How enhancers regulate wavelike gene expression patterns

Article activity feed

1. 

   Version published to 10.7554/elife.84969 on eLife

   Jul 11, 2023
2. 

   eLife

   Jun 12, 2023

   Author Response

   Reviewer #1 (Public Review):

   The Introduction starts by setting up a straw-man argument, claiming that the assumption is that gene expression is set up as stable expression domains that undergo little or no subsequent change. I don't think that any current developmental biologist thinks this is true. The references used to support this claim are from the 1990s up to the early 2000s. There are numerous examples since then that show that developmental gene expression is dynamic as a rule.

   Our argument might seem like a strawman for certain sector of developmental biologists who work in the field of pattern formation, or aware of the latest advances in the field. However, a look at current publications on developmental enhancers reveals that the dominant model with which enhancer biologists interpret their data is still …

   Author Response

   Reviewer #1 (Public Review):

   The Introduction starts by setting up a straw-man argument, claiming that the assumption is that gene expression is set up as stable expression domains that undergo little or no subsequent change. I don't think that any current developmental biologist thinks this is true. The references used to support this claim are from the 1990s up to the early 2000s. There are numerous examples since then that show that developmental gene expression is dynamic as a rule.

   Our argument might seem like a strawman for certain sector of developmental biologists who work in the field of pattern formation, or aware of the latest advances in the field. However, a look at current publications on developmental enhancers reveals that the dominant model with which enhancer biologists interpret their data is still the French Flag model (specifically, the eve-stripe-2 model of enhancer function). We meant to address this audience, and attempted to clarify this from the very beginning by stating that “Much of our models of how enhancers work during development relies on the assumption that …”. Please, note here that we are talking about “models of how enhancers work”, not models of pattern formation in general.

   The Introduction then continues as a rather detailed review of enhancers, Tribolium methodology, tools for identifying enhancers, and more. The Introduction cites 99 references, which seems excessive for what is essentially an experimental paper. Significant parts of the Introduction can be trimmed or removed. There is no need to mention all the tools available for Tribolium if they are not used in the described experiments. A thorough analysis of the advantages and disadvantages of different modes of ATAC-seq is also beyond the scope of the Introduction. The authors should explain why they chose the tools they chose without excessive background.

   In the revised manuscript, we shortened the discussion of Tribolium methodologies and imaging techniques. However, we think that the paragraph discussing ATAC-seq strategies are important to justify our choices as why we took the effort to cut the embryos to perform tissue-specific ATAC-seq analysis, instead of performing whole-embryo ATAC-seq.

   Having said that, the Introduction actually overlooks a lot of significant work that is relevant to the subject of the paper. Specifically, the authors completely ignore all of the work on development in hemimetabolous insects such as Oncopeltus and Gryllus - the omission is glaring. There has been a lot of relevant work on dynamic gene expression patterns coming out of these species.

   You are right indeed. We apologize for that. We added now citations to relevant works from those to insect to the manuscript.

   The experimental setup involves cutting embryos into three sections at two time points. The results then discuss differences in "space" and "time" but there is no discussion of the embryological meaning of these terms. What is happening at the two time points from a developmental perspective? What is the difference between the three sections? There is a lot of relevant development going on at these stages and important regional differences, which have been well-studied in Tribolium and in other insects but are not even mentioned.

   A good point. Correlating chromatin landscape changes with embryological events is an interesting point that needs further analysis and the application of ATAC-seq to further timepoints. We chose leaving this to future work (possibly using single cell ATAC-seq). In this work, we restricted our analysis to the benefits of applying time- and tissue-specific ATAC-seq in predicting active enhancers. We added a note on this point in the discussion.

   In the preliminary results of the ATAC-seq analysis, it is clear that there are significant differences between the sections, which should come as no surprise, but fairly minor differences between the same section at the two time points. This could be because the two time points are pretty close together at a stage when there is a lot of repetitive patterning going on. A possible interpretation, which the authors don't mention because it goes against their main thesis, is that maybe most of the processes that are taking place at this stage are not dynamic enough to show up at the temporal resolution they have applied. This is worth at least a mention.

   We agree with this observation. We would like to draw the reviewer’s attention to our statement “Together, our findings indicate that changes in chromatin accessibility in Tribolium at this developmental stage are primarily associated with space rather than time…””. Detailed analysis of the chromatin dynamics across time would need taking more datapoints, which is something we plan to do in future work.

   The authors link each accessible site to the nearest gene when looking at putative enhancer function. This is a risky assumption since there are many examples of enhancer sites that are far upstream or downstream of the target gene and often closer to an unrelated gene than to the target gene. The authors should at least acknowledge this problem with their functional annotation.

   The reviewer is correct in that, in particular for large eukaryotic genomes, enhancers are often located far away from their target genes. We have no comprehensive enhancer-target data that would enable us to perform a more accurate analysis. Furthermore, the assumption that at least for some of the enhancers the nearest genes will also be their targets, and hence, provide insight into the function of the enhancers themselves seems reasonable given the relatively compact organization of the Tribolium genome. In any case, the analysis was just presented as one of several sanity checks for our ATAC-seq data; for the sake of streamlining the manuscript we no longer include this analysis in the current version of the manuscript.

   In the Discussion, the authors claim that contrary to how it may seem, the question they are addressing is not a "fringe problem". Once again, I think this is a straw man. No active researcher thinks that the question of dynamic regulation of gene expression during development is a fringe problem. On the contrary, most researchers will accept that this is one of the most interesting and important questions in current developmental biology.

   This whole argument was removed from the Discussion in the revised manuscript.

   Perhaps the most significant problem with the manuscript is that it is all built around the premise of enhancer switching between dynamic enhancers and static enhancers. The authors find one site that is consistent with their prediction for a dynamic enhancer and one site - regulating a different gene - that is consistent with their prediction for a static enhancer and claim that they have provided support for their model. I think this claim is grossly exaggerated. They present data that can be seen as consistent with their model but are a long way from providing evidence for it.

   We actually thought we were cautious enough about this. Nowhere in our text did we mention that our data “support” the enhancer switching model. We stated quite early (in the abstract, actually) that:

   “We found our data consistent with a model in which the timing of gene expression during embryonic pattern formation is mediated by a balancing act between enhancers that induce rapid changes in gene expressions (that we call ‘dynamic enhancers’) and enhancers that stabilizes gene expressions (that we call ‘static enhancers’).”

   To make this message clearer, we added the following sentence to the abstract of the revised manuscript: “However, more data is needed for a strong support for this or any other alternative models.” And again at the end of the Introductions: “While these data are in line with our Enhancer Switching model, more data is needed as a strong support for the model.” Also, at the end of the Results section examining runB enhancer dynamics, we stated: “However, this merely shows that runB activity dynamics are consistent with our model, but is still far from strongly supporting the model (more on that in the Discussion).” Also for the Results section on enhancer hbA dynamics: “Again, this merely shows that hbA activity dynamics are consistent with our model, but is still far from strongly supporting it.”.

   Moreover, in the opening paragraph of the Discussion, we explicitly and quite openly addressed this point, and suggested what kind of observations and experiments needed in the future to qualify as a “strong support” for the model. We even ran simulations for what kind of observation should one expect in enhancer deletion experiments if the model is correct (Figure 7).

   But it seems like discussing the enhancer switching model in detail gives the impression of its central importance to the paper. In our view, our experimental system is quite general and does not depend on that model, but the point of mentioning it is that it is an example of how could an alternative model of enhancer regulation be of relevance to the problem of dynamic gene expression. This wouldn’t be obvious without this or a similar model that is showing this, even if it is hypothetical. But since our presentation is obviously giving the impression that our claims are stronger that they really are, we altered our phrasing in the introduction of the revised manuscript to make our point clearer:

   “Despite its potential inaccuracies, the Enhancer Switching model exemplifies the type of alternative frameworks we need to explore in order to elucidate the mechanisms driving the generation of gene expression waves during development. Consequently, an appropriate model system is required, allowing us to test not only the Enhancer Switching model but also any other prospective model that provides a satisfactory explanation for the initiation of gene expression waves at the enhancer level.”

   We hope that this addresses the reviewer’s quite legitimate concerns.

   Like the Introduction, the Discussion includes long paragraphs (lines 450-480) that are more suitable for a review/hypothesis paper. The data presented in this manuscript has little relevance to the question of kinematic vs. trigger waves, and therefore there is no real reason for the question to be discussed here.

   We have now significantly shortened the discussion.

   Reviewer #2 (Public Review):

   Open questions:

   What happens with the runB enhancer at later stages of embryogenesis? With what kind of dynamics do the anterior-most stripes fade and does that agree with the model? Do they show the same dynamics throughout segmentation? I think later stages need to be shown because the prediction from the model would be that the dynamics are repeated with each wave. I am not so sure about the prediction for ageing stripes – yet it would have been interesting to see the model prediction and the activity of the static enhancer.

   Yes, the dynamics repeats in the germband. This is shown in Supplementary Figure 8. The dynamics in germband were shown by visualizing yellow mRNA and intronic probes. MS2 imaging was not possible to be used because the embryo dive into the yolk for a while, and then it becomes difficult to capture the germband in the right orientation for imaging. We are currently working to use light sheet microscopy for imaging germband stages.

   I understand that the mRNA of the reporter gene yellow is more stable than the runt mRNA. This might interfere with the possibility to test your prediction for static enhancers: The criterion is that the stripes should increase in strength as the wave migrates towards the anterior. You show this for runB – but given that yellow has a more stable transcript – could this lead to a “false positive” increase in intensity with the slower migration and accumulation of transcripts? I would feel more comfortable with the statement that this is a static enhancer if you could exclude that the signal is blurred by an artifact based on different mRNA stability. What about re-running the simulation (with the p–rameters that have shown to well reflect endogenous –unt mRNA levels) but i“creasing the parameter for the stability of the mRNA? Are static and dynamic enhancers still distinguishable? The claim of having found a static enhancer rests on this increase in signal, hence, other explanations need to be excluded carefully.

   Good questions. Note that runB reporter dynamics were examined not only by visualizing yellow mRNAs (which indeed seem to be more stable than endogenous run mRNA; see Supplementary Figure 10), but also using MS2 (with virtually zero mRNA stability; although stability was simulated in the shown movies to show virtual mRNA dynamics), and intronic yellow mRNA (showing de novo transcription; Supplementary Figure 10; you will need to zoom in to see intronic de novo transcripts). The expected dynamics of a static enhancer reporter is quite unique: it progressively increases initially as it propagates from posterior to anterior, then it progressively decreases as it slows down and stabilizes at the anterior. Then they eventually fade. These full range of dynamics is obvious in germband embryos stained for intronic yellow to show de novo transcription of runB enhancer reporter (Supplementary Figure 10; you will need to zoom in to see intronic de novo transcripts).

   Running the simulation for the model using different degradation rates for the enhancer reporter made the static enhancer’s expression either less or more persistent, but gave the same overall result: the static enhancer expression has diminished expression at the very posterior, but high expression as its expression wave exiting the growth-zone/SAZ. This is consistent with not only yellow mRNA expressions of runB, but with its intronic expression as well (Supplementary Figure 10; you will need to zoom in to see intronic de novo transcripts).

   What about the head domain of the runB enhancer (e.g. Fig. 6A lowest row): This seems to be different from endogenous expression in your work and in Choe et al. Is that aspect different from endogenous expression and can this be reconciled with your model?

   Yes, indeed this aspect cannot be explained by our model. We believe that head patterning in insects is regulated by a different regulatory network. This network might be (de)-activated by missing repressors in the selected DNA segment for runB enhancer. We mentioned this issue in the revised manuscript.

   The claim of similar dynamics of expression visualized by in situ and MS2 in vivo relies on comparing Fig. 6C with 6A. To compare these two panels, I would need to know to what stage in A the embryo in C should be compared. Actually, the stripe in 6C appears more crisp than the stripes in 6A.

   Were the enhancer dynamics tested in vivo at later stages as well? I would appreciate a clear statement on what stages can be visualized and where the technical boundaries are because this will influence any considerations by others using this system.

   One really cannot be that super-precise about the timing of a very dynamic process in space and time like this one we are studying. We believe that Figure 6D shows clearly that runB activity dynamics are similar to endogenous run expression.

   How do the reported accessibility dynamics of runA enhancer correlate with the activity of the reporter: E.g. is the enhancer open in the middle body region but closed at the posterior part of the embryo? Or is it closed at the anterior – and if so: why is there a signal of the reporter in the head?

   You show that chromatin accessibility dynamics help in identifying active enhancers. Is this idea new or is it based on previous experience with Drosophila (e.g. PMID: 29539636 or works cited in https://doi.org/10.1002/bies.201900188)? Or in what respect is this novel?

   Our manuscript contributes to the growing body of evidence confirming that accessibility per se does not imply activity. Of course, this is not a new idea, but given the widely use of accessibility as a proxy for enhancer activity in the genomics community, we do feel it is important to reiterate the message. As the reviewer correctly indicates, several published findings point to a correlation between accessibility dynamics and enhancer activity. However, to our knowledge, this is the first example in Tribolium. It is important to point out that what “dynamic” means strongly depends on the experimental design. Even in Drosophila, not enough studies have been conducted to fully understand the relationship (e.g., ideally, this should be done on a continuous time scale and at single cell level). We acknowledge in the manuscript that this relationship has been observed before in other species (and have added the references suggested by the reviewer, for which we are very grateful), but still believe that our observations are highly significant to the Tribolium community.

   Reviewer #3 (Public Review):

   I have two major concerns: First, the claim about differential accessibility being related to enhancer activity is not really established from the presented data, in my view. This needs to be clarified. (I do believe in the claim to some extent, but not based on presented evidence.)

   We agree with the reviewer that more data – and, more importantly, independent replication – are necessary to confirm this finding. Please, refer to our response to your comment regarding the statistical significance of the findings.

   Second, the evidence in support of the Enhancer Switching model for runt should be accompanied by identification of and spatiotemporal profiling of the “speed regulator”, if this is not established yet.

   Experiments supporting the role of Cad as a speed regulator for both pair-rule and gap genes have been published in El-Sherif et al PLOS Genetics 2014 and Zhu et al PNAS 2017. We added a comment stressing this fact.

   In addition to these two concerns, the simulations of the Enhancer Switching model need to be described, at least in the outline, in the Methods section.

   Done

   Read the original source
3. 

   eLife

   Jun 8, 2023

   eLife assessment

   The authors describe a sophisticated method to follow enhancer activity in both live embryos and fixed embryos in Tribolium and present important data about the function of a number of enhancers in early development. They show that some of the enhancers are "dynamic" and others are "static" and use this to provide support for the "enhancer-switching" model of gene regulation suggested by some of these authors in the past. However, the evidence they provide is incomplete and although it is consistent with the model, it does not directly support it.

   Read the original source
4. 

   eLife

   Jun 8, 2023

   Reviewer #1 (Public Review):

   The manuscript by Mau et al. describes a sophisticated method to follow enhancer activity in both live embryos and fixed embryos in Tribolium. The authors identified putative enhancers via comparative ATAC-seq of embryos divided into different regions and at different developmental time points. As an experimental piece of work, this is excellent. However, the framing and presentation in this manuscript would need to be improved to avoid misrepresentation of existing ideas and over-interpretation of results. The manuscript would require significant re-writing. This can be done without additional data or analyses, but simply more careful writing.

   The Introduction starts by setting up a straw-man argument, claiming that the assumption is that gene expression is set up as stable expression domains that undergo …

   Reviewer #1 (Public Review):

   The manuscript by Mau et al. describes a sophisticated method to follow enhancer activity in both live embryos and fixed embryos in Tribolium. The authors identified putative enhancers via comparative ATAC-seq of embryos divided into different regions and at different developmental time points. As an experimental piece of work, this is excellent. However, the framing and presentation in this manuscript would need to be improved to avoid misrepresentation of existing ideas and over-interpretation of results. The manuscript would require significant re-writing. This can be done without additional data or analyses, but simply more careful writing.

   The Introduction starts by setting up a straw-man argument, claiming that the assumption is that gene expression is set up as stable expression domains that undergo little or no subsequent change. I don't think that any current developmental biologist thinks this is true. The references used to support this claim are from the 1990s up to the early 2000s. There are numerous examples since then that show that developmental gene expression is dynamic as a rule.

   The Introduction then continues as a rather detailed review of enhancers, Tribolium methodology, tools for identifying enhancers, and more. The Introduction cites 99 references, which seems excessive for what is essentially an experimental paper. Significant parts of the Introduction can be trimmed or removed. There is no need to mention all the tools available for Tribolium if they are not used in the described experiments. A thorough analysis of the advantages and disadvantages of different modes of ATAC-seq is also beyond the scope of the Introduction. The authors should explain why they chose the tools they chose without excessive background. Having said that, the Introduction actually overlooks a lot of significant work that is relevant to the subject of the paper. Specifically, the authors completely ignore all of the work on development in hemimetabolous insects such as Oncopeltus and Gryllus - the omission is glaring. There has been a lot of relevant work on dynamic gene expression patterns coming out of these species.

   The experimental setup involves cutting embryos into three sections at two time points. The results then discuss differences in "space" and "time" but there is no discussion of the embryological meaning of these terms. What is happening at the two time points from a developmental perspective? What is the difference between the three sections? There is a lot of relevant development going on at these stages and important regional differences, which have been well-studied in Tribolium and in other insects but are not even mentioned.

   In the preliminary results of the ATAC-seq analysis, it is clear that there are significant differences between the sections, which should come as no surprise, but fairly minor differences between the same section at the two time points. This could be because the two time points are pretty close together at a stage when there is a lot of repetitive patterning going on. A possible interpretation, which the authors don't mention because it goes against their main thesis, is that maybe most of the processes that are taking place at this stage are not dynamic enough to show up at the temporal resolution they have applied. This is worth at least a mention.

   The authors link each accessible site to the nearest gene when looking at putative enhancer function. This is a risky assumption since there are many examples of enhancer sites that are far upstream or downstream of the target gene and often closer to an unrelated gene than to the target gene. The authors should at least acknowledge this problem with their functional annotation.

   In the Discussion, the authors claim that contrary to how it may seem, the question they are addressing is not a "fringe problem". Once again, I think this is a straw man. No active researcher thinks that the question of dynamic regulation of gene expression during development is a fringe problem. On the contrary, most researchers will accept that this is one of the most interesting and important questions in current developmental biology.
   Perhaps the most significant problem with the manuscript is that it is all built around the premise of enhancer switching between dynamic enhancers and static enhancers. The authors find one site that is consistent with their prediction for a dynamic enhancer and one site - regulating a different gene - that is consistent with their prediction for a static enhancer and claim that they have provided support for their model. I think this claim is grossly exaggerated. They present data that can be seen as consistent with their model but are a long way from providing evidence for it.
   Like the Introduction, the Discussion includes long paragraphs (lines 450-480) that are more suitable for a review/hypothesis paper. The data presented in this manuscript has little relevance to the question of kinematic vs. trigger waves, and therefore there is no real reason for the question to be discussed here.

   Read the original source
5. 

   eLife

   Jun 8, 2023

   Reviewer #2 (Public Review):

   Embryonic development requires differential gene expression, which is regulated by enhancer elements. Regulatory proteins bind to these DNA elements to regulate close-by promoters. Key insights into the molecular mechanisms of enhancer function have been gained by studying fly segmentation, where a hierarchical cascade of gene regulation subdivides the embryo into ever smaller units. However, segmentation in other insects and arthropods as well as in vertebrates relies on a much more dynamic process where repetitive gene expression patterns appear to migrate across tissues similar to waves. Only recently, models have been proposed that make predictions on the underlying gene regulatory networks (GRN) and the properties of the respective enhancers. Specifically, the previously suggested model of the authors, …

   Reviewer #2 (Public Review):

   Embryonic development requires differential gene expression, which is regulated by enhancer elements. Regulatory proteins bind to these DNA elements to regulate close-by promoters. Key insights into the molecular mechanisms of enhancer function have been gained by studying fly segmentation, where a hierarchical cascade of gene regulation subdivides the embryo into ever smaller units. However, segmentation in other insects and arthropods as well as in vertebrates relies on a much more dynamic process where repetitive gene expression patterns appear to migrate across tissues similar to waves. Only recently, models have been proposed that make predictions on the underlying gene regulatory networks (GRN) and the properties of the respective enhancers. Specifically, the previously suggested model of the authors, the enhancer switching model, predicted that each gene expression wave should actually be regulated by two GRNS - one based on a "dynamic enhancer", which directs the early wave-like pattern and the other involving a "static enhancer", which directs the more stable expression defining the segment anlagen at the end of each cycle. However, these predicted enhancer types have not been described so far. In flies, where the respective methodology would be available, the segmentation does not show prominent wave-like patterns. In beetles, where pronounced wave-like patterns have been described, the respective methodology has been missing.

   With this work, the authors establish a genomic resource and a transgenic line in the red flour beetle in order to establish it as a model system to tackle questions on enhancers driving dynamic expressions during development. First, they determine the open chromatin at early embryonic stages thereby generating a valuable resource for enhancer detection. They did so by dissecting the embryos of two temporal stages into three parts (head, middle part, and growth zone) and then determined open chromatin via ATAC-seq. This setup allowed for a comparison across tissues and stages to identify dynamically regulated chromatin. Indeed, Mau et al. find that dynamic chromatin regulation is a good criterion to enrich for active enhancers.

   Second, they established an enhancer reporter system, which allows for visualization of de novo transcription by both in situ hybridization and in vivo. This MS2 system has for the first time been implemented in this beetle and the authors convincingly show its functionality. Indeed, the expression dynamics can be very nicely visualized in vivo at blastoderm stages.

   Combing these two resources, they predicted enhancers based on the criterion of dynamic chromatin regulation (from their ATAC-seq resource) and tested them using their novel MS2 system. Out of 9 tested enhancers located close to the gap genes hunchback and Krüppel and the pair-rule gene runt, 4 drove expression. Combining these data with previously published beetle enhancers, they show that DNA regions with differential accessibility were indeed enriched in active enhancers (appr. 60%), providing a good selection criterion.

   Finally, they characterize two of the newly identified enhancers that reflect wave-like expression patterns in fixed embryos and in vivo by using the MS2 system to test predictions of the enhancer switching model. The results are compared with an elaboration of their previously suggested enhancer-switching model, which predicts different patterns for static vs. dynamic enhancers. Indeed, they think that the runB enhancer fits the predictions of a static enhancer.

   The authors have generated a genomic resource that will be of very high value to the community in the future. The fact that they dissected the embryos for that purpose makes it even more precise and valuable. Likewise, the transgenic system that allows for testing enhancer activity in vivo will be very valuable for the highly active research field dealing with the prediction and analyses of enhancers.

   The analysis of the identified enhancers provides partial confirmation for their model. As the authors state in the discussion, finding at least one pair of such enhancers for one gene would be a great test of their hypothesis - finding pairs of static and dynamic enhancers in several genes would be strong support. Unfortunately, they found only one of the two enhancer types in runt and one in hunchback, respectively. Hence, the prediction of the model remains to be tested in the future.

   The authors provide a lot of high-quality data visualized nicely in the figures. The text would profit from some re-formulation, re-structuring, and shortening.

   Open questions:
   What happens with the runB enhancer at later stages of embryogenesis? With what kind of dynamics do the anterior-most stripes fade and does that agree with the model? Do they show the same dynamics throughout segmentation? I think later stages need to be shown because the prediction from the model would be that the dynamics are repeated with each wave. I am not so sure about the prediction for ageing stripes - yet it would have been interesting to see the model prediction and the activity of the static enhancer.
   I understand that the mRNA of the reporter gene yellow is more stable than the runt mRNA. This might interfere with the possibility to test your prediction for static enhancers: The criterion is that the stripes should increase in strength as the wave migrates towards the anterior. You show this for runB - but given that yellow has a more stable transcript - could this lead to a "false positive" increase in intensity with the slower migration and accumulation of transcripts? I would feel more comfortable with the statement that this is a static enhancer if you could exclude that the signal is blurred by an artifact based on different mRNA stability. What about re-running the simulation (with the parameters that have shown to well reflect endogenous runt mRNA levels) but increasing the parameter for the stability of the mRNA? Are static and dynamic enhancers still distinguishable? The claim of having found a static enhancer rests on this increase in signal, hence, other explanations need to be excluded carefully.
   What about the head domain of the runB enhancer (e.g. Fig. 6A lowest row): This seems to be different from endogenous expression in your work and in Choe et al. Is that aspect different from endogenous expression and can this be reconciled with your model?
   The claim of similar dynamics of expression visualized by in situ and MS2 in vivo relies on comparing Fig. 6C with 6A. To compare these two panels, I would need to know to what stage in A the embryo in C should be compared. Actually, the stripe in 6C appears more crisp than the stripes in 6A.
   Were the enhancer dynamics tested in vivo at later stages as well? I would appreciate a clear statement on what stages can be visualized and where the technical boundaries are because this will influence any considerations by others using this system.
   How do the reported accessibility dynamics of runA enhancer correlate with the activity of the reporter: E.g. is the enhancer open in the middle body region but closed at the posterior part of the embryo? Or is it closed at the anterior - and if so: why is there a signal of the reporter in the head?
   You show that chromatin accessibility dynamics help in identifying active enhancers. Is this idea new or is it based on previous experience with Drosophila (e.g. PMID: 29539636 or works cited in https://doi.org/10.1002/bies.201900188)? Or in what respect is this novel?

   Read the original source
6. 

   eLife

   Jun 8, 2023

   Reviewer #3 (Public Review):

   The authors study the spatio-temporal dynamics of gap and pair-rule pattern formation in the Tribolium embryo. Their main contributions are (1) to perform DNA accessibility profiles at multiple time points and in three domains along the A/P axis, (2) to establish a reporter gene system to examine reporter gene expression driven by candidate enhancers (including live imaging), (3) identify at least three new enhancers, and (4) provide some evidence in favor of the "Enhancer Switching" model.

   This is an interesting study that marks solid progress towards an organizing principle of pattern formation. The two practical contributions of the work are impactful: (1) germband region-specific accessibility profiling provides a novel view of the epigenome, especially when combined with profiling of temporal variation. …

   Reviewer #3 (Public Review):

   The authors study the spatio-temporal dynamics of gap and pair-rule pattern formation in the Tribolium embryo. Their main contributions are (1) to perform DNA accessibility profiles at multiple time points and in three domains along the A/P axis, (2) to establish a reporter gene system to examine reporter gene expression driven by candidate enhancers (including live imaging), (3) identify at least three new enhancers, and (4) provide some evidence in favor of the "Enhancer Switching" model.

   This is an interesting study that marks solid progress towards an organizing principle of pattern formation. The two practical contributions of the work are impactful: (1) germband region-specific accessibility profiling provides a novel view of the epigenome, especially when combined with profiling of temporal variation. (2) the live imaging system has been powerful in Drosophila studies and this work establishes this system for Tribolium, which has certain advantages as a model.

   I have two major concerns: First, the claim about differential accessibility being related to enhancer activity is not really established from the presented data, in my view. This needs to be clarified. (I do believe in the claim to some extent, but not based on presented evidence.) Second, the evidence in support of the Enhancer Switching model for runt should be accompanied by identification of and spatiotemporal profiling of the "speed regulator", if this is not established yet. In addition to these two concerns, the simulations of the Enhancer Switching model need to be described, at least in the outline, in the Methods section.

   Read the original source
7. 

   Version published to 10.1101/2022.09.09.507237v2 on bioRxiv

   Nov 16, 2022
8. 

   Version published to 10.1101/2022.09.09.507237v1 on bioRxiv

   Sep 10, 2022