Mechanisms of cilia regeneration in Xenopus multiciliated epithelium in vivo

  1. Venkatramanan G. Rao
  2. Vignesharavind Subramanianbalachandar
  3. Magdalena M. Magaj
  4. Stefanie Redemann
  5. Saurabh S. Kulkarni

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Cilia regeneration is a physiological event, and while studied extensively in unicellular organisms, it remains poorly understood in vertebrates. In this study, using Xenopus multiciliated cells (MCCs) as a model, we demonstrate that, unlike unicellular organisms, deciliation removes the transition zone (TZ) and the ciliary axoneme. While MCCs immediately begin the regeneration of the ciliary axoneme, surprisingly, the assembly of TZ is delayed. However, ciliary tip proteins, Sentan and Clamp, localize to regenerating cilia without delay. Using cycloheximide (CHX) to block new protein synthesis, we show that the TZ protein B9d1 is not a component of the cilia precursor pool and requires new transcription/translation, providing insights into the delayed repair of TZ. Moreover, MCCs in CHX treatment assemble fewer (∼ 10 vs. ∼150 in controls) but near wild-type length (ranging between 60 to 90%) cilia by gradually concentrating ciliogenesis proteins like IFTs at a select few basal bodies. Using mathematical modeling, we show that cilia length compared to cilia number influences the force generated by MCCs more. In summary, our results question the requirement of TZ in motile cilia assembly and provide insights into how cells determine organelle size and number.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    The work would have significant impact in the cilia community, if the conclusion is correct. This reviewer, however, has a concern about the authors concluding the presence/absence of TZ, based on only B9D1 and the H-shaped body among nine doublet microtubules. First, is it really established how the structure of Xenopus embryo TZ is? While Chlamydomonas is well known to have a H-shaped TZ, other species have different form inside the 9+0 doublet, or no feature (Comparison of TZ from various species in Dennis Diener https://doi.org/10.1016/B978-0-12-822508-0.00007-1). Fig.2B of this manuscript shows visible densities in the panel "Pre", but it does not look like an H-shape. The tomogram of TZ before deciliation seems clearer (but judging from wavy MTs and membrane in this tomogram, there could be unevenness of embedding and staining), while the tomogram after deciliation is thin and does not cover the entire width. Therefore it is not sure that absence of TZ can be concluded. If the author claims Xenopus embryo cilia have a H-shaped TZ, they have to provide multiple micrographs (ideally tomogram or serial section TEM to cover the entire TZ structure) and/or past literature on Xenopus embryo TZ. B9D1 is likely a membrane associated protein (according to their deciliation by detergent and mechanical force). This may mean B9D1 is located on or near the membrane, in vicinity to TZ, and thus binds to TZ after the main part of TZ is built. In this case, it is risky to judge presence of TZ based on B9D1. Also in this point, TEM imaging will be helpful to confirm the authors' conclusion.

    RESPONSE: We appreciate the reviewer’s thoughtful comments on the loss of TZ upon deciliation and its absence during the initial regeneration period. The reviewer is right in their assessment that the TZ of Xenopus cilia has not been well defined before in any manuscript. We want the reviewer to consider that our goal was not to define the TZ in Xenopus but to study deciliation and how cilia regenerate in a vertebrate model system for the first time. We unexpectedly discovered that cilia are deciliated distal to the basal body at the plasma membrane, and the “H-shaped structure,” similar to TZ, was also removed and did not come back for first hour during regeneration. Given this surprising observation, we felt obliged to study and explain our results. To that end, we explored different resources (antibodies and markers of TZ) and different methods over 6 years trying to define TZ in Xenopus.

    Our conclusion about the TZ structure came from multiple lines of evidence from our experiments and published literature, including the similarity in structure compared to other organisms and its physical location in the cilium. Specifically, 1) In a review of the basal bodies, Mitchell indirectly suggested that the electron-dense “H-shaped” structure could be a TZ in Xenopus. 2) The electron-dense “H” shaped structure in Chlamydomonas is similar, if not identical, to that shown in Xenopus cilia. 3) The physical location of TZ is always shown to be distal to the basal body and transition fibers (except in clubmoss Phylloglosum) while proximal to the central pair. The electron-dense “H-shaped” structure in Xenopus fulfills these criteria, suggesting that this structure is the TZ in Xenopus. 4) The TZ bonafide protein B9D1 is localized distal to Chibby, which labels the distal end of the basal body, suggesting that the TZ is localized distal to the basal body. Moreover, the loss of an “H-shaped” structure determined using TEM and tomograms corresponds to the loss of the B9D1 signal, further strengthening the conclusion that the H-shaped structure is the TZ.

    We will include serial sectioning and imaging of multiple Xenopus cilia in control and 0hr (after deciliation) to address this reviewer's concerns further. Our preliminary data has suggested that the ciliary membrane is tightened around this electron-dense structure, similar to what has been shown before for other organisms like Chlamydomonas. and thus boosts our confidence that this structure likely corresponds to the TZ in Xenopus.

    The reviewer has raised a concern that “the tomogram after deciliation is thin and does not cover the entire width. Therefore, it is not sure that absence of TZ can be concluded”. We note that even if the tomograms do not go through the entire cilium (supplementary videos 2 and 3), it does go through more than the center of cilium as seen by the presence of central pair microtubules and we can observe that the electron-dense “H-shaped” structure is not present in these cilia. Further, in the supplementary videos 5 and 6, even if the tomogram again only covers half of the cilia, we can see the presence of the structure, confirming that our tomograms can demonstrate the presence or absence of the H-shaped structure confidently. We have also provided TEM sections in addition to the tomograms to show the same result.

    The Reviewer has commented that “B9D1 is located on or near the membrane, in vicinity to TZ, and thus binds to TZ after the main part of TZ is built”. This reviewer is correct in their assessment. This is why we argue that the presence or absence of B9D1 may be a good marker for understanding the presence or absence of TZ assembly.

    TIMELINE: We are performing additional serial TEM in the control and deciliated (0hr.) embryos to address the reviewer’s concern. We will need 1 month to finish these experiments.

    Their discussion about length/number of cilia and force generated by cilia is interesting, but in the context of this research, this reviewer is skeptical about its value. The calcium induced deciliation is not a physiological phenomenon, but an artificial event (please correct if I am wrong). The argument how length and number of cilia are regulated upon deciliation makes sense only in case deciliation happens regularly and the species must optimize themselves to survive. The argument about possible passway of protein transport to control ciliary number and length (Line408-) seems, although it is an interesting topic in general, not suitable in this manuscript. For this reviewer's view, it is relatively straightforward to interpret the result of cilia number/length under normal growth, without new protein expression (CHX), with protein degradation blocked. Cilia will extend when components are provided. Growth will slow down when it is exhausted. Existing cilia start degrading, when they lack proteins, which are necessary for turn-over. With the current experimental output, there is no point to describe redistribution of proteins.

    RESPONSE: We appreciate the reviewer’s comment; however, we would like to argue that different methods of deciliation have been used in different model systems, such as Chlamydomonas, to study cilia regeneration. Although this reviewer may not find some of the experiments and conclusions appropriate for this manuscript, other research groups have found these results interesting. For example, reviewer 2 states, “To support their observations that cilia length is favored over cilia number under conditions of limiting ciliary precursor availability, the authors use a mathematical model that leads to the conclusion that force generation is optimized by increasing cilia length. This is a convincing conclusion and in agreement with other comparable modeling studies performed in the field.” We have already had great discussions about these results with many cilia researchers at multiple conferences. Therefore, we prefer to keep these experiments and results in the manuscript and let readers come to their own conclusions about their importance.

    Minor points:

    Line65: do they mean "selected few basal bodies"? – we have removed the word “select”

    Line73: extracellular flow is not limited to developmental system. – we have altered the statement to add “growth, development and homeostasis”

    Line124: alpha-tubulin signal and SEM image – we have added “and scanning electron microscopy (SEM)”

    Line139: Could you define explicitly the two hypotheses? – Now, we have reworded the sentences to clarify the two hypotheses. “Therefore, we considered two hypotheses: First, Xenopus MCCs regenerate cilia or second, Xenopus depend on stem cell-based replacement of damaged MCCs.”

    Line164: 10,31-33 are not suitable citation for the location of calcium induced deciliation in Chlamydomonas. cite Sanders and Salisbury JCB 108, 1751 – We have changed the citation.

    Line181: Later -> latter – We have changed the text.

    Line195: by mechanical shearing, B9D1 remained with cilia. They concluded that TZ stays with the axoneme by deciliation. How can they exclude the possibility that mechanical separation works differently from calcium shock? – We do not intend to claim that both calcium-based and mechanical ripping of cilia from cells adopt the same deciliation mechanism, and we have mentioned in line 193 that ‘we adopted an alternative approach of mechanical deciliation’. Using these two methods as complimentary to each other, our aim was to show that TZ is lost by both ciliation methods. For the calcium method, because the membrane is ripped with detergent, we show the loss of TZ by examining the MCCs devoid of cilia. In the mechanical deciliation protocol, since no detergent is involved, we can examine cilia that are likely to have intact membranes and thus maintain a B9D1 signal.

    Line214: 1.33uM -> 1.33um - We have made these changes to the text.

    __RESPONSE: __All the minor points in the manuscript are addressed.

    Overall, the results are well presented and allow strong conclusions to be drawn. The results are based on both immunofluorescence studies and EM analysis. To support their observations that cilia length is favored over cilia number under conditions of limiting ciliary precursor availability, the authors use a mathematical model that leads to the conclusion that force generation is optimized by increasing cilia length. This is a convincing conclusion, and in agreement with other comparable modeling studies performed in the field. It would be fascinating to be able to measure the flow parameters at the cell surface during cilia regeneration to see whether this regeneration actually leads to an increase in the overall flow or force generated by the cilia. But as the authors explain, this is probably a difficult experiment to carry out and appears to be optional in the context of this study.

    __RESPONSE: __We thank the reviewer for recognizing and stating that “the results are well presented and allow strong conclusions to be drawn”. We also want to sincerely thank the reviewer for understanding the technical difficulties in performing these experiments.

    The authors are apparently only able to detect a single TZ protein, B9D1, to follow the fate of the TZ during the deciliation and reciliation process. In some ways, this provides an incomplete demonstration that all the TZ is indeed removed during deciliation, although this is supported by EM observations. It also provides a limited understanding of the time course of TZ re-formation during reciliation. Given the limitations of antibody availability, could it be possible to express tagged proteins in the animal cap system to track more TZ proteins? In particular, would it be possible to track for example Cby and NPHP proteins. What is the behavior of Cep290? This would greatly reinforce the conclusions on the molecular reorganisation of the TZ after deciliation and during cilia regeneration.

    __RESPONSE: __We appreciate this reviewer’s brilliant questions on understanding the time course of TZ re-formation during reciliation. When we started this project and observed that TZ was lost upon deciliation in our preliminary TEM experiment, our first goal was to confirm this outstanding result. Thus, we did more TEMs and EM tomography, used bonafide TZ protein B9D1 to label the structure, and observed its loss upon deciliation. Taken together, we feel highly confident that TZ is lost upon deciliation. To address this reviewer’s concerns, we will performing additional serial TEMs to confirm the loss of TZ after deciliation.

    Our next goal was to understand what the reviewer has mentioned, the TZ assembly time course. We started with TEMs at different time points and again saw a surprising result: TZ assembly was delayed compared to cilia axoneme. We were driven by this question of understanding how cilia “put together” the complex structure of TZ structurally and molecularly using EM and fluorescence data. We first attempted a few antibodies, including B9D1, CEP290, MKS5, and NPHP4, to localize to the TZ in the Xenopus cilia. Despite our efforts with different fixation strategies, only B9D1 appeared to localize to the TZ, whereas others did not give any signal or localized at the basal body. Next, we tried localizing TMEM216, TMEM67, and NPHP4 using fluorescent tags, but we again found the same result: they localized to the basal body but not at the TZ. We are perplexed by this result and are pursuing the reasons behind them. However, these experiments are out of the scope of this paper. We want to note that we have used Chibby in our experiments and that it is not lost upon deciliation (Fig S1). This is because Chibby is a distal transition fiber protein (distal end of basal body) and does not extend up to the transition zone.

    TIMELINE: To address the reviewer's concern, we are performing additional serial TEM in the control and deciliated (0hr.) embryos. We will attempt to localize CEP290-GFP, requiring approximately 1 month to finish the experiment. However, we would like to note that we cannot guarantee that this experiment will work, as similar experiments with other TZ markers have failed before.

    Minor comments

    Figure 4: The images are poorly defined, and it is difficult to distinguish individual basal bodies and cilia. Therefore, it is not clear how the authors can confidently quantify the number of basal bodies in each condition to construct the graph at the bottom of the figure. In addition, it would be interesting to label the basal body with a centriolar marker to better define it. - Figure 4 labels the Transition Zone protein B9D1 and cilia marker acetylated tubulin and not basal bodies. The graph represents the number of cells with the presence or absence of elongated B9d1 signal.

    Figure 5: not clear why the graph on the lower right does not include the control at 3 and 6 hrs? Is it because the number is too high and difficult to quantify? – Yes, the reviewer is right. Cilia become too long and too many to quantify their number reliably.

    References: I would like to draw the authors' attention to studies of deciliation in Paramecia that could be cited in the introduction or discussion of the conservation of this pathway through evolution. – We have added multiple references to paramecia throughout the manuscript. Specifically, we mention that deciliation and regeneration in unicellular models like paramecia have added to our understanding of ciliogenesis. Line 102 “While it is important to remember that regeneration of cilia may not be identical to de novo assembly, cilia regeneration studies in Chlamydomonas *reinhardtii, Paramecium and Tetrahymena etc., *have provided significant insights into ciliogenesis, g., cargo transport, the presence of precursor pool, regulation of ciliary gene expression.18,23–26”. Further, we also added the reference to paramecia in results, line164 “Next, we determined the location where the deciliation treatment severed cilia. Unicellular models such as *Chlamydomonas, Paramecium *and Tetrahymena lose cilia distal to the TZ and below the central pair (CP) microtubules33.”. We also add discussion on the importance of TZ in paramecia, line 203 “Interestingly in Paramecium also a unicellular multiciliated cell, displays constant shedding of cilia when TZ proteins are depleted.25”. These statements have been supported by the following studies that are now cited in the manuscript: Machemer and Ogura 1979 Journal of Cell Physiology (10.1113/jphysiol.1979.sp012990) and Gogenddeau et al., Plos Biology (10.1371/journal.pbio.3000640).

    RESPONSE: All the minor points in the manuscript are addressed.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Rao et al. focuses on determining the mechanism of cilia regeneration using Xenopus mucociliary epithelium. The authors employ a simple yet powerful approach to trigger deciliation of multiciliated cells, enabling them to study the mechanism of cilia regeneration. This research has a significant impact on the field of cilia biology and enhances our understanding of ciliopathies. Through detailed cell biological methodologies, the authors obtained intriguing results, including the finding that deciliation removes the transition zone and that cilia repair precedes the transition zone assembly. Additionally, the authors demonstrate that IFT proteins involved in cilia construction concentrate at selected basal bodies. Although there are open questions that the authors also highlight, this manuscript provides solid, pioneering insights into the process of cilia regeneration in vivo.

    Significance

    The manuscript characterizes the mechanism of cilia regeneration, providing new insights into processes that could be harnessed to restore ciliary function in patients suffering from chronic respiratory diseases.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary

    This manuscript investigates how cilia regenerate in multi-ciliated cells. The authors have exploited an original multi-ciliated cell system derived from the Xenopus embryonic cap and use chemical and mechanical deciliation to understand the different steps of cilia regeneration. In this model, they show that cilia are excised just above the BB and below the ciliary transition zone. Their results indicate that during ciliary regeneration, axoneme reassembly precedes TZ formation and that ciliary reassembly relies on de novo protein synthesis. In the context of limited protein synthesis, cells regenerate fewer cilia, but of almost the same size as control cells, suggesting the existence of a cell control system to maximise force generation. Mathematical modelling of the forces exerted by defined numbers of cilia of different lengths supports this hypothesis.

    Major comments

    Overall, the results are well presented and allow strong conclusions to be drawn. The results are based on both immunofluorescence studies and EM analysis. To support their observations that cilia length is favored over cilia number under conditions of limiting ciliary precursor availability, the authors use a mathematical model that leads to the conclusion that force generation is optimized by increasing cilia length. This is a convincing conclusion, and in agreement with other comparable modeling studies performed in the field. It would be fascinating to be able to measure the flow parameters at the cell surface during cilia regeneration to see whether this regeneration actually leads to an increase in the overall flow or force generated by the cilia. But as the authors explain, this is probably a difficult experiment to carry out and appears to be optional in the context of this study.

    The authors are apparently only able to detect a single TZ protein, B9D1, to follow the fate of the TZ during the deciliation and reciliation process. In some ways, this provides an incomplete demonstration that all the TZ is indeed removed during deciliation, although this is supported by EM observations. It also provides a limited understanding of the time course of TZ re-formation during reciliation. Given the limitations of antibody availability, could it be possible to express tagged proteins in the animal cap system to track more TZ proteins? In particular, would it be possible to track for example Cby and NPHP proteins. What is the behavior of Cep290? This would greatly reinforce the conclusions on the molecular reorganisation of the TZ after deciliation and during cilia regeneration.

    Minor comments

    Figure 4: The images are poorly defined and it is difficult to distinguish individual basal bodies and cilia. It is therefore not clear how the authors can confidently quantify the number of basal bodies in each condition to construct the graph at the bottom of the figure. In addition, it would be interesting to label the basal body with a centriolar marker to better define the basal body.

    Figure 5: not clear why the graph on the lower right does not include the control at 3 and 6 hrs? Is it because the number is too high and difficult to quantify?

    References: I would like to draw the authors' attention to studies of deciliation in Paramecia that could be cited in the introduction or discussion of the conservation of this pathway through evolution.

    Significance

    The mechanisms of deciliation and re-ciliation have mostly been studied in protozoa (Chlamydomonas, Paramecia) or in primary ciliated cell cultures. Only a few studies have described deciliation in multiciliated cells, such as sea urchins, or physiological deciliation in the oviduct. The Xenopus deciliation system described here has already been used to determine the dynamics of IFT proteins during ciliogenesis or to define the ciliary proteome. In this study, the authors go one step further by describing more precisely which part of the cilium is shed upon induction of deciliation and the dynamics of the recruitment of the Tip and of the TZ proteins.

    This study provides a completely new perspective on the deciliation process:

    1. the authors show that, contrary to what is generally accepted from protozoan studies, the deciliation process, in Xenopus multiciliated cells, expels the TZ, leaving only the basal body in the cell;
    2. While ciliogenesis is described in various models to begin with the formation of the TZ, in this Xenopus system the TZ maturates after the onset of axonemal elongation, calling into question the precise function of the TZ in axonemal elongation. The observations could be further strengthened by analyzing more TZ proteins to better understand the time course of events involved in the deciliation-reciliation program.

    The protocol used to deciliate Xenopus multiciliated cells has been described in previous manuscripts. Its use here reveals striking differences in the deciliation-reconciliation pathways from what is known in the field. It provides new conceptual perspectives for researchers working on the basic mechanisms of ciliogenesis. Note that, as a geneticist and specialist in ciliogenesis using various model organisms, I am not fully competent to critically evaluate the mathematical models developed in this study.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript entitled "Machanisms of cilia regeneration in Xenopus multiciliated epithelium in vivo", the authors mostly focus on the question, whether TZ (transition zone of cilia) plays an essential role for ciliogenesis during cilia regeneration in multiciliated cells. They used Xenopus embryo as a system to examine this question. While cilia regeneration has been actively studies in unicellular green algae, Chlamydomonas reinhardtii, the mechanism of cilia regeneration is not known yet. Their approach is to investigate cells after deciliation by calcium shock, based on a TZ protein B9D1, as well as ultrastructure observation using conventional electron microscopy.

    The authors observed loss of signal from B9D1 and H-shaped objects, which is typical for TZ, upon deciliation induced by calcium and also during the following re-growth of cilia. Based on these experiments they concluded that TZ formation is not necessary for cilia regeneration in multiciliated cells, differently from Chlamydomonas. They further conducted experiments to pursue source of component proteins for re-generation. They compared CHX-treated cells (lacking new protein production) and CHX/MG132 (reduced protein degradation) treated cells to find how the massive amount of protein components upon re-ciliation for multiple cilia will be supplied and regulated. This reviewer found the results of the experiments clearly presented and conducted properly.

    The work would have significant impact in the cilia community, if the conclusion is correct. This reviewer, however, has a concern about the authors concluding the presence/absence of TZ, based on only B9D1 and the H-shaped body among nine doublet microtubules. First, is it really established how the structure of Xenopus embryo TZ is? While Chlamydomonas is well known to have a H-shaped TZ, other species have different form inside the 9+0 doublet, or no feature (Comparison of TZ from various species in Dennis Diener https://doi.org/10.1016/B978-0-12-822508-0.00007-1). Fig.2B of this manuscript shows visible densities in the panel "Pre", but it does not look like an H-shape. The tomogram of TZ before deciliation seems clearer (but judging from wavy MTs and membrane in this tomogram, there could be unevenness of embedding and staining), while the tomogram after deciliation is thin and does not cover the entire width. Therefore it is not sure that absence of TZ can be concluded. If the author claims Xenopus embryo cilia have a H-shaped TZ, they have to provide multiple micrographs (ideally tomogram or serial section TEM to cover the entire TZ structure) and/or past literature on Xenopus embryo TZ. B9D1 is likely a membrane associated protein (according to their deciliation by detergent and mechanical force). This may mean B9D1 is located on or near the membrane, in vicinity to TZ, and thus binds to TZ after the main part of TZ is built. In this case, it is risky to judge presence of TZ based on B9D1. Also in this point, TEM imaging will be helpful to confirm the authors' conclusion.

    Their discussion about length/number of cilia and force generated by cilia is interesting, but in the context of this research, this reviewer is skeptical about its value. The calcium induced deciliation is not a physiological phenomena, but an artificial event (please correct if I am wrong). The argument how length and number of cilia are regulated upon deciliation makes sense only in case deciliation happens regularly and the species must optimize themselves to survive. The argument about possible passway of protein transport to control ciliary number and length (Line408-) seems, although it is an interesting topic in general, not suitable in this manuscript. For this reviewer's view, it is relatively straightforward to interpret the result of cilia number/length under normal growth, without new protein expression (CHX), with protein degradation blocked. Cilia will extend when components are provided. Growth will slow down when it is exhausted. Existing cilia start degrading, when they lack proteins, which are necessary for turn-over. With the current experimental output, there is no point to describe redistribution of proteins.

    Minor points:

    Line65: do they mean "selected few basal bodies"?

    Line73: extracellular flow is not limited to developmental system.

    Line124: alpha-tubulin signal and SEM image

    Line139: Could you define explicitly the two hypotheses?

    Line164: 10,31-33 are not suitable citation for the location of calcium induced deciliation in Chlamydomonas. cite Sanders and Salisbury JCB 108, 1751

    Line181: Later -> latter

    Line195: by mechanical shearing, B9D1 remained with cilia. They concluded that TZ stays with the axoneme by deciliation. How can they exclude the possibility that mechanical separation works differently from calcium shock?

    Line214: 1.33uM -> 1.33um

    Significance

    The work would have significant impact in the cilia community, if the conclusion is correct. Their discussion about length/number of cilia and force generated by cilia is interesting, but in the context of this research, this reviewer is skeptical about its value.

  5. Arcadia Science

    Super interesting result, particularly the clear demonstration that the site of mechanical deciliation is proximal to the transition zone.

    One thing I’d be careful about is the claim that delay in regeneration is due to the need for protein synthesis of B9d1 on the basis of antibody staining with chx treatment. While it’s clear from figure 4 that this TZ component does increase its expression upon deciliation, antibody staining is likely insufficiently sensitive to make a claim of this protein being limiting for ciliary assembly in the precursor pool.

  6. Version published to 10.1101/2023.06.14.544972v2 on bioRxiv
  7. Version published to 10.1101/2023.06.14.544972v1 on bioRxiv