Reviewer #1 (Public Review):

Summary:

Zhou and colleagues developed a computational model of replay that heavily builds on cognitive models of memory in context (e.g., the context-maintenance and retrieval model), which have been successfully used to explain memory phenomena in the past. Their model produces results that mirror previous empirical findings in rodents and offers a new computational framework for thinking about replay.

Strengths:

The model is compelling and seems to explain a number of findings from the rodent literature. It is commendable that the authors implement commonly used algorithms from wakefulness to model sleep/rest, thereby linking wake and sleep phenomena in a parsimonious way. Additionally, the manuscript's comprehensive perspective on replay, bridging humans and non-human animals, enhanced its theoretical contribution.

Weaknesses:

This reviewer is not a computational neuroscientist by training, so some comments may stem from misunderstandings. I hope the authors would see those instances as opportunities to clarify their findings for broader audiences.

(1) The model predicts that temporally close items will be co-reactivated, yet evidence from humans suggests that temporal context doesn't guide sleep benefits (instead, semantic connections seem to be of more importance; Liu and Ranganath 2021, Schechtman et al 2023). Could these findings be reconciled with the model or is this a limitation of the current framework?

(2) During replay, the model is set so that the next reactivated item is sampled without replacement (i.e., the model cannot get "stuck" on a single item). I'm not sure what the biological backing behind this is and why the brain can't reactivate the same item consistently. Furthermore, I'm afraid that such a rule may artificially generate sequential reactivation of items regardless of wake training. Could the authors explain this better or show that this isn't the case?

(3) If I understand correctly, there are two ways in which novelty (i.e., less exposure) is accounted for in the model. The first and more talked about is the suppression mechanism (lines 639-646). The second is a change in learning rates (lines 593-595). It's unclear to me why both procedures are needed, how they differ, and whether these are two different mechanisms that the model implements. Also, since the authors controlled the extent to which each item was experienced during wakefulness, it's not entirely clear to me which of the simulations manipulated novelty on an individual item level, as described in lines 593-595 (if any).

As to the first mechanism - experience-based suppression - I find it challenging to think of a biological mechanism that would achieve this and is selectively activated immediately before sleep (somehow anticipating its onset). In fact, the prominent synaptic homeostasis hypothesis suggests that such suppression, at least on a synaptic level, is exactly what sleep itself does (i.e., prune or weaken synapses that were enhanced due to learning during the day). This begs the question of whether certain sleep stages (or ultradian cycles) may be involved in pruning, whereas others leverage its results for reactivation (e.g., a sequential hypothesis; Rasch & Born, 2013). That could be a compelling synthesis of this literature. Regardless of whether the authors agree, I believe that this point is a major caveat to the current model. It is addressed in the discussion, but perhaps it would be beneficial to explicitly state to what extent the results rely on the assumption of a pre-sleep suppression mechanism.

(4) As the manuscript mentions, the only difference between sleep and wake in the model is the initial conditions (a0). This is an obvious simplification, especially given the last author's recent models discussing the very different roles of REM vs NREM. Could the authors suggest how different sleep stages may relate to the model or how it could be developed to interact with other successful models such as the ones the last author has developed (e.g., C-HORSE)? Finally, I wonder how the model would explain findings (including the authors') showing a preference for reactivation of weaker memories. The literature seems to suggest that it isn't just a matter of novelty or exposure, but encoding strength. Can the model explain this? Or would it require additional assumptions or some mechanism for selective endogenous reactivation during sleep and rest?

(5) Lines 186-200 - Perhaps I'm misunderstanding, but wouldn't it be trivial that an external cue at the end-item of Figure 7a would result in backward replay, simply because there is no potential for forward replay for sequences starting at the last item (there simply aren't any subsequent items)? The opposite is true, of course, for the first-item replay, which can't go backward. More generally, my understanding of the literature on forward vs backward replay is that neither is linked to the rodent's location. Both commonly happen at a resting station that is further away from the track. It seems as though the model's result may not hold if replay occurs away from the track (i.e. if a0 would be equal for both pre- and post-run).

(6) The manuscript describes a study by Bendor & Wilson (2012) and tightly mimics their results. However, notably, that study did not find triggered replay immediately following sound presentation, but rather a general bias toward reactivation of the cued sequence over longer stretches of time. In other words, it seems that the model's results don't fully mirror the empirical results. One idea that came to mind is that perhaps it is the R/L context - not the first R/L item - that is cued in this study. This is in line with other TMR studies showing what may be seen as contextual reactivation. If the authors think that such a simulation may better mirror the empirical results, I encourage them to try. If not, however, this limitation should be discussed.

(7) There is some discussion about replay's benefit to memory. One point of interest could be whether this benefit changes between wake and sleep. Relatedly, it would be interesting to see whether the proportion of forward replay, backward replay, or both correlated with memory benefits. I encourage the authors to extend the section on the function of replay and explore these questions.

(8) Replay has been mostly studied in rodents, with few exceptions, whereas CMR and similar models have mostly been used in humans. Although replay is considered a good model of episodic memory, it is still limited due to limited findings of sequential replay in humans and its reliance on very structured and inherently autocorrelated items (i.e., place fields). I'm wondering if the authors could speak to the implications of those limitations on the generalizability of their model. Relatedly, I wonder if the model could or does lead to generalization to some extent in a way that would align with the complementary learning systems framework.

Here the author explains another reason for the decline in TV recaps. I believe that overstimulation plays a huge role in this as well. There are a lot more shows to recap, however we have to manage our time well and refrain from overconsumption.

The passage reflects on the evolution of television criticism and audience engagement, highlighting the importance of discussion and analysis in a variety of formats while emphasizing the joy and affection that drive these conversations.

I find that some shows that still release episodically are popular so I believe its still possible.

I would say the "The Recap" is still very much alive and thriving. The part of it that has died is it being text based, it's all on video now. Youtube and TikTok are flooded with users doing recaps for tv shows.

Sometimes us as writers we tend to overthink what we are going to write about, and come to the conclusion that we dont have nothing to write. In reality, we just need to put some words on the paper, and the ideas will start flowing.

Something like grief is very important in our journey to grow and living because no war was ever won without losses. However, when it comes to unexpected losses, we normally don't comprehend what just happened so sometimes people use coping skills like writing to make sense of what they lost.

Author response:

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In their paper, Zhan et al. have used Pf genetic data from simulated data and Ghanaian field samples to elucidate a relationship between multiplicity of infection (MOI) (the number of distinct parasite clones in a single host infection) and force of infection (FOI). Specifically, they use sequencing data from the var genes of Pf along with Bayesian modeling to estimate MOI individual infections and use these values along with methods from queueing theory that rely on various assumptions to estimate FOI. They compare these estimates to known FOIs in a simulated scenario and describe the relationship between these estimated FOI values and another commonly used metric of transmission EIR (entomological inoculation rate).

This approach does fill an important gap in malaria epidemiology, namely estimating the force of infection, which is currently complicated by several factors including superinfection, unknown duration of infection, and highly genetically diverse parasite populations. The authors use a new approach borrowing from other fields of statistics and modeling and make extensive efforts to evaluate their approach under a range of realistic sampling scenarios. However, the write-up would greatly benefit from added clarity both in the description of methods and in the presentation of the results. Without these clarifications, rigorously evaluating whether the author's proposed method of estimating FOI is sound remains difficult. Additionally, there are several limitations that call into question the stated generalizability of this method that should at minimum be further discussed by authors and in some cases require a more thorough evaluation.

Major comments:

(1) Description and evaluation of FOI estimation procedure.

a. The methods section describing the two-moment approximation and accompanying appendix is lacking several important details. Equations on lines 891 and 892 are only a small part of the equations in Choi et al. and do not adequately describe the procedure notably several quantities in those equations are never defined some of them are important to understand the method (e.g. A, S as the main random variables for inter-arrival times and service times, aR and bR which are the known time average quantities, and these also rely on the squared coefficient of variation of the random variable which is also never introduced in the paper). Without going back to the Choi paper to understand these quantities, and to understand the assumptions of this method it was not possible to follow how this works in the paper. At a minimum, all variables used in the equations should be clearly defined. 

We thank the reviewer for this useful comment. We plan to clarify the method, including all the relevant variables in our revised manuscript. The reviewer is correct in pointing out that there are more sections and equations in Choi et al., including the derivation of an exact expression for the steady-state queue-length distribution and the two-moment approximation for the queue-length distribution. Since only the latter was directly utilized in our work, we included in the first version of our manuscript only material on this section and not the other. We agree with the reviewer on readers benefiting from additional information on the derivation of the exact expression for the steady-state queue-length distribution. Therefore, we will summarize the derivation of this expression in our revised manuscript. Regarding the assumptions of the method we applied, especially those for going from the exact expression to the two-moment approximation, we did describe these in the Materials and Methods of our manuscript. We recognize from this comment that the writing and organization of this information may not have been sufficiently clear. We had separated the information on this method into two parts, with the descriptive summary placed in the Materials and Methods and the equations or mathematical formula placed in the Appendix. This can make it difficult for readers to connect the two parts and remember what was introduced earlier in the Materials and Methods when reading the equations and mathematical details in the Appendix. For our revised manuscript, we plan to cover both parts in the Materials and Methods, and to provide more of the technical details in one place, which will be easier to understand and follow.

b. Additionally, the description in the main text of how the queueing procedure can be used to describe malaria infections would benefit from a diagram currently as written it's very difficult to follow. 

We thank the reviewer for this suggestion. We will add a diagram illustrating the connection between the queueing procedure and malaria transmission.

c. Just observing the box plots of mean and 95% CI on a plot with the FOI estimate (Figures 1, 2, and 10-14) is not sufficient to adequately assess the performance of this estimator. First, it is not clear whether the authors are displaying the bootstrapped 95%CIs or whether they are just showing the distribution of the mean FOI taken over multiple simulations, and then it seems that they are also estimating mean FOI per host on an annual basis. Showing a distribution of those per-host estimates would also be helpful. Second, a more quantitative assessment of the ability of the estimator to recover the truth across simulations (e.g. proportion of simulations where the truth is captured in the 95% CI or something like this) is important in many cases it seems that the estimator is always underestimating the true FOI and may not even contain the true value in the FOI distribution (e.g. Figure 10, Figure 1 under the mid-IRS panel). But it's not possible to conclude one way or the other based on this visualization. This is a major issue since it calls into question whether there is in fact data to support that these methods give good and consistent FOI estimates. 

There appears to be some confusion on what we display in some key figures. We will clarify this further both here and in the revised text. In Figures 1, 2, and 10-14, we displayed the bootstrapped distributions including the 95% CIs. These figures do not show the distribution of the mean FOI taken over multiple simulations. We estimated mean FOI on an annual basis per host in the following sense. Both of our proposed methods require either a steady-state queue length distribution, or moments of this distribution for FOI inference. However, we only have one realization or observation for each individual host, and we do not have access to either the time-series observation of a single individual’s MOI or many realizations of a single individual’s MOI at the same sampling time. This is typically the case for empirical data, although numerical simulations could circumvent this limitation and generate such output. Nonetheless, we do have a queue length distribution at the population level for both the simulation output and the empirical data, which can be obtained by simply aggregating MOI estimates across all sampled individuals. We use this population-level queue length distribution to represent and approximate the steady-state queue length distribution at the individual level. Such representation or approximation does not consider explicitly any individual heterogeneity due to biology or transmission. The estimated FOI is per host in the sense of representing the FOI experienced by an individual host whose queue length distribution is approximated from the collection of all sampled individuals. The true FOI per host per year in the simulation output is obtained from dividing the total FOI of all hosts per year by the total number of all hosts. Therefore, our estimator, combined with the demographic information on population size, is for the total number of Plasmodium falciparum infections acquired by all individual hosts in the population of interest per year.

We evaluated the impact of individual heterogeneity on FOI inference by introducing individual heterogeneity into the simulations. With a considerable amount of transmission heterogeneity across individuals (namely 2/3 of the population receiving more than 90% of all bites whereas the remaining 1/3 receives the rest of the bites), our two methods exhibit a similar performance than those of the homogeneous transmission scenarios.

Concerning the second point, we will add a quantitative assessment of the ability of the estimator to recover the truth across simulations and include this information in the legend of each figure. In particular, we will provide the proportion of simulations where the truth is captured by the entire bootstrap distribution, in addition to some measure of relative deviation, such as the relative difference between the true FOI value and the median of the bootstrap distribution for the estimate. This assessment will be a valuable addition, but please note that the comparisons we have provided in a graphical way do illustrate the ability of the methods to estimate “sensible” values, close to the truth despite multiple sources of errors. “Close” is here relative to the scale of variation of FOI in the field and to the kind of precision that would be useful in an empirical context. From a practical perspective based on the potential range of variation of FOI, the graphical results already illustrate that the estimated distributions would be informative.

d. Furthermore the authors state in the methods that the choice of mean and variance (and thus second moment) parameters for inter-arrival times are varied widely, however, it's not clear what those ranges are there needs to be a clear table or figure caption showing what combinations of values were tested and which results are produced from them, this is an essential component of the method and it's impossible to fully evaluate its performance without this information. This relates to the issue of selecting the mean and variance values that maximize the likelihood of observing a given distribution of MOI estimates, this is very unclear since no likelihoods have been written down in the methods section of the main text, which likelihood are the authors referring to, is this the probability distribution of the steady state queue length distribution? At other places the authors refer to these quantities as Maximum Likelihood estimators, how do they know they have found the MLE? There are no derivations in the manuscript to support this. The authors should specify the likelihood and include in an appendix an explanation of why their estimation procedure is in fact maximizing this likelihood, preferably with evidence of the shape of the likelihood, and how fine the grid of values they tested is for their mean and variance since this could influence the overall quality of the estimation procedure. 

We thank the reviewer for pointing out these aspects of the work that can be further clarified. We will specify the ranges for the choice of mean and variance parameters for inter-arrival times as well as the grid of values tested in the corresponding figure caption or in a separate supplementary table. We maximized the likelihood of observing the set of individual MOI estimates in a sampled population given steady queue length distributions (with these distributions based on the two-moment approximation method for different combinations of the mean and variance of inter-arrival times). We will add a section to either the Materials and Methods or the Appendix in our revised manuscript including an explicit formulation of the likelihood.

We will add example figures on the shape of the likelihood to the Appendix. We will also test how choices of the grid of values influence the overall quality of the estimation procedure. Specifically, we will further refine the grid of values to include more points and examine whether the results of FOI inference are consistent and robust against each other.

(2) Limitation of FOI estimation procedure.

a. The authors discuss the importance of the duration of infection to this problem. While I agree that empirically estimating this is not possible, there are other options besides assuming that all 1-5-year-olds have the same duration of infection distribution as naïve adults co-infected with syphilis. E.g. it would be useful to test a wide range of assumed infection duration and assess their impact on the estimation procedure. Furthermore, if the authors are going to stick to the described method for duration of infection, the potentially limited generalizability of this method needs to be further highlighted in both the introduction, and the discussion. In particular, for an estimated mean FOI of about 5 per host per year in the pre-IRS season as estimated in Ghana (Figure 3) it seems that this would not translate to 4-year-old being immune naïve, and certainly this would not necessarily generalize well to a school-aged child population or an adult population. 

The reviewer is indeed correct about the difficulty of empirically measuring the duration of infection for 1-5-year-olds, and that of further testing whether these 1-5-year-olds exhibit the same distribution for duration of infection as naïve adults co-infected with syphilis. We will nevertheless continue to use the described method for duration of infection, while better acknowledging and discussing the limitations this aspect of the method introduces. We note that the infection duration from the historical clinical data we have relied on, is being used in the malaria modeling community as one of the credible sources for this parameter of untreated natural infections in malaria-naïve individuals in malaria-endemic settings of Africa (e.g. in the agent-based model OpenMalaria, see 1).

It is important to emphasize that the proposed methods apply to the MOI estimates for naïve or close to naïve patients. They are not suitable for FOI inference for the school-aged children and the adult populations of high-transmission endemic regions, since individuals in these age classes have been infected many times and their duration of infection is significantly shortened by their immunity. To reduce the degree of misspecification in infection duration and take full advantage of our proposed methods, we will emphasize in the revision the need to prioritize in future data collection and sampling efforts the subpopulation class who has received either no infection or a minimum number of infections in the past, and whose immune profile is close to that of naïve adults, for example, infants. This emphasis is aligned with the top priority of all intervention efforts in the short term, which is to monitor and protect the most vulnerable individuals from severe clinical symptoms and death.

Also, force of infection for naïve hosts is a key basic parameter for epidemiological models of a complex infectious disease such as falciparum malaria, whether for agent-based formulations or equation-based ones. This is because force of infection for non-naïve hosts is typically a function of their immune status and the force of infection of naïve hosts. Thus, knowing the force of infection of naïve hosts can help parameterize and validate these models by reducing degrees of freedom.

b. The evaluation of the capacity parameter c seems to be quite important and is set at 30, however, the authors only describe trying values of 25 and 30, and claim that this does not impact FOI inference, however it is not clear that this is the case. What happens if the carrying capacity is increased substantially? Alternatively, this would be more convincing if the authors provided a mathematical explanation of why the carrying capacity increase will not influence the FOI inference, but absent that, this should be mentioned and discussed as a limitation. 

Thank you for this question. We will investigate more values of the parameter c systematically, including substantially higher ones. We note however that this quantity is the carrying capacity of the queuing system, or the maximum number of blood-stage strains that an individual human host can be co-infected with. We do have empirical evidence for the value of the latter being around 20 (2). This observed value provides a lower bound for parameter c. To account for potential under-sampling of strains, we thus tried values of 25 and 30 in the first version of our manuscript.

In general, this parameter influences the steady-state queue length distribution based on the two-moment approximation, more specifically, the tail of this distribution when the flow of customers/infections is high. Smaller values of parameter c put a lower cap on the maximum value possible for the queue length distribution. The system is more easily “overflowed”, in which case customers (or infections) often find that there is no space available in the queuing system/individual host upon their arrival. These customers (or infections) will not increment the queue length. The parameter c has therefore a small impact for the part of the grid resulting in low flows of customers/infection, for which the system is unlikely to be overflowed. The empirical MOI distribution centers around 4 or 5 with most values well below 10, and only a small fraction of higher values between 15-20 (2). When one increases the value of c, the part of the grid generating very high flows of customers/infections results in queue length distributions with a heavy tail around large MOI values that are not supported by the empirical distribution. We therefore do not expect that substantially higher values for parameter c would change either the relative shape of the likelihood or the MLE.

Reviewer #2 (Public Review):

Summary:

The authors combine a clever use of historical clinical data on infection duration in immunologically naive individuals and queuing theory to infer the force of infection (FOI) from measured multiplicity of infection (MOI) in a sparsely sampled setting. They conduct extensive simulations using agent-based modeling to recapitulate realistic population dynamics and successfully apply their method to recover FOI from measured MOI. They then go on to apply their method to real-world data from Ghana before and after an indoor residual spraying campaign.

Strengths:

(1) The use of historical clinical data is very clever in this context. 

(2) The simulations are very sophisticated with respect to trying to capture realistic population dynamics. 

(3) The mathematical approach is simple and elegant, and thus easy to understand. 

Weaknesses: 

(1) The assumptions of the approach are quite strong and should be made more clear. While the historical clinical data is a unique resource, it would be useful to see how misspecification of the duration of infection distribution would impact the estimates. 

We thank the reviewer for bringing up the limitation of our proposed methods due to their reliance on a known and fixed duration of infection from historical clinical data. Please see our response to reviewer 1 comment 2a.

(2) Seeing as how the assumption of the duration of infection distribution is drawn from historical data and not informed by the data on hand, it does not substantially expand beyond MOI. The authors could address this by suggesting avenues for more refined estimates of infection duration. 

We thank the reviewer for pointing out a potential improvement to the work. We acknowledge that FOI is inferred from MOI, and thus is dependent on the information contained in MOI. FOI reflects risk of infection, is associated with risk of clinical episodes, and can relate local variation in malaria burden to transmission better than other proxy parameters for transmission intensity. It is possible that MOI can be as informative as FOI when one regresses the risk of clinical episodes and local variation in malaria burden with MOI. But MOI by definition is a number and not a rate parameter. FOI for naïve hosts is a key basic parameter for epidemiological models. This is because FOI of non-naïve hosts is typically a function of their immune status and the FOI of naïve hosts. Thus, knowing the FOI of naïve hosts can help parameterize and validate these models by reducing degrees of freedom. In this sense, we believe the transformation from MOI to FOI provides a useful step.

Given the difficulty of measuring infection duration, estimating infection duration and FOI simultaneously appears to be an attractive alternative, as the referee pointed out. This will require however either cohort studies or more densely sampled cross-sectional surveys due to the heterogeneity in infection duration across a multiplicity of factors. These kinds of studies have not been, and will not be, widely available across geographical locations and time. This work aims to utilize more readily available data, in the form of sparsely sampled single-time-point cross-sectional surveys.

(3) It is unclear in the example how their bootstrap imputation approach is accounting for measurement error due to antimalarial treatment. They supply two approaches. First, there is no effect on measurement, so the measured MOI is unaffected, which is likely false and I think the authors are in agreement. The second approach instead discards the measurement for malaria-treated individuals and imputes their MOI by drawing from the remaining distribution. This is an extremely strong assumption that the distribution of MOI of the treated is the same as the untreated, which seems unlikely simply out of treatment-seeking behavior. By imputing in this way, the authors will also deflate the variability of their estimates. 

We thank the reviewer for pointing out aspects of the work that can be further clarified. It is difficult to disentangle the effect of drug treatment on measurement, including infection status, MOI, and duration of infection. Thus, we did not attempt to address this matter explicitly in the original version of our manuscript. Instead, we considered two extreme scenarios which bound reality, well summarized by the reviewer. First, if drug treatment has had no impact on measurement, the MOI of the drug-treated 1-5-year-olds would reflect their true underlying MOI. We can then use their MOI directly for FOI inference. Second, if the drug treatment had a significant impact on measurement, i.e., if it completely changed the infection status, MOI, and duration infection of drug-treated 1-5-year-olds, we would need to either exclude those individuals’ MOI or impute their true underlying MOI. We chose to do the latter in the original version of the manuscript. If those 1-5-year-olds had not received drug treatment, they would have had similar MOI values than those of the non-treated 1-5-year-olds. We can then impute their MOI by sampling from the MOI estimates of non-treated 1-5-year-olds.

The reviewer is correct in pointing out that this imputation does not add additional information and can potentially deflate the variability of MOI distributions, compared to simply throwing or excluding those drug-treated 1-5-year-olds from the analysis. Thus, we can include in our revision FOI estimates with the drug-treated 1-5-year-olds excluded in the estimation.

- For similar reasons, their imputation of microscopy-negative individuals is also questionable, as it also assumes the same distributions of MOI for microscopy-positive and negative individuals. 

We imputed the MOI values of microscopy-negative but PCR-positive 1-5-year-olds by sampling from the microscopy-positive 1-5-year-olds, effectively assuming that both have the same, or similar, MOI distributions. We did so because there is a weak relationship in our Ghana data between the parasitemia level of individual hosts and their MOI (or detected number of var genes, on the basis of which the MOI values themselves were estimated). Parasitemia levels underlie the difference in detection sensitivity of PCR and microscopy.

We will elaborate on this matter in our revised manuscript and include information from our previous and on-going work on the weak relationship between MOI/the number of var genes detected within an individual host and their parasitemia levels. We will also discuss potential reasons or hypotheses for this pattern.

Reviewer #3 (Public Review):

Summary: 

It has been proposed that the FOI is a method of using parasite genetics to determine changes in transmission in areas with high asymptomatic infection. The manuscript attempts to use queuing theory to convert multiplicity of infection estimates (MOI) into estimates of the force of infection (FOI), which they define as the number of genetically distinct blood-stage strains. They look to validate the method by applying it to simulated results from a previously published agent-based model. They then apply these queuing theory methods to previously published and analysed genetic data from Ghana. They then compare their results to previous estimates of FOI. 

Strengths: 

It would be great to be able to infer FOI from cross-sectional surveys which are easier and cheaper than current FOI estimates which require longitudinal studies. This work proposes a method to convert MOI to FOI for cross-sectional studies. They attempt to validate this process using a previously published agent-based model which helps us understand the complexity of parasite population genetics. 

Weaknesses: 

(1) I fear that the work could be easily over-interpreted as no true validation was done, as no field estimates of FOI (I think considered true validation) were measured. The authors have developed a method of estimating FOI from MOI which makes a number of biological and structural assumptions. I would not call being able to recreate model results that were generated using a model that makes its own (probably similar) defined set of biological and structural assumptions a validation of what is going on in the field. The authors claim this at times (for example, Line 153 ) and I feel it would be appropriate to differentiate this in the discussion. 

We thank the reviewer for this comment, although we think there is a mis-understanding on what can and cannot be practically validated in the sense of a “true” measure of FOI that would be free from assumptions for a complex disease such as malaria. We would not want the results to be over-interpreted and will extend the discussion of what we have done to test the methods. We note that for the performance evaluation of statistical methods, the use of simulation output is quite common and often a necessary and important step. In some cases, the simulation output is generated by dynamical models, whereas in others, by purely descriptive ones. All these models make their own assumptions which are necessarily a simplification of reality. The stochastic agent-based model (ABM) of malaria transmission utilized in this work has been shown to reproduce several important patterns observed in empirical data from high-transmission regions, including aspects of strain diversity which are not represented in simpler models.

In what sense this ABM makes a set of biological and structural assumptions which are “probably similar” to those of the queuing methods we present, is not clear to us. We agree that relying on models whose structural assumptions differ from those of a given method or model to be tested, is the best approach. Our proposed methods for FOI inference based on queuing theory rely on the duration of infection distribution and the MOI distribution among sampled individuals, both of which can be direct outputs from the ABM. But these methods are agnostic on the specific mechanisms or biology underlying the regulation of duration and MOI.

Another important point raised by this comment is what would be the “true” FOI value against which to validate our methods. Empirical MOI-FOI pairs for FOI measured directly by tracking cohort studies are still lacking. There are potential measurement errors for both MOI and FOI because the polymorphic markers typically used in different cohort studies cannot differentiate hyper-diverse antigenic strains fully and well (5). Also, these cohort studies usually start with drug treatment. Alternative approaches do not provide a measure of true FOI, in the sense of the estimation being free from assumptions. For example, one approach would be to fit epidemiological models to densely sampled/repeated cross-sectional surveys for FOI inference. In this case, no FOI is measured directly and further benchmarked against fitted FOI values. The evaluation of these models is typically based on how well they can capture other epidemiological quantities which are more easily sampled or measured, including prevalence or incidence. This is similar to what is done in this work. We selected the FOI values that maximize the likelihood of observing the given distribution of MOI estimates. Furthermore, we paired our estimated FOI value for the empirical data from Ghana with another independently measured quantity EIR (Entomological Inoculation Rate), typically used in the field as a measure of transmission intensity. We check whether the resulting FOI-EIR point is consistent with the existing set of FOI-EIR pairs and the relationship between these two quantities from previous studies. We acknowledge that as for model fitting approaches for FOI inference, our validation is also indirect for the field data.

Prompted by the reviewer’s comment, we will discuss this matter in more detail in our revised manuscript, including clarifying further certain basic assumptions of our agent-based model, emphasizing the indirect nature of the validation with the field data and the existing constraints for such validation.

(2) Another aspect of the paper is adding greater realism to the previous agent-based model, by including assumptions on missing data and under-sampling. This takes prominence in the figures and results section, but I would imagine is generally not as interesting to the less specialised reader. The apparent lack of impact of drug treatment on MOI is interesting and counterintuitive, though it is not really mentioned in the results or discussion sufficiently to allay my confusion. I would have been interested in understanding the relationship between MOI and FOI as generated by your queuing theory method and the model. It isn't clear to me why these more standard results are not presented, as I would imagine they are outputs of the model (though happy to stand corrected - it isn't entirely clear to me what the model is doing in this manuscript alone). 

We thank the reviewer for this comment. We will add supplementary figures for the MOI distributions generated by the queuing theory method (i.e., the two-moment approximation method) and our agent-based model in our revised manuscript.

In the first version of our manuscript, we considered two extreme scenarios which bound the reality, instead of simply assuming that drug treatment does not impact the infection status, MOI, and duration of infection. See our response to reviewer 2 point (3). The resulting FOI estimates differ but not substantially across the two extreme scenarios, partially because drug-treated individuals’ MOI distribution is similar to that of non-treated individuals (or the apparent lack of drug treatment on MOI as pointed by the referee). We will consider potentially adding some formal test to quantify the difference between the two MOI distributions and how significant the difference is. We will discuss which of the two extreme scenarios reality is closer to, given the result of the formal test. We will also discuss in our revision possible reasons/hypotheses underlying the impact of drug treatment on MOI from the perspective of the nature, efficiency, and duration of the drugs administrated.

Regarding the last point of the reviewer, on understanding the relationship between MOI and FOI, we are not fully clear about what was meant. We are also confused about the statement on what the “model is doing in this manuscript alone”. We interpret the overall comment as the reviewer suggesting a better understanding of the relationship between MOI and FOI, either between their distributions, or the moments of their distributions, perhaps by fitting models including simple linear regression models. This approach is in principle possible, but it is not the focus of this work. It will be equally difficult to evaluate the performance of this alternative approach given the lack of MOI-FOI pairs from empirical settings with directly measured FOI values (from large cohort studies). Moreover, the qualitative relationship between the two quantities is intuitive. Higher FOI values should correspond to higher MOI values. Less variable FOI values should correspond to more narrow or concentrated MOI distributions, whereas more variable FOI values should correspond to more spread-out ones. We will discuss this matter in our revised manuscript.

(3) I would suggest that outside of malaria geneticists, the force of infection is considered to be the entomological inoculation rate, not the number of genetically distinct blood-stage strains. I appreciate that FOI has been used to explain the latter before by others, though the authors could avoid confusion by stating this clearly throughout the manuscript. For example, the abstract says FOI is "the number of new infections acquired by an individual host over a given time interval" which suggests the former, please consider clarifying. 

We thank the reviewer for this helpful comment as it is fundamental that there is no confusion on the basic definitions. EIR, the entomological inoculation rate, is closely related to the force of infection but is not equal to it. EIR focuses on the rate of arrival of infectious bites and is measured as such by focusing on the mosquito vectors that are infectious and arrive to bite a given host. Not all these bites result in actual infection of the human host. Epidemiological models of malaria transmission clearly make this distinction, as FOI is defined as the rate at which a host acquires infection. This definition comes from more general models for the population dynamics of infectious diseases in general. (For diseases simpler than malaria, with no super-infection, the typical SIR models define the force of infection as the rate at which a susceptible individual becomes infected).  For malaria, force of infection refers to the number of blood-stage new infections acquired by an individual host over a given time interval. This distinction between EIR and FOI is the reason why studies have investigated their relationship, with the nonlinearity of this relationship reflecting the complexity of the underlying biology and how host immunity influences the outcome of an infectious bite.

We agree however with the referee that there could be some confusion in our definition resulting from the approach we use to estimate the MOI distribution (which provides the basis for estimating FOI). In particular, we rely on the non-existent to very low overlap of var repertoires among individuals with MOI=1, an empirical pattern we have documented extensively in previous work (See 2, 3, and 4). The method of var_coding and its Bayesian formulation rely on the assumption of negligible overlap. We note that other approaches for estimating MOI (and FOI) based on other polymorphic markers, also make this assumption (reviewed in _5). Ultimately, the FOI we seek to estimate is the one defined as specified above and in both the abstract and introduction, consistent with the epidemiological literature. We will include clarification in the introduction and discussion of this point in the revision.

(4) Line 319 says "Nevertheless, overall, our paired EIR (directly measured by the entomological team in Ghana (Tiedje et al., 2022)) and FOI values are reasonably consistent with the data points from previous studies, suggesting the robustness of our proposed methods". I would agree that the results are consistent, given that there is huge variation in Figure 4 despite the transformed scales, but I would not say this suggests a robustness of the method. 

We will modify the relevant sentences to use “consistent” instead of “robust”.

(5) The text is a little difficult to follow at times and sometimes requires multiple reads to understand. Greater precision is needed with the language in a few situations and some of the assumptions made in the modelling process are not referenced, making it unclear whether it is a true representation of the biology. 

We thank the reviewer for this comment. As also mentioned in the response to reviewer 1’s comments, we will reorganize and rewrite parts of the text in our revision to improve clarity.

References and Notes

(1) Maire, N. et al. A model for natural immunity to asexual blood stages of Plasmodium falciparum malaria in endemic areas. Am J Trop Med Hyg., 75(2 Suppl):19-31 (2006).

(2) Tiedje, K. E. et al. Measuring changes in Plasmodium falciparum census population size in response to sequential malaria control interventions. eLife, 12 (2023).

(3) Day, K. P. et al. Evidence of strain structure in Plasmodium falciparum var gene repertoires in children from Gabon, West Africa. Proc. Natl. Acad. Sci. U.S.A., 114(20), 4103-4111 (2017).

(4) Ruybal-Pesántez, S. et al. Population genomics of virulence genes of Plasmodium falciparum in clinical isolates from Uganda. Sci. Rep., 7(11810) (2017).

(5) Labbé, F. et al. Neutral vs. non-neutral genetic footprints of Plasmodium falciparum multiclonal infections. PLoS Comput Biol 19(1) (2023).

Reviewer #1 (Public Review):

Summary:

In their paper, Zhan et al. have used Pf genetic data from simulated data and Ghanaian field samples to elucidate a relationship between multiplicity of infection (MOI) (the number of distinct parasite clones in a single host infection) and force of infection (FOI). Specifically, they use sequencing data from the var genes of Pf along with Bayesian modeling to estimate MOI individual infections and use these values along with methods from queueing theory that rely on various assumptions to estimate FOI. They compare these estimates to known FOIs in a simulated scenario and describe the relationship between these estimated FOI values and another commonly used metric of transmission EIR (entomological inoculation rate).

This approach does fill an important gap in malaria epidemiology, namely estimating the force of infection, which is currently complicated by several factors including superinfection, unknown duration of infection, and highly genetically diverse parasite populations. The authors use a new approach borrowing from other fields of statistics and modeling and make extensive efforts to evaluate their approach under a range of realistic sampling scenarios. However, the write-up would greatly benefit from added clarity both in the description of methods and in the presentation of the results. Without these clarifications, rigorously evaluating whether the author's proposed method of estimating FOI is sound remains difficult. Additionally, there are several limitations that call into question the stated generalizability of this method that should at minimum be further discussed by authors and in some cases require a more thorough evaluation.

Major comments:

(1) Description and evaluation of FOI estimation procedure.

a. The methods section describing the two-moment approximation and accompanying appendix is lacking several important details. Equations on lines 891 and 892 are only a small part of the equations in Choi et al. and do not adequately describe the procedure notably several quantities in those equations are never defined some of them are important to understand the method (e.g. A, S as the main random variables for inter-arrival times and service times, aR and bR which are the known time average quantities, and these also rely on the squared coefficient of variation of the random variable which is also never introduced in the paper). Without going back to the Choi paper to understand these quantities, and to understand the assumptions of this method it was not possible to follow how this works in the paper. At a minimum, all variables used in the equations should be clearly defined.

b. Additionally, the description in the main text of how the queueing procedure can be used to describe malaria infections would benefit from a diagram currently as written it's very difficult to follow.

c. Just observing the box plots of mean and 95% CI on a plot with the FOI estimate (Figures 1, 2, and 10-14) is not sufficient to adequately assess the performance of this estimator. First, it is not clear whether the authors are displaying the bootstrapped 95%CIs or whether they are just showing the distribution of the mean FOI taken over multiple simulations, and then it seems that they are also estimating mean FOI per host on an annual basis. Showing a distribution of those per-host estimates would also be helpful. Second, a more quantitative assessment of the ability of the estimator to recover the truth across simulations (e.g. proportion of simulations where the truth is captured in the 95% CI or something like this) is important in many cases it seems that the estimator is always underestimating the true FOI and may not even contain the true value in the FOI distribution (e.g. Figure 10, Figure 1 under the mid-IRS panel). But it's not possible to conclude one way or the other based on this visualization. This is a major issue since it calls into question whether there is in fact data to support that these methods give good and consistent FOI estimates.

d. Furthermore the authors state in the methods that the choice of mean and variance (and thus second moment) parameters for inter-arrival times are varied widely, however, it's not clear what those ranges are there needs to be a clear table or figure caption showing what combinations of values were tested and which results are produced from them, this is an essential component of the method and it's impossible to fully evaluate its performance without this information. This relates to the issue of selecting the mean and variance values that maximize the likelihood of observing a given distribution of MOI estimates, this is very unclear since no likelihoods have been written down in the methods section of the main text, which likelihood are the authors referring to, is this the probability distribution of the steady state queue length distribution? At other places the authors refer to these quantities as Maximum Likelihood estimators, how do they know they have found the MLE? There are no derivations in the manuscript to support this. The authors should specify the likelihood and include in an appendix an explanation of why their estimation procedure is in fact maximizing this likelihood, preferably with evidence of the shape of the likelihood, and how fine the grid of values they tested is for their mean and variance since this could influence the overall quality of the estimation procedure.

(2) Limitation of FOI estimation procedure.

a. The authors discuss the importance of the duration of infection to this problem. While I agree that empirically estimating this is not possible, there are other options besides assuming that all 1-5-year-olds have the same duration of infection distribution as naïve adults co-infected with syphilis. E.g. it would be useful to test a wide range of assumed infection duration and assess their impact on the estimation procedure. Furthermore, if the authors are going to stick to the described method for duration of infection, the potentially limited generalizability of this method needs to be further highlighted in both the introduction, and the discussion. In particular, for an estimated mean FOI of about 5 per host per year in the pre-IRS season as estimated in Ghana (Figure 3) it seems that this would not translate to 4-year-old being immune naïve, and certainly this would not necessarily generalize well to a school-aged child population or an adult population.

b. The evaluation of the capacity parameter c seems to be quite important and is set at 30, however, the authors only describe trying values of 25 and 30, and claim that this does not impact FOI inference, however it is not clear that this is the case. What happens if the carrying capacity is increased substantially? Alternatively, this would be more convincing if the authors provided a mathematical explanation of why the carrying capacity increase will not influence the FOI inference, but absent that, this should be mentioned and discussed as a limitation.

I agree with the term problem spotting since it's not solving yet just pointing at the problem

This statement stood out to me because it states how writing is more than just a form of expression. Its more complex meaning it forces us to analyze different things when we're writing.

i agree with this because personally most of the time i want my first draft is perfect but in all honesty it's called a draft for a reason it's so when can just throw out ideas and fix it up later.

Welcome back.

This demo is going to bring together some really important theory and architecture that you've learned over the past few lessons.

What we're starting this demo lesson with is this architecture.

We have our VPC, the Animals for Life VPC in US East 1.

It uses the 10.16.0.0/16 side range.

It has 12 subnets created inside it, over three AZs with four tiers, Reserve, DB, Application and Web.

Now currently all the subnets are private and can't be used for communication with the internet or the AWS public zone.

In this demo we want to reconfigure the VPC to allow that.

So the first step is to create an internet gateway and attach it.

To do that, I'm going to move across to my desktop.

Now to do this in your environment, you'll need the VPC and subnet configuration as you set it up in the previous demo lesson.

So that configuration needs to be in place already.

You need to be logged in as the I am admin user of the management account of the organization and have the Northern Virginia region selected, so US - East - 1.

So go ahead and move across to the VPC console.

Now this should already be in the recently visited services because you were using this in the previous demo lesson, but if it's not visible just click in the services drop down, type VPC and then click to move to the VPC console.

Now if you do still have the configuration as it was at the end of the previous demo lesson, you should be able to click on subnets on the menu on the left and see a list of lots of subnets.

You'll see the ones for the default VPC without a name.

And if you have the correct configuration, you should see a collection of 12 subnets, 3 application subnets, 3 database subnets, 3 reserved subnets and then 3 web subnets.

So all of these should be in place within the Animals for Life VPC in order to do the tasks within this demo lesson.

So I'm going to assume from this point onwards that you do have all of these subnets created and configured.

Now what we're going to be doing in this demo lesson is configuring the 3 web subnets, so web A, web B and web C, to be public subnets.

Being a public subnet means that you can launch resources into the subnet, have them allocated with a public IP version 4 address and have connectivity to and from the public IP version 4 internet.

And in order to enable that functionality, there are a number of steps that we need to perform and I want you to get the practical experience of implementing these within your own environment.

Now the first step to making subnets public is that we need an internet gateway attached to this VPC.

So internet gateways, as I talked about in the previous theory lesson, are highly available gateway objects which can be used to allow public routing to and from the internet.

So we need to create one.

So let's click on internet gateways on the menu on the left.

They'll already be an internet gateway in place for the default VPC.

Remember when you created the default VPC, all this networking infrastructure is created and configured on your behalf.

But because we created a custom VPC for animals for life, we need to do this manually.

So to do that, go ahead and click on create internet gateway.

We're going to call the internet gateway A4L, so animals for life, VPC 1, which is the VPC we're going to attach it to and then IGW for internet gateway.

So A4L-VPC1-IGW.

Now that's the only information that we need to enter, so scroll down and click on create internet gateway.

Internet gateways are initially not attached to a VPC and we can tell that because it's initially in.

We need to attach this to the animals for life VPC.

So click on actions and then attach to VPC inside the available VPCs box.

Just click and then select A4L-IVAN-VPC1.

Once selected, go ahead and click on attach internet gateway and that will attach our brand new internet gateway to the animals for life VPC.

And that means that it's now available within that VPC as a gateway object, which gives the VPC the capability to communicate to and receive communications from the public internet and the AWS public zone.

Now the next step is that we want to make all the subnets in the web tier public, so the services deployed into these subnets can take advantage of this functionality.

So we want the web subnets to be able to communicate to and receive communications from the public internet and AWS public services.

Now there are a number of steps that we need to do to accomplish this.

We need to create a route table for the public subnets.

We need to associate this route table with the three public subnets, so web A, web B and web C and then we need to add two routes to this route table.

One route will be a default route for IP version 4 traffic and the other will be a default route for IP version 6 traffic.

And both of these routes for their target will be pointing at the internet gateway that you've just created and attached to this VPC.

Now this will configure the VPC router to forward any data intended for anything not within our VPC to the internet gateway.

Finally, on each of these web subnets will be configuring the subnet to auto assign a public IP version 4 address and that will complete the process of making them public.

So let's perform all of these sets of configuration.

So now that we're back at the AWS console, we need to create a route table.

So go ahead and click on route tables on the menu on the left and then we're going to create a new route table.

First we'll select the VPC that this route table will belong to and it's going to be the animals for life.

If a VPC, so go ahead and select that VPC and then we're going to give this route table a name.

And I like to keep the naming scheme consistent, so we're going to use A4, L4, animals for life and then a hyphen.

VPC1 because this is the VPC the route table will belong to and then hyphen RT for route table and then hyphen and then web because this route table is going to be used for the web subnets.

So go ahead and create this route table and click route tables on the menu on the left.

If we select the route table that we've just created, so that's the one that's called A4, L hyphen VPC1 hyphen RT hyphen web and then just expand this overview area at the bottom.

We'll be able to see all the information about this route table.

Now there are a number of areas of this which are important to understand.

One is the routes area which lists all the routes on this route table and the other is subnet associations.

This determines which subnets this route table is.

So let's go to subnet associations and currently we can see that it's not actually associated with any subnets within this VPC.

We need to adjust that so go ahead and edit those associations and we're going to associate it with the three web subnets.

So you need to select web A, web B and web C.

Now notice how all those are currently associated with the main route table of the VPC.

Remember a subnet can only be associated with one route table at a time.

If you don't explicitly associate a route table with a subnet then it's associated with the main route table.

We're going to change that.

We're going to explicitly associate this new route table with the web A, web B and web C subnets.

So go ahead and say that.

So now this route table has been associated with web A, web B and web C.

Those subnets are no longer associated with the main route table of the VPC.

So now we've configured the association as a route to routes.

And we can see that this route table has two local routes.

We've got the IP version 4 side of the VPC and the app.

IP version 6 side of the VPC.

So these two routes on this route table will mean that web A, web B and web C will know how to direct traffic towards any other IP version 4 or IP version 6 addresses within this VPC.

Now these local routes can never be adjusted or removed, but what we can do is add additional routes.

So we're going to add two routes, a default route for IP version 4 and a default route for IP version 6.

So we'll do that.

We'll start with IP version 4.

So we'll edit those routes and then we'll add a route.

The format for the IP version 4 default route is 0.0.0.0/0.

And this means any IP addresses.

Now I've talked elsewhere in the course how there is a priority to routing.

Within a VPC there's a more specific route always takes priority.

So this route, the /16 is more specific than this default route.

So this default route will only affect IP version 4 traffic, which is not matched by this local route.

So essentially anything which is IP version 4, which is not destined for the VPC, will use this default route.

Now we need to pick the internet gateway as the target for this route.

So click in the target box on this row, select internet gateway.

There should only be one that's highlighted.

That's the Animals for Life internet gateway you created moments ago.

So select that and that means that any IP version 4 traffic which is not destined for the VPC side of it. [siren] Range will be sent to the Internet Gateway.

Now we're going to do the same for IPv6.

So go ahead and add another route.

And the format for IPv60 default routes is double colon, forward slash zero.

And this is the same architecture. [siren] It essentially means this matches all IPv6 addresses, but it's less specific than the IPv6 and version 6 local route on this top row.

So this will only be used for any IPv6 addresses which are not in the IPv6 VPC side range.

So go ahead and select Target, go to Internet Gateway, and select the Animals for Life Internet Gateway.

And once you've done both of those, go ahead and click on Save Changes.

Now this means that we now have two default routes, an IPv4 default route, and an IPv6 default route.

So this means that anything which is associated with these route tables will now send any unknown traffic towards the Internet Gateway.

But what we need to do before this works is we need to ensure that any resources launched into the Web A, Web B, or Web C subnets are allocated with public IPv4 addresses.

To do that, go ahead and click on Subnets.

In the list, we need to locate Web A, Web B, and Web C.

So we'll start with Web A, so select Web A, click on Actions, and then Edit Subnet Settings.

And this time, we're going to modify this subnet so that it automatically assigns a public IPv4 address.

So check this box into the Save, and that means that any resources launched into the Web A subnet will be allocated with a public IPv4 address.

Now we need to follow the same process for the other web subnets, so select the Web B subnet, click on Actions, and then Edit Subnet Settings.

Enable IPv4, click on Save, and then do that same process for Web C.

So locate Web C, click on Actions, and then Edit Subnet Settings.

And then enable public IPv4 addresses and click on Save.

So that's all the network configuration done.

We've created an Internet Gateway.

We've associated the Internet Gateway with the Animals for IPPC.

We've created a Routetable for the web subnets.

We've associated this Routetable with the web subnets.

We've added default routes onto this Routetable, pointing at the Internet Gateway as a default IPv4 and IPv6 route.

And then we've enabled the allocation of public IPv4 addresses for Web A, Web B, and Web C.

Okay, so this is the end of Part 1 of this lesson.

It was getting a little bit on the long side, and so I wanted to add a break.

It's an opportunity just to take a rest or grab a coffee.

Part 2 will be continuing immediately from the end of Part 1.

To go ahead, complete the video, I'm ready, join me in part two.

I think saying that the english language is in a decline doesn't make sense for this reason; maybe it's not a decline, perhaps just the slow movement of our language transitioning naturally and we shouldn't think of it as a collapse.

I think it's really interesting how there's data on the enslavers and not just the Africans. Usually we don't see that side of history

Sometimes the things that our parents do, we tend to follow those exact ways. From personal experience, my parents always use a plastic glove to throw away the garbage bags, so I feel it is necessary to use gloves everytime it’s my turn to throw away the garbage.

It's really important to recognize just how significant it means to pass down dishes from generation to generation because sometimes foods and entire cultural meals can be forgotten because the next generation was never taught

I still am not comfortable suggesting that we can even leave this to AI. Just because it can generate proper syntax and readable prose, it doesn't mean it's qualified to EXPLAIN it because it still doesn't understand it.

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1. Point-by-point description of the revisions

This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

Reviewer #1 (Evidence, reproducibility, and clarity (Required)): This is an interesting manuscript from the Jagannathan laboratory, which addresses the interaction proteome of two satellite DNA-binding proteins, D1 and Prod. To prevent a bias by different antibody affinities they use GFP-fusion proteins of D1 and Prod as baits and purified them using anti GFP nanobodies. They performed the purifications in three different tissues: embryo, ovary and GSC enriched testes. Across all experiments, they identified 500 proteins with surprisingly little overlap among tissues and between the two different baits. Based on the observed interaction of prod and D1 with members of the canonical piRNA pathway the authors hypothesized that both proteins might influence the expression of transposable elements. However, neither by specific RNAi alleles or mutants that lead to a down regulation of D1 and Prod in the gonadal soma nor in the germline did they find an effect on the repression of transposable elements. They also did not detect an effect of a removal of piRNA pathway proteins on satellite DNA clustering, which is mediated by Prod and D1. However, they do observe a mis-localisation of the piRNA biogenesis complex to an expanded satellite DNA in absence of D1, which presumably is the cause of a mis-regulation of transposable elements in the F2 generation.This is an interesting finding linking satellite DNA and transposable element regulation in the germline. However, I find the title profoundly misleading as the link between satellite DNA organization and transgenerational transposon repression in Drosophila has not been identified by multi-tissue proteomics but by a finding of the Brennecke lab that the piRNA biogenesis complex has a tendency to localise to satellite DNA when the localisation to the piRNA locus is impaired. Nevertheless, the investigation of the D1 and Prod interactome is interesting and might reveal insights into the pathways that drive the formation of centromeres in a tissue specific manner.

We thank the reviewer for the overall positive comments on our manuscript. As noted above, we have performed a substantial number of revision experiments and improved our text. We believe that our revised manuscript demonstrates a clear link between our proteomics data and the transposon repression. We would like to make three main points,

1. Our proteomics data identified that D1 and Prod co-purified transposon repression proteins in embryos. To test the functional significance of this association, we have used Drosophila genetics to generate flies lacking embryonic D1. In adult ovaries from these flies, we observe a striking elevation in transposon expression and Chk2-dependent gonadal atrophy. Along with the results from the control genotypes (F1 D1 mutant, F2 D1 het), our data clearly indicate that embryogenesis (and potentially early larval development) are a period when D1 establishes heritable TE silencing that can persist throughout development.
2. Based on the newly acquired RNA-seq and small RNA seq data, we have edited our title to more accurately reflect our data. Specifically, we have substituted the word 'transgenerational' with 'heritable', meaning that the presence of D1 during early development alone is sufficient to heritably repress TEs at later stages of development.
3. In addition, our RNA seq and small RNA seq experiments demonstrate that D1 makes a negligible contribution to piRNA biogenesis and TE repression in adults, despite the significant mislocalization of the RDC complex. In this regard, our results are substantially different from the recent Kipferl study from the Brennecke lab (Baumgartner et al. 2022).

   Major comments Unfortunately, the proteomic data sets are not very convincing. Not even the corresponding baits are detected in all assays. I wonder whether the extraction procedure really allows the authors to analyze all functionally relevant interactions of Prod and D1. It would be good to see a western blot or an MS analysis of the soluble nuclear extract they use for purification compared to the insoluble chromatin. It may well be that a large portion of Prod or D1 is lost in this early step. I also find the description of the proteomic results hard to follow as the authors mostly list which proteins the find as interactors of Prod and D1 without stating in which tissue or with what bait they could purify them (i.e. p7: Importantly, our hits included known chromocenter-associated or pericentromeric heterochromatin-associated proteins, such as Su(var)3-9[52], ADD1[23,24], HP5[23,24],mei-S332[53], Mes-4[23], Hmr[24,39,54], Lhr[24,39], and members of the chromosome passenger complex, such as borr and Incenp[55]). It would be interesting to at least discuss tissue specific interactions.

Out of six total AP-MS experiments in this manuscript (D1 x 3, Prod x 2 and Piwi), we observe a strong enrichment of the bait in 5/6 attempts (log2FC between 4-12). In the initial submission, the lack of a third high-quality biological replicate for the D1 testis sample meant that only the p-value (0.07) was not meeting the cutoff. To address this, we have repeated this experiment with an additional biological replicate (Fig. S2A), and our data now clearly show that D1 is significantly enriched in the testis sample.

As suggested by the reviewer, we have also assessed our lysis conditions (450mM NaCl and benzonase) and the solubilization of our baits post-lysis. In Fig. S1D, we have blotted equivalent fractions of the soluble supernatant and insoluble pellet from GFP-Piwi embryos and show that both GFP-Piwi and D1 are largely solubilized following lysis. In Fig. S1E, we also show that our IP protocol works efficiently.

GFP-Prod pulldown in embryos is the only instance in which we do not detect the bait by mass spectrometry. Here, one reason could be relatively low expression of GFP-Prod in comparison to GFP-D1 (Fig. S1E), which may lead to technical difficulties in detecting peptides corresponding to Prod. However, we note that Prod IP co-purified proteins such as Bocks that were previously suggested as Prod interactors from high-throughput studies (Giot et al. 2003; Guruharsha et al. 2011). In addition, Prod IP from embryos also co-purified proteins known to associate with chromocenters such as Hcs and Saf-B. Finally, the concordance between D1 and Prod co-purified proteins from embryo lysates (Fig. 2A, C) suggest that the Prod IP from embryos is of reasonable quality.

We also acknowledge the reviewer's comment that the description of the proteomic data was hard to follow. Therefore, we have revised our text to clearly indicate which bait pulled down which protein in which tissue (lines 148-156). We have also highlighted and discussed bait-specific and tissue-specific interactions in the text (lines 162-173).

Minor comments The authors may also want to provide a bit more information on the quantitation of the proteomic data such as how many values were derive from the match-between runs function and how many were imputed as this can severely distort the quantification.

Figure 1: Distribution of data after imputation in embryo (left), ovary (middle) and testis (right) datasets. Imputation is performed with random sampling from the 1% least intense values generated by a normal distribution.

To ensure the robustness of our data analysis, we considered only those proteins that were consistently identified in all replicates for at least one bait (GFP-D1, GFP-Prod or NLS-GFP). This approach resulted in a relative low number of missing values. However, it is also important to bear in mind that in an AP-MS experiment, the number of missing values is higher, as interactors are not identified in the control pulldown. Importantly, the imputation of missing values during the data analysis did not alter the normal distribution of the dataset (Fig. 1, this document). Detailed information regarding the imputed values is also provided (Table 1, this document). The coding script used for the data analysis is available in the PRIDE submission of the dataset (Table 2, this document). This information has been added to our methods section under data availability.

Table 1: ____Number of match-between-runs and imputations for embryo, ovary and testis datasets

Dataset

#match-between-runs

%match-between-runs

%imputation

Embryo

5541/27543

20.11%

8.36%

Ovary

1936/9530

20.30%

8.18%

Testis

1748/7168

24.39%

3.12%

Table 2: ____Access to the PRIDE submission of the datasets

Name

ID PRIDE

UN reviewer

PW reviewer

IP-MS of D1 from Testis tissue

PXD044026

reviewer_pxd044026@ebi.ac.uk

ydswDQVW

IP-MS of Piwi from Embryo tissue

PXD043237

reviewer_pxd043237@ebi.ac.uk

TMCoDsdx

IP-MS of Prod and D1 proteins from Ovary tissue

PXD043236

reviewer_pxd043236@ebi.ac.uk

VOHqPmaS

IP-MS of Prod and D1 proteins from Embryo tissue

PXD043234

reviewer_pxd043234@ebi.ac.uk

L77VXdvA

**Referee Cross-Commenting** I agree with the two other reviewers that the connection between the interactome and the transgenerational phenotype is unclear. This is also what I meant i my comment that the title is somewhat misleading. A systematic analysis of the D1 and Prod knock down effect on mRNAs and small Rnas would indeed be helpful to better understand the interesting effect.

As suggested by the reviewer, we have performed RNA seq and small RNA seq in control and D1 mutant ovaries (Fig. 4) to fully understand the contribution of D1 in piRNA biogenesis and TE repression. Briefly, the mislocalization of RDC complex in D1 mutant ovaries does not significantly affect TE-mapping piRNA biogenesis (Fig. 4C, E). In addition, loss of D1 does not substantially alter TE expression in the ovaries (Fig. 4B) or alter the expression of genes involved in TE repression (Fig. 4F). Along with the results presented in Fig. 5 and Fig. 6, our data clearly indicate that embryogenesis (and potentially early larval development) is a critical period during which D1 makes an important contribution to TE repression.

Reviewer #1 (Significance (Required)): Nevertheless, the investigation of the D1 and Prod interactome is interesting and might reveal insights into the pathways that drive the formation of centromeres in a tissue specific manner. It may be mostly interesting for the Drosophila community but could also be exiting for a broader audience interested in the connection of heterochromatin and its indirect effect on the regulation of transposable elements.

We thank the reviewer again for the helpful and constructive comments, which have enabled us to significantly improve our study. We are excited by the results from our study, which illuminate unappreciated aspects of transcriptional silencing in constitutive heterochromatin.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): Chavan et al. set out to enrich our compendium of pericentric heterochromatin-associated proteins - and to learn some new biology along the way. An ambitious AP-Mass baited with two DNA satellite-binding proteins (D1 and Prod), conducted across embryos, ovaries, and testes, yielded hundreds of candidate proteins putatively engaged at chromocenters. These proteins are enriched for a restricted number of biological pathways, including DNA repair and transposon regulation. To investigate the latter in greater depth, the authors examine D1 and prod mutants for transposon activity changes using reporter constructs for multiple elements. These reporter constructs revealed no transposon activation in the adult ovary, where many proteins identified in the mass spec experiments restrict transposons. However, the authors suggest that the D1 mutant ovaries do show disrupted localization of a key member of a transposon restriction pathway (Cuff), and infer that this mislocalization triggers a striking, transposon derepression phenotype in the F2 ovaries.

The dataset produced by the AP-Mass Spec offers chromosome biologists an unprecedented resource. The PCH is long-ignored chromosomal region that has historically received minimal attention; consequently, the pathways that regulate heterochromatin are understudied. Moreover, attempting to connect genome organization to transposon regulation is a new and fascinating area. I can easily envision this manuscript triggering a flurry of discovery; however, there is quite a lot of work to do before the data can fully support the claims.

We appreciate the reviewer taking the time to provide thoughtful comments and constructive suggestions to improve the manuscript. We believe that we have addressed all the comments raised to the significant advantage of our paper.

Major comments 1. The introduction requires quite a radical restructure to better highlight the A) importance of the work and B) limit information whose relevance is not clear early in the manuscript. A. Delineating who makes up heterochromatin is a long-standing problem in chromosome biology. This paper has huge potential to contribute to this field; however, it is not the first. Others are working on this problem in other systems, for example PMID:29272703. Moreover, we have some understanding of the distinct pathways that may impact heterochromatin in different tissues (e.g., piRNA biology in ovaries vs the soma). Also, the mutant phenotypes of prod and D1 are different. Fleshing out these three distinct points could help the reader understand what we know and what we don't know about heterochromatin composition and its special biology. Understanding where we are as a field will offer clear predictions about who the interactors might be that we expect to find. For example, given the dramatically different D1 and prod mutant phenotypes (and allele swap phenotypes), how might the interactors with these proteins differ? What do we know about heterochromatin formation differences in different tissues? And how might these differences impact heterochromatin composition?

The reviewer brings up a fair point and we have significantly reworked our introduction. We share the reviewer's opinion that improved knowledge of the constitutive heterochromatin proteome will reveal novel biology.

1. The attempt to offer background on the piRNA pathway and hybrid dysgenesis in the Introduction does not work. As a naïve reader, it was not clear why I was reading about these pathways - it is only explicable once the reader gets to the final third of the Results. Moreover, the reader will not retain this information until the TE results are presented many pages later. I strongly urge the authors to shunt the two TE restriction paragraphs to later in the manuscript. They are currently a major impediment to understanding the power of the experiment - which is to identify new proteins, pathways, and ultimately, biology that are currently obscure because we have so little handle on who makes up heterochromatin.

We agree with this suggestion. We have introduced the piRNA pathway in the results section (lines 205 - 216), right before this information is needed. We've also removed the details on hybrid dysgenesis, since our new data argues against a maternal effect from the D1 mutant.

The implications of the failure to rescue female fertility by the tagged versions of both D1 and Prod are not discussed. Consequently, the reader is left uneasy about how to interpret the data.

We understand this point raised by the reviewer. However, in our proteomics experiments, we have used GFP-D1 and GFP-Prod ovaries from ~1 day old females (line 579, methods). These ovaries are morphologically similar to the wild type (Fig. S1C) and their early germ cell development appears to be intact. Moreover, chromocenter formation in female GSCs is comparable to the wildtype (Fig. 1C-D). These data, along with the rescue of the viability of the Prod mutant (Fig. S1A-B), suggest that the presence of a GFP tag is not compromising D1 or Prod function in the early stages of germline development and is consistent with published and unpublished data from our lab. In addition, D1 and Prod from ovaries co-purify proteins such as Rfc38 (D1), Smn (D1), CG15107 (Prod), which have been identified in previous high-throughput screens (Guruharsha et al. 2011; Tang et al. 2023). For these reasons, we believe that GFP-D1 and GFP-Prod ovaries are a good starting point for the AP-MS experiment. We speculate that the failure to completely rescue female fertility may be due to improper expression levels of GFP-D1 or GFP-Prod flies at later stages of oogenesis, which are not present in ovaries from newly eclosed females and therefore unlikely to affect our proteomic data.

1. How were the significance cut-offs determined? Is the p-value reported the adjusted p-value? As a non-expert in AP-MS, I was surprised to find that the p-value, at least according to the Methods, was not adjusted based on the number of tests. This is particularly relevant given the large/unwieldy(?) number of proteins that were identified as signficant in this study. Moreover, the D1 hit in Piwi pull down is actually not significant according to their criteria of p We used a standard cutoff of log2FC>1 and p2FC and low p-value) since these are more likely to be bona fide interactors. Third, we have used string-DB for functional analyses where we can place our hits in existing protein-protein interaction networks. Using this approach, we detect that Prod (but not D1) pulls down multiple members of the RPA complex in the embryo (RPA2 and RpA-70, Fig. S2B) while embryonic D1 (but not Prod) pulls down multiple components of the origin recognition complex (Orc1, lat, Orc5, Orc6, Fig. S2C) and the condensin I complex (Cap-G, Cap-D2, barr, Fig. S2D). Altogether, these filtering strategies allow us to eliminate as many false positives as possible while making sure to minimize the loss of true hits through multiple testing correction.

How do we know if the lack of overlap across tissues is indeed germline- or soma-specialization rather than noise?

To address this part of the comment, we have amended our text (lines 162-173) as follows,

'We also observed a substantial overlap between D1- and Prod-associated proteins (yellow points in Fig. 2A, B, Table S1-3), with 61 hits pulled down by both baits (blue arrowheads, Fig. 2C) in embryos and ovaries. This observation is consistent with the fact that both D1 and Prod occupy sub-domains within the larger constitutive heterochromatin domain in nuclei. Surprisingly, only 12 proteins were co-purified by the same bait (D1 or Prod) across different tissues (magenta arrowheads, Fig. 2C, Table S1-3). In addition, only a few proteins such as an uncharacterized DnaJ-like chaperone, CG5504, were associated with both D1 and Prod in embryos and ovaries (Fig. 2D). One interpretation of these results is that the protein composition of chromocenters may be tailored to cell- and tissue-specific functions in Drosophila. However, we also note that the large number of unidentified peptides in AP-MS experiments means that more targeted experiments are required to validate whether certain proteins are indeed tissue-specific interactors of D1 and Prod.'

To make this inference, conducting some validation would be required. More generally, I was surprised to see no single interactor validated by reciprocal IP-Westerns to validate the Mass-Spec results, though I am admittedly only adjacent to this technique. Note that colocalization, to my mind, does not validate the AP-MS data - in fact, we would a priori predict that piRNA pathway members would co-localize with PCH given the enrichment of piRNA clusters there.

Here, we would point out that we have conducted multiple validation experiments with a specific focus on the functional significance of the associations between D1/Prod and TE repression proteins in embryos. While the reviewer suggests that piRNA pathway proteins may be expected to enrich at the pericentromeric heterochromatin, this is not always the case. For example, Piwi and Mael are present across the nucleus (pulled down by D1/Prod in embryos) while Cutoff, which is present adjacent to chromocenters in nurse cells, was not observed to interact with either D1 or Prod in the ovary samples.

Therefore, for Piwi, we performed a reciprocal AP-MS experiment in embryos due to the higher sensitivity of this method (GFP-Piwi AP-MS, Fig. 3B). Excitingly, this experiment revealed that four largely uncharacterized proteins (CG14715, CG10208, Ugt35D1 and Fit) were highly enriched in the D1, Prod and Piwi pulldowns and these proteins will be an interesting cohort for future studies on transposon repression in Drosophila (Fig. 3C).

Furthermore, we believe that determining the localization of proteins co-purified by D1/Prod is an important and orthogonal validation approach. For Sov, which is implicated in piRNA-dependent heterochromatin formation, we observe foci that are in close proximity to D1- and Prod-containing chromocenters (Fig. 3A).

As for suggestion to validate by IP-WBs, we would point out that chromocenters exhibit properties associated with phase separated biomolecular condensates. Based on the literature, these condensates tend to associate with other proteins/condensates through low affinity or transient interactions that are rarely preserved in IP-WBs, even if there are strong functional relationships. One example is the association between D1 and Prod, which do not pull each other down in an IP-WB (Jagannathan et al. 2019), even though D1 and Prod foci dynamically associate in the nucleus and mutually regulate each other's ability to cluster satellite DNA repeats (Jagannathan et al. 2019). Similarly, IP-WB using GFP-Piwi embryos did not reveal an interaction with D1 (Fig. S4B). However, our extensive functional validations (Figures 4-6) have revealed a critical role for D1 in heritable TE repression.

The AlphaFold2 data are very interesting but seem to lack of negative control. Is it possible to incorporate a dataset of proteins that are not predicted to interact physically to elevate the impact of the ones that you have focused on? Moreover, the structural modeling might suggest a competitive interaction between D1 and piRNAs for Piwi. Is this true? And even if not, how does the structural model contribute to your understanding for how D1 engages with the piRNA pathway? The Cuff mislocalization?

In the revised manuscript, we have generated more structural models using AlphaFold Multimer (AFM) for proteins (log2FC>2, p0.5 and ipTM>0.8), now elaborated in lines 175-177. Despite the extensive disorder in D1 and Prod, we identified 22 proteins, including Piwi, that yield interfaces with ipTM scores >0.5 (Table S4, Table S8). These hits are promising candidates to further understand D1 and Prod function in the future.

For the predicted model between Prod/D1 and Piwi (Fig. S4A), one conclusion could indeed be competition between D1/Prod and piRNAs for Piwi. Another possibility is that a transient interaction mediated by disordered regions on D1/Prod could recruit Piwi to satellite DNA-embedded TE loci in the pericentromeric heterochromatin. These types of interactions may be especially important in the early embryonic cycles, where repressive histone modifications such as H3K9me2/3 must be deposited at the correct loci for the first time. We suggest that mutating the disordered regions on D1 and Prod to potentially abrogate the interaction with Piwi will be important for future studies.

The absence of a TE signal in D1 and Prod mutant ovaries would be much more compelling if investigated more agnostically. The observation that not all TE reporter constructs show a striking signal in the F2 embryos makes me wonder if Burdock and gypsy are not regulated by these two proteins but possibly other TEs are. Alternatively, small RNA-seq would more directly address the question of whether D1 and Prod regulate TEs through the piRNA pathway.

We completely agree with this comment from the reviewer. We have performed RNA seq on D1 heterozygous (control) and D1 mutant ovaries in a chk26006 background. Since Chk2 arrests germ cell development in response to TE de-repression and DNA damage(Ghabrial and Schüpbach 1999; Moon et al. 2018), we reasoned that the chk2 mutant background would prevent developmental arrest of potential TE-expressing germ cells and we observed that both genotypes exhibited similar gonad morphology (Fig. 4A). From our analysis, we do not observe a significant effect on TE expression in the absence of D1, except for the LTR retrotransposon tirant (Fig. 4B). We also do not observe differential expression of TE repression genes (Fig. 4F).

We have complemented our RNA seq experiment with small RNA profiling from D1 heterozygous (control) and D1 mutant ovaries. Here, overall piRNA production and antisense piRNAs mapping to TEs were largely unperturbed (Fig. 4C-E).

Overall, our data is consistent with the TE reporter data (Fig. S7) and suggests that zygotic depletion of D1 does not have a prominent role in TE repression. However, we have uncovered that the presence of D1 during embryogenesis is critical for TE repression in adult gonads (Fig. 6, Fig. S9).

I had trouble understanding the significance of the Cuff mis-localization when D1 is depleted. Given Cuff's role in the piRNA pathway and close association with chromatin, what would the null hypothesis be for Cuff localization when a chromocenter is disrupted? What is the null expectation of % Cuff at chromocenter given that the chromocenter itself expands massively in size (Figure 4D). The relationship between these two factors seems rather indirect and indeed, the absence of Cuff in the AP would suggest this. The biggest surprise is the absence of TE phenotype in the ovary, given the Cuff mutant phenotype - but we can't rule out given the absence of a genome-wide analysis. I think that these data leave the reader unconvinced that the F2 phenotype is causally linked to Cuff mislocalization.

We apologize that this data was not more clearly represented. In a wild-type context, Cuff is distributed in a punctate manner across the nurse cell nuclei, with the puncta likely representing piRNA clusters (Fig. 5A-B). We find that a small fraction of Cuff (~5%) is present adjacent to the nurse cell chromocenter (inset, Fig. 5A and Fig. 5D). In the absence of D1, the nurse cell chromocenters increase ~3-4 fold in size. However, the null expectation is still that ~5% of total Cuff would be adjacent to the chromocenter, since the piRNA clusters are not expected to expand in size. In reality, we observe ~27% of total Cuff is mislocalized to chromocenters. Our data indicate that the satellite DNA repeats at the larger chromocenters must be more accessible to Cuff in the D1 mutant nurse cells. This observation is corroborated by the significant increase in piRNAs corresponding to the 1.688 satellite DNA repeat family (Fig. 4E).

The lack of TE expression in the F1 D1 mutant was indeed surprising based on the Cuff mislocalization but as the reviewers pointed out, we only analyzed two TE reporter constructs in the initial submission. In the revised manuscript, we have performed both RNA seq and small RNA seq. Surprisingly, our data reveal that the Cuff mislocalization does not significantly affect piRNA biogenesis (Fig. 4C, D) and piRNAs mapping to TEs. As a result, both TE repression (Fig. 4B) and fertility (Fig. 6D) are largely preserved in the absence of D1 in adult ovaries.

Finally, we thank the reviewer for their excellent suggestion to incorporate the F2 D1 heterozygote (Fig. S9) in our analysis! This important control has revealed that the maternal effect of the D1 mutant is negligible for gonad development and fertility (Fig. 6B-D). Rather, our data clearly emphasize embryogenesis (or early larval development) as a key period during which D1 can promote heritable TE repression. Essentially, D1 is not required during adulthood for TE repression if it was present in the early stages of development.

Apologies if I missed this, but Figure 5 shows the F2 D1 mutant ovaries only. Did you look at the TM6 ovaries as well? These ovaries should lack the maternally provisioned D1 (assuming that females are on the right side) but have the zygotic transcription.

As mentioned above, this was a great suggestion and we've now characterized this important control in the context of germline development and fertility, to the significant advantage of our paper.

Minor comments 9. Add line numbers for ease of reference

We apologize for this. Line numbers have been added in the full revision.

1. The function of satellite DNA itself is still quite controversial - I would recommend being a bit more careful here - the authors could refer instead to genomic regions enriched for satellite DNA are linked to xyz function (see Abstract line 2 and 7, for example.)

The abstract has been rewritten and does not directly implicate satellite DNA in a specific cellular function. However, we have taken the reviewer's suggestion in the introduction (line 57-58).

"Genetic conflicts" in the introduction needs more explanation.

This sentence has been removed from the introduction in the revised manuscript.

"In contrast" is not quite the right word. Maybe "However" instead (1st line second paragraph of Intro)

Done. Line 57 of the revised manuscript.

Results: what is the motivation for using GSC-enriched testis?

We use GSC-enriched testes for practical reasons. First, they contain a relatively uniform population of mitotically dividing germ cells unlike regular testes which contain a multitude of post-mitotic germ cells such as spermatocytes, spermatids and sperm. Second, GSC-enriched testes are much larger than normal testes and reduced the number of dissections that were needed for each replicate.

1. Clarify sentence about the 500 proteins in the Results section - it's not clear from context that this is the union of all experiments.

Done. Lines 145-149 in the revised manuscript.

The data reported are not the first to suggest that satellite DNA may have special DNA repair requirements. e.g., PMID: 25340780

We apologize if we gave the impression that we were making a novel claim. Specialized DNA repair requirements at repetitive sequences have indeed been previously hypothesized(Charlesworth et al. 1994) and studied and we have altered the text to better reflect this (lines 193-195). We have cited the study suggested by the reviewer as well as studies from the Chiolo(Chiolo et al. 2011; Ryu et al. 2015; Caridi et al. 2018) and Soutoglou(Mitrentsi et al. 2022) labs, which have also addressed this fascinating question.

Page 10: indicate-> indicates.

Done.

1. Page 14: revise for clarity: "investigate a context whether these interactions could not take place"

We've incorporated this suggestion in the revised text (lines 383-386).

1. Might be important to highlight the 500 interactions are both direct and indirect. "Interacting proteins" alone suggests direct interactions only.

Done. Lines 145-149.

The effect of the aub mutant on chromocenter foci did not seem modest to me - however, the bar graphs obscure the raw data - consider plotting all the data not just the mean and error?

Done. This data is now represented by a box-and-whisker plot (Fig. S5), which shows the distribution of the data.

Reviewer #2 (Significance (Required)):

The dataset produced by the AP-Mass Spec offers chromosome biologists an unprecedented resource. The PCH is long-ignored chromosomal region that has historically received minimal attention; consequently, the pathways that regulate heterochromatin are understudied. Moreover, attempting to connect genome organization to transposon regulation is a new and fascinating area. I can easily envision this manuscript triggering a flurry of discovery; however, there is quite a lot of work to do before the data can fully support the claims.

This manuscript represents a significant contribution to the field of chromosome biology.

We thank the reviewer for the positive evaluation of our manuscript, and we really appreciate the great suggestion for the F2 D1 heterozygote control! Overall, we believe that our manuscript is substantially improved with the addition of RNA seq, small RNA seq and important genetic controls. Moreover, we are excited by the potential of our study to open new doors in the study of pericentromeric heterochromatin.

Reviewer #3 (Evidence, reproducibility and clarity (Required)): In the manuscript entitled "Multi-tissue proteomics identifies a link between satellite DNA organization and transgenerational transposon repression", the authors used two satellite DNA-binding proteins, D1 and Prod, as baits to identify chromocenter-associated proteins through quantitative mass spectrometry. The proteomic analysis identified ~500 proteins across embryos, ovaries, and testes, including several piRNA pathways proteins. Depletion of D1 or Prod did not directly contribute to transposon repression in ovary. However, in the absence of maternal and zygotic D1, there was a dramatic increase of agametic ovaries and transgenerational transposon de-repression. Although the study provides a wealth of proteomic data, it lacks mechanistic insights into how satellite DNA organization influence the interactions with other proteins and their functional consequences.

We thank the reviewer for highlighting that this study will be a valuable resource for future studies on the composition and function of pericentromeric heterochromatin. Based on the reviewer's request for more mechanistic knowledge into how satellite DNA organization affects transposon repression, we have performed RNA seq and small RNA seq, added important genetic controls and significantly improved our text. In the revised manuscript, our data clearly demonstrate that embryogenesis (and potentially early larval development) is a critical time period when D1 contributes to heritable TE repression. Flies lacking D1 during embryogenesis exhibit TE expression in germ cells as adults, which is associated with Chk2-dependent gonadal atrophy. We are particularly excited by these data since TE loci are embedded in the satellite DNA-rich pericentromeric heterochromatin and our study demonstrates an important role for a satellite DNA-binding protein in TE repression.

Major____ comments 1. While the identification of numerous interactions is significant, it would be helpful to acknowledge that lots of these proteins were known to bind DNA or heterochromatin regions. To strengthen the study, I recommend conducting functional validation of the identified interactions, in addition to the predictions made by Alphfold 2.

We are happy to take this comment on board. In fact, we believe that the large number of DNA-binding and heterochromatin-associated proteins identified in this study are a sign of quality for the proteomic datasets. In the revised manuscript, we have highlighted known heterochromatin proteins co-purified by D1/Prod in lines 148-151 as well as proteins previously suggested to interact with D1/Prod from high-throughput studies in lines 153-156 (Guruharsha et al. 2011; Tang et al. 2023). In this study, we have focused on the previously unknown associations between D1/Prod and TE repression proteins and functionally validated these interactions as presented in Figures 3-6.

The observation of transgenerational de-repression is intriguing. However, to better support this finding, it would be better to provide a mechanistic explanation based on the data presented.

We appreciate this comment from the reviewer, which is similar to major comment #6 raised by reviewer #2. To generate mechanistic insight into the underlying cause of gonad atrophy in the F2 D1 mutant, we have performed RNA seq, small RNA seq and analyzed germline development and fertility in the F2 D1 heterozygous control (Fig. S9).

For the RNA seq, we used D1 heterozygous (control) and D1 mutant ovaries in a chk26006 background. Since Chk2 arrests germ cell development in response to TE de-repression and DNA damage(Ghabrial and Schüpbach 1999; Moon et al. 2018), we reasoned that the chk2 mutant background would prevent developmental arrest of potential TE-expressing germ cells and we observed that both genotypes exhibited similar gonad morphology (Fig. 4A). From our analysis, we do not observe a significant effect on TE expression in the absence of D1, except for the LTR retrotransposon tirant (Fig. 4B). We also do not observe differential expression of TE repression genes (Fig. 4F).

We have complemented our RNA seq experiment with small RNA profiling from D1 heterozygous (control) and D1 mutant ovaries. Here, overall piRNA production and antisense piRNAs mapping to TEs were largely unperturbed (Fig. 4C-E). Together, these data are consistent with the TE reporter data (Fig. S7) and suggests that zygotic depletion of D1 does not have a prominent role in TE repression.

However, we have uncovered that the presence of D1 during embryogenesis is critical for TE repression in adult gonads (Fig. 6, Fig. S9). Essentially, either only maternal deposited D1 (F1 D1 mutant) or only zygotically expressed D1 (F2 D1 het) were sufficient to ensure TE repression and fertility. In contrast, a lack of D1 during embryogenesis (F2 D1 mutant) led to elevated TE expression and Chk2-mediated gonadal atrophy.

Our results also explain why previous studies have not implicated either D1 or Prod in TE repression, since D1 likely persists during embryogenesis when using depletion approaches such as RNAi-mediated knockdown or F1 generation mutants.

Minor____ comments 3. Given the maternal effect of the D1 mutant, in Figure 4, I suggest analyzing not only nurse cells but also oocytes to gain a more comprehensive understanding.

We agree with the reviewer that this experiment can be informative. In the F2 D1 mutant ovaries, germ cell development does not proceed to a stage where oocytes are specified, thus limiting microscopy-based approaches. Nevertheless, we have gauged oocyte quality in all the genotypes using a fertility assay (Fig. 6D) since even ovaries that have a wild-type appearance can produce dysfunctional gametes. In this experiment, we observe that fertility is largely intact in the F1 D1 mutant and F2 D1 heterozygote strains, suggesting that it is the presence of D1 during embryogenesis (or early larval development) that is critical for heritable TE repression and proper oocyte development.

The conclusion that D1 and Prod do not directly contribute to the repression of transposons needs further analysis from RNA-seq data. Alternatively, the wording could be adjusted to indicate that D1 and Prod are not required for specific transposon silencing, such as Burdock and gypsy.

Agreed. We have performed RNA-seq in D1 heterozygous (control) and D1 mutant ovaries in a chk26006 background (Fig. 4A, B) as described above.

As D1 mutation affects Cuff nuclear localization, it would be insightful to sequence the piRNA in the ovaries.

Agreed. We have performed small RNA profiling from D1 heterozygous (control) and D1 mutant ovaries. Despite the significant mislocalization of the RDC complex, overall piRNA production and antisense piRNAs mapping to TEs were largely unaffected (Fig. 4C-E). However, we observed significant changes in piRNAs mapping to complex satellite DNA repeats (Fig. 4D), but these changes were not associated with a maternal effect on germline development or fertility (F2 D1 heterozygote, Fig. 6B-D).

**Referee Cross-Commenting**

I appreciate the valuable insights provided by the other two reviewers regarding this manuscript. I concur with their assessment that substantial improvements are needed before considering this manuscript for publication.

1. The proteomics data has the potential to be a valuable resource for other scientific community. However, I share the concerns raised by reviewer 1 about the current quality of the data sets. Addressing this, it will augment the manuscript with additional data to show the success of the precipitation. Moreover, as reviewer 2 and I suggested, additional co-IP validations of the IP-MS results are needed to enhance the reliability.

In the revised manuscript, we have performed multiple experiments to address the quality of the MS datasets based on comments raised by reviewer #1. They are as follows,

Out of six total AP-MS experiments in this manuscript (D1 x 3, Prod x 2 and Piwi), we observe a strong enrichment of the bait in 5/6 attempts (log2FC between 4-12, Fig. 2A, B, Fig. S2A, Table S1-S3, Table S7). In the D1 testis sample from the initial submission, the lack of a third biological replicate meant that only the p-value (0.07) was not meeting the cutoff. To address this, we have repeated this experiment with an additional biological replicate (Fig. S2A), and our data now clearly show that D1 is also significantly enriched in the testis sample.

As suggested by the reviewer #1, we have assessed our lysis conditions (450mM NaCl and benzonase) and the solubilization of our baits post-lysis. In Fig. S1D, we have blotted equivalent fractions of the soluble supernatant and insoluble pellet from GFP-Piwi embryos and show that both GFP-Piwi and D1 are largely solubilized following lysis. In Fig. S1E, we also show that our IP protocol works efficiently.

The only instance in which we do not detect the bait by mass spectrometry is for GFP-Prod pulldown in embryos. Here, one reason could be relatively low expression of GFP-Prod in comparison to GFP-D1 (Fig. S1E), which may lead to technical difficulties in detecting peptides corresponding to Prod. However, we note that Prod IP from embryos co-purified proteins such as Bocks that were previously suggested as Prod interactors from high-throughput studies (Giot et al. 2003; Guruharsha et al. 2011). In addition, Prod IP from embryos also co-purified proteins known to associate with chromocenters such as Hcs(Reyes-Carmona et al. 2011) and Saf-B(Huo et al. 2020). Finally, the concordance between D1 and Prod co-purified proteins from embryo lysates (Fig. 2A, C) suggest that the Prod IP from embryos is of reasonable quality.

As for the IP-WB validations, we would point out that chromocenters exhibit properties associated with phase separated biomolecular condensates. In our experience, these condensates tend to associate with other proteins/condensates through low affinity or transient interactions that are rarely preserved in IP-WBs, even if there are strong functional relationships. One example is the association between D1 and Prod, which do not pull each other down in an IP-WB (Jagannathan et al. 2019), even though D1 and Prod foci dynamically associate in the nucleus and mutually regulate each other's ability to cluster satellite DNA repeats (Jagannathan et al. 2019). Similarly, IP-WB using GFP-Piwi embryos did not reveal an interaction with D1 (Fig. S4B). However, our extensive functional validations (Figures 4-6) have revealed a critical role for D1 in heritable TE repression.

I agree with reviewer 2 that the present conclusion is not appropriate regarding D1 and Prod do not contribute to transposon silencing. As reviewer 2 and I suggested, the systematical analysis by using both mRNA-seq and small RNA-seq is required to draw the conclusion.

Agreed. We have performed RNA seq and small RNA seq as elaborated above. Our conclusions on the role of D1 in TE repression are now much stronger.

1. The transgenerational phenotype is an intriguing aspect of the study. I agree with reviewer 2 that the inclusion of TM6 ovaries would be a nice control. Further, it is hard to connect this phenotype with the interactions identified in this manuscript. It would be appreciated if the author could provide a mechanistic explanation.

We have significantly improved these aspects of our study in the revised manuscript. Through the analysis of germline development in the F2 D1 heterozygotes as suggested by reviewer #2, in addition to the recommended RNA seq and small RNA seq, we have now identified embryogenesis (and potentially early larval development) as a time period when D1 makes an important contribution to TE repression. Loss of D1 during embryogenesis leads to TE expression in adult germline cells, which is associated with Chk2-dependent gonadal atrophy. Taken together, we have pinpointed the specific contribution of a satellite DNA-binding protein to transposon repression.

Reviewer #3 (Significance (Required)):

Although this study successfully identified several interactions, the authors did not fully elucidate how these interactions contribute to the transgenerational silencing of transposons or ovary development.

We thank the reviewer for the thoughtful comments and overall positive evaluation of our study, especially the proteomic dataset. We are confident that the revised manuscript has improved our mechanistic understanding of the contribution made by a satellite DNA-binding protein in TE repression.

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Referee #2

Evidence, reproducibility and clarity

Chavan et al. set out to enrich our compendium of pericentric heterochromatin-associated proteins - and to learn some new biology along the way. An ambitious AP-Mass baited with two DNA satellite-binding proteins (D1 and Prod), conducted across embryos, ovaries, and testes, yielded hundreds of candidate proteins putatively engaged at chromocenters. These proteins are enriched for a restricted number of biological pathways, including DNA repair and transposon regulation. To investigate the latter in greater depth, the authors examine D1 and prod mutants for transposon activity changes using reporter constructs for multiple elements. These reporter constructs revealed no transposon activation in the adult ovary, where many proteins identified in the mass spec experiments restrict transposons. However, the authors suggest that the D1 mutant ovaries do show disrupted localization of a key member of a transposon restriction pathway (Cuff), and infer that this mislocalization triggers a striking, transposon derepression phenotype in the F2 ovaries.

The dataset produced by the AP-Mass Spec offers chromosome biologists an unprecedented resource. The PCH is long-ignored chromosomal region that has historically received minimal attention; consequently, the pathways that regulate heterochromatin are understudied. Moreover, attempting to connect genome organization to transposon regulation is a new and fascinating area. I can easily envision this manuscript triggering a flurry of discovery; however, there is quite a lot of work to do before the data can fully support the claims.

Major

1. The introduction requires quite a radical restructure to better highlight the A) importance of the work and B) limit information whose relevance is not clear early in the manuscript. A. Delineating who makes up heterochromatin is a long-standing problem in chromosome biology. This paper has huge potential to contribute to this field; however, it is not the first. Others are working on this problem in other systems, for example PMID:29272703. Moreover, we have some understanding of the distinct pathways that may impact heterochromatin in different tissues (e.g., piRNA biology in ovaries vs the soma). Also, the mutant phenotypes of prod and D1 are different. Fleshing out these three distinct points could help the reader understand what we know and what we don't know about heterochromatin composition and its special biology. Understanding where we are as a field will offer clear predictions about who the interactors might be that we expect to find. For example, given the dramatically different D1 and prod mutant phenotypes (and allele swap phenotypes), how might the interactors with these proteins differ? What do we know about heterochromatin formation differences in different tissues? And how might these differences impact heterochromatin composition? B. The attempt to offer background on the piRNA pathway and hybrid dysgenesis in the Introduction does not work. As a naïve reader, it was not clear why I was reading about these pathways - it is only explicable once the reader gets to the final third of the Results. Moreover, the reader will not retain this information until the TE results are presented many pages later. I strongly urge the authors to shunt the two TE restriction paragraphs to later in the manuscript. They are currently a major impediment to understanding the power of the experiment - which is to identify new proteins, pathways, and ultimately, biology that are currently obscure because we have so little handle on who makes up heterochromatin.
2. The implications of the failure to rescue female fertility by the tagged versions of both D1 and Prod are not discussed. Consequently, the reader is left uneasy about how to interpret the data.
3. How were the significance cut-offs determined? Is the p-value reported the adjusted p-value? As a non-expert in AP-MS, I was surprised to find that the p-value, at least according to the Methods, was not adjusted based on the number of tests. This is particularly relevant given the large/unwieldy(?) number of proteins that were identified as signficant in this study. Moreover, the D1 hit in Piwi pull down is actually not significant according to their criteria of p <0.05 (D1 is p=0.05).
4. How do we know if the lack of overlap across tissues is indeed germline- or soma-specialization rather than noise? To make this inference, conducting some validation would be required. More generally, I was surprised to see no single interactor validated by reciprocal IP-Westerns to validate the Mass-Spec results, though I am admittedly only adjacent to this technique. Note that colocalization, to my mind, does not validate the AP-MS data - in fact, we would a priori predict that piRNA pathway members would co-localize with PCH given the enrichment of piRNA clusters there.
5. The AlphaFold2 data are very interesting but seem to lack of negative control. Is it possible to incorporate a dataset of proteins that are not predicted to interact physically to elevate the impact of the ones that you have focused on? Moreover, the structural modeling might suggest a competitive interaction between D1 and piRNAs for Piwi. Is this true? And even if not, how does the structural model contribute to your understanding for how D1 engages with the piRNA pathway? The Cuff mislocalization?
6. The absence of a TE signal in D1 and Prod mutant ovaries would be much more compelling if investigated more agnostically. The observation that not all TE reporter constructs show a striking signal in the F2 embryos makes me wonder if Burdock and gypsy are not regulated by these two proteins but possibly other TEs are. Alternatively, small RNA-seq would more directly address the question of whether D1 and Prod regulate TEs through the piRNA pathway.
7. I had trouble understanding the significance of the Cuff mis-localization when D1 is depleted. Given Cuff's role in the piRNA pathway and close association with chromatin, what would the null hypothesis be for Cuff localization when a chromocenter is disrupted? What is the null expectation of % Cuff at chromocenter given that the chromocenter itself expands massively in size (Figure 4D). The relationship between these two factors seems rather indirect and indeed, the absence of Cuff in the AP would suggest this. The biggest surprise is the absence of TE phenotype in the ovary, given the Cuff mutant phenotype - but we can't rule out given the absence of a genome-wide analysis. I think that these data leave the reader unconvinced that the F2 phenotype is causally linked to Cuff mislocalization.
8. Apologies if I missed this, but Figure 5 shows the F2 D1 mutant ovaries only. Did you look at the TM6 ovaries as well? These ovaries should lack the maternally provisioned D1 (assuming that females are on the right side) but have the zygotic transcription.

Minor

1. Add line numbers for ease of reference
2. The function of satellite DNA itself is still quite controversial - I would recommend being a bit more careful here - the authors could refer instead to genomic regions enriched for satellite DNA are linked to xyz function (see Abstract line 2 and 7, for example.)
3. "Genetic conflicts" in the introduction needs more explanation.
4. "In contrast" is not quite the right word. Maybe "However" instead (1st line second paragraph of Intro)
5. Results: what is the motivation for using GSC-enriched testis?
6. Clarify sentence about the 500 proteins in the Results section - it's not clear from context that this is the union of all experiments.
7. The data reported are not the first to suggest that satellite DNA may have special DNA repair requirements. e.g., PMID: 25340780
8. Page 10: indicate-> indicates.
9. Page 14: revise for clarity: "investigate a context whether these interactions could not take place"
10. Might be important to highlight the 500 interactions are both direct and indirect. "Interacting proteins" alone suggests direct interactions only.
11. The effect of the aub mutant on chromocenter foci did not seem modest to me - however, the bar graphs obscure the raw data - consider plotting all the data not just the mean and error?

Significance

The dataset produced by the AP-Mass Spec offers chromosome biologists an unprecedented resource. The PCH is long-ignored chromosomal region that has historically received minimal attention; consequently, the pathways that regulate heterochromatin are understudied. Moreover, attempting to connect genome organization to transposon regulation is a new and fascinating area. I can easily envision this manuscript triggering a flurry of discovery; however, there is quite a lot of work to do before the data can fully support the claims.

This manuscript represents a significant contribution to the field of chromosome biology.

Reviewer #1 (Public Review):

The question of whether eyespots mimic eyes has certainly been around for a very long time and led to a good deal of debate and contention. This isn't purely an issue of how eyespots work either, but more widely an example of the potential pitfalls of adopting 'just-so-stories' in biology before conducting the appropriate experiments. Recent years have seen a range of studies testing eye mimicry, often purporting to find evidence for or against it, and not always entirely objectively. Thus, the current study is very welcome, rigorously analysing the findings across a suite of papers based on evidence/effect sizes in a meta-analysis.

The work is very well conducted, robust, objective, and makes a range of valuable contributions and conclusions, with an extensive use of literature for the research. I have no issues with the analysis undertaken. The results and conclusions are compelling. It's probably fair to say that the topic needs more experiments to really reach firm conclusions but the authors do a good job of acknowledging this and highlighting where that future work would be best placed.

Author response:

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

Public Reviews: 

Reviewer #1 (Public Review):

Summary:

The question of whether eyespots mimic eyes has certainly been around for a very long time and led to a good deal of debate and contention. This isn't purely an issue of how eyespots work either, but more widely an example of the potential pitfalls of adopting 'just-so-stories' in biology before conducting the appropriate experiments. Recent years have seen a range of studies testing eye mimicry, often purporting to find evidence for or against it, and not always entirely objectively. Thus, the current study is very welcome, rigorously analysing the findings across a suite of papers based on evidence/effect sizes in a meta-analysis.

Strengths:

The work is very well conducted, robust, objective, and makes a range of valuable contributions and conclusions, with an extensive use of literature for the research. I have no issues with the analysis undertaken, just some minor comments on the manuscript. The results and conclusions are compelling. It's probably fair to say that the topic needs more experiments to really reach firm conclusions but the authors do a good job of acknowledging this and highlighting where that future work would be best placed.

Weaknesses:

There are few weaknesses in this work, just some minor amendments to the text for clarity and information.

We greatly appreciate Reviewer 1’s positive comments on our manuscript. We also revised our manuscript text and a figure in accordance with Reviewer 1’s recommendations.

Reviewer #2 (Public Review):

Many prey animals have eyespot-like markings (called eyespots) which have been shown in experiments to hinder predation. However, why eyespots are effective against predation has been debated. The authors attempt to use a meta-analytical approach to address the issue of whether eye-mimicry or conspicuousness makes eyespots effective against predation. They state that their results support the importance of conspicuousness. However, I am not convinced by this.

There have been many experimental studies that have weighed in on the debate. Experiments have included manipulating target eyespot properties to make them more or less conspicuous, or to make them more or less similar to eyes. Each study has used its own set of protocols. Experiments have been done indoors with a single predator species, and outdoors where, presumably, a large number of predator species predated upon targets. The targets (i.e, prey with eyespot-like markings) have varied from simple triangular paper pieces with circles printed on them to real lepidopteran wings. Some studies have suggested that conspicuousness is important and eye-mimicry is ineffective, while other studies have suggested that more eye-like targets are better protected. Therefore, there is no consensus across experiments on the eye-mimicry versus conspicuousness debate.

The authors enter the picture with their meta-analysis. The manuscript is well-written and easy to follow. The meta-analysis appears well-carried out, statistically. Their results suggest that conspicuousness is effective, while eye-mimicry is not. I am not convinced that their meta-analysis provides strong enough evidence for this conclusion. The studies that are part of the meta-analysis are varied in terms of protocols, and no single protocol is necessarily better than another. Support for conspicuousness has come primarily from one research group (as acknowledged by the authors), based on a particular set of protocols.

Furthermore, although conspicuousness is amenable to being quantified, for e.g., using contrast or size of stimuli, assessment of 'similarity to eyes' is inherently subjective. Therefore, manipulation of 'similarity to eyes' in some studies may have been subtle enough that there was no effect.

There are a few experiments that have indeed supported eye-mimicry. The results from experiments so far suggest that both eye-mimicry and conspicuousness are effective, possibly depending on the predator(s). Importantly, conspicuousness can benefit from eye-mimicry, while eye-mimicry can benefit from conspicuousness.

Therefore, I argue that generalizing based on a meta-analysis of a small number of studies that conspicuousness is more important than eye-mimicry is not justified. To summarize, I am not convinced that the current study rules out the importance of eye-mimicry in the evolution of eyespots, although I agree with the authors that conspicuousness is important.

We understand Reviewer 2’s concerns and have addressed them by adding some sentences in the discussion part (L506- 508, L538-L540). In addition, our findings, which were guided by current knowledge, support the conspicuousness hypothesis, but we acknowledge the two hypotheses are not mutually exclusive (L110-112). We also do not reject the eye mimicry hypothesis. As we have demonstrated, there are still several gaps in the current literature and our understanding (L501-553). Our aim is for this research to stimulate further studies on this intriguing topic and to foster more fruitful discussions.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Minor comments

Lines 59/60: "it is possible that eyespots do not involve mimicry of eyes..."

The sentence was revised (L59). To enhance readability, we have integrated Reviewer 1's suggestions by simplifying the relevant section instead of using the suggested sentence.

Line 61: not necessarily aposematism. They might work simply through neophobia, unfamiliarity, etc even without unprofitability

We changed the text in line with the comment from Reviewer 1 (L61-63).

Lines 62/63 - this is a little hard to follow because I think you really mean both studies of real lepidopterans as well as artificial targets. Need to explain a bit more clearly.

We provided an additional explanation of our included primary study type (L64-65).

Lines 93/94 - not quite that they have nothing to do with predator avoidance, but more that any subjective resemblance to eyes is coincidental, or simply as a result of those marking properties being more effective through conspicuousness in their own right.

Line 94 - similarly, not just aposematism. You explain the possible reasons above on l92 as also being neophobia, etc.

We agreed with Reviewer 1’s comments and added more explanations about the conspicuousness hypothesis (L96-97). We have also rewritten the sentences that could be misleading to readers (L428).

Line 96 - this is perhaps a bit misleading as it seems to conflate mechanism and function. The eye mimicry vs conspicuousness debate is largely about how the so-called 'intimidation' function of eyespots works. That is, how eyespots prevent predators from attacking. The deflection hypothesis is a second function of eyespots, which might also work via consciousness or eye mimicry (e.g. if predators try to peck at 'eyes') but has been less central to the mimicry debate.

The explanations and suggestions from Reviewer 1 are very helpful. We revised this part of our manuscript (L103-108) and Figure 1 and its legend to make it clearer that the eyespot hypothesis and the conspicuousness hypothesis explain anti-predator functions from a different perspective than the deflection hypothesis.

There is a third function of eyespots too, that being as mate selection traits. Note that Figure 1 should also be altered to reflect these points.

We wanted to focus on explaining why eyespot patterns can contribute to prey survival. Therefore, we did not state that eyespot patterns function as mate selection traits in this paragraph. Alternatively, we have already mentioned this in the Discussion part (L455-L465) and rewrote it more clearly (L456).

Were there enough studies on non-avian predators to analyse in any way? 

We found a few studies on non-avian predators (e.g. fish, invertebrates, or reptiles), but not enough to conduct a meta-analysis.

Line 171/72 - why? Can you explain, please.

The reason we excluded studies that used bright or contrasting patterns as control stimuli in our meta-analysis is to ensure comparability across primary studies. We added an explanation in the text (L180-181).

Line 177 - can you clarify this?

Without control stimuli, it is challenging to accurately assess the effect of eyespots or other conspicuous patterns on predation avoidance. Control stimuli allow for a comparison of the effect of eyespots or patterns. We added a more detailed explanation to clarify here (L186-188).

Line 309 - presumably you mean 33 papers, each of which may have multiple experiments? I might have missed it, but how many individual experiments in total? 

There were 164 individual experiments. We have now added that information in the manuscript (L320).

Line 320 - paper shaped in a triangle mostly?

We cannot say that most artificial prey were triangular. After excluding the caterpillar type, 57.4% were triangular, while the remaining 43.6% were rectangular (Figure 2b).

Line 406: Stevens.

We fixed this name, thank you (L417).

Discussion - nice, balanced and thorough. Much of the work done has been in Northern Europe where eyespot species are less common. Perhaps things may differ in areas where eyespots are more prevalent.

We appreciate Reviewer 1’s kind words and comments. We agree with your comments and reflected them in our manuscript (L542-545).

Line 477 - True, and predators often have forward-facing eyes making it likely both would often be seen, but a pair of eyes may not be absolutely crucial to avoidance since sometimes a prey animal may only see one eye of a predator (e.g. if the other is occluded, or only one side of the head is visible).

We were grateful for Reviewer 1's comment. We added a sentence noting that the eyespots do not necessarily have to be in pairs to resemble eyes (L490-L492).

This is a big deal with different friend groups, there are always different humors. It's not just about the humor, it's how you want to fit in with them. If you do something that puts you in the spotlight, there would be two ways to deal with it, either the friend group would ignore it or get mad at you. We dislike having the thought that other people dislike us so we try to blend in and do whatever we can to be a part of them even though it would be uncomfortable for us. Humans want to be noticed and have friends, not be left alone.

Just before the final invite, I'd insert a section that speaks to immediate, short-term and long-term wins. You'd structure it in 3 columns titled As soon as you join..., In a month..., in 6 months... That's where you outline what they can achieve/experience/feel at each stage. It's super powerful and hopefully invites readers to opt for the annual membership because you highlight the long-term transformation.

Speaking of summaries, AI worse than humans at summaries studies show.

Succinct reason why by David Chisnall:

LLMs are good at transforms that have the same shape as ones that appear in their training data. They're fairly good, for example, at generating comments from code because code follows common structures and naming conventions that are mirrored in the comments (with totally different shapes of text).

In contrast, summarisation is tightly coupled to meaning. Summarisation is not just about making text shorter, it's about discarding things that don't contribute to the overall point and combining related things. This is a problem that requires understanding the material, because it's all about making value judgements.

This is another example of how we need more women in STEM. There are so many officially desegregated organizations. But segragation is embedded in behavior and that is what needs coaching.

From what I've understand it's people who want to go to college without needing to work and just focusing on college and enjoying life. They don't need to do part time or so.

Al has been used way more than just for educational purposes. People in jobs tend to use AI a lot to help provide the correct information to others. AI is a technology based tool and all jobs that use technology have a sense of when AI can be helpful in projects or articles and when you should use AI

From my understanding, Culture is more than just meets the eye, and for that, I agree with this statement. Culture can. Be as simple as the way one moves as part of one's lifestyle to as deep as those "intangible qualities" of a person's life. Because it's intangible Some examples of these can be traditions or knowledge about culture.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This paper conducted a GWAS meta-analysis for COVID-19 hospitalization among admixed American populations. The authors identified four genome-wide significant associations, including two novel loci (BAZ2B and DDIAS), and an additional risk locus near CREBBP using cross-ancestry meta-analysis. They utilized multiple strategies to prioritize risk variants and target genes. Finally, they constructed and assessed a polygenic risk score model with 49 variants associated with critical COVID-19 conditions.

Strengths:

Given that most of the previous studies were done in European ancestries, this study provides unique findings about the genetics of COVID-19 in admixed American populations. The GWAS data would be a valuable resource for the community. The authors conducted comprehensive analyses using multiple different strategies, including Bayesian fine mapping, colocalization, TWAS, etc., to prioritize risk variants and target genes. The polygenic risk score (PGS) result demonstrated the ability of the cross-population

PGS model for COVID-19 risk stratification.

Thank you very much for the positive comments and the willingness to revise this manuscript.

Weaknesses:

(1) One of the major limitations of this study is that the GWAS sample size is relatively small, which limits its power.

(2) The fine mapping section is unclear and there is a lack of information. The authors assumed one causal signal per locus, and only provided credible sets, but did not provide posterior inclusion probabilities (PIP) for the variants to be causal.

(3) Colocalization and TWAS used eQTL data from GTEx data, which are mainly from European ancestries. It is unclear how much impact the ancestry mismatch would have on the result. The readers should be cautious when interpreting the results and designing follow-up studies.

We agree with that the sample size is relatively small. Despite that, it was sufficient to reveal novel risk loci supporting the robustness of the main findings. We have indicated this limitation at the end of the discussion section.

Thank you for rising this point. As suggested, we have also used SuSIE, which allows to assume more than one causal signal per locus. However, in this case the results were not different from those obtained with the original Bayesian colocalization performed with corrcoverage. Regarding the PIP, at the fine mapping stage we are inclined to put more weight on the functional annotations of the variants in the credible set than on the statistical contributions to the signal. This is the reason why we prefer not to put weight on the PIP of the variants but prioritize variants that were enriched functional annotations.

This is a good point regarding the lack of diversity in GTEx data. We have also used data from AMR populations (GALA II-SAGE models), although it was only available for blood tissue. Regarding the ancestry mismatch between datasets, several studies have attempted to explore the impact. Gay et al. (PMID: 32912333) studied local ancestry effects on eQTLs from the GTEx consortium and concluded that adjustment of eQTLs by local ancestry only yields modest improvement over using global ancestry (as done in GTEx). Moreover, the colocalization results between adjusting by Local Ancestry and Global Ancestry were not significantly different. Besides, Mogil et al. (PMID: 30096133) observed that genes with higher heritability share genetic architecture between populations. Nevertheless, both studies have evidenced decreased power and poorer predictive performances regarding gene expression because of reduced diversity in eQTL analyses. As consequence of the ancestry mismatch, we now warn the readers that this may compromise signal detection (Discussion, lines 531-533). 

Reviewer #2 (Public Review):

This is a genome-wide association study of COVID-19 in individuals of admixed American ancestry (AMR) recruited from Brazil, Colombia, Ecuador, Mexico, Paraguay, and Spain. After quality control and admixture analysis, a total of 3,512 individuals were interrogated for 10,671,028 genetic variants (genotyped + imputed). The genetic association results for these cohorts were meta-analyzed with the results from The Host Genetics Initiative (HGI), involving 3,077 cases and 66,686 controls. The authors found two novel genetic loci associated with COVID-19 at 2q24.2 (rs13003835) and 11q14.1 (rs77599934), and other two independent signals at 3p21.31 (rs35731912) and 6p21.1 (rs2477820) already reported as associated with COVID-19 in previous GWASs. Additional meta-analysis with other HGI studies also suggested risk variants near CREBBP, ZBTB7A, and CASC20 genes.

Strengths:

These findings rely on state-of-the-art methods in the field of Statistical Genomics and help to address the issue of a low number of GWASs in non-European populations, ultimately contributing to reducing health inequalities across the globe.

Thank you very much for the positive comments and the willingness to revise this manuscript.

Weaknesses:

There is no replication cohort, as acknowledged by the authors (page 29, line 587), and no experimental validation to assess the biological effect of putative causal variants/genes. Thus, the study provides good evidence of association, rather than causation, between the genetic variants and COVID-19. Lastly, I consider it crucial to report the results for the SCOURGE Latin American GWAS, in addition to its meta-analysis with HGI results, since HGI data has a different phenotype scheme (Hospitalized COVID vs Population) compared to SCOURGE (Hospitalized COVID vs Non-hospitalized COVID).

We essentially agree with the reviewer in that one of the main limitations of the study is the lack of a replication stage because of the use of all available datasets on a one-stage analysis. To contribute to the interpretation of the findings in the absence of a replication stage, we now assessed the replicability of the novel loci using the Meta-Analysis Model-based Assessment of replicability (MAMBA) approach (PMID: 33785739) and included the posterior probabilities of replication in Table 2. We also explored further the potential replicability of signals in other populations. We agree that the results should be interpreted in terms of associations given the lack of functional validation of main findings, so we have slightly modified the discussion.

As suggested, the SCOURGE Latin American GWAS summary is now accessible by direct request to the Consortium GitHub repository (https://github.com/CIBERER/Scourge-COVID19) (lines 797-799). We have also included the results from the SCOURGE GWAS analysis for the replication of the 40 lead variants in the Supplementary Table 12. Results from the SCOURGE GWAS for the lead variants in the AMR meta-analysis with HGI were already included in the Supplementary Table 2. As note, we have not been able to conduct the meta-analysis with the same hospitalization scheme as in the HGI study since the population-specific results for those analyses were not publicly released. However, sensitivity analyses included within the supplementary material from the COVID-19 Host Genetics Initiative (2021) stated that there were no significant differences in effects (Odds Ratios) between analyses using population controls or just non-hospitalized COVID-19 patients.

Reviewer #3 (Public Review):

Summary:

In the context of the SCOURGE consortium's research, the authors conduct a GWAS meta-analysis on 4,702 hospitalized individuals of admixed American descent suffering from COVID-19. This study identified four significant genetic associations, including two loci initially discovered in Latin American cohorts. Furthermore, a trans-ethnic meta-analysis highlighted an additional novel risk locus in the CREBBP gene, underscoring the critical role of genetic diversity in understanding the pathogenesis of COVID-19.

Strengths:

(1) The study identified two novel severe COVID-19 loci (BAZ2B and DDIAS) by the largest GWAS meta-analysis for COVID-19 hospitalization in admixed Americans.

(2) With a trans-ethnic meta-analysis, an additional risk locus near CREBBP was identified.

Thank you very much for the positive comments and the willingness to revise this manuscript.

Weaknesses:

(1) The GWAS power is limited due to the relatively small number of cases.

(2) There is no replication study for the novel severe COVID-19 loci, which may lead to false positive findings.

We agree with that the sample size is relatively small. Despite that, it was sufficient to reveal novel risk loci supporting the robustness of the main findings. We have indicated this limitation at the end of the discussion section.

Regarding the lack of a replication study, we now assessed the replicability of the novel loci using the Meta-Analysis Model-based Assessment of replicability (MAMBA) approach (PMID: 33785739). We have included the posterior probabilities of replication in Table 2.

(3) Significant differences exist in the ages between cases and controls, which could potentially introduce biased confounders. I'm curious about how the authors treated age as a covariate. For instance, did they use ten-year intervals? This needs clarification for reproducibility.

Thank you for rising this point. Age was included as a continuous variable. This has been now indicated in line 667 (within Material and Methods).

(4)"Those in the top PGS decile exhibited a 5.90-fold (95% CI=3.29-10.60, p=2.79x10-9) greater risk compared to individuals in the lowest decile". I would recommend comparing with the 40-60% PGS decile rather than the lowest decile, as the lowest PGS decile does not represent 'normal controls'.

Thank you. In the revised version, the PGS categories was compared following the recommendation (lines 461-463).

(5) In the field of PGS, it's common to require an independent dataset for training and testing the PGS model. Here, there seems to be an overfitting issue due to using the same subjects for both training and testing the variants.

We are sorry for the misunderstanding. In fact, we have followed the standard to avoid overfitting of the PGS model and have used different training and testing datasets. The training data (GWAS) was the HGI-B2 ALL meta-analysis, in which our AMR GWAS was not included. The PRS model was then tested in the SCOURGE AMR cohort. However, it is true that we did test the combination of the PRS adding the new discovered variants in the SCOURGE cohort. To avoid potential overfitting by adding the new loci, we have excluded from the manuscript the results on which we included the newly discovered variants.

(6) The variants selected for the PGS appear arbitrary and may not leverage the GWAS findings without an independent training dataset.

Again, we are sorry for the misunderstanding. The PGS model was built with 43 variants associated with hospitalization or severity within the HGI v7 results and 7 which were discovered by the GenOMICC consortium in their latest study and were not in the latest HGI release. The variants are included within the Supplementary Table 14, but we have now annotated the discovery GWAS.

(7) The TWAS models were predominantly trained on European samples, and there is no replication study for the findings as well.

This is a good point regarding the lack of diversity in GTEx data. We have also used data from AMR populations (GALA II-SAGE models), although it was only available for blood tissue. Regarding the ancestry mismatch between datasets, several studies have attempted to explore the impact. Gay et al. (PMID: 32912333) studied local ancestry effects on eQTLs from the GTEx consortium and concluded that adjustment of eQTLs by local ancestry only yields modest improvement over using global ancestry (as done in GTEx). Moreover, the colocalization results between adjusting by Local Ancestry and Global Ancestry were not significantly different. Besides, Mogil et al. (PMID: 30096133) observed that genes with higher heritability share genetic architecture between populations. Nevertheless, both studies have evidenced decreased power and poorer predictive performances regarding gene expression because of reduced diversity in eQTL analyses. As consequence of the ancestry mismatch, we now warn the readers that this may compromise signal detection (Discussion, lines 531-533). 

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) The authors mentioned the fine mapping method did not converge for the locus in chr 11. I would consider trying a different fine-mapping method (such as SuSiE or FINEMAP). It would be helpful to provide posterior inclusion probabilities (PIP) for the variants in fine mapping results and plot the PIP values in the regional association plots.

As suggested, we have also used SuSIE, which allows to assume more than one causal signal per locus. However, in this case the results were not different from those obtained with the original Bayesian colocalization performed with corrcoverage. SuSIE’s fine-mapping for chromosome 11 prioritized a single variant, which is likely due to the rare frequency. Thus, we have maintained the fine-mapping as it was originally indicated in the previous version of the manuscript but have now included the credible set in Supplementary Table 6.

Regarding the PIP, at the fine mapping stage we are inclined to put more weight on the functional annotations of the variants in the credible set than on the statistical contributions to the signal. This is the reason why we prefer not to put weight on the PIP of the variants but prioritize variants that were enriched functional annotations.

(2) Please provide more detailed information about the VEP and V2G analysis and how to interpret those results. My understanding of V2G is that it includes different sources of information (such as molecular QTLs and chromatin interactions from different tissues/cell types, etc.). It is unclear what sources of information and weight settings were used in the V2G model.

Thank you for rising this point. As suggested, we have clarified the basis for VEP and V2G and the interpretation (lines 732-743).

(3) The authors identified multiple genes with different strategies, e.g. FUMA, V2G, COLOC, TWAS, etc. How many genes were found/supported by evidence provided by multiple methods? It could be helpful to have a table summarizing the risk genes found by different strategies, and the evidence supporting the genes. e.g. which genes are found by which methods, and the biological functions of the genes, etc.

Thank you for rising this point. As suggested, we now added a new figure (Figure 5) to summarize the findings with the multiple methods used.

(4) It would be helpful to make the code/scripts available for reproducibility.

As suggested, the SCOURGE Latin American GWAS summary and the analysis scripts (https://github.com/CIBERER/Scourge-COVID19/tree/main/scripts/novel-risk-hosp-AMR-2024) are now accessible in the Consortium GitHub repository (https://github.com/CIBERER/Scourge-COVID19) (lines 806-807).

(5) The fonts in some of the figures (e.g. Figure 2) are hard to read.

Thank you. We have now included the figures as SVG files.

Reviewer #2 (Recommendations For The Authors):

- The abstract lacks a conclusion sentence.

Thank you. As suggested, we have included two additional sentences with broad conclusions from the study. We preferred to avoid relying on conclusions related to known or new biological links of the prioritized genes given the lack of functional validation of main findings.

- Regarding the association analysis (page 27, line 677), I wonder if some of the 10 principal components (PCs) are capturing information about the recruitment areas (countries). It may be relevant to test for multicollinearity among these variables.

Since we acknowledge that some of the categories might be correlated with a certain PC but not all of them do, we have calculated GVIF values for the main variables to assess the categorical variable as a single entity. The scaled GVIF^1(1/2*Df)) value for the categorical variable is 1.52. Thus, if we square this value, we obtain 2.31, which can be then used for applying usual rule-of-thumb for VIF values.

- Still on the topic of association analysis, did the authors adjust the logistic model for comorbidities variables from Table 1? Given these comorbidities also have a genetic component and their distribution differs between non-hospitalized vs hospitalized, I am concerned that comorbidities might be confounding the association between genetic variants and COVID.

We did not adjust by comorbidities since HGI studies were not adjusted either and we aimed to be as aligned as possible with HGI. However, as suggested, we have now tested the association between each of the comorbidities in Table 1 and each of the variants in Table 2, using the comorbidities as dependent variables and adjusting for the main covariables (age, sex, PCs and country of recruitment). None of the variants were significantly associated to the comorbidities (line 333).

- If I understood correctly, the 49 genetic variants used to develop the polygenic risk score model (PRS) were based on the HGI total sample size (data release 7), which is predominantly of European ancestry. I am concerned about the prediction accuracy in the AMR population (PRS transferability issue).

We have explored literature in search of other PRS to compare the associated OR in our cohort with ORs calculated in European populations. Horowitz et al. (2022) reported an OR of 1.38 for the top 10% with respect to hospitalization risk in European individuals using a GRS with 12 variants.

We acknowledge that this might be an issue and is now explained in discussion of the revised version (lines 561-568). However, as this is the first time a PRS for COVID-19 is applied to a relatively large AMR cohort, we believe that this analysis will be of value for further analyses regarding PRS transferability, providing a source for comparison in further studies.    

- On page 23, line 579, the authors acknowledge their "GWAS is underpowered". This sentence requires a sample/power calculation, otherwise, I suggest using "is likely underpowered".

Thanks for the input. We have modified the sentence as suggested.

Reviewer #3 (Recommendations For The Authors):

I wonder if the authors have an approximate date when the GWAS summary statistic will be available. I reviewed some manuscripts in the past, and the authors claimed they would deposit the data soon, but in fact it would not happen until 2 years later.

The summary statistics are already available from the SCOURGE Consortium repository https://github.com/CIBERER/Scourge-COVID19 (lines 806-807).

It's really crazy how prices changed exponentially like that so the lower class men can't eat such "rich food", But I'm glad they overpriced food that in my opinion just sounds gross except clams, which may taste good with lime

So President Cleveland sent in the military using tax dollars instead of just passing legislation to meet the union's request? It's clear to see that the government was in the hands of the business owners and not the people who elected them into power.

this is important because it's showing the affect of people who take notes on their laptop. i can somewhat say i would agree with this research. coming into college, i thought it was so cool to take notes on my laptop but i did notice that i wasn't retaining any of the information, i was just focused on typing.

for - question - nilhism - nondualism - is fighting evil a contradiction? - Rupert Spira - Bernado Kastrup - question - nilhism - how do we prevent falling into?

question - nondualism - is fighting evil a contradiction? - Pondering this idea raises the question: - Is fighting evil a contradiction? - Do we fall into duality if we fight evil? - Does nonduality imply not creating categories of morality of good and evil? - This question has no answer because - If you understand the question, you are already - a language user - applying some morality - We are already post category and post linguistic - we can never undo this and get back to pre-category and pre-linguistic - Fighting evil cannot conquer it because - in fighting evil, this implies using (deadly) force - deadly force results in death, the most extreme form of suffering - It is tantamount to abuse and justifying death is the greatest act of separation, causing great suffering to the other - In effect, we have the same result as the abuser and this can create a new generation of abused

question - nilhism - how do we prevent falling into? - Rather, what is needed is to PENETRATE moral relativism / dualism altogether to re-discover the common sacred ground both moral categories are based upon - The use of force as a form of vengeance - is the perpetuation of the abused-abuser cycle

Author response:

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

Reviewer #1 (Public Review):

My main point of concern is the precision of dissection. The authors distinguish cells isolated from the tailbud and different areas in the PSM. They suggest that the cell-autonomous timer is initiated, as cells exit the tailbud.

This is also relevant for the comparison of single cells isolated from the embryo and cells within the embryo. The dissection will always be less precise and cells within the PSM4 region could contain tailbud cells (as also indicated in Figure 1A), while in the analysis of live imaging data cells can be selected more precisely based on their location. This could therefore contribute to the difference in noise between isolated single cells and cells in the embryo. This could also explain why there are "on average more peaks" in isolated cells (p. 6, l. 7).

This aspect should be considered in the interpretation of the data and mentioned at least in the discussion. (It does not contradict their finding that more anterior cells oscillate less often and differentiate earlier than more posterior ones.)

Reviewer #1 rightly points out that selecting cells in a timelapse is more precise than manual dissection. Manual dissection is inherently variable but we believe in general it is not a major source of noise in our experiments. To control for this, we compared the results of 11 manual dissections of the posterior quarter of the PSM (PSM4) with those of the pooled PSM4 data. In general, we did not see large differences in the distributions of peak number or arrest timing that would markedly increase the variability of the pooled data above that of the individual dissections (Figure 1 – supplement figure 7). We have edited the text in the Results to highlight this control experiment (page 6, lines 13-17).

It is of course possible that we picked up adjacent TB cells when dissecting PSM4, however the reviewer’s assertion that inclusion of TB cells “could also explain why there are "on average more peaks" in isolated cells” is incorrect. Later in the paper we show that cells from the TB have almost identical distributions to PSM4 (mean ± SD, PSM4 4.36 ± 1.44; TB 4.26 ± 1.35; Figure 4 _ supplement 1). Thus, inadvertent inclusion of TB cells while dissecting would in fact not increase the number of peaks.

Here, the authors focus on the question of how cells differentiate. The reverse question is not addressed at all. How do cells maintain their oscillatory state in the tailbud? One possibility is that cells need external signals to maintain that as indicated in Hubaud et al. 2014. In this regard, the definition of tailbud is also very vague. What is the role of neuromesodermal progenitors? The proposal that the timer is started when cells exit the tailbud is at this point a correlation and there is no functional proof, as long as we do not understand how cells maintain the tailbud state. These are points that should be considered in the discussion.

The reviewer asks “How do cells maintain their oscillatory state in the tailbud?”. This is a very interesting question, but as recognized by the reviewer, beyond the scope of our current paper.

We now further emphasize the point “One possibility is that cells need external signals to maintain … as indicated in Hubaud et al. 2014” in the Discussion and added a reference to the review Hubaud and Pourquié 2014 (Signalling dynamics in vertebrate segmentation. Nat Rev Mol Cell Biol 15, 709–721 (2014). https://doi.org/10.1038/nrm3891) (page 18, lines 19-22).

To clarify the definition of the TB, we have stated more clearly in the results (page 15, lines 8-12) that we defined TB cells as all cells posterior to the notochord (minus skin) and analyzed those that survived

>5 hours post-dissociation, did not divide, and showed transient Her1-YFP dynamics.

The reviewer asks: What is the role of neuromesodermal progenitors? In responding to this, we refer to Attardi et al., 2018 (Neuromesodermal progenitors are a conserved source of spinal cord with divergent growth dynamics. Development. 2018 Nov 9;145(21):dev166728. doi: 10.1242/dev.166728).

Around the stage of dissection in zebrafish in our work, there is a small remaining group of cells characterized as NMPs (Sox2 +, Tbxta+ expression) in the dorsal-posterior wall of the TB. These NMPs rarely divide and are not thought to act as a bipotential pool of progenitors for the elongating axis, as is the case in amniotes, rather contributing to the developing spinal cord. How this particular group of cells behaves in culture is unclear as we did not subdivide the TB tissue before culturing. It would be possible to specifically investigate these NMPs regarding a role in TB oscillations, but given the results of Attardi et al., 2018 (small number of cells, low bipotentiality), we argue that it would not be significant for the conclusions of the current work. To indicate this, we included a sentence and a citation of this paper in the results towards the beginning of the section on the tail bud (page 15, lines 8-12).

The authors observe that the number of oscillations in single cells ex vivo is more variable than in the embryo. This is presumably due to synchronization between neighbouring cells via Notch signalling in the embryo. Would it be possible to add low doses of Notch inhibitor to interfere with efficient synchronization, while at the same time keeping single cell oscillations high enough to be able to quantify them?

It is a formal possibility that Delta-Notch signaling may have some impact on the variability in the number of oscillations. However, we argue that the significant amount of cell tracking work required to carry out the suggested experiments would not be justified, considering what has been previously shown in the literature. If Delta-Notch signaling was a major factor controlling the variability of the intrinsic program that we describe, then we would expect that in Delta-Notch mutants the anterior- posterior limits of cyclic gene expression in the PSM would extend beyond those seen in wildtype embryos. Specifically, we might expect to see her1 expression extending more anteriorly in mutants to account for the dramatic increase in the number of cells that have 5, 6, 7 and 8 cycles in culture (Fig. 1E versus Fig. 1I). However, as shown in Holley et al., 2002 (Fig. 5A, B; her1 and the notch pathway function within the oscillator mechanism that regulates zebrafish somitogenesis. Development. 2002 Mar;129(5):1175-83. doi: 10.1242/dev.129.5.1175), the anterior limit of her1 expression in the PSM in DeltaD mutants (aei) is not different to WT. Thus, Delta-Notch signaling may exert a limited control over the number of oscillations, but likely not in excess of one cycle difference.

In the same direction, it would be interesting to test if variation is decreased, when the number of isolated cells is increased, i.e. if cells are cultured in groups of 2, 3 or 4 cells, for instance.

This is a great proposal – however the culture setup used here is a wide-field system that doesn’t allow us to accurately follow more than one cell at a time. Cells that adhere to each other tend to crawl over each other, blurring their identity in Z. This is also why we excluded dividing cells in culture from the analysis. Experiments carried out with a customized optical setup will be needed to investigate this in the future.

It seems that the initiation of Mesp2 expression is rather reproducible and less noisy (+/- 2 oscillation cycles), while the number of oscillations varies considerably (and the number of cells continuing to oscillate after Mesp2 expression is too low to account for that). How can the authors explain this apparent discrepancy?

The observed tight linkage of the Mesp onset and Her1 arrest argue for a single timing mechanism that is upstream of both gene expression events; indeed, this is one of the key implications of the paper. However, the infrequent dissociation of these events observed in FGF-treated cells suggests that more than one timing pathway could be involved, although there are other interpretations. We’ve added more discussion in the text on one vs multi-timers (page 17, lines 19-23 – page 18, line 1 - 8)., see next point.

The observation that some cells continue oscillating despite the upregulation of Mesp2 should be discussed further and potential mechanism described, such as incomplete differentiation.

This is an infrequent (5 out of 54 cells) and interesting feature of PSM4 cells in the presence of FGF. We imagine that this disassociation of clock arrest from mesp on-set timing could be the result of alterations in the thresholds in the sensing mechanisms controlling these two processes. Alternatively - as reviewer 2 argues - it might reflect multiple timing mechanisms at work. We have added a discussion of these alternative interpretations (page 17, lines 19-23 – page 18, line 1 - 8).

Fig. 3 supplement 3 B missing

It’s there in the BioRxiv downloadable PDF and full text – but seems to not be included when previewing the PDF. Thanks for the heads up.

Reviewer #2 (Public Review):

The authors demonstrate convincingly the potential of single mesodermal cells, removed from zebrafish embryos, to show cell-autonomous oscillatory signaling dynamics and differentiation. Their main conclusion is that a cell-autonomous timer operates in these cells and that additional external signals are integrated to tune cellular dynamics. Combined, this is underlying the precision required for proper embryonic segmentation, in vivo. I think this work stands out for its very thorough, quantitative, single-cell real-time imaging approach, both in vitro and also in vivo. A very significant progress and investment in method development, at the level of the imaging setup and also image analysis, was required to achieve this highly demanding task. This work provides new insight into the biology underlying embryo axis segmentation.

The work is very well presented and accessible. I think most of the conclusions are well supported. Here a my comments and suggestions:

The authors state that "We compare their cell-autonomous oscillatory and arrest dynamics to those we observe in the embryo at cellular resolution, finding remarkable agreement."

I think this statement needs to be better placed in context. In absolute terms, the period of oscillations and the timing of differentiation are actually very different in vitro, compared to in vitro. While oscillations have a period of ~30 minutes in vivo, oscillations take twice as long in vitro. Likewise, while the last oscillation is seen after 143 minutes in vivo, the timing of differentiation is very significantly prolonged, i.e.more than doubled, to 373min in vitro (Supplementary Figure 1-9). I understand what the authors mean with 'remarkable agreement', but this statement is at the risk of being misleading. I think the in vitro to in vivo differences (in absolute time scales) needs to be stated more explicitly. In fact, the drastic change in absolute timescales, while preserving the relative ones, i.e. the number of oscillations a cell is showing before onset of differentiation remains relatively invariant, is a remarkable finding that I think merits more consideration (see below).

We have changed the text in the abstract (page 1, line 28) to clarify that the agreement is in the relative slowing, intensity increases and peak numbers.

One timer vs. many timers

The authors show that the oscillation clock slowing down and the timing of differentiation, i.e. the time it takes to activate the gene mesp, are in principle dissociable processes. In physiological conditions, these are however linked. We are hence dealing with several processes, each controlled in time (and thereby space). Rather than suggesting the presence of ‘a timer’, I think the presence of multiple timing mechanisms would reflect the phenomenology better. I would hence suggest separating the questions more consistently, for instance into the following three:

a.  what underlies the slowing down of oscillations?

b.  what controls the timing of onset of differentiation?

c.  and finally, how are these processes linked?

Currently, these are discussed somewhat interchangeably, for instance here: “Other models posit that the slowing of Her oscillations arise due to an increase of time-delays in the negative feedback loop of the core clock circuit (Yabe, Uriu, and Takada 2023; Ay et al. 2014), suggesting that factors influencing the duration of pre-mRNA splicing, translation, or nuclear transport may be relevant. Whatever the identity, our results indicate the timer ought to exert control over differentiation independent of the clock.”(page 14). In the first part, the slowing down of oscillations is discussed and then the authors conclude on 'the timer', which however is the one timing differentiation, not the slowing down. I think this could be somewhat misleading.

To help distinguish the clock’s slowing & arrest from differentiation, we have clarified the text in how we describe our experiments using her1-/-; her7-/- cells (page 10, lines 9-20).

From this and previous studies, we learn/know that without clock oscillations, the onset of differentiation still occurs. For instance in clock mutant embryos (mouse, zebrafish), mesp onset is still occurring, albeit slightly delayed and not in a periodic but smooth progression. This timing of differentiation can occur without a clock and it is this timer the authors refer to "Whatever the identity, our results indicate the timer ought to exert control over differentiation independent of the clock." (page 14). This 'timer' is related to what has been previously termed 'the wavefront' in the classic Clock and Wavefront model from 1976, i.e. a "timing gradient' and smooth progression of cellular change. The experimental evidence showing it is cell-autonomous by the time it has been laid down,, using single cell measurements, is an important finding, and I would suggest to connect it more clearly to the concept of a wavefront, as per model from 1976.

We have been explicit about the connection to the clock & wavefront in the discussion (page 17, line 12-17).

Regarding question a., clearly, the timer for the slowing down of oscillations is operating in single cells, an important finding of this study. It is remarkable to note in this context that while the overall, absolute timescale of slowing down is entirely changed by going from in vivo to in vitro, the relative slowing down of oscillations, per cycle, is very much comparable, both in vivo and in vivo.

We have now pointed out the relative nature of this phenomenon in the abstract, page 1, line 28.

To me, while this study does not address the nature of this timer directly, the findings imply that the cell-autonomous timer that controls slowing down is, in fact, linked to the oscillations themselves. We have previously discussed such a timer, i.e. a 'self-referential oscillator' mechanism (in mouse embryos, see Lauschke et al., 2013) and it seems the new exciting findings shown here in zebrafish provide important additional evidence in this direction. I would suggest commenting on this potential conceptual link, especially for those readers interested to see general patterns.

While we posit that the timer provides positional info to the clock to slow oscillations and instruct its arrest – we do not believe that “the findings imply that the cell-autonomous timer that controls slowing down is, in fact, linked to [i.e., governed by] the oscillations themselves.”. As we show, in her1-/-; her7-/- embryos lacking oscillations, the timing / positional information across the PSM still exists as read-out by Mesp expression. Is this different positional information than that used by the clock? – possibly – but given the tight linkage between Mesp onset and the timing/positioning of clock arrest, both cell-autonomously and in the embryo, we argue that the simplest explanation is that the timing/positional information used by the clock and differentiation are the same. Please see page 10, lines 9-20, as well as the discussion (page 17, lines 19-23; page 18. Lines 1-8 ).

We agree that the timer must communicate to the clock– but this does not mean it is dependent on the clock for positional information.

Regarding question c., i.e. how the two timing mechanisms are functionally linked, I think concluding that "Whatever the identity, our results indicate the timer ought to exert control over differentiation independent of the clock." (page 14), might be a bit of an oversimplification. It is correct that the timer of differentiation is operating without a clock, however, physiologically, the link to the clock (and hence the dependence of the timescale of clock slowing down), is also evident. As the author states, without clock input, the precision of when and where differentiation occurs is impacted. I would hence emphasize the need to answer question c., more clearly, not to give the impression that the timing of differentiation does not integrate the clock, which above statement could be interpreted to say.

As far as we can tell, we do not state that “without clock input, the precision of when and where differentiation occurs is impacted”, and we certainly do not want to give this impression. In contrast, as mentioned above, the her1-/-; her7-/- mutant embryo studies indicate that the lack of a clock signal does not change the differentiation timing, i.e. it does not integrate the clock. Of course, in the formation of a real somite in the embryo, the clock’s input might be expected to cause a given cell to differentiate a little earlier or later so as to be coordinated with its neighbors, for example, along a boundary. But this magnitude of timing difference is within one clock cycle at most, and does not match the large variation seen in the cultured cells that spans over many clock cycles.

A very interesting finding presented here is that in some rare examples, the arrest of oscillations and onset of differentiation (i.e. mesp) can become dissociated. Again, this shows we deal here with interacting, but independent modules. Just as a comment, there is an interesting medaka mutant, called doppelkorn (Elmasri et al. 2004), which shows a reminiscent phenotype "the Medaka dpk mutant shows an expansion of the her7 expression domain, with apparently normal mesp expression levels in the anterior PSM.". The authors might want to refer to this potential in vivo analogue to their single cell phenotype.

Thank you, we had forgotten this result. Although we do not agree that this result necessarily means there are two interacting modules, we have included a citation to the paper, along with a discussion of alternative explanations for the dissociation (page 18, lines 2-14).

One strength of the presented in vitro system is that it enables precise control and experimental perturbations. A very informative set of experiments would be to test the dependence of the cell-autonomous timing mechanisms (plural) seen in isolated cells on ongoing signalling cues, for instance via Fgf and Wnt signaling. The inhibition of these pathways with well-characterised inhibitors, in single cells, would provide important additional insight into the nature of the timing mechanisms, their dependence on signaling and potentially even into how these timers are functionally interdependent.

We agree and in future experiments we are taking advantage of this in vitro system to directly investigate the effect of signaling cues on the intrinsic timing mechanism.

I like this example of how the author described it. I liked this analogy and will try to use it in my writings.

This is an interesting rule to give to the robots because it makes it so that the robots will fight for their "life" in a similar way that humans do. It seems like this rule was put in place to make sure the robots don't just destroy themselves, but giving them this thought can potentially give the robot a thought that it is valuable and with higher forms of AI, maybe it could develop it's own feelings surrounding their life.

Welcome back.

And in this lesson, I want to talk about how routing works within a VPC and introduce the internet gateway, which is how we configure a VPC so that data can exit to and enter from the AWS public zone and public internet.

Now, this lesson will be theory where I'm going to introduce routing and the internet gateway to the architecture behind both those things, as well as jump boxes also known as Bastion hosts.

In the demo lesson, which immediately follows this one, you'll get the opportunity to implement an internet gateway yourself and the animals for life at VPC and fully configure the VPC with public subnet that allow you to connect to that jump box.

So let's get started.

We've got a lot to cover.

A VPC router is a highly available device which is present in every VPC, both default or custom, which moves traffic from somewhere to somewhere else.

It runs in all the availability zones that the VPC uses and never needs a way about its availability.

It simply works.

The router can be networked in every subnet, and the network is just one address of the subnet.

By default in a custom VPC, without any other configuration, the VPC router will simply route traffic between subnets in that VPC.

If an EC2 instance in one subnet wants to communicate with something in another subnet, the VPC router is the thing that moves the traffic between subnets.

Now, the VPC router is controllable.

You create route tables which influence what's to do with traffic when it leaves a subnet.

So just to be clear, the route table that's associated with a subnet defines what the VPC router will do when data leaves that subnet.

A VPC is created with what's known as a main route table.

If you don't explicitly associate a custom route table with a subnet, it uses the main route table of the VPC.

If you do associate a route table that you create with a subnet, then when you associate that, the main route table is disassociated.

A subnet can only have one route table associated with it at any one time, but a route table can be associated with many subnets.

A route table looks like this in the user interface.

In this case, this is the main route table for this specific VPC.

And a route table is just a list of routes.

This is one of those routes.

When traffic leaves the subnet that this route table is associated with, the VPC router reviews the IP packets.

And remember, I said that a packet had a source address and a destination address, as well as some data.

The VPC router looks at the destination address of all packets leaving the subnet.

And when it has that address, it looks at the route table and it identifies all the routes which match that destination address.

And it does that by checking the destination field at the route.

This destination field determines what destination the route matches.

Now, the destination field on a route could match exactly one specific IP address.

It could be an IP with a /32 prefix.

And remember, that means that it matches one single IP.

But the destination field on a route could also be a network match.

So matching an entire network of which that IP is part.

Or it could be a default route.

Remember, for IP version 4, I mentioned that 0.0.0.0.0/0 matches all IP version 4 IP addresses.

That's known as a default route, a catchall.

In the case where traffic leaving a subnet only matches one route, then that one route is selected.

If multiple routes match, so maybe there's a specific /32 IP match, maybe there's a /16 network match, and maybe there's a 0.0.0.0/0 default match, well then the prefix is used as a priority.

The higher the prefix value, the more specific the route is and the higher priority that that route has.

So the higher the prefix, all the way up to the highest priority of /32, that is used to select which route applies when traffic leaves a subnet.

Once we get to the point where a single rule in a route table is selected, either the sole route that applies or the one with the highest priority, then the VPC router forwards that traffic through to its destination, which is determined by the target field on the route.

And the target field will either point at an AWS gateway, or it will say, as with this example, local.

And local means that the destination is in the VPC itself, so the VPC router can forward the traffic directly.

All route tables have at least one route, the local route.

This matches the VPC side range, and it lets the VPC router know that traffic destined for any IP address in the VPC side range is local and it can be delivered directly.

If the VPC is also IPv6 enabled, people also have another local route matching the IPv6 side for the VPC.

As is the case with this example, that bottom route, beginning 2600, that is an IPv6 local route.

That's the IPv6 side of this specific VPC.

Now, these local routes can never be updated.

They're always present, and the local routes always take priority.

They're the exception to that previous rule about the more specific the route is, the higher the priority.

Local routes always take priority.

For the exam, remember the route tables are attached to zero or more subnets.

A subnet has to have a route table.

It's either the main route table of the VPC or a custom one that you've created.

A route table controls what happens to data as it leaves the subnet or subnets that that route table is associated with.

Local routes are always there, uneditable, and match the VPC IPv4 or VPC side range.

For anything else, higher, prefix values are more specific than they take priority.

The way the route works is it matches a destination IP, and for that route, it directs traffic towards a specific target.

Now, a default route, which I'll talk about shortly, is what happens if nothing else matches.

Now, an internet gateway is one of the most important add-on features available within a VPC.

It's a regionally resilient gateway which can be attached to a VPC.

I've highlighted the words "region" and "resilience" because it always comes up in the exam.

You do not need a gateway per availability zone.

The internet gateway's resilient by design.

One internet gateway will cover all of the availability zones in the region which the VPC is using.

Now, there's a one-to-one relationship between internet gateways and the VPC.

A VPC can have no internet gateways which makes it entirely private, or it can have one internet gateway.

Those are the two choices.

An internet gateway can be created and not attached to a VPC, so it can have zero attachments, but it can only ever be attached to one VPC at a time, at which point it's valid in all of the availability zones that the VPC uses.

Now, the internet gateway runs from the border of the VPC and the AWS public zone.

It's what allows services inside the VPC, which are allocated with public IP version 4 addresses or IP version 6 addresses, to be reached from the internet and to connect to the AWS public zone or the internet.

Of course, the AWS public zone is used if you're accessing S3, SQS, SNS, or any other AWS public services from the VPC.

Now, it's a managed gateway and so AWS handles the performance.

From your perspective as an architect, it simply works.

Now, using an internet gateway within a VPC, it's not all that complex.

It's a simplified VPC diagram.

First, we create and attach an internet gateway to a VPC.

This means that it's available for use inside the VPC.

We can use it as a target within route tables.

So then we create a custom route table and it's within route tables.

So then we create a custom route table and we associate it with a web subnet.

Then we add IP version 4 and optionally IP version 6 default routes to the route table with the target being the internet gateway.

Then finally, we configure the subnet to allocate IP version 4 addresses and optionally IP version 6 by default.

And at that point, once we've done all of those actions together, the subnet is classified as being a public subnet and any services inside that subnet with public IP addresses can communicate to the internet and vice versa and they can communicate with the AWS public zone as long as there's no other security limitations that are in play.

Now, don't worry if this seems complex.

You'll get to experience it shortly in the upcoming demo lesson.

But before that, I want to talk about how IP version 4 addressing actually works inside the VPC because I've seen quite a few difficult questions on the exam based around IP version 4 addressing and I want to clarify exactly how it works.

So conceptually, this is how an EC2 instance might look if it's using IP version 4 to communicate with a software update server of some kind.

So we've got the instance on the left with an internet gateway in between and let's say it's a Linux EC2 instance trying to do some software updates to a Linux update server that's located somewhere in the public internet zone.

So the instance has a private IP address of let's say 10.16.16.20 and it also has an IP version 4 public address that's assigned to it of 43.250.192.20.

Only that's not how it really works.

This is another one of those little details which I try to include in my training courses because it really comes invaluable for the exam.

What actually happens with public IP version 4 addresses is that they never touch the actual services inside the VPC.

Instead, when you allocate a public IP version 4 address, for example, to this EC2 instance, a record is created which the internet gateway maintains.

It links the instance's private IP to its allocated public IP.

So the instance itself is not configured with that public IP.

That's why when you make an EC2 instance and allocate it a public IP version 4 address, inside the operating system, it only sees the private IP address.

Keep this in mind for the exam that there are questions which will try to trip you up on this one.

For IP version 4, it is not configured in the OS with the public IP address.

So let's look at the flow of data.

How does this work?

Well, when the Linux instance wants to communicate with the Linux software update server, it creates a packet of data.

Now obviously it probably creates a lot of packets.

Well, let's focus on one for now because it keeps the diagram nice and simple.

The packet has a source address of the EC2 instance and a destination address of the Linux software update server.

So at this point, the packet is not configured with any public addressing.

This packet would not be routable across the public internet.

It could not reach the Linux update server.

That's really important to realize.

Now the packet leaves the instance and because we've configured a default route, it arrives at the internet gateway.

The internet gateway sees that this packet is from the EC2 instance because it analyzes the source IP address of that packet.

And it knows that this instance has an associated public IP version 4 address.

And so it adjusts the packet.

It changes the packet's source IP address to the public IP address that's allocated to that instance.

And this IP address, because it's public, is routable across the internet.

So the internet gateway then forwards the updated packet onto its destination.

So as far as the Linux software update server is concerned, it's receiving a packet from a source IP address of 43.250.192.20.

It knows nothing of the private IP address of the EC2 instance.

Now on the way back, the inverse happens.

The Linux software update server wants to send a packet back to our EC2 instance.

But as far as it's concerned, it doesn't know about the private address.

It just knows about the public address.

So the software update server sends a packet back, addressed to the instance's public IP address with its source address.

So it thinks that the real IP address of the instance is this 43.250.192.20 address.

Now this IP address actually belongs to the internet gateway.

And so it travels over the public internet and it arrives at the internet gateway, which then modifies this packet.

It changes it.

It changes the destination address of this packet from the 43 address to the original IP address of the EC2 instance.

And it does this because it's got a record of the relationship between the private IP and the allocated public IP.

So it just changes the destination to the private IP address of the instance.

And then it forwards this packet through the VPC network to the original EC2 instance.

So the reason I wanted to highlight this is because at no point is the operating system on the EC2 instance aware of its public IP.

It just has a private IP.

Don't fall for any exam questions which try to convince you to assign the public IP version 4 address of an EC2 instance directly to the operating system.

It has no knowledge of this public address.

Configuring an EC2 instance appropriately using IP version 4 means putting the private IP address only.

The public address never touches the instance.

For IP version 6, all addresses that AWS users are natively, publicly, routable.

And so in the case of IP version 6, the operating system does have the IP address version 6 address configured upon it.

That's the publicly routable address.

And all the internet gateway does is pass traffic from an instance to an internet server.

And then back again, it doesn't do any translation.

Now, before we implement this in the demo lesson, I just want to briefly touch upon bastion hosts and jump boxes.

At a high level, bastion hosts and jump boxes are one and the same.

Essentially, it's just an instance in a public subnet inside of VPC.

And architecturally, they're used to allow incoming management connections.

So all incoming management connections arrive at bastion hosts or jump box.

And then once connected, you can then go on to access internal only VPC resources.

So bastion hosts and jump boxes are generally used either as a management point or as an entry point for private only VPCs.

So if your VPC is a highly secure private VPC, you'll generally have a bastion host or a jump box being the only way to get access to that VPC.

So it's essentially just an inbound management point.

And you can configure these bastion hosts or jump boxes to only accept connections from certain IP addresses, to authenticate with SSH, or to integrate with your on-premises identity servers.

You can configure them exactly how you need, but at a real core architectural level, they are generally the only entry point to a highly secure VPC.

And historically, they were the only way to manage private VPC instances.

Now, there are alternative ways to do that now, but you will still find bastion hosts and jump boxes do feature on the exam.

OK, so that's all the theory that I wanted to cover in this lesson.

It's now time for a demo.

In the next lesson, we're going to implement the Internet gateway in the Animals for Life VPC.

We'll create it, we'll attach it to the VPC, we'll create a custom route table for the web subnets, we'll create two routes in that route table, one for IP version 4 and one for IP version 6.

And both of these will point at the Internet gateway as a target.

We'll associate that route table with the web tier subnets, configure those subnets to allocate public IP version 4 addresses, and then launch a bastion host into one of those web subnets.

And if all goes well, we will be able to connect to that instance using our SSH application.

So I think this demo lesson is going to be really interesting and really exciting.

It's the first time that we're going to be stepping through something together that we could qualify as production like.

Something that you could implement and would implement in a production ready VPC.

So go ahead, complete this video, and when you're ready, join me in the demo lesson.

Welcome back and in this demo lesson you're going to create all of the subnets within the custom VPC for animals for life.

So we're going to create the subnets as shown on screen now.

We're going to be creating four subnets in each availability zone.

So that's the web subnet, the application subnet, the database subnet and the reserved subnet.

And we're going to create each of those four in availability zone A, B and C.

Now before we get started, attached to this lesson is a link to this document.

Now this is a list of all the details of the subnet you're going to create in this demo lesson.

So we've got a reserved subnet, a database subnet, an app subnet and a web subnet.

And we've got each of those in availability zone A, B and C.

Now in terms of what this document contains, we have a subnet name, then we have the IP range that subnet will use, the availability zone that subnet is within and then this last value and we'll talk about what this is very shortly.

This relates to IP version 6.

Now you'll notice that for each subnet this is a unique value.

You're going to need this to configure the IP version 6 configuration for each of the subnets.

Now let's get started.

So let's move to the AWS console and you need to be within the VPC console.

So if you're not already there, go ahead and type that in the search box at the top and then click to move to the VPC console and then click on subnets.

And once you've done that, go ahead and click on create subnet.

Now the newest version of the user interface allows you to create multiple subnets at a time and so we're going to create all four of the subnets in each availability zone.

So we'll do this three times, one for availability zone A, one for B and one for C.

So we'll get started with availability zone A and first we need to select the VPC to use.

So click in the VPC ID drop down and select the animals for live VPC.

Once you've done that, we're going to start with subnet one of one.

So let's move to the subnet's document.

So it's these subnets that we're going to create and we'll start with the reserved subnet.

So copy the name of the subnet into your clipboard and paste it into subnet name.

Then change the availability zone to AZA and then make sure IPv4 is set to manual and move back to the subnet's document and copy the IP range that we're going to use and paste that into this box.

Then scroll down again and make sure manual input is selected for IPv6.

Then click the drop down and select the IPv6 range for the VPC.

Now the VPC uses a /56 IPv6 range.

Because we need our subnet to fit inside this, we're going to make the individual subnet ranges much smaller.

So what I'll need you to do and you'll need to do this each time is to click on the down arrow and you'll need to click on the down arrow twice.

The first time will change it to a /60 and the second time to a /64.

Now note in my case how I have 9, 6, 0, 0 and if I click on this right arrow, it increments this value by one each time.

Now this value corresponds to the value in the subnet's document, so in this case 0, 0.

By changing this value each time, it means that you're giving a unique IPv6 range to each subnet.

So in this case, start off by leaving this set to 0, 0.

And once you've done that, you can click on add new subnet.

And we're going to create the next subnet.

So in this case, it's sn-db-a, so enter that name, change the availability zone to A, manual input for IPv4, copy the IP range for DBA into your clipboard, paste that in, manual for IPv6, change the VPC range in the drop down, click the down arrow twice to set this to /64, and then change this value to 0, 1.

And again, this matches the IPv6 value in the subnet's document.

Then we'll do the same process for the third subnet, so we're going to add a new subnet.

This time the name is sn-app-a, enter that, availability zone A, manual for IPv4, and then paste in the IP range, manual for IPv6, and select the VPC range, and then change the subnet block to /64 by clicking the down arrow twice.

And then click the right arrow twice to set 0, 2 as the unique value for the subnet.

And again, this matches the value in the subnet's document.

Then lastly, we're going to do the same thing for the last subnet in availability zone A, so we're going to add a new subnet.

This time it's web A, so copy and paste that into the subnet name box, set availability zone A, manual for IPv4, copy and paste the range from the subnet's document, manual for IPv6, select the IPv6 range from the VPC, click the down arrow twice to set the appropriate size for the subnet IPv6 range, and then click on the right arrow to change this value to 0, 3.

Now that's all the subnet's created, all four of them in availability zone A, so we can scroll all the way down to the bottom, and click on create subnet, and that's going to create all four of those subnets.

We can see those in this list.

Now we're going to follow that same process for availability zone B, so click on create subnet, change the VPC to the animals for IPvc, and now we're going to start moving through quicker.

So first we're going to do the reserve B subnet, so copy the name, paste that in, set the availability zone this time to B, manual for IPv4, paste in the range, manual for IPv6, select the VPC range in the drop-down, click on the down arrow twice to set the /64, and then click on the right arrow and set to 0, 4 as the unique value.

Scroll down, click add new subnet, next is DBB, enter that, availability zone B, manual for IPv4, enter the appropriate range, and just take note of the IPv6 value because then we don't have to keep switching backwards and forwards to this document in this case it's 0, 5, paste in the IPv4 range, manual for IPv6, select the VPC range in the drop-down, down arrow twice, and then the left arrow and set to 0, 5, which is the unique value for the subnet, click add new subnet, this time it's app B, enter that name, availability zone B, manual for IPv4, you'll need to enter the IP range for app B, and again pay attention to the fact that the unique value for the subnet is 0, 6, manual for IPv6, select the VPC range in the drop-down, down arrow twice, right arrow until it says 0, 6, and then add new subnet again, we're going to do the last one, this time it's WebB, enter that name, availability zone B, and drop-down, manual for IPv4, copy and paste the IP range, and pay attention to 0, 7, which is the IPv6 unique value, enter the IPv4 subnet range in this box, manual for IPv6, select the VPC range in the drop-down, down arrow twice, and then right arrow until it says 0, 7, and now we've got all four subnets in AZB, so click on create subnet, and then we're going to do this one last time for availability zone C, so click on create subnet, select the animals for IPPC in the drop-down, and we're going to follow the same process, so for subnet 1 it will be SN_reserved-C, availability zone C, you'll need to enter the IPv4 range, pay attention to the IPv6 unique value, which is 0, 8, paste in that range in the box, manual for IPv6, select the VPC range, down arrow twice, set it to /64, and then right arrow until it says 0, 8, scroll down, add a new subnet, next is DVC, so enter that, availability zone C, and do the same thing as before, we'll need the IP range, and the IPv6 unique value, so 0, 9, enter that, manual for IPv6, select the VPC range in the drop-down, down arrow twice, and then click the right arrow until 0, 9 is selected, add a new subnet, then the application subnet, copy that, paste it in, availability zone C, get the IPv4 range and the unique value of IPv6, now note this is hexadecimal, so 0, 8 directly follows 0, 9, so pay attention to that, go back, paste in the IPv4 range, manual for IPv6, select the VPC range, down arrow twice to select /64, and then right arrow until you get 0, 8, then one last time, click on add new subnet, go back to the subnet document, Web C, availability zone C, get the IPv4 range and note the unique IPv6 value, paste that in, select the IPv6 range for the VPC, down arrow twice to select /64, and then right arrow all the way through to 0, B, at that point you can go ahead and click on create subnet, and that's created all four subnets in availability zone C, and all of the subnets now that are within the annuals for IPv6, at least those in AZA, AZB, and AZC, once again we're not going to create the ones in AZD which are reserved for future growth, now there's one final thing that we need to do to all of these subnets, so each of these subnets is allocated with an IPv6 range, however it's not set to auto-allocate IPv6 addressing to anything created within each of these subnets, now to do that go ahead and select SN-AP-A, click on actions, and then edit subnet settings, and I want you to check the box to say enable auto-assign IPv6 addresses, once you've done that scroll to the bottom and click on save, so that's one subnet that you've done that for, next I want you to do it for app B, follow the same process, actions, edit subnet settings, auto-assign IPv6, click on save, notice how we're not touching the IPv4 setting, we'll be changing that as appropriate later, select SN-AP-AP-A and see and again edit subnet settings, enable auto-assign IPv6, and click on save, then we're going to do the same for the database subnets, so dba, edit subnet settings, enable IPv6 and save, then dbb, check this box, save, then dbc, edit subnet settings, check this box, save, now we'll do the reserve, so reserve A, then reserve B, and then reserve C, and then finally we're going to do the web subnets, so we'll start with A, again make sure you're only changing the IPv6 box, say that, do the same with web B, and then once that's done we'll scroll down and do the final subnet, so web C, same process, IPv6, and save, and at this point you've gone through the very manual process of creating 12 subnets across three availability zones using the architecture that's shown on screen now, now in production usage in the real world you would automate this process, you wouldn't do this manually each and every time, but I think it's important that you understand how to do this process manually, so you can understand exactly what to select when configuring automation to achieve the same end goal, so whenever I'm using automation I always like to understand how it works manually, so that I can fully understand what it is that automation is doing, now at this point that is everything that I wanted to cover in this demo lesson, we're going to be continually evolving this design as we move through this section of the course, but at this point that is everything I wanted to do, so go ahead and complete this video and when you're ready, I'll look forward to you joining me in the next.

Welcome to this lesson where I'm going to be talking about S3 access points, which is a feature of S3, which improves the manageability of S3 buckets, especially when you have buckets which are used by many different teams or users, or when buckets store objects with a wide range of functions.

Now we have a good amount to cover, so let's just jump in and get started.

S3 access points simplify the process of managing access to S3 buckets and objects.

I want you to imagine an S3 bucket with billions of objects using many different prefixes.

Imagine this bucket is accessed by hundreds of different teams within business.

Now by default you would have one bucket with one really complex bucket policy.

It would be hard to manage and prone to errors.

Access points allow you to conceptually split this.

You can create many access points for a bucket and each of these can have different policies, so different access controls from a permissions perspective.

But also, each access point can be limited in terms of where they can be accessed from.

So for example, a VPC or the internet.

Every access point has its own endpoint address and these can be given to different teams.

So rather than using the default endpoint for S3 and accessing the bucket as a whole, users can use a specifically created access point, along with that specific endpoint address, and get access to part of that bucket or the whole bucket, but with certain restrictions.

Now you can create access points using either the console UI, or you can use the CLI or ABI using create-access-point.

And it's important that you remember this command.

Please try and remember create-access-point.

Now it's going to be easier for you to understand this architecture if we look at it visually.

Let's explore...

Or how everything fits together using an architecture diagram.

And we're going to use a typical example.

Let's say that we have an S3 bucket and this bucket stores sensitive health information for the animals for life organization.

Now this includes health information about the animals, but also the staff working for the business, such as medical conditions and any vaccination status.

Now we have three different sets of staff.

We have admin staff, field workers and vet staff.

The admin staff look after the organization's employees.

The field workers actually visit other countries doing wildlife studies and helping animals.

And then the vet staff look after the medical needs of any animals which the business takes care of.

Now in this example, the business are also using a VPC, with some instances and other resources performing data analysis functions.

If we weren't able to use access points, then we'd have to manage the bucket as a monolithic entity, managing a large and complex bucket policy to control access for identities within this account and potentially other accounts.

And this can become unwieldy very quickly.

One option that we have is to use access points.

And you can sexually think of these as mini-buckets or views on the bucket.

So we might have three access points for our three types of users and then one for the VPC.

Now each of these access points will have a unique DNS address for accessing it and it's this DNS address that we would give to our staff.

But more than this, each access point also has its own policy.

And you can think about this as functionally equivalent to a bucket policy, in that it controls access to the objects in a bucket when using that access point.

So this can control access to certain objects, prefixes or certain tags on objects.

And so it's super powerful.

So now we have a unique DNS name and a unique policy.

Essentially we have mini-buckets which are independently controlled.

And this makes it much easier to manage the different use cases for the main bucket and our staff can access it via the access points.

From the VPC side, access points can be set to only allow a VPC origin, which means that the access point is tied to a specific VPC.

This will need a VPC endpoint in the VPC and the two can be tied together so that the S3 endpoint in the VPC can be configured to only allow access via the S3 access point.

Now one really crucial thing to understand permissions-wise is that any permissions defined on an access point need to be also defined on the bucket policy.

So in this example, if the...

That's Stafford Grant.

And an access via an access point policy, then the same would need to also be granted via the bucket policy.

Now you can do delegation where on the bucket policy you grant wide open access via the access point.

So as long as the access point is used, any action on that bucket's objects is allowed.

And then you'll be fine, more granular control over access to objects in that bucket using the access point policies.

And that's a pretty common permissions architecture to make things simpler to manage.

Now I've included a link attached to this lesson with more details on permissions delegation together with some example access point policies.

You won't need this for the exam, but if you do want to do a bit of extra reading, do make sure you check out the links included with this lesson.

Now at this point, that's everything I wanted to cover.

You just need to have an overview of how this works.

At this point, that's everything.

So thanks for watching.

Go ahead and complete this video.

And when you're ready, I look forward to you joining me in the next.

Welcome back in this lesson, I want to cover a really important feature of S3 in Glacier that you need to be aware of for the exam.

Now you don't need to understand the implementation just the architecture.

S3 Select and Glacier Select are ways that you can retrieve parts of objects rather than the entire object.

Now I expect that this will feature only in the minor way in the exam and it will be focused on architecture and features but I do want you to be aware of exactly how this feature works.

So let's jump in and step through this architecture and what benefits it provides.

Now you know by now that both S3 and Glacier are super scalable services.

You can use them both to store huge quantities of data.

S3 for example can store objects up to five terabytes in size and can store an infinite number of those objects.

Now often when you're interacting with objects inside S3 you intentionally want to interact with that full object so you might want to retrieve that full five terabyte object.

What's critical to understand as a solutions architect is that logically if you retrieve a five terabyte object then it takes time and it consumes that full five terabytes of transfer.

So if you're downloading a five terabyte object from S3 into your application then you consume five terabytes of data you're accessing that full object and it takes time.

Now you can filter this on the client side but this occurs after the figurative damage is done.

You've already consumed that capacity, you've already downloaded that data, filtering it at the client side just means throwing away the data that you don't need.

S3 and Glacia provide services which allow you to access partial objects so that's what's provided by the S3 select and Glacia select services and the way that you do this is that both services allow you to create a SQL like statement so cut down SQL statement.

So you create this, you supply it to that service and then the service uses this SQL like statement to select part of that object and this part and only this part is sent to the client in a pre-filtered way so you only consume the pre-filtered part of that object, the part that you select so it's faster and it's cheaper.

Now both S3 select and Glacia select allow you to operate on a number of file formats with this level of functionality so examples of this include comma separated values JSON can even use visa to compression comma separated values and for JSON so it's a really flexible service.

Now visually this is how it looks.

Now at the top we have the architecture without using S3 or Glacia select and at the bottom we have the architecture when we utilize these services so in both cases we have an application which stores its data on S3.

So when an application interacts with S3 to retrieve any data the entire object or stream of objects are delivered to the application so the application receives everything.

It can either accept it as a whole or it can perform its own filtering but the critical thing to understand is that any filtering performed by the application is performed inside the application.

It doesn't impact the cost or performance.

The only data which is filtered out is simply discarded but it's still billed for and it still takes time.

Now contrast this to using S3 select with the same architecture so we still have the same application that interacts with the same S3 bucket.

It places the filter point inside the S3 service itself and this allows us to use a SQL-like expression and provide this to the S3 select service.

The S3 select service can then use the SQL-like expression and it can apply this to the raw data in S3 so in effect it's taking the raw data filtering it down but this is occurring inside the S3 service so the data that the application receives is the pre-filtered data and this means that we can achieve faster speeds and a significant reduction in cost.

Once the filtering occurs before it's transferred to our application it means that we get substantial benefits both in speed and in cost and this is because the S3 service doesn't have to load all of the data and deliver it to the application.

We're applying this filter at the source, the source of the data which is the S3 service itself.

Now this is a feature which our applications will need to explicitly use but as a solutions architect it's a powerful feature that you need to understand to improve the levels of performance in any systems that you design.

Now at this point that's everything I wanted to cover on to keep it brief because it's a product that I only expect to feature in a very very minor way in the exam and I do want you to be aware of its existence.

So thanks for watching, go ahead, complete this video and when you're ready I look forward to you joining me in the next lesson.

Welcome back.

And in this lesson, I want to talk about S3 Object Lock.

Now, this is something which is really important to understand for the SysOps certification, but equally, if you're developing applications running in AWS or architecting solutions for AWS, you also need to have an awareness.

Now, we've got a fair amount to cover, so let's jump in and get started.

S3 Object Lock is actually a group of related features which I'll talk about in this lesson, but it's something that you enable on new S3 buckets.

If you want to turn on Object Lock for existing buckets, then you need to contact AWS support.

And this is probably going to change over time, but at the time of creating this lesson, this is the current situation.

Now, when you create a bucket with Object Lock enabled, versioning is also enabled on that bucket.

Once you create a bucket with Object Lock and enable it, you cannot disable Object Lock or suspend versioning on that bucket.

Object Lock implements a right once, read many architecture, and this is known as Worm.

It means that you can set it so that Object Versions once created can't be overwritten or deleted.

And just to reiterate, since this is a pretty important point, the feature requires versioning to be enabled on a bucket.

And because of this, it's actually individual Object Versions which are locked.

Now, when we talk about Object Lock, there are actually two ways it manages Object Retention.

Retention periods and legal holds.

An Object Version can have both of these, one or the other or none of them.

And it's really important for the exam and for the real world to think of these as two different things.

So, S3 Object Lock Retention and S3 Object Lock Legal Hold.

Now, just like with bucket default encryption settings, these can be defined on individual Object Versions, or you can define bucket defaults for all of the Object Lock features.

Now, this is just the feature at the high level.

Next, I want to quickly step through the key points of the different retention methods.

So, Retention Period and Legal Hold.

And we're going to start with Retention Period.

With the Retention Period style of object locking, when you create the Object Lock, you specify a Retention Period in days and/or years.

One year means the Retention Period will end one year from when it's applied.

Now, there are two modes of Retention Period Lock which you can apply.

And it's really, really important that you understand how these work and the differences between the two.

One, because it matters for the exam, and two, because if you get it wrong, it will cause a world of pain.

The first mode is Compliance Mode.

If you set a Retention Period on an object using Compliance Mode, it means that an Object Version cannot be deleted or overwritten for the duration of the Retention Period.

But, it also means that the Retention Period itself cannot be reduced and the Retention Mode cannot be adjusted during the Retention Period.

So, no changes at all to the Object Version or Retention Period settings.

And this even includes the account root user.

So, no identity in the account can make any changes to Object Versions, delete Object Versions, or change the Retention Settings until the Retention Period expires.

So, this is serious business.

This is the most strict form of Object Lock.

Don't set this unless you really want that Object Version to stay around in its current form until the Retention Period expires.

Now, you've used this mode as the name suggests for compliance reasons.

An example of this might be medical or financial data.

If you have compliance laws stating that you have to keep data, for example, for three years, with no exceptions, then this is the mode that you set.

Now, a less strict version of this is Governance Mode.

With this mode, you still set a Retention Period, and while active, the Object Version cannot be deleted or changed in any way.

But you can grant special permissions to allow these to be changed.

So, if you want a specific group of identities to be able to change settings and Object Versions, then you can provide them with the permission S3 colon Bypass Governance Retention.

And as long as they have that permission and they provide a header along with their request, which is X-AMZ-BYPASS-GOVERNANCE-RETENTION, then they can override the Governance Mode of Retention.

Now, an important point to understand is this last header, so X-AMZ-HIVEN, and then all the rest.

This is actually the default for the console UI.

And so, using the console UI, you have the S3 colon Bypass Governance Retention, you will be able to make changes to Governance Mode Retention Locks.

So, Governance Mode is useful for a few things.

One, if you want to prevent accidental deletion.

Two, if you have process reasons or governance reasons to keep Object Versions.

Or lastly, you might use it as a test of settings before picking the Compliance Mode.

So, that's Governance Mode.

These are both modes that can be used when using the Retention Period feature of S3 Object Locking.

So, please make sure you understand how they both work and the differences between the two before we finish with this lesson.

It's really, really critical that you understand.

Now, the last overview that I want to give is S3 Object Lock Legal Hold.

With this type, you don't actually set a Retention Period at all.

Instead, for an Object Version, you set Legal Hold to be on or off.

To repeat, there's no concept of retention.

This is a binary.

It's either on or off.

While Legal Hold is enabled on an Object Version, you can't delete or overwrite that specific Object Version.

An extra permission is required, which is S3 colon Put Object Legal Hold.

And this is required if you want to add or remove the Legal Hold feature.

And this type of Object Locking can be used to prevent accidental deletions of Object Versions, or for actual legal situations when you need to flag specific Object Versions as critical for a given case or a project.

Now, this point, let's take a moment to summarise and look visually at how all these different features work.

Let's start with Legal Hold.

We start with a normal Object and we upload it to a bucket with a setting Legal Hold Status to On.

And this means that the Object Version is locked until the Legal Hold is removed.

In this state, the Object Version can't be deleted or changed, but you can set the Legal Hold Status to Off, at which point normal Commissions apply and the Object Version can be deleted or replaced as required.

It's a binary.

It's either on or off, and that isn't the concept of Retention Period.

Next, we have the S3 Object Locks that use the Retention Period architecture.

First, we have Governance, so we put an Object into a bucket with a Lock Configuration of Governance and specify a Retention Period.

This creates a locked Object Version for a given number of days or years, and while it's in this state, it cannot be deleted or updated.

With Governance Mode, this can be bypassed if you have the permissions and specify the correct header.

And once again, this header is the default in the console, so you can adjust or remove the Object Lock or delete or replace the Object Version.

So the important thing to realise here is while an Object is locked for a given Retention Period using the Governance Mode, you can't make any changes to Object Versions or delete them, but you can be provided with the S3 colon bypass and Governance Retention Permission, and as long as you have that and specify the X-AMZ-VIPAS-GOVERNANCE-RETENTION-TRUEHEADER, then you can override the Governance Mode Object Lock during the Retention Period.

Then lastly, we have Compliance, which is the same architecture.

We upload an Object.

We specify Compliance Mode together with the Retention Period, and this creates an Object Version, which is locked for a certain period in days and years.

The difference though is that this can't be changed.

An Object Version can't be deleted or updated.

The Retention Period cannot be shortened.

The Compliance Model can't be changed to something else even by the account root user.

This is permanent.

Only once the Retention Period expires can the Object Version or the Retention Settings be updated.

And for all of these, they can be set on Object Versions or what defaults can be fine.

And that's the architecture of S3 Object Lock.

It's critical that you understand this.

If it takes a few watches at this lesson, then that's OK.

Make sure you understand it in detail, including how each type differs from the others.

And remember, they can be used in conjunction with each other, so the effects can overlap.

You might use Legal Hold together with either Governance or Compliance, and if you do, then the effects of this could overlap, so you need to understand all of this in detail.

But at this point, that's everything I wanted to cover in this lesson, so go ahead and complete the video.

I mean, you ready?

I look forward to you joining me in the next.

Welcome to this mini project where you're going to get the experience of creating S3 multi-region access points.

Now multi-region access points give you the ability to create a single S3 global endpoint and point this at multiple S3 buckets.

It's an effective way to use a single endpoint throughout requests to the closest S3 service.

Now in order to do this mini project you need to be logged in to an AWS account with admin permissions.

If you're using one of my courses then you should use the IAM admin user of the general AWS account which is the management account of the organization.

If you're not using my courses make sure you're using an identity with admin permissions.

You'll also need to select two different AWS regions before this mini project we're going to create S3 buckets in two regions.

I'm going to use AP, South East 2 or the Sydney region and CA Central 1 or the Canada region.

Now the first thing to do is to move to the S3 console so type S3 in the search box at the top and then open that in a new tab.

Once you're there we're going to create two buckets so first go ahead and click on create bucket.

Now we'll keep the bucket naming consistent so we'll use multi-heiven region -demo -heiven and then the region that you're in.

So in my case Sydney and then at the end I want you to append on a random number.

In my case 1-3-3-7.

Remember S3 bucket names need to be globally unique and this will ensure both of our buckets are.

Once you put the name make sure you set the region correctly everything else can be left as default apart from we need to enable bucket versioning.

So set this box under versioning to enable.

Now scroll to the bottom and click on create bucket.

Then we need to follow that same process again for the second bucket.

So click on create bucket.

Use the same bucket naming so multi-heiven region -demo -heiven and then the region name in this case Canada.

And make sure you append your random number and set the region.

Then scroll down, enable bucket versioning again and create the bucket.

Now once you've got these two buckets we're going to create the multi-region access point.

So click on multi-region access point on the menu on the left and click create multi-region access point.

For the name you can pick whatever you want it doesn't need to be globally unique only unique within an AWS account.

I'm going to pick really really critical cat data and then scroll down and add the buckets that you've just created.

These can't be added or edited after creation so we need to do it now.

Now we're going to add buckets.

Select the two buckets and then click on add buckets to confirm.

Once you've done that scroll down to the bottom and click create multi-region access point.

Now this process can take worst case up to 24 hours to complete but typically it creates much faster, generally around 10 to 30 minutes.

Now we do need this to be created before we continue.

So go ahead and pause the video, wait for the status on this to change to ready and then you're good to continue.

Okay so now that we've got this multi-region access point configured and it's ready to go.

Now that we've got this multi-region access point configured we need to configure replication between the two buckets because anyone using this multi-region access point will be directed to the closest S3 bucket and we need to make sure that the data in both matches.

So to do that go ahead and click on the multi-region access point name and go inside there and you'll see that the access point has an Amazon resource name as well as an alias.

Now you should probably note down the Amazon resource name because we might need it later on.

Once you've done that click on the replication and failover tab and you'll be able to see a graphical representation of any replication or failover configuration.

If we click on the replication tab you'll see there's no replication configured.

If we click on the failover tab you can see that we've got these two S3 buckets in different AWS regions configured as an active active failover configuration which means any requests made to this multi-region access point will be delivered to either of these S3 buckets as long as they're available.

Now we can click on one and click on edit routing status and configure it as passive which means it will only be used if no active buckets exist.

But in our case we want it to be active active so we'll leave both of these set to active.

Now we want to configure replication between the buckets so we're going to scroll down to replication rules and click create replication rule.

Now there are two templates available to start with, replicate objects amongst all specified buckets and replicate objects from one or more source buckets to one or more destination buckets.

Now which of these you pick depends on the architecture that you're using but because we have an active active configuration we want all the buckets to be the same.

So we're going to pick the replicate objects among all specified bucket template so this is replicating between every bucket and every other bucket.

Essentially it creates a set of buckets which contain exactly the same data all fronted by a multi-region access point.

So go ahead and make sure this template is selected and then click to select both of the buckets that you created.

In my case Sydney and Canada.

Once we've done that scroll down you can set whether you want the status to be enabled or disabled when created we're going to choose enable and you get to adjust the scope so you can either have it configured so that you can replicate objects using one or more filters or you can apply to all objects in the bucket.

Now we want to make sure the entire bucket is replicated so we're going to use apply to all objects in the bucket.

Now you're informed that an Ion role or roles will be generated based on your configuration and this will provide S3 with the permissions that it needs to replicate objects between the buckets.

Now this is informational we don't need to do anything so let's move on.

Now you're also told what encryption settings are used as well as the destination storage class so because of the template that we picked above we don't get to change the destination storage class and that's okay.

If we scroll down to the bottom we have additional replication options, we have replication time control which applies in SLA to the replication process, we have replication metrics and notifications to provide additional rich information and we can choose whether to replicate the lead markers and whether to replicate modifications.

Now for this mini project we're not going to use replication time control we don't need that level of SLA.

We are going to make sure that replication metrics and notifications is selected.

We don't want to replicate the lead markers and we do want to make sure that replica modifications sync is checked.

So we only want replication metrics and notifications and replica modifications sync.

So make sure that both of those are checked and then click on create replication roles.

Now at this point all the buckets within this multi-region access point are now replicating with each other.

In our case it's only the two, in my case it's Canada and Sydney.

So go ahead and click on close and we can see how this graphical representation has changed showing us that we now have two-way replication in my case between Sydney and Canada.

Now at this point we need to test out the multi-region access point and rather than having you configure your local command line interface we're going to do that with Cloud Shell.

Now what I want you to do is to go ahead and move to a different AWS region so not the same AWS regions that either of your buckets are created in.

What I do want you to do though is make a region close to one of your buckets.

Now I'm going to start off with Sydney and in order to test this I'm going to switch across to the Tokyo region which is relatively close to Sydney, at least from a global perspective.

So I'm going to click on the region drop down at the top and change it from Sydney to Tokyo.

And what's on there I'm going to click on this icon which starts the Cloud Shell.

If this is the first time you're using it in this region you'll probably get the welcome to AWS Cloud Shell notification.

Just either click on close or check this box and then click on close if you don't want to see this notification again.

Now all these commands that we're going to be running are in the instructions which are attached to this video.

The first thing that we're going to do is to create a test file that we're going to upload to S3.

We're going to do that using the DD command.

So we're going to have an input of /dev/urandom which just gives us a stream of random data.

And then for the output using the OF option we're going to create a file called test1.file.

This is going to have a block size of 1 meg and a count of 10 which means that it's going to create a 10 meg file called test1.file.

So run that command.

Now once you've done that just go back to the tab that you've got open to S3.

Scroll to the top, click on multi-region access points, check the access point that you've created and then just click on copy ARN to copy the ARN for this access point into your clipboard and then go back to Cloud Shell.

Next I'm going to do an LS making sure I just have a file created within Cloud Shell, I do.

And now I'm going to run this command so aws space S3, space CP for copy, space test1.file.

So this is the local file we created within Cloud Shell and then space and then S3 colon, double forward slash and then the ARN of the multi-region access point.

Now this command is going to copy the file that we created to this multi-region access point and this multi-region access point is going to direct us towards the closest S3 location that it serves which should be the bucket within the Sydney region.

So go ahead and run that command.

It's going to take a few moments to upload but when it does switch back to S3, go to buckets.

In my case I'm going to go to the Sydney bucket and I should see the file created in this bucket.

I do, that's good, so I'm going to go back to buckets and go to Canada and I don't yet see the object created in the Canada bucket and that's because replication can take a few minutes to replicate from the bucket where the object was stored through to the destination bucket.

If we just give this a few moments and keep hitting refresh, after a few moments we should see the same S1.file which has been replicated from the Sydney region through to the Canada region.

Now S3 replication isn't guaranteed to complete in a set time, especially if you haven't configured the replication time control option.

So it's fairly normal to see a small delay between when the object gets written to one bucket and when it's replicated to another.

Now we're going to try this with a different region, so go back to Cloud Shell and then click on the region drop down and we're going to pick a region which is close to our other bucket but not in the same region.

So the other bucket is created in the Canada region, so I'm going to pick a close region.

In this case I'm going to pick US East 2 which is in Ohio.

Once I've done that I'm going to go back to Cloud Shell.

Once I've done that I should be in Cloud Shell in a different AWS region.

So I'll need to recreate the test file.

In this case I'm going to call it test2.file and I'm going to use all the same options.

And again this command is contained in the instructions attached to this video.

So run that command and it will take a few moments to complete and then we're going to follow the same process.

We're going to upload this file to our S3 buckets using the multi-region access point.

So again just make sure you've got the ARN for the access point in your clipboard and then in the Cloud Shell type AWS space S3, space CP, space test2.file, space S3, colon, forward slash forward slash and then the ARN of the multi-region access point.

Again we're going to press enter, wait for this to upload and then check the buckets.

So run that command and then go back to S3, go to buckets.

I'm going to go to the bucket in Canada first and I'm going to hit refresh and we should see test2.file in this bucket which is good.

Then go to buckets and go to Sydney and we probably won't see that file just yet because it will take a few moments to replicate.

So keep hitting refresh and eventually you should see test2.file arrives in the S3 bucket.

Now this time we're going to run the same test but we're going to pick a region that is relatively in the middle between these two regions where our S3 buckets are created.

In my case I'm going to change the region to APSALV1 which is the Mumbai region.

And I'm going to follow exactly the same process and move to Cloud Shell.

I'm going to generate a new file so in this case it's test3.file.

Now before we upload this we're going to go to the S3 console, go to buckets and just make sure that we have a tab open for both the Canada and the Sydney bucket.

This test is going to be uploaded from a region that's in the middle of these two buckets and so we need to be able to quickly check which bucket receives the upload directly and which bucket receives the replicated copy.

So once you've got a tab open for both those buckets, go back to Cloud Shell and run this command.

So aws space S3 space CP, space test3.file, space S3//// and then the name of the S3 multi-region access point.

Once you've done that go ahead and run that command, it will take a few moments to upload and then move straight to the tabs that you've got open to the S3 buckets and refresh both of them.

If I look at Sydney it looks as though that receives the file straight away.

So this multi-region access point is directed us to Sydney.

If I move to Canada and hit refresh we can see that the file's not yet arrived so this is receiving the replicated copy.

And it does that after a few minutes we can see test3.file arrives in this bucket.

Now this one we're going to run a test to show the type of problem that can occur if you're using this type of global replicated configuration using multi-region access points.

What I want you to do is to open up two different Cloud Shells.

I want one Cloud Shell open in the Canada region so the region may have one of your buckets and I want the other Cloud Shell open in the AP Southeast two region, so the Sydney region.

So we've got one Cloud Shell open in the same region as each of our buckets.

Now once we've done that in one region, in my case I'm going to use the Canada region, I'm going to generate a file called test4.file.

The command is going to be the same as we've used in previous steps.

Then I'm going to copy the ARN of the multi-region access point into my clipboard and I'm going to type this command again in the Canada Cloud Shell.

So this command is going to copy this file to the multi-region access point.

Now because I'm doing this in the Canada region, it's almost guaranteed that the multi-region access point is going to direct us to the bucket which is also in the Canada region.

And so the bucket in this region is going to get the direct copy of the object and the bucket in the Sydney region is going to receive the replicated copy.

So I'm going to fully type out this command and before I run it I'm going to move it to the Sydney region and type out a slightly different command.

This command is also contained in the instructions attached to this lesson.

This command is AWS space S3 space CP space and then S3 colon forward slash forward slash and then the name of the multi-region access point and then forward slash test4.file and then space and then a period.

Now this command when we run it which we're not going to do yet is going to copy the test4.file object from the multi-region access point into our Cloud Shell.

Remember we haven't created this object yet.

Now because this Cloud Shell is in the Sydney region, the multi-region access point is almost certainly going to redirect us to the bucket in the Sydney region.

So let's move back to the Canada Cloud Shell, run this command which is probably going to copy this object into the Canada bucket, then move back to the Cloud Shell in the Sydney region and then run this command to copy the object from the multi-region access point into our Cloud Shell and we receive this error.

Now we're getting this error because the object test4.file doesn't exist within the bucket that the multi-region access point is directing us to.

So just to reiterate what we've done, we've created this object in the Canada region using the multi-region access point which is going to have created the object in the closest S3 resource which is the bucket also in the Canada region.

So the Canada region bucket has the direct copy of the object.

This will then take a few minutes to replicate to the Sydney bucket because we're using this reverse copy command in the Sydney region.

It's attempting to copy the test4.file object from the multi-region access point to our Cloud Shell but because this replication won't have occurred yet and because this is going to direct us at the bucket in the Sydney region, we get this object does not exist error.

And this is one of the issues you can experience when you're using multi-region access points in that there's a consistency lack.

You have to wait for the replication to occur before you can retrieve replicated objects from different buckets using the multi-region access point.

Now this process can be improved by using the RTC option when setting up replication but this does come with additional costs.

So this is something to keep in mind when you're using this type of architecture.

And if we keep running this command we'll see that it keeps failing over and over again until the replication process finishes and then we can copy down the object.

Now that is everything which I wanted to cover in this brief mini project in the multi-region access points.

At this point all that remains is for us to clean up the account and return it to the same state as it was at the start of this mini project.

So to do that we need to move back to the S3 console, go to multi-region access points, select the access point you created for this mini project and click delete.

And then you need to copy and paste the name and click delete to confirm.

Then we need to go ahead and move to buckets.

Select each of the buckets in turn, first click on empty and you need to copy and paste or type permanently delete and then click to confirm that empty process.

Once that's finished click on exit, select the same bucket again, this time click delete, copy and paste or type the bucket name and then click to confirm.

And then do the same process with the other buckets so first empty it, confirm that and then delete it and confirm that.

And then at that point all the billable elements created as part of this mini project have been deleted and we go to finish off this mini project.

So that's everything I wanted to cover, I hope it's been useful and I hope it's given you some practical experience of how to use multi-region access points together with S3 replication.

At this point that's everything to go ahead and complete this video and I hope you'll join me soon for another exciting mini project.

Welcome back.

In this brief demo lesson, you're going to get some experience working with S3 pre-signed URLs.

Now, as you learned in the theory lesson, a pre-signed URL is a type of URL which can be used to grant access to certain objects within an S3 bucket where the credentials for accessing that object are encoded on the URL.

I want to explain exactly what that means and how you can use it in the real world.

Now, to do that, we're not going to need to create any infrastructure using CloudFormation.

Instead, we're going to do it manually.

So first, I want you to make sure that you're logged in to the general AWS account as the I am admin user.

And as always, please make sure that you have the Northern Virginia region selected.

Assuming that's all good, go ahead and type S3 in the search box at the top and open that in a new tab.

We're going to create an S3 bucket within the general AWS account.

So go ahead and click on create bucket to be in that process.

Now, I want you to call the bucket animals for life media.

And because of the unique naming requirements of S3 buckets, you'll need to add some randomness onto the end of this name.

We'll need to select us - east - one for the region.

We can scroll past the bucket settings for block public access.

We don't need to enable bucket versioning.

We won't be using any form of encryption.

Just go ahead and click on create bucket.

Now I want you to go inside the bucket that you've created and click on the upload button to upload an object.

Now, at this point, we need to upload an image file to this bucket.

Any image file will do.

But I've included a sample one attached to this lesson if you don't have one available.

So click the link for the image download.

That will download an image called all5.jpeg.

Once that's downloaded to your local machine, click on add files, select the file, open it, and then go ahead and upload the file to the S3 bucket.

Once that's finished, you can go ahead and click on close.

And you'll see now that we've got our S3 bucket and one object uploaded to that bucket called all5.jpeg.

Next, go ahead and click on that object.

Now, I want to demonstrate how you can interact with this object in a number of different ways.

And the detail really matters here.

So we really need to be sure of exactly what differences there are between these different methods.

The first thing I want you to do is towards the top right of the screen, click on the open button.

You might get a pop-up notification if you do just allow pop-ups.

And what that's going to do is open a jpeg object in a new tab.

Now, I want to point out a number of really important points about how this object has been opened.

If you take a moment to review the URL that's been used to open this object, you'll note that a number of pieces of information have been specified on the URL, including AMZ-SecurityToken.

So essentially, a form of authentication has been provided on the URL which allows you to access this object.

Now, I want you to contrast this by what happens if we go back to the S3 bucket and just copy down the object URL into our clipboard and note how this does not contain any additional authentication information.

It's just the raw URL for this object.

So copy that into your clipboard and then open a new tab and paste that in.

What you're going to see is an access denied message.

And this makes sense because we're now attempting to access this object as an unauthenticated identity, just like any internet user, anyone browsing to this bucket would be doing.

We're not providing any authentication.

And so the only way that we can access the object in this way by not providing any authentication is if we made the bucket public and the bucket currently isn't public, which is why we're getting this access denied message.

Just to reiterate, we can access it by using the previous method because by opening it from within the console, the console is intelligent enough to add authentication information onto the URL which allows us to access this object.

So those are the differences between these two methods.

One is providing authentication and the other isn't.

So right now the only entity that is able to access any of the objects within this bucket is this AWS account and specifically the I am admin user.

The object has no public access.

So now what we're going to do is we're going to work with the scenario that you want to grant access to this object to somebody else for a limited amount of time.

So you don't want to provide the URL that includes authentication information, you want to provide a URL which allows access to that object for a limited amount of time.

That's important.

So we're going to generate a time limited pre-sign URL.

So go ahead and click on the Cloud Shell icon and this is going to open a Cloud Shell using the identity that you're currently logged in at.

So it's going to open a shell much like the one you'll see when you're connected to an EC2 instance but the credentials that you have in the shell are going to be the credentials of the identity that you're currently logged into AWS using in our case the I am admin user with a general AWS account.

You'll see a message saying preparing your terminal and then you'll be logged in to what looks like an EC2 instance prompt.

You can use the AWS CLI tool so I'm able to run an AWS space S3 space LS and this shell will interact with your current AWS account using your current credentials.

So in my case I'm able to see the Animals for Life media bucket which is in my general AWS account because I'm currently logged into this Cloud Shell using the credentials of my I am admin user.

Now to generate a pre-sign URL we have to use this command.

So use AWS to use the command line tools and then a space S3 because we're using the S3 service and then a space and then the word pre-sign because we want to generate a pre-signed URL and then a space and then we need the S3 URI to this object.

Now we can get that from the S3 console.

For every object you can see this unique URI so go ahead and copy this into your click mode go back to the Cloud Shell paste it in and then we'll need space and then we'll use double hyphen expires hyphen in and then a space and then we need to provide the number of seconds that this pre-signed URL will be valid for.

In this case we're going to use 180 which is a total of three minutes so this is in seconds so three minutes is 180 seconds.

So go ahead and press enter and this will generate you a unique pre-signed URL.

So go ahead and copy this into your click board and it is a really long URL so make sure that you get everything including HTTPS all the way to the end of this URL.

Copy that into your click board and then I want you to open a new tab and load that URL and there you go you can see this object loads up using this pre-signed URL.

Now this URL is valid only for 180 seconds.

To demonstrate that I'm going to skip ahead 180 seconds and demonstrate exactly what happens when this URL expires.

After 180 seconds when a next refresh we see this access denied page with a message request has expired.

So you can see how pre-signed URLs are a really effective way of granting access to objects within an S3 bucket for a limited amount of time.

Now there are a number of really interesting aspects to pre-signed URLs that you really need to understand as an architect, a developer or an engineer and I want to go through these really interesting aspects of pre-signed URLs before we finish up with this demo lesson.

Now first just to make everything easier to see I'm going to close down any of these tabs that we got open to this all five object.

I'm going to go back to the cloud shell and I'm going to generate a new pre-signed URL but this time I'm going to use a much larger expires in time.

So I'm going to press the up arrow to return to the previous command.

I'm going to delete this 180 second expiring time and instead I'm going to use 604,800 which is a pretty high number but this is something that we can pick so that we won't have any unintentional expires at the URL as part of this demo lesson.

So pick something crazily large just to make sure that it doesn't expire until we're ready.

So we're generating another URL and so this is an additional unique pre-signed URL.

So I'm going to select all of this URL and you need to do the same go ahead and copy that into your clipboard and open it in a new tab.

Now we can see that that pre-signed URL has opened.

Keep in mind that we've generated this using the identity that we're currently logged into AWS using.

So next what I'm going to do is move back to the AWS console and I'm going to click on the services drop down and move across to IAM.

So I'm going to open IAM up in a new tab.

I'm going to select the users option, select the IAM admin user, I'm going to add an inline policy to this user, select JSON.

Now attached to this lesson is another link for a file called deny s3.json.

Go ahead and click that link and then you'll need to get the contents of the file.

So copy all the contents of the file into your clipboard and then select all of this JSON and paste in the contents of that file and this is an explicit deny policy which denies our user so IAM admin any access to s3.

So this essentially prevents our IAM admin user any level of access to s3.

Go ahead and click on review policy for name, call it deny s3 and click on create policy and this will attach this as an inline policy to our IAM admin user.

Now I'm going to clear the screen within this cloud shell to make it easier to see and then I'm going to run an AWS space s3 space ls and press enter.

Now we get an access deny when accessing the s3 service because we just added an explicit deny onto the IAM admin user and remember the rule that applies to permissions deny allow deny and explicit deny always overrules everything else and so even though the IAM admin user has administrative permissions by applying this deny s3 policy which has an s3 explicit deny this wins so currently we have no access to s3.

Now let's return to the tab where we have this pre-signed url open.

So remember this pre-signed url was generated at the time when we had access to s3 so now let's refresh this page and now we get an access denied message and this is one of those interesting aspects of pre-signed urls.

When you generate a pre-signed url using an identity such as an IAM user that url has your access permissions so you generate a pre-signed url and anyone using that pre-signed url for the duration that it's active will be interacting with that one object on s3 as though they were using your identity.

Now if you adjust the permissions on your identity as we've just done by denying access to s3 it means that that pre-signed url will also have that deny s3 permissions and so this pre-signed url now no longer has any access to anything in s3 including the object that it was configured to provide access to so now let's go back to our cloud shell and what we're going to do now remember we are still denied access to s3.

Let's press the app arrow and move back to the command which we used to generate this pre-signed url.

Now let's regenerate another pre-signed url.

Now note how even though we are denied access to any part of s3 we can generate a pre-signed url which points at this one specific object all five dot jpeg so we're not prevented from generating a pre-signed url for something that we have no access to.

Now if we copy this into our clipboard move to the tab that we already have open and just replace this url with the pre-signed url that you just generated.

Note that we're still denied we're still denied because although we could generate this pre-signed url we've generated it using an identity which has no access to s3 so the next interesting fact to pre-signed urls that I want to demonstrate if we go back to the iam console and now we remove this deny s3 policy from our iam admin user now our iam admin user once again has access to s3 so if we go back to the cloud shell we can run natal us space s3 space ls and press enter and now we can access s3 again from this cloud shell remember the cloud shell is using commissions based on our iam admin user now let's go back to the pre-signed url and now if we refresh this now we can access this object again so I'm doing this to illustrate that when you generate a pre-signed url that pre-signed url is linked to the identity that generates it whatever the permissions are of that identity when the pre-signed url is used that is the permissions that that pre-signed url has so for the duration it always has the same permissions that the identity which generated it has at that very moment so that's something that you need to keep in mind now more interestingly is that you can actually generate a pre-signed url for a non-existent object there's nothing preventing that from occurring so if we go back to the cloud shell we press the up arrow and a number of times to bring up this pre-signed url command and this time we try to generate a pre-signed url for an object which doesn't exist so if I change this all 5 to all 1 3 3 7 and press enter it will generate a pre-signed url that pre-signed url will be valid to access an object called all 1 3 3 7 .jpeg inside this bucket but because there's no such object in that bucket if I try to use it I won't be able to do so so if I open that new invalid pre-signed url I'll get the message that the specified key does not exist but we can generate pre-signed urls for non-existent objects now one more interesting thing about pre-signed urls which I'm not going to demonstrate is if you generate a pre-signed url using temporary credentials you get by issuing a roll so for example if we logged into an ec2 instance which had an instance roll on that instance and then we generated a pre-signed url even if we set a huge expiry time so 604800 that pre-signed url would stop working when those temporary credentials for that roll also stopped working now it is possible to generate a pre-signed url from the console UI this is a relatively recent change from the object if you click on the object actions drop down you can click share with the pre-signed url you have to set the same settings so what you want the expiry to be in this particular case let's say 60 minutes and then I can go ahead and click on create pre-signed url now that's automatically copied into my clipboard and I can go ahead and move to a different tab paste that in and we can open the console UI generated pre-signed url so that's just an alternative way of doing the same process that we just used the cloud shell for now that's everything that I wanted to demonstrate in this demo lesson about pre-signed url and don't worry we're going to be talking about these more later in the course as well as looking at some alternatives what we need to do to tidy up this lesson is go back to the AWS console move to the S3 console and then just go ahead and empty and delete the bucket that you created so select animals for live media click on empty type or paste in permanently delete and then confirm or once that's successfully empty click on exit the bucket should still be selected and then go ahead and click on delete delete confirm that with the name of the bucket and then go ahead and delete the bucket and at this point we've cleaned up the account and the resources back in the same state as they were at the start of the lesson so I hope you've enjoyed this brief demo lesson go ahead complete the video and when you're ready I look forward to you joining me in the next video.

Welcome back and in this lesson I want to talk to you about S3 pre-signed URLs.

Pre-signed URLs are a way that you can give another person or application access to an object inside an S3 bucket using your credentials in a safe and secure way.

Let's have a look at how this works architecturally.

To illustrate how pre-signed URLs work, let's use this example architecture, an S3 bucket which doesn't have any public access configured.

So it's still in a default private configuration.

This means that in order to access the bucket or any resources in it an IAM user such as IAM admin would have to authenticate to AWS and be authorized to access the resource.

IAM admin would send credentials along with the access request, AWS would validate them at the time that the request is made and only then grant access to the object in S3.

Now one issue that we have is that because the bucket is private only authenticated users are able to access it.

Our masked man here has no way of providing authentication information to AWS because he doesn't have any and so any request that's unauthenticated would fail.

Now if giving the mystery user access to S3 is an essential requirement for the business then there are three common solutions at this point and none of them are ideal.

Number one is to give the mystery user an AWS identity.

Number two is to give the mystery user some AWS credentials to use or number three is to make the bucket or the object public.

Now none of these are ideal.

If the user only needs short term access to the bucket and objects the effort of supplying an identity seems excessive.

Just giving some credentials to the user appears on the surface to be a security risk and definitely if it's somebody else's credentials that's just bad practice.

Making the bucket public for anything which has sensitive data in it also appears to be less than ideal.

So one solution that AWS offers is to use pre-signed URLs and let's look at how this would work with an architecture example.

IAM admin is an AWS identity with some access rights granted via a permissions policy.

So IAM admin can make the request to S3 to generate a pre-signed URL.

She would need to provide her security credentials, specify a bucket name, an object key and an expiry date and time as well as indicate how the object would be accessed.

And S3 will create a pre-signed URL and return it.

This URL will have encoded inside it the details that IAM admin provided.

So which bucket is for which object is for it will be encoded with the fact that the IAM admin user generated it and it will be configured to expire at a certain date and time as requested by the IAM admin user.

The URL could then be passed to our mystery user and he or she could use it to access a specific object in the specific S3 bucket up until the point at which it expires.

When the pre-signed URL is used the holder of that URL is actually interacting with S3 as the person who generated it.

So for that specific object in that specific bucket until the timer expires our masked man is actually IAM admin.

Pre-signed URLs can be used for both downloads from S3 so get operations or uploads to S3 known as put operations.

So this type of architecture might be useful for the animals for life remote workers if they don't have access to AWS accounts or accessing from a secure location and just need to upload one specific object.

Now there's another type of architecture which commonly uses pre-signed URLs and I've mentioned this earlier in the course when I was talking about some benefits of S3.

I want you to think about a traditional application architecture.

On the left we have an application user with a laptop.

We've got an application server in the public cloud in the middle which hosts the application and for example say let's say this is a video processing application for wildlife videos that the animals for life organization manages.

We've learned that one of the real strengths of S3 is its ability to host the large media files and so the large wildlife video files have been migrated from the application server to a media S3 bucket.

But by doing this we've introduced a problem.

Previously the videos were hosted on the application server and it could control access to the video files.

If we host them on an S3 bucket then either every user needs an AWS identity so an IAM user to access the videos or we need to make the videos public so that the user running the web application can download them into her browser and neither of those are ideal.

Luckily pre-signed URLs offer a solution.

With pre-signed URLs we can keep the bucket private.

Then we can create an IAM user in our AWS account for this application.

Remember when I talked about choosing between an IAM user or a role I said that if you could visualize how many of a certain thing that would be using an identity then it's likely to suit an IAM user.

Well in this case we have one application and application service accounts are a common scenario where IAM users are used.

So for this example we create an IAM user for the application.

So when our application user interacts with a web application she makes a request and that's step one.

The request might be for a page of information on a bushfire which is currently happening in Australia which has a video file associated with it.

The application that's running on the server knows that it can directly return the information that the request is asking for but that the video is hosted on the private S3 bucket.

So it initiates a request to S3 asking for it to generate a pre-signed URL for that particular video object using the permissions of the IAM user that the application is using.

So IAM app one.

The S3 service then creates a URL which has encoded within it the authentication information for the IAM app one user.

The access to one object in the bucket for a short time limited basis maybe two to three hours.

And the S3 service then returns that to the application server and through to the end user.

The web application that's running on the user's laptop then uses this pre-signed URL to securely access the particular object that's stored on the media bucket.

Pre-signed URLs are often used when you offload media into S3 or as part of serverless architectures where access to a private S3 bucket needs to be controlled and you don't want to run thick application servers to broke at that access.

And we'll look at serverless architectures later in the course.

For now I just want to be sure that you understand what pre-signed URLs do and in the next lesson which is a demo lesson you're going to get the chance to experiment yourself and generate a pre-signed URL.

Pre-signed URLs can be used to access an object in a private S3 bucket with the access rights of the identity which generates them.

They're time limited and they encode all of the authentication information needed inside.

And they can be used to upload objects and download objects from S3.

Now I want to show you this in a demo because it's far easier to actually do it.

But before we do that I want to step through some exam power-ups.

There are a couple of really interesting facts about generating pre-signed URLs which might help you out in some exam questions.

Now first and this is a fairly odd behavior but you can create a pre-signed URL for an object that you have no access to.

The only requirement for generating a pre-signed URL is that you specify a particular object and an expiry date and time.

If you don't have access to that object you can still generate a pre-signed URL which also because it's linked to you will have no access to that object.

So there aren't many use cases where this is applicable but you do need to be aware that it is possible to generate a pre-signed URL when you don't have any access.

Now when you're using the URL, so when you utilize a URL and attempt to access an object, the permissions that you have access to match the identity that generated it.

And it's important to understand that it matches the permissions that the identity has right now.

So at the point when you use the URL, the URL has the same permissions as the identity that generated it has right now.

So if you get an access denied error when you attempt to use a pre-signed URL to access an object, it could be that the identity that generated that URL never had access or it could be that it simply doesn't have access right now.

And they're two very important nuances to understand about pre-signed URLs.

So when you're using a URL to access an object, it matches the current permissions of the identity that generated that URL.

Now that's fairly okay for an IM user.

As I demonstrated on the previous example of the application, generally you would create an IM user for the application and this IM user would have fairly static permissions.

So the permissions generally wouldn't change between when you created the URL and when your customer is using that URL within an application.

But don't generate pre-signed URLs based on an IM role.

You can in theory assume an IM role, which remember gives you temporary credentials, and then you can use those temporary credentials to generate a pre-signed URL.

A pre-signed URL can have a much longer validity period than those temporary credentials.

So those temporary credentials will generally expire well before a pre-signed URL does.

So if you generate a pre-signed URL using a role and then those temporary credentials expire, those credentials are no longer valid.

And so that URL will stop working.

And so it's almost never a good idea to generate a pre-signed URL using an IM role.

You should always use long-term identities.

So generally an IM user.

With that being said though, that is everything that I wanted to cover in this theory lesson.

So go ahead, complete the video, and when you're ready, I'll look forward to you joining me in the next.

Scrapping nearly all the writing assignments takes away so much of the chances to learn, experiment and improve on their skills. Especially when many other professors aren't going to follow this example, either.

This does completely shut down usage of Generative AI, but at the cost of an effective class. Whether it's the fault of AI usage or the teacher is up to anyone, but I'm not on the teacher's side on this.

While I understand where the administrators are coming from, it's important to keep accessibility in mind. Let me provide an example on this:

You're teaching someone to drive in your car, and your car has cameras that covers blind spots and the rear view. Do you make the student check their blind spots manually? If not, how will they know to do so in a car without cameras?

I believe you should teach students to develop these skills without AI's help. If you don't, how will they function without AI?

Author response:

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

Reviewer #1 (Public Review):

Weakness 1. Enhancing Reproducibility and Robustness: To enhance the reproducibility and robustness of the findings, it would be valuable for the authors to provide specific numbers of animals used in each experiment. Explicitly stating the penetrance of the rod-like neurocranial shape in dact1/2-/- animals would provide a clearer understanding of the consistency of this phenotype. 

In Fig. 3 and Fig. 4 animal numbers were added to the figure and figure legend (line 1111). In Fig. 5 animal numbers were added to the figure. We now state that dact1/2-/- animals exhibit the rod-like neurocranial shape that is completely penetrant (Line 260). 

Weakness 2. Strengthening Single-Cell Data Interpretation: To further validate the single-cell data and strengthen the interpretation of the gene expression patterns, I recommend the following: 

-Provide a more thorough explanation of the rationale for comparing dact1/2 double mutants with gpc4 mutants.

-Employ genotyping techniques after embryo collection to ensure the accuracy of animal selection based on phenotype and address the potential for contamination of wild-type "delayed" animals.

-Supplement the single-cell data with secondary validation using RNA in situ or immunohistochemistry techniques. 

An explanation of our rationale was added to the results section (Lines 391403) and a summary schematic was added to Figure 6 (panel A).

Genotyping of the embryos was not possible but quality control analysis by considering the top 2000 most variable genes across the dataset showed good clustering by genotype, indicating the reproducibility of individuals in each group (See Supplemental Fig. 4).

The gene expression profiles obtained in our single-cell data analysis for gpc4, dact1, and dact2 correlate closely with our in situ hybridization analyses. Further, our data is consistent with published zebrafish single-cell data. We validated our finding of increased capn8 expression in dact1/2 mutants by in situ hybridization. Therefore we are confident in the robustness of our single-cell data.  

Weakness 3. Directly Investigating Non-Cell-Autonomous Effects: To directly assess the proposed non-cell-autonomous role of dact1/2, I suggest conducting transplantation experiments to examine the ability of ectodermal/neural crest cells from dact1/2 double mutants to form wild-type-like neurocranium.  

The reviewer’s suggestion is an excellent experiment and something to consider for future work. Cell transplant experiments between animals of specific genotypes are challenging and require large numbers. It is not possible to determine the genotype of the donor and recipient embryos at the early timepoint of 1,000 cell stage where the transplants would have to be done in the zebrafish. So that each transplant will have to be carried out blind to genotype from a dact1+/-; dact2+/- or dact1-/-; dact2+/- intercross and then both animals have to be genotyped at a subsequent time point, and the phenotype of the transplant recipient be analyzed. While possible, this is a monumental undertaking and beyond the scope of the current study.

Weakness 4. Further Elucidating Calpain 8's Role: To strengthen the evidence supporting the critical role of Calpain 8, I recommend conducting overexpression experiments using a sensitized background to enhance the statistical significance of the findings. 

We thank the reviewer for their suggestion and have now performed capn8 overexpression experiments in embryos generated from dact1/2 double heterozygous breeding. We found a statistically significant effect of capn8 overexpression in the dact1+/-,dact2+/- fish (Lines 462-464 and Fig. 8C,D). 

Minor Comments:  

Comment: Creating the manuscript without numbered pages, lines, or figures makes orientation and referencing harder.  

Revised

Comment: Authors are inconsistent in the use of font and adverbs, which requires extra effort from the reader. ("wntIIf2 vs wnt11f2 vs wnt11f2l"; "dact1/2-/- vs dact1/dact2 -/-"; "whole-mount vs wholemount vs whole mount").  

Revised throughout.

Comment: Multiple sentences in the "Results" belong to the "Materials and Methods" or the "Discussion" section. 

We have worked to ensure that sentences are within the appropriate sections of the manuscript.

Comment: Abstract:

"wnt11f2l" should be "wnt11f2"  

Revised (Line 24).

Comment: Main text:

Page 5 - citation Waxman, Hocking et al. 2004 is used 3x without interruption any other citation. 

Revised (Line 112).

Page 9 - "dsh" mutant is mentioned once in the whole manuscript - is this a mistake?

Revised, Rewritten (Line 196).

Page 10 - Fig 2B does not show ISH.

Revised (Line 229).

Page 11 - "kyn" mutant is mentioned here for the first time but defined on page 15.

Revised (Line 245). Now first described on page 4.

Page 14 - "cranial CNN" should be CNCC.

Revised. (Line 334)

Page 16 - dact1/dact2/gpc4: Fig. 5C is used but it should be Fig 5E.

Revised. (Line 381)

Page 18 - dact1/2-/- or dact1-/-, dact2-/-. 

Revised. (Line 428)

Comment: Methods:

Page 24 - ZIRC () "dot" is missing. ChopChop ")" is missing. "located near the 5' end of the gene" - In the Supplementary Figure 1 looks like in the middle of the gene.

Revised. (Lines 600, 609, 611, respectively).

Page 25 - WISH -not used in the main text.

Revised. (Line 346).

Page 26 - 4% (v/v) formaldehyde; at 4C - 4{degree sign}C; 50% (v/v) ethanol; 3% (w/v) methylcellulose.

Revised. (Lines 659, 660, 662).

Page 27 - 0.1% (w/v) BSA. 

Revised. (Line 668).

Comment: Discussion:

The overall discussion requires more references and additional hypotheses. On page 20, when mentioning 'as single mutants develop normally,' does this refer to the entire animals or solely the craniofacial domain? Are these mutants viable? If they are, it's crucial to discuss this phenomenon in relation to prior morpholino studies and genetic compensation.

Observing how the authors interpret previously documented changes in nodal and shh signaling would be beneficial. While Smad1 is discussed, what about other downstream genes? Is shh signaling altered in the dact1/2 double mutants? 

We have revised the Discussion to include more references (Lines 473, 476, 483, 488, 491, 499, 501, 502, 510, 515, 529, 557, 558) and additional hypotheses (Lines 503-505, 511-519, 522-525). We have added more specific information regarding the single mutants (Lines 270-275, 480-493, Fig. S3). We have added discussion of other downstream genes, including smad1 (Lines 561-572) and shh (Lines 572-580).

Comment: Figures:

Appreciating differences between specimens when eyes were or were not removed is quite hard.

Yes this was an unfortunate oversight, however, the key phenotype is the EP shown in the dissections.

Fig 1. - wntIIf2 vs wnt11f2? C - Thisse 2001 - correct is Thisse et al. 2001.

Revised typo in Fig 1. (And Line 1083).

Fig 1E: These plots are hard to understand without previous and detailed knowledge. Authors should include at least some demarcations for the cephalic mesoderm, neural ectoderm, mesenchyme, and muscle. Missing color code.

We have moved this data to supplementary figure S1 and have added labels of the relevant cell types and have added the color code.

Comment:- Fig 2 - In the legend for C - "wildtype and dact2-/- mutant" and "dact1/2 mutant"; in the picture is dact1-/-, dact2-/-.

Revised (Line 1105).

Fig 2 - B - it is a mistake in 6th condition dact1: 2x +/+, heterozygote (+/-) is missing.

Revised Figure 2B.

Fig 4. - Typo in the legend: dact1/"t"2-/- .

Revised. (Line 1127).

Fig 8C - In my view, when the condition gfp mRNA says "0/197, " none of the animals show this phenotype. I assume the authors wanted to say that all the animals show this phenotype; therefore, "197/197" should be used.

We have removed this data from the figure as there were concerns by the reviewers regarding reproducibility. 

Fig S1 - Missing legend for the 28 + 250, 380 + 387 peaks? RT-qPCR - is not mentioned in the Materials and Methods. In D - ratio of 25% (legend), but 35% (graph).

Revised.(Line 1203, Line 625, Line 1213, respectively).

Fig S2 - The word "identified" - 2x in one sentence. 

Revised. (Line 1230).

Reviewer #2 (Public Review):

Weakness(1) While the qualitative data show altered morphologies in each mutant, quantifications of these phenotypes are lacking in several instances, making it difficult to gauge reproducibility and penetrance, as well as to assess the novel ANC forms described in certain mutants.  

In Fig. 3 and Fig. 4 animal numbers were added to the figure legend. In Fig. 5 animal numbers were added to the figure to demonstrate reproducibility. We now state that dact1/2-/- animals exhibit the rod-like neurocranial shape that is completely penetrant (Line 260). As the altered morphologies that we report are qualitatively significant from wildtype we did not find it necessary to make quantitative measurements. For experiments in which it was necessary to in-cross triple heterozygotes (Fig 3, Fig. 5), we dissected and visually analyzed the ANC of at least 3 compound mutant individuals. At least one individual was dissected for the previously published or described genotypes/phenotypes (i.e. wt, wntllf2-/-, dact1/2-/-, gpc4-/-, wls/-). We realize quantitative measurements may identify subtle differences between genotypes. However, the sheer number of embryos needed to generate these relatively rare combinatorial genotypes and the amount of genotyping required prevented quantitative analyses. 

Weakness 2) Germline mutations limit the authors' ability to study a gene's spatiotemporal functional requirement. They therefore cannot concretely attribute nor separate early-stage phenotypes (during gastrulation) to/from late-stage phenotypes (ANC morphological changes). 

We agree that we cannot concretely attribute nor separate early and latestage phenotypes. Conditional mutants to provide temporal or cell-specific analysis are beyond the scope of this work. Here we speculate based on evidence obtained by comparing and contrasting embryos with grossly similar early phenotypes and divergent late-stage phenotypes. We believe our findings contribute to the existing body of literature on zebrafish mutants with both early convergent extension defects and craniofacial abnormalities.   

Weakness (3) Given that dact1/2 can regulate both canonical and non-canonical wnt signaling, this study did not specifically test which of these pathways is altered in the dact1/2 mutants, and it is currently unclear whether disrupted canonical wnt signaling contributes to the craniofacial phenotypes, even though these phenotypes are typical non-canonical wnt phenotypes. 

Previous literature has attributed canonical wnt, non-canonical wnt, and nonwnt functions to dact, and each of these likely contributes to the dact mutant phenotype (Lines 87-89). We performed cursory analyses of tcf/lef:gfp expression in the dact mutants and did not find evidence to support further analysis of canonical wnt signaling in these fish. Single-cell RNAseq did not identify differential expression of any canonical or non-canonical wnt genes in the dact1/2 mutants.

Further research is needed to parse out the intracellular roles of dact1 and dact2 in response to wnt and tgf-beta signaling. Here we find that dact may also have a role in calcium signaling, and further experiments are needed to elaborate this role.      

Weakness (4) The use of single-cell RNA sequencing unveiled genes and processes that are uniquely altered in the dact1/2 mutants, but not in the gpc4 mutants during gastrulation. However, how these changes lead to the manifested ANC phenotype later during craniofacial development remains unclear. The authors showed that calpain 8 is significantly upregulated in the mutant, but the fact that only 1 out of 142 calpainoverexpressing animals phenocopied dact1/2 mutants indicates the complexity of the system. 

To further test whether capn8 overexpression may contribute to the ANC phenotype we performed overexpression experiments in the resultant embryos of dact1/dact2 double het incross. We found the addition of capn8 caused a small but statistically significant occurrence of the mutant phenotype in dact1/2 double heterozygotes (Fig.8D). We agree with the reviewer that our results indicate a complex system of dysregulation that leads to the mutant phenotype. We hypothesize that a combination of gene dysregulation may be required to recapitulate the mutant ANC phenotype. Further, as capn8 activity is regulated by calcium levels, overexpression of the mRNA alone likely has a small effect on the manifestation of the phenotype. 

Weakness (5) Craniofacial phenotypes observed in this study are attributed to convergent extension defects but convergent extension cell movement itself was not directly examined, leaving open if changes in other cellular processes, such as cell differentiation, proliferation, or oriented division, could cause distinct phenotypes between different mutants. 

Although convergent extension cell movements were not directly examined, our phenotypic analyses of the dact1/2 mutant are consistent with previous literature where axis extension anomalies were attributed to defects in convergent extension (Waxman 2004, Xing 2018, Topczewski 2001). We do not attribute the axis defect to differentiation differences as in situ analyses of established cell type markers show the existence of these cells, only displaced relative to wildtype (Figure 1). We agree that we cannot rule out a role for differences in apoptosis or proliferation however, we did not detect transcriptional differences in dact1/2 mutants that would indicate this in the single-cell RNAseq dataset. Defects in directed division are possible, but alone would not explain that dact1/2 mutant phenotype, particularly the widened dorsal axis (Figure 1).

Major comments:  

Comment (1) The author examined and showed convergent extension phenotype (CE) during body axis elongation in dact1/dact2-/- homozygous mutants. Given that dact2-/- single mutants also displayed shortened axis, the authors should either explain why they didn't analyze CE in dact2-/- (perhaps because that has been looked at in previously published dact2 morphants?) or additionally show whether CE phenotypes are present in dact1 and dact2 single mutants.  

The authors should quantify the CE phenotype in both dact2-/- single mutants and dact1/dact2-/- double mutants, and examine whether the CE phenotypes are exacerbated in the double mutants, which may lend support to the authors' idea that dact1 can contribute to CE. The authors stated in the discussion that they "posit that dact1 expression in the mesoderm is required for dorsal CE during gastrulation through its role in noncanonical Wnt/PCP signaling". However, no evidence was presented in the paper to show that dact1 influences CE during body axis elongation.  

Because any axis shortening in shortening in dact2-/- single mutants was overcome during the course of development and at 5 dpf there was no noticeable phenotype, we did not analyze the single mutants further.  

We have added data to demonstrate the resulting phenotype of each combinatorial genotype to provide a more clear and detailed description of the single and compound mutants (Fig. S3). 

Our hypothesis that dact1 may contribute to convergent extension is based on its apparent ability to compensate (either directly or indirectly) for dact2 loss in the dact2-/- single mutant. 

Comment (2) Except in Fig. 2, I could not find n numbers given in other experiments. It is therefore unclear if these mutant phenotypes were fully or partially penetrant. In general, there is also a lack of quantifications to help support the qualitative results. For example, in Fig. 4, n numbers should be given and cell movements and/or contributions to the ANC should be quantified to statistically demonstrate that the second stream of CNCC failed to contribute to the ANC.  

Similarly, while the fan-shaped and the rod-shaped ANCs are very distinct, the various rod-shaped ANCs need to be quantified (e.g. morphometry or measurements of morphological features) in order for the authors to claim that these are "novel ANC forms", such as in the dact1/2-/-, gpc4/dact1/2-/-, and wls/dact1/2-/- mutants (Fig. 5).  

We have added n numbers for each experiment and stated that the rod-like phenotype of the dact1/2-/- mutant was fully penetrant. 

Regarding CNCC experiments, we repeated the analysis on 3 individual controls and mutants and did not find evidence that CNCC migration was directly affected in the dact1/2 mutant. Rather, differences in ANC development are likely secondary to defects in floor plate and eye field morphometry. Therefore we did not do any further analyses of the CNCCs.

Regarding figure 5, we have added n numbers. We dissected and analyzed a minimum of three triple mutants (dact1/2-/-,gpc4-/- and dact1/2-/-,wls-/-) and numerous dact1/s double mutants and found that the triple mutant ANC phenotype was consistent and recognizably different enough from the dact1/2-/-, or gpc4 or wls single mutant that morphometry measurements were not needed. Further, the triple mutant phenotype (narrow and shortened) appears to be a simple combination of dact1/2 (narrow) and gpc4/wls (shortened) phenotypes. As we did not find evidence of genetic epistasis, we did not analyze the novel ANC forms further.

Comment (3): The authors have attributed the ANC phenotypes in dact1/2-/- to CE defects and altered noncanonical wnt signaling. However, no evidence was presented to support either. The authors can perhaps utilize diI labelling, photoconversionmediated lineage tracing, or live imaging to study cell movement in the ANC and compare that with the cell movement change in the gpc4-/- , and gpc4/dact1/2-/- mutants in order to first establish that dact1/2 affect CE and then examine how dact1/2 mutations can modulate the CE phenotypes in gpc4-/- mutants.  

Concurrently, given that dact1 and dact2 can affect (perhaps differentially) both canonical and non-canonical wnt signaling, the authors are encouraged to also test whether canonical wnt signaling is affected in the ANC or surrounding tissues, or at minimum, discuss the potential role/contribution of canonical wnt signaling in this context.  

Given the substantial body of research on the role of noncanonical wnt signaling and planar cell polarity pathway on convergent extension during axis formation (reviewed by Yang and Mlodzik 2015, Roszko et al., 2009) and the resulting phenotypes of various zebrafish mutants (i.e. Xing 2018, Topczewski 2001), including previous research on dact1 and 2 morphants (Waxman 2004), we did not find it necessary to analyze CE cell movements directly.  

Our finding that CNCC migration was not defective in the dact1/2 mutants and the knowledge that various zebrafish mutants with anterior patterning defects (slb, smo, cyc) have a similar craniofacial abnormality led us to conclude that the rod-like ANC in the dact1/2 mutant was secondary to an early patterning defect (abnormal eye field morphology). Therefore, testing dact1/2 and convergent extension or wnt signaling in the ANC itself was not an aim of this paper.  

Comment (4) The authors also have not ruled out other possibilities that could cause the dact1/2-/- ANC phenotype. For example, increased cell death or reduced proliferation in the ANC may result in the phenotype, and changes in cell fate specification or differentiation in the second CNCC stream may also result in their inability to contribute to the ANC. 

We agree that we cannot rule out whether cell death or proliferation is different in the dact1/2 mutant ANC. However, because we do not find the second CNCC stream within the ANC, this is the most likely explanation for the abnormal ANC shape. Because the first stream of CNCC are able to populate the ANC and differentiate normally, it is most likely that the inability of the second stream to populate the ANC is due to steric hindrance imposed by the abnormal cranial/eye field morphology. These hypotheses would need to be tested, ideally with an inducible dact1/2 mutant, however, this is beyond the scope of this paper.     

Comment (5) The last paragraph of the section "Genetic interaction of dact1/2 with Wnt regulators..." misuses terms and conflates phenotypes observed. For instance, the authors wrote "dact2 haploinsuffciency in the context of dact1-/-; gpc4-/- double mutant produced ANC in the opposite phenotypic spectrum of ANC morphology, appearing similar to the gpc4-/- mutant phenotype". However, if heterozygous dact2 is not modulating phenotypes in this genetic background, its function is not "haploinsuffcient". The authors then said, "These results show that dact1 and dact2 do not have redundant function during craniofacial morphogenesis, and that dact2 function is more indispensable than dact1". However this statement should be confined to the context of modulating gpc4 phenotypes, which is not clearly stated. 

Revised (Lines 380, 382).   

Comment (6) For the scRNA-seq analysis, the authors should show the population distribution in the UMAP for the 3 genotypes, even if there are no obvious changes. The authors are encouraged, although not required, to perform pseudotime or RNA velocity analysis to determine if differentiation trajectories are changed in the NC populations, in light of what they found in Fig. 4. The authors can also check the expression of reporter genes downstream of certain pathways, e.g. axin2 in canonical wnt signaling, to query if these signaling activities are changed (also related to point #3 above). 

We have added population distribution data for the 3 genotypes to Supplemental Figure 4. Although RNA velocity analysis would be an interesting additional analysis, we would hypothesize that the NC population is not driving the differences in phenotype. Rather these are likely changes in the anterior neural plate and mesoderm. 

Comment (7) While the phenotypic difference between gpc4-/- and dact1/2-/- are in the ANC at a later stage, ssRNA-seq was performed using younger embryos. The authors should better explain the rationale and discuss how transcriptomic differences in these younger embryos can explain later phenotypes. Importantly, dact1, dact2, and capn8 expression were not shown in and around the ANC during its development and this information is crucial for interpreting some of the results shown in this paper. For example, if dact1 and dact2 are expressed during ANC development, they may have specific functions during that stage. Alternatively, if dact1 and dact2 are not expressed when the second stream CNCCs are found to be outside the ANC, then the ANC phenotype may be due to dact1/2's functions at an earlier time point. The author's statement in the discussion that "embryonic fields determined during gastrulation effect the CNCC ability to contribute to the craniofacial skeleton" is currently speculative. 

We have reworded our rationale and hypothesis to increase clarity (Lines 391-405). We believe that the ANC phenotype of the dact1/2 mutants is secondary to defective CE and anterior axis lengthening, as has been reported for the slb mutant (Heisenberg 1997, 2000). We utilized the gpc4 mutant as a foil to the dact1/2 mutant, as the gpc4 mutant has defective CE and axis extension without the same craniofacial phenotype.

We have added dact1 and dact2 WISH of 24 and 48 hpf (Fig1. D,E) to show expression during ANC development. 

Comment (8) The functional testing of capn8 did not yield a result that would suggest a strong effect, as only 1 in 142 animals phenocopied dact1/2. Therefore, while the result is interesting, the authors should tone down its importance. Alternatively, the authors can try knocking down capn8 in the dact1/2 mutants to test how that affects the CE phenotype during axis elongation, as well as ANC morphogenesis. 

As overexpression of capn8 in wildtype animals did not result in a significant phenotype, we tested capn8 overexpression in compound dact1/2 mutants as these have a sensitized background. We found a small but statistically significant effect of exogenous capn8 in dact1+/-,dact2+/- animals. While the effect is not what one would expect comparing to Mendelian genetic ratios, the rod-like ANC phenotype is an extreme craniofacial dysmorphology not observed in wildtype or mRNA injected embryos hence significant. The experiment is limited by the available technology of over-expressing mRNA broadly without temporal or cell specificity control. It is possible that if capn8 over-expression was restricted to specific cells (floor plate, notochord or mesoderm) and at the optimal time period during gastrulation/segmentation that the aberrant ANC phenotype would be more robust. We agree with the reviewer that although the finding of a new role for capn8 during development is interesting, its importance in the context of dact should be toned down and we have altered the manuscript accordingly (Lines 455-467).  

Comment (9) A difference between the two images in Fig. 8B is hard to distinguish.

Consider showing flat-mount images. 

We have added flat-mount images to Fig. 8B

Minor comments:

Comment (1) wnt11f2 is spelled incorrectly in a couple of places, e.g. "wnt11f2l" in the abstract and "wntllf2" in the discussion. 

Revised throughout.

Comment (2) For Fig. 1D, the white dact1 and yellow dact2 are hard to distinguish in the merged image. Consider changing one of their colors to a different one and only merge dact1 and dact2 without irf6 to better show their complementarity.  

We agree with the reviewer that the expression patterns of dact1 and dact2 are difficult to distinguish in the merged image. We have added outlines of the cartilage elements to the images to facilitate comparisons of dact1 and dact2 expression (Fig 1F). 

Comment (3) For Fig. 1E, please label the clusters mentioned in the text so readers can better compare expressions in these cell populations.  

We have moved this data to supplementary figure S1 and have added labels.

Comment (4) The citing and labelling of certain figures can be more specific. For example, Fig. S1A, B, and Fig. S1C should be used instead of just Fig. S1 (under the section titled dact1 and dact2 contribute to axis extension...". Similarly, Fig. 4 can be better labeled with alphabets and cited at the relevant places in the text.  

We have modified the labeling of the figures according to the reviewer’s suggestion (Fig S2 (previously S1), Fig4) and have added reference to these labels in the text (Lines 202, 204, 212, 328, 334, 336). 

Comment (5) For Fig. 2B, the (+/+,-/-) on x-axis should be (+/-,-/-).  

Revised in Figure 2B.

Comment (6) Several figures are incorrectly cited. Fig. 2C is not cited, and the "Fig. 2C" and "Fig. 2D" cited in the text should be "Fig. 2D" and "Fig. 2E" respectively. Similarly, Fig. 5C and D are not cited in the text and the cited Fig. 5C should be 5E. The VC images in Fig. 5 are not talked about in the text. Finally, Fig. 7C was also not mentioned in the text.  

We have corrected the labeling and have added descriptions of each panel in the Results (Fig.2 Line 231, 237, 242, Fig 5 Line 373, 381, Fig 7 line 431). 

Comment (7) In the main text, it is indicated that zebrafish at 3ss were used for ssRNAseq, but in the figure legend, it says 4ss. 

Revised (Line 682)

Comment (8) No error bars in Fig. S1B and the difference between the black and grey shades in Fig. S1D is not explained.  

Error bars are not included in the graphs of qPCR results (now Fig S2C) as these are results of a pool of 8 embryos performed one time. We have added a legend to explain the gray vs. black bars (now Fig S2E). 

Reviewer #3 (Public Review):  

Weaknesses: The hypotheses are very poorly defined and misinterpret key previous findings surrounding the roles of wnt11 and gpc4, which results in a very confusing manuscript. Many of the results are not novel and focus on secondary defects. The most novel result of overexpressing calpain8 in dact1/2 mutants is preliminary and not convincing.  

We apologize for not presenting the question more clearly. The Introduction was revised with particular attention to distinguish this work using genetic germline mutants from prior morpholino studies. Please refer to pages 4-5, lines 106-121.

Weakness 1) One major problem throughout the paper is that the authors misrepresent the fact that wnt11f2 and gpc4 act in different cell populations at different times. Gastrulation defects in these mutants are not similar: wnt11 is required for anterior mesoderm CE during gastrulation but not during subsequent craniofacial development while gpc4 is required for posterior mesoderm CE and later craniofacial cartilage morphogenesis (LeClair et al., 2009). Overall, the non-overlapping functions of wnt11 and gpc4, both temporally and spatially, suggest that they are not part of the same pathway.  

We have reworded the text to add clarity. While the loss of wnt11 versus the loss of gpc4 may affect different cell populations, the overall effect is a shortened body axis. We stressed that it is this similar impaired axis elongation phenotype but discrepant ANC morphology phenotypes in the opposite ends of the ANC morphologic spectrum that is very interesting and leads us to investigate dact1/2 in the genetic contexts of wnt11f2 and gpc4.  Pls refer to page 4, lines 73-84. Further, the reviewer’s comment that wnt11 and gpc4 are spatially and temporally distinct is untested. We think the reviewer’s claim of gpc4 acting in the posterior mesoderm refers to its requirement in the tailbud (Marlow 2004). However this does not exclude gpc4 from acting elsewhere as well. Further experiments would be necessary. Both wnt11f2 and gpc4 regulate non-canonical wnt signaling and are coexpressed during some points of gastrulation and CF development (Gupta et al., 2013; Sisson 2015). This data supports the possibility of overlapping roles. 

Weakness 2) There are also serious problems surrounding attempts to relate single-cell data with the other data in the manuscript and many claims that lack validation. For example, in Fig 1 it is entirely unclear how the Daniocell scRNA-seq data have been used to compare dact1/2 with wnt11f2 or gpc4. With no labeling in panel 1E of this figure these comparisons are impossible to follow. Similarly, the comparisons between dact1/2 and gpc4 in scRNA-seq data in Fig. 6 as well as the choices of DEGs in dact1/2 or gpc4 mutants in Fig. 7 seem arbitrary and do not make a convincing case for any specific developmental hypothesis. Are dact1 and gpc4 or dact2 and wnt11 coexpressed in individual cells? Eyeballing similarity is not acceptable.  

We have moved the previously published Daniocell data to Figure S1 and have added labeling. These data are meant to complement and support the WISH results and demonstrate the utility of using available public Daniocell data. Please recommend how we can do this better or recommend how we can remediate this work with specific comment. 

Regarding our own scRNA-seq data, we have added rationale (line 391-403) and details of the results to increase clarity (Lines 419-436). We have added a panel to Figure 6 (panel A) to help illustrate or rationale for comparing dact1/2 to gpc4 mutants to wt. The DEGs displayed in Fig.7A are the top 50 most differentially expressed genes between dact1/2 mutants and WT (Figure 7 legend, line 422-424).   

We have looked at our scRNA-seq gene expression results for our clusters of interest (lateral plate mesoderm, paraxial mesoderm, and ectoderm). We find dact1, dact2, and gpc4 co-expression within these clusters. Knowing whether these genes are coexpressed within the same individual cell would require going back and analyzing the raw expression data. We do not find this to be necessary to support our conclusions. The expression pattern of wnt11f2 is irrelevant here.   

Weakness 3) Many of the results in the paper are not novel and either confirm previous findings, particularly Waxman et al (2004), or even contradict them without good evidence. The authors should make sure that dact2 loss-of-function is not compensated for by an increase in dact1 transcription or vice versa. Testing genetic interactions, including investigating the expression of wnt11f2 in dact1/2 mutants, dact1/2 expression in wnt11f2 mutants, or the ability of dact1/2 to rescue wnt11f2 loss of function would give this work a more novel, mechanistic angle.

We clarified here that the prior work carried out by Waxman using morppholinos, while acceptable at the time in 2004, does not meet the rigor of developmental studies today which is to generate germline mutants. The reviewer’s acceptance of the prior work at face value fails to take the limitation of prior work into account. Further, the prior paper from Waxman et al did not analyze craniofacial morphology other than eyeballing the shape of the head and eyes. Please compare the Waxman paper and this work figure for figure and the additional detail of this study should be clear. Again, this is by no means any criticism of prior work as the prior study suffered from the technological limitations of 2004, just as this study also is the best we can do using the tools we have today. Any discrepancies in results are likely due to differences in morpholino versus genetic disruption and most reviewers would favor the phenotype analysis from the germline genetic context. We have addressed these concerns as objectively as we can in the text (Lines 482-493). The fact that dact1/2 double mutants display a craniofacial phenotype while the single mutants do not, suggests compensation (Lines 503-505), but not necessarily at the mRNA expression level (Fig. S2C). 

This paper tests genetic interaction through phenotyping the wntll/dact1/dact2 mutant.

Our results support the previous literature that dact1/2 act downstream of wnt11 signaling. There is no evidence of cross-regulation of gene expression. We do not expect that changes in wnt11 or dact would result in expression changes in the others.

RNA-seq of the dact1/2 mutants did not show changes in wnt11 gene expression. Unless dact1 and/or dact2 mRNA are under expressed in the wnt11 mutant, we would not expect a rescue experiment to be informative. And as wnt11 is not a focus of this paper, we have not performed the experiment.  

Weakness 4) The identification of calpain 8 overexpression in Dact1/2 mutants is interesting, but getting 1/142 phenotypes from mRNA injections does not meet reproducibility standards.

As the occurrence of the mutant phenotype in wildtype animals with exogenous capn8 expression was below what would meet reproducibility standards, we performed an additional experiment where capn8 was overexpressed in embryos resulting from dact1/dact2 double heterozygotes incross (Fig. 8). We reasoned that an effect of capn8 overexpression may be more robust on a sensitized background. We found a statistically significant effect of capn8 in dact1/2 double heterozygotes, though the occurrence was still relatively rare (6/80). These data suggest dysregulation of capn8 contributes to the mutant ANC phenotype, though there are likely other factors involved. 

Comment: The manuscript title is not representative of the findings of this study.  

We revised the title to strictly describe that we generated and carried out genetic analysis in loss of function compound mutants (Genetic requirement) and that we found capn8 was important which modified this requirement.

Introduction: p.4:

Comment: Anterior neurocranium (ANC) - it has to be stated that this refers to the combined ethmoid plate and trabecular cartilages. 

Thank you, we agree that the ANC and ethmoid plate terminology has been confusing in the literature and we should endeavor to more clearly describe that the phenotypes in question are all in the ethmoid plate and the trabeculae are not affected. ANC has been replaced with ethmoid plate (EP) throughout the manuscript and figures. We also describe that all the observed phenotypes affect the ethmoid plate and not the trabeculae, (pages 13, Lines 265-267).

Comment: Transverse dimension is incorrect terminology - replace with medio-lateral.

Revised (Lines 69, 74).

Comment: Improper way of explaining the relationship between mutant and gene..."Another mutant knypek, later identified as gpc4..." a better  way to explain this would be that the knypek mutation was found to be a non-sense mutation in the gpc4 gene.  

Revised (Line 71)

Comment: "...the gpc4 mutant formed an ANC that is wider in the transverse dimension than the wildtype, in the opposite end of the ANC phenotypic spectrum compared to wnt11f2...These observations beg the question how defects in early patterning and convergent extension of the embryo may be associated with later craniofacial morphogenesis."

This statement is broadly representative of the general failure to distinguish primary from secondary defects in this manuscript. Focusing on secondary defects may be useful to understand the etiology of a human disease, but it is misleading to focus on secondary defects when studying gene function. The rod-like ethmoid of slb mutant results from a CE defect of anterior mesoderm during gastrulation(Heisenberg et al. 1997, 2000), while the wide ethmoid plate of kny mutants results from CE defects of cartilage precursors (Rochard et al., 2016). Based on this evidence, wnt11f2 and gpc4 act in different cell populations at different times.  

It is true that the slb mutant craniofacial phenotype has been stated as secondary to the CE defect during gastrulation and the kny phenotype as primary to chondrocyte CE defects in the ethmoid, however the direct experimental evidence to conclude only primary or only secondary effects does not yet exist. There is no experiment to our knowledge where wnt11f2 was found to not affect ethmoid chondrocytes directly. Likewise, there is no experiment having demonstrated that dysregulated CE in gpc4 mutants does not contribute to a secondary abnormality in the ethmoid. 

Here, we are analyzing the CE and craniofacial phenotypes of the dact1/2 mutants without any assumptions about primary or secondary effects and without drawing any conclusions about wnt11f2 or gpc4 cellular mechanisms.     

Comment: "The observation that wnt11f2 and gpc4 mutants share similar gastrulation and axis extension phenotypes but contrasting ANC morphologies supports a hypothesis that convergent extension mechanisms regulated by these Wnt pathway genes are specific to the temporal and spatial context during embryogenesis."

This sentence is quite vague and potentially misleading. The gastrulation defects of these 2 mutants are not similar - wnt11 is required for anterior mesoderm CE during gastrulation and has not been shown to be active during subsequent craniofacial development while gpc4 is required for posterior mesoderm CE and craniofacial cartilage morphogenesis (LeClair et al., 2009). Here again, the non-spatially overlapping functions of wnt11 and gpc4 suggest that are not part of the same pathway.  

Though the cells displaying defective CE in wnt11f2 and gpc4 mutants are different, the effects on the body axis are similar. The dact1/2 showed a similar axis extension defect (grossly) to these mutants. Our aim with the scRNA-seq experiment was to determine which cells and gene programs are disrupted in dact1/2 mutants. We found that some cell types and programs were disrupted similarly in dact1/2 mutants and gpc4 mutants, while other cells and programs were specific to dact1/2 versus gpc4 mutants. We can speculate that these that were specific to dact1/2 versus gpc4 may be attributed to CE in the anterior mesoderm, as is the case for wnt11. 

p.5

Comment: "We examined the connection between convergent extension governing gastrulation, body axis segmentation, and craniofacial morphogenesis." A statement focused on the mechanistic findings of this paper would be welcome here, instead of a claim for a "connection" that is vague and hard to find in the manuscript.  

We have rewritten this statement (Line 125).

p.7 Results:

Comment: It is unclear why Farrel et al., 2018 and Lange et al., 2023 are appropriate references for WISH. Please justify or edit.  

This was a mistake and has been edited (Page 9).

Comment: " Further, dact gene expression was distinct from wnt11f2." This statement is inaccurate in light of the data shown in Fig1A and the following statements - please edit to reflect the partially overlapping expression patterns.  

We have edited to clarify (Lines 142-143).

p.8

Comment: "...we examined dact1 and 2 expression in the developing orofacial tissues. We found that at 72hpf..." - expression at 72hpf is not relevant to craniofacial morphogenesis, which takes place between 48h-60hpf (Kimmel et al., 1998; Rochard et al., 2016; Le Pabic et al., 2014).  

We have included images and discussion of dact1 and dact2 expression at earlier time points that are important to craniofacial development (Lines 160-171)(Fig 1D,E). 

Comment: "This is in line with our prior finding of decreased dact2 expression in irf6 null embryos". - This statement is too vague. How are th.e two observations "in line".  

We have removed this statement from the manuscript.

Comment: Incomplete sentence (no verb) - "The differences in expression pattern between dact1 and dact2...".  

Revised (Line 172).

Comment: "During embryogenesis..." - Please label the named structures in Fig.1E.

Please be more precise with the described expression time. Also, it would be useful to integrate the scRNAseq data with the WISH data to create an overall picture instead of treating each dataset separately.  

We have moved the previously published Daniocell data to supplementary figure S1 and have labeled the key cell types. 

p.9

Comment: "The specificity of the gene disruption was demonstrated by phenotypic rescue with the injection of dact1 or dact2 mRNA (Fig. S1)." - please describe what is considered a phenotypic rescue.

-The body axis reduction of dact mutants needs to be documented in a figure. Head pictures are not sufficient. Is the head alone affected, or both the head and trunk/tail? Fig.2E suggests that both head and trunk/tail are affected - please include a live embryos picture at a later stage.  

We have added a description of how phenotypic rescue was determined (Line 208). We have added a figure with representative images of the whole body of dact1/2 mutants. Measurements of body length found a shortening in dact1/2 double mutants versus wildtype, however differences were not found to be significantly different by ANOVA (Fig. 3C, Fig. S3, Line 270-275).

p. 11

Comment: "These dact1-/-;dact2-/- CE phenotypes were similar to findings in other Wnt mutants, such as slb and kny (Heisenberg, Tada et al., 2000; Topczewski, Sepich et al., 2001)." The similarity between slb and kny phenotypes should be mentioned with caution as CE defects affect different regions in these 2 mutants. It is misleading to combine them into one phenotype category as wnt11 and gpc4 are most likely not acting in the same pathway based on these spatially distinct phenotypes.  

Here we are referring to the grossly similar axis extension defects in slb and kny mutants. We refer to these mutants to illustrate that dact1 and or 2 deficiency could affect axis extension through diverse mechanisms. We have added text for clarity (Lines 249-252).  

Comment: "No craniofacial phenotype was observed in dact1 or dact2 single mutants. However, in-crossing to generate [...] compound homozygotes resulted in dramatic craniofacial deformity."

This result is intriguing in light of (1) the similar craniofacial phenotype previously reported by Waxman et al (2004) using morpholino- based knock-down of dact2, and the phenomenon of genetic compensation demonstrated by Jakutis and Stainier 2001 (https://doi.org/10.1146/annurev-genet-071719-020342). The authors should make sure that dact2 loss-of-function is not compensated for by an increase in dact1 transcription, as such compensation could lead to inaccurate conclusions if ignored.  

We agree with the reviewer that genetic compensation of dact2 by dact1 likely explains the different result found in the dact2 morphant versus CRISPR mutant. We found increased dact1 mRNA expression in the dact2-/- mutant (Fig S2X) however a more thorough examination is required to draw a conclusion. Interestingly, we found that in wildtype embryos dact1 and dact2 expression patterns are distinct though with some overlap. It would be informative to investigate whether the dact1 expression pattern changes in dact2-/- mutants to account for dact2 loss.   

Comment: "Lineage tracing of NCC movements in dact1/2 mutants reveals ANC composition" - the title is misleading - ANC composition was previously investigated by lineage tracing (Eberhardt et al., 2006; Wada et al., 2005).  

This has been reworded (Line 292)

p.13

Comment: There is no frontonasal prominence in zebrafish.  

This is true, texts have been changed to frontal prominence.  (Lines 293,

299, 320)

Comment: The rationale for investigating NC migration in mutants where there is a gastrula-stage failure of head mesoderm convergent extension is unclear. The whole head is deformed even before neural crest cells migrate as the eye field does not get split in two (Heisenberg et al., 1997; 2000), suggesting that the rod-like ethmoid plate is a secondary defect of this gastrula-stage defect. In addition, neural crest migration and cartilage morphogenesis are different processes, with clear temporal and spatial distinctions.  

We carried out the lineage tracing experiment to determine which NC streams contributed to the aberrantly shaped EP, whether the anteromost NC stream frontal prominence, the second NC stream of maxillary prominence, or both.  We found that the anteromost NCC did contribute to the rod-like EP, which is different from when hedgehod signaling is disrupted,  So while it is possible that the gastrula-effect head mesoderm CE caused a secondary effect on NC migration, how the anterior NC stream and second NC stream are affected differently between dact1/2 and shh pathway is interesting.  We added discussion of this observation to the manuscript (page 23, Lines 514-520). 

p. 14-16

Comment: Based on the heavy suspicion that the rod-like ethmoid plate of the dact1/2 mutant results from a gastrulation defect, not a primary defect in later craniofacial morphogenesis, the prospect of crossing dact1/2 mutants with other wnt-pathway mutants for which craniofacial defects result from craniofacial morphogenetic defects is at the very least unlikely to generate any useful mechanistic information, and at most very likely to generate lots of confusion. Both predictions seem to take form here.  

However, the ethmoid plate phenotype observed in the gpc4-/-; dact1+/-; dact2-/- mutants (Fig. 5E) does suggest that gpc4 may interact with dact1/2 during gastrulation, but that is the case only if dact1+/-; dact2-/- mutants do not have an ethmoid cartilage defect, which I could not find in the manuscript. Please clarify.  

The perspective that the rod-like EP of the dact1/2 is due to gastrulation defect is being examined here. Why would other mutants such as wnt11f2 and gpc4 that have gastrulation CE defects have very different EP morphology, whether primary or secondary NCC effect?  Further dact1 and dact2 were reported as modifiers of Wnt signaling, so it is logical to genetically test the relationship between dact1, dact2, wnt11f2, gpc4 and wls. The experiment had to be done to investigate how these genetic combinations impact EP morphology. This study found that combined loss of dact1, dact2 and wls or gpc4 yielded new EP morphology different than those previously observed in either dact1/2, wls, gpc4, or any other mutant is important, suggesting that there are distinct roles for each of these genes contributing to facial morphology, that is not explained by CE defect alone.   

Comment: I encourage the authors to explore ways to test whether the rod-like ethmoid of dact1/2 mutants is more than a secondary effect of the CE failure of the head mesoderm during gastrulation. Without this evidence, the phenotypes of dact1/2 -gpc4 or - wls are not going to convince us that these factors actually interact.  

Actually, we find our results to support the hypothesis that the ethmoid of the dact1/2 mutants is a secondary effect of defective gastrulation and anterior extension of the body axis. However, our findings suggest (by contrasting to another mutant with impaired CE during gastrulation) that this CE defect alone cannot explain the dysmorphic ethmoid plate. Our single-cell RNA seq results and the discovery of dysregulated capn8 expression and proteolytic processes presents new wnt-regulated mechanisms for axis extension.    

p. 20 Discussion

Comment: "Here we show that dact1 and dact2 are required for axis extension during gastrulation and show a new example of CE defects during gastrulation associated with craniofacial defects."

Waxman et al. (2004) previously showed that dact2 is involved in CE during gastrulation.

Heisenberg et al. (1997, 2000), previously showed with the slb mutant how a CE defect during gastrulation causes a craniofacial defect.  

The Waxman paper using morpholino to disrupt dact2 is produced limited analysis of CE and no analysis of craniofacial morphogenesis. We generated genetic mutants here to validate the earlier morpholino results and to analyze the craniofacial phenotype in detail. We have removed the word “new” to make the statement more clear (Line 475).

Comment: "Our data supports the hypothesis that CE gastrulation defects are not causal to the craniofacial defect of medially displaced eyes and midfacial hypoplasia and that an additional morphological process is disrupted."

It is unclear to me how the authors reached this conclusion. I find the view that medially displaced eyes and midfacial hypoplasia are secondary to the CE gastrulation defects unchallenged by the data presented. 

This statement was removed and the discussion was reworded.

Comment: The discussion should include a detailed comparison of this study's findings with those of zebrafish morpholino studies.  

We have added more discussion to compare ours to the previous morpholino findings (Lines 476-484).

Comment: The discussion should try to reconcile the different expression patterns of dact1 and dact2, and the functional redundancy suggested by the absence of phenotype of single mutants. Genetic compensation should be considered (and perhaps tested).  

The different expression patterns of dact1 and dact2 along with our finding that dact1 and dact2 genetic deficiency differently affect the gpc4 mutant phenotype suggest that dact1 and dact2 are not functionally redundant during normal development. This is in line with the previously published data showing different phenotypes of dact1 or dact2 knockdown. However, our results that genetic ablation of both dact1 and dact2 are required for a mutant phenotype suggests that these genes can compensate upon loss of the other. This would suggest then that the expression pattern of dact1 would be changed in the dact2 mutant and visa versa. We find that this line of investigation would be interesting in future studies. We have addressed this in the Discussion (Lines 485498).

Comment: "Based on the data...Conversely, we propose...ascribed to wnt11f2 "

Functional data always prevail overexpression data for inferring functional requirements.  

This is true.

p.21

Comment: "Our results underscore the crucial roles of dact1 and dact2 in embryonic development, specifically in the connection between CE during gastrulation and ultimate craniofacial development."

How is this novel in light of previous studies, especially by Waxman et al. (2004) and Heisenberg et al. (1997, 2000). In this study, the authors fail to present compelling evidence that craniofacial defects are not secondary to the early gastrulation defects resulting from dact1/2 mutations.  p. 22

We have not claimed that the craniofacial defects are not secondary to the gastrulation defects. In fact, we state that there is a “connection”. Further, we do not claim that this is the first or only such finding. We believe our findings have validated the previous dact morpholino experiments and have contributed to the body of literature concerning wnt signaling during embryogenesis. 

Comment: The section on Smad1 discusses a result not reported in the results section. Any data discussed in the discussion section needs to be reported first in the results section.  

We have added a comment on the differential expression of smad1 to the results section (Lines 446-448).

This section to me is the framework of what data is that will launch into doing cool things this semester definitely things to revisit for sure to get a better understanding if we don’t read it all the way through the first time

Welcome back.

In this lesson, I want to talk about S3 replication, the feature which allows you to configure the replication of objects between a source and destination S3 bucket.

Now there are two types of replication supported by S3.

The first type, which has been available for some time, is cross-region replication or CRR, and that allows the replication of objects from source buckets to one or more destination buckets in different AWS regions.

The second type of replication announced more recently is same-region replication or SRR, which as the name suggests is the same process, but where both the source and destination buckets are in the same AWS region.

Now the architecture for both types of replication is pretty simple to understand once you've seen it visually.

It only differs depending on whether the buckets are in the same AWS accounts or different AWS accounts.

Both types of replication support both, so the buckets could be in the same or different AWS accounts.

In both cases, replication configuration is applied to the source bucket.

The replication configuration configures S3 to replicate from the source bucket to a destination bucket, and it specifies a few important things.

The first is logically the destination bucket to use as part of that replication, and another thing that's configured in the replication configuration is an IAM role to use for the replication process.

The role is configured to allow the S3 service to assume it, so that's defined in its trust policy.

The role's permissions policy gives it the permission to read objects on the source bucket and permissions to replicate those objects to the destination bucket.

And this is how replication is configured between source and destination buckets, and of course that replication is encrypted.

Now the configuration does define a few other items, but I'll talk about them on the next screen for now, and just focusing on this basic architecture.

There is one crucial difference between replication which occurs in the same AWS accounts versus different AWS accounts.

Inside one account, both S3 buckets are owned by the same AWS account, so they both trust that same AWS account that they're in.

That means that they both trust IAM as a service, which means that they both trust the IAM role.

For the same account, that means that the IAM role automatically has access to the source and the destination buckets as long as the role's permission policy grants the access.

If you're configuring replication between different AWS accounts, though, that's nothing off.

The destination bucket, because it's in a different AWS account, doesn't trust the source account or the role that's used to replicate the bucket contents.

So in different accounts, remember that the role that's configured to perform the replication isn't by default trusted by the destination account because it's a separate AWS account.

So if you're configuring this replication between different accounts, there's also a requirement to add a bucket policy on the destination bucket, which allows the role in the source account to replicate objects into it.

So you're using a bucket policy, which is a resource policy, to define the role in a separate account can write or replicate objects into that bucket.

Once this configuration is applied, so either the top configuration is the same account or the bottom, if it's different accounts, then S3 can perform the replication.

Now let's quickly review some of the options available for replication configuration, so that might actually come in handy for you to know.

The first important option is what to replicate.

The default is to replicate an entire source bucket to a destination bucket, so all objects, all prefixes, and all packs.

You can, though, choose a subset of objects, so you can create a rule that has a filter, and the filter can filter objects by prefix or tags or a combination of both, and that can define exactly what objects are replicated from the source to the destination.

You can also select which storage class the objects in the destination bucket will use.

Now the default is to use the same class, but you can pick a cheaper class if this is going to be a secondary copy of data.

Remember when I talked about the storage classes that are available in S3, I talked about infrequent access or one-zone infrequent access classes, which could be used for secondary data.

So with secondary data, you're able to tolerate a lower level of resiliency, so we could use one-zone infrequent access for the destination bucket objects, and we can do that because we've always got this primary copy in the source bucket, so we can achieve better economies by using a lower cost storage class in the destination.

So remember this is the example, the default is to use the same storage class on the destination as is used on the source, but you can override that in the replication configuration.

Now you can also define the ownership of the objects in the destination bucket.

The default is that they will be owned by the same account as the source bucket.

Now this is fine if both buckets are inside the same account.

That will mean that objects in the destination bucket will be owned by the same as the source bucket, which is the same account, so that's all good.

However, if the buckets are in different accounts, then by default the objects inside the destination bucket will be owned by the source bucket account, and that could mean when you end up in a situation where the destination account can't read those objects because they're owned by a different AWS account.

So with this option you can override that and you can set it so that anything created in the destination bucket is owned by the destination account.

And lastly there's an extra feature that can be enabled called replication time control or RTC, and this is a feature which adds a guaranteed 15-minute replication SLA onto this process.

Now without this, it's a best efforts process, but RTC adds this SLA, it's a guaranteed level of predictability, and it even adds additional monitoring so you can see which objects are queued for replication.

So this is something that you would tend to only use if you've got a really strict set of requirements from your business, and make sure that the destination bucket and source buckets are in sync as closely as possible.

If you don't require this, if this is just performing backups or it's just for a personal project, or if the source and destination buckets aren't required to always be in sync within this 15-minute window, then it's probably not worth adding this feature.

It's something to keep in mind and be aware of for the exam.

If you do see any questions that mention 15 minutes for replication, then you know that you need this replication time control.

Now there are some considerations that you should be aware of, especially for the exam.

These will come up in the exam, so please pay attention and try to remember these points.

The first thing is that by default replication isn't retroactive.

You enable replication on a pair of buckets of source and destination, and only from that point onward are objects replicated from source to destination.

So if you enable replication on a bucket which already has objects, those objects will not be replicated.

And related to this, in order to enable replication on a bucket, both the source and destination bucket need to have versioning enabled.

You'll be allowed to enable versioning as part of the process of enabling replication, but it is a requirement to have it on, so a bucket cannot be enabled for replication without versioning.

Now you can use S3 batch replication to replicate existing objects, but this is something that you need to specifically configure.

If you don't, just remember by default replication is not retroactive.

Secondly, it is a one-way replication process only.

Objects are replicated from the source to the destination.

If you add objects manually in the destination, they will not be replicated back to the source.

This is not a bi-directional replication.

It's one-way only.

Now more recently, AWS did add the feature which allows you to add bi-directional replication, but just be aware that this is an additional setting which you need to configure.

By default, replication is one-way, and enabling bi-directional replication is something you need to specifically configure, so keep that in mind.

Now in terms of what does get replicated from source to destination, replication is capable of handling objects which are unencrypted, so if you don't have any encryption on an object, it's capable of handling objects which are encrypted using S3 and S3, and it's even capable of handling objects which are encrypted using S3 and KMS, but this is an extra piece of configuration that you'll need to enable.

So there's configuration and there's extra permissions which are required, because of course KMS is emolved.

Now more recently, AWS did add the ability to replicate objects encrypted with SSE-C, so that's server-side encryption with custom-managed keys, but this is a relatively recent addition.

Historically, SSE-C was incompatible with cross or same-region replication.

Replication also requires that the owner of the source bucket needs permissions on the objects which will replicate, so in most cases, if you create a bucket in an account and you add those objects, then the owner of the object will be the source account, but if you grant cross-account access to a bucket, if you add a resource policy allowing all the AWS accounts to create objects in a bucket, it's possible that the source bucket account will not own some of those objects, and the style of replication can only replicate objects where the source account owns those objects.

So keep that in mind, and the limitation is it will not replicate system events, so if any changes are made in the source bucket by lifecycle management, they will not be replicated to the destination bucket, so only user events are replicated, and in addition to that, it can't replicate any objects inside a bucket that are using the Belysia or Glacia Deep Archive storage classes.

Now that makes sense, because Glacia and Belysia Deep Archive, while they are shown as being inside an S3 bucket, you need to conceptually think of them as separate storage products, so they cannot be replicated using this process.

And then lastly, it's important to understand that by default, deletes are not replicated between buckets, so the adding of a delete marker, which is how object deletions are handled for a version-enabled bucket, by default, these delete markers are not replicated.

Now you can enable that, but you need to be aware that by default, this isn't enabled.

So one of the important things I need to make sure you're aware of in terms of replication is why you would use replication.

What are some of the scenarios that you'll use replication for?

So for the same region replication specifically, you might use this process for log aggregation, so if you've got multiple different S3 buckets which store logs for different systems, then you could use this to aggregate those logs into a single S3 bucket.

You might want to use the same region replication to configure some sort of synchronization between production and test accounts.

Maybe you want to replicate data from prod to test periodically, or maybe you want to replicate some testing data into your prod account.

This can be configured in either direction, but a very common use case for same region replication is this replication between different AWS accounts, different functions, so prod and test, or different functional teams within your business.

You might want to use same region replication to implement resilience if you have strict sovereignty requirements.

So there are companies in certain sectors which cannot have data leaving a specific AWS region because of sovereignty requirements, so you can have same region replication replicating between different buckets and different accounts, and then you have this account isolation for that data.

So having a separate account with separate logins isolated to make an audit team or a security team replicates it into that account, it provides this account level isolation.

Obviously, if you don't have those sovereignty requirements, then you can use cross region replication and use replication to implement global resilience improvements, so you can have backups of your data copied to different AWS regions to cope with large scale failure.

You can also replicate data into different regions to reduce latency.

So if you have, for example, a web application or your application loads data, then obviously it might be latency sensitive, so you can replicate data from one AWS region to another, so the customers in that remote region can access the bucket that's closest to them, and that reduces latency generally gives them better performance.

Now that is everything I want to cover in this video, so go ahead and complete the video, and when you're ready, I look forward to you joining me in the next.

Companies, half of an anecdotal sample of some 20 US CDOs, have turned Copilot off / restricting it strongly. This as it surfaces info in summaries etc that employees would not have direct access to. No access security connection between Copilot and results. So data governance is blocking its roll-out.

for - question - what would Federic Faggin think of this? - question - multi-scale communication - question - are Tibetan Rainbow body and knowing time of death examples of multi-scale communications? question - what would Federic Faggin think of this? - He comes from an experiential perspective, not just an intellectual one.

question - what would Federic Faggin think of this? - I don't think Michael Levin provides a satisfactory answer to this and this is related to the meaning crisis modernity finds itself in - when traditional religions no longer suffice, - but there is nothing in modernity that can fill the gap yet, if mortality salience is a big issue - I don't think an intellectual answer can meet the needs of people suffering in the meaning crisis, although it is necessary, it is not sufficient - I think they are after some kind of nonverbal, nondual transformative experience

question - multi-scale communication - This is also a question about multi-scale communication - I've recently used a metaphor to compare - the unitary, monatic experience of consciousness to - an elected government - The trillions of cells "elect" consciousness" as the high level government to oversea them - but we seem to be in the situation of the government being out of touch with the citizens - At one time in our history, was it common to be able for - high level consciousness to communicate directly with - low level cells and subcellular structures? - If so, why has this practice disappeared and - how can we re-establish it?

question - Are Tibetan Rainbow body and knowing time of death examples of multi-scale communications? - In some older spiritual traditions such as found in the East, it seems deep meditative practitioners are able to achieve a degree of communications with parts of their body that is unconventional and surprising to modern researchers - For example, Tibetan meditators report of having the abiity to predict the time of their death by recognizing subtle bodily, interoceptive signals - Rare instances also occur of the Rainbow Body, when great meditators in the Dzogchen tradition whose body at time of death can disappear in a body of light

Excellent point

The extension policy is fair and reasonable. I agree that it's important for both the student and the professor to be organized and informed in advance. This also allows both to benefit and maintain a good relationship. Particularly, I wanted to thank the extension granted due to my flight cancellation!

Opposing view against the teaching values professors have installed to students.

-to desire a life with work and no enjoyment cannot even be considered living at all. Further they cannot live to a full potential in thinking in such a linear way

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

In this study, the authors used a stopped-flow method to investigate the kinetics of substrate translocation through the channel in hexameric ClpB, an ATP-dependent bacterial protein disaggregase. They engineered a series of polypeptides with the N-terminal RepA ClpB-targeting sequence followed by a variable number of folded titin domains. The authors detected translocation of the substrate polypeptides by observing the enhancement of fluorescence from a probe located at the substrate's C-terminus. The total time of the substrates' translocation correlated with their lengths, which allowed the authors to determine the number of residues translocated by ClpB per unit time.

Strengths:

This study confirms a previously proposed model of processive translocation of polypeptides through the channel in ClpB. The novelty of this work is in the clever design of a series of kinetic experiments with an engineered substrate that includes stably folded domains. This approach produced a quantitative description of the reaction rates and kinetic step sizes. Another valuable aspect is that the method can be used for other translocases from the AAA+ family to characterize their mechanism of substrate processing.

Weaknesses:

The main limitation of the study is in using a single non-physiological substrate of ClpB, which does not replicate the physical properties of the aggregated cellular proteins and includes a non-physiological ClpB-targeting sequence. Another limitation is in the use of ATPgammaS to stimulate the substrate processing. It is not clear how relevant the results are to the ClpB function in living cells with ATP as the source of energy, a multitude of various aggregated substrates without targeting sequences that need ClpB's assistance, and in the presence of the co-chaperones.

Indeed, we agree that our RepA-Titinx substrates are not aggregates but are model, soluble, substrates used to reveal information about enzyme catalyzed protein unfolding and translocation.  Our substrates are similar to RepA-GFP and GFP-SsrA used by multiple labs including Wickner, Horwich, Sauer, Baker, Shorter, Bukua, to name only a few.  The fact that “this is what everyone does” does not make the substrates physiological or the most ideal. However, this is the technology we currently have until we and others develop something better. In the meantime, we contend that  the results presented here do advance our knowledge on enzyme catalyzed protein unfolding

Part of what this manuscript seeks to accomplish is presenting the development of a single-turnover experiment that reports on processive protein unfolding by AAA+ molecular motors, in this case, ClpB.  Importantly, we are treating translocation on an unfolded polypeptide chain and protein unfolding of stably folded proteins as two distinct reactions catalyzed by ClpB. If these functions are used to disrupt protein aggregates, in vivo, then this remains to be seen.

We contend that processive ClpB catalyzed protein unfolding has not been rigorously demonstrated prior to our results presented here.  Avellaneda et al mechanically unfolded their substrate before loading ClpB (Avellaneda, Franke, Sunderlikova et al. 2020).  Thus, their experiment represents valuable observations reflecting polypeptide translocation on a pre-unfolded protein.  Our previous work using single-turnover stopped-flow experiments employed unstructured synthetic polypeptides and therefore reflects polypeptide translocation and not protein unfolding (Li, Weaver, Lin et al. 2015).  Weibezahn et al used unstructured substrates in their study with ClpB (BAP/ClpP), and thus their results represent translocation of a pre-unfolded polypeptide and not enzyme catalyzed protein unfolding (Weibezahn, Tessarz, Schlieker et al. 2004). 

Many studies have reported the use of  GFP with tags or RepA-GFP and used the loss of GFP fluorescence to conclude protein unfolding.  However, such results do not reveal if ClpB processively and fully translocates the substrate through its axial channel.  One cannot rule out, even when trapping with “GroEL trap”, the possibility that ClpB only needs to disrupt some of the fold in GFP before cooperative unfolding occurs leading to loss of fluorescence.  Once the cooperative collapse of the structure occurs and fluorescence is lost it has not been shown that ClpB will continue to translocate on the newly unfolded chain or dissociate. In fact, the Bukau group showed that folded YFP remained intact after luciferase was unfolded (Haslberger, Zdanowicz, Brand et al. 2008).  Our approach, reported here, yields signal upon arrival of the motor at the c-terminus or within the PIFE distance thus we can be certain that the motor does arrive at the c-terminus after unfolding up to three tandem repeats of the Titin I27 domain.

ATPgS is a non-physiological nucleotide analog.  However, ClpB has been shown to exhibit curious behavior in its presence that we and others, as the reviewer acknowledges, do not fully understand (Doyle, Shorter, Zolkiewski et al. 2007).  Some of the experiments reported here are seeking to better understand that fact.  Here we have shown that ATPgS alone will support processive protein unfolding. With this assay in hand, we are now seeking to go forward and address many of the points raised by this reviewer. 

The authors do not attempt to correlate the kinetic step sizes detected during substrate translocation and unfolding with the substrate's structure, which should be possible, given how extensively the stability and unfolding of the titin I27 domain were studied before. Also, since the substrate contains up to three I27 domains separated with unstructured linkers, it is not clear why all the translocation steps are assumed to occur with the same rate constant.

We assume that all protein unfolding steps occur with the same rate constant, ku.  We conclude that we are not detecting the translocation rate constant, kt, as our results support a model where kt is much faster than ku.  We do think it makes sense that the same slow step occurs between each cycle of protein unfolding.

We have added a discussion relating our observations to mechanical unfolding of tandem repeats of Titin I27 from AFM experiments  (Oberhauser, Hansma, Carrion-Vazquez and Fernandez 2001). Most interestingly, they report unfolding of Titin I27 in 22 nm steps.  Using 0.34 nm per amino acids this yields ~65 amino acids per unfolding step, which is comparable to our kinetic step-size of 57 – 58 amino acids per step.

Some conclusions presented in the manuscript are speculative:

The notion that the emission from Alexa Fluor 555 is enhanced when ClpB approaches the substrate's C-terminus needs to be supported experimentally. Also, evidence that ATPgammaS without ATP can provide sufficient energy for substrate translocation and unfolding is missing in the paper.

In our previous work we have used fluorescently labeled 50 amino acid peptides as substrates to examine ClpB binding (Li, Lin and Lucius 2015, Li, Weaver, Lin et al. 2015).  In that work we have used fluorescein, which exhibits quenching upon ClpB binding.  We have added a control experiment where we have attached alexa fluor 555 to the 50 amino acid substrate so we can be assured the ClpB binds close to the fluorophore.  As seen in supplemental Fig. 1 A  upon titration with ClpB, in the presence of ATPγS, we observe an increase in fluorescence from AF555, consistent with PIFE.  Supplemental Fig. 1 B shows the relative fluorescence enhancement at the peak max increases up to ~ 0.2 or a 20 % increase in fluorescence, due to PIFE, upon ClpB binding.   

Further, peak time is our hypothesized measure of ClpB’s arrival at the dye. Our results indicate that the peak time linearly increases as a function of an increase in the number of folded TitinI27 repeats in the substrates which also supports the PIFE hypothesis. Finally, others have shown that AF555 exhibits PIFE and we have added those references.

The evidence that ATPγS alone can support translocation is shown in Fig. 2 and supplemental Figure 1.  Fig. 2 and supplemental Figure 1 are two different mixing strategies where we use only ATPgS and no ATP at all.  In both cases the time courses are consistent with processive protein unfolding by ClpB with only ATPγS.

Reviewer #2 (Public Review):

Summary:

The current work by Banwait et al. reports a fluorescence-based single turnover method based on protein-induced fluorescence enhancement (PIFE) to show that ClpB is a processive motor. The paper is a crucial finding as there has been ambiguity on whether ClpB is a processive or non-processive motor. Optical tweezers-based single-molecule studies have shown that ClpB is a processive motor, whereas previous studies from the same group hypothesized it to be a non-processive motor. As co-chaperones are needed for the motor activity of the ClpB, to isolate the activity of ClpB, they have used a 1:1 ratio ATP and ATPgS, where the enzyme is active even in the absence of its co-chaperones, as previously observed. A sequential mixing stop-flow protocol was developed, and the unfolding and translocation of RepA-TitinX, X = 1,2,3 repeats was monitored by measuring the fluorescence intensity with the time of Alexa F555 which was labelled at the C-terminal Cysteine. The observations were a lag time, followed by a gradual increase in fluorescence due to PIFE, and then a decrease in fluorescence plausibly due to the dissociation from the substrate allowing it to refold. The authors observed that the peak time depends on the substrate length, indicating the processive nature of ClpB. In addition, the lag and peak times depend on the pre-incubation time with ATPgS, indicating that the enzyme translocates on the substrates even with just ATPgS without the addition of ATP, which is plausible due to the slow hydrolysis of ATPgS. From the plot of substrate length vs peak time, the authors calculated the rate of unfolding and translocation to be ~0.1 aas-1 in the presence of ~1 mM ATPgS and increases to 1 aas-1 in the presence of 1:1 ATP and ATPgS. The authors have further performed experiments at 3:1 ATP and ATPgS concentrations and observed ~5 times increase in the translocation rates as expected due to faster hydrolysis of ATP by ClpB and reconfirming that processivity is majorly ATP driven. Further, the authors model their results to multiple sequential unfolding steps, determining the rate of unfolding and the number of amino acids unfolded during each step. Overall, the study uses a novel method to reconfirm the processive nature of ClpB.

Strengths:

(1) Previous studies on understanding the processivity of ClpB have primarily focused on unfolded or disordered proteins; this study paves new insights into our understanding of the processing of folded proteins by ClpB. They have cleverly used RepA as a recognition sequence to understand the unfolding of titin-I27 folded domains.

(2) The method developed can be applied to many disaggregating enzymes and has broader significance.

(3) The data from various experiments are consistent with each other, indicating the reproducibility of the data. For example, the rate of translocation in the presence of ATPgS, ~0.1 aas-1 from the single mixing experiment and double mixing experiment are very similar.

(4) The study convincingly shows that ClpB is a processive motor, which has long been debated, describing its activity in the presence of only ATPgS and a mixture of ATP and ATPgS.

(5) The discussion part has been written in a way that describes many previous experiments from various groups supporting the processive nature of the enzyme and supports their current study.

Weaknesses:

(1) The authors model that the enzyme unfolds the protein sequentially around 60 aa each time through multiple steps and translocates rapidly. This contradicts our knowledge of protein unfolding, which is generally cooperative, particularly for titinI27, which is reported to unfold cooperatively or utmost through one intermediate during enzymatic unfolding by ClpX and ClpA.

We do not think this represents a contradiction.  In fact, our observations are in good agreement with mechanical unfolding of tandem repeats of Titin I27 using AFM experiments (Oberhauser, Hansma, Carrion-Vazquez and Fernandez 2001).  They showed that tandem repeats of TitinI27 unfolded in steps of ~22 nm.  Dividing 22 nm by 0.34 nm/Amino Acid gives ~65 amino acids per unfolding event.  This implies that, under force, ~65 amino acids of folded structure unfolds in a single step.  This number is in excellent agreement with our kinetic step-size of 65 AA/step. 

Importantly, the experiments cited by the reviewer on ClpA and ClpX are actually with ClpAP and ClpXP.  We assert that this is an important distinction as we have shown that ClpA employs a different mechanism than ClpAP (Rajendar and Lucius 2010, Miller, Lin, Li and Lucius 2013, Miller and Lucius 2014).  Thus, ClpA and ClpAP should be treated as different enzymes but, without question, ClpB and ClpA are different enzymes.

(2) It is also important to note that the unfolding of titinI27 from the N-terminus (as done in this study) has been reported to be very fast and cannot be the rate-limiting step as reported earlier(Olivares et al, PNAS, 2017). This contradicts the current model where unfolding is the rate-limiting step, and the translocation is assumed to be many orders faster than unfolding.

Most importantly, the Olivares paper is examining ClpXP and ClpAP catalyzed protein unfolding and translocation and not ClpB.  These are different enzymes.  Additionally, we have shown that ClpAP and ClpA translocate unfolded polypeptides with different rates, rate constants, and kinetic step-sizes indicating that ClpP allosterically impacts the mechanism employed by ClpA to the extent that even ClpA and ClpAP should be considered different enzymes (Rajendar and Lucius 2010, Miller, Lin, Li and Lucius 2013).  We would further assert that there is no reason to assume ClpAP and ClpXP would catalyze protein unfolding using the same mechanism as ClpB as we do not think it should be assumed ClpA and ClpX use the same mechanism as ClpAP and ClpXP, respectively. 

The Olivares et al paper reports a dwell time preceding protein unfolding of ~0.9 and ~0.8 s for ClpXP and ClpAP, respectively.   The inverse of this can be taken as the rate constant for protein unfolding and would yield a rate constant of ~1.2 s-1, which is in good agreement with our observed rate constant of 0.9 – 4.3 s-1 depending on the ATP:ATPγS mixing ratio.  For ClpB, we propose that the slow unfolding is then followed by rapid translocation on the unfolded chain where translocation by ClpB must be much faster than for ClpAP and ClpXP.  We think this is a reasonable interpretation of our results and not a contradiction of the results in Olivares et al. Moreover, this is completely consistent with the mechanistic differences that we have reported, using the same single-turnover stopped flow approach on the same unfolded polypeptide chains with ClpB, ClpA, and ClpAP (Rajendar and Lucius 2010, Miller, Lin, Li and Lucius 2013, Miller and Lucius 2014, Li, Weaver, Lin et al. 2015).

(3) The model assumes the same time constant for all the unfolding steps irrespective of the secondary structural interactions.

Yes, we contend that this is a good assumption because it represents repetition of protein unfolding catalyzed by ClpB upon encountering the same repeating structural elements, i.e. Beta sheets. 

(4) Unlike other single-molecule optical tweezer-based assays, the study cannot distinguish the unfolding and translocation events and assumes that unfolding is the rate-limiting step.

Although we cannot, directly, distinguish between protein unfolding and translocation we have logically concluded that protein unfolding is likely rate limiting. This is because the large kinetic step-size represents the collapse of ~60 amino acids of structure between two rate-limiting steps, which we interpret to represent cooperative protein unfolding induced by ClpB.  It is not an assumption it is our current best interpretation of the observations that we are now seeking to further test. 

Reviewer #3 (Public Review):

Summary:

The authors have devised an elegant stopped-flow fluorescence approach to probe the mechanism of action of the Hsp100 protein unfoldase ClpB on an unfolded substrate (RepA) coupled to 1-3 repeats of a folded titin domain. They provide useful new insight into the kinetics of ClpB action. The results support their conclusions for the model setup used.

Strengths:

The stopped-flow fluorescence method with a variable delay after mixing the reactants is informative, as is the use of variable numbers of folded domains to probe the unfolding steps.

Weaknesses:

The setup does not reflect the physiological setting for ClpB action. A mixture of ATP and ATPgammaS is used to activate ClpB without the need for its co-chaperones, Hsp70. Hsp40 and an Hsp70 nucleotide exchange factor. This nucleotide strategy was discovered by Doyle et al (2007) but the mechanism of action is not fully understood. Other authors have used different approaches. As mentioned by the authors, Weibezahn et al used a construct coupled to the ClpA protease to demonstrate translocation. Avellaneda et al used a mutant (Y503D) in the coiled-coil regulatory domain to bypass the Hsp70 system. These differences complicate comparisons of rates and step sizes with previous work. It is unclear which results, if any, reflect the in vivo action of ClpB on the disassembly of aggregates.

We agree with the reviewer, there are several strategies that have been employed to bypass the need for Hsp70/40 or KJE to simplify in vitro experiments.  Here we have developed a first of its kind transient state kinetics approach that can be used to examine processive protein unfolding.  We now seek to go forward with examining the mechanisms of hyperactive mutants, like Y503D, and add the co-chaperones so that we can address the limitations articulated by the reviewer.   In fact we already began adding DnaK to the reaction and found that DnaK induced ClpB to release the polypeptide chain (Durie, Duran and Lucius 2018).  However, the sequential mixing strategy developed here was needed to go forward with examining the impact of co-chaperones. 

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Line 1: I recommend changing the title of the paper to remove the terms that are not clearly defined in the text: "robust" and "processive". What are the Authors' criteria for describing a molecular machine as "robust" vs. "not robust"? A definition of processivity is given in equation 2, but its value for ClpB is not reported in the text, and the criteria for classifying a machine as "processive" vs. "non-processive" are not included. Besides, the Authors have previously reported that ClpB is non-processive (Biochem. J., 2015), so it is now clear that a more nuanced terminology should be applied to this protein. Also, Escherichia coli should be fully spelled out in the title.

The title has been changed.  We have removed “robust” as we agree with the reviewer, there is no way to quantify “robust”.  However, we have kept “processive” and have added to the discussion a calculation of processivity since we can quantify processivity.  Importantly, the unstructured substrates used in our previous studies represent translocation and not protein unfolding.  here, on folded substrates, we detect rate-limiting protein unfolding followed by rapid translocation.  Thus, we report a lower bound on protein unfolding processivity of 362 amino acids. 

Line 20: The comment about mitochondrial SKD3 should be removed. SKD3, like ClpB, belongs to the AAA+ family, and it is simply a coincidence that the original study that discovered SKD3 termed it an Hsp100 homolog. The similarity between SKD3 and ClpB is limited to the AAA+ module, so there are many other metazoan ATPases, besides SKD3, that could be called homologs of ClpB, including mitochondrial ClpX, ER-localized torsins, p97, etc.

Removed.

Lines 133-139. Contrary to what the authors state, it is not clear that the "lag-phase" becomes significantly shorter for subsequent mixing experiments (Figure 1E) perhaps except for the last one (2070 s). It is clear, however, that the emission enhancement becomes stronger for later mixes. This effect should be discussed and explained, as it suggests that the pre-equilibrations shorter than ~2000 sec do not produce saturation of ClpB binding to the substrate.

We have added supplemental figure 2, which represents a zoom into the lag region.  This better illustrates what we were seeing but did not clearly show to the reader.  In addition, we address all three changes in the time courses, i.e. extend of lag, change in peak position, and the change in peak height. 

Line 175. The hydrolysis rate of ATPgammaS in the presence of ClpB should be measured and compared to the hydrolysis rate with ATP/ATPgammaS to check if the ratio of those rates agrees with the ratio of the translocation rates. These experiments should be performed with and without the RepA-titin substrate, which could reveal an important linkage between the ATPase engine and substrate translocation. These experiments are essential to support the claim of substrate translocation and unfolding with ATPgammaS as the sole energy source.

The time courses shown in figure 2 and supplemental Figure 1 are collected with only ATPgS and no ATP.  The time courses show a clear increase in lag and appearance of a peak with increasing number of tandem repeats of titin domains.  We do not see an alternate explanation for this observation other than ATPγS supports ClpB catalyzed protein unfolding and translocation.  What is the reviewers alternate explanation for these observations?

We agree with the reviewer that the linkage of ATP hydrolysis to protein unfolding and translocation is essential and we are seeking to acquire this knowledge.  However, a simple comparison of the ratio of rates is not adequate. We contend that a complete mechanistic study of ATP turnover by ClpB is required to properly address this linkage and such a study is too substantial to be included here but is currently underway. 

All that said, the statement on line 175 was removed since we do not report any ATPase measurements in this paper.

Line 199: It is an over-simplification to state that "1:1 mix of ATP to ATPgammaS replaces the need for co-chaperones". This sentence should be corrected or removed. The ClpB co-chaperones (DnaK, DnaJ, GrpE) play a major role in targeting ClpB to its aggregated substrates in cells and in regulating the ClpB activity through interactions with its middle domain. ATPgammaS does not replace the co-chaperones; it is a chemical probe that modifies the mechanism of ClpB in a way that is not entirely understood.

We agree with the reviewer.  The sentence has been modified to point out that the mix of ATP and ATPγS activates ClpB.

Figure 3B, Supplementary Figure 5A. The solid lines from the model fit cannot be distinguished from the data points. Please modify the figures' format to clearly show the fits and the data points.

Done.

Lines 326, 329. It is not clear why the authors mention a lack of covalent modification of substrates by ClpB. AAA+ ATPases do not produce covalent modifications of their substrates.

The issue of covalent modification was presented in the introduction lines 55 – 60 pointing out that much of what we have learned about protein unfolding and translocation catalyzed by ClpA and ClpX is from the observations of proteolytic degradation catalyzed by the associated protease ClpP.  However, this approach is not possible for ClpB/Hsp104 as these motors do not associate with a protease unless they have been artificially engineered to do so. 

Lines 396-399. I am puzzled why the authors try to correlate the size of the detected kinetic step with the length of the ClpB channel instead of the size characteristics of the substrate.

We are attempting to discuss/rationalize the observed large kinetic step-size which, in part, is defined by the structural properties of the enzyme as well as the size characteristics of the substrate.  We have attempted to clarify this and better discuss the properties of the substrate as well as ClpB.

As I mentioned in the Public Review, it is essential to demonstrate that the emission increase used as the only readout of the ClpB position along the substrate is indeed caused by the proximity of ClpB to the fluorophore. One way to accomplish that would be to place the fluorophore upstream from the first I27 domain and determine if the "lag phase" in the emission enhancement disappears.

Alexa Fluor 555 is well established to exhibit PIFE.  However, as in the response to the public review, we have included an appropriate control showing this in supplemental Fig. 1.

Finally, the authors repetitively place their results in opposition to the study of Weibezahn et al. published in 2004 which first demonstrated substrate translocation by engineering a peptidase-associated variant of ClpB. It should be noted that the field of protein disaggregases has moved since the time of that publication from the initial "from-start-to-end" translocation model to a more nuanced picture of partial translocation of polypeptide loops with possible substrate slipping through the ClpB channel and a dynamic assembly of ClpB hexamers with possible subunit exchange, all of which may affect the kinetics in a complex way. However, the present study confirmed the "start-to-end" translocation model, albeit for a non-physiological ClpB substrate, and that is the take-home message, which should be included in the text.

It is not clear to us that the field has “moved on” since Weibezahn et al 2004.  Their engineered construct that they term “BAP” with ClpP is still used in the field despite us reporting that proteolytic degradation is observed in the absence of ATP with that system  (Li, Weaver, Lin et al. 2015) and should, therefore, not be used to conclude processive energy driven translocation. The “partial translocation” by ClpB is also grounded in observations of partial degradation catalyzed by ClpP with BAP from the same group (Haslberger, Zdanowicz, Brand et al. 2008). It is not clear to us that the idea of subunit exchange leading to the possibility of assembly around internal sequences is being considered.  We do agree that this is an important mechanistic possibility that needs further interrogation. We agree with the reviewer, all these factors are confounding and lead to a more nuanced view of the mechanism.

All that said, we have removed some of the opposition in the discussion.

Reviewer #2 (Recommendations For The Authors):

(1) It is assumed that the lag phase will be much longer than the phase in which we see a gradual increase in fluorescence, as the effect of PIFE is significant only when the enzyme is very close to the fluorophore. Particularly for RepA-titin3, the enzyme has to translocate many tens of nm before it is closer to the C-terminus fluorophore. However, in all cases, the lag time is lower or similar to the gradual increase phase (for example, Figure 3B). Could the authors explain this?

The extent of the lag, or time zero until the signal starts to increase, is interpreted to indicate the time the motor moves from it’s initial binding site until it gets close enough to the fluorophore that PIFE starts to occur.  In our analysis we apply signal change to the last intermediate and dissociation or release of unfolded RepA-TitinX.  The increase in PIFE is not “all or nothing”.  Rather, it is starting to increase gradually.  Further, because these are ensemble measurements, and each molecule will exhibit variability in rate there is increased breadth of the peak due to ensemble averaging. 

(2) Although the reason for differences in the peak position (for example, Figure 1E, 2B) is apparent, the reason for variations in the relative intensities has to be given or speculated.

We have addressed the reason for the different peak heights in the revised manuscript.  It is the consequence of the fact that each substrate has slightly different fluorescent labeling efficiencies.  Thus, for each sample there is a mix of labeled and unlabeled substrates both of which will bind to ClpB but the unlabeled ClpB bound substrates do not contribute to the fluorescence signal, but will represent a binding competitor.  Thus, for low labeling efficiency there is a lower concentration of ClpB bound to fluorescent RepA-Titinx and for higher labeling efficiency there is higher concentration of ClpB bound to RepA-Titinx leading to an increased peak height.  RepA-Titin2 has the highest labeling efficiency and thus the largest peak height.

Reviewer #3 (Recommendations For The Authors):

The authors should make it clear that they and previous authors have used different constructs or conditions to bypass the physiological regulation of ClpB action by Hsp70 and its co-factors as mentioned above. In particular, the construct used by Avellaneda et al should be explained when they challenge the findings of those authors.

Minor points:

The lines fitting the experimental points are difficult or impossible to see in Figures 2B, 3B, and s5B.

Fixed

Typo bottom of p6 - "averge"

Fixed

Avellaneda, M. J., K. B. Franke, V. Sunderlikova, B. Bukau, A. Mogk and S. J. Tans (2020). "Processive extrusion of polypeptide loops by a Hsp100 disaggregase." Nature.

Doyle, S. M., J. Shorter, M. Zolkiewski, J. R. Hoskins, S. Lindquist and S. Wickner (2007). "Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity." Nature structural & molecular biology 14(2): 114-122.

Durie, C. L., E. C. Duran and A. L. Lucius (2018). "Escherichia coli DnaK Allosterically Modulates ClpB between High- and Low-Peptide Affinity States." Biochemistry 57(26): 3665-3675.

Haslberger, T., A. Zdanowicz, I. Brand, J. Kirstein, K. Turgay, A. Mogk and B. Bukau (2008). "Protein disaggregation by the AAA+ chaperone ClpB involves partial threading of looped polypeptide segments." Nat Struct Mol Biol 15(6): 641-650.

Li, T., J. Lin and A. L. Lucius (2015). "Examination of polypeptide substrate specificity for Escherichia coli ClpB." Proteins 83(1): 117-134.

Li, T., C. L. Weaver, J. Lin, E. C. Duran, J. M. Miller and A. L. Lucius (2015). "Escherichia coli ClpB is a non-processive polypeptide translocase." Biochem J 470(1): 39-52.

Miller, J. M., J. Lin, T. Li and A. L. Lucius (2013). "E. coli ClpA Catalyzed Polypeptide Translocation is Allosterically Controlled by the Protease ClpP." Journal of Molecular Biology 425(15): 2795-2812.

Miller, J. M. and A. L. Lucius (2014). "ATP-gamma-S Competes with ATP for Binding at Domain 1 but not Domain 2 during ClpA Catalyzed Polypeptide Translocation." Biophys Chem 185: 58-69.

Oberhauser, A. F., P. K. Hansma, M. Carrion-Vazquez and J. M. Fernandez (2001). "Stepwise unfolding of titin under force-clamp atomic force microscopy." Proc Natl Acad Sci U S A 98(2): 468-472.

Rajendar, B. and A. L. Lucius (2010). "Molecular mechanism of polypeptide translocation catalyzed by the Escherichia coli ClpA protein translocase." J Mol Biol 399(5): 665-679.

Weibezahn, J., P. Tessarz, C. Schlieker, R. Zahn, Z. Maglica, S. Lee, H. Zentgraf, E. U. Weber-Ban, D. A. Dougan, F. T. Tsai, A. Mogk and B. Bukau (2004). "Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB." Cell 119(5): 653-665.

I observe everyone agrees this is an unfortunate scene that had to happen. You can feel what it's their hearts is disappointment and potentially fear. These trials happened all over Protestant Europe, not just in Salem. Learning about the past based on this I understand that everyone had a hard time even suggesting this idea. This statement was made from emotion showing that regardless of the situation. Accusing people of witchcraft and then prosecuting them if found guilty would put a red pin within the township, leaving all people effected even after the trial is over.

Reviewer #1 (Public Review):

Summary:<br /> The authors used multiple approaches to study salt effects in liquid-liquid phase separation (LLPS). Results on both wild-type Caprin1 and mutants and on different types of salts contribute to a comprehensive understanding.

Strengths:<br /> The main strength of this work is the thoroughness of investigation. This aspect is highlighted by the multiple approaches used in the study, and reinforced by the multiple protein variants and different salts studied.

Weaknesses:<br /> (1) The multiple computational approaches are a strength, but they're cruder than explicit-solvent all-atom molecular dynamics (MD) simulations and may miss subtle effects of salts. In particular, all-atom MD simulations demonstrate that high salt strengthens pi-types of interactions (ref. 42 and MacAinsh et al, https://www.biorxiv.org/content/10.1101/2024.05.26.596000v3).

(2) The paper can be improved by distilling the various results into a simple set of conclusions. By example, based on salt effects revealed by all-atom MD simulations, MacAinsh et al. presented a sequence-based predictor for classes of salt dependence. Wild-type Caprin1 fits right into the "high net charge" class, with a high net charge and a high aromatic content, showing no LLPS at 0 NaCl and an increasing tendency of LLPS with increasing NaCl. In contrast, pY-Caprin1 belongs to the "screening" class, with a high level of charged residues and showing a decreasing tendency of LLPS.

(3) Mechanistic interpretations can be further simplified or clarified. (i) Reentrant salt effects (e.g., Fig. 4a) are reported but no simple explanation seems to have been provided. Fig. 4a,b look very similar to what has been reported as strong-attraction promotor and weak-attraction suppressor, respectively (ref. 50; see also PMC5928213 Fig. 2d,b). According to the latter two studies, the "reentrant" behavior of a strong-attraction promotor, CL- in the present case, is due to Cl-mediated attraction at low to medium [NaCl] and repulsion between Cl- ions at high salt. Do the authors agree with this explanation? If not, could they provide another simple physical explanation? (ii) The authors attributed the promotional effect of Cl- to counterion-bridged interchain contacts, based on a single instance. There is another simple explanation, i.e., neutralization of the net charge on Caprin1. The authors should analyze their simulation results to distinguish net charge neutralization and interchain bridging; see MacAinsh et al.

(4) The authors presented ATP-Mg both as a single ion and as two separate ions; there is no explanation of which of the two versions reflects reality. When presenting ATP-Mg as a single ion, it's as though it forms a salt with Na+. I assume NaCl, ATP, and MgCl2 were used in the experiment. Why is Cl- not considered? Related to this point, it looks like ATP is just another salt ion studied and much of the Results section is on NaCl, so the emphasis of ATP ("Diverse Roles of ATP" in the title is somewhat misleading.

Reviewer #3 (Public Review):

Summary:

Cheng, Liu, Dong, et al. demonstrate that anterior endoderm cells can arise from prechordal plate progenitors, which is suggested by pseudo time reanalysis of published scRNAseq data, pseudo time analysis of new scRNAseq data generated from Nodal-stimulated explants, live imaging from sox17:DsRed and Gsc:eGFP transgenics, fluorescent in situ hybridization, and a Cre/Lox system. Early fate mapping studies already suggested that progenitors at the dorsal margin give rise to both of these cell types (Warga) and live imaging from the Heisenberg lab (Sako 2016, Barone 2017) also pretty convincingly showed this. However, the data presented for this point are very nice, and the additional experiments in this manuscript, however, further cement this result. Though better demonstrated by previous work (Alexander 1999, Gritsman 1999, Gritsman 2000, Sako 2016, Rogers 2017, others), the manuscript suggests that high Nodal signaling is required for both cell types, and shows preliminary data that suggests that FGF signaling may also be important in their segregation. The manuscript also presents new single-cell RNAseq data from Nodal-stimulated explants with increased (lft1 KO) or decreased (ndr1 KD) Nodal signaling and multi-omic ATAC+scRNAseq data from wild-type 6 hpf embryos but draws relatively few conclusions from these data. Lastly, the manuscript presents data that SWI/SNF remodelers and Ripply1 may be involved in the anterior endoderm - prechordal plate decision, but these data are less convincing. The SWI/SNF remodeler experiments are unconvincing because the demonstration that these factors are differentially expressed or active between the two cell types is weak. The Ripply1 gain-of-function experiments are unconvincing because they are based on incredibly high overexpression of ripply1 (500 pg or 1000 pg) that generates a phenotype that is not in line with previously demonstrated overexpression studies (with phenotypes from 10-20x lower expression). Similarly, the cut-and-tag data seems low quality and like it doesn't support direct binding of ripply1 to these loci.

In the end, this study provides new details that are likely important in the cell fate decision between the prechordal plate and anterior endoderm; however, it is unclear how Nodal signaling, FGF signaling, and elements of the gene regulatory network (including Gsc, possibly ripply1, and other factors) interact to make the decision. I suggest that this manuscript is of most interest to Nodal signaling or zebrafish germ layer patterning afficionados. While it provides new datasets and observations, it does not weave these into a convincing story to provide a major advance in our understanding of the specification of these cell types.

Major issues:

(1) UMAPs: There are several instances in the manuscript where UMAPs are used incorrectly as support for statements about how transcriptionally similar two populations are. UMAP is a stochastic, non-linear projection for visualization - distances in UMAP cannot be used to determine how transcriptionally similar or dissimilar two groups are. In order to make conclusions about how transcriptionally similar two populations are requires performing calculations either in the gene expression space, or in a linear dimensional reduction space (e.g. PCA, keeping in mind that this will only consider the subset of genes used as input into the PCA). Please correct or remove these instances, which include (but are not limited to):<br /> p.4 107-110<br /> p.4 112<br /> p.8 207-208<br /> p.10 273-275

(2) Nodal and lefty manipulations: The section "Nodal-Lefty regulatory loop is needed for PP and anterior Endo fate specification" and Figure 3 do not draw any significant conclusions. This section presents a LIANA analysis to determine the signals that might be important between prechordal plate and endoderm, but despite the fact that it suggests that BMP, Nodal, FGF, and Wnt signaling might be important, the manuscript just concludes that Nodal signaling is important. Perhaps this is because the conclusion that Nodal signaling is required for the specification of these cell types has been demonstrated in zebrafish in several other studies with more convincing experiments (Alexander 1999, Gritsman 1999, Gritsman 2000, Rogers 2017, Sako 2016). While FGF has recently been demonstrated to be a key player in the stochastic decision to adopt endodermal fate in lateral endoderm (Economou 2022), the idea that FGF signaling may be a key player in the differentiation of these two cell types has strangely been relegated to the discussion and supplement. Lastly, the manuscript does not make clear the advantage of performing experiments to explore the PP-Endo decision in Nodal-stimulated explants compared to data from intact embryos. What would be learned from this and not from an embryo? Since Nodal signaling stimulates the expression of Wnts and FGFs, these data do not test Nodal signaling independent of the other pathways. It is unclear why this artificial system that has some disadvantages is used since the manuscript does not make clear any advantages that it might have had.

(3) ripply1 mRNA injection phenotype inconsistent with previous literature: The phenotype presented in this manuscript from overexpressing ripply1 mRNA (Fig S11) is inconsistent with previous observations. This study shows a much more dramatic phenotype, suggesting that the overexpression may be to a non-physiological level that makes it difficult to interpret the gain-of-function experiments. For instance, Kawamura et al 2005 perform this experiment but do not trigger loss of head and eye structures or loss of tail structures. Similarly, Kawamura et al 2008 repeat the experiment, triggering a mildly more dramatic shortening of the tail and complete removal of the notochord, but again no disturbance of head structures as displayed here. These previous studies injected 25 - 100 pg of ripply1 mRNA with dramatic phenotypes, whereas this study uses 500 - 1000 pg. The phenotype is so much more dramatic than previously presented that it suggests that the level of ripply1 overexpression is sufficiently high that it may no longer be regulating only its endogenous targets, making the results drawn from ripply1 overexpression difficult to trust.

(4) Ripply1 binding to sox17 and sox32 regulatory regions not convincing: The Cut and Tag data presented in Fig 6J-K does not seem to be high quality and does not seem to provide strong support that Ripply 1 binds to the regulatory regions of these genes. The signal-to-noise ratio is very poor, and the 'binding' near sox17 that is identified seems to be even coverage over a 14 kb region, which is not consistent with site-specific recruitment of this factor, and the 'peaks' highlighted with yellow boxes do not appear to be peaks at all. To me, it seems this probably represents either: (1) overtagmentation of these samples or (2) an overexpression artifact from injection of too high concentration of ripply1-HA mRNA. In general, Cut and Tag is only recommended for histone modifications, and Cut and Run would be recommended for transcriptional regulators like these (see Epicypher's literature). Given this and the previous point about Ripply1 overexpression, I am not convinced that Ripply1 regulates endodermal genes. The existing data could be made somewhat more convincing by showing the tracks for other genes as positive and negative controls, given that Ripply1 has known muscle targets (how does its binding look at those targets in comparison) and there should be a number of Nodal target genes that Ripply1 does not bind to that could be used as negative controls. Overall this experiment doesn't seem to be of high enough quality to drive the conclusion that Ripply1 directly binds near sox17 and sox32 and from the data presented in the manuscript looks as if it failed technically.

(5) "Cooperatively Gsc and ripply1 regulate": I suggest avoiding the term "cooperative," when describing the relationship between Ripply1 and Gsc regulation of PP and anterior endoderm - it evokes the concept of cooperative gene regulation, which implies that these factors interact with each biochemically in order to bind to the DNA. This is not supported by the data in this manuscript, and is especially confusing since Ripply1 is thought to require cooperative binding with a T-box family transcription factor to direct its binding to the DNA.

(6) SWI/SNF: The differential expression of srcap doesn't seem very remarkable. The dot plots in the supplement S7H don't help - they seem to show no expression at all in the endoderm, which is clearly a distortion of the data, since from the violin plots it's obviously expressed and the dot-size scale only ranges from ~30-38%. Please add to the figure information about fold-change and p-value for the differential expression. Publicly available scRNAseq databases show scrap is expressed throughout the entire early embryo, suggesting that it would be surprising for it to have differential activity in these two cell types and thereby contribute to their separate specification during development. It seems equally possible that this just mildly influences the level of Nodal or FGF signaling, which would create this effect.

The multiome data seems like a valuable data set for researchers interested in this stage of zebrafish development. However, the presentation of the data doesn't make many conclusions, aside from identifying an element adjacent to ripply1 whose chromatin is open in prechordal plate cells and not endodermal cells and showing that there are a number of loci with differential accessibility between these cell types. That seems fairly expected since both cell types have several differentially expressed transcriptional regulators (for instance, ripply1 has previously been demonstrated in multiple studies to be specific to the prechordal plate during blastula stages). The manuscript implies that SWI/SNF remodeling by Srcap is responsible for the chromatin accessibility differences between these cell types, but that has not actually been tested. It seems more likely that the differences in chromatin accessibility observed are a result of transcription factors binding downstream of Nodal signaling.

Minor issues:

Figure 2 E-F: It's not clear which cells from E are quantitated in F. For instance, the dorsal forerunner cells are likely to behave very differently from other endodermal progenitors in this assay. It would be helpful to indicate which cells are analyzed in Fig F with an outline or other indicator of some kind. Or - if both DFCs and endodermal cells are included in F, to perhaps use different colors for their points to help indicate if their fluorescence changes differently.

Fig 3 J: Should the reference be Dubrulle et al 2015, rather than Julien et al?

References:<br /> Alexander, J. & Stainier, D. Y. A molecular pathway leading to endoderm formation in zebrafish. Current biology : CB 9, 1147-1157 (1999).<br /> Barone, V. et al. An Effective Feedback Loop between Cell-Cell Contact Duration and Morphogen Signaling Determines Cell Fate. Dev. Cell 43, 198-211.e12 (2017).<br /> Economou, A. D., Guglielmi, L., East, P. & Hill, C. S. Nodal signaling establishes a competency window for stochastic cell fate switching. Dev. Cell 57, 2604-2622.e5 (2022).<br /> Gritsman, K. et al. The EGF-CFC protein one-eyed pinhead is essential for nodal signaling. Cell 97, 121-132 (1999).<br /> Gritsman, K., Talbot, W. S. & Schier, A. F. Nodal signaling patterns the organizer. Development (Cambridge, England) 127, 921-932 (2000).<br /> Kawamura, A. et al. Groucho-associated transcriptional repressor ripply1 is required for proper transition from the presomitic mesoderm to somites. Developmental cell 9, 735-744 (2005).<br /> Kawamura, A., Koshida, S. & Takada, S. Activator-to-repressor conversion of T-box transcription factors by the Ripply family of Groucho/TLE-associated mediators. Molecular and cellular biology 28, 3236-3244 (2008).<br /> Sako, K. et al. Optogenetic Control of Nodal Signaling Reveals a Temporal Pattern of Nodal Signaling Regulating Cell Fate Specification during Gastrulation. Cell Rep. 16, 866-877 (2016).<br /> Rogers, K. W. et al. Nodal patterning without Lefty inhibitory feedback is functional but fragile. eLife 6, e28785 (2017).<br /> Warga, R. M. & Nüsslein-Volhard, C. Origin and development of the zebrafish endoderm. Development 126, 827-838 (1999).

If you're after a fun creative project that you ACTUALLY take from idea to custom art journey, then get FREE instant access now.

And I'd make the button somewhat less doubtful ;-) SIGN ME UP NOW

I never thought of this as a way that arts help learning! It just felt like such an easy and obvious way of memorizing the alphabet. It's incredible ow much art can enhance learning, and I look forward of discovering and creating new ways of applying this method of teaching in my future classroom.

These results paired with the runtimes are really quite impressive! But the contrast between the results for KF distances as compared to RF distances are interesting, and seems like they may be worth unpacking.

In particular, it's notable that the RF distances at greater tree sizes for PF+FastME seem to converge with FastME, being greater than seen for IQTree/FastTree, with the difference increasing along with tree size.

As you say, RF is just the sum of differences in bipartitions between two trees, whereas KF considers both differences in topology and branch length. You find that PF+FastME consistently infers trees with lower or equivalent KF distances to IQTree and FastTree. But, as tree size increases, RF distances increase for PF+FastME at a high rate, exceeding those of FastTree and IQTree starting at relatively small trees (~20 tips).

Together, these results would suggest that PF+FastME estimates branch lengths well. This is maybe expected but a great thing to see, since PF is effectively trained to infer those evolutionary distances that FastME uses to infer branch lengths! However, despite accurately inferring branch-lengths, there seems to be a larger number of topological errors in the larger trees inferred by PF+FastME as compared to the other methods.

Do you have any intuition as to why this discrepancy arises? Or any thoughts on how you might modify the model/model architecture to better account for and mitigate this effect?

Funny how a natural wonder, in some mental image sense, is only as beautiful and marvelous as society accumulates it to be. postcards, paintings, articles, etc set a general expectation, turning a visit to see said natural wonder into a visit purely just to visit, and our thoughts at the sight of it are inseparable from another person's description of it's beauty. Like a blind ritual of a sort, something we people just do to do it. To go and see and have some life changing, awe-inspiring moment because we simply must. And then imagine it's a rainy day and the sight of whatever natural wonder is ruined. Maybe we'd want to be disappointed. Maybe we wouldn't allow ourslves to be.

I feel like it's a little alienating to say the only instrument. We don't have kazoos in orchestras (usually) and I don't think it would hurt to just say that it's widely excluded from symphonies and orchestras.

This must be very frustrating for teachers who truly care about the students and their progression, but they are just trying to get through another class. I don't think I ever really thought about instructors in this scenario because I feel like I haven't ever really had a teacher truly passionate enough about me and my education. That has made it a bit harder for me to actually put my best effort in their classes because if they aren't invested in me then why should I be invested in their class. This is mostly for all of my high school classes, but could be applicable to higher education as well.

This is interesting, I didn't know that Grammarly used generative AI. Now that I think about it it makes sense, but it is a bit unnerving that we could be using AI for assignments and not even know about it.

Are there laws that require businesses to state up-front that they are using AI? If not, should students still be held accountable?

A very strong tie-in to the word choice for the book title. Again, it's blue jeans, not just jeans. This is also an excellent indicator that the book will globalize its discussion, which I commented on earlier with regard to its specifically American bent.

It can be lonely at times and sometimes have you questioning your own sanity, but it's a journey that every person is walking at their own pace in their own time. With awareness or without it.

worth noting, that its not just publishing, but mass media and transmissions, not unlike the global village as mccluhan predicted. its in every area of life.

the gatekeeping previously at least had certain checks and balances, and its part of why platforms like tiktok and the platform's spread of misinformation has become such a lightning rod issue

on the other hand UMG for example, had to make a deal though to, admitting that the platform had created and validated a lucrative market

and that's the thing.

Blurred lines. Music, news, sports, and in every aspect of life, there's grey. Do you want to be positive or not?

UMG, a public company, has a publishing division. They publish music, and they often have to work with newsrooms to license out music, if say they want a particular song as ESPN might, for example - instead of "library music" -- which is another type of company, that ultimately, functions, as a publisher.

Linda Villarosa, is quite the accomplished multi-hyphenate, and that includes publisher. It's not the first thing she discloses or usually is referred to as, but she has a publishing company and she published her own novel under it. Whether or not she partnered with a University Press or a for-profit company, or printed and bounded it all by herself, her books, were published via Linda's publishing and she is a publisher

This is a new term to me. I think it's bit like PUE in that you want the number to be as close to 1 as possible (i.e. for 1 server's worth of utilisation, you want to have provision 1 server, not 5)

So, I think this is the term you might use to talk about how a hyperscaler datacentre might be full of individually efficient servers, but only have 5% of them serving workloads - the rest is just spare capacity ready to sell, or give away as credits

Some people only look at nature for the look or it, or for the postcard view. I think that nature should be appreciated for what it is. I think it's good to add this in the story though, because it shows that side of thinking.

economy was shifted cause of it

Lol, it's quite literally a cycle. That or writing, print, spoken word, and computers are intrinsically linked. It's impossible to leverage one over the other, as all are needed in a constantly shifting society.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this paper, the researchers aimed to address whether bees causally understand string-pulling through a series of experiments. I first briefly summarize what they did:

- In experiment 1, the researchers trained bees without string and then presented them with flowers in the test phase that either had connected or disconnected strings, to determine what their preference was without any training. Bees did not show any preference.

- In experiment 2, bees were trained to have experience with string and then tested on their choice between connected vs. disconnected string.

- experiment 3 was similar except that instead of having one option which was an attached string broken in the middle, the string was completely disconnected from the flower.

- In experiment 4, bees were trained on green strings and tested on white strings to determine if they generalize across color.

- In experiment 5, bees were trained on blue strings and tested on white strings.

- In experiment 6, bees were trained where black tape covered the area between the string and the flower (i.e. so they would not be able to see/ learn whether it was connected or disconnected).

- In experiments 2-6, bees chose the connected string in the test phase.

- In experiment 7, bees were trained as in experiment 3 and then tested where the string was either disconnected or coiled i.e. still being 'functional' but appearing different.

- In experiment 8, bees were trained as before and then tested on a string that was in a different coiled orientation, either connected or disconnected.

- In experiments 7 and 8 the bees showed no preference.

Strengths:

I appreciate the amount of work that has gone into this study and think it contains a nice, thorough set of experiments. I enjoyed reading the paper and felt that overall it was well-written and clear. I think experiment 1 shows that bees do not have an untrained understanding of the function of the string in this context. The rest of the experiments indicate that with training, bees have a preference for unbroken over broken string and likely use visual cues learned during training to make this choice. They also show that as in other contexts, bees readily generalize across different colors.

Weaknesses:

(1) I think there are 2 key pieces of information that can be taken from the test phase - the bees' first choice and then their behavior across the whole test. I think the first choice is critical in terms of what the bee has learned from the training phase - then their behavior from this point is informed by the feedback they obtain during the test phase. I think both pieces of information are worth considering, but their behavior across the entire test phase is giving different information than their first choice, and this distinction could be made more explicit. In addition, while the bees' first choice is reported, no statistics are presented for their preferences.

We agree with the reviewer that the first choice is critical in terms of what the bumblebees have learned from the training phase. We analyzed the bees’ first choice in Table 1, and we added the tested videos. The entire connected and disconnected strings were glued to the floor, the bees were unable to move either the connected or disconnected strings, and avoid learning behavior during the tests. We added the data of bee's each choice in the Supplementary table.

(2) It seemed to me that the bees might not only be using visual feedback but also motor feedback. This would not explain their behavior in the first test choice, but could explain some of their subsequent behavior. For example, bees might learn during training that there is some friction/weight associated with pulling the string, but in cases where the string is separated from the flower, this would presumably feel different to the bee in terms of the physical feedback it is receiving. I'd be interested to see some of these test videos (perhaps these could be shared as supplementary material, in addition to the training videos already uploaded), to see what the bees' behavior looks like after they attempt to pull a disconnected string.

We added supplementary videos of testing phase. As noted in General Methods, both connected and disconnected strings were glued to the floor to prevent the air flow generated by flying bumblebees’ wings from changing the position of the string during the testing phase. The bees were unable to move either the connected or disconnected strings during the tests, and only attempted to pull them. Therefore, the difference in the friction/weight of pulling the both strings cannot be a factor in the test.

(3) I think the statistics section needs to be made clearer (more in private comments).

We changed the statistical analysis section as suggested by the reviewer.

(4) I think the paper would be made stronger by considering the natural context in which the bee performs this behavior. Bees manipulate flowers in all kinds of contexts and scrabble with their legs to achieve nectar rewards. Rather than thinking that it is pulling a string, my guess would be that the bee learns that a particular motor pattern within their usual foraging repertoire (scrabbling with legs), leads to a reward. I don't think this makes the behavior any less interesting - in fact, I think considering the behavior through an ecological lens can help make better sense of it.

Here we respectfully disagree. The solving of Rubik’s cube by humans could be said to be version of finger-movements naturally required to open nuts or remove ticks from fur, but this is somewhat beside the point: it’s not the motor sequences that are of interest, but the cognition involved. A general approach in work on animal intelligence and cognition is to deliberately choose paradigms that are outside the animals’ daily routines-this is what we have done here, in asking whether there is means-end comprehension in bee problem solving. Like comparable studies on this question in other animals, the experiments are designed to probe this question, not one of ecological validity.

Reviewer #2 (Public Review):

Summary:

The authors wanted to see if bumblebees could succeed in the string-pulling paradigm with broken strings. They found that bumblebees can learn to pull strings and that they have a preference to pull on intact strings vs broken ones. The authors conclude that bumblebees use image matching to complete the string-pulling task.

Strengths:

The study has an excellent experimental design and contributes to our understanding of what information bumblebees use to solve a string-pulling task.

Weaknesses:

Overall, I think the manuscript is good, but it is missing some context. Why do bumblebees rely on image matching rather than causal reasoning? Could it have something to do with their ecology? And how is the task relevant for bumblebees in the wild? Does the test translate to any real-life situations? Is pulling a natural behaviour that bees do? Does image matching have adaptive significance?

We appreciate the valuable comment from the reviewer. Our explanation, which we have now added to the manuscript, is as follows:

“Different flower species offer varying profitability in terms of nectar and pollen to bumblebees; they need to make careful choices and learn to use floral cues to predict rewards (Chittka, 2017). Bumblebees can easily learn visual patterns and shapes of flower (Meyer-Rochow, 2019); they can detect stimuli and discriminate between differently coloured stimuli when presented as briefly as 25 ms (Nityananda et al., 2014). In contrast, causal reasoning involves understanding and responding to causal relationships. Bumblebees might favor, or be limited to, a visual approach, likely due to the efficiency and simplicity of processing visual cues to solve the string-pulling task. ”

As above, it worth noting that our work is not designed as an ecological study, but one about the question of whether causal reasoning can explain how bees solve a string-pulling puzzle. We have a cognitive focus, in line with comparable studies on other animals. We deliberately chose a paradigm that is to some extent outside of the daily challenges of the animal.

Reviewer #3 (Public Review):

Summary:

This paper presents bees with varying levels of experience with a choice task where bees have to choose to pull either a connected or unconnected string, each attached to a yellow flower containing sugar water. Bees without experience of string pulling did not choose the connected string above chance (experiment 1), but with experience of horizontal string pulling (as in the right-hand panel of Figure 4) bees did choose the connected string above chance (experiments 2-3), even when the string colour changed between training and test (experiments 4-5). Bees that were not provided with perceptual-motor feedback (i.e they could not observe that each pull of the string moved the flower) during training still learned to string pull and then chose the connected string option above chance (experiment 6). Bees with normal experience of string pulling then failed to discriminate between connected and unconnected strings when the strings were coiled or looped, rather than presented straight (experiments 7-8).

Weaknesses:

The authors have only provided video of some of the conditions where the bees succeeded. In general, I think a video explaining each condition and then showing a clip of a typical performance would make it much easier to follow the study designs for scholars. Videos of the conditions bees failed at would be highly useful in order to compare different hypotheses for how the bees are solving this problem. I also think it is highly important to code the videos for switching behaviours. When solving the connected vs unconnected string tasks, when bees were observed pulling the unconnected string, did they quickly switch to the other string? Or did they continue to pull the wrong string? This would help discriminate the use of perceptual-motor feedback from other hypotheses.

We added the test videos as suggested by the reviewer, and we added the data for each bee's choice. However, both connected and disconnected strings were glued to the floor, and therefore perceptual-motor feedback was equal and irrelevant between the choices during the test.

The experiments are also not described well, for my below comments I have assumed that different groups of bees were tested for experiments 1-8, and that experiment 6 was run as described in line 331, where bees were given string-pulling training without perceptual feedback rather than how it is described in Figure 4B, which describes bees as receiving string pulling training with feedback.

We now added figures of Experiment 6 and 7 in the Figure 1B, and we mentioned that different groups of bees were tested for Experiments 1-9.

The authors suggest the bees' performance is best explained by what they term 'image matching'. However, experiment 6 does not seem to support this without assuming retroactive image matching after the problem is solved. The logic of experiment 6 is described as "This was to ensure that the bees could not see the familiar "lollipop shape" while pulling strings....If the bees prefer to pull the connected strings, this would indicate that bees memorize the arrangement of strings-connected flowers in this task." I disagree with this second sentence, removing perceptual feedback during training would prevent bees memorising the lollipop shape, because, while solving the task, they don't actually see a string connected to a yellow flower, due to the black barrier. At the end of the task, the string is now behind the bee, so unless the bee is turning around and encoding this object retrospectively as the image to match, it seems hard to imagine how the bee learns the lollipop shape.

We agree with the reviewer that while solving the task in the last step during training, the bees don't actually see a string connected to a yellow flower, due to the black barrier. Since the full shape is only visible after the pulling is completed and this requires the bee to “check back” on the entire display after feeding, to basically conclude “ this is the shape that I need to be looking for later”.

Another possibility is that bumblebees might remember the image of the “lollipop shape” while training the bees in the first step, in which the “lollipop shape” was directly presented to the bumblebee in the early step of the training.

We added the experiment suggested by the reviewer, and the result showed that when a green table was placed behind the string to obscure the “lollipop shape” at any point during the training phase, the bees were unable to identify the connected string. The result further supports that bumblebees learn to choose the connected string through image matching.

Despite this, the authors go on to describe image matching as one of their main findings. For this claim, I would suggest the authors run another experiment, identical to experiment 6 but with a black panel behind the bee, such that the string the bee pulls behind itself disappears from view. There is now no image to match at any point from the bee's perspective so it should now fail the connectivity task.

Strengths:

Despite these issues, this is a fascinating dataset. Experiments 1 and 2 show that the bees are not learning to discriminate between connected and unconnected stimuli rapidly in the first trials of the test. Instead, it is clear that experience in string pulling is needed to discriminate between connected and unconnected strings. What aspect of this experience is important? Experiment 6 suggests it is not image matching (when no image is provided during problem-solving, but only afterward, bees still attend to string connectivity) and casts doubt on perceptual-motor feedback (unless from the bee's perspective, they do actually get feedback that pulling the string moves the flower, video is needed here). Experiments 7 and 8 rule out means-end understanding because if the bees are capable of imagining the effect of their actions on the string and then planning out their actions (as hypotheses such as insight, means-end understanding and string connectivity suggest), they should solve these tasks. If the authors can compare the bees' performance in a more detailed way to other species, and run the experiment suggested, this will be a highly exciting paper

We appreciate the valuable comment from the reviewer. We compared the bees' performance to other species, and conducted the experiment as suggested by the reviewer.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Smaller comments:

Line 64: is the word 'simple' needed here? It could also be explained by more complex forms of associative learning, no?

We deleted “simple”.

Methods:

Line 230: was it checked that this was high-contrast for the bees?

We added the relevant reference in the revised manuscript.

Line 240: how much sucrose solution was present in the flowers?

We added 25 microliters sucrose solution in the flowers. We added the information in the revised manuscript.

Line 266: check grammar.

We checked the grammar as follows: “During tests, both strings were glued to the floor of the arena to prevent the air flow generated by flying bumblebees’ wings from changing the position of the string.”

Statistical analysis:

- What does it mean that "Bees identity and colony were analyzed with likelihood ratio tests"?

Bees identity and colony was set as a random variable. We changed the analysis methods in the revised manuscript, and results of the all the experiments did not changed.

- Line 359: do you mean proportion rather than percentage?

We mean the percentage.

- "the number of total choices as weights" - this should be explained further. This is the number of choices that each bee made? What was the variation and mean of this number? If bees varied a lot in this metric, it might make more sense to analyze their first choice (as I see you've done) and their first 10 choices or something like that - for consistency.

This refers to the total number of choices made by each bumblebee. We added the mean and standard error of each bee’s number of choices in Table 1. Some bees pulled the string fewer than 10 times; we chose to include all choices made by each bee.

- More generally I think the first test is more informative than the subsequent choices, since every choice after their first could be affected by feedback they are getting in that test phase. Or rather, they are telling you different things.

All the bees were tested only once, however, you might be referring to the first choice. We used Chi-square test to analyze the bumblebees’ first choices in the test. It is worth noting that both connected and disconnected strings were glued to the floor. The bees were unable to move either the connected or disconnected strings during the tests, and only attempted to pull them. Therefore,the feedback from pulling either the connected or disconnected strings is the same.

- Line 362: I think I know what you mean, but this should be re-phrased because the "number of" sounds more appropriate for a Poisson distribution. I think what you are testing is whether each individual bee chose the connected or the disconnected string - i.e. a 0 or 1 response for each bee?

We agree with the reviewer that each bee chose the connected or the disconnected string - i.e. a 0 or 1 response for each bee, but not the number. We clarify this as: “The total number of the choices made by each bee was set as weights.” 

- Line 364-365: here and elsewhere, every time you mention a model, make it clear what the dependent and independent variables are. i.e. for the mixed model, the 'bee' is the random factor? Or also the colony that the bee came from? Were these nested etc?

We clarify this in the revised manuscript. The bee identity and colony is the random factor in the mixed model.

- Line 368: "Latency to the first choice of each bee was recorded" - why? What were the hypotheses/ predictions here?

The latency to the first choice was intended to see if the bumblebees were familiarizing with the testing pattern. A shorter delay time might indicate that the bumblebees were more familiar with the pattern.

- Line 371: "Multiple comparisons among experiments were.." - do you mean 'within' experiments? It seems that treatments should not be compared between different experiments.

We mean multiple comparisons among different experiments; we clarify this in the revised manuscript.

Results

Experiment 1: From the methods, it sounded like you both analyzed the bees' first choice and their total no. of choices, but in the results section (and Figure 1) I only see the data for all choices combined here.

In table 1 and in the text you report the number of bees that chose each option on their first choice, but there are no statistical results associated with these results. At the very least, a chi square or binomial test could be run.

Line 138: "Interestingly, ten out of fifteen bees pulled the connected string in their first choice" - this is presented like it is a significant majority of bees, but a chi-square test of 10 vs 5 has a p-value = 0.1967

We used the Chi square test to analyzed of the bees’ first choice. We also added the analyzed data in the Table 1.

Line 143: "It makes sense because the bees could see the "lollipop shape" once they pulled it out from the table." - this feels more like interpretation (i.e. Discussion) rather than results.

We moved the sentence to the discussion.

Line 162: again this feels more like interpretation/ conjecture than results.

We removed the sentence in the results.

Line 184: check grammar.

We checked the grammar. We changed “task” to “tasks”.

Figures

I really appreciated the overview in Figure 5 - though I think this should be Figure 1? Even if the methods come later in eLife, I think it would be nice to have that cited earlier on (e.g. at the start of the results) to draw the reader's attention to it quickly, since it's so helpful. It also then makes the images at the bottom of what is currently Figure 1 make more sense. I also think that the authors could make it clearer in Figure 5 which strings are connected vs disconnected in the figure (even if it means exaggerating the distance more than it was in real life). I had to zoom in quite a bit to see which were connected vs. not. Alternatively, you could have an arrow to the string with the words "connected" "disconnected" the first time you draw it - and similar labels for the other string conditions.

We appreciate the valuable comment from the reviewer. We changed Figure 5 to Figure 2, and Figure 4 to Figure 1. We cited the Figures at the start of the results. We also changed the gap distance between the disconnected strings. Additionally, we added arrows to indicate “connected” and “disconnected” strings in the Figure.

Figure 1 - I think you could make it clearer that the bars refer to experiments (e.g. have an x-axis with this as a label). Also, check the grammar of the y-axis.

We added the experiments number in the Figures. Additionally, we checked the grammar of the y-axis. We changed “percentages” to “parentage”. 

I also think it's really helpful to see the supplementary videos but I think it would be nice to see some examples of the test phase, and not just the training examples.

We added Supplementary videos of the testing phase.

Reviewer #2 (Recommendations For The Authors):

Below are also some minor comments:

L40: "approaches".

We changed “approach” to “approaches”.

L42: but likely mainly due to sampling bias of mammals and birds.

We changed the sentence as follows: String pulling is one of the most extensively used approaches in comparative psychology to evaluate the understanding of causal relationships (Jacobs & Osvath, 2015), with most research focused on mammals and birds, where a food item is visible to the animal but accessible only by pulling on a string attached to the reward (Taylor, 2010; Range et al., 2012; Jacobs & Osvath, 2015; Wakonig et al., 2021).

L64: remove "in this study"

We removed “in this study”.

L64: simple associative learning of what? Isn't your image matching associative too?

We removed “ simple”.

L97: remove "a" before "connected".

We removed “a” before “connected”.

L136-138: but maybe they could still feel the weight of the flower when pulling?

Because both strings were glued to the floor in the test phase, the feedback was the same and therefore irrelevant. This information is noted in the General Methods.

L161: what are these numbers?

We removed the latency in the revised manuscript.

L167/ Table 1: I realise that the authors never tried slanted strings to check if bumblebees used proximity as a cue. Why?

This was simply because we wanted to focus on whether bumblebees could recognize the connectivity of the string.

Discussion: Why did you only control for colour of the string? What if you had used strings with different textures or smells? Unclear if the authors controlled for "bumblebee smell" on the strings, i.e., after a bee had used the string, was the string replaced by a new one or was the same one used multiple times?

We used different colors to investigate featural generalization of the visual display of the string connected to the flower in this task. We controlled for color because it is a feature that bumblebees can easily distinguish.

Both the flowers and the strings were used only once, to prevent the use of chemosensory cues. We clarify this in the revised manuscript.

L182: since what?

We deleted “since” in the revised manuscript.

L182-188: might be worth mentioning that some crows and parrots known for complex cognition perform poorly on broken strings (e.g., https://doi.org/10.1098/rspb.2012.1998 ; https://doi.org/10.1163/1568539X-00003511 ; https://doi.org/10.1038/s41598-021-94879-x ) and Australian magpies use trial and error (https://doi.org/10.1007/s00265-023-03326-6).

We added the following sentences as suggested by the reviewer: “It is worth noting that some crows and parrots known for complex cognition perform poorly on the broken string task without perceptual feedback or learning. For example, New Caledonian crows use perceptual feedback strategies to solve the broken string-pulling task, and no individual showed a significant preference for the connected string when perceptual feedback was restricted (Taylor et al., 2012). Some Australian magpies and African grey parrots can solve the broken string task, but they required a high number of trials, indicating that learning plays a crucial role in solving this task (Molina et al., 2019; Johnsson et al., 2023).”

L193: maybe expand on this to put the task into a natural context?

We added the following sentences as suggested by the reviewer:

“Different flower species offer varying profitability in terms of nectar and pollen to bumblebees; they need to make careful choices and learn to use floral cues to predict rewards (Chittka, 2017). Bumblebees can easily learn visual patterns and shapes of flower (Meyer-Rochow, 2019); they can detect stimuli and discriminate between differently coloured stimuli when presented as briefly as 25 ms (Nityananda et al., 2014). In contrast, causal reasoning involves understanding and responding to causal relationships. Bumblebees might favor, or be limited to, a visual approach, likely due to the efficiency and simplicity of processing visual cues to solve the string-pulling task. ”

L204: is causal understanding the same as means-end understanding?

Means-end understanding is expressed as goal-directed behavior, which involves the deliberate and planned execution of a sequence of steps to achieve a goal. Includes some understanding of the causal relationship (Jacobs & Osvath, 2015; Ortiz et al., 2019). .

L235: this is a very big span of time. Why not control for motivation? Cognitive performance can vary significantly across the day (at least in humans).

Bumblebee motivation is understood to be rather consistent, as those that were trained and tested came to the flight arena of their own volition and were foragers looking to fill their crop load each time to return it to the colony.

L232: what is "(w/w)" ? This occurs throughout the manuscript.

“w/w” represents the weight-to-weight percentage of sugar.

L250: this sentence sounds odd. "containing in the central well.." ?? Perhaps rephrase? Unclear what central well refers to? Did the flowers have multiple wells?

We rephrased the sentence as follows: For each experiment, bumblebees were trained to retrieve a flower with an inverted Eppendorf cap at the center, containing 25 microliters of 50% sucrose solution, from underneath a transparent acrylic table

L268: why euthanise?

The reason for euthanizing the bees is that new foragers will typically only become active after the current ones were removed from the hive.

L270: chemosensory cues answer my concern above. Maybe make it clear earlier.

We moved this sentence earlier in the result.

L273: did different individuals use different pulling strategies? Do you have the data to analyse this? This has been done on birds and would offer a nice comparison.

We analyzed the string-pulling strategies among different individuals, and provided Supplementary Table 1 to display the performances of each individual in different string-pulling experiments.

L365: unclear why both models. Would be nice to see a GLM output table.

The duration of pulling different kinds of strings were first tested with the Shapiro-Wilk test to assess data normality. The duration data that conforms to a normal distribution was compared using linear mixed-effects models (LMM), while the data that deviates from normality were examined with a generalized linear-mixed model (GLMM). We added a GLM and GLMM output table in the revised manuscript.

L377: should be a space between the "." and "This".

We added a space between the “.” and “This”.

L383-390: some commas and semicolons are in the wrong places.

We carefully checked the commas and semicolons in this sentence.

Reviewer #3 (Recommendations For The Authors):

Minor comments

Line 32: seems to be missing a word, suggest "the bumblebees' ability to distinguish".

we added “the” in the revised manuscript.

Line 47: it would be good to reference other scholars here, this is the central focus of all work in comparative psychology.

We added the reference in the revised manuscript.

Line 50-61: I think the string-pulling literature could be described in more detail here, with mention of perceptual-motor feedback loops as a competing hypothesis to means-end understanding (see Taylor et al 2010, 2012). It seems a stretch to suggest that "String-pulling studies have directly tested means-end comprehension in various species", when perceptual-motor feedback is a competing hypothesis that we have positive evidence for in several species.

We mentioned the perceptual-motor feedback in the introduction as follow:

“Multiple mechanisms can be involved in the string-pulling task, including the proximity principle, perceptual feedback and means-end understanding (Taylor et al., 2012; Wasserman et al., 2013; Jacobs & Osvath, 2015; Wang et al., 2020). The principle of proximity refers to animals preferring to pull the reward that is closest to them (Jacobs & Osvath, 2015). Taylor et al. (2012) proposed that the success of New Caledonian crows in string-pulling tasks is based on a perceptual-motor feedback loop, where the reward gradually moves closer to the animal as they pull the strings. If the visual signal of the reward approaching is restricted, crows with no prior string-pulling experience are unable to solve the broken string task (Taylor et al., 2012).

However, when a green table was placed behind the string to obscure the “lollipop” structure during the training, the bees could not see the “lollipop” during the initial training stage or after pulling the string from under the table. In this situation, the bees were unable to identify the connected string, further proving that bumblebees chose the connected string based on image matching.

Line 68: suggest remove 'meticulously'.

We removed “meticulously”.

Line 99: This is an exciting finding, can the authors please provide a video of a bee solving this task on its first trial?

We added videos in the supplementary materials.

Line 133: perceptual-motor feedback loops should be introduced in the introduction.

We introduced perceptual-motor feedback loops in the revised manuscript.

Line 136: please clarify the prior experience of these bees, it is not clear from the text.

We clarified the prior experience of these bees as follow: Bumblebees were initially attracted to feed on yellow artificial flowers, and then trained with transparent tables covered by black tape (S7 video) through a four-step process.

Line 138: from the video it is not possible to see the bee's perspective of this occlusion. Do the authors have a video or image showing the feedback the bees received? I think this is highly important if they wish to argue that this condition prevents the use of both image matching and a perceptual-motor feedback loop.

We prevented the use of image matching: the bees were unable to see the flower moving towards them above the table during the training phase in this condition. But the bees may receive visual image both after pulling the string out from the table and in the initial stages of training in this condition.

Line 147: please clarify what experience these bees had before this test.

We added the prior experience of bumblebees before training as follow: We therefore designed further experiments based on Taylor et al. (2012) to test this hypothesis. Bumblebees were first trained to feed on yellow artificial, and then trained with the same procedure as Experiment 2, but the connected strings were coiled in the test.

Line 155: This is a highly similar test to that used in Taylor et al 2012, have the authors seen this study?

We mentioned the reference in the revised manuscript as follows: We therefore designed further experiments based on Taylor et al. (2012) to test this hypothesis.

Line 183: This sentence needs rewriting "Since the vast majority of animals, including dogs 183 (Osthaus et al., 2005), cats (Whitt et al., 2009), western scrub-jays (Hofmann et al.,2016) and azure-winged magpies (Wang et al., 2019) are failing in such tasks spontaneously".

We changed the sentence as suggested by the reviewer as follow:  Some animals, including dogs (Osthaus et al., 2005), cats (Whitt et al., 2009), western scrub-jays (Hofmann et al., 2016) and azure-winged magpies (Wang et al., 2019) fail in such task spontaneously.

Line 186: "complete comprehension of the functionality of strings is rare" I am not sure the evidence in the current literature supports any animal showing full understanding, can the authors explain how they reach this conclusion?

We wished to say that few animal species could distinguish between connected and disconnected strings without trial and error learning. We revised the sentence as follows:

It is worth noting that some crows and parrots known for complex cognition perform poorly on broken string task without perceptual feedback or learning. For example, New Caledonian crows use perceptual feedback strategies to solve broken string-pulling task, and no individual showed a significant preference for the connected string when perceptual feedback is restricted (Taylor et al., 2012). Some Australian magpies and African grey parrots can solve the broken string task, but it required a high number of trials, indicating that learning plays a crucial role in solving this task (Molina et al., 2019; Johnsson et al., 2023).

Line 190: the authors need to clarify which part of their study provides positive evidence for this conclusion.

We added the evidence for this conclusion as follows: Our findings suggest that bumblebees with experience of string pulling prefer the connected strings, but they failed to identify the interrupted strings when the string was coiled in the test.

Line 265: was the far end of the string glued only?

The entire string was glued to the floor, not just the far ends of the string.

you heard weird al.dont pirate or download his song!!!

I really like this angle (and it's why I highlighted the "narrative turn" on pg. 230. This article engages in a "selective reading" (I love that term) of Branson's narrative (literally, his autobiography). But every account of entrepreneurs and their successes (and failures/struggles to succeed) can be understood as a story. We're not just reading the story of that individual (though that's often the focus); we are also encountering the story of entrepreneurship and the process of its ongoing fashioning/re-imagining. When I ask you to consider how you're entrepreneurial, I'm asking you to re-write your own story...

all of the success stories surrounding entrepreneurs encourage us to want to be entrepreneurs... and not just the heroic version either! All the narratives of people with side-hustles teach us to desire a similar pursuit, selling a gig-economy and precarious labour under the guise of passion and self-improvement... It's not just that we are all entrepreneurs (Szeman), but that we desire to be... (because we all DESIRE (or ought to desire, supposedly) more success, more freedom, more independence (financially and otherwise)...)

And maybe, the more we desire and dream, the more we might do (converting our desires into action and making our dreams reality !!)

not just in Branson's case either. I think it's reasonable to suggest that these are potentially pillars in any entrepreneurial mindset.

I think this is a very powerful concept -- as a former student said, entrepreneurs must have cruel optimism, individuals must be able to adapt and ride out whatever wave or obstacle comes around.

Berlant uses the term cruel optimism to refer to our our investments in “compromised conditions of possibility whose realization is discovered to be impossible, sheer fantasy.” (i.e., we keep cheering for a team we know will lose; we maintain hope in an unattainable romantic ideal promulgated by Hollywood or pursue happiness based on unrealistic beauty standards; we engage in small acts of environmental stewardship like recycling or buying a hybrid in the face of potentially unstoppable climate change...) Berlant basically means that the thing we seek to achieve, the thing (or state of being) that we desire (or the act of seeking and desiring itself) might actually threaten our well-being (that's what makes it cruel!). As she put it succinctly, “a relation of cruel optimism exists when something you desire is actually an obstacle to your flourishing.”

This relates to entrepreneurialism in so many ways: Engaging in the gig economy or a side-hustle as a way to increase one's income (or security) in uncertain times is cruel and optimistic. Similarly, we encounter aspirational labour in the form of internships or any form of unpaid labour while looking for a "real" job. Perhaps you feel the pressure of cultivating a sense of employability. According to Frayne (2015), today, students are expected “to improve their prospects by training, acquiring educational credentials, networking, learning how to project the right kind of personality, and gaining life experiences that match up with the values sought by employers.” In other words, they have to act entrepreneurially even to get a non-entrepreneurial job. As Hawzen et al. (2018) assert, this incites anxiety and results in a colonization of one’s entire life by work-related demands as students feel the need to separate themselves from the competition, doing things like volunteering to gain an advantage or to get a "foot in the door"... We also see it to a certain extent in the example of entrepreneurial vloggers in the sense that the fantasy of a “good life” through fame and fortune is rarely realized. The cruel conditions of precocity are, for most, more of a reality than the fantasy... and we take up this theme explicitly in two weeks hence with digital 'autopreneurs'

Overall, this also highlights one of the reasons I chose this article -- rather than just highlighting how entrepreneurs are certain types of people (or motivated by certain types of things), it emphasizes how entrepreneurship is a mental orientation, not just a business concept but a way of living. But it's not all sunshine and happiness. Cruel optimism, indeed!

What about you? Are you familiar with the feeling of 'cruel optimism'? Does it define the current times or your current disposition?

Remember at the beginning of the article (when Szeman says "we are all entrepreneurs now") (p. 472)? He doesn't mean that we are all creating business start-ups. Rather, he's suggesting that there is a spirit-of-the-times wherein entrepreneurship has become this new common-sense reality. It is both a dominant way of thinking about how we ought to act, AND an informal rulebook for how economies (and other forms of practice) ought to function too... In other words, entrepreneurship isn't just about undertaking profit-making (and risk-inducing) economic practices in capitalism. Rather, it's about undertaking a new subjectivity, a new identity when it comes to how we think of ourselves, how we relate to others, and how we respond to our wider social, cultural, political, and economic environment.

I felt this way during the pandemic as well. There was a shift in the way classes were taught and in the quality of work students were expected to do. I always wanted to do my best, and I was always pushing myself. However, during the pandemic, it felt like teachers didn't care as much if you succeeded or not, and they didn't care if you actually learned.

Im not sure that it is as simple as this. There is more that goes into students ability to persist through challenges than just a love for learning. Sometimes we have students that love learning but are unable to persist through challenge because of embarrassment or what has been previously told to them about how they are as a student. As teachers we want to build up students identities as learners to help them persist. I don't think it's always as simple as the student loving what they are doing.

Author response:

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

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

Glaser et al present ExA-SPIM, a light-sheet microscope platform with large volumetric coverage (Field of view 85mm^2, working distance 35mm), designed to image expanded mouse brains in their entirety. The authors also present an expansion method optimized for whole mouse brains and an acquisition software suite. The microscope is employed in imaging an expanded mouse brain, the macaque motor cortex, and human brain slices of white matter. 

This is impressive work and represents a leap over existing light-sheet microscopes. As an example, it offers a fivefold higher resolution than mesoSPIM (https://mesospim.org/), a popular platform for imaging large cleared samples. Thus while this work is rooted in optical engineering, it manifests a huge step forward and has the potential to become an important tool in the neurosciences. 

Strengths: 

- ExA-SPIM features an exceptional combination of field of view, working distance, resolution, and throughput. 

- An expanded mouse brain can be acquired with only 15 tiles, lowering the burden on computational stitching. That the brain does not need to be mechanically sectioned is also seen as an important capability. 

- The image data is compelling, and tracing of neurons has been performed. This demonstrates the potential of the microscope platform. 

Weaknesses: 

- There is a general question about the scaling laws of lenses, and expansion microscopy, which in my opinion remained unanswered: In the context of whole brain imaging, a larger expansion factor requires a microscope system with larger volumetric coverage, which in turn will have lower resolution (Figure 1B). So what is optimal? Could one alternatively image a cleared (non-expanded) brain with a high-resolution ASLM system (Chakraborty, Tonmoy, Nature Methods 2019, potentially upgraded with custom objectives) and get a similar effective resolution as the authors get with expansion? This is not meant to diminish the achievement, but it was unclear if the gains in resolution from the expansion factor are traded off by the scaling laws of current optical systems. 

Paraphrasing the reviewer: Expanding the tissue requires imaging larger volumes and allows lower optical resolution. What has been gained?

The answer to the reviewer’s question is nuanced and contains four parts. 

First, optical engineering requirements are more forgiving for lenses with lower resolution. Lower resolution lenses can have much larger fields of view (in real terms: the number of resolvable elements, proportional to ‘etendue’) and much longer working distances. In other words, it is currently more feasible to engineer lower resolution lenses with larger volumetric coverage, even when accounting for the expansion factor. 

Second, these lenses are also much better corrected compared to higher resolution (NA) lenses. They have a flat field of view, negligible pincushion distortions, and constant resolution across the field of view. We are not aware of comparable performance for high NA objectives, even when correcting for expansion.

Third, although clearing and expansion render tissues ‘transparent’, there still exist refractive index inhomogeneities which deteriorate image quality, especially at larger imaging depths. These effects are more severe for higher optical resolutions (NA), because the rays entering the objective at higher angles have longer paths in the tissue and will see more aberrations. For lower NA systems, such as ExaSPIM, the differences in paths between the extreme and axial rays are relatively small and image formation is less sensitive to aberrations. 

Fourth, aberrations are proportional to the index of refraction inhomogeneities (dn/dx). Since the index of refraction is roughly proportional to density, scattering and aberration of light decreases as M^3, where M is the expansion factor. In contrast, the imaging path length through the tissue only increases as M. This produces a huge win for imaging larger samples with lower resolutions. 

To our knowledge there are no convincing demonstrations in the literature of diffraction-limited ASLM imaging at a depth of 1 cm in cleared mouse brain tissue, which would be equivalent to the ExA-SPIM imaging results presented in this manuscript.  

In the discussion of the revised manuscript we discuss these factors in more depth. 

- It was unclear if 300 nm lateral and 800 nm axial resolution is enough for many questions in neuroscience. Segmenting spines, distinguishing pre- and postsynaptic densities, or tracing densely labeled neurons might be challenging. A discussion about the necessary resolution levels in neuroscience would be appreciated. 

We have previously shown good results in tracing the thinnest (100 nm thick) axons over cm scales with 1.5 um axial resolution. It is the contrast (SNR) that matters, and the ExaSPIM contrast exceeds the block-face 2-photon contrast, not to mention imaging speed (> 10x).  

Indeed, for some questions, like distinguishing fluorescence in pre- and postsynaptic structures, higher resolutions will be required (0.2 um isotropic; Rah et al Frontiers Neurosci, 2013). This could be achieved with higher expansion factors.

This is not within the intended scope of the current manuscript. As mentioned in the discussion section, we are working towards ExA-SPIM-based concepts to achieve better resolution through the design and fabrication of a customized imaging lens that maintains a high volumetric coverage with increased numerical aperture.  

- Would it be possible to characterize the aberrations that might be still present after whole brain expansion? One approach could be to image small fluorescent nanospheres behind the expanded brain and recover the pupil function via phase retrieval. But even full width half maximum (FWHM) measurements of the nanospheres' images would give some idea of the magnitude of the aberrations. 

We now included a supplementary figure highlighting images of small axon segments within distal regions of the brain.  

Reviewer #2 (Public Review): 

Summary: 

In this manuscript, Glaser et al. describe a new selective plane illumination microscope designed to image a large field of view that is optimized for expanded and cleared tissue samples. For the most part, the microscope design follows a standard formula that is common among many systems (e.g. Keller PJ et al Science 2008, Pitrone PG et al. Nature Methods 2013, Dean KM et al. Biophys J 2015, and Voigt FF et al. Nature Methods 2019). The primary conceptual and technical novelty is to use a detection objective from the metrology industry that has a large field of view and a large area camera. The authors characterize the system resolution, field curvature, and chromatic focal shift by measuring fluorescent beads in a hydrogel and then show example images of expanded samples from mouse, macaque, and human brain tissue. 

Strengths: 

I commend the authors for making all of the documentation, models, and acquisition software openly accessible and believe that this will help assist others who would like to replicate the instrument. I anticipate that the protocols for imaging large expanded tissues (such as an entire mouse brain) will also be useful to the community. 

Weaknesses: 

The characterization of the instrument needs to be improved to validate the claims. If the manuscript claims that the instrument allows for robust automated neuronal tracing, then this should be included in the data. 

The reviewer raises a valid concern. Our assertion that the resolution and contrast is sufficient for robust automated neuronal tracing is overstated based on the data in the paper. We are hard at work on automated tracing of datasets from the ExA-SPIM microscope. We have demonstrated full reconstruction of axonal arbors encompassing >20 cm of axonal length.  But including these methods and results is out of the scope of the current manuscript. 

The claims of robust automated neuronal tracing have been appropriately modified.  

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors): 

Smaller questions to the authors: 

- Would a multi-directional illumination and detection architecture help? Was there a particular reason the authors did not go that route?

Despite the clarity of the expanded tissue, and the lower numerical aperture of the ExA-SPIM microscope, image quality still degrades slightly towards the distal regions of the brain relative to both the excitation and detection objective. Therefore, multi-directional illumination and detection would be advantageous. Since the initial submission of the manuscript, we have undertaken re-designing the optics and mechanics of the system. This includes provisions for multi-directional illumination and detection. However, this new design is beyond the scope of this manuscript. We now mention this in L254-255 of the Discussion section.

- Why did the authors not use the same objective for illumination and detection, which would allow isotropic resolution in ASLM? 

The current implementation of ASLM requires an infinity corrected objective (i.e. conjugating the axial sweeping mechanism to the back focal plane). This is not possible due to the finite conjugate design of the ExA-SPIM detection lens.

More fundamentally, pushing the excitation NA higher would result in a shorter light sheet Rayleigh length, which would require a smaller detection slit (shorter exposure time, lower signal to noise ratio). For our purposes an excitation NA of 0.1 is an excellent compromise between axial resolution, signal to noise ratio, and imaging speed. 

For other potentially brighter biological structures, it may be possible to design a custom infinity corrected objective that enables ASLM with NA > 0.1.

- Have the authors made any attempt to characterize distortions of the brain tissue that can occur due to expansion? 

We have not systematically characterized the distortions of the brain tissue pre and post expansion. Imaged mouse brain volumes are registered to the Allen CCF regardless of whether or not the tissue was expanded. It is beyond the scope of this manuscript to include these results and processing methods, but we have confirmed that the ExA-SPIM mouse brain volumes contain only modest deformation that is easily accounted for during registration to the Allen CCF. 

- The authors state that a custom lens with NA 0.5-0.6 lens can be designed, featuring similar specifications. Is there a practical design? Wouldn't such a lens be more prone to Field curvature? 

This custom lens has already been designed and is currently being fabricated. The lens maintains a similar space bandwidth product as the current lens (increased numerical aperture but over a proportionally smaller field of view). Over the designed field of view, field curvature is <1 µm. However, including additional discussion or results of this customized lens is beyond the scope of this manuscript.

Reviewer #2 (Recommendations For The Authors): 

• System characterization: 

- Please state what wavelength was used for the resolution measurements in Figure 2.

An excitation wavelength of 561 nm was used. This has been added to the manuscript text.

- The manuscript highlights that a key advance for the microscope is the ability to image over a very large 13 mm diameter field of view. Can the authors clarify why they chose to characterize resolution over an 8diameter mm field rather than the full area? 

The 13 mm diameter field of view refers to the diagonal of the 10.6 x 8.0 mm field of view. The results presented in Figure 1c are with respect to the horizontal x direction and vertical y direction. A note indicating that the 13 mm is with respect to the diagonal of the rectangular imaging field has been added to the manuscript text. The results were presented in this way to present the axial and lateral resolution as a function of y (the axial sweeping direction).

- The resolution estimates seem lower than I would expect for a 0.30 NA lens (which should be closer to ~850 nm for 515 nm emission). Could the authors clarify the discrepancy? Is this predicted by the Zemax model and due to using the lens in immersion media, related to sampling size on the camera, or something else? It would be helpful if the authors could overlay the expected diffraction-limited performance together with the plots in Figure 2C. 

As mentioned previously, the resolution measurements were performed with 561 nm excitation and an emission bandpass of ~573 – 616 nm (595 nm average). Based on this we would expect the full width half maximum resolution to be ~975 nm. The resolution is in fact limited by sampling on the camera. The 3.76 µm pixel size, combined with the 5.0X magnification results in a sampling of 752 nm. Based on the Nyquist the resolution is limited to ~1.5 µm. We have added clarifying statements to the text.

- I'm confused about the characterization of light sheet thickness and how it relates to the measured detection field curvature. The authors state that they "deliver a light sheet with NA = 0.10 which has a width of 12.5 mm (FWHM)." If we estimate that light fills the 0.10 NA, it should have a beam waist (2wo) of ~3 microns (assuming Gaussian beam approximations). Although field curvature is described as "minimal" in the text, it is still ~10-15 microns at the edge of the field for the emission bands for GFP and RFP proteins. Given that this is 5X larger than the light sheet thickness, how do the authors deal with this? 

The generated light sheet is flat, with a thickness of ~ 3 µm. This flat light sheet will be captured in focus over the depth of focus of the detection objective. The stated field curvature is within 2.5X the depth of focus of the detection lens, which is equivalent to the “Plan” specification of standard microscope objectives.

- In Figure 2E, it would be helpful if the authors could list the exposure times as well as the total voxels/second for the two-camera comparison. It's also worth noting that the Sony chip used in the VP151MX camera was released last year whereas the Orca Flash V3 chosen for comparison is over a decade old now. I'm confused as to why the authors chose this camera for comparison when they appear to have a more recent Orca BT-Fusion that they show in a picture in the supplement (indicated as Figure S2 in the text, but I believe this is a typo and should be Figure S3). 

This is a useful addition, and we have added exposure times to the plot. We have also added a note that the Orca Flash V3 is an older generation sCMOS camera and that newer variants exist. Including the Orca BT-Fusion. The BT-Fusion has a read noise of 1.0 e- rms versus 1.6 e- rms, and a peak quantum efficiency of ~95% vs. 85%. Based on the discussion in Supplementary Note S1, we do not expect that these differences in specifications would dramatically change the data presented in the plot. In addition, the typo in Figure S2 has been corrected to Figure S3.

- In Table S1, the authors note that they only compare their work to prior modalities that are capable of providing <= 1 micron resolution. I'm a bit confused by this choice given that Figure 2 seems to show the resolution of ExA-SPIM as ~1.5 microns at 4 mm off center (1/2 their stated radial field of view). It also excludes a comparison with the mesoSPIM project which at least to me seems to be the most relevant prior to this manuscript. This system is designed for imaging large cleared tissues like the ones shown here. While the original publication in 2019 had a substantially lower lateral resolution, a newer variant, Nikita et al bioRxiv (which is cited in general terms in this manuscript, but not explicitly discussed) also provides 1.5-micron lateral resolution over a comparable field of view. 

We have updated the table to include the benchtop mesoSPIM from Nikita et al., Nature Communications, 2024. Based on this published version of the manuscript, the lateral resolution is 1.5 µm and axial resolution is 3.3 µm. Assuming the Iris 15 camera sensor, with the stated 2.5 fps, the volumetric rate (megavoxels/sec) is 37.41.

- The authors state that, "We systematically evaluated dehydration agents, including methanol, ethanol, and tetrahydrofuran (THF), followed by delipidation with commonly used protocols on 1 mm thick brain slices. Slices were expanded and examined for clarity under a macroscope." It would be useful to include some data from this evaluation in the manuscript to make it clear how the authors arrived at their final protocol. 

Additional details on the expansion protocol may be included in another manuscript.

General comments: 

• There is a tendency in the manuscript to use negative qualitative terms when describing prior work and positive qualitative terms when describing the work here. Examples include: 

- "Throughput is limited in part by cumbersome and error-prone microscopy methods". While I agree that performing single neuron reconstructions at a large scale is a difficult challenge, the terms cumbersome and error-prone are qualitative and lacking objective metrics.

We have revised this statement to be more precise, stating that throughput is limited in part by the speed and image quality of existing microscopy methods.

- The resolution of the system is described in several places as "near-isotropic" whereas prior methods were described as "highly anisotropic". I agree that the ~1:3 lateral to axial ratio here is more isotropic than the 1:6 ratio of the other cited publications. However, I'm not sure I'd consider 3-fold worse axial resolution than lateral to be considered "near" isotropic.

We agree that the term near-isotropic is ambiguous. We have modified the text accordingly, removing the term near-isotropic and where appropriate stating that the resolution is more isotropic than that of other cited publications.

- exposures (which in the caption is described as "modest"). I'd suggest removing these qualitative terms and just stating the values.

We agree and have changed the text accordingly.

• The results section for Figure 5 is titled "Tracing axons in human neocortex and white matter". Although this section states "larger axons (>1 um) are well separated... allowing for robust automated and manual tracing" there is no data for any tracing in the manuscript. Although I agree that the images are visually impressive, I'm not sure that this claim is backed by data.

We have now removed the text in this section referring to automated and manual tracing.

Some of the world’s first cities were organized without the need for rulers or hierarchies, challenging the idea that urbanization leads to inequality.

It must be hard to pick something to recap considering how many shows people watch now.

I think this is a very powerful concept -- as a former student said, entrepreneurs must have cruel optimism, individuals must be able to adapt and ride out whatever wave or obstacle comes around.

Berlant uses the term cruel optimism to refer to our our investments in “compromised conditions of possibility whose realization is discovered to be impossible, sheer fantasy.” (i.e., we keep cheering for a team we know will lose; we maintain hope in an unattainable romantic ideal promulgated by Hollywood or pursue happiness based on unrealistic beauty standards; we engage in small acts of environmental stewardship like recycling or buying a hybrid in the face of potentially unstoppable climate change...) Berlant basically means that the thing we seek to achieve, the thing (or state of being) that we desire (or the act of seeking and desiring itself) might actually threaten our well-being (that's what makes it cruel!). As she put it succinctly, “a relation of cruel optimism exists when something you desire is actually an obstacle to your flourishing.”

This relates to entrepreneurialism in so many ways: Engaging in the gig economy or a side-hustle as a way to increase one's income (or security) in uncertain times is cruel and optimistic. Similarly, we encounter aspirational labour in the form of internships or any form of unpaid labour while looking for a "real" job. Perhaps you feel the pressure of cultivating a sense of employability. According to Frayne (2015), today, students are expected “to improve their prospects by training, acquiring educational credentials, networking, learning how to project the right kind of personality, and gaining life experiences that match up with the values sought by employers.” In other words, they have to act entrepreneurially even to get a non-entrepreneurial job. As Hawzen et al. (2018) assert, this incites anxiety and results in a colonization of one’s entire life by work-related demands as students feel the need to separate themselves from the competition, doing things like volunteering to gain an advantage or to get a "foot in the door"... We also see it to a certain extent in the example of entrepreneurial vloggers in the sense that the fantasy of a “good life” through fame and fortune is rarely realized. The cruel conditions of precocity are, for most, more of a reality than the fantasy... and we take up this theme explicitly in two weeks hence with digital 'autopreneurs'

Overall, this also highlights one of the reasons I chose this article -- rather than just highlighting how entrepreneurs are certain types of people (or motivated by certain types of things), it emphasizes how entrepreneurship is a mental orientation, not just a business concept but a way of living. But it's not all sunshine and happiness. Cruel optimism, indeed!

What about you? Are you familiar with the feeling of 'cruel optimism'? Does it define the current times or your current disposition?

Remember at the beginning of the article (when Szeman says "we are all entrepreneurs now") (p. 472)? He doesn't mean that we are all creating business start-ups. Rather, he's suggesting that there is a spirit-of-the-times wherein entrepreneurship has become this new common-sense reality. It is both a dominant way of thinking about how we ought to act, AND an informal rulebook for how economies (and other forms of practice) ought to function too... In other words, entrepreneurship isn't just about undertaking profit-making (and risk-inducing) economic practices in capitalism. Rather, it's about undertaking a new subjectivity, a new identity when it comes to how we think of ourselves, how we relate to others, and how we respond to our wider social, cultural, political, and economic environment.

Welcome back, this is part two of this lesson.

We're going to continue immediately from the end of part one.

So let's get started.

Now let's talk about the first of the Glacier Storage Classes, S3 Glacier Instant Retrieval.

If I had to summarize this storage class, it's like S3 standard in frequent access, except it offers cheaper storage, more expensive retrieval costs, and longer minimums.

Standard IA is designed for when you need data instantly, but not very often, say once a month.

Glacier Instant Retrieval extends this, so data where you still want instant retrieval, but where you might only access it say once every quarter.

In line with this, it has a minimum storage duration charge of 90 days versus the 30 days of standard in frequent access.

This class is the next step along the path of access frequency, as the access frequency of objects decrease, you can move them gradually from standard, then to standard in frequent access, and then to Glacier Instant Retrieval.

The important thing to remember about this specific S3 Glacier class is that you still have instant access to your data.

There's no retrieval process required, you can still use it like S3 standard and S3 standard in frequent access.

It's just that it costs you more if you need to access the data, but less if you don't.

Now let's move on to the next type of S3 Glacier Storage Class.

And the next one I want to talk about is S3 Glacier Flexible Retrieval, and this storage class was formally known as S3 Glacier.

The name was changed when the previously discussed Instant Retrieval class was added to the lineup of storage classes available within S3.

So Glacier Flexible Retrieval has the same three availability zone architecture as S3 standard and S3 standard in frequent access.

It has the same durability characteristics of 11-9s, and at the time of creating this lesson, S3 Glacier Flexible Retrieval has a storage cost which is about one-sixth of the cost of S3 standard.

So it's really cost effective, but there are some serious trade-offs which you have to accept in order to make use of it.

For the exam, it's these trade-offs which you need to be fully aware of.

Conceptually, I want you to think of objects stored with the Glacier Flexible Retrieval class as cold objects.

They aren't warm, they aren't ready for use, and this will form a good knowledge anchor for the exam.

Now because they're cold, they aren't immediately available, they can't be made public.

Well, you can see these objects within an S3 bucket, they're now just a pointer to that object.

To get access to them, you need to perform a retrieval process.

That's a specific operation, a job which needs to be run to gain access to the objects.

Now you pay for this retrieval process.

When you retrieve objects from S3 Glacier Flexible Retrieval, they're stored in the S3 standard in frequent access storage class on a temporary basis.

You access them and then they're removed.

You can retrieve them permanently by changing the class back to one of the S3 ones, but this is a different process.

Now retrieval jobs come in three different types.

We have expedited, which generally results in data being available within one to five minutes, and this is the most expensive.

We've got standard where data is usually accessible in three to five hours, and then a low cost bulk option where data is available in between five and 12 hours.

So the faster the job type, the more expensive.

Now this means that S3 Glacier Flexible Retrieval has a first byte latency of minutes or hours, and that's really important to know for the exam.

So while it's really cheap, you have to be able to tolerate, you can't make the objects public anymore, either in the bucket or using static website hosting, and two, when you do access the objects, it's not an immediate process.

So you can see the object metadata in the bucket, but the data itself is in chilled storage, and you need to retrieve that data in order to access it.

Now S3 Glacier Flexible Retrieval has some other limits, so a 40 kb minimum available size and a 90 day minimum available duration.

For the exam, Glacier Flexible Retrieval is for situations where you need to store archival data where frequent or real-time access isn't needed.

For example, yearly access, and you're OK with minutes to hours for retrieval operations.

So it's one of the cheapest forms of storage in S3, as long as you can tolerate the characteristics of the storage class, but it's not the cheapest form of storage.

That honor goes to S3 Glacier Deep Archive.

Now S3 Glacier Deep Archive is much cheaper than the storage class we were just discussing.

In exchange for that, there are even more restrictions which you need to be able to tolerate.

Conceptually, where S3 Glacier Flexible Retrieval, which data in a chilled state, Glacier Deep Archive is data in a frozen state.

Objects have minimum, so 40 kb minimum available size and 180 day minimum available duration.

Like Glacier Flexible Retrieval, objects cannot be made publicly accessible.

Access to the data requires a retrieval job.

Just like Glacier Flexible Retrieval, the jobs temporarily restore to S3 standard and frequent access, but those retrieval jobs take longer.

Standard is 12 hours and bulk is up to 48 hours, so this is much longer than Glacier Flexible Retrieval, and that's the compromise that you agree to.

The storage is a lot cheaper in exchange for much longer restore times.

Glacier Deep Archive should be used for data which is archival, which rarely, if ever, needs to be accessed, and where hours or days is tolerable for the retrieval process.

So it's not really suited to primary system backups because of this restore time.

It's more suited for secondary long-term archival backups or data which comes under legal or regulatory requirements in terms of retention length.

Now this being said, there's one final type of storage class which I want to cover, and that's intelligent tearing.

Now intelligent tearing is different from all the other storage classes which I've talked about.

It's actually the storage class which contains five different storage tiers.

With intelligent tearing, when you move objects into this class, there are a range of ways that an object can be stored.

It can be stored within a frequent access tier or an infrequent access tier, or for objects which are accessed even less frequently, there's an archive instant access, archive access, or deep archived set of tiers.

You can think of the frequent access tier like S3 standard and the infrequent access tier like S3 standard infrequent access, and the archive tiers are the same price of performance as S3, Glacier, instant retrieval, and flexible retrieval.

And the deep archive tier is the same price of performance as Glacier Deep Archive.

Now unlike the other S3 storage classes, you don't have to worry about moving objects between tiers.

With intelligent tearing, the intelligent tearing system does this for you.

Let's say that we have an object, say a picture of whiskers which is initially kind of popular and then not popular, and then it goes super viral.

Well if you store this object using the intelligent tearing storage class, it would monitor the usage of the object.

When the object is in regular use, it would stay within the frequent access tier and would have the same costs as S3 standard.

If the object isn't accessed for 30 days, then it would be moved automatically into the infrequent tier where it would stay while being stored at a lower rate.

Now at this stage you could also add configuration, so based on a bucket, prefix or object tag, any objects which are accessed less frequently can be moved into the three archive tiers.

Now there's a 90 day minimum for archive instant access, and this is fully automatic.

Think of this as a cheaper version of infrequent access for objects which are accessed even less frequently.

Crucially this tier, so archive instant access, still gives you access to the data automatically as and when you need it, just like infrequent access.

In addition to this, there are two more entirely optional tiers, archive access and deep archive.

And these can be configured so that objects move into them when they haven't been accessed for 98 to 270 days for archive access, or 180 through to 730 days for deep archive.

Now these are entirely optional, and it's worth mentioning that when objects are moved into these tiers, getting them back isn't immediate.

There's a retrieval time to bring them back, so only use these tiers when your application can tolerate asynchronous access patterns.

So archive instant access requires no application or system changes, it's just another tier for less frequently accessed objects with a lower cost.

Archive access and deep archive changes things, your applications must support these tiers because retrieving objects requires specific API calls.

Now if objects do stay in infrequent access or archive instant access, when the objects become super viral in access, these will be moved back to frequent access automatically with no retrieval charges.

Intelligent tiering has a monitoring and automation cost per 1000 objects instead of the retrieval cost.

So essentially the system manages the movement of data between these tiers automatically without any penalty for this management fee.

The cost of the tiers are the same as the base S3 tiers, standard and infrequent access, there's just the management fee on top.

So it's more flexible than S3 standard and S3 infrequent access, but it's more expensive because of the management fee.

Now intelligent tiering is designed for long-lived data where the usage is...

[Sounds of S3 storage] Changing or unknown, if the usage is static either frequently accessed or infrequently accessed, then you're better using the direct S3 storage class, either standard or infrequent access.

Intelligent tiering is only good if you have data where the pattern changes or you don't know it.

Now with that being said, that's all of the S3 storage classes which I want to cover.

That's at least enough technical information and context which you'll need for the exam and to get started in the real world.

So go ahead and complete the video and when you're ready, I look forward to you joining me in the next.

Welcome back and in this demo lesson I just want to give you the opportunity to gain some practical experience of how S3 handles encryption.

So what we're going to do is create an S3 bucket and into that bucket we're going to put a number of objects and for each one we're going to utilize a different type of server-side encryption.

So we'll be uploading one object using SSE-S3 so S3 managed encryption and then one object using SSE-KMS which will utilize KMS for key management.

So once we've uploaded those objects we'll experiment with some permissions changes just to see how each of these different encryption types work.

So let's get started.

Now the first thing you'll need to check is that you're logged into the IAM admin user of the general AWS account and you need to have the Northern Virginia region selected.

Then let's move to S3 so click in the services drop-down type S3 and then open that in a new tab and then click to move to the S3 console.

Now I want you to go ahead and click on create bucket and then just create a bucket called catpicks and then put some random text on the end.

You should use something different and something that's unique to you.

Leave the region set as US East Northern Virginia scroll all the way down to the bottom and click on create bucket.

Next change across to the key management service console that will either be in your history or you'll have to type it in the services drop-down.

Once you hear go ahead and click on create key, pick symmetric key then expand advanced options, make sure KMS is selected as well as single region key.

Click next.

The alias we'll be using is catpicks, so type catpicks and click next.

Don't select anything for define key administrative permissions.

We'll not set any permissions on the key policy this time so just click next and then on this screen define key usage permissions just click next again without selecting anything.

So the key policy that's going to be created for this KMS key only trusts the account so only trust the account user of this specific AWS account and that's what we want.

So go ahead and click on finish.

At this point move back across to the S3 console, go into the catpicks bucket that you just created and I'll let you to go ahead and download the file that's attached to this lesson and then extract that file and inside the resultant folder are some objects that you're going to be uploading to this S3 bucket.

So go ahead and do that and then click on upload and files then locate the folder that you just extracted and go into it and you should see three images in that folder default -merlin.jpg, sse -kms -ginny.jpg and sse -s3 -dwees.jpg.

So that's good so the first one that we want to upload need to do these one by one because we're going to be configuring the encryption type to use.

So the first one is sse -s3 -dwees.jpg so select that and click on open, expand properties and then scroll down to serve aside encryption and it's here where you can specify to accept the bucket defaults by not specifying an encryption key or you can specify an encryption key.

Now when you pick to specify an encryption key you're again offered the ability to use the bucket default settings for encryption or you can override bucket settings and choose between two different types either Amazon s3 key which is sse -s3 or AWS key management service which is sse -kms.

Now for this upload we're going to use sse -s3 so Amazon s3 managed keys so select this option scroll all the way down to the bottom and click upload.

Wait for this upload process to complete and then click on close.

Now we're going to follow that same process again so click on upload again, add files.

This time sse -kms -ginny.

So select that click on open, expand properties and then scroll down to serve aside encryption then click specify an encryption key override bucket settings for default encryption and this time we're going to use sse -kms so select that and then select choose from your AWS kms keys and then you can either use the AWS managed key so this is the service default key so AWS forward slash s3 or you can choose your own kms key to use for this object.

What I'm going to do first is select this AWS managed key so the default key for this service and scroll all the way down to the bottom and click on upload.

Wait for this upload process to complete and then click on close so that will encrypt the object using this default s3 AWS managed kms key that's now inside kms.

I wanted to do that just to demonstrate how it automatically creates it so now let's go ahead and re-upload this object so click on upload, add files, select sse -kms -ginny.jpg and click on open, scroll down, expand properties, scroll down again, for service side encryption select specify an encryption key, select override bucket settings for default encryption, pick AWS key management service key so sse -kms, select choose from your AWS kms keys, click in the drop down and then select captics that we created earlier.

Once you've done that scroll all the way down to the bottom click on upload, wait for that process to complete and then click on close.

Now at this point we've got two different objects in this bucket and we're going to open both of these.

We're going to start with sse -s3 -dwees so let's click on it and then click on open and that works and then let's try the other object sse -kms -ginny so click on that and click on open and that also opens okay because IAM admin is a full administrator of the entire AWS account so that includes s3 and all the services including KMS.

Next what we're going to do is apply a deny policy on the IAM admin user which prevents us from using KMS so we'll stay as being a full account administrator and a full s3 administrator but we're going to block off the KMS service entirely and I want to demonstrate exactly what that does to our ability to open these three objects.

So click on services and either open the IAM console from the history or type it in the find services box and then click it.

Once we're here click on users select your IAM admin user, click add permissions and then create inline policy, click on the JSON tab and then delete the skeleton template that's in this box and then paste in the contents of the deny kms.json file and this is contained in the folder you extracted from this lessons download link and it's also attached to this lesson.

This is what it should look like the effect is to deny it denies any actions KMS call on star so any KMS actions on all resources so essentially this blocks off the entire KMS service for this user.

So go ahead and click on review policy call it deny KMS and click on create policy and this now means that IAM admin can no longer access KMS so now if we go back to the s3 console go inside the capex bucket we should still be able to open sse-s3 dweez.jpeg object.

If we click that click on open because this is encrypted using sse-s3 and this is completely internal to the s3 product we should have no problems owning this object because we have the permissions inside s3 to do anything in s3 but something different happens if we try to open the sse-kms-ginning object.

Now just to explain what will occur when I click on this open link s3 will then have to liaise with KMS and get KMS to decrypt the data encryption key that encrypts this object so we need to retrieve the encrypted data encryption key for this object and request that KMS decrypts it.

Now if that worked we'd get back the plain text version of that key and we would use it to decrypt this object and it would open up in a tab without any issues because we've got full rights over s3 we have permission to do almost all of that process but what we don't have permission to do is to actually get KMS to decrypt this encrypted data encryption key.

We don't have that permission because we just added a deny policy to the IAM admin user and as we know by now deny allow deny deny always wins and explicit deny always overrules everything else.

So when I click on open and s3 retrieves this encrypted data encryption key and gives it to KMS and says please decrypt this and give me the plain text back KMS is going to refuse and what we see when we do that is we get an access deny.

So now we've implemented this role separation so even though we have full s3 admin rights so if I went back to this bucket and I clicked on the sse-kms-ginning file and deleted it I would be able to delete that object because I have full control over s3 but I can't open it because I've prevented us accessing KMS and that's how we implement role separation.

So sse-kms is definitely the encryption type that you want to use if you've got these really extensive regulations or any security requirements around key control.

So let's go ahead and just remove that restriction so just go back to the IAM console.

I just want to do this before we forget and have problems later in the course.

Click on users click on IAM admin check the box next to deny KMS and then click remove and confirm that removal and that will allow us to access KMS again.

We can verify that by moving to the KMS console and we can bring up this list which proves that we've got some access to KMS again so that's good.

Now if we just go ahead and click on the AWS managed keys option on the left here this is where you'll be able to see this default encryption key that's used when you upload an object using sse-kms-incription but don't pick a particular key so this is now the default.

Now if we open this because it's an AWS managed key we don't have the ability to set any key rotation we can see the key policy here but we can't make any changes to it this is set by AWS when it creates it so that it only allows accesses from s3 so this is a fixed key policy but we can't control anything about this key.

Now contrast that with the customer managed keys that we've got and if we go into cat pics this is the key that we created now we can edit the key policy we could switch to policy view and make changes we've got the ability to control the key rotation so if you face any exam questions where you need to fully manage the keys that are used as part of the s3 encryption process then you've got to use sse-kms.

Now if we just return to the s3 console there's just one more thing that I want to demonstrate go into the cat pics bucket again click on properties locate default encryption and then click on edit and this is where you get the option to specify the default encryption to use for this bucket.

Now again this isn't a restriction this does not prevent anyone uploading objects to the bucket using a different type of encryption all it does is specify what the default is if the upload itself does not specify an encryption method so we could select Amazon s3 key which is sse-s3 and you might also see this referred to elsewhere as AES 256 it's also known by that name but we could also select the AWS key management service key known as sse-kms and this is where we can either choose to use the default key or pick a customer managed key that we want to use as the default for the bucket.

So let's just demonstrate that go ahead and select the cat pics key to use for this bucket then scroll down and click on save changes and that will set the defaults for the bucket and we can demonstrate that let's go ahead and click on the objects tab and we're going to upload a new object to this bucket so click on upload add files then go ahead and select the default hyphen Merlin object and click open scroll down click on upload and even though we didn't pick a particular encryption method for this object it's going to use the default settings that we picked for the bucket and now we can see that default hyphen Merlin.jpg object has been uploaded so if we open up default hyphen Merlin we can see it's using sse-kms as the service side encryption type and it's using the KMS key that we set in the default encryption settings on the bucket.

Okay well that's everything I wanted to cover in this demo lesson so let's just tidy up to make sure that we don't experience any charges go back to the Amazon S3 console select the bucket that you've created and then click on empty you need to confirm to empty that bucket once that process is completed and the bucket's emptied and then follow that same process but this time click on delete to delete the bucket from your AWS account click on key management service and we'll just mark the key that were created for deletion so select customer managed keys select cat pics click on key actions schedule key deletion set this to 7 which is the minimum check the box and click on schedule deletion with that being said though that's everything I wanted to cover in this demonstration I hope it's been fun and useful go ahead mark this video is complete and when you're ready I'll see you in the next video.

Automatic thinking causes us to simplify problems and see them through narrow frames. We fi ll in miss- ing information based on our assumptions about the world and evaluate situations based on associations that automatically come to mind and belief systems that we take for granted. In so doing, we may form a mistaken picture of a situation, just as looking through a small window overlooking an urban park could mis- lead someone into thinking he or she was in a more bucolic place. page 12

I think that the results from the research conducted on culting stigmatized identity affecting students' performances made me realize how much of a mental toll stereotypes can play on people. It's disheartening and ironic simultaneously to see how the high and low sides of the caste-system groups collectively performed worse when they were told their respective roles. It influences the way that I think as a student going to an international school because it is interesting to see how these ideas can parallel to students around me. Regardless, this passage relates to today's inquiry question because it can be used and argued to reflect how poor people shape individual economic actions due to neglect and stereotypes affecting their life subconsciously.

Welcome back and in this lesson I want to talk about S3 encryption.

Now we're going to be focusing on server-side encryption known as SSE, which I will be coaching on client-side encryption and how that's different.

Now we've got a lot to get through so let's jump in and get started.

Now before we start there's one common misconception which I want to fix right away, and that's that buckets aren't encrypted, objects are.

You don't define encryption at the bucket level.

There's something called bucket default encryption, but that's different and I'll cover that elsewhere in the course.

For now, understand that you define encryption at the object level, and each object in a bucket might be using different encryption settings.

Now before we talk about the ways that S3 natively handles encryption for objects, I think it's useful to just review the two main architectures of encryption which can be used with the product.

There's client-side encryption and server-side encryption, and both of these refer to what method is used for encryption at rest, and this controls how objects are encrypted as they're written to disk.

It's a method of ensuring that even if somebody were to get the physical disks from AWS which your data is on, they would need something else, a type of key to access that data.

So visually this is how a transaction between a group of users or an application and S3 looks like.

The users of the application on the left are loading data to an S3 endpoint for a specific bucket which gets stored on S3's base storage hardware.

Now it's a simplistic overview, but for this lesson it's enough.

I want to illustrate the difference between client-side encryption and server-side encryption.

So on the top we have client-side encryption, and on the bottom we have server-side encryption.

Now this is a really, really important point which often confuses students.

What I'm talking about in this lesson is encryption at rest, so how data is stored on disk in an encrypted way.

Both of these methods also use encryption in transit between the user-side and S3.

So this is an encrypted tunnel which means that you can't see the raw data inside the tunnel.

It's encrypted.

So ignoring any S3 encryption, ignoring how data is encrypted as it's written to disk, data transferred to S3 and from S3 is generally encrypted in transit.

Now there are exceptions, but use this as your default and I'll cover those exceptions elsewhere in the course.

So in this lesson when we're talking about S3 encryption, we're focusing on encryption at rest and not encryption in transit, which happens anyway.

Now the difference between client-side encryption and server-side encryption is pretty simple to understand when you see it visually.

With client-side encryption, the objects being uploaded are encrypted by the client before they ever leave, and this means that the data is ciphertexted the entire time.

From AWS's perspective, the data is received in a scrambled form and then stored in a scrambled form.

AWS would have no opportunity to see the data in its plain text form.

With server-side encryption known as SSE, it's slightly different.

Here, even though the data is encrypted in transit using HTTPS, the objects themselves aren't initially encrypted, meaning that inside the tunnel, the data is in its original form.

Let's assume it's animal images.

So you could remove the HTTP encrypted tunnel somehow and the animal pictures would be in plain text.

Now once the data hits S3, then it's encrypted by the S3 servers, which is why it's referred to as server-side encryption.

So to high level, the differences are with client-side encryption, everything is yours to control.

You take on all of the risks and you control everything, which is both good and bad.

You take the original data, you are the only one who ever sees the plain text version of that data, you generate a key, you hold that key and you manage that key.

You are responsible for recording which key is used for which object, and you perform the encryption process before it's uploaded to S3, and this consumes CPU capacity on whatever device is performing the encryption.

You just use S3 for storage, nothing else.

It isn't involved in the encryption process in any way, so you own and control the keys, the process and any tooling.

So if your organization needs all of these, if you have real reasons that AWS cannot be involved in the process, then you need to use client-side encryption.

Now with server-side encryption known as SSE, you allow S3 to handle some or all of that process, and this means there are parts that you need to trust S3 with.

How much of that process you trust S3 with and how you want the process to occur and determine which type of server-side encryption you use as there are multiple types.

Now AWS has recently made server-side encryption mandatory, and so you can no longer store objects in an unencrypted form on S3.

You have to use encryption at rest.

So let's break apart server-side encryption and review the differences between each of the various types.

There are three types of server-side encryption available for S3 objects, and each is a trade-off of the usual things, trust, overhead, cost, resource consumption and more.

So let's quickly step through them and look at how they work.

The first is SSE-C, and this is server-side encryption with customer-provided keys.

Now don't confuse this with client-side encryption because it's very different.

The second is SSE-S3, which is server-side encryption with Amazon S3 managed keys, and this is the default.

The last one is an enhancement on SSE-S3, which is SSE-KMS, and this is server-side encryption with KMS keys stored inside the AWS Key Management Service, known as KMS.

Now the difference between all of these methods is what parts of the process you trust S3 with and how the encryption process and key management is handled.

At a high level, there are two components to server-side encryption.

First, the encryption and decryption process.

This is the process where you take plain text, a key and an algorithm, and generate cyber text.

It's also the reverse, so taking that cyber text and a key and using an algorithm to output plain text.

Now this is symmetrical encryption, so the same key is used for both encryption and decryption.