Rootletin Fiber Dynamics Integrate Cytoskeletal Programs to Shape Neuroepithelial Architecture

  1. Axelle Wilmerding
  2. Glòria Casas Gimeno
  3. Paula Espana-Bonilla
  4. Susana Usieto
  5. Murielle Saade

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Abstract

Central nervous system (CNS) architecture is established early by the organization and proliferative behavior of neuroepithelial (NE) cells, which form a pseudostratified epithelium during neural tube (NT) formation. In neurogenesis, newborn neurons have to detach and exit the neuroepithelium, in a process that requires the coordinated disassembly of apical junctions and the centrosome-cilia module. Although Rootletin—the structural component of the ciliary rootlet and centriolar linker—is classically viewed as a static mechanical element, its behavior in NE cells has not been described in detail. Here, we uncover a conserved, dynamic form of Rootletin fiber organization that remodels in synchrony with NE cell morphogenesis. We show that, in NE cells in interphase, Rootletin fibers extend from the basal body through the apical process toward the nucleus, and that Rootletin maintains its fibrous conformation throughout mitosis. As NE cells initiate apical constriction, Rootletin fibers retract from the apical process and assemble into an anisotropic rim-like structure that aligns with the apical junctional complex. This remodeling is coordinated with microtubule stabilization in low-tension apical endfeet and occurs prior to Lzts1 expression and increased actomyosin contractility. Forced neuronal delamination via Neurogenin-2 or Lzts1 promotes Rootletin rim formation and Rootletin alignment with the apical endfoot cortex. Finally, we show that Zika virus NS5 protein can aberrantly associate with all conformational states of Rootletin fibers, providing a potential mechanical link between ZikaV infection and premature delamination. Together, our findings identify Rootletin as a dynamically regulated cytoskeletal scaffold that orchestrates apical surface remodeling in NE cells and identify a potential mechanism by which ZikaV disrupts neurodevelopment.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    This study characterizes the localization of Rootletin, the major molecular component of the ciliary rootlet, in radial glial cells (RGCs) of the developing cortex. The authors show that in addition to connecting the two centrioles, Rootlet also forms a ring-like structure in RGCs while their apical endfeet constrict (Fig 1-2). This ring-like structure is also observed when delamination and neuronal differentiation are forced by overexpressing Lzts1 and Ngn2 in RGCs (Fig 3-4). Finally, the authors characterize the interaction between the rootlet and the Zika virus protein NS5 (Fig 5).

    Major comments:

    (1) It is difficult to extract the information on the actual function of Rootletin from this manuscript. There is no data demonstrating its active function in RGCs. All figures are descriptive, illustrating where Rootletin localizes and showing that its intensity increases during apical endfoot constriction. Most major claims are unsupported by data. For example:

    i) Which specific data support the claim that the Rootletin "shapes neuroepithelial architecture"? There is no such data. Most figures in the manuscript point to a single conclusion: Rootletin intensity increases at the apical endfeet concurrently with the shrinkage of the apical surface area. Does Rootletin actively drive apical surface constriction, or does the constriction of the apical endfeet cause the now one-end-free rootlet fiber to passively coil into a circular structure? It seems that the latter explanation is more plausible.

    ii) How does the Zika virus protein NS5 "hijack" the rootlet to trigger premature neuronal delamination. The data in Figure 5 attempt to show colocalization between GFP-Rootletin and NS5; no functional data demonstrate how the "hijacking" work. In addition, the claimed co-localization is not unconvincing either.

    Overall, the manuscript is crammed with data, but none of the data support the major claim.

    (2) Most images are oversaturated and over-processed; the only acceptable figure is Figure 2F. The remaining figures lack sufficient detail, and could be artifacts from excessive image processing. Due to this overall low quality, the conclusions drawn from these images could be misleading. For example:

    i) The pattern in Fig 2I is typical image processing artifact, and may not represent the true biological structure.

    ii) Most of the GFP-Rootlet images in Figures 3 and 5 are oversaturated and over-processed, obscuring the details of the true endogenous structure.

    iii) Images in these figures are low quality and does not contain the necessary details to support the conclusion: Figure 3I-K, 4F-P.

    (3) Most conclusions are made from over-expression of GFP-Rootletin. However, the GFP-Rootletin does not resemble endogenous Rootlet: in FigEV3H, Rootletin antibody does not recognize electroplated GFP-Rootletin; In fig EV3A-F, none of the endogenous Rootlet show the rim-like structure that is observed in GFP-Rootletin. Therefore, most data on over-expressed GFP-Rootletin could be overexpression artifacts.

    (4) Other critical stuctures appear to be inaccurately represented. For instance, at the apical endfeet, acetylated-tubulin staining typically highlights a wheel-like structure with a circular ring and radial spokes (Kasioulis et al., 2017 eLife). However, this structure is not observed in Figures 3G-N.

    Also in Figures 3G-N, the claimed primary cilium does not represent ciliary structure. Another non-microtubule marker is needed to label primary cilia in these neuroepithelial cells.

    The Drebrin staining in Figure3Q is also different from what is usually observed (such as in Kasioulis et al., 2017 eLife). The model showing that it localizes to the corner is misleading.

    These issues reinforce the concern that images in this manuscript are either overprocessed or of insufficient quality to accurately resolve true biological structures.

    Minor comments:

    Some quantification does not make sense: for example, Figure 3B: The majority of the data points are clustered in the left corner of the plot. Given this distribution, it is statistically unsound to infer a negative correlation based on a few sporadic outliers residing from the main cluster. The same is true for Figure 4G and 4Q.

    Significance

    The topic addressed in this manuscript is significant and of general interest. However, there are serious concerns regarding the methodology, data quality, and interpretation of the results. There is a serious disconnect between the presented data and the authors' major claims.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    During CNS development, neuroepithelial (NE) cells committed to neural differentiation detach from the ventricular surface. This detachment involves dynamic cytoskeleton and membrane remodelling including downregulation of adherens junction proteins, actomyosin contractility and microtubules reorganisation at the apical end-foot of the NE cell. Infection by the Zica virus induces premature apical constriction and NE cells delamination, which could explain the microcephaly observed in embryos from mothers infected by the virus. Saade et al. (2020) previously identified Rootletin, a component of the ciliary rootlet involved in cilium stability, as an interactor of Zica NS5 protein.
    Here, the authors investigate the dynamics of rootletin subcellular localisation and remodelling during NE cells delamination using the surface epithelium of the developing chick neural tube electroporated to express GFP-Rootletin as a model. They describe several Rootletin organisation pattern, suggesting that Rootletin fibres are dynamic structures. In mitotic cells, GFP-Rootletin associates with either one or both spindle poles. While in interphase NE cells GFP-Rootletin fibres present an extended apico-basal organisation that reorganises into a rim-like structure at the apical foot before and during delamination of NE cell. They show that this rim-like rearrangement precedes apical foot constriction and investigate the connection between cytoskeleton and Rootletin remodelling. This leads the authors to propose a model of sequential spatiotemporal reorganisation of Rootletin, microtubule and acto-myosin cytoskeleton during apical foot contraction. Importantly, this Rootletin fibre organisation is also observed in embryonic mouse neural tube, human 3D neural organoids. Last, the authors investigate how Zika-NS5 and GFP-Rootletin dynamically interact during NE cell delamination.

    Major comments:

    Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them?

    1.. The first part of this manuscript consists in the description of rootletin organisation in NE cells, mainly on fixed tissues. Although the authors present endogenous localisation in the mouse embryonic neural tube and in human neural organoid, the anti rootletin staining of the chicken neural tube is very noisy and hence difficult to interpret (EV3). This, and the aim to study the dynamics of Root organisation, led the authors to use mis-expression of GFP-Root in the chicken neural epithelium as a model. A validation that the ectopic expression of GFP-Root is not inducing artificial Root structures is missing. Specifically, the manuscript relies on the observation that Rootletin adopts several organisations from inter-centriolar, basally directed, fibres to fibres oriented in the apical plane first in a "coma" shape and then adopting a rim-like structure. Although en face imaging of the embryonic mouse neural tube are shown, these rootletin patterns are not studied. This would however constitute a validation that the GFP-Rootletin structures described are not artificially induced by their ectopic GFP-Root expression. Alternatively, the use of another anti Rootletin antibody with less background noise in the chicken neural epithelium could be used. (Of note, the "co-localisation" of GFP-Root and "endogenous" Root labelled with the antibody presented in EV3 only shows that the anti rootletin recognises the GFP-Root, unless the epitope was mutated, which should then be mentioned).

    1. There is an overall lack of quantification. There is no indication of the number at which the different Root patterns are observed. Again, the whole work relies on these static observations to propose that Rootletin fibres reorganise before and during apical constriction of the NE cells. Indications of the frequency of these events, and the number of independent biological replicates used to obtain these data, is thus mandatory to support the model. This concerns particularly the events described in Figure 1C, 1H, 2A-F, 3C-E, I-K, Q-R. If indeed root forms a rim before apical constriction, this should be supported by quantifications. Also, in GFP-root expressing cells, is there an apical foot area below which root forms a rim, but is still not constricted by the apical foot perimeter? That information is not present with the different ratios integrated by the authors (SAM, AE) and should be analysed (see comment below) to better describe the process.
    2. the authors aim to determine if rootletin remodelling precedes neuronal delamination by inducing differentiation or constriction with ectopic expression of respectively Neurog2 and Lzts1. As far as I understand, this section only shows that i) neurog2 induces apical constriction and neuronal differentiation as previously published (panels B, E), premature accumulation of Lzts1 (panel J-L), which surprisingly does not lead to apical constriction in all cells since there is a population of cells with large apical surface areas and Lzts1 while in control condition such population is not found (4L black dots and 3F). But this experiment is not detailed enough to provide information regarding rootletin organisation. Quantification in 4G does not differ from 3F in the distribution of cell population with low or high rootletin occupancy. A relevant information would be to analyse if the cells that have a large apical area and Lzts1 also present rooteltin as a rim or not. If not, that would show that Lzts1 expression is not sufficient to trigger root reorganisation, nor is neurog2 induction, and that Root reorganisation is controlled by a different, potentially earlier, mechanism. Similarly, Lzts1 mis-expression only shows that it can induce apical foot constriction independently of cell differentiation to neuronal fate. Again, one should look at the shape of root fibres in the "not natural" population, ie large apical surface with Lzts1. Unless this specific population presents a different behaviour of Root fibres than large apical surface cells without Lzts1, I do not see the point of this part. Dynamics of root fibres organisation with respect to apical foot surface already suggest that remodelling precedes delamination. This could be further supported if remodelling could be triggered independently of differentiation and induce apical foot constriction. With this in mind, does mis-expression of GFP-Root trigger more apical constriction than mis-expression of a mock GFP construct? Going back to the point, does mis-expression of GFP-Root artfectualy trigger GFP-Root shapes?
    3. In a previous work (Saade 2020), the authors showed that mis-expression of Zika virus NS5 protein localises at the centriole and nuclei of E cells and promotes their delamination. They had further identified Root as an interactor of ZikV-NS5 and showed that upon ZikV-NS5 mis-expression in human cells, cilia associated pool of endogenous Rootletin decreases. Here, the authors aim to further characterise ZikV-NS5 Rootelin interaction, with the idea to propose a model for ZikV behaviour. However, they did not describe any lagging localisation for ZikV-NS5 basal to the centriole, as shown in Figure 5C-G, in their previous work. This raises the question whether the observed localisation here could be an artefact due to the mis expression of GFP-Root itself. Same mis-expression experiments using a GFP mock plasmid in place of GFP-Root is necessary to verify that the observed localization is not a consequence of GFP-Root mis-expression triggering ectopic localisation of ZikV-NS3 but a real "hacking" of the rootletin. More, they show here an accumulation of GFP-Root at the apical foot of NE cells, in contradiction with previous work showing a decrease of Root centriolar localisation. It is surprising that such accumulation has not been observed before. To exclude any artefact due to GFP-Root misexpression, endogenous Root should be monitored.

    Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated time investment for substantial experiments.

    Are the data and the methods presented in such a way that they can be reproduced?

    yes, except that the type of image shown in the figures is not specified: single confocal plane versus Z projection should be mentioned when relevant. As well the line along which plots were drawn in figures 1 and 2 are missing and should be shown.

    Are the experiments adequately replicated and statistical analysis adequate?

    there is no information regarding numbers of events analysed for Rootletin localisation pattern, nor about biological replicates. these should be added or when necessary biological replicates performed.

    Minor comments:

    Specific experimental issues that are easily addressable.

    1. Lines 143-146 : live imaging of GFP-Root "fibres" revealing a movement "reminiscent of interkinetic nuclear migration". As H2B-RFP was co electroporated with Root-GFP, showing nuclei could be informative as this would show if the rootletin structure is connected to and moves together with nuclei or has independent movements.
    2. In several figures, co-stainings are necessary to support the claims regarding localisation patterns. As well IF stainings are often saturated, which deteriorates the analysis. Please improve the image quality

    a. Figure 1F: showing ZO1 (membrane label) and centrin (centriole) in the same colour is confusing, especially since one of the staining seems dotty. Show centrioles and membrane in two different colours as in Fig 2A-F.

    b. Figure 1T,V: add a centrin label to support the claim of intercentriolar localisation of Rootletin

    c. Figure 3G: Acetylated tubulin staining saturated. Image should be improved. As well, a costaining, with Arl13B for example, is necessary to identify the presence of cilia.

    1. Figure 3G-M: the panels with Acetylated tubulin staining are not convincing. The stainings are saturated and, based on the text, it seems that they correspond to z-projection of several focal planes (Fig 3G: there is a mention of "basally oriented bundles"). If so, this should me mentioned in the figure legend. Otherwise, how can you decipher bundles in the plane of the epithelium from basally oriented? New images should be provided to better support the microtubules rearrangements. Again here, there is a need for quantification of number of events observed.

    Are prior studies referenced appropriately?

    yes

    Are the text and figures clear and accurate?

    The text is overall clear however some sentences are over-interpretations and should be either removed or rephrased.

    1. Lines 155-160 "In some apical end-feet, Rootletin is exclusively localized at the centrosome (Figs. 1H, I (upper), J (left)). In others, Rootletin appears as a rim-like structure, extending from the centrosome towards, and aligning partially with, the apical junction, where the adherens junctions (AJs), tight junctions (TJs)- and the polarity complex are localised (Figs. 1 H, I (lower), J (right), K)" this centrosome localisation could in fact be fibres with an apico-basal orientation, as shown in fig 1C. This part should be modified to propose this alternative.
    2. Lines 261-263: there are no evidence of a mechanical contribution of Rootletin to apical constriction. the sentence "likely playing a mechanical role in apical constriction...morphogenesis" should be either removed or clearly presented as a hypothesis of the authors.
    3. Lines 283-293: with fig 2 A-F and Fig 3C-E, the authors show that Rootletin fibre remodelling occurs before apical constriction and Lzts1 accumulation since rim-like structures are observed in not-constricted apical foot with no Lzts1 staining. However, there are no data showing that once Rootletin forms a rim, it always leads to apical constriction. Conclusion on a tight association between remodelling and constriction should thus be removed.
    4. Lines 333-336: the "uniform" distribution of debrin in constricted apical feet could be the consequence of tricellular junctions being very close to each other and not resolved with the imaging set used here. Hence the mention (line 364) that Debrin "redistributes" along apical endfoot cortex seems to be an overinterpretation of the observation.

    Figures

    1. As a general comment, IF stainings are saturated, which deteriorates the analysis. Please improve the image quality. Specifically Fig. 3I-K.
    2. Figure 1J,W,X: show the region/ line used to generate these profiles.
    3. Figure 2H-I: quality of image very degraded. provide better images to support measure of Root length.
    4. Figure 4L and 3B should be shown in the same orientation for comparison purposes. Same for 3F,4G and 4Q
    5. Figure legends: We lack necessary information regarding the imaging modalities, specifically to conclude on apico-distal localisation. Are the images shown Z-projections? How are cell outlined?

    Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    1. the authors aim to quantify the Rootletin reorganisation using two read-outs: the ratio between root fibre length and cell membrane periphery (SAM) and the ratio between Root area and apical foot area (AE). These read-outs are not fully convincing. First, measure of Root fibre length appears subject to high variability due to the precision of the images and the way Root fibres seem to coil as apical foot surface reduces. Second, these read-outs do not account for the formation of rim-like structures preceding the constriction of the apical foot surface. Analysing the categories of root structure for different surface coverage (AE) could be more informative.

    Significance

    General assessment:

    this work provides a description of Root dynamics of localisation, as reconstructed mainly from still images, but also with the support of a few movies. It shows that Root fibres are dynamics and actively reorganise during apical constriction and NE cells delamination. This work however lacks quantification of the events described to strongly support the model proposed. As well, this work is grounded on mis expression of GFP-Root. The model proposed could benefit from a similar description of the reorganisation of endogenous rootletin fibres during apical foot constriction in mouse neural tube.

    Advance:

    Until know, Rootletin fibres have been considered as a static structure providing mechanical resistance to ciliated cells. A recent work based on Cryo ET has shown that Rootlet is constituted of flexible fibres. Here, the authors show that Rootletin fibres change shape during a dynamic cellular process using still and live images. As such, it provides a conceptual advance that a structure often considered as static might be dynamic, at least in some cellular contexts, and could thus mediate other cellular functions than mechanical support.

    Audience:

    this work will be of interest for a specialized audience in the neurodevelopment field but also in the field of ciliogenesis. It will open a new perspective on a structure often seen as a static anchor of the cilium.

    Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    I am expert in ciliogenesis, with a strong background in genetics and imaging. Regarding the analysis of the quantified data (length or area of Rootletin, Lzts1 intensity) in relation with area of the apical foot, I lack expertise in the correlation analysis but I am surprised that correlations with a R2 below 0.4 come as biologicaly relevant.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, based on previous observations from their own and other groups, Wilmerding and collaborators attempt a thorough description of the dynamic localization, as well as possible interactions and functional roles, of the cilia-associated protein Rootletin during the process of neurogenesis in the early chick embryo neural tube. Through a combination of electroporation of constructs containing fluorescent reporters and immunostaining, the authors show, mainly in the chick embryo but also in mouse embryos and in human ESC-derived organoids, that Rootletin localization is highly dynamic in relation to the neuroepithelial cells cell cycle, and particularly, in correlation with neurogenic apical abscission events. All the observations are supported by labeling with several other cell markers, especially those highlighting the structure and dynamics of NE cells apical processes, by time-lapse in vivo imaging of the neurogenic NE, and by extensive quantifications. They reinforce the initially observed correlation by inducing apical abscission by overexpression of the transcription factor Neurogenin2 (that induces neurogenesis) and the microtubule-associated protein Lzts1 (that induces apical detachment but not neurogenesis). Lastly, they demonstrate a partial but very suggestive co-localization of the Zika virus NS5 protein with Rootletin during the process of apical abscission, a process that is known to be accelerated by viral infection.

    I have comments only on minor issues that are listed below with no particular order (except of appearance in the manuscript):

    1. A general issue has to do with figure accessibility to visually impaired persons (these are suggestions only):

    a- Some figures, particularly Fig. 1, and to a lesser extent Fig. 3, are huge, with a lot of images and letters that, when compressed to fit a pdf page, will result too small. If possible, please consider dividing these figures in two (this is very straightforward in the case of Fig. 1, which is already composed of 2 easily divided parts: chick on one side, mammals on the other).

    b- Colors: although many microscopy images are fine, there are some others using red in fluorescence, which should always be avoided considering color-blind persons. There is a possibility of choosing color-blind friendly palettes as plugins for Fiji (see for example https://github.com/bruvellu/color-blind-luts).

    1. Line 95: What is the "surface epithelium of the developing chick NT"? Maybe you meant "the apical surface of the epithelium..."? NE cells in chick are a simple layer of cells, forming a pseudostratified epithelium but with all cells contacting apical and basal surfaces.
    2. Abbreviation of "electroporation" to "EP". The whole term is first mentioned in line 126, and there is a sudden switch to the abbreviation from line 382. The explanation to abbreviation only appears at Fig. 1 legend, please clarify this earlier in the main text.
    3. Lines 483-485: It is claimed that "...apically accumulated ZIKV-NS5 actively recruit Rootletin fibers...". This seems to be an assumption based on a partial co-localization of the overexpressed proteins in the apical process, hence a correlation that gives no causal evidence. Please revise.
    4. Figure 1C legend, an error repeated in several similar cases in most figure legends: Where it reads "...transversal view of the apical process of electroporated NE cells...", the authors probably mean something like "... view of the apical process...on a transverse section of the NT...". The cell, and the process, are actually shown in a longitudinal view, what is transversal is the section of the neural tube.
    5. Fig. 1D legend: Correct duplication of "(AU)".
    6. Fig. 1D and J legend, and repeated in other cases, two comments:

    a- More important (and very often seen in the figure legends): Fluorescence intensity is shown with a statistical analysis, which is excellent. However, many times it is not clarified if the central line means "average/mean" (most probably) or another parameter. And, in most cases SEM is shown instead SD. Is there a reason for choosing SEM? Since variability in these data are most probably due to biological diversity than to experimental error, would it not be better to show the SD to convey a better idea of this (expected) intrinsic variability?

    b- Although very minor, it could help the reader if the authors draw a line on the example images to illustrate where fluorescence intensity was measured.

    1. Fig. 1P legend: What is illustrated with the lines on the graph; mean/average, median?
    2. Fig. 2G legend: Where it reads "L1 and L2 parameters were used to measure ... SAM...", the authors probably meant "... to calculate..."?
    3. Fig. 2M legend: I am slightly confused by what is shown in the graphs. Rather than "SAM occupancy", which is a calculated percent, is it not directly shown the extension of the Rootletin signal as an outline? And in the case of the "Apical area", is it not the "apical outline" as well?
    4. Fig. 3F: Where it reads "...they show stronger correlation...", I suggest writing "stronger negative correlation".
    5. Fig. 4C: What are the parameters shown? What is the "n"?
    6. Fig. 4L: The "white arrow" is cyan.
    7. Fig. EV3: Since the images are from NE at the same developmental stage, why is it that in A,B Rootletin immunodetection is only seen at the centriolar liker and in B,C along the ciliary rootlet fibers extended inside the apical processes? Is it a matter of signal intensity and different microscopy settings? Something else? This should be clarified.
    8. Fig. EV3E: Why "orthogonal views"? According to the outlines drawn by the authors, it would look like these are "oblique views", since we can see the apical surface and the end of the apical processes inside the NE.

    Significance

    The observations are novel and very interesting for the field of neural development, addressing a process that is still little understood at the cellular and molecular level: how neural progenitor/early neuronal cells eventually detach from a surface (in this case, the apical neuroepithelial surface) to begin differentiation. The results are in general well analyzed and support the conclusions. The manuscript is very well written and organized, in a way that can be readily understood by a wide readership.

  5. Version published to 10.64898/2025.12.09.693208 on bioRxiv