Multiciliated cells adapt the mechanochemical Piezo1-Erk1/2-Yap1 cell proliferation axis to fine-tune centriole number

  1. Vani Narayanan
  2. Venkatramanan G Rao
  3. Angelo Arrigo
  4. Saurabh S Kulkarni

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Abstract

Multiciliated cells (MCCs) are specialized epithelial cells that undergo massive amplification of centrioles, constructing several motile cilia to propel fluid flow. The abundance of cilia is critical for efficient fluid flow, yet how MCCs regulate centriole/cilia numbers remains a major knowledge gap. We have shown that mechanical tension plays a central role in regulating apical area and centriole number in MCCs. Here, we demonstrate that centriole amplification is controlled by a mechanochemical pathway essential for cell proliferation in cycling cells. Specifically, MCCs under tension use Piezo1-mediated calcium signaling to drive Erk½ phosphorylation via PKC and subsequent Yap1 activation. Remarkably, MCCs use this pathway to activate a cilia-specific transcription program, influencing the expression of Foxj1, a master regulator of motile ciliogenesis. Our work is the first to identify a novel function for an important mechanochemical pathway in centriole amplification in MCCs, offering new insights into ciliopathies and cancer, where aberrant centriole numbers are implicated.

Teaser

This study demonstrates that multiciliated cells utilize the mechanochemical Piezo1-Erk1/2-Yap1 cell proliferation axis to activate the cilia-specific transcriptional factor Foxj1 and amplify centrioles in a tension- dependent manner.

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    Reply to the reviewers

    Manuscript number: RC-2025-03094

    Corresponding author(s): Saurabh S. Kulkarni

    1. General Statements

    We thank the reviewers for their strong praise of the manuscript, highlighting its rigor, depth, and conceptual importance. They consistently described the study as a beautiful, fascinating, and conceptually strong piece of work that addresses a timely question in multiciliated cells. They also noted the high quality of the data, careful quantification, and the use of multiple genetic and pharmacological approaches, all of which improve the reproducibility and credibility of the findings. Importantly, they emphasized the novelty of discovering a direct mechanistic link between Piezo1-mediated mechanotransduction and Foxj1-driven transcriptional control of multiciliation, representing a significant breakthrough for both the cilia field and mechanobiology more broadly. Collectively, these strengths highlight the manuscript’s wide impact and make it highly suitable for publication in a high-impact journal.

    2. Description of the planned revisions

    Reviewer #1:

    There are two experiments that would significantly strengthen these claims.

    • First if their model is correct then even short term treatment with Yoda1 should induce the pathway and effect centriole numbers. While I appreciate the challenge of long term Yoda1 treatment its not clear to me why it would be needed if short term treatment is setting off the transcriptional cascade. Yoda is used throughout the paper to induce all the pathways but we don't know if it actually induces the phenotype. I think this should be addressed with either short term treatments or a dose response to find a dose that does not lead to skin pealing. It is hard to ignore this obvious deficiency.
    • Second, the model predicts that all of this is to regulate Foxj1 levels to regulate the subtle balance between cell size and centriole number. If this is correct, then the overexpression of Foxj1 should have a profound effect on centriole number in multiciliated cells. This is such an easy experiment that would validate many of the claims. RESPONSE:

    We recognize that the reviewer is asking us to test the sufficiency of the pathway with these comments: “If their model is correct, then they should be able to activate the pathway in one way or another to stimulate centriole number. This is a significant limitation to their overall model.” And “If this is correct, then the overexpression of Foxj1 should have a profound effect on centriole number in multiciliated cells.”

    To address reviewers’ suggestions, we will perform the following experiments.

    1. A brief exposure (15 and 30 mins) to Yoda1 and wait for 3 hours to examine changes in centriole amplification. This will avoid skin peeling from long-term exposure.
    2. A brief exposure to Yoda1 (15 mins) followed by a 30-minute wait period, and the cycle repeats a total of 4 times for a total of 3 hours to examine centriole amplification.
    3. The above two experiments will also be done in a constitutively active-Yap background to increase the probability that synergistic activation can lead to centriole amplification.
    4. Although Foxj1 is essential for multiciliogenesis, it is not sufficient to induce multiciliogenesis, as shown by multiple previous studies. Therefore, we do not expect overexpression of Foxj1 to have a profound effect on centriole number. While we will conduct the experiments because we truly want to address the suggestions and gain insight into the answers ourselves, we respectfully ask the Reviewer to consider the following responses to their concern.

    Yoda1 sufficiency: We agree that testing whether acute Yoda1 treatment can induce centriole amplification is an important question. We will conduct experiments with short-pulse and cyclic Yoda1 exposure, including in a constitutively active-YAP background (listed above), to address this possibility. However, several challenges complicate interpretation: (i) PIEZO1 adapts and desensitizes upon activation, (ii) transient signaling may be sufficient to cause secondary signaling but insufficient to drive stable transcriptional programs required for amplification, and (iii) centriole number is inherently variable, making modest effects difficult to resolve. However, we must recognize that failure to observe sufficiency under these conditions would not invalidate the model for two reasons: 1) absence of evidence is not evidence of absence, and thus, we may not have found the right experimental design. 2) PIEZO1–YAP is a necessary input but not sufficient on its own, as elaborated below. For both reasons, we are very careful about the interpretation of results in the manuscript, which shows that this pathway is necessary for centriole amplification using loss-of-function approaches.

    Foxj1 overexpression: Foxj1 is a well-established regulator essential for motile and multiciliogenesis across species (Xenopus, zebrafish, mouse). Loss of Foxj1 reduces cilia number in MCCs, but its activation alone does not have a profound effect on ciliogenesis/cilia number in MCCs. This is because Foxj1 is a part of a larger network essential for multiciliogenesis. This parallels the behavior of other transcriptional regulators, such as Myb, where loss of function impairs centriole amplification, but overexpression does not drive the formation of supernumerary centrioles. Both studies are seminal discoveries in the field of ciliogenesis, but they did not demonstrate the sufficiency of these molecules/pathways. Thus, our results, demonstrating that Foxj1 is necessary to induce tension-dependent centriole amplification, are significant, as the reviewer mentioned. The lack of Foxj1 sufficiency to induce centriole amplification is not a deficiency of the study, but rather evidence that Foxj1 is a part of a larger network essential for tension-dependent centriole amplification.

    Necessity versus sufficiency: We respectfully emphasize that sufficiency is not a prerequisite for demonstrating the significance of a pathway. Mechanochemical signaling is inherently complex, involving many mechanosensitive proteins and pathways. In our case, mechanical stretch increases centriole amplification, with PIEZO1–YAP signaling identified as a key mediator. However, we do not claim that PIEZO1–YAP alone is sufficient. Other pathways, including cadherin-mediated junctions, F-actin–myosin contractility, integrin–focal adhesion signaling, and nuclear mechanotransduction, likely contribute and may regulate unique downstream effectors that collectively promote centriole amplification. Therefore, PIEZO1–YAP should be regarded as one essential component within a larger network.

    __TIMELINE: __We will perform these additional proposed experiments. Since the first author, a postdoctoral researcher on this manuscript, has started a new job and will be coming in on weekends to complete the experiments, we estimate it will take approximately 2-3 months to finish them.

    Reviewer #2:

    Considering the Yap-piezo mechanism of action, the authors' logic for the selection of myb, foxj, plk4 and ccno as transcriptional targets is clear, but the HCR-derived signal and the differences seen in the yap morphants are not very strong, notwithstanding the statistical significance. There appear to be distinct subgroups within the treated populations (in Figure S6B, although these data seem quite different in Fig. 7H, so a comment on the technical differences might be helpful), so that the extent to which Yap1 regulates (Myb-)Foxj1 expression in MCCs is not clearly demonstrated by this experiment. Related to this point, it is unclear why 20-25% of the yap1/ piezo1 MO-treated embryos do not show a decline in FOXj1 in Fig. 6, given the qualitative nature of the scoring. Assuming the KD penetrance would vary on a cell-to-cell basis, rather than an embryo-to-embryo basis, this may suggest that there are additional relevant targets (some of which are discussed by the authors). Single-cell analysis might be a way to address this; however, this is not a trivial experiment, it might be sufficient to include a caveat in the text. Furthermore, the conclusion that Foxj1 regulates centriole amplification in a tension-dependent manner is well-supported by the data.

    RESPONSE: We appreciate the reviewer’s thoughtful observation. Differences in the expression of Foxj1 from experiment to experiment are possible due to a combination of factors, including heterogeneity in MCC development across embryos, slightly different embryonic stages, differences in embryo quality between fertilizations, and variability in morpholino delivery and knockdown penetrance, which can occur both across embryos and on a cell-to-cell basis within an embryo. We also note that technical aspects of HCR RNA-FISH, such as proteinase K treatment and washing steps, can affect signal intensity, potentially contributing to the appearance of distinct subgroups within treated populations.

    We agree that single-cell analysis would be a powerful way to dissect these differences, but as the reviewer notes, this is not a trivial experiment and is beyond the scope of the present study. We have therefore added clarifications in the text and discussion to acknowledge these sources of variability and to highlight the possibility of parallel pathways regulating foxj1 expression.

    ********************************************

    Controls for the knockdowns by the various MOs should be provided.

    RESPONSE: We appreciate the reviewer’s comment. The piezo1 MO has been previously established in Kulkarni et al. (2021). Additionally, the current manuscript includes MO control experiments for both erk2 and yap1, through KD at the 1-cell stage using the MO oligonucleotide, followed by mosaic-rescue with the respective WT RNA constructs (mCherry-ERK2 and yap1-GFP) and a nuclear tracer molecule such as H2B-RFP (Fig. 5, E-H, Fig. S5, C&D, Fig. 3, D-F). The mosaic-rescue is a robust experiment that provides an internal control within the same embryo, thereby avoiding differences that may arise due to embryo-to-embryo variability, embryo quality, or differences in fertilization batches. This approach also serves as a valuable tool for detecting cell-autonomous effects, providing a clear readout against uninjected neighboring cells, as the injected cells are labeled with a tracer. We will perform a similar mosaic-rescue experiment for the foxj1 MO.

    TIMELINE: We will conduct mosaic-rescue experiments for the foxj1 MO. We will need 1 month to complete the experiment.

    ********************************************

    __Minor comments:

    __

    Autocorrection of ERK1/2 or MEK1/2 pathways to 1/2 should be avoided. – We are unclear on this comment. Can reviewer please clarify what they mean.

    Reviewer # 3

    Major concerns

    1- The presented data do not yet establish a specific, direct pathway linking mechanotransduction to centriole number, because the molecular players tested (PIEZO1, Ca²⁺, PKC, ERK, YAP, Foxj1) are highly pleiotropic. As such, the observed centriole number phenotypes, and some of the major conclusions, could be indirect. It is therefore critical to test the specificity and causality of the proposed pathway. This could be done with the authors' own strategies and/or with the following potential approaches:

    • Genetic dependency and sufficiency tests: It could be shown that Yoda1 has no effect in PIEZO1 loss-of-function MCCs, and that wild-type PIEZO1, but not conductance-ad PIEZO1 pore mutants restores Yoda1 responsiveness across centriole number, pERK, and YAP readouts. For example, PIEZO1 C terminus was shown to govern Ca²⁺ influx and ERK1/2 activation. Comparing full length PIEZO1 with a C terminal deletion in MCC restricted rescue; loss of rescue of centriole amplification and ERK/YAP activation with the C terminal deletion can provide a genetics anchored specificity test beyond broad inhibitors.

    RESPONSE:

    • To address the reviewer’s concern, we will test whether Yoda1 affects ERK and Yap activation when Piezo1 is depleted. We appreciate the reviewer’s thoughtful suggestion to employ genetic rescue experiments with Piezo1 mutants. Unfortunately, these are not technically feasible in Xenopus, as the Piezo1 coding sequence is exceptionally large (~7.5 kb)____, and repeated attempts by our group to generate and express stable, translatable transcripts have been unsuccessful. To address genetic dependency and specificity despite these technical barriers, we have employed a combination of orthogonal strategies that together provide strong genetic and mechanistic evidence:
    1. Mosaic loss-of-function experiments (Fig. 1) demonstrate that Piezo1 regulates centriole number in a cell-autonomous manner, ruling out global epithelial or indirect tissue-wide effects.
    2. Pharmacological activation/inhibition with Piezo1-specific agonist (Yoda1) and inhibitors (GSMTx4, gadolinium) produced consistent phenotypes, including activation of downstream ERK and YAP readouts. Notably, Yoda1 is a Piezo-specific agonist, not a broad pharmacological agent.
    3. Downstream pathway dissection (calcium chelation, PKC inhibition, ERK2 depletion, and YAP1 knockdown/rescue) consistently converges on the same phenotypes, reduced centriole amplification and altered Foxj1 expression, providing multiple independent lines of evidence that the Piezo1–Ca²⁺–PKC–ERK–YAP axis specifically controls centriole number.
    4. Positive feedback regulation of Piezo1 expression by YAP/Foxj1 (Fig. 7) further strengthens the argument for a pathway-specific role rather than pleiotropic, indirect effects. Taken together, while full-length Piezo1 rescue experiments are technically not possible in Xenopus due to gene size constraints, our data employ state-of-the-art genetic, pharmacological, and orthogonal functional assays to rigorously test pathway specificity. These complementary approaches provide compelling evidence for the causal role of Piezo1-mediated mechanotransduction in centriole number control in MCCs.
    • Downstream bypass/rescue experiments: In PIEZO1 loss-of-function or BAPTA conditions, can enforcing MEK/ERK activation or YAP rescue centriole number defect? Conversely, can MEK inhibitors block Yoda1-induced effects.

    RESPONSE: We appreciate the reviewer’s insightful questions.

    • We will express CA Yap in the Piezo1 KD background to assess if we can rescue centriole number. We also note that the converse experiment has already been performed in our study: 1) PKC inhibition abolishes Yoda1-induced ERK phosphorylation and nuclear localization (Fig. 2), 2) both MEK inhibition and ERK2 depletion block Yoda1-induced Yap activation and nuclear entry (Figs. 4, S2). Thus, we have directly demonstrated that MEK inhibition prevents Yoda1-induced effects, satisfying this aspect of the reviewer’s concern.

    ********************************************

    2- Image quantification and analysis must be described in greater detail in the Methods section, as they are central to the major conclusions of the manuscript. For example, the authors should explain how nuclear, cytoplasmic, and centriole segmentation were performed, and how relative protein levels in the nucleus versus the cytoplasm (e.g., YAP, volume- or area-based) were quantified. Specifically, the thresholds and segmentation criteria applied to different cellular structures under various conditions, as well as the use of Imaris and other software, should be clearly detailed.

    RESPONSE: We will describe the methods in greater detail.

    ********************************************

    3- PIEZO1 mRNA was shown to incrase in a Foxj1 linked feedback loop. Does this increase translate into an increase in total protein levels?

    RESPONSE: If the reviewer is referring to Figure 7B, that is the Piezo1 antibody, so yes, the Piezo1 protein levels have increased.

    If the reviewer is referring to Figure 7C and D, we show that loss of Foxj1 leads to a reduction in Piezo1 mRNA expression.

    ********************************************

    4- Is the proposed signaling cascade active in mammalian multiciliated cells (e.g., airway epithelium). If possible, testing this by using one of the major players of the pathway as a readout such as as ERK phosphorylation, YAP nuclear localization in mammalian MCCs will reveal whether regulation of centriole number through this pathway is conserved and would strengthen the generality.

    RESPONSE: We agree with the reviewer that testing conservation of this pathway in mammalian MCCs is of great interest. Indeed, another group is currently investigating the role of Yap in the mammalian airway epithelium; in their temporally controlled Yap knockout model (the global Yap KO being embryonic lethal), they observed that Yap loss led to a reduction in centriole number. To avoid overlap and direct competition with this ongoing work, we chose to focus our efforts on Xenopus.

    Importantly, Xenopus has become a widely recognized and powerful system for MCC biology, enabling mechanistic dissection of centriole amplification and ciliogenesis. Several key discoveries in the field, including the identification of MCIDAS as a master regulator of MCC fate, were first made in Xenopus before being validated in mammals. Similarly, our study provides a mechanistic framework in Xenopus that can inform and guide ongoing studies in the mammalian airway.

    ********************************************

    5- Throughout the results section, there are multiple times where authors raised specific hypothesis about their data (e.g. foxj1 regulation of number control, apical actin/YAP). However, they have not tested them. These hypothesis are very exciting and if possible, testing experimentally, would strengthen the conclusions associated with them.

    RESPONSE: We are not sure what the reviewer means here by “authors raised specific hypothesis about their data (e.g., foxj1 regulation of number control, apical actin/YAP). However, they have not tested them”,

    BECAUSE:

    • Foxj1 regulation of centriole number: We demonstrate a clear reduction in centriole number upon Foxj1 depletion, and importantly, we extend this finding by showing that the reduction is tension-dependent (Fig. 6). We will perform a rescue assay to demonstrate the specificity.
    • Foxj1 and YAP: We never claimed that Foxj1 regulates YAP expression, and this is not part of our proposed model. Instead, our data show that Piezo1–ERK–YAP signaling regulates Foxj1
    • Foxj1 and apical actin: Foxj1 regulation of apical F-actin has already been established in prior work, and in our study, we clearly observe reduced apical actin intensity in Foxj1-depleted MCCs (Fig. 6). To further strengthen this conclusion, we will provide a quantitative analysis of apical actin intensity in Foxj1 morphants. ********************************************

    __TIMELINE: __We will perform these additional proposed experiments. Since the first author, a postdoc on this manuscript, has started a new job and will be coming in on weekends to finish the experiments, we estimate it will take approximately 2-3 months to complete them.

    Minor comments

    MCC vs non MCC identification (Fig. 1): Clarify how non MCCs were distinguished from MCCs (e.g. markers/criteria). – Can the reviewer please clarify which panel or panels? Or provide more specific text that needs to be changed.

    Add the Kintner group reference linking motile cilia number and centriole number in Xenopus MCCs.– Can the reviewer clarify where and which reference? Thank you.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    Reviewer 2

    Major comments:

    1. It should be clarified whether the immunoblots and the related quantitations in Figs. 2 and S2 are all from separate blots/ exposures. If so, they are not useful as controls, and these blots should be repeated with the relevant samples analyzed in parallel. Size markers and labels should be included (2B, 2G; S2B and S2G). An increase in total ERK would alter the interpretation of the increase in nuclear pERK in the IF experiments. RESPONSE: We thank the reviewer for raising this important point regarding clarification of the immunoblots. All experimental groups were analyzed in parallel with their corresponding controls. Because the primary antibodies for pERK and ERK were both raised in rabbit, we optimized our workflow to prevent protein loss during stripping and to ensure accurate visualization. Specifically, lysates from each experimental group were loaded in duplicate on the same gel, separated by a molecular weight ladder that served as a reference point. After transfer, the blot was cut along the ladder, and the two halves were processed in parallel: one probed with anti-pERK and the other with anti-ERK. This strategy ensured that all samples from a single experiment (e.g., Control and Yoda1-treated groups) were analyzed under identical conditions, with staining and imaging performed together at the same exposure. To enhance clarity, we have provided this data as __uncut, full-length __as Supplemental Figure 7 (Figure S7) in the revised revision.

    ********************************************

    Minor comments:

    1. Reference list should be checked for completeness; some citations lack journal/ volume/ page/ year details. – We have corrected the references.

    An 'overexposed' version of the image selected for centrioles in Figure 5F might be included with the Chibby-BFP at the same level as in the other figures. At present, the Yap KD cell in the image appears to have normal centrioles; this is potentially confusing, even though the authors clearly explain the matter in the text. – __We have added a new panel to Fig. 5F to avoid confusion.

    __

    1. It might be clearer to present injected/ uninjected in the same orientation in Fig. 6A and B. – __Unfortunately, that is not possible because the injected and uninjected sides are left and right, and they cannot be in the same orientation.

    __

    1. Figure 7B lacks the schematic described in the figure legend. – We have removed the Schematic sentence from the figure legend. That was an error on our side. Thank you for catching it.

    Reviewer 3

    1. Abstract: "how MCCs regulate centriole/cilia numbers remains a major knowledge gap" overstates the field; please soften to reflect recent advances (mechanics/apical area scaling; PIEZO1 implication). – We changed the text to “incompletely understood”.

    GsMTx4 rationale: State that GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (including PIEZO1) and justify its use alongside Yoda1.– GsMTx4 was used in the previous manuscript, and its use was justified there. Here, we are only comparing the results. However, we have added a sentence describing what GSMTx4 is. We have also included a sentence explaining the use of Yoda1. “GsMTx4, a spider venom peptide used in our previous study, inhibits cationic mechanosensitive channels, including Piezo1.”

    “For this experiment, we used the Piezo1 channel-specific chemical agonist, Yoda1, to increase the sensitivity of Piezo1 and upregulate calcium entry into cells”

    Timeline statement: "Centriole amplification to migration and apical docking takes ~4-5 h (personal observation)" is not appropriate; either cite time lapse literature or include your own time lapse data.– We have added a reference that showed imaging for 2 hours, but it was not enough to capture the entire process from intercalation to maturation, so we also kept “personal observation” still in the manuscript. We are unaware of any study that has done time-lapse imaging for 4 hours to capture the entire process of centriole amplification.

    Redundancy: The description of Yoda1 as a channel specific agonist is repeated; keep only once.- Removed

    "WT yap1 GFP construct previously used by Dr. Lance Davidson ..." should move construct description to Methods and keep only the citation in Results.– We moved it to Methods.

    "(Unpublished data; Dr. Mahjoub)" should be removed unless data are shown.- Removed

    Replace "as shown previously in our eLife paper" with "as we previously showed or shown previously (Kulkarni et al., 2021)".– We have made the change.

    The two hypotheses for how Foxj1 could regulate number under tension (actin remodeling vs. transcriptional control of amplification genes) belong in the Discussion unless tested. Moreover, the part on the discussion on yap sequestration by apical actin and the two possibilities presented also should go do discussion. – We have moved both to the discussion section.

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    Reviewer 3

    1- The hypothesis about the centriole pool of Piezo as the mechnosensor for centriole number regulation is very exciting and novel. Can localization controlled variants be used to test whether a centriole associated pool directly senses tension for number control (for example, centrosome targeted PIEZO1 via a PACT tag). Alternatively, broad cellular Ca sensors (GcaMP) or centrosome proximal Ca sensors (e.g., PACT GCaMP) can be used detect local calcium microdomains during tethering or Yoda1 treatment.

    RESPONSE: We appreciate the reviewer's curiosity and excitement; however, these experiments will not alter the conclusion of this paper and will be part of the next study, which aims to delve deeper into how different pools of Piezo1 at centrioles versus cell junctions function in MCCs. To that point, we had thought about these experiments. As mentioned earlier, the Piezo1 coding sequence is exceptionally large (~7.5 kb)____, and repeated attempts by our group to generate and express stable, translatable transcripts have been unsuccessful. Thus, the idea of centrosome-targeted PIEZO1 via a PACT is very exciting; however, it is not technically feasible. Beyond size, PIEZO1 is a trimeric, large plasma-membrane mechanosensitive channel that requires proper ER processing and bilayer incorporation. PACT localizes cargo to the centriole/pericentriolar material, not a membrane compartment; thus, a PACT-anchored PIEZO1 would be membrane-mismatched and almost certainly nonfunctional even if expressed/

    Second, Centrosome-proximal GCaMP (PACT-GCaMP) would show correlation, not causation. This experiment does not address the question “centriole pool of Piezo as the mechanosensor for centriole number regulation”. It will only show if the Ca2+ influx is happening at the basal bodies, but not whether and how that Ca2+ is essential for centriole amplification. For this purpose, we will need to find a way to block Ca2+ influx specifically at basal bodies, rather than junctions, which will require extensive controls.

    We do not claim that any specific Piezo1 or Ca2+ pool is critical for controlling centriole number and thus the suggested experiment would not alter the manuscript's conclusions. We therefore view the above as exciting future directions rather than prerequisites.

    ********************************************

    2- Because the proposed pathway is tension-sensing and YAP pathway is tightly linked to the actin cytoskeleton, the role of actin cysoskeleton in the proposed pathway should be tested directly. The authors mention different hypothesis around actin but has not tested them in the manuscript. For example, actin-depedent sequestration of Yap at the apical surface is intriguing. Does actin polymerization induced by drugs release Yap from the apical surface?

    RESPONSE: We would like to thank the reviewer for their suggestion. As per the reviewers' suggestion, we have moved this section to discussion, stating that “In the future, we plan to address this question by examining how Yap is sequestered by apical actin.”.

    However, we appreciate the reviewer’s enthusiasm and would like to share some experiments we are thinking/planning of to test the hypothesis.

    We plan to examine if the actin polymerization or contractility is responsible for Yap sequestration/release from the apical surface with the following experiments: 1) if the Yap is displaced by Jasplakinolide treatment, which stabilizes filamentous actin, 2) use of ROCK inhibitor to decrease contractility in the absence or presence of Yoda1, 3) Use genetic constructs such as Shroom3 to increase ROCK-mediated contractility to observe changes in Yap localization and dynamics.

    Although these experiments are interesting, they do not alter the conclusion of the current manuscript, and they represent future directions for our research.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    This manuscript investigates how mechanical tension is transduced into centriole amplification in Xenopus multiciliated cells (MCCs). Building on prior work that centriole number scales with MCC apical area and that this scaling depends on PIEZO1, the study proposes that MCCs repurpose a canonical mechanochemical axis-PIEZO1 → Ca²⁺/PKC → ERK1/2 → YAP → Foxj1-to regulate centriole number rather than mitosis. The authors use tethered vs. untetheredanimal cap explants to modulate tissue tension, combine pharmacologic perturbations with genetic loss of function and rescue, quantititative image analysis and present a model in which tension gated PIEZO1 activates ERK/YAP, influences Foxj1, and tunes centriole number in MCCs.

    The manuscript tackles an important and timely problem with clear disease relevance. It major advance is their presented model that posits that post mitotic MCCs repurpose a canonical mechanotransduction module to regulate organelle number rather than proliferation. It is a conceptually strong study addressing an important problem with a clean mechanical paradigm. However, to support the central claim that centriole number control is a specific, direct consequence of the PIEZO1-Ca²⁺-ERK/YAP pathway within MCCs, the revision should establish specificity and causality and provide experimental data for some of the major conclusions as detailed below. Addressing these points are critical to support the mechanistic conclusions and impact.

    Major concerns:

    1. The presented data do not yet establish a specific, direct pathway linking mechanotransduction to centriole number, because the molecular players tested (PIEZO1, Ca²⁺, PKC, ERK, YAP, Foxj1) are highly pleiotropic. As such, the observed centriole number phenotypes, and some of the major conclusions, could be indirect. It is therefore critical to test the specificity and causality of the proposed pathway. This could be done with the authors' own strategies and/or with the following potential approaches:

    • Genetic dependency and sufficiency tests: It could be shown that Yoda1 has no effect in PIEZO1 loss-of-function MCCs, and that wild-type PIEZO1, but not conductance-dead PIEZO1 pore mutants restores Yoda1 responsiveness across centriole number, pERK, and YAP readouts. For example, PIEZO1 C terminus was shown to govern Ca²⁺ influx and ERK1/2 activation. Comparing full length PIEZO1 with a C terminal deletion in MCC restricted rescue; loss of rescue of centriole amplification and ERK/YAP activation with the C terminal deletion can provide a genetics anchored specificity test beyond broad inhibitors.

    • Downstream bypass/rescue experiments: In PIEZO1 loss-of-function or BAPTA conditions, can enforcing MEK/ERK activation or YAP rescue centriole number defect? Conversely, can MEK inhibitors block Yoda1-induced effects.

    1. The hypothesis about the centriole pool of Piezo as the mechnosensor for centriole number regulation is very exciting and novel. Can localization controlled variants be used to test whether a centriole associated pool directly senses tension for number control (for example, centrosome targeted PIEZO1 via a PACT tag). Alternatively, broad cellular Ca sensors (GcaMP) or centrosome proximal Ca sensors (e.g., PACT GCaMP) can be used detect local calcium microdomains during tethering or Yoda1 treatment.

    2. Because the proposed pathway is tension-sensing and YAP pathway is tightly linked to the actin cytoskeleton, the role of actin cysoskeleton in the proposed pathway should be tested directly. The authors mention different hypothesis around actin but has not tested them in the manuscript. For example, actin-depedent sequestration of Yap at the apical surface is intriguing. Does actin polymerization induced by drugs release Yap from the apical surface?

    3. Image quantification and analysis must be described in greater detail in the Methods section, as they are central to the major conclusions of the manuscript. For example, the authors should explain how nuclear, cytoplasmic, and centriole segmentation were performed, and how relative protein levels in the nucleus versus the cytoplasm (e.g., YAP, volume- or area-based) were quantified. Specifically, the thresholds and segmentation criteria applied to different cellular structures under various conditions, as well as the use of Imaris and other software, should be clearly detailed.

    4. PIEZO1 mRNA was shown to incrase in a Foxj1 linked feedback loop. Does this increase translate into an increase in total protein levels?

    5. Is the proposed signaling cascade active in mammalian multiciliated cells (e.g., airway epithelium). If possible, testing this by using one of the major players of the pathway as a readout such as as ERK phosphorylation, YAP nuclear localization in mammalian MCCs will reveal whether regulation of centriole number through this pathway is conserved and would strengthen the generality.

    6. Throughout the results section, there are multiple times where authors raised specific hypothesis about their data (e.g. foxj1 regulation of number control, apical actin/YAP). However, they have not tested them. These hypothesis are very exciting and if possible, testing experimentally, would strengthen the conclusions associated with them.

    Minor concerns:

    1. Abstract: "how MCCs regulate centriole/cilia numbers remains a major knowledge gap" overstates the field; please soften to reflect recent advances (mechanics/apical area scaling; PIEZO1 implication).

    2. MCC vs non MCC identification (Fig. 1): Clarify how non MCCs were distinguished from MCCs (e.g. markers/criteria).

    3. GsMTx4 rationale: State that GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (including PIEZO1) and justify its use alongside Yoda1.

    4. Timeline statement: "Centriole amplification to migration and apical docking takes ~4-5 h (personal observation)" is not appropriate; either cite time lapse literature or include your own time lapse data.

    5. Redundancy: The description of Yoda1 as a channel specific agonist is repeated; keep only once.

    6. "WT yap1 GFP construct previously used by Dr. Lance Davidson ..." should move construct description to Methods and keep only the citation in Results.

    7. "(Unpublished data; Dr. Mahjoub)" should be removed unless data are shown.

    8. Add the Kintner group reference linking motile cilia number and centriole number in Xenopus MCCs.

    9. Replace "as shown previously in our eLife paper" with "as we previously showed or shown previously (Kulkarni et al., 2021)".

    10. The two hypotheses for how Foxj1 could regulate number under tension (actin remodeling vs. transcriptional control of amplification genes) belong in the Discussion unless tested. Moreover, the part on the discussion on yap sequestration by apical actin and the two possibilities presented also should go do discussion.

    Significance

    This manuscirpt dissects Piezo1-mediated mechanotransduction to regulation of centriole number in Xenopus multiciliated cells (MCCs) via Ca²⁺, ERK/YAP, and Foxj1. While Piezo1 and its downstream effectors have been implicated broadly in mechanosensation, cellular tension responses, and transcriptional regulation, their specific role in centriole nubmer control in MCCs has been unknown By integrating pharmacological manipulation, genetic perturbation, and functional readouts, the authors demonstrate that this pathway directly influences centriole number.

    The findings extend published knowledge in two main ways:

    (1) they connect a mechanosensitive ion channel to the transcriptional program governing Foxj1 expression and multiciliation, a mechanistic link not previously defined, and

    (2) they highlight the pleiotropic yet coordinated nature of Piezo1 signaling in organelle biogenesis. This work will be of broad interest to cell and developmental biologists studying ciliogenesis, epithelial differentiation, and mechanotransduction, as well as to biomedical researchers interested in multicilaited cells and ciliopathies. By situating a well-studied mechanosensor within the context of MCC biology, the study opens new directions for understanding how tissue-level forces shape organelle number control and function.

    At the same time, the impact of the study is weakened by concerns regarding the causability and specificity of the pathway, since the signaling components examined are highly pleiotropic and it remains challenging to separate direct effects on centriole number from broader cellular consequences. The causal relationships among Piezo1 activity, downstream signaling, and Foxj1 expression require stronger substantiation, and the extent to which this pathway operates in mammalian multiciliated cells remains an open question. Addressing these limitations would strengthen the robustness, generality, and translational relevance of the conclusions.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Narayanan, Kulkami and colleagues here examine how the Piezo-Erk-Yap pathway is involved in centriole numerical control in multiciliated cells (MCCs). Using reverse genetic and pharmacological methods in Xenopus embryos, they show that Piezo-mediated ERK signalling through to Yap regulates tension-sensitive centriole number, through a mechanism that involves Foxj1, very likely acting as a transcription factor. The data are carefully controlled, robustly analysed and well presented. Statistical analyses are notably thorough.

    Main points:

    1. It should be clarified whether the immunoblots and the related quantitations in Figs. 2 and S2 are all from separate blots/ exposures. If so, they are not useful as controls, and these blots should be repeated with the relevant samples analysed in parallel. Size markers and labels should be included (2B, 2G; S2B and S2G). An increase in total ERK would alter the interpretation of the increase in nuclear pERK in the IF experiments.

    2. Considering the Yap-piezo mechanism of action, the authors' logic for the selection of myb, foxj, plk4 and ccno as transcriptional targets is clear, but the HCR-derived signal and the differences seen in the yap morphants are not very strong, notwithstanding the statistical significance. There appear to be distinct subgroups within the treated populations (in Figure S6B, although these data seem quite different in Fig. 7H, so a comment on the technical differences might be helpful), so that the extent to which Yap1 regulates (Myb-)Foxj1 expression in MCCs is not clearly demonstrated by this experiment. Related to this point, it is unclear why 20-25% of the yap1/ piezo1 MO -treated embryos do not show a decline in FOXj1 in Fig. 6, given the qualitative nature of the scoring. Assuming the KD penetrance would vary on a cell-to-cell basis, rather than an embryo-to-embryo basis, this may suggest that there are additional relevant targets (some of which are discussed by the authors). Single-cell analysis might be a way to address this; however, this is not a trivial experiment, it might be sufficient to include a caveat in the text. Furthermore, the conclusion that Foxj1 regulates centriole amplification in a tension-dependent manner is well-supported by the data.

    3. Controls for the knockdowns by the various MOs should be provided.

    Minor points:

    1. Autocorrection of ERK1/2 or MEK1/2 pathways to 1/2 should be avoided.

    1. Reference list should be checked for completeness; some citations lack journal/ volume/ page/ year details.

    2. An 'overexposed' version of the image selected for centrioles in Figure 5F might be included with the Chibby-BFP at the same level as in the other figures. At present, the Yap KD cell in the image appears to have the normal centrioles; this is potentially confusing, even though the authors clearly explain matters in the text.

    3. It might be clearer to present injected/ uninjected in the same orientation in Fig. 6A and B.

    4. Figure 7B lacks the schematic described in the figure legend.

    Significance

    This study presents novel insight into the developmentally important process of ciliogenesis in multiciliated cells that will be of specific interest to the fields of cilium biology and mechanobiology, with additional general interest in calcium signalling and cell biology.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript from Narayanan addresses the fascinating question of how Multiciliated cells regulate centriole number to scale with cell size. They have generated a tremendous amount of high quality data that supports a model in which mechanosensitive signaling via piezo1 leads to an increase in intracellular Ca++ that leads to an activation of the Erk pathway which in turn activates the Yap pathway that in turn regulates FoxJ1 levels which they propose regulates centriole number. This is complicated but they have strong quantifiable data that supports most of the claims. I think this is a beautiful study that adds significantly to the field. There is a lot of evidence that disrupting these pathways has a negative consequence on centriole number. What is lacking is a positive connection showing a role of these processes in fine tuning the centriole number as the title suggests. Several key experiments would significantly strengthen their claims.

    • The data is presented in a way that proposes that the ultimate role of these pathways is to regulate Foxj1 levels to fine tune centriole number based on the level of tension. There are two experiments that would significantly strengthen these claims. First if their model is correct then even short term treatment with Yoda1 should induce the pathway and effect centriole numbers. While I appreciate the challenge of long term Yoda1 treatment its not clear to me why it would be needed if short term treatment is setting off the transcriptional cascade. Yoda is used throughout the paper to induce all the pathways but we don't know if it actually induces the phenotype. I think this should be addressed with either short term treatments or a dose response to find a dose that does not lead to skin pealing. It is hard to ignore this obvious deficiency. Second, the model predicts that all of this is to regulate Foxj1 levels to regulate the subtle balance between cell size and centriole number. If this is correct, then the overexpression of Foxj1 should have a profound effect on centriole number in multiciliated cells. This is such an easy experiment that would validate many of the claims.

    Minor issues:

    • The authors attempt to measure an effect of plk4 and ccno in the Yap MO experiment. However, the fact that they could not be scored means the experiment wasn't really performed. I think it is more appropriate to leave out rather than risk giving the impression that these genes were unaffected.

    • The authors indicate that the foxj1 result suggests two alternatives, one that foxj1 regulates actin (pan 2007) and the other that it is a transcription factor. I think the evidence for foxj1 being a transcription factor is extremely well established and while it is possible for it to have an additional unrelated role my interpretation of the Pan paper is that the failed apical docking leads to disrupted actin which is also well established. I don't think there is a lot of evidence for foxj1 being anything other than a TF.

    Significance

    • This is a really beautiful paper that will be well appreciated by the cilia community but also should be appreciated by the broader cell biology community.

    • The strengths of this paper are a high level of rigor in which they perform detailed quantification of a wide range of processes. For many experiments they have multiple methods for disrupting function which again adds to the rigor. They have successfully linked Piezo1, Erk, Yap and FoxJ1 function to proper centriole biogenesis, which is a significant advance.

    • The limitation is that all their perturbations negatively effect centriole number which could be indirect. If their model is correct then they should be able to activate the pathway in one way or another to stimulate centriole number. This is a significant limitation to their overall model.

  5. Version published to 10.1101/2025.01.21.634139 on bioRxiv