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Referee #1

Evidence, reproducibility and clarity

In this manuscript, Flemr et al. characterize the roles that proteins Nrde2 and CCDC174 play in mammalian splicing regulation. The authors perform native (nTAP-MS) and cross-linking (xTAP-MS) based IP-MS methods to identify the NRDE2 and NRDE2 mutant interactomes, and demonstrate NRDE2's enriched interactions with splicing factors. The authors further develop a RNA footprinting method (BCLIPseq) in order to capture NRDE2 and CCDC174 binding patterns on RNA, revealing a preference for binding unspliced introns close to the 5' SS. Furthermore, upon NRDE2 knockdown, the authors note a significant increase in alternative 5'SS splicing events, making NRDE2 a putative regulator of cryptic 5'SS. Following up on this observation through luciferase reporter assays, the authors demonstrate how NRDE2 and CCDC174 work together to inhibit cryptic splicing at weak 5'SSs. Finally, the authors demonstrate that NRDE2 and CCDC174 interact with U1 snRNA (but not core U1 snRNP protein components), providing a basis for their interactions with 5'SS. Overall, the authors thoroughly characterize protein and RNA interactions with NRDE2, demonstrating its role in mammalian pre-mRNA splicing, and its concerted role with CCDC174 in regulating splicing at some weak 5' splice sites.

The study would be greatly improved through additional controls and more careful analyses. For instance, many controls are missing for the cell lines used throughout the study, making interpretation of the data more difficult. Other issues include that techniques developed within the study are presented without validation experiments, and key analyses, including microscopy experiments and rMATs analysis of RNAseq data, are performed without proper quantification, weakening the authors' conclusions from these experiments. Finally, major conclusions of the paper, such as the potential role of NRDE2 in a non-canonical U1 snRNP complex, would be greatly strengthened by additional experiments. However, overall, it appears that many of the major concerns should be readily addressable.

Major comments

1. Data are not present to demonstrate that cell lines were validated and compared properly:
   • a. The authors say "We assessed potential consequences of the tagging approach on protein function by comparing RNA-seq gene expression profiles with that of untagged cells, which remained unchanged for the proteins we report hereinafter" (p.6). This analysis should be made available in the supplement. They should additionally show western blots of tagged/untagged protein, in order to demonstrate similar expression levels for endogenous and engineered proteins.
   • b. Knockdown (KD) levels of all proteins from engineered lines should be shown over the KD timeline used in the study. For instance, no westerns in the paper show the degradation efficiency of the dTAG KD lines.
   • c. The authors should address why knockdown lines were made in different ways for different proteins (ex. only one cell line is a dTAG degradation line) and why knockdowns were performed for different amounts of time for every protein.
   • d. The authors should consider that, since knockdowns are performed for different amounts of time, results between protein knockdowns may not be directly comparable. For instance, in figure 3D, Ccdc174 dTAG lines have less misspliced target introns than the other knockdown lines. However, this may simply be because the knockdown period is shorter for the dTAG line than the other knockdown lines, and the length of treatment affects the overall number of introns affected.
2. Nuclear localization experiments would benefit from further controls and quantification:
   • a. The authors conclude that "NRDE2 localisation to nuclear speckles depends on active pre-mRNA splicing" (p.7), which seems to contradict their result that "Chemical inhibition of splicing with Thailanstatin A (Liu et al., 2013) resulted in...wild-type NRDE2 remaining concentrated in enlarged NSs (Figure 1H and S1J)" (p.8).
   • b. Since the splicing inhibitor Thailanstatin A also changes the localization patterns of U2AF2 (Fig. 1G-H), it is unclear if U2AF2 is still a reliable nuclear speckle marker in the presence of the drug. Additional controls (such as staining for other nuclear speckle markers) are necessary to make this assertion.
   • c. To make the conclusion that "NRDE2-D174R accumulated in nucleoli" (p. 8), the authors should also include a nucleoli marker in their microscopy experiments.
   • d. Signal quantification of NRDE2 distribution/overlap with U2AF2 signal would strengthen the conclusions in Fig. 1G-H.
   • e. Quantification would again be helpful in Fig. 5C to demonstrate changes in NS localization. In addition, it looks like Nrde2-KO does not just lead to lack of CCDC174 accumulation, but to a decrease in its overall expression. The authors should comment on this observation, or quantify CCDC174 signal in both images to demonstrate that the overall levels remain the same.
3. Since BCLIPseq is a technique developed by the authors, a more in-depth discussion of the technique development and quality control of the resulting data is warranted.
   • a. The authors mention that BCLIPseq offers a "streamlined and sensitive alternative to existing CLIP techniques" (p.9), but they don't provide any specifics into the ways they improve existing CLIP techniques in the main text. In what ways is it more streamlined and sensitive? This should be discussed in the main text rather than just the discussion, in order for the assertions made to be backed up with (supplementary) figures. A comparison of the coverage provided by a BCLIPseq library for NRDE2 to a CLIP library, for instance, would help to support these assertions.
   • b. The authors should address or provide evidence for why on-bead polyadenylation is preferable/more efficient than adapter ligation, especially as polyadenylation may be variable across transcripts. For instance, the authors could show more controls demonstrating the efficiency of on-bead polyadenylation or cite papers that have already extensively tested on-bead polyadenylation.
   • c. Many other RNA footprinting techniques (eCLIP, RIPseq) have noted significant nonspecific background in the resulting libraries, and usually use input controls to filter for this nonspecific background. The authors should clearly state if their BCLIPseq libraries also suffer from the same nonspecific background, and if so, what quality control steps exist in their analysis pipeline to minimize this background.
   • d. Related to the previous point, there is a high amount of rRNA reads in all the BCLIP libraries except EIF4A3. The authors suggest it is likely background, but if they are using a FLAG antibody for all of these, I'm not sure why there would be so much more background for some and not the others. If it's because EIF4A3 pulls down much more RNA with it because it binds most exon-exon junctions, whereas binding of the others is more rare, then isn't it possible that the mRNA reads are also partially background? This could explain why there is a very small overlap between the BCLIP bound loci and the affected 5'SS. An input control would help to determine what is indeed background.
4. The conclusion that "This [U1 snRNA binding] leads to the provocative idea that NRDE2 could potentially mediate the formation of a non-canonical U1 snRNP" (p.20) is a very intriguing conclusion that would largely benefit from additional experiments to strengthen the claim.
   • a. Depletion of a U1-specific protein (U1-70k, U1C) and analysis of the effect (or lack thereof) on Nrde-U1 snRNA interactions would strengthen the assertion that Nrde-U1 snRNA interactions are independent of core U1 snRNP components.
   • b. Depletion of a U1-specific protein (U1-70k, U1C) and analysis of the effect (or lack therefore) on Nrde2-KO sensitive introns would also strengthen the assertion that Nrde2 regulates introns as part of a non-canonical U1 snRNP.
   • c. Overall, a schematic in the last figure that depicts the splicing model presented in the discussion would be helpful for describing the Nrde2/Ccdc-174 model proposed.
   • d. The authors show that the majority of ms-snRNA reads map to U1 snRNA. However, U1 snRNA is generally more abundant than other snRNAs (Dvinge et al. 2019), so the authors should show how the distribution shown in Fig. 6B compares to the input distribution of snRNA levels in the cell line used. Also, relative levels of U1 snRNA detected by IP-Northern Blot (Fig. 6C) don't seem to match the results shown in Fig. 6A and B, as U1 snRNA seems most abundant in the NRDE2 IP by Northern Blot and most abundant in NRDE-Δ200 by BCLIP-seq.
   • e. In Figs. 6D, E and F, the authors suggest that NRDE2 and CCDC174 contact U1 snRNA at multiple positions based on observing highest enrichment over SL2 and SL3. However, snRNAs are highly structured and modified, which may interfere with reverse transcription during library preparation and lead to uneven signal throughout the gene body. To show that the proteins of interest are really enriched at these positions, the authors could perform the same experiment for a protein that is known to bind at a different location on U1 snRNA.
5. The rMATs analysis performed is very lenient; notably, there is no reported filtering for splicing events with some minimum coverage across replicates, and the inclusion level difference threshold of >0 (rather than >0.1 etc) is extremely low. As the rMATs analysis is key to the authors conclusion that there is "frequent cryptic 5'SS upon Nrde2 knockout" (p. 13), it seems important that this analysis is performed with more stringency in order to capture robust and meaningful splicing changes.

Minor comments

1. Some parts of the paper are organized in a confusing manner:
   • a. It is unclear why development of the xTAP-MS protocol is under the section "NRDE2 Localization to Nuclear Speckles Depends on Active pre-mRNA Splicing"
   • b. The section "Nrde2 and Mtrex Knockouts Induce a 2C-like State", while interesting, seems to be outside the scope of the paper
2. It would be interesting for the authors to look into the BCLIPseq data to see if there are any enriched RBP binding motifs for the proteins studied.
3. Western blots for IPs (ex. Fig 1F) should show the input for both the IP bait and prey proteins, not just the prey. In addition, input and IP'ed protein should be displayed in the same western blot image (without cropping in-between).
4. Previous studies (Boehm et al 2018) have found that other EJC-associated proteins also are important for regulation of 5' cryptic splice site usage. It would be interesting for the authors to compare the 5' cryptic splice sites identified in these earlier studies to look for overlap between the 5' cryptic splice sites regulated by these proteins vs. NRDE2.
5. The luciferase reporter assays are an especially strong portion of the paper, and are a nice orthogonal validation of the link between Nrde2, splicing regulation, and SS strength.
6. It would be interesting for the authors to investigate the effect of the mutations in the luciferase reporter constructs on the binding patterns of Nrde2 on construct transcripts. This may help provide a mechanistic basis for the chosen cryptic 5' SSs.
7. "Thus, NRDE2 promotes splicing from the most upstream of a series of 5'SSs" (p. 16) is an interesting conclusion, but this statement is far too general given the low number of genes surveyed using the luciferase assay. The statement should be rewritten to reflect that this statement has only been shown to be true for the few genes tested.
8. The statement "NRDE2 and CCDC174 promote splicing at many of the same weak 5'SSs" (p. 16) would be stronger if it was not just based on the genes studied through the luciferase assay, but based on splicing changes analyzed genome-wide through rMATs analysis. Do NRDE2 and CCDC174 promote splicing of the same weak splice sites globally?
9. R squared values should be added to the correlations presented in Fig. S2B to support the claim that replicates "correlated strongly", since that is the basis for merging replicates in subsequent analyses.
10. In Fig. 3A, the four clusters should be annotated on the figure to increase clarity.

Significance

Overall, the study is thorough in its approaches to studying NRDE2 biology and makes a strong case for the exciting role of NRDE2 and CCDC174 in 5'SS selection. Combined IP-MS and RNA footprinting approaches compellingly demonstrate NRDE2's associations with splicing factors and splice sites in vivo. In addition, the combination of genome-wide approaches (RNAseq) with targeted analyses (luciferase reporter assays) allow for detailed analyses of cryptic splice site choice in the absence of NRDE2, NRDE2 mutants, MTREX, or CCDC174. These experiments support the novel role of NRDE2 and its associated proteins in splice site choice.

Interesting.

Aviv, it's a great call to arms followed by a great framework for the dimensions of accountability. My first thought was that it's a little "too good": are there (large-scale) platforms/communities that succeed by your standards on any of those dimensions? I that kind of forum possible not just on Facebook but on the internet? It's tough enough in face-to-face venues. I don't want to add bloat, but in this or a companion piece it would be fantastic if you'd provide diverse cases offering examples/hopes of success for each dimension.

Yes... this is the problem. It's easier to make rubrics "work" (by twisting the learning experience to fit) than to develop and engage in authentic assessment.

Reviewer #1 (Public Review):

This work is an important contribution to simulator-based inference, substantially improving over previous work by Fengler et al. (2021) with ideas from other modern work in likelihood-free inference (largely driven by the authors' group).

The authors provide a technique, Mixed Neural Likelihood Estimator (MNLE) to efficiently build a likelihood emulator (i.e., an approximation of the likelihood function) for simulator-based models for which typically the likelihood is unavailable. The strength of this approach is that then the emulated likelihood then can be flexibly plugged in whenever a likelihood is needed, using *any* desired inference schema and hierarchical structure at inference time. Moreover, it is important to note that unlike other likelihood-free inference approaches, this method is learning an emulator of the likelihood per trial (or per observation), i.e., there are no summary statistics involved and modulo approximation errors this method could match exact inference (unlike methods based on non-sufficient summary statistics).

Another thing to note is that, unlike previous work from the authors, this approach amortizes the training of the likelihood emulator (which needs to be done once per model), but does not amortize inference itself (i.e., the modeller still needs to run MCMC or any other inference method).

MNLE is similar in spirit to the likelihood approximation networks (LANs) proposed by Fengler et al. (2021), but arguably better in any aspect. As well-argued by this paper, both in principle and empirically, the main issue of LANs is that they use density estimation to estimate the likelihood *independently* for each parameter setting, and then train a neural network to effectively interpolate between these density estimates. Instead, MNLE uses *conditional* density estimation which trains a density network while sharing information across different parameters settings, an approach that is orders of magnitude more efficient. MNLE performs conditional estimation with mixed observations (discrete and continuous) by first learning a model of the discrete variables and then a model of the continuous variables conditioned on the discrete observations.

On top of the humongous gain in efficiency of the training (10^5 simulations required for training MNLE vs 1011 for LAN), the paper shows that MNLE perform at least as well (and often better) than LANs on a variety of quality-of-approximation and quality-of-inference metrics (e.g., error, calibration, etc.). The authors also show results with a very useful technique, simulation-based calibration (SBC; Talts et al. 2018), which should become a gold standard.

Important limitations that are worth highlighting and could perhaps be discussed a bit more explicitly in the paper are:<br /> - The current example models (drift-diffusion models) have a very low-dimensional observation structure (one binary observation + one continuous observation per trial). This limitation is not necessarily a problem as many models in cognitive neuroscience have a very low-dimensional observation structure just like the one used in this example (one discrete + one continuous variable), but it is worth mentioning.<br /> - The method works for i.i.d. data. Any additional structure/dependence (e.g., adding parameters to characterize the stimulus shown in the trial) effectively increases the dimensionality of the likelihood approximation the network needs to learn. For reference, the current examples explore models with medium-low dimensionality (4-5 dimensions). This is mentioned briefly in Section 4.4.<br /> - Related to the two points above, the study does not truly discuss nor explore issues of scalability of the method. While previous related work (by some of the authors) has shown remarkable results, such as the ability to infer posteriors up to ~30 parameters (with carefully selected and tuning of the neural architecture; Gonçalves et al., 2020), the scalability of the current approach is not analyzed here.<br /> - Also related, like any neural-network based approach, it seems there is some art (and brute search) required in selecting the hyperparameters and architecture of the network, and it's unclear how much the method can be applied out-of-the-box for different models. For example, in Section 4.5 the authors say that they started with standard hyperparameter choices, but ended up having to perform a hyperparameter search over multiple dimensions (number of hidden layers, hidden units, neural spline transforms, spline bins) to achieve the results presented in the paper. In short, while training an MNLE *given the hyperparameters* might require a small number of simulations (10^5), in practice we would need to account for the further cost of finding the correct hyperparameters for training the emulator (presumably, requiring an exploration of at least 10-100 hyperparameter setting).

Notably, all these limitations also apply to LANs, so while it is important to acknowledge these, it is also clear that MNLE is a radical improvement over the previous approach along any axis.

In short, this is a strong contribution to the field of computational methods for statistical inference in the sciences (here applied to a common class of models in cognitive neuroscience) and I expect this method and others built on top of or inspired by it will have a large impact in the field, even more so since all code has been made available by the authors.

This is arguably a good thing in society, even if social media companies take it on the chin in lost revenue.

I don't think it's that they can't cope, they're just the first ones who have tried to do anything about it

There's deep ties between environmental justice and racial justice. It's sad how impoverished communities have to suffer/be picked on by big businesses because they aren't able to fight. More and more people are coming to realize this, so I'm eager to see how this may change things. Just because you don't have enough money, doesn't mean you should have to deal with exposure to hazardous chemical toxins from the landfills and power plants near your communities.

I'll bet you there was a lot of fluff and drivel at any given period you're thinking of -- but that it's been rightly discarded since.

I despise invocations of the incoherent concept of virtue signaling, but anyone contrasting Bezos and Scott wins points with me.

Iv'e never understood why people have the need to bash other people because of there economic status. It's one of the most lowest points you can go. I know some people believe that money is everything but its not. Money does not determine who you are as a person. Money will satisfy who you are as a person and It will not fulfill your life because eventually you will want something that money can't buy. Making fun of someone's situation serves no other purpose than just finding ways to bring other people down.

Beanstalk governance attack

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Reply to the reviewers

Major comments:

Are the key conclusions convincing?

We discuss 4 key conclusions.

__# 1 __A PRC of the segmentation clock was constructed.

Although the authors have produced an interesting phase map, the regulation function F(\phi) of the circle map does not give the phase response curve (PRC) (Hoppensteadt & Keener 1982, Guevara & Glass 1982). This holds only when the system is stimulated with very short pulses (ideally Dirac delta), but the experimental pulses here are a quarter of the intrinsic period.

There are several definitions of the PRC (Dirac pulses PRCs, linear PRCs, etc.). We use the general definition from Izhikievich, 2007: “In contrast to the common folklore, the function PRC (θ) can be measured for an arbitrary stimulus, not necessarily weak or brief. The only caveat is that to measure the new phase of oscillation perturbed by a stimulus, we must wait long enough for transients to subside“.

The corresponding equation from Izhikievich (section 10.1.3) is

PRC(θ)= θ_new-θ

which is equivalent to our Equation 1.

Hence, the key assumption we make is that after perturbing the system, we are back on the limit cycle as pointed out by Izhikievich. We think this is a reasonable assumption, because the perturbation we impose is relatively weak, despite pulsing for almost one quarter of the intrinsic period. The concentrations of DAPT we used in this current study are just enough to elicit a measurable response, and further lowering the concentration does not result in entrainment within our experiment time (0.5uM, Figure S7B in submitted version of the manuscript). Additionally, we previously reported that periodic pulsing with 2uM DAPT did not result in change of the Notch signaling activity with respect to control samples (Sonnen et al., 2018). Along similar lines, the DAPT drug concentrations we used are much lower compared to what has been used in previous studies aiming to perturb signaling levels, e.g. 100uM and 50uM used in study of segmentation clock in zebrafish embryos (Özbudak and Lewis, 2008 and Liao et al., 2016, respectively), and 25uM used in study of the segmentation clock in mouse PSM cells (Hubaud et al., 2017). Combined, we reason that we apply weak perturbations that allow to extract the PRC of the segmentation clock during entrainment. Additional evidence that indeed we have revealed a meaningful PRC is provided below, please see our response to point #3.

__# 2 __Furthermore, in eq. 1 T_ext must be the winding number, and the modulus must be in units of

phase, either one or two pi, for the circle map to be correct. Thus, calling the measured response of the system a PRC is not convincing.

We thank the reviewer for pointing this out. We indeed rescaled everything to express the PRC in units of phase. We made this more explicit and updated equations throughout the text.

__# 3 __The system is being entrained. Technically, It would also be easier to get the stroboscopic maps

in the quasi-periodic regime since all the points in the circle will be sampled. Since no quasi-periodic response was demonstrated, the claim of entrainment is not convincing.

While, in principle, PRC can be indeed obtained from responses in the “quasi-periodic” regime, such an approach is, in practice, challenging due to the intrinsic noise. The closest approximation to this is the phase response after the first pulse, that we reproduce below and compare to our inferred PRC, where we indeed clearly see a high noise level. Nevertheless, also the PRC based on the 1st pulse is in agreement with the PRC we derived from the entrainment data.

In the entrained regime, one can get a much more reliable estimate of the phase response despite the noise. The level of noise in the stroboscopic map lowers as the samples approach entrainment (Figure S12), and the entrainment phase itself is a reliable statistical quantity that can be used to infer regions of the PRC as the detuning is varied.

In addition, and maybe even more importantly, we identify several key features characteristics of entrainment, such as the change of entrainment phase as a function of detuning (Figure 7, Figure S6-S7 in submitted version of the manuscript) and the dependency of the time to entrainment as a function of initial phase (Figure 6). While additional features can be linked, in theory, with entrainment, i.e. period-doubling, higher harmonics (Figure 5), quasi-periodicity, we do not agree with the reviewer that all of these need, or in fact, can be found in the experimental data, in particular because of the influence of the noise. Conversely the positive experimental evidence that we provide for the presence of entrainment, combined with the theoretical framework we develop, justifies, in our view, the conclusions we make.

__# 4 __The response of the system to external pulses is compatible with a SNIC. This is compatible, but

it is equally compatible with other explanations. Assuming that the PRC is the same as the regulation function F(\phi), the PRC in Kotani 2012 (PRL 2012 fig. 3C) would be a similar shape as that shown by the authors. Similar models to that in Kotani et al., have been studied, but a SNIC has not been found (an der Heiden & Mackey 1982). It is relatively straightforward to construct a phenomenological model with a SNIC, but having underlying biological insight is not guaranteed. No argument for choosing a SNIC is given, so this emphasis of the paper is not convincing.

It is true that the mapping of PRCs to oscillators is undetermined, in the sense that many systems could potentially give rise to similar PRCs. That said, there is value in parsimonious models, which often generalize very well despite their simplicity. This explains why in neuroscience, constant sign PRCs are generally associated with SNIC. There is a mathematical reason for this : 1-D oscillators with resetting (such as the quadratic fire-and-integrate model) are the simplest models displaying constant sign PRCs, and are the “normal” form for SNICs. In other words, SNIC bifurcations are among the simplest ones compatible with constant sign PRCs, and we think it is informative to point this out. In our manuscript, we go one step further by actually fitting the experimental PRC with a simple, analytical model that allows us to compute Arnold tongue for any values of the perturbation (contrary to more complex models).

Other models such as Kotani 2012 can display similar PRC shapes, but they are of mathematically higher complexity, and furthermore it is not clear how such systems might behave when entrained. For instance that model in particular uses delayed differential equations, and as such contains long term couplings, so that a perturbation might have effects over many cycles, which is not consistent with the hypothesis we here make of a relatively rapid return to the limit cycle. Furthermore, for more complex models, PRCs are analytical only in the linear regime, while our model is analytical for all perturbations. That said, we agree that other types of oscillators can be associated with constant sign PRCs, and we have given more details in this part, in particular we better emphasize the Class I vs Class II oscillators as a way to broaden our discussion on PRC, and emphasize the “infinite period” bifurcation category which is more intuitive and further includes saddle node homoclinic bifurcations.

__# 5 __The work demonstrates coarse graining of complex systems.

This conclusion is correct, but coarse graining theory-driven analysis and control of dynamical systems has been established for many years. What is new here is that it is applied specifically to the in vitro culture system of the mouse segmentation clock.

We agree it is new to successfully apply coarse-graining analysis and, importantly, control, to the in vitro culture system of the mouse segmentation clock. We also agree that such an approach has been pioneered and established for many years, especially in (theoretical) physics, but indeed, the key question is whether and how this can be applied to complex biological systems. Insights coming from theoretical considerations on idealized physical systems might not necessarily apply to biology, as already pointed out by Winfree.

There are still very few examples in biology with coarse graining similar to what we do here. We think there is immense value in demonstrating that quantitative insights, and control of the biological systems, can be obtained without precise knowledge of molecular details, which is still counter-intuitive to many biologists. In this sense, we think our report will be of interest to both colleagues within the field of the segmentation clock and also to anyone interested to in the question, how theory and physics guided approaches can enable novel insight into biological complexity.

Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

Following on the points above, each of these needs to be corrected or re-done, and/or the conclusions need to be modified accordingly.

We have modified the manuscript in response to all those points.

# 6 Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. If the authors wish to make the strong claim of determining a true PRC, Dirac delta-like perturbation needs to be applied, or approximated by short time duration pulses compared to the intrinsic period.

Please refer to our response to point #1 and #3..

# 7 *Are the suggested experiments realistic in terms of time and resources? It would help if you could *

add an estimated cost and time investment for substantial experiments.

It's not clear to this reviewer if it is feasible to deliver a very short pulse and record a response. But this may not be relevant, see above.

Please refer to our response to point #1 and #3 .

Are the data and the methods presented in such a way that they can be reproduced?

Yes.

Are the experiments adequately replicated and statistical analysis adequate?

Yes.

Minor comments:

Specific experimental issues that are easily addressable.

No issues.

Are prior studies referenced appropriately?

Yes.

# 8 Are the text and figures clear and accurate?

Figure 1D illustrates how a PRC should be obtained, but doesn't show the experimental protocol applied in the paper.

Figure 1D is a general introduction on the phase description of oscillators and phase response. It demonstrates how a perturbation can change the phase and is not supposed to represent the experimental protocol. We describe how data are analyzed and how phases are extracted in Supplementary Note 1.

__# 9 __In Figure 5B, 10 uM DAPT, the traces are already synchronized before the pulse train starts,

which makes the subsequent behavior difficult to interpret.

It appears here that by chance, the samples were already almost synchronized. We notice however that the establishment of a stable rhythm with the pulses (which here is not a multiple of the natural period) supports entrainment, and is already evident when looking at the timeseries with respect to the perturbation. The temporal evolution of the instantaneous period further confirms this, showing a change in period close to ½ zeitgeber period (which is very different from the natural period of ~140 mins). This also relates to point #35, in reply to both comments we have further expanded this figure to better show the 2:1 entrainment, adding statistics on the measured period and period evolution for a zeitgeber period of 300 mins.

# 10 Do you have suggestions that would help the authors improve the presentation of their data and Conclusions? The text includes several paragraphs reviewing broad principles of coarse graining and making general conclusions. This is confusing, because, as mentioned above, there is no new general advance in this paper. The interesting contributions here are specific to the applications to the segmentation clock, and the text should be focused on this aspect.

As commented above for #3 , we respectfully disagree that there is no “new general advance” in this paper. It is far from obvious that a complex ensemble of coupled oscillators implicated in embryonic development would be amenable to such coarse-graining theory. Of note, we still do not have a full understanding of neither the core oscillators in individual cells, nor what slows these down and eventually stops the oscillations, and multiple recent works suggest that both phenomena are under transient nonlinear control (e.g. our own work in Lauschke 2013). It is remarkable that despite this lack of detailed mechanistic insight, general entrainment theory can be applied to the segmentation process at the tissue level. We further show that classical entrainment theory alone is not sufficient to account for the experimental findings. Specifically, we need to account for a period change that we interpret as an internal feedback, an insight that would be impossible without our coarse-graining approach. While the results might of course be specific to the segmentation process, we think our approach motivated by coarse-graining theory and leading to new insights into the process is of general interest. We tried to make these points explicit in our conclusion.

Reviewer #1 (Significance (Required)):

Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

Description of the complex mouse segmentation clock in terms of a simple model and its PRC is an interesting, original and non-trivial result. The proposal that the segmentation clock is close to a SNIC bifurcation provides a consistent dynamical explanation of slowing behavior that has been recognized for some time, but not fully understood. This proposal also raises a hypothesis about the behavior of the underlying molecular regulatory networks, which may be tested in the future. The increase or decrease of the intrinsic period due to the zeitgeber period is not expected from theory, pointing to structures in internal biochemical feedback loops, an idea which again may be tested in the future. Also surprising from a theoretical perspective, the spatial gradient of period in the system persisted after entrainment. Although the categorization of the generic behavior is interesting, by its nature there is little from this that might give a typical developmental biologist any conclusions about pathways or molecules. The successes and limits of the theoretical description do nevertheless focus future attention on interesting behaviors.

# 11 Place the work in the context of the existing literature (provide references, where appropriate).

Such an analysis of the segmentation clock is based strongly on the experimental system and results in Sonnen et al., 2018, and goes well beyond it in terms of the dynamical analysis. It provisionally categorizes the mouse segmentation clock as a Class I excitable system, allowing its dynamics at a coarse grained level to be compared to other oscillatory systems. In this aspect of simplification, it is similar to approach of Riedel-Kruse et al., 2007 who used a mean-field model of oscillator coupling to explain the synchrony dynamics observed in the zebrafish segmentation clock in response to blockade of coupling pathways, thereby allowing a high-level comparison to other synchronizing systems.

It is interesting the reviewer sees similarities with the work of Riedel-Kruse et al, which uses a mean-field variable Z that corresponds to a classical approach, as described in Pikovsky’s textbook, to quantify synchronization of oscillators. In our view, while of course we work in the same context of coupled oscillators in the PSM, our approach based on perturbing and monitoring the system’s PRC in real-time provides a novel strategy to gain insight. This is evidenced by the fact that our quantifications of synchronization and insight into the PRC is the basis to exert precise control of the pace and rhythm of segmentation.

State what audience might be interested in and influenced by the reported findings.

Developmental biologists, biophysicists

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.

Developmental biology, somitogenesis, dynamical systems theory, biophysics, cell signaling

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

Summary: This is a beautifully elegant study that tests how previously published theoretical predictions about entraining nonlinear oscillators applies to a biological oscillator, the segmentation clock. The authors use a combination of state of the art experimental techniques, signal processing and analytical theory to reach a series of interesting and novel conclusions.

They show that the segmentation clock period can be entrained through Notch inhibitor (DAPT) pulses acting as an external clock (referred to as zeitgeber) using a previously developed and sophisticated microfluidic perfusion system. Pulsing DAPT every 120 to 180min can change the internal clock period while entrainment beyond this range leads to higher order coupling to the zeitgeber period, i.e. entrainment of every other pulse. They then perform entrainment experiments where the concentration of DAPT is changed to elicit a change in the strength of interaction between the internal clock and the external stimulus (referred to as zeitgeber strength); interestingly at low strength response to entrainment is more variable leading to entrainment occurring in some samples while others remain unaffected (Figure 4A); overall, higher concentration leads to faster entrainment (Figure 4C). The experimental data is then analysed using stroboscopic maps to reveal that a stable entrainment phase shift is achieved between the internal clock and the external zeitgeber. Phase response curve (PRC) analysis indicates that the system response is not sinusoidal but predominantly characterised by negative PRC, a behaviour consistent with saddle-node on invariant cycle (SNIC); it also reveals that the intrinsic period changes in a non-linear way and that this effect is reversible when external stimulation stops. Finally, a theoretical model is proposed to represent the segmentation clock as a dynamical system; this is based upon Radial Isochron Cycle with Acceleration (ERICA), an extension motivated by the PRC analysis results which are incompatible with a Radial Isochron Cycle (RIC); this model has predictive capability and could be used to design new control strategies for entrainment of the segmentation clock.

This study makes a series of key conclusions which are of particular importance in understanding the dynamic response of a biological oscillators. Firstly, given it's the characteristics of the dynamic response to entrainment, the segmentation clock is likely close to a SNIC bifurcation and this can explain the tendency for relaxation of the period over time. Secondly, the clock period was changed in a non-linear way in the direction of the zeitgeber period, a finding which is interpreted to indicate the presence of feedback of the segmentation clock onto itself, potentially via Wnt. This makes an excellent prediction that if tested experimentally would greatly improve the impact of the study. It is also noted that the entrainment of the segmentation clock does not abolish spatial periodicity and phase wave emergence suggesting that single cell oscillators can adjust to periodic perturbation while maintaining emergent properties. This is also a significant result that would need to be followed up with experiments and computation however would be best suited to a separate study.

Major comments:

__# 12 __The coarse graining is a major point that would need to be clarified since the rest of the analysis

and theoretical modelling in the paper flow from this. Firstly, the interpretation of the schematic in Figure 1A on experimental data collection is not immediately obvious to the reader, lacks a clear flow between the different panels or steps (which could be numbered for example) and does not have a legend to indicate the different colour mapping.

We are grateful to the reviewer for this comment. We have implemented in Figure 1A all the changes suggested by the reviewer: we numbered the different steps and have added a colour mapping. In addition we have rephrased the caption of Fig 1A to better connect the experimental steps.

__# 13 __Secondly, Figure 2A which explicitly addresses coarse graining is not clear enough. Is the

message here that by excluding the inner parts of the sample with a radial ROI, a similar dynamic response is observed over time?

Yes, indeed this is the point and we have adjusted the figure and text to explain this better. Our goal is to focus on the quantification of segmentation pace and rhythm. This is best captured by reporters such as LuVeLu, which has maximum intensity in regions where segment forms, and which dynamics is known to be strongly correlated to segmentation (Aulehla et al., 2007; Lauschke and Tsiairis et al., 20132). The global ROI is thus expected to precisely capture these segmentation and clock dynamics and we have now included more validation data and have also edited the text to make this very important point clearer:

“To perform a systematic analysis of entrainment dynamics, we first introduced a single oscillator description of the segmentation clock. We used the segmentation clock reporter LuVeLu, which shows highest signal levels in regions where segments form \cite{Aulehla_A_2007}. Hence, we reasoned that a global ROI quantification, averaging LuVeLu intensities over the entire sample, should faithfully report on the segmentation rate and rhythm, essentially quantifying 'wave arrival' and segment formation in the periphery of the sample.”

Figure 2A indeed shows that the dynamics (from the timeseries) is very similar when considering the entire field of view (global ROI) or when considering only the periphery of the 2D-assay (excluding central regions). We modified Figure 2A to clarify this point by indicating each measurement as either global ROI or global ROI minus the diameter of the excluded circular region (e.g. global ROI - 50px). We also emphasized in the caption that timeseries are obtained using global ROI, unless otherwise specified. We included a link (https://youtu.be/fRHsHYU_H2Q) in the caption to a movie of 2D-assay subjected to periodic pulses of DAPT (or DMSO) and corresponding timeseries from global ROI.

Since the inner part of the sample corresponds to the posterior side how do we interpret similarities and differences between signals with different ROIs?

As stated above, the global ROI measurements essentially capture the signal at the periphery where segments form and faithfully mirrors segmentation rate and rhythm. We have now included a comparison to the center ROI, also in response to reviewer’s comments, see our response #34.

The result shows that the period and PRC in the center matches the one found in the periphery, i.e. global ROI. We have shown previously that center and periphery differ in their oscillation phase by 2pi, i.e., one full cycle (Lauschke et al., 2013). We interpret these findings as confirmation of our analysis strategy, i.e. the global ROI allows a very reproducible, unbiased quantification that reports on segmentation clock and period.

__# 14 __A quantitative analysis of essential coarse-grained properties such as period and amplitude

should be performed for different ROIs and across multiple samples. As this effectively masks any spatial differences, limitations of this approach should be clearly stated in the Discussion. For example in lines 466-470 where it is difficult to interpret the slowing down tendency and relate back to single cell level.

As outlined in our response to comment #13 and also #34, we chose an analysis that allows to determine the segmentation pace and rhythm, i.e. segment formation, which is well captured by LuVeLu signal and a global ROI analysis. We agree that a spatially resolved analysis of dynamic behaviour is important (and indeed a gradient of amplitude might be relevant in such context), but we think this is beyond the scope of the current study focused on the system level segmentation clock behaviour. We have revised the discussion as suggested by the reviewer to make this point approach and the need for future studies clearer.

__# 15 __The functional characterisation of the sample using LFNG, AXIN2 and MESP2 is unclear. The

images included in Figure 2D representing expression observed when tissue explants are grown within the microfluidic chip are difficult to interpret and would require a more detailed description of anterior-posterior, pillars etc; it is also difficult to view the bright-field since it is presented as a merged image.

It is particularly difficult to see the somite boundaries for the same reason. In lines 113-117 the authors state that the global oscillation period matches the periodic boundary formation. How do we reach this conclusion from these images? What is the variability between samples?

If these two issues would be addressed it would increase confidence in the coarse graining argument and thus would strengthen the importance of the findings in the study.

We thank the reviewer for this feedback, and we have added more quantifications to address this point directly in the modified Figure 2. Importantly, we added the quantification of the rate of segmentation in multiple samples based on segment boundary formation (new Figure 2D) and compared this to the global ROI quantifications using the reporter lines LuVeLu. This data provides clear evidence that the quantification of global ROI reporter intensities closely matches the rate of morphological segment boundary formation. In addition, we show that segment formation and also Wnt-signaling oscillations (Axin2-Achilles) and the segmentation marker Mesp2 (Mesp2-GFP) are all entrained to the zeitgeber period. We have also revised the text to clarify this important validation of our quantitative approach.

In addition, we provide, in the revised Figure Suppl. 2, details of entrained samples, focusing on the segmenting regions. The brightfield and reporter channels were separated, emphasizing the segment boundaries and the expression pattern of the reporters. For ease of visualization, these samples were also re-oriented so that the tissue periphery (corresponding to anterior PSM) is at the top while the tissue center (corresponding to the posterior PSM) is at the bottom. This now additionally better shows the localization of the different reporters with respect to the segment boundary. We also included supplementary movies showing timelapse of samples expressing either Axin2-GSAGS-Achilles or Mesp2-GFP that were subjected to periodic DAPT pulses, with their respective controls.

Several minor points could be addressed to improve the manuscript and are listed below:

# 16 Figure 1 A the colormap and axes for the oscillatory traces should be defined

We thank the reviewer, and we have modified the figure accordingly (related to point # 12). A colormap and axes for the illustrated timeseries are now included.

# 17 Strength of zeitgeber is not defined and there is no analytical expression provided; how does it

relate to DAPT concentration? Is the fact that low DAPT concentration corresponds to weak strength expected or is it a result?

Zeitgeber strength generally refers to the magnitude of the perturbation periodically applied to an oscillator. With DAPT pulses, our expectation was that both the duration of the pulse and the drug concentration could influence the strength. Practically, the pulse duration was kept constant for all experiments and the concentration was varied. We thus expected that DAPT concentration would indeed be correlated to zeitgeber strength. We have discussed multiple evidence supporting this assumption in the main text, and this is indeed a result. In particular, as explained in the section “The pace of segmentation clock can be locked to a wide range of entrainment periods”, higher DAPT concentration gives rise to faster and better entrainment, as expected from classical theory. In the context of Arnold tongue, weaker zeitgeber strength corresponds to narrower entrainment region, which is experimentally observed (Fig 8F, showing regions where the clock is entrained).

From a modelling standpoint, Zeitgeber strength corresponds to parameter A which is the amplitude of the perturbation. Possible zeitgeber strength was inferred from the model by matching the experimental entrainment phase with that obtained from the model isophases. As explained in Supplementary Note 2, we tested four concentrations of DAPT (0.5, 1, 2, and 3 uM) respectively corresponding to A values of 0.13, 0.31,0.43, 0.55. As we can see, those A values are not linear in DAPT concentrations, which is expected since multiple effects (such as saturation) can occur.

__# 18 __In some figures it looks like the amplitude of oscillations may change with DAPT concentration

and hence zeitgeber strength? Is this expected?

We have not systematically analyzed the amplitude effect and have, intentionally, focused on the period and phase readout as most robust and faithful parameters to be quantified. Regarding the amplitude of LuVeLu reporter, we are cautious given that it is influenced, potentially, by the (artificial) degradation system that we included in LuVeLu, i.e. a PEST domain. This effect concerns the amplitude, but not the phase and period, explaining our strategy.

That said, we agree with the referee that DAPT concentrations might change the amplitude of oscillations. Such change could even play a role in the change of intrinsic period (in fact a similar mechanism drives overdrive suppression for cardiac oscillators, Kunysz et al., 1995). But since the change of period can be more easily measured and inferred, we prefer to directly model it instead of introducing a new hypothesis on amplitude/period coupling, at least for this first study of entrainment.

__# 19 __Figure 2A including the black area creates confusion and it is unclear which ROI is used in the

rest of the study; consider moving this to a supplementary figure perhaps

We thank the reviewer for this feedback (related to point #13), and we have modified the figure accordingly. As we responded to point # 13: We modified Figure 2A, by indicating each measurement as either global ROI or global ROI minus the diameter of the excluded circular region (e.g. global ROI - 50px). We also emphasized in the caption that timeseries are obtained using global ROI, unless otherwise specified.

__# 20 __What type of detrending is used in Figure 2 and throughout (include info in the figure legend)?

We used sinc-filter detrending, described and validated in detail previously (Mönke et al., 2020), as specified in Supplementary Note 1: Materials and methods > H. Data analysis > Monitoring period-locking and phase-locking: In this workflow, timeseries was first detrended using a sinc filter and then subjected to continuous wavelet transform. We thank the reviewer for pointing out that this detail is lacking in the figure captions, and we have modified the captions accordingly.

__# 21 __Figure 2D merged images are difficult to read/interpret (see major comments)

We thank the reviewer for this comment, and we have modified the figure accordingly (please see response to related point #15).

__# 22 __Kuramoto order parameter is used to quantify the level of synchrony across the different samples

however it is not defined in the text. Is it also possible to assess variability in each sample? For example how quickly does entrained occur in each sample? How faithfully the peaks of expression beyond 80min (to exclude initial unsynchronised state) match with zeitgeber time? This would help make the point that weak strength leads to a more variable response which is an interesting finding.

We have now added a mathematical definition of the Kuramoto parameter in Supplementary Note 1.

A high order parameter corresponds to coherence between samples, as also elaborated in respective figure captions (e.g. in the caption for polar plots in Figure 4D).

In terms of variability in response to entrainment, we thank the reviewer for the comments, which has prompted us to perform an additional analysis, now included as Figure S13 in the Supplement.

Briefly, we represent below figures showing how different samples get synchronized with the zeitgeber. To do this, we first represent the zeitgeber signal as a continuous uniformly increasing phase (“zeitgeber time”) with period : . The initial condition for is chosen so that the zeitgeber phase at the moment of last pulse is matching the experimental entrainment phase for each . We plot for each sample (dotted lines) and the zeitgeber phase (magenta line). To quantify how well each sample is following the zeitgeber time, we compute the Kuramoto parameter: . By the end of experiment most samples reach , indicating entrainment. Most samples need zeitgeber cycles to become entrained. For min the entrainment takes much longer (edge of the Arnold tongue). For min there is much variability, which can be explained by the horizontal region in the PRC around the entrainment phase. As suggested by the referee, synchronization is faster for higher DAPT concentration. So those dynamics are indeed consistent with the expectation from classical PRC theory.

# 23 Do samples change period to Tzeit in similar ways - i.e. patterns over time. It looks like the

kuramoto order parameter and period drop initially - why?

We do not have a direct answer as to why the Kuramoto first order parameter and the period drop for the condition the reviewer specified. It has to be noted though that because of how wavelet analysis is done (cross-correlation of the timeseries with wavelets), the period and phase determination at the boundaries of the time series are less reliable (edge effects, see Mönke et al., 2020). Because of this, we should take caution when considering data to and from the first and last pulses, respectively. This was explicitly stated in the generation of stroboscopic maps: “As wavelets only partially overlap the signal at the edges of the timeseries, resulting in deviations from true phase values (Mönke et al., 2020), the first and last pulse pairs were not considered in the generation of stroboscopic maps.”

# 24 In Figure 4C why is the Kuramoto order parameter already higher in the 2uM DAPT conditions at

the start of the experiment?

Samples can, by chance, start synchronously and this results in a high Kuramoto first order parameter. Because of this likelihood, it is thus important to interpret the entrainment behaviour of multiple samples using various readouts, in addition to a high Kuramoto first order parameter. We investigated entrainment of the samples based on several measures: multiple samples remaining (or becoming more) synchronous (because each sample actively synchronizes with the zeitgeber), period-locking (where the pace of the samples match the pace of the zeitgeber, which can be distinct from natural pace), and phase-locking (where there is an establishment of a stable phase relationship between the samples and the zeitgeber).

# 25 Figure 3C and Figure S2 require statistical testing between CTRL and DAPT in each condition

p-values were calculated for the specified conditions and were added in the caption of the figures. These values are enumerated here:

• Figure 3C
• 170-min 2uM DAPT (vs DMSO control): p
• Figure S2
• 120-min 2uM DAPT (vs DMSO control): p = 0.064
• 130-min 2uM DAPT (vs DMSO control): p = 0.003
• 140-min 2uM DAPT (vs DMSO control): p = 0.272
• 150-min 2uM DAPT (vs DMSO control): p = 0.001
• 160-min 2uM DAPT (vs DMSO control): p To calculate p-values, two-tailed test for absolute difference between medians was done via a randomization method (Goedhart, 2019). This confirms that the period of samples subjected to pulses of DAPT is not equal to the controls, except for the 140-min condition (where the zeitgeber period is equal to the natural period, i.e. 140 mins).

# 26 Figure 3A gray shaded area not clearly visible on the graph

We have decided to remove the interquartile range (IQR) in the specified figure as it does not serve a crucial purpose in this case. By removing it in Figure 3A, the timeseries of individual samples are now clearer.

# 27 Figure 6C colour maping of time progression is not clearly visible on the graph; the interpretation

of this observation is unclear in the text and the figure

We agree that the low quality of the image is unfortunate, and it seems that our file was greatly compressed upon submission. We have checked the proper quality of figures in the resubmitted version of the manuscript.

Regarding the interpretation of Figure 6C, we conclude that in our experiments the entrainment phase is an attractor or stable fixed point, in line with theory (Granada and Herzel, 2009; Granada et al., 2009),. We had elaborated this in the text (lines 248-252 of the submitted version of the manuscript): at the same zeitgeber strength and zeitgeber period, faster (or slower) convergence towards this fixed point (i.e. entrainment) was achieved when the initial phase of the endogenous oscillation (φinit) was closer or farther to φent.

# 28 Figure 7A circular spread not clearly visible on the graph

Similar to point #27, we have provided a high resolution graph for the re-submission and hopefully resolved this issue.

# 29 Figure S7A difficult to see the difference between colours

See point #28.

# 30 Is it possible to compare the PRC and the plots of period over time during entrainment? The PRC

is mainly negative (Fig 8A1,A2), in my understanding this means a delay, however the periods seem to decrease over time before entraining to the Tzeit (Fig 3B). Is this reflective of a decrease in Kuramoto parameter and potential de-synchronisation of single cells before re-synchronisation at Tzeit?

To address this question, we now plot the Phase response with colors indicating pulse number in new Supplementary Figure S13. While capturing the entire PRC as a function of time would require many more experiments (in particular to sample the phases far from entrainment phase), we still clearly see that the PRCs appear to translate vertically as the oscillator is being entrained, i.e. the latter time points are shifted up (down) for T_zeit = 120 (170) min, respectively.

# 31 Fig 8A What is the importance/meaning of the PRC being similar shape between different

entrainment periods? Does this reflect that the underlying gene network is the same?

If one single gene network is responsible for oscillations, we expect from dynamical systems theory that the PRC are not only of similar shape but actually the same, independent of the entrainment period. What is surprising is that the PRC for different entrainment periods do not overlap, and the simplest explanation for this is that the intrinsic period changes with entrainment, all things being kept equal (including the underlying gene networks). This relates to the previous point since we indeed observe that the PRC “translates” vertically with the pulse number for longer periods. The change of period might be due to a long-term regulation as detailed in the discussion.

# 32 The spatial period gradient and wave propagation under DAPT (Figure S8) should be included in

the results and not just the discussion.

We fully agree with the reviewer that both the establishment and the maintenance of a spatial phase gradient is of great interest. However, many more experiments would be required to fully quantify and understand the processes at play here, which we believe to be out of the scope of the current manuscript. To keep the focus of the paper on the global segmentation clock itself, we prefer to keep this figure in Supplement.

Reviewer #2 (Significance (Required)):

We currently do not have a detailed understanding of how biological oscillators integrate local signals from their neighbours as well global external signals to give rise to complex patterning that is important for embryonic development. Main bottlenecks that hinder our understanding are lack of real-time endogenous dynamic response together with known global inputs as well as comprehensive models that can explain emergent behaviour in a variety of tissues.

This study goes a long way in addressing these bottlenecks in the embryonic tissue responsible for somite formation, a dynamical and oscillatory system also known as the segmentation clock. Firstly, they rely on a state-of-the-art previously developed system to entrain endogenous response in live tissue explants using precise microfluidic control. They test the complete range of exogenous perturbation periods and use an existing live reporter (LuVeLu) to monitor endogenous response. They also identify higher order coupling relationships whereby every other LuVeLu peak is entrained through external stimulation.

As the stimulation system does not control but rather perturb the endogenous response, the observations from LuVeLu provide a unique opportunity in understanding input-output relationships and thus describing the dynamic response of the segmentation clock. Authors propose to study dynamic behaviour of the clock using coarse-graining and focus on describing the overall response over time while amalgamating spatial information. Appropriate coarse-graining is an important strategy in addressing complex problems and is widely used. They use sophisticated methodology such as phase response curves and Arnold tongue mapping to make several important observations. For example the nonlinear shortening and elongation of the period in response to stimulation is particularly interesting since this may indicates a feedback of the clock onto itself potentially via Wnt. Another key observation is that the spatial periodicity and phase wave activity persists in the perturbed conditions suggesting that individual single cell oscillators can adjust their behaviour to external input while retaining coordination with their neighbours. Finally, the authors go on to construct a general dynamical model of the segmentation clock and use this to conclude that the intrinsic period of the oscillator is altered and that the oscillator can be considered excitable.

This work sheds light onto mechanisms of coordination of Notch activity in assemblies of cells observed in living tissue, an area of research that is important not only for somitogenesis but also for understanding gene expression patterning in many other tissues where Notch plays a critical role, for example in the development of the neural system and organs. As a study of a real-world nonlinear oscillator this work is directly of interest to theoreticians and synthetic biology experts interested in understanding complex patterning and emergence.

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

In this manuscript, authors studied the system-level responses of the somite segmentation clock by the coarse-grained theoretical-experimental approach, applying the theory of entrainment to understanding the phase responses of mouse pre-somitic mesoderm (PSM) tissues in the presence of periodic perturbation of Notch inhibitor DAPT generated by micro-fluidics technique. It was demonstrated that the segmentation clock is responsive to diverse range of the perturbation-periods from 120 to 180 min, can be period- and phase-locked, and the efficiency is dependent of the DAPT concentration (input-strength). The authors also observed two cycles of the segmentation-clock ticking in single cycles of 300 or 350 min period-perturbation, suggesting that higher order (2:1 mode) entrainment. They also applied stroboscopic maps to analysis and found that entrainment-phases are dependent of period of DAPT pulses, which is recapitulating theoretical predictions. The estimation of the phase response curve (PRC) of the segmentation clock revealed that the inferred PRC is an asymmetrical and mainly negative function, which represents characteristic features in oscillators that emerge after saddle-node on invariant cycle (SNIC) bifurcation. These results also indicated that the the segmentation clock changed the intrinsic period during entrainment.

Major comments:

# 33 I have major concerns about the relevance of the global time-series analysis proposed in Fig.2

and conclusion about the changes of the intrinsic period during entrainment. The validity of the global time-series analysis should be carefully analyzed, because it could bring artifacts in estimated values of the intrinsic period. The authors concluded (page 3, line 172) that the period calculated by the global analysis represents similar values with the rate of segment formation, but there is no data about the quantification of the periods of segmentation, such as the frequency of Mesp2 reporter expression.

We thank the reviewer for this feedback. We have now added the quantification of the period of segment formation (new Figure 2E) and show its strong correspondence to the dynamics of reporters used (Lfng, Axin2, and Mesp2). Please see also our response to point #15 with additional comments regarding the validation of the global time-series analysis.

# 34 Another related issue is the presence of spatial period gradient as mentioned (page 13, line 524).

One possible approach to circumvent this issue would be "local" time-series analysis; for instance, just focusing on the "putative posterior" regions that are close to source-positions of waves. Authors can re-compute and estimate PRCs by using such a method.

We thank the reviewer for this suggestion and have accordingly now included the analysis of a localized ROI at the center (center ROI) of the 2D-assays (new Figures S5-S6). We also computed the PRC from center ROIs as shown below. We note strong correspondence between the global ROI and the center ROI.

# 35 I have another major concern about the evidence of higher order entrainment shown in Fig.5. If

the 1:2 entrainment is successful, we can expect that the values of observed period is close to the half of the period of pulses; However, the period shown in Fig.5B looks like 185 min longer than the half of 350 min. Is this gap due to the temporal accuracy of time-lapse movies?

We do not think the discrepancy comes from a problem of temporal accuracy as the temporal accuracy is the same for all movies and there is no reason why there would be a specific issue for this set of experiments. In addition, we have re-analyzed the data to calculate the period from the stroboscopic maps. Mathematically speaking, we take the stroboscopic map as (see PDF) and use this to estimate the period of oscillation in entrained samples , in particular inverting the formula for 1:2 entrainment we have : see PDF.

The advantage of this method is that it gives a more ``instantaneous” estimation of the period.

The results are as follows:

350 10uM: 187 +- 8 min (average across entrained samples from the last zeitgeber period)

350 5uM: 193 +- 13 min (average across entrained samples from the last zeitgeber period)

300 2uM: 148 +- 8 min (averaged across entrained samples and from two last periods)

This additional analysis is in agreement with the wavelet analysis.

The reviewer is right that for 350 minutes, entrained samples show an observed period that is higher than expected, also based on this new additional analysis. The reason for this is not known. One explanation is the relatively short observation time, especially considering for pulses separated by as much as 350-minutes, i.e. only 3 pulses are applied. [We notice that for 300 minutes pulses, the period converges to 150 mins between the 3rd and the 4th pulse]. We have adjusted the text in the results section to reflect that for 350min entrained samples, the observed period ‘approaches’ the predicted value, while for 300min entrained samples, the observed period is very close to it, i.e. 147mins In addition, we comment that the phase distribution narrows with time, another indication supporting higher order entrainment.

# 36 Also, authors showed the period evolution towards 1:2 locking with just one condition (350 min).

Authors can show the data for multiple conditions as in Fig. 3D, at least for 300 min and 325 min pulses and add the data about final entrained period with statistic analysis that supports the difference between the entrained period and the natural period (140 min).

We thank the reviewer for this feedback and have modified the figure accordingly. In particular, in Figure 5A, we have added the period evolution plot for samples subjected to 300-min periodic pulses of 2uM DAPT (or DMSO for control). Additionally, we have added Figure 5D, which plots the average period in the 300-min and 350-min conditions. We summarize the median average period here with computed p-values:

• 300-min pulses of 2uM DAPT (or DMSO for control): p-value = 0.191
• CTRL: 130.39 mins

• DAPT: 146.45 mins

• 350-min pulses of 5uM DAPT (or DMSO for control): p-value = 0.049

• CTRL: 127 mins

• DAPT: 174.86 mins

• 350-min pulses of 10uM DAPT (or DMSO for control): p-value = 0.016

• CTRL: 142.82 mins
• DAPT: 185.12 mins

Minor comments:

# 37 The authors can draw vertical lines indicating the T_zeit in Fig.3B, Fig.4B and Fig.5B in order to

help comparisons between T_zeit and patterns of period (solid lines).

We thank the reviewer for this comment. We have accordingly added a horizontal line indicating Tzeit in Figures 3B, 4B, S4A, and S5A (figure panel numbers based on the submitted version of the manuscript). We similarly added a horizontal line indicating 0.5Tzeit in the period evolution plots of 300-min and 350-min conditions in Figures 5A and 5B, respectively.

# 38 In Fig.5A, the authors can show period evolution in the case of 300 min DAPT-pulses as shown

in Fig.5B.

We thank the reviewer for this feedback (related to point #36), and we have modified the figure accordingly.

# 39 In Fig.6B DAPT panel, the authors can draw the points of phi_ent as shown in Fig.7A.

We thank the reviewer for this comment, and we have modified the figure accordingly.

# 40 In Fig. 8F, authors can put the information about DAPT concentration at the right y-axis.

This is a similar comment as point #17, see above. In brief, we do not know the precise relation between the strength of the perturbation in our model and DAPT concentration, zeitgeber strength was inferred from the model by matching the experimental entrainment phase with that obtained from the model isophases.

# 41 In Fig. 8G, the PRC in the panel "170 mins" does not have any fixed point (cross sections with

horizontal lines of "0" phase response). If entrainment is successful, there should be stable and unstable fixed points, but those are absent, although 170 min pulses succeeded in the entrainment as shown in Fig.3D. Authors can explain where the fixed points are.

The fixed points are indeed defined by the intersection with a horizontal line, but not with the ‘0’ line. They are found where the phase response compensates for the detuning/period mismatch, not at ‘0’ phase response. (See PDF for more details).

Note however on Fig 8G that we further observe a vertical shift of the PRC, which prompted us to propose a change of the intrinsic period with (as explained in the text when we introduce Figs 8A1-2).

Another way to visualize fixed points is offered in Fig 16 D-E, where we plot the inferred corrected PTC and the stroboscopic maps: there, fixed points correspond to intersections with the diagonal.

Reviewer #3 (Significance (Required)):

Although the phase-analysis has been widely applied to various biological systems, such as circadian clocks, cardiac tissues and neurons, this paper represents the first detailed experimental analysis of the segmentation clock based on the theory of phase dynamics. The major results are inline with theoretical predictions, whereas the suggestion about the SNIC bifurcation is attractive not only to the theoretical researchers but also to the experimental biologists; it has been believed that the segmentation clock consists of negative-feedback oscillator that emerge by Hopf bifurcation, whereas this paper proposes another possibility of the molecular network structure for the clockwork. This issue is related to recently proposed hypothesis about the excitable system in the segmentation clock based on the Yap signaling (Hubaud et al. Cell 171, 668 (2017)). However, unfortunately, discussion about detailed molecular networks are not abundant.

# 42 Thus, maybe the main readers are computational biologists and systems biologists.

We thank the reviewer for his/her significance comment. We have added comments on the bifurcation structure of the segmentation clock and on excitable systems in the discussion. While our focus is on coarse-graining so that we do not and cannot infer precise molecular details, we can still infer some properties of the underlying networks. In particular we now cite several papers explaining how systems with tunable periods/excitable are indicative of the interplay between positive and negative feedbacks. We think those considerations are of interest to a broad range of biologists interested in connecting experiments to theory.

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Referee #2

Evidence, reproducibility and clarity

Summary:

This is a beautifully elegant study that tests how previously published theoretical predictions about entraining nonlinear oscillators applies to a biological oscillator, the segmentation clock. The authors use a combination of state of the art experimental techniques, signal processing and analytical theory to reach a series of interesting and novel conclusions.

They show that the segmentation clock period can be entrained through Notch inhibitor (DAPT) pulses acting as an external clock (referred to as zeitgeber) using a previously developed and sophisticated microfluidic perfusion system. Pulsing DAPT every 120 to 180min can change the internal clock period while entrainment beyond this range leads to higher order coupling to the zeitgeber period, i.e. entrainment of every other pulse. They then perform entrainment experiments where the concentration of DAPT is changed to elicit a change in the strength of interaction between the internal clock and the external stimulus (referred to as zeitgeber strength); interestingly at low strength response to entrainment is more variable leading to entrainment occurring in some samples while others remain unaffected (Figure 4A); overall, higher concentration leads to faster entrainment (Figure 4C). The experimental data is then analysed using stroboscopic maps to reveal that a stable entrainment phase shift is achieved between the internal clock and the external zeitgeber. Phase response curve (PRC) analysis indicates that the system response is not sinusoidal but predominantly characterised by negative PRC, a behaviour consistent with saddle-node on invariant cycle (SNIC); it also reveals that the intrinsic period changes in a non-linear way and that this effect is reversible when external stimulation stops. Finally, a theoretical model is proposed to represent the segmentation clock as a dynamical system; this is based upon Radial Isochron Cycle with Acceleration (ERICA), an extension motivated by the PRC analysis results which are incompatible with a Radial Isochron Cycle (RIC); this model has predictive capability and could be used to design new control strategies for entrainment of the segmentation clock.

This study makes a series of key conclusions which are of particular importance in understanding the dynamic response of a biological oscillators. Firstly, given it's the characteristics of the dynamic response to entrainment, the segmentation clock is likely close to a SNIC bifurcation and this can explain the tendency for relaxation of the period over time. Secondly, the clock period was changed in a non-linear way in the direction of the zeitgeber period, a finding which is interpreted to indicate the presence of feedback of the segmentation clock onto itself, potentially via Wnt. This makes an excellent prediction that if tested experimentally would greatly improve the impact of the study. It is also noted that the entrainment of the segmentation clock does not abolish spatial periodicity and phase wave emergence suggesting that single cell oscillators can adjust to periodic perturbation while maintaining emergent properties. This is also a significant result that would need to be followed up with experiments and computation however would be best suited to a separate study.

Major comments:

The coarse graining is a major point that would need to be clarified since the rest of the analysis and theoretical modelling in the paper flow from this. Firstly, the interpretation of the schematic in Figure 1A on experimental data collection is not immediately obvious to the reader, lacks a clear flow between the different panels or steps (which could be numbered for example) and does not have a legend to indicate the different colour mapping. Secondly, Figure 2A which explicitly addresses coarse graining is not clear enough. Is the message here that by excluding the inner parts of the sample with a radial ROI, a similar dynamic response is observed over time? Since the inner part of the sample corresponds to the posterior side how do we interpret similarities and differences between signals with different ROIs? A quantitative analysis of essential coarse-grained properties such as period and amplitude should be performed for different ROIs and across multiple samples. As this effectively masks any spatial differences, limitations of this approach should be clearly stated in the Discussion. For example in lines 466-470 where it is difficult to interpret the slowing down tendency and relate back to single cell level.

The functional characterisation of the sample using LFNG, AXIN2 and MESP2 is unclear. The images included in Figure 2D representing expression observed when tissue explants are grown within the microfluidic chip are difficult to interpret and would require a more detailed description of anterior-posterior, pillars etc; it is also difficult to view the bright-field since it is presented as a merged image. It is particularly difficult to see the somite boundaries for the same reason. In lines 113-117 the authors state that the global oscillation period matches the periodic boundary formation. How do we reach this conclusion from these images? What is the variability between samples?

If these two issues would be addressed it would increase confidence in the coarse graining argument and thus would strengthen the importance of the findings in the study.

Several minor points could be addressed to improve the manuscript and are listed below: -Figure 1 A the colormap and axes for the oscillatory traces should be defined -strength of zeitgeber is not defined and there is no analytical expression provided; how does it relate to DAPT concentration? Is the fact that low DAPT concentration corresponds to weak strength expected or is it a result? - In some figures it looks like the amplitude of oscillations may change with DAPT concentration and hence zeitgeber strength? Is this expected? -Figure 2A including the black area creates confusion and it is unclear which ROI is used in the rest of the study; consider moving this to a supplementary figure perhaps -what type of detrending is used in Figure 2 and throughout (include info in the figure legend) -Figure 2D merged images are difficult to read/interpret (see major comments) -Kuramoto order parameter is used to quantify the level of synchrony across the different samples however it is not defined in the text. Is it also possible to assess variability in each sample? For example how quickly does entrained occur in each sample? How faithfully the peaks of expression beyond 80min (to exclude initial unsynchronised state) match with zeitgeber time? This would help make the point that weak strength leads to a more variable response which is an interesting finding. - Do samples change period to Tzeit in similar ways - i.e. patterns over time. It looks like the kuramoto order parameter and period drop initially - why? -In Figure 4C why is the Kuramoto order parameter already higher in the 2uM DAPT conditions at the start of the experiment? -Figure 3C and Figure S2 require statistical testing between CTRL and DAPT in each condition -Figure 3A gray shaded area not clearly visible on the graph -Figure 6C colour maping of time progression is not clearly visible on the graph; the interpretation of this observation is unclear in the text and the figure -Figure 7A circular spread not clearly visible on the graph -Figure S7A difficult to see the difference between colours -Is it possible to compare the PRC and the plots of period over time during entrainment? The PRC is mainly negative (Fig 8A1,A2), in my understanding this means a delay, however the periods seem to decrease over time before entraining to the Tzeit (Fig 3B). Is this reflective of a decrease in Kuramoto parameter and potential de-synchronisation of single cells before re-synchronisation at Tzeit? -Fig 8A What is the importance/meaning of the PRC being similar shape between different entrainment periods? Does this reflect that the underlying gene network is the same? -The spatial period gradient and wave propagation under DAPT (Figure S8) should be included in the results and not just the discussion.

Significance

We currently do not have a detailed understanding of how biological oscillators integrate local signals from their neighbours as well global external signals to give rise to complex patterning that is important for embryonic development. Main bottlenecks that hinder our understanding are lack of real-time endogenous dynamic response together with known global inputs as well as comprehensive models that can explain emergent behaviour in a variety of tissues.

This study goes a long way in addressing these bottlenecks in the embryonic tissue responsible for somite formation, a dynamical and oscillatory system also known as the segmentation clock. Firstly, they rely on a state-of-the-art previously developed system to entrain endogenous response in live tissue explants using precise microfluidic control. They test the complete range of exogenous perturbation periods and use an existing live reporter (LuVeLu) to monitor endogenous response. They also identify higher order coupling relationships whereby every other LuVeLu peak is entrained through external stimulation.

As the stimulation system does not control but rather perturb the endogenous response, the observations from LuVeLu provide a unique opportunity in understanding input-output relationships and thus describing the dynamic response of the segmentation clock. Authors propose to study dynamic behaviour of the clock using coarse-graining and focus on describing the overall response over time while amalgamating spatial information. Appropriate coarse-graining is an important strategy in addressing complex problems and is widely used. They use sophisticated methodology such as phase response curves and Arnold tongue mapping to make several important observations. For example the nonlinear shortening and elongation of the period in response to stimulation is particularly interesting since this may indicates a feedback of the clock onto itself potentially via Wnt. Another key observation is that the spatial periodicity and phase wave activity persists in the perturbed conditions suggesting that individual single cell oscillators can adjust their behaviour to external input while retaining coordination with their neighbours. Finally, the authors go on to construct a general dynamical model of the segmentation clock and use this to conclude that the intrinsic period of the oscillator is altered and that the oscillator can be considered excitable. This work sheds light onto mechanisms of coordination of Notch activity in assemblies of cells observed in living tissue, an area of research that is important not only for somitogenesis but also for understanding gene expression patterning in many other tissues where Notch plays a critical role, for example in the development of the neural system and organs. As a study of a real-world nonlinear oscillator this work is directly of interest to theoreticians and synthetic biology experts interested in understanding complex patterning and emergence.

What’s the point of sending the child Back to their family when the situation would remain same rather they live in foster care or with parents who are working 24/7 just to pay off foster care bills. It’s much more frustrating for a child to live in a family like that where parents don’t have time for them.

El amor es caprichoso, que surga con alguien no significa que ese alguien sea tu pareja perfecta, el amor surge con la minima sobresimpatia, provocado por una lluvia de quimicos que acrecentan lo real. No solo hay que buscar en una pareja que haya amor, sino que ha de haber complicidad, union, comodidad, confianza, risas, poder hablar de lo que sea, sumarte, apoyo...

It's interesting that just analyzing the musical or rhythmic modes and musical structure isn't enough to explain the huge response to Umm Kulthum's songs, that even very sophisticated musicians weren't as interested in that.

This deeply resonates with me. Love the classification of Type II fun.

Huge concept to understand_ ROAS 26% BUT just because it's not negative doesn't mean it's giving you a good return. Remember this for 26/100, 26%, or/and 0.26. So for every $1 you invest that means you only made $0.26 of income.

Never Ignore Margins!!!

I find the tendency of people to frame their thinking in terms of other people's involvement in the projects in question being to make money (as with GitHubbers "contributing"/participating by filing issues only because they're trying to help themselves on some blocker they ran into at work) pretty annoying.

The comments here boil down to, "You say it should be rewarding? Yeah, well, if you're getting paid for it, you shouldn't have any expectations that the the act itself will be rewarding."

I actually agree with this perspective. It's the basis of my comments in Nobody wants to work on infrastructure that "if you get an infusion of cash that leaves you with a source of funding for your project[...] then the absolute first thing you should start throwing money at is making sure all the boring stuff that [no] one wants to work". I just don't think it's particularly relevant to what Kartik is arguing for here.

Open source means that apps are home-cooked meals. Some people get paid to cook, but most people don't. Imagine, though, if the state of home cooking were such that it took far more than one hour's effort (say several days, or a week) before we could reap an appreciable reward—tonight's dinner—and that this were true, by and large, for almost every one of your meals. That's not the case with home cooking, fortunately, but that is what open source is like. The existence of professional chefs doesn't change that. There's still something wrong that could stand to be fixed.

No. Again, this is exactly what I don't want. (I don't mean as an author—e.g. burdened with hand-coding my own website—I mean as a person whose eyes will land on the pages that other people have authored with far more frequency than I do look at my own content.)

As I mentioned—not just in my earlier responses to this piece, but elsewhere—the constant hammering on about how much control personal homepages give the author over the final product is absolutely the wrong way to appeal to people. In the first place, it only appeals to probably about a tenth of as many people as the people advocating think it does. In the second place, the results can and usually are not good, cf MySpace.

The best thing about Facebook, Twitter, etc? Finally getting people to separate content from presentation. Hand-coded CSS doesn't get you there alone. That's basically a talisman (false shibboleth). Facebook content is in a database somewhere. The presentation gets layered on through subsequent transformations into different HTML+CSS views.

I don't think it is as easy as she makes it sound. As we learned in last week's article, there are some kids who don't even understand the order of taking pre-algebra before algebra simply because they don't have people in their lives to explain that to them. It may seem easy to her because it is a path a lot of people in her family have taken or that her family holds strong values to school so she knows more about it.

.

I find this to be true in my experience. I have worked with some professors who baulk at the idea of spending time creating or searching for a course banner image. There's other examples related to this, but I personally think that something as simple as finding or creating a course banner image can excite students. Or, if it's a corporate training hosted through Rise or Storyline, this may just be little visual elements and images that add a little something to the visual experience.

It's terrible how many awful, degrading names they had for people with disabilities. This just goes to show how they were almost never viewed as human beings who are capable of feeling emotions. It just raises the question for me of why they weren't more professional about it? Why were people in these professions giving such hurtful names as opposed to clinical terms? Language and word choice has such a huge impact on things and I feel as though things would have been different today if they were more professional in diagnosing people with disabilities. I've actually looked into the origin of the word "idiot" and it's been around since the 14th century. Therefore, that just shows us how they took that word and used it knowing its hurtful meaning.

Author Response

Reviewer #1 (Public Review):

This study provides data suggesting that tonic presynaptic a7 nicotinic receptor activity enhances corticostriatal input-mediated excitation of striatal medium spiny neurons; the data also suggest that tonic a4b2 nicotinic receptor activity on PV-fast spiking GABA interneurons inhibits striatal medium spiny neurons. These data advance our understanding about the complex cholinergic regulation of striatal neuronal circuits.

The presented data are generally clean and high quality; but there are some problems that require the authors' attention.

We thank the Reviewer for their insightful comments. We have addressed each point below with additional data and/or text. We believe these revisions have made the manuscript significantly stronger.

1. In this study, ADP is a key parameter manipulated by several pharmacological treatments. But it is not clearly defined. The authors indicate EPSP and ADP are distinct by stating "LED pulse of increasing intensity generates excitatory postsynaptic potentials (EPSPs), or an AP followed by an after depolarization (ADP)." But the data (e.g. Fig. 1B) indicates that much of the ADP is probably EPSP. Please clarify. If much of the ADP is indeed EPSP, how are the data interpretation and the overall conclusion affected?

We apologize for the oversight. The main focus of our study is on how tonic nAChR activation controls the timing of striatal output; our justification for including the ADP in our experimental analysis was simply corroborative, in that it represents an additional, easily measured parameter of the postsynaptic response to convergent cortical stimulation that 1) can be modulated by similar local inhibitory circuits that we show to mediate the effect of tonic nAChR activation and 2) is positioned (as opposed to EPSPs) to influence subsequent spiking, should the appropriate synaptic cues be present (which are deliberately omitted in our study). That said, under our experimental conditions EPSPs and ADPs were similar in both their kinetics and modulation by mecamylamine, suggesting that they represent mechanistically similar responses to cortical afferents. The defining difference (besides ADPs exhibiting larger amplitudes) is that they appear either in the absence of or following a spike. For these reasons we ultimately decided that reporting changes in both ADPs and EPSPs would be redundant, and limited our analyses to ADPs. Text has been added to the first paragraph of the results section to address these points.

In Fig. 1F, ADP is absent. Why? Please clarify.

Figure 1F shows an example of a SPN held at a mimicked ‘up-state’, achieved by injecting positive somatic current to produce a ‘resting’ membrane potential of -55-50mV. In this scenario, the ‘up-state’ membrane potential is higher than what would be reached during most ADPs evoked from Vrest, preventing the observation of ADPs in many trials. Text has been added to the end of the first paragraph in the results section to clarify this point.

If ADP is distinct from EPSP here in MSNs, has it been reported in the literature, and how is it generated?

Under our experimental conditions, we do not see any major differences between EPSPs and what we term ADPs (other than amplitude), at least in terms of kinetics and modulation by mecamylamine. That said, we have added text to the first paragraph of the results section that references previous work (Flores-Barrera et al.) describing suprathreshold depolarizations proceeding SPN spikes, which shaped our reasoning for including this measure in our study.

1. In Fig. 1F, the holding potential for mecamylamine is a few mV more negative than the control, but the spike latency is shorter under mecamylamine. This is hard to understand because membrane potential (current-injection-induced depolarization + EPSP) determines spike firing and latency. If the holding potential is the same, then it's easy to understand (larger EPSP under mycamylamine).

Thanks for pointing this out! We agree that this might seem counter-intuitive in terms of Vrest and EPSP amplitude only. Given that mecamylamine reduces GABAergic inputs to SPNs, the reduction in spike latency in this case is consistent with a reduction of GABA receptor mediated shunting. We have added this point to the text in the 3rd paragraph of the results section, which we think strengthens our justification to look at GINs as the potential mediators of mecamylamine’s effect on spike latency.

1. Data in Fig. 2D, E are weak. The spiking ability of whole-cell recorded neurons often declines over time (evidence: the AP duration for the red trace is longer); recovery/partial recovery from MLA is needed for the data to be reliable. Fig. 2E shows 8 cells: 6 had no response, 2 increased. Sample size needs to increase.

We appreciate this comment. Our initial justification for this experiment was from previous reports that alpha-7 nAChRs reduce corticostriatal glutamate release probability. We have now added additional data (Figure 2 supplemental data) showing that blockade of tonically activated alpha-7 nAChRs with the more specific antagonist MLA was not sufficient to change corticostriatal synaptic strength or release probability. In parallel, as we began increasing the sample size of the experiment testing the effect of MLA on spike latency, we noticed that the effect size became smaller than what we initially reported, which was already modest. Given the modest effect size of MLA on spike latency (with no presynaptic mechanism to offer), we reason that it would likely have minimal impact compared to the larger effect of mecamylamine. For this reason, we have backed off our conclusion that TONIC activation of presynaptic alpha-7 nAChRs on corticostriatal axon terminals will have a meaningful physiological impact on SPN spike timing. Accordingly, we removed previous figure 2D/E, but supplemented Figure 2A/B/C with new data (figure 2 supplement) demonstrating the lack of effect of tonic nAChR activation on corticostriatal synapse release probability. The title of the manuscript has been altered to reflect this.

1. Fig. 7: the data on DhbE increasing AP duration is not convincing: no effect in 4 neurons, increase in 4 other neurons, and decrease in other neurons. Data ismore important than p<0.05. How do you interpret DhbE increasing AP duration?

Point taken. We shouldn’t let a statistical calculation dominate the interpretation of a mostly mixed population result. Furthermore, upon revisiting this figure we realized that the main points pertinent to our conclusions (mecamylamine hyperpolarizes PV-FSI Vrest) were obscured by data that were of limited relevance. We have re-focused this figure to highlight data that are directly pertinent to our interpretation. This included removing the AP duration data set in question, which does not add to or inform our conclusions. We have further strengthened our conclusion that PV-FSIs are a primary mediator of the effect of tonic nAChR activation on spike latency by adding new data showing that pharmacologically blocking cortical activation of PV-FSIs occludes the effect of mecamylamine (new figure 8, see comments to Reviewer 2).

Fig. 7F shows AP duration for PV-FSI is around 1.75 ms (some are over 2 ms, recorded at 35 C). This is unusually long. Also, the AP rise time is around 1.4 ms, very long. 1.75 ms total rise time vs. 1.4 ms for just rise: they do not add up?

Please see our response to the above point.

Reviewer #2 (Public Review):

This manuscript examines one aspect of how acetylcholine influences striatal microcircuit function. While striatal cholinergic interneurons are known to be engaged in key events and tasks related to the basal ganglia in vivo, and pharmacological studies indicate cholinergic signaling is complex and critical to striatal function, the mechanistic details by which acetylcholine regulates individual cell types within the striatum, as well as how these integrate to shape striatal output, remain largely unknown. This work thus addresses an important problem in the basal ganglia field, with likely relevance to both normal function and disease-related dysfunction. The authors used a brain slice preparation in which a large number of excitatory cortical inputs to the striatum are activated, and they could measure the resulting activation of striatal projection neurons (SPNs). Their primary finding was that in this preparation, blocking nicotinic acetylcholine signaling resulted in more rapid activation of SPNs. They then explored some of the potential mechanisms for this phenomenon, and conclude that in their preparation, cholinergic interneurons are engaged both tonically and phasically, resulting in recruitment of local GABAergic interneurons that provide feedforward inhibition onto SPNs. They show that one striatal GABAergic interneuron subclass, PV-FSI, are modestly excited by tonic nicotinic signaling, and suggest this may be one contributor to their primary finding.

Strengths of the study include the focus on cholinergic signaling across multiple striatal cell types, careful and clearly displayed slice electrophysiology, good writing, and a methodical approach to pharmacology.

Weaknesses include reliance on the Thy1-ChR2 line to activate excitatory cortical inputs to the striatum (this line may be less specific to cortical pyramidal neurons than a specific Cre recombinase mouse line used with Cre-dependent ChR2, and thus have unintended influences on the results), and despite a strong start, a fairly weak mechanistic exploration of what GABAergic neuron subclasses might contribute to their original phenomenon.

We thank the Reviewer for their thoughtful and constructive comments. The Reviewer identified two weakness of our study, as presented. The first weakness was our reliance on a transgenic mouse line (Thy1-ChR2) to activate cortical inputs to the striatum. Specifically, how a potential lack of specificity/ectopic expression of ChR2 in non-glutamatergic cortical neurons may impact our interpretation of the data. The second is that we did not make an effort to identify the specific subclass(es) of GINs that contribute to the phenomenon we describe. We have addressed both of these comments with new experiments, which we will describe individually below.

1) Specificity of corticostriatal afferent activation in Thy1-ChR2 mice. As the Reviewer keenly points out, although Thy1-ChR2 mice are often used as a tool to specifically activate excitatory corticostriatal nerve terminals with optogenetic stimuli, there is concern that ChR2 expression is not exclusively limited to glutamatergic cortical neurons. If present, direct optogenetic activation of non-cortical striatal afferents would influence our results and impact our interpretation. We have addressed this issue experimentally by adding two new types of experiments (and related text, pages 7-8).

We have added new data using immunohistochemical staining to survey for ectopic expression of ChR2 in the cortex. Staining for GAD, to broadly identify GABAergic neurons, displayed no overlap with ChR2-expressing cortical neurons in Thy1-ChR2 mice. Since a population of GABAergic somatostatin-expressing cortical neurons (particularly in the auditory cortex), have been shown to directly innervate the striatum (Rock et al., 2016), we also show that we found no evidence for somatostatin-ChR2 colocalization in our mice. Furthermore, we report no evidence for somatic expression of ChR2 in the striatum. We do report somatic expression of ChR2 in a population of globus pallidus soma, and add text to describe the above data (figure 3 supplement ) as well as published data identifying ChR2 in axons of the substantia nigra. Together, these data suggest that cortical expression of ChR2 is limited to non-GABAergic neurons, though do not eliminate the possibility of a direct monosynaptic GABAergic input to the striatum form non-cortical (and extrastriatal) brain regions. We describe newly added experimental data below to address this possibility.

We have added new data to directly test if the optogenetic stimulation protocol used in this study induces a monosynaptic GABAergic current in SPNs (figure 3 supplement). We report that an optogenetically-evoked monosynaptic GABAergic current is indeed detected in SPNs, though it is unlikely to affect our results or interpretations for two reasons. First, based on the newly added histological data, the source of this GABAergic current is non-cortical and extrastriatal. Second, and more importantly, this input is insensitive to mecamylamine (new data, figure 3 supplement) and as such would not be modulated by the key manipulations presented in this study. Finally, experiments described below – instructed by a suggestion made by Reviewer 2 (see below) – show that blocking glutamatergic synaptic activation of a class of striatal GINs eliminates the effect of mecamylamine on SPN spike latency, ruling out the involvement of a monosynaptic GABAergic input in mediating the phenomenon.

2) Identification of the key GIN subclass that mediates the phenomenon. Our initial manuscript included data demonstrating the feasibility of PV-FSIs in participating in the phenomenon we described, but we agree with the Reviewer that we stopped well short of identifying the class of GINs that are actually involved. We have added two new data sets to the manuscript that now corroborate both the involvement and necessity of PV-FSIs in mediating this phenomenon. First, we have added data showing that striatal SOM+ interneurons respond to mecamylamine differently than PV-FSIs do: while mecamylamine hyperpolarizes PV-FSIs, it depolarizes the average membrane potential of SOM+ interneurons and has no effect on their spontaneous firing frequency, making them unlikely candidates to mediate the phenomenon we describe. Second, we have added data showing that pharmacologically preventing cortical activation of PV-FSIs both mimics and occludes the effect of mecamylamine on spike latency and ADP amplitude (new figure 8). This data also rules out the involvement of certain other classes of GINs, such as PLTS interneurons, as the pharmacological manipulation we performed (blockade of calcium-permeable GluA2-lacking AMPA receptors) does not affect their response to cortical inputs (Gittis et al., 2010).

Reviewer #3 (Public Review):

The manuscript by Matityahu et al., investigated the role of tonic activation of AChRs on the spike timing of striatal spiny projection neurons (SPNs) in acute striatal slices. By selectively activation of corticostrialal projections using optogenetic tools (ChR2), they find that pharmacological blockade of presynaptic α7 nAChRs delays SPN spikes, whereas blockade of α4β2 nAChRs on GABAergic interneurons advances SPN spikes. The work is carefully done with proper control experiments, and the main conclusions are mostly well supported by data.

Although they only constitute ~1% of the total striatal neurons in rodents and humans, cholinergic interneurons (ChINs) are gatekeepers of striatal circuitry because of their extensively arborized axons and varicosities which tonically release ACh. Whereas the role of muscarinic AChRs (mAChRs) in modulating striatal output has been well established, the role of nAChRs (especially the tonic activation) remains to be elucidated. The study is solid and the results are new and convincing. The data suggest that tonic activation of nAChRs may place a "brake" on SPN activity, and the lift of this brake during pauses of ChIN firing in response to salient stimuli may be critical for striatal information processing and learning. The findings from this study will enhance our understanding of the role of tonic nAChR activation in controlling SPNs and striatal output.

We thank the reviewer for their careful reading of our manuscript and for their kind words and helpful suggestions.

Unjustified Conclusions and Suggestions:

1) The change of the SPN spike timing by AChR modulation is on a few milliseconds time scale. To make the current study more significant, the authors should design and perform additional experiments to demonstrate the functional consequence in controlling striatal output and learning. For example, will activation or blockade of nAChRs have effects on striatal STDP?

We too would be thrilled to see the results of such experiments. Unfortunately our early attempts to perform such tests (e.g., crossing Thy1-ChR2 mice with ChAT-Cre mice to selectively express halorhodopsin in CINs, and combine cortical excitation with silencing of CINs) have been plagued by technical challenges, and would require time and resources that we feel are pragmatically beyond the scope of this study. That said, we’ve included new text (particularly, page 15) discussing how our results may fit with a newly published study on the role of CINs in corticostriatal LTP (Reynolds et al., 2022).

2) Modulation of striatal circuitry is complex. The addition of a diagram illustrating the hypothesis and key results would help.

Excellent suggestion. We have added a summary diagram, which is now figure 9.

looking for a callback that you can register to happen after current transaction is committed, not just after_commit of model -- though actually, that might fire precisely when current transaction is committed, too (except that it might only get triggered for nested transactions, not the top-most transaction), so it could maybe go there ... but I think the problem is just that it doesn't belong there, because it's not specific to the model...

I guess the OP said it best:

I am not looking for model based after commits on update/create/etc, I want to be able to dynamically define a block that will be executed only if the current (top-most) transaction passes:

Forecasting Rules

(Kicking Off a New Quarter) * There is often a delay of 3-4 days at the beginning of the quarter for all revenue data to appear in the tool. Your quota for the quarter is typically not assigned until a week into the quarter, and the Sales Forecast Tool will update several days after it is assigned. * In the Client Forecast, enter the amount of revenue you expect the client to spend outside of any Opportunities in Stages 0, 1, 2 or 3 you are working on. * The Client Forecast can be thought of as the amount of revenue you can safely consider “locked in” for the quarter.

(Account Ownership & Forecasting) * The Client Forecast is inclusive of all Sub-Account and Product spend. * The Sales Forecast Tool will show you all the Parents you own, regardless of whether or not it has an Opportunity, has spent, etc. * The Client Forecast should be entered based on what you believe the client will spend for the given quarter, regardless of whether or not you will own the account for the full quarter. In the case where you only own the Account for part of the quarter:

• The Rep who is losing the account will have their My Forecast replaced with the revenue that spent on the account while they owned it
• The rep gaining the account will have their My Forecast be the Client Forecast minus any revenue that was spent while they weren’t the owner

• In this way, we use the Client Forecast and then “pro-rate” it based on the revenue that you will actually get credit for. This is why we use your My Forecast as the number we compare against your quota.

• Note: When accounts transition to a new owner, it will take a day in order to start showing revenue data as well as the My Forecast for the new owner

• Note: For new reps, we need a quota and team to be assigned in order to display revenue information which can sometimes take several weeks
• Parent ownership is basically determined by whichever rep is assigned to the most Sub-Accounts under that Parent.

(When to Update the Forecast) * The Client Forecast should be updated every Monday morning (unless otherwise instructed by your Director). * The revenue information in the tool is a guideline only - you are responsible for making sure the forecast reasonably reflects what you expect the client to spend. * Your market leaders will tell you whether you are responsible for entering the Client Forecast for Agency Sub-Accounts. In some markets this will be the Direct Rep, in others the Agency Rep. * When entering the Client forecast for the Direct Parent, you should enter the total amount of Direct spend AND Agency spend. When entering the Agency Sub-Account spend, you should only enter the Agency spend. * Direct reps will only see their Direct Parents by default. To see Agencies, select the “Show Agency Accts” checkbox.

• You can still reference the Account Executive Report, the PIT Reports, etc for additional information about your client’s spend, if needed.

(Managing Closed-Won Opportunities) * Essentially, the single number for the Client Forecast replaces the need to ever adjust any Closed-Won Opportunity. * You no longer need to update your Closed-Won Opportunities. Instead, enter and update the single number for the Client Forecast.

(Navigation Basics) * When you make updates to your Client Forecast, there is a “Save” button you need to click in order to save these changes. It’s at the bottom of the page, so isn’t always obvious depending on where you’re looking. * If my Forecast and the numbers in the Summary don't change, just refresh of the page and you should see that information updated!

(Keep in mind - data) * When you refresh the Sales Forecast Tool, you will see your Pipeline and Forecast (Client Forecast, My Forecast, etc) in real-time, Einstein will update in the next 5 hours or less. * All the revenue reporting we provide you is updated as close to real-time as possible, but that is different for each system: Adcentral is very close to real-time, Einstein is up to 5 hours delayed, and the Sales Forecast Tool is approximately 1 day delayed.

• Additionally, most reports you have access to are pulling commissionable revenue (the revenue you actually get credit for while you owned the account), whereas the data in the Sales Forecast Tool is based on what the Account spent, regardless of when you owned it.
• The revenue data in the Sales Forecast Tool (QTD Revenue, Straight Line Projection, Projection Category, Last Revenue Date) is approximately 1 day delayed. All other data in the tool will be in real-time (just make sure to refresh the page!)

In a relatively closed and small school environment, everyone is vulnerable to attention. People are also more susceptible to stereotypes, especially if they are in the minority. The child is constantly subjected to stereotypes and even accepts them. The world outside of school has its own pressures from home. The parents think it is normal for her to do well in school and help the family achieve better living conditions in the future.

• ABOUT property "Report broken translators"

Representation matters across disciplines

I like this! We always say that this utopic vision is impossible, but to me, it is possible ... if everyone does the work.

I have found myself doing this while reading dystopias. Dystopias, even though they represent “bad places,” do give me a sense of comfort knowing that the world I currently reside in isn’t horrid like the text or movies I’ve consumed. It isn’t the best rational for dystopias, because that idea is contingent on the fact that the world remains the habitable place it currently is without drastic change. The world could actually get as macabre as dystopias; it just hasn’t come to pass yet.

Science is the only ideology that can be easily backed up with proof, which is why it's so hard to consider it an idea as opposed to just fact

In the earlier context, I thought about this in a bad context- that everyone just has to live life dragging by whether we like it or not. In this context, it's used in hopes that things will get better rather than worse. So let's all try our best

I definitely agree that being digitally literate is much more than being able to punch some phrase into a web browser, and get results; sometimes finding a particular webpage or book is tougher than just knowing their name. As someone who has to frequently consult Google while programming, knowing how to piece together my confusion into a question is very helpful - it's like, you need to find the sweet spot between being vague and specific.

it is not exactly clear if the number is representative of the entire population of Americans or just those who had COVID. it could be assumed that it's just those who had COVID but it is unclear

Reviewer #1 (Public Review):

Tiedemann et al. investigated internal generative models as a mechanistic explanation of one-shot learning of visual categories. To achieve this, they designed a clever paradigm where participants draw new Variations of a category based on a single presented shape (the Exemplar). They found that participants could successfully generate new Variations based on an Exemplar (Exp 1), that these vary in Similarity to the Exemplar (Exp 2), and are more than just mere copies of the Exemplar (Exp 2b). Next, they demonstrated that Variations are readily attributed to the correct corresponding Exemplar 'category' by naïve participants (Exp 3). Having established that novel Variations in a category can be generated as a result of one-shot learning, the authors turned to establishing what features drive these novel categories. To this end, they asked naïve participants to view pairings of a single Exemplar and Variation, and label overlapping features (Exp 4), or to view single Exemplars or Variations and label their distinctive parts (Exp 5). The authors find that each shape had distinctive features that were reliably reported across participants, that most features were shared among an Exemplar and its Variations, and that few features were shared with Variations of other Exemplars. To confirm the importance of these distinctive features, shapes were produced where the distinctive parts from two shapes were traded. Naïve participants sorting decisions now appeared to be biased toward the Exemplar that matched the swapped-in diagnostic feature, not the remainder of the shape (Exp 6).

These experiments were thoroughly run, they establish a rich basis for exploring one-shot learning, provide novel methodological approaches for quantifying information about complex shape comparisons, and contribute to our understanding of how novel categories can be extrapolated from individual objects - through internal generation driven by distinctive features. The conclusions drawn here are mostly consistent with what the data demonstrated, though could use some clarification and further justification. There are some instances where within- and across-experiment comparisons could have been used to drive further insights, allowing some of the proposed mechanisms to be clarified and better supported.

1) The authors present an interesting proposal for how the generative model operates when producing shapes in Fig 6, as well as some alternative strategies in Fig 7. It is not clear what evidence supports the idea that shapes are first broken down into parts, then modified and recombined. It is obvious from the data that distinctive features are preserved (in some cases), but some clarification on the rest would be useful. For instance, is it possible that conjunctions or combinations of features are processed in concert? What determines whether critical features are added or subtracted to the shape during generation? Some more justification for this proposed model is needed, as well as for how the exceptions and alternate strategies were determined.

2) Some claims are made in the manuscript about large changes being made to Variations without consequence to effective categorization. However, these appeal to findings derived from collapsing across all Variations, when it could be informative to investigate the edge cases in more detail. There is a broad range in the similarity of Variations to Exemplars, and this could have been profitably considered in some analyses, especially zooming in on the 'Low Similarity' Variations. For example, this would help determine whether classification performance and the confusion matrix change in predictable ways for high-, relative to medium- and low-similarity Variations. It could also indicate whether the features and feature overlap can tell us anything about how likely a Variation is to be perceived as from the correct category.

3) The authors cross-referenced data from Experiments 4 and 5 to draw the conclusion that the most distinct features are preserved in Variations. This was very compelling and raised the idea that there are further opportunities to perform cross-experiment comparisons to better support the existing claims. For example, perhaps the correspondence percentages in Exp 4, or the 'distinctive feature-ness' in E5, allow prediction of the confusion proportions in Exp 3.

4) The Variation generation task did not require any explicit discrimination between objects to establish category learning, which is a strength of the work that the authors highlighted. However, it's worth considering that discrimination may have had some lingering impact on Variation generation, given that participants were tasked with generating Variations for multiple exemplars. Specifically, when they are creating Variations for Exemplar B after having created Variations for Exemplar A, are they influenced both by trying to generate something that is very like Exemplar B but also something that is decidedly not like Exemplar A? A prediction that logically follows from this would be that there are order effects, such that metrics of feature overlap and confusion across categories decreases for later Exemplars.

good to see governments control here. in the certain controlled substances it's better to have the governmentn monitoring, just in case some agency abuse them.

a fellow ADHDer! I definitely get the feeling of wanting to try EVERYTHING.

Huh, that's actually pretty interesting. I didn't even think that there would be two different versions of TickTick. I don't know if the names are just different or if there are actual feature differences, but it's still interesting.

I do like how this user emphasizes that just because they pointed out the connections to China does not mean that it means anything bad.

This is a great question and I ask it frequently with many different answers.

I've not seen specific numbers, but I suspect that the majority of Hypothes.is users are annotating in small private groups/classes using their learning management system (LMS) integrations through their university. As a result, using it and hoping for a big social experience is going to be discouraging for most.

Of course this doesn't mean that no one is out there. After all, here you are following my RSS feed of annotations and asking these questions!

I'd say that 95+% or more of my annotations are ultimately for my own learning and ends. If others stumble upon them and find them interesting, then great! But I'm not really here for them.

As more people have begun using Hypothes.is over the past few years I have slowly but surely run into people hiding in the margins of texts and quietly interacted with them and begun to know some of them. Often they're also on Twitter or have their own websites too which only adds to the social glue. It has been one of the slowest social media experiences I've ever had (even in comparison to old school blogging where discovery is much higher in general use). There has been a small uptick (anecdotally) in Hypothes.is use by some in the note taking application space (Obsidian, Roam Research, Logseq, etc.), so I've seen some of them from time to time.

I can only think of one time in the last five or so years in which I happened to be "in a text" and a total stranger was coincidentally reading and annotating at the same time. There have been a few times I've specifically been in a shared text with a small group annotating simultaneously. Other than this it's all been asynchronous experiences.

There are a few people working at some of the social side of Hypothes.is if you're searching for it, though even their Hypothes.is presences may seem as sparse as your own at present @tonz.

Some examples:

@peterhagen Has built an alternate interface for the main Hypothes.is feed that adds some additional discovery dimensions you might find interesting. It highlights some frequent annotators and provide a more visual feed of what's happening on the public Hypothes.is timeline as well as data from HackerNews.

@flancian maintains anagora.org, which is like a planet of wikis and related applications, where he keeps a list of annotations on Hypothes.is by members of the collective at https://anagora.org/latest

@tomcritchlow has experimented with using Hypothes.is as a "traditional" comments section on his personal website.

@remikalir has a nice little tool https://crowdlaaers.org/ for looking at documents with lots of annotations.

Right now, I'm also in an Obsidian-based book club run by Dan Allosso in which some of us are actively annotating the two books using Hypothes.is and dovetailing some of this with activity in a shared Obsidian vault. see: https://boffosocko.com/2022/03/24/55803196/. While there is a small private group for our annotations a few of us are still annotating the books in public. Perhaps if I had a group of people who were heavily interested in keeping a group going on a regular basis, I might find the value in it, but until then public is better and I'm more likely to come across and see more of what's happening out there.

I've got a collection of odd Hypothes.is related quirks, off label use cases, and experiments: https://boffosocko.com/tag/hypothes.is/ including a list of those I frequently follow: https://boffosocko.com/about/following/#Hypothesis%20Feeds

Like good annotations and notes, you've got to put some work into finding the social portion what's happening in this fun little space. My best recommendation to find your "tribe" is to do some targeted tag searches in their search box to see who's annotating things in which you're interested.

To a great extent, Hypothes.is has such a small footprint of users (in comparison to massive platforms like Twitter, Facebook, etc.) that it's never been a performative platform for me. As a design choice they have specifically kept their social media functionalities very sparse, so one also doesn't generally encounter the toxic elements that are rampant in other locations. This helps immensely. I might likely change my tune if it were ever to hit larger scales or experienced the Eternal September effect.

Beyond this, I mostly endeavor to write things for later re-use. As a result I'm trying to write as clearly as possible in full sentences and explain things as best I can so that my future self doesn't need to do heavy work or lifting to recreate the context or do heavy editing. Writing notes in public and knowing that others might read these ideas does hold my feet to the fire in this respect. Half-formed thoughts are often shaky and unclear both to me and to others and really do no one any good. In personal experience they also tend not to be revisited and revised or revised as well as I would have done the first time around (in public or otherwise).

Occasionally I'll be in a rush reading something and not have time for more detailed notes in which case I'll do my best to get the broad gist knowing that later in the day or at least within the week, I'll revisit the notes in my own spaces and heavily elaborate on them. I've been endeavoring to stay away from this bad habit though as it's just kicking the can down the road and not getting the work done that I ultimately want to have. Usually when I'm being fast/lazy, my notes will revert to highlighting and tagging sections of material that are straightforward facts that I'll only be reframing into my own words at a later date for reuse. If it's an original though or comment or link to something important, I'll go all in and put in the actual work right now. Doing it later has generally been a recipe for disaster in my experience.

There have been a few instances where a half-formed thought does get seen and called out. Or it's a thought which I have significantly more personal context for and that is only reflected in the body of my other notes, but isn't apparent in the public version. Usually these provide some additional insight which I hadn't had that makes the overall enterprise more interesting. Here's a recent example, albeit on a private document, but which I think still has enough context to be reasonably clear: https://hypothes.is/a/vmmw4KPmEeyvf7NWphRiMw

There may also be infrequent articles online which are heavily annotated and which I'm excerpting ideas to be reused later. In these cases I may highlight and rewrite them in my own words for later use in a piece, but I'll make them private or put them in a private group as they don't add any value to the original article or potential conversation though they do add significant value to my collection as "literature notes" for immediate reuse somewhere in the future. On broadly unannotated documents, I'll leave these literature notes public as a means of modeling the practice for others, though without the suggestion of how they would be (re-)used for.

All this being said, I will very rarely annotate things privately or in a private group if they're of a very sensitive cultural nature or personal in manner. My current set up with Hypothesidian still allows me to import these notes into Obsidian with my API key. In practice these tend to be incredibly rare for me and may only occur a handful of times in a year.

Generally my intention is that ultimately all of my notes get published in something in a final form somewhere, so I'm really only frontloading the work into the notes now to make the writing/editing process easier later.

Where

My annotations broadly flow into two spaces:

Obsidian

My private Obsidian-based vault is where I collect the notes and actively work on, modify, edit, and expand them if and when necessary. This is also the space where I'm broadly attempting to densely interlink them together for future use and publication in other venues. If I could, I would publish these all on the web, but I've yet to find a set up with a low enough admin tax that I can publish them inexpensively in a way I'd like them to appear (primarily with properly linked [[WikiLinks]]) while still owning them in my own space.

I've been experimenting around with using Blot.im as a solution to display them here https://notes.boffosocko.com/, but at present it's a very limited selection of my extant notes and doesn't include Webmention or other niceties I'd like to add. As it's a very alpha stage experiment I don't recommend anyone follow or use it and it may disappear altogether in the coming months.

WordPress

My main website uses WordPress. To a great extent, this is (now) primarily a back up location and the majority of the annotations are unpublished to the public, but are searchable to me on the back end.

I do, however, use it occasionally for quickly publishing and syndicating select annotations which I think others may find interesting or upon which I'm looking for comments/feedback and don't expect that the audience I'd like these from will find them natively on Hypothes.is' platform. An example of this might be a paper I was reading this weekend on Roland Barthes which discusses his reasonably well documented zettelkasten-like note taking practice. The article can be found here: https://culturemachine.net/wp-content/uploads/2019/01/373-604-1-PB.pdf with the annotations seen here: https://docdrop.org/pdf/The-Card-Index-as-Creativity-Ma---Wilken-Rowan-upq8g.pdf/. To tip off others in the space, I made a post on my site with a bit of a puzzle and syndicated it to Twitter. A few hours later I posted a follow up with some additional details and links to my notes on hypothesis which got some useful feedback from Matthias Melcher on the Barthes paper as well as on a related paper I mentioned by Luhmann, particularly about German translation, with which I have little facility.

Another recent illustrative example was this annotation on the Library of Congress website about Vladimir Nabokov which was picked up by my website (though unpublished/not public) but which I syndicated to Twitter primarily to be able to send a notification to Eleanor Konik who I know is interested in the idea of World Building using historical facts and uses Obsidian in her work. (The @mention in the tweet is hiding in the image of the index card so that I could save text space in the main tweet.) Several others interested in note taking and zettelkasten for writing also noticed it and "liked" it. Not being on Hypothes.is to my knowledge much less following me there, neither Eleanor nor the others would have seen it without the Tweet.

Nabokov used index cards for his research & writing. In one index card for Lolita, he creates a "weight-heigh-age table for girls of school age" to be able to specify Lolita's measurements. He also researched the Colt catalog of 1940. #WorldBuildinghttps://t.co/i16Yc7CbJ8 pic.twitter.com/JSjXV50L3M

— Chris Aldrich (@ChrisAldrich) April 10, 2022

How

Obsidian

Getting annotations from Hypothes.is to Obsidian is a short two-step process which is reasonably well automated so that I don't spend a lot of time cutting/pasting/formatting.

I start with an IFTTT recipe that takes the RSS output of Hypothes.is and creates text files directly into my Obsidian vault. The results are quite rudimentary and only include the title of the document, the permalink of the Hypothes.is post, the highlighted text, and my annotation. It doesn't include the tags as RSS doesn't have a specification for these.

Second, I've set up Hypothesidian which has a much higher fidelity dovetail with the Hypothes.is API to get all the data and even the formatting set up I'm looking for. A reasonably well laid out set of instructions with a low/no code approach for it can be found at https://forum.obsidian.md/t/retrieve-annotations-for-hypothes-is-via-templater-plugin-hypothes-idian/17225. It allows importing annotations by a variety of methods including by date and by document URL. I've also made a small modification to it so that tags on Hypothes.is are turned into [[wikilinks]] in Obsidian instead of #tags which I only use sparingly.

All the IFTTT annotations will be ported individually into a specific Obsidian folder where I'll process them. I can then quickly use Hypothesidian to import the properly laid out version (using templates) of the notes with just a few keystrokes and then focus my time on revising my notes if necessary and then linking them to the appropriate notes already in my system. Finally I'll move them into the appropriate folder based on their content—typically one of the following: zettelkasten, wiki, commonplace, dictionary, or sources (for bibliographic use). Careful watchers will notice that I often use Hypothes.is' "page notes" functionality to create a bookmark-like annotation into which I will frequently post the URL of the page and occasionally a summary of a piece, these are imported into my system and are used as source/bibliographic information. I also have some dovetailing with Zotero as a bibliographic set up which feeds into this data as well.

This version which I've cobbled together works well for me so that I'm not missing anything, but there are definitely other similar processes available out there both for Obsidian (with plugins or scripts) as well as for other platforms. If I'm not mistaken, I think Readwise (a paid solution) has a set up for note transfer and formatting.

WordPress

As there isn't an extant Micropub client for Hypothes.is I initially used RSS as a transport layer to get my notes from Hypothes.is into WordPress. The fidelity isn't great in part because RSS doesn't include any tags. To get some slightly better presentation I set up a workflow using RSS output from Hypothes.is as input into an IFTTT workflow which outputs to a webhook that stands in as a Micropub client targeting my websites Micropub server. Some of the display on my site is assisted by using the Post Kinds plugin, which I know you've been working around yourself. The details may be above some, but I've outlined most of the broad strokes of how this is done in a tutorial at https://boffosocko.com/2020/01/21/using-ifttt-to-syndicate-pesos-content-from-social-services-to-wordpress-using-micropub/. In that example, I use the service Pocket as an example, but Hypothes.is specific information could easily be swapped out on a 1-1 basis.

A custom stand-alone or even an integrated micropub client for Hypothes.is would be a fantastic project if someone wanted to dig into the details and dovetail it with the Hypothes.is API.

Why

Ideally, I'm hoping that small pieces loosely joined and IndieWeb building blocks will allow me to use the tools and have the patterns I'm looking for, without a lot of work, so that I can easily make annotations with Hypothes.is but have and share (POSSE) my content on my own site in a way that works much the way many IndieWeb sites dovetail with Twitter or Mastodon.

I'm doing some portions of it manually at present, without a lot of overhead, but it would be fun to see someone add micropub and webmention capabilities to Hypothes.is or other IndieWeb building blocks. (I suspect it won't be Hypothes.is themselves as their team is very small and they're already spread thin on multiple other mission critical projects.)

In the end, I'm using Hypothes.is as a well designed and convenient tool for quickly making notes on digital documents. All the data is flowing to one of two other locations where I'm actually making use of it. While there is some social layer there, I'm getting email notifications through the Hypothes.is settings and the data from my responses just gets rolled back into my spaces which I try to keep open and IndieWeb friendly by default. At the same time, for those who want or need it, Hypothes.is' interface is a great way of reading, searching, sorting, and interacting with my notes in public, particularly until I get something specific and user friendly up to do it on my own domain.

My first view, even before any of the potential social annotation angle, is that in annotating or taking notes, I'm simultaneously having a conversation with the author of the work and/or my own thoughts on the topic at hand. Anything beyond that for me is "gravy".

I occasionally find that if I'm writing as I go that I'll have questions and take a stab only to find that the author provides an answer a few paragraphs or pages on. I can then look back at my thought to see where I got things right, where I may have missed or where to go from there. Sometimes I'll find holes that both the author and I missed. Almost always I'm glad that I spent the time thinking about the idea critically and got to the place myself with or without the author's help. I'm not sure that most others always do this, but it's a habit I've picked up from reading mathematics texts which frequently say things like "we'll leave it to the reader to verify or fill in the gaps" or "this is left as an exercise". Most readers won't/don't do this, but my view is that it's almost always where the actual engagement and learning from the material stems.

Sometimes I may be writing out pieces to clarify them for myself and solidify my understanding while other times, I'm using the text as a prompt for my own writing. My intention most often is to add my own thoughts in a significantly well-thought out manner such that I can in the near future reuse these annotations/notes in essays or other writing. Some of this comes from broad experience of keeping a commonplace book for quite a while, and some of it has been influenced on reading about the history of note taking practices by others. One of the best summations of the overall practice I've seen thus far is Sönke Ahrens' How to Take Smart Notes (Create Space, 2017), though I find there are some practical steps missing that can only be found by actually practicing his methods in a dedicated fashion for several months before one sees changes in their thought patterns, the questions they ask, and the work that stems from it all. And by work, I mean just that. The whole enterprise is a fair amount of work, though I find it quite fun and very productive over time.

In my youth, I'd read passages and come up with some brilliant ideas. I might have underlined the passage and written something like "revisit this and expand", but I found I almost never did and upon revisiting it I couldn't capture the spark of the brilliant idea I had managed to see before. Now I just take the time out to write out the entire thing then and there with the knowledge that I can then later revise it and work it into something bigger later. Doing the work right now has been one of the biggest differences in my practice, and I'm finding that projects I want to make progress on are moving forward much more rapidly than they ever did.

• VERY LIMITED
• I have got PDFs in different locations!
• I WANT to link to the absolute location at import

Sometimes this phenomenon can be happened, which is uncomfortable. This could present the author's argument that it's not just stereotypes, it's the embodiment of America's special cultural, national structure, and history.

While note cards have a certain portability about them for writing almost anywhere, aren't notebooks just as easily portable? In fact, with a notebook, one doesn't need to worry about spilling and unordering the entire enterprise.

There are, however, other benefits. By using small atomic pieces on note cards, one can be far more focused on the idea and words immediately at hand. It's also far easier in a creative and editorial process to move pieces around experimentally.

Similarly, when facing Hemmingway's White Bull, the size and space of an index card is fall smaller. This may have the effect that Twitter's short status updates have for writers who aren't faced with the seemingly insurmountable burden of writing a long blog post or essay in other software. They can write 280 characters and stop. Of if they feel motivated, they can continue on by adding to the prior parts of a growing thread. Sadly, Twitter doesn't allow either editing or rearrangements, so the endeavor and analogy are lost beyond here.

It'd be interesting to make a list of what is Actually Doable on the internet and what is Not

In addition to using his card index for producing his published works, Barthes also used his note taking system for teaching as well. His final course on the topic of the Neutral, which he taught as a seminar at Collège de France, was contained in four bundles consisting of 800 cards which contained everything from notes, summaries, figures, and bibliographic entries.

Given this and the easy portability of index cards, should we instead of recommending notebooks, laptops, or systems like Cornell notes, recommend students take notes directly on their note cards and revise them from there? The physicality of the medium may also have other benefits in terms of touch, smell, use of colors on them, etc. for memory and easy regular use. They could also be used physically for spaced repetition relatively quickly.

Teachers using their index cards of notes physically in class or in discussions has the benefit of modeling the sort of note taking behaviors we might ask of our students. Imagine a classroom that has access to a teacher's public notes (electronic perhaps) which could be searched and cross linked by the students in real-time. This would also allow students to go beyond the immediate topic at hand, but see how that topic may dovetail with the teachers' other research work and interests. This also gives greater meaning to introductory coursework to allow students to see how it underpins other related and advanced intellectual endeavors and invites the student into those spaces as well. This sort of practice could bring to bear the full weight of the literacy space which we center in Western culture, for compare this with the primarily oral interactions that most teachers have with students. It's only in a small subset of suggested or required readings that students can use for leveraging the knowledge of their teachers while all the remainder of the interactions focus on conversation with the instructor and questions that they might put to them. With access to a teacher's card index, they would have so much more as they might also query that separately without making demands of time and attention to their professors. Even if answers aren't immediately forthcoming from the file, then there might at least be bibliographic entries that could be useful.

I recently had the experience of asking a colleague for some basic references about the history and culture of the ancient Near East. Knowing that he had some significant expertise in the space, it would have been easier to query his proverbial card index for the lived experience and references than to bother him with the burden of doing work to pull them up.

What sorts of digital systems could help to center these practices? Hypothes.is quickly comes to mind, though many teachers and even students will prefer to keep their notes private and not public where they're searchable.

Another potential pathway here are systems like FedWiki or anagora.org which provide shared and interlinked note spaces. Have any educators attempted to use these for coursework? The closest I've seen recently are public groups using shared Roam Research or Obsidian-based collections for book clubs.

This work has been peer reviewed in GigaScience (https://doi.org/10.1093/gigascience/giac028), which carries out open, named peer-review.

These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer 1: Zehong Ding

In this manuscript, Qi et al. assembled two chromosome-scale haploid genomes in African cassava TME204, validated the structural and phasing accuracy of haplotigs by BACs and high-density genetic map, revealed extensive chromosome re-arrangements and abundant intra-genomic and inter-genomic divergent sequences, analyzed the allele-specific expression patterns in different tissues, and built a cassava pan-genome and demonstrated its importance in down-stream omics analysis. Overall, this work is of crucial importance and should be sufficient to publish in the GigaScience Journal. However, I found that this manuscript lacks the basic logical and some analyses have major flaws. Please see the details below:

1) According to Supplementary table10, there were at least 9 different tissues of the TME204 Illumina RNA-seq data. However, when the authors performing analysis of 'Tissue specific differentially expressed transcripts (Line 393)', why just compared between leaf and stem but ignore the remaining tissues? This is illogical.

2) Two cassava haplotypes (H1 and H2) were constructed in this study. In Table 4 and Supplementary figure 9, why the authors performed analysis between 'TME204 H1 vs. AM560' but did not mention the comparison between 'TME204 H2 vs. AM560' at all? Similarly, in Fig. 8 and Fig. 10c, the analysis was also performed in 'TME204 H1' but not in 'TME204 H2'.

3) in Fig.7C, ASE should be the expression level comparisons between H1 and H2, why the legends still are H1 (red bar) and H2 (blue bar)? I cannot understand. Also in Fig. 7D, it's very difficult to understand this figure. E.g., what's the meaning of labels (e.g., "leaf_H1" and "Stem_H1; Leaf_H1") on x-axis? Logically, there are "stem_H1; leaf_H1", "stem_H1; leaf_H2", "stem_H2; leaf_H2", then where is the "stem_H2; leaf_H1"?

4) Fig6d, Line 110-111, "The transcriptome comparison between TME204 leaf and stem tissues identified gene loci with associated transcripts that were differentially regulated in one haplotype only." This statement is not true because the comparison between leaf and stem cannot conclude that the transcripts were differentially regulated in one haplotype only. Thus, the sentences in Line 407-408 also need to be revised.

Other suggestions to the authors:

• Fig6a, what's meaning of Het_Uniq, Het_Dup, Hom_Uniq, and Hom_Dup.

• Fig6d, what's the meaning of legend bar? Log2(leaf/stem) or log2(stem/leaf)? - ref30 cannot be cited because it is still under preparation.

• In 'Conclusions section', the statement "The haplotype-resolved genome allows the first systematic view of the heterozygous diploid genome organization in cassava." is inaccurate, because two haplotypes in heterozygous cassava genome have already been published in Hu et al. (2021, Molecular Plant, 10.1016/j.molp.2021.04.009)

• The title is also suggested to be changed because it is not attractive.

• The citation of 'Figure 10b' (Line 497) and 'Figure 10c' (Line 502) are wrong.

The fact that Khan obviously wants the foreign investment piece (and other protections) to be removed as "nice-to-haves" and insist on one single hypothesis about density is irritating. Why is this the only contrary voice being presented. It almost reads like an opinion piece by Khan which could have amounted to a single paragraph stating that we need to turn the entire country into as densely urban as possible - which won't even work in most towns / cities which simply don't have the infrastructure. Waterloo is already collapsing under the increases in density and it will only increase significantly. This is a short-sighted solution that insists on ignoring the other solutions that need to be addressed. It is not an effective analysis of the actual budget because the most he will do is compare each suggestion to urban density? It's just ineffective.

Why does every left-leaning proposal seem to insist on density?

Toronto, Brampton, Scarborough and other dense cities are not the pinnacle of development. They are just the largest. Smaller cities like Waterloo, North Bay, Oshawa, Coburg, Newmarket, Barrie, and Aurora do in fact house a very large number of people and they don't need to be anywhere near as dense as Brampton or Toronto and GTA. These communities seem just as effective, with generally happier residents (in my experience). There is no reason that we cannot simply expand the countless municipalities throughout Canada (and probably build more) instead of insisting we turn the entire country into massive metropolises with ultra-dense housing.

This example makes me feel sorry and sad.two young man have totally different developments due to their different family backgrounds. This seems to be cruel, however it’s just reality. People from privileged don’t need to suffer from the hardness of surviving. All they need to do is to focus on their education. However, people from less privileged family are too busy to pay same amount of attention because they got too much to worry about.

Performance in SAT tests are also determined by how much effort each student puts into preparing. Students at Troy spend much more time than most other students and as a result, they have much higher score than many other students.

Is "see" here not as much figurative as literal? It refers to the novelist's duty to perceive clearly and completely and to translate the nature of that perception to the reader. An an exhortation, it's normative as well as epistemological -- not just "see" but "see properly."

It's going to be difficult for me to use my voice and share an opinion on this topic because I recognize my white privilege and I understand that my perspective is very different from a person of color. My opinion is that I should be learning and listening. My feedback for this article will mostly be just sharing how shocking this information is. The fact that this was ever accepted and that people even thought this way is so wrong and just truly unbelievable.

And as you should choose your own boundaries and decide for yourself. You make the choices if you want to be someone with a lot of money you work for it or someone that wants to be known you make yourself known instead of just envying the people who has all of that stuff. You can decide what you want to do or what can happen in your future

Connecting the essay to Hanson's arguments

Uncle Ghillar Michael Anderson told a story of how his mob went into a museum and transferred the knowledge from sacred objects in the museum and then took the spirts out of there and moved them back in country. The curators didn't understand the process at all or how they had corrupted the sacred objects.

I wanted to emphasize the privilege of having a access to a private tutor. This is because specially for the SAT's the education is made built to help high income students succeed due to the access of having the financial access to private tutors. I went to a low-income high school where there were 97% hispanic students and when it came to SAT tutoring. The school started to provide tutoring just one week away before taking the standardized test. Fortunately, I was accepted into an internship where I was able to obtain a scholarship and have access to a private tutor. If it wasn't for me actively seeking opportunities, the financial disadvantage would have affected me.

Ha ha! Skepticism of public square discourse!

This quote stood out to me because we hear numbers as high as this all the time, but it can be hard to actually understand how much that is because it's not a number we can easily visualize. The comparison that stood out to me the most was that the amount of e-waste produced per year could cover an area the size of Manhattan. This helps put into perspective just how much e-waste we're actually creating annually.

https://www.youtube.com/watch?v=67Uy-6uQOTQ&t=4s

This video talks about how eWaste is contaminating our environment and the dangers it is presenting to our health.

David: -Ideally we would have the video start automatically when the page opens -On mobile the video is too small, can we make it bigger somehow? -For all videos we should allow the option to increase the speed, in case someone just wants to fo over everything quickly. I don't see any downside doing only avoiding people that lose patience like me lol -The calls to action buttons should be A LOT more visible, almost as if you had no other choice but to click :) -We are missing Tania's feedback, she is hot it will add some value if it's good

This makes a good point that it makes because jobs can't require the age like 65. So it brings the issue is that is this proposal unfair.

It's interesting because this became one of their major shortcomings; the working class did not have the qualifications or resources to effectively run a social democracy in a nation that has always had a central figure or figures in charge. I find it impressive that the Commune was as successful as it was, though for a short time, and that social democracy could be partially achieved where an absolute monarchy was in power just a century before.

This is a perfect example of how media outlets can put out their idea of the "truth" of a story without knowing all the facts. Posting about peoples livelihood without knowing every detail is reckless and can be extremely detrimental to someone's character and image. Amanda Knox is just one example of many people whom have been falsely reported on. It's irresponsible to write about someone if you are not completely aware of the story through all angles. It's evident that many outlets were just "piggy backing" off on one another and simply just trying to hook new consumers and gain traffic.

Unfortunately this happens far too often with media outlets. News platforms want to just put out a strong article with strong headlines in order to catch the attention of consumers. It's evident that not all the facts were aware and media outlets were only putting out one sided stories. Without all the facts out there, people will form opinions without knowing every detail that may have gone in the story as whole. This unfortunately was extremely detrimental towards Amanda Know's image.

As a student, I have always preferred that teachers tell me what they expect. I think that there are often teachers who have an idea of what they expect students to turn and when that is not turned in, the student gets marked down. As a student who had a teacher that set little to no expectations, it was frustrating at times because we were still getting graded on a rubric, we just were not aware of what it was. Now, if the aspect of grades were removed, then an argument for not needing to tell students what exactly is expected of them could be helpful. It would provide students creative freedom and thinking outside the box.

Reviewer #1 (Public Review):

Antibiotics of different classes often have the unfortunate property of inhibiting mitochondrial protein synthesis in humans. To overcome these off-target activities, it would be helpful to have high-resolution views of how these antibiotics bind the mitochondrial ribosome to enable future medicinal chemistry efforts. However, to date, no such high-resolution views are available, even for the analogous bacterial system. In this manuscript, Itoh et al. present a cryo-EM reconstruction of the human mitochondrial small ribosomal subunit bound to streptomycin, at a local resolution of ~2.2 Å. With this resolution, the authors are able to define the binding interactions between the compound and the binding pocket in the ribosome. Notably, the antibiotic seems to adopt the gem-diol form in the streptose ring, and also to involve a nearby magnesium ion with one inner-sphere coordination to an rRNA phosphate group. This structure provides an important advance in our understanding of how streptomycin binds the mitochondrial ribosome that could aid future efforts to improve the therapeutic properties of streptomycin derivatives. There are a few issues the authors should address, however, as described below.

First, the authors suggest that adding streptomycin to the human cells prior to ribosome preparation ensures the complex is more physiological. It's not clear that they would have observed a different result had they purified mitochondrial small subunits and subsequently added the antibiotic in vitro. Rather, the more important point the authors could make is that the off-rate of the antibiotic must be rather slow for it to remain bound through the purification steps. (It would help to know just how much washing was done and with what volumes and times.) Is there evidence for slow off-rates, for example from wash-out experiments?

Second, the authors present an "unbound" structure in Figure 1c on the right. Where are the data for this structure? It also needs to be described and deposited.

Third, it is quite puzzling how the central streptose ring could be so flexible, given that the first and third rings are so well constrained by the binding pocket. Could the authors tell us whether it is possible that there is a mixture of aldehyde and gem-diol? It would help to see the aldehyde form modeled and fit to the density. The authors should present correlations for density fit as well.

misallignment

transferwise

As a rejoinder, have you ever undertaken any serious endeavor to work out exactly how difficult it would be to pay for the software you use that happens to be open source? Massively.

It's hard enough getting people to who sell SaaS/PaaS stuff to let you pay them a fair price for letting you use their "free" tier if none of their other (often pricey, enterprise-/B2B-oriented) plans offer you anything of value—and these are entities that already have payment processing infrastructure set up!

It's always interesting to me how people can be so blinded to the reality of another's situation. I guess in some ways it's to be expected. I just hope situations like these are indicative of how the majority of politicians operate because their constituents would suffer greatly if that were the case

I think it's very ignorant for people to think this way. This sort of reminds me of when individuals suggest homeless people to work so that they’re not homeless anymore. Like just because things are offered does not mean they are easily reachable.

Very important piece right here that I'm glad the author pointed out. I knew that there was certain core topics that must be taught in any particular class and that the teachers generally had autonomy over how they covered these topics, but I was unaware just how great an effect their biases had on the material. How much of what I learned growing up, or lack thereof, came about as a result of personal biases harbored by my educators? I think it's important to recognize that this is an issue and deserves a lot more recognition. It'll help provide a more holistic approach to learning as a whole

Through my experiences with different professors at UCI, you can tell how much more passionate they are teaching then high school teachers. I can see the joyment on the professor's face when students participate and unmute themselves to talk/ask a question on zoom. It's sad to think that professors feel like students don't appreciate them when in reality most of the students are engaged into the class because they genuinely want to learn about the subject and it is much more helpful when the professor engaged more the students instead of just lecturing through a screen.

Reviewer #3 (Public Review):

Murphy et al. further develop the linked selection model of Elyashiv et al. (2016) and apply it to human genetic variation data. This model is itself an extension of the McVicker et al. (2009) paper, which developed a statistical inference method around classic background selection (BGS) theory (Hudson and Kaplan, 1995, Nordborg et al., 1996). These methods fit a composite likelihood model to diversity data along the chromosome, where the level of diversity is reduced by a local factor from some initial "neutral" level π0 down to observed levels. The level of reduction is determined by a combination of both BGS and the expected reduction around substitutions due to a sweep (though the authors state that these models are robust to partial and soft sweeps). The expected reduction factor is a function of local recombination rates and genomic annotation (such as exonic and phylogenetically conserved sequences), as well as the selection parameters (i.e. mutation rates and selection coefficients for different annotation classes).

Overall, this work is a nice addition to an important line of work using models of linked selection to differentiate selection processes. The authors find that positive selection around substitutions explains little of the variation in diversity levels across the genome, whereas a background selection model can explain up to 80% of the variance in diversity. Additionally, their model seems to have solved a mystery of the McVicker et al. (2009) paper: why the estimated deleterious mutation rate was unreasonably high. Throughout the paper, the authors are careful not only in their methodology but also in their interpretation of the results. For example, when interpreting the good fit of the BGS model, the authors correctly point out that stabilizing selection on a polygenic trait can also lead to BGS-like reductions.

Furthermore, the authors have carefully chosen their model's exogenous parameters to avoid circularity. The concern here is that if the input data into the model - in particular the recombination maps and segments liked to be conserved - are estimated or identified using signals in genetic variation, the model's good fit to diversity may be spurious. For example, often recombination maps are estimated from linkage disequilibrium (LD) data which is itself obtained from variation along the chromosome. Murphy et al. use a recombination map based on ancestry switches in African Americans which should prevent "information leakage" between the recombination map and the BGS model from leading to spuriously good fits. Likewise, the authors use phylogenetic conservation maps rather than those estimated from diversity reductions (such as McVicker et al.'s B maps) to avoid circularity between the conserved annotation track and diversity levels being modeled. Additionally, the authors have carefully assessed and modified the original McVicker et al. algorithm, reducing relative error (Figure A2).

One could raise the concern that non-equilibrium demography confounds their results, but the authors have a very nice analysis in Section 7 of the supplementary material showing that their estimates are remarkably stable when the model is fit separately in different human populations (Figure A35). Supporting previous work that emphasizes the dependence between BGS and demography, the authors find evidence of such an interaction with a clever decomposition of variance approach (Figure A37). The consistency of BGS estimates across populations (e.g. Figures A35 and A36) is an additional strong bit of evidence that BGS is indeed shaping patterns of diversity; readers would benefit if some of these results were discussed in the main text.

I have three major concerns about this work. First, it's unclear how accurate the selection coefficient estimates are given the non-equilibrium demography of humans (pre-Out of Africa split, and thus not addressed by the separate population analyses). The authors do not make a big point about the selection coefficient estimates in the main section of the paper, so I don't find this to be a big problem. Still, some mention of this issue might be helpful to readers trying to interpret the results presented in the supplementary text.

Second, I'm curious whether the composite likelihood BGS model could overfit any variance along the chromosome - even neutral variance. At some level, the composite likelihood approach may behave like a sort of smoothing algorithm, albeit with a functional form and parameters of a BGS model. The fact that there is information sharing across different regions with the same annotation class should in principle prevent overfitting to local noise. Still, there are two ways I think to address this overfitting concern. First, a negative neutral control could help - how much variation in diversity along the chromosome can this model explain in a purely neutral simulation? I imagine very little, likely less than 5%, but I think this paper would be much stronger with the addition of a negative control like this. Second, I think the main text should include the R2 values from out-sample predictions, rather than just the R2 estimates from the model fit on the entire data. For example, one could fit the model on 20 chromosomes, use the estimated θΒ parameters to predict variation on the remaining two. The authors do a sort of leave-one-out validation at the window level (Figure A31); however, this may not be robust to linkage disequilibrium between adjacent windows in the way leaving out an entire chromosome would be.

Finally, I feel like this paper would be stronger with realistic forward simulations. The deterministic simulations described in the supplementary materials show the implementation of the model is correct, but it's an exact simulation under the model - and thus not testing the accuracy of the model itself against realistic forward simulations. However, this is a sizable task and efforts to add selection to projects like Standard PopSim are ongoing.

a hypercube house

30-50% of women diagnosed with depression being misdiagnosed is such a large margin of error that it can't possibly be true. Unfortunately it is true, and that just goes to show how women continue to be misdiagnosed. You would think that in todays day-in-age, and modern medicine, that there wouldn't be any medical misdiagnoses, but the continual medical biases failing women prove that to be false.

Reviewer #2 (Public Review):

The authors present genetic and cellular evidence for a role for Shh in repressing EMT, such that in the absence of Shh cells undergo EMT and are replaced with a novel epithelial cell population of unknown origin. The methodology is straightforward and although the paper is essentially a standard mutant phenotype analysis, given the relative lack of knowledge about how this important structure develops these results make a potentially significant contribution to field.

The first part of the study investigates how Shh signaling normally is deployed during larynx development, using an allele of Shh that expresses both GFP and Cre, allowing both real-time gene expression reporter and lineage trace functions in the presence of normal SHH signaling.They then investigate whether complete loss of Shh impacts these same phenotypes. They show using IHC and RNA-seq the upregulation broadly of EMT-associated markers, and the localized cadherin switch and vimentin expression consistent with EMT, as well as the morphological appearance of what appear to be extruded cells within the lumen. They then document the ontogeny of this cadherin switch, and confirm the origin of these cells using the ShhCre lineage trace, as well as demonstrating they undergo apoptosis at high rates after leaving the epithelium. These cells also downregulate Foxa2, a transcription factor known to suppress EMT. These data are all straightforward, quantified, and reasonably interpreted.

The more interesting aspect of this mutant analysis is their investigation of why this increase in EMT and apoptosis doesn't result in loss of epithelial integrity. They revisit Figure 4 and point out that only the ventral pharynx is Shh lineage-positive at E10.5, even though it is initially expressed throughout the pharynx. Their conclusion that this result implies "that foregut epithelial cells undergo dynamic regional cellular rearrangements during this timepoint" is a bit vague, and it isn't actually clear what they mean - what kind of rearrangements, and where do these non-Shh lineage cells come from? They then show that there are very few Shh lineage positive cells in the mutants at this stage (although the quantification isn't quite correct - 80% positive to 20% positive is a loss of 75% of the Shh lineage cells - and the image shown only has a couple of TdT+ cells, not consistent with the 20% their quantification in panel L shows).

The authors then explore where these Shh lineage-negative cells come from. The authors start by suggesting that perhaps they are dorsal-identity cells that spread, and use Sox2 and Nkx2.1 as dorsal and ventral identity markers. However, both of these markers are reduced in the mutants, leading them to conclude that Shh is required for regional identity of both dorsal and ventral cells. However, this doesn't answer the question of whether dorsal cells spread ventrally, and the shape of the pharynx and the epithelium itself are both really abnormal, so it's just unclear what is happening here.

They then look at RNA-seq data again for a clue and find that these cells have up-regulated pharynx-related genes most of all, the highest of which is Pax1, normally a pharyngeal pouch-specific transcription factor. They propose several possibilities for the origin of these cells, but do not address this question. As written, this is an interesting observation but not really explored.

The prevailing thought in my head while reading this was not simply just the appalling extent of the erasure, but the fact that it has innocuously expanded and its narratives still stand today. While learning history in school, the innovations made during the Scientific Revolution were touted as "the best of their time" - was it to assert western supremacy, or are we synonymizing innovation for the best? If it's the latter, it is a benign mistake that ultimately perpetuates the petty goals of erasure in history.

explanation

This point catches my eye for a few reasons. Part of me thinks that it's unreasonable to question someone who has done so much for the field and contributed in ways that led to receiving the Nobel prize. However, I also appreciate that everyone is held to the same standard, regardless of your past accomplishments and contributions. It not only keeps the field competitive, but allows newcomers to have just as much of a shot.

The Aboriginal flag was developed in 1971 by Luritja artist Harold Thomas. Centering its importance to Aboriginal Australians, the sun appears in the middle of the flag.

It's subtle here, as in other instances, but notice that Hamacher gives the citation to the Indigenous artist that developed the flag and simultaneously underlines the source of visual information that is associated with the flag and the sun. It's not just the knowledge of the two things which are associated to each other, but they're also both associated with a person who is that source of knowledge.

Is this three-way association common in all Indigenous cultures? While names may be tricky for some, the visual image of a particular person's face, body, and presence is usually very memorable and thereby easy to attach to various forms of knowledge.

Does the person/source of knowledge form or act like an 'oral folder' for Indigenous knowledge?

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

Reviewer #1 (Evidence, reproducibility and clarity):

The authors present further investigation of the Sox transcription factors in the model Cnidarian Hydractinia. They showcase the Hydractinia as now a relatively technically advanced model system to study animal stem cells, regeneration and the control of differentiation in animal cells. In this study they characterise the neural cells in hydractinia using FACS and sing cell transcriptome sequencing, investigate the sequential expression of SoxB genes in the i-cells and presumptive lineage giving rise to i-cells and investigate the neuronal regeneration making good use of transgenic rules. Finally, they investigate the role of SoxB genes in embryonic neurogenesis.

There are no major or minor issues effecting the conclusions

Reviewer #1 (Significance):

This study helps to confirm the role of an important group of transcription factors is conserved across the metazoan as well as showcasing an exciting model organism for regeneration and stem cell biology. This will of interest to a broad audience of developmental and biologists.

My own research is in the same field, using a different model system

Referees cross-commenting

I agree with the comments from the other reviewers, and am sure the authors can address these adequately with further explanation.

Reviewer #2 (Evidence, reproducibility and clarity):

Summary

Chrysostomou et al. investigate the role of three putative SoxB genes in embryonic neurogenesis in the colonial hydrozoan Hydractinia. They show that SoxB1 is co-expressed with Piwi in the multipotent i-cells and, using transgenics, they show that these Piwi/SoxB1 cells become neurons and gametes, consistent with the cell types that differentiate from i-cells. They further suggest that SoxB2 and SoxB3 are expressed downstream of SoxB1 in the progeny of the i-cells and, using shRNAs, investigate the role of SoxB genes on embryonic neurogenesis. The primary conclusions center on the similarity between neural differentiation in humans and Hydractinia as both systems pattern neurons using sequential expression of SoxB genes during the differentiation of neurons. The manuscript presents a large and diverse set of data derived from analysis of transgenic animals, single-cell sequencing, and investigation of gene function; despite this, the conclusions are either not particularly novel or not well-supported. The co-expression of SoxB1 in Piwi-expressing i-cells appears to be both novel and significant but the implications are not clearly indicated. Additional specific concerns are detailed below.

Major comments

1. SoxB genes act sequentially<br /> Knockdown of SoxB2 has already been shown to result in the loss of SoxB3, so the sequential action of SoxB genes in this animal does not seem to be a terribly novel conclusion.

Sequential expression of Soxb1-Soxb2 has not been demonstrated previously. Flici et al. did show some data on Soxb1 expression but these were not detailed. Furthermore, they have not shown in vivo transition to Soxb2. Our new single-molecule fluorescence in situ hybridization, and the transgenic reporter animals have been developed to address these issues.

While this manuscript does appear to report the most comprehensive analysis of SoxB1 expression, the evidence for sequential activation of SoxB1 and then SoxB2 in the same lineage (Figure 4) is a bit troubling. Panel A of this figure appears to show complete overlap between SoxB1 and SoxB2, suggesting all the cells in this field are synchronously passing through the transition point from SoxB1 to SoxB2 expression. While this may reflect reality, it would be more convincing to see adjacent cells expressing SoxB1 only or SoxB2 only, reflecting the dynamic progression of cell type specification along the main body axis.

As shown in Figures 1, Soxb1 is expressed by i-cells (together with Piwi1) in the lower body column of feeding polyps and in germ cells in sexual polyps. These cells do not express Soxb2. Figure 2 shows that Soxb2 is expressed more orally in a population of putative i-cell progeny as they migrate towards the head. These cells still express Soxb1. In the upper part of the body column, just under the tentacle line, there are Soxb2+ cells that do not express Soxb1. Therefore, cells expressing Soxb1 but not Soxb2 are present in the basal part of the polyp, Soxb1+/Soxb2+ double positive cells in the mid body region (i.e., the interface between the two domains where Soxb1+ cells start to express Soxb2 and downregulate Soxb1.), and cells expressing Soxb2 but not Soxb1 in the upper part of the polyp, just under the tentacle line. In Figure 4, we show the interface between these two domains using in vivo imaging of double transgenic reporter animals to visualize the Soxb1 to Soxb2 transition. Indeed, in the mid body area, most Soxb1+ cells also express Soxb2 (Figure 2). Hence, Figure 4 should be seen keeping Figure 2’s data in mind. At the mRNA level, the overlap between the Soxb1 and Soxb2 domains is smaller (Figure 2) than the one shown in Figure 4 because the latter constitutes a lineage tracing, showing fluorescent proteins with a long half-life. Therefore, when i-cells downregulate Soxb1 while starting to express Soxb2, the long half-life of tdTomato results in red fluorescence persisting longer than the mRNA encoding it. We have added cartoons to Figure 4 to indicate the position along the main body axis that are depicted.

Panel B is more concerning; while the authors have highlighted a cell that does appear to transition from SoxB1+ to SoxB1+/SoxB2+, there are several cells in the background that appear to gain SoxB2 expression without first expressing SoxB1. Do these cells constitute a fundamentally different, SoxB1-indpenendent, lineage of SoxB2+ cells? This would be noteworthy but is not mentioned or characterized.

The panels included in Figure 4 constitute selected confocal slices of stacks acquired in vivo. During imaging, cells move in three dimensions, making them appear and disappear in given optical planes over time. In other words, the individual time frames shown (T0-T5) were not always found in the same plane due to cell migration in the Z dimension. The cells that appear to gain Soxb2+ w/o having expressed Soxb1 first are an example of such cells. They are probably Soxb2+ cells that had already downregulated Soxb1 and migrated into the respective plane of image. We have added the explanation to Figure 4's legend.

Figure 7 shows the effect of SoxB1 knockdown (by shRNA) on the number of Piwi-expressing cells, nematocytes, etc but why not show that SoxB2 and SoxB3 are also knocked down in these experiments? Figure S11 shows no effect of SoxB2 and SoxB3 knockdown on SoxB1 expression but why wasn't the reciprocal experiment performed? If SoxB2 and SoxB3 are really downstream of SoxB1, the authors should demonstrate that with the shRNA experiments.

Our data show that Soxb1 is expressed in i-cells and its KD reduces the number of these stem cells (assessed by expression of Piwi1, an i-cell marker). Because i-cells give rise to all Hydractinia somatic lineages (and to germ cells), focusing specifically on Soxb2+ cells would provide no further insight because all cell types are expected to be affected. Indeed, injection of shRNA targeting Soxb1 resulted in smaller animals with multiple defects, including but not limited to the neural lineage.

1. Knockdown of SoxB genes resulted in complex defects in embryonic neurogenesis<br /> The manuscript aims to detail the roles of SoxB1, SoxB2, and SoxB3 in embryogenesis but only one of the main figures even shows pre-polyp life stages (Figure 7) and the results presented in in this figure are confusing. The authors suggest that knockdown of SoxB3 had no effect on embryonic neurogenesis but another interpretation of these data is that the SoxB3 shRNA simply did not work. The authors should provide additional support to show that this reagent is working as expected.

This information is included in Figure S11. Using mRNA in situ hybridization, we show that injection of shRNA targeting Soxb3 causes transcriptional downregulation of Soxb3 but not of Soxb2. The figure also shows the specificities of the shRNAs targeting Soxb1 and Soxb2.

Further, the results for SoxB1 and SoxB2 knockdown do not support the previous investigation of the role of SoxB2 in neurogenesis (Flici et al 2017). If SoxB1 is upstream of SoxB2, how does knockdown of SoxB1 have such a dramatic effect on RFamide neurons and nematocytes but knockdown of SoxB2 has an effect only on RFamide neurons? Is it possible the SoxB2 shRNA also wasn't working as expected? Can the results of the Flici et al 2017 paper showing SoxB2 knockdown in polyps be recapitulated using these shRNAs? If the point is to argue that embryos and adults (polyps) use fundamentally different mechanisms to drive neurogenesis, then the results presented in Figures 1-6 (which investigate SoxB genes in polyps) can't really be used to make inferences about embryonic neurogenesis. I think the authors have more work to do to demonstrate that embryonic and adult neurogenesis fundamentally differ.

The Soxb2 shRNA specificity is shown in Figure S11 (i.e., it KD Soxb2 but not Soxb1). We were equally surprised to discover that Soxb2 KD resulted in somewhat different phenotypes than the ones obtained by Flici et al. (2017) in polyps. At this stage, we cannot explain the difference. However, one could speculate that it resulted from slightly different regulation logic between embryonic and adult neurogenesis. More specifically, we propose different priorities for generating neural subtypes as explanation. Unfortunately, shRNAs work only with embryos, and long dsRNA mediated KD works only with polyps. CRISPR/Cas9-mediated KO is feasible in Hydractinia, but knocking out developmental genes, such as these Sox genes, would likely cause embryonic lethality. Other conditional KO/KD approaches are not available for Hydractinia. We believe we have made all possible efforts to clarify the roles of these genes using currently available techniques. Neurogenesis is a complex process that is only partially conserved among different animals and poorly studied in non-bilaterians. Furthermore, it is not possible to answer all questions in one study. As many studies before, our work contributes to the understanding of neurogenesis but also raises new questions. Addressing them is matter for future research. We have toned down the statement in the last sentence of the results and in the discussion and do not claim that embryonic and adult neurogenesis are fundamentally different.

Minor comments

Methods: A large bit of data from this manuscript relies on quantitative analysis of cell number but there's not enough information in the methods to understand how quantification was performed. How many slices from the z-stack were analyzed? Were counts made relative to the total tissue area in the X/Y dimension or relative to the number of total nuclei in the same section? How many individuals were examined for each analysis?

All cell counting analysis was performed using ImageJ/Fiji software. Counts were made relative to the total tissue area in the X/Y dimension (for the shRNA experiments). A Z-stack covering the whole depth of each larva was obtained. Counting was performed on cells positive for the respective cell type marker based on antibody staining and numbers were compared between shControl and shSoxb1/2/3 animals. At least 4 animals were counted per condition.

Page 11 - "Piwi2low cells, which are presumably i-cell progeny" - how were "high" and "low quantified?

“High” and “low” were not quantified. This is because i-cells progressively downregulate Piwi genes (i.e., Piwi1 and Piwi2) as they differentiate but this is a continuous process. Hence, it is difficult to put a threshold of Piwi1/Piwi2 protein level below which a cell ceases to be an i-cell while becoming a committed progeny. This is a similar process that is well documented in other animals where stemness markers are gradually downregulated during differentiation.

Page 13 - "a role in maintaining stemness" - this comment is not totally clear to me. Why would the number of EdU+ cells increase if the role of SoxB1 is to maintain stemness? Wouldn't SoxB1 knockdown then force stem cells to exit their program, resulting in early differentiation of i-cell progeny? This should be clarified.

KD of Soxb1 resulted in a decrease in the number of i-cells (i.e., Piwi1+ ones), suggesting that the gene is required for stemness maintenance. The increase in the numbers of cells in S-phase in this context was not related to i-cells because most of them were Piwi1-negative (Figure 7B). The identity of the cells in S-phase remains unknown, but a plausible explanation is that i-cell progeny (e.g., nematoblasts; see also next comment) increase their proliferative activity when i-cells numbers are low as a compensatory mechanism. This is merely a speculation. We have rephrased the paragraph to increase clarity.

Page 13 - "if progenitors are limiting" - if progenitors are limited why would there be an increase in nematocytes?

We do not have a definitive answer to this question but speculate that nematoblasts (i.e., stinging cell progenitors) account, at least in part, for the excessive proliferation seen under Soxb1 KD. This may constitute a mechanism allowing a depleted i-cell population to recover by self-renewal (instead of differentiation), moving temporarily the proliferation task to committed progeny (e.g., nematoblasts) until i-cell numbers return to normal. However, in the absence of evidence we refrain from expanding on this in the text.

Figures 1 and 2 claim to show "partial overlap" but they look perfectly overlapping to me. This makes the situation in Figure 4B difficult to interpret.

Figure 1 shows full overlap between Piwi1 and Sox1 expression and this is reflected in the text. Figure 2 shows no overlap between Soxb1 and Soxb2 in the lower body column (where only Soxb1 is expressed), overlap in the mid body region, and Soxb2 only expressing cells in the upper part of the body, just under the tentacle line. Similarly, the figure shows overlap between Soxb2/Soxb3 under the tentacle line, and predominantly Soxb3 above it in the head region. The small cartoons at the left side of each panel indicate its position along the oralaboral axis. See also our reply to the second part of comment #1.

Figure 4 - No indication of which part of the animal or which stage is shown in these images.

We have added cartoons to indicate the area in the polyp from where the images were taken.

Figure 5 - No indication of where these dissociated cells came from - polyps? Larvae?

All tissue samples were taken from feeding polyps; this is now mentioned in the Materials and Methods section.

Panel D is a bit perplexing - what are the "progeny" of Piwi+ cells if not SoxB2+ cells and their derivatives?

In Panel D, we show three cell fractions. One constitutes i-cells, based on high Piwi1 expression (green fluorescence of the Piwi1::GFP reporter transgene) and morphology; one fraction includes nematocytes, based on the characteristic nematocyst capsule, and one constitutes a mixture of other i-cell progeny. The latter includes different cell types, given that i-cells are thought to contribute to all lineages. They have only dim GFP fluorescence because the Piwi1 promoter-driven GFP shuts down upon i-cell differentiation. Soxb2+ cells are also among them but are not the only i-cell progeny.

Why are nematocytes but not neurons indicated?

Neurons are shown on Panels E & F. See also next comment.

Piwi seems to be maintained in Ncol-expressing cells but not in SoxB2- or RFamide-expressing cells? Does this suggest that Piwi is turned on in i-cells, off in SoxB2-expressing cells, and on again in terminally differentiating nematocytes? This would be quite surprising and should be verified with antibody labeling/imaging in Piwi transgenics to confirm the result. The resolution for Panel M is too low to evaluate this part of the figure.

The Piwi1i gene is downregulated upon i-cell differentiation. In the Piwi1:GFP reporter animal, residual GFP fluorescence persists post differentiation due to GFP's long half-life. The brightness of which depends on the time elapsed since differentiation. Because nematocytes are short living cells with high turnover, most nematocytes have recently differentiated and are therefore relatively bright green in the Piwi1::GFP animal. Neuron turnover is lower, making most neurons in the same transgenic animal appear dim. The resolution of the imaging flow cytometer is limited because the machine images 1000s of cells per second through all optical channels. However, it is high enough to allow the identification of features such as cell shape, some organelles (e.g., nematocytes), nuclear size and shape, and fluorescence intensity.

Figure 7 - the low magnification images provide nice overall context but the authors should also provide high magnification panels for the same images. Without them it is not possible to assess "defects in ciliation" or to determine if there are defects in GLWamide neurons from these knockdowns (e.g., neurite vs cell body defects). There's no mention of the fact that SoxB1 knockdown resulted in complete loss of RFamide cells, which is strange. Are there SoxB2-independent populations of RFamide? Panel B could be interpreted multiple ways - downregulation of Piwi in SoxB1 shRNA or upregulation in SoxB2/B3. The authors should provide an image of control shRNA-injected larvae with the same co-labeling of Piwi/EdU for context. From the images, it's not clear that there were differential effects of SoxB2 and SoxB3 on nematocytes.

The resolution of the images is, in fact, high, allowing it to be blown up on the screen. Even higher magnification of ciliation can be seen in Figure S12. KD of Soxb1 resulted in complete or nearly complete loss of Rfamide+ neurons. We have added this statement to the text as requested. Panel B shows the relative difference in Piwi1+ and S-phase cells between shSoxb1, shSoxb2, and shSoxb3-treated animals. The quantification relative to the control is presented in Figure 7C.

Figures 6 and S9 - why piwi2 and not piwi1?

In Figure 6, we co-stained the regenerates with two antibodies: one was a rabbit anti-GFP (to visualize the RFamide+ neurons), and the other was a guinea pig anti-Piwi2 (to visualize icells). The anti-Piwi1 antibody that was used in other images to visualize i-cells was raised in rabbit and could not be used in conjunction with the anti-GFP one.

Figure S1 - Kayal et al 2018 is the most recent phylogeny of cnidarians and should probably be cited in place of Zapata throughout the manuscript. Independent of this, the polytomy in Figure S1 panel A is not supported by either Zapata or Kayal and should be fixed.

We have cited Kayal et al. 2018 and revised the tree in Figure S1 as pointed.

Figure S3 - is this mRNA? Protein? Panels E-G are too small to interpret. Please provide stage/time for cartoons in panel H.

As per the legend, Panels A, B, D, E, F refer to protein; C is lectin staining (DSA), and G is EdU. The resolution of Panels E-G is actually high, allowing blowing up of the images on the screen to view the details. The stages of the cartoon in Panel H are now provided in the figure legend.

Figure S11 - please provide images of whole larvae as shown for Piwi knockdown in Fig S9 and some additional support (e.g., qPCR) to demonstrate the shRNAs are actually working.

Figure S9 represents immunostaining using the anti-Piwi1 antibody. In Figure S11, we show the specificity of the shRNA treatments; we used highly sensitive single-molecule mRNA in situ hybridization. Whole animal imaging is not informative due to the punctuated nature of the single-molecule staining.

Figure S12 - it's not clear what ciliary "defects" are being shown.

In the control, cilia are uniformly distributed along the oral-aboral axis whereas in the shSoxb1-injected animals, the pattern is patchy. Additionally, shSoxb1-injected larvae could not swim (planulae swim by coordinated cilia beat).

Reviewer #2 (Significance):

Generally, the results are either equivocal or the conclusions are not well supported by the results (as detailed above). The significance of this work to vertebrate neurobiology is somewhat weak. (Especially considering the orthology of these genes to bilaterian SoxB genes is not well supported.) Why not compare these results to other cnidarians - the expression patterns of SoxB1 and SoxB2 in corals and sea anemones seem to differ quite a lot (Shinzato et al 2008; Magie et al 2005), suggesting these genes are almost certainly not behaving in the same way across cnidarians. This is exciting! What's happening in Hydra? Seems like it should be possible to mine the single-cell data set from Siebert et al to test these hypothesized relationships between the Sox genes in another hydrozoan which constantly makes new neurons.

We have modified the concluding section in the discussion, in line with this comment. See also comment to Reviewer #3.

Reviewer #3 (Evidence, reproducibility and clarity):

This paper characterizes the role of Soxb genes in neurogenesis in Hydractinia. The authors use cutting edge approaches including FISH, transgenics, image flow cytometry, FACS and shRNA knock downs to characterize SoxB in Hydractinia. The images are beautiful, the data is sound and the interpretation of the data is appropriate.

I have only minor suggested listed by section below:

Abstract<br /> - The abstract and introduction should make clear that this is a colonial animal and the cell migration occurs from the aboral to the oral end of the polyp (not the animal, as there are many oral ends). This is relevant to the interpretation of the data as the polyps do not act in isolation as they interconnected and may communicate via the stolonal network that connects the polyps in the colony.

We have added a section to the Introduction to address the reviewer's comment. The Abstract, however, is too short to include this explanation.

• The human disease justification is a relatively weak one and does not need to be included. Using Hydractinia to understand the role of SoxB in the evolution of neurogenesis in animals is enough justification for the study.

We have adopted the reviewer's comment and modified the statement in the discussion (see also comment to Reviewer #2).

Introduction<br /> - Instead of Sox phylogenies (the term phylogeny is more appropriate for species trees), consider substituting, for Sox gene trees. And instead of "phylogenetic relation" use the term "orthology"

This has been done.

• The number of times the sentences that have the sentiment "....remain unknown." "....little is known.." "...unclear..." , "....difficult to establish...." etc. is distracting and detracts from what IS known about these genes. It is not necessary to continually justify the study throughout the introduction. Instead a clearer description of the background and setting up the question/hypothesis of SoxB paralog subfuctionalization in space and time - would be more informative to the reader.

We have reduced the number of occasions as recommended.

• The authors state that there are three SoxB genes in the Hydractinia genome? What genome? For several years there has been multiple papers published by subsets of these authors have used unpublished genome data, but the complete genome has yet to be released to the public. This is especially egregious because they cite their NSF funded EDGE proposal to CEF and UF which is supposed to develop tools to the community, and yet the community at large doesn't have access to the genome. If these data came from the genome, then the genome should be released. If these data came from a previously published transcriptome as in the previous SoxB paper then this should be stated explicitly.

The Hydractinia genome assembly, annotation, RNA-seq data, and genome browser are now available in the Hydractinia genome project portal at the National Human Genome Research Institute (NIH) website (https://research.nhgri.nih.gov/hydractinia/). The raw data have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA807936. This information has been added to the 'Resource availability' section.

Results<br /> - I assume there was no expression of Soxb2 and Soxb3 in the reproductive polyps? This should be stated explicitly.

Soxb2 expression in sexual polyps was consistent with the nervous system and with maternal deposition in oocytes. It was not detected in male germ cells. We have added a new in situ hybridization image of Soxb2 to Figure 12.

• The word "progeny" is used throughout to describe terminally differentiated cells. However, progeny implies offspring, but these are actually later stages of differentiation of the in a cell's ontogeny, thus the term should be changed to "differentiated cells"

We used "progeny" to indicate that the corresponding cells derived from a specific progenitor cell type. We did try replacing it with "differentiated cells" but this completely changes the meaning of the sentence: first, it does not include the cell of origin info and second, not all progeny are already fully differentiated.

• Typo on page 11 "This predictable generation of many new neurons provides an opportunity to study neurogenesis in [a ]regeneration." - Remove the "a"

Corrected.

• While the regeneration study is interesting, there is nothing revealed about the role of Soxb and there is not a lot of new information revealed about regenerations. Authors should better justify this section or consider omitting.

These sections demonstrate de novo neurogenesis in head regeneration. This was not known in this animal before.

Discussion<br /> - The authors assume that in the transgenic lineage, the fluorescent marker in differentiated cells is due to retention of fluorescence, but it is unclear if they can rule out that Soxb2 is still being expressed in those cells" Please clarify.

We conclude this by comparing the mRNA expression (Figures 1 & 2) with the fluorescent proteins (Figure 3).

• How did the authors determine that the shSoxb3 knockdown worked? Please discuss relevant controls and validation (either in discussion or methods). This is particularly important given that it didn't have an apparent phenotypic effect.

The efficacy of all shRNAs determined by in situ hybridization, showing that each shRNA downregulates its own target mRNA but not the others (Figure S11).

• Again, the connection to human health is a bit of a stretch. Instead, what is most interesting is the similarity of Soxb paralogs acting sequentially as has been found in vertebrates. This suggests a highly conserved mechanism of subfunctionization following gene duplication at the base of animals.

We agree. This is now also better highlighted in the discussion.

Figures<br /> - Its very hard to distinguish the overall abundance of Soxb2 and Soxb3 expression along the polyp body axis from the panels figure 2. A lower magnification or larger area in each region would be helpful

In Figure 2, we performed single-molecule in situ hybridization. While highly sensitive, this method generates spotty images because they highlight single molecules and are not coupled to an enzymatic reaction as in other methods. They mostly looks poor when showing low magnification images. Because a previous study (Flici et al. 2017) has already shown the general expression pattern, we aimed at providing the details of the transition.

• Figure 4 - either the figure is upside down or the text is upside down. It is also difficult to see the double staining (if any).

The figure is oriented to position the oral end up. The resolution of the panels is high, enabling blowing-up on the screen. The quality of in vivo time lapse images cannot match that of fixed and antibody stained ones, or of single in vivo images. This is because the animals are imaged for many hours during which they tend to bleach.

• Figure 5M is difficult to read due to the small print. Consider enlarging and moving it to Supplementary Material

The size of the text is small but the resolution is very high, enabling blowing up the image on the screen. We thought that the information was important enough to be presented in the main text and given that most readers would use the electronic version we preferred this option on another supplemental figure on top of the 12 we already have.

Reviewer #3 (Significance):

This is an interesting and important study because although it is well known that SoxB genes function in neurogenesis in animals, it is unclear how and if subfunctionalization occurs outside of vertebrates. Hydractinia is an excellent model to study SoxB genes because of its colonial organization and continuous development of nerve cells throughout the life of the animal. In addition, it is part of the early diverging cnidarian lineage and thus can provide insight into the relative conservation of SoxB genes across animals.

Reviewer #1 (Public Review):

Overview

This is a well-conducted study and speaks to an interesting finding in an important topic, whether ethological validity causes co-variation in gamma above and beyond the already present ethological differences present in systemic stimulus sensitivity.

I like the fact that while this finding (seeing red = ethnologically valid = more gamma) seems to favor views the PI has argued for, the paper comes to a much simpler and more mechanistic conclusion. In short, it's good science.

I think they missed a key logical point of analysis, in failing to dive into ERF <----> gamma relationships. In contrast to the modeled assumption that they have succeeded in color matching to create matched LGN output, the ERF and its distinct features are metrics of afferent drive in their own data. And, their data seem to suggest these two variables are not tightly correlated, so at very least it is a topic that needs treatment and clarity as discussed below.

Minor concerns

In generally, very well motived and described, a few terms need more precision (speedily and staircased are too inaccurate given their precise psychophysical goals)

I got confused some about the across-group gamma analysis:

"The induced change spectra were fit per participant and stimulus with the sum of a linear slope and up to two Gaussians." What is the linear slope?

To me, a few other analyses approaches would have been intuitive. First, before averaging peak-aligned data, might consider transforming into log, and might consider making average data with measures that don't confound peak height and frequency spread (e.g., using the FWHM/peak power as your shape for each, then averaging).

Moderate

I. I would like to see a more precise treatment of ERF and gamma power. The initial slope of the ERF should, by typical convention, correlate strongly with input strength, and the peak should similarly be a predictor of such drive, albeit a weaker one. Figure 4C looks good, but I'm totally confused about what this is showing. If drive = gamma in color space, then these ERF features and gamma power should (by Occham's sledgehammer...) be correlated. I invoke the sledgehammer not the razor because I could easily be wrong, but if you could unpack this relationship convincingly, this would be a far stronger foundation for the 'equalized for drive, gamma doesn't change across colors' argument...(see also IIB below)...

...and, in my own squinting, there is a difference (~25%) in the evoked dipole amplitudes for the vertically aligned opponent pairs of red- and green (along the L-M axis Fig 2C) on which much hinges in this paper, but no difference in gamma power for these pairs. How is that possible? This logic doesn't support the main prediction that drive matched differences = matched gamma...Again, I'm happy to be wrong, but I would to see this analyzed and explained intuitively.

II. As indicated above, the paper rests on accurate modeling of human LGN recruitment, based in fact on human cone recruitment. However, the exact details of how such matching was obtained were rapidly discussed-this technical detail is much more than just a detail in a study on color matching: I am not against the logic nor do I know of a flaw, but it's the hinge of the paper and is dealt with glancingly.

A. Some discussion of model limitations

B. Why it's valid to assume LGN matching has been achieved using data from the periphery: To buy knowledge, nobody has ever recorded single units in human LGN with these color stimuli...in contrast, the ERF is 'in their hands' and could be directly related (or not) to gamma and to the color matching predictions of their model.

Audrey Watters starts out a piece on the history of school bells with just the sort of falsehood that she's probably aiming to debunk. Perhaps she would have been better off with George Lakoff's truth sandwich model as starting off with the false story is too often has the opposite effect and leads readers down the road to inculcating the idea further into the culture.

She doesn't reveal the falsehood until the end of the third graph at which time one's brain has been stewing in falsehood for far too long.

This is a big gotcha. We need to close this, or else the OS won't understand that the input we don't care about isn't being used, and will continue listening from that unused input.

It's super important to think about implementing different means of engagement when in a classroom setting. We all learn differently and I know personally I have been in classes before where my style of learning was rarely or never used making it hard for me to just sit there and listen to a professor talk the entire class.

All students deserve access to higher education. This Gannon's inclusive design, all students will benefit. He goes on to mention how this does not just help out students who have been "disenfranchised". It is important to note that this is not just helping out some people, but everyone.

They probably just believe it's more feminine in their country.

Some discussion of doing research on zettelkasten methods and workflows.

What do note taking methods and processes look like for individual people?

What questions would one ask for this sort of research in an interview setting (compared to how one would look at extant physical examples in document-based research)? #openquestions

Link this to the work of Earle Havens on commonplace books through portions of history.

The commonly used terms for what would become the "sets" were "mannigfaltig (manifold, or as it was rendered by Clifford's translation of Riemann's habilitation, "manifoldness", and which was what Cantor called sets in the paper where he introduced the concept of ordinals) and "inbegriff (tough to translate, but it isn't just "collection" --- this was used in Cantors paper on algebraic numbers)", and "notion (used by Clifford)" and even a "multiplicity". It wasn't until Cantor's 1885 paper on "menge" that sets were clearly defined as collections of definite mathematical objects.

I write this because there is a tendency among mathematicians to rewrite mathematical history so as to make the current accepted practices seem logically inevitable. This is so common that it's largely unnoticed. But the ways we think about mathematics today are the result of historical processes, and are not eternal or necessary.

You can in fact do all of mathematics without a notion of collection --- if you use the notion of "type" (type, concept, notion, kind, sort...). This is almost a distinction without a difference, because the type theoretic theories of sets (where a set is a type for which the identification of elements is a proposition, or in the case of a subset is equated with its membership predicate) is equivalent to the set theoretic theory of sets (where sets are collections). But still, it narrows a students' view of the broadness of mathematics to say that the idea of a collection is necessary to do mathematics.

I appreciate the time given to describing how formalizing reasoning is largely a design problem, but I also think it is worth mentioning that there are actually different logics that reach different conclusions --- it's just that one logic is overwhelmingly standard. Ultimately, that's a historical accident, and not a mathematical fact.

I feel that the positive way to say this would be clearer: any WFF is of the forms (a) (b) or (c). (This is, of course, really a hidden way to state the mapping property of the set of WFFs.)

Just in case, let me state that mapping property, both in the set theoretic and in the "type theoretic" styles, so it's clear what I mean.

The set theoretic induction principle says that the set of WFFs is the smallest set containing the atomics and closed under the connectives. Explicitly, if X is a set containing the atomic formulas and closed under the operations, then X contains the set of WFFs.

The type theoretic induction principle says that to construct any mathematical object c assuming as given a WFF F, it suffices to construct c in the cases that F is atomic, that F is of the form ~G for G a WFF, that F is of the form G&H etc.

Why not, the category theoretic way to state this induction principle is that if X is any set and for every atomic a we have an associated element f_0(a) in X and for every x in X we have an element ~x in X and for every x and y in X we have an element x&y in X etc, then there is a unique function assigning each WFF F to an element f(F) of X so that atomics a are assigned to f_0(a), ~F is assigned to ~f(F), etc...

Taxonomies by folks - Folksonomy - Analogie zur Entwicklung des Wörterbuchs - spannende Beobachtung und spannende These

Honestly, no. Technology has been a part of my education since the beginning, and I have always been interested in it. That being said, I never considered that it was essential. It makes sense that it is, I just never took the time to stop and think about why it's always been there.

What so high about it. It's just a proper agreement, on paper.

I wonder if customers themselves actually believed that these silicon wristbands can cause some improvements in energy, flexibility, and balance, or did the customers already know and realise that these cannot have any real effect but it's just a "fun" thing to get? Same question about the people who support Flat Earth theory despite of huge amount of evidence that says otherwise. Do all these people sincerely believe in above mentioned things, or do they simply deceive themselves and deep inside realise that? Can this phenomenons' roots come from childhood when (almost) all of us had been fueled with countless fairytales, so that, consequently, now we have that internal desire to have something divine/magical in our lives, to be a part of something special?

Just test.

Hang on, I'm kind of lost. I thought this person was on the side of "There are too many Fire Emblem characters in Smash?"

We see everyone having their own opinion on the situation. No it isn’t a bad thing to have a more modernized living area but also it takes out the history of the place and what the people saw growing up. Yet I believe that the people should have a say rather than just the government.

Google Scholar is a recommended branch of the Google Search engine that is dedicated to locating only scholarly sources and bases the relevancy of an article. This is my first time seeing this word.

I need to understand the academic publication process and the structure of scholarly articles tells me a lot about how to find, read and use these sources.

The horse strikes the audience just as strikingly as it's rider, with it's mane and tail clean and braided, no less green, but intertwined with gold ribbons. The mane was then wrapped up into topknots, and secured with a green ribbons, decorated with gemstones. The same was done with the tail, and both were also hung with bright gold bells that jingled with every step. This helped complete the overall "otherness" feel that the Green Knight was projecting. Maybe a man in all green could be handled as an eccentric creature, but a steed to match tips this encounter into the fantastical. This gives pause to everyone in the banquet hall, and sets the stage for what further eccentricities the Green Knight concocts later on in the story. Laurel McCormick states that the symbolism of the green colors state to the wildness of the Knight character by saying, " The Green Knight has many things that link him to the free natural world; his greenness, his green horse, the holly branch, the axe and the Green Chapel. These objects reflect his wildness of character" (McCormick).

McCormick, Laurel. "Symbolic Color in Sir Gawain and the Green Knight." http://blogs.cofc.edu/seamanm-engl360-s17/2017/04/12/paper-proposal-symbolic-color-in-sir-gawain-and-the-green-knight/#:~:text=The%20colors%20green%20and%20gold,of%20nature%20and%20inner%20morality.

This is all so wrong that... I guess I'm gonna just unfollow this?

• Her mother was enslaved, and her father was a chieftain, so that matters to making it make sense as to why her hand would be a subject of negotiation.
• There is no debate about whether the semi-historical figure of Brigid of Kildare "identified as Pagan", holy shit, what do you think it means to be an abbess, like what do you think the meaning of the word is
• There is a story of her going to study with the druids and the druids patting her on the head and saying "you're meant for different things" and if you can't understand there was more going on with these dynamics than "England and Scotland were pressuring them to convert" then maybe don't write about this
• Brigid! Has! Her own! Feast day! St. Patrick's Day! Is for! St. Patrick! It's not like there aren't interesting pagan angles on St. Patrick!
• "Celtic" and "Christian" are not points of contrast, like imagine saying "Oh well her mom is Christian and her dad is French"

h

Huh! I like the idea of folding some pieces of clothing, like t-shirts -- but I like heaps of socks (then again, all my socks are black.)

Although I'm open to it, I'm not sure I'm into the idea of applying the concept of dignity to socks somehow. Perhaps it's better to save the concept for sentient beings only?

Aimee Benedict It's interesting to think of postmodernism as being like Narcissus' reflection in the water, disintegrating when one grasps it because it is simultaneously saying postmodernism is both reproduction and reflection- both re- words. It reminds me of a song I can't remember which's lyrics-"the world's just a shadow of what went before; the world gives off none of its own light". its kind of a similar idea. "Western Sky" by Mark Eitzel.

SUMMARY:

This article gives some basic information on Chabacano but doesn't focus on Zamboangueño. Lots of information is present on why it exists in the first place, with emphasis on Spanish's failure to take over and also how most Filipinos aren't aware of its existence. That being said, Chabacano is still very much alive, with close to a half million speakers present. It's more than just a broken form of Spanish, although it is often confused as such, making its origins hard to pinpoint.

Only Spanish-Creole language in Asia, some argue that it's just a reflexification (substitution of most/all vocabulary of one language to the other without changing gramatical structure) of Portuguese.

Author Response:

Reviewer #1:

The authors perform very careful growth speed and growth fluctuation analysis of microtubules growing in vitro in the presence of either GMPCPP or GTP. This is essentially a re-examination of highly cited work published by Gardner et al in 2011. The quality of the current analysis is improved compared to previous work, because the authors use a label-free imaging method providing higher signal-to-noise-ratio data and allowing longer imaging at higher time resolution, and because the fluctuation analysis is technically more advanced. The main conclusions are that growth fluctuations are lower than previously published by Gardner et al., however in the presence of GTP they are still higher than expected, as reported previously, but less dramatically different than proposed previously. The authors propose a kinetic model that includes the possibility of GTP hydrolysis causing a hypothetical (but plausible) slowdown of tubulin addition when a GDP tubulin is exposed at the microtubule end to explain the larger growth fluctuations in the presence of GTP. This is an important study proposing a new model for the origin of the natural growth fluctuations of microtubules. In the future, this work will also have an impact on our understanding of how regulators of microtubule polymerization act. Overall this is a carefully performed study, with especially the experimental and data analysis part being of very high quality.

Questions that the authors might want to address:

1. Can the measured growth fluctuations in the presence of GMPCPP be explained by an even simpler 1-dimensional single protofilament growth model? Or is indeed a 2-dimensional model required that the authors use here.

We thank the reviewer for their point that is now addressed as part of the broader explanation of the introduction. This helps give context to the need for the 2D model in the first place in lieu of the canonical 1D polymer model for growth. Earlier work (Gardner et al., “Rapid microtubule assembly kinetics”, Cell 2011) also demonstrated that 1D models are more limited in the magnitude of fluctuations that they can produce.

1. Can the measured taper of growing microtubule ends be used to further constrain the fits to the data?

This is an excellent point and should be achievable in principle. However, in the context of our simple model, we were unable to identify a set of parameters that could simultaneously recapitulate growth rates, growth fluctuations, and end taper. This is a limitation of our study that we acknowledge. We suspect that at least one additional state in the model will be required to improve its ability to predict end taper. This will be the subject of future work in our laboratories.

1. The authors mention that they choose the optimal kon from the fits to the GMPCPP data also for the fits to the GDP data, if this reviewer understood correctly. Is this justified, given that the longitudinal interactions are probably different in a GMPCPP and a GTP lattice?

The reviewer does understand the choice correctly: we used the same on-rate constant for fitting to the growth rates in GTP and GMPCPP. We think this is well- justified. First, 1D analyses (see Fig 1C and 3B) of the concentration-dependent growth rates yields apparent on-rate constants of 3.1 μM-1s-1 ± 0.6 and 2.3 μM-1s-1 ± 1.2 for microtubules grown with GMPCPP and GTP, respectively. These apparent on-rate constants fall within error of each other. Second, large changes in affinity like we observed between GMPCPP and GTP, are commonly assumed to manifest themselves in off-rate constants, not on-rate constants. Third, in the absence of evidence to the contrary, it just seems simpler to assume that GTP- and GMPCPP- bound tubulin will have similar on-rate constants for binding to the microtubule end. We added a sentence to be more this more explicit about this point.

1. How reliably can the kinetic model of the authors predict the GTP hydrolysis rate at growing microtubule ends and how does this rate compare to previously published measurements or models?

This is an interesting question from the reviewer. The GTPase rate constant we used here (0.08 s-1) falls at the lower end of the rather large range of values obtained in prior studies (range: 0.07 - 1 s-1). As we and others have noted previously, the relatively simple biochemical model we used does not capture the observed dependence of catastrophe frequency on tubulin concentration (e.g. Kim and Rice, MBoC 2019; also VanBuren et al., 2005). More complex models are better able to recapitulate this concentration-dependence, and in principle one could use measured catastrophe frequencies and/or GTP cap sizes as constraints on model fits. However, in the present work we chose to use the simplest model, and this is why we focused on trends with GTPase rate as opposed to one specific rate. We appreciate the opportunity to clarify this point, and we added a sentence to emphasize that we focused on trends with increasing GTPase rate rather than on a particular value of the GTPase rate.

Reviewer #3:

This paper applies rigorous quantitative microscopy to an open problem in biophysics, namely the kinetics of microtubule dynamic instability. Previous studies that analyzed these kinetics found them to be "fast", which is to say that tubulin binds very frequently to the end of a microtubule, but falls off almost as frequently (Gardner et al. Cell 2011). This "rapid self-assembly kinetics" is arguably the prevailing conceptual framework for microtubule polymerization. In contrast, the present study finds the kinetics of polymerization to be "slow", with infrequent binding events that persist for longer periods of time. The conceptual shift from "fast" to "slow" has significant implications, in particular for the mechanisms of microtubule polymerases.

The difference in results from Gardner et al. Cell 2011 comes from 2 places. First, the authors use interference reflection microscopy (IRM) instead of fluorescence. Using IRM allows them to image growing microtubules for long time intervals at high frame rates. Thus, a single microtubule can generate a long plot of length versus time, in contrast to Gardner, who concantenated many short traces together to create a long plot. Second, the authors apply sub-pixel drift correction to their movies and show conclusively that pixel-based drift correction contributes to the appearance of "fast kinetics". Figure 1 (and its supplements) are an outstanding example of technical rigor, where different analyses are displayed side-by-side to justify the conclusion of slow kinetics, particularly for the growth of GMPCPP-tubulin.

With GTP-tubulin in the reaction, growth is significantly more variable. To explain the increased variability, the authors use a computational model to test a particular hypothesis, namely that the tubulin at the very end of a microtubule can be in the GDP state, and that these terminal GDP-subunits have a reduced affinity for incoming dimers. In other words, the simulations argue that exposure of a GDP subunit at tip could "poison" that protofilament, and because that protofilament now lags behind the others, the microtubule end position fluctuates. But the manuscript is missing an experimental corrolary for their model of GDP exposure. And there are other potential explanations for why GTP-tubulin growth could be more variable than GMPCPP-tubulin growth. For example, we know that GMPCPP microtubules are stiff and uniformly 14-pf. Perhaps growth fluctuations are linked to tubulin's flexibility, which is included as a parameter in some computational models (e.g., Zakharov Biophys J 2015). The modeling here has demonstrated that GDP exposure is sufficient to explain growth variation, but they have not demonstrated that it is necessary, which would require experiments. The authors should spend part of their discussion considering alternative models and arguing explicitly for why trans-acting nucleotide makes sense.

We added a sentence to the ‘Limitations of the model’ section to provide additional kinds of model alterations than were already listed.

We also added a sentence to be more explicit about why we favor trans-acting GTP.

The idea that GDP exposure could "poison" a protofilament end reminded me of eribulin and Doodhi et al., Curr Biol 2016. After all, eribulin is a bona fide poison (err, microtubule-targeting anti-cancer drug). Doodhi et al. defined the binding site for eribulin as the terminal end of b-tubulin, meaning that it blocks incoming subunits. They showed that the drug perturbed dynamic instability significantly, induced catastrophes, created "split EB comets", etc. Is the poisoning effect of eribulin related to the poisoning effect of GDP-exposure? Are eribulin and GDP-exposure both explainable as alterations in longitudinal affinity? A discussion of this comparison would be interesting.

These are interesting questions. It’s not clear (to us, at least) that eribulin can be taken as equivalent to GDP-exposure. Indeed, there are interesting differences in the effects observed from different plus-end modulating compounds (GDP, eribulin, and even Darpin). These different modulators have the ability to limit protofilament elongation by blocking the terminal β-tubulin interface but give rise to different effects that probably depend on the lifetime of the blocked state and perhaps also other allosteric effects. For the sake of simplicity, we would prefer not to incorporate these ideas into the manuscript.

Lastly, the relationship of to the authors' previous computational work (Piedra et al. MBoC 2016) needs further elaboration. In Piedra et al., their model allows GTP exchange into the poisoned GDP-terminal subunit. In this manuscript, the exchange is disallowed, which is the same as saying that its rate is 0. Is this reasonable? In Fig. 3B of Piedra, they plot how catastrophe frequency is affected by the rate of GDP->GTP exchange. If exchange is slow, then the impact of exchange on catastrophes is minimal. Is the same true for growth? The current manuscript should be viewed as an opportunity to elaborate on Piedra to the extent possible. It's clear in Piedra that the GTPase rate itself matters in terms of the sensitivity of catastrophes to GDP->GTP exchange rates. The authors write "a finite rate of exchange would only modulate the amount of GDP on the microtubule end for a given GTPase rate; it would not eliminate the 'poisoning' effect of GDP exposure that increases fluctuations in growth rate." But the interesting question is the sensitivity of the growth rate to the finite rate of GDP->GTP exchange.

As one might expect, if the rate of GDP->GTP exchange is too fast, the effects on growth rate and fluctuations vanish (because exchange effectively becomes instantaneous). If the rate of exchange is too slow, there is no change from the ‘no exchange’ simulations. At intermediate rates of exchange, the magnitudes of the effects on growth rate and fluctuations decreases as the exchange rate increases. We saw no evidence for a regime where growth rates but not growth fluctuations (or vice versa) were affected. We prefer to not dwell on this in the present manuscript, but we hope to revisit the question experimentally in the future.

A memo to myself last week on this topic: people—even smart ones—still have trouble wrangling the implications of natural selection.

Natural selection does not mean survival of the fittest. It means survival of the good enough.

There was more to it than that, but funnily enough, there's this part:

Dan Luu writes of Twitter and companies generally that they should continue to hire so long as it makes financial sense, and what makes financial sense is defined by an equation where savings at the margin is one component

Understanding this is important, not just for the reasons that Dan mentions in the linked ("I could do that in a weekend!") piece, but because it gives companies a budget for inefficiency.

Companies can make non-optimal decisions. (Deleterious ones, even.a^1 a^2)

Instant death is not really a thing, although most misunderstandings demand that it is (as in the case of the parable of the "non-existent" 20-dollar bill). This is the timeless fallacy.

When i was working this into a starter for a potential standalone piece, i drafted the following passage, hoping to use it as an intro:

This is not a piece about biology. It is not even a piece about humans' understanding of biology. It is a piece about humans and humans' inability to grapple with particularly counterintuitive concepts (like natural selection).

i also jotted down the phrase "cognitive fluency".