is clearly demonstrated. We have added additional discussion in Line 396-403 of our revised manuscript.

(9) Fig. 5D needs improvement (the magnification is poor) and further explanation and discussion. mi6012 and CcBurs-R seem to be expressed in complementary tissues--do we see internal tissues also (see problem under point 2)? Again, the magnification is not high enough to understand and appreciate the relationships discussed.

Thanks for your valuable suggestion. In order to enhance the resolution of the magnified images, we conducted FISH co-localization of miR-6012 and CcBurs-R in 3rd instar nymphs and obtained detailed zoomed-in images. As shown in the magnified view of Figure 5D, miR-6012 and CcBurs-R appear to exhibit complementary expression patterns in tissues. During the FISH assays, epidermis transparency of C. chinensis was achieved via decolorization treatment. Noteworthy observations from Figure 3G and Figure 5E reveal an inverse correlation in the expression profiles of CcBurs-R and miR-6012. Consequently, the FISH results distinctly highlight a significant disparity in the expression levels of CcBurs-R and miR-6012 within the same tissue. We have added related explanation and discussion in Line 291-293 of our revised manuscript.

(10) The schematic in Fig. 7 is a useful summary, but there is a part of the logic that is unsupported by the data, specifically in terms of environmental influence on cuticle formation (i.e., plasticity). What is the evidence that lower temperatures influence expression of miR-6012? The study measures its expression over life stages, whether with an agonist or not, over a single temperature. Measuring levels of expression under summer form-inducing temperature is necessary to test the dependence of miR-6012 expression on temperature. Otherwise, this result cannot be interpreted as polyphenism control, but rather the control of a specific trait.

Thanks for your great suggestion. We actually conducted the assessment of miR-6012 expression at specific time intervals (3, 6, 9, 12, 15 days) under different temperatures of 10°C and 25°C. As depicted in Figure 5E, the expression levels of miR-6012 were notably reduced at 10°C compared to 25°C. Additionally, the evaluation of agomir-6012 expression level of C. chinensis under 25°C conditions at various time points (3, 6, 9, 12, 15 days) revealed no significant changes. Hence, we suggest that the impact of miR-6012 on the seasonal morphological transition is influenced upon temperature.

Recommendations for the authors:

The authors report a novel role of Bursicon and its receptor in regulating the seasonal polyphenism of Cacopsylla chinensis. They found that low temperature treatment (10°C) activated the Bursicon signaling pathway during the transition from summer-form to winter-form, which influences cuticle pigment content, cuticle chitin content, and cuticle thickness. Moreover, the authors identified miR-6012 and show that it targets CcBurs-R, thereby modulating the function of Bursicon signaling pathway in the seasonal polyphenism of C. chinensis. This discovery expands our knowledge of multiple roles of neuropeptide bursicon action in arthropod biology. However, the m

anuscript does have several major weaknesses, described under "Public review", which the authors need to address.

Major issues:

(1) L152-154 Fig S2E and S2F: Bursicon has been shown to be expressed in the CNS in a specific set of neurons. For example, In the larval CNS of Manduca sexta, bursicon expression is restricted to the subesophageal ganglion (SG), thoracic ganglia, and first abdominal ganglion. Pharate pupae and pharate adults show expression of this heterodimer in all ganglia. In Drosophila larvae, expression of a bursicon heterodimer is confined to abdominal ganglia. The additional neurons in the ventral nerve cord express only burs. In pharate adults, bursicon is produced by neurons in the SG and abdominal ganglia. I am wondering where bursicon subunits are expressed in the C. chinensis CNS? Since the authors have the antibodies, it would be useful to include immunocytochemical staining of bursicon alpha and beta in the CNS. The qPCR results from head or other tissues (Fig S2E and S2F) is not the most informative way to document localization of gene expression. Regarding the qPCR results, they show that the cuticle and the fat body express CcBurs-α and CcBurs-β. Can the authors confirm this unexpected results independently?

Thanks for your insightful comment. In this study, we did not directly used antibodies targeting bursicon subunits, instead, the bursicon subunits along with a histidine tag were integrated into the expression vector pcDNA3.1 using homologous recombination. The experimental procedures were executed as follows: initially, the histidine tag was fused to the pcDNA3.1-mCherry vector through homologous recombination to generate the recombinant plasmid pcDNA3.1-his-mCherry. Subsequently, the amino acid sequences of the two bursicon subunits were introduced into the pcDNA3.1-his-mCherry vector via homologous recombination to produce the recombinant plasmids pcDNA3.1-CcBurs-α-his-mCherry and pcDNA3.1-CcBurs-β-his-mCherry. Finally, the P2A sequence was incorporated into the vector using reverse PCR to yield the recombinant plasmids pcDNA3.1-CcBurs-α-his-P2A-mCherry and pcDNA3.1-CcBurs-β-his-P2A-mCherry. Consequently, the bursicon subunits, along with the histidine tag, were capable of generating fusion proteins with the histidine tag. Western blot analysis was conducted using antibodies targeting the histidine tag, enabling the detection of histidine expression, which corresponds to the expression of the bursicon subunits. However, they are not suitable to conduct the in vivo immunocytochemical staining of bursicon alpha and beta in the CNS.

Due to the diminutive size of the C. chinensis nymphs, dissection of the central nervous system (CNS) was unfeasible, precluding specific assessment of bursicon expression in the CNS. Prior literature has documented the expression of bursicon subunits in the epidermis and fat body of C. chinensis. Studies suggest that bursicon subunits not only play a role in the melanization and sclerotization processes of insect epidermis but also have significant roles in insect immunity (An et al., 2012). The presence of bursicon subunits in the epidermis, gut, and fat body of C. chinensis may indicate their crucial roles in the immune functions of these tissues. Further investigation is required to elucidate the specific immune functions they perform, hinting at the potential expression of these bursicon subunits in these two tissues.

An, S., Dong, S., Wang, Q., Li, S., Gilbert, L. I., Stanley, D., & Song, Q. (2012). Insect neuropeptide bursicon homodimers induce innate immune and stress genes during molting by activating the NF-κB transcription factor Relish. PloS one, 7(3), e34510. https://doi.org/10.1371/journal.pone.0034510

(2) L222: "CcBurs-R is the Bursicon receptor of C. chinensis". Is this statement supported by affinity binding assay results?

Thanks for your excellent suggestion. We employed a fluorescence-based assay to quantify calcium ion concentrations and investigate the binding affinities of bursicon heterodimers and homodimers to the bursicon receptor across varying concentrations. Our findings suggest that activation of the receptor by the burs α-β heterodimer leads to significant alterations in intracellular calcium ion levels, whereas stimulation with burs α-α and burs β-β homodimers, in conjunction with Adipokinetic hormone (AKH), maintains consistent intracellular calcium ion levels. Consequently, this research definitively identifies CcBurs-R as the bursicon receptor. For further details, please refer to the Materials and Methods (Lines 493-504), Results (Lines 231-239), and Discussion (Lines 377-384) of our revised manuscript.

(3) L245 Figure 4I-4J: Since knockdown of bursicon and its receptor cause a decrease pigment accumulation in the cuticle, it would be useful to examine 1-2 rate limiting enzyme-encoding genes in the bursicon regulated cuticle darkening process if possible (as was done for genes involved in cuticle thickening).

Thanks for your excellent comment. Following the further study, a thorough analysis was conducted to evaluate the impact of bursicon and its receptor on the expression levels of Lactase, Tyrosine hydroxylase, Dopa decarboxylase, Acetyltransferase, and the effects of RNA interference targeting these genes on the seasonal morphological transition. The findings underscored their role in the bursicon-mediated cuticle darkening process. However, as this section is slated for inclusion in an upcoming manuscript intended for submission, it is deemed unsuitable for incorporation into the current manuscript.

Minor issues:

(1) L75 "stronger resistance (Ge et al., 2019; Tougeron et al., 2021)". Stronger resistance to what? Stronger resistance to environmental stress or weather condition? Please clarify.

Thanks for your excellent suggestion. We have changed the statement to “stronger resistance to weather condition” in Line 75 of our revised manuscript.

(2) L132 Figure 1A and 1B: Bursicon sequence was first identified and functionally characterized in Drosophila melanogaster: is there any reason why Drosophila bursicon sequences were not included in the comparison?

Thanks for your excellent comment. We have added the sequence of Burs-α and Burs-β of D. melanogaster in the sequence alignment results of Figure 1A and 1B of our revised manuscript.

(3) Although the authors clearly identify and validate the function for the bursicon genes and its receptor's, there is no mention of whether duplicates of this gene are also present in the pear psyllid. This has been known to happen in otherwise conserved hormone pathways (e.g., insulin receptor in some insects), so a formal check of this should be done.

Thanks for your excellent comment. As shown in Figure S2A-S2B and 3B, there are two bursicon subunit genes and only one bursicon receptor gene in our selected insect species, for examples Drosophila melanogaster, Diaphorina citri, Bemisia tabaci, Nilaparvata lugens, and Sogatella furcifera. In our transcriptome database of C. chinensis, we also only identified two bursicon subunit genes and only one bursicon receptor gene.

(4) Line 41: Here, as in the title, "fascinating" is a subjective judgement that does not improve a study's presentation.

Thanks for your great comment. We have changed "fascinating" to "transformation" in Line 41 and also revised the title of our revised manuscript.

(5) Line 44: What makes some fields "cutting-edge" and others not?

Thanks for your excellent suggestion. The expression of "in cutting-edge fields" has been deleted in Line 44 of our revised manuscript.

(6) Line 97: This is a peculiar choice of reference for the concept of slower development in cold temperatures. The concept of degree-days and growth rates is old and widespread in entomology.

Thanks for your insightful comment. The reference of Nyamaukondiwa et al., 2011 in Line 95 has been deleted in our revised manuscript.

(7) Lines 149-150: What justifies the assumption that higher levels of expression mean a more important role? This gene might be just as necessary for development of the summer form, even if expressed at lower levels.

Thanks for your excellent suggestion. This sentence has been revised to “Increased gene expression levels may potentially contribute to the transition from summer-form to winter-form in C. chinensis.” in Line 168-169 of our revised manuscript.

(8) The blue arrow in Fig. 7 is confusing.

Thanks for your excellent suggestion. In Figure 7, the blue arrow represents the down-regulated expression of miR-6012. We have added a description about the blue arrow in Figure 7 of our revised manuscript.