Something to keep in mind about everyday life.

This reminds me of an episode of the Saved By the Bell reboot where a character who is a Dominicana tries to speak Spanish in Spanish class, and her (white) Spanish teacher tells her that the way that she's speaking it is "wrong," even though it's not "wrong," it's just different than the way that Spanish speakers from Spain would say it.

(This is a good annotation because it makes a relevant connection to the course material from outside of the class to extend the point and make it more concrete. It would be great to also include a link to the clip if you have one!)

It's crazy to think of how early in schooling is your college education influenced by. I personally remember taking a placement test in fifth grade to determine which students would be allowed to enroll in "honors" courses once we moved to the middle school. Most of my friends and I had been placed into both the honors math and English/History tracks, but I had one friend who was not placed into honors math. She had no lower scores than the rest of us did in the school year, but had forgotten some of the math we had learned earlier in the year, lowering her placement score and ultimately excluding her out of the honors track. By the time we reached high school, my friends who were placed in the honors track (based on a placement test we took in the fifth grade without any studying) were given the opportunity to take free extra classes to finish AP Calc A/B by junior year, while my friend, who was technically a year's level lower (since middle school) was now two years lower. It's just crazy the difference in opportunity and confidence this created for students onwhat we would be judged on in our college apps. Additionally, I had a lot of friends who were in the honors track and therefore took many APs in high school, but also plenty of friends who were never in honors and excelled in all of the APs they took. I think students should be able to elect taking "advanced" classes rather than being forced or excluded out of them, as that system contributes to the "self-fulfilling prophecy" that was mentioned earlier.

Like it is mentioned later in this paragraph, I think it's so strange that in many of the "first-world countries" that America likes to compare itself to have established socialism-based welfare programs including public education, yet national healthcare programs--a welfare program, just the same--is so polarizing in the States.

Author Response

Reviewer #2 (Public Review):

This paper combines neuroimaging, behavioral experiments, and computational modeling to argue that (a) there is a network of brain areas that represent physical stability, (b) these areas do so in a way that generalizes across many kinds of instability (e.g., not only a tower of blocks about to fall over, but also a person about to fall off a ladder), and (c) that this supports a simulation account of physical reasoning, rather than one based on feedforward processing; this last claim arises through a comparison of humans to CNNs, which do an OK job classifying physical instability but not in a way that transfers across these different stability classes. In my opinion, this is a lovely contribution to the literatures on both intuitive physical reasoning and (un)humanlike machine vision. At the same time, I wasn't sure that the broader conclusions followed from the data in the way the authors preferred, and I also had some concerns about some of the methodological choices made here.

1. The following framing puzzled me a bit, and even seemed to raise an unaddressed confound in the paper: "Here we investigate how the brain makes the most basic prediction about the physical world: whether the situation in front of us is stable, and hence likely to stay the same, or unstable, and hence likely to change in the immediate future".

Consider the following minor worry, which sets up a more major one: This framing, which connects 'stability' to 'change' and which continues throughout the paper, seems to equivocate on the notion of 'stability'. One meaning of 'stable' is, roughly, 'unchanging'. Another meaning is 'unlikely to fall over'. The above quotation, along with others like it, makes it seem like the authors are investigating the former, since that's the only meaning that makes this quotation make sense. But in fact the experiments are about the latter -- towers falling down, people falling off ladders, etc. But these aren't the same thing! So there's a bit of wordplay happening here, it seemed to me.

This sets up the more serious worry. As this framing reveals, unstable scenes (in the likely-to-fall-over sense) are, by their nature, scenes where something is likely to change. In that case, how do we know that the brain areas this project has identified aren't representing 'likeliness to change', rather than physical stability? There are, of course, many objects and scenes that might be highly likely to change without being at all physically unstable. Even the first example in the paper ("a dog about to give chase") is about likely changes without any physical instability. But isn't this a confound? All of the examples of physical instability explored here also involve likeliness to change! So these could be 'likely to change' brain areas, not 'physically unstable' brain areas. Right? Or if not, what am I missing?

The caption of Figure 1 seems to get at this a bit, but in a way I admit I just found a bit confusing. If authors do after all intend "physically unstable" to mean "likely to change", then many classes of scenarios that are unexplored here seem like they would be relevant: a line of sprinters about to dash off in a race, someone about to turn off all the lights in a home, a spectacular chemical reaction about to start, etc. But the authors don't intend those scenarios to fall under the current project, right?

The reviewer is correct that "stability" has (at least) these two different meanings, and also correct that we are investigating here the situation in which a configuration is not changing now but would be likely to change with just the slightest perturbation. Our hypothesis is that the “Physics Network” will be sensitive to the likelihood that a physical configuration will change for physical (not social) reasons. That is what our data show: we do not find the same univariate and multivariate effects for situations that are likely to change because of the behavior of an animal. This indicates that what we are decoding is not general ‘likeliness to change’ but rather physical instability in particular.

(Also: Is stability really 'the most basic prediction' we make about the world? Who is to say that stable vs. unstable is a more basic judgment than, say, present vs. absent, or expected vs. unexpected, or safe vs. unsafe, etc? I know this is mostly just trying to get the reader excited about the results, but I stumbled there.)

We have now modified the sentence to say: “…how the brain makes a fundamental prediction about the physical world: whether the situation in front of us is stable, and hence likely to stay the same, or unstable, and hence likely to change in the immediate future.”

1. Laying out these issues in terms of feedforward processing vs. simulation felt a bit misleading and/or unfair to those views, given the substance of what this paper is actually doing. In particular, the feedforward view ends up getting assimilated to "what CNNs do"; but these are completely different hypotheses (or at least can be). Note, for example, that many vision researchers who don't think CNNs are good models of human vision nevertheless do think that lots of what human vision does is feedforward; that view could only be coherent if there are kinds of feedforward processing that are un-CNN-like. It would be better not to conflate these two and just say that the pattern of results rules out CNN-like feedforward processing without ruling out feedforward processing in general.

This is a fair point, and we certainly agree that we cannot rule out all feedforward models. We have tried to be clear about this claim, e.g., here (in the Discussion: “Three lines of evidence from the present study indicate that pattern recognition alone – as instantiated in feedforward CNNs and the ventral visual pathway – is unlikely to explain physical inference in humans, at least for the case of physical stability."

3a. I wasn't sure how impressed to be by the fact that, say, 60% classification accuracy one class of stable/unstable scenes doesn't lead to above-chance performance on another class of stable/unstable scenes. Put differently, it seems that the CNNs simply didn't do a great job classifying physical stability in the first place; in that case, how general should we expect their representations to be anyway? Now, on one hand, I could see this worry only further supporting the authors' case, since you could think of this as all the more evidence that CNNs won't have representations of stability in them. But since (a) the claims the authors are making are about feedforward processing in principle, not just in one or two CNNs, and (b) the purpose of this paper is to explore the issue of generality per se, rather than just stability, this seems inadequate. It could be that a CNN that does achieve high accuracy on physical stability judgments (90%?) would actually show this kind of general transfer; but we don't know that from the data presented here, because it's possible that the lack of generality arises from poor performance to begin with.

You are correct in noting that CNNs don’t do a great job in classifying physical stability, which reinforces our point that pattern recognition systems are not very good at discerning physical stability. In fact, the classification accuracy that we have reported is close to the baseline performance in literature (Lerer et al 2016). Interestingly, training on the block tower dataset itself could only bring up the stability classification accuracy to 68.8% on the real-world block tower images. While this is true of the current best model of stability detection, we think that CNNs trained on large-scale datasets of stability under varying scenarios may in future be able to potentially generalize to other natural scenarios. However, to our knowledge no such datasets exist.

3b. I wasn't sure how to think about whether showing CNNs stable and unstable scenes is a fair test of their ability to represent physical stability. Do we know that stability is all that these images have in common? Maybe the CNN is doing a great job learning some other representation. This sort of thing comes up in some recent discussions of 'shortcuts' and/or the 'fairness' of comparisons between human and machine vision, including some recent theoretical papers (see author recommendations for specific suggestions here).

If our point were that CNNs do a great job at representing physical stability, we would indeed have to worry about low-level image confounds or “shortcuts” enabling this performance. But our point is that they do badly. If some of their already bad performance is due to image confounds/shortcuts then they are in fact doing even worse, and that only makes our point stronger.

4a. I didn't really follow this passage, which is relied on to interpret greater activity for unstable vs stable scenes: "we reasoned that if the candidate physics regions are engaged automatically in simulating what will happen next, they should show a higher mean response when viewing physically unstable scenes (because there is more to simulate) than stable scenes (where nothing is predicted to happen)." It seems true enough that, once one knows that a scene is stable, one doesn't then need a dynamically updated representation of its unfolding. But the question that this paper is about is how we determine, in the first place, that a scene is stable or not. The simulations at issue are simulations one runs before one knows their outcome, and so it wasn't clear at all to me that there is always more to simulate in an unstable scene. Stable scenes may well have a lot to simulate, even if we determine after those hefty simulations that the scene is stable after all. And of course unstable scenes might well have very little to simulate, if the scene is simple and the instability is straightforwardly evident. Can the authors say more about why it's easier to determine that a stable scene is stable than that an unstable scene is unstable? They may have a good answer! It would just be better to see it in the paper.

The idea here is that forward simulation happens in all cases but stops if no change has occurred since the last frame. That stopping, both represents the stability of the configuration and produces less activity. This idea is akin to the “sleep state” used for nonmoving objects in a physics engine: they do not need to be re-simulated or re-rendered if they have not moved since the last frame (Ullman et al, 2017 TICS).

4b. I was confused a bit by the Animals-People condition, and whether to think of it as a control condition or not. The image of it in Figure 1a makes it seem like it is meant to be interpreted along the usual "physical stability" lines, just like falling towers and people on ladders, and the caption seems to say this too; it also makes intuitive sense since the man in the boat looks like he'll fall if and when the alligator attacks. But then in the main text the authors predict that the representations of stability would not extend to the Animals-People condition, because they are just supposed to be about peril but not stability. Why not? And then the results themselves are equivocal, with some findings generalizing to Animals-People and some not. I don't have much more to say here other than that I found this hard to follow.

We used the Animals-People as a control for peril/instability that is not caused by the physical situation (but rather by another agent). Our hypothesis was that the “Physics Network” would hold information about physical stability, not just any kind of propensity for change for any reason. Hence, we predicted, that any brain region responding (only) to physical stability should not respond in a similar way to peril/non-peril conditions in the Animals-People scenario as they involve a more biological-agent driven interaction. That is what we found.

Reviewer #2 (Public Review):

This paper combines neuroimaging, behavioral experiments, and computational modeling to argue that (a) there is a network of brain areas that represent physical stability, (b) these areas do so in a way that generalizes across many kinds of instability (e.g., not only a tower of blocks about to fall over, but also a person about to fall off a ladder), and (c) that this supports a simulation account of physical reasoning, rather than one based on feedforward processing; this last claim arises through a comparison of humans to CNNs, which do an OK job classifying physical instability but not in a way that transfers across these different stability classes. In my opinion, this is a lovely contribution to the literatures on both intuitive physical reasoning and (un)humanlike machine vision. At the same time, I wasn't sure that the broader conclusions followed from the data in the way the authors preferred, and I also had some concerns about some of the methodological choices made here.

1. The following framing puzzled me a bit, and even seemed to raise an unaddressed confound in the paper: "Here we investigate how the brain makes the most basic prediction about the physical world: whether the situation in front of us is stable, and hence likely to stay the same, or unstable, and hence likely to change in the immediate future".

Consider the following minor worry, which sets up a more major one: This framing, which connects 'stability' to 'change' and which continues throughout the paper, seems to equivocate on the notion of 'stability'. One meaning of 'stable' is, roughly, 'unchanging'. Another meaning is 'unlikely to fall over'. The above quotation, along with others like it, makes it seem like the authors are investigating the former, since that's the only meaning that makes this quotation make sense. But in fact the experiments are about the latter -- towers falling down, people falling off ladders, etc. But these aren't the same thing! So there's a bit of wordplay happening here, it seemed to me.

This sets up the more serious worry. As this framing reveals, unstable scenes (in the likely-to-fall-over sense) are, by their nature, scenes where something is likely to change. In that case, how do we know that the brain areas this project has identified aren't representing 'likeliness to change', rather than physical stability? There are, of course, many objects and scenes that might be highly likely to change without being at all physically unstable. Even the first example in the paper ("a dog about to give chase") is about likely changes without any physical instability. But isn't this a confound? All of the examples of physical instability explored here also involve likeliness to change! So these could be 'likely to change' brain areas, not 'physically unstable' brain areas. Right? Or if not, what am I missing?

The caption of Figure 1 seems to get at this a bit, but in a way I admit I just found a bit confusing. If authors do after all intend "physically unstable" to mean "likely to change", then many classes of scenarios that are unexplored here seem like they would be relevant: a line of sprinters about to dash off in a race, someone about to turn off all the lights in a home, a spectacular chemical reaction about to start, etc. But the authors don't intend those scenarios to fall under the current project, right?

(Also: Is stability really 'the most basic prediction' we make about the world? Who is to say that stable vs. unstable is a more basic judgment than, say, present vs. absent, or expected vs. unexpected, or safe vs. unsafe, etc? I know this is mostly just trying to get the reader excited about the results, but I stumbled there.)

2. Laying out these issues in terms of feedforward processing vs. simulation felt a bit misleading and/or unfair to those views, given the substance of what this paper is actually doing. In particular, the feedforward view ends up getting assimilated to "what CNNs do"; but these are completely different hypotheses (or at least can be). Note, for example, that many vision researchers who don't think CNNs are good models of human vision nevertheless do think that lots of what human vision does is feedforward; that view could only be coherent if there are kinds of feedforward processing that are un-CNN-like. It would be better not to conflate these two and just say that the pattern of results rules out CNN-like feedforward processing without ruling out feedforward processing in general.

3a. I wasn't sure how impressed to be by the fact that, say, 60% classification accuracy one class of stable/unstable scenes doesn't lead to above-chance performance on another class of stable/unstable scenes. Put differently, it seems that the CNNs simply didn't do a great job classifying physical stability in the first place; in that case, how general should we expect their representations to be anyway? Now, on one hand, I could see this worry only further supporting the authors' case, since you could think of this as all the more evidence that CNNs won't have representations of stability in them. But since (a) the claims the authors are making are about feedforward processing in principle, not just in one or two CNNs, and (b) the purpose of this paper is to explore the issue of generality per se, rather than just stability, this seems inadequate. It could be that a CNN that does achieve high accuracy on physical stability judgments (90%?) would actually show this kind of general transfer; but we don't know that from the data presented here, because it's possible that the lack of generality arises from poor performance to begin with.

3b. I wasn't sure how to think about whether showing CNNs stable and unstable scenes is a fair test of their ability to represent physical stability. Do we know that stability is all that these images have in common? Maybe the CNN is doing a great job learning some other representation. This sort of thing comes up in some recent discussions of 'shortcuts' and/or the 'fairness' of comparisons between human and machine vision, including some recent theoretical papers (see author recommendations for specific suggestions here).

4a. I didn't really follow this passage, which is relied on to interpret greater activity for unstable vs stable scenes: "we reasoned that if the candidate physics regions are engaged automatically in simulating what will happen next, they should show a higher mean response when viewing physically unstable scenes (because there is more to simulate) than stable scenes (where nothing is predicted to happen)." It seems true enough that, once one knows that a scene is stable, one doesn't then need a dynamically updated representation of its unfolding. But the question that this paper is about is how we determine, in the first place, that a scene is stable or not. The simulations at issue are simulations one runs before one knows their outcome, and so it wasn't clear at all to me that there is always more to simulate in an unstable scene. Stable scenes may well have a lot to simulate, even if we determine after those hefty simulations that the scene is stable after all. And of course unstable scenes might well have very little to simulate, if the scene is simple and the instability is straightforwardly evident. Can the authors say more about why it's easier to determine that a stable scene is stable than that an unstable scene is unstable? They may have a good answer! It would just be better to see it in the paper.

4b. I was confused a bit by the Animals-People condition, and whether to think of it as a control condition or not. The image of it in Figure 1a makes it seem like it is meant to be interpreted along the usual "physical stability" lines, just like falling towers and people on ladders, and the caption seems to say this too; it also makes intuitive sense since the man in the boat looks like he'll fall if and when the alligator attacks. But then in the main text the authors predict that the representations of stability would not extend to the Animals-People condition, because they are just supposed to be about peril but not stability. Why not? And then the results themselves are equivocal, with some findings generalizing to Animals-People and some not. I don't have much more to say here other than that I found this hard to follow.

5. "Interestingness" ratings felt like a not-quite-adequate approach for evaluating how attention-grabbing the towers were. A Bach concerto is more interesting than a gunshot (and would be rated that way, I imagine), but the gunshot is surely more attention-grabbing. Why not use a measure like how much they distract from another task? That's the sort of thing I'd have expected, in any case.

It's crazy how much culture changes like that in just 10 years. Now it seems like we all see the downsides of hypergrowth and spending billions on marketing (e.g. Uber), and often swing to the complete opposite -- taking no funding at all.

So, not that I don't want to believe it's 12,000 years old but. What if it was just a museum. apparently not much of it has been excavated... there's literally an engraving of a dinosaur there.Other thoughts are on accelerated carbon decomposition... and holy $hiT Gods real.. he's moving people through time. (The picts artwork is staggeringly similar and mysterious)

I think it's really neat that you developed a mathematical model that actually predicts temperature increase. I do think this section is much more equation heavy than any of the previous ones, so as I mentioned above, I think you should prepare the reader gradually for the section. Having a short section about ratios and proportions for example, as well as just dealing with algebraic equations might be helpful.

How to use Relational Databases to process logs

what do we say, "it's not okay to _" on ?

managing emotions is a form of repression... with an intellectual component... "we will usually do anger and sadness first, then probably fear, then joy, bliss, peace. All of that stuff starts coming"

"The negative emotions need to move first and then the positive emotions. Oftentimes people don’t understand they are repressing the positive emotions."

I think repressing the positive emotions is where a lot of the people around me get stuck! They don't know how to react or respond to someone who is in a positive emotional state.

pendulating through the cycle

Reviewer 1. Stian Soiland-Reyes

This review is also available at Description The article describes a mechanism to add the BLAST+ functionality to the Galaxy workflow system. This is a very useful feature, and so in principle I would want to see this article published. I do however have some concerns with the aspects of reproducibility and documentation, which are detailed below.

Major Compulsory Revisions

I am afraid I will have to ask for major compulsory revisions as I was unable to reproduce any the claims of the paper.

1: Docker image is not BLAST enabled

p5.

the command docker ... start a BLAST enabled Galaxy instance I tried the docker image. It starts up fine, and presents a Galaxy that includes a list of BLAST tools - so the BLAST tools have been installed. The docker instance is however not BLAST enabled, as the BLAST tools requires further configuration/download of the external BLAST reference database to align against. This procedure is loosely documented at https://registry.hub.docker.com/u/bgruening/galaxy-blast/ - but I was unable to follow through with this installation as it was quite complicated and seems to require manual downloading and configuration of many GB of reference data spread over more than 300 files. I was assuming that a docker image would be 'usable out of the box' - but this is far from the truth in this case. Accessing "NCBI BLAST+ database info" gives an empty dropdown list in The article mentions that the public Galaxy instance usegalaxy.com does not provide the BLAST tools by default due to concerns over computational load - but I am also worried if it could be because configuring the BLAST+ tools is quite a complicated job. The article does not mention at all the excessive amount of system administraton that is required in order to finalize the BLAST installation, and the docker image does not provide any helper scripts to assist with this. In fact, the example database configuration files uses a totally different path, e.g. /depot/data2/galaxy/blastdb/nt/nt.chunk - while the docker image would require these under /data/nt/nt.chunk. The article or Docker README does not mention which subset of the databases would commonly need to be downloaded - or even the fact that all of the numbered fragments need to be downloaded. The dataset referenced from the example configuration, e.g. nt.chunk and wgs.chunk do not exist on ftp://ftp.ncbi.nlm.nih.gov/blast/db/ - only non-chunk version exist. I tried to download a subset of the datasets from ftp://ftp.ncbi.nlm.nih.gov/blast/db/ stain@biggie-utopic:/galaxy_store/data/blast_databases$ ls human_genomic.00.nhd nt.00.nhd refseq_genomic.148.nhr refseq_protein.00.pin refseq_protein.15.pnd wgs.00.nhi human_genomic.00.nhi nt.00.nhi refseq_genomic.148.nin refseq_protein.00.pnd refseq_protein.15.pni wgs.00.nhr human_genomic.00.nhr nt.00.nhr refseq_genomic.148.nnd refseq_protein.00.pni refseq_protein.15.pog wgs.00.nin human_genomic.00.nin nt.00.nin refseq_genomic.148.nni refseq_protein.00.pog refseq_protein.15.ppd wgs.00.nnd human_genomic.00.nnd nt.00.nnd refseq_genomic.148.nog refseq_protein.00.ppd refseq_protein.15.ppi wgs.00.nni human_genomic.00.nni nt.00.nni refseq_genomic.148.nsd refseq_protein.00.ppi refseq_protein.15.psd wgs.00.nog human_genomic.00.nog nt.00.nog refseq_genomic.148.nsi refseq_protein.00.psd refseq_protein.15.psi wgs.00.nsd human_genomic.00.nsd nt.00.nsd refseq_genomic.148.nsq refseq_protein.00.psi refseq_protein.15.psq wgs.00.nsi human_genomic.00.nsi nt.00.nsi refseq_genomic.148.tar.gz refseq_protein.00.psq refseq_protein.15.tar.gz wgs.00.nsq human_genomic.00.nsq nt.00.nsq refseq_genomic.nal refseq_protein.15.phr refseq_protein.pal wgs.nal human_genomic.nal nt.nal refseq_protein.00.phr refseq_protein.15.pin wgs.00.nhd and configured these in blastdb.loc according to the Docker readme. The readme says: you need to add the paths to your blast databases and they need to look like /export/swissprot/swissprot but I have followed the instructions three lines above which mounted the datasets at /data - hence I used /data/ instead of /export. Some consistency would help here. stain@biggie-utopic:/galaxy_store/data/blast_databases$ grep -v ^# /tmp/galaxy/galaxy-central/tool-data/blastdb*loc /tmp/galaxy/galaxy-central/tool-data/blastdb.loc:nt_02_Dec_2009 nt 02 Dec 2009 /data/nt /tmp/galaxy/galaxy-central/tool-data/blastdb.loc:wgs_30_Nov_2009 wgs 30 Nov 2009 /data/wgs/wgs /tmp/galaxy/galaxy-central/tool-data/blastdb.loc:refseq_genomic_148 refseq 148 /data/refseq_genomic /tmp/galaxy/galaxy-central/tool-data/blastdb.loc: /tmp/galaxy/galaxy-central/tool-data/blastdb.loc: /tmp/galaxy/galaxy-central/tool-data/blastdb_p.loc:nt_02_Dec_2009 nt 02 Dec 2009 /data/nt /tmp/galaxy/galaxy-central/tool-data/blastdb_p.loc:wgs_30_Nov_2009 wgs 30 Nov 2009 /data/wgs/wgs /tmp/galaxy/galaxy-central/tool-data/blastdb_p.loc:refseq_protein refseq protein /data/refseq_genomic /tmp/galaxy/galaxy-central/tool-data/blastdb_p.loc: /tmp/galaxy/galaxy-central/tool-data/blastdb_p.loc: A BLAST Data Manager is available at at https://github.com/peterjc/galaxy_blast/ - in theory this can download and populate the blastdb data table. This is mentioned as "Future work" in the article, so presumably it is not yet production ready. This data manager does not appear under Data Libraries in the docker image, and it is not included in the installation at https://registry.hub.docker.com/u/bgruening/galaxy-blast/dockerfile/

2: Galaxy Tool Shed not working with the docker image

The article says: The recently published Galaxy Tool Shed [9] allows anyone hosting a Galaxy instance to install tools and defined dependencies with a few clicks right from the Galaxy web application itself. I am unable to verify this claim using the provided Docker image. I am unable to install any tools from the Galaxy Tool Shed from the web interface of the Docker image. I am logged in as admin@galaxy.org according to the instructions, but if I go to Admin -> Search and browse tool sheds http://localhost:8080/admin_toolshed/browse_tool_sheds and click the dropdown list for "Browse valid sheds", this hangs for a while before failing with "Can't find the server". On the console I get many error messages like: URLError: <urlopen error [Errno -2] Name or service not known> tool_shed.util.shed_util_common ERROR 2015-01-19 10:22:08,698 Error attempting to get tool shed status for installed repository ncbi_blast_plus:

<urlopen error [Errno -2] Name or service not known> Traceback (most recent call last): File "lib/tool_shed/util/shed_util_common.py", line 772, in get_tool_shed_status_for_installed_repository encoded_tool_shed_status_dict = common_util.tool_shed_get( app, tool_shed_url, url ) File "lib/tool_shed/util/common_util.py", line 345, in tool_shed_get response = urlopener.open( uri ) File "/usr/lib/python2.7/urllib2.py", line 404, in open response = self._open(req, data) File "/usr/lib/python2.7/urllib2.py", line 422, in _open '_open', req) File "/usr/lib/python2.7/urllib2.py", line 382, in _call_chain result = func(*args) File "/usr/lib/python2.7/urllib2.py", line 1214, in http_open return self.do_open(httplib.HTTPConnection, req) File "/usr/lib/python2.7/urllib2.py", line 1184, in do_open raise URLError(err) Inspecting the internal frame I see the link is http://toolshed.g2.bx.psu.edu/repository/browse_valid_categories?galaxy_url=http://localhost:8080 I somehow feel that toolshed.g2.bx.psu.edu will try to connect to my galaxy instance at http://localhost:8080 - which is not going to work. The actual toolshed site is unavailable http://www.downforeveryoneorjustme.com/toolshed.g2.bx.psu.edu It's not just you! http://toolshed.g2.bx.psu.edu looks down from here. ..so this might be an temporary network problem that is not related to the "localhost" bit. I am nevertheless unable to verify the claim of the ease of using the Tool Shed to install the BLAST+ tool because of this. If it is true that the Docker image does not work with the Galaxy Tool Shed - which now host most of the tools required in a Galaxy installation, then this should be duely noted in the article and the README of the Docker image.

3: Supporting data has no usage instructions

The article links to https://github.com/peterjc/galaxy_blast as the supporting data - but this website has no instructions on how to install/use with Galaxy or the Galaxy docker image. I could execute .travis.yml "by hand" - but I do not feel this is sufficient documentaton for a supporting data set. I have therefore not been able to verify that the supporting data actually supports the article, beyond inspecting the Travis-CI build logs at https://travis-ci.org/peterjc/galaxy_blast/builds .. which except for a single error seem to be verifying the tools. https://travis-ci.org/peterjc/galaxy_blast/builds/45137901 OperationalError: (OperationalError) unable to open database file None None

Minor Essential Revisions

4: Provenance and update issue not addressed

Workflow systems are commonly praised in bioinformatics because they enable reproducibility and sharing of analytical pipelines. One challenge in this aspect is that the domain of bioinformatics commonly update software tools and reference datasets. In fact, BLAST+ 2.2.30 was released just 6 weeks ago [ ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/ ] and the latest BLAST reference dataset taxdb.tar.gz was updated today [ ftp://ftp.ncbi.nlm.nih.gov/blast/db/ ]. The blast FTP site does not seem to contain any version number of the dataset, and as datasets are split over multiple files, it would be difficult to know if you have downloaded half an old dataset and half a new dataset. (A new version of the dataset could be released in the middle of your lengthy download). The sanity of the dataset could potentially be verified by downloading the *.md5 files both before and after the large download -- but this should be automated by a script to be done correctly. I would say the main challenges are in this respect, assuming a successful workflow run using the described Galaxy BLAST tools: a) Which version of the BLAST tool was used? b) Which reference data set was used? c) Which version? d) Was the install complete/sane? (ref. MD5 files and updates) e) Are there any later versions of tool or reference data set? How do I keep my Galaxy instance up to date? (going through the lengthy database download+config again?) f) How can a Galaxy workflow using BLAST+ be shared with another Galaxy instance (supposedly easily started with Docker), when manual download and configuration of databases are required? Your article does not mention how the BLAST+ tool for Galaxy addresses any of these concerns. The use of the Galaxy Tool Shed should in theory allow for automatic updating of the tool - and I believe the BLAST tools would output log information that includes at least version number. I am worried that the dataset description that is entered manually by the system administrator into /galaxy-central/tool-data/blastdb.loc and friends contain an element of "manual versioning", as the example contains

nt_02_Dec_2009 nt 02 Dec 2009 /depot/data2/galaxy/blastdb/nt/nt.chunk

wgs_30_Nov_2009 wgs 30 Nov 2009

/depot/data2/galaxy/blastdb/wgs/wgs.chunk This sounds very errorprone, and as older datasets not available from NCBI, definitiely not reproducible. I would expect the article to at least acknowledge these concerns - and ideally for the tooling to support this (e.g. through the BLAST Data Manager and additional provenance output from the BLAST+ tools, e.g. in W3C PROV format).

5: Results workflows unavavailable

We now describe some use-cases and workflows combining these tools within Galaxy. The first two examples:

• Assessing a de novo assembly
• Finding genes of interest in a de novo assembly do not link to any actual Galaxy workflow descriptions, but are only described as bullet point lists. The descriptions do not link to any examples for "Upload ** sequence" or of the expected outputs. "Identifying candidate genes clusters" is described in more detail, but the workflow is only included as a visual Figure, and not in the supporting data or uploaded/linked to an external repository like the mentioned myExperiment. The citation for this workflow, [22] http://dx.doi.org/10.1021/ja501630w is not Open Access, and I was required to use the University of Manchester. The article does not mention the word "workflow" once, and do not seem to contain any data citations for the workflow, only for the sequence. The only supporting information provided at http://pubs.acs.org/doi/suppl/10.1021/ja501630w is a PDF with tables, graphs and sequence views. Again the word "workflow" is not mentioned. A direct link to the workflow definition should be included for all three examples.

Discretionary Revisions

Spelling corrections for product/company names p2:

• MyExperiment -> myExperiment
• Amazon Inc. -> Amazon AWS
• "Cloud Computing" -> Cloud Computing
• Galaxy "CloudMan" -> Galaxy CloudMan
• "Galaxy Tool Shed" -> Galaxy Tool Shed p5:
• "such FASTA format" -> "such as FASTA format"
• Docker Inc. -> Docker Inc. (https://www.docker.com/)
• Galaxy "CloudMan" -> Galaxy CloudMano Useful hyperlinks:
• whose functional tests are then run. -> whose ..then run (https://travis-ci.org/peterjc/galaxy_blast).
• The Galaxy-P project -> The Galaxy-P project https://usegalaxyp.org/

Nothing too tricky from a linguistic perspective, just wanted to highlight this fun image: "carnival of confetti," all those punched holes, that stick to his clothes like sand in your shoes after a day at the beach...

But why is it in his head? He's not imagining it — it's quite literal. Is it that his brain has been reduced to little fragmented, meaningless bits by his boring, repetitive job?

Man kann also diese clade unterscheiden von anderen, die tatsächlich existiert haben, und sagen, dass sie bestimmte Eigenschaften hat, durch die sie diesen anderen überlegen war, wobei dies nicht unbedingt Eigenschaften einzelner Exemplare sein müssen. Hier ist die Eigenschaft, die genannt wird, die Diversität.

—Bruno Winck

Reminder to look up Roland Claude. I couldn't find his work in the usual spaces in English or French.

This image deserves some attention, as there's a strange redundancy. Both "clochettes" and "grelots" mean "bells." So the silver bells on her wrists shake her bells? Gainsbourg is too good a writer to produce such a clumsy phrase.

So I went looking for double meanings. It seems that "avoir des grelots" means "tremble with fears" ("grelotter" has the same meaning). So there's no redundancy: it's just that the literal bells she wears act out the literal meaning behind the expression "avoid des grelots" — she advances with trepidation to pronounce the words that ends her relationship with Serge. Whether out of nervousness or out of cowardice — out of sympathy for Serge, or fear of leaving her rich husband — I'll leave to you...

By looking at the risk to women and girls from males. That has to be the starting point. I can't see why the precautionary principle should not be invoked here: if you want to claim that some males (the ones who want to be treated as if they were female) are no threat to females (not that it's just about feeling or being threatened), then the burden is on the advocates of allowing males into female spaces to show that the risk is - at best - minimal. It is usually accepted that some women are a threat to other women (even if the vast majority of those who are a threat are male), then it has to be accepted that at least some of those males who want to be treated as if they were female are also a threat. The only rebuttal of that is to posit that all men who want to be treated as if they were female are a lower risk to women than even women are. I can't see how anyone could be so credulous.

ok!

• si se crea una tree-structure, luego es "incomodo" moverse, o no se "recuerda" donde estaba;
• o se mete todo en una "bolsa de basura", XOR no se utilizan los bookmarks!

This piece claims to be about free software but is actually full of sparkling social insight, which is probably illustrative of how you shouldn't be trying to do software at any kind of scale absent efforts at social insight.

Quite interesting: Amazon is not the first platform you think of when considering to “make your voice heard”.

Totally agree. The point is not to get obsessive about each decision and try to think through every consequence, but to be aware that big things could happen because of it.

That also works in the positive direction -- e.g. tweeting often probably won't get you a large audience by itself, but potentially you become a person who connects well with people, that builds a great product, and build an audience as a result of that. Just one example of the many things that can happen by doing things with some uncertainty.

Isn't this also just hypothesized? It's not 100% certain that this is what caused the extinction, right?

Just as Western historians erase indigenous ideas as misunderstandings or fabrications or outright appropriation of those ideas as pre-existing ideas in European culture, is it possible that we do the same thing with orality and memory? Are medievalists seeing mnemotechniques of the time and not properly interpreting them by not seeing them in their original contexts and practices?

The idea of talking rocks, as an example, is dismissed as lunacy, crazy, or some new-age hokum, but in reality it's at the far end of the spectrum. It's so unknowable for Western audiences that it's wholly dismissed rather than embraced, extended, and erased.

What does the spectrum of potentially appropriated ideas look like? What causes their adoption or not, particularly in cases of otherwise cultural heterodoxy?

revisit this... it's an important point, particularly when looking at complex ideas with potentially emergent properties

chỉ viết: từ khi chuyển sang học từ xa, hầu hết các sinh viên đều không thể cân bằng thời gian học tập của mình, ngay cả bản thân tôi cũng như thế. Họ có vô số lý do như nhà mất mạng, khi ở nhà sẽ có em nhỏ hoặc những tiếng ồn bên ngoài, chưa kể là có những bạn ngủ quá nhiều, vừa học vừa lướt mạng xã hội, không tập trung vào bài giảng dẫn đến không hiểu bài .

Tangential to the article, but this seems like a powerful idea. I wonder what the best importance filter is if thinking is faulty.

Possibly it's time that creates concrete evidence for importance. But connecting past dots is thinking, and you can't notice reoccurrences of something you don't know. You need to believe something is important to prove that it really is.

Maybe the quote is not such a powerful idea after all, just a reminder to question our heuristics sometimes.

The final piece of writing is a critical reflection about your work that uses the ideas and texts in the course to support, and highlight, your ideas.

This piece will also be published in SCALAR for future use; that is, the final piece should say something about how you've encountered the material in the course so as to instruct future users/readers. Your writing—your readings of the texts; your telling of your experience with ideas—will be great guides for students.

Think broadly, first. Ruminate on the following and record it for yourselves somewhere (I use a black notebook for this and write longhand; no technology, other than a Sharpie, for thinking—that works best for me):

Explain and describe what the texts help you see and understand, even if this means further confusion, or the creation of more questions that are yet unanswerable for you. Here, begin to not rely on invisibility and slow violence; think beyond these very large themes.

How have these texts helped you delve deeper into the questions of environmental justice—and expanding the meaning, on confusing it, making it, perhaps, more nuanced?

How does environmental justice affect your view of what you need to examine about your goals going forward?

This preamble, the questions, should be seen as lofty goals to guide your thinking and your writing. More specific "how-to" is below.

The GOAL of this piece is to describe, discuss, and even argue ideas that will flow smoothly into an answer to the following question: Knowing what you know now, how are you going to approach the rest of your life?

This question doesn't have a definitive answer; it's about perspective, point of view, attitude. It's also about responsibility — the ability to respond: where will you find the space(s) necessary to give yourself enough time to respond to what comes your way—unexpectedly? how do you do this? how will your education help, with examples, referring to texts, in this course and others you've encountered, that have affected the way you think? after summarizing the texts in this course, which singular text, which one of these, is something you know you'll take with you, meaning you know that it has affected you and the ideas found in this text are important guides? how does this text help you see yourself better?

I taught an FYS back in the fall of 2015 and the class had a similar assignment. Students asked that I too write the assignment and provide a model. You guys didn't take this course, but I'm sharing with you what I wrote (since published) (Links to an external site.) so that you can see a model for the work I'm asking you to do. Notice how I contextualize the texts, give the reading of each I need for my argument/description, and use this to describe a native characteristic of American culture. I'm not asking that you be as lengthy. I'm not expecting this sort of reading of American culture; these courses are different. I'm basically wanting to know what it is you see now that the course is over, or nearly over. This model (linked) is simply a sketch for you, and an outline that allows you to see a way into the work, and a way through—a way to organize.

I want you to be creative about your approach. You can tell a story and use the texts, for instance. You can use your experiences as a way through this and use the texts. And so on ...

I have created a SCALAR PAGE (Links to an external site.) for each of you.

I want you to consider following these guidelines for writing:

Go back to your mapping exercise: How did your plan turn out? Where are you now? [this is something we will have already spoken about in our f2f meetings, so you want to have notes from that]. This shouldn't be written, When I look at my mapping...or I said in my mapping that ... Rather, it should be something along the lines of, My writing interests in this course suggest (ideas + support from essays) ... or A central focus of my essays (or thinking) has been ... (examples)...or Engaging the texts in this course, I started to think about ... (examples) ... I thought ... and now I'm thinking that ... (examples)  — This section should be short (no more than a tight paragraph) and strategically placed.
As preparation for writing, without looking at the texts, just referring to their titles for inspiration, see what you recall: write out a paragraph or so about each text encountered in the course—this is for your own use and a way to organize before writing; this is done to determine what you recall, which is important and what you should focus on because that's instinct talking; eventually open the texts to make sure you have examples for citing [any material from outside the syllabus you wish to cite is fine, too, especially since I've sent you a lot of reading from the popular media].
Find a central idea or theme you want to explore. Set it down somewhere so you can see it and read it back to yourself. Yes, there will be the tendency to speak about invisibility and slow violence, I get that; however, these should be ideas that help illustrate a central idea or theme that's your very own and based on what your reading of the courses' texts tells you about the nature of society, as it is now, the challenges we face, and, definitely, where you're situated, in the texts and the challenges we face. Thus, invisibility and slow violence should not be the central ideas/themes of your work, rather instruments/conditions/truths you found along the way and you're using these to pry open a deeper, richer understanding of your relationship to these ideas and environmental justice.
Write a draft and start sharing it with your group. Likewise, you want to make sure that you and I sit with your piece, letting me comment on it before it's due  (even numerous times) so that we have the piece you want. You definitely want to conference with me about your piece so I can help you get as deep as possible into the subject—in other words: making sure each of you writes something you're really moved by and proud of.
Since this is on SCALAR, make sure you have relevant images, links, where appropriate and necessary, and any other media (clips, sound, etc), you may wish to insert creatively to lift your piece.

7

Reviewer #2 (Public Review):

The manuscript by Carrasquilla and colleagues applied Mendelian Randomization (MR) techniques to study causal relationship of physical activity and obesity. Their results support the causal effects of physical activity on obesity, and bi-directional causal effects of sedentary time and obesity. One strength of this work is the use of CAUSE, a recently developed MR method that is robust to common violations of MR assumptions. The conclusion reached could potentially have a large impact on an important public health problem.

Major comments:

(1) While the effect of physical activity on obesity is in line with earlier studies, the finding that BMI has a causal effect on sedendary time is somewhat unexpected. In particular, the authors found this effect only with CAUSE, but the evidence from other MR methods do not reach statistical significance cutoff. The strength of CAUSE is more about the control of false positive, instead of high power. In general, the power of CAUSE is lower than the simple IVW method. This is also the case in this setting, of high power of exposure (BMI) but lower power of outcome (sedentary time) - see Fig. 2B of the CAUSE paper.

It does not necessarily mean that the results are wrong. It's possible for example, by better modeling pleiotropic effects, CAUSE better captures the causal effects and have higher power. Nevertheless, it would be helpful to better understand why CAUSE gives high statistical significance while others not. Two suggestions here:

(a) It is useful to visualize the MR analysis with scatter plot of the effect sizes of variants on the exposure (BMI) and outcome (sedentary time). In the plot, the variants can be colored by their contribution to the CAUSE statistics, see Fig. 4 of the CAUSE paper. This plot would help show, for example, whether there are outlier variants; or whether the results are largely driven by just a small number of variants.

(b) CAUSE is susceptible to false positives when the value of q, a measure of the proportion of shared variants, is high. The authors stated that q is about 0.2, which is pretty small. However, it is unclear if this is q under the causal model or the sharing model. If q is small under the sharing model, the result would be quite convincing. This needs to be clarified.

(2) Given the concern above, it may be helpful to strengthen the results using additional strategy. Note that the biggest worry with BMI-sedantary time relation is that the two traits are both affected by an unobserved heritable factor. This hidden factor likely affects some behavior component, so most likely act through the brain. On the other hand, BMI may involve multiple tissue types, e.g. adipose. So the idea is: suppose we can partition BMI variants into different tissues, those acted via brain or via adipose, say; then we can test MR using only BMI variants in a certain tissue. If there is a causal effect of BMI on sedentary time, we expect to see similar results from MR with different tissues. If the two are affected by the hidden factor, then the MR analysis using BMI variants acted in adipose would not show significant results.

While I think this strategy is feasible conceptually, I realize that it may be difficult to implement. BMI heritability were found to be primarily enriched in brain regulatory elements [PMID:29632380], so even if there are other tissue components, their contribution may be small. One paper does report that BMI is enriched in CD19 cells [PMID: 28892062], though. A second challenge is to figure out the tissue of origin of GWAS variants. This probably require fine-mapping analysis to pinpoint causal variants, and overlap with tissue-specific enhancer maps, not a small task. So I'd strongly encourage the authors to pursue some analysis along this line, but it would be understandable if the results of this anlysis are negative.

Minor comments

- The term "causally associated" are confusing, e.g. in l32. If it's causal, then use the term "causal".

Good to reflect on. Even if you never use the note again, you should probably make the effort to create it in the moment when it first seemed interesting. Because you never know what the future will bring, you never know if the note will be useful or not.

This means that you need to strike a balance between going down the rabbit hole of trying to capture everything you read (just in case it's useful), and capturing nothing (because maybe it will never be used again).

Today, the number of on-demand service apps is incredible. It’s difficult to imagine an industry that isn’t affected by them. Taxi, food delivery, shopping, cleaning, reservations, and more are available with just a few clicks.

2020 boosted the popularity of such apps. People got stuck at home, so the necessity for on-demand services became high as ever. Moreover, new industries try themselves in providing people on-demand services, so the market is flourishing and evolving.

If this scenario looks attractive and you want to offer an on-demand service app, here’s a comprehensive guide on how to create one. It will ensure that you’re going in the right direction.

Colin Davis. (2021, December 20). Update for 20th Dec. The trend line still reflects 1.8 day doubling (it’s 1.7 days if we look at just the last week). Today’s number is down, but I wouldn’t read too much into that at this point. Https://t.co/kOCjxhRbop [Tweet]. @ProfColinDavis. https://twitter.com/ProfColinDavis/status/1472969632705392640

Author Response:

Reviewer #1:

This manuscript by Silver, et al., details work investigating the relationship between season of conception and DNA methylation differences at sites across the genome, measured by widely-used arrays, in two cohorts of children using Fourier regression. They find that season of conception is associated with persistent methylation differences at several hundred CpG sites, and that these CpG are enriched for properties, compared to sets of control sites, that suggest that methylation at these sites is influenced very early in development/during conception and that these sites are positioned in genomic regions relevant for gene activation and regulation. Additional analyses investigated the effects of genetic variation of these sites, and found no evidence for single nucleotide polymorphisms nor child sex confounding the associations between season of conception and DNA methylation. As the number of sites measures by these arrays are a very small amount of total sites across the genome, the authors suggest that these findings indicate there may be many more sensitive methylation 'hotspots' in the genome that are not captured by these arrays but could impact on health/development.

The key strengths of this manuscript include the use of two cohorts of children at different ages, providing evidence that these effects of season of conception appear to attenuate by 8-9 years of age; and comparison with control sites and additional analyses investigating confounding to build the evidence for these relationships reflecting true, biological associations rather than statistical artefacts or the result of confounding.

However, the conclusions around the potential functional importance of these methylation differences are limited by a lack of evidence for a relationship between methylation of these season-of-conception-associated sites and child growth/development, so while this manuscript builds compelling evidence for the effects of season of conception on methylation, it's functional relevance is unclear. Additionally, there are some choices made in the analyses where the rationale for those choices should be made more clear, such as the use of CpG sites above or below a certain estimated effect size for different analyses.

Overall, the approach taken here to demonstrate different levels of evidence for true relationships between early development exposures and differences in DNA methylation is a compelling one, and the manuscript delivers clear evidence for its primary conclusions.

We are currently researching links between several SoC-CpGs and health-related outcomes including measures of growth, and we have prepared/submitted other papers with different groups of authors (e.g. the EMPHASIS team) relating to other phenotypes. We consider a detailed analysis of links between SoC-CpGs and diverse outcome measures in Gambian children to be beyond the scope of the current study and would argue that such an analysis would dilute the central focus of this paper that is already long and complex. We do already refer to two existing studies linking Gambian SoC or nutrition-associated CpGs to health outcomes in non-Gambians (child & adult obesity/POMC, Kuhnen et al Cell Metab 2016; cancer/VTRNA2-1, Silver et al, Gen Biol 2015) in the current manuscript. The VTRNA2-1 locus does not overlap any SoC-CpGs and we already speculate that this may be due to SoC effect attenuation, since the previous association was observed in younger (3-9mth) infants. We have additionally referenced a recently published paper linking another SoC-associated locus to thyroid volume and function in Gambian children (Candler et al Sci Adv 2021) and highlighted that neither this nor the POMC locus overlap the array background analysed in this study. Finally we had already included an analysis of overlaps between SoC-CpGs and traits in published EWAS and GWAS catalogues.

Regarding our use of different SoC amplitude thresholds for one analysis, our original motivation for analysing all 768 ‘SoC-associated CpGs’ with FDR<5% in the ENID 2yr analysis, including those with amplitude < 4%, was to explore the degree to which the strength / amplitude of SoC effects could be explained by proximity to ERV1 over the wider range of amplitudes represented by the larger set of loci. However we agree that this approach is open to question and have removed this analysis (previous Fig. 6B and Supp. Fig. 11, and text in section headed ‘Enrichment of transposable elements and transcription factors associated with genomic imprinting’). We have also removed the definition of ‘SoC- associated CpGs’ (which included CpGs with SoC amplitude < 4%) from Table 2 and Methods to aid clarity and avoid confusion.

Reviewer #2:

This is a very interesting manuscript, which will be of interest for a broader readership. The authors have analysed an unique cohort, which is of importance to understand the impact of environmental factors on DNA methylation.

The performed analysis is well balanced, and the conclusions are justified by the presented data. It is a strength of this study, that results from the initial ENID study have been re-evaluated in the EMPHASIS study. Unfortunately, DNA methylation has been analysed using HM450 and EPIC arrays. Both methods are providing only a limited view on methylome-wide DNA methylation.

Another limitation (as already addressed by the authors) is the lack of longitudinal samples. This would potentially have helped to gain further knowledge about the identified attenuation of DNA methylation levels at SoC associated CpGs.

Finally, I am not entirely sure, that one confounding factor has been completely ruled out: It is known, that blood composition may cause methylation variability. In general, the authors addressed this point and analysed blood compositions (supplementary Figure 16) of both cohorts. Here, no marked seasonal differences between and within both cohorts have been identified. However, the participants of the EMPHASIS cohort have a very similar age (8-9 years). For this reason, I am wondering if methylation variability/ differences and in addition the attenuation of methylation levels might be influenced by the younger age of ENID participants compared to EMPHASIS study individuals.

We agree that the necessary restriction of our analysis to data derived from Illumina 450k and EPIC arrays means that we can only obtain a limited view of DNAm loci associated with Gambian season of conception. We expect that there will be many more such hotspots across the human methylome. We have commented on this in the Discussion.

Regarding the lack of longitudinal data to confirm the potential attenuation of SoC effects with age observed between unrelated cohorts, we are pleased to report that we have now acquired an additional EPIC array dataset covering a subset of n=138 individuals from the ENID cohort included in the main analysis. This subset had methylation measured in blood at age 5-7yrs enabling us to conduct an investigation of longitudinal methylation changes in these individuals. This analysis strongly supports the circumstantial evidence of SoC effect attenuation with age suggested by our previous comparison of the independent ENID (2yr) and EMPHASIS (7-9yr) cohorts, with:

a) strong correlation of conception date methylation maximum between age 2yr and 5- 7yrs at SoC-CpGs in these 138 individuals (Figs. 3A, 4A); and

b) evidence of SoC effect size attenuation at the majority of SoC-CpGs (Fig. 3B; Wilcoxon signed rank sum p=10-12).

We note that this additional longitudinal dataset has a different confounding structure with respect to biological and technical covariates (Supp Tables 15-17) and date of sample collection (Supp. Fig. 1B), lending strong support to our previous two-cohort cross-sectional analysis.

Regarding the potential for confounding by differences in blood cell composition, we have performed an additional sensitivity analysis with Houseman estimated blood cell counts added directly to the linear regression model for the ENID cohort (see ST1s). 518 out of the 520 estimated Fourier regression coefficients from the main analysis (1 pair of sine and cosine terms for each of the 259 SoC-CpGs) fall within the 95% confidence interval obtained in the Houseman-adjusted analysis, confirming that cell composition effects did not unduly influence SoC effect estimates in the original analysis. We have added a brief note on this and the other sensitivity analyses (batch, cell composition and village effects) in Results to the manuscript, with more details in Methods.

If the reviewer is referring to the possibility that the SoC effect attenuation with age could be driven by different cell composition effects in the older cohort, we think that the replication of the timing of SoC effects across the 3 datasets analysed (including the additional longitudinal data; Fig. 4A), all of which have different confounding structures with respect to season of sample collection (Fig. 2A; Supp Fig. 1B), together with additional evidence of SoC effect attenuation with age in the longitudinal analysis (Fig. 3B) support this being a genuine age attenuation effect.

Reviewer #3:

Silver et al. Investigate the influence of seasonal variation (nutrition, infection, environment) on blood DNA methylation in two cohorts of children (233 [2y] and 289 [8y-9y]) from the same sustenance farming communities in rural Gambia. One cohort (450K,233) was extensively studied before in multiple publications, the second dataset (850k,289) is unpublished. Using cosinor modeling they find 768 CpGs with a significant seasonal pattern(SoC-CpG, FDR<0.05) in the probes that overlap between the 450k and 850k arrays. Look-up of these 768 SoC-CpGs in the second sample showed 61 SoC-CpGs with FDR 0.05 (no mention is made if the direction of effect is consistent, but we assume it is so).

In fact we did report that the ‘direction’ of the effect (conception date at methylation maximum) is highly consistent with increased DNAm in conceptions at the peak of the rainy season across the two cohorts at the 61 SoC-CpGs with FDR<0.05 – see Fig. 2C.

The authors notice that most SoCs seem to be attenuated in the 8-9y sample. Then the authors select out of the 768 SoC-CpG the FDR<0.05 and >=4% seasonal amplitude in this discovery sample: 257 which they bring further in (enrichment) analyses. It is unclear if all 257 are (nominally) significant in the replication sample.

We did not check this because of evidence that, despite strong replication of effect direction (Fig. 4A), the amplitude of the SoC effect attenuates with age (Fig. 2E). This means that it would not be surprising if one or more SoC-CpGs failed to achieve nominal significance in the older cohort. This is now strongly supported by our additional analysis of longitudinal data confirming SoC effect attenuation with age and consistency of SoC effect direction (Figs. 3B and 4A).

These SoC-CpGs are enriched for imprinted and oocyte germline loci. Roughly 10% of SoC-CpGs overlap with so-called meta-stable epialleles (MEs), on which the authors have published greatly. This is a large fold enrichment, and subsequently the main focus of the Results and Discussion. Indeed, it skews the Discussion heavily and one wonders what could have been found in the other 90%?

Our strategy throughout the Results and Discussion was to focus on characteristics including metastability, parent of origin-specific methylation, histone modifications and gametic and early embryo methylation patterns that suggest a link to establishment of methylation states in the early embryo at SoC-CpGs. For these analyses all SoC-CpGs were considered at every stage and metastability was not the primary focus. However, as the reviewer suggests, we do repeatedly point out that many of the above contextual characteristics that are associated with SoC-CpGs have also been associated with metastability which we consider to be worthy of note, in part because it suggests that many SoC-CpGs may in fact be MEs, despite not having been previously identified as such. We have further cause to believe this could be the case because of i) the typically small sample size of multi-germ layer/tissue datasets used to screen for MEs, meaning that published screens for human MEs are likely to be underpowered and will hence fail to capture most MEs; and ii) the evidence that we present suggesting that environmentally-driven inter-individual variation at loci exhibiting ME-like properties may diminish with age, again suggesting that ME screens, which largely analyse adult tissues, will miss metastable loci present in infancy and early childhood.

We had already made the point ii) above in the Discussion. However, given the reviewer’s concerns we have added an additional comment on point i).

The Discussion is heavily geared to interpretation within their MEs focus and does little to discuss study weaknesses and strengths, to which the tail of the Results suggest there are multiple. For at the end of the Results and in the Methods we find additional sensitivity analyses and discussion points on a very strong enrichment for CpGs with a mean difference in methylation between the sexes (>1/3 of the 257), adjustments for genetic confounding and a high inflation factor in the discovery cohort.

We have added an additional comment on the need for further functional analysis in cell and/or animal models at the end of our discussion on possible mechanisms underpinning the observed strong enrichment for sex effects at loci associated with periconceptional environment. We have performed an additional analysis of SoC effects on global methylation using predicted LINE1 and Alu element methylation to address the issue of genomic inflation in the discovery cohort (Methods ‘Inflation of test statistics’ and additional Supp. Fig. 14). We have commented on the potential for residual genetic confounding and the limitation of a lack of genetic data in the discovery cohort in the Discussion. We have also provided an additional comment on the potential influence of unmeasured inter-relatedness in our study population.

Indeed, despite the strong and good flow of the Result section and the impressive (albeit somewhat one-side) look-up of SoC-CpGs in published datasets; the tail and Methods section leaves this reader with a strong suspicion of possible methodological issues on the measurement level already identified prior.

The authors reports that the discovery cohort is biased in the collection of conception months (figure 2A), has a strong inflation of 1.3 (no QQ-plot is shown to assess bias in addition to inflation), no adjustment for genetic background could be made (which is false, as the 450k array contains several dedicated SNP probes, even hundreds when extracted with the omicsPrint package) and > 1/3 of SoC-CpGs is a sex CpG. For the latter observation the authors regressed out sex and repeated the analysis, noting no difference. However, regressing out sex does not help if sex is heavily correlated with confounding biological/sampling/technical covariates.

The authors reason that the inflation is nothing to worry about citing single cohort studies on global effects on DNAm of methyl donors. Global DNAm is indeed often association with methyl donor intake but generally these studies investigate ALU or SINES repetitive elements and the PACE consortium reported only modest effects on select 450K array loci for prenatal folate supplementation, showing that their reasoning might hold on the ME loci (in/close to repetitive elements) but not the genome-wide analysis per se.

The authors should convince the reader that their (discovery) data is valid. The data they do show in Supplemental tables 16 and 17 show that after functional normalization a strong effect of batches remains, while from my own experience these are normally nicely mitigated via functional normalization. Normally only strong cell type correlations remain in the first PCAs of the normalized data. But for ENID we see a remainder of sentrix row, often the strongest batch effect, and slide and plate remaining. Also, the biological, season and cohort specific variables are not noted here. We just must assume that the blank correction for the first 6 PCAs, rather than the actual adjustment for the measured batch/confounding effects, does not remove (or over adjusts) for biological/study design (village, genetic ancestry) effects. In addition to these observations figure 2C seems to indicate that the controls CpGs (elegantly selected by the authors) also show seasonal variation, just not as much as the SoC-CpGs. This leaves the reader to wonder: is there bias in their sample randomization across plates, rows and slides? This feeling is amplified by the fact that almost all SoC-CpGs seem to show an increase in DNAm in jul-aug (Suppl Fig. S5 and Figure 1B). [An observation that is not given enough prominence in the Results]. Which might or might not hint to a correlation with a batch effect (like sentrix row?).

Our addition of a third longitudinal dataset with a very different confounding structure provides strong reassurance of the robustness of the reported SoC effects. However we recognise many of the concerns raised by the review and have therefore substantially extended our analysis of potential confounders in our analysis, including additional sensitivity analyses (see Supplementary Tables ST1p-1s).

In our extended analysis of possible confounding of technical and biological covariates by SoC, we note that the majority of batch and biological covariates are categorical so that it was not possible to report correlation rho’s. We have instead reported p-values for corresponding association tests – see Supplementary Tables for further details of tests that were carried out. Also note that for simplicity season of conception is modelled as a binary variable (Dry: Jan-Jun; Rainy: July-Dec). We consider this to be a valid approximation to the main cosinor (Fourier) regression analysis since this showed a clear relationship between DNAm and dichotomised (Dry/Rainy) season of conception (Figs 2D & 4A). Note that we have not included month of collection as this completely confounds season of conception in the main ENID (2yr) analysis and cannot confound the EMPHASIS (7-9yr) analysis, as discussed in the manuscript (Fig. 2A). This is a key reason why we compared SoC effects across these two cohorts. Note that the month of collection also cannot confound the ENID 5-7yr (longitudinal) analysis as all samples are collected in the rainy season (additional Supp. Fig. 1B).

The covariate correlation analysis confirms:

• No correlation between SoC and all considered batch and biological covariates including principal components across all three analysed datasets (Supp Table, ST1p- 1r).

• No correlation between sex and all considered batch and biological covariates; weak correlations with PC4 and PC3 in EMPHASIS and ENID 5-7yr datasets respectively (ST1q,1r); note also that the sex sensitivity analysis previously reported in the manuscript used methylation values that were pre-adjusted for sex using a regression model that included sex as the only adjustment covariate, alleviating concerns that there may be residual confounding due to strong correlations between technical/biological/sampling covariates and sex. We have added some additional comments on this to Results.

• Expected strong correlations between SoC, month of conception and month of birth in all datasets (ST1p-1r).

• Functional Normalisation (FN) removed most but not all of the effects of technical batch effects (sample plate, slide etc) from the DNAm array data used in the main ENID analysis (ST1p).

• Samples are not perfectly randomised across 450k sample plate (month of birth [mob] and conception [moc]) and slide (mob and village) for the ENID 2yr cohort (ST1p).

The last point raises the possibility of potential residual confounding due to array batch effects in the ENID analysis. We checked for this in two ways. First, we performed sensitivity analyses with batch and village ID variables included directly in the linear regression models, in addition to the PCs that served as proxies for batch variables in our original analysis. This suggested no residual confounding due to array batch or village ID effects (ST1s: ‘batch adjusted model’ and ‘village adjusted model’). Second, we confirmed that neither mob, moc nor village ID were associated with batch or any other covariates in the EMPHASIS or new ENID 5-7yr analyses (ST1q, ST1r). The tight correspondence of date of methylation maximum across all three datasets (cross-cohort and longitudinal analyses) (Figs. 2C, 3A and 4A) with different confounding structures (ST1p-1r) strongly suggests that the reported SoC associations are not driven by residual confounding.

In summary, this analysis provides strong reassurance that our main analysis is not confounded by residual associations with technical and/or biological covariates considered in this analysis, and that the observed enrichment for previously identified sex-associations amongst SoC-CpGs is not driven by residual confounding due to sex.

We have made multiple amendments to the manuscript to incorporate the longitudinal analysis; in the Introduction (lines 58-9); in the first section of Results; and we have made particular reference to the alignment of SoC effects across 3 datasets with different confounding structures. We have also amended several figure captions to distinguish the ENID 2yr and 5-7yr datasets and added the longitudinal dataset to Methods and to the study design schematic (revised Fig. 1), and visualised key results from this additional analysis in Figs. 3 and 4A. Finally we have added additional text on the sensitivity analyses in the main text and in Methods.

Maybe Satan caused the inmate to forget about Joseph because Joseph had mentioned that only his true God was the one worth believing in. Whether it's religion or our own arguments we are all like Joseph. We all want someone to believe our story or be on our side or just be understanding. When we add religion to the mix, it's a different kind of understanding that people want to receive. It's not so simple as saying "believe in my god." (Itani, T. (n.d.). Quran in English - Clear and Easy to Read. Quran in English. https://www.clearquran.com/012.html) CC BY-NC

I have noticed in some anthropological literature that it appears that the authors completely missed the boat as the result of the lack of ability to communicate with their subjects or better understand their broader basic contexts.

Particular examples of this: -1930s: A. Irving Hallowell conversations with William Berens, Chief of the Berens River Anishinaabe about rocks

• Robin Wall Kimmerer mentions in Braiding Sweetgrass that the new American immigrants looked down on the indigenous people for not "giving thanks" for their food, when in fact it was so embedded into their general culture that it should never have been in question. The immigrants just didn't possess the ability to see the how the thanks had been given.

Illustration by MIKKI LEE

The Good-Morrow is an aubade, which is a dawn love song. It's the wholesome moment in the morning that this song is sung. How about reading as if the speaker has just woken up from the bed and is whispering directly to the lover's ears with a subtle and gentle physical touch.

Wow, ok so he is telling me that basic free-rider problem with some probability of defection is why he gets libertarianism doesn't work ... Great, that was easy.

Basically it's as simple as waving a big sign saying "public goods".

I want to keep this description of me playing basketball because I feel this is the highlight of my essay. It is packed with concrete details that I enjoyed writing about. I picked good details to mention because a lot happened in that hour I was playing. One thing I did add was what I was listening to as I was playing. That was originally not included but I liked adding that small detail because it adds more personality to my work. I am a bit self-conscious about my music taste which is why I did not include it at first. However, now that it's there it gives more insight into my character.

This was a section I added in my revised piece. I wanted to be more honest with my reader about my thoughts on the food in Eagle's Nest. Although it was important to include details on how much it reminded me of home, I felt it would be a dishonour to my reader and myself to not be honest enough to talk about how I actually felt about the food and its taste. My aim with this section was to make my reader understand that it is still important to me to have the rice and beans in Eagle's Nest, but it is no where near the actual taste of what I am used to having at home, it's just the closest thing I have to it at the moment.

As Mark Twain once said “If it's your job to eat a frog, it's best to do it first thing in the morning. And if it's your job to eat two frogs, it's best to eat the biggest one first.” ... Eating the frog means to just do it, otherwise the frog will eat you meaning that you'll end up procrastinating it the whole day.

I believe that all data has error in it to some extent it's just a matter of limiting that. Human error needs to be accounted for in the creation of work.

Someone should turn this into a poster: Working Hard It's not just a dial you turn up to 11.

1. Understand the shape of real work.
2. See clearly what kind of work you're best suited for.
3. Aim as close to the true core of the problem as you can.
4. Accurately judge each moment both by both what you're capable of and how you're doing
5. Put as many hours each day as you can without harming the quality of the result.

Glad the author highlights this caveat. It's very easy to say "well, I'm supremely passionate about this thing, so why don't I just going to 'out work' everyone else who's also passionate about that thing?" This seems like a recipe for burnout and may result in a net loss of "work done" in the long-term.

Reminds me of the quality line/preference curve mentioned in: https://mindingourway.com/half-assing-it-with-everything-youve-got/

A list of books about how higher education has lost its soul.

Are these just complaining or do any of them work on a solution for making things better?

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Reply to the reviewers

Response to reviewers

Reviewer #1

I believe that this is a very sound and authoritative study. The analysis of all data seems appropriate and robust, and many connections between the data (and subsets of data) and their possible interpretations have been considered. In fact, in the massive Results section, some interpretations are supported by cited references (this is not meant as a critique). However, I wonder about the length of the Results section, and the balance between it and the relatively short Discussion section. It is difficult for me to nail down any part of Results that might be shortened, as I could not find clear redundancies. I also think that the level of speculation is absolutely warranted, and I did not find excessive claims being made to this or that end. Rather, I suggest to broaden the perspective somewhat (in their Discussion; see below under Significance), which might allow people with a less mechanistic perspective to grasp the potential relevance of this work for non-model plant systems studied mostly by evolutionary geneticists.

Response: We thank the reviewer for their kind remarks. We have spent a very large amount of time trying to streamline the results section and we are not sure if it would be possible to shorten it any further without removing critical details.

We appreciate the reviewer’s comment to add more detail to the discussion to make it more appealing to evolutionary geneticists and we have added the following lines to the discussion section: “The WISO or “weak inbreeder/strong outcrosser” model (Brandvain & Haig, 2005) emerges from the dynamics of parental conflict and parent-of-origin effects. Under this model, a parent from populations with higher levels of outcrossing is exposed to higher levels of conflict and can thus dominate the programming of maternal resource allocation in a cross with an individual from a population with lower levels of outcrossing. Such a phenomenon has been observed in numerous clades including Dalechampia, Arabidopsis, Capsella and Leavenworthia (Brandvain & Haig, 2018; İltaş et al., 2021; Lafon-Placette et al., 2018; Raunsgard et al., 2018). Intriguingly, loss of function phenotypes in the RdDM pathway are more severe in recently outcrossing species than in A.thaliana (Grover et al., 2018; Wang et al., 2020) and suggests that RNA Pol IV functions are more elaborate and important in these species. This raises the possibility that the role for RNA Pol IV and RdDM in parental conflict that we describe in A.thaliana here is likely heightened in and mediates the elevated level of parental conflict in species that are currently or have been recently outcrossing.”

One aspect that might warrant more scrutiny is the mapping of sRNA reads to the reference genome. I found the short section of this (M&M section, page 20, lines 23-25) to be too brief. It is not clear to me which of ShortStack's v3 weighting scheme the authors used, which is relevant for multi-mapping reads (see NR Johnson et al. 2016, G3). In addition, it is not mentioned whether zero mismatches were allowed. Perhaps this is described in more detail in Erdmann et al. (2017), but even if so, it deserves to be clarified here.

Response: Small RNA reads were aligned after allowing two mismatches. This was indicated in the bowtie command (‘bowtie -v 2’ where v 2 indicates two mis-matches). We have added text to expand on the meaning of the commands.

We have also expanded the commands used for ShortStack. We used the “Placement guided by uniquely mapping reads (-u)” option to divide the multi-mapping reads.

The manuscript is well-written and concise, despite the length of the Results section. The verbal clarity and absence of typos or grammatical issues is superb. I did find some of the Figures to be somewhat "un-intuitive", in the sense that it takes acute concentration for an outsider (of sorts) to gather and interpret the underlying data. This is probably due to the many cross-comparisons of differences between two genotypes on one axis and those of a different pair of genotypes on the other axis. I am not sure how this issue can be ameliorated (nor whether this is really necessary); however, from a technical point of view, all Figures and Suppl. Figures are really well-done.

Response: We thank the reviewer for their kind remarks. We have strived to make the figures easier to understand but we are aware that the figures do require a lot of concentration. We haven’t found an easy way to fix this. We thank the reviewer for patiently going through the figures.

The list of references seems adequate in terms of citing relevant (both older and very recent) publications. However, almost all cited papers concern Arabidopsis or other model species; I suggest to consider adding a few relevant studies on non-Brassicaceae (whether considered model taxa or not), in conjunction with my suggestion (in Significance) to potentially broaden the scope by searching for natural phenomena that also involve parent-of-origin effects on endosperm/seed development. Curiously, many of the references are "incomplete" in the sense of stopping with the journal's name, then stating the doi, i.e. they lack volume numbers and page/article numbers. This should be harmonized throughout.

Response: We have added references to non-Brassicaceae species and have also fixed the references.

Reviewer #2: This manuscript provides evidence that a loss of either the maternal or paternal copy of NRPD1 have different, and sometime opposite, effects on the accumulation of small RNAs and on expression of a subset of genes, with a loss of the maternal copy having more substantial effects. The manuscript is well written, and the conclusions, as far as they go, are justified by the data, which are effectively presented. The overall effect is subtle but informative and according to the authors support a parental conflict model for imprinting. The experiments failed to find a smoking gun in the form of a mechanism to explain how or why the maternal and paternal alleles have different effects and the explanation for a lack of clear phenotypic differences was reasonable, but untested. I would have like to see it tested by looking in a plant species that is outcrossing and highly polymorphic. However, I do appreciate that the observation that the male and female alleles can have distinct effect when mutant is an important clue. My specific comments below may reflect confusion on my part, rather than real issues. If that is that I hope that confusion can aid in clarifying what are in places quite subtle points.

Response: We thank the reviewer for their comments. We agree that it would be potentially informative to do similar experiments in an outcrossing species but that this is beyond the scope of this manuscript. Additionally, loss of NRPD1 or other components of the RdDM pathway has dramatic effects on gametogenesis in some examined outcrossing species(Grover et al., 2018; Wang et al., 2020), which could prevent the detection of subtle parent-of-origin effects on seed development.

Page 6, last paragraph: "Because the endosperm is triploid, in these comparisons there are 3 (wild-type), 2 (pat nrpd1+/-), 1 (mat nrpd1+/-) and 0 (nrpd1-/-) functional NRPD1 alleles in the endosperm. However, NRPD1 is a paternally expressed imprinted gene in wild-type Ler x Col endosperm and the single paternal allele contributes 62% of the NRPD1 transcript whereas 38% comes from the two maternal alleles (Pignatta et al., 2014). Consistent with paternal allele bias in NPRD1 expression, mRNA-Seq data shows that NRPD1 is expressed at 42% of wild- type levels in pat nrpd1+/- and at 91% of wild-type levels in mat nrpd1+/- (Supplementary Table 6)". I would think this would really complicate the analysis. Should all of the dosage values include NPRD1 imprinting values? That is to say, expressed in terms of expression values? This is also a bit confusing. The maternal copies together express 38% of the transcript, so why isn't the mat nrpd1 at 68%, rather than 91%? In any event, given this imprinting and differences in dosage of the male and female it appears that two variables, parental origin and expression levels are being compared. Since 91% is awfully close to 100%, are the mat pat comparisons really just comparing low with nearly normal expression of NRPD1? And actually, given that, the outsized effect of the mat nrpd1 +/- is even more striking.

Response: We included the details of dosage rather than imprinting values because the potential for buffering of expression upon loss of one allele could not be discounted. Indeed, we do find that the endosperm transcriptome buffers against the loss of the maternal or paternal alleles (Supplementary Table 6). The reviewer is correct in pointing out that the outsized effect of mat nrpd1+/- on gene expression is even more striking, and strongly supports our view that these effects are parental rather than endospermic.

To reduce confusion in this section, we removed the details about 38% maternal allele transcripts obtained from our previous study, and instead report only the observed values from this study (which are also consistent with the previously reported paternally-biased expression of NRPD1 in endosperm).

Page 4, Line 16. I'm afraid it's still a bit difficult to understand what was being compared what in this section. Please clarify.

Response: The authors in this previously published study compared sRNAs obtained from wild-type whole seeds (which consists of three different tissues, including endosperm) with mutant endosperm. We are pointing out that the difference in tissue composition makes the effect of nrpd1 mutation hard to disentangle from the tissue differences between the two genotypes.

Page 5, Line 5. I'm sure this is fine, but it's not entirely clear what is from the previously published paper and what is reanalysis here. All the crosses and measurements were made then, but not organized in this way?

Response: This data was indeed previously published. In that analysis, we had pooled results from different crosses and calculated significance between genotypes using chi-square tests. During a later study (Satyaki and Gehring, 2019), we realized that we were losing information by ignoring the seed abortion values per cross. So, a reanalysis of that data on a cross by cross basis allowed us to find strong evidence for maternal and paternal effects.

Page 6, Line 26. This is an excellent dosage series, but it's complicated by imprinting. So it's not 3, 2, 1, 0 effective copies. If we set the paternal copy at ~1 and each maternal at ~0.1, then it's 1.2 (wild type), 0.20 (pat nrpd1+/-), 1 (mat nrpd1+/-), and 0 (nrpd1-/-).

Response: At the genomic DNA level, its 3, 2,1 and 0 doses. The reviewer’s comment on the transcriptional dose is not clear to us. Based on measured gene expression levels, relative wild-type NRPD1 transcriptional dose =1, pat nrpd1+/- is 0.42, and mat nrpd1+/- is 0.91.

Page 6, line 31. Is the main thing we are comparing the difference between expression at 42% verses 91% of wild type?

Response: We are using the small RNA-seq data alongside the mRNA-seq data to argue that loss of mat and pat nrpd1+/- have no impact on overall Pol IV activity in endosperm (as measured by small RNA production). A nrpd1 heterozygous endosperm has almost the same small RNA profile as a wild-type endosperm. Thus any effects seen in the endosperm, including the effects on mRNA expression described later in the manuscript, are likely parental rather than zygotic endospermic effects.

Page 7, line 11. So, the overall effect in either direction on smRNA gene targets was really quite small, and I'm guessing the effect on gene expression was even smaller.

Response: The effects of loss of maternal or paternal Pol IV on sRNAs was indeed small (Fig. 1/Fig. S3). Effect of loss of maternal Pol IV on gene expression was substantially large and distinct from the relatively small impacts observed upon loss of paternal Pol IV (Fig. 3) This observation supports the view that Pol IV mediates parent-of-origin effects on gene expression.

Page 7, line 17. I take it that it is this difference, rather than the overall numbers that is of interest.

Response: Correct. The lack of a relationship between sRNAs impacted upon loss of mat and pat nrpd1 is additionally suggestive of parent-of-origin effects

Page 9, line 2. Really interesting, since one might expect that these are methylated loci that would be expected to be fed into any existing embryo maintenance methylation pathway. Surprising that they are maintained independently.

Response: It is indeed surprising that Pol IV activity in parents can have different impacts on sRNAs in the endosperm. It should be noted though, that as described in Erdmann et al 2017 and in this paper later on, many endosperm sRNA loci are in fact not associated with endosperm DNA methylation. In addition, sRNA loci that are dependent on paternal Pol IV activity are more likely to be associated with DNA methylation than are sRNA loci associated with maternal Pol IV activity. These points have been described in Figure S8.

Page 9, line 22. Proportion of total imprinted genes? Did the mutant obviate/enhance the imprinting?

Response: We have modified the manuscript to describe effects on imprinted genes: “ The expression of imprinted genes is known to be regulated epigenetically in endosperm. In mat nrpd1+/- imprinted genes were more likely to be mis-regulated than expected by chance (hypergeometric test p-15) – 15 out of 43 paternally expressed and 45 out of 128 maternally expressed imprinted genes were mis-regulated in mat nrpd1+/- while two maternally expressed imprinted genes but no paternally expressed imprinted genes were mis-regulated in pat nrpd1+/- (Table S6).” We have also added a new supplementary figure (Fig. S6) that describes the impacts of NRPD1 loss of imprinted gene expression.

Page 9, line 27. How could 2) occur in the homozygous mutant?

Response: Loss of NRPD1 may impact gene expression in both parents. When the nrpd1-/- mutant endosperm is investigated, we are also examining the consequences of the inheritance of these disrupted gene expression states. We refer to this as epistatic interactions of mat and pat nrpd1.

Page 10, line 9. Interesting!

Response: We strongly agree!

Page 10, line 11. Is this 2.7 versus 2.18 significant because it's statistically significant, or because it's conceptually significant?

Response: We are pointing out that the 2.7-fold value is quite similar to the predicted value of 2.18-fold, which is arrived at by simply summing the effects of mat nrpd1 and pat nrpd1. This is a conceptually significant point.

Are the examples in 3D representative, or the most convincing examples? And a big difference in ROS1 is of some concern, since that may well be expected to affect imprinting indirectly. I know I'm being picky here, but the pattern is so intriguing I'd be worried about confirmation bias.

Response: The examples in 3D are representative for those genes with significant changes in expression in both mat and pat nrpd1, and other genes also behave similarly. The antagonistic effect described for 3D can also be observed as a much broader trend affecting hundreds of genes to varying extents in Fig 3C and 3E-H. The concern about ROS1 is not clear to us but we agree that an effect of ROS1 may be one way that NRPD1 controls gene expression.

Page 10, line 18. Ok, but 0.123 is a pretty subtle negative correlation. Although I do appreciate that it clearly is not a positive correlation. If I'm understanding correctly, this was the "aha" moment, because it's exactly what one might expect of NRPD1 from the mother and father or working at cross purposes. But the numbers are getting awfully small here.

Response: It is unclear how to calibrate our expectations of effect sizes considering that our study is the first (to our knowledge) to make such a measurement involving gene expression in parental conflict. A review of the few empirical examples of parental conflict’s impact on seeds shows that parental conflict may drive small changes in seed size (Brandvain and Haig, 2018).

The evolution of quantitative traits maybe driven by selection for large effects at a small number of loci and/or by selection of small effects at a large number of loci. In a similar vein, parental conflict can impact seed phenotypes either via large effects at a few loci or via small effects at a large number of loci. Our analysis described in Fig 3D-H can fit either possibility. Large effects can be found at a few loci such as SUC2 and PICC (Fig. 3D). Smaller antagonistic effects can be found at hundreds of loci as shown in Figure 5A. The negative correlation described in this figure can be observed even upon dropping the genes that show a statistically significant differential expression in both mat and pat nrpd1+/- (slope after dropping genes significantly mis-regulated in both mat and pat nrpd1+/- is -0.126). In summary, a correlation of -0.123 strongly supports the existence of a widespread antagonistic regulatory effect.

Page 10, line 29. The point simply being that that other phenomenon is also significant even if the differences are that large?

Response: We are pointing out that the magnitude of the effects we see are similar to that observed for phenomenon such as dosage compensation.

Page 12. So, there is no effect on cleavage and no obvious effect on flanking siRNA clusters. The suspense is building...

Page 12, line 24. And not in potential regulatory regions? CNSs?

Response: We did not identify a significant enrichment for differentially methylated regions in regulatory regions. We used the relative distance function in bedtools (https://bedtools.readthedocs.io/en/latest/content/tools/reldist.html) to calculate the relationship between the genomic location of DMRs and genomic location of a differentially expressed gene. This analysis was chosen as it does not make a priori assumptions about the size of the regulatory region of a gene. A broad association between DMRs and differentially expressed genes would be indicated by a frequency far greater than 0.02. We show the results of this analysis in Fig. S8F; we find no evidence for significant enrichment of DMRs in the regulatory regions of differentially expressed genes.

Page 12, line 28. I guess it depend on whether or not the changes are in regulatory sequences no immediately apparent as part of the gene, doesn't it?

Response: We examined DNA methylation over genes here because in endosperm, unlike in other tissues, many small RNAs are genic. Moreover, DNA methylation within the gene may control transcript abundance (Eimer et al., 2018; Klosinska et al., 2016). We have also examined regulatory regions adjacent to genes in Fig S8F and found no effect.

Line 13, line 2. Not sure it's that important, but couldn't you chop all of these genes in half and see if they are no longer significant collectively?

Response: We do not think that this will provide a useful insight.

Page 14, line 15. I'm afraid I'm getting confused here with the terms cis and trans here. Just to be clear, cis means a direct effect of small RNAs that are dependent on NRPD1 on a gene and trans means anything else? But in this context, it's not clear that is what is meant. Do you mean that gene expression is determined and preset in the gametophyte? What are the levels of expression of NRPD1 in the two gametophytes?

Response: The reviewer’s interpretation of cis and trans is correct. However, the cis imprints may be preset in gametophytes or in the sporophytic tissues that surround or give rise to the gametophyte. Pol IV is known to be active either in gametophyte or in related sporophytic tissues in both the mother and the father(Kirkbride et al., 2019; Long et al., 2021; Olmedo-Monfil et al., 2010).

Page 14, line 19. Prior to fertilization?

Response: Yes, that is the idea. As described in the manuscript, Pol IV activity in either the parental sporophyte or gametophyte prior to fertilization could impact gene expression in the endosperm after fertilization.

Page 14, line 27. Do you mean driven by, or just associated with?

Response: In response to the comment, we have replaced the phrase “driven by” with “due to” for increased clarity. In wild-type, DOG1 is predominantly expressed from the paternal allele. In mat nrpd1+/-, the paternal allele is somewhat upregulated but the maternal allele, which is almost silent in wild-type, is highly expressed in mat nrpd1+/-.

Page 15, line 26. And this is really the issue. The primary conclusion, backed up by the lack (I'm assuming) of phenotypic differences between mat NRPD1 -/+ and pat NRPD1 +/- suggests that the observed differences in expression are not particularly important, unless the exceptional cases are informative.

Response: We are not sure whether the reviewer means “issue” in a negative, neutral, or positive light. Seed phenotypes are often subtle and we have not examined phenotypic differences in sufficient detail to comment.

Page 15, line 12. Yes, but I'm not at all clear what the mechanism for this is.

Response: We have tested and falsified multiple hypotheses to explain how Pol IV can regulate gene expression in endosperm. Considering the complex genetics and the difficulty of isolating endosperm, we have concluded that this is a matter for a future study. The point of this study is the discovery of Pol IV’s parental effects.

Page 15, line 23. Since this is a very small subset of genes, are these genes that you might expect to play a role in parental conflict?

Response: The functions of most genes in endosperm remain unknown. However, some have a likely role in conflict. SUC2 is antagonistically regulated by parental Pol IV (Fig. 3D). SUC2 transports sucrose, the key form of carbon imported into seeds from the mother (Sauer & Stolz, 1994).

Page 15, line 33. Indeed, these could be the informative exceptions.

Response: We believe the reviewer means that the identify of strongly antagonistically regulated genes may be informative in terms of thinking about these results in the context of parental genetic conflict, which we agree with.

Page 15, line 29. Hardly surprising, given that the paternal copy of NRPD1 is expressed at a higher level than the maternal copies, is it?

Response: It is actually somewhat surprising since we show in Fig. 2 that the sRNA production in mat and pat nrpd1+/- are comparable to that of wild-type. The higher contribution of NRPD1 from the paternal copy does not really explain the methylation differences

Page 16, line 1. So this is what you mean by in cis. Presetting?

Response: The reviewer’s previous interpretation of cis (acting directly at a target gene) is correct. However, the cis imprints may be preset in gametophyte or in the sporophytic tissues that surround or give rise to the gametophyte. Pol IV is known to be active in gametophytes and in related sporophytic tissues in both the mother and the father.

These are intriguing results that would benefit from a test of the hypothesis by comparing these result with those obtained in an outcrossing plant species.

Response: We agree that it would interesting and informative to perform similar experiments in an outcrossing species. However, loss of NRPD1 or other components of the the RdDM pathway have dramatic effects on gametogenesis in outcrossing species (Grover et al., 2018; Wang et al., 2020), preventing the detection of subtle parent-of-origin effects on seed development. Additionally, this would be a separate study.

Reviewer #3

We thank the reviewer for their comments.

• Expression of NRPD1 was 42% of WT in paternal nrpd1 and 91% of WT in maternal nrpd1, yet throughout the paper the effect of maternal nrpd1 was far stronger than paternal nrpd1. The authors may also want to confirm that protein levels follow the same pattern, in case protein degradation or post-transcriptional regulation may play a role.

Response: We show in Fig. 2 that sRNA production in mat and pat nrpd1+/- are similar to wild-type endosperm. This strongly suggests that NRPD1 protein is produced at functionally equivalent levels in wild-type, mat and pat nrpd1+/-. The finding that mat nrpd1+/- has a stronger effect on gene expression and small RNAs, despite having higher levels of NRPD1 transcript in endosperm, is consistent with our conclusion that the effects we are observing in heterozygous endosperm are due to NRPD1 action before fertilization.

P. 9 line 1 - this only seems to be true for maternal ISRs, not paternal ISRs; this claim should be narrowed.

Response: Accordingly, we have modified the text here to : “In summary, these results indicate that most maternally and some paternally imprinted sRNA loci in endosperm are dependent on Pol IV activity in the parents and are not established de novo post-fertilization.”

A small number of sRNA loci become highly depleted in maternal nrpd1 but not paternal nrpd1 (Fig. 1D, F, Fig. 2C) - are these siren loci?

Response: This is an interesting question. Siren loci have not been defined in Arabidopsis but are described as loci with high levels of sRNAs in ovules, seed coat, endosperm and embryo (Grover et al., 2020). Loci losing sRNAs in maternal nrpd1+/- include a large number of maternally expressed imprinted sRNAs (mat ISRs). We do not know if mat ISR loci are expressed in the ovule. In Erdmann et al (2017), we excluded loci that were also expressed in the seed coat from mat ISRs. Thus, these loci meet only some of the conditions for being defined as siren loci.

Fig. 2 suggests that many of the downregulated sRNA regions in maternal nrpd1 are maternally biased to begin with. Related, are genic sRNAs more likely to be affected by maternal or paternal nrpd1 than non-genic or TE sRNAs?

Response: As described in Fig. 1B and S3, loss of maternal NRPD1 has more impacts on the sRNA landscape. As a percentage of total loci, genes are more likely to be affected than TEs.

For the sRNA loci shown in Fig. 2C, how is % maternal affected in maternal vs. paternal nrpd1? These ISRs are normally maternal or paternal biased, does this change in maternal or paternal nrpd1?

Response: We assess the allelic bias of ISRs only when they have at least ten reads in the genotypes being compared. In mat nrpd1+/-, most mat ISRs lose almost all their reads (Fig. 2) and we can assess allelic bias only at 107/366 mat ISRs. As seen in the Rev. comment. Fig1, these 107 lose their maternal bias. In pat nrpd1+/-, loci with maternally biased sRNAs show somewhat increased expression (Fig 2E) but do not show an appreciable change in maternal bias (Figure Review 1). All paternal ISRs do not show any dramatic impacts on allelic bias in mat or pat nrpd1+/-. We have not added this additional datapoint to our paper because we were worried that the paper was becoming too dense – a concern also voiced by reviewer 1. However, we can add this to the manuscript if the reviewer prefers.

• Might have missed this, but I didn't see the gene ontology results (p9 line 16) shown anywhere? Would like to see significance values, fold enrichments, etc. In particular, the group of paternal nrpd1 up-regulated genes seems too small to have much confidence for GO enrichment analysis.

Response: We have added a Supplementary Table 7 with outputs of GO analyses.

• I would suggest expanding the analysis in Fig. 3D-H to explore whether the additive model is more predictive of nrpd1-/- expression levels than other potential models (epistatic, etc.) in general at all genes, or only at the subsets of genes shown, independently of whether the effects are large enough to pass the arbitrary significance cutoffs used in E-H. Identifying specifically which genes do and don't follow this additive pattern could help dissect mechanism. For example, genes following this pattern might share a TF binding site for a TF that is regulated by Pol IV.

Response: While we are interested, we currently cannot explore other models such as epistasis as this would require knock-down of NRPD1 in the endosperm and we plan to do this as part of a future study.

1. 13 line 26 - how do changes in CG methylation in maternal or paternal nrpd1 compare to changes in dme or ros1? Do either set of DMRs significantly overlap dme or ros1 DMRs? Could some of these be explained by changes in ROS1 expression, since ROS1 is a Pol IV target?

Response: Yes. It’s entirely possible that a subset of observed gene expression changes are linked to changes in ROS1 expression. However, there are no comparable methylation data for ROS1 in the endosperm. A potential role for ROS1 has been discussed on Page 11, line 4. Comparison with DMRs in the dme endosperm is difficult. dme mutant endosperm has low non-CG methylation (Ibarra et al., 2012). We have unpublished data showing that the expression of genes involved in RNA-directed DNA methylation (RdDM) is reduced in the dme endosperm. It is therefore difficult to understand if and how DME-mediated demethylation may impact RdDM.

P. 10 line 3 - is the overlap of 36 out of 51 genes unlikely to occur by chance

Response: A hypergeometric test indicates that this is indeed significant. We have added it to text on Page 9, line 34.

In sRNA and mRNA-seq libraries, what was the overall maternal/paternal ratio in each library? Did loss of Pol IV affect this?

The graphs above show the maternally derived fraction of mRNA and sRNA libraries for different genotypes. Please note that the Ler nrpd1 mutant was generated by backcrossing Col-0 nrpd1+/- into Ler. Some Col-0 regions remain in this background and are called “hold-outs”. Reads mapping to these hold-outs have been excluded while calculating the maternal fraction of each library described in the graph above. We cannot confidently judge if the overall maternal fraction of the mRNA transcriptome is affected by loss of NRPD1 as we likely need more replicates. However, we find that loss of all NRPD1-dependent sRNAs (as in the nrpd1 null mutant) leaves behind sRNAs that roughly reflect the genomic 2:1 ratio.

P. 9 line 22 - how many paternally and maternally expressed imprinted genes were considered? Were imprinted genes statistically more likely to be misregulated in mat nrpd1?

Response: We considered 128 maternally and 43 paternally expressed genes that had been previously been identified as imprinted in Col x Ler crosses (Pignatta et al 2014). We have modified the manuscript to describe effects on imprinted genes: “ The expression of imprinted genes is known to be regulated epigenetically in endosperm. In mat nrpd1+/- imprinted genes were more likely to be mis-regulated than expected by chance (hypergeometric test p-15) – 15 out of 43 paternally expressed and 45 out of 128 maternally expressed imprinted genes were mis-regulated in mat nrpd1+/- while two maternally expressed imprinted genes but no paternally expressed imprinted genes were mis-regulated in pat nrpd1+/- (Table S6). “ We have also added a supplementary figure (Figure S6) that focuses on genic mRNA imprinting in NRPD1 heterozygotes and homozygous mutants.

References cited in the response

Brandvain, Y., & Haig, D. (2005). Divergent Mating Systems and Parental Conflict as a Barrier to Hybridization in Flowering Plants. The American Naturalist, 166(3), 330–338. https://doi.org/10.1086/432036

Brandvain, Y., & Haig, D. (2018). Outbreeders pull harder in a parental tug-of-war. Proceedings of the National Academy of Sciences, 115(45), 11354–11356. https://doi.org/10.1073/pnas.1816187115

Eimer, H., Sureshkumar, S., Singh Yadav, A., Kraupner-Taylor, C., Bandaranayake, C., Seleznev, A., Thomason, T., Fletcher, S. J., Gordon, S. F., Carroll, B. J., & Balasubramanian, S. (2018). RNA-Dependent Epigenetic Silencing Directs Transcriptional Downregulation Caused by Intronic Repeat Expansions. Cell. https://doi.org/10.1016/j.cell.2018.06.044

Grover, J. W., Burgess, D., Kendall, T., Baten, A., Pokhrel, S., King, G. J., Meyers, B. C., Freeling, M., & Mosher, R. A. (2020). Abundant expression of maternal siRNAs is a conserved feature of seed development. Proceedings of the National Academy of Sciences of the United States of America, 117(26), 15305–15315. https://doi.org/10.1073/pnas.2001332117

Grover, J. W., Kendall, T., Baten, A., Burgess, D., Freeling, M., King, G. J., & Mosher, R. A. (2018). Maternal components of RNA ‐directed DNA methylation are required for seed development in Brassica rapa. The Plant Journal, 94(4), 575–582. https://doi.org/10.1111/tpj.13910

Ibarra, C. A., Feng, X., Schoft, V. K., Hsieh, T.-F., Uzawa, R., Rodrigues, J. A., Zemach, A., Chumak, N., Machlicova, A., Nishimura, T., Rojas, D., Fischer, R. L., Tamaru, H., & Zilberman, D. (2012). Active DNA Demethylation in Plant Companion Cells Reinforces Transposon Methylation in Gametes. Science, 337(6100), 1360–1364. https://doi.org/10.1126/science.1224839

İltaş, Ö., Svitok, M., Cornille, A., Schmickl, R., & Lafon Placette, C. (2021). Early evolution of reproductive isolation: A case of weak inbreeder/strong outbreeder leads to an intraspecific hybridization barrier in Arabidopsis lyrata. Evolution, 75(6), 1466–1476. https://doi.org/10.1111/evo.14240

Kirkbride, R. C., Lu, J., Zhang, C., Mosher, R. A., Baulcombe, D. C., & Chen, Z. J. (2019). Maternal small RNAs mediate spatial-temporal regulation of gene expression, imprinting, and seed development in Arabidopsis. Proceedings of the National Academy of Sciences, 116(7), 2761–2766. https://doi.org/10.1073/pnas.1807621116

Klosinska, M., Picard, C. L., & Gehring, M. (2016). Conserved imprinting associated with unique epigenetic signatures in the Arabidopsis genus. Nature Plants, 2, 16145. https://doi.org/10.1038/nplants.2016.145

Lafon-Placette, C., Hatorangan, M. R., Steige, K. A., Cornille, A., Lascoux, M., Slotte, T., & Köhler, C. (2018). Paternally expressed imprinted genes associate with hybridization barriers in Capsella. Nature Plants, 4(6), 352–357. https://doi.org/10.1038/s41477-018-0161-6

Long, J., Walker, J., She, W., Aldridge, B., Gao, H., Deans, S., Vickers, M., & Feng, X. (2021). Nurse cell­–derived small RNAs define paternal epigenetic inheritance in Arabidopsis. Science, 373(6550). https://doi.org/10.1126/science.abh0556

Olmedo-Monfil, V., Durán-Figueroa, N., Arteaga-Vázquez, M., Demesa-Arévalo, E., Autran, D., Grimanelli, D., Slotkin, R. K., Martienssen, R. A., & Vielle-Calzada, J.-P. (2010). Control of female gamete formation by a small RNA pathway in Arabidopsis. Nature, 464(7288), 628–632. https://doi.org/10.1038/nature08828

Raunsgard, A., Opedal, Ø. H., Ekrem, R. K., Wright, J., Bolstad, G. H., Armbruster, W. S., & Pélabon, C. (2018). Intersexual conflict over seed size is stronger in more outcrossed populations of a mixed-mating plant. Proceedings of the National Academy of Sciences, 115(45), 11561–11566. https://doi.org/10.1073/pnas.1810979115

Sauer, N., & Stolz, J. (1994). SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker’s yeast and identification of the histidine-tagged protein. The Plant Journal, 6(1), 67–77. https://doi.org/10.1046/j.1365-313X.1994.6010067.x

Wang, Z., Butel, N., Santos-González, J., Borges, F., Yi, J., Martienssen, R. A., Martinez, G., & Köhler, C. (2020). Polymerase IV Plays a Crucial Role in Pollen Development in Capsella. The Plant Cell, 32(4), 950–966. https://doi.org/10.1105/tpc.19.00938

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Referee #2

Evidence, reproducibility and clarity

This manuscript provides evidence that a loss of either the maternal or paternal copy of NRPD1 have different, and sometime opposite, effects on the accumulation of small RNAs and on expression of a subset of genes, with a loss of the maternal copy having more substantial effects. The manuscript is well written, and the conclusions, as far as they go, are justified by the data, which are effectively presented. The overall effect is subtle but informative and according to the authors support a parental conflict model for imprinting. The experiments failed to find a smoking gun in the form of a mechanism to explain how or why the maternal and paternal alleles have different effects and the explanation for a lack of clear phenotypic differences was reasonable, but untested. I would have like to see it tested by looking in a plant species that is outcrossing and highly polymorphic. However, I do appreciate that the observation that the male and female alleles can have distinct effect when mutant is an important clue. My specific comments below may reflect confusion on my part, rather than real issues. If that is that I hope that confusion can aid in clarifying what are in places quite subtle points.

Specific comments:

Page 6, last paragraph: "Because the endosperm is triploid, in these comparisons there are 3 (wild-type), 2 (pat nrpd1+/-), 1 (mat nrpd1+/-) and 0 (nrpd1-/-) functional NRPD1 alleles in the endosperm. However, NRPD1 is a paternally expressed imprinted gene in wild-type Ler x Col endosperm and the single paternal allele contributes 62% of the NRPD1 transcript whereas 38% comes from the two maternal alleles (Pignatta et al., 2014). Consistent with paternal allele bias in NPRD1 expression, mRNA-Seq data shows that NRPD1 is expressed at 42% of wild- type levels in pat nrpd1+/- and at 91% of wild-type levels in mat nrpd1+/- (Supplementary Table 6)".

I would think this would really complicate the analysis. Should all of the dosage values include NPRD1 imprinting values? That is to say, expressed in terms of expression values? This is also a bit confusing. The maternal copies together express 38% of the transcript, so why isn't the mat nrpd1 at 68%, rather than 91%? In any event, given this imprinting and differences in dosage of the male and female it appears that two variables, parental origin and expression levels are being compared. Since 91% is awfully close to 100%, are the mat pat comparisons really just comparing low with nearly normal expression of NRPD1? And actually, given that, the outsized effect of the mat nrpd1 +/- is even more striking.

Page 4, Line 16. I'm afraid it's still a bit difficult to understand what was being compared what in this section. Please clarify.

Page 5, Line 5. I'm sure this is fine, but it's not entirely clear what is from the previously published paper and what is reanalysis here. All the crosses and measurements were made then, but not organized in this way?

Page 6, Line 26. This is an excellent dosage series, but it's complicated by imprinting. So it's not 3, 2, 1, 0 effective copies. If we set the paternal copy at ~1 and each maternal at ~0.1, then it's 1.2 (wild type), 0.20 (pat nrpd1+/-), 1 (mat nrpd1+/-), and 0 (nrpd1-/-).

Page 6, line 31. Is the main thing we are comparing the difference between expression at 42% verses 91% of wild type?

Page 7, line 11. So, the overall effect in either direction on smRNA gene targets was really quite small, and I'm guessing the effect on gene expression was even smaller.

Page 7, line 17. I take it that it is this difference, rather than the overall numbers that is of interest.

Page 9, line 2. Really interesting, since one might expect that these are methylated loci that would be expected to be fed into any existing embryo maintenance methylation pathway. Surprising that they are maintained independently.

Page 9, line 22. Proportion of total imprinted genes? Did the mutant obviate/enhance the imprinting?

Page 9, line 27. How could 2) occur in the homozygous mutant?

Page 10, line 9. Interesting!

Page 10, line 11. Is this 2.7 versus 2.18 significant because it's statistically significant, or because it's conceptually significant? Are the examples in 3D representative, or the most convincing examples? And a big difference in ROS1 is of some concern, since that may well be expected to affect imprinting indirectly. I know I'm being picky here, but the pattern is so intriguing I'd be worried about confirmation bias.

Page 10, line 18. Ok, but 0.123 is a pretty subtle negative correlation. Although I do appreciate that it clearly is not a positive correlation. If I'm understanding correctly, this was the "aha" moment, because it's exactly what one might expect of NRPD1 from the mother and father or working at cross purposes. But the numbers are getting awfully small here.

Page 10, line 29. The point simply being that that other phenomenon is also significant even if the differences are that large?

Page 12. So, there is no effect on cleavage and no obvious effect on flanking siRNA clusters. The suspense is building...

Page 12, line 24. And not in potential regulatory regions? CNSs?

Page 12, line 28. I guess it depend on whether or not the changes are in regulatory sequences no immediately apparent as part of the gene, doesn't it?

Line 13, line 2. Not sure it's that important, but couldn't you chop all of these genes in half and see if they are no longer significant collectively?

Page 14, line 15. I'm afraid I'm getting confused here with the terms cis and trans here. Just to be clear, cis means a direct effect of small RNAs that are dependent on NRPD1 on a gene and trans means anything else? But in this context, it's not clear that is what is meant. Do you mean that gene expression is determined and preset in the gametophyte? What are the levels of expression of NRPD1 in the two gemetophytes?

Page 14, line 19. Prior to fertilization?

Page 14, line 27. Do you mean driven by, or just associated with?

Page 15, line 26. And this is really the issue. The primary conclusion, backed up by the lack (I'm assuming) of phenotypic differences between mat NRPD1 -/+ and pat NRPD1 +/- suggests that the observed differences in expression are not particularly important, unless the exceptional cases are informative.

Page 15, line 12. Yes, but I'm not at all clear what the mechanism for this is.

Page 15, line 23. Since this is a very small subset of genes, are these genes that you might expect to play a role in parental conflict?

Page 15, line 33. Indeed, these could be the informative exceptions.

Page 15, line 29. Hardly surprising, given that the paternal copy of NRPD1 is expressed at a higher level than the maternal copies, is it?

Page 16, line 1. So this is what you mean by in cis. Presetting?

Page 16, line 9. So ideally, one would want to look at a highly polymorphic out-crosser. I'm not suggesting that for this paper, but would this be a good test of the hypothesis? How about maize?

Page 16, line 15. But the pat and mat heterozygotes looked the same. No differences in phenotype?

Page 17, line 22. I'm confused, since aren't most 24 nt smRNAs dependent on POLIV (Figure S2)? Do you mean differentially regulated smRNAs? Expression of POLIV specifically in one or the other parent?

Page 17, line 23. How are you defining important here? Important because at least in the female NPRD1 is not expressed in the central cell? But not important, since this mutant has no effect on phenotype except in an imbalanced cross.

Page 18, line 13. For this reason, it would be nice to know much more about these genes. Mutant phenotypes, for instance. And how many of these have this feature conserved?

Significance

These are intriguing results that would benefit from a test of the hypothesis by comparing these result with those obtained in an outcrossing plant species.

Referee Cross-commenting

I agree that the other comments seem both fair and reasonable.

akin in the game

project funder

altruistic markets

This part came directly from the first freewrite back in August. I wrote this in response to the prompt about losing and finding yourself, but I liked it enough that I decided to keep it, almost word for word. I thought while my focus of this revised piece expanded beyond just losing and finding yourself, this was still a true statement about me that relates well to the topic of the paragraph it's in.

The morally superior tone here is really something else. Clearly he doesn't respect his colleagues at all.

Again, where is the line between "alternatives" and misinformation? I don't think lying to the public is acceptable, and I've seen plenty of chiropractors on Twitter calling themselves Doctor and doing just that. Unfortunately with no citations it's difficult to assess what he's talking about.

I felt like my conclusion needed a little more nuance. I didn't want to just tell a feel-good story that everyone's heard a thousand times before. Previously I had written that everything had gotten better and my attempts at fixing my anxiety fully worked. Obviously, that isn't the truth. It's never that easy. So, instead, I was brutally honest with myself and the reader by saying that I am constantly working on it, but it's hard. I won't always succeed. This adds a level of reality to my essay that makes it more relatable. I think that it is a much more powerful way to end because the struggle continues beyond the page. I used my knowledge of effective conclusions to leave the reader with a way that they can think about their own struggles. It's impossible to do it all in one day, but you can work at it over time.

According to all known laws of aviation,

there is no way a bee should be able to fly.

Its wings are too small to get its fat little body off the ground.

The bee, of course, flies anyway

because bees don't care what humans think is impossible.

Yellow, black. Yellow, black. Yellow, black. Yellow, black.

Ooh, black and yellow! Let's shake it up a little.

Barry! Breakfast is ready!

Ooming!

Hang on a second.

Hello?

• Barry?
• Adam?

• Oan you believe this is happening?
• I can't. I'll pick you up.

Looking sharp.

Use the stairs. Your father paid good money for those.

Sorry. I'm excited.

Here's the graduate. We're very proud of you, son.

A perfect report card, all B's.

Very proud.

Ma! I got a thing going here.

• You got lint on your fuzz.
• Ow! That's me!

• Wave to us! We'll be in row 118,000.
• Bye!

Barry, I told you, stop flying in the house!

• Hey, Adam.
• Hey, Barry.

• Is that fuzz gel?
• A little. Special day, graduation.

Never thought I'd make it.

Three days grade school, three days high school.

Those were awkward.

Three days college. I'm glad I took a day and hitchhiked around the hive.

You did come back different.

• Hi, Barry.
• Artie, growing a mustache? Looks good.

• Hear about Frankie?
• Yeah.

• You going to the funeral?
• No, I'm not going.

Everybody knows, sting someone, you die.

Don't waste it on a squirrel. Such a hothead.

I guess he could have just gotten out of the way.

I love this incorporating an amusement park into our day.

That's why we don't need vacations.

Boy, quite a bit of pomp... under the circumstances.

• Well, Adam, today we are men.
• We are!

• Bee-men.
• Amen!

Hallelujah!

Students, faculty, distinguished bees,

please welcome Dean Buzzwell.

Welcome, New Hive Oity graduating class of...

...9:15.

That concludes our ceremonies.

And begins your career at Honex Industries!

Will we pick ourjob today?

I heard it's just orientation.

Heads up! Here we go.

Keep your hands and antennas inside the tram at all times.

• Wonder what it'll be like?
• A little scary.

Welcome to Honex, a division of Honesco

and a part of the Hexagon Group.

This is it!

Wow.

Wow.

We know that you, as a bee, have worked your whole life

to get to the point where you can work for your whole life.

Honey begins when our valiant Pollen Jocks bring the nectar to the hive.

Our top-secret formula

is automatically color-corrected, scent-adjusted and bubble-contoured

into this soothing sweet syrup

with its distinctive golden glow you know as...

Honey!

• That girl was hot.
• She's my cousin!

• She is?
• Yes, we're all cousins.

• Right. You're right.
• At Honex, we constantly strive

to improve every aspect of bee existence.

These bees are stress-testing a new helmet technology.

• What do you think he makes?
• Not enough.

Here we have our latest advancement, the Krelman.

• What does that do?
• Oatches that little strand of honey

that hangs after you pour it. Saves us millions.

Oan anyone work on the Krelman?

Of course. Most bee jobs are small ones. But bees know

that every small job, if it's done well, means a lot.

But choose carefully

because you'll stay in the job you pick for the rest of your life.

The same job the rest of your life? I didn't know that.

What's the difference?

You'll be happy to know that bees, as a species, haven't had one day off

in 27 million years.

So you'll just work us to death?

We'll sure try.

Wow! That blew my mind!

"What's the difference?" How can you say that?

One job forever? That's an insane choice to have to make.

I'm relieved. Now we only have to make one decision in life.

But, Adam, how could they never have told us that?

Why would you question anything? We're bees.

We're the most perfectly functioning society on Earth.

You ever think maybe things work a little too well here?

Like what? Give me one example.

I don't know. But you know what I'm talking about.

Please clear the gate. Royal Nectar Force on approach.

Wait a second. Oheck it out.

• Hey, those are Pollen Jocks!
• Wow.

I've never seen them this close.

They know what it's like outside the hive.

Yeah, but some don't come back.

• Hey, Jocks!
• Hi, Jocks!

You guys did great!

You're monsters! You're sky freaks! I love it! I love it!

• I wonder where they were.
• I don't know.

Their day's not planned.

Outside the hive, flying who knows where, doing who knows what.

You can'tjust decide to be a Pollen Jock. You have to be bred for that.

Right.

Look. That's more pollen than you and I will see in a lifetime.

It's just a status symbol. Bees make too much of it.

Perhaps. Unless you're wearing it and the ladies see you wearing it.

Those ladies? Aren't they our cousins too?

Distant. Distant.

Look at these two.

• Oouple of Hive Harrys.
• Let's have fun with them.

It must be dangerous being a Pollen Jock.

Yeah. Once a bear pinned me against a mushroom!

He had a paw on my throat, and with the other, he was slapping me!

• Oh, my!
• I never thought I'd knock him out.

What were you doing during this?

Trying to alert the authorities.

I can autograph that.

A little gusty out there today, wasn't it, comrades?

Yeah. Gusty.

We're hitting a sunflower patch six miles from here tomorrow.

• Six miles, huh?
• Barry!

A puddle jump for us, but maybe you're not up for it.

• Maybe I am.
• You are not!

We're going 0900 at J-Gate.

What do you think, buzzy-boy? Are you bee enough?

I might be. It all depends on what 0900 means.

Hey, Honex!

Dad, you surprised me.

You decide what you're interested in?

• Well, there's a lot of choices.
• But you only get one.

Do you ever get bored doing the same job every day?

Son, let me tell you about stirring.

You grab that stick, and you just move it around, and you stir it around.

You get yourself into a rhythm. It's a beautiful thing.

You know, Dad, the more I think about it,

maybe the honey field just isn't right for me.

You were thinking of what, making balloon animals?

That's a bad job for a guy with a stinger.

Janet, your son's not sure he wants to go into honey!

• Barry, you are so funny sometimes.
• I'm not trying to be funny.

You're not funny! You're going into honey. Our son, the stirrer!

• You're gonna be a stirrer?
• No one's listening to me!

Wait till you see the sticks I have.

I could say anything right now. I'm gonna get an ant tattoo!

Let's open some honey and celebrate!

Maybe I'll pierce my thorax. Shave my antennae.

Shack up with a grasshopper. Get a gold tooth and call everybody "dawg"!

I'm so proud.

• We're starting work today!
• Today's the day.

Oome on! All the good jobs will be gone.

Yeah, right.

Pollen counting, stunt bee, pouring, stirrer, front desk, hair removal...

• Is it still available?
• Hang on. Two left!

One of them's yours! Oongratulations! Step to the side.

• What'd you get?
• Picking crud out. Stellar!

Wow!

Oouple of newbies?

Yes, sir! Our first day! We are ready!

Make your choice.

• You want to go first?
• No, you go.

Oh, my. What's available?

Restroom attendant's open, not for the reason you think.

• Any chance of getting the Krelman?
• Sure, you're on.

I'm sorry, the Krelman just closed out.

Wax monkey's always open.

The Krelman opened up again.

What happened?

A bee died. Makes an opening. See? He's dead. Another dead one.

Deady. Deadified. Two more dead.

Dead from the neck up. Dead from the neck down. That's life!

Oh, this is so hard!

Heating, cooling, stunt bee, pourer, stirrer,

humming, inspector number seven, lint coordinator, stripe supervisor,

mite wrangler. Barry, what do you think I should... Barry?

Barry!

All right, we've got the sunflower patch in quadrant nine...

What happened to you? Where are you?

• I'm going out.
• Out? Out where?

• Out there.
• Oh, no!

I have to, before I go to work for the rest of my life.

You're gonna die! You're crazy! Hello?

Another call coming in.

If anyone's feeling brave, there's a Korean deli on 83rd

that gets their roses today.

Hey, guys.

• Look at that.
• Isn't that the kid we saw yesterday?

Hold it, son, flight deck's restricted.

It's OK, Lou. We're gonna take him up.

Really? Feeling lucky, are you?

Sign here, here. Just initial that.

• Thank you.
• OK.

You got a rain advisory today,

and as you all know, bees cannot fly in rain.

So be careful. As always, watch your brooms,

hockey sticks, dogs, birds, bears and bats.

Also, I got a couple of reports of root beer being poured on us.

Murphy's in a home because of it, babbling like a cicada!

• That's awful.
• And a reminder for you rookies,

bee law number one, absolutely no talking to humans!

All right, launch positions!

Buzz, buzz, buzz, buzz! Buzz, buzz, buzz, buzz! Buzz, buzz, buzz, buzz!

Black and yellow!

Hello!

You ready for this, hot shot?

Yeah. Yeah, bring it on.

Wind, check.

• Antennae, check.
• Nectar pack, check.

• Wings, check.
• Stinger, check.

Scared out of my shorts, check.

OK, ladies,

let's move it out!

Pound those petunias, you striped stem-suckers!

All of you, drain those flowers!

Wow! I'm out!

I can't believe I'm out!

So blue.

I feel so fast and free!

Box kite!

Wow!

Flowers!

This is Blue Leader. We have roses visual.

Bring it around 30 degrees and hold.

Roses!

30 degrees, roger. Bringing it around.

Stand to the side, kid. It's got a bit of a kick.

That is one nectar collector!

• Ever see pollination up close?
• No, sir.

I pick up some pollen here, sprinkle it over here. Maybe a dash over there,

a pinch on that one. See that? It's a little bit of magic.

That's amazing. Why do we do that?

That's pollen power. More pollen, more flowers, more nectar, more honey for us.

Oool.

I'm picking up a lot of bright yellow. Oould be daisies. Don't we need those?

Oopy that visual.

Wait. One of these flowers seems to be on the move.

Say again? You're reporting a moving flower?

Affirmative.

That was on the line!

This is the coolest. What is it?

I don't know, but I'm loving this color.

It smells good. Not like a flower, but I like it.

Yeah, fuzzy.

Ohemical-y.

Oareful, guys. It's a little grabby.

My sweet lord of bees!

Oandy-brain, get off there!

Problem!

• Guys!
• This could be bad.

Affirmative.

Very close.

Gonna hurt.

Mama's little boy.

You are way out of position, rookie!

Ooming in at you like a missile!

Help me!

I don't think these are flowers.

• Should we tell him?
• I think he knows.

What is this?!

Match point!

You can start packing up, honey, because you're about to eat it!

Yowser!

Gross.

There's a bee in the car!

• Do something!
• I'm driving!

• Hi, bee.
• He's back here!

He's going to sting me!

Nobody move. If you don't move, he won't sting you. Freeze!

He blinked!

Spray him, Granny!

What are you doing?!

Wow... the tension level out here is unbelievable.

I gotta get home.

Oan't fly in rain.

Oan't fly in rain.

Oan't fly in rain.

Mayday! Mayday! Bee going down!

Ken, could you close the window please?

Ken, could you close the window please?

Oheck out my new resume. I made it into a fold-out brochure.

You see? Folds out.

Oh, no. More humans. I don't need this.

What was that?

Maybe this time. This time. This time. This time! This time! This...

Drapes!

That is diabolical.

It's fantastic. It's got all my special skills, even my top-ten favorite movies.

What's number one? Star Wars?

Nah, I don't go for that...

...kind of stuff.

No wonder we shouldn't talk to them. They're out of their minds.

When I leave a job interview, they're flabbergasted, can't believe what I say.

There's the sun. Maybe that's a way out.

I don't remember the sun having a big 75 on it.

I predicted global warming.

I could feel it getting hotter. At first I thought it was just me.

Wait! Stop! Bee!

Stand back. These are winter boots.

Wait!

Don't kill him!

You know I'm allergic to them! This thing could kill me!

Why does his life have less value than yours?

Why does his life have any less value than mine? Is that your statement?

I'm just saying all life has value. You don't know what he's capable of feeling.

My brochure!

There you go, little guy.

I'm not scared of him. It's an allergic thing.

Put that on your resume brochure.

My whole face could puff up.

Make it one of your special skills.

Knocking someone out is also a special skill.

Right. Bye, Vanessa. Thanks.

• Vanessa, next week? Yogurt night?
• Sure, Ken. You know, whatever.

• You could put carob chips on there.
• Bye.

• Supposed to be less calories.
• Bye.

I gotta say something.

She saved my life. I gotta say something.

All right, here it goes.

Nah.

What would I say?

I could really get in trouble.

It's a bee law. You're not supposed to talk to a human.

I can't believe I'm doing this.

I've got to.

Oh, I can't do it. Oome on!

No. Yes. No.

Do it. I can't.

How should I start it? "You like jazz?" No, that's no good.

Here she comes! Speak, you fool!

Hi!

I'm sorry.

• You're talking.
• Yes, I know.

You're talking!

I'm so sorry.

No, it's OK. It's fine. I know I'm dreaming.

But I don't recall going to bed.

Well, I'm sure this is very disconcerting.

This is a bit of a surprise to me. I mean, you're a bee!

I am. And I'm not supposed to be doing this,

but they were all trying to kill me.

And if it wasn't for you...

I had to thank you. It's just how I was raised.

That was a little weird.

• I'm talking with a bee.
• Yeah.

I'm talking to a bee. And the bee is talking to me!

I just want to say I'm grateful. I'll leave now.

• Wait! How did you learn to do that?
• What?

The talking thing.

Same way you did, I guess. "Mama, Dada, honey." You pick it up.

• That's very funny.
• Yeah.

Bees are funny. If we didn't laugh, we'd cry with what we have to deal with.

Anyway...

Oan I...

...get you something?

• Like what?

I don't know. I mean... I don't know. Ooffee?

I don't want to put you out.

It's no trouble. It takes two minutes.

• It's just coffee.
• I hate to impose.

• Don't be ridiculous!
• Actually, I would love a cup.

Hey, you want rum cake?

• I shouldn't.
• Have some.

• No, I can't.
• Oome on!

I'm trying to lose a couple micrograms.

• Where?
• These stripes don't help.

You look great!

I don't know if you know anything about fashion.

Are you all right?

No.

He's making the tie in the cab as they're flying up Madison.

He finally gets there.

He runs up the steps into the church. The wedding is on.

And he says, "Watermelon? I thought you said Guatemalan.

Why would I marry a watermelon?"

Is that a bee joke?

That's the kind of stuff we do.

Yeah, different.

So, what are you gonna do, Barry?

About work? I don't know.

I want to do my part for the hive, but I can't do it the way they want.

I know how you feel.

• You do?
• Sure.

My parents wanted me to be a lawyer or a doctor, but I wanted to be a florist.

• Really?
• My only interest is flowers.

Our new queen was just elected with that same campaign slogan.

Anyway, if you look...

There's my hive right there. See it?

You're in Sheep Meadow!

Yes! I'm right off the Turtle Pond!

No way! I know that area. I lost a toe ring there once.

• Why do girls put rings on their toes?
• Why not?

• It's like putting a hat on your knee.
• Maybe I'll try that.

• You all right, ma'am?
• Oh, yeah. Fine.

Just having two cups of coffee!

Anyway, this has been great. Thanks for the coffee.

Yeah, it's no trouble.

Sorry I couldn't finish it. If I did, I'd be up the rest of my life.

Are you...?

Oan I take a piece of this with me?

Sure! Here, have a crumb.

• Thanks!
• Yeah.

All right. Well, then... I guess I'll see you around.

Or not.

OK, Barry.

And thank you so much again... for before.

Oh, that? That was nothing.

Well, not nothing, but... Anyway...

This can't possibly work.

He's all set to go. We may as well try it.

OK, Dave, pull the chute.

• Sounds amazing.
• It was amazing!

It was the scariest, happiest moment of my life.

Humans! I can't believe you were with humans!

Giant, scary humans! What were they like?

Huge and crazy. They talk crazy.

They eat crazy giant things. They drive crazy.

• Do they try and kill you, like on TV?
• Some of them. But some of them don't.

• How'd you get back?
• Poodle.

You did it, and I'm glad. You saw whatever you wanted to see.

You had your "experience." Now you can pick out yourjob and be normal.

• Well...
• Well?

Well, I met someone.

You did? Was she Bee-ish?

• A wasp?! Your parents will kill you!
• No, no, no, not a wasp.

• Spider?
• I'm not attracted to spiders.

I know it's the hottest thing, with the eight legs and all.

I can't get by that face.

So who is she?

She's... human.

No, no. That's a bee law. You wouldn't break a bee law.

• Her name's Vanessa.
• Oh, boy.

She's so nice. And she's a florist!

Oh, no! You're dating a human florist!

We're not dating.

You're flying outside the hive, talking to humans that attack our homes

with power washers and M-80s! One-eighth a stick of dynamite!

She saved my life! And she understands me.

This is over!

Eat this.

This is not over! What was that?

• They call it a crumb.
• It was so stingin' stripey!

And that's not what they eat. That's what falls off what they eat!

• You know what a Oinnabon is?
• No.

It's bread and cinnamon and frosting. They heat it up...

Sit down!

...really hot!

• Listen to me!

We are not them! We're us. There's us and there's them!

Yes, but who can deny the heart that is yearning?

There's no yearning. Stop yearning. Listen to me!

You have got to start thinking bee, my friend. Thinking bee!

• Thinking bee.
• Thinking bee.

Thinking bee! Thinking bee! Thinking bee! Thinking bee!

There he is. He's in the pool.

You know what your problem is, Barry?

I gotta start thinking bee?

How much longer will this go on?

It's been three days! Why aren't you working?

I've got a lot of big life decisions to think about.

What life? You have no life! You have no job. You're barely a bee!

Would it kill you to make a little honey?

Barry, come out. Your father's talking to you.

Martin, would you talk to him?

Barry, I'm talking to you!

You coming?

Got everything?

All set!

Go ahead. I'll catch up.

Don't be too long.

Watch this!

Vanessa!

• We're still here.
• I told you not to yell at him.

He doesn't respond to yelling!

• Then why yell at me?
• Because you don't listen!

I'm not listening to this.

Sorry, I've gotta go.

• Where are you going?
• I'm meeting a friend.

A girl? Is this why you can't decide?

Bye.

I just hope she's Bee-ish.

They have a huge parade of flowers every year in Pasadena?

To be in the Tournament of Roses, that's every florist's dream!

Up on a float, surrounded by flowers, crowds cheering.

A tournament. Do the roses compete in athletic events?

No. All right, I've got one. How come you don't fly everywhere?

It's exhausting. Why don't you run everywhere? It's faster.

Yeah, OK, I see, I see. All right, your turn.

TiVo. You can just freeze live TV? That's insane!

You don't have that?

We have Hivo, but it's a disease. It's a horrible, horrible disease.

Oh, my.

Dumb bees!

You must want to sting all those jerks.

We try not to sting. It's usually fatal for us.

So you have to watch your temper.

Very carefully. You kick a wall, take a walk,

write an angry letter and throw it out. Work through it like any emotion:

Anger, jealousy, lust.

Oh, my goodness! Are you OK?

Yeah.

• What is wrong with you?!
• It's a bug.

He's not bothering anybody. Get out of here, you creep!

What was that? A Pic 'N' Save circular?

Yeah, it was. How did you know?

It felt like about 10 pages. Seventy-five is pretty much our limit.

You've really got that down to a science.

• I lost a cousin to Italian Vogue.
• I'll bet.

What in the name of Mighty Hercules is this?

How did this get here? Oute Bee, Golden Blossom,

Ray Liotta Private Select?

• Is he that actor?
• I never heard of him.

• Why is this here?
• For people. We eat it.

You don't have enough food of your own?

• Well, yes.
• How do you get it?

• Bees make it.
• I know who makes it!

And it's hard to make it!

There's heating, cooling, stirring. You need a whole Krelman thing!

• It's organic.
• It's our-ganic!

It's just honey, Barry.

Just what?!

Bees don't know about this! This is stealing! A lot of stealing!

You've taken our homes, schools, hospitals! This is all we have!

And it's on sale?! I'm getting to the bottom of this.

I'm getting to the bottom of all of this!

Hey, Hector.

• You almost done?
• Almost.

He is here. I sense it.

Well, I guess I'll go home now

and just leave this nice honey out, with no one around.

You're busted, box boy!

I knew I heard something. So you can talk!

I can talk. And now you'll start talking!

Where you getting the sweet stuff? Who's your supplier?

I don't understand. I thought we were friends.

The last thing we want to do is upset bees!

You're too late! It's ours now!

You, sir, have crossed the wrong sword!

You, sir, will be lunch for my iguana, Ignacio!

Where is the honey coming from?

Tell me where!

Honey Farms! It comes from Honey Farms!

Orazy person!

What horrible thing has happened here?

These faces, they never knew what hit them. And now

they're on the road to nowhere!

Just keep still.

What? You're not dead?

Do I look dead? They will wipe anything that moves. Where you headed?

To Honey Farms. I am onto something huge here.

I'm going to Alaska. Moose blood, crazy stuff. Blows your head off!

I'm going to Tacoma.

• And you?
• He really is dead.

All right.

Uh-oh!

• What is that?!
• Oh, no!

• A wiper! Triple blade!
• Triple blade?

Jump on! It's your only chance, bee!

Why does everything have to be so doggone clean?!

How much do you people need to see?!

Open your eyes! Stick your head out the window!

From NPR News in Washington, I'm Oarl Kasell.

But don't kill no more bugs!

• Bee!
• Moose blood guy!!

• You hear something?
• Like what?

Like tiny screaming.

Turn off the radio.

Whassup, bee boy?

Hey, Blood.

Just a row of honey jars, as far as the eye could see.

Wow!

I assume wherever this truck goes is where they're getting it.

I mean, that honey's ours.

• Bees hang tight.
• We're all jammed in.

It's a close community.

Not us, man. We on our own. Every mosquito on his own.

• What if you get in trouble?
• You a mosquito, you in trouble.

Nobody likes us. They just smack. See a mosquito, smack, smack!

At least you're out in the world. You must meet girls.

Mosquito girls try to trade up, get with a moth, dragonfly.

Mosquito girl don't want no mosquito.

You got to be kidding me!

Mooseblood's about to leave the building! So long, bee!

• Hey, guys!
• Mooseblood!

I knew I'd catch y'all down here. Did you bring your crazy straw?

We throw it in jars, slap a label on it, and it's pretty much pure profit.

What is this place?

A bee's got a brain the size of a pinhead.

They are pinheads!

Pinhead.

• Oheck out the new smoker.
• Oh, sweet. That's the one you want.

The Thomas 3000!

Smoker?

Ninety puffs a minute, semi-automatic. Twice the nicotine, all the tar.

A couple breaths of this knocks them right out.

They make the honey, and we make the money.

"They make the honey, and we make the money"?

Oh, my!

What's going on? Are you OK?

Yeah. It doesn't last too long.

Do you know you're in a fake hive with fake walls?

Our queen was moved here. We had no choice.

This is your queen? That's a man in women's clothes!

That's a drag queen!

What is this?

Oh, no!

There's hundreds of them!

Bee honey.

Our honey is being brazenly stolen on a massive scale!

This is worse than anything bears have done! I intend to do something.

Oh, Barry, stop.

Who told you humans are taking our honey? That's a rumor.

Do these look like rumors?

That's a conspiracy theory. These are obviously doctored photos.

How did you get mixed up in this?

He's been talking to humans.

• What?
• Talking to humans?!

He has a human girlfriend. And they make out!

Make out? Barry!

We do not.

• You wish you could.
• Whose side are you on?

The bees!

I dated a cricket once in San Antonio. Those crazy legs kept me up all night.

Barry, this is what you want to do with your life?

I want to do it for all our lives. Nobody works harder than bees!

Dad, I remember you coming home so overworked

your hands were still stirring. You couldn't stop.

I remember that.

What right do they have to our honey?

We live on two cups a year. They put it in lip balm for no reason whatsoever!

Even if it's true, what can one bee do?

Sting them where it really hurts.

In the face! The eye!

• That would hurt.
• No.

Up the nose? That's a killer.

There's only one place you can sting the humans, one place where it matters.

Hive at Five, the hive's only full-hour action news source.

No more bee beards!

With Bob Bumble at the anchor desk.

Weather with Storm Stinger.

Sports with Buzz Larvi.

And Jeanette Ohung.

• Good evening. I'm Bob Bumble.
• And I'm Jeanette Ohung.

A tri-county bee, Barry Benson,

intends to sue the human race for stealing our honey,

packaging it and profiting from it illegally!

Tomorrow night on Bee Larry King,

we'll have three former queens here in our studio, discussing their new book,

Olassy Ladies, out this week on Hexagon.

Tonight we're talking to Barry Benson.

Did you ever think, "I'm a kid from the hive. I can't do this"?

Bees have never been afraid to change the world.

What about Bee Oolumbus? Bee Gandhi? Bejesus?

Where I'm from, we'd never sue humans.

We were thinking of stickball or candy stores.

How old are you?

The bee community is supporting you in this case,

which will be the trial of the bee century.

You know, they have a Larry King in the human world too.

It's a common name. Next week...

He looks like you and has a show and suspenders and colored dots...

Next week...

Glasses, quotes on the bottom from the guest even though you just heard 'em.

Bear Week next week! They're scary, hairy and here live.

Always leans forward, pointy shoulders, squinty eyes, very Jewish.

In tennis, you attack at the point of weakness!

It was my grandmother, Ken. She's 81.

Honey, her backhand's a joke! I'm not gonna take advantage of that?

Quiet, please. Actual work going on here.

• Is that that same bee?
• Yes, it is!

I'm helping him sue the human race.

• Hello.
• Hello, bee.

This is Ken.

Yeah, I remember you. Timberland, size ten and a half. Vibram sole, I believe.

Why does he talk again?

Listen, you better go 'cause we're really busy working.

But it's our yogurt night!

Bye-bye.

Why is yogurt night so difficult?!

You poor thing. You two have been at this for hours!

Yes, and Adam here has been a huge help.

• Frosting...
• How many sugars?

Just one. I try not to use the competition.

So why are you helping me?

Bees have good qualities.

And it takes my mind off the shop.

Instead of flowers, people are giving balloon bouquets now.

Those are great, if you're three.

And artificial flowers.

• Oh, those just get me psychotic!
• Yeah, me too.

Bent stingers, pointless pollination.

Bees must hate those fake things!

Nothing worse than a daffodil that's had work done.

Maybe this could make up for it a little bit.

• This lawsuit's a pretty big deal.
• I guess.

You sure you want to go through with it?

Am I sure? When I'm done with the humans, they won't be able

to say, "Honey, I'm home," without paying a royalty!

It's an incredible scene here in downtown Manhattan,

where the world anxiously waits, because for the first time in history,

we will hear for ourselves if a honeybee can actually speak.

What have we gotten into here, Barry?

It's pretty big, isn't it?

I can't believe how many humans don't work during the day.

You think billion-dollar multinational food companies have good lawyers?

Everybody needs to stay behind the barricade.

• What's the matter?
• I don't know, I just got a chill.

Well, if it isn't the bee team.

You boys work on this?

All rise! The Honorable Judge Bumbleton presiding.

All right. Oase number 4475,

Superior Oourt of New York, Barry Bee Benson v. the Honey Industry

is now in session.

Mr. Montgomery, you're representing the five food companies collectively?

A privilege.

Mr. Benson... you're representing all the bees of the world?

I'm kidding. Yes, Your Honor, we're ready to proceed.

Mr. Montgomery, your opening statement, please.

Ladies and gentlemen of the jury,

my grandmother was a simple woman.

Born on a farm, she believed it was man's divine right

to benefit from the bounty of nature God put before us.

If we lived in the topsy-turvy world Mr. Benson imagines,

just think of what would it mean.

I would have to negotiate with the silkworm

for the elastic in my britches!

Talking bee!

How do we know this isn't some sort of

holographic motion-picture-capture Hollywood wizardry?

They could be using laser beams!

Robotics! Ventriloquism! Oloning! For all we know,

he could be on steroids!

Mr. Benson?

Ladies and gentlemen, there's no trickery here.

I'm just an ordinary bee. Honey's pretty important to me.

It's important to all bees. We invented it!

We make it. And we protect it with our lives.

Unfortunately, there are some people in this room

who think they can take it from us

'cause we're the little guys! I'm hoping that, after this is all over,

you'll see how, by taking our honey, you not only take everything we have

but everything we are!

I wish he'd dress like that all the time. So nice!

Oall your first witness.

So, Mr. Klauss Vanderhayden of Honey Farms, big company you have.

I suppose so.

I see you also own Honeyburton and Honron!

Yes, they provide beekeepers for our farms.

Beekeeper. I find that to be a very disturbing term.

I don't imagine you employ any bee-free-ers, do you?

• No.
• I couldn't hear you.

• No.
• No.

Because you don't free bees. You keep bees. Not only that,

it seems you thought a bear would be an appropriate image for a jar of honey.

They're very lovable creatures.

Yogi Bear, Fozzie Bear, Build-A-Bear.

You mean like this?

Bears kill bees!

How'd you like his head crashing through your living room?!

Biting into your couch! Spitting out your throw pillows!

OK, that's enough. Take him away.

So, Mr. Sting, thank you for being here. Your name intrigues me.

• Where have I heard it before?
• I was with a band called The Police.

But you've never been a police officer, have you?

No, I haven't.

No, you haven't. And so here we have yet another example

of bee culture casually stolen by a human

for nothing more than a prance-about stage name.

Oh, please.

Have you ever been stung, Mr. Sting?

Because I'm feeling a little stung, Sting.

Or should I say... Mr. Gordon M. Sumner!

That's not his real name?! You idiots!

Mr. Liotta, first, belated congratulations on

your Emmy win for a guest spot on ER in 2005.

Thank you. Thank you.

I see from your resume that you're devilishly handsome

with a churning inner turmoil that's ready to blow.

I enjoy what I do. Is that a crime?

Not yet it isn't. But is this what it's come to for you?

Exploiting tiny, helpless bees so you don't

have to rehearse your part and learn your lines, sir?

Watch it, Benson! I could blow right now!

This isn't a goodfella. This is a badfella!

Why doesn't someone just step on this creep, and we can all go home?!

• Order in this court!
• You're all thinking it!

Order! Order, I say!

• Say it!
• Mr. Liotta, please sit down!

I think it was awfully nice of that bear to pitch in like that.

I think the jury's on our side.

Are we doing everything right, legally?

I'm a florist.

Right. Well, here's to a great team.

To a great team!

Well, hello.

• Ken!
• Hello.

I didn't think you were coming.

No, I was just late. I tried to call, but... the battery.

I didn't want all this to go to waste, so I called Barry. Luckily, he was free.

Oh, that was lucky.

There's a little left. I could heat it up.

Yeah, heat it up, sure, whatever.

So I hear you're quite a tennis player.

I'm not much for the game myself. The ball's a little grabby.

That's where I usually sit. Right... there.

Ken, Barry was looking at your resume,

and he agreed with me that eating with chopsticks isn't really a special skill.

You think I don't see what you're doing?

I know how hard it is to find the rightjob. We have that in common.

Do we?

Bees have 100 percent employment, but we do jobs like taking the crud out.

That's just what I was thinking about doing.

Ken, I let Barry borrow your razor for his fuzz. I hope that was all right.

I'm going to drain the old stinger.

Yeah, you do that.

Look at that.

You know, I've just about had it

with your little mind games.

• What's that?
• Italian Vogue.

Mamma mia, that's a lot of pages.

A lot of ads.

Remember what Van said, why is your life more valuable than mine?

Funny, I just can't seem to recall that!

I think something stinks in here!

I love the smell of flowers.

How do you like the smell of flames?!

Not as much.

Water bug! Not taking sides!

Ken, I'm wearing a Ohapstick hat! This is pathetic!

I've got issues!

Well, well, well, a royal flush!

• You're bluffing.
• Am I?

Surf's up, dude!

Poo water!

That bowl is gnarly.

Except for those dirty yellow rings!

Kenneth! What are you doing?!

You know, I don't even like honey! I don't eat it!

We need to talk!

He's just a little bee!

And he happens to be the nicest bee I've met in a long time!

Long time? What are you talking about?! Are there other bugs in your life?

No, but there are other things bugging me in life. And you're one of them!

Fine! Talking bees, no yogurt night...

My nerves are fried from riding on this emotional roller coaster!

Goodbye, Ken.

And for your information,

I prefer sugar-free, artificial sweeteners made by man!

I'm sorry about all that.

I know it's got an aftertaste! I like it!

I always felt there was some kind of barrier between Ken and me.

I couldn't overcome it. Oh, well.

Are you OK for the trial?

I believe Mr. Montgomery is about out of ideas.

We would like to call Mr. Barry Benson Bee to the stand.

Good idea! You can really see why he's considered one of the best lawyers...

Yeah.

Layton, you've gotta weave some magic

with this jury, or it's gonna be all over.

Don't worry. The only thing I have to do to turn this jury around

is to remind them of what they don't like about bees.

• You got the tweezers?
• Are you allergic?

Only to losing, son. Only to losing.

Mr. Benson Bee, I'll ask you what I think we'd all like to know.

What exactly is your relationship

to that woman?

We're friends.

• Good friends?
• Yes.

How good? Do you live together?

Wait a minute...

Are you her little...

...bedbug?

I've seen a bee documentary or two. From what I understand,

doesn't your queen give birth to all the bee children?

• Yeah, but...
• So those aren't your real parents!

• Oh, Barry...
• Yes, they are!

Hold me back!

You're an illegitimate bee, aren't you, Benson?

He's denouncing bees!

Don't y'all date your cousins?

• Objection!
• I'm going to pincushion this guy!

Adam, don't! It's what he wants!

Oh, I'm hit!!

Oh, lordy, I am hit!

Order! Order!

The venom! The venom is coursing through my veins!

I have been felled by a winged beast of destruction!

You see? You can't treat them like equals! They're striped savages!

Stinging's the only thing they know! It's their way!

• Adam, stay with me.
• I can't feel my legs.

What angel of mercy will come forward to suck the poison

from my heaving buttocks?

I will have order in this court. Order!

Order, please!

The case of the honeybees versus the human race

took a pointed turn against the bees

yesterday when one of their legal team stung Layton T. Montgomery.

• Hey, buddy.
• Hey.

• Is there much pain?
• Yeah.

I...

I blew the whole case, didn't I?

It doesn't matter. What matters is you're alive. You could have died.

I'd be better off dead. Look at me.

They got it from the cafeteria downstairs, in a tuna sandwich.

Look, there's a little celery still on it.

What was it like to sting someone?

I can't explain it. It was all...

All adrenaline and then... and then ecstasy!

All right.

You think it was all a trap?

Of course. I'm sorry. I flew us right into this.

What were we thinking? Look at us. We're just a couple of bugs in this world.

What will the humans do to us if they win?

I don't know.

I hear they put the roaches in motels. That doesn't sound so bad.

Adam, they check in, but they don't check out!

Oh, my.

Oould you get a nurse to close that window?

• Why?
• The smoke.

Bees don't smoke.

Right. Bees don't smoke.

Bees don't smoke! But some bees are smoking.

That's it! That's our case!

It is? It's not over?

Get dressed. I've gotta go somewhere.

Get back to the court and stall. Stall any way you can.

And assuming you've done step correctly, you're ready for the tub.

Mr. Flayman.

Yes? Yes, Your Honor!

Where is the rest of your team?

Well, Your Honor, it's interesting.

Bees are trained to fly haphazardly,

and as a result, we don't make very good time.

I actually heard a funny story about...

Your Honor, haven't these ridiculous bugs

taken up enough of this court's valuable time?

How much longer will we allow these absurd shenanigans to go on?

They have presented no compelling evidence to support their charges

against my clients, who run legitimate businesses.

I move for a complete dismissal of this entire case!

Mr. Flayman, I'm afraid I'm going

to have to consider Mr. Montgomery's motion.

But you can't! We have a terrific case.

Where is your proof? Where is the evidence?

Show me the smoking gun!

Hold it, Your Honor! You want a smoking gun?

Here is your smoking gun.

What is that?

It's a bee smoker!

What, this? This harmless little contraption?

This couldn't hurt a fly, let alone a bee.

Look at what has happened

to bees who have never been asked, "Smoking or non?"

Is this what nature intended for us?

To be forcibly addicted to smoke machines

and man-made wooden slat work camps?

Living out our lives as honey slaves to the white man?

• What are we gonna do?
• He's playing the species card.

Ladies and gentlemen, please, free these bees!

Free the bees! Free the bees!

Free the bees!

Free the bees! Free the bees!

The court finds in favor of the bees!

Vanessa, we won!

I knew you could do it! High-five!

Sorry.

I'm OK! You know what this means?

All the honey will finally belong to the bees.

Now we won't have to work so hard all the time.

This is an unholy perversion of the balance of nature, Benson.

You'll regret this.

Barry, how much honey is out there?

All right. One at a time.

Barry, who are you wearing?

My sweater is Ralph Lauren, and I have no pants.

• What if Montgomery's right?
• What do you mean?

We've been living the bee way a long time, 27 million years.

Oongratulations on your victory. What will you demand as a settlement?

First, we'll demand a complete shutdown of all bee work camps.

Then we want back the honey that was ours to begin with,

every last drop.

We demand an end to the glorification of the bear as anything more

than a filthy, smelly, bad-breath stink machine.

We're all aware of what they do in the woods.

Wait for my signal.

Take him out.

He'll have nauseous for a few hours, then he'll be fine.

And we will no longer tolerate bee-negative nicknames...

But it's just a prance-about stage name!

...unnecessary inclusion of honey in bogus health products

and la-dee-da human tea-time snack garnishments.

Oan't breathe.

Bring it in, boys!

Hold it right there! Good.

Tap it.

Mr. Buzzwell, we just passed three cups, and there's gallons more coming!

• I think we need to shut down!
• Shut down? We've never shut down.

Shut down honey production!

Stop making honey!

Turn your key, sir!

What do we do now?

Oannonball!

We're shutting honey production!

Mission abort.

Aborting pollination and nectar detail. Returning to base.

Adam, you wouldn't believe how much honey was out there.

Oh, yeah?

What's going on? Where is everybody?

• Are they out celebrating?
• They're home.

They don't know what to do. Laying out, sleeping in.

I heard your Uncle Oarl was on his way to San Antonio with a cricket.

At least we got our honey back.

Sometimes I think, so what if humans liked our honey? Who wouldn't?

It's the greatest thing in the world! I was excited to be part of making it.

This was my new desk. This was my new job. I wanted to do it really well.

And now...

Now I can't.

I don't understand why they're not happy.

I thought their lives would be better!

They're doing nothing. It's amazing. Honey really changes people.

You don't have any idea what's going on, do you?

• What did you want to show me?
• This.

What happened here?

That is not the half of it.

Oh, no. Oh, my.

They're all wilting.

Doesn't look very good, does it?

No.

And whose fault do you think that is?

You know, I'm gonna guess bees.

Bees?

Specifically, me.

I didn't think bees not needing to make honey would affect all these things.

It's notjust flowers. Fruits, vegetables, they all need bees.

That's our whole SAT test right there.

Take away produce, that affects the entire animal kingdom.

And then, of course...

The human species?

So if there's no more pollination,

it could all just go south here, couldn't it?

I know this is also partly my fault.

How about a suicide pact?

How do we do it?

• I'll sting you, you step on me.
• Thatjust kills you twice.

Right, right.

Listen, Barry... sorry, but I gotta get going.

I had to open my mouth and talk.

Vanessa?

Vanessa? Why are you leaving? Where are you going?

To the final Tournament of Roses parade in Pasadena.

They've moved it to this weekend because all the flowers are dying.

It's the last chance I'll ever have to see it.

Vanessa, I just wanna say I'm sorry. I never meant it to turn out like this.

I know. Me neither.

Tournament of Roses. Roses can't do sports.

Wait a minute. Roses. Roses?

Roses!

Vanessa!

Roses?!

Barry?

• Roses are flowers!
• Yes, they are.

Flowers, bees, pollen!

I know. That's why this is the last parade.

Maybe not. Oould you ask him to slow down?

Oould you slow down?

Barry!

OK, I made a huge mistake. This is a total disaster, all my fault.

Yes, it kind of is.

I've ruined the planet. I wanted to help you

with the flower shop. I've made it worse.

Actually, it's completely closed down.

I thought maybe you were remodeling.

But I have another idea, and it's greater than my previous ideas combined.

I don't want to hear it!

All right, they have the roses, the roses have the pollen.

I know every bee, plant and flower bud in this park.

All we gotta do is get what they've got back here with what we've got.

• Bees.
• Park.

• Pollen!
• Flowers.

• Repollination!
• Across the nation!

Tournament of Roses, Pasadena, Oalifornia.

They've got nothing but flowers, floats and cotton candy.

Security will be tight.

I have an idea.

Vanessa Bloome, FTD.

Official floral business. It's real.

Sorry, ma'am. Nice brooch.

Thank you. It was a gift.

Once inside, we just pick the right float.

How about The Princess and the Pea?

I could be the princess, and you could be the pea!

Yes, I got it.

• Where should I sit?
• What are you?

• I believe I'm the pea.
• The pea?

It goes under the mattresses.

• Not in this fairy tale, sweetheart.
• I'm getting the marshal.

You do that! This whole parade is a fiasco!

Let's see what this baby'll do.

Hey, what are you doing?!

Then all we do is blend in with traffic...

...without arousing suspicion.

Once at the airport, there's no stopping us.

Stop! Security.

• You and your insect pack your float?
• Yes.

Has it been in your possession the entire time?

Would you remove your shoes?

• Remove your stinger.
• It's part of me.

I know. Just having some fun. Enjoy your flight.

Then if we're lucky, we'll have just enough pollen to do the job.

Oan you believe how lucky we are? We have just enough pollen to do the job!

I think this is gonna work.

It's got to work.

Attention, passengers, this is Oaptain Scott.

We have a bit of bad weather in New York.

It looks like we'll experience a couple hours delay.

Barry, these are cut flowers with no water. They'll never make it.

I gotta get up there and talk to them.

Be careful.

Oan I get help with the Sky Mall magazine?

I'd like to order the talking inflatable nose and ear hair trimmer.

Oaptain, I'm in a real situation.

• What'd you say, Hal?
• Nothing.

Bee!

Don't freak out! My entire species...

What are you doing?

• Wait a minute! I'm an attorney!
• Who's an attorney?

Don't move.

Oh, Barry.

Good afternoon, passengers. This is your captain.

Would a Miss Vanessa Bloome in 24B please report to the cockpit?

And please hurry!

What happened here?

There was a DustBuster, a toupee, a life raft exploded.

One's bald, one's in a boat, they're both unconscious!

• Is that another bee joke?
• No!

No one's flying the plane!

This is JFK control tower, Flight 356. What's your status?

This is Vanessa Bloome. I'm a florist from New York.

Where's the pilot?

He's unconscious, and so is the copilot.

Not good. Does anyone onboard have flight experience?

As a matter of fact, there is.

• Who's that?
• Barry Benson.

From the honey trial?! Oh, great.

Vanessa, this is nothing more than a big metal bee.

It's got giant wings, huge engines.

I can't fly a plane.

• Why not? Isn't John Travolta a pilot?
• Yes.

How hard could it be?

Wait, Barry! We're headed into some lightning.

This is Bob Bumble. We have some late-breaking news from JFK Airport,

where a suspenseful scene is developing.

Barry Benson, fresh from his legal victory...

That's Barry!

...is attempting to land a plane, loaded with people, flowers

and an incapacitated flight crew.

Flowers?!

We have a storm in the area and two individuals at the controls

with absolutely no flight experience.

Just a minute. There's a bee on that plane.

I'm quite familiar with Mr. Benson and his no-account compadres.

They've done enough damage.

But isn't he your only hope?

Technically, a bee shouldn't be able to fly at all.

Their wings are too small...

Haven't we heard this a million times?

"The surface area of the wings and body mass make no sense."

• Get this on the air!
• Got it.

• Stand by.
• We're going live.

The way we work may be a mystery to you.

Making honey takes a lot of bees doing a lot of small jobs.

But let me tell you about a small job.

If you do it well, it makes a big difference.

More than we realized. To us, to everyone.

That's why I want to get bees back to working together.

That's the bee way! We're not made of Jell-O.

We get behind a fellow.

• Black and yellow!
• Hello!

Left, right, down, hover.

• Hover?
• Forget hover.

This isn't so hard. Beep-beep! Beep-beep!

Barry, what happened?!

Wait, I think we were on autopilot the whole time.

• That may have been helping me.
• And now we're not!

So it turns out I cannot fly a plane.

All of you, let's get behind this fellow! Move it out!

Move out!

Our only chance is if I do what I'd do, you copy me with the wings of the plane!

Don't have to yell.

I'm not yelling! We're in a lot of trouble.

It's very hard to concentrate with that panicky tone in your voice!

It's not a tone. I'm panicking!

I can't do this!

Vanessa, pull yourself together. You have to snap out of it!

You snap out of it.

You snap out of it.

• You snap out of it!
• You snap out of it!

• You snap out of it!
• You snap out of it!

• You snap out of it!
• You snap out of it!

• Hold it!
• Why? Oome on, it's my turn.

How is the plane flying?

I don't know.

Hello?

Benson, got any flowers for a happy occasion in there?

The Pollen Jocks!

They do get behind a fellow.

• Black and yellow.
• Hello.

All right, let's drop this tin can on the blacktop.

Where? I can't see anything. Oan you?

No, nothing. It's all cloudy.

Oome on. You got to think bee, Barry.

• Thinking bee.
• Thinking bee.

Thinking bee! Thinking bee! Thinking bee!

Wait a minute. I think I'm feeling something.

• What?
• I don't know. It's strong, pulling me.

Like a 27-million-year-old instinct.

Bring the nose down.

Thinking bee! Thinking bee! Thinking bee!

• What in the world is on the tarmac?
• Get some lights on that!

Thinking bee! Thinking bee! Thinking bee!

• Vanessa, aim for the flower.
• OK.

Out the engines. We're going in on bee power. Ready, boys?

Affirmative!

Good. Good. Easy, now. That's it.

Land on that flower!

Ready? Full reverse!

Spin it around!

• Not that flower! The other one!
• Which one?

• That flower.
• I'm aiming at the flower!

That's a fat guy in a flowered shirt. I mean the giant pulsating flower

made of millions of bees!

Pull forward. Nose down. Tail up.

Rotate around it.

• This is insane, Barry!
• This's the only way I know how to fly.

Am I koo-koo-kachoo, or is this plane flying in an insect-like pattern?

Get your nose in there. Don't be afraid. Smell it. Full reverse!

Just drop it. Be a part of it.

Aim for the center!

Now drop it in! Drop it in, woman!

Oome on, already.

Barry, we did it! You taught me how to fly!

• Yes. No high-five!
• Right.

Barry, it worked! Did you see the giant flower?

What giant flower? Where? Of course I saw the flower! That was genius!

• Thank you.
• But we're not done yet.

Listen, everyone!

This runway is covered with the last pollen

from the last flowers available anywhere on Earth.

That means this is our last chance.

We're the only ones who make honey, pollinate flowers and dress like this.

If we're gonna survive as a species, this is our moment! What do you say?

Are we going to be bees, orjust Museum of Natural History keychains?

We're bees!

Keychain!

Then follow me! Except Keychain.

Hold on, Barry. Here.

You've earned this.

Yeah!

I'm a Pollen Jock! And it's a perfect fit. All I gotta do are the sleeves.

Oh, yeah.

That's our Barry.

Mom! The bees are back!

If anybody needs to make a call, now's the time.

I got a feeling we'll be working late tonight!

Here's your change. Have a great afternoon! Oan I help who's next?

Would you like some honey with that? It is bee-approved. Don't forget these.

Milk, cream, cheese, it's all me. And I don't see a nickel!

Sometimes I just feel like a piece of meat!

I had no idea.

Barry, I'm sorry. Have you got a moment?

Would you excuse me? My mosquito associate will help you.

Sorry I'm late.

He's a lawyer too?

I was already a blood-sucking parasite. All I needed was a briefcase.

Have a great afternoon!

Barry, I just got this huge tulip order, and I can't get them anywhere.

No problem, Vannie. Just leave it to me.

You're a lifesaver, Barry. Oan I help who's next?

All right, scramble, jocks! It's time to fly.

Thank you, Barry!

That bee is living my life!

Let it go, Kenny.

• When will this nightmare end?!
• Let it all go.

• Beautiful day to fly.
• Sure is.

Between you and me, I was dying to get out of that office.

You have got to start thinking bee, my friend.

• Thinking bee!
• Me?

Hold it. Let's just stop for a second. Hold it.

I'm sorry. I'm sorry, everyone. Oan we stop here?

I'm not making a major life decision during a production number!

All right. Take ten, everybody. Wrap it up, guys.

I had virtually no rehearsal for that.

Does it? It's pretty much just as easy or easier than doing it the way that everyone else insists is correct. I'm more than half convinced, in fact, that the npm install way being unnatural is the reason why it's sacrosanct. You can't just let things be easy—people dislike any state of affairs where their experience/participation isn't some combination of necessary and valuable.

It's enraging to hear that this individual could not feel safe in their OWN hometown and because of that they had to move to another place in order to express themselves. Sadly, I am sure there are a lot more cases like these, it's saddening how this society is.

It's not about identity, it's about data.

1. Cf. Schmidt, ‘Luhmanns Zettelkasten’, 7. The heading of this file card is formulated in form of a question: ‘Geist im Kasten?’ (‘Does Spirit hide in the filing cabinet?’). Obviously, the answer is no. Many thanks to Johannes Schmidt for providing the image of this file card.

In a zettel in his system entitled "Does Spirit hide in the filing cabinet", Niklas Luhmann wrote the note: "People come, they see everything and nothing more than that, just like in porn movies; consequently, they leave disappointed." This is a telling story about the simplicity of the idea of a slip box (zettelkasten, card catalog, or commonplace book).

Niklas Luhmann, Zettelkasten II, index card no. 9/8,3

It's also a testament to the fact that the value of it is in the upfront work that is required in making valuable notes and linking them. Many end up trying out the simple looking system and then wonder why it isn't working for them. The answer is that they're not working for it.

I feel like it would be everyones responsibility. Just so everyone can get some good valid information, and can even continue their research from those hyperlinks.

because women are givers and robbers... just like when in the traffic of women it talked about how women are the ones that develop culture because they teach things from their culture for generations

I see that adding humor into writing is much more complex than it seems. Especially since you can't physically hear it, everything you write has to be precise in order for your joke to go through. Like the context, the timing of it, the words that you use etc.

Originally, I read this lesson plan for Dixieland Jazz to be a deductive lesson because it was so fact heavy at the start in relaying information and having the students absorb rather than conceptualize. One of the history standards that can be connected with music in this lesson is "Students should understand how new cultural movements reflected and changed American society," and the music standard would be "[students] support personal interpretation of contrasting programs of music and explain how creators or performers apply the elements of music and expressive qualities, within genres, cultures, and historical periods to convey expressive intent."

As you can see, there is a lot of discussion by the teacher listed, which makes me believe that this would be lecture heavy. What I would like to alter to make this lesson more inductive is the information that the students would be interacting with physically. Incorporating the student handouts (newspaper article excerpts, music reviews, history article, photographs etc.) and having them read first and discuss second can help scaffold what the students are learning by allowing them to view the information in multiple ways rather than just a lecture format. Through the lecture and handouts, the teacher is identifying and teaching the critical content that students must know (p.56). After reviewing the historical standpoints of this era, we would listen to musical excepts that would align with the music standard listed to have the students identify why the music may sound a certain way (sad - maybe it's a blues/folk based song, happy - celebratory march, etc.). This application of knowledge will help them connect with the music to better understand how history and music interact with each other (p. 35). Through these lessons, students should be able to connect concepts and topics from the history lesson to the interpretations of the music we will listen to (p. 56).

importance of representation and also some comfortability with stereotypes

Building a well-functional Contentful-powered website is easy if you follow the steps listed below. The usual development process with Contentful looks like that:

In my opinion I can relate to this because in the very beginning of Covid when I had nothing to do other then just stay home I would stay up late at night on my electronics

Reviewer #2 (Public Review):

This manuscript presents a herculean effort in extracting and modeling Imaging Derived Phenotypes (IDPs; Alfaro-Almagro, 2018) from anatomical MRI data of the human brain from a large sample of 58,836 images corresponding to individuals of ages 2-100, compiled from a total of 82 previously existing datasets. The objective of this modeling was to define standard (authors refer to this as "normative" and add a disclaimer in the discussion that the term should be avoided) distributions of a set of target IDPs across the lifespan. The manuscript shows the clinical utility of the model on a transdiagnostic psychiatric sample (N=1,985), in that "individual-level deviations provide complementary information to the group effects." The full extent of this application is however left for further work based on this paper. Finally, in the discussion, it is highlighted that the model "can easily be transferred to new sites," which indeed is a fundamental aspect, as the model should generalize to data coming from new (unseen by the model) acquisition sites - a major culprit of almost every current MRI study.

## Strengths<br> 1. The problem is important, and the establishment of these standard distributions of IDPs across time is critical to better describe the healthy brain development trajectories while providing clinicians with a powerful differential tool to aid diagnosis of atypical and diseased brains.<br> 2. The proposed analysis entails a massive feature extraction approach that, albeit based on the widely-used FreeSurfer software (i.e., not implemented by the authors themselves), requires very careful tracking of the computational execution of neuroimaging workflows and their outcomes.<br> 3. The choice of model seems reasonable and it is effectively shown that it indeed resolves the problem at hand.<br> 4. The manual quality control (QC) by author SR also deserves recognition, visually assessing and (I'm assuming as nothing is said otherwise) manually bookkeeping the QC annotations of thousands of subjects.<br> 5. Prior work noted by the Editors is referenced, and the results are discussed in relation to that prior work.<br> 6. The manuscript is well-written - the organization, accessibility, length, clarity, and flow are overall very adequate.

## Weaknesses<br> 1. The evidence that the model will generalize ("transfer" as per the authors) to new, unseen sites, is very limited. To robustly support the claim that the model generalizes to data from new sites, a cross-validation evaluation with a "leave-one-site-out" (or leave-K-sites-out) folding strategy seems unavoidable, so that at each cross-validation split completely unseen sites are tested (for further justification of this assertion, please refer to Esteban et al., (2017)). The "transferability" of the model is left very weakly supported by figures 3 and 4, which interpretation is very unclear. This point is further developed below, regarding the over-representation of the UK Biobank dataset.<br> 2. If I understand the corresponding tables correctly, it seems that UK biobank data account for roughly half of the whole dataset. If the cross-validation approach is not considered, at the very (very) least, more granular analyses of the evaluation on the test set should be provided, for example, plotting the distribution of prediction accuracy per site, to spot whether the model is just overfitted to the UKB sample. For instance, in Figure 4 it would be easy to split row 2 into UKB and "other" sites to ensure both look the same.<br> 3. Beyond the outstanding work of visually assessing thousand of images, the Quality Control areas of the manuscript should be better executed, and particularly lines 212-233):<br> 3.a. The overall role of the mQC dataset is unclear. QC implies a destructive process in which subpar examples of a given dataset (or a product) are excluded and dropped out of the pipeline, but that doesn't seem the case of the mQC subset, that seems a dataset with binary annotations of the quality of the FreeSurfer outcomes and the image.<br> 3.b The visual assessment protocol is insufficiently described for any attempt to reproduce: (i) numbers of images rated by author SR and reused from the ABCD's accept/reject ratings; (ii) of those rated by author SR, state how the images were selected (randomly, stratified, etc.) and whether site-provenance, age, etc. were blinded to the rater; (iii) protocol details such as whether the rater navigated through slices, whether that was programmatic or decided per-case by the rater, average time eyeballing an image, etc; (iv) rating range (i.e., accept/reject) and assignment criteria; (v) quality assurance decisions (i.e., how the quality annotations are further used)<br> 3.c Similarly, the integration within the model and/or the training/testing of the automated QC is unclear.

## Additional comments<br> - Repeated individuals: it seems likely that there are repeated individuals, at least within the UKB and perhaps ABCD. This could be more clearly stated, indicating whether this is something that was considered or, conversely, that shouldn't influence the analysis.<br> - Figure 3 - the Y-axis of each column should have a constant range to allow the suggested direct comparison.<br> - Tables 5 through 8 are hard to parse - They may be moved to CSV files available somewhere under a CC-BY or similarly open license, and better interpreted with figures that highlight the message distilled from these results.<br> - Lines 212-214 about the QA/QC problem in neuroimaging are susceptible to misinterpretation. That particular sentence tries to bridge between the dataset description and the justification for the mQC sample and corresponding experiments. However, it fails in that objective (as I noted as a weakness, it's unclear the connection between the model and QC), and also misrepresents the why and how of QC overall.<br> - The fact that the code or data are accessible doesn't mean they are usable. Indeed, the lack of license on two of the linked repositories (https://github.com/predictive-clinical-neuroscience/braincharts and https://github.com/saigerutherford/lifespan\_qc\_scripts) effectively preempts reuse. The journal guidelines Please state a license on them. We provide some food for thought about how to choose a license, and why we set the licenses we use in our projects here: https://www.nipreps.org/community/licensing/.<br> - Figure 1 - caption mentions a panel E) that seems missing in the figure.<br> - There is no comment on the adaptations taken to execute FreeSurfer on the first age range of the sample (2-7 yo.).<br> - Following up on weakness 3.c, while scaling and centering is a sensible thing to do, it's likely that those pruned outliers actually account for much of the information under investigation. Meaning, EC is a good proxy for manual rating - but Rosen et al. demonstrate this on human, neurotypical, adult brains. Therefore, general application must be dealt with care. For example, elderly and young populations will, on average, show substantially more images with excessive motion. These images will go through FreeSurfer, and often produce an outlier EC, while a few will yield a perfectly standard EC. Typically, these cases with standard ECs are probably less accurate on the IDPs being analyzed, for example, if prior knowledge biased more the output for the hidden properties of this subject. In other words, in these cases, a researcher would be better off actually including the outliers.<br> - Title: "high precision" - it is unclear what precision this is qualifying as high. Is it spatial granularity for a large number of ROIs being modeled or is it because the spread of the normative charts is narrow along the lifespan and as compared to some standard of variability.

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

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Reply to the reviewers

We thank the reviewers for their helpful, detailed and insightful comments. We have modified the figures and rewritten large sections of the manuscript following the reviewers’ suggestions. In addition, we have incorporated new data throughout the manuscript and figures to clarify and better support our conclusions. All of these changes have significantly improved the coherence, consistency and clarity of our data, and have allowed us to better communicate the advance our findings represent for the fields of splicing and muscle development.

Please find a point-by-point response to the reviewers’ comments below. The reviewers’ comments are in black and italics.

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

Rbfox proteins regulate skeletal muscle splicing and function and in this manuscript, Nikonova et.al. sought to investigate the mechanisms by which Rbfox1 promotes muscle function in Drosophila.

Using a GFP-tagged Rbfox1 line, the authors showed that Rbfox1 is expressed in all muscles examined but differentially expressed in tubular and fibrillar (IFM)muscle types, and expression is developmentally regulated. Based on RNA-seq data from isolated muscle groups, the authors showed that Rbfox1 expression is much higher in TDT (jump muscle) than IFM.

Using fly genetics authors developed tools to reduce expression of Rbfox1 at different levels and the highest levels of muscle-specific Rbfox1 knockdown was lethal and displayed eclosion defects (deGradFP > Rbfox1-IRKK110518 > Rbfox1-RNAi > Rbfox1-IR27286). Consistently, Rbfox1 knockdown flies have reduced jumping and climbing phenotypes, due to tubular muscle defect where Rbfox1 is expressed at higher levels. Rbfox1 knockdown in IFM caused flight defects which have been shown previously. Further characterization of IFM and tubular muscles demonstrated a requirement of Rbfox1 for the development of myofibrillar structures in both fibrillar (IFM) and tubular fiber-types in Drosophila. Interestingly, knockdown or overexpression of Rbfox1 displayed hypercontraction phenotypes in IFMs which is often an end result of misregulation of acto-myosin interactions which was rescued by expression of force-reduction myosin heavy chain (Mhc, P401S), in the context of Rbfox1 knockdown (the rescue experiment could not be performed with Rbfox1 overexpression due to complex genetics).

Authors also performed computation analyses of the Rbfox binding motifs in the fly genome and identified GCAUG motif in 3,312, 683, and 1184 genes in the intronic, 5'UTR, and 3'UTR, respectively. These genes are enriched for factors that play important roles in muscle function including transcription factors (exd, Mef2, Salm), RNA-binding proteins (Bru1), and structural proteins (TnI, encoded by wupA). Many of these gene transcripts and proteins are affected in flies with reduction or overexpression of Rbfox1. Using fly genetics, authors propose and test different mechanisms (co-regulation of gene targets by Rbfox1 and Bru1), and regulators of muscle function (exd, Me2, Salm) and structural proteins (TnI, Mhc, Zasp52, Strn-Mlck, Sls) by which these changes could affect the muscle function.

*Overall, the characterization of Rbfox1 phenotypes and myofibrillar structure is very well elucidated, mechanisms by which Rbfox1 affects muscle function are not clear and remain largely speculative. We thank the reviewer for the positive evaluation of our phenotypic analysis of Rbfox1 knockdown in multiple muscle fiber types. This manuscript is the first detailed characterization of Rbfox1 in Drosophila muscle, extending far beyond our previous finding that Rbfox1-IR flies are flightless. Beyond behavioral and cellular phenotypes, we report that there are regulatory interactions between Rbfox1, Bruno1 and Salm and identify other Rbfox1 targets in flies. We acknowledge that there are molecular and biochemical details of specific regulatory mechanisms that remain to be elucidated, but this paper provides many foundational observations to guide future biochemical experiments and is thus important to the muscle field.

\*Major comments**

*1. The varying level of Rbfox1 knockdown (deGradFP > Rbfox1-IRKK110518 > Rbfox1-RNAi > Rbfox1-IR27286) was achieved by different strategies without validation at the protein level (likely due to lack of a Rbfox1 antibody). It is important to show different Rbfox1 protein level (at least with different RNAi), especially when authors propose that autoregulation of Rbfox1 causes increased level Rbfox1 transcript in case of Rbfox1-RNAi (mild knockdown). Autoregulation of Rbfox1 in mammalian cells may not be similar in flies.

To address this comment, we have toned-down the discussion of level-dependent regulation throughout the manuscript, and have removed claims of Rbfox1 autoregulation. We appreciate the reviewer’s point that it would be ideal to be able to determine the protein levels of Rbfox1 in the different knockdown conditions. We have tested the published antibody against DmRbfox1, but it is very dirty and we see multiple bands in Western Blot. This background partially obscures the bands from 80-90 kDa at the molecular weight where we expect Rbfox1, and prevents accurate quantification (see Reviewer Figure 1). Verification of protein levels of Rbfox1 will require generation of a new antibody which is beyond the scope of this study. As we do not have a good antibody, we performed two experiments to demonstrate our ability to tune knockdown efficiency. First, we crossed Rbfox1-IRKK110518 and Rbfox1-IR27286 to UAS-Dcr2, Mef2-Gal4 and demonstrated we could enhance the phenotype (Figure 2A, B). Second, we performed knockdown with the same hairpins at different temperatures and demonstrate that stronger knockdown at higher temperature leads to stronger phenotypes with the same hairpin

(Figure 2B). This data supports our knockdown series interpretation.

Reviewer Figure 1. Western Blot of whole fly with anti-Rbfox1 (A2BP1) (Shukla et al., 2017). Tubulin was blotted as a loading control.

• TnI and Act88F protein levels are inversely correlated with Rbfox1 level in IFM but did not correlate with the RNA level. Using RIP authors showed that Rbfox1 was shown to bound to wupA transcripts (has Rbfox binding sites) but not Act88F transcripts (does not have Rbfox binding sites). Authors performed Rbfox1 IP and identified co-IP of components of cellular translational machinery and propose that wupA (TnI) levels are regulated by translation or NMD (non-sense mediated decay). A follow up experiment was not performed to identify the mechanism by which TnI level is regulated by Rbfox1. *

Further biochemical and genetic verification of the underlying mechanisms of Rbfox1 regulation in Drosophila muscle will be addressed in a future manuscript, as in vivo modulation of translation or NMD in an Rbfox1 knockdown background involves recombination to coordinate multiple genetic elements. We have modified the text to reflect this hypothesis remains to be explored in future experiments (Line 473-474).

We have further added RT-PCR data for wupA transcript levels in IFM and TDT with Rbfox1-IRKK110518 knockdown (Figure S4 A), but as in Rbfox1-RNAi flies, there is not a significant change in expression. We do see significant downregulation of Act88F when we overexpress Rbfox1 in IFM (Figure S4 B), as well as in TDT when we knockdown Rbfox1 with either Rbfox1-IRKK110518 or Rbfox1-IR27286.

It was known that TnI mutations (affects splice site, fliH or Mef2 binding site, Hdp-3) led to a reduction in TnI level and hypercontraction. Authors showed rescue of hypercontraction phenotype in hdp-3 background by knocking down Rbfox1, likely due to increase in wupA transcription (Mef2-dependent or independent manner). However, no rescue was observed in the fliH background. Reduced level of Rbfox1 in fliH background would be expected to cause worsening of phenotype as splicing of remaining wupA transcripts would be affected with reduced Rbfox1 level. The splicing of wupA of exon 4 is not affected in Rbfox1 knockdown (fig. 6U), it's not clear if the splicing of exon 6b1 is affected in Rbfox1 knockdown.

We thank the reviewer for pointing out our lack of clarity regarding exon 6b1 and IFM-specific isoform 6b1. To address this comment and validate our previous data, we performed additional Sanger sequencing on RT-PCR products, added a diagram of the wupA gene region in Figure 4 A and improved the clarity of our discussion of the fliH and hdp3 alleles and our results in the text.

To directly respond to the reviewer, first, it is unclear if the reduced level of Rbfox1 in a fliH background should actually cause a more severe phenotype. Our data suggests that Rbfox1 represses TnI expression through binding the 3’-UTR, and can likely indirectly regulate wupA expression level via Mef2. Thus, arguably, the reduced level of Rbfox1 in the fliH background might not affect splicing, as the mutations in the regulatory element should rather make wupA insensitive to increased Mef2 expression in the Rbfox-RNAi background.

Second, we confirmed via Sanger sequencing of RT-PCR products that both IFM and TDT in control and Rbfox1-IR flies use exon 6b1 (current exon 7). The IFM isoform contains exon 3, 6b1 and 9, while the TDT isoform contains exon 3 and 6b1, but skips exon 9 (see Figure 4 A). In other tubular muscles, wupA isoforms skip exons 3 and 9, and use exon 6b2 instead of 6b1. Thus, to directly answer the reviewer’s question, no, splicing of exon 6b1 itself is not affected by Rbfox1. However, Rbfox1 does influence expression of the ”6b1 isoform”, or the wupA isoforms in IFM and TDT containing exon 6b1 and exon 3. Additionally, our data shows that Bru1, not Rbfox1, regulates alternative splicing of wupA exon 9 (Fig. S6 T).

What the reviewer has correctly identified with this comment is that the effect on splicing in the hdp-3 allele also appears to be complex and to have not been fully clarified. Although hdp-3 results from mutation of a splice site in exon 6b1 (which based on (Barbas et al., 1993) results in aberrant use of 6b2 in IFM), it also results in a near complete absence of the longer isoform containing exon 3 in adult flies. hdp-3 is reported in the same paper to affect both IFM and TDT, which both express isoforms containing exon 3 and 6b1. It is not known how mis-splicing of exon 6b1 leads to loss of isoforms containing exon 3, but our data indicate that Rbfox1 is somehow involved. It is purely speculation and beyond the scope of this manuscript, but perhaps selection of alternative exons in wupA are not independent events (ie that the splicing of exon 3 depends on correct splicing of exon 6b1). This could be mediated with interactions with chromatin, the PolII complex or through a larger splicing factor complex (something like LASR, for example (Damianov et al., 2016)), that restricts choice in alternative events through higher-order interactions. Another possible mechanism is that a second mutation exists in the hdp-3 allele that affects splicing of exon 3, although this was not indicated in the extensive sequencing data in (Barbas et al., 1993).

Bruno1 was identified as a co-regulator of Rbfox1 in different IFM and tubular muscle types. However, except Mhc, other Rbfox1 targets seem to be regulated by either Rbfox1 or Bruno1, not both. Analyses of RNA-seq datasets from single and double knockouts should identify additional targets to support the claim that - Rbfox1 and Bruno1 co-regulate alternative splice events in IFMs. Phenotypic changes with reduced Rbfox1 and Bruno1 double knockdowns are very severe, but the mechanistic basis of such genetic interaction resulting in synergistic phenotypes in IFMs is lacking as splicing changes in single vs double knockout is similar.

We agree with the reviewer that RNA-seq data would be useful to obtain a genome-wide perspective on the regulatory interactions between Rbfox1 and Bru1, and we plan to generate this data as part of a future manuscript. However, the tissue-specific dissections to isolate enough material from all of the necessary genotypes will take months to complete, and are not realistic to wait to include in this manuscript. Instead, to address the reviewer’s question, we have expanded our RT-PCR experiments to cover a wider panel of events in 12 sarcomere genes (see new data in Figures 6 and S6 and summary in Figure 8). We now can show that splice events in Fhos and Zasp67 are Rbfox1 dependent, while events in sls, Strn-Mlck and wupA are Bru1 dependent. An event in Zasp66 responds to both Rbfox1 and Bru1, but in opposite directions. Events in Mhc, Tm1 and Zasp52 are regulated by both Rbfox1 and Bru1 (or are sensitive to changes in Bru1 expression in the Rbfox1 background), and change in the same direction. This data provides a clearer mechanistic basis for the synergistic phenotype observed between Rbfox1 and Bru1 in IFM.

Rbfox1 is expressed at a high level in tubular muscle whereas Bruno1 is expressed at a high level in IFM. Rbfox1 binds to Bruno1 transcript and inversely regulates Bru1-RB level but knockdown of Bru1 does not affect Rbfox1 level (Fig. S5 G,I,J). Overexpression of Bruno1 decreased the Rbfox1 level, however, it's difficult to interpret these results as overexpression of Bruno1 may have other effects on IFM gene expression.

The reviewer correctly pointed out that we did not observe significant changes in Rbfox1 mRNA levels in the mutant bru1M3 background, however, in the original version of this manuscript, we also showed a significant decrease in Rbfox1 expression in IFM from the bru1-IR background at both 72 h APF and 1 d adult in mRNA-Seq data. To clarify differences in Rbfox1 levels between bru1-IR and our bru1 mutant backgrounds, we have performed additional RT-PCR experiments. We examined Rbfox1 levels after knockdown of bru1 (bru1-IR), and we now show that Rbfox1 levels are significantly decreased in IFM and TDT after bru1-IR (Fig. 5S, Fig S5 I). We see a weaker effect in the bru1M2 hypomorphic mutant, which likely reflects differences in Bru1 expression levels in bru1-IR and the bru1M2 allele. These results are consistent with the mRNA-seq data we presented previously (now in Fig. 5R). These additional data suggest that loss as well as gain of Bru1 affects Rbfox1 expression levels.

A dose-dependent effect of Rbfox1 knockdown was shown to regulate the expression of transcription factors that are important for muscle type specification and function including exd, Mef2, and Salm. However, it is not clear how Rbfox1 mechanistically regulates the expression of these transcription factors.

We present two pieces of data suggesting possible regulatory mechanisms for Mef2. First, RIP data suggest Rbfox1 can directly bind the 3’-UTR region of Mef2, and this region contains two binding motifs identified in both the oRNAment database and in our PWMScan dataset. Second, we show that use of the 5’-UTR regions of Mef2 is altered in Rbfox1-IR muscle. Although not definitive, this suggests that regulation of alternative 5’-UTR use may influence transcript stability or translation efficiency. We feel the many experiments to elucidate the detailed mechanism of regulation (and indeed to determine the likely contribution of multiple, layered regulatory processes) are beyond the scope of this paper, and are better left for future studies. This manuscript is the first in-depth characterization of Rbfox1 function in Drosophila muscle, and we provide multiple lines of evidence suggesting that different regulatory mechanisms exist as a basis for future experiments to explore these interesting and important regulatory interactions.*

**Minor comments**

1. It is not described if the rescue of Rbfox1 knockout by expression of force-reduction myosin heavy chain (Mhc, P401S) led to rescue of phenotypes (jumping, climbing, flight). *

Force-reduction myosin heavy chain MhcP401S is a mutation at the endogenous Mhc locus that results in a headless myosin and was previously characterized to be flightless (Nongthomba et al., 2003). It is however able to rescue jumping and walking defects observed with the hdp2 TnI allele, and supports largely normal myofibril assembly (Nongthomba et al., 2003). It is also important to note that fibrillar muscle function is very finely tuned, such that alterations that result in flightlessness in many cases do not alter myofibril structure as detected by confocal microscopy (Schnorrer et al., 2010). We therefore looked at myofiber and sarcomere structure as a more sensitive read-out of the rescue ability in the Rbfox1 knockdown, to be able to detect a partial-rescue of myofibrillar structure that may not be evident in a behavioral assay.

Immunofluorescence (IF) and Western blotting are different techniques, and Bruno1 antibody was validated for specificity in IF but not in Western blots. Figure 5L and S5 E should include muscle samples from Bru1M2.

We have added a Western Blot panel in Figure S5 D including bru1-IR, bru1M2 and samples of different wild-type tissues including abdomen, ovaries, testis and IFM.

To quantify alternative splicing or percent spliced in (PSI), primers are typically designed in the exons flanking the alternative exons. A better primer design along with PSI calculation by RT-PCR will robustly validate alternative splicing changes in different genetic background (Fig 6U and S6 U).

We do not yet have RNA-Seq data from these Rbfox1 knockdown samples to facilitate calculation of transcriptome-wide PSI values; thus, we rely on the results from our RT-PCR experiments. Our primers used to detect alternative splice events are indeed located within flanking exons or as close to the alternative exons as possible based on sequence design limitations (see schemes in Figure 6 and Figure S6). Many of the events we are detecting are complex, and not a simple “included” or “excluded” determination, and are therefore not amenable to RT-qPCR. To increase the robustness of our validation, we now provide RT-PCR gel-based quantification of exon use for the events we tested in Zasp52, Zasp66, Zasp67, wupA and Mhc (Figure 6 U-W and Figure S6 T-U).*

Reviewer #1 (Significance (Required)):

Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

Understanding how muscle fiber type splicing and gene expression is regulated will conceptually move the field forward. How transcriptional and posttranscriptional programs coordinate to specify muscle fiber type gene expression is still lacking.

Place the work in the context of the existing literature (provide references, where appropriate). Multiple RNA binding proteins and splicing factors have been shown to affect muscle function along with hundreds of gene expression and splicing changes in a complex fashion. Linking phenotypes with gene expression changes is still challenging as RNA binding proteins or RBPs are multifunctional and affect the function of other regulators that are important for muscle biology. *We thank the reviewer for recognizing the conceptual advance our findings represent, as well as the complexity in the regulatory network we are seeking to understand. A detailed understanding of the coordination of transcriptional and posttranscriptional programs is enabled by our work and will be the subject of future investigation.

* State what audience might be interested in and influenced by the reported findings.

Fly genetics, alternative splicing regulation, muscle specification and function.

Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

Regulation and function of alternative splicing in muscle. I do not have a thorough knowledge of Drosophila genetics.

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

**Summary**

This paper reports analysis of the function of RbFox1, an RNA-binding protein, best known for roles in the regulation of alternative splicing. It uses Drosophila as its in vivo model system, one that is highly suited to the analysis in vivo of complex biological events. In general, the authors present a very thorough approach with an impressive range of molecular analysis, genetic experiments and phenotypic assays. *We thank the reviewer for recognizing the suitability of our model organism as well as the time investment and diversity of experiments that were performed in this work. We have added and revised multiple experiments during this revision, which has greatly improved the manuscript.

* The authors report that Rbfox1 is expressed in all Drosophila muscle types, and regulated in both a temporal and muscle type specific manner. Using inhibitory RNA to knock down gene function, they show that Rbfox1 is required in muscle for both viability and pupal eclosion, and contributes to both muscle development and function. A Bioinformatic approach then identifies muscle genes with Rbfox1-binding motifs. They show Rbfox1 regulates expression of both muscle structural proteins and the splicing factor Bruno1, interestingly preferentially targeting the Bruno1-RB isoform. They report functional interaction between Rbfox1 and Bruno1 and that this is expression level-dependent. Lastly, they report that Rbfox1 regulates transcription factors that control muscle gene expression.

They conclude that the effect on muscle function of RbFox1 knock down is through mis-regulation of fibre type specific gene and splice isoform expression. Moreover, "Rbfox1 functions in a fibre-type and level-dependent manner to modulate both fibrillar and tubular muscle development". They propose that it does this by "binding to 5'-UTR and 3'-UTR regions to regulate transcript levels and binding to intronic regions to promote or inhibit alternative splice events." They also suggest that Rbfox1 acts "also through hierarchical regulation of the fibre diversity pathway." They provide further evidence to the field that Rbfox1's role in muscle development is conserved.

**MAJOR COMMENTS**

Are key conclusions convincing?

In terms of presentation, I suggest ensuring a clear demarcation throughout of the evidence behind the main conclusions. This can get somewhat lost as a great deal of information is presented, including all the parallels with prior findings in other systems. I am not saying this is a major problem, just highlighting the importance of clarity. Conclusions to clearly evidence include: Rbfox1 functions in a fibre-type manner to modulate both fibrillar and tubular muscle development (e.g. L664); Rbfox1 functions in a level-dependent manner (e.g. L664); Rbfox1 functions by binding to 5'-UTR and 3'-UTR regions to regulate transcript levels (e.g. L670); Rbfox1 functions by binding to intronic regions to promote or inhibit alternative splice events" (e.g. L670); "Bru1 can regulate Rbfox1 levels in Drosophila muscle, and likely in a level-dependent manner" (L488) - Clearly evidence the level effect; "first evidence for negative regulation for fine tuning acquisition of muscle-type specific properties. Depending on its expression level, Rbfox1 can either promote or inhibit expression of" muscle regulators (L797). Lastly, the controlled stoichiometry of muscle structural proteins is known to be important, but all mechanisms are not known, so again make the supporting evidence as clear as possible for the interesting point of a role for Rbfox1 in this (e.g. L787). *Using the above comments from the reviewer as a guide, we have rewritten the manuscript, including large portions of the discussion, introduction and results. We thank the reviewer for pointing out where we could more effectively communicate our results, support our conclusions and highlight the significance of our findings.

* Should some claims be qualified as preliminary or removed?

P301 "complicated genetic recombination" - seems a bit weak to include. Either do it or don't include? *

We have removed this statement from the text.*

*

Also, see section below on "adequate replication of experiments"

Are additional exps essential? (if so realistic in terms of time and cost) None essential in my view. It depends on the authors' goals, but for the most impact of the project then following up these suggestions are possible. L369-372: mutate putative Rbfox1 binding site and ask does binding still occur or not. If it doesn't, then ask if this mutation affects the expression of the putative target gene. L775-777 "Our data thus support findings that Rbfox1 modulates transcription, but introduce a novel method of regulation, via regulating transcription factor transcript stability." It would be good to demonstrate this.

We thank the reviewer for these suggestions, and agree they are indeed interesting experiments, but beyond the scope of this manuscript. We plan to pursue the detailed molecular and biochemical mechanisms of regulation in a future project including exploring Rbfox1 binding through use of reporters, identification of direct targets via CLIP and investigation of post-transcriptional regulation of translation or NMD.*

Presented in such a way as to be reproduced

Yes

Are exps adequately replicated?

A main area I would address is the authors frequent use of "may", "tend", "trend". This is confusing the picture they present. What is statistically significant and what is not? Only the former can be used as evidence. Examples include: L170: "may display preferential exon use" - does it or doesn't it? L272: "myofibrils tended to be thicker" - were they or weren't they? L350 "wupA mRNA levels tend towards upregulation in Rbfox1-RNAi". L353 "but tended towards upregulation (Fig. S4A)" L466 "Correspondingly, we see a trend towards increased protein-level expression of Bru1-PA" L474 "both Bru1-PA and Bru1-PB tend to increase" L485 "Overexpression of Bru1 in TDT with Act79B-Gal4 also tends to reduce Rbfox1" L595 "Rbfox1-IR27286 tended towards increased exd levels in IFM (Fig. 7A)" L614 "and a trend towards increased use of Mef2-Ex20 " Also, L487 "suggesting that Bru1 can also negatively regulate Rbfox1" - one cannot use a non-significant observation to suggest something. *

We have modified the text to limit use of “may”, “tend” and “trend”, and have removed discussion of non-significant results. We thank the reviewer for the very helpful and detailed list of sentences to modify.

\*MINOR COMMENTS**

*

Although individual samples are not significant, in aggregate there is a trend….

* Specific exp issues that are easily addressable

L162: "dip in Rbfox1 expression levels around 50h APF". The Fig indicates as early as 30h. Is this significantly less than the 24h data point? Comparisons in Figure 1G that are significant based on DESeq2 differential expression analysis with an adjusted p-value L427 "this staining was lost after Rbfox1 knockdown". This conflicts with Fig 5K which says no significant difference. Again in L429 "Rbfox1 knockdown leads to a reduction of Bru1 protein levels in IFMs and TDT." Fig says no significant difference in TDT. *

We thank the reviewer for pointing out this inconsistency. We have revised the text accordingly. Our Western Blot (Figure 5L, M) and RT-PCR (Figure 5N, O) do show changes of Bru1 protein and mRNA expression levels after knockdown of Rbfox1KK110518. *

Are prior studies referenced appropriately?

This m/s is an authoritative presentation of the field as a whole with a comprehensive, impressive reference list. However, a point related to this area is one of the main things I would consider tackling. This is to have more clarity in the demarcation of what this study has found that adds to prior knowledge. It is worthwhile in itself to demonstrate the many similarities with previous work in other systems, as part of establishing the Drosophila system with all its analytical advantages for in vivo molecular genetics as an excellent model for future study in this area of research. However, the impact/strength of this m/s would be enhanced by clarity in presenting what is new to the field in all organisms. *We thank the reviewer for this suggestion. We have rewritten large portions of the manuscript, including the introduction and discussion, to improve the clarity of our findings and their importance to the field.

* Are the text and Figs clear and accurate?

TEXT

L156: more precise language than "in a pattern consistent with the myoblasts" - maybe a simple co-expression with a myoblast marker? *

We have revised this phrasing in the text. Rbfox1 expression in myoblasts was previously reported by (Usha and Shashidhara, 2010). *

L181: at first use define difference between RNAi and IR*

We use IR as an abbreviation for RNAi. In particular, we are trying to distinguish the two hairpins obtained from stock centers (27286 and KK110518) from the third, homemade RNAi hairpin, originally named UAS-dA2BP1RNAi, that was generated by Usha and Shashidhara (Usha and Shashidhara, 2010). We have better defined this in the text and methods. *

L205: maybe clearly explain the link between eclosion and tubular muscle?? *

We have added a sentence explaining the link between eclosion and tubular muscle (see Line 331).*

L231: "Sarcomeres were not significantly shorter at 90h APF with the stronger Mef2-Gal4" - not clear why this is the case when the less strong knockdown conditions have shorter sarcomeres. *

We have modified the text as well as the figure labeling to clarify that the other samples were tested in 1 d adult, while the KK110518 hairpin was tested at 90 h APF. This likely indicates that the short sarcomeres observed in 1 d adults reflect hypercontraction, which in IFM is classically first apparent after eclosion when the flies actively try to use the flight muscles. The difference in timing is due to pupal lethality of the KK110518 hairpin line, so we could not evaluate adult flies.*

L234: "classic hypercontraction mutants in IFMs display a similar phenotype" - presumably not similar to the not significantly shorter sarcomeres of the previous sentence. *

We have modified the text to clarify this statement. The change in sarcomere length from 90 h APF to 1 d adult is actually the relevant observation, as this reflects the progressive shortening of sarcomeres observed in classic hypercontraction mutants.*

L244: "90h", should be "90h APF"? *

Yes, we have modified the text.*

L273: "Myofibrils in Act88F-Gal4 mediated knockdown only showed mild defects (Fig. 3 G, H, Fig. S2 C, D) despite adult flies being flight impaired". This seems worthy of discussion - the functional defect is not due to overt structure change? *

In our own experience as well as observations included in a genome-wide RNAi screen in muscle (Schnorrer et al., 2010), there are a rather large number of knockdown conditions where few if any structural defects are observed at the level of light microscopy, but flies are completely flightless. We interpret this to reflect the narrow tuning of IFM function, where slight alterations in calcium regulation or sarcomere gene isoform expression result in dysfunction and a lack of flight. Ultrastructural evaluation might reveal defects in these cases, but the defect could also be with the dynamics of tropomyosin complex function, calcium regulation, mitochondrial function or even neuro-muscular junction structure. We have added a sentence to the text to discuss and clarify the Act88F result.*

L281 "also known as Zebra bodies" - helpful to indicate these on the Fig, they are not. *

We have added arrows to the figure to mark the Zebra bodies, and updated the figure legend.*

L282: "we were unable to attempt a rescue of these defects" - I may have missed something, but what about rescue undertaken of the defects on previous pages? *

This is the first point in the text where we introduced overexpression of Rbfox1, as preceding experiments where knockdown or using a GFP-tagged protein trap line at the endogenous locus. We have revised the sentence to focus on the overexpression phenotype with UH3-Gal4.*

L283: "Over-expression of Rbfox1 from 40h APF" - this is the first over-expression experiment, so introduce why done now (and perhaps not earlier), and also explain the use of a different Gal4 driver.*

We have reworded this section of the text. The UH3-Gal4 driver is restricted to expressing in IFM from 40h APF, so is first expressed after myofibrils have been generated and selectively in IFM. This avoids lethality observed from pan-muscle expression with Mef2-Gal4 (presumably due to severe defects in tubular muscles), and also allows us to image IFM tissue from adult flies. Later experiments with Mef2-Gal4 were performed with a later temperature shift to avoid this early lethality.*

L290 "Interestingly, both Rbfox1 knockdown and Rbfox1 over-expression produce similar hypercontraction defects" - this could be interesting, worthy of discussion/explanation. *

The most logical explanation is that Rbfox1 regulates the balance in fiber-type specific isoform expression. Loss of Rbfox1 would cause a shift in the relative ratio of the isoforms of structural genes, and overexpression of Rbfox1 would likely cause a similar shift in the opposite direction. This is supported by our RT-PCR panel, where we see co-regulation of different events with Bru1, and we see fiber-type specific difference in regulation of alternative splicing (Figure 8). Overexpression of Rbfox1 would be expected to make IFM look more like TDT, which would result in an isoform imbalance and lead to the observed hypercontraction phenotype. Interestingly, loss and overexpression of Bru1 also result in the same hypercontraction phenotype, similar to what we observe with Rbfox1. We have added a paragraph in the discussion about level-dependent regulation, to address this reviewer comment.*

P305: Bioinformatic analysis. It is not clear what is taken as a potentially interesting result. On average a specific 5 base motif is found every 1000bps - so what is being looked for? How many sites in what length or position? A range of examples are described in the next pages of the m/s. For example: L337 "Bruno1.... contains 42 intronic and 2 5'-UTR Rbfox1 binding motifs" and L591 "exd contains three Rbfox1 binding sites," *

We have redone the bioinformatic analysis completely, relying on data from oRNAment and the in-vitro determined PWM. We have also rewritten all portions of the text related to this analysis and no longer focus on the number of observed motifs in a given gene. As we unfortunately do not have RNA CLIP data, we do not know genome-wide which motifs are bound in muscle. Clustering of motifs may reflect binding, but a single, strong motif can also be bound, as we demonstrate via RIP of the wupA transcript. Thus, we identified interesting targets to test based on 1) a previously described role in the literature in myofibril assembly or contractility and 2) the presence of any Rbfox1 motif in that gene. A more elegant selection method of direct and indirect target exons will be designed for a future manuscript after integrating CLIP and mRNA-Seq data that have not yet been collected.

L315: "many of these genes have binding or catalytic activity". "catalytic activity" seems very vague.

For the original supplemental figure panel, we relied on Panther high-level ontology terms, which can unfortunately be rather vague, ie “catalytic activity” or “binding activity”. We have redone this analysis and rely rather on GO terms in the biological process and molecular function categories (Figure S3 B).

L317 "When we look in previously annotated gene lists" - be more specific. What are they?

This section of the text has been rewritten, and the “previously annotated gene lists” are described in greater detail in the Methods. *

L327 "may also affect the neuro-muscular junction" - maybe better left for the Discussion? *

We have removed this sentence from the Results.*

L333 "extradenticle (exd) and Myocyte enhancer factor 2 (Mef2) contain 3 and 7 Rbfox1 motifs," Discuss the number and position of multiple motifs found in known targets? *

We have removed the discussion of the number of binding sites for different target genes, instead incorporating this information graphically in Figure S3 C. It is not clear that the number of binding sites per gene has any influence on whether it is regulated in Rbfox1 knockdown. Thus, we have de-emphasized discussion of the number of binding sites throughout the text.*

L350 "wupA mRNA levels " - clearer to stick to using TroponinI or WupA? *

We have updated instances throughout the text to consistently refer to the protein as Troponin-I (TnI) and the gene or mRNA as wupA. *

L376 "To check whether Rbfox1 regulates some target mRNAs such as wupA....." The suggestion here is more of a further indication than a "check". *

We have reworded this section of the results to make the link between post-transcriptional regulation and our mass spectrometry results more salient.*

L544 "In IFMs, knockdown of Rbfox1 and loss of Bru1 results in...." clarify if this is the two genes separately or the two genes together? *

We have rewritten this entire section and present an expanded list of tested alternative events. We have taken care in this revision to clearly denote if the genotype is Rbfox1-IR or bru1M2 or a double knockdown background.*

L580 "Our bioinformatic analysis identified Rbfox1 binding motifs in more than 40% of transcription factors genes" - is this all TFs or just "muscle" TF genes? *

We have redone this analysis and changed this sentence in the text.*

L598, what would be the mechanism of some decrease in Rbfox1 increasing mRNA levels and more of a decrease resulting in a decrease of the mRNA? The authors say "the nature of this regulation requires further investigation". *

We have added more data to this section of the manuscript and repeated several of these experiments. After adding more biological replicates and additional data points, we have more consistent results that also demonstrate the variability in bru1 expression levels after Rbfox1 knockdown. Overall levels of bru1 assayed with a primer set in exons 14 and 17 now consistently show an increase in bru1 expression after Rbfox1 knockdown between all three hairpins (Rbfox1-RNAi, Rbfox1-IRKK110518 and Dcr2, Rbfox1-IR27286) (Figure 5 N).

The relationship between expression level of Rbfox1 and expression level of bru1 and Bru1 protein isoforms is more complex. We now report a novel splice event in the annotated isoform bru1-RB that skips exon 7, resulting in a frame shift and generation of a protein that lacks all RRM domains, which we call bru1-RBshort (Figure S5). This short isoform is preferentially used in TDT, while the long isoform encoding the full-length protein is preferentially used in IFM (Figure 5 P). Presumably, this provides a mechanism, in addition to the use of different promoters, for muscle cells to regulate expression levels of different Bru1 isoforms. Knockdown of Rbfox1 in IFM results in a significant increase in the use of the long mRNA isoform, but paradoxically a decrease in the corresponding protein isoform (Figure 5, S5). We interpret this to mean that Rbfox1 regulates alternative splicing of Bru1, and likely independently a translational/post-translational mechanism regulates the expression level of Bru1-RB. This in theory could be mediated by interaction with translational machinery, post-translational modification, increased P-granule association, etc., and given the depth and breadth of experiments (as well as the multitude of isoform-specific expression reagents) required to isolate the responsible pathway, we deem it beyond the scope of this manuscript to biochemically demonstrate this specific regulatory mechanism. *

L609 "The short 5'-UTR encoded by Mef2-Ex17". Ensure all abbreviations are defined. What does "Ex" mean here? Not straightforward to relate to the diagram in the Supplemental material that indicates the Mef2 gene has many fewer than 17 exons. In Fig7 legend too. *

We have changed “Ex” to “exon” in the text. We apologize for the confusion. We have also added a diagram to Figure 7 E of the 5’-UTR region of Mef2, and a complete diagram of the locus in Figure S3 C. Based on the current annotation, Mef2 exons are numbered 1 to 21, corresponding to at least 16 distinct regions of the genome (18 if you include the variable 3’-UTR lengths). Exons sometimes will have more than one number in the annotation if a particular splice event causes a shift in the ORF, or if alternative splice sites or poly-adenylation sites are used. Mef2 is also on the minus strand, so as exons are numbered based on the genome scaffold, the exon numbering goes in reverse (ie exon 1 is the 3’-UTR).

We strongly believe in following the numbering provided in the annotation, to increase reproducibility and transparency in working with complex gene loci for many different genes. Another researcher can go to Flybase, look-up the exon number from a given gene from a specific annotation, and get the exact location and sequence of the exons we name. It is incredibly challenging and time intensive to go through older papers and figure out which exon or splice event corresponds to those in the current annotation, and we aim to alleviate this difficulty (we illustrate this in Figure 4 A for the wupA locus, where we verified exon numbers in annotation FB2021_05 by BLASTing each individual sequence and primer provided in (Barbas et al., 1993).*

L617 "Levels of Mef2 are known to affect muscle morphogenesis but not production of different isoforms" - clarify what is meant here by "different isoforms". *

We have revised this section of the text. This statement was meant to reflect that Mef2 affects muscle morphogenesis through regulation of transcription levels, but not at the level of alternative splicing.*

L638 "Salm levels were significantly increased in IFM from Rbfox1-RNAi animals, but significantly decreased in IFMs from flies with Dcr2 enhanced Rbfox1-IR27286 or Rbfox1-IRKK110518". This is worth discussion or further analysis. Normally would expect an allelic series, with an effect becoming more apparent with increased loss-of-function. *

Dcr2, Rbfox1-IR27286 and Rbfox1-IRKK110518 produce a stronger knockdown than Rbfox1-RNAi, and indeed produce significantly decreased levels of salm, thus following the allelic series. We repeated this experiment, but obtained the same results. *

L641 "This suggests that Rbfox1 can regulated Salm". How, if there are no Rbfox1 binding sites? Deserves further analysis? *

Our new bioinformatic analysis suggests a possible answer, in that it identified possible Rbfox1 motifs in a salm exon and a site in an intron. Previously, we had focused on introns and UTR regions. In addition, using the PWM we now recover Rbfox1 binding sites of the canonical TGCATGA as well as AGCATGA sites. The intron site in salm is an AGCATGA site. Further experiments will be required to determine if Rbfox1 directly binds to salm mRNA, if it interacts with the transcriptional machinery to regulate salm expression, or if this regulation occurs through yet a different mechanism, and are beyond the scope of this manuscript.*

L674: "We found the valence of several regulatory interactions..." I'm not sure the meaning of "valence" here and elsewhere will be readily understood. *

Thank you for pointing this out. We have used a different phrasing throughout the text.*

FIGURES

Fig 1 it is difficult to see the green in A-F. Can this be improved? It is clearer in I-L. *

We have replaced the images with better examples and increased the levels to make the green channel better visible. *

Fig 2 legend (others too), say what the clusters of small black ellipses in P and Q are. *

Thank you for pointing out this oversight. All boxplots are plotted with Tukey whiskers, such that they are drawn to the 25th and 75th percentile plus 1.5 the interquartile range. Dots represent outlying datapoints outside of this range. We have added statements in the relevant figure legends, as well as a more detailed explanation in the Methods. *

Fig 3 it is not easy to see a shorter sarcomere in D, as the arrow partially obscures what is being indicated. Also, the data in G indicates that sarcomeres are not shorter in Mef2 GAL4 > KK110518, although the legend says this is shown in D. *We have rephrased the statement in the legend. The arrows are pointing to frayed or torn myofibrils.

Fig 5 legend "-J). Bru1 signal is reduced with Rbfox1-IRKK110518 (C, F, I)". Clarify that this is only in IFM. It is not significant in TDT or Abd-M.

Done.*

Fig 7 legend "quantification of the fold change in exd transcript levels" - only KK110518 in IFM is significant. *

This panel was moved to Figure S7. The relevant regions of the text and figure legend were modified to reflect that only Rbfox1-IRKK110518 results in a significant change in exd levels. C - "indicates Rbfox1 binds to Mef2 mRNA" - it is not easy to see the band.

We replaced the image and adjusted the levels to make the band more visible. D - what do the different lanes on the gel below the histogram in D correspond to? We adjusted the labeling on the figure panel. The gel is a representative image of RT-PCR results that are quantified above in the histogram.

*Suggestions that would help the presentation of their data and conclusion **

There is a lot of good, thorough work here, but overall there is the impression that some of the presentation/writing could be improved (also see the above lists on clarity and accuracy). I admire the authors for their comprehensive presentation of what has already been found out in this field. As the authors summarise, a lot is already known in many other species, so (as also indicated above) it is crucial to emphasise what new is found in this work that advances overall knowledge in this field. This can be obscured in many places where they say because of what was found in vertebrate systems we looked in Drosophila. These include: L417: "This led us to investigate if Rbfox1 might regulate Bru1 in Drosophila." L452: "and we were curious if these interactions are evolutionarily conserved in flies." L528 "Thus, we next checked if Rbfox1 and Bru1 co-regulate alternative splicing in Drosophila muscle." L677 "Moreover, as in vertebrates, Rbfox1 and Bru1 exhibit cross-regulatory interactions" L683 "Rbfox1 function in muscle development is evolutionarily conserved" L697 "Here we extend those findings and show that as in vertebrates......" L702 "our observations are consistent with observations in vertebrates" L707 "Studies from both vertebrates and C. elegans suggest that Rbfox1 modulates developmental isoform switches." L746 "We see evidence for similar regulatory interactions between Rbfox1 and the CELF1/2 homolog Bru1 in our data from Drosophila." *We thank the reviewer for this honest and helpful assessment of the manuscript. Upon rereading the original text and with the guidance of the list of sentences above, we agreed with the reviewer and we have rewritten large segments of the manuscript. In particular in the introduction and discussion, we now better emphasize what is new in our findings and how they advance overall knowledge in this field.

L185 paragraph. The knockdown series is important for the study. A lot is presented in this paragraph, especially for a non-specialist and it could be easier to follow. Perhaps present the four genetic conditions in the order of the severity of their phenotype on viability. Also, clearly state what each Gal4 driver is used for. What is the nature of the RNAi/IR lines such that Dcr2 could enhance their action? Also comment on off targets - are any predicted?

We have rewritten this paragraph as the reviewer requested. The hairpins are ordered by decreasing phenotypic severity, and we have more clearly described each Gal4 driver as well as Dicer2. This information is also available in the Methods, along with the off targets for the hairpins. KK110518 has one predicted off-target ichor, but this gene is not expressed in IFM, TDT or leg based on mRNA-Seq data. 27286 has no predicted off-targets. *

L227: "In severe examples". Be as clear as possible. Are the "severe examples" using the stronger RNAi line or are they the most severe examples with a single line? I'd suggest including the result in the main Fig rather than in the Supplemental. However, as I read more of the m/s I realise there is a great deal of important information in the Supplemental Figs, and so the case is not much stronger for this example than many others. The balance of what is included where could be looked at, because it is not straightforward for the reader to read the paper and quickly flick between the main and supplemental Figs. Later in the m/s is a substantial section that starts L450 (finishes L489) and which only refers to Supplemental Figs. L503 is another area where it is necessary, and difficult, for the reader to move between main Figs and supplemental Figs. *We have reorganized the figure panels in several figures, notably Figures 4, 5, 6, 7 and 8 and the corresponding supplementary figures, including moving panels from the supplemental figures to the main figures and generating more comprehensive quantification panels. In the specific case referenced here for Fig. S1 P and Q, we chose to keep the most representative images of the phenotype in the main figure (Fig. 2 I, N), and have reworded the text to reflect that the most severe phenotypic instances are in the supplement. As we do not have CLIP data, we chose to keep the bioinformatics analysis in the supplement and have shortened the paragraph in the results devoted to Figure S3. We hope our reorganization and rewriting have better streamlined the text and figures.

L258: - perhaps a Table summarising this and other phenotype trends with the different RNA conditions might be helpful. It gets quite difficult to follow.

We have revised the text and several figure panels to make the phenotypic trends with the different RNAi conditions easier to follow.*

Reviewer #2 (Significance (Required)):

The advance reported is mechanistic.

The authors already do a very good job of placing their work in the context of prior research (see comment is Section A).

Muscle biologists interested in its development and function will be interested in this work. More broadly, those intrigued by alternative splicing will be interested. Despite its very widespread occurrence, much about alternative splicing is still poorly understood in terms of regulation and significance. This is especially the case in vivo, and this paper uses an excellent in vivo model system (Drosophila) for the genetic and mechanistic analysis of complex biological problems. My field of expertise: cell differentiation, gene expression, muscle development, Drosophila.

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

**SUMMARY**

This manuscript characterizes the role of splicing factor Rbfox1 in Drosophila muscle and explores its ability to modulate expression of genes important for fibrillar and tubular muscle development. The authors hypothesize that Rbfox1 binds directly to 5'-UTR and 3'-UTR regions to regulate transcript levels, and to intronic regions to promote or inhibit alternative splicing events. Because some of the regulated genes encode transcriptional activators and other splicing factors such as Bru1, the effects of Rbfox1 may encompass a complex regulatory network that fine-tunes transcript levels and alternative splicing patterns that shape developing muscle. Most likely the authors' hypothesis is correct that Rbfox1 is critical for muscle development in Drosophila, but overall the interesting ideas presented here are too often based only on correlations without further experimental validation. *

We respectfully disagree with the reviewer that our hypothesis that Rbfox1 is critical for muscle development in Drosophila is based only on correlation without further experimental validation. In this manuscript we extensively characterize the knockdown phenotype of 3 RNAi hairpins against Rbfox1 as well as a GFP-tagged Rbfox1 protein in both fibrillar flight muscle and tubular abdominal and jump muscle. All hairpins produce similar phenotypes with defects in myofiber and myofibril structure and result in behavioral defects in climbing, flight and jumping, confirming this phenotype is due to loss of Rbfox1 and not a random off-target gene. We also convincingly demonstrate that Rbfox1 regulates Bru1, another splicing factor known to be critical for fibrillar specific splice events in IFM. Moreover, Rbfox1 and Bru1 genetically interact selectively in IFM and our RT-PCR data for 12 select structural genes reveals fiber-type specific alternative splicing defects regulated by Rbfox1 selectively, by Bru1 selectively, or by both Rbfox1 and Bru1. Thus, we conclude that Rbfox1 is indeed critical for muscle development, and this is the first report to demonstrate this requirement in Drosophila.*

**MAJOR COMMENTS**

The hypothesis that Rbfox1 plays an important role in regulating muscle development is based on previous studies in other species and supported by much new data in this manuscript. Initial bioinformatic analysis showed that many Drosophila genes, including 20% of all RNA-binding proteins, 40% of transcription factors, etc. have the motifs in introns or UTR regions. However, I think a deeper analysis is required. Any hexamer might be present about once every 4kb, and we do not expect all UGCAUG motifs are necessarily functional, so one might ask whether the association of Rbfox motifs with muscle development genes is statistically significant? Are the motifs conserved in other Drosophila species, which might support a functional role in muscle? Are the intronic motifs located as expected for regulatory effects, that is, proximal to alternative exons that exhibit changes in splicing when Rbfox1 expression is decreased or increased? *

We appreciate the point of the reviewer that it would be ideal to distinguish genome-wide motifs that are actually bound directly by Rbfox1 from those that are unused, but our behavioral and phenotypic characterization of the knockdown phenotype in this manuscript is also valid without this data. The most effective approach to identify direct targets is to perform cross-linking immunoprecipitation, or CLIP, but we unfortunately do not have CLIP data from Drosophila muscle and it is beyond the scope of the current study to generate this data. It is not trivial to obtain the amount of material necessary to identify tissue-specific binding sites, as we would also likely expect differences in targeting specificity between tubular and fibrillar muscle. Genome-wide analysis of the evolutionary conservation of binding site motifs is also not trivial and is beyond the scope of this paper.

Despite these limitations and to address the reviewer’s comment, we have done the following:

1. We have completely redone our bioinformatic analysis using transcriptome data from the oRNAment database (Benoit Bouvrette et al., 2020), as well as searching genome-wide for instances of the in vitro determined PWM using PWMScan, to capture possible sites in introns (Figure S3). The oRNAment database was shown to reasonably predict peaks identified in eCLIP from human cell lines, which we assume would translate to a similar predictive capacity in the Drosophila
2. We have calculated the expected distribution of Rbfox1 sites in a random gene list for Figure S3, and indeed the number of Rbfox1 sites in sarcomere genes is significantly enriched.
3. We have looked more carefully at the distribution of Rbfox1 and Bru1 motifs in the transcriptome (in the oRNAment data), and find not only that these motifs frequently occur in the same muscle phenotype genes, but also that they are closer together than is expected by chance (Fig. S4 J).
4. We marked the location of Rbfox1 and Bru1 motifs in the vicinity of select alternative splice events we tested via RT-PCR on the provided summary diagrams (Fig. 6, Fig. S6).
5. We have tested additional alternative splice events in total from 12 structural genes, and of the 9 events misregulated after Rbfox1 or Bru1 knockdown, all but 1 are flanked by Rbfox1 or Bru1 binding motifs. This indicates that the motifs are indeed located as expected for a regulatory effect. Is it possible to knock out an Rbfox motif and show that splicing of the alternative exon is altered, or regulation of transcript levels is abrogated?


The construction and mutation of reporter constructs is possible, but would take longer than the recommended revision time-frame, in particular to generate reporters that can be evaluated in vivo. We intend to address the biochemical mechanism(s) of Rbfox1 regulation with future experiments in a separate manuscript.

Also, what was the background set of genes used for the GO enrichment analysis? Genes expressed in muscle or all genes?

The background set of genes for GO enrichment (now Figure S3 B) was all annotated genes for the “all genes” label and all muscle phenotype genes for the “Muscle phenotype” label.

The data on cross regulation between Rbfox1 and Bru1 are confusing and inconsistent, since mild knockdown and stronger knockdown of Rbfox1 seem to have different effects on Bru1 expression. New data suggest that Rbfox1 can positively regulate Bru1 protein levels (Fig.5), but this seems inconsistent with the lab's earlier studies indicating opposite temporal mRNA expression profiles for Rbfox1 and Bru1 across IFM development. 


We apologize for the confusion, but the relationship between Rbfox1 and bru1 levels across IFM development has not been published previously. We previously generated that mRNA-Seq data, but presented here (now in Figure 5Q) is a new analysis of that data, specifically focused on Rbfox1 and bru1 expression. We have corrected the phrasing in the text.

To address this comment, along with points raised above by Reviewer 2, we have revised this part of the manuscript, added more data to this section of the manuscript and repeated several of these experiments. After adding more biological replicates and additional data points, we have more consistent results that also demonstrate the variability in bru1 expression levels after Rbfox1 knockdown. Overall levels of bru1 assayed with a primer set in exons 14 and 17 now consistently show an increase in bru1 expression after Rbfox1 knockdown between all three hairpins (Rbfox1-RNAi, Rbfox1-IRKK110518 and Dcr2, Rbfox1-IR27286) (Figure 5 N). This is consistent with our observations of inversely correlated mRNA levels during IFM development, as when Rbfox1 levels decrease, bru1 transcripts increase.

We agree with the reviewer that the relationship between the expression level of Rbfox1 and expression level of bru1 mRNA and Bru1 protein isoforms is more complex. We now report a novel splice event in the annotated isoform bru1-RB that skips exon 7, resulting in a frame shift and generation of a protein that lacks all RRM domains, which we call bru1-RBshort (Figure S5). Unknowingly, we had previously used a primer set from exon 7 to exon 8 as “common”, which lead to some confusion. This short isoform is preferentially used in TDT, while the long isoform encoding the full-length protein is preferentially used in IFM (Figure 5 P). Presumably, this provides a mechanism, in addition to the use of different promoters, for muscle cells to regulate expression levels of different Bru1 isoforms. Knockdown of Rbfox1 in IFM results in a significant increase in the use of the long mRNA isoform, but paradoxically a decrease in the corresponding protein isoform (Figure 5, S5). We interpret this to mean that Rbfox1 regulates alternative splicing of Bru1, and likely independently a translational/post-translational mechanism regulates the expression level of Bru1-RB. This in theory could be mediated by interaction with translational machinery, post-translational modification, increased P-granule association, etc., and given the depth and breadth of experiments (as well as the multitude of isoform-specific expression reagents) required to isolate the responsible pathway, we deem it beyond the scope of this manuscript to biochemically demonstrate this specific regulatory mechanism. *

*

Both Rbfox1 and Bru1 gene have many Rbfox motifs, but they are both large genes (>100kb) and would be expected to have many copies of all hexamers. How do we know whether any of them are functional?

We do not know if all of the Rbfox1 binding sites in the Bru1 and Rbfox1 loci are bound, but the CLIP data required to assess this is beyond the scope of this manuscript, as discussed above. We do show, however, that changes in the expression level of Rbfox1 affect the expression of Bru1 on both the mRNA transcript and protein level, and changes in the expression level of Bru1 also can affect the expression level of Rbfox1. The direct or indirect nature of this regulation remains to be fully elucidated, although we do provide RIP data showing we can detect bru1 transcript bound to Rbfox1-GFP (Figure S4 I). We have modified the text to address this comment.

Figure S4, section I, J: if changes in Bru1-RB isoform expression are correlated with Rbfox1 knockdown, it seems reasonable to test whether the Bru1-RB promoter can drive expression of GFP in an Rbfox1-dependent manner. But if I understand correctly, the assay as described on p. 19 uses the promoter region upstream of Bru1-RA. What is the logic for this experiment? It is not surprising that no effect was observed. The end result is that we have no idea whether Rbfox1 directly regulates bru1-RB. Even if it does, bru-Rb appears to be a minor component of Bru expression in IFM.

Upon reevaluating this experiment and with respect to the reviewer’s comment, we have removed it from the manuscript to avoid confusion. Our new data indicate a switch in use of the bru1-RBlong and bru1-RBshort isoforms (Figure 5 N-P), suggesting that Rbfox1 regulation is on the level of splicing.

Further experiments will be necessary to refine the indirect versus direct regulatory effects of Rbfox1 on Bru1, but our data do demonstrate that Bru1 levels are regulated in Rbfox1 knockdown conditions. We also provide a RIP experiment (Figure S4 I) showing that Rbfox1-GFP does directly bind bru1 mRNA, but we did not determine if this was isoform-specific. Multiple additional experiments would be necessary to distinguish between regulation of alternative splicing, direct binding to regulate transcript translation or stability, or transcriptional regulation via regulation of Salm, or some combination of these possible mechanisms. The data presented here are important to the field as they are the first report of isoform-specific regulation of Bru1 in muscle, even if we do not conclusively show if this regulation by Rbfox1 is direct or indirect.

In the section "Rbfox1 and Bruno1 co-regulate alternative splice events in IFMs", the data show that splicing of several genes is altered by knockdown or over-expression of Rbfox1 and Bru1. The interesting conclusion is for a complex regulatory dynamic where Rbfox1 and Bru1 co-regulate some alternative splice events and independently regulate other events in a muscle-type specific manner. However, if we are to conclude that these activities are due to direct binding of Rbfox1 and Bru1 to the adjacent introns, we need information about the location of flanking Rbfox and/or Bru1 motifs. Do upstream or downstream binding sites correlate with enhancer or silencer activity, as reported in previous studies of these splicing factors in other species? For wupA, Figure S3 shows an intronic Rbfox site, but exon 4 is not labeled so the reader cannot correlate this information with the diagram in Figure 6U.

As mentioned above, we have marked the location of Rbfox1as well as Bru1 binding motifs in the diagrams in Figure 6 and Figure S6. We have tested additional alternative splice events, and can now show events regulated only in the Rbfox1 knockdown, only after bru1 knockdown, or in double knockdown flies (Figure 8). 8 out of 9 events where we see clear changes in splicing are flanked by potential Rbfox1 or Bru1 motifs. Demonstration of direct binding and assay of genome-wide binding sites through CLIP studies is beyond the scope of this manuscript and will be pursued in the future.

The evidence that Rbfox1 directly affects expression of transcription factor Exd seems to be based only a correlation between Rbfox1 knockdown and decreased expression of Exd. The observation that binding of Rbfox1 to the Exd 3'UTR in RIP experiments further weakens the case.

We agree with the reviewer and have moved the data related to exd to the supplement (Figure 7 and S7). We still mention exd in the text as it is significantly decreased after knockdown with Rbfox1-IRKK110518, but we have removed it from larger claims of transcriptional regulation as well as from the summary in Figure 8. Also, just to note that although we failed to detect Rbfox1-GFP bound to exd, this experiment was performed with adult flies. Since Exd is functionally important early in pupal development during fate specification of the IFMs, it is possible we might detect binding to exd mRNA at a different developmental timepoint.

Similarly, there is a correlation of Rbfox1 knockdown with expression of alternative 5'UTRs in the Mef2 gene. However, the changes in UTR expression appear mostly not statistically significant. Do the authors have a model to explain what mechanism might allow Rbfox to regulate expression of alternative 5'UTRs, which would seem to be a transcriptional process?

Mef2 transcript levels are significantly increased after knockdown with Rbfox1-RNAi and decreased after overexpression of Rbfox1, and we can detect direct binding of Rbfox1-GFP to Mef2 RNA via RIP. This establishes Mef2 as a likely direct target of Rbfox1 regulation, likely through the two Rbfox1 motifs in the 3’-UTR (Figure S3 C). In addition to this regulation, we made an observation that has not been previously reported in the literature, that IFM expresses a particular isoform of Mef2 that uses a short promoter encoded by Exon 17. We see both tissue-specific use of Exon 17 (Figure 7 F) as well as developmental regulation of Exon 17 use in IFM (Figure S7 C). Surprisingly, we saw that use of exon 17 in the Mef2 promoter is altered in Rbfox1 knockdown muscle. We now provide a quantification of this data, to show the change is statistically significant. We also provide a scheme of the Mef2 locus and RT-PCR primers with exons 17, 20 and 21 labelled (Figure 7 E). We have also rewritten this section of the text to increase the impact and clarity of our finding.

For Salm, there apparently are no Rbfox motifs in the gene, and there are statistically significant but apparently inconsistent changes in Salm expression when it is knocked down in IFM by Rbfox1-RNAi (Salm increases) vs knockdown by Rbfox1-IR27286 or Rbfox1-IRKK110518 (Salm decreases). These are potentially interesting observations but more data would be needed to make stronger conclusions. How would regulation occur in the absence of Rbfox motifs?


The best explanation we can provide for why salm expression is increased with the weak hypomorph Rbfox1-RNAi condition, but decreased with the stronger hypomorph Rbfox1-IRKK110518 or Dcr2, Rbfox1-IR27286 conditions is that salm regulation is sensitive to Rbfox1 expression or activity level. We now discuss this in a new section of the discussion. We further attempted several experiments to address this question, including obtaining an endogenously tagged Salm-GFP line, as well as a UAS-Salm line (kindly provided by F. Schnorrer). Disappointingly, there is no GFP expressed in the Salm-GFP line, either live, by immunostaining or in Western Blot of multiple developmental stages, indicating that the line has fallen apart and we have not yet redone the CRISPR targeting to generate a new line. The UAS-Salm construct works (too well), in that overexpression with Mef2-Gal4 results in early lethality and we have not yet managed to optimize the experiment and obtain enough pupal muscle where we can evaluate the effect on Bru1 or Rbfox1 levels.

Our new bioinformatic analysis further revealed possible Rbfox1 motifs in a salm exon and a site in an intron. Previously, we had focused on introns and UTR regions. Now, using the in vitro determined PWM, we can recover Rbfox1 binding sites of the canonical TGCATGA as well as AGCATGA sites. The intron site in salm is an AGCATGA site. Further experiments will be required to determine if Rbfox1 directly binds to salm pre-mRNA, if it interacts with the transcriptional machinery to regulate salm expression, or if this regulation occurs through yet a different mechanism. We feel the many required experiments are beyond the scope of the current manuscript. Our data provides an experimental basis for future studies on this topic.

\*MINOR COMMENTS**

1. In several figures there is a misalignment of the transcriptional driver information with the phenotype data in the bar graphs above. Please correct the alignments to make interpretation easier. *

We have revised the layout of labels for many plots throughout the manuscript to avoid a category label associated with a genotype label at a 45-degree angle, and to make interpretation easier.

On p. 14 Brudno et al. is cited as ref for Fox motifs near muscle exons, but this paper only focused on brain-specific exons.

In addition to brain-specific exons, Brudno et al. also analyzed a set of muscle-specific exons, and thus this is the appropriate reference. For instance, from the Brudno paper, “As an additional control in some experiments we analyzed a smaller sample of muscle-specific alternative exons that were collected exactly as described above for the brain-specific exons” and “UGCAUG was also found at a high frequency downstream of a smaller group of muscle-specific exons.” Further details of the muscle-specific exon analysis can be found in (Brudno et al., 2001).

For Mef2, why do exons described as 5'UTR have numbers 17, 20, and 21? One would normally expect these to be exon 1, 2 or 1A, 1B, etc.

We rely on the Flybase annotation and numbering system to refer to exons. Per Flybase, all exons are labeled in the 5’ to 3’ direction of the sequenced genome, even for genes, such as Mef2 or wupA, that are encoded on the reverse strand. We strongly believe in following the numbering provided in the annotation, to increase reproducibility and transparency in working with complex gene loci for many different genes. Another researcher can go to Flybase, look-up the exon number from a given gene from a specific annotation, and get the exact location and sequence of the exons we name. It is incredibly challenging and time intensive to go through older papers and figure out which exon or splice event corresponds to those in the current annotation. We illustrate this in Figure 4 A for the wupA locus, where we verified exon numbers in annotation FB2021_05 by BLASTing each individual sequence and primer provided in (Barbas et al., 1993). The Mhc locus is even more complex, in particular regarding alternative 3’-UTR regions and historic versus current exon designations (Nikonova et al., 2020). For clarity and reproducibility, we therefore rely on the current Flybase designations.

Fig 8: "regulation of regulators" seems to imply the Rbfox1 is impacting transcription?? Is there precedence for this type of regulation by Rbfox1? Yes, indeed, there is precedence for Rbfox1 impacting transcription, as we presented in the Discussion. Rbfox2 is reported to interact with the Polycomb repressive complex 2 to regulate gene transcription in mouse (Wei et al., 2016) and in flies Rbfox1 interacts with transcription factors including Cubitus interruptus and Suppressor of Hairless to regulate transcription downstream of Hedgehog and Notch signaling (Shukla et al., 2017; Usha and Shashidhara, 2010). In addition, Rbfox1 regulates splicing of Mef2A and Rbfox1 and Rbfox1 cooperatively regulate splicing of Mef2D during C2C12 cell differentiation (Gao et al., 2016). Our results provide a further piece of evidence implicating Rbfox1 either directly or indirectly in transcriptional regulation as well as regulation of alternative splicing.

* Reviewer #3 (Significance (Required)):

**SIGNIFICANCE**

These studies of a major tissue-specific RNA binding protein, Rbfox1, are definitely important for our understanding of functional differences between muscle subtypes, and between muscle and nonmuscle tissues. The broad outlines of Rbfox1 alternative splicing regulation are known, but there is very little specific detail about the important targets in muscle subtypes that might help explain functional differences between subtypes. If more experimental validation can be obtained for regulation of transcript levels by binding 3'UTRs, this would also represent new information. *

We thank the reviewer for recognizing the significance of our work and our detailed analysis of Rbfox1 phenotypes in different muscle fiber-types. Experimental validation of 3’-UTR binding will be a significant time investment in terms of building and testing in-vivo reporter constructs, assaying NMD and translation effects and performing the CLIP studies necessary for identification of directly-bound 3’-UTR regions, extending beyond the scope of this manuscript and the time allotted for revision. The data we present here represent an important advance in our understanding how Rbfox1 contributes to muscle-type specific differentiation, and form the basis for future experiments to explore the molecular and biochemical mechanisms underlying this regulation. *

I am reviewing based on my experience studying alternative splicing in vertebrate systems, with an emphasis on Rbfox genes. Therefore I am unable to evaluate the functional data on different subtypes of muscle in Drosophila.

*

Reviewer Response References

Barbas, J. A., Galceran, J., Torroja, L., Prado, A. and Ferrús, A. (1993). Abnormal muscle development in the heldup3 mutant of Drosophila melanogaster is caused by a splicing defect affecting selected troponin I isoforms. Mol Cell Biol 13, 1433–1439.

Benoit Bouvrette, L. P., Bovaird, S., Blanchette, M. and Lécuyer, E. (2020). oRNAment: a database of putative RNA binding protein target sites in the transcriptomes of model species. Nucleic Acids Research 48, D166–D173.

Brudno, M., Gelfand, M. S., Spengler, S., Zorn, M., Dubchak, I. and Conboy, J. G. (2001). Computational analysis of candidate intron regulatory elements for tissue-specific alternative pre-mRNA splicing. Nucleic Acids Res 29, 2338–2348.

Damianov, A., Ying, Y., Lin, C.-H., Lee, J.-A., Tran, D., Vashisht, A. A., Bahrami-Samani, E., Xing, Y., Martin, K. C., Wohlschlegel, J. A., et al. (2016). Rbfox Proteins Regulate Splicing as Part of a Large Multiprotein Complex LASR. Cell 165, 606–619.

Gao, C., Ren, S., Lee, J.-H., Qiu, J., Chapski, D. J., Rau, C. D., Zhou, Y., Abdellatif, M., Nakano, A., Vondriska, T. M., et al. (2016). RBFox1-mediated RNA splicing regulates cardiac hypertrophy and heart failure. J Clin Invest 126, 195–206.

Nikonova, E., Kao, S.-Y. and Spletter, M. L. (2020). Contributions of alternative splicing to muscle type development and function. Semin. Cell Dev. Biol.

Nongthomba, U., Cummins, M., Clark, S., Vigoreaux, J. O. and Sparrow, J. C. (2003). Suppression of muscle hypercontraction by mutations in the myosin heavy chain gene of Drosophila melanogaster. Genetics 164, 209–222.

Schnorrer, F., Schönbauer, C., Langer, C. C. H., Dietzl, G., Novatchkova, M., Schernhuber, K., Fellner, M., Azaryan, A., Radolf, M., Stark, A., et al. (2010). Systematic genetic analysis of muscle morphogenesis and function in Drosophila. Nature 464, 287–291.

Shukla, J. P., Deshpande, G. and Shashidhara, L. S. (2017). Ataxin 2-binding protein 1 is a context-specific positive regulator of Notch signaling during neurogenesis in Drosophila melanogaster. Development 144, 905–915.

Usha, N. and Shashidhara, L. S. (2010). Interaction between Ataxin-2 Binding Protein 1 and Cubitus-interruptus during wing development in Drosophila. Dev Biol 341, 389–399.

Wei, C., Xiao, R., Chen, L., Cui, H., Zhou, Y., Xue, Y., Hu, J., Zhou, B., Tsutsui, T., Qiu, J., et al. (2016). RBFox2 Binds Nascent RNA to Globally Regulate Polycomb Complex 2 Targeting in Mammalian Genomes. Mol Cell 62, 875–889.

Eric: I will define phenomenal consciousness by examples and folk psychology!

Phenomenal consciousness is the most folk psychologically obvious thing or feature that the positive examples possess and that the negative examples lack. I do think that there is one very obvious feature that ties together sensory experiences, imagery experiences, emotional experiences, dream experiences, and conscious thoughts and desires. They’re all conscious experiences. None of the other stuff is experienced (lipid absorption, the tactile smoothness of your desk, etc.).

Bakker: Which part of our brains is making the "obvious" verdict? Not folk psychology, but folk philosophy!

Typically, recognition of experience-qua-experience is thought to be an intellectual achievement of some kind, a first step toward the ‘philosophical’ or ‘reflective’ or ‘contemplative’ attitude. Shouldn’t we say, rather, that phenomenal consciousness is the most obvious thing or feature these examples share upon reflection, which is to say, philosophically?

Eric: Also, any theory of phenomenal consciousness must explain why it is obvious, but we are struck with wonder and confusion when we stop to think about it.

If the reduction of phenomenal consciousness to something physical or functional or “easy” is possible, it should take some work. It should not be obviously so, just on the surface of the definition. We should be able to wonder how consciousness could possibly arise from functional mechanisms and matter in motion. Call this the wonderfulness condition.

Bakker: Yeah, I understand that. It's just like St Augustine with time

“What, then, is time? If no one asks of me, I know; if I wish to explain to him who asks, I know not.”

And I can explain both the same way! The reason is that we know how to use time, but not how to theorize abstractly concerning time. Similarly, we know how to use phenomenal consciousness, but not how to theorize abstractly about it.

Why?

Evolution! We die if we don't know how to use time, but nothing bad would happen if we can't theorize time. Similarly for phenomenal consciousness.

Why would we die if we don't know how to use phenomenal consciousness? Bakker doesn't explain, though presumably it is important for something (otherwise evolution would not have produced it).

Why is there such a difference between using and theorizing? That brings us to two families of cognitive processes.

Family 1: source sensitive. Such a process is evolved to deal with causal mechanisms (basically, doing science, asking "how", although in an unschooled, primitive way). Because it deals with causal mechanisms, and everything has a previous cause, the process always has "open slots" where more information can be sent in for processing.

This family is more versatile and it is responsible for the dramatic scientific progress.

Family 2: source insensitive. A member of this family would have "fixed input format" as an analogy. It would not request for information on "and what happened before that?" or "and what is inside this black box function?".

This family is inflexible, and it is responsible for the endless philosophical debates.

In fact, they are blind to these further information, and blind to their blindness. The result is that subjectively, when thinking using such source-insensitive cognition processes, the feeling is a sense of sufficiency: there's nothing lacking. No puzzling fact that requires expanding. No "and what happened before that?"

Phenomenal consciousness seems to be made of family 2 processes. That's why it feels so complete even if it is so information-poor.

Clickup seems to feature all these small little features that seem really useful for teams. It seems like they create whatever their users tend to need! I wonder how each piece fits into one another.

Seems like note-taking and task management are closely linked. What about these other features?

CON: It might be gimmicky, and long-term it seems like a maintainability nightmare. Unless there is a team focussed on this type of tool, (i.e. it is core to the product lifecycle) then it is likely to become outdated over time.

Three tips for making meaningful and engaging personal pieces

1. Create from who you are and what you genuinely care about. Think about these two questions to try to apply this tip: When do you feel most like yourself? What do you create (or have you created in the past) that best expresses who you are?

2. Focus on something small to tell a larger story.

A small story about, say, making brownies with your stepbrother at 3 a.m. can speak volumes about the experience of living in a blended family during quarantine. You don’t always have to reach for a “deep meaning” — often that meaning is inherent in the details of the story itself, or in the way you tell it.

3. Find a unique way to approach your topic by playing with genre, voice, tone, the use of detail and other craft tools.

Although it’s hard to come up with a topic that’s original. The good news is that you don’t have to — you just need to put your own special spin on it.

what crypto winter looks like? how to survive through the winter is key topic.

Great one

Remember.

Using ethically charged words such as evil and crime shift the paradigm.

Is there any research on global heating resilient agriculture? Camilo Mora has done some research on this.

Bakker proposes to construct the Ontological Difference (a concept in ontology) with Neuroscience (a theory of certain ontic things). This is a circular construction, since ontic things are defined using the ontology.

But it's the best we could do, since directly constructing ontology without ontic concepts gave us unreadable crap like Heidegger's later books. Humans are just too bad at finding general truths through any route except science.

I had a feeling that he was thinking above just everyone else because look where it led him to but he had the privilege of that money when he sold his estates. Now his wealth has increased and his business is making him more money and people start to perceive him as a selfish person. It's interesting how people of all levels of society are introduced in this play you have starving women selling their children for two silver currency and then you have a man that goes to owning banks and factories.

I agree that sport is part of a culture but I think that there are many more aspects to a culture then just sports especially in the U.S. However, in Canada this might be different. That is why it is always good to learn about other countries other then our own because each country does have their unique characteristics that make up their culture that we might not understand.

Personalization is a process of tailoring the content for various segments of the target audience.

A mainstream NPR podcast briefly discusses DAOs in the context of the bid from "ConstitutionDAO" to bid on and purchase an authentic copy of the US Constitution via Sotheby's. Amusingly, her description repeats the word "little", like "little governance token" and "little voting body".

so my understanding is that it's a general term for when you model both the mean (w/ something more than just an intercept) and covariance terms of a multivariate normal in a GP setting -- it's usually used in spatial autocorrelation models but I've heard it used for temporal autocorrelation settings too. AFAIK, though, complicated covariance functions and complicated mean functions are non-identifiable, so you have to pick one or other (some might even say trying to put a trend on the mean is non-identifiable with the the covariance function -- which it sort of is, in that a GP can pick up on a trend in-sample no problem -- but I think if there is a trend identifying it can help tighten up variance, and so is worth trying to include + it's helpful for interpretability).

Author Response:

Reviewer #1 (Public Review):

The model proposed here is the first large-scale model that actually performs a cognitive task, which in this case is working memory but could easily extend to decision making in general as is acknowledged by the authors. Briefly, each of the 30 areas are simulated as a rate, Wong-Wang circuit (i.e. two excitatory pools inhibit each other through a third, inhibitory population). The authors use previously collected anatomical data to constrain the model and show qualitatively match with the data, in particular how mnemonic activity emerges somewhat abruptly along the brain hierarchy.

Strengths Previous models have focused on neural dynamics during the so-called "resting state", in which subjects are not performing any cognitive task - thus, resting. This study is therefore an important improvement in the field of large-scale modelling and will certainly become an influential reference for future modelling efforts. As typically done in large-scale modelling, some anatomical data is used to constrain the model. The model shows several interesting characteristics, in particular how distributed working memory is more resilient to distractors and how the global attractors can be turned off by inhibition of only top areas.

Weaknesses Some of these results are not clear how they emerge, and some "biological constraints" do not seem to constrain. Moreover, some claims are slightly exaggerated, in particular how the model matches the data in the literature (which in some cases it does not) or how somatosensory working memory can be simulated by simply stimulating the "somatosensory cortex".

This paper has two different models, one being a simplified version of the main model. However, it is not very clear what the simplified model adds the main findings, if not to show that the empirical anatomical connectivity does not constrain the full model.

We thank the reviewer for this evaluation, and for appreciating the innovative character of our study in implementing a cognitive function in a data-constrained large-scale brain model. We hope that it will be useful for future studies planning to add cognitive functions to their large-scale models, and also for experimentalists who might benefit from this insight.

In response to the detailed comments of the reviewer, and to address the weaknesses identified above, we have rewritten parts of the text, clarified important concepts and included a new simulations. Briefly:

-We have clarified the nature and effects of the ‘biological constraints’ that we use. The full model that we use is indeed data-constrained, in the sense that we use real data to determine the values of many parameters. Having a data-constrained model, however, does not mean that all the results will be equally constrained. Some model results will critically depend on (some) data used to constrain the model, while other results will be more robust to changes in these parameters. We have highlighted this point and we also added explanations for each of the results presented.

-We have corrected several claims along the text to make it more in line with experimental evidence, and included the new references suggested by the reviewer to this effect. For example, for the case of somatosensory WM mentioned by the reviewer, we have indicated that the existence of a ‘gating’ mechanism (explored in a supplementary figure) is important for achieving an accurate match with the experimentally observed effects of somatosensory stimulation.

-Finally, we have highlighted the complementary benefits of the full and simplified models, and improved our motivation for the latter. Briefly, the simplified model allows us to identify the key ingredients needed for distributed WM (useful to generalize to other animal models), while the full model ensures that the main findings are still present when more realistic assumptions are made. A good example is the counterstream inhibitory bias, which is in principle not necessary for a simplified model but becomes a crucial factor to implement the distributed WM mechanism in our macaque model.

Reviewer #2 (Public Review):

There is a lot to like about this manuscript. It provides a large-scale model of a well-known phenomenon, the "delay activity" underlying working memory, our oldest and most enduring model of a cognitive function. The authors correctly state that despite the ubiquity of delay activity, there is little known about the macro and micro circuitry that produces it. The authors offer a computational model with testable hypotheses that is rooted in biology. I think this will be of interest to a wide variety of researchers just as delay activity is studied across a variety of animal models, brain systems, and behavior. It is also well-written.

My main concern is the authors may be self-handicapping the impact of their model by not taking into account newer observations about delay activity. For a number of years now, evidence has been building that working memory is more complicated than "persistent activity" alone. Stokes, Pasternak, Dehaene, Miller and others have been mounting considerable evidence for more complex dynamics and for "activity-silent" mechanisms where memories are briefly held in latent (non-active) forms between bouts of spiking. There is also mounting evidence that the thalamus plays a key role in working memory (and attention). In particular, higher thalamic nuclei are critical for regulating cortical feedback. Cortical feedback plays a central role in the model presented here. The model presented in this manuscript just deals with persistent attractor states and the cortex alone.

This is not to say that this manuscript does not have good value as is. No one disputes that some form of elevated, sustained, activity underlies working memory. This work adds insights into how that activity gets sustained and the role of, and interactions between, different cortical areas. The observation that the prefrontal and parietal cortex are more critical than other areas, that there are "hidden" attractor states, and "counterstream inhibitory bias" are important insights (and, importantly, testable). They will likely remain relevant even as the field is moving beyond persistent attractor states alone as the model for working memory. The new developments do not argue against the importance of delay activity in working memory. They show that it is more to the story, as inevitably happens in brain science.

The authors do include a paragraph in the Discussion referencing the newer developments. Kudos to them for that. However, it presented as "new stuff to address in the future". Well, that future is now. These "newer" developments have been mounting over the past 10 years. The worry here is that by relying so heavily on the older persistent attractor dynamics model and presenting it as the only model, the authors are putting an early expiration date on their work, at least in terms of how it will be received and disseminated.