Novel analytical tools reveal that local synchronization of cilia coincides with tissue-scale metachronal waves in zebrafish multiciliated epithelia

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    Evaluation Summary:

    This is a comprehensive, in vivo study of motile cilia dynamics, organisation and coordination in the larval zebrafish nose. The authors used a combination of highly quantitative imaging methods and transgenics to visualise the properties of multiciliated cells in this model organism - with particular emphasis on measuring the spatiotemporal coherence and organisation of cilia across the organ, and on the discovery of large-scale metachronal waves. With the aid of a computational model, the authors also explored the implications of metachronal ciliary action for fluid pumping.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Motile cilia are hair-like cell extensions that beat periodically to generate fluid flow along various epithelial tissues within the body. In dense multiciliated carpets, cilia were shown to exhibit a remarkable coordination of their beat in the form of traveling metachronal waves, a phenomenon which supposedly enhances fluid transport. Yet, how cilia coordinate their regular beat in multiciliated epithelia to move fluids remains insufficiently understood, particularly due to lack of rigorous quantification. We combine experiments, novel analysis tools, and theory to address this knowledge gap. To investigate collective dynamics of cilia, we studied zebrafish multiciliated epithelia in the nose and the brain. We focused mainly on the zebrafish nose, due to its conserved properties with other ciliated tissues and its superior accessibility for non-invasive imaging. We revealed that cilia are synchronized only locally and that the size of local synchronization domains increases with the viscosity of the surrounding medium. Even though synchronization is local only, we observed global patterns of traveling metachronal waves across the zebrafish multiciliated epithelium. Intriguingly, these global wave direction patterns are conserved across individual fish, but different for left and right noses, unveiling a chiral asymmetry of metachronal coordination. To understand the implications of synchronization for fluid pumping, we used a computational model of a regular array of cilia. We found that local metachronal synchronization prevents steric collisions, i.e., cilia colliding with each other, and improves fluid pumping in dense cilia carpets, but hardly affects the direction of fluid flow. In conclusion, we show that local synchronization together with tissue-scale cilia alignment coincide and generate metachronal wave patterns in multiciliated epithelia, which enhance their physiological function of fluid pumping.

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  1. Evaluation Summary:

    This is a comprehensive, in vivo study of motile cilia dynamics, organisation and coordination in the larval zebrafish nose. The authors used a combination of highly quantitative imaging methods and transgenics to visualise the properties of multiciliated cells in this model organism - with particular emphasis on measuring the spatiotemporal coherence and organisation of cilia across the organ, and on the discovery of large-scale metachronal waves. With the aid of a computational model, the authors also explored the implications of metachronal ciliary action for fluid pumping.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  2. Reviewer #1 (Public Review):

    The authors examined using various optical live-imaging techniques the beat properties and coordination of motile cilia across the whole surface of the zebrafish nose embryo. As far as I know, this is a level of detail that had never been explored. This is an important "model organism" to understand vertebrates, and motile cilia and their carpets are a fascinating system, with aspects that one expects to be conserved across species.

    The experimental data is really impressive to me. I think there is in fact a wealth of data, and the analysis of it here is just one part of what can be extracted. The theory and the specific question are also posed clearly, and are a strength. I do wonder why the experiments did not include visualization of the fluid flow, on the same fish - this is actually a much easier experiment I think, and would have given an important other spatial map to relate to the cilia dynamics.

    The theoretical analysis backing the data allows the authors to discuss on what length scale cilia are strictly synchronized to each other, versus the (longer) scale over which there is an element of coordinated dynamics. This is an important conceptual point that is discussed here very clearly.

    I think the authors have made a clear point on the degree of coherence in the in-vivo system, and on the consequences on longer range coordination and fluid transport.

    Motile cilia are one of the most fascinating and conserved structures across eukaryotes, and motile cilia carpets deliver critical and poorly understood physiological fluid transport. Here the authors present a new level of detail in-vivo zebrafish embryo nose data on the coordination of cilia, and discuss it in the context of what is known from other systems and from basic physical models. I think this paper will have strong impact on the specific field of motile cilia, and is generally an important result in development. Both physicists and biologists will use this and build on it.

    I do wonder about the title: "Local synchronization of cilia and tissue-scale cilia alignment are sufficient for global metachronal waves". This is indeed what they find, but this title will not be very clear to many biologists. I would recommend naming the species. In fact I think a better title would focus on having discovered that there is a certain behavior in zebrafish... it sounds less "general" but represents the work better.

  3. Reviewer #2 (Public Review):

    The strength of this work is the quality and quantitative nature of the experimental data. Despite the complexity of the zebrafish olfactory pit architecture, the analysis resolution achieved is remarkable. The data reveals for the first time that cilia beating is heterogeneous and patchy, yet still becomes ordered across the entire tissue.

    The main weakness is the disconnect between the theoretical contribution and the experiments. There is some overinterpretation here - the authors should revise their conclusions accordingly. The main output of the theory is a comparison between pumping direction, rates and efficiencies for different wave and lattice parameters. The theory does not yet explain how global metachronism arises from local synchrony, what sets the wave direction etc... but only the implications of this once it does.

    A key (and unexpected) finding is the asymmetry between the left and right nose in terms of metachronal wave direction (detectable due to the quality and rigor of the analysis). The manuscript leaves open how this can arise, since the model does not help explain why the wave direction can differ, despite cilia being oriented similarly.

    I think the present title does not reflect the key findings presented in the paper and should be changed - see specific recommendations below. This is fundamentally a detailed study and mapping of cilia coordination in the zebrafish nose - the organism name should really appear in the title, there is no need to over-emphasize the implications for metachronal coordination in general. I suspect these conclusions may be specific to this organism, and so the overall message may not apply to other ciliated tissues.