Long-Range Coupling of Posterior Cell Addition and Anterior Vacuolation Provides Robustness in Notochord Elongation

  1. Carlos Camacho-Macorra
  2. Alberto Ceccarelli
  3. Dillan Saunders
  4. Guillermo Serrano Nájera
  5. Osvaldo Chara
  6. Benjamin Steventon

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Abstract

Robust tissue growth control requires long-range communication between the rate of progenitor addition and tissue expansion. However, the regulatory mechanisms that couple these two processes are unknown. In zebrafish, notochord morphogenesis is a principal driver of axis extension through the combined actions of posterior progenitor addition and anterior vacuolation. To elucidate how progenitor dynamics and vacuole-driven cell expansion interact to shape notochord development, we first generated a mathematical model that links progenitor addition rate to the progressive expansion of cells from anterior-to-posterior to simulate vacuolation rate. Comparing this with empirical measurements, we find that progenitor incorporation together with vacuolation, produces a linear gradient in nearest neighbour distance. We next explored the role of YAP/TAZ in regulating the rate of progenitor addition in mutants for YAP/TAZ inhibitor vgll4b . We find that vgll4b expression and YAP activity are enriched in posterior midline progenitors. Loss of vgll4b elevates YAP signaling, enhances progenitor addition, restricts vacuole expansion, and—after a transient buffering phase—compromises A-P axis elongation. These results support a long-range feedback mechanism linking progenitor recruitment to vacuolation, enabling the notochord to balance cellular input with volumetric expansion, thereby maintaining tissue proportions.

A . Schematic representation of the authors’ proposed model illustrating how increased YAP activation in notochord progenitors of vgll4b mutants leads to enhanced progenitor addition to the notochord. This increased incorporation subsequently compromises the ability of notochord cells to undergo proper vacuolation, resulting in reduced axial elongation compared with control embryos.

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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    This work focuses on zebrafish notochord morphogenesis during axial elongation. In particular it dissects the role of YAP signalling on regulating the balance between caudal cell addition with the cell enlargement occurring rostrally through vacuolation.

    The article is timely to the field and includes several important experiments. The overall presentation and written style are good, citations are adequate and there is a clear effort to integrate experiments and mathematical modelling from the outset. The logic behind experiments is sound and the conclusion coherent (even if not totally unexpected given the literature): YAP affects progenitor addition which in turn changes packing, vacuolation and axis length. I just have a few points that could make the article clearer and more persuasive.

    We thank the reviewer for these positive comments about our manuscript. We would like to reiterate the two main unexpected findings based on our results:

    • While YAP mutants display a defective notochord (Kimelman et al., 2017; eLife) it has not been clear what specific role that YAP signalling is playing during notochord development. Therefore, the finding that Yap signalling plays a role in controlling the rate to notochord progenitor addition and represents a novel discovery.
    • The observation that the notochord can buffer its elongation rate against an increased influx of progenitors is novel and counter intuitive. Our current understanding of tissue elongation depends on the central idea that the addition of progenitors directly impacts elongation rate. Here we show for the first time that this has minimal impact at the tissue level using the notochord as an example. Major points

    - Last section of results is difficult and confusing. After analysing vgll4b loss-of-function line, effectively over-activating YAP, the focus is on YAP inhibition using Verteporfin.

    o Concerns on Verteporfin: the molecule has been widely used to module YAP, but there are also plenty of studies suggesting it is non-specific (also degrades YAP, has 14-4-3σ dependency and induces stress). I would consider an alternative: truncated TEAD, LATS over-expression or gain-of-function phosphomimetic versions of YAP.

    o Presentation: regardless of point above, Verteporfin's role on YAP should be verified in the system. As such it is crucial to include: images of 4xGTIIC, noto and YAP stains after treatment. Only then inspect the effects on vacuolation and different treatments.

    As suggested by the reviewer, we have added a supplementary figure validating the verteporfin treatment, including quantification of GFP reduction across the three tissues and quantification of notochord staining. We did not include Yap1 immunostaining data because the signal quality was insufficient for reliable analysis.

    A simple over-expression experiment will not allow the spatial and temporal control required to test our hypothesis. Yap has a known function in gastrulation, so we need experiments that allow us to perturb Yap activity only at posterior body elongation stages. This has been achieved with the vgl4b experiments shown in the manuscript, as this gene is specifically expressed in the tailbud at these stages. In addition to the full verification of verteporfin's impact on YAP activity, we feel this is sufficient evidence to support our conclusions.

    - In Fig 3F, noto HCR staining is taken as evidence for progenitor exhaustion/ faster depletion. Other scenarios would be possible without more direct demonstration. Evidence (either experimental or literature) that YAP is not involved in self-renewal or induction of these progenitors at these stages should be discussed.

    We have concluded that the smaller volume of noto expressing cells is consistent with the faster depletion of the progenitor pool based on the direct observation of increased progenitor addition rate from photo-labelling experiments (Figure 3A,B). As suggested by the reviewer, we have now quantified cell divisions within the midline progenitor population and found no significant differences between mutant and control embryos. These data have now been included in Supplementary figure 3.

    - Individual datapoints in Fig 3C and 4D should be shown.

    These data have now been added to the figures

    Additional justification is needed as to why spinal cord is the best to benchmark displacement. Additionally looking at this with respect to mesoderm migration could capture another set of progenitors and behaviour/ displacements.

    Photolabels within the pre-somitic mesoderm are difficult to interpret as the high amount of cell rearrangement in this tissue leads to a spreading out of the labelled clone in a manner that then makes it difficult to assess tissue displacement (see Figure 2D,E; Thomson et al., (2021) Cells and Development). In contrast, aprevious paper has shown that notochord-spinal cord displacements can be mapped in a reliable manner across the anterior-posterior axis which motivated our choice here (McLaren and Steventon (2021) Development).

    - Plotting vacuole area in Fig.4I vs A-P position (similar to plots 1H, 2F-H) could further strengthen the point of gradual (linear) vacuolation.

    As suggested by the reviewer, we have plotted vacuole area as a function of position for the verteporfin treatment experiments, and these data have now been included in Figure 5.

    Minor points:

    - Scheme of Fig1A could benefit from having the info of zebrafish timeline (hpf)

    The scheme has been modified indicating zebrafish timeline

    - Figure 3B, what was time 0?

    Timepoints have now been included in the text and figure legend

    - The authors should address whether Verteporfin-treated mutants are rescued or whether the compound overwhelms the genetic effect.

    Given that verteporfin will impact Yap signalling in a global manner, whereas the vgl4b have a localised over-activation of Yap signalling, we think this experiment would be difficult to interpret and would likely be non-informative.

    - Cell density is an elegant measure but quite abstract. A plot of cells detected at each AP position would be quite valuable to reinforce more cells are being added to a relatively constant area.

    As suggested by the reviewer, we have now plotted these data for mutant and controls and also for verteporfin treatments. These data have now been included in supplementary figures 3 and 7.

    Reviewer #1 (Significance (Required)):

    Significance included above.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary

    Camacho-Macorra et al. investigate the mechanisms of axis extension in zebrafish embryos, focusing on the notochord and its two key elongation processes: progenitor addition (occurring early and posteriorly) and vacuolization (occurring later and in an anterior to posterior sequence). The authors first develop a mathematical model to predict notochord elongation dynamics by integrating these processes. They demonstrate that the YAP signaling pathway is active in both the notochord and its progenitors during axial extension. Their analysis reveals that vgll4b, an inhibitor of YAP, is expressed in the same regions. Knockdown of vgll4b results in YAP hyperactivation in the notochord and posterior progenitor regions, leading to increased progenitor recruitment into the notochord and a reduction in the progenitor pool. The effects of this mutation on extension are most pronounced during the late phase, which is dominated by vacuolization. The authors observe smaller vacuoles in mutants during this phase. However, early (but not late) YAP inhibition decreases notochord cell density and increases vacuole size, suggesting that YAP primarily regulates notochord progenitor uptake, which indirectly affect vacuolization.

    Major Comments

    The authors propose that YAP activity mediates a long-range feedback mechanism linking posterior progenitor addition to anterior vacuolization. Two lines of evidence are presented to support this idea. First, there appears to be compensation for tissue length during Phase 2, when both progenitor addition and vacuolization occur. Second, temporal YAP inhibition experiments show that early, but not late, YAP inactivation affects both cell addition and vacuolization. While these observations are intriguing, they do not conclusively demonstrate spatial long-range coordination. Instead, the global decrease of vacuole size could be a simple delayed consequence of cell density increase or cell disorganization at the posterior end without involving a long-range feedback along AP axis. Claiming that such long-range feedback is taking place would require a more precise characterization and/or the identification of its nature (chemical, mechanical).

    We would like to thank this reviewer for this point, that we feel requires further clarification. As they suggest, the increased additional rate of posterior progenitors leads to a later impact on vacuolation, once these cells have reached more anterior parts of the body axis- creating an effective long-range feedback mechanism to link the two processes. However, this is not a direct propagation of a signal (mechanical or otherwise) across the length of the notochord, as may have been interpreted to be based on the previous framing of our conclusions. We have modified the title of our manuscript to place less emphasis on the 'long-range feedback', and included an additional discussion paragraph to make this point clearer.

    Furthermore, there are several caveats with the interpretations of the claims cited above. The authors do not show quantification of vacuole area using notochord cell segmentation as described in Fig 1C in vgll4b mutants at stages when progenitor addition is increased.

    This is an important point highlighted by the reviewer. We have now included analysis at 24 hpf, where we do see a significant reduction in vacuole area within the anterior part of the notochord during the buffering phase in vgl4b mutants- consistent with our model that reduced anterior vacuolation compensates for increased progenitor addition rate during this phase of notochord elongation (Figure 4E).

    The slope of internuclear distances in Supplementary Figure 4A at 27 hours post-fertilization suggests that vacuolization is initially normal (similar to wt context in Fig 1H), arguing against an early defect in vacuolation dynamics along the Anterior to Posterior axis that could compensate for extra addition of progenitors.

    We have revised Supplementary Figure 4 to present a direct comparison between mutant and control embryos at each time point analyzed. This analysis shows that within the mid-trunk region of the notochord, differences in cell size first emerge at the developmental stage when vacuolation becomes the primary driver of axis elongation. In addition, we observe a progressive decoupling of the scaling relationship in mutant embryos over time. As mentioned above- there is a significant difference in vacuole size within more anterior regions at 22.5 hpf that is consistent with the model that this is buffering against increase posterior addition.

    Finally, the timing of the analysis of the effect of Verteporfin treatments is unclear. According to the legend of Figure 4F, analyses for Treatment A (16-27 hpf) and Treatment B (27-38 hpf) were done at 24 hpf and 30 hpf, respectively. If this is the case, the 3-hour window for Treatment B may not allow sufficient time to reveal effects on vacuolization.

    We agree that the information regarding the verteporfin experiments was not clearly presented in the original figure, and we have therefore revised the schematic accordingly.

    To strengthen the claim of long-range coupling, the authors could:

    Provide direct measurements of vacuolization A-P dynamics/area during Phase 2, before the effect on notochord length in the mutant, to see if there is indeed a compensatory effect on notochord length for the additional accretion of notochord progenitors in the Vgll4b mutant.

    As suggested by the reviewer, we have added an earlier time point to the A-P area dynamics plot in phase 2, corresponding to a stage at which the effect on notochord length in the mutant is not yet detectable. At this stage, we observed no difference in vacuole area between mutants and controls. We have also included an earlier time point analysis in the anterior region of the axis, which shows a similar cell size difference to that observed later in a more posterior region (Figure 4F; see above response).

    Clarify the analysis timing of Treatment B to confirm that YAP inhibition during the vacuolization phase truly has no effect.

    This has now been clarified.

    Additionally, as a non-specialist, I found the distinction between the two modeling hypotheses difficult to follow. Specifically, it is unclear why the first hypothesis assumes YAP affects vacuolation rate, while the second assumes it affects vacuolation front speed. It is also not intuitive how front speed can be independent of vacuolation rate, as one would expect that if cells form vacuoles more slowly, the front should progress more slowly as well. Therefore, it could be good to clarify these aspects of the modeling part.

    We thank the reviewer for this comment and apologise for the lack of clarity in our description of the model. In our framework, the cell size profile along the AP axis of the notochord is governed by two distinct processes: (i) the addition of progenitors at the posterior tip, and (ii) vacuolation, which increases cell size and proceeds from anterior to posterior. We model the latter as a propagating wave with velocity vf​, such that cells begin to vacuolate when the wave front reaches their position.

    Importantly, in the model these two aspects of vacuolation are decoupled: the front velocity vf​ determines when a given cell starts vacuolating, whereas the vacuolation rate J determines how fast the cell increases in size once the process has started. Biologically, this corresponds to distinguishing between the propagation of a trigger or competence signal along the tissue, and the execution of vacuole growth within each cell. Our reasoning was that they need not be strictly proportional: a signalling wave could propagate at a given speed even if the downstream cellular response is slower or faster.

    This is why we considered two alternative hypotheses: either YAP modulates the propagation of the vacuolation front (affecting vf​), or it modulates the growth dynamics within each cell (affecting J). Our quantitative comparison with the experimental data supports the former scenario. This has now been clarified in the main text.

    Minor Comments

    While the study is technically sound, a few areas could benefit from improved clarity or additional data.

    An intriguing but puzzling finding is the reduction in the noto-expressing progenitor domain in vgll4b mutants, despite elevated YAP activity in progenitors. Intuitively, if YAP promotes progenitor maintenance or expansion, one might expect the noto+ domain to increase, not shrink. This paradox suggests that YAP may not only simply maintain progenitors but instead accelerates their differentiation or migration into the notochord (as stated in the manuscript and graphical abstract). Alternatively, YAP could only deplete the noto+ pool by driving premature entry into the notochord, though the lack of clear YAP upregulation in this domain would imply a non-cell autonomous role of YAP for this interpretation. The authors should discuss these possibilities more explicitly in the Discussion section and could consider including additional markers, such as proliferation assays or apoptosis markers, to clarify whether YAP affects progenitor proliferation, differentiation, or migration.

    As also suggested by the reviewer, we have included a cell proliferation analysis in Supplementary Figure 3 and have revised the Discussion section accordingly.

    In Figure 2B, the YAP activity reporter signal in the posterior floor plate is not immediately obvious. The authors should consider providing higher-magnification insets.

    As suggested by the reviewer, we have included higher-magnification insets in Figure 2

    In Figure 2C, the differences in tail shape between wild-type and mutant embryos are visually striking. If these differences have not been quantified or discussed, a brief comment in the text would be helpful.

    We did not see a consistent impact on the morphology of the posterior body, this has now been clarified in the main text.

    Supplementary Figure 6 describes embryo length differences in mutants but does not include a representative image. Adding one would strengthen the phenotypic description.

    As suggested by the reviewer, we have modified Supplementary Figure 6

    Figure 1C is not cited in the text as not associated with a result, but just a description of the approach that is used later in Fig 4I

    We have modified the text to include the appropriate figure reference.

    Finally, the authors might consider citing Michaud & Pourquié (2025) when presenting the role of hydrostatic pressure in axis elongation in the Introduction.

    We have now modified the text to include this citation which we agree is relevant to this work.

    Reviewer #2 (Significance (Required)):

    This study by Camacho-Macorra et al. presents a fascinating exploration of how YAP signaling and its inhibition by vgll4b coordinate progenitor addition and vacuolization during zebrafish notochord elongation. The work is well executed, with clear results and integration of mathematical modeling and experimental data. The findings shed new light on the molecular and mechanical regulation of axis extension, a fundamental process in vertebrate development. However, while the study is innovative and rigorously conducted, the central claim of "long-range coupling" between progenitor addition and vacuolization requires further substantiation. Addressing the points discussed below will make the study more convincing and accessible to developmental biologists and mechanobiologists alike.

    reviewer expertise: developmental biologist specialised in morphogenesis

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    In the studies conducted by Camacho-Macorra et al., the authors examine the extension of the body axis is zebrafish, focusing on the notochord. They specifically compare timepoints where progenitor addition to the notochord and vacuolization are important to drive axis extension. They generate a simple mathematical model of notochord extension and show that it recapitulates observations in vivo where progenitor addition and vacuolation drive tissue elongation. They further perturb the system by showing that YAP activity is localized to the midline progenitors of the notochord where when the competitive inhibitor of YAP vgll4b is perturbed it increases YAP signaling and results in increase progenitor addition to the notochord. They further describe a possible indirect-feedback mechanism linking YAP driven progenitor addition to the notochord with anterior vacuolation which when perturbed (i.e. increased YAP) results in reduced notochord elongation.

    Major Comments:

    NA

    Minor comments:

    1.Figure 1B - please put the model equation in the figure or at least point out what variables of the equation refer to each part of the schematic.

    As suggested by the reviewer, we have modified the scheme in Figure 1

    2.Figure 1F - smooth line is misleading, please include individual embryo measurement points. This comment could be applied to several figures

    We agree with the reviewer that the graphs in the original manuscript could be improved, and we have therefore modified all figures to better represent data dispersion within each group.

    3.Figure 2C/D - To make this manuscript more accessible to individuals who are not familiar with the anatomy of zebrafish tail, please include zoom in panels of the region of interest where arrows are pointing out increased YAP signaling in the floor plate and hypochord.

    As suggested by the reviewer, we have included higher-magnification insets in Figure 2

    4.In discussion - "In vgll4b mutants, increased progenitor incorporation initially does not alter overall notochord length due to a buffering mechanism for natural variation in progenitor addition" - this is not directly tested in terms of buffering for variation and is an assumption. Please either cite a paper or reword

    This point has been clarified in the revised discussion.

    Reviewer #3 (Significance (Required)):

    Overall, the logic and experiments conducted in these studies are well defined. However, the significance of the work is minimal and makes only a small contribution to the advancement of the field of developmental biology. Regardless, the studies are well done and worth publication.

    Strengths:

    -The study does a good job of incorporating and testing a computational model in a way that proves/disproves their hypothesis

    -The manuscript is well written and follows a logical order, making it easy for readers to understand the main findings

    -The study uses multiple routes of YAP inhibition (genetic and drug) to show effect on progenitor addition to the notochord and shortened body axis

    -The discussion does aa very good job of giving the context of the study's results.

    Weaknesses:

    -The study is minimal and fails to illuminate the mechanism that connects progenitor addition to vacuolization, claiming only an indirect relationship with YAP signaling. However, this is admitted by the authors and not overstated

    The study provides a minimal advancement to the field by investigating an unexplored area of zebrafish notochord extension. It provides a small step toward connecting mechanical/morphogenic mechanisms with signalling in zebrafish body axis extension.

    The audience of this work is a specialized basic research group of developmental biology scientists. The research is of particular relevance to individuals studying zebrafish or axis elongation. While the authors make comparisons to other systems, due to the unique nature of the zebrafish body extension, this generates a narrow field of focus for the manuscript.

    We have previously discussed the uniqueness of zebrafish posterior body elongation in light of critical differences in the degree to which posterior growth from self-renewing tailbud progenitor populations contribute to the mechanisms of axis elongation (Sambasivan and Steventon (2021) Frontiers in cell and dev. Biol; Steventon and Martinez Arias (2017) Developmental Biology). Here too, we think zebrafish provide an important system to explore differences in the mechanisms that drive notochord elongation, and we envisage that this study will provoke a similar cross species comparison that takes into account differences in the relative timing of progenitor addition and anterior notochord expansion (that occurs much later in amniotes, for example). It is only by considering these species-specific differences across experimental organisms that we can arrive at the fundamental principles that drive developmental processes, and how evolution has acted upon these to drive change in adult body plans. We therefore respectfully disagree with the review about the scope and importance of this work for these reasons.

    In addition, we feel that the principles by which dynamic processes are coupled across an organ are broadly applicable and will illuminate further research into understanding organ growth control.

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    Referee #3

    Evidence, reproducibility and clarity

    In the studies conducted by Camacho-Macorra et al., the authors examine the extension of the body axis is zebrafish, focusing on the notochord. They specifically compare timepoints where progenitor addition to the notochord and vacuolization are important to drive axis extension. They generate a simple mathematical model of notochord extension and show that it recapitulates observations in vivo where progenitor addition and vacuolation drive tissue elongation. They further perturb the system by showing that YAP activity is localized to the midline progenitors of the notochord where when the competitive inhibitor of YAP vgll4b is perturbed it increases YAP signaling and results in increase progenitor addition to the notochord. They further describe a possible indirect-feedback mechanism linking YAP driven progenitor addition to the notochord with anterior vacuolation which when perturbed (i.e. increased YAP) results in reduced notochord elongation.

    Major Comments: NA

    Minor comments: 1.Figure 1B - please put the model equation in the figure or at least point out what variables of the equation refer to each part of the schematic.

    2.Figure 1F - smooth line is misleading, please include individual embryo measurement points. This comment could be applied to several figures

    3.Figure 2C/D - To make this manuscript more accessible to individuals who are not familiar with the anatomy of zebrafish tail, please include zoom in panels of the region of interest where arrows are pointing out increased YAP signaling in the floor plate and hypochord.

    4.In discussion - "In vgll4b mutants, increased progenitor incorporation initially does not alter overall notochord length due to a buffering mechanism for natural variation in progenitor addition" - this is not directly tested in terms of buffering for variation and is an assumption. Please either cite a paper or reword

    Significance

    Overall, the logic and experiments conducted in these studies are well defined. However, the significance of the work is minimal and makes only a small contribution to the advancement of the field of developmental biology. Regardless, the studies are well done and worth publication.

    Strengths:

    • The study does a good job of incorporating and testing a computational model in a way that proves/disproves their hypothesis
    • The manuscript is well written and follows a logical order, making it easy for readers to understand the main findings
    • The study uses multiple routes of YAP inhibition (genetic and drug) to show effect on progenitor addition to the notochord and shortened body axis
    • The discussion does aa very good job of giving the context of the study's results.

    Weaknesses:

    • The study is minimal and fails to illuminate the mechanism that connects progenitor addition to vacuolization, claiming only an indirect relationship with YAP signaling. However, this is admitted by the authors and not overstated

    The study provides a minimal advancement to the field by investigating an unexplored area of zebrafish notochord extension. It provides a small step toward connecting mechanical/morphogenic mechanisms with signalling in zebrafish body axis extension.

    The audience of this work is a specialized basic research group of developmental biology scientists. The research is of particular relevance to individuals studying zebrafish or axis elongation. While the authors make comparisons to other systems, due to the unique nature of the zebrafish body extension, this generates a narrow field of focus for the manuscript.

  3. Review Commons

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

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    Camacho-Macorra et al. investigate the mechanisms of axis extension in zebrafish embryos, focusing on the notochord and its two key elongation processes: progenitor addition (occurring early and posteriorly) and vacuolization (occurring later and in an anterior to posterior sequence). The authors first develop a mathematical model to predict notochord elongation dynamics by integrating these processes. They demonstrate that the YAP signaling pathway is active in both the notochord and its progenitors during axial extension. Their analysis reveals that vgll4b, an inhibitor of YAP, is expressed in the same regions. Knockdown of vgll4b results in YAP hyperactivation in the notochord and posterior progenitor regions, leading to increased progenitor recruitment into the notochord and a reduction in the progenitor pool. The effects of this mutation on extension are most pronounced during the late phase, which is dominated by vacuolization. The authors observe smaller vacuoles in mutants during this phase. However, early (but not late) YAP inhibition decreases notochord cell density and increases vacuole size, suggesting that YAP primarily regulates notochord progenitor uptake, which indirectly affect vacuolization.

    Major Comments

    The authors propose that YAP activity mediates a long-range feedback mechanism linking posterior progenitor addition to anterior vacuolization. Two lines of evidence are presented to support this idea. First, there appears to be compensation for tissue length during Phase 2, when both progenitor addition and vacuolization occur. Second, temporal YAP inhibition experiments show that early, but not late, YAP inactivation affects both cell addition and vacuolization. While these observations are intriguing, they do not conclusively demonstrate spatial long-range coordination. Instead, the global decrease of vacuole size could be a simple delayed consequence of cell density increase or cell disorganization at the posterior end without involving a long-range feedback along AP axis. Claiming that such long-range feedback is taking place would require a more precise characterization and/or the identification of its nature (chemical, mechanical). Furthermore, there are several caveats with the interpretations of the claims cited above. The authors do not show quantification of vacuole area using notochord cell segmentation as described in Fig 1C in vgll4b mutants at stages when progenitor addition is increased. The slope of internuclear distances in Supplementary Figure 4A at 27 hours post-fertilization suggests that vacuolization is initially normal (similar to wt context in Fig 1H), arguing against an early defect in vacuolation dynamics along the Anterior to Posterior axis that could compensate for extra addition of progenitors. Finally, the timing of the analysis of the effect of Verteporfin treatments is unclear. According to the legend of Figure 4F, analyses for Treatment A (16-27 hpf) and Treatment B (27-38 hpf) were done at 24 hpf and 30 hpf, respectively. If this is the case, the 3-hour window for Treatment B may not allow sufficient time to reveal effects on vacuolization. To strengthen the claim of long-range coupling, the authors could: Provide direct measurements of vacuolization A-P dynamics/area during Phase 2, before the effect on notochord length in the mutant, to see if there is indeed a compensatory effect on notochord length for the additional accretion of notochord progenitors in the Vgll4b mutant. Clarify the analysis timing of Treatment B to confirm that YAP inhibition during the vacuolization phase truly has no effect. Additionally, as a non-specialist, I found the distinction between the two modeling hypotheses difficult to follow. Specifically, it is unclear why the first hypothesis assumes YAP affects vacuolation rate, while the second assumes it affects vacuolation front speed. It is also not intuitive how front speed can be independent of vacuolation rate, as one would expect that if cells form vacuoles more slowly, the front should progress more slowly as well. Therefore, it could be good to clarify these aspects of the modeling part.

    Minor Comments

    While the study is technically sound, a few areas could benefit from improved clarity or additional data. An intriguing but puzzling finding is the reduction in the noto-expressing progenitor domain in vgll4b mutants, despite elevated YAP activity in progenitors. Intuitively, if YAP promotes progenitor maintenance or expansion, one might expect the noto+ domain to increase, not shrink. This paradox suggests that YAP may not only simply maintain progenitors but instead accelerates their differentiation or migration into the notochord (as stated in the manuscript and graphical abstract). Alternatively, YAP could only deplete the noto+ pool by driving premature entry into the notochord, though the lack of clear YAP upregulation in this domain would imply a non-cell autonomous role of YAP for this interpretation. The authors should discuss these possibilities more explicitly in the Discussion section and could consider including additional markers, such as proliferation assays or apoptosis markers, to clarify whether YAP affects progenitor proliferation, differentiation, or migration. In Figure 2B, the YAP activity reporter signal in the posterior floor plate is not immediately obvious. The authors should consider providing higher-magnification insets. In Figure 2C, the differences in tail shape between wild-type and mutant embryos are visually striking. If these differences have not been quantified or discussed, a brief comment in the text would be helpful. Supplementary Figure 6 describes embryo length differences in mutants but does not include a representative image. Adding one would strengthen the phenotypic description. Figure 1C is not cited in the text as not associated with a result, but just a description of the approach that is used later in Fig 4I Finally, the authors might consider citing Michaud & Pourquié (2025) when presenting the role of hydrostatic pressure in axis elongation in the Introduction.

    Significance

    This study by Camacho-Macorra et al. presents a fascinating exploration of how YAP signaling and its inhibition by vgll4b coordinate progenitor addition and vacuolization during zebrafish notochord elongation. The work is well executed, with clear results and integration of mathematical modeling and experimental data. The findings shed new light on the molecular and mechanical regulation of axis extension, a fundamental process in vertebrate development. However, while the study is innovative and rigorously conducted, the central claim of "long-range coupling" between progenitor addition and vacuolization requires further substantiation. Addressing the points discussed below will make the study more convincing and accessible to developmental biologists and mechanobiologists alike.

    reviewer expertise: developmental biologist specialised in morphogenesis

  4. Review Commons

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

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    Referee #1

    Evidence, reproducibility and clarity

    This work focuses on zebrafish notochord morphogenesis during axial elongation. In particular it dissects the role of YAP signalling on regulating the balance between caudal cell addition with the cell enlargement occurring rostrally through vacuolation.

    The article is timely to the field and includes several important experiments. The overall presentation and written style are good, citations are adequate and there is a clear effort to integrate experiments and mathematical modelling from the outset. The logic behind experiments is sound and the conclusion coherent (even if not totally unexpected given the literature): YAP affects progenitor addition which in turn changes packing, vacuolation and axis length. I just have a few points that could make the article clearer and more persuasive.

    Major points

    • Last section of results is difficult and confusing. After analysing vgll4b loss-of-function line, effectively over-activating YAP, the focus is on YAP inhibition using Verteporfin.
      • Concerns on Verteporfin: the molecule has been widely used to module YAP, but there are also plenty of studies suggesting it is non-specific (also degrades YAP, has 14-4-3σ dependency and induces stress). I would consider an alternative: truncated TEAD, LATS over-expression or gain-of-function phosphomimetic versions of YAP.
      • Presentation: regardless of point above, Verteporfin's role on YAP should be verified in the system. As such it is crucial to include: images of 4xGTIIC, noto and YAP stains after treatment. Only then inspect the effects on vacuolation and different treatments.
    • In Fig 3F, noto HCR staining is taken as evidence for progenitor exhaustion/ faster depletion. Other scenarios would be possible without more direct demonstration. Evidence (either experimental or literature) that YAP is not involved in self-renewal or induction of these progenitors at these stages should be discussed.
    • Individual datapoints in Fig 3C and 4D should be shown. Additional justification is needed as to why spinal cord is the best to benchmark displacement. Additionally looking at this with respect to mesoderm migration could capture another set of progenitors and behaviour/ displacements.
    • Plotting vacuole area in Fig.4I vs A-P position (similar to plots 1H, 2F-H) could further strengthen the point of gradual (linear) vacuolation.

    Minor points:

    • Scheme of Fig1A could benefit from having the info of zebrafish timeline (hpf)
    • Figure 3B, what was time 0?
    • The authors should address whether Verteporfin-treated mutants are rescued or whether the compound overwhelms the genetic effect.
    • Cell density is an elegant measure but quite abstract. A plot of cells detected at each AP position would be quite valuable to reinforce more cells are being added to a relatively constant area.

    Significance

    Significance included above.

  5. Version published to 10.64898/2026.02.17.706348 on bioRxiv