PGENETICS-D-26-00018

Yap1 regulates motility and vertebral development and prevents kyphoscoliosis in zebrafish

PLOS Genetics

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Pablo Wappner

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Aimée Dudley

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PLOS Genetics

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PLOS Genetics

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

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Reviewer #1: This article investigates the role of Hippo signaling in zebrafish through mutations in yap1 and wwtr1. The authors show that yap1 and wwtr1 are expressed in somatic muscle, and that ablation of each gene individually produces only minor defects in larval trunk movements or mobility, with no obvious defects in muscle development or gross architecture. Functionally, wwtr1 mutants display normal larval swimming velocity, whereas yap mutants exhibit impaired swimming, indicating that yap contributes to functional swimming behaviors. Additionally, yap mutants raised at the restrictive temperature develop scoliosis in some individuals, despite an absence of obvious musculature defects. The onset of abnormal body curvature occurs at late larval stages and is accompanied by subtle morphologic changes in the vertebral bodies in adulthood. Finally, yap is required for normal larval growth (including overall body length) independent of scoliosis. While the study does not reveal a clear mechanism for scoliosis onset, its quantitative approach suggests that small differences in growth trajectories may destabilize tissues required to maintain a straight spine, with yap playing a contributing role. I would recommend acceptance of the manuscript after revisions, as suggested by other reviewers.

Concerns to address:

Introduction: Please re-think the statement that scoliosis reduces quality of life for over 3% of people. This is at face value simply not true. In truth only about 40K surgeries are performed in the US for scoliosis per year so the number are just not correct here. In fact, while some people due experience psychosocial distress at having a curvy spine and may require bracing or surgery, in truth most people with scoliosis have no noticeable effects associated with scoliosis. Moreover, I have met people with scoliosis that have challenged my own language about how this disorder is a ‘burden’, so please be mindful of your language.

Figure 2: Makes a claim that wwtr1 mutants are ‘defective in early larval movement’. However, swimming velocity is not affected at 5 or 11 dpf? Thus, this claim stands only on the evidence of a reduction in trunk angle measures in larval fish after electrical stimulation. I would question, if this change in trunk angle does not attribute to a functional measure of movement such as swimming velocity, if the conclusion is well supported?

Figure 3: Makes a claim that yap1 mutants are ‘defective in neuromuscular function’. Again I don’t think this conclusion is supported by the evidence. Instead, I only see changes in trunk angle measure, that are now supported by reduction in larval movement. This suggest that instead yap1 mutants are defective in larval movement, as stated for Figure 2. I think you would need to demonstrate that muscle or the neuromuscular junctions were effected to make the claim above.

Line 285-286: ‘yap1 and wwtr1 have significant motility defects’ As I argued for Figure 2 , I don’t think an issue with trunk angle measures can translate to significant motility defects for wwtr1 mutants, if they functional are able to swim at a velocity also attributed with wild type animals. How is this conclusion supported by evidence?

Lines 269 and 279: “staged in days, not dpf” Can you explain your metrics for staging? Standard length, myotome number, ect? It is very important to be concise about this so that others can learn what you did and perhaps do the same one day if they needed to.

Given that Yap1 has hydrocephalus in mice and that central canal cilia defects are associated with scoliosis in zebrafish. I am not sure that these cilia are not contributing to the larval onset of scoliosis, potentially destabilizing the Reissner fiber and causing scoliosis. IT is not the same to look at multiciliate cells in the brain as these cilia do not even form in zebrafish until much later than 13dpf (PMID: 26991819). Plus, the more phenotypic yap1 mutant embryos do display phenotypes associated with cilia defects, including edema and curved bodies. It may be interesting to look at the central canal motile cilia in these mutants.

Figure 4: The cartoon schematic is difficult to read. Please use white lettering or pull them out from the blue vertebral body background to make them easily seen. I would also suggest a bracket outside the vertebral body to represent the posterior height metric.

Reviewer #2: The manuscript by Williams-Ward et al Williams-Ward et. al unravels how YAP1 determines the motility and development of kyphoscoliosis in zebrafish. Scoliosis affects over 3% of people and is influenced by genetic, environmental, and mechanical factors. This study investigated the role of the mechanosensitive Hippo pathway genes yap1 and wwtr1 in zebrafish. The Authors found that loss of yap1 impaired growth, caused early motility defects, and led to vertebral abnormalities. Although initial defects were mild, many mutants later developed kyphoscoliosis, likely due to a positive mechanical feedback process. The findings suggest that Yap1 plays a key role in mechanosensory feedback between muscle, bone, and tendon to regulate vertebral growth and prevent spinal deformities.

The manuscript is interesting and addresses the role of the Yap1 and Wwtr1 in scoliosis. The Authors do provide some intriguing findings, although the mechanistic aspects are weak. Overall, the manuscript may be revised to address the specific comments below and reconsidered.

Major comments

1. The representative data for the wwtr1kg133/+ genotype should be included in Figure 2A. Are the gaps between muscle fibers increased in the wwtr1kg133 genotype at 3 and 5 dpf?

2. At the functional level, YAP and TAZ are effectors of the Hippo pathway, leading to binding to TEAD and target gene activation; are the effects shown here mediated by TEAD?

3. Have the Authors checked if Yap1 expression changes in the Wwtr1 mutant and vice versa?

4. The authors mention that yap1kg151 mutants show no possible defects in the notochord formation. However, are the vacuolated and epithelial cells able to secrete collagen and ECM comparable to wild types. Can authors comment on the expansion of these cells?

5. Is the shortening of the embryonic axis dependent on Dstyk (dual serine/threonine and tyrosine protein kinase) activity?

6. The authors have shown the expression profiles of myod and myog in the flat mounts (Fig S5B). Are the expression profiles of myosin heavy chain genes affected in the yap1kg151 mutants?

7. Previous studies have shown the importance of Reisnerr’s fibers (RF) in axial development (H Lu, 2020; Troutwine BR, 2020). Is the formation of RF normal in the mutants?

8. Is the sarcomere organisation defective in yap1kg151 mutants?

Minor comment:

I would highly recommend authors to change the arrangement of figures to sequential manner.

Reviewer #3: In their study, Williams-Ward and colleagues have used global deletion mutants of yap1 and wwtr1 to describe a kyphoscoliosis phenotype in adults (most pronounced in yap1 mutants). Compound genetic analysis confirm existing data that these factors have some degree of redundant function. While the manuscript is well written and the data are high quality, the study falls short of making a significant contribution to understanding the cell types that require yap1/wwtr1 for proper spine development. Given the less than full penetrance of the yap1 spine phenotype, the authors conclude non-genetic factors impact the manifestation. Solid ideas are proposed in the discussion (such as altered mechanical forces - perhaps impacted by ECM production or muscle cell function). Evaluating those issues as well as the cell autonomy would go a long way to increase the impact of this study. As it stands, this work might be better suited for a more specialized journal.

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: Yes:Sam J Mathew

Reviewer #3: No

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Dear Simon,

We are pleased to inform you that your manuscript entitled "Yap1 regulates motility and vertebral development and prevents kyphoscoliosis in zebrafish" has been editorially accepted for publication in PLOS Genetics. Congratulations! Thank you for carefully considering the reviewer's comments and for adding interesting further data to the manuscript.

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Pablo Wappner

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PLOS Genetics

Aimée Dudley

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PLOS Genetics

Anne Goriely

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PLOS Genetics

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Comments from the reviewers (if applicable):

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