The Reissner fiber under tension in vivo shows dynamic interaction with ciliated cells contacting the cerebrospinal fluid

  1. Celine Bellegarda
  2. Guillaume Zavard
  3. Lionel Moisan
  4. Françoise Brochard-Wyart
  5. Jean-François Joanny
  6. Ryan S Gray
  7. Yasmine Cantaut-Belarif
  8. Claire Wyart

  • Curated by eLife

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    This exceptional work substantially advances our understanding of the mechanics of the Reissner's fibre (RF) by performing in-vivo experiments that track and analyze the behavior of the RF when it is cut and the behavior of ciliated cells touching the RF when contact is interrupted. The data is valuable and the conclusions are compelling. The work will be of broad interest to many research communities including developmental neuroscience and cilia biology.

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Abstract

The Reissner fiber (RF) is an acellular thread positioned in the midline of the central canal that aggregates thanks to the beating of numerous cilia from ependymal radial glial cells (ERGs) generating flow in the central canal of the spinal cord. RF together with cerebrospinal fluid (CSF)-contacting neurons (CSF-cNs) form an axial sensory system detecting curvature. How RF, CSF-cNs and the multitude of motile cilia from ERGs interact in vivo appears critical for maintenance of RF and sensory functions of CSF-cNs to keep a straight body axis, but is not well-understood. Using in vivo imaging in larval zebrafish, we show that RF is under tension and resonates dorsoventrally. Focal RF ablations trigger retraction and relaxation of the fiber’s cut ends, with larger retraction speeds for rostral ablations. We built a mechanical model that estimates RF stress diffusion coefficient D at 5 mm 2 /s and reveals that tension builds up rostrally along the fiber. After RF ablation, spontaneous CSF-cN activity decreased and ciliary motility changed, suggesting physical interactions between RF and cilia projecting into the central canal. We observed that motile cilia were caudally-tilted and frequently interacted with RF. We propose that the numerous ependymal motile monocilia contribute to RF’s heterogenous tension via weak interactions. Our work demonstrates that under tension, the Reissner fiber dynamically interacts with motile cilia generating CSF flow and spinal sensory neurons.

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  1. Version published to 10.7554/elife.86175 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    This manuscript provides novel and intriguing experiments that aim to elucidate the mechanical properties of the Reissner fiber (RF) and to probe its interactions with the motile cilia in the central canal of the spinal cord. Using in vivo imaging in larval zebrafish, the authors show that the RF is under tension and oscillates dorsoventrally. Importantly, ablation of the RF triggered retraction and relaxation of the fiber cut ends. The retraction speed depends on where the fiber was ablated, with fastest retraction in the rostral side, indicating that tension in the RF builds up rostrally. The authors, based on observations from live imaging of intact and ablated RF and central canal, conjecture that numerous ependymal motile monocilia, that are tilted caudally and interact frequently with the RF, contribute to RF heterogenous tension via weak interactions.

    The work is important. The experiments are thorough and intricate. The findings are fascinating and open up the prospect for future investigations and models. I'm particularly curious as to what future experiments can be used to test the hypothesis put forward by the authors about the role of ciliafiber interactions in the RF mechanical properties and function.

    We thank Reviewer#1 for showing enthusiasm and support.

    Reviewer #2 (Public Review):

    The present manuscript by the Claire Wyart group analyses the behaviour of Reissner's fibre (RF) when it is cut, as well as the behaviour of cells touching RF when contact is interrupted. They show that RF is under tension that is higher in the rostral than in the caudal spinal cord. One of the proposed mechanisms is a caudally oriented movement of the cilia of ependymal radial glials cells (ERG) that is inherent rather than caused by the contact with RF. Kolmer Agduhr neurons that are also CSF contacting (CSF-cN), alter their activity when contact is lost through laser ablation of RF.

    This is an interesting paper - RF has long been proposed to be a source of signalling molecules in the development and physiological function of neural cells in the spinal cord. Cilia are the main centre of signalling activity in ciliated cells (e.g. for sonic hedgehog signalling) and the fact that ERG cilia are in direct contact with RF is intriguing. Presumably, signalling molecules could be directly transferred from RF to ERG at the contact points.

    Functionally, CSF-cN are augmenting spinal cord intrinsic sensory feedback on body curvature. This had been shown in vitro/ex vivo, but not clearly evaluated in the living animal. The data shown here demonstrate a possible mechanism for how the feedback can be mediated through contact with RF. This is of fundamental interest to understand the functioning of a locomotor network that is under evolutionary pressure to function early, since fish hatch at 3 days post fertilisation.

    We thank Reviewer#2 for the interest in our work.

    Interestingly, the authors propose (and discuss against the relevant literature) that the presence of RF in the central canal can influence the flow of the CSF, which should be investigated in further work.

    To bring readers back in the context of the existing literature:

    When using beads to track particles in the flow in the presence or in the absence of RF, we have not seen major difference in the bidirectional dorsoventral profile of the embryonic CSF flow (Cantaut-Belarif et al CB 2018 ; Sternberg et al., Nature Comm 2019 ; Thouvenin et al., eLife 2020).

    However, we cannot exclude that there could be a very local impact of the RF on CSF flow, due to the fact that the flow has to be null on the surface of the fiber (of 200 nm diameter). With our methods for tracking fluorescent particles in single planes at a time (Cantaut-Belarif et al CB 2018 ; Sternberg et al., Nature Comm 2019 ; Thouvenin et al., eLife 2020), we are likely missing the fiber in the plane and the fine analysis of the domain surrounding the fiber is not resolved. However, a null flow at the surface of the RF would impose a sharp gradient around the fiber.

    Note that our results estimating the effect of cutting the fiber on the beating frequency of motile cilia were not consistent across fish – half the cilia showing an increase while the rest show a decrease, making it hard to conclude. A finer analysis with higher temporal and spatial resolution in 3D will be necessary to decipher the role of the fiber on the beating of cilia and local CSF flow.

    Overall, the results are clearly presented, and methods are thoroughly given, including some indication on the reduction of bias (by blinding movies before analysis). The authors also clearly state the limitations of their work, mostly derived from optical limitation (size of the RF in the larval fish, and speed of the recording in the laser-equipped microscope). This doesn't affect the fundamental statements.

    Thank you again for your appreciation of our work.

    Reviewer #3 (Public Review):

    This manuscript by Bellegarda et al. examined the in vivo dynamic behavior of the Reissner fiber and its interactions with cilia and sensory neurons in the central canal of zebrafish larvae. The authors accomplished this by performing live imaging with a transgenic reporter zebrafish line in which the fiber is GFP-tagged and by finely tracking the movement of the fiber. Interestingly, they discovered that the fiber undergoes a dynamic vibratory-like movement along the dorsoventral axis. The authors then utilized a pulsed laser to precisely cut the fiber, which frequently resulted in a fast retraction behavior and a loss of calcium activity in sensory neurons in the central canal called CSFCNs. Mechanical modeling of the elastic properties of the fiber indicated that the fiber is a soft elastic rod with graded tension along the rostrocaudal axis. Finally, by performing live imaging of motile cilia and the fiber in the central canal, they found that the two interact in close proximity and that cilia motility is affected when the fiber was cut. The authors concluded that the Reissner fiber is a dynamic structure under tension that interacts with sensory neurons and cilia in the central canal.

    Strengths:

    1. The study utilizes state-of-the-art microscopy techniques and beautiful transgenic zebrafish tools to characterize the in vivo behavior of the Reissner fiber and found that it exhibits surprising dynamic movements along the dorsal-ventral axis. This observation has important implications for the physiology and function of the Reissner fiber.
    1. By performing a series of clever laser cutting experiments, the authors reveal that the Reissner fiber is under tension in the central canal of zebrafish. This finding provides direct experimental evidence to support the hypothesis that the Reissner fiber functions in a biomechanical manner during spinal cord development and body axis straightening.
    1. By developing a mechanical model of the Reissner fiber and its retraction behavior, the authors estimate the elastic properties of the fiber and found that it is more akin to an elastic polymer rather than a stiff rod. This is a useful finding that illuminates the biophysical properties of the fiber.
    1. Through calcium and cilia imaging studies, the authors demonstrate that the Reissner fiber likely interacts with motile cilia and regulates the activity of ciliated sensory neurons (CSF-CNs). The authors propose a model in which fiber-cilia interactions may occur via weak interactions or frictional forces. This model is plausible and opens several new doors for additional investigation.

    We thank Reviewer#3 for the support.

    Weaknesses:

    1. All the live imaging experiments appear to be performed with animals paralyzed via the injection of a chemical agent (bungarotoxin). Does paralysis and/or bungarotoxin negatively impact the behavior of the Reissner fiber? Some data from non-paralyzed animals would ameliorate this concern.

    We performed very few experiments on non paralyzed fish as the position of the Reissner fiber were difficult / impossible to analyze in 3D. In a movie added to our revision as Movie 3, it is obvious that skeletal muscle contractions result in very large jumps of the fiber that cannot be corrected for using single plane imaging. Without being able to monitor and correct for muscle contractions, an accurate estimation of the fiber motion in this context would be artefactual.

    1. Although the authors convincingly demonstrate that the Reissner fiber is under graded tension, it remains unclear what is the relevance and function of tension on this structure. The photoablation data presented do not delineate between the relevance of the fiber being intact or tension on the fiber as cutting the fiber impacts both. Is fiber tension required for body straightening? At the site of fiber photoablation, does a spinal curvature develop? If cultured, do the ablated animals exhibit a scoliotic phenotype?

    We thank Reviewer#3 for asking these important questions. We did ask ourselves the same questions, but had to restrain the ambition of our study as for technical reasons, the ablation experiments performed on an inverted microscope required to mount the fish closed to the bottom coverslip and were extremely difficult to perform while safely removing the animal from the imaging cuvette and not affecting the alignment of its body axis.

    1. One of the most potentially impactful conclusions of the paper is that the Reissner fiber interacts with cilia, but the evidence is insufficient to support this. Although some motile cilia are near the fiber (Figure 3A), many cilia are not near the fiber. The provided images and videos do not clearly demonstrate that cilia physically contact or influence the behavior of the Reissner fiber. Further, the data is lacking to conclude that the Reissner fiber directly impacts cilia motility as they did not observe an overall statistically significant difference before and after ablation (Supplemental Figure 1A). Higher magnification, higher resolution, higher acquisition rate and/or colocalization analyses of fiber-cilia interactions could alleviate this concern.

    We agree with the reviewer but could not yet perform for technical reasons more spatially- and temporally- resolutive experiments. Further analysis of cilia and RF translational motion is displayed on the Figure 4 - Supplemental Figure 2 and presented in the Results sections.. We observed that for 7 out of 15 dorsal cilia and 4 out of 9 ventral cilia, the preferred position of the cilium was correlated with a position of the fiber – suggesting that they could interact. However, our current dataset in 2 D is too incomplete to draw strong conclusions on the nature of interactions between fiber and cilia. A future study relying on 3D analysis of the fiber and cilia should resolve how collective interactions of cilia may determine the position of the fiber.

    1. Similarly, how does the Reissner fiber interact with CSF-CN sensory neurons? The authors suggest that the fiber interacts with CSF-CN sensory neurons by modulating their spontaneous calcium activity via weak interactions or frictional forces from motile ciliated ependymal radial glial cells. While the calcium imaging data of the CSF-CNs is convincing and sound, the exact nature of the fiber-neuron interaction is unclear. Do cilia or apical extensions on CSF-CN sensory neurons sense the fiber or forces through a mechanosensing or chemosensing mechanism?

    This question is of great interest to us and will be the topic of a future investigation, as it is very difficult to image CSF-cN motile cilium (see Bohm et al., Nature Comm 2016) and even more with the Reissner fiber.

    There is some additional confusion as the authors appear to focus their cilia experiments on ependymal radial glial cells in section 4, rather than CSF-CNs. The addition of an illustrative cartoon would add clarity.

    We agree and we added a schematic in the last figure (Figure 4A).

    Overall, the conclusions of the study are well supported by the data presented. However, the strength of the conclusions could be enhanced by additional controls, alternative experimental approaches and clarifications.

    This manuscript is an important contribution to the fields of spinal cord development and body axis development, which are fundamental questions in neurobiology, developmental biology, and musculoskeletal biology. In recent years, the Reissner fiber and motile cilia function have been linked to cerebrospinal fluid flow signaling and body straightening, but the precise form and function of the fiber remain unclear. This study provides new insight into the dynamic and biophysical properties of the Reissner fiber in vivo in zebrafish and proposes a model in which the fiber interacts with cilia and sensory neurons. This study provides novel insight into the cellular mechanisms that underlie the pathogenesis of disorders such as idiopathic scoliosis.

    We thank the Reviewer #3 and added further analysis of cilia and RF motion displayed on the figures below added as well as extended data figures in the main manuscript.

  3. eLife

    eLife assessment

    This exceptional work substantially advances our understanding of the mechanics of the Reissner's fibre (RF) by performing in-vivo experiments that track and analyze the behavior of the RF when it is cut and the behavior of ciliated cells touching the RF when contact is interrupted. The data is valuable and the conclusions are compelling. The work will be of broad interest to many research communities including developmental neuroscience and cilia biology.

  4. eLife

    Reviewer #1 (Public Review):

    This manuscript provides novel and intriguing experiments that aim to elucidate the mechanical properties of the Reissner fiber (RF) and to probe its interactions with the motile cilia in the central canal of the spinal cord. Using in vivo imaging in larval zebrafish, the authors show that the RF is under tension and oscillates dorsoventrally. Importantly, ablation of the RF triggered retraction and relaxation of the fiber cut ends. The retraction speed depends on where the fiber was ablated, with fastest retraction in the rostral side, indicating that tension in the RF builds up rostrally. The authors, based on observations from live imaging of intact and ablated RF and central canal, conjecture that numerous ependymal motile monocilia, that are tilted caudally and interact frequently with the RF, contribute to RF heterogenous tension via weak interactions.

    The work is important. The experiments are thorough and intricate. The findings are fascinating and open up the prospect for future investigations and models. I'm particularly curious as to what future experiments can be used to test the hypothesis put forward by the authors about the role of cilia-fiber interactions in the RF mechanical properties and function.

  5. eLife

    Reviewer #2 (Public Review):

    The present manuscript by the Claire Wyart group analyses the behaviour of Reissner's fibre (RF) when it is cut, as well as the behaviour of cells touching RF when contact is interrupted. They show that RF is under tension that is higher in the rostral than in the caudal spinal cord. One of the proposed mechanisms is a caudally oriented movement of the cilia of ependymal radial glials cells (ERG) that is inherent rather than caused by the contact with RF. Kolmer Agduhr neurons that are also CSF contacting (CSF-cN), alter their activity when contact is lost through laser ablation of RF.

    This is an interesting paper - RF has long been proposed to be a source of signalling molecules in the development and physiological function of neural cells in the spinal cord. Cilia are the main centre of signalling activity in ciliated cells (e.g. for sonic hedgehog signalling) and the fact that ERG cilia are in direct contact with RF is intriguing. Presumably, signalling molecules could be directly transferred from RF to ERG at the contact points.

    Functionally, CSF-cN are augmenting spinal cord intrinsic sensory feedback on body curvature. This had been shown in vitro/ex vivo, but not clearly evaluated in the living animal. The data shown here demonstrate a possible mechanism for how the feedback can be mediated through contact with RF. This is of fundamental interest to understand the functioning of a locomotor network that is under evolutionary pressure to function early, since fish hatch at 3 days post fertilisation.

    Interestingly, the authors propose (and discuss against the relevant literature) that the presence of RF in the central canal can influence the flow of the CSF, which should be investigated in further work.

    Overall, the results are clearly presented, and methods are thoroughly given, including some indication on the reduction of bias (by blinding movies before analysis). The authors also clearly state the limitations of their work, mostly derived from optical limitation (size of the RF in the larval fish, and speed of the recording in the laser-equipped microscope). This doesn't affect the fundamental statements.

  6. eLife

    Reviewer #3 (Public Review):

    This manuscript by Bellegarda et al. examined the in vivo dynamic behavior of the Reissner fiber and its interactions with cilia and sensory neurons in the central canal of zebrafish larvae. The authors accomplished this by performing live imaging with a transgenic reporter zebrafish line in which the fiber is GFP-tagged and by finely tracking the movement of the fiber. Interestingly, they discovered that the fiber undergoes a dynamic vibratory-like movement along the dorsoventral axis. The authors then utilized a pulsed laser to precisely cut the fiber, which frequently resulted in a fast retraction behavior and a loss of calcium activity in sensory neurons in the central canal called CSF-CNs. Mechanical modeling of the elastic properties of the fiber indicated that the fiber is a soft elastic rod with graded tension along the rostrocaudal axis. Finally, by performing live imaging of motile cilia and the fiber in the central canal, they found that the two interact in close proximity and that cilia motility is affected when the fiber was cut. The authors concluded that the Reissner fiber is a dynamic structure under tension that interacts with sensory neurons and cilia in the central canal.

    Strengths:
    1. The study utilizes state-of-the-art microscopy techniques and beautiful transgenic zebrafish tools to characterize the in vivo behavior of the Reissner fiber and found that it exhibits surprising dynamic movements along the dorsal-ventral axis. This observation has important implications for the physiology and function of the Reissner fiber.

    2. By performing a series of clever laser cutting experiments, the authors reveal that the Reissner fiber is under tension in the central canal of zebrafish. This finding provides direct experimental evidence to support the hypothesis that the Reissner fiber functions in a biomechanical manner during spinal cord development and body axis straightening.

    3. By developing a mechanical model of the Reissner fiber and its retraction behavior, the authors estimate the elastic properties of the fiber and found that it is more akin to an elastic polymer rather than a stiff rod. This is a useful finding that illuminates the biophysical properties of the fiber.

    4. Through calcium and cilia imaging studies, the authors demonstrate that the Reissner fiber likely interacts with motile cilia and regulates the activity of ciliated sensory neurons (CSF-CNs). The authors propose a model in which fiber-cilia interactions may occur via weak interactions or frictional forces. This model is plausible and opens several new doors for additional investigation.

    Weaknesses:
    1. All the live imaging experiments appear to be performed with animals paralyzed via the injection of a chemical agent (bungarotoxin). Does paralysis and/or bungarotoxin negatively impact the behavior of the Reissner fiber? Some data from non-paralyzed animals would ameliorate this concern.

    2. Although the authors convincingly demonstrate that the Reissner fiber is under graded tension, it remains unclear what is the relevance and function of tension on this structure. The photoablation data presented do not delineate between the relevance of the fiber being intact or tension on the fiber as cutting the fiber impacts both. Is fiber tension required for body straightening? At the site of fiber photoablation, does a spinal curvature develop? If cultured, do the ablated animals exhibit a scoliotic phenotype?

    3. One of the most potentially impactful conclusions of the paper is that the Reissner fiber interacts with cilia, but the evidence is insufficient to support this. Although some motile cilia are near the fiber (Figure 3A), many cilia are not near the fiber. The provided images and videos do not clearly demonstrate that cilia physically contact or influence the behavior of the Reissner fiber. Further, the data is lacking to conclude that the Reissner fiber directly impacts cilia motility as they did not observe an overall statistically significant difference before and after ablation (Supplemental Figure 1A). Higher magnification, higher resolution, higher acquisition rate and/or colocalization analyses of fiber-cilia interactions could alleviate this concern.

    4. Similarly, how does the Reissner fiber interact with CSF-CN sensory neurons? The authors suggest that the fiber interacts with CSF-CN sensory neurons by modulating their spontaneous calcium activity via weak interactions or frictional forces from motile ciliated ependymal radial glial cells. While the calcium imaging data of the CSF-CNs is convincing and sound, the exact nature of the fiber-neuron interaction is unclear. Do cilia or apical extensions on CSF-CN sensory neurons sense the fiber or forces through a mechanosensing or chemosensing mechanism? There is some additional confusion as the authors appear to focus their cilia experiments on ependymal radial glial cells in section 4, rather than CSF-CNs. The addition of an illustrative cartoon would add clarity.

    Overall, the conclusions of the study are well supported by the data presented. However, the strength of the conclusions could be enhanced by additional controls, alternative experimental approaches and clarifications.

    This manuscript is an important contribution to the fields of spinal cord development and body axis development, which are fundamental questions in neurobiology, developmental biology, and musculoskeletal biology. In recent years, the Reissner fiber and motile cilia function have been linked to cerebrospinal fluid flow signaling and body straightening, but the precise form and function of the fiber remain unclear. This study provides new insight into the dynamic and biophysical properties of the Reissner fiber in vivo in zebrafish and proposes a model in which the fiber interacts with cilia and sensory neurons. This study provides novel insight into the cellular mechanisms that underlie the pathogenesis of disorders such as idiopathic scoliosis.

  7. Version published to 10.1101/2023.02.22.529498v1 on bioRxiv